Prevalence of XMRV nucleic acid and antibody in HIV-1-Infected men and in men at risk for HIV-1 Infection.

Xenotropic MLV-Related Virus (XMRV) was recently reported to be associated with prostate cancer and chronic fatigue syndrome (CFS). Infection was also reported in 3.7% of healthy individuals. These highly reported frequencies of infection prompted concerns about the possibility of a new, widespread retroviral epidemic. The Multicenter AIDS Cohort Study (MACS) provides an opportunity to assess the prevalence of XMRV infection and its association with HIV-1 infection among men who have sex with men. Reliable detection of XMRV infection requires the application of multiple diagnostic methods, including detection of human antibodies to XMRV and detection of XMRV nucleic acid. We, therefore, tested 332 patient plasma and PBMC samples obtained from recent visits in a subset of patients in the MACS cohort for XMRV antibodies using Abbott prototype ARCHITECT chemiluminescent immunoassays (CMIAs) and for XMRV RNA and proviral DNA using a XMRV single-copy qPCR assay (X-SCA). Although 9 of 332 (2.7%) samples showed low positive reactivity against a single antigen in the CMIA, none of these samples or matched controls were positive for plasma XMRV RNA or PBMC XMRV DNA by X-SCA. Thus, we found no evidence of XMRV infection among men in the MACS regardless of HIV-1 serostatus.

Optimal neural differentiation and extension of hybrid neuroblastoma cells (NDC) for nerve-target evaluations using a multifactorial approach.

In vitro models of tissues, such as the cornea, represent systems for modeling cell-to-cell interactions and tissue function. The objective of this study was to develop an optimized nerve differentiation medium to incorporate into a 3D in vitro model to study innervation and cell targeting. A hybrid neuroblastoma cell line (NDC) was examined for its ability to differentiate into neurons, produce neurites, and functionally contact target cells. Neuronal differentiation of NDCs was optimized through a combinatorial approach which involved culturing cells in the presence of various extracellular matrices and soluble factors. A serum-free medium containing nerve growth factor (NGF), dimethyl sulfoxide (DMSO), or dexamethasone resulted in the greatest proportion of NDCs demonstrating a neuronal morphology. Similarly, with supplementation of cyclic AMP (cAMP) or NGF, neurite extension was optimized. Combining these factors generated an optimized differentiation and extension medium, relative to the individual components alone. In co-culture with epithelial cells, NDC neurites generated in the optimized medium formed contacts with epithelial targets and produced substance P. Similarly, NDCs seeded into a collagen matrix produced neurites that projected through the matrix to target epithelial cells, promoted epithelial stratification, and increased the rate of epithelial wound healing. As well, differentiated NDCs could target and alter acetylcholine receptor clustering in mouse C2C12 myotubes, demonstrating synaptic plasticity. Our data supports the use of NDCs, in combination with optimized medium, for generating an innervated in vitro model.

Isolation and characterizaton of Edwardsiella tarda from pacu Myleus micans / Isolamento e caracterização de Edwardsiella tarda em pacu Myleus micans

Investigaram-se as causas da mortalidade de peixes ocorrida em janeiro de 2005 na bacia do Rio São Francisco, Brasil. Edwardsiella tarda foi isolada dos rins de pacu Myleus micans. O isolado, denominado Et-LIS, caracterizado por bastonetes Gram negativos móveis, foi identificado por testes bioquímicos e confirmado pelo kit comercial Bactray. A susceptibilidade a 10 drogas das 12 testadas foi determinada pelo método de difusão de discos, enquanto as características de virulência foram avaliadas mediante inoculação experimental em Cyprinus carpio e em Oreochromis spp. Ambas as espécies desafiadas apresentaram sinais compatíveis com infecção por E. tarda. As tilápias (Oreochromis spp.) morreram 48h após a inoculação, enquanto as carpas (Cyprinus carpio) sobreviveram por 72h. Este é o primeiro relato da ocorrência de E. tarda em pacu.

