Elevated senescence in the bone marrow mesenchymal stem cells of acquired aplastic anemia patients: A possible implication of DNA damage responses and telomere attrition.

BACKGROUND: Bone marrow mesenchymal stem cells (BM-MSC) are an integral part of the BM niche that is essential to maintain hematopoietic homeostasis. In aplastic anemia (AA), a few studies have reported phenotypic defects in the BM-MSC, such as reduced proliferation, imbalanced differentiation, and apoptosis; however, the alterations at the molecular level need to be better characterized. Therefore, the current study aims to identify the causative factors underlying the compromised functions of AA BM-MSC that might eventually be contributing to the AA pathobiology. METHODS: We performed RNA sequencing (RNA-Seq) using the Illumina platform to comprehend the distinction between the transcriptional landscape of AA and control BM-MSC. Further, we validated the alterations observed in senescence by Senescence- associated beta-galactosidase (SA -ß-gal) assay, DNA damage by γH2AX staining, and telomere attrition by relative telomere length assessment and telomerase activity assay. We used qRT-PCR to analyze changes in some of the genes associated with these molecular mechanisms. RESULTS: The transcriptome profiling revealed enrichment of senescence-associated genes and pathways in AA BM-MSC. The senescent phenotype of AA BM-MSC was accompanied by enhanced SA -ß-gal activity and elevated expression of senescence associated genes TP53, PARP1, and CDKN1A. Further, we observed increased γH2AX foci indicating DNA damage, reduced telomere length, and diminished telomerase activity in the AA BM-MSC. CONCLUSION: Our results highlight that AA BM-MSC have a senescent phenotype accompanied by other cellular defects like DNA damage and telomere attrition, which are most likely driving the senescent phenotype of AA BM-MSC thus hampering their hematopoiesis supporting properties as observed in AA.

A Rapid Method for Determination of Serum Methotrexate Using Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry and Its Application in Therapeutic Drug Monitoring.

Objectives Methotrexate (MTX) has anticancer therapeutic potential with multiple doses-related adverse effects and toxicities. Immunoassays for therapeutic monitoring of serum MTX have their own limitations. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered as the reference method; however, commercially availability of them is limited. We aimed to adapt/develop an in-house LC-MS/MS method for therapeutic monitoring of serum MTX. Materials and Methods Serum protein precipitation was performed using acetonitrile-water containing 250 µM solution of aminoacetophenone as internal standard (IS). Chromatographic separation was achieved on a C18 column with mobile phase of 0.1% solution of formic acid (solvent A) and acetonitrile (solvent B) at a flow rate of 0.4 mL/min. MS was performed under positive ion mode with mass transition for MTX and IS as m/z 455.1→308.1 and 136.2→94.1, respectively. The method was validated by following Bioanalytical Method Validation Guidance for Industry, 2018 and applied on leukemia patients' samples on MTX therapy. Results The correlation coefficient of eight serially diluted calibration standards of 0.09 to 12.5 µM was >0.99 and had linearity with > 95% precision and accuracy at analytical quality control levels. The lower limit of MTX quantification achieved was 0.09 µM with good intensity and sharp peak as compared with blank sample. The total run time of the assay was 5 minutes. The serum MTX levels obtained by this method in leukemia patients exhibited clinical correlation and an excellent agreement with commercial immunoassay used in parallel. Conclusion We were able to develop a rapid, sensitive, and cost-effective LC-MS/MS method suitable for therapeutic drug monitoring of MTX in routine clinical diagnostic laboratories.

Effect of glucocorticoids on the development of COVID-19-associated pulmonary aspergillosis: A meta-analysis of 21 studies and 5174 patients.

