Pyridylnidulin exerts anti-diabetic properties and improves non-alcoholic fatty liver disease in diet-induced obesity mice.

Introduction: Non-alcoholic fatty liver disease (NAFLD) is one of the metabolic disorders related to the pathophysiology of type 2 diabetes mellitus (T2DM). Therapeutic strategies are focused on the improvement of energy balance and lifestyle modification. Additionally, the derivative of the bioactive fungal metabolite is of interest to provide health benefits, especially in obese and pre-diabetic conditions. In our screening of anti-diabetic compounds from fungal metabolites and semisynthetic derivatives, a depsidone derivative, namely pyridylnidulin (PN), showed potent glucose uptake-inducing activity. The present study aimed to investigate the liver lipid metabolism and anti-diabetic properties of PN in diet-induced obesity mice. Methods: Male C57BL/6 mice were induced obesity and pre-diabetic conditions by dietary intervention with a high-fat diet (HFD) for 6 weeks. These obese mice were orally administered with PN (40 or 120 mg/kg), metformin (150 mg/kg), or vehicle for 4 weeks. Glucose tolerance, plasma adipocytokines, hepatic gene and protein expressions were assessed after treatment. Results: Improved glucose tolerance and reduced fasting blood glucose levels were found in the PN and metformin-treated mice. Additionally, hepatic triglyceride levels were consistent with the histopathological steatosis score regarding hepatocellular hypertrophy in the PN and metformin groups. The levels of plasma adipocytokines such as tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) were reduced in the PN (120 mg/kg) and metformin-treated mice. In addition, hepatic gene expression involved in lipid metabolism, including lipogenic enzymes was significantly reduced in the PN (120 mg/kg) and metformin-treated mice. The increased protein expression levels of phosphorylated AMP-activated protein kinase (p-AMPK) was also found in PN and metformin-treated mice. Discussion: Considering the increased p-AMPK protein expression levels in PN and metformin-treated mice were revealed as the underlying mechanisms to improve metabolic parameters. These results suggested that PN provided the health benefit to slow the progression of NAFLD and T2DM in obese and pre-diabetic conditions.

A systematic analysis of ultrastructural lesions in the Plasmodium coatneyi splenectomized rhesus macaque model of severe malaria.

Plasmodium falciparum remains one of the world's deadliest diseases and with ongoing concerns of evolving drug resistance, there is a need for continued refinement of the Plasmodium coatneyi infection model in macaques to study severe malaria. As such, the systemic ultrastructural lesions associated with P. coatneyi infection in splenectomized rhesus macaques was evaluated in 6 animals. Autopsy samples from multiple areas of the central nervous system (CNS), kidneys, heart, liver, and lungs of all 6 animals were processed for electron microscopy. A systematic analysis of the ultrastructural changes associated with the plasmodium was undertaken by multiple pathologists to ensure consensus. All tissues exhibited marked sequestration of infected red blood cells comprised either of cytoadherence to endothelium or rosette formation, associated with variable degrees of host cell damage in a range of tissues that in severe cases resulted in necrosis. This is the first complete systemic evaluation of ultrastructural tissue lesions in P. coatneyi-infected rhesus macaques, and the findings have important implications evaluating of the use of this model for the study of severe malaria caused by P. falciparum in humans.

Interactions of duck Tembusu virus with Aedes aegypti and Aedes albopictus mosquitoes: Vector competence and viral mutation.

Duck Tembusu virus (DTMUV) is an emerging flavivirus that causes severe disease in avian hosts, while also affecting mammalian hosts; however, information on viral interaction with mosquito vectors for mammalian hosts is limited. Vector competence of Aedes (Ae.) aegypti and Aedes albopictus mosquitoes for DTMUV were investigated. Both Aedes mosquito species were orally infected with DK/TH/CU-1 strain of Thai DTMUV and isolated DTMUV from BALB/c mouse. Genomes of the viruses isolated from hosts and vectors were analyzed and compared with the positive virus. Findings showed that both Aedes mosquito species could serve as vectors for DTMUV with minimum viral titer in blood meal of 106 TCID50/mL. After taking blood meal with viral titer at 107 TCID50/mL, vector competence of the mosquitoes was significantly different from the lower titer in both species. Both Aedes species did not support development of the isolated viruses from mouse. A point mutation of nucleotide and amino acid was found in all isolated DTMUV from Ae. aegypti saliva, while other viruses were similar to the positive virus. Our findings demonstrated that both Ae. aegypti and Ae. albopictus had potential to transmit the virus and play important roles in the viral transmission cycle in mammalian hosts, while viral mutation occurred in Ae. aegypti mosquitoes.

