Intravenous thrombolysis for multi-ethnic Asians with acute ischaemia stroke in Malaysian public primary stroke centres versus acute stroke ready hospitals: Comparison of real-world clinical outcomes.

INTRODUCTION: Intravenous thrombolysis (IVT) with recombinant tissue plasminogen activator is beneficial in acute ischaemic stroke (AIS). We aim to compare the realworld clinical outcomes and service efficiency of IVT in Malaysian primary stroke centres (PSCs) versus acute stroke ready hospitals (ASRHs). MATERIALS AND METHODS: We conducted a multi-centre cohort study involving 5 PSCs and 7 ASRHs in Malaysia. Through review of medical records of AIS patients who received IVT from 01 January 2014 to 30 June 2021, real-world data was extracted for analysis. Univariate and multivariate regression models were employed to evaluate the role of PSCs versus ASRHs in post-IVT outcomes and complications. Statistical significance was set at p<0.05. RESULTS: A total of 313 multi-ethnic Asians, namely 231 from PSCs and 82 from ASRHs, were included. Both groups were comparable in baseline demographic, clinical, and stroke characteristics. The efficiency of IVT delivery (door-toneedle time), functional outcomes (mRS at 3 months post- IVT), and rates of adverse events (intracranial haemorrhages and mortality) following IVT were comparable between the 2 groups. Notably, 46.8% and 48.8% of patients in PSCs and ASRHs group respectively (p=0.752) achieved favourable functional outcome (mRS≤1 at 3 months post-IVT). Regression analyses demonstrated that post-IVT functional outcomes and adverse events were independent of the role of PSCs or ASRHs. CONCLUSION: Our study provides real-world evidence which suggests that IVT can be equally safe, effective, and efficiently delivered in ASRHs. This may encourage the establishment of more ASRHs to extend the benefits of IVT to a greater proportion of stroke populations and enhance the regional stroke care.

2,3,5,4'-Tetrahydroxystilbene-2-O-ß-glucoside potentiates self-renewal of human dental pulp stem cells via the AMPK/ERK/SIRT1 axis.

AIM: To evaluate the effect of 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside (THSG) on cell proliferation and examine the mechanisms of THSG-enhanced proliferative potential in human dental pulp stem cells (hDPSC). METHODOLOGY: After treatment with THSG, hDPSC were collected. Cell viability was determined by MTS assay, while messenger RNA (mRNA) expressions of proliferation and stem cell markers were analyzed using real-time PCR. Flow cytometry was also conducted to analysis protein expression of stem cell markers. A colony-forming unit assay of hDPSC was carried out. Cellular telomerase activity was also identified using real-time PCR. In addition, proliferation-related proteins involved in the effects of THSG on hDPSC were analyzed by Western blotting. Data were analyzed using one-way analysis of variance and two-tailed Student's t-test. RESULTS: Cell viability, colony-forming rates and telomerase activities of hDPSCs were enhanced after THSG treatment. mRNA expressions of proliferation markers (including expressions of NAD+-dependent histone deacetylase sirtuin 1 (SIRT1), proliferating cell nuclear antigen (PCNA), cyclin D1 and ribonucleotide reductase subunit M2 (RRM2)) increased significantly after THSG treatment (P < 0.05). Treatment with THSG for 3 h significantly augmented SIRT1 protein expression (P < 0.05). Furthermore, activities of proliferation-related proteins (including AMP-activated protein kinase (AMPK) and extracellular signal-regulated kinase (ERK) had also significantly increased at 3 h (P < 0.05). After THSG treatment, increased gene and protein expressions of pluripotent-like stem cell markers (including NANOG, OCT4, and SOX2) were observed. CONCLUSIONS: 2,3,5,4'-Tetrahydroxystilbene-2-O-ß-glucoside treatment enhanced the renewal ability and proliferative potential of hDPSCs via the AMPK/ERK/SIRT1 axis, which may provide a novel autogenic cell-based therapeutic strategy in regenerative dentistry.

Effect of high glucose, Porphyromonas gingivalis lipopolysaccharide and advanced glycation end-products on production of interleukin-6/-8 by gingival fibroblasts.

