Biomedical subjects

S Quan

Publications and source records attributed to S Quan.

At least 19 recordsLinked to original sources

PubMed2007

Impact of silencing HO-2 on EC-SOD and the mitochondrial signaling pathway.

The contribution of heme oxygenase HO-2, the primary source of bilirubin and carbon monoxide (CO) under physiological conditions, to the regulation of vascular function has remained largely unexplored. Using siRNA HO-2, we examined the effect of suppressed levels of HO-2 on vascular antioxidant and survival proteins. In vivo HO-2 siRNA treatment decreased the basal levels of EC-SOD, pAKT proteins (serine-473 and threonine-308), without changing Akt protein expression. HO-2 siRNA treatment increased 3-nitrotyrosine (3-NT) and apoptotic signaling kinase-1 (ASK-1) (P < 0.01). HO activity was decreased by the use of siRNA HO-2. We extended these studies to the mitochondria, examining for the presence of HO-1 and its role in the regulation of pro- and anti-apoptotic proteins. HO activity was increased by the administration of CoPP resulting in the translocation of HO-1 into the mitochondria, mainly to the inner face of the mitochondrial inner membrane. These findings suggest that HO-2 is critical in the maintenance of heme homeostasis and also the regulation of apoptosis by controlling levels of EC-SOD, Akt, 3-NT, and ASK-1. In addition, localization of HO-1 in the mitochondrial compartment plays a critical role in mitochondria-mediated apoptosis.

Animals↗

PubMed2001

Regulation of human heme oxygenase in endothelial cells by using sense and antisense retroviral constructs.

Our objective was to determine whether overexpression and underexpression of human heme oxygenase (HHO)-1 could be controlled on a long-term basis by introduction of the HO-1 gene in sense (S) and antisense (AS) orientation with an appropriate vector into endothelial cells. Retroviral vector (LXSN) containing viral long terminal repeat promoter-driven human HO-1 S (LSN-HHO-1) and LXSN vectors containing HHO-1 promoter (HOP)-controlled HHO-1 S and AS (LSN-HOP-HHO-1 and LSN-HOP-HHO-1-AS) sequences were constructed and used to transfect rat lung microvessel endothelial cells (RLMV cells) and human dermal microvessel endothelial cells (HMEC-1 cells). RLMV cells transduced with HHO-1 S expressed human HO-1 mRNA and HO-1 protein associated with elevation in total HO activity compared with nontransduced cells. Vector-mediated expression of HHO-1 S or AS under control of HOP resulted in effective production of HO-1 or blocked induction of endogenous human HO-1 in HMEC-1 cells, respectively. Overexpression of HO-1 AS was associated with a long-term decrease (45%) of endogenous HO-1 protein and an increase (167%) in unmetabolized exogenous heme in HMEC-1 cells. Carbon monoxide (CO) production in HO-1 S- or AS-transduced HMEC-1 cells after heme treatment was increased (159%) or decreased (50%), respectively, compared with nontransduced cells. HO-2 protein levels did not change. These findings demonstrate that HHO-1 S and AS retroviral constructs are functional in enhancing and reducing HO activity, respectively, and thus can be used to regulate cellular heme levels, the activity of heme-dependent enzymes, and the rate of heme catabolism to CO and bilirubin.

3T3 Cells↗

PubMed2001

Functional expression and stabilization of horseradish peroxidase by directed evolution in Saccharomyces cerevisiae.

Biotechnology applications of horseradish peroxidase (HRP) would benefit from access to tailor-made variants with greater specific activity, lower K(m) for peroxide, and higher thermostability. Starting with a mutant that is functionally expressed in Saccharomyces cerevisiae, we used random mutagenesis, recombination, and screening to identify HRP-C mutants that are more active and stable to incubation in hydrogen peroxide at 50 degrees C. A single mutation (N175S) in the HRP active site was found to improve thermal stability. Introducing this mutation into an HRP variant evolved for higher activity yielded HRP 13A7-N175S, whose half-life at 60 degrees C and pH 7.0 is three times that of wild-type (recombinant) HRP and a commercially available HRP preparation from Sigma (St. Louis, MO). The variant is also more stable in the presence of H(2)O(2), SDS, salts (NaCl and urea), and at different pH values. Furthermore, this variant is more active towards a variety of small organic substrates frequently used in diagnostic applications. Site-directed mutagenesis to replace each of the four methionine residues in HRP (M83, M181, M281, M284) with isoleucine revealed no mutation that significantly increased the enzyme's stability to hydrogen peroxide.

