A nitroalkene derivative of salicylate alleviates diet-induced obesity by activating creatine metabolism and non-shivering thermogenesis.

Obesity-related type II diabetes (diabesity) has increased global morbidity and mortality dramatically. Previously, the ancient drug salicylate demonstrated promise for the treatment of type II diabetes, but its clinical use was precluded due to high dose requirements. In this study, we present a nitroalkene derivative of salicylate, 5-(2-nitroethenyl)salicylic acid (SANA), a molecule with unprecedented beneficial effects in diet-induced obesity (DIO). SANA reduces DIO, liver steatosis and insulin resistance at doses up to 40 times lower than salicylate. Mechanistically, SANA stimulated mitochondrial respiration and increased creatine-dependent energy expenditure in adipose tissue. Indeed, depletion of creatine resulted in the loss of SANA action. Moreover, we found that SANA binds to creatine kinases CKMT1/2, and downregulation CKMT1 interferes with the effect of SANA in vivo. Together, these data demonstrate that SANA is a first-in-class activator of creatine-dependent energy expenditure and thermogenesis in adipose tissue and emerges as a candidate for the treatment of diabesity.

Trypanosoma cruzi Isolates Naturally Adapted to Congenital Transmission Display a Unique Strategy of Transplacental Passage.

Chagas disease is mainly transmitted by vertical transmission (VT) in nonendemic areas and in endemic areas where vector control programs have been successful. For the present study, we isolated natural Trypanosoma cruzi strains vertically transmitted through three generations and proceeded to study their molecular mechanism of VT using mice. No parasitemia was detected in immunocompetent mice, but the parasites were able to induce an immune response and colonize different organs. VT experiments revealed that infection with different strains did not affect mating, pregnancy, or resorption, but despite low parasitemia, VT strains reached the placenta and resulted in higher vertical transmission rates than strains of either moderate or high virulence. While the virulent strain modulated more than 2,500 placental genes, VT strains modulated 150, and only 29 genes are shared between them. VT strains downregulated genes associated with cell division and replication and upregulated immunomodulatory genes, leading to anti-inflammatory responses and tolerance. The virulent strain stimulated a strong proinflammatory immune response, and this molecular footprint correlated with histopathological analyses. We describe a unique placental response regarding the passage of T. cruzi VT isolates across the maternal-fetal interphase, challenging the current knowledge derived mainly from studies of laboratory-adapted or highly virulent strains. IMPORTANCE The main findings of this study are that we determined that there are Trypanosoma cruzi strains adapted to transplacental transmission and completely different from the commonly used laboratory reference strains. This implies a specific strategy for the vertical transmission of Chagas disease. It is impressive that the strains specialized for vertical transmission modify the gene expression of the placenta in a totally different way than the reference strains. In addition, we describe isolates of T. cruzi that cannot be transmitted transplacentally. Taken together, these results open up new insights into the molecular mechanisms of this insect vector-independent transmission form.

Jejunum-derived NF-κB reporter organoids as 3D models for the study of TNF-alpha-induced inflammation.

Inflammation is an important process for epithelial barrier protection but when uncontrolled, it can also lead to tissue damage. The nuclear factor-kappa light chain enhancer of activated B cells (NF-κB) signaling pathway is particularly relevant in the intestine, as it seems to play a dual role. Whereas NF-κB protects intestinal epithelium against various noxious stimuli, the same pathway mediates intestinal inflammatory diseases by inducing pro-inflammatory gene expression. The availability of appropriate in vitro models of the intestinal epithelium is crucial for further understanding the contribution of NF-κB in physiological and pathological processes and advancing in the development of drugs and therapies against gut diseases. Here we established, characterized, and validated three-dimensional cultures of intestinal organoids obtained from biopsies of NF-κB-RE-Luc mice. The NF-κB-RE-Luc intestinal organoids derived from different intestine regions recreated the cellular composition of the tissue and showed a reporter responsiveness similar to the in vivo murine model. When stimulated with TNF-α, jejunum-derived NF-κB-RE-Luc-reporter organoids, provided a useful model to evaluate the anti-inflammatory effects of natural and synthetic compounds. These reporter organoids are valuable tools to explore the epithelial TNF-α-induced NF-κB contribution in the small intestine, being a reliable alternative method while helping to reduce the use of laboratory animals for experimentation.

