bluuebunny/crossref_metadata_2025 huggingface dataset:
The common heat shock protein receptor CD91 is up-regulated on monocytes of advanced melanoma slow progressors
Authors: STEBBING J , BOWER M , GAZZARD B , WILDFIRE A , PANDHA H , DALGLEISH A , SPICER J
Published: October 2004

Despite advances in our understanding of tumour immunology there is no therapy of proven survival benefit for advanced melanoma. Nevertheless, disease progression is slow in a small proportion of patients with metastatic melanoma, suggesting a contribution to outcome from host factors. Recent data have indicated the importance of the heat shock protein receptor CD91 in immune responses to, and progression of, infectious disease. Here we investigate the relationship between CD91 expression and outcome in malignancy. Rare melanoma patients were recruited with advanced disease that was progressing unusually slowly. CD91 expression on their monocytes was compared with control patients with more typical rapidly advancing metastatic disease. Th1 and Th2 cytokines, as well as innate and adaptive immune subsets, were also measured in the two groups. A significant increase in median CD91 expression levels was observed in slow progressors (P = 0·006). There were no differences in other immune subset markers or inflammatory cytokines. The ability of CD91 to internalize and cross-present tumour antigens through the major histocompatibility complex class I pathway may maintain CD8-positive cytotoxic T cell responses and contribute to slow progression of advanced melanoma.
Allogenic donor splenocytes pretreated with antisense peptide against B7 prolong cardiac allograft survival
Authors: CHEN J , HE Q , ZHANG R , CHU Y , WANG Y , LIU Q , XIONG S
Published: October 2004

The interaction of T cell CD28/CTLA-4 receptors with B7 on antigen-presenting cells (APCs) represents an important co-stimulatory pathway in T cell activation or anergy. Our previous study indicated that recipients immunized with allogenic donor immature dendritic cells (DCs) or resting B cells could induce specific immune tolerance and prolong allograft survival. A possible mechanism for this observation is that the expression of B7 molecules is either at a low level or lacking on these cells. The present study investigates whether blockade of B7 molecules on donor splenocytes with a B7 antisense peptide (B7AP), i.e. a peptide analogue of the CD28-binding region, could induce specific immune tolerance and prolong allograft survival in the recipients. Both the lymphocyte proliferation reaction and the mice pinna cardiac allograft experiment were performed to evaluate the role of B7AP in inducing specific immune tolerance in recipients in vitro and in vivo. The results showed that 56·65% and 20·52% of C57BL/6 splenocytes expressed B7.1 and B7.2 molecules, respectively, on their cell surface. There were no significant changes of the B7 expression on such splenocytes after being treated by the B7AP (53·28% and 19·06%, respectively). B7AP inhibited the mixed lymphocyte reaction by up to 38·4% and a dose-response correlation was observed for inhibition. The recipients (BALB/c) immunized with B7AP-pretreated C57BL/6 splenocytes induced a specific immune hypo-response (43%versus control) and notably prolonged survival of the C57BL/6 cardiac allograft by up to 20·3 days. In contrast to the normal saline group (average: 8·6 days) and FTD10 control peptide group (<4 days), the cardiac allograft survival of the test group was extended for an additional 11·7 days. These results strongly support the notion that immunization with donor splenocytes, which had been pretreated with B7AP, induced specific immune tolerance and prolonged allograft survival in the recipients.
Gene expression analysis of peripheral T cells in a subgroup of common variable immunodeficiency shows predominance of CCR7– effector-memory T cells
Authors: HOLM A M , SIVERTSEN E A , TUNHEIM S H , HAUG T , BJERKELI V , YNDESTAD A , AUKRUST P , FRØLAND S S
Published: October 2004

Common variable immunodeficiency (CVID) represents a heterogeneous group of antibody deficiency syndromes, characterized by defective antibody production in which T cell deficiency may play a pathogenic role. A subgroup of CVID patients has impaired in vitro T cell proliferation. Using microarray analyses of T cells from these patients, we found a gene expression pattern different from healthy controls and patients with X-linked agammaglobulinaemia. The profile of the differentially expressed genes suggests enhanced cytotoxic effector functions, antigen experienced or chronically activated T cells and a predominance of CCR7– T cells. Further experiments using flow cytometry revealed a striking predominance of CCR7– T cells in a subgroup of CVID patients, and an association with impaired T cell proliferation. Our observations indicate that a predominance of CCR7– T cells with effector-memory cell features and with reduced proliferative capacity may characterize a subgroup of CVID.
Interleukin‐15 is not required for the induction or maintenance of orally induced peripheral tolerance
Authors: Millington Owain R. , Wei Xiao‐Qing , Garside Paul , Mowat Allan MC I.
Published: October 2004

Orally induced tolerance is a physiologically relevant form of peripheral tolerance, which is believed to be important for the prevention of pathological immune responses in the gut. Of several mechanisms proposed to mediate oral tolerance, one that has received much attention recently is the concept of regulatory CD4+ T cells. As recent studies have suggested that interleukin (IL)‐15 may be important for the differentiation and maintenance of regulatory CD4+ T cells, we have examined the role of IL‐15 in oral tolerance, using a soluble form of the IL‐15 receptor (sIL‐15R) which blocks the biological effects of IL‐15 in vivo. Oral tolerance induced by feeding mice ovalbumin (OVA) in a low‐dose regimen believed to induce regulatory T cell activity was not affected by the administration of sIL‐15R during either the induction or maintenance phase of tolerance. Thus, oral tolerance does not involve an IL‐15‐dependent mechanism.
Ovine dendritic cells transduced with an adenoviral CTLA4eEGFP fusion protein construct induce hyporesponsiveness to allostimulation
Authors: Newland Ashley , Kireta Svjetlana , Russ Graeme , Krishnan Ravi
Published: October 2004

