Parvovirus B19 infection in patients with hemophilia

BACKGROUND: The long-term consequences of parvovirus B19 infection in transfusion recipients are not known, and thus the value of B19 screening of blood donors remains unresolved. Hemophiliacs, at risk for B19 through their chronic exposure to clotting factor concentrates, have frequent, close medical follow-up and thereby constitute an ideal group in which to study the hematologic sequelae of B19 infection. STUDY DESIGN AND METHODS: An enzyme-linked immunosorbent assay was used to detect B19 IgG and IgM and the polymerase chain reaction was used to detect B19 DNA in frozen, stored plasma samples, obtained between 1987 and 1994, from 136 subjects with hemophilia, including 71 who were human immunodeficiency virus (HIV)-positive and 65 who were HIV- negative. Then the results of the tests were compared with clinical hematological data and blood product usage data. RESULTS: B19 seroprevalence in the hemophilic cohort was 81.6 percent (111/136), including 74.6 percent (53/71) of HIV-positive and 89.2 percent (58/65) of HIV-negative hemophiliacs. It was not affected by age, type or severity of hemophilia, HIV status, CD4 number, or yearly blood product usage. Only 1 (0.7%) of the 136 samples was positive for B19 IgM and none was positive in polymerase chain reaction for B19 DNA. After adjusting for HIV status, there were no differences between B19- positive and B19-negative hemophiliacs in hematologic values, CD4 counts, or blood product use. CONCLUSION: Although B19 IgG seroprevalence in this hemophilic cohort is high and indicative of past B19 infection, there is no detectable B19 viral activity or any associated long-term clinical or hematologic sequelae.     

Polymorphic glycoprotein-1 on mouse platelets: possible role of Pgp-1 and LFA-1 in antibody-dependent platelet cytotoxicity involving complement

The presence of the Pgp-1 glycoprotein on mouse platelets is demonstrated by antibody-binding techniques, by immunoprecipitation, and by transblotting using the monoclonal antibody (MoAb) C71/26 against Pgp-1. C71/26 immunoprecipitates as a broad band of mol wt 87,000 to 100,000 as determined by radioiodination of the platelet cell surface and by the 3H-sodium borohydride labeling technique. Immunoblotting showed Pgp-1 expression on platelets to be quantitatively similar to its presence on macrophages and resolved platelet Pgp-1 into two bands of mol wt 87,000 and 97,000 whereas Pgp-1 on parasite-elicited peritoneal macrophages showed 82,000 and 87,000 mol wt species. Platelets and monocyte/macrophage cells from either peripheral blood or from the peritoneal cavity showed homogeneous binding of Pgp-1 antibody to greater than 97% of cells by flow cytometry. In contrast, lymphocytes from peripheral blood or from the spleen showed a heterogeneous binding pattern with 20% to 30% of cells being negative, and the majority weakly positive. In functional studies, MoAbs against CR1 and CR3 substantially inhibited platelet immune adherence, whereas C71/26 showed only marginal inhibitor. In contrast, C71/26 and other MoAbs against Pgp-1 inhibited platelet- dependent cytotoxicity of antibody-coated sheep erythrocytes in the presence of C5-deficient mouse plasma whereas M1/70 against CR3 showed no effect. In this assay, MoAbs against the alpha- and beta-subunits of leukocyte functional molecule LFA-1 also inhibited platelet cytotoxicity. These results show that the platelet cell surface moieties Pgp-1 and LFA-1 are involved in or closely associated with antibody-dependent cellular cytotoxicity by platelets.     

Human immunodeficiency virus-related conditions in children and adults with hemophilia: rates, relationship to CD4 counts, and predictive value

To further elucidate the natural history of human immunodeficiency virus (HIV) infection, we studied intermediate HIV-related conditions occurring before acquired immunodeficiency syndrome (AIDS) in a prospectively observed multicenter cohort of 738 HIV-infected persons with hemophilia. We analyzed the frequency in adults and children of common HIV-related conditions and the relative risk of AIDS after occurrence of these conditions, controlling for age at seroconversion and the percentage of CD4+ lymphocytes. Thrombocytopenia was the most frequently observed condition with cumulative incidences of 43% +/- 7% in adults and 27% +/- 6% in children and adolescents by 10 years after seroconversion. Oral candidiasis, fever, weight loss, and non-AIDS pneumonia were two to four times more common in adults than children, whereas herpes zoster risk was similar in the two age groups. HIV- related conditions were infrequent during the first 4 years of infection, particularly in children. With the exception of thrombocytopenia, mean CD4 counts were less than 350 cells/microL at the onset of the conditions. The relative hazard of AIDS after oral candidiasis was 18 in children and 3.8 in adults. Relative hazard in adults was also increased after persistent fever (10), weight loss (3.2), and non-AIDS pneumonia (2.2). Herpes zoster and thrombocytopenia were not significantly associated with AIDS in either age group. We conclude that intermediate HIV-related conditions occur more frequently in adults than in children with hemophilia. Persistent fever is the strongest predictor of AIDS in adults, whereas oral candidiasis is the strongest predictor in children. These findings should facilitate the design and conduct of clinical trials as well as the management of HIV- infected children and adults.     

