Effects of IFN-γ, TNF-α, IL-10 and TGF-ß on Neospora caninum infection in rat glial cells.

Neospora caninum causes abortion in cattle and neurological disorders in dogs. The immunological response to this parasite has been described as predominantly of the Th1 type. However, infected primary glial cell cultures release IL-10 and IL-6 but not IFN-γ. This suggests a rather protective response of the glia to avoid inflammatory damage of the nervous tissue. In this study, we investigated the effects of pro-inflammatory cytokines in primary mixed cultures of rat astrocytes and microglia infected with N. caninum. The cells were treated with either IFN-γ, TNF-α, anti-IL-10 or anti-TGF-ß antibodies and were infected with parasite tachyzoites 24h later. Trypan Blue exclusion and MTT assays were performed to test cell viability. It was observed that cytokines, antibody treatment and in vitro infection did not reveal significant cell death in the various culture conditions. Treatment with 50, 150 and 300 IU/mL of either IFN-γ or TNF-α reduced tachyzoites numbers in cultures by 36.7%, 54.8% and 63.8% for IFN-γ and by 27.6%, 38.4% and 29.7% for TNF-α, respectively. In the absence of IL-10 and TGF-ß, tachyzoite numbers were reduced by 52.8% and 41.5%, respectively. While IFN-γ (150 and 300 IU/mL) increased the nitrite levels in uninfected cells, parasite infection seemed to reduce the nitrite levels, and this reduction was more expressive in IFN-γ-infected cells, thereby suggesting an inhibitory effect on its production. However, TNF-α, IL-10 and TGF-ß did not affect the nitrite levels. Basal PGE(2) levels also increased by 17% and 25%; 78% and 13% in uninfected and infected cells treated with IFN-γ or anti-TGF-ß, respectively. Nevertheless, the antibody neutralization of IL-10 reduced PGE(2) release significantly. These results highlight the possibility of a combined effect between the IFN-γ and parasite evasion strategies and show that the IFN-γ, TNF-α, IL-10 and TGF-ß cytokines participate in parasite proliferation control mechanisms.

[Embryology of the heart walls]. / Embryologie des cloisons du cœur.

Although anatomically simple structures, the atrial septum and the ventricular septum have complex embryological origins. Recent findings in molecular biology allowed better comprehension of their formation. As soon as the heart tube is formed, cells migrate from several cardiogenic fields to take part in the septation. Elongation, ballooning, and later inflexion of the heart tube create chamber separating grooves, facing the future septa. The systemic venous tributaries conflate at the venous pole of the heart; it will partially involute while contributing to the atrial septum. The primary atrial septum grows from the atrial roof towards the atrioventricular canal. It fuses there with the atrioventricular cushions, while its upper margin breaks down to form the ostium secundum. Then a deep fold develops from the atrial roof and partly covers the ostium secundum, leaving a flap-like interatrial communication through the oval foramen. It will close at birth. The interventricular septum has three embryological origins. The ventricular septum primum, created during the ballooning process, origins from the primary heart tube. It will form the trabecular septum and the inlet septum. The interventricular ring, surrounding the interventricular foramen, will participate in the inlet septum and also form the atrioventricular conduction axis. The outflow cushions will separate the outflow tract in the aorta and pulmonary artery, and grow to create the outlet septum. After merging with the atrioventricular cushions, they will also be part of the membranous septum.

Neospora caninum: early immune response of rat mixed glial cultures after tachyzoites infection.

Neospora caninum causes neurologic disease in dogs and abortion in cattle. Little is known about the immune response of the CNS against this protozoan. The aim of this study was to evaluate production of IL-6, IL-10, TNF-alpha, IFN-gamma, and NO in rat mixed glial cell cultures infected by N. caninum. IFN-gamma was not observed. The mean cytokine released after 24 and 72 h of infection were 3.8+/-0.6 and 3.7+/-0.6 pg TNF-alpha/mg protein and 2.7+/-0.69 and 4.1+/-0.64 pg IL-10/mg protein, respectively, and more than 8.0 pg IL-6/mg protein for both time points. NO levels increased 24h post-infection (2.3+/-0.8 pg/mg protein) until 72 h (4.2+/-1.1 pg/mg protein) and the number of tachyzoites reduced with the time. Our results show high levels of regulatory cytokines that may suppress the harmful effects of IFN-gamma; high levels of TNF-alpha and NO may represent an effective response by infected glial cells against N. caninum.

Microtubule-associated proteins and in vitro astrocyte differentiation.