Cloning, Molecular↗

PubMed2001

Penetrating capacity of human spermatozoa cool preserved in electrolyte-free solution.

OBJECTIVE: To evaluate whether human spermatozoa preserved in electrolyte-free (EF) solution at 4 degrees C possess normal penetrating capacity. STUDY DESIGN: The acrosomal status of human spermatozoa cool preserved in EF solution was evaluated before preservation and before and after reinitiation by using chlortetracycline staining. The zona-free hamster egg sperm penetration test was performed using spermatozoa cool preserved in EF solution. RESULTS: The percentages of capacitated and acrosome-reacted spermatozoa cool preserved in EF solution before reinitiation were similar to those of fresh spermatozoa, but they significantly increased after reinitiation. The penetration rate and fertility index of spermatozoa cool preserved in EF solution were comparable to those of fresh spermatozoa (48.3% vs. 50.8% and 1.37 +/- 0.15 vs. 1.29 +/- 0.10, respectively). CONCLUSION: Human spermatozoa cool preserved in EF solution for one week can possess as much penetrating capacity as fresh spermatozoa.

Acrosome↗

PubMed2000

Activation of the pro-drug ethionamide is regulated in mycobacteria.

The anti-tuberculosis drug ethionamide (ETH), which is a structural analog of isoniazid (INH), is known to strongly inhibit mycolic acid synthesis in Mycobacterium tuberculosis. Although several targets have been identified for INH, only speculative information is available concerning ETH. Mutations within the promoter and the coding region of enoyl-acyl carrier protein reductase (InhA) were found to confer resistance to both drugs, thus leading to the impression that INH and ETH may share a common mode of action. However, a notable distinction between the two drugs lies in the lack of cross-resistance in clinical isolates. This may be attributed in part to the fact that the pro-drug INH must be activated via KatG, and no activation step for ETH has yet been described. Here we report the identification of an activator for ETH. The ETH activator (Rv3854c), which we have termed EthA, was found to be homologous to various monooxygenases and induced ETH sensitivity when overexpressed in mycobacteria. Interestingly, the neighboring open reading frame (Rv3855), which was found homologous to transcriptional repressors of the tetR family, led to ETH resistance when overexpressed. In addition, chromosomal inactivation of this gene by transposition led to ETH hypersensitivity. These data strongly suggest that Rv3855, which we have termed EthR, regulates the production of EthA, which subsequently activates the pro-drug ETH. This study opens up new avenues of research relating to ETH activation in mycobacteria, possibly leading to an improved efficacy of ETH and to the generation of new anti-mycobacterial agents.

Amino Acid Sequence↗

PubMed2000

CXC chemokine receptor 3 expression on CD34(+) hematopoietic progenitors from human cord blood induced by granulocyte-macrophage colony-stimulating factor: chemotaxis and adhesion induced by its ligands, interferon gamma-inducible protein 10 and monokine induced by interferon gamma.

