Bone marrow-derived mesenchymal stem cells alleviate paclitaxel-induced mechanical allodynia in rats.

Anticancer drug paclitaxel (PTX) frequently causes painful peripheral neuropathy; however, no medication has been shown to be effective in the treatment of this debilitating side effect. We aimed to investigate the efficacy of two different doses of allogeneic bone marrow-derived mesenchymal stem cells (BM-MSCs) on PTX-induced mechanical allodynia and spinal cytokine levels and their localization to target tissues such as the spinal cord and sciatic nerve. After the development of mechanical allodynia with repeated PTX administration, two different doses of rat BM-MSCs, low or high (1 × 106 -5 × 106 ), were transplanted into rats and the evaluation continued for 30 days. Interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and IL-10 levels in spinal cord samples of animals were analyzed by enzyme-linked immunosorbent assay. PTX-induced mechanical allodynia was relieved significantly 15 days after the transplantation of high-dose of BM-MSCs. Both MSCs doses were effective in alleviating allodynia, but the onset of effect was earlier with the high dose. High-dose of BM-MSCs significantly decreased spinal IL-1ß and TNF-α levels compared to the PTX group. Fluorescent dye-labeled BM-MSCs were observed much more frequently in the sciatic nerve and spinal cord samples of the high-dose BM-MSCs transplanted group than in the low-dose group animals. In conclusion, we found that the antiallodynic effects of BM-MSCs appeared earlier when high-dose of cells were administered. We think that other mechanisms may play a role in the effects of MSCs, besides localization to damaged tissues and reducing spinal inflammatory cytokine levels. We show that BM-MSCs can be a novel approach in PTX-induced mechanical allodynia.

Flow cytometric analysis of platelet surface glycoproteins in the diagnosis of thirty-two Turkish patients with Glanzmann thrombasthenia: a multicenter experience

Background/aim: Glanzmann thrombasthenia (GT) is a rare autosomal recessively inherited bleeding disorder characterized by the quantitative (type 1 and type 2) or qualitative (type 3) deficiency in platelet membrane glycoprotein (GP) IIb/IIIa (CD41a/CD61) fibrinogen receptors. In type 1, 2, and 3, CD41a/CD61 expression is 5%, 5%­20% and above 20%, respectively. In this study, diagnosis of GT was confirmed and subgroups were identified in 32 Turkish patients by flow cytometry analysis. Materials and methods: CD41a/CD61 expression levels in platelet-rich plasma (PRP) obtained from peripheral venous EDTA blood samples were analyzed with a BD FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). GT subgroup analysis was performed by counting 50,000 events in the BD FACSDiva Software v6.1.3 program of the instrument. Results: In the present study, in blood samples of 32 patients from 23 families with GT and 22 healthy controls, co-expression levels of CD41a and CD61 in PRP was analyzed. 12 out of 23 families were consistent with type 1 GT (52.2%), 4 were consistent with type 2 GT (17.4%), and 7 were consistent with type 3 GT (30.4%). Conclusion: Especially due to consanguineous marriages, GT with various glycoprotein levels may be detected. As a result of the flow cytometry analysis of the present study with the highest GT patient population in Turkey, type 1 GT patients were the most common subgroup. In the determination of the GT subgroups; especially in the detection of type 3 GT, flow cytometry is the most sensitive glycoprotein analysis method. In addition to light transmission aggregometry, CD41a/CD61 study by flow cytometer confirms diagnosis when mutation analysis cannot be performed.

Diagnostic Modalities Based on Flow Cytometry for Chronic Granulomatous Disease: A Multicenter Study in a Well-Defined Cohort.

BACKGROUND: Chronic granulomatous disease (CGD) is characterized by defective microbial killing due to mutations affecting subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. Definitive genetic identification of disease subtypes may be delayed or not readily available. OBJECTIVE: Sought to investigate the role of intracellular staining of NADPH oxidase enzyme subunits in predicting the respective genetic defects in patients with CGD and carriers. METHODS: Thirty-four patients with genetically inherited CGD, including 12 patients with X-linked CGD (gp91phagocyte oxidase (phox) deficiency due to cytochrome b-245, beta polypeptide [CYBB] mutations) and 22 patients with autosomal-recessive CGD (p22phox, p47phox, and p67phox deficiency due to cytochrome b-245, alpha polypeptide [CYBA], neutrophil cytosolic factor 1 [NCF1] and NCF2 mutations, respectively) were recruited from different immunology centers and followed up prospectively. Dihydrorhodamine testing and NADPH oxidase subunit expression in white blood cells were determined by flow cytometry. RESULTS: gp91phox and p22phox defects, which result in simultaneous loss of both proteins due to their complex formation, were differentiated only by comparative analysis of patients' and mothers' intracellular staining. p47phox and p67phox protein expression was almost undetectable in patients compared with carrier mothers and healthy controls. The expression values of the respective subunits were found to be significantly higher in all controls as compared with carrier mothers, which in turn were higher than those of patients. CONCLUSIONS: Analysis of NADPH oxidase enzyme subunits by flow cytometry in patients and carriers is useful in the rapid prediction of the genetic defect of patients with CGD, thus guiding targeted sequencing and aiding in their early diagnosis.

