Reconstruction of cranial cruciate ligament in rabbits using polyester implants saturated with PRP, antlerogenic stem cells MIC-1 and their homogenate.

AIM OF THE STUDY: The attempt to limit the negative effects of polyester implants on the articular cavity by using preparations containing growth factors. MATERIALS AND METHODS: Polyester implants used for the reconstruction of a rabbit's cranial cruciate ligament (CCL) were saturated with autogenic platelet-rich plasma (PRP), antlerogenic stem cells MIC-1 and their homogenate prior to the surgery. Six months after CCL reconstruction, morphological, and biochemical blood tests were carried out, including proteinogram and acute phase proteins. The knee joints were also examined macro- and microscopically. RESULTS: The results, compared to the control group, showed a favorable effect of the PRP and homogenate of antlerogenic cells on limiting the inflammation caused by the presence of polyester implant in the knee joint. The addition of growth factors caused covering the implant faster with the recipient's connective tissue, thus contributing to reducing the inflammatory reaction of the articular capsule to the presence of polyester. At the same time, no enhanced local or general reaction of the rabbit organism was observed to the presence of xenogenic antlerogenic stem cells MIC-1 homogenate which, like the PRP, may provide an easily available source of growth factors, increasingly often used in regenerative medicine. CONCLUSIONS: Applying antlerogenic stem cells, their homogenate or PRP increases the volume of connective tissue that surrounds and intertwines polyester CCL implant, separating it from synovial cavity environment.

Extracellular magnesium and calcium blockers modulate macrophage activity.

Magnesium (Mg) possesses anti-inflammatory properties, partly because it antagonizes calcium (Ca) and inhibits L-type Ca channels. Our aim was to determine the effects of different concentrations of extracellular Mg, with or without Ca-channel blockers, in macrophages. A macrophage-like cell line J774.E was cultured in different concentrations of extracellular Mg and exposed to i) the phorbol ester PMA to induce the production of reactive oxygen species ii) lipopolysaccharide to induce the production of pro-inflammatory cytokines, or iii) ovalbumin to study endocytosis. The Ca antagonists verapamil and/or TMB-8 were used to interfere with Ca homeostasis. Different concentrations of extracellular Mg did not impact on endocytosis, while Ca antagonists markedly decreased it. Low extracellular Mg exacerbated, whereas Ca antagonists inhibited, PMA-induced production of free radicals. Ca blockers prevented lipopolysaccharide-induced transcription and release of IL-1ß, IL-6 and TNF-α, while extracellular Mg had only a marginal effect. Ca channel inhibitors markedly reduced the activity of J774.E cells, thus underscoring the critical role of Ca in the non-specific immune response, a role which was, in some instances, also modulated by extracellular Mg.

Antlerogenic stem cells: molecular features and potential in rabbit bone regeneration.

AIM: (i) To assess the expression profiles of stem cell-associated markers including Oct4, Sox2, Klf4, Nanog, C-myc, Stat3 and Cd9, (ii) analyze the nanotopography of the MIC-1 stem cells and (iii) evaluate the efficiency of live stem cell implants and stem cell culture derivatives on the regeneration of bone deficiencies in rabbit mandibles. MATERIALS AND METHODS: The expression profiles of stem cell-associated genes, including Oct4, Sox2, Klf4, Nanog, C-myc, Stat3 and CD9 were assessed using reverse transcription polymerase chain reaction and flow cytometry. Nanotopography of the antlerogenic MIC-1 cell lineage was analyzed using atomic force microscopy. The effect of MIC-1 stem cells, their homogenate and supernatant on the regeneration of bone deficiencies in rabbit mandibles was evaluated using histological analysis. The effect of MIC-1 stem cells and stem cell-based derivatives on the immune responses of the animals was assessed by analyses of acute phase protein levels (haptoglobin and fibrinogen). RESULTS: We found that the MIC-1 cells isolated from the apical regions of growing antlers exhibited molecular features that were characteristics of pluripotent stem cells. Using atomic force microscopy, we determined the details of the cell surface morphologies with a particular emphasis on the patterns of formation of plasma extensions for interlinking adjacent cells. We also demonstrated that not only implanted stem cells but also cell homogenates and cell post-culture supernatants have potential in the regeneration of bone deficiencies in the rabbit mandible. CONCLUSIONS: Our findings indicate that the use of both antlerogenic stem cell implants and the preparations derived from the cells offer alternative approaches to those based on autologous stem cells in the biological stimulation of osteogenesis and in bone regeneration.

