Association of leptin and insulin with childhood obesity and retinal vessel diameters.

OBJECTIVE: Childhood obesity is associated with an impaired retinal microcirculation. The aim of the study was to investigate the association between specific obesity-related biomarkers, physical fitness and retinal vessel diameters in school children. DESIGN AND SUBJECTS: We studied 381 children aged 10-11 years (body mass index (BMI): 19.3±3.7 kg m(-2)) in a school-based setting. MEASUREMENTS: Anthropometric measurements and blood sampling were conducted using standard protocols for children. The serum biomarkers leptin, adiponectin, insulin as well as interleukin-6 (IL-6) were analyzed. Physical fitness was determined by a six-item-test battery and physical activity by use of a questionnaire. Central retinal arteriolar equivalent (CRAE), central retinal venular equivalent (CRVE) and the arteriolar-to-venular diameter ratio (AVR) were assessed with a non-mydriatic vessel analyzer (SVA-T) using a computer-based program. RESULTS: Compared with normal weight children (n=254), obese children (n=39) showed higher leptin (P<0.001), higher insulin (P<0.001), higher IL-6 (P<0.001) and lower adiponectin levels (P=0.013). Obese children demonstrated wider CRVE (P=0.041) and lower AVR (P<0.001). Higher leptin levels were associated with wider CRVE (P=0.032) and lower AVR (P=0.010), that was BMI dependent. Insulin levels were associated with arteriolar (P=0.045) and venular dilatation (P=0.034) after adjustment for BMI. No significant associations between adiponectin levels, IL-6 levels, physical fitness or physical activity and retinal vessel diameter were observed. Lower leptin levels were independently correlated with higher physical fitness (r=-0.33; P<0.001). CONCLUSION: Leptin and insulin levels are associated with changes of the retinal microcirculation. Especially insulin seems to be a good target marker for the cardiometabolic risk assessment in children since elevated insulin levels are independently associated with microvascular end-organ alterations at an early stage. Lifestyle intervention studies are warranted to examine whether improvement of physical fitness or weight reduction can affect cardiometabolic risk markers and reverse alterations of the retinal microcirculation.

[Do injuries of the upper extremity in geriatric patients end up in helplessness? A prospective study for the outcome of distal radius and proximal humerus fractures in individuals over 65]. / Frakturen der oberen Extremität beim geriatrischen Patienten - Harmlose Monoverletzung oder Ende der Selbstständigkeit? Eine prospektive Studie zum Outcome nach distaler Radius- und proximaler Humerusfraktur bei über 65-jährigen.

In a prospective study 104 patients >or=65 years with distal radius fractures (DRF; n=52) and proximal forearm fractures (PHF; n=52) were followed up for a period of 4 months after injury. As an inception- cohort study, influence on treatment pattern was not part of the examination. A total of 53% of the DRF and 74% of the PHF patients underwent surgery. There were no significant changes in the ability of daily living management (IADL) with either fracture form. Functional outcome was better in PHF than DRF patients. PHF patients showed a high incidence in "fear of falling" throughout the whole study, whereas fear of falling rose significantly in DRF patients. 4% of DRF and 9.6% of PHF patients died during the observation period, while 6% of DRF and even 17% of PHF patients had to give up their own housekeeping. One third of both patient groups did not receive physiotherapy. In only 12% of DRF and 6% of PHF patients was osteoporosis treated. In both groups of patients there was a significant worsening in the ability of walking after injury, leading to two or more new falls in 24% of DRF and 28% of PHF patients.

Identification of yeasts associated with milk products using traditional and molecular techniques.

