In vitro megakaryocytopoietic and thrombopoietic activity of c-mpl ligand (TPO) on purified murine hematopoietic stem cells

Recently, the ligand for c-mpl has been identified and cloned. Initial studies of this molecule indicate that it is the platelet regulatory factor, thrombopoietin (TPO). Previous work has indicated that c-mpl is expressed in very immature hematopoietic precursors and thus raised the possibility that TPO may act directly on the hematopoietic stem cell. Therefore, in these studies, we investigate the effects of TPO on hematopoietic stem cell populations isolated from the murine fetal liver and bone marrow. Cocultivation of stem cells with fetal liver stroma give rise to multilineage expansion of the stem cells but with little or no megakaryocytopoiesis. Addition of TPO to these cocultures gives significant megakaryocyte production. This production is enhanced in combination with Kit ligand or interleukin-3. The addition of TPO to stem cell suspension cultures produces a dynamic thrombopoietic system in which stem cells undergo differentiation to produce megakaryocytes and proplatelets. These experiments show that the megakaryocytopoietic and thrombopoietic activities of TPO are initiated at the level of an early progenitor cell or upon the hematopoietic stem cell.     

Measurement of ploidy distribution in megakaryocyte colonies obtained from culture: with studies of the effects of thrombocytopenia

Microdensitometric measurement of the DNA content of individual megakaryocytes was performed using megakaryocyte colonies obtained following culture, in soft agar, of hematopoietic cells from C57BL/6J mice. Two types of colonies were detected. After 7 days of culture, the big cell type contained 16 /+- 2.3 acetylcholinesterase (AChE) positive cells/colony, with a mean ploidy level of 16.8 /+- 0.8/cell and the ploidy distribution characteristic of recognizable megakaryocytes in bone marrow. The heterogeneous type contained 44 /+- 9.6 cells/colony (some of which were AChE negative), with a mean ploidy level of 6.8 /+- 0.7/cell. The ploidy distribution of heterogeneous colonies differed markedly from big cell colonies, with preponderance of 2N and 4N cells. Colony-forming cells, obtained 4-5 days after induction of acute thrombocytopenia, gave big cell colonies with a marked increase in DNA content. Mean ploidy level increased to 21.5 /%- 1.8/cell; the frequency of 32N cells increased from 17% to 30% and 64N cells from 0% to 6%. This is the pattern of change observed in bone marrow, in vivo, 24 to 48 hr after induction of acute thrombocytopenia. The number of cells/colony did not increase. In contrast, acute thrombocytopenia did not alter the ploidy of heterogeneous colonies. The different responses to the stimulus of acute thrombocytopenia suggest that there are at least two types of Meg-CFC. The delayed appearance of altered Meg-CFC that produced big cell colonies indicates that the pool of stem cells, from which committed megakaryocyte precursors are derived, may respond indirectly to the stimulus of platelet depletion.     

A fatal case of AIDS-defining meningoencephalitis by C. Neoformans,sensitive to antifungal therapy

Cryptococcus neoformans is the most common cause of life threatening meningoencephalitis in HIV-infected patients. Diagnosis is based on tests for cryptoccocal antigen in serum and cerebrospinal fluid, and on culture of the organism. We present a case of AIDS-related cryptococcal meningoencephalitis unresponsive to antifungal combination therapy, despite of evidence of fungal susceptibility in vitro. Significant decreases in cryptococcal antigen titers in serum and cerebrospinal fluid did not correlate with progress in disease and fatal outcome.     

The classification and diagnostic algorithm for primary lymphatic dysplasia: an update from 2010 to include molecular findings

Historically, primary lymphoedema was classified into just three categories depending on the age of onset of swelling; congenital, praecox and tarda. Developments in clinical phenotyping and identification of the genetic cause of some of these conditions have demonstrated that primary lymphoedema is highly heterogenous. In 2010, we introduced a new classification and diagnostic pathway as a clinical and research tool. This algorithm has been used to delineate specific primary lymphoedema phenotypes, facilitating the discovery of new causative genes. This article reviews the latest molecular findings and provides an updated version of the classification and diagnostic pathway based on this new knowledge.     

Tricortical Calcaneal Bone Graft and Management of the Donor Site

The clinical outcomes of 19 patients requiring autogenous grafts for foot surgery were followed up until healing at the donor site occurred. In all cases, tricortical bone was extracted from the calcaneus for use at another pedal site. The first cohort of 9 patients had the calcaneal deficit replaced with allogenic cubes. The second cohort received no tissue replacement. Patients were reviewed postoperatively with a questionnaire and clinical examination to evaluate the outcome of the operations. Radiographic outcomes were observed at the donor and recipient sites in both groups until healing was confirmed as bridging trabeculation. Incorporation of the graft at the donor site was also reviewed. Clinical outcomes, namely pain, local sensory function, and return to footwear, were satisfactory in all patients and were not significantly different between groups. One patient from each group sustained a heel fracture. The donated autogenous grafts at the recipient sites were all incorporated uneventfully at 6 months. In the first cohort, allogenic graft incorporation in the calcaneus was complete in only 2 patients at the 12-month stage. The remaining 7 cases showed reduction of the deficit by new bone formation arising from the calcaneus. Donor sites with allogenic bone replacement healed at a median of 18 months (interquartile range, 18-18 months). In the group without replacement, healing occurred at a median of 6 months (interquartile range, 6-12 months), a highly statistically significant difference (P < .001). In the second cohort without allogenic graft replacement, radiographic filling at the donor site was complete within a 12-month period. Tricortical bone can be successfully harvested from the calcaneus, but there may an associated risk of heel fracture. The role of replacement allogenic bone in assisting healing at the donor site is unclear.     

Double sodium and potassium sulphates revealed by microprobe analysis in dried cervical mucus: a mid-cycle crystallographic index

The use of physical techniques such as scanning electron microscopyand microprobe analysis permits the analysis of valuable detailsof the structure of dried samples of mid-cycle cervical mucusduring the period of maximum ferning. From a crystallographicpoint of view, particular attention was paid to the location,morphology and chemical composition of anisotropic organic sulphatemicrostructures. As distinct from the isotropic sodium chloridepattern described by Papanicolaou, these appear in a narrowperiod of time corresponding to the middle of the period ofmaximum ferning. Anisotropic microstructures were revealed tobe tiny isolated crystals or polycrystalline aggregates composedof double salts of potassium and sodium. The potassium/sodiumcation ratio, equal to 3/1, was constant in the best specimens.Using a simple polarizing microscope, the detection of anisotropicmicrostructures may be useful to determine the period of maximumfertility more accurately. In addition, experiments have beencarried out in order to determine the relative importance ofsulphate anisotropic structures as compared with the whole crystallizedsurface and to investigate precisely their transience afterthey appear.