Evaluation of antiviral activities of Houttuynia cordata Thunb. extract, quercetin, quercetrin and cinanserin on murine coronavirus and dengue virus infection.

OBJECTIVE: To evaluate the in vitro activities of the ethyl acetate (EA) fraction of Houttuynia cordata (H. cordata) Thunb. (Saururaceae) and three of its constituent flavonoids (quercetin, quercitrin and rutin) against murine coronavirus and dengue virus (DENV). METHODS: The antiviral activities of various concentrations of the EA fraction of H. cordata and flavonoids were assessed using virus neutralization tests against mouse hepatitis virus (MHV) and DENV type 2 (DENV-2). Cinanserin hydrochloride was also tested against MHV. The EA fraction of H. cordata was tested for acute oral toxicity in C57BL/6 mice. RESULTS: The EA fraction of H. cordata inhibited viral infectivity up to 6 d. Cinanserin hydrochloride was able to inhibit MHV for only 2 d. The 50% inhibitory concentrations (IC50) of the EA fraction of H. cordata added before the viral adsorption stage were 0.98 µg/mL for MHV and 7.50 µg/mL for DENV-2 with absence of cytotoxicity. The mice fed with the EA fraction up to 2000 mg/kg did not induce any signs of acute toxicity, with normal histological features of major organs. Certain flavonoids exhibited comparatively weaker antiviral activity, notably quercetin which could inhibit both MHV and DENV-2. This was followed by quercitrin which could inhibit DENV-2 but not MHV, whereas rutin did not exert any inhibitory effect on either virus. When quercetin was combined with quercitrin, enhancement of anti-DENV-2 activity and reduced cytotoxicity were observed. However, the synergistic efficacy of the flavonoid combination was still less than that of the EA fraction. CONCLUSIONS: The compounds in H. cordata contribute to the superior antiviral efficacy of the EA fraction which lacked cytotoxicity in vitro and acute toxicity in vivo. H. cordata has much potential for the development of antiviral agents against coronavirus and dengue infections.

Combination therapy with hepatocyte growth factor and oseltamivir confers enhanced protection against influenza viral pneumonia.

Frequent outbreaks caused by influenza viruses pose considerable public health threats worldwide. Virus-inflicted alveolar damage represents a major contributor of acute lung injury in influenza. We have previously demonstrated that hepatocyte growth factor (HGF) produced by macrophages enhances alveolar epithelial proliferation during influenza infection. Here, we investigated the therapeutic efficacy of recombinant human HGF (rhHGF) and an antiviral agent (oseltamivir) alone or in combination to treat influenza viral pneumonia in macrophage-depleted BALB/c mice. Combination therapy of infected mice significantly reduced lung pathology and mortality compared to other animal groups that received either treatment alone. Combination treatment with rhHGF induced alveolar type II (AT2) epithelial hyperplasia more prominently in the distal airways, evident by increased cells with double-positive staining for surfactant protein-C and proliferating cell nuclear antigen within the alveolar epithelial lining. Similarly, rhHGF supplementation also induced stem cell antigen-1 (SCA-1) transcriptional expression at 5 days post-infection (dpi), but mRNA levels of both SCA-1 and its receptor c-KIT were decreased by 10 dpi. Microarray and pathway analyses indicated that rhHGF administration may act by accelerating tissue repair and suppressing inflammatory processes to minimize damage by infection and to restore lung function by earlier repair. These results reveal that transient administration of rhHGF may confer synergistic effects in enhancing pulmonary repair by promoting AT2 cell proliferation. Thus, the combination of rhHGF and oseltamivir may represent a promising therapeutic option against influenza pneumonia to improve existing antiviral treatment regimens.

Neutrophils infected with highly virulent influenza H3N2 virus exhibit augmented early cell death and rapid induction of type I interferon signaling pathways.