COVID-19-associated pulmonary aspergillosis (CAPA) remains a high mortality mycotic infection throughout the pandemic, and glucocorticoids (GC) may be its root cause. Our aim was to evaluate the effect of systemic GC treatment on the development of CAPA. We systematically searched the PubMed, Google Scholar, Scopus and Embase databases to collect eligible studies published until 31 December 2022. The pooled outcome of CAPA development was calculated as the log odds ratio (LOR) with 95% confidence intervals (CI) using a random effect model. A total of 21 studies with 5174 patients were included. Of these, 20 studies with 4675 patients consisting of 2565 treated with GC but without other immunomodulators (GC group) and 2110 treated without GC or other immunomodulators (controls) were analysed. The pooled LOR of CAPA development was higher for the GC group than for the controls (0.54; 95% CI: 0.22, 0.86; p < .01). In the subgroups, the pooled LOR was higher for high-dose GC (0.90; 95% CI: 0.17, 1.62: p = .01) and dexamethasone (0.71; 95% CI: 0.35, 1.07; p < .01) but had no significant difference for low-dose GC (0.41; 95% CI: -0.07, 0.89; p = .09), and non-dexamethasone GC (0.21; 95% CI: -0.36, 0.79; p = .47), treated patients versus controls. GC treatment increases the risk of CAPA development, and this risk is particularly associated with the use of high-dose GC or dexamethasone treatment.

Analysis of Immunophenotypic Changes during Ex Vivo Human Erythropoiesis and Its Application in the Study of Normal and Defective Erythropoiesis.

Erythropoiesis is a highly regulated process and undergoes several genotypic and phenotypic changes during differentiation. The phenotypic changes can be evaluated using a combination of cell surface markers expressed at different cellular stages of erythropoiesis using FACS. However, limited studies are available on the in-depth phenotypic characterization of progenitors from human adult hematopoietic stem and progenitor cells (HSPCs) to red blood cells. Therefore, using a set of designed marker panels, in the current study we have kinetically characterized the hematopoietic, erythroid progenitors, and terminally differentiated erythroblasts ex vivo. Furthermore, the progenitor stages were explored for expression of CD117, CD31, CD41a, CD133, and CD45, along with known key markers CD36, CD71, CD105, and GPA. Additionally, we used these marker panels to study the stage-specific phenotypic changes regulated by the epigenetic regulator; Nuclear receptor binding SET Domain protein 1 (NSD1) during erythropoiesis and to study ineffective erythropoiesis in myelodysplastic syndrome (MDS) and pure red cell aplasia (PRCA) patients. Our immunophenotyping strategy can be used to sort and study erythroid-primed hematopoietic and erythroid precursors at specified time points and to study diseases resulting from erythroid dyspoiesis. Overall, the current study explores the in-depth kinetics of phenotypic changes occurring during human erythropoiesis and applies this strategy to study normal and defective erythropoiesis.

New insights into development and mortality of COVID-19-associated pulmonary aspergillosis in a homogenous cohort of 1161 intensive care patients.

BACKGROUND: COVID-19-associated pulmonary aspergillosis (CAPA) has been widely reported but homogenous large cohort studies are needed to gain real-world insights about the disease. METHODS: We collected clinical and laboratory data of 1161 patients hospitalised at our Institute from March 2020 to August 2021, defined their CAPA pathology, and analysed the data of CAPA/non-CAPA and deceased/survived CAPA patients using univariable and multivariable models. RESULTS: The overall prevalence and mortality of CAPA in our homogenous cohort of 1161 patients were 6.4% and 47.3%, respectively. The mortality of CAPA was higher than that of non-CAPA patients (hazard ratio: 1.8 [95% confidence interval: 1.1-2.8]). Diabetes (odds ratio [OR] 1.92 [1.15-3.21]); persistent fever (2.54 [1.17-5.53]); hemoptysis (7.91 [4.45-14.06]); and lung lesions of cavitation (8.78 [2.27-34.03]), consolidation (9.06 [2.03-40.39]), and nodules (8.26 [2.39-28.58]) were associated with development of CAPA by multivariable analysis. Acute respiratory distress syndrome (ARDS) (2.68 [1.09-6.55]), a high computed tomography score index (OR 1.18 [1.08-1.29]; p < .001), and pulse glucocorticoid treatment (HR 4.0 [1.3-9.2]) were associated with mortality of the disease. Whereas neutrophilic leukocytosis (development: 1.09 [1.03-1.15] and mortality: 1.17 [1.08-1.28]) and lymphopenia (development: 0.68 [0.51-0.91] and mortality: 0.40 [0.20-0.83]) were associated with the development as well as mortality of CAPA. CONCLUSION: We observed a low but likely underestimated prevalence of CAPA in our study. CAPA is a disease with high mortality and diabetes is a significant factor for its development while ARDS and pulse glucocorticoid treatment are significant factors for its mortality. Cellular immune dysregulation may have a central role in CAPA from its development to mortality.

