Functional polymorphisms of interleukin 4 and interleukin 10 may predict evolution and functional outcome of an ischaemic stroke.

BACKGROUND AND PURPOSE: Inflammatory cytokines are involved in the systemic inflammation, which precedes an ischaemic stroke (IS), and also participate into brain ischaemia-reperfusion injury. We sought to investigate whether functional polymorphisms of two anti-inflammatory molecules, interleukin (IL)4-589C>T and IL10-1082G>A, might be associated with the occurrence, clinical course and functional outcome of an acute IS. METHODS: We genotyped 290 subjects (145 consecutive IS cases and 145 age- and sex-matched controls) using a real-time PCR technology, prototypically designed for these mutations. Patients were evaluated with the Scandinavian Stroke Scale, and definitions of severity grouping and stroke progression were applied based on international agreements. Follow-up on months 1, 3, and 6 included registration of disease relapses, deaths and functional outcome measured by the Barthel Index. RESULTS: IL4-589 and IL10-1082 genotypes did not significantly differ between cases and controls. The presence of IL4-589 T allele was associated with total IS recurrences [OR (95% CI) = 3.34 (1.18-9.45)], adjusted for age, sex and conventional risk factors. IL10-1082 GG genotype was found to significantly predict early stroke progression [OR (95% CI) = 3.72 (1.28-10.76)] and functional outcome by months 1 and 3 [OR (95% CI) = 5.03 (1.15-21.94) and 5.84 (1.07-31.85), respectively], after further corrections for stroke severity and TOAST categories. CONCLUSIONS: The functional IL4-589C>T and IL10-1082G>A polymorphisms seem not to be associated with occurrence of an IS, but may predict IS relapses, progressing strokes and functional outcome, independently of conventional risk factors. Our results merit further confirmation in future studies.

Pharmacokinetics and clinical activity of very low-dose alemtuzumab in transplantation for acute leukemia.

The optimal dose of in vivo-administrated alemtuzumab in the allogeneic transplantation setting has not been defined. We report our experience on 37 patients with high-risk diseases, mainly acute leukemia (AML 23, ALL 10 patients), who underwent sibling (49%) or unrelated (51%) PBSCT (35 patients), and received a total dose of only 10-20 mg Campath-1H as part of the conditioning, and post-transplant CYA without MTX. The neutrophil and especially the platelet engraftment were rapid. There were only two grade III-IV acute GvHD cases, which occurred in unrelated transplants in the Campath-10 cohort. Chronic GvHD developed in six cases (17%) and was limited to skin in five of them. After a median follow-up of 371 days (59-1191), 70% patients are alive and in CR (Karnofsky 100%), and 11 died (TRM n=6, relapse n=5). From the five patients relapsed, three were at advanced stage at transplant and four underwent sibling HCT with the higher (20 mg) alemtuzumab dose. With the 10 mg alemtuzumab schedule (5 mg/day at days -2 and -1) we achieve at day of transplantation low but still lymphotoxic alemtuzumab serum concentrations (176 ng/mL), whereas levels declined fast thereafter, and at engraftment nearly no Campath antibody remained in the patient's serum.

Serum adiponectin acutely after an ischemic stroke: implications for a long-lasting, suppressed anti-inflammatory role.

OBJECTIVE: Past ischemic stroke (IS) patients display suppressed adiponectin (ADPN) levels a few months after disease onset. It is still unclear whether hypoadiponectinemia is already present by the early stages of stroke or occurs as a delayed effect of the acute ischemic reaction. In the present study we investigated ADPN levels acutely after an IS. MATERIALS AND METHODS: Serum ADPN was measured in 82 consecutive acute IS patients, and 30 stroke-free subjects of similar age and sex distributions. RESULTS: Patients had significantly lower ADPN levels than controls. Higher ADPN was significantly associated with reduced odds for IS accounting for age, sex and high-density lipoproteins. This association was strengthened after further adjustments for potential confounders. ADPN levels remained suppressed even 6 months after stroke. CONCLUSIONS: ADPN is significantly suppressed already by the early phases of stroke, and remains unchanged 6 months later. We propose a stable-over-time anti-inflammatory role of ADPN in IS, unrelated to the acute ischemic reaction.

