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A major surface glycoprotein of Leishmania parasites, gp63, is highly conserved among species and is expressed in both infective and intracellular stages. Although much is known about its role in entry and survival in host macrophages, patient antibody responses to this glycoprotein have not been well characterized. The prevalence of anti-gp63 antibody in sera of leishmaniasis patients was evaluated by ELISA. Sera from most acute visceral leishmaniasis patients (84%) from Brazil and Sudan had notably high levels of antibody to recombinant (r) gp63. Sera from other forms of leishmaniasis and from other diseases did not contain significantly elevated levels of anti-gp63 antibody. These results indicate that rgp63 might be a useful constituent of a defined serologic test for visceral leishmaniasis.

The major surface glycoprotein of Leishmania (GP63) is present on all known species of Leishmania and likely plays an integral role during the infection of macrophages in the mammalian host. To identify regions of GP63 which may be of functional significance, the nucleotide sequence of a gene encoding GP63 of Leishmania donovani was determined and compared to the sequences reported for GP63 genes of Leishmania major and Leishmania chagasi. The GP63 nucleotide and predicted protein sequence was highly conserved among the 3 species despite their diverse geographical distribution. L. donovani GP63 is encoded by a multigene family and the gene locus contains at least 7 tandemly repeated genes and at least 3 genes which are dispersed from the tandem array. In addition, polymerase chain reaction and Southern blot analyses demonstrated that there was size heterogeneity within the pro-peptide coding regions of the multiple GP63 genes of L. donovani and that such genes were expressed concurrently in the promastigote life stage.

Toward the future development of a defined subunit vaccine against leishmaniasis is, high levels of recombinant GP63 for diverse species of Leishmania were produced in Escherichia coli. Several features of Leishmania GP63 genes were simultaneously modified with the polymerase chain reaction (PCR) using either cloned genes or total genomic DNA from Leishmania as template DNA for the PCR amplification reactions. The PCR products included only the coding region for the predicted mature form of GP63 that occurs on the surface of Leishmania, flanked by the appropriate translation signals and cloning sites for the production of recombinant GP63 as nonfusion protein in E. coli. When the codon usage in the GP63 gene was modified to reduce the guanine and cytosine content for the codons adjacent to the ATG initiation codon, rGP63 represented about 50% of total protein in E. coli. Mouse monoclonal antibodies raised against purified Leishmania major rGP63 had equivalent immunoblotting characteristics for native GP63 and recombinant GP63 with respect to linear determinants on GP63 expressed in diverse species of Leishmania. Human T cell lines and clones were derived from a patient infected with Leishmania braziliensis panamensis using rGP63 purified from an L. major GP63 expression clone as antigen.

The Initial Blood Storage Experiment (IBSE) probed the behavior of human red cells, white cells, and platelets during exposure to microgravity for 6 days and 2 hours on a National Aeronautics and Space Administration (NASA) shuttle mission, named STS 61-C, which was launched on January 12, 1986. IBSE involved carefully controlled comparisons between two identical sets of blood cells, one exposed to microgravity and the other held on the ground. Specially designed and fabricated, electrically powered environmental chambers provided appropriate environmental temperatures and air flow to support cell metabolism throughout the experiment. To circumvent the need for constant agitation of platelets during storage, a new thin-layer compression method for platelet preservation was developed. Blood cell samples were allocated to the two arms of the experiment, microgravity and earth gravity, by blind assignment. Moreover, to ensure unbiased assessment of the experiment's findings, postexperiment samples for measurement were identified by code. To optimize the chances of detecting possible gravitational effects, a wide array of measurements of cellular function, morphology, metabolism, and immunology were made. Analysis of variance was used in analyzing the data. The most striking finding was that platelets displayed markedly superior structural and functional integrity at microgravity. Granulocytes held on the ground were preserved slightly better than those that orbited in the shuttle, whereas red cells displayed few effects that were attributable to the gravitational variable. Polyvinylchloride-di-(2-ethylhexyl)phthalate (PVC-DEHP) was the plastic of choice for storage of red cells, while PVC-trioctyltrimellitate (TOTM) was superior to PVC-DEHP and polyolefin (PO) for platelets.

A stable (nonradioactive) isotope of selenium in a chemical form common in foods (selenomethionine) or inorganic selenite was taken orally (200 micrograms/d) for 3 wk to label deep body pools. By deep body pools we mean selenium compartments that are large and/or have a slow turnover (exchange) rate. Blood plasma was removed, stored for 11 mo, and later reinfused as a labeled tracer dose with the selenium label in all of the biologically significant chemical forms. Accessible tissues such as red blood cells were highly labeled (20-25%) in the subjects receiving selenomethionine. Selenium from deep body pools is excreted primarily via the urine (80%). Reexcretion of previously absorbed selenium back into the gastrointestinal tract can be measured, avoiding a major source of error in conventional balance studies used to estimate nutrient absorption.

