Elif Azarsiz

PON1 activities and oxidative markers of LDL in patients with angiographically proven coronary artery disease

BACKGROUND There is growing evidence that ox-LDL plays an important role during the atherosclerosis process and PON1 can significantly inhibit generation of lipid peroxidation during LDL oxidation and thus play a role in the in vivo protection by HDL against atherosclerosis. METHODS Twenty-four healthy volunteers and one-hundred and one patients were taken into study, sixty-eight patients had coronary artery disease which was confirmed by coronary angiography. Serum PON1, erythrocyte superoxide dismutase and catalase activities, oxidative markers of LDL were determined along with the routine biochemical parameters in all groups. RESULTS The indicators of oxidative stress were higher in the patients compared with the controls. No statistically significant difference in any of parameters were observed between the patients who had obstruction with different degrees except for erythrocyte TBARS [24.5 nM/g Hb in patients with one vessel disease (VD) (n=22), 29.6 nM/g Hb in patients with two VD (n=26) and 33.5 nM/g Hb in patients with three VD (n=20)]. Basal and stimulated diene levels were higher in patients who had more diseased vessels than those who had less. CONCLUSION The increase in erythrocyte TBARS and CRP levels with the severity of disease supports the reports that showed the inflammatory and oxidative nature of atherosclerosis. In the light of the fact that the well-known classical risk factors for atherosclerosis are closely associated with increased oxidative stress, we propose that the elevation in TBARS levels might be a more marked indicator for the degree of atherosclerosis than the insufficiency in antioxidant enzymes such as SOD and PON1.

Antioxidant enzymes and paraoxonase show a co-activity in preserving low-density lipoprotein from oxidation.

Abstract Oxidative modification of low-density lipoprotein in the artery wall plays a crucial role in the development of atherosclerosis. This physiopathological mechanism is clearly inhibited by high-density lipoprotein possibly via paraoxonase enzyme activity, present in high-density lipoprotein. In this study we determined the in vitro susceptibility of low-density lipoprotein to oxidation and the effect of various factors, such as paraoxonase phenotypes, on this process. Low-density lipoprotein from healthy volunteers (n=66) was isolated using the precipitant reagent and the oxidation was evaluated by measuring the malonyl dialdehyde and diene levels. Low-density lipoprotein cholesterol and phospholipid, vitamin E, serum cholesterol, high-density lipoprotein and low-density lipoprotein cholesterol levels, and erythrocyte antioxidant enzymes were also determined. There was no difference among the parameters with regard to gender. Low-density lipoprotein samples obtained from subjects with the AA allele were more prone to oxidation, as observed by their higher stimulated conjugated diene (P=0.041) and thiobarbituric acid-related substance (P=0.042) levels, than samples from subjects with AB or BB alleles. The subjects with the BB allele had higher superoxide dismutase (P=0.021) and catalase (insignificant increase) activities, while their conjugated diene (P=0.000) levels were lower. In conclusion, our results revealed that the high low-density lipoprotein oxidation is related to the high low-density lipoprotein cholesterol content and low phospholipid content. The present study demonstrated an increase in superoxide dismutase and catalase activities, asl well as PON1 activities, in subjects with the BB allele. Since these enzymes all show activity against low-density lipoprotein oxidation, we propose that future investigations on atherosclerotic processes should address PON1 polymorphism as well as PON1 and other antioxidant enzymes.

CD4+CD25+Foxp3+ T regulatory cells, Th1 (CCR5, IL-2, IFN-γ) and Th2 (CCR4, IL-4, Il-13) type chemokine receptors and intracellular cytokines in children with common variable immunodeficiency