Central nervous system lesions caused by canine distemper virus in 4 vaccinated dogs.

We examined the cerebellum and cerebrum of 4 vaccinated dogs, 3-60-mo-old, that displayed clinical signs of canine distemper virus (CDV) infection, and died 7-40 d after developing neurologic signs. The main histologic lesions were demyelination, gliosis, meningitis, perivascular lymphocytic cuffing, and inclusion bodies. These lesions were similar in all 4 cases regardless of the time since vaccination, except that meningoencephalitis and gliosis were subacute in 3 dogs and chronic in 1 dog. However, these differences did not appear to be related to their vaccination status. Immunohistologically, a CDV-positive immunoreaction was seen mainly in astrocytes, neurons and their axons, lymphocytes around and in the blood vessels of the pia mater and choroid plexus, ependymal cells of each ventricle, and the cells of the choroid plexus. The histologic and immunohistologic changes were similar in the cerebellum and cerebrum. The genetic characterization of the virus strains in 2 of these naturally occurring canine distemper cases confirmed that they were South American wild-type strains (Kiki and Uy251) belonging to the EU1/SA1 lineage. These strains are not included in the commercial CDV vaccines available in Uruguay.

Intramural Vascular Edema in the Brain of Goats With Clostridium perfringens Type D Enterotoxemia.

Enterotoxemia caused by Clostridium perfringens type D is an important disease of sheep and goats with a worldwide distribution. Cerebral microangiopathy is considered pathognomonic for ovine enterotoxemia and is seen in most cases of the disorder in sheep. However, these lesions are poorly described in goats. In this article, we describe the vasculocentric brain lesions in 44 cases of caprine spontaneous C. perfringens type D enterotoxemia. Only 1 goat had gross changes in the brain, which consisted of mild cerebellar coning. However, 8 of 44 (18%) cases showed microscopic brain lesions, characterized by intramural vascular proteinaceous edema, a novel and diagnostically significant finding. The precise location of the edema was better observed with periodic acid-Schiff, Gomori's, and albumin stains. Glial fibrillary acidic protein and aquaporin 4 immunostaining revealed strong immunolabeling of astrocyte foot processes surrounding microvessels. The areas of the brain most frequently affected were the cerebral cortex, corpus striatum (basal ganglia), and cerebellar peduncles, and both arterioles and venules were involved. Most of the goats of this study showed lesions in the intestine (enteritis, colitis, and typhlitis), although pulmonary congestion and edema, hydrothorax, hydropericardium, and ascites were also described. Although the intramural edema described, for the first time, in these caprine cases is useful for the diagnosis of enterotoxemia when observed, its absence cannot exclude the disease.

Sensitivity of histology for the detection of the amphibian chytrid fungus Batrachochytrium dendrobatidis.

Histology is often underappreciated for the detection of the amphibian pathogenic fungus Batrachochytrium dendrobatidis, the cause of the potentially lethal skin disease chytridiomycosis. We evaluated the sensitivity of histology to detect chytrids in 20 wild specimens of 2 frog species from Uruguay that were clinically normal, but confirmed by PCR to be infected by B. dendrobatidis. We detected maturing and sporulated sporangia in 15 of 20 (75%) frogs, which is more sensitive than previously reported for histology. The effort needed to identify chytrids in histologic skin sections of Physalaemus henselii and Pleurodema bibroni required examination of 3.2 and 8.7 mm of skin sections for each frog species, respectively.

Subcutaneous extraskeletal chondroblastic osteosarcoma in a cat.

