Margarita Bofill

HIV transfer between CD4 T cells does not require LFA-1 binding to ICAM-1 and is governed by the interaction of HIV envelope glycoprotein with CD4

BackgroundCell-to-cell HIV transmission requires cellular contacts that may be in part mediated by the integrin leukocyte function antigen (LFA)-1 and its ligands intercellular adhesion molecule (ICAM)-1, -2 and -3. The role of these molecules in free virus infection of CD4 T cells or in transinfection mediated by dendritic cells (DC) has been previously described. Here, we evaluate their role in viral transmission between different HIV producing cells and primary CD4 T cells.ResultsThe formation of cellular conjugates and subsequent HIV transmission between productively infected MOLT cell lines and primary CD4 T cells was not inhibited by a panel of blocking antibodies against ICAM-1, ICAM-3 and α and β chains of LFA-1. Complete abrogation of HIV transmission and formation of cellular conjugates was only observed when gp120/CD4 interactions were blocked. The dispensable role of LFA-1 in HIV transmission was confirmed using non-lymphoid 293T cells, lacking the expression of adhesion molecules, as HIV producing cells. Moreover, HIV transmission between infected and uninfected primary CD4 T cells was abrogated by inhibitors of gp120 binding to CD4 but was not inhibited by blocking LFA-1 binding to ICAM-1 or ICAM-3. Rather, LFA-1 and ICAM-3 mAbs enhanced HIV transfer. All HIV producing cells (including 293T cells) transferred HIV particles more efficiently to memory than to naive CD4 T cells.ConclusionIn contrast to other mechanisms of viral spread, HIV transmission between infected and uninfected T cells efficiently occurs in the absence of adhesion molecules. Thus, gp120/CD4 interactions are the main driving force of the formation of cellular contacts between infected and uninfected CD4 T cells whereby HIV transmission occurs.

Treatment of monocytes with interleukin (IL)-12 plus IL-18 stimulates survival, differentiation and the production of CXC chemokine ligands (CXCL)8, CXCL9 and CXCL10

During inflammation, interleukin (IL)‐12 and IL‐18 are produced by macrophages and other cell types such as neutrophils (IL‐12), keratinocytes and damaged endothelial cells (IL‐18). To explore the role of IL‐12 and IL‐18 in inflammatory innate immune responses we investigated their impact on human peripheral blood monocytes and mature bronchoalveolar lavage (BAL) macrophages. IL‐12 and IL‐18 together, but not alone, prevented spontaneous apoptosis of cultured monocytes, promoted monocyte clustering and subsequent differentiation into macrophages. These morphological changes were accompanied by increased secretion of CXC chemokine ligands (CXCL)9, CXCL10 (up to 100‐fold, P < 0·001) and CXCL8 (up to 10‐fold, P < 0·001) but not CCL3, CCL4 or CCL5. Mature macrophages (from BALs) expressed high basal levels of CXCL8, that were no modified upon stimulation with IL‐12 and IL‐18. In contrast, the basal production of CXCL9 and CXCL10 by BALs was increased by 10‐fold (P < 0·001) in the presence of either IL‐12 or IL‐18 alone and by 50‐fold in the presence of both cytokines. In conclusion, our results indicate a relevant role for IL‐12 and IL‐18 in the activation and resolution of inflammatory immune responses, by increasing the survival of monocytes and by inducing the production of chemokines. In particular, those that may regulate angiogenesis and promote the recruitment of monocytes, activated T cells (CXCL9 and CXCL10) and granulocytes (CXCL8).

HIV endocytosis after dendritic cell to T cell viral transfer leads to productive virus infection.

Contacts between HIV-producing T cells and primary CD4+ T cells may induce the uptake of HIV by target cells that are endocytosed into trypsin-resistant compartments. We have now compared the mechanism of virus transmission from T cell-to-T cell versus infected dendritic cells (DCs)-to-T cell. In cocultures of HIV-1-infected DCs with primary CD4+ T cells, virus transmission to the target cells was resistant to trypsin treatment and could only be prevented by the anti-SUgp120 antibody IgGb12 but not by TAK-779, C34 or AZT. Importantly, upon stimulation of purified HIV-1-loaded CD4+ T cells with PHA/IL-2, cells became productively infected as measured by intracellular CAp24 staining and antigen determination in the cell supernatant. These results suggest that the viral endocytic transfer may represent a escape mechanism in the presence of drugs targeting HIV-1 entry or the host immune system.

Epidemiological data of different human papillomavirus genotypes in cervical specimens of HIV-1-infected women without history of cervical pathology.

