Ultrasound-guided aspiration and corticosteroid injection compared to horizontal therapy for treatment of knee osteoarthritis complicated with Baker's cyst: a randomized, controlled trial.

BACKGROUND: Combining different therapies, physical therapy agents, pharmacological and physical therapies, generally produces better outcomes for symptoms of knee osteoarthritis (OA) than do isolated therapies. AIM: To demonstrate if horizontal therapy (HT) and aspiration alone and corticosteroid injection alone or in combination determine pain relief and functional improvement in a group of patients with knee OA complicated with Baker's cyst (BC). DESIGN: We designed a randomized controlled trial (RCT). SETTING: Outpatients. POPULATION: Sixty patients with a knee OA and diagnosis of BC confirmed by means of standard ultrasound (US) evaluation. METHODS: The trial was conducted as a randomized, controlled trial. Patients who satisfied the inclusion criteria were randomized to either the US-guided (Ultrasound Guided BC aspiration and corticosteroid injection group (Group A), the Horizontal Therapy group (Group B) or the US-guided BC aspiration and corticosteroid injection plus Horizontal therapy group (Group C). Outcome measures included: 1) pain reduction as measured by visual analogue scale (VAS); 2) functional improvement, as measured by WOMAC; and 3) US evaluation at baseline (T0), at one (T1) and four (T2) weeks follow-up. RESULTS: A total of 60 patients were randomized into group A (N.=20), group B (N.=20) or Group C (N.=20). Patients in group A and in group C, but not those in group B maintained lower pain level at T2 than at baseline, with significant lower VAS values in Group C. As regards US measurements, the maximum axial area did not change as a consequence of the treatment in any of the three groups (P=0.259). Contrarily, sagittal area measurements were influenced by time (P<0.01). CONCLUSION: Our results show that the group with the best performance for pain, functionality and dimension of BC was that in which combined use was made of horizontal and corticosteroid injection therapies. CLINICAL REHABILITATION IMPACT: In this study we want to demonstrate the effectiveness of Horizontal Therapy in the treatment of knee OA complicated by BC.

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.

Monoclonal antibodies with broad specificity for hepatitis C virus hypervariable region 1 variants can recognize viral particles.

The hypervariable region 1 (HVR1) of the E2 protein of hepatitis C virus (HCV) is a highly heterogeneous sequence that is promiscuously recognized by human sera via binding to amino acid residues with conserved physicochemical properties. We generated a panel of mAbs from mice immunized with HVR1 surrogate peptides (mimotopes) affinity-selected with sera from HCV-infected patients from a phage display library. A high number of specific clones was obtained after immunization with a pool of nine mimotopes, and the resulting mAbs were shown to recognize several 16- and 27-mer peptides derived from natural HVR1 sequences isolated from patients with acute and chronic HCV infection, suggesting that HVR1 mimotopes were efficient antigenic and immunogenic mimics of naturally occurring HCV variants. Moreover, most mAbs were shown to bind HVR1 in the context of a complete soluble form of the E2 glycoprotein, indicating recognition of correctly folded HVR1. In addition, a highly promiscuous mAb was able to specifically capture bona fide viral particles (circulating HCV RNA) as well as rHCV-like particles assembled in insect cells expressing structural viral polypeptides derived from an HCV 1a isolate. These findings demonstrate that it is possible to induce a broadly cross-reactive clonal Ab response to multiple HCV variants. In consideration of the potentially important role of HVR1 in virus binding to cellular receptor(s), such a mechanism could be exploited for induction of neutralizing Abs specific for a large repertoire of viral 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.

GB virus type C infection in patients treated for childhood acute lymphoblastic leukemia.

BACKGROUND: The purpose of this study was to determine the prevalence of GB virus type C (GBV-C) infection in subjects treated for childhood acute lymphoblastic leukemia (ALL) or non-Hodgkin's lymphoma. STUDY DESIGN AND METHODS: One hundred forty patients (82 males) aged 4 to 27 years (median, 11) diagnosed with ALL between 1976 and 1993, were prospectively followed for a median of 5 years (range, 0.1-17) after completion of therapy. Stored sera were tested for antibody to hepatitis C virus (HCV), HCV RNA, antibody to GBV-C E2 (anti-E2), and GBV-C RNA. RESULTS: Thirty-eight patients (27%) were exposed to GBV-C: 30 were positive for GBV-C RNA (mostly type 2) and 8 were positive for anti-E2. Anti-E2 and GBV-C RNA were mutually exclusive: 61 patients (43%) were positive for HCV RNA, 16 (11%) were coinfected with GBV-C and HCV. Alanine aminotransferase (ALT) levels were increased (>35 mU/mL) in 32 (23%) of 137: 3 of 20 who were positive for GBV-C and negative for HCV, 7 of 15 who were positive for GBV-C and HCV, 15 of 44 who were negative for GBV-C and positive for HCV, and 7 of 58 who were negative for GBV-C and HCV (p<0.001). Median ALT values were significantly higher in patients positive for GBV-C and HCV than in those who were positive for GBV-C and negative for HCV (35 vs. 13 mU/mL, p = 0.003). Thirty-one of 38 patients with GBV-C markers were retested: GBV-C RNA was lost in 16 of 30 tested, but 7 were still GBV-C RNA positive up to 50 months later, 3 tested positive for anti-E2 up to 27 months later, and 1 was positive for GBV-C RNA and anti-E2 26 months later, while 20 tested negative for both. CONCLUSION: GBV-C did not behave as a liver pathogen, because ALT alterations were unrelated to GBV-C status, but, rather, were related to HCV infection or coinfection. GBV-C RNA was frequently lost over a relatively short period, though in some cases, it was retained for a longer time. Anti-E2 rarely coexisted with GBV-C RNA and might be short-term.