CASE SUMMARY: An 11-year-old neutered male cat was presented with a fixed, subcutaneous mass in the left hindlimb. The neoplasm was surgically removed and determined to be a 2 × 2 × 9 cm mass that extended over the plantar surface of the left hindlimb from the tarsus to the phalanges. It was independent from the skeletal system but firmly attached to the adjacent connective tissue. Microscopically, the neoplasm was composed of highly proliferative mesenchymal neoplastic cells that formed both osseous and cartilaginous tissues with associated production of chondroid, osteoid and associated matrixes. This neoplasia was diagnosed as an extraskeletal chondroblastic osteosarcoma. Extraskeletal osteosarcomas, especially the chondroblastic subtype, are extremely rare in cats. Consequently, little is known concerning their course and prognosis. In this case, excision with wide margins appeared to be successful as, at the time of writing, 24 months after limbectomy, the cat is healthy with no evidence of recurrence or metastasis. RELEVANCE AND NOVEL INFORMATION: To our knowledge, this is the first report of an appendicular large extraskeletal chondroblastic osteosarcoma occurring in a domestic cat. As these neoplasms are rare, it should be considered as a less likely cause of soft tissue appendicular neoplasms in domestic cats.

Amphibian parasites of the Order Dermocystida (Ichthyosporea): current knowledge, taxonomic review and new records from Brazil.

Amphibian parasites of the Order Dermocystida (Ichthyosporea) are widespread pathogens known mainly from Europe and North America, which cause primarily a disease of skin and subcutaneous tissue in their hosts. The taxonomy of these organisms has been problematic given their conserved morphology, similar clinical disease and pathology. Currently recognized taxa belong to the three closely related genera, Amphibiocystidium, Amphibiothecum, and Rhinosporidium, whereas species of Dermocystidium and Sphaerothecum destruens include fish parasites. Here, we review the taxonomy of Dermocystida based on a molecular phylogenetic analysis, principally of amphibian parasites, including DNA sequences obtained from amphibian hosts collected in the central-eastern region of South America. A new taxonomic arrangement is proposed, which includes the designation of type material for Dermocystidium pusula, synonymization of Amphibiothecum with Dermocystidium, and the restriction of Amphibiocystidium to its type species A. ranae. We also review the taxonomic status of Dermosporidium hylarum until the present work included in the synonymy of the human and animal pathogen R. seeberi, and considered herein as a valid taxon, however. In addition, a new species of Sphaerothecum parasitic to amphibians is described, being the first record of this genus in the southern hemisphere and in an amphibian host.

Calbindin D28k distribution in neurons and reactive gliosis in cerebellar cortex of natural Rabies virus-infected cattle.

Rabies has been an enigmatic disease because microscopic findings in central nervous system tissues do not always correlate well with the severity of the clinical illness. Immunohistochemical staining of the calcium-binding protein calbindin (specifically CbD28k) seems to be the technique most used to identify Purkinje neurons under normal and pathological conditions. In the present work, we evaluated CbD28k immunoreactivity in the cerebellar cortex of normal and natural Rabies virus (RABV)-infected cattle. We examined brains from 3 normal cows and from 6 crossbreed cattle with a histologic diagnosis of rabies. Samples were taken from the cerebral cortex, cerebellum, hippocampus, and brainstem. Immunohistochemistry was carried out using the following primary antibodies: anti-RABV, anti-GFAP, and anti-CbD28k. In the cerebellar cortex, RABV infection caused the loss of CbD28k immunostaining in Purkinje cells; some large interneurons in the granular layer maintained their positive CbD28k immunoreaction. The identification of this loss of CbD28k reactivity in cerebellar Purkinje cells of RABV-infected cattle presents a potentially valuable tool to explore the impairment of Ca(2+) homeostasis. In addition, this may become a useful method to identify specific molecular alterations associated with the higher prevalence of Negri bodies in Purkinje cells of cattle. Furthermore, we detected the presence of rabies viral antigens in different regions of the central nervous system, accompanied by microglial proliferation and mild reactive astrogliosis.

