Peripheral blood natural killer cell count is associated with clinical outcome in patients with aaIPI 2-3 diffuse large B-cell lymphoma.

BACKGROUND: Lymphocytopenia is a prognostic factor in Hodgkin's disease. In diffuse large B-cell lymphoma (DLBCL), data are much less established, in spite of numerous reports on immune system-lymphoma interactions. This study addresses the prognostic value of blood lymphocyte subsets at diagnosis in DLBCL. PATIENTS AND METHODS: Absolute values of blood lymphocyte subsets and monocytes were prospectively determined by flow cytometry in 140 patients with 2 or 3 adverse age-adjusted International Prognostic Index (aaIPI) factors included in a Groupe d'Etude des Lymphomes de l'Adulte protocol (LNH98B3). Absolute cell counts at diagnosis and aaIPI were evaluated with regard to clinical outcome. RESULTS: Low median cell counts of 337, 211, and 104/mul were evidenced for the CD4+, CD8+ T, and natural killer (NK) cells, respectively. In univariate analysis, only NK cell count [odds ratio (OR) = 1.81 (1.27, 2.57), P = 0.001] and aaIPI [OR = 2.29 (0.95, 5.45), P = 0.06] were associated with induction treatment response. Low NK cell count [Hazard ratio (HR) = 1.27 (1.06, 1.52), P = 0.01] and aaIPI 3 [HR = 1.95 (1.20, 3.16), P = 0.01] were also associated with a shorter event free survival (EFS). In multivariate analysis, NK cell count was associated with response [OR = 1.77 (1.24, 2.54), P = 0.002] and EFS [HR = 1.25 (1.04, 1.50) P = 0.02] independently of aaIPI. CONCLUSIONS: This study shows an association between circulating NK cell number and clinical outcome in DLBCL, possibly important in the context of the broadening use of rituximab, a likely NK-dependent therapy.

Use of allogenic epidermal sheets for difficult wound healing: selection and testing of relevant growth factors.

The clinical interest of using allogenic epidermal sheets (AES) has largely been shown [1,2,3]. As well as covering, they also stimulate healing, by simultaneously secreting numerous growth factors (GFs), although little is known on their mechanism of action. Our objectives were to: (a) devise a test for the efficacy of AES release, (b) select keratinocyte-secreting strains and optimal culture conditions. Three GFs were selected: IL-1alpha, IL-8 and VEGF. Three different keratinocyte strains were cultured for 3 and 6 days after confluence for 3 passages. Assays were performed after 3 h and 24 h+3 h after dispase treatment (AES conservation for 24 h then change of medium and sampling after 3 h). AES were found to secrete GFs in DMEM and the amounts were greater when cultured for 6 rather than 3 days after confluence. Each strain had different secretory patterns depending on passage and time in culture, this variability being explained by inter-individual heterogeneity.

An in vitro model to evaluate the cytokine response in Echinococcus infections.

With the aim of proposing an alternative model to animal experimentation, we investigated cytokine production in response to antigens in an in vitro system. This is a co-culture system of healthy human leukocytes and enterocyte-like Caco-2 cells. The antigens tested, EgA31, EgTrp, and FABP1, are candidates for vaccines in infections caused by Echinococcus spp. in the gut. All three have previously been described in the protoscolex stage and belong to protein families which confer protective immunity against several helminths. In this study, we evaluate the Th1/Th2 profile (Th1: IL-12, IFN-gamma; Th2: IL-6, IL-10) in response to protoscoleces, EgA31 and the mixture of EgA31, EgTrp and FABP1. No cytokine production was detected in response to protoscoleces. Neither IFN-gamma nor IL-6, but a significant IL-10 and IL-12 concentration was detected in response to both types of antigens. These findings suggest that EgA31 and the mixture EgA31/EgTrp/FABP1 generated an immunogenic response associated with a mixed Th1/Th2 cytokine.

[Preliminary results in standardization of flow cytometry protocols for monocytic HLA-DR measurement and their application in the follow up of septic shock]. / Standardisation de la mesure de l'antigène HLA-DR monocytaire par cytométrie en flux: résultats préliminaires et application dans le suivi des chocs septiques.

