CN1353573A - Anti-inflammatory therapy for inflammatory mediated infection - Google Patents

Abstract

Provided are methods for inhibiting the progression of an inflammatory mediated mucosal infection. The methods include administering an effective amount of an anti-inflammatory agent. Also provided are compositions and articles of manufacture for preventing, and inhibiting the activation and progression of a mucosal infection.

Description

Anti-inflammatory therapy to inflammatory mediated infection

Invention field

The present invention relates to treat retroviral infection with antiinflammatory agent.The treatment that physical relationship of the present invention infects to HIV.

Background of invention

HIV infectious cycle originates in virus and enters target cell.It is believed that people CD4 is the major receptors of HIV identification on the T cell.HIV envelope glycoprotein (env) causes the fusion of virus and cell membrane with the CD4 receptors bind, and then promotes that virus enters the host.The final expression of evn on the host cell surface that HIV infects merged this cell and not infected CD-4 positive cell, thus virus spread.But HVI also can enter other cells such as monocyte, B cell and dendritic cell, though they do not express CD4, can be used as the storage place of virus.Known cell factor affects duplicating of HIV.The proinflammatory sexual cell factor can promote that HIV duplicates (Fauci, Nature 384:529-534,1996), beta-chemokine then suppress obligate utilize CCR5 virus duplicate that (Moore waits the people, J.Viorl.70:551-562,1996), and improve the duplicating of the viral isolates utilize CXCR4 people such as (, AIDS 12:183-190,1998) Dolei.

In the development of TD humoral immunity, the physical property contact (being subjected to for example mediation of CD4, TXi Baoshouti and MHC II quasi-molecule) in the body between helper cell and the B cell is necessary.Interaction between CD40 and the CD40 part is immunologic development and the center of keeping.CD40 is the saturating membrane glycoprotein of a kind of 45kDa, and it is the member (people such as Armitage, Nature 357:80-82,1992) in the surface molecular family with born of the same parents' intracellular domain homology of trk C, TNF acceptor, Fas, CD17 and CD30.On prematurity and ripe bone-marrow-derived lymphocyte, identified CD40, when it induces the B cell proliferation (people such as Vall, Eur.J.Immunol.19:1464-1467,1989) on monocyte, dendritic cell, the thymic epithelial cells when antibody linked.The function of CD40 on the antigen presenting cell (APC) is the collaborative costimulatory receptor (summary is seen people such as Clark, Adv.Immunol.63:43-68,1996) that promotes the T cells with antigenic specificity activation.Also identified part (being also referred to as gp39, CD40 part or the CD40L) (people such as Armitage of CD40 from molecule, 1992, the same), and find that it expresses (people such as Spriggs on the CD4+Th cell of activation, J.Exp.Med.176:1543-1550,1992).The cell of expressing gp39 protein can inducing B cell proliferation, and can induce antibody hyperplasia (people such as Armitage, 1992, the same) by other stimulus signals.Report the expression that CD40L and engaging of CD40 will be improved CD80 and CD86 and the secretion of the proinflammatory sexual cell factor in addition.

The characteristics of normal enteron aisle are that low-level mild inflammation is arranged, and it is (Shanahan and Anton, the Gut Peptides that is supported by merocrine chemotactic factor (CF) of foundation level and cell factor, J.Walsh edits (RavenPress, Ltd, New York, 1994, the 851 pages; People such as Schreiber, Gastroenterology101:1020 (1991); People such as MacDermott, Inflammatory Bowel Diseases 4,54 (1998); Luster, N.Engl.J.Med.338:436 (1998)).For the contrast of health, known stomach lymphocyte and peripheral blood lymphocyte different on function and phenotype (people such as Allison, Gastroenterology99:421 (1990); People such as Jarry, Eur.J.Immunol.20:1097 (1990); People such as McGowan, Neuroimmunomodulation 4:70 (1997)).In fact, all mucous membrane CD4+ lymphocytes are all expressed the activation mark, and all are CD45RO+ memory subgroup people such as (, J.Virol.149:2816 (1992)) Schieferdecker.In the HIV course of infection, the lymphocytic various phenotypes of described intestines T unusual normal relevant with the lymphocytic disappearance of CD4+ people such as (, Clin.Exp.Immunol.95:430 (1994)) Schnieder.

If there is not effective vaccine, the number that infected by HIV will continue to increase.In addition, owing to lack effective methods of treatment, the individuality that major part is infected by HIV all will develop into aids (AIDS), and all will die from the malignant tumour due to opportunistic infect or the immune system depletion.Though now existing anti-HIV medicines can slow down advancing of disease, still press for more effective methods of treatment and drug regimen.

Summary of the invention

Basis of the present invention is to find that retrovirus (as human immunodeficiency virus (HIV)) causes a kind of inflammatory conditions, this with thought in the past HIV be a kind of static state or the viewpoint of progressive immune deficiency state opposite.This inflammatory conditions is by replenishing other inflammatory cells containing the inflammation part that HIV infects required acceptor, and helps HIV by the infected inflammatory cell of activation (generation virion) and infect and spread.

In one embodiment, the invention provides the method that a kind of inflammation mediated sexuality that suppresses mucosal tissue is dyed, promptly contact by this tissue is united with the antiinflammatory agent that suppresses effective dose or with antiviral agent.Inflammation mediated infection may be by virus, and institute causes as retrovirus (as slow virus (lentivirus), as being selected from the Immunodeficiency virus of 1 type human immunodeficiency virus (HIV), 2 type HIV and simian immunodeficiency virus (SIV)).Make mucosal tissue and antiinflammatory agent separately or with antiviral agent unite contact can be in vivo, external or in vitro.Mucosal tissue is normally mammiferous, is preferably people's mucosal tissue.The example of this mucosal tissue comprises urogenital tissue (as vagina tissue), stomach tissue, following intestines and stomach tissue and nose-pharynx tissue etc.Antiinflammatory agent can the part or whole body give, as respectively by topical, intravenous administration, oral or parenteral.When antiinflammatory agent and antiviral agent coupling, can be before giving antiviral agent, simultaneously or give afterwards.Antiinflammatory agent can be any antiinflammatory agent, if can reduce the generation that inflammatory cell replenished, reduced chemotactic factor (CF), those antiinflammatory agents that reduce proinflammatory production of cytokines or chemokine inhibiting or cytokine receptor and its ligand interaction.These antiinflammatory agents can give separately also can unite and give, and also can give separately or coupling with other antiviral compounds.

In another embodiment, the invention provides a kind of contact separately or unites to contact by the antiinflammatory agent that makes the cell that is infected by the virus and suppress virus activation dosage with antiviral agent suppress the method that retrovirus activates.Autocrine and paracrine action by the proinflammatory conditioning agent activate the variation that inflammatory cell will cause inflammation part inflammatory cell transcriptional regulatory.Activate the retrovirus that inflammatory cell will cause activating latent infection by this process.These retrovirus comprise that slow virus is as 1 type Immunodeficiency virus HIV, 2 type HIV and simian immunodeficiency virus (SIV).Make cell and antiinflammatory agent separately or with antiviral agent unite contact can be in vivo, external or in vitro.When antiinflammatory agent and antiviral agent coupling, can be before giving antiviral agent, simultaneously or give afterwards.Cell can be a mucomembranous cell, and is normally mammiferous, is preferably people's mucosal tissue.The example of these mucomembranous cells comprises urogenital tissue (as vagina tissue), stomach tissue, the mucomembranous cell organized etc. of intestines and stomach tissue, mouth-BT and nose-pharynx down.

In another embodiment, the invention provides and a kind ofly unite the method that contacts the mucosal infections that suppresses inflammation mediated in the object separately or with antiviral agent by the antiinflammatory agent that makes patient and effective dose.Described inflammation mediated mucosal infections can be caused by virus or other cause of diseases.Virus can be retrovirus, as slow virus as 1 type Immunodeficiency virus HIV, 2 type HIV, simian immunodeficiency virus (SIV).When this contact in vivo the time, can give antiinflammatory agent by part or whole body (as respectively by topical, intravenous, oral or parenteral).When with the antiviral agent coupling, antiinflammatory agent can be before giving antiviral agent, simultaneously or give afterwards.The object of administration is generally mammal, is preferably the people.

In another embodiment, the mucosal infections that the invention provides the mediation of a kind of inflammation-inhibiting is propagated method to another object from suffering from the object that maybe may suffer from inflammation mediated mucosal infections, contact by the antiinflammatory agent of suffering from the object that maybe may suffer from inflammation mediated mucosal infections and effective dose is united separately or with antiviral agent, thereby the mucosal infections of inflammation-inhibiting mediation is propagated to other objects.Described inflammation mediated mucosal infections can be caused by virus or other cause of diseases.Described virus can be retrovirus, as slow virus as 1 type Immunodeficiency virus, 2 type HIV and simian immunodeficiency virus (SIV).When suffering from the object contact medicine that maybe may suffer from inflammation mediated mucosal infections is in vivo the time, can give antiinflammatory agent by part or whole body (as respectively by topical, intravenous, oral or parenteral).When with the antiviral agent coupling, antiinflammatory agent can be before giving antiviral agent, simultaneously and give afterwards.Suffer from the administration object that maybe may suffer from inflammation mediated mucosal infections and be generally mammal, be preferably the people.

In another embodiment, the invention provides a kind of antiinflammatory agent by making patient and effective dose and unite the method that the mucosal infections that suppresses inflammation mediated in the object develops that contact separately or with antiviral agent.Described inflammation mediated mucosal infections can be caused by virus or other cause of diseases, or relevant with the infection of viral or other cause of diseases.Virus can be retrovirus, as slow virus as 1 type Immunodeficiency virus HIV, 2 type HIV, simian immunodeficiency virus (SIV).When this contact in vivo the time, can give antiinflammatory agent by part or whole body (for example respectively by topical, intravenous, oral or parenteral).When with the antiviral agent coupling, antiinflammatory agent can be before giving antiviral agent, simultaneously or give afterwards.The administration object is generally mammal, is preferably the people.

In another embodiment, the invention provides a kind of prevention or reduce object by human immunodeficiency virus infection's possibility, antiinflammatory agent by having the object prevention effective dose that HIV infects separately or with the antiviral agent coupling, by reducing the quantity of the inflammatory cell that exists in the specified tissue (as urogenital tissue, stomach or other mucosal tissues), suppress duplicating, activate or developing of HIV.

In another embodiment, the invention provides a kind of prevention or reduce the human immunodeficiency virus is propagated possibility to another object from the object that is infected by HIV, antiinflammatory agent by giving to infect the object effective dose separately or with the antiviral agent coupling, suppress duplicating, activate or developing of HIV by the quantity that reduces the inflammatory cell that exists in the specified tissue (as urogenital tissue, stomach or other mucosal tissues), thus prevention or reduce HIV and propagate possibility to other objects from infecting object.

A kind of pharmaceutical composition also is provided, it send the antiinflammatory agent of passing the treatment effective dose that contains at least one dosage in the pharmaceutically acceptable carrier being designed for to mucosal tissue, and this dosage is the amount that can effectively suppress or reduce Immunodeficiency virus development, infection or diffusibility.

In another embodiment, the invention provides a kind of goods, comprise at least a antiinflammatory agent and this medicine operation instructions in suppressing immunodeficiency virus infection.These goods can comprise at least a antiinflammatory agent (separately), or make up with antiviral agent.This goods comprise for example condom, cotton wool, barrier film, pessary, pesseulum, suppository and enema.Operation instruction can be included in these goods, the operation instruction of prevention of immunodeficiency virus infection for example, or prevention or suppress Immunodeficiency virus and propagate operation instruction to another object from an object.

In following accompanying drawing and description, listed the detailed description of one or more embodiment of the present invention.Other features of the present invention, purpose and advantage can be obvious from narration, accompanying drawing and the claim of this paper.

The accompanying drawing summary

Fig. 1 has shown the CCR5 acceptor of expressing on blood and the casing slime CD+4 lymphocyte.The flow cytometry scatter diagram shows the lymphocyte subgroup analysis of the quantitative percentage of the cell of expressing CCR5 and/or CD4 in its representative object blood (A) and the intestines (B).Express the lymphocytic percentage of CD4+ of CCR5 in this object of the quantitaes of each figure right upper quadrant.(C) each data point is represented six objects, and data are from the blood and the intestines of each object.The intestines sample of all 6 objects is compared with blood has higher CCR5+CD4+ cell percentage (P=0.03); Disparity range 2.0-5.4 doubly.

Fig. 2 has shown the CCR5 acceptor quantity of each cell on blood and the casing slime CD4+ lymphocyte.The amount that CCR5 expresses on the CD4+ lymphocyte of one of 6 objects of streaming counting frequency curve demonstration, (A) expression blood and (B) expression intestines.Moving to right of mean fluorecence index shows that the lymphocytic CCR5 acceptor quantity of each CD4+ increases in the CCR5+CD4+ mucomembranous cell.The molecular amounts of the CCR5 that each CCR5+CD4+ lymphocyte is expressed in this individual blood of numeric representation on A and the B post and the intestines.(C) the intestines sample of all six objects has higher CCR5 and expresses (P=0.03) with comparing with blood; Disparity range is 1.4-3.5 times.Used identical among everyone symbol and Fig. 1.

Fig. 3 has shown the CXCR4 receptor expression on blood and the casing slime CD4+ lymphocyte.The flow cytometry scatter diagram shows the lymphocyte subgroup analysis of the quantitative percentage of the cell of expressing CXCR4 and/or CD4 in its representative blood samples of patients (A) and the intestines (B).Express the lymphocytic percentage of CD4+ of CXCR4 in this object of the quantitaes of each figure right upper quadrant.(C) each data point is represented six objects.Percentage between two groups of parameters does not have difference (P=0.3).Used identical among everyone symbol and Fig. 1.

Fig. 4 has shown with after HIVSX and the HIVNL4-3 infection, the micromicrogram amount of the p24 that MMC and PBMC produce.Line Chart represents to use M-tropism HIVSX (A) or T-tropism HIVNL4-3 (B) to infect after 3 hours, in the output (per 10 of the 18th, 72 and 130 hour p24 ⁴Individual CD4+ lymphocyte, micromicrogram).After 72 and 130 hours, exist the MMC culture supernatant liquor ratio of 20IU/ml IL-2 (●) that (zero) is arranged or do not have the PBMC culture supernatant of () 20IU/ml IL-2 to contain the p24 of higher concentration.The mucomembranous cell of cultivating produces more p24 and shows that they are than PBMC transreplication M-or T-tropism's HIV-1 more.

Fig. 5 has shown with the clostridiopetidase A of routine/dispase digestion and has compared that the monocytic 3 days/IL-2 of the mucous membrane of separation culture has produced CD45+, CD3+, CD4+ and CD8+ cell that quantity increases.The monocyte group who separates with each method does not show notable difference in their T cell subgroup is formed.

Fig. 6 has shown that pre-amplification processing biopsy sample causes the 5-10%RNA forfeiture.The seronegativity sample is sample (right side) after extracting with standard LTR sequence point sample (left side) parallel sample before 250 parts of extractions.The quantitative 32P emission of digitlization shows before the extraction and there is 5-10% difference in extraction back adding same amount LTR RNA.Standard items show that assay sensitivity is 10 parts of copies.

Fig. 7 has shown the internal consistency between the sample that the different parts at the colon of same circumference level (30cm) obtains.The duplicate leakage of electricity swimming of every duplicate samples.Difference average out to 0.2log SD between each individual sample.All do not detect virus in all object blood plasma.

Fig. 8 has shown the quantitative assay of HIV in the rectum biopsy.Never detect in the duplicate biopsy of object of blood plasma HIV RNA and extracted DNA.Carry out the qPCR of HIV LTR sequence, and accurately detected the copy number (figure below) of 2 duplicate samples of each registering object.(4 different objects are labeled as " sample #1-4 " respectively).2 duplicate samples of object to each are carried out the beta-globin quantitative assay in triplicate and have been drawn calibration curve (result who has only shown a biopsy is in last figure).The calculated value of HIV DNA copy is with per 2 * 10 ⁶Individual beta-globin copy (1 * 10 ⁶Individual cell) report.

Fig. 9 has shown that this separating method does not change the expression of associated receptor.Shown in trunnion axis and vertical axis, last figure is the streaming figure that directly uses the peripheral blood lymphocytes (PBMC) of CD4, CD8, CCR5 or CXCR4 antibody staining.Figure below is presented at contact and is used for behind the mucous membrane monocyte separating step result to the parallel dyeing of same individual PBMC.

Figure 10 has shown that comparing the CCR5 acceptor with blood expresses on the mucous membrane CD4+T cell of obvious higher percentage.The CD4 of seronegativity contrast (n=6) sample that dyeing is healthy and the initial Open valve of CCR5 mucous membrane sample: CD45 and sidescattering).The percentage of the cd4 t cell of CCR5 acceptor is expressed in numeric representation in the right upper quadrant.

Figure 11 shown with same target sample shown in Figure 10 in, compare with the blood cd4 t cell, the CCR5 acceptor quantity of each cell obviously increases on the mucous membrane cd4 t cell.Histogram shows that the average channel fluorescence volume relevant with each cell receptor quantity increase in the mucous membrane prepared product significantly moves to right.

