The expanding toolbox for hepatitis C virus research.

Hepatitis C virus is a major global health concern with 170 million people chronically infected. Despite the availability of potent antiviral agents targeting multiple HCV proteins and cure rates above 90%, global treatment availability, the likelihood of emerging drug-resistant viral variants and the unavailability of a protective vaccine underline the many unresolved questions remaining to be answered. Model systems allowing the dissection of individual HCV life cycle steps have previously been developed and span noninfectious and infectious means of assessing HCV entry and replication, multiple cellular systems enabling host/pathogen interaction studies as well as in vivo model systems for basic as well as translational HCV research. This review provides an overview of available systems and a comparative summary of assays and models.

Bioluminescence assay for the highly sensitive detection of botulinum neurotoxin A activity.

This article describes a novel bioluminescence assay for detecting the proteolytic activity of Botulinum NeuroToxins (BoNT) in complex matrices. The assay is capable of detecting traces of BoNT in blood samples as well as in food drinks. The assay was responsive to BoNT/A subtypes 1 to 5, and serotype E3 in buffered solutions. It was responsive to filtered Clostridium botulinum supernatants and BoNT/A1 in complex with neurotoxin associated proteins in bouillon and milk (3.8% fat) down to 400 fM after 4 h RT incubation and in bouillon at concentrations down to 120 fM after 21 h RT incubation. In combination with an immunocapture/enrichment step it could detect BoNT/A1 in citrated plasma at concentrations down to 30 fM (1.2 mouse LD50 per mL). The simplicity of the assay, combined with a demonstrated ability to lyophilize the reagents, demonstrates its usefulness for detection of BoNT in non-specialised analytical laboratories.

Necrotizing endometritis and isolation of an alpha-toxin producing strain of Clostridium septicum in a wild sooty mangabey from Côte d'Ivoire.

Few lethal pathogens in wild-living primates have been described, and little is known about infectious diseases of the reproductive tract and their possible impact on health and reproduction. This report describes the pathology and isolation of an alpha-toxin producing strain of Clostridium septicum in a case of necrotizing endometritis in a wild sooty mangabey found dead in a tropical rainforest of West Africa.

Binding properties of immunoglobulin combining sites specific for terminal or nonterminal antigenic determinants in dextran.

Binding constants of the dextran-reactive BALB/c mouse IgA myeloma proteins W3129 and QUPC 52 have been determined for each member of the isomaltose series of oligosaccharides and for methyl alphaDglucoside. Protein W3129 has maximum complementarity for isomaltopentaose (IM5) deltaf degrees = 7,180 cal/mol) with 55-60% of the total binding energy directed against methylalphaDglucoside. Protein QUPC 52 gives maximum binding with isomaltohexaose (IM6) (deltaF degrees = -5,340 cal/mol) and has about 70% of its total binding energy for isomaltotriose (IM3), but at most only 5% for isomaltose (IM2) or methyl alphaDglucoside. Protein W3129 precipitates with branched dextrans high in alpha (1 yields 6) linkages and reacts with but does not precipitate a synthetic alpha (1 yields 6)-linked linear dextran. Protein QUPC 52 precipitates both branched and linear dextrans. Thus, the immunodominant group for protein W3129 is mimicked by methyl alphaDglucoside and this protein reacts exclusively at the terminal nonreducing ends of alpha (1 yields 6)-linked dextran chains. Protein QUPC 52 has an immunodominant group which is expressed by IM3 but not smaller oligosaccharides and this protein can react at nonterminal locations along alpha (1 yields 6)-linked dextran chains. Precipitation of linear dextran seems to be a valid although not quantitative assay for antidextrans with nonterminal specificity. Quantitative precipitin reactions with branched and linear dextrans suggest that alpha (1 yields 6)-specific human antidextrans are mixtures of molecules having terminal and nonterminal specificities and that the fraction of each type can vary among individuals. Rabbit antisera against IM3 or IM6 coupled to bovine serum albumin also appear to contain antibodies with nonterminal specificity for dextran chains although a large fraction has terminal specificity. Low molecular weight clinical dextran N-150N (congruent to 60,000) reacted more like linear dextran than like its parent native-branched dextran B512. This is thought to result from an abundance of nonterminal determinants in clinical dextran N-150N but a very small number of functional terminal determinants per molecule. An appreciation of terminal and nonterminal specificities and of the different immunodominant structures in isomaltosyl chains has proven to be of a great value in understanding the immunochemical reactions of dextrans. Moreover, certain previous findings with fructosan-reactive mouse myeloma proteins and human antilevans (55, 84) also suggest terminal and nonterminal specificities for levan chains.