Primary cultures of mouse brain astrocytes have been used to identify the microtubule-associated proteins (MAPs) present in this cell type at different stages of in vitro differentiation. The MAPs of the astrocyte have been identified by polyacrylamide gel electrophoresis and immunological detection. Two antisera were raised against two brain MAPs, tau and MAP-2. These antisera were also used to label the microtubular network in the intact astrocytes at different stages of the culture. The mature astrocyte contains a variety of MAP-like proteins. Anti-MAP-2 serum detected several proteins of high molecular weight (380,000, 260,000, 205,000 and 165,000 mol wt) and one microheterogeneous peak of 83,000 mol wt. Anti-tau also detected high molecular weight components (380,000 to approximately 200,000 mol wt) but not the 165,000-mol-wt peak; in addition two microheterogeneous peaks of 83,000 and 62,000 mol wt were detected by the anti-tau serum. The 62,000-mol-wt peak was therefore detected only by the anti-tau serum whereas the 83,000-mol-wt component cross-reacted with both antisera. At early stages of the culture the immature cell contained about two times less immunoreactive material than at mature stages. Qualitative changes of the high molecular weight components were also observed. In the intact cell both antisera revealed a dense fibrous network. At early stages of the culture the astroblasts were stained by the antisera but the reaction was very diffuse in the cytoplasm; few fibrous cells were intensively stained. Morphological differentiation, which began after serum deprivation and which was accelerated by forskolin (a drug that induces cyclic AMP accumulation), led to high labeling of both the cell body and the cellular processes. In the presence of colchicine the staining regressed, the processes shortened, and the cell returned to a less-apparently differentiated state.

The flavonoid rutin induces astrocyte and microglia activation and regulates TNF-alpha and NO release in primary glial cell cultures.

Astrocyte and microglia cells play an important role in the central nervous system (CNS). They react to various external aggressions by becoming reactive and releasing neurotrophic and/or neurotoxic factors. Rutin is a flavonoid found in many plants and has been shown to have some biological activities, but its direct effects on cells of the CNS have not been well studied. To investigate its potential effects on CNS glial cells, we used both astrocyte primary cultures and astrocyte/microglia mixed primary cell cultures derived from newborn rat cortical brain. The cultures were treated for 24 h with rutin (50 or 100 micromol/L) or vehicle (0.5% dimethyl sulfoxide). Mitochondrial function on glial cells was not evidenced by the MTT test. However, an increased lactate dehydrogenase activity was detected in the culture medium of both culture systems when treated with 100 micromol/L rutin, suggesting loss of cell membrane integrity. Astrocytes exposed to 50 micromol/L rutin became reactive as revealed by glial fibrillary acidic protein (GFAP) overexpression and showed a star-like phenotype revealed by Rosenfeld's staining. The number of activated microglia expressing OX-42 increased in the presence of rutin. A significant increase of nitric oxide (NO) was observed only in mixed cultures exposed to 100 micromol/L rutin. Enhanced TNFalpha release was observed in astrocyte primary cultures treated with 100 micromol/L rutin and in mixed primary cultures treated with 50 and 100 micromol/L, suggesting different sensitivity of both activated cell types. These results demonstrated that rutin affects astrocytes and microglial cells in culture and has the capacity to induce NO and TNFalpha production in these cells. Hence, the impact of these effects on neurons in vitro and in vivo needs to be studied.

Alkaloids from Prosopis juliflora leaves induce glial activation, cytotoxicity and stimulate NO production.

Prosopis juliflora is used for feeding cattle and humans. Intoxication with the plant has been reported, and is characterized by neuromuscular alterations and gliosis. Total alkaloidal extract (TAE) was obtained using acid/basic-modified extraction and was fractionated. TAE and seven alkaloidal fractions, at concentrations ranging 0.03-30 microg/ml, were tested for 24h on astrocyte primary cultures derived from the cortex of newborn Wistar rats. The MTT test and the measure of LDH activity on the culture medium, revealed that TAE and fractions F29/30, F31/33, F32 and F34/35 were cytotoxic to astrocytes. The EC(50) values for the most toxic compounds, TAE, F31/33 and F32 were 2.87 2.82 and 3.01 microg/ml, respectively. Morphological changes and glial cells activation were investigated through Rosenfeld's staining, by immunocytochemistry for the protein OX-42, specific of activated microglia, by immunocytochemistry and western immunoblot for GFAP, the marker of reactive and mature astrocytes, and by the production of nitric oxide (NO). We observed that astrocytes exposed to 3 microg/ml TAE, F29/30 or F31/33 developed compact cell body with many processes overexpressing GFAP. Treatment with 30 microg/ml TAE and fractions, induced cytotoxicity characterized by a strong cell body contraction, very thin and long processes and condensed chromatin. We also observed that when compared with the control (+/-1.34%), the proportion of OX-42 positive cells was increased in cultures treated with 30 microg/ml TAE or F29/30, F31/33, F32 and F34/35, with values raging from 7.27% to 28.74%. Moreover, incubation with 3 microg/ml F32, 30 microg/ml TAE, F29/30, F31/33 or F34/35 induced accumulation of nitrite in culture medium indicating induction of NO production. Taken together these results show that TAE and fractionated alkaloids from P. juliflora act directly on glial cells, inducing activation and/or cytotoxicity, stimulating NO production, and may have an impact on neuronal damages observed on intoxicated animals.

