Brain Infection by Hepatitis E Virus Probably via Damage of the Blood-Brain Barrier Due to Alterations of Tight Junction Proteins.

Extrahepatic injury, particularly neurologic dysfunctions such as Guillain-Barré syndrome, neurologic amyotrophy, and encephalitis/meningoencephalitis/myositis were associated with HEV infection, which was supported by both clinical and laboratory studies. Thus, it is crucial to figure out how the virus invades into the central nervous system (CNS). In this study, CNS lesions were determined in rabbits and Mongolian gerbils inoculated with genotype 4 HEV. Junctional proteins were detected in HEV infected primary human brain microvascular cells (HBMVCs). Viral encephalitis associated perivascular cuffs of lymphocytes and microglial nodules were observed in HEV infected rabbits. Both positive- and negative-strand of HEV RNA was detected in brain and spinal cord in rabbits intraperitoneally infected with HEV at 28 dpi (days postinoculation), but not in rabbits gavaged with HEV. HEV ORF2 protein was further examined in both brain and spinal cord sections of infected rabbits, with positive signals located mainly in neural cells and perivascular areas. Ultrastructural study showed thickened and reduplicated basement membranes of capillary endothelium in HEV RNA positive brain tissues. In vitro study showed loss of tight junction proteins including Claudin5, Occludin, and ZO-1 (zonula occludens-1) in HBMVCs inoculated with HEV for 48 h. These findings indicated that disruption of the blood-brain barrier (BBB) might be potential mechanisms of HEV invasion into the CNS. It provides new insights to further study HEV associated neurologic disorders and will be helpful for seeking potential therapeutics for HEV infection in the future.

Oral cancer via the bargain bin: The risk of oral cancer associated with a smokeless tobacco product (Naswar).

In the wake of smokeless tobacco (SLT) being advocated as a mean of tobacco harm reduction, it is pertinent to establish individual health risks associated with each SLT product. This case-control study was aimed at assessing the risk of oral cancer associated with a smokeless tobacco product (Naswar). The study was conducted from September 2014 till May 2015 in Khyber Pakhtunkhwa, Pakistan. Exposure and covariate information was collected through a structured questionnaire. Conditional logistic regression was used to calculate odds ratios (OR) along with their 95% confidence intervals (CI). 84 oral cancer cases (62% males) and 174 age- and sex-matched controls were recruited. Ever users of Naswar had more than a 20-fold higher risk of oral cancer compared to never-users (OR 21.2, 95% CI 8.4-53.8). Females had a higher risk of oral cancer with the use of Naswar (OR 29.0, 95% CI 5.4-153.9) as compared to males (OR 21.0, 95% CI 6.1-72.1). Based on this result, 68% (men) and 38% (women) of the oral cancer burden in Pakistan is attributable to Naswar. The risk estimates observed in this study are comparable to risk estimates reported by previous studies on other forms of SLT use and the risk of oral cancer in Pakistan. The exposure-response relationship also supports a strong role of Naswar in the etiology of oral cancer in Pakistan. Although still requiring further validation through independent studies, these findings may be used for smokeless tobacco control in countries where Naswar use is common.

Hepatitis E Virus Genotype 4 Sequences Detected in Sewage from Treatment Plants of China.

The aim of this study was to investigate the occurrence of hepatitis E virus (HEV) in sewage samples in Shen Zhen, China. Sewage samples were collected from 152 sewage plants including livestock sewage, domestic sewage and treated sewage from May to July of 2015. Two of 152 samples were HEV positive (1.32%) from the livestock sewage plants. Partial ORF2 fragments of HEV were sequenced and a phylogenetic tree was constructed using MEGA5.1. Blast and phylogenetic analyses showed that both of these two sequences belonged to HEV Genotype 4. To the best of our knowledge, this is the first study on the molecular characterization of HEV in wastewater in China and the first time to detect Genotype 4 in the sewage. Results from this study indicate that the possibilities of sporadic infections of HEV should be emphasized because virus still has the possibility to be circulating in the sewage in China.

Antimicrobial activity and safety evaluation of peptides isolated from the hemoglobin of chickens.