Figure 12 is presented at the CCR5 expression ratio blood CD4+T cellular expression that detects mucous membrane CD4+ cell in normal struvite and the sample that HIV infects to be increased.Shown that seronegativity normal healthy controls (n=6), inflammatory contrast (n=4) and HIV infect (n=8) person's the CD4+ and the average percent of the two staining cells of CCR5+.Significant difference in clinical group of the P value representation under the group of objects x axle between blood and the enterocyte.Mucous membrane is expressed the significance level between the cd4 t cell of CCR5 between clinical group of the P value representation on figure top.

Figure 13 A and 13B shown in IBD and HIV (A) blood and (B) in the intestines CCR5+CD4:CD8 ratio descend.Left figure shows that healthy person, seronegativity contrast, seronegativity inflammation collator and HIV infect the relative expression of CCR5 in the object blood.What show in the square frame under the figure is the ratio of expressing CD4+T cell with the CD8+T cell of expressing CCR5 of CCR5.(A) shown the lymphocytic similar pattern of mucous membrane in.

Figure 14 has shown with peripheral blood lymphocytes (PBMC) and has compared that the amount of p24 in the mucous membrane monocyte (MMC) (index that HIV produces) is obviously higher.

Figure 15 A and 15B have shown that the CD8+ cell increases in the HIV infected patient colon.Biopsy is from (A) HIV (-) and (B) HIV (+) colon.The CD8+ cell is brown (being dark-coloured in black-and-white photograph).

Figure 16 A and 16B have shown the increase of CCR5+ cell in the HIV infected patient colon.Biopsy is from (A) HIV (-) and (B) HIV (+) colon.The CCR5+ cell is brown (being dark-coloured in black-and-white photograph).

Figure 17 A has shown in HIV (+) patient of the HIV (+) of the infected not, the low amount of mucosal virus and mucosal virus a large amount (A) RANTES, (B) IFN γ and (C) amount of THF to 17D.(D) shown the increase (shown in the HLA-DR that arrives increases because HIV has induced the increase of the proinflammatory sexual cell factor) of activation cd4 cell quantity.The y axle represents that the cd4 cell % that activates, x axle represent the amount of virus.

Figure 18 has shown the increase of virus (Nlegfp) amount that is produced by mucomembranous cell (MMC) and haemocyte (PBMC), and egfp is expressed as shown in the increase when duplicating in cell as Nlegfp.

Figure 19 has shown the influence that Asacol (aminosalicylic acid) duplicates HIV.With shown in the 5-ASA or the AZT of amount handle the cultured cell infected by HIV.Expression (amount that its expression HIV duplicates) with the plain enzyme of luminous photometer quantitative fluorescence.Shown data represented 9 independently tests.

Detailed Description Of The Invention

Must notice, unless otherwise indicated, the singulative in this paper and the claims " ", " with " and " being somebody's turn to do " comprise plural number. So for example " a kind of cell " also comprises many this cells.

Unless otherwise indicated, all technology used herein are all identical with the common implication of understanding of those skilled in the art with scientific terminology. Although also can be used for implementing or test the present invention with similar any method, device and material described herein, describe method for optimizing, device and material here.

For describing and open clone, antibody and methodological purpose, this paper includes the publication of all references in as a reference, the use that combines that may describe with the present invention described in these publications. When if conflict is arranged, comprise with specification of the present invention being defined as contrast. On to address herein described publication all be their disclosed contents before being provided at separately the application and proposing. This paper can not be interpreted as allowing the inventor to have the right to say the disclosed date of existing invention than actual Zao.

Term used herein " inhibition " or " inhibition " but refer to lower active, function or characteristic with measured quantity, as be reduced by at least 30% or more than. When there being multiple different when may be repressed active (as prevent that cell from replenishing, generation, cell or the viral activation of proinflammatory conditioning agent, virus replication or virus development or propagation), any single reduction of planting active (have or without other activity) all is enough to be included in the range of definition of the present invention. In addition, when giving one or more medicines and suppress activity, drug-inducedly anyly singly plant active reduction or any singly plant active reduction and all be enough to be included in the range of definition of the present invention by what drug regimen caused by a kind of. " inflammation amount of suppression " refers to regulate, suppress or suppress the amount of the required anti-inflammatory agent of inflammatory reaction or symptom.

Term used herein " activation " (when being used for virus) refers to the increase of whole body or local virion or virus quantity, or virus protein or the synthetic increase of nucleic acid in the cell. This increase is normally induced by the variation of infection cell transcriptional state, causes cell to produce the increase of virus. This increase may not rely on this variation of transcriptional state yet and occurs. The variation of infection cell transcriptional state may be induced by cell factor, chemotactic factor (CF) and/or other molecules of regulating cell proliferation, differentiation or activity (chemotaxis). The variation of infection cell transcriptional state also may be induced by virus or other cause of diseases (bacterium, fungi, mycobacterium etc.), perhaps owing to contact immune regulative antigen (such as LPS) is induced. The cell quantity increase that common activation will cause virus quantity to increase and then can cause being infected by the virus is also referred to as " virus diffusion ".

Term used herein " inflammation mediated " refers to make in the reaction of object to inflammatory response infection, disease, disorder or illness development or diffusion or accelerates or worsen when being used for infection, disease, disorder or illness. In the example that retrovirus (such as HIV) infects, PD may occur, such as the inflammation part that exists to infection cell and virus by target (infecting) cell migration. The cell that replenishes provides new target cell for virus infections, infects development thereby cause the virus diffusion to make. Inflammation mediated situation also comprises when the cell (as being in latence) that infects and immune modulatory molecules or other signaling molecules (such as the molecule of proinflammatory cell factor or other enhancing inflammatory responses) when contacting, regulate the cell transcription state, thereby stimulate or increase the amount that cell produces virus.

Term used herein " mucosal tissue " refers to any tissue that can find mucomembranous cell, such as comprising stomach-intestinal tissue (such as stomach, small intestine, large intestine, rectum), urogenital tissue (such as vagina tissue, penile tissue, urethra), nose-larynx tissue (such as nose tissue, larynx tissue), mouthful (BT) etc. Other mucosal tissues are known, and those skilled in the art are not difficult to determine.

Inventor of the present invention finds that retroviral infection (infecting such as immunodeficiency virus infection such as HIV) is a kind of inflammation that exists in the object tissue (such as mucosal tissue). Many reports emphasize this be a kind of mucous membrane medium size lymphocyte reduce or " anti-inflammatory " state and the infected's blood in to carry out finding similar. Because do not exist CD4+ cell activation that quantity increases, Memorability, expression coreceptor in the mucosal tissue of infected individuals in health, very high by the infectibility of these tissues. In the typical case who infects was replied, mucosal immunity cell (most of such as CD8+T lymphocyte and macrophage) was secreted the level rise of proinflammatory chemotactic factor (CF) and cell factor and is replenished other T-lymphocytes to the position of mucosal infections. This rising reply or the new target cell effect that provides quantity obviously to increase to infection is provided " inflammatory response " (although attempt be restriction infect), and be conducive to the diffusion of virus. So, the present invention relates to process multiextent retroviral infection, disease and disorder that reverse transcription is relevant, by alone anti-inflammatory agent or with the antiviral agent coupling, with prevention or suppress the Inflamed tissue that other permissive cells replenish object, thereby the prevention inflammatory cell is by inflammation mechanism activation inflammatory cell, with other proinflammatory media by suppressing these approach and prevent other objects (as do not infect or infection) propagation that contacts with mucosal tissue. Can comprise that with the retroviral infection of method treatment of the present invention the retrovirus that is caused by many retrovirus is disorderly.

For example, in the macaque that SIV infects, intestines and stomach is the main position of early stage CD4+ Lymphocyte depletion and virus replication, thereby to infect may mainly be a kind of mucomembranous immune system disease (people such as Veazey, Science 280:427 (1998) to prompting SIV; MacDonald and Spencer, " stomach and liver immunology ", R.H. Heatley edits (Cambridge University Press, 1994). For the people, HIV infects and also comprises mucomembranous immune system, directly be recovered to infectious viral particle from the mucous membrane sample, original position research has shown that lamina propria T lymphocyte belongs at first infected cell (people such as Koteler, Am.J.Pathol.139:823 (1991); The people such as Heise, J.Infect.Dis.169:1116 (1994); The people such as Heise, Am.J.Pathol.142:1759 (1993); The people such as SmitMcBride, J.Virol.72:6646 (1998); The people such as Clayton, Gastroenterology 103:919 (1992); The people such as Ellakay, Am.J.Clin .Pathol., 153:481 (1998). In addition, inserting rectosigmoid mucous membrane inwall in the property sexual intercourse process at anus is the virus main position of introducing people such as (, Am.J.Pathol.153:481 (1998)) Patterson.

Inflammation

Inflammation is to be caused by many many single reactions or reactions of relevant cascade that caused by proinflammatory medium (comprising cell factor, prostaglandin, leukotrienes, chemotactic factor (CF), adhesion molecule (such as LFA-1) and other media well known by persons skilled in the art). For example, chemokine receptors plays key effect in allowing cell entry CD4+ cell. The part of these acceptors (being called chemotactic factor (CF)) is also extremely important. These chemotactic factor (CF)s and Preinflammtory cytokine all are the main stimulants of cell, but also play other effects in the expansion of cascade of response of inflammation. These solubility inflammatory mediators mainly produce from the CD8+ cell. In case produce they just can paracrine or the autocrine mode work and further activate near the cell their and raise other T cells to inflammation part. These lymphocytes self that replenish are activated, thereby have enlarged cascade of response of inflammation.

Inflammation causes stimulating lymphocyte and macrophage (CD4+ cell).To induce when those contain the inflammatory cell irriate of HIV and produce a large amount of virus.(even patient of healthy not infected by HIV) keeping a kind of low-level physiological inflammatory conditions in the intestines, and this is that potential pathogen of protecting the intestines inwall to avoid its surface contact is invaded necessary.The most of lymphocyte and the macrophage that constitute this infiltration expression activate.The main target cell of HIV (virus can be duplicated most effectively therein) is the CD4+ cell that activates.Such cells fill stomach mucous membrane.The increase of virus replication causes HIV to pass through bigger diffusion of mucous membrane and higher mucous membrane HIV quantity of viruses.The main purpose of anti-HIV treatment is to reduce the ability that HIV duplicates, thereby reduces its diffusion between CD4+ cell (finally by virus damage).When duplicating, HIV reduced the sudden change that causes the enantiopathy cytotoxic drug to tolerate to cause virus in its genetic material, not produce effectively.Immunosuppressive activity used herein refers to suppress or reduces the respond of B and T cell and do not raised inflammation part or become activating cell.NSAID can reduce the synthetic of prostaglandin (PG).PG is the cell arrestant.

Signals of other activation inflammatory cells comprise that adhesion receptor such as LFA-1 (CD11a and CD18) are incorporated into its pairing acceptor such as ICAM-1 (CD54) (people such as Staunton, 1990, Cell 61:243-254).If secondary signal is blockaded, then T cells with antigenic specificity will be dead by apoptosis, or enter cell anergy state.Block this interaction with the monoclone antibody of LFA-1 and ICAM-1 and will increase the time-to-live (people such as Isobe, 1992, Science 255:I 125-1 127) of accepting heart heteroplastic transplantation mouse.

The stomach mucous membrane is an adenoid part, and the evidence prompting of growth all involves mucous membrane at all stage HIV of disease.Intestines and stomach is not only the path that most of patient disease is propagated, and also is maximum lymphatic tissue.As mentioned above, the feature of stomach mucous membrane is low-level physiology inflammatory conditions, and its most of lymphocyte activates.The proinflammatory cell factor that is present in the natural high concentration in the mucous membrane be it seems and increased duplicating of this position HIV, causes the new stomach target CD4+ cell of mucous membrane HIV virus quantity height and consecutive infection.

The present inventor finds that most of stomach CD4+ cellular expression HIV enters necessary chemokine receptors.Most lymphocytes of stomach mucous membrane are expressed CCR5 and CXCR4.As if for CCR5, mucous membrane monocyte (MMC) is expressed the monocyte height (calculating with each cell) that the level of this receptor is derived than blood.The present inventor finds that also external mucomembranous cell more is subject to HIV than peripheral blood cells and infects.

Can in the mucous membrane of the individuality that most of HIV infects, find HIV nucleic acid; Human gag Auele Specific Primers such as Kotler detect HIV DNA by PCR in its 70% patient who studies (20).Even the present inventor finds the patient who does not detect virus in the blood plasma virus of just duplicating is arranged also in their mucous membrane.No matter macaque is to infect by intestines or by parenteral route, all finds a large amount of SIV in the stomach mucous membrane, and this shows that the congenital height of mucous membrane is subject to HIV and infects.After the infection, these macaques show in early days (in 7-21 days) stomach mucous membrane CD4+ cell and seriously lose.Do not observe the active signal of so vigorous HIV at other lymphatic tissue positions.The mankind, after existing people is described in chronically infected early stage asymptomatic stage and clinical AIDS morbidity, the disappearance of mucous membrane CD4+ cell in colon and the duodenum.

The present inventor finds retroviral infection, as the infection relevant with Immunodeficiency virus, especially HIV with inflammatory conditions.Inflammation can be cellularity, solubility or both.Inflammation can occur in the tissue (as mucosal tissue) of any amount susceptible viral infection.Confirm inflammatory process and HIV infect relevant and and the effect of chemotactic factor (CF) and chemokine receptors (its function is the coreceptor of HIV) about the method for this retroviral infection that is caused by IDAV (as HIV-1/2) of a kind of novel treatment is provided.

The inflammatory cell of in the mucosal inflammation zone, finding (comprising most of CD4-positive t lymphocytes) be activation, the memory phenotype, express the cell of the essential coreceptor of high-level HIV, be the preferred target cell of HIV.The coreceptor (as the acceptor of beta-chemokine) that comprises chemokine receptors etc. is a normal part of endogenous inflammatory immune response function.These chemotactic factor (CF)s and acceptor initiation cyclicity inflammatory cell thereof significantly add to mucosal sites when activation, cause cellularity solubility inflammation.Infect activation, development and the diffusion that (owing to existing inflammation to strengthen) will provide a kind of effective method to reduce this mucosal tissue disease by suppressing mucosal tissue with antiinflammatory agent.

Therefore, the employing antiinflammatory agent provides a kind of useful method to relax, control or has reduced the inflammatory response that retroviral infection causes.Because inflammation often (comprises by the proinflammatory medium, but be not restricted to prostaglandin, leukotrienes, cell factor, chemotactic factor (CF) and other media well known by persons skilled in the art) cause the additional and activation of other inflammatory cells, so reduce this quantity that can reduce the available CD-4 positive cell of HIV infection of replenishing and activate.

So, can be used for antiinflammatory agent of the present invention and comprise that those can reduce inflammatory cell and replenish, reduce medicine that chemotactic factor (CF) and proinflammatory cell factor (keeping the inflammation cascade reaction) produce and chemokine inhibiting or cytokine receptor (as the interaction by chemokine inhibiting acceptor and its part) and come the medicine of prevention of inflammation signal propagation.These medicines comprise " anti-inflammatory antibody ", and they can be incorporated into the protein molecule that above-mentioned antibody discerns and suppress its biologically active.These antibody comprise designed can with the antibody of cell factor, cytokine receptor, chemotactic factor (CF), chemokine receptors reaction, design them and reduce or patient (for example people) is replied or offered to prevention of inflammation.

The present invention has also considered various pharmaceutical compositions, and they can be blocked duplicating of reverse transcription and Immunodeficiency virus or block the secretion of cell factor in to the reaction of HIV replication.Pharmaceutical composition of the present invention can be prepared into the form (as pharmaceutically acceptable carrier) that is fit to give object by peptide, nucleotide sequence or other antiinflammatory agents or the medicine of the present invention with antibody, separation with carrier, excipient and additive or adjuvant.Normally used carrier or adjuvant comprise magnesium carbonate, titanium dioxide, lactose, mannitol and other sugar, talcum powder, milk protein, gelatin, starch, vitamin, cellulose and derivative thereof, animal and plant oil, polyethylene glycol and solvent (as sterile water, alcohol, glycerine and polyalcohol).Intravenous vehicles comprises liquid and nutritious supplementary pharmaceutical.Preservative comprises antimicrobial, antioxidant, intercalating agent and inert gas.Other pharmaceutically acceptable carriers comprise the aqueous solution, non-toxic excipients, comprise salt, preservative, buffer etc., referring to Remington ' sPharmaceutical Sciences, the 15th edition, Easton:Mack Publishing Co., 1405-1412,1416-1487,1975 and The National Formulary XIV, the 14th edition, Washington:American Pharmaceutical Association, 1975, this paper fits into as a reference in them.Regulate the pH and the accurate concentration of various compositions in the pharmaceutical composition by the ordinary skill in the art.Referring to the The Pharmacological Basis for Therapeutics of Goodman and Gilman, the 7th edition.These pharmaceutical compositions can comprise one or more antiinflammatory agents and one or more antiviral agents of associating.

