First Membrane Proximal External Region-Specific Anti-HIV1 Broadly Neutralizing Monoclonal IgA1 Presenting Short CDRH3 and Low Somatic Mutations.

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.

Delivery of antigen to nasal-associated lymphoid tissue microfold cells through secretory IgA targeting local dendritic cells confers protective immunity.

BACKGROUND: Transmission of mucosal pathogens relies on their ability to bind to the surfaces of epithelial cells, to cross this thin barrier, and to gain access to target cells and tissues, leading to systemic infection. This implies that pathogen-specific immunity at mucosal sites is critical for the control of infectious agents using these routes to enter the body. Although mucosal delivery would ensure the best onset of protective immunity, most of the candidate vaccines are administered through the parenteral route. OBJECTIVE: The present study evaluates the feasibility of delivering the chemically bound p24gag (referred to as p24 in the text) HIV antigen through secretory IgA (SIgA) in nasal mucosae in mice. RESULTS: We show that SIgA interacts specifically with mucosal microfold cells present in the nasal-associated lymphoid tissue. p24-SIgA complexes are quickly taken up in the nasal cavity and selectively engulfed by mucosal dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin-positive dendritic cells. Nasal immunization with p24-SIgA elicits both a strong humoral and cellular immune response against p24 at the systemic and mucosal levels. This ensures effective protection against intranasal challenge with recombinant vaccinia virus encoding p24. CONCLUSION: This study represents the first example that underscores the remarkable potential of SIgA to serve as a carrier for a protein antigen in a mucosal vaccine approach targeting the nasal environment.

Major influence of CD4 count at the initiation of cART on viral and immunological reservoir constitution in HIV-1 infected patients.

BACKGROUND: A persistent immune activation is observed in gut during HIV-1 infection, which is not completely reversed by a combined antiretroviral therapy (cART). The impact of the time of cART initiation may highly influence the size of the viral reservoir and the ratio of CD4(+)/CD8(+) T cells in the gut. In this study, we analyzed the characteristics of HIV rectal reservoir of long-term treated patients, regarding their blood CD4(+) T cells count at the time of cART initiation. RESULTS: Twenty-four consenting men were enrolled: 9 exhibiting a CD4(+) T cells count >350/mm(3) ("high-level CD4 group") and 15 < 350/mm(3) ("low-level CD4 group") in blood, at the start of cART. An immunophenotypical analysis of T and B cells subpopulations was performed in blood and rectal biopsies. HIV cell-associated DNA loads and qualitative intra-cellular RNA were determined in both compartments. The ratio of CD4(+)/CD8(+) T cells was significantly decreased in the blood but not in the rectum of the "low-level CD4 group" of patients. The alteration in ß7(+) CD4(+) T cells homing was higher in this group and was correlated to a low ratio of CD4(+)/CD8(+) T cells in blood. An initiation of cART in men exhibiting a low-level CD4 count was also associated with an alteration of B cells maturation. HIV blood and gut DNA reservoirs were significantly lower in the "high-level CD4 group" of men. A high HIV DNA level was associated to a detectable intracellular HIV RNA in rectum. CONCLUSIONS: An early initiation of cART could significantly preserve gut immunity and limit the viral reservoir constitution.

A Difficult and Rare Diagnosis of Autoimmune Enteropathy in a Patient Affected by Down Syndrome.

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.

Secretory IgA as a vaccine carrier for delivery of HIV antigen to M cells.

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.

Dectin-1 is essential for reverse transcytosis of glycosylated SIgA-antigen complexes by intestinal M cells.

Intestinal microfold (M) cells possess a high transcytosis capacity and are able to transport a broad range of materials including particulate antigens, soluble macromolecules, and pathogens from the intestinal lumen to inductive sites of the mucosal immune system. M cells are also the primary pathway for delivery of secretory IgA (SIgA) to the gut-associated lymphoid tissue. However, although the consequences of SIgA uptake by M cells are now well known and described, the mechanisms whereby SIgA is selectively bound and taken up remain poorly understood. Here we first demonstrate that both the Cα1 region and glycosylation, more particularly sialic acid residues, are involved in M cell-mediated reverse transcytosis. Second, we found that SIgA is taken up by M cells via the Dectin-1 receptor, with the possible involvement of Siglec-5 acting as a co-receptor. Third, we establish that transcytosed SIgA is taken up by mucosal CX3CR1⁺ dendritic cells (DCs) via the DC-SIGN receptor. Fourth, we show that mucosal and systemic antibody responses against the HIV p24-SIgA complexes administered orally is strictly dependent on the expression of Dectin-1. Having deciphered the mechanisms leading to specific targeting of SIgA-based Ag complexes paves the way to the use of such a vehicle for mucosal vaccination against various infectious diseases.

