Localization of IFN-gamma-activated Stat1 and IFN regulatory factors 1 and 2 in breast cancer cells.

The aim of the present work was to evaluate the induction and localization of Stat1, interferon (IFN) regulatory factor-1 (IRF-1), and IRF-2 after IFN-gamma exposure of human breast cancer cell lines, SKBR3, MDA468, MCF7, and BT20. Results from growth assays, Western staining, electrophoretic mobility shift assay (EMSA), and immunohistochemical staining were collated to test our hypothesis that immunohistochemical analysis of Stat1, IRF-1, and IRF-2 would provide additional information about the functionality of the IFN-gamma signaling pathway in human tumor lines. EMSA results showed that in each of four cell lines, Stat1 expression was increased and demonstrated functional activity after IFN-gamma stimulation. Western and EMSA analysis showed upregulation of IRF-1 but not IRF-2 in each cell line. Confocal microscopy of cells stained for Stat1, IRF-1, and IRF-2 confirmed the results and also provided novel information about the intracellular localization of proteins and intercellular variations in responses. The proportion of cells with IRF-1 stimulation and translocation was positively correlated with the IFN-gamma growth suppression in vitro. In conclusion, using four independent assays, we have demonstrated that heterogeneity in IFN-gamma-mediated upregulation of signal transduction proteins can be detected in vitro and that these differences can explain distinct cellular growth effects.     

How clients with religious or spiritual beliefs experience psychological help‐seeking and therapy: A qualitative study

This qualitative study explored the process of help‐seeking and therapy among clients with religious or spiritual beliefs. Ten clients who were currently in, or had recently finished, therapy were interviewed. Participants reported using their religious or spiritual beliefs to cope with their psychological problems before and during therapy. Prior to therapy, they were worried that secular‐based help might weaken their faith. However, the experience of having psychological distress and the process of receiving therapy were both perceived as strengthening to faith and ultimately part of a spiritual journey. Contrary to expectations, a match between the spirituality or religious affiliation of the therapist and client was not considered important. This implies that the ‘religiosity gap’ between secular therapists and clients with religious/spiritual beliefs is bridgeable. Copyright © 2007 John Wiley & Sons, Ltd.     

Quantitative nucleic acid sequence-based assay as a new molecular tool for detection and quantification of Leishmania parasites in skin biopsy samples

Currently available methods for the diagnosis of cutaneous leishmaniasis (CL) have low sensitivities or are unable to quantify the number of viable parasites. This constitutes a major obstacle for the diagnosis of the disease and for the study of the effectiveness of treatment schedules and urges the development of improved detection methods. In this study, quantitative nucleic acid sequence-based amplification (QT-NASBA) technology was used to detect and quantify Leishmania parasites in skin biopsy samples from CL patients. The assay is based on the detection of a small subunit rRNA (18S rRNA), which may allow for the detection of viable parasites. The QT-NASBA assay was evaluated using in vitro-cultured promastigotes and amastigotes and 2-mm skin biopsy samples from Old and New World CL patients. The study demonstrated that the lower detection limit of the QT-NASBA was two parasites per biopsy sample. Parasites could be quantified in a range of 2 to 11,300,000 parasites per biopsy sample. The QT-NASBA could detect levels of parasites 100-fold lower than those detected by conventional PCR. Test evaluation revealed that the QT-NASBA had a sensitivity of 97.5% and a specificity of 100% in the present study. The QT-NASBA is a highly sensitive and specific method that allows quantification of both Old and New World Leishmania parasites in skin biopsy samples and may provide an important tool for diagnosis as well as for monitoring the therapy of CL patients.     

Genome-wide search for atopy susceptibility genes in Dutch families with asthma

BACKGROUND: Atopy is a phenotype associated with asthma that has a heritable component. However, the role of atopysusceptibility genes in the development and expression of asthma and allergic disorders is not understood. OBJECTIVE: We sought to study the familial aggregation and co-occurrence of atopic phenotypes within family members of patients with asthma and to identify chromosomal regions that may contain genes that regulate different atopic phenotypes. METHODS: In 200 families (n = 1174) ascertained through a proband with asthma, genome-wide screen and linkage analysis was performed for the following atopic phenotypes: (1) specific IgE to common aeroallergens (Phadiatop assay); (2) specific IgE to Der p 1; (3) positive skin test responses to house dust mite; (4) positive skin test responses to 1 or more of 16 allergens; and (5) peripheral blood eosinophils. Results were compared with the linkage results for total serum IgE levels. RESULTS: There was clear familial aggregation of atopy. A high total serum IgE level in combination with a positive Phadiatop result or a normal total IgE level in combination with a negative Phadiatop result was found in 56.1% of the probands and 66.9% of the offspring. Several chromosomal regions that showed evidence for linkage to an atopic phenotype (ie, 2q, 6p, 7q, and 13q) also showed evidence of linkage with total serum IgE (Xu et al. Am J Hum Genet 2000;67:1163-73). Specific regions of interest for atopic traits were also detected on chromosomes 11q, 17q, and 22q. CONCLUSIONS: Atopic phenotypes show familial aggregation, although family members may differ in expression of atopy. Specific chromosomal regions appear to be important in susceptibility to different phenotypes of atopic responsiveness.     

