Eculizumab for Treatment of Refractory Antibody-Mediated Rejection in Kidney Transplant Patients: A Single-Center Experience.

Antibody-mediated rejection (AMR) is responsible for up to 20%-30% of acute rejection episodes after kidney transplantation. In several cases, conventional therapies including plasmapheresis, intravenous immunoglobulin, and anti-CD20 therapy can resolve AMR successfully. But in some cases the load of immunoglobulins that can activate complement cascade may submerge the routine desensitization therapy and result in the formation of membrane attack complexes. Eculizumab, a monoclonal antibody against C5, was reported to be an option in cases with severe AMR that are resistant to conventional therapy. Here, we present 8 cases that were resistant to conventional therapy and in which eculizumab was given as a salvage treatment. Given the bad prognosis for renal transplants displaying acute injury progressing rapidly to cortical necrosis on the biopsy, the prompt use of eculizumab could have the advantage of immediate effects by stopping cellular injury. This can provide a therapeutic window to allow conventional treatment modalities to be effective and prevent early graft loss.

Activation of the JAK/STAT pathway in Behcet's disease.

Th1/Th17-type T-cell responses are upregulated in Behcet's disease (BD). However, signaling pathways associated with this aberrant immune response are not clarified. Whole-genome microarray profiling was performed with human U133 (Plus 2.0) chips using messenger RNA of isolated CD14(+) monocytes and CD4(+) T cells from peripheral blood mononucleated cell (PBMC) in patients with BD (n = 9) and healthy controls (HCs) (n = 9). Flow cytometric analysis of unstimulated (US) and stimulated (phytohaemagglutinin) signal transducer and activator of transcription (STAT3) and pSTAT3 expressions of PBMCs were also analyzed (BD and HC, both n = 26). Janus family of kinase (JAK1) was observed to be upregulated in both CD14(+) monocytes (1.95-fold) and CD4(+) T lymphocytes (1.40-fold) of BD patients. Using canonical pathway enrichment analysis, JAK/STAT signaling was identified as activated in both CD14(+) monocytes (P = 9.55E-03) and in CD4(+) lymphocytes (P =8.13E-04) in BD. Interferon signaling was also prominent among upregulated genes in CD14(+) monocytes (P = 5.62E-05). Glucocorticoid receptor signaling and interleukin (IL-6) signaling were among the most enriched pathways in differentially expressed genes in CD14+ monocytes (P = 2.45E-09 and 1.00E-06, respectively). Basal US total STAT3 expression was significantly higher in BD (1.2 vs 3.45, P < 0.05). The JAK1/STAT3 signaling pathway is activated in BD, possibly through the activation of Th1/Th17-type cytokines such as IL-2, interferon (IFN-γ), IL-6, IL-17 and IL-23.

Eculizumab for salvage treatment of refractory antibody-mediated rejection in kidney transplant patients: case reports.

Antibody-mediated rejection (AMR) in a group of preoperatively desensitized patients may follow a dreadful course and result in loss of the transplanted kidney. In several cases, conventional therapies including plasmapheresis, intravenous immunoglobulin, and anti-CD 20 therapy can resolve AMR successfully. But in some cases the load of immunoglobulins that can activate complement cascade may submerge the routine desensitization therapy and result in the formation of membrane attack complexes. Eculizumab, monoclonal antibody against C5, was reported to be an option in cases with severe AMR that are resistant to conventional therapy. Here, we present two cases of acute-onset AMR in preoperatively desensitized patients. Eculizumab was used as a salvage agent in addition to conventional therapy. Given the bad prognosis for renal transplants displaying acute injury progressing rapidly to cortical necrosis on the biopsy, the prompt use of eculizumab could have the advantage of immediate effects by stopping cellular injury. This can provide a therapeutic window to allow conventional treatment modalities to be effective and prevent early graft loss.

Effects of malnutrition on oxidative burst functions and infection episodes in children with acute lymphoblastic leukemia.