We developed a model of influenza virus infection of neutrophils by inducing differentiation of the MPRO promyelocytic cell line. After 5 days of differentiation, about 20-30% of mature neutrophils could be detected. Only a fraction of neutrophils were infected by highly virulent influenza (HVI) virus, but were unable to support active viral replication compared with MDCK cells. HVI infection of neutrophils augmented early and late apoptosis as indicated by annexin V and TUNEL assays. Comparison between the global transcriptomic responses of neutrophils to HVI and low virulent influenza (LVI) revealed that the IFN regulatory factor and IFN signaling pathways were the most significantly overrepresented pathways, with activation of related genes in HVI as early as 3 h. Relatively consistent results were obtained by real-time RT-PCR of selected genes associated with the type I IFN pathway. Early after HVI infection, comparatively enhanced expression of apoptosis-related genes was also elicited.

Comparison of neutralizing antibody and cell-mediated immune responses to pandemic H1N1 2009 influenza virus before and after H1N1 2009 influenza vaccination of elderly subjects and healthcare workers.

BACKGROUND: The recent H1N1 pandemic virus that emerged in 2009 resulted in high morbidity rates mainly in younger individuals, albeit with relatively low mortality. We investigated both humoral and cellular immune responses against the pandemic H1N1 2009 virus before and after immunization with inactivated H1N1 2009 vaccine. METHODS: We obtained paired blood specimens from a cohort of participants from nursing homes (n=108) and a public hospital (n=60) in Singapore. Serum samples were tested for neutralizing antibodies against H1N1 2009 using microneutralization assays, while peripheral blood mononuclear cells were subjected to interferon-γ enzyme-linked immunosorbent spot (ELISPOT) assays for whole virus-specific T-cell responses. RESULTS: We observed significant increases in geometric mean titers of neutralizing antibodies after H1N1 2009 vaccination (from 23.6 pre-vaccination to 94.7 post-vaccination). Approximately 77% and 54% of the cohort exhibited ≥2-fold and ≥4-fold increases in neutralizing antibody titers following vaccination; 89.9% of the cohort had a post-vaccination antibody titer of ≥32. Adjusted for gender, participants aged ≥60 years were less likely to have a ≥4-fold increase in antibody titers after vaccination than those aged <60 years (0.48; 95% confidence interval (95% CI) 0.32-0.71, p=0.007). There was a 1.4-fold elevation in H1N1 2009-specific T-cell responses after vaccination (p<0.05). Adjusted for gender, age ≥60 years was positively associated with a greater increase in T-cell response (ß=4.9, 95% CI 1.58-8.29, p=0.018). No significant correlation was observed between humoral and cellular immune responses. CONCLUSIONS: Influenza vaccination elicits significant neutralizing antibody and T-cell responses to pandemic H1N1 2009 influenza virus. However, in response to vaccination, increases in neutralizing antibody titers were comparatively lower but T-cell responses were higher in older participants. Therefore, our study suggests that memory T-cells may play a crucial role in protecting older individuals against pandemic H1N1 2009 infection.

Comparative seroepidemiology of pertussis, diphtheria and poliovirus antibodies in Singapore: waning pertussis immunity in a highly immunized population and the need for adolescent booster doses.

BACKGROUND: We assessed the seroepidemiology of pertussis, diphtheria and poliovirus antibodies in a cohort of highly immunized children, together with the burden of these diseases in Singapore. METHODS: Hospital residual sera collected between August 2008 and July 2010 from 1200 children aged 1-17 years were tested for the prevalence of IgG antibodies against Bordetella pertussis, diphtheria toxoid, and all three poliovirus types by enzyme-linked immunosorbent assays. RESULTS: We found an overall seroprevalence of 99.4% (95% CI 98.8-99.7%) for diphtheria, and 92.3% (95% CI 90.6-93.6%) for poliomyelitis, along with no indigenous cases of these diseases since 1993. However, the seroprevalence for pertussis was 60.8% (95% CI 58.0-63.5%) only. Among the subjects who had completed three doses of pertussis vaccination by the age of 2 years (n=1092), the pertussis seroprevalence was 85.0% (95% CI 79.7-89.2%) in those who received the last vaccination within a year before the study, and it decreased to 75.0% (95% CI 64.5-83.2%) and 63.1% (95% CI 50.9-73.8%) in those who had the last vaccination 1 year and 2 years before the study, respectively. The seroprevalence remained at about 50% for those whose last pertussis vaccination was administered 4 years and longer before the study. CONCLUSIONS: The high seroprevalence for poliomyelitis and diphtheria confer solid herd immunity to eliminate these diseases in Singapore. In contrast, immunity against pertussis waned considerably over time, and routine boosters should be given to adolescents to ensure sustained immunity against pertussis.

