Lack of evidence of prolonged human immunodeficiency virus infection before antibody seroconversion

Recently, considerable concern has been raised regarding the possibility that antibody-based screening tests for the human immunodeficiency virus (HIV) may fail to detect certain high-risk individuals for prolonged periods of time. It has been proposed that testing for HIV-related antigen may be a necessary procedure to detect such individuals. To address this issue, we longitudinally studied two groups of homosexual men: direct sexual partners of acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC) patients and individuals who ultimately sero-converted. There was no evidence of prolonged infection with HIV in the absence of detectable antibody in these two groups. It appears at this time that, even among subjects at very high risk for HIV infection, currently available antibody-based assays are sufficient to identify infected individuals.     

Progressive dysfunction of monocytes associated with iron overload and age in patients with thalassemia major

We evaluated phagocytic and lytic activities of peripheral blood monocytes (PBMo) from patients with thalassemia major (ThP) using C pseudotropicalis as the target. PBMo from ThP showed decreased lytic activity (P less than .001), whereas the phagocytic activity did not differ from that of the controls. Significant inverse correlations were found between lytic activity of PBMo and age of patients (r2 = .47; P less than .01) and also between lytic activity and serum ferritin levels (r2 = .65; P less than .001). No association was found between lytic activity and other variables (blood transfusion regimens, therapy with desferrioxamine, liver damage, and the presence of sHBAg). Splenectomy showed no positive effect on PBMo functions from ThP. Our results suggest that PBMo from ThP have an intracellular defect in their microbicidal mechanisms associated with iron overload. This cell dysfunction could be responsible, at least in part, for the increased susceptibility to infections reported in ThP.     

Mechanisms of adenosine 5'-monophosphate catabolism in human erythrocytes

Uncertainties regarding the role of pyrimidine nucleotidase (PyrNase) in AMP catabolism were resolved by studies of erythrocytes from normal controls, controls with young mean cell ages, and patients with hereditary hemolytic anemia due to severe deficiency of PyrNase. Hemolysates from the latter exhibited undiminished capacity to dephosphorylate AMP over a broad range of pH, indicating that PyrNase was not directly involved. In each subject group, the rates of AMP dephosphorylation between pH 5.1 and 8.3 were indistinguishable from those of IMP, suggesting a potential role for AMP-deaminase, an erythrocyte enzyme that was stimulated by coformycin at pH 7.2. Quantitative analysis of catabolites in incubated hemolysates confirmed that AMP degradation preferentially occurred via deamination to IMP with subsequent dephosphorylation by another erythrocyte nucleotidase isozyme, deoxyribonucleotidase. Both AMP-deaminase and deoxyribonucleotidase have acidic pH optima with minimal activities at physiologic pH, suggesting that this pathway of AMP catabolism could accelerate depletion of the adenine nucleotide pool and thereby mediate the demise of senescent erythrocytes sequestered in the spleen.     

Effect of cleavage of the heavy chain of human plasma kallikrein on its functional properties

Human plasma kallikrein consists of an N-terminal heavy chain of molecular weight (mol wt) 52,000, linked by disulfide bonds to two light chain variants (mol wt 36,000 or 33,000). Although the active catalytic site of kallikrein resides on the C-terminal light chain, the role of the N-terminal heavy chain is less clear. We therefore studied an enzyme designated beta-kallikrein, containing a single cleavage in the heavy chain (mol wt 28,000 + 18,000) and compared it to the enzyme, alpha-kallikrein, with an intact heavy chain. The rates of inactivation by C1 inhibitor of plasma alpha- and beta-kallikreins were kinetically identical, as measured by residual amidolytic activity, after various times of incubation with the inhibitor. Both enzymes reacted completely with C1 inhibitor after 18 hours and formed identical C1 inhibitor- kallikrein complexes of mol wt 195,000. The rate of activation of factor XII by alpha-kallikrein and beta-kallikrein was similar. In contrast, the rate of cleavage of high molecular weight kininogen (HMWK) by alpha-kallikrein was at least fivefold faster and the ratio of coagulant activity to amidolytic activity was fourfold greater than for beta-kallikrein. Plasma alpha-kallikrein, at concentrations potentially achievable in plasma, induced aggregation of neutrophils, but beta-kallikrein failed to elicit this response. In addition, human neutrophils pretreated with cytochalasin B released 2.46 +/- 0.10 microgram/10(7) cells of elastase antigen, but beta-kallikrein released only 0.25 +/- 0.10 micrograms/10(7) cells. These observations suggest that cleavage of the heavy chain influences the rate of cleavage of HMWK and decreases its coagulant activity. Moreover, an intact heavy chain appears to be requisite to support the ability of kallikrein to aggregate neutrophils and release elastase.     

