Distinct Inflammatory Phenotypes are associated with subclinical and clinical Cardiovascular disease in People living with HIV.

Despite inflammation being implicated in cardiovascular disease (CVD) in people with HIV (PWH), considerable heterogeneity within populations of PWH exists. Stratifying CVD risk based on inflammatory phenotype could play an important role. Using principal component analyses and unsupervised hierarchical clustering, we examined 38 biomarkers to identify inflammatory phenotypes in two independent cohorts of PWH. We identified three distinct inflammatory clusters present in both cohorts that associated with altered risk of both subclinical CVD (cohort 1) and prevalent clinical CVD (cohort 2) after adjusting for CVD risk factors. These data support precision medicine approaches to enhance CVD risk assessment in PWH.

The TLR9 agonist MGN1703 triggers a potent type I interferon response in the sigmoid colon.

Toll-like receptor 9 (TLR9) agonists are being developed for treatment of colorectal and other cancers, yet the impact of these drugs on human intestines remains unknown. This, together with the fact that there are additional potential indications for TLR9 agonist therapy (e.g., autoimmune and infectious diseases), led us to investigate the impact of MGN1703 (Lefitolimod) on intestinal homeostasis and viral persistence in HIV-positive individuals. Colonic sigmoid biopsies were collected (baseline and week four) from 11 HIV+ individuals on suppressive antiretroviral therapy, who received MGN1703 (60 mg s.c.) twice weekly for 4 weeks in a single-arm, phase 1b/2a study. Within sigmoid mucosa, global transcriptomic analyses revealed 248 modulated genes (false discovery rate<0.05) including many type I interferon (IFN)-stimulated genes. MGN1703 increased the frequencies of cells exhibiting MX1 (P=0.001) and ISG15 (P=0.014) protein expression. No changes were observed in neutrophil infiltration (myeloperoxidase; P=0.97). No systematic effect on fecal microbiota structure was observed (analysis of similarity Global R=-0.105; P=0.929). TLR9 expression at baseline was inversely proportional to the change in integrated HIV DNA during MGN1703 treatment (P=0.020). In conclusion, MGN1703 induced a potent type I IFN response, without a concomitant general inflammatory response, in the intestines.

A comprehensive review of the nasal microbiome in chronic rhinosinusitis (CRS).

Chronic rhinosinusitis (CRS) has been known as a disease with strong infectious and inflammatory components for decades. The recent advancement in methods identifying microbes has helped implicate the airway microbiome in inflammatory respiratory diseases such as asthma and COPD. Such studies support a role of resident microbes in both health and disease of host tissue, especially in the case of inflammatory mucosal diseases. Identifying interactive events between microbes and elements of the immune system can help us to uncover the pathogenic mechanisms underlying CRS. Here we provide a review of the findings on the complex upper respiratory microbiome in CRS in comparison with healthy controls. Furthermore, we have reviewed the defects and alterations of the host immune system that interact with microbes and could be associated with dysbiosis in CRS.

Gut dendritic cell activation links an altered colonic microbiome to mucosal and systemic T-cell activation in untreated HIV-1 infection.

HIV-1-associated disruption of intestinal homeostasis is a major factor contributing to chronic immune activation and inflammation. Dendritic cells (DCs) are crucial in maintaining intestinal homeostasis, but the impact of HIV-1 infection on intestinal DC number and function has not been extensively studied. We compared the frequency and activation/maturation status of colonic myeloid DC (mDC) subsets (CD1c(+) and CD1c(neg)) and plasmacytoid DCs in untreated HIV-1-infected subjects with uninfected controls. Colonic mDCs in HIV-1-infected subjects had increased CD40 but decreased CD83 expression, and CD40 expression on CD1c(+) mDCs positively correlated with mucosal HIV-1 viral load, with mucosal and systemic cytokine production, and with frequencies of activated colon and blood T cells. Percentage of CD83(+)CD1c(+) mDCs negatively correlated with frequencies of interferon-γ-producing colon CD4(+) and CD8(+) T cells. CD40 expression on CD1c(+) mDCs positively associated with abundance of high prevalence mucosal Prevotella copri and Prevotella stercorea but negatively associated with a number of low prevalence mucosal species, including Rumminococcus bromii. CD1c(+) mDC cytokine production was greater in response to in vitro stimulation with Prevotella species relative to R. bromii. These findings suggest that, during HIV infection, colonic mDCs become activated upon exposure to mucosal pathobiont bacteria leading to mucosal and systemic immune activation.

