Metformin treatment may be associated with decreased levels of NT-proBNP in patients with type 2 diabetes.

PURPOSE: Individuals with type 2 diabetes (T2DM) are at increased risk of cardiovascular disease, including heart failure (HF). In patients with T2DM elevated serum concentrations of the N-terminal fragment of pro-B-type natriuretic peptide (NT-proBNP) correlate with cardiovascular morbidity and mortality. We aimed to identify predictors of increased serum NT-proBNP levels in patients with T2DM. METHODS: The study included 185 patients with T2DM treated with either oral antidiabetic agents (49.7%) or insulin (17.8%), or both (32.5%). We divided the patients into two groups: with high (>200 pg/mL) and low (≤200 pg/mL) NT-proBNP concentrations. RESULTS: We found differences between the patients with high and low NT-proBNP levels including age, prevalence of dyslipidemia and HF, history of previous myocardial infarction (MI), heart rate, hemoglobin level, platelet count, creatinine, urea and uric acid concentrations, use of beta-blockers, loop diuretics, metformin and insulin. In a multivariate analysis metformin was a negative predictor of increased NT-proBNP concentration. Age, history of HF and decreased estimated glomerular filtration rate (eGFR) were positive predictors. We found no correlation between NT-proBNP serum concentration and insulin treatment or history of coronary artery disease or MI. CONCLUSION: Metformin correlates with lower concentrations of NT-proBNP in patients with T2DM.

Association of dopamine receptor gene polymorphisms with the clinical course of Wilson disease.

BACKGROUND: Dopamine receptor D2 (DRD2) polymorphisms are proposed to be important factors in the presentation of neuropsychiatric symptoms in many disorders, including decreased striatum levels of dopamine D2 receptors in Wilson disease. The present study investigated the association between DRD2 gene polymorphisms and clinical manifestation of Wilson disease. METHODS: Analyzing data from 97 symptomatic Wilson disease patients, we investigated the DRD2 gene polymorphisms rs1800497, rs1799732, and rs12364283. We assessed the polymorphisms impact on the phenotypic presentation of the disease. RESULTS: Generally, the DRD2 gene polymorphisms had no impact on the hepatic or neuropsychiatric clinical presentation of Wilson disease. However, rs1799732 deletion allele carriers with neuropsychiatric symptoms had earlier onset of WD symptoms by almost 6 years compared with individuals without this allele (22.5 vs. 28.3 years; P < 0.05). This unfavorable effect of the rs1799732 polymorphism was even more pronounced among adenosine triphosphatase 7B gene (ATP7B) p.H1069Q homozygous patients, in whom carriership of the deletion allele was related to earlier initial neuropsychiatric manifestation by 14 years (18.4 vs. 32.2 years; P < 0.01). CONCLUSIONS: Genetic variation of DRD2, specifically the rs1799732 polymorphism, may produce an earlier clinical presentation of Wilson disease neuropsychiatric symptoms and signs that occur in the course of dopaminergic system impairment due to copper accumulation in the brain. We speculate that this effect may be due to the impact of DRD2 polymorphism on dopamine D2 receptor density, but further studies are needed to understand the mechanisms of such phenotypic effects.

Genetic determinants of platelet reactivity during acetylsalicylic acid therapy in diabetic patients: evaluation of 27 polymorphisms within candidate genes.