BACKGROUND AND OBJECTIVE: It is known that chronic periodontal infection can magnify the cytokine responses in patients with diabetes. Hyperglycemia increases the proinflammatory status, including the levels of advanced glycation end-products (AGEs), in patients with periodontitis. However, whether AGEs have additional effects on the production of those proinflammatory cytokines in diabetic patients with periodontitis is still unknown. To examine in vitro the effect of hyperglycemia and AGEs on the amounts of interleukin (IL)-6 and IL-8 produced in periodontally infected gingiva, human gingival fibroblasts (HGFs) were stimulated with glucose, AGE-modified bovine serum albumin (AGE-BSA) and Porphyromonas gingivalis LPS in the present study. MATERIAL AND METHODS: Primary culture of HGFs was incubated with various concentrations of AGE-BSA (0, 50, 100 and 200 µg/mL) and LPS (0, 10, 100 or 1000 ng/mL) at two different glucose concentrations - normal glucose (5 mm) and high glucose (25 mm). The amounts of IL-6 and IL-8 produced by HGFs were evaluated using ELISA. Expression of the AGE receptor on HGFs was determined by flow cytometry. RESULTS: High glucose stimulated a significant increase in the production of IL-6 and IL-8 by HGFs compared with normal glucose. This enhanced production of IL-6 and IL-8 could also be observed in the presence of LPS and/or AGE-BSA. When both LPS and AGE-BSA were present, especially at high concentrations (≥ 500 µg/mL of LPS and ≥ 25 µg/mL of AGE-BSA), a synergistic effect on IL-8 production was found in the high-glucose condition. CONCLUSIONS: A synergistic effect of the production of IL-8 could be induced in HGFs with the combination of high glucose, LPS and AGEs.

Accurate coding in sepsis: clinical significance and financial implications.

Sepsis is a major healthcare problem and leading cause of death worldwide. UK hospital mortality statistics and payments for patient episodes of care are calculated on clinical coding data. The accuracy of these data depends on the quality of coding. This study aimed to investigate whether patients with significant bacteraemia are coded for sepsis and to estimate the financial costs of miscoding. Of 54 patients over a one-month period with a significant bacteraemia, only 19% had been coded for sepsis. This is likely to lead to falsely high calculated hospital mortality. Furthermore, this resulted in an underpayment of £21,000 for one month alone.

A Salvia miltiorrhiza ethanol extract ameliorates tissue destruction caused by experimental periodontitis in rats.

BACKGROUND AND OBJECTIVE: Salvia miltiorrhiza Bunge (Labiatae), or Danshen, is a Chinese medicine used for treatment of cardiac diseases by improving blood circulation and inhibiting inflammatory responses. In this study, we aimed to determine whether an ethanol extract of S. miltiorrhiza can ameliorate tissue damage caused by periodontitis. MATERIAL AND METHODS: An ethanolic extract of S. miltiorrhiza roots was prepared, and its major constituents were determined by HPLC analysis, by comparison with known standards for the major bioactive components. The activity of the extract was evaluated in a rat model in which periodontitis was induced by ligation of a silk suture around the neck of molar teeth. The effects of the S. miltiorrhiza extract on periodontitis were assessed by dental radiography, micro-computed tomography and histology. RESULTS: The cemento-enamel junction-bone distances among the four different groups of rats were significantly different: the distance was shorter in groups treated with ligation + S. miltiorrhiza extract than in the group treated with ligation only, but was longer than in the nonligated group, regardless of the radiographic methods used. Histology and histometry also indicated a similar trend of less gingival inflammation and alveolar bone destruction in the histological sections from the S. miltiorrhiza extract groups than in those from the ligation group. CONCLUSION: Because the S. miltiorrhiza extract reduced tissue damage and bone loss caused by ligation-induced periodontitis in rats, we suggest that the S. miltiorrhiza extract might have an ameliorative effect on periodontal tissue destruction during the process of periodontitis.