The presence of hepatitis C virus (HCV) antibody in human immunodeficiency virus-positive hemophilic men undergoing HCV "seroreversion"

Hepatitis C virus (HCV) is a major cause of transfusion-induced chronic liver disease in hemophiliacs, with 70% to 90% being anti-HCV positive. Seroreversion or loss of antibody response to HCV has been observed in a small proportion of human immunodeficiency virus-positive [HIV(+)] anti-HCV(+) hemophilic men. Despite the seroreversion to an anti-HCV- negative state, such patients continue to show serum alanine aminotransferase (ALT) elevations and biopsy evidence of cirrhosis and/or chronic active hepatitis. To determine the cause for the loss of anti-HCV antibody, we compared first- and second-generation anti-HCV enzyme immunosorbent assay (EIA 1.0 and 2.0), second-generation recombinant immunoblot (RIBA 2.0), and HCV-RNA amplification using polymerase chain reaction (PCR) in 19 "seroreverters" before and after seroreversion. There was no difference between 19 seroreverters and 59 persistently anti-HCV-positive hemophiliacs in mean ALT (1.1 +/- 0.1 XUL v 2.0 +/- 0.2 XUL; chi 2 = 1.80, P > .05), in mean CD4 (188 +/- 36/microL v 232 +/- 28/microL; t = 0.965, P > .05), or in the rate of progression to acquired immunodeficiency syndrome (13 of 19 [68.4%] v 30 of 59 [50.9%]; chi 2 = .987, P > .05, respectively). Before seroreversion, all 19 seroreverters (100%) were positive for EIA 1.0 and 2.0 and PCR, and all but 2 of 19 (89.5%) were RIBA 2.0 positive, whereas, after seroreversion, none were positive for EIA 1.0, 15 of 19 (78.9%) were positive for EIA 2.0, 8 of 18 (44.4%) were positive for RIBA 2.0, and 18 of 19 (94.7%) were positive for PCR. There was a lower CD4 lymphocyte number after seroreversion in those who were RIBA 2.0 negative as compared with those who were RIBA 2.0 positive (32 +/- 10/microL v 171 +/- 52/microL; t = 2.638, P > .05). These results indicate that HIV(+) anti-HCV(+) hemophilic men who undergo "HCV seroreversion" are truly infectious and anti-HCV positive by second- generation tests. Anti-HCV detection in immunosuppressed hosts is significantly improved by second-generation EIA and RIBA assays.     

Erythropoietin concentration during the development and recovery from iron deficiency in the rat

The concentration of plasma erythropoietin was determined by radioimmunoassay during the progression of and subsequent recovery from iron-deficiency anemia in the rat. During the development of anemia, the plasma erythropoietin level rose as the hemoglobin (Hgb) concentration declined, reaching maximal levels when the Hgb was lowest. During the recovery from iron-deficiency anemia after institution of the control diet, the plasma erythropoietin concentration rapidly declined to baseline or below baseline levels even before the Hgb had completely returned to control values. This early fall in the erythropoietin level was associated with a sustained decrease in blood oxygen affinity (increase in P50). The rise in P50 was associated with an increase in the number of circulating reticulocytes in addition to and independently of an increase in the concentration of 2,3-diphosphoglycerate (DPG) in red cells. Therefore, reticulocytosis may play a part in the recovery from anemia, not only by replenishing the red cell pool but also by temporarily facilitating oxygen delivery to the tissues.     

Clinical spectrum of clonal proliferations of T-large granular lymphocytes: a T-cell clonopathy of undetermined significance?

