The actual burden and causative agent of tuberculous lymphadenitis (TBLN) cases is not well known due to lack of strong surveillance system and diagnostic facilities in Ethiopia. This study was conducted to determine the prevalence of TBLN, its causative agent and risk factors for acquiring this infection.
A cross-sectional study was conducted from April to May 2012 at four main hospitals and one diagnostic clinic located in northern Ethiopia. Fine needle aspirates (FNAs) from TBLN suspects were taken for acid fast bacilli (AFB) microscopy, culture and molecular typing.
Among 437 aspirates, culture yielded AFB in 226 (51.7%) of cases. Sixty one culture negative cases (30.5% of 200 cases) were positive by Xpert MTB/RIF test. Moreover, a rifampicin resistant AFB was detected from culture negative cases. The overall prevalence of FNAs positive TBLN cases was 65.8 %. The BacT/AlerT 3D system proved to be a more rapid method with higher recovery rate than Lowenstein-Jensen (L-J) and/or Gottsacker media (P<0.0001). Molecular typing identified all culture positive isolates as M.tuberculosis. The main risk factors for TBLN were pediatric age (OR 2.8, 95% CI, 1.09- 7.05) and cough (OR 2, 95%CI, 1.09-3.7).
The results of this study revealed a high prevalence of TBLN in the study sites and that pediatric age and cough are key predictors of the disease. TBLN is an important public health problem that needs to be addressed in the area. It is important to note that MDR strains of TB could be involved and aetiological confirmation and drug sensitivity testing of TBLN isolates should be expanded. Further studies on the M.tuberculosis lineages, circulating strains and transmission dynamics, are recommended.
The interplay between immune and nervous systems plays a pivotal role in the pathophysiology of depression. In depressive episodes, patients show increased production of pro-inflammatory cytokines such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α. There is limited information on the effect of antidepressant drugs on cytokines, most studies report on a limited sample of cytokines and none have reported effects on IL-22. We systematically investigated the effect of three antidepressant drugs, citalopram, escitalopram and mirtazapine, on secretion of cytokines IL-1β, IL-2, IL-4, IL-6, IL-17, IL-22 and TNF-α in a whole blood assay in vitro, using murine anti-human CD3 monoclonal antibody OKT3, and 5C3 monoclonal antibody against CD40, to stimulate T and B cells respectively. Citalopram increased production of IL-1β, IL-6, TNF-α and IL-22. Mirtazapine increased IL-1β, TNF-α and IL-22. Escitalopram decreased IL-17 levels. The influence of antidepressants on IL-2 and IL-4 levels was not significant for all three drugs. Compared to escitalopram, citalopram led to higher levels of IL-1β, IL-6, IL-17 and IL-22; and mirtazapine to higher levels of IL-1β, IL-17, IL-22 and TNF-α. Mirtazapine and citalopram increased IL-22 production. The differing profile of cytokine production may relate to differences in therapeutic effects, risk of relapse and side effects.
cytokines; depression; antidepressants; citalopram; escitalopram; mirtazapine
Extensive research in recent years suggests that exposure to xenobiotic stimuli plays a critical role in autoimmunity induction and severity and that the resulting response would be exacerbated in individuals with an infection-aroused immune system. In this context, heavy metals constitute a prominent category of xenobiotic substances, known to alter divergent immune cell responses in accidentally and occupationally exposed individuals, thereby increasing the susceptibility to autoimmunity and cancer, especially when accompanied by inflammation-triggered persistent sensitization. This perception is learned from experimental models of infection and epidemiologic studies and clearly underscores the interplay of exposure to such immunomodulatory elements with pre- or postexposure infectious events. Further, the TH17 cell subset, known to be associated with a growing list of autoimmune manifestations, may be the “superstar” at the interface of xenobiotic exposure and autoimmunity. In this review, the most recently established links to this nomination are short-listed to create a framework to better understand new insights into TH17's contributions to autoimmunity.
Although Ethiopia ranks seventh among the world’s 22 high-burden tuberculosis (TB) countries, little is known about strain diversity and transmission. In this study, we present the first in-depth analysis of the population structure and transmission dynamics of Mycobacterium tuberculosis strains from Northwest Ethiopia.
In the present study, 244 M. tuberculosis isolates where analysed by mycobacterial interspersed repetitive unit - variable number tandem repeat 24-loci typing and spoligotyping methods to determine phylogenetic lineages and perform cluster analysis. Clusters of strains with identical genotyping patterns were considered as an indicator for the recent transmission.
