Abstract Cadmium is a heavy metal, toxic even in trace amounts and its biological function in the human body has not been described to date. It is assumed that cadmium manifests in dose-dependent genotoxic and cytotoxic effects on many organs and tissue types. In this study, we have analyzed the biochemical parameters in the serum of Japanese quails (Coturnix japonica) after chronic in vivo exposure to cadmium. Adult animals were exposed to cadmium in the form of CdCl2 dissolved in water (0.20 mg/L) for 20 days. Significant differences between controls and exposed animals were found in 12 out of 13 analyzed biochemical parameters. Total bilirubin concentrations did not show any significant differences between the two groups. Exposure to cadmium has resulted in a significant increase in lactate dehydrogenase activity, sodium and chloride concentration, as well as significant reductions in total proteins, albumins, globulins, glucose, triglycerides, cholesterol, calcium concentration, and alkaline phosphatase activity. In this sense, chronic in vivo exposure to low doses of cadmium has induced severe changes in the levels of observed biochemical parameters and enzyme activity. Additionally, evident cytogenetic changes in the liver were also noted, where hepatocyte damage and even lack of organized nuclei, including nuclear fragmentation, clearly indicated ongoing apoptotic processes.

Copper sulfate has been used for many years as an algaecide and parasite treatment. The usage of copper has certain issues as there is a thin line that separates effective treatment levels from overdoses. Copper sulfate can be extremely toxic to fish under certain conditions. This study is focused on the effects of copper sulfate on some blood indices in Oncorhynchus mykiss during a 24 hour period. Our research used one procedure 40 Oncorhynchus mykiss with the average weight of 220 ±10 g. The experimental group had 20 fish that were treated with the blue vitriol (dose of 0,012 g/40l), during a 24 hour period. The control group had 20 fish. The presence of copper in the water leads to the significant increase of erythrocyte, hemoglobin, MCHC and leukocyte levels while the MCV levels were noted to be considerably low. Monocytes, unsegmented and segmented granylocites were significantly increased in the experimental group of fish. Lymphocyte count was considerably reduced in the same group of fish.

Objectives This study aims to investigate the low-resolution human leukocyte antigen (HLA)-B locus polymorphisms between unrelated healthy individuals and patients with diagnosis of seronegative spondyloarthropathies and determine risky and protective allelic groups and genotypes. Patients and methods The study included 104 healthy control individuals (52 males, 52 females; median age 43 years; range 2 to 76 years) and 96 patients (43 males, 53 females; median age 28.5 years; range 2 to 67 years) diagnosed with: ankylosing spondylitis (AS) (n=19), reactive arthritis (n=19), psoriatic arthritis (n=28) and undifferentiated spondyloarthropathies (n=30). Genomic deoxyribonucleic acid was extracted from peripheral blood to detect allelic groups of HLA class I and II. Single-specific-primer polymerase chain reaction was used for HLA genotyping and visualization of products after their separation on 1.5% agarose gel for horizontal gel electrophoresis. Results Significantly increased frequency was found for HLA-A*02 and HLA-B*27 allelic variants in all groups of patients. The increased frequency of the HLA-B*35 allelic group in the control group represents the protective gene variant for the occurrence of AS. The predisposing genotype (HLA-B*27/B*44 and B*27/B*51) for the onset of disease was only found in AS patients. Conclusion This study shows the strong association of HLA-B*27 antigen with spondyloarthropathies, which is considered a risk variant of the gene for the onset of disease. Protective and risky allelic variants and genotypes are rare and their detection as well as increased frequency are possible if larger numbers of patients are involved.

