Vesna Barac-Latas

Patterns of oligodendrocyte pathology in coronavirus‐induced subacute demyelinating encephalomyelitis in the lewis rat

Intracerebral infection of rats with JHM coronavirus induces a chronic inflammatory demyelinating disease, which in many respects mimicks the pathology of multiple sclerosis. We investigated the patterns of demyelination and oligodendrocyte pathology in this model. In early stages of the disease infection of oligodendrocytes was associated with a downregulation of expression of mRNA for proteolipid protein in the absence of myelin destruction. When demyelinating lesions were formed infected oligodendrocytes were destroyed by necrosis, whereas oligodendrocytes that did not contain detectable virus antigen or RNA were in part dying by apoptosis. At this stage of the disease remyelination of the lesions was pronounced. At later stages after infection virus antigen was nearly completely cleared from the lesions. In spite of the lack of detectable virus, ongoing demyelination and unspecific tissue destruction occurred, and oligodendrocytes were mainly destroyed by apoptosis. These late lesions revealed only minimal central remyelination, but they were frequently repaired by Schwann cells. Our studies suggest that the mechanisms of myelin destruction in this model of virus‐induced demyelination are complex and that the patterns of tissue damage may change during the course of the disease. Glia 19:1–7, 1997

No association of CCR5D32 gene mutation with multiple sclerosis in Croatian and Slovenian patients

Several studies investigating the role of the CCR5D32 mutation in multiple sclerosis (MS) have reported varied, often contradictory results. Therefore in the present study we have analysed whether the CCR5D32 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 CCR5D32 mutations between MS and control subjects, indicating that this mutation does not influence susceptibility to MS. Furthermore, we did not observe that CCR5D32 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 CCR5D32 mutation is neither protective of, nor a risk factor, for MS development.

Coronavirus Infection and Demyelination

Coronaviruses display a large phenotypic variability, which may be an important factor for diversification and selection. Previous studies have demonstrated that the S-protein is an essential determinant of virulence and pathogenicity. Therefore we studied the S-gene as an indicator molecule for selection processes employing two different MHV-JHM variants. First, Lewis-rats were infected with MHV-JHM-Pi, a variant that causes demyelinating disease after several weeks p. i. It was not possible to isolate infectious MHV-JHM-Pi from such rats, although viral proteins were expressed. The S-gene was rescued directly from brain tissue employing RT-PCR technology. The amplicons were sequenced in bulk or at the level of single clones. We detected no evidence for an increase of S-gene mutants during the length of time. Only few mutations were found at the clonal level. The changes were distributed throughout the analysed S-gene fragments without a predilection in their location. The frequency of mutation remained low within a range of 0.03 to 0.5 mutations per thousand nucleotides. As a second approach, we sequenced the S-genes of viruses isolated from brain tissue infected with MHV-JHM-ts43. Infection of adult Lewis rats with that mutant resulted several weeks to months p.i. in demyelinating encephalomyelitis. The S-gene of this virus contains an insertion of 423 bp in the SI region, which is identical to a polymorphic region described for MHV-4. In contrast to JHM-Pi, infectious MHV-JHM-ts43 was readily to iso Coronaviruses and Arteriviruses, edited by Enjuanes et al. Plenum Press, New York, 1998 late from brain tissue. The S-gene sequences of virus isolated 45–106 days p.i. from diseased rats were identical with that of the input virus. These results show, that during a persistent infection of Lewis-rats the S-gene was highly conserved.

Coronavirus-induced encephalomyelitis: balance between protection and immune pathology depends on the immunization schedule with spike protein S

The neurotropic mouse hepatitis virus MHV-JHM induces central nervous system (CNS) demyelination in Lewis rats that pathologically resembles CNS lesions in multiple sclerosis. The mechanisms of MHV-JHM-induced demyelination remain unclear and several studies have implicated the role of the immune response in this process. We have shown previously that protective immunity against MHV-JHM-induced encephalomyelitis was induced by immunization with a vaccinia virus (VV) recombinant expressing MHV-JHM S-protein (VV-S). Here, we present evidence that the time of MHV-JHM challenge after immunization with VV-S plays a critical role in protective immunity. The induction of virus-neutralizing S-protein-specific antibodies prior to the MHV-JHM challenge modulates the disease process and a subacute encephalomyelitis based on a persistent virus infection developed. Typical pathological alterations were lesions of inflammatory demyelination. In addition, the results indicate that after seroconversion, CD8+ T cells were no longer essential for virus elimination in contrast to their role in protection during acute encephalomyelitis.

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.

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 Freunds adjuvant or only with complete Freunds 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.

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.

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 × 5 µg/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.

Analysis of the elective curriculum in undergraduate medical education in Croatia

Medical Education 2010: 44: 387–395

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 Freunds 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.