Tamjanika, a Balkan native variety of Vitis vinifera L.: Chemical characterization, antibacterial, and anti-dermatomycosis potential of seed oil.

This study was designed to explore functional food properties of edible seed oil obtained from Tamjanika seeds-autochthonous grape variety of Balkan Peninsula. In order to accomplish our goals, seed oil was isolated by Soxhlet apparatus and chemically characterized regarding fatty acids, carotenoids, tocopherols, and tocotrienols. Antimicrobial activity of the isolated oil was tested by microdilution method. For that purposes, six bacterial species were used, belonging to human infectious agents and food contaminants. Furthermore, the activity of the oil was investigated against clinical isolates of dermatomycetes. Our study has shown that oil of Vitis vinifera L. Tamjanika variety was an abundant source of polyunsaturated fatty acids (81.43%) with predominant linoleic acid. HPLC analysis revealed the presence of carotenoid lutein (0.15 mg/100 g). The seed oil was rich in tocotrienols (85.04 mg/100 g) predominating over tocopherols (8.37 mg/100 g). The oil possessed microbicidal activity against all the tested microbes. Bacteria were more sensitive to the effect of the oil (minimum inhibitory concentration [MIC] 7.7-15.4) when compared with oil effect on tested dermatomycetes (MIC 20-40). Our investigation has shown for the first time that grape oil could be active against wide spectrum of bacteria and clinically isolated dermatomycetes. The significance of this study lies in the fact that it pointed out the functional food properties of grape seed oil that was fully chemically characterized.

Chemical and Biological Investigations of Allium scorodoprasum L. Flower Extracts.

This study was designed to investigate the impact of different extraction solvent systems on the chemical composition and biological activities of Allium scorodoprasum L. (Amaryllidaceae)-the medicinal plant that was traditionally used as a remedy in the medieval period in the Balkans. Targeted chemical analysis of nine different extracts was performed by UHPLC(-)HESI-QqQ-MS/MS. Antimicrobial and antibiofilm activities of the extracts were investigated on sixteen clinical isolates of bacteria, yeasts and dermatomycetes, all isolated from infected human skin and corneal formations. Cytotoxicity and wound-healing properties were tested on human immortalized keratinocytes (HaCaT cell line). Antioxidant activity was assessed by six different assays, while beneficial potential against certain neurodegenerative diseases and type 2 diabetes was determined in selected enzyme inhibition assays coupled with molecular modeling. The results showed that the obtained extracts were rich in phenolic compounds, especially flavonoid glycosides such as rutin and kaempferol 3-O-glucoside. All of the extracts showed antimicrobial, wound-healing, antioxidant and anti-enzymatic properties. This study is the first of its kind, linking the medieval medicinal use of wild-growing flowers of A. scorodoprasum with contemporary in vitro scientific approaches.

Exploring immunogenicity of tick salivary AV422 protein in persons exposed to ticks: prospects for utilization.

In order to determine whether conserved tick salivary protein AV422 is immunogenic, the goal of our study was to detect specific IgG response within at-risk populations. Study groups included 76 individuals, differing in occurrence of recently recorded tick bites and health status. Western blotting with recombinant (r) protein derived from Ixodes ricinus (Ir) was performed. IgG response to Borrelia/Rickettsia, as indicators of previous tick infestations, was also assessed. Additionally, a detailed in silico AV422 protein sequence analysis was performed, followed by modelling of the interactions between peptides and corresponding MHC II molecules by molecular docking. Anti-rIrAV422 seroprevalences among individuals exposed to ticks were high (62.5, 57.9 and 66.7%) and anti-Borrelia/Rickettsia seroprevalences were 54.2, 15.8 and 44.4% among individuals with/without recent tick bite and patients suspected of tick-borne disease, respectively. In silico analysis of AV422 protein sequence showed a high level of conservation across tick genera, including also the predicted antigenic determinants specific for T and B cells. Docking to the restricted MHC II molecules was performed for all predicted AV422 T cell epitopes, and the most potent (highly immunogenic) epitope determinants were suggested. The epitope prediction reveals that tick salivary protein AV422 may elicit humoral immune response in humans, which is consistent with the high anti-rIrAV422 seroprevalence in tested at-risk subjects. Tick-borne diseases are a growing public health concern worldwide, and AV422 is potentially useful in clinical practice and epidemiological studies.