The Activity of Neutral α-Glucosidase and Selected Biochemical Parameters in the Annual Cycle of Breeding Carp (Cyprinus carpio L.).

The aim of the study was to demonstrate seasonal changes in the hydrolytic and transferase activity of neutral α-glucosidase, the level of glucose, cholesterol, triglycerides and total protein in the annual breeding cycle of the carp. The study was conducted on fish from a fish farm in Lower Silesia (Poland). Blood serum was collected from the heart in: June, September and December of two consecutive years. The results of the study show that the hydrolytic and transferase activity of neutral α-glucosidase, as well as the results of basic biochemical parameters are highest in summer, when the fish seek and intake food intensively. The lowest values were observed in spring, when carp have the lowest metabolism after the wintering period.

Extracellular Mg concentration and Ca blockers modulate the initial steps of the response of Th2 lymphocytes in co-culture with macrophages and dendritic cells.

Magnesium is highly involved in the metabolic network such that even subtle disturbances in its homeostasis affect many cellular functions, including calcium homeostasis, signal transduction, energy metabolism, membrane stability and cell proliferation. Recently, magnesium level has been proposed to modulate the priming and activity of immune cells. We studied the behavior of antigen-presenting cells (APCs) and T lymphocytes after altering the magnesium/calcium balance. We used two different populations of primary APCs, i.e. bone marrow-derived dendritic cells and bone marrow-derived macrophages, while D10.G4.1 cells served as a model of responding Th2 cells. Our principal findings are the following: (i) the extracellular magnesium concentration had no significant impact on endocytosis by bone marrow-derived APCs, (ii) high concentrations of extracellular magnesium, with or without calcium antagonists, significantly decreased IL-4 and IL-10 secretion by Th2 cells in a co-culture system of APCs and Th2 lymphocytes, (iii) proliferation of Th2 cells in co-culture systems was significantly inhibited by calcium antagonists independently from extracellular magnesium concentrations. Our results suggest that alterations of magnesium and calcium homeostasis impact on some crucial steps of the immune response.

Métodos para la determinación del estado del magnesio en humanos / Methods of assessment of magnesium status in humans / Métodos para a determinação do estado do magnésio em humanos

En la actualidad, datos epidemiológicos sugieren que, en países occidentales, la ingesta de magnesio no satisface la ingesta recomendada, lo que apoya un riesgo de deficiencia de magnesio latente en estas poblaciones. La evaluación del estado de magnesio sigue siendo un desafío para el laboratorio clínico ya que el magnesio se encuentra distribuido mayoritariamente en el hueso y tejidos blandos. Existe la necesidad de conciliación entre una prueba de fácil acceso, rápida, sensible y representativa del magnesio intracelular. La utilidad de diferentes biomarcadores en sujetos sanos ha sido evaluada; se ha reportado que el magnesio en plasma, eritrocitos y orina parecen ser biomarcadores sensibles a la ingesta dietética y útiles como biomarcadores en la población general. Sin embargo, esto no es concluyente, ya que se resalta que aún se requieren estudios mejor diseñados, que impliquen factores como mayor población empleada, dosis y tiempo de suplementación. El progreso en la genética y la genómica abren perspectivas interesantes en la búsqueda de estos biomarcadores que permitan cuantificar los niveles de magnesio celular así como también las reservas de todo el cuerpo, para poder así establecer recomendaciones dietéticas mejor ajustadas a la población.

