Caffeine induces metformin anticancer effect on fibrosarcoma in hamsters.

OBJECTIVE: We investigated the effect of metformin and caffeine on fibrosarcoma in hamsters. MATERIALS AND METHODS: 32 Syrian golden hamsters of both sexes, weighing approximately 100 g, were randomly allocated to 3 experimental and 2 control groups, with a minimum of 6 animals per group. 2 x 106 BHK-21/C13 cells in 1 ml were injected subcutaneously into the animals' back in 4 groups. The first experimental group started peroral treatment with metformin 500 mg/kg daily, the second with caffeine 100 mg/kg daily and the third with a combination of metformin 500 mg/kg and caffeine 100 mg/kg daily, via a gastric probe 3 days before tumor inoculation. After 2 weeks, when the tumors were approximately 2 cm in the control group, all animals were sacrificed. The blood was collected for glucose and other analyses. The tumors were excised and weighed and their diameters were measured. The tumor samples were pathohistologically (HE) and immunohistochemically (Ki-67, CD 31, COX IV, GLUT-1, iNOS) assessed and the main organs toxicologically analyzed, including the control animals that had received metformin and caffeine. Tumor volume was determined using the formula LxS2/2, where L was the longest and S the shortest diameter. Ki-67-positive cells in the tumor samples were quantified. Images were taken and processed by software UTHSCSA Image Tools for Windows Version 3.00. Statistical significances were determined by the Student's t-test. RESULTS: The combination of metformin and caffeine inhibited fibrosarcoma growth in hamsters without toxicity. CONCLUSIONS: Administration of metformin with caffeine might be an effective and safe approach in novel nontoxic adjuvant anticancer treatment.

Respiratory and Cardiopulmonary Nematode Species of Foxes and Jackals in Serbia.

As part of routine monitoring of foxes (Vulpes vulpes) and jackals (Canis aureus) on the territory of Vojvodina province (northern Serbia), an analysis of respiratory and cardiopulmonary parasitic nematodes was conducted. Both host species harbored Eucoleus aerophilus, E. boehmi and Crenosoma vulpis, whereas Angiostrongylus vasorum was found only in foxes. A high prevalence of infection (72.6 %) was noted for E. aerophilus in foxes. The remaining parasite species occurred less frequently in both host species. In all species where it could be quantified, a high degree of parasite aggregation within host individuals was noted. Single species infections were most common, whereas two and three species infections occurred less frequently in both host species. The distribution of abundance of E. aerophilus was affected by host sex, with abundances higher in male foxes. Sampling site and year influenced abundance variation in E. boehmi.

Effect of metformin on fibrosarcoma in hamsters.

OBJECTIVE: We investigated the effect of metformin on an in vivo solid tumor model of fibrosarcoma in hamsters. MATERIALS AND METHODS: 33 Syrian golden hamsters of both sexes, weighing approximately 100 g, were randomly allocated to 3 experimental and 2 control groups. 2 x 106 BHK-21/C13 cells in 1 ml were injected subcutaneously into the animals' back in 4 groups. The first experimental group (7 animals) started peroral treatment with metformin 500 mg/kg daily via a gastric probe 7 days before tumor inoculation, the second (8 animals) 3 days before inoculation and the third (6 animals) immediately after inoculation. After 2 weeks, when the tumors were approximately 2-3 cm in the control group with tumors (6 hamsters), all animals were sacrificed. The blood was collected for glucose and other analyses. The tumors were excised and weighed and their diameters were measured. The tumor samples were histologically assessed and the main organs toxicologically analyzed, including 6 control animals that had received metformin without tumor inoculation. Tumor volume was determined using the formula Lx S2/2, where L was the longest and S the shortest diameter. Ki-67-positive cells in the tumor samples were quantified; images were taken and processed by software UTHSCSA Image Tools for Windows Version 3.00. Statistical significances of differences in tumor weight, volume, number of Ki-67-positive cells and other parameters were determined by the Student´s t-test. RESULTS: Metformin inhibited fibrosarcoma growth in hamsters without toxicity. The seven-day pretreatment was important for the statistically significant effect. CONCLUSIONS: Administration of metformin as an anti-tumor drug might be an effective and safe therapeutic approach in novel non-toxic therapies for human sarcomas.

Use of eriochrome cyanine R in routine histology and histopathology: is it time to say goodbye to hematoxylin?

