Samuel J. Pirruccello

X-linked lymphoproliferative disease: twenty-five years after the discovery.

The X-linked lymphoproliferative disease (XLP), one of six described X-linked immunodeficiencies, stems from a mutation at Xq25 which renders males impotent to mount an effective immune response to the ubiquitous EBV. Purtilo, who first observed this disease in 1969, established a Registry in 1980 to serve as a worldwide resource for the diagnosis, treatment, and research of this condition. Since Purtilos death in late 1992, the Registry and research unit have not only continued to function as a worldwide consultative service, but have contributed the following. First, the number of affected boys has continued to grow; some 272 among 80 kindreds have been identified. Second, some boys (10%) who inherit the mutated XLP gene are immunologically abnormal even before evidence of EBV exposure. Third, the search for the XLP gene has been narrowed to a small region on Xq25. Its identification is near at hand; once cloned, this gene may well illustrate how the body orchestrates the complex immune response to EBV. Therein lies the justification for the quest for this gene, not only for the benefit of the few surviving boys and those to be born to female carriers, but also for defining its role in defending the body against a ubiquitous DNA virus.

Toward Testing the Hypothesis that Group B Coxsackieviruses (CVB) Trigger Insulin-Dependent Diabetes: Inoculating Nonobese Diabetic Mice with CVB Markedly Lowers Diabetes Incidence

ABSTRACT Insulin-dependent (type 1) diabetes mellitus (T1D) onset is mediated by individual human genetics as well as undefined environmental influences such as viral infections. The group B coxsackieviruses (CVB) are commonly named as putative T1D-inducing agents. We studied CVB replication in nonobese diabetic (NOD) mice to assess how infection by diverse CVB strains affected T1D incidence in a model of human T1D. Inoculation of 4- or 8-week-old NOD mice with any of nine different CVB strains significantly reduced the incidence of T1D by 2- to 10-fold over a 10-month period relative to T1D incidences in mock-infected control mice. Greater protection was conferred by more-pathogenic CVB strains relative to less-virulent or avirulent strains. Two CVB3 strains were employed to further explore the relationship of CVB virulence phenotypes to T1D onset and incidence: a pathogenic strain (CVB3/M) and a nonvirulent strain (CVB3/GA). CVB3/M replicated to four- to fivefold-higher titers than CVB3/GA in the pancreas and induced widespread pancreatitis, whereas CVB3/GA induced no pancreatitis. Apoptotic nuclei were detected by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay in CVB3/M-infected pancreata but not in CVB3/GA-infected pancreata. In situ hybridization detected CVB3 RNA in acinar tissue but not in pancreatic islets. Although islets demonstrated inflammatory infiltrates in CVB3-protected mice, insulin remained detectable by immunohistochemistry in these islets but not in those from diabetic mice. Enzyme-linked immunosorbent assay-based examination of murine sera for immunoglobulin G1 (IgG1) and IgG2a immunoreactivity against diabetic autoantigens insulin and HSP60 revealed no statistically significant relationship between CVB3-protected mice or diabetic mice and specific autoimmunity. However, when pooled sera from CVB3/M-protected mice were used to probe a Western blot of pancreatic proteins, numerous proteins were detected, whereas only one band was detected by sera from CVB3/GA-protected mice. No proteins were detected by sera from diabetic or normal mice. Cumulatively, these data do not support the hypothesis that CVB are causative agents of T1D. To the contrary, CVB infections provide significant protection from T1D onset in NOD mice. Possible mechanisms by which this virus-induced protection may occur are discussed.

Human Immunodeficiency Virus Type 1 Pathobiology Studied in Humanized BALB/c-Rag2−/−γc−/− Mice

ABSTRACT The specificity of human immunodeficiency virus type 1 (HIV-1) for human cells precludes virus infection in most mammalian species and limits the utility of small animal models for studies of disease pathogenesis, therapy, and vaccine development. One way to overcome this limitation is by human cell xenotransplantation in immune-deficient mice. However, this has proved inadequate, as engraftment of human immune cells is limited (both functionally and quantitatively) following transplantation of mature human lymphocytes or fetal thymus/liver. To this end, a human immune system was generated from umbilical cord blood-derived CD34+ hematopoietic stem cells in BALB/c-Rag2−/−γc−/− mice. Intrapartum busulfan administration followed by irradiation of newborn pups resulted in uniform engraftment characterized by human T-cell development in thymus, B-cell maturation in bone marrow, lymph node development, immunoglobulin M (IgM)/IgG production, and humoral immune responses following ActHIB vaccination. Infection of reconstituted mice by CCR5-coreceptor utilizing HIV-1ADA and subtype C 1157 viral strains elicited productive viral replication and lymphadenopathy in a dose-dependent fashion. We conclude that humanized BALB/c-Rag2−/−γc−/− mice represent a unique and valuable resource for HIV-1 pathobiology studies.

