Accidental discovery of Trypanosoma theileri in the in vitro culture of the heifer lymphocytes.

The diagnostics of the Trypanosoma sp. invasion by means of the classic methods i.e. the methods of thin smears or thick drop or even the microhematocrite method, especially when intensity of infection is low, is very difficult. In our climatic zone, trypanosomosis is usually considered as an exotic disease. An opportunistic model of the infection with the parasite and a lack of current data on the prevalence of T. theileri in the cattle in Poland cause that it is neglected as a potential reason of contamination of tissue cultures in cattle. We showed the presence of T. theileri in culture of isolated lymphocytes from one of six heifers examined. It seems that the prevalence of the invasion of the parasite is not very intense but it should be considered as a possible threat for bovine cell culture. It is also worth including this parasitosis in the differential diagnostics of other diseases that are infectious and/or proceed with symptoms of immunosuppression.

The relationship between concentrations of vitamin D-binding protein (DBP) in serum and colostrum of mares and in serum of their foals in the neonatal period.

Vitamin D-binding protein (DBP) participates in the actin scavenger system, it is a carrier of vitamin D and its derivatives, it manifests the capacity to bind mainly monounsaturated and saturated fatty acids, it binds to the surface of several cells and enhances chemotactic activity of C5a of the complement. The present study was aimed at answering the question whether serum DBP level in mares is related to levels of this protein in colostrum and in serum of its progeny. For this purpose, sera from 77 mares, colostra from 72 mares and sera from 69 Thoroughbred foals were collected. Mother's age, number of deliveries experienced in the past, month of delivery, feeding of foals with colostra were recorded. Blood of the foals was sampled from the umbilical vein during delivery (0h) and 36-48 h after delivery from the external jugular vein, colostra of the mares were obtained after delivery and blood of the mares was sampled 36-48 h after delivery. Concentration of DBP was estimated by a self-designed ELISA. In the present study, DBP concentrations in newborn's serum were found independent of their concentrations in mother's serum, her age and number of parities experienced in the past. Colostrum DBP level was found to be lower than that in the mare's serum and was not correlated to the concentration of this protein in mare's serum. There was no effect of colostrum feeding on DBP level in the foal serum. These results indicate that serum DBP concentration in newborn foals depends on factors which act directly on the foal. Because of the lack of correlation between plasma and colostrum concentrations of DBP, it can be assumed that DBP is synthesised in the mammary gland and/or specific transport mechanisms exist in the mammary gland.

High fructose consumption combined with low dietary magnesium intake may increase the incidence of the metabolic syndrome by inducing inflammation.

The metabolic syndrome is a cluster of common pathologies: abdominal obesity linked to an excess of visceral fat, insulin resistance, dyslipidemia and hypertension. This syndrome is occurring at epidemic rates, with dramatic consequences for human health worldwide, and appears to have emerged largely from changes in our diet and reduced physical activity. An important but not well-appreciated dietary change has been the substantial increase in fructose intake, which appears to be an important causative factor in the metabolic syndrome. There is also experimental and clinical evidence that the amount of magnesium in the western diet is insufficient to meet individual needs and that magnesium deficiency may contribute to insulin resistance. In recent years, several studies have been published that implicate subclinical chronic inflammation as an important pathogenic factor in the development of metabolic syndrome. Pro-inflammatory molecules produced by adipose tissue have been implicated in the development of insulin resistance. The present review will discuss experimental evidence showing that the metabolic syndrome, high fructose intake and low magnesium diet may all be linked to the inflammatory response. In many ways, fructose-fed rats display the changes observed in the metabolic syndrome and recent studies indicate that high-fructose feeding is associated with NADPH oxidase and renin-angiotensin activation. The production of reactive oxygen species results in the initiation and development of insulin resistance, hyperlipemia and high blood pressure in this model. In this rat model, a few days of experimental magnesium deficiency produces a clinical inflammatory syndrome characterized by leukocyte and macrophage activation, release of inflammatory cytokines, appearance of the acute phase proteins and excessive production of free radicals. Because magnesium acts as a natural calcium antagonist, the molecular basis for the inflammatory response is probably the result of a modulation of the intracellular calcium concentration. Potential mechanisms include the priming of phagocytic cells, the opening of calcium channels, activation of N-methyl-D-aspartate (NMDA) receptors, the activation of nuclear factor-kappaB (NFkB) and activation of the renin-angiotensin system. Since magnesium deficiency has a pro-inflammatory effect, the expected consequence would be an increased risk of developing insulin resistance when magnesium deficiency is combined with a high-fructose diet. Accordingly, magnesium deficiency combined with a high-fructose diet induces insulin resistance, hypertension, dyslipidemia, endothelial activation and prothrombic changes in combination with the upregulation of markers of inflammation and oxidative stress.

