Reversal of nonstructural protein 3-specific CD4(+) T cell dysfunction in patients with persistent hepatitis C virus infection.

Hepatitis C virus (HCV)-specific T cell responses are essential for HCV control, and chronic infection is characterized by functionally altered antigen-specific T cells. It has been proposed that the early inactivation of specific CD4(+) T cell responses may be involved in establishment of HCV persistence. We have investigated whether HCV-specific CD4(+) T cells dysfunction can be reversed in vitro. Nonstructural protein 3 (NS3) and core-specific CD4(+) T cells from eight chronically infected and eight spontaneously resolved HCV individuals were selected through transient CD154 (CD40 ligand) expression, and their functional profile (IFN-γ, IL-2, TNF-α, IL-10 and IL-4 production by enzyme-linked immunospot assay, cytometric bead array and intracellular cytokine staining, and proliferation by carboxy-fluorescein diacetate succinimidyl ester dilution assay) was determined both ex vivo and after in vitro expansion of sorted CD154-expressing cells in the absence of specific antigen in IL-7/IL-15-supplemented medium. Ex vivo bulk CD4(+) T cells from chronic patients expressed CD154 in most cases, albeit at lower frequencies than those of resolved patients (0.11%vs 0.41%; P = 0.01), when stimulated with NS3, but not core, although they had a markedly impaired capacity to produce IL-2 and IFN-γ. Antigen-free in vitro expansion of NS3-specific CD154(+) cells from chronic patients restored IFN-γ and IL-2 production and proliferation to levels similar to those of patients with spontaneously resolved infection. Hence, NS3-specific CD4(+) T cell response can be rescued in most chronic HCV patients by in vitro expansion in the absence of HCV-specific antigen. These results might provide a rationale for adoptive immunotherapy.

IL28B genetic variation and hepatitis C virus-specific CD4(+) T-cell responses in anti-HCV-positive blood donors.

Epidemiological, viral and host factors are associated with the outcome of hepatitis C virus (HCV) infection, and strong host immune responses against HCV favour viral clearance. Recently, genome-wide association studies have shown a strong correlation between single-nucleotide polymorphisms (SNPs) near the interleukin-28B (IL28B) gene and spontaneous or treatment-induced HCV clearance. We have investigated whether protective IL28B genetic variants are associated with HCV-specific T-cell responses among Spanish blood donors. The rs12979860 IL28B haplotype was determined in 69 anti-HCV-positive blood donors (21 HCV RNA negative and 48 HCV RNA positive) and 30 seronegative donors. In all cases, HCV-specific CD4(+) T-cell responses to HCV recombinant proteins (core, NS3 and NS3 helicase) were assessed by ex vivo interferon-γ ELISpot assay. The rs12979860-CC genotype was highly overrepresented in donors with spontaneous HCV clearance when compared to those with chronic infection (76.2%vs 29.2%, P < 0.001; odds ratio, 7.77; 95% confidence interval, 2.4-25.3, P < 0.001). HCV-specific CD4(+) T-cell responses were detected in 16 (76.2%) spontaneous resolvers especially towards nonstructural proteins, but with no correlation with IL28B genotype. Chronic individuals had a significantly lower overall T-cell response again irrespective of IL28B genotype. When spontaneous resolvers and chronic individuals were stratified according to their IL28B genotype, significantly stronger T-cell responses were only observed among those with non-CC haplotypes. Although the protective rs12979860 IL28B CC genotype is associated with spontaneous HCV clearance, stronger CD4(+) T-cell responses towards NS3 were only evident among those with non-CC haplotypes.

A multicentre evaluation of the Elecsys® HIV Duo assay.