Epidemiological studies suggest that dietary magnesium in the Western countries does not meet the recommended intake, supporting a risk of latent magnesium deficiency with Western diet behavior. Assessment of magnesium status remains a major challenge for the clinical laboratory, since, magnesium storage is mostly found in bone and soft tissues. The conciliation between an easy obtained sample, rapid and robust laboratory test, and the parameter representative for intracellular magnesium is extremely difficult to reach. In a current systematic review study, the usefulness of magnesium status biomarkers in healthy subjects has been evaluated. It is proposed that plasma and erythrocyte magnesium, and urinary magnesium excretion which respond to dietary manipulation appear to be useful biomarkers in the general population. However, it is emphasized that well-designed studies of sufficient size with varying doses and duration of magnesium supplementation are still required. The development of specific and sensible biomarkers, making it possible to obtain cell magnesium levels as well as body magnesium pool evaluation, relevant to study individuals, small and large populations, remains a major challenge for the assessment of magnesium status. A progress in genetics and genomics opens new interesting perspectives in the search of these biomarkers.

Na atualidade, dados epidemiológicos sugerem que, nos países ocidentais, a ingestão de magnésio não supre a ingestão recomendada, o que apoia um risco de deficiência de magnésio latente nestas populações. A avaliação do estado do magnésio continua sendo um desafio para o laboratório clínico, visto que o magnésio se encontra distribuído principalmente no osso e nos tecidos moles. Há a necessidade de conciliar evidência facilmente acessível, rápida, sensível e representativa do magnésio intracelular. A utilidade de vários biomarcadores em indivíduos saudáveis foi avaliada, e foi relatado que o magnésio em plasma, eritrócitos e urina parecem ser biomarcadores sensíveis à ingestão dietética e úteis como biomarcadores na população geral. No entanto, esta não é conclusiva, uma vez que se destaca que são requeridos ainda estudos melhor desenhados, envolvendo fatores como utilização de maior população, dosagem e tempo de suplementação. Um avanço na genética e na genômica abre perspectivas interessantes na busca desses biomarcadores para poder quantificar os níveis de magnésio celular bem como as reservas do corpo inteiro, e assim poder estabelecer melhores recomendações na dieta adaptadas à população.

Métodos para la determinación del estado del magnesio en humanos / Methods of assessment of magnesium status in humans / Métodos para a determinaþÒo do estado do magnésio em humanos

En la actualidad, datos epidemiológicos sugieren que, en países occidentales, la ingesta de magnesio no satisface la ingesta recomendada, lo que apoya un riesgo de deficiencia de magnesio latente en estas poblaciones. La evaluación del estado de magnesio sigue siendo un desafío para el laboratorio clínico ya que el magnesio se encuentra distribuido mayoritariamente en el hueso y tejidos blandos. Existe la necesidad de conciliación entre una prueba de fácil acceso, rápida, sensible y representativa del magnesio intracelular. La utilidad de diferentes biomarcadores en sujetos sanos ha sido evaluada; se ha reportado que el magnesio en plasma, eritrocitos y orina parecen ser biomarcadores sensibles a la ingesta dietética y útiles como biomarcadores en la población general. Sin embargo, esto no es concluyente, ya que se resalta que aún se requieren estudios mejor diseñados, que impliquen factores como mayor población empleada, dosis y tiempo de suplementación. El progreso en la genética y la genómica abren perspectivas interesantes en la búsqueda de estos biomarcadores que permitan cuantificar los niveles de magnesio celular así como también las reservas de todo el cuerpo, para poder así establecer recomendaciones dietéticas mejor ajustadas a la población.(AU)

Epidemiological studies suggest that dietary magnesium in the Western countries does not meet the recommended intake, supporting a risk of latent magnesium deficiency with Western diet behavior. Assessment of magnesium status remains a major challenge for the clinical laboratory, since, magnesium storage is mostly found in bone and soft tissues. The conciliation between an easy obtained sample, rapid and robust laboratory test, and the parameter representative for intracellular magnesium is extremely difficult to reach. In a current systematic review study, the usefulness of magnesium status biomarkers in healthy subjects has been evaluated. It is proposed that plasma and erythrocyte magnesium, and urinary magnesium excretion which respond to dietary manipulation appear to be useful biomarkers in the general population. However, it is emphasized that well-designed studies of sufficient size with varying doses and duration of magnesium supplementation are still required. The development of specific and sensible biomarkers, making it possible to obtain cell magnesium levels as well as body magnesium pool evaluation, relevant to study individuals, small and large populations, remains a major challenge for the assessment of magnesium status. A progress in genetics and genomics opens new interesting perspectives in the search of these biomarkers.(AU)