Of 244 isolates, 59.0% were classified into nine previously described lineages: Dehli/CAS (38.9%), Haarlem (8.6%), Ural (3.3%), LAM (3.3%), TUR (2.0%), X-type (1.2%), S-type (0.8%), Beijing (0.4%) and Uganda II (0.4%). Interestingly, 31.6% of the strains were grouped into four new lineages and were named as Ethiopia_3 (13.1%), Ethiopia_1 (7.8%), Ethiopia_H37Rv like (7.0%) and Ethiopia_2 (3.7%) lineages. The remaining 9.4% of the isolates could not be assigned to the known or new lineages. Overall, 45.1% of the isolates were grouped in clusters, indicating a high rate of recent transmission.
This study confirms a highly diverse M. tuberculosis population structure, the presence of new phylogenetic lineages and a predominance of the Dehli/CAS lineage in Northwest Ethiopia. The high rate of recent transmission indicates defects of the TB control program in Northwest Ethiopia. This emphasizes the importance of strengthening laboratory diagnosis of TB, intensified case finding and treatment of TB patients to interrupt the chain of transmission.
Mycobacterium tuberculosis; Molecular epidemiology; Transmission dynamics
Objective: Resistance to drugs is due to particular genomic mutations in the specific genes of Mycobacterium tuberculosis. Timely genetic characterization will allow identification of resistance mutations that will optimize an effective antibiotic treatment regimen. We determine the magnitude of gene mutations conferring resistance to isoniazid (INH), rifampicin (RMP) and ethambutol (EMB) among tuberculosis (TB) lymphadenitis patients.
Methods: A cross sectional prospective study was conducted among 226 M.tuberculosis isolates from culture positive lymph node aspirates collected from TB lymphadenitis patients between April 2012 and May 2012. Detection of mutations conferring resistance to drugs was carried out using GenoType® MTBDRplus and GenoType® MTBDRsl assay.
Results: Out of the 226 strains, mutations conferring resistance to INH, RMP, multidrug resistance tuberculosis (MDR-TB) and EMB were 8, 3, 2 and 2 isolates, respectively. There was no isolated strain that showed mutation in the inhA promoter region gene. All INH resistant strains had mutations in the katG gene at codon 315 with amino acid change of S315T1. Among rifampicin resistant strains, two isolates displayed mutations at codon 531 in the rpoB gene with amino acid change of S531L and one isolate was by omission of wild type probes at Q513L. According to mutations associated with ethambutol resistance, all of the isolates had mutations in the embB gene with aminoacid change of M306I. All isolates resistant to INH, RMP and MDR using BacT/AlerT 3D system were correctly identified by GenoType® MTBDRplus assay.
Conclusion: We observed mutations conferring resistance to INH at S315T1 of the katG gene, RMP at S531L and Q513L in the rpoB genes and EMB at M306I of the embB gene. In the absence of conventional drug susceptibility testing, the effort to develop easy, rapid and cost effective molecular assays for drug resistance TB monitoring is definitely desirable and the GenoType® MTBDRplus assay was found to be a useful method for diagnosis of resistance to INH, RMP and MDR from lymph node aspirates. Further molecular cluster analysis to determine transmission dynamics of mutated strain is required.
Isoniazid; Rifampicin; Ethambutol; Mutation; M.tuberculosis; aspirates; Ethiopia.
Antibody assessment is an essential part in the serological diagnosis of autoimmune diseases. However, different diagnostic strategies have been proposed for the work up of sera in particular from patients with systemic autoimmune rheumatic disease (SARD). In general, screening for SARD-associated antibodies by indirect immunofluorescence (IIF) is followed by confirmatory testing covering different assay techniques. Due to lacking automation, standardization, modern data management, and human bias in IIF screening, this two-stage approach has recently been challenged by multiplex techniques particularly in laboratories with high workload. However, detection of antinuclear antibodies by IIF is still recommended to be the gold standard method for antibody screening in sera from patients with suspected SARD. To address the limitations of IIF and to meet the demand for cost-efficient autoantibody screening, automated IIF methods employing novel pattern recognition algorithms for image analysis have been introduced recently. In this respect, the AKLIDES technology has been the first commercially available platform for automated interpretation of cell-based IIF testing and provides multiplexing by addressable microbead immunoassays for confirmatory testing. This paper gives an overview of recently published studies demonstrating the advantages of this new technology for SARD serology.
