Characterization of immunoregulatory T lymphocytes during ageing by monoclonal antibodies.

Monoclonal antibodies of the OKT series were used to identify T lymphocytes (OKT3+) and their inducer (OKT4+) and suppressor-cytotoxic (OKT8+) subsets in the peripheral blood mononuclear cells (PBMC) of 32 healthy old-aged people more than 70 years old (16 men and 16 women) compared to 47 adults (29 men, 18 women) less than 40 years old. The absolute lymphocyte count in the peripheral blood was not significantly influenced by age or sex. Both the proportions and the absolute numbers of T3+ and T4+ cells were significantly lower in aged than in young participants. The proportions but not the absolute counts of OKT8+ cells were higher in the elderly. Most interesting is the influence of sex and these parameters. Old women have normal numbers and proportions of T3+, T4+ and T8+ cells when compared to young women. The latter have a significantly higher proportion of T8+ cells than young adult males. Old men have a striking reduction of both the numbers and proportions of OKT3+ and OKT4+ cells when compared with young men and with women. In addition, old men have an elevated proportion, but a normal absolute number, of OKT8+ cells. The responses of PBMC to phytohaemagglutinin extent (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) are reduced to the same extent in ageing male and female subjects when compared to young adults. In the older group, the magnitude of the lymphocyte response to PHA and Con A but not to PWM is negatively correlated with the proportions of OKT8+ cells. Surprisingly, these correlations are observed only in old women but not in old men. The latter finding excludes the possibility that the age-associated decline of the lymphocyte response to T cell mitogens is secondary to an imbalance between T4+ and T8+ lymphocytes.

Characterization of immunoregulatory T lymphocytes in juvenile diabetes by monoclonal antibodies

info:eu-repo/semantics/nonPublished

Development and characterization of anti-nitr9 antibodies.

The novel immune-type receptors (NITRs), which have been described in numerous bony fish species, are encoded by multigene families of inhibitory and activating receptors and are predicted to be functional orthologs to the mammalian natural killer cell receptors (NKRs). Within the zebrafish NITR family, nitr9 is the only gene predicted to encode an activating receptor. However, alternative RNA splicing generates three distinct nitr9 transcripts, each of which encodes a different isoform. Although nitr9 transcripts have been detected in zebrafish lymphocytes, the specific hematopoietic lineage(s) that expresses Nitr9 remains to be determined. In an effort to better understand the role of NITRs in zebrafish immunity, anti-Nitr9 monoclonal antibodies were generated and evaluated for the ability to recognize the three Nitr9 isoforms. The application of these antibodies to flow cytometry should prove to be useful for identifying the specific lymphocyte lineages that express Nitr9 and may permit the isolation of Nitr9-expressing cells that can be directly assessed for cytotoxic (e.g. NK) function.

Evaluation of HER2, p53, bcl-2, topoisomerase II-alpha, heat shock proteins 27 and 70 in primary breast cancer and metastatic ipsilateral axillary lymph nodes.

BACKGROUND: Breast cancer is a heterogeneous disease. Predictive biological markers (BM) of responsiveness to therapy need to be identified. Evaluation of BM is mainly done at the primary site. However, in the adjuvant therapy of breast cancer, the main goal is control of micrometastases. It is still unknown whether heterogeneity in the expression of BM between the primary site and its micrometastases exists. OBJECTIVE: To evaluate the expression of some BM with potential predictive value from the primary breast cancer site and metastatic ipsilateral axillary lymph nodes. PATIENTS AND METHODS: Focality (percentage of positive cells) and intensity staining scores were evaluated for each marker. Freshly cut sections (4 microm) from embedded blocks of breast cancer fixed in formalin or bouin were put onto superfrost slides (Menzel-Gläser). Protein expression was evaluated immunohistochemically (IHC) using monoclonal antibodies against: topo II-alpha (clone KiS1, 1 microg/ml, Roche) with a trypsine pre-treatment (P); HSP27 (clone G3.1, 1/60, Biogenex), HSP70 (clone BRM.22, 1/80, Biogenex) and HER2 (clone CB11, 1/40, Novocastra; without P); p53 (clone D07, 1/750, Dako) and bcl-2 (clone 124, 1/60, Dako) with citrate buffer as P. RESULTS: Overall, the percentage of discordant marker status in the primary tumour and its metastatic lymph nodes was 2% for HER2, 6% for p53, 15% for bcl-2, 19% for topoisomerase II-alpha, 24% for HSP27 and 30% for HSP70. For the subgroup of patients with positive BM in the primary tumour, the percentage of discordance was 6% for HER2, 7% for p53, 14% for bcl-2, 19% for HSP70, 21% for topoisomerase II-alpha and 36% for HSP27. For the subgroup of patients with positive BM in the lymph nodes, the percentage of discordance was 9% for bcl-2, 15% for HER2 and p53, 21% for topoisomerase II-alpha, 22% for HSP27 and 25% for HSP70. CONCLUSIONS: 1) No biological marker had 100% concordant results. 2) Although some discordant cases might be explained by the limitations of the IHC technique, future studies aiming to evaluate the predictive value of BM in the adjuvant therapy of breast cancer should take into account a possible difference in BM expression between the primary and the metastatic sites.