Na atualidade, dados epidemiológicos sugerem que, nos países ocidentais, a ingestÒo de magnésio nÒo supre a ingestÒo recomendada, o que apoia um risco de deficiÛncia de magnésio latente nestas populaþ§es. A avaliaþÒo do estado do magnésio continua sendo um desafio para o laboratório clínico, visto que o magnésio se encontra distribuído principalmente no osso e nos tecidos moles. Há a necessidade de conciliar evidÛncia facilmente acessível, rápida, sensível e representativa do magnésio intracelular. A utilidade de vários biomarcadores em indivíduos saudáveis foi avaliada, e foi relatado que o magnésio em plasma, eritrócitos e urina parecem ser biomarcadores sensíveis O ingestÒo dietética e úteis como biomarcadores na populaþÒo geral. No entanto, esta nÒo é conclusiva, uma vez que se destaca que sÒo requeridos ainda estudos melhor desenhados, envolvendo fatores como utilizaþÒo de maior populaþÒo, dosagem e tempo de suplementaþÒo. Um avanþo na genética e na gen¶mica abre perspectivas interessantes na busca desses biomarcadores para poder quantificar os níveis de magnésio celular bem como as reservas do corpo inteiro, e assim poder estabelecer melhores recomendaþ§es na dieta adaptadas O populaþÒo.(AU)

Blocking the rise of intracellular calcium inhibits the growth of cells cultured in different concentrations of magnesium.

Divalent cations, especially calcium and magnesium, have been shown to play an important regulatory role in endothelial and immune cells. To learn more about the interaction of these two metals in the regulation of cell growth, we altered the calcium/magnesium ratio by culturing human endothelial cells, macrophages, and T lymphocytes in media containing different concentrations of magnesium. We observed that the growth of the three cell types was retarded in low extracellular magnesium, and this retardation is particularly evident in highly proliferating cells. High concentrations of magnesium does not exert any effect on cell growth. When (i) calcium influx was blocked by adding the calcium antagonist verapamil, and (ii) calcium release from intracellular stores was inhibited by exposure to TMB-8, the growth of endothelial cells, macrophages, and T lymphocytes was inhibited. In particular, the release of calcium from intracellular stores seems to be more important than its influx in sustaining cell proliferation. Our results indicate that calcium plays a crucial role in mediating cell proliferation independently from the extracellular concentrations of magnesium.

Magnesium deficiency and metabolic syndrome: stress and inflammation may reflect calcium activation.

Magnesium (Mg) intake is inadequate in the western diet and metabolic syndrome is highly prevalent in populations around the world. Epidemiological studies suggest that high Mg intake may reduce the risk but the possibility of confounding factors exists, given the strong association between Mg and other beneficial nutriments (vegetables, fibers, cereals). The concept that metabolic syndrome is an inflammatory condition may explain the role of Mg.Mg deficiency results in a stress effect and increased susceptibility to physiological damage produced by stress. Stress activates the hypothalamic-pituitary-adrenal axis (HPA) axis and the sympathetic nervous system. The activation of the renin-angiotensin-aldosterone system is a factor in the development of insulin resistance by increasing oxidative stress. In both humans and rats, aldosteronism results in an immunostimulatory state and leads to an inflammatory phenotype. Stress response induces the release of large quantities of excitatory amino acids and activates the nuclear factor NFkappaB, promoting translation of molecules involved in cell regulation, metabolism and apoptosis. The rise in neuropeptides is also well documented. Stress-induced HPA activation has been identified to play an important role in the preferential body fat accumulation but evidence that Mg is involved in body weight regulation is lacking. One of the earliest events in the acute response to stress is endothelial dysfunction. Endothelial cells actively contribute to inflammation by elaborating cytokines, synthesizing chemical mediators and expressing adhesion molecules. Experimental Mg deficiency in rats induces a clinical inflammatory syndrome characterized by leukocyte and macrophage activation, synthesis of inflammatory cytokines and acute phase proteins, extensive production of free radicals. An increase in extracellular Mg concentration decreases inflammatory effects, while reduction in extracellular Mg results in cell activation. The effect of Mg deficiency in the development of insulin resistance in the rat model is well documented. Inflammation occurring during experimental Mg deficiency is the mechanism that induces hypertriglyceridemia and pro-atherogenic changes in lipoprotein metabolism. The presence of endothelial dysfunction and dyslipidemia triggers platelet aggregability, thus increasing the risk of thrombotic events. Oxidative stress contributes to the elevation of blood pressure. The inflammatory syndrome induces activation of several factors, which are dependent on cytosolic Ca activation. Recent findings support the hypothesis that the Mg effect on intracellular Ca2+ homeostasis may be a common link between stress, inflammation and a possible relationship to metabolic syndrome.

