A metaphyseal fracture rat model for mechanistic studies of osteoporotic bone healing.

Most osteoporotic fractures occur at metaphyseal regions of long bones. The present study proposed a clinically relevant animal model that satisfied: i) induction of osteoporosis, ii) unilateral complete osteotomy at metaphysis, iii) internal fixation. 6 months old female Sprague-Dawley rats (n = 64) were randomly divided into the ovariectomised-metaphyseal osteotomy (OVX, n = 32) and metaphyseal osteotomy (SHAM, n = 32) groups. The metaphyseal-osteotomy model was created with a plate-fixation of the osteotomy and assessed by X-ray, micro-computed tomography, histomorphometry and mechanical testing at weeks 1, 3 and 6. X-ray results showed complete healing of metaphyseal osteotomy at week 6. Histology showed 3 stages of metaphyseal healing. Stage 1 was characterised by fibrous tissue, consisting of disorganised orientation of collagen fibres, and infiltration of immune cells. At stage 2, a transitional zone consisting of maturing fibrous tissue and differentiating mesenchymal cells with early trabecular bone formation and disorganised woven bone were observed. During stage 3, cortical bone ends unified and woven bone underwent transformation to lamellar bone. OVX group healing was significantly delayed when compared to SHAM samples. The study demonstrated that healing of osteoporotic osteotomy at the metaphyseal region was delayed in terms of radiography, histomorphometry and mechanical strength. These quantitative evaluations, along with histological features, may provide key references for future studies. The animal model may provide additional clinical relevance as most osteoporotic fracture in humans occurs at metaphyseal regions.

Candida catenulata Candidaemia and Possible Endocarditis in a Cirrhotic Patient Successfully De-escalated to Oral Fluconazole.

WHAT IS KNOWN AND OBJECTIVE: Candida catenulata is a fungus commonly found in Australian cheeses. C. catenulata has been identified as the causative pathogen for one report of onychomycosis and one report of candidaemia. CASE DESCRIPTION: A 37-year-old male underwent surgery for an incarcerated umbilical hernia repair and bowel obstruction and presented with severe abdominal pain and ascitic fluid draining from the surgical site. C. catenulata was isolated in blood cultures. The patient was treated with antifungal therapy for approximately 6 weeks. WHAT IS NEW AND CONCLUSION: To our knowledge, this is the first case describing successful treatment of possible fungal endocarditis caused by C. catenulata.

Attitudes, barriers and facilitators to the conduct of research in government hospitals: a cross-sectional study among specialists in government hospitals, northern states of Malaysia.

INTRODUCTION: Specialists constitute a major 'driving force' and catalyst for growth of research in their speciality. A clearer understanding is required as to what motivates their participation in research as well as the barriers they faced. This research aims to study the attitudes, barriers and facilitators faced by specialists and to identify strategies to promote and sustain research activities in their hospitals. METHODOLOGY: A cross-sectional survey using selfadministered questionnaires was conducted among all specialists working in government specialist hospitals in the northern states of Malaysia. RESULTS: Out of 733 questionnaires distributed, 467 were returned giving a response rate of 63.7%. Ninety-nine percent of the respondents believed that research benefits patients while 93.3% think research helps in their professional development. However, 34.8% think that under their present working conditions, it is unlikely they will participate in research. The major barriers identified were lack of funds for research (81%); lack access to expertise, software or statistical analysis (78.4%); interference with daily work schedule (75.1%) and inconsistent manpower in their department (74.2%). There are three barriers with statistically significant difference between hospitals with CRC compared to hospitals without CRC; lack of funds, mentors and access to expertise, software or statistical analysis. The demographic factors, attitudes and barriers contributing to involvement in research also investigated. The main facilitators for the conduct of research are potential to benefit patients and potential for professional development. CONCLUSION: Taking note of the findings, the Ministry of Health can implement appropriate strategies to improve specialist participation in research.

Histone deacetylase inhibitor induces DNA damage, which normal but not transformed cells can repair.