BACKGROUND: Hemoglobin is a rich source of biological peptides. As a byproduct and even wastewater of poultry-slaughtering facilities, chicken blood is one of the most abundant source of hemoglobin. RESULTS: In this study, the chicken hemoglobin antimicrobial peptides (CHAP) were isolated and the antimicrobial and bactericidal activities were tested by the agarose diffusion assay, minimum inhibitory concentration (MIC) analysis, minimal bactericidal concentration (MBC) analysis, and time-dependent inhibitory and bactericidal assays. The results demonstrated that CHAP had potent and rapid antimicrobial activity against 19 bacterial strains, including 9 multidrug-resistant bacterial strains. Bacterial biofilm and NaCl permeability assays, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were further performed to detect the mechanism of its antimicrobial effect. Additionally, CHAP showed low hemolytic activity, embryo toxicity, and high stability in different temperatures and animal plasma. CONCLUSION: CHAP may have great potential for expanding production and development value in animal medication, the breeding industry and environment protection.

Antigen detection and apoptosis in Mongolian gerbil's kidney experimentally intraperitoneally infected by swine hepatitis E virus.

We examined the effect of hepatitis E virus (HEV) on the renal tissue pathogenesis, morphological damages and related molecular mechanisms following swine HEV suspension intraperitoneally inoculation in Mongolian gerbils. The microscopic and ultramicroscopic analyses of kidney tissue structure were carried out at different points after inoculation of HEV. The immunohistochemistry, real-time PCR and Western blot were performed to explore the molecular mechanisms associated with HEV presence in the renal tissues. Real-time PCR revealed that the copies of HEV RNA in the kidney were detected at 7 dpi, and peaked at 14 dpi at a concentration was 7.18 logs g(-1), with detection of HEV ORF2 antigen by immunohistochemistry. Hematoxylin and eosin (HE) staining showed pathological lesions including glomerular atrophy, degeneration, edema and necrosis of renal tubular epithelial cells and Mallory and Sirius red staining indicated the presence of collagen fibers and fibrosis in kidney tissues of inoculated gerbils. Ultrastructural studies of basal membrane of renal tubules demonstrated the rough and uneven with mitochondria swelling and vacuolation in the tissues of HEV inoculated animals. Similarly, significantly higher number of (TUNEL)-positive cells were seen in renal tubule tissues compared to control group. Moreover, immuno histochemical results indicated that significant increase expression of the B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax), FAS and Caspase-3 in HEV inoculated Mongolian gerbils at each time points. Relative mRNA expression by real-time PCR revealed a significantly higher (P<0.05) mRNA level of BAX, Bcl-2 and caspase-3 transcription in HEV inoculated Mongolian gerbils. Our results demonstrates that activation of mitochondria and Caspase-3 protease might be induced the apoptosis which subsequently cause the necrosis and cell death of renal epithelial cells during acute phase of HEV infection in HEV inoculated Mongolian gerbils.

Effect of Oral Pathway on Charged Particles Deposition in Human Bronchial Airways.

BACKGROUND: In vitro studies to investigate the effect of charged particle deposition in the oral pathway of human adults have demonstrated substantial increases in deposition due to an induced charge effect. In the current study, charged particle deposition in the oral pathway was incorporated in the stochastic human airway generation model IDEAL (Inhalation, Deposition, and Exhalation of Aerosols in the Lung) to quantify their effect on bronchial airways deposition. METHODS: Calculation of increased oral deposition due to charged particles was performed by a modified version of IDEAL for oral pathway, whereas deposition in the bronchial airways was carried out by the already employed efficiency equation. Deposition calculations were performed for 3, 4.5, and 6 µm particles at flow rates of 15 and 30 L/min. RESULTS: The enhancement in deposition is found to be 40 times higher in oral pathway and 6 times higher in bronchial airways for 3 µm size particles carrying 2500 elementary charges. For particles larger than 3 µm, deposition by impaction dominates over deposition by particle charges, and hence higher deposition in oral pathway is observed primarily due to impaction. As a consequence of this increased oral deposition, bronchial airways deposition decreases. CONCLUSION: By controlling breathing, aerosol properties, and electrostatic charge, targeted deposition in the human airways can be improved. Hence, charged particles can therefore be utilized to give better control on regional drug delivery in the lungs or to filter out toxic constituents.

Case Report Associated with Aspergillosis and Hepatitis E Virus Coinfection in Himalayan Griffons.