Role of lymphokines in immunoregulatory function of mucosal T cells in humans and nonhuman primates

The findings presented above and in other studies provide substantial evidence that lymphocytes in the intestinal lamina propria differ from lymphocyte populations in the circulation or in other tissue sites in a number of ways. First, lamina propria lymphocytes are phenotypically distinct and have evidence of activation. Lymphocytes in the intestinal lamina propria are different in their potential for expression of lymphokine gene products, since activated cells from the lamina propria have high expression of mRNA for IL-2, IL-4, IL-5 and IFN-gamma in comparison to circulating lymphocytes. Mesenteric lymph node T cells also differ from circulating lymphocytes in their high expression of IL-4 and IL-5 mRNA. A further difference between mesenteric lymph node and lamina propria T cells is that the former are capable of proliferating in response to IL-4, whereas the latter are not. These phenotypic and mRNA differences of lamina propria lymphocytes also correlate well with their high helper activity in vitro for immunoglobulin synthesis in the pokeweed mitogen system. Finally, lamina propria T cells at a site of inflammation are able to provide high helper activity in response to specific antigens. These observations are all consistent with the conclusion that T cells in the lamina propria are pleomorphic, but are highly enriched for subpopulations of activated memory cells that are geared for effector functions. These functions are likely to be critical in maintaining normal host defense in the mucosal environment.     

Characterization of creatinine error in ketotic patients. A prospective comparison of alkaline picrate methods with an enzymatic method

Creatinine measurement by alkaline picrate reagents is subject to positive interference by acetoacetate. Enzymatic reagents avoid this interference and have been adapted to instruments such as the Ektachem-400 (Kodak). By documenting the discrepancy between alkaline picrate and Ektachem determinations for creatinine, the authors prospectively identified ketotic patients in whom the presence of ketones was responsible for a significant creatinine error. During their three-month survey, they identified 50 such ketotic inpatients. Those admitted to the medicine service represented almost 5% of all medicine admissions over this time. Of the total specimens, the mean discrepancy was 14 +/- 8 mg/L with a range of 4-44 mg/L. The greater the ketosis, the greater the discrepancy. Two-thirds of the samples were normal on the Ektachem but greater than normal by picrate methods. In addition to diabetes or ethanol abuse, 17% of the ketotic patients had severe or terminal illness that was generally associated with malnutrition.     

Effect of hydrostatic pressure on ADH induced osmotic water flow in toad bladder

The effect of hydrostatic pressure (HP) on antidiuretic hormone (ADH) stimulated osmotic water flow (Jv) across the toad urinary bladder was evaluated. Jv for ADH-stimulated bladders was significantly reduced by an elevation of the serosal HP gradient to 1 cm H₂O. Subsequent elimination of the HP gradient resulted in a recovery of Jv. Serosal HP also caused a reversible increase in sucrose permeability (P sucrose). For ADH-treated bladders fixed with glutaraldehyde during serosal HP exposure, subsequent exposure to a mucosal or serosal HP gradient caused acceleration or inhibition of Jv, respectively. The reduction in ADH-associated Jv with serosal HP was apparently caused by a back-flux of water through a paracellular pathway. Jv and P sucrose were not affected by mucosal HP during ADH stimulation. The results suggest a specific sensitivity of a paracellular pathway to a small serosal HP gradient in bladders with ADH-stimulated water flow. The reversibility of this effect on P sucrose suggests that the elements comprising the apical junctions are dynamic structures capable of recovering at least some of their permeability properties.     

Immunohistochemical localization of pepsinogen A and C containing cells in Barrett's oesophagus

Summary No data are available on the localization of Pepsinogen A (PGA=PG I) and Pepsinogen C (PGC=PG II) positive cells in Barrett's epithelium. Endoscopic biopsy specimens were taken from the columnar epithelium from 23 patients (n=93), and in addition from the cardia from eight healthy control subjects (n=38). The tissue was stained by the immunoperoxidase technique with specific anti-pepsinogen antisera, and double immunostained for PGA and PGC. In the Barrett's epithelium PGA was found in 28 out of 93 biopsy specimens (30.1%) and PGC in 55 out of 93 (59.1%). Chief cells always stained both for PGA and PGC, while clear mucous cells were often PGA– and PGC+. PGA+ and PGC+ cells were found each in 100% of the biopsy specimens with fundic type epithelium, in 21.7% and 70.7% of biopsy specimens with junctional type, in 0% and 26.1% of biopsy specimens with specialized epithelium and in 12.5% and 43.5% of biopsy specimens with mixed junctional/specialized features respectively. Dysplastic epithelium stained always negatively with both anti-pepsinogen antisera. In most control cardia biopsy specimens PGA as well as PGC were demonstrable; occasionally clear mucous glands were PGA– and PGC+.It is concluded that pepsinogen-containing cells can be accurately identified in the Barrett's epithelium; their presence seems related to the histological cell type. Identification of pepsinogen positive cells may contribute to a more accurate morphological classification of the Barrett's epithelium.Presented in part at the Annual Meeting of the American Gastroenterological Association, San Francisco, May 1986     

