Thrombocytopoiesis--analysis by membrane tracer and freeze-fracture studies on fresh human and cultured mouse megakaryocytes.

The origin of platelets (Pt) from megakaryocytes (MK) is beyond question, but the mechanism whereby Pts are released from the precursor cell is still debated. A widely-held theory claims that the MK plasma membrane invaginates to form demarcation membranes (DMS), which delineate Pt territories. Accordingly, Pts would be derived mostly from the periphery of the MK, and the MK and Pt plasma membranes would have to be virtually identical. Since, on morphologic grounds, this theory is untenable, several aspects of thrombocytopoiesis were reexamined with the help of membrane tracer and freeze-fracture analyses of freshly-collected human and cultured mouse MK. To our surprise, freeze-cleavage of the MK plasma membrane revealed that the vast majority of intramembranous particles (IMP) remained associated with the protoplasmic leaflet (P face), whereas the partition coefficient of IMPs of the platelet membrane was the reverse. This is the first time that any difference between MK and Pt membranes has been determined. Replicas of freeze-fractured MK that were in the process of thrombocytopoiesis revealed an additional novel phenomenon, i.e., numerous areas of membrane discontinuity that appeared to be related to Pt discharge. When such areas were small, the IMP were lined up along the margin of the crevice. At a later phase, a labyrinth of fenestrations was observed. Thin sections of MK at various stages of differentiation showed that Pt territories were fully demarcated before connections of the DMS with the surface could be found. Therefore, the Pt envelope is probably not derived from invaginations of the MK plasma membrane. When living, MK were incubated with cationic ferritin or peroxidase at 37 degrees C, the tracers entered into the DMS but did not delineate all membranes with which the DMS was in continuity, suggesting the existence of distinctive membrane domains. Interiorization of tracer was not energy-dependent, but arrested at low temperatures. At 4 degrees C the DMS remained empty, unless there was evidence that Pts had been released. In such instances, the tracers outlined infoldings of peripheral cytoplasm that was devoid of organelles. Thus, the majority of Pts seem to originate from the interior of the MK, and the surface membranes of the two cells differ in origin and structure. The observations do not only throw new light on the process of thrombocytopoiesis, but also strengthen the possibility that MKs and Pts may be subject to different stimuli.

Transient knock-down of kcna2 reduces sleep in larval zebrafish.

In the current study we set out to determine the effects of morpholino oligonucleotide (MO) knock-down of kcna2 on sleep-wake cycles in zebrafish. The results were compared to a non-overlapping MO injection, Dec2, who's mutant is also linked with a short sleep phenotype. Four groups of fish were used in the experiment: naïve fish, and fish injected with either control, kcna2, or Dec2 MO. All groups underwent 24-h behavioral monitoring of sleep-wake cycles at four and seven days-post-fertilization (dpf). First, we established an immobility dependent, sleep related, increase in arousal thresholds at both 4 and 7 dpf. Secondly, we show that kcna2 MO injected fish exhibit significantly less sleep behavior than controls and naïve fish, whereas Dec2 MO injections had similar but less severe effects. Finally, using kcna2 MO injected fish only, we turn to local field recordings at the level of the telencephalon and tectum opticum and rule out that the knock-down resulted in a non-specific increase in neural excitability that would mask sleep behavior.

Comparative effects of thrombopoietic-stimulatory factor and spleen cell-conditioned medium on megakaryocytopoiesis in a short-term bone marrow liquid culture system.

