Lack of association between C282Y and H63D polymorphisms in the hemochromatosis gene and risk of multiple sclerosis: A meta-analysis.

Increasing evidence supports the potential role of iron metabolism in multiple sclerosis (MS). Previous studies examining the association between polymorphisms of the hemochromatosis gene (HFE) and susceptibility to MS have yielded inconsistent results. In the present study, a meta-analysis of 7 studies was performed conducted in populations of Caucasian origin using the Comprehensive Meta-analysis 3.0 software. The strength of association between the C282Y and H63D polymorphisms in HFE and MS risk was estimated by odds ratios with 95% confidence intervals. Cochran's Q statistic and I2 tests were applied to quantify heterogeneity between studies. An Egger's test was used to estimate publication bias. The C282Y and H63D polymorphisms had no significant association with increased MS risk (all P≥0.05) in the following genetic comparison models: Dominant model (YY + CY vs. CC or DD + HD vs. HH) and allele contrast (Y vs. C or D vs. H). No apparent publication bias or significant heterogeneity was found between studies. These results suggest that the HFE polymorphisms C282Y and H63D are not associated with susceptibility to MS in populations of Caucasian origin. Further studies should be performed in a larger series of MS patients to evaluate the contribution of HFE and other genetic variants associated with iron regulation in the development and progression of MS.

Hepcidin and metallothioneins as molecular base for sex-dependent differences in clinical course of experimental autoimmune encephalomyelitis in chronic iron overload.

Multiple sclerosis is a chronic demyelinating disease of the central nervous system characterised by inflammatory and degenerative changes. It is considered that disease arises from the influence of environmental factors on genetically susceptible individuals. Recent researches, using magnetic resonance imaging, connected iron deposits in different brain regions with demyelinating process in multiple sclerosis patients. Although iron is an essential trace element important for many biological functions it could be harmful because iron excess can induce the production of reactive oxygen species, development of oxidative stress and lipid peroxidation which leads to demyelination. In experimental autoimmune encephalomyelitis model, the most common experimental animal model for multiple sclerosis, we recently found that chronic iron overload influences the clinical course of disease in Dark Agouti rats. In female rats iron overload accelerated the onset of disease, while in male rats it accelerated the progression of disease and increased mortality rate. We hypothesize that those differences arise on molecular level in different expression of stress response proteins hepcidin and metallothioneins in male and female iron overloaded rats. They are both upregulated by metal ions in both sexes. Hepcidin is additionally upregulated by estrogen in female rats and therefore causes higher degradation of iron exporter ferroportin and sequestration of iron in the cells, lowering the possibility for the development of oxidative stress. Antioxidative effect of metallothioneins could be increased in female rats because of their ability to reversibly exchange metal ions with the estrogen receptor. In case of iron excess metallothioneins release zinc, which is normally bound to them. Zinc binds to estrogen receptor and leaves metallothioneins binding domains free for iron, causing at least provisional cytoprotective effect. To test this hypothesis, we propose to determine and compare serum levels of hepcidin and estrogen using ELISA essay as well as expression and distribution of acute stress response proteins hepcidin and metallothioneins, iron and estrogen receptor in the brain and spinal cord tissue using immunohistochemistry in control and chronic iron overloaded male and female rats in experimental autoimmune encephalomyelitis model. It would be also possible to perform the same immunohistochemistry in the brain tissue of multiple sclerosis patients post mortem. The results of experiments could contribute to better understanding of cytoprotective mechanisms in chronic iron overload that could have possible therapeutic applications in iron disturbances. In order to elucidate whether common measure of systemic iron status, like ferritin, haemoglobin concentration and transferrin saturation levels, may be used to distinguish physiologic from potentially harmful iron levels in local disease, for example multiple sclerosis and Still's disease, well-designed clinical trials would be of great interest.

Chronic iron overload induces gender-dependent changes in iron homeostasis, lipid peroxidation and clinical course of experimental autoimmune encephalomyelitis.

To analyze iron- and gender-dependent mechanisms possibly involved in pathogenesis of multiple sclerosis (MS) in this study we evaluated the effects of iron overload (IO) on iron status and lipid peroxidation processes (LPO) in tissues of female and male DA rats during chronic relapsing experimental autoimmune encephalomyelitis, a well-established MS animal model. Rats were treated by iron sucrose (75mg/kg bw/day) or with saline solution during two weeks before the sensitization with bovine brain homogenate in complete Freund's adjuvant. Clinical signs of EAE were monitored during 29 days. Serum and tissues of CNS and liver were sampled before immunization and at day 13th post immunization (during acute phase of EAE). The determination of ferritin, iron, malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) and evaluation of histopathology were performed by ELISA, ICP spectrometry and immunohistochemistry. Results showed that IO in female EAE rats accelerated the onset of disease. In contrast, in male rats it accelerated the progression of disease and increased the mortality rate. During acute phase of EAE female IO rats sequestered more Fe in the liver, spinal cord and in the brain and produced more ferritin than male EAE rats. Male rats, however, reacted on IO by higher production of MDA or 4-HNE in the neural tissues and showed greater signs of plaque formation and gliosis in spinal cord. The data point to sexual dimorphism in mechanisms that regulate peripheral and brain iron homeostasis and imply that men and women during MS might be differentially vulnerable to exogenous iron overload.

