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Hybrids of tobacco mosaic virus (TMV) were constructed with the use of fusion to the coat protein peptides of 10 or 15 amino acids, containing the 5B19 epitope from the spike protein of murine hepatitis virus (MHV) and giving rise to TMV-5B19 and TMV-5B19L, respectively. The TMV hybrids were propagated in tobacco plants, and the virus particles were purified. Immunogold labeling, with the use of the monoclonal MAb5B19 antibody, showed specific decoration of hybrid TMV particles, confirming the expression and display of the MHV epitope on the surface of the TMV. Mice were immunized with purified hybrid viruses after several regimens of immunization. Mice that received TMV-5B19L intranasally developed serum IgG and IgA specific for the 5B19 epitope and for the TMV coat protein. Hybrid TMV-5B19, administered by subcutaneous injections, elicited high titers of serum IgG that was specific for the 5B19 epitope and for coat protein, but IgA that was specific against 5B19 was not observed. Mice that were immunized with hybrid virus by subcutaneous or intranasal routes of administration survived challenge with a lethal dose (10 x LD50) of MHV strain JHM, whereas mice administered wild-type TMV died 10 d post challenge. Furthermore, there was a positive correlation between the dose of administered immunogen and protection against MHV infection. These studies show that TMV can be an effective vaccine delivery vehicle for parenteral and mucosal immunization and for protection from challenge with viral infection.

Coat protein-mediated resistance (CP-MR) has been widely used to protect transgenic plants against virus diseases. To characterize the mechanisms of CP-MR to tobacco mosaic tobamovirus (TMV) we developed mutants of the coat protein that affected subunit-subunit interactions. Mutant CPs were expressed during TMV replication as well as in transgenic Nicotiana tabacum plants. The mutation T42-->W increased protein aggregation and T28-->W abolished aggregation and assembly, while the mutations T28-->W plus T42-->W and T89-->W altered normal CP subunit-subunit interactions. The mutant T28W was unable to assemble virus-like particles (VLPs) during infection and in transgenic plants failed to aggregate; this protein conferred no protection against challenge of transgenic plants by TMV. The mutant T42W had strong CP subunit-subunit interactions and formed VLPs but not infectious virions. Transgenic lines with this protein exhibited stronger protection against TMV infection than transgenic plants that contained the wild-type (wt) CP. It is proposed that increased resistance conferred by the T42W mutant results from strong interaction between transgenic CP subunits and challenge virus CP subunits. CP carrying the mutation T89-->W formed flexuous and unstable VLPs whereas the double mutant T28W:T42W formed open helical structures that accumulated as paracrystalline arrays. In transgenic plants, T89W and the double mutant CPs showed reduced ability to aggregate and provided lower protection against TMV infection than wt CP. A strong correlation between normal CP subunit-subunit interactions and CP-MR is observed, and a model for CP-MR involving interactions between the transgenic CP and the CP of the challenge virus as well as interference with virus movement is discussed.

Diagnosis of human immunodeficiency virus-1 (HIV-1) infection requires the collection of either serum or oral fluid that is subsequently tested for the presence of antibodies to HIV-1. The effective use of oral fluid for the detection of HIV antibodies is contingent on stabilization of immunoglobulins in the sample through the use of preservatives. Oral fluid preservatives also contain agents that can disrupt and inactivate viruses. This study demonstrates the virucidal activity of a commercially available oral fluid preservative against HIV-1 using a sensitive 28-day cell culture assay designed to detect infectious virus. The results demonstrate that a 5-log reduction in viral titer is obtained when equal volumes of HIV-1 viral stocks and the preservative are mixed. The data provide strong evidence that preserved oral fluid samples from infected individuals are noninfectious for HIV-1.