To study the function and maturation of the human hematopoietic and immune system without endangering individuals, translational human-like animal models are needed. We compare the efficiency of CD34+ stem cells isolated from cryopreserved cord blood from a blood bank (CCB) and fresh cord blood (FCB) in generating highly engrafted humanized mice in NOD-SCID IL2Rγnull (NSG) rodents. Interestingly, the isolation of CD34+ cells from CCB results in a lower yield and purity compared to FCB. The purity of CD34+ isolation from CCB decreases with an increasing number of mononuclear cells that is not evident in FCB. Despite the lower yield and purity of CD34+ stem cell isolation from CCB compared to FCB, the overall reconstitution with human immune cells (CD45) and the differentiation of its subpopulations e.g., B cells, T cells or monocytes is comparable between both sources. In addition, independent of the cord blood origin, human B cells are able to produce high amounts of human IgM antibodies and human T cells are able to proliferate after stimulation with anti-CD3 antibodies. Nevertheless, T cells generated from FCB showed increased response to restimulation with anti-CD3. Our study reveals that the application of CCB samples for the engraftment of humanized mice does not result in less engraftment or a loss of differentiation and function of its subpopulations. Therefore, CCB is a reasonable alternative to FCB and allows the selection of specific genotypes (or any other criteria), which allows scientists to be independent from the daily changing birth rate.
Most infants with long-gap esophageal atresia receive an esophageal replacement with tissue from stomach or colon, because the native esophagus is too short for true primary repair. Tissue-engineered esophageal conducts could present an attractive alternative. In this paper, circular decellularized porcine esophageal scaffold tissues were implanted subcutaneously into Sprague-Dawley rats. Depending on scaffold cross-linking with genipin, glutaraldehyde, and carbodiimide (untreated scaffolds : positive control; bovine pericardium : gold standard), the number of infiltrating fibroblasts, lymphocytes, macrophages, giant cells, and capillaries was determined to quantify the host response after 1, 9, and 30 days. Decellularized esophagus scaffolds were shown to maintain native matrix morphology and extracellular matrix composition. Typical inflammatory reactions were observed in all implants; however, the cellular infiltration was reduced in the genipin group. We conclude that genipin is the most efficient and best tolerated cross-linking agent to attenuate inflammation and to improve the integration of esophageal scaffolds into its surrounding tissue after implantation.
The emergence of drug resistance is one of the most important threats to tuberculosis control programs. This study was aimed to analyze the frequency of gene mutations associated with resistance to isoniazid (INH), rifampicin (RMP) and ethambutol (EMB) among Mycobacterium tuberculosis isolates from Northwest Ethiopia, and to assess the performance of the GenoType® MTBDRplus and GenoType® MTBDRsl assays as compared to the BacT/ALERT 3D system.
Two hundred sixty Mycobacterium tuberculosis isolates from smear positive tuberculosis patients diagnosed between March 2009 and July 2009 were included in this study. Drug susceptibility tests were performed in the Institute of Medical Microbiology and Epidemiology of Infectious Diseases, University Hospital of Leipzig, Germany.
Of 260 isolates, mutations conferring resistance to INH, RMP, or EMB were detected in 35, 15, and 8 isolates, respectively, while multidrug resistance (MDR) was present in 13 of the isolates. Of 35 INH resistant strains, 33 had mutations in the katG gene at Ser315Thr 1 and two strains had mutation in the inhA gene at C15T. Among 15 RMP resistant isolates, 11 had rpoB gene mutation at Ser531Leu, one at His526Asp, and three strains had mutations only at the wild type probes. Of 8 EMB resistant strains, two had mutations in the embB gene at Met306Ile, one at Met306Val, and five strains had mutations only at the wild type probes. The GenoType® MTBDRplus assay had a sensitivity of 92% and specificity of 99% for INH resistance, and 100% sensitivity and specificity to detect RMP resistance and MDR. The GenoType® MTBDRsl assay had a sensitivity of 42% and specificity of 100% for EMB resistance.
The dominance of single gene mutations associated with the resistance to INH and RMP was observed in the codon 315 of the katG gene and codon 531 of the rpoB gene, respectively. The GenoType® MTBDRplus assay is a sensitive and specific tool for diagnosis of resistance to INH, RMP and MDR. However, the GenoType® MTBDRsl assay shows limitations in detecting resistance to EMB.
Mycobacterium tuberculosis; Drug resistance; Gene mutation
Suitable biomarkers are essential for therapeutic strategies in personalized medicine in terms of diagnosis as well as of prognosis. With highly specific biomarkers, it is possible, for example, to identify patients with poor prognosis, which enables early intervention and intensive treatment. The aim of this study was to identify and validate biomarkers and possible combinations for a prospective use in immunoscintigraphy, which may improve diagnosis of rheumatoid arthritis (RA) patients with consideration of inflammatory activity in the affected joints. Therefore, we tested several monoclonal antibodies (mAbs) directed against cellular-surface molecules on cells likely to be involved in the pathogenesis of RA.