CTLA4 (CD152) is a transmembrane molecule expressed on activated T cells and functions as a negative regulator of T cell activation upon binding to the costimulatory molecules CD80/86. In this study, CTLA4eEGFP constructs were engineered by cloning the extracellular domains of ovine and human CTLA4 (CTLA4e) ‘in frame’ with the enhanced green fluorescent protein (EGFP). Recombinant adenoviral vectors were generated by incorporation of the CTLA4eEGFP sequence into the adenoviral genome using homologous recombination in Esherichia coli. The functional activity of the adenoviral vectors was shown by the secretion of the CTLA4eEGFP upon infection of ovine fibroblasts and the binding of the fusion protein to the target ovine and human dendritic cells expressing CD80/86 receptors by flow cytometry. The EGFP tag facilitated molecular size determinations and quantification of the secreted ovine CTLA4 fusion protein by immunoprecipitation and enzyme‐linked immunosorbent assay (ELISA), respectively, using anti‐GFP mAbs. Ovine dendritic cells obtained from pseudoafferent lymphatic cannulation of sheep were characterized based on high major histocompatibility complex (MHC) class II expression and cross‐reactivity with monoclonal antibodies to the human dendritic cell markers, CD83 and CMRF‐56. In addition, ovine dendritic cells (DC) were transfected with the adenoviral CTLA4eEGFP and when used as stimulators in a mixed lymphocyte reaction showed a reduced capacity to induce allogeneic lymphocyte proliferation. This study verifies that the ovine CTLA4eEGFP fusion protein functions similarly to its human homologue and that DC modified with adenoviral CTLA4‐EGFP may provide an effective therapeutic approach in targeting alloreactive T cells to prolong allograft acceptance in a preclinical ovine model of renal transplantation.
Transcription of major histocompatibility complex class I (Kb) and transporter associated with antigen processing 1 and 2 genes is up‐regulated with age
Authors: Assounga Alain G. , Warner Carol M.
Published: October 2004

The transporter associated with antigen processing 1 and 2 (TAP1 and TAP2) genes belong to the ATP‐binding cassette family of transporter genes. They provide peptides necessary for the assembly of major histocompatibility complex (MHC) class I molecules by transporting these peptides into the endoplasmic reticulum. As MHC class I protein expression increases with age, we have explored the effect of age on the transcription of MHC class I genes (Kb) and TAP1 and TAP2 genes in C57BL/6 mice. Blood and spleen lymphocytes were isolated from mice aged from 3 months to over 24 months. RNA was extracted and mRNA for Kb, TAP1, TAP2 was quantified using slot‐blot hybridization followed by densitometry. There was a parallel age‐related increase (1·5‐fold) in blood lymphocyte mRNA of these genes from 3 months to 21 months. In mice over 24 months old there was a decrease in Kb and TAP1 mRNA, but an increase in TAP2 mRNA. In spleen lymphocytes an age‐related increase in all three mRNA species occurred throughout life. While MHC class I and Tap genes underwent very similar age‐related changes, MHC class I mRNA was about 50 times more abundant than either TAP1 or TAP2 mRNA.
Interleukin‐10 secreted by B‐1 cells modulates the phagocytic activity of murine macrophages in vitro
Authors: Popi Ana Flavia , Lopes José Daniel , Mariano Mario
Published: October 2004

As demonstrated previously in our laboratory, B‐1 cells migrate from the peritoneal cavity of mice and home to a distant site of inflammation to become macrophage‐like cells. However, the influence that these cells might have on the kinetics and fate of the inflammatory process is not known. Considering that macrophages are pivotal in the inflammatory reaction, we decided to investigate the possible influence B‐1 cells could have on macrophage activities in vitro. Our results show that peritoneal macrophages from Xid mice, a mouse strain deprived of B‐1 cells, have higher phagocytic indexes for zymozan particles when compared with macrophages from wild‐type mice. Moreover, macrophages from wild‐type mice have a lower ability to release nitric oxide and hydrogen peroxide when compared with macrophages from Xid mice. Experiments using cocultures of B‐1 cells and macrophages from Xid mice in transwell plates demonstrated that B‐1 cells down‐regulate macrophage activities. These observations also indicate that this phenomenon is not due to a physical interaction between these two cell populations. As B‐1 cells are one of the main sources of interleukin (IL)‐10, we demonstrate in this study that adherent peritoneal cells from Xid mice produce significantly less amounts of this cytokine in culture when compared with IL‐10 production by cells from wild‐type mice. When B‐1 cells from IL‐10 knock‐out mice and macrophages from wild‐type mice were cocultured in transwell plates, the phagocytic index of macrophages was not altered demonstrating that B‐1 cells can influence the effector functions of macrophages in vitro via IL‐10 secretion.
Immunosuppression, interleukin‐10 synthesis and apoptosis are induced in rats inoculated with Cryptococcus neoformans glucuronoxylomannan
Authors: Chiapello Laura S. , Baronetti José L. , Aoki María P. , Gea Susana , Rubinstein Héctor , Masih Diana T.
Published: October 2004

