Induction of immune tolerance in patients with hemophilia A and inhibitors treated with porcine VIIIC by home therapy

Home therapy with porcine factor VIIIC was safe and effective when administered to five hemophilic patients over periods of 8 1/2, 6, 4, 3 1/2, and 2 years. No significant transfusion reactions occurred. Before treatment with porcine factor VIIIC, all five had high-level, high- responding anti-human VIIIC inhibitors initially lacking anti-porcine factor VIIIC activity. Although specific anti-porcine VIIIC inhibitors arose in all patients, these were generally transient, and only one patient became refractory to treatment. We believe that porcine factor VIIIC is the treatment of choice in patients whose inhibitors do not cross-react. All five patients lost their original anti-human VIIIC inhibitors after starting treatment with porcine VIIIC, permitting the reintroduction of human VIIIC in three of them. There has been no recurrence of anti-human VIIIC inhibitor activity during 2 to 3 years of regular treatment with human VIIIC in these patients. This suggests that tolerance to human VIIIC has arisen as a result of treatment with porcine VIIIC. Porcine VIIIC may have a role in the desensitization of some factor VIIIC inhibitor patients.     

Factor V Quebec revisited

Factor V Quebec has been described as a bleeding disorder that exhibits an autosomal dominant inheritance pattern and presents severe bleeding after trauma. Two members of a fourth-generation (IV.13 and IV.15) Canadian family have been studied in detail and are the subject of this report. Their clinical presentations and histories have been described previously (Tracy et al: J Clin Invest 74:1221, 1984). Persistent abnormalities include mild thrombocytopenia and defective platelet factor V. Plasma factor V is present at near normal concentration and is fully functional. Thus, the bleeding diathesis appears to reflect the absence of platelet factor V activity. The recent report (Hayward et al: Blood 84:110a, 1994 [suppl, abstr]) of multimerin deficiency in these individuals led us to reevaluate these patients. Western blot analyses of platelet lysates developed with a variety of monoclonal antibodies show that the alpha-granule proteins, fibrinogen, von Willebrand factor, factor V and osteonectin are decreased in concentration and significantly degraded in the platelets of these patients. Thrombospondin, while not degraded, is substantially decreased. In contrast, platelet factor 4 and beta-thromboglobulin do not appear to be affected. These observations suggest that the alpha- granules are correctly assembled but the contents are subsequently subjected to proteolytic degradation. The results indicate that factor V Quebec disorder is probably associated with a generalized defect that leads to degradation of most proteins of the alpha-granules.     

Abnormal function of the bone marrow microenvironment in chronic myelogenous leukemia: role of malignant stromal macrophages

The bone marrow microenvironment supports and regulates the proliferation and differentiation of hematopoietic cells. Dysregulated hematopoiesis in chronic myelogenous leukemia (CML) is caused, at least in part, by abnormalities in CML hematopoietic progenitors leading to altered interactions with the marrow microenvironment. The role of the microenvironment itself in CML has not been well characterized. We examined the capacity of CML stroma to support the growth of long-term culture-initiating cells (LTC-IC) obtained from normal and CML marrow. The growth of normal LTC-IC on CML stroma was significantly reduced compared with normal stroma. This did not appear to be related to abnormal production of soluble factors by CML stroma because normal LTC- IC grew equally well in Transwells above CML stroma as in Transwells above normal stroma. In addition, CML and normal stromal supernatants contained similar quantities of both growth-stimulatory (granulocyte colony-stimulating factor (CSF), interleukin-6, stem cell factor, granulocyte-macrophage CSF, and interleukin-1 beta) and growth- inhibitory cytokines (transforming growth factor-beta, macrophage inflammatory protein-1 alpha, and tumor necrosis factor-alpha). The relative proportion of different cell types in CML and normal stroma was similar. However, polymerase chain reaction and fluorescence in situ hybridization studies showed the presence of bcr-abl-positivo cells in CML stroma, which were CD14+ stromal macrophages. To assess the effect of these malignant macrophages on stromal function, CML and normal stromal cells were separated by fluorescence-activated cell sorting into stromal mesenchymal cell (CD14-) and macrophage (CD14+) populations. CML and normal CD14- cells supported the growth of normal LTC-IC equally well. However, the addition of CML macrophages to normal or CML CD14- mesenchymal cells resulted in impaired progenitor support. This finding indicates that the abnormal function of CML bone marrow stroma is related to the presence of malignant macrophages. In contrast to normal LTC-IC, the growth of CML LTC-IC on allogeneic CML stromal layers was not impaired and was significantly better than that of normal LTC-IC cocultured with the same CML stromal layers. These studies demonstrate that, in addition to abnormalities in CML progenitors themselves, abnormalities in the CML marrow microenvironment related to the presence of malignant stromal macrophages may contribute to the selective expansion of leukemic progenitors and suppression of normal hematopoiesis in CML.     

