Hepatitis C virus (HCV) genotype, tissue HCV antigens, hepatocellular expression of HLA-A,B,C, and intercellular adhesion-1 molecules. Clues to pathogenesis of hepatocellular damage and response to interferon treatment in patients with chronic hepatitis C.

To obtain information on the mechanisms of hepatocellular damage and the determinants of response to interferon, hepatitis C virus (HCV) genotype, tissue HCV antigens, hepatocellular expression of HLA-A,B,C and intercellular adhesion-1 molecules, and the number of lobular T lymphocytes were studied in 38 anti-HCV-positive patients. 14 patients did not show a primary response to interferon treatment. HCV genotype 1b was detected in 11 of them. They displayed higher scores of HCV-positive hepatocytes, HLA-A,B,C, and ICAM-1 molecules expression than with the responders. HCV-infected hepatocytes maintained the capacity to express HLA-A,B,C and ICAM-1 molecules. CD8-positive T cells in contact with infected hepatocytes and Councilman-like bodies were observed. A significant correlation was found between the number of lobular CD8-positive T cells and alanine amino transferase levels. No differences were observed in clinical, biochemical, and histological features between patients with high and low number of hepatocytes containing HCV antigens. These data suggest a prominent role of T cell-mediated cytotoxicity in the genesis of hepatocellular damage. The high expression of interferon-inducible antigens like HLA-A,B,C molecules suggests the presence of strong activation of the interferon system possibly related to high HCV replication in nonresponder patients infected with genotype 1b.

Autologous marrow recovery following allogeneic marrow transplantation in a patient with severe aplastic anemia.

A 45-year-old woman with severe idiopathic marrow failure was prepared for marrow transplantation by administration of cyclophosphamide (cy) 50 mg/kg on each of four successive days. She then received an intravenous infusion of 20 X 10(9) nucleated marrow cells from an HL-A matched and mixed lymphocyte culture (MLC) non-reactive sister. There was evidence for minimal marrow recovery in 1-2 months and a second marrow infusion was carried out 69 days after the first without additional immunosuppression. There was a continued slow recovery of peripheral blood counts with complete reconstitution of erythropoiesis, return of the white blood cell count to between 3 and 4000/mm3, with 50% granulocytes, and platelets to 60--70,000/mm3, 11 months after the initial grafting attempt. Red cell antigens and gamma globulin allotypes were of recipient type. The MLC and the indirect cell mediated lympholysis (CML) test became positive possibly indicating cellular sensitization to non HL-A antigens. This report of a patient with severe marrow failure documents autologous recovery of marrow function after receiving a large dose of cy and allogeneic marrow. The implications of this are discussed.

Hepatocellular expression of HLA-A, B, C molecules predicts primary response to interferon in patients with chronic hepatitis C.

Primary response rate to alpha-interferon (IFN) is about 50% in patients with chronic hepatitis C. Criteria for predicting a positive primary response are lacking. HLA-A,B,C molecule expression is known to be stimulated by viral infections. In 36 consecutive interferon-treated anti-HCV positive patients with an available frozen liver biopsy sample, the predictive value of liver HLA-A,B,C expression, and of histologic, clinical, and biochemical parameters was evaluated. Response to treatment was defined by normalization of transaminases, and disappearance of serum HCV-RNA within 3 months. According to these criteria, 17 patients were classified as nonresponders and 19 were classified as responders. The pattern of HLA-A,B,C hepatocellular positivity varied from normal (negative or occasional faint staining of hepatocellular membranes) to diffuse, strong "honeycomb" positivity. The highest scores of positivity were found in nonresponder patients. The discriminant capacity of HLA-A,B,C scores of positivity was compared with clinical, biochemical and histologic parameters by discriminant analysis. HLA-A,B,C expression was found to be the main discriminant parameter, in addition to alkaline phosphate (ALP) and gamma-glutamyl-transpeptidase (GGT) which added little additional information. The higher hepatocellular expression of class I MHC molecules in nonresponder cases may reflect a different viral effect on hepatocytes, which is induced by different HCV genotypes or levels of viremia. From a clinical point of view, the pretreatment HLA-A,B,C pattern of positivity represents a powerful tool in the selection of patients for interferon treatment.