INTRODUCTION: The aim of this study was to determine the effect of malnutrition on oxidative burst functions (OBF) of neutrophils in children with acute lymphoblastic leukemia (ALL). MATERIALS AND METHODS: Twenty-eight patients with ALL and thirty healthy controls were enrolled to the study. Thirteen patients with ALL were found to have malnutrition. While neutrophil OBF of ALL patients without malnutrition were studied both before induction chemotherapy and 3 months after, the same functions in ALL patients with malnutrition were studied both before induction chemotherapy and when the nutritional status improved. Control group were studied at admission and 3 months later. RESULTS: The OBF of ALL patients with and without malnutrition before induction chemotherapy were found to be significantly lower than the control group (P = 0.009), whereas the OBF were found to be similar in both patient groups with ALL (P = 0.27). The median infection episode rate and the duration of antibiotics therapy during the study period were similar in both patient groups with ALL. The repeated OBF of both patient groups with ALL were shown to increase to similar values with the control group in the third month of chemotherapy (P = 0.002). The median infection episode rate during the first month of chemotherapy was shown to decrease significantly during the third month of chemotherapy in both patient with ALL groups (P < 0.001). CONCLUSIONS: We have not been able to demonstrate an overt effect of malnutrition on OBF. However, our results still need to be verified via further larger scaled studies of OBF in leukemic children with and without malnutrition.

Dietary whey supplementation in experimental models of wound healing.

Whey is a dairy product containing milk serum proteins with diverse biological effects. In this study, the effect of dietary whey supplementation on wound healing was investigated. Rats were fed a standard or whey-supplemented diet for three weeks. Wound healing parameters, glutathione, and lipid peroxide levels were determined three days after the application of two different models of wound healing, i.e. laparotomy and colonic anastomosis. Dietary whey supplementation significantly increased glutathione levels and suppressed lipid peroxidation after experimental laparotomy and colonic anastomosis. Bursting pressures, hydroxyproline, and cytokine levels were not changed. Our results show that dietary whey supplementation increases glutathione synthesis and cellular antioxidant defense. Long-term effects of whey feeding on wound healing remains to be investigated.

CD8+CD28-, suppressive T cells in systemic lupus erythematosus.

Recent studies show that a CD8+CD28- phenotype of T-cell population inhibits the reactivity of T-helper cells either by a contact-dependent mechanism or with secreting suppressive cytokines. In this study, we have explored the peripheral blood CD8+CD28- T-cell population in 53 patients with systemic lupus erythematosus (SLE) in comparison to healthy and diseased control groups. The distribution of CD28- cells within CD8+ population has been found significantly lower in patients with SLE than in healthy individuals. While there were no significant differences in the expression of costimulatory molecules CD80 and CD86, the CD40 expression on monocytes was found significantly lower and there was a slight decrease of expression of Interleukin-10 (IL-10) in CD8+CD28- population in patients with SLE. The Transforming growth factor-beta (TGF-beta) mRNA expression was found higher in CD8+CD28- T cells. Neither activation induced nor time-dependent change in the frequency of CD8+CD28- cells has been observed following stimulation at various time-points indicating that the control of CD28 expression was not dependent on the presence of sustained stimulations. Decrease in CD8+CD28- T cells which normally produce TGF-beta and their low-level IL-10 content may reflect impaired T-cell suppression and accordingly, increased T cell help to autoreactive B cells in patients with SLE.

Thalidomide has both anti-inflammatory and regulatory effects in Behcet's disease.