Cutting edge: New chimeric NOD2/TLR2 adjuvant drastically increases vaccine immunogenicity.

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.

Comparison of Screening Dilution and Automated Reading for Antinuclear Antibody Detection on HEP2 Cells in the Monitoring of Connective Tissue Diseases.

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.

Cell-free RNA content in urine as a possible molecular diagnostic tool for clear cell renal cell carcinoma.

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.

Association between pharmacokinetics of adalimumab and mucosal healing in patients with inflammatory bowel diseases.

BACKGROUND & AIMS: Little is known about the association between pharmacokinetic features of adalimumab and mucosal healing in patients with inflammatory bowel disease (IBD). METHODS: We conducted a cross-sectional study of 40 patients with Crohn's disease (CD) or ulcerative colitis (UC) who received adalimumab maintenance therapy and underwent endoscopic evaluation of disease activity and pharmacokinetic analysis (measurements of trough levels and antibodies against adalimumab). Patients in clinical remission were identified based on CD activity index scores less than 150 or Mayo scores less than 3 (for those with UC). Patients with mucosal healing were identified based on Mayo endoscopic scores less than 2 (for UC) or the disappearance of all ulcerations (for CD). RESULTS: The median trough level of adalimumab was higher in patients in clinical remission (6.02 µg/mL) than in patients with active disease (3.2 µg/mL; P = .012). Trough levels of adalimumab were also higher in patients with mucosal healing (6.5 µg/mL) than in patients without (4.2 µg/mL; P < .005). These results did not vary with type of IBD. On multivariate analysis, trough levels of adalimumab (relative risk, 0.62; 95% confidence interval, 0.40-0.94; P = .026) and duration of adalimumab treatment (relative risk, 0.82; 95% confidence interval, 0.68-0.97; P = .026) were associated independently with healing mucosa. An absence of mucosal healing was associated with trough levels of adalimumab less than 4.9 µg/mL (likelihood ratio, 4.3; sensitivity, 66%; specificity, 85%). CONCLUSIONS: Trough levels of adalimumab are significantly higher in IBD patients who are in clinical remission and in those with mucosal healing. Detection of antibodies against adalimumab predicts a lack of mucosal healing.

Serum miR-210 as a novel biomarker for molecular diagnosis of clear cell renal cell carcinoma.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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 (p<0.001). 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). CONCLUSION: Serum mi-210 may have a potential as a novel noninvasive biomarker for the detection of cRCC.

Aerosol immunization with NYVAC and MVA vectored vaccines is safe, simple, and immunogenic.

Each year, approximately five million people die worldwide from putatively vaccine-preventable mucosally transmitted diseases. With respect to mass vaccination campaigns, one strategy to cope with this formidable challenge is aerosol vaccine delivery, which offers potential safety, logistical, and cost-saving advantages over traditional vaccination routes. Additionally, aerosol vaccination may elicit pivotal mucosal immune responses that could contain or eliminate mucosally transmitted pathogens in a preventative or therapeutic vaccine context. In this current preclinical non-human primate investigation, we demonstrate the feasibility of aerosol vaccination with the recombinant poxvirus-based vaccine vectors NYVAC and MVA. Real-time in vivo scintigraphy experiments with radiolabeled, aerosol-administered NYVAC-C (Clade C, HIV-1 vaccine) and MVA-HPV vaccines revealed consistent mucosal delivery to the respiratory tract. Furthermore, aerosol delivery of the vaccines was safe, inducing no vaccine-associated pathology, in particular in the brain and lungs, and was immunogenic. Administration of a DNA-C/NYVAC-C prime/boost regime resulted in both systemic and anal-genital HIV-specific immune responses that were still detectable 5 months after immunization. Thus, aerosol vaccination with NYVAC and MVA vectored vaccines constitutes a tool for large-scale vaccine efforts against mucosally transmitted pathogens.

Improvement of malignant serous effusions diagnosis by quantitative analysis of molecular claudin 4 expression.

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.

CA9 level in renal cyst fluid: a possible molecular diagnosis of malignant tumours.

