Gut-derived acetate promotes B10 cells with antiinflammatory effects.

Autoimmune diseases are characterized by a breakdown of immune tolerance partly due to environmental factors. The short-chain fatty acid acetate, derived mostly from gut microbial fermentation of dietary fiber, promotes antiinflammatory Tregs and protects mice from type 1 diabetes, colitis, and allergies. Here, we show that the effects of acetate extend to another important immune subset involved in tolerance, the IL-10-producing regulatory B cells (B10 cells). Acetate directly promoted B10 cell differentiation from mouse B1a cells both in vivo and in vitro. These effects were linked to metabolic changes through the increased production of acetyl-coenzyme A, which fueled the TCA cycle and promoted posttranslational lysine acetylation. Acetate also promoted B10 cells from human blood cells through similar mechanisms. Finally, we identified that dietary fiber supplementation in healthy individuals was associated with increased blood-derived B10 cells. Direct delivery of acetate or indirect delivery via diets or bacteria that produce acetate might be a promising approach to restore B10 cells in noncommunicable diseases.

A case report of puffer fish poisoning in singapore.

Although many Asians regard puffer fish as a delicacy since ancient times, puffer fish (Lageocephalus scitalleratus) is also a well-known source of possibly lethal food poisoning. The fish is gaining popularity in Singapore and can be found in quite a few restaurants now. Puffer fish contains tetrodotoxin (TTX), a potent poison affecting the neural pathway. Puffer fish poisoning may cause a constellation of symptoms, such as giddiness, numbness and tingling sensation of the mouth, paresthesia, and muscle weakness. Severe cases may present with respiratory depression, circulatory failure, and death. TTX poisonings have been reported in Japan, Taiwan, Hong Kong, Bangladesh, and the United States (Haque et al. 2008). We report a case of mild poisoning and suggest observation for such cases.

Azacitidine fails to eradicate leukemic stem/progenitor cell populations in patients with acute myeloid leukemia and myelodysplasia.

Epigenetic therapies demonstrate significant clinical activity in acute myeloid leukemia (AML) and myelodysplasia (MDS) and constitute an important new class of therapeutic agents. However hematological responses are not durable and disease relapse appears inevitable. Experimentally, leukemic stem/progenitor cells (LSC) propagate disease in animal models of AML and it has been postulated that their relative chemo-resistance contributes to disease relapse. We serially measured LSC numbers in patients with high-risk AML and MDS treated with 5'-azacitidine and sodium valproate (VAL-AZA). Fifteen out of seventy-nine patients achieved a complete remission (CR) or complete remission with incomplete blood count recovery (CRi) with VAL-AZA therapy. There was no significant reduction in the size of the LSC-containing population in non-responders. While the LSC-containing population was substantially reduced in all patients achieving a CR/CRi it was never eradicated and expansion of this population antedated morphological relapse. Similar studies were performed in seven patients with newly diagnosed AML treated with induction chemotherapy. Eradication of the LSC-containing population was observed in three patients all of whom achieved a durable CR in contrast to patients with resistant disease where LSC persistence was observed. LSC quantitation provides a novel biomarker of disease response and relapse in patients with AML treated with epigenetic therapies. New drugs that target this cellular population in vivo are required.

A review of elderly injuries seen in a Singapore emergency department.

INTRODUCTION: The elderly population in Singapore is steadily increasing, thus increasing the stress on healthcare provision and financing. Elderly injuries result in significant mortality and morbidity. This study aimed to identify the injury patterns, specific risk factors involved and needs of the elderly so that the current emergency model of care for the injured elderly can be improved and injury prevention strategies devised. METHODS: We conducted a retrospective study of all elderly aged 65 years and older seen for trauma in an emergency department over six months. Data captured in the real-time computer system was studied with regard to patient profile, mechanism of injury and patient disposition. RESULTS: 720 patients aged 65 years and older were seen for trauma in the first six months of 2005, accounting for 10.4 percent of the total attendance for that age group. Home injuries (67.9 percent) were the most common, followed by road-related injuries (21.2 percent). 85.3 percent of the injuries were due to falls. 49.9 percent of the patients were admitted to hospital. We also examined the underlying causes of the injuries and the common injuries sustained. CONCLUSION: Injuries in the elderly is a significant problem. Most of the injuries occur at home and falling is the commonest cause. Many of the injuries are potentially preventable. Several possible injury prevention strategies and improvements to the current emergency model of care of the injured elderly are discussed. The establishment of a national elderly injury surveillance database is advocated.

