Breaking the reproductive barrier of divergent species to explore the genomic landscape.

Background: Climate change will have significant consequences for species. Species range shifts induce the emergence of new hybrid zones or the spatial displacement of pre-existing ones. These hybrid zones may become more porous as alleles are passed from one species to another. Currently, hybridization between highly divergent species living in sympatry seems extremely limited. Indeed, this phenomenon involves breaking two barriers. The first is the pre-mating barrier, related to the reproductive phenology of the two species. The second is the post-zygotic barrier, related to the genetic divergence between these species. Here, we were interested in identifying new hybridization patterns and potential implications, especially in the context of environmental modifications. Methods: We sampled Telestes souffia and Parachondrostoma toxostoma wild specimens from different locations across France and genotyped them for SNP markers. We identified discriminant loci using F1-hybrid specimens and parental species and performed principal component analysis and Bayesian model-based clustering to analyze phylogenetic information. Furthermore, we assessed deviation in allele frequency from F1 to F2 and for Hardy-Weinberg equilibrium for F2 and assessed gene function associated with two F2 cohorts. Results: We demonstrate that by breaking the ecological barrier, massive introgressive hybridization is possible between two endemic lineages of Cyprinidae belonging to two distinct genera. For both cohorts studied (=2 cm and >2 cm), a large majority of loci (>88%) presented no deviation in allele frequency and no departure from the Hardy-Weinberg equilibrium. For individuals beyond the 2 cm stage, two phenomena were observed. The first was an allelic imbalance in favor of P. toxostoma, for some genomic regions, with genes involved in developmental regulatory processes, cytoskeletal organization, and chromosome organization. The second was an excess of heterozygous loci coupled with an equilibrium of allelic frequencies for genes involved in immune response and kidney/liver development. Moreover, the 2 cm-sized specimens with high mortality yielded a particular genomic signature. Conclusion: Our study displayed important results for understanding the early stages of hybridization between divergent lineages and predicting the emergence of future hybrid zones in the wild. Moreover, this hybridization generates a wide spectrum of hybrids that are a potential source of important evolutionary novelties.

Unusual co-infection of severe malaria by Plasmodium vivax and dengue virus in Mexico.

Malaria and dengue fever are among the most common mosquito-borne diseases worldwide; however, reports of coinfection are rare. We present a case of severe malaria and dengue coinfection in a 16-yearold female patient presenting with fever, thrombocytopenia, pleural effusion, myopericarditis, and acute respiratory distress syndrome. Dengue infection was confirmed by the presence of immunoglobin M antibodies and nonstructural protein 1, while malaria was confirmed by the presence of Plasmodium vivax in thick and thin blood smears. This is the first report of a dengue/malaria coinfection in Mexico.

Unusual co-infection of severe malaria by Plasmodium vivax and dengue virus in Mexico

@#Malaria and dengue fever are among the most common mosquito-borne diseases worldwide; however, reports of coinfection are rare. We present a case of severe malaria and dengue coinfection in a 16-yearold female patient presenting with fever, thrombocytopenia, pleural effusion, myopericarditis, and acute respiratory distress syndrome. Dengue infection was confirmed by the presence of immunoglobin M antibodies and nonstructural protein 1, while malaria was confirmed by the presence of Plasmodium vivax in thick and thin blood smears. This is the first report of a dengue/malaria coinfection in Mexico.

Retroviral vector integration in post-transplant hematopoiesis in mice conditioned with either submyeloablative or ablative irradiation.

X-linked chronic granulomatous disease (X-CGD) is an inherited immunodeficiency with absent phagocyte NADPH-oxidase activity caused by defects in the gene-encoding gp91(phox). Here, we evaluated strategies for less intensive conditioning for gene therapy of genetic blood disorders without selective advantage for gene correction, such as might be used in a human X-CGD protocol. We compared submyeloablative with ablative irradiation as conditioning in murine X-CGD, examining engraftment, oxidase activity and vector integration in mice transplanted with marrow transduced with a gamma-retroviral vector for gp91(phox) expression. The frequency of oxidase-positive neutrophils in the donor population was unexpectedly higher in many 300 cGy-conditioned mice compared with lethally irradiated recipients, as was the fraction of vector-marked donor secondary CFU-S12. Vector integration sites in marrow, spleen and secondary CFU-S12 DNA from primary recipients were enriched for cancer-associated genes, including Evi1, and integrations in or near cancer-associated genes were more frequent in marrow and secondary CFU-S12 from 300 cGy-conditioned mice compared with fully ablated mice. These findings support the concept that vector integration can confer a selection bias, and suggest that the intensity of the conditioning regimen may further influence the effects of vector integration on clonal selection in post-transplant engraftment and hematopoiesis.