Glucuronoxylomannan (GXM) is the major Cryptococcus neoformans capsular polysaccharide and represents the main virulence factor of this fungus. In in vitro studies we have demonstrated previously that this acidic and high‐molecular‐weight polysaccharide suppresses lymphoproliferation, modulates cytokine production and promotes apoptosis in spleen mononuclear (Spm) cells from rats. In this study we demonstrate that these phenomena also occur in vivo after the intracardiac inoculation of GXM into normal Wistar rats. The results of this study show suppression of the proliferative response Spm cells to concanavalin A (Con A) or heat‐killed C. neoformans (HKCn) in the first 2 weeks after polysaccharide administration. In addition, increased levels of interleukin (IL)‐10 were produced by Con A‐stimulated Spm cells, coinciding with immunohistochemical GXM detection in the white pulp of spleen. In particular, high production of IL‐10 with diminution of IL‐2, interferon (IFN)‐γ and tumour necrosis factor (TNF)‐α synthesis were detected 14 days after GXM administration. In situ cell death detection by TdT‐mediated biotin–dUTP nick‐end labelling (TUNEL) reaction in sections of spleen, lung and liver demonstrates apoptosis in tissues with deposits of GXM. These data demonstrate the in vivo ability of GXM to modify cytokine synthesis by Spm cells and to promote host cell apoptosis.
Analysis of the mechanisms involved in the stimulation of neutrophil apoptosis by tumour necrosis factor‐α
Authors: Salamone Gabriela , Trevani Analía , Martínez Diego , Vermeulen Mónica , Gamberale Romina , Fernández‐Calotti Paula , Raiden Silvina , Giordano Mirta , Geffner Jorge
Published: October 2004

We have previously reported that human neutrophils pretreated with tumour necrosis factor‐α (TNF‐α) and then exposed to a variety of agents such as immune complexes, zymosan, phorbol 12‐myristate 13‐acetate (PMA), C5a, fMLP, or granulocyte–macrophage colony‐stimulating factor (GM‐CSF), undergo a dramatic stimulation of apoptosis, suggesting that TNF‐α is able to prime an apoptotic death programme which can be rapidly triggered by different stimuli. We report here that this response involves the participation of Mac‐1 (CD11b/CD18), is dependent on caspases 3, 8 and 9, and is associated with both a loss of mitochondrial transmembrane potential and a down‐regulation in expression of the anti‐apoptotic protein, Mcl‐1. Interestingly, we also found that the anti‐apoptotic cytokine interleukin‐1 (IL‐1) improves the ability of TNF‐α to promote apoptosis, supporting the notion than TNF‐α, acting together with IL‐1, may favour the depletion of neutrophils from the inflammatory areas during the course of acute inflammation.
CD27− CD4+ memory T cells define a differentiated memory population at both the functional and transcriptional levels
Authors: Schiött Åsa , Lindstedt Malin , Johansson‐Lindbom Bengt , Roggen Erwin , Borrebaeck Carl A. K.
Published: October 2004

The memory T‐cell population is a heterogeneous population, including both effector cells, which exert a direct secondary immune response, and resting or intermediate cells, which serve as a reservoir and exert a possible regulatory role. To further dissect the T‐cell memory population residing in the CD4+ CD45RO+ T‐cell pool, we studied the functional properties of memory populations identified by the CD27 marker. This marker clearly divides the memory population into two groups. One group consists of effector cells lacking CD27 and displaying a high antigen recall response. The other group consists of an intermediate memory population, displaying CD27. This latter group lacks an antigen recall response and requires costimulation for T‐cell receptor triggering. To evaluate the function of the CD27+ memory pool, we analysed the transcriptional profile, using high‐density microarray technology. These gene data strongly support the different functional profiles of CD27+ and CD27– memory populations, in terms of protein expression and the capacity to respond to antigen.
Interleukin‐10 suppression of myeloid cell activation — a continuing puzzle
Authors: Williams Lynn M. , Ricchetti Giuseppe , Sarma Usha , Smallie Timothy , Foxwell Brian M. J.
Published: October 2004

Efforts to identify the signal transduction pathways used by interleukin‐10 (IL‐10) have resulted in limited success. The anti‐inflammatory effects elicited by IL‐10, and the mechanisms by which these are mediated, are still relatively unknown. Understanding the signalling mechanisms behind the suppression of cytokine expression by IL‐10 could be of potential therapeutic interest. Although the consensus is that the Janus kinase, Jak1, as well as the signal transducer and activator of transcription STAT3 are central, much controversy exists about the participation and roles of many other signalling pathways targeted by IL‐10. The mechanisms of cytokine suppression proposed by various groups have included transcriptional, post‐transcriptional and post‐translational regulation of IL‐10 target genes; nevertheless no unifying model has emerged thus far. Here we would like to highlight novel findings and discuss their implications in the context of current understanding of IL‐10 signalling.
Immunoreactive properties of peptide fractions of cow whey milk proteins after enzymatic hydrolysis
Authors: Wróblewska Barbara , Karamać Magdalena , Amarowicz Ryszard , Szymkiewicz Agata , Troszyńska Agnieszka , Kubicka Ewa
Published: October 2004

Commercial whey protein concentrate (WPC) was hydrolysed with either Alcalase 2.4 FG (Novo Nordisk), or papain (Sigma) (in one‐step process) or with two enzymes (in two‐step process) to determine the changes in the immunoreactivity of α‐lactalbumin and β‐lactoglobulin. Enzymatic hydrolysis of WPC was performed by pH‐stat method. Hydrolysates were analysed using sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, immunoblotting and size‐exclusion chromatography (SE‐HPLC). Immunoreactive properties of peptide fractions separated from the hydrolysates by fast protein liquid chromatography (FPLC) were determined using dot‐immunobinding and enzyme‐linked immunosorbent assay (ELISA) methods. Finally the sensory analysis was used to confirm organoleptic changes resulting from the application of different enzymes. The ‘two‐step’ process was observed to be the most effective however allergenic epitopes were still present, as it was found by ELISA with anti‐α‐la and anti‐β‐lg antibodies. The addition of papain as the second enzyme in the hydrolysis process contributed to the improvement of the sensory properties of WPC hydrolysate as compared with the Alcalase hydrolysate. Alcalase‐papain partially hydrolysated WPC can be found a promising base for production of the tolerogenic formula.
RIDASCREEN® FAST PEANUT, a rapid and safe tool to determine peanut contamination in food
Authors: Immer Ulrike , Reck Bernhard , Lindeke Stella , Koppelman Stef
Published: October 2004

