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1.  Molecular epidemiology and transmission dynamics of Mycobacterium tuberculosis in Northwest Ethiopia: new phylogenetic lineages found in Northwest Ethiopia 
BMC Infectious Diseases  2013;13:131.
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/1471-2334-13-131
PMCID: PMC3605317  PMID: 23496968
Mycobacterium tuberculosis; Molecular epidemiology; Transmission dynamics
2.  Xenogenic Esophagus Scaffolds Fixed with Several Agents: Comparative In Vivo Study of Rejection and Inflammation 
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.
doi:10.1155/2012/948320
PMCID: PMC3312382  PMID: 22505820
3.  Analysis of gene mutations associated with isoniazid, rifampicin and ethambutol resistance among Mycobacterium tuberculosis isolates from Ethiopia 
Background
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1186/1471-2334-12-37
PMCID: PMC3378438  PMID: 22325147
Mycobacterium tuberculosis; Drug resistance; Gene mutation
4.  Identification and evaluation of novel synovial tissue biomarkers in rheumatoid arthritis by laser scanning cytometry 
Introduction
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/ar3682
PMCID: PMC3392796  PMID: 22251373
5.  Rate of Recovery of Mycobacterium tuberculosis from Frozen Acid-Fast-Bacillus Smear-Positive Sputum Samples Subjected to Long-Term Storage in Northwest Ethiopia ▿ 
Journal of Clinical Microbiology  2011;49(7):2557-2561.
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.
doi:10.1128/JCM.00059-11
PMCID: PMC3147816  PMID: 21562105
6.  Seroprevalence of HIV, HBV, HCV and syphilis infections among blood donors at Gondar University Teaching Hospital, Northwest Ethiopia: declining trends over a period of five years 
BMC Infectious Diseases  2010;10:111.
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/1471-2334-10-111
PMCID: PMC2881920  PMID: 20459703
7.  Magnitude and determinants of nonadherence and nonreadiness to highly active antiretroviral therapy among people living with HIV/AIDS in Northwest Ethiopia: a cross - sectional study 
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/1742-6405-7-2
PMCID: PMC2845546  PMID: 20180959
8.  Treatment outcome of tuberculosis patients at Gondar University Teaching Hospital, Northwest Ethiopia. A five - year retrospective study 
BMC Public Health  2009;9:371.
Background
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1186/1471-2458-9-371
PMCID: PMC2765962  PMID: 19799801
9.  Effectiveness of cytopenia prophylaxis for different filgrastim and pegfilgrastim schedules in a chemotherapy mouse model 
Objectives
Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is widely used to treat neutropenia during cytotoxic chemotherapy. The optimal scheduling of rhG-CSF is unknown and can hardly be tested in clinical studies due to numerous therapy parameters affecting outcome (chemotherapeutic regimen, rhG-CSF schedules, individual covariables). Motivated by biomathematical model simulations, we aim to investigate different rhG-CSF schedules in a preclinical chemotherapy mouse model.
Methods
The time course of hematotoxicity was studied in CD-1 mice after cyclophosphamide (CP) administration. Filgrastim was applied concomitantly in a 2 × 3-factorial design of two dosing options (2 × 20 μg and 4 × 10 μg) and three timing options (directly, one, and two days after CP). Alternatively, a single dose of 40 μg pegfilgrastim was applied at the three timing options. The resulting cytopenia was compared among the schedules.
Results
Dosing and timing had a significant influence on the effectiveness of filgrastim schedules whereas for pegfilgrastim the timing effect was irrelevant. The best filgrastim and pegfilgrastim schedules exhibited equivalent toxicity. Monocytes dynamics performed analogously to granulocytes. All schedules showed roughly the same lymphotoxicity.
Conclusion
We conclude that effectiveness of filgrastim application depends heavily on its scheduling during chemotherapy. There is an optimum of timing. Dose splitting is better than concentrated applications. Effectiveness of pegfilgrastim is less dependent on timing.