An integrated approach including phenotypic (morphological, biochemical and physiological characterization) and genotypic (RAPD-PCR, sequencing of D1/D2 domain of 26S rRNA encoding gene) methods was used for the identification of yeasts isolated from different milk products. There were 513 isolates in all, 460 ascomycetous and 53 basidiomycetous yeasts. The yeast isolates were characterized on the basis of their biochemical and physiological properties, and the D1/D2 domain of 26S rDNA was sequenced in selected strains. Relying on the obtained results from both the data-sets, corresponding type strains were selected and compared with the respective yeast isolates from milk products by RAPD fingerprinting. The strains showing a degree of similarity >80% were considered conspecific. By means of the applied techniques it was possible to identify 92% yeast isolates at species level. Debaryomyces hansenii, Geotrichum candidum, Kluyveromyces marxianus, Yarrowia lipolytica and Candida zeylanoides are the most frequently isolated species. The majority of the yeasts were isolated from fresh and sour curd cheese. A comparison of the results obtained by phenotypic and genotypic investigation revealed that the identification based on classical methods was supported by genotypic characterization in only 54% of examined isolates. The results described in this work show that the applied molecular identification is a reliable approach to the identification of yeasts associated with milk products in contrast to the conventional biochemical and physiological tests. The identification of new yeast species requires additional genetic markers such as sequencing of different genes or DNA:DNA hybridization.

Characterization of Listeria monocytogenes isolates from 50 small-scale Austrian cheese factories.

One hundred eighty-one small-scale cheese factories (annual production < 100,000 kg) were tested for the presence of Listeria monocytogenes in cheese and smear samples from 1997 to 2000. In total, 2615 samples were drawn. Fifty (27.6%) of 181 enterprises yielded L. monocytogenes. From 14 of the cheese-making facilities, we obtained more than four L. monocytogenes isolates. A total of 182 mostly cheese- and smear-borne L. monocytogenes strains were characterized by serotyping and pulsed-field gel electrophoresis. In 12 of 14 cheese factories, over half of the L. monocytogenes isolates were genetically indistinguishable by pulsetype. On average, genetically indistinguishable isolates were recovered for 11.9 months. Regarding serotypes, 27.3% of the isolates were of serovar 4b. Inadequate personal hygiene could explain the high prevalence of serovar 4b isolates in small-scale cheesemaking facilities. Forty-two percent of the serovar 4b isolates recovered from epidemiologically unlinked facilities (in comparison to 40 and 29% of the 1/2a and 1/2b isolates, respectively) were genetically indistinguishable from at least one other isolate. Indistinguishable serovar 1/2a and 1/2b isolates belonged to five and six different pulsetypes, respectively, whereas serovar 4b isolates belonged to only two pulsetypes. This finding suggested a wide distribution of genetically homologous serovar 4b isolates among the facilities tested in our study.

Ex vivo analysis of phenotype and TCR usage in relation to CD45 isoform expression on cytomegalovirus-specific CD8+ T lymphocytes.

Human cytomegalovirus (CMV) is a ubiquitous pathogen which sets up a lifelong persistent infection and which can lead to significant disease in the immunosuppressed. The immunological mechanisms controlling CMV in the long term are not defined completely, but CD8+ T lymphocytes are thought to play an important role. Antiviral CD8+ T lymphocytes may exist in very large pools in healthy individuals. Although the detailed composition of these pools is not completely understood, there is known to be heterogeneity, in particular of CD45 isoform expression. We have therefore investigated the CD8+ T-lymphocyte response against CMV directly ex vivo using Class I tetramers combined with stains for a range of phenotypic markers followed by four-colour flow cytometric analysis. In particular, we examined expression of these phenotypic markers in relation to the expression of CD45 isoforms. We found that a spectrum of phenotypes exists stably, from CD45R0(high)/RA(low) through CD45RA(high)/R0(low), and that expression of other surface markers such as CD28 and CD62L, and also TCR usage, may vary in parallel with CD45 isoform expression. In some individuals, expansions of antigen-specific CD8+ T lymphocytes bearing specific TCR Vbeta chains were restricted to cells of particular CD45 isoforms. Immunity against CMV comprises a large population of CD8+ T lymphocytes with heterogeneous potential, a spectrum in which CD45 isoform expression may play a central role.

Direct ex vivo comparison of the breadth and specificity of the T cells in the liver and peripheral blood of patients with chronic HCV infection.