Comparison of the Cardiomyogenic Potency of Human Amniotic Fluid and Bone Marrow Mesenchymal Stem Cells.

BACKGROUND AND OBJECTIVES: Most studies in cardiac regeneration have explored bone marrow mesenchymal stem cells (BM-MSC) with variable therapeutic effects. Amniotic fluid MSC (AF-MSC) having extended self-renewal and multipotent properties may be superior to bone marrow MSC (BM-MSC). However, a comparison of their cardiomyogenic potency has not been studied yet. METHODS: The 5-azacytidine (5-aza) treated AF-MSC and BM-MSC were evaluated for the expression of GATA-4, Nkx2.5 and ISL-1 transcripts and proteins by quantitative RT-PCR and Western blotting, respectively as well as for the expression of cardiomyogenic differentiation markers cardiac troponin-T (cTNT), beta myosin heavy chain (ßMHC) and alpha sarcomeric actinin (ASA) by immunocytochemistry. RESULTS: The AF-MSC as compared to BM-MSC had significantly higher expression of GATA-4 (183.06±29.85 vs. 9.80±0.05; p<0.01), Nkx2.5 (8.3±1.4 vs. 1.82±0.32; p<0.05), and ISL-1 (39.59±4.05 vs. 4.36±0.39; p<0.01) genes as well as GATA-4 (2.01±0.5 vs. 0.6±0.1; p<0.05), NKx2.5 (1.9±0.14 vs. 0.8±0.2; p<0.01) and ISL-1 (1.7±0.3 vs. 0.9±0.1; p<0.05) proteins. The AF-MSC also had significantly elevated expression of cTNT (5.0×104±0.6×104 vs. 3.5×104±0.8×104; p<0.01), ß-MHC (15.7×104±0.9×104 vs. 8.2×104±0.6×104; p<0.01) and ASA (18.6×104±4.9×104 vs. 13.1×104±3.0×104; p<0.05) than BM-MSC. CONCLUSIONS: Our data suggest that AF-MSC have greater cardiomyogenic potency than BM-MSC, and thus may be a better source of MSC for therapeutic applications in cardiac regenerative medicine.

Altered Expression of Hematopoiesis Regulatory Molecules in Lipopolysaccharide-Induced Bone Marrow Mesenchymal Stem Cells of Patients with Aplastic Anemia.

We have investigated the expression of RNA transcripts of hematopoiesis regulatory molecules, viz., macrophage inflammatory protein (MIP)-1α, tumor necrosis factor (TNF)-α, granulocyte colony-stimulating factor (G-CSF), stromal cell-derived factor (SDF)-1α, stem cell factor (SCF), and transforming growth factor (TGF)-ß in lipopolysaccharide-induced bone marrow mesenchymal stem cells (BM-MSCs) and levels of their soluble forms in the culture supernatants of BM-MSCs and BM plasma of patients with acquired aplastic anemia (AA) (n = 29) and controls (n = 29). The BM-MSCs of AA patients as compared to controls had markedly lower expression of MIP-1α transcripts (p < 0.001), higher expression of TNF-α (p < 0.001), G-CSF (p < 0.001), and SDF-1α (p < 0.01) transcripts, and no difference in the expression of SCF and TGF-ß transcripts. The culture supernatants of BM-MSCs and BM plasma of AA patients in comparison to controls also had lower levels of MIP-1α (p < 0.01 and p < 0.001, respectively) and higher levels of TNF-α (p < 0.05 for both) and G-CSF (p < 0.05 and p < 0.001, respectively) but with no difference in the levels of SDF-1α and SCF. The levels of TGF-ß were although similar in culture supernatants of BM-MSCs of both the groups, but they were significantly lower in BM plasma of the patients than controls (p < 0.001). Our data shows that BM-MSCs of AA patients have altered expression of hematopoiesis regulatory molecules suggesting that they may have a role in the pathogenesis of the disease.