Synovial tissue from patients with long-standing RA (accompanied by synovitis with varying states of current activity) and patients with acute non-RA arthritis were stained for surface molecules on different cell types by using fluorochrome-labeled antibodies. Tissue analysis was done by laser scanning cytometry (LSC), and statistical evaluation, by discriminant analysis and ROC analysis.
CD11b, HLA-DR, CD90, and CD64 revealed significant differences between tissues from patients with RA and acute non-RA arthritis. Especially with the expression of CD64, both patient cohorts could be discriminated with high sensitivity and specificity. RA classification was improved by simultaneously investigating the expression of two or three different surface proteins, such as HLA-DR, CD90, and CD29 in the tissue. The simultaneous analysis of CD64 together with CD304 or the combination of CD11b and CD38 was suitable for the identification of RA patients with high current activity in synovitis.
In this study, we showed that LSC is a novel reliable method in biomarker prevalidation in RA. Hence, identified mAbs in situ may allow their potential use in in vivo approaches. Moreover, we proved that biomarker-combination analysis resulted in better discrimination than did single-marker analysis. Combinations of these markers make a novel and reliable panel for the discrimination between RA and acute non-RA arthritis. In addition, further expedient combinations may be novel promising biomarker panels to identify current activity in synovitis in RA.
Tuberculosis is a major public health problem in Ethiopia. The diagnosis and treatment of drug-resistant tuberculosis remain a challenge in the country. This study aimed to assess whether single morning sputum samples could be stored at −20°C for extended periods of time at remote settings and then transported and successfully cultured for Mycobacterium tuberculosis. Single morning sputum samples were collected from all smear-positive tuberculosis patients diagnosed at Gondar Hospital, Gondar Health Center, Metemma Hospital, Bahir Dar Hospital, and Debre Markos Hospital in Northwest Ethiopia between March and July 2009. Specimens were stored at the study sites and sent to the mycobacteriology laboratory at the University Hospital, Leipzig, Germany, where specimens were processed and inoculated into the BacT/Alert 3D system and Lowenstein-Jensen and Gottsacker media. Ice packs were added in the package of the specimens during transport. A total of 319 patients were enrolled in this study. The median specimen storage time was 132 days (range, 16 to 180 days). Of all specimens, 283 (88.7%) were culture positive by any of the three culturing systems. M. tuberculosis isolates from four contaminated specimens in all culturing systems were successfully isolated on Middlebrook 7H10 agar; thereby, the recovery rate increased to 287 (90.0%). The length of time of sputum storage had no significant effect on the rate of recovery of M. tuberculosis in all culturing systems. In conclusion, single morning sputum specimens collected at remote settings stored at −20°C for long periods of time without the addition of preservatives can yield a high recovery rate. These findings suggest a simple and cost-effective alternative method of sputum storage for epidemiological and drug resistance studies in low-resource countries.
Peptide patterns of bronchoalveolar lavage fluid (BALF) were assumed to reflect the complex pathology of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) better than clinical and inflammatory parameters and may be superior for outcome prediction.
A training group of patients suffering from ALI/ARDS was compiled from equal numbers of survivors and nonsurvivors. Clinical history, ventilation parameters, Murray's lung injury severity score (Murray's LISS) and interleukins in BALF were gathered. In addition, samples of bronchoalveolar lavage fluid were analyzed by means of hydrophobic chromatography and MALDI-ToF mass spectrometry (MALDI-ToF MS).
Receiver operating characteristic (ROC) analysis for each clinical and cytokine parameter revealed interleukin-6>interleukin-8>diabetes mellitus>Murray's LISS as the best outcome predictors. Outcome predicted on the basis of BALF levels of interleukin-6 resulted in 79.4% accuracy, 82.7% sensitivity and 76.1% specificity (area under the ROC curve, AUC, 0.853). Both clinical parameters and cytokines as well as peptide patterns determined by MALDI-ToF MS were analyzed by classification and regression tree (CART) analysis and support vector machine (SVM) algorithms. CART analysis including Murray's LISS, interleukin-6 and interleukin-8 in combination was correct in 78.0%. MALDI-ToF MS of BALF peptides did not reveal a single identifiable biomarker for ARDS. However, classification of patients was successfully achieved based on the entire peptide pattern analyzed using SVM. This method resulted in 90% accuracy, 93.3% sensitivity and 86.7% specificity following a 10-fold cross validation (AUC = 0.953). Subsequent validation of the optimized SVM algorithm with a test group of patients with unknown prognosis yielded 87.5% accuracy, 83.3% sensitivity and 90.0% specificity.
