Real-time PCR methods for independent quantitation of TTV and TLMV

There is considerable interest in the possible clinical effects of the human circoviruses TT virus (TTV) and TTV-like mini virus (TLMV). Most people appear to have at least one of these viruses replicating actively in their bodies, thus mere correlation of the presence of virus and disease states are probably less informative than a quantitative analysis of viraemia. Real-time PCR based methods, with either SYBR Green or TaqMan probe, designed to quantitate selectively TTV and TLMV are described. The suggested TaqMan-based protocols were suitable for quantitation of viruses in the range of 10(2)-10(9) copies/ml of sample; and proved, by sequencing of PCR products, to be specific for each of the two viruses.     

Analysis of aberrant somatic hypermutation (SHM) in non-Hodgkin's lymphomas of patients with chronic HCV infection

Hepatitis C virus (HCV) and aberrant somatic hypermutation (SHM) have each been suggested to contribute to the development of B-cell non-Hodgkin's lymphoma (NHL). The incidence of PIM-1, PAX-5, RhoH/TTF, and c-MYC mutations in tumour biopsy specimens from 32 HCV-infected B-cell NHL patients was analysed to determine whether the extent of aberrant SHM among these patients differed from that previously reported for HCV-negative B-cell NHL patients. Mutation of PIM-1, PAX-5, RhoH/TTF, and c-MYC was detected in 4 (13%), 5 (16%), 4 (13%), and 4 (13%) of 32 samples, respectively. In HCV-positive B-cell NHL patients, the frequency of aberrant SHM was lower than that already found in HCV-negative B-cell NHL patients. This indicates that, unlike B-cell lymphomas from HCV-negative patients, aberrant SHM may not contribute significantly to malignant transformation in HCV-associated B-cell lymphomas.     

Circulating inflammatory mediators during start of fever in differential diagnosis of gram-negative and gram-positive infections in leukopenic rats

Gram-negative and gram-positive infections have been considered the most important causes of morbidity and mortality in patients with leukopenia following chemotherapy. However, discrimination between bacterial infections and harmless fever episodes is difficult. Because classical inflammatory signs of infection are often absent and fever is frequently the only sign of infection, the aim of this study was to assess the significance of serum interleukin-6 (IL-6), IL-10, macrophage inflammatory protein-2 (MIP-2), procalcitonin (PCT), and C-reactive protein (CRP) patterns in identifying bacterial infections during start of fever in normal and cyclophosphamide-treated (leukopenic) rats following an injection of lipopolysaccharide (LPS) or muramyl dipeptide (MDP) as a model for gram-negative and gram-positive bacterial infections. We found that, compared to normal rats, immunosuppressed animals exhibited significantly higher fevers and lesser production of all mediators, except IL-6, after toxin challenge. Moreover, compared to rats that received MDP, both groups of animals that received an equivalent dose of LPS showed significantly higher fevers and greater increase in serum cytokine levels. Furthermore, in contrast to those in immunocompetent rats, serum levels of IL-6 and MIP-2 were not significantly changed in leukopenic animals after MDP injection. Other serum markers such as PCT and CRP failed to discriminate between bacterial stimuli in both groups of animals. These results suggest that the use of the analyzed serum markers at an early stage of fever could give useful information for the clinician for excluding gram-negative from gram-positive infections.     

Antibody response to a synthetic peptide derived from the human papillomavirus type 6/11 L2 protein in recurrent respiratory papillomatosis: Correlation between southern blot hybridization,polymerase chain reaction,and serology

Recurrent respiratory papillomatosis (RRP) is the most common benign tumour of the larynx, affecting both children and adults. We present a series of 25 patients, including 10 cases of juvenile multiple, 8 cases of adult solitary, and 7 cases of adult multiple RRP. Biopsy tissue from each patient was screened by Southern blot hybridization and polymerase chain reaction for the presence of human papillomavirus (HPV) DNA. Sera from patients and age- and sex-matched controls were tested for the presence of HPV-specific antibodies using a synthetic pep-tide derived from the minor capsid protein (L2) of HPV 6/11. By Southern blot hybridization and/or polymerase chain reaction, biopsies from all patients were positive for HPV 6/11 DNA. There was no difference in antibody response between cases and controls. Female cases and controls had significantly higher antibody titers than male subjects. A correlation was observed between the HPV-specific antibody level and the number of surgery-necessitating recurrences. © 1994 Wiiey-Liss, Inc.     