Sequence family variant loss from the AZFc interval of the human Y chromosome, but not gene copy loss, is strongly associated with male infertility.

BACKGROUND: Complete deletion of the complete AZFc interval of the Y chromosome is the most common known genetic cause of human male infertility. Two partial AZFc deletions (gr/gr and b1/b3) that remove some copies of all AZFc genes have recently been identified in infertile and fertile populations, and an association study indicates that the resulting gene dose reduction represents a risk factor for spermatogenic failure. METHODS: To determine the incidence of various partial AZFc deletions and their effect on fertility, we combined quantitative and qualitative analyses of the AZFc interval at the DAZ and CDY1 loci in 300 infertile men and 399 control men. RESULTS: We detected 34 partial AZFc deletions (32 gr/gr deletions), arising from at least 19 independent deletion events, and found gr/gr deletion in 6% of infertile and 3.5% of control men (p>0.05). Our data provide evidence for two large AZFc inversion polymorphisms, and for relative hot and cold spots of unequal crossing over within the blocks of homology that mediate gr/gr deletion. Using SFVs (sequence family variants), we discriminate DAZ1/2, DAZ3/4, CDY1a (proximal), and CDY1b (distal) and define four types of DAZ-CDY1 gr/gr deletion. CONCLUSIONS: The only deletion type to show an association with infertility was DAZ3/4-CDY1a (p = 0.042), suggesting that most gr/gr deletions are neutral variants. We see a stronger association, however, between loss of the CDY1a SFV and infertility (p = 0.002). Thus, loss of this SFV through deletion or gene conversion could be a major risk factor for male infertility.

Variable correction of host defense following gene transfer and bone marrow transplantation in murine X-linked chronic granulomatous disease.

Chronic granulomatous disease (CGD) is an inherited immunodeficiency in which the absence of the phagocyte superoxide-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase results in recurrent bacterial and fungal infections. A murine model of X-linked CGD (X-CGD) was used to explore variables influencing reconstitution of host defense following bone marrow transplantation and retroviral-mediated gene transfer. The outcomes of experimental infection with Aspergillus fumigatus, Staphylococcus aureus, or Burkholderia cepacia were compared in wild-type, X-CGD mice, and transplanted X-CGD mice that were chimeric for either wild-type neutrophils or neutrophils with partial correction of NADPH oxidase activity after retroviral-mediated gene transfer. Host defense to these pathogens was improved in X-CGD mice even with correction of a limited number of neutrophils. However, intact protection against bacterial pathogens required relatively greater numbers of oxidant-generating phagocytes compared to protection against A fumigatus. The host response also appeared to be influenced by the relative level of cellular NADPH oxidase activity, particularly for A fumigatus. These results may have implications for developing effective approaches for gene therapy of CGD. (Blood. 2001;97:3738-3745)

Long-term correction of phagocyte NADPH oxidase activity by retroviral-mediated gene transfer in murine X-linked chronic granulomatous disease.