Histone deacetylase inhibitors (HDACi) developed as anti-cancer agents have a high degree of selectivity for killing cancer cells. HDACi induce acetylation of histones and nonhistone proteins, which affect gene expression, cell cycle progression, cell migration, and cell death. The mechanism of the tumor selective action of HDACi is unclear. Here, we show that the HDACi, vorinostat (Suberoylanilide hydroxamic acid, SAHA), induces DNA double-strand breaks (DSBs) in normal (HFS) and cancer (LNCaP, A549) cells. Normal cells in contrast to cancer cells repair the DSBs despite continued culture with vorinostat. In transformed cells, phosphorylated H2AX (gammaH2AX), a marker of DNA DSBs, levels increased with continued culture with vorinostat, whereas in normal cells, this marker decreased with time. Vorinostat induced the accumulation of acetylated histones within 30 min, which could alter chromatin structure-exposing DNA to damage. After a 24-h culture of cells with vorinostat, and reculture without the HDACi, gammaH2AX was undetectable by 2 h in normal cells, while persisting in transformed cells for the duration of culture. Further, we found that vorinostat suppressed DNA DSB repair proteins, e.g., RAD50, MRE11, in cancer but not normal cells. Thus, the HDACi, vorinostat, induces DNA damage which normal but not cancer cells can repair. This DNA damage is associated with cancer cell death. These findings can explain, in part, the selectivity of vorinostat in causing cancer cell death at concentrations that cause little or no normal cell death.

Paraquat poisoning: experience in hospital taiping (year 2008 - october 2011).

INTRODUCTION: Paraquat is a quaternary nitrogen herbicide which is highly toxic to human. Death is usually from respiratory failure and may occur within days up to a month after exposure. It is easily available and commonly abused to commit suicide. METHODOLOGY: This is a retrospective study describing the demographic characteristics, clinical features and outcomes of paraquat poisoning cases admitted to Hospital Taiping from 1st January 2008 to 30th October 2011. Medical records of 79 patients were reviewed. RESULT: Majority were of the Indian ethnicity (72.2%) followed by Chinese (13.9%) and Malay (10.1%). Majority was male (73.4%) and between 20 to 29 years old (34.2%). The median age of the patients was 30 years old. The mean length of stay was 6.2 days. Most exposures were intentional (69.6%) and presented to the hospital early at less than 6 hours after exposure (72.2%). Patients with positive urine paraquat result had significantly higher mortality rate compared to patients with negative results (47.4% vs 15.2% respectively). We found that neither hemofiltration nor immunosuppressive therapies help to improve survival. CONCLUSION: The non-survivor characteristics of patients with paraquat poisoning are intentional exposure, delay from exposure to hospital admission, urine paraquat positivity and manifestation of respiratory failure. The demographic characteristics, reasons for exposure and mortality rate are similar to previous reports. Urine paraquat may be used to assess severity of the exposure as well as prognosis. Hemofiltration and immunosuppression therapy do not improve patients' survival and paraquat remains a lethal killer.

A systematic review of current osteoporotic metaphyseal fracture animal models.

OBJECTIVES: The treatment of osteoporotic fractures is a major challenge, and the enhancement of healing is critical as a major goal in modern fracture management. Most osteoporotic fractures occur at the metaphyseal bone region but few models exist and the healing is still poorly understood. A systematic review was conducted to identify and analyse the appropriateness of current osteoporotic metaphyseal fracture animal models. MATERIALS AND METHODS: A literature search was performed on the Pubmed, Embase, and Web of Science databases, and relevant articles were selected. A total of 19 studies were included. Information on the animal, induction of osteoporosis, fracture technique, site and fixation, healing results, and utility of the model were extracted. RESULTS: Fracture techniques included drill hole defects (3 of 19), bone defects (3 of 19), partial osteotomy (1 of 19), and complete osteotomies (12 of 19). Drill hole models and incomplete osteotomy models are easy to perform and allow the study of therapeutic agents but do not represent the usual clinical setting. Additionally, biomaterials can be filled into drill hole defects for analysis. Complete osteotomy models are most commonly used and are best suited for the investigation of therapeutic drugs or noninvasive interventions. The metaphyseal defect models allow the study of biomaterials, which are associated with complex and comminuted osteoporotic fractures. CONCLUSION: For a clinically relevant model, we propose that an animal model should satisfy the following criteria to study osteoporotic fracture healing: 1) induction of osteoporosis, 2) complete osteotomy or defect at the metaphysis unilaterally, and 3) internal fixation.Cite this article: R. M. Y. Wong, M. H. V. Choy, M. C. M. Li, K-S. Leung, S. K-H. Chow, W-H. Cheung, J. C. Y. Cheng. A systematic review of current osteoporotic metaphyseal fracture animal models. Bone Joint Res 2018;7:6-11. DOI: 10.1302/2046-3758.71.BJR-2016-0334.R2.

Autocrine stimulation of VEGFR-2 activates human leukemic cell growth and migration.