BACKGROUND: Fourth generation HIV assays, which detect both HIV p24 antigen and HIV antibodies are widely used in HIV screening. The combination of markers enables the fourth generation assays to shorten the window of detection, which is important in real-world testing scenarios. The Elecsys® HIV Duo assay is a fourth generation assay, which provides an overall result based on both the detection of the p24 antigen and HIV antibodies, and lists the sub-results for the antibody and antigen units. OBJECTIVES AND STUDY DESIGN: The performance of the Elecsys® HIV Duo assay was assessed at five international centres and compared with other available fourth generation assays. RESULTS: The specificity of the Elecsys® HIV Duo assay in 13,328 blood donor samples was 99.87% (95% confidence interval [CI] 99.80-99.93) and was 100% (95% CI 99.63-100) in 1000 routine diagnostic samples. Sensitivity was assessed in 139 seroconversion panels; the Elecsys® HIV Duo assay detected a greater number of positive samples/number of bleeds compared with other assays investigated. An individual analysis of those seroconversion panels also shows that the Elecsys® HIV Duo assay compared to other fourth generation assays detected HIV up to 2 days earlier than other assays. The Elecsys® HIV Duo assay also detected 125/130 'early seroconversion' samples assessed, which was greater than the number detected with comparator fourth generation assays. CONCLUSION: These results indicate that the Elecsys® HIV Duo assay is appropriate for use in the diagnosis of HIV and for screening of blood donations and is sensitive for the early detection of HIV.

Receptor binding properties and biologic activity in vitro of biosynthetic human insulin.

The interaction of biosynthetic human insulin with human cultured lymphocytes, human circulating erythrocytes, and isolated rat fat cells was examined. The binding of the biosynthetic insulin to human cells was identical to that of native human or pork insulins, with respect to affinity, kinetics of association and dissociation, negative cooperativity, and downregulation of lymphocyte receptors. The biosynthetic insulin also had equal potency in stimulating the incorporation of [3H]-glucose into the lipids of isolated rat fat cells. These data suggest that the structure of the biosynthetic insulin has been integrally reconstituted.

Determination of total mercury in human hair samples by cold vapor atomic absorption spectrometry.

A procedure for the determination of mercury in human hair by cold-vapor atomic absorption spectrometry with a new reaction vessel was developed and evaluated. To eliminate the so-called matrix effect, calibration was carried out in an isomedium under identical operating conditions. Under these conditions, the recovery percentage of mercury from human hair digest samples using the peak height method, was 102 +/- 2.2%. The method has been used for measuring the mercury level in hair of different groups of people living in the Nantes, France area.

An in vitro comparison of capillary flow dialyzer performances on a single needle system (double headpump).

The ultrafiltration, clearance and priming characteristics of six capillary dialyzers (C-DAK 2.5 D, C-DAK 1.8 D, Tri-Ex 1, CF M 1500, N16F160 and HF 150) were studied in single needle dialysis by way of the double headpump (BL 760, Bellco Ltd). A good control of the ultrafiltration by an appropriate choice of the mean bloodcompartment pressures (Pbo) is present in all dialyzers. After correction of the clearance for ultrafiltration the clearances of urea, creatinine, sodiumiothalamate-l125 and cyanocobalamine-Co58 (vit. B 12) rise with increasing mean pressures in the bloodcompartment of all dialyzers. We assume that a better use of available surface area due to the characteristics of the double headpump such as TMP mainly influenced by Pbo pressures and other such as tidal flow are responsible for the excellent in vitro clearances of these dialyzers with this type of single needle system.

A study of the influence of single needle dialysis on the principal parameters of haemodialyser performance.

The clearance and ultrafiltration characteristics of flat plate and hollow fibre hemodialysers have been established using conventional two needle dialysis and unipuncture with a Bellco BL 760 double head pump system. The results obtained are comparable with no detriment to the performance when in the unipuncture mode. Furthermore, the unipuncture system used for our studies enables control of ultrafiltration to be achieved enabling the use of high flux dialysers without the need for special equipment.

Absence of a beneficial haemodynamic effect of bicarbonate versus acetate haemodialysis.

The present study compares data on blood pressure and clinical tolerance, obtained consecutively in the same patients during acetate and bicarbonate haemodialysis. Twenty-one patients were followed over an equal period of acetate and bicarbonate dialysis, averaging more than 30 months per patient. Absolute and relative blood pressure changes were noted. Contrary to what often has been claimed previously, it is concluded from the present long-term study that, bicarbonate haemodialysis has no specific beneficial effect on blood pressure in stabilised chronic patients. As far as vomiting and nausea are concerned, clinical tolerance is, however, significantly better than for acetate haemodialysis.