Chronic granulomatous disease (CGD) is an inherited deficiency of the superoxide-generating phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, resulting in recurrent, severe bacterial and fungal infections. The X-linked form of this disorder (X-CGD) results from mutations in the X-linked gene for gp91(phox), the larger subunit of the oxidase flavocytochrome b(558). In this study, we used a murine model of X-CGD to examine the long-term function of retroviral vectors for expression of gp91(phox) based on the murine stem cell virus (MSCV) backbone. NADPH oxidase activity was reconstituted in neutrophils and macrophages for up to 18 to 24 months posttransplantation of transduced X-CGD bone marrow into lethally irradiated syngeneic X-CGD mice. Southern blot analysis and secondary transplant data showed proviral integration in multilineage repopulating cells. Although relatively small amounts of recombinant gp91(phox) (approximately 5% to 10% of wild-type levels) were detected in neutrophils after retroviral-mediated gene transfer, superoxide-generating activity was approximately 20% to 25% of wild-type mouse neutrophils. Expression of gp91(phox) is normally restricted to mature phagocytes. No obvious toxicity was observed in other hematopoietic lineages in transplant recipients, and provirus-marked cells were capable of reconstituting secondary transplant recipients, who also exhibited NADPH oxidase-positive neutrophils. MSCV-based vectors for long-term expression of gp91(phox) may be useful for gene therapy of human CGD targeted at hematopoietic stem cells.

Retroviral-mediated gene transfer of gp91phox into bone marrow cells rescues defect in host defense against Aspergillus fumigatus in murine X-linked chronic granulomatous disease.

The X-linked form of chronic granulomatous disease (X-CGD), an inherited deficiency of the respiratory burst oxidase, results from mutations in the X-linked gene for gp91phox, the larger subunit of the oxidase cytochrome b. The goal of this study was to evaluate the impact of retroviral-mediated gene transfer of gp91phox on host defense against Aspergillus fumigatus in a murine model of X-CGD. Retrovirus vectors constructed using the murine stem cell virus (MSCV) backbone were used for gene transfer of the gp91phox cDNA into murine X-CGD bone marrow cells. Transduced cells were transplanted into lethally irradiated syngeneic X-CGD mice. After hematologic recovery, superoxide production, as monitored by the nitroblue tetrazolium (NBT) test, was detected in up to approximately 80% of peripheral blood neutrophils for at least 28 to 35 weeks after transplantation. Neutrophil expression of recombinant gp91phox and superoxide production were significantly less than wild-type neutrophils. However, 9 of 9 mice with approximately 50% to 80% NBT+ neutrophils after gene transfer did not develop lung disease after respiratory challenge with 150 to 500 A fumigatus spores, doses that produced disease in 16 of 16 control X-CGD mice. In X-CGD mice transplanted with mixtures of wild-type and X-CGD bone marrow, > or = 5% wild-type neutrophils were required for protection against A fumigatus challenge. These data suggest that expression of even low levels of recombinant gp91phox can substantially improve phagocyte function in X-CGD, although correction of very small percentage of phagocytes may not be sufficient for protection against A fumigatus.

Internal autocrine regulation by erythropoietin of erythroleukemic cell proliferation.

Antisense oligomers (18 mers) corresponding to the erythropoietin and erythropoietin receptor 5' coding sequences cause marked suppression of proliferation of several lines of erythroleukemic cells. In these systems, phosphorothioate protected sense oligomers are inhibitory, while the unmodified sense oligomers have no significant effect on cell growth. These data indicate that proliferation of some erythroleukemic cells is under internal autocrine regulation by erythropoietin and its receptor.

High-level reconstitution of respiratory burst activity in a human X-linked chronic granulomatous disease (X-CGD) cell line and correction of murine X-CGD bone marrow cells by retroviral-mediated gene transfer of human gp91phox.

The X-linked form of chronic granulomatous disease (X-CGD) results from mutations in the gene encoding gp91phox, a 91-kD membrane glycoprotein that is the larger subunit of the respiratory burst oxidase cytochrome b. In this study, a new retroviral vector for expression of human gp91phox, MSCV-h91Neo, based on murine stem cell virus vectors, was evaluated using a human X-CGD myeloid cell line (X-CGD PLB-985 cells) and murine bone marrow cells. Expression of recombinant gp91phox in transduced X-CGD PLB-985 cells was substantially improved compared with levels achieved previously using a different retroviral construct, and respiratory burst oxidase activity was fully reconstituted in the majority of clones analyzed. Expression of gp91phox transcripts was also observed in primary and secondary murine colony-forming unit-spleen derived from transduced bone marrow cells. Furthermore, respiratory burst activity was restored to granulocyte-monocyte progeny of transduced X-CGD mice bone marrow cells cultured in vitro. This observation is the first reported use of gene transfer to correct the enzymatic defect in murine CGD phagocytes and is also consistent with the high conservation of the oxidase complex among different species. Taken together, these data suggest that the MSCV-h91Neo vector may be useful for gene replacement therapy in X-linked CGD, in which high-level reconstitution of phagocyte oxidase activity may be important for full correction of phagocyte microbicidal function.