The effects of diallyl sulfide upon Porphyromonas gingivalis lipopolysaccharide stimulated proinflammatory cytokine expressions and nuclear factor-kappa B activation in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Diallyl sulfide (DAS), a flavor compound from garlic, has varied potential therapeutic activities. Periodontitis is a disease that develops because of host-mediated inflammation to periodontal pathogens. In this study, the effects of DAS on the common proinflammatory cytokines and nuclear factor-kappa B (NF-κB) in human gingival fibroblasts (HGFs) being stimulated with lipopolysaccharide from Porphyromonas gingivalis, a potent periodontal pathogen, were evaluated. MATERIAL AND METHODS: Cytotoxicities of DAS and lipopolysaccharide on HGFs were measured with MTS assay. The mRNA and protein expressions of proinflammatory cytokines, including interleukin (IL)-1ß, IL-6 and tumor necrosis factor (TNF)-α, from the HGFs treated with lipopolysaccharide with and without DAS were examined with reverse transcription-polymerase chain reaction and immunocytochemistry, respectively. In addition, the activation and nuclear translocation of NF-κB with and without DAS were compared. RESULTS: DAS and lipopolysaccharide treatments within 3 mm and 10 µg/mL, respectively, did not affect the survival rate of HGFs. Lipopolysaccharide (1 µg/mL) significantly increased the mRNA expressions of IL-1ß, IL-6 and TNF-α; however, DAS (1 mm) inhibited these expressions. The protein expressions of TNF-α, IL-1ß, as well as the NF-κB nuclear translocation were increased after lipopolysaccharide treatment, but decreased when there was a DAS pretreatment. CONCLUSION: DAS diminished P. gingivalis lipopolysaccharide-stimulated cytokine expression and NF-κB activation in HGFs; we therefore suggest DAS may be beneficial on periodontal inflammation.

Cyclosporine-A inhibits MMP-2 and -9 activities in the presence of Porphyromonas gingivalis lipopolysaccharide: an experiment in human gingival fibroblast and U937 macrophage co-culture.

BACKGROUND AND OBJECTIVE: Studies have shown that bacterial plaque and the associated gingival inflammation increase the severity of gingival overgrowth induced by cyclosporine-A (CsA). This in vitro study aimed to evaluate the effect of CsA on the activities of MMPs from the co-culture of human gingival fibroblasts and U937 macrophages in the presence or absence of Porphyromonas gingivalis lipopolysaccharide (LPS). MATERIAL AND METHODS: Activities of pro-MMP-2, MMP-2 and pro-MMP-9 in the supernatants of independent cultures and co-cultures were examined by zymography. RT-PCR was selected to evaluate the expression of mRNA for membrane type-1 (MT1) MMP in the co-cultures. RESULTS: Activities of MMPs in the co-cultures were significantly greater when compared with any of the independent cultures. Lipopolysaccharide significantly increased the MMP activities in a dose-dependent manner in the co-cultures, whereas CsA inhibited these activities. In the presence of both CsA and LPS, the MMP activities inhibited by CsA could still be observed in the co-cultures. In the individual cultures, in contrast, the CsA-inhibited MMP activities, in the presence of LPS, were minimally detected. The mRNA expression of MT1-MMP was significantly enhanced after LPS treatment; however, this enhancement was inhibited by CsA. CONCLUSION: This study demonstrated that, in co-cultures of human gingival fibroblasts and U937 macrophages, CsA could inhibit MMP activities in the presence of P. gingivalis LPS. It might be part of the underlying reason for the persistent overgrowth of gingiva seen when bacterial plaque and local inflammation are present during CsA therapy.

Nrf-2 regulates cyclosporine-stimulated HO-1 expression in gingiva.

Cyclosporine-A (CsA) stimulates heme oxygenase-1 (HO-1) expression in the gingiva, but the regulation and the role of HO-1 in gingival overgrowth are not well-understood. HO-1 is regulated by several transcription factors, such as nuclear factor-κB (NF-κB) and nuclear factor erythroid-2-related factor 2 (Nrf-2). The aim of this study was to examine the role of Nrf-2 in the regulation of CsA-stimulated HO-1 expression in human gingival fibroblasts. Nrf-2 siRNA (siNrf-2), NF-κB, kinase inhibitors, and sulforaphane (SFN) were used to examine the nuclear translocation of Nrf-2 and expression of HO-1 and transforming growth factor-ß1 (TGF-ß1) in cells. Treatment with siNrf-2, but not with an NF-κB inhibitor, reduced CsA-stimulated HO-1 mRNA expression. ERK inhibition significantly decreased CsA-stimulated Nrf-2 nuclear translocation and HO-1 mRNA expression. Pre-treatment with SFN showed that HO-1 plays a role in attenuating CsA-mediated TGF-ß1 expressions. These findings suggest that CsA-stimulated HO-1 expression is mediated through the activation of ERK, and that Nrf-2 plays a protective role against CsA-induced gingival fibrosis by modulating collagen turnover-related genes.

