Spontaneous Rosette Formation Research Articles

Стан Т- і В-клітинної ланок специфічного захисту у собак при пухлинах молочних залоз

The purpose of the research was to determine the activity of Tand B-cell specific protection in dogs 9–13 years old age with benign and malignant tumors of the mammary glands to assess the level of immune response to the pathological process. Assessment of the activity of cellular factors of immunity has been carried out by determining the number of T-lymphocytes and their subpopulations and B-lymphocytes in the peripheral blood of dogs in the reaction of spontaneous rosette formation with ram erythrocytes. The clinical classification and determination of the stage of breast tumors were carried out according to the TNM system. The studies have shown that the occurrence of breast tumor diseases in dogs has a significant effect on the activity of T- and B-cellular link of specific immunity mechanisms, especially the number of T-lymphocytes and their populations in animals with malignant neoplasia. In particular, in the blood of animals with malignant neoplasia of the mammary glands, the number of Tlymphocytes (common and, especially, theophylline resistant) decreases accompanied with an increase in the number of T-suppressors, which is an important criterion for predicting the course of the disease. These changes in the number of Tlymphocytes in the blood of dogs of the experimental groups have led to a decrease in the amount of immunoregulatory index, which indicates a decrease in lymphocytic activity. In the blood of dogs with malignant neoplasia, a tendency to increase the number of antigenic-dependent B-lymphocytes has been shown. Similar changes, only of a lesser extent, are also have been demonstrated in the blood of dogs with benign breast neoplasia. Thus, the results of the research showed that the development of mammary gland tumors in dogs was accompanied by an immunosuppressive effect on the specific defense mechanisms, especially the T-cell link of the immune response in animals with malignant neoplasms.

Indicators of Immunity in Associated Mycotoxicosis of Cows

The issue of cattle reproduction was and still is one of the main tasks in cattle breeding. Losses of farms from infertility of cows are quite significant and range within 3.19-5.41 per 1 day of infertility. Mycotoxins produced by fungi of the Fusarium family, namely deoxynivalenol (DON) and zearalenone (ZEN) adversely affect not only the functioning of all organs and systems of the cow’s body, but also produce an immunosedative effect. The purpose of this study was to establish the effect of the DON and ZEN complex on the main indicators of the immune response of cows and its correction in a comparative aspect using a feed additive based on zeolite and organic acids and recombinant α-, g-interferons. The study material was the blood of cows (serum and stabilized) sick with mycotoxicosis caused by the association of DON and ZEN. Methods used: photonephelometric using E. coli test culture, spontaneous rosette formation with sheep red blood cells according to M. Jondal, modified method of rosette formation according to M. Wansbrough-Jones, R. Limatibul’s method, simple radial immunodiffusion in gel according to G. Mancini, precipitation in a polyethylene glycol solution according to M. Digeon. Experimental studies were performed on black-spotted cows in farms of the Sumy Oblast. The dynamics of the immune indicators of cows during the development of mycotoxicosis and upon treatment with products zeolite-based, organic acids, and an aqueous solution of recombinant α-, g-interferons were studied. It was found that the indicator of bactericidal, lysozyme, complementary, and phagocytic activity of cow blood serum under treatment increased to the indicator inherent in healthy animals. The dynamics of immunoglobulins in the treatment with zeolite and organic acids and recombinant α-, g-interferons was investigated, and an increase to the level of intact cows was established. It was proved that the indicators of the immune response of cows upon using a feed additive based on zeolite and organic acids at a dose of 2.5 kg per tonne of fodder and a preparation based on an aqueous solution of recombinant α-, g-interferons at a dose of 3 ml per animal were significantly higher