TCR V alpha- and V beta-gene segment use in T-cell subcultures derived from a type-III bare lymphocyte syndrome patient deficient in MHC class-II expression.

Previously, we and others have shown that MHC class-II deficient humans have greatly reduced numbers of CD4+CD8- peripheral T cells. These type-III Bare Lymphocyte Syndrome patients lack MHC class-II and have an impaired MHC class-I antigen expression. In this study, we analyzed the impact of the MHC class-II deficient environment on the TCR V-gene segment usage in this reduced CD4+CD8- T-cell subset. For these studies, we employed TcR V-region-specific monoclonal antibodies (mAbs) and a semiquantitative PCR technique with V alpha and V beta amplimers, specific for each of the most known V alpha- and V beta-gene region families. The results of our studies demonstrate that some of the V alpha-gene segments are used less frequent in the CD4+CD8- T-cell subset of the patient, whereas the majority of the TCR V alpha- and V beta-gene segments investigated were used with similar frequencies in both subsets in the type-III Bare Lymphocyte Syndrome patient compared to healthy control family members. Interestingly, the frequency of TcR V alpha 12 transcripts was greatly diminished in the patient, both in the CD4+CD8- as well as in the CD4-CD8+ compartment, whereas this gene segment could easily be detected in the healthy family controls. On the basis of the results obtained in this study, it is concluded that within the reduced CD4+CD8- T-cell subset of this patient, most of the TCR V-gene segments tested for are employed. However, a skewing in the usage frequency of some of the V alpha-gene segments toward the CD4-CD8+ T-cell subset was noticeable in the MHC class-II deficient patient that differed from those observed in the healthy family controls.

Sensitivity of HER-2/neu antibodies in archival tissue samples of invasive breast carcinomas. Correlation with oncogene amplification in 160 cases.

Overexpression and amplification of the HER-2 oncogene in patients with breast cancer has correlated with early onset of metastasis, resistance to hormonal therapy and some forms of chemotherapy, and shortened survival. Therefore, evaluation of this putative prognostic or predictive factor seems critical. Because different antibodies are used for the detection of the 185-kd HER-2 oncoprotein, we studied the sensitivity of 3 frequently used antibodies. Immunohistochemistry results were correlated with gene amplification level as assessed by fluorescence in situ hybridization. Protein overexpression was found in 17.2% and 12.5% of cases using antibodies against the external (TAB250) and internal (CB11) domains of the protein, respectively, and in 38.0% of cases using a rabbit polyclonal antibody. Fluorescence in situ hybridization was successful in all 160 tumors, and amplification was found in 37 tumors (23.1%). The monoclonal antibody TAB250 had the lowest misclassification rate, 9.6% (sensitivity, 67%; specificity, 97.5%).

Critical role of interleukin 5 and eosinophils in concanavalin A-induced hepatitis in mice.