In another embodiment, the present invention relates to blocking-up or suppress HIV replication or diffusion, or in to the reaction of Immunodeficiency virus, block or suppress the method for cytokine secretion.This method comprises the pharmaceutical composition that contains The compounds of this invention and pharmaceutically acceptable carrier that gives object treatment effective dose." giving " pharmaceutical composition of the present invention can realize by any method known to those skilled in the art." object " refers to any mammal, especially refers to the people.

Preferably prepare pharmaceutical composition and give with dosage unit.Solid dosage unit is tablet, capsule and suppository.For the treatment patient,, need different daily doses according to character and the order of severity, patient's age and the body weight of the activity of compound, administering mode, disease.And in some cases, higher or lower daily dose may be suitable.Giving of daily dose can the single dose form single gives or is divided into less several times dosage, repeatedly gives with the specific interval time.

Dosage can not be greatly to causing adverse side effect, as bad cross reaction, allergy etc.Usually, dosage should be different by the degree of patient's age, illness, sex and disease or infection, can be determined by those skilled in the art.If can regulate dosage by patient's doctor when contraindication is arranged, this need not too much test and just can determine.In any case, the validity of treatment can be determined by the HIV rna level or the dna virus load of for example monitoring immunodeficiency virus infection patient inflammation part (as mucosal tissue), or comprise that by additive method measuring inflammatory mediator, cell factor, chemotactic factor (CF) and/or CD4+ cell determines.In the tissue minimizing of the relative populations of CD4+ cell, cell factor, proinflammatory medium or chemotactic factor (CF) level or stable should be relevant with the level of inflammation in individual or the tissue.

Pharmaceutical composition of the present invention gives with part, intravenous, oral or parenteral mode usually, or implants.Rectum and vagina administration proof are more effective, because they are the positions that at first contact virus and mucosal tissue inflammation usually.Medicine can contact these positions and infect or propagation with prevention or inhibition.The suitable solid-state or liquid drug dosage form such as the injection solution of particle, powder, tablet, sugar coated tablet, little micella, suppository, syrup, emulsion, suspending agent, emulsifiable paste, gel, aerosol, dropping liquid or ampoule form, also can be prepared into the reactive compound of slowly-releasing, use excipient, conditioning agent and/or adjuvant such as disintegrant, bond, coating agent, swelling agent, lubricant, flavor enhancement, sweetener or solubilizer in the preparation as mentioned above usually.This pharmaceutical composition is fit to use various drug delivery system.Existing medicine send the brief overview of the method for passing referring to Langer, Science, and 249:1527-1533,1990, this paper includes it as a reference in.

Pharmaceutical composition of the present invention can the part or whole body give." treatment effective dose " refers to prevent or treat or prevent or reduce to small part the amount of disease symptoms and the necessary The compounds of this invention of complication thereof.So the effective dose of using depends on the order of severity of disease or infection and the body weight and the overall state of object certainly.Usually, the dosage of external use can provide original position to give the guide that this pharmaceutical composition has consumption, and animal model can be used for determining the effective dose of treatment particular disorder.The various factors that will consider have been described, Goodman that edits as people such as Gilman and " the Pharmacological Bases of Therapeutics " the 8th edition of Gilman, Pergamon Press, 1990; And Remington ' s Pharmaceutical Science, the 17th edition, Mack Publishing Co., Easton, Pa, 1990, this paper includes them as a reference in respectively.HIV RNA or the dna level or the quantity of viruses of the inflammation part (as mucosal tissue) by determining the immunodeficiency virus infection patient; Minimizing by one or more symptoms relevant with infection; Or comprise that by additive method the method known with those of ordinary skills measures the amount of inflammatory mediator, cell factor, chemotactic factor (CF) and/or CD4+ cell, monitor the validity of dosage.

Immunotherapy method of the present invention comprises the method for being prevented by immunodeficiency virus infection danger person being in.For example, be used for method to the people who is in the HIV risk of infection." prevention effectively " amount of antiinflammatory agent refers to reply by minimizing, inhibition or prevention of inflammation and can suppress the amount that HIV duplicates, activates or develops.HIV propagates by at least three kinds of known paths: sexual intercourse, blood transfusion (blood products) and placenta.Infection by blood comprises the propagation between the intravenous drug user.Because contact HIV not necessarily causes Symptomatic infection (measuring as seroconversion), everyone has potential danger, so be considerable with methods of treatment of the present invention as prophylactic treatment.

Anti-inflammatory composition of the present invention can be used for prophylactic activity or pre-preventing transmission, comprises the condom as scribbling antiinflammatory agent or lubricating with antiinflammatory agent; The condom that comprises capsule, capsule discharge antiinflammatory agent after being used to break; The vagina articles for use comprise barrier film, pessary, cotton wool, scribble or with the lubricated ring of antiinflammatory agent; Vaginal washing fluid, emulsifiable paste, lubricationg jelly, suppository, foam or contain smart gel of killing of antiinflammatory agent and other compositions and the goods that the antiinflammatory agent part sent the mucosal tissue that contacts when passing sexual intercourse well known by persons skilled in the art.These goods can be used for prevention or suppress the patient infecting to other people, or are used for prevention or suppress the sufferer and propagate to other people.

Composition described herein and that use in the methods of the invention can be before being subjected to Immunodeficiency virus (promptly prophylactically) or infect at any stage as described below and initial stage after give the patient, or after infection to the propagation that is prevented subsequently.For example, HIV infects and can be divided into following process:

(1) about 15% the infected has acute disease, is characterized as fever, fash and enlargement of lymph nodes and contacts the interior meningitis that takes place of six weeks of HIV.After this acute infection, these individualities become asymptomatic.

(2) remaining HIV the infected can not have symptom several years.

(3) some individual continuation general lymphadenopathy (PLG) that takes place is characterized as neck, groin and axillary gland swelling.The individuality of 5-10%PGL will revert back to asymptomatic.

(4) any of these individuality may develop into the relevant syndrome (ARC) of AIDS, and ARC patient can not revert back to no disease symptoms attitude.

(5) ARC and PGL patient and asymptomatic person final (after several months or several years) will develop into AIDS, and must cause death.

Antiinflammatory agent

" antiinflammatory agent " used herein refers to can reduce, prevent or regulate the medicine of inflammatory reaction, as replenishing by the minimizing inflammatory cell; Reduce the generation of chemotactic factor (CF); Reduce the generation of proinflammatory chemotactic factor (CF); Reduce the proinflammatory production of cytokines; Or the interaction of chemokine inhibiting or cell factor and its acceptor.These medicines comprise as anti-inflammatory antibody (as the antibody of antibacterial agent, anti-acceptor), peptide (as the agonist or the antagonist of inflammatory mediator, the antagonist of cell factor such as IL-1 or acceptor such as IL-1 acceptor, or soluble TNF acceptor), nucleic acid (as the nucleic acid of coding antiinflammatory agent, anti-inflammatory peptides, ribozyme or antisense molecule), steroids (as prednisone), nonsteroidal anti-inflammatory agent (as aspirin), 5-ASA product, normally used antiinflammatory agent and their combination.

The example that is used for anti-inflammatory antibody of the present invention comprises the antibody of cell factor and acceptor thereof, as the antibody of anti-IL-8 acceptor, the antibody of antibacterial agent (as the antibody of anti-THF, REMICAD  as the Centocor manufacturing), anti-chemotactic factor (CF) antibody is (referring to people such as Olson, J.of Virol.73 (5): 4145-4155 (1999), this paper includes it as a reference in) antibody (as anti-CCR5 or anti-CXCR4 receptor antibody) and their combination of anti-chemokine receptors.Other antibody comprise the antibody at the enzyme of the enzyme path that produces the proinflammatory medium, as U.S. Patent No. 5,767, and the antibody of 1 type phosphate A2 described in 249, this paper includes this patent as a reference in).

The example that is used for anti-inflammatory nucleic acid of the present invention comprise coding anti-inflammatory peptide nucleic acid, cutting coding proinflammatory polypeptide (as cell factor or chemotactic factor (CF)) RNA ribozyme, can with the antisense molecule of the nucleic acid array hybridizing of coding proinflammatory medium (as cell factor, cytokine receptor, chemotactic factor (CF), chemokine receptors, other inflammatory peptide or acceptors disclosed herein) and their combination, or those skilled in the art's antisense molecule of being not difficult to identify.

The example of anti-inflammatory peptide comprises: LFA adhesion molecule antagonist, cytokine receptor antagonist, transcription factor, soluble T HF-α receptor polypeptides.Other anti-inflammatory peptides comprise for example transcription factor such as NF-kappa B (people such as Schottelius AJ, Int J Colorectal Dis 1999 Feb; 14 (1): 18-28), the peptide (U.S. Patent No. 5,776,892, this paper includes it as a reference in) of platelet factor 4 and based on U.S. Patent No. 5,766, the peptide of the disclosed CD14 of 593 (this paper includes it as a reference in).

The example of anti-inflammatory cytokine comprises as cell factor and transcription factor.Anti-inflammatory cytokine comprises (Watson ML, Am J Respir Cell Mol Biol on May 1st, 1999 as IL-13; 20 (5): 1007-1012); IL-4 and IL-10 (people such as Jarvelainen HA, Hepatology in May, 1999; 29 (5): 1503-10), IL-6 (people such as Klimiuk PA, J Immunol.1999 1:162 in April (7): 429-4299) and other anti-inflammatory cytokine well known by persons skilled in the art.

The example that is used for antiinflammatory agent of the present invention comprises various steroid classes, non-steroid class, salicylic acid is water-soluble and water-insoluble drug and their acid-addition salts or slaine.As long as antiinflammatory agent keeps its medical value, also can adopt its organic and mineral salt.Antiinflammatory agent can be selected from the therapeutic agent of broad range and the mixture of therapeutic agent (can slowly-releasing or continuous action form give).

Nonsteroidal anti-inflammatory agent (NASID) comprises the compound of various chemical constitutions.It is believed that if not whole its major parts all have common mechanism of action, nearly all is organic monoacid.This big compounds can be divided into 2 groups, carboxylic acid (R-COOH) and bmap acid (R-COH).Also can further divide into groups by chemical constitution.The main grouping of bmap acid is pyrazolone such as phenylbutazone, oxyphenbutazone, analgin and isopyrin, and the former times health (xicam) that comprises piroxicam and Mi Luo former times health (miloxicam).The carboxylic acid subgroup comprises salicylate, as acetylsalicylic acid (aspirin); Propionic acid such as brufen and naproxen; Anthranilic acid such as meclofenamic acid; Phenylacetic acid such as acetaminophen; Amino-nicotinic acid such as fluorine ammonia nicotinic acid (flunixin); With indoline such as Indomethacin.

With regard to the known NASID of those mechanism of action, found most ofly to suppress the formation of arachidonic acid metabolite, thereby reduced the inflammation of these metabolites mediations by suppressing cyclooxygenase and lipoxygenase path.Cyclooxygenase changes into annular endoperoxide, PGG2 and PGH2 (being also referred to as PGs) with arachidonic acid.By the effect of other enzyme-specifics, these compounds change into inflammatory mediator family, and it comprises different members-eicosanoid of PGE2 and PGI2.Yet the structure of cyclooxygenase has nothing in common with each other in the different tissues, and the ability of NASID and these enzymes associating also has nothing in common with each other, and this has just explained effectiveness and the kind difference between replying.

The non-limitative illustration example of nonsteroidal anti-inflammatory agent (standard volume and chemical constitution) with trade (brand) name, generic name, dosage, comprise following medicine: brufen (as MOTRIN  300,400,600,800mg, ADVIL  200mg) (±)-2-(right-isobutyl phenenyl) propionic acid; Tolmetin (TOLECTIN ) 5-(right-toluoyl)-1-methylpyrrole-2-acetate; Naproxen (as ALEVE , ANAPRFX  or NAPROSYN ) 250,375 and 500mg, 6-methoxyl group-alpha-methyl-2-methyl, (+); Flurbiprofen (ANSAID ), 50mg and 100mg, 2-fluoro-Alpha-Methyl-[1,1 '-diphenyl]-4-acetate, (±); Sulindac (CLINORN ) 150 and 250mg, 5-fluoro-2-methyl isophthalic acid-[[right-(methyl sulfenyl) phenyl]-methylene]-1H-indenes-3-acetate; Diflunisal (FLOVACIL ) 250 and 500mg, 2 ', 4 '-two fluoro-4-hydroxyls-[1,1 '-diphenyl)-the 3-carboxylic acid; Piroxicam (FELDENE ) 4-hydroxy-2-methyl-N-2-pyridine radicals-2H-1,2-benzothiazine-3-carboxamide 1,1-dioxide; Indomethacin (INDOCIN ) 25 and 50mg, 1-(4-chlorobenzoyl)-5-methoxyl group-2-Methyl-1H-indole-3-acetate; Etodolac (ULTRADOL ) 1,8-takes off ethyl-1,3,4,9-oxinane-[3,4-b] indoles-1-acetate; Meclofenamate sodium (MECLOMEN ) 50 and 100mg, N-(2,6-two chloro-M-tolyls) anthranilic acid sodium salt, monohydrate; Fenoprofen and fenoprofen calcium (NALFON ) be as dihydrate, 200mg and 300mg, the derivative of Arylacetic acids, Alpha-Methyl-3-phenoxy group phenylacetic acid; Ketorolac tromethamine; Ketoprofen (ORUDIS ), 25,50 and 75mg, 2-(3-benzoyl phenyl)-propionic acid; The fragrant sodium acid of first chlorine sodium; Mefenamic acid (BONABOL ) N-(2, the 3-xylyl) anthranilic acid; Nabumetone; Ketorolac tromethamine; C14H10Cl2NNaO2 (PROPHENATIN ) 2-[(2, the 6-dichlorophenyl) amino] phenylacetic acid list sodium salt; Bromfenac sodium; Phenylbutazone (BUTAZOLIDIN ) 100mg, 4-butyl-1,2-diphenyl-3,5-pyrrolidine-diones; Other cox 2 inhibitors are as match sieve comparable (celocoxib), Meloxicam, aulin and Luo Fei comparable (rofecoxib); Suprofen; Fragrant brufen (fenbuprofen); Fluprofen; Midol-PMS, acetaminophen, 500mg; Tylenol Extra Strength, acetaminophen, 500mg; Neurosedyn; The Evil promazine; Contain bigcatkin willow ester compound and evening primrose oil (containing have an appointment 72% linoleic acid and about 9% gamma-linoleic acid), its single isomer and their combination.

The non-limiting instantiation of salicylate antiinflammatory agent comprises following medicine: bismuth subsalicylate; Salsalate; Salicylic acid and salicyclic acid derivatives are as thiosalicylic acid sodium, choline salicylate, magnesium salicylate, Diflunisal, brufen, NAP, sulindac, Diflunisal, disalicylic acid, choline three magnesium salicylates, acetylsalicylic acid, salsalate, sodium salicylate and their combination; 5-aminosalicylic acid (5-ASA) and contain product or the compound of 5-ASA, as: oral aminosalicylic acid is (by Procter ﹠amp; The ASACOL  that GamblePharmaceuticals makes, the PENTASA  that Roberts Pharmaceuticals makes), aminosalicylic acid rectal enema or foam or suppository, sulfasalazine, Balsalazide, ipsalazide and olsalazine (the DEPENTUM  that Pharmacia Upjohn makes) and their mixture.

Steroidal anti-inflammatory drugs comprises glucocorticoid.The non-limiting instantiation of steroidal anti-inflammatory drugs comprises following medicine: flunisolide, fluoxyprednisolone, acetone fluoxyprednisolone, beclomethasone dipropionate, BDP, hydrocortisone, cortisone, dexamethasone, budesonide, metacortandracin, methylprednisolone, prednisolone, the ester of these compounds and their combination.Other limiting examples of antiinflammatory agent comprise Thalidomide (being made by Celgene).

Retrovirus

Retrovirus is a kind of RNA viruses, and promptly Bing Du genome is RNA.When host cell was reversed the record virus infections, its geneome RNA reverse transcription was the DNA intermediate product, and this DNA product is integrated in the chromosomal DNA of infected cell efficiently.The DNA intermediate product that is integrated is called provirus.Retrovirus family comprises strand coating RNA viruses, infects mammal such as ox, monkey, sheep and people usually.Retrovirus is very unique in RNA viruses because they duplicate the DNA copy that comprises synthetic RNA, subsequently it is integrated as the genome of infected cell in.