Combined mass spectrometric methods for the characterization of human hemoglobin variants localized within alpha T9 peptide: identification of Hb Villeurbanne alpha 89 (FG1) His-->Tyr.

Mutation-induced amino acid exchanges occurring on the large T9 peptide of the alpha-chain of human hemoglobin (residues 62-90) are difficult to identify. Despite their high m/z value (around m/z 3000), collision-induced dissociation spectra of liquid secondary ion mass spectrometrically generated protonated alpha T9 peptides were performed successfully. In parallel electrospray mass spectrometry (MS) was used both to measure the molecular mass of the intact proteins and to determine the number of protonatable sites in the alpha T9 peptides. Peptide ladder sequencing using carboxypeptidase digestions and analysis of the truncated peptides by matrix-assisted laser desorption ionization time-of-flight MS confirmed the interpretation. This set of methods allowed the characterization of three hemoglobin variants, with amino acid exchanges located in the alpha T9 part of the sequence. Two of them, Hb Aztec [alpha 76(EF5) Met-->Thr] and Hb M-Iwate [alpha 87(F8) His-->Tyr] were already known. The third [alpha 89(FG1) His-->Tyr] was novel and named Hb Villeurbanne.

Handedness effects in the detection of dichotically-presented words and emotions.

Left-handed and right-handed subjects were given two dichotic listening tasks using identical material. In one task, they were asked to determine whether or not a specific target word was present (verbal task). In the other task, subjects were asked to indicate whether or not one of the dichotically-competing words was spoken in a particular affective tone (emotion task). Overall, a right-ear advantage (REA) was found with the verbal task and a left-ear advantage (LEA) with the emotion task. Left-handers showed a slightly smaller REA than right-handers on the verbal task, but a slightly larger LEA on the emotion task. This finding suggests that left- and right-hemispheric functions are not related in a complementary fashion and that handedness effects for nonverbal tasks are different from those seen with verbal tasks. The emotional LEA was much larger for angry stimuli than for happy, sad, or neutral stimuli. Rather than providing evidence for a hemisphere by affective valence interaction, such results suggest a stimulus-specific effect.

Cleavage by protease from Staphylococcus aureus V8: an improvement in the sequence analysis of human hemoglobin variants.

Protease from Staphylococcus aureus V8 cleaves either at glutamic residues or at both aspartic and glutamic residues, depending on the experimental conditions. In structural analyses of human hemoglobin variants, the specificity of this enzyme is of considerable interest to localize substitutions occurring in medium or large size peptides as it cleaves in smaller fragments which may be unambiguously characterized. It may also recognize the replacement of an acidic residue by the corresponding amide, or vice versa, avoiding protein sequence analysis. The various aspects of the use of protease V8 are illustrated by the study of four alpha chain hemoglobin variants concerning peptides alpha T-9 and alpha T-12b.

Teachers' opinions of the whole language approach to reading instruction.

This article reports the findings of a study of the opinions of first- and first/second-grade teachers of certain comments made about the whole language approach to reading instruction by leading advocates of this procedure. The investigation revealed that these teachers rejected as "false" more of these comments about the whole language approach than they accepted as "true." The findings of this study, apparently the first of their kind that have been reported, suggest that less progress has been made in persuading teachers that the whole language approach to reading instruction is the preferred teaching procedure than some leaders of the whole language movement previously have claimed.

Incidence of insulin dependent diabetes mellitus in children 0-14 years old in the Veneto Region, Italy.

The objective of this study was to provide reliable data on the incidence of type 1 diabetes, in children aged 0-14 years, in the Veneto region, and to better understand its geographical variability throughout Italy and Europe. All new cases of type 1 diabetes diagnosed between 1st January 1993 and 31st December 1994 among residents in Veneto were recorded. Clinical records from 33 hospitals in the region were used as the primary source for this study. The number of disease-related free-of-charge prescriptions served as a secondary source. The completeness of ascertainment was estimated at 89%. The mean incidence estimated over the 1993-1994 period was 10.7 per 100,000/year. The Veneto region appears to have a relatively high incidence of IDDM among non-insular Italian regions. No significant sex-related difference in incidence was noted; the male/female ratio was 1.5.