Efficacy of community-based multidisciplinary disease management of chronic heart failure.

INTRODUCTION: A multidisciplinary disease management (DM) programme in chronic heart failure (CHF) improves clinical outcome. The efficacy of such a programme in a heterogeneous Asian community is not well established. Therefore, we undertook the evaluation of the efficacy of the multidisciplinary community-based DM CHF programme. METHODS: This was a prospective study involving 154 patients (54 percent male) with a primary diagnosis of CHF, New York Heart Association functional class III/IV CHF, with left ventricular ejection fraction (LVEF) less than 40 percent. The mean age was 65 +/- 12 years and mean LVEF was 27 +/- 9 percent. We evaluated CHF hospitalisation, quality of life, activity status and quality of care (percentage of patients who received ACE inhibitors/angiotensin receptor blockers (ARB) and beta blockers after a period of six months. RESULTS: At six months, there was improvement in the quality of life and activity status (p < 0.001). ACE inhibitors/ARB were maintained in 97 percent of the patients and there was an increased usage of beta blockers (p-value equals 0.001). The rate of CHF hospitalisation was reduced by 68 percent (p-value is less than 0.001) and there was no mortality. CONCLUSION: The multidisciplinary DM of CHF in a heterogeneous Asian community showed significant improvement in quality of life, quality of care and reduction in CHF hospitalisation.

Genetic and pharmacological analyses of involvement of Src-family, Syk and Btk tyrosine kinases in platelet shape change. Src-kinases mediate integrin alphaIIb beta3 inside-out signalling during shape change.

Platelet shape change was found to be associated with an increase in protein tyrosine phosphorylation upon stimulation of thrombin-, ADP- and thromboxane A2-G-protein coupled receptors in human platelets and thromboxane A2 receptors in mouse platelets. By using PP1 and PD173956, two structurally unrelated specific inhibitors of Src-family tyrosine kinases, and mouse platelets deficient in the Src-kinase Fyn or Lyn, we show that Src-family kinases cause the increase in protein tyrosine phosphorylation. We further detected that the non-Src tyrosine kinase Syk was activated during shape change in a manner dependent on Src-family kinaseactivation. The pharmacological experiments and the studies on Fyn-, Lyn- and Syk-deficient mouse platelets showed that neither Src-family kinases nor Syk are functionally involved in shape change. Also human platelets deficient of the tyrosine kinase Btk showed a normal shape change. Binding of PAC-1 that recognizes activated integrin alphaIIb beta3 complexes on the platelet surface was enhanced during shape change and blocked by inhibition of Src-kinases. We conclude that the activation of Src-kinases and the subsequent Syk stimulation upon activation of G-protein coupled receptors are not involved in the cytoskeletal changes underlying shape change of human and mouse platelets, but that the stimulation of this evolutionary conserved pathway leads to integrin alphaIIb beta3 exposure during shape change.

Fyn and Lyn phosphorylate the Fc receptor gamma chain downstream of glycoprotein VI in murine platelets, and Lyn regulates a novel feedback pathway.

Activation of platelets by collagen is mediated by the complex glycoprotein VI (GPVI)/Fc receptor gamma (FcR gamma chain). In the current study, the role of 2 Src family kinases, Fyn and Lyn, in GPVI signaling has been examined using murine platelets deficient in one or both kinases. In the fyn(-/-) platelets, tyrosine phosphorylation of FcR gamma chain, phopholipase C (PLC) activity, aggregation, and secretion are reduced, though the time of onset of response is unchanged. In the lyn(-/-) platelets, there is a delay of up to 30 seconds in the onset of tyrosine phosphorylation and functional responses, followed by recovery of phosphorylation and potentiation of aggregation and alpha-granule secretion. Tyrosine phosphorylation and aggregation in response to stimulation by collagen-related peptide is further attenuated and delayed in fyn(-/-)lyn(-/-) double-mutant platelets, and potentiation is not seen. This study provides the first genetic evidence that Fyn and Lyn mediate FcR immune receptor tyrosine-based activation motif phosphorylation and PLC gamma 2 activation after the ligation of GPVI. Lyn plays an additional role in inhibiting platelet activation through an uncharacterized inhibitory pathway. (Blood. 2000;96:4246-4253)

Evidence of a role for SHP-1 in platelet activation by the collagen receptor glycoprotein VI.

