EV71:TLLcho virus murine model of enterovirus A71 neurological disease does not exhibit neurogenic pulmonary oedema.

Small animal models play an important role in investigating and revealing the molecular determinants and mechanisms underlying neuro-virulence of enterovirus A71 (EV-A71). In our previous study, we successfully developed two mouse cell-line replication competent EV-A71 strains (EV71:TLLm and EV71:TLLmv) which were capable of inducing neuro-invasion in BALB/c mice. The more virulent EV71:TLLmv exhibited ability to induce acute encephalomyelitis accompanied by neurogenic pulmonary oedema. EV71:TLLcho virus strain was generated from EV71:TLLm by a series of passages in CHO-K1 cells. EV71:TLLcho demonstrated a broader range of infectivity across various mammalian cell lines and exhibited complete cytopathic effects (CPE) within 48 hours post-inoculation in comparison to EV71:TLLm or EV71:TLLmv. EV71:TLLcho consistently yielded higher levels of viral replication at all time points examined. In comparison to EV71:TLLm, EV71:TLLcho consistently induced more severe disease and increased mortality in one-week old BALB/c mice. However, unlike mice challenged with EV71:TLLmv, none of the mice challenged with EV71:TLLcho progressed to severe acute encephalomyelitis and developed neurogenic pulmonary oedema.

Patterns and risk factors for locoregional failures after mastectomy for breast cancer: an International Breast Cancer Study Group report.

BACKGROUND: Rates and risk factors of local, axillary and supraclavicular recurrences can guide patient selection and target for postmastectomy radiotherapy (PMRT). PATIENTS AND METHODS: Local, axillary and supraclavicular recurrences were evaluated in 8106 patients enrolled in 13 randomized trials. Patients received chemotherapy and/or endocrine therapy and mastectomy without radiotherapy. Median follow-up was 15.2 years. RESULTS: Ten-year cumulative incidence for chest wall recurrence of >15% was seen in patients aged <40 years (16.1%), with ≥4 positive nodes (16.5%) or 0-7 uninvolved nodes (15.1%); for supraclavicular failures >10%: ≥4 positive nodes (10.2%); for axillary failures of >5%: aged <40 years (5.1%), unknown primary tumor size (5.2%), 0-7 uninvolved nodes (5.2%). In patients with 1-3 positive nodes, 10-year cumulative incidence for chest wall recurrence of >15% were age <40, peritumoral vessel invasion or 0-7 uninvolved nodes. Age, number of positive nodes and number of uninvolved nodes were significant parameters for each locoregional relapse site. CONCLUSION: PMRT to the chest wall and supraclavicular fossa is supported in patients with ≥4 positive nodes. With 1-3 positive nodes, chest wall PMRT may be considered in patients aged <40 years, with 0-7 uninvolved nodes or with vascular invasion. The findings do not support PMRT to the dissected axilla.

TROG 14.04: Multicentre Study of Feasibility and Impact on Anxiety of DIBH in Breast Cancer Patients.

AIMS: The aim of TROG 14.04 was to assess the feasibility of deep inspiration breath hold (DIBH) and its impact on radiation dose to the heart in patients with left-sided breast cancer undergoing radiotherapy. Secondary end points pertained to patient anxiety and cost of delivering a DIBH programme. MATERIALS AND METHODS: The study comprised two groups - left-sided breast cancer patients engaging DIBH and right-sided breast cancer patients using free breathing through radiotherapy. The primary end point was the feasibility of DIBH, defined as left-sided breast cancer patients' ability to breath hold for 15 s, decrease in heart dose in DIBH compared with the free breathing treatment plan and reproducibility of radiotherapy delivery using mid-lung distance (MLD) assessed on electronic portal imaging as the surrogate. The time required for treatment delivery, patient-reported outcomes and resource requirement were compared between the groups. RESULTS: Between February and November 2018, 32 left-sided and 30 right-sided breast cancer patients from six radiotherapy centres were enrolled. Two left-sided breast cancer patients did not undergo DIBH (one treated in free breathing as per investigator choice, one withdrawn). The mean heart dose was reduced from 2.8 Gy (free breathing) to 1.5 Gy (DIBH). Set-up reproducibility in the first week of treatment assessed by MLD was 1.88 ± 1.04 mm (average ± 1 standard deviation) for DIBH and 1.59 ± 0.93 mm for free breathing patients. Using a reproducibility cut-off for MLD of 2 mm (1 standard deviation) as per study protocol, DIBH was feasible for 67% of DIBH patients. Radiotherapy delivery using DIBH took about 2 min longer than for free breathing. Anxiety was not significantly different in DIBH patients and decreased over the course of treatment in both groups. CONCLUSION: Although DIBH was shown to require about 2 min longer per treatment slot, it has the potential to reduce heart dose in left-sided breast cancer patients by nearly a half, provided careful assessment of breath hold reproducibility is carried out.

