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Metabolic control during preimplantation mammalian development.

Metabolic control during preimplantation development may be either intrinsic to the embryo or extrinsic, i.e. mediated via the environment within the female tract. Intrinsic control is exhibited by early preimplantation embryos, which are undifferentiated, largely autonomous and show no growth. The best characterized intrinsic metabolic control mechanism is the switch from pyruvate to glucose as major energy substrate during mouse preimplantation development. A shift to extrinsic control occurs during the later preimplantation stages, when differentiation into trophectoderm and inner cell mass occurs, net growth begins and the embryo prepares for implantation. Thus, the environment provided by the oviduct, in which the embryos of most mammalian species spend their first 3 days, may be considered as facilitatory rather than obligatory for early embryo development, whereas the uterine environment is required to provide a more complex milieu. The 'facilitators' provided by the oviduct include physical factors, such as ciliary and muscular action, and chemical factors, including oxygen, nutrients, ions and macromolecules. Oviduct fluid secretion is under adrenergic influence, defects in which could contribute to impaired mucosal defence and tubal blockage in women. Preimplantation embryos exhibit metabolic adaptation, whereby their metabolism responds to changes in the external environment. Such adaptation explains the ability of early embryos to develop in a variety of culture media, a situation similar to that which they will experience in the female tract. Thus, the provision of a variety of media in vitro is likely to ensure the development of embryos in a physiological manner.

Adaptation, Physiological↗

Opposing roles of NF-kappaB family members in the regulation of NK cell proliferation and production of IFN-gamma.

It is well established that the nuclear factor-kappaB (NF-kappaB) family of transcription factors participates in the regulation of many aspects of innate and adaptive immunity. The majority of these reports have focused on the role of NF-kappaB in accessory cell and T or B cell function, but less is known about the role of NF-kappaB in NK cells. However, several studies have demonstrated that these transcription factors are required for NK cell production of IFN-gamma and proliferation. The studies presented here examine the role of two NF-kappaB members, c-Rel and p50, in NK cell function. In vitro data revealed that in the absence of c-Rel, NK cells have a defect in their ability to secrete IFN-gamma, but remain unaffected in their capacity to proliferate. In contrast, p50-/- NK cells have enhanced proliferative and IFN-gamma responses compared with wild-type NK cells. The latter findings suggest a role for p50 as a negative regulator of NK cell production of IFN-gamma and chromatin immunoprecipitation assays demonstrated the association of p50 with the IFN-gamma promoter of resting NK cells. Consistent with the in vitro studies, in vivo studies with NF-kappaB gene-deficient mice infected with Toxoplasma gondii revealed that the absence of p50 leads to enhanced NK cell proliferation and production of IFN-gamma. Together, these studies define distinct roles for c-Rel and p50 in the function of NK cells.

Animals↗

Use of recombinant activated factor VII to control bleeding in a young child with qualitative platelet disorder: a case report.

Defects of platelet adhesion, aggregation, secretion, or procoagulant activities can lead to bleeding diathesis of variable severity. We used recombinant activated factor VII (rFVIIa) in the treatment of uncontrolled epistaxis in a patient with a qualitative platelet disorder. We aimed to assess the efficacy of a single rFVIIa dose (100 microg/kg) in the control of mild and severe refractory epistaxis, and evaluate the influence of rFVIIa on markers of platelet adhesion and aggregation during a period of hematological stability (i.e. non-bleeding, no medication). The efficacy study showed mild episodes of epistaxis could be successfully managed using a single rFVIIa (100 microg/kg) dose; however, severe bleedings were not well controlled, and platelet transfusion was required to achieve hemostasis. Hematological investigations showed ADP-induced and collagen-induced platelet aggregation increases from 20 to 34% and 16 to 30%, respectively, following rFVIIa administration. There were no differences between pre-dose and post-dose concentrations of membrane glycoproteins. rFVIIa may therefore induce platelet aggregation by activating a glycoprotein-independent aggregation pathway. rFVIIa may have a role in managing mild bleeding episodes not controlled using conventional measures in patients with a qualitative platelet disorder. Further research is needed to determine the mechanism of action, efficacy, and safety of rFVIIa in this population.

Child, Preschool↗

Pathogenesis of diarrhea in ulcerative colitis: new views on an old problem.

