Human defensins

Antimicrobial peptides are small, cationic, amphiphilic peptides of 12–50 amino acids with microbicidal activity against both bacteria and fungi. The eukaryotic antimicrobial peptides may be divided into four distinct groups according to their structural features: cysteine-free -helices, extended cysteine-free -helices with a predominance of one or two amino acids, loop structures with one intramolecular disulfide bond, and -sheet structures which are stabilised by two or three intramolecular disulfide bonds. Mammalian defensins are part of the last-mentioned group. The mammalian defensins can be subdivided into three main classes according to their structural differences: the -defensins, -defensins and the recently described -defensins. Mammalian -defensins are predominantly found in neutrophils and in small intestinal Paneth cells, whereas mammalian -defensins have been isolated from both leukocytes and epithelial cells. Recently, two novel human -defensins, human beta-defensin-3 (HBD-3), and human beta-defensin-4 (HBD-4) have been discovered. Similar to HBD-1 and HBD-2, HBD-3 has microbicidal activity towards the Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli) and the yeasts Candida albicans and Malassezia furfur. In addition, HBD-3 kills Gram-positive bacteria such as Streptococcus pyogenes or Staphylococcus aureus, including multi-resistant S. aureus strains, and even vancomycin-resistant Enterococcus faecium. In contrast to HBD-1 and HBD-2, significant expression of HBD-3 has been demonstrated in non-epithelial tissues, such as leukocytes, heart and skeletal muscle. HBD-4 is expressed in certain epithelia and in neutrophils. Its bactericidal activity against P. aeruginosa is stronger than that of the other known -defensins. Here we present an overview of human antimicrobial peptides with some emphasis on their antifungal properties.J.J. Schneider and A. Unholzer contributed equally to this work     

Immunization with recombinant protein: conditions for cytotoxic T cell and/or antibody induction

Safe vaccines should optimally induce both cell-mediated and humoral immunity. Recently, it has been shown that protective cytotoxic T cells (CTLs) can be induced not only with live vaccines, but also with recombinant viral proteins. This report shows in C57BL/6 (H-2^b) mice that the recombinant nucleoprotein (N) of vesicular stomatitis virus (VSV) induced protective CTLs but no neutralizing antibodies in mice, whereas the recombinant glycoprotein (G) of VSV alone induced neutralizing antibodies but no CTLs. If the N and G of VSV were coinjected, both CTLs and a long-lasting neutralizing IgG response was measurable, demonstrating that mixed vaccines can be used to induce protective CTLs and antibodies with an efficiency comparable to live virus. In an attempt to define optimal conditions for CTL priming, the intravenous, intraperitoneal and subcutanous route of injection were compared. Intravenous injection of recombinant VSV-N induced up to 30 times higher responses than the latter two routes. Finally, we tried to define conditions inducing only CTLs and no antibodies binding to the native protein form, or vice versa, only antibodies and no CTLs. Intravenous injection of boiled VSV-N induced a CTL response but no antibodies specific for the native VSV-N, whereas VSV-N injected subcutanously in incomplete Freund's adjuvant induced high amounts of anti-VSV-N antibodies but virtually no CTLs. The conditions defined here permit vaccines to be designed which would function along selected and defined immunological effector pathways.     

Duchenne muscular dystrophy and idiopathic hyperCKemia segregating in a family

A 7-month-old boy with gross motor delay and failure to thrive presented with rhabdomyolysis following an acute asthmatic episode. During hospitalization an electrocardiographic conversion to a Wolff-Parkinson-White type 1 (WPW) pattern took place. Duchenne muscular dystrophy (DMD) was suspected based on elevated creatine kinase (CK) serum levels, muscle biopsy, and family history. The diagnosis was confirmed by molecular analysis, which documented a deletion corresponding to cDNA probe 1-2a in the dystrophin gene, in the propositus and in an affected male cousin of his mother. “Idiopathic” hyperCKemia was found in the propositus, his father, and 5 of his relatives. We suggest that the unusually early and severe manifestations of DMD in this patient may be related to the coincidental inheritance of the maternal DMD gene and of a paternal gene, causing hyperCKemia. © 1995 Wiley-Liss, Inc.     