A skin suction blister model in hairless rats: application to the study of anti-inflammatory and immunomodulatory drugs.

A suction blister model was developed in the hairless rat, in order to study the effects of various agents on the migration of polymorphonuclear leukocytes (PMN). A standardized abrasion, a suction blister, was formed by applying negative pressure to the skin and then separating the epidermis from the dermis. A migration chamber containing serum as the chemoattractant was placed over the wound. After 6 h of migration, the cells in the chamber were harvested, counted and identified. We evaluated PMN migration after treating the animals with active compounds: niflumic acid, and anti-inflammatory drug, and RU 41740, an immunomodulator. This in vivo model provided reproducible data and could be used to study further the functional properties of PMN. In addition, because this assay can also be used in man, a drug found to be effective in the animal system could then be tested for its activity in man.

Efficient translation of rotavirus mRNA requires simultaneous interaction of NSP3 with the eukaryotic translation initiation factor eIF4G and the mRNA 3' end.

In contrast to the vast majority of cellular proteins, rotavirus proteins are translated from capped but nonpolyadenylated mRNAs. The viral nonstructural protein NSP3 specifically binds the 3'-end consensus sequence of viral mRNAs and interacts with the eukaryotic translation initiation factor eIF4G. Here we show that expression of NSP3 in mammalian cells allows the efficient translation of virus-like mRNA. A synergistic effect between the cap structure and the 3' end of rotavirus mRNA was observed in NSP3-expressing cells. The enhancement of viral mRNA translation by NSP3 was also observed in a rabbit reticulocyte lysate translation system supplemented with recombinant NSP3. The use of NSP3 mutants indicates that its RNA- and eIF4G-binding domains are both required to enhance the translation of viral mRNA. The results reported here show that NSP3 forms a link between viral mRNA and the cellular translation machinery and hence is a functional analogue of cellular poly(A)-binding protein.

Human insulin prepared by recombinant DNA techniques and native human insulin interact identically with insulin receptors.

Human insulin synthesized from A and B chains separately produced in Escherichia coli from cloned synthetic genes (prepared by the Eli Lilly Research Laboratories, Indianapolis, IN) was characterized by examining its interaction with human cultured lymphocytes, human circulating erythrocytes in vitro, and isolated rat fat cells. The binding behavior of the biosynthetic insulin with human cells was indistinguishable from that of native human or porcine insulins, with respect to affinity, association and dissociation kinetics, negative cooperativity, and the down-regulation of lymphocyte receptors. Similarly, the biosynthetic insulin was as potent as the native insulins in stimulating lipogenesis in isolated rat fat cells. We also examined the receptor binding characteristics of 125I-labeled human and porcine insulins monoiodinated solely at Tyr-A14, which were obtained by means of high-performance liquid chromatography of the iodination reaction mixture (this material was prepared by B. Frank, Eli Lilly Research Laboratories). In all aspects studied, the pure [TyrA14-125I]iodoinsulins were superior as tracers to the monoiodoinsulin purified by the more conventional method of gel filtration.

Identification of the RNA-binding, dimerization, and eIF4GI-binding domains of rotavirus nonstructural protein NSP3.

The rotavirus nonstructural protein NSP3 is a sequence-specific RNA binding protein that binds the nonpolyadenylated 3' end of the rotavirus mRNAs. NSP3 also interacts with the translation initiation factor eIF4GI and competes with the poly(A) binding protein. Deletion mutations and point mutations of NSP3 from group A rotavirus (NSP3A), expressed in Escherichia coli, indicate that the RNA binding domain lies between amino acids 4 and 149. Similar results were obtained with NSP3 from group C rotaviruses. Data also indicate that a dimer of NSP3A binds one molecule of RNA and that dimerization is necessary for strong RNA binding. The dimerization domain of NSP3 was mapped between amino acids 150 and 206 by using the yeast two-hybrid system. The eukaryotic initiation factor 4 GI subunit (eIF-4GI) binding domain of NSP3A has been mapped in the last 107 amino acids of its C terminus by using a pulldown assay and the yeast two-hybrid system. NSP3 is composed of two functional domains separated by a dimerization domain.