AIMS: The preoperative differentiation of malignant renal cystic tumours from benign lesions is critical, and it remains a common diagnostic problem. The aim was to examine if the Carbonic anhydrase 9 (CA9) level in cyst fluid can provide a molecular diagnosis of malignant cyst. METHODS AND RESULTS: Twenty-eight patients with a cystic renal mass were included. Fine-needle aspiration was performed to obtain the fluid. Postoperative pathology confirmed that there were 16 cystic renal cell carcinomas. Twelve benign cystic tumours were used as controls. One hundred microlitres of supernatant of cyst fluid was used to measure the CA9 protein level, which was measured by an enzyme-linked immunosorbent assay technique. CA9 was strongly detected and considered as positive in the cyst fluid of all 16 cystic malignant tumours (>1000 pg/ml), whereas its expression was negative in 11/12 benign cystic tumours (<300 pg/ml). The difference in percentages of positive CA9 between malignant and benign renal cystic tumours was significant (P < 0.001). CONCLUSION: The fluid of malignant cystic renal tumours contains a high level of CA9 protein. The measurement of CA9 level in cyst fluid may be used as a molecular diagnosis for differentiation between malignant and benign renal cystic masses.

A high mucosal blocking score is associated with HIV protection.

BACKGROUND: Early steps of HIV infection are mediated by the binding of the envelope to mucosal receptors as α4ß7 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 α4ß7, DC-SIGN and/or langerinin HESN. New cell-based assays were developed to analyze whether antibodies that can alter gp120 binding to α4ß7, 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/α4ß7, 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 α4ß7 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 α4ß7 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.

Serum carbonic anhydrase 9 level is associated with postoperative recurrence of conventional renal cell cancer.

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 <0.001 and p = 0.001, respectively). The mean serum carbonic anhydrase 9 level in patients with metastatic conventional renal cell cancer was significantly higher than in those with localized disease (p = 0.004). Of patients with localized disease those with recurrence had a significantly higher serum carbonic anhydrase 9 than those without recurrence (p = 0.001). On univariate analysis serum carbonic anhydrase 9, tumor stage, tumor grade and tumor size were associated with recurrence. The recurrence-free survival curve indicates that patients with a high serum carbonic anhydrase 9 level had a significantly higher recurrence rate than those with a low serum carbonic anhydrase 9 (p = 0.001). CONCLUSIONS: Our data suggest that serum carbonic anhydrase 9 is increased as the tumor progression occurs. A high carbonic anhydrase 9 level is associated with postoperative recurrence.

Opposite immune reactivity of serum IgG and secretory IgA to conformational recombinant proteins mimicking V1/V2 domains of three different HIV type 1 subtypes depending on glycosylation.

The V1/V2 domain of the HIV-1 gp120 envelope protein has been shown to contribute to viral cell tropism during infection and also to viral recognition by neutralizing monoclonal antibodies. However, this domain has been poorly investigated. Carbohydrates have been demonstrated to dramatically influence immune reactivity of antisera to viral glycoprotein antigens. In this study, DNA sequences coding for V1/V2 domains from HIV-1 primary isolates of three subtypes (A, B, and C) were subcloned into a secretion vector and used to transfect CHO cells that are able to achieve the glycosylation of proteins. The structure of purified recombinant V1/V2 proteins was tested using two anti-V1/V2 monoclonal antibodies directed against either a linear or a conformational and glycosylation-dependent epitope (8.22.2 and 697-D). Serum or saliva of 14/82 seropositive patients with anti-V1/V2 reactivity demonstrated good recognition of the recombinant proteins. Deglycosylation of the recombinant proteins was found to increase the reactivity of the serum IgG to the clade A and C but not to clade B V1/V2 domain demonstrating that the recognition of glycosylation sites by serum IgG is clade dependent. When considering SIgA from parotid saliva, deglycosylation of all recombinant proteins tested decreased the reactivity, suggesting that glycosylation plays an important role in the recognition of V1/V2 domain target epitopes by this class of antibodies. In conclusion, these results suggest the influence of carbohydrate moieties on the specificity of the antibodies to the V1/V2 domain produced during HIV infection and the potential importance of viral glycans in vaccine responses after mucosal administration.

Elevated serum-circulating RNA in patients with conventional renal cell cancer.

