Dog introduction alters the home dust microbiota.

Research has largely reported that dog exposure is associated with reduced allergic disease risk. Responsible mechanism(s) are not understood. The goal was to investigate whether introducing a dog into the home changes the home dust microbiota. Families without dogs or cats planning to adopt a dog and those who were not were recruited. Dust samples were collected from the homes at recruitment and 12 months later. Microbiota composition and taxa (V4 region of the 16S rRNA gene) were compared between homes that did and did not adopt a dog. A total of 91 dust samples from 54 families (27 each, dog and no dog; 17 dog and 20 no dog homes with paired samples) were analyzed. A significant dog effect was seen across time in both unweighted UniFrac and Canberra metrics (both P = .008), indicating dog introduction may result in rapid establishment of rarer and phylogenetically related taxa. A significant dog-time interaction was seen in both weighted UniFrac (P < .001) and Bray-Curtis (P = .002) metrics, suggesting that while there may not initially be large relative abundance shifts following dog introduction, differences can be seen within a year. Therefore, dog introduction into the home has both immediate effects and effects that emerge over time.

Rearrangement of a large novel Pseudomonas aeruginosa gene island in strains isolated from a patient developing ventilator-associated pneumonia.

Bacterial gene islands add to the genetic repertoire of opportunistic pathogens. Here, we perform comparative analyses of three Pseudomonas aeruginosa strains isolated sequentially over a 3-week period from a patient with ventilator-associated pneumonia (VAP) who received clindamycin and piperacillin-tazobactam as part of their treatment regime. While all three strains appeared to be clonal by standard pulsed-field gel electrophoresis, whole-genome sequencing revealed subtle alterations in the chromosomal organization of the last two strains; specifically, an inversion event within a novel 124-kb gene island (PAGI 12) composed of 137 open reading frames [ORFs]. Predicted ORFs in the island included metabolism and virulence genes. Overexpression of a gene island-borne putative ß-lactamase gene was observed following piperacillin-tazobactam exposure and only in those strains that had undergone the inversion event, indicating altered gene regulation following genomic remodeling. Examination of a separate cohort of 76 patients with VAP for integration at this tRNA(lys) recombination site demonstrated that patients exhibiting evidence of integration at this site had significantly higher 28-day mortality. These findings provide evidence that P. aeruginosa can integrate, rapidly remodel, and express exogenous genes, which likely contributes to its fitness in a clinical setting.

Role of mineralocorticoid receptor/Rho/Rho-kinase pathway in obesity-related renal injury.

OBJECTIVE: We examined whether aldosterone/Rho/Rho-kinase pathway contributed to obesity-associated nephropathy. SUBJECTS: C57BL/6J mice were fed a high fat or low fat diet, and mice on a high fat diet were treated with a mineralocorticoid receptor antagonist, eplerenone. RESULTS: The mice on a high fat diet not only developed obesity, but also manifested renal histological changes, including glomerular hypercellularity and increased mesangial matrix, which paralleled the increase in albuminuria. Furthermore, enhanced Rho-kinase activity was noted in kidneys from high fat diet-fed mice, as well as increased expressions of inflammatory chemokines. All of these changes were attenuated by eplerenone. In high fat diet-fed mice, mineralocorticoid receptor protein levels in the nuclear fraction and SGK1, an effector of aldosterone, were upregulated in kidneys, although serum aldosterone levels were unaltered. Furthermore, aldosterone and 3ß-hydroxysteroid dehydrogenase in renal tissues were upregulated in high fat diet-fed mice. Finally, in cultured mesangial cells, stimulation with aldosterone enhanced Rho-kinase activity, and pre-incubation with eplerenone prevented the aldosterone-induced activation of Rho kinase. CONCLUSION: Excess fat intake causes obesity and renal injury in C57BL/6J mice, and these changes are mediated by an enhanced mineralocorticoid receptor/Rho/Rho-kinase pathway and inflammatory process. Mineralocorticoid receptor activation in the kidney tissue and the subsequent Rho-kinase stimulation are likely to participate in the development of obesity-associated nephropathy without elevation in serum aldosterone levels.

Viable bacterial colonization is highly limited in the human intestine in utero.