Conditioning and aversion to toxic Solanum bonariense ("naranjillo") leaves in calves / Condicionado e aversão a folhas tóxicas de Solanum bonariense ("naranjillo") em bezerros

ABSTRACT: Solanum bonariense is a perennial poisonous shrub that induces cerebellar cortical degeneration when eaten by cattle. The aim of this research was to outline a protocol to induce a conditioned aversion to this plant. During the pre-conditioning period ten calves (126±12kg BW) were maintained at half of their normal energy intake with lucerne hay and water ad libitum, to stimulate consumption of S. bonariense. Every two days they were offered 100g ofS. bonariense leaves for 5 minutes. Calves began eating the target plant on day 10 and consumed all the plant material on day 12. The conditioning period began after each calf consumed the entire amount of S. bonariense for three consecutive sessions. Five animals were randomly selected for conditioning, and after ingestion ofS. bonariense they were dosed by oral gavage with lithium chloride (LiCl) at 200mg kg-1 BW (treated group), while the other five received a similar volume of water by oral gavage (control group). After 2 doses of LiCl the treated group ate no S. bonariense while the control group consumed the entire 100g. We confirmed that LiCl is a powerful tool to induce conditioned aversions against S. bonariense in calves, which persists for at least 3 months.

RESUMO: Solanum bonariense é uma planta tóxica, arbustiva e perene que induz a degeneração cortical cerebelar quando ingerida por bovinos. O presente trabalho objetiva criar um protocolo para induzir aversão condicionada para essa planta em bovinos. Durante o período de adaptação às condições experimentais, dez bezerros (126±12kg) foram mantidos com metade de seu fornecimento alimentar energético normal que incluiu feno de alfafa e água ad libitum, para estimular o consumo de S. bonariense. A cada dois dias, foram fornecidas oralmente 100g de folhas de S. bonariense por cinco minutos. Os bezerros começaram a ingerir a planta no 10o dia de experimento e consumiram toda a planta no 12o dia. O período condicionado começou após cada bezerro ter consumido a quantidade total de S. bonariense por três sessões consecutivas. Cinco animais foram aleatoriamente selecionados para aversão condicionada e, após ingestão da planta, receberam solução oral de 200mg kg-1 de peso vivo de cloreto de lítio (LiCl) (grupo tratado), enquanto que os outros cinco bovinos receberam dosagem oral com volume similar de solução fisiológica (grupo controle). Após duas doses of LiCl, o grupo tratado não comeu folhas de S. bonariense, enquanto que o grupo controle consumiu toda a quantidade fornecida. Os resultados do presente trabalho demonstram que LiCl é uma ferramenta útil para induzir aversão condicionada contra S. bonariense em bezerros, que persiste por pelo menos 3 meses.

A novel pathogenic mechanism for cerebellar lesions produced by Solanum bonariense in cattle.

Intoxication with Solanum bonariense in cattle causes cerebellar cortical degeneration with perikaryal vacuolation, axonal swelling, and death primarily of Purkinje cells, with accumulation of electron-dense residual storage bodies in membrane-bound vesicles. The pathogenesis of this disease is not fully understood. Previously, we proposed that inhibition of protein synthesis in Purkinje cells among other altered metabolic pathways could lead to cytoskeletal alterations, subsequently altering cell-specific axonal transport. In the present study, immunohistochemical and histochemical methods were used to identify neuronal cytoskeletal alterations and axonal loss, demyelination, and astrogliosis in the cerebellum of intoxicated bovines. Samples of cerebellum from 3 natural and 4 experimental cases and 2 control bovines were studied. Immunoreactivity against neurofilament (NF)-200KDa confirmed marked loss of Purkinje neurons, and phospho-NF protein, ß-tubulin, and affinity reaction against phalloidin revealed an altered perikaryal distribution of neuronal cytoskeletal proteins in the remaining Purkinje cells in intoxicated cattle. Reactive astrogliosis in every layer of the cerebellar cortex was also observed with anti-glial fibrillary acidic protein immunohistochemistry. In affected cattle, demyelination and axonal loss in the cerebellar white matter, as well as basket cell loss were demonstrated with Klüver-Barrera and Bielschowsky stains, respectively. Based on these results, we propose that neuronal cytoskeletal alterations with subsequent interference of the axonal transport in Purkinje cells may play a relevant role in the pathogenesis of this neurodegenerative disorder, and also that demyelination and axonal loss in the cerebellar white matter, as well as astrogliosis in the gray matter, likely occur secondarily to Purkinje cell degeneration and death.