Cardiomyogenic Heterogeneity of Clonal Subpopulations of Human Bone Marrow Mesenchymal Stem Cells.

We have evaluated the cardiomyogenic potential of clonal populations of human bone marrow mesenchymal stem cells (BM-MSC). Four rapidly proliferating clones of BM-MSC were obtained from the BM of a healthy donor which were then treated with 5-azacytidine and evaluated for the expression of GATA-4, NKx-2.5, FOG-2, TDGF-1, ß-MHC, MEF2D and NPPA genes and cTnT, Desmin and ß-MHC proteins. Of the four clones (i) Clone-1 had high expression of GATA-4 (1.89 fold (p<0.05), Nkx2.5 (2.29 fold; p<0.05), FOG2 (2.76 fold; p<0.05), TDGF1 (6.97 fold, p<0.005), ßMHC (10.22 fold; p<0.005), MEF-2D (1.91 fold; p<0.005) and NPPA (1.65 fold; p<0.005); (ii) clone-2 had up-regulation of Nkx2.5 (1.98 fold; p<0.05) but down-regulation of rest of the genes; (iii) clone-3 had up-regulation of Nkx2.5 (2.11 fold; p<0.05), TDGF1 (1.88 fold; p<0.05), MEF-2D (1.30 fold; p<0.05) and NPPA (1.21 fold; p<0.05), down regulation of GATA-4 and Fog-2 but no change in ßMHC gene; and (iv) clone-4 had up-regulation of MEF-2D (1.17 fold; p<0.05) and down regulation of GATA-4, Nkx2.5 but no change in other genes compared to untreated cells of the clones. At the protein level, clone-1 expressed cTnT, Desmin, and ßMHC; clone-2 Desmin only while clones-3 and 4 each expressed cTnT, Desmin, and ßMHC. Our data shows that BM-MSC are a heterogenous population of stem cells with sub-populations exhibiting a marked difference in the expression of cardiac markers both at gene and protein levels. This highlights that administering selected sub-populations of BM-MSC with a cardiomyogenic potential may be more efficacious than whole population of cells for cardiac regeneration.

Fetal kidney stem cells ameliorate cisplatin induced acute renal failure and promote renal angiogenesis.

AIM: To investigate whether fetal kidney stem cells (fKSC) ameliorate cisplatin induced acute renal failure (ARF) in rats and promote renal angiogenesis. METHODS: The fKSC were isolated from rat fetuses of gestation day 16 and expanded in vitro up to 3(rd) passage. They were characterized for the expression of mesenchymal and renal progenitor markers by flow cytometry and immunocytochemistry, respectively. The in vitro differentiation of fKSC towards epithelial lineage was evaluated by the treatment with specific induction medium and their angiogenic potential by matrigel induced tube formation assay. To study the effect of fKSC in ARF, fKSC labeled with PKH26 were infused in rats with cisplatin induced ARF and, the blood and renal tissues of the rats were collected at different time points. Blood biochemical parameters were studied to evaluate renal function. Renal tissues were evaluated for renal architecture, renal cell proliferation and angiogenesis by immunohistochemistry, renal cell apoptosis by terminal deoxynucleotidyl transferase nick-end labeling assay and early expression of angiogenic molecules viz. vascular endothelial growth factor (VEGF), hypoxia-inducible factor (HIF)-1α and endothelial nitric oxide synthase (eNOS) by western blot. RESULTS: The fKSC expressed mesenchymal markers viz. CD29, CD44, CD73, CD90 and CD105 as well as renal progenitor markers viz. Wt1, Pax2 and Six2. They exhibited a potential to form CD31 and Von Willebrand factor expressing capillary-like structures and could be differentiated into cytokeratin (CK)18 and CK19 positive epithelial cells. Administration of fKSC in rats with ARF as compared to administration of saline alone, resulted in a significant improvement in renal function and histology on day 3 (2.33 ± 0.33 vs 3.50 ± 0.34, P < 0.05) and on day 7 (0.83 ± 0.16 vs 2.00 ± 0.25, P < 0.05). The infused PKH26 labeled fKSC were observed to engraft in damaged renal tubules and showed increased proliferation and reduced apoptosis (P < 0.05) of renal cells. The kidneys of fKSC as compared to saline treated rats had a higher capillary density on day 3 [13.30 ± 1.54 vs 7.10 ± 1.29, capillaries/high-power fields (HPF), P < 0.05], and on day 7 (21.10 ± 1.46 vs 15.00 ± 1.30, capillaries/HPF, P < 0.05). In addition, kidneys of fKSC treated rats had an up-regulation of angiogenic proteins hypoxia-inducible factor-1α, VEGF and eNOS on day 3 (P < 0.05). CONCLUSION: Our study shows that fKSC ameliorate cisplatin induced ARF in rats and promote renal angiogenesis, which may be an important therapeutic mechanism of these stem cells in the disease.