Further study of internal autocrine regulation of multipotent hematopoietic cells.

We have extended the study of the effects of antisense oligodeoxynucleotides on hematopoietic colony formation to include the effects of antisense to granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and macrophage colony-stimulating factor (M-CSF) on bone marrow cultures. GM-CSF antisense and GM-CSF receptor antisense cause an increase in mixed erythroid:nonerythroid colonies and a decrease in mixed nonerythroid colonies, which is an effect opposite to that described previously for erythropoietin (Epo) and Epo receptor antisense. The effect of GM-CSF antisense oligomer is not abrogated by the presence of the ligand in the culture. Antisense oligomers to G-CSF and M-CSF have no effect. When Epo and GM-CSF antisense oligomers are added simultaneously, the effects seem to be independent, with the GM-CSF antisense predominating. These data support the hypothesis of internal autocrine regulation of multipotent hematopoietic precursor cells, and extend the concept to myeloid as well as erythroid differentiation.

An autocrine role for erythropoietin in mouse hematopoietic cell differentiation.

Erythropoietin (epo) is the primary regulator of the rate of red blood cell formation in mammals. Because it is formed in the kidney and acts on the bone marrow, its action is classically endocrine. We have shown by PCR that marrow cells contain epo mRNA and that antisense oligodeoxynucleotides, to both epo and its receptor, act on multipotent hematopoietic cells to cause a decrease in mixed erythroid:nonerythroid colonies. The antisense oligonucleotides also cause an increase in mixed nonerythroid colonies with no effect on erythroid burst formation. Sense oligonucleotides have no effect. The antisense suppression is not due to adherent cells, cycling late differentiated cells or lymphocytes, and not reversed by exogenous epo. We conclude that normal erythroid differentiation may have an early phase that is dependent on an internal autocrine mechanism involving epo and its receptor.

[Treatment of tachyarrhythmia in a patient with long QT syndrome by autologous heart transplantation and atrial stimulation controlled by the sinoatrial node]. / Lechenie takhiaritmii u bolnoi s sindromom udlinennogo intervala Q-T s pomoshch'iu autotransplantatsii serdtsa i stimuliatsii predserdii, upravliaemoi sinusovym uzlom.

The authors described an operation performed in a female patient aged 37 years who was first examined in 1980. That year she experienced the first ventricular fibrillation episode. The duration of the Q-T interval was 600 msec. She was examined by a comprehensive protocol which enabled coronary abnormalities to be excluded. The operation was made in July 1987 in the Charite Hospital by Dr. Warnke. The operation involved dissection of the nerve connections nearest the heart by cutting the pulmonary artery, aorta, both atria and subsequently suturing by the scheme used in cardiac grafting. The patient rapidly recovered after the surgery. She had a normal Q-T interval and no recurrences of ventricular tachycardias. Following the surgery she displayed disturbances of intraatrial conduction and pacemaker displacement into the lower segments of the right atrium, so she was implanted a DDD-mode pacemaker.

Interleukin 3 can compensate for the deficiency in erythroid burst-forming cells in anemic mice of genotype W/Wv.

Bone marrow cells from mice with the W/Wv mutation have about one-third the erythroid burst-forming cells (BFU-E) found with wild-type littermates. The mutant cells are also less responsive in vitro to exogenous erythropoietin (epo) than are the wild-type cells. Addition of interleukin 3 (IL-3) to the culture medium largely corrects the deficit in burst formation and in responsiveness to epo. When mutant cells are incubated for 2 days in the absence of epo but with IL-3 present, burst formation is the same as with wild-type cells, suggesting that IL-3 acts on maintenance in vitro and/or proliferation of primitive precursor cells of both mutant and wild-type mice.