Gut dysbiosis is associated with acceleration of lupus nephritis.

The gut microbiota (GM) exerts a strong influence over the host immune system and dysbiosis of this microbial community can affect the clinical phenotype in chronic inflammatory conditions. To explore the role of the GM in lupus nephritis, we colonized NZM2410 mice with Segmented Filamentous Bacteria (SFB). Gut colonization with SFB was associated with worsening glomerulonephritis, glomerular and tubular immune complex deposition and interstitial inflammation compared to NZM2410 mice free of SFB. With SFB colonization mice experienced an increase in small intestinal lamina propria Th17 cells and group 3 innate lymphoid cells (ILC3s). However, although serum IL-17A expression was elevated in these mice, Th17 cells and ILC3s were not detected in the inflammatory infiltrate in the kidney. In contrast, serum and kidney tissue expression of the macrophage chemoattractants MCP-1 and CXCL1 were significantly elevated in SFB colonized mice. Furthermore, kidney infiltrating F4/80+CD206+M2-like macrophages were significantly increased in these mice. Evidence of increased gut permeability or "leakiness" was also detected in SFB colonized mice. Finally, the intestinal microbiome of SFB colonized mice at 15 and 30 weeks of age exhibited dysbiosis when compared to uncolonized mice at the same time points. Both microbial relative abundance as well as biodiversity of colonized mice was found to be altered. Collectively, SFB gut colonization in the NZM2410 mouse exacerbates kidney disease, promotes kidney M2-like macrophage infiltration and overall intestinal microbiota dysbiosis.

Effects of twice-weekly follicular punctures of ovaries with or without the corpus luteum on follicular and luteal dynamics.

We investigated the effects of twice-weekly follicular punctures of ovaries with or without corpus luteum (CL) on follicular and luteal dynamics. A cross-over design was used, with each cow (seven Japanese Black beef cows) being assigned to one of the three groups at 2-month intervals. Follicular punctures were performed twice weekly for three consecutive weeks until day 20 (oestrus = day 0). All visible follicles (diameter >3 mm) in the ovaries bearing CL (ipsilateral group) or those in the contralateral ovaries (contralateral group) were aspirated. As a control, all visible follicles in both ovaries were aspirated (bilateral group). Follicular development, CL formation and progesterone concentrations in each cow were monitored from days 0 to 30. Follicular growth profiles in the punctured ovaries during/after puncture treatment were similar, irrespective of the presence of follicles in the unpunctured ovary and the CL in the punctured or unpunctured ovaries. After puncture, two cows (28.6%) each in the ipsilateral and bilateral groups did not exhibit behavioural oestrus until day 30, whereas all cows in the contralateral group exhibited oestrus. CL growth and increase in progesterone concentrations after the last follicular puncture in the bilateral group were delayed when compared with those in the ipsilateral group. Our results indicate that the presence of follicles in the unpunctured ovary and the CL in the punctured or unpunctured ovaries does not significantly influence follicular growth in punctured ovaries during/after puncture treatment. However, follicular puncture in ovaries bearing CL may disturb or delay oestrus occurrence after treatment.

Differential gene expression of vascular endothelial growth factor isoforms and their receptors in the development of the rat masseter muscle.

The capillary network in the masseter muscle develops dramatically with the differentiation of muscle fibres after birth, especially around weaning. Here, developmental changes in mRNA expression for four splicing variants of vascular endothelial growth factor (VEGF) and for two distinct VEGF receptors (Fms-like tyrosine kinase (Flt-1) and kinase insert domain-containing receptor/fetal liver kinase-1 (KDR/Flk-1)) were studied in rat masseter. The relative abundance of VEGF (120) mRNA was the highest, representing 35% of total VEGF mRNA on day 7 after birth and gradually decreased with age to become approximately 5% on day 37. In contrast, VEGF (188) mRNA was very low in the newborn rat, but increased sharply before weaning and reached 40-50% of the total on day 50. Neither VEGF (144) nor VEGF(164) mRNA showed any significant change in abundance after birth. The expression of KDR/Flk-1 mRNA was transiently high in the early postnatal stage and gradually decreased with age, Flt-1 mRNA was stably expressed at a constant level after birth. These findings suggest that different combinations of VEGF isoforms and their receptors regulate angiogenesis in the development of the masseter muscle.

Invasive pulmonary aspergillosis resulting in respiratory failure during neutrophil recovery from postchemotherapy neutropenia in three patients with acute leukaemia.