CXC chemokine receptor 3 (CXCR3), which is known to be expressed predominately on memory and activated T lymphocytes, is a receptor for both interferon gamma (IFN-gamma)-inducible protein 10 (gamma IP-10) and monokine induced by IFN-gamma (Mig). We report the novel finding that CXCR3 is also expressed on CD34(+) hematopoietic progenitors from human cord blood stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF) but not on freshly isolated CD34(+) progenitors. Freshly isolated CD34(+) progenitors expressed low levels of CXCR3 messenger RNA, but this expression was highly up-regulated by GM-CSF, as indicated by a real-time quantitative reverse transcriptase-polymerase chain reaction technique. gamma IP-10 and Mig induced chemotaxis of GM-CSF-stimulated CD34(+) progenitors by means of CXCR3, since an anti-CXCR3 monoclonal antibody (mAb) was found to block gamma IP-10-induced and Mig-induced CD34(+) progenitor chemotaxis. These chemotactic attracted CD34(+) progenitors are colony-forming units-granulocyte-macrophage. gamma IP-10 and Mig also induced GM-CSF-stimulated CD34(+) progenitor adhesion and aggregation by means of CXCR3, a finding confirmed by the observation that anti-CXCR3 mAb blocked these functions of gammaIP-10 and Mig but not of chemokine stromal cell-derived factor 1 alpha. gamma IP-10-induced and Mig-induced up-regulation of integrins (CD49a and CD49b) was found to play a crucial role in adhesion of GM-CSF-stimulated CD34(+) progenitors. Moreover, gamma IP-10 and Mig stimulated CXCR3 redistribution and cellular polarization in GM-CSF-stimulated CD34(+) progenitors. These results indicate that CXCR3-gamma IP-10 and CXCR3-Mig receptor-ligand pairs, as well as the effects of GM-CSF on them, may be especially important in the cytokine/chemokine environment for the physiologic and pathophysiologic events of differentiation of CD34(+) hematopoietic progenitors into lymphoid and myeloid stem cells, subsequently immune and inflammatory cells. These processes include transmigration, relocation, differentiation, and maturation of CD34(+) hematopoietic progenitors. (Blood. 2000;96:1230-1238)

Antigens, CD34↗

PubMed2000

CXCR3 expression and activation of eosinophils: role of IFN-gamma-inducible protein-10 and monokine induced by IFN-gamma.

CXC chemokine receptor 3 (CXCR3), predominately expressed on memory/activated T lymphocytes, is a receptor for both IFN-gamma-inducible protein-10 (gamma IP-10) and monokine induced by IFN-gamma (Mig). We report a novel finding that CXCR3 is also expressed on eosinophils. gamma IP-10 and Mig induce eosinophil chemotaxis via CXCR3, as documented by the fact that anti-CXCR3 mAb blocks gamma IP-10- and Mig-induced eosinophil chemotaxis. gamma IP-10- and Mig-induced eosinophil chemotaxis are up- and down-regulated by IL-2 and IL-10, respectively. Correspondingly, CXCR3 protein and mRNA expressions in eosinophils are up- and down-regulated by IL-2 and IL-10, respectively, as detected using flow cytometry, immunocytochemical assay, and a real-time quantitative RT-PCR technique. gamma IP-10 and Mig act eosinophils to induce chemotaxis via the cAMP-dependent protein kinase A signaling pathways. The fact that gamma IP-10 and Mig induce an increase in intracellular calcium in eosinophils confirms that CXCR3 exists on eosinophils. Besides induction to chemotaxis, gamma IP-10 and Mig also activate eosinophils to eosinophil cationic protein release. These results indicate that CXCR3-gamma IP-10 and -Mig receptor-ligand pairs as well as the effects of IL-2 and IL-10 on them may be especially important in the cytokine/chemokine environment for the pathophysiologic events of allergic inflammation, including initiation, progression, and termination in the processes.

Blood Proteins↗

PubMed2000

Eotaxin induces degranulation and chemotaxis of eosinophils through the activation of ERK2 and p38 mitogen-activated protein kinases.