The effect of partially purified thrombopoietic stimulatory factor (TSF) on megakaryocytopoiesis was studied using the soft-gel colony-forming assay and a short-term marrow liquid culture system (STLC) and compared to the effects of megakaryocyte colony-stimulating activity present in pokeweed mitogen-stimulated spleen cell-conditioned medium (PWCM). Nonadherent cells from STLC were sampled daily for acetylcholinesterase-positive cells and megakaryocyte progenitor cells (CFU-M). CFU-M were assayed in the soft-gel colony-forming system using PWCM as a source of colony-stimulating activity. Proliferative capacity of CFU-M obtained from liquid culture was determined from megakaryocyte colony size (number of megakaryocytes per colony) following plating of cells in a secondary colony-forming assay. Megakaryocytes were grouped into four maturation classes and megakaryocyte diameter was determined on acetylcholinesterase-stained cytocentrifuged cells using an eye-piece micrometer. TSF produced no CFU-M-derived colonies in the soft-gel colony-forming assay. Addition of TSF to STLC had no effect on the total number of CFU-M, megakaryocyte colony size, or total number of megakaryocytes compared to unstimulated STLC. However, on days 4-9 there was a significant increase in megakaryocyte diameter and the proportion of mature (stage III, IV) megakaryocytes obtained from TSF containing STLC compared to unstimulated STLC. In contrast, 5 days after addition of PWCM to STLC a sixfold increase in the total number of CFU-M per flask and a threefold increase in megakaryocytes was observed compared to unstimulated STLC. However, megakaryocyte colony size and megakaryocyte size were significantly reduced and a greater number of immature (stage I, II) megakaryocytes were present in STLC containing PWCM compared to unstimulated STLC. These results indicate that TSF accelerates the maturation of megakaryocytes in vitro and that a factor or factors present in spleen cell-conditioned medium, in addition to influencing megakaryocyte progenitor cell proliferation, also affect(s) megakaryocyte size.

Inhibition of HIV replication by amino-sugar derivatives.

The plant alkaloids castanospermine, dihydroxymethyldihydroxypyrrolidine and deoxynojirimycin have recently been shown to have potential anti-HIV activity [(1987) Proc. Natl. Acad. Sci. USA 84, 8120-8124; (1987) Nature 330, 74-77; (1987) Lancet i, 1025-1026]. They are thought to act by inhibiting alpha-glucosidase I, an enzyme involved in the processing of N-linked oligosaccharides on glycoproteins. We report here the relative efficacy of a spectrum of amino-sugar derivatives as inhibition of HIV cytopathicity. Several alpha-glucosidase inhibitors and alpha-fucosidase inhibitors were found to be active at concentrations which were non-cytotoxic.

Diagnostic value of candida precipitins determined by counterimmunoelectrophoresis in patients with acute leukemia: a prospective study.

Thirty-nine patients receiving chemotherapy for acute leukemia had weekly determinations of Candida precipitins by use of counterimmunoelectrophoresis. The only two patients who had proven disseminated candidiasis both had diagnostic (greater than or equal to 1/8) titers. However, most patients with "possible" disseminated candidiasis did not have diagnostic titers. The test's specificity was high (90%), and only one of five patients infected with other fungi had diagnostic titers. However, because there were so few "true positives" in this small series, the relatively small number of "false positives" resulted in a low predictive value for this test. Based on a review of the literature and the authors' experience, the value of this test for patients who have acute leukemia does not appear to be well established.

Primary central nervous system lymphoma: long-term survival following treatment with radiation and methotrexate.

Primary lymphoma of the central nervous system is a rare disease with poor response to therapy. A 37-year-old man presented with primary cerebral lymphoma diagnosed by stereotactic brain biopsy. He was initially treated with whole brain irradiation but subsequently developed recurrent disease in the spinal cord manifested by paraplegia. Combined modality treatment with spinal cord irradiation, intrathecal methotrexate and 19 courses of high-dose systemic methotrexate with urinary alkalinization, resulted in stabilization of his neurologic status. No further disease progression has been observed and the patient remains free of disease 62 months after beginning chemotherapy. Methotrexate therapy may offer an effective means of treating recurrent primary central nervous system lymphomas.

New evidence for the mechanism of the tin(II) chloride catalyzed reactions of vicinal diols with diazodiphenylmethane in 1,2-dimethoxyethane.