Identification of leukemia-associated inhibitory activity as acidic isoferritins. A regulatory role for acidic isoferritins in the production of granulocytes and macrophages.

Acidic isoferritins have been identified as leukemia-associated inhibitory activity (LIA), which suppresses colony and cluster formation of colony-forming unit-granulocyte macrophages from normal donors but not from patients with leukemia. LIA was detected in all ferritin preparations tested, including ferritin isolated from normal heart, spleen, liver, and placental tissues, and from the spleens of patients with chronic myelogenous leukemia and Hodgkin's disease. Purified preparations of LIA were composed almost entirely of acidic isoferritins, as determined by immunoassay, radioimmunoassay, and isoelectric focusing. The inhibitory activity in the LIA and ferritin samples was inactivated by a battery of antisera specific for ferritin, including those prepared against acidic isoferritins from normal heart and spleen tissues from patients with Hodgkin's disease, and those previously absorbed with basic isoferritins. Antisera absorbed with acidic isoferritins did not inactivate the inhibitory activity. Separation of LIA and chronic myelogenous leukemia and normal spleen ferritin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing confirmed that the regions of peak inhibitory activity corresponded in each to an apparent molecular weight of approximately 550,000 and to a pI value of 4.7. Similar physicochemical characteristics included inactivation by methods that dissociate ferritin molecules into subunits and by treatment with trypsin, chymotrypsin, pronase, and periodate. The purified preparations were extremely stable to heat treatment. The glycoprotein nature of the inhibitory activity was substantiated because it bound to concanavalin A-Sepharose and was eluted off by alpha-methyl mannose. Inhibitory activity of the activity of the acidic isoferritins was detected at concentrations as low as 10(-17)-10(-19) M and iron saturation did not appear to be necessary for its action. These results implicate acidic isoferritins in the regulation of normal myelopoiesis and suggest a role for them in the progression of leukemia.

Studies on human antibodies. VI. Selective variations in subgroup composition and genetic markers.

The composition of various isolated antibodies was determined by quantitative analyses for heavy chain subgroups and light chain types. Certain antibodies such as anti-tetanus toxoid and anti-A isoagglutinins were predominantly of the major gammaG1-type. However, a high preponderance of molecules of the minor gammaG2-subgroup was found for antibodies to dextran, levan, and teichoic acid. These findings explain some unusual features previously noted for anti-dextrans such as weak PCA reactions and lack of Gm antigens. Studies of several isolated antibodies from single heterozygous individuals showed a selective absence of genetic markers in certain antibodies and their presence in others. The "allelic exclusion" principle was clearly evident in the isolated antibodies of two different individuals. Large differences in the ratio of kappa to lambda light chains were observed for the same type of antibody from different individuals. Subfractionation of dextran antibodies by affinity for specific glycosidic linkage or combining site size produced marked changes in the ratios. The isomaltohexaose eluates of the dextran antibodies from two subjects were primarily kappa and the isomaltotriose eluates were predominantly lambda. The one anti-levan antibody studied was uniquely homogeneous, consisting exclusively of gammaG2-heavy chains and kappa light chains. By these criteria as well as others, it closely resembled myeloma proteins.

Studies on human antibodies. V. Amino acid composition of antidextrans of the same and of differing specificities from several individuals.

Human dextran-antidextran-specific precipitates from individuals immunized with dextrans containing either one or two antigenic determinants, were analyzed for their content of various amino acids. Large differences in certain amino acids were found among alpha-(1 --> 6)-specific antidextrans. Wide variations were also observed in antidextran fractions from a given individual specific for alpha-(1 --> 6)- and alpha-(1 --> 2)-linked glucose residues.

Ferritin synthesis by human T lymphocytes.

Mononuclear cells from peripheral blood of normal humans, unselected spleen cells from patients with Hodgkin's disease, and selected T and non-T lymphoid cells from normal peripheral blood and from the spleens of Hodgkin's disease patients were examined for de novo synthesis and secretion of ferritin. After precipitation of labeled lysates and supernatants from unseparated and selected T cells with antiserum to human liver ferritin, two bands were visible on sodium dodecyl sulfate-polyacrylimide gel analysis. The two bands were detected in molecular weight regions 19,000 and 21,000, which are thought to represent the L and H subunits of the ferritin molecule, respectively. The slower band (subunit H) was more radioactive than the faster band (subunit L). The H subunit is found in greater amounts in the serum of some tumor patients, but its cellular origin has not been established. The present findings indicate that cells of the immune system contribute to the synthesis and secretion of a ferritin molecule with a high proportion of H subunits.