An altered intestinal mucosal microbiome in HIV-1 infection is associated with mucosal and systemic immune activation and endotoxemia.

Human immunodeficiency virus-1 (HIV-1) infection disrupts the intestinal immune system, leading to microbial translocation and systemic immune activation. We investigated the impact of HIV-1 infection on the intestinal microbiome and its association with mucosal T-cell and dendritic cell (DC) frequency and activation, as well as with levels of systemic T-cell activation, inflammation, and microbial translocation. Bacterial 16S ribosomal DNA sequencing was performed on colon biopsies and fecal samples from subjects with chronic, untreated HIV-1 infection and uninfected control subjects. Colon biopsies of HIV-1-infected subjects had increased abundances of Proteobacteria and decreased abundances of Firmicutes compared with uninfected donors. Furthermore at the genus level, a significant increase in Prevotella and decrease in Bacteroides was observed in HIV-1-infected subjects, indicating a disruption in the Bacteroidetes bacterial community structure. This HIV-1-associated increase in Prevotella abundance was associated with increased numbers of activated colonic T cells and myeloid DCs. Principal coordinates analysis demonstrated an HIV-1-related change in the microbiome that was associated with increased mucosal cellular immune activation, microbial translocation, and blood T-cell activation. These observations suggest that an important relationship exists between altered mucosal bacterial communities and intestinal inflammation during chronic HIV-1 infection.

Neurodegeneration and ageing in the HAART era.

Cognitive impairment and neurodegeneration still occur despite highly active antiretroviral therapy (HAART). While there are many potential reasons for this, there is increasing evidence that such impairment occurs in the absence of a clear cause. Furthermore, there are data that some neurodegenerative diseases, especially Alzheimer's or an Alzheimer-like illness, are becoming more common in the context of HAART-treated human immunodeficiency virus (HIV) disease. This review will critically examine the evidence underpinning these observations. Potential mechanisms will be discussed with particular emphasis on the effect of ageing and how it overlaps with the effects of HIV disease itself thereby leading to neurodegeneration. The nature of this overlap will then be explored for its potential role in the facilitated expression and development of neurodegenerative diseases. Lastly, there will be a brief discussion of interventions to minimize such neurodegeneration including optimization of HAART for brain entry.

In vitro cell-mediated immune responses of human immunodeficiency virus-infected and -uninfected individuals to whole cytomegalovirus antigens and their subunits.

The aim of this study was to optimize the ability to detect cytomegalovirus (CMV)-specfic cell-mediated immunity (CMI) in human immunodeficiency virus (HIV)-infected individuals by comparing different assays (the lymphocyte proliferation assay [LPA] and assays for gamma interferon [IFN-gamma] and interleukin-2 [IL-2] production) and CMV antigenic preparations. Thresholds discriminating positive from negative CMI results were developed with specimens from 36 CMV-seropositive and 21 CMV-seronegative healthy individuals. The analysis showed that the CMI elicited by any of the four CMV whole lysates tested in this study tended to be more robust and sensitive than the responses to the subunit antigens gB and pp65. LPA and inducible IFN-gamma but not IL-2 were highly sensitive measures of CMV-specific CMI in HIV-infected and -uninfected individuals. The ability to detect CMV-specific LPA or IFN-gamma responses in HIV-infected individuals significantly increased with higher CD4 cell numbers. Nevertheless, the proportion of HIV-infected subjects with CD4 counts of >or=500 cells/mul who had a detectable CMV-specific CMI remained significantly lower than that of healthy adults. The ability to detect CMV-specific CMI in HIV-infected individuals decreased with higher levels of HIV replication, with discriminative thresholds of 10(3) to 10(4) HIV RNA copies/ml of plasma, for LPA or inducible IFN-gamma production elicited by different antigens. The LPA responses obtained with CMV whole lysate and phytohemagglutinin were significantly correlated in HIV-infected subjects but not uninfected controls, indicating a novel characteristic of the CMI defect caused by HIV. The intrasubject variabilities of the CMV-specific CMI were similar in HIV-infected and -uninfected individuals. These data show that LPA and the inducible IFN-gamma production elicited by CMV whole lysates may be used to assess modifications of the immune competency of HIV-infected individuals.