Aim:To study the epidemiology of different human papillomavirus (HPV) genotypes in cervical samples of HIV-1-infected women with normal Papanicolau smears. Design:Retrospective analysis of a prospective cohort. Patients and Methods:We selected HIV-1-infected women with 2 consecutive normal Papanicolau smears at baseline and at least 1 baseline and 1 follow-up cervical sample. HPV infection was assessed by second-generation hybrid capture (HC-2) and multiplex polymerase chain reaction (mPCR). HPV genotypes were determined by mPCR. Results:From a cohort of 139 women followed up to 4 years, 93 women meeting the inclusion criteria were analyzed. The mean period between samples was 20 months (range, 6-44 months). HPV baseline prevalence was 63% [59/93; 95% confidence interval (CI), 53% to 73%] using polymerase chain reaction and 41% (38/93; 95% CI, 31% to 51%) using HC-2, P = 0.007 (kappa, 0.45; P = 0.001). The most prevalent high oncogenic risk genotypes (HR-HPV) were HPV-16 (28%), HPV-33 (18%), HPV-52 (12%), HPV-58 (11%), and HPV-39 (11%). Infection with multiple HPV genotypes was detected in >40% of women. HPV infection persisted at follow-up in 86% (51/59; 95% CI, 77% to 95%) by polymerase chain reaction and 76% (29/38; 95% CI, 62% to 90%) by HC-2. HPV infection persisted in 55% of women with samples available beyond 3 years. The actuarial probabilities of clearance and incidence of HPV infection at 36 months were 16% and 45%, respectively. Conclusions:HPV infection is highly prevalent and persistent among HIV-1-infected women with normal Papanicolau smears. HR-HPV genotypes other than HPV-16 (HPV-33, HPV-52) are frequently detected in HIV-infected women. mPCR provides better surveillance of HPV infection than HC-2 methods.

Differential expression of the cytokine receptors for human interleukin (IL)-12 and IL-18 on lymphocytes of both CD45RA+ and CD45RO+ phenotype from tonsils, cord and adult peripheral blood

The objective of this study was to demonstrate the variable expression of cytokine receptors on naive versus memory human CD4+ T cell subpopulations in tonsillar tissue, cord blood and adult blood. We prove that the receptors for both interleukin (IL)‐12 and IL‐18 are expressed exclusively on memory T cells. This observation was seen not only on the CD45RO+ memory T cells but also on a significant percentage of the CD45RA+, CD62L–, CD27– and CCR7– populations. Furthermore, CD45RA+ CD62L+, CD27+ or CCR7+ CD4+ T cells that expressed IL‐12Rβ1 and IL‐18Rα did not express CD31, a marker for recent thymic emigrants. We reveal that cord blood lymphocytes do not express IL‐12Rβ1 whereas IL‐18Rα expression was detected at low levels. Importantly, the IL‐12Rβ2 signalling chain, which is absent in all resting T cells, was up‐regulated in both CD45RA+ and CD45RO+ T cells as a result of stimulation with anti‐CD3 and anti‐CD28 in vitro. This observed up‐regulation was, however, restricted to 80% of the total CD4+ population. Finally, a very small proportion of the CD4+ CD45RO+ tonsillar T cells expressed the IL‐12 and IL‐18 receptors, thereby establishing the differential expression of these receptors between peripheral and tonsillar memory T cell subpopulations.

Human papillomavirus 16 integration and risk factors associated in anal samples of HIV-1 infected men.

Background: The integration of HPV-16 DNA into the host genome is considered an important event in the progression of premalignant cervical lesions to cervical cancer. The aim of our study was to assess the prevalence of HPV-16 integration in anal cytologic specimens of HIV-1 infected men and its association with risk factors. Patients Methods: This cross-sectional study included 269 HIV-infected males. Detection and typing of HPV-infection was done by multiplex PCR, and integration of HPV-16 by real-time PCR. Results: The overall anal HPV-infection prevalence was 78% (209/269), 29% (77/269) for HPV-16 infection, and 9% (25/269) for HPV-16 integration. In HPV-16 infected group, the integration prevalence represented 32% (25/77). The only risk factor associated with HPV-16 integration was the time since HIV diagnosis (OR = 1.2, 95% CI: 1.0–1.3; P = 0.010). The risk factors associated with abnormal cytology results were: HPV infection (OR = 17.8, 95% CI: 6.8–46.6), HPV-16 infection (OR = 4.6, 95% CI: 2.5–8.4), and presence of HPV-16 integrated forms (OR = 11.7, 95% CI: 1.5–93.5). Moreover, in the multivariate analysis, the HPV-16 integration continued representing the most important risk factor (OR = 20, 95% CI: 1.6–226) for anal cytologic abnormalities. Conclusion: HPV-16 infection and its integration in anal cells were highly prevalent in HIV-infected men. The assessment of HPV-16 integration rather than HPV-infection could be a good biomarker for predicting anal precancerous lesions in HIV-positive men.

