Treatment of mice with the anticoccidial drug Toltrazuril does not interfere with the development of a specific cellular intestinal immune response to Eimeria falciformis

Immunity against Eimeria-infections is highly specific and it depends on cell-mediated effector mechanisms. Infections of BALB/c mice with 1000 sporulated oocysts of Eimeria falciformis led to protection against challenge infections. Treatment with the anticoccidium Toltrazuril, during primary infection, terminated the ongoing disease and did not interfere with the establishment of protective immunity against challenge infections. Mesenteric lymph node cells of infected, treated as well as non-treated and challenged BALB/c mice, showed a similar proliferation upon stimulation with parasite antigen. In contrast, neither cells of the Peyers patches, intraepithelial lymphocytes, nor spleen cells responded to stimulation with parasite antigens. Cells from all compartments and of all investigated groups proliferated and released the cytokines IFN- and IL-4 in response to the mitogen Concanavalin A. The number of cells releasing IFN- or IL-4 was not dependent on the status of infection or previous treatment with Toltrazuril. The serum IgG response against total sporozoite antigens of individual mice showed that in addition, a systemic humoral response developed in infected mice, independent of a previous drug treatment, although the specific IgG antibody concentration was higher in non-treated mice. Thus, Toltrazuril does not impair the parasite specific intestinal cellular and systemic antibody response and does not prevent the development of protection against challenge infection.     

Correcting signal biases and detecting regulatory elements in STARR-seq data

High-throughput reporter assays such as self-transcribing active regulatory region sequencing (STARR-seq) have made it possible to measure regulatory element activity across the entire human genome at once. The resulting data, however, present substantial analytical challenges. Here, we identify technical biases that explain most of the variance in STARR-seq data. We then develop a statistical model to correct those biases and to improve detection of regulatory elements. This approach substantially improves precision and recall over current methods, improves detection of both activating and repressive regulatory elements, and controls for false discoveries despite strong local correlations in signal.

Gene regulation is of foundational importance to nearly all biological processes, and variation in gene regulatory activity plays a major role in human disease risk (Lee and Young 2013; Parker et al. 2013; Finucane et al. 2015). A major step toward measuring regulatory activity across the human genome has been the development of high-throughput reporter assays such as STARR-seq (Arnold et al. 2013) that allow regulatory element activity to be quantified with high-throughput sequencing rather than with optical detection of a fluorescent or luminescent signal.High-throughput reporter assays create substantial analytical challenges that are distinct from other sequencing-based genomic assays. There is significant local variation in high-throughput reporter assay signal. We show here that, across data from several laboratories, most of that variation can be explained by features of the underlying genomic sequence and experimental procedures rather than by regulatory element activity. For example, nucleotide composition can alter PCR efficiency leading to under- and overrepresentation of some sequences. Meanwhile, highly repetitive sequences often do not align uniquely to the human reference genome, also biasing signal estimates. Additional analytical challenges include that STARR-seq signals can be both positive and negative, reflecting activation and repression, and the boundaries of regulatory elements are typically unknown and must therefore be estimated from the data. Those challenges together impact signal representations, hinder estimation of regulatory element activity, and cause false positives and false negatives when left unaddressed.Taken together, key requirements of statistical methods to analyze STARR-seq data are the ability to identify and estimate the effect of both activating and repressing regulatory elements while also correcting for underlying sequence biases in high-throughput reporter assays. A statistical model was recently introduced that corrects technical biases and detects regulatory elements in STARR-seq, but the model is limited to detecting only activating regulatory elements (Lee et al. 2020). Considering repression is a crucial gene regulation mechanism (Courey and Jia 2001), overlooking repressive elements may limit understanding of gene regulation with STARR-seq. To overcome that challenge, our correcting reads and analysis of differentially active elements (CRADLE) model takes a two-step approach. First, CRADLE uses a generalized linear regression model to estimate and correct major biases that we have identified in STARR-seq data. Next, CRADLE detects regions with statistically significant regulatory activity from the bias-corrected signals while rigorously controlling FDR. In doing so, CRADLE substantially improves the use of STARR-seq by providing a robust estimation of regulatory activity and improved visualization of raw signals.     