Doxycycline treatment attenuates acute lung injury in mice infected with virulent influenza H3N2 virus: involvement of matrix metalloproteinases.

Acute respiratory distress syndrome, a severe form of acute lung injury (ALI), is a major cause of death during influenza pneumonia. We have provided evidence for the involvement of recruited neutrophils, their toxic enzymes such as myeloperoxidase and matrix metalloproteinases (MMPs), and neutrophil extracellular traps in aggravating alveolar-capillary damage. In this study, we investigated the effects of doxycycline (DOX), an inhibitor of MMPs, on influenza-induced ALI. BALB/c mice were infected with a sublethal dose of mouse-adapted virulent influenza A/Aichi/2/68 (H3N2) virus, and administered daily with 20mg/kg or 60 mg/kg DOX orally. The effects of DOX on ALI were determined by measuring inflammation, capillary leakage, and MMP activities. Furthermore, levels of T1-α (a membrane protein of alveolar type I epithelium) and thrombomodulin (an endothelial protein) in the bronchoalveolar lavage fluid were evaluated by Western blot analysis. Our results demonstrate significantly decreased inflammation and protein leakage in the lungs after DOX treatment. Levels of MMP-2 and MMP-9 activity, T1-α and thrombomodulin were also diminished in the DOX-treated group. These findings were corroborated by histopathologic analyses, which demonstrated significant reduction in lung damage. Although DOX treatment reduced ALI, there were no effects on virus titers and body weights. Taken together, these results demonstrate that DOX may be useful in ameliorating ALI during influenza pneumonia. Further studies are warranted to determine whether DOX can be used in combination with anti-viral agents to alleviate severe influenza pneumonia.

Comparative Seroepidemiologic Analysis of Chlamydophila Pneumoniae Infection using Microimmunofluorescence, Enzyme Immunoassay and Neutralization Test: Implications for Serodiagnosis.

A seroepidemiologic study using the microimmunofluorescence (MIF) technique was conducted to determine the prevalence of Chlamydophila pneumoniae IgG antibodies among 205 healthy Singapore university undergraduates using the MRL Diagnostics MIF test kit. The overall seroprevalence was 35.1% with significantly higher seropositivity rates among males than females (48.2 vs. 18.7%, P < 0.001). A comparative study using the Labsystems MIF test kit was conducted on sera from 192 students. Using the MRL MIF test as the reference, the sensitivity and specificity of Labsystems MIF test were 92.6 and 87.9%, respectively. A total of 78 samples comprising 15 MIF-negative and 63 MIF-positive samples were also tested for complement-independent neutralizing antibodies in vitro. All the 78 samples and 11 additional MIF-negative samples were also tested for IgM, IgG and IgA against C. pneumoniae by enzyme immunoassay (EIA) using the Labsystems EIA test kit. None of these 89 samples were seropositive for IgM. The percentages of IgG and IgA seropositivity increased with increasing grades of MIF-positivity. Among the IgG seropositive samples, 69.1% were also positive for IgA, suggesting that a high proportion of infected individuals also had IgA antibodies denoting chronicity. Neutralizing antibodies were detected in 22.2% of MIF-positive sera, but only in 6.7% of MIF-negative sera. 26.4 and 34.2% of samples which were IgG and IgA seropositive respectively also exhibited neutralizing activity. The percentages of MIF-positive sera with neutralizing activity increased with the grade of MIF positivity, i.e. 0% (1+), 7.1% (2+), 18.8% (3+), and 63.6% (4+). High-grade MIF positivity (particularly with MRL MIF kits) may represent a useful serologic marker of predictive value for neutralizing activity.

The largest outbreak of hand; foot and mouth disease in Singapore in 2008: the role of enterovirus 71 and coxsackievirus A strains.