AIMS: Decreased platelet responsiveness to acetylsalicylic acid (ASA) reported previously in diabetic patients could be attributed to patient-based, clinical, genetic and cellular factors. The objective of the present study was to investigate the effect of the genomic polymorphism on the platelet reactivity in diabetic patients treated with ASA. METHODS AND RESULTS: The study cohort consisted of 295 Caucasians with diabetes type 2 who had been taking ASA tablets at the dose of 75 mg per day for at least 3 months for primary or secondary prevention of myocardial infarction (MI). Platelet reactivity analyzes were performed using VerifyNow ASA and PFA-100 assays. Genotyping for the selected 27 single nucleotide polymorphisms (SNPs) within 19 genes was performed using a Sequenom iPLEX platform. The results indicate that the statistically significant differences in platelet reactivity were observed in the PFA-100 assay for SNPs in following genes: TXBA2R (rs1131882), ADRA2A (rs4311994), PLA2G7 (rs7756935) and 9p21.3 (rs10120688) (P = 0.02, P = 0.03, P = 0.02, P = 0.03, respectively, all significance levels corrected for multiple comparisons). When using the VerifyNow ASA test, a weak nominal statistical significance (i.e. before multiple comparison testing) was observed for two SNPs in the GPVI gene: rs1671152 and rs1613662 [P = 0.025 (0.5) for both SNPs, corrected for multiple comparisons test]. CONCLUSIONS: The results from the present study suggest that the four analyzed genes may contribute to platelet reactivity measured with the PFA-100 assay in the diabetic population treated with ASA.

The impact of age and gender on the striatal astrocytes activation in murine model of Parkinson's disease.

OBJECTIVE: The aim of the present study was to determine how aging and gender influence the response of astrocytes to 1-methyl-4-phenyl-1,2,3,6-tetrahydropiridine (MPTP) intoxication. MATERIALS AND METHODS: To asses the MPTP-induced astrocytes activation in nigro-striatal system, we measured the temporal changes in mRNA and protein expression of the specific astrocytic marker, glial fibrillary acidic protein (GFAP; by RT-PCR and Western blot), in the striatum of male and female C57BL/6 mice (2 and 12-month old) after 6 h and 1, 3, 7, 14 and 21 days post-intoxication. RESULTS: We observed the increases of GFAP mRNA level post-MPTP intoxication in both young and aging males only at early time points, whereas in females (both ages) also at later time points. We noticed maximal increase of GFAP protein content on the 3rd day post-intoxication in young and aged males, whereas in females at the 7-daytime point. CONCLUSIONS: The present results provide additional information of potential relevance to understand the mechanisms of gender and age-related difference in susceptibility of nigro-striatal system to MPTP insult.

Decreased inflammation and augmented expression of trophic factors correlate with MOG-induced neuroprotection of the injured nigrostriatal system in the murine MPTP model of Parkinson's disease.

The response of the immune system during injury of the central nervous system may play a role in protecting neurons. We have previously reported that immunization with MOG 35-55 prior to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced injury of the dopaminergic system promotes less dopamine depletion and less dopaminergic damage of neurons in mice. In this study, we evaluate the influence of MOG immunization on the inflammatory reaction that occurs at the place of injury. C57Bl male mice, 2 and 12 months old, received i.p. injections of MPTP (40 mg/kg) and some groups animals also received an additional injection with myelin oligodendrocyte glycoprotein (MOG) 35-55 in CFA 6 days before MPTP administration. MPTP caused a common inflammatory reaction characterized by microglial activation, infiltration of T cells into the substantia nigra and striatum and increased expression of mRNA encoding pro-inflammatory cytokines (IL-1 beta, TNFalpha, INF gamma) and trophic factors (TGFbeta, GDNF). MOG immunization prior to MPTP administration significantly diminished the microglial reaction and reduced the levels of infiltrating CD8+ lymphocytes. The number of CD4+ T cells remained at the same level as in the MPTP group. Expression of pro-inflammatory cytokines was diminished. The mRNA expression of GDNF was significantly higher in the MOG pretreated mice relative to the MPTP group, both in the 2 month old and 12 month old groups. Since MOG immunization prior to MPTP intoxication appears to prevent nigrostriatal injury, the observed decrease of inflammation and increase of GDNF mRNA expression in the injured areas might represent one of the mechanisms of observed neuroprotection.

MPTP-induced central dopamine depletion exacerbates experimental autoimmune encephalomyelitis (EAE) in C57BL mice.

