Human blood-brain barrier disruption by retroviral-infected lymphocytes: role of myosin light chain kinase in endothelial tight-junction disorganization

The blood-brain barrier (BBB), which constitutes the interface between blood and cerebral parenchyma, has been shown to be disrupted during retroviral associated neuromyelopathies. Human T cell leukemia virus (HTLV-1)-associated myelopathy/tropical spastic paraparesis is a slowly progressive neurodegenerative disease, in which evidence of BBB breakdown has been demonstrated by the presence of lymphocytic infiltrates in the CNS and plasma protein leakage through cerebral endothelium. Using an in vitro human BBB model, we investigated the cellular and molecular mechanisms involved in endothelial changes induced by HTLV-1-infected lymphocytes. We demonstrate that coculture with infected lymphocytes induces an increase in paracellular endothelial permeability and transcellular migration, via IL-1alpha and TNF-alpha secretion. This disruption is associated with tight junction disorganization between endothelial cells, and alterations in the expression pattern of tight junction proteins such as zonula occludens 1. These changes could be prevented by inhibition of the NF-kappaB pathway or of myosin light chain kinase activity. Such disorganization was confirmed in histological sections of spinal cord from an HTLV-1-associated myelopathy/tropical spastic paraparesis patient. Based on this BBB model, the present data indicate that HTLV-1-infected lymphocytes can induce BBB breakdown and may be responsible for the CNS infiltration that occurs in the early steps of retroviral-associated neuromyelopathies.     

Identification of monoclonal antibody defined epitopes on human leukocyte antigens utilizing phage display peptide libraries

Summary Utilizing phage display peptide libraries, we have identified and mapped the antigenic determinants recognized by mouse monoclonal antibodies (mAb) on two sets of immunologically important molecules, HLA class I and class II antigens. Anti-HLA class I mAb TP25.99 recognizes a conformational and a linear determinant on distinct regions of the HLA class I α3 domain. Anti-HLA class I mAb HO-4 recognizes a conformational determinant on the α2 domain of HLA-A2 and A28 allospecificities. Anti-HLA-DR1,-DR4,-DR6,-DR8,-DR9 mAb SM/549 recognizes a conformational determinant on the β chain of HLA class II antigens. These results indicate the versatility of phage display peptide libraries to characterize antigenic determinants recognized by anti-HLA mAb.     

Characterization of reemerging chikungunya virus

An unprecedented epidemic of chikungunya virus (CHIKV) infection recently started in countries of the Indian Ocean area, causing an acute and painful syndrome with strong fever, asthenia, skin rash, polyarthritis, and lethal cases of encephalitis. The basis for chikungunya disease and the tropism of CHIKV remain unknown. Here, we describe the replication characteristics of recent clinical CHIKV strains. Human epithelial and endothelial cells, primary fibroblasts and, to a lesser extent, monocyte-derived macrophages, were susceptible to infection and allowed viral production. In contrast, CHIKV did not replicate in lymphoid and monocytoid cell lines, primary lymphocytes and monocytes, or monocyte-derived dendritic cells. CHIKV replication was cytopathic and associated with an induction of apoptosis in infected cells. Chloroquine, bafilomycin-A1, and short hairpin RNAs against dynamin-2 inhibited viral production, indicating that viral entry occurs through pH-dependent endocytosis. CHIKV was highly sensitive to the antiviral activity of type I and II interferons. These results provide a general insight into the interaction between CHIKV and its mammalian host.     

Synthesis and in vitro activity of 2-thiazolylhydrazone derivatives compared with the activity of clotrimazole against clinical isolates of Candida spp

In this paper, we report on the synthesis of a novel series of 2-thiazolylhydrazone derivatives and the influence of the substituents on the thiazole ring on antifungal activity. All synthesized compounds were screened for their in vitro activities against 22 clinical isolates of Candida spp., representing six different species, compared to clotrimazole as a reference compound. Some of the tested compounds were found to possess significant antifungal activity when compared to clotrimazole, in particular compound 14 which exhibited higher potency against most of the Candida spp. considered. The compounds that were most active as anti-Candida agents were also submitted to cytotoxic screening by the Trypan Blue dye exclusion assay and in general they were shown to induce low cytotoxic effects.     

Bladder Cancer Biomarker Discovery Using Global Metabolomic Profiling of Urine

Bladder cancer (BCa) is a common malignancy worldwide and has a high probability of recurrence after initial diagnosis and treatment. As a result, recurrent surveillance, primarily involving repeated cystoscopies, is a critical component of post diagnosis patient management. Since cystoscopy is invasive, expensive and a possible deterrent to patient compliance with regular follow-up screening, new non-invasive technologies to aid in the detection of recurrent and/or primary bladder cancer are strongly needed. In this study, mass spectrometry based metabolomics was employed to identify biochemical signatures in human urine that differentiate bladder cancer from non-cancer controls. Over 1000 distinct compounds were measured including 587 named compounds of known chemical identity. Initial biomarker identification was conducted using a 332 subject sample set of retrospective urine samples (cohort 1), which included 66 BCa positive samples. A set of 25 candidate biomarkers was selected based on statistical significance, fold difference and metabolic pathway coverage. The 25 candidate biomarkers were tested against an independent urine sample set (cohort 2) using random forest analysis, with palmitoyl sphingomyelin, lactate, adenosine and succinate providing the strongest predictive power for differentiating cohort 2 cancer from non-cancer urines. Cohort 2 metabolite profiling revealed additional metabolites, including arachidonate, that were higher in cohort 2 cancer vs. non-cancer controls, but were below quantitation limits in the cohort 1 profiling. Metabolites related to lipid metabolism may be especially interesting biomarkers. The results suggest that urine metabolites may provide a much needed non-invasive adjunct diagnostic to cystoscopy for detection of bladder cancer and recurrent disease management.     

