Cumulative effect of arsenic on the number of mouse offspring and the female reproductive hormones in mice.

In this study, we evaluated the cumulative effects of arsenic (III) oxide on the number of mouse offspring over three consecutive generations and monitored changes in levels of the reproductive hormones, oestradiol and progesterone in female mice during the dioestrus phase of the cycle. The control group received water from the mains. In two experimental groups, mice were given drinking water containing dissolved arsenic (III) oxide at concentrations of 10.6 mg L-1 and 106 mg L-1, respectively. These concentrations represent the values converted from a human model to an animal model (mice) thus correspond to the arsenic content of the groundwater in the southern part of the Pannonian Basin, in the province of Vojvodina, in the Banat region, in particular in the town of Zrenjanin. The average number of newborn mice in both experimental groups decreased for three consecutive generations. The total arsenic content of day-old mice did not show significant differences between the experimental groups. Arsenic (III) oxide affected the reproductive hormone levels of female mice at both concentrations.

Diclofenac and metformin synergistic dose dependent inhibition of hamster fibrosarcoma, rescued with mebendazole.

We examined whether combinig diclofenac and metformin in doses equivalent to human doses would synergize their anticancer activity on fibrosarcoma inoculated to hamsters and in vitro. Rescue experiment was performed to examine whether the prosurvival NF-κB stimulation by mebendazole can reverse anticancer effects of the treatment. BHK-21/C13 cell culture was subcutaneously inoculated to Syrian golden hamsters randomly divided into groups (6 animals per group): 1) untreated control; treated daily with 2) diclofenac; 3) metformin; 4) combinations of diclofenac and metformin at various doses; 5) combination of diclofenac, metformin and mebendazole; 6) mebendazole. Dose response curves were made for diclofenac and metformin combination. Tumor growth kinetics, biophysical, pathological, histological and immunohistochemical characteristics of excised tumors and hamster organs as well as biochemical and hematological blood tests were compared among the groups. Single treatments had no anticancer effects. Diclofenac (60 mg/kg/day) exhibited significant (P < 0.05) synergistic inhibitory effect with metformin (500 mg/kg/day) on all tumor growth parameters, without toxicity and influence on biochemical and hematological blood tests. The same results were obtained with double doses of diclofenac and metformin combination. The addition of mebendazole to the diclofenac and metformin combination rescued tumor expansion. Furthermore, diclofenac with metformin demonstrated antiproliferative effects in hamster fibrosarcoma BHK-21/C13, human lung carcinoma A549 (CCL-185), colon carcinoma HT-29 (HTB-38) and cervical carcinoma HeLa (CCL-2) cell cultures, with markedly lower cytotoxicity in the normal fetal lung MRC-5 cells. In conclusion, diclofenac and metformin combination may be recommended for potential use in oncology, due to synergistic anticancer effect in doses achievable in humans.

Transcriptional Profiles of Mitochondrial Dynamics Markers Are Disturbed in Adrenal Glands of Stressed Adult Male Rats.

Mitochondrial dynamics plays a significant role in shaping the mitochondrial network and maintaining mitochondrial function. Imbalanced mitochondrial dynamics can cause mitochondrial dysfunction leading to a wide range of diseases/disorders. The aim of this study was to investigate the expression of mitochondrial dynamics markers and regulatory molecules in whole adrenal glands, cortices, and medullae obtained from adult male rats exposed to acute and repeated psychophysical stress, the most common stress in human society. The transcriptional profiles of most of the mitochondrial dynamics markers investigated here were altered: 81%-(17/21) in the whole adrenal gland, 76.2%-(16/21) in the adrenal cortex, and 85.7%-(18/21) in the adrenal medulla. Changes were evident in representatives of every process of mitochondrial dynamics. Markers of mitobiogenesis were changed up to 62.5%-(5/8) in the whole adrenal gland, 62.5%-(5/8) in the adrenal cortex, and 87.5%-(7/8) in the adrenal medulla. Markers of mitofusion were changed up to 100%-(3/3) in the whole adrenal gland, 66.7%-(5/8) in the adrenal cortex, and 87.5%-(7/8) in the adrenal medulla, while all markers of mitofission and mitophagy were changed in the adrenal glands. Moreover, almost all markers of mitochondrial functionality were changed: 83.3%-(5/6) in the whole adrenal, 83.3%-(5/6) in the cortex, 66.7%-(4/6) in the medulla. Accordingly, the study highlights the significant impact of acute and repeated stress on mitochondrial dynamics in the adrenal gland.