New molecular method for the detection of human papillomavirus type 16 integration

Human papillomavirus (HPV) infection is the cause of cervical cancer. Integration of HPV-16 DNA in cervical cells is considered to be a key event in the progression towards invasive cancer, but little is known about this event in anal carcinogenesis. The integration could be a useful biomarker for cancer progression. Optimized assays are needed to determine the value of real-time detection of HPV integration in longitudinal studies, and this approach is only possible with a high-throughput assay. The aim of this study was to develop a new multiplex real-time PCR assay based on simultaneous amplification of the E2 and E6 HPV open reading frames (ORFs) in order to assess the physical status (episomal and/or integrated) of HPV-16 in anal cells of HIV-positive men. The comparative threshold (Ct) cycle values for E2 and E6 obtained for SiHA cells and artificial mixtures of episomal and integrated DNA were as expected: similar Ct for episomal forms and absence of E2 amplification for integrated forms. The multiplex real-time PCR was tested in 77 consecutive samples from individual HIV-infected patients with HPV-16 anal infection. The integration of HPV-16 was detected in 25 (32%) patients: 23 as mixed (episomal and integrated) and two as completed integrated forms. The integration occurs in the early stage of anal lesions and was associated with the severity of the lesions (p 0.004). The multiplex real-time PCR assay developed in the course of this study was shown to be a simple, sensitive, specific and inexpensive technique which may be applied routinely to detect HPV-16 integration.

Tacrolimus treatment of plasmacytoid dendritic cells inhibits dinucleotide (CpG-)-induced tumour necrosis factor-alpha secretion

Tacrolimus is a widely used immunosuppressive agent. Although T cells are the main targets of these pharmacological drugs, antigen presentation may also be affected. Among antigen‐presenting cells, plasmacytoid dendritic cells (PDCs) are the main source of type I interferons upon microbial challenge, and are involved in several diseases and autoimmune disorders. The aim of this study was to evaluate whether tacrolimus can modulate the function of PDCs in vitro. Maturation and function of PDCs was determined using flow cytometry, enzyme‐linked immunosorbent assay and cytometry bead arrays. The effect of tacrolimus on PDCs was observed mainly when the cells were pretreated with the immunosuppressive agent before activation. Upon dinucleotide–oligodeoxynucleotide (CpG–ODN) activation, tacrolimus pretreated PDCs showed a significant reduction in the surface expression of co‐stimulatory molecules and human leucocyte antigen D‐related (HLA‐DR) and secreted reduced levels of tumour necrosis factor (TNF)‐α. These results show that tacrolimus treatment of PDCs impairs CpG‐induced activation, which could affect the outcome of the immune response.

Induction of interleukins IL-6 and IL-8 by siRNA

The HIV‐1 co‐receptor CCR5 has been thought a relevant target for small interfering RNA (siRNA)‐based therapeutics. However, recent findings suggest that siRNA can stimulate innate cytokine responses in mammals. All siRNA agents tested were able to down‐regulate the expression of CCR5, albeit with different efficiency (51–74% down‐regulation), block HIV‐induced syncytia formation between HIV‐1 BaL‐infected and uninfected CD4+ cells or block single‐round HIV‐1 infection as measured by a luciferase reporter assay (46–83% inhibition). Conversely, siRNA directed against CCR5 did not affect replication of a vesicular stomatitis virus (VSV) pseudotyped virus, suggesting that inhibition of HIV replication was specific to CCR5 down‐regulation. However, two of four siRNA tested were able to induce the production of interleukin (IL) IL‐6 (sixfold induction) and IL‐8 (ninefold induction) but no interferon (IFN)‐α, IFN‐β, IFN‐γ, tumour necrosis factor (TNF)‐α, monocyte chemoattractant protein (MCP)‐1, macrophage inflammatory protein (MIP)‐1α, MIP‐1β, RANTES, IL‐1β, IL‐10 or IL‐12p70 cytokine induction was noted. In the absence of detectable IFN‐α, IL‐6 or IL‐8 may represent markers of non‐specific effects triggered by siRNA.

The reconstitution of the thymus in immunosuppressed individuals restores CD4‐specific cellular and humoral immune responses

Infection with HIV‐1 frequently results in the loss of specific cellular immune responses and an associated lack of antibodies. Recombinant growth hormone (rGH) administration reconstitutes thymic tissue and boosts the levels of peripheral T cells, so rGH therapy may be an effective adjuvant through promoting the recovery of lost cellular and T‐cell‐dependent humoral immune responses in immunosuppressed individuals. To test this concept, we administered rGH to a clinically defined group of HIV‐1‐infected subjects with defective cellular and serological immune responses to at least one of three commonly employed vaccines (hepatitis A, hepatitis B or tetanus toxoid). Of the original 278 HIV‐1‐infected patients entering the trial, only 20 conformed to these immunological criteria and were randomized into three groups: Group A (n = 8) receiving rGH and challenged with the same vaccine to which they were unresponsive and Groups B (n = 5) and C (n = 7) who received either rGH or vaccination alone, respectively. Of the eight subjects in Group A, five recovered CD4 cellular responses to vaccine antigen and four of these produced the corresponding antibodies. In the controls, three of the five in group B recovered cellular responses with two producing antibodies, whereas three of the seven in Group C recovered CD4 responses, with only two producing antibodies. Significantly, whereas seven of ten patients receiving rGH treatment in Group A (six patients) and B (one patient) recovered T‐cell responses to HIVp24, only two of six in Group C responded similarly. In conclusion, reconstitution of the thymus in immunosuppressed adults through rGH hormone treatment restored both specific antibody and CD4 T‐cell responses.