The Src homology (SH)2 domain-containing protein-tyrosine phosphatase SHP-1 is tyrosine phosphorylated in platelets in response to the glycoprotein VI (GPVI)-selective agonist collagen-related peptide (CRP), collagen, and thrombin. Two major unidentified tyrosine-phosphorylated bands of 28 and 32 kDa and a minor band of 130 kDa coprecipitate with SHP-1 in response to all three agonists. Additionally, tyrosine-phosphorylated proteins of 50-55 and 70 kDa specifically associate with SHP-1 following stimulation by CRP and collagen. The tyrosine kinases Lyn, which exists as a 53 and 56-kDa doublet, and Syk were identified as major components of these bands, respectively. Kinase assays on SHP-1 immunoprecipitates performed in the presence of the Src family kinase inhibitor PP1 confirmed the presence of a Src kinase in CRP- but not thrombin-stimulated cells. Lyn, Syk, and SLP-76, along with tyrosine-phosphorylated 28-, 32-, and 130-kDa proteins, bound selectively to a glutathione S-transferase protein encoding the SH2 domains of SHP-1, suggesting that this is the major site of interaction. Platelets isolated from motheaten viable mice (mev/mev) revealed the presence of a heavily tyrosine-phosphorylated 26-kDa protein that was not found in wild-type platelets. CRP-stimulated mev/mev platelets manifested hypophosphorylation of Syk and Lyn and reduced P-selectin expression relative to controls. These observations provide evidence of a functional role for SHP-1 in platelet activation by GPVI.

Phosphatidylinositol 3,4,5-trisphosphate regulates Ca(2+) entry via btk in platelets and megakaryocytes without increasing phospholipase C activity.

The role of phosphatidylinositol 3,4,5-trisphosphate (PI3,4,5P(3)) and Btk in signalling by the collagen receptor glycoprotein VI was investigated. PI3,4,5P(3) was increased in platelets from mice deficient in the SH2 domain-containing inositol 5-phosphatase (SHIP), in response to collagen related peptide (CRP). Tyrosine phosphorylation and activation of phospholipase Cgamma2 (PLCgamma2) were unaltered in SHIP(-/-) platelets, whereas Btk was heavily tyrosine phosphorylated under basal conditions and maximally phosphorylated by low concentrations of CRP. There was an increase in basal Ca(2+), maximal expression of P-selectin, and potentiation of Ca(2+) and aminophospholipid exposure to CRP in SHIP(-/-) platelets in the presence of Ca(2+) (1 mM). Microinjection of PI3,4, 5P(3) into megakaryocytes caused a 3-fold increase in Ca(2+) in response to CRP, which was absent in X-linked immunodeficiency (Xid) mice, which have a mutation in the PH domain of Btk. There was a corresponding partial reduction in the sustained level of intracellular Ca(2+) in response to CRP in Xid mice but no change in PLC activity. These results demonstrate a novel pathway of Ca(2+) entry that involves PI3,4,5P(3) and Btk, and which is independent of increased PLC activity.

Thrombopoietin potentiates collagen receptor signaling in platelets through a phosphatidylinositol 3-kinase-dependent pathway.

Collagen activates platelets through a tyrosine kinase-dependent pathway, involving phospholipase Cgamma2. Functional responses such as aggregation and secretion induced by collagen are potentiated by preincubation with thrombopoietin (TPO). In this study, we show that collagen and thrombopoietin activate the phosphatidylinositol 3-kinase (PI 3-kinase) pathway and that this contributes to their respective actions. The structurally distinct inhibitors of PI 3-kinase, wortmannin, and LY294002, completely inhibit formation of phosphatidylinositol 3,4,5-trisphosphate by collagen. This leads to a substantial reduction in the formation of inositol phosphates and phosphatidic acid, 2 indices of PLC activity, and the consequent inhibition of intracellular Ca(++) [Ca(++)](i), aggregation and secretion. Potentiation of the collagen response by TPO is prevented in the presence of wortmannin and LY294002. However, when the 2 PI 3-kinase inhibitors are given after the addition of TPO but before the collagen, recovery of potentiation is observed. This suggests that potentiation is mediated through activation of PI 3-kinase. TPO stimulates aggregation of platelets from a low percentage of donors and this is also blocked by wortmannin. These results suggest that the PI 3-kinase pathway plays an important role in signaling by collagen and in the priming action of TPO.