Circadian desynchrony disturbs the function of rat spermatozoa.

Decreased male fertility is a growing health problem that requires a better understanding of molecular events regulating reproductive competence. Here the effects of circadian desynchrony on the rat spermatozoa functionality were studied. Circadian desynchrony was induced in rats that lived for 2 months under disturbed light conditions designed to mimic shiftwork in humans (two days of constant light, two days of continual dark, and three days of 14:10 h light:dark schedule). Such a condition abolished circadian oscillations in the rats' voluntary activity, followed by a flattened transcriptional pattern of the pituitary gene encoding follicle stimulating hormone subunit (Fshb), and genes important for germ cell maturation (Tnp1 and Prm2) as well as the clock in seminiferous tubules. However, the number of spermatozoa isolated from the epididymis of the rats suffering from circadian desynchrony did not deviate from the controls. Nevertheless, spermatozoa functionality, estimated by motility and progesterone-induced acrosome reaction, was reduced compared to the control. These changes were associated with the altered level of main markers of mitochondrial biogenesis (Pprgc1a/PGC1A, Nrf1/NRF1, Tfam, Cytc), decreased mitochondrial DNA copy number, ATP content, and clock genes (Bmal1/BMAL1, Clock, Cry1/2, and Reverba). The principal-component-analysis (PCA) points to a positive association of the clock and mitochondrial biogenesis-related genes in spermatozoa from rats suffering circadian desynchrony. Altogether, the results show the harmful effect of circadian desynchrony on spermatozoa functionality, targeting energetic homeostasis.

Subcutaneous Taenia crassiceps Cysticercosis in a Ring-Tailed Lemur (Lemur catta) in a Serbian Zoo.

BACKGROUND: Different rodent species serve as natural intermediate hosts for carnivore tapeworm Taenia crassiceps. However, this cestode occasionally infects various dead-end hosts including humans and other primates and may cause serious pathological implications with potentially fatal outcome. In this paper, we present subcutaneous cysticercosis caused by T. crassiceps, found in a previously healthy 17-years-old male ring-tailed lemur (Lemur catta) in a Serbian Zoo. CASE PRESENTATION: The animal was presented to a veterinarian with a history of periarticular subcutaneous swelling in medial right knee region. After fine needle aspiration revealed cycticerci-like structures, a surgery was performed for complete extraction of the incapsulated multicystic mass containing numerous cysticerci. Collected material was sent for parasitological, histological and molecular analysis. One month after surgery, the lemur died due to respiratory failure unrelated to cysticercosis. Based on morphological features of large and small hooks and characteristic proliferation of cysticerci, a metacestode of T. crassiceps was identified, which was confirmed after sequencing of obtained amplicons and comparing them to the GenBank database. CONCLUSIONS: This is one of the few reported cases of T. crassiceps cysticercosis in a ring-tailed lemur, and the first one in Serbia. This endangered species seem to be more sensitive for T. crassiceps than other non-human primates, which represents serious conservation challenge for captive animals. Due to zoonotic nature of the parasite, challenging diagnosis, severity of the disease, difficult treatment and possible fatalities, high biosecurity measures are of particular importance, especially in endemic regions.

Cell numbers in the reflected blade of CA3 and their relation to other hippocampal principal cell populations across seven species.

