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The homing of lymphocytes to the mucosa is mainly controlled by α4β7 integrin, and it is amplified during gut chronic inflammation, as occurs with HIV and/or inflammatory bowel diseases. We designed and applied an improved immunization strategy based on an innovative selection process to isolate new α4β7 lymphocyte-specific monoclonal antibodies that are able to prevent their migration into inflamed gut tissues and/or to counteract HIV infection in vitro. First, 5 monoclonal antibodies (1 IgA, 1 IgM, and 4 IgGs) were selected based on their capacity to recognize α4 or β7 homodimers and α4β7 heterodimers in transfected human cells. Their ability to block gp120/α4β7 or MAdCAM-1/α4β7 interactions was then measured in vitro with human T and B lymphocytes. In vitro, the anti-α4β7 IgA isotype was found to have the highest affinity for the α4β7 heterodimer, and it significantly reduced HIV replication in retinoic acid-treated α4β7 CD4 human T cells. This α4β7-specific IgA also displayed a high avidity for human and mouse α4β7 lymphocytes in both mouse and human inflammatory colitis tissues. These new antibodies, and in particular those with mucosa-targeting isotypes such as IgA, could therefore be potential novel therapeutic tools for treating HIV and inflammatory bowel disease.

International audience; BACKGROUND:Early steps of HIV infection are mediated by the binding of the envelope to mucosal receptors as α4β7 and the C-type lectins DC-SIGN and langerin. Previously Env-specific B-cell responses have been reported in highly exposed seronegative individuals (HESN).METHOD:Here, we studied gp120-specific antibodies ability to block HIV interaction with α4β7, DC-SIGN and/or langerinin HESN. New cell-based assays were developed to analyze whether antibodies that can alter gp120 binding to α4β7, DC-SIGN and/or langerin are induced in HESN. A mucosal blocking score (MBS) was defined based on the ability of antibodies to interfere with gp120/α4β7, gp120/DC-SIGN, and gp120/langerin binding. A new MBS was evaluated in a cohort of 86 HESN individuals and compared with HIV+ patients or HIV- unexposed healthy individuals.RESULTS:Antibodies reducing gp120 binding to both α4β7 and DC-SIGN were present in HESN serum but also in mucosal secretions, whereas antibodies from HIV+ patients facilitated gp120 binding to DC-SIGN. Any correlation was observed between MBS and the capacity of antibodies to neutralize infection of α4β7 CD4+ T cells with primary isolates.CONCLUSIONS:MBS is significantly associated with protection in HESN and might reflect altered HIV spreading to mucosal-associated lymphoid tissues.

Resume Les maladies chroniques inflammatoires intestinales sont des pathologies auto-immunes relativement frequentes dans la population. La clinique, la radiologie, l’endoscopie et la biologie doivent etre associees afin de poser le diagnostic positif de ces pathologies, et de permettre leurs suivis. Ainsi la biologie est a la fois une aide au diagnostic de maladies chroniques inflammatoires intestinales, mais aussi une aide pour la distinction entre maladie de Crohn et rectocolite hemorragique. Ces marqueurs trouvent aussi leurs interets dans la prevention des rechutes, des complications, le suivi de la remission et rentrent dans des algorithmes decisionnels de prise en charge therapeutique. Differents types de marqueurs seront decrits et developpes dans cette revue tels que les anticorps seriques, les proteines fecales ainsi que le suivi pharmacologique des biotherapies. L’adaptation therapeutique (optimisation des doses, suspension de traitements) et l’espacement dans le temps de gestes invasifs telle l’endoscopie correspondent a des avancees majeures permises notamment grace a ces marqueurs biologiques.

Background Indirect immunofluorescence plays a major role in the detection of antinuclear antibodies (ANAs) and follow-up of their titers in the context of connective tissue diseases. Given the numerous unfavorable features of the conventional manual reading of HEP2 slides (need of time and expert morphologists for the reading, lack of standardization, subjectivity of the interpretation), the biomedical industry has developed automated techniques of slide preparation and microscope reading. Methods We collected 49 sera beforehand analyzed by the conventional reading of slides. They were prepared again by QUANTA-Lyser® and reanalyzed in four different conditions: two dilutions of screening (1/40 and 1/80), two different systems of analysis, NOVA View® automated reading (INOVA Diagnostics), then confirmation by the operator, and conventional manual reading by two different qualified operators. The analysis was realized in blind of the first interpretation and clinical diagnosis. The sera were classified in four groups, on the basis of the results of the first analysis: negative sera (titer < 1/160; 11 patients), low positives (titer at 1/160; 18 patients), moderated positives (titers between 1/320 and 1/640; 10 patients), and strong positives (titers between 1/1,280 and 1/2,560; 10 patients). Results Among the 49 patients, 13 presented a connective tissue disease including 4 systemic scleroderma (SS), 3 rheumatoid arthritis (RA), 2 Goujerot-Sjogren (GS), 2 systemic lupus erythematosus (SLE), 1 polymyositis (PM), 1 Raynaud's syndrome (RS), and 1 CREST syndrome. One patient presented both an SLE and an SS. Regarding the screening dilution, the 1/40 dilution is less specific than the 1/80 dilution for both the systems of analysis (5.6% vs. 16.7% for the manual reading, and 27.8% vs. 50% for the automated reading). It also generates statistically more false positives (P = 0.037 for the conventional analysis and P = 0.003 for the automated system). The automated NOVA View® reading of slides allows a gain in specificity for both dilutions, and also statistically less false positives (P = 0.002 at the 1/40 and P = 0.0006 at the 1/80), and detriment of the sensitivity at the highest dilution (84.6% vs. 92.3% with manual reading). Thus, according to our analysis of 49 sera, the automated NOVA View® system of reading of slides at the dilution 1/80 seems to be a successful condition for the detection of ANAs on HEP2 cells, close to the significance (P = 0.067). Conclusion The automated NOVA View® reading of slides allows saving time, and an improvement in the standardization. Nevertheless, it requires a confirmation by a qualified operator, to interpret mixed patterns in particular.

Patients with Down syndrome are more susceptible to autoimmune pathologies, in particular endocrine or digestive diseases such as celiac disease. Autoimmune enteropathy is another form of digestive autoimmune disease, non-gluten-dependant, more often diagnosed in male neonates with immunodysregulation and polyendocrinopathy such as the Immunodysregulation, Polyendocrinopathy, Enteropathy, X-linked syndrome. It also exists in the adult, but this pathology is less known and therefore frequently under-diagnosed. Clinical manifestations are similar to celiac disease, but not improved after a gluten-free diet. Autoimmune enteropathy is frequently associated with other autoimmune diseases, such as thyroiditis, myasthenia gravis, lupus or immune deficiencies, as Common Variable Immunodeficiency. Pathological analysis of intestinal biopsies can frequently distinguish autoimmune enteropathy and celiac disease. Autoimmune enteropathy usually has an important lymphoplasmacytic infiltration of the mucosa and a lack of intraepithelial lymphocytes in the gastrointestinal mucosal surface, while celiac disease usually has a polymorph infiltration of the mucosa and an important intraepithelial lymphocytes infiltration. Nevertheless, the two pathological patterns may overlap. Here we report the first case of a patient with Down syndrome associated to autoimmune enteropathy (initially diagnosed as celiac disease), chronic pancreatitis and cutaneous lupus erythematosus. Even if autoimmune pathologies are much more common in patients with Down syndrome, we would like to report on this rare and original association found in our patient.

