Immune responses induced by the Leishmania (Leishmania) donovani A2 antigen,but not by the LACK antigen,are protective against experimental Leishmania (Leishmania) amazonensis infection

Leishmania amazonensis is one of the major etiologic agents of a broad spectrum of clinical forms of leishmaniasis and has a wide geographical distribution in the Americas, which overlaps with the areas of transmission of many other Leishmania species. The LACK and A2 antigens are shared by various Leishmania species. A2 was previously shown to induce a potent Th1 immune response and protection against L. donovani infection in BALB/c mice. LACK is effective against L. major infection, but no significant protection against L. donovani infection was observed, in spite of the induction of a potent Th1 immune response. In an attempt to select candidate antigens for an American leishmaniasis vaccine, we investigated the protective effect of these recombinant antigens (rLACK and rA2) and recombinant interleukin-12 (rIL-12) against L. amazonensis infection in BALB/c mice. As expected, immunization with either rA2-rIL-12 or rLACK-rIL-12 induced a robust Th1 response prior to infection. However, only the BALB/c mice immunized with rA2-rIL-12 were protected against infection. Sustained gamma interferon (IFN-gamma) production, high levels of anti-A2 antibodies, and low levels of parasite-specific antibodies were detected in these mice after infection. In contrast, mice immunized with rLACK-rIL-12 displayed decreased levels of IFN-gamma and high levels of both anti-LACK and parasite-specific antibodies. Curiously, the association between rA2 and rLACK antigens in the same vaccine completely inhibited the rA2-specific IFN-gamma and humoral responses and, consequently, the protective effect of the rA2 antigen against L. amazonensis infection. We concluded that A2, but not LACK, fits the requirements for a safe vaccine against American leishmaniasis.     

Endogenous levels of mRNA for IFNs and IFN-related genes in hepatic biopsies of chronic HCV-infected and non-alcoholic steatohepatitis patients

To investigate the intra-hepatic activation of the IFN system in patients affected by chronic HCV-infection in comparison with that observed in a non-infectious liver disease such as non-alcoholic steatohepatitis, we measured the liver steady state mRNA levels of interferon-alpha, interferon-beta and interferon-gamma as well as of IFN-related genes (IFNAR-1, STAT1alpha, PKR, 2-5 AS, IRF-1, ICE and IL-18). In HCV-infected subjects, possible correlations of these parameters with viral load and liver injury were also analyzed. Twenty-four chronic untreated HCV-infected subjects and seven patients with non-alcoholic steatohepatitis were enrolled in the study. Liver biopsies were graded according to Knodell scores. Intra-hepatic mRNA levels of IFNs and related genes were assessed by semi-quantitative RT-PCR. In comparison with non-alcoholic steatohepatitis, in HCV-infected subjects IFN-alpha and -beta mRNA levels were significantly lower, whereas IFN-gamma, IFNAR-1, STAT1alpha IRF-1, and IL-18 mRNA were upregulated. Moreover, IFN-gamma mRNA steady state levels were correlated positively with those of IFNAR-1, IRF-1, and IL-18, suggesting a coordinated induction of these genes. Although plasma viral load was correlated inversely with IL-18-specific mRNA, viral load was not related to liver injury. IFN-gamma and IRF-1 mRNA levels were correlated positively with ALT, but not with the grading or staging. Conversely, IFN-alpha and -beta mRNA levels were higher in livers with lower staging scores. These findings support the hypothesis that in chronic HCV infection there is an imbalance between an upregulated IFN-gamma system and a downregulated IFN-alpha and -beta system, probably due to a mixed effect exerted by HCV-specific and inflammatory non-specific factors.     

BODE-Index vs HADO-Score in Chronic Obstructive Pulmonary Disease: Which one to use in general practice?

Background  

Forced expiratory volume in one second (FEV₁) is used to diagnose and establish a prognosis in chronic obstructive pulmonary disease (COPD). Using multi-dimensional scores improves this predictive capacity.Two instruments, the BODE-index (Body mass index, Obstruction, Dyspnea, Exercise capacity) and the HADO-score (Health, Activity, Dyspnea, Obstruction), were compared in the prediction of mortality among COPD patients.     

Detection of clarithromycin-resistant Helicobacter pylori in stool samples

The recognition of the role of Helicobacter pylori in gastric diseases has led to the widespread use of antibiotics in the eradication of this pathogen. The most advocated therapy, triple therapy, often includes clarithromycin. It is well known that clarithromycin resistance is one of the major causes of eradication failure. The development of a rapid noninvasive technique that could easily be performed on fecal samples and that could also provide information about the antibiotic resistance of this microorganism is therefore advisable. Previous findings have demonstrated that clarithromycin resistance is due to a single point mutation in the 23S rRNA. All the mutations described have been associated with specific restriction sites, namely BsaI (A2143G), MboII (A2142C/G), and HhaI (T2717C). On this basis we have developed a new method, a seminested PCR, allowing screening for clarithromycin resistance of H. pylori directly on stool samples. This method furnished a 783-bp fragment of the 23S rRNA, which was subsequently digested by MboII, BsaI, and HhaI, in order to identify single point mutations associated with clarithromycin resistance. Of a total of 283 stool samples examined, 125 were H. pylori positive and two of them were shown to contain clarithromycin-resistant strains due to the presence of a mutation at position 2717, whereas no PCR products contained mutations at position 2142 or 2143. In order to evaluate the reliability of the new system, we compared the results of restriction analysis of the PCR products with the MICs shown by the H. pylori isolates by culturing gastric biopsies from the same patients.     