The hippocampus of many mammals contains a histoarchitectural region that is not present in laboratory mice and rats-the reflected blade of the CA3 pyramidal cell layer. Pyramidal cells of the reflected blade do not extend dendrites into the hippocampal molecular layer, and recent evidence indicates that they, like the proximal CA3 pyramids in laboratory rats and mice, partially integrate functionally with the dentate circuitry in pattern separation. Quantitative assessments of phylogenetic or disease-related changes in the hippocampal structure and function treat the reflected blade heterogeneously. Depending on the ease with which it can be differentiated, it is either assigned to the dentate hilus or to the remainder of CA3. Here, we investigate the impact that the differential assignment of reflected blade neurons may have on the outcomes of quantitative comparisons. We find it to be massive. If reflected blade neurons are treated as a separate entity or pooled with dentate hilar cells, the quantitative makeup of hippocampal cell populations can differentiate between species in a taxonomically sensible way. Assigning reflected blade neurons to CA3 greatly diminishes the differentiating power of all hippocampal principal cell populations, which may point towards a quantitative hippocampal archetype. A heterogeneous assignment results in a differentiation pattern with little taxonomic semblance. The outcomes point towards the reflected blade as either a major potential player in hippocampal functional and structural differentiation or a region that may have cloaked that hippocampi are more similarly organized across species than generally believed.

Disulfiram and metformin combination anticancer effect reversible partly by antioxidant nitroglycerin and completely by NF-κB activator mebendazole in hamster fibrosarcoma.

We investigated the anticancer effect of disulfiram and metformin combination on fibrosarcoma in hamsters. Hamsters of both sexes (~ 70 g) were randomly allocated to control and experimental groups (8 animals per group). In all 10 groups, 2 × 106 BHK-21/C13 cells in 1 ml were injected subcutaneously into the animals' backs. Peroral treatments were carried out with disulfiram 50 mg/kg daily, or with metformin 500 mg/kg daily, or with their combination. Validation and rescue grups were treated by double doses of the single therapy and by the combination with addition of rescue daily doses of ROS inhibitor nitroglycerin 25 mg/kg or NF-κB stimulator mebendazole 460 mg/kg, via a gastric probe after tumor inoculation. After 19 days all animals were sacrificed. Blood samples were collected for hematological and biochemical analyses, the tumors were excised and weighed, and their diameters and volumes were measured. The tumor samples were pathohistologically and immunohistochemically assessed (Ki-67, PCNA, CD34, CD31, COX4, Cytochrome C, GLUT1, iNOS), and the main organs were toxicologically tested. The combination of disulfiram and metformin significantly inhibited fibrosarcoma growth in hamsters without toxicity, compared to monotherapy or control. The single treatments did not show significant antisarcoma effect. Co-treatment with nitroglycerin partly rescued tumor progression, probably by ROS inhibition, while mebendazole completely blocked anticancer activity of the disulfiram and metformin combination, most likely by NF-κB stimulation. Combination of disulfiram with metformin may be used as an effective and safe candidate for novel nontoxic adjuvant and relapse prevention anticancer therapy.

Intestinal helminth infections in the golden jackal (Canis aureus L.) from Vojvodina: Hotspot area of multilocular echinococcosis in Serbia.

In the present study, 64 golden jackals were examined for intestinal helminths in three regions of Vojvodina, Serbia. Among the examined jackals 57.8% were infected with at least one parasite species. Using the intestinal scraping technique (SCT), eight species of intestinal helminths were found: Alaria alata (7.8%), Toxascaris leonina (9.4%), Toxocara canis (4.7%), Uncinaria stenocephala (20.3%), Echinococcus multilocularis (14.1%), Mesocestoides sp. (42.2%), Taenia pisiformis, and Taenia hydatigena (the overall prevalence of Taenia infection was 6.3%). To the best of our knowledge, this is the first report of T. leonina in jackals from Serbia. In comparison with the SCT results, coprological tests were less sensitive and specific for parasite identification, as only two nematode species (T. leonina and T. canis) as well as ancylostomatid and taeniid eggs were identified. The total prevalence of intestinal helminths was higher in males (71.9% males, 45% females), but the difference was not statistically significant (χ 2 = 3.76; P = 0.052). Co-infection with two species of intestinal helminths was found in 35% of the examined golden jackal individuals, three-species co-infection was demonstrated in 21.6%, whereas four-species co-infection was detected in 2.7% of the golden jackals examined. Echinococcus multilocularis has previously been recorded in jackals and foxes in Serbia, but only in Vojvodina. Our results corroborate the findings of previous studies, and indicate that the Vojvodina Province, more specifically the Srem region, is probably a high-risk area for E. multilocularis transmission to humans.