BACKGROUND & AIMS: Eosinophils are observed in several liver diseases, but their contribution in the pathogenesis of these disorders remains poorly investigated. Concanavalin A (Con A)-induced hepatitis is an experimental model of immune-mediated liver injury in which natural killer T (NKT) cells play a critical role through the production of interleukin (IL)-4 and the expression of Fas ligand (FasL). Because activated NKT cells also produce IL-5, a critical cytokine for eosinophil maturation and function, the role of IL-5 was investigated in this model. METHODS: IL-5-deficient mice, eosinophil depletion in wild-type (WT) mice, and NKT cell transfer from WT- or IL-5-deficient mice into NKT cell-deficient mice were used to assess the role of IL-5 and eosinophils. RESULTS: Liver eosinophil infiltrate and IL-5 production were observed after Con A challenge. Liver injury was dramatically reduced in IL-5-deficient or eosinophil-depleted mice. In addition, residual hepatitis observed in Fas-deficient mice was abolished after IL-5 neutralization. Finally, we showed that NKT cells constituted a critical source of IL-5. Indeed, transfer of WT NKT cells to mice lacking NKT cells restored liver injury, whereas transfer of IL-5-deficient NKT cells did not. CONCLUSIONS: These observations highlight the pathologic role of IL-5 and eosinophils in experimental immune-mediated hepatitis.

Comparison of ELISA for tissue transglutaminase autoantibodies with antiendomysium antibodies in pediatric and adult patients with celiac disease.

BACKGROUND: Tissue transglutaminase (t-TG) is the main autoantigen recognized by the endomysium antibodies (EMA) observed in patients with celiac disease (CD). The aim of the study was to assess an ELISA method for t-TG antibodies (t-TGA) with respect to EMA IF assay in pediatric and adult patients. METHODS: t-TGA were analyzed by ELISA in 220 sera samples: 82 patients with biopsy-proven untreated CD (23 adults and 59 children), 14 CD children on gluten-free diet, 18 asymptomatic relatives of CD patients, and 106 age-matched control patients with gluten-unrelated gastrointestinal diseases (58 adults and 48 children). Serum IgA EMA were tested on umbilical cord sections in all patients. RESULTS: The great majority (92.7%) of untreated CD patients (both adults and children) were t-TGA positive (values ranging from 20.1 to > 300 AU). None of the child control patients and only two out of 58 (3.4%) of the adults with unrelated gastrointestinal diseases had serum t-TGA positivity; two out of 18 first-degree relatives with biopsy-proved silent CD were t-TGA (as well as EMA) positive. Finally, two out of 14 CD children, assuming a gluten-free diet, had serum t-TGA (as well as EMA). A highly significant correlation (P < 0.001) was observed between t-TGA concentrations and EMA. t-TGA showed a sensitivity of 87% and 95%, a specificity of 97% and 100% for adults and children, respectively. CONCLUSION: The method is highly sensitive and specific in the diagnosis of CD and is promising as a tool for routine diagnostic use and population screening, especially in children.

Optimal assessment of the ability of children with recurrent respiratory tract infections to produce anti-polysaccharide antibodies.

Specific anti-polysaccharide antibody deficiency (SPAD) is an immune disorder. Diagnostic criteria have not yet been defined clearly. One hundred and seventy-six children evaluated for recurrent respiratory tract infections were analysed retrospectively. For each subject, specific anti-pneumococcal antibodies had been measured with two enzyme-linked immunosorbent assays (ELISAs), one overall assay (OA) using the 23-valent pneumococcal polysaccharide vaccine (23-PPSV) as detecting antigen and the other purified pneumococcal polysaccharide serotypes (serotype-specific assay, SSA) (serotypes 14, 19F and 23F). Antibody levels were measured before (n = 176) and after (n = 93) immunization with the 23-PPSV. Before immunization, low titres were found for 138 of 176 patients (78%) with OA, compared to 20 of 176 patients (11%) with the SSA. We found a significant correlation between OA and SSA results. After immunization, 88% (71 of 81) of the patients considered as responders in the OA test were also responders in the SSA; 93% (71 of 76) of the patients classified as responders according to the SSA were also responders in the OA. SPAD was diagnosed in 8% (seven of 93) of patients on the basis of the absence of response in both tests. Thus, we propose to use OA as a screening test for SPAD before 23-PPSV immunization. After immunization, SSA should be used only in case of a low response in OA. Only the absence of or a very low antibody response detected by both tests should be used as a diagnostic criterion for SPAD.

Reliability of antitransglutaminase antibodies as predictors of gluten-free diet compliance in adult celiac disease.

