Skin bioengineering in the diagnosis of occupational protein contact dermatitis.

Protein contact dermatitis (PCD) often presents as chronic hand eczema (CHE) with an immediate hypersensitivity to protein proved by a positive skin prick test or by the presence of specific immunoglobulin E. This is frequently induced by occupational exposure to proteins in food workers, farmers, animal breeders, veterinarians and healthcare professionals. While skin barrier impairment is crucial in the pathogenesis of PCD, methods to assess skin barrier function such as trans-epidermal water loss and stratum corneum hydration are not widely used in clinical settings. We describe the diagnostic workup of occupational PCD due to Argentinean shrimps and discuss how the use of skin bioengineering methods including assessment of corneocytes morphology by Scanning Electron Microscopy provides with insightful information on skin barrier function. Diagnosis of PCD is time-consuming and a multidisciplinary team contributes to early diagnosis and proper occupational rehabilitation.

Enhanced protection conferred by mucosal BCG vaccination associates with presence of antigen-specific lung tissue-resident PD-1+ KLRG1- CD4+ T cells.

BCG, the only vaccine licensed against tuberculosis, demonstrates variable efficacy in humans. Recent preclinical studies highlight the potential for mucosal BCG vaccination to improve protection. Lung tissue-resident memory T cells reside within the parenchyma, potentially playing an important role in protective immunity to tuberculosis. We hypothesised that mucosal BCG vaccination may enhance generation of lung tissue-resident T cells, affording improved protection against Mycobacterium tuberculosis. In a mouse model, mucosal intranasal (IN) BCG vaccination conferred superior protection in the lungs compared to the systemic intradermal (ID) route. Intravascular staining allowed discrimination of lung tissue-resident CD4+ T cells from those in the lung vasculature, revealing that mucosal vaccination resulted in an increased frequency of antigen-specific tissue-resident CD4+ T cells compared to systemic vaccination. Tissue-resident CD4+ T cells induced by mucosal BCG displayed enhanced proliferative capacity compared to lung vascular and splenic CD4+ T cells. Only mucosal BCG induced antigen-specific tissue-resident T cells expressing a PD-1+ KLRG1- cell-surface phenotype. These cells constitute a BCG-induced population which may be responsible for the enhanced protection observed with IN vaccination. We demonstrate that mucosal BCG vaccination significantly improves protection over systemic BCG and this correlates with a novel population of BCG-induced lung tissue-resident CD4+ T cells.

Regulation of interleukin-8 gene expression after phagocytosis of zymosan by human monocytic cells.

Monocyte phagocytosis of pathogens or inflammatory debris leads to chemokine secretion and heralds the influx of leukocytes to the site of injury. Persistent chemokine secretion can lead to tissue damage. However, the mechanisms by which phagocytosis regulates chemokine synthesis remain poorly understood. As a first step, we have studied regulation of interleukin (IL) 8 gene expression after interaction with zymosan or latex. IL-8 secretion was consistently one- or twofold higher after incubation with zymosan than with latex. Nuclear factor (NF) kappaB translocation to the nucleus was induced by zymosan but not latex, indicating that its translocation is dependent on the nature of the phagocytic stimulus. NFkappaB activation coincided with IkappaBalpha degradation but had no effect on processing of NFkappaB1/p105, the precursor of the NFkappaB protein p50. The NFkappaB inhibitor gliotoxin abrogated zymosan-induced IL-8 synthesis in peripheral blood monocytes, further demonstrating that the induction of IL-8 mRNA by zymosan is NFkappaB dependent. SB203580 inhibition of the p38 mitogen-activated protein kinase (MAPK) pathway significantly decreased zymosan-induced IL-8 mRNA accumulation. Inhibitors of protein kinases A and C or tyrosine kinases had no significant effect on zymosan-induced IL-8 synthesis. These data indicate that p38 MAPK and NFkappaB are critical in controlling zymosan-induced IL-8 secretion.

Improvement of BCG protective efficacy with a novel chimpanzee adenovirus and a modified vaccinia Ankara virus both expressing Ag85A.

A replication-deficient chimpanzee adenovirus expressing Ag85A (ChAdOx1.85A) was assessed, both alone and in combination with modified vaccinia Ankara also expressing Ag85A (MVA85A), for its immunogenicity and protective efficacy against a Mycobacterium tuberculosis (M.tb) challenge in mice. Naïve and BCG-primed mice were vaccinated or boosted with ChAdOx1.85A and MVA85A in different combinations. Although intranasally administered ChAdOx1.85A induced strong immune responses in the lungs, it failed to consistently protect against aerosol M.tb challenge. In contrast, ChAdOx1.85A followed by MVA85A administered either mucosally or systemically, induced strong immune responses and was able to improve the protective efficacy of BCG. This vaccination regime has consistently shown superior protection over BCG alone and should be evaluated further.

