Whole-blood flow-cytometric analysis of antigen-specific CD4 T-cell cytokine profiles distinguishes active tuberculosis from non-active states

T-cell based IFN-γ release assays do not permit distinction of active tuberculosis (TB) from successfully treated disease or latent M. tuberculosis infection. We postulated that IFN-γ and IL-2 cytokine profiles of antigen-specific T cells measured by flow-cytometry ex vivo might correlate with TB disease activity in vivo. Tuberculin (PPD), ESAT-6 and CFP-10 were used as stimuli to determine antigen-specific cytokine profiles in CD4 T cells from 24 patients with active TB and 28 patients with successfully treated TB using flow-cytometry. Moreover, 25 individuals with immunity consistent with latent M. tuberculosis infection and BCG-vaccination, respectively, were recruited. Although the frequency of cytokine secreting PPD reactive CD4 T cells was higher in patients with active TB compared to patients with treated TB (median 0.81% vs. 0.39% of CD4 T cells, p?=?0.02), the overlap in frequencies precluded distinction between the groups on an individual basis. When assessing cytokine profiles, PPD specific CD4 T cells secreting both IFN-γ and IL-2 predominated in treated TB, latent infection and BCG-vaccination, whilst in active TB the cytokine profile was shifted towards cells secreting IFN-γ only (p<0.0001). Cytokine profiles of ESAT-6 or CFP-10 reactive CD4 T cells did not differ between the groups. Receiver operator characteristics (ROC) analysis revealed that frequencies of PPD specific IFN-γ/IL-2 dual-positive T cells below 56% were an accurate marker for active TB (specificity 100%, sensitivity 70%) enabling effective discrimination from non-active states. In conclusion, a frequency lower than 56% IFN-γ/IL-2 dual positive PPD-specific circulating CD4 T-cells is strongly indicative of active TB.     

Continuous versus cyclic progesterone exposure differentially regulates hippocampal gene expression and functional profiles

This study investigated the impact of chronic exposure to continuous (CoP4) versus cyclic progesterone (CyP4) alone or in combination with 17β-estradiol (E2) on gene expression profiles targeting bioenergetics, metabolism and inflammation in the adult female rat hippocampus. High-throughput qRT-PCR analyses revealed that ovarian hormonal depletion induced by ovariectomy (OVX) led to multiple significant gene expression alterations, which were to a great extent reversed by co-administration of E2 and CyP4. In contrast, co-administration of E2 and CoP4 induced a pattern highly resembling OVX. Bioinformatics analyses further revealed clear disparities in functional profiles associated with E2+CoP4 and E2+CyP4. Genes involved in mitochondrial energy (ATP synthase α subunit; Atp5a1), redox homeostasis (peroxiredoxin 5; Prdx5), insulin signaling (insulin-like growth factor I; Igf1), and cholesterol trafficking (liver X receptor α subtype; Nr1h3), differed in direction of regulation by E2+CoP4 (down-regulation relative to OVX) and E2+CyP4 (up-regulation relative to OVX). In contrast, genes involved in amyloid metabolism (β-secretase; Bace1) differed only in degree of regulation, as both E2+CoP4 and E2+CyP4 induced down-regulation at different efficacy. E2+CyP4-induced changes could be associated with regulation of progesterone receptor membrane component 1(Pgrmc1). In summary, results from this study provide evidence at the molecular level that differing regimens of hormone therapy (HT) can induce disparate gene expression profiles in brain. From a translational perspective, confirmation of these results in a model of natural menopause, would imply that the common regimen of continuous combined HT may have adverse consequences whereas a cyclic combined regimen, which is more physiological, could be an effective strategy to maintain neurological health and function throughout menopausal aging.     

Spectral Sensitivity of the Planarian Ocellus

The ocellar potential (OP) of planaria was recorded using microelectrode techniques. The action spectrum and spectral sensitivity of the OP are described. Maximum OP sensitivity was found with 508 nm light. A moderate increase in sensitivity to blue light was observed. This is typical of many invertebrate photoreceptors and was shown, by selective chromatic adaptation, not to indicate the presence of a second pigment.     

Selective photoreactions in a programmable array microscope (PAM): photoinitiated polymerization, photodecaging, and photochromic conversion.

BACKGROUND: Innovative thinking and experimentation were the hallmarks of Mack Fulwyler's approach to research. This report summarizes some of the ideas and their early realizations that he pursued in the field of imaging cytometry, work that was not published before his untimely death, although he composed the initial draft of this report. METHODS: Included are related experiments implemented in the programmable array microscope (PAM) devised for patterned illumination and detection, the instrument that Mack Fulwyler employed during a sabbatical leave in G?ttingen in 1998. Despite being the originator of instrumentation for flow cytometry and sorting, Mack Fulwyler was intensely interested in imaging systems, recognizing their ability to resolve cellular details obscured by the whole cell signals generally acquired in flow. At one point, these interests merged with those of two other authors (I.T.Y. and T.M.J.), leading to the Image Cytometry and Sorting (ICAS) strategy and project. A major goal was uncomplicated rare cell detection and isolation using a sequential process of cellular labeling via suitable probes, whole field imaging, and selective area-restricted photoinduced reactions designed to encapsulate and/or chemically or physically tag cells in a manner permitting subsequent fractionation by bulk techniques. RESULTS AND CONCLUSION: This publication features photoinduced polymerization, photodecaging, photoactivation, and photochromic conversion reactions carried out by Fulwyler and/or the other authors with the PAM, employing operator designated patterns and locations in various samples. Photopolymerization of polyethylene glycol-diacrylate to a gel-like structure allowing the specific selection of objects (cells) for further analysis and processing techniques was the approach explored personally by Mack Fulwyler in relation to the ICAS concept.     

