Emergence of resistant hepatitis B virus strains during long-term lamivudine therapy in human immunodeficiency virus co-infected patients

Seven patients co-infected with hepatitis B virus (HBsAg and HBeAg carriers, quantifiable HBV DNA with the bDNA technic) and human immunodeficiency virus received a triple antiretroviral combination therapy, including lamivudine (150 mg twice a day). Hepatitis B viral load rapidly became undetectable in 6/7 patients. It remained below the level of detection in 2 subjects, after 20 and 22 months of treatment, with one of them achieving HBeAg/anti-HBe seroconversion. However, in the other 4 individuals, hepatitis B viremia increased again after 8 to 16 months of lamivudine-containing regimen. The last patient was a non-responder. The 4 relapsers developed a double mutation Leu(528) for Met(528) and Met(552) for Val(552), on hepatitis B virus polymerase, either concomitant (M8 and M16) with a hepatitis B virus DNA increase, or 2 months earlier (M10 and M12). The high frequency of hepatitis B virus resistance to lamivudine emphasizes the necessity of identifying more effective strategies, such as double combination therapies.     

Isolation of erythropoietin-sensitive cells from Friend virus-infected marrow cultures: characteristics of the erythropoietin response

Murine erythroid precursor cells, stimulated to proliferate in vitro in the absence of added erythropoietin (EP) by the anemia strain of Friend virus (FVA), will subsequently respond to EP by complete erythrocyte differentiation. If not exposed to EP, the erythroid cells divide for about 120 hr in culture, and they maintain the potential for full differentiation in response to EP added at any time during the period from 72 to 120 hr. Between 96 and 120 hr of culture without added EP, the EP-sensitive erythroid precursor cells that have formed discrete erythroid bursts can be isolated in relatively large numbers from such cultures by plucking with a Pasteur pipette. The addition of EP initiates the final stages of erythroid differentiation, including heme synthesis in 70%-80% of these isolated cells. With respect to homogeneity of the precursor cells, quantity of EP-responsive cells obtainable, and uniformity of EP responsiveness, this system is uniquely favorable for biochemical studies of the late differentiation effects of EP. The overall changes in gene expression accompanying EP- induced terminal differentiation were examined by two-dimensional gel electrophoresis of proteins labeled for a short time with radioactive amino acids. Several new proteins are synthesized in these erythroid cells during terminal differentiation, but the number is a very small percentage of the total number of proteins being made. Thus, in this system, the effect of EP is to initiate expression of a small group of genes, including those for globins, spectrin, and other proteins involved in the final stages of erythroid differentiation.     

Identification of the receptor for erythropoietin by cross-linking to Friend virus-infected erythroid cells.

Erythropoietin (Epo) is a glycoprotein hormone that regulates erythroid development and interacts with surface receptors on developing erythroid cells. In this laboratory, a cell system with a relatively pure population of erythroid cells that respond to Epo has been developed. Immature erythroid cells are obtained from the spleens of mice infected with the anemia strain of Friend virus. The binding of 125I-labeled Epo (125I-Epo) to plasma membranes from these cells was studied in this investigation. 125I-Epo binding reached equilibrium within 20 min at 37 degrees C. Twenty percent of the receptors bound 125I-Epo with a Kd of 0.08 X 10(-9) M, while the remaining receptors bound the hormone with a Kd of 0.6 X 10(-9) M. In this study, a receptor for Epo was identified by cross-linking 125I-Epo to the receptor in intact cells and plasma membrane preparations using disuccinimidyl suberate. Polyacrylamide gel electrophoresis revealed two labeled bands of 100 and 85 kDa. The 85-kDa band was more heavily labeled (65%) than the 100-kDa band. Both bands were equally decreased when increasing amounts of unlabeled Epo were included in the binding mixture, indicating a specific interaction of 125I-Epo with the receptor.     