Comparison of GB virus C, HIV, and HCV infection markers in hemophiliacs exposed to non-inactivated or inactivated factor concentrates.

BACKGROUND: Until the mandatory introduction of viral inactivation techniques of blood plasma products in the early 1980s many recipients of these products were infected with various viral pathogens. OBJECTIVES: To determine the rate of transmission of GB virus C/hepatitis G virus (GBV-C/HGV) HCV, and HIV through non-virus-inactivated clotting factor concentrates in hemophiliacs, as well as the relation between amount of administered clotting factor and risk for GBV-C/HGV infection. STUDY DESIGN: In this cross-sectional study, we determined retrospectively the rates of infection markers for GBV-C/HGV, HCV, and HIV in a German cohort of hemophiliacs treated with documented amounts of non-virus-inactivated clotting factor concentrates (group A) and in a second group of hemophiliacs who were treated exclusively with virus-inactivated clotting factor (group B). The presence of anti-virus antibodies was determined by ELISA. Viral RNA was detected by RT-PCR. Markers for viral infections were compared to amounts of administered non-virus-inactivated clotting factor. RESULTS: Among hemophiliacs treated with documented amounts of non-virus-inactivated clotting factor the prevalence for GBV-C/HGV, HCV, and HIV was 40.3%, 98.6%, and 56.3%, respectively. In contrast to HIV, the rate of GBV-C/HGV infections did not increase with increasing amounts of consumed non-inactivated clotting factor. Even in the subgroup of heavily treated hemophiliacs the rate of GBV-C/HGV infection markers did not exceed 45%. CONCLUSIONS: The amount of non-virus-inactivated clotting factor is not predictive for the risk of GBV-C/HGV infection in hemophiliacs. Despite repeated parenteral exposure more than 55% of hemophiliacs were not infected with GBV-C/HGV. Our findings indicate a high frequency of host factors preventing parenteral transmission of GBV-C/HGV.     

GLUT-4 expression is not consistently higher in type-1 than in type-2 fibres of rat and human vastus lateralis muscles; an immunohistochemical study

In whole muscle homogenates, the glucose transporter-4 (GLUT-4) content is reported to be higher in muscles consisting predominantly of oxidative (type-1) muscle fibres than in muscles consisting predominantly of glycolytic (type-2) fibres. From these findings, it has been deduced that in rat muscle, oxidative fibres have an intrinsically higher level of GLUT-4 protein than glycolytic fibres. No data is available concerning human muscle. Moreover, the fibre-type-specific expression of GLUT-4 has not yet been examined directly. In this study, the relative abundance of GLUT-4 protein expression in individual fibres of different types within a muscle was compared directly in immunohistochemical assays. The human vastus lateralis muscle and a selection of rat muscles were studied using a novel GLUT-4 antiserum. It is concluded that the pattern of fibre-type-specific GLUT-4 expression differs between human and rats and varies between the different muscles studied, indicating that non-fibre-type-specific factor(s) affect expression of GLUT-4. The observation that within a muscle a fibre-type-specific expression of GLUT-4 was observed indicates that fibre-type-specific factors contribute to GLUT-4 expression as well. Thus, it can be postulated that both fibre-type-dependent and fibre-type-independent factors affect GLUT-4 expression.     

Differentiation of human single-positive fetal thymocytes in vitro into IL-4- and/or IFN-{gamma}-producing CD4+ and CD8+ T cells