Inherited thrombophilia screening in Greek women with recurrent fetal loss.

OBJECTIVE: The present study was designed to determine the prevalence of factor V Leiden (FVL), prothrombin gene G20210A (PTG) and methylenetetrahydrofolate reductase (MTHFR C677T) mutations in women from South-Western Greece with recurrent fetal loss (RFL) and negative personal thromboembolic history. MATERIALS AND METHODS: 212 women with RFL and 181 women with at least two pregnancies with normal outcome and no history of pregnancy loss were investigated for the commonest thrombophilic mutations (FVL, PTG, MTHFR C677T). Comparisons between groups were performed by Pearson's chi-square test and odd ratios were calculated. RESULTS: An abnormal genotype was detected in 49 women of the study group (23.1%) and in 41 women of the control group (22.6%). CONCLUSION: Inherited thrombophilia screening is not indicated as an initial approach in Greek women with RFL and negative personal thromboembolic history.

Prognostic value of phagocytic activity of neutrophils and monocytes in sepsis. Correlation to CD64 and CD14 antigen expression.

The role of the phagocytic function of monocytes and neutrophils in sepsis has been poorly investigated. The present study evaluated the impact of the phagocytic activity of neutrophils and monocytes on the outcome of patients with severe sepsis. Thirty-one patients and 30 healthy individuals were enrolled in the study. The phagocytic activity of monocytes and neutrophils was evaluated during 24 h after admission and the results were correlated to the expression of CD64 on neutrophils and monocytes, CD14 antigen on monocytes, the Simplified Acute Physiology Score II and the patients' survival. A reduced phagocytic activity of neutrophils during the first 24 h after admission was a negative predictor for survival. Increased expression of CD64 antigen on polymorphonuclear cells (PMNs) and monocytes was favourably correlated to the patients' survival. In multivariate analysis the phagocytic activity of PMNs was the only independent predictor factor for survival. Patients with PMN phagocytic activity <37% had lower expression of CD64 on monocytes and PMNs and worse outcome, while those with phagocytic activity >37% had higher expression of CD64 on monocytes and PMNs and better outcome. Reduced phagocytic activity of neutrophils may represent a state of neutrophil inactivation similar to that previously described for monocytes during the compensatory anti-inflammatory response.

c-Jun N-terminal kinase activation failure is a new mechanism of anthracycline resistance in acute myeloid leukemia.

Chemotherapy resistance is a major challenge in acute myeloid leukemia (AML). Besides the P-glycoprotein efflux, additional cellular factors may contribute to drug resistance in AML. c-Jun N-terminal kinase (JNK) is activated after exposure of cells to chemotherapeutics. We asked whether chemoresistance in AML is attributed to intrinsic failure of the AML blasts to activate JNK. In vitro treatment of U937 AML cell line with anthracyclines induced a rapid and robust JNK phosphorylation and apoptosis. In contrast, the anthracyline-resistant derivative cell lines U937R and URD40 showed no JNK activation after exposure to anthracyclines, also at doses that resulted in high accumulation of the drug within the cells. RNA interference-based depletion of JNK1 in drug-sensitive U937 cells made them chemoresistant, whereas selective restoration of the inactive JNK pathway in the resistant U937R cells sensitized them to anthracyclines. Short-term in vitro exposure of primary AML cells (n=29) to daunorubicin showed a strong correlation between the in vitro pharmacodymanic changes of phospho-JNK levels and the response of patients to standard induction chemotherapy (P=0.012). We conclude that JNK activation failure confers another mechanism of anthracycline resistance in AML. Elucidating the ultimate mechanisms leading to JNK suppression in chemoresistant AML may be of major therapeutic value.