BACKGROUND: During 2008, Singapore experienced its largest ever outbreak of hand, foot and mouth disease (HFMD), resulting in 29686 cases, including four cases of encephalitis and one fatality. METHODS: A total of 51 clinical specimens from 43 patients with suspected HFMD at the National University Hospital, Singapore were collected for virus isolation and identification by reverse transcription polymerase chain reaction (RT-PCR) and sequencing. RESULTS: Enteroviruses were identified in 34 samples (66.7%), with 11 samples (21.6%) being positive for enterovirus 71 (EV71). Other non-EV71 enteroviruses (including coxsackievirus A4, A6, A10, and A16) were identified in 23 samples (45.1%). The most prevalent virus serotypes were CA6, CA10, and EV71. CA6 and CA10 accounted for 35.3% of all HFMD cases, which may explain the high transmissibility and low fatality that characterized this unprecedented epidemic associated with relatively mild disease. Phylogenetic analyses of 10 circulating EV71 strains indicated that they belonged to two subgenogroups, i.e., B5 (80%) and C2 (20%). The VP1 sequences of the 2008 EV71 strains also exhibited continuous mutations during the outbreak, reflecting the relatively high mutation rate of the EV71 capsid protein, which may have implications for future vaccine development. CONCLUSIONS: A safe and effective vaccine against EV71 is certainly warranted in view of its potential neurovirulence and its role in HFMD epidemics of recurring frequency with resultant fatalities in Asia, as well as other parts of the world.

Inactivated trivalent seasonal influenza vaccine induces limited cross-reactive neutralizing antibody responses against 2009 pandemic and 1934 PR8 H1N1 strains.

BACKGROUND: In June 2009, we conducted a prospective study in Singapore on 51 individuals to determine their serologic responses before and following receipt of the 2009 Southern Hemisphere seasonal influenza vaccine. MATERIALS AND METHODS: Paired serum samples were obtained before and 3-4 weeks after vaccination. Virus microneutralization assays were performed to quantify antibodies against A/Brisbane/59/2007 vaccine, pandemic H1N1-2009 and A/Puerto Rico/08/34 H1N1 strains. RESULTS: Post-vaccination, 43%, 12% and 24% of subjects displayed a 4-fold or greater rise in neutralizing antibody titers against the three strains, respectively. There was a positive correlation among individuals who showed increased titers to both pandemic H1N1-2009 and A/Puerto Rico/08/34 (p<0.001). However, this correlation was not observed for A/Brisbane/59/2007 with either strain. The relative conservation and accessibility of predicted B-cell epitopes may explain the limited cross-reactivity of the antibodies directed against common H1N1 epitopes. CONCLUSIONS: These results suggest that seasonal influenza vaccination confers a certain degree of cross-protection to other H1N1 strains. The correlation in cross-reactive antibody titers to A/Puerto Rico/08/34 and pandemic H1N1-2009 implies that previous exposure to pre-1957 H1N1 strains may confer some protection against the 2009 pandemic strain.

MCP-1 antibody treatment enhances damage and impedes repair of the alveolar epithelium in influenza pneumonitis.

Recent studies have demonstrated an essential role of alveolar macrophages during influenza virus infection. Enhanced mortalities were observed in macrophage-depleted mice and pigs after influenza virus infection, but the basis for the enhanced pathogenesis is unclear. This study revealed that blocking macrophage recruitment into the lungs in a mouse model of influenza pneumonitis resulted in enhanced alveolar epithelial damage and apoptosis, as evaluated by histopathology, immunohistochemistry, Western blot, RT-PCR, and TUNEL assays. Abrogation of macrophage recruitment was achieved by treatment with monoclonal antibody against monocyte chemoattractant protein-1 (MCP-1) after sub-lethal challenge with mouse-adapted human influenza A/Aichi/2/68 virus. Interestingly, elevated levels of hepatocyte growth factor (HGF), a mitogen for alveolar epithelium, were detected in bronchoalveolar lavage samples and in lung homogenates of control untreated and nonimmune immunoglobulin (Ig)G-treated mice after infection compared with anti-MCP-1-treated infected mice. The lungs of control animals also displayed strongly positive HGF staining in alveolar macrophages as well as alveolar epithelial cell hyperplasia. Co-culture of influenza virus-infected alveolar epithelial cells with freshly isolated alveolar macrophages induced HGF production and phagocytic activity of macrophages. Recombinant HGF added to mouse lung explants after influenza virus infection resulted in enhanced BrdU labeling of alveolar type II epithelial cells, indicating their proliferation, in contrast with anti-HGF treatment showing significantly reduced epithelial regeneration. Our data indicate that inhibition of macrophage recruitment augmented alveolar epithelial damage and apoptosis during influenza pneumonitis, and that HGF produced by macrophages in response to influenza participates in the resolution of alveolar epithelium.