Multiple supernumerary teeth associated with an impacted maxillary central incisor: Surgical and orthodontic management

Various anomalies in the size, shape, number, structure and eruption of the teeth are often observed clinical conditions. Supernumerary teeth can be found in almost any region of the dental arch, and most of the times they are asymptomatic, and are routinely found during radiographic evaluation. The most common cause of impacted maxillary incisors is the presence of the supernumerary teeth. This paper describes a case of multiple supernumerary teeth associated with an impacted permanent maxillary central incisor in an 11-year old child along with its surgical and orthodontic management.     

RecA.oligonucleotide filaments bind in the minor groove of double-stranded DNA.

Escherichia coli RecA protein, in the presence of ATP or its analog adenosine 5'-[gamma-thio]triphosphate, polymerizes on single-stranded DNA to form nucleoprotein filaments that can then bind to homologous sequences on duplex DNA. The three-stranded joint molecule formed as a result of this binding event is a key intermediate in general recombination. We have used affinity cleavage to examine this three-stranded joint by incorporating a single thymidine-EDTA.Fe (T*) into the oligonucleotide part of the filament. Our analysis of the cleavage patterns from the joint molecule reveals that the nucleoprotein filament binds in the minor groove of an extended Watson-Crick duplex.     

Hematopoietic growth factors for graft failure after bone marrow transplantation: a randomized trial of granulocyte-macrophage colony- stimulating factor (GM-CSF) versus sequential GM-CSF plus granulocyte- CSF

Delay in hematologic recovery after bone marrow transplantation (BMT) can extend and amplify the risks of infection and hemorrhage, compromise patients' survival, and increase the duration and cost of hospitalization. Because current studies suggest that granulocyte- macrophage (GM) colony-stimulating factor (CSF) may potentiate the sensitivity of hematopoietic progenitor cells to G-CSF, we performed a prospective, randomized trial comparing GM-CSF (250 micrograms/m2/d x 14 days) versus sequential GM-CSF x 7 days followed by G-CSF (5 micrograms/kg/d x 7 days) as treatment for primary or secondary graft failure after BMT. Eligibility criteria included failure to achieve a white blood cell (WBC) count > or = 100/microL by day +21 or > or = 300/microL by day +28, no absolute neutrophil count (ANC) > or = 200/microL by day +28, or secondary sustained neutropenia after initial engraftment. Forty-seven patients were enrolled: 23 received GM-CSF (10 unrelated, 8 related allogeneic, and 5 autologous), and 24 received GM- CSF followed by G-CSF (12 unrelated, 7 related allogeneic, and 5 autologous). For patients receiving GM-CSF alone, neutrophil recovery (ANC > or = 500/microL) occurred between 2 and 61 days (median, 8 days) after therapy, while those receiving GM-CSF+G-CSF recovered at a similar rate of 1 to 36 days (median, 6 days; P = .39). Recovery to red blood cell (RBC) transfusion independence was slow, occurring 6 to 250 days (median, 35 days) after enrollment with no significant difference between the two treatment groups (GM-CSF: median, 30 days; GM-CSF+G- CSF; median, 42 days; P = .24). Similarly, platelet transfusion independence was delayed until 4 to 249 days (median, 32 days) after enrollment, with no difference between the two treatment groups (GM- CSF: median, 28 days; GM-CSF+G-CSF: median, 42 days; P = .38). Recovery times were not different between patients with unrelated donors and those with related donors or autologous transplant recipients. Survival at 100 days after enrollment was superior after treatment with GM-CSF alone. Only 1 of 23 patients treated with GM-CSF died versus 7 of 24 treated with GM-CSF+G-CSF who died 16 to 84 days (median, 38 days) after enrollment, yielding Kaplan-Meier 100-day survival estimates of 96% +/- 8% for GM-CSF versus 71% +/- 18% for GM-CSF+G-CSF (P = .026). These data suggest that sequential growth factor therapy with GM-CSF followed by G-CSF offers no advantage over GM-CSF alone in accelerating trilineage hematopoiesis or preventing lethal complications in patients with poor graft function after BMT.(ABSTRACT TRUNCATED AT 400 WORDS)     