During the last years allergenic substances in food have gained more and more attention. Stricter rules for a peanut free food labelling demand a peanut contamination less than 5 ppm. The maintenance of a high quality of food products with regards to allergenic exposure is preferable warranted with rapid tests that can be utilized as an aid for suppliers to control raw materials, to avoid cross contamination during production and to check the final products. A rapid sandwich peanut ELISA was developed, which covers a measuring range of 2.5–20 ppm peanut (detection limit of 1.5 ppm). This test was successfully evaluated in an independent study under the supervision of the AOAC Research Institute, getting the AOAC certificate No. 030404. In‐house validation and independent validation data demonstrate the reliability, accuracy and precision of the test.
Fluorescent polarization immunoassay for sulphadiazine using a high specificity antibody
Authors: Murtazina Nailya R. , Eremin Sergei A. , Mozoleva Olga V. , Everest Sally J , Jim Brown A , Jackman Roy
Published: October 2004

A rapid and sensitive fluorescent‐labelled polarization immunoassay for sulphadiazine (SDZ) is reported. Fluorescein‐labelled tracers were synthesized and a specific SDZ antibody has been used in the development of the method. The influence of the fluorescent label type on the immunoassay sensitivity was investigated. Homologous and non‐homologous tracer – antiserum combinations were varied to provide increased sensitivity of the immunoassay. A detection limit of 0.2 ng mL−1 for SDZ in 50 μL aqueous sample has been achieved. The cross‐reactivity of other sulphonamide drugs has also been examined.
Immunobiosensor determination of β‐agonists in urine using integrated immunofiltration clean‐up
Authors: Annette Johansson Marie , Hellenäs Karl‐Erik
Published: October 2004

A fast immunobiosensor assay for screening of β‐agonists in urine, using integrated immunofiltration clean‐up with 30 kDa cutoff centrifugal filter, was developed. The same antibody aliquot was used for clean‐up and biosensor analysis, and no organic solvent was needed. Matrix interference from urine was efficiently reduced. Two antibodies with different cross‐reactivities were compared. Although both antibodies were able to detect clenbuterol (CBL) below 1 ng mL−1, a polyclonal antibody directed against salbutamol was chosen because of its ability to recover several β‐agonists. The decision limit (CCα) of the final assay was 0.29 ng mL−1, and an α‐error of 1% gave a β‐error of <1% for twenty reference bovine samples spiked with 1 ng mL−1 CBL.
Clinical efficacy of carprofen as an adjunct to the antibacterial treatment of bovine respiratory disease
Authors: Elitok B. , Elitok Ö. M.
Published: October 2004

A clinical trial was undertaken to investigate the efficacy of a single dose of carprofen (CPF) in the treatment of bovine respiratory disease in cattle. Tilmicosin was used as a basal treatment in all animals. Six hours after dosing, body temperature and respiratory rates in animals treated with CPF–tilmicosin had decreased and were significantly lower than in the animals treated with tilmicosin alone (P < 0.05). Over the period of clinical observation, CPF–tilmicosin treatment produced a clinical resolution of the pneumonia similar to treatment with tilmicosin alone. However, it is significant from an animal welfare perspective that over the period of the study after treatment, CPF–tilmicosin therapy produced significantly greater symptomatic improvement than tilmicosin.
Cefotaxime kinetics in plasma and synovial fluid following intravenous administration in horses
Authors: Orsini J. A. , Moate P. J. , Engiles J. , Norman T. , Poppenga R. , Benson C. E. , Boston R. C.
Published: October 2004

Cefotaxime powder was diluted with sterile water to a concentration of 100 mg/mL. The volume of solution was adjusted for each experimental horse to provide a total dose of 15, 20, and 25 mg/kg and was administered by infusion through a jugular vein catheter over a 10‐min period. All three doses were administered to each of the six experimental horses at three different times. Cefotaxime concentrations in plasma and synovial fluid samples were measured by high‐performance liquid chromatography (HPLC). Standard compartmental analysis techniques and theWinSAAMmodeling program were used to determine standard pharmacokinetic parameters for cefotaxime. The plasma and synovial fluid data from the five horses administered the 25 mg/kg dose was analyzed. Plasma cefotaxime concentrations appeared to be linearly related to dose infused and declined in parallel, suggesting linear drug kinetics. Moreover, cefotaxime concentrations declined monotonically suggesting that its disposition kinetics could essentially be described by a one‐compartment model rather than the fact that sampling occurred after the infusion was discontinued. Maximum concentration of cefotaxime in plasma occurred immediately after cessation of the infusion. Minimum inhibitory concentrations were determined forStaphylococcus aureus,Escherichia coli,Pseudomonas aeruginosa, andKlebsiella pneumoniae, common isolates from septic arthritis in horses. Based on our pharmacokinetic data, a regimen of 25 mg/kg administered i.v. every 6 h appears appropriate for susceptible joint infections in adult horses.
Plasma profile and pharmacokinetics of dextromethorphan after intravenous and oral administration in healthy dogs
Authors: KuKanich B. , Papich M. G.
Published: October 2004