Ichthyophonus sp. (Ichthyophonae, Ichthyophonida) infection in a South American amphibian, the hylid frog Hypsiboas pulchellus.

We report infection by Ichthyophonus sp. in a South American amphibian, the hylid frog Hypsiboas pulchellus in Uruguay. This frog had a large subcutaneous mass over the urostyle and dorsal musculature comprised of parasitic cysts with mild granulomatous inflammation but otherwise appeared healthy.

Cryptosporidium spp. in bursa of Fabricius of broiler chickens from Uruguay / Cryptosporidium spp. em bursa de Fabrícius de frangos de corte no Uruguai

The presence of Cryptosporidium spp. was detected in broiler chickens of 32-40 days of age, colonizing the bursa of Fabricius. The histologic study with haematoxylin and eosin staining was done on specimens of trachea, complete intestinal tract and bursa. Samples of intestinal content were also studied using Kinyoun staining technique. All birds with signs of illness were positive to the presence of parasite, showing a hypertrophic bursa of Fabricius, filled with caseous content. The bursal epithelial cells were full of different developmental stages of Cryptosporidium spp. This note describing the presence of Cryptosporidium spp. in industrial flocks of broiler chickens, is the first report of this parasite in Uruguay.

A presença de Cryptosporidium spp. foi detectada em frangos de corte de 32-40 dias de idade, colonizando a bursa de Fabrícius. O estudo histológico com hematoxilina e eosina foi feito em amostras de traqueia, trato intestinal completo e bursa. As amostras de conteúdo intestinal também foram estudadas utilizando a técnica de coloração de Kinyoun. Todas as aves com sinais de doença foram positivas para a presença do parasita, mostrando uma bursa de Fabrícius hipertrófica com grande quantidade de conteúdo caseoso. As células epiteliais bursais estavam repletas de diferentes estágios de desenvolvimento de Cryptosporidium spp. Esta nota, que descreve a presença de Cryptosporidium spp. em lotes industriais de frangos de corte, é o primeiro relato deste parasita no Uruguai.

Dermocystid-chytrid coinfection in the neotropical frog Hypsiboas pulchellus (Anura: Hylidae).

We present gross and histologic evidence of coinfection in amphibians by fungal-like parasites of the order Dermocystidia (Amphibiocystidium sp.) and the fungus Batrachochytrium dendrobatidis. The condition was observed in frogs Hypsiboas pulchellus (Hylidae) from Uruguay in 2009 to 2012. This report is the first of dermocystids in Neotropical amphibians since 1940.

Cerebellar degeneration in cattle grazing Solanum bonariense ("Naranjillo") in Western Uruguay.

Cattle in western Uruguay that were eating Solanum bonariense developed periodic episodes of ataxia, hypermetria, hyperesthesia, head and thoracic limb extension, opisthotonus, nystagmus, and falling to the side or backward. Similar clinical signs were experimentally reproduced in cattle by administration of S. bonariense via rumen cannula at a dose of 1,024 g/kg body mass. No significant gross lesions were observed in field cases or experimentally induced cases. Spontaneous and induced histologic lesions were similar and included vacuolation, degeneration, and loss of Purkinje cells. Axonal spheroids, microcavitations, and other changes of wallerian-type degeneration in cerebellar white matter were also observed. Ultrastructural changes included increased number of electron-dense residual storage bodies in membrane-bound vesicles in affected Purkinje cells, and similar vesicles and mitochondria in axonal spheroids. No histologic lesions were detected in the other examined tissues. The Purkinje-cell swelling and vacuolation with subsequent cerebellar degeneration are suggestive of Purkinje-cell specific toxin that produces abnormal lysosome function and cell specific axonal transport. This is the first report of S. bonariense toxicity.