TRIO amplification and abundant mRNA expression is associated with invasive tumor growth and rapid tumor cell proliferation in urinary bladder cancer

Studies by comparative genome hybridization have suggested that 5p amplification is related to tumor progression in urinary bladder cancer. In this study seven genes (TAS2R, ADCY2, DNAH5, CTNND2, TRIO, ANKH, and MYO10) located to 5p15.31-5p15.1 were analyzed by fluorescence in situ hybridization using a tissue microarray containing samples from tumors and cell lines with known 5p amplification by comparative genome hybridization. Amplification frequency was highest for TRIO, which maps to 5p15.2 and encodes a protein with a putative role in cell-cycle regulation. To further investigate the role of TRIO amplification in bladder cancer, a tissue microarray containing samples from 2317 bladder tumors was used for fluorescence in situ hybridization analysis. TRIO amplification was strongly associated with invasive tumor phenotype, high tumor grade, and rapid tumor cell proliferation (Ki67 LI) (P < 0.0001 each). Only 7 of 456 pTaG1/G2 tumors (1.5%) but 62 of 485 pT1-4 carcinomas (12.8%) had TRIO amplification. TRIO amplification was not associated with poor prognosis. Using a frozen bladder tumor tissue microarray RNA in situ hybridization confirmed that TRIO is up-regulated in amplified tumors. It is concluded that TRIO up-regulation through amplification has a potential role in bladder cancer progression.     

Heat shock protein 60 from Chlamydia pneumoniae elicits an unusual set of inflammatory responses via Toll-like receptor 2 and 4 in vivo

Heat shock protein 60 (HSP60) from Chlamydia pneumoniae was described to trigger in vitro inflammatory and cytokine responses including TNF and IL-12p40. Although it can be found in atherosclerotic plaques of patients, the stimulatory potential of chlamydial and other HSP60 in vivo is unclear. We now report that chlamydial HSP60 fails to induce TNF expression in vivo, and significant serum levels of IL-12p40 are only found upon intraperitoneal injection of high doses of HSP60 or after intravenous application. Upon purification of chlamydial HSP60 with polymyxin B-agarose columns, its ability to induce TNF secretion in vitro is much reduced. However, purified chlamydial HSP60 causes increased serum levels of the CXC chemokines KC and MIP2 in vivo, as well as a strong accumulation of polymorphonuclear neutrophils (PMN) in the peritoneal cavity upon intraperitoneal challenge. With respect to PMN accumulation, chlamydial HSP60 is more potent than endotoxin or the CpG oligonucleotide 1668. The responses observed are completely abolished in Toll-like receptor (TLR)2/4-double-deficient mice, while single-deficient mice respond almost normally. Furthermore, KC induction and PMN accumulation are largely dependent on MyD88. In conclusion, HSP60 from C. pneumoniae triggers inflammatory responses in vivo that differ from responses induced by endotoxin or CpG oligonucleotides and are dependent on TLR2 and 4.     

Stage-specific protein synthesis by asexual blood stage parasites of Plasmodium knowlesi

P. knowlesi parasites with a maximum age distribution of 3 h were metabolically labelled with [³⁵S]methionine during 9 sequential non-overlapping intervals, from young rings to mature segmented schizonts. The proteins synthesised at the different stages were compared using sodium dodecyl sulphate-polyacrylamide gel electrophoresis; more than 40 polypeptides (M_r 20 000 to over 200 000) were identified in the different parasite preparations. The major polypeptides synthesised by rings and trophozoites of different ages were similar, but differences in minor polypeptides could always be recognised. At the onset of schizogony ring and trophozoite specific proteins ceased to be synthesised and proteins specific to schizogony emerged. In general, schizont-specific proteins were of higher molecular weight than ring stage proteins. Details of the morphological changes which occurred during the metabolic labelling episode permits correlation between parasite structure and synthesis of particular polypeptides. Comparison of parasite components metabolically labelled with [³H]glucosamine during defined periods of development also revealed stage-specific synthesis of glycoproteins. The extent to which proteins are altered after synthesis has been investigated by pulse-chase experiments.     

LTD4 augments TNF releasein vivo andin vitro

In vivo data suggest a role of LTD₄ in mediating endotoxin (LPS)-inducible liver injury in galactosamine-sensitized mice. Leukotriene D₄ (LTD₄) was shown to synergize in this model with subtoxic amounts of LPS in inducing hepatitis. Mice challenged i.v. with a subtoxic dose of LPS [50 ng/kg] showed significant TNF serum levels 90 min later which were sixfold increased by coadministration of 50 μg/kg LTD₄. When rat Kupffer cells were challenged with LPS, TNF-α measured in the supernatant was significantly increased by LTD₄ [100 pg–100 ng/ml]. Addition of LTD₄ alone did not result in any detectable TNF formation.Since Kupffer cells are known producers of small amounts of LTD₄, it seems feasible that LTD₄ represents an autocrine stimulus of nonparenchymal liver cells. In fact, different LTD₄ synthesis inhibitors and receptor antagonists attenuated LPS-inducible TNF release of rat Kupffer cells supporting the conclusion that LTD₄ acts as an endogenous autocrine enhancer of liver macrophage TNF release.     