Retrovirus family is made up of three classes: foamy virus (or foam-like virus) is Human foamy spumavirus (HFV) for example; Slow virus such as visna virus; And oncogenic virus (though not all virus all is carcinogenic in this group).Its conventional meaning of term used herein " slow virus " is used to describe a Tobamovirus that contains revertase.Slow virus comprises " Immunodeficiency virus ", and it comprises 1 type and 2 type human immunodeficiency viruses (HIV) and simian immunodeficiency virus (SIV).When lacking effective methods of treatment, the individuality of most of infected person Immunodeficiency virus will develop into aids (AIDS) and die from opportunistic infections and because the malignant tumour that immune system is damaged or viral direct effect causes.Oncogenic virus can according to the form of particle (as in the virus maturation process under electron microscope finding) further be divided into category-A, category-B, C class and D class.The category-A particle is the B-seen in the cytoplasm of infected cell and the immature particle of D-type virus.These particles do not have infectivity.The category-B particle is wrapped in cytoplasmic membrane by intracytoplasmic category-A particle and sprouts from film as the virion of maturation.They have the toroid core of a 75mm on film, and long glycoprotein spike thing is projection thus.After sprouting, the category-B particle contains a cipher telegram daughter nucleus heart that is positioned at eccentric position.Prototype category-B virus is mouse mammary tumor virus (MMTV).In the cell that the C viroids infects, do not observe the endochylema endoparticle.On the contrary, ripe particle directly sprouts from cell surface by the cohesion of crescent shape ' C ' shape, and closed in itself is surrounded by cytoplasmic membrane then.The envelope glycoprotein thrust can be seen with the uniform cipher telegram daughter nucleus heart.Sprout intracellular vacuole may take place from the cytoplasmic membrane on surface or directly enter.The C viroids is the most generally studied, and comprises many birds and muroid leukemia virus.The form that bovine leukemia virus (BLV) and I and II type adult T-cell leukosis virus (HTL-I/II) are sprouted from cell surface because of them classifies as C class particle similarly.But they also have the hexagon form of rule, and have more complicated genome structure than prototype C viroids (as murine leukemia virus (MLV)).D type particle and B type particles are similar, because their structures (sprouting from cell surface) in the form of a ring in infected cells, but virion has mixed short surface glycoprotein projection.The cipher telegram daughter nucleus heart also is positioned at eccentric position in the particle.The luxuriant and rich with fragrance simian virus of wheat (MPMV) is a prototype D viroids.

Retrovirus is to define according to the mode that they duplicate genetic material.In reproduction process, RNA changes into DNA.Behind the cell infection,,, generate the duplex molecule of DNA by being called the molecule processing of reverse transcription by 2 molecular dnas that are carried in the virion.Dna form is integrated in the host cell gene group as the provirus covalency, expresses viral RNA by cell intrinsic factor and/or virokine.The viral RNA of expressing is packaged into particle, and discharges as infectious virus particle.

Retroviral particle is made of two kinds of identical RNA molecules.Each genome is the sense single stranded rna molecule, and it adds cap and at 3 ' end polyamides thuja acid tail is arranged at 5 ends.The dliploid virion contains 2 the RNA chains compound with gag albumen, viral enzyme (pol gene outcome) and host tRNA molecule (in ' core ' structure of gag albumen).Around with this capsid of protection be a lipid bilayer, it produces from host cell membrane and contains virus envelope protein.Envelope protein is incorporated into the cell receptor of virus, and virion enters host cell by receptor-mediated encytosis and/or film fusion usually.

After the peplos conductively-closed, RNA is copied into DNA by retrovirus.This is enzymatic by the coded reverse transcription in pol zone, and utilizes the host cell tRNA that is packaged into virion as the synthetic primer of DNA.The rna gene group is transformed into the DNA genome thus.

Before being integrated into the host cell gene group, the double-stranded linear DNA that reverse transcription produces may also may not need cyclisation in cell nucleus.This moment, provirus had 2 identical repetitive sequences at each end, was called long terminal repeat (LTR).Connection between the LTR sequence of two joints has produced the site of pol product (integrase protein that catalysis is integrated) identification, thereby provirus always is incorporated into 2 base-pairs (bp) of the LTR end of host DNA.Can see bipartite cell sequence at the end of these two LTR, make the people associate the integration mode of swivel base genetic elements.It is believed that integration takes place substantially at random in the target cell genome.

The transcribing of the viral DNA of integrating, RNA montage and translation are mediated by host cell proteins matter.Produce various montage transcripts.For human reverse transcript virus HIV-1/2 and HTLV-I/II, virus protein also is used for the expression of regulatory gene.The latence and the time series (viral gene expression is wherein arranged) of the interaction partners control virus between cell and the virokine are important.

But retrovirus level and up rightness are propagated.Retroviral effective contagiosity is propagated and is needed the expression of its acceptor on target cell (envelope protein of its specific recognition virus), though virus can enter (efficient is lower) with the non-specific approach of acceptor dependent/non-dependent.In addition, after viral combination and infiltration (nucleic acid of virus enters cell), the type of target cell must be able to be supported all stages of replicative cycle.When the kind of going into the host when the genome conformity of virus is, up rightness takes place propagate.Though provirus is a gene in the cell, it passes a generation with a generation.Therefore, endogenous provirus has been established, and it is normally hidden, but can activate when the host contacts with the suitable factor.But any stage in the present composition and the method in the used antiviral agent target virus life cycle.

Oncogenic virus (being commonly referred to the RNA tumour virus) is further divided into following two class cause of diseases, is called the retrovirus of acute conversion and chronic conversion.

The retrovirus of acute conversion can transform cultured cells and cause the susceptible animal disease rapidly.These viruses carry an oncogene (v-onc) usually in viral genome, it directly is responsible for their tumorigenicity, and has nothing in common with each other in each viroids.Having derived from the cytogene that obtains virus obtains viral oncogene, may be the result who comprises cell RNA in the virion.Reorganization takes place between virus and the cell RNA during reverse transcription subsequently the sequence of cell is mixed in the viral genome, and the unit that this is new send among the DNA that passs host cell.If the gene of transduction plays central role usually in growth of control cell and differentiation, the variation of coded sequence and/or expression control (it has experienced and has mixed viral genome) may be given its carcinogenicity.By under the control (quantitative and sequential) that places new decision virus transcription and/or by coded sequence being done lasting key sudden change, the prototype oncogene (c-onc) of these cells may become oncogene.Yet, cell transformation completely need express v-onc usually and unite with in the target cell other heredity with after become to sexually revise.

Chronic conversion retrovirus does not contain the oncogene of ' classics ' usually.It is believed that shifts to new management mechanisms comprise that provirus is inserted in the coding region of cell prototype oncogene or near, be called insertion mutation.Strong promoter among the virus LTR and enhancer sequence can be from the distance performance transcriptions of several thousand base-pairs of distance proto-oncogene.The normal regulating of cellular gene expression is destroyed, and the expression of overexpression or inappropriate time may cause transforming.

HTLV-I is a kind of chronic transforming virus, usually relevant with adult T-cell leukemia (ATL), but it may promote the T cell transformation by different approach (the adjusting albumen, the especially p40tax that relate to encoding viral, the expression of its trans-activation cellular proto-oncogene).Also pointed out HIV-1 and 2 the two direct and indirect various types of malignant tumour (as the Kaposi sarcoma) that promote, much higher among the frequency ratio general population that these tumours take place in AIDS patient.Yet the direct effect of HIV in malignant tumour transforms is still open to suspicion, because manyly increase because of other infection or treatment (as transplanting the recipient) cause the ratio that immunosuppressant patient produces tumour.

Though MPMV is initial relevant with the tumor of breast of macaque, D viroids and malignant tumour do not have the teiology relation.The D viroids causes the immunosupress of primate ape by the mechanism of the unknown.Immunosupress also is a kind of feature that slow virus (as HIV and SIV) and feline leukaemia virus (FeLV) dissociant infect.After HIV and FeLV infection (n), observed a large amount of proviral DNAs of not integrating, this may be relevant with pathogenesis.

It is relevant with the chronic progressive disease that causes immunosupress and neurological disorders to comprise that HIV-1/2 and sheep take off the slow virus of ridge sheath leukoencephalitis virus.HIV is the virulence factor that is widely known by the people of aids (AIDS).

Pharmaceutical composition of the present invention and method comprise single with antiinflammatory agent (antibody, peptide, simulating peptide (peptidomimetic), Chemical composition that etc.) or with antiviral agent (as antibody, peptide, virus protein, enzyme inhibitor etc.) coupling, treat and suffer from or dangerous trouble Immunodeficiency virus (as HIV) relevant disease, or propagate the people that maybe may propagate the HIV relevant disease.AIDS and ARC are the object lessons of these diseases.Have recognized that the HIV relevant disease is mainly seen in " danger " crowd and comprises the homosexual active male sex, intravenous pharmacy user, blood or blood product recipient and some crowd in area, the middle not sum Caribbean.This syndrome is also found in the heterosexual companion who owns " danger " crowds and infected mother's baby.

Retrovirus is relevant with numerous disease, comprises anaemia, neurological disorders, immunosupress and malignant tumour.For example HTLV-I is relevant with tropical spastic paraparesis, this be a kind of in some aspects with the similar disease of multiple sclerosis.

Antiviral agent

" antiviral agent " used herein refers to that each step or the stage that can suppress, reduce, prevent or regulate virus infections or viral life cycle produce viral medicine, the initial step by inhibition, minimizing, prophylaxis of viral infections for example, as virus by cell surface acceptor or do not rely on cell surface receptor and be blended in cell, viral nucleic acid enters cell (" entry inhibitor ") subsequently; The reverse transcription of viral nucleic acid (" reverse transcriptase inhibitors "); The viral nucleic acid of reverse transcription is integrated into the genome (" integration inhibitor ") of cell; Provirus transcribed nucleic acid or duplicate; The translation or the formation of ripe virus protein; Formation/the assembling of infectious virus particle; Reducing ripe virion sprouts or discharges from cell; With reduce merge with virus or infect, duplicate, maturation or sprout or discharge the activity and the quantity of relevant enzyme from cell.

The example of antiviral agent comprises polypeptide or its functional analogies, as in conjunction with the part of soluble cell surface receptor peptide, the cell surface receptor of virus, in conjunction with the antibody or the antibody fragment of cell surface receptor or virion, thus the combination between the cell surface receptor that prevents to exist on virus and the cell.The attractive especially virus protein of enantiopathy poison target comprises envelope polypeptides: gp120 or pg41.The especially attractive cell surface receptor of enantiopathy poison target comprises X4 and R5 acceptor.

Term used herein " functional analogies " refers to the molecule through chemistry or structural modification, but it has the activity (being function) or even active raising of one or more unmodified molecules.So with regard to the functional analogies of antiviral agent, these analogies have kept one or more antiviral activities of this antiviral agent.The particular type of functional analogies is polypeptide or peptide mimics (" peptide mimics "), and it is a kind of compound of simulating peptide three-dimensional structure, the i.e. imitation of simulation meaning.Can assist the design of peptide mimics by the three dimensional computer modeling method, it can be designed to have other characteristics (improving treatment uses).For example, antisense molecule can be designed to contain non-natural nucleotides or carry out chemical modification, to suppress this antisense molecule of nucleic acid in vivo enzymic digestion.

Other examples of antiviral agent are cell or the viral enzymes that suppress important to viral life cycle, as virus protease, revertase or integrase.The object lesson of antiviral agent comprises: for example viral fusion inhibitor such as T20 and T20 analogies (Trimeris, Inc.); Entry inhibitor; Integrase inhibitor; Protease inhibitors (as inverase, Ritonavir, indinavir, nelfinavir, amprenavir); Nucleosides reverse transcription inhibitor (as Zidovudine (AZT), stavudine (d4T), Lamivudine (larnivudine) (3TC), dideoxycytidine (DDI), zalcitabine (ddC), Abacavir (abacavir)); Non-nucleosides reverse transcription inhibitor (as Nevirapine, delavirdine, Ai Faweizi (efavirenz)); Suppressing virus maturation becomes the inhibitor of infective virus (as " agent of zinc pointed injection (zinc finger iniectors) ", a class suppresses the inhibitor of suitable viral α NP matter assembling, thereby prevents the formation of infectious virus particle); With their mixture.Can suppress that virus is sprouted or discharge with the medicine that suppresses virus maturation or virus protein maturation from cell.

The control of mucosal inflammation

The high activity antiviral therapy (HAART) that the patient of most of infected by HIV uses prolongs patient's life expectancy of infected by HIV.But unfortunately,, manyly studies show that HIV is still constantly duplicating in the lymphoid organ although the plasma viral amount is reduced to the level that can not record.Studies show that 88% patient's plasma viral load can not be checked through but in their stomach mucous membrane, have can be quantitative HIV nucleic acid.

So though the plasma viral load is reduced to can not the inspection level for HARRT, overwhelming majority patient plasma viral mass formed by blood stasis bounce-back property rising when treatment stops, supposition are because blood exists virus outward stores.Found in lymphatic tissue that this virus stores major part (98%) lymphocyte of the body of wherein residing.Because the stomach mucous membrane contains most of lymphocyte (40-65%) of organism, it represents the maximum storage of HIV probably, and therefore is the main target of anti-HIV treatment.In view of mucosal inflammation state in the HIV disease raises, an emphasis of concrete grammar of the present invention reduces mucosal inflammation exactly.The also anti-virus generation drug resistance sudden change in advance of duplicating by proinflammatory cell factor and the pre-anti-virus enhancing of chemotactic factor (CF) level energy in the treatment reduction mucous membrane compartment.In addition, reduce the solubility inflammation and can also weaken the mucous membrane that new CD4+ cell replenishes the HIV infection, thus prevention or the hide diffusion of inhibition HIV in the body lymphocyte populations.

Identify the validity of antiinflammatory agent

Can adopt many methods well known by persons skilled in the art assess external and body in antiinflammatory agent to suppress, treatment or reduce retroviral activation or Influence and Development in mucomembranous cell, tissue and the object.These methods are applicable to that the antiinflammatory agent of broad range and mucomembranous cell infect.For example, with method as described below, those skilled in the art can assess the effect of antiinflammatory agent to the mucosal tissue of HIV infection and object in the mucosal tissue.Only in order to illustrate, below summarized containing the appraisal procedure of Thalidomide and 5-ASA compound or goods effect, but these embodiment only play a part explanation to scope of the present invention without any restriction.Also shown with aminosalicylic acid (Asacol) and suppressed the data that HIV duplicates in the cell.

Thalidomide

Thalidomide is a kind of strong antiinflammatory agent.Its immunoregulation effect major part depends on the ability that its downward modulation TNF α produces, and it seems that it has influence on a plurality of sites of inflammation cascade antisense.Other anti-inflammatory mechanisms of generally acknowledging comprise and weaken T cell proliferation, the level of downward modulation lymphocyte activation, suppress the lymphocyte chemotaxis and regulate the level of proinflammatory cell factor.By reducing HIV whole body and duplicating in the stomach mucous membrane, Thalidomide proves a kind of composition of the HIV of being of value to treatment.In view of this, the Thalidomide adjuvant that can be used as HAART give the plasma viral load check less than the patient.

With Thalidomide treatment the HIV replication capacity in helping the lymph environment that HIV duplicates is reduced, this can have lower HIV RNA by the object of receiving treatment and organize level to be proved.

Study in the external effect that infected by HIV with regard to the Thalidomide pair cell.Evaluation is also convened the patient who suffers from moderate or serious activity ileitis disease and begin to treat with Thalidomide, assists the effect of distinguishing the nonspecific inflammation reaction and infecting.Adopt the mucous membrane of these objects and haemocyte to assess Thalidomide and reduce infective ability.

The existence that HIV infects has increased inflammatory conditions, and feature is that the level of proinflammatory cell factor and chemotactic factor (CF) raises.These solubility media are attracted at the CD4+ cell chemotaxis that will carry chemokine receptors and play important effect in the mucous membrane.These target cells of HIV are further activated by the solubility medium in the mucous membrane environment, thereby increase duplicating of HIV.For the inflammatory conditions of further clear and definite mucous membrane, many proinflammatory chemotactic factor (CF)s (TNF α, IL-1, IL-6, γ-IFN) and chemotactic factor (CF) (MIP 1a, MIP 1b, RANTES) have been measured.

Convened the negative HIV seropositivity patient of plasma viral load, but their mucosal virus load positive.These object stable disease, cd4 cell is counted greater than 250, does not have significant mucosal inflammation or gastrointestinal symptom.These objects are considered as the baseline of endoscope assessment.Obtain the biopsy through endoscope of standard level (30cm) by these patients.The mucous membrane biopsy is used for the separation of mucous membrane monocyte (MMC), the PCR and the quantitatively property graphical analysis (QIA) of the test of RNA enzyme protection (RPA), HIV RNA and proviral DNA.The flow cytometry MMC that will dye measures CD45, CD4, CD8, HLA-DR, CD45RO, CXCR4, CCR5, LFA-1 and ICAM-1.

The PBMC that separates with autoblood from mucous membrane isolated M MC is carried out flow cytometry, form with the baseline of measuring these compartment medium size lymphocyte groups.Pay close attention to the lymphocytic state of activation and remember condition, and the expression of chemokine receptors and adhesion label.With analyzing these baseline parameters after the Thalidomide treatment.

(Riboquant Pharmingen) comes quantitative assay beta-chemokine and cell factor with a plurality of probe RPA.This sensitivity and special test can quantitative assay chemotactic factor (CF) from the RNA that freezing biopsy extracts and the mRNA of cell factor.Quantity and the GADPH (as housekeeping gene) of various mRNA are compared.If chemotactic factor (CF) and cell factor can not be used the PRA quantitative assay, then can measure chemotactic factor (CF) and cell factor in the MMC supernatant that irriate cultivates with ELISA.

