Platelet-rich plasma efficacy in alopecia areata patients with normal and elevated levels of antibodies against thyroglobulin and thyroid peroxidase.

AIM: To evaluate and compare the efficacy of platelet-rich plasma (PRP) therapy in alopecia areata (AA) patients with normal and with elevated levels of anti-thyroglobulin antibodies and/or anti-thyroid peroxidase antibodies.

Desmoglein autoantibodies and disease severity in pemphigus patients - correlations and discrepancies.

AIM: To assess the correlation between the levels of anti-desmoglein-1 and anti-desmoglein-3 autoantibodies and disease severity in pemphigus patients.

A concise review of flow cytometric methods for minimal residual disease assessment in childhood B-cell precursor acute lymphoblastic leukemia.

Minimal residual disease refers to a leukemia cell population that is resistant to chemotherapy or radiotherapy and leads to disease relapse. The assessment of MRD is crucial for making an accurate prognosis of the disease and for the choice of optimal treatment strategy. Here, we review the advantages and disadvantages of the available genetic and phenotypic methods and focus on the multiparametric flow cytometry as a promising method with greater sensitivity, speed, and standardization options. In addition, we discuss how the application of automated data analysis outweighs the use of complex combinations of windows and gates in classical analysis, thus eliminating subjective evaluation.

Antidepressant-like effect of salidroside and curcumin on the immunoreactivity of rats subjected to a chronic mild stress model.

Deregulated cytokines' production is found in depressed patients. Salidroside and curcumin both have been described with potential antidepressant-like activities. The present study investigated the effect of pure salidroside, curcumin and their combination on the immunoreactivity of animals, subjected to a chronic mild stress (CMS) model, followed by lipopolysaccharide (LPS)-induced inflammation. Wistar male rats were separated in the following six groups: control, CMS model, fluoxetine (2.5 mg/kg, oral), salidroside (5 mg/kg, oral), curcumin (20 mg/kg, oral) and salidroside + curcumin (5 mg/kg + 20 mg/kg, oral). Changes in glucose preference, spatial learning and exploratory behavior were recorded. The IL-6 levels in the rats' sera and of the TNF-α levels in the rats' sera and the brain tissue homogenate were evaluated. The groups exposed to stress and treated with fluoxetine, salidroside, curcumin or salidroside + curcumin showed increase in the glucose preference and locomotor activity, as well as, decrease in the escape latency and the cytokines' levels compared to the CMS model group. The chronic stress induced behavioral alternations and increased cytokines' levels in rats which were reversed by administration of salidroside and curcumin, suggesting antidepressant-like effects comparable to that of fluoxetine and potential synergistic interaction regarding the anti-inflammatory and anti-stress effects.

Association between Type 2 Diabetes, Obesity and Key Immunological Components of IgE-mediated Inflammation.

BACKGROUND: Changes in lifestyle and obesity in recent decades have brought about a dramatic increase in type 2 diabetes mellitus (DM2) and allergic diseases. Clinical and epidemiological studies associate obesity with epidemics of allergic diseases. The link between obesity and DM2 with immunological components of IgE-mediated allergic inflammation is not yet conclusively established. AIM: To examine the key immunological components of IgE-mediated allergic inflammation in patients with DM2 and their relationship with glycemic control and anthropometric indicators. MATERIALS AND METHODS: Fifty-five patients with DM2 and 32 healthy controls with normal weight and body mass index (BMI) of 18-24.9 kg/m2 were included in the study. Th2-cytokine profile (serum levels of IL-4 and IL-5, pg/ml) and total serum IgE IU/ml were assessed in all participants in the study using ELISA. In patients with DM2, levels of glycated hemoglobin (HbA1c%) in the blood were also measured. RESULTS: Serum levels of IL-4 and IL-5 are significantly higher in patients with DM2 compared to the control group. Serum levels of IL-4 and IL-5 positively correlated with BMI as well as serum levels of IL-4 with waist circumference. Total serum IgE positively correlated with HbA1c. CONCLUSION: Obesity and poor glycemic control in patients with DM2 affect key immunological components of IgE-mediated allergic inflammation and possibly alter the immune response to allergens and antigens.