MALDI-ToF MS peptide patterns of BALF, evaluated by appropriate mathematical methods can be of value in predicting outcome in pneumonia induced ALI/ARDS.
Background: This study explores effects of instrumental music on the hormonal system (as indicated by serum cortisol and adrenocorticotropic hormone), the immune system (as indicated by immunoglobulin A) and sedative drug requirements during surgery (elective total hip joint replacement under spinal anesthesia with light sedation). This is the first study investigating this issue with a double-blind design using instrumental music. Methodology/Principal Findings: Patients (n = 40) were randomly assigned either to a music group (listening to instrumental music), or to a control group (listening to a non-musical placebo stimulus). Both groups listened to the auditory stimulus about 2 h before, and during the entire intra-operative period (during the intra-operative light sedation, subjects were able to respond lethargically to verbal commands). Results indicate that, during surgery, patients of the music group had a lower propofol consumption, and lower cortisol levels, compared to the control group. Conclusion/Significance: Our data show that listening to music during surgery under regional anesthesia has effects on cortisol levels (reflecting stress-reducing effects) and reduces sedative requirements to reach light sedation.
emotion; music; hormones; immunology; anesthesia; cortisol; IgA; ACTH
Transfusion-transmissible infectious agents such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV) and syphilis are among the greatest threats to blood safety for the recipient. This study aimed to determine the seroprevalence, risk factors and trends of HIV, HBV, HCV and syphilis infections among blood donors over a period of five years at Gondar University Teaching Hospital, Northwest Ethiopia.
A retrospective analysis of consecutive blood donors' records covering the period between January 2003 and December 2007 was conducted. Logistic regression analysis was used to determine risk factors associated with HIV, HBV, HCV and syphilis infections.
From the total of 6361 consecutive blood donors, 607 (9.5%) had serological evidence of infection with at least one pathogen and 50 (0.8%) had multiple infections. The overall seroprevalence of HIV, HBV, HCV and syphilis was 3.8%, 4.7%, 0.7%, and 1.3% respectively. Among those with multiple infections, the most common combinations were HIV - syphilis 19 (38%) and HIV - HBV 17 (34%). The seropositivity of HIV was significantly increased among female blood donors, first time donors, housewives, merchants, soldiers, drivers and construction workers. Significantly increased HBV seropositivity was observed among farmers, first time donors and age groups of 26 - 35 and 36 - 45 years. Similarly, the seroprevalence of syphilis was significantly increased among daily labourers and construction workers. Statistically significant association was observed between syphilis and HIV infections, and HCV and HIV infections. Moreover, significantly declining trends of HIV, HCV and syphilis seropositivity were observed over the study period.
A substantial percentage of the blood donors harbour HIV, HBV, HCV and syphilis infections. Strict selection of blood donors and comprehensive screening of donors' blood using standard methods are highly recommended to ensure the safety of blood for recipient.
Adequate antiretroviral drug potency is essential for obtaining therapeutic benefit, however, the behavioral aspects of proper adherence and readiness to medication, often determine therapeutic outcome. Therefore, this study aimed to assess the level and determinants of nonadherence and nonreadiness to highly active antiretroviral therapy (HAART) among people living with HIV/AIDS (PLWHA) at Gondar University Teaching Hospital and Felege Hiwot Hospital in Northwest Ethiopia.
A cross-sectional study was conducted between July and September 2008 using structured interviewer-administered questionnaire. All consecutive adult outpatients who were receiving antiretroviral treatment for at least three months, seen at both hospitals during the study period and able to give informed consent were included in the study. Multivariate logistic regression was used to determine factors associated with nonadherence and nonreadiness.
A total of 504 study subjects were included in this study. The prevalence rates of nonadherence and nonreadiness to HAART were 87 (17.3%) and 70 (13.9%) respectively. Multivariate logistic regression analysis revealed that medication adverse effects, nonreadiness to HAART, contact with psychiatric care service and having no goal had statistically significant association with nonadherence. Moreover, unwillingness to disclose HIV status was significantly associated with nonreadiness to HAART.
In this study the level of nonadherence and nonreadiness to HAART seems to be encouraging. Several factors associated with nonadherance and nonreadiness to HAART were identified. Efforts to minimize nonadherence and nonreadiness to HAART should be integrated in to regular clinical follow up of patients.