Female genital schistosomiasis as an evidence of a neglected cause for reproductive ill-health: a retrospective histopathological study from Tanzania

Background  

Schistosomiasis affects the reproductive health of women. Described sequelae are ectopic pregnancy, infertility, abortion, and cervical lesions and symptoms mimicking cervical cancer and STIs. There are indications that cervical schistosomiasis lesions could become co-factors for viral infection such as HIV and HPV.     

p16INK4A and p15INK4B gene deletions in primary leukemias

The 9p21 locus has been deleted at a high frequency in a wide variety of tumors. Recently, two genes, p16INK4A and p15INK4B (also called MTS1 and MTS2), have been localized in close proximity at the 9p21 locus, encoding cyclin-dependent kinases 4/6 inhibitors of relative molecular mass 16 kD and 15 kD, respectively and also found to be deleted at a high frequency in tumor cell lines. We analyzed p16INK4A and p15INK4B genes in 178 cases of primary leukemias including 81 cases of chronic lymphocytic leukemia (CLL), seven of hairy cell leukemia (HCL), seven of chronic myelogenous leukemia (CML), 43 of acute myelogenous leukemia (AML), 27 of acute lymphoblastic leukemia (ALL), and 13 of myelodysplastic syndrome (MDS) by Southern blot analyses. The ALL cases showed a relatively high frequency of homozygous deletions (22%, 6 of 27) at the p16INK4A gene locus. Interestingly, of the six cases with p16INK4A homozygous deletions, only three showed homozygous deletions at the p15INK4B gene. In 81 CLL patients, we detected one homozygous and five heterozygous deletions at both the p16INK4A and p15INK4B genes and two heterozygous deletions at the p16INK4A gene alone. Deletion of these two genes in AML cases is relatively low (9%). We did not detect deletions in any of the MDS, HCL, and CML cases examined. Sequence analyses of p16INK4A gene of six CLL cases with heterozygous deletion at this locus showed a 27-bp deletion at the splice acceptor site of intron 1 in one case and changes in the coding sequence in three other cases. The data presented in this report showed that (1) p16INK4A and p15INK4B genes are preferentially deleted homozygously in ALL and heterozygously in CLL cases with frequent mutation in the second allele, and (2) p16INK4A gene appears to be more frequently deleted than p15INK4B gene.     

Antiidiotypic IgG crossreactive with Rh alloantibodies in red cell autoimmunity

IgG autoantibodies eluted from RBCs of antiglobulin positive normal blood donors contained at least two antibody populations, an IgG autoantibody (Ab 1), and an IgG population (Ab 2) that agglutinated RBCs coated with some Rh(D) alloantibodies. Eight of 24 autoantibody eluates tested agglutinated 3 of 10 anti-Rh(D) sensitized RBCs. The agglutinating activity was inhibited specifically by preincubation of the autoantibody eluate with the reactive anti-D. The reaction did not require the Fc domain of the anti-Rh(D), since autoantibody eluates agglutinated RBCs coated with F(ab')2 prepared from the reactive anti-D sera. These findings indicate that the RBCs of some antiglobulin- positive blood donors contain an immunoglobulin auto-antiidiotype (Ab 2) against the RBC autoantibody (Ab 1) which is demonstrable through its cross-reactivity with selected Rh(D) alloantibodies. Identification of auto-antiidiotypes in RBC autoimmunity lends support to the idiotype- antiidiotype network hypothesis of immune regulation and is consistent with the bizarre and complex serology of autoimmune hemolytic anemia. The absence of clinical hemolysis in antiglobulin-positive normal blood donors suggests that immunoglobulin idiotype-antiidiotype interactions may play a role in modulating the effects of RBC autoimmunity.     

Immune reconstitution in severe combined immunodeficiency disease after lectin-treated, T-cell-depleted haplocompatible bone marrow transplantation