Beneficial Effect of Chronic Treatment with Extracts from Rhodiola Rosea L. and Curcuma Longa L. on the Immunoreactivity of Animals Subjected to a Chronic Mild Stress Model.

BACKGROUND: Recent studies have suggested increased levels of pro-inflammatory cytokines in depression. AIM: The present study aimed to evaluate the effect of extracts from Rhodiola and Curcuma on immunoreactivity of animals subjected to a chronic mild stress (CMS) model followed by lipopolysaccharide-induced inflammation. MATERIALS AND METHODS: Male Wistar rats (n=56) divided in 7 groups were treated orally with: distilled water 10 ml/kg (control and CMS model groups); Rhodiola 250 mg/kg; Rhodiola 500 mg/kg; Curcuma 250 mg/kg; Curcuma 500 mg/kg, Rhodiola 250 mg/kg and Curcuma 250 mg/kg. All groups except the control were stressed daily according to a CMS protocol. Changes in glucose preference, weight gain and locomotor activity were recorded. In the sixth week the animals were challenged with LPS and rats' sera were obtained for ELISA evaluation of TNF-α and IL-6 levels. RESULTS: The animals from the model group decreased their weight gain, glucose preference and locomotor activity compared to controls. The groups exposed to stress and treated with Rhodiola 500 mg/kg, Curcuma 500 mg/kg and their combination increased their locomotor activity compared to the model group. High expression of the pro-inflammatory cytokines TNF-α and IL-6 were found in all groups exposed to CMS and challenged by LPS. CONCLUSIONS: The groups exposed to the stress procedure showed a variety of depression-like behavioral changes. In addition, ELISA tests showed that CMS is affecting rats' immunity by increasing the cytokines' levels. These changes could be reversed by administration of Rhodiola and Curcuma in combination suggesting synergic interaction regarding their anti-inflammatory and anti-stress effects.

Carbapenemase Production of Clinical Isolates Acinetobacter baumannii and Pseudomonas aeruginosa from a Bulgarian University Hospital.

BACKGROUND: Production of Bla OXA-23, OXA-24, OXA-58 and hyperexpression of OXA-51 due to ISAba1 insertion sequence are the leading causes of carbapenem resistance in Acinetobacter baumannii. The loss of OprD transmembrane protein and the overexpression of some effl ux pumps are considered to be the main factors for carbapenem resistance in Pseudomonas aeruginosa whereas metallo-enzymes' production has a secondary role. AIM: Тo examine the carbapenem resistance due to carbapenemase production among clinically signifi cant Gram-negative non-fermenters from St George University hospital, Plovdiv: A. baumannii and P. aeruginosa. MATERIALS AND METHODS: Forty three A. baumannii and 43 P. aeruginosa isolates, resistant or with intermediate resistance to imipenem and/or meropenem were included in the study. They were collected from patients admitted in 14 various hospital wards between 2010 and 2014. Both phenotypic and genetic methods were used for identifi cation and antimicrobial susceptibility testing. RESULTS: All A. baumannii demonstrated carbapenemase production determined by a modifi ed Hodge test whereas P. aeruginosa isolates did not show this phenomenon. OXA-23 genes were determined in 97.7% (42 out of 43) of A. baumannii isolates indistinguishable from the sequence of the classical ARI-1 gene. OXA-24, OXA-58 and overexpression of OXA-51 were not registered in any of the isolates. All P. aeruginosa were negative for blaVIM and blaIMP genes. CONCLUSION: The leading cause of carbapenem resistance in A. baumannii isolates from our hospital is the carbapenemase production due to the expression of OXA- 23 gene, whereas in P. aeruginosa - the loss of transmembrane OprD protein and the effl ux pumps' hyperexpression are suspected to be the main mechanisms.

Immunophenotypic Modulation of the Blast Cells in Childhood Acute Lymphoblastic Leukemia Minimal Residual Disease Detection.