Mutations of CD40 ligand in two patients with hyper-IgM syndrome

Two patients with the X-linked form of the hyper-IgM syndrome have been studied. Both patients present: 1. Mutations in the CD40L gene (a nonsense point mutation that introduces a termination codon at the extracellular domain of the protein, and a deletion that eliminates exon 4 as consequence of an abnormal splicing). 2. Lack of CD40L expression on the lymphocyte surface after stimulation with ionomycin and PMA. 3. Altered lymphocytic proliferation in response to anti-CD3. 4. Hyper IgM, low IgG and IgA levels and neutropenia. One of the patients shows, in addition, low Natural Killer cell numbers and severe herpetic infections, which distinguishes this case from the common hyper-IgM syndrome phenotype. Finally, a hyper-IgM stable phenotype has been immortalized by Herpes virus Saimiri for the first time.     

Portal hypertension in Williams syndrome: report of two patients

Williams or Williams-Beuren syndrome (WBS) is a developmental disorder with multisystemic manifestations characterized by distinctive facial features, mental disability with unique cognitive and personality profiles, vascular stenoses, growth retardation, and occasional infantile hypercalcemia, caused by haploinsufficiency for genes deleted in chromosome band 7q11.23. However, with the exception of arterial stenoses caused by haploinsufficiency for the elastin gene (ELN), no specific implication of any other gene in the phenotype has been established. We present two patients with portal hypertension leading to splenomegaly and pancytopenia carrying the common 1.5 Mb WBS deletion. We propose this is an additional severe vascular complication of ELN deficiency and discuss the specific characteristics of the portal venous tract that could explain the impact of ELN deficiency in that venous territory. This complication is potentially lethal and should thus be considered in any patient with WBS and splenomegaly.     

Single step enrichment of blood dendritic cells by positive immunoselection

Dendritic cells (DC) for cancer immunotherapy protocols are generated most commonly by in vitro differentiation of monocytes with exogenous cytokines (Mo-DC). However, Mo-DC differ in their molecular phenotype and function from blood DC (BDC). Clinical isolation of BDC has been limited to the use of density gradients, which result in low yields of variable purity. We have developed a DC enrichment platform, which uses the CMRF-44 (IgM) or CMRF-56 (IgG) monoclonal antibodies (mAb) to select BDC that express these antigens after a short overnight incubation. After culture of peripheral blood mononuclear cells (PBMC) in autologous/AB serum, biotinylated CMRF-44 was used to select DC in a single step immuno-magnetic bead procedure; this produced populations containing up to 99% CMRF-44(+) cells, including up to 67% CMRF-44(+) CD14(-) CD19(-) DC, from an initial starting population of approximately 0.5%. We observed consistent differences in the purities obtained from individual donors with a mean of 54% CMRF-44(+) cells (range 19-99%). Similar results were obtained using biotinylated CMRF-56 mAb, an antibody identifying a comparable population in cultured PBMC. We recovered an average of 54% and 66% of the available BDC in separations performed with the CMRF-44 and CMRF-56 mAb, respectively. The reproducibility of the procedure and the ability to perform it in a closed sterile system makes it suitable for clinical use. Larger scale preparations starting from apheresis derived PBMC will produce sufficient BDC for immunotherapy protocols. The purified BDC elicited strong allogeneic mixed leukocyte reactions and HLA classes II- and I-restricted antigen-specific primary immune responses.     

Increased cyclin D3 expression significantly correlates with p27 nuclear positivity in gastrointestinal stromal tumors

Gastrointestinal stromal tumors, the most common mesenchymal tumors of the gastrointestinal tract, are characterized by strong expression of c-Kit protein. Recently, it has been shown that gastrointestinal stromal tumors may also contain alterations of genes involved in the regulation of cell cycle. In this study, we evaluate the prevalence and clinical significance of cyclin D1 and D3, Ki-67, p27, and retinoblastoma protein expression in a group of 50 human gastrointestinal stromal tumors selected from the files of the Moffitt Cancer Center. Tissue sections from each case were subjected to immunostaining using the avidin-biotin complex method. Cyclin D1 nuclear positivity was detected in 21 of 50 (42%) and cyclin D3 in 24 of 50 (48%) cases. p27 high immunoreactivity and negative or decreased retinoblastoma protein expression were identified in 33 of 50 (66%) gastrointestinal stromal tumors. In 19 of 50 (38%) tumors, Ki-67 had high labeling index. Direct correlation was observed between cyclin D3 and p27 expression (P < .0001), and between cyclin D1 and retinoblastoma protein (P = .03). Coexpression of cyclin D3 and p27 was demonstrated by immunofluorescence. The p27 protein expression inversely correlated with tumor size (P = .004), but was not correlated with tumor grade (P = .12). Ki-67 directly correlated with both tumor size (P = .03) and tumor grade (P = .008). We report a direct correlation between cyclin D3 and p27 expression in gastrointestinal stromal tumors. Additional alterations in cyclin D1, Ki-67, and retinoblastoma protein expression indicate a disregulated cell cycle in these tumors.