Eotaxin and other CC chemokines acting via CC chemokine receptor-3 (CCR3) are believed to play an integral role in the development of eosinophilic inflammation in asthma and allergic inflammatory diseases. However, little is known about the intracellular events following agonist binding to CCR3 and the relationship of these events to the functional response of the cell. The objectives of this study were to investigate CCR3-mediated activation of the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase-2 (ERK2), p38, and c-jun N-terminal kinase (JNK) in eosinophils and to assess the requirement for MAP kinases in eotaxin-induced eosinophil cationic protein (ECP) release and chemotaxis. MAP kinase activation was studied in eotaxin-stimulated eosinophils (more than 97% purity) by Western blotting and immune-complex kinase assays. ECP release was measured by radioimmunoassay. Chemotaxis was assessed using Boyden microchambers. Eotaxin (10(-11) to 10(-7) mol/L) induced concentration-dependent phosphorylation of ERK2 and p38. Phosphorylation was detectable after 30 seconds, peaked at about 1 minute, and returned to baseline after 2 to 5 minutes. Phosphorylation of JNK above baseline could not be detected. The kinase activity of ERK2 and p38 paralleled phosphorylation. PD980 59, an inhibitor of the ERK2-activating enzyme MEK (MAP ERK kinase), blocked phosphorylation of ERK2 in a concentration-dependent manner. The functional relevance of ERK2 and p38 was studied using PD98 059 and the p38 inhibitor SB202 190. PD98 059 and SB202 190 both caused inhibition of eotaxin-induced ECP release and chemotaxis. We conclude that eotaxin induces a rapid concentration-dependent activation of ERK2 and p38 in eosinophils and that the activation of these MAP kinases is required for eotaxin-stimulated degranulation and directed locomotion. (Blood. 2000;95:1911-1917)

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed2000

Light inhibits rifampicin inactivation and reduces rifampicin resistance due to a cloned mycobacterial ADP-ribosylation gene.

Rifampicin is a principal drug used to combat infections by mycobacteria and related organisms. Most strains of Mycobacterium are able to inactivate this antibiotic by ribosylation via an ADP-ribosylated intermediate. We found that this inactivation was inhibited by light at levels similar to those prevailing in laboratory environments. Rifampicin resistance arising from the cloned ADP-ribosyl transferase was also greatly diminished at these light levels. The cloned Rhodococcus equi monooxygenase which inactivates this antibiotic by a different mechanism was, in contrast, not inhibited by light.

Antibiotics, Antitubercular↗

PubMed2000

Serotyping strip immunoblot assay for assessing hepatitis C virus strains in dialysis patients.

Recent accumulated evidence shows that dialysis patients are a high-risk group for hepatitis C virus (HCV) infection. Assessment of HCV genotype distribution among dialysis patients may be important because specific viral genotypes are associated with different clinical manifestations, disease progression, and response to antiviral therapy. However, polymerase chain reaction-based methods are cumbersome and unsuitable for analyzing large cohorts of dialysis patients with HCV. Instead, this information can be obtained by using a novel recombinant immunoblot assay (RIBA) recently developed for determining HCV serotype. The RIBA HCV serotyping strip immunoblot assay (SIA; Chiron Corporation, Emeryville, CA), is based on an immunoblot strip with five lanes of immobilized serotype-specific HCV peptides from the nonstructural (NS4) and core regions of the genomes of HCV types 1, 2, and 3. HCV serotype is deduced by determining the greatest intensity of reactivity to the NS4 serotype-specific HCV peptide band in relation to the internal control band (human immunoglobulin G) intensity on each strip. HCV core peptide reactivity is used only in the absence of NS4 reactivity. We compared RIBA HCV serotyping SIA with genotyping using sera from a large (n = 107) cohort of HCV-infected patients undergoing chronic hemodialysis (HD). We successfully serotyped 79 of 107 patients (74%) undergoing HD. We found a remarkable concordance (65 of 70 results; 93%) between RIBA HCV serotyping SIA and genotyping (line probe assay [LiPA]) techniques (kappa = 0.786) with sera from viremic patients infected with a known genotype. Only 5 of 70 patients (7%) had apparently discordant results. In a subset of patients (28 of 107 patients; 26%) not typed by RIBA HCV serotyping SIA, most (24 of 28 patients; 86%) were successfully genotyped by LiPA technology. It was possible to assess serotype reactivity in some patients (9 of 107 patients; 7%) who could not be genotyped. The distribution of HCV serotypes was associated with the antibody response against HCV proteins and the patterns of reactivity by RIBA HCV 2.0 SIA. In conclusion, (1) we found good agreement between serotyping and genotyping methods in our large cohort of dialysis patients infected with HCV; (2) the impaired immunocompetence conferred by uremia may limit serotyping analysis in some HCV-infected patients undergoing HD; (3) RIBA HCV serotyping SIA may be useful in tracking transmission routes for HD patients who cleared the virus and have only anti-HCV antibody; and (4) the distribution of HCV serotypes was associated with the antibody response against HCV proteins and the patterns of reactivity by RIBA HCV 2.0 SIA. Assessment of HCV strains appears to be very useful in the routine clinical activity of nephrologists within HD units because consistent biological differences among HCV strains exist. RIBA serotyping SIA is a simple, inexpensive, and highly reproducible assay to obtain information about HCV types in the HD setting.