QIA accurately measures lymphocytic quantity and the anatomical position that carries CD4, CD8, CD38, HLA-DR, CD45RO CXCR4 and CCR5 in the biopsy with the paraffin-embedded tissue of immunohistochemical staining.In addition, it can be measured, and those cells produce cell factor and beta-chemokine in the biopsy, and the tissue concentration of these factors.In the seropositive biopsy of HIV, can measure the level of organizing of p24.Computer generated image analyzer and special-purpose software be can obtain and staining cell, chemotactic factor (CF), cell factor and p24 level in the whole tissue regions assessed.

Result's proof organizes chemotactic factor (CF) and cytokine levels to increase to being characterized as of mucous membrane responsing reaction of HIV.This will cause the moderate of mucous membrane inflammatory infiltration to increase.By observing chemotactic factor (CF) and cell factor, will confirm that by simple check cell index " solubility " inflammation can ignore.

With Thalidomide (a kind of strong immunomodulator) treatment, HIV the infected's mucosal infections will be reduced.This shows as HIV replication capacity in this maximum lymphoid organ probably and weakens.Cause virus in mucous membrane and the minimizing of may general duplicating by Thalidomide, may prevent the generation of antiviral agent drug resistance HIV sudden change and improve the long-term effect of HAART.As a kind of adjuvant of HAART treatment, Thalidomide can further improve the life expectancy that the whole world is subjected to millions of patients of this virus infections.

Blood plasma can not detect HIV's and experience the object that the baseline biopsy detects, and the oral dose of 200 gram Thalidomides treated for 16 weeks with every day.After 4 and 16 weeks of treatment, carry out repetition endoscopic biopsy tissue examination and repetition venesection for these objects, obtain PBMC, and carry out identical biomolecule experiment as mentioned above.In addition, each time point is gathered the mucosal virus load that biopsy is used to analyze the patient, and the blood plasma that each time point venesection obtains is analyzed with Roche hypersensitive test (detection level=40 a HIVRNA copy).Carry out RT-and DNA-PCR to analyze the variation between baseline and treatment back mucous membrane and the plasma viral load.For the statistics assessment, unique index of effectiveness is the minimizing of mucous membrane HIV quantity of viruses, and the increase that does not have the plasma viral load to accompany.

In addition, checked with before the Thalidomide treatment and after, the Thalidomide treatment is to separating MMC and the external influence to M-tropism and T-tropism HIV neurological susceptibility of PBMC from the regional enteritis patient.This research can be assessed the external influence of HIV being infected the lymphocyte ability that derives from other struvite membrane diseases of Thalidomide.These data will be associated with the discovery of flow cytometry better, and it relates to the expression of chemotactic factor (CF) and the variation of activation levels and mucosal virus load.For experimental infection, the M tropism HIVsx of known titer and T tropism's HIVNL4-3 are cultivated with MMC and the PBMC that baseline place and Thalidomide treatment obtained during 4 and 16 weeks.Collect the p24 of supernatant as ELISA come quantitative assay 0 hour, 36 hours, 3 days and 7 days.Before treatment and match T afterwards and check the variation of the MMC neurological susceptibility of analyzing IBD patient.

To change the inflammation environment of stomach mucous membrane with Thalidomide treatment HIV patient, cause the minimizing of solubility inflammatory mediator (being proinflammatory cell factor, chemotactic factor (CF)), and then reduce the additional of immunocyte that HIV can infect.Reduce the p24 generation that inflammation will influence PBMC and MMC in the Infection in Vitro experiment with Thalidomide treatment.The main benefit of expection Thalidomide treatment is that the replenishing of CD4+T cell (target of virus) that carries coreceptor by minimizing causes, but also may be that adhesion molecule expression reduces role owing to weakened solubility inflammatory mediator in the mucous membrane.

5-ASA compound or product

Aminosalicylic acid (as Asacol) is a kind of with mucous membrane antiinflammatory agent oral or by bowel lavage, foam or suppository (can be used for topical therapeutic) administration.By duplicating of HIV in the minimizing stomach mucous membrane, it is a kind of composition of the HIV of being of value to treatment.Make great efforts to make mucosal inflammation to minimize with aminosalicylic acid, can also therefore reduce replenishing and activation of HIV target cell by the concentration of proinflammatory cell factor and chemotactic factor (CF) in the minimizing mucous membrane compartment, and be of value to treatment.By reducing concentration and the activity of replicability HIV, can delay HIV and produce using the drug resistance of antiviral agent always.So aminosalicylic acid may be a kind of potent adjuvant that Topically active is arranged, be used for the treatment of, suppress the virus multiplication/diffusion in the HIV object, and suppress or prevention is in the infected by HIV of the object in the risk of infection and suppresses or prevention HIV the infected gives another object (do not infect or infect) with virus disseminating.

Those have according to clinical, endoscope and tissue examination that known moderate activity infects with research, but do not accept aminosalicylic acid or the 4 week treatment of other 5-ASA medicines, and do not accept the patient of immunoregulation medicament or steroid class (oral or local) treatment in 3 months.The control patients of no colitis and colitis patient (their colon does not have symptom and endoscopy NIP) carry out age-matched.Colitis patient carries out the evaluation of 2 baseline endoscopes when treatment the last week and treatment beginning.No colitis control patients will be carried out 2 endoscopies, is separated by for 1 week.Relatively ulcerative colitis and no colitis contrast the solubility and the cellularity inflammatory conditions of mucous membrane sample.

Obtain 15 parts of endoscopic biopsy tissues (3.3mm OD) from each patient from standard level (30cm).In these biopsies, 12 parts are used for MMC and separate; 1 part is used for RPA; 2 parts are used for QIA.The MMC that is used for flow cytometry does dyeing and measures CD45, CD4, CD8, CD38, HLA-DR, CD45RO, CCR5 and CXCR4.

With the ELISA quantitative assay in containing the medium of IL-2, cultivate beta-chemokine and cell factor in the supernatant that obtains behind MMC and the PBMC.Adopt 10,000 cells of the every 1ml culture fluid of 6 orifice plates.Cultivate after 18 hours, take out the 200ml culture fluid and measure RANTES, MIP-1a, MIP-1b, IL-1b, IL-12, TNF-α and IFN-γ.If desired, can extract nucleic acid from the biopsy of liquid nitrogen frozen, (Riboquant Pharmingen) measures the mRNA level of these cell factors and chemotactic factor (CF) with a plurality of probe RPA.Quantity and the GADPH (as housekeeping gene) of various mRNA are compared.

QIA adopts the FF or paraffin-embedded tissue of immunohistochemical staining accurately to measure lymphocytic quantity and the anatomical position that carries CD4, CD8, CD38, HLA-DR, CD45RO and CCR5 in the biopsy.In addition, it can be measured, and those cells produce cell factor and beta-chemokine in the biopsy, the tissue concentration of these factors, and infer of the influence of these parts near the expression of the chemokine receptors of cell.Computer generated image analyzer and special-purpose software be can obtain and the staining cell of whole cell compartment, chemotactic factor (CF) and cell factor (being expressed as the % zone) assessed.

The feature of the ulcerative colitis that moderate is serious is that chemotactic factor (CF) and cell factor " solubility inflammation " level raise and known cellular inflammation increases.The feature of the mucous membrane of ulcerative colitis is that solubility and cellularity inflammation are obviously more serious than non-colitis contrast.Detected the ability of aminosalicylic acid reduction solubility and cellularity inflammatory mediator.

With aminosalicylic acid treatment reduced cell factor and chemotactic factor (CF) level, the CD4+ cell of band CCR5 is replenished and the mucous membrane lymphocyte in the sideways diffusion of HIV minimize; The beta-chemokine secretion that aminosalicylic acid causes reduces the amount of the part that may accept energy blocking virus coreceptor, and this helps the diffusion of HIV; Aminosalicylic acid suppresses the proinflammatory cell factor according to qualifications, has caused suppressing the blocking-up of the not obvious weakening chemokine receptors of additional while of T cell.It seems that this be best result when treatment HIV infected patient.By 16 weeks of patient of suffering from moderate activity inflammatory bowel disease with the aminosalicylic acid treatment, and the variation of inspection mucous membrane lymphocyte subgroup, chemokine receptor expression and the mucomembranous cell factor and beta-chemokine level, check possible result.

Treat moderate activity ulcerative colitis (experience 2 baseline biopsies check) object 16 weeks with the aminosalicylic acid of 4.8 gram dosage every day.Receive treatment 4 week backs and after 16 weeks, treatment was finished, they accept the check of endoscopic biopsy tissue once more, and sample after treating is repeated to test.These researchs comprise the expression carrying out flow cytometry and come quantitative assay lymphocyte subgroup and chemokine receptors, carry out RPA comes the quantitative assay beta-chemokine and carries out QIA and do the original position analysis.The patient will continue to accept aminosalicylic acid treatment, or determine treatment by their clinician.

Treat the inflammation environment that IBD patient will change the stomach mucous membrane with aminosalicylic acid, cause solubility and cellularity inflammatory mediator to reduce.

HIV infects the CD4+ of activation, the cell of band chemokine receptors causes HIV to duplicate raising.The feature of this inflammation environment is that the stomach mucous membrane may induce vigorous HIV to duplicate.With the aminosalicylic acid treatment, by reducing inflammation, with the replication capacity of downward modulation HIV in the stomach mucous membrane that infects.

For the effect of checking aminosalicylic acid treatment that HIV is duplicated in the mucous membrane monocyte, with there being or not having the neurological susceptibility that check MMC and PBMC (from untreated patients of ulcerative colitis) in the culture of gradient dosage aminosalicylic acid infect M tropism and T tropism HIV.After the patient accepted 4 and 16 all aminosalicylic acid treatments, the cell with these patients carried out similarly infectious experiment again.

In infectivity experiment, with the M tropism HIVSX of known titer and T tropism HIVNL4-3 (have and do not have gradient) in containing physiological concentration aminosalicylic acid (according to known mucosal tissue concentration), MMC and PBMC that cultivation obtains at the baseline time point.Duplicate situation by the HIV that measures more every part of these samples of generation of HIV protein p24 in the culture supernatant.The MMC and the PBMC of object after 4 weeks of treatment and 16 weeks are cultivated with M and T tropism HIV, do not add aminosalicylic acid.The p24 result that these samples are collected compares with baseline sample.Supernatant is all collected in these tests at every turn, with the p24 of ELISA quantitative assay 0 hour, 36 hours, 3 days and 7 days.The T that matches check is to analyze the variation of these cells to the HIV neurological susceptibility.

As we had shown, because the CCR5+ phenotype of activation, MMC was than PBMC susceptible HIV more.The aggravation of mucosal inflammation further helps HIV infection MMC among the IBD.Reduce inflammation with the aminosalicylic acid treatment, the influence comparison PBMC that MMC is produced p24 is higher.

Proinflammatory cell factor and chemotactic factor (CF) concentration raise and show that the mucous membrane environment of HIV is an inflammatory.Aminosalicylic acid can obviously reduce these solubility media, and has therefore reduced HIV and infected immigration mucous membrane and other target cells immigration mucous membrane and activation.

Embodiment

In the multiple coreceptor that HIV-1 can utilize, CCR5 and CXCR4 play a major role, and preponderate in initial infection CCR5-tropism virus, and CXCR4-tropism's virus is more in the disease late, and heterozygosis person CCR5 makes the virus survival longer.The difference of CCR5 on mucous membrane CD4+T lymphocyte expressed and may be propagated M tropism's virus according to qualifications.For the relatively expression of coreceptor on mucous membrane and cyclicity lymphocyte, obtain mucous membrane monocyte (MMC) from the endoscopic biopsy separation of proctosigmoid, and obtain unprovoked venesection sample from HIV-1 seronegativity healthy individual.The expression of coreceptor on the CD4+ cell that we have used the flow cytometry quantitative assay.

Consistent with research, lymphocytic 23% intermediate value of all CD4+ (the quartile interval is the 18-30% scope) is expressed CCR5 in the blood.As shown in Figure 1, the lymphocytic intermediate value 71% of intestines CD4+ (being the 50-87% scope) is expressed CCR5, than high 2.8 times (P=0.03) of blood percentage.Mucous membrane CD4+ lymphocyte is also than the obviously more CCR5 acceptor of each cellular expression of CD4+ lymphocyte of the expression CCR5+ of its correspondence in the blood.As shown in Figure 2, the lymphocytic intermediate value CCR5 acceptor quantity of each CD4+ mucous membrane is that 6,946 molecules (are that scope is 6,306-10,416), with each CD4+ blood lymphocyte about 3, the individual CCR5 acceptor of 841 (promptly 3,259-4,441 scopes) is compared high 2.2 times (P=0.03).Integrate and see, this makes the overall CCR5 acceptor of expressing of intestines CD4+ lymphocyte higher 6.2 times than blood, and the CCR5 acceptor to be virus can enter the CD4+ lymphocyte may need.These find prompting mucous membrane CD4+ lymphocyte, and cell is easier is infected by M tropism's HIV-1 than their blood correspondence.

Blood and intestines CD4+ lymphocyte that nearly all (97%) expresses CCR5 all are Memorability CD45RO+ phenotype cells.Consistent with the research of having delivered, we find to compare with blood CD4+ lymphocyte cell (intermediate value 4%, i.e. 38-53% scope), and the ratio of CD45RO+ memory cell increases (intermediate value 95%, i.e. 90-97% scope) in the intestines CD4+ lymphocyte.

Existing reporting: most of periphery T cell CXCR4 is expressed in originally on the CD45RO, rather than on memory CD4+RO+T cell.So initial institute estimates that the high-level CCR5 expression of MMC and CD45RO+ phenotype are preponderated may provide explanation dominant dissection of M tropism's virus disseminating and cell mechanism in accepting the coitus in ano process at least.Yet, also find the cell that high level expression CXCR4 is arranged as shown in Figure 3 in blood (intermediate value 83%, i.e. 75-87% scope) and intestines (intermediate value 64%, i.e. 59-79% scope).Be percentage (P=0.03) between these two compartments or all do not have notable difference with the fluorescence intensity of anti-CXCR4 antibody staining.The expression of the sufficient cell analysis CXCR4 that provides with three blood donors on memory CD4+ lymphocyte, and proof blood memory cell (intermediate value 69%, be scope 57-73%) and intestines memory cell (intermediate value 62%, i.e. scope 57-65%) but solvent express the CXCR4 of detection level.By the fluorescence intensity that CXCR4 on blood memory (CD45RO+) and natural (CD45RO-) CD4+ cell relatively expresses, estimate to show that the CXCR4 level of memory CD4+ cellular expression is more lower slightly than the level of original CD4+ cellular expression.Our experimental result shows that memory CD45RO+ cell not only can express CCR5 and also can express CXCR4, and expression levels is enough to help infect.

We find opposite with the research of carrying out with macaque (identifying the cell of a small amount of expression CXCR4 in rectum and colon's section), have HIV to infect required high-caliber coreceptor in the human body and express.The T cell and the macrophage that carry CCR5 in the macaque research are more sparse.This species diversity may reflect the difference that ethnic difference XOR tissue obtains.Our discovery is to be based upon on the living cells basis of separating of adopting the method acquisition of preserving CCR5 and CXCR4 cell surface expression.

In order to assess the neurological susceptibility that the mucous membrane monocyte infects HIV, we will infect with the PBMC external use HIV that separates from healthy HIV-seronegativity volunteer from the MMC of endoscopic biopsy separate tissue.Exist the zoo virus strain (M-tropism HIVSX or T tropism HIVNL4-3) of using HIV under the 20IU interleukin 2 (IL-2) to infect mucomembranous cell, shown in data, need IL-2 to keep mucomembranous cell group's viability.In contrast, known IL-2 can raise CCR5 and can improve duplicating of virus, has also infected same patient's PBMC, adds and do not add IL-2.At the 18th, 72 and 130 hour, the output of p24 in the quantitative assay supernatant was expressed as per 10 ⁴Individual CD4+ lymphocyte produces the pg amount of p24.As shown in Figure 4, in representativeness experiment (in 2 times 1 time), compare with the PBMC that has or do not have an IL-2, the mucous membrane monocyte can be supported vigorous virus replication in cultivation.The PBMC that infects during no IL-2 can not support duplicating of HIVSX or HIVNL4-3.Compare with the PBMC of similar cultivation when having IL-2, mucomembranous cell infects than obvious M tropism and the T tropism HIV of more being subject to of PBMC.For example, the 72nd hour, the supernatant p24 level of M tropism HIVSX was per 10 in the MMC culture ⁴Individual CD4+ lymphocyte 164pg/ml, the PBMC culture is 51pg/ml in contrast to this.For T tropism's HIVNL4-3, virus is grown in whole process and is all quickened, and is 1194pg/ml (per 10 the 130th hour MMC culture supernatants p24 level ⁴Individual CD4+ lymphocyte), the p24 level of comparing with it in PBMC culture can not detect.These data show that the neurological susceptibility that HIV is infected improves, and prompting mucous membrane CD4+ lymphocyte coreceptor phenotype makes the infection of cell in external M of being subject to and T tropism HIV-1 Strain from function.