Dextromethorphan is an N‐methyl‐d‐aspartate (NMDA) noncompetitive antagonist which has been used as an antitussive, analgesic adjunct, probe drug, experimentally to attenuate acute opiate and ethanol withdrawal, and as an anticonvulsant. A metabolite of dextromethorphan, dextrorphan, has been shown to behave pharmacodynamically in a similar manner to dextromethorphan. The pharmacokinetics of dextromethorphan were examined in six healthy dogs following intravenous (2.2 mg/kg) and oral (5 mg/kg) administration in a randomized crossover design. Dextromethorphan behaved in a similar manner to other NMDA antagonists upon injection causing muscle rigidity, ataxia to recumbency, sedation, urination, and ptyalism which resolved within 90 min. One dog repeatedly vomited upon oral administration and was excluded from oral analysis. Mean ± SD values for half‐life, apparent volume of distribution, and clearance after i.v. administration were 2.0 ±0.6 h, 5.1 ± 2.6 L/kg, and 33.8 ± 16.5 mL/min/kg. Oral bioavailability was 11% as calculated from naïve pooled data. Free dextrorphan was not detected in any plasma sample, however enzymatic treatment of plasma with glucuronidase released both dextromethorphan and dextrorphan indicating that conjugation is a metabolic route. The short half‐life, rapid clearance, and poor bioavailability of dextromethorphan limit its potential use as a chronic orally administered therapeutic.
Integrated pharmacological assessment of flubendazole potential for use in sheep: disposition kinetics, liver metabolism and parasite diffusion ability1
Authors: Moreno L. , Alvarez L. , Mottier L. , Virkel G. , Sanchez Bruni S. , Lanusse C.
Published: October 2004

Flubendazole (FLBZ) is a broad spectrum benzimidazole methylcarbamate anthelmintic widely used in poultry and swine. However, there is no information available on the pharmacological behaviour of FLBZ in ruminants. The work reported here was addressed to evaluate the potential of FLBZ for use in sheep. The integrated assessment included evaluation of FLBZ and metabolites plasma disposition kinetics, liver metabolism and ex vivo ability to diffuse into the cestode parasite Moniezia benedeni. In a cross‐over kinetic study, six healthy Corriedale sheep were treated with FLBZ by intravenous (i.v.) (4% solution) and intraruminal (i.r.) (4% suspension) administrations at the same dosage (5 mg/kg) with a 21‐day washout period between treatments. Blood samples were collected between 0 and 72 h post‐treatments. Sheep liver microsomes were incubated with 40 μm FLBZ and specimens of the cestode parasite M. benedeni, collected from untreated animals, were incubated (5–120 min) with FLBZ and its reduced (R‐FLBZ) metabolite (5 μm). Samples of plasma, microsomal incubations and parasite material were prepared and analyzed by high‐performance liquid chromatography to measure FLBZ and its metabolites. FLBZ parent drug showed a fast disposition being detected in the bloodstream up to 36 h after its i.v. administration. Both R‐FLBZ and hydrolyzed FLBZ (H‐FLBZ) metabolites were recovered in plasma as early as 5 min after the i.v. treatment in sheep. The plasma AUC ratios for R‐FLBZ and FLBZ (AUCR‐FLBZ/AUCFLBZ) were 4.07 i.v. and 5.55 i.r., respectively. R‐FLBZ achieved a significantly higher (P < 0.01) Cmax value (0.14 μg/mL at 17.3 h post‐treatment) than that observed for the parent drug FLBZ (0.04 μg/mL at 14.4 h post‐treatment). Low plasma concentrations of FLBZ parent drug were measured between 6 and 48 h, and only trace concentrations of H‐FLBZ were detected during a short period of time after the i.r. treatment. Consistently, sheep liver microsomes metabolized FLBZ into its reduced metabolite at a rate of 9.46 ± 2.72 nmol/mg/h. Both FLBZ and R‐FLBZ demonstrated a similar ability to quickly diffuse through the tegument of the cestode parasite. The data on FLBZ pharmacological behaviour presented here contribute to evaluate its potential to be developed as an anthelmintic for broad spectrum parasite control in ruminants.
Hepatitis C virus infection in asymptomatic male volunteer blood donors in Karachi, Pakistan
Authors: Akhtar S. , Younus M. , Adil S. , Jafri S. H. , Hassan F.
Published: October 2004

Summary.  The objectives of this study were to assess the proportion of hepatitis C virus (HCV) reactors and to identify risk factors associated with HCV infection in volunteer blood donors in Karachi, Pakistan. Between 1 January 1998 and 31 December 2002, consecutive blood donations tested at two blood banks were used to assess the proportion of HCV sero‐reactors donors. To evaluate the potential risk factors, a case‐control study design was implemented to select cases and controls between 15 October 2001 and 15 March 2002. The overall seroprevalence of HCV in these blood donors was 1.8% (6349/35 1309). Trend analysis revealed a significant (P < 0.001) linear increase in proportions of HCV‐seropositive donors from 1998 to 2002. Final multivariate logistic regression model showed that the cases were more likely than controls to have reported past hospitalization or to have received multiple injections. When a glass syringe was used to give therapeutic injections, it increased the adjusted odds of being HCV seropositive significantly more among cases than in controls and this relationship was stronger when injection was given by general medical practitioner than if the injection was given in hospital setting. Cases were more likely than controls to have reported sexual contact with multiple sexual partners. Primary prevention programmes focused on identified risk factors might help to curtail the spread of HCV infection in this community and in other similar settings in developing countries.
Is it possible to diagnose acute hepatitis C virus (HCV) infection by a rising anti‐HCV titre rather than by seroconversion?
Authors: Lu S.‐N. , Tung H.‐D. , Chen T.‐M. , Lee C.‐M. , Wang J.‐H. , Hung C.‐H. , Chen C.‐H. , Changchien C.‐S.
Published: October 2004

