Maria Paola Perrone

Multispecific T cell response and negative HCV RNA tests during acute HCV infection are early prognostic factors of spontaneous clearance

Background/Aims: Hepatitis C virus (HCV) infection results in a high frequency of chronic disease. The aim of this study was to identify early prognostic markers of disease resolution by performing a comprehensive analysis of viral and host factors during the natural course of acute HCV infection. Methods: The clinical course of acute hepatitis C was determined in 34 consecutive patients. Epidemiological and virological parameters, as well as cell mediated immunity (CMI) and distribution of human leukocyte antigens (HLA) alleles were analysed. Results: Ten out of 34 patients experienced self-limiting infection, with most resolving patients showing fast kinetics of viral clearance: at least one negative HCV RNA test during this phase predicted a favourable outcome. Among other clinical epidemiological parameters measured, the self-limiting course was significantly associated with higher median peak bilirubin levels at the onset of disease, and with the female sex, but only the latter parameter was independently associated after multivariate analysis. No significant differences between self-limiting or chronic course were observed for the distribution of DRB1 and DQB1 alleles. HCV specific T cell response was more frequently detected during acute HCV infection, than in patients with chronic HCV disease. A significantly broader T cell response was found in patients with self-limiting infection than in those with chronic evolving acute hepatitis C. Conclusion: The results suggest that host related factors, in particular sex and CMI, play a crucial role in the spontaneous clearance of this virus. Most importantly, a negative HCV RNA test and broad CMI within the first month after onset of the symptoms represent very efficacious predictors of viral clearance and could thus be used as criteria in selecting candidates for early antiviral treatment.

Monocyte-derived dendritic cells generated after a short-term culture with IFN-α and granulocyte-macrophage colony-stimulating factor stimulate a potent epstein-barr virus-specific CD8+ T cell response

Cellular immune responses are crucial for the control of EBV-associated lymphoproliferative diseases. To induce an anti-EBV cell-mediated immunity, we have used dendritic cells (DCs) generated by a 3-day culture of human CD14+ monocytes in the presence of GM-CSF and type I IFN (IFN-DCs) and pulsed with peptides corresponding to CTL EBV epitopes. The functional activity of IFN-DCs was compared with that of APCs differentiated by culturing monocytes for 3 days with GM-CSF and IL-4 and indicated as IL-4-DCs. Stimulation of PBLs from EBV-seropositive donors with EBV peptide-pulsed autologous IFN-DCs resulted in a stronger expansion of specific T lymphocytes producing IFN-γ with respect to stimulation with peptide-loaded IL-4-DCs, as assessed by ELISPOT assays. When purified CD8+ T cells were cocultured with EBV peptide-pulsed IFN-DCs or IL-4-DCs, significantly higher levels of specific cytotoxic activity were observed in CD8+ T cell cultures stimulated with IFN-DCs. Injection of peptide-pulsed IFN-DCs into SCID mice transplanted with autologous PBLs led to the recovery of a significantly greater number of EBV-specific human CD8+ T cells from the spleen and the peritoneal cavity with respect to that recovered from mice injected with peptide-pulsed IL-4-DCs. Moreover, a significant delay in lymphoma development was observed when peptide-pulsed IFN-DCs were injected into SCID mice reconstituted with PBMCs endowed with a high capability of lymphoma induction, whereas injection of unpulsed IFN-DCs was ineffective. Our results indicate that IFN-DCs efficiently promote in vitro and in vivo the expansion of CD8+ T lymphocytes acting as cytotoxic effectors against EBV-transformed cells.

Early impairment of hepatitis C virus specific T cell proliferation during acute infection leads to failure of viral clearance

Background and aims: Cellular mediated immunity (CMI) is thought to play a key role in resolution of primary hepatitis C virus (HCV) infection. However, CD4+ and CD8+ T cell responses are also generated during acute infection in individuals who become chronic, suggesting that they developed a defective CMI. The aim of this study was to verify if and when such immune dysfunction is established by measuring the breadth, magnitude, function, and duration of CMI in a large cohort of subjects during the natural course of acute HCV infection. Methods: CMI was comprehensively studied by prospective sampling of 31 HCV acutely infected subjects enrolled at the onset of infection and followed for a median period of one year. Results: Our results indicated that while at the onset of acute HCV infection a measurable CMI with effector function was detected in the majority of subjects, after approximately six months less than 10% of chronically infected individuals displayed significant CMI compared with 70% of subjects who cleared the virus. We showed that progressive disappearance of HCV specific T cells from the peripheral blood of chronic patients was due to an impaired ability to proliferate that could be rescued in vitro by concomitant exposure to interleukin 2 and the antigen. Conclusion: Our data provide evidence of strong and multispecific T cell responses with a sustained ability to proliferate in response to antigen stimulation as reliable pharmacodynamic measures of a protective CMI during acute infection, and suggest that early impairment of proliferation may contribute to loss of T cell response and chronic HCV persistence.