Fetal Kidney Cells Can Ameliorate Ischemic Acute Renal Failure in Rats through Their Anti-Inflammatory, Anti-Apoptotic and Anti-Oxidative Effects.

Fetal kidney cells may contain multiple populations of kidney stem cells and thus appear to be a suitable cellular therapy for the treatment of acute renal failure (ARF) but their biological characteristics and therapeutic potential have not been adequately explored. We have culture expanded fetal kidney cells derived from rat fetal kidneys, characterized them and evaluated their therapeutic effect in an ischemia reperfusion (IR) induced rat model of ARF. The fetal kidney cells grew in culture as adherent spindle shaped/polygonal cells and expressed CD29, CD44, CD73, CD90, CD105, CD24 and CD133 markers. Administration of PKH26 labeled fetal kidney cells in ARF rats resulted in a significant decrease in the levels of blood urea nitrogen, creatinine, and neutrophil gelatinase-associated lipocalin and decreased tubular necrosis in the kidney tissues (p<0.05 for all). The injected fetal kidney cells were observed to engraft around injured tubular cells, and there was increased proliferation and decreased apoptosis of tubular cells in the kidneys (p<0.05 for both). In addition, the kidney tissues of ARF rats treated with fetal kidney cells had a higher gene expression of renotropic growth factors (VEGF-A, IGF-1, BMP-7 and bFGF) and anti-inflammatory cytokine (IL10); up regulation of anti-oxidative markers (HO-1 and NQO-1); and a lower Bax/Bcl2 ratio as compared to saline treated rats (p<0.05 for all). Our data shows that culture expanded fetal kidney cells express mesenchymal and renal progenitor markers, and ameliorate ischemic ARF predominantly by their anti-apoptotic, anti-inflammatory and anti-oxidative effects.

Enhanced adipogenicity of bone marrow mesenchymal stem cells in aplastic anemia.

Fatty bone marrow (BM) and defective hematopoiesis are a pathologic hallmark of aplastic anemia (AA). We have investigated adipogenic and osteogenic potential of BM mesenchymal stem cells (BM-MSC) in 10 AA patients (08 males and 02 females) with median age of 37 years (range: 06 to 79 years) and in the same number of age and sex matched controls. It was observed that BM-MSC of AA patients had a morphology, phenotype, and osteogenic differentiation potential similar to control subjects but adipocytes differentiated from AA BM-MSC had a higher density and larger size of lipid droplets and they expressed significantly higher levels of adiponectin and FABP4 genes and proteins as compared to control BM-MSC (P < 0.01 for both). Thus our data shows that AA BM-MSC have enhanced adipogenicity, which may have an important implication in the pathogenesis of the disease.

Comparison of phenotypic markers and neural differentiation potential of multipotent adult progenitor cells and mesenchymal stem cells.