Pathogenesis of Thai duck Tembusu virus in BALB/c mice: Descending infection and neuroinvasive virulence.

Duck Tembusu virus (DTMUV) is an emerging flavivirus that causes systemic disease in an avian host. The predominant cluster of DTMUV circulating in Thailand was recently classified as cluster 2.1. The pathogenesis of this virus has been extensively studied in avian hosts but not in mammalian hosts. Six-week-old BALB/c mice were intracerebrally or subcutaneously inoculated with Thai DTMUV to examine clinical signs, pathological changes, viral load and virus distribution. Results demonstrated that the virus caused disease in BALB/c mice by the intracerebral inoculation route. Infected mice demonstrated both systemic and neurological symptoms. Pathological changes and virus distribution were observed in all tested organs. Viral load in the brain was significantly higher than in other organs (p < .05), and the virus caused acute death in BALB/c mice. The virus was disseminated in all parts of the body, but no virus shedding was recorded in saliva and faeces. Findings highlighted the potential of Thai DTMUV to transmit disease in mammalian hosts.

Liver- and Spleen-Specific Immune Responses in Experimental Leishmania martiniquensis Infection in BALB/c Mice.

Leishmania martiniquensis is a neglected cause of an emerging leishmaniasis in many countries, including France, Germany, Switzerland, the United States of America, Myanmar, and Thailand, with different clinical manifestations ranging from asymptomatic, cutaneous (CL), visceral (VL), and atypically disseminated CL and VL. The persistence of parasites and the recurrence of the disease after treatment are challenges in controlling the disease. To explore efficient prophylaxis and therapy, this study aimed to investigate infection outcome and organ-specific immune responses after inoculation with L. martiniquensis (MHOM/TH/2011/PG; 5 x 106 promastigotes) in BALB/c mice via intravenous and intraperitoneal routes. A quantitative PCR technique, targeting L. martiniquensis ITS1, was primarily established to estimate the parasite burden. We found that the infection in the liver resolved; however, persistent infection was observed in the spleen. Histopathology with Leishmania-specific immunostaining revealed efficient hepatic granuloma formation, while splenic disorganization with parasitized macrophages at different locations was demonstrated. The mRNA expression of Th1 cytokines (IFN-γ, TNF-α, IL-12p40) and iNOS in the liver and spleen was upregulated. In addition, high expression of IL-10 was observed in the spleen in the chronic phase, revealing a significant moderate correlation with the parasite persistence [r(12) = 0.72, P = 0.009]. Further clarification of the mechanisms of persistent infection and experimental infection in immunosuppressed murine models are warranted.

Epizootic of multi-centric, squamous cell carcinomas in populations of Indo-Pacific humpbacked dolphins Sousa chinensis in Thai waters.

Over the span of several years, 3 Indo-Pacific humpbacked dolphins died and were necropsied in Thailand. These 3 animals were all captive-bred at Oasis Sea World (Chanthaburi, Thailand), and displayed similar macroscopic progressive cutaneous lesions diagnosed as squamous cell carcinomas. In 2 of the 3 animals, necropsy revealed a severe fibrinosuppurative tracheitis and pneumonia secondary to metastasis of a cutaneous squamous cell carcinoma which extended from the head throughout the trunk and flippers. The tumors were characterized by coalescing botryoid masses with severe areas of cutaneous erosion, ulceration and necrohemorrhagic dermatitis. There was evidence of metastasis to the lungs and hilar lymph nodes. Necropsy of the third animal revealed similar progressive cutaneous squamous cell carcinomas but without evidence of metastasis. DNA molecular analysis of homogenized neoplastic tissue was conducted using polymerase chain reaction for both herpesvirus and papillomavirus in 2 of the 3 cases. In the first case, the tissues were positive for a herpesvirus alone, and this was phylogenetically classified as an alphaherpesvirus. This new herpesvirus has been tentatively named Sousa chinensis alphaherpesvirus. The second animal was negative for this novel herpesvirus and the third was not analyzed. In addition to the captive population, there is photographic evidence from 2 separate wild populations of Indo-Pacific humpbacked dolphins in the Gulf of Thailand, of a macroscopically identical proliferative and ulcerative process suspected to be squamous cell carcinomas.

Efficacy of bubaline fibrin glue on full-thickness pinch and punch skin grafting in a pig.

