Effect of regional lung inflation on ventilation heterogeneity at different length scales during mechanical ventilation of normal sheep lungs

Heterogeneous, small-airway diameters and alveolar derecruitment in poorly aerated regions of normal lungs could produce ventilation heterogeneity at those anatomic levels. We modeled the washout kinetics of (13)NN with positron emission tomography to examine how specific ventilation (sV) heterogeneity at different length scales is influenced by lung aeration. Three groups of anesthetized, supine sheep were studied: high tidal volume (Vt; 18.4 ± 4.2 ml/kg) and zero end-expiratory pressure (ZEEP) (n = 6); low Vt (9.2 ± 1.0 ml/kg) and ZEEP (n = 6); and low Vt (8.2 ± 0.2 ml/kg) and positive end-expiratory pressure (PEEP; 19 ± 1 cmH(2)O) (n = 4). We quantified fractional gas content with transmission scans, and sV with emission scans of infused (13)NN-saline. Voxel (13)NN-washout curves were fit with one- or two-compartment models to estimate sV. Total heterogeneity, measured as SD[log(10)(sV)], was divided into length-scale ranges by measuring changes in variance of log(10)(sV), resulting from progressive filtering of sV images. High-Vt ZEEP showed higher sV heterogeneity at <12- (P < 0.01), 12- to 36- (P < 0.01), and 36- to 60-mm (P < 0.05) length scales compared with low-Vt PEEP, with low-Vt ZEEP in between. Increased heterogeneity was associated with the emergence of low sV units in poorly aerated regions, with a high correlation (r = 0.95, P < 0.001) between total heterogeneity and the fraction of lung with slow washout. Regional mean fractional gas content was inversely correlated with regional sV heterogeneity at <12- (r = -0.67), 12- to 36- (r = -0.74), and >36-mm (r = -0.72) length scales (P < 0.001). We conclude that sV heterogeneity at length scales <60 mm increases in poorly aerated regions of mechanically ventilated normal lungs, likely due to heterogeneous small-airway narrowing and alveolar derecruitment. PEEP reduces sV heterogeneity by maintaining lung expansion and airway patency at those small length scales.     

The distal region and receptor tyrosines of the Epo receptor are non-essential for in vivo erythropoiesis.

The erythropoietin receptor (EpoR) is required for the proliferation and survival of committed erythroid lineage cells. Previous studies have utilized receptor mutations to show the requirement for the distal half of the cytoplasmic domain of the EpoR and receptor tyrosines for activation of signaling pathways potentially critical to Epo function. To extend these studies to in vivo erythropoiesis, we have created two mutant strains of mice. One strain (H) contains a truncation of the distal half of the cytoplasmic domain, while the second strain (HM) contains the same truncation as well as the mutation of the residual tyrosine (Y(343)) to a phenylalanine. Strikingly, both strains of mice are viable, with only slight alterations in constitutive erythropoiesis or in in vitro assays of red cell lineage function. Challenging H mutant mice with continuous injections of Epo results in an erythrocytosis that is not seen in HM mice. The results demonstrate that neither the distal region nor receptor tyrosines are essential for in vivo EpoR function, but contribute to receptor function in a subtle manner.     

Identification of B-Cell Epitopes on Virus-Like Particles of Cutaneous Alpha-Human Papillomaviruses