Eriochrome cyanine R (ECR) is a synthetic anionic dye that forms complexes with cations such as iron. We found that an iron-ECR (Fe-ECR) mixture provided either nuclear or myelin staining depending on the differentiator used. Selective nuclear staining was obtained by differentiation in an aqueous HCl solution, pH 0.95, followed by a wash in slightly alkaline tap water; the pH difference facilitated control of differentiation. When used with an eosin B counterstain, results were nearly indistinguishable from standard hematoxylin and eosin (H & E) staining. Nuclear staining with Fe-ECR provides tinctorial features similar to regressive aluminum-hemateins as well as resistance to acidic solutions such as those of iron hemateins. Fe-ECR also stained selectively intestinal cells of the diffuse neuroendocrine system (DNES). In addition to its use as an H & E substitute, acid differentiated Fe-ECR produced acid-resistant and selective nuclear counterstaining in combination with Alcian blue, and in the Papanicolaou and van Gieson techniques. With alkali differentiation, Fe-ECR produced selective myelin staining, which was compatible with neutral red counterstaining. Myelin sheaths were stained aqua blue. Fe-ECR could be used for both cytological and histological samples, and was suitable for use in automated tissue stainers. ECR also is less expensive than hematoxylin. Hematoxylin still may be preferred as a nuclear counterstain for some immunostaining methods for which Fe-ECR mixtures probably are too acidic.

Vitamin C Depletion in Prenatal Guinea Pigs as a Model of Lissencephaly Type II.

Humans and guinea pigs are unable to produce vitamin C, with deficiency resulting in a well-known disorder of collagen synthesis. Pial basement membrane structure preservation is essential in the proper migration of neurons. In our study, intrauterine deprivation of vitamin C in guinea pig fetuses led to a collagen synthesis disorder, weakness, and finally a breach of pial basement membrane. We found excessive migration of the external germinal layer cells into the subarachnoid space of the cerebellum through defects in the pial basement membrane. The changes ranged from focal rupture of pial basement membranes to their complete disintegration. The loss of proper folia formation resulted in macroscopically visible flattening of the cerebellar surface. Different grades of dysplastic changes in the folia of the cerebellar cortex were observed in 2 experimental groups assigned different limits to mark the time of commencement and duration of vitamin C deprivation. The most severe form of dysplastic changes was characterized by marked irregularity of the cerebellar cortex similar to that in lissencephaly type II. Thus, prenatal vitamin C deficiency represents a novel animal model to study the effects of collagen synthesis on development of breaches in the pial basement membrane, disordered migration of neurons, dysplasia of cerebellar cortex, and the pathogenesis of lissencephaly.

Acute toxic effects of single dose dacarbazine: hematological and histological changes in an animal model.

Treatment of advanced soft tissue sarcoma usually includes dacarbazine (DTIC), an alkylating agent that methylates DNA and is active during all phases of the cell cycle. Common side effects of DTIC include nausea, vomiting, impaired liver and kidney function, myelosuppression, and pneumonia. There are no accounts, however, of histological and hematological changes caused by DTIC. We investigated acute hematological and morphological changes in different organs and in tumors that were caused by a single dose of DTIC. Adult Syrian golden hamsters were inoculated with a suspension of tumorigenic baby hamster kidney (BHK) cells by subcutaneous injection. On day 14 after inoculation, doses of 1.4, 1.6, 1.8 or 2.0 g/m(2) DTIC were injected intraperitoneally into the hamsters. Hamsters in the control group were injected with physiological saline in the same way. Seven days after drug or saline injection the animals were sacrificed and samples of blood, heart, kidney, liver, lungs, spleen, small intestine and tumor were excised, processed and analyzed. Mitoses were counted using an ocular extension with engraved frame. Anemia, thrombocytopenia and leukocytosis were found in the control group of hamsters with fibrosarcoma, whereas animals with fibrosarcoma treated with DTIC developed anemia, thrombocytopenia and leukopenia. Severe pneumonia and moderate hepatitis were detected in all DTIC treated groups. Effects of DTIC on tumor cells included rounding and enlargement of nuclei and rarefaction of chromatin. The number of mitoses was reduced with increasing doses of DTIC. Hepatitis, myelosuppression, pneumonia, and dose-related inhibition of tumor cell proliferation were observed after a single dose of DTIC.

Molecular diversity and evolutionary history of rabies virus strains circulating in the Balkans.

Molecular studies of European classical rabies viruses (RABV) have revealed a number of geographically clustered lineages. To study the diversity of Balkan RABV, partial nucleoprotein (N) gene sequences were analysed from a unique panel of isolates (n = 210), collected from various hosts between 1972 and 2006. All of the Balkan isolates grouped within the European/Middle East Lineage, with the majority most closely related to East European strains. A number of RABV from Bosnia & Herzegovina and Montenegro, collected between 1986 and 2006, grouped with the West European strains, believed to be responsible for the rabies epizootic that spread throughout Europe in the latter half of the 20th Century. In contrast, no Serbian RABV belonged to this sublineage. However, a distinct group of Serbian fox RABV provided further evidence for the southwards wildlife-mediated movement of rabies from Hungary, Romania and Serbia into Bulgaria. To determine the optimal region for evolutionary analysis, partial, full and concatenated N-gene and glycoprotein (G) gene sequences were compared. Whilst both the divergence times and evolutionary rates were similar irrespective of genomic region, the 95 % highest probability density (HPD) limits were significantly reduced for full N-gene and concatenated NG-gene sequences compared with partial gene sequences. Bayesian coalescent analysis estimated the date of the most common recent ancestor of the Balkan RABV to be 1885 (95 % HPD, 1852-1913), and skyline plots suggested an expansion of the local viral population in 1980-1990, which coincides with the observed emergence of fox rabies in the region.

