The quantitative humoral immune response to the hepatitis C virus is correlated with disease activity and response to interferon-alpha

BACKGROUND/AIM: Virus-host interactions may have pathogenetic significance in chronic hepatitis. Thus the humoral immune response was evaluated during the clinical course of HCV-infected patients. METHODS: Eighteen selected chronic HCV patients received three doses of 3 or 6 MU interferon-alpha 2a weekly for 6 to 12 months and were followed up for 6 to 60 months. Anti-HCV antibody levels were serially measured either in end-point diluted sera with the Matrix-Assay or with quantitative anti-HC34-IgG and -IgM ELISA. Circulating immune complexes were assessed by flow cytometry and the results were correlated with histology, quantitative HCV-RNA levels and genotypes. RESULTS: Nine complete responders (CR; genotypes 1a n = 4; 1b n = 1; 2a n = 1; 3a n = 3) showing sustained virus elimination and ALT normalisation had low HCV-RNA pretreatment levels (mean 14 x 10(3) copies/ml) compared to six nonresponders and three partial responders (NR/PR; genotypes 1a n = 2; 1b n = 7) who had significantly higher HCV-RNA pretreatment levels (mean 254 x 10(3) copies/ml; p < 0.01). In untreated NR/PR the HC34 core-antigen was most immunogenic, in CR the NS3-derived HC29-antigen. Pre-treatment levels of anti-HC 34-IgG and -IgM antibody levels in NR/PR were higher than in CR (IgM/IgG p = 0.05, n.s.) and these differences became significant during or after therapy (3 months therapy: IgM p < 0.02/IgG p < 0.07; end of therapy: IgM 0.006/IgG p < 0.04; 6 months post-therapy: IgM p < 0.002/IgG p < 0.004). The PR patients showed recurrent anti-HC34 antibody levels that preceded disease reactivation and detectable HCV-RNA in serum. Immune complex formation increased in some patients during treatment but did not correlate with disease activity, quantitative viraemia, antibody levels or therapy outcome. CONCLUSION: Anti-HC34 antibodies, i.e. of the IgM-subtype, correlated quantitatively with viraemia and disease activity. Monitoring the antibody levels may predict the long-term therapy outcome during interferon-alpha treatment.     

Human humoral immune response to Dirofilaria species

BACKGROUND: Keloids are relatively common sequelae of trauma to the skin of the head and neck. A wide variety of treatment approaches developed over the years document the difficulty in eradicating these lesions. OBJECTIVE: To review the senior author's (W.H.L) 15-year experience in treating keloids both medically and surgically. DESIGN: A retrospective analysis of 202 patients with histologically documented keloids of the head and neck with at least a 2-year follow-up. RESULTS: A combination of precise surgical excision, postoperative steroid infiltration, silicone sheeting, and conservative auricular radiotherapy has resulted in an acceptable 15% recurrence rate overall. CONCLUSIONS: The treatment of facial keloids remains a challenge for the facial plastic and reconstructive surgeon. Precise surgical techniques with adjuvant therapies have resulted in a relatively low recurrence rate.     

The equine immune response to endometrial cups

Out of all the areas of comparative immunological study in the horse, the field of reproductive immunology has proven to be one of the most fertile and exciting. Maternal immunological interactions with the fetus involve a set of events which prevent maternal rejection of trophoblastic tissue invading the uterus, and at the same time control this invasion to regulate growth and prevent damage to maternal tissues. Unique features of equine placentation make it exceptionally well-suited to studying these immunological interactions.     