Research has demonstrated that oral mucosal transudate (OMT), a serum-derived fluid that enters saliva from the gingival crevice and across oral mucosal surfaces, can be preferentially concentrated by a novel collecting system to yield detectable levels of immunoglobulins (i.e., IgG and IgM antibodies) against various bacterial and viral diseases. Assays based on OMT can aid in the diagnosis of disease and in the management of therapeutic drugs. A reliable and accurate OMT-based test to detect human immunodeficiency virus (HIV) antibodies is commercially available. Additional tests based on similar technologies may aid in the diagnosis of viral hepatitis, measles, mumps, and rubella as well as in monitoring levels of therapeutic drugs such as theophylline. The future use of OMT-based testing will likely increase because of the inherent advantages of this technology: convenience; avoidance of inadvertent transmission of blood-borne pathogens; ease of use in pediatric and geriatric populations; as well as the potential for blood-free home and workplace collection of patient samples.

Objective: To determine accuracy of a human immunodeficiency virus type 1 (HIV-1) antibody testing system using a device to collect and stabilize oral mucosal transudate (OMT), a fluid with increased levels of IgG; an enzyme immunoassay (EIA) screening test optimized for OMT; and a Western blot confirmatory test designed for use with OMT., Design: The OMT specimens were tested by EIA and, if indicated, confirmatory Western blot according to a standard testing algorithm. The OMT results were compared with true HIV status as determined by serum testing and/or clinical diagnosis., Patients: Specimens from 3570 subjects (2382 at low risk, 698 at high risk, 242 with acquired immunodeficiency syndrome [AIDS], and 248 "nonspecificity" [persons with diseases associated with an increased frequency of false-positive results in HIV testing]) were collected at 11 geographically diverse sites (including blood banks, public health clinics, general medical clinics, HIV clinics, sexually transmitted disease clinics, and a hemophilia center) in the United States., Main Outcome Measures: Overall accuracy of testing OMT for HIV-1 antibodies compared with true HIV-1 antibody status; sensitivity and specificity of OMT EIA and Western blot., Results: Sensitivity of OMT EIA testing in 673 true-positive subjects was 99.9% (672/673). The OMT Western blot results in the 673 true-positive subjects were positive in 665 and indeterminate in 8. The EIA followed by Western blot (if EIA was repeatedly reactive) yielded a negative result in 99.9% (2893/2897) of OMT samples from true negatives and an indeterminate result in 4. The OMT testing system provided the correct result or would trigger appropriate follow-up testing in 3569 (>99.9%) of 3570 cases., Conclusion: HIV-1 antibody testing of OMT samples is a highly accurate alternative to serum testing.

S-Adenosyl-L-methionine:scoulerine-9-O-methyltransferase (SMT) catalyzes the transfer of the S-methyl group of S-adenosyl-L-methionine to the 9-hydroxyl group of scoulerine during the biosynthesis of berberine. We have isolated functionally active cDNA clones (pCJSMTs) from a cDNA library prepared from cultured cells of Coptis japonica. The longest cDNA insert (pCJSMT1) had an open reading frame that encoded 351 amino acids, but the calculated molecular mass (38,364 Da) of the deduced product was slightly lower than the experimentally determined molecular mass of purified SMT. Rapid amplification of the 5' end of the cDNA indicated that the full-length cDNA of SMT consisted of 1,458 nucleotides that encoded 381 amino acids. When the full-length cDNA was expressed in E. coli, the molecular mass of the expressed SMT was greater than that of native SMT in Coptis cells. This result suggests that SMT might be produced in a pre-mature form and processed post-translationally. SMT was also found to exhibit sequence homology to other O-methyltransferases from plants and N-terminal region of the SMT polypeptide appeared to be necessary for enzymatic activity.

We tested the utility of a novel collection system that allows measurement of theophylline in oral mucosal transudate (OMT) to calculate serum theophylline concentrations. In 25 adult patients, theophylline levels in OMT correlated better than expectorated saliva with serum theophylline (r = 0.927 for OMT versus r = 0.831 for expectorated saliva, each p < 0.0001). In a subsequent study of 128 patients (118 adults and 10 children aged 4 to 12 yr), OMT and serum theophylline were measured and polynomial regression analysis performed to allow calculation of serum level for any given OMT level. Theophylline levels calculated from OMT values closely followed measured serum theophylline in two normal subjects after administration of either intravenous or oral theophylline. OMT samples collected by 24 patients at home were mailed to the laboratory for testing. Theophylline values from the home collection samples correlated closely (r = 0.930, p < 0.0001) with serum theophylline levels obtained at the same dose of theophylline. These findings suggest that once the relationship of serum to OMT theophylline is established in a given laboratory, the latter can be used to monitor outpatient theophylline therapy in adults (and possibly children) at times of the day otherwise inaccessible to serum sampling.