BHK-21 cell culture rabies vaccine: immunogenicity of a candidate vaccine for humans.

Veterinary rabies vaccines produced in BHK-21/C13 permanent cell cultures have been used for a long period of time and have been proven as efficacious and safe. A candidate vaccine for human use (YU BHK Rabivak) was developed at the Pasteur Institute, Novi Sad, Serbia on the basis of the fixed rabies virus strain "L. Pasteur 2061/Vero 15 pas" using BHK 21/C13 as a cell substrate for vaccine production. To test the vaccine immunogenicity, a clinical trial was conducted involving 164 subjects between 18 and 60 years of age, immunized either with the YU BHK Rabivak vaccine candidate orwith a commercially available vaccine (Rabipur). Three groups of subjects were immunized with either vaccine by intramuscular administration in the deltoid region, following a pre-exposure regimen on days 0, 7 and 21, or the Essen or Zagreb post-exposure regimens. Rabies virus neutralizing antibodies (VNA) titres were determined by rapid fluorescent focus inhibition test (RFFIT) 21 and 30-45 days post vaccination. A protective titre of VNAs (>0.5 IU/ml) was found in all subjects vaccinated. Dynamics of the immune response showed that 96.4% of the subjects developed protective VNA titres after two doses, 99.3% after three doses and 100% after four and five doses of the candidate YU BHK Rabivak vaccine. There was a low reactogenicity without serious adverse events indicating a satisfactory safety profile in humans. Results obtained in this study indicate that BHK 21 cells offer the possibility of producing an efficacious and safe cell-culture rabies vaccine for humane use.

Molecular epidemiology of rabies viruses in Europe.

Several different strains of classical rabies virus co-circulate in Europe. In order to investigate the roles of the host species and topography on the molecular epidemiology of these viruses, a 400 bp region of the nucleoprotein gene was sequenced and compared with more than 500 European virus isolates. Viruses from 21 European countries were represented including some unique panels of archived isolates from the former Republic of Yugoslavia, Estonia, the Czech Republic, Poland and Austria. Phylogenetic analysis of 198 unique sequences demonstrated numerous groups of viruses clustered at both geographical and host-species levels.

[Clinical trial of YU BHK Rabivak vaccine against rabies in volunteers]. / Klinicko ispitivanje YU BHK RABIVAK vakcine protiv besnila na dobrovoljcima.

The BHK (baby hamster kidney) continual cell line has been used for years in the production of rabies vaccine for animals. Becouse of the rich harvesting of rabies virus from this cell substrate, there is no need of the an additional virus concentration, so that the vaccine production can be organised in small local Pasteur Institutes as well. Although its long-range use in the veterinary medicine has proved that the BHK line is safe and that the vaccine is immunogenic, its use in the human medicine has been only recently studied. According to the latest recommendations of the Expert Committee for the Biological Standardisation of the WHO from 1998, residual cellular DNA in the vaccine is not a potential risk because the inactivation of the vaccine by the beta-propiolactone completely inactivates its possible biologic activity. In the work, the authors report the results of the third phase of the clinical trail of the YU BHK Rabivak vaccine on volunteers. The rabies vaccine studied, named YU BHK Rabivak, produced by Novi Sad Pasteur Institute, caused the generation of protective titre of virus-neutralising antibodies in all examinees: in 96.4% after 2 doses, in 99% after 3 doses, and in 100% after 4 and 5 doses of the vaccine. The registered side effects were expected, mild, did not need medical treatment, and were not more frequent than in the case of other vaccines from cell culture.

[10 years' of production and use of human rabies immunoglobulin in Yugoslavia]. / Deset godina proizvodnje i primene humanog imunoglobulina protiv besnila u Jugoslaviji.

Application of the rabies immunoglobuline is a compulsory part of the prophylaxis of rabies in all severe, transdermal lesions caused by rabies infected animals. Sylvatic rabies has spread in the past few years throughout the whole Yugoslavia, and human cases of rabies have also been reported in other East European countries. In order to achieve the highest level of rabies prophylaxis, apart from postinfective rabies vaccination, it is necessary to provide passive immunization using specific antibodies against rabies. After successful immunization of the young, healthy volunteers in 1990, National Blood Transfusion Institute, in cooperation with the Pasteur Institute from Novi Sad, prepared the first quantities of immunized plasma by plasmapheresis procedure and human rabies immunoglobuline. Without national production, sufficient quantities of human rabies immunoglobuline could not be provided, since the price on the world market is rather high (over $1000 per patient).