Metallothionein I+II expression as an early sign of chronic relapsing experimental autoimmune encephalomyelitis in rats.

Metallothioneins (MTs) are small, cysteine-rich proteins which have been implicated in various forms of stress providing cytoprotective action against oxidative injury, DNA damage and apoptosis. Owing to their high affinity for physiological metals, such as zinc and copper MTs are also critical components of regulatory proteins involved in cell growth and multiplication, as well as in the maintenance of immune homeostasis. To elucidate the role of MTs in the pathomechanisms of autoimmune CNS disorders we estimated the expression of MT I+II proteins and the content of free Zn ions in the brain, spinal cord and in the liver early in the course of chronic relapsing experimental autoimmune encephalomyelitis (CR-EAE) pathogenesis, i.e. before the onset of any clinical symptoms. Disease was induced in the genetically susceptible Dark Agouti (DA) rats by subcutaneous injection of bovine brain homogenate in CFA. Control animals were treated with CFA alone. The data, obtained by immuno-histochemistry and in situ fluorescent labeling of free zinc ions, have shown that in the presymptomatic phase of CR-EAE (on the seventh postimmunization day) MTs I+II were markedly upregulated in the cells that form blood-brain and blood-cerebrospinal fluid barriers, as well as in the cerebellar parenchyma and hippocampal dentate gyri. Furthermore, we found that the liver also becomes a site of extensive MTs I+II synthesis shortly after immunization. Simultaneously, tissue content of free zinc ions increased at the sites of MTs induction, reflecting their antioxidative activity. The data, described in this paper point to regulatory and neuroprotective role of MTs in the pathogenesis of CR-EAE.

Metallothioneins I/II expression in rat strains with genetically different susceptibility to experimental autoimmune encephalomyelitis.

OBJECTIVES: Compared to the Dark Agouti (DA), the Albino Oxford (AO) rat strain exhibits lower susceptibility to the induction of experimental autoimmune encephalomyelitis (EAE). Here, we investigated the potential contribution of the heavy metal-binding proteins metallothioneins (MTs) I/II to these effects. METHODS: Rats were immunized with bovine brain homogenate emulsified in complete Freund's adjuvant or only with complete Freund's adjuvant. The expression patterns of MTs mRNA and proteins and tissue concentrations of Zn2+ and Cu2+ were estimated in the brain and in the liver on days 7 and 12 after immunization, by real-time PCR, immunohistochemistry and inductively coupled plasma spectrometry, respectively. Additionally, the hepatic transforming growth factor beta and nuclear factor kappa B immunoreactivities were tested. RESULTS: Clinical signs of EAE were not induced in AO rats, but they upregulated the expression of MT I/II proteins in the brain (hippocampus and cerebellum) and in the liver, similarly as DA rats. The transcriptional activation of MT-I occurred, however, only in DA rats, which accumulated also more zinc in the brain and in the liver. In contrast, intact AO rats had greater hepatic MT-I mRNA immunoreactivity and more Cu2+ in the hippocampus. Besides, in immunized AO rats a high upregulation of transforming growth factor beta and nuclear factor kappa B immunoreactivities was found in several hepatic structures (vascular endothelium, Kupffer cells and hepatocytes). CONCLUSIONS: Our data show that AO and DA rats differ in constitutive and inductive MT-I gene expression in the brain and in the liver, as well as in the hepatic cytokine profile, suggesting that these mechanisms may contribute to the discrepancy in the susceptibility to EAE.

Influence of a vertical subject on research in biomedicine and activities of The Cochrane Collaboration branch on medical students' knowledge and attitudes toward evidence-based medicine.