Monitoring of laying capacity, immunoglobulin Y concentration, and antibody titer development in chickens immunized with ricin and botulinum toxins over a two-year period.

One of the key benefits in using chickens for immunization is the high yield of antibodies obtainable. It is known that egg production decreases over time, while animal maintenance costs remain stable. It would, however, be desirable to keep hens as long as possible to obtain maximal amounts of antibodies. To identify a suitable length of time that animals can be kept and to optimize the cost:yield ratio, we monitored the number of eggs laid, the total amount of chicken IgY, and the specific antibody titer from individually prepared eggs over a 2-yr period. The plant toxin ricin and the Clostridium botulinum neurotoxins type A and B were used to immunize 4 chickens. The number of eggs laid in 2 yr was approximately 600 per hen (about 80% of the maximum egg number), yielding about 20 to 40 g of total IgY per hen. A stable antibody titer of 1:100,000 to 1:1,000,000, as measured by ELISA, was obtained following up to 11 injections of 10 to 20 microg of immobilized native toxin. Laying capacities were found to decrease, on average, from 7 eggs/wk at the point of first immunization to 2 eggs/wk after more than 2 yr. In parallel, the yield of total and specific IgY increased over time, so that the antibody recovery remained high, even after prolonged immunization times. Using purified IgY preparations, classical immunological assays such as ELISA and Western blotting were performed. Furthermore, the IgY showed neutralizing capacity when used to block the functional activity of the toxins both in vitro and in vivo. Analysis of the total IgY content over time demonstrated a complex biological oscillation (and the antigen-specific titer), with a shorter time period of around 7 d (circaseptan rhythm). In summary, we successfully immunized chickens with ricin and botulinum neurotoxins and monitored laying capacity, IgY concentration, and specific antibody titer over an extended period of 2 yr.

3D microfluidic liver cultures as a physiological preclinical tool for hepatitis B virus infection.

With more than 240 million people infected, hepatitis B virus (HBV) is a major health concern. The inability to mimic the complexity of the liver using cell lines and regular primary human hepatocyte (PHH) cultures pose significant limitations for studying host/pathogen interactions. Here, we describe a 3D microfluidic PHH system permissive to HBV infection, which can be maintained for at least 40 days. This system enables the recapitulation of all steps of the HBV life cycle, including the replication of patient-derived HBV and the maintenance of HBV cccDNA. We show that innate immune and cytokine responses following infection with HBV mimic those observed in HBV-infected patients, thus allowing the dissection of pathways important for immune evasion and validation of biomarkers. Additionally, we demonstrate that the co-culture of PHH with other non-parenchymal cells enables the identification of the cellular origin of immune effectors, thus providing a valuable preclinical platform for HBV research.

T cell immunity to Zika virus targets immunodominant epitopes that show cross-reactivity with other Flaviviruses.

Zika virus (ZIKV) Infection has several outcomes from asymptomatic exposure to rash, conjunctivitis, Guillain-Barré syndrome or congenital Zika syndrome. Analysis of ZIKV immunity is confounded by the fact that several related Flaviviruses infect humans, including Dengue virus 1-4, West Nile virus and Yellow Fever virus. HLA class II restricted T cell cross-reactivity between ZIKV and other Flaviviruses infection(s) or vaccination may contribute to protection or to enhanced immunopathology. We mapped immunodominant, HLA class II restricted, CD4 epitopes from ZIKV Envelope (Env), and Non-structural (NS) NS1, NS3 and NS5 antigens in HLA class II transgenic mice. In several cases, ZIKV primed CD4 cells responded to homologous sequences from other viruses, including DENV1-4, WNV or YFV. However, cross-reactive responses could confer immune deviation - the response to the Env DENV4 p1 epitope in HLA-DR1 resulted in IL-17A immunity, often associated with exacerbated immunopathogenesis. This conservation of recognition across Flaviviruses, may encompass protective and/or pathogenic components and poses challenges to characterization of ZIKV protective immunity.

Evidence for import of a lysyl-tRNA into marsupial mitochondria.