Positive selection of cytotoxic T lymphocyte escape variants during acute hepatitis C virus infection.

Cellular immune responses are induced during hepatitis C virus (HCV) infection and acute‐phase CD8+ T cells are supposed to play an important role in controlling viral replication. In chimpanzees, failure of CD8+ T cells to control HCV replication has been associated with acquisition of mutations in MHC class I‐restricted epitopes. In humans, although selection of escape mutations in an immunodominant CTL epitope has been recently described, the overall impact of immune escape during acute HCV infection is unclear. Here, by performing an in depth analysis of the relationship between early cellular immune responses and viral evolution in a chronically evolving HCV acutely infected individual, we demonstrate: (i) the presence of a potent and focused CD8+ T cell response against a novel epitope in the NS3 protein, (ii) the elimination of the quasi‐species harboring the original amino acid sequence within this epitope, and (iii) the selection for a virus population bearing amino acid changes at a single residue within the cytotoxic T cell epitope that strongly diminished T cell recognition. These results support the view that acute‐phase CD8+ T cell responses exert a biologically relevant pressure on HCV replication and that viruses escaping this host response could have a significant survival advantage.

Pre-transplant prognostic factors for patients with high-risk leukemia undergoing an unrelated cord blood transplantation

Summary:From July 1995 to December 2001, 42 patients with leukemia aged 1–42 years underwent cord blood transplant (CBT) from unrelated, ⩽2 antigen HLA mismatched donors. In all, 26 patients were in ⩽2nd complete remission and 16 in more advanced phase. Conditioning regimens, graft-versus-host disease (GVHD) prophylaxis and supportive policy were uniform for all patients. The cumulative incidence of engraftment was 90% (95% CI: 0.78–0.91). The cumulative incidence of III–IV grade acute- and chronic-GVHD was 9% (95% CI: 0.04–0.24) and 35% (95% CI: 0.21–0.60), respectively. The 4-year cumulative incidence of transplant-related mortality (TRM) and relapse was 28% (95% CI: 0.17–0.47) and 25% (95% CI: 0.14–0.45), respectively. The 4-year overall survival (OS), leukemia-free survival (LFS) and event-free survival (EFS) were 45% (95% CI: 0.27–0.63), 47% (95% CI: 0.30–0 .64) and 46% (95% CI: 0.30–0.62), respectively. In multivariate analysis, the most important factor affecting outcomes was the CFU-GM dose, associated with CMV serology (P=0.003 and 0.04, respectively) in influencing OS and with patient sex (P=0.008 and 0.03, respectively) in influencing LFS. Finally, CFU-GM dose was the only factor that affected EFS significantly (P=0.02). In conclusion, the infused cell dose expressed as in vitro progenitor cell growth is highly predictive of outcomes after an unrelated CBT and should be considered the main parameter in selecting cord blood units for transplant.

Histopathological and molecular features of persistent polyclonal B-cell lymphocytosis (PPBL) with progressive splenomegaly

Five cases of persistent polyclonal B‐cell lymphocytosis (PPBL) with progressive splenomegaly are reported; three were splenectomized. BCL2/IGH rearrangements were found in three cases; HLA‐DRB1*07 in all. Bone marrow (BM) trephines showed a moderate lymphoid infiltrate with intrasinusoidal distribution resembling a splenic marginal‐zone lymphoma. Splenic white pulp revealed an enlargement of the marginal‐zone area; red pulp was infiltrated by the same lymphocytes engulfing the sinuses. Splenic and BM B‐lymphocytes were CD79a+/CD20+/IgM+/IgD+/bcl‐2+/CD27+/DBA.44−/CD31− and polyclonal by immunophenotype/polymerase chain reaction. PPBL features an expansion of splenic marginal‐zone B‐lymphocytes, which infiltrate BM sinusoids and circulate in the blood with no evidence of clonality, even in cases with progressive splenomegaly.