Dissection of human humoral immune response against hepatitis C virus E2 glycoprotein by repertoire cloning and generation of recombinant Fab fragments

Demonstration of antibodies inhibiting key viral functions is the basis for the design of an effective vaccine. Dissection of the human antibody response by repertoire cloning may be a powerful means to address this issue. In this study, a panel of human monoclonal recombinant Fab fragments specific for hepatitis C virus (HCV) E2 envelope protein was generated. The selection procedure was designed to select for cross-genotype reactive antibodies. Sequences coding five different human recombinant Fabs specific for the HCV/E2 protein were cloned and characterized. The ability of the cloned antibody fragments to inhibit adhesion of recombinant envelope E2 protein to target cells was assayed. While affinity of the different antibody fragments appeared similar, activity in inhibiting E2 binding to target cells varied considerably from one Fab fragment to another. Two Fabs were not able to inhibit E2 binding at high concentration (40 microg/mL), while three other Fab clones were active in neutralizing 50% of the E2 binding at concentrations ranging from 3 to 0.35 microg/mL.     

Expression and immune response to hepatitis C virus core DNA-based vaccine constructs

Primary antiphospholipid syndrome (pAPS) can be experimentally induced in mice by immunization with anti-cardiolipin (aCL) antibodies (Abs). Recently, we have pointed to the pathogenic role of antiphosphatidylserine (aPS) Abs by inducing an experimental model of pAPS in naive mice following passive transfer of human aPS to the tail vein of ICR mice. The aim of the present study was to induce experimental pAPS in mice following active immunization with aPS. Mice were immunized with IgG and IgM aPS, purified from two patients with pAPS. The sera of the mice were examined for the presence of different antiphospholipid Abs, and the beta 2 GPI dependency of aPS, using an enzyme-linked immunosorbent assay (ELISA). Inhibition ELISA studies, with silica beads coated with phospholipids, were used to detect cross-reactivity of aPS or aCL to other phospholipids. The mice were also tested for the presence of thrombocytopenia, prolonged activated partial thromboplastin time (APTT), and the percentage of fetal resorptions. The purified IgG aPS but not IgM aPS had a strong lupus anticoagulant activity. Both IgG and IgM aPS were beta 2 GPI dependent and did not bind PS in the absence of the glycoprotein. The mice immunized with IgG aPS, but not IgM aPS, developed high titers of mouse aPS. The mice generated polyspecific Abs, cross-reacting with aCL and aPS, as well as individual aPS or aCL Abs. Only the mice immunized with IgG aPS developed clinical parameters of pAPS: prolonged APTT, thrombocytopenia, and an increased fetal resorption rate. In conclusion, active immunization with IgG, but not IgM, aPS induces experimental pAPS in naive mice, thus pointing to the participation of aPS in the idiotypic network.     

Preliminary studies on humoral immune response of sheep to wohlfahrtiosis

The humoral immune response of sheep to wohlfahrtiosis was studied. An enzyme-linked immunosorbent assay was developed to compare four different types of antigens obtained from the third-stage larvae of Wohlfahrtia magnifica. The antigen prepared from salivary glands detected a humoral response in all 35 infested sheep and was more specific in the ELISA than cuticular, intestinal or whole larval antigens. The level of the humoral response in sheep to wohlfahrtiosis differed according to the location of the wounds.     

Dysregulation of the humoral immune response in old mice

The increase in autoantibodies with age of both experimental animals and humans has been thought to reflect a shift in the antibody repertoire from foreign to self antigens. In mice, before immunization, the age-associated increase in antibodies reactive with a prototypic autoantigen, bromelain-treated autologous erythrocytes (BrMRBC), reflected a 3-fold increase in serum IgM and the number of IgM-secreting spleen cells in old compared with young mice. However, the percentage of the IgM-secreting spleen cell repertoire reactive with BrMRBC in old mice was actually approximately 50% that in young mice. In contrast, after immunization with sheep erythrocytes (SRBC), old mice showed a 5-fold increase in the percentage of IgM-secreting cells reactive with BrMRBC while young mice showed no significant increase. The converse is true for the percentage of IgM-secreting spleen cells in old mice specific for SBRC, which is 10% the number generated by young mice. The increased autoantibody response of old mice is not, however, linked to their poor response to the nominal antigen. Thus, immunization with phosphorylcholine (PC) conjugated keyhole limpet hemocyanin, an antigen that induces a comparable anti-PC response in old and young mice, also induced more autoantibody forming cells in old than young mice. The increased autoantibody response of old mice after immunization can be accounted for by both an increased number of Ig-secreting spleen cells as well as an increased percentage of the expressed repertoire of IgM-secreting spleen cells that react with autoantigens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteolytic processing of the replicase ORF1a protein of equine arteritis virus