Patterns of rapd markers and heavy metal concentrations in Perna viridis (L.), collected from metal-contaminated and uncontaminated coastal waters: are they correlated with each other?

Genetic variation due to heavy metal contamination has always been an interesting topic of study. Because of the numerous contaminants being found in coastal and intertidal waters, there is always much discussion and argument as to which contaminant(s) caused the variations in the genetic structures of biomonitors. This study used a Single Primer Amplification Reaction (SPAR) technique namely Random Amplified Polymorphic DNA (RAPD) to determine the genetic diversity of the populations of the green-lipped mussel Perna viridis collected from a metal-contaminated site at Kg. Pasir Puteh and those from four relatively' uncontaminated sites (reference sites). Heavy metal levels (Cd, Cu, Pb and Zn) were also measured in the soft tissues and byssus of the mussels from all the sites. Cluster analyses employing UPGMA done based on the RAPD makers grouped the populations into two major clusters; the Bagan Tiang, Pantai Lido, Pontian and Kg. Pasir Puteh populations were in one cluster, while the Sg. Belungkor population clustered by itself. This indicated that the genetic diversity based on bands resulting from the use of all four RAPD primers on P. viridis did not indicate its potential use as a biomarker of heavy metal pollution in coastal waters. However, based on a correlation analysis between a particular metal and a band resulting from a specific RAPD primer revealed some significant (P < 0.01) correlations between the primers and the heavy metal concentrations in the byssus and soft tissues. Thus, the correlation between a particular metal and the bands resulting from the use of a specific RAPD primer on P. viridis could be used as biomonitoring tool of heavy metal pollution.

Genetic diversity of enterovirus 71 isolated from cases of hand, foot and mouth disease in the 1997, 2000 and 2005 outbreaks, Peninsular Malaysia.

All known field isolates of enterovirus 71 (EV71) can be divided into three distinct genogroups (A, B, C) and 10 subgenogroups (A, B1-5, C1-4) based on VP1 gene sequences. We examined VP1 gene sequences of 10, 12 and 11 EV71 strains isolated in peninsular Malaysia during the outbreaks of hand, foot and mouth disease in 1997, 2000 and 2005 respectively. Four EV71 strains isolated in the hand, foot and mouth disease outbreak of 2006 in Sarawak (Malaysian Borneo) were included to describe their genetic relationship. Four subgenogroups (C1, C2, B3 and B4) of EV71 co-circulated and caused the outbreak of hand, foot and mouth disease in peninsular Malaysia in 1997. Two subgenogroups (C1 and B4) were noted to cause the outbreak in 2000. In the 2005 outbreak, besides EV71 strains of subgenogroup C1, EV71 strains belonged to subgenogroup B5 were isolated but formed a cluster which was distinct from EV71 strains of the subgenogroup B5 isolated in 2003. The four EV71 strains isolated from clinical specimens of patients with hand, foot and mouth disease in the Sarawak outbreak in early 2006 also belonged to subgenogroup B5. Phylogenetic analysis of the VP1 gene sequences showed that the four Sarawak EV71 isolates belonged to the same cluster as the EV71 strains that were isolated in peninsular Malaysia as early as May 2005. The data suggested that the EV71 strains causing the outbreak in Sarawak could have originated from peninsular Malaysia.

Upregulation of vascular endothelial growth factor by angiotensin II in rat heart endothelial cells.

Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells and a vascular permeability factor. In this study we found that the addition of angiotensin II (AII) to rat heart endothelial cells induced VEGF mRNA production. VEGF mRNA levels reached a plateau within 2 h after the addition of AII and decreased after 4 h. The induction was superinduced by cycloheximide and blocked by actinomycin D. Losartan, an AT1 receptor antagonist, abolished the induction of VEGF mRNA by AII, whereas PD 123319, an AT2 receptor antagonist, had no effect on VEGF mRNA induction. H7, a protein kinase C inhibitor, blocked the induction. RT-PCR experiments showed two mRNA species (VEGF 120 and VEGF 164) in these cells and both species were stimulated by AII. Transient transfection experiment showed that VEGF promoter activity was increased 2.2-fold upon AII stimulation. Electrophoretic mobility shift assay revealed an enhanced binding of transcription factors AP-1 and NF-kappa B. Immunoblot analysis showed that the amount of secreted VEGF was elevated in the medium 8 h after AII stimulation. Our results demonstrate for the first time that the upregulation of VEGF by AII may play a significant role in AII-induced hyperpermeability.

Regulation of endothelin-1 mRNA by angiotensin II in rat heart endothelial cells.

In a series of experiments carried out in cultured endothelial cells derived from rat hearts (RHE), angiotensin II (AII) is shown to stimulate preproendothelin-1 mRNA in a dose- and time-dependent manner. The induction of preproendothelin-1 mRNA is rapid, reaching a maximal level 1 h after the addition of AII (1 x 10(-8) M). The mRNA levels decline rapidly to basal levels in 4 h. The addition of Losartan (Dup 753; 1 x 10(-6) M), an AII receptor (type I) antagonist, blocks the AII effect. Calphostin C, a potent protein kinase C inhibitor, is able to abolish this effect of AII suggesting that the induction of preproendothelin-1 mRNA is mediated by a protein kinase C-dependent pathway. Since endothelial cells line the inner surface of the myocardium and blood vessels and sense the rise of AII associated with renovascular hypertension at the endothelial surface, these data suggest that endothelin which is produced by RHE cells in response to AII could be an important mediator which may play a role in modulating gene expression in AII-mediated cardiac hypertrophy.

Angiotensin II induces TGF-beta 1 production in rat heart endothelial cells.

Angiotensin II (AII) was found to stimulate TGF-beta 1 gene expression in rat heart endothelial cells in a dose- and time-dependent manner. The maximal induction of TGF-beta 1 mRNA was achieved by 6 h after the addition of AII. This induction was blocked by losartan, an AT1 receptor antagonist and by calphostin C, a protein kinase C inhibitor. Addition of actinomycin D and cycloheximide abolished the induction. TGF-beta 1 promoter activities were stimulated 5-fold by AII. TGF-beta 1 secreted by the rat heart endothelial cells in response to AII was in a latent form and could be activated by mild heat treatment. These results suggest that AII stimulates TGF-beta 1 production by a protein kinase C-dependent pathway which is dependent upon de novo RNA synthesis and protein synthesis. Since endothelial cells line the blood vessels and sense the rise in AII associated with hypertension, the release of TGF-beta 1 by these cells may provide the initial trigger leading to cardiac fibrosis in angiotensin-renin-dependent hypertension.

Regulation of thrombospondin-1 production by angiotensin II in rat heart endothelial cells.

Thrombospondin-1 (TSP-1) is synthesized, secreted, and incorporated into the extracellular matrix by a variety of cells, including the endothelial cells. Addition of angiotensin II (AII) significantly induced TSP-1 mRNA in rat heart-derived endothelial cells. TSP-1 mRNA levels reached a plateau within 2 h after the addition of AII and decreased after 5 h. The induction was superinduced by cycloheximide and blocked by actinomycin D. Losartan, an AT1 receptor antagonist, could abolish the induction of TSP-1 mRNA by AII. Phorbol 12-myristate 13-acetate (TPA) was found to enhance TSP-1 mRNA level whereas a protein kinase C inhibitor, H7, was shown to block the induction. Immunoblot analysis revealed that TSP-1 was detectable in the medium 4 h after AII stimulation. Our results suggest that the upregulation of TSP-1 by AII represents an important mechanism leading to perivascular fibrosis in the heart.