Evaluation of the impact of highly active antiretroviral therapy on immune recovery in antiretroviral naive patients.

OBJECTIVES: To examine the extent of immune reconstitution in treatment-naive patients with CD4 T-cell counts <500 cells/microL following 48 weeks of highly active antiretroviral therapy (HAART). METHODS: Thirteen antiretroviral naive patients were evaluated longitudinally for 48 weeks on HAART utilizing immune functional and lymphocyte phenotyping assays, including lymphocyte proliferation assay, flow cytometric evaluation of cell surface markers, and delayed type hypersensitivity skin tests. Virologic responses were monitored using commercially available viral load assays and gag/pol mRNA quantification using simultaneous immunophenotyping/UltraSensitive fluorescence in situ hybridization (ViroTect In Cell HIV-1 Detection Kit; Invirion, Frankfort, MI). Thymic function was evaluated for a subset of four patients using real-time polymerase chain reaction (PCR) for T-cell receptor excision circle (TREC) quantification and thymic scans using computerized axial tomography (CT) of the thymus. RESULTS: HAART initiation resulted in a significant decline in plasma viremia and percentage of infected peripheral blood cells, and a rise in CD4 T cells from a baseline median of 207 cells/microL to a week-48 median of 617 cells/microL. The rise was predominately in CD4 memory cells. Naive T cells also increased in number, but at a slower rate. Activated (HLA-DR CD38) CD4 and CD8 T cells were elevated at baseline (24 and 62%, respectively) and declined by week 48 (17 and 36%, respectively) but did not reach normal levels. The number of Fas CD4 T cells increased from a baseline median of 169 to 381 cells/microL at week 48. Both soluble interleukin (IL)-2 and tumour necrosis factor (TNF) II receptors declined by week 48. HIV p24 lymphocyte proliferation assay responses were transiently detected in three patients. TREC values increased from a median 6400 copies/microg at baseline to a week-48 median value of 26 697 copies/microg. CONCLUSION: Immune functional reconstitution was not achieved in these HAART naive patients.

Determinants of HIV-1 shedding in the genital tract of women.

BACKGROUND: Plasma HIV-1 RNA concentration has been the best predictor for risk of heterosexual and perinatal transmission. However, direct contact with HIV-1 present locally in the genital tract might be necessary for transmission. We aimed to assess the relation between HIV-1 shedding (RNA or culturable virus) in female genital secretions and other factors that might affect HIV-1 shedding. METHODS: This was a cross-sectional study within the Women's Interagency HIV Study (WIHS), a prospective longitudinal cohort study of HIV-infected women. We enrolled 311 HIV positive women from Jan 30, 1997 to July 1, 1998. We did clinical assessments, cultured HIV-1, and measured RNA in peripheral blood mononuclear cells (PBMC) and genital secretions. We compared the results with univariate and multivariate analyses. Presence of HIV-1 RNA or culturable virus in genital secretions was defined as HIV-1 shedding. FINDINGS: HIV-1 RNA was present in genital secretions of 57% (152/268) of women whereas infectious virus was detected only in 6% (17/271). Genital tract HIV-1 shedding was found in 80% (130/163) of women with detectable plasma RNA and 78% (116/148) of women with positive PBMC cultures. 33% (27/83) of women with less than 500 copies/mL plasma RNA and 39% (35/90) of those with negative PBMC cultures also had genital tract shedding. INTERPRETATION: Plasma RNA concentration, both qualitatively and quantitatively, was the most important factor in predicting genital HIV-1 shedding, even among women receiving potent antiretroviral therapy. However, HIV-1 shedding did occur in women with less than 500 copies/mL plasma HIV-1 RNA. This finding suggests that a separate reservoir of HIV-1 replication may exist in some women.