Mucosal immunity develops in the human fetal intestine by 11-14 weeks of gestation, yet whether viable microbes exist in utero and interact with the intestinal immune system is unknown. Bacteria-like morphology was identified in pockets of human fetal meconium at mid-gestation by scanning electron microscopy (n = 4), and a sparse bacterial signal was detected by 16S rRNA sequencing (n = 40 of 50) compared to environmental controls (n = 87). Eighteen taxa were enriched in fetal meconium, with Micrococcaceae (n = 9) and Lactobacillus (n = 6) the most abundant. Fetal intestines dominated by Micrococcaceae exhibited distinct patterns of T cell composition and epithelial transcription. Fetal Micrococcus luteus, isolated only in the presence of monocytes, grew on placental hormones, remained viable within antigen presenting cells, limited inflammation ex vivo and possessed genomic features linked with survival in the fetus. Thus, viable bacteria are highly limited in the fetal intestine at mid-gestation, although strains with immunomodulatory capacity are detected in subsets of specimens.

Transplantation of Human-Induced Pluripotent Stem Cell-Derived Neural Precursors into Early-Stage Zebrafish Embryos.

Induced pluripotent stem cells (iPS cells) generated from somatic cells through reprogramming hold great promises for regenerative medicine. However, how reprogrammed cells survive, behave in vivo, and interact with host cells after transplantation still remains to be addressed. There is a significant need for animal models that allow in vivo tracking of transplanted cells in real time. In this regard, the zebrafish, a tropical freshwater fish, provides significant advantage as it is optically transparent and can be imaged in high resolution using confocal microscopy. The principal goal of this study was to optimize the protocol for successful short-term and immunosuppression-free transplantation of human iPS cell-derived neural progenitor cells into zebrafish and to test their ability to differentiate in this animal model. To address this aim, we isolated human iPS cell-derived neural progenitor cells from human fibroblasts and grafted them into (a) early (blastocyst)-stage wild-type AB zebrafish embryos or (b) 3-day-old Tg(gfap:GFP) zebrafish embryos (intracranial injection). We found that transplanted human neuronal progenitor cells can be effectively grafted and that they differentiate and survive in zebrafish for more than 2 weeks, validating the model as an ideal platform for in vivo screening experiments. We conclude that zebrafish provides an excellent model for studying iPS cell-derived cells in vivo.

Regeneration of articular cartilage defects in the temporomandibular joint of rabbits by fibroblast growth factor-2: a pilot study.

The purpose of this study was to investigate the therapeutic usefulness of fibroblast growth factor-2 (FGF-2) in rabbit temporomandibular joints (TMJ) with osteoarthritis. A 10-mm(3) defect was bored in the surface of the mandibular condyle head. The animals were divided into four groups: two test groups in which the defect was filled with lyophilized collagen containing 0.1 or 1.0microg of FGF-2, and two control groups, in which the defects were filled with lyophilized collagen without FGF-2 or left empty. The defective sites were examined under a light microscope 3 weeks after surgery. Initiation of cartilage formation was observed in the defects filled with 0.1microg of FGF-2, but only a small amount of cartilage was found in the defects of the 1.0-mug FGF-2- treated group. In the control groups, soft-tissue repair only or no tissue repair was found. In vivo, a dose of 0.1microg of FGF-2 can stimulate articular cartilage restoration in defects of the TMJ in rabbits, although determining the effective concentration range of FGF-2 may be difficult. The present results suggest that an optimum concentration of FGF-2 could restore defects of TMJ articular cartilage clinically.

Lactobacillus johnsonii supplementation attenuates respiratory viral infection via metabolic reprogramming and immune cell modulation.