This study involved a death which occurred in four Himalayan griffons housed in Beijing zoo, China. Based on pathogen identification and the pathological changes observed, we did characterize the fungi and Hepatitis E virus (HEV) in four dead Himalayan griffons. Pathological changes were severe. Membranous-like material was observed on the surface of the internal organs. Spleen was necrotic. Focal lymphocyte infiltration in the liver and many sunflower-like fungi nodules were evident in the tissues, especially in the kidney. PCR was used to identify the pathogen. Based on the 18SrRNA genomic sequence of known fungi, the results confirmed that all four dead Himalayan griffons were infected with Aspergillus. At the same time the detection of HEV also showed positive results. To the best of our knowledge, this work appears to be the first report of concurrent presence of Aspergillosis and Hepatitis E virus in rare avian species.

Effect of swine hepatitis E virus on the livers of experimentally infected Mongolian gerbils by swine hepatitis E virus.

Previous studies have shown that hepatitis E virus (HEV) can be transmitted between rats, pigs, cattle, rabbits, chicken, cats, and deer. Because wild and domestic rodents have anti-HEV antibodies, they are considered potential reservoirs of HEV. In the current study, Mongolian gerbils were experimentally infected with swine hepatitis E virus and the effects of this infection were investigated. After inoculation with HEV, the liver-to-body weight ratio increased at 7 dpi. Mongolian gerbils demonstrated significant increase (p<0.05) in Aspartate Transaminase (AST), alanine transaminase (ALT) and total bilirubin (T-BIL) concentrations in the sera, and HEV IgG was detected at 21 days post-inoculation (dpi). Real-time PCR revealed that the copies of HEV RNA in the liver were detected at 7 dpi, and peaked at 28 dpi at a concentration of 7.73 logs g(-1). Using both light and electron microscopy, hepatic lesions were observed in the HEV inoculated animals. In the experimental group, characteristic viral hepatitis lesions were prominent in the liver. HEV antigen was detected in the liver by immunohistochemistry, and HEV ORF3 antigen was detectable in liver by Western blot. These results clearly demonstrate that viral load of HEV in livers was dynamic, and ultrastructural hepatic injury in HEV infected Mongolian gerbils and anti-HEV IgG positive seroconversion were observed during infection.

Fatal disease associated with Swine Hepatitis E virus and Porcine circovirus 2 co-infection in four weaned pigs in China.

BACKGROUND: In recent decades, Porcine circovirus 2 (PCV2) infection has been recognized as the causative agent of postweaning multisystemic wasting syndrome, and has become a threat to the swine industry. Hepatitis E virus (HEV) is another high prevalent pathogen in swine in many regions of the world. PCV2 and HEV are both highly prevalent in pig farms in China. CASE PRESENTATION: In this study, we characterized the HEV and PCV2 co-infection in 2-3 month-old piglets, based on pathogen identification and the pathological changes observed, in Hebei Province, China. The pathological changes were severe, and general hyperemia, hemorrhage, inflammatory cell infiltration, and necrosis were evident in the tissues of dead swine. PCR was used to identify the pathogen and we tested for eight viruses (HEV, Porcine reproductive and respiratory syndrome virus, PCV2, Classical swine fever virus, Porcine epidemic diarrhea virus, Transmissible gastroenteritis coronavirus, Porcine parvovirus and Pseudorabies virus) that are prevalent in Chinese pig farms. The livers, kidneys, spleens, and other organs of the necropsied swine were positive for HEV and/or PCV2. Immunohistochemical staining showed HEV- and PCV2-antigen-positive signals in the livers, kidneys, lungs, lymph nodes, and intestine. CONCLUSION: HEV and PCV2 co-infection in piglets was detected in four out of seven dead pigs from two pig farms in Hebei, China, producing severe pathological changes. The natural co-infection of HEV and PCV2 in pigs in China has rarely been reported. We speculate that co-infection with PCV2 and HEV may bring some negative effect on pig production and recommend that more attention should be paid to this phenomenon.

FREQUENCY OF LINGUAL NERVE INJURY IN MANDIBULAR THIRD MOLAR EXTRACTION: A COMPARISON OF TWO SURGICAL TECHNIQUES.