T cell receptor excision circle (TREC) content following maximum HIV suppression is equivalent in HIV-infected and HIV-uninfected individuals.

BACKGROUND: The adult human thymus contributes to de novo T cell synthesis; such synthesis can be assessed by analyzing T cell receptor excision circles (TREC). METHODS: TREC levels were measured in total peripheral blood mononuclear cells (PBMC) and CD4- and CD8-enriched cells of 29 HIV-positive patients with maximal viral suppression. The expression of CD45RA+CD45RO-, CD45RA+CD62L+, CD45RO-CD27+CD95low and HLA-DR+CD38+ was assessed using three-color flow cytometric analysis of whole blood. Thymic index score was based on computed tomographic scans of the thymus. The relationship of TREC with thymic index and the expression of the naive phenotypes was evaluated. RESULTS: TREC expression was not statistically different in these HIV-positive patients from that in age-matched HIV-negative controls. Among HIV-positive patients with CD4 cell count of > 500 x 10(6) cells/l after antiretroviral therapy (n = 15), PBMC TREC levels correlated with the expression of CD45RA+CD45RO- and CD45RA+CD62L+ naive phenotypes, and inversely correlated with the expression of HLA-DR+CD38+. The change between pre- and post-therapy CD4 cell counts for these 15 patients significantly correlated with both thymic index and expression of the CD45RA+CD45RO- phenotype. CONCLUSIONS: The finding that TREC expression was equivalent between HIV-positive patients after therapy and HIV-negative donors suggests that there is no reduction in thymic output among HIV-positive individuals after therapy. Given that TREC is inversely correlated with HLA-DR/CD38 expression, its analysis in studies of thymopoiesis should be evaluated in the context of maximum viral suppression to reduce HIV-mediated immune activation and/or by normalizing for cell turnover.

Variation of human immunodeficiency virus type 1 viral RNA levels in the female genital tract: implications for applying measurements to individual women.

The short-term detection and variability of human immunodeficiency virus type 1 (HIV-1) RNA level was assessed in the blood plasma and genital tracts of 55 HIV-1-infected women. Specimens were collected weekly for 8 weeks from the endocervical canal with wicks and cytobrushes and from the ectocervix and vagina with cervicovaginal lavage. In all, 48 women (87.3%) had detectable genital tract HIV-1 RNA at > or =1 collection times. HIV-1 RNA levels varied least in specimens from endocervical canal wick and most in cervicovaginal lavage samples. The within-subject variation for genital-tract virus level was greater than that for blood. Overall, the odds for viral RNA detection in the genital tract approximately tripled for each 10-fold increase in plasma viral RNA concentration (P<.001) or with concomitant genital tract infection (P=.003). Endocervical canal wicks should be considered as an adjunct to cervicovaginal lavage, to improve the sensitivity and precision of HIV-1 RNA detection.

Persistence of intracellular HIV-1 mRNA correlates with HIV-1-specific immune responses in infected subjects on stable HAART.