Emerging data suggest that VEGF receptors are expressed by endothelial cells as well as hematopoietic stem cells. Therefore, we hypothesized that functional VEGF receptors may also be expressed in malignant counterparts of hematopoietic stem cells such as leukemias. We demonstrate that certain leukemias not only produce VEGF but also express functional VEGFR-2 in vivo and in vitro, resulting in the generation of an autocrine loop that may support leukemic cell survival and proliferation. Approximately 50% of freshly isolated leukemias expressed mRNA and protein for VEGFR-2. VEGF(165) induced phosphorylation of VEGFR-2 and increased proliferation of leukemic cells, demonstrating these receptors were functional. VEGF(165) also induced the expression of MMP-9 by leukemic cells and promoted their migration through reconstituted basement membrane. The neutralizing mAb IMC-1C11, specific to human VEGFR-2, inhibited leukemic cell survival in vitro and blocked VEGF(165)-mediated proliferation of leukemic cells and VEGF-induced leukemic cell migration. Xenotransplantation of primary leukemias and leukemic cell lines into immunocompromised nonobese diabetic mice resulted in significant elevation of human, but not murine, VEGF in plasma and death of inoculated mice within 3 weeks. Injection of IMC-1C11 inhibited proliferation of xenotransplanted human leukemias and significantly increased the survival of inoculated mice. Interruption of signaling by VEGFRs, particularly VEGFR-2, may provide a novel strategy for inhibiting leukemic cell proliferation.

Purification and reconstitution studies of the nucleoside transporter from pig erythrocytes.

The pig erythrocyte nucleoside transporter has been identified as a band 4.5 polypeptide (Mr 64,000) on the basis of photoaffinity labelling experiments with the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR). This protein was purified 140-fold by treatment of haemoglobin-free erythrocytes 'ghosts' with EDTA (pH 11.2) to remove extrinsic proteins, extraction of the protein-depleted membranes with n-octyl-glucoside and subsequent gradient-elution ion-exchange chromatography on DEAE-cellulose. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified material revealed the presence of only two detectable protein bands, one which co-migrated with the radiolabelled NBMPR-binding protein, and a lower molecular weight species with an Mr of 43,000. The latter protein may be a degradation product of the band 3 anion-exchange transporter. The overall purification of the NBMPR-binding protein with respect to the Mr 64,000 band was 350-fold. Reversible NBMPR-binding to the partially-purified band 4.5 preparation was saturable (apparent Kd 7.2 nM). Adjustment of the chromatography conditions to allow elution of the NBMPR-binding protein along with the majority of solubilised membrane phospholipid reduced the apparent Kd value to 3.0 nM. Purification of reversible NBMPR-binding activity during ion-exchange chromatography was paralleled by an increase in the specific activity of nitrobenzylthioguanosine (NBTGR) -sensitive uridine transport as assayed in proteoliposomes reconstituted by a freeze-thaw-sonication procedure.

Relationship between smooth pursuit eye-tracking and cognitive performance in schizophrenia.

The relationship between measures of smooth pursuit and neuropsychological performance was assessed in 20 unmedicated schizophrenics. Eye-tracking measures included gain, catch-up saccade parameters, and rate of saccadic intrusions. Neuropsychological measures included tests generally considered as "frontal": Wisconsin Card Sorting Test (WCST), Consonant Trigram Test (CTT), and Controlled Oral Word Association Test (COWAT). The Digit Symbol Test (DST), which is generally considered to be a measure of global functioning, was also included. Gain and other pursuit measures were significantly correlated with the DST and the COWAT, but were not correlated with the WCST or the CTT.

Structural organization of the rat mitogen-activated protein kinase phosphatase 2 gene.

Mitogen-activated protein kinase (MAPK) phosphatases (MKPs) are dual specificity protein phosphatases that specifically inactivate MAPKs. Regulated expression of MKPs plays a key role in determining their physiological function. However, little is known about the molecular mechanism of the activation of MKP genes. In this study, we cloned the rat MKP-2 gene and characterized its structure. The MKP-2 gene has four exons and three introns. The organization of exons of the MKP-2 gene is very similar to that of the MKP-1 gene, suggesting that MKP-1 and MKP-2 are derived from the same ancestral gene. We identified multiple transcription start sites (TSSs) for the MKP-2 gene. There is no functional TATA motif in the 5' proximal region of the TSSs. Instead, this region is highly GC-rich and has two putative Sp1 sites. A 1.8 kb 5' flanking region of the MKP-2 gene is sufficient to mediate transcriptional activation of the luciferase reporter gene by phorbol ester in GH3 cells. These results provide essential information about structural organization and regulatory sequences of the MKP-2 gene for further investigation of the molecular mechanisms of MKP-2 induction by extracellular stimuli.