OBJECTIVE: Strict lifelong compliance to a gluten-free diet (GFD) minimizes the long-term risk of mortality, especially from lymphoma, in adult celiac disease (CD). Although serum IgA antitransglutaminase (IgA-tTG-ab), like antiendomysium (IgA-EMA) antibodies, are sensitive and specific screening tests for untreated CD, their reliability as predictors of strict compliance to and dietary transgressions from a GFD is not precisely known. We aimed to address this question in consecutively treated adult celiacs. METHODS: In a cross-sectional study, 95 non-IgA deficient adult (median age: 41 yr) celiacs on a GFD for at least 1 yr (median: 6 yr) were subjected to 1) a dietician-administered inquiry to pinpoint and quantify the number and levels of transgressions (classified as moderate or large, using as a cutoff value the median gluten amount ingested in the overall noncompliant patients of the series) over the previous 2 months, 2) a search for IgA-tTG-ab and -EMA, and 3) perendoscopic duodenal biopsies. The ability of both antibodies to discriminate celiacs with and without detected transgressions was described using receiver operating characteristic curves and quantified as to sensitivity and specificity, according to the level of transgressions. RESULTS: Forty (42%) patients strictly adhered to a GFD, 55 (58%) had committed transgressions, classified as moderate (< or = 18 g of gluten/2 months; median number 6) in 27 and large (>18 g; median number 69) in 28. IgA-tTG-ab and -EMA specificity (proportion of correct recognition of strictly compliant celiacs) was 0.97 and 0.98, respectively, and sensitivity (proportion of correct recognition of overall, moderate, and large levels of transgressions) was 0.52, 0.31, and 0.77, and 0.62, 0.37, and 0.86, respectively. IgA-tTG-ab and -EMA titers were correlated (p < 0.001) to transgression levels (r = 0.560 and R = 0.631, respectively) and one to another (p < 0.001) in the whole patient population (r = 0.834, N = 84) as in the noncompliant (r = 0.915, N = 48) group. Specificity and sensitivity of IgA-tTG-ab and IgA-EMA for recognition of total villous atrophy in patients under a GFD were 0.90 and 0.91, and 0.60 and 0.73, respectively. CONCLUSIONS: In adult CD patients on a GFD, IgA-tTG-ab are poor predictors of dietary transgressions. Their negativity is a falsely secure marker of strict diet compliance.

Antiendomysial and antihuman recombinant tissue transglutaminase antibodies in the diagnosis of coeliac disease: a biopsy-proven European multicentre study.

OBJECTIVE: To investigate the value of serum antitissue transglutaminase IgA antibodies (IgA-TTG) and IgA antiendomysial antibodies (IgA-EMA) in the diagnosis of coeliac disease in cohorts from different geographical areas in Europe. The setting allowed a further comparison between the antibody results and the conventional small-intestinal histology. METHODS: A total of 144 cases with coeliac disease [median age 19.5 years (range 0.9-81.4)], and 127 disease controls [median age 29.2 years (range 0.5-79.0)], were recruited, on the basis of biopsy, from 13 centres in nine countries. All biopsy specimens were re-evaluated and classified blindly a second time by two investigators. IgA-TTG were determined by ELISA with human recombinant antigen and IgA-EMA by an immunofluorescence test with human umbilical cord as antigen. RESULTS: The quality of the biopsy specimens was not acceptable in 29 (10.7%) of 271 cases and a reliable judgement could not be made, mainly due to poor orientation of the samples. The primary clinical diagnosis and the second classification of the biopsy specimens were divergent in nine cases, and one patient was initially enrolled in the wrong group. Thus, 126 coeliac patients and 106 controls, verified by biopsy, remained for final analysis. The sensitivity of IgA-TTG was 94% and IgA-EMA 89%, the specificity was 99% and 98%, respectively. CONCLUSIONS: Serum IgA-TTG measurement is effective and at least as good as IgA-EMA in the identification of coeliac disease. Due to a high percentage of poor histological specimens, the diagnosis of coeliac disease should not depend only on biopsy, but in addition the clinical picture and serology should be considered.

Anti-CTLA-4 treatment induces IL-10-producing ICOS+ regulatory T cells displaying IDO-dependent anti-inflammatory properties in a mouse model of colitis.

