Home About us Contact
|
Home About us Contact | ||
|
|
||
Low Titer (low + titer)
Selected AbstractsHuman CD4+ T-cell epitope repertoire on the C2 domain of coagulation factor VIIIJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 8 2003M. T. Reding Summary., Approximately 25% of severe hemophilia A patients develop antibodies (Ab) that neutralize the procoagulant function of factor (F)VIII (inhibitors). Autoimmune FVIII inhibitors may develop in individuals without congenital FVIII deficiency and cause acquired hemophilia. Low titers of anti-FVIII Ab may be present in hemophilia A patients without inhibitors and in healthy blood donors. FVIII-specific CD4+ T-cells drive the synthesis of anti-FVIII Ab. We examined the epitope repertoire of CD4+ T-cells from 15 healthy subjects, 10 hemophilia A patients without inhibitors, 11 hemophilia A patients with inhibitors, and six acquired hemophilia patients. Blood CD4+ T-cells were challenged in proliferation assays with a panel 16 overlapping synthetic peptides, spanning the sequence of the FVIII C2 domain. The sequence region 2291,2330 contained the most frequently and strongly recognized peptides in each of the four subject groups. Crystallographic B factor data and the location of these peptides within the three-dimensional structure of the C2 domain confirm that this region has a high degree of solvent exposure and flexibility within the peptide backbone, which are structural features typical of immunodominant universal CD4+ epitopes. Furthermore, this sequence region overlaps inhibitor-binding sites, suggesting that CD4+ T-cells recognizing peptide sequences within this region might be involved in inhibitor synthesis. The sequence regions 2191,2210 (recognized strongly by each study group except hemophilia A patients with inhibitors) and 2241,2290 (recognized primarily by acquired hemophilia patients and healthy subjects) share the same structural features, and also overlap inhibitor-binding sites. Although similar, there appear to be important differences in the CD4+ epitope repertoires of congenital and acquired hemophilia patients. [source] Erythema dyschromicum perstans and hepatitis C virus infectionINTERNATIONAL JOURNAL OF DERMATOLOGY, Issue 5 2001George J. Kontochristopoulos MD A 48-year-old woman with a 10-month history of widespread, hyperpigmented, slightly pruritic macules, with a red border, involving the trunk and the proximal limbs (Fig. 1) was referred to our outpatient department. The oral mucosa, palms, soles, scalp, and nails were normal. Figure 1. Multiple hyperpigmented macules with an active border on the trunk Laboratory tests showed elevated liver enzymes [alanine aminotransferase (ALT), 68 IU/L (normal value, <,40 IU/L); aspartate aminotransferase (AST), 41 IU/L (normal value, <,40 IU/L)], the presence of antibodies to hepatitis C virus (anti-HCV) and HCV RNA (Amplicor Roche). In addition, cryoglobulinemia type III (IgM,,,, IgG,,,) was detected with a high cryocrit value, and there was detectable C-reactive protein, rheumatoid factor, and a low titer of antinuclear antibodies (1 : 80). A percutaneous liver biopsy showed changes compatible with mild chronic hepatitis (grade, 6; stage, 0). The possible source of infection was unknown, as the patient had no history of parenteral transmission (e.g. blood transfusions, intravenous illicit drug use). A skin biopsy specimen from the active border of a lesion showed hyperkeratosis, parakeratosis, and hydropic degeneration of the basal cell layer, with the formation of colloid bodies in the epidermis. A moderate perivascular lymphohistiocytic infiltrate with melanophages and free melanin granules was observed in the upper dermis (Fig. 2). Immunostaining of paraffin-embedded tissue sections with the TORDJT-22 IgG1 mouse monoclonal antibody to HCV (Biogenex, Son Ramon, USA), which is specific for the nonstructural region of HCV (NS3-NSH, C100 antigen) using the avidin,biotin,peroxidase complex (ABC) as well as the alkaline phosphatase antialkaline phosphatase (APAAP) methods, failed to detect HCV in the lesion of erythema dyschromicum perstans (EDP) (Nakopoulou L, Manolaki N, Lazaris A et al. Tissue immunodetection of C100 hepatitis C virus antigen in major thalassemic patients. Hepato-Gastroenterol 1999; 46: 2515,2520). Direct immunofluorescence showed IgG, IgM, IgA, and fibrinogen deposits on colloid bodies. EDP was diagnosed on the basis of these clinical and laboratory findings. Figure 2. Hydropic degeneration of the basal cell layer with colloid bodies in the epidermis. Moderate perivascular lymphohistiocytic infiltrate with melanophages and free melanin granules in the upper dermis (hematoxylin and eosin, ×,200) The patient was treated with interferon-,2b (Intron-A, Schering Plough Athens, Greece), 3 MU thrice weekly subcutaneously for 12 months, with additional topical steroid application. There was no response to this treatment with new lesions appearing in previously unaffected areas of the trunk and extremities. HCV RNA remained persistently positive. Thus, a modified regimen with interferon-,2b, 6 MU thrice weekly for 6 