Changes in fatty acid profile of Bovec sheep milk due to different pasture altitude.

Sheep rearing on mountain pastures is an ancestral tradition in northwestern Slovenia. The indigenous Bovec sheep are widespread there and are well adapted to the rough Alpine rearing conditions. Every year, after weaning, the sheep start grazing in the lowlands (L) and then gradually move to mountain pastures, and finally, to the highland (H) pastures of the Alps. Grazing positively affects the fatty acid (FA) composition in sheep milk fat with increased availability of polyunsaturated FA (PUFA) in grass, and subsequently, in milk. Consequently, the objective of this work was to study the FA profile in sheep milk during grazing in four geographical areas in the Alps. A total of 15 ewes of the Bovec sheep breed were randomly selected and milk samples from these ewes were taken at four different pasture locations that differed with regard to altitude: the L pasture location at an altitude of 480 m, the mountain pastures (M1 and M2) at altitudes of 1100 to 1300 m and 1600 to 1900 m, respectively, and the H pastures at altitudes of 2100 to 2200 m. Milk samples from the ewes were taken during the grazing season from April to September. The chemical and FA composition of the milk samples from each pasture location were determined. There were significant differences in the concentrations of FA among the L, M1, M2 and H milk samples. We observed decreases of the concentrations of saturated FA (SFA) in milk from L to H pastures. The concentration of α-linolenic FA, monounsaturated FA (MUFA), PUFA and n-3 PUFA in milk were increased significantly with pasture altitude. The n-6/n-3 PUFA ratio was reduced by the change of pasture altitude with the lowest value at the M1 pasture (1.5). The concentrations of total SFA decreased significantly and was lowest at the L pasture. Our results underline the importance of the effect of grazing in the Alpine region associated with pasture altitude on the FA profile of sheep milk. The first variation in FA concentration in sheep milk occurred between L and M1, although it was more evident on H pastures in the Alpine mountains. Changes of the FA profile in sheep milk due to pasture altitude were related to variation in FA concentration in the pasture and the botanical composition of the pasture location.

Hypervariable region 1 of hepatitis C virus: immunological decoy or biologically relevant domain?

The hypervariable region 1 (HVR1) of the E2 protein of hepatitis C virus (HCV) is highly heterogeneous and is responsible for significant inter- and intra-individual variation of the infecting virus, which may represent an important pathogenetic mechanism leading to escape and persistent infection. Moreover, a binding site for neutralizing antibodies (Ab) has been allegedly identified in this region. Prospective studies of serological responses to synthetic oligopeptides derived from HVR1 sequences of patients with acute and chronic HCV infection showed extensive serological cross-reactivity for unrelated HVR1 peptides in the majority of the patients. A significant correlation was found between HVR1 sequence variation, and intensity, and cross-reactivity of humoral immune responses providing strong evidence in support of the contention that HCV variant selection is driven by the host immune pressure. Monoclonal Ab (mAb) generated following immunization of mice with peptides derived from natural HVR1 sequences also showed cross-reactivity for several HVR1 sequences attesting to the existence of conserved amino acid motifs among different variants. These findings suggest that it is possible to induce a broadly cross-reactive immune response to HVR1 and that this mechanism can be used to generate protective immunity for a large repertoire of HCV variants.

Antibody responses to hepatitis C virus hypervariable region 1: evidence for cross-reactivity and immune-mediated sequence variation.

Sequence heterogeneity of hepatitis C virus (HCV) is unevenly distributed along the genome, and maximal variation is confined to a short sequence of the HCV second envelope glycoprotein (E2), designated hypervariable region 1 (HVR1), whose biological function is still undefined. We prospectively studied serological responses to synthetic oligopeptides derived from HVR1 sequences of patients with acute and chronic HCV infection obtained at baseline and after a defined follow-up period. Extensive serological cross-reactivity for unrelated HVR1 peptides was observed in the majority of the patients. Antibody response was restricted to the IgG1 isotype and was focused on the carboxyterminal end of the HVR1 region. Cross-reactive antibodies could be readily elicited following immunization of mice with multiple antigenic peptides carrying HVR1 sequences derived from our patients. The vigor and heterogeneity of cross-reactive antibody responses were significantly higher in patients with chronic hepatitis compared with those with acute hepatitis and in patients infected with HCV type 2 compared with patients infected with other viral genotypes (predominantly type 1), which suggest that higher time-related HVR1 sequence diversification previously described for type 2 may result from immune selection. The finding of a statistically significant correlation between HVR1 sequence variation, and intensity, and cross-reactivity of humoral immune responses provided stronger evidence in support of the contention that HCV variant selection is driven by the host's immune pressure.

