A pilot randomized controlled trial of dual-plasmid HBV DNA vaccine mediated by in vivo electroporation in chronic hepatitis B patients under lamivudine chemotherapy.

A DNA vaccine against the hepatitis B virus (HBV), enhanced by IL-2/IFN-γ fusion protein expression from a plasmid construct and mediated by in vivo electroporation, was evaluated in a total of 39 HBeAg-positive patients with chronic hepatitis B (CHB). The six of 39 patients with a serum alanine aminotransferase (ALT) value of 1-2 times upper limit of normal (ULN) were assigned to the open-label arm (Group01) receiving vaccine monotherapy; the remaining 33 patients with an ALT of more than two times ULN were enroled to the randomized and controlled arm (Group02) receiving lamivudine (LAM) monotherapy (LAM+placebo) or combined therapy (LAM+DNA vaccine) in 1:2 ratio. In Group01, a significant elevation of HBV-specific IFN-γ-secreting T-cell counts in comparison with baseline was observed. In Group02, the proportion of patients with HBV DNA suppression was higher with LAM+DNA vaccine than with LAM monotherapy at each visit time point after the final injection of DNA vaccine at week 36, revealing a significant difference between the two groups (P = 0.03) at week 60. The incidence of dual-site mutations of rtM204/I/S+rtL180M was significantly lower (P = 0.03) with an identified lower virological breakthrough (VBT) rate (P = 0.03) in patients receiving LAM+DNA vaccine than LAM monotherapy, accompanied with a significant higher positive T-cell response rate in patients receiving LAM+DNA vaccine (P = 0.03). In conclusion, this study provides evidence that HBV DNA vaccination is safe and immunologically effective, and that the HBV-specific T-cell responses induced by DNA vaccination under LAM chemotherapy showed a correlation with the suppression of viral replication in patients with CHB.

A new record of longicorn beetle, Acanthophorus rugiceps, from India as a root borer on physic nut, Jatropha curcas, with a description of life stages, biology, and seasonal dynamics.

Longicorn beetle, Acanthophorus rugiceps Gahan (Coleoptera: Cerambycidae), is reported for the first time as a confirmed host on physic nut, Jatropha curcas L. (Malpighiales: Euphorbiaceae), from India, causing extensive damage to roots. Plants of three years age and above were prone to attack by this pest. In a six year study beginning in 2005, about 11.3 percent of plants in a 16.25 acre physic nut plantation were severely damaged by A. rugiceps. Life stages of A. rugiceps, including egg, larvae, pupae, and adult, are described with a note on their habitat, biology, and behavior. Strategies to manage this pest on physic nut are discussed.

Crystallization and preliminary X-ray analysis of crinumin, a chymotrypsin-like glycosylated serine protease with thrombolytic and antiplatelet activity.

Crinumin, a novel glycosylated serine protease with chymotrypsin-like catalytic specificity, was purified from the medicinally important plant Crinum asiaticum. Crinumin is a 67.7 kDa protease with an extraordinary stability and activity over a wide range of pH and temperature and is functional in aqueous, organic and chaotropic solutions. The purified protease has thrombolytic and antiplatelet activity. The use of C. asiaticum extracts has also been reported for the treatment of a variety of disorders such as injury, joint inflammation and arthritis. In order to understand its structure-function relationship, the enzyme was purified from the plant latex and crystallized by the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from a single crystal and processed to 2.8 Å resolution. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 121.61, b = 95.00, c = 72.10 Å, α = γ = 90, ß = 114.19°. The Matthews coefficient was 2.81 Å(3) Da(-1), corresponding to a solvent content of 56%, assuming one molecule in the asymmetric unit. Structure determination of the enzyme is in progress.

Efficacy and tolerability of sertaconazole nitrate 2% cream vs. miconazole in patients with cutaneous dermatophytosis.

Sertaconazole is a new antifungal agent. To compare the efficacy and tolerability of sertaconazole and miconazole cream in cutaneous dermatophytosis, this prospective, randomized, multicentric comparative, phase 4 study was undertaken in 260 patients with cutaneous dermatophytosis after approvals from Institutional Ethics Committees. Patients were assigned to sertaconazole cream (2%) or miconazole cream (2%) topically twice daily for 2 weeks after obtaining informed consent. Efficacy variables included changes in mean scores of erythema, pruritus, desquamation, erythema/itching, burning/weeping, scaling/pustule and overall global assessment. Safety and tolerability were also assessed. A total of 122 patients in the sertaconazole group and 128 in the miconazole group completed the study with 10 drop-outs. There was a significant decrease (P < 0.05) in mean symptom scores and total scores from the first week onwards, sustained till 2 weeks and statistically significant (P < 0.05) in favour of sertaconazole. Moreover, 62.3% patients had complete clinical cure in the sertaconazole group (P < 0.05) compared with 44.6% in miconazole users. Both drugs were well tolerated and five patients in the sertaconazole group and nine in the miconazole group reported mild to moderate adverse events. Therapy with sertaconazole cream (2%) provided a better efficacy and tolerability compared with the miconazole cream (2%) and could thus be a therapeutic option in cutaneous dermatophytosis.