History of Rabies Incidence and Rabies Control in Serbia in Support of the Zero by 2030 Campaign to Eliminate Dog-Mediated Human Rabies.

Urban (principally canine-mediated) rabies has been a public health risk for people living in Serbia for centuries. The first legal act in urban rabies prevention in Serbia was established in 1834 by introducing high taxes for pet dog owners. Five years later in 1839, the first set of literature describing rabies prevention was issued by the health department from The Serbian Ministry of Interior. An overview of cauterization of rabies wounds was presented as the principal method of rabies post exposure prophylaxis. In 1890, a human rabies vaccination was introduced in Serbia with the royal government directive which ordered patients to be treated at the Pasteur Institute in Budapest in receipt of rabies vaccination. Urban (canine) rabies was eliminated during the 1980s, but sylvatic (principally fox-mediated) rabies still prevailed. The last human rabies case was recorded in the Province of Kosovo and Metohija in 1980. Sylvatic rabies in Serbia is in the final stages of elimination by orally vaccinating foxes (Vulpes vulpes). The only published finding of a lyssavirus among Serbian bats was made in 1954 by Dr Milan Nikolic in the vicinity of Novi Sad. In 2006, a comprehensive two-year active surveillance program of lyssaviruses in bats in Serbia was undertaken. In this single study, all of the bats from Serbia tested negative for a lyssavirus.

Co-treatment with nitroglycerin and metformin exhibits physicochemically and pathohistologically detectable anticancer effects on fibrosarcoma in hamsters.

We investigated the effect of nitroglycerin with metformin on fibrosarcoma in hamsters. Syrian golden hamsters of both sexes, weighing approximately 60 g, were randomly allocated to control and experimental groups, with 8 animals per group. In all groups, 2 × 106 BHK-21/C13 cells in 1 ml were injected subcutaneously into the animals' backs. Peroral treatment carried out with nitroglycerin 25 mg/kg daily, or with metformin 500 mg/kg daily, or with a combination of nitroglycerin 25 mg/kg and metformin 500 mg/kg daily. Later validation experiments were conducted with double doses of the single therapy and additional rescue doses of mebendazole 460 mg/kg daily, via a gastric probe after tumor inoculation. After 2 weeks, when the tumors were approximately 2-3 cm in the control group, all animals were sacrificed. Blood samples were collected for hematological and biochemical analyses, the tumors were excised and weighed, and their diameters and volumes were measured. The tumor samples were pathohistologically and immunohistochemically assessed for proliferation marker protein Ki-67, proliferating cell nuclear antigen PCNA, hematopoietic progenitor cell antigen CD34, cluster of differentiation 31 (CD31), cytochrome c oxidase subunit 4 (COX4), mitochondria marker Cytochrome C, glucose transporter 1 (GLUT1) and inducible nitric oxide synthase (iNOS), and the main organs were toxicologically tested. The Ki-67 and PCNA positivity and the cytoplasmic marker (CD34, CD31, COX4, Cytochrome C, GLUT1, iNOS) immunoexpression in the tumor samples were quantified. The combination of nitroglycerin and metformin significantly inhibited fibrosarcoma growth in hamsters without toxicity, compared to monotherapy or control. The results were validated and confirmed in the subsequently accomplished experiment with doubled doses of the single drug therapy and in the rescue experiment with addition of mebendazole. The single treatments did not show significant antisarcoma effect, regardless of the dose. Co-treatment with mebendazole inhibited anticancer activity of the nitroglycerin and metformin combination. Mebendazole rescued tumor progression suppressed by the combination of nitroglycerin and metformin. Administration of nitroglycerin with metformin might be an effective and safe approach in novel nontoxic adjuvant and relapse prevention anticancer treatment.

Molecular evaluation of chronic restrain stress in mice model of non metastatic fibrosarcoma.