Effect of phytobiotic - Germivit on the functional state of cattle

Plant-based feed additives, also known as phytobiotics, show great promise to compensate deficiencies of important biologically active substances in the diet. Therefore, this study aimed to evaluate the effect of Germivit on the feed supplement of cattle of different ages. In the first experiment, four groups of 9-month-old Simmental calves (10 animals in each group) were fed with Germivit at 0.0, 0.5, 0.7, and 0.9 g kg-1 live body weight, respectively. In the second experiment, three groups of pregnant cows (10 animals in each group) received Germivit at 0.0, 0.25, and 0.50 kg-1 live body weight, respectively. The morphological composition of the collected blood was studied using a PCE-90vet automatic hematology analyzer. The blood biochemical composition was studied using a Stat Fax 1904 biochemical analyzer, and the immunological status of the animals was evaluated using generally accepted methods in veterinary medicine: immunocompetent cells by spontaneous rosette formation; immunoglobulins by the radial immunodiffusion method; phagocytosis by using S. aureus culture; serum lysozyme activity by the photoelectrocolorimetric method; and bactericidal activity of serum by determining the degree of growth inhibition of the mixture of daily culture of E. coli in a nutritive culture broth. Germivit contributed to the improvement of the biochemical and immunological parameters in the calves. In the cows, an increase in the morphological parameters of blood was observed, their immune status improved, and their calves were born with high rates of natural resistance and health. Germivit had a positive effect on the functional state of cattle.

Вплив вітамінів Е і С на кількість і функціональну активність τ- ι β-лімфоцитів крові курчат-бройлерів

Abstract . Separate indices of T- and B-cell immunity of broiler chickens were investigated during the period of their growth with additional introduction of tocopherol acetate and ascorbic acid into their diet. The studies were conducted in one of the farms in the Lviv region on four groups of 100 broiler chickens each, ranging from 1- to 41 days of age. Chickens in the control group received a standard diet, the first experimental diet with the addition of tocopherol acetate, the second experimental - ascorbic acid, and the third - a complex of these vitamins. In whole blood, the relative number of T and B lymphocytes and their individual populations were determined in the reaction of spontaneous rosette formation with ram erythrocytes, differentiated calculation of rosette lymphocytes with different degree of functional activity was performed. For research, blood was collected from broiler chickens after slaughter at different ages: 27-, 34-, and 41-day-olds. During the work, the bioethical requirements of the animals were complied with according to the current legislation. Studies have shown that the additional introduction of chickens to their diet tocopherol acetate and ascorbic acid has a stimulating effect on the number of T and B lymphocytes and the functional activity of T and B cell units of immunity. This is evidenced by the higher number of (-lymphocytes (common, active, theophylline-resistant) and Β-lymphocytes in the blood of broiler chickens, which were supplemented with vitamins E and C. Τ- and Β-lymphocyte apparatus - an increase in the number of cells with low and medium degrees of aperture and a decrease in the inactive function of Τ- and Β-lymphocytes in the blood. These changes were more pronounced in the blood of chickens at 27 days of age due to the action of vitamin C, as well as the complex of tocopherol acetate and ascorbic acid. Therefore, on the basis of the conducted researches it is possible to confirm a regulatory role of vitamins E and C in formation of a cellular link of specific immune response of chickens-broilers. The action of the investigated vitamins, and especially of ascorbic acid, revealed an increase in the number and increase in the functional activity of T- and B-lymphocytes of the blood. These changes are probably due to the antioxidant action of vitamins in providing metabolic homeostasis of membranes and the receptor apparatus of immunocompetent cells. Key words: vitamins E and C, broiler chickens, specific immunity, T and B lymphocytes, functional activity of immunocompetent cells

Вплив кормової добавки на основі амаранту на показники клітинного імунітету кролів за еймеріозу