Characterization of patients with an increased susceptibility to bacterial infections and a genetic deficiency of leukocyte membrane complement receptor type 3 and the related membrane antigen LFA-1

Three children from two unrelated families had a history of recurrent bacterial infections, and their neutrophils were shown to have deficient phagocytic and respiratory responses and possible deficiencies in chemotaxis or adherence. Their neutrophils were strikingly deficient in the ability to ingest or give a respiratory burst in response to unopsonized bakers' yeast or zymosan (Z). Tests for neutrophil and monocyte CR1 (C3b/iC3b receptor) and CR3 (iC3b receptor) demonstrated rosettes with both EC3b and EC3bi. However, EC3bi were bound only to CR1, and not to CR3, because EC3bi rosettes were inhibited completely by anti-CR1. Neutrophils, monocytes, and natural killer (NK) cells also did not fluorescence stain with monoclonal antibodies specific for the alpha-chain of CR3 (anti-Mac-1, anti-Mol, OKM1, and MN-41). Quantitation of C receptors with 125I monoclonal anti-CR1 and anti-CR3 indicated that neutrophils from each patient expressed normal amounts of CR1 per cell but less than 10% of the normal amount of CR3. Examination of neutrophils by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that a normal glycoprotein of approximately 165,000 daltons was missing. Immunoblotting of these gels indicated that the missing band was the alpha-chain of CR3. Subsequent analysis of all three patients' cells also demonstrated a deficiency of LFA-1 alpha-chain and the common beta- chain that is shared by the CR3/LFA-1/p150,95 membrane antigen family. The deficiency of LFA-1 probably explained the absent NK cell function, as normal NK cell activity is inhibited by anti-LFA-1 but not by anti- CR3. The reduced phagocytic and respiratory responses to Z were probably due to CR3 deficiency, because treatment of normal neutrophils with anti-CR3, but not anti-FLA-1, inhibits responses to Z by 80% to 90%. Ingestion of Staphylococcus epidermidis by normal neutrophils was shown to be partially inhibited by monoclonal antibodies to the alpha- chain of either CR3 or LFA-1, and monoclonal antibody to the common beta-chain inhibited ingestion by 75%. Thus, both CR3 and LFA-1 may have previously unrecognized functions as phagocyte receptors for bacteria. The absence of this type of nonimmune recognition of bacteria by these children's neutrophils may be one of the reasons for their increased susceptibility to bacterial infections.     

Use of a promoter-trap retrovirus to identify and isolate genes involved in differentiation of a myeloid progenitor cell line in vitro

Anaplastic large cell lymphoma (ALCL) is a distinct clinicopathologic variant of intermediate grade non-Hodgkin's lymphomas (NHL) composed of large pleomorphic cells that usually express the CD30 antigen and interleukin (IL)-2 receptors, and is characterized by frequent cutaneous and extranodal involvement. With variable frequency ALCL bear the t(2;5)(p23;q35) chromosomal translocation that fuses the nucleophosmin (NPM) gene on chromosome 5q35 to a novel protein kinase gene, Anaplastic Lymphoma Kinase (ALK), on chromosome 2p23. We determined the frequency of this translocation with a novel DNA polymerase chain reaction (PCR) technique using 0.5 microgram of genomic DNA, 5'-primers derived from the NPM gene and 3'-primers derived from the ALK gene and hybridization with internal probes. The presence of amplifiable DNA in the samples was tested with the inclusion in the PCR reaction of oligonucleotide primers designed to amplify a 3016-bp fragment from the beta-globin locus. NMP-ALK fusion amplicons were detected using DNA isolated either from all three ALCL cell lines tested, or from all four primary ALCL tumors known to contain the t(2;5)(p23;q35) translocation. Nested amplicons were detected by hybridization in 100% of specimens diluted 10(4)-fold and in 20% of those diluted 10(5)-fold. We subsequently examined archival genomic DNA from 20 patients with ALCL, 39 with diffuse large cell, 2 with mantle cell, 20 with peripheral T cell, 13 with low-grade NHL, 31 with Hodgkin's disease (HD), and 6 with lymphomatoid papulosis. Fusion of the NPM and ALK genes was detected in three of 18 patients with ALCL who had amplifiable DNA (17%, 95% confidence intervals 4% to 41%), but not in any patients with other NHL, HD, or lymphomatoid papulosis. The amplicon sizes were different in all cell lines and patients reflecting unique genomic DNA breakpoints. We conclude that with genomic DNA-PCR the rearrangement of the NPM and ALK loci is restricted to patients with ALCL. Further studies are needed to determine the prognostic significance of the NPM-ALK rearrangement, to determine whether its detection can aid in the differential diagnosis between ALCL. Hodgkin's disease, and lymphomatoid papulosis, and to establish the usefulness of the genomic DNA PCR in the monitoring of minimal residual disease in those patients whose tumors bear the t(2;5).     