From the Cover: Erosion of functional independence early in the evolution of a microbial mutualism

Many species have evolved to function as specialized mutualists, often to the detriment of their ability to survive independently. However, there are few, if any, well-controlled observations of the evolutionary processes underlying the genesis of new mutualisms. Here, we show that within the first 1,000 generations of initiating independent syntrophic interactions between a sulfate reducer (Desulfovibrio vulgaris) and a hydrogenotrophic methanogen (Methanococcus maripaludis), D. vulgaris frequently lost the capacity to grow by sulfate respiration, thus losing the primary physiological attribute of the genus. The loss of sulfate respiration was a consequence of mutations in one or more of three key genes in the pathway for sulfate respiration, required for sulfate activation (sat) and sulfate reduction to sulfite (apsA or apsB). Because loss-of-function mutations arose rapidly and independently in replicated experiments, and because these mutations were correlated with enhanced growth rate and productivity, gene loss could be attributed to natural selection, even though these mutations should significantly restrict the independence of the evolved D. vulgaris. Together, these data present an empirical demonstration that specialization for a mutualistic interaction can evolve by natural selection shortly after its origin. They also demonstrate that a sulfate-reducing bacterium can readily evolve to become a specialized syntroph, a situation that may have often occurred in nature.From flowering plants and their pollinators to the microbial endosymbionts of insects, there are many examples in nature of obligate mutualists (1, 2), or species dependent upon a mutually beneficial interaction for their survival or reproduction. How these interactions evolve is a mystery because much theory predicts that cooperative interactions should be unstable (3) and because of the difficulty of inferring evolutionary events that occurred in the distant past (4). Although there are few, if any, empirical observations of evolution toward dependence on mutualism, there are now several examples of mutualisms evolving de novo in the laboratory (5–8). This advancement has provided researchers an experimental framework to study populations and ecological conditions in the early stages of evolution (5–8).Here, we describe our observations of rapid and repeated evolution of increased dependency on a mutualism through natural selection. This interaction is similar to a widespread relationship between prokaryotes that plays a pivotal role in the decomposition of carbon in many oxygen-free environments. In these syntrophic mutualisms, bacteria ferment organic acids, producing hydrogen or formate as by-products, which are then used by hydrogen-consuming species, often methanogenic archaea (9). Removal of hydrogen and formate benefits the bacteria because the free energy (ΔG) available decreases with increasing concentrations of these products (9). A variety of bacterial species have been described that seem to be specialized for fermenting organic acids in syntrophic association with hydrogen-consuming species (10–13). Notably, most clades of characterized syntrophs share a recent common ancestry with sulfate reducers (10, 14). Some retain vestiges of the sulfate-reducing pathway, and several lines of evidence hint at the possibility that specialized syntrophs were once sulfate reducers (11, 14).Sulfate-reducing bacteria gain energy from organic acids, such as lactate, in the absence of oxygen by coupling their oxidation to the reduction of sulfate to sulfide. These bacteria play a critical role in sulfur and carbon cycling, contribute to corrosion in the petroleum industry and wastewater treatment plants, and have been used for bioremediation of toxic heavy metals (15). The ability of sulfate reducers to grow in syntrophic association with methanogens was first demonstrated in laboratory studies (16) and is now generally recognized to be of environmental relevance (17–19). Many sulfate reducers would therefore be better described as facultative syntrophs, well adapted to environments of fluctuating electron acceptor availability (17, 20). Past evolutionary transitions of sulfate-reducing bacteria between obligate and facultative syntrophs is also indicated by comparative analyses indicating horizontal transfer of genes in the pathway of sulfate respiration (21, 22). Thus, the evolutionary adaptive flexibility of sulfate-reducing bacteria suggests that they offer an attractive experimental system to study the evolution of mutualism.To understand how mutualisms, and syntrophic interactions in particular, might evolve from their origin, we paired the sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough, with the archaeon, Methanococcus maripaludis S2, and propagated 22 initially isogenic planktonic cocultures for 1,000 generations in medium with lactate but no sulfate or added hydrogen. In this environment, neither species can gain energy from the oxidation of lactate without syntrophic cooperation.Within the first 300 generations of evolution, the cocultures evolved increased stability, higher yields, and higher growth rates, with both species contributing to these changes (6), a trend that continued through 1,000 generations. We describe a common evolutionary outcome of these experiments. Many of the independently evolved D. vulgaris accumulated loss-of-function mutations in genes required for the reduction of sulfate, suggesting strong selection for mutations resulting in loss of the ability to respire sulfate during evolution in syntrophy.