Thalidomide is shown to be an effective treatment for mucocutaneous symptoms of Behcet's disease (BD). In this study, the effects of thalidomide on peripheral blood mononuclear cells were investigated ex vivo. In an open prospective study, ten patients were given 200 mg/day thalidomide for 12 weeks and cluster of differentiation 4 (CD4), CD8, CD11a, CD11b, CD16, CD18, CD28, CD44, CD45RO, CD45RA, CD56, CD120a and gammadelta+ T cells were analysed with flow cytometry at 0, 3, 7, 30 and 90 days. Two patients were excluded from the analysis for attacks of uveitis within the first 2 weeks. At day 7, tumour necrosis factor-alpha (TNF-alpha) receptor+ (CD120a; 12% vs 5%), CD8/CD11b+ (12% vs 6%) and CD16/CD56+ (16% vs 9%) cells decreased in BD patients compared to day 0. On the other hand, CD4+CD45RO+ T cells (24% vs 34%) at day 30 and gammadelta+ T cells (11% vs 21%) at day 90 increased after treatment. These results suggest that thalidomide tends to decrease TNF-alpha receptor levels, CD8/CD11b+ T cells and natural killer cells in early treatment and increases CD4+CD45RO+ memory T and gammadelta+ T cells later in BD.

Activation of neutrophils by testosterone in Behçet's disease.

OBJECTIVE: Gender differences, especially in disease severity, are observed in Behçet's disease (BD), a systemic vasculitis of unknown etiology. To determine the putative role of testosterone on neutrophil activity exhibited by patients with BD, peripheral blood neutrophils were examined in vitro before and after treatment with testosterone. METHODS: Peripheral blood neutrophils of 49 patients with BD (26M:23F), 33 patients with ankylosing spondylitis (AS) (23M:10F), 8 female patients with hirsutism and 31 healthy individuals (19M:12F) were analyzed by flow cytometry. Neutrophil-ROS-generating capacity, anti-CD66b, anti-CD16, Phi-philux and PI staining techniques were used for evaluating neutrophil activation and apoptosis. RESULTS: Gender differences were striking not only in the mean oxidative burst response but also in the rate of apoptosis. Male BD patients manifested increased burst response before testosterone treatment compared with females (8.0 +/- 4.9 vs. 4.9 +/- 3.3, p < 0.01). Consistent with oxidative burst results, baseline percentages of CD66b (99.1 +/- 0.9 vs. 94.7 +/- 5.3 p < 0.04) and CD16 expressing cells were greater in male BD patients. A decreased apoptosis ratio was observed using phi-philux and PI staining in BD patients. This was especially significant in male compared to female BD patients (2.4 +/- 1.4 vs. 6.8 +/- 5.8, p < 0.002). BD itself rather than the gender was found to be the most important predictor of this altered apoptosis ratio in BD determined by linear regression analysis. CONCLUSION: Our results suggest that a contribution of testosterone to the BD pathogenesis cannot be ruled out. However, causative factors for disturbed apoptosis especially seen in male BD patients need to be further evaluated.

The effects of iron deficiency on neutrophil/monocyte apoptosis in children.

OBJECTIVES: Iron is essential for DNA synthesis; its deficiency may lead to impaired DNA synthesis and subsequent alterations in levels of apoptosis. Here, we have aimed to investigate effects of iron deficiency anaemia (IDA) on apoptotic response of phagocytic cells and to understand whether the effect is reversible after iron supplementation. MATERIALS AND METHODS: Forty-nine IDA patients and 26 healthy controls, aged between 6 months and 12 years with similar demographic status, were considered. Neutrophil- and monocyte-apoptotic responses of IDA patients and the control group were compared by flow cytometry. Then, IDA patients were provided with oral iron supplementation. On day 15 of iron therapy, neutrophil- and monocyte-apoptotic responses of IDA patients were rechecked and were compared to those of control group. RESULTS: Neutrophil- and monocyte-apoptotic responses in terms of early and late percentages of apoptosis, and percentages of necrotic cells, were significantly less in IDA patients compared to the control group. The significantly low apoptotic responses of IDA patients rose to levels of the control group by day 15 of iron therapy. Besides, the effect of IDA on apoptotic responses was found to be more enhanced in severe IDA patients that those of mild IDA patients. CONCLUSION: Correction of differences after iron supplementation therapy implies that IDA might be a cause for changes in neutophil- and monocyte-apoptotic responses. The impact of this diminution of apoptotic cellular function in IDA should be further investigated, with longitudinal studies, in order to document the impact of any severe and/or long-lasting IDA on autoimmunity and malignancy.