In septic shock, the diminished HLA-DR expression on monocytes has been proposed as a marker of immunoparalysis that correlates with an increased risk for fatal outcome. The present study was designed to determine whether some differences in protocol procedures could lead to discrepant results in HLA-DR measurement. After establishing a reliable protocol, the second objective was to illustrate the immunoparalysis in patients with septic shock. HLA-DR measurement on monocytes was determined by means of flow cytometry in 54 healthy donors and 16 patients with septic shock. We demonstrated that storage temperature, storage duration before staining and red cells lysis constitute crucial steps in HLA-DR measurement. The precision results with coefficients of variation below 5%, were quite convincing for a manual immunoassay. At 48 hours after diagnosis of septic shock, we found severely decreased percentages of monocytes expressing HLA-DR in septic patients (24 +/- 4%, mean +/- SEM) in comparison with healthy donors (90 +/- 1%), p < 0.001). Furthermore, the persistence of a low level of monocytic HLA-DR (less than 50 %) at day 9 after admittance was associated with patients who died. This study illustrates the state of immunoparalysis in patients with septic shock and supports the potential interest in measuring HLA-DR expression on monocytes. However, multicenter studies are now needed to validate this parameter. Based on our analytical results, we conclude that a critical issue in such studies will be the capacity in each center to perform standardized measurement of HLA-DR. It should be remembered that this determination requires the definition of a common analytical procedure between laboratories participating in the trial.

Human gammadelta T cells express a higher TCR/CD3 complex density than alphabeta T cells.

The aim of our study was to compare CD3 expression on gammadelta T cells and alphabeta T cells in human patients. The antigen density of TCR and CD3 on both subsets was assessed by a quantitative method in eight patients. In parallel, we developed and validated a reliable direct tricolor staining protocol that we tested on samples from hospitalized and healthy individuals (n = 60). Our results demonstrate that human gammadelta T cells constitutively express approximately twofold more of the TCR/CD3 complex than alphabeta T cells. We suggest that this enhanced expression of the TCR/CD3 complex could contribute to the higher reactivity of gammadelta T cells compared to alphabeta T cells. These clinical laboratory results confirm the fundamental data described elsewhere. gammadelta T cells deserve further clinical investigations to understand their precise role in human immunity.

Serum levels of YKL-40 and C reactive protein in patients with hip osteoarthritis and healthy subjects: a cross sectional study.

BACKGROUND: YKL-40 is a 40 kDa glycoprotein secreted by chondrocytes and synoviocytes. It has been suggested that it is a surrogate marker of synovial inflammation and joint destruction in rheumatoid arthritis (RA) and osteoarthritis (OA) and related to C reactive protein (CRP) serum levels in RA. OBJECTIVE: To study serum levels of YKL-40 in patients with hip OA and its relation with CRP. METHODS: YKL-40 and CRP were assayed in serum samples from 45 patients (24 women, 21 men, mean age 65) with symptomatic OA of the hip and 33 healthy controls. YKL-40 was assayed by immunoassay and CRP by ultrasensitive immunonephelometry. OA severity was assessed by the measurement of joint space width with a computer analysis system of digitised hip radiographs. Statistical analysis was performed to determine correlations between serum markers and radiological joint space width. RESULTS: The mean (standard error) YKL-40 level was 90.3 (8.2) ng/ml in patients with hip OA and 66.9 (8.2) ng/ml in controls (p=0.03). The mean CRP level was 2.93 (3.03) mg/l in OA and 1.40 (1.61) mg/l in controls (p=0.006). The serum levels of YKL-40 and CRP increased with age and were significantly correlated (Spearman test: r(s)=0.42, p=0.005) in patients but not in controls. Neither YKL-40 nor CRP correlated with radiographic joint space width. CONCLUSIONS: Serum YKL-40 was significantly increased in patients with hip OA. The correlation between YKL-40 and CRP suggests that YKL-40 may be a marker of joint inflammation in OA. Longitudinal studies are required to assess the usefulness of YKL-40 in the monitoring of patients with hip OA.

[Standardization and automation of HLA B27 typing by flow cytometry: validation and comparison with microlymphocytotoxicity]. / Standardisation et automatisation du typage HLA B27 par une technique cytofluorimétrique: validation par comparaison à la microlymphocytoxicité.

One of the strongest known association between human leukocyte antigen (HLA) phenotype and disease is that of ankylosing spondylitis and HLA-B27. Thus, the determination of HLA-B27 status is an useful tool in the diagnosis of ankylosing spondylitis. To date, the 2 reference methods for HLA typing (microlymphocytotoxicity and molecular biology techniques), are costly in terms of both technician time and materials, and require a great deal of experience. In total, these techniques are not well-suited for routine application in clinical immunology laboratories. Use of flow cytometry has recently been applied for HLA-B27 typing. Nevertheless, it requires an extensive validation protocol. We developed a flow cytometry technique as standardized as possible (whole blood, automated lysing system, automated photomultiplier voltage calibration, definition of thresholds stable with time) and validated our results by comparison with microlymphocytotoxicity. In total, 326 samples were analyzed. We found 99% of concordant results between the 2 techniques, and neither false positive results nor false negative results with flow cytometry could be observed. These results illustrate the reliability of the protocol. It should be remembered that reference technique remains necessary to confirm the few results (< 1%) found in "grey zone" by flow cytometry. Standardization of flow cytometry techniques, as described in this work for HLA B27, seems to be a reasonable goal for the next decade in clinical immunology laboratories.