In this study we have investigated the capacity of human fetalthymocytes to differentiate in vitro into subsets of T cellswith polarized T_h1 or T_h2 cytokine profiles. Stimulation offreshly isolated human fetal thymocytes with anti-CD3 mAb, cross-linkedonto CD32,CD58,CD80-expressing mouse fibroblasts and subsequentculture in the presence of exogenous rIL-2 for 6 days, inducedthe production of both IL-4 and IFN-, which was mainly producedby CD4⁺ single-positive (SP) and CD8⁺ SP cells respectively.Addition of rIL-4 during priming augmented IL-4 production incultures of human fetal thymocytes, which was mainly due toan increased production of IL-4 by CD8SP cells. In contrast,addition of IL-4 to the cultures only slightly enhanced IL-4production and had little effect on frequencies of IL-4-producingCD4SP cells. Both CD4SP and CD8SP cells produced IL-5, IL-10and IL-13 at comparable levels, following priming in the presenceof rIL-4. Priming in the presence of rIL-12 strongly enhancedthe production of IFN- in both CD4SP and CD8SP cells. No correlationbetween expression of CD27, CD30 and CD60, and a particularcytokine profile of differentiated thymocytes could be demonstrated.Together, these results demonstrate the full capacity of fetalhuman thymocytes to differentiate into cytokine-producing Tcells in a priming milieu with appropriate stimulatory moleculesand exogenous cytokines. In addition, CD4SP thymocytes rapidlydifferentiate into polarized T_h2 cells following stimulationin vitro in the absence of exogenous rIL-4.     

Detection of perforin and granzyme A mRNA in infiltrating cells during infection of mice with lymphocytic choriomeningitis virus

The analysis of gene expression in cytotoxic T cells by in situ hybridization of serial liver and brain sections from mice infected with lymphocytic choriomeningitis virus (LCMV) and immunostaining with T cell marker- and virus-specific antibodies revealed a close histological association of infiltrating lymphocytes expressing the perforin and granzyme A genes with virally infected cells. Maximal frequency of perforin and granzyme A mRNA-containing cells on liver sections preceded by about 2 days maximal LCMV-specific cytotoxicity of the lymphoid liver infiltrating cells. These results are most consistent with an involvement of perforin and granzyme A in cell-mediated cytotoxicity in vivo.     

Influence of calcium and ionophore 23187 on tubular phosphate reabsorption

In previous studies it has been demonstrated that a decline of plasma calcium concentration accounts for the decrease of phosphate reabsorption in thyroparathyroidectomized (TPTX) rats undergoing phosphate loading.Microinfusion studies were performed in TPTX rats in order to discriminate between a systemic effect of calcium an a direct renal effect.Thyroparathyroidectomized animals were infused with a phosphate solution continuously. When plasma calcium concentration fell below 1.30 mmol/l, proximal convoluted tubules were microinfused with a phosphate tracer solution for 42 min. After 18 min a calcium chloride-containing solution was applied superficially (superfused) to the area of the microinfused tubule. This elevation of peritubular calcium concentration led to an immediate increase of phosphate reabsorption up to 12% of the microinfused phosphate load within 24 min.In another series of experiments, the calcium specific ionophore A 23187 — a substance which is known to increase intracellular calcium — was superfused on the microinfused tubule. This resulted again in an increase of fractional phosphate reabsorption of about 15% after 24 min. In contrast, when calcium chloride-free as well as ionophore-free solutions were superfused fractional phosphate reabsorption decreased (7%).From these data we conclude that 1. calcium has a direct renal effect on phosphate reabsorption in the absence of parathyroid hormone and 2. intracellular calcium appears to be a major parameter in the regulation of renal phosphate transport under these conditions.This study was supported by Dr. Legerlotz StiftungParts of this study were presented at the fall meeting of the Nephrologische Gesellschaft in Bonn, 1977 and at the spring meeting of the Deutsche Physiologische Gesellschaft in Göttingen, 1978     

On the peripheral bladder control system of the dog and urodynamics:In vivo characterisation and hybrid computer simulation

A quantitative model of the peripheral bladder control system of the dog is developed. This model is based on the experimental characterisation of the detrusor muscle and the urethra in 25 female dogs. Intravesical pressure, urinary flow and bladder volume were simultaneously recorded during electrical bladder stimulation, and, from these data, a dynamic model of the bladder control system has been deduced. A general equation for predicting urinary flow patterns is developed, and the hybrid computer EAI-690 is used to simulate the evacuation of the bladder. The effect of different parameters of the system on the micturition pattern is investigated in detail. This work may have interesting clinical applications: for example, in the development of adequate techniques for the evacuation of the paraplegic bladder. Also, the experimental procedures elaborated in this study could be applied in the diagnosis of bladder function and urethral obstruction. Indeed, we have been able to characterise the detrusor muscle and the urethra independently using clinically accessible variables.