Origin and primary dispersal of the Mycobacterium tuberculosis Beijing genotype: clues from human phylogeography

We suggest that the evolution of the population structure of microbial pathogens is influenced by that of modern humans. Consequently, the timing of hallmark changes in bacterial genomes within the last 100,000 yr may be attempted by comparison with relevant human migrations. Here, we used a lineage within Mycobacterium tuberculosis, a Beijing genotype, as a model and compared its phylogeography with human demography and Y chromosome-based phylogeography. We hypothesize that two key events shaped the early history of the Beijing genotype: (1) its Upper Palaeolithic origin in the Homo sapiens sapiens K-M9 cluster in Central Asia, and (2) primary Neolithic dispersal of the secondary Beijing NTF::IS6110 lineage by Proto-Sino-Tibetan farmers within east Asia (human O-M214/M122 haplogroup). The independent introductions of the Beijing strains from east Asia to northern Eurasia and South Africa were likely historically recent, whereas their differential dissemination within these areas has been influenced by demographic and climatic factors.     

Expression and characterization of recombinant soluble human CD3 molecules: presentation of antigenic epitopes defined on the native TCR-CD3 complex

The TCR-CD3 complex consists of the clonotypic disulfide-linked TCRalphabeta or TCRdeltagamma heterodimers, and the invariant CD3delta, epsilon, gamma and zeta chains. We generated plasmid constructs expressing the extracellular domains of the CD3delta, epsilon or gamma subunits fused to human IgG1 Fc. Recombinant fusion proteins consisting of individual CD3delta, epsilon or gamma subunits reacted poorly with anti-CD3 mAb including G19-4, BC3, OKT3 and 64.1. Co-expression of the CD3epsilon-Ig with either the CD3delta-Ig (CD3epsilondelta-Ig) or the CD3gamma-Ig (CD3epsilongamma-Ig) resulted in fusion proteins with much increased binding to G19-4. A brief acid treatment of the purified CD3epsilondelta-Ig fusion protein substantially improved its binding to BC3, OKT3 and 64.1. Surface plasmon resonance analysis revealed that the dissociation constants for CD3epsilondelta-Ig and anti-CD3 mAb ranged from 10(-8) to 10(-9) M. Based on these results, a single-chain (sc) construct encoding the CD3delta chain linked to the CD3epsilon chain with a flexible linker followed by human IgG1 Fc was expressed. The sc CD3deltaepsilon-scIg reacted with anti-CD3 mAb without requiring acid treatment. Moreover, anti-CD3 mAb bound CD3epsilondelta-Ig at a higher affinity than CD3epsilongamma-Ig, suggesting potential structural differences between the CD3epsilondelta and CD3epsilongamma subunits. In summary, we report the expression of soluble recombinant CD3 proteins that demonstrate structural characteristics of the native CD3 complex expressed on the T cell surface. These CD3 fusion proteins can be used to further analyze the structure of the TCR-CD3 complex, and to identify molecules that can interfere with TCR-CD3-mediated signal transduction by disrupting the interaction between CD3 and TCR subunits.     

Comparison of methods for identifying resistant herpes simplex virus and measuring antiviral susceptibility.

BACKGROUND: A number of in vitro assays are used to determine susceptibility of HSV to antiviral agents, but results from these in vitro assays do not necessarily correlate with treatment outcome. OBJECTIVES: A method with improved capability for identifying an isolate as acyclovir (ACV) or penciclovir (PCV) resistant when resistance is borderline could greatly improve the management of HSV disease. STUDY DESIGN: A comparative evaluation of four in vitro assays, plaque reduction (PRA), DNA hybridization, plating efficiency (PEA) and plaque autoradiography (PAR) was performed to accurately identify and measure resistance of a TK-altered clinical HSV isolate (HSV-1 N4) from a patient who was non-responsive to ACV treatment. Two established criteria for the prediction of antiviral resistance, IC(50)> or =2.0 microg/ml or an IC(50) greater than 10x above a sensitive virus IC(50), as well as testing in human (MRC-5) and nonhuman (Vero and CV-1 monkey kidney) cell lines were evaluated. RESULTS: The PRA and DNA hybridization assays accurately identified HSV-1 N4 as ACV(r) in human cells when using the 10x above sensitive virus IC(50) resistance criterion. Moreover, the PEA and PAR assays failed to classify HSV-1 N4 as drug resistant and indicate that these technologies alone are inadequate for identifying resistant virus. CONCLUSIONS: The data presented herein indicate that the PRA and DNA hybridization assays most accurately identified an otherwise borderline-resistant isolate as drug resistant: (i) when a sensitive virus is used within each individual assay as a control, (ii) when ACV and PCV susceptibility is evaluated in human cells, and (iii) when the 10x above sensitive IC(50) criterion is used to classify a virus as drug-resistant. Testing of additional clinical samples is warranted to further confirm these findings.     