Chemoattractant-induced NADPH oxidase activity in human monocytes is terminated without any association of receptor-ligand complex to cytoskeleton

When the chemotactic peptide formylmethionyl-leucyl-phenylalanine binds to its cell surface receptor, a transmembrane signal is generated that activates the superoxide-producing NADPH oxidase of human phagocytes. Comparing monocytes and neutrophils with regard to the production of superoxide anion induced by the peptide, we found a similar time-course for both types of cells. In neutrophils, ligand binding induced a conversion of the receptor to a high-affinity form, a change suggested to be due to an association of the receptor-ligand complex to the Triton X-100-insoluble cytoskeleton. This event has been hypothesized to terminate the signal that activates the NADPH oxidase and thereby results in cessation of the cellular production of superoxide anion. Neutrophils preincubated with the cytoskeleton-disrupting drug cytochalasin B showed an increased and prolonged superoxide anion production after activation with the peptide, thus indicating that the cytoskeleton is involved in terminating this response. Formylmethionyl-leucyl-phenylalanine was also found to induce polymerization of actin in monocytes; however, cytochalasin B had no effect on the peptide-induced generation of superoxide anion in these cells. Furthermore, also in monocytes, ligand binding induced a conversion of the receptor to a high-affinity form; however, the receptor-ligand complex did not coisolate with the Triton X-100-insoluble cytoskeleton. These results indicate that, in monocytes, the NADPH oxidase activating pathway is terminated without any association of the receptor-ligand complex to the Triton X-100-insoluble cytoskeleton.     

An intact heavy chain at the actin-subfragment 1 interface is required for ATPase activity of scallop myosin

Summary Scallop S1 has a region sensitive to tryptic hydrolysis not found thus far in S1s of other species, located 65K from theN-terminus as determined by SDS/polyacrylamide-gel electrophoresis. In the presence of actin the S1 heavy chain is preferentially cleaved at this site. The high-salt EDTA and calcium ATPase activities of the nicked 65K–31K S1 are abolished. This inactivation is not due to denaturation, conformational effects of actin, or to light chain dissociation. The unique proteolytic site of scallop S1 is adjacent to a peptide involved in actin-S1 interaction in scallop and rabbit but it is far removed from the nucleotide-binding site in the linear amino acid sequence. We conclude that proteolysis inactivates the high-salt ATPase activities through a connection mediated by tertiary interactions. Such a connection provides a structural correlate for the known reciprocal relationship between the nucleotide and actin affinities of myosin.Abbreviations S1 papain subfragment 1 containing both light chains - S1^–LC papain subfragment 1 prepared in the absence of bound regulatory light chain containing only a part of the essential light chain and having a shorter heavy chain - HC heavy chain - LC light chain - DTT dithiothreitol - TLCK 7-amino-1-chloro-3-l-tosylamidoheptan-2-one - TPCK 1-chloro-4-phenyl-3-l-toluene-p- sulphonamidobutan-2-one - K kilodalton     

Immunoglobulins in the egg,embryo and young chick

Current knowledge of the immunoglobulin classes identified in some avian species is reviewed. The distribution and fate of passively acquired immunoglobulins or specific antibodies in compartments of the egg and of the developing embryo and in the newly hatched chick are described, together with the ontogeny of active Ig biosynthesis.     

Hyperplastic-like colon polyps that preceded microsatellite-unstable adenocarcinomas

We compared hyperplastic-like polyps that preceded microsatellite-unstable adenocarcinomas to incidental hyperplastic polyps to identify distinguishing morphologic criteria. The study group included 106 hyperplastic-like, nonadenomatous, serrated polyps, most from the ascending colon in 91 patients; the control group included 106 rectosigmoid hyperplastic polyps from 106 patients in whom adenocarcinoma did not develop. Study group polyps had an expanded crypt proliferative zone, a serrated architectural outline that became apparent in the basilar crypt regions, basilar crypt dilation, inverted crypts, and a predominance of dysmaturational crypts (crypts with minimal cell maturation). In contrast, control group polyps had a proliferative zone confined to the basal crypt region, serrated architecture that became apparent in the superficial crypt region, rare to no basilar crypt dilation, and rare or no dysmaturational crypts. Hyperplastic-like polyps that preceded microsatellite-unstable adenocarcinomas had a distinctive constellation of morphologic features related to altered and decreased cell function and control that resulted in dysmaturational crypts. Dysmaturation constitutes a range of morphologic alterations, some of which overlap with incidental-type innocuous hyperplastic polyps. The morphologic features described herein provide initial guidelines to identify this potentially important subset of premalignant serrated-like polyps.     

Monoaminergic Fluorescence in Frog Skin

The Falck-Hillarp fluorescence technique was employed in an attempt to determine the distribution of sympathetic innervation in frog skin. No evidence was found of a direct monoaminergic nerve supply to the cells of the non-glandular epithelium in the epidermis. Instead, specific fluorescence was mainly confined to the vicinity of the skin glands. Fluorescent fibers were observed surrounding the mucous type of gland. The secretory content of this gland was not fluorescent. In the granular type of gland the main source of fluorescence was the secretory granules filling the lumen. These developed a fluorescence in the spectral range of 5-hydroxytryptamine. The brightness of the fluorescence indicated a very high content of this amine. Fluorimetric analysis showed that no catecholamines were present in the secretion. In glands devoid of secretory granules there were some indications of a monoaminergic innervation of the secretory epithelium, but this was hard to determine because of the abundant nonspecific fluorescence. Sparse dots of specific fluorescence were found close to the surrounding smooth muscle cells. — These findings rule out the possibility of a direct sympathetic nervous control of the non-glandular epithelium in frog skin but indicate that this is instead confined to the skin glands.