AIM: To compare the phenotypic and neural differentiation potential of human bone marrow derived multipotent adult progenitor cells (MAPC) and mesenchymal stem cells (MSC). METHODS: Cultures of MAPC and MSC were established in parallel from same samples of human bone marrow (n = 5). Both stem cell types were evaluated for expression of pluripotency markers including Oct-4 and Nanog by immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) and expression of standard mesenchymal markers including CD14, CD34, CD44, CD45, CD73, CD90, CD105 and human leukocyte antigen (HLA)-ABC by flow cytometry. After treatment with neural induction medium both MAPC and MSC were evaluated for expression of neural proteins [neuronal filament-200 (NF-200) and glial fibrillar acidic protein (GFAP)] by immunocytochemistry and Western blotting and neural genes [NF-200, GFAP, Tau, microtubule-associated protein (MAP)-1B, MAP-2, neuron-specific enolase (NSE) and oligodendrocyte-1 (Olig-1)] by quantitative real-time-PCR. RESULTS: MAPC had small trigonal shaped while MSC had elongated spindle-shaped morphology. The MAPC expressed Oct-4 and Nanog both at gene and protein levels, whereas MSC were negative for these pluripotent markers. MAPC were negative for HLA-ABC while MSC had high expression of HLA-ABC. In addition, MAPC as compared to MSC had significantly lower expression of CD44 (36.56% ± 1.92% vs 98.23% ± 0.51%), CD73 (15.11% ± 2.24% vs 98.53% ± 2.22%) and CD105 (13.81% ± 3.82% vs 95.12% ± 5.65%) (P < 0.001, for all) MAPC cultures compared to MSC cultures treated with neural induction medium had significantly higher fold change expression of NF-200 (0.64), GFAP (0.52), Tau (0.59), MAP-2 (0.72), Olig-1 (0.18) and NSE (0.29) proteins (P < 0.01 for Olig-1 and P < 0.001 for rest) as well as higher fold change expression of genes of NF-200 (1.34), GFAP (1.12), Tau (1.08), MAP-1B (0.92), MAP-2 (1.14) and NSE (0.4) (P < 0.001 for all). CONCLUSION: MAPC can be differentially characterized from MSC as Oct-4 and Nanog positive stem cells with no expression of HLA-ABC and low expression of mesenchymal markers CD44, CD73 and CD105 and when compared to MSC they possess greater predilection for differentiation into neuro-ectodermal lineage.

Fetal cardiac mesenchymal stem cells express embryonal markers and exhibit differentiation into cells of all three germ layers.

AIM: To study the expression of embryonal markers by fetal cardiac mesenchymal stem cells (fC-MSC) and their differentiation into cells of all the germ layers. METHODS: Ten independent cultures of rat fC-MSC were set up from cells derived from individual or pooled fetal hearts and studies given below were carried out at passages 3, 6, 15 and 21. The phenotypic markers CD29, CD31, CD34, CD45, CD73, CD90, CD105, CD166 and HLA-DR were analyzed by flow cytometry. The expression of embryonal markers Oct-4, Nanog, Sox-2, SSEA-1, SSEA-3, SSEA-4, TRA-1-60 and TRA 1-81 were studied by immunocytochemistry. The fC-MSC treated with specific induction medium were evaluated for their differentiation into (1) adipocytes and osteocytes (mesodermal cells) by Oil Red O and Alizarin Red staining, respectively, as well as by expression of lipoprotein lipase, PPARγ2 genes in adipocytes and osteopontin and RUNX2 genes in osteocytes by reverse-transcription polymerase chain reaction (RT-PCR); (2) neuronal (ectodermal) cells by expression of neuronal Filament-160 and Glial Fibrillar Acidic Protein by RT-PCR and immunocytochemistry; and (3) hepatocytic (endodermal) cells by expression of albumin by RT-PCR and immunocytochemistry, glycogen deposits by Periodic Acid Schiff staining and excretion of urea into the culture supernatant. RESULTS: The fC-MSC expressed CD29, CD73, CD90, CD105, CD166 but lacked expression of CD31, CD34, CD45 and HLA-DR. They expressed embryonal markers, viz. Oct-4, Nanog, Sox-2, SSEA-1, SSEA-3, SSEA-4, TRA-1-81 but not TRA-1-60. On treatment with specific induction media, they differentiated into adipocytes and osteocytes, neuronal cells and hepatocytic cells. CONCLUSION: Our results together suggest that fC-MSC are primitive stem cell types with a high degree of plasticity and, in addition to their suitability for cardiovascular regenerative therapy, they may have a wide spectrum of therapeutic applications in regenerative medicine.

Antigenic targets and pathogenicity of anti-aortic endothelial cell antibodies in Takayasu arteritis.

