Real time detection and quantification of Epstein Barr virus in different grades of oral gingivitis and periodontitis patients.

BACKGROUND: Periodontal diseases are of microbial etiology and are globally causing loss of teeth in adult population. Many severe oral diseases have been recently associated to Herpes viruses, of which Epstein Barr Virus (EBV) and human cytomegalovirus (HCMV) have been indicated in the etiology of periodontal diseases. AIM: The purpose of the study was to compare the effect of EBV in different types of periodontal diseases namely acute gingivitis, chronic gingivitis, acute and chronic, localized and generalized aggressive (juvenile) periodontitis and apical periodontitis. MATERIAL AND METHOD: 70 individuals were included in this study. Supragingival plaque and plaque from two deepest sites of the periodontal pockets were collected then stored at 70° c and prepared for nucleic acid extraction. For EBV detection, DNA were extracted from the plaque samples with the QIAamp DNA mini kit. Q-PCR was performed by targeting the non-polymorphic Epstein-Barr nuclear antigen-1 (EBNA-1) gene using Corbett Research 6000 Q-PCR instrument and Rotor gene 6000 software. RESULTS: Overall prevalence of EBV in the disease group was 60% (27/45 patients) as compared to only 8% (4/25 people) in the normal population. The mean copy number of EBV DNA was found to be significantly higher in periodontitis (2234 ± 1811.34) when compared to gingivitis (554 ± 537.64, p = .001) and normal patients (370 ± 161.03, p < .001). CONCLUSION: Here, we found that the prevalence of EBV as well as copy number of EBV was significantly higher in periodontitis patients as compared to gingivitis patients or normal population.

Reversal of effects of intra peritoneally administered beryllium nitrate by tiron and CaNa3DTPA alone or in combination with alpha-tocopherol.

To evaluate therapeutic efficacy of chelating agents tiron (Sodium-4,5-dihydroxy-1,3-benzene disulphonate) and CaNa3DTPA (Calcium trisodium diethylene triamine pentaacetic acid) in presence of alpha-tocopherol against beryllium induced toxicity, adult female albino rats were exposed to beryllium nitrate for 28 days followed by therapy with tiron (471 mg/kg, i.p.) and CaNa3DTPA (35 mg/kg, i.p.) alone and in combination with alpha-tocopherol (25 mg/kg, p.o.). Results revealed non-significant fall in haemoglobin and total serum protein content while significant fall in blood sugar level and activity of serum alkaline phosphatase. On the other hand, significant rise in the activity of serum transaminases and LDH was noticed after beryllium administration. Significant increase in total and esterified cholesterol was found in liver and kidney after toxicity. Significant increase in lipid peroxidation and decreased level of reduced glutathione in both the organs showed oxidative stress due to beryllium exposure. Histopathological and ultrastructural observations of liver and kidney revealed lesions due to beryllium toxicity followed by recovery due to combined therapy. CaNa3DTPA showed moderate therapeutic efficacy; however, its effectiveness was enhanced with alpha-tocopherol to some extent. Tiron in combination with alpha-tocopherol exerted statistically more beneficial effects in reversal of beryllium induced biochemical, histopathological and ultrastructural alterations.

Kinetics of cytokine profile during intraperitoneal inoculation of Japanese encephalitis virus in BALB/c mice model.

