Clinical utility of viral load measurements in individuals with chronic hepatitis C infection on antiviral therapy.

SUMMARY: Both absolute viral load and log decline in viral load from baseline were found clinically useful in predicting sustained virological response and lack of sustained virological response (non-sustained virological response, NSVR) to treatment. We assessed the clinical utility of hepatitis C virus (HCV) RNA quantitation and changes in viral load using the VERSANT HCV RNA 3.0 Assay (bDNA) in 351 HCV-infected individuals treated with interferon plus ribavirin. We show that viral load decision thresholds provided negative predictive values (NPVs) of >95% at week 4 using a 100 000 IU/mL cut-off and at weeks 8 and 12 using 10 000 IU/mL cut-offs. A 2-log decline from baseline provided NPVs >95% at weeks 8 and 12. Combinations of absolute viral loads and changes in viral load from baseline did not enhance the performance of the decision rules for predicting NSVR. The positive predictive values (PPVs) at weeks 8 and 12 were 59.1 and 67.3%. This study highlights the critical importance of viral quantitation in gauging therapeutic response in patients with chronic HCV infection on antiviral therapy. Early changes in viral load, measured as absolute viral loads or change in viral load from baseline, are highly predictive of NSVR at 8 and 12 weeks. PPVs are modest but these data may provide encouragement to patients who are in the early phases of treatment when side effects are frequent. Additionally, we demonstrated the need for cautious interpretation of stopping rules when the values are at or near the decision thresholds.

Transcription-mediated amplification is more sensitive than conventional PCR-based assays for detecting residual serum HCV RNA at end of treatment.

OBJECTIVE: In patients chronically infected with hepatitis C virus (HCV) undergoing antiviral therapy, sustained virologic response is suggested by viral clearance by end of treatment (EOT). Viral clearance is defined by nondetection of serum HCV RNA, usually by qualitative PCR-based assays with limits of detection ranging from 100 to 1000 copies/ml. However, some individuals relapse after achieving apparent viral clearance by EOT. These individuals may have low levels of viremia not detected by current PCR methods. The aim of this retrospective study was to determine whether the Bayer HCV RNA Qualitative Assay, which employs Transcription Mediated Amplification (TMA) and detects 50 HCV RNA copies/ml, could detect residual serum HCV RNA in patients who achieved apparent viral clearance by EOT and subsequently relapsed. METHODS: Samples were obtained at EOT (wk 24 or 48) and follow-up (wk 24-26 posttreatment) from 97 patients treated for HCV (78 relapsing patients, 19 sustained responders). All samples in which HCV RNA was not detected by PCR were tested in a blinded manner for HCV RNA by the TMA-based assay. RESULTS: HCV RNA was detected by the TMA-based assay in 27 (34.6%) EOT and 76 (97.4%) follow-up samples from relapsing patients, but not in any of the EOT or follow-up samples from sustained responders. CONCLUSION: Residual serum HCV RNA was detected by the TMA-based assay in EOT samples from 34.6% of patients that had achieved apparent viral clearance by PCR. The detection of HCV RNA by the TMA-based assay could help redefine EOT response and assist in the antiviral management of HCV infection.

Assessment of hepatitis B virus DNA and hepatitis C virus RNA in the common bedbug (Cimex lectularius L.) and kissing bug (Rodnius prolixus).

OBJECTIVE: Historical clinical studies suggest the potential for insect-borne transmission of human hepatitis viruses. Studies of hepatitis B virus (HBV) persistence in insects were performed before the advent of molecular techniques, and studies to assess possible insect-borne transmission of hepatitis viruses have not yet been performed. The aim of this study was to determine, using molecular techniques, whether HBV and hepatitis C virus (HCV) persist in and are excreted in the feces of the bedbug Cimex lectularius L. and kissing bug Rodnius prolixus after an infectious meal. METHODS: Blood-feeding insects from the insect order Hemiptera (Cimex lectularius L. and Rhodnius prolixus) were fed on blood from infected patients with high titers of HBV, HCV, and control uninfected patients. Insects and insect excrement were collected at weekly intervals and tested for HBV DNA and HCV RNA using the polymerase chain reaction. RESULTS: HBV DNA was detected in bedbugs and excrement up to 6 wk after feeding on an infectious meal. HBV DNA was also detected in most kissing bugs and excrement up to 2 wk after feeding. HCV RNA was not detected in bedbugs at any time after feeding. CONCLUSIONS: We did not detect HCV RNA in bedbugs after feeding on an infectious meal. Our data provide molecular evidence to suggest that HBV may persist in Hemiptera. Additional studies are ongoing to determine whether this viral persistence is capable of infection.

