Gordana Rubeša

Characteristics of Perforin Expressing Lymphocytes Within the First Trimester Decidua of Human Pregnancy

PROBLEM: The number of perforin (P)‐positive cells in decidua of pregnancy is larger than that observed in any other pathological condition. The aim was to investigate the distribution and the phenotype of P+ cells.

Perforin-expressing lymphocytes in peripheral blood and decidua of human first-trimester pathological pregnancies

PROBLEM: We have shown previously that the decidua of first‐trimester human pregnancy is heavily infiltrated with perforin‐positive cells. The aim was to detect expression of perforin in both decidual lymphocytes (DL) and peripheral blood lymphocytes (PBL) in the first trimester of pathological pregnancies: Anembryonic pregnancy and missed abortion.

Perforin expression in peripheral blood lymphocytes in rejecting and tolerant kidney transplant recipients

Perforin (P) is a cytolytic molecule expressed in the granules of cytolytic T cells and natural killer cells. Although cytotoxic cells have been implicated in graft rejection, no prospective clinical study has been published that examines the dynamics of perforin expressing cells in peripheral blood lymphocytes of transplanted patients. The cytofluorimetric assay developed in our laboratory previously for the simultaneous detection of intracellular perforin together with cell surface molecules was used for posttransplantation monitoring of patients, for the assessment of the efficiency of immunosuppressive treatment, and for the prediction of acute kidney transplant rejection and the stability of tolerance to long lived kidney transplants. Immunosuppression for the purpose of allotransplantation causes a decline in the number of perforin-expressing cells in peripheral blood. In contrast, in patients with clinical signs of acute rejection, the total number of perforin-expressing lymphocytes was increased in comparison with nonrejecting patients. Analyzing perforin-expressing subsets, rejection crises were accompanied by a relative decrease of perforin expression in the CD4+ subpopulation while increasing in the CD8+ subset. In the CD56+ and CD16+ NK subpopulations changes in perforin expression were mixed. In nonrejecting patients the ratio of perforin expression in CD4+ cells was high compared with CD8+ cells. Intensive therapy of acute rejection episodes with high doses of corticosteroids (methylprednisolonet [Solumedrol] bolus) strongly and significantly decreased the percentage of both, the subpopulations of perforin-positive T cells and the subpopulation of CD56+P+ NK cells. The lowest level of perforin expression, including low frequencies of perforin among CD8+ and CD4+ cells, was found in the group of patients tolerating transplanted kidneys for several years. These changes in perforin protein expression in peripheral blood can be used to discriminate between immunosuppressed patients who are immunologically quiescent and those who undergo transplant rejection. Our results confirm the hypothesis that cytotoxicity mediated by perforin may be an important effector mechanism in the rejection of allografted kidneys.

Decidual-trophoblast interactions: decidual lymphoid cell function in normal, anembryonic, missed abortion and ectopic human pregnancy

This study was designed to investigate the consequences of decidua-trophoblast interactions on the phenotype, spontaneous and induced proliferation and immunoregulatory potential of decidual leukocytes in normal pregnancies (NP), anembryonic pregnancies (AP), missed abortions (MA) and ectopic pregnancies (EP). Spontaneous proliferation of decidual non-adherent cells (NAD) from pregnancies with viable trophoblast inside the uterus is significantly higher than proliferation of peripheral blood lymphocytes (PBL) from the same groups (P < 0.001 for NP; P < 0.05 for AP). Spontaneous proliferation of decidual NAD cells from NP was higher (P < 0.001) when compared with AP and EP. The induced (PHA and Con A) responses of PBL from women with normal and pathological pregnancies were significantly higher than that of decidual NAD cells (P < 0.001). Higher proliferation of NAD decidual cells was obtained when Con A-stimulated NP were compared with MA and EP (P < 0.01). The interaction of viable trophoblast with intrauterine decidua appears to be a prerequisite for the activation of NAD suppressor cells, since NAD cells from MA produced stimulation instead of suppression, and NAD cells from EP had no suppressive effect. On the contrary, both NAD and adherent (AD) decidual leukocytes from NP and AP produced very strong suppression of PHA or alloantigen-induced PBL proliferation. The contact between trophoblast and AD decidual leukocytes is not necessary for their suppressive function, since even higher suppression is obtained with the cells from ectopic pregnancies.