BACKGROUND: Surgical removal of impacted mandibular third molar is associated with a number of complications including postoperative bleeding, dry socket, postoperative infection, and injury to regional nerves. Lingual nerve damage is one of the main complications. To prevent this complication different techniques had been used. Lingual flap reflection is one of these procedures in which lingual soft tissue is reflected and retracted deliberately, the nerve is identified and is kept out of the surgical field. The objective of this study was to evaluate a surgical technique for third molar removal which is associated with minimum frequency of lingual nerve damage. METHODS: A randomized controlled trial was performed. A total of 380 patients with impacted mandibular third molars were included in this study. Each patient was allotted randomly by blocked randomization to group A where procedure was performed by reflection and retraction of lingual flap in addition to buccal flap and group B where procedure was performed by retraction of buccal flap only. RESULTS: Lingual nerve damage occurred in 8.94% in Group A in which lingual flap retraction was performed but damage was reversible. In group B, 2.63% lingual nerve damage was observed and nature of damage was permanent. The difference was statistically significant (p=0.008). CONCLUSIONS: Lingual flap retraction poses 3.4 times increased risk of lingual nerve damage during extraction of mandibular third molar when lingual flap is retracted but the nature of damage is reversible.

Acute testicular toxicity induced by melamine alone or a mixture of melamine and cyanuric acid in mice.

Eight-week-old male Kunming mice were administered either melamine (MA, 30, 140, or 700 mg/kg/day), a melamine and cyanuric acid mixture (MC, each at 15, 70, or 350 mg/kg/day), or vehicle (control) for 3 consecutive days. Testicular toxicity was evaluated on days 1 and 5 after the final exposure. The testicular and epididymal weights and serum testosterone level were significantly decreased in the highest MC group (350 mg/kg/day). Histopathologically, both MA and MC caused obvious lesions in the testis and epididymis, with significant increases in sperm abnormalities. By TEM, the blood-testis barrier was damaged dose dependently. TUNEL staining showed that both MA and MC induced increases in germ cell apoptosis. The Sertoli cell vimentin was collapsed in the treated animals as detected by immunohistochemical staining and Western blotting. This study demonstrated that both MA and MC treatments could disrupt the blood-testis barrier and cause a clear testicular toxicity.

Detection and localization of rabbit hepatitis e virus and antigen in systemic tissues from experimentally intraperitoneally infected rabbits.

Rabbit hepatitis E virus (HEV) is a novel genotype of HEV, and is considered to pose a risk of zoonotic transmission. Research into the systemic distribution of rabbit HEV in rabbits during different periods of infection has rarely been reported. To better understand this virus, we infected rabbits with second-passage rabbit HEV via an intraperitoneal route. After inoculation, the infection showed two types, temporary and constant infection. The detection of HEV RNA in the feces varied with time, and serum antigen correlated with fecal HEV RNA. Viremia only appeared 72 days after inoculation. The rabbits remained antibody negative throughout the experimental period. When HEV was localized, several organs besides the liver were HEV RNA positive. Tissue antigen was observed immunohistochemically in the different cells of various organs, especially in parts of the small intestine and the characteristic rabbit gut-associated lymphoid tissue. These data provide valuable information for future research into the pathogenesis of HEV.

Exposure assessment and associated lung deposition calculations for vehicular exhaust in four metropolitan cities of Pakistan.

Ambient aerosol concentrations along the roadside of metropolitan cities of Pakistan were measured using a Grimm 1.109 dust monitor. Considering the high ambient aerosol concentrations, regional lung deposition of aerosol particles in the human respiratory tract was calculated to assess extent of exposure. Lung deposition was computed in terms of mass concentration and the associated surface area for 12 male traffic wardens using the latest version of the stochastic lung deposition code Inhalation, Deposition, and Exhalation of Aerosols in the Lung. The results have revealed 4 to 10 times higher concentrations than recommended by WHO guidelines. The deposition results derived from the model disclose that extrathoracic deposition is in the range of 22 to 28 % with total lung deposition ranging from 40 to 44 % for the scanned particle window of 0.25-10 µm. Considering an average 8-h shift per day and an average breathing rate of 1.3 m(3) h(-1), it is approximated that in a worker, up to 1.6 mg of inhalable particle mass can deposit per day.

