The HLA-DP2 protein binds the immunodominant epitope from myelin basic protein, MBP85-99, with high affinity

Myelin basic protein (MBP) is a candidate autoantigen in multiple sclerosis (MS). The immunodominant epitope for T-cell responses is assigned to the amino acid sequence MBP84-102, which binds to human leukocyte antigen (HLA)-DR2a (DRB5*0101) and HLA-DR2b (DRB1*1501) of the HLA-DR2 haplotype carrying the strongest genetic association with MS. In contrast with HLA-DR and -DQ molecules, HLA-DP molecules are poorly characterized with respect to the binding of self-peptides. We show here that HLA-DP2 binds MBP85-99 with high affinity, and that the amino acid residues in position MBP91, MBP92 and MBP93 are influencing the binding, as shown by alanine scans. We further used a series of truncated peptides to identify the core of the binding. Moving the frame along the peptide from residues 87-97 to 89-99 progressively decreased the binding affinity for HLA-DP2, while moving further towards the C-terminal completely abrogated the binding of peptides to HLA-DP2. The data suggest that the docking of the MBP85-99 peptide into the HLA-DP2 groove is dependent on MBP88V and MBP89V and may use either of them as primary anchor for the p1 position. HLA-DP2 might thus present the MBP85-99 peptide in the same register as the HLA-DRB1*1501, where the MBP89V is preferred as the p1 anchor. Notably, full-length MBP was able to compete for peptide binding with an affinity similar to that seen for the high-affinity binding peptides, DRα170-83 and IIP53-65. In summary, the HLA-DP2 molecule binds the immunodominant epitope in MS, MBP85-99, possibly in more than one register.     

Chromosome painting in Tragulidae facilitates the reconstruction of Ruminantia ancestral karyotype

Although Tragulidae, as the basal family in Ruminantia phylogenetic tree, is the key taxon for understanding the early chromosome evolution of extant ruminants, comparative molecular cytogenetic data on the tragulids are scarce. Here, we present the first genome-wide comparative map of the Java mouse deer (Tragulus javanicus, Tragulidae) revealed by chromosome painting with human and dromedary probes. Together with the published comparative maps of major representative cetartiodactyl species established with the same set of probes, our results allowed us to reconstruct a 2n = 48 Ruminantia ancestral karyotype, which is similar to the cetartiodactyl ancestral karyotype. The karyotype evolution of T. javanicus has involved multiple rearrangements, most of which appear to be apomorphic and have not found in karyotype evolution of pecoran species (i.e., Ruminantia excluding Tragulidae). The rate of chromosome evolution of the mouse deer was rather low—0.4 R/Ma, while the estimated tempo of chromosome changes on the lineages leading from Cetartiodactyla ancestor to Ruminantia and from Ruminantia to Pecora were roughly the same (about 1.2 R/Ma).     

Diagnosis of autoimmune pancreatitis by core needle biopsy: application of six microscopic criteria

Autoimmune pancreatitis (AIP) has been established as a special entity of chronic pancreatitis (CP). However, its clinical distinction from pancreatic cancer and other types of CP is still difficult. The aim of this study was to evaluate the efficacy of pancreatic core needle biopsy for the diagnosis of AIP. In 44 core needle biopsy specimens, we assessed the following microscopic features: granulocytic epithelial lesions (GELs), more than ten IgG4-positive plasma cells/HPF, more than ten eosinophilic granulocytes/HPF, cellular fibrosis with inflammation, lymphoplasmacytic infiltration, and venulitis. All biopsies that showed four or more of the six features (22 of 44) were obtained from 21 of 26 patients whose clinical diagnosis and follow-up were consistent with AIP. All non-AIP CP patients (n = 14) showed three or less than three of the features in their biopsies. GELs were only observed in biopsy specimens from AIP patients. In conclusion, our data indicate that the six criteria we applied were able to recognize AIP in 76% of biopsy specimens using a cut-off level of four. When the specimens that revealed only three features but showed GELs were added, the sensitivity rose to 86%. Pancreatic core needle biopsy can therefore make a significant contribution to the diagnosis of AIP.     