Transfer of maternal passive immunity to kids in goat herd.

The efficiency of tranfer of maternal immunity and its infuence on the kids' health was observed in a herd in which kids (n=20) had whole contact with the dam (n=13). The factors associated with dam, kid and human, which influence the efficiency of passive transfer were observed. It was estimated that the single-born kids reached higher serum gamma-globulin level (mean 23.14 g/dm3) than twin kids (mean 18.2 g/dm3) (p < 0.05). The gamma-globulin level was the highest in single-born kids at 48 h, and in twin kids in 24 h of life. The IgG class antibodies to herd-homologous strains of Pasteurella multocida and Escherichia coli were estimated using ELISA in sera, colostrum and milk whey samples of dams and sera of kids. It was found that maternal antibodies specific to these two facultative pathogens decreased in kids sera rapidly and the self humoral immune response occurred within the period of observation. Two kids delivered by goats with lowest hierarchic position in the herd showed failure of passive transfer, and died at the 10th and 12th weeks of life due to chronic infections induced by both above mentioned bacterial strains.

Differential effects of heparin on NO and tumor necrosis factor-alpha production in bovine blood mononuclear cells stimulated with Salmonella typhimurium lipopolisaccharide.

We investigated the influence of heparin, one of the extracellular matrix (ECM) components, on nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production by bovine peripheral blood mononuclear cells (PBMC) and monocytes left to adhere for 2 (freshly adherent monocytes) and 48 h (resting monocytes), activated with Salmonella typhimurium lipopolysaccharide (LPS). After 24-h stimulation with LPS, heparin (100 microg/ml) increased (by about 40%) NO production by peripheral blood mononuclear cells and by freshly adherent monocytes. However, it did not change NO synthesis by the resting monocytes. Unlike its influence on NO level, heparin diminished TNF-alpha production by PBMC and monocytes stimulated with LPS. Microscopical examination of PBMC stained with biotin-labeled heparin, showed that both lymphocytes and monocytes were able to bind this glycosaminoglycan. We suggest that heparin, as a component of ECM, modulates the early response of monocytes to exogenous stimuli.

Inflammatory response following acute magnesium deficiency in the rat.

The importance of inflammatory processes in the pathology of Mg deficiency has been recently reconsidered but the sequence of events leading to the inflammatory response remains unclear. Thus, the purpose of the present study was to characterize more precisely the acute phase response following Mg deficiency in the rat. Weaning male Wistar rats were pair-fed either a Mg-deficient or a control diet for either 4 or 8 days. The characteristic allergy-like crisis of Mg-deficient rats was accompanied by a blood leukocyte response and changes in leukocytes subpopulations. A significant increase in interleukin-6 (IL-6) plasma level was observed in Mg-deficient rats compared to rats fed a control diet. The inflammatory process was accompanied by an increase in plasma levels of acute phase proteins. The concentrations of alpha2-macroglobulin and alpha1-acid glycoprotein in the plasma of Mg-deficient rats were higher than in control rats. This was accompanied in the liver by an increase in the level of mRNA coding for these proteins. Moreover, Mg-deficient rats showed a significant increase in plasma fibrinogen and a significant decrease in albumin concentrations. Macrophages found in greater number in the peritoneal cavity of Mg-deficient rats were activated endogenously and appeared to be primed for superoxide production following phorbol myristate acetate stimulation. A high plasma level of IL-6 could be detected as early as day 4 for the Mg-deficient diet. Substance P does not appear to be the initiator of inflammation since IL-6 increase was observed without plasma elevation of this neuropeptide. The fact that the inflammatory response was an early consequence of Mg deficiency suggests that reduced extracellular Mg might be responsible for the activated state of immune cells.

[The use of the Richard's dynamic hip screw and hip Zespol fixator in the treatment of proximal femoral fracture]. / Zastosowanie sruboplytki Richardsa i stabilizatora biodrowego Zespol w leczeniu zlaman blizszego konca kosci udowej.

A series of 49 patients (27 females, 22 males) aged 24-82 operated on due to proximal end of the femur fracture between 1990 and 1995 were included in this study. There were 23 femoral neck fractures and 26 trochanteric fractures. DHS was used in 35 patients and Zespol hip fixator in 14 cases. Good results have been achieved in 85.7%. Neither method proved superior to the other. Poor results occurred in subcapital neck fractures. DHS seems to offer advantage in trochanteric fractures and Zespol method in femoral neck fractures.