Analysis of modern literary sources concluded that amaranth has high biological value and that eimeriosis is widespread across the rabbit farms. Therefore, to increase the effectiveness of natural feed additive usage, it is necessary to develop scientifically based systems for its use, taking into account animals’ biological characteristics and their disease. The animals of the experimental group were fed by an amaranth oilcake with the same feed as in the control group for one month. The infestation intensity (II) was determined by the method of McMaster. B- and T-lymphocytes count was determined by the spontaneous rosette formation method with sheep erythrocytes. It was found that feed additive based on amaranth oilcake reduced II by 2.58 times in the experimental group, while II in the control group was increased by 1.25 times. It has also decreased leukocyte count due to a drop-in lymphocytes regarding the preparatory period and control group, by 1.50 times and 1.29 times respectively. During this period, rabbits from the experimental group compared to the control and preparatory period had a lower number of neutrophilic metamyelocytes (by 3.62 and 2.24 times, respectively) and eosinophils (by 1.91 and 2.26 times, respectively). Monocytes and basophils in the rabbits’ experimental group decreased by 1.79 and 3.40 times compared with the control ones. It was found that in the control, during the month, there was an increase in the number of neutrophilic metamyelocytes by 2.00 times, monocytes by 3.09 times and basophils by 1.89 times compared to the decrease of eosinophils by 1.55 times. In the blood of experimental group animals, relative to the preparatory period and control, a decrease in the number of B-lymphocytes (by 1.36 and 1.53 times, respectively), O-lymphocytes (by 2.26 and 4.39 times), T-helpers (by 1.69 and 1.45 times), T-active (2.35 and 1.73 times), IRI (at 1.81 times and 1.32 times), while the percentage of T-lymphocytes increased (by 1.23 times) than in the control group. The percentage of T-suppressors in the experimental group increased by 1.93 times against a decrease in T-active lymphocytes by 1.17 times compared to the control. The amaranth oilcake showed an eimeriostatic effect when feeding it to animals for a month. The feed additive suppressed the inflammatory processes in the intestinal mucosa, that aroused by the parasite Eimeria spp. Prospects for further research are to determine the effect of this oilcake on the level of rabbits’ blood A, G, M immunoglobulins during eimeriosis.

Normalization of the immune system of large cattle with the help of “Immunopheron” and “Ribotan” medicines

This article presents the results of studying the effect of immunostimulants “Immunoferon” and “Ribotan” on the indices of specific and non-specific resistance of calves. To this end, according to the principle of analogues, 3 groups of calves were formed, with 5 animals each. Calves of the third group served as a control. In the blood of calves, the content of immunoglobulins A, M, G was determined on the analyzer “Uniplan”. The number of T-lymphocytes in the blood was determined by the method of spontaneous rosette formation with erythrocytes of a ram, B-lymphocytes - by the method of complementary rosette formation using standard rabbit hemolysin and as a complement - fresh serum of cows. The bactericidal activity of the blood serum (BASK) was determined by O.V. Smirnova and TA. Kuzminoy, lysozyme activity of blood serum (LASK) - according to KA. Kagramanova and Z.V. Ermolieva, phagocytic index and number (FH and FI) - according to V.S. Gostevu. Experiments showed that the calves of the experimental groups showed an increase in serum levels of immunoglobulin A, M and G, as well as bactericidal and lysozyme activity of blood serum, a phagocytic number and an index. The relative and absolute levels of B-lymphocytes and T-lymphocytes were also significantly higher in the calves of the test groups than in the control group. And the most significant changes were noted with the use of the drug “Immunoferon”.

Rosette formation by Plasmodium coatneyi-infected erythrocytes of the Japanese macaque (Macaca fuscata).

We studied in vitro the spontaneous rosette formation by red blood cells of a Japanese macaque infected with Plasmodium coatneyi, which occurred after 30 hr of incubation. Rosette formation involved 88% of parasitized red blood cells (PRBCs). Spontaneous rosettes were formed when the ring-stage parasites developed into late trophozoites or schizonts. A rosette usually consisted of a PRBC surrounded by three or more uninfected erythrocytes. Electron microscopic examination revealed that interaction with adjacent uninfected erythrocytes in rosettes appeared to be mediated by knobs of PRBCs. Protruding ends of these knobs attached to the membranes of adjacent uninfected erythrocytes. In the present study, we have also obtained evidence that another pattern of cell adhesion was mediated by flat and focal electron-dense knobs that had formed on the membranes of PRBCs.

Investigation of Conditions Optimum for Spontaneous Rosette Formation by Lymphocytes of Yellowtail, Seriola quinqueradiata

Investigation of Conditions Optimum for Spontaneous Rosette Formation by Lymphocytes of Yellowtail, Seriola quinqueradiata

The Squirrel Monkey as an Experimental Model for Plasmodium falciparum Erythrocyte Rosette Formation