The mitochondrial tRNA gene for lysine was analyzed in 11 different marsupial mammals. Whereas its location is conserved when compared with other vertebrate mitochondrial genomes, its primary sequence and inferred secondary structure are highly unusual and variable. For example, eight species lack the expected anticodon. Because the corresponding transcripts are not altered by any RNA-editing mechanism, the lysyl-tRNA gene seems to represent a mitochondrial pseudogene. Purification of marsupial mitochondria and in vitro aminoacylation of isolated tRNAs with lysine, followed by analysis of aminoacylated tRNAs, show that a nuclear-encoded tRNA(Lys) is associated with marsupial mitochondria. We conclude that a functional tRNA(Lys) encoded in the nuclear genome is imported into mitochondria in marsupials. Thus, tRNA import is not restricted to plant, yeast, and protozoan mitochondria but also occurs also in mammals.

Biochemistry of S-layers.

During evolution prokaryotes have developed different envelope structures exterior to the cell wall proper. Among these surface components are regularly arranged S-layers and capsules. The structural characterization and the detailed chemical analysis of these surface molecules is a prerequisite to understand their biosynthesis and functional role(s) at the molecular level. Of particular interest are the glycosylated S-layer proteins which belong to the first prokaryotic glycoproteins ever described. Their characterization was performed on strains belonging to the thermophilic Bacillaceae and included structural studies and experiments to learn about the pathways for the glycan biosynthesis of S-layer glycoproteins. As an example for non-glycosylated S-layer proteins those of Lactobacillus helveticus strains are described in detail. Recently, a novel type of bacterial glycoconjugate was observed in the cell envelope of the extremely halophilic archaeon Natronococcus occultus which consists of a glycosylated polyglutamyl polymer. Beside the conventional biochemical techniques for the analysis new sophisticated instrumental methods such as X-ray photoelectron spectroscopy and matrix-assisted laser desorption ionization or electrospray ionization mass spectrometry have been introduced for the analysis of the protein and glycan portions of these cell surface macromolecules.

Studies on structure-function relationships of human choriogonadotropins with C-terminally shortened alpha-subunits. I. Receptor binding and immunologic properties.

Recombination products composed of the native beta-subunit and an alpha-subunit with an enzymatically shortened C-terminal region showed a diminished (less than 5 amino acids removed) or - in the case of des-(88-92)-alpha/native beta - a completely abolished ability to bind to testicular LH/hCG receptors of the rat. An antigenic determinant which is present in native hCG but not in the isolated subunits was not or incompletely expressed in the modified hormone species. Antigenic determinants which are characteristic for the isolated alpha-subunit, however, were not affected by removal of the C-terminal residues 88-92. The immunologic experiments indicate that hCG containing an alpha-subunit with a shortened C-terminal region differs from native hCG in its conformation. These conformational changes are probably responsible for the loss in receptor-binding ability.

Relative subunit composition of the ferritin synthesized by selected human lymphomyeloid cell populations.

Biosynthesis of ferritin subunits by cell sets isolated from normal human peripheral blood, spleens of Hodgkin's disease patients, and tumor cell lines were investigated. Normal mature hematopoietic cells made a ferritin with more H (21K) than L (19K) subunits. The reverse was found for a promyelocytic tumor cell line and tumor cell lines derived from other tissues. Two dimensional electrophoresis indicated H has a lower pI than L. Therefore relative proportions of the two subunits contribute to the electrophoretically distinct forms of the isoferritins. In response to increasing concentrations of iron in vitro, a selected monocyte population synthesized more H than L; L biosynthesis however increased more than H. Some possible regulatory implications of these observations are discussed.

RNA editing in metazoan mitochondria: staying fit without sex.

RNA editing subsumes a number of functionally different mechanisms which have in common that they change the nucleotide sequence of RNA transcripts such that they become different from what would conventionally be predicted from their gene sequences. RNA editing has now been found in the organelles of numerous organisms as well as in a few nuclear transcripts. Most recently, it was shown to affect tRNAs in the mitochondria of several animals. The occurrence and evolutionary persistence of RNA editing is perplexing since backmutations in the genes might be assumed rapidly to eliminate the need for 'correction' of the gene sequences at the post-transcriptional level. Here, we review the recent RNA editing systems discovered in animal mitochondria and propose that they have arisen as a mechanism counteracting the accumulation of mutations that occurs in asexual genetic system.

Transcription of the Trypanosoma brucei spliced leader RNA gene is dependent only on the presence of upstream regulatory elements.