Lymphocyte reconstitution after allogeneic blood stem cell transplantation for hematologic malignancies

Forty-one patients were studied at set times after allogeneic blood stem cell transplantation (alloBSCT) for recovery of lymphocyte numbers and function. Cells were mobilized with G-CSF from HLA-matched related donors and cryopreserved. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine and methotrexate; G-CSF was administered post-transplant. Median time to absolute lymphocyte count (ALC) ⩾500/μl was 17 days vs 41 and 49 days in historical alloBMT patients with G-CSF (n = 23) or no cytokine (n = 29) post-transplant, respectively (P < 0.0001). CD4/CD8+ ratio was 1.9 on day 28 after alloBSCT, then gradually declined to 0.8 at 1 year due to more rapid CD8+ cell recovery. Mean phytohemagglutinin-induced T cell responses were lower than normal on day +28 (P < 0.05), then tended to recover towards normal values. Natural-killer cytotoxicity remained low from day +28 to 1 year post-alloBSCT, but considerable lymphokine-activated killer cytotoxicity was induced from cells already obtained on day +28. Faster lymphocyte recovery correlated with better survival in alloBSCT patients (median follow-up 287 days, P = 0.002), ALC recovery was not affected by acute GVHD, CMV infections or doses of infused cells. ALC recovery did not correlate with survival in either historical alloBMT group. These data suggest that after alloBSCT lymphocyte reconstitution is faster than after alloBMT, and that quicker lymphocyte recovery predicts better survival in the alloBSCT setting.

Group B coxsackievirus myocarditis and pancreatitis: Connection between viral virulence phenotypes in mice

The group B coxsackieviruses (CVB) induce experimental pancreatitis and myocarditis in mice and are established agents of human myocarditis, especially in children. We tested the hypothesis that the development of CVB‐induced myocarditis is linked to CVB‐induced pancreatitis by studying the replication of different CVB strains in mice. Eight of nine genotypically different type 3 CVB (CVB3) strains induced acute pancreatitis in mice; of these, three viruses also induced acute myocarditis. One CVB3 strain was avirulent for both organs. Myocarditis was not observed in the absence of pancreatitis. The results obtained by inoculation of mice with strains of other CVB serotypes were consistent with these data. Infectious virus titers were measured in serum, pancreas, and heart as a function of time after inoculation of mice with three CVB3 strains. Each strain was representative of one of the three viral virulence phenotypes: avirulent, pancreovirulent only, and cardiovirulent. All strains replicated well and persisted in the pancreas through 8 days post‐inoculation, but the cardiovirulent CVB3 strain tended to replicate to higher titer earlier and persist longer in sera, pancreatic, and cardiac tissues than the noncardiovirulent strains. Replication of the CVB3 strains were studied in two human pancreatic tumor lines and in primary human endothelial cell cultures derived from cardiac artery. Cardiovirulent strains, both individually and as a group, tended to replicate to titers as high as, or higher than, noncardiovirulent strains did in cell culture. The data are consistent with the possibility of an etiologic link between CVB‐induced pancreatic and heart disease. J. Med. Virol. 62:70–81, 2000.

Immunovirological studies of fatal infectious mononucleosis in a patient with X-linked lymphoproliferative syndrome treated with intravenous immunoglobulin and interferon-α

We have studied a 19-year-old male with X-linked lymphoproliferative syndrome (XLP) and infectious mononucleosis (IM) who was treated with high-dose immunoglobulin (500 mg/kg/day) and recombinant interferon (IFN)-alpha (2 x 10(6) IU/m2/day). Fulminant hepatitis was delayed; however, virus-associated hemophagocytic syndrome, cholestatic jaundice, and renal failure occurred terminally. Initially, nonspecific natural killer (NK) cell activity against K562 cells was normal but it gradually decreased. Although reactive T cells were markedly increased in his blood during the acute phase, spontaneous EBV-positive cell lines were easily established. Additionally, his mononuclear cells produced IFN-gamma but not IFN-alpha prior to treatment. Based on results of in vitro studies, we conclude that both IFN-alpha and IFN-gamma production are likely necessary for inhibiting EBV immortalization in vitro. Both IFN-alpha and -gamma were produced in cultures of B95-8 EBV-infected mononuclear cells from EBV-seropositive healthy individuals. These results suggest that defective EBV-specific cytotoxic T cell activity accompanied with defective or discordant IFN-alpha and -gamma production permitted the development of fatal IM in this patient. Combined treatment with immunoglobulin and IFN-alpha appeared to be partially effective during the early stage of this disease.