BACKGROUND: Whereas water movement into the intestinal lumen occurs secondary to Cl secretion in secretory diarrheal diseases, defects in key transport processes lead to profound decreases in colonic Na, Cl, and water absorption in ulcerative colitis. STUDIES AND RESULTS: Recent studies indicate reduced expression/activity of apical Na channels and basolateral Na, K-ATPase, leading to loss of electrogenic Na absorption in the distal colon and rectum. There is also likely to be a decrease in electroneutral NaCl cotransport, which is present throughout the colon. Preliminary work on basolateral K channel abundance and activity in colonic epithelial cells suggests that whole-cell K conductance is decreased in ulcerative colitis, leading to epithelial cell depolarization, and further limitation of Na absorption. In addition, there is a marked reduction in colonic epithelial resistance, which reflects a decrease in the integrity of intercellular tight junctions and the presence of apoptotic foci. CONCLUSIONS: Impaired Na and Cl transport, combined with enhanced epithelial "leakiness," results in a profound decrease in the capacity of the inflamed colon to absorb salt and water. Transport abnormalities in ulcerative colitis may at least partly reflect the effects of proinflammatory cytokines, raising the possibility of novel approaches to the restoration of colonic absorptive capacity in this disease.

Biological Transport↗

VimA is part of the maturation pathway for the major gingipains of Porphyromonas gingivalis W83.

The authors have shown previously that the vimA gene, which is part of the bcp-recA-vimA operon, plays an important role in protease activation in Porphyromonas gingivalis. The gingipain RgpB proenzyme is secreted in the vimA-defective mutant P. gingivalis FLL92. An important question that is raised is whether the vimA gene product could directly interact with the proteases for their activation or regulate a pathway responsible for protease activation. To further study the mechanism(s) of VimA-dependent protease activation, the vimA gene product was further characterized. A 39 kDa protein consistent with the size of the predicted VimA protein was purified. In protein-protein interaction studies, the VimA protein was shown to interact with gingipains RgpA, RgpB and Kgp. Immune sera from mice immunized with P. gingivalis immunoreacted with the purified VimA protein. Taken together, these data suggest an interaction of VimA with the gingipains and further confirm the role of this protein in their regulation or maturation.

Adhesins, Bacterial↗

A role for exocytosis in the spatial regulation of actin organization.

The efficient organization of the actin cytoskeleton is important for many cellular functions. However, how the local actin organization is regulated in a cell is not well understood. By using yeast mutants defective in actin organization and secretion, we demonstrated that exocytosis plays a role in the spatial regulation of actin organization. Our findings suggest that the actin cytoskeleton, exocytosis, and perhaps endocytosis, may depend on each other for efficiency and reinforce each other.

Actins↗

Oct-2, although not required for early B-cell development, is critical for later B-cell maturation and for postnatal survival.

Oct-2, a POU homeo domain transcription factor, is believed to stimulate B-cell-restricted expression of immunoglobulin genes through binding sites in immunoglobulin gene promoters and enhancers. To determine whether Oct-2 is required for B-cell development or function, or has other developmental roles, the gene was disrupted by homologous recombination. Oct-2-l- mice develop normally but die within hours of birth for undetermined reasons. Mutants contain normal numbers of B-cell precursors but are somewhat deficient in IgM+ B cells. These B cells have a marked defect in their capacity to secrete immunoglobulin upon mitogenic stimulation in vitro. Thus, Oct-2 is not required for the generation of immunoglobulin-bearing B cells but is crucial for their maturation to immunoglobulin-secreting cells and for another undetermined organismal function.

Animals↗

Anchoring fibrils and type VII collagen are absent from skin in severe recessive dystrophic epidermolysis bullosa.

Skin of patients with severe generalized recessive dystrophic epidermolysis bullosa (SGRDEB) was studied by immunoelectron microscopy and immunoblotting with antibodies to type VII collagen, a major structural component of anchoring fibrils. In normal skin, the protein was localized to the dermoepidermal junction zone below the basement membrane and was extractable from the papillary dermis after artificial epidermolysis. In SGRDEB skin, neither immunoreactive material below the basement membrane nor identifiable anchoring fibrils could be recognized and neither the tissue form nor the specific proteolytic fragments of type VII collagen were found in extracts of SGRDEB skin. Very low amounts of type VII collagen alpha-chains could be detected in cultures of SGRDEB-fibroblasts, whereas normal fibroblasts synthesized more of this collagen. These results suggest that a genetic defect in the correct synthesis, secretion, or in the molecular assembly of type VII collagen may underlie SGRDEB.