The Mr 63,000 membrane polypeptide (gp63) is one of the Leishmania receptors for host macrophages and has been shown to protect mice from infection. The gene encoding gp63, the major Mr 63,000 surface glycoprotein of L. major promastigotes, has been expressed as a fusion protein with the enzyme glutathione S- transferase encoded by the parasitic helminth Schistosoma japonicum. This fusion protein was recognized by polyclonal antibodies to the native Leishmania gp63 polypeptide. The insoluble gp63 fusion protein was purified by SDS-PAGE and electroelution and was used to raise antibodies in rabbits. These rabbit anti-gp63 antibodies recognized the fusion protein and the denatured parasite gp63 on immunoblots and by immunofluorescence on fixed promastigotes, but did not recognize the native molecule on live organisms. However, antibodies raised against native promastigote glycoproteins, affinity purified on solid-phase gp63 fusion protein, recognized both native and denatured gp63, suggesting the presence of native determinants in the recombinant protein. The gp63 fusion protein did not protect mice of either healer or nonhealer phenotype from challenge infection with live promatigotes. The implications of these results for the engineering of recombinant DNA-produced molecular vaccines are discussed.

Leishmania exist as extracellular promastigotes which multiply in the gut of the sandfly insect vector and as intracellular amastigotes which divide in the phagolysosome of mononuclear phagocytic cells of the mammalian host. Promastigotes express a major surface glycoprotein of 63 kDa, referred to as GP63. The expression of GP63 in both Leishmania life stages was studied using rabbit antibodies against native GP63 as well as rabbit antibodies against recombinant GP63 that was synthesized in an Escherichia coli expression system. Immunofluorescence staining detected GP63 in intracellular amastigotes contained within a macrophage cell line and within freshly isolated lesion amastigotes. Western blot analysis using anti-recombinant GP63 antibodies also demonstrated that amastigotes synthesize GP63 which may undergo differential post-translational processing as compared to promastigote GP63.

A preliminary serotherapeutic trial was undertaken with a monoclonal antibody designated antibody 89 (Ab 89) directed against a lymphoma-associated antigen. In vitro studies demonstrated that Ab 89 could mediate complement-dependent lysis and macrophage adherence but not antibody-dependent cell-mediated cytotoxicity. To evaluate toxicity and therapeutic efficacy, two courses of Ab 89 were administered to a patient with an Ab 89-reactive tumor. Transient decreases in the number of circulating tumor cells and the appearance of circulating dead cells were noted with the infusion of Ab 89. Following administration of 150 mg or more of Ab 89, small amounts of antibody could be demonstrated on circulating tumor cells at a time when no free antibody was found in the serum. The inability to deliver a significant amount of Ab 89 to tumor cells in vivo is thought to be secondary to a circulating tumor antigen. Following each infusion, the amount of this blocking antigen decreased but could not be entirely cleared from the serum. This study provides preliminary evidence for the lack of clinical toxicity of a monoclonal antibody and identifies circulating blocking antigens as a significant obstacle to serotherapy.

Leishmania were isolated from Peruvian patients with uta or espundia; genomic DNA was examined for restriction fragment length differences by Southern blot analysis using DNA probes for beta-tubulin and for the major surface antigen gp63. Using 5 different restriction endonucleases, Peruvian isolates show homogeneity when examined at the beta-tubulin locus. In contrast, the organisms demonstrated heterogeneity both within and between disease groups when examined for restriction site differences within the gp63 locus. The differences observed did not correlate with the 2 disease groups. Comparison of these Peruvian isolates to New World reference strains of the Leishmania braziliensis complex reveals no consistent pattern of identity with either L. b. guyanensis, L. b. panamensis, or L. b. braziliensis.

Two methods of determining the survival of stored red cells are described: one using a double label and one using a single label. It is not suggested that one is superior, but rather both methods are presented in the hope that the personnel of laboratories engaged in determining the effectiveness of anticoagulant-preservation will follow one of the protocols presented. Were this to occur, many of the interlaboratory variations that have decreased the value of data from red cell survival studies would be decreased.

Twenty-one children with chronic thrombocytopenia of obscure etiology, 19 of whom had normal numbers of bone marrow megakaryocytes, were evaluated in order to determine if factors extrinsic to the platelets themselves (presumably antibodies) caused the thrombocytopenia. The rapid destruction of platelets by such antibodies has been demonstrated to cause idiopathic thrombocytopenic purpura (ITP). History, physical examination, and routine laboratory studies were unable to provide an exact diagnosis in these patients. Measurement of platelet survival, using sodium chromate Cr 51 labeled homologous platelets obtained from unrelated donors, permitted a clear separation of these children into two groups. One group (11 patients) had a markedly shortened platelet life span, and was considered to have chronic ITP. The other group (10 patients) had a normal or only slightly reduced platelet life span. Thus, the destruction of platelets by extrinsic factors could be ruled out. Since the diagnosis of chronic ITP was excluded, splenectomy could be avoided in these patients since it is rarely useful in children with nonimmune forms of chronic thrombocytopenia. Homologous platelet survival measurement is a useful diagnostic tool in certain children with chronic thrombocytopenia.