Transgenic plants carrying nucleotide sequences derived from plant viruses can exhibit increased resistance to viral disease. Many viral sequences confer some level of either resistance to infection or suppression of disease symptoms (tolerance). These include segments of viral genomes encoding capsid or coat proteins, sequences encoding proteins that are or may be subunits of the viral replicase, sequences incapable of encoding proteins, entire genomes of defective interfering viruses and satellite viruses, and complete genomes of mild strains of virus. The transgene may act on initiation of infection, replication of virus, spread of the infection throughout the plant, and symptom development. More than one of these processes can be impaired by a single transgene derived from a single viral gene. The level of protection ranges from very low to high, while the breadth of protection ranges from very narrow, where protection is only observed against closely related strains of the virus from which the transgene was derived, to moderately broad, extending to other viruses. Data are insufficient to establish a molecular mechanism of resistance for most of the described examples. In addition, although the use of a particular segment of the viral genome confers resistance in one virus-host system, analogous sequences from a different virus in another host may be ineffective.

5-Methylthioribose (MTR) is an intermediate in the methionine recycling pathway of organisms containing the enzyme MTR kinase. Analogs of MTR have been proposed as a new class of antimicrobial agents because of their ability to perturb the growth of MTR kinase-containing pathogens through inhibition of methionine salvage or by conversion to toxic products. One such analog, 5-trifluoromethylthioribose (TFMTR), has demonstrated potent inhibitory effects on the growth of Klebsiella pneumoniae (A. G. Gianotti, P. A. Tower, J. H. Sheley, P. A. Conte, C. Spiro, J. H. Fitchen, and M. K. Riscoe, J. Biol. Chem. 265:831-837, 1990). Although the mode of action of TFMTR has yet to be determined, it is believed that the drug is converted to the toxic products trifluoromethionine or carbonothioic difluoride via MTR kinase and the methionine recycling pathway. On the basis of this assumption, we theorized that blocking de novo methionine synthesis would increase dependence on the methionine salvage pathway and lead to an increased rate of synthesis of toxic metabolites from TFMTR. In this report, we show that three separate inhibitors of de novo methionine synthesis (1,2,4-triazole, azaserine, and propargylglycine) act synergistically with TFMTR in inhibiting the growth of K. pneumoniae.

To explore the role of individual residues in the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), small subunits with single amino acid substitutions in three regions of relative sequence conservation were produced by directed mutagenesis of the rbcS gene from Anabaena 7120. These altered small subunits were cosynthesized with large subunits (from an expressed Anabaena rbcL gene) in Escherichia coli. Mutants were analyzed for effects on quaternary structure and catalytic activity. Changing Glu-13S (numbering used is that of the spinach enzyme) to Val, Trp-67S to Arg, Pro-73S to His, or Tyr-98S to Asn prevented accumulation of stable holoenzyme. Interpretation of these results using a model for the three-dimensional structure of the spinach enzyme based on x-ray crystallographic data suggests that our small subunit mutants containing substitutions at positions 13S and 67S probably do not assemble because of mispairing or nonpairing of charged residues on the interfacing surfaces of the large and small subunits. The failure of small subunits substituted at positions 73S or 98S to assemble correctly may result from disruption of intersubunit or intrasubunit hydrophobic pockets, respectively.