Human leukocyte antigen class II and III alleles and severity of hepatitis C virus-related chronic liver disease.

Hepatitis C outcome is likely related both to viral factors and host's immune responses. We correlated the severity of liver disease with human leukocyte antigen (HLA) genes (C4A, C4B, TNFA, TNFB, DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, TAP1, and TAP2) in three groups of subjects: 99 patients with chronic hepatitis, 41 asymptomatic carriers, and 179 uninfected controls. Patients with grade/stage 3 to 4 hepatitis significantly differentiated for their low frequency of alleles TNFB*1, DRB1*1104, and DRB3*03, which had a protective role, and high frequency of allele DRB1*1001, which was associated with disease severity. HLA-DRB*11 subtypes were differentially distributed: DRB1*1104 was most frequent in carriers, whereas DRB1*1101 was more frequent in patients. The TAP1C,2A haplotype was also underrepresented in patients with respect to controls. Finally, a decrease of heterozygous subjects was observed in patients with respect to carriers at the DQB1 locus. Multivariate analysis by correspondence analysis and multiple logistic regression indicated that age, sex, and hepatitis C virus (HCV) type were the strongest risk factors; however, some immunogenetic variables (TNFB*1, DRB1*1104, and DRB3*03) showed an independent contribution, especially in comparing patients with extreme manifestations of disease. The involvement of different genes in various HLA subregions suggests that anti-HCV responses are modulated by a complex gene interplay rather than by single alleles.

Clonality and specificity of cryoglobulins associated with HCV: pathophysiological implications.

BACKGROUND/AIMS: Hepatitis C virus (HCV) infection plays a central role in the pathogenesis of mixed cryoglobulinemia through molecular mechanisms which remain to be elucidated. The aim of this study was to investigate the role of antibody responses to HCV in the pathogenesis of cryoglobulinemia through characterization of the anti-HCV specificity and immunochemical characteristics of the immunoglobulins involved in cryoprecipitation. METHODS: Sera from 50 consecutive patients with chronic HCV infection (RNA positive) were screened for the presence of cryoglobulins. The two major components of cryoprecipitates, IgM rheumatoid factors and IgG, were separated by high performance liquid chromatography and analyzed for immunochemical composition by immunoblotting and antibody specificity by ELISA and immunoblotting using recombinant HCV proteins and synthetic peptides as antigens. RESULTS: Cryoprecipitates were observed in 27 patients and characterized by immunofixation: 13 (48%) were classified as type II and 14 (52%) as type III. Monoclonal immunoglobulins were detected by immunoblotting in 20 cryoprecipitates: IgM in 14 samples and IgG in 14, with a clear preponderance of IgG3 (12/14). Specificity studies on sera and purified IgM and IgG fractions from cryoprecipitates revealed enrichment in cryoglobulins, predominantly polyclonal IgG1, reactive with the HCV structural proteins, whereas specificities for nonstructural viral proteins were relatively less represented compared to whole serum. No restricted pattern of fine specificity was observed. IgG3 subclass was apparently not involved in HCV nucleoprotein binding. CONCLUSIONS: Our findings do not support a direct link between monoclonal cryoglobulins and immune response to HCV According to the proposed pathogenetic model, HCV infection can induce the formation of cryoprecipitable rheumatoid factors, sustain their production, and eventually lead to monoclonal B-cell expansion through several cooperative mechanisms.

Hepatitis C virus infection among institutionalised psychiatric patients: a regression analysis of indicators of risk.