Recent advances in the treatment of patients with thalassemia major have centered around the removal of iron from individuals already overloaded due to repeated transfusions. In this report we present therapeutic maneuvers designed to decrease the rate of iron accumulation. We demonstrate that the persistent maintenance of hematocrits above 35% ("supertransfusion") is not associated with an increased transfusion requirement because it produces a decrease in whole blood volume (21% +/- 2%). Supertransfusion is also associated with normalization or even prolongation of plasma iron turnover. In addition, we describe a method for obtaining units of blood from normal donors that contain primarily young red cells ("neocytes"). These cells have prolonged in vivo survival as measured by the interval between transfusions (30 +/- 2.5 days to 43 +/- 4.5 days) and 51Cr red cell survival (43.8 days versus 27.8 days). Supertransfusion with neocytes is effective in decreasing the rate of iron accumulation in thalassemia.

DNA fragments isolated from the left end of chromosome III in the yeast Saccharomyces cerevisiae are recognized as telomeres in yeast, since plasmids constructed with such fragments are replicated as linear molecules in yeast. The fragments have a 1.18-kb region homologous with the type X regions at yeast telomeres that contain ARS elements that allow the autonomous replication of plasmids in yeast. The X region contains a functional ARS element including the ARS consensus sequence and a distal 200-bp region that enhances ARS function. Distal to the type X region, the fragments have a region of tandemly repeated DNA sequence defined by the formula 5′-C(1–3)A-3′, and designated as the T region at yeast telomeres. Although the terminus of the chromosome was removed in the procedure used to molecularly isolate the left end of chromosome III, the fragments containing the X and T region are recognized as chromosome ends in yeast. Plasmids with inverted repeats of the chromosome III end fragments are resolved in yeast to produce linear plasmids with telomeres that are similar in length and heterogeneity with the natural left end on chromosome III. Fragments with progressive deletions from the distal end of chromosome III were prepared and used to study the X – T region requirements for distinguishing the fragments as telomeres in yeast.

The functional properties of formed elements of whole blood were studied following irradiation doses of 500 to 20,000 rads. Irradiated lymphocytes retained only 1.5 per cent of their 3H thymidine uptake after a 5,000-rad exposure and none after 7,500 rads. Red blood cells stored for 21 days and then irradiated with 5,000 rads had the same survival as nonirradiated controls. In contrast, 5,000 rads reduced platelet yields. However, transfused irradiated platelets produced the expected increases in platelet counts and controlled hemostasis in thrombocytopenic patients. After 5,000 rads, granulocytes had normal bacterial killing capacity, chemotactic mobility, and normal superoxide production after high-dose stimulation. Nitroblue tetrazolium reduction and ingestion stimulated by complement opsonized oil droplets were not diminished by 5,000- and 10,000-rad irradiation. The functional qualities of cellular blood components other than lymphocytes are not compromised by 5,000 rads. This irradiation dose may be an effective means of controlling incidence of graft-vs-host disease in immunosuppressed patients.

The major surface glycoprotein of Leishmania, gp63, is encoded by a small multi-gene family of tandemly linked genes which map to a single chromosome. For Leishmania major, there are five 3.1 kilobasepair (kb) direct repeat units which include a 1.8-kb open reading frame and a 1.3-kb intergenic or spacer region. In addition, there is a single gene copy linked as a direct repeat but separated from the tandem array of gp63 genes by about 8 kb. The restriction enzyme map of the repeat unit is highly conserved among the gene copies. The regions which flank the tandemly repeated genes diverge outside of the 3.1-kb repeat unit. Transcription of the gp63 gene locus is constitutive as the 3-kb transcript was present in promastigotes as well as in amastigotes. A minor 6-kb gp63 gene transcript was also detected in Northern blot analysis which could signify the transcription of the genes as a polycistronic or multigene precursor RNA.

Frozen red cells have a definite place in the mangement of a transfusion service even if one does not include its originally intended use -- inventory control. Undoubtedly, the benefits achieved from frozen red cells can be accomplished in less expensive ways. However the growth in transsplantion and intensive chemotherapy with its concommiatant uses of granulocyte and platelet support predict a greater upsurge in the use of frozen red cells in the very near future.

Simultaneous red blood cell (RBC) and plasma volume determinations were obtained in 16 patients with cystic fibrosis (CF) and moderately severe pulmonary involvement. Hypervolemia with an increase in both RBC and plasma volumes was observed. Changes in blood volume were marked when values were indexed by weight but less significant when indexed by height. Decreasing systemic arterial oxygen saturation was associated with a progressive increase in RBC mass, hematocrit value, and hemoglobin level and a decrease in mean corpuscular hemoglobin concentration. RBC and total blood volumes were highest in patients with cor pulmonale and congestive heart failure. However, the compensatory polycythemic response in patients with CF was inadequate when compared with the response to hypoxemia in patients with cyanotic congenital heart disease. The insufficient oxygen-carrying capacity may compromise tissue oxygen delivery and necessitate treatment.