Effects of substerilizing doses of gamma radiation on adult longevity and level of inherited sterility in Teia anartoides (Lepidoptera: Lymantriidae).

The effects of substerilizing doses of gamma radiation on the longevity and level of inherited sterility in the Australian moth Teia anartoides Walker were determined. Six day-old male pupae were treated with 0, 100, and 160 Gy of gamma radiation by using a 1.25 MeV Cobalt60 irradiation source. Laboratory studies of male longevity showed that radiation had little impact in adult moths of the P1, F1, and F2 generations. Inherited deleterious effects resulting from irradiation were observed in the progeny of F1 and F2 generations. Outcrosses between substerile parental males or their highly sterile male progeny to wild-type females did not affect female fecundity. However, adverse effects were observed for these crosses in the rates of successful egg hatch and postembryonic development. Fertility was always greater in out-crosses involving a P1 male than in any of the F1 out-crosses. F1 males were always more sterile than F1 females, and the level of sterility for the F1 and F2 generations was higher than that of the controls. The incidence of larval and pupal mortality was higher in the F2 than the F1 generation. A dose of 100 Gy had the highest success in inducing deleterious effects that were inherited through to the F2 generation. Our results indicated that the use of partially sterilizing doses of radiation has good potential as a selective strategy for management or eradication of T. anartoides.

Fixed-dose pegfilgrastim is safe and allows neutrophil recovery in patients with non-Hodgkin's lymphoma.

Twenty-nine patients with non-Hodgkin's lymphoma received a single subcutaneous injection of 6 mg pegfilgrastim approximately 24 h after the start of CHOP chemotherapy. The safety of pegfilgrastim in this patient population was determined by reports of adverse events. The pharmacokinetics of pegfilgrastim were characterized and the duration of grade 4 neutropenia, time to absolute neutrophil count (ANC) recovery to > or = 2.0 x 10(9)/l, neutrophil nadir, and incidence of febrile neutropenia were determined in the first 21-day chemotherapy cycle. The incidence of grade 4 neutropenia in cycle 1 was 43% with a mean (SD) duration of grade 4 neutropenia value of 1.0 (1.4) day. No apparent relationship between the duration of grade 4 neutropenia and body weight was observed. The median [quartiles] time to ANC recovery was 10 [9, 11] days. The incidence of febrile neutropenia was 11%. No unexpected adverse events were reported and no patient developed antibodies to pegfilgrastim. Serum concentration of pegfilgrastim reached a maximum (median [quartiles]) of 128 [58, 159] ng/ml at approximately 24 h after administration, and was followed by a second smaller peak (median [quartiles]) of 10.6 [3.0, 20.5] ng/ml at the time of the neutrophil nadir. After the second peak, concentration of pegfilgrastim declined linearly with a median terminal half-life of approximately 42 h.

Randomized, multicenter, open-label study of pegfilgrastim compared with daily filgrastim after chemotherapy for lymphoma.

PURPOSE: The primary objective was to assess the duration of grade 4 neutropenia (neutrophil count < 0.5 x 10(9)/L) after one cycle of chemotherapy with etoposide, methylprednisolone, cisplatin, and cytarabine in patients randomly assigned to receive one dose of pegfilgrastim or daily filgrastim after chemotherapy. Febrile neutropenia, neutrophil profiles, time to neutrophil recovery, pharmacokinetics, and safety were also assessed. PATIENTS AND METHODS: An open-label, randomized, phase II study was designed to compare the effects of a single subcutaneous injection of pegfilgrastim (sustained-duration filgrastim) 100 micro g/kg per chemotherapy cycle (n = 33) with daily subcutaneous injections of filgrastim 5 micro g/kg (n = 33) in patients receiving salvage chemotherapy for relapsed or refractory Hodgkin's or non-Hodgkin's lymphoma. RESULTS: The incidence of grade 4 neutropenia in the pegfilgrastim and filgrastim groups was 69% and 68%, respectively. In addition, the mean duration of grade 4 neutropenia was similar in both groups (2.8 and 2.4 days, respectively). The results for the two groups were also not significantly different for febrile neutropenia, neutrophil profile, time to neutrophil recovery, or toxicity profile. A single subcutaneous injection of pegfilgrastim 100 micro g/kg produced a sustained serum concentration relative to daily subcutaneous injections of filgrastim. Filgrastim-treated patients received a median of 11 injections per cycle. CONCLUSION: Pegfilgrastim was safe and well tolerated in this patient population. A single injection of pegfilgrastim per chemotherapy cycle provided neutrophil support with safety and efficacy similar to that provided by daily injections of filgrastim. Once-per-cycle administration of pegfilgrastim simplifies the management of neutropenia and may have important clinical benefits for patients and healthcare providers.