Respiratory failure is a severe complication of invasive pulmonary aspergillosis (IPA). Its pathogenesis is not well understood. We herein describe three cases of subacute respiratory failure that occurred during the recovery phase of neutropenia following induction chemotherapy for acute leukaemia with IPA. In each case, severe neutropenia (19-85 days), high-grade fever, severe anaemia, the use of granulocyte-colony-stimulating factor and increasing infusion volume were noted. As the neutrophil count was recovering, the shadows on the chest X-ray expanded with progressing hypoxia. We should pay attention to the respiratory failure during the recovery phase of neutropenia in patients with IPA.

Differential expression of human beta-defensin 2 in keratinized and non-keratinized oral epithelial lesions; immunohistochemistry and in situ hybridization.

Human beta-defensin(hBD)-2, an antimicrobial peptide, is produced by various epithelial cells. Because hBD-2 expression in the oral epithelium has not been assessed, we investigated its localization in normal oral epithelium and epithelial lesions. hBD-2 expression was studied using immunohistochemistry and in situ hybridization on formalin-fixed, paraffin-embedded tissue sections from 30 cases of squamous cell carcinoma and 6 cases of leukoplakia. Immunostaining for hBD-2 was more intense in hyperkeratinized than in ortho- or non-keratinized epithelium. In contrast, signals for hBD-2 mRNA were frequently stronger in non-keratinized epithelium than in hyper- or ortho-keratinized epithelium. The results suggest that keratinization in oral epithelium plays an important role in the biological function of hBD-2 both at the mRNA level and in the retention of the peptide in the epithelium.

Developmental expression of vascular endothelial growth factor in the masseter muscle of rats.

Developmental changes in vascular endothelial growth factor (VEGF) in rat masseter after birth were investigated. VEGF was extracted efficiently and reproducibly from muscle homogenate with low concentrations of non-ionic detergents, such as Triton X-100, Nonidet P-40, and Tween 20. The amount of VEGF measured by enzyme-linked immunosorbent assay (ELISA) increased markedly by approximately 9-fold, from day 8 to 35 after birth. The increase in VEGF was closely correlated with the development of the capillary network, as shown by the capillary to muscle fibre ratio (C/F ratio). Immunoblotting revealed that the predominant molecular species of VEGF concentrated with heparin-sepharose beads was VEGF(188). These results suggest that VEGF plays an important part in the development and maintenance of the capillary network in the rat masseter.

Interaction of SNARE proteins in rat parotid acinar cells.

The protein-protein interaction between [soluble NSF attachment protein (SNAP) receptor] (SNARE) proteins found in the lysate of parotid acinar cells was investigated. Immunoblotting analysis showed that parotid acini contain both syntaxin-4 and SNAP-23, plausible candidates of target membranes (t-) SNAREs in non-neuronal cells. However, when vesicle-associated membrane protein (VAMP)-2 was immunoprecipitated from lysates of parotid acinar cells, syntaxin-4 and SNAP-23 were not coprecipitated with VAMP-2, although syntaxin-1 and SNAP-25, t-SNAREs in neuronal cells, were clearly coprecipitated with VAMP-2 from brain lysates. Inversely, when syntaxin-4 was immunoprecipitated from parotid lysates, SNAP-23, Munc18c, and N-ethylmaleimide-sensitive fusion protein (NSF) were coprecipitated, but VAMP-2 was again undetectable. When proteins in the crude secretory-granule fraction were biotinylated and then immunoprecipitated with anti-VAMP-2, 35- and 80-kDa proteins were coprecipitated along with VAMP-2. These results suggest that the interaction between syntaxin-4, SNAP-23 and VAMP-2 is fairly weak and their concentrations in the cell lysate are insufficient to make a readily detectable complex, and that bindings between these proteins are hindered by other proteins in parotid acinar cells.

Ovariectomy causes cell proliferation and matrix synthesis in the growth plate cartilage of the adult rat.

The in vivo effects of ovariectomy in rats have been studied on cell proliferation and matrix synthesis in the growth plate cartilage by assessing immunohistochemically the levels of proliferating cell nuclear antigen and chondroitin sulphate proteoglycan(s). The serum levels of insulin-like growth factor-I and growth hormone were also measured by radioimmunoassay procedures. At 5 weeks after ovariectomy, the serum levels of the growth factor were significantly higher than those in sham-operated rats. In contrast, the level of growth hormone was lower. The nuclear staining of proliferating cell nuclear antigen was generally seen in the zone of proliferative chondrocytes from both groups of rats. Whereas almost all chondrocytes in the proliferative zone of ovariectomized rats expressed proliferating cell nuclear antigen immunoreactivity, fewer did so in that of the sham rats. Quantitative image analysis by ACAS 570 laser cytometry demonstrated that the nuclear antigen-positive sites in ovariectomized rats had significantly higher integrated values (staining intensity), areas and perimeters than those in sham rats. In addition, the number of chondroitin sulphate proteoglycan-immunoreactive cells in the proliferative chondrocytes was also higher in ovariectomized rats than in sham ones. These results suggest that ovariectomy significantly stimulates the cell proliferation and matrix synthesis in the growth plate cartilage, probably through the higher serum level of insulin-like growth factor-I.