Common variable immunodeficiency (CVID) is a heterogeneous group of primary antibody deficiencies characterized by decreased serum immunoglobulin G along with a decrease in serum IgA and/or IgM, defective specific antibody production, and recurrent bacterial infections. Abnormal lymphocyte trafficking, dysregulated cellular responses to chemokines, and uncontrolled T cell polarization may be involved in the pathogenesis and may help to understand the clinical complications. We evaluated T helper cell subsets (chemokine receptors CCR4, CCR5, and CCR7), expressions on T lymphocytes, intracellular cytokines – IL-2, IL-4, IL-13, IFN- γ-on CD4+ T cells, and expression of CD4+CD25+Foxp3+ regulatory T cells of 20 CVID patients and 26 healthy controls. Autoimmune clinical findings and other complications were also determined. Percentages and absolute numbers of CD4+CD25+ Foxp3+ cells did not show any significant difference between CVID cases and healthy controls nor between severe and moderate disease patients. The only significant difference regarding Th1 and Th2 type intracellular cytokines was the decreased absolute numbers of CD3+CD4+IL4+ cells in CVID cases. There were some findings about T helper cell type dominance in CVID patients such as positive correlation between hepatomegaly and high IL-2 and IFN-γ in CD3+CD4+ cells and very high expression of CCR5 (Th1) on CD3+CD4+ cells in patients with granuloma. Th1 (CCR5) and Th2 (CCR4) type chemokine receptors did not show any dominance in CVID cases. However, frequencies of CCR7 expressing CD3+ T cells, CD3+CD4+ T helper cells and CD3+CD8+ T cytotoxic cells were significantly lower in severe CVID patients. In addition, presence of autoimmune clinical findings was negatively correlated with CCR7+ cells. As CCR7 is a key mediator balancing immunity and tolerance in the immune system, the abnormality of this mediator may contribute to the profound immune dysregulation seen in CVID. In addition, Th1 cells seem to be more involved in the disease pathogenesis than Th2 cells.

Does OM-85 BV prophylaxis trigger autoimmunity in IgA deficient children?

BACKGROUND IgA deficiency (IgAD) is the most common primary antibody deficiency. Although two-third of the cases are reported to be asymptomatic, some IgAD children may have frequent infections that urge the clinicians to search for prophylactic measures. OM-85 BV is one of these agents that is known to stimulate mucosa associated lymphoid tissue, and upregulate Th-1 response. This study was performed to determine a possible role of OM-85 BV in triggering autoimmunity in IgAD children within a four-year-follow up period. METHODS Sixty-three children (34 males (54%), 29 females (46%)) with sporadic IgAD and recurrent febrile infections were included. Patients were carefully screened for autoimmunity both on admission and in follow-up: Those with autoimmune features or under immunosuppressant treatment were excluded. Patients were randomly divided into two groups: Group I received bacterial lysate propylaxis (OM-85 BV) (n:37), and Group 2 received no prophylactic regimen (n:26). Development of clinical autoimmune findings or autoantibodies (anti-nuclear antibody (ANA), ANA profile (14 antigens), anti-cytoplasmic antibodies (ANCA), anti-cardiolipin antibodies IgG and IgM (aCL), rheumatoid factor (RF), direct Coombs test, anti-thyroglobulin (anti-T) and anti-thyroid microsomal antigen (anti-M)) were evaluated. RESULTS Mean age of the study group, age at the onset of infectious symptoms and at admission were 102.9±42.2, 27.1±24.9, and 55.2±25.1 months, respectively. Follow-up duration of the whole study group was 48.3±23.1 months. Number of infections was 6.2±2.7 per year in the whole study group. Sixteen patients (25.4%) of the whole study group showed ANA positivity in different patterns and titers. Frequency of ANA, ANCA and RF positivity was 24.3%, 5.4%, 2.7% in Group 1, and 26.9%, 11.5%, 3.8% in Group 2, respectively. Statistical comparisons revealed no significant difference between the two groups. CONCLUSION Significant clinical or laboratory markers for autoimmunity in follow-up were not observed between receivers or non-receivers of OM-85 BV. Frequency of ANA positivity was comparable to the previously reported values in IgAD children which was not affected by OM-85 BV usage. Possible effect of triggering autoimmunity with repeated cures of bacterial lysates needs to be further clarified. Side effects requiring the cessation of treatment were not recorded.

A Clinical and Laboratory Approach to the Evaluation of Innate Immunity in Pediatric CVID Patients