UNLABELLED: Early clearance of leukemic cells during induction therapy of childhood acute lymphoblastic leukemia (ALL) is a basis for treatment optimization. Currently, the most widely used methods for the detection of minute residual malignant cells in the bone marrow and/or peripheral blood, minimal residual disease (MRD), are PCR and flow cytometry (FCM). Immunophenotypic modulation (IM) is a well known factor that can hamper the accurate FCM analysis. AIM: To report the IM detected by 8-color FCM during the BFM-type remission induction in 24 consecutive MRD-positive samples of children with B-cell precursor ALL and the possible implications for MRD detection. PATIENTS AND METHODS: Between 2010 and 2012 we prospectively followed up the MRD on days 15 and 33 of induction treatment in bone marrow (BM) samples and on day 8 in peripheral blood (PB). The IM was assessed by comparative analyses of the changes in the mean fluorescence intensity of 7 highly relevant antigens expressed by the leukemic cells and normal B-lymphocytes. RESULTS: IM occurred, to different extents, in all analyzed day 15 BM and in most day 33 BM samples. Statistically significant changes in the MFI-levels of four CDs expressed by the leukemic blasts were observed: downmodulation of CD10, CD19 and CD34 and upmodulation of CD20. No changes in the expression of CD38, CD58 and CD45 were noticed. CONCLUSIONS: Measuring the MRD by standardized 8-color flow cytometry helps improve the monitoring of the disease, leading to better therapeutic results. However, the IM of the different antigens expressed by the leukemic blasts should be taken into consideration and cautiously analyzed.

Micromeritic procedures in assessing antinuclear antibody patterns in immunofluorescent assay.

AIM: The aim of this study was to introduce a micromeritic procedure (a statistical approach for small objects) in indirect immunofluorescence assay (IFA) to find objective quantitative parameters of antinuclear antibody (ANA) patterns which could support a diagnosis of auto-immune diseases. MATERIALS AND METHODS: Sera of patients with systemic autoimmune diseases, McCoy-Plovdiv serum-free cell line, goat anti-human immunoglobulin-G FITC-conjugate, fluorescent microscope, computer-assisted digital image processing, analysis using a micromeritic procedure, ANOVA. RESULTS: Three ANA fluorescent patterns (homogeneous, rim and speckled) were analyzed by the micromeritic procedure. Parameters for the image brightness of the pixels (pixel grey value) were obtained and discussed as objective characteristics of fluorescent patterns: maximum ANA-linkage volume and surface density were established for the objects with speckled localization pattern. CONCLUSION: The micromeritic method for getting objective quantitative values of ANA fluorescent patterns in indirect immunofluorescent assay might be a valuable tool aiding in immunological diagnosing if integrated in a laboratory software package.

Levels of certain endothelial biomarkers during the acute phase and convalescence in patients with different severity of Mediterranean spotted fever.

INTRODUCTION: Mediterranean spotted fever (MSF) is a tick-borne disease caused by Rickettsia conorii subspp. conorii. It is transmitted by the bite of the tick Rhipicephalus sanguineus. Modified by the rickettsial invasion, the micro-vascular endothelium acquires an activated inflammatory phenotype and initiates secretion of cytokines and expression of cell adhesion molecules (CAMs) and chemoattractans. AIM: This study aims at investigating the alterations in the soluble cellular adhesion molecules (sCAMs) and chemokine MCP-1 levels in patients with MSF of varying severity in the acute and convalescence stage in order to assess their diagnostic and prognostic value. MATERIALS AND METHODS: The soluble forms of cellular adhesion molecules (sCAMs)--sE-selectin and sP-selectin, the intercellular (sICAM-1) and vascular (sVCAM-1) adhesion molecules as well as the monocyte chemoattractant protein-1 (MCP-1) were studied in the sera of 80 patients with MSF. The presence of MSF was confirmed serologically by indirect fluorescence assay (IFA). In order to study disease dynamics, serum samples from 80 patients were drawn on day 1 following the onset of rash; in 60 patients (part of the surveyed 80) a second sample was taken in the convalescence period--14 days post hospital discharge. The investigation was focused on mild, moderate and severe forms of MSF. Enzyme linked immune-sorbent assay was used for sCAMs determination (Quantikine IVD colorimetric RESULTS: Overall, in the acute stage, patients presented with increased levels of sE-selectin, sICAM-1, sVCAM-1 and MCP-1, whereas sP-selectin level was decreased. The levels of sE-selectin, sICAM-1 and sVCAM-1 were significantly elevated in mild, moderate and severe forms of the disease with sE-selectin level exhibiting a plateau tendency and sICAM and sVCAM levels demonstrating an upward trend from mild towards severe MSF forms. MCP-1 level was elevated only in severe MSF. In all forms of MSF, in the convalescence period, sICAM-1, sVCAM-1 and MCP-1 concentration returned to reference levels whereas sE-selectin level persisted elevated. In the convalescence stage, sP- selectin concentration also showed an upward tendency, which in severe forms of MSF slightly exceeded the level in controls. sP-selectin levels correlated directly with platelet count, whereas sICAM-1 and sVCAM-1 levels showed a reverse correlation, sE-selectin, sICAM-1 and MCP-1 levels directly correlated with aminotransferase activity (ALT and/or AST). CONCLUSION: The soluble forms of CAMs reflect the endothelial inflammatory potential. There is evidence that endothelium activation is more potent in severe forms of MSF. Assessment of the endothelial response in the course of the disease is an important predictor of the outcome, the choice of therapeutic approach and disease prognosis.