Experimental cerebral malaria was recently found to occur in the squirrel monkey Saimiri sciureus when infected with the human malaria parasite Plasmodium falciparum. This report is concerned with the existence of spontaneous rosette formation ex vivo (infected blood samples) and in vitro (cultured parasites) between red blood cells (RBC) infected with squirrel monkey-adapted P. falciparum isolates and normal squirrel monkey RBC. Transfer of P. falciparum with high rosette formation tendencies (90-100 R+) from one donor monkey to several recipients gave rise to parasites that varied extensively in their ex vivo rosette formation capacity (4-96% R+). However, all individual parasites readily form rosettes after 24 hr of in vitro culture (60-95% R+). Host factors may be involved in the modulation of rosette formation, although it is found to occur both in splenectomized and spleen-intact animals. Cross-rosette formation is seen between parasitized human RBC and normal squirrel monkey RBC and vice versa, and rosettes formed by RBC of the two hosts are similarly affected by pH, temperature, EDTA, trypsin, as well as squirrel monkey and African human hyperimmune IgG. These characteristics of rosette formation are preserved after long-term in vitro culture in human RBC. Rosettes formed by some isolates are highly sensitive to heparin while others are not, suggesting at least two distinct mechanisms of rosette formation. This idea is also supported by the observation that specific squirrel monkey antisera to heparin-sensitive strains does not dissociate rosettes formed by a heparin-resistant strain. The results suggest that rosettes and anti-rosette formation antibodies formed by squirrel monkeys and humans exhibited similar characteristics, and that the squirrel monkey is therefore a good experimental model to study erythrocyte rosette formation and cerebral malaria.

Preparation and characterization of a monoclonal antibody against bovine CD5 lymphocyte surface antigen

The M1 monoclonal antibody (mAb) was proved to recognize 51–70% of Bovine peripheral blood lymphocytes (PBL). The M1+ cells were SIg-. In spleen and lymph nodes, the M1 positive lymphocytes were located within the T cell areas. All the lymphoid follicles remained negative. In the thymus, 10% of thymocytes were M1+, most of them were located in the medulla. The M1 mAb did not inhibit spontaneous rosette formation by sheep erythrocytes and bovine lymphocytes. On the other hand, biochemical analysis of membrane antigen with bovine thymic tumor cell line LB203 gave a molecular weight of 75 kDa. Despite a slight difference in biochemical results (75 vs 67–69 kDa). Our data permit us to consider M1 mAb as a possible homologous of human anti-CD5 mAb. Finally, M1 cross-reacted with sheep peripheral blood T lymphocytes.

H19, a surface membrane molecule involved in T-cell activation, inhibits channel formation by human complement

Here we compare the properties of leukocyte antigens H19 and CD59 with those of the PI-linked 18,000–20,000 M r molecules which inhibit lysis of human cells by the autologous terminal complement components C5b-9. H19, a 19,000 M r protein found on human erythrocytes, monocytes, neutrophils, T-lymphocytes and other cells, is one of the ligands involved in the spontaneous rosette formation between human T-lymphocytes and erythrocytes. Recent evidence indicates that H19 also participates in T-cell activation. CD59 is a widely distributed 18,000–25,000 M r protein anchored to the cell membrane by phosphatidylinositol (PI). The function of CD59 is unknown. Affinity-purified H19 incorporates into cell membranes and inhibits channel formation by human C5b-9 on guinea pig erythrocytes. Significant inhibition is achieved with picogram quantities of H19, corresponding to approximately 600 molecules per erythrocyte. H19 is most effective when C9 is limiting but quite active when C5b-7 or C8 are limiting, indicating that it may interact with several of the structurally related terminal complement components. The inhibitory activity is blocked by mAbs to either CD59 or to H19. H19 is PI-anchored: it is released from the cell membrane by treatment with PI-specific phospholipase C, and it is absent from cells from a patient with paroxysmal nocturnal hemoglobinuria (PNH). Analysis of PNH erythrocytes after treatment with terminal complement proteins shows that the H19-negative erythrocytes are more susceptible to C5b-9-mediated lysis. Treatment of normal human erythrocytes with either anti-H19 or anti-CD59 renders them more susceptible to lysis by human C5b-9. We conclude that H19 and CD59 are probably the same molecule and are identical or closely related to the recently described inhibitors of C5b-9 channel formation.