months, was tried. At the end of the treatment course, the eruption of EDP had greatly improved. Liver enzymes were normal (ALT, 22 IU/L; AST, 24 IU/L) and HCV RNA had become negative. Four months later, however, cutaneous lesions reappeared and hepatitis C relapsed. At this time point, combination therapy of interferon-,2b, 3 MU thrice weekly, with ribavirin, 1000 mg daily, was given. Six months later, liver enzymes were normal (ALT, 42 IU/L; AST, 39 IU/L), HCV RNA was negative, and the lesions of EDP had resolved. [source] Antiphospholipid antibodies and hepatitis C virus infection in Iranian thalassemia major patientsINTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 1 2008S. KASHEF Summary Although the precise nature of Antiphospholipid antibodies is still not clearly defined, they are known to have association with thromboembolic events and have been found in hepatitis C virus (HCV) infection. Moreover, high prevalence of HCV infection and thrombotic risk is described in thalassemia. We aimed at investigating the prevalence of anticardiolipin antibodies (aCLAbs), lupus anticoagulant (LA), and their relation with HCV infection in Iranian thalassemic patients. Presence of anti-HCV antibody, serum HCV-RNA, aCLAbs, and LA activity was determined in 131 patients with thalassemia major (male/female: 63/68 aged 3,29 years) registered at thalassemia unit, Dastgheib Hospital, Shiraz, Iran. Sixty-one healthy controls were also included. Anti-HCV antibody was positive in 24 (18.3%), IgG aCLAbs in 56 (42.7%), and LA activity in 9 (6.9%) patients. 87.5% of patients positive for aCLAbs had a low titer of aCLAbs. Although none of the participants had a previous history of thrombosis, higher prevalence of aCLAbs was detected in thalassemic patients compared with controls. No significant difference in the prevalence of aCLAbs was found between HCV-infected and noninfected patients. A high prevalence of aCLAbs, the majority in low titers, was detected in Iranian thalassemic patients irrespective of previous history of thrombosis and presence of HCV infection. [source] Humoral immunity in natural infection by tick-borne encephalitis virusJOURNAL OF MEDICAL VIROLOGY, Issue 4 2009Giulietta Venturi Abstract Tick-borne encephalitis (TBE) virus is one of the most important flaviviruses associated with neurological disease in Europe. Cross-reactive antibodies elicited by different flaviviruses can make difficult the interpretation of ELISA and hemagglutination-inhibition (HI) tests for the diagnosis of TBE. Neutralization tests, which are more specific, are not in common use because they are difficult to perform and standardize. A plaque reduction neutralization test (PRNT), optimized previously in vaccinated children, was evaluated in sera from acute cases of TBE, collected for diagnostic purposes, and from healthy human population and wild ruminants, collected for serosurvey purposes. The PRNT results were compared with the results of ELISA and HI tests. In acute TBE disease, most sera were positive for IgM antibodies by ELISA and with high HI antibody titers; neutralizing antibodies were detected in 71.4% of patients, at a very low titer (1:10 NT50) in almost all cases. Seroprevalences of 8% and 6.5% for anti-TBE ELISA antibodies were found in healthy subjects and wild ruminants, respectively. Among anti-TBE positive healthy subjects, a very low 1:10 NT50 titer was detected in 17.4% of cases, while NT80 titers ranging from 1:10 to 1:80 were detected in 65.2% of cases. Among wild ruminants, 90.9% of ELISA and HI positive samples showed a positive, ,1:10 NT80 titer. In conclusion, neutralization assays can be useful for the diagnosis and serosurveys of TBE. J. Med. Virol. 81:665,671, 2009 © 2009 Wiley-Liss, Inc. [source] Generation of high rapamycin producing strain via rational metabolic pathway-based mutagenesis and further titer improvement with fed-batch bioprocess optimizationBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2010Xiangcheng Zhu Abstract Rapamycin is a triene macrolide antibiotic produced by Streptomyces hygroscopicus. Besides its wide application as an effective immunosuppressive agent, other important bioactivities have made rapamycin a potential drug lead for novel pharmaceutical development. However, the low titer of rapamycin in the original producer strain limits further industrialization efforts and restricts its use for other applications. Predicated on knowledge of the metabolic pathways related to rapamycin biosynthesis in S. hygroscopicus, we have rationally designed approaches to generate a rapamycin high producer strain of S. hygroscopicus HD-04-S. These have included alleviation of glucose repression, improved tolerance towards lysine and shikimic acid, and auxotrophy of tryptophan and phenylalanine through the application of stepwise UV mutagenesis. The resultant strain produced rapamycin at 450,mg/L in the shake flask scale. These fermentations were further scaled up in 120 and 20,000,L fermentors, respectively, at the pilot plant. Selected fermentation factors including agitation speed, pH, and on-line supplementation were systematically evaluated. A fed-batch strategy was established to maximize rapamycin production. With these efforts, an optimized fermentation process in the larger scale fermentor was developed. The final titer of rapamycin was 812,mg/L in the 120,L fermentor and 783,mg/L in the 20,000,L fermentor. This work highlights a high rapamycin producing strain derived by mutagenesis and subsequent screening, fermentation optimization of which has now made it feasible to produce rapamycin on an industrial scale by fermentation. The strategies developed here should also be applicable to titer improvement of other important microbial natural products on an industrial scale. Biotechnol. Bioeng. 