Regulation of respiratory mucosal immunity by microbial-derived metabolites has been a proposed mechanism that may provide airway protection. Here we examine the effect of oral Lactobacillus johnsonii supplementation on metabolic and immune response dynamics during respiratory syncytial virus (RSV) infection. L. johnsonii supplementation reduced airway T helper type 2 cytokines and dendritic cell (DC) function, increased regulatory T cells, and was associated with a reprogrammed circulating metabolic environment, including docosahexanoic acid (DHA) enrichment. RSV-infected bone marrow-derived DCs (BMDCs) from L. johnsonii-supplemented mice had altered cytokine secretion, reduced expression of co-stimulatory molecules, and modified CD4+ T-cell cytokines. This was replicated upon co-incubation of wild-type BMDCs with either plasma from L. johnsonii-supplemented mice or DHA. Finally, airway transfer of BMDCs from L. johnsonii-supplemented mice or with wild-type derived BMDCs pretreated with plasma from L. johnsonii-supplemented mice reduced airway pathological responses to infection in recipient animals. Thus L. johnsonii supplementation mediates airway mucosal protection via immunomodulatory metabolites and altered immune function.

Preliminary laboratory based diagnostic criteria for immune thrombocytopenic purpura: evaluation by multi-center prospective study.

BACKGROUND: We proposed diagnostic criteria for immune thrombocytopenic purpura (ITP) by modifying the existing guidelines for diagnosis of ITP and by incorporating laboratory tests found useful for predicting its diagnosis, for example erythrocyte count, leukocyte count, anti-GPIIb/IIIa antibody-producing B cells, platelet-associated anti-GPIIb/IIIa antibodies, percentage of reticulated platelets, and plasma thrombopoietin. OBJECTIVE AND METHODS: To validate our criteria, we conducted a multi-center prospective study involving 112 patients with thrombocytopenia and a morphologically normal peripheral blood film at the first visit. Each patient underwent a physical examination, routine laboratory tests, and specialized tests for the anti-GPIIb/IIIa antibody response and platelet turnover. RESULTS: Ninety-one patients (81%) satisfied the proposed criteria at first visit. Clinical diagnosis was made by skilled hematologists > 6 months after the first visit; ITP was diagnosed in 88 patients and non-ITP disorders in 24. The proposed criteria had 98% sensitivity, 79% specificity, a 95% positive predictive value, and a 90% negative predictive value. A relatively low specificity appears to be attributed to a few patients who had both ITP and aplastic anemia or myelodysplastic syndrome. CONCLUSIONS: Our preliminary diagnostic criteria based on ITP-associated laboratory findings were useful for the differential diagnosis of ITP, but additional evaluations and modifications will be necessary to develop criteria that can be used routinely.

Comparison of human mesenchymal stem cells derived from bone marrow, synovial fluid, adult dental pulp, and exfoliated deciduous tooth pulp.

Populations of pluripotent stem cells were isolated from bone marrow, synovial fluid, adult dental pulp, and exfoliated deciduous teeth and their multipotentiality properties compared. Osteogenic, chondrogenic, adipogenic, and neurogenic differentiation potentials were examined. Bone marrow mesenchymal stem cells (BMMSCs) and synovial fluid-derived cells (SFCs) showed the highest levels of osteogenesis as expressed by alkaline phosphatase (ALP) activity (0.54±0.094 U/mg protein and 0.57±0.039 U/mg protein, respectively; P=0.60) and by osteocalcin (BGLAP; determined by real-time RT-PCR). SFCs showed the highest levels of chondrogenesis as expressed by ALP activity (1.75±0.097 U/mg protein) and of COL2A1 and COL10A1 by real-time PCR. In terms of adipogenesis, lipid vesicles were observed in the BMMSCs and SFCs. Dental pulp stem cells (DPSCs) and stem cells from human exfoliated deciduous teeth (SHED) exhibited neurogenesis potential, as shown by increases in expression of class III ß-tubulin (TUBB3) and microtubule-associated protein 2 (MAP2) on RT-PCR. Variability was found in the differentiation potential corresponding to the tendency of the original tissue to differentiate. It is suggested that the cell type should be selected depending on the regenerative treatment regimen.

Tissue reaction at the implantation of recombinant human bone morphogenetic protein-2 into the skeletal muscle.