Selective Cytotoxicity to Human Leukemic Myeloblasts Produced by Oligodeoxyribonucleotide Phosphorothioates Complementary to p53 Nucleotide Sequences

Cells were treated in vitro with oligodeoxyribonucleotide phosphorothioates (ODNs) complementary to sites common to both wild-type and mutant p53 nucleotide sequences. Acute myelogenous leukemia (AML) blasts from peripheral blood were exposed to four different p53 ODNs and showed anti-leukemic effects in suspension culture. This effect continued after removal of the ODN from the medium. Blocking of self-renewal of the leukemic blast stem cells in secondary plating of cells from cloning assays by two of the p53 ODNs was also observed. Control ODNs had no effect on leukemic blasts. Treatment of normal bone marrow cells with the four p53 ODNs did not influence their growth, nor was there any effect by the p53 ODNs on the leukemic cell-line, HL60, that does not express p53. These data suggest that p53 ODNs are selectively toxic to primary myelogenous blasts and may be therapeutically useful in AML.

Myeloblast Phenotypic Changes in Myelodysplasia

We used a new method of data presentation and analysis, termed antigen mapping, to characterize recurring myeloblast phenotypic abnormalities in a series of 28 cases of myelodysplastic syndrome (MDS), including refractory anemia with ringed sideroblasts (RARS), refractory anemia with multilineage dysplasia (RCMLD), and refractory anemia with excess blasts (RAEB). Abnormal patterns of CD34 and CD117 expression were present in 50% of RARS, 68% of RCMLD, and 100% of RAEB cases. The presence of decreased myeloblast CD45 density, increased CD13 and CD34 density, and increased expression of CD11c and CD4dim were MDS grade-related. There was a direct relationship between the number of myeloblast phenotypic abnormalities (phenotypic score) and MDS grade. The myeloblast phenotypic scores also were correlated highly with International Prognostic Scoring System scores and risk categories. We found the antigen mapping technique to be an efficient data presentation and analysis method for the detection of MDS-associated abnormalities of antigen distribution and density.

Age-related changes in naive and memory CD4+ T cells in healthy human children

Subsets of CD4+ T cells originally identified functionally as suppressor-inducer and helper-inducer populations have recently been reinterpreted as naive and memory maturational states. The subsets can be identified by the surface expression of CD45R and CDw29, respectively. Using two-color flow cytometric analysis, we measured these CD4+ T cell subsets in two samples of cord blood and in 26 healthy children between the ages of 1 and 19 years. As has been reported by others, we observed that the majority of CD4+ T cells in cord blood consist predominantly of the CD45R+ subset. With aging we could demonstrate a gradual acquisition of CDw29+, CD4+ T cells and a concomitant gradual decrease in the percentage of CD45R+, CD4+ T cells. These age-related changes are consistent with the concept of naive (CD45R+) and memory (CDw29+) subsets. Further, because of the dynamic changes, their utilization as prognostic indicators in immunologic disease states cannot be applied to children in the same manner as adults.

Clinical and immunologic studies in reticular erythematous mucinosis and Jessner's lymphocytic infiltrate of skin.

BACKGROUND Little is understood about reticular erythematous mucinosis and Jessners lymphocytic infiltrate of skin. OBJECTIVE Our purpose was to define reticular erythematous mucinosis and Jessners lymphocytic infiltrate of skin further with focus on immunologic studies. METHODS In patients with reticular erythematous mucinosis and Jessners lymphocytic infiltrate of skin, we measured circulating immune complexes before, during, and after therapy. We examined natural killer cells in a functional assay; we performed direct immunofluorescence and T- and B-cell marker studies in skin biopsy specimens. RESULTS The infiltrate in reticular erythematous mucinosis is composed of helper T cells. Circulating immune complexes are increased in both reticular erythematous mucinosis and Jessners lymphocytic infiltrate of skin and decrease with hydroxychloroquine therapy and clinical clearing. Natural killer cell function is decreased in reticular erythematous mucinosis and Jessners lymphocytic infiltrate of skin. CONCLUSION Changes in circulating immune complex titers accompanying therapy with hydroxychloroquine and clinical clearing, with recurrence of the condition and increase in circulating immune complexes on discontinuation of treatment, point to a possible relation between these events.