Production of human monoclonal antibodies to hepatitis C virus and their characterization.

Human monoclonal antibodies (hMAb) provide novel ways to probe the B-cell repertoire in health and disease. However, the development of hMAb technology has met with several difficulties owing to the instability of the cell lines, the low level of specific antibody secretion, and the poor cloning efficiency, particularly when using lymphoblastoid cells (1,2). In order to overcome these problems, some investigators have fused human B lymphocytes with human/mouse myeloma heterohybrids. However, in such systems, human chromosomes are unstable and may occasionally be deleted. Despite the potential emergence of technical pitfalls, B-cell immortalization with EBV has been extensively used for hMAb production, because of its simplicity and because EBV can bind to and penetrate in virtually all B lymphocytes, theoretically allowing the exploration of the whole B-cell repertoire. The most recent protocols have made use of techniques aimed at expanding the population of antigen-specific B-cell precursors and improving the capacity of B-cells to grow at low density. These methods will be discussed below.

HGV/GBV-C infection in liver transplant recipients: antibodies to the viral E2 envelope glycoprotein protect from de novo infection.

BACKGROUND/AIMS: Liver transplantation for endstage liver cirrhosis provides a useful model to investigate the pathogenetic role of hepatotropic viral agents. Recently, a new member of the Flaviviridae family, provisionally named HGV/GBV-C virus, has been associated with acute and chronic non A-E hepatitis. We studied 136 patients with cirrhosis consecutively transplanted at our institution for evidence of hepatitis G virus infection and correlation with the patients' clinical course. METHODS: All patients survived for at least 6 months after transplantation (median follow-up 44 months) and underwent routine liver biopsies. Hepatitis G virus infection was studied using both direct viral RNA identification by RT-PCR and indirect detection of antibodies to the E2 glycoprotein. RESULTS: There was a high frequency of the hepatitis G virus among patients undergoing liver transplantation, with HGV RNA and anti-E2 prevalence rates of 18.4% and 26.5%, respectively. HGV RNA prevalences significantly increased after transplantation (47.8%), with 47.3% rate of new infections in susceptible subjects. Anti-E2 antibodies were significantly more prevalent among patients transplanted for HCV-related cirrhosis and represented a strong protective factor against hepatitis G virus reinfection or recurrent infection. No correlation was found between HGV RNA or anti-E2 prevalences and survival after transplantation or rates of recurrent liver damage. CONCLUSIONS: All available evidence suggests that, although liver transplant patients are heavily exposed to hepatitis G virus both before and after transplantation, hepatitis G virus does not induce liver disease in this setting. Most infections appear to be self-limited and induce a protective immunity which is marked by the presence of anti-E2 antibodies.

Antibody-negative chronic hepatitis C virus infection in immunocompetent children.

Antibody-negative hepatitis C virus (HCV) infection, defined by the presence of HCV viremia in the absence of a serologic response to HCV, was detected in two immunocompetent and symptom-free children; each had a history of exposure to blood products. HCV infection may occasionally explain cryptogenic elevation of aminotransferases, even in the absence of serum anti-HCV. HCV-RNA should be investigated in these cases, particularly in the presence of previous exposure to blood products.

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.

Hepatitis C virus genotypes and risk of hepatocellular carcinoma in cirrhosis: a prospective study.