[A case of hyperthyroidism due to functioning metastasis of differentiated thyroid carcinoma. Discussion and literature review]. / Un cas d'hyperthyroïdie par métastases sécrétantes de cancer thyroïdien différencié. Revue de la littérature.

We report on a very rare case of hyperthyroidism due to multiple autonomously functioning bone metastasis of papillary thyroid cancer in a 79-year-old woman. This situation remains extremely uncommon, as shown by our review of the literature; only 47 similar cases have been published from 1946 to 2005. The pathogenic mechanism remains largely unknown in spite of several hypotheses (conjunction in volume and differentiation, auto-antibodies). Hyperthyroidism can be severe, and often T3 levels are markedly more elevated than T4 levels. Apart from hyperthyroidism caused by the hormone-production, clinical features are similar to that of usual metastatic thyroid cancer, occurring in elderly women in most cases, and of follicular type on pathology. Metastases mostly occur in bones and lungs. Treatment relies mainly on radio-iodine ((131)I), which is efficient on hormonal disorders, and prognosis appears to be correlated with the ability of the metastatic sites to concentrate radio-iodine.

Astroglial cells in primary culture: a valid model to study Neospora caninum infection in the CNS.

The protozoan Neospora caninum has a veterinary importance because it causes abortion in cattle and neuromuscular alterations in dogs. We infected rat astrocytes, in vitro, with different concentrations of N. caninum. Astrocytes responded to infection by producing the pro-inflammatory cytokine TNF-alpha and the neurotoxic-free radical NO, 24 and 72 h post-infection. These data suggest that astrocytes, which are essential for brain function, are targets for the parasite and this represents a practical and valid model to study the effects of N. caninum on the CNS.

Purification and properties of 4-aminobutyrate 2-ketoglutarate aminotransferase from pig liver.

4-Aminobutyrate-transaminase (4-aminobutyrate: 2-oxoglutarate amino-transferase, EC 2.6.1.19) from pig liver has been purified to electrophoretic homogeneity. It has a molecular weight of about 110 000 and is composed of two subunits of the same molecular weight but of different charges. Two forms of pig liver 4-aminobutyrate-transaminase were isolated by DEAE-cellulose chromatography and designated as 4-aminobutyrate-transaminase I and 4-aminobutyrate-transaminase II, corresponding to a cationic and anionic form. Some physical and kinetic properties of liver enzyme were compared to those of brain enzyme and no significant difference were found, except for their sedimentation coefficients and the charges of their subunits. The role of 4-aminobutyrate-transaminase in liver remains a matter of speculation, but could be related to a metabolic function.

Subjective sleep and cognitive complaints in 65 year old subjects: a significant association. The PROOF cohort.

OBJECTIVE: The prevalence of subjective sleep and cognitive complaints increases with age. The purpose of this study was to investigate the link between subjective cognitive and sleep complaints in a population aged 65. DESIGN AND SETTING: analysis of a cohort of 1011 subjects aged 65 years old at time of inclusion. METHODS: Older people underwent a cognitive tests battery and a nocturnal polygraphy recording. Subjective cognitive difficulties were scored on the McNair and Kahn Scale. Subjective sleep complaints were evaluated according to the St. Mary's Hospital Sleep Questionnaire and the Epworth Sleepiness Scale score. RESULTS: In a 65 years old population, an association between subjective cognitive difficulties and poor sleep quality was observed. This remained significant after adjustment on gender, depression score, anxiety, educational level, medication intake, Apnea/Hypopnea index, Body Mass Index and Mini-Mental State Examination (OR = 2.1; p = 0.0002). Similar significant association was demonstrated between subjective cognitive difficulties and daytime sleepiness (OR = 2.6; p = 0.0007). CONCLUSION: There was a significant association between subjective cognitive and sleep complaints, and daytime sleepiness in our population of older people.