A Monoclonal Antibody Specific for Bovine T Cell Surface Antigen Associated with the E‐Rosette Receptor

From mice immunized with T lymphocyte-enriched bovine peripheral blood mononuclear cells (PBMC), a monoclonal antibody termed BLMo-12 was obtained. BLMo-12 reacted with the antigen of Mr 56,000 in lysate of T lymphocytes. This mAb was found to inhibit spontaneous rosette formation by T-bovine lymphocytes with sheep red blood cells but it did not react with B lymphocytes, monocytes, neutrophils or eosinophils. In frozen section of the thymus, BLMo-12 showed a positive staining both the cortex and the medulla. In lymph nodes, the mAb stained the T-dependent paracortex. BLMo-12 reacted with 49.9% of PBMC and 82.5% of thymocytes. Recognition of the bovine homologue of CD2 on the T lymphocyte surface by this mAb was discussed.

Spontaneous rosette formation of carp peripheral leucocytes with mammalian red blood cells.

The spontaneous rosette formation of peripheral leucocytes of carp with red blood cells of various mammals was investigated. Spontaneous rosette formation was observed with SRBCs, HRBCs and BRBCs with the frequency of rosette formation being higher with SRBCs and HRBCs than with BRBCs. Variations in surface morphology, in cell size of the rosette forming cells and in rosette formations were observed under light and scanning electron microscope. However, by examining Giemsa-stained preparates, the RFCs were identified as lymphocytes. The spontaneous rosette formation occurred gradually depend-ing on the incubation time. The spontaneous rosette formation was not specific to SRBCs and was observed at a temperature range of 0°C to 37°C. In these respects the rosette formation of carp lymphocytes differed from the earlier reported rosette formation of mam-malian lymphocytes.

The E2 antigen, a 32 kd glycoprotein involved in T-cell adhesion processes, is the MIC2 gene product.

E2 is a 32 kd human T-cell surface glycoprotein involved in spontaneous rosette formation with erythrocytes. A 1.11 kb cDNA was isolated from a lambda gt11 expression library by screening with monoclonal antibodies directed against E2. The primary structure of E2, deduced from the nucleotide sequence of its gene, comprises 185 amino acids and is devoid of N-linked glycosylation sites. The E2 protein is rich in proline residues and displays an organization typical of an integral membrane protein. Northern blotting showed a good correlation between mRNA abundance, E2 surface density and the level of T cell differentiation. In fact, nucleotide sequencing revealed that E2 is the MIC2 gene product, previously identified with the 12E7 Mab. Xg(a-) female individuals have no E2 molecule on the surface of their red cells, in contrast with Xg(a+) individuals, but have the molecule in their cytoplasm, in the form of the 28 kd precursor. These findings show that the E2 antigen, a cell surface molecule involved in T cell adhesion processes, is the product of the MIC2 gene, the only pseudoautosomal gene to be described in man.

A T cell surface molecule different from CD2 is involved in spontaneous rosette formation with erythrocytes.

Increasing evidence indicates that rosettes which spontaneously from between human T cells and E might be of physiologic relevance. We show here that another T cell-surface molecule than CD2 is involved in rosette formation. Four mAb have been obtained reacting with human T cells that block rosettes with E from many species, including autologous cells. They react with a molecule, we termed E2, which is actively synthetized by T and monocytic cells. Immunoprecipitation revealed a major 32-kDa band. Immunoblots revealed a major 32-kDa band and a minor 20-kDa band. This molecule was detected on all T cells tested--and present at high densities on corticothymocytes, but at low densities on medullary thymocytes. It was also found on monocytes but not on B cells. However B-CLL cells did carry this molecule. E2 molecules were also detected on nonhematologic cells. Together with the recent evidence that 3 molecules from the erythrocyte surface are also involved for rosettes, intricate molecular interactions would account for adhesion of T cells to autologous E and possibly autologous nucleated cells.

Marmoset red blood cell receptor for membrane-associated complement components is not related to human CR1: Partial characterization of the C3-binding proteins responsible for the spontaneous rosette formation between marmoset red blood cells and human leukocytes