Bone morphogenetic protein (BMP) is a unique cytokine that induces bony tissue in soft tissue. Tissue reactions at and around the implantation of recombinant human BMP-2 (rhBMP-2) into the soft tissue of rats and nonhuman primates were investigated. At the osteoinduced site of rats, massive trabeculae-lined osteoblasts and rich marrow were observed. At and around the nonosteoinduced sites of nonhuman primates, large clear nuclei were observed in reaction to rhBMP-2 implantation. The surrounding area was visually classified into zones 1, 2 and 3. Zone 3 was near the center of the implant. The area of nuclei, the major axis, the minor axis and the ratio of minor axis per major axis were image-analyzed in the histological views. In zones 1, 2 and 3, the nuclear areas were 18.0 (3.1) mean (SD); unit micron2, 33.4 (5.61) and 110.1 (23.7), respectively. The major axes of nuclear ellipses were 7.45 (0.22) (unit micron), 7.76 (0.26), and 13.9 (1.88), respectively. The minor axes were 3.07 (0.53), 5.59 (0.95) and 10.1 (1.35), respectively. The ratios of minor axis per major axis of nuclear ellipses were 0.4 (0.57), 0.72 (0.11) and 0.73 (0.11) in zones 1, 2 and 3, respectively. These results showed that in zones 2 and 3 cell and tissue reactions were marked against rhBMP-2 implantation.

Contribution of salt bridge in the protease inhibitor SSI (Streptomyces subtilisin inhibitor) to its inhibitory action.

The tertiary structure of proteinaceous protease inhibitors is considered to be maintained by various interactions in the molecule that prevent degradation by protease. In this study, the Arg29 of Streptomyces subtilisin inhibitor (SSI) forming a salt bridge with the carboxyl group of carboxyl-terminal Phe113 was replaced with Ala, Met or Lys by cassette mutagenesis to clarify the role of Arg29 in the function of SSI. The inhibitory activity of each mutated SSI decreased with increasing incubation time after mixing with subtilisin, indicating that the SSI was changed into a temporary inhibitor upon mutation. This decrease was shown by SDS polyacrylamide gel electrophoresis to be due to cooperative degradation of the mutated SSI by subtilisin. In addition, the denaturation temperature of the Ala or Met mutant was decreased by ten degrees and that of the Lys mutant by 1.5 degrees, suggesting that the destabilization of SSI may be related to its temporary inhibition. Thus, interaction in the protease inhibitor molecule for maintaining the tertiary structure, such as that of Arg29 in SSI, was shown to be required for the inhibitory action.

Functional Ca2+ channel produced by purified platelet membrane glycoprotein IIB-IIIA complex incorporated into planar phospholipid bilayer.

The ability of the platelet membrane glycoprotein (GP) IIb-IIIa complex to function as a Ca2+ channel was investigated by electrophysiological methods. The GPIIb-IIIa complex was purified with an electrically silent detergent, CHAPS, and reconstituted into liposomes. After incorporation of these liposomes to a planar phospholipid bilayer, Ba(2+)-permeable channel currents were detected. Since neither residual detergent nor dissociated GPIIb and IIIa produced any currents, the observed channel currents were attributed to the GPIIb-IIIa complex. These channel currents showed similar electrical properties with the Ca2+ channel previously described in platelet plasma membrane; a single channel conductance of approximately 10 pS in 53 mM Ba2+ solution and voltage-independency. It was concluded that the purified GPIIb-IIIa complex can act as a divalent cation-permeable channel in the artificial phospholipid bilayer.

Expression of platelet membrane glycoprotein V in human megakaryocytes and megakaryocytic cell lines: a study using a novel monoclonal antibody against GPV.

Glycoprotein V (GPV) is a platelet membrane protein with a molecular weight of 82 kD, and one of the leucine rich glycoproteins (LRG). By reverse transcription-polymerase chain reaction (RT-PCR), GPV cDNA was amplified from mRNA of platelets and megakaryocytic cell lines. However, since there are few reports indicating whether GPV protein is expressed in megakaryocytes as a lineage and maturation specific protein, we studied the GPV expression at the protein level by using a novel monoclonal antibody (1D9) recognizing GPV. Flow cytometric and immunohistochemical analysis indicated that GPV was detected on the surface and in the cytoplasm of only the megakaryocytes in bone marrow aspirates. In a megakaryocytic cell line UT-7, GPV antigen increased after treatment with phorbol-12-myristate-13-acetate (PMA). These data indicate that only megakaryocytes specifically express the GPV protein among hematopoietic cells and that the expression of GPV increases with differentiation of the megakaryocyte as GPIb-IX complex.