The Fanconi anemia complementation group C protein corrects DNA interstrand cross-link-specific apoptosis in HSC536N cells

Fanconi anemia (FA) cells are hypersensitive to cytotoxicity, cell cycle arrest, and chromosomal aberrations induced by DNA cross-linking agents, such as mitomycin C (MMC) and nitrogen mustard (HN2). Although MMC hypersensitivity is complemented in a subset of FA cells (complementation group C [FA-C]) by wild-type FAC cDNA, the cytoprotective mechanism is unknown. In the current study, we tested the hypothesis that FAC protein functions in the suppression of DNA interstand cross-link (ISC)-induced cell cycle arrest and apoptosis. Comparison of HN2-induced cell cycle arrest and apoptosis with those of its non-cross-linking analogs, diethylaminoethyl chloride and 2- dimethylaminoethyl chloride, delineated the DNA ISC specificity of FAC- mediated cytoprotection. Overexpression of wild-type FAC cDNA in FA-C lymphoblasts (HSC536N cell line) prevented HN2-induced growth inhibition, G2 arrest, and DNA fragmentation that is characteristic of apoptosis. In contrast cytoprotection was not conferred against the effects of the non-cross-linking mustards. Our data show that DNA ISCs induce apoptosis more potently than do DNA monoadducts and suggest that FAC suppresses specifically DNA ISC-induced apoptosis in the G2 phase of the cell cycle.     

Subtypes of cutaneous T-cell lymphoma defined by expression of leu-1 and Ia

Monoclonal antibodies were used to characterize immunohistologically the expression of cellular antigens in 25 patients with cutaneous T- cell lymphoma (CTCL). Although all cases expressed the Leu-2a-/Leu-3a+ immunophenotype characteristic of helper T cells, four subtypes were defined based on variable expression of Leu-1 and Ia. In individual patients, the immunophenotype was constant irrespective of body compartment sampled or interim therapy. Ia+ non T-cells typically constituted one-third of the cellular infiltrate. Along with neoplastic cells, Ia+/T6+ dendritic cells were observed within Pautrier microabscesses, dermis, and individually throughout the epidermis. It will be important to determine if different CTCL immunophenotypes represent different biologic subsets of disease or have prognostic relevance. Prospective studies will be facilitated by single- and double-label immunohistologic techniques that allow the simultaneous evaluation of cellular antigen expression and architectural detail.     

Interleukin-4 induces association of the c-fes proto-oncogene product with phosphatidylinositol-3 kinase

We have previously demonstrated that interleukin-4 (IL-4) induces tyrosine phosphorylation of a protein closely related or identical to the c-fes proto-oncogene product (FES) and association of this protein with the IL-4 receptor alpha chain (IL-4R alpha). IL-4 is known to induce association of phosphatidylinositol-3 (PI3) kinase with the IL- 4R alpha. Since FES contains the consensus motifs for PI3 kinase binding, we tested the possibility that FES may associate with PI3 kinase upon IL-4 stimulation. We demonstrate herein that IL-4 stimulation induced rapid association of FES or a related protein with PI3 kinase in mouse T-cell lines. We also show an association of human FES (hFES) with the src homology 2 (SH2) domain of PI3 kinase in a COS7 cell expression system. The in vitro PI3 kinase assay using COS7 cells suggested that hFES partly contributes to the association between the hIL-4R alpha and PI3 kinase. We have further identified the important region in the cytoplasmic domain of the hIL-4R alpha for association of tyrosine-phosphorylated hFES with the hIL-4R alpha and SH2 domain of PI3 kinase using a COS7 cell expression system. These results suggest that FES or a related protein/PI3 kinase pathway may play a role in the pleiotropic effects of IL-4.     

Tacrolimus (FK506) alone or in combination with methotrexate or methylprednisolone for the prevention of acute graft-versus-host disease after marrow transplantation from HLA-matched siblings: a single-center study

The pharmacokinetics, safety, and efficacy in marrow transplantation of FK506-based immunosuppression for graft-versus-host disease (GVHD) prophylaxis was evaluated in an open label pilot study of 18 patients. Patients more than 12 years of age (median, 35 years; range, 15 to 50 years) with advanced hematologic malignancies receiving HLA-matched sibling marrow grafts were randomized to receive FK506 alone, FK506 and methotrexate (MTX), or FK506 and methyl-prednisolone. Of 17 evaluable patients, all had evidence of sustained marrow engraftment. The median time to an absolute neutrophil count of greater than 500/microL was 15 days for patients receiving FK506 alone or FK506 plus methylprednisolone and 23 days for FK506 plus short MTX. Pharmacokinetic studies did not show any significant difference in clearance of FK506 when administered alone or in combination with methylprednisolone or MTX. The mean bioavailability after oral administration in these same three groups was 0.49 +/- 0.1, 0.27 +/- 0.12, and 0.16 +/- 0.08, respectively (P = .003). The decrease in bioavailability may have resulted from an exacerbation of radiation- induced gastroenteritis by MTX. The most significant adverse effect associated with the administration of FK506 was nephrotoxicity, which occurred in 14 of 18 patients (78%). The mean glomerular filtration rate, determined by clearance of (99MTc)DTPA, decreased to 56% (+/- 18%) of the pretransplant baseline level by week 8 (P = .002). Eight of 18 patients (44%) developed grades II-IV acute GVHD, predominantly of the skin and gastrointestinal tract. The actuarial probability of transplant-related mortality during the first 100 days was 24%. The actuarial probability of 1-year disease-free survival was 39%. In conclusion, although bioavailability of FK506 may be affected in patients receiving MTX, this study suggests that FK506 may have a role in the management of patients after allogeneic marrow transplantation.