Development of a method for assessing the immunological reactivity of the animal body to prevent the development of pathology in the early stages of postnatal development

The study of immunological relationships in the functional system “mother-placenta-offspring” can make a significant contribution to the solution of the issue of improving the safety of the population of newborn animals and poultry. The pathology of the antenatal development of animals has not been sufficiently studied. Antenatal pathology of animals is more often manifested in the form of congenital malformations (anomalies) of development. Congenital defects are usually called those that occur during intrauterine development. However, congenital malformations are also observed in the period of postnatal development – as a consequence of a violation of the further formation of organs in animals. There is a need to develop a method for determining the immunological reactivity of the animal body, which would allow to assess the combined functional state of cell-mediated immunological reactivity of innate and acquired immunity in the functional system “mother-placenta-offspring” and determine the factors that cause immunosuppression. Determination of the immunological reactivity of the animal body includes a blood test by conducting a biological test, which uses the biological activity of red blood cells in the Nitroblue Tetrazolium Test and according to their sorption activity-more than 40% - animals are classified as individuals with reduced immunological reactivity among similar ones.

Antioxidant Vitamins Are Correlated with Different Aspects of Phagocytic Processes in Healthy Nigerians: Benefits As Supplements During Antimicrobial Treatment

Background: Antioxidant vitamins are important for the immune system to function efficiently through several mechanisms. However, according to several previous studies, individual step of leucocyte phagocytosis is not correlated with different antioxidant vitamins. Methods: This study included 50 healthy Nigerians whose cellular phagocytic mechanism such as percentage leucocyte migration (%LM) and intracellular killing (%Nitroblue Tetrazolium Test) were determined by microscopy, neutrophil chemokines [plasma interleukin 8 (IL-8)] was determined using ELISA, and respiratory burst indices [plasma catalase (CAT), superoxide dismutase (SOD), myeloperoxidase (MPO), hydrogen peroxide (H2O2), and nitric oxide (NO)] were determined by spectrophotometry. While the plasma antioxidant vitamins (Vitamins A, C, and E) were determined using HPLC, the phagocytic indices, chemokines, and respiratory burst indices were correlated with plasma antioxidant vitamins using Spearman’s Correlation analysis at α0.05. Results: The results show that although among the healthy Nigerian adults, vitamin C was significantly and positively correlated with %NBT, it was negatively correlated with CAT activity. Vitamin A showed a significantly positive correlation with SOD while Vitamin E showed a significantly negative correlation with MPO. Conclusions: These findings suggest that antioxidant vitamins affect different stages of phagocytosis. It is advisable to use a combination of antioxidant vitamins as supplements with recommended treatment strategies against intracellular micro-organisms or inflammatory diseases. Keywords: Antioxidants, Intracellular microbial killing, Vitamins

COMPARISON OF THREE DIFFERENT TECHNIQUES FOR ISOLATION OF NEUTROPHILS FROM BLOOD AND THEIR UTILITY IN PERFORMING NITROBLUE TETRAZOLIUM TEST

Background & Objectives: Assays for neutrophils constitute an important component of screening tests in clinical immunology. There are no standard protocols for performing many of these tests and procedures vary from one laboratory to another. In addition, normal ranges for these assays in healthy Indian population have not been defined. Hence, an attempt is made to evaluate and present a simple technique for WBC isolation and NBT test. Methods: The study involved participation of 30 healthy adult volunteers. Ten of blood sample collected from each subject was subjected to three different procedures for isolation of WBCs - Ficoll-Hypaque gradient, dextran sedimentation and gelatin sedimentation methods. Cells isolated from these procedures were then used to perform NBT test. Smears were prepared, stained with Giemsa and results were expressed as % of stimulated and unstimulated cells. Results: The mean cell yield from both dextran and gelatin methods was comparable (2921.67cells vs 2806.67cells/cu mm). The cell yield from Ficoll-Hypaque method was much lower (1408.33 cells/cu mm). In NBT test, the mean readings of stimulated (61%) and unstimulated cells (18%) were almost similar in all three procedures of cell isolation. Conclusions: Comparison of procedures show that gelatin and dextran sedimentation methods yield high amount of relatively purified WBCs. The efficacy of cells isolated from all three procedures in NBT test was almost similar. The range of stimulated and unstimualted cells in the subjects were within expected levels. Gelatin sedimentation is economical, easy to perform and can be adopted to any clinical laboratory for WBC isolation.