Immunological responses in patients who have spontaneously eradicated hepatitis C virus infection.

We examined the proliferative responses of peripheral blood mononuclear cells obtained from 60 untreated patients who were seropositive by enzyme immunoassay, but negative for hepatitis C virus (HCV) RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). We used second- and third-generation recombinant immunoblot assay (RIBA) for further serological characterization. In vitro HCV-specific proliferative responses of mononuclear cells were compared to those of both untreated chronic HCV patients and patients who showed sustained virological response to interferon-alpha monotherapy, in order to assess the relative contribution of the immune response to the eradication of HCV. We found that frequency of responses to nonstructural proteins showed statistically significant differences, which were attributable to vigorous, polyspecific responses by cells from the RIBA-positive patients. In this group, core-specific proliferation was significantly associated with intravenous drug use as route of acquisition. Both other patient groups and the RIBA-indeterminate patients showed indistinguishable frequencies of proliferative responses. No association was detected between residual humoral responses, as determined from the RIBA results, and elapsed time since infection. The frequency of antibodies to NS5 differs between spontaneous cure and chronically infected patients. Cell-mediated and humoral immunity appear to be maintained in this population of patients.

Regulation of cytokine gene expression in experimental autoimmune encephalomyelitis.

We previously reported that recovery of Lewis rats from experimental autoimmune encephalomyelitis (EAE) is associated with the appearance of suppressor T cells (Ts). These Ts secrete TGF-beta which down-regulates the production of inflammatory cytokines by the effector T cells that mediate this disease. In the present study, we immunized Lewis rats with myelin basic protein (MBP)+CFA, and evaluated purified T cells and MBP-activated spleen cells (SpC) during the paralytic phase (day 12) and after recovery (days 30-33) for TGF-beta and interferon (IFN)-gamma mRNA. We used reverse transcriptase-polymerase chain reaction (RT-PCR), quantitated on the basis of beta-actin mRNA. Abundant IFN-gamma mRNA was present in MBP-activated SpC obtained on day 12. In contrast, only trace IFN-gamma mRNA was detected in day 30 activated SpC, and no IFN-gamma mRNA was present in purified, nonactivated T cells obtained at either time. The level of IFN-gamma mRNA correlated with secretion of IFN-gamma as determined by ELISA on SpC culture supernatants, and with severity of adoptively transferred EAE by the activated SpC. Thus, it appears that IFN-gamma mRNA is both transcribed and translated in response to antigen activation, resulting in secretion of IFN-gamma by the disease-inducing Te. In contrast, when we used RT-PCR to investigate the expression of TGF-beta mRNA, we found the transcript present in isolated T cells and MBP-activated SpC obtained from rats at both days 12 and 30. The presence of TGF-beta mRNA at time points corresponding to both clinical EAE and recovery suggests post-transcriptional regulation of the production of this immunoregulatory cytokine.

Regional differences in lymphocyte function following resuscitated hemorrhagic shock.

Although it is well known that hemorrhagic shock causes immunosuppression, there have been few attempts to define these changes in the various immune compartments. Accordingly, male rats were bled into severe hemorrhagic shock for 60 minutes (mean arterial pressure 35 +/- 5 mm Hg). Twenty-four hours following resuscitation, splenic, mesenteric, and peripheral lymphocytes were harvested for cell population analysis and mitogen stimulation assays. Cell marker analysis revealed no changes in B-cell or T-cell subpopulations in any immune compartment after shock. The splenic and peripheral lymphocytes showed marked depression of mitogen-induced stimulation after shock. In contrast, mesenteric lymphocyte responses to both T-cell and B-cell mitogens were not depressed after shock. Regional variability in mitogen responses after shock occur without change in B-cell or T-cell subpopulations in any immune compartment tested. The mechanism or mechanisms involved warrant further investigation.