Comparison of Frontal EMG Biofeedback and Several Types of Relaxation Instructions in Reducing Multiple Indices of Arousal

During the training phase, 96 subjects were given one of four types of relaxation instructions (single instructions, repeated instructions, relaxation training, no instructions) and in addition either did or did not receive frontal EMG biofeedback training. Results indicated that each of the instructions and biofeedback procedures were equally effective in reducing frontal EMG, but that none of these procedures had any effect on subjective anxiety or autonomic indices of arousal (pulse rate, skin temperature, and finger pulse volume). During the generalization/stress phase, subjects were threatened with electric shock and were told to apply the relaxation techniques they learned during the training phase even though no additional instructions and/ or biofeedback training would be provided. To assess the effectiveness of the shock manipulation, a no-threat control group was included. Results indicated that: a) the shock manipulation was effective in increasing arousal, b) previous instructions and/or biofeedback were equally effective in reducing frontal EMG levels, but that c) only relaxation training was consistently effective in reducing subjective and autonomic indices of arousal. These findings: a) suggest that in stressful situations, relaxation training may be more effective than either EMG biofeedback or simple relaxation instructions in producing a general relaxation effect as opposed to a specific EMG effect; and b) indicate the importance of assessing the effectiveness of relaxation procedures during stressful situations during which subjects’ levels of arousal are elevated above resting baseline levels.     

Sunday Canyon virus, a new ungrouped agent from the tick Argas (A.) cooleyi in Texas.

A new arbovirus was isolated from Texas, U.S.A., populations of the Cliff Swallow parasits Argas (Argas) cooleyi Kohls and Hoogstraal, 1960. The virus, named Sunday Canyon, is serological urelated to any of 185 arbovirus strains or 20 other viral agents with which it was compared. Morphologically it resembles Bunyamwera viruses and, in common with them, is sensitive to lipid solvents and acid pH, and apparently possesses RNA. Although considerably resistant to a temperature of 41.5 degrees C, it rapidly loses infectivity when incubated at 56 degrees C. It is lethal for newborn white mice and infective for the Vero and Antheraea eucalypti cell lines. Sundays Canyon virus is the second tick-associated, Bunyamwera virus-like agent known from North America and the third virus to be reported from A. cooleyi in Texas.     

Working with community mental health professionals: a survey among general practitioners.

Links between general practitioners and mental health professionals, such as counsellors, psychiatrists, community psychiatric nurses, clinical psychologists and social workers, are increasing in number and type. The aim of this survey was to elicit general practitioners' attitudes to these workers, comparing those with a link with a mental health worker and those without. General practitioners in two district health authorities were surveyed and a response rate of 70% was obtained. General practitioners linked to a mental health professional were more likely to have made a referral to that service in the previous three months and, on the whole, were more satisfied with that service. The commonest problem reported by respondents was the length of waiting lists. Regarding liaison with social workers, inadequate feedback and difficulty with contact were the problems mentioned most by doctors. A number of general practitioners expressed a desire for closer contact with all these mental health services. While caution is required in ascribing causality to these relationships, it is clear that a closer working relationship between general practitioners and mental health workers is productive and is valued by general practitioners. The challenge for policy makers is to structure mental health provision in such a way that more general practitioners are able to benefit than at present.     

New fimbrial antigenic type (E8775) that may represent a colonization factor in enterotoxigenic Escherichia coli in humans.

An enterotoxigenic strain of Escherichia coli O25:H42 (strain E8775), isolated from a patient in Bangladesh with diarrhea, caused mannose-resistant hemagglutination (MRHA) of human and bovine erythrocytes. The strain did not show slide agglutination or immunodiffusion precipitin lines with antiserum specific for the colonization factor antigen CFA/I or CFA/II. A variant E. coli strain, E8775-B, did not cause MRHA or produce enterotoxin. Electron microscopy revealed the presence of fimbriae on the surface of strain E8775 but not strain E8775-B. When strain E8775 was grown at 22 degrees C, it became MRHA negative and fimbriae were absent. An antiserum prepared against strain E8775 was absorbed with strain E8775-B to make an antiserum specific for the fimbrial antigen. Using this absorbed antiserum, we found the fimbrial antigen in 48 of 742 enterotoxigenic E. coli strains. The 48 strains belonged to serogroups O25, O115, and O167. It is suggested by analogy to the properties of previously described colonization factors that these fimbriae may play a part in the colonization of the intestinal epithelium.