Background Nearly 40% of patients who failed on ADA have therapeutic residual trough levels (pharmacodynamic failure). It is then recommended to change the therapeutic class to a non-anti-TNF biological. In this situation, however, a quarter of patients respond to an ADA dose optimisation. The purpose of this work was to compare these two strategies and identify predictors of response for each strategy. Methods This is a bi-centre, retrospective study based on a clinical and biological database that included patients followed from 2015 to 2016 with an IBD in maintenance treatment with ADA as monotherapy (40 mg/sc/14 days) and in loss of response (CDAI > 220 with faecal calprotectin > 250 µg/g stool for CD and Mayo score > 6 with endoscopic sub-score > 1 for UC). All patients included had residual trough ADA levels > 5 µg/ml. Relapsed patients were treated either with dose optimisation (ADA: 40 mg/sc/7 days) or other biotherapy (ustekinumab or vedolizumab, according to classic regimens and dosages). The primary endpoint was the relapse-free survival rate (defined as clinical and biological or endoscopic activity requiring therapeutic change). The survival curve analysis was performed using the Kaplan–Meier method and the log-rank test. Results In total, 125 patients (mean age: 38 years, 75% CD, sex ratio M/F = 57%, previous infliximab = 42%) were included and 50 were treated with another biological (Vedolizumab: 30 patients, Ustekinumab: 20 patients). The characteristics of the two groups were comparable except for significantly higher levels of CRP and faecal calprotectin at inclusion in the biological change arm. The duration without relapse was significantly longer after the class change (p = 0.0005; HR = 0.39[0.23–0.68]). The relapse-free survival rates at 54 weeks were 35% for the optimised arm and 66% for a class change (p = 0.04). In multivariate analysis, the only long-term response predictor was biotherapy class change vs. ADA dose optimisation (p = 0.042; HR = 0.3[0.02–0.95]). Previous use of IFX or type of IBD were not associated with relapse. For the dose optimisation strategy, in multivariate analysis, only age < 40 years was associated with a progression without relapse (p = 0.007; HR = 0.09[0.01–0.52]). Residual ADA levels were not predictive of response to optimisation (p = 0.73) and no ADA quartile was significantly associated with a more favourable clinical response. For the class change strategy, no relapse predictors were isolated in univariate analysis. Conclusion In patients on ADA who are losing response despite therapeutic rates, the change of class to non-anti-TNF biotherapy allows a significantly greater lasting response than a dose optimisation strategy for ADA.

Integrins are a large family of heterodimeric cell adhesion molecules that are key regulators in multiple biological functions. They orchestrate cell-cell and cell-extracellular matrix (ECM) adhesive interactions from embryonic development to mature tissue function, and are thus involved in cell migration, proliferation, differentiation, and survival. As such, they are also involved in human diseases, such as thrombotic diseases, inflammation, cancer, fibrosis and infectious diseases. Integrins are exciting pharmacological targets because they are exposed on the cell surface. Indeed, several compounds have been developed that block integrins function, and five have been approved as therapeutic drugs for use in clinic. This review will detail the role of α4β7, an integrin of particular relevance for mucosal diseases such as IBD (inflammatory bowel disease) and also, as reported more recently, HIV infection.

International audience; Traditionally, IBD diagnosis is based on clinical, radiological, endoscopic, and histological criteria. Biomarkers are needed in cases of uncertain diagnosis, or to predict disease course and therapeutic response. No guideline recommends the detection of antibodies (including ASCA and ANCA) for diagnosis or prognosis of IBD to date. However, many recent data suggest the potential role of new serological markers (anti-glycan (ACCA, ALCA, AMCA, anti-L and anti-C), anti-GP2 and anti-GM-CSF Ab). This review focuses on clinical utility of these new serological markers in diagnosis, prognosis and therapeutic monitoring of IBD. Literature review of anti-glycan, anti-GP2 and anti-GM-CSF Ab and their impact on diagnosis, prognosis and prediction of therapeutic response was performed in PubMed/MEDLINE up to June 2014. Anti-glycan, anti-GP2 and anti-GM-CSF Ab are especially associated with CD and seem to be correlated with complicated disease phenotypes even if results differ between studies. Although anti-glycan Ab and anti-GP2 Ab have low sensitivity in diagnosis of IBD, they could identify a small number of CD patients not detected by other tests such as ASCA. Anti-glycan Abs are associated with a progression to a more severe disease course and a higher risk for IBD-related surgery. Anti-GP2 Ab could particularly contribute to better stratify cases of pouchitis. Anti-GM-CSF Ab seems to be correlated with disease activity and could help predict relapses. These new promising biomarkers could particularly be useful in stratification of patients according to disease phenotype and risk of complications. They could be a valuable aid in prediction of disease course and therapeutic response but more prospective studies are needed.

HIV transmission and spread in the host are based on the survival of the virus or infected cells present in mucosal secretions, and the virus' ability to cross the epithelial barrier and access immune target cells, which leads to systemic infection. Therefore, HIV-specific immunity at mucosal sites is critical for control of infection. Although mucosal delivery would ensure the best onset of protective immunity, most candidate vaccines are administered through the parenteral route. Remarkably, secretory IgA (SIgA) interacts specifically with mucosal microfold (M) cells present in gut-associated lymphoid tissues. Here we evaluate the feasibility of delivering chemically bound p24HIV antigen via SIgA into the intestinal mucosae in mice. After oral administration, p24-SIgA complexes are quickly delivered into the tissue and selectively captured by CX3CR1(+) dendritic cells. Oral immunization with p24gag linked to SIgA (p24-SIgA) adjuvanted with E. coli heat labile enterotoxin (HLT) elicits both humoral and cellular immune responses against p24 at the systemic and mucosal levels and induces efficient protection against rectal challenge with a recombinant vaccinia virus encoding gag. This is the first study which underscores the remarkable potential of SIgA to serve as a vaccine carrier for an HIV antigen in mucosal administration targeting the gastrointestinal environment.

TLR ligands are critical activators of innate immunity and are being developed as vaccine adjuvants. However, their usefulness in conjunction with NOD-like receptor agonists remains poorly studied. In this study, we evaluated a new ligand that targets both TLR2 and NOD2 receptors. We assessed its ability to enhance dendritic cell maturation in vitro in addition to improving systemic and mucosal immune responses in mice. The chimeric NOD2/TLR2 ligand induced synergistic upregulation of dendritic cell maturation markers, costimulatory molecules, and secretion of proinflammatory cytokines compared with combinations of separate ligands. Furthermore, when coadministered with biodegradable nanoparticles carrying a model Ag, the ligand was able to induce high Ag-specific IgA and IgG titers at both systemic and mucosal sites after parenteral immunizations. These findings point out the potential utility of chimeric molecules TLR/NOD as adjuvants for vaccines to induce systemic and mucosal immune responses.