Clinical and laboratory study of pro-inflammatory and antiinflammatory cytokines in women with multiple sclerosis.

UNLABELLED: Multiple sclerosis (MS) is an autoimmune demyelinating disorder of the central nervous system characterised with a complex system of interactions between proinflammatory and anti-inflammatory cytokines in its course. AIM: The aim of the present study was to investigate the serum levels of cytokines TNF-a, IFN-gamma, IL-4 and IL-10 in female patients with MS and healthy individuals, the changes occurring in the relapse and remission phases of the disease and their correlation with the severity of the neurological deficit. PATIENTS AND METHODS: Thirty-five women with relapsing-remitting MS were examined. The patients' age ranged between 18 and 50 years and MS was verified clinically and by magnetic resonance imaging according to the McDonald criteria. Thirteen of the patients were treated with interferon-beta-1b. The serum concentrations of TNF-a, IFN-y, IL-4 and IL-10 were determined twice - in relapse and in remission - using an enzyme-linked immunosorbent assay (ELISA). The control group consisted of 35 age-matched healthy females. RESULTS: The comparison of cytokine serum concentrations during the two phases of the disease showed significant elevation of the TNF-alpha serum levels in the relapse phase and of IL-4 - in the remission phase. The comparison between the patients and the healthy control subjects demonstrated statistically significant lower concentrations of TNF-a in remission patients and higher concentrations of IL-10 in relapse patients. The patients with interferon-beta-lb treatment showed different profile of cytokine secretion from the patients without interferon-beta-1b treatment. Interferon-beta-1b-treated patients showed significantly lower serum levels of TNF-a and IFN-gamma during the relapse phase and higher TNF-a and IL-10 serum levels during the remission phase compared with the untreated patients. CONCLUSIONS: Serum levels of TNF-a and IL-4 objectively reflect the immune response during relapse and remission of the disease. The severity of neurological deficit as estimated with the expanded disability status scale (EDSS) does not depend on the serum levels of TNF-a, IL-10 and IFN-gamma in the two phases of MS.

Dynamics in serum cytokine responses during acute and convalescent stages of Mediterranean spotted fever.