Summary.  The diagnosis of acute hepatitis C virus (HCV) infection relies on documented positive‐seroconversion of HCV antibody (anti‐HCV). Because of the detection of seroconversion at an earlier stage by second or third generation anti‐HCV enzyme immunoassays (EIA), the diagnosis of acute hepatitis C (AHC) may be underestimated. The aim of this study was to evaluate whether rising anti‐HCV titre could be used to diagnose AHC or not. Eighteen patients with a clinical diagnosis of acute hepatitis C were enrolled, including eight cases with documented seroconversion to anti‐HCV and 10 cases with clinically suspected acute hepatitis C. Four chronic hepatitis C patients with acute exacerbation were selected as a control group. Serial sera were assayed with a third generation anti‐HCV (AxSYM, version 3.0; Abbott, Chicago, IL, USA) and recombinant immunoblot assays (RIBA; Chiron HCV 3.0 Strip; Immunoblot, Emeryville, CA, USA) and the titre of anti‐HCV expressed as signal/cutoff (S/CO) ratio and the RIBA patterns were correlated. Seven of eight documented AHC (one lacking the initial serum) and five of 10 clinically suspected AHC showed a rising pattern of S/CO values. The initial S/CO values on the first visit were less than 40 in 14 of 18 cases. The RIBA pattern shifted from negative/indeterminate to positive in five of seven documented AHC and 4 of 10 clinically suspected AHC cases. Fifteen of 18 cases had seroconversions of at least one antibody, whilst 85.7% showed a rising S/CO ratio. On the contrary, the S/CO ratio and RIBA pattern remained unaltered in chronic hepatitis C with acute exacerbation. The rise in S/CO was usually accompanied with an increase in the number of RIBA reactive bands and their intensity in acute hepatitis C patients. The rise in S/CO ratios using a third generation anti‐HCV assay and the RIBA pattern might be used as a supplemental diagnostic criterion for acute HCV infection.
A randomized trial of prolonged high dose of interferon plus ribavirin for hepatitis C patients nonresponders to interferon alone
Authors: Fattovich G. , Zagni I. , Ribero M. L. , Castagnetti E. , Minola E. , Lomonaco L. , Scattolini C. , Fabris P. , Boccia S. , Giusti M. , Abbati G. , Felder M. , Rovere P. , Redaelli A. , Tonon A. , Tomba A. , Montanari R. , Paternoster C. , Distasi M. , Fornaciari G. , Tositti G. , Rizzo C. , Suppressa S. , Pantalena M. , Noventa F. , Tagger A.
Published: October 2004

Summary. Retreatment of chronic hepatitis C patients nonresponders to interferon (IFN) alone with the standard dose of IFN [3 million units (MU) thrice weekly (TIW)] plus ribavirin for 24 weeks has yielded low sustained virological response (SVR), averaging 8%. The aim of the present, open‐labelled, randomized study was to evaluate the efficacy of IFN induction therapy followed by prolonged high dose of IFN plus ribavirin in nonresponders. One hundred and fifty‐one patients were randomized to receive 5 MU daily of IFN alfa‐2b (group 1,n = 73) or 5 MU TIW of IFN alfa 2b (group 2,n = 78) for 4 weeks followed by IFN (5 MU TIW) plus ribavirin (1000/1200 mg/daily) for 48 weeks in both groups. In an intention‐to‐treat analysis, the sustained virological response (SVR) at 24‐week follow‐up was 33 and 23% for group 1 and 2, respectively (P = 0.17). The overall SVR was 52 and 18% in patients with genotype 2/3 and 1/4, respectively. Among genotype 1/4 patients the SVR was 29 and 11% for age younger or older than 40 years. Compared with genotype 2/3 patients, the risk (95% confidence interval) of nonresponse to retreatment was 3.0‐fold (1.17–8.0) in younger genotype 1/4 patients and 8.4‐fold (3.0–23.29) in older genotype 1/4 patients. In conclusion these results suggest that retreatment with a reinforced regimen should be focused in nonresponder genotype 2/3 patients and younger genotype 1/4 patients, who are most likely to benefit. Induction therapy does not improve SVR.
High levels of p53 protein expression do not correlate with p53 mutations in hepatocellular carcinoma
Authors: Anzola M. , Saiz A. , Cuevas N. , Lopez‐Martinez M. , Martinez de Pancorbo M. A. , Burgos J. J.
Published: October 2004

Summary.  To determine the relationship between p53 altered expression and p53 mutations in hepatocellular carcinoma (HCC), we analysed p53 protein immunohistochemically and assessed the presence of mutations in exons 4–8 of the p53 gene using SSCP assay in 117 HCCs corresponding to 78 patients. We also determined the relationship of p53 expression with cellular proliferation by immunostaining with monoclonal antibodies to Ki‐67. We found significant levels of p53 protein expression in 23.1% of the 117 cases studied, but identified mutations in only 12 cases (10.3%). Only four of the p53‐positive cases had mutations in the regions analysed. Six of the cases that displayed mutations at p53 gene were negative for immunohistochemical analysis (IHC) and two cases showed positive immunoreactivity in the cytoplasm of the cell.