There is limited research on cell-free RNA (cf-RNA) in the urine of cancer patients. The present study was performed to detect the cf-RNA in the urine of patients with clear cell renal cell carcinoma (ccRCC). Ninety-five urine samples from ccRCC patients and 50 urine samples from control subjects were analyzed. The cf-RNA integrity index was calculated by using quantitative real-time RT-PCR assays of the small-sized fragment (106 bp) and the big-sized fragment (416 bp) in GAPDH mRNA. The initial analysis showed that cf-RNA was stable and detectable in the urine. The mean cf-RNA integrity index was significantly lower in the urine of ccRCC patients (mean: 0.07, 95%CI: 0.05-0.10) when compared with the urine from control subjects (mean: 0.25, 95%CI: 0.16-0.33) (p < 0.001). The value of the area under the receiver-operating characteristic curve by using the cf-RNA integrity index for the diagnosis of ccRCC was 0.858 with a sensitivity of 68.0% and a specificity of 92.6%. Moreover, the small-sized VEGF mRNA fragment (98 bp) was detected in 31 of 50 urine samples of patients with ccRCC and in only 2 of 50 urine samples of control subjects (p < 0.001) while the detection of the big-sized (420 bp) VEGF mRNA fragment was an infrequent event. Our findings suggest that the small-sized cf-RNA in urine was more abundant in cancer patients. The tumor-related gene VEGF mRNA fragment was detectable in the urine of cancer patients. Our finding may provide a new molecular assay for the diagnosis of renal cell carcinoma.

International audience; OBJECTIVES:Several decision algorithms based on the measurement of infliximab (IFX) trough levels and antibodies to IFX have been proposed. Whether such algorithms can be extrapolated to the pharmacokinetics of adalimumab (ADA) has yet to be determined.METHODS:A prospective study included all consecutive patients with inflammatory bowel disease (IBD) having a disease flare while being on ADA 40 mg every 2 weeks were included. All patients were primary responders to ADA therapy and were anti-tumor necrosis factor (TNF) naive. ADA trough levels and antibodies against ADA (AAA) were measured blinded to clinical data (Elisa LISA-Tracker, Theradiag). All patients were optimized with ADA 40 mg weekly. Four months later, in the absence of clinical remission (CR; Crohn's disease activity index 4.9 μg/ml; group B, ADA10 ng/ml.RESULTS:A total of 82 patients were included (55% CD; mean age=43 years, disease duration=7.4 years, duration of ADA therapy=17 months). After optimization of ADA treatment, 29.2% of patients achieved CR in group A (N=41), 67% in group B (N=24), and 12% in group C (N=17; P4.9 μg/ml are associated with failure of two anti-TNF agents (ADA and IFX) in 90% of cases, and switching to another drug class should be considered.

BACKGROUND: A previous meta-analysis suggested that 6-thioguanine nucleotides levels are associated with clinical remission in inflammatory bowel disease. It was criticized because of the relatively small number of patients included in this meta-analysis and heterogeneity between studies. Recent studies provided conflicting results, and the source of those discrepancies has yet to be explored. METHODS: A comprehensive, computerized literature search was conducted in Medline, ISI Web of Science, and EMBASE until December 31, 2012. A combined odd ratio with its 95% confidence interval was calculated using a fixed effects model based on the Mantel-Haenszel method. Between-study heterogeneity was assessed using Cochran's Q statistic. RESULTS: Seventeen studies enrolling 2049 patients with inflammatory bowel disease were analyzed. A significant heterogeneity was found in the overall analysis (P = 0.005). As heterogeneity among studies could be explained by differences in metabolite assay methods, an analysis including only studies using the reference method by Lennard et al (N = 10) was performed, and the pooled odds ratio for clinical remission among patients with 6-thioguanine nucleotides levels over a cut-off value between 230 and 260 pmol/8.10^8 RBC was 3.15 (95% confidence interval, 2.41-4.11). CONCLUSIONS: This meta-analysis clearly establishes an association between 6-thioguanine nucleotides levels and clinical remission rates in patients with inflammatory bowel disease and explains the heterogeneity of results among selected studies. The lack of standardization in 6-thioguanine nucleotides assays is responsible for recent contradictory results. Whether therapeutic drug monitoring of thiopurines should be systematically used in clinical practice in inflammatory bowel disease to improve disease outcomes will require further investigation.

Background Data on the value of therapeutic drug monitoring of infliximab (IFX) to predict mucosal healing (MH) in inflammatory bowel diseases (IBD) are scarce. Methods All consecutive patients with IBD receiving ongoing IFX (5 mg/kg) treatment and developing secondary failure to IFX were enrolled in a prospective study between June 2010 and May 2011. IFX trough levels, antibodies to IFX concentrations, C-reactive protein levels, and fecal calprotectin were measured before IFX optimization and at week 8. A proctosigmoidoscopy was performed on the day of first IFX optimization and at week 8 in all patients with ulcerative colitis (UC). MH was defined by fecal calprotectin 0.5 μg/mL was associated with MH (sensitivity [se], 0.88; specificity [sp], 0.77; P = 0.0001, area under the receiver operating characteristic curve, 0.89). On multivariate analysis, the only factor associated with MH after IFX optimization was a delta IFX >0.5 µg/mL (likelihood ratio = 2.02; 95% confidence interval, 1.01–4.08; P = 0.048) in patients with IBD. Conclusions Therapeutic drug monitoring of IFX strongly predicts the likelihood of achieving MH following IFX dose intensification in both CD and UC.

Our objective was to evaluate the levels of miR-210 in tumor and serum samples of conventional renal cell cancer (cRCC) patients to explore whether circulating miR-210 in serum can be used as a biomarker for the detection of cRCC.The paired samples from primary cRCC tumors and adjacent non-tumoral renal parenchyma were collected from 32 patients with cRCC. Serum samples were obtained from 68 patients with a cRCC before surgery, 10 samples after one week of surgery, and 42 healthy individuals were included in this study. Real-time PCR was used to measure the microRNA level. The expression of miRNAs was normalized using the dCT method. Expression levels of miR-210 were compared using the Mann-Whitney U test or Wilcoxon test. Diagnostic performance of serum miR-210 level was calculated by using the receiver operating characteristic (ROC) curve.The average miR-210 level was higher in primary cRCC tissues than in normal tissue (p=0.004). For serum samples, the average level of miR-210 was significantly higher in cRCC patients than in controls (p0.001). The serum miR-210 level yielded an AUC (the areas under the ROC curve) of 0.874 with a sensitivity of 81.0% and a specificity of 79.4%. Furthermore, the average serum level of miR-210 was significantly decreased in the patients one week after the operation (p=0.001).Serum mi-210 may have a potential as a novel noninvasive biomarker for the detection of cRCC.

Mucosal HIV-1–specific IgA have been described as being able to neutralize HIV-1 and to block viral transcytosis. In serum and saliva, the anti-HIV IgA response is predominantly raised against the envelope of HIV-1. In this work, we describe the in vivo generation of gp41-specific IgA1 in humanized α1KI mice to produce chimeric IgA1. Mice were immunized with a conformational immunogenic gp41-transfected cell line. Among 2300 clones screened by immunofluorescence microscopy, six different gp41-specific IgA with strong recognition of gp41 were identified. Two of them have strong neutralizing activity against primary HIV-1 tier 1, 2, and 3 strains and present a low rate of somatic mutations and autoreactivity, unlike what was described for classical gp41-specific IgG. Epitopes were identified and located in the hepted repeat 2/membrane proximal external region. These Abs could be of interest in prophylactic treatment to block HIV-1 penetration in mucosa or in chronically infected patients in combination with antiretroviral therapy to reduce viral load and reservoir.