Adenoma-derived antibody, Adnab-9 recognizes a membrane-bound glycoprotein in colonic tissue and effluent material from patients with colorectal neoplasia.

The binding in pre-colonoscopic effluent of Adnab-9, a monoclonal antibody raised against colonic adenomas, was evaluated for specificity in the diagnosis of colorectal cancer. A heterogeneous group of 58 patients was evaluated by ELISA. Effluent samples and tissue extracts were subjected to Western blotting or ELISA to confirm specificity. Immunohistochemistry was performed on the cancer tissue sections. The proportion of positive effluent binding was higher in the cancer when compared to the normal group (P = 0.036). A dominant 87 M(r) band was found in adenoma extracts and some effluent samples. Adnab-9 binding in effluent samples predominated in membrane-bound fractions. Immunohistochemistry showed no specific staining in the cancer cells. The antigen recognised is a glycoprotein shown by effects of N-glycanase digestion and not cross-reactive with carcinoembryonic antigen. Non-gastro-intestinal tissue extracts did not bind Adnab-9. The major 87 M(r) adenoma-derived antigen may be found in effluent material, particularly in the membrane-bound fraction.

Carrier-specific T cells sufficient for the expression of multiple isotypes in B cell cultures.

A modified splenic fragment assay was used to assess the role of antigen-specific helper T cells in B cell isotype expression. Limiting numbers of carrier-specific helper T cells from lines or clones were injected along with a source of B cells into lethally irradiated unprimed recipients. The incidence of lodging of the T cell lines in recipient spleens at 18 h was determined by autoradiography to be 1.5 to 4.3% of the injected cells. These T cells were necessary and sufficient for the generation of T-dependent B cell responses within splenic fragments cultured in vitro with specific antigen. A comparison of isotypic responses from splenic and Peyer's patch B cells generated with the same T cell population revealed that a high proportion of the response from Peyer's patch B cells consisted of IgA antibody exclusively (46-57%) while the percentage of such responses from splenic B cells was much lower (7-10%). Thus, the isotype pattern of the response reflected the B cell source. Experiments in which cloned hemocyanin-specific T cells provided help to T-depleted spleen cells within splenic fragments from athymic recipients indicated that a single specificity of helper T cell is both necessary and sufficient to support the generation of antibody responses consisting of multiple isotypes. Isotype-specific T cells do not appear to be required in this system.

Ontogeny of interleukin 2 responsive cells in the fetal thymus.

The ontogeny of IL 2-responsive cells in the thymus of CBA/J mice was examined in neonatal animals and in fetuses at 14, 16, and 18 to 20 days gestation. The thymocytes were tested for responsiveness to 2 micrograms/ml Con A, TCGF, IL 2, and co-stimulation by Con A plus TCGF or IL 2. These responses were compared with those of thymocytes of 6- to 8-wk-old CBA/J. Thymocytes (1 X 10(5)) were cultured, and the reaction was measured at maximum response (96 hr). Neonatal animals gave an unusually high response to TCGF or partially purified IL 2 alone, approximately five times greater than the adult. A low but significantly enhanced proliferation, stimulated by partially purified IL 2 alone, was observed with 14-day fetal thymocytes, even though cultures of these cells in medium alone had higher background proliferation than any other age tested. In the co-stimulator reaction, proliferation significantly above background was measured at 16 days of gestation with Con A plus TCGF. The magnitude of the co-stimulator reaction increased with age, especially between the 16th and 18th day of gestation and immediately after birth.

The cellular stimuli for the rejection of established islet allografts.

Previous work from our laboratory has indicated that the transplantation of pancreatic islets is a feasible approach to the problem of diabetes. A major obstacle to transplantation is presented by passenger leucocytes, which contaminate the preparations and can lead to the prompt rejection of fresh islets. We have extended our previous studies on the rejection of islet allografts by challenging transplanted animals with enriched lymphoid cell populations prepared from animals both syngeneic to the transplanted islets and third party. Rapid and complete rejection was observed when the challenge peritoneal exudate cell population was syngeneic with the transplanted islets; rejection was determined by both functional and histologic criteria. Peritoneal exudate cells from a third-party rat strain induced delayed and variable effects upon the function of the transplant. In contrast, splenic T-cells were capable of inducing rejection, regardless of the strain of origin, though the time course of T-cell-induced rejection was slower than that observed by syngeneic peritoneal exudate cells. Finally, splenic B-cells completely failed to induce rejection. Our data indicate that at least two mechanisms exist by which the rejection of islet allografts may be triggered. The first is a haplotype-specific mechanism initiated by a cell type present at high frequency in peritoneal exudate cells; these are probably macrophages. The second mechanism is initiated by immunocompetent T-cells; this mechanism shows no haplotype specificity. We suggest that both macrophages and T-cells must be considered when devising protocols for the removal of passenger leucocytes from allografts.