Reactivity and assay restriction profiles of monoclonal and polyclonal antibodies to acid phosphatases: a preliminary study.

The development of secure diagnostic immunoassays requires, among others, rigorous characterisation of potential antibody reagents. The reactivity profiles of seven antibodies (six monoclonal [MAb] and one polyclonal [PAb]) with putative specificity for tartrate-resistant acid phosphatase (TRAP) and/or osteoclasts were evaluated in enzyme-linked immunosorbent assay (ELISA) and/or immunocytochemistry. MAbs 2H1, 4E6 and 5Cl demonstrated assay restriction: exhibiting reactivity only in ELISA. The remaining three MAbs (G211D, G312G and V35B) and the PAb 8023 recognised recombinant TRAP (rTRAP) in ELISA and native acid phosphatases in selected tissues and cell lines. The latter were cytochemically assessed for both tartrate-sensitive acid phosphatase (TSAP) and TRAP. V35B showed reactivity against the monocytic leukaemia cell line U937 and guinea pig kidney tissue (both TSAP+ and TRAP+) and ECV304 (TSAP+) cells. Interestingly, the reactivity of MAb G211D co-localised with TRAP activity in the membrane of osteoclasts but also detected cytoplasmic components in U937 cells and human embryonic lung fibroblasts (TRAP+ and TRAP+). G211D exhibited immunoreactivity against placental trophoblasts (positive for total AP). Intriguingly, MAbs 2H1, 4E6, 5Cl and PAb 8023 cross-reacted with potato acid phosphatase in ELISA, suggesting reactivity to conformationally similar epitopes. Thus, some of these reagents could be used in the development of standardised diagnostic immunoassays or as drug-targeting agents for conditions in which the pathological process involves bone resorption, the MAbs G211D, 2H1, 4E6, 5Cl and PAb 8023 being useful in ELISA but not immunocytochemical detection of TRAP.

A comparative study of the ability of calcitonin gene-related peptide and adrenomedullin(13 - 52) to modulate microvascular but not thermal hyperalgesia responses.

Calcitonin gene-related peptide (CGRP), a neuropeptide, is a potent vasodilator. Adrenomedullin (ADM) is suggested to be produced by vascular cells in inflamed tissue. ADM shares some structural homology with CGRP. We have compared the ability of CGRP and ADM to modulate microvascular and thermal hyperalgesic responses in rat skin. Vasodilator activity was assessed by laser Doppler flowmetry, inflammatory oedema by the extravascular accumulation of intravenously-injected labelled albumin, and neutrophil accumulation by tissue myeloperoxidase, in dorsal skin. Hyperalgesia was assessed by a thermal hyperalgesimeter in paw skin. ADM (10-300 pmol) was 3 fold less potent than CGRP (3-100 pmol) as a direct vasodilator. CGRP (30 pmol) potentiated oedema formation induced by mediators of increased microvascular permeability, as expected (P<0.01). However, ADM (30-100 pmol) was without a potentiating effect, although ADM (300 pmol) was effective (P<0.01). By comparison ADM (100 pmol) potentiated neutrophil accumulation induced by interleukin-1beta (P<0.05), whereas CGRP (30 pmol) did not. No thermal hyperalgesia was observed to either CGRP or ADM, when given as single or repeated treatments. Thus despite a dilator activity neither CGRP nor ADM appears to mediate hyperalgesic activity in the periphery. However ADM, like CGRP, has the ability to potentiate inflammatory oedema formation and, in addition, ADM can potentiate neutrophil accumulation. ADM may, as suggested for CGRP, act as a modulator of the vascular phases of inflammation. The property of the two compounds of evoking differential microvascular responses and neutrophil accumulation may be due to differing mechanisms of action.

Ebstein's anomaly appearing in the neonate. A new surgical approach.