Supplemental Digital Content is Available in the Text., Objective: Correlation between GALT homing markers on lymphocytes and the low blood CD4 T-cell reconstitution in immunological nonresponders (INRs) has been studied. Design: Thirty-one INRs, 19 immunological responders (IRs), and 12 noninfected controls were enrolled in this study. INRs were defined by an undetectable plasma viral load RNA less than 40 copies per milliliter and CD4+ T-cell count

CA9 is proven to be a powerful marker for clear cell renal cell carcinoma. The studies on CA9 have been limited to solid renal cell carcinomas (RCC). We have conducted a study of CA9 expression in renal cystic tumors. The purpose of the present study was to extend the utility of CA9 for cystic renal tumors.Immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) were used to detect CA9 expression in cystic renal tumors. Forty-three cystic renal tumors (22 benign and 21 malignant) were included for the immunohistochemical staining. Thirty-six patients with a cystic renal mass (20 malignant and 16 benign cystic tumors) were studied to measure CA9 level in the fluid by ELISA. Sixteen cysts (9 malignant and 7 benign cysts) were subjected both to immunohistochemistry and CA9 measurement in the fluid.Using immunohistochemical staining, all the benign cystic renal tumors including the 18 simple cyst and 4 benign multilocular cystic nephromas did not express CA9. All 13 cystic clear cell RCC were scored as strong staining for CA9. For 8 multilocular clear cell RCC, 7 were scored as strong staining for CA9 and the other one was negative. There was a significant difference in positive percentage (P0.001) between the 2 groups of malignant and benign cysts. For the 16 benign cysts, the mean concentration of CA9 in the fluid of cyst was 162 ± 133 pg/ml (median: 0 pg/ml; range: 0-2140 pg/ml). For the 20 malignant renal cystic tumors, the mean concentration of CA9 in the fluid of cyst was 2043 ± 62 pg/ml (median: 2,140 pg/ml; range: 1,112-2,140 pg/ml). There was a significant difference in mean concentration of CA9 between the two groups of malignant and benign cysts (P0.001). The presence or absence of CA9 expression measured by immunohistochemistry and ELISA test was concordant in 14 out of 16 cases (88%).Malignant cystic renal tumors expressed strongly CA9 while the benign renal cysts did not express CA9. CA9 can be detected in the fluid of malignant cystic renal tumors. CA9 is a promising molecular marker to differentiate the malignant cystic renal tumors from the benign cysts.

Claudin gene expression is frequently altered in human cancers. Our aim was to improve the cytology diagnosis of malignancy in serous fluids with the quantification of claudins compared with various classic molecular markers using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method. Peritoneal or pleural effusions of 56 patients were assessed as malignant from histological analysis and 19 as benign. Claudin 4 was the most significantly upregulated (68%) marker in patients with malignant effusions. In cytologically negative malignant effusions, claudin 4 was found increased in 8/18 fluids. Quantitative RT-PCR is a sensitive method for the detection of free cancer cells in serous effusions.

The development of a preventive vaccine against human immunodeficiency virus type-1 (HIV-1) provides hope for control of the pandemic over the coming years. Nevertheless, it is clear that one of the greatest difficulties in achieving this vaccine is the high mutation rate of the virus, which enables it to evade the host's immune response. The production of neutralizing antibodies (NAb) against the HIV-1 envelope proteins is believed to play an important role in controlling the infection and in providing effective protection following vaccination. Several studies have shown that the V1/V2 domain of the HIV-1 gp120 envelope protein is involved in viral tropism during infection, in masking conserved neutralizing epitopes, in the conformational changes occurring after coreceptor binding, and in NAb induction. Nonetheless, this domain has been poorly investigated. However, because the V1/V2 domain is highly glycosylated, numerous studies have determined the influence of carbohydrates on NAb production. The present review focuses on the importance of NAb directed against epitopes of the variable regions, mainly V1/V2, their importance in protecting against HIV-1 infection, and the role these regions play in evading the immune response. Lastly, we will discuss the importance of NAb in the search for an effective vaccine against HIV-1.

Objective: The objective of this paper was to evaluate the presence and the neutralizing activity of antibodies directed against the complex formed between the two heptad repeat regions (HR1 and HR2) of HIV-1 gp41 in sera of HIV-1-infected patients. Research designs and methods: The HR1 region was represented by the peptide N36 and the maltose-binding protein (MBP)-HR1, the HR2 region by the peptide C34 and MBP44. Antibodies directed to the HR1/HR2 complex were purified from sera by affinity chromatography using MBP-HR1/C34 adsorbed onto a resin. Results: First, we demonstrated that human monoclonal antibodies, which are directed specifically to the HR1/HR2 complex recognized in enzyme-linked immunosorbent assay the MBP-HR1/C34 and MBP44/N36 mixtures but not the proteins or the peptides individually. We investigated the ability of 50 sera of HIV-1-infected patients to react with the MBP-HR1/C34 and MBP44/N36 complexes. We found that the majority of sera of HIV-1-infected patients recognized the HR1/HR2 complexes but not or to a lower extent the proteins or the peptides individually. Antibodies purified from sera by affinity chromatography using MBP-HR1/C34 adsorbed to a resin neutralized different primary HIV-1 isolates. Conclusion: The presence of antibodies directed to the HR1/HR2 complex in sera of HIV-infected patients highlights the immunogenic character of the complex, whereas the neutralizing activity of these antibodies suggests that immunogens representing HIV-1 HR1/HR2 complexes might be used in anti-HIV vaccine.

Purpose: We explored the clinical usefulness of serum carbonic anhydrase 9 as a potential biomarker for conventional renal cell cancer.Materials and Methods: This study included 91 patients with conventional renal cell cancer and 32 healthy individuals. Enzyme linked immunosorbent assay was used to measure the carbonic anhydrase 9 level. A followup (median 38 months) was performed to track early recurrence after surgery for patients with localized disease. Recurrence-free survival curves were calculated by the Kaplan-Meier method and compared using the log rank test.Results: The mean serum carbonic anhydrase 9 level in patients with metastatic conventional renal cell cancer (216.68 ± 67.02 pg/ml) or localized conventional renal cell cancer (91.65 ± 13.29 pg/ml) was significantly higher than in healthy individuals (14.59 ± 6.22 pg/ml, p

Many cancers cause malignant effusions. The presence of malignant cells in effusions has implications in diagnosis, tumour staging and prognosis. The detection of malignant cells currently presents a challenge for cytopathologists. New adjunctive methods are needed. Although the effusions provide excellent materials for molecular assay, the available molecular markers are extremely limited, which hinders its clinical application. MN/CA9 has proved to be a valuable marker in many cancers such as lung, breast, colon, kidney, etc. The present study was to evaluate MN/CA9 as a new molecular marker for the detection of cancer cells in pleural effusions. Seventy-one pleural effusions including 59 malignant effusions from patients with cancer, and 12 patients with benign diseases as a control, were subjected to RT-PCR for detection of MN/CA9 gene expression. MN/CA9 gene expression was detected in 53/59 (89.8%) pleural effusions from cancer patients (15/16 for breast cancers, 10/11 for lung cancers, 4/4 for ovary cancers, 2/3 for colon-rectal cancers, 5/6 for cancers of unknown site, 7/8 for mesothelioma and 10/11 for other cancers). Furthermore, MN/CA9 was positive in 13/18 (72.2%) of cytologically negative effusions of cancer patients. MN/CA9 was detected in only 1/12 (8.3%) effusions from the control patients (p < 0.01). The sensitivity and specificity of MN/CA9 gene expression were, respectively, 89.8% and 91.7%. Our preliminary results suggest that MN/CA9 could be a potential marker for the detection of malignant cells in effusions. A large-scale study is needed to confirm these results.