A lump on the tongue: a diagnostic dilemma?

This paper is a case report of an oral lesion detected in a female patient in her twenties. The aetiology and pathology was not immediately clear-cut but the patient's history provided a clue to its origins and histopathological examination confirmed the diagnosis. It was a "Pregnancy Tumour" which had failed to regress after parturition. This case report will make medical and dental practitioners more aware of lumps and lesions in the mouth. It will also provide a more rational and scientific approach to the management of "Pregnancy Tumours".

Comparable efficacy and safety profiles of once-per-cycle pegfilgrastim and daily injection filgrastim in chemotherapy-induced neutropenia: a multicenter dose-finding study in women with breast cancer.

BACKGROUND: Neutropenia is common in patients receiving myelotoxic chemotherapy. Pegfilgrastim, a sustained-duration filgrastim is a once-per-cycle therapy for prophylactic neutrophil support. PATIENTS AND METHODS: Women, treated with four cycles of doxorubicin/docetaxel chemotherapy every 21 days, received pegfilgrastim or filgrastim 24 h after chemotherapy as a single subcutaneous injection per chemotherapy cycle (pegfilgrastim 30, 60 or 100 microg/kg) or daily subcutaneous injections (filgrastim 5 microg/kg/day). Safety, efficacy and pharmacokinetics were analyzed. RESULTS: The incidence of grade 4 neutropenia in cycle 1 was 95, 90 and 74%, in patients who received pegfilgrastim 30, 60 and 100 microg/kg, respectively, and 76% in patients who received filgrastim. Mean duration of grade 4 neutropenia in cycle 1 was 2.7,2 and 1.3 days for doses of pegfilgrastim, and 1.6 days for filgrastim. The pharmacokinetics of pegfilgrastim were non-linear and dependent on both dose and neutrophil count. Pegfilgrastim serum concentration was sustained until the neutrophil nadir occurred then declined rapidly as neutrophils started to recover, consistent with a self-regulating neutrophil-mediated clearance mechanism. The safety profiles of pegfilgrastim and filgrastim were similar. CONCLUSIONS: A single subcutaneous injection of pegfilgrastim 100 microg/kg provided neutrophil support and a safety profile comparable to daily subcutaneous injections of filgrastim during multiple chemotherapy cycles.

Telomere dysfunction increases mutation rate and genomic instability.

The increased tumor incidence in telomerase null mice suggests that telomere dysfunction induces genetic instability. To test this directly, we examined mutation rate in the absence of telomerase in S. cerevisiae. The mutation rate in the CAN1 gene increased 10- to 100-fold in est1Delta strains as telomeres became dysfunctional. This increased mutation rate resulted from an increased frequency of terminal deletions. Chromosome fusions were recovered from est1Delta strains, suggesting that the terminal deletions may occur by a breakage-fusion-bridge type mechanism. At one locus, chromosomes with terminal deletions gained a new telomere through a Rad52p-dependent, Rad51p-independent process consistent with break-induced replication. At a second locus, more complicated rearrangements involving multiple chromosomes were seen. These data suggest that telomerase can inhibit chromosomal instability.

Salmonella enterica serovar typhi uses type IVB pili to enter human intestinal epithelial cells.