A kinetic study of the tin(II) chloride catalyzed reaction of diazodiphenylmethane with ethylene glycol in dimethoxyethane is reported. The preparation and characterization of ethylene glycol monodiphenylmethyl ether, the main product from this reaction, is also reported as well as the preparation of the two diphenylmethyl monoethers of methyl 4,6-O-benzylidene-alpha-D-glucopyranoside. An unexpected relationship between the concentration of ethylene glycol and the pseudo first-order rate constant, k', was observed in these reactions. For low concentrations of ethylene glycol (below 0.06 M), k' increases with increasing concentration of the diol. This trend is reversed for high concentrations of ethylene glycol (from about 0.06 to about 0.2 M). The apparent rate constant was also inversely related to the initial concentration of diazodiphenylmethane for the concentrations investigated. These results make the previously proposed involvement of a 1,3,2-dioxastannolane intermediate very unlikely [Petursson, S.; Webber, J.M. Carbohydr. Res. 1982, 103, 41-52]. The results suggest that more likely intermediates for these reactions involve tin(II) chloride complexes in a dynamic equilibrium with the diol.

Metastatic medullary thyroid carcinoma. Complete response to combination chemotherapy with dacarbazine and 5-fluorouracil.

A 20-year-old woman had a sporadic case of medullary thyroid carcinoma (MTC) metastatic to the lungs. After a transient response to streptozotocin and doxorubicin, new subcutaneous lesions appeared on the left chest wall and there was progression of pulmonary disease. Because MTC is one of the amine precursor uptake and decarboxylation (APUD) tumors, treatment was undertaken with agents active in these diseases. Dacarbazine and 5-fluorouracil, given daily for 5 days every 4 weeks, resulted in complete resolution of pulmonary and subcutaneous lesions and a sharp decrease in tumor marker levels that lasted 10 months. Recurrence of the pulmonary disease lead to her death 21 months after presentation. Thus, the chemo-responsiveness of MTC may be akin to that of other APUD carcinomas (APUDomas) and treatment of metastatic MTC and other APUDomas with the combination of dacarbazine and 5-fluorouracil appears to merit further study.

Adenoid cystic carcinoma of the esophagus. Complete response to combination chemotherapy.

A 55-year-old man had adenoid cystic carcinoma of the esophagus metastatic to the lungs and right supraclavicular fossa. He was treated with local radiation therapy to the esophagus and supraclavicular fossa, followed by combination chemotherapy with doxorubicin, mitomycin C, and 5-fluorouracil (5-FU). After a modest initial response, disease progression was noted in the pulmonary nodules. He was then treated with cisplatin, cyclophosphamide, vincristine, and doxorubicin. After two cycles of this regimen, there was complete regression of his pulmonary nodules, which was sustained for 5 months. A review of 44 literature cases of esophageal adenoid cystic carcinoma contrasted with adenoid cystic carcinoma of salivary gland origin indicated that the esophageal adenoid cystic carcinomas have a high tendency to metastasize (76% of cases) and a much poorer prognosis, with only 23% 1-year survival rate. It was concluded that esophageal adenoid carcinoma is clinopathologically distinct from the salivary gland variant, and that combination chemotherapy may be an effective treatment modality for this cancer.

Effect of spleen cell conditioned medium on megakaryocytopoiesis in a short-term bone marrow culture system.

The effect of pokeweed mitogen-stimulated spleen cell conditioned medium (PWCM) on the proliferation of megakaryocytes and megakaryocyte progenitor cells (CFU-M) was studied with a short-term liquid culture (STLC) system. Adherent and nonadherent cells were sampled daily for acetylcholinesterase-positive cells and CFU-M. The proliferative capacity of CFU-M was determined by culturing cells from STLC in secondary methylcellulose cultures and counting the number of megakaryocytes per colony. Positive dose-related effects were observed between the number of megakaryocytes and CFU-M in liquid culture and the concentration of PWCM in the culture. In contrast, the proliferative capacity of CFU-M was lower in cultures containing high concentrations of PWCM compared with cultures containing low concentrations of PWCM. Furthermore, mean megakaryocyte diameter was significantly smaller in cultures containing high levels of PWCM compared with cultures with low concentrations. These data suggest that at low levels of conditioned medium, megakaryocytopoiesis is characterized by production of fewer CFU-M with a higher proliferative capacity and fewer large megakaryocytes. In turn, high concentrations of PWCM promote the production of a greater number of CFU-M with reduced proliferative capacity and an increased number of small megakaryocytes.