Kinetics of Antibody Responses in Rickettsia africae and Rickettsia conorii Infections

African tick-bite fever, caused by Rickettsia africae, is the most common tick-borne rickettsiosis in sub-Saharan Africa. Mediterranean spotted fever due to Rickettsia conorii also occurs in the region but is more prevalent in Mediterranean countries. Using microimmunofluorescence, we compared the development of immunoglobulin G (IgG) and IgM titers in 48 patients with African tick-bite fever and 48 patients with Mediterranean spotted fever. Doxycycline treatment within 7 days from the onset of disease significantly prevented the development of antibodies to R. africae. In patients with African tick-bite fever, the median times to seroconversion with IgG and IgM were 28 and 25 days, respectively, after the onset of symptoms. These were significantly longer by a median of 6 days for IgG and 9 days for IgM than the times for seroconversion in patients with Mediterranean spotted fever (P < 10⁻²). We recommend that sera collected 4 weeks after the onset of signs of patients with suspected African tick-bite fever should be used for the definitive serological diagnosis of R. africae infections.     

In vitro susceptibility of Microsporum canis and other dermatophyte isolates from veterinary infections during therapy with terbinafine or griseofulvin.

We investigated the in vitro activity of terbinafine against fresh veterinary isolates of Microsporum canis and the potential of this organism to develop resistance in vivo during oral therapy. Dermatophyte cultures (n = 300) were obtained from naturally infected cats and dogs undergoing oral therapy with terbinafine or griseofulvin. M. canis comprised 92% of isolates; other species included Microsporum gypseum and Trichophyton mentagrophytes. Minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) of terbinafine and griseofulvin were determined by broth macrodilution assay. Terbinafine was highly active against all three species with MIC90< or =0.03 microg ml(-1), in agreement with published data. However, terbinafine exhibited primary cidal activity against 66% of Microsporum isolates (n = 275) in contrast to the almost complete cidal effect in Trichophyton (n = 18). Griseofulvin was significantly less active than terbinafine (MIC90 = 4 microg ml(-1)) but had a primary cidal action on about 40% of the isolates. The data were analysed for changes in MIC and MFC during the course of therapy, which could be indicative for development of acquired resistance. Oral treatment of 37 animals with terbinafine for up to 39 weeks caused no increase in MIC or MFC of terbinafine, either in individual patients or in the whole group.     

Demonstration of strong enterobacterial reactivity of CD4+CD25- T cells from conventional and germ-free mice which is counter-regulated by CD4+CD25+ T cells

Unfractionated CD4+ T cells from the gut-associated lymphoid tissue (GALT) and peripheral lymph nodes are unresponsive when exposed to enterobacterial antigens in vitro. Under similar conditions, CD4+ T cells depleted in vivo or in vitro of CD4+CD25+ T cells proliferate extensively. The CD4+CD25- T cell reactivity depends on MHC class II presentation, specific TCR stimulation, CD4 ligation, and antigen processing by antigen-presenting cells. The CD4+CD25- T cells respond to autologous and heterologous enterobacterial antigens, but not to antigens from the feces of germ-free mice. Surprisingly, CD4+CD25- T cells obtained from the GALT of germ-free mice also proliferate when exposed to enterobacterial antigens, and adding back the conventional or germ-free CD4+CD25+ T cells to the enteroantigen-stimulated CD4+CD25- T cells abolishes proliferation. As judged from carboxyfluorescein diacetate succinimidyl ester-labeling experiments, 4-5% of the CD4+CD25- T cells respond to enteroantigen. The data show for the first time that CD4+CD25- T cells with reactivity towards the enterobacterial flora and regulatory CD4+CD25- T cells are present in both conventional and germ-free mice. The data suggest that a significant proportion of the peripheral pool of CD4+CD25- T cells express anti-enterobacterial reactivity, which, due to the presence of regulatory CD4+CD25+ T cells, is kept in a quiescent state.     