Mechanism of transforming growth factor-beta1-induced expression of vascular endothelial growth factor in murine osteoblastic MC3T3-E1 cells.

Transforming growth factor-beta1 (TGF-beta1), an abundant growth factor in bone matrix, has been shown to be involved in bone formation and fracture healing. The mechanism of action of the osteogenic effect of TGF-beta1 is not clearly understood. In this study, we found that the addition of TGF-beta1 to murine osteoblastic MC3T3-E1 cells induced vascular endothelial growth factor (VEGF) mRNA production. VEGF mRNA levels reached a plateau within 2 h after the addition of TGF-beta1. The induction was superinduced by cycloheximide and blocked by actinomycin D. Ro 31-8220, a protein kinase C inhibitor, abrogated the induction. In addition, curcumin, an inhibitor for transcription factor AP-1, also blocked the induction. Electrophoretic mobility shift assay revealed an enhanced binding of transcription factors AP-1 and NF-kappaB. Transient transfection experiment showed that VEGF promoter activity increased 3.6-fold upon TGF-beta1 stimulation. Immunoblot analysis showed that the amount of secreted VEGF was elevated in the medium 4 h after TGF-beta1 stimulation. Our results therefore suggest that at least part of the osteogenic activity of TGF-beta1 may be attributed to the production of VEGF.

Upregulation of endothelin-1 production by lysophosphatidic acid in rat aortic endothelial cells.

Addition of lysophosphatidic acid (LPA) to rat aorta-derived endothelial cells significantly induced preproendothelin-1 (preproET-1) mRNA expression. PreproET-1 mRNA levels reached a plateau within 1 h after the addition of 0.5 microM LPA and declined after 2 h. The induction was superinduced by cycloheximide and was blocked by actinomycin D. Suramin, an LPA receptor antagonist, abolished the induction of preproET-1 mRNA by LPA. Protein kinase C inhibitors, H7 and bisindolylmaleimide, were able to block the induction. Transient transfection experiment revealed that the elevated preproET-1 mRNA was a result of the activation of ET-1 gene activity. Electrophoretic mobility shift assay revealed that LPA stimulated the binding of AP-1. The secreted level of ET-1 was elevated 2.3-fold after 12 h of stimulation with LPA. Our results suggest that the upregulation of preproET-1 by LPA may serve to augment and prolong the vasoconstriction action of LPA.

Regulation of endothelin-1 production by a thromboxane A2 mimetic in rat heart smooth muscle cells.

Thromboxane A2 (TXA2) and ET-1 have been known to play important roles in modulating vascular contraction and growth. The present study was undertaken to examine the effect of TXA2 on the induction of endothelin-1 (ET-1) mRNA and protein levels in smooth muscle cells derived from rat heart. U-46619, a stable TXA2 mimetic, superinduced preproET-1 mRNA in the presence of cycloheximide in these cells. This effect could be blocked by SQ-29548, a TXA2/prostaglandin H2 receptor antagonist and by actinomycin D, and RNA synthesis inhibitor. In addition, H7, a protein kinase C inhibitor, could abolish the induction. Transient transfection experiment revealed that the elevated ET-1 mRNA level after U-46619 treatment was a result of the activation of ET-1 gene activity. The elevated ET-1 message level was accompanied by increased ET-1 release into the cultured medium. These results show that the short-lived TXA2 can induce potent and long-lived ET-1. These findings support a potential role for ET-1 in the pathogenesis of coronary atherosclerosis and hypertension evoked by TXA2.

Angiotensin II induces TIMP-1 production in rat heart endothelial cells.