Hepatoprotective Effect of Mixture of Dipropyl Polysulfides in Concanavalin A-Induced Hepatitis.

The main biologically active components of plants belonging to the genus Allium, responsible for their biological activities, including anti-inflammatory, antioxidant and immunomodulatory, are organosulfur compounds. The aim of this study was to synthetize the mixture of dipropyl polysulfides (DPPS) and to test their biological activity in acute hepatitis. C57BL/6 mice were administered orally with DPPS 6 h before intravenous injection of Concanavalin A (ConA). Liver inflammation, necrosis and hepatocytes apoptosis were determined by histological analyses. Cytokines in liver tissue were determined by ELISA, expression of adhesive molecules and enzymes by RT PCR, while liver mononuclear cells were analyzed by flow cytometry. DPPS pretreatment significantly attenuated liver inflammation and injury, as evidenced by biochemical and histopathological observations. In DPPS-pretreated mice, messenger RNA levels of adhesion molecules and NADPH oxidase complex were significantly reduced, while the expression of SOD enzymes was enhanced. DPPS pretreatment decreased protein level of inflammatory cytokines and increased percentage of T regulatory cells in the livers of ConA mice. DPPS showed hepatoprotective effects in ConA-induced hepatitis, characterized by attenuation of inflammation and affection of Th17/Treg balance in favor of T regulatory cells and implicating potential therapeutic usage of DPPS mixture in inflammatory liver diseases.

A Forgotten Episode of Marburg Virus Disease: Belgrade, Yugoslavia, 1967.

In 1967, several workers involved in poliomyelitis vaccine development and production fell ill at three different locations in Europe with a severe and often lethal novel disease associated with grivets (Chlorocebus aethiops) imported from Uganda. This disease was named Marburg virus disease (MVD) after the West German town of Marburg an der Lahn, where most human infections and deaths had been recorded. Consequently, the Marburg episode received the most scientific and media attention. Cases that occurred in Frankfurt am Main, West Germany, were also described in commonly accessible scientific literature, although they were less frequently cited than those pertaining to the Marburg infections. However, two infections occurring in a third location, in Belgrade, Yugoslavia, have seemingly been all but forgotten. Due in part to their absence in commonly used databases and in part to the fact that they were written in languages other than English, the important articles describing this part of the outbreak are very rarely cited. Here, we summarize this literature and correct published inaccuracies to remind a younger generation of scientists focusing on Marburg virus and its closest filoviral relatives of this important historical context. Importantly, and unfortunately, the three episodes of infection of 1967 still represent the best in-depth clinical look at MVD in general and in the context of "modern" medicine (fully resourced versus less-resourced capacity) in particular. Hence, each individual case of these episodes holds crucial information for health care providers who may be confronted with MVD today.

Molecular Detection and Serological Evidence of Tick-Borne Encephalitis Virus in Serbia.

Tick-borne encephalitis (TBE) is a zoonotic flaviviral infection that is a growing public health concern in European countries. The aims of this research were to detect and characterize tick-borne encephalitis virus (TBEV) in Ixodes ricinus ticks at presumed natural foci in Serbia, and to determine seroprevalence of TBEV IgG antibodies in humans and animals. A total of 500 I. ricinus ticks were examined for the presence of TBEV by real-time RT-PCR, and conventional nested PCR and sequencing. To determine TBEV seroprevalence, 267 human sera samples were collected, as were 200 sera samples from different animal species. All sera samples were examined by ELISA for the presence of anti-TBEV antibodies. To exclude cross-reactivity, all sera samples were tested for anti-West Nile virus (WNV) antibodies and all human sera samples were also tested for anti-Usutu virus antibodies by ELISA. Results of this preliminary study indicated TBEV activity in Serbia at two microfoci. Several decades after the previous documentation of TBEV in Serbia, we have demonstrated the presence of TBEV in I. ricinus questing nymphs (prevalence 2% and 6.6% at the two different localities) and anti-TBEV antibodies in humans (seroprevalence 0.37%). Moreover, we show for the first time TBEV seroprevalence in several animal species in Serbia, including dogs (seroprevalence 17.5%), horses (5%), wild boars (12.5%), cattle (2.5%), and roe deer (2.5%). None of the goats tested was positive for anti-TBEV IgG antibodies. TBEV isolate from I. ricinus tick in this study belonged to the Western European subtype. To understand the true public health concern in Serbia, detailed epidemiological, clinical, virological, and acarological research are required. This is important for implementation of effective control measures to reduce the incidence of TBE in Serbia.