Regulation and function of WASp in platelets by the collagen receptor, glycoprotein VI.

Wiskott Aldrich syndrome (WAS) is an X-linked recessive disorder associated with abnormalities in platelets and lymphocytes giving rise to thrombocytopenia and immunodeficiency. WAS is caused by a mutation in the gene encoding the cytoskeletal protein (WASp). Despite its importance, the role of WASp in platelet function is not established. WASp was recently shown to undergo tyrosine phosphorylation in platelets after activation by collagen, suggesting that it may play a selective role in activation by the adhesion molecule. In the present study, we show that WASp is heavily tyrosine phosphorylated by a collagen-related peptide (CRP) that binds to the collagen receptor glycoprotein (GP) VI, but not to the integrin alpha2beta1. Tyrosine phosphorylation of WASp was blocked by Src family kinase inhibitors and reduced by treatment with wortmannin and in patients with X-linked agammaglobulinemia (XLA), a condition caused by a lack of functional expression of Btk. This indicates that Src kinases, phosphatidylinositol 3-kinase (PI 3-kinase), and Btk all contribute to the regulation of tyrosine phosphorylation of WASp. The functional importance of WASp was investigated in 2 WAS brothers who show no detectable expression of WASp. Platelet aggregation and secretion from dense granules induced by CRP and thrombin was slightly enhanced in the WAS platelets relative to controls. Furthermore, there was no apparent difference in morphology in WAS platelets after stimulation by these agonists. These observations suggest that WASp does not play a critical role in intracellular signaling downstream of tyrosine kinase-linked and G protein-coupled receptors in platelets.

LAT is required for tyrosine phosphorylation of phospholipase cgamma2 and platelet activation by the collagen receptor GPVI.

In the present study, we have addressed the role of the linker for activation of T cells (LAT) in the regulation of phospholipase Cgamma2 (PLCgamma2) by the platelet collagen receptor glycoprotein VI (GPVI). LAT is tyrosine phosphorylated in human platelets heavily in response to collagen, collagen-related peptide (CRP), and FcgammaRIIA cross-linking but only weakly in response to the G-protein-receptor-coupled agonist thrombin. LAT tyrosine phosphorylation is abolished in CRP-stimulated Syk-deficient mouse platelets, whereas it is not altered in SLP-76-deficient mice or Btk-deficient X-linked agammaglobulinemia (XLA) human platelets. Using mice engineered to lack the adapter LAT, we showed that tyrosine phosphorylation of Syk and Btk in response to CRP was maintained in LAT-deficient platelets whereas phosphorylation of SLP-76 was slightly impaired. In contrast, tyrosine phosphorylation of PLCgamma2 was substantially reduced in LAT-deficient platelets but was not completely inhibited. The reduction in phosphorylation of PLCgamma2 was associated with marked inhibition of formation of phosphatidic acid, a metabolite of 1,2-diacylglycerol, phosphorylation of pleckstrin, a substrate of protein kinase C, and expression of P-selectin in response to CRP, whereas these parameters were not altered in response to thrombin. Activation of the fibrinogen receptor integrin alpha(IIb)beta(3) in response to CRP was also reduced in LAT-deficient platelets but was not completely inhibited. These results demonstrate that LAT tyrosine phosphorylation occurs downstream of Syk and is independent of the adapter SLP-76, and they establish a major role for LAT in the phosphorylation and activation of PLCgamma2, leading to downstream responses such as alpha-granule secretion and activation of integrin alpha(IIb)beta(3). The results further demonstrate that the major pathway of tyrosine phosphorylation of SLP-76 is independent of LAT and that there is a minor, LAT-independent pathway of tyrosine phosphorylation of PLCgamma2. We propose a model in which LAT and SLP-76 are required for PLCgamma2 phosphorylation but are regulated through independent pathways downstream of Syk.