AIM: To investigate whether the introduction of a vertical subject on research in biomedicine and founding of The Cochrane Collaboration branch at the University of Split School of Medicine influenced students' knowledge and attitudes toward evidence-based medicine (EBM), including the use of research literature. METHODS: We used a 26-item questionnaire on EBM knowledge and attitudes to survey 1232 medical students of all study years in 3 medical schools in Croatia (Split, Rijeka, Osijek) and the Croatian-speaking medical school in Mostar (Bosnia and Herzegovina). RESULTS: Students from the University of Split School of Medicine who had been exposed to the vertical subject on research in biomedicine and activities of The Cochrane Collaboration at the school had better knowledge and more positive attitudes toward EBM. In general, students rarely searched for evidence; 28% of students searched for evidence more than once a month and 96% of students used only textbooks in Croatian and teachers' handouts, even though 74% of students agreed that articles from scholarly journals were an important supplement for textbooks. CONCLUSION: Building up an environment that fosters EBM may be beneficial for students' knowledge and attitudes toward EBM. Teachers should encourage and require using evidence during all the courses in medical school.

Time-course expression of metallothioneins and tissue metals in chronic relapsing form of experimental autoimmune encephalomyelitis.

To elucidate the role of metallothioneins (MTs) in the pathomechanisms of autoimmune CNS disorders we estimated the expression of MTs I+II and the tissue concentrations of Zn²+ and Cu²+ in the brain, spinal cord (SC) and in the liver during the periods of attacks and remissions in chronic relapsing experimental autoimmune encephalomyelitis (CR-EAE). Disease was induced in the genetically susceptible Dark Agouti (DA) rats by subcutaneous injection of bovine brain homogenate in CFA. Control rats were treated with CFA. The data, obtained by clinical assessment, immunohistochemistry and inductivity coupled plasma spectrometry, have shown that during the first attack (on the 12th day) MTs I+II were markedly upregulated in subarachnoid regions and perivascular space on astrocytes, microglia and on spinal neurons. Simultaneously, the concentrations of zinc in the SC and zinc and copper in the liver have found to be increased. During the second attack (on the 22nd day) a new overexpression of MTs was found in the cerebellum, in sulcus hippocampi, in spinal neurons and particularly in hepatocytes around the central vein. Concomitantly, in the brain and SC the concentration of copper increased. The data point to a neuroprotective role of MTs and to an important regulatory role of essential metals and hepatic MTs in the pathogenesis of CR-EAE.

Analysis of the elective curriculum in undergraduate medical education in Croatia.

CONTEXT: Elective courses are a significant part of undergraduate medical education throughout the world, but the value provided by these courses and the reasons for choosing particular elective courses have not been studied extensively. OBJECTIVES: The aim of this study was to investigate medical and dental students' experiences of elective courses in undergraduate medical education in Croatia and to gather students' recommendations for the improvement of elective courses. METHODS: Medical and dental students studying under the Bologna curriculum were given a questionnaire in which they were asked for their opinions of elective courses and their suggestions as to how they might be improved. Data on elective courses were obtained from medical schools' administrative offices. RESULTS: The survey response rate was 92% (834/903). Medical students gave elective courses an average grade of 3.44 out of 5, whereas dental students gave a lower average of 3.15. Students' suggestions for change included introducing more practical work and recognising international student exchanges and attendance at conferences as elective options. A third of students indicated that teachers should be given additional training in leading elective courses. Analysis of the curriculum showed that elective courses in Croatian medical schools are very heterogeneous in terms of their content and the number of credits and assessment methods they involve, and are very conservative in that only structured courses are offered. Students cannot design their own courses or take more elective courses than represent 10% of their total number of credits. CONCLUSIONS: Student opinion should be consulted when medical schools venture into the elective curriculum so that students can feel that they are really benefiting from these subjects. Students would welcome new and personally designed strands. Elective courses are a significant part of medical education and therefore their quality and purpose need to be assessed regularly in order to ensure that they meet students' needs.

The influence of pregnancy on development and course of chronic relapsing experimental autoimmune encephalomyelitis in rats: implications for multiple sclerosis.

Multiple sclerosis is a chronic, autoimmune disease of the central nervous system, which mainly affects young women during a reproductive period of life. Since, its symptoms might be significantly affected by pregnancy, in this study we investigated the development and kinetics of disease in the model of chronic relapsing experimental autoimmune encephalomyelitis (CR-EAE), induced in genetically susceptible Dark Agouti (DA) strain of rats. They were sensitized with bovine brain white matter homogenate (BBH) in complete Freund's adjuvant during the first, second or third week of pregnancy, and the disease scores were compared between treatment groups, and identically treated nongravid females. Additionally, the susceptibility to the induction of EAE was tested in offspring of mothers that during the pregnancy were sensitized with BBH. The data have shown that pregnancy does not block the induction of EAE, but that it significantly changes the course of diseases, depending on time of immunization. In rats sensitized during the first week of gestation the onset of the clinical signs was delayed, but after the delivery the intensity of disease significantly increased. Similar aggravation, with appearance of monophasic form of disease was observed in the group of rats sensitized during the third week of gestation. On the contrary, in rats sensitized during the second week of gestation the beneficial effects were observed, with later onset of attacks, and lower disease score. Furthermore, offspring of these rats after immunization with BBH developed a monophasic form of EAE of lower intensity, suggesting that some protective factors might be transferred across the placenta.