To study the proteolytic processing of the equine arteritis virus (EAV) replicase open reading frame 1a (ORF1a) protein, specific antisera were raised in rabbits, with six synthetic peptides and a bacterial fusion protein as antigens. The processing of the EAV ORF1a product in infected cells was analyzed with Western blot (immunoblot) and immunoprecipitation techniques. Additional information was obtained from transient expression of ORF1a cDNA constructs. The 187-kDa ORF1a protein was found to be subject to at least five proteolytic cleavages. The processing scheme, which covers the entire ORF1a protein, results in cleavage products of approximately 29, 61, 22, 31, 41, and 3 kDa, which were named nonstructural proteins (nsps) 1 through 6, respectively. Pulse-chase experiments revealed that the cleavages at the nsp1/2 and nsp2/3 junctions are the most rapid processing steps. The remaining nsp3456 precursor is first cleaved at the nsp4/5 site. Final processing of the nsp34 and nsp56 intermediates is extremely slow. As predicted from previous in vitro translation experiments (E. J. Snijder, A. L. M. Wassenaar, and W. J. M. Spaan, J. Virol. 66:7040-7048, 1992), a cysteine protease domain in nsp1 was shown to be responsible for the nsp1/2 cleavage. The other processing steps are carried out by the putative EAV serine protease in nsp4 and by a third protease, which remains to be identified.     

An antigen valence theory to explain the evolution and organization of the humoral immune response

The three modes of antibody production, natural, T independent (TI) and T dependent (TD) are conserved among vertebrate species suggesting an important role for each in protection against pathogens. Here, I use an artificial 'universe' to argue that the three modes of antibody production represent layers that evolved to deal optimally with antigens of different valence. Thus, the apparently more sophisticated TD response has not superseded the natural and TI components of the humoral immune response. Furthermore, the characteristic differences in isotype, somatic mutation and memory displayed by each antibody layer are appropriate for their targeted range of surface structures. It is also suggested that the TD and TI activation arms are at the extremes of a continuum, with signal integration of antigen and T cell-derived signals contributing to B cell decisions about isotype selection, proliferation and secretion that minimize the time to protection.     

Group selection for adaptation to multiple-hen cages: humoral immune response

A selected line of White Leghorns, which has shown improved survivability and reduced feather loss in large multiple-hen cages, was evaluated for humoral immune response to SRBC under both stressed and unstressed conditions. Three lines of chickens (selected, control, and commercial) were housed in either single- (1 hen) or multiple-hen cages (12 hens, social competition) and subjected to a cold ambient temperature (0 C) at 33 wk of age and to two heating episodes (38 C) at 44 wk of age. Each hen was challenged intravenously with 1 mL of a 7% saline suspension of SRBC at the time that cold exposure was initiated. Hens subjected to high ambient temperatures had been exposed previously to a cold temperature, but were not challenged with SRBC until 16 to 18 h following the end of the second heating episode. Exposure to cold caused immunosuppression in single-caged hens, but not in hens in colony cages. Single- vs colony-caged hens of the control environment challenged with SRBC at 33 wk of age had similar primary hemagglutinin responses to SRBC. Hens subjected to heat experienced immunosuppression at 9 and 12 d following challenge to SRBC when compared to the controls. Hens of multiple-bird cages challenged with antigen at 44 wk of age had a significantly lower hemagglutinin response to SRBC than those reared in single-bird cages. The three lines of genetic stock had similar primary hemagglutinin responses to SRBC; the interactions of genetic stock with cage size or environmental temperature were not significant. It was concluded that genetically selecting hens for survival in multiple-hen cages did not affect their humoral immune response to SRBC.     