Regulation of CREB phosphorylation by cAMP and Ca2+ in parotid acinar cells.

Since various secretory stimuli regulate not only secretion but also protein, RNA, and DNA syntheses in salivary glands, we evaluated the effect of secretory stimuli on the phosphorylation state of CREB (cAMP response element-binding protein). Isoproterenol, forskolin, and CPS-cAMP markedly stimulated the phosphorylation of CREB in parotid acinar cells, and PKA inhibitors H-8 and H-89 dose-dependently inhibited it. In contrast, carbachol (CCH) and A23187 decreased CREB phosphorylation, but CCH did not decrease it in the absence of extracellular Ca2+. Although protein phosphatase inhibitor calyculin A alone markedly increased the phosphorylation, it could not prevent CCH-induced dephosphorylation of CREB. CaM kinase IV, a putative protein kinase for CREB in response to Ca2+ elevation, was undetectable in parotid acinar cells.

Dephosphorylation of cofilin in parotid acinar cells.

Cofilin is an actin-depolymerizing protein, whose depolymerizing activity is supposed to be regulated in part by phosphorylation and dephosphorylation. Thus, we studied the phosphorylation states of cofilin in rat parotid acinar cells during stimulation for amylase exocytosis. Isoproterenol and carbachol induced rapid and extensive dephosphorylation of cofilin; 60-70% dephosphorylation was clearly detectable within 1 min. Membrane-permeable cyclic AMP (CPS-cAMP), phorbol ester (PMA), and Ca ionophore A23187 mimicked the effect of isoproterenol and carbachol. Protein phosphatase inhibitors (calyculin A or FK506 plus cyclosporin A) did not block the dephosphorylation in response to isoproterenol or carbachol. Furthermore, calyculin A alone strongly dephosphorylated cofilin. Although no exogenous protein phosphatases tested dephosphorylated cofilin in the homogenate, the cofilin that was isolated by immunoprecipitation was clearly dephosphorylated by protein phosphatases 1, 2A, and 2C.

Induction of amylase release from rat parotid acinar cells by cooling in vitro.

As amylase exocytosis from parotid acinar cells is an energy-dependent process, it was supposed that it could be terminated by cooling on ice. Unexpectedly, however, the cooling itself markedly induced amylase release from parotid acini. The release finished within 1 min and prolonged incubation on ice did not cause further release. The cold-induced amylase release was observed in the absence of extracellular calcium, and calcium ionophore A23187 neither enhanced nor inhibited it. Treatments with cytochalasin D, phalloidin, or taxol did not disturb the release. Cold treatment did not increase the leakage of lactate dehydrogenase, a cytosolic enzyme, and the acini still maintained normal responsiveness to isoproterenol. Electron-microscopic observation revealed that the plasma membrane and zymogen granules of cold-exposed acini were intact, but many acinar lumina were distended with secretory materials. These results suggest that the cold treatment induces transient amylase release by the fusion of plasma membrane and the zymogen granules that have been closely docked at the luminal membrane.

Effect of genistein on amylase release and protein tyrosine phosphorylation in parotid acinar cells.

We evaluated the role of protein tyrosine phosphorylation in amylase exocytosis from parotid acinar cells by using genistein, a tyrosine kinase inhibitor. Amylase release stimulated by isoproterenol was dose-dependently inhibited by genistein. Genistein also inhibited the exocytosis evoked by dibutyryl- or 8-chlorophenylthio-cAMP. Daidzein, a negative control agent of genistein, elicited no inhibitory effect. Isoproterenol had dual effects on protein tyrosine phosphorylation; it increased that phosphorylation of 190- and 210-kDa proteins and decreased that of a 90-kDa one. The phosphorylation was dose-dependently inhibited by genistein but not by daidzein. These results suggest that protein tyrosine phosphorylation plays a role in the process of amylase exocytosis from parotid acinar cells.

Catalytic subunit of protein kinase A induces amylase release from streptolysin O-permeabilized parotid acini.