DNA sequencing upstream of the Salmonella enterica serovar Typhi pilV and rci genes previously identified in the ca. 118-kb major pathogenicity island (X.-L. Zhang, C. Morris, and J. Hackett, Gene 202:139-146, 1997) identified a further 10 pil genes apparently forming a pil operon. The product of the pilS gene, prePilS protein (a putative type IVB structural prepilin) was purified, and an anti-prePilS antiserum was raised in mice. Mutants of serovar Typhi either lacking the whole pil operon or with an insertion mutation in the pilS gene were constructed, as was a strain in which the pilN to pilV genes were driven by the tac promoter. The pil(+) strains synthesized type IVB pili, as judged by (i) visualization in the electron microscope of thin pili in culture supernatants of one such strain and (ii) the presence of PilS protein (smaller than the prePilS protein by removal of the leader peptide) on immunoblotting of material pelleted by high-speed centrifugation of either the culture supernatant or sonicates of pil(+) strains. Control pil mutants did not express the PilS protein. A pilS mutant of serovar Typhi entered human intestinal INT407 cells in culture to levels only 5 to 25% of those of the wild-type strain, and serovar Typhi entry was strongly inhibited by soluble prePilS protein (50% inhibition of entry at 1.4 microM prePilS).

Frequency of human leukocyte antigen-A 24 alleles in patients with melanoma determined by human leukocyte antigen-A sequence-based typing.

The analysis of immune responses of patients with melanoma has led to the identification of melanoma-associated antigens targeted by T cells. Cytotoxic T lymphocytes recognize peptides from melanoma-associated antigens presented on the cancer cell surface in the context of HLA class I molecules. Immunodominant melanoma-associated antigen epitopes are being evaluated for their ability to immunize patients with advanced melanoma. However, these vaccination efforts are limited by the extensive polymorphism of the HLA class I heavy chain, which occurs in functional domains of the molecule. Patients with melanoma with the HLA-A-24 phenotype were recruited for vaccination with the peptide AFLPWHRLF from the melanoma-associated antigen tyrosinase. This peptide is recognized in association with HLA-A*2402. The HLA-A24 family includes at least 15 alleles whose frequency and ability to present the same peptide are unknown. The distribution of HLA-A24 alleles was studied in a melanoma population for the practical purpose of identifying patients suitable for vaccination with HLA-A*2402 epitopes. An HLA-A locus-specific polymerase chain reaction method followed by sequencing was developed to determine the HLA-A alleles in genomic DNA. HLA-A 24 was also typed in healthy persons of various ethnic backgrounds to further explore the HLA-A24 family. In white persons, the HLA-A*2402 allele was most common (in 85% of white persons and in 97% of the patients with melanoma). Fewer persons carried the HLA-A*2403 allele (13% in all samples, 3% in melanoma patients). Finally, two new alleles, HLA-A*2422 and HLA-A*24 null, were identified. These results suggest that vaccination with HLA-A*2402-associated epitopes has the potential for broad use in this patient population.

Natural variation of the expression of HLA and endogenous antigen modulates CTL recognition in an in vitro melanoma model.

Increasing attention has been devoted to elucidating the mechanism of lost or decreased expression of MHC or melanoma-associated antigens (MAAs), which may lead to tumor escape from immune recognition. Loss of expression of HLA class I or MAA has, as an undisputed consequence, loss of recognition by HLA class I-restricted cytotoxic T cells (CTLs). However, the relevance of down-regulation remains in question in terms of frequency of occurrence. Moreover the functional significance of epitope down-regulation, defining the relationship between MHC/epitope density and CTL interactions, is a matter of controversy, particularly with regard to whether the noted variability of expression of MHC/epitope occurs within a range likely to affect target recognition by CTLs. In this study, bulk metastatic melanoma cell lines originated from 25 HLA-A*0201 patients were analyzed for expression of HLA-A2 and MAAs. HLA-A2 expression was heterogeneous and correlated with lysis by CTLs. Sensitivity to lysis was also independently affected by the amount of ligand available for binding at concentrations of 0.001 to 1 mM. Natural expression of MAA was variable, independent from the expression of HLA-A*0201, and a significant co-factor determining recognition of melanoma targets. Thus, the naturally occurring variation in the expression of MAA and/or HLA documented by our in vitro results modulates recognition of melanoma targets and may (i) partially explain CTL-target interactions in vitro and (ii) elucidate potential mechanisms for progressive escape of tumor cells from immune recognition in vivo.