Fibrin glue, which is formed from the action of thrombin (a serine protease) on fibrinogen, has been developed for use as an adhesive to increase the success of skin graft surgery. The objective of this study was to evaluate if bubaline fibrin glue would promote skin graft survival in pigs. The grafting was divided into two steps. First, granulation wound preparation was performed in a healthy swine by creating four full-skin depth wounds (3 × 12 cm2) at the dorsal part of the loin area on each side. Second, pinch and punch skin grafting, where eight skin discs (0.6 cm diameter) were regularly placed (0.6 cm distance apart) in the granulation tissue bed of each wound, was performed 5 days later. The bubaline fibrin glue was added prior to application of the 16 skin graft discs in two of the wounds, while no glue was added to the other 16 skin graft discs in the other two wounds. The number of surviving graft pieces and histological examination was evaluated after 3, 7, and 14 days post-operation and compared by pairing between the control and the bubaline fibrin glue groups. The number of grafts that remained at 3 and 7 days post-operation and the number of new microvessels at 3 days post-operation were significantly higher ( p < 0.05) in the bubaline fibrin glue group than in the control group. However, there was no significant difference in the number of fibroblasts, the intensity of scarring and the intensity of inflammation between the two groups, except for the significantly lower intensity of inflammation at 7 days post-operation in the bubaline fibrin glue group. In conclusion, bubaline fibrin glue has the advantage of decreasing the skin graft loss by approximately 31.3-37.5% compared with the control group and also promotes angiogenesis.

Acute and subacute pulmonary toxicity caused by a single intratracheal instillation of colloidal silver nanoparticles in mice: pathobiological changes and metallothionein responses.

To study the acute and subacute pulmonary toxicity of colloidal silver nanoparticles (Ag-NPs), 0 or 100 ppm of Ag-NPs were instilled intratracheally in mice. Cellular and biochemical parameters in bronchoalveolar lavage fluid (BALF) and histological alterations were determined 1, 3, 7, 15, and 30 days after instillation. Ag-NPs induced moderate pulmonary inflammation and injury on BALF indices during the acute period; however, these changes gradually regressed in a time-dependent manner. Concomitant histopathological and laminin immunohistochemical findings generally correlated to BALF data. Superoxide dismutase and metallothionein expression occurred in particle-laden macrophages and alveolar epithelial cells, which correlated to lung lesions in mice treated with Ag-NPs. These findings suggest that instillation of Ag-NPs causes transient moderate acute lung inflammation and tissue damage. Oxidative stress may underlie the induction of injury to lung tissue. Moreover, the expression of metallothionein in tissues indicated the protective response to exposure to Ag-NPs.

Optimization of a Der p 2-based prophylactic DNA vaccine against house dust mite allergy.

DNA vaccines encoding allergens are promising immunotherapeutics to prevent or to treat allergy through induction of allergen-specific Th1 responses. Despite anti-allergy effects observed in small rodents, DNA-based vaccines are weak immunogens in primates and humans and particularly when administered by conventional injection. The goal of the present study was to improve the immunogenicity of a prophylactic vaccine encoding the major house dust mite allergen Der p 2. In this context, we evaluated the influence of different DNA backbones including notably intron and CpG enriched sequence, the DNA dose, the in vivo delivery by electroporation as well as the heterologous prime boost regimen on the vaccine efficiency. We found that a minimal allergen expression level threshold must be reached to induce the production of specific antibodies but beyond this limit, the intensity of the immune response was independent on the DNA dose and allergen expression. The in vivo DNA delivery by electroporation drastically enhanced the production of specific antibodies but not the IFNg secretion. Vaccination of naïve mice with DNA encoding Der p 2 delivered by electroporation even at very low dose (2µg) prevented the development of house dust mite allergy through Th1-skewed immune response characterized by the drastic reduction of allergen-specific IgE, IL-5 and lung inflammation together with the induction of strong specific IgG2a titers and IFNg secretion. CpG cassette in the DNA backbone does not play a critical role in the efficient prophylaxis. Finally, comparable protective immune responses were observed when using heterologous DNA prime/protein boost or homologous DNA prime/boost. Taken together, these data suggest that the potent Th1 response induced by DNA-based vaccine encoding allergens through electroporation provides the rationale for the evaluation of DNA encoding Der p 2 into HDM allergy clinical trials.

Short-term exposure of Nile Tilapia (Oreochromis niloticus) to mercury: histopathological changes, mercury bioaccumulation, and protective role of metallothioneins in different exposure routes.