Bridging student health risks and academic achievement through comprehensive school health programs.

In the National Action Plan for Comprehensive School Health Education, representatives for over 40 health, education, and social service organizations viewed education and health as independent systems. Participation concluded that healthy children learn better, and they cautioned that no curriculum can compensate for deficiencies in student health status. While literature confirms the complexity of health issues confronting today's students, schools face enormous pressure to improve academic skills. Local school leaders and stakeholders often remain unconvinced that improving student health represents a means to achieving improved academic outcomes. A rich body of literature confirms a direct link between student health risk behavior and education outcomes, education behavior, and student attitudes about education. This article summarizes relevant information concerning the health risk behavioral categories of intentional injuries; tobacco; alcohol, and other drugs; dietary, physical activity, and sexual risk behaviors.

HbSC hemoglobinopathy suspected by chest x-ray and red blood cell morphology.

Thorax scan was performed for elucidation of a pulmonary problem in a Nigerian immigrant. The aspect of the vertebrae suggested sickle cell disease, of course without specification of the genotype. Routine hematological tests seemed compatible with an HbSC disease, showing typical laboratory features, namely a significant proportion of hyperchromic RBC, corresponding to secondary, non hereditary spherocytosis, presence of numerous target cells and occasional HbC crystals on Pappenheim stained blood films. The diagnosis of HbSC disease was confirmed by HPLC, iso-electric focusing and citrate agar electrophoresis of hemoglobin and by reverse phase HPLC of globin-chains. This case illustrates the importance of screening for hemoglobin anomalies as it is performed in a multiethnic country such as the Grand Duchy of Luxembourg

Hb Melusine [alpha 114(GH2)Pro-->Ser]: a new neutral hemoglobin variant.

Hb Melusine [alpha 114(GH2)Pro-->Ser] was found in an Algerian patient during a systematic screening for hemoglobinopathies performed in Luxembourg. The abnormal hemoglobin was suspected when a thickening of the Hb A band was observed by isoelectrofocusing. The mutant hemoglobin was silent in all other electrophoretic methods used for presumptive diagnosis with the exception of globin electrophoresis in the presence of Triton X-100. This technique revealed an alpha chain considerably more hydrophobic than normal. The structural abnormality of Hb Melusine concerns position alpha 114(GH2) that belongs to a cluster of hydrophobic residues localized in the N-terminal half of the alpha T-12b tryptic peptide. It has been shown in the case of another variant of that position (Hb Nouakchott), that the replacement of the Pro GH2 by a Leu was responsible for a dramatic increase in the retention time of the alpha polypeptide chain during reversed phase high performance liquid chromatography, much higher than that reported for similar substitutions in other regions of the hemoglobin molecule.

Hb Luxembourg [alpha 24(B5) Tyr----His]: a new unstable variant.

Hb Luxembourg [alpha 24(B5)Tyr----His] was found in association with mild hemolytic anemia and increased indirect bilirubinemia in a family originating from the Netherlands. The slight instability of this variant may be the consequence of an indirect effect of the substitution on the alpha 1 beta 1 contact since position alpha 24 (B5) is internal and in contact with several residues involved in this interface.

Specific reactivity of fluorescein isothiocyanate-conjugated separated IgG and IgA from celiac disease sera on human tissues.

Study of the reaction of celiac disease-related antibodies with human substrates has been hampered by the immunological cross-reactivity between fluorescein isothiocyanate-conjugated anti-human immunoglobulins and immunoglobulins normally present in human tissues. In order to overcome this problem we extracted IgG from 2 celiac disease sera (positive for specific IgG and IgA antibodies) using a genetically engineered recombinant form of streptococcal protein G covalently immobilized on 4% agarose beads. The separated IgG and IgA was conjugated with fluorescein isothiocyanate and used in direct immunofluorescence on 0 blood group human duodenum, liver, and myocardium. Staining of the lamina propria beneath the villous epithelium, the endomysium of smooth muscle layers in the duodenum, and the liver sinusoidal and pericellular myocardial matrix was observed. The reactivity of celiac disease-related antibodies with various human tissues indicates that these antibodies are the result of a systemic immune response directed against all tissues containing matrix proteins.