Female↗

PubMed2000

Effects of preservation of mouse spermatozoa in electrolyte-free solution at 4 degrees C on the outcome of mouse in vitro fertilization.

PURPOSE: The aim was to assess the fertilizing capacity of spermatozoa cool-preserved in electrolyte-free (EF) solution. METHODS: Mouse spermatozoa were cool-preserved in EF solution and the acrosomal status of the spermatozoa was compared before and after preservation using chlortetracycline stain. Mouse oocytes were inseminated by spermatozoa cool-preserved in EF solution for 2, 4, or 7 days and fertilization and blastocyst rates were evaluated. RESULTS: Acrosomal status of spermatozoa cool-preserved in EF solution was not different from spermatozoa before preservation, but the capacitated and acrosome-reacted spermatozoa significantly increased after reinitiation. Cool-preservation in EF solution for up to 4 days did not affect fertilization rate. Blastocyst rate of embryos derived from spermatozoa cool-preserved for 4 or 7 days in EF solution was significantly lower than that of embryos derived from fresh spermatozoa. CONCLUSIONS: Mouse spermatozoa cool-preserved in EF solution possesses as much fertilizing capacity as fresh spermatozoa. However, prolonged preservation affects the embryonic development.

Acrosome Reaction↗

PubMed2000

CXC chemokine receptor 4 expression and stromal cell-derived factor-1alpha-induced chemotaxis in CD4+ T lymphocytes are regulated by interleukin-4 and interleukin-10.

We report that interleukin (IL)-4 and IL-10 can significantly up- or down-regulate CXC chemokine receptor 4 (CXCR4) expression on CD4+ T lymphocytes, respectively. Stromal cell-derived factor-1alpha (SDF-1alpha)-induced CD4+ T-lymphocyte chemotaxis was also correspondingly regulated by IL-4 and IL-10. IL-4 and IL-10 up- or down-regulated CXCR4 mRNA expression in CD4+ T lymphocytes, respectively, as detected by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). Scatchard analysis revealed a type of CXCR4 with affinity (Kd approximately 6.3 nM), and approximately 70,000 SDF-1alpha-binding sites per cell, among freshly isolated CD4+ T lymphocytes, and two types of CXCR4 with different affinities (Kd1 approximately 4.4 nM and Kd2 approximately 14.6 nM), and a total of approximately 130,000 SDF-1alpha-binding sites per cell, among IL-4-stimulated CD4+ T lymphocytes. The regulation of CXCR4 expression in CD4+ T lymphocytes by IL-4 and IL-10 could be blocked by a selective inhibitor of protein kinase (staurosporine) or by a selective inhibitor of cAMP- and cGMP-dependent protein kinase (H-8), indicating that these cytokines regulate CXCR4 on CD4+ T lymphocytes via both cAMP and cGMP signalling pathways. The fact that cyclosporin A or ionomycin were able to independently change the CXCR4 expression and block the effects of IL-4 and IL-10 on CXCR4 expression implied that the capacity of IL-4 and IL-10 to regulate CXCR4 on CD4+ T lymphocytes is not linked to calcium-mobilization stimulation. These results indicate that the effects of IL-4 and IL-10 on the CXCR4-SDF-1 receptor-ligand pair may be of particular importance in the cytokine/chemokine environment concerning the inflammatory processes and in the progression of human immunodeficiency virus (HIV) infection.