PMCID: PMC2726061  PMID: 19707393
rhG-CSF; chemotherapy toxicity; mice; cyclophosphamide; cytopenia; neutropenia
10.  Allogeneic Non-Adherent Bone Marrow Cells Facilitate Hematopoietic Recovery but Do Not Lead to Allogeneic Engraftment 
PLoS ONE  2009;4(7):e6157.
Background
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.
Methodology/Principal Findings
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.
Conclusions/Significance
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.
doi:10.1371/journal.pone.0006157
PMCID: PMC2701999  PMID: 19582154
11.  Association of MICA with rheumatoid arthritis independent of known HLA-DRB1 risk alleles in a family-based and a case control study 
Introduction
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/ar2683
PMCID: PMC2714103  PMID: 19409079
12.  Neuronal hypoxia in vitro: Investigation of therapeutic principles of HUCB-MNC and CD133+ stem cells 
BMC Neuroscience  2008;9:91.
Background
The therapeutic capacity of human umbilical cord blood mononuclear cells (HUCB-MNC) and stem cells derived thereof is documented in animal models of focal cerebral ischemia, while mechanisms behind the reduction of lesion size and the observed improvement of behavioral skills still remain poorly understood.
Methods
A human in vitro model of neuronal hypoxia was used to address the impact of total HUCB-MNC (tMNC), a stem cell enriched fraction (CD133+, 97.38% CD133-positive cells) and a stem cell depleted fraction (CD133-, 0.06% CD133-positive cells) of HUCB-MNC by either direct or indirect co-cultivation with post-hypoxic neuronal cells (differentiated SH-SY5Y). Over three days, development of apoptosis and necrosis of neuronal cells, chemotaxis of MNC and production of chemokines (CCL2, CCL3, CCL5, CXCL8, CXCL9) and growth factors (G-CSF, GM-CSF, VEGF, bFGF) were analyzed using fluorescence microscopy, FACS and cytometric bead array.
Results
tMNC, CD133+ and surprisingly CD133- reduced neuronal apoptosis in direct co-cultivations significantly to levels in the range of normoxic controls (7% ± 3%). Untreated post-hypoxic control cultures showed apoptosis rates of 85% ± 11%. tMNC actively migrated towards injured neuronal cells. Both co-cultivation types using tMNC or CD133- reduced apoptosis comparably. CD133- produced high concentrations of CCL3 and neuroprotective G-CSF within indirect co-cultures. Soluble factors produced by CD133+ cells were not detectable in direct co-cultures.
Conclusion
Our data show that heterogeneous tMNC and even CD133-depleted fractions have the capability not only to reduce apoptosis in neuronal cells but also to trigger the retaining of neuronal phenotypes.
doi:10.1186/1471-2202-9-91
PMCID: PMC2569042  PMID: 18803816
13.  Evidence for neuroprotective properties of human umbilical cord blood cells after neuronal hypoxia in vitro 
BMC Neuroscience  2008;9:30.
Background
One of the most promising options for treatment of stroke using adult stem cells are human umbilical cord blood (HUCB) cells that were already approved for therapeutic efficacy in vivo. However, complexity of animal models has thus far limited the understanding of beneficial cellular mechanisms. To address the influence of HUCB cells on neuronal tissue after stroke we established and employed a human in vitro model of neuronal hypoxia using fully differentiated vulnerable SH-SY5Y cells. These cells were incubated under an oxygen-reduced atmosphere (O2< 1%) for 48 hours. Subsequently, HUCB mononuclear cells (MNC) were added to post-hypoxic neuronal cultures. These cultures were characterized regarding to the development of apoptosis and necrosis over three days. Based on this we investigated the therapeutic influence of HUCB MNC on the progression of apoptotic cell death. The impact of HUCB cells and hypoxia on secretion of neuroprotective and inflammatory cytokines, chemokines and expression of adhesion molecules was proved.