Ito cell heterogeneity: desmin-negative Ito cells in normal rat liver.

The presence of desmin is used to identify Ito cells in rat liver and to evaluate the purity of separated and cultured Ito cells. Heterogeneity of the normal Ito cell population has been suggested; this could include variations in the content of cytoskeletal components. For these reasons we decided to reevaluate the use of desmin staining as a phenotypical marker of Ito cells in normal rat liver. Our approach was to combine desmin staining with identification of vitamin A (autofluorescence), lipid droplets (Sudan III), vimentin, laminin and tenascin, using cryostat sections: Immunofluorescence, double-immunofluorescence or immunoperoxidase techniques were used. All the techniques described corroborate the existence of desmin-negative Ito cells, mainly located in pericentral areas. In fact, lobular desmin-positive cells showed uneven distribution because they were more frequent in periportal than in pericentral areas. On the contrary, Ito cells identified on the basis of morphological criteria or positivity for laminin were evenly distributed. Double immunofluorescence confirmed this observation, showing nearly complete codistribution of laminin and desmin in periportal areas. Outside this area, positivity for desmin was observed only in about 50% of laminin-positive cells. Our observations suggest that desmin cannot be viewed as a phenotypical marker but rather is a differentiation marker of Ito cells, possibly indicating a specific functional state.

[Thrombocyte replacement with HL-A typed non-related single donors]. / Thrombozytenersatz mit HL-A-typisierten nicht-verwandten Einzelspendern

Thrombocytopenic patients who have become refractory to platelet transfusions from random donors will usually respond to HL-A compatible platelets with normal increments and hemostatic improvement. In our experience with unrelated HL-A typed donors, HL-A identity is unnecessary. Donors without excess antigen are compatible and even many donors with 1 excess antigen can be used, at any rate in immunosuppressed patients. Thus successful substitution of platelet becomes possible in refractory patients when family donors cannot be used.

Structural determination of a new electrophoreticaly silent variant: hemoglobin Alzette, beta 104(G6)Arg --> Lys.

A new electrophoreticaly neutral hemoglobin variant was found by ion-exchange high-performance liquid chromatography (HPLC). The molecular mass of the beta-chain was shifted down 28 mass units. The modification was found in the beta T-11 peptide that co-elutes with beta T-14 in the tryptic HPLC profile. Collision-induced decomposition of the protonated modified peptide indicated the Arg --> Lys exchange at the C-terminus. This modifies the fragmentation pattern as charge-remote processes induced by the strong basicity of arginine were replaced by charge-induced mechanisms. The exchanged 104Arg is one of the chloride binding sites in the central cavity of Hb.

Hepatocellular codistribution of c100, c33, c22, and NS5 hepatitis C virus antigens detected by using immunopurified polyclonal spontaneous human antibodies.

Hepatitis C virus (HCV) antigens in liver biopsy have been detected by immunohistochemistry using both spontaneous human IgG and murine monoclonal or rabbit polyclonal monospecific reagents. Conflicting results have been obtained in different studies. This was probably because of the incapacity of single experimental antibodies, raised against synthetic or recombinant peptides, to recognize native tissue antigens. To overcome this possibility, we immunopurified monospecific spontaneous polyclonal human Ig, therefore induced by native antigens, from the single antigen-containing bands of RIBA 3 strips. Antibodies to c100, c33, c22, and NS5 antigens were obtained from the serum of a patient affected by chronic hepatitis C. The IgG fraction of this serum had proved to stain tissue HCV antigens. Eight biopsies were selected on the basis of strong hepatocellular reactivity with the whole IgG fraction in a variable number (from 5% to 75%) of cells. The four antigens were detected in all biopsies; a clear cellular codistribution was observed on serial sections. These data demonstrate that the possibility to identify HCV antigens in liver biopsies is higher when using human antibodies induced by native antigens rather than experimental antibodies. The approach of immunopurification of human antibodies can be extended to other HCV-related epitopes to obtain reagents useful for the selection and optimization of monoclonal or polyclonal antibodies.