Similar hypotensive responses to resistance exercise with and without blood flow restriction

Low intensity resistance exercise (RE) with blood flow restriction (BFR) has gained attention in the literature due to the beneficial effects on functional and morphological variables, similar to those observed during traditional RE without BFR, while the effects of BFR on post-exercise hypotension remain unclear. The aim of the present study was to compare the blood pressure (BP) response of trained normotensive individuals to RE with and without BFR. In this cross-over randomized trial, eight male subjects (23.8 ± 4 years, 74 ± 3 kg, 174 ± 4 cm) completed two exercise protocols: traditional RE (3 x 10 repetitions at 70% one-repetition maximum [1-RM]) and low intensity RE (3 x 15 repetitions at 20% 1-RM) with BFR. Blood pressure measurements were performed after 15 min of seated rest (0), immediately after and 10 min, 20 min, 30 min, 40 min, 50 min and 60 min after the experimental sessions. Similar hypotensive effects for systolic BP (SBP) were observed for both protocols (P < 0.05) after exercise, with no differences between groups (P > 0.05) and no statistically significant difference for diastolic BP (P > 0.05). These results suggest that in normotensive trained individuals, both traditional RE and RE with BFR induce hypotension for SBP, which is important to prevent cardiovascular disturbances.     

Transfection of Reaggregating Embryonic Chicken Retinal Cells with an Antisense 5'-DNA Butyrylcholinesterase Expression Vector Inhibits Proliferation and Alters Morphogenesis

Abstract: The function of the enzyme butyrylcholinesterase (BChE) in the developing and mature brain is still unclear. We have inserted 577 bp of the 5' upstream region plus 106 bp of the exon 1 of the rabbit BChE gene in reverse orientation under control of an SV40 early promoter derivative in an expression vector. This vector was introduced by calcium phosphate-mediated transfection into embryonic chicken retina cells during the first days of reaggregation culture. Depending on the retinal origin, the transfected cell population forms histotypic retina-like spheres, so-called rosetted or stratified retinospheroids. We show that antisense 5'-BChE gene expression decreased the steady-state mRNA level of BChE and the translation of the BChE protein, inhibited proliferation, and accelerated histogenesis in both cellular systems. The pronounced effects of antisense 5'-BChE transfection of spheroids document a key role of BChE during the early reaggregation process of retinal cells, most likely by regulating their growth and differentiation.     

Tracking human migrations by the analysis of the distribution of HLA alleles, lineages and haplotypes in closed and open populations

The human leucocyte antigen (HLA) system shows extensive variation in the number and function of loci and the number of alleles present at any one locus. Allele distribution has been analysed in many populations through the course of several decades, and the implementation of molecular typing has significantly increased the level of diversity revealing that many serotypes have multiple functional variants. While the degree of diversity in many populations is equivalent and may result from functional polymorphism(s) in peptide presentation, homogeneous and heterogeneous populations present contrasting numbers of alleles and lineages at the loci with high-density expression products. In spite of these differences, the homozygosity levels are comparable in almost all of them. The balanced distribution of HLA alleles is consistent with overdominant selection. The genetic distances between outbred populations correlate with their geographical locations; the formal genetic distance measurements are larger than expected between inbred populations in the same region. The latter present many unique alleles grouped in a few lineages consistent with limited founder polymorphism in which any novel allele may have been positively selected to enlarge the communal peptide-binding repertoire of a given population. On the other hand, it has been observed that some alleles are found in multiple populations with distinctive haplotypic associations suggesting that convergent evolution events may have taken place as well. It appears that the HLA system has been under strong selection, probably owing to its fundamental role in varying immune responses. Therefore, allelic diversity in HLA should be analysed in conjunction with other genetic markers to accurately track the migrations of modern humans.     

Comparison of two expression platforms in respect to protein yield and quality: Pichia pastoris versus Pichia angusta

The methylotrophic yeasts Pichia pastoris and Pichia angusta (Hansenula polymorpha) were used for the comparative heterologous production of two model mammalian proteins of pharmaceutical interest, the NK1-fragment (22 kDa) of human hepatocyte growth factor and the extracellular domain (28 kDa) of mouse tissue factor (MTF). Both recombinant proteins were engineered to contain an N-terminal Strep- (WSHPQFEK) and a C-terminal His₆-tag. In addition, both proteins contained the pre-pro-sequence of Saccharomyces cerevisiae mating factor alpha to allow secretion. Following vector construction, transformation and zeocin amplification, the best Pichia producers were identified in a screening procedure using Western blot and a Luminex xMAP™ based high-throughput method. Recombinant NK1-fragment and MTF were purified from culture supernatants of the best producers by affinity chromatography (Ni–nitrilotriacetic acid columns). Using P. pastoris as a host for the synthesis of NK1-fragment a protein yield of 5.7 mg/l was achieved. In comparable expression experiments P. angusta yielded 1.6 mg/l of NK1-fragment. NK1-fragment apparently was not glycosylated in either system. For the production of MTF, P. pastoris was also the superior host yielding 1.2 mg/l glycosylated recombinant protein whereas P. angusta was clearly less efficient (<0.2 mg/l MTF). For both expression systems no correlation between the amount of recombinant protein and the copy number of the chromosomally integrated heterologous genes was found. In P. pastoris strains less degradation of the two model recombinant proteins was observed. Altogether, this paper provides a structured protocol for rapidly identifying productive Pichia strains for the synthesis of full-length recombinant proteins.