In Gondar University Teaching Hospital standardized tuberculosis prevention and control programme, incorporating Directly Observed Treatment, Short Course (DOTS) started in 2000. According to the proposal of World Health Organization (WHO), treatment outcome is an important indicator of tuberculosis control programs. This study investigated the outcome of tuberculosis treatment at Gondar University Teaching Hospital in Northwest Ethiopia.
We analyzed the records of 4000 tuberculosis patients registered at Gondar University Teaching Hospital from September 2003 to May 2008. Treatment outcome and tuberculosis type were categorized according to the national tuberculosis control program guideline. Multivariate analysis using logistic regression model was used to analyse the association between treatment outcome and potential predictor variables.
From the total of 4000 patients, tuberculosis type was categorized as extrapulmonary in 1133 (28.3%), smear negative pulmonary tuberculosis in 2196 (54.9%) and smear positive pulmonary tuberculosis in 671 (16.8%) cases. Of all patients, treatment outcome was classified as successfully treated in 1181(29.5%), defaulted in 730 (18.3%), died in 403 (10.1%), treatment failed in six (0.2%) and transferred out in 1680 (42.0%) patients. Males had the trend to be more likely to experience death or default than females, and the elderly were more likely to die than younger. The proportion of default rate was increased across the years from 97(9.2%) to 228(42.9%). Being female, age group 15-24 years, smear positive pulmonary tuberculosis and being urban resident were associated with higher treatment success rate.
The treatment success rate of tuberculosis patients was unsatisfactorily low (29.5%). A high proportion of patients died (10.1%) or defaulted (18.3%), which is a serious public health concern that needs to be addressed urgently.
Non adherent bone marrow derived cells (NA-BMCs) have recently been described to give rise to multiple mesenchymal phenotypes and have an impact in tissue regeneration. Therefore, the effects of murine bone marrow derived NA-BMCs were investigated with regard to engraftment capacities in allogeneic and syngeneic stem cell transplantation using transgenic, human CD4+, murine CD4−/−, HLA-DR3+ mice.
Bone marrow cells were harvested from C57Bl/6 and Balb/c wild-type mice, expanded to NA-BMCs for 4 days and characterized by flow cytometry before transplantation in lethally irradiated recipient mice. Chimerism was detected using flow cytometry for MHC-I (H-2D[b], H-2K[d]), mu/huCD4, and huHLA-DR3). Culturing of bone marrow cells in a dexamethasone containing DMEM medium induced expansion of non adherent cells expressing CD11b, CD45, and CD90. Analysis of the CD45+ showed depletion of CD4+, CD8+, CD19+, and CD117+ cells. Expanded syngeneic and allogeneic NA-BMCs were transplanted into triple transgenic mice. Syngeneic NA-BMCs protected 83% of mice from death (n = 8, CD4+ donor chimerism of 5.8±2.4% [day 40], P<.001). Allogeneic NA-BMCs preserved 62.5% (n = 8) of mice from death without detectable hematopoietic donor chimerism. Transplantation of syngeneic bone marrow cells preserved 100%, transplantation of allogeneic bone marrow cells 33% of mice from death.
NA-BMCs triggered endogenous hematopoiesis and induced faster recovery compared to bone marrow controls. These findings may be of relevance in the refinement of strategies in the treatment of hematological malignancies.
The gene MICA encodes the protein major histocompatibility complex class I polypeptide-related sequence A. It is expressed in synovium of patients with rheumatoid arthritis (RA) and its implication in autoimmunity is discussed. We analyzed the association of genetic variants of MICA with susceptibility to RA.
Initially, 300 French Caucasian individuals belonging to 100 RA trio families were studied. An additional 100 independent RA trio families and a German Caucasian case-control cohort (90/182 individuals) were available for replication. As MICA is situated in proximity to known risk alleles of the HLA-DRB1 locus, our analysis accounted for linkage disequilibrium either by analyzing the subgroup consisting of parents not carrying HLA-DRB1 risk alleles with transmission disequilibrium test (TDT) or by implementing a regression model including all available data. Analysis included a microsatellite polymorphism (GCT)n and single-nucleotide polymorphisms (SNPs) rs3763288 and rs1051794.