Results
Hypoxic cultivation of neurons initially induced a rate of 26% ± 13% of apoptosis. Hypoxia also caused an enhanced expression of Caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP). Necrosis was only detected in low amounts. Within the next three days rate of apoptosis in untreated hypoxic cultures cumulated to 85% ± 11% (p ≤ 0.001). Specific cytokine (VEGF) patterns also suggest anti-apoptotic strategies of neuronal cells. Remarkably, the administration of MNC showed a noticeable reduction of apoptosis rates to levels of normoxic control cultures (7% ± 3%; p ≤ 0.001). In parallel, clustering of administered MNC next to axons and somata of neuronal cells was observed. Furthermore, MNC caused a pronounced increase of chemokines (CCL5; CCL3 and CXCL10).
Conclusion
We established an in vitro model of neuronal hypoxia that affords the possibility to investigate both, apoptotic neuronal cell death and neuroprotective therapies. Here we employed the therapeutic model to study neuroprotective properties of HUCB cells.
We hypothesize that the neuroprotective effect of MNC was due to anti-apoptotic mechanisms related to direct cell-cell contacts with injured neuronal cells and distinct changes in neuroprotective, inflammatory cytokines as well as to the upregulation of chemokines within the co-cultures.
doi:10.1186/1471-2202-9-30
PMCID: PMC2294131  PMID: 18312640
14.  Experimental treatment of stroke in spontaneously hypertensive rats by CD34+ and CD34- cord blood cells 
Human umbilical cord blood as a source of stem cells has recently been reported in experimental treatment of cerebral disorders. However, little is known about the nature of cells and cellular mechanisms leading to neurofunctional improvement. Here we investigated the potential of separated CD34+ versus CD34- human umbilical cord blood cells (HUCBC) to promote functional recovery following stroke. The experiments were performed in spontaneously hypertensive (SH) rats, known for a risk profile comparable to stroke patients.
After three weeks of behavioral training in the RotaRod and Beamwalk test arrays, stroke was induced by permanent middle cerebral artery occlusion (MCAO). For cell therapy, 1x106 cryopreserved cells were administered systemically between 8 and 10 hours after MCAO. The behavioral tests were performed together with a neurological severity score (mNSS) until day 29 to assess neurofunctional disabilities. Nearly complete functional remission was observed with both subpopulations CD34+ as well as CD34- cells. To localize cells histologically, they were labeled with a fluorescence dye (CFSE) before injection. Again, after administration of CD34+ as well as CD34- cells, CFSE labelled cells were found that accumulated in the border zone between the central necrosis of the ischemic lesion and functional brain tissue, thus indicating active attraction towards the lesion for both cell populations. Immunohistology with anti-CD68 and antibodies to human neuronal markers (NF-L, chromogranin) indicated an accumulation of human and rat monocytes in the border zone of the lesion while neuronal cells of human origin could not be detected in host brains.
PMCID: PMC2703252  PMID: 19675726
behavioral test; cord blood; middle cerebral artery occlusion; stem cell; stroke; transplantation
15.  Expression of cytokine mRNA and protein in joints and lymphoid organs during the course of rat antigen-induced arthritis 
Arthritis Research & Therapy  2005;7(3):R445-R457.