Enzyme-linked immunosorbent assay for serological survey of equine arteritis virus in racehorses

To examine antibodies against equine arteritis virus (EAV), an enzyme-linked immunosorbent assay (ELISA) using purified virus antigen was developed. The results of ELISA were compared with those of serum neutralization (SN) tests. The ELISA absorbance values and the SN titers in sera collected weekly from EAV-infected horses showed a similar pattern. The ELISA could detect antibody to EAV in horses experimentally infected with not only a homologous virus strain, which was used as the ELISA antigen, but also a heterologous strain. Using the ELISA, serum samples collected in 1996 from racehorses in three prefectures (Hokkaido, Ibaraki, and Shiga) were examined and there was no evidence of recent EAV infection among these racehorse populations in Japan. The ELISA should be a simple and highly specific method for rapid screening of EAV infection in racehorses.     

Genetic analysis of the humoral immune response of White Leghorn chicks

Total agglutinin antibody titers, 2-mercapto ethanol (2-ME) sensitive and 2-ME resistant antibody titers were determined in 598 White Leghorn chicks after intramuscular injection with sheep red blood cells. Antibody titers were determined on day 0 and on days 3, 7, 10, 13 postinjection. Mean total tier (5.2, log2 value) was highest on day 7. Females showed a significantly higher response to injection with sheep red blood cells than males. Also, significant hatch effects were noted. Heritability estimates generally varied from 0 to .5 for all parameters. In the earlier stages of the immune response the sire estimate of heritability for total and 2-ME sensitive antibody titer was higher than the dam estimate. Additive genetic correlations between 2-ME sensitive (days 3 to 13) and resistant (days 7 to 13) antibody titers were negative, varying from -.30 to -.93. The response to selection for total antibody titer is, therefore, not easily predicted.     

Detection of antibodies to equine arteritis virus by enzyme linked immunosorbant assays utilizing G(L), M and N proteins expressed from recombinant baculoviruses

On the experience of 73 patients, the authors state their guidelines on the treatment of bone metastases on the cervical spine. Most of the cases on which no vertebral collapse occur neither neurological deficit, radiation therapy and external support are suggested. Surgery is necessary on case of severe bone destruction, collapse with or without subsequent neurological impairment. Anterior excision is considered the best approach, sometimes complemented by posterior stabilisation.     

Genetic immunization generates cellular and humoral immune responses against the nonstructural proteins of the hepatitis C virus in a murine model

Exposure to hepatitis C virus (HCV) is associated with a high prevalence of persistent viral infection and the development of chronic liver disease and hepatocellular carcinoma. Recovery from acute infection may depend upon the generation of broad-based cellular immune responses to viral structural and nonstructural proteins. We used the DNA-based immunization approach in BALB/c mice to determine whether the HCV nonstructural proteins NS3, NS4, and NS5 will induce Ab responses, CD4+ Th cell proliferation, and cytokine release in response to stimulation by recombinant proteins as well as generate CD8+ CTL activity both in vitro and in vivo. We found that the nonstructural proteins were particularly good immunogens and produced cellular immune responses when administered as a DNA construct. Indeed, a tumor model was established following inoculation of syngenic SP2/0 cells stably transfected with NS5. We observed protection against tumor formation and growth only in mice immunized with the NS5-encoding DNA construct, establishing the generation of significant CTL activity in vivo by this technique. The results indicate that genetic immunization may define the cellular immune response of the host to HCV nonstructural proteins and is a promising approach for vaccine development.     