We identified 68 patients with clonal T-large granular lymphocyte (T- LGL) proliferations who were seen at the Mayo Clinic between 1984 and 1992. Nineteen (28%) were asymptomatic at diagnosis, while the rest experienced fatigue (60%), B-symptoms (12%), and recurrent infections (15%). Associated comorbid conditions included rheumatoid arthritis (RA) in 26%. Severe anemia (hemoglobin [Hb] < 8g/dL) and neutopenia (absolute neutrophil count [ANC] < 500/microL) were seen in 19% and 40% of patients, respectively. Immunophenotypic studies showed CD3+, CD8+ phenotype in the majority (72%). Twenty-one patients (31%) have required no therapy, and remain relatively stable with a median follow- up period of 50 months. Treatment was required at either diagnosis (36 patients) or at subsequent follow-up (11 patients). Initial response rates were similar in patients treated with cyclophosphamide (CTX) with or without prednisone (69%), or prednisone alone (73%). Overall, 61 patients (90%) are alive with a median follow-up of 44 months. Actuarial median survival of this entire cohort is 161 months. The presence of anemia or symptoms does not appear to correlate with the tumor burden. In patients requiring therapy, a lower ANC and the presence of B-symptoms/infection were independently associated with a significantly lower probability of achieving a molecular or hematologic complete remission (H-CR). Intermittent immunosuppressive therapy is effective in achieving durable responses in a number of patients. T-LGL proliferations are associated with a favorable prognosis and response to therapy. However, significant heterogeneity exists in clinical presentation and associated comorbid conditions. These disorders should be included in the differential diagnosis of patients with unexplained cytopenias, particularly in the setting of RA and other autoimmune disorders. Analogous to the situation with monoclonal gammopathies, a term such as T-cell clonopathy of undetermined significance (TCUS) may be more appropriate to describe these patients.     

Toxicity induced by nanoparticles

Nanoparticle research is currently an area of intense scientific interest due to a wide variety of potential applications. Human beings have been exposed to airborne nanosized particles throughout their evolutionary stages, and such exposures have increased dramatically over the last century. Nanoparticle can modify the physicochemical properties of the material as well as create the opportunity for increased uptake and interaction with biological tissues through inhalation, ingestion, and injection. This combination of effects can generate adverse biological effects in living cells. Nanoparticles have proved toxic to human once in the blood stream, nanoparticles, spleen, bone marrow and nervous system can be transported around the body and be taken up by organs tissue and cell cultures, resulting in increased oxidative stress, inflammatory cytokine production and cell death. Unlike larger particles, nanoparticles may be taken up by cell mitochondria and the cell nucleus studies demonstrate the potential for nanoparticles to cause DNA mutation and induce major structural damage to mitochondria, even resulting in cell death. Size is therefore a key factor in determining the potential toxicity of a particle. How these nanoparticles behave inside the body is still a major question that needs to be resolved. There is a responsibility to test and optimize these new nanomaterials early during the development process to eliminate or ameliorate identified toxic characteristics.     

Expression of granulocyte colony-stimulating factor and granulocyte colony-stimulating factor receptor genes in partially overlapping monoclonal B-cell populations from chronic lymphocytic leukemia patients

B lymphocytes were purified from the peripheral blood of 30 B-cell chronic lymphocytic leukemia (B-CLL) patients and tested for the ability to produce granulocyte colony-stimulating factor (G-CSF) in vitro. Fifteen Staphylococcus aureus Cowan I (SAC)-stimulated, but not unstimulated, B-cell suspensions produced G-CSF in short-term cultures. Accordingly, G-CSF mRNA was detected only in SAC-stimulated B cells. Five CLL B-cell fractions that released G-CSF following exposure to SAC were also incubated with CD40 or anti-mu antibodies in the presence or absence of recombinant (r) interleukin-2 (IL-2) or IL-4. The 5 cell suspensions produced G-CSF only on culture with CD40 monoclonal antibody in combination with rIL-2 or rIL-4. CD5+ B lymphocytes, which represent the normal counterparts of most B-CLL proliferations, did not produce G-CSF under any of the above culture conditions. G-CSF produced by leukemic B lymphocytes was biologically active, because conditioned media of SAC-stimulated cells supported the in vitro growth of myeloid colonies from normal bone marrow progenitors. The colony stimulating activity of CLL B-cell supernatants was ascribed to both G-CSF and granulocyte-macrophage colony stimulating factor. G-CSF receptors (G- CSFRs) were detected on freshly isolated B lymphocytes from 7 of 11 B- CLL patients; 5 of these cell suspensions produced G-CSF in culture, whereas 2 did not. rG-CSF rescued 3 of the 7 G-CSFR+ cell fractions from spontaneous apoptosis but had no effect on their in vitro proliferation.