OBJECTIVE: To determine if low level, persistent, HIV-1 replication within specific immune cells contributes to HIV-1-specific immune responsiveness. DESIGN: We analyzed 59 HIV-1-infected subjects on stable highly active antiretroviral therapy (HAART) therapy (not including zidovudine) with suppressed plasma viremia (< 400 copies/ml) for phenotypic and lymphoproliferative correlates of immune function. METHODS: Peripheral blood mononuclear cells were collected for immunophenotyping, lymphoproliferative assays, and simultaneous immunophenotyping/ultrasensitive in situ hybridization. Plasma was collected for plasma viral load as determined by the Ultra Sensitive Roche Amplicor RT-PCR. Descriptive statistics (mean and SD, median, first and third quartiles) were determined for all variables in two groups defined as having persistent viral replication present or absent. The two-sided Wilcoxon test (continuity correction, 0.5) was used to compare lymphocyte phenotypes, lymphoproliferative assay responses, intracellular gag-pol mRNA, lowest CD4 counts and CD4% of these two groups. RESULTS: HIV-1 replication in CD4, CD45RO memory T lymphocytes persists in spite of undetectable plasma viral load. Patients (n = 24) with persistent intracellular expression of HIV-1 mRNA (> 0.3%) showed significant in vitro proliferative responses to HIV-1 p24 (stimulation index > or = 10) compared to patients (n = 35) without persistent intracellular replication. The group with persistent HIV-1 replication in cells showed no significant response to the recall antigen tetanus toxoid but a trend toward higher responses to pathogen antigens. There were no differences between the groups in the prevalence of AIDS or occurrences of opportunistic infections; however, the high viral persistence group was more HAART experienced (P < 0.05). CONCLUSIONS: These results suggest that HIV-1-specific immune responses correlate with evidence of ongoing HIV-1 replication.

Multiple CD4+ cell kinetic patterns and their relationships with baseline factors and virological responses in HIV type 1 patients receiving highly active antiretroviral therapy.

This exploratory analyses characterizes patterns of lymphocyte recovery in HIV-1-infected patients treated with highly active antiretroviral therapy (HAART) and investigates their relationship with baseline indices and virologic responses. We modeled kinetics of total CD4+ lymphocytes, as well as naive (CD45RA+ CD62L+), and memory (CD45RA- CD45RO+) subsets in 48 patients treated with AZT/3TC/Ritonavir for 48 weeks in ACTG protocol 315. Cell kinetic indices were estimated by nonlinear regression methods and were correlated with baseline factors and virologic responses. Five different kinetic patterns were identified, including biphasic growth, growth-plateau, growth-depletion, decay-recovery, and biphasic decay. Although overall mean lymphocyte responses showed a biphasic increase in cell number, a careful investigation reveals that only one-third of patients actually followed the biphasic growth pattern in CD4+ cell response, while 44% of 48 patients from this study followed the growth-depletion pattern. CD4+ cell recovery during the first phase and the 48-week study period were negatively correlated with baseline CD4+ cell counts, and positively correlated with baseline viral load. Memory CD4+ cell recovery during the first phase was also negatively correlated with baseline memory CD4+ and total CD4+ cell number, but the recovery rate of memory CD4+ cells during the second phase was positively correlated with baseline CD4+ cell number. Patients with a decay in CD4+ cell count during treatment were more likely to have experienced virological rebound (58%) than patients with nondecay patterns (24%). The rate and magnitude of the absolute increase in total CD4+ and memory CD4+ cell number (but not naive CD4+ cells) during the second phase were lower in patients with viral rebound compared with patients with persistent viral suppression. These results show that the kinetics of lymphocyte reconstitution in response to potent antiretroviral therapy in individual patients vary considerably from the "classic" biphasic increase that characterizes the mean or median response pattern. Pattern analysis of lymphocyte kinetics may be useful for testing relationships among factors that modulate the response to treatment.

A menstrual cycle pattern for cytokine levels exists in HIV-positive women: implication for HIV vaginal and plasma shedding.