Enhanced tumor necrosis factor-alpha production following endotoxin challenge in rats is an early event during magnesium deficiency.

Magnesium (Mg) plays an essential role in fundamental cellular reactions and the importance of the immuno-inflammatory processes in the pathology of Mg deficiency has been recently reconsidered. The purpose of the present study was to assess the effect of different stages of Mg deficiency on endotoxin response and tumor necrosis factor-alpha (TNF alpha) production. Weaning male Wistar rats were pair fed either a Mg-deficient or a control diet. At day 7, lipopolysaccharide (LPS) induced no lethal effects in control rats but resulted in 70% mortality in Mg-deficient rats within 3 h. The vulnerability of Mg-deficient rats to LPS was associated with higher TNF alpha plasma values. Mg-deficient animals that received magnesium supplementation before endotoxin challenge had significantly increased survival. At day 2, control and Mg-deficient rats were also subjected to endotoxin challenge with or without magnesium pre-treatment. A significant increase in TNF alpha plasma level was observed in Mg-deficient rats compared to rats fed the control diet. Mg-deficient rats that received magnesium replacement therapy before endotoxin challenge had significantly lower TNF alpha plasma values than those receiving saline before endotoxin. Thus, the results of this experiment suggest that the activated or primed state of immune cells is an early event occurring in Mg deficiency.

Accelerated thymus involution in magnesium-deficient rats is related to enhanced apoptosis and sensitivity to oxidative stress.

Experimental Mg deficiency leads to alterations in the immune response. Reduction of thymus weight and histological changes were previously observed in Mg-deficient rats after several weeks on a deficient diet, suggesting that functions of this immune organ may be affected by Mg deficiency. More recently, changes in the immune system during early Mg deficiency were shown. Thus, in the present study we examined modifications in the thymus during the early stages of Mg deficiency in weanling rats. From our results, it appears that Mg deficiency accelerates thymus involution. The assessment of apoptosis (enumeration of apoptotic cells on the basis of morphological criteria and intranucleosomal degradation of genomic DNA) showed greater values in thymuses from Mg-deficient rats as compared with controls. This was observed very early, since a significant difference was shown on the second day of deficiency, before reduced weight of thymus, which was recorded in the later period. These results indicate the relationship of accelerated thymus involution with an active process of cell death. Mg deficiency led to histological changes in the thymus. In the early stage of deficiency (second day) the presence of inflammatory cells was shown, suggesting that the inflammatory process was already occurring in the tissue studied. Later (eighth day) an increased proportion of epithelial reticular cells in the cortex was shown, indicating a remodelling process occurring in this period. Enhanced susceptibility to peroxidation also occurred very early during Mg deficiency. It may be hypothesized that disturbances in Mg status of short duration could have cellular effects with various deleterious consequences.

Early morphological and immunological alterations in the spleen during magnesium deficiency in the rat.

Dietary magnesium deficiency in rodents, and especially in rats, causes inflammation and leads to alterations in the immune response. One of the characteristics of magnesium deficiency in the rat is a marked enlargement of the spleen. Considering the importance of the spleen for the immune response, in this study we have evaluated histological, cytological and immunological changes in this organ of rats in early stages of this deficiency. For this purpose, male weaning Wistar rats were pair-fed with either control or magnesium-deficient diet, for 2, 4 or 8 days. Results indicate that after 8 days on the deficient diet rats presented clinical signs of inflammation, splenomegalia and leukocytosis. As shown by histometrical analysis, both the red and white spleen pulps of deficient rats displayed an increased incidence of polymorphonuclear leukocytes and macrophages in all studied stages of deficiency. Concomitantly, the relative number of lymphocytes decreased. This observation was confirmed by the analysis of the cell suspension obtained from the spleen. The greater number of adherent cells in the cell suspension from deficient rats provides an additional confirmation of the increased number of macrophages in the spleen of these rats. Analysis of lymphocyte populations demonstrated a reduced proportion of CD5+ and CD8+ cells after 8 days of deficiency. The reduction in the number of CD8+ cells in deficient rats could be related to the observed decrease in IFN-gamma concentration in the spleen homogenate. In short, this study shows that magnesium deficiency causes early cytological and immunological modifications in the spleen which appeared before macroscopical changes in this organ and before clinical symptoms of inflammation. These changes could be related to the altered immune response of deficient animals.