Rotavirus RNA-binding protein NSP3 interacts with eIF4GI and evicts the poly(A) binding protein from eIF4F.

Most eukaryotic mRNAs contain a 5'cap structure and a 3'poly(A) sequence that synergistically increase the efficiency of translation. Rotavirus mRNAs are capped, but lack poly(A) sequences. During rotavirus infection, the viral protein NSP3A is bound to the viral mRNAs 3' end. We looked for cellular proteins that could interact with NSP3A, using the two-hybrid system in yeast. Screening a CV1 cell cDNA library allowed us to isolate a partial cDNA of the human eukaryotic initiation factor 4GI (eIF4GI). The interaction of NSP3A with eIF4GI was confirmed in rotavirus infected cells by co-immunoprecipitation and in vitro with NSP3A produced in Escherichia coli. In addition, we show that the amount of poly(A) binding protein (PABP) present in eIF4F complexes decreases during rotavirus infection, even though eIF4A and eIF4E remain unaffected. PABP is removed from the eIF4F complex after incubation in vitro with the C-terminal part of NSP3A, but not with its N-terminal part produced in E.coli. These results show that a physical link between the 5' and the 3' ends of mRNA is necessary for the efficient translation of viral mRNAs and strongly support the closed loop model for the initiation of translation. These results also suggest that NSP3A, by taking the place of PABP on eIF4GI, is responsible for the shut-off of cellular protein synthesis.

Quality of perinatal death registration. A study in Hainaut, Belgium.

The quality of national perinatal mortality statistics was evaluated from a survey in nine maternity hospitals in Hainaut, Belgium (total births: 7862). The overall completeness of perinatal death registration was 86%. Under-registration was especially frequent in low birth weight babies. In 69% of cases, the birth weight value reported on death certificates was in exact agreement with the value in hospital records. Using detailed categories of causes, there was, in 37% of cases, agreement between the underlying cause on death certificates and the main cause identified in hospital records. Using gross categories of causes, the level of agreement was 56%. Disagreement was mostly due to the lack of specificity of the underlying cause on death certificates. The authors suggest ways to improve the quality of registration.

Endocytosis of MHC molecules by B cell-B lymphoma and B cell-T lymphoma hybrids.

Different cells and different cell surface determinants of the same cells take up liposomes, bound to them via monoclonal antibodies, with variable efficiency. We have previously reported that T and B lymphocytes differ in the extent to which they take up liposomes bound to MHC class I molecules; T cells endocytose class I molecules rapidly, but B cells endocytose class I molecules much less efficiently, although their endocytosis of class II molecules is rapid. An approach toward understanding the molecular basis for this difference was made by evaluating the internalization patterns of somatic cell hybrids of B and T cells. Hybrid cells were constructed between LPS-stimulated purified B cell blasts from C57BL/6 mice (H-2b) and the HAT-sensitive AKR T lymphoma BW 5147 (H-2k). Hybrids between the BALB/c B lymphoma M12.4.1 (H-2d) and B cell LPS blasts from B10.BR (H-2k) mice were also evaluated. In all cases, for hybrid tumor cells, liposomes that were bound to class I molecules encoded by genes from the B cell donor were endocytosed as efficiently as liposomes bound to the class I molecules of the recipient lymphoid cell. T cell tumors efficiently internalized their own class I molecules and those donated by B cells; B cell tumors internalized liposomes that were bound to their own and the donor B cell class I molecules poorly. Thus, our results suggest that the internalization of an MHC molecule is not an intrinsic property of the molecule, but rather of the cell in which it is found.

Cessation of intensive treatment with recombinant human erythropoietin is followed by secondary anemia.