OBJECTIVES: To evaluate the effect of the menstrual cycle in HIV-positive women on plasma and genital cytokine levels, interrelationships between vaginal and plasma cytokines, CD4 and CD8 T cell fluctuations, and genital and plasma viral loads. METHODS: Plasma and cervicovaginal lavage specimens were collected from 55 HIV-positive women with CD4 cell counts < 350 cells/microl during phases of the menstrual cycle. Samples were assayed for IL-1beta, IL-6, IL-4, IL-8, IL-10, TGFbeta, TNFalpha, INFgamma, MIP1alpha, MIP1beta, RANTES, and TNFR-II using enzyme-linked immunosorbent assays. CD4 and CD8 T cell expression was evaluated by flow cytometry. Repeated measures regression models were used to assess the effect of the menstrual cycle on cytokines and viral load. Multivariate repeated regression models were used to assess the correlation among selected cytokines and between selected cytokines and HIV viral load. RESULTS: Vaginal IL-1beta, IL-4, IL-6, IL-8, IL-10, MIP1beta, RANTES, TGFbeta, and TNFR-II were significantly elevated during menses but were not altered during other phases. Plasma cytokine levels were not altered during the menstrual cycle. A positive Candida culture increased vaginal IL-8 during menses, whereas vaginal discharge was associated with a reduction in vaginal IL-4, IL-10, and RANTES. CD4 and CD8 cell numbers did not vary with the menstrual cycle. Vaginal cytokine levels correlated only with vaginal viral load, in a sampling method-dependent manner. CONCLUSION: We provide evidence of elevated vaginal cytokine levels during menses, which appear to regulate vaginal and not plasma HIV shedding, suggesting that a menstrual cycle pattern exists for cytokine production in HIV-positive women impacting vaginal shedding of HIV.

Upregulation of CD4 on CD8+ T cells: CD4dimCD8bright T cells constitute an activated phenotype of CD8+ T cells.

Aside from an intermediate stage in thymic T-cell development, the expression of CD4 and CD8 is generally thought to be mutually exclusive, associated with helper or cytotoxic T-cell functions, respectively. Stimulation of CD8+ T cells, however, induces the de novo expression of CD4. We demonstrate that while superantigen (staphylococcal enterotoxin B, SEB) and anti-CD3/CD28 costimulation of purified CD8+ T cells induced the expression of CD4 on CD8+ T cells by 30 and 17%, respectively, phytohaemagglutinin (PHA) stimulation did not induce CD4 expression on purified CD8+ T cells but significantly induced the expression of both CD4 on CD8 (CD4dimCD8bright) and CD8 on CD4 (CD4brightCD8dim) T cells in unfractionated peripheral blood mononuclear cells (PBMC). The level of the PHA-mediated induction of CD4dimCD8bright and CD4brightCD8dim was at 27 and 17%, respectively. Depletion of CD4+ T cells from PBMC abrogated this PHA-mediated effect. Autologous CD4+ and CD8+ T-cell co-cultures in the presence of PHA induced this CD4dimCD8bright T-cell expression by 33%, demonstrating a role for CD4 cells in the PHA-mediated induction of the double positive cells. The induction of CD4dimCD8bright was independent of a soluble factor(s). Phenotypic analysis of CD4dimCD8bright T cells indicated significantly higher levels of CD95, CD25, CD38, CD69, CD28, and CD45RO expression than their CD8+CD4- counterparts. CD4dimCD8bright T cells were also negative for CD1a expression and were predominantly T-cell receptor (TCR) alphabeta cells. Our data demonstrate that CD4dimCD8bright T cells are an activated phenotype of CD8+ T cells and suggest that CD4 upregulation on CD8+ T cells may function as an additional marker to identify activated CD8+ T cells.

A potential role for interleukin-7 in T-cell homeostasis.

Interleukin (IL)-7 is known to up-regulate thymopoietic pathways of T-cell regeneration. Recent work also has shown it to potently enhance thymic-independent peripheral expansion and to restore immunocompetence in athymic T-cell-depleted hosts. We hypothesized that endogenous IL-7 could contribute to the restoration of T-cell homeostasis following T-cell depletion. To analyze this, we evaluated circulating IL-7 levels and lymphocyte subsets in multiple clinical cohorts with T-cell depletion of varying etiologies. In pediatric (n = 41) and adult (n = 51) human immunodeficiency virus-infected CD4-depleted patients, there were strong inverse correlations between IL-7 levels and CD4 counts (r = -0.77, P <.0001, and r = -0.68, P <.0001). Declines in IL-7 were temporally correlated with recovery of CD4 counts. Similar patterns were observed in CD4-depleted patients receiving cancer chemotherapy (r = -0.65, P =.009). Therefore, in 2 disparate clinical scenarios involving CD4 depletion, IL-7 levels dynamically respond to changes in CD4 T-cell number, making this cytokine uniquely suited as a candidate regulator of T-cell homeostasis. Furthermore, in patients with idiopathic CD4 lymphopenia, a much weaker relationship between IL-7 levels and peripheral blood CD4 counts was observed, suggesting that an impaired IL-7 response to CD4 depletion may contribute to the impaired lymphocyte homeostasis observed in this population. In light of the known effects of IL-7 on T-cell regeneration, we postulate that increased availability of IL-7 could play a critical role in restoring T-cell homeostasis following T-cell depletion.