Chronic stress is regarded as a significant factor in the etiology of the many diseases. Numerous methods have been developed through which the effect of chronic stress is examined. The aim of this study is to demonstrate the new experimental model for analysis of immuno-suppression induced by chronic restraint stress, through challenge with conditionally tumorigenic cell line BHK-21/C13. 20 male NMRI mice were randomly divided into 2 groups-control and experimental. Each mouse was subcutaneously inoculated with BHK-21/C13 cells. Stress in the experimental group was induced for 20 days. After the experiment, tumor masses were removed, and analyzed using histology and immunohistochemistry techniques. We found a statistically significant difference (p = 0.034) in tumor expression and tumor volumes (p = 0.0061) between groups, as well as in immunopositivity on Ki67, cytochrome C and matrix metalloproteinase 9. Absence of immune infiltrate was noticed in experimental, and the presence of inflammatory infiltrate at tumor invasion front in control group.

Significance of the red fox as a natural reservoir of intestinal zoonoses in Vojvodina, Serbia.

In the present study, 223 foxes were collected from various localities in the northern part of the Republic of Serbia (Vojvodina province) and examined for intestinal helminths. Among the examined foxes 178 (79.8%) were infected. The most frequently identified parasites were Mesocestoides spp. (49.3%) and Toxascaris leonina (36.3%). The parasite with the lowest prevalence was Pterygodermatites affinis (0.9%), and this is the first confirmed finding in Serbia. The other recovered species were Alaria alata (25.6%), Taenia spp. (6.3%), Echinococcus multilocularis (13%), Toxocara canis (16.6%) and Uncinaria stenocephala (14.8%). The highest number of foxes infected with E. multilocularis were in the Srem area. The results of this study indicate the presence of helminth species in red foxes in Vojvodina which may also infect humans.

Physicochemical and pathohistological changes in experimental fibrosarcoma tumors of hamsters treated with metformin and itraconazole.

The anticancer effects of metformin (an antihyperglycaemic agent) and itraconazole (an antifungal agent), which are established non-oncologic drugs, were investigated in the present study. The weight, diameter, volume, density, surface, surface to volume ratio and immunohistochemistry of experimental fibrosarcoma tumors were investigated in hamsters treated with metformin and itraconazole. Briefly, the hamsters were injected with BHK-21/C13 cells in order to induce fibrosarcoma, and the animals were treated daily with metformin, itraconazole or a combination of the two drugs. Subsequently, blood samples were obtained for biochemical analyses and the tumors were excised, weighed and measured. The tumor samples were pathohistologically and immunohistochemically assessed for proliferation marker protein Ki-67, hematopoietic progenitor cell antigen CD34, cytochrome c oxidase subunit 4 (COX4), glucose transporter 1 (GLUT1) and inducible nitric oxide synthase (iNOS), and vital organs were toxicologically tested. Ki-67-positivity and cytoplasmic marker (CD34, COX4, GLUT1, iNOS) immunoexpression in the tumor samples were quantified. The results revealed that the combination of metformin and itraconazole significantly altered the physicochemical and pathohistological characteristics of the hamster fibrosarcoma tumors, including absolute and relative weight, volume, density, length, surface area, surface to volume ratio, Ki-67-positivity and the immunoexpression of cytoplasmic markers, without indications of toxicity. Furthermore, metformin with itraconazole demonstrated antiproliferative functions in cervical carcinoma HeLa, colon carcinoma HT-29, lung carcinoma A549 and fibrosarcoma BHK-21/C13 cells, with markedly lower cytotoxicity in the normal fetal lung MRC-5 cells. In conclusion, the administration of metformin in combination with itraconazole may inhibit the growth of fibrosarcoma tumors in vivo and the proliferation of various malignant cell lines in vitro, suggesting that this may be an effective and safe approach as a nontoxic anticancer adjuvant and relapse prevention therapy.

Mysterious path of Borrelia spielmanii: spreading without morphological alteration of collagen type I and IV.

The majority of suggested mechanisms of Borrelia spreading inside erythema migrans (EM) are developed from in vitro studies and animal models. This report is the first to describe pathomorphological substrate of EM caused by Borrelia spielmanii in humans, addressing the hypothesis of enhanced Borrelia penetration through extracellular matrix. In the process of ruling out of atypical Masters' disease, we conducted a punch biopsy of suspected EM and a two-tier serology testing for Lyme borreliosis, where we registered antibodies against B. spielmanii. Skin biopsy showed CD4+ and CD8+ lymphocyte involvement and high activity of matrix metalloproteinase 9. No alterations were detected in distribution and morphology of collagen type I and IV. Therefore, it is suggested that other mechanisms should be considered as major contributing factors to local spreading of B.spielmanii.