5'-Deoxy-5'-methylthioadenosine (MTA), an important intermediate in methionine recycling, can be metabolized by one of two mechanisms that appear to be mutually exclusive. In human cells, MTA is degraded in one step to adenine and 5-methylthioribose 1-phosphate (MTR-1-P) via MTA phosphorylase. In contrast, certain microbes metabolize MTA in two steps: first to 5-methylthioribose (MTR) followed by conversion to MTR-1-P. The enzymes involved in this two-step conversion are MTA nucleosidase and MTR kinase. In both cases, MTR-1-P is subsequently recycled to methionine. Because MTR kinase is "unique" to microbes (it is also found in plant tissue) and since it is essential to microbial methionine salvage, we hypothesized that MTR kinase is a promising target for chemotherapeutic exploitation. We demonstrate that 5-trifluoromethylthioribose (TFMTR), a structural analog of MTR, is a potent inhibitor of the MTR kinase-containing organism Klebsiella pneumoniae. TFMTR not only inhibits the growth of K. pneumoniae in a dose-dependent manner (50% inhibition at approximately 40 nM) but also competitively inhibits MTR kinase activity (Ki approximately 7 microM). Furthermore, TFMTR is shown to be a substrate for MTR kinase (Km = 1.7 microM), suggesting that the drug could be converted to toxic products (e.g. trifluoromethionine or carbonothionic difluoride) in enzyme-containing organisms. Structural analogs of MTR represent a new class of compounds with the potential for treating diseases caused by MTR kinase-containing microorganisms.

We treated mice with high-dose busulfan to produce a residual or "latent" hematopoietic injury characterized by persistent normal or near-normal peripheral blood counts and marrow cellularity but moderate to marked reduction in hematopoietic stem cells. Control and "latent" mice were then challenged with glucan, a granulopoietic stimulant. We found that glucan administration induced a comparable increase in granulocytic differentiation in control and "latent" mice. This differentiational expansion was not accompanied by a further depletion of pluripotent hematopoietic stem cells. Our results fail to demonstrate competition between self-renewal and differentiation at the level of the CFUS, at least in this model. Stimuli for granulopoiesis act primarily on more distal, committed populations which are apparently unaffected by prior exposure to busulfan.

We studied myeloid progenitor cells (CFUc) from peripheral blood mononuclear leukocytes (MNL) of normals and patients with severe aplastic anaemia (AA). Baseline CFUc averaged 4.4 +/- 1.5 (range 0--17)/10(6) MNL in fifteen normals and 0 +/- 1/10(6) MNL in six patients with severe AA (P less than 0.05). To assess CFUc proliferative capacity, 10(7) MNL were put into liquid culture in Marbrook chambers with colony-stimulating activity and sub-cultured in agar at intervals up to 10 days. CFUc from normal MNL increased from 44 +/- 15/chamber at Day 0 to 156 +/- 33/chamber at peak value (P less than 0.02). In contrast, CFUc from AA MNL remained undetectable throughout the period of liquid culture (AA peak v. normal peak; P less than 0.001). These results indicate a marked decrease in circulating CFUc in patients with severe AA, and a profound abnormality in CFUc proliferation in vitro. This abnormality could be due to defective replication of CFUc, a lack of feed-in from more primitive precursors, or both.

This article focuses on the chemotherapeutic agents which alter purine metabolism as a means to achieve selective killing of leukemic cells. We present an overview of purine metabolism in order to highlight enzymatic steps which are targeted by antileukemic drugs. Purine antimetabolites used in the treatment of leukemia can be grouped into three classes: (1) structural analogs of normal purines (6-mercaptopurine and 6-thioguanine); (2) inhibitors of de novo purine biosynthesis (methotrexate and hydroxyurea); and (3) inhibitors of purine salvage (2'-deoxycoformycin). In addition, a number of investigational drugs (trimetrexate, fludarabine and 2'-chlorodeoxyadenosine) have been recently introduced and show promise in early clinical trials. Purine antimetabolites are active in a variety of lymphoid and myeloid leukemias and represent an important component of the therapy of these disorders. Several of the drugs have been developed with the specific intent of perturbing enzymes involved in purine metabolism. Refinements in our understanding of purine biochemistry in normal and leukemic cells may aid future efforts to design more effective drugs.