Black Hispanics have a worse cardiovascular risk profile than mixed Hispanics in Venezuela

In order to characterize components of the metabolic syndrome (MS) in Venezuelan black Hispanics and compare these metabolic abnormalities with those found in the predominant mixed Hispanic population, 2336 mixed Hispanics (69% women) and 281 black Hispanics (60% women), aged 20-78 years, without prior history of diabetes and/or cardiovascular disease were evaluated in a population-based study in Zulia State, Venezuela. Blood pressure (BP), waist circumference, as well as fasting insulin, fasting blood glucose (FBG), triglycerides (TG) and high-density lipoprotein cholesterol (HDL-C) levels were measured. The criteria proposed by the National Cholesterol Education Program/Adult Treatment Panel III (NCEP/ATP III) to identify those with metabolic abnormalities were used. We found that black Hispanics showed higher frequency of age-adjusted elevated BP than mixed Hispanics in both men (66.9% vs. 52.3%, p < 0.01) and women (39.3% vs. 30.4%, p < 0.05). In men, elevated FBG was also more frequent in black Hispanics (32.7%) than in mixed Hispanics (22.3%), despite the lack of significant differences in fasting insulin, HOMA-insulin resistance and HOMA-beta cell function values. In women low HDL-C and higher abdominal obesity were more common in black Hispanics (71.8% and 54.1%, respectively) than in mixed Hispanics (56.2% and 44.5%, respectively), despite the greater frequency of high TG in mixed Hispanics (22.6%) when compared to black Hispanics (13.3%). Furthermore, in logistic regression analysis black Hispanic race was independently associated with higher risk for hypertension, fasting hyperglycemia, and low HDL-C. These results suggest that black Hispanics have worse cardiovascular risk profile than mixed Hispanics in Zulia State, with higher BP, higher FBG, more abdominal obesity, and lower HDL-C. Identification and intervention of these high-risk subjects are important strategies for diabetes and cardiovascular disease prevention in Venezuela.     

Assessment of Clostridium difficile Infections by Quantitative Detection of tcdB Toxin by Use of a Real-Time Cell Analysis System