Little information is available on the evolution of erythropoiesis after interruption of recombinant human erythropoietin (rHuEpo) therapy. Iron-overloaded rats received 20 daily injections of rHuEpo. During treatment, reticulocytes, soluble transferrin receptor (sTfR), and hematocrit increased progressively. This was accompanied by a substantial expansion of spleen erythropoiesis but a decrease in the bone marrow. Five weeks after treatment, rats developed a significant degree of a regenerative anemia. Erythropoietic activity, as assessed by reticulocytes, sTfR, erythroid cellularity, iron incorporation into heme, and the number of erythroid colonies, was severely depressed 3 weeks after cessation of rHuEpo. This was followed by regeneration of erythroblasts and reticulocytes at weeks 6 to 7 post-Epo, but erythroid progenitors recovered only partially by that time. The anemia was definitely corrected 2 months after cessation of rHuEpo treatment. Serum Epo levels remained elevated for several weeks, but the sensitivity of marrow erythroid precursors to Epo was preserved. No rat antibodies to rHuEpo were detected, and serum from post-Epo animals did not exert any inhibitory activity on erythropoiesis. In conclusion, after cessation of intensive rHuEpo therapy, there was a strong inhibition of erythropoietic activity with secondary anemia followed by late recovery. This was not due to antibodies or other soluble inhibitory factors, a defect in endogenous Epo production, or a loss of sensitivity to Epo. This may rather represent intrinsic erythroid marrow exhaustion, mostly at the level of erythroid progenitors but also at later stages of erythropoiesis.

Cap-Poly(A) synergy in mammalian cell-free extracts. Investigation of the requirements for poly(A)-mediated stimulation of translation initiation.

The 5' cap and 3' poly(A) tail of eukaryotic mRNAs cooperate to stimulate synergistically translation initiation in vivo, a phenomenon observed to date in vitro only in translation systems containing endogenous competitor mRNAs. Here we describe nuclease-treated rabbit reticulocyte lysates and HeLa cell cytoplasmic extracts that reproduce cap-poly(A) synergy in the absence of such competitor RNAs. Extracts were rendered poly(A)-dependent by ultracentrifugation to partially deplete them of ribosomes and associated initiation factors. Under optimal conditions, values for synergy in reticulocyte lysates approached 10-fold. By using this system, we investigated the molecular mechanism of poly(A) stimulation of translation. Maximal cap-poly(A) cooperativity required the integrity of the eukaryotic initiation factor 4G-poly(A)-binding protein (eIF4G-PABP) interaction, suggesting that synergy results from mRNA circularization. In addition, polyadenylation stimulated uncapped cellular mRNA translation and that driven by the encephalomyocarditis virus internal ribosome entry segment (IRES). These effects of poly(A) were also sensitive to disruption of the eIF4G-PABP interaction, suggesting that 5'-3' end cross-talk is functionally conserved between classical mRNAs and an IRES-containing mRNA. Finally, we demonstrate that a rotaviral non-structural protein that evicts PABP from eIF4G is capable of provoking the shut-off of host cell translation seen during rotavirus infection.

Longitudinal evaluation of the structure of replicating and circulating hepatitis C virus quasispecies in nonprogressive chronic hepatitis C patients.

In previous cross-sectional studies, we demonstrated that, in most patients with chronic hepatitis C, the composition and complexity of the circulating hepatitis C virus (HCV) population do not coincide with those of the virus replicating in the liver. In the subgroup of patients with similar complexities in both compartments, the ratio of quasispecies complexity in the liver to that in serum (liver/serum complexity ratio) of paired samples correlated with disease stage. In the present study we investigated the dynamic behavior of viral population parameters in consecutive paired liver and serum samples, obtained 3 to 6 years apart, from four chronic hepatitis C patients with persistently normal transaminases and stable liver histology. We sequenced 359 clones of a genomic fragment encompassing the E2(p7)-NS2 junction, in two consecutive liver-serum sample pairs from the four patients and in four intermediate serum samples from one of the patients. The results show that the liver/serum complexity ratio is not stable but rather fluctuates widely over time. Hence, the liver/serum complexity ratio does not identify a particular group of patients but a particular state of the infecting quasispecies. Phylogenetic analysis and signature mutation patterns showed that virtually all circulating sequences originated from sequences present in the liver specimens. The overall behavior of the circulating viral quasispecies appears to originate from changes in the relative replication kinetics of the large mutant spectrum present in the infected liver.