5'-Methylthioadenosine (MTA) is a naturally occurring nucleoside which is degraded by MTA phosphorylase (MTAase) to adenine and methylthioribose-1-phosphate in all normal mammalian cells. These products of the phosphorylytic cleavage of MTA are recycled to the nucleotide pool and methionine, respectively. Thus, supplemental MTA could theoretically be utilized by MTAase-containing cells as a source of methionine and adenine. In fact, in vitro experiments have shown that MTAase-containing cells proliferate normally in methionine-free medium if MTA is added to the cultures (M. K. Riscoe and A. J. Ferro, J. Biol. Chem., 259: 5465-5471, 1984). In contrast, MTAase-deficient malignant cell lines do not proliferate under these conditions. In light of these observations and the recent demonstration (N. Kamatani et al., Blood, 60: 1387-1391, 1982) that a proportion of acute lymphoblastic leukemias lack MTAase, we wished to determine if this enzyme deficiency occurs in a variety of human neoplasms. Accordingly, malignant cells from eight patients with acute nonlymphocytic leukemia and ten patients with various solid tumors were assayed for MTAase activity. Samples from one of the eight acute nonlymphocytic leukemia patients and three of the 10 solid tumor patients (one with melanoma, one with squamous cell lung cancer, and one with adenocarcinoma of the rectum) had undetectable MTAase activity. In contrast, erythrocytes, neutrophils, and monocytes isolated from normal subjects and from patients with immunodeficiency syndromes or cancer all contained enzyme activity. In addition, the methods of preservation, storage, and cell disruption did not affect MTAase activity. These observations confirm and extend the findings of Kamatani et al. (Blood, 60: 1387-1391, 1982) by demonstrating that MTAase deficiency occurs in a variety of human malignancies including acute nonlymphocytic leukemia and solid tumors. This metabolic difference between normal and malignant cells may be therapeutically exploitable.

We used hybridoma-derived monoclonal antibody to H-2Kk antigens and xenoantibodies to beta 2-microglobulin (beta 2 m) to study the expression of the H-2 antigenic molecular complex on murine hematopoietic stem cells and the effect of long-term culture in vitro on the expression of these antigens. Monoclonal anti-H2Kk antibody produced potent complement-dependent inhibition of pluripotent (CFU-S), myeloid (CFU-C), and erythroid (CFU-E) stem cells from the bone marrow of C3H and AKR(H-2k) mice and was without effect on stem cells from Balb/C (H-2d) mice. Anti-beta 2m xenoantibodies inhibited stem cells from all three strains. Stem cells could not be 'rescued' from the inhibitory effects of the antibodies by the addition of thymocytes to marrow cells after antibody treatment. Both the anti-H-2Kk monoclonal antibody and the anti-beta 2m xenoantibodies produced potent inhibition of AKR (H-2k) CFU-C that had been maintained in culture for up to 6 weeks. These results indicate that murine CFU-S, CFU-C, and CFU-E express H-2 antigens and that the expression of these antigens by CFU-C is not altered during long-term culture.

Aplastic anemia is characterized by decreased bone marrow function with inadequate production of erythrocytes, granulocytes, and platelets. Marrow failure may be caused by absence of or defects in hematopoietic stem cells, abnormalities of the bone marrow microenvironment, ineffective cell-to-cell interactions, or immune disorders. Although most patients with aplastic anemia have normal immunity, some have abnormalities of T- and B-lymphocytes. Rare patients have an immune cause of marrow failure. Treatment of aplastic anemia involves blood transfusions and withdrawal of potential causal factors. Efforts to stimulate hematopoiesis with androgens, corticosteroids, and other drugs have been largely unsuccessful. Some patients may recover after treatment with antithymocyte globulin or other immunosuppressive agents. Bone marrow transplantation is the preferred treatment for patients with severe aplastic anemia who have a human-leukocyte-antigen-identical related donor. Transplants of hematopoietic stem cells obtained from alternative sources, such as fetal liver cells or stem cells from long-term, in-vitro cultures, also may be useful.