We explored the use of a real-time cell analysis (RTCA) system for the assessment of Clostridium difficile toxins in human stool specimens by monitoring the dynamic responses of the HS27 cells to tcdB toxins. The C. difficile toxin caused cytotoxic effects on the cells, which resulted in a dose-dependent and time-dependent decrease in cell impedance. The RTCA assay possessed an analytical sensitivity of 0.2 ng/ml for C. difficile toxin B with no cross-reactions with other enterotoxins, nontoxigenic C. difficile, or other Clostridum species. Clinical validation was performed on 300 consecutively collected stool specimens from patients with suspected C. difficile infection (CDI). Each stool specimen was tested in parallel by a real-time PCR assay (PCR), a dual glutamate dehydrogenase and toxin A/B enzyme immunoassay (EIA), and the RTCA assay. In comparison to a reference standard in a combination of the three assays, the RTCA had a specificity of 99.6% and a sensitivity of 87.5% (28 of 32), which was higher than the EIA result (P = 0.005) but lower than the PCR result (P = 0.057). In addition, the RTCA assay allowed for quantification of toxin protein concentration in a given specimen. Among RTCA-positive specimens collected prior to treatment with metronidazole and/or vancomycin, a significant correlation between toxin protein concentrations and clinical CDI severities was observed (R² = 0.732, P = 0.0004). Toxin concentrations after treatment (0.89 ng/ml) were significantly lower than those prior to the treatment (15.68 ng/ml, Wilcoxon P = 0.01). The study demonstrates that the RTCA assay provides a functional tool for the potential assessment of C. difficile infections.Clostridium difficile is recognized as the leading cause of infectious diarrhea that develops in patients after hospitalization and/or in patients receiving antibiotic treatment (3, 12). Moreover, the recently emerged, highly virulent strain BI-NAP1-027 has been associated with increased morbidity and mortality (14, 16). A definitive diagnosis depends on the detection of C. difficile-specific toxin B production in the laboratory, which allows for prompt treatment and isolation procedures to prevent further nosocomial spread of infection (12, 19). There are a number of methods available that have been used for the laboratory diagnosis of C. difficile infection (CDI). The well-accepted standard is cytotoxigenic culture, which is conducted by culturing C. difficile from the stool and then performing a cytotoxin assay on the isolate (21). This standard is labor-intensive, subjective, and time-consuming and therefore is not widely used in the clinical setting. Several enzyme immunoassays (EIAs) detect C. difficile antigens in stool, including glutamate dehydrogenase (GDH), as well as toxins A and B (7, 19, 20, 22-24, 28, 29). PCR-based molecular assays that detect toxin A or B, or both, have shown promising performance (4, 10, 18, 24, 30).Currently, recommended therapies for CDI include oral administration of metronidazole and/or vancomycin for 10 to 14 days (6). However, increased percentages of patients experience infection relapse after completion of treatment (3, 12, 17). The emergence and spread of resistance in C. difficile are complicating treatment and prevention (9). While new antibiotics and therapeutic methods are available, providing a rapid and accurate laboratory tool for monitoring disease severity and therapy efficacy is clinically desirable. The C. difficile toxin assay, which detects cell toxicity caused by toxin B, is a direct determination of whether a functional C. difficile toxin exists in stool. However, this assay is labor- and time-intensive and provides only qualitative results.A real-time cell analysis (RTCA) assay (ACEA Biosciences, San Diego, CA) was developed for monitoring the cell status using electronic impedance technology. Utilizing a dimensionless parameter called the cell index (CI), the RTCA system detects the changes to the cell layers cultured on gold microelectrodes on the glass substrates integrated in the bottom of the microelectronic plates (25). This technology has been applied in a number of cell-based assays, including cytotoxicity, cell adhesion and spreading, functional monitoring of receptor-mediated signaling, and cell invasion and migration (2, 11, 31, 32). In this study, we adapt the system for assessment of C. difficile toxin directly from stool specimens. Analytical and diagnostic sensitivities and specificities of this system for the diagnosis and monitoring of CDI were determined. We also explored the assessment of clinical CDI severities and therapeutic efficacies by using toxin concentrations in stool specimens as determined by the RTCA assay.(This study was presented in part at the 110th American Society for Microbiology Annual Meeting, San Diego, CA, 23 to 27 May 2010.)     

A hybrid form of myeloid/NK-cell acute leukemia and myeloid/NK-cell precursor acute leukemia

Natural killer (NK)-cell leukemia/lymphoma is a rare entity that has been defined only in recent years. In the Revised European-American Lymphoma and World Health Organization classifications, only the mature NK-cell malignancies are included. However, at least 3 types of precursor NK-cell neoplasms have been reported in the literature. These include myeloid/NK-cell acute leukemia, myeloid/NK-cell precursor acute leukemia, and blastic NK-cell lymphoma/leukemia. These leukemias are characterized by the presence of blasts, which express CD56, in the peripheral blood, bone marrow, lymph nodes, and/or extranodal tissues. We report a case that is morphologically consistent with myeloid/NK-cell acute leukemia but immunologically is myeloid/NK-cell precursor acute leukemia. This case is unique in its cutaneous presentation without involvement of the peripheral blood. Extensive flow cytometric studies were performed on the skin biopsy and bone marrow aspirate specimens, which included many markers that had not been tested before in these entities. The clinical implications of these findings are discussed.