OBJECTIVE: Anti-endothelial cell antibodies are considered to have an important role in the pathogenesis of Takayasu arteritis (TA). Previously, these antibodies were detected using human umbilical vein endothelial cells, which do not completely represent the antigenicity/functions of aortic endothelial cells, the specific targets in TA. To delineate the precise role of antigenic targets, we investigated such targets as well as the pathogenic mechanism of antibodies directed against aortic endothelial cells (AAECAs) in TA. METHODS: AAECAs were detected using a cellular enzyme-linked immunosorbent assay (ELISA), and their antigenic targets were detected by immunoblotting. AAECA-mediated induction of endothelial adhesion molecule expression and cytokine production was studied by ELISA, and apoptosis was studied using the TUNEL method. RESULTS: AAECAs were detected in 86% of patients with TA and in 9% of controls. Sera obtained from AAECA-positive patients with TA recognized a total of 9 antigens ranging in size from 18 kd to 200 kd, of which the 60-65-kd triplet was recognized most often. The aortic endothelial cell reactivity of Hsp60-absorbed sera was reduced by approximately 50% as compared with that of unabsorbed sera (mean +/- SD 0.488 +/- 0.08 versus 0.838 +/- 0.116). Sera from AAECA-positive patients with TA, compared with sera from AAECA-negative patients with TA and that from controls, induced increased expression of E-selectin (mean +/- SD 0.833 +/- 0.063 versus 0.217 +/- 0.081 and 0.221 +/- 0.101 optical density [OD] units, respectively) and vascular cell adhesion molecule 1 (0.620 +/- 0.144 versus 0.165 +/- 0.005 and 0.177 +/- 0.055 OD units, respectively) and increased production of interleukin-4 (IL-4) (6.8 +/- 2.4 versus 1.2 +/- 1.6 and 1.3 +/- 2.5 pg/ml, respectively), IL-6 (24.3 +/- 2.4 versus 4.5 +/- 6.7 and 5.9 +/- 5.1 pg/ml, respectively), and IL-8 (36.8 +/- 10.3 versus 10.1 +/- 6.7 and 7.8 +/- 2.1 pg/ml, respectively). Sera from AAECA-positive patients with TA induced 29 +/- 6% (median +/- SEM) apoptosis of aortic endothelial cells. CONCLUSION: Our data show that the AAECAs that are present in patients with TA are directed mainly against 60-65-kd antigen(s) and may cause vascular dysfunction by inducing expression of endothelial adhesion molecules, cytokine production, and apoptosis.

T-cell receptor repertoire of circulating gamma delta T-cells in Takayasu's arteritis.

We studied T-cell receptor (TCR) repertoire of circulating gamma delta (gammadelta) T-cells in 20 patients with Takayasu's arteritis (TA), 20 healthy controls (HC), 7 follow up TA patients, and 10 patients with rheumatoid arthritis (RA) and 5 Wegener's granulomatosis (WG) patients as disease controls. Patients with TA (8.1 +/- 5.1%) compared to HC (3.7 +/- 2.1%, P = 0.014), RA (4.8 +/- 0.6%, P = 0.032), and WG (4.2 +/- 0.8%, P = 0.030) as well as active TA compared to inactive TA (13.9 +/- 4.1% vs. 4.9 +/- 1.5%; P < 0.001) had higher number of gammadelta T-cells. The numbers of Vdelta1+ cells were significantly higher in patients with TA (40.0 +/- 20.8%) than HC (13.1 +/- 8.0%; P = 0.001), RA (19.5 +/- 1.8%, P = 0.004), and WG (17.0 +/- 3.9%, P = 0.007). The numbers of gammadelta T-cells normalized in all the 7 patients after 180 days of follow up (13.9 +/- 4.1% vs. 6.9 +/- 2.5%; P = 0.001). We also observed higher number of activated and IFN-gamma producing gammadelta T-cells in active TA. Our data show that gammadelta T-cells particularly those bearing Vdelta1 TCR may have an important role in the immunopathogenesis of TA.

High TNF-alpha and low IL-2 producing T cells characterize active disease in Takayasu's arteritis.