It is obvious that the central nervous system plays a role in the regulation of an immune response. However, the mechanisms of this regulation are poorly understood. The goal of the present study was to examine the role of one of the neurotransmitters - dopamine, in this process. We used experimental autoimmune encephalomyelitis (EAE), an autoimmune disease with its effector phase in the CNS, as a model to study the effect of central dopamine depletion on the development of an immune response. Dopamine depletion was achieved by treatment with 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropiridine (MPTP; 40 mg/kg), whereas EAE was elicited by immunization with MOG 35-55 (150 microg) in complete Freund's adjuvant (CFA), supplemented with Mycobacterium tuberculosis. As determined by HPLC, striatal dopamine contents in mice treated with MPTP were significantly lower compared to vehicle-treated controls. Remarkably, striatal depletion of dopamine prior to EAE induction resulted in an earlier onset of the disease and an augmentation of its clinical signs. Moreover, the striatal dopamine-depleted mice demonstrated an increased concentration of IL-1beta and decreased concentration of TGFbeta in the spinal cord, compared to EAE mice. Since MPTP itself does not have any direct effect on immune cells, it strongly suggests that the observed changes in EAE induction and progression after MPTP administration depended on lower dopamine level. Further studies are required to find out the cellular mechanism of the dopamine action.

Immunization with myelin oligodendrocyte glycoprotein and complete Freund adjuvant partially protects dopaminergic neurons from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced damage in mouse model of Parkinson's disease.

The concept of neuroprotective immunity identifies a new role of autoimmune cells in the CNS pathology. Specifically, immune cells infiltrating the CNS during an injury may help in a regeneration process and prevent the secondary degeneration of neurons. The objectives of our study were to determine the role of autoimmune and peripheral immune enhancement in neurodegeneration process, and to compare the results between young adult and aging animals. C57Bl mice were immunized with either myelin oligodendrocyte glycoprotein (MOG) 35-55 combined with complete Freund adjuvant (CFA), or CFA alone. Following 6 days, the animals were injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine to produce a damage of the nigrostriatal dopaminergic system. Although immunization with MOG 35-55 combined with CFA resulted in autoimmune encephalomyelosis, it substantially enhanced neuronal survival after the toxic insult. The immunization with CFA alone was also effective in preventing neuronal cell death, but the magnitude of the neuroprotective effect was smaller. Interestingly, the neuroprotective effect of MOG 35-55 and CFA was more pronounced in aging (i.e. 10-month-old) compared with young (i.e. 2-month-old) mice. Our results indicate that an increased immune activation may be beneficial for neurodegenerative processes following the CNS injury, but the mechanisms of such immune neuroprotection and of age differences need further investigation.

A prospective study of the financial costs of multiple sclerosis at different stages of the disease.

The aim of our study was to estimate the costs of multiple sclerosis (MS) in Poland according to severity of disease. Total, direct and indirect costs were compared in 148 patients divided into three groups categorized by disease severity: stage I Expanded Disability Status Scale (EDSS <3.5), stage II (EDSS 4.0-6.0) and stage III (EDSS >6.5). Cost evaluation was performed from the societal perspective and covered the 5-month period. Simple sensitivity analysis was performed by varying the tariffs and valuing caregiving at 40% of the average wage. The mean total cost/patient for 5 months was estimated at 10,955, 15, 603 and 18, 464 PLN for stage I, II and III, respectively [exchange rate: 4 PLN=1 EUR; purchasing power pariety: 1 EUR=2.05 PLN] (P <0.0001). Regardless of EDSS stage indirect costs exceeded direct costs. Both direct and indirect costs increased with MS progression. For indirect cost the main item was productivity loss. This study confirms that MS represents a high economic burden, with indirect costs greatly exceeding direct costs. As costs increase with disease progression, treatment efforts should focus on patients in the early stages of MS.

Increase of matrix metalloproteinase-9 in peripheral blood of multiple sclerosis patients treated with high doses of methylprednisolone.