Cytokine expression was assessed during antigen-induced arthritis (AIA) in synovial membrane (SM), inguinal lymph node (LN), and spleen using competitive RT-PCR and sandwich ELISA. In the SM, early elevations of IL-1β and IL-6 mRNA (by 6 hours; 450- and 200-fold, respectively) correlated with the joint swelling; a 6-fold increase in tumor necrosis factor α (TNFα) was not significant. Not only IL-2 and IFN-γ (which increased 10,000-fold and 200-fold, respectively), but also IL-5 and IL-10, increased acutely (6 hours – day 1; 3-fold and 35-fold, respectively) in the SM. In general, the protein levels in the SM for IL-1β, IL-6, TNFα, IFN-γ, IL-4, and IL-10 (increase from 4-fold to 15-fold) matched the course of mRNA expression. In the inguinal LN, there were early mRNA elevations of IL-6 (a 2.5-fold increase by 6 hours, which correlated positively with the joint swelling) and IL-2 (4-fold by 6 hours), as well as later rises of IL-4 and IL-5 (2.5- and 4-fold, respectively, by day 3). No significant elevations of the corresponding proteins in this tissue were observed, except for IL-1β (by day 6) and IL-10 (by day 1). In the spleen, there were significant mRNA elevations at 6 hours of IL-1β (1.5-fold), IL-6 (4-fold; positively correlated with the joint swelling), IFN-γ (3-fold), and IL-2 (7- to 10-fold). IL-5 and IL-10 (2- and 3-fold, respectively) peaked from 6 hours to day 3 in the spleen. Increases of the corresponding proteins were significant in comparison with day 0 only in the case of IL-2 (day 6). By day 6 (transition to the chronic phase), the mRNA for cytokines declined to or below prearthritis levels in all the tissues studied except for IL-1β in the SM and IL-6 in the spleen. AIA is thus characterized by four phenomena: early synovial activation of macrophages, T helper (Th)1-like, and Th2-like cells; late, well-segregated Th2-like responses in the inguinal LN; late, overlapping Th1-like/Th2-like peaks in the spleen; and chronic elevation of synovial IL-1β mRNA and spleen IL-6 mRNA.
doi:10.1186/ar1689
PMCID: PMC1174936  PMID: 15899031
16.  Differential clinical efficacy of anti-CD4 monoclonal antibodies in rat adjuvant arthritis is paralleled by differential influence on NF-κB binding activity and TNF-α secretion of T cells 
Arthritis Research  2002;4(3):184-189.
The aim of this study was to analyze the differential effects of three anti-CD4 monoclonal antibodies (mAbs) (with distinct epitope specifities) in the treatment of rat adjuvant arthritis (AA) and on T-cell function and signal transduction. Rat AA was preventively treated by intraperitoneal injection of the anti-CD4 mAbs W3/25, OX35, and RIB5/2 (on days -1, 0, 3, and 6, i.e. 1 day before AA induction, on the day of induction [day 0], and thereafter). The effects on T-cell reactivity in vivo (delayed-type hypersensitivity), ex vivo (ConA-induced proliferation), and in vitro (mixed lymphocyte culture) were assessed. The in vitro effects of anti-CD4 preincubation on T-cell receptor (TCR)/CD3-induced cytokine production and signal transduction were also analyzed. While preventive treatment with OX35 and W3/25 significantly ameliorated AA from the onset, treatment with RIB5/2 even accelerated the onset of AA by approximately 2 days (day 10), and ameliorated the arthritis only in the late phase (day 27). Differential clinical effects at the onset of AA were paralleled by a differential influence of the mAbs on T-cell functions, i.e. in comparison with OX35 and W3/25, the 'accelerating' mAb RIB5/2 failed to increase the delayed-type hypersentivity (DTH) to Mycobacterium tuberculosis, increased the in vitro tumor necrosis factor (TNF)-α secretion, and more strongly induced NF-κB binding activity after anti-CD4 preincubation and subsequent TCR/CD3-stimulation. Depending on their epitope specificity, different anti-CD4 mAbs differentially influence individual proinflammatory functions of T cells. This fine regulation may explain the differential efficacy in the treatment of AA and may contribute to the understanding of such treatments in other immunopathologies.
PMCID: PMC111020  PMID: 12010568
adjuvant arthritis; anti-CD4 monoclonal antibody; TNF-alpha; NF-kappaB
17.  Cytokine mRNA and protein expression in primary-culture and repeated-passage synovial fibroblasts from patients with rheumatoid arthritis 
Arthritis Research  2001;4(2):117-125.
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.