Analysis of platelet cytoskeleton assembly during platelet activation in Hermansky-Pudlak syndrome and thrombasthenia.

Time course change of the platelet cytoskeletal protein component in the Triton X-100 insoluble fraction after stimulation was analyzed in Hermansky-Pudlak syndrome and thrombasthenia. In Hermansky-Pudlak syndrome (HPS), a 31 kDa protein, myosin, actin, and a 100 kDa protein assembled as in the normal platelets at the shape change and release reaction phases after ADP or collagen stimulation, suggesting that, the deficient dense granule content do not lead to an abnormal platelet cytoskeletal protein assembly. In thrombasthenia (Type I), myosin increased at the shape change and release reaction phases as it does in normal platelets, but actin and the 100 kDa protein increased only at the initial activation phase, and then subsequently decreased to the level of the resting phase. The actin-binding protein (ABP) and the 31 kDa protein increased a little following stimulation. Similar cytoskeletal protein change after stimulation were found in normal platelets which were prevented from the aggregation process by chelating the external Ca2+ or by using synthetic decapeptide of fibrinogen gamma-chain of carboxyl terminus. The decreased platelet cytoskeletal protein assembly in thrombasthenia or in platelets stimulated without aggregation, was derived from a loss of the platelet aggregation process due to the defect of GP IIb-IIIa complex or an interaction failure between GP IIb-IIIa complex and fibrinogen. The interaction between platelets and either fibrinogen or fibrin can induce a more stable platelet cytoskeletal protein assembly, however, agonistic stimulation without these interactions cannot do it directly.

Molecular and genetic analysis of two patients with Bernard-Soulier syndrome--identification of new mutations in glycoprotein Ib alpha gene.

We investigated two unrelated patients with Bernard-Soulier syndrome (BSS) by performing molecular and genetic analysis. A flow cytometric and immunoblotting analysis showed GP Ib alpha to be absent from the platelet membrane of both patients. Other glycoproteins that formed GP Ib/IX/V complex were present on the platelets, but in decreased amounts. Therefore, GP Ib alpha gene from both cases was sequenced after PCR amplification and subcloning. We identified a homozygous mutation of a dinucleotide deletion within the TGTG repeat at cDNA number 972 to 975 in GP Ib alpha gene from Case 1. In Case 2, compound heterozygosity was demonstrated in GP Ib alpha gene; an insertion of a single base (T) at cDNA number 1,418 in one allele, and a deletion of a single base (A) within the 7-adenine repeat at cDNA number 1,438 to 1,444 in another allele. The three new mutations in both patients appeared to cause a frameshift, which created a new termination codon shortly thereafter, and thus lead to a GP Ib alpha deficiency on the platelet membrane. Truncated mutant proteins could be detected in the plasma and platelets of Case 2, but not of Case 1. According to these findings, it is thus supposed that the properties and conformation of additional COOH-terminal peptides, which were supposedly synthesized as results of the mutations, may have an important role on the processing of mutant GP Ib alpha in megakaryocytes and platelets.

Heterogeneous expression of glycoprotein Ib, IX and V in platelets from two patients with Bernard-Soulier syndrome caused by different genetic abnormalities.

Bernard-Soulier syndrome (BSS) is a rare inherited bleeding disorder, which is caused by deficiency or decrease of the platelet GPIb/IX/V complex. Analysis of two patients with BSS by flow cytometry of the blood revealed different expression patterns of the components of the GPIb/IX/V complex. In case 1, GPIX was completely absent but residual amounts of GPIb alpha and GPV were detectable; in case 2, GPIb alpha was completely absent. We amplified the coding regions of GPIb alpha, GPIb beta, GPV, and GPIX from the patients' genomic DNA with the polymerase chain reaction (PCR) and sequenced the PCR products. in case 1, we identified a point mutation in the GPIX coding region that changes the codon for tryptophan-126 (TGG) to a nonsense codon (TGA). In case 2, we found a deletion of nucleotide within seven adenine repeats at the position of 1932 to 1938 in the coding region of GPIb alpha, which causes a frame shift that results in 58 altered amino acids and a premature stop codon. These genetic changes alter the transmembrane domain of GPIX or GPIb alpha and, therefore, would prevent proper insertion of the proteins in the plasma membrane. Thus, abnormality of a single component protein (GPIX or GPIb alpha) alters the assembly of the GPIb/IX/V complex and causes heterogeneous surface expression of GPIb alpha, GPV and GPIX.