Method for predicting the development of severe nummular microbial eczema

Key words: nummular microbial eczema, phagocytic index, spontaneous redox activity of neutrophils in the nitroblue tetrazolium test

Features of cellular mechanisms affecting the pathogenesis and course of chronic catarrhal gingivitis

Aim. To investigate the cellular mechanisms of chronic catarrhal gingivitis to develop the appropriate treatment schemes based on the analysis of obtained data. Methods. 57 patients aged 16 to 22 years were observed and divided into the control group (18 subjects with the intact periodontium with no orthopedic constructions) and the study group of patients with chronic catarrhal gingivitis (39 subjects, group 1). The degree of gingival inflammation was evaluated by papillo-marginal alveolar index as well as by gingival bleeding index. In addition, laboratory parameters were measured: spontaneous nitroblue tetrazolium test and zymosan-stimulated nitroblue tetrazolium test. Leukocyte-modulating activity of oral fluid was determined by measurement of luminol-dependent chemiluminescence. The activity of lysosomal enzymes - acid deoxyribonuclease and acid phosphatase - was determined in whole oral fluid, supernatant fraction of oral fluid and in sediment, obtained by centrifugation. Antioxidant activity was determined in whole oral fluid by biochemiluminescent method with hydrogen peroxide. Results. The performed investigations demonstrated that the development of chronic catarrhal gingivitis is accompanied by a sharp increase of the number of neutrophils and macrophages with a significant increase of their functional activity. At the same time, more than two-fold increase of the activity of lysosomal enzymes, lysosomal membrane labilization and release of enzymes outside the organelles and cells were observed, that indicated the increase of destructive potential in chronic catarrhal gingivitis. Use of indometacin in the complex treatment of chronic catarrhal gingivitis contributed to normalization of clinical parameters, increased neutrophil functional activity and enhanced their functional reserve, reduced the activity of phlogogenic factors and normalized the antioxidant activity of oral fluid. Pro- and anti-inflammatory factors secreted into the oral cavity during inflammatory periodontal diseases collectively have leukocyte-modulating activity. Conclusion. The obtained data indicate the important role of both high oxygen-dependent and oxygen-independent biocidal activity of neutrophils and macrophages in the pathogenesis and clinical course of chronic catarrhal gingivitis.

Conjunctival leishmaniasis in a case of disseminated cutaneous leishmaniasis

A 15-year-old female patient presented with numerous, small, papulonodular skin lesions, and hepatosplenomegaly 9 months after a treated biopsy proved cutaneous leishmaniasis. In ocular examination there were two yellowish, raised gelatinous conjunctival lesions in the left eye. The exisional conjunctival lesion biopsy revealed many Leishman bodies inside tissue histiocytes. The patient had no systemic immunologic problems (normal serum immunoglobulins, nitroblue-tetrazolium test, complement CH50 test and flow cytometry of leukocytes). The indirect immunofluorescent antibody test for Leishmania tropica (titre of 1:1024) and the leishmanin skin test were positive. DNA of L. tropica was detected by a specific polymerase chain reaction on whole blood, bone marrow and skin biopsy specimens. The skin and conjunctival lesions disappeared with miltefosine and no intraocular tissue penetration of organism happened. Conjunctival leishmaniasis should be considered in the differential diagnosis of raised conjunctival lesions in a disseminated cutaneous leishmaniasis patient and needs proper systemic therapy.

Effect of smoking on neutrophil oxidative metabolism

Alterations in neutrophil function by tobacco products may play a central role in the pathogenesis of periodontal diseases and several smoking-related systemic diseases. The aim of the study was to evaluate the effect of smoking on neutrophil oxidative metabolism. Materials and methods. The study included 17 smoking men free of systemic diseases who were referred for treatment of various odontological diseases to outpatient department of Kaunas University of Medicine Hospital. The age of subjects varied from 22 to 43 years. All subjects answered the questions about smoking habits. Clinical examination included assessment of oral hygiene status according to the OHI-s index and periodontal status according to Russell and Ramfjord indices. To evaluate the oxidative metabolism of neutrophils, luminol- and liucigenin-dependent chemiluminescence and nitroblue tetrazolium test were used. Results. After smoking, extracellular liucigenin-dependent chemiluminescence response was higher as compared to the response before smoking, but total (intra- and extracellular) luminol-dependent chemiluminescence response was the same both before and after smoking. Exposure of neutrophils to smoking caused a significant increase in nitroblue tetrazolium reduction. Conclusion. The release of reactive oxygen species in neutrophils exposed to smoking may alter the pathogenic processes in periodontal diseases.