Amylase release from parotid acinar cells is a typical model of cAMP-mediated exocytosis. To obtain unequivocal data concerning the role of cAMP-dependent protein kinase (PKA) in amylase exocytosis, we undertook the direct introduction of the PKA catalytic subunit into the parotid acini by permeabilization with streptolysin O (SLO). In the presence of 100 hemolytic units/ml SLO, cAMP increased amylase release in a time- and dose-dependent manner. PKI-(5-24)-peptide, a specific PKA inhibitor, markedly inhibited amylase release, but the extent of inhibition was approximately 50%. On the other hand, the PKA catalytic subunit highly purified from bovine hearts significantly induced amylase release. The release was strictly dependent on the presence of SLO and the catalytic activity of PKA added. The catalytic subunit dose dependently induced amylase release, but the heat-inactivated subunit had no stimulatory effect. PKI-(5-24)-peptide completely blocked amylase release evoked by the subunit. These results clearly demonstrate that the catalytic subunit of PKA regulates cAMP-mediated amylase release through phosphorylation of unidentified protein(s) directly or indirectly involved in the process of exocytosis.

Evidence for the involvement of protein phosphorylation in cyclic AMP-mediated amylase exocytosis from parotid acinar cells.

We evaluated the role of protein phosphorylation in cAMP-mediated amylase exocytosis from parotid acinar cells by using H89, a new protein kinase A (PKA) inhibitor, which is more lipophilic and 25 times more potent than H8. In our previous studies, H8 markedly inhibited protein phosphorylation without decreasing amylase release [Takuma, T. (1988) Biochem. J. 256, 867-871]. These findings were completely reproduced even in the small acini that were prepared by trypsin treatment before collagenase digestion. In the present study, however, H89 strongly inhibited both amylase release and protein phosphorylation in a dose-dependent manner. The inhibitory effect was specific for PKA at least up to 33 microM, since 33 microM H89 did not block amylase release stimulated by PMA. H85, a closely related compound of H89 without inhibitory effect on PKA, did not prevent amylase release or protein phosphorylation at least up to 33 microM. These results suggest that protein phosphorylation by PKA is involved in cAMP-mediated amylase exocytosis. The inhibition of protein phosphorylation by H8 might be insufficient or inadequate for blocking of amylase release.

Protein phosphatase inhibitor calyculin A induces hyperphosphorylation of cytokeratins and inhibits amylase exocytosis in the rat parotid acini.

Calyculin A, a protein phosphatase inhibitor with a chemical structure completely different from that of okadaic acid, reproduced the inhibitory effect of okadaic acid on cyclic AMP-mediated amylase release from rat parotid acinar cells. Calyculin A markedly enhanced phosphorylation of cytokeratins in the cytoskeletal fraction of the cells, whereas cAMP had apparently no effect on the phosphorylation. Microscopic observations showed that parotid acini incubated with 100 nM calyculin A for 15 min had large vacuoles in the cytoplasm and conspicuous blebs on the basal plasma membrane. K252a, a nonselective protein kinase inhibitor, clearly reduced calyclin A-induced phosphorylation of cytokeratins, and it markedly blocked the inhibition of amylase release and morphological changes evoked by calyculin A. These results suggest that hyperphosphorylation of cytokeratins profoundly affects the morphology and secretory activity of parotid acinar cells.

Effects of valinomycin on osmotic lysis of zymogen granules and amylase exocytosis from parotid acini.

The role of osmotic swelling of the secretory granules in adenosine 3',5'-cyclic monophosphate (cAMP)-mediated amylase exocytosis was evaluated by use of isolated zymogen granules and saponin-permeabilized acini of the rat parotid gland. The osmotic lysis of the isolated granules was markedly enhanced by the addition of valinomycin (> 10(-9) M) in the presence of isosmotic KSCN or KI medium. However, valinomycin (up to 10(-5) M) did not increase the granule lysis in KCl medium, although the granules were slightly less stable in KCl medium than in K2SO4 or potassium gluconate medium. Guanosine 5'-O-(3-thiotriphosphate) did not affect the granule lysis. Valinomycin alone had no effect on amylase release from saponin-permeabilized parotid acini incubated in KCl medium, but completely abolished cAMP-mediated amylase release in all K+ media used. The inhibition was clearly detected at 0.1 microM valinomycin in KCl medium, not blocked by the addition of 1 mM MgATP to the medium, and was greatly reduced in NaCl medium. cAMP-evoked amylase release was completely inhibited by SCN- and I- (permeant anions), the mean inhibitory dosages of which were approximately 25 and 50 mM, respectively. These results suggest that 1) the membrane of parotid zymogen granules has no detectable Cl- channels responsible for osmotic swelling of the granules, and 2) increase in K+ or anion conductance of the granule does not enhance but inhibits cAMP-mediated amylase exocytosis from parotid acini.