Ebstein's anomaly appearing during the neonatal period carries a high mortality rate. These infants exhibit cyanosis, acidosis, and congestive heart failure. The pathophysiologic characteristics consist of severe tricuspid regurgitation and functional pulmonary atresia. As a result of the inability of the right ventricle to generate forward flow through the pulmonary arteries, these infants remain dependent on ductal patency. Since May 1988, five newborn infants with severe Ebstein's anomaly have been admitted for treatment at our institution. At initial examination, they weighed 3.6 +/- 1.8 kg and had a mean oxygen tension of 29.6 +/- 2.3 mm Hg and a mean pH of 7.20 +/- 0.05. Chest roentgenography demonstrated a mean cardiothoracic ratio of 0.81 +/- 0.02. As determined by echocardiography, the right atria were massively enlarged, severe tricuspid regurgitation was present in all patients, and the pulmonary valves were not opening. All infants were dependent on prostaglandin E1 and attempts to wean them from this drug were unsuccessful. Palliative treatment consisted of tricuspid closure with autologous pericardium and an aortopulmonary shunt of 4 mm polytetrafluoroethylene tubing. There were no operative or late deaths. At discharge, mean oxygen tension was 42.2 +/- 0.85 mm Hg and mean systemic oxygen saturation was 83.2% +/- 1.94%. Infants have grown satisfactorily during the follow-up period. Three infants have since returned for further surgical intervention. One infant, at 11 months of age, underwent a Glenn anastomosis for progressive oxygen desaturation. Two infants have returned, at ages 23 and 22 months, for Fontan procedures, which represent their definitive operative management. We believe this new procedure offers excellent palliative treatment for Ebstein's anomaly in critically ill neonates. Feasibility of later definitive correction is demonstrated by the good results obtained with the Fontan procedure in two infants.

T cell receptor V beta expression by mucosal T cells.

Immunohistochemistry with monoclonal antibodies to the T cell receptor V beta regions 5, 6, 8 and 12 was used to determine whether normal intestinal lymphocytes that are potentially exposed to many bacterially derived superantigens show any preferential expression of particular V beta regions compared with the blood. No difference between V beta expression in the mucosa and the blood was observed, suggesting that they share a common pool of alpha beta T cells and that there is no expansion of alpha beta T cells in response to bacterial "superantigens" in the gut. The T cell receptor V beta expressed by the activated T cells in the lamina propria of bowel from patients with Crohn's disease was also studied. There was no increase in V beta 8 expression in these cells, suggesting that the increase in V beta 8 observed in the blood and mesenteric nodes of patients with Crohn's disease is not of primary importance in the aetiology of the disease. Finally, V beta expression by mucosal T cells in coeliac disease was studied. There was no difference in V beta use by T cells in coeliac disease and those in the blood and normal jejunum.

Increased expression of laminin/collagen receptor (VLA-1) on epithelium of inflamed human intestine.

Immunohistochemical techniques were used to investigate the epithelial expression of VLA-1 in inflammatory bowel disease in six patients with Crohn's disease, in four patients with ulcerative colitis, and in one patient with indeterminate colitis, and compared with that in the small intestine and colons of 10 normal controls. In normal small bowel VLA-1 was expressed on crypt epithelial cells and only weakly or not at all on surface epithelium. VLA-1 was again expressed weakly in normal colon, except in one case, a 1 year old child with diarrhoea but no histological abnormalities. In small and large intestine affected with Crohn's disease, ulcerative colitis, or indeterminate colitis, there was increased expression of VLA-1 on the basolateral aspects of crypt cells and de novo expression on surface epithelium. It is suggested that this is an adaptive response to prevent epithelial cell loss as a result of inflammation in the underlying lamina propria.

Histopathology and immunohistochemistry of the caecum in children with the Trichuris dysentery syndrome.

Caecal biopsy specimens from Jamaican children with the Trichuris dysentery syndrome (TDS) and age matched Jamaican controls were investigated by immunohistochemistry and by light microscopy. Biopsy specimens from all children (with TDS and controls) showed a mild to moderate increase in inflammatory cells. Except in the vicinity of the worm, where the epithelium was flattened, there was no other epithelial abnormality. Compared with controls, children with TDS had increased IgM lamina propria plasma cells and decreased intraepithelial T cells. There was also an increase in crypt epithelial cell proliferation. Lamina propria T cells (both activated and non-activated) were no more common in children with the Trichuris syndrome than controls. Epithelial cell HLA-DR and VLA-1 expression (which are increased in other colitides) were the same in both groups. Despite the presence of large worm burdens and chronic dysentery, therefore, only minor changes were seen in the caecal mucosa of children with TDS.

Liposomal mediated transfection of mature rat astrocytes in vitro: a comparison of ten reagents.

Ten cationic liposomes were tested for their ability to transfect mature, astrocyte monolayers in vitro using the eucaryotic expression vector plasmid p cytomegalovirus (CMV)-beta. Liposomal agents were examined for optimum length of exposure and optimum cDNA/lipid ratios. Lipofectin demonstrated the highest transfection efficiencies of all agents tested (3.3%). When examined at 3 days following transfection, 24-h exposures yielded higher efficiencies compared to 6 h exposures (1.9%, P=0.07). Although expression appeared to decline by up to 80%, positive cells were still detected up to 2 weeks after transfection with all reagents. Lipofectin represents a useful tool for transfecting mature astrocytes for investigation of gene transfer in vitro.