A prospective study was performed to establish whether infection with specific hepatitis C virus (HCV) genotypes was associated with an increased risk of development of hepatocellular carcinoma (HCC) in cirrhosis. A cohort of 163 consecutive hepatitis C virus antibody (anti-HCV)-positive cirrhotic patients was prospectively evaluated for the development of HCC at 6-month intervals by ultrasound (US) scan and alpha-fetoprotein (AFP) concentration. HCV genotypes were determined according to Okamoto. Risk factors associated with cancer development were analyzed by univariate and multivariate statistics. At enrollment, 101 patients (62%) were infected with type 1b, 48 (29.5%) were infected with type 2a/c, 2 (1.2%) were infected with type 3a, 1 (0.6%) was infected with type 1a, 3 (1.8%) had a mixed-type infection, and, in 8 patients (4.9%), genotype could not be assigned. After a 5- to 7-year follow-up (median, 68 months), HCC developed in 22 of the patients, 19 infected with type 1b and 3 with type 2a/c (P < .005). Moreover, HCC developed more frequently in males (P < .01), patients with excessive alcohol intake (P < .01), those over 60 years of age (P < .02), and in patients who did not receive interferon treatment (P < .02). Multivariate analysis showed that type 1b was the most important risk factor associated with tumor development (odds ratio 6.14, 1.77-21.37 95% confidence interval). Other independent risk factors were older age and male sex. Cirrhotic patients infected with HCV type 1b carry a significantly higher risk of developing HCC than patients infected by other HCV types. The latter may require a less intensive clinical surveillance for the early detection of neoplasia.

Recombinant human antibodies specific for hepatitis C virus proteins.

Human antibodies to hepatitis C virus core, NS4A and NS3 were cloned in a prokaryotic vector and expressed as soluble Fab fragments and as phage-displayed Fabs. The recombinant Fabs were shown to be a suitable tool for immunohistochemistry, since they recognize the cognate antigen expressed in mammalian cells. The nucleotide sequence of the cDNA for the variable domains of these antibodies was determined and the V-gene usage was derived. On the basis of the deduced amino acid sequence, a structural model of the V domains of the Fabs was constructed.

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.

A human antibody specific for hepatitis C virus core protein: synthesis in a bacterial system and characterization.

The cDNA coding for the Fab fragment of the human B12.F8 antibody (Ab), directed against the putative nucleocapsid component (core protein) of hepatitis C virus (HCV), was cloned in the prokaryotic phagemid vector, pHEN-1, to obtain its expression in Escherichia coli. The functionality and specificity of the recombinant Ab, called B12Fab, were examined by Western blot and ELISA using recombinant HCV core protein as antigen. The specificity of B12Fab was further confirmed by ELISA with the 33-mer peptide epitope recognized by the original whole B12.F8 Ab. By immunofluorescence, the recombinant B12Fab was shown to recognize HCV core protein produced in cells transfected with HCV cDNA, indicating that the recombinant B12Fab is suitable as a diagnostic tool for tissue localization of the virus. The B12Fab also functioned when displayed on phage particles, providing the basis for future experiments of in vitro affinity maturation and selection of mutants. The variable chain coding regions of the recombinant B12Fab clone were sequenced and the V-gene usage was determined by comparison with the V kappa and VH germline sequences. The B12Fab V kappa chain belongs to the subgroup II and shows the highest degree of homology with the A3 germline gene, whereas the sequence of the VH chain is strictly related to that of the Humhv3019b18 gene of the VH3 family. These results are, to our knowledge, the first report of molecular cloning and characterization of a functional human Ab specific for an HCV antigen.

Occurrence of antibodies reactive with more than one variant of the putative envelope glycoprotein (gp70) hypervariable region 1 in viremic hepatitis C virus-infected patients.

The hepatitis C virus (HCV) is a frequent cause of chronic liver disease. A mechanism proposed as being responsible for virus persistence is evasion of the host immune response through a high mutation rate in crucial regions of the viral genome. We have sequenced the hypervariable region 1 (HVR1) of the virus isolated from three serum samples, collected during 18 months of follow-up, from an asymptomatic HCV-infected patient. A synthetic peptide of 27 amino acids, corresponding to the HVR1 sequence found to be predominant in both the second and third samples, was used as the antigen for detection of antibodies by enzyme-linked immunosorbent assay (ELISA). We observed reactivity against this HVR1 sequence in the first serum sample before the appearance of the viral isolate in the bloodstream; the reactivity increased in the second and third samples while the cognate viral sequence became predominant. Moreover, our results show that antibodies from all three samples recognize a region mapping at the carboxyl-terminal part of the HVR1 and are cross-reactive with the HVR1 sequence previously found in the same patient. The presence of anti-HVR1 antibodies was investigated in a further 142 HCV patients: 121 viremic and 21 nonviremic. Two synthetic peptides were used, the first corresponding to the sequence derived from the patient described above and the second one synthesized according to the sequence of the HCV BK strain. A high frequency of positive reactions against both HVR1 variants was detected in the samples from the viremic individuals. Finally, antibodies cross-reactive with both variants were shown to be present by competitive ELISA in 6 of 10 viremic patients. The potential negative implications of this observation for the host are discussed.

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.