Infection with Japanese encephalitis virus (JEV) is mostly asymptomatic/subclinical in 90% of the individuals. Host immune response during subclinical JEV infection is poorly understood. We assessed iNOS, IFN-gamma, TNF-alpha, IL-10 and IL-4 production in spleen, brain and sera of intraperitoneally challenged BALB/c mice by RT-PCR and ELISA along with brain histopathology at different days post inoculation (d.p.i.). In spleen of virus infected mice, expression of all cytokines including iNOS mRNA were upregulated till 5d.p.i. followed by decline. At 5d.p.i., IL-10 expression outcompeted TNF-alpha, IFN-gamma and IL-4. However, in the virus infected mice sera, IL-4 production predominated over TNF-alpha and IL-10 at 5d.p.i. Conversely, cytokines expression and iNOS mRNA remained unchanged in the brain of virus infected mice from 1 to 7d.p.i. A significant increase in the cytokine expression was observed at 11d.p.i. (P<0.05) in virus infected mice brain, with the predominance of IL-10 along with the presence of meningeal inflammation and viral RNA by histology and RT-PCR, respectively. We report a biased pattern of cytokine production in sera, brain and spleen of mice intraperitoneally challenged with JEV. IL-10 exerts neuroprotective function during JEV and regulates deleterious effects of proinflammatory cytokines; however, its mechanism needs further investigation.

Secretion of the chemokine interleukin-8 during Japanese encephalitis virus infection.

Japanese encephalitis (JE) virus infection induces infiltration of neutrophils in neural as well as extraneural tissues in patients. As interleukin-8 (IL-8) has inflammatory properties, the present study was undertaken to investigate the IL-8 concentrations in cerebrospinal fluid (CSF) and serum from patients with JE and correlate them with neutrophil counts. IL-8 was measured in the CSF or serum of 30 patients with confirmed JE. The majority (92%) of the acute CSF samples showed raised levels of IL-8 with raised numbers of polymorphonuclear leucocytes. Similarly, significantly higher serum IL-8 concentrations were detected in the acute phase of illness than in convalescent JE patients or normal healthy controls. Twenty-one of 25 patients with high concentrations of IL-8 showed significantly increased neutrophil counts in acute phase sera. A gradual decline in neutrophil counts was observed in the convalescent phase of patients who recovered. There was a significant correlation between IL-8 level and the severity of illness, as all severely ill and fatal cases showed higher levels of IL-8 in acute CSF or serum than the levels found in those who recovered. IL-8 concentrations remained high for a longer period in patients with prolonged severe illness than in those who made a complete recovery.

Inhibition of Japanese encephalitis virus infection by diethyldithiocarbamate is independent of its antioxidant potential.

Diethyldithiocarbamate (DDTC), a low molecular weight dithiol, has been described as an immunomodulator and modifier of diverse biological actions in human and animal models, and has also been shown to be effective in several disease conditions. Therefore, we studied the therapeutic aspect of DDTC in providing inhibition of Japanese encephalitis virus (JEV) infection. DDTC tested at various doses (10-100 micromol/kg) revealed that administration at low concentration (10 micromol/kg; i.p.) on alternate days prolonged the average survival time (AST) of mice infected with lethal dose of JEV (102 LD50, i.c.) and delayed progression of the disease. The low dose also provided > 80% survival in sub-clinical (10(5) LD50, i.c.) JEV infection. Administration of DDTC to JEV-infected mice enhanced the inducible nitric oxide synthase (iNOS) activity in brain and level of serum tumour necrosis factor-alpha (TNF-alpha). We have recently demonstrated the production of nitric oxide (NO) via induction of iNOS activity is meditated by circulating macrophage-derived factor (MDF), which may be responsible for the delayed progression of the disease. DDTC-mediated inhibition of JEV is believed to involve the augmentation of protective role of MDF as evidenced by the observation that pretreatment with anti-MDF antibody significantly decreased the AST of mice and together with the inhibition of iNOS activity. Interestingly, DDTC alone did not stimulate iNOS and TNF-alpha in mock-infected normal mice. These results show that DDTC may have a possible therapeutic role during JEV infection.

Comparative effectiveness of CaNa3DTPA and tiron along with alpha-tocopherol against beryllium-induced biochemical alterations in rats.