Disodium Cromolyn and Anti-podoplanin Antibodies Strongly Inhibit Growth of BHK 21/C13-derived Fibrosarcoma in a Chick Embryo Chorioallantoic Membrane Model.

AIM: To characterize baby hamster kidney fibroblast (BHK 21/C13) cells and test the effects of antibodies against podoplanin and disodium cromolyn on BHK 21/C13 cell line-derived tumors grown on chick embryo chorioallantoic membrane (CAM). MATERIAL AND METHODS: BHK 21/C13 cell-derived fibrosarcomas developed in hamsters were implanted on CAM and treated with anti-podoplanin antibodies and disodium cromolyn. BHK 21/C13 cell immunophenotype was assessed. RESULTS: Fibrosarcoma cells were positive for vimentin, CD117, smooth muscle actin, vascular endothelial growth factor epidermal growth factor receptor, homebox prospero gene 1 and negative for platelet-derived growth factor B, neuron-specific enolase, S100, CD34, Ewing sarcoma and podoplanin. CAM-grown fibrosarcomas were highly sensitive to disodium cromolyn and anti-podoplanin antibodies. CONCLUSION: Immunophenotyping BHK 21/C13 cells and their response to drugs represent the first step in revealing cell line utility and a reliable tool for experimental cancer research.

Drug repurposing: mebendazole as effective antitumor agent. Are we seeing the whole story?

PURPOSE: To examine the antitumor effects of Mebendazole (MZ) in a model of experimental fibrosarcoma induced by inoculation of BHK-21/C13 cells in Syrian golden hamster. METHODS: Hamsters were inoculated with a suspension of BHK cells by subcutaneous injection and randomly divided into 5 experimental and 2 control groups. Treatment started on the 10th day after inoculation, when the tumor grew to a diameter of 5mm. The experimental design was based on distributing the total amount of drug MZ(z) in different protocols and approaches (oral/intraperitoneal) to the 5 experimental groups. The positive control group received doxorubicin intraperitoneally. Negative control group received olive oil orally. The total amount of MZ(z) was chosen to be the highest for the animal to survive during the experiment. For antitumor effect evaluation, the main parameters were tumor size, number of mitoses, cytochrome-C immunopositivity and tumor tissue morphology incuding cytoarchitecture and percentage of preserved tumor tissue in stereologically reconstructed tumor mass. RESULTS: The results of this study showed absence of objective MZ antitumor effect on experimental fibrosarcoma. MZ does not exhibit activity similar to DNA-damaging agents on the fibrosarcoma model. CONCLUSIONS: It might be postulated that soft tissue tumors on animal models could show high level of resistance to MZ effect.

Detection of Toxoplasma gondii in naturally infected domestic pigs in Northern Serbia.

Insufficiently cooked pork is considered as an important source of human infection with Toxoplasma gondii. The aim of our study was to investigate the presence of T. gondii in pigs intended for human consumption from Northern Serbia. Blood and diaphragm samples were collected from 182 naturally infected market-weight pigs, originating from both commercial farms and smallholdings. Sera were examined using modified agglutination test (MAT), and diaphragms from seropositive, as well as from some MAT-negative pigs, were bioassayed in mice. In addition, digests were examined for the presence of T. gondii DNA using a real-time polymerase chain reaction (qPCR) which was targeted at the 529 bp repetitive element of the T. gondii genome. The overall seroprevalence of T. gondii in pigs was 17% (31/182), with no difference between pigs from large commercial farms (17.8%) and those raised on smallholdings (16.3%). However, the seroprevalence in farm pigs was largely influenced by the findings on a single farm, where all examined animals tested positive. Parasites and/or parasite DNA were detected in the tissues of 15 of the 45 (25 seropositive and 20 seronegative) animals examined by either direct method. Tissue cysts were isolated in eight bioassays and an additional bioassay was positive by serology; all nine were confirmed positive by qPCR. All positive bioassays originated from seropositive pigs, but no correlation was observed between isolation rate and antibody titer. T. gondii DNA was detected in diaphragm tissues of eight pigs, of which three were seronegative. The results of our study provide further evidence for pork as a source of human T. gondii infection.