The main circulatory medium of echinoderms is the coelomic fluid. Biochemistry of coelomic fluid is very complex and slightly different from seawater. The main aim of this research was to analyse concentration of electrolytes (potassium, calcium, magnesium, chlorine and sodium), heavy metals (lead, chromium, cadmium and cobalt) and iron in coelomic fluid of sea urchin, Arbacia lixula as an indirect indicator of seawater contamination. After precise statistical evaluation it was observed that electrolyte concentrations were; Na 189.20±11.41 mmol/l, Cl 134.06±37.08 mmol/l, Mg 4.24±1.08 mmol/l, Ca 3.04±0.84 mmol/l. Biochemical content of heavy metals in coelomic fluid was; Co 1.292±0.879 ppm, Pb 0.644±0.131 ppm, Cr 0.116±0.055 ppm, Cd 0.031±0.017 ppm and iron 0.259±0.058 ppm, and it depends on the potential accumulation level in the organism. The composition of electrolyte and heavy metal content varies depending on the composition of seawater. Obtained values are within the range of values most commonly found in low polluted areas of the Adriatic sea. Similar models may be applied for monitoring of heavy metal contamination in marine environment.

Thermal changes in water cause many metabolic changes that manifest themselves in physiological fish adaptations. The analysis of hematologic and biochemical blood parameters provides important information on environmental influences on the health status of fish. The hematocrit (HCT) (l/l), hemoglobin concentration (Hb) (g/l), mean corpuscular volume (MCV) (fl), mean corpuscular hemoglobin (MCH) (pg), mean corpuscular hemoglobin concentration (MCHC) (g/l) and red blood cells (RBC) (x1012/L) were analyzed. Animals were grouped into two groups: control (n=10) and experimental (n=16). The experimental fish were exposed to 28°C for 30 min. Puncture of the heart was done and the blood without anticoagulant was analyzed. During hyperthermia, an increase in hematological parameters was observed, except for MCV values that were low. Significant differences were established only for the number of erythrocytes and the hematocrit values (p<0.05). The results showed a decrease in MCV and an increase in the value of other erythrocyte parameters. Significant changes in the number of erythrocyte and hematocrit values were found. Some hematological parameters such as erythrocyte and MCV values are significant stress indicators and can serve us as important factors for physiological adaptations of fish. The carp shows excellent ability to adjust to temperature variations that can be seen through the analysis of hematological status.

Abstract Emperipolesis is considered a physiological phenomena often present in various pathophysiological conditions, but its etiology is still unknown. In this study, we analyzed the number of megakaryocytes and the percentage of emperipoletic cells in the sternal and femoral bone marrow of Wistar rats. Five types in the thrombopoiesis lineage (megakaryoblasts, promegakaryocytes and megakaryocytes - acidophilic, basophilic and thrombocytogenic) were determined. Except for basophilic megakaryocytes, significant differences were found for number of thrombopoietic cells in the sternal and femoral bone marrow. A larger number of thrombocytogenic megakaryocytes were present in the sternal bone marrow. Emperipoletic cells were significantly present in the femoral compared to the sternal bone marrow. Emperipolesis was typical for lymphocytes and neutrophils individually, while emperipolesis with two or more cells within thrombopoietic cell was also present (1-7 %) and significant differences between the sternal and femoral bone marrow were detected. Emperipolesis was found in all analysed rats and it most commonly occured within mature megakaryocytes and rarely megakaryoblasts, while it was not recorded in the promegakaryocytes. The high incidence of megakaryocytes with emperopolesis in rats could be a consequence of “normal” cell retention in the cytoplasm of megakaryocytes while passing blood cells to circulation or related to haematopoietic response due to high incidence of inbreeding.