Defective adaptive immune responses are well studied in common variable immunodeficiency (CVID) patients; however, more focus is needed on innate immune system defects to explain CVID’s clinical and laboratory heterogeneity. This is the first study comparing migratory function of granulocytes, oxidative burst activity of phagocytic cells, surface integrin expressions on neutrophils and lymphocytes, natural killer (NK) cell numbers and cytotoxic activity, natural killer T cells, lymphocyte subsets such as CD8+CD28+, CD4+CTLA-4+ cells in CVID patients (n: 20) and healthy controls (n: 26). The relationship between laboratory findings and some clinical was also investigated. CD3+CD8+ T cytotoxic cells were found to be elevated in CVID patients, but CD3+CD8+CD28+ or CD3+CD8+CD28− cells did not show any significant difference. CD4+CTLA-4+ cell percentages were significantly lower in CVID patients compared to healthy controls. Severe CVID patients had decreased percentages of NK cells with increased NK cell cytotoxicity suggesting possibly increased activation. Furthermore, CD3−CD16+CD56+CD28+ cells of CVID patients were elevated while percentage of CD28− NK cells was decreased. Neutrophil migration percentages were lower but and oxidative burst activity was not affected. CD11a expressions on these cells were depressed in contrast to increased expression of CD18. Innate immunity defects may affect the extent of recurrence and severity of infections in CVID. Our observations highlight some of these associations and indicate the need for further similar studies for improving better innate system evaluation batteries for these patients. Further phenotypic correlations of these analyses will help clinicians reach a more definitive target for the molecular genetic diagnostic of pediatric CVID patients.

Do elevated serum IgM levels have to be included in probable diagnosis criteria of patients with ataxia-telangiectasia?

Ataxia-telangiectasia (AT) is a rare multisystem, neurodegenerative genetic disorder that is characterised by progressive neurological abnormalities, oculocutaneous telangiectasias and immunodeficiency. Delay in diagnosis or misdiagnosis is probable due to its wide clinical heterogeneity in infancy. Recurrent sinopulmonary infections are often the only presenting symptom and usually patients have decreased immunoglobulins. A total 10% of patients who present with decreased serum immunoglobulin G and A and with normal or elevated immunoglobulin M levels are often misdiagnosed as hyperimmunoglobulin M syndrome. Definitive diagnosis is made if a patient with progressive cerebellar ataxia has a disease causing mutation on the ATM gene. Ataxia-telangiectasia guideline of the European Society for Immunodeficiencies defines the probable diagnosis criteria. We evaluated twenty ataxia-telangiectasia patients (mean age 13.8±4.1 years) retrospectively who were followed-up for a mean of 38.6±27.0 months. Twelve patients had a family history of consanguinity. A total of 80% patients suffered from various infections. Neoplasms occurred in three of them. Patients showed immunological abnormalities as low IgG (45%), low IgA (65%) and elevated IgM (60%) levels. CD3+CD4+ T lymphocyte frequency was low in 45% patients. The mean AFP concentration at the diagnosis was 191.9±140.1 ng/ mL and the raised IgM values did not show any statistically significant relationship with high AFP concentrations. Frequency of the elevated IgM concentrations in (60%) patients raises the concerns about thinking this finding has to be accepted as a probable diagnosis criterium.

Does Intravenous Immunoglobulin Therapy Prolong Immunodeficiency in Transient Hypogammaglobulinemia of Infancy

Transient hypogammaglobulinemia of infancy (THI) is characterized by recurrent infections and one or more reduced serum immunoglobulin levels. Typically, THI patients recover spontaneously, mostly within 30-40 months of age, but sometimes recovery may be delayed until 5-6 years of age. The use of intravenous immunoglobulin (IVIg) as an alternative to antibiotic prophylaxis remains contraversial also in symptomatic THI patients. In fact, some authors believe that IVIg therapy may cause a delay in the maturation of the humoral immune system because of the interference from passively transfered antibodies. The aim of this study was to investigate the effect of IVIg replacement on recovery from immunodeficiency in THI patients and determine new parameters in order to include these patients in IVIg therapy groups. In this retrospective study, 43 patients (65%) received IVIg replacement therapy while 23 patients (34.8%) showed spontaneous normalization without IVIg. The percentages of patients who had more than six times the number of febrile infections in a year decreased from 91% to 21% in the group receiving IVIg treatment. At admission, before being recruited to IVIg therapy, serum immunoglobulin G (IgG) levels and anti-hemophilus B (Hib) antibody titers were found to be significantly low in cases who were selected for IVIg replacement. The percentages of patients who did not have protective levels of anti-Hib, anti-rubella or anti-rubeola-IgG were also significantly high in IVIg cases. There was no statistically significant difference in the age at which IgG levels normalized between the IVIg and the non-IVIg group. Patients in the IVIg group and non-IVIg group reached normal IgG levels at the age of 42.9±22.0 and 40.7±19.8 months, respectively. In conclusion, IVIg infusions do not cause a delay in the maturation of the immune system in THI patients. Besides the well-established criteria, very low and non-protective specific antibody responses against previously applied vaccines are important factors to consider when selecting patients for IVIg therapy.