The role of intrahepatic lymphocytes in the control of hepatitis C virus (HCV) infection and the pathology associated with it is not understood; most studies of the immunology of this infection use peripheral blood lymphocyte populations. To address this further, we examined in detail the IHL from HCV-infected patients and controls, focusing on the antigen-specific CD8(+) T lymphocyte component. Individual T cells from needle liver biopsies and peripheral blood were isolated from patients with chronic HCV infection and examined directly ex vivo. We used RT-PCR spectratyping to compare the breadth of the T cell receptor usage in the liver in comparison with the peripheral blood, and applied MHC class I tetramer technology to investigate the numbers of HCV-specific CD8(+) cells in the two compartments. T cell receptor usage in the liver of HCV-infected patients was broad, comparable with that in the peripheral blood of the same patients. A much higher proportion of liver CD8(+) cells expressed receptors specific for HCV antigens compared with paired peripheral blood CD8(+) cells. A greater proportion of the liver tetramer-positive cells expressed the activation marker CD69, compared with those in the periphery or other CD8(+) cells in the liver. In the course of chronic HCV infection, HCV-specific CD8 cells, which have been recently activated, appear to accumulate specifically in the livers of infected patients but are present in much lower numbers in the peripheral circulation. Further studies are needed to determine the function of these cells and their role in protection and immunopathology.

Sustained dysfunction of antiviral CD8+ T lymphocytes after infection with hepatitis C virus.

Hepatitis C virus (HCV) sets up persistent infection in the majority of those exposed. It is likely that, as with other persistent viral infections, the efficacy of T-lymphocyte responses influences long-term outcome. However, little is known about the functional capacity of HCV-specific T-lymphocyte responses induced after acute infection. We investigated this by using major histocompatibility complex class I-peptide tetrameric complexes (tetramers), which allow direct detection of specific CD8+ T lymphocytes ex vivo, independently of function. Here we show that, early after infection, virus-specific CD8+ T lymphocytes detected with a panel of four such tetramers are abnormal in terms of their synthesis of antiviral cytokines and lytic activity. Furthermore, this phenotype is commonly maintained long term, since large sustained populations of HCV-specific CD8+ T lymphocytes were identified, which consistently had very poor antiviral cytokine responses as measured in vitro. Overall, HCV-specific CD8+ T lymphocytes show reduced synthesis of tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma) after stimulation with either mitogens or peptides, compared to responses to Epstein-Barr virus and/or cytomegalovirus. This behavior of antiviral CD8+ T lymphocytes induced after HCV infection may contribute to viral persistence through failure to effectively suppress viral replication.

CD8+ cytotoxic lymphocyte responses against cytomegalovirus after liver transplantation: correlation with time from transplant to receipt of tacrolimus.

The effects of the immunocompromised state after liver transplantation on the frequency of cytomegalovirus-specific cytotoxic T lymphocytes (CTL) were investigated in 93 patients by using HLA class I tetrameric complexes corresponding to HLA-A*0201, HLA-B*0702, HLA-B*0801, and HLA-B*3501 refolded with peptides from the ppUL83 matrix protein. ppUL83 CTL frequencies were suppressed during the first 6 months after transplantation. Patients with >1 HLA-restricted response detected had high correlation among ppUL83 CD8(+) CTL frequencies restricted by different HLA haplotypes (Spearman's rho=.67; P<.0001). There was an inverse correlation among levels of the calcineurin inhibitor, tacrolimus, and ppUL83 CD8(+) CTL frequencies (r=-.31; P=.005), which is consistent with the presence of a large proportion (70%) of activated (CD38(+)) ppUL83 CD8(+) CTL within the population of HLA class I tetramer-positive cells.

Studies of human antiviral CD8+ lymphocytes using class I peptide tetramers.