Abeta-degrading endopeptidase,neprilysin, in mouse brain: synaptic and axonal localization inversely correlating with Abeta pathology

Metabolism of amyloid-beta peptide (Abeta) is closely associated with the pathology and etiology of Alzheimer's disease (AD). Since neprilysin is the only rate-limiting catabolic peptidase proven by reverse genetics to participate in Abeta metabolism in vivo, we performed detailed immunohistochemical analysis of neprilysin in mouse brain using neprilysin-deficient mice as a negative control. The aim was to assess, at both the cellular and subcellular levels, where Abeta undergoes neprilysin-dependent degradation in the brain and how neprilysin localization relates to Abeta pathology in amyloid precursor protein (APP)-transgenic mice. In hippocampus, neprilysin was present in the stratum pyramidale and stratum lacunosum-moleculare of the CA1-3 fields and the molecular layer of the dentate gyrus. Confocal double immunofluorescence analyses revealed the subcellular localization of neprilysin along axons and at synapses. This observation suggests that after synthesis in the soma, neprilysin, a type II membrane-associated protein, is axonally transported to the terminals, where Abeta degradation is likely to take place. Among various cell types, GABAergic and metabotropic glutamate 2/3 receptor-positive neurons but not catecholaminergic or cholinergic neurons, expressed neprilysin in hippocampus and neocortex, implying the presence of a cell type-specific mechanism that regulates neprilysin gene expression. As expected, Abeta deposition correlated inversely with neprilysin expression in TgCRND8 APP-transgenic mice. These observations not only support the notion that neprilysin functions as a major Abeta-degrading enzyme in the brain but also suggest that down-regulation of neprilysin activity, which may be caused by aging, is likely to elevate local concentrations of Abeta at and around neuronal synapses.     

Foreign bodies,exogenous & endogenous triggering mechanical effects,good/bad,in the airways. The anesthesiologist as the diagnostician

Fifty odd years ago, in his historic book, 'The Silent World,' Jacques Cousteau told us that scuba divers breathing compressed air were apt to get a 'high,' which he called, 'Rapture of the Deep,' and a form of cerebral arrest, which he termed, 'Nitrogen Narcosis'. Furthermore, these submarine soloists, also like persons under general anesthesia, are prone to mysterious sudden death. All this reminds us of 'ether parties,' and contemporary snorters and huffers, sniffing such foreign bodies as, glue, cocaine, paint-thinner, gasoline, spray-can-propellant, etc. Relevant is the fact that the endogenous and other soporifics which arise within the body proper (endo-integumentarily) do not induce general anesthesia, which is cerebral arrest, until they have entered the extra-integumentary mucosal compartments called airways. Nitrogen is an inert gas, so its actions are neither toxic nor chemical, but mechanical. The effects of endogenous soporifics such as acetone, carbon dioxide, alcohol, and ammonia, are usually assumed to be toxic and endo-integumentary. Therefore, I must emphasize the fact that they do not cause 'Highs,' somnolence, nor deaths until/unless they have entered the extra-integumentary airways. Certainly those agents may also have some endo-integumentary actions. I must also stress the fact that foreign bodies in the airways which do not pierce the integument to enter the body proper, such as endotracheal or tracheostomy tubes, lumps of meat, mucus plugs (boogers), etc., can also occasionally trigger cerebral, respiratory and even cardiac arrests locally and mechanically in the airways, depending on which trigger points they stimulate and how powerfully. Four points explain the mechanism of general anesthesia: (a) Endogenous soporifics do not induce sleep until they have entered the extra-integumentary airways, and contacted the mural mucosa; (b) however administered, intravenously, rectally, or by inhalation, exogenous general agents do not anesthetize until they enter the airways and contact the extra-integumentary mucosa; (c) other foreign bodies, e.g., mishandled endotubes in the airways, which cannot pierce the integument to enter the body proper, can and do trigger cerebral/respiratory/cardiac arrest locally, mechanically, and extra-integumentarily. These effects are neither toxic, nor chemical; (d) Some agents, which can and do pierce the integument, having induced anesthesia, will later exit the body chemically unchanged by their endo-integumentary sojourn, which suggests that their modus operandi is mechanical.