Phagocyte priming by low magnesium status: input to the enhanced inflammatory and oxidative stress responses.

Epidemiological and experimental studies underline the role of magnesium in inflammation. Several data indicate an enhanced response of phagocytes (granulocytes, macrophages) derived from magnesium-deficient animals or cultured under low magnesium conditions to the inflammatory mediators' stimulation. On the contrary, it was pointed out that high extracellular Mg2+ concentration might partially attenuate the activation of phagocyte leukocytes. Thus, it is likely that magnesium-deficient conditions lead to the priming (pre-activation) of phagocytic cells. Magnesium status is an important modulator of the phagocyte response to immune stimuli and consequently could be implicated in a wide range of pathophysiological issues, e.g. those related to the production of radical oxygen species (ROS). It is likely that magnesium directly modulates phagocyte priming by its calcium antagonism and indirectly by its effect on the immunoinflammatory processes, the source of the priming mediators.

High-magnesium concentration and cytokine production in human whole blood model.

The potential influence of magnesium (Mg) on inflammatory responses was assessed using an ex vivo model--human whole blood incubated with and without lipopolysaccharide (LPS). Addition of LPS leads to higher levels of cytokines including TNF-alpha and IL-6. No significant effect of Mg was observed following LPS stimulation whereas high concentration of Mg inhibited the baseline level (without LPS) of TNF-alpha and IL-6 production. This observation contrasts with that of a previous one on Mg-deficient animals. Therefore, the weak efficiency of increasing Mg concentration in this study on the whole blood from healthy volunteers suggests that the efficiency of Mg supplementation on cytokine production induced by endotoxin challenge depends on Mg status.

Serum and peritoneal evaluation of vitamin D-binding protein in women with endometriosis.

INTRODUCTION: Vitamin D-binding protein (also called DBP or Gc-globulin) is recognized as a multifunctional protein involved in the action scavenger system, the transport of vitamin D sterols, and the modulation of immune and inflammatory responses. This study evaluated total serum and peritoneal concentrations of vitamin D-binding protein in women with endometriosis, known as an inflammation-associated disease. MATERIALS/METHODS: The total concentration of DBP was measured with an enzyme-linked immunosorbent assay (ELISA) using a polyclonal antibody raised in a goat immunized with human DBP. Serum and peritoneal fluid were collected from women with endometriosis (n=26) and from patients with benign gynecological conditions serving as a control group (n=17). RESULTS: In general, the vitamin D-binding protein concentration was higher in serum than in peritoneal fluid. Women with endometriosis had higher serum but lower peritoneal levels of DBP compared with the control group; however, no significance was noted. When the endometriosis group was divided with regard to severity, an insignificantly higher serum level of DBP was observed in advanced endometriosis compared with the mild form of the disease, whereas the peritoneal concentration was not dependent on disease severity. CONCLUSIONS: It is concluded that serum and peritoneal DBP concentrations are not affected in women with endometriosis; however, based on the latest published data, it is possible that both the serum and peritoneal concentrations of vitamin D-binding protein may be dependent on Gc genotype, which results in differential modulation of monocyte/macrophage activity.

[Magnesium in skin allergy]. / Wplyw magnezu na reakcje alergiczne skóry.