We describe our 9-year experience with lectin-treated T-cell-depleted haplocompatible parental bone marrow transplantation (BMT) for 24 patients with severe combined immunodeficiency disease (SCID). Nineteen of 21 evaluable patients had T-cell engraftment; 2 of 11 patients tested had B-cell and monocyte engraftment. Fourteen of 24 (58%) patients are alive 7 months to 9.8 years post-BMT. Seventeen of 24 patients received pretransplant conditioning with chemotherapy and/or total body irradiation, and 8 of 24 received more than one transplant. Patients who received conditioning had a survival rate of 61% versus 57% for those who received no conditioning. None received graft-versus- host disease (GVHD) prophylaxis and no patient had acute or chronic GVHD greater than grade I. Kinetics and follow-up of immune recovery were analyzed in 14 patients who are greater than 1 year from transplant. Half of the patients showed evidence of T-cell function by 3 months and normal T-cell function by 4 to 7 months post-BMT. On average, T-cell numbers and subsets became normal 10 to 12 months posttransplant. Recovery of B-cell function was more delayed, although in most patients B-cell numbers and IgM levels were normal by 12 months post-BMT. B-cell function, as determined by isohemagglutinin titers or specific antibodies to pneumococcal polysaccharide, keyhole limpet hemocyanin, or tetanus toxoid, became normal in 10 of 14 patients 2 to 8 years post-BMT. Seven of the 14 are off gammaglobulin therapy. Production of isohemagglutinins tended to predict recovery of antibody response to pneumococcal polysaccharide (P < .064). Based on these results, we believe that haplocompatible BMT is an effective, curative treatment for patients with SCID who lack an HLA-matched related donor.     

Platelet glycoproteins IIb and IIIa as a calcium channel in liposomes

Human platelet membrane glycoproteins IIb and IIIa (GPIIb and IIIa) were incorporated into phospholipid vesicles by the reverse-phase technique to assess the ability of GPIIb and IIIa to function as a Ca2+ channel. Movement of Ca2+ across the lipid bilayer was quantitated by injection of proteoliposomes with encapsulated Fura-2 into Ca2+ buffers and measurement of Fura-2 fluorescence as an indicator of Ca2+ influx. Reciprocally, to assess the function of proteins in an inside-out orientation, Ca2+-loaded vesicles were injected into Ca2+-free buffer and Ca2+ efflux monitored by a calcium electrode. Incorporation of the IIb-IIIa complex produced significant facilitation of Ca2+ movement across the lipid bilayer. No net transmembrane Ca2+ movement was seen with dissociated IIb and IIIa. Movement of Ca2+ was proportional to the transmembrane Ca2+ gradient. Ca2+ movement into the vesicles was inversely proportional to extravesicular NaCl from 25 to 150 mmol/L, analogous to several studies in the intact platelet. Adenosine triphosphate had no effect on Ca2+ movement into or out of the vesicles. Specific inhibition of a Ca2+ shift into the vesicles was seen with M148, a monoclonal antibody to IIb/IIIa, while no inhibition was observed with a panel of other anti-IIb/IIIa monoclonal antibodies. This suggests that a specific site on the complex or orientation of the complex is essential for calcium channel function. These data demonstrate that the GPIIb/IIIa complex can serve as a passive Ca2+ channel across a phospholipid bilayer and has the potential to play a role in Ca2+ flux across the platelet plasma membrane.     

Activated protein C resistance: molecular mechanisms based on studies using purified Gln506-factor V

Gln506-factor V (FV) was purified from plasma of an individual homozygous for an Arg506Gln mutation in FV that is associated with activated protein C (APC) resistance. Purified Gln506-FV, as well as Gln506-FVa generated by either thrombin or FXa, conveyed APC resistance to FV-deficient plasma in coagulation assays. Clotting assay studies also suggested that APC resistance does not involve any abnormality in FV-APC-cofactor activity. In purified reaction mixtures, Gln506-FVa in comparison to normal FVa showed reduced susceptibility to APC, because it was inactivated approximately 10-fold slower than normal Arg506-FVa. It was previously reported that inactivation of normal FVa by APC involves an initial cleavage at Arg506 followed by phospholipid- dependent cleavage at Arg306. Immunoblot and amino acid sequence analyses showed that the 102-kD heavy chain of Gln506-FVa was cleaved at Arg306 during inactivation by APC in a phospholipid-dependent reaction. This reduced but measurable susceptibility of Gln506-FVa to APC inactivation may help explain why APC resistance is a mild risk factor for thrombosis because APC can inactivate both normal FVa and variant Gln506-FVa. In summary, this study shows that purified Gln506- FV can account for APC resistance of plasma because Gln506-FVa, whether generated by thrombin or FXa, is relatively resistant to APC.