Interleukin-1.

Interleukin 1 (IL1) is a primary regulator of inflammatory and immune responses. Via its type I receptor it activates specific protein kinases, including the NF kappa B inducing kinase (NIK) and three distinct mitogen-activated protein (MAP) kinase cascades. These modulate a number of transcription factors including NF kappa B, AP1 and CREB each of which regulate a plethora of immediate early genes central to the inflammatory response. Phase I clinical trials of the soluble type I receptor and IRAP indicate that these have potential anti-inflammatory effects.

Enhancement of lymphocyte proliferation induced by interleukin-12 and anti-interleukin-10 in HIV-infected patients during highly active antiretroviral therapy.

Based on the potentially important role of IL-10 and IL-12 in the pathogenesis of HIV infection, we have examined the effect of highly active antiretroviral therapy (HAART) on the production of these two cytokines, and whether addition of IL-12 or anti-IL-10 in vitro could improve the proliferative response in peripheral blood mononuclear cells (PBMC) from HIV-infected patients during such therapy. Our findings are: (i) After initiating HAART there were no significant changes in PHA- or MAC-PPD-stimulated IL-10 and IL-12 levels in PBMC supernatants in the patient group as a whole. (ii) However, while a decline in IL-10 synthesis was shown in patients with high baseline MAC-PPD- and PHA-stimulated IL-10 levels, IL-10 increased in patients with lower baseline levels. A similar pattern was seen for MAC-PPD-stimulated IL-12 levels. (iii) Exogenously added IL-12 and anti-IL-10 markedly and additively improved MAC-PPD-stimulated PBMC proliferation in vitro. Thus, a loss of cell-mediated immune response exists in HIV-infected patients also during apparently successful HAART and this can be significantly improved by addition of IL-12 and anti-IL-10, at least in vitro. These results suggest that further exploration of both IL-10 and IL-12 as targets for immunomodulating therapy in HIV-infected patients in addition to HAART might be important.

c-Rel and p65 trans-activate the monocyte chemoattractant protein-1 gene in interleukin-1 stimulated mesangial cells.

BACKGROUND: The chemokine monocyte chemoattractant protein-1 (MCP-1) is secreted by human glomerular mesangial cells in response to interleukin-1 (IL-1) and has a central role in amplifying the inflammatory response during glomerulonephritis. However, the mechanism by which IL-1 regulates its transcription is not understood. Specific members of the nuclear factor kappaB/rel (NF-kappaB) proteins may regulate MCP-1 expression in a stimulus- and tissue-specific manner. METHODS: Electrophoretic mobility shift assays and Western blot analysis characterized the members of the NF-kappaB family that bound the two NF-kappaB sites of the MCP-1 enhancer (A1 and A2) in vitro. Trans-activation of the MCP-1 gene was investigated by transfer of the MCP-1 enhancer DNA to mesangial cells. RESULTS: Primary human mesangial cells contained in addition to p50 (NF-kappaB1) and p65 (Rel A) NF-kappaB proteins, the oncoprotein c-rel, and Rel B, but not p52 (NF-kappaB2). IL-1 induced c-rel to form a complex with p65, which bound the MCP-1 A2 site but not the A1 or IL-6 NF-kappaB sites in vitro. IL-1 up-regulated transfected MCP-1 enhancer activity. Cotransfer of the MCP-1 enhancer together with individual members of the NF-kappaB family showed that the heterodimer c-relp65 or (p65)2 can selectively trans-activate the MCP-1 gene via its A1 and A2 sites in mesangial cells. CONCLUSIONS: This study demonstrates for the first time that the c-rel oncoprotein can enhance MCP-1 transcription in mesangial cells and suggests that it may have an important role in amplifying gene expression in the inflamed glomerulus.

Bioflavonoid quercetin inhibits interleukin-1-induced transcriptional expression of monocyte chemoattractant protein-1 in glomerular cells via suppression of nuclear factor-kappaB.