Regulation of murine B cells through surface immunoglobulin. I. Monoclonal anti-delta antibody that induces allotype-specific proliferation.

We describe the identification of a monoclonal antibody that recognizes a determinant on the delta chain of mice of the Iga, allotype groups. The monoclonal Ig in soluble form induces allotype-specific proliferation by splenic B lymphocytes from normal animals of these haplotypes. Spleen cells from mice bearing the X-linked defect of CBA/N mice fail to respond, although they bear the determinant. Proliferation is independent of T lymphocytes. The data indicate a direct triggering function for sIgD.

Immunoglobulin subclass-specific immunodeficiency in mice with an X-linked B-lymphocyte defect.

CBA/N mice express an X-linked deficiency in their antibody response to many bacterial carbohydrates; we have shown recently that these antigens normally elicit antibody responses predominantly of the IgM and IgG3 isotypes. Here we demonstrate that mice, with the CBA/N phenotype have perferential deficiencies of IgM and IgG3 immunoglobulin expression, both when measured in serum and in cells secreting these isotypes, and that this deficiency is only partially corrected by polyclonal activation of B cells. This suggests that CBA/N mice may lack a subpopulation of B cells that contain most of the IgG3 precursors.

Activation of mouse lymphocytes by anti-immunoglobulin. I. Parameters of the proliferative response.

Spleen cell cultures from young adult mice of a variety of strains were stimulated to incorporate tritiated thymidine ([3H]TdR) by a goat anti-mouse IgM antiserum and by purified anti-mu antibodies prepared from this serum. This stimulation was shown to depend upon the anti-mu activity of the antiserum. In addition, ultracentrifuged anti-mu and F(ab')2 fragments of anti-mu were shown to be stimulatory. The anti-mu preparation lacked detectable endotoxin contamination and was also shown to stimulate response by two strains (C57BL/10ScCr and C3H/HeJ) which are unresponsive to the mitogenic effects of endotoxin, while it failed to stimulate a response by cells from a mouse strain (CBA/N) which responds to endotoxin. In addition purified goat anti-mouse gamma, kappa antibodies and rabbit anti-mouse kappa-antib odies stimulated uptake of [3H]TdR by mouse spleen cells, although to a lesser degree than the anti-mu preparation. The cell density, culture requirements, and kinetics of the response are presented.

The role of surface IgD in the response to thymic-independent antigens.

An alloanti-delta antibody was prepared by immunizing C57BL/Ka mice with BALB/c spleen cells. Its specificity for delta-chain was demonstrated by immunoprecipitation and SDS-PAGE of 125I-labeled membrane proteins from BC8 spleen cells. BC8 mice possess C57BL/Ka "background" genes and BALB/c IgH genes. The anti-delta reagent without complement inhibited the primary in vitro anti-TNP antibody response to TNP-AECM-Ficoll by BC8 spleen cells, although it had no effect of the anti-TNP response of congenic C57BL/Ka spleen cells, which lack the delta-allotype identified by this antibody. On the other hand, the anti-delta antibody had no effect on the anti-TNP response of BC8 spleen cells to TNP-BA, except at limiting antigen concentrations. Both TNP-AECM-Ficoll and TNP-BA are T-I antigens, but they differ in that TNP-AECM-Ficoll fails to stimulate in vitro responses by immunologically defective CBA/N and neonatal spleen cells whereas TNP-BA can cause responses from both these animals. These results suggest that the IgD receptor is critical to T-I antibody responses initiated by TNP-AECM-Ficoll but that it is not required for T-I responses stimulated by TNP-BA.