Procleix Ultrio transcription-mediated amplification vs. serological blood screening in south-western Greece.

We present here our overall experience after 27 months of performance of the Procleix Ultrio [HIV-1, hepatitis C virus (HCV), hepatitis B virus (HBV)] transcription-mediated amplification (TMA) assay. The aim of this report is to assess the impact of nucleic acid testing (NAT) implementation in blood screening of south-western Greek blood donors. We processed 38,264 units of blood as neat samples with the Procleix Ultrio TMA assay (Chiron/GenProbe, Emeryville/San Diego, CA, USA) between 1 January 2005 and 31 March 2007. NAT results were compared to those obtained from routine serology tests and quantitative polymerase chain reaction (PCR) assays. Overall, 52 units of blood tested positive for HBV (1.4 per thousand), 8 for HCV (0.2 per thousand) and none for HIV or multiple infections. The yield of TMA was 0.183 per thousand for HBV (7/38 264) and 0 for HCV. The TMA HBV-positive donations were tested for HBV DNA by a quantitative PCR assay and were found negative (below the detection limit of the method, 200 copies/mL). Follow-up testing showed that the TMA HBV-positive donations were positive for anti-hepatitis B core antigen immunoglobulin G antibodies. Implementation of the TMA assay in the individual donation configuration increased HBV detection compared to serological screening or a commonly used quantitative PCR assay. Follow-up studies will determine the impact of NAT implementation in HBV transmission in countries with an intermediate HBV incidence.

T helper 1 (Th1)/Th2 cytokine expression shift of peripheral blood CD4+ and CD8+ T cells in patients at the post-acute phase of stroke.

Local humoral and cellular immune responses modulate the inflammatory processes involved in the development of atherosclerotic lesions, as well as in the evolution of brain infarcts in stroke patients. The role of systemic adaptive immunity on the progression of such disease manifestations is less clear. In the current study, we evaluated the percentages of T helper 1 (Th1) [interleukin (IL)-2, interferon (IFN)-gamma] and Th2 (IL-4, IL-10) cytokine-producing peripheral blood CD4+ and CD8+ T cells in 23 patients with a history of ischaemic stroke (IS) at the chronic stable phase of the disease (median post-stroke time 34.5 months). Seven stroke-free individuals matched for age and vascular risk factors (matched controls, MC) were collected for comparison. To measure cytokine values at baseline and after stimulation, we used a flow cytometry method of intracellular cytokine staining. Intrinsic Th1 and Th2 cytokine production in unstimulated T cells was negligible in all study participants. Following mitogenic stimulation with phorbol 12-myristate13-acetate/ionomycin, both the IS and the MC groups exhibited a similarly strong Th1 response; IL-2 production predominated in the CD4+ T cells and IFN-gamma in the CD8+ T cells. However, when measuring the Th2 cytokine-production capacity post-stimulation, a significant increase in the percentage of IL-4-producing T cells was observed in the IS groups, compared with the MC group, resulting in a significantly lower ratio of IFN-gamma-/IL-4-producing T cells. No such Th2 enhancement could be confirmed for the case of IL-10. We propose that in IS patients there is a systemic shift of the immune system towards Th2 responses at the late post-acute phase of stroke.

Effect of non-operative management (NOM) of splenic rupture versus splenectomy on the distribution of peripheral blood lymphocyte populations and cytokine production by T cells.