Smallpox as an actual biothreat: lessons learned from its outbreak in ex-Yugoslavia in 1972.

Variola (smallpox) virus is classified as class A of potential biological weapons, due to its microbiological, genetic, antigenic and epidemiological characteristics. The potential danger is more real because vaccination against smallpox has stopped since disease eradication in 1979. That is why we want to share our unique, rich experience and acquired knowledge in the fight against this highly contagious and deadly disease during the smallpox outbreak in ex-Yugoslavia in 1972. It was the largest postwar outbreak in Europe when there were officially registered 175 ill patients, 35 of them with lethal outcome. This outbreak was specific by the time of its occurrence, the affected territory, dimensions and some epidemiological characteristics, but also by the well-organized, synchronized and efficient reaction of the competent state services in the fight against it.

Prevalence of Panton-Valentine leukocidin genes in community-associated methicillin-resistant Staphylococcus aureus in the District of Pomoravlje.

BACKGROUND/AIM: Community-associated methicillin-resistant Staphjlococcus aureus (CA-MRSA) strains appear to have rapidly disseminated among population in the community without established risk factors for MRSA worldwide. Panton-Valentine leukocidin (PVL) is a cytolytic toxin, encoded by the lukF-PV and lukF-PV genes. PVL may be the key toxin responsible for enhanced virulence of CA-MRSA. The aim of this study was to detect the genes encoding PVL in CA-MRSA isolates from healthy people from the District of Pomoravlje. METHODS: We took throat and nose swabs from healthy, employed persons with mandatory sanitary examinations and analyzed the presence of MRSA, between January 2011 and December 2012 in the District of Pomoravlje. Susceptibility of isolated strains to cefoxitin was investigated by using disc diffusion according to the recommendation of CLSI (Clinical Laboratory Standard Institute), and by E test. The presence of penicillin-binding protein 2a (PBP2a) in Stapbylococd was detected using latex agglutination Slidex@MRSA Detection test. The gold standard, polymerase chain reaction (PCR) assay, was used for detection of mecA gene and PVL gene, and typing of SCCmec region. RESULTS: Our investigation showed that staphylococcal carrier state was present in 2.58% of 52,910 throat and nasal swabs, and in 50 of them (3.67%) MRSA was isolated. Among these MRSA, 2 (4/6) isolates were PVL-positive. CONCLUSION: The prevalence of CA-MRSA and the presence of PVL gene among healthy, employed population in the District of Pomoravlje were low. The values obtained in this study show that, our region is not significantly different from the other parts of our country, nor from the other European countries.

Antimicrobial susceptibility and ß-lactamase production in Bacillus cereus isolates from stool of patients, food and environment samples.