Roles of potassium and chloride ions in cAMP-mediated amylase exocytosis from rat parotid acini.

The roles of potassium and chloride ions in cAMP-mediated amylase exocytosis were studied using intact and saponin-permeabilized parotid acini. Cyclic AMP-evoked amylase release from saponin-permeabilized parotid acini decreased markedly when KCl in the incubation medium was isoosmotically replaced by K-glutamate, NaCl, Na-isothionate, or mannitol. Quinidine and barium, K+ channel blockers, clearly inhibited amylase release from the permeabilized acini, but not from intact ones. The chloride channel blocker DPC (diphenylamine-2-carboxylate) also inhibited amylase release, while DIDS (4,4'-diisothiocyanostilben-2,2'-disulfonate) or bumetanide had little effect, if any, on the exocytosis. Hyperosmolarity with mannitol markedly reduced amylase release from permeabilized acini. These results suggest that potassium and chloride ions play important roles in cAMP-mediated amylase exocytosis, and that these ions act on secretory granules inside the acinar cells.

Cyclic AMP antagonist Rp-cAMPS inhibits amylase exocytosis from saponin-permeabilized parotid acini.

Rp-cAMPS, the Rp-diastereomer of adenosine 3',5'-phosphorothioate, is often referred to as a cAMP antagonist, since it binds to the regulatory subunit of cAMP-dependent protein kinase without dissociation of free catalytic subunits. To evaluate the role of cAMP-dependent protein kinase in amylase exocytosis, we examined the effect of Rp-cAMPS on amylase release from rat parotid acini. Rp-cAMPS did not stimulate amylase release from saponin-permeabilized parotid acini, whereas its Sp-isomer strongly evoked amylase release. Rp-cAMPS dose-dependently inhibited amylase release stimulated by Sp-cAMPS. In the presence of Rp-cAMPS, the dose-response curve of Sp-cAMPS was shifted to the right. The inhibitory effect of Rp-cAMPS on isoproterenol-induced amylase release was not detected in intact acini, but was clearly observed in the permeabilized ones. Rp-cAMPS markedly inhibited protein phosphorylation evoked by Sp-cAMPS, indicating that Rp-cAMPS prevents the dissociation of cAMP-dependent protein kinase. These results, taken together with synergistic increase in amylase release by the combination of site-selective cAMP analogues [T. Takuma (1990) J. Biochem. 108, 99-102], suggest that cAMP-dependent protein kinase is involved in the exocytosis of amylase from parotid acini.

Okadaic acid inhibits amylase exocytosis from parotid acini stimulated by cyclic AMP.

To evaluate the role of protein phosphorylation in amylase exocytosis, we studied the effects of okadaic acid, a potent inhibitor of protein phosphatase types 1 and 2A, on amylase release and protein phosphorylation in rat parotid acini. Although okadaic acid by itself weakly stimulated amylase release, it did not potentiate amylase release stimulated by half-maximum doses of isoproterenol or cAMP, and markedly inhibited their maximum effects. Okadaic acid dose-dependently increased cAMP-independent phosphorylation of some proteins and enhanced cAMP-dependent phosphorylation of 21- and 26-kDa proteins. These results indicate that increase in protein phosphorylation does not necessarily enhance the exocytosis of amylase from parotid acini.

Tumor necrosis factor-alpha, transforming growth factor-beta, and tetradecanoylphorbol acetate: competence factors for ML-1 human myeloblastic leukemia cell differentiation.

ML-1 human myeloblastic leukemia cells, suspended in RPMI-1640 medium, differentiated to monocyte or macrophage-like cells when either tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta), or tetradecanoylphorbol acetate (TPA) was added prior to or simultaneously with fetal bovine serum (FBS). When FBS was applied first, and followed, after washing, by the cytokines or by TPA, maturation did not occur. A 77 kDa glycoprotein (DF77), isolated from human leukocyte-conditioned medium and present in FBS, was capable of replacing FBS for induction of differentiation. Thus, in this cell system, TNF-alpha, TGF-beta, and TPA acted as competence factors, whereas DF77 acted as the progression signal. Optimal competence was established after exposure of the cells to TPA or to either of the cytokines for approximately 2 or 30 min, respectively. After removal of the factors, competence was retained for approximately 3 hr before it declined. These results demonstrate that the initiation of ML-1 human myeloblastic leukemia cell differentiation relied upon the sequential and ordered input of competence and progression signals.