BACKGROUND/AIMS: Institutionalised psychiatric patients are at increased risk of developing chronic infection with hepatitis B virus (HBV). However, little information is available on transmission and epidemiology of hepatitis C virus (HCV) in this setting. The aim of this study was to identify potential risk factors of acquiring HCV infection in two large psychiatric institutions in northern Italy. METHODS: We designed a case-control study using randomly selected controls from the same study database, consisting of a total of 1180 patients, in order to satisfy the principle that both cases and controls should be representative of the same base experience. A multiple regression logistic analysis was used to identify features that could predict exposure to HCV as evidenced by the presence of circulating anti-HCV antibodies. RESULTS: Anti-HCV was detected in 79 patients (6.7%). The prevalence of viraemia and the distribution of genotypes were very similar to those found in subjects with chronic HCV infection drawn from the same geographical area. Multivariate analysis indicated that a diagnosis of psychosis and a history of trauma were statistically significant independent risk factors associated with a positive anti-HCV result (OR 2.615, 1.273-5.373 95% CI and OR 2.096, 1.133-3.877 95% CI, respectively). CONCLUSIONS: The findings of this large epidemiological study show for the first time that prolonged residence in psychiatric institutions does not entail per se a significant risk of acquiring HCV infection. Since transmission of HCV in this setting appears to occur predominantly via classical parenteral routes, simple prophylactic measures appear to be adequate to prevent infection.

Antibody responses to the hepatitis C virus E2 protein: relationship to viraemia and prevalence in anti-HCV seronegative subjects.

A small proportion of patients with chronic hepatitis C virus (HCV) infection show no serological responses to the HCV polypeptides incorporated in commercial III generation immunoassays. To determine whether sera from these subjects contain antibodies to the highly immunoreactive second envelope polypeptide E2, which is not included in current anti-HCV assays, we studied 59 anti-HCV negative subjects who were found consistently to be HCV RNA positive by polymerase chain reaction (PCR). Controls included 167 anti-HCV seropositive patients with or without serum HCV RNA and normal subjects. Antibodies to the E2 region were sought for by ELISA using the following antigens: a full length E2 protein expressed in insect cells using a baculovirus vector and extracted under denaturing conditions (dE2), and a C-terminal truncated soluble E2 (sE2) protein (a.a. 390-683), also expressed with a baculovirus vector, containing a signal peptide of rabies virus G protein which allows its secretion into the culture supernatant. Sera from only two (3.4%) of the 59 anti-HCV negative, HCV RNA positive patients recognised sE2 and none dE2. In sharp contrast, 82% of seropositive, viraemic patients recognised sE2 and 60% dE2, the difference in immunoreactivity being statistically significant (P < 0.0003). A significantly lower proportion of sera from anti-HCV positive, HCV RNA negative subjects recognised either sE2 or dE2 (16% and 13%, respectively, P < 0.000001). Healthy controls were consistently negative. These results indicate that antibody responses to predominantly conformational epitopes on the HCV E2 protein are common in patients with chronic HCV infection and are strictly related to the presence of circulating viral genomes. In contrast, only a minor proportion of HCV RNA positive patients, but anti-HCV seronegative by commercial immunoassays, have humoral immune responses to the HCV E2 region.

Comparative evaluation of two serologic typing methods for hepatitis C virus.

Serologic methods of typing for hepatitis C virus offer advantages over PCR-based typing methods in terms of speed and simplicity of sample preparation and in the use of standard laboratory equipment. We examined the sensitivities and specificities of two hepatitis C virus serotyping assays which use sets of type-specific antigenic peptides derived from the core or the nonstructural 4 (NS4) regions and compared the results with those of molecular typing with type-specific primers from the core region. Although there was a good concordance between serotyping by either assay and genotyping, we found that the sensitivities of both serologic assays were less than optimal compared with that of molecular typing, with only about 50% of samples being unequivocally typed. Moreover, amino acid sequence similarities within the regions of the genome used for serotyping preclude differentiation into subtypes, which may have important clinical and therapeutic implications.

Molecular epidemiology of hepatitis C virus infection among intravenous drug users.

BACKGROUND/AIMS: The clinico-pathological features of hepatitis C virus infection in intravenous drug users are different from those found in other hepatitis C virus-infected patients. Our aim was to test whether specific viral variants circulate within this particular patient population. METHODS: We studied the distribution of hepatitis C virus genotypes in 90 drug addicts and 484 controls, according to the method described by Okamoto. RESULTS: Hepatitis C virus type 1a and 3a infections were more frequent among intravenous drug users than in 125 age-matched controls (48.8% and 21.1% vs 17.6% and 11.2%), accounting for the majority of infections in intravenous drug users. Analysis of hepatitis C virus genotypes according to age showed that, in the general population, hepatitis C virus types 1a and 3a were more prevalent among patients younger than 40 years of age than in older individuals (17.6% and 11.2% vs 1.4% and 0.6%). CONCLUSIONS: These findings suggest that hepatitis C virus types 1a and 3a were recently introduced in Italy, presumably via needle-sharing among intravenous drug users, and from this reservoir they are extending to the general population, particularly among younger subjects.