Transgenic rat model of Huntington's disease

Huntington's disease (HD) is a late manifesting neurodegenerative disorder in humans caused by an expansion of a CAG trinucleotide repeat of more than 39 units in a gene of unknown function. Several mouse models have been reported which show rapid progression of a phenotype leading to death within 3-5 months (transgenic models) resembling the rare juvenile course of HD (Westphal variant) or which do not present with any symptoms (knock-in mice). Owing to the small size of the brain, mice are not suitable for repetitive in vivo imaging studies. Also, rapid progression of the disease in the transgenic models limits their usefulness for neurotransplantation. We therefore generated a rat model transgenic of HD, which carries a truncated huntingtin cDNA fragment with 51 CAG repeats under control of the native rat huntingtin promoter. This is the first transgenic rat model of a neurodegenerative disorder of the brain. These rats exhibit adult-onset neurological phenotypes with reduced anxiety, cognitive impairments, and slowly progressive motor dysfunction as well as typical histopathological alterations in the form of neuronal nuclear inclusions in the brain. As in HD patients, in vivo imaging demonstrates striatal shrinkage in magnetic resonance images and a reduced brain glucose metabolism in high-resolution fluor-deoxy-glucose positron emission tomography studies. This model allows longitudinal in vivo imaging studies and is therefore ideally suited for the evaluation of novel therapeutic approaches such as neurotransplantation.     

Peritubular capillary loss is associated with chronic tubulointerstitial injury in human kidney: altered expression of vascular endothelial growth factor

Chronic tubulointerstitial injury (CTI) including tubular atrophy and interstitial fibrosis represents one major determinant for the progression of chronic renal disease regardless of cause. Although peritubular capillaries (PTCs) are essential to maintain the normal structure and function of renal tubules, little is known about the role of PTCs in the development of CTI. The integrity of PTCs seems to be regulated by growth factors. Vascular endothelial cell growth factor (VEGF) has recently been recognized as a potent regulator of angiogenesis, vascular survival, and vascular permeability. Knowledge of the role of VEGF in renal disease is still rudimentary, and its role in CTI has not been explored. We analyzed the morphologic changes of PTCs and correlated them with other morphologic parameters of CTI in 32 human kidneys with various types of chronic tubulointerstitial disease. The VEGF expression was immunohistochemically evaluated. Compared with normal kidney, PTC loss (41% to 55% of control) and reduced size of PTCs (55% to 88% of control) were noted in kidneys with CTI. The PTC density was positively correlated with the proximal tubular density (r = 0.66, P <.0001), proximal tubular size (r = 0.54, P <.001), and negatively correlated with interstitial volume (r = -0.84, P <.0001). Compared with normal kidney, where podocytes were the only cell type that constantly expressed VEGF, an interesting pattern of increased VEGF expression by renal tubules, especially morphologically intact or hypertrophic ones, was shared by all cases with CTI. Loss of VEGF in sclerotic glomeruli was noted. PTC injury is pathogenetically linked to tubular atrophy, tubular loss, and interstitial fibrosis in human kidneys with CTI and might be a key factor for the progression of chronic tubulointerstitial disease. The characteristic and uniform pattern of altered VEGF expression in kidneys with CTI may result from ischemia induced by PTC loss and represent a protective mechanism against further PTC injuries. HUM PATHOL 31:1491-1497.     

Neutralization of adenovirus toxins: Specificity and antigenic relationships

Summary Early cytopathic or toxic activity was found in all human adenovirus types investigated, taken from all four subgroups. In neutralization tests with rabbit immune sera low homologous titres were observed. In subgroup III (types 1, 2, 5, 6) the cross-neutralization was entirely subgroup-specific. In subgroup I, toxins of types 7, 14, 16 and 21 reacted type-specifically, while type 3 toxin was also neutralized by type 7 antiserum and type 11 toxin by type 3, 7 and 14 antisera. Toxins of subgroup II (types 9, 15, 19 and 9–15) reacted mainly type-specifically with a crossing of types 9, 9–15 and 8. There was a neutralization by homologous dodecon antiserum, but none by hexon or fibre antiserum. The partial purification of type 2, 3 and 7 toxins did not alter their cross-neutralization pattern.Aided by a grant from the Deutsche Forschungsgemeinschaft.     