Magnesium is involved in many biological processes within the body. Magnesium deficiency causes many disorders, including impairment of immunity. This review summarizes present knowledge on the relationship between magnesium and skin allergy reactions. Special focus is on allergy types I and IV. At present the best knowledge is on allergy I. Magnesium deficiency in experimental animals, mainly rats, leads to characteristic hyperemia, an increase in IgE, neutrophilia and eosinophilia, an increase in the level of proinflammatory cytokines, mastocyte degranulation, histaminemia, and splenomegaly. These symptoms observed in hypomagnesemic rats are similar to those in atopic patients. Data on the relationship between magnesium and other types of allergy are scarce. Clinical observations show the beneficial effect of topical and oral administration of magnesium salts in patients with skin allergy. All the presented data point to an important role of magnesium in allergy reactions. Other studies are needed to better understand the mechanism of magnesium's action. Well-controlled clinical protocols should also be conducted to assess the efficiency of magnesium supplementation in patients with skin allergy.

Magnesium and the inflammatory response: potential physiopathological implications.

The purpose of this review is to summarize experimental findings showing that magnesium modulates cellular events involved in inflammation. Experimental magnesium deficiency in the rat induces after a few days a clinical inflammatory syndrome characterized by leukocyte and macrophage activation, release of inflammatory cytokines and acute phase proteins, excessive production of free radicals. Increase in extracellular magnesium concentration, decreases inflammatory response while reduction in the extracellular magnesium results in cell activation. Because magnesium acts as a natural calcium antagonist, the molecular basis for inflammatory response is probably the result of modulation of intracellular calcium concentration. The priming of phagocytic cells, the opening calcium channel and activation of N-methyl-d-aspartate (NMDA) receptors, the activation of nuclear factor-kappa B (NFkappaB) have been considered as potential mechanisms. Moreover, magnesium deficiency induces a systemic stress response by activation of neuro endocrinological pathways. As nervous and immune systems interact bidirectionally, the roles of neuromediators have also been considered. Magnesium deficiency contributes to an exaggerated response to immune stress and oxidative stress is the consequence of the inflammatory response. Inflammation contributes to the pro-atherogenic changes in lipoprotein metabolism, endothelial dysfunction, thrombosis, hypertension and explains the aggravating effect of magnesium deficiency on the development of metabolic syndrome. Further studies are still needed to assess more accurately the role of magnesium in immune response in humans, but these experimental findings in animal models suggest that inflammation is the missing link to explain the role of magnesium in many pathological conditions.

Megalin and cubilin are endocytic receptors involved in renal clearance of hemoglobin.

The kidney is the main site of hemoglobin clearance and degradation in conditions of severe hemolysis. Herein it is reported that megalin and cubilin, two epithelial endocytic receptors, mediate the uptake of hemoglobin in renal proximal tubules. Both receptors were purified by use of hemoglobin-Sepharose affinity chromatography of solubilized renal brush-border membranes. Apparent dissociation constants of 1.7 microM for megalin and 4.1 microM for cubilin were determined by surface plasmon resonance analysis. The binding was calcium dependent in both cases. Uptake of fluorescence-labeled hemoglobin by BN-16 cells was inhibited by anti-megalin and anti-cubilin antibodies as well as by receptor-associated protein, a chaperone for LDL-receptor family proteins. Partial inhibition by myoglobin was observed, whereas bovine serum albumin, intrinsic factor-cobalamin complexes, and beta2-microglobulin did not affect the uptake. By use of immunohistochemistry, it was demonstrated that uptake of hemoglobin in proximal tubules of rat, mouse, and dog kidneys occurs under physiologic conditions. Studies on normal and megalin knockout mouse kidney sections showed that megalin is responsible for physiologic clearance of hemoglobin. Labeling intensities in kidneys from normal and cubilin-malexpressing dogs were similar, which suggests that, in the normal state, the role of cubilin in uptake of hemoglobin is rather limited. However, cubilin is likely to assist hemoglobin endocytosis in settings of hemoglobinuria. In conclusion, the study provides a molecular explanation for long-standing observations of hemoglobin uptake in renal proximal tubules that involve the endocytic receptors megalin and cubilin. The findings may prove to be essential for further research on the pathophysiology of hemoglobinuric acute renal failure and proteinuria-associated tubulointerstitial nephritis.