The spliced leader (SL) RNA plays a key role in mRNA maturation in trypanosomatid protozoa by providing the SL sequence, which is joined to the 5' end of every mRNA. As a first step towards a better understanding of the biogenesis and function of the SL RNA, we expressed a tagged SL RNA gene in a cell-free system of procyclic Trypanosoma brucei cells. Transcription initiates at + 1 can be detected as early as 1 min after addition of extract. Transcription of the SL RNA gene in vitro, as well as in permeable cells, is mediated by an alpha-amanitin/tagetitoxin resistant complex, suggesting a promoter that is intermediate between a classical RNA polymerase II and RNA polymerase III promoter. An analysis of the promoter architecture of the SL RNA gene revealed that regulatory elements are located upstream of the coding region and that the SL sequence, in contrast to the nematode SL sequence, is not required for T. brucei SL RNA gene transcription.

Significance of antral pH for gastrin release by insulin hypoglycemia in duodenal ulcer patients.

The significance of antral pH for the basal serum level of immunoreactive gastrin and for the release of gastrin during insulin hypoglycemia has been studied in duodenal ulcer (DU) patients. To permit paired comparisons, 14 DU patients underwent two or three tests with insulin. Venous blood samples were collected at fixed intervals for determination of gastrin (radioimmunoassay). In the first insulin test, the gastric juice was aspirated; in the second test, the stomach was perfused with citrate-phosphate buffer, pH 7.0; and in the third test the stomach was perfused with 0.1M HCl, pH 1.0. The rate of buffer or acid perfusion was adjusted, and the pH of the perfusate was kept above 5.0 and below 1.3, respectively. Gastric perfusion with buffer or acid for 1 hour did not affect the basal serum gastrin level, nor did perfusion with buffer for 3 hours. Insulin hypoglycemia stimulated acid secretion and produced a significant integrated serum gastrin response during gastric aspiration, but the gastrin response was four times greater during buffer perfusion. Acid perfusion abolished the gastrin response. From our previous and present findings, it is concluded that the gastrin in serum during basal conditions is of extra-antral origin and is independent of antral pH. Insulin hypoglycemia releases antral gastrin by a pH-sensitive mechanism in DU patients; the release is suppressed at pH 1.3 or less and also is markedly inhibited when the gastric juice is aspirated.

Determination of cefalexin pharmacokinetics and dosage adjustments in relation to renal function.

Following oral administration of a single 500-mg dose of cefalexin, serum and urine levels of the antibiotic were determined comparatively in ten normal subjects, ten patients with renal impairment, and ten patients with chronic nephritis on maintenance hemodialysis. In normal subjects, mean serum peak levels (12.0 +/- 0.8 mcg/ml) were observed 2 hours after drug administration. Absorption half-time (Ta1/2) averaged 0.82 hour and mean serum half-life (T 1/2) was 1.03 hours. Urinary recovery of cefalexin over a 6-hour period amounted to 64 per cent of the ingested dose. The renal clearance of the drug was 214 ml/min. In patients with renal impairment and in patients on maintenance hemodialysis, total elimination rate constant (Ke) was markedly lower (CrCl=0; Ke=Km=0.0766), whereas serum half-life (T 1/2) was significantly increased, reaching theoretically 8.47 hours in patinets with creatinine clearance of 0 ml/min. A correlation was established between Ke values and the creatinine clearances of the patients under study (Ke=0.0766 + 0.0060 CrCl). Initial loading doses, maintenance doses, and intervals adjusted to creatinine clearances were calculated from these data; accurate dosage schedules well adjusted to the renal status of each individual patient were derived from the calculated values.

Effect of middle-term gliclazide treatment on insulin secretion in non-insulin dependent diabetics.

Insulin secretion was studied in 12 non-insulin dependent diabetics during middle-term administration of the sulphonylurea gliclazide. Blood sugar, C-peptide and glucagon were also estimated during the intravenous glucose tolerance and arginine tests performed before and after therapy. After 3 months of gliclazide therapy (240 mg/day) in addition to a low carbohydrate diet, the intravenous glucose tolerance test showed a significant reduction in blood sugar levels and in the partial and total areas under the blood sugar curve, as well as an improvement in early insulin secretion, characterized by a significant increase in plasma C-peptide at 4, 10 and 20 minutes. Plasma glucagon levels were not affected by the sulphonylurea therapy. In the arginine test, blood sugar levels were lower at the end of the treatment period; plasma insulin, C-peptide and glucagon did not change significantly. In this study, plasma C-peptide has proved to be a better indicator of stimulated insulin secretion than plasma insulin levels. The scarcity of hypoglycaemic episodes during therapy with gliclazide may be related to the selective stimulation of early insulin secretion by this drug.