Cells from all the human B-lymphoblastoid cell lines tested and most human monocytes form rosettes with marmoset red blood cells (MaRBC). Because previous reports suggested the involvement of complement components in this phenomenon, the mechanism of rosette formation and the eventual similarities between the MaRBC receptor and the CR1 receptor present on human erythrocytes have been analyzed herein. The binding of MaRBC to human leukocytes strongly differs from the immune adherence phenomenon: rabbit anti-human CR1 did not react with MaRBC and the MaRBC receptor-binding activity is Ca 2+-dependent. Rosette formation required intact energy metabolism and cytoskeleton integrity of leukocytes. Our attempts to purify the receptor from MaRBC membranes revealed the absence of CR1. Nevertheless, C3-binding proteins were isolated by selective desorption by Sepharose iC3 column chromatography. A three-band pattern was observed under reduced conditions with 74,000, 70,000, and 53,000 molecular weights. It was not possible to further separate these components. This protein complex inhibited the rosette phenomenon between MaRBC and both Raji and U-937 cells, exhibited a very poor cofactor activity, and had no decay-accelerating activity toward the classical C3 convertase. This material did not cross-react with antibodies directed to human proteins. These results showed that erythrocytes from new world monkeys do not express a receptor analogous to the human CR1, but expressed C3-binding protein with low cofactor activity that could recognize membrane-associated complement components.

Antilymphocytic antibodies in various human diseases as a factor depressing suppressor T cell function

The total number of rosette-forming T cells (T-RFC) was determined by the spontaneous rosette formation method with sheep's red blood cells (SRBC) [5]~ Theophylline-sensitive and theophylline-resistant T lymphocytes (T-TSL and T-TRL respectively) were determined by the method in [8]. Suppressor T cells were induced in CBA mice by intravenous injection of a supraoptimal dose (5"109 ) of SRBC [2]; the splenocytes of these mice, i0 days after immunization in a dose

Immunological abnormalities in patients with cervical carcinoma

The cellular immune response of 58 patients affected by cervical cancer was evaluated with the aim of investigating abnormalities in T-cell populations. Subjects were divided into three groups: (a) severe dysplasia or carcinoma in situ, (b) microinvasive carcinoma, and (c) invasive carcinoma. T cells were assessed by a spontaneous rosette formation test with sheep red blood cells and by reactivity with specific monoclonal antibodies of OKT series (OKT4 and OKT8 positive lymphocytes). A significant T-cell depression was observed in all patient groups; the lowest T-cell level was observed in group (b), which revealed an inverted OKT4 +/OKT8 + ratio too. The results support the hypothesis that cell-mediated immunity is a key factor in premalignant status and in the early development of cervical cancer.

Human autologous rosette-forming cells: III. Binding of erythrocytes from different species to the T-cell receptors for autologous red blood cells

Spontaneous rosette formation in humans is restricted to a subpopulation of the circulating T cells. We have previously shown that the interaction between lymphocytes and autologous red blood cells (auto-RBC) is not mediated by a self-recognition mechanism, since allogeneic (allo-) RBC bind to T cells through the same receptors. In this work, we have extended these observations to thymocytes. Using a mixed-rosette assay in which one type of erythrocyte was identified by FITC labeling, we have shown that almost all the thymocytes which attached auto-RBC could also fix allo-RBC. However, as for the peripheral blood lymphocytes (PBL), binding of human RBC to thymocytes occurred with varying affinities according to the erythrocyte's origin. In order to further study the specificity of the erythrocyte to lymphocyte binding in rosette formation, PBL were mixed with auto-RBC and erythrocytes of xenogeneic (xeno-) origin. Although very disparate incidences of rosettes were found according to the species from which the RBC were derived, most of the autorosetting lymphocytes also had receptors for xeno-RBC. In addition, preincubation of PBL with monoclonal antibody OKT11A (directed against the sheep RBC receptors on T cells) completely abrogated rosette formation with all the erythrocytes tested (human auto- and allo-, sheep, pig, and rabbit) except mouse RBC. Taken together these data strongly suggest that human auto- or allo-, as well as sheep or some other xeno-RBC, bind to T lymphocytes by a single receptor and that the combining sites are expressed with different densities or varying affinities depending upon the RBC's origin. Therefore, spontaneous autorosettes may represent T lymphocytes having high-affinity receptors for sheep RBC.

Study of spontaneous rosette formation and changes of sialic acid content in the surface glycoproteins of macrophages induced by hamster transplantable melanomas

The influence of two kinds of transplantable melanomas on the content of sialic acids in the surface glycoproteins of hamster peritoneal macrophages was studied, and the ability of these cells to form spontaneous rosettes. It has been found that the intensification of rosette formation by macrophages with SRBC depends on the melanoma line. No correlation has, however, been found between changes in the sialic acids content and the ability of macrophages to form rosettes under the influence of melanomas differing in malignancy.