Exacerbated immune stress response during experimental magnesium deficiency results from abnormal cell calcium homeostasis.

The aim of this study was to assess the potential mechanism underlying the enhanced inflammatory processes during magnesium deficit. In this study, exacerbated response to live bacteria and platelet activating factors was shown in rats fed a magnesium-deficient diet. Peritoneal cells from these animals also showed enhanced superoxide anion production and calcium mobilising potency following in vitro stimulation. The latter effect occurred very early in the course of magnesium deficiency. These studies first showed that an abnormal calcium handling induced by extracellular magnesium depression in vivo may be at the origin of exacerbated inflammatory response.

Magnesium deficiency in rats induces a rise in plasma nitric oxide.

Magnesium deficiency in rats leads to an oxidative stress involving an increased production of radical oxygen species. The present study was designed to examine the effect of experimental magnesium deficiency on plasma nitric oxide (NO) level and nitric oxide synthases (NOS) activities in rats. The data show that the concentration of NO is markedly increased in plasma of magnesium-deficient rats. This rise in plasma NO results from activation of inducible nitric oxide synthase (iNOS) rather than of the constitutive form (cNOS) of the enzyme. These data are in agreement with previous observations indicating that inflammation occurs during magnesium-deficiency and provide an additional cause of oxidative lesions through formation of peroxynitrite from nitric oxide and superoxide anion.

Triglyceride-rich lipoproteins from magnesium-deficient rats are more susceptible to oxidation by cells and promote proliferation of cultured vascular smooth muscle cells.

An important characteristic of hyperlipaemia associated with magnesium deficiency in rats is the postprandial accumulation of triglyceride-rich lipoproteins (TGRLP). The present investigation was performed to determine the susceptibility of TGRLP isolated from magnesium-deficient rats to metal ion and cell dependent peroxidation and the effect of these lipoproteins on cultured vascular smooth muscle cells (VSMC). TGRLP were isolated by sequential ultracentrifugation from plasma of control and magnesium-deficient rats fed for 8 d on adequate or magnesium-deficient diets. Magnesium-deficient animals were hypertriglyceridemic as compared to control rats. After exposure to oxidative stress in vitro, Cu2+ or activated macrophages, conjugated diene production was significantly higher in TGRLP isolated from magnesium-deficient rats. Culture of VSMC with TGRLP from magnesium-deficient rats resulted in more important cell growth than with lipoproteins isolated from control animals. After incubation with VSMC, TGRLP from magnesium-deficient rats showed higher conjugated diene production than those from control rats. These results support the atherogenic properties of magnesium deficiency, which is accompanied by hyperlipaemia and which affects two important linked pathways in atherosclerosis, lipoprotein peroxidation and VSMC proliferation.

Frequency of interferon-alpha-secreting blood leukocytes in irradiated and bone-marrow-grafted pigs.

The effects of irradiation were studied on porcine interferon-alpha (IFN-alpha) secreting cells (IFN-alpha SC). IFN-alpha SC were characterized by an ELISPOT assay on non-adherent PBMC following incubation with the transmissible gastroenteritis coronavirus. In vitro irradiation of PBMC was followed by a decrease in the number of IFN-alpha SC while IFN-gamma production and cell viability were not affected. These data indicate that porcine IFN-alpha SC are relatively radiosensitive. Indeed, the frequency of blood IFN-alpha SC decreased markedly and rapidly after in vivo whole body or partial lymphoid irradiation. In addition, within several days of compatible bone-marrow engraftment in the irradiated animals, the number of blood IFN-alpha SC returned to normal values. These data demonstrate that circulating porcine IFN-alpha SC are derived from bone-marrow progenitors.

Age-related increase of porcine natural interferon alpha producing cell frequency and of interferon yield per cell.

Porcine blood mononuclear cells (PBMC) were shown to secrete interferon alpha (IFN-alpha) after induction by a coronavirus, the transmissible gastroenteritis virus (TGEV). IFN-alpha producing cells, referred to as natural interferon alpha producing (NIP) cells, were detected by an ELISPOT assay using anti-porcine IFN-alpha monoclonal antibodies. The frequency of NIP cells among blood cells is low, at most 40-110 per 10(5) PBMC and each NIP cell was found to produce several units of IFN. We have shown that NIP cell frequency and IFN yield per cell gradually increased with the age of the donor animals, from the neonatal period to the adult age, with a significant increase around puberty. Our present results also indicate that NIP cells may be influenced by physiological and genetic factors; thus (1) NIP cell frequency and IFN yield per cell were decreased during lactation; (2) Chinese (Meishan) pigs were found to have higher NIP cell frequency and IFN yield per cell than European (Large White) animals.