The therapeutic efficacy of chelating agents CaNa3DTPA (calcium trisodium diethylene triamine penta acetic acid) and Tiron (sodium-4,5-dihydroxy-1,3-benzene disulphonate) with and without antioxidant, alpha-Tocopherol was evaluated in the treatment of beryllium-induced toxicity in female albino rats. The animals were exposed to beryllium (as beryllium nitrate) at a dose of 1 mg/kg (ip) once a day for 28 consecutive days followed by chelation therapy by CaNa3DTPA (0.1 mM/kg, ip) and Tiron (471 mg/kg, ip) with and without alpha-Tocopherol (25 mg/kg, orally) for 5 consecutive days after toxicant administration. Tissue biochemistry revealed severe alterations in liver and kidney. A significant fall in total protein and glycogen contents, alkaline phosphatase, adenosine tri-phosphatase and succinic dehydrogenase level was noticed. On the contrary, an elevation in acid phosphatase was recorded. The significant rise in hepatic lipid peroxidation and decreased level of hepatic reduced glutathione showed toxicity due to beryllium. CaNa3DTPA with alpha-Tocopherol showed moderate therapeutic efficacy while Tiron in combination with alpha-Tocopherol exerted statistically more beneficial effects to reverse biochemical alterations in different variables altered due to beryllium intoxication.

In vitro evaluation of microbiological flora of orofacial infections.

OBJECTIVE: To assess the most common micro-organisms causing odontogenic infections and their antimicrobial susceptibility. METHODS: The study was conducted in 80 patients with orofacial infection. The pus sample was collected, cultured (aerobically and anaerobically) and stained for morphological study of the isolates. Antibiotic sensitivity test for the isolates were performed. RESULTS: A total of 109 micro-organisms were isolated, no pathogenic organism were isolated in 3 cases. Out of 109 micro-organism isolated, 107 bacteria and 2 fungi were identified. Pure aerobes were identified in 28(35%) of cases, pure anaerobes in 18(22.5%), mixed aerobes and anaerobes in 10(12.5%), mixed aerobes in 15(18.75%) and mixed anaerobes were isolated in 6(7.5%) cases. Among the entire pure gram positive isolates, ofloxacin was the most sensitive drug 83.33% followed by ciprofloxacin 76.2% and sparfloxacin 76.2%. The most resistant drugs were amoxicillin (92.85%) and ampicillin (92.85%). Cefotaxime was found sensitive in 75% of pure gram negative isolates. CONCLUSION: Ofloxacin was the most sensitive drug followed by ciprofloxacin and sparfloxacin for pure gram positive isolates. The most resistant drugs were amoxicillin and ampicillin. The gram negative colonies were sensitive to Cefotaxime.

Trend of Japanese encephalitis in North India: evidence from thirty-eight acute encephalitis cases and appraisal of niceties.

BACKGROUND: In the year 2005, an epidemic of Japanese encephalitis (JE) occurred in the northern states of India. The present study was planned to reconfirm the circulation of JE in the area and to assess the trend of the disease to slow down the burden of JE. METHODOLOGY: Surveillance was conducted to identify patients with acute encephalitis. Blood and cerebrospinal fluid specimens from suspected cases underwent pathological, serological, and demographic investigations. Viral testing for evidence of Japanese encephalitis virus (JEV) infection was also performed, either by IgM capture ELISA/RT-PCR or both. To identify circulating JEV strains, RT-PCR, sequencing and phylogenetic analysis was performed. Based on clinical cases reported between 1992 and 2008, the trend of JE infection in the state was analyzed to examine the dynamics of infection. RESULTS: Our investigations (n = 38) revealed that only 55.3% cases were positive for JE. Pathological examination revealed marked pleocytosis in CSF (90+/-76.9 cells/mm(3)), and peripheral leucocytosis (64.7+/-8.86% neutrophils) with mild anemia. Males were more susceptible than females with a ratio of 1.63:1 and significant gender difference (P<0.05) was observed in patients below six years. In the patient group younger than six years, the rate of infection per million was six-fold higher (P<0.005) in males as compared to females. Our phylogenetic study suggests that the circulating strain during the 2005 JE epidemic was close to GP78, and in the future a larger epidemic may occur. CONCLUSIONS: The 2005 JE epidemic was possibly caused by JEV GP78 and it is spreading into newer areas. The trend of JE suggests that the problem in North India is escalating and larger epidemics may occur in the future; therefore, serious steps are necessary to combat JE, including the development of more efficient surveillance methods and differential diagnosis.