Understanding the interactions between a host and a pathogen relies crucially on quantitative measurements of immune responses. Until recently, measurements of the levels of cellular immune responses, i.e. those mediated by CD4+ and CD8+ T lymphocytes have depended largely on culture in vitro and subsequent measurement of specific functions (such as cytolysis). More recently, new technologies based around tetrameric class I peptide complexes (tetramers) have allowed immunologists to measure CD8+ T lymphocyte levels directly ex vivo and independently of function. Since CD8+ lymphocytes play a key role in a number of important human viral infections, these tools have yielded useful insights into the dynamics, phenotype and function of human antiviral lymphocyte populations. In this review we describe some of the basic aspects of the biology of virus-specific CD8+ lymphocytes, and the current methods available to detect them. The use of tetramers has, in just four years, transformed our understanding of the immune responses against HIV, HTLV-1, HBV, HCV, CMV and EBV, and holds promise in a number of areas where quantitative analysis of the antiviral response in terms of both number and function is critical.

CD8+ T lymphocyte responses are induced during acute hepatitis C virus infection but are not sustained.

Cellular immune responses are likely to play a key role in determining the clinical outcome in acute infection with hepatitis C virus (HCV), but the dynamics of such responses and their relationship to viral clearance are poorly understood. In a previous study we have shown highly activated, multispecific cytotoxic T lymphocyte responses arising early and persisting in an individual who subsequently cleared the virus. In this study the HCV-specific CD8+ lymphocytes response has been similarly analyzed, using peptide-HLA class I tetramers, in a further nine individuals with documented acute HCV infection, six of whom failed to clear the virus. Significant populations of virus-specific CD8+ lymphocytes were detected at the peak of acute hepatic illness (maximally 3.5% of CD8+ lymphocytes). Frequencies were commonly lower than those seen previously and were generally not sustained. Early HCV-specific CD8+ lymphocytes showed an activated phenotype in all patients (CD38+ and HLA class II+), but this activation was short-lived. Failure to sustain sufficient numbers of activated virus-specific CD8+ lymphocytes may contribute to persistence of HCV.

Antibodies to the junctional adhesion molecule cause disruption of endothelial cells and do not prevent leukocyte influx into the meninges after viral or bacterial infection.

A hallmark of infectious meningitis is the invasion of leukocytes into the subarachnoid space. In experimental meningitis triggered by tumor necrosis factor-alpha and interleukin-1beta, the interaction of leukocytes with endothelial cells and the subsequent migration of the cells through the vessel wall can be inhibited by an antibody to the junctional adhesion molecule (JAM). In contrast to the cytokine-induced meningitis model, anti-JAM antibodies failed to prevent leukocyte influx into the central nervous system after infection of mice with Listeria monocytogenes or lymphocytic choriomeningitis virus. Furthermore, in bacterial meningitis, anti-JAM IgG antibodies, but not Fab fragments, caused disruption of the endothelium. Likewise complement-dependent antibody-mediated cytotoxicity was observed in cultured brain endothelial cells treated with anti-JAM IgG but not with its Fab fragment.

Characterization of a novel respiratory syncytial virus-specific human cytotoxic T-lymphocyte epitope.

Respiratory syncytial virus (RSV) infection is a major cause of morbidity in childhood worldwide. The first human RSV-specific cytotoxic T-lymphocyte epitope to be defined is described. This HLA B7-restricted epitope in nucleoprotein (NP) was detectable in four healthy, B7-positive adult subjects using B7-RSV-NP tetrameric complexes to stain CD8(+) T cells.

Analysis of successful immune responses in persons infected with hepatitis C virus.

Although hepatitis C virus (HCV) infection is very common, identification of patients during acute infection is rare. Consequently, little is known about the immune response during this critical stage of the disease. We analyzed the T lymphocyte response during and after acute resolving HCV infection in three persons, using interferon (IFN)-gamma enzyme-linked immunospot (ELISPOT) and human histocompatibility leukocyte antigen (HLA) peptide tetramer assays. Acute infection was associated with a broadly directed T helper and cytotoxic T lymphocyte (CTL) response, which persisted after resolution of clinical hepatitis and clearance of viremia. At the earliest time point studied, highly activated CTL populations were observed that temporarily failed to secrete IFN-gamma, a "stunned" phenotype, from which they recovered as viremia declined. In long-term HCV-seropositive persons, CTL responses were more common in persons who had cleared viremia compared with those with persistent viremia, although the frequencies of HCV-specific CTLs were lower than those found in persons during and after resolution of acute HCV infection. These studies demonstrate a strong and persistent CTL response in resolving acute HCV infection, and provide rationale to explore immune augmentation as a therapeutic intervention in chronic HCV infection.