Flavonoids are semiessential food components that possess anti-inflammatory properties. This report describes a novel potential of bioflavonoid quercetin as an inhibitor of monocyte chemoattractant protein-1 (MCP-1) in glomerular cells. Cultured mesangial cells as well as isolated glomeruli expressed MCP-1 mRNA in response to interleukin-1beta (IL-1beta). Quercetin dramatically inhibited the cytokine-triggered MCP-1 expression. To explore the mechanisms involved, effects of quercetin on the putative transcriptional activators of MCP-1, nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1), were examined. Exposure of the cells to IL-1beta caused activation of NF-kappaB without significant upregulation of AP-1 activity. NF-kappaB inhibitor MG132 diminished the IL-1-induced expression of MCP-1 in mesangial cells and isolated glomeruli, whereas c-Jun/AP-1 inhibitor curcumin did not affect this process. Consistently, NF-kappaB-inactive mesangial cells expressing a super-repressor mutant of IkappaBalpha showed blunted expression of MCP-1 by IL-1beta. In contrast, AP-1-inactive mesangial cells expressing a dominant-negative mutant of c-Jun exhibited the same level of MCP-1 mRNA as that in control cells. These results suggest that: (1) quercetin has the ability to attenuate activation of NF-kappaB; and (2) it inhibits IL-1-triggered MCP-1 expression via suppression of NF-kappaB, but not AP-1, in glomerular cells.

Interleukin 1-induced phosphorylation of MAD3, the major inhibitor of nuclear factor kappa B of HeLa cells. Interference in signalling by the proteinase inhibitors 3,4-dichloroisocoumarin and tosylphenylalanyl chloromethylketone.

The regulation of the inhibitor of nuclear factor kappa B (I kappa B) by interleukin 1 (IL1) was investigated in HeLa cells. Two forms of I kappa B were resolved by ion-exchange chromatography. The major form (75%) was identified as MAD3 by specific antisera. IL1 generated rapidly (6 min) an electrophoretically retarded form of MAD3 that was stable in acid and was converted into the unmodified form by phosphatase 2A. It thus corresponded to a phosphorylation of the protein on serine or threonine. IL1 also caused the disappearance of MAD3 from the cells, which was complete 15 min after stimulation and coincided with a 46% reduction of cellular I kappa B activity. Newly-synthesized MAD3 accumulated to pre-stimulation levels between 60 and 90 min after stimulation and this coincided with the down-regulation of the phosphorylating activity. The serine proteinase inhibitors 3,4-dichloroisocoumarin (DCI) and tosylphenylalanyl chloromethylketone (TPCK) prevented phosphorylation and disappearance of MAD3. At the same concentrations (10-100 microM), they also increased basal phosphorylation of the small heat shock protein (hsp27) and prevented the IL1- and phorbol 12-myristate 13-acetate-induced increases of its phosphorylation. The inhibitors were thus interfering with protein kinases when blocking degradation of MAD3. Recombinant MAD3 phosphorylated in vitro by protein kinase C was not electrophoretically retarded, suggesting that MAD3 was phosphorylated by another kinase in IL1-stimulated cells. Our results suggest that the IL1-induced phosphorylation of MAD3 on serine or threonine leads to its degradation. DCI and TPCK blocked phosphorylation mechanisms and it could not be concluded that serine proteinases were involved in the breakdown of MAD3.

Isolation, culture and characterization of human peritoneal mesothelial cells.

This study establishes a reproducible technique for the culture of human peritoneal mesothelial cells. Direct explants, as well as enzymatically degraded specimens, of human omentum have been used as the source of cells. Cells were grown on collagen and gelatin coated matrices and were maintained in supplemented Ham's F-12 medium containing 10% (vol/vol) Fetal calf serum. Morphologically and ultrastructurally, the cells formed a homogeneous population. They were polygonal when confluent and devoid of contaminating fibroblasts, endothelial cells and macrophages. Cultured mesothelial cells co-expressed cytokeratin and vimentin and synthesized laminin, fibronectin, mesosecrin, non-specific esterase and collagen Types I and III but not Type IV. Ultrastructural features included numerous surface microvilli, cytoplasmic vesicles and an abundant endoplasmic reticulum. The stimulation of mesothelial cells by the calcium ionophore A23187 demonstrated that the two major products of arachidonic acid metabolism were prostacyclin and prostaglandin E2. The peritoneal mesothelial cell may be pivotal in the initiation of the inflammatory response during peritonitis and its establishment in culture will provide the basis for an in vitro model of peritoneal inflammation.

Complex effects of interferon-alpha on the cytokine network in HIV infection--possible contribution to immunosuppression.