Post-traumatic splenectomy is associated with increased postoperative morbidity and mortality and long-term impairment of humoral and cellular immunity. Alternatives to surgery have been developed to minimize or avoid the immediate and/or long-term complications of splenectomy. Herein we investigated the long-term effect of non-operative management (NOM) of the traumatic rupture of the spleen on the distribution of peripheral blood (PB) lymphocyte populations and cytokine production by T cells. PB samples were drawn from six NOM patients, 13 age-matched adults who had undergone splenectomy after trauma (SP patients) and 31 age-matched controls. Cellular phenotypes and the intracellular production of interferon (IFN)-gamma, interleukin (IL)-2, IL-4 and IL-10 cytokines in T cells were determined in whole blood +/- mitogens by flow cytometry. NOM patients did not show any changes in the absolute numbers of lymphocytes or the distribution of their subsets, compared to the controls. In contrast, SP patients showed a sustained increase in the percentage and/or absolute numbers of lymphocytes, CD8 T cells, activated CD8 T cells, natural killer (NK) T cells, NK cells and gammadelta T cells, and a reduction in naive CD4 T cells. The constitutive or induced cytokine production by T cells of the NOM group was similar to the control group, whereas SP patients had increased percentages of constitutive IL-2- and IFN-gamma-producing CD8 T cells and IFN-gamma-producing CD4 T cells. Our findings indicate collectively that the healing process in NOM does not affect the architecture of the spleen to such an extent that it would lead to long-term alterations of the proportions of PB lymphocytes or the T cell cytokine profiles.

Combined inherited thrombophilia and adverse pregnancy outcome.

Inherited thrombophilia has been suggested as a possible condition of increased susceptibility to adverse pregnancy outcomes. In our prospective study, we investigated the association between combined inherited thrombophilia and adverse pregnancy outcome in the South-Western Greek population. Three hundred and ninety-six healthy Greek women with spontaneous pregnancies were investigated for combinations of the three commonest thrombophilic mutations (Factor II G20210A, Factor V Leiden and MTHFR C677T) and followed for adverse pregnancy outcomes. Statistical analysis was performed by Pearson's chi-square test. Four women (1%) had the FV Leiden/MTHFR T677T double genotype and two women (0.5%) had the FII G20210A/MTHFR T677T double genotype. Although the small number of cases of combined inherited thrombophilia, it seems that the presence of FV Leiden/MTHFR T677T double genotype increases the risk for placental abruption.

In vitro study of the long-term effects of post-traumatic splenectomy on cellular immunity.

The purpose of this study was to investigate the effect of splenectomy on cellular immunity. We studied the cellular phenotype and type 1 [interferon-gamma, interleukin-2 (IL-2)] and type 2 (IL-4 and IL-10) cytokine-producing peripheral blood CD4+ and CD8+ T lymphocytes in 22 healthy adults who had undergone post-traumatic splenectomy about 1 to 35 years ago. Splenectomy resulted in a long-term reduction of the percentage of CD4+CD45RA+ cells and a late increase of the percentage and absolute numbers of T-cell receptor gamma/delta cells. Stimulation with Staphylococcal enterotoxin B resulted in normal IL-2 production by CD4+ T cells, indicating that the naïve cells were not anergic. Splenectomy also resulted in long-term priming of both CD4+ and CD8+ T cells. During the first 8 years, both type 1 and type 2 CD4+ T cells were primed to varying degrees. About 8 years later, the percentage of primed type 2 CD4+ T cells subsided, but that of type 1 CD4+ T cells, although decreased, remained detectable over a longer period. Priming of CD8+ T cells persisted throughout the study period. The long-term priming of type 1 CD4+ and CD8+ T cells, which may result in partial impairment of T-cell functions, may explain reported defects of immune responses to recall antigens in splenectomized individuals. In addition, changes in the profile of primed CD4+ T cells with time may be clinically relevant to relapses in autoimmune thrombocytopenia after splenectomy.

Cytokine production and monocyte HLA-DR expression as predictors of outcome for patients with community-acquired severe infections.