Characterization of cytotoxic T-lymphocyte epitopes and immune responses to SARS coronavirus spike DNA vaccine expressing the RGD-integrin-binding motif.

Integrins are critical for initiating T-cell activation events. The integrin-binding motif Arg-Gly-Asp (RGD) was incorporated into the pcDNA 3.1 mammalian expression vector expressing the codon-optimized extracellular domain of SARS coronavirus (SARS-CoV) spike protein, and tested by immunizing C57BL/6 mice. Significant cell-mediated immune responses were characterized by cytotoxic T-lymphocyte (51)Cr release assay and interferon-gamma secretion ELISPOT assay against RMA-S target cells presenting predicted MHC class I H2-Kb epitopes, including those spanning residues 884-891 and 1116-1123 within the S2 subunit of SARS-CoV spike protein. DNA vaccines incorporating the Spike-RGD/His motif or the Spike-His construct generated robust cell-mediated immune responses. Moreover, the Spike-His DNA vaccine construct generated a significant antibody response. Immunization with these DNA vaccine constructs elicited significant cellular and humoral immune responses. Additional T-cell epitopes within the SARS-CoV spike protein that may contribute to cell-mediated immunity in vivo were also identified.

Adaptation of human influenza H3N2 virus in a mouse pneumonitis model: insights into viral virulence, tissue tropism and host pathogenesis.

Most pandemic influenza virus strains undergo adaptation or reassortment before they acquire the ability to cause fatal infections in a new host species. The pathologic changes and tissue tropism during virus adaptation are not fully understood. Here we investigated pathologic changes and tissue tropism by serial lung-to-lung passaging of human influenza virus strain A/Aichi/2/68 (H3N2) in a BALB/c mouse model. Enhanced pulmonary lesions and systemic virus infection were observed during adaptation. Late passage 10 (P10) virus caused extra-pulmonary spread with necrotic and inflammatory lesions in the brain, heart, spleen and intestine of infected animals, in contrast to infection with earlier passage viruses which were restricted to lungs. Non-conservative mutations in the hemagglutinin (Gly218Glu) and non-structural 1 (Asp125Gly) proteins were identified in P10 virus which exhibited high virulence. Virus growth kinetics showed enhanced replication ability of P10 virus in different cell lines. P10 virus also exhibited the ability to bind to erythrocytes of different host species. These results demonstrate extra-pulmonary spread of influenza virus during adaptation with enhanced replication ability in a new host. This mouse adaptation model may provide a basis for understanding cross-species adaptability corresponding to increased virulence of the influenza A virus, a phenomenon of relevance to the emergence of future highly pathogenic strains.

Identification of human metapneumovirus and Chlamydophila pneumoniae in children with asthma and wheeze in Singapore.

INTRODUCTION: The aim of our study was to determine if human metapneumovirus (hMPV) and Chlamydophila pneumoniae (CP) could be detected in Singaporean asthmatic children and wheezing infants during an acute asthma attack. METHODS: The study was performed on 30 older children (mean age 9.8 years) and 30 young children (mean age 1.3 years), who were admitted with an acute exacerbation of wheezing. Nasopharyngeal aspirates were collected and tested by polymerase chain reaction for CP, and for a panel of viruses (hMPV, respiratory syncytial virus, adenovirus, influenza virus types A and B, parainfluenza virus types 1 and 3, and rhinovirus). RESULTS: hMPV was isolated in eight out of 60 children (13.3 percent), while CP was isolated in two cases. Overall, 48/60 (80 percent) samples were positive for the presence of viruses. CONCLUSION: In most of the children admitted because of acute wheezing, a virus could be detected. hMPV was isolated for the first time in Singapore in children who were admitted with an acute asthma attack.

Measurement of serum leptin concentrations in university undergraduates by competitive ELISA reveals correlations with body mass index and sex.