Spinal cord involvement in leukemia: a review of the literature and a case of Ph1+ acute myeloid leukemia presenting with a conus medullaris syndrome.

A 21-year-old male with Ph1 positive acute myeloid leukemia (AML) became constipated, experienced pain in the sacral area and retained urine. These were the principle presenting complaints of his disease. With local radiotherapy and systemic chemotherapy, all neurologic symptoms and signs disappeared within three days. A conus medullaris syndrome is uncommon in leukemia, as we were able to find only three other such patients in the literature and three others with the closely related cauda equina syndrome. A literature review of spinal cord involvement in leukemia in general revealed 67 reasonably well-described cases. Compilation of data from these suggests it is most often seen in patients with AML and that it is most common in the second decade of life. Tumors extrinsic to but compressing the cord are the most common finding and the thoracic cord is the most common site of involvement.

Effects of hypoxia on megakaryocytopoiesis and granulopoiesis.

Previous studies have reported that exposure of mice to hypoxic conditions results in a decrease in blood platelets. To further explore the effect of hypoxia on megakaryocytopoiesis, megakaryocytes and their progenitor cells (CFU-M) were studied in hypoxic mice. Mice were exposed to hypoxia by enclosure in cages covered with dimethyl-silicone rubber membranes for up to 10 d. At various times during the hypoxic and ex-hypoxic periods the total megakaryocytes and CFU-M were determined in the humerus and spleen. CFU-M were assayed in the soft gel colony forming assay using pokeweed mitogen-stimulated spleen cell conditioned medium (PWCM) as a source of colony stimulating activity. After 10 d of hypoxia, packed red cell volume (PRCV) increased to 148% of control levels and blood platelets decreased to 40% of controls. Total megakaryocytes and CFU-M per humerus decreased to 18% and 50% of controls respectively. 4 d into the ex-hypoxic phase, PRCV was still increased at 128% of controls while marrow megakaryocytes and CFU-M increased to normal levels. Platelet recovery was somewhat slower, returning to normal by d 6. In contrast to the findings in the marrow, total spleen megakaryocytes and CFU-M increased to about 3- and 5-fold of control levels respectively by 6 d of hypoxia. During the exhypoxic phase, CFU-M decreased to normal on d 4, followed by a rebound of 3-fold control values on d 8. Spleen megakaryocytes decreased more slowly, returning to normal by d 10. A marked granulocytosis was observed during the hypoxic phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Megakaryocytopoiesis and granulopoiesis in W/Wv mice: comparisons between bone marrow and spleen.

To determine how megakaryocyte progenitor cells (CFU-M) of W/Wv mice are affected by the hematopoietic stem cell abnormality, megakaryocytopoiesis was studied in the spleen and marrow of these genetically anemic W/Wv mice. CFU-M were assayed in the soft gel colony-forming system. Megakaryocyte colony size was determined by counting the number of megakaryocytes per colony, and megakaryocyte diameter was determined on acetylcholinesterase-stained cytocentrifuged cell preparations with use of an eyepiece micrometer. In spite of normal blood platelet levels, megakaryocyte level was reduced in the spleen and humerus to about 60% that of +/+ littermates. Megakaryocyte diameters were increased in W/Wv mice. CFU-M in W/Wv mice were reduced to 40% the number seen in the spleen of +/+ mice and to 62% in the humerus. In cell cycle studies, significantly fewer marrow CFU-M were in DNA synthesis in W/Wv mice compared with +/+ animals, but similar numbers of cells were in cycle in the spleen for both genotypes. No difference was observed between W/Wv and +/+ CFU-M in their requirement for exogenous colony-stimulating activity or in the distribution of colony sizes. However, CFU-M-derived colonies cloned from adherent cell-depleted marrow cells were significantly smaller compared with those cultured from unfractionated marrow cells. Results for granulocytes and granulocyte-macrophage progenitor cells (CFU-GM) were similar to those obtained for the megakaryocyte series, indicating that the abnormalities are present in different cell lineages. These results suggest that the macromegakaryocytosis of W/Wv mice appears to be a compensation for the megakaryocytopenia. Cells in the progenitor cell compartment appeared not to be involved in this compensation. Furthermore, adherent cells appear to elaborate a factor regulating megakaryocyte development. These findings are compatible with two-level regulation of megakaryocyte formation and a complex mechanism of blood platelet level regulation.