L-Carnitine ameliorates methotrexate-induced oxidative organ injury and inhibits leukocyte death.

Methotrexate (MTX), a folic acid antagonist widely used for the treatment of a variety of tumors and inflammatory diseases, affects normal tissues that have a high rate of proliferation, including the hematopoietic cells of the bone marrow and the gastrointestinal mucosal cells. To elucidate the role of free radicals and leukocytes in MTX-induced oxidative organ damage and the putative protective effect of L-carnitine (L-Car), Wistar albino rats were administered a single dose of MTX (20 mg/kg) followed by either saline or L-Car (500 mg/kg) for 5 days. After decapitation of the rats, trunk blood was obtained, and the ileum, liver, and kidney were removed for histological examination and for the measurement of malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) activity, and collagen content. Our results showed that MTX administration increased the MDA and MPO activities and collagen content and decreased GSH levels in all tissues, while these alterations were reversed in L-Car-treated group. The elevated serum TNF-alpha level observed following MTX treatment was depressed with L-Car. The oxidative burst of neutrophils stimulated by Annexin V was reduced in the saline-treated MTX group, while L-Car abolished this inhibition. Similarly, flow cytometric measurements revealed that leukocyte apoptosis was increased in MTX-treated animals, while L-Car reversed these effects. Severe degeneration of the intestinal mucosa, liver parenchyma, and glomerular and tubular epithelium observed in the saline-treated MTX group was improved by L-Car treatment. These results suggest that L-Car, possibly via its free radical scavenging and antioxidant properties, ameliorates MTX-induced oxidative organ injury and inhibits leukocyte apoptosis. Thus, supplementation with L-Carnitine as an adjuvant therapy may be promising in alleviating the systemic side-effects of chemotherapeutics.

Oxidative burst response to monosodium urate crystals in patients with Behçet's syndrome.

OBJECTIVE: An erythematous response to intradermal injection of monosodium urate crystals (MSU) has been demonstrated in Behçet's syndrome (BS). To further elucidate the pathogenesis of this response, the effects of MSU on in vitro oxidative burst reaction of neutrophils and monocytes were investigated. METHODS: Peripheral blood mononuclear cells from patients with Behçet's syndrome (BS), rheumatoid arthritis (RA), familial Mediterranean fever (FMF) and healthy controls (HC) were incubated with 100 ng/ml phorbol myristate acetate (PMA) and MSU at different dosages (25-500 microg/ml). Oxidative burst reaction was evaluated in neutrophils and monocytes by flow cytometry. RESULTS: In patients with BS, oxidative burst of neutrophils was significantly increased compared to HC at 125 microg/ml and 250 microg/ml dosages of MSU (p < or = 0.001 and 0.004 respectively). In patients with FMF; there was also an increased oxidative burst reaction at 75 microg/ml, 250 g/ml and 500 microg/ml (p < or = 0.007; 0.001 and 0.004 respectively). In patients with BS, oxidative burst of monocytes was increased only at 125 g/ml dosage of MSU (p < or = 0.002). However, in patients with FMF monocyte burst response was increased at 25 microg/ml, 75 microg/ml and 125 g/ml (p < or = 0.004; < 0.0001; < 0.0001 and 0.002 respectively). In RA group, stimulation with PMA resulted in a higher oxidative burst reaction than FMF and BS (p < or = 0.000 and p < or = 0.008). No correlation was observed between oxidative burst of neutrophils or monocytes and intradermal responses to MSU crystals. CONCLUSION: Oxidative burst reaction with MSU is augmented in neutrophils and monocytes of BS. However, the response is not specific and is unassociated with skin dermal test which has a high specificity for BS.

Clinical and immunological effects of azithromycin in Behçet's disease.