INCREASED PERFORIN EXPRESSION IN MULTIPLE SCLEROSIS PATIENTS DURING EXACERBATION OF DISEASE IN PERIPHERAL BLOOD LYMPHOCYTES

The expression of perforin (P) in subpopulations of the PBL of multiple sclerosis (MS) patients in stable and active phase of disease was investigated, by simultaneous detection of P (intracellular molecule) and cell surface antigens. A significant increase of CD4+P+ (p < 0.02) and CD16+P+ (p < 0.001), and decrease of CD56+P+ (p < 0.05) cells in active MS was found. In active disease there is a highly significant increase (p < 0.001) of average fluorescence intensity (AFI) for P in CD4(dim+) cells, and these cells are larger in size and have higher granularity (p < 0.05) compared to CD4(bright+) p(dim+) cells. Surprisingly, there were no CD25+P+ cells in either group of MS patients. These results show that CD4+P+ cells are upregulated in active disease in cell number, in the level of P expression per cell, and in the level of cell activation (increase in cell size and granularity). It is suggested that CD4+P+ cytotoxic cells may play a role in the pathogenetic mechanisms of MS.

Down-regulated expression of perforin-positive/CD16+ cells in the peripheral blood lymphocytes in the first trimester of pregnancy and up-regulation at the end of pregnancy.

PROBLEM: Immunophenotypic profiles of perforin (P)‐positive peripheral blood lymphocytes in the first trimester and at the term of human pregnancy were analyzed.

BanI polymorphism of cytosolic phospholipase A2 gene is associated with age at onset in male patients with schizophrenia and schizoaffective disorder

The enzymes phospholipases A2 are believed to be involved in the pathology of schizophrenia. We investigated allelic and genotype frequencies of PLA2G4A BanI polymorphism and the rs4375 in PLA2G6A in Croatian schizophrenic patients (n=81) and controls (n=182), using PCR/RFLP. Genotype and allelic frequencies of both loci, alone or in combination did not show significant difference (chi2-test). Allele-wise and genotype-wise meta-analyses of BanI polymorphism in case-control and family-based studies also revealed no significant association with schizophrenia. Multiple logistic regression analyses revealed statistically significant association between several items from PANSS general psychopathology scale and BanI polymorphism in PLA2G4A. BanI polymorphism further showed a significant impact on mean age of the onset of disease in males (betaA1=0.351, P=0.021; Spearmans rA1=0.391, P=0.010) indicating lower mean age at admission in homozygous A2A2 males.

Angiotensin-converting enzyme gene insertion/deletion polymorphism is not associated with schizophrenia in a Croatian population.