Angiotensin II (AII) was found to upregulate tissue inhibitor of metalloproteineses-1 (TIMP-1) gene expression in rat heart endothelial cells in a dose and time-dependent manner. The maximal stimulation of TIMP-1 mRNA was achieved by 2 h after the addition of AII. This effect was blocked by losartan, an AT1 receptor antagonist and by calphostin C, a protein kinase C inhibitor. Addition of cycloheximide superinduced and actinomycin D abolished the induction. These results suggest that AII stimulates TIMP-1 production by a protein kinase C dependent pathway which is dependent upon de novo RNA synthesis. Immunoprecipitation experiment showed an enhanced band of 28 kDa from the conditioned medium of AII-treated cultures. Immunoblot analysis revealed that TIMP-1 was detectable in the conditioned medium 4 h after AII stimulation. Since endothelial cells line the blood vessels and sense the rise in AII associated with hypertension, the TIMP-1 released by these cells may provide an initial trigger leading to cardiac fibrosis in angiotensin-renin dependent hypertension.

Upregulation of vascular endothelial growth factor by H2O2 in rat heart endothelial cells.

Hydrogen peroxide (H2O2) is a reactive oxygen species generated by several metabolic pathways in mammalian cells. Endothelial cells are extremely susceptible to oxidative stress. H2O2 has been reported to increase the permeability in these cells. Using rat heart endothelial cell culture as a model system, we examined the effect of H2O2 on the gene expression of vascular endothelial growth factor (VEGF), a potent mitogen of endothelial cells and a vascular permeability factor. By Northern blot analysis we found that VEGF mRNA responded to H2O2 in a dose-and time-dependent manner. The induction was superinduced by cycloheximide and blocked by actinomycin D. N-Acetylcysteine, a synthetic antioxidant, was able to suppress the induction. H7, a protein kinase C inhibitor, could also block the induction. Electrophoretic mobility shift assay revealed an enhanced binding of transcription factors, AP-1 and NF-kappaB. Immunoblot analysis showed that the amount of secreted VEGF was elevated in the medium 4 h after H2O2 stimulation. Our results demonstrate that VEGF gene expression is upregulated by H2O2 in these endothelial cells.

Overexpression of CuZnSOD in coronary vascular cells attenuates myocardial ischemia/reperfusion injury.

Superoxide dismutase scavenges oxygen radicals, which have been implicated in ischemia/reperfusion (I/R) injury in the heart. Our experiments were designed to study the effect of a moderate increase of copper/zinc superoxide dismutase (CuZnSOD) on myocardial I/R injury in TgN(SOD1)3Cje transgenic mice. A species of 0.8 kb human CuZnSOD mRNA was expressed, and a 273% increase in CuZnSOD activity was detected in the hearts of transgenic mice with no changes in the activities of other antioxidant enzymes. Furthermore, immunoblot analysis revealed no changes in the levels of HSP-70 or HSP-25 levels. Immunocytochemical study indicated that there was increased labeling of CuZnSOD in the cytosolic fractions of both endothelial cells and smooth muscle cells, but not in the myocytes of the hearts from transgenic mice. When these hearts were perfused as Langendorff preparations for 45 min after 35 min of global ischemia, the functional recovery of the hearts, expressed as heart rate x LVDP, was 48 +/- 3% in the transgenic hearts as compared to 30 +/- 5% in the nontransgenic hearts (p <.05). The improved cardiac function was accompanied by a significant reduction in lactate dehydrogenase release from the transgenic hearts. Our results demonstrate that overexpression of CuZnSOD in coronary vascular cells renders the heart more resistant to I/R injury.

Retrospective review of serologic rubella activity in University Hospital Kuala Lumpur.

A retrospective review of rubella serological results carried out in the Virus Diagnostic Unit, University Hospital Kuala Lumpur (UHKL) from January 1993 to September 1999 showed the presence of rubella infection annually which appeared to increase periodically every two to three years. There was no statistical significant difference in the rubella positive rate between male and female population aged 14 to 48 years. Congenital rubella infections (CRI) occurred in babies delivered in UHKL yearly from 1993 to 1998 with an average incidence rate of 48 per 100,000 deliveries. Peaks of rubella cases appeared to be followed by an increase in incidence of CRI cases 6 to 9 months later. The study showed that only 50.8% clinically diagnosed rubella was confirmed by laboratory finding. This study also showed an increase of rubella activity for the months of July, August and September 1999 and this may herald an increase of CRI cases in the coming millennium.