Genetic variability of Eucoleus aerophilus from domestic and wild hosts.

Eucoleus aerophilus (syn. Capillaria aerophila) is a trichuroid nematode affecting domestic and wild carnivores and, sometimes, humans. This parasite has a worldwide distribution and may cause significant clinical disease in pet animals. The present paper investigates the sequence variation in partial mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of E. aerophilus isolates from pets and wild animals from different countries. Forty-four egg pools of E. aerophilus were collected from dogs, cats and foxes from Italy, while seventeen adult stages of E. aerophilus were obtained from red foxes and beech martens from Portugal, Romania, Serbia and UK. Fifteen different haplotypes were characterized and five were shared between pets in Italy and wildlife from Europe. The remaining haplotypes were either confined only in hosts or countries, or in a given host from a country. The phylogenetic analysis showed that all haplotypes clustered as a monophyletic group with a strong nodal support, indicating that all sequence types represented E. aerophilus. The results here presented have implications for a better understanding of the epidemiology, phylo-geography and clinical impact of E. aerophilus. In particular, the geographic distribution of E. aerophilus haplotypes in different host species and geographic regions, and their variation in terms of pathogenic impact and zoonotic role, warrant further investigations.

The opposing roles of nitric oxide and cGMP in the age-associated decline in rat testicular steroidogenesis.

The molecular mechanism of the aging-associated dysfunction of Leydig cells (LCs) is complex and poorly understood. In this study, we analyzed the contribution of nitric oxide (NO) and cGMP signaling to the age-dependent decline in LC function. Significant (>50%) decreases in serum, intratesticular, and LC androgens in aging rats (15-24 months) were accompanied by a proportional increase in NO production, an up-regulation of cGMP levels, and the expression of soluble guanylyl cyclase-1B and protein kinase G1 in LCs. In contrast, LC cAMP levels decreased with age, most likely reflecting the up-regulation of cAMP-specific phosphodiesterase expression. Moreover, the expression of genes encoding enzymes responsible for cholesterol transport and its conversion to T were reduced. Exposing LCs from aged animals to NO further increased cGMP levels and decreased cAMP and androgen production, whereas the addition of cell-permeable 8-bromoguanosine-cGMP alone had the opposite effect. In vivo inhibition of cGMP-specific phosphodiesterase-5 for 3 and 6 months in aged rats led to a partial restoration of androgens, NO, and cyclic nucleotide levels, as well as the expression of steroidogenic and NO/cGMP signaling genes. These results indicate that a progressive increase in NO production contributes to the age-dependent decrease in steroidogenesis in a cGMP-independent manner, whereas the sustained elevation in cGMP levels significantly slows the decline in LC function.

High infection rate of zoonotic Eucoleus aerophilus infection in foxes from Serbia.

The respiratory capillariid nematode Eucoleus aerophilus (Creplin, 1839) infects wild and domestic carnivores and, occasionally, humans. Thus far, a dozen of human infections have been published in the literature but it cannot be ruled out that lung capillariosis is underdiagnosed in human medicine. Also, the apparent spreading of E. aerophilus in different geographic areas spurs new studies on the epidemiology of this nematode. After the recognition of the first human case of E. aerophilus infection in Serbia, there is a significant merit in enhancing knowledge on the distribution of the nematode. In the present work the infection rate of pulmonary capillariosis was investigated in 70 red foxes (Vulpes vulpes) from the northern part of Serbia by autopsy. The estimated infection rate with Eucoleus aerophilus was 84%. In contrast, by copromicroscopic examination only 38% of foxes were positive. In addition, 10 foxes were investigated for the closely related species in nasal cavity, Eucoleus boehmi, and nine were positive. Our study demonstrates one of the highest infection rates of pulmonary capillariosis in foxes over the world.