A point mutation in glycoprotein IX coding sequence (Cys73 (TGT) to Tyr(TAT)) causes impaired surface expression of GPIb/IX/V complex in two families with Bernard-Soulier syndrome.

Bernard-Soulier syndrome (BSS) is a rare inherited bleeding disorder which is caused by abnormal expression or function of the glycoprotein (GP) Ib/IX/V complex, a platelet major receptor for von Willebrand factor. We studied four BSS patients in two unrelated families in which the same and novel mutation was found. Flow cytometric analysis showed that GPIX was completely absent but residual amounts of GPIb alpha and GPV were detectable in these patients. We analyzed all coding regions of GPIb alpha, GPIb beta, GPV and GPIX which were amplified from the patients' genomic DNA by the polymerase chain reaction (PCR). In all four cases, we identified a point mutation in the GPIX coding region that changes the codon for cysteine 73 (TGT) to a codon for tyrosine (TAT). Furthermore, we confirmed by a transient expression study that the mutation caused the loss of adequate surface expression of GPIX. Since cysteine might be important for the secondary structure, this mutation of GPIX gene would lead to a dramatic conformational change of GPIX protein, resulting in the reduced surface expression. We concluded that this novel point mutation of the GPIX gene was responsible for BSS in these families.

Two distinct novel splice site mutations in a compound heterozygous patient with protein S deficiency.

Genetic analysis revealed two distinct novel splice site mutations in a compound heterozygous patient with protein S deficiency. The paternal mutation was a G-to-T transition at position-1 of the acceptor splice site of intron N (Mutation I), and the maternal mutation was a G-to-C transversion at position-1 of the donor splice site of intron C (Mutation II). Both splice site mutations decreased the mutated mRNA accumulation to the same extent, approximately 40% of the normal mRNA. However, the mutations were associated with different phenotypical expressions: the paternal mutant protein S was not detected in vivo, while the maternal mutant protein S was present in the plasma in reduced quantity. Because Mutation I caused a cryptic splicing in the mutated mRNA, resulting in a reading frameshift and premature termination, the predicted mutant protein S might be highly unstable. In contrast. Mutation II led to the substitution of Va146 by Leu, which might be much less deleterious for the synthesis, secretion and stability of the predicted mutant protein S. It was supposed that the different post-translational metabolisms produced the distinct phenotypical expressions of the mutations.

Modulation by drugs acting on the autonomic nervous system of platelet-activating factor in the stomach of rats.

Platelet-activating factor (PAF), an ether linked choline glycerophospholipid, is a potent initiator of diverse physiological and pathological processes. We have reported that gastric endogenous PAF levels were reduced and the contents of each of its molecular species changed during water-immersion stress in rats (Sugatani J et al., FASEB J 3: 65-70, 1989 and Sugatani J et al., Lipids 26: 1347-1353, 1991). In this study, we determined the effects of autonomic drugs on the level of gastric PAF, its molecular heterogeneity and formation of gastric erosions in unstressed rats and those subjected to water-immersion stress. Atropine, an anticholinergic drug, suppressed both the stress-induced changes and development of gastric lesions. 6-Hydroxydopamine-induced sympathectomy induced a small decrease in the gastric PAF levels and the addition of stress further decreased the PAF levels and development of gastric lesions. Carbamylcholine induced a transient decrease in the gastric PAF level of normal rats, which was not associated with gastric erosion formation. In contrast, the endogenous gastroprotective factor dopamine evoked transient dose- and time-dependent increases in the gastric PAF levels. These observations indicate that cholinergic muscarinic-receptor activation in rats led to decreases in gastric PAF levels and a prolonged and marked decrease in its level was associated with the development of gastric lesions, and that dopamine increases gastric PAF levels. Gastric endogenous PAF levels are closely associated with the autonomic nervous system and should be considered further in investigations of gastric function.