Structural and antigenic variance between novel influenza A/H1N1/2009 and influenza A/H1N1/2008 viruses.

BACKGROUND: The emergence of influenza A/H1N1/2009 is alarming. The severity of previous epidemics suggests that the susceptibility of the human population to H1N1 is directly proportional to the degree of changes in hemagglutinin/HA and neuraminidase/NA; therefore, H1N1/2009 and H1N1/2008 were analyzed for their sequence as well as structural divergence. METHODOLOGY: The structural and sequence divergence of H1N1/2009 and H1N1/2008 strains were analyzed by aligning HA and NA amino acid sequences by using ClustalW and ESyPred3D software. To determine the variations in sites of viral attachment to host cells, a comparison between amino acid sequences of HA and NA glycosylation sites was performed with NetNGlyc software. The antigenic divergence was executed by CTL epitope prediction method. RESULTS: The amino acid homology levels of H1N1/2009 were 20.32% and 18.73% compared to H1N1/2008 for HA and NA genes, respectively. In spite of the high variation in HA and NA amino acid composition, there was no significant difference in their structures. Antigenic analysis proposes that great antigenic differences exist between both the viral strains, but no addition of a new site of glycosylation was observed. CONCLUSIONS: To our knowledge, this is the first report suggesting that the circulating novel influenza virus A/H1N1/2009 attaches to the same glycosylation receptor sites as its predecessor influenza A/H1N1/2008 virus, but is antigenically different and may have the potential for initiating a significant pandemic. Our study may facilitate the development of better therapeutics and preventive strategies, as well as impart clues for novel H1N1 diagnostic and vaccine development.

Influence of alpha-tocopherol, propolis and piperine on therapeutic potential of tiferron against beryllium induced toxic manifestations.

The therapeutic potential of the chelator tiferron (sodium-4,5-dihydroxy-1,3-benzene disulphonate; 300 mg kg(-1), i.p.) and adjuvants, i.e. alpha-tocopherol (25 mg kg(-1), p.o.), propolis (a honey-bee hive product; 200 mg kg(-1), p.o.) and piperine (10 mg kg(-1), p.o.) were evaluated individually and in combination against beryllium induced biochemical alterations and oxidative stress consequences. Female albino rats were exposed to beryllium nitrate (1 mg kg(-1), i.p.) daily for 28 days followed by treatment with the above mentioned therapeutic agents for 5 consecutive days. Administration of beryllium altered blood biochemical variables with significant depletion in hemoglobin, blood sugar, total serum protein, albumin and significant enhancement in the release of serum transaminases. A significantly increased lipid peroxidation and a decreased level of glutathione after beryllium exposure indicated oxidative stress in the liver and kidney. Beryllium exposure decreased total protein and glycogen contents, whereas triglycerides and cholesterol increased significantly in liver and kidney. Individual administration of all the four compounds showed significant therapeutic potential in reverse of some of the biochemical parameters mentioned above. Furthermore, the combination of tiferron with alpha-tocopherol, propolis or piperine, respectively, could reverse all the variables significantly more towards the control. None of the test compounds showed any significant change in choleretic activity (bile flow and bile solids), indicating that these compounds had no adverse effects at these dose levels. It was concluded that all the combinations of tiferron and adjuvants played a beneficial role in reducing beryllium induced systemic toxicity at relatively lower doses and the combination of tiferron and propolis showed a more pronounced therapeutic potential.

Pharmacological intervention of tiferron and propolis to alleviate beryllium-induced hepatorenal toxicity.