We have investigated intracellular production by T cells and plasma levels of TNF-alpha, IL-2 and IFN-gamma in 12 active and 10 inactive Takayasu's arteritis (TA) patients and 12 healthy controls. The active TA compared to inactive TA and controls had higher TNF-alpha (52.7 +/- 22.3% vs. 32.9 +/- 14.2% and 35.2 +/- 14.5%, respectively; P = 0. 020), lower IL-2 (19.6 +/- 13.2% vs. 36.1 +/- 10.1% and 31.2 +/- 10.3%, respectively; P = 0.010) and comparable IFN-gamma (38.6 +/- 13.9% vs. 34.2 +/- 12.4% and 34.9 +/- 11.1%, respectively; P = 0.581) producing CD3+ T cells. There was no difference in the plasma levels of the cytokines between active TA, inactive TA and controls (TNF-alpha: 79.1 +/- 94.5 vs. 72.9 +/- 120.0 and 9.5 +/- 6.7 pg/ml, P = 0.110; IL-2: 4.3 +/- 4.8 vs. 6.6 +/- 4.7 and 8.6 +/- 4.5 pg/ml, P = 0.094 and IFN-gamma: 10.1 +/- 11.3 vs. 8.8 +/- 8.7 and 8.2 +/- 6.5 pg/ml, P = 0.871, respectively). The data show an important role of these high TNF-alpha and low IL-2 producing T cells in TA.

Bone marrow and blood plasma levels of IL-8 in aplastic anemia and their relationship with disease severity.

Interleukin-8 (IL-8), a CXC chemokine, is also a potent inhibitor of myelopoiesis, the hematopoietic process that is severely impaired in aplastic anemia (AA). To elucidate its role in the disease, we have investigated levels of IL-8 by quantitative enzyme-linked immunosorbent assay in bone marrow and peripheral blood plasma of 27 AA patients and in the marrow of 16 controls and blood of 20 controls. Significantly increased levels of IL-8 were observed in the marrow and blood of patients as compared to controls (470.4 +/- 549.6 vs. 37.5 +/- 30.3; P < 0.0001) and (247.3 +/- 286.3 vs. 7.9 +/- 5.5; P < 0.0001), respectively. Among the patients, the IL-8 levels were higher in patients with severe AA than those with nonsevere AA in the marrow (568.8 +/- 586.9 vs. 126.3 +/- 102.5; P < 0.005) as well as in the blood (296.6 +/- 305.5 vs. 75.0 +/- 84.4; P < 0.008) plasma. The marrow and blood of 74% (20/27) of the patients had increased levels of IL-8 compared to 12% (2/16; P < 0.001) and 10% (2/20, P < 0.001) of the controls, respectively. These results suggest that IL-8 may have an important role in the pathogenesis of AA.

Antimonocyte antibodies in Takayasu's arteritis: prevalence of and relation to disease activity.

OBJECTIVE: To investigate the prevalence of antimonocyte antibodies (AMA) in Takayasu's arteritis (TA) and their relationship with disease activity. METHODS: IgG-AMA were studied in the sera of 60 patients with TA (29 active disease, 31 inactive) and 43 controls by a cellular ELISA using glutaraldehyde fixed U-937 cells or peripheral blood monocytes as antigen. Relationship of AMA with disease activity was evaluated by measuring titers of these antibodies in followup sera of 15 AMA positive patients with active TA undergoing immunosuppressive therapy. RESULTS: Twenty-six of 60 TA patients (43%) compared to 4 of 43 controls (9%) (p < 0.001) and 20 of 29 patients with active disease (69%) compared to 6 of 31 patients with inactive disease (19%) (p < 0.001) were positive for AMA. The antibody titers were significantly higher in patients with active disease than those with inactive disease (0.396 +/- 0.172 vs 0.232 +/- 0.096; p < 0.001). In the followup study of 15 patients with active disease who received immunosuppressive therapy, we observed normalization of AMA titers in 6 of the 7 patients who became inactive, compared to only one of the 8 patients whose disease remained active during followup (p < 0.01). CONCLUSION: AMA are present in a significant proportion of patients with TA and correlate with disease activity, suggesting a possible pathogenic role of these antibodies in TA.