Rapid assay for simultaneous detection and differentiation of immunoglobulin G antibodies to human immunodeficiency virus type 1 (HIV-1) group M, HIV-1 group O, and HIV-2.

A rapid immunodiagnostic test that detects and discriminates human immunodeficiency virus (HIV) infections on the basis of viral type, HIV type 1 (HIV-1) group M, HIV-1 group O, or HIV-2, was developed. The rapid assay for the detection of HIV (HIV rapid assay) was designed as an instrument-free chromatographic immunoassay that detects immunoglobulin G (IgG) antibodies to HIV. To assess the performance of the HIV rapid assay, 470 HIV-positive plasma samples were tested by PCR and/or Western blotting to confirm the genotype of the infecting virus. These samples were infected with strains that represented a wide variety of HIV strains including HIV-1 group M (subtypes A through G), HIV-1 group O, and HIV-2 (subtypes A and B). The results showed that the HIV genotype identity established by the rapid assay reliably (469 of 470 samples) correlates with the HIV genotype identity established by PCR or Western blotting. A total of 879 plasma samples were tested for IgG to HIV by a licensed enzyme immunoassay (EIA) (470 HIV-positive samples and 409 HIV-negative samples). When they were tested by the rapid assay, 469 samples were positive and 410 were negative (99.88% agreement). Twelve seroconversion panels were tested by both the rapid assay and a licensed EIA. For nine panels identical results were obtained by the two assays. For the remaining three panels, the rapid assay was positive one bleed later in comparison to the bleed at which the EIA was positive. One hundred three urine samples, including 93 urine samples from HIV-seropositive individuals and 10 urine samples from seronegative individuals, were tested by the rapid assay. Ninety-one of the ninety-three urine samples from HIV-seropositive individuals were found to be positive by the rapid assay. There were no false-positive results (98.05% agreement). Virus in all urine samples tested were typed as HIV-1 group M. These results suggest that a rapid assay based on the detection of IgG specific for selected transmembrane HIV antigens provides a simple and reliable test that is capable of distinguishing HIV infections on the basis of viral type.

Detection and differentiation of HIV-1 group O sera from HIV-1 group M and HIV-2 using recombinant antigens and peptides.

Recombinant antigens and peptides were used to develop an HIV slot immunoblot assay to confirm and differentiate infection by HIV-1 group M, HIV-1 group O or HIV-2. Recombinant antigens from the gag, pol or env regions of HIV-1 and HIV-2, in addition to synthetic peptides from the immunodominant region (IDR) of transmembrane proteins gp41 (HIV-1) or gp36 (HIV-2), were blotted on nitrocellulose strips and used as a substitute for competitive Western blots. Evaluation of a large number of samples (N = 440) from various regions of the world, using the immunoblot, showed effective differentiation of HIV-1 group M, HIV-1 group O and HIV-2. The immunoblot identified correctly all (24/24) HIV-1 group O samples that were confirmed subsequently by PCR and sequence analysis. The immunoblot is a useful tool for identifying HIV-1 group O seropositive samples and has the potential to identify other serological HIV variants that may represent detection problems for HIV screening assays using HIV-1 group M subtype B reagents.

Search for retrovirus in patients with multiple sclerosis.