In contrast to the other investigated polymorphisms, the non-synonymously coding SNP MICA-250 (rs1051794, Lys196Glu) was strongly associated in the first family cohort (TDT: P = 0.014; regression model: odds ratio [OR] 0.46, 95% confidence interval [CI] 0.25 to 0.82, P = 0.007). Although the replication family sample showed only a trend, combined family data remained consistent with the hypothesis of MICA-250 association independent from shared epitope (SE) alleles (TDT: P = 0.027; regression model: OR 0.56, 95% CI 0.38 to 0.83, P = 0.003). We also replicated the protective association of MICA-250A within a German Caucasian cohort (OR 0.31, 95% CI 0.1 to 0.7, P = 0.005; regression model: OR 0.6, 95% CI 0.37 to 0.96, P = 0.032). We showed complete linkage disequilibrium of MICA-250 (D' = 1, r2= 1) with the functional MICA variant rs1051792 (D' = 1, r2= 1). As rs1051792 confers differential allelic affinity of MICA to the receptor NKG2D, this provides a possible functional explanation for the observed association.
We present evidence for linkage and association of MICA-250 (rs1051794) with RA independent of known HLA-DRB1 risk alleles, suggesting MICA as an RA susceptibility gene. However, more studies within other populations are necessary to prove the general relevance of this polymorphism for RA.
Glyoxalases (Glo1 and Glo2) are involved in the glycolytic pathway by detoxifying the reactive methylglyoxal (MGO) into D-lactate in a two-step reaction using glutathione (GSH) as cofactor. Inhibitors of glyoxalases are considered as anti-inflammatory and anti-carcinogenic agents. The recent finding that various polyphenols modulate Glo1 activity has prompted us to assess curcumin's potency as an Glo1 inhibitor.
Cultures of whole blood cells and tumor cell lines (PC-3, JIM-1, MDA-MD 231 and 1321N1) were set up to investigate the effect of selected polyphenols, including curcumin, on the LPS-induced cytokine production (cytometric bead-based array), cell proliferation (WST-1 assay), cytosolic Glo1 and Glo2 enzymatic activity, apoptosis/necrosis (annexin V-FITC/propidium iodide staining; flow cytometric analysis) as well as GSH and ATP content. Results of enzyme kinetics revealed that curcumin, compared to the polyphenols quercetin, myricetin, kaempferol, luteolin and rutin, elicited a stronger competitive inhibitory effect on Glo1 (Ki = 5.1±1.4 µM). Applying a whole blood assay, IC50 values of pro-inflammatory cytokine release (TNF-α, IL-6, IL-8, IL-1β) were found to be positively correlated with the Ki-values of the aforementioned polyphenols. Moreover, whereas curcumin was found to hamper the growth of breast cancer (JIMT-1, MDA-MB-231), prostate cancer PC-3 and brain astrocytoma 1321N1 cells, no effect on growth or vitality of human primary hepatocytes was elucidated. Curcumin decreased D-lactate release by tumor cells, another clue for inhibition of intracellular Glo1.
The results described herein provide new insights into curcumin's biological activities as they indicate that inhibition of Glo1 by curcumin may result in non-tolerable levels of MGO and GSH, which, in turn, modulate various metabolic cellular pathways including depletion of cellular ATP and GSH content. This may account for curcumin's potency as an anti-inflammatory and anti-tumor agent. The findings support the use of curcumin as a potential therapeutic agent.
A case of tracheobronchopathia osteochondroplastic (TPO) was diagnosed in a 69-year old male with prolonged cough. TPO is a rare condition of unknown cause and only sporadic cases have been reported. The condition is benign, characterized by submucosal nodules growing from the submucosal layer of the airways, protruding into the bronchial lumen. The bronchscopic view together with bronchial cartilage with abnormal distributed mineralization of the histologic examination of theses nodules leads to the correct diagnosis. Mild cases are treated symptomatically, whereas we tried an inhaled corticosteroid. Prominent protrusions in the trachea or the bronchi must be removed. In most cases the disease is stable over years but progressive forms have been reported. TPO may cause chronic refractory cough, which eventually is the only prominent symptom of this disease.
Acute appendicitis (AA) is a common surgical problem that is associated with an acute-phase reaction. Previous studies have shown that cytokines and acute-phase proteins are activated and may serve as indicators for the severity of appendicitis. The aim of this study was to compare diagnostic value of different serum inflammatory markers in detection of phlegmonous or perforated appendicitis in children.
Data were collected prospectively on 211 consecutive children. Laparotomy was performed for suspected AA for 189 patients. Patients were subdivided into groups: nonsurgical abdominal pain, early appendicitis, phlegmonous or gangrenous appendicitis, perforated appendicitis.
White blood cell count (WBC), serum C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), acid α1-glycoprotein (α1GP), endotoxin, and erythrocyte sedimentation reaction (ESR) were estimated ad the time of admission. The diagnostic performance was analyzed using receiver operating characteristic (ROC) curves.
WBC count, CRP and IL-6 correlated significantly with the severity of appendiceal inflammation. Identification of children with severe appendicitis was supported by IL-6 or CRP but not WBC. Between IL-6 and CRP, there were no significant differences in diagnostic use.