A role for Bruton's tyrosine kinase (Btk) in platelet activation by collagen.

Bruton's tyrosine kinase (Btk) is essential for normal B-cell receptor signalling. The lack of expression of functional Btk in humans leads to the B-cell deficiency X-linked agammaglobulinaemia (XLA). We report here that Btk is also important for signalling via the collagen receptor glycoprotein VI (GPVI) in platelets. GPVI is coupled to the Fc receptor gamma chain (FcRgamma). The FcRgamma-chain contains a consensus sequence known as the immune-receptor tyrosine-based activation motif (ITAM). Tyrosine phosphorylation of the ITAM upon GPVI stimulation is the initial step in the regulation of phospholipase C gamma2 (PLCgamma2) isoforms via the tyrosine kinase p72(Syk) (Syk) in platelets. Here we show that collagen and a collagen-related peptide (CRP), which binds to GPVI but does not bind to the integrin alpha2beta1, induced Btk tyrosine phosphorylation in platelets. Aggregation, dense granule secretion and calcium mobilisation were significantly diminished but not completely abolished in platelets from XLA patients in response to collagen and CRP. These effects were associated with a reduction in tyrosine phosphorylation of PLCgamma2. In contrast, aggregation and secretion stimulated by thrombin in Btk-deficient platelets were not significantly altered. Our results demonstrate that Btk is important for collagen signalling via GPVI, but is not essential for thrombin-mediated platelet activation.

A novel inhibitory action of wheat germ agglutinin on phospholipase C in HEL and MEG-01 cell lines.

Stimulation of HEL megakaryocytic cells by Fc gammaRIIA crosslinking is associated with tyrosine phosphorylation of syk and phospholipase C gamma2 (PLCgamma2) and is accompanied by formation of inositol phosphates and release of intracellular Ca2+. These responses are inhibited by the kinase inhibitors, staurosporine and ST271. In contrast, the G-protein receptor agonist, thrombin induces formation of inositol phosphates and release of intracellular calcium without an increase in tyrosine phosphorylation. The plant lectin wheat germ agglutinin (WGA) stimulates tyrosine phosphorylation of syk and PLCgamma2 but surprisingly does not stimulate formation of inositol phosphates and induce release of intracellular Ca2+. WGA also inhibited formation of inositol phosphates and release of intracellular Ca2+ by Fc gammaRIIA crosslinking and thrombin-stimulation. A similar inhibitory effect of WGA was observed against elevation of Ca2+ by the same two stimuli in MEG-01 megakaryotic cells. The results demonstrate a novel pathway of inhibition of PLC on crosslinking of cell surface proteins that is not present in platelets.

Glue sniffing deaths in Singapore--volatile aromatic hydrocarbons in post-mortem blood by headspace gas chromatography.

Over a period from 1983 to 1991, of a total of 19,000 post-mortems, 33 were found to have at least one aromatic hydrocarbon (benzene, toluene or xylenes) in the blood. Of the 33 deceased, 22 had a history of toluene or petrol abuse while most of the remaining 11 were suspected to be glue sniffers through evidence found at the scene. This number, which represented 0.17 per cent of all the unnatural deaths, is considered small for a nation having a glue sniffing epidemic. The low death rate, as compared to 2.1 per cent through drug and chemical poisoning during the same period, is attributed to the timely intervention by the Government who outlawed glue sniffing and the effectiveness of compulsory rehabilitation. The male gender predominates (81.8 per cent) among the 33 deceased with a mean age of 20.1 years (range 15 to 33). The mean age for the female gender is 17.7 years (range 16 to 20). The blood toluene levels were found to be in the range 0.2 to 92 micrograms per ml blood. The causes of death are: 63.6 per cent due to falling or suicide by jumping; 18.2 per cent drowning; 6.1 per cent hanging; 6.1 per cent homicide; and 6.1 per cent acute toluene poisoning. The high proportion of traumatic deaths are discussed. Headspace gas chromatography with a suitable GC column was used for the analysis. Calibration blood standards were prepared in situ or in bulk stabilized by 10 per cent (v/v) methanol to overcome the hydrophobic and volatile nature of the aromatic hydrocarbons.(ABSTRACT TRUNCATED AT 250 WORDS)