BACKGROUND AND AIMS: Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) has been shown to act as a negative regulator of T cell function and has been implicated in the regulation of T helper 1 (Th1)/Th2 development and the function of regulatory T cells. Tests were carried out to determine whether anti-CTLA-4 treatment would alter the polarisation of naive T cells in vivo. METHODS: Mice were treated with anti-CTLA-4 monoclonal antibody (mAb) (UC10-4F10) at the time of immunisation or colonic instillation of trinitrobenzene sulfonic acid (TNBS). The cytokines produced by lymph node cells after in vitro antigenic stimulation and the role of indoleamine 2,3 dioxygenase (IDO) and of interleukin-10 (IL-10) were tested, and the survival of mice was monitored. RESULTS: Injection of anti-CTLA-4 mAb in mice during priming induced the development of adaptive CD4(+) regulatory T cells which expressed high levels of ICOS (inducible co-stimulator), secreted IL-4 and IL-10. This treatment inhibited Th1 memory responses in vivo and repressed experimental intestinal inflammation. The anti-CTLA-4-induced amelioration of disease correlated with IDO expression and infiltration of ICOS(high) Foxp3(+) T cells in the intestine, suggesting that anti-CTLA-4 acted indirectly through the development of regulatory T cells producing IL-10 and inducing IDO. CONCLUSIONS: These observations emphasise the synergy between IL-10 and IDO as anti-inflammatory agents and highlight anti-CTLA-4 treatment as a potential novel immunotherapeutic approach for inducing adaptive regulatory T cells.

HER-2/neu evaluation by immunohistochemistry and fluorescence in situ hybridization in breast cancer: implications for daily laboratory practice.

BACKGROUND: HER-2/neu status was determined by immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH) methods in more than 300 paraffin-embedded primary breast cancer samples. MATERIALS AND METHODS: HER-2/neu status was determined by FISH using the PathVysion kit (Vysis) and by IHC using either a monoclonal antibody CB11 or a cocktail of antibodies: the monoclonal TAB250 and the polyclonal pAb1. RESULTS: Of the 324 cases evaluable by IHC, 65 out of 318 (20%) and 24 out of 324 (7%) were scored as positive when using the antibody cocktail and the CB11, respectively. HER-2/neu gene amplification occured in 64 out of 324 cases (20%). Concordance of FISH and IHC was found in 285 out of 318 cases (90%) and 278 out of 324 cases (86%) using the cocktail and the CB11, respectively. CONCLUSION: The cost-effectiveness analysis revealed that the use of a sensitive IHC method followed by confirmation of positive results by FISH considerably decreased the FISH costs and may become standard practice for HER-2/neu evaluation.

Estimating the magnitude of trastuzumab effects within patient subgroups in the HERA trial.

BACKGROUND: Trastuzumab (Herceptin(R)) improves disease-free survival (DFS) and overall survival for patients with human epidermal growth factor receptor 2 (HER2)-positive early breast cancer. We aimed to assess the magnitude of its clinical benefit for subpopulations defined by nodal and steroid hormone receptor status using data from the Herceptin Adjuvant (HERA) study. PATIENTS AND METHODS: HERA is an international multicenter randomized trial comparing 1 or 2 years of trastuzumab treatment with observation after standard chemotherapy in women with HER2-positive breast cancer. In total, 1703 women randomized to 1-year trastuzumab and 1698 women randomized to observation were included in these analyses. Median follow-up was 23.5 months. The primary endpoint was DFS. RESULTS: The overall hazard ratio (HR) for trastuzumab versus observation was 0.64 [95% confidence interval (CI) 0.54-0.76; P < 0.0001], ranging from 0.46 to 0.82 for subgroups. Estimated improvement in 3-year DFS in subgroups ranged from +11.3% to +0.6%. Patients with the best prognosis (those with node-negative disease and tumors 1.1-2.0 cm) had benefit similar to the overall cohort (HR 0.53, 95% CI 0.26-1.07; 3-year DFS improvement +4.6%, 95% CI -4.0% to 13.2%). CONCLUSIONS: Adjuvant trastuzumab therapy reduces the risk of relapse similarly across subgroups defined by nodal status and steroid hormone receptor status, even those at relatively low risk for relapse.

Differential inducibility of HLA class I and II antigens by r-IFN gamma in type III bare lymphocyte syndrome.

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