CD4-Positive T-Lymphocytes↗

PubMed2000

Use of third-generation hepatitis C virus (HCV) enzyme immunoassay (EIA) to resolve second-generation HCV EIA-reactive and second-generation recombinant immunoblot assay-indeterminate blood samples: data to support current Food and Drug Administration guidance on HCV lookback.

Explore the source record for details and available documents.

Blood Donors↗

PubMed2000

Lookback on donors who are repeatedly reactive on first-generation hepatitis C virus assays:justification and rational implementation.

BACKGROUND: The purpose of this study was to explore strategies to minimize the number of unwarranted consignee notifications resulting from hepatitis C virus (HCV) first-generation (single-antigen) enzyme immunoassay (EIA 1.0) targeted lookback. STUDY DESIGN AND METHOD: The four blood centers participating in this study contributed data on 3753 HCV EIA 1.0-repeatably reactive (RR) donations. The analysis focused on 1) statistical evaluation of HCV EIA 1.0 signal-to-cutoff (S/CO) ratios versus HCV second-generation recombinant immunoblot assay (RIBA 2.0) interpretation from all participating blood centers and 2) RNA testing using transcription-mediated amplification on all HCV EIA 1.0 RR/RIBA 2. 0-positive or -indeterminate specimens and a subset of RIBA 2. 0-negative donations for which specimens were available. RESULTS: Analysis of HCV EIA 1.0 S/CO ratios versus RIBA 2.0 indicated that 1180 (89%) of 1326 RIBA 2.0-positive specimens had an S/CO ratio >2. 5, while 146 (11%) had a ratio </=2.5. In contrast, of 2253 RIBA 2. 0-negative specimens, 299 (13%) had an S/CO ratio >2.5, while 1954 (87%) had a ratio </=2.5. Of 248 HCV EIA 1.0-RR/RIBA 2.0-positive samples with stored specimens available for additional testing, 198 (80%) were HCV RNA positive; 15 (7.5%) of these specimens had an S/CO ratio </=2.5, while 183 (92%) had a ratio >2.5. HCV RNA was detected in only 2 (1.5%) of 137 HCV EIA 1.0-RR/RIBA 2.0-negative specimens: 1 of these 2 specimens had an S/CO >2.5, while the other had an S/CO </=2.5. CONCLUSION: A highly significant (<0.0001) correlation was found between the S/CO ratio on HCV EIA 1.0- and RIBA 2.0-positive or -negative results. An S/CO ratio >2.5 yielded an 89- percent sensitivity for RIBA 2.0-positive specimens, and donations with an S/CO ratio >2.5 had a 75-percent probability of being RIBA 2.0 positive. A policy recommendation to use the S/CO ratio to triage lookback would prevent unwarranted notification of 87 percent of recipients of blood from RIBA 2.0-negative donors and would result in a failure to notify only 5 to 10 percent of recipients potentially exposed to infectious units.

Blood Donors↗

PubMed2000

Performance of second- and third-generation RIBAs for confirmation of third-generation HCV EIA-reactive blood donations. Retrovirus Epidemiology Donor Study.