Up-regulation of retinoblastoma protein phosphorylation in gingiva after cyclosporine A treatment: an in vivo and in vitro study.

BACKGROUND AND OBJECTIVE: Cyclosporine A can induce gingival cell proliferation; however, the precise molecular regulation of the proliferation is uncertain. Therefore, this study was carried out to examine, in vivo and in vitro, the expression of genes and proteins associated with gingival cell proliferation after treatment with cyclosporine A. MATERIAL AND METHODS: Forty Sprague Dawley rats with right maxillary posterior edentulous gingivae were assigned to a cyclosporine A group (30 mg/kg daily of cyclosporine A, administered orally) or a control group (administered mineral oil only). The animals were killed 4 wk after treatment. The edentulous gingivae were dissected out and analyzed for the expression of proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin-dependent kinase 4 (CDK4) and retinoblastoma protein (Rb1) mRNA and/or protein, and phosphorylated Rb1 (pRb1), by real-time RT-PCR or immunohistochemistry. In human gingival fibroblast (HGF) cultures, the expression of PCNA, CDK4, cyclin D1 and Rb1 proteins and Rb1 phosphorylation were determined by western blotting after cyclosporine A treatment (0-10(4) ng/mL). RESULTS: Proliferating cell nuclear antigen and cyclin D1 mRNAs (Pcna and Ccnd1, respectively) were expressed more strongly in the gingivae of cyclosporine A-treated animals than in the gingivae of the controls. Immunohistochemical analyses showed that a greater number of gingival cells stained positive for cyclin D1, CDK4 and pRb1 in the cyclosporine A group than in the control group. Increased expression of cyclin D1, CDK4 and PCNA proteins was observed in HGFs after cyclosporine A treatment. The phosphorylation of Rb1 was enhanced in HGFs after treatment with cyclosporine A at concentrations of 10(2)-10(3) ng/mL. CONCLUSION: The increases in cyclin D1, PCNA and CDK4, together with the enhanced phosphorylation of Rb1, suggest that cyclosporine A promotes cell-cycle progression through the G(1)/S transition in the gingiva.

Cyclosporine A enhances apoptosis in gingival keratinocytes of rats and in OECM1 cells via the mitochondrial pathway.

BACKGROUND AND OBJECTIVE: We reported previously that cyclosporine A induces a high level of expression of p21 in rat gingival keratinocytes and in OECM1 cells. In this study, the apoptosis of gingival keratinocytes after treatment with cyclosporine A was evaluated using the same models. MATERIAL AND METHODS: Forty Sprague-Dawley rats with right edentulous ridges were assigned into cyclosporine A (30 mg/kg) and control groups. Four weeks later, gingivae were screened for expression of apoptotic genes using microarray analyses and DNA fragmentation. The expression of bcl2-associated X protein (Bax), apoptosis-inducing factor (AIF) and Caspase 3 mRNAs, and the expression of Bax, AIF, Caspase 9 and Fas proteins, were analyzed using the reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. Apoptosis in OECM1 cells (keratinocytes of a gingival carcinoma cell line), after treatment with cyclosporine A, was evaluated by 4',6-diamidino-2-phenylindole (DAPI) staining and flow cytometry, whereas the expression of Bax, AIF, Caspase 3 and 8, Bcl-2 and Fas proteins were examined using western blotting. RESULTS: According to microarray analyses, the expression of certain apoptotic genes was altered in the gingiva of rats who received cyclosporine A, and increased number of DNA fragments were detected. Expression of mRNA or protein for Bax, AIF and Caspase 3 and 9 in the gingivae of rats increased after treatment with cyclosporine A. An increased number of apoptotic bodies and of OECM1 cells in the sub-G1 phase was observed after treatment with cyclosporine A. Increased expression of AIF, Bax and Caspase 3 protein, but not of bcl-2, Caspase 8 or Fas protein, was observed in cells after treatment with cyclosporine A. CONCLUSION: Based on the above findings, we suggest that cyclosporine A might enhance the apoptosis of gingival keratinocytes, mainly via the mitochondrial pathway.