Seven chronic granulomatous disease cases in a single-center experience and a review of the literature.

BACKGROUND: Chronic granulomatous disease (CGD) is a rare primary immunodeficiency caused by defects in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme system. This disease causes the disordered functioning of phagocytic cells. It is characterized by life-threatening and/or recurrent infections by bacteria and fungi. CGD has both an X-linked recessive (X-CGD) and autosomal recessive (AR-CGD) phenotypes. AR form have four subtypes including defects with one of these NADPH oxidase components (p22, p40, p47 and p67phox). OBJECTIVES: To report the clinical and laboratory characteristics of seven CGD patients based on their genetic characteristics. METHODS: Seven boys with CGD were reviewed based on clinical findings and genetic results. Dihydrorhodamine-1,2,3 (DHR) assay was used as a diagnostic test. Genetic analysis was conducted to establish moleculer diagnoses in all patients. RESULTS: The age of diagnosis varied between 1.5 years and 15 years. The most frequent clinical presentation was pneumonia, and two patients had BCG-itis. Four patients had the AR-CGD phenotype, and three patients had the X-CGD phenotype. Severe invasive infections due to Aspergillus, Staphylococcus, and Serratia species were reported. Frequent lung and lymph node involvement was observed during follow-up of the cases. CONCLUSIONS: CGD is life-threatening disease that involves deep-seated infection. In our patients, the most commonly affected organs were the lungs and lymph nodes. Phagocytic disorders should be considered in cases of recurrent infectious diseases, invasive fungal diseases, BCG complications that are not self-limiting, unexplained lymphadenitis or osteomyelitis, and chronic inflammatory disorders.

Performance of Galactomannan Antigen, Beta-d-Glucan, and Aspergillus-Lateral-Flow Device for the Diagnosis of Invasive Aspergillosis.

Aspergillus lateral-flow device (LFD) was recently introduced as a practical tool for the diagnosis of invasive aspergillosis (IA). We investigated the performance of Aspergillus-LFD as a point-of-care test for the diagnosis of IA. Serum samples were collected twice weekly from patients who received intensive chemotherapy for acute leukemia, or recepients of allogeneic stem cell transplantation. Aspergillus galactomannan (GM) antigen, 1,3-beta-d-glucan and Aspergillus-LFD tests were carried out according to manufacturers' recommendations. GM testing was repeated with a modified procedure which was proven to increase the sensitivity. Aspergillus-LFD was performed without applying any pretreatment procedure to allow the kit to fit as a point-of-care test. Fungal infections were categorized according to European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) criteria. A total of 75 neutropenia episodes in 64 patients were prospectively followed between February 2012 and January 2013. Probable IA was diagnosed in 11 patients, probable pulmonary fungal disease was diagnosed in one patient, and rhinocerebral aspergillosis was diagnosed in one patient. Fungemia was detected in two patients. Aspergillus-LFD was positive in serum of a patient with probable IA and in the bronchoalveolar lavage fluid of an other patient with probable IA. Aspergillus-LFD was false positive in serum of two patients. Although there was no radiological finding of IA or documented fungemia, fever resolved after empirical caspofungin therapy in one of these patients. The sensitivity of Aspergillus-LFD as a point-of-care test without any pretreatment of serum sample is low.

Increased plasma soluble human leukocyte antigen-G in persistent wheezy infants.