Matrix metalloproteinases (MMPs) are believed to play a role in the pathogenesis of multiple sclerosis (MS). As methylprednisolone is the treatment of choice for a relapse, we investigated the effect of methylprednisolone on blood levels of MMPs. Baseline TIMP-1 and MMP-2 levels were lower in MS patients than in healthy controls. MMP-9 levels tended to be elevated. During therapy, MMP-9 levels demonstrate a dose-dependent increase. No effect was noted on TIMP-1 and MMP-2 levels. The short-lived increase of MMP-9 plasma levels may be at least in part due to an activation and an increase of granulocytes and monocytes by methylprednisolone.

Indomethacin protects against neurodegeneration caused by MPTP intoxication in mice.

The anti-inflammatory agents are postulated to be effective in treating neurodegenerative disorders. In this study, we showed that indomethacin (IND) in the dose of 1 mg/kg protected neurons against toxic damage caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice model of Parkinson's disease. IND also diminished microglial activation and lymphocytic infiltration in the injured areas. These observations suggest that anti-inflammatory properties of IND may play a role in the neuron's protection in this model. However, diminished inflammatory reaction may be secondary to less neuronal damage.

MHC class II positive microglia and lymphocytic infiltration are present in the substantia nigra and striatum in mouse model of Parkinson's disease.

We have studied MHC class II antigen expression and lymphocytic infiltration during dopaminergic neurone degeneration produced by intoxication with 1-methyl-4-phenyl-1,2,3,6-tetrahydropiridine (MPTP). Microglial activation was observed in the striatum and in the substantia nigra (SN) in this model. We noticed a marked increase of MHC class II antigen expression on microglia and T-cell recruitment in these regions after MPTP treatment. B-lymphocytes were not observed. T-cell infiltration predominantly consisted of CD8+ cells at every time point but CD4+ cells were present too. More than a half of the observed lymphocytes showed strong staining of CD44 antigen. Our findings suggest a possible immune system involvement in the pathological process following MPTP intoxication.

The inflammatory reaction following 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine intoxication in mouse.

In degenerative disorders of the CNS an immune system involvement in the pathological process is postulated. The MPTP model of Parkinson's disease seem to be a good model for studying an inflammation following toxic neurodegeneration. In this model, microglial and astroglial reactions were previously found around impaired neurons. In the present work we showed an immune reaction, including lymphocytic infiltration of CD4+ and CD8+ T cells in the substantia nigra and striatum and elevated MHC class I and II antigens expression on microglia. Many activated lymphocytes were present, showing increased LFA-1 and CD44 antigen expression. We found also that ICAM-1 expression increased on the endothelium and appeared on microglia in the injured regions. Treatment with dexamethasone inhibited T-cell infiltration and MHC class II expression, lessened the glial reaction, and also diminished neuronal impairment. These findings suggest that an immune mechanism may contribute to the neuronal damage following MPTP administration.

Phenotyping analysis of peripheral blood leukocytes in patients with multiple sclerosis.