UNLABELLED: Mediterranean spotted fever (MSF) is a re-emerging rickettsiosis in Bulgaria after 20 years of absence (1972-1992), and it has since been affecting many people annually in the endemic regions of the country. The role of cytokines in MSF is still in the focus of research due to their complex participation in the immune pathogenesis of the disease. AIM: To study the changes in the serum cytokine concentrations in MSF patients. PATIENDS AND METHODS: Eighty patients with MSF and 20 healthy controls were enrolled in the study. The pro-inflammatory and immunoregulatory cytokines IL-1beta, TNF-a, IL-6, IL-8, IL-12, IFN-gamma, IL-2, and IL-10 were studied in the burst of disease, at clinical recovery stage, and two weeks later. The disease etiology was verified by indirect IFA in the Referral Rickettsiosis Laboratory. The cytokine levels were determined by ELISA (BioSource Europe S.A). RESULTS: In the disease flare up patients showed a manifold increase in the activity of IL-1beta (p < 0.01), TNF-alpha (p < 0.001), IL-6 (p < 0.001), and IL-8 (p < 0.001) compared with the controls. Significant elevation in IFN-gamma and IL-12 values (p < 0.001) was also found. The increase in the immunoregulatory IL-10 also reached statistical significance (p < 0.001), while the rise in IL-2 did not (p > 0.05). Followed in dynamics, only IL-1beta and IL-6 measured up the control levels at the time of clinical recovery. Two weeks later, in the early convalescence IL-12 and TNF-alpha further diminished but did not normalize their values. CONCLUSION: Our findings show that MSF is characterized by a Th1 cytokine profile. The patient's immune system responds by proinflammatory and immunoregulatory cytokine production that accompanies the rickettsial vasculitis and contributes to the healing process. The latter is probably not fully achieved in the early convalescent period, according to our data concerning some pro-inflammatory cytokines' elevation at this period.

A comparative study of immunological methods for determination of serum antinuclear antibodies.

INTRODUCTION: Several immunological methods are used to determine the serum antinuclear antibodies (ANA). Indirect immunofluorescence assay (IFA) with tissue slices or HEp-2 cells is the standard technique considered the gold standard for their screening. Serum-free McCoy-Plovdiv cell line may also be used as substrate for IFA. Another method for detection of total and specific ANA is the enzyme-linked immunosorbent assay (ELISA). Immunoblot is also applied in specific ANA confirmation. The aim of the current study was to determine and propose a justified immunological approach for identification of clinically significant ANA by comparing the screening tests - ANA-IFA on serum-free McCoy-Plovdiv cell substrate with ELISA for total ANA, and confirmative methods for specific ANA-ELISA with immunoblot. MATERIALS AND METHODS: Serum samples from 38 patients screened for totalANA by ELISA (Trinity Biotech, NY, USA) and IFA-ANA with McCoy-Plovdiv cell line, were included in the study. Positive samples were confirmed by immunoblot (Orgentec Diagnostika, Germany) and ELISA for specificity of confirmed ANA. RESULTS: No significant difference (P > 0.05) and very good agreement were found between the two screening tests. Very good agreement for specific antibodies against SS-A, SS-B, dsDNA, moderate for anti-Sm and anti-Sm/RNP and fair for anti-histone/nucleosomal antibodies was found between confirmative methods. No agreement was found for anti-Scl-70 antibodies. CONCLUSION: IFA-ANA with serum-free McCoy-Plovdiv cell line and screening ELISA may be recommended for determination of total ANA, and immunoblot and ELISA- for confirmation and identification of specific ANA.

Immunopathological characteristics of cryptoglandular and Crohn's anorectal fistulas.

Anorectal fistulas are quite common proctologic disorders. They can be either of cryptoglandular origin or can be associated with Crohn's disease and chronic ileocolitis. Mechanical obstruction and local infections are prime causes of this pathological condition. Genetic predisposition and inadequate immune response with overproduction of pro-inflammatory cytokines appear prominently in the course of Crohn's disease. Interferon-gamma, a Th1 type cytokine, reflecting the engagement of cellular immune mechanisms, is the first to be produced in the intestinal mucosa. The inflammatory process in the colon mucosa induced by the abundant microbial flora is sustained and turned chronic by the gradual elevation of the local TNF-a and regulatory cytokines levels (interleukin-10, transforming growth factor-beta). The number of activated local memory T cells CD4+CD45RBl0 increases significantly. The regulatory CD4+CD25+ T lymphocytes producing interleukin-10 increase also trying to counterbalance the cytokine reaction. The chronic inflammatory infiltrates of the colon mucosa are represented by lymphocytes, plasma cells, macrophages. The long-term activation of macrophages by the released interferon-gamma leads to tissue damage and potentiation of angiogenesis--a risk factor for carcinoma development. Management of anorectal abscesses and fistulas is complex aiming to alleviate the symptoms, prevent relapses, reduce the risk of sphincter damage and improve quality of life. The main approach (surgery) should be combined with antimicrobial infection control and immunomodulation by intravenous or local administration of anti-TNF-alpha antibodies.