This study was performed to evaluate the impact of pro- and anti-inflammatory molecules and human leukocyte antigen DR (HLA-DR) expression as markers of immune status for the final outcome of septic patients. The study included 30 patients with severe sepsis due to community-acquired infections. Concentrations of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), IL-8, IL-10, and transforming growth factor beta1 (TGF-beta1) in serum, as well as monocyte HLA-DR expression, were determined on admission and on days 3, 10, 13, and 17 during hospitalization. Of the 30 patients enrolled, 13 survived, while 17 died during their hospital stay. All patients had significantly lower HLA-DR expression and higher pro- and anti-inflammatory cytokine levels than healthy individuals. HLA-DR expression was significantly decreased in nonsurvivors at almost all time points. In nonsurvivors, higher levels in serum of TNF-alpha on days 13 and 17; IL-6 levels on day 3; and IL-10 on days 3, 10, and 13 were found. Baseline levels of TGF-beta1 were significantly higher in survivors. Independent risk factors of mortality were IL-10 levels on days 3 and 10, while monocyte HLA-DR expression on admission was a good predictor for survival. Several pro- and anti-inflammatory cytokines are oversynthesized during severe infections, especially in patients with a poor outcome. Monocyte HLA-DR expression is an early and constant predictive marker for survival in severe sepsis, while serum IL-10 levels on days 3 and 10 have negative prognostic value for the final outcome.

Th1 and Th2 cytokines in a patient with Evans' syndrome and profound lymphopenia.

A case of Evans' syndrome with IgM deficiency and lymphopenia was studied before and after splenectomy. The lymphopenia was as a result of profound reduction of CD4 and CD8 cells. Study of cytokine secretion before splenectomy revealed a spontaneous Th1- and Th2-type cytokine production, and complete suppression of transforming growth factor (TGF)-beta. After splenectomy, the patient achieved clinical remission, the natural killer (NK) cell number increased and the pattern of cytokine production showed normalization of interleukin (IL)-2, IL-4, IL-10, TGF-beta and abolition of interferon (IFN)-gamma production. We conclude that splenectomy had a beneficial effect owing to an increase in NK cells and an associated increase in TGF-beta production.

Association between sarcoidosis and lymphoma revisited.

AIMS: To investigate the relation between sarcoidosis and lymphoma. METHODS: The hospital notes of five patients with sarcoidosis and a lymphoproliferative disorder were reviewed. Histological material on which the diagnoses of sarcoidosis and lymphoma were made was re-analysed. RESULTS: Four of the five patients had well documented sarcoidosis preceding the development of lymphoma by 18 months to 28 years; the fifth patient had lymphoplasmacytic lymphoma with a reactive granuloma reaction. Two patients had chronic sarcoidosis and three were treated with prednisolone. The types of lymphoma were: Hodgkin's disease (n = 1), B cell lymphoma (n = 2) (mantle cell and lymphoplasmacytic/local plasmacytoma) and large granular lymphocyte leukaemia (T cell) (n = 1). CONCLUSIONS: The association between sarcoidosis and lymphoma is confirmed, suggesting that sarcoidosis may be a predisposing factor for the development of a lymphoid malignancy due to disturbance of the immune system. All types of lymphoma may develop. The first case of T cell granular lymphocytic leukaemia in a patient with sarcoidosis has been documented.

Adhesion molecule expression on CD34+ progenitor cells from normal and aplastic anaemia bone marrow.

Aplastic anaemia (AA) is a disease of bone marrow failure. Evidence has been produced for both a stem cell and a stromal cell defect in this disease. The contribution of deficient or defective cell adhesion molecules (CAMs) has not been determined. CAMs have been shown to be important in stem cell-stromal cell interactions and maintenance of haemopoiesis. In this study the expression of CAMs (LFA-1, LFA-3, ICAM-1. VLA-4, CD44, sLex and L-selectin) on CD34+ progenitor cells from 10 normal donors and eight patients with AA was investigated using double immunofluorescence. There was no significant difference in the percentage of CD34+ cells that were CAM+ between normal and AA bone marrow, suggesting that abnormal CAM expression on AA progenitor cells is not responsible for nor contributes to the pathogenesis of the disease. However, these findings do not exclude abnormal CAM function on progenitor cells, or abnormal expression or function of CAM ligands or counter-receptors on AA stromal cells.