To investigate effects of short-term mercury (Hg) exposure in tilapia (Oreochromis niloticus) including histopathological changes, Hg bioaccumulation, and protective role of metallothionein (MT) in different exposure routes, adult tilapias were intraperitoneally injected, orally intubated, or semistatically exposed to 0.5, 1, 2, 5 µg/g mercuric chloride. Histopathology, autometallography (AMG), inductive coupled plasma-atomic emission spectrometry (ICP-AES), and MT immunohistochemistry were determined at 0, 3, 6, 9, 12, and 15 days postexposure. Microscopic lesions were observed in the kidney, hepatopancreas, spleen, and intestine. AMG positive grains were found in renal tubule epithelium, melanomacrophage centers (MMCs), and intestinal epithelium of treated tilapias. Hg concentrations measured by ICP-AES in abdominal visceral organs were significantly higher than in other organs. All exposure routes caused lesions of increasing severity and Hg accumulations in a dose-dependent manner. Semistatic groups produced the highest intensity of lesions, AMG positive staining, as well as total Hg concentrations. Positive MT expression in renal tubule epithelium, pancreatic acini, and splenic MMCs was observed only in semistatic groups. The semistatic exposure route demonstrated the most significant microscopic lesions, Hg bioaccumulation, and MT expression.

An evaluation of acute toxicity of colloidal silver nanoparticles.

Tests for acute oral toxicity, eye irritation, corrosion and dermal toxicity of colloidal silver nanoparticles (AgNPs) were conducted in laboratory animals following OECD guidelines. Oral administration of AgNPs at a limited dose of 5,000 mg/kg produced neither mortality nor acute toxic signs throughout the observation period. Percentage of body weight gain of the mice showed no significant difference between control and treatment groups. In the hematological analysis, there was no significant difference between mice treated with AgNPs and controls. Blood chemistry analysis also showed no differences in any of the parameter examined. There was neither any gross lesion nor histopathological change observed in various organs. The results indicated that the LD(50) of colloidal AgNPs is greater than 5,000 mg/kg body weight. In acute eye irritation and corrosion study, no mortality and toxic signs were observed when various doses of colloidal AgNPs were instilled in guinea pig eyes during 72 hr observation period. However, the instillation of AgNPs at 5,000 ppm produced transient eye irritation during early 24 hr observation time. No any gross abnormality was noted in the skins of the guinea pigs exposed to various doses of colloidal AgNPs. In addition, no significant AgNPs exposure relating to dermal tissue changes was observed microscopically. In summary, these findings of all toxicity tests in this study suggest that colloidal AgNPs could be relatively safe when administered to oral, eye and skin of the animal models for short periods of time.

Ultrastructural changes of the air-blood barrier in mice after intratracheal instillation of lipopolysaccharide and ultrafine carbon black particles.

Epidemiological studies have indicated associations between exposure to increased concentrations of ambient ultrafine particles and adverse health effects especially in susceptible individuals. To ellucidate the mechanisms underlying the findings from epidemiological studies, mice pretreated with lipopolysaccharide (LPS) (acute lung injury model) were intratracheally instilled with ultrafine carbon black particles (UFCB), and the air-blood barrier was observed to examine the translocation pathway of UFCB from the lung into the systemic circulation. In addition, lung toxicity induced by the intratracheal instillation of LPS and UFCB was studied with the use of electron microscope. LPS treatment induced acute inflammatory changes with increased number of activated macrophages and neutrophils in the degenerated alveolar walls. UFCB were demonstrated on or in the denuded basement membrane in the air-blood barrier; these findings were associated with edematous changes and fragmentation of the cytoplasms of alveolar epithelial cell type 1, and the damages of alveolar epithelial cell type 1 were frequently observed in the close vicinity of the clumps of UFCB. These findings suggest that translocation of the exposed ultrafine particles may be enhanced in the lung tissues with acute inflammatory changes.

Role of metallothionein in lung inflammation induced by ozone exposure in mice.

Metallothionein (MT) is a free radical scavenger induced by inflammatory stimuli; however, its roles in inflammation have not been fully investigated. In the present study, we genetically determined the role of MT in ozone (O(3))-induced lung inflammation using MT-I/II null (-/-) mice. Subacute (65 h) exposure to O(3) (0.3 ppm) induced lung inflammation and enhanced vascular permeability, which was significantly greater in MT(-/-) than in corresponding wild-type mice. Electron microscopically, O(3) exposure induced vacuolar degeneration of pulmonary endothelial and epithelial cells, and interstitial edema with focal loss of the basement membrane, which was more prominent in MT(-/-) than in wild-type mice. O(3) -induced lung expression of interleukin-6 was significantly greater in MT(-/-) than in wild-type mice; however, lung expression of the chemokines examined was comparable in both genotypes of mice in the presence of O(3). Following O(3) exposure, the formation of oxidative stress-related molecules/adducts, such as heme oxidase-1, inducible nitric oxide synthase, 8-hydroxy-2'-deoxyguanosine, and nitrotyrosine, in the lung was significantly greater in MT(-/-) than in wild-type mice. Collectively, MT protects against O(3)-induced lung inflammation, at least partly, via the regulation of pulmonary endothelial and epithelial integrity and its antioxidative property.