Life, activation and death of intrahepatic lymphocytes in chronic hepatitis C.

The healthy liver of adult humans has little or no lymphocyte component and the histological finding of intrahepatic lymphocytes (IHL) is evidence of liver pathology. In a liver injured by chronic hepatitis C, the most common chronic liver disease, most IHL are activated/pro-inflammatory cells, which are particularly enriched for effectors of innate immunity (natural killer (NK), natural T, and other NK-like T cells). IHL do not undergo clonal expansion in the liver but migrate from extrahepatic sites to the chronically infected liver, where they display effector function and subsequently die, suggesting that maintenance of the IHL pool depends on continuous lymphocyte migration. The cytotoxic and inflammatory functions of these IHL have three potential outcomes: 1) they could be helpful in clearing the virus (a rare case in hepatitis C virus (HCV) infection); 2) they could be useless and have no effect on the infection; or 3) they could be harmful, whereby overaggressive lymphocyte responses destroy the liver in a continuous and unsuccessful attempt to clear the virus. Unfortunately, we do not know as of yet which of these possibilities is the case and, therefore, a more complete picture of the intrahepatic immune response will be relevant to the development of new therapeutic strategies against HCV. Additionally and from a more general perspective, due to the availability of biopsied material and the high prevalence (approximately 3%) of HCV infection worldwide, studying the chronically inflamed liver of hepatitis C patients is an ideal model to investigate the poorly understood processes of lymphocyte trafficking, activation and death to non-lymphoid sites of chronic inflammation in man.

Why do cytotoxic T lymphocytes fail to eliminate hepatitis C virus? Lessons from studies using major histocompatibility complex class I peptide tetramers.

Hepatitis C virus (HCV) infection is a major public health problem, affecting an estimated 3% of the world's population, and over 10% in some countries. Infection in most cases becomes persistent, and can lead to hepatic inflammation, fibrosis and liver failure. The T lymphocyte reponse, in particular that mediated by cytotoxic T lymphocytes (CTLs), is likely to be involved in determining the outcome of infection, although its overall role is not clear. The use of major histocompatibility complex (MHC) class I peptide tetrameric complexes (tetramers) to study antiviral CTL responses has revolutionized our approach to the study of human infection. We have used a panel of MHC class I tetramers to analyse immune responses in HCV-infected individuals at various stages of disease. We find that the CTL response against HCV is vigorous in its early phases but dwindles over time both in terms of lymphocyte number and function. A number of potential explanations for this 'CTL failure' are discussed.

Growth of facultatively heterofermentative lactobacilli on starter cell suspensions.

The growth of facultatively heterofermentative lactobacilli (FHL) on cell suspensions of the homofermentative Lactobacillus helveticus was investigated. Osmotic lysis of L. helveticus led to a significant increase of ribose. It decreased steadily in parallel with the growth of FHL, strongly suggesting that the bacteria used ribose as a growth substrate.

Specificity of CTL interactions with peptide-MHC class I tetrameric complexes is temperature dependent.