Detection of human papilloma virus 16 and 18 DNA sequences by southern blot hybridization in oral leukoplakia and squamous cell carcinoma.

BACKGROUND: The etiopathological role of human papilloma virus (HPV) in the causation of oral cancer is till a subject of speculation. METHODS: We used the technique of Southern blot hybridization to detect the presence of HPV types 16 & 18 in biopsy specimens from oral cancer and leukoplakia patients as well as normal oral mucosal biopsies. RESULTS: The prevalence of either HPV type 16 or 18 was found in 64.5% (29/45) of oral cancer, 40%(12/30) of leukoplakia and 20%(9/45) of normal oral mucosal biopsies. No association could be demonstrated between tobacco usage habits or a history of genital warts with HPV prevalence. CONCLUSION: A significant finding was that none of the oral cancer patients were negative for both: a history of tobacco usage as well as presence of HPV infection, on Southern blot hybridization.

Prevalence of Helicobacter pylori in asymptomatic subjects--a nested PCR based study.

The aim of the study was to see the prevalence of Helicobacter pylori in asymptomatic children and adults by using nested PCR which is considered to be more specific than serological methods. Saliva and stool samples of 137 healthy children (aged 8 months to 16 y) and 108 asymptomatic adults (aged 17-60 y) were collected. PCR with primers targeting Hsp60 gene sequence of H. pylori was used. H. pylori positivity with nested PCR was observed in 45.7% (112/245) of the saliva and 42.8% (105/245) of the stool specimens. Prevalence of H. pylori in saliva was found to be 2.1%, 22.7%, 55.9%, 56.0%, 68.9% and 62.9% in the age groups of < 5 y, 6-10 y, 11-16 y, 17-30 y , 31-45 y and 45-60 y, respectively. The detection rates in stool were 4.25% in < 5 y, 13.64% in 6-10 y, 50% in 11-16 y, 64% in 17-30 y, 58.62% in 31-45 y and 61.1% in 45-60 y of age groups. The most favourable age group for acquiring the infection was 11-16 y. H. pylori positivity increased with lowering of socioeconomic status. There was no gender bias in prevalence of the bacterium.

Evaluation of nested PCR in detection of Helicobacter pylori targeting a highly conserved gene: HSP60.

OBJECTIVE: To comparatively evaluate a new nested set of primers designed for the detection of Helicobacter pylori targeting a highly conserved heat shock protein gene (Hsp60). METHODS: A total of 60 subjects having peptic ulcer diseases were tested for the detection of H. pylori using rapid urease test (RUT), histology, culture, and polymerase chain reaction (PCR) in their antral biopsy specimens. A newly designed Hsp60 gene-based primer set was evaluated against commonly used PCR primers for detection of H. pylori. RESULTS: Forty-six of the 60 study subjects were found positive for culture isolation and all the 46 culture-positive specimens were also positive with Hsp60 gene PCR. Of the 46 culture-positive specimens, 44 were positive for 16S rRNA gene, ureC gene, RUT, and histology whereas only 29 were positive with ureA gene PCR. Of the 14 culture-negative subjects, 10 were positive with 16S rRNA gene, 4 were positive with ureC (glmM) gene PCR, and 2 were positive with RUT and 1 was positive on histology. CONCLUSION: This study shows that nested amplification targeting Hsp60 gene is the most sensitive and specific with LR+ and LR- values of proportional, variant and 0, respectively, when compared with the other three PCR methods. Also, HSP60 gene-specific nested protocol was the most appropriate for detection of H. pylori in clinical specimens. This is particularly valuable because it can be used as a noninvasive method for detecting H. pylori infection in young children and also, in follow-up studies with peptic ulcer patients, on samples like feces and saliva.

Detection of Helicobacter pylori using nested polymerase chain reaction in gastric biopsy samples.