Megakaryocytopoiesis and granulopoiesis following cyclophosphamide.

CTX is a marrow-suppressant drug that has been reported to have a "sparing effect" on blood platelets. To investigate whether CTX spares platelet precursor cells as well as platelets, blood platelets, megakaryocytes, and CFU-M were studied in mice after the injection of CTX. The soft gel in vitro culture system was utilized to assay for CFU-M in marrow and spleen. CTX produced only a mild thrombocytopenia to 88% to 95% of control values. This was followed by a modest, but significant thrombocytosis of 120% of controls on day 10. Despite the slight effect on blood platelets, striking changes were observed in megakaryocytes and CFU-M. Megakaryocytes and CFU-M were decreased to 10% to 25% of control values within 24 hr of CTX administration. This was followed by a recovery of spleen megakaryocytes and CFU-M to normal within 6 days, followed by a 24-fold increase above control values in spleen megakaryocytes and a 29-fold increase in spleen CFU-M. In contrast, recovery of megakaryocytes and CFU-M in the marrow was delayed for 2 weeks. An increase in marrow CFU-M above control values was not observed, although megakaryocytes increased to 150% of control. Granulocytes and their progenitor cells (CFU-GM) responded in a similar manner after CTX administration. These results indicate that blood platelet levels are only slightly decreased after a single injection of CTX whereas CFU-M and megakaryocytes are markedly decreased. The recovery and increased production of megakaryocytes and CFU-M in the face of a near-normal platelet level suggests that factors other than the platelet level are responsible for production of CFU-M and megakaryocytes.

Megakaryocytopoiesis and granulopoiesis of W/Wv mice studied in long-term bone marrow cultures.

Megakaryocytopoiesis and granulopoiesis of marrow cells from W/Wv mice were studied using a continuous liquid marrow culture system. Cells in the suspension phase were assayed weekly over a 16-week period for total nucleated cells, megakaryocytes, granulocytes, megakaryocytes and granulocyte-macrophage progenitor cells (CFU-Ms, CFU-GMs), and spleen colony-forming cells (CFU-Ss). Without hydrocortisone supplementation, proliferation of megakaryocytes, granulocytes, and their progenitor cells was significantly less in W/Wv cultures than in +/+ cultures. These cells became undetectable in both W/Wv and +/+ cultures at seven to 11 weeks in culture, after which only monocytes and macrophages proliferated in the cultures. Treatment of cultures with hydrocortisone improved megakaryocytopoiesis and granulopoiesis in both W/Wv and +/+ cultures. Following an initial lag phase of three to four weeks, proliferation of megakaryocytes, granulocytes, and their progenitor cells in W/Wv cultures equalled that observed in +/+ cultures and was sustained for 16 to 24 weeks. This improvement was associated with a sustained reduction in monocytes and macrophages. Despite improvements in megakaryocytopoiesis and granulopoiesis, production of macroscopic and microscopic spleen colonies by cells from W/Wv cultures remained severely reduced or absent. Studies of DNA synthesis rates of fresh marrow cells indicated that significantly fewer CFU-Ms and CFU-GMs were in cycle in W/Wv mice compared with +/+ mice. However, in hydrocortisone-treated W/Wv cultures, DNA synthesis rates of CFU-Ms and CFU-GMs increased markedly and equalled those observed for +/+ cultures. These results suggest that the improvements in megakaryocytopoiesis and granulopoiesis in hydrocortisone-treated liquid cultures is associated with a reduction in monocytes and macrophages and that progenitor cells of W/Wv mice have a proliferative defect that is correctable by hydrocortisone treatment in vitro.

Incidence and range of microscopic hematuria in patients with indwelling urinary catheters.