2010;107: 506,515. © 2010 Wiley Periodicals, Inc. [source] CHO gene expression profiling in biopharmaceutical process analysis and designBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010Jochen Schaub Abstract Increase in both productivity and product yields in biopharmaceutical process development with recombinant protein producing mammalian cells can be mainly attributed to the advancements in cell line development, media, and process optimization. Only recently, genome-scale technologies enable a system-level analysis to elucidate the complex biomolecular basis of protein production in mammalian cells promising an increased process understanding and the deduction of knowledge-based approaches for further process optimization. Here, the use of gene expression profiling for the analysis of a low titer (LT) and high titer (HT) fed batch process using the same IgG producing CHO cell line was investigated. We found that gene expression (i) significantly differed in HT versus LT process conditions due to differences in applied chemically defined, serum-free media, (ii) changed over the time course of the fed batch processes, and that (iii) both metabolic pathways and 14 biological functions such as cellular growth or cell death were affected. Furthermore, detailed analysis of metabolism in a standard process format revealed the potential use of transcriptomics for rational media design as is shown for the case of lipid metabolism where the product titer could be increased by about 20% based on a lipid modified basal medium. The results demonstrate that gene expression profiling can be an important tool for mammalian biopharmaceutical process analysis and optimization. Biotechnol. Bioeng. 2010; 105: 431,438. © 2009 Wiley Periodicals, Inc. [source] Virion half-life in chronic hepatitis B infection is strongly correlated with levels of viremia,HEPATOLOGY, Issue 4 2008Maura Dandri Analysis of hepatitis B virus (HBV) kinetics with mathematical models may disclose new aspects of HBV infection and host response mechanisms. To determine the kinetics of virion decay from the blood of patients in different phases of chronic infection, we applied mathematical modeling to real-time polymerase chain reaction assays, which enable quantification of viremia and intrahepatic HBV productivity by measuring both copy number and activity of covalently closed circular DNA (relaxed circular DNA/covalently closed circular DNA) in the liver of 80 untreated chronically active HBV carriers (38 hepatitis B e antigen [HBeAg]-positive and 42 HBeAg-negative individuals). We found that the half-life of circulating virions is very fast (median 46 and 2.5 minutes in HBeAg-positive and HBeAg-negative individuals, respectively) and strongly related to viremia, with clearance rates significantly accelerating as viral loads decrease. To investigate whether immune components can influence the kinetics of virion decay, we analyzed viral dynamics in immunodeficient urokinase-type plasminogen activator chimera mice. Virion half-life in mice (range, 44 minutes to >4 hours) was comparable to estimates determined in high viremic carriers, implying that clearance rates in these patients are mostly determined by common nonspecific mechanisms. Notably, the lack of correlation between virion half-life and viremia in mice indicated that immune components significantly accelerate virion clearance rates in individuals with low titers. Conclusion: Our analyses suggest that both host defense mechanisms and levels of circulating virions affect the kinetics of HBV decay assessed in the serum of chronic carriers. Identification of the factors affecting clearance rates will be important for future antiviral drug developments and it may give insights into the mechanisms involved in clearance of other chronic infections, such as human immunodeficiency virus and hepatitis C virus. (HEPATOLOGY 2008.) [source] Vaccination trial with HPV16 L1E7 chimeric virus-like particles in women suffering from high grade cervical intraepithelial neoplasia (CIN 2/3)INTERNATIONAL JOURNAL OF CANCER, Issue 12 2007Andreas M. Kaufmann Abstract Persistent infection with human papillomaviruses (HPV) is a prerequisite for the development of cervical cancer. Vaccination with virus-like particles (VLP) has demonstrated efficacy in prophylaxis but lacks therapeutic potential. HPV16 L1E7 chimeric virus-like particles (CVLP) consist of a carboxy-terminally