In conclusion, strong IHC reactivity for p53 protein is not an indicator of the presence of p53 gene mutations at exons 4–8 in HCC. Thus, p53 loss of function in HCC should be evaluated both by p53 mutation analysis and p53 protein expression, as both give complementary information about p53 status.
Survival in HIV‐infected patients is associated with hepatitis C virus infection and injecting drug use since the use of highly active antiretroviral therapy in the Lyon observational database
Authors: Voirin N. , Trépo C. , Miailhes P. , Touraine J. L. , Chidiac C. , Peyramond D. , Livrozet J. M. , Ritter J. , Chevallier P. , Fabry J. , Allard R. , Vanhems P.
Published: October 2004

Summary.  Highly active antiretroviral therapy (HAART) has reduced the incidence of death in HIV‐infected patients but various rates of survival have been reported due to the infection with hepatitis C virus (HCV) and the use of injecting drugs (IDU). A survival analysis was performed to estimate and compare the death rates in HIV‐positive patients infected by IDU and/or positive for HCV antibodies in the pre‐HAART and HAART periods in Lyon (France) between 1992 and 2002. Patients were stratified into four groups (G): HCV−/IDU−(G1), HCV+/IDU−(G2), HCV+/IDU−(G3), HCV+/IDU+ (G4) and adjusted death rates in the pre‐HAART era (< 1996) and the HAART era (≥ 1996) were compared. The aHR of progression to death was 1.05 (95% CI 0.75–1.47, P = 0.75) for G2, 1.09 (95% CI 0.54–2.22, P = 0.81) for G3 and 0.90 (95% CI 0.65–1.24, P =0.51) for G4 compared with G1 in the pre‐HAART era. The aHR of progression to death was 0.76 (95% CI 0.28–2.08, P = 0.59) for G2, 1.23 (95% CI 0.17–8.86, P = 0.84) for G3 and 2.90 (95% CI 1.62–5.20, P < 0.001) for G4, compared with G1 in the HAART era. HAART management of HCV+/IDU+ patients needs to be optimized for them to achieve a similar benefit as observed among other individuals.
Localization of the sporulation protein SpoIIE in Bacillus subtilis is dependent upon the cell division protein FtsZ
Authors: Anne Levin Petra , Losick Richard , Stragier Patrick , Arigoni Fabrizio
Published: October 2004

SpoIIE is an integral membrane protein that governs the establishment of cell‐specific gene transcription during the process of sporulation in Bacillus subtilis. Synthesis of SpoIIE commences shortly after the onset of sporulation, after which the protein localizes at sites of potential cell division near both ends of the sporangium. We now show that, within the limits of resolution of immunofluorescence microscopy, this bipolar pattern of localization observed in early‐sporulating cells was superimposable with the bipolar pattern of localization of the cell division protein FtsZ. The localization of SpoIIE was dependent upon FtsZ because little or no localization was observed along the length of filaments that were generated by depleting sporulating cells for the cell division protein. In contrast, SpoIIE and FtsZ were found to co‐localize at regularly spaced intervals in filaments generated by the use of a temperature‐sensitive mutant of the cell division gene divIC. Finally, in cells engineered to synthesize SpoIIE during growth, SpoIIE localized at the mid‐cell position, coincident with the position of FtsZ, which exhibits a medial pattern of localization in cells undergoing binary fission. These results suggest that the bipolar pattern of localization of SpoIIE is dictated by the sporulation‐induced switch in the position of FtsZ or of other, FtsZ‐associated, cell division proteins. Thus, it appears that B. subtilis has co‐opted the cell division machinery as a means of localizing a cell fate determinant to the polar septum during sporulation.
Positive control of the two‐component RcsC/B signal transduction network by DjlA: a member of the DnaJ family of molecular chaperones in Escherichia coli
Authors: Kelley William L. , Georgopoulos Costa
Published: October 2004

The membrane‐anchored DjlA protein represents the third member of the DnaJ ‘J‐domain’ family of Escherichia coli that includes DnaJ and CbpA. DjlA possesses a J‐domain at its extreme C‐terminus but shares no additional homology with DnaJ. Our genetic analysis suggests that DjlA acts in concert with the RcsB/C two‐component signal transduction system to augment induction of the cps (capsular polysaccharide) operon and synthesis of colanic acid mucoid capsule. The DjlA J‐domain is essential for the observed stimulation of this pathway as deletion, or introduction of the mutation H233Q, within the highly conserved HPD tripeptide abolished all inducing activity. Deletion of the transmembrane anchor sequence also abolished all inducing activity. djlA is not an essential gene under all conditions tested, nor is it essential for mucoid capsule biosynthesis; however, strong overexpression leads to rapid loss of cell viability suggesting that the gene is normally tightly regulated. Northern analysis revealed that djlA message was extremely unstable but could be induced or stabilized in response to cold shock. The activation of the cps operon by DjlA is dependent upon both DnaK(Hsp70) and GrpE, and therefore we propose a role for DjlA, together with this chaperone machine, as a novel regulator of a two‐component histidine kinase signal transduction pathway.
Point mutations in the transmembrane domain of DjlA, a membrane‐linked DnaJ‐like protein, abolish its function in promoting colanic acid production via the Rcs signal transduction pathway
Authors: Clarke David J. , Holland I. B. , Jacq Annick
Published: October 2004

DjlA is a novel DnaJ‐like protein localized to the inner membrane of Escherichia coli through the single transmembrane domain (TMD) found at the N‐terminus. The overproduction of DjlA activates expression of the cps operon, controlling synthesis and export of the extracellular polysaccharide colanic acid via the Rcs/B two‐component signal transduction pathway. We now show that both the TMD and the J‐region are essential for the induction of cps expression observed with the overproduction of DjlA. Furthermore, we describe the isolation and characterization of different point mutations in the TMD that completely or partially block the induction of cps expression associated with overproduction of DjlA. These mutations were shown not to affect the localization, stability or topology of the mutant DjlA proteins. We propose that these mutations are affecting specific interactions between the TMD of DjlA and its substrate protein(s), for example RcsC, the membrane sensor kinase partner of the Rcs/B signal transduction pathway.
Direct evidence for active segregation of oriC regions of the Bacillus subtilis chromosome and co‐localization with the Spo0J partitioning protein
Authors: Lewis Peter J. , Errington Jeffery
Published: October 2004