A modification of the microcytotoxicity assay of Terasaki & McClelland was used to screen for serum inhibitors of myeloid progenitor cells (CFU-C). Sera from 104 patients with neutropenia or bone marrow failure and from 104 controls, including 30 normal subjects and 74 patients with various disorders or multiple transfusions, were studied. Inhibitors of CFU-C were found in 19 of the 104 patients with neutropenia or marrow failure: three with acquited neutropenia and 16 with aplastic anaemia. In 17 of the 19 patients the inhibitor was either complement dependent or chavincingly associated with clinical remission of disease in one patient. In another patient, the inhibitor was an IgG antibody directed against non-HL1 antigens present on CFU-C of all 50 normal target marrows tested; however, it was not clearly autoreactive. Inhibitors were also found in one of nine patients with lupus erythematosus, one with breast carcinoma, one patient with renal transplant rejection, and six of 46 multiply transfused patients without marrow failure. Our findings indicate that most serum inhibitors are associated with multiple transfusion: convincing evidence of pathogenetically important autoreactive antibody was present in only one of 19 patients with inhibitors.

Methylthioadenosine (MTA), a coproduct of polyamine biosynthesis, is known to inhibit proliferation in a variety of cell culture systems. In this paper, we show that while MTA inhibits the growth of the human promyelocytic cell line HL-60, it does not interfere with retinoic acid-induced granulocytic or phorbol ester-induced monocytic differentiation of these cells. MTA also inhibits proliferation induced by colony stimulating activity of normal human granulocytic precursor cells grown in suspension culture but does not suppress terminal differentiation of these cells. In contrast to the lack of effect of MTA on granulocytic differentiation which we report here, others have shown that MTA prevents terminal differentiation of murine erythroleukemia cells. That MTA is a normal cellular constituent which inhibits proliferation but not differentiation of normal granulopoietic cells and may have opposing effects on immature cells of erythroid lineage suggests a possible role for this compound in the regulation of hematopoiesis. In addition, MTA may be useful for studying the process of differentiation in the absence of cell proliferation in granulopoietic cells.

A previous report has suggested an antigenic relationship between beta 2-microglobulin (beta 2 mu) and granulocyte colony-stimulating activity (CSA). Since human myeloid progenitor cells (CFU-C) express HLA antigens and beta 2 mu is a known molecular component of HLA antigens, we wondered whether the reported effect of anti-beta 2 mu heteroantisera on in vitro granulopoiesis might result from cytotoxicity to CFU-C rather than from cross-reactivity with CSA. To test this, we used rabbit antibody reactive with human and murine beta 2 mu (anti-beta 2 mu). Treatment of human and murine bone marrow cells with anti-beta 2 mu and complement resulted in 95+% inhibition of CFU-C colony formation compared to controls. To test for an effect on CSA, anti-beta 2 mu was incubated with human and murine sources of CSA. After addition of goat anti-rabbit Ig antiserum to precipitate immune complexes and unbound anti-beta 2 mu, the supernatant fluid retained CSA but was no longer cytotoxic to CFU-C. These results indicate that human and murine CFU-C express membrane beta 2 mu and that anti-beta 2 mu antibody does not cross-react with human or murine CSA.

Bone marrow failure in aplastic anemia (AA) could result from abnormalities of hematopoietic stem cells, abnormal control of hematopoiesis, or abnormalities of the hematopoietic environment. Bone marrow transplantation, in vitro marrow culture techniques, and studies in animal models of marrow failure have provided insights on the possible pathogenetic mechanisms underlying AA. Studies in man and in murine models suggest that most often AA results from injuries to hematopoietic stem cells. Despite the intriguing report of abnormal regulatory cells in congenitally anemic mice, instances of marrow failure due to defective humoral or cellular control of hematopoiesis have not been identified in man. In vitro studies employing allogeneic marrow targets have suggested that immune suppression of hematopoiesis may occasionally mediate AA in man. Marrow failure due to abnormalities of the hematopoietic microenvironment has been suggested by experience with bone marrow transplantation, but no direct study of this possibility has been reported. Based on available evidence, it seems likely that AA will prove to be many diseases that share common clinical and morphologic features.

A case of a patient with otherwise typical multiple myeloma whose myeloma cells were phagocytic is described. Phagocytosis of erythrocytes, erythrocytic precursors, lymphocytes, and platelets was found. Ingestion of latex particles by myeloma cells was demonstrated in vitro.