A retrospective review of mucocutaneous infections by human herpesvirus 1 and 2 in an urban population in Malaysia.

This is a 10-year retrospective review of mucocutaneous infection by human herpesvirus 1 (HHV1) and human herpesvirus 2 (HHV2) carried out by the virus diagnostic unit of University Malaya Medical Centre (UMMC). A total of 504 specimens from UMMC and a private clinic in the same city (KLSC) were tested; 198 samples from patients with oral lesions and 306 from patients with genital lesions. HHV1 was found to be responsible for 98.4% of oral lesions whereas HHV2 was the cause of 83.6% of all genital lesions. Detailed analysis showed no statistical difference by age group, race or gender among the patients with oral and genital lesions. Two laboratory methods were used in this study. Of the total 504 specimens tested, 18.0% specimens were positive by direct immunofluorescence (IF), 55.0% by virus isolation and 56.5% when both methods were used in combination. Although IF can provide a more rapid diagnosis, it is, however, less sensitive and can be attributed partly to inadequate collection of specimens.

Hand, foot and mouth disease: University Malaya Medical Centre experience.

The prevalence of HFMD as well as the causative agents was unknown in peninsular Malaysia prior to May 1997. From May 1997 to June 2001, 585 patients suspected to have enterovirus infections, with 467 patients clinically diagnosed as having HFMD, were investigated in the diagnostic virology unit of the University Malaya Medical Centre. Data from this study showed that HFMD is endemic in Malaysia with the occurrence of two outbreaks during the study period. In each outbreak, a number of viruses were isolated but enterovirus 71 was the main virus isolated in both outbreaks. Echovirus 7 (Eo7) was isolated from 5 patients with HFMD in the second outbreak, a clinical entity that has not been attributed to it previously. Children aged 4 years and below, particularly those between 1 and 2 years of age, were in the main group of patients affected by the illness. HFMD by itself and without neurological involvement was relatively benign and self-limiting. There was no significant difference in the virus isolation rate with respect to gender and ethnic groups. Virus isolation was attempted in a total of 764 clinical specimens consisting of 342 stool specimens, 285 oral secretions specimens and 137 vesicular fluid specimens. Oral specimens gave the highest virus isolation rate (33.3%) followed by vesicular specimens (27.0%). Stool specimens only yielded an isolation rate of 14.0%.

Seroprevalence of human parvovirus B19 infection in an urban population in Malaysia.

A seroepidemiological study carried out on 800 stored serum samples collected between January 1999 to December 2000 derived from an urban population in Malaysia showed that the overall seropositive rate of human paravovirus B19 infection was 37.6%, with an overall geometric mean titre (GMT) of 18.3 IU. The seropositive rates of B19 among the male and female populations were 39.0% and 36.3% respectively. The seropositive rates among the racial groups were 37.2%, 38.2%, 38.1% and 29.4% respectively for the Malays, Chinese, Indians and other races. There was no statistical significant gender and racial differences in the B19 seropositive rates. When compared with the seroprevalence of B19 infection in other Asian countries, the seropositive rate of B19 in Malaysia was low in the younger age group and increased steadily with age. The unusual finding in this study was the presence of a high seropositive rate in those between six months to five years of age, especially in children in the one year old age group.

A review of the management of ductal carcinoma in situ following breast conserving surgery.

Ductal carcinoma in situ (DCIS) is a heterogeneous, pre-malignant disease accounting for 10-20% of all new breast tumours. Evidence shows a statistically significant local control benefit for adjuvant radiotherapy (RT) following breast conserving surgery (BCS) for all patients. The baseline recurrence risk of individual patients varies according to clinical-pathological criteria and in selected patients, omission of RT may be considered, following a discussion with the patient. The role of adjuvant endocrine therapy remains uncertain. Ongoing studies are attempting to define subgroups of patients who are at sufficiently low risk of recurrence that RT may be safely omitted; investigating RT techniques and dose fractionation schedules; and defining the role of endocrine therapy. Future directions in the management of patients with DCIS will include investigation of prognostic and predictive biomarkers to inform individualised therapy tailored to the risk of recurrence.