Cyclosporine A inhibits the expression of membrane type-I matrix metalloproteinase in gingiva.

BACKGROUND AND OBJECTIVE: Membrane type-I matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase-2 (TIMP-2) regulate the activation of MMP-2; however, their roles in the activation of MMP-2 in gingiva during treatment with cyclosporine A are still unknown. Therefore, the expressions of membrane type-I MMP and TIMP-2, as well as MMP-2, in gingivae upon treatment with cyclosporine A were examined in vivo and in vitro. MATERIAL AND METHODS: Thirty-four rats were divided into two groups after edentulous ridges were established. The experimental group received 30 mg/kg/d of cyclosporine A and the control group received vehicle. At the end of the experimental period, the rats were killed, the gingivae were obtained and the expression of mRNA and protein of membrane type-I MMP, TIMP-2 and MMP-2 in gingiva were examined using real-time polymerase chain reaction and immunohistochemistry. In human gingival fibroblasts, the activity of MMP-2 and the expression of MMP-2, membrane type-I MMP and TIMP-2 mRNAs were examined (using zymography and reverse transcription-polymerase chain reaction, respectively) after treatment with cyclosporine A. RESULTS: In gingivae of rats, cyclosporine A significantly decreased the expression of mRNA and protein of membrane type-I MMP, but not of TIMP-2. The expression of MMP-2 mRNA was unaffected but the expression of MMP-2 protein showed a significant decrease upon treatment with cyclosporine A. In fibroblast culture medium, the presence of cyclosporine A induced a decrease in MMP-2 activity in a dose-dependent manner. The expression of MMP-2, membrane type-I MMP and TIMP-2 mRNAs in fibroblasts was not significantly affected by cyclosporine A; however, in fibroblasts the ratio of mRNA expression of membrane type-I MMP to that of TIMP-2 decreased as the cyclosporine A dose was increased. CONCLUSION: Cyclosporine A inhibits the expression of membrane type-I MMP in gingiva and it may further reduce the activation of MMP-2.

Expression and bioactivities of endothelin-1 in gingiva during cyclosporine A treatment.

BACKGROUND AND OBJECTIVE: This study aimed to evaluate the expression and bioactivities of endothelin-1 (ET-1) in gingiva during cyclosporine A (CsA) treatment. MATERIAL AND METHODS: After establishing edentulous ridges, experimental rats were fed 30 mg/kg/day CsA while control animals received mineral oil for 4 weeks, after which a reverse transcription-polymerase chain reaction (RT-PCR) and/or immunohistochemistry was used to examine the expression of ET-1, its receptors, proliferating cell nuclear antigen (PCNA) and inducible nitric oxide synthase (iNOS) in gingivae. The roles of the endothelin receptors A and B (ET(A) and ET(B)) in CsA-enhanced expression of PCNA and iNOS were examined in cultured human gingival fibroblasts pretreated with receptor antagonists, by immunocytochemistry and RT-PCR, respectively. RESULTS: The mRNA expression of ET-1, ET(A) and ET(B), as well as of PCNA and iNOS, was significantly greater in edentulous gingiva that received CsA compared with control gingiva. Immunohistochemistry revealed more cells positively stained for ET-1 and its receptors in the tissues of CsA-treated rats than in those of control rats. In fibroblast cultures, enhanced mRNA expression of ET-1, ET(A) and ET(B) was observed after CsA treatment at the concentrations of 10 and 100 ng/mL. Cyclosporine A-enhanced PCNA expression was somewhat reduced by blockade of ET(A), but not ET(B), whereas iNOS expression was somewhat reduced by blockade of ET(B). CONCLUSION: Based on the present findings, we suggest that: (1) CsA upregulates the gingival expression of ET-1 and its receptors; and (2) ET(A) and ET(B) have different bioactivities, ET(A) being involved in cell proliferation and ET(B) being associated with iNOS expression.