BACKGROUND: Human leukocyte antigen (HLA)-G is a non-classical major histocompatibility complex class I antigen characterized by limited polymorphism in its coding region, unique tissue expression pattern in physiologic conditions and immunomodulatory properties. Recently, the level of soluble (s)HLA-G was found to be higher in atopic asthma and allergic rhinitis, but this remains to be clarified in wheezy infants. The aim of the present study was therefore to investigate sHLA-G in wheezy infants. METHODS: The subjects consisted of infants with persistent wheezing and positive modified asthma predictive index (mAPI; n = 30; persistent group) and those with transient wheezing and negative mAPI (n = 17; transient group). sHLA-G was measured in plasma using enzyme-linked immunosorbent assay. Total immunoglobulin E (IgE) and eosinophil count were measured, and skin testing was performed with a battery of 13 antigens with appropriate positive and negative controls. RESULTS: sHLA-G was significantly higher in the persistent wheezing (positive mAPI) group compared with the transient wheezing (negative mAPI) group (P = 0.008). There was no significant difference in peripheral blood eosinophil count and total IgE between the groups. CONCLUSIONS: The increased sHLA-G in infants with persistent wheeze suggests that sHLA-G may be able to be used to distinguish persistent from transient wheeze. Further comprehensive studies are needed on this topic.

In the diagnosis of neonatal sepsis importance of gelsolin and relationship with mortality and morbidity.

In spite of advances in neonatal care and the new generation of antibiotics, neonatal sepsis is still a major cause of morbidity and mortality. Early diagnosis of neonatal sepsis is difficult because clinical signs are non-specific. Thus, new biomarkers are still needed for diagnosis. Gelsolin is an actin-binding plasma protein. Furthermore, extracellular gelsolin binds lipopolysaccharide and lipoteichoic acid, which are major virulence factors of Gram-negative and Gram-positive bacteria. The result of this binding is the inhibition of gelsolin's F-actin depolymerizing activity. Thus, gelsolin inhibits the release of IL-8 from human neutrophils subjected to lipoteichoic acid, lipopolysaccharide and heat-inactivated bacteria treatment. Our hypothesis is that pGSN levels decrease in neonatal infants with sepsis and this decrease might be used as a reliable biological marker. Forty patients who were diagnosed with severe sepsis at a neonatal intensive care unit were enrolled in the sepsis group. Twenty patients who were followed for prematurity were enrolled in the control group. The pGSN level at the time of diagnosis in the sepsis group was 33.98±11.44µg/ml, which was significantly lower than that of control group (60.05±11.3µg/ml, P<0.001) and after treatment (53.38±31.26µg/ml, P=0.003). Area under ROC curve was 0.96 (p: 0.0001, 95% CI; 0.90-0.99). Sensitivity was 90.32 (95% CI; 74.2-97.8), specificity was 95 (95% CI; 75.1-99.2). Plasma gelsolin significantly decreased in septic patient and recovery of decreased gelsolin levels correlated with clinical improvement. Thus, plasma gelsolin may be a usable marker for severe sepsis.

The association of endothelial progenitor cell markers with arteriovenous fistula maturation in hemodialysis patients.

AIMS: Arteriovenous fistula (AVF) failure is one of the most important clinical problems in end-stage renal disease. Endothelial progenitor cells (EPCs) have a role on vascular angiogenesis and endothelialization. We aimed to investigate the association markers of EPCs on AVF maturation by measuring the surface expressions of CD34, CD309 and CD133 on the monocytes. METHODS: This prospective observational study was conducted in 54 voluntary patients with end-stage renal disease who were admitted for their first renal replacement therapy and were available for AVF creation. Venography was performed in all patients before AVF creation. Six patients were excluded due to inadequate veins after venographic imaging, and also seven patients were excluded due to postoperative thrombosis. The blood samples were analyzed a day before the fistula operation, and the expressions of CD34, CD133 and CD309 on the surface of monocytes were measured. RESULTS: Patients were divided into two groups after the evaluation of AVF maturation, as the mature group and the failure group. The CD309 expression level on the monocytes was 338.00 (35.00-479.00) in the mature group; however, it was 36.00 (5.50-237.00) (p 0.031) in the failure group. Multiple logistic regression analyses showed that both BMI and the mean fluorescence intensity level of CD309 expression on monocytes independently predicted AVF maturation. CONCLUSIONS: The presence of DM and increased BMI negatively correlated with AVF maturation. High intensity of CD309 expression on monocytes was observed in patients with successful AVF maturation.

Serum Visfatin Levels in Ulcerative Colitis.