Tetrameric peptide-MHC class I complexes ("tetramers") are proving invaluable as reagents for characterizing immune responses involving CTLs. However, because the TCR can exhibit a degree of promiscuity for binding peptide-MHC class I ligands, there is potential for cross-reactivity. Recent reports showing that the TCR/peptide-MHC interaction is dramatically dependent upon temperature led us to investigate the effects of incubation temperature on tetramer staining. We find that tetramers rapidly stain CTLs with high intensity at 37 degrees C. We examine the fine specificity of tetramer staining using a well-characterized set of natural epitope variants. Peptide variants that elicit little or no functional cellular response from CTLs can stain these cells at 4 degrees C but not at 37 degrees C when incorporated into tetramers. These results suggest that some studies reporting tetramer incubations at 4 degrees C could detect cross-reactive populations of CTLs with minimal avidity for the tetramer peptide, especially in the tetramer-low population. For identifying specific CTLs among polyclonal cell populations such as PBLs, incubation with tetramers at 37 degrees C improves the staining intensity of specific CTLs, resulting in improved separation of tetramer-high CD8+ cells. Confocal microscopy reveals that tetramers incubated at 37 degrees C can be rapidly internalized by specific CTLs into vesicles that overlap with the early endocytic compartment. This TCR-specific internalization suggests that coupling of tetramers or analogues with toxins, which are activated only after receptor internalization, may create immunotoxins capable of killing CTLs of single specificities.

Phenotypic and genotypic identification of yeasts from cheese.

Eighty-five years strains isolated from different cheeses of Austria, Denmark, France, Germany, and Italy were identified using physiological methods and genotypically using random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis. Good congruence was found between the phenotypic and genotypic data for 39 of the isolates. However, 26 isolates of Geotrichum could only be identified to the species level using the genotypic methods and 7 isolates were correctly identified to the genus level only using phenotypic identification methods. The phenotypic identification did not agree with the genotypic data for 14 yeast isolates. Using ubiquinone analysis, yeast cell wall sugars and the diazonium blue B test 5 incorrectly identified isolates with phenotypic methods could be identified genotypically. In addition the 7 isolates identified only to the genus level by the phenotypic methods and the 26 Geotrichum strains were identified to the species level using the polyphasic molecular approach mentioned above. Eleven strains remained unidentified. The 76 identified yeast isolates were assigned to 39 species, the most frequent assignments were made to Debaryomyces hansenii, Geotrichum candidum, Issatchenkia orientalis, Kluyveromyces lactis, K. marxianus, Saccharomyces cerevisiae, Yarrowia lipolytica, and Candida catenulata. It is proposed that Debaryomyces hansenii (Zopf) Lodder et Kregervan Rij and Debaryomyces fabryi Ota should be reinstated. The RAPD-PCR data reinforced the view that the species Galactomyces geotrichum is heterogeneous with all of the Geotrichum isolates from cheese products being assigned G. geotrichum group A sensu M.T. Smith. It is suggested that the name Geotrichum candidum be conserved for this rather common species.

Inducible nitric oxide synthase is expressed in joints of goats in the late stage of infection with caprine arthritis encephalitis virus.

We have studied the expression of the inducible form of nitric oxide synthase (iNOS) in joints of goats infected with the caprine arthritis encephalitis virus (CAEV). Nitric oxide generated by iNOS is thought to play an important role in the pathogenesis of various types of arthritis, especially rheumatoid arthritis (RA) in humans. Surprisingly, iNOS immunoreactivity was found only in joints of long-term infected goats with severe clinical arthritis, whereas-despite the presence of high numbers of inflammatory cells in the synovial tissue-no iNOS immunoreactivity was detected in mildly arthritic and in short-term experimentally infected goats. Most iNOS-positive cells expressed neither MHC class II nor CD68, which suggests that they were fibroblast-like synoviocytes. In situ hybridization studies showed that there was no correlation between iNOS immunoreactivity and detectable virus expression in the joint. In addition, infection of macrophages in vitro-the major host cells of CAEV in vivo-did not lead to increased iNOS mRNA expression. In response to stimulation, similar levels of iNOS expression were observed in infected and in uninfected macrophages. These findings suggest that the expression of iNOS is a feature of late-stage chronic arthritis and is not involved in the development of the inflammatory lesions. Both the lack of co-localization of iNOS protein and viral transcripts in the joint and the finding that CAEV does not stimulate the expression of iNOS in vitro further suggest that iNOS is not directly induced by the virus or the anti-viral immune response in the joint, that it may well, however, be involved in tissue remodelling or scar formation.