[Manifestations of visceral and ocular symptoms of toxocariasis in a 6-year-old boy]. / Pojava visceralne i okularne klinicke slike toksokarijaze kod sestogodisnjeg decaka.

In the past 10 years we have examined 137 cases of toxocariasis, predominantly in children. Three cases were with unilateral ocular involvement. The article reviews a 6-year-old boy with left side strabismus and granulomatous chorioretinitis. Laboratory examinations revealed blood eosinophylia 24% and IgG against Toxocara canis larvae in titer 1:320 by indirect immunofluorescent assay. In epidemiologic anamnesis we concluded that about 4, 5-year-long geophagia was the source of infection.

Immunogenicity of BHK-rabies vaccine in human volunteers.

The production of rabies vaccine on baby hamster kidney (BHK-21) cells for human use is discussed. Long term experience in application of this vaccine in animals, without any noticeable complications and findings of inactivation of contaminated DNA from the cell substrate by beta-propiolactone have justified its recommendation for human use. Preliminary results of applying this simple, adjuvant vaccine in volunteers, confirmed its good tolerability and immunogenicity.

[Prevention of human rabies in Yugoslavia]. / Profilaksa besnila ljudi u FR Jugoslaviji.

Over the last two decades human rabies prophylaxis in Yugoslavia was successful and not a single case of the disease was registered. However, every year there are registered cases of those injured by animals in which rabies was confirmed by laboratory evidence. In FR Yugoslavia every year 800-1000 people are vaccinated against rabies, which makes 10% of all the people injured by animals in Yugoslavia. During 1997 there were 10.266 patients injured by animals; 816 (7.95%) were vaccinated whereas 52 were injured by rabid animals or rabies-suspected animals; 662 were injured by unfamiliar or wild animals, while 102 patients were injured by familiar or preventively vaccinated animals. In other patients vaccination was avoided by a 10-day veterinarian surveillance of the dog. New literature estimated application of reduced so-called "Zagreb-2-1-1" vaccination protocol in severe injuries as unsuccessful in children because of active immune response suppression by simultaneously given immunoglobulin, and that is why this protocol must be abandoned.

[Epizootiologic situation of rabies in 1997. Urbanization of sylvatic rabies in Yugoslavia]. / Epizootioloska situacija besnila u 1997. godini. Urbanizacija silvaticnog besnila u Jugoslaviji?

The Federal Republic of Yugoslavia is situated on the border of a great sylvatic rabies epizootics occurring in central and eastern Europe. That is the reason why a relatively small number of rabid animals have been registered. In 1997, 124 rabid animals, mainly red foxes, were identified. In recent years red fox rabies epizootics in Yugoslavia has annually spread 15-20 km to the south. Rabies in domestic animals, especially dogs, was characterized with furious clinical forms and dog-to-dog transmission typical of urban rabies. Sylvatic rabies has been almost completely eradicated in some countries of western Europe in the last years, mostly due to the method of oral vaccination of foxes-the main vector of rabies.

[Rabies in dogs and efficacy of active immunoprophylaxis in Yugoslavia]. / Besnilo pasa i efikasnost aktivne imunoprofilakse u FR Yugoslavia.

In this study we analyzed 36 cases of dog rabies diagnosed at the Pasteur Institute in Novi Sad during the last 10 years as well as the quality of postvaccination immunity. Most dogs had the paralytic form, more common in sylvatic rabies than furious clinical picture. 11% of dogs were aggressive, whereas 44% had injured humans, including their owners. Postvaccination immunity was examined in a group of dogs vaccinated with three domestic and one vaccine imported from abroad. Vaccination was successful in more than 70% of dogs and it is a sufficient percentage of seroconversion in dog rabies eradication. However, in dogs vaccinated only once during lifetime, the immunity was not satisfactory. That is why in primary vaccination of dogs compulsory revaccination 30 days after the first vaccine dose is suggested.

[Economical production of rabies vaccine on cell cultures]. / Mogucnosti ekonomicne proizvodnje vakcine protiv besnila sa kulture celija.

By producing the rabies vaccine from cell culture, this vaccination has become safe, with minimal postvaccinal reactions. The first vaccine according to this technology was produced by Pavle (Paul) Fenje, former chief of department of the Pasteur Institute in Novi Sad. Many cell cultures have been introduced so far for the rabies virus multiplication: primary hamster kidney, fetal bovine kidney, chick embryo, continuous cell line monkey kidney (VERO), human diploid cell (HDC), etc. Some possibilities of an economical rabies vaccine production from a continuous BHK-21 cell line have been discussed and recommended.