Intervention of chelating agent tiferron (sodium-4,5-dihydroxy-1,3-benzene disulfonate; 300 mg/kg, intraperitoneal) with propolis (honey beehive product; 200 mg/kg, p.o.) was evaluated to encounter the characteristic biochemical and ultra-morphological alterations following subchronic exposure to beryllium. Female albino rats were challenged with beryllium nitrate (1 mg/kg, i.p.) daily for 28 days followed by treatment of the above-mentioned therapeutic agents either individually or in combination for five consecutive days. Exposure to beryllium increased its concentration in the serum, liver and kidney, and caused significant alterations in cytochrome P450 activity, microsomal lipid peroxidation and proteins. Activities of alkaline phosphatase, lactate dehydrogenase, gamma-glutamyl transpeptidase, bilirubin, creatinine and urea in the serum and activity of acid phosphatase, alkaline phosphatase, adenosine triphosphatase, glucose-6-phophatase and succinic dehydrogenase in the liver and kidney were significantly altered after beryllium administration. Beryllium exposure also induced severe hepatorenal alterations at histopathological and ultra-morphological level. Tiferron along with propolis dramatically reversed the alterations in all the variables more towards control rather than their individual treatment. The study concludes that pharmacological intervention of tiferron and propolis is beneficial in attenuating beryllium-induced systemic toxicity.

Amelioration of beryllium induced alterations in hepatorenal biochemistry and ultramorphology by co-administration of tiferron and adjuvants.

Influence of adjuvants i.e., alpha-tocopherol (25 mg/kg, p.o.) and piperine (10 mg/kg, p.o.) on therapeutic potential of chelator tiferron (300 mg/kg, i.p.) was evaluated to encounter toxicogenic events of beryllium exposure. Albino rats were exposed to beryllium nitrate (1 mg/kg, i.p.) daily for 28 days followed by treatment of aforesaid therapeutic agents for 5 consecutive days. Results were considered to be significant at p < or =0.01 and p < or =0.05. Exposure to beryllium increased its concentration in liver, kidney and serum causing significant alterations in the activity of CYP-450 2E1 system, microsomal lipid peroxidation and protein; alkaline phosphtase, lactate dehydrogenase, gamma-glutamyl transpeptidase, bilirubin, creatinine and urea in serum; activity of acid phosphatase, alkaline phosphatase, adenosine triphosphatase, glucose-6-phosphatase and succinic dehydrogenase in liver and kidney. Beryllium exposure also induced severe alterations in histopathology and ultramorphology of liver and kidney proving its toxic consequences at cellular level. Tiferron along with adjuvants dramatically reversed alterations of all variables more towards control rather than individual treatment. Study concluded that tiferron in combination with alpha-tocopherol and piperine respectively was beneficial in diluting beryllium induced systemic toxicity; however, combination of tiferron and piperine presented more pronounced therapeutic potential.

Alteration in plasma glucose levels in Japanese encephalitis patients.

A unique factor, human T cell hypoglycaemic factor (hTCHF), has been shown to produce hypoglycaemia during the convalescent stage in the plasma of patients with Japanese encephalitis virus (JEV) infection. The present study was undertaken to investigate the ability of T cells from fresh peripheral blood mononuclear cells (PBMC) of such patients to produce hTCHF. The PBMC, as well as the individual subpopulations, were cultured for 24 h and the culture supernatants (CS) were assayed for hypoglycaemic activity. The activity was observed in the CD8+ T cells. The hypoglycaemia in JE-confirmed patients coincided with the gradual rise in circulating glucagon level, with no significant alterations in insulin, growth hormone and cortisol levels. The hTCHF was purified by ion exchange chromatography and the purified protein was observed as a approximately 25 kDa band on SDS-PAGE. Secretory hTCHF in the sera of patients and T cell CS was present in 88% of convalescent serum samples. We conclude that during the convalescent stage of JEV infection, a unique factor, hTCHF, is secreted by activated CD8+ T cells from patients and that this is responsible for the development of hypoglycaemia.