BACKGROUND: Licensure of an enhanced HCV screening assay (HCV 3.0 EIA) without concurrent licensure of a complementary supplemental assay (i.e., RIBA HCV 3.0 strip immunoblot assay [RIBA-3]) decoupled screening and supplemental testing. In March 1998, the FDA Center for Biologics Evaluation and Research (CBER) recommended the use of RIBA-3 on RIBA HCV 2.0 strip immunoblot assay (RIBA-2)-indeterminate units screened with HCV EIA 3.0. STUDY DESIGN AND METHODS: The sensitivity of RIBA-2 and RIBA-3 was compared in tests on HCV 3.0 EIA-repeatably reactive (RR) units identified immediately after the implementation of HCV 3.0 EIA screening. Two protocols were evaluated: parallel testing of HCV 3.0 EIA-RR units by RIBA-2 and RIBA-3 and reflex testing of HCV 3.0 EIA-RR and RIBA-3-confirmed-positive units by RIBA-2. All specimens with discordant RIBA-2 and RIBA-3 results and a representative sampling with concordant RIBA results were tested by PCR. RESULTS: In the parallel testing protocol, 99,777 donations were screened, with 245 HCV 3.0 EIA-RR specimens included in the study. Of 166 RIBA-2-positive samples, 165 tested positive in RIBA-3 (1 sample reacted to the control superoxide dismutase antigen in RIBA-3). Thirty-two (74%) of 43 RIBA-2-indeterminate specimens and 4 (11%) of 36 RIBA-2-negative specimens tested positive in RIBA-3. HCV RNA was identified in 5 (16%) of 32 RIBA-2-indeterminate/RIBA-3-positive donations, as well as in 26 (70%) of 37 concordant RIBA-2/RIBA-3-positive donations. In the reflex testing protocol, 292,459 donations were screened, with 709 HCV 3.0 EIA-RR specimens included in the study. RIBA-3 testing yielded 517 (73%) positive specimens, of which 50 (9.7%) tested indeterminate and 15 (2.9%) tested negative in RIBA-2. Among the RIBA-discordant specimens, 10 (20%) RIBA-2-indeterminate specimens and 1 (7%) RIBA-2-negative specimens tested positive in PCR; in comparison, 60 (77%) of 78 concordant RIBA-2/RIBA-3-positive units tested positive in PCR. CONCLUSIONS: RIBA-3 is significantly more sensitive than RIBA-2 in testing of HCV 3.0 EIA-screened donations. During the review process of this manuscript, the FDA licensed the RIBA-3 test.

Blood Donors↗

PubMed2000

Chemokine stromal cell-derived factor 1alpha activates basophils by means of CXCR4.

BACKGROUND: The CXC chemokine receptor 4 (CXCR4) is predominantly expressed on inactivated naive T lymphocytes, B lymphocytes, dendritic cells, and endothelial cells. CXC chemokine stromal cell-derived factor 1alpha (SDF-1alpha) is the only known ligand for CXCR4. To date, the CXCR4 expression and function of SDF-1alpha in basophils are unknown. OBJECTIVE: The purpose of this study was to investigate the expression of CXCR4 and functions of SDF-1alpha in basophils and to characterize the role of the CXCR4-SDF-1alpha receptor ligand pair in the allergic inflammation. METHODS: Basophil purification, flow cytometry, real-time quantitative RT-PCR assay, Northern blotting, intracellular free Ca(2+) change, chemotaxis assay, and histamine release assay were used. RESULTS: CXCR4 is abundantly expressed on peripheral blood resting basophils (91%). Likewise, CXCR4 messenger (m)RNA is expressed in resting basophils (3.2 x 10(3) copies per 2 x 10(2) cells). The existence of CXCR4 mRNA was also confirmed in basophils by means of Northern blot analysis. SDF-1alpha induces an increase in intracellular free Ca(2+) in basophils. SDF-1alpha activates basophils to chemotaxis (chemotactic index = 3.8) and histamine release (36% of total content) through CXCR4 on the cells. The chemokines SDF-1alpha, eotaxin, RANTES, monocyte chemoattractant protein (MCP) 1, and macrophage inflammatory protein (MIP) 1alpha have been demonstrated at different potencies in induction of chemotaxis (eotaxin > SDF-1alpha > RANTES congruent with MCP-1 >> MIP-1alpha) and histamine release (MCP-1 congruent with SDF-1alpha > eotaxin > RANTES > MIP-1alpha). The optimal concentration seen for SDF-1alpha effects (chemotaxis and histamine release) on basophils was 100 ng/mL. CONCLUSION: These results indicate that the CXCR4-SDF-1alpha receptor ligand pair may be important for the recruitment and activation of the basophils, which is a characteristic effector cell of the allergic inflammation.