Integrins are a large family of heterodimeric cell adhesion molecules that are key regulators in multiple biological functions. They orchestrate cell-cell and cell-extracellular matrix (ECM) adhesive interactions from embryonic development to mature tissue function, and are thus involved in cell migration, proliferation, differentiation, and survival. As such, they are also involved in human diseases, such as thrombotic diseases, inflammation, cancer, fibrosis and infectious diseases. Integrins are exciting pharmacological targets because they are exposed on the cell surface. Indeed, several compounds have been developed that block integrins function, and five have been approved as therapeutic drugs for use in clinic. This review will detail the role of α4β7, an integrin of particular relevance for mucosal diseases such as IBD (inflammatory bowel disease) and also, as reported more recently, HIV infection.

Mucosal surfaces are a major portal of entry for many pathogens that are the cause of infectious diseases. Therefore, effective vaccines that induce a protective immune response at these sites are much needed. However, despite early success with the live attenuated oral polio vaccine over 50 years ago, only a few new mucosal vaccines have been subsequently licensed. Development of new adjuvants, comprising antigen delivery platforms and immunostimulatory molecules, are critical for the successful development of new mucosal vaccines. Among them, biodegradable nanoparticle delivery systems are promising and NOD-like receptors are considered as potential new targets for immunostimulatory molecules. In this work, different NOD1 and NOD2 ligands were encapsulated in polylactic acid (PLA) nanoparticles, coated with HIV-1 gag p24 antigen. We showed that these new formulations are able to induce proliferation of HIV-specific T cells from HIV(+) individuals as well as autophagy. In vivo, these formulations highly enhanced p24-specific systemic and mucosal immune responses in mice not only after mucosal administration but also after immunization via the parenteral route. Our results provide a rational approach for combining nanosized particulate carriers and encapsulated NOD receptor ligands as potent synergistic tools for induction of specific mucosal immunity.

International audience; BACKGROUND AND AIMS: The usefulness of anti-glycan antibodies alone or combined with anti-Saccharomyces cerevisiae [ASCA] or perinuclear antineutrophil cytoplasmic [pANCA] antibodies for diagnosis of inflammatory bowel disease [IBD], differentiation between Crohn's disease [CD] and ulcerative colitis [UC], disease stratification including IBD phenotype, and also for determination of the course of the disease, remain unclear. METHODS: A large panel of serological anti-glycan carbohydrate antibodies, including anti-mannobioside IgG antibodies [AMCA], anti-chitobioside IgA [ACCA], anti-laminaribioside IgG antibodies [ALCA], anti-laminarin [anti-L] and anti-chitine [anti-C] were measured in the serum from a cohort of 195 patients with IBD] [107 CD and 88 UC]. The respective accuracy of isolated or combined markers for diagnosis, disease differentiation, stratification disease phenotype, and severity of the disease course, defined by a wide panel of criteria obtained from the past medical history, was assessed. RESULTS: The positivity of at least one anti-glycan antibody was detected in a significant higher proportion of CD and UC compared with healthy controls [p \textless 0.0001 and p \textless 0.0007, respectively]. Whereas ASCA and ANCA antibody status had the highest efficacy to be associated with CD in comparison with UC (area under receiver operating characteristic curve [AUROC] = 0.70 for each], the adjunction of anti-laminarin antibody substantially improved the differentiation between CD and UC [AUROC = 0.77]. Titres of ACCA [\textgreater 51U/ml] and anti-laminarin [\textgreater 31U/ml] were significantly linked with a higher association with steroid dependency (odds ratio [OR] =2.0 [1.0-4.0], p = 0.03 and OR = 2.4 [1.1-5.2], p = 0.02, respectively]. We further defined the respective performance of anti-glycan antibodies to discriminate between patients with severe or not severe CD and UC course and determined the associated optimal cut-off values: severe CD course was significantly more likely in case of AMCA \textgreater 77U/ml [OR = 4.3; p = 0.002], ASCA \textgreater 63U/ml [OR = 3.5; p \textless 0.009] and at a lesser degree ACCA \textgreater 50U/ml [OR = 2.8; p \textless 0.02] and severe UC course was significantly associated with AMCA \textgreater 52U/ml [OR = 3.4; p = 0.04] and ACCA \textgreater 25U/ml [OR = 3.0; p \textless 0.04]. CONCLUSIONS: Anti-glycan antibodies are valuable serological markers, especially AMCA antibodies that may help clinicians to promptly classify patients into high risk for severe disease.

International audience; BACKGROUND:The magnitude of the efficacy of anti-tumour necrosis factor (TNF) therapy in preventing and treating postoperative Crohn's disease recurrence has yet to be determined.METHODS:We searched MEDLINE, the Cochrane Library, and EMBASE. The primary endpoints, and clinical and endoscopic recurrence, were analysed using the Mantel-Haenszel and DerSimonian and Laird methods.RESULTS:Nine controlled trials (n=362) that evaluated the efficacy of anti-TNF therapy in preventing (n=7) or treating (n=2) postoperative recurrence were included. Anti-TNF therapy was more effective at preventing (n=6) endoscopic recurrence than the control arms (odds ratio 0.05; 95% confidence interval 0.02-0.13, P

BACKGROUND: Indirect immunofluorescence plays a major role in the detection of antinuclear antibodies (ANAs) and follow‐up of their titers in the context of connective tissue diseases. Given the numerous unfavorable features of the conventional manual reading of HEP2 slides (need of time and expert morphologists for the reading, lack of standardization, subjectivity of the interpretation), the biomedical industry has developed automated techniques of slide preparation and microscope reading. METHODS: We collected 49 sera beforehand analyzed by the conventional reading of slides. They were prepared again by QUANTA‐Lyser(®) and reanalyzed in four different conditions: two dilutions of screening (1/40 and 1/80), two different systems of analysis, NOVA View(®) automated reading (INOVA Diagnostics), then confirmation by the operator, and conventional manual reading by two different qualified operators. The analysis was realized in blind of the first interpretation and clinical diagnosis. The sera were classified in four groups, on the basis of the results of the first analysis: negative sera (titer < 1/160; 11 patients), low positives (titer at 1/160; 18 patients), moderated positives (titers between 1/320 and 1/640; 10 patients), and strong positives (titers between 1/1,280 and 1/2,560; 10 patients). RESULTS: Among the 49 patients, 13 presented a connective tissue disease including 4 systemic scleroderma (SS), 3 rheumatoid arthritis (RA), 2 Goujerot‐Sjogren (GS), 2 systemic lupus erythematosus (SLE), 1 polymyositis (PM), 1 Raynaud's syndrome (RS), and 1 CREST syndrome. One patient presented both an SLE and an SS. Regarding the screening dilution, the 1/40 dilution is less specific than the 1/80 dilution for both the systems of analysis (5.6% vs. 16.7% for the manual reading, and 27.8% vs. 50% for the automated reading). It also generates statistically more false positives (P = 0.037 for the conventional analysis and P = 0.003 for the automated system). The automated NOVA View(®) reading of slides allows a gain in specificity for both dilutions, and also statistically less false positives (P = 0.002 at the 1/40 and P = 0.0006 at the 1/80), and detriment of the sensitivity at the highest dilution (84.6% vs. 92.3% with manual reading). Thus, according to our analysis of 49 sera, the automated NOVA View(®) system of reading of slides at the dilution 1/80 seems to be a successful condition for the detection of ANAs on HEP2 cells, close to the significance (P = 0.067). CONCLUSION: The automated NOVA View(®) reading of slides allows saving time, and an improvement in the standardization. Nevertheless, it requires a confirmation by a qualified operator, to interpret mixed patterns in particular.