These data have proposed 3 interesting aspects.At first, can obtain safely easily and the chorista immunocyte by the mucous membrane internal layer (a kind of reproducible tissue-derived) of intestines.Because the emphasis of HIV-1 study of pathogenesis more and more pays attention to organizing continuing and propagation research of compartment, the easy and adenoid technology of safety contact is essential.Lymph node and tonsil resection/absorption method is the research method of normal report.The research of carrying out with these methods provides the discovery of illustrative change idea, comprises that when blood plasma HIV horizontal stable maybe can not detect the activity of HIV-1 continues to exist in the lymphatic tissue.These tissue-derived secondarys that disclosed in the HIV course of infection are organized the biology activity that is taken place in the lymph node structure, but need the invasive operation to obtain tissue.On the contrary, the intestinal mucosa lymphatic tissue abundant, easily obtain, healing rapidly, self can replenish and directly visual.Endoscopic biopsy is organized test safety, rapid, painless, provides to contact the lymph compartment method that contains lymphocyte 100% sample.Biopsy has kept structural approach, and can carry out histological examination, maybe can dissociate to be used for flow cytometry and to carry out tissue culture assessment as herein described.

These second interesting aspects researching and proposing are that CCR5 obviously improves in the lymphocytic expression of people's mucous membrane CD4+, and the percentage of comparing total CD4+ lymphocyte and each cell CCR5 expression with peripheral blood cells is all high.These mucous membranes T cell has been supported higher levels of virus replication (Fig. 4) than blood CD4+ lymphocyte.Owing to be and the maximally related coreceptor of HIV-1 at initial infection CCR5, and intestines and stomach is to propagate one of modal position and is the main lymphoid organ of human body that important hint is sent out and treated to detected CCR5 expression to propagation, primary infection, the part of HIV.M tropism HIV-1 can obtain the mathematic(al) mode of visibly different progression of disease to the supposition of the contagiosity (according to the expression of CCR5 in the haemocyte) of T cell.When CD4 was not limiting factor, the minimum 700-2000 of each a cell CCR5 acceptor was enough to make the neurological susceptibility maximum that HIV is infected.By our calculating, the quantity of CCR acceptor (intermediate value is about 3000/cell) will be in this scope of Infection in Vitro on the blood T cell.And mucous membrane level (intermediate value is about 7000 acceptor/cells) is considerably beyond this minimum zone.Comprise among the MMC b-chemotactic factor (CF) yield level and whether exist factors such as cell activation factor also may influence the ability that these cell supports are duplicated.Find high-caliber cell factor (comprising TNF-a and IL-2) in intestinal mucosa, they also may make the virus replication in the intestines increase.

The 3rd interesting aspect is to compare with PBMC, and the susceptibility that the mucous membrane lymphocyte infects CCR5-and the CXCR4-tropism's strain of HIV-1 raises.There is IL-2 but further during irritation cell, produces unusual high-caliber HIVNL4-3 in the MMC culture, show that mucous membrane T cell is the microenvironment that provides rich of duplicating of HIV-1 mutation (arbitrary main coreceptor of originating enters).Why utilize the mutation of CCR5-to be had neurological susceptibility to remain major issue to be answered to this amphitypy virus infections by preferential propagation the (though sufficient CD4+ lymphocyte being arranged) and these lymphocytes at the position of mucous membrane propagation HIV-1.Whether our discovery: utilizing CXCR4 at first to be propagated as the HIV-1 mutation of coreceptor, may be to pass through immunologic mechanism but be eliminated if having proposed following problem.In addition, when the mutation that utilizes CCR5 is diffused into PBMC, utilize the mutation of CXCR-4 might stay the position of propagation.

Our result shows the quantitative information that can obtain with the biopsy sample of intestinal mucosa about immunity and viral factor of determination (may influence HIV-1 propagation and morbidity).To the potential neurological susceptibility that the primary of stomach mucous membrane and continuation HIV-1 infect, property contact and the internal layer that is subject to wound are to introduce to note.This neurological susceptibility is included in the lymphocytic advantage of memory CD4+ of the activation of this mucosal sites.Our result shows that also CCR5 and CXCR4 highly are expressed on healthy people HIV-1 seronegativity patient's the mucous membrane CD4+ lymphocyte, and these mucomembranous cells are external more much higher to the neurological susceptibility that infects than blood cell.

Patient group: collected six healthy individual, 3 male 3 woman (45 years old mean age, scope is 24-68 year) study lymphocyte phenotype.Collected other 2 male sex and made viral culture experiment (as follows), had blood in stool and get the permission before history or the conventional polyp examination carrying out selectivity endoscope check.Nobody suffers from the history of diarrhoea or enteritis or infectious diseases.Same area gather through the biopsy of haematine and eosin dyeing biopsy as research usefulness, showing does not have pathology, and when stomach surgery virologist all be normal performance during with blind method form reinspection.This research is by be put to the test protective committee (Human SubjectsProtection Committee) approval of UCLA people.All samples is gathered at conventional 30cm position in proctosigmoid, to avoid because of wound or caused potential the mixing property inflammation of infectious rectitis.Obtain mucous membrane monocyte (MMC) from each one four endoscopy biopsies separation.With the 3.3OD tweezers biopsy is collected in the 15ml tissue culture medium (TCM) (RPMI 1640, Irvine Scientific).Room temperature maintains biopsy on the turntable, up to separating (about 20-60 minute), move in 10 * 35mm culture dish (having the sample test locellus) that phosphate buffered saline (PBS) (PBS) (containing 1mM EDTA and 50mM 2 mercapto ethanol) is housed with the 18G pin then.Cultivated broken tissue 20 minutes with the oscillatory type water-bath for 37 ℃.After centrifugal, at 37 ℃ of mixtures (Boehringer Mannheim#269638 with clostridiopetidase A and dispase; 0.1mg/ml, in RPMI) and digested tissue sample 1 hour.Sample is carried out further fragmentation by the syringe that a series of pins number of successively decreasing are housed.Remove fragment with 70 microns cell screen clothes (Falcon#2350).The cell that obtains is resuspended among the RPMI that contains 10% hyclone.Comprise former generation epithelial cell and leukocytic monocyte with the visual calculating of hemacytometer, and calculate the ratio of monocyte (being leucocyte).Per four parts of biopsies obtain mean value 1.3 * 10 ⁶± 1.1 * 10 ⁶S.D. the cell of (n=6), about 20% monocyte is a leucocyte.Trypan blue repels measures vigor＞90%.With the blood collecting of donor in EDTA, with the dyeing of whole blood colouring method.

The monoclone antibody of buying comprises CD4-fluorescence isothiocyanates and the green fibroin of CD45-stegophyll (BDIS), CD8-allophycocyanin (Caltag) and anti--CXCR4-R-phycoerythrin (PE; Pharmingen).(Cambridge, Dr.Walter Newman MA) provides anti--CCR5, and has been prepared 1: 1 the conjugate of BDIS with PE by KennethDavis and Noel doctor Earner by Leukosite.On FACSCaliburo (BDIS), analyze, and with Cell Questo software analysis.With sidescattering and CD45 fluorescence isolated M MC is carried out initial gate pulse (gating), carry out forward scatter and sidescattering gate pulse then.Identify the leukoceratosis group who clearly defines and separate, account for initial monokaryon sample group's 10-50%.Wherein 20-40% is that CD4+ lymphocyte 26-41% is the CD8+ cell.

In order to measure the lymphocytic CCR5 molecular number of each CD4+, multiply by viewed CCR4 relative intensity of fluorescence (RFI) with calibration factor (the FACSCalibur mensuration for us is specially 44).This calibration factor is the PE molecular number that every RFI port number is detected.For the monoclonal antibody of preparation and 1: 1 conjugate of PE, the RFI port number can be multiply by this calibration factor with estimate each cell combination but anti-number.CXCR4 is not carried out this calculating, because can not obtain 1: 1 conjugate.

In order to ensure to the intestines biopsy with clostridiopetidase A/dispase separating step non-degradable strip surface antigen, separate to obtain peripheral blood lymphocytes (PBMC) with the Ficoll-Hypaque separating method, then dyeing or the percentage of directly expressing with flow cytometry CD45, CD4, CD8, CCR5 and CXCR4 or process (clostridiopetidase A/dispase processing) by the mucous membrane separating method and dye then and analyze antigen.Be used for the conventional PBMC that separates of all antibody researchs 25% and do not show recognizable difference with those separate the enzyme contact with mucous membrane PBMC.Therefore, the increase (comparing with haemocyte) of viewed enterocyte CCR5 percentage and expression is not due to the separating method, because handle the expression that does not increase CCR5 with clostridiopetidase A/dispase.

After the mechanicalness fragmentation, separate the MMC that obtains each healthy people HIV-seronegativity volunteer from 4 endoscopic mucous membrane biopsies, in being supplemented with Iscove ' the sDMEM medium (containing 10mg/ml gentamicin, penicillin, streptomycin and glutamine) of 10% human serum, cultivated 3 days then.Add every ml 20IU interleukin 2 (IL-2, Amgen).Infecting back 3 hours with 50mgHIVSX or PBMC, is 10 with total amount ⁵Individual mucous membrane monocyte and PBMC place 96 orifice plates (100 microlitre medium).Before placing flat board, wash these cells 2 times to remove the p24 of free virus and adhesion.Gather 30 microlitre supernatants at each time point 18 hours, the 3rd day (the 72nd hour) and the 5th day (the 130th hour), measure p24 with ELISA (Coulter).With the percentage of flow cytometry mensuration CD4+, determine the lymphocytic quantity of CD4+ in the culture with it.Because the cell of results is sufficient inadequately, so do not measure the expression of the coreceptor on these people's enterocytes.

In order to increase the monocytic harvest yield of mucous membrane that is used for functional, contagiosity and flow cytometry research, other separating methods have been used.The endoscopy biopsy of fresh collection is minced, directly be added in the 10ml Iscove medium (being supplemented with 20 units/ml IL-2) in 100 * 300mm culture dish 37 ℃, 5%CO ₂Cultivated 3 days.By 70 μ m cell grid harvestings, with the visual tally sheet nucleus of hemacytometer total recovery.Measure the yield of CD45+, CD3+, CD4+ and CD8+ cell with the TruCount pearl, and with compare from the biopsy productive rate of same individual collection with conventional clostridiopetidase A/dispase scheme.The lymphocytic yield of mucous membrane has increased by 6 times (Fig. 5).

The quantitative assay of HIV in the tissue

The RNA that extracts from the proctosigmoid biopsy can be recovered to＞95% organize RNA.We have developed a kind of quantitative PCR test that is used to organize the quantitative RT PCR test of RNA and is used for tissue DNA, and this is by the breadboard method improvement of above-mentioned Chen.Our PRELIMINARY RESULTS shows that available RTPCR comes quantitative assay to be low to moderate the HIV-1 RNA (Fig. 6) of 10 parts of copy levels.

With the sample homogenate immediately of freezing state (with Powergen 125 Potter-Elvehjem Tissue Grinders), Trizol extracts the separator that contains RNA and DNA.Further extract RNA with the Rneasy post.Quantitatively comparative study confirms that the RNA degraded is minimum, and does not have DNA to pollute (PCR of RNA template).Come quantitative assay HIV RNA copy number with having HIV LTR Auele Specific Primer 667/AA55 (design is used for catching the HIV RNA of montage of not montage/repeatedly) improved rTTHRNA PCR kit (Perkin-Elmer).With the 127bp sequence generation linear standard curve of 667/AA55 primer to identification.With precipitation with alcohol (washing 2 times with 0.1M sodium citrate/10% ethanol buffer solution at least) DNA isolation.To HIV DNA, use identical primer in the pcr amplification to (667/AA55).Produce linear standard curve with the beta-globin primer.

For the methodological standardization that makes us and make the closer amount of HIV RNA in the antimer of the result that obtains, before the separate nucleic acid and in the seronegativity biopsy, add the HIV LTR RNA of known quantity afterwards.HIV LTR RNA (with the dilution of 0.5 μ g/ μ l Hela-cell total rna) with purifying produces linear standard curve, carries out RT-PCR as mentioned above.The quantitative assay sample replenishes before the LTR and the difference between the back, the minimum that finds differences (＞95% reclaims).

For the qualitative assessment recovering state, with the fluorescein enzyme dna of known quantity, contain the unknown human autoploidy the bacterium sequence come quantitative assay tissue DNA recovering state (usually＞75%); The seronegativity sample has obtained the recovery (＞95%) that the LTR HIV sequence of known quantity is come quantitative assay RNA.

The plasma viral load detect less than patient's proctosigmoid biopsy in detect HIV-RNA repeatedly

Carried out making great efforts proving with other biopsies that identical peripheral level (30cm) intestinal mucosa from same patient obtains simultaneously and compared a biopsy result's repeatability.Will from the plasma viral load detect less than the biopsy (every part of 10mg) of object freezing, extract RNA as mentioned above and with LTR-Auele Specific Primer 667/AA55 amplification.Each biopsy is on average gathered in the crops 25 μ g RNA, and wherein 1/100 is used for quantitative assay.Fig. 7 result is presented in the sensitivity tests has repeatability with rectosigmoid biopsy quantitative assay RNA viruses load.Data show baseline sigmoidoscopy plasma viral load detect less than the object process in organize HIV RNA viruses load from what 2 parts of biopsies obtained.It was reported that these individual plasma viral load detect less than＞1 year.Difference between the same target sample is average＜0.2log10.These data show come quantitative assay to organize the repeatability of quantity of viruses and the minimum change of sample and sample room with biopsy.It is also important that proof blood plasma HIV RNA detect less than its tissue of object but the HIV RNA (being generally every μ g RNA is 102-103) of detection level is arranged.

The plasma viral load detect less than the proctosigmoid biopsy of object in repeatability detect HIV-DNA.

The plasma viral load detect less than the discrete group of object in, with quantitative property pcr amplification HIVDNA, wherein use the specificity 667/AA55 primer in LTR zone and be used for the b-globin Auele Specific Primer of inner linear standard.Fig. 8 has shown the duplicate experimental result of the triplicate experiment and the quantitative assay of HIV proviral DNA that are used to analyze b-globin Auele Specific Primer.Though the lower limit that detects is 3 parts of copies, 10 parts of copies have been used at our following cut off.This figure shows the actual copy number of quantitative assay and the calculating copy number of counting according to b-globin-dependent cell.These results show our method can be used to detect the plasma viral load detect less than the patient be low to moderate 10 parts proviral DNA copy.

The expression of CCR5 coreceptor on mucous membrane CD4 T cell

The monocytic separation of mucous membrane does not change the phenotypic expression of CD4, CD8, CCR5 and CXCR4.

From collator's blood health, seronegative (peripheral blood lymphocytes: PBMC) and intestinal mucosa (the mucous membrane monocyte: MMC) sample separation, to set up the expression of baseline CCR5 and CXCR4 in these two kinds of compartments.Fig. 9 shows that our separating method neither removes relevant acceptor (CD4, CD8, CCR5, CXCR4) and do not change their surface expression again.

Compare with PBMC, the mucous membrane of CCR5 on CD4 T cell expressed greatly and increased.Seronegativity by health contrasts mucous membrane monocyte and the peripheral blood lymphocytes that object obtains separating, and assesses to determine to express in each compartment the lymphocytic relative percentage of CD4 T of CCR5 acceptor.With 1: 1 ratio with 2D7 CCR5 antibody coupling in phycoerythrin; Calibrate used flow cytometer to detect the 44 phycoerythrin molecules (according to the antibody quantity of standardized CD4 expression and each cell combination) in each RFI passage.Subsequently, the antibodies against CCR 5 number of each cell combination can be translated into the acceptor number (supposition antibody unit price is incorporated into acceptor) of each cell.

Compare with haemocyte (11%), the percentage that enterocyte (87%) is expressed the CD4+ cell of CCR5 obviously increases (p=0.0019) (Figure 10).

Further increase the neurological susceptibility that HIV infects, Figure 11 shows that comparing each cell of (mean value 2700) mucous membrane CD4 T cell with blood CD4 T cell also expresses obviously more acceptor (mean value 8500) (p=0.007).

The CCR5 of mucous membrane cd4 cell expresses almost, and selectivity is a Memorability subgroup cell.With the indicant of CD45RO antibody as memory subgroup cell, dyeing counting the blood and the mucous membrane sample that obtain from identical seronegativity normal healthy controls person, measure the relative distribution of CCR5 staining cell.After CD4+ fluorescence gate pulse, to compare with the groups of cells of mating in the blood (24%), 91% CD4+CD45RO+ mucomembranous cell is expressed CCR5 (p=0.017).

The PBMC with in the inflammatory sample that infects with HIV compares, and the CCR5 of mucous membrane CD4 T cell expresses still higher.

Tentatively determined the baseline that CCR5 expresses in the mucous membrane of healthy seronegativity object and the blood CD4 T cell, the CCR5 that has assessed on chronic HIV the infected's cd4 cell expresses, to check following hypothesis: the expression of comparing CCR5 in the mucous membrane with haemocyte is still higher, and this helps HIV and duplicates.Comprise that the inflammatory control patients is differentiated and HIV infects not directly related variation.The inflammatory control patients is that those clearly have the especially object of ulcerative colitis of inflammatory bowel disease (IBD).These objects are in the paracmasis (mucosal inflammation lasting but control is arranged) clinically, only give the 5-ASA antiinflammatory agent, and do not use any steroid class or immunosuppressive drug.HIV the infected's peripheral blood CD4 counting is 200-700 cell/mm ³Plasma viral load scope (detect less than n=2 with hypersensitive test; Plasma viral load scope is between 200-2000 part copy/ml: n=2; Plasma viral load scope is 20, and 000-40 is between 000 part of copy/ml: n=4).

