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; Spearman's rA1=0.391, P=0.010) indicating lower mean age at admission in homozygous A2A2 males.

Changes of cytolytic cells and perforin expression in patients with posttraumatic stress disorder.

AIM: To define phenotypic characteristics of cytotoxic T lymphocytes (CTL) and natural killer cells (NK) in peripheral blood, frequency of somatic symptoms, and level of anxiety and depression in 25 patients clinically diagnosed with chronic post-traumatic stress disorder (PTSD). METHODS: Patients were divided into two sub-groups according to the stressor: 18 PTSD patients with the battlefield experience and 7 PTSD patients with battlefield experience who were tortured as the prisoners of war (POW) in Bosnian-Serbian camps. The control group consisted of 15 healthy volunteers matched to the patients by sex and age. We tested all patients using Becks depression inventory, Spielberger anxiety test, and somatic disturbance list, and analyzed their peripheral blood lymphocytes using flow cytometry with the double fluorescence staining of cell surface antigens (CD3, CD4, CD8, CD16, and CD56) and intracellular cytolytic molecule perforin (P), a mediator of cytolytic action at the molecular level. RESULTS: All PTSD patients showed a significant level of anxiety, depression, and numerous somatic symptoms. The only significant difference between PTSD patients with and without POW experience was in the anxiety level (median, 71; range 61-79; vs median, 65; range, 49-77). PTSD patients with POW experience had significantly higher levels of CD16+ cells (median, 37%; range, 16-55%) than those without it (median, 12%; range, 5-37%). Double labeling for intracellular P antigen and cell surface antigens showed the highest levels of CD16+P+ (median, 33%; range, 15-40%; vs median, 10%; range, 3-29%) and CD56+P+ (median, 21%; range, 11-40%; vs median 8%; range, 1-30%) cells in PTSD-POW patients. CONCLUSION: Chronic PTSD patients who survived concentration camps show the most numerous alterations in PBL phenotype, the highest number of perforin-containing cells, and a significantly higher level of anxiety.

Age-related decline of perforin expression in human cytotoxic T lymphocytes and natural killer cells.

In this study a flow cytometric technique for detecting cytoplasmic perforin (P) has been used to quantify age-related changes in perforin expression in human peripheral blood lymphocytes (PBL). Proportions of P+ lymphocytes increased after birth, but declined rapidly after the age of 70 years. This was true for both T cells and CD16(+) and CD56(+) natural killer (NK) cells. Children showed in addition to high levels of perforin positive CD8(+) cells a much higher proportion of CD4(+)P+ cells than the other age groups. In elderly individuals there was also a highly significant reduction in mean levels of perforin per cell as compared with all other groups (P < .05 to .001). Adult women had consistently higher mean levels of perforin per cell than adult men for all P+ cell phenotypes. Functional tests clearly showed the deficiency in early spontaneous cytotoxic potential of PBL from elderly persons due to relative P deficiency, which can be corrected by stimulation of cytolytic cells with target cells and interleukin-2 (IL-2). The deficiency in cytolytic activity on the contact with target cells may have implications for antiviral and antitumor immunity in elderly persons.

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. METHOD OF STUDY: Perforin expression in peripheral blood lymphocyte subsets was measured by simultaneous detection of P (intracellular antigen) and cell-surface antigens (CD3, CD4, CD8, CD16, and CD56) by flow cytometry in nonpregnant (NP) and pregnant women in the first trimester (FTP) and at the time of parturition (TP). RESULTS: The percentage of total P+ cells in peripheral blood compared to nonpregnant women was slightly lower in the FTP but significantly higher at TP. Perforin-positive cells were significantly elevated in T lymphocyte subsets (CD3+P+, CD4+P+, CD8+P+) in both the FTP and TP groups, as was the percentage of CD56+P+ cells. Profound changes in the CD16+ subpopulation were found in the FTP group compared to both the NP and TP groups (a drastic decrease of CD16+P+ cells; CD16+ cells among P+ cells; P+ cells among CD16+ cells). A considerable part of CD3+ cells in both the FTP and TP groups are CD3+CD56+P+. The average fluorescence intensity (AFI) for P (a measure of P content per cell) was significantly decreased in FTP and increased in TP groups. CONCLUSIONS: The CD16 molecule is Fc gamma RIIIA which is the only Fc receptor responsible for antibody dependent cell-cytotoxicity (ADCC) of NK and T-cells. In the first-trimester human pregnancy this mechanism is severely down-regulated compared to both the NP and TP groups.