* Correspondence: suljevic.damir@gmail.com A great success in the evolution of animal behavior and physiology is the ability to survive seasonally unfavorable conditions, especially when it comes to variable and extremely low temperatures (Roots, 2006). Environmental variations lead animals to a state of hibernation, which causes depression of metabolic and physiological processes, as well as a slow development and reproduction so that the animal can survive (Dugbartey and Henning, 2013). Life activities of the gastropods depend on environmental factors throughout the whole year. Shells have great importance because they reduce water loss during summer days. The formation of the calcareous epiphragm, with which they bind to substrates, is an additional protection mechanism of evaporation (Elmslie, 1998). Snail hibernation includes shell closing by the secretion of the epiphragm, while energy is gained from stored food reserves during this process (Holtz and Von Brand, 1940). In experimental conditions, aestivation can last for years, which suggests that the metabolic rate depends on both exogenous and endogenous factors (Nowakowska, 2011). Respiration is reduced during hibernation and about 10% of the fluid is lost, which affects heart rate, oxygen consumption, and energy requirements (Nicolai et al., 2011). Metabolic changes during hibernation affect the value of circulating hemocytes. The phagocytic activity of hemocytes and lectins, as a defense mechanism, takes place thanks to the presence of calcium ions in the snail hemolymph. Consequently, the changes in the values of calcium ions affect the hemocyte number (Saleddin and Wilbur, 1984). Many studies confirmed the importance of hemocytes in immune responses, antibody recognition, phagocytosis, encapsulation, and cytotoxic reactions (Yoshino et al., 2001). Two types of hemocytes, granulocytes and hyalinocytes, have been identified in the hemolymph of Biomphalaria (Ratcliffe, 1985). Seasonal factors and different experimental tests affect the total number, type, and behavior of hemocytes (Oliver and Fisher, 1995). The role of hemocytes today is most widely considered as an immunological response to infections. Seta et al. (1996) reported that the phagocytic potential of hyalinocytes is questionable and that only granulocytes are crucial in the immune response. Vinaud et al. (2008), Delgado et al. (2001), and Matricon-Gondran and Letocart (1999) identified four types of hemocytes based on their size (small, medium, large, and gigantic cells) in the hemolymph of Biomphalaria glabrata. According to morphological criteria, there are two types, round cells and spread cells (Sminia, 1981), while in the study of Adamowicz and Bolaczek (2003), they refer to these cells as type I and type II. Wojtaszek et al. Abstract: The total number of hemocytes and their morphological characteristics were analyzed in Helix pomatia Linnaeus, 1758, by light microscopy during different phases of hibernation. Three types of hemocytes were identified: hyalinocytes, agranulocytes, and granulocytes. Total number of hemocytes decreased significantly during hibernation. Most intensive changes in cell size, morphology, and appearance of special structures in granulocytes happened during the hibernation period itself. Termination of hibernation coincides with very important granulocyte transformation and the appearance of specific gigantic short-lived cells. All of the changes in cell number and morphology are characteristics of different hibernation phases and have a major role in adaptive hypothermia. Granulocytes are hemocytes of great importance in immune response while specific functions of hyalinocytes and agranulocytes remain largely unexplored.

Hemolymph of snail Helix pomatia was analyzed in this study to determine the presence of neurons. The animals were sampled at different localities from April to June 2018. Locations differ in many ecological features, especially in insolation, humidity and altitude. Neurons were recorded in snail's hemolymph from one locality (neurone presence in hemolymph in 40% of specimens). Two types of neurons were identified: unipolar and multipolar. This physiological phenomenon is characterized by the presence of different forms of neurons, both individual and related, as well as the formation of a neuronal network. Neurons are very heterogeneous both in terms of number and size, as well as morphological characteristics. Some neurons did not have dendrites or had short axon, while some had giant axons. The presence of neurons in hemolymph still does not have physiological explanation, but the interaction of hemolymph and nervous system as well as seasonal influences may have great significance for this physiological phenomenon which opens up a significant topic for future research.

Total number and types of hemocytes were analysed in hemolymph of Roman snail (Helix pomatia L.). Significant differences in the number and types of hemocytes during the season at different locations were identified. The average number of hemocytes ranged from 154 to 288 per 10 μL of hemolymph. The highest number of hemocytes was recorded in snails at higher altitudes. Significant intergroup differences between all sites were identified. The most numerous hemocytes were granulocytes, followed by agranulocytes, while hyalinocytes were the least common in the post-hibernation period. Post hoc analysis showed certain intragroup differences for most locations except for comparison between wet habitat and habitats with anthropogenic influence, and between wet habitats and urban place with wet vegetation. A large number of transformed hemocytes in the encapsulated phase were identified in snail hemolymph infected with flat worms. Our results represent the first record of data for the number of hemocytes in molluscs and the first data for hemocytes of Helix pomatia. Further investigations and development of a uniform classification of hemocytes and their role in the immune response would be necessary in the future.