Anti-CCR5 antibodies in sera of HIV-positive individuals.

One of the proposed mechanisms for resistance to human immunodeficiency virus-1 (HIV-1) infection is the presence of antibodies against receptor for CC-chemokines (CCR5). These antibodies, detected in sera of uninfected individuals exposed to HIV, have been shown to downmodulate surface CCR5 in vivo and are able to neutralize the infectivity of CCR5 strains in vitro. To address the potential role of anti-CCR5 antibodies in HIV infection, we analyzed anti-CCR5 antibody levels in plasma from HIV-infected patients who present a wide range of CD4(+) T-cell counts and viral load. Increased levels of anti-CCR5 antibodies were found in plasma from 13/46 HIV-positive donors compared with healthy controls (0/36). However, antibody levels were not associated with disease stage evaluated by CD4(+) T-cell counts and viral load.

Quantitative single cell methods that identify cytokine and chemokine expression in dendritic cells.

Two techniques based upon flow cytometry (FCM) and in situ image analysis were developed for quantification of intracellular cytokine and chemokine protein expression at the single cell level in dendritic cells (DCs). The qualitative and quantitative differences between the two methods were evaluated. In vitro differentiated DCs were stimulated with lipopolysaccaride (LPS) and thereafter stained for either IL-8, which is secreted through the Golgi-organelle, or IL-1ra, which localises diffusely in the cytoplasm. Microscopic examination, both for fluorophore and enzymatically stained cells, showed that DCs expressed IL-8 and IL-1ra with two different staining patterns. FCM analysis showed high frequencies of IL-1ra producing cells (76+/-13%), which was similar to the frequency obtained by in situ imaging. However, in contrast to IL-1ra, the incidence of IL-8 expressing DCs showed high variability between the donors. The numbers of positive cells were 19+/-19% as measured by FCM. The detection of IL-8 analysed by in situ imaging revealed higher frequencies (26+/-14%). The addition of brefeldin-A, leading to cytoplasmic accumulation of proteins secreted through the Golgi endoplasmatic route, generated a significantly increased signal intensity and incidence of producer cells, resulting in similar frequencies for both methods. FCM has the advantage of being less time consuming than image analysis and is also able to facilitate multiple colour analysis. However, FCM is less accurate in detecting and quantifying cytokines and chemokines with a preserved juxtanuclear staining pattern. The correct choice of detection technique therefore depends on the study question.

Leukemia inhibitory factor inhibits HIV-1 replication and is upregulated in placentae from nontransmitting women.

The placenta may play a critical role in inhibiting vertical transmission of HIV-1. Here we demonstrate that leukemia inhibitory factor (LIF) is a potent endogenous HIV-1-suppressive factor produced locally in placentae. In vitro, LIF exerted a potent, gp130-LIFRbeta-dependent, HIV coreceptor-independent inhibition of HIV-1 replication with IC50 values between 0.1 pg/ml and 0.7 pg/ml, depending on the HIV-1 isolate. LIF also inhibited HIV-1 in placenta and thymus tissues grown in ex vivo organ culture. The level of LIF mRNA and the incidence of LIF protein-expressing cells were significantly greater in placentae from HIV-1-infected women who did not transmit HIV-1 to their fetuses compared with women who transmitted the infection, but they were not significantly different from placentae of uninfected mothers. These findings demonstrate a novel pathway for endogenous HIV suppression that may prove to be an effective immune therapy for HIV infection.