Still keep the CCR5 expression difference between the viewed mucous membrane and blood CD4 T cell among the seronegativity normal healthy controls person in (Figure 12) inflammation collator (p=0.012) and HIV the infected (p=0.04).Consistent with our hypothesis, as shown in the drawingly compare with normal control, a kind of trend is arranged, obviously identify the expression decreased of mucous membrane CCR5 on CD4 T cell when HIV.

CD4 in the T cell of blood and intestines expression CCR5 when HIV and IBD: CD8 ratio reduces

CCR5 acceptor on the CD4 T cell is also expressed on the CD8+T cell.For whether the expression of further determining mucous membrane CD4 T cell CCR5 really reduces, assessed the relative distribution of CCR5 acceptor between the CD4 and CD8 T lymphocyte in three kinds of these two kinds of compartments of clinical setting.Figure 13 shows the CD4 that expresses the CCR5 cell: the ratio of CD8 is drift downwards obviously, and it is about 50% that blood and intestines sample reduce when IBD, reduces 70～90% when HIV.This may be that a kind of protectiveness downward modulation of CCR5 is to suppress the HIV diffusion.In view of similar tendency is also arranged in the inflammation collator, the preliminary stimulation that reduces surface expression may be relevant with inflammatory microenvironment.Processed sample raises with proof b-chemotactic factor (CF) level in both of these case, but in the HIV sample even than higher in the IBD sample.Except that the hypothesis of supporting us,, there is special movable inflammatory mucous membrane state (determining) during HIV by the active of chemotactic factor (CF) although these find that also prompting histology report lymphocyte reduces relatively.

Tissue concentration with QIA quantitative assay β chemotactic factor (CF)

For the hypothesis (HIV infects and causes significant mucous membrane inflammatory response) of further assessing us, carried out pilot study with doctor JanAndersson at Karolinska Institute, with the chemotactic factor (CF) concentration in the quantitative assay tissue.Will be on aluminium foil the endoscopy biopsy of directed rapidly and snap frozen, deliver to Sweden with dry ice and make freezing microtome section (7 μ m), and carry out quantitative property immunohistochemical staining with evaluation RANTES, MIP-1a and MIP-1b with antibody.And carry out quantitative image analysis (QIA) with CD4, CD8 and CCR5 antibody.Studied the sample of taking from healthy, seronegativity collator and HIV the infected's (can detect the quantity of viruses of blood plasma).The result represents with the percentage of the tissue test overall area that accounts for the peroxidase labeled antibodies stained positive.Find that in normal healthy controls person RANTES, MIP-1a and MIP-1b are respectively 2.04%, 1.39% and 1.65%.In HIV the infected's sample, these values increase to 15.1%, 6.2% and 12.1% respectively.The function of these chemotactic factor (CF)s is that the inflammatory cell that replenishes is convened in the mucous membrane of inflammation.This tangible increase that table 1 provides the HIV relevant with mucosal inflammation to infect.

Table 1

Chemotactic factor (CF)	Normal control	Infected by HIV
			????RANTES	????2.04％	????15.1％
????MIP-1a	????1.39％	????6.2％
			????MIP-1b	????1.65％	????12.1％

External mucous membrane monocyte (MMC) is obvious easier to be more infected than PBMC

Preliminary observation shows that mucomembranous cell partly is because the increase that coreceptor is expressed to the increase of the neurological susceptibility of HIV infection.In this hypothesis of external check, use from same individual isolated M MC and PBMC, to cultivate 2 hours with M tropism's HIVSX, washing was also cultivated 3-10 days.Collect the equal portions supernatant in the indicated time, and detect p24 generation (as the evidence that infects).Compare with the PBMC that cultivates simultaneously, obviously increase (being 4000ng p24/ml among the MMC, is 550ng p24/ml) in PBMC in the generation of first time point (3 days) p24.Institute's summary data has been supported following hypothesis among Figure 14: the increase that mucous membrane CD4 T cell coreceptor is expressed has improved the HIV infectivity.

Analyze CD8+ and CCR5 cell in the colon

Usually the main cell in the compartment is CD8+ in epithelium, but main lamina propria lymphocyte is CD4+.In order to measure the relative number of CD8+ cell in the colon, analyzed whether the CD8+ cell exists in the mucous membrane of colon biopsy.The result of Figure 15 shows that CD8+ dye levels (the brown cell is dark-coloured in the black/white imaging) in HIV the infected's the biopsy is than high in the negative biopsy of HIV (comparison diagram A and figure B).Especially, most of cell is CD8+ rather than CD4+ in HIV the infected's lamina propria.In addition, the CD4+ cell quantity seriously reduces in HIV the infected's mucous membrane of colon.In this case, if biopsy microexamination is that it can be described as the normal cell shape rather than inflammatory, this is because the increase of cd8 cell, its balance the HIV cd4 cell of inducing be killed and the losing of the cd4 cell that causes.Why this possible explanation observes mucous membrane in early days can not detect the inflammation that is subjected in the mucosal tissue that HIV influences.This is the relevant cellularity inflammation of cd8 cell.

Aforesaid b-chemotactic factor (CF) (raising because of HIV as above-mentioned) convenes the cell that has the CCR5 acceptor to replenish mucous membrane (Figure 16).The CCR5 acceptor is the main coreceptor that HIV is used to enter these cells.So HIV guarantees that it can further breed by inducing the inflammation in this environment, to small part by producing the proinflammatory cell factor, replenish the target cells that infect thereby convene more.

Tissue concentration with pcr analysis quantitative assay beta-chemokine

Figure 17 (A-C) has shown that further evidence: HIV is a kind of mucosal inflammation disease and therefore can treats with antiinflammatory agent.Data show proinflammatory cell factor in the HIV patient's that comparatively high amts is arranged in the HIV patient of a small amount of virus and the mucous membrane the mucous membrane biopsy and the concentration of chemotactic factor (CF) (mRNA), RNATES, IFN γ and TNF (RNA) arranged in normal health control patients, the mucous membrane.Can from these researchs, be clear that: compare with the patient of health, higher levels of proinflammatory chemotactic factor (CF) RANTES, interferon-(IFN γ) and TNF are arranged in HIV patient's the mucous membrane.In addition, the patient that virus quantity higher H IV patient is lower than virus quantity in the mucous membrane in the mucous membrane has much higher RNATES, IFN γ and TNF level.These relatively in the significance,statistical that relatively has between HIV patient and healthy normal person (RANTES is respectively p=0.008 and 0.001; IFN γ is respectively, p=0.0008 and 0.002; Be respectively p=0.002 and 0.01 with TNF).

The load increase of HIV produces CD4+ cell activation and viral offspring

Figure 17 D shows the increase along with the mucosal virus load, and the percentage of the CD4+ cell that is activated in the mucous membrane also increases (as shown in the expression increase of HLA-DR).As shown in figure 18, in case the infected more virus that just will produce of mucomembranous cell.This green fluorescent protein that is the acceptor virus (Nlegfp) of T tropism when duplicating is expressed in cell increases shown.The percentage that these results show the mucomembranous cell that produces virus high 300 times than in the haemocyte.

So these results prove that HIV increases the level of proinflammatory cell factor, it causes the activation (this is infected by HIV to chop up born of the same parents' type really) of CD4+ cell.The CD4+ cell that infects and then the viral offspring of generation can infect the various proinflammatory cell factors and the chemotactic factor (CF) of other CD4+ cells and generation and convene additional cell, thereby infect diffusion.

Antiinflammatory agent 5-ASA suppresses intracellular virus replication

In order to assess the ability that 5-ASA suppresses external HIV virus replication, when the 5-ASA that has gradient dosage (3,30,300,3,000 μ M), with 1 * 10 ⁶The PBMC of activation carries out the research that HIV infects.PBMC comprises the CD4 that has chemotactic factor (CF) and cytokine receptor that is present in the medium and the heterogeneous population of cd8 cell.It is believed that used 5-ASA dosage range represents the physiological range seen in patient's stomach mucous membrane of oral 4.8g 5-ASA every day.Infect with the HIV pNLluc Δ Bgl pseudotype virus that does not have replication capacity that has M-tropism JRFL or T tropism LAI coating.Assess the HIV of cultivation after 4 days with the expression of luciferase and duplicate degree.The culture of treated with medicaments is compared with the sample of not accepting medicine.With 7-AAD dyeing, use the death of flow cytometry cell after 4 days.

Fig. 2 result shows: 5-ASA handles the expression that suppresses the 3-67% luciferase, suppresses significantly (p＜0.005) (N=7 infection/processed group) at maximum dose level.It seems that cell death be not because viewed inhibition (the dead %=7.8% of untreated control, 30 μ M5-ASA=7.3%, 300 μ M 5-ASA=9.4% and 3000 μ M 5-ASA=13.3%).

Table 2

M-tropism HIV	Variation % with respect to the culture that is untreated	T-tropism HIV	Variation % with respect to the culture that is untreated
				???ASA?3μM	????46.2	??ASA?2μM	????3.2
???ASA?30μM	????50.3	??ASA?20μM	????22.4
				???ASA?300μM	????48.9	??ASA?200μM	????26.0
???ASA?3000μM	????67.6 *	??ASA?2000μM	????66.7 *
				???AZT?5μM	????80.0 *	??AZT?5μM	????99.3 *

^*P＜0.05

Figure 19 shows that can suppress HIV with Asacol (aminosalicylic acid) (a kind of local anti-inflammatory medicine) duplicates.The cell culture (but not doing any processing) that the HIV of expressing luciferase infects when duplicating in contrast, Asacol lowest dose level (30 and 300 microgram) can suppress duplicating of about 20%HIV.When 3000 micrograms dose, amount of suppression is higher, but this part is (about 30% cell death and 13-15% cell death when 30 and 300 micrograms dose because the cytotoxicity of this dose drug; 5 μ g dosage AZT produce about 16% cell death).These results and more effective known direct antiviral AZT are compared, and latter's blocking virus reverses its genetic material (RNA) ability of typing DNA (being integrated into subsequently among the DNA of cell).Though the anti-inflammatory activity of Asacol not as AZT like that the strong HIV of inhibition duplicate, this activity still has significance,statistical.

In a word, data show 5-ASA (Asacol) under the physiological concentration of inferring suppresses duplicate (during the maximum dose level) of HIV, and active it seems of inhibition do not rely on drug-induced cytotoxicity.The inhibition of 5-ASA is active may (to small part) to be because its anti-inflammatory activity.So these results show and can use antiinflammatory agent to treat HIV widely.

A large amount of embodiments of the invention have been described.Yet be appreciated that the various improvement that can not break away from spirit and scope of the invention.

Claims (129)

1. a method that suppresses the retrovirus activation is characterized in that, described method comprises that the cell that makes this virus infections contacts with antiviral agent with the antiinflammatory agent that suppresses viral volume of activation.

2. the method for claim 1 is characterized in that, described retrovirus is a slow virus.

3. method as claimed in claim 2 is characterized in that described slow virus is an Immunodeficiency virus.

4. method as claimed in claim 3 is characterized in that, described Immunodeficiency virus is selected from: 1 type human immunodeficiency virus HIV, 2 type HIV and simian immunodeficiency virus SIV.

5. the method for claim 1 is characterized in that, described contact is in vivo.

6. the method for claim 1 is characterized in that, described contact is external.

7. the method for claim 1 is characterized in that, described cell is a mammalian cell.

8. method as claimed in claim 7 is characterized in that, described mammalian cell is people's cell.

9. the method for claim 1 is characterized in that, described antiviral agent suppresses virus and is blended in or enters cell, the reverse transcription or the nucleic acid replication of virus, and viral integrase is gone in the cell DNA, and virus is sprouted or is discharged from cell, produces infective virus; Or inhibition and viral fusion or infection, reverse transcription or nucleic acid replication, viral integrase are gone in the cell DNA, virus is sprouted or discharge or produce the relevant enzyme of infective virus from cell.

10. the method for claim 1 is characterized in that, described antiviral agent is polypeptide or its functional analogies.

11. method as claimed in claim 10 is characterized in that, described polypeptide or its functional analogies are incorporated into virus or cell surface receptor.

12. method as claimed in claim 10 is characterized in that, described polypeptide is part, virus receptor, antibody or their segment.

13. method as claimed in claim 9 is characterized in that, described enzyme is protease, revertase or integrase.

14. the method for claim 1 is characterized in that, described antiviral agent is selected from: protease inhibitors, nucleoside reverse transcriptase inhibitor, non-nucleoside reverse transcriptase inhibitor, integrase inhibitor and their combination.

15. method as claimed in claim 14, it is characterized in that described nucleosidic inhibitors is: Zidovudine (AZT), stavudine (d4T), Lamivudine (3TC), dideoxycytidine (DDI), zalcitabine (ddC), Abacavir and their combination.

16. method as claimed in claim 14 is characterized in that, described non-nucleosidic inhibitors is selected from: Nevirapine, delavirdine and Ai Faweizi.

17. the method for claim 1 is characterized in that, described antiviral agent is a protease inhibitors.

18. method as claimed in claim 17 is characterized in that, described protease inhibitors is inverase, Ritonavir, indinavir, nelfinavir or amprenavir.

19. the method for claim 1 is characterized in that, described antiinflammatory agent reduces the interaction of the generation that replenishes, reduces chemotactic factor (CF), minimizing proinflammatory production of cytokines or chemokine inhibiting acceptor and its part of inflammatory cell.

20. the method for claim 1, it is characterized in that described antiinflammatory agent is selected from: the antibody of anti-inflammatory, anti-inflammatory peptide, anti-inflammatory cytokine, anti-inflammatory chemotactic factor (CF), anti-inflammatory nucleic acid, steroid class, nonsteroidal anti-inflammatory agent, 5-ASA product and their combination.

21. method as claimed in claim 20 is characterized in that, described anti-inflammatory antibody is selected from: anti-cytokine antibodies, antibacterial agent receptor antibody, anti-chemotactic factor (CF) antibody, anti-chemokine receptors antibody, anti-proinflammatory peptide antibody and their combination.

22. method as claimed in claim 20 is characterized in that, described anti-inflammatory peptide is selected from: LFA adhesion molecule antagonist, cytokine receptor antagonist, transcription factor and soluble TNF-α receptor polypeptides.

23. method as claimed in claim 20 is characterized in that, described anti-inflammatory cytokine is selected from: IL-4, IL-10, IL-13, IL-16 and their combination.

24. method as claimed in claim 20 is characterized in that, described anti-inflammatory nucleic acid is selected from: nucleic acid, antisensenucleic acids and their combination of ribozyme, coding anti-inflammatory peptide.

25. method as claimed in claim 24 is characterized in that, the nucleic acid hybridization of described antisensenucleic acids and coding chemokine receptors, inflammatory cytokine, chemokine receptors or chemotactic factor (CF).

26. method as claimed in claim 20 is characterized in that, described steroid class is a glucocorticoid.

27. method as claimed in claim 20, it is characterized in that described steroid class is selected from: flunisolide, fluoxyprednisolone, acetone fluoxyprednisolone, beclomethasone dipropionate, BDP, hydrocortisone, cortisone, dexamethasone, budesonide, metacortandracin, methylprednisolone, prednisolone and their combination.

28. method as claimed in claim 20; it is characterized in that; described nonsteroidal anti-inflammatory agent is selected from: salicyclic acid derivatives comprises salicylic acid, thiosalicylic acid sodium, choline salicylate, magnesium salicylate, Diflunisal, brufen, NAP, sulindac, Diflunisal, disalicylic acid, choline three magnesium salicylates, acetylsalicylic acid, salsalate, sodium salicylate and their combination.

29. method as claimed in claim 20, it is characterized in that described nonsteroidal anti-inflammatory agent is selected from: the fragrant sodium acid of Flurbiprofen, fenoprofen, Nabumetone, Ketoprofen, piroxicam, Indomethacin, tolmetin, first chlorine, mefenamic acid, Etodolac, ketorolac tromethamine, Diclofenac, Evil promazine, bromfenac sodium, Luo Fei are comparable, suprofen, fragrant brufen, Fluprofen, Thalidomide, evening primrose oil, their single isomer and their combination.

30. method as claimed in claim 20 is characterized in that, described 5-ASA product is selected from: aminosalicylic acid, Balsalazide, ipsalazide, olsalazine, sulfasalazine and their combination.

31. the method that the mucosal tissue of an inflammation-inhibiting mediation infects is characterized in that, described method comprises makes mucosal tissue contact with antiviral agent with the antiinflammatory agent that suppresses effective dose.

32. method as claimed in claim 31 is characterized in that, described inflammation mediated infection is caused by virus.

33. method as claimed in claim 32 is characterized in that, described virus is retrovirus.

34. method as claimed in claim 33 is characterized in that, described retrovirus is a slow virus.

35. method as claimed in claim 34 is characterized in that, described slow virus is an Immunodeficiency virus.