Human papillomavirus (PV) (HPV) types 2, 27, and 57 are closely related and, hence, represent a promising model system to study the correlation of phylogenetic relationship and immunological distinctiveness of PVs. These HPV types cause a large fraction of cutaneous warts occurring in immunocompromised patients. Therefore, they constitute a target for the development of virus-like particle (VLP)-based vaccines. However, the immunogenic structure of HPV type 2, 27, and 57 capsids has not been studied yet. Here we provide, for the first time, a characterization of the B-cell epitopes on VLPs of cutaneous alpha-HPVs using a panel of 94 monoclonal antibodies (MAbs) generated upon immunization with capsids from HPV types 2, 27, and 57. The MAbs generated were characterized regarding their reactivities with glutathione S-transferase-L1 fusion proteins from 18 different PV types, the nature of their recognized epitopes, their isotypes, and their ability to neutralize HPV type 2, 27, 57, or 16. In total, 33 of the 94 MAbs (35%) showed type-specific reactivity. All type-specific MAbs recognize linear epitopes, most of which map to the hypervariable surface loop regions of the L1 amino acid sequence. Four of the generated MAbs neutralized pseudovirions of the inoculated HPV type efficiently. All four MAbs recognized epitopes within the BC loop, which is required and sufficient for their neutralizing activity. Our data highlight the immunological distinctiveness of individual HPV types, even in comparison to their closest relatives, and they provide a basis for the development of VLP-based vaccines against cutaneous alpha-HPVs.Recently licensed prophylactic vaccines confer efficient protection against infections by human papillomavirus (PV) (HPV) types 16 and 18, thereby aiming to prevent approximately 70% of all cervical cancer cases (17, 39). These vaccines are composed of virus-like particles (VLPs), which spontaneously assemble from the major capsid protein L1 via 72 pentamers (capsomeres) as subunits (2, 23, 26).In the process of vaccine development, monoclonal antibodies (MAbs) proved to be valuable tools for the immunological analysis of recombinantly produced capsids and capsomeres (51) as well as for serological studies (25, 49, 56). Moreover, the identification and characterization of many neutralizing epitopes of HPV types 11 and 16 have been facilitated by the employment of MAbs (6, 11, 30-32, 41, 42, 55). Such epitopes to neutralizing antibodies are mostly conformation dependent, but a few neutralizing MAbs that recognize linear epitopes have also been generated (16, 18). Most neutralizing MAbs are HPV type specific due to the hypervariable nature of their respective epitopes, which typically reside in the surface-exposed loop regions of the L1 protein (10). In contrast, cross-reactive MAbs targeting rather conserved L1 epitopes are generally nonneutralizing.HPV types 2, 27, and 57 are the three members of Alphapapillomavirus species 4 (20). They are very closely related, and HPV types 2 and 27 hardly fulfill the requirement of more than 10% nucleotide variation in the L1 open reading frame to be classified as distinct types (8). Therefore, they represent a promising model system to study the immunological distinctiveness of closely related HPV types. Pathologically, HPV types 2, 27, and 57 infect primarily the cutaneous epithelia, thereby causing common skin warts, which often occur ubiquitously and confluently in immunocompromised patients (1, 24, 28). It is our long-term goal to develop a prophylactic L1 VLP-based vaccine to alleviate the burden provoked by HPV-induced skin lesions in these patients. However, to date, neither the structure nor the immunogenicity of HPV type 2, 27, and 57 capsids has been elucidated.The purpose of the present study was twofold. First, we sought to generate MAbs specific for HPV types 2, 27, and 57 as tools for type-specific diagnostic assays. Second, we aimed to exploit the generated MAbs for an investigation of the B-cell epitopes on capsids of HPV types 2, 27, and 57.     

Chondrogenic differentiation potential of osteoarthritic chondrocytes and their possible use in matrix-associated autologous chondrocyte transplantation

Introduction  

Autologous chondrocyte transplantation (ACT) is a routine technique to regenerate focal cartilage lesions. However, patients with osteoarthritis (OA) are lacking an appropriate long-lasting treatment alternative, partly since it is not known if chondrocytes from OA patients have the same chondrogenic differentiation potential as chondrocytes from donors not affected by OA.     

In Vivo Generation of Flavoproteins with Modified Cofactors

To understand flavoprotein mechanisms and reactivity, biochemical and biophysical methods are usually employed, and differences between wild-type and mutated proteins with altered primary structures are placed under specific consideration. Alternatively, the cofactor can be modified, and modified flavoproteins can be studied accordingly. Here we present an efficient and general method for modifying the cofactor of flavoproteins in vivo. The modified cofactor is incorporated into apoprotein during protein biosynthesis in a riboflavin-auxotrophic Escherichia coli strain, which expresses a bacterial riboflavin transporter to import flavins from the medium. This system was used to introduce roseoflavin into the riboflavin-binding protein dodecin and into microbial blue-light photoreceptors of the BLUF (blue-light sensors using FAD) and LOV (light oxygen voltage) families. The modified photoreceptors showed absorption and fluorescence different from those of proteins carrying their natural cofactor or chromophores in solution, but did not show any photochemical reaction as implied by former physiological studies.     

Cognitive behavioural therapy versus supportive therapy for persistent positive symptoms in psychotic disorders: The POSITIVE Study, a multicenter, prospective, single-blind, randomised controlled clinical trial

Background

It has been demonstrated that cognitive behavioural therapy (CBT) has a moderate effect on symptom reduction and on general well being of patients suffering from psychosis. However, questions regarding the specific efficacy of CBT, the treatment safety, the cost-effectiveness, and the moderators and mediators of treatment effects are still a major issue. The major objective of this trial is to investigate whether CBT is specifically efficacious in reducing positive symptoms when compared with non-specific supportive therapy (ST) which does not implement CBT-techniques but provides comparable therapeutic attention.

Methods/Design

The POSITIVE study is a multicenter, prospective, single-blind, parallel group, randomised clinical trial, comparing CBT and ST with respect to the efficacy in reducing positive symptoms in psychotic disorders. CBT as well as ST consist of 20 sessions altogether, 165 participants receiving CBT and 165 participants receiving ST. Major methodological aspects of the study are systematic recruitment, explicit inclusion criteria, reliability checks of assessments with control for rater shift, analysis by intention to treat, data management using remote data entry, measures of quality assurance (e.g. on-site monitoring with source data verification, regular query process), advanced statistical analysis, manualized treatment, checks of adherence and competence of therapists. Research relating the psychotherapy process with outcome, neurobiological research addressing basic questions of delusion formation using fMRI and neuropsychological assessment and treatment research investigating adaptations of CBT for adolescents is combined in this network. Problems of transfer into routine clinical care will be identified and addressed by a project focusing on cost efficiency.