Adolescent↗

Multiple platelet defects identified by flow cytometry at diagnosis in acute myeloid leukaemia.

Summary Previous findings of megakaryocytic hypogranulation and dysmegakaryocytopoietic features in acute myeloid leukaemia (AML) strongly indicate defects in platelet production. The bleeding tendency of these patients may result from dysregulated platelet production, resulting in thrombocytopenia as well as qualitative platelet defects. The present study examined platelet function at diagnosis in 50 AML patients by whole blood flow cytometry. Following in vitro platelet agonist stimulation, platelet activation markers were analysed and compared with 20 healthy individuals. To detect recent in vivo platelet activation, plasma soluble P-selectin (sP-selectin) was measured. Flow cytometric analysis of platelet activation markers demonstrated reduced CD62P [35.6 vs. 118.5 x 10(3) molecules of equivalent soluble fluorochrome (MESF); P < 0.0001], CD63 (11.3 vs. 50.7 x 10(3) MESF; P < 0.0001), and PAC-1 (41.5 vs. 90.5%; P = 0.0001) while reductions in CD42b were abnormal (45.6 vs. 70%; P < 0.0001). sP-selectin levels were similar in patients and healthy controls (0.04 vs. 0.27 fg/platelet; P = 0.84). The presented data indicate that AML pathogenesis may result in multiple platelet defects, involving adhesion, aggregation, and secretion and demonstrate that flow cytometry is a feasible method for platelet function analysis in patients with thrombocytopenia.

Acute Disease↗

Expression of two type 2N von Willebrand disease mutations identified in exon 18 of von Willebrand factor gene.

Type 2N von Willebrand disease (VWD) is characterized by a markedly decreased affinity of von Willebrand factor (VWF) for factor VIII (FVIII). The FVIII binding site has been localized within the first 272 amino acid residues of mature VWF, encoded by exons 18-23. Two substitutions in exon 18 of VWF gene, inducing candidate mutations Y795C and C804F were identified in the heterozygous state in two French patients who also displayed the frequent R854Q mutation in exon 20. Expression studies in Cos-7 cells showed that these abnormalities, which implicate cysteine residues, induced secretion, multimerization and FVIII binding defects of corresponding recombinant VWF. Results from transfection experiments with R854Q, performed to reproduce the hybrid VWF present in patient plasma, were in agreement with those obtained for patient's plasma VWF. These findings confirm the importance of the VWF D' domain in FVIII binding. In addition, this work shows that exon 18 should preferentially be sequenced in type 2N VWD patients when the frequent R854Q mutation in exon 20 has been excluded or detected in the heterozygous state.

Adult↗

Suppression of plasma ACTH concentration by angiotensin II infusion in normal humans and in a subject with a steroid 17 alpha-hydroxylase defect.

Six healthy subjects were infused with angiotensin II and plasma concentrations of angiotensin, ACTH and cortisol were measured before, during and after the infusion. In all cases the plasma ACTH concentration fell as plasma angiotensin increased and rose again, sometimes to higher than basal levels, when the angiotensin infusion was terminated. These effects were most marked at the highest rate of infusion (8 pmol/kg/min) and, at the lower rates (2 and 4 pmol/kg/min), there was some recovery of ACTH levels during the infusion period in some subjects. Plasma ACTH concentrations also fell when angiotensin was infused into a patient with high ACTH levels due to a steroid 17 alpha-hydroxylation defect. The inhibition of ACTH secretion is not due to a rise in plasma cortisol operating a negative feedback inhibition. It could be a direct effect of the infused angiotensin on the brain-hypothalamus-pituitary complex or an effect on the metabolism of ACTH.

Adrenal Hyperplasia, Congenital↗

Alterations in circulating thyroid hormones and thyroxine binding globulin in chronic alcoholism.

The present study concerns eighteen chronic alcoholics with minimal liver damage. A significant reduction in total serum T4 with an accompanying drop in circulating TBG was observed in these otherwise euthyroid patients. During alcohol withdrawal, we observed a rapid increase in T4 and TBG into the normal range. We suggest that the fall in the level of circulating thyroxine-binding globulin is related to a defect in its synthesis or secretion by the liver due to ethanol consumption. Caution is recommended in the interpretation of thyroid function tests in chronic alcoholics.