Comparison of stochastic lung deposition fractions with experimental data.

Deposition fractions of inhaled particles predicted by different computational models vary with respect to physical and biological factors and mathematical modeling techniques. These models must be validated by comparison with available experimental data. Experimental data supplied by different deposition studies with surrogate airway models or lung casts were used in this study to evaluate the stochastic deposition model Inhalation, Deposition and Exhalation of Aerosols in the Lung at the airway generation level. Furthermore, different analytical equations derived for the three major deposition mechanisms, diffusion, impaction, and sedimentation, were applied to different cast or airway models to quantify their effect on calculated particle deposition fractions. The experimental results for ultrafine particles (0.00175 and 0.01) were found to be in close agreement with the stochastic model predictions; however, for coarse particles (3 and 8 µm), experimental deposition fractions became higher with increasing flow rate. An overall fair agreement among the calculated deposition fractions for the different cast geometries was found. However, alternative deposition equations resulted in up to 300% variation in predicted deposition fractions, although all equations predicted the same trends as functions of particle diameter and breathing conditions. From this comparative study, it can be concluded that structural differences in lung morphologies among different individuals are responsible for the apparent variability in particle deposition in each generation. The use of different deposition equations yields varying deposition results caused primarily by (i) different lung morphometries employed in their derivation and the choice of the central bifurcation zone geometry, (ii) the assumption of specific flow profiles, and (iii) different methods used in the derivation of these equations.

Frequency of unerupted mandibular third molar in mandibular angle fractures.

BACKGROUND: Fractures of the mandibular angle are common and comprise 31% of all mandibular fractures. Multiple recent studies report a 2-3 fold increased risk for mandibular angle fractures when un-erupted mandibular third molars are present. The objective of this study was to assess the frequency of un-erupted mandibular third molar in mandibular angle fractures. METHODS: This cross-sectional study was conducted at the Department of Oral and Maxillofacial Surgery, Ayub Medical College, Abbottabad from April to October 2009. One hundred and two patients were included both from the outdoor and ward on consecutive non-probability sampling base. Data were recorded on a structured Performa and analysed using SPSS-16. RESULTS: A hemi-mandible containing un-erupted mandibular third molar was seen to have a 1.41 times the risk of mandibular angle fracture then a hemi-mandible containing an erupted mandibular third molar. CONCLUSION: The presence of unerupted mandibular third molar is associated with an increased risk for mandibular angle fracture.

Vitamin D deficiency and rickets in the Eastern Province of Saudi Arabia.

BACKGROUND: Nutritional rickets remains prevalent in many developing countries, despite the availability of ample sunlight. The aim of this study was to investigate the clinical features and chemical pathology in a group of children with rickets and to compare them with a control group. SUBJECTS AND METHODS: In a case-control study over a 1-year period (March 2004 to February 2005), children clinically diagnosed with rickets (n=61) were age- and sex-matched with controls (n=58). In addition to routine chemical pathology, 25 (OH) vitamin D3 and parathormone (PTH) were determined. Controls were children without clinical rickets attending hospital for other blood investigations. RESULTS: The mean age of children with rickets was 14.8 mths and of controls was 16.5 mths. Mean (SD) body mass index of the children with rickets [16.8 (1.86)] was not significantly different from that of the controls [17.02 (3.16)]. Mean (SD) head circumference of rachitic children [45.41 (3.64) cm] was greater than that of controls [44.39 (5.07) cm, p=0.03]. Eighty per cent of the children with rickets were breastfed compared with 67% of controls. Thirty per cent of children with rickets were hypocalcaemic vs <7% of controls, 89% had phosphorus values <1.5 mmol/L vs 34.5% of controls and 75% had alkaline phosphatise levels >500 IU/L vs 28% of controls. Seventy-five per cent of children with rickets had serum 25 (OH) D3 <20 nmol/L vs 25% of controls. Mean (SD) PTH level was 23.59 (19.03) pmol/L in the rachitic group and 1.9 (1.05) pmol/L in controls (p<0.05). Lack of exposure to sunlight was recorded in 90% of the children with rickets and in 37% of the controls. CONCLUSION: Apparently healthy children living in areas where rickets is prevalent have risk factors for rickets and a small proportion will have evidence of biochemical rickets.