Multiple sclerosis (MS) patient samples were screened for known or novel retroviruses using an ultrasensitive technique, IMx PERT, that detects the presence of reverse transcriptase (RT). This procedure has 10(5)- to 10(7)-fold greater sensitivity than conventional RT assays and is capable of detecting 10 to 50 virions. Moreover, IMx PERT is at least as sensitive as polymerase chain reaction, and requires no previous knowledge of viral nucleotide sequence. The MS specimens analyzed in this study included 136 sera from 79 patients and 128 cerebrospinal fluid samples from 53 patients with relapsing or chronic progressive disease. In addition, peripheral blood mononuclear cells from 19 MS patients were cultured in an attempt to amplify or induce expression of low-copy number or cell-associated retrovirus. No evidence of retrovirus was found in any of the specimens obtained from MS patients.

In vitro selection of a high affinity antibody to oestradiol using a phage display human antibody library.

We have isolated a monoclonal antibody binding to oestradiol with high affinity (3.7 nM), and exhibiting a better than 1000-fold selectivity in binding to other steroids. A high affinity antibody with good specificity is essential for the accurate determination of circulating oestradiol levels. To date, conventional hybridoma technology has not yielded a reagent of sufficiently high affinity and specificity for this ligand. The aim of this study was to investigate whether such a reagent was accessible through the engineering of antibodies on the surface of filamentous phage. Antibodies were isolated from a large repertoire of single chain Fv fragments (scFv) derived from non-immunised human donors, with selection and screening procedures biased to favour those binding to free oestradiol. This resulted in an antibody with nanomolar affinity for oestradiol, while affinities for related steroids are in the micromolar range. The relative lack of reactivity for steroids substituted at either end of the molecule suggests that this antibody is unique among anti-steroid monoclonal antibodies in lacking a 'blind-spot'. Our results demonstrate that phage display can provide solutions to problems that have so far proved intractable using conventional hybridoma technology.

Collagen-assisted healing of facial wounds after Mohs surgery.

Allowing selected full-thickness skin defects to heal by secondary intention offers the advantages of optimal cancer surveillance, simplified wound management, and avoidance of reconstructive procedures with their associated costs and potential complications. The topical use of bovine collagen has been suggested as a method of enhancing wound closure and final cosmetic appearance. This study evaluated the effect of bovine collagen on wound healing in patients undergoing facial Mohs surgery using the fresh-tissue technique. A total of 111 consecutive patients were assigned to a collagen or no-collagen group. Wound care was identical except for the weekly addition of bovine collagen to the wound of patients in the collagen group. Evaluation was at weekly intervals until the wound epithelialized, then bimonthly for at least 6 months. There was no difference in the rate of wound epithelialization or final cosmetic appearance. This study provides no evidence that the topical use of bovine collagen in a facial wound after Mohs surgery enhances wound epithelialization or the final cosmetic appearance.

Locus-specific analysis of human leukocyte antigen class I expression in melanoma cell lines.

Surface expression of human leukocyte antigen (HLA) class I antigens on melanoma lines was evaluated by locus-specific monoclonal antibodies (mAbs) with three different techniques: Fluorescence-activated cell sorting (FACS), immunohistochemistry with cytospin preparation (ICP), and complement-mediated cytotoxicity (CMC). Eleven HLA class I-expressing cell lines developed from metastases were used. Specific expression of HLA loci was examined under routine culture conditions and after 48-h incubation in interferon-gamma (IFN-gamma; 500 U/ml). Loss of allelic expression was seen in one line (586-MEL): Products of genes coding for HLA-A29 and -B44, in strong linkage disequilibrium, were not detectable. HLA-A antigens were consistently detected by all methodologies and minimally affected by pretreatment with IFN-gamma. HLA-B antigens were detectable in 8 of 11 lines by ICP and 3 of 11 lines by CMC. By FACS the supratypic specificity HLA-Bw6 was expressed at low levels in most lines (mean fluorescence 47.2 +/- 13.4 and rose to 259.8 +/- 45.9 after incubation with IFN-gamma; p < 0.001). HLA-Cw antigen detection by CMC correlated with HLA-B (p < 0.01), suggesting that down-regulation and sensitivity to IFN-gamma are shared by the two loci. This low expression of the HLA-B antigens may play a role in the evasion of the host immune response and its up-regulation may be useful in allowing tumor antigen recognition.