Basophils↗

PubMed2000

Humoral immune response to hepatitis C after liver transplantation: assessment of a new recombinant immunoblot assay.

OBJECTIVE: The immune control of infection with hepatitis C virus (HCV) is poorly understood; vigorous antibody responses to viral proteins seem to coexist with the virus and thus whether they are neutralizing remains controversial. HCV-related liver failure is the leading indication for orthotopic liver transplantation (OLT) worldwide. Attenuated antibody responses in immunosuppressed patients and decreased reliability compared to assessment of HCV RNA has hampered the use of antibody testing post-OLT. The goals of this current analysis were twofold: to determine the sensitivity of a prototype strip immunoblot assay (RIBA 3.0, Chiron Diagnostics) for the diagnosis of HCV post-OLT; to determine if there was a correlation between antibody response and severity of histological recurrence. METHODS: The study was comprised of 76 HCV-positive individuals divided into three patient groups: liver allograft recipients with evidence of mild or no histological recurrence (n = 52), liver allograft recipients with evidence of severe HCV recurrence and allograft cirrhosis (n = 12), and nontransplant patients being enrolled in an induction interferon trial (n = 12). All transplant patients had histological follow-up of at least 1 yr. RESULTS: Sixty of the 64 (94%) HCV-positive OLT recipients had 1+ reactivity to two or more recombinant antigens; three of the patients who lacked a detectable response had minimal histological recurrence and one had severe recurrence. All nontransplant patients demonstrated 4+ reactivity to at least two antigens, and 55/64 (86%) OLT recipients demonstrated this same level of reactivity. Seven of the nine patients lacking this high level of reactivity had evidence of minimal recurrence. Furthermore, the mean (+/- SEM) level of antibody reactivity for c100 (p = 0.04) and NS5 (p = 0.01) were significantly lower for patients with mild recurrence after OLT, compared to the other groups. The level of antibody reactivity was unrelated to HCV genotype or viral load. CONCLUSIONS: The recently developed RIBA 3.0 assay for detection of antibodies to HCV appears to be highly sensitive for the diagnosis of HCV post-OLT. In general, the level of antibody reactivity was comparable in the transplant patients and in nonimmunosuppressed controls. The pathogenic implications of the relatively diminished humoral response in patients with mild recurrence post-OLT are discussed.

Antibody Formation↗

PubMed2000

[Measurement of macrophage colony-stimulating factor during in-vitro fertilization and embryo transfer cycles].

OBJECTIVE: To explore the influences of macrophage colony-stimulating factor (M-CSF) on follicle development, ovulation, fertilization and embryo quality during in-vitro fertilization and embryo transfer (IVF-ET) cycles. METHODS: 36 patients underwent 54 cycles of controlled ovarian hyperstimulation by gonadotropin-releasing hormone agonist-human menopausal goudotropin-human chorionic gonadotropin for IVF-ET program. M-CSF concentrations in serum were measured at early follicular phase, mid-follicular phase and the day of oocyte retrieval by enzyme-linked immunosorbent assay. Follicular fluid (FF) on the day of egg retrieval was also measured for M-CSF concentration. RESULTS: M-CSF concentration in serum gradually increased throughout ovarian hyperstimulation and reached a peak on the day of oocyte retrieval. M-CSF concentration in FF was significantly higher than that in serum on the same day (P < 0.01). The M-CSF concentration in FF from follicles where oocytes could be retrieved, fertilized or with a diameter > or 16 mm, volume > or 2 ml, was significantly higher than those from follicles where oocyte could not be retrieved, fertilized or with a diameter < 16 mm, volume < 2 ml, respectively (P < 0.01). However, there was no significant difference in M-CSF concentration of FF between high and low embryo grade groups (P > 0.05). CONCLUSIONS: M-CSF may play an important role as an intraovarian regulator in the process of follicle development, maturation and ovulation. Therefore, M-CSF may exert an effect on fertilization process but not on the embryo quality.

Embryo Transfer↗