BACKGROUND: The aim of this study was to evaluate the effects of azithromycin on mucocutaneous manifestations, oral health and immune response in Behçet's disease (BD). METHODS: Eight BD patients with active mucocutaneous symptoms were treated with azithromycin for 4 weeks. Oral health, clinical manifestations and in vitro interleukin (IL)-12, interferon (IFN)-gamma, IL-10 and monocyte chemotactic protein (MCP)-1 responses were evaluated before and after treatment. RESULTS: The number of folliculitic lesions, healing time of oral ulcers and scores of plaque indexes (PLIs) were lower after azithromycin treatment (P<0.05). Scores of PLIs correlated positively with the healing time of oral ulcers (P=0.02). Although a trend towards increased stimulated IL-10 responses with azithromycin was observed, no statistically significant difference was found. Stimulated and unstimulated MCP-1, IFN-gamma and IL-12 responses were similar before and after treatment (P>0.05). CONCLUSION: Azithromycin was observed to be effective in decreasing folliculitic lesions and fastening the healing time of oral ulcers in BD.

T and NK cell subset changes with microbial extracts and human HSP60-derived peptides in Behçet's disease.

OBJECTIVE: Microorganisms such as streptococcus and autoantigens such as 60 kD heat-shock protein (HSP60) are implicated in the etiopathogenesis of Behçet's disease (BD). METHODS: Peripheral blood mononuclear cells from patients with BD (n = 16) and healthy controls (HC) (n = 11) were cultured for 5 days with extracts of S. sanguis-KTH-1 (SS), E. coli (EC) and a mixed peptide combination from human HSP60 (aa 136-50, 179-97, 224-58 and 336-51) reported to be associated with BD. T and NK cell subset changes were determined by flow cytometry. RESULTS: In unstimulated 5-day cultures gammadelta+ (both CD4+gammadelta+ and CD8+gammadelta+), CD8+alphabeta+, CD4+CD56+ and CD8+CD11b+ cells were increased in BD compared to HC. In antigen-stimulated cultures of BD patients CD3+ and alphabeta+ T cells responded to HSP60 peptides whereas EC stimulated only CD16/ CD56+ NK cells. In the control group, similar to BD, alphabeta+ and CD4+ T cells responded to HSP60 peptides, however SS and EC mainly activated cytotoxic T cell subsets (CD8+CD11b and CD4+CD56+ T cells). CONCLUSION: Significant increases in unstimulated T cell subsets suggest the presence of an in vivo T cell activation in BD. In both patients and controls similar patterns of responses were observed against different microorganisms, however the role of human HSP60 peptides as immunodominant, crossreactive antigens could not be demonstrated.

Echocardiographical characteristics of healthy young subjects with physiological mitral regurgitation.

We aimed to analyse the echocardiographical characteristics of healthy subjects and determine the causal mechanism of the development of physiological mitral regurgitation (PMR). A total of 130 healthy subjects were divided into two groups according to whether or not PMR was detected. There were no statistical differences between the two groups in terms of the mean values of the systolic and diastolic left ventricular internal diameters and the left atrial diameter. The mean values of the length and thickness of the anterior mitral valve (AMV) and the mitral annular diameter (MAD) from subjects in group 1 (with PMR) were statistically different from those of group 2 (without PMR). In conclusion, the AMV was slightly longer and thicker and the MAD was smaller in subjects with PMR than in subjects without PMR. The changes observed in the AMV and the mitral annulus may play a causal role in the development of PMR.

Assessment of interventricular septal motion using colour tissue Doppler imaging in adult patients with atrial septal defect.

We aimed to characterize changes in interventricular septum (IVS) motion and any relationship between them and the pulmonary-to-systemic flow ratio (Qp/Qs) in adult patients with atrial septal defect (ASD). Patients and controls were studied using colour tissue Doppler imaging (TDI). The septum length (SL) and distance from the colour change point (CCP) on the IVS to the aortic valve (the CCP distance) were measured on parasternal long axis views. Values normalized for body surface area, and the CCP distance to SL ratio, were calculated. Qp/Qs values were correlated with CCP distance, normalized CCP distance and CCP distance:SL ratio. Statistically significant differences in CCP distance, normalized CCP distance and CCP distance:SL ratio were found between the two groups. In the ASD group, there was no correlation between Qp/Qs and the echocardiographic measurements. The point between the motions in two different directions from the IVS shifted toward the apex in ASD patients compared with controls, and may be a mechanism involved in paradoxical septal motion.