Hybridomas derived from the fusion of murine myeloma cells with splenocytes from mice immunized with human cultured lymphoid cells secreted monoclonal antibodies to human cell surface antigens. Serologic and immunochemical assays showed that 4 monoclonal antibodies (Ab Q2/47, Q2/61, Q2/70, Q2/80) recognize framework determinants of Ia-like antigens and 1 monoclonal antibody (Ab Q1/28) reacts with determinants expressed on the heavy chain of HLA-A,B antigens. Both anti-HLA-A,B and anti-Ia-like antigen monoclonal antibodies caused complement-dependent inhibition of granulocyte-macrophage colony formation by human bone marrow grown in soft agar. Mixing experiments excluded the possibility of an indirect effect on progenitor cells by lysis of auxiliary cells. These results indicate that human myeloid progenitor cells express HLA-A,B and Ia-like antigens.

Recent studies using cytotoxic and cell separation techniques have identified differentiation-related antigens on human hematopoietic cells. These results, combined with bone marrow transplantation studies, have yielded a picture of antigenic modulation from the pluripotent hematopoietic stem cell through the differentiated end cells.

5'-Methylthioadenosine (MTA), a naturally occurring nucleoside, inhibited in vitro colony formation by murine erythroid (CFU-E) and granulocyte-macrophage (CFU-GM) progenitor cells in a dose-dependent fashion with maximal inhibition at concentrations of 2 X 10(-3) M and 1 X 10(-4) M, respectively. The inhibitory effect was reversible after up to 8 h of exposure to MTA but was irreversible after 24 h. MTA also inhibited hematopoietic progenitors in vivo. In mice given daily intraperitoneal injections of MTA for 28 days, CFU-GM were maximally reduced on day 14 to 51% of control. CFU-GM returned toward control levels by day 28 despite the continued administration of MTA. Hematocrit and leukocyte count were not reduced until day 28 and then only to 90% and 70% of control, respectively. MTA reached peak plasma levels of 2.8 X 10(-5) M 5 min after a single intraperitoneal injection of 75 mg/kg and was almost completely cleared by 60 min. These findings indicate that MTA produces reversible inhibition of murine hematopoietic progenitors both in vitro and in vivo. Despite the inhibitory effect on progenitors there is little effect on peripheral blood counts, which suggests that MTA inhibits hematopoietic proliferation without affecting hematopoietic differentiation.

Mice treated with high-dose busulfan develop a 'latent' form of bone marrow failure characterized by near-normal peripheral blood counts and marrow cellularity, but marked reductions in marrow pluripotent stem cells (CFUs) and myeloid progenitor cells (CFUc). Spleen cell suspensions from control and 'latent' mice were placed in liquid culture in the presence of colony-stimulating activity. Cells were harvested at intervals up to 14 days and sub-cultured in agar to assay for CFUc. Baseline splenic CFUc did not differ significantly between control and 'latent' mice. Splenic CFUc from control mice increased 50-fold and reached a peak at day 10 in liquid culture. In contrast, splenic CFUc from 'latent' mice increased only 7-fold and reached a peak at day 3. Our results indicate that although splenic CFUc are present in normal numbers in 'latent' mice, their proliferative capacity is markedly reduced, either as the result of defective CFUc self-renewal or defective feed-in from CFUs or both.

Since drug resistance and toxicity limit the use of available antiprotozoal agents, it is important that new drugs be developed as soon as possible. In this study, the method by which several protozoa degrade 5'-methylthioadenosine (MTA) was shown to differ from MTA catabolism in human cells. To exploit this metabolic difference, two analogs of methylthioribose (MTR), an MTA catabolite, were synthesized and found to be cytocidal to Plasmodium falciparum, Giardia lamblia, and Ochromonas malhamensis in vitro. In contrast, these analogs had no effect on cultured mammalian cells. Analogs of MTR represent a potential new class of antiprotozoal drugs.

A correlation was demonstrated between bone-marrow graft rejection and pretransplant lymphocytotoxins. This finding might prove useful as a test for identification of patients at high risk of rejection who might benefit from more intensive immunosuppression.