The female genital tract is an important site for numerous pathogens entry. Local immunization, generating specific mucosal IgA and systemic IgG, represents an interesting alternative immunization pathway. However, such a vaccine strategy needs mucosal adjuvants to obtain the best immune response. Considering that the immunization process is mainly dependent on the capture and on the transport of the antigen by Langerhans cells, we evaluated potential adjuvant molecules by analysing their effects on the CCL20 secretion by endocervical and exocervical/vaginal epithelial cells as well as on dendritic cell and Langerhans cell maturation. We demonstrated that DC-Chol and Zymosan are the most efficient mucosal candidate immunoadjuvants that generate a strong increase of CCL20 secretion by the two epithelial cell lines and the maturation of dendritic and Langerhans cells, respectively.

Objective: Small solid renal tumors are increasingly encountered. It is important to determine the malignancy of solid renal tumors for the choice of treatment. MN/CA9 is expressed in malignant renal cells but absent in normal cells. MN/CA9 is one of the most powerful gene markers available for RCC. The objective of this pilot study is to utilize MN/CA9 gene expression in FNA biopsy to determine the malignancy of imaging-indeterminate solid renal tumors. Methods: A total of 35 patients with an imaging-indeterminate solid renal mass entered into this study. The molecular protocol consisted of a rapid column extraction of RNA and one-step RT-PCR for the detection of MN/CA9 gene expression. The preoperative molecular diagnosis was compared with postoperative pathology. Results: There were 28 RCCs (19 clear cell carcinomas, 7 papillary carcinomas and 2 chromophobe carcinomas) and 7 benign tumors proved by postoperative pathology. The overall sensitivity and specificity for MN/ CA9 were respectively 68% and 100%. MN/CA9 was positive in 16/19 (84%) FNA biopsies of clear cell RCCs. No false positive appeared for MN/CA9 gene expression. Moreover, MN/CA9 gene expression was positive in 8/13 (62%) of false negative or suspect cytology. Conclusion: Detection of MN/CA9 gene expression in FNA biopsy is possible. Its detection can be helpful in identifying the malignancy among renal tumors.

Purpose: Our main goal was to evaluate a panel of molecular markers for the detection of cancer cells in serous effusions and to determine their value as an adjunctive reverse transcription-PCR (RT-PCR) test to cytologic examination. Experimental Design: One hundred fourteen serous effusions from 71 patients with tumors and 43 patients with benign diseases were subjected to RT-PCR for expression of carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (Ep-CAM), E-cadherin, mammaglobin, mucin 1 (MUC1) isoforms MUC1/REP, MUC1/Y, and MUC1/Z, calretinin, and Wilms' tumor 1 susceptibility gene. Results: CEA, Ep-CAM, E-cadherin, and mammaglobin were specifically expressed in malignant effusions. The sensitivity of RT-PCR in cytologically negative malignant effusions was 63.1% combining CEA and Ep-CAM (with 100% specificity) and reached 78.9% adding MUC1/Y or MUC1/Z (with 93% specificity). In the whole population of effusions, the combination of cytology with RT-PCR of CEA and Ep-CAM yielded a 90.1% sensitivity, a specificity and a positive predictive value of 100%, and a 86% negative predictive value for malignancy. Adding MUC1/Y or MUC1/Z to the panel, the sensitivity was 94.5% with 93% specificity, 95.7% PPV, and 90.9% negative predictive value. Moreover, CEA and mammaglobin were specifically expressed in epithelial malignancies, and mammaglobin was mainly expressed in effusions from breast carcinoma (97.3% of specificity). Conclusions: A combination of cytology and RT-PCR analysis of CEA and Ep-CAM significantly improved the detection sensitivity of tumor cells in serous effusions. RT-PCR analysis of CEA, Ep-CAM, and mammaglobin in serous effusions could be a beneficial adjunct to cytology for the diagnosis of malignancy.

Aim The aim of this study is to evaluate the S100A1 and KIT as gene markers for the differentiation of common subtypes of renal tumours. Methods Fifty-five tissue samples (15 clear cell RCCs, 15 papillary RCCs, 7 chromophobe RCCs, 8 oncocytomas and 10 normal renal tissues) were studied The gene expressions of S100A1 and KIT were analysed by one-step RT-PCR by using the specific primers. Results S100A1 was expressed in 2/15 clear cell RCCs, 11/15 papillary RCCs, 7/8 oncocytomas and in 0/7 chromophobe RCCs. KIT gene was expressed in 6/7 chromophobe RCCs and 7/8 oncocytomas while 0/15 clear cell RCCs and 1/15 papillary RCCs expressed kit gene. Normal tissue expressed neither S100A1 nor KIT gene. Conclusion S100A1 and KIT can be used as gene markers for the differentiation of common subtypes of renal tumours.

Background Routine CD4/CD8 T-cell phenotyping may shows a small fraction of CD4dimCD8+ T cells with a homogeneous appearance as described for lymphoproliferative syndromes or chronic infections. The aim of this study was to elucidate the significance of CD4dimCD8+ T cells and their degree of diversity. Methods Phenotyping was performed in 272 samples from healthy donors, elderly patients, and immunocompromised (human immunodeficiency virus or renal transplantation) patients. Results The CD4dimCD8+ T cells had decreased fluorescence intensity for CD4 but not for CD8. The frequency of patients with CD4dimCD8+ T cells (>20 cells/μl; 10.3% of patients with human immunodeficiency virus and 7.7% with renal transplantation) was not significantly different when compared with healthy donors (9.7%). The CD4dimCD8+ T cells did not express the activation marker CD69. The CD8 of CD4dimCD8+ T cells expressed the heterodimeric (β) isoform. In 13 of 26 samples, the apparently highly homogeneous CD4dimCD8+ T cells expressed one predominant T-cell receptor Vβ clonotype. These predominant clonotypes were widely distributed among patients: Vβ 5.2, 17, 2, 3, 5.1, 13.1, 14, and 20. Conclusions Whether these findings demonstrate an oligoclonal reaction to chronic inflammation or an emerging lymphoproliferative disorder must be elucidated in a long-term longitudinal study. By analogy to monoclonal gammopathy, we propose to name this phenomenon “oligoclonal clonopathy of undetermined significance.” © 2005 Wiley-Liss, Inc.