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. METHOD: Decidual tissue from a normal pregnancy group and from pathological pregnancies was obtained by vaginal curettage. Perforin (an intracellular antigen) and the cell surface antigens CD3, CD4, CD8, CD16, CD56, CD11c, and CD45RA were quantified simultaneously by flow cytometric analysis. RESULTS: In the missed abortion group, we found: 1) a relative decrease in the frequency of both CD4+P+ cells and CD56+P+ cells as well as the mean fluorescence intensity for perforin; 2) a relative increase of CD16+P+ PBL cells; and 3) a relative increase of CD4+ cells in PBL compared with anembryonic pregnancy and normal pregnancy. There was also a significant relative decrease in the proportion of CD4+ and CD8+ cells among perforin-positive PBL in both anembryonic pregnancy and missed abortion. CONCLUSION: Our results show that significant decreases in the prevalence of perforin-positive lymphoid cells, their subpopulations, and mean fluorescence intensity for perforin are associated with pregnancy failure.

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.

Expression of membrane form of the pregnancy associated protein TJ6 on decidual lymphocytes in the first trimester of pregnancy.

TJ6, a newly described protein produced locally in the uterine decidua during pregnancy, may be involved in maintaining a unique immunological environment at the maternal-fetal interface. The aim of this study was to determine whether TJ6 is expressed as membrane form on decidual lymphocytes (DL), to define the phenotypes of TJ6m (membrane form TJ6) expressing cells and to analyze the fluorescence intensity of TJ6m expression. Peripheral blood lymphocytes (PBL) and DL were obtained from first trimester pregnancies undergoing elective termination and immunophenotyped for TJ6m and other cell surface antigens (CD3, CD8, CD19, CD56, CD16) by flow cytometry. This is the first study showing that TJ6 molecules are present on decidual lymphocytes in human pregnancy. TJ6m expression on PBL was not different from that of DL. However, a significantly higher percentage of double positive (TJ6m+CD3+, TJ6m+,CD8+,TJ6m+CD19+) cells were found in PBL when compared to DL. The average fluorescence intensity (AFI) for the TJ6m marker among cells with CD8+, CD19+ and CD56+ double positive was significantly higher in DL as compared with those of PBL. The AFI for granularity of double positive DL was significantly higher than observed in PBL.

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.

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. METHOD: Decidual tissue was obtained from the first trimester vaginal termination of pregnancy. Tissue distribution of P+ cells was analyzed by immunohistochemistry. The method for simultaneous measurement of P and cell surface is presented. RESULTS: There is no difference in number and distribution of P+ cells between decidua basalis (DB) and decidua parietalis (DP). The percentage of P+ decidual lymphocytes (DL) is two times higher than in peripheral blood lymphocytes (PBL) (55% vs. 27%), and the prevalent phenotype is CD3- CD4- CD8- CD2+ (95%) CD11c+ (68%) and CD56+ (82%). CD56bright+ DL are also Pbright+ and this is the largest DL subpopulation (42.4% DL). Two different subpopulations of CD8+ DL exist: 1) CD8bright+, which are CD3+ CD56- P- and 2) CD8dim+, which are CD3- CD56+ P+. CONCLUSION: P expressing DL are prevalently nonclassical NK cells (CD16-) with low cytolytic activity but fully equipped with potent cytolytic machinery (Pbright+). There are no classical cytotoxic lymphocytes (CTL) (CD3+ CD8+ P+) in the decidua, and all CD8+ P+ cells are CD3- CD56+. The number of P+ cells is even higher in DP in the vicinity of noninvasive trophoblast, than in DB.

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.