BACKGROUND: Reliable serum biomarkers for differential diagnosis of conventional renal cell carcinoma (RCC) are highly desirable. Recent studies have confirmed the stability of circulating RNA in serum of cancer patients. The purpose of our study was to evaluate whether the amounts of circulating RNA could discriminate between conventional renal cancer patients and healthy individuals as a tumor marker. PATIENTS AND METHODS: A total of 71 patients with conventional RCC, 12 with renal oncocytomas and 44 healthy individuals entered into this study. Serum samples were taken and subjected to RNA extraction. The amount of RNA was quantified spectrophotometrically. Additionally, 9 serum samples from conventional RCC were also studied one week after nephrectomy. Diagnostic performance of RNA concentration was calculated through the receiver operating characteristic (ROC) curve to distinguish between conventional RCC and healthy individuals. RESULTS: The mean level of RNA in conventional RCC (1414.19 +/- 91.95 ng/ml) was significantly higher than that in healthy individuals (520.49 +/- 39.75 ng/ml, p<0.0001) and these with renal oncocytomas (560.71 +/- 69.54 ng/ml, p<0.0001). Among the conventional RCC, there was no significant difference in circulating RNA levels in terms of tumor stage, grade or size. The area under the ROC curve was 0.956 (95% confidence interval, 0.923 to 0.989), indicating an acceptable sensitivity and specificity as a tumor marker. For conventional RCC, the RNA level was reduced significantly (p<0.0001) one week after nephrectomy. CONCLUSION: The data suggest that elevated circulating RNA may be a valuable diagnostic tool for discriminating conventional RCC patients from normal individuals or from these with renal oncocytoma. Elevated serum circulating RNA provides a new research area as biomarker for the diagnosis of conventional RCC.

A flow cytometry technique to study intracellular signals NF-kappaB and STAT3 in peripheral blood mononuclear cells.

BACKGROUND: Cytokines have essential roles on intercellular communications and are effective in using a variety of intracellular pathways. Among this multitude of signalling pathways, the NF-kappaB (nuclear factor kappaB) and STAT (signal transducer and activator of transcription) families are among the most frequently investigated because of their importance. Indeed, they have important role in innate and adaptive immunity. Current techniques to study NF-kappaB and STAT rely on specific ELISAs, Western Blots and--most recently described--flow cytometry; so far, investigation of such signalling pathways are most commonly performed on homogeneous cells after purification. RESULTS: The present investigation aimed at developing a flow cytometry technique to study transcription factors in various cellular types such as mixtures of B-cells, T-lymphocytes and monocytes/macrophages stimulated in steady state conditions (in other words, as peripheral blood mononuclear cells). To achieve this goal, a two step procedure was carried out; the first one consisted of stimulating PBMCs with IL1beta, sCD40L and/or IL10 in such a manner that optimal stimulus was found for each cell subset (and subsequent signal transduction, therefore screened by specific ELISA); the second step consisted of assessing confirmation and fine delineation of technical conditions by specific Western-Blotting for either NF-kappaB or STAT products. We then went on to sensitize the detection technique for mixed cells using 4 color flow cytometry. CONCLUSION: In response to IL1beta, or IL10, the levels of phosphorylated NF-kappaB and STAT3--respectively--increased significantly for all the studied cell types. In contrast, B-cells and monocytes/macrophages--but, interestingly, not T-lymphocytes (in the context of PBMCs)--responded significantly to sCD40L by increasing phosphorylated NF-kappaB.

CA9 gene expression in conventional renal cell carcinoma: a potential marker for prediction of early metastasis after nephrectomy.

About 30-40% of patients with renal cell carcinoma (RCC) will develop metastasis after curative nephrectomy. There is a strong need to identify the early metastasis with conventional and molecular risk factors. The present study aimed to test if analysis of the CA9 gene can provide useful information to predict early metastasis after nephrectomy. This study included 63 patients with a conventional RCC. Ten tumors were N+ or/and M+ at diagnosis. The mean follow-up was 43 months (range, 4-67 months). About 11 M0N0 patients were found to have a metastasis during the follow-up. Quantitative RT-PCR of CA9 gene expression was performed. The metastasis-free survival curve was established according to the Kaplan-Meier method with comparison by the Log-Rank test. At diagnosis, the average of CA9 gene expression was significantly lower (p = 0.004) in metastatic tumors (N+ or/and M+) than in non-metastatic tumors (N0M0). For the follow-up of M0N0 patients, the metastasis-free survival rate was significantly higher (p = 0.005) in the high CA9 group than in the low-CA9 group. When combined with CA9, the metastasis-free survival rates, in terms of stage (p = 0.015) or grade (p = 0.010) were significantly different. When the stage, grade, and CA9 were combined, there was a significant difference (p = 0.004) in metastasis-free survival rates (T1T2 + G1G2 + high expression of CA9 versus T3 + G3G4 + low expression of CA9). Finally, the multivariate regression analysis identified CA9 expression (p = 0.036) as an independent predictor of early metastasis. Our study confirms that the expression level of CA9 gene in conventional RCC is related to metastasis. CA9 may be a potential marker for the prediction of early metastasis after nephrectomy and to guide post-operative follow-up and treatment.