A novel disease-causing CD40L mutation reduces expression of CD40 ligand, but preserves CD40 binding capacity

Mutations in CD40 ligand (CD40L) that permit residual CD40L expression typically impair binding of CD40. We report a male patient who presented with recurrent bacterial respiratory tract infections, normal IgM, decreased IgG, absent IgA levels, and CD40L expression at ~50% of the level observed in the normal control. He subsequently developed autoimmunity, inflammatory bowel disease, severe opportunistic infections suggestive of a combined immunodeficiency, and a cervical spine schwannoma. Whole exome sequencing of the patients genomic DNA revealed a novel missense mutation (p.H47Y) in CD40L. Although this mutation was predicted to be benign in silico, flow cytometry at 13 years of age demonstrated markedly decreased CD40L expression (~32% of normal control) that retained the capacity to bind soluble CD40-Ig, suggesting that the mutation impairs CD40L surface expression without affecting its affinity for CD40. This case highlights the variability in the clinical evolution and phenotype of CD40L deficiency.

Serum paraoxonase 1 activity and oxidative markers of LDL in patients with cardiac syndrome X.

Objective — Myocardial ischaemia in cardiac syndrome X (CSX) is believed to be due to microvascular dysfunction. Increased oxidative stress is one of the suspected mechanisms of microvascular dysfunction. The aim of this study was to evaluate the oxidative status in patients with CSX, by determining serum paraoxonase-1 (PON1) activity in addition to LDL-oxidation markers. Methods and results — This cross-sectional study consisted of patients with CSX (group I, n = 30), patients with coronary artery disease (group II, n = 31), and healthy controls (group III, n = 32). Lipid parameters, PON-1 activity, and LDL oxidation markers (conjugated-diene and thiobarbituric acid-reactive substance-TBARS) were measured. Endothelium-dependent vasodilatation was determined by brachial artery ultrasonography.There were no significant differences in serum LDL, apolipopro-tein-B, baseline LDL-diene, and LDL-TBARS levels between groups.There were no differences in both apolipoprotein-A1 and HDL levels between group I and group III. Apolipoprotein-A1 and HDL levels were significantly lower in group II than group I patients (P < 0.001). PON-1 activity was lowest in group II patients. Average PON-1 activity in group I was in between of group II and group III.The percent change of LDL-diene levels after stimulation was significantly higher in group II than in groups I and III (P = 0.005 and P = 0.02, respectively).The percent change of LDL-TBARS levels was lowest in group I (P = 0.03). There was a moderate correlation between endothelium-dependent vasodilatation and PON-1 activity in group I (r = 0.43, P = 0.04). Conclusions — Enhanced oxidative stress might be one of the causes of impaired endothelial functions resulting in myocardial ischaemia and chest pain in patients with CSX.The relatively preserved HDL and apolipoprotein-A1 levels in patients with CSX might be a protective mechanism against progression of coronary microvascular dysfunction to atherosclerotic coronary artery disease.

Reference values for B-cell surface markers and co-receptors associated with primary immune deficiencies in healthy Turkish children

In order to evaluate B-lymphocyte subsets of patients with primary immunodeficiencies, the normal values for national healthy children have to be used as a reference. Recently, B-cell co-receptor markers (CD19, CD21, and CD81) and CD20, CD22, and CD27 deficiencies have been reported in relation with different primary immunodeficiency diseases. The objective of this study was to establish national reference values for B-lymphocyte co-receptors and some surface markers, CD20, CD22, CD27, as well as classic lymphocyte subsets in the peripheral blood of healthy children. A total of 90 healthy children were included in this study. Complete blood counts were performed and cells with CD3, CD4, CD8, CD19, CD16/56, CD20, CD21, CD22, CD27, and CD81 surface markers were simultaneously detected by flow cytometry. The children were evaluated in three age subgroups, 0–1, 1–6, and >6 years, and minimum, maximum, mean, mean minus standard deviation, and 2.5–97.5 percentile values were all determined. By establishing reliable reference ranges for these surface markers, we hoped to help identifying and classifying some primary immunodeficiency patients, especially those defined as unclassified hypogammaglobulinemia and those without definite diagnosis.