Hemolytic Anemia and Thrombocytopenia Induced by Cyanidanol

Five patients who received cyanidanol for 4-36 months are presented. Three developed both hemolytic anemia and thrombocytopenia, while 2 had only thrombocytopenia. After suspending the drug the hematological values returned to normal in all of the patients. Drug-dependent platelet antibodies were detected in 4 of the 5 patients and cyanidanol-dependent red blood cell antibodies were present in 3. There are various mechanisms involved in the cyanidanol-induced immune cytopenias and, as in the present study, were sometimes simultaneously observed in the same patient.

A metastatic breast carcinoma presenting as autoimmune hemolytic anemia.

The authors describe the case of a 75-year-old female who was hospitalized for anemia of unknown origin. Physical examination revealed a swelling in the right mammary region, where a mastectomy scar was present from surgery for a breast carcinoma. On admission, laboratory tests disclosed anemia (Hb, 8.5 g/dl), with a reticulocyte count of 65,000/mm3 and slightly increased bilirubin. Immunohematologic study revealed the presence of a red cell autoantibody with anti-D specificity in the serum and in the eluate from the patients erythrocytes. A biopsy of the swelling was performed and histologic examination showed the presence of metastatic cells of breast carcinoma. The patient was given chemotherapy and radiotherapy. At this writing the anemia was absent, the Immunohematologic study was negative, the swelling was greatly reduced, and no other metastatic lesions of breast carcinoma were present.

Idarubicin intensified BUCY2 regimen in allogeneic unmanipulated transplant for high-risk hematological malignancies

Twenty-nine consecutive patients with high-risk hematological malignancy aged from 3 to 58 years underwent an unmanipulated graft from an HLA-identical sibling after an irradiation-free preparative regimen consisting of idarubicin (IDA), 21 mg/m2/day administered by continuous infusion on days −12 and −11, followed by busulphan (BU), 4 mg/kg/day orally from day −7 to −4, and cyclophosphamide (CY), 60 mg/kg/day intravenously on days −3 and −2 (IDA-BUCY2). Most clinically relevant extra-hematological regimen-related toxicities consisted of stomatitis observed in all subjects and hemorrhagic cystitis occurred in five cases (17%) within 100 days after transplant. Six patients (21%) developed a grade 2 acute graft-versus-host disease (GVHD) and three (10%) a grade 3 or 4; extensive chronic GVHD was assessed in nine of 22 (41%) evaluable patients. So far, 12 patients have died and 17 are alive, 16 of whom disease-free, 5–41 months after transplant (median, 15 months). The causes of death were related to GVHD in three patients, to sepsis in one and to disease recurrence in the remaining eight. At present, only one of nine relapsed patients is alive. For all patients the actuarial probability of survival (OS) at 1 and 2 years ± standard error (s.e.) was 63 ± 9% and 52 ± 10%, respectively. The actuarial probabilities of disease-free survival (DFS), relapse and transplant-related mortality (TRM) at both 1 and 2 years ± s.e. were 53 ± 9%, 35 ± 9% and 16 ± 7%, respectively. These results are encouraging but not substantially different from those obtained in 28 patients with malignancy in advanced phase transplanted after the standard BUCY2 regimen, who had an actuarial probability of OS, DFS, relapse and TRM projected at 10 years ± s.e. of 54 ± 10%, 57 ± 9%, 36 ± 9% and 11 ± 6%, respectively. Although the retrospective comparison between the two groups does not seem to show any advantage in the use of the IDA intensified regimen, only a prospective randomized trial could answer this question.

Impact of viral selected mutations on T cell mediated immunity in chronically evolving and self limiting acute HCV infection

The ability of HCV to mutate in response to cytotoxic T lymphocyte (CTL) pressure is increasingly recognized, but the influence of such a mechanism in viral persistence and final disease outcome has not been ascertained. In this study, we performed a detailed longitudinal analysis of cell mediated immunity and HCV evolution in two self limiting and two chronically evolving HCV acutely infected patients, one of whom transiently controlled viremia. Amino acid mutations in immunodominant regions of viruses were observed in all patients, although they conferred viral escape from CTL responses only in chronically infected individuals. Resurgence of viremia coincided with the replacement of the original virus quasispecies with mutant viruses that had escaped recognition by primary CD8(+) T cell responses and infection persisted in the presence of variant viruses which were less efficiently recognized by preexisting and de novo induced T cell responses.