We have developed methods for labelling regions of the Bacillus subtilis chromosome with the nucleotide analogue 5‐bromodeoxyuridine (BrdU) and for subcellular visualization of the labelled DNA. Examination of oriC‐labelled chromosomes in outgrowing spores has provided direct evidence for active segregation of sister chromosomes. Co‐immunodetection of Spo0J and BrdU‐labelled DNA has directly confirmed the expected close association between this chromosome partitioning protein and the oriC region of the chromosome. The results provide further support for the notion that bacterial cells use an active mitotic‐like mechanism to segregate their chromosomes.
Assembly and interactions of cotJ‐encoded proteins, constituents of the inner layers of the Bacillus subtilis spore coat
Authors: Seyler Jr Richard W. , Henriques Adriano O. , Ozin Amanda J. , Moran Jr Charles P.
Published: October 2004

During Bacillus subtilis endospore formation, a complex protein coat is assembled around the maturing spore. The coat is made up of more than two dozen proteins that form an outer layer, which provides chemical resistance, and an inner layer, which may play a role in the activation of germination. A third, amorphous layer of the coat occupies the space between the inner coat and the cortex, and is referred to as the undercoat. Although several coat proteins have been characterized, little is known about their interactions during assembly of the coat. We show here that at least two open reading frames of the cotJ operon (cotJA and cotJC) encode spore coat proteins. We suggest that CotJC is a component of the undercoat, since we found that its assembly onto the forespore is not prevented by mutations that block both inner and outer coat assembly, and because CotJC is more accessible to antibody staining in spores lacking both of these coat layers. Assembly of CotJC into the coat is dependent upon expression of cotJA. Conversely, CotJA is not detected in the coats of a cotJC insertional mutant. Co‐immunoprecipitation was used to demonstrate the formation of complexes containing CotJA and CotJC 6 h after the onset of sporulation. Experiments with the yeast two‐hybrid system indicate that CotJC may interact with itself and with CotJA. We suggest that interaction of CotJA with CotJC is required for the assembly of both CotJA and CotJC into the spore coat.
Characterization of a ferric‐binding protein mutant in Haemophilus influenzae
Authors: Kirby Shane D. , Gray‐Owen Scott D. , Schryvers Anthony B.
Published: October 2004

Ferric‐binding proteins (FbpA) have been implicated in the transferrin receptor‐mediated iron acquisition pathways of Haemophilus influenzae and Neisseria spp. These proteins are believed to function by shuttling iron from outer membrane transferrin receptors to a specific inner membrane permease complex. However, the role of these proteins has not been conclusively resolved, as attempts at creating isogenic mutants in the fbpA genes of both species have been unsuccessful, prompting the hypothesis that FbpA may play a critical role in H. influenzae and Neisseria spp. This study describes the construction and characterization of an H. influenzae isogenic fbpA mutant. It is demonstrated that this mutant is deficient in its ability to use human transferrin as a sole iron source, even though the strain is still competent for binding human transferrin. It is also demonstrated that this mutant is impaired in its ability to use ferric citrate as an iron source, and grows at a reduced rate relative to wild type in broth supplemented with protoporphyrin rather than haemin.
Identification and characterization of an aliphatic amidase in Helicobacter pylori
Authors: Skouloubris Stéphane , Labigne Agnès , De Reuse Hilde
Published: October 2004

We report, for the first time, the presence in Helicobacter pylori of an aliphatic amidase that, like urease, contributes to ammonia production. Aliphatic amidases are cytoplasmic acylamide amidohydrolases (EC 3.5.1.4) hydrolysing short‐chain aliphatic amides to produce ammonia and the corresponding organic acid. The finding of an aliphatic amidase in H. pylori was unexpected as this enzyme has only previously been described in bacteria of environmental (soil or water) origin. The H. pylori amidase gene amiE (1017 bp) was sequenced, and the deduced amino acid sequence of AmiE (37 746 Da) is very similar (75% identity) to the other two sequenced aliphatic amidases, one from Pseudomonas aeruginosa and one from Rhodococcus sp. R312. Amidase activity was measured as the release of ammonia by sonicated crude extracts from H. pylori strains and from recombinant Escherichia coli strains overproducing the H. pylori amidase. The substrate specificity was analysed with crude extracts from H. pylori cells grown in vitro; the best substrates were propionamide, acrylamide and acetamide. Polymerase chain reaction (PCR) amplification of an internal amiE sequence was obtained with each of 45 different H. pylori clinical isolates, suggesting that amidase is common to all H. pylori strains. A H. pylori mutant (N6‐836) carrying an interrupted amiE gene was constructed by allelic exchange. No amidase activity could be detected in N6‐836. In a N6–urease negative mutant, amidase activity was two‐ to threefold higher than in the parental strain N6. Crude extracts of strain N6 slowly hydrolysed formamide. This activity was affected in neither the amidase negative strain (N6‐836) nor a double mutant strain deficient in both amidase and urease activities, suggesting the presence of an independent discrete formamidase in H. pylori. The existence of an aliphatic amidase, a correlation between the urease and amidase activities and the possible presence of a formamidase indicates that H. pylori has a large range of possibilities for intracellular ammonia production.