Synthesized mainly in adipocytes, leptin is a peptide hormone that plays a key role in the regulation of body weight and composition. The serum leptin concentrations of 193 Singapore university medical and bioscience undergraduates aged 19-26 yr were measured using a competitive ELISA kit, and their leptin levels were correlated with sex and body mass index (BMI). Mean leptin levels were more than twice as high in females than in males of corresponding weight status, especially among females of healthy weight who exhibited levels that were 5.7 times higher. Overweight individuals generally demonstrated higher circulating leptin concentrations than healthy-weight and underweight participants. The differences in mean leptin levels between underweight and overweight males (P = 0.006), as well as between healthy-weight and overweight males (P = 0.011) were statistically significant. Comparison tests of leptin levels between healthy-weight and underweight females were highly significant (P = 0.001). Highly significant linear correlations between BMI and the logarithm of leptin concentration were observed in the female (r = 0.44) and male (r = 0.36) groups. These results reiterate the impact of gonadal steroids as mediators of the apparent sexual dimorphism in circulating leptin. The findings also corroborate evidence that adiposity determines leptin levels. This laboratory exercise has educational value for undergraduates by determining their BMIs, by alluding to the importance of maintaining healthy body composition, and by emphasizing the molecular mechanisms of body weight regulation and obesity, with special reference to leptin. This practical study also exemplifies the principles and applications of the competitive ELISA technique and integrates certain key concepts of physiology, molecular biology, immunology, and medicine.

Seroprevalence of IgG antibodies against Chlamydia pneumoniae in Chinese, Malays and Asian Indians in Singapore.

BACKGROUND: Chlamydia pneumoniae, a bacterium that causes respiratory infections, is probably under-diagnosed. There is also interest in its possible role in the aetiology of coronary heart disease. This is the first population-based seroprevalence survey of C. pneumoniae infection in Singapore. METHODS: A random sample of 1,068 people aged 18-69 years was selected from the participants of the Singapore National Health Survey conducted in 1998. Sera and data on certain clinical measurements and conditions had been collected. IgG antibodies for C. pneumoniae were detected using an indirect microimmunofluorescence test and positivity graded. Seropositivity was defined as IgG titre >/=1:16. RESULTS: There were no statistically significant differences in the prevalence rates of seropositivity to C. pneumoniae for age group 18-69 years among the three ethnic groups, i.e. Chinese (males 76.7%, females 68.3%), Malays (males 75.4%, females 59.1%), and Asian Indians (males 74.6%, females 59.4%). The seropositivity rate for people aged 18-69 years in Singapore was 75.0% for males and 65.5% for females (difference of 9.5%, P < 0.001). In both genders combined, seropositivity increased from 46.5% in the age group 18-29 to reach a plateau of 78.9% in the age group 40-49, which remained stable to 60-69 years. There was no association of seropositivity with smoking, diabetes mellitus, hypertension or body mass index after adjustment for age and gender. CONCLUSION: The high prevalence rates in our study population and the higher rate in males compared to females are consistent with studies from other parts of the world. No significant difference in prevalence rates was observed among Chinese, Malays and Indians. The pattern of rising and levelling off of seropositivity with age suggests that C. pneumoniae infection occurs early in life, and in older ages the high level of seropositivity is probably maintained by re-infections or chronic infections. Chlamydia pneumoniae infection was not found to be associated with the cardiovascular risk factors examined.

Direct detection of enterovirus 71 (EV71) in clinical specimens from a hand, foot, and mouth disease outbreak in Singapore by reverse transcription-PCR with universal enterovirus and EV71-specific primers.

A recent outbreak of hand, foot, and mouth disease in Singapore in 2000 affected several thousand children and resulted in four deaths. The aim of this study was to determine the applicability of reverse transcription-PCR (RT-PCR) with universal pan-enterovirus primers and enterovirus 71 (EV71) type-specific primers for the direct detection of enteroviruses in clinical specimens derived from this outbreak. With the universal primers, EV71 RNA sequences were successfully detected by RT-PCR and direct sequencing in 71% of positive specimens. Three pairs of EV71 type-specific primers were evaluated for rapid detection of EV71 directly from clinical specimens and cell culture isolates. By using a seminested RT-PCR strategy, specific identification of EV71 sequences directly in clinical specimens was achieved, with a detection rate of 53%. In contrast, cell culture could isolate EV71 in only 20% of positive specimens. EV71 was detected directly from brain, heart, and lung specimens of two deceased siblings. Although more than one type of enterovirus was identified in clinical specimens from this outbreak, 90% of the enteroviruses were confirmed as EV71. The data demonstrate the clinical applicability of pan-enterovirus and seminested RT-PCR for the detection of EV71 RNA directly from clinical specimens in an outbreak situation.