Helicobacter pylori remains a controversial organism with regards to humans, its epidemiology still unclear nearly two decades after discovery. The present study was undertaken to estimate the prevalence of the organism in the gastrointestinal tract in symptomatic and asymptomatic subjects to understand its precise natural history in India. A total of 154 specimens were a part of the study. These included gastric biopsies from peptic ulcer disease and Non ulcer dyspepsia subjects, as visualized on endoscopy, saliva and stool samples from apparently normal healthy adults. Nested polymerase chain reaction was performed using the primers Hp1, Hp2, Hp3 targeting 16S rRNA gene. A prevalence of 65.1%, 100%, 66.7%, and 73.3% respectively was observed by polymerase chain reaction. No association was observed between the H.pylori status and the disease condition of the patient.

Detection of Helicobacter pylori in stool specimens: comparative evaluation of nested PCR and antigen detection.

BACKGROUND: Efficacy of Helicobacter pylori stool antigen enzyme immunoassay (HpSA) and stool PCR was evaluated, before and after treatment, in a country with a high prevalence of H. pylori infection. METHODOLOGY: A total of 52 patients with dyspeptic symptoms were included in the study. Antral biopsy was collected during pre- and post-therapy periods for rapid urease test (RUT) and PCR. Similarly stool specimens for PCR and HpSA test were collected during both the periods from all 52 patients. Biopsy, PCR and RUT results together were considered the "gold standard." RESULTS: On the basis of gold standard tests, 40/52 patients were H. pylori positive. The sensitivity and specificity of HpSA test were 80% and 83.3% respectively in untreated patients. On the other hand, the sensitivity and specificity of stool PCR in untreated patients were 72.5% and 100% respectively. After eradication therapy, the results of both RUT and biopsy PCR were negative in 87.5% and positive in 12.5% of the patients. Although post treatment sensitivity of HpSA and stool PCR was equal (60%), specificity of HpSA and stool PCR were 68.6% and 97.1% respectively. CONCLUSION: The H. pylori stool tests represent a non-invasive concept for diagnosis of infection. Both HpSA and stool PCR seem to be satisfactory tests for pre-eradication as well as assessment of infection. But stool PCR is a better indicator than HpSA test in the post-eradication assessment of infection.

Oxidative stress in neonatal hyperbilirubinemia.

We investigated the role of bilirubin as an antioxidant in neonatal hyperbilirubinemia (NNH) by measuring malondialdehyde (MDA) levels, a marker of oxidative stress and key antioxidant enzymes viz., superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) in otherwise healthy 70 term newborns with NNH and 20 control newborns without jaundice. Jaundiced newborns had significantly lower MDA but higher SOD, catalase and GPx levels. Furthermore, plasma bilirubin showed significant negative correlation with MDA but positive correlation with antioxidant enzyme activities. It is concluded that NNH is associated with lower oxidative stress.

Oral rehydration for viral gastroenteritis in adults: a randomized, controlled trial of 3 solutions.

BACKGROUND: Pedialyte and Gatorade are advocated for the treatment of dehydration in viral gastroenteritis, but there is limited evidence to support their use. We examine the efficacy, safety, and palatability of Pedialyte, Gatorade, and a New Oral Rehydration Solution (N-ORS). This was a randomized double-blind trial conducted in an inpatient, community hospital. Seventy-five consecutive adult patients (male, 42; female, 33) admitted with viral gastroenteritis were randomized to receive Gatorade, Pedialyte, or N-ORS for 48 hours. A yogurt/rice diet was allowed ad libitum. Stool and urine output, electrolytes, fluid intake, body weight, hematocrit, and palatability of solutions were measured. RESULTS: Sixty completed the study. Stool frequency, consistency, and body weight improved (p < .001) in all 3 groups, but there was no difference between groups. Likewise, urine output, hematocrit, and correlations between fluid ingested, stool weight, or urine output were similar. At admission and 24 and 48 hours later, hypokalemia was observed in 7, 10, and 8 patients with Gatorade; 3, 2, and 1 with N-ORS; and 2, 2, and 1 with Pedialyte, respectively. Similarly, hyponatremia was observed in 6, 9, and 3 patients with Gatorade; 5, 3, and 4 with N-ORS; and 4, 5, and 4 with Pedialyte. Tastewise, Gatorade and N-ORS were rated higher (p < .05) than Pedialyte. Limitations were a smaller sample size and higher dropout (20%). CONCLUSIONS: Gatorade and N-ORS seem to be as effective as Pedialyte in correcting dehydration and in improving bowel symptoms. All 3 solutions were safe. Unlike other groups, hypokalemia persisted in the Gatorade group. Gatorade and N-ORS may be effective in the treatment of dehydration associated with mild viral gastroenteritis.

Role of malondialdehyde and superoxide dismutase in cataractogenesis.