Objectives: The common subtypes of renal tumors are conventional or clear cell carcinoma, papillary carcinoma, chromophobe carcinoma and oncocytoma. Each subtype has its distinct histogenesis and clinical evolution. DNA ploidy is viewed as a marker of gross genomic aberrations. The aim of this study is to evaluate the DNA ploidy in the common subtypes of renal tumors to increase our understanding of renal tumor biology and to broaden clinical application of DNA ploidy. Methods: 38 renal tumor samples (13 clear cell RCCs, 12 papillary RCCs, 7 chromophobe RCCs, and 6 oncocytomas) were studied. Five biopsies of different parts of each fresh tumor were subjected to a flow cytometric analysis of DNA ploidy. Results: All tumors except one papillary RCC generated interpretable DNA histograms. Flow cytometric analysis of oncocytomas showed the diploid pattern (29/30 frequencies) while the chromophobe RCC never showed the diploid pattern (0/55 frequencies) (p < 0.01). 3/7 chromopbobe RCCs possessed the hypodiploid stemline. The hypodiploid stemline appeared neither in conventional RCCs (0/63 frequencies) nor in papillary RCCs (0/50 frequencies). The diploid pattern was dominant in conventional and papillary RCCs. 10/13 (76.9%) of clear cell RCCs and 9/11 (81.8%) of papillary RCCs possessed a homogeneous DNA ploidy pattern while only 1/7 (14.3%) has a homogeneous DNA ploidy pattern. 6/7 chromophobe RCCs had multiple aneuploid stemlines. Conclusions: Flow cytometric analysis reveals that conventional and papillary RCCs are more homogeneous than chromophobe RCC. Each subtype of renal tumors possesses a specific DNA ploidy pattern. The analysis of DNA ploidy is useful for the differentiation of common subtypes of renal tumors in morphologically difficult cases.

We studied isotype profiles of anti-HIV antibodies (Ab) in HIV-1-infected African patients with high viral loads and major B cell dysfunction. We focussed on IgG1, IgG3, and IgA as these classes and subclasses tend to support neutralizing functions against HIV. Total IgG1, IgG3 and IgA were detected in the plasma of both HIV-1-infected and HIV-negative African individuals, but there was significantly more IgG3, a rare subclass, in HIV-1-infected patients (P

Objective: To evaluate the presence of IgA directed to the CD4-binding domain of gp120 and to a conserved region of gp41 (the Kennedy epitope) in serum and parotid saliva of HIV-1-seropositive patients. Methods: IgA were separated from IgG by anion-exchange chromatography and protein G treatment. The reactivity of IgA was tested against peptides and fusion proteins of the maltose-binding protein (MBP) and the CD4-binding site (MBP24) and MBP and the Kennedy epitope (MBP42). The capacity of serum and saliva IgA to interfere with the gp120-soluble CD4 (sCD4) interaction was examined. IgA were also purified by affinity chromatography using the MBP proteins adsorbed to a resin. Results: Peptides representing the CD4-binding domain and the Kennedy epitope were recognized by serum and saliva IgA of HIV-1-seropositive patients. Of the sera and saliva samples tested, 6/26 serum IgA and 5/25 saliva IgA inhibited the gp120-sCD4 interaction by approximately 50%. The gp120-sCD4 interaction was inhibited by MBP24 affinity-purified IgA but not by MBP42 affinity-purified IgA. Conclusion: Immunogens capable of eliciting IgA antibodies that inhibit gp120-CD4 binding might be efficiently used in vaccine to prevent mucosal transmission of HIV-I.

Background In routine CD3/CD4/CD8 T-cell analysis, a CD3 bright population of lymphocytes is frequently observed. The aim of the present study was to identify the immunological significance of such CD3 bright lymphocytes. Methods We analyzed samples from 31 healthy adult volunteers, 78 human immunodeficiency virus (HIV)-positive, and 78 renal transplanted patients. Results A clearly distinct CD3 bright (frequently CD4-/CD8-) T-cell fraction was observed in 84% of donors and was directly correlated with the fraction of gammadelta T cells (r2 = 0.64). CD3 overexpression on gammadelta T cells was confirmed by a combination of monoclonal antibody staining (CD3-ECD, gammadeltaTCR-FITC, and alphabetaTCR-PE-Cy5) or immunomagnetic purification of gammadelta T cells (i.e., MdFI 20 vs 8.86). The gammadelta T cells expressed CD8 polypeptide chains (alpha and beta) in all possible combinations. The largest proportion, surprisingly, were cells expressing CD8betabeta homodimers (43.8 +/- 16.5%). CD8alphaalpha homodimers were expressed on 14.2% (+/- 12.3) of total gammadelta T cells, whereas CD8alphabeta heterodimers were expressed on 12.2% (+/- 7.5). We also observed a bimodal distribution of the intensity of CD3 fluorescence of gammadelta T cells in immunocompromised patients with a threshold at 105 cell/microl. CD3 bright gammadelta T cells were more frequently observed in HIV patients (29%) compared with renal transplant patients (11%) and healthy donors (3%; chi2 test: P = 0.0007). Conclusions The simple observation of a CD3 bright T-cell subset on CD3/CD4/CD8 routine analysis suggests a high gammadelta T-cell fraction and, in our opinion, should be followed by a complementary analysis to determine precisely the number of gammadelta T cells and to identify their CD8alpha/beta phenotype. When CD3 bright T cells/microl were more than 40%, high gammadelta T cells were detected in more than 87% of cases, with a specificity of 76%. Occasionally, the CD3 bright subset appeared to be strongly homogeneous, suggesting an oligoclonal proliferation that could possibly reveal a chronic localized stimulation or an early lymphoproliferative disorder. Because the gammadelta T cells have interesting immunological peculiarities, the clinical significance of their quantitative abnormality should be clarified in diseases such as HIV, organ transplantation, autoimmunity and lymphoma.

We aimed at examining NFkappaB translocation in B lymphocytes during in vitro activation through the specific receptor for antigen using a technique convenient in most laboratories such as flow cytometry. We present here an original, convenient, and reproducible technique to study B cell activation events through NFkappaB translocation by means of a novel, specific flow cytometry assay. Intranuclear translocation of NFkappaB p65 was induced after a 45min stimulation; the highest signal was detected for a 10 ng/ml stimulus compared to the unstimulated condition (P0.05). Purified CD19+ B cells--cultured in the presence of optimal concentrations of anti-micro fragment Abs (10ng/ml) for 45min at 37 degrees C--induced a mean 60% (range: 45-67%) MFI-- and thus, nuclear NFkappaB translocation-increase. We observed a one-pike profile of NFkappaB staining in PBMC B cells and a two-pike profile of NFkappaB staining in using tonsil B cells. B cells are susceptible to various dysregulations leading to minor to severe pathology (including immunoproliferative disorders). Studies of signal transduction carried out specifically in human B cells, using a novel technique gave considerable advantages: feasibility, sensitivity, reproducibility, ease.