The presence and number of red blood cells (RBCs) in the urine after catheterization were ascertained in 40 patients without urinary tract pathology or other cause for hematuria. Before catheterization, 90% of the patients had less than or equal to 1 RBC/high power field (HPF), a figure similar to that for normal volunteers. Two days after catheterization, however, 61,5% of the patients had 2 RBC/HPF with a range between zero and "too numerous to count." The incidence of "hematuria" (greater than or equal to 2 RBC/HPF) declined with time. Neither urinary specific gravity nor urinary tract infection could be correlated with hematuria. At any time after catheterization, 90% of the patients had greater than or equal to 50 RBC/HPF. These data may be helpful in differentiating hematuria caused by the catheter from that due to drug reactions or disease states.

Megakaryocytopoiesis and granulopoiesis after murine cytomegalovirus infection.

Thrombopoiesis and granulopoiesis following murine cytomegalovirus infection were investigated by studying changes in megakaryocytes, megakaryocyte and granulocyte-macrophage progenitor cells, and spleen colony-forming cells. The soft gel in vitro culture system was used to assay for megakaryocyte and granulocyte-macrophage progenitor cells in marrow and spleen. Murine cytomegalovirus produced a mild thrombocytopenia to 90% of control values 1 day after infection at a time when marrow megakaryocyte levels were normal, suggesting a mild direct toxic effect of the virus on platelets. A reduction of megakaryocytes, megakaryocyte and granulocyte-macrophage progenitor cells, and spleen colony-forming cells to 40% to 60% of control values occurred within 24 to 48 hours of infection in association with an additional decrease in platelets to 58% of control levels on day 4. In vitro inoculation of marrow cell cultures with murine cytomegalovirus also resulted in a reduction of megakaryocyte- and granulocyte-macrophage colony-forming cells within 24 to 48 hours, suggesting that murine cytomegalovirus-induced thrombocytopenia and granulocytopenia may be in part caused by direct infection of precursor cells. The recovery of cells in the spleen was followed by a striking seven- to 10-fold increase in spleen colony-forming cells and megakaryocyte and granulocyte-macrophage progenitor cells in the spleen. These marked increases followed significant increases in spleen cell production of colony-stimulating activities within 2 days of murine cytomegalovirus infection, suggesting that hematopoietic cell recovery is mediated by increased local production of colony-stimulating activities in the spleen.

Long-term survival after allogeneic bone marrow transplantation complicated by trichosporosis.

We report a case of sepsis due to Trichosporon cutaneum in a 20-year-old patient with acute promyelocytic leukemia. Neutropenia with a hypocellular marrow persisted for 90 days after two courses of induction chemotherapy with mitoxantrone and ara-C. Amphotericin B, fluconazole, and granulocyte-macrophage colony-stimulating factors were administered. Neutropenia (ANC < 1,000/microL) resolved 14 days after HLA-identical bone marrow transplantation. The patient is in remission, with a performance status of 100%, more than 1 year after transplantation.

The acyl-enzyme mechanism of beta-lactamase action. The evidence for class C Beta-lactamases.

Methanol or ethanol can replace water in the action of certain chromosomal beta-lactamases on benzylpenicillin: the products are alpha-methyl or alpha-ethyl benzylpenicilloate. The beta-lactamases were from a mutant of Pseudomonas aeruginosa 18S that produces the enzyme constitutively [Flett, Curtis & Richmond (1976) J. Bacteriol. 127, 1585-1586; Berks, Redhead & Abraham (1982) J. Gen. Microbiol. 128, 155-159] and from Escherichia coli K12 (the ampC beta-lactamase) [Lindström, Boman & Steele (1970) J. Bacteriol. 101, 218-231]. The variation of the rates of alcoholysis and hydrolysis with concentration of alcohol show that the rate-determining step is breakdown of an intermediate. This intermediate is likely to be the acyl-enzyme. The esters, alpha-methyl or alpha-ethyl benzylpenicilloate, are themselves substrates for the Pseudomonas beta-lactamase, benzylpenicilloic acid being formed. Thus this beta-lactamase can be an esterase. The kinetics for the hydrolysis of cloxacillin by the Pseudomonas beta-lactamase are consistent with the acyl-enzyme, formed by acylation of serine-80, being an intermediate in the overall hydrolysis.