36. method as claimed in claim 35 is characterized in that, described Immunodeficiency virus is selected from: 1 type human immunodeficiency virus HIV, 2 type HIV and simian immunodeficiency virus SIV.

37. method as claimed in claim 31 is characterized in that, described contact is in vivo.

38. method as claimed in claim 31 is characterized in that, described contact is external.

39. method as claimed in claim 31 is characterized in that, described contact is in vitro.

40. method as claimed in claim 31 is characterized in that, described tissue is a mammalian tissues.

41. method as claimed in claim 40 is characterized in that, described mammalian tissues is people's a tissue.

42. method as claimed in claim 31 is characterized in that, described mucosal tissue is vagina tissue, stomach tissue, nose tissue or following intestines and stomach tissue.

43. method as claimed in claim 31 is characterized in that, described contact be before giving antiviral agent, simultaneously or afterwards, part or whole body give antiinflammatory agent.

44. method as claimed in claim 43 is characterized in that, described administration is that the part contact by topical realizes.

45. method as claimed in claim 43 is characterized in that, described whole body administration is by intravenous, oral or parenteral realization.

46. method as claimed in claim 31 is characterized in that, described antiviral agent suppresses virus and is blended in or enters cell, the reverse transcription or the nucleic acid replication of virus, and viral integrase is gone in the cell DNA, and virus is sprouted or is discharged from cell, produces infective virus; Or inhibition and viral fusion or infection, reverse transcription or nucleic acid replication, viral integrase are gone in the cell DNA, virus is sprouted or discharge or produce the relevant enzyme of infective virus from cell.

47. method as claimed in claim 31 is characterized in that, described antiviral agent is polypeptide or its functional analogies.

48. method as claimed in claim 47 is characterized in that, described polypeptide or its functional analogies are incorporated into virus or cell surface receptor.

49. method as claimed in claim 47 is characterized in that, described polypeptide is part, virus receptor, antibody or their segment.

50. method as claimed in claim 46 is characterized in that, described enzyme is protease, revertase or integrase.

51. method as claimed in claim 31 is characterized in that, described antiviral agent is: protease inhibition, nucleoside reverse transcriptase inhibitor, non-nucleoside reverse transcriptase inhibitor, integrase inhibitor and their combination.

52. method as claimed in claim 51, it is characterized in that described nucleosidic inhibitors is selected from: Zidovudine (AZT), stavudine (d4T), Lamivudine (3TC), dideoxycytidine (DDI), zalcitabine (ddC), Abacavir and their combination.

53. method as claimed in claim 51 is characterized in that, described non-nucleosidic inhibitors is selected from: Nevirapine, delavirdine and Ai Faweizi.

54. method as claimed in claim 31 is characterized in that, described antiviral agent is protease inhibitors, reverse transcriptase inhibitors or integrase inhibitor.

55. method as claimed in claim 54 is characterized in that, described protease inhibitors is inverase, Ritonavir, indinavir, nelfinavir or amprenavir.

56. method as claimed in claim 31 is characterized in that, described antiinflammatory agent reduces the interaction of the generation that replenishes, reduces chemotactic factor (CF), minimizing proinflammatory production of cytokines or chemokine inhibiting acceptor and its part of inflammatory cell.

57. method as claimed in claim 31, it is characterized in that described antiinflammatory agent is selected from: anti-inflammatory antibody, anti-inflammatory peptide, anti-inflammatory cytokine, anti-inflammatory chemotactic factor (CF), anti-inflammatory nucleic acid, steroid class, nonsteroidal anti-inflammatory agent, 5-ASA product and their combination.

58. method as claimed in claim 57, it is characterized in that the antibody of described anti-inflammatory is selected from: anti-cytokine antibodies, antibacterial agent receptor antibody, anti-chemotactic factor (CF) antibody, anti-chemokine receptors antibody, anti-proinflammatory peptide antibody and their combination.

59. method as claimed in claim 57 is characterized in that, described anti-inflammatory peptide is selected from: LFA-1 antagonist, cytokine receptor antagonist, transcription factor and soluble TNF-α acceptor.

60. method as claimed in claim 57 is characterized in that, described anti-inflammatory cytokine is selected from: IL-4, IL-10, IL-13, IL-16 and their combination.

61. method as claimed in claim 57 is characterized in that, described anti-inflammatory nucleic acid is selected from: nucleic acid, antisensenucleic acids and their combination of ribozyme, coding anti-inflammatory peptide.

62. method as claimed in claim 61 is characterized in that, the nucleic acid hybridization of described antisensenucleic acids and coding chemokine receptors, inflammatory cytokine, chemokine receptors or chemotactic factor (CF).

63. method as claimed in claim 57 is characterized in that, described steroid class is a glucocorticoid.

64. method as claimed in claim 57, it is characterized in that described steroid class is selected from: flunisolide, fluoxyprednisolone, acetone fluoxyprednisolone, beclomethasone dipropionate, BDP, hydrocortisone, cortisone, dexamethasone, budesonide, metacortandracin, methylprednisolone, prednisolone and their combination.

65. method as claimed in claim 57; it is characterized in that; described nonsteroidal anti-inflammatory agent is selected from: salicyclic acid derivatives comprises salicylic acid, thiosalicylic acid sodium, choline salicylate, magnesium salicylate, Diflunisal, brufen, NAP, sulindac, Diflunisal, disalicylic acid, choline three magnesium salicylates, acetylsalicylic acid, salsalate, sodium salicylate and their combination.

66. method as claimed in claim 57, it is characterized in that described nonsteroidal anti-inflammatory agent is selected from: the fragrant sodium acid of Flurbiprofen, fenoprofen, Nabumetone, Ketoprofen, piroxicam, Indomethacin, tolmetin, first chlorine, mefenamic acid, Etodolac, ketorolac tromethamine, Diclofenac, Evil promazine, bromfenac sodium, Luo Fei are comparable, suprofen, fragrant brufen, Fluprofen, Thalidomide, evening primrose oil, their single isomer and their combination.

67. method as claimed in claim 57 is characterized in that, described 5-ASA product is selected from: aminosalicylic acid, Balsalazide, ipsalazide, olsalazine, sulfasalazine and their combination.

68. a reduction has the object of suffering from inflammation mediated mucosal infections danger to suffer from the method for the possibility of inflammation mediated mucosal infections, it is characterized in that described method comprises makes this object contact with antiviral agent with the antiinflammatory agent of effective dose.

69., it is characterized in that described inflammation mediated infection is caused by virus as the described method of claim 68.

70., it is characterized in that described virus is retrovirus as the described method of claim 69.

71., it is characterized in that described retrovirus is a slow virus as the described method of claim 70.

72., it is characterized in that described slow virus is an Immunodeficiency virus as the described method of claim 71.

73., it is characterized in that described Immunodeficiency virus is selected from as the described method of claim 70: 1 type human immunodeficiency virus HIV, 2 type HIV and simian immunodeficiency virus SIV.

74., it is characterized in that described contact is in vivo as the described method of claim 68.

75. as the described method of claim 68, it is characterized in that, in the described body contact be before giving antiviral agent, simultaneously or afterwards, part or whole body give antiinflammatory agent.

76., it is characterized in that described administration is that the part contact by topical realizes as the described method of claim 75.

77., it is characterized in that described whole body administration is by intravenous, oral or parenteral realization as the described method of claim 75.

78. as the described method of claim 68, it is characterized in that, described to liking mammal.

79., it is characterized in that described mammal is the people as the described method of claim 78.

80., it is characterized in that described antiviral agent suppresses virus and is blended in or enters cell as the described method of claim 68, the reverse transcription or the nucleic acid replication of virus, viral integrase is gone in the cell DNA, and virus is sprouted or is discharged from cell, produces infective virus; Or inhibition and viral fusion or infection, reverse transcription or nucleic acid replication, viral integrase are gone in the cell DNA, virus is sprouted or discharge or produce the relevant enzyme of infective virus from cell.

81., it is characterized in that described antiviral agent is polypeptide or its functional analogies as the described method of claim 68.

82., it is characterized in that described polypeptide or its functional analogies are incorporated into virus or cell surface receptor as the described method of claim 81.

83., it is characterized in that described polypeptide is part, virus receptor, antibody or their segment as the described method of claim 81.

84., it is characterized in that described enzyme is protease, revertase or integrase as the described method of claim 80.

85., it is characterized in that described antiviral agent is selected from as the described method of claim 68: protease inhibitors, nucleoside reverse transcriptase inhibitor, non-nucleoside reverse transcriptase inhibitor, integrase inhibitor and their combination.

86. as the described method of claim 85, it is characterized in that described nucleosidic inhibitors is: Zidovudine (AZT), stavudine (d4T), Lamivudine (3TC), dideoxycytidine (DDI), zalcitabine (ddC), Abacavir and their combination.

87., it is characterized in that described non-nucleosidic inhibitors is selected from as the described method of claim 85: Nevirapine, delavirdine and Ai Faweizi.

88., it is characterized in that described antiviral agent is protease inhibitors, reverse transcriptase inhibitors or integrase inhibitor as the described method of claim 68.

89., it is characterized in that described protease inhibitors is inverase, Ritonavir, indinavir, nelfinavir or amprenavir as the described method of claim 88.

90., it is characterized in that described antiinflammatory agent reduces the interaction of the generation that replenishes, reduces chemotactic factor (CF), minimizing proinflammatory production of cytokines or chemokine inhibiting acceptor and its part of inflammatory cell as the described method of claim 68.

91. as the described method of claim 68, it is characterized in that described antiinflammatory agent is selected from: anti-inflammatory antibody, anti-inflammatory peptide, anti-inflammatory cytokine, anti-inflammatory chemotactic factor (CF), anti-inflammatory nucleic acid, steroid class, nonsteroidal anti-inflammatory agent, 5-ASA product and their combination.

92. as the described method of claim 91, it is characterized in that the antibody of described anti-inflammatory is selected from: anti-cytokine antibodies, antibacterial agent receptor antibody, anti-chemotactic factor (CF) antibody, anti-chemokine receptors antibody, anti-proinflammatory peptide antibody and their combination.

93., it is characterized in that described anti-inflammatory peptide is selected from as the described method of claim 91: LFA-1 antagonist, cytokine receptor antagonist, transcription factor and soluble TNF-α acceptor.

94., it is characterized in that described anti-inflammatory cytokine is selected from as the described method of claim 91: IL-4, IL-10, IL-13, IL-16 and their combination.

95., it is characterized in that described anti-inflammatory nucleic acid is selected from as the described method of claim 91: nucleic acid, antisensenucleic acids and their combination of ribozyme, coding anti-inflammatory peptide.

96., it is characterized in that the nucleic acid hybridization of described antisensenucleic acids and coding chemokine receptors, inflammatory cytokine, chemokine receptors or chemotactic factor (CF) as the described method of claim 95.

97. as the described method of claim 91, it is characterized in that described steroid class is selected from: flunisolide, fluoxyprednisolone, acetone fluoxyprednisolone, beclomethasone dipropionate, BDP, hydrocortisone, cortisone, dexamethasone, budesonide, metacortandracin, methylprednisolone, prednisolone and their combination.

98. as the described method of claim 91; it is characterized in that; described nonsteroidal anti-inflammatory agent is selected from: salicyclic acid derivatives comprises salicylic acid, thiosalicylic acid sodium, choline salicylate, magnesium salicylate, Diflunisal, brufen, NAP, sulindac, Diflunisal, disalicylic acid, choline three magnesium salicylates, acetylsalicylic acid, salsalate, sodium salicylate and their combination.

99. as the described method of claim 91, it is characterized in that described nonsteroidal anti-inflammatory agent is selected from: the fragrant sodium acid of Flurbiprofen, fenoprofen, Nabumetone, Ketoprofen, piroxicam, Indomethacin, tolmetin, first chlorine, mefenamic acid, Etodolac, ketorolac tromethamine, Diclofenac, Evil promazine, bromfenac sodium, Luo Fei are comparable, suprofen, fragrant brufen, Fluprofen, Thalidomide, evening primrose oil, their single isomer and their combination.

100., it is characterized in that described 5-ASA product is selected from as the described method of claim 91: aminosalicylic acid, Balsalazide, ipsalazide, olsalazine, sulfasalazine and their combination.

101. a method that suppresses the retrovirus activation is characterized in that, described method comprises and will be contacted with the antiinflammatory agent that suppresses volume of activation by the cell of virus infections that wherein antiinflammatory agent is not 5-ASA or ASA.

102., it is characterized in that described antiinflammatory agent is selected from as the described method of claim 101: anti-inflammatory antibody, anti-inflammatory peptide, anti-inflammatory cytokine, anti-inflammatory chemotactic factor (CF), anti-inflammatory nucleic acid, steroid class, nonsteroidal anti-inflammatory agent and their combination.

103. a method that suppresses the inflammation mediated infection of mucosal tissue is characterized in that, described method comprises makes this tissue contact with the antiinflammatory agent that suppresses effective dose, and wherein antiinflammatory agent is not 5-ASA or ASA.

104., it is characterized in that described antiinflammatory agent is selected from as the described method of claim 103: anti-inflammatory antibody, anti-inflammatory peptide, anti-inflammatory cytokine, anti-inflammatory chemotactic factor (CF), anti-inflammatory nucleic acid, steroid class, nonsteroidal anti-inflammatory agent and their combination.

105. an inhibition has the object of suffering from inflammation mediated mucosal infections danger to suffer from the method for inflammation mediated mucosal infections, it is characterized in that described method comprises makes this object contact with the antiinflammatory agent of effective dose, wherein antiinflammatory agent is not 5-ASA or ASA.

106., it is characterized in that described antiinflammatory agent is selected from as the described method of claim 105: anti-inflammatory antibody, anti-inflammatory peptide, anti-inflammatory cytokine, anti-inflammatory chemotactic factor (CF), anti-inflammatory nucleic acid, steroid class, nonsteroidal anti-inflammatory agent and their combination.

107. one kind is suppressed the method that disorder relevant with HIV in the object of carrier's Immunodeficiency virus develops, and it is characterized in that, described method comprises the antiinflammatory agent of the inhibition HIV virus development of the object treatment effective dose that gives this trouble HIV virus infections.

108., it is characterized in that described antiinflammatory agent is not ASA or 5-ASA as the described method of claim 107.

109., it is characterized in that described method also comprises and gives antiviral agent as the described method of claim 107.

110. one kind is prevented or reduces to have by the method for the object generation possibility of infection of HIV risk of infection, it is characterized in that, described method comprises the antiinflammatory agent that gives this object prevention effective dose, and described antiinflammatory agent suppresses or reduce the possibility of this object infected by HIV.

111., it is characterized in that described antiinflammatory agent is not ASA or 5-ASA as the described method of claim 110.

112., it is characterized in that described method also is included in and gives before the antiinflammatory agent, simultaneously or give antiviral agent afterwards as the described method of claim 110.

113., it is characterized in that described antiinflammatory agent is a topical administration as claim 107 or 110 described methods.

114., it is characterized in that described antiinflammatory agent is that whole body gives as claim 107 or 110 described methods.

115. a pharmaceutical composition is characterized in that, described composition is included in the antiinflammatory agent of the treatment effective dose of at least one dosage in the pharmaceutically acceptable carrier, and wherein this dosage is to suppress or to reduce by the effective dose of immunodeficiency virus infection possibility.

116., it is characterized in that described composition also comprises antiviral agent as the described pharmaceutical composition of claim 115.

117., it is characterized in that described pharmaceutical composition also comprises pharmaceutically acceptable gel, emulsifiable paste, foaming agent or suppository as the described pharmaceutical composition of claim 115.

118. goods is characterized in that, described goods comprise that at least a antiinflammatory agent and this medicine are used for the treatment of, prevent or reduce by the specification of immunodeficiency virus infection possibility.

119., it is characterized in that described goods also comprise antiviral agent as the described goods of claim 118.

120., it is characterized in that described goods are selected from as the described goods of claim 118: condom, cotton wool, barrier film, pessary, pesseulum, suppository and enema.

121. an inhibition be is characterized in that by the method for retrovirus activation in the tissue of virus infections described method comprises makes this tissue contact with the antiinflammatory agent that suppresses the activation effective dose.

122., it is characterized in that described antiinflammatory agent is not ASA or 5-ASA as the described method of claim 121.

123., it is characterized in that described method also is included in and gives before the antiinflammatory agent, simultaneously or described tissue is contacted with antiviral agent as the described method of claim 121.

124. an inhibition be is characterized in that by the method for retrovirus activation in the object of virus infections described method comprises makes this object contact with the antiinflammatory agent that suppresses the activation effective dose.

125., it is characterized in that described antiinflammatory agent is not ASA or 5-ASA as the described method of claim 124.

126., it is characterized in that described method also is included in and gives before the antiinflammatory agent, simultaneously or described object is contacted with antiviral agent as the described method of claim 124.

127. goods is characterized in that, described goods comprise the specification that at least a antiinflammatory agent and this antiinflammatory agent are used for the prevention of immunodeficiency virus infection usage.

128., it is characterized in that described goods also comprise antiviral agent as the described goods of claim 127.

129., it is characterized in that described goods are selected from as the described goods of claim 127: condom, cotton wool, barrier film, pessary, pesseulum, suppository and enema.

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