Adult↗

Differential expression of IL-10 receptor by epithelial cells and alveolar macrophages.

BACKGROUND: Interleukin (IL)-10 is a pleiotropic cytokine with a broad spectrum of immunosuppressive and anti-inflammatory effects. IL-10 secretion from alveolar macrophages is defective in patients with asthma and lower concentrations of IL-10 are found in bronchoalveolar lavage (BAL) from asthmatic patients than in normal control subjects. Reduced IL-10 may result in exaggerated and more prolonged inflammatory responses in asthmatic airways. IL-10 acting through the IL-10 receptor (IL-10R) stimulates the transcription factors STAT1 and STAT3. METHODS: We investigated IL-10 and IL-10R expression in normal and asthmatic bronchial epithelium and BAL macrophages using reverse transcription-polymerase chain reaction, immunohistochemistry and Western blotting. The functional effect of IL-10 was examined using granulocyte-macrophage-colony stimulating factor, enzyme-linked immunosorbent assay and Western blotting for phosphorylated STAT1 and STAT3. RESULTS: IL-10 was not expressed in epithelial cells; furthermore these cells did not express the IL-10R and had no functional response to exogenous IL-10. Bronchial epithelial cells expressed variable levels of phosphorylated STAT1 and STAT3 with no change in expression between normal subjects and asthmatics. IL-10 protein and IL-10R expression was detected in alveolar macrophages from all subjects. CONCLUSION: Our study suggests that the bronchial epithelium is not a source of IL-10 and cannot respond to exogenous IL-10 because of a lack of IL-10R expression.

Adult↗

Contact with cultured epithelial cells stimulates secretion of Salmonella typhimurium invasion protein InvJ.

Contact of Salmonella typhimurium with cultured epithelial cells results in the assembly of surface appendages termed invasomes which are presumably required for the internalization of these organisms into host cells. The assembly of these structures requires the function of a dedicated protein secretion system encoded in the inv locus. We show in this report that contact of wild-type S. typhimurium with cultured Henle-407 cells stimulated the secretion of InvJ, a recently identified target of the inv-encoded type III protein secretion system. Stimulation of InvJ secretion also occurred upon bacterial contact with bovine calf serum-coated culture dishes but did not occur upon S. typhimurium contact with glutaraldehyde-fixed Henle-407 cells. The stimulation of InvJ secretion did not require de novo protein synthesis. Invasion-defective invC and invG mutants of S. typhimurium failed to secrete InvJ upon contact with live Henle-407 cells. In contrast, contact-dependent secretion of InvJ in S. typhimurium invE mutants occurred at levels equivalent to those of the wild type. These results indicate that the presence of Henle-407 cells and/or serum is capable of activating the type III secretion system encoded in the inv locus, further supporting the notion that Salmonella entry into cultured cells is the result of a biochemical cross-talk between the bacteria and the host cells.

Antigens, Bacterial↗

Disruption of the phagosomal membrane and egress of Legionella pneumophila into the cytoplasm during the last stages of intracellular infection of macrophages and Acanthamoeba polyphaga.

Although the early stages of intracellular infection by Legionella pneumophila are well established at the ultrastructural level, a detailed ultrastructural analysis of late stages of intracellular replication has never been done. Here we show that the membrane of the L. pneumophila-containing phagosome (LCP) is intact for up to 8 h postinfection of macrophages and Acanthamoeba polyphaga. At 12 h, 71 and 74% of the LCPs are disrupted within macrophages and A. polyphaga, respectively, while the plasma membrane remains intact. At 18 and 24 h postinfection, cytoplasmic elements such as mitochondria, lysosomes, vesicles, and amorphous material are dispersed among the bacteria and these bacteria are considered cytoplasmic. At 18 h, 77% of infected macrophages and 32% of infected A. polyphaga amoebae harbor cytoplasmic bacteria. At 24 h, 99 and 78% of infected macrophages and amoebae, respectively, contain cytoplasmic bacteria. On the basis of lysosomal acid phosphatase staining of infected macrophages and A. polyphaga, the lysosomal enzyme is present among the bacteria when host vesicles are dispersed among bacteria. Our data indicate that bacterial replication proceeds despite physical disruption of the phagosomal membrane. We also show that an lspG mutant that is defective in the type II secretion system and therefore does not secrete the hydrolytic enzymes metalloprotease, p-nitrophenol phosphorylcholine hydrolase, lipase, phospholipase A, and lysophospholipase A is as efficient as the wild-type strain in disruption of the LCP. Therefore, L. pneumophila disrupts the phagosomal membrane and becomes cytoplasmic at the last stages of infection in both macrophages and A. polyphaga. Lysosomal elements, mitochondria, cytoplasmic vesicles, and amorphous material are all dispersed among the bacteria, after phagosomal disruption, within both human macrophages and A. polyphaga. The disruption of the LCP is independent of the hydrolytic enzymes exported by the type II secretion system.