All three-dimensional in vitro mucosal models constructed, thus far, have only been reconstituted by epithelial cells. We have developed a reconstructed oral and vaginal epithelium that integrates Langerhans' cells (LC), the dendritic cells (DC) of malpighian epithelia. The epithelium was composed of gingival or vaginal keratinocytes seeded on a de-epidermized dermis (DED) and grown in submerged culture for 2 weeks. LC precursors, obtained after differentiation of cord blood-derived CD34+ hematopoietic progenitor cells (CD34+HPC) by granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta) and Flt3-ligand (Flt3-L), were introduced after 6-8 days of culture into the reconstituted epithelium. The in vitro reconstituted mucosal epithelium formed a multilayered, well-differentiated epithelial structure, confirmed by the immunohistochemical expression of cytokeratins 4, 6, 10, 13, 14, 16 and involucrin. LC were identified in the basal and suprabasal epithelial layers by CD1a antigen, S100 protein and Langerin/CD207 expression, and by transmission electron microscopy. Type IV collagen was expressed at the chorio-epithelial junction, and most ultrastructural features of this junction were visualized by electron microscopy. This in vitro reconstructed gingiva or vagina integrating LC represents interesting models very similar to native tissues. Because LC play an important role in the mucosal immune system, our models could be useful for conducting studies on interactions with pathogenic agents (viruses, bacteria etc.), as well as in pharmacological, toxicological and clinical research.

Background: Tumor-infiltrating macrophages secrete cytokines, including Fas ligand, tumor necrosis factor- (TNF-), and TNF-related apoptosis-inducing ligand (TRAIL). TRAIL induces apoptosis in tumor cells but not in normal cells; however, regulation of TRAIL and its receptors in cancer patients is relatively uncharacterized. We investigated whether macrophages from cancer patients produce TRAIL and whether apoptosis in cultured colon adenocarcinoma cells involves TRAIL and its receptors. Methods: Macrophages isolated from pleural effusions of nine cancer patients and five control patients with congestive heart failure (whose effusions contained no tumor cells) were cultured. Levels of TRAIL, TNF-, interferon , and Fas ligand in conditioned medium were measured by enzyme-linked immunosorbent assays. Apoptosis of human colon adenocarcinoma cell lines, including Colo 205, was determined by the Annexin V method and terminal deoxynucleotidyltransferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL). Cell-surface TRAIL receptors were measured by flow cytometry. Results: Conditioned culture medium from macrophages isolated from pleural effusions containing 1%–5% tumor cells (CM-A) contained TRAIL at 980– 1300 pg/mL, whereas that from macrophages from pleural effusions containing more than 50% tumor cells or containing no tumor cells (CM-B) contained TRAIL at 0–50 pg/mL. When cultured with medium containing 50% CM-A, 40% (95% confidence interval [CI] = 30% to 50%) of Colo 205 cells underwent apoptosis; when cultured with 50% CM-B, 8% (95% CI = 3% to 13%) underwent apoptosis. When Colo 205 cells were cultured with 50% CM-A, cell-surface expression of TRAIL death receptors DR5 and DR4 increased 13fold and sixfold, respectively, compared with that of untreated Colo 205 cells. Recombinant TRAIL induced 90% (95% CI = 85% to 95%) of Colo 205 cells to undergo apoptosis and acted synergistically with TNF- to induce apoptosis. Conclusion: Macrophages from cancer patients appear to be activated by tumor cells to produce TRAIL and to increase the expression of TRAIL death receptors DR4 and DR5 on tumor cells. [J Natl Cancer Inst 2003;95:611–21]

SUMMARYHIV1-gp160 holds promises in anti-HIV vaccinal strategies. However, this molecule has been described to exhibit superantigenic activities. The present study aimed at examining the effect(s) of HIV1-gp160 on human B cells and in particular on B cells originating from HIV– donors. We purified human B cells of various origins, i.e. from blood and from tonsils (representing a mucosal-type origin), and we tested these cells (stimulated with a polyclonal B cell activator, interleukin (IL)-2 and IL-10 as cytokines, and recombinant HIV1-gp160) for the production of IgG and IgA in an in vitro model. Gp160 induced significantly less total IgG by blood − but not tonsil-originating − B cells and did not affect total IgA production. Further, HIV1-gp160 up-regulated IL-2-, IL-4- and IL-10-mRNA levels in stimulated blood B cells (these cytokines are known to be active on B cell activation and differentiation). Interestingly, HIV1-gp160 also up-regulated IL-1β-, transforming growth factor (TGF)-β-, interferon (IFN)-γ- and IL-12-mRNA levels in stimulated mucosal-type, tonsil-originating, B cells. As these latter cytokines are involved in proinflammatory activities, HIV-gp160 delivery at the mucosal sites would be compatible with an adjuvant activity.

Epidermis and surface epithelium-dendritic cells comprise of immature cells termed Langerhans cells (LCs), which express characteristically the Birbeck granules, along with surface markers such as CD1a. These cells can capture a pathogen and then migrate and differentiate to a more mature stage. During this maturation process, dentritic cells express surface markers differentially. In physio-pathological models of infection where LCs are involved, it is critically important to ensure that the LCs tested in vitro are still immature and are not artefactually matured-dentritic cells. For experimental purposes, LCs were isolated from skin epidermis obtained from patients undergoing plastic surgery. This work thus aimed at collecting fresh LCs ex vivo and at testing the cells for phenotypic and functional characteristics of the immature stage. After mechanic disruption of the epidermis and proceeding for single cell suspension obtaining, two methods for purification were tested in parallel: (a) a positive immuno-magnetic separation by anti-CD1a-coated beads and (b) a purely mechanic purification system based on a three-step Ficoll floatation process. Both systems were equally efficient in terms of purification and yield. By using flow cytometry phenotyping, we have demonstrated that the use of magnetic beads led to some degree of maturation of CD1a+ LCs, contrary to the repeated Ficoll floatation. This work calls attention for the use of certain monoclonal antibodies such as anti-CD1a to purify immature dendritic cells as they pre-activate these cells. Pre-activation would render a number of assays on the early events of LC physiology invalid, contrary to the purification of fresh skin epidermis LCs by means of a repeated Ficoll floatation.

Objective To investigate the presence of the genome of GB virus C/hepatitis G virus (GBV-C/HGV) in semen and saliva from HIV-1-infected men. Methods Samples of blood from 33 men seropositive for HIV-1 were tested for the presence of GBV-C/HGV markers of infection, RNA by RT-PCR, and anti-E2 antibodies by ELISA, respectively. The cell-free fractions of seminal fluid and saliva samples of the patients with positive blood samples for GBV-C/HGV RNA or anti-E2 antibodies were then analyzed for the presence of the RNA of this virus. In addition, six semen samples and 11 saliva samples from GBV-C/HGV-negative men were tested. Results The GBV-C/HGV RNA tested by RT-PCR was recovered from blood in 11 patients of 33 (33.3%), and the antibodies to E2 envelope protein were detected in six patients (18.2%). Since no patient was positive for both markers, the overall prevalence of GBV-C/HGV infection was 51.5% in the studied population. Four—all belonging to the homosexual risk group—of the 17 men with markers to GBV-C/HGV in blood were found to be positive for GBV-C/HGV RNA in mucosal samples: two of them exhibited genomic RNA in both semen and saliva, and two others were positive for semen only. The absence of inhibitors of the PCR technique was confirmed in all mucosal fractions found negative for GBV-C/HGV RNA, except for one saliva sample and one seminal fluid sample. Conclusion These results confirm the high prevalence of GBV-C/HGV infection in patients infected with HIV-1 by sexual exposure and the presence of GBV-C/HGV RNA in seminal fluid and saliva of men with markers of this virus in the blood, suggesting that mucosal fluids could be a potential source for the spread of the GBV-C/HGV infection.