Development and evaluation of an ELISA using recombinant fusion protein to detect the presence of host antibody to equine arteritis virus

Over the past 12 years, the poxvirus vector technology has provided scientists with valuable reagents to achieve high-level expression of proteins, to address questions of structure-function relationship of specific polypeptides, to investigate the immunobiology of specific pathogens, and to develop recombinant vaccine candidates. It is this last role that has drawn enthusiasm from the medical community because of the potential this technology has to provide novel approaches for addressing urgent needs in human and veterinary medicine. From one perspective, the safety issues surrounding the use of vaccinia-based vaccine candidates have been addressed with the development of the NYVAC and ALVAC vectors. Evaluation of these novel poxvirus vectors are in progress to determine their potential impact on cancer and infectious disease.     

Characterization of the humoral immune response to Klebsiella species in inflammatory bowel disease and ankylosing spondylitis

This study was carried out to characterize the antibody class response by ELISA to seven Klebsiella pneumoniae serotypes (K2, K3, K17, K21, K26, K36, K50) in five different groups, 40 HLA-B27-positive ankylosing spondylitis (AS) patients, 46 patients with Crohn's disease (CD), 38 patients with ulcerative colitis (UC), 50 patients with active anti-endomysial antibody-positive coeliac disease and 40 healthy controls, using whole bacteria and capsular polysaccharide. IgG antibody levels were significantly elevated in AS patients to K17, K36, K50; IgA to K2, K3, K21, K26, K36 and K50; and IgM to serotype K21 when compared to normal controls. Furthermore, IgG antibody levels were significantly elevated in CD patients to K2, K17, K21, K26, K36 and K50; IgA to K2, K3, K21, K26, K36 and K50; and IgM to K2, K3, K17, K21 and K50. Increased IgG antibody levels in the UC group were limited only to K17, K36 and K50. No antibody class was increased to any of the K. pneumoniae serotypes in the coeliac disease group. The immune responses in AS patients also involve Klebsiella bacteria having capsular serotypes other than K26, K36 and K50. The similarity in the immune responses between CD and AS groups suggests that many AS patients may have occult bowel inflammation.     

Cross-talk between gammadelta T lymphocytes and immune cells in humoral response

The role of gamma delta T cells in immunoregulation is largely unknown. In the current study we noted that gamma delta T cells play a positive role in the humoral response. These positively acting gamma delta T cells are required for the successful adoptive cell transfer of the humoral response, as well as for in vitro generation of plaque-forming cells (PFC). The presented results show that gammadelta T cells cause an increase in interleukin-10 (IL-10) production, which partly elucidates the mechanism of action of these cells. However, experiments with cell culture inserts strongly suggest that direct cell-cell contact between immune and gamma delta H-2-compatible regulatory T cells is critical to the exertion of the positive immunoregulatory function of gamma delta cells. The mechanism of cross-talk between these two cell populations is still not clear but we regard as most likely that the positively acting gamma delta T cells may interact with a complex of heat-shock protein-non-polymorphic MHC (IB) on the surface of T helper type 2 and/or B cells. This could provide, by direct cell-cell contact, the cognate recognition between gamma delta T-cell receptors and heat-shock protein-MHC that leads to positive internal signalling in the immune cells.     

Serologic and molecular characterization of an abortigenic strain of equine arteritis virus isolated from infective frozen semen and an aborted equine fetus

A virus isolated from an aborted equine fetus was determined to be antigenically distinct from several other strains of equine arteritis virus (EAV) by use of a neutralization assay with a large panel of neutralizing monoclonal antibodies. The virus was readily neutralized by polyclonal equine anti-EAV serum. Comparative nucleotide and amino acid sequence analyses indicated that the virus (WA97) isolated from the aborted fetus was virtually identical to a virus (S1971) isolated from imported semen used to inseminate another mare on the farm. Phylogenetic analysis indicated that the WA97/S1971 virus was more related to European than to North American strains of EAV. These sensitive molecular procedures may be useful for epidemiologic investigations of EAV infections. Screening and certification of stallions and frozen equine semen would prevent dissemination of pathogenic strains of EAV.