In this study we analyzed the effects of NaOCl solution on the blood and biochemical parameters of the rainbow trout Oncorhynchus mykiss (Walbaum, 1792). The treatment lasted for three days with prior dechlorination of the water. Significant differences in the concentration of K+, Ca2+, Cl-, proteins, glucose were detected and the largest variations were found for creatinine concentration (P<0.05). Significant differences were found Hct, Hb concentration, RBC, WBC, MCH and MCHC values, percentage ratio of lymphocytes, segmented granulocytes and the total number of neutrophil granulocytes (P<0.05). Small doses of NaOCl alter the acid-base balance, suggesting a very low-level adaption in the rainbow trout.

BACKGROUND: AFP serum levels are considered as diagnostic and specific for hepatocellular carcinoma (HCC) in patients with liver cirrhosis (LC). AIM: This study aimed to examine the diagnostic value of AFP in the distinguishing of patients with HCC from patients with LC, and to analyse the potential correlation between AFP levels and liver disease stages. MATERIAL AND METHODS: Fifty patients with LC and fifty patients with HCC were included in this study. The majority of the patients were males, while the HBV aetiology was dominant. RESULTS: Significant differences between LC and HCC patients were detected for AST, ALT, GGT, bilirubin, AFP and AP. Patients with HCC had higher AFP values compared to LC. There was no significant correlation between the size of the tumour lesion and serum AFP levels. A positive correlation between AFP concentration and GGT activity was determined, as was the negative correlation between AFP and age of the subjects. The AFP value of 23.34 ng/m showed high sensitivity (84%) and specificity (82%). CONCLUSION: The size of the surface below the ROC curve (AUC) was 0.877 (0.80-0.95), which makes AFP a good biomarker and this diagnostic test is sufficient to separate patients with HCC and LC.

Zubčević, N., M. Fočak and D. Suljević, 2018. Highly specifi c hemagglutination activity of plant lectins in specifi c species: case of Fabaceae and Solanaceae. Bulg. J. Agric. Sci., 24 (3): 391–397 Lectins are carbohydrate-binding proteins present in most of the plants and in some animals. They possess the ability to agglutinate erythrocytes with known carbohydrate specifi city since they have at least one non-catalytic domain that binds reversibly specifi c monosaccharides or oligosaccharides. This study investigated the presence of lectins in the plant species of the family Fabaceae and Solanaceae. The results of our study have shown that 6 of 10 plant lectins caused agglutination, and 4 of them did not cause agglutination of human erythrocytes. Blood agglutination activity against A, B, AB and 0 groups was shown after exposing blood to lectin extracts obtained from 80% of tested plants in family Fabaceae, and 20% of tested plants in family Solanaceae. The highest degree of agglutination was obtained in Phaseolus vulgaris (+4) and the highest protein concentration was obtained in Lens esculenta within the family Fabaceae. There was no statistically signifi cant difference for protein concentration and specifi c lectin activity between two compared families (p>0.05).

Peripheral blood and haematopoiesis in bone marrow and liver tissue were investigated in Bufo bufo during the sexual cycle. A combined touch and imprinting tissue method were applied for the haematopoietic cell isolation. The liver is the primary site of erythropoiesis and partial lymphopoiesis. There was a positive correlation between body mass and liver mass, but it was not significant (P > 0.05). The primary role of yellow bone marrow is the maturation of haematopoietic cells, while red bone marrow is responsible for the production of blood cells. The largest surface, and therefore the largest dimensions, was established for erythrocytes. Significant differences in the number of leukocytes and their size were established between males and females (P < 0.05). Small numbers of leukocytes but very large cells were characteristic for females. The highest percentages of leukocytes were for lymphocytes and eosinophils. The reproductive period showed significant impact on the haematopoietic intensity in the bone marrow and liver, resulting in changes in peripheral blood, regarding the ratio of mature blood cells.