PMCID: PMC83845  PMID: 11879547
cytokines; inflammation; mRNA; rheumatoid arthritis; synovial fibroblasts
18.  Mosaic chromosomal aberrations in synovial fibroblasts of patients with rheumatoid arthritis, osteoarthritis, and other inflammatory joint diseases 
Arthritis Research  2001;3(5):319-330.
Chromosomal aberrations were comparatively assessed in nuclei extracted from synovial tissue, primary-culture (P-0) synovial cells, and early-passage synovial fibroblasts (SFB; 98% enrichment; P-1, P-4 [passage 1, passage 4]) from patients with rheumatoid arthritis (RA; n = 21), osteoarthritis (OA; n = 24), and other rheumatic diseases. Peripheral blood lymphocytes (PBL) and skin fibroblasts (FB) (P-1, P-4) from the same patients, as well as SFB from normal joints and patients with joint trauma (JT) (n = 4), were used as controls. Analyses proceeded by standard GTG-banding and interphase centromere fluorescence in situ hybridization. Structural chromosomal aberrations were observed in SFB (P-1 or P-4) from 4 of 21 RA patients (19%), with involvement of chromosome 1 [e.g. del(1)(q12)] in 3 of 4 cases. In 10 of the 21 RA cases (48%), polysomy 7 was observed in P-1 SFB. In addition, aneusomies of chromosomes 4, 6, 8, 9, 12, 18, and Y were present. The percentage of polysomies was increased in P-4. Similar chromosomal aberrations were detected in SFB of OA and spondylarthropathy patients. No aberrations were detected in i) PBL or skin FB from the same patients (except for one OA patient with a karyotype 45,X[10]/46,XX[17] in PBL and variable polysomies in long-term culture skin FB); or ii) synovial tissue and/or P-1 SFB of normal joints or of patients with joint trauma. In conclusion, qualitatively comparable chromosomal aberrations were observed in synovial tissue and early-passage SFB of patients with RA, OA, and other inflammatory joint diseases. Thus, although of possible functional relevance for the pathologic role of SFB in RA, these alterations probably reflect a common response to chronic inflammatory stress in rheumatic diseases.
PMCID: PMC64845  PMID: 11549374
osteoarthritis; rheumatoid arthritis; spondylarthropathy; synovial fibroblasts; trisomy/polysomy 7
19.  Isolation and characterization of rheumatoid arthritis synovial fibroblasts from primary culture — primary culture cells markedly differ from fourth-passage cells 
Arthritis Research  2000;3(1):72-76.
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.
PMCID: PMC17827  PMID: 11178129
isolation; phenotype/function; primary culture; rheumatoid arthritis; synovial fibroblasts
20.  Age-Dependent Levels of Select Immunological Mediators in Sera of Healthy Children 
Serum cytokine levels were measured in 275 healthy children of different ages (3 to 17 years). Interleukin-1 receptor antagonist (IL-1RA), soluble IL-2R (sIL-2R) (sCD25), IL-6, IL-8, tumor necrosis factor alpha (TNF-α), soluble TNF receptor type II (sTNF-RII) (sCD120b), gamma interferon (IFN-γ), soluble intercellular adhesion molecule 1 (sICAM-1) (sCD54), soluble E selectin (sE-selectin) (ELAM-1; sCD62E), sCD14, and neopterin were measured with commercial test kits. The mean levels of IL-1RA, sIL-2R, TNF-α, sICAM-1, sE-selectin, and sCD14 were higher than in healthy adults. In contrast, IFN-γ and IL-8 were hardly detectable in children and thereby significantly lower than in adults. In the case of TNF-α, sICAM-1, sE selectin, and sCD14, there was a high interindividual variability, apparently unrelated to disease. The profiles of some cytokines, i.e., IL-1RA, IL-6, and TNF-α, showed age-related increases that overlapped with known patterns of physical growth. Of note, sIL-2R and sE-selectin instead declined with time. Because of the remarkable age-dependent variability in healthy pediatric subjects, disease-related changes, as well as therapy-dependent alterations, should be considered with caution.
PMCID: PMC121386  PMID: 9455875

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