Malondialdehyde (MDA) and superoxide dismutase (SOD) in lens and plasma of patients with age-related and myopic cataract were assessed. Higher levels of lens MDA in myopic patients showed a greater lipid peroxidation process than in patients with age-related cataract indicating higher oxidative stress in myopic lens. The difference in plasma MDA of myopic cataract and age-related cataract patients was not significant. The SOD level was lower in myopic patients than in patients with age-related cataract.

Identification of a novel pulmonary oedema producing toxin from Indian red scorpion (Mesobuthus tamulus) venom.

The experiments were conducted to identify the toxin that produces pulmonary oedema in Mesobuthus tamulus (BT) envenomed animals. Crude BT venom was subjected to Sephadex gel filtration (G-75) and the fractions were screened for optical density (OD), neurotoxicity (prolongation of compound action potential in frog sciatic nerve) and lethality. All these parameters exhibited a peak between 54-94 ml eluates. Fractions of this peak were pooled (SP) and loaded on to carboxymethyl cellulose column. The column was then eluted with increasing buffer concentrations at constant pH and temperature. Eluates were screened for neurotoxicity and OD. Four peaks of neurotoxic activity (T1-T4) were detected. T2 and T3 were lethal whereas T1 and T4 were non-lethal. T2 exhibited mainly neurotoxicity and failed to augment phenyldiguanide (PDG)-induced reflex response or to produce pulmonary oedema. T3 was having minimal neurotoxic actions but augmented PDG-reflex and produced pulmonary oedema. The effects of T3 persisted even after dialysis with 8 kDa cut-off filter but not those of T2. The T3 effects resembled toxic manifestations of BT venom and were blocked by aprotinin pre-treatment. T3 demonstrated a band at approximately 100 kDa in SDS-PAGE. The results demonstrate the presence of a lethal, high molecular weight, pulmonary oedema producing toxin in BT venom.

Right prosubiculum amyloid plaque density correlates with anosognosia in Alzheimer's disease.

BACKGROUND: Anosognosia is a common manifestation of Alzheimer's disease. There is an association between impaired awareness and frontal-executive cognitive deficits. Anosognosia is also correlated with decreased metabolism in the right hemisphere, particularly in frontal lobe regions. OBJECTIVE: To investigate pathological correlates of anosognosia in Alzheimer's disease. DESIGN: 41 subjects followed longitudinally in the University of Pittsburgh memory disorders clinic and with necropsy verified Alzheimer's disease were divided into two groups, based on previous clinical assessment: +Aware (n = 23) and -Aware (n = 18). A subset analysis matching subjects for dementia severity using mini-mental state examination scores was also carried out (13 +Aware; 13 -Aware). Histopathological data from necropsy brain tissue consisted of senile plaque (SP) and neurofibrillary tangle (NFT) counts (regional density) from four different brain regions in the right and left hemispheres: superior and middle frontal gyri (SMF), superior temporal isocortex (ST), the prosubiculum of the hippocampus (PRO), and the entorhinal cortex (EC). RESULTS: SP density was greater in the right PRO region of -Aware subjects (F = 6.54, p = 0.015) than +Aware subjects. Significant differences between SP or NFT density were not observed in any other regions. In the subset analysis matching for dementia severity, SP density was again greater in the right PRO region of -Aware subjects than in the other regions (F = 12.72, p = 0.002). CONCLUSIONS: Increased SP density in the right PRO region suggests that selective pathological involvement of this area contributes to awareness deficits in Alzheimer's disease. The putative role of the PRO in self appraisal may reflect its interconnections with other medial temporal and prefrontal regions.

A novel autophosphorylation mediated regulation of nitrite reductase in Candida utilis.

The assimilatory nitrite reductase catalyses the conversion of nitrite to ammonia. The enzyme from Candida utilis has been previously purified to homogeneity and shown to be a heterodimer consisting of 58 kDa and 66 kDa subunits. The enzyme has also been shown to be induced by nitrate and repressed by ammonium ions. The levels of nitrite reductase mRNA, its protein and the enzyme activity were modulated together indicating that the primary level of regulation of this enzyme existed at the transcriptional level. Here we report that the 58 kDa and 66 kDa subunits of the enzyme were differentially phosphorylated under the induced and repressed conditions, indicating a second level of regulation. The highly phosphorylated 66 kDa subunit was shown to be dephosphorylated by calf intestinal alkaline phosphatase. The enzymatic activity associated with the native enzyme also decreased due to the dephosphorylation. Each of the subunits could undergo autophosphorylation at serine/threonine residues as demonstrated by thin layer chromatography and recognition by antibodies to phosphoamino acids. The presence of similar phosphorylated subunits under in vivo conditions has also been demonstrated. A model has been proposed to explain the post-translational regulation of the enzyme.