Linoleic and linolelaidic acids differentially influence proliferation and apoptosis of MOLT-4 leukaemia cells.

The effects of varying concentrations of linoleic acid and its transisomer linolelaidic acid on the proliferation the ultrastructural morphology of MOLT-4 T-lymphoblastic leukaemia cells were investigated. At 2 and 4 days after exposure to the fatty acids, the cells were counted by flow cytometry and observed by electron microscopy. After 4 days of treatment, linoleic acid was growth stimulatory at concentrations of 200 microM or less, but was markedly inhibitory at 400 microM. In contrast, linolelaidic acid stimulated proliferation at concentrations of 100 and 200 microM, but inhibited cell growth at 400 microM. Cells treated with 400 microM linoleic acid displayed dense accumulations of characteristic lipid globules and glycogen granules, and exhibited ultrastructural evidence of apoptosis including vacuolization, membrane blebbing and chromatin margination at the nuclear periphery. These results support the notion that geometrical isomerism and concentration of polyunsaturated fatty acids influence the proliferative destiny of cancer cells. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed a previously documented larger alternatively spliced p53 gene transcript in MOLT-4 cells cultured under reduced serum conditions. However, only wild-type p53 transcripts were amplified by RT-PCR of MOLT-4 cells exposed to phytohaemagglutinin, linoleic acid or linolelaidic acid.

Diagnosis of nipah virus encephalitis by electron microscopy of cerebrospinal fluid.

BACKGROUND: between 1998 and 1999, an outbreak of potentially fatal viral encephalitis erupted among pig farm workers in West Malaysia, and later spread to Singapore where abattoir workers were afflicted. Although Japanese encephalitis virus was initially suspected, the predominant aetiologic agent was subsequently confirmed to be Nipah virus, a novel paramyxovirus related to but distinct from Hendra virus. OBJECTIVE: to describe a case of Nipah virus encephalitis in a pig farm worker from Malaysia. STUDY DESIGN: the clinical, laboratory and radiological findings of this patient were scrutinized. Special emphasis was placed on the electron microscopic analysis of the cerebrospinal fluid (CSF) specimen from this patient. RESULTS: the neurological deficits indicative of cerebellar involvement were supported by the magnetic resonance imaging that showed prominent cerebellar and brainstem lesions. CSF examination provided further evidence of viral encephalitis. Complement fixation and/or RT-PCR assays were negative for Japanese encephalitis, herpes simplex, measles and mumps viruses. ELISA for detecting IgM and IgG antibodies against Hendra viral antigens were equivocal for the CSF specimen, and tested initially negative for the first serum sample but subsequently positive for the repeat serum sample. Transmission electron microscopy of negatively-stained preparations of CSF revealed enveloped virus-like structures fringed with surface projections as well as nucleocapsids with distinctive helical and herringbone patterns, features consistent with those of other paramyxoviruses, including Hendra virus. CONCLUSION: this case report reiterates the relevant and feasible role of diagnostic electron microscopy for identifying and/or classifying novel or emerging viral pathogens for which sufficiently specific and sensitive tests are lacking.

A recent epidemic of acute hemorrhagic conjunctivitis.

The coxsackievirus A24 variant was implicated in four of six major acute hemorrhagic conjunctivitis outbreaks in Singapore since its discovery in 1970. Hela cell suspension in 24-well flat-bottom tissue culture plates was a satisfactory alternative to monolayer cells grown in test tubes for virus isolation. Respiratory illness occurred in 20 of 98 patients with acute hemorrhagic conjunctivitis. Apart from conjunctival secretions, respiratory and oral transmission of coxsackievirus A24 variant would explain the rapid and extensive spread of acute hemorrhagic conjunctivitis during an outbreak.