Differential distribution of hepatitis C virus genotypes in patients with and without liver function abnormalities.

Hepatitis C virus (HCV) infection persists for an indefinite length of time in a major proportion of patients, inducing chronic liver lesions that evolve to cirrhosis and hepatocellular carcinoma (HCC) in approximately 20% of cases. We studied HCV viremia and genotypes by reverse transcription-polymerase chain reaction (RT-PCR) in 341 consecutive anti-HCV-positive patients. Of these, 167 patients had persistently normal or near normal alanine aminotransferase (ALT) levels (fluctuations < or = 5 IU above the upper limit of normal); the remaining 174 patients presented with elevated ALT and histological evidence of chronic liver disease. Seventy percent of patients with normal ALT values had circulating HCV RNA despite the absence of biochemical indicators of liver damage and mild histological forms of chronic hepatitis were detected in most patients who underwent liver biopsy. Isolated genotype III infection was significantly more prevalent in this patient group with respect to control patients with abnormal ALT values (70% vs. 39%; P < .001). Conversely, isolated genotype II was more frequently found in patients with elevated ALT values and evidence of chronic liver disease (45% vs. 23%; P < .01) and it was progressively more represented in advanced liver disease, such as cirrhosis and HCC. Virological features of HCV infection might be associated with different clinical manifestations, suggesting a potential prognostic significance on disease outcome.

Hepatitis C virus (HCV) core serotypes in chronic HCV infection.

Recently, two distinct hepatitis C virus (HCV) serologic types have been identified on the basis of amino acid variations in the core region. The two serologic types can readily discriminate between genotypes I-II-V (serotype 1) and III-IV (serotype 2), according to the Okamoto classification. We compared HCV core serotyping with genotyping with sera from 363 anti-HCV-positive patients (309 HCV RNA positive by PCR) using a synthetic core peptide-based enzyme immunoassay and PCR amplification of core region sequences with type-specific primers, respectively. Serologic responses to HCV serotypes were successfully identified in 164 (45%) patients, of whom 153 were viremic. Eighty-nine patients had evidence of exposure to serotype 1: 8 of these were infected with genotype I, 50 were infected with genotype II, 2 were infected with genotype III, 7 were infected with genotype V, 13 had infections with mixed genotypes, 3 were infected with an indeterminate genotype, and 6 were nonviremic. Seventy-four patients had been exposed to serotype 2: 64 were infected with genotype III, 3 were infected with mixed genotypes, 2 were infected with an indeterminate genotype, and 5 were nonviremic. The serum of one patient, infected with genotype III, showed reactivity to both serotypes. Comparative evaluation of HCV core region serotyping and genotyping with sera from 294 viremic patients infected with a known HCV genotype showed a remarkable concordance between HCV core region genotyping and serotyping, with only 2 apparently discordant serum samples (both from patients with genotype III infection) of 148 (1.4%) successfully serotyped samples. Serotype 1 infection was more frequently observed in patients with overt chronic liver disease and accounted for all successfully serotyped samples from intravenous drug abusers. In contrast, serotype 2 was more prevalent in subjects with biochemically silent HCV infection (alanine aminotransferase, < 45 U/liter), in agreement with previous findings at the molecular level. HCV core serologic typing is a simple, inexpensive, and highly reproducible assay that can be applied to more than 50% of viremic HCV antibody carriers prior to the use of more sophisticated molecular typing techniques. Moreover, it may be helpful in tracking transmissions routes, particularly for incorrectly stored samples in which the RNA has degraded or for subjects who have cleared the virus and therefore have only antibodies remaining to testify to a remote infection. The lack of recognition of the core sequence from residues 67 to 81, which contains a minor B-cell epitope used to detect type-specific immunoreactivity, may explain the negative serologic findings for half of the patients.