Since interleukin (IL-)2, IL-10 and IL-12 may contribute to the pathogenesis of human immune deficiency virus (HIV) infection we examined the effect of interferon (IFN)-alpha on these cytokines in cultures of various subsets of peripheral blood mononuclear cells (PBMC) in ten HIV-infected patients and ten healthy controls. Our main findings were: (1) IFN-alpha markedly enhanced IL-10 levels in a dose-dependent manner in both lipopolysaccharide (LPS)- and phytohaemagglutinin (PHA)-stimulated PBMC, as well as in anti-CD3- and anti-CD3/anti-CD28-stimulated T cells in both HIV-infected patients and controls. (2) In contrast, IFN-alpha had a downregulatory effect on IL-10 levels in Candida -stimulated PBMC,with particularly strong suppressive effect in HIV-infected patients. (3) Furthermore, IFN-alpha had a significant but modest stimulatory effect on IL-2 levels in PHA- and Candida -stimulated PBMC and anti-CD3-stimulated T cells. (4) IFN-alpha enhanced IL-12 levels in a dose-dependent manner in LPS-stimulated PBMC in both patients and controls. Our findings that IFN-alpha markedly enhanced IL-10 and modestly enhanced IL-2 and IL-12, suggest a net immunosuppressive effect of IFN-alpha in HIV-infected patients, possibly contributing to progression of immunodeficiency in these patients.

Selective sensitization to tumor necrosis factor-alpha-induced apoptosis by blockade of NF-kappaB in primary glomerular mesangial cells.

Recent data have implicated nuclear factor kappaB (NF-kappaB) in the prevention of apoptosis in transformed cell lines exposed to tumor necrosis factor alpha (TNF-alpha). However, it is obscure whether NF-kappaB plays an anti-apoptotic role in nontransformed cells, and it is not clear whether NF-kappaB inhibits apoptosis triggered by other mediators. We investigated the effect of specific inhibition of NF-kappaB on cytokine-induced apoptosis of glomerular mesangial cells, which is important in determining the outcome of glomerulonephritis. Cultured rat mesangial cells were stably transfected with the dominant negative mutant inhibitor of NF-kappaB (IkappaBalphaM). IkappaBalphaM was resistant to stimulus-dependent degradation and suppressed NF-kappaB activation induced by TNF-alpha (10 ng/ml) or IL-1beta (10 ng/ml). IkappaBalphaM significantly sensitized mesangial cells to TNF-alpha-induced apoptosis in a dose- and time-dependent manner but had no significant effects on the level of apoptosis in the presence of proinflammatory or apoptosis-inducing stimuli including Fas ligand, IL-1alpha, IL-1beta, hydrogen peroxide, lipopolysaccharide, cycloheximide, or serum deprivation. Moreover, IkappaBalphaM-mediated sensitization to TNF-alpha overcame the protective effect of mesangial cell survival factors present in serum, which usually inhibit killing of mesangial cells by the proapoptotic stimuli used. These data show that inhibition of NF-kappaB selectively sensitizes primary adult glomerular mesangial cells to TNF-induced apoptosis but not to other mediators of cell death including the Fas ligand.

IL-10 in HIV infection: increasing serum IL-10 levels with disease progression--down-regulatory effect of potent anti-retroviral therapy.

To examine the potential pathogenic role of IL-10 in HIV infection, we measured serum IL-10 levels in 51 HIV-infected patients and 23 healthy controls both on cross-sectional and longitudinal testing. All clinical groups (Centers for Disease Control (CDC) categories) of HIV-infected patients had significantly higher circulating IL-10 levels than controls, with the highest levels among the AIDS patients, particularly in patients with ongoing Mycobacterium avium complex (MAC) infection. Among 32 HIV-infected patients followed with longitudinal testing (median observation time 39 months), patients with disease progression had increasing IL-10 levels in serum, in contrast to non-progressing patients where levels were stable. While both IL-10 and tumour necrosis factor-alpha (TNF-alpha) increased in patients with disease progression, the IL-10/TNF-alpha ratio decreased in these patients, suggesting imbalance between these two cytokines. Finally, we found that highly active anti-retroviral therapy (HAART) induced a significant, gradual decrease in IL-10 levels but without normalization. These findings suggest a pathogenic role for IL-10 in HIV infection, and may suggest a possible role for immunomodulating therapy which down-regulates IL-10 activity in addition to concomitant potent anti-retroviral therapy in HIV-infected patients.

Interferons and interferon (IFN)-inducible protein 10 during highly active anti-retroviral therapy (HAART)-possible immunosuppressive role of IFN-alpha in HIV infection.