Expression of KIR and C-type lectin receptors in Behcet's disease.

OBJECTIVE: Behçet's disease (BD) is a multisystemic disorder with a possible underlying pathology of immune-mediated vasculitis. Genetic susceptibility associated with HLA-B*51 and B*2702 has been implicated in its pathogenesis. Considering the recently defined regulatory mechanisms of NK cells through HLA class I binding receptors, we hypothesized that interactions of NK and T cells through the NK receptors may be important in the pathogenesis of BD. METHODS: The impact of different expression patterns of HLA-recognizing receptors on NK or T cells was analysed in 51 patients with BD and 32 healthy controls. We used flow cytometry to investigate the expression of KIR3DL1 from the polymorphic killer immunoglobulin-like receptor (KIR) family, which binds a shared HLA-Bw4 motif on HLA-B51 and *2702 alleles, and CD94 from the conserved C-type lectin receptor family, which binds HLA-E. Thirty-three of the BD patients and 19 of the controls carried the same HLA-Bw4 motif. RESULTS: CD3(+) T cells were increased in patients with BD compared with controls (81 vs 75%, P = 0.001), whereas the NK cells did not show any difference between the two groups. Increased expression of CD94 in BD was observed on CD16(+)CD56(+) cells (66 vs 57, P = 0.04) and on CD3(+) (7.7 vs 4.0, P < 0.001) and CD3(+)CD56(+) (44 vs 35, P = 0.02) T cells. KIR3DL1 expression on the NK and T cells was not statistically different between the two groups. No effect of HLA-Bw4 motif was observed on the expression of CD94 and KIR3DL1 in both the patients and the controls. CONCLUSION: The absence of a correlation between KIR3DL1 expression and HLA-Bw4 motif confirms previous work reporting that the expression of these molecules is regulated separately. Increased expression of CD94 may suggest that NK receptors play a pathogenic or regulatory role in BD.

Wellens' syndrome: a case report.

The pattern of clinical findings and electrocardiography (ECG) changes known as Wellens' syndrome is associated with significant stenosis of the proximal left anterior descending coronary artery. Cases can be classified according to the ECG pattern into type 1 (biphasic T waves) or type 2 (deeply inverted T waves, especially in leads V2 and V3). We present here an unusual case of Wellens' syndrome in which the ECG pattern changed from type 2 to type 1 during observation, and in which the coronary lesion was in the middle rather than the proximal part of the left anterior descending artery.

Role of homocysteine for thromboembolic complication in patients with non-valvular atrial fibrilation.

Thromboembolism is the most important complication in patients with atrial fibrilation (AF). Homocysteine is a toxic amino acid that has been recently accepted as a risk factor for atherosclerosis and stroke. The aim of the present study is to show whether there is a relation between hyperhomocysteinemia and thromboembolic complications in patients with non-valvular AF. We admitted 38 patients with non-valvular AF. The patients were divided into two groups: group A (n = 20; mean age, 75.7 +/- 10.4 years; three males/17 females), and group B (n = 18; mean age, 68.0 +/- 10.6 years; 11 males/seven females). While group A consisted of the patients with AF and stroke, group B was composed of the patients with AF but without stroke. The patients having sinus rhythm (15 subjects) were used as the reference group to obtain the cut-off value. Homocysteine was measured by the immunoassay method. The means of the homocysteine levels were 12.4 +/- 3.3 micromol/l in group A, 8.3 +/- 2.3 micromol/l in group B and 9.3 +/- 1.8 micromol/l in the reference group. The cut-off value was 10.6 micromol/l. Group A had a statistically higher homocysteine level than not only group B, but also the reference group (P < 0.05). While 60% of group A (n = 12) had the elevated homocysteine level, the rate was only 22% for group B (n = 4). In conclusion, hyperhomocysteinemia may be one of the explanations for the increased rate of thromboembolic complications in older patients with AF.