Multiple sclerosis (MS) is a central nervous disease thought to be elicited by an autoimmune process. Many studies in recent years have concentrated on finding the alterations in the peripheral blood immune profile in MS patients that would reflect disease activity. In the present study, we investigated surface antigen expression on lymphocytes and granulocytes from MS patients and control subjects. We have studied 29 patients suffering from relapsing-remitting or relapsing-progressive forms of MS. The disease was diagnosed in all patients at least 12 months before inclusion into the study. All patients had no attack at the study entry date or within a previous month. The control group included 29 age-matched subjects. Phenotyping of peripheral blood leukocytes was carried out with different fluorescence-conjugated murine monoclonal antibodies. The analysis was performed with three-color flow cytometry. The following antigens were determined [cluster of definition (CD)]: leukocyte common antigen (LCA) (B220, T 200, Ly-5), CD45; LPS-R (lipopolysaccharide receptor), CD14; found on all T cells, CD3; LFA-2 (lymphocyte function associated antigen, T 11), CD2; coreceptor for MHC class II molecules, found on helper T cells, CD4; coreceptor for MHC class I molecules, found on suppressor/cytotoxic T cells, CD8; B4, found on all human B cells, CD19; NCAM (neural cell adhesion molecule), CD56; integrin beta2 subunit, associated with CD11a (CD11a/CD18, LFA-1, alphaLbeta2) and CD11b (CD11b/CD18, Mac-1,CR3, alphaMbeta2), CD18; alphaL, alpha subunit of integrin LFA-1 (alphaLbeta2, CD11a/CD18), CD11a; alphaM, alpha subunit of integrin Mac-1 (CR3, alphaMbeta2, CD11b/CD18), CD11b; ICAM-1 (intercellular adhesion molecule), CD54; H-CAM, Hermes antigen, Pgp-1, CD44; AIM (activation inducer molecule), early activation antigen, CD69; T-cell receptor gammadelta, TCR gammadelta. In the MS group, we have found a significant increased expression of CD54 and CD44 antigens on lymphocytes, and higher percentage CD54(+) and CD11a+CD54(+) lymphocytes out of all lymphocytes compared with the control group. We have also found a significant increased expression of CD11a, CD18 and CD54 antigens on granulocytes, and higher percentage CD11b+CD18(+) granulocytes out of all granulocytes in MS patients compared with control. Higher levels of expression of the adhesion molecules may reflect the activation state of leukocytes in MS patients.

Ultrastructural changes in substantia nigra and striatum observed on a mouse model of Parkinson's disease induced by MPTP administration.

The study was carried out on a mouse model of Parkinson's disease induced by the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-neurotoxin which damages dopaminergic neurons in substantia nigra. Occurrence of dark degenerated neurons was the most prominent ultrastructural change. They were characterized by the progressive condensation of cytoplasm and nuclear chromatin as well as by the light mitochondria and dilated cisternae of Golgi apparatus. Dark degenerated neurons were found particularly often on the 7th day after toxication, however on the last day of the observation, only a few neurons showed the features of dark degeneration. It is likely that degenerative changes led to death in the part of neurons only.

Microglial and astrocytic involvement in a murine model of Parkinson's disease induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

We have studied the reaction of glial cells in mice treated with an intraperitoneal administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a selective neurotoxin of dopaminergic nigrostriatal neurons. Signs of injury to the dopaminergic neurons started on the 1st day after MPTP administration and progressed up to the end of the observation time (21st day). A transient microglial reaction was demonstrated from the 1st until the 14th day in the substantia nigra (SN) and striatum. The cells showed an increase in number and changes in morphology. At the ultrastructural level, signs of phagocytosis and features indicating the secretion of biologically active substances were observed. Astrocytosis followed the microglial reaction by one day and was noticed until the end of the observation time. Interleukin-6 immunoreactivity was observed within microglia and astrocytes in the SN on days 2 and 3. There were no signs of depletion of dopaminergic cells or glial activation after the administration of MPTP simultaneously with pargyline, an inhibitor of monoamine oxidase-B that prevents MPTP neurotoxicity. Our study indicates that microglia and astrocytes are involved in the pathological process in the nigrostriatal system following MPTP administration. MPTP alone is not responsible for glial cell activation but its metabolite MPP+ and/or agents released by injured neurons may participate in this process.

Microglial reaction in MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) induced Parkinson's disease mice model.

We studied the microglial reaction in mice using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced model for Parkinson's disease (PD). Microglial cells were identified by means of the Griffonia simplicifolia lectin (GSA-I-B4). Dopaminergic neurons were marked by tyrosine hydroxylase antibodies. Microglial activation was demonstrated by an increase in cellular number and changes of morphology (increased lectin staining, larger cell bodies and thicker processes) were seen in the substantia nigra from the 1st to the 14th day and in the striatum from the 1st to the 4th day after intoxication. Depletion of dopaminergic neurons was most pronounced 7 and 14 days following the treatment. The results suggest that microglial activation may be involved in the sequence of pathological changes that lead to dopaminergic neuronal damage after MPTP intoxication.