The development of new and effective ontiprotozool drugs has been difficult because of the close metabolic relationship between protozoa and mammalian cells. In this article, Michael Riscoe, Al Ferro and john Fitchen present their hypothesis for chemotherapeutic exploitation of methylthioribose (MTR) kinase, an enzyme critical to methionine salvage in certain protozoa. They propose that analogues of MTR if properly designed, would be converted to toxic products in organisms that contain MTR kinase but not in mammalian cells, which lack this enzyme.

We describe an antigen(s) characterized by a heteroantiserum raised in rabbits against mature human granulocytes. This antigen was found on neutrophils, monocytes, platelets, acute and chronic myelocytic leukemia cells and on granulocyte-macrophage progenitor cells grown in agar. It was not found on lymphocytes, eosinophils, erythrocytes, or erythroid progenitor cells. On the basis of tissue distribution and absorption studies, the antigen (tentatively designated the "myelo-monocytic" antigen) is distinct from antigens previously identified on human neutrophils. Restriction of the "myelo-monocytic" antigen to normal and malignant cells of the myelo-monocytic series suggests that it may represent a normal differentiation antigen of the myelo-monocytic lineage.

We studied the effect of the H2-receptor antagonists, cimetidine and metiamide, on in vitro myeloid colony formation by bone marrow cells from seven normal volunteers and two patients with a cimetidine-associated neutropenia. A cimetidine concentration of 500 microgram/ml produced 50% inhibition of normal granulocyte-macrophage colony formation, and 1000 microgram/ml of cimetidine completely suppressed proliferation. The inhibitory effect of metiamide occurred at lower concentrations: 50% inhibition at 250 microgram/ml and 95% inhibition at 350 microgram/ml. Cimetidine had a similar inhibitory effect on colony formation by recovery marrow from the two patients with cimetidine-associated neutropenia. Treatment of autologous and allogeneic marrows with patients' acute phase sera and cimetidine failed to show evidence of antibody-mediated suppression of granulopoiesis. Our results indicate that at sufficiently high concentrations, cimetidine and metiamide inhibit human bone marrow myeloid colony formation in vitro. These findings are consistent with the hypothesis that H2-receptor antagonists may produce neutropenia in a dose-related fashion by injury to granulocytic progenitor cells in vivo.

We studied recovery of peripheral blood- and bone marrow-derived myeloid progenitor cells (CFU-G,M) in 29 patients who received bone marrow transplants 2 mo to 8.5 yr previously. All patients had normal levels of peripheral blood neutrophils, normal bone marrow cellularity, and a normal myeloid-erythroid ratio. Both peripheral blood- and bone marrow-derived CFU-G,M were markedly reduced compared with normal controls and bone marrow donors [5 +/- 1/10(6) vs. 37 +/- 4/10(6) (P less than 0.001) and 23 +/- 5/2 x 10(5) vs. 170 +/- 21/2 x 10(5) (P less than 0.001)]. Five patients had no detectable CFU-G,M even when 10(6) bone marrow cels were plated. These abnormalities of CFU-G,M were unrelated to age, sex, diagnosis, conditioning regimen, dose of bone marrow cells transplanted, and presence or absence of graft-vs.-host disease. Patients who received either autotransplants or transplants from identical twins also had decreased or absent CFU-G,M indicating that allogeneic factors and posttransplant immune suppressor with methotrexate or corticosteroids were not major determinants of this abnormality. Co-culture of normal or donor peripheral blood or bone marrow mononuclear cells with recipients peripheral blood or bone marrow mononuclear cells, purified T cells, or serum failed to show any evidence of active CFU-G,M suppression. Furthermore, the abnormality of CFU-G,M could not be corrected by the addition of normal syngeneic (donor) hematopoietic cells or serum. Depletion of T-cells from recipient bone marrow by physical techniques resulted in marked increase in CFU-G,M (36 +/- 13 vs. 138 +/- 36; P less than 0.05). The abnormality could be reproduced in vitro by readdition of autologous T cells. In contrast to results with T cell depletion by physical techniques, T cell depletion with a monoclonal anti-T antibody (B7) and complement had no effect. These data indicate that most-transplant recipients have a marked abnormality in CFU-G,M when these cells are cultured in vitro. In at least some of these patients, the decreased cloning efficiency of CFU-G,M appears to be mediated by a suppressive effect of autologous T cells.