Background/Aim: Bacillus cereus (B. cereus) usually ingested by food can cause two types of diseases: vomiting due to the presence of emetic toxin and diarrheal syndrome, due to the presence of diarrheal toxins. Systemic manifestations can also occur. The severe forms of disease demand antibiotic treatmant. The aim of this study was to determine the differences in antibiotic susceptibility and ß-lactamase activity of B. cereus isolates from stools of humans, food and environment. Methods: Identification of B. cereus was performed with selective medium, classical biochemical test and polymerase chain reaction (PCR) with primers specific for bal gene. Thirty isolates from each group were analysed for antibiotic susceptibility using the disk-diffusion assay. Production of ß-lactamase was determined by cefinase test, and double-disc method. Results: All strains identified as B. cereus using classical biochemical test, yielded 533 bp fragment with PCR. Isolates from all the three groups were susceptible to imipenem, vancomycin, and erythromycin. All isolates were susceptible to ciprofloxacin but one from the environment. A statistically significant difference between the groups was confirmed to tetracycline and trimethoprim-sulphamethoxazole sensitivity. A total of 28/30 (93.33%) samples from the foods and 25/30 (83.33%) samples from environment were approved sensitive to tetracycline, while 10/30 (33.33%) isolates from stools were sensitive. Opposite to this result, high susceptibility to trimethoprim-sulphamethoxazole was shown in samples from stools (100%), while isolates from foods (63.33%) and from environment (70%) had low susceptibility. All samples produced ß-lactamases. Conclusion: The strains of B. cereus from all the three groups showed high rate of sensitivity to most tested antibiotics, except to tetracycline in samples from human stool and to trimethoprim-sulphamethoxazole in samples from food and environment. The production of ß-lactamases was confirmed in all the strains.

Seroprevalence of Borrelia burgdorferi in occupationally exposed persons in the Belgrade area, Serbia.

Lyme disease (LD) is a natural focal zoonotic disease caused by Borrelia burgdorferi, which is mainly transmitted through infected Ixodes ricinus tick bites. The presence and abundance of ticks in various habitats, the infectivity rate, as well as prolonged human exposure to ticks are factors that may affect the infection risk as well as the incidence of LD. In recent years, 20% to 25% of ticks infected with different borrelial species, as well as about 5,300 citizens with LD, have been registered in the Belgrade area. Many of the patients reported tick bites in city's grassy areas. The aim of this study was to assess the seroprevalence of B. burgdorferi in high-risk groups (forestry workers and soldiers) in the Belgrade area, and to compare the results with healthy blood donors. A two-step algorithm consisting of ELISA and Western blot tests was used in the study. Immunoreactivity profiles were also compared between the groups. The results obtained showed the seroprevalence to be 11.76% in the group of forestry workers, 17.14% in the group of soldiers infected by tick bites and 8.57% in the population of healthy blood donors. The highest IgM reactivity was detected against the OspC protein, while IgG antibodies showed high reactivity against VlsE, p19, p41, OspC, OspA and p17. Further investigations in this field are necessary in humans and animals in order to improve protective and preventive measures against LD.

Seroprevalence of Borrelia burgdorferi in occupationally exposed persons in the Belgrade area, Serbia

Lyme disease (LD) is a natural focal zoonotic disease caused by Borrelia burgdorferi, which is mainly transmitted through infected Ixodes ricinus tick bites. The presence and abundance of ticks in various habitats, the infectivity rate, as well as prolonged human exposure to ticks are factors that may affect the infection risk as well as the incidence of LD. In recent years, 20% to 25% of ticks infected with different borrelial species, as well as about 5,300 citizens with LD, have been registered in the Belgrade area. Many of the patients reported tick bites in city’s grassy areas. The aim of this study was to assess the seroprevalence of B. burgdorferi in high-risk groups (forestry workers and soldiers) in the Belgrade area, and to compare the results with healthy blood donors. A two-step algorithm consisting of ELISA and Western blot tests was used in the study. Immunoreactivity profiles were also compared between the groups. The results obtained showed the seroprevalence to be 11.76% in the group of forestry workers, 17.14% in the group of soldiers infected by tick bites and 8.57% in the population of healthy blood donors. The highest IgM reactivity was detected against the OspC protein, while IgG antibodies showed high reactivity against VlsE, p19, p41, OspC, OspA and p17. Further investigations in this field are necessary in humans and animals in order to improve protective and preventive measures against LD.

A case of human monocytic ehrlichiosis in Serbia.