Acanthamoeba↗

Conditional loss of Nkx3.1 in adult mice induces prostatic intraepithelial neoplasia.

The homeodomain-containing transcription factor NKX3.1 is a putative prostate tumor suppressor that is expressed in a largely prostate-specific and androgen-regulated manner. Loss of NKX3.1 protein expression is common in human prostate carcinomas and prostatic intraepithelial neoplasia (PIN) lesions and correlates with tumor progression. Disruption of the murine Nkx3.1 gene results in defects in prostate branching morphogenesis, secretions, and growth. To more closely mimic the pattern of NKX3.1 loss that occurs in human prostate tumors, we have used Cre- and loxP-mediated recombination to delete the Nkx3.1 gene in the prostates of adult transgenic mice. Conditional deletion of one or both alleles of Nkx3.1 leads to the development of preinvasive lesions that resemble PIN. The pattern of expression of several biomarkers (Ki-67, E-cadherin, and high-molecular-weight cytokeratins) in these PIN lesions resembled that observed in human cases of PIN. Furthermore, PIN foci in mice with conditional deletion of a single Nkx3.1 allele lose expression of the wild-type allele. Our results support the role of NKX3.1 as a prostate tumor suppressor and indicate a role for this gene in tumor initiation.

Animals↗

Abnormal glucose homeostasis and pancreatic islet function in mice with inactivation of the Fem1b gene.

Type 2 diabetes mellitus is a disorder of glucose homeostasis involving complex gene and environmental interactions that are incompletely understood. Mammalian homologs of nematode sex determination genes have recently been implicated in glucose homeostasis and type 2 diabetes mellitus. These are the Hedgehog receptor Patched and Calpain-10, which have homology to the nematode tra-2 and tra-3 sex determination genes, respectively. Here, we have developed Fem1b knockout (Fem1b-KO) mice, with targeted inactivation of Fem1b, a homolog of the nematode fem-1 sex determination gene. We show that the Fem1b-KO mice display abnormal glucose tolerance and that this is due predominantly to defective glucose-stimulated insulin secretion. Arginine-stimulated insulin secretion is also affected. The Fem1b gene is expressed in pancreatic islets, within both beta cells and non-beta cells, and is highly expressed in INS-1E cells, a pancreatic beta-cell line. In conclusion, these data implicate Fem1b in pancreatic islet function and insulin secretion, strengthening evidence that a genetic pathway homologous to nematode sex determination may be involved in glucose homeostasis and suggesting novel genes and processes as potential candidates in the pathogenesis of diabetes mellitus.

Animals↗

Vitamin B12 absorption--a study of intraluminal events in control subjects and patients with tropical sprue.

The intraluminal fate of orally administered radioactive vitamin B12 has been studied in control subjects with normal vitamin B12 absorption and those with vitamin B12 malabsorption due to tropical sprue. In control subjects 1 to 21% of the dose was bound to sedimentable material and 37 to 75% was bound to immunoreactive intrinsic factor. In subjects with vitamin B12 malabsorption due to tropical sprue, the results were identical with the control subjects. Bacteriological studies showed a statistically significant correlation between both the number of flora in the jejunum and the number of bacteroides in both the jejunum and ileum and vitamin B12 malabsorption. In patients with tropical sprue who have normal intrinsic factor secretion, the vitamin B12 absorptive defect is not due to binding of the vitamin to bacteria or to alteration to the intrinsic factor vitamin B12 complex in the intestinal lumen. The lesion appears to be one of the mucosal cell receptors or of the cells themselves, possibly caused by bacterial toxins.

Bacteroides↗