Laboratory results should be considered to be integrated within the clinical assessment. If used critically, CRP and IL-6 equally provide surgeons with complementary information in discerning the necessity for urgent operation.
The R122H mutation of the cationic trypsinogen was found in patients with hereditary pancreatitis. A transgenic animal carrying this mutation could be useful as a genetic model system of pancreatitis.
Mice transgenic for the human R122H cationic trypsinogen were generated using the -205 fragment of the rat elastase promoter. The presence of the transgene was assayed in the DNA, in pancreatic mRNA and in zymogen granule lysates. Serum levels of amylase, lipase and cytokines (MCP-1, IL-6) were monitored and the histological appearance of the tissue was investigated. Pancreatitis was induced by 7 hourly injections of 50 μg/kg cerulein. The procedure was repeated twice weekly for 10 consecutive weeks. The animals were sacrificed 24 (n = 8) and 48 hours (n = 8) after the first injection and at the end of the whole treatment (n = 7).
The transgene was detected at the genomic level and in pancreatic mRNA. The corresponding protein was found in low amounts in zymogen granule lysates. R122H mice showed elevated pancreatic lipase, but there was no spontaneous development of pancreatitis within 18 months. After induction of pancreatitis, levels of lipase (after 24 hours) and amylase (after 48 hours) were higher in R122H mice compared to controls. Repeated treatment with cerulein resulted in a slightly more severe pancreatitis in R122H animals. Amylase, lipase, and the cytokine levels were similar to controls.
The R122H transgenic mouse failed to develop a spontaneous pancreatitis but a repeatedly provoked cerulein-induced pancreatitis led to a slightly more severe pancreatitis. The rather small difference in comparison to controls could be due to the low expression of the transgene in the mouse pancreas.
Constitutive mRNA expression and secretion of proinflammatory and anti-inflammatory cytokines was comparatively analyzed in rheumatoid arthritis (RA) synovial fibroblasts (SFB), isolated from primary culture or derived by repeated passage; normal-skin fibroblasts were used as controls. First-passage RA-SFB (n = 3) secreted large amounts of IL-6 (15,800 ± 2,110 pg/ml; mean ± SEM), but only limited amounts of tumor necrosis factor (TNF)-α (22.1 ± 1.1 pg/ml) or IL-10 (35.7 ± 34.2 pg/ml; only one of three samples was positive). IL-1β, IL-15, and IL-18 were not detectable at the protein level and showed very low mRNA levels by semiquantitative RT-PCR. In repeated-passage RA-SFB (tenth passage), protein secretion was significantly lower for IL-6 (one-twentieth of the initial level) and TNF-α (two-thirds), and markedly reduced for IL-10 (one-quarter, with only one of three samples positive). While the decrease of IL-10 protein from first to tenth passage was paralleled by a corresponding decrease of mRNA, the relative mRNA levels for IL-6 and TNF-α were actually increased (20-fold and 300-fold, respectively), indicating post-transcriptional and/or post-translational regulation of these cytokines. Due to highly variable levels among individual patients, however, no significant differences were observed for any cytokine mRNA between primary-culture and repeated-passage RA-SFB (ninth passage). Likewise, no significant differences were detectable between RA-SFB and normal-skin fibroblasts (primary-culture and repeated-passage). By producing high amounts of IL-6 and limited amounts of TNF-α, RA-SFB may contribute to the (im)balance of proinflammatory and anti-inflammatory cytokines in the inflamed joint.
cytokines; inflammation; mRNA; rheumatoid arthritis; synovial fibroblasts
To reduce culture artifacts by conventional repeated passaging and long-term culture in vitro, the isolation of synovial fibroblasts (SFB) was attempted from rheumatoid arthritis (RA) synovial membranes by trypsin/collagenase digest, short-term in vitro adherence (7 days), and negative isolation using magnetobead-coupled anti-CD14 monoclonal antibodies. This method yielded highly enriched SFB (85% prolyl-4-hydroxylase+/74% Thy-1/CD90+ cells; <2% contaminating macrophages; <1% leukocytes/endothelial cells) that, in comparison with conventional fourth-passage RA-SFB, showed a markedly different phenotype and significantly lower proliferation rates upon stimulation with platelet-derived growth factor and IL-1β. This isolation method is simple and reliable, and may yield cells with features closer to the in vivo configuration of RA-SFB by avoiding extended in vitro culture.
isolation; phenotype/function; primary culture; rheumatoid arthritis; synovial fibroblasts