Interferons play an important, but incompletely understood role in HIV-related disease. We investigated the effect of HAART on plasma levels of IFN-alpha, IFN-gamma, neopterin and interferon-inducible protein 10 (IP-10) in 41 HIV-infected patients during 78 weeks of therapy. At baseline HIV-infected patients had raised levels of both IP-10 and IFN-alpha compared with healthy controls (n = 19), with particularly high levels in advanced disease. HAART induced a marked decrease in levels of both IFN-alpha, neopterin and IP-10, though not to normal concentrations. In contrast, IFN-gamma levels were low throughout the study, and not different from controls. While neopterin and IP-10 remained significantly decreased compared with baseline levels throughout the study, IFN-alpha levels returned to baseline at the end of the study. Persistently high IP-10 and IFN-alpha levels were associated with immunological treatment failure and even high baseline levels of IFN-alpha appeared to predict immunological relapse. Furthermore, we found a markedly suppressive effect of exogenously added IFN-alpha on phytohaemagglutinin-stimulated lymphocyte proliferation in both patients and controls, and this suppressive effect seemed not to involve enhanced lymphocyte apoptosis. Our findings suggest a pathogenic role of IFN-alpha in HIV infection, which may be a potential target for immunomodulating therapy in combination with HAART.

Raised serum levels of interleukin-18 is associated with disease progression and may contribute to virological treatment failure in HIV-1-infected patients.

To gain further insight into the possible role of interleukin (IL)-18 in HIV-1 infection we examined serum levels of IL-18 in various clinical and immunological stages of HIV-1 infection during cross-sectional (n = 41) and longitudinal testing (n = 20) and during HAART (n = 21, 24 months follow-up). Our main findings were that HIV-1-infected patients had significantly raised IL-18 levels comparing healthy controls, particularly in those with advanced disease, that while HAART induced a marked decline in IL-18, virological treatment failure was associated with persistently raised IL-18 levels during such therapy and that our in vitro experiments showed an IL-18-mediated up-regulation of the HIV-1 coreceptor CXCR4 and the pro-apoptotic mediator TRAIL in PBMC from HIV-1-infected patients receiving HAART. HIV-1 infection appears to be characterized by persistently raised IL-18 levels and during HAART, such a pattern was associated with virological treatment failure, possibly contributing to immunodeficiency and HIV-1 replication in these patients.

Source of peritoneal proteoglycans. Human peritoneal mesothelial cells synthesize and secrete mainly small dermatan sulfate proteoglycans.

This study describes experiments that compare the proteoglycans (PGs) extracted from the dialysate from patients receiving continuous peritoneal ambulatory dialysis (CAPD) with those secreted by metabolically labeled human peritoneal mesothelial cells in vitro. The PGs isolated from both sources were predominantly small chondroitin sulfate/dermatan sulfate PGs. Western blot of the core proteins obtained after chondroitin ABC lyase treatment with specific antibodies identified decorin and biglycan. With [35S]sulfate and [35S]methionine as labeling precursors it was shown that dermatan sulfate rather than chondroitin sulfate were the major glycosaminoglycan chains and that decorin was the predominant species. These data provide the first evidence that human peritoneal mesothelial cells may be the principal source of PGs in the peritoneum. Given the proposed functions of decorin and biglycan, the results suggest that these PGs may be involved in the control of transforming growth factor-beta activity and collagen fibril formation in the peritoneum.

Interleukin 1 induces NF-kappa B through its type I but not its type II receptor in lymphocytes.

It is not known whether one or both of the interleukin 1 (IL1) receptors mediates the induction of the DNA-binding protein NF-kappa B. Nuclear extracts of the murine lines EL4.NOB.1 and 70Z/3, which bear the type I (80 kDa) and type II (67 kDa) IL1 receptor, respectively, were analyzed by an electrophoretic mobility shift assay. A 265-base pair sequence of the human serum amyloid A gene or a synthetic oligonucleotide each containing the NF-kappa B site were used as the DNA probes. IL1 induction of NF-kappa B was rapid (optimal at 15-30 min) and transient in both cell types. The IL1 receptor antagonist (IL1ra), which binds strongly to the type I receptor, inhibited the NF-kappa B response in both cell lines. IL1ra did not bind to the type II receptor on 70Z/3 cells as judged by competition for binding with 125I-IL1 alpha. When 125I-IL1ra binding to 70Z/3 cells was measured, a small number (10/cell) of high affinity sites (Kd = 5 x 10(-12) M) were detected. These were likely to have been type I receptor because an antibody to this inhibited the NF-kappa B induction in 70Z/3 cells (as well as EL4). Potential signal transduction mechanisms involving protein kinase C or oxygen radicals were studied. Phorbol 12-myristate 13-acetate induced NF-kappa B with a similar time course to IL1 in 70Z/3 but only after 4 h in EL4.IL1 was unaffected by a protein kinase C inhibitor (staurosporine). H2O2 did not mimic IL1, and IL1 was not inhibited by an antioxidant. The type I receptor mediates the induction of NF-kappa B in response to IL1 via a signaling mechanism that still remains to be identified.