Enrichment of coronavirus-induced interferon-producing blood leukocytes increases the interferon yield per cell: a study with pig leukocytes.

Porcine peripheral blood mononuclear cells, which secrete IFN alpha in response to a coronavirus, transmissible gastroenteritis virus, were detected by a filter immunoplaque assay (ELISPOT). IFN alpha-producing cells (IPC), which are present at a low frequency in the blood, could be enriched up to 100-fold by sequential depletion of plastic-adherent cells and cell fractionation on metrizamide density gradients. IPC were present in the non-adherent low-density cell subpopulation. Cell selection experiments using antibody (Ab)-coated immunomagnetic beads revealed that porcine IPC could be positively selected by anti-CD4 or -SLA-class-II Ab, but not by anti-CD2 or -CD8 Ab. The estimated IFN yield per IPC was found to increase when IPC were assayed at higher concentrations. These data suggest that IPC represent a unique and distinct cell population in the blood, which could secrete higher amounts of IFN following its accumulation at a site of viral infection.

The bactericidal capacity of peripheral blood monocytes from HIV positive patients may collapse very soon after the infection.

The activity of peripheral blood monocytes from HIV positive patients was measured by the intensity of the chemoluminescence those cells emit when they ingest a foreign particle. In most patients (84%) that value was impaired. Such an alteration may occur very early in the course of the disease. The anti-p24 antibody titer was correlated with monocyte phagocytic potential as measured by the chemoluminescent value thus indicating the need for adequate monocyte activity in order to obtain antibody formation. The severity of opportunistic infections that HIV positive subjects may develop is a clear indication that their immune systems are abnormal. The most frequently affected cells are those which bear the viral receptor, the CD4 antigen. Those cells are mainly the helper T cells and monocytes. The monocytes are the immune system phagocytic cells which actively control infections, in part by the release oxidative radicals. Those radicals can easily be measured by the chemoluminescence (CL) the cells emit when they ingest a foreign particle. This study examines the CL emitted by peripheral blood monocytes from HIV-positive and control subjects while they phagocytose opsonized zymosan in vitro and correlates these values with other laboratory parameters.

[Changes in the aortic arch of calves with bacterial infections]. / Zmiany w luku aorty cielat w przebiegu infekcji bakteryjnych.

The autopsy and histopathological changes in the wall of aortic arch were presented in calves died of pulmonary and intestinal disorders. The bacteriological examinations of the internal organs demonstrated: E. coli, Pseudomonas aeruginosa Corynebacterium et al. Gelatinous edematous++ and hemorrhagic inflammatory centers of aortic arch adventitia were distinctly visible during thorax examination. In aortic arch the diagonal fissures of endothelium and deeper layers of the wall were found. In microscopical examination the heavy degenerative changes in the arch endothelium and deeper layers were observed. The fissures were from 0.5 to several millimeters in length and penetrated 1/3 to 1/2 thickness at the wall. The authors suggested that starting point of this changes are Gram-negative bacillary inflammatory centers in lungs. The bacterial endotoxins and vasoactive substances liberated by altered cells penetrated the aortic arch by the tissues continuity and in contact way.

Elimination of goat haemoglobin and its complexes with goat haptoglobin from goat and rat circulation.

The rate of elimination from rat and goat circulation of 125I-labelled goat haemoglobin and its complexes with human and goat haptoglobin in goat circulation, was studied. The half-life time t1/2 of haemoglobin in rat circulation was 1.5h, whereas in goat circulation -- 4 h, and was 10 times shorter than t1/2 for its complexes.

Immunoglobulins of colostrum--VI. Comparative studies of cytophilic properties of bovine serum and colostral IgG.

In our previous studies, using physical-chemical and serological methods, substantial differences between bovine serum and colostral IgG, especially IgG2, have been shown. The structural differences were localized in the Fc region of immunoglobulins studied. The present comparative studies were undertaken to determine whether structural differences in the Fc region of bovine serum and colostral IgG are reflected in the interaction of these immunoglobulins with the guinea-pig peritoneal macrophage Fc gamma receptor. It was found that binding of bovine serum and colostral IgG1 was a saturable process and only quantitative differences in the mode of binding to the Fc receptor were observed. There is, however, a big difference in cytophilic activity of bovine IgG2--no saturable and reversible binding is observed in the case of bovine serum IgG2.