Cytokine profiles of HeLa and human diploid cells induced by different fractions of Vibrio parahaemolyticus cultures exposed to stress conditions

Vibrio (V.) parahaemolyticus is an aquatic halophilic bacteria which produces gastroenteritis and in rare cases septicaemia after the consumption of raw or under-cooked contaminated seafood.The severity of diarrheal illness caused by this bacterium is closely related to the presence of two types of hemolysins (the thermostable direct hemolysin-TDH and TDH related hemolysin-TRH) and also of type III secretion system (TTSS) proteins. The TTSS type 1 induces a wide array of effects on infected HeLa cells such as autophagy, oncosis, cell rounding and lysis. Previous studies have shown that heat shock proteins have the ability to stimulate the production of interleukins in different cellular cultures. In our studies we have stimulated two cellular lines (HeLa and human diploid cells) with different V. parahaemolyticus culture fractions in order to observe the effect on cytokines production. Thus, the purpose of this study was to analyze the expression of IL-1, IL-2, IL-4, IL-6, IL-10 and TNF-alpha induced by the cell treatment with total cellular lysate, periplasmic fractions and culture supernatants extracted from V. parahaemolyticus exposed to normal and also to stress conditions. The ELISA assay of the cytokine profile of the HeLa and HDC cell lines stimulated with different bacterial fractions revealed that in the V. parahemolyticus cultures submitted to osmotic and heat shock stress are accumulating factors (probably heat shock proteins) which are exhibiting immunomodulatory activity, responsible for the induction of a pro-inflammatory response associated with increased levels of IL-6 and TNF-alpha expression, however balanced by the stimulation of the anti-inflammatory cytokine IL-4 synthesis.     

Use of Quantitative PCR Detection Methods to Study Biocontrol Agents and Phytopathogenic Fungi and Oomycetes in Environmental Samples

Quantitative polymerase chain reaction (qPCR) is a versatile technique for the accurate, sensitive, reliable and high‐throughput detection and quantification of target DNA in various environmental samples, and in recent years, it has greatly contributed to the advancement of knowledge in the plant pathology field. Indeed, this technique is ideal to evaluate inoculum threshold levels and to study the epidemiology, biology and ecology of phytopathogenic fungi and oomycetes, thus opening up new research opportunities to investigate host–pathogen interactions and to address tasks related to quarantine, eradication and biosecurity. Moreover, it can be a useful tool in breeding programs. The present review analyses the most relevant applications of qPCR for the detection and quantification of filamentous fungi and oomycetes within host tissues and in soil, air and water, along with brief paragraphs focusing on new application fields such as the detection and quantification of mycotoxigenic fungi and biocontrol agents. The high potentiality of qPCR for present and future applications is highlighted together with a critical analysis of major drawbacks that need to be corrected to definitively confirm it as a preferential routine quantitative detection method.     

Specific heparan sulfate structures modulate FGF10-mediated submandibular gland epithelial morphogenesis and differentiation

FGF10, a heparan sulfate (HS)-binding growth factor, is required for branching morphogenesis of mouse submandibular glands (SMGs). HS increases the affinity of FGF10 for FGFR2b, which forms an FGF10.FGFR2b.HS ternary signaling complex, and results in diverse biological outcomes, including proliferation and epithelial morphogenesis. Defining the HS structures involved in specific FGF10-mediated events is critical to understand how HS modulates growth factor signaling in specific developmental contexts. We used HS-deficient BaF3/FGFR2b cells, which require exogenous HS to proliferate, to investigate the HS requirements for FGF10-mediated proliferation and primary SMG epithelia to investigate the structural requirements of HS for FGF10-mediated epithelial morphogenesis. In BaF3/FGFR2b cells, heparin with at least 10 saccharides and 6-O-, 2-O-, and N-sulfates were required for maximal proliferation. During FGF10-mediated SMG epithelial morphogenesis, HS increased proliferation and end bud expansion. Defined heparin decasaccharide libraries showed that 2-O-sulfation with either an N-or 6-O-sulfate induced end bud expansion, whereas decasaccharides with 6-O-sulfation alone induced duct elongation. End bud expansion resulted from increased FGFR1b signaling, with increased FGFR1b, Fgf1, and Spry1 as well as increased Aqp5 expression, a marker of end bud differentiation. Duct elongation was associated with expression of Cp2L1, a marker of developing ducts. Collectively, these findings show that the size and sulfate patterns of HS modulate specific FGF10-mediated events, such as proliferation, duct elongation, end bud expansion, and differentiation, and provide mechanistic insight as to how the developmental localization of specific HS structures in tissues influences FGF10-mediated morphogenesis and differentiation.     

The co-evolution of language and emotions

We argue that language evolution started like the evolution of reading and writing, through cultural evolutionary processes. Genuinely new behavioural patterns emerged from collective exploratory processes that individuals could learn because of their brain plasticity. Those cultural-linguistic innovative practices that were consistently socially and culturally selected drove a process of genetic accommodation of both general and language-specific aspects of cognition. We focus on the affective facet of this culture-driven cognitive evolution, and argue that the evolution of human emotions co-evolved with that of language. We suggest that complex tool manufacture and alloparenting played an important role in the evolution of emotions, by leading to increased executive control and inter-subjective sensitivity. This process, which can be interpreted as a special case of self-domestication, culminated in the construction of human-specific social emotions, which facilitated information-sharing. Once in place, language enhanced the inhibitory control of emotions, enabled the development of novel emotions and emotional capacities, and led to a human mentality that departs in fundamental ways from that of other apes. We end by suggesting experimental approaches that can help in evaluating some of these proposals and hence lead to better understanding of the evolutionary biology of language and emotions.