Because angiotensin converting enzyme (ACE) plays an important role in dopamine system functioning in the brain and the insertion/deletion (I/D) polymorphism of a 287 nucleotide fragment of the ACE gene correlates with enzyme activity, several studies have investigated the role of ACE I/D polymorphism in psychiatric diseases (Segman et al., 2002 ; Crescenti et al., 2009). Two recent studies yielded contradictory results: the D allele was identified as a protective factor in a Spanish population, while a protective effect toward schizophrenia and bipolar disorder was attributed to the I allele in a Turkish population (Crescenti et al., 2009 ; Kucukali et al., 2010). We tested whether schizophrenia risk was associated with ACE I/D polymorphism in a Croatian population, and examined its possible impact on schizophrenia symptom severity. Our study group consisted of 211 Croatian patients (115 male, 96 female) who met DSM-IV criteria for schizophrenia (n = 187) and schizoaffective disorder (n = 24), and 270 healthy blood donors (135 male, 135 female). All participants gave informed consent for the analysis. The study was approved by the Ethics Committee of the School of Medicine, University of Rijeka, Croatia. Genotyping was performed by polymerase chain reaction (PCR) as previously described (Rigat et al., 1990). To exclude mistyping of the heterozygotes as DD homozygotes, all DD genotype samples were confirmed with insertion-specific PCR (Shanmugan et al., 1993). The significance of differences in genotype and allele frequencies between patients and controls was determined using the χ2 test. Potential correlation between Positive and Negative Symptom Scale (PANSS) scores and I/D polymorphism was tested using linear regression analysis (stepwise selection), adjusted for age at PANSS assessment and sex, in patients with schizophrenia. PANSS evaluation was performed during the last hospitalization due to a psychotic episode. P-values < 0.01 were considered statistically significant. Allelic and genotypic frequencies of I/D polymorphisms were consistent with Hardy-Weinberg equilibrium and were not significantly different between groups. Allele frequencies were 230 (D) and 192 (I) in the patient group, and 285 (D) and 255 (I) in the control group. Genotype frequencies were 0.299 (DD = 63), 0.493 (ID = 104) and 0.208 (II = 44) in the patient group, and 0.274 (DD = 74), 0.507 (ID = 137) and 0.219 (II = 59) in the control group. Therefore, our data did not support results from Spanish or Turkish samples. However, after adjusting for sex and age at PANSS assessment we observed a significant correlation between ACE genotype and psychopathology evaluated by PANSS. Increased negative and total PANSS scores were significantly correlated with the number of D alleles (b = 0.26, F = 7.803 ; P = 0.006 and b = 0.29, F = 7.557 ; P = 0.002, respectively). Increased symptom severity of the general psychopathology scale in males with schizophrenia could also be attributed to the number of D alleles (b = 0.37, F = 9.910 ; P = 0.008). This is the first study reporting significant correlation between clinical expression of illness and I/D polymorphic variants. I/D polymorphism may affect symptom severity by modulating ACE expression/activity. Further studies in other populations/ethnic groups may help clarify the relationship between ACE activity and schizophrenia.

HFE mutations and transferrin C1/C2 polymorphism among Croatian patients with schizophrenia and schizoaffective disorder

The aim of this study was to investigate the possible influence of hemochromatosis gene mutations (HFE-C282Y and H63D) and transferrin gene C2 variant (TF-C2) on susceptibility to schizophrenia and schizoaffective disorder and/or age at first hospital admission. Genotyping was performed in 176 Croatian patients and 171 non-psychiatric Croatian controls using PCR-RFLP analyses. Regarding the H63D mutation, allele and genotype frequencies reached boundary statistical significance. Other allele and genotype distributions were not significantly different between two groups. We also analyzed age at first hospital admission as a continuous variable using the non-parametric Mann–Whitney U-test and Kruskal–Wallis test, and multiple regression analysis. The results of these tests were negative. We concluded that investigated HFE mutations and TF-C2 variant are not high-risk genetic variants for schizophrenia/schizoaffective disorder in our population. Also our data do not support their impact on age at onset of the first psychotic symptoms.

Craniofacial morphologic and anthropometric features of Croatian schizophrenia patients and non-psychiatric controls - a pilot study

AIM to evaluate differences in craniofacial morphologic features and several anthropometric measures between schizophrenia patients and non-psychiatric controls, and to find the best-fit model to differentiate between two groups. METHODS 40 morphologic features of the head and face, and 5 craniofacial anthropometric measures were evaluated using the Lane Dysmorphology Scale in 58 patients and 46 controls. Total MPA score and subscores for different craniofacial regions were calculated. Individual items were examined using logistic regression analyses to define a model that can discriminate between patient vs. control status. RESULTS total MPA score, and several subscores (general asymmetry, nasal, lip, ear and tongue) were significantly higher in the patient group. Patients were distinguished by significantly higher measures of maxillary and mandibular facial arcs, general and subtle facial asymmetries presented as deviation of facial landmarks from the vertical facial midline and horizontals, more variable vermilion of the upper lip, tongue surface, frenulum and anterior hair margin, and more adherent and underdeveloped earlobes. A final regression model including maxillary are, facial asymmetry, and adherent earlobes as independent predictors proved useful to efficiently recognize schizophrenia patients (specificity and positive prediction value of 100% when all the three items were present in an individual) or to exclude risk for schizophrenia (sensitivity and negative prediction value of 96.6% and 84.6%, respectively, in cases no one of the three items was present). CONCLUSIONS schizophrenia patients evidenced significantly more craniofacial dysmorphology than controls. The model revealed in the study needs to be verified in larger samples and other populations.