Discussion

This clinical trial is part of efforts to intensify psychotherapy research in the field of psychosis in Germany, to contribute to the international discussion on psychotherapy in psychotic disorders, and to help implement psychotherapy in routine care. Furthermore, the study will allow drawing conclusions about the mediators of treatment effects of CBT of psychotic disorders.

Trial Registration

Current Controlled Trials ISRCTN29242879     

Receptor specific downregulation of cytokine signaling by autophosphorylation in the FERM domain of Jak2

The tyrosine kinase, Janus kinase-2 (Jak2), plays a pivotal role in signal transduction through a variety of cytokine receptors, including the receptor for erythropoietin (Epo). Although the physiological relevance of Jak2 has been definitively established, less is known about its regulation. In studies assessing the roles of sites of tyrosine phosphorylation, we identified Y(119) in the FERM (band 4.1, Ezrin, radixin and moesin) domain as a phosphorylation site. In these studies, we demonstrate that the phosphorylation of Y(119) in response to Epo downregulates Jak2 kinase activity. Using a phosphorylation mimic mutation (Y(119)E), downregulation is shown to involve dissociation of Jak2 from the receptor complex. Conversely, a Y(119)F mutant is more stably associated with the receptor complex. Thus, in cytokine responses, ligand binding induces activation of receptor associated Jak2, autophosphorylation of Y(119) in the FERM domain and the subsequent dissociation of the activated Jak2 from the receptor and degradation. This regulation occurs with the receptors for Epo, thrombopoietin and growth hormone but not with the receptor for interferon-gamma.     

Interleukin 3: A differentiation and growth factor for the mouse mast cell that contains chondroitin sulfate E proteoglycan

Mouse mast cells were differentiated and grown by culturing bone marrow cells in medium containing 2 X 10(-10) M purified interleukin 3 (IL 3). The cells obtained were similar in ultrastructure, membrane antigen phenotype, proteoglycan type, and lipid products generated upon immunologic activation to mast cells differentiated in culture by WEHI-3-conditioned medium (WEHI-3-CM) and by concanavalin A (Con A) splenocyte-conditioned medium. Phenotypically, these cells expressed IgE receptors and H-2 antigens and were recognized by a monoclonal antibody (B23.1) that did not react with mouse serosal heparin-containing mast cells. The classic phenotypic markers of mouse T cells or macrophages were not detected. The mouse mast cells differentiated with IL 3 as well as those differentiated in WEHI-3-CM incorporated [35S]sulfate into a nonheparin proteoglycan of 150,000 to 200,000 m.w. Most of the 35S-labeled macromolecules were degraded by chondroitinase ABC to yield only two disaccharides, which co-chromatographed on ascending thin layer chromatography with delta Di-4S and delta Di-diSE; thus, the proteoglycan in these cells is composed of chondroitin sulfate E glycosaminoglycans. After sensitization with monoclonal IgE, washing, and antigen activation, the IL 3 differentiated cells released the preformed mediator beta-hexosaminidase and generated and released two major classes of lipid mediators. The quantities of leukotriene C4 (LTC4), leukotriene B4 (LTB4), and platelet-activating factor (PAF-acether) generated/10(6) cells were 17, 3.0, and 3.1 ng, respectively. The ratio of these three lipid mediators was similar to that obtained from mast cells differentiated in WEHI-3-CM and in Con A-conditioned medium. Thus, T cell-derived IL 3 is the component present in the conditioned media that is required for differentiation and growth of the subclass of mast cells containing chondroitin sulfate E proteoglycan, designated E-MC. The IL 3-dependent E-MC may represent the in vitro counterpart of the T-cell-dependent mucosal mast cell, suggesting in turn that the production of LTC4 and LTB4 and of PAF-acether may play a role in adaptive intestinal immunity to helminthic parasites.     

Thrombocyte counting with the Laborscale (PSL-1/PSA-1) particle-counting instrument by Medicor (Budapest/Hungary) and thrombocyte volume analysis in the diagnosis of selected thrombocytopathies

A thrombocyte counting technique is presented in comparison with the chambre counting according to the German Book of Medicaments (DAB 7 D.L.) GDR on the particle counting device "Laborscale" (PSL-1/PSA-1) of Medicor by using a thrombofuge (70 g). The composition of reagents accounts for problems of measurement which are caused by the device and influences the separation of particles. The possibility of recording the curve of thrombocyte volume distribution and calculating the average thrombocyte volume (VM) is demonstrated by means of a normal collective of test persons as well as by intravascular disorders of haemostasis and during cytostatic therapy.