BACKGROUND: Previous studies have suggested that adipokines play a role in inflammatory bowel disease by inducing proinflammatory cytokines, but it is uncertain whether visfatin is causally involved in ulcerative colitis (UC). We evaluated visfatin levels in patients who presented with UC flares before and after treatment. METHODS: In this cohort study, we assessed 31 patients with UC in the activation period and remission in the same patients after treatment, and a healthy control group, consisting of 29 persons, at a single academic medical centre between 2010 and 2013. Disease severity was evaluated clinically using Trulove and Witt's criteria. RESULTS: Serum visfatin levels did not vary according to the extent of disease and were significantly higher in patients in the activation period (7.77 ± 2.41 ng/ml) than in remission (6.18 ± 2.04 ng/ml) and the healthy controls (6.54 ± 2.20 ng/ml; P < 0.01 and < 0.05, respectively). In a comparison of patients in the inactive period with the control group, there was no statistically significant difference (P > 0.05). To assess activation of the disease, a visfatin cut-off point for active UC was determined as 6.40, with sensitivity, specificity, positive predictive value (PPV) and negative predictive values (NPV) of 72%, 52%, 66.7% (43.0-85.4) and 50.0% (29.1-70.9), respectively. CONCLUSIONS: The visfatin level was higher in the active group than in post-treatment remission and the healthy control group. Sensitivity and specificity were similar to other inflammatory markers for assessing clinical activity, which did not improve clinical outcomes in patients with acute respiratory distress syndrome (ARDS). These findings did not provide a rationale for assessment of UC activation.

Evaluation of two different adjuvants with immunogenic uroplakin 3A-derived peptide for their ability to evoke an immune response in mice.

RATIONALE: Organ- or tissue-specific antigens produced by normal tissue or by cancer cells could be used in cancer immunotherapy, to target the tumor. In our previous study, we induced T-cell-mediated, bladder-specific autoimmunity by targeting the bladder-specific protein Uroplakin 3A (UPK3A). UPK3A is a well-chosen target for developing an autoimmune response against bladder cancer since the antigen is also expressed in bladder tumors. To use this peptide, which was derived from the UPK3A protein in a bladder cancer vaccine study, it is necessary to induce a strong immune response. In this study, we aimed to develop a robust immune response in BALB/c mice using the well-characterized keyhole limpet hemocyanin (KLH)-conjugated peptide antigen (UPK3A 65-84) conjugated with an immunogenic carrier protein. In combination with the peptide, we used either Freund's complete adjuvant (CFA) or CpG (cytosine-phosphate-guanine oligonucleotides) as effective adjuvants in order to overcome tumor tolerance. OBJECTIVES: The immune response evoked by UPK3A 65-84 peptide, using two different adjuvants, was compared by detection of changes in the proliferative response of immune cells, in the cytokine profile, and in the immune cell populations. FINDINGS: We demonstrated that CpG, combined with KLH-UPK3A 65-84, promoted a more robust immune response, via induction of higher IL-2, IFN-γ, TNF-α, IL-17 production and activation of more immune cells (CD4(+) T cells, CD8(+) T cells, NK cells CD11b, CD45), than CFA and the KLH- UPK3A 65-84. CONCLUSION: CpG as an adjuvant combined with KLH-UPK3A 65-84 could be used in preclinical models of bladder cancer for the development of cancer immunotherapy strategies.

Decreased levels of lipoxin A4 and annexin A1 in wheezy infants.

OBJECTIVE: Wheezing is a common and challenging health issue in infancy and early childhood. Asthma diagnosis is frequent in patients with a history of recurrent wheezing. A relationship has been reported between asthma and anti-inflammatory mediators such as lipoxin A4 and annexin A1. However, this remains uncertain in wheezy infants. The aim of the present study was to determine lipoxin A4 and annexin A1 levels in wheezy infants. MATERIALS AND METHODS: Eighty-seven patients aged 6-36 months were included in this study. Demographic characteristics, clinical features, laboratory data, clinical diagnoses, and treatments, if present, were recorded. Patients were divided into 2 groups: patients with wheezing (n = 59) and healthy controls (n = 28). Blood samples were taken and lipoxin A4 and annexin A1 levels were evaluated by ELISA. RESULTS: Lipoxin A4 and annexin A1 levels were significantly lower in the wheezing group than in the control group (p < 0.05). A significant correlation was found between the serum total immunoglobulin E (IgE) level and the percentage and absolute number of eosinophils (p < 0.05). No significant correlation was found in terms of lipoxin A4 and annexin A1 levels, the serum total IgE level, and the percentage and absolute number of eosinophils among groups (p > 0.05). CONCLUSION: This is the first study to assess lipoxin A4 and annexin A1 levels in wheezy infants. The levels of lipoxin A4 and annexin A1 were found to be low in wheezy infants. We hope that these results will lead to novel therapeutic options for asthma in cases where an optimal treatment modality is lacking.