Demographics and motives of medical school applicants in Croatia.

BACKGROUND: According to data regarding number of physicians per 100,000 inhabitants, Croatia is below the European average. Under those circumstances, more attention needs to be devoted to Croatian medical schools and their applicants. AIMS: This study sought to investigate admission trends of applicants to Croatian medical schools, analyse their demographics and motives for medical school enrollment. METHODS: We collected admissions data of applicants to Croatian medical schools from 1979 to 2006. Motives for and against medical school enrollment were assessed in a survey of 1146 applicants (response rate 84%, 966/1146) and 98 final-year medical students (response rate 82%, 80/98) during July 2006. RESULTS: The number of applicants to Croatian medical schools had been declining until 1995, it was lowest during the 1991-1995 war in Croatia and it has been rising from 1996 onwards. Majority of applicants in 2006/07 were women (69%). Most of the applicants attended general high schools. The applicants profess choosing a certain medical school for its quality and reputation, but we showed that they actually chose the closest school. The main motives for medical school enrollment were humanitarian and scientific, while main reasons against were perceived difficulty and financial burden. We showed that final-year medical students profess significantly lower interest in science and that they are less interested in altruistic aspects of medicine. Instead, great number of them would reconsider choosing medical studies again because of the corruption in medicine, fear of mistakes and uncertainty of employment. CONCLUSIONS: Following the admission trends in medical schools on a national level gives insight into the prospects of health care. Analysis of motives for and against medical school enrollment can provide guidelines for their improvement. Unless Croatia and other countries in transition devote more attention to recruitment, education and retention of physicians, the prospects of our healthcare are poor.

No association of CCR5delta32 gene mutation with multiple sclerosis in Croatian and Slovenian patients.

Several studies investigating the role of the CCR5delta32 mutation in multiple sclerosis (MS) have reported varied, often contradictory results. Therefore in the present study we have analysed whether the CCR5delta32 mutation is associated with the risk of/or disease process in Croatian and Slovene MS patients. Three hundred and twenty-five MS patients and 356 healthy controls were genotyped by the polymerase chain reaction method. Our results showed no significant differences in the distribution of CCR5delta32 mutations between MS and control subjects, indicating that this mutation does not influence susceptibility to MS. Furthermore, we did not observe that CCR5delta32 carrier-status could modulate age of disease onset or progression of the disease. It is therefore our conclusion that the present study indicates that the CCR5delta32 mutation is neither protective of, nor a risk factor, for MS development.

Induction of experimental allergic encephalomyelitis in a low-susceptible Albino Oxford rat strain by somatostatin analogue SMS 201-995.

OBJECTIVE: The effect of the somatostatin analogue SMS 201-995 (octreotide; OCT) on the course of experimental allergic encephalomyelitis (EAE) in the relatively resistant Albino Oxford (AO) strain of rats was studied. METHODS: Animals were actively immunized with bovine brain homogenate in complete Freund's adjuvant. OCT was given subcutaneously in the hind legs on days 7, 8 and 9 after immunization, at a dose of 3 x 5 microg/kg/day. Rats in control groups were treated with saline or were left untreated. EAE was scored clinically and immunophenotypically, estimating by flow cytometry the changes in the popliteal lymph nodes (PLN) and spleen and monitoring immunohistologically the brain sections of rats recovered from disease. RESULTS: In control AO rats, EAE was induced in only 2 of 22 rats (9%). In OCT-treated rats, however, EAE developed in 11 of 20 rats (55%), in comparison with 3 of 17 saline-treated animals (17%) (p <0.05). In PLN of OCT-treated rats during the clinical course of EAE, a decreased proportion of OX8+ cells was seen, followed by increases in OX39+ and W3/25+ cells on days 17 and 26. In spleen, OCT decreased the proportion of OX1+, OX39+ and OX8+ cells (on days 12 and/or 17), and increased the proportion of OX39+ cells on days 26 and 31. In the brain sections of saline-treated rats recovered from EAE, numerous Mac-1+, Mac-3+ and OX8+ cells were found. These cells were, however, absent in OCT-treated rats; instead, several W3/25+ cells were noticed. CONCLUSIONS: These data imply that OCT increases the susceptibility of AO rats to EAE, interfering with specific and/or nonspecific defense mechanisms operating in both the initial and recovery phase of EAE.