Human peritoneal mesothelial cell prostaglandin synthesis: induction of cyclooxygenase mRNA by peritoneal macrophage-derived cytokines.

Increasing evidence suggests that the mesothelial cell contributes to the control of inflammation in both the normal and inflamed peritoneal cavity. The present study examines the regulation of prostaglandin production by human peritoneal mesothelial cells (HPMC) following stimulation with peritoneal macrophage-conditioned medium and the cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha). IL-1 beta and TNF-alpha stimulated significant release of prostaglandin above background levels in a time and dose dependent manner. Stimulation of HPMC with IL-1 beta (500 pg/ml) or TNF-alpha (100 pg/ml) for 24 hours resulted in the release of 24.5 +/- 4.3 (N = 11) (z = 3.40, P < 0.001 vs. control) and 19.4 +/- 4.5 (N = 10; z = 3.29, P < 0.001 vs. control) pg 6-keto-PGF1 a/micrograms cellular protein, respectively. Pretreatment of HPMC with dexamethasone (10(-6) to 10(-9) M) inhibited both constitutive and cytokine stimulated prostaglandin synthesis in a dose dependent manner. Both PMø-CM and PMø-S.epiCM stimulated 6-keto-PGF1 alpha and PGE2 synthesis by HPMC in a time and dose dependent manner (PMø-S.epiCM >> PMø-CM). Co-incubation of HPMC with PMø-S.epiCM in the presence of anti-IL-1 beta and/or anti-TNF-alpha antibody, interleukin-1 receptor antagonist or soluble TNF receptor (TNF p75) significantly reduced the capacity of these supernatants to stimulate prostaglandin synthesis. Exposure of HPMC to cytokines or PMø-S.epiCM resulted in the time dependent increase in the levels of both Cox-1 and Cox-2 mRNA as assessed by RT/PCR analysis with the greatest increase being seen for Cox-2. These data demonstrate specific stimulation of eicosanoid metabolism in HPMC by peritoneal macrophage derived cytokines, indicating the possible importance of these mediators in the activation of intraperitoneal prostaglandin synthesis. HPMC prostaglandins might act as important pro/anti-inflammatory mediators contributing to a cytokine network in the peritoneal cavity during CAPD peritonitis.

Effects of interferon-alpha on gene expression of chemokines and members of the tumour necrosis factor superfamily in HIV-infected patients.

We examined the effect of interferon (IFN)-alpha on the expression of 375 genes relevant to inflammatory and immunological reactions in peripheral blood mononuclear cells (PBMC) from HIV-infected patients by cDNA expression array and real-time quantitative RT-PCR. Our main findings were: (i) IFN-alpha induced up-regulation of several genes in the tumour necrosis factor (TNF) superfamily including the ligands APRIL, FasL, TNF-alpha and TRAIL, with particularly enhancing effects on the latter in HIV-infected patients. (ii) While IFN-alpha markedly up-regulated the expression of anti-angionetic ELR- CXC-chemokines (e.g. MIG and IP-10), it suppressed the expression of angiogenic ELR+ CXC-chemokines (e.g. GRO-alpha, IL-8 and ENA-78), with similar patterns in both patients and controls. (iii) IFN-alpha induced a marked increase in gene expression of the HIV co-receptor CCR5 in both patients and controls. We suggest that these effects may contribute to both the therapeutic and toxic effects of IFN-alpha. Moreover, our findings underscore that the biological effects of IFN-alpha in HIV infection are complex and that the clinical net effects of IFN-alpha treatment may be difficult to predict. However, the potent enhancing effect of IFN-alpha on several pro-apoptotic genes in the TNF superfamily and the enhancing effect on CCR5 expression suggest a possible pathogenic role of IFN-alpha in the progression of HIV-related immunodeficiency and suggests caution in the therapeutic use of IFN-alpha in HIV-infected -individuals.