Phenylethylidenehydrazine, a novel GABA-transaminase inhibitor, reduces epileptiform activity in rat hippocampal slices

Phenylethylidenehydrazine (PEH), an analog of the monoamine oxidase inhibitor, beta-phenylethylhydrazine (phenelzine), inhibits the gamma-aminobutyric acid (GABA) catabolic enzyme GABA-transaminase and increases brain levels of GABA. GABA is the predominant fast inhibitory transmitter counteracting glutamatergic excitation, and increased neural GABA could influence a wide range of synaptic and circuit properties under both physiologic and pathophysiologic conditions. To examine the scope of these effects, we applied PEH (or vehicle) to rat hippocampal slices and measured basal glutamatergic transmission, synaptic plasticity, and epileptiform activity using extracellular field and whole cell patch clamp recordings. In vitro pre-treatment with PEH (100 microM) increased the GABA content of hippocampal slices by approximately 60% over vehicle-treated controls, but it had no effect on basal field excitatory postsynaptic potentials, tonic GABA currents, paired-pulse facilitation, or long-term potentiation. In contrast, pre-incubation with PEH caused a dose- and time-dependent reduction in epileptiform burst frequency induced by superfusion with Mg2+-free or high-K+ artificial cerebrospinal fluid. Thus, the inhibitory effects of PEH are state-dependent: hyper-excitation during epileptiform bursting was reduced, whereas synaptic transmission and plasticity were unaffected.     

New thrombolytic agents

Tissue Plasminogen Activator (t-PA), Single Chain Urokinase Plasminogen Activator or pro-Urokinase (scu-PA or pro-UK) and acyl enzymes are new thrombolytic agents, characterized by a high fibrin affinity, so that they provoke only mild systemic fibrinolytic effect. Their infusion would allie good thrombolytic activity and reduced hemorragic risks, usually related to fibrinogen and others coagulation factors degradation t-PA and scu-PA are natural, physiological substances, obtained by recombinant DNA technology. t-PA infusion in acute myocardial infusion (AMI) has been shown to be at least as efficient than intracoronary Streptokinase (SK) administration, but fibrinogenolysis was much lower as compared to SK. In vitro studies have shown that scu-PA was an efficient thrombolytic agent and has a relative fibrin specificity, at least as similar to t-PA, but much superior to classical Urokinase. The acyl-enzyme APSAC or Eminase is a SK-plasminogen complex in which the proteolytic site has been inactivated with an anisoic-acid. This acyl enzyme has a longer half-life than t-PA and scu-PA, and can be injected as a bolus. Its administration in AMI have shown that APSAC is as effective as SK, but can also provoke severe fibrinogenolysis. These 3 agents seem to have similar thrombolytic activities on coronary thrombi. However, further studies are required to evaluate the bleeding incidence and coronary reocclusion rates associated with their utilisation.     

Quantitative differences in lipid raft components between murine CD4<Superscript>+</Superscript> and CD8<Superscript>+</Superscript> T cells

Background  

Lipid rafts have been shown to play a role in T cell maturation, activation as well as in the formation of immunological synapses in CD4⁺ helper and CD8⁺ cytotoxic T cells. However, the differential expression of lipid raft components between CD4⁺ and CD8⁺ T cells is still poorly defined. To examine this question, we analyzed the expression of GM1 in T cells from young and aged mice as well as the expression of the glycosylphosphatidylinositol (GPI)-linked protein Thy-1 and cholesterol in murine CD4⁺ and CD8⁺ T cell subpopulations.     

Engagement of class I major histocompatibility complex molecules by cell surface CD8 delivers an activation signal.

Recent evidence has demonstrated that cross-linking class I major histocompatibility complex (MHC) molecules on human T cells with monoclonal antibodies (mAb) triggers T cell activation. The only known natural ligand for MHC class I molecules is CD8. Therefore, the possibility that CD8+ T cells might provide activation signals to other T cells by engaging MHC class I molecules was examined by culturing CD4+ peripheral blood T cells with Chinese hamster ovary cells (CHO) cells that had been transfected with the alpha chain or alpha and beta chains of CD8 and assessing interleukin (IL)-2 production. CD4+ T cells did not secrete IL-2 when cultured alone, with control or CD8+ CHO cells. In contrast, CD4+ T cells produced IL-2 when cultured with CD8+ CHO cells and co-stimulated with phorbol myristate acetate (PMA) or mAb to CD3 or CD28. PMA stimulated substantially less IL-2 when control CHO cells were employed and the mAb to CD3 and CD28 did not stimulate IL-2 production in the presence of control CHO cells. The co-stimulatory activity of CD8+ CHO cells was completely eliminated by mAb to CD8 or MHC class I molecules. The data demonstrate that CD8 can interact with MHC class I molecules expressed on T cells and deliver a costimulatory signal that increases IL-2 production. Thus, engagement of MHC class I molecules by its natural ligand, CD8, provides an activation signal to T cells. Under some circumstances, such interactions may amplify the responses of T cells.