INTRODUCTION: Ehrlichiosis is a bacterial zoonosis transmitted by hematophagous arthropods--ticks. In humans, it occurs as monocytic, granulocytic, and ewingii ehrlichiosis. Pathological process is based on parasitic presence of Ehrlichia organisms within peripheral blood cells--monocytes and granulocytes. CASE OUTLINE: Fifty-two year old patient was admitted to hospital due to high fever of over 40 degrees C that lasted two days, accompanied with chills, muscle aches, malaise, loss of appetite, headache, confusion, breathing difficulties, and mild dry cough. The history suggested tick bite that occurred seven days before the onset of disease. Doxycycline was introduced and administered for 14 days, causing the disease to subside. Indirect immunofluorescence assay was used to analyze three serum samples obtained from this patient for Ehrlichia chaffeensis antibodies, and peripheral blood smear was evaluated for the presence of Ehrlichia and Ehrlichia aggregation into morulae. CONCLUSION: Ehrlichiosis should be considered in each case where there is a history of tick bite together with the clinical picture (high fever, chills, muscle aches, headache, generalized weakness and malaise, and possible maculopapular rash). The presence of Ehrlichia chaffeensis antibodies was confirmed in a patient with the history of tick bite, appropriate clinical picture and indirect immunofluorescence assay. This confirmed the presence of human monocytotropic ehrlichiosis, a disease that is uncommonly identified in our country.

Epidemiological characteristics of brucellosis in Serbia, 1980-2008.

AIM: To analyze the epidemiological characteristics of human brucellosis in Serbia from 1980 to 2008 and the most important factors affecting its emergence and spread. METHODS: Public sources of data on brucellosis were used, including official reports of infectious diseases and epidemics, as well as monthly and annual reports of the Serbia and Vojvodina Institutes of Public Health. RESULTS: From 1980 through 2008, there were 1521 human brucellosis cases in Serbia. The annual number ranged from 2 in 2000 to 324 in 1991. Infections occurred more often in men (67% of cases) than in women (odds ratio, 2.17; 95% confidence interval, 1.57-3.00; chi2=24.52, P<0.001). The largest number of patients over the entire study period (1184) was recorded in Kosovo and Metohija, which accounted for 78% of the total number of patients. The maximum incidence rate in Kosovo and Metohija was 12 per 100,000 in 1991. In Vojvodina, the first autochthonous human cases of brucellosis were recorded in 1999, and 101 affected persons were registered by the end of 2008. During the period 1994-2008, the largest number of patients in Serbia was recorded from June to September (310 of 623 cases, 50%). The disease was most prevalent among people aged 30-49 years, accounting for 81 of 177 (46%) of the cases in Serbia from 1999 to 2008. CONCLUSION: Brucellosis has been a significant public health concern in Serbia. This problem may be solved by joint efforts of all relevant factors, first of all human and veterinary medical services.

The use of original ex vivo immunoadsorption and "multi-manner" apheresis in ABO/H-mismatched kidney transplants--A phase II clinical study.

Anti-A/B depletion efficacy and clinical outcome for 22 ABO-incompatible kidney transplants were investigated. Preconditioning by anti-CD20 therapeutic plasma exchange (TPE) by Cobe-Spectra (CaridianBCT USA) and simplified extracorporeal immunoadsorption (ECIA) as well as triple immunosuppression (tacrolimus/mycophenolate-mofetil/steroid) were performed. The use of TPE with ECIA resulted in a high in vivo anti-A/B depletion, 94.23±4.2% for IgG and 95.26±3.2% for IgM. The mean anti-A/B titers on day 0 were: IgG=1.27±1.03 and IgM=2.20±1.47. One HLA cross-match positive patient (beside ABO-incompatibility) subjected to double-dose anti-CD20 and intensive TPE-treatment had no allograft rejection. The level of serum creatinine ranged from 100 to 156 µmol/L in the entire group of patients during postoperative follow-up (up to 36 months). One recipient (with sepsis and multi organ distress syndrome) lost kidney function in early posttransplant period. ABO-incompatible (n=2) and ABO-compatible (n=3) kidney recipients had severe anemia and bleeding episodes. They were efficiently treated using original "multi-manner" apheresis. Our study represents a clear demonstration that the combination of TPE with ECIA and anti-CD20 is effective in anti-A/B depletion. This therapeutic approach is feasible in clinical setting showing satisfactory short-term results although verification of long-term effects needs to be confirmed in a larger study. The rapid beneficial outcome of "multi-manner" apheresis strongly supports the future use of this therapeutic modality for efficient oxygenation and advanced engraftment.