truncated HPV16L1 protein fused to the amino-terminal part of the HPV16 E7 protein and self-assemble by recombinant expression of the fusion protein. The CVLP are able to induce L1- and E7-specific cytotoxic T lymphocytes. We have performed a first clinical trial to gain information about the safety and to generate preliminary data on the therapeutic potential of the CVLP in humans. A randomized, double blind, placebo-controlled clinical trial has been conducted in 39 HPV16 mono-infected high grade cervical intraepithelial neoplasia (CIN) patients (CIN 2/3). Two doses (75 ,g or 250 ,g) of CVLP were applied. The duration of the study was 24 weeks with 2 optional visits after another 12 and 24 weeks. The vaccine showed a very good safety profile with only minor adverse events attributable to the immunization. Antibodies with high titers against HPV16 L1 and low titers against HPV16 E7 as well as cellular immune responses against both proteins were induced. Responses were equivalent for both vaccine concentrations. A trend for histological improvement to CIN 1 or normal was seen in 39% of the patients receiving the vaccine and only 25% of the placebo recipients. Fifty-six percent of the responders were also HPV16 DNA-negative by the end of the study. Therefore, we demonstrated evidence for safety and a nonsignificant trend for the clinical efficacy of the HPV16 L1E7 CVLP vaccine. © 2007 Wiley-Liss, Inc. [source] Antiphospholipid antibodies and hepatitis C virus infection in Iranian thalassemia major patientsINTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 1 2008S. KASHEF Summary Although the precise nature of Antiphospholipid antibodies is still not clearly defined, they are known to have association with thromboembolic events and have been found in hepatitis C virus (HCV) infection. Moreover, high prevalence of HCV infection and thrombotic risk is described in thalassemia. We aimed at investigating the prevalence of anticardiolipin antibodies (aCLAbs), lupus anticoagulant (LA), and their relation with HCV infection in Iranian thalassemic patients. Presence of anti-HCV antibody, serum HCV-RNA, aCLAbs, and LA activity was determined in 131 patients with thalassemia major (male/female: 63/68 aged 3,29 years) registered at thalassemia unit, Dastgheib Hospital, Shiraz, Iran. Sixty-one healthy controls were also included. Anti-HCV antibody was positive in 24 (18.3%), IgG aCLAbs in 56 (42.7%), and LA activity in 9 (6.9%) patients. 87.5% of patients positive for aCLAbs had a low titer of aCLAbs. Although none of the participants had a previous history of thrombosis, higher prevalence of aCLAbs was detected in thalassemic patients compared with controls. No significant difference in the prevalence of aCLAbs was found between HCV-infected and noninfected patients. A high prevalence of aCLAbs, the majority in low titers, was detected in Iranian thalassemic patients irrespective of previous history of thrombosis and presence of HCV infection. [source] Factors affecting proliferation and differentiation of lepidopteran midgut stem cells,ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 1 2010Marcia J. Loeb Midgut stem cells of last instar larvae and pupae of Heliothis virescens, Lymantria dispar and several other Lepidopteran species have been cultured in vitro and have been induced to proliferate using low titers of ecdysteroids and the 77-Kda peptide fragment, ,-arylphorin, isolated and identified from pupal fat body tissue. The insulin-related hormone, Bombyxin, also induced mitosis in cultured midgut stem cells; it appeared to be fast-acting and quickly inactivated, while ,-arylphorin was slower to act and had a longer lasting effect in vitro, indicating different functions for these proliferation agents. Changes in Calcium ion concentration within or outside the cells discretely affected stem cell differentiation, indicating a role for second messenger participation in peptide regulation of this process. Four different peptides (MDFs 1,4) that induced midgut stem cells to differentiate to mature midgut cell types in vitro were isolated and characterized from conditioned media and hemolymph of H. virescens and L. dispar. However, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and all-trans retinoic acid (RA) from vertebrate sources induced differentiation to non-midgut cell types as well. MDF1 was located in basal areas of columnar cells of midgut epithelium, although MDF2 was observed in all of the cytoplasm of columnar cells and in droplets of antibody positive material in the midgut lumen, suggesting a digestive function as well for this peptide. Anti-MDF-3 stained the central areas of cultured midgut columnar cells and the bases of columnar cells of midgut epithelium in vivo. Midgut secretory cells stained with anti-MDF-4; streams of MFD-4-positive material were observed extending from secretory cells facing the epithelial lumen, and as a layer on the hemolymph-facing side, suggesting an endocrine or paracrine function for this or an immunologically similar peptide. Published 2010 Wiley Periodicals, Inc. [source] | |||||||||||||