Translocation pathway of the intratracheally instilled ultrafine particles from the lung into the blood circulation in the mouse.

Recently, it has been demonstrated that ultrafine particles (UFPs) are able to translocate from the lung into the systemic circulation. Precise mechanisms of the anatomical translocation (crossing the air-blood barrier) of inhaled UFPs at the alveolar wall are not fully understood. In this study, we examined the translocation pathway of the intratracheally instilled ultrafine carbon black (UFCB) from the lung into the blood circulation in mouse. Electron microscopy demonstrated accumulation of intratracheally instilled UFCB in the large-sized gaps developing between the cytoplasmic processes of the alveolar epithelial cells, possibly as a result of shrinkage of cytoplasm, by receiving stimulus/signals generated and released following UFCB attachment on the alveolar epithelial cells. Occasional penetration of the accumulated UFCB into the alveolar basement membrane, exposing to the air space, was observed at the gap. These results suggest that inhaled UFPs may, in part, pass the air-blood barrier through the large-sized gap formed between the alveolar epithelial cells.

Acute and subacute pulmonary toxicity of low dose of ultrafine colloidal silica particles in mice after intratracheal instillation.

To study the acute and subacute lung toxicity of low dose of ultrafine colloidal silica particles (UFCSs), mice were intratracheally instilled with 0, 0.3, 3, 10, 30 or 100 microg of UFCSs. Cellular and biochemical parameters in bronchoalveolar lavage fluid (BALF), histological alteration and the body weight were determined at 3 days after instillation. Exposure to 30 or 100 microg of UFCSs produced moderate to severe pulmonary inflammation and tissue injury. To investigate the time response, mice were instilled with 30 microg of UFCSs and sacrificed at intervals from 1 to 30 days post-exposure. UFCSs induced moderate pulmonary inflammation and injury on BALF indices at acute period; however, these changes gradually regressed until recovery during the experiment. Concomitant histopathological and laminin immunohistochemical findings generally correlated to BALF data. TUNEL analyses in UFCSs-treated animals showed a significant increase of the apoptotic index in lung parenchyma at all observation times. 8-OHdG expression occurred in lung epithelial cells and activated macrophages, which correlated to lung lesions in UFCSs-treated mice. These findings suggest that instillation of a small dose of UFCSs causes transient acute moderate lung inflammation and tissue damage. Oxidative stress and apoptosis may underlie the lung tissue injury induction.

Acute pulmonary toxicity caused by exposure to colloidal silica: particle size dependent pathological changes in mice.

To compare the pulmonary toxicity between ultrafine colloidal silica particles (UFCSs) and fine colloidal silica particles (FCSs), mice were intratracheally instilled with 3 mg of 14 nm UFCSs and 230 nm FCSs and pathologically examined from 30 minutes to 24 hour postexposure. Histopathologically, lungs exposed to both sizes of particles showed bronchiolar degeneration and necrosis, neutrophilic inflammation in alveoli with alveolar type II cell swelling and particle-laden alveolar macrophage accumulation. UFCSs, however, induced extensive alveolar hemorrhage compared to FCSs from 30 minutes onwards. UFCSs also caused more severe bronchiolar epithelial cell necrosis and neutrophil influx in alveoli than FCSs at 12 and 24 hours postexposure. Laminin positive immunolabellings in basement membranes of bronchioles and alveoli of UFCSs treated animals was weaker than those of FCSs-treated animals in all observation times. Electron microscopy demonstrated UFCSs and FCSs on bronchiolar and alveolar wall surface as well as in the cytoplasm of alveolar epithelial cells, alveolar macrophages and neutrophils. Type I alveolar epithelial cell erosion with basement membrane damage in UFCSs treated animals was more severe than those in FCSs-treated animals. At 12 and 24 hours postexposure, bronchiolar epithelial cells in UFCSs-treated animals showed more intense vacuolation and necrosis compared to FCSs-treated animals. These findings suggest that UFCSs have greater ability to induce lung inflammation and tissue damages than FCSs.