Soniya Nityanand

Intravenous Autologous Bone Marrow Mononuclear Stem Cell Therapy for Ischemic Stroke: A Multicentric, Randomized Trial

Background and Purpose— Pilot studies have suggested benefit from intravenous administration of bone marrow mononuclear stem cells (BMSCs) in stroke. We explored the efficacy and safety of autologous BMSCs in subacute ischemic stroke. Methods— This was a phase II, multicenter, parallel group, randomized trial with blinded outcome assessment that included 120 patients. Patients with subacute ischemic stroke were randomly assigned to the arm that received intravenous infusion of autologous BMSCs or to control arm. Coprimary clinical efficacy outcomes were Barthel Index score and modified Rankin scale at day 180. Secondary outcomes were change in infarct volume, National Institute of Health Stroke Scale (NIHSS) at day 90 and 180. Main safety outcomes were adverse events, any new area of 18fluorodeoxyglucose positron emission tomography uptake in any body part over 365 days. Results— Fifty-eight patients received a mean of 280.75 million BMSCs at median of 18.5 days after stroke onset. There was no significant difference between BMSCs arm and control arm in the Barthel Index score (63.1 versus 63.6; P=0.92), modified Rankin scale shift analysis (P=0.53) or score >3 (47.5% versus 49.2%; P=0.85), NIHSS score (6.3 versus 7.0; P=0.53), change in infarct volume (−11.1 versus −7.36; P=0.63) at day 180. Adverse events were also similar in the 2 arms, and no patient showed any new area of 18fluorodeoxyglucose uptake. Conclusions— With the methods used, results of this hitherto first randomized controlled trial indicate that intravenous infusion of BMSCs is safe, but there is no beneficial effect of treatment on stroke outcome. Clinical Trial Registration— URLs: http://ctri.nic.in/Clinicaltrials and http://www.clinicaltrials.gov. Unique identifiers: CTRI-ROVCTRI/2008/091/0004 and NCT0150177.

Cellular and humoral immune responses to mycobacterial heat shock protein-65 and its human homologue in Takayasu's arteritis

Expression of heat shock protein (HSP)‐65 as well as infiltration of T‐cells in arterial lesions and raised levels of circulating antibodies against mycobacterial HSP65 (mHSP65) led us to the concept that mHSP65 or its human homologue (hHSP60) might be involved in the etiopathogenesis of Takayasus arteritis (TA). Therefore, we investigated mHSP65 and hHSP60 reactive peripheral blood T‐cell subsets by BrdU incorporation assay and flow cytometry as well as investigating the different isotypes of anti‐mHSP65 and hHSP60 antibodies by ELISA. Eighty‐four percent (22/26) of the TA patients were observed to show T‐cell proliferation to mHSP65 and hHSP60 whereas only 16% (3/18) healthy controls showed such proliferation (P < 0·001). Both HSPs induced proliferation of exclusively CD4+ T‐cells and not CD8+ T‐cells. We also observed a significantly higher prevalence of only the IgG isotype reactive to mHSP65 and hHSP60 in TA as compared to HC (mHSP65: 92% TA versus 11% HC, P < 0·0001 and hHSP60: 84% versus 22%, P < 0·001). Our data show a significant correlation between mHSP65 and hHSP60 reactive T‐cells (CD3+: r = 0·901; CD4+: r = 0·968) as well as anti‐mHSP65 and anti‐hHSP60 IgG antibodies (r = 0·814) suggesting an infection induced autoimmunity in TA, possibly induced by molecular mimicry between mHSP65 and hHSP60 or other tissue specific antigens.

Cytokine mRNA repertoire of peripheral blood mononuclear cells in Takayasu's arteritis

We have investigated constitutive and phytohaemagglutinin (PHA) + phorbol 12‐myristate 13‐acetate (PMA)‐induced gene expression of tumour necrosis factor (TNF)‐α, interferon (IFN)‐γ, interleukin (IL)‐2, IL‐3, IL‐4, IL‐10, IL‐12 and granulocyte macrophage colony‐stimulating factor (GM‐CSF) in peripheral blood mononuclear cells (PBMCs) of 10 patients with Takayasus arteritis (TA) and 10 healthy controls by semiquantitative reverse transcriptase polymerase chain reaction (RT‐PCR). The constitutive mRNA expression of TNF‐α (69·0 ± 4·0%versus 27·5 ± 18·0%; P = 0·001) and IL‐4 (60·0 ± 10·0%versus 0%; P = 0·001) was significantly higher in patients than controls; that of IL‐3 was comparable in both groups (38·0 ± 6·0%versus 32·0 ± 5·0%; P = 0·651) while no constitutive mRNA expression was observed for the other cytokines studied. The stimulated PBMCs of patients, as compared with the controls, had higher mRNA gene expression of TNF‐α (127·0 ± 16·0%versus 54·0 ± 6·0%; P = 0·001), IFN‐γ (93·0 ± 13·0%versus 57·0 ± 5·0%; P = 0·032), IL‐2 (109·0 ± 13·0%versus 68·0 ± 6·0%; P = 0·015), IL‐3 (60·0 ± 8·0%versus 21·2 ± 3·0%; P = 0·045) and IL‐4 (68·0 ± 7·0%versus 27·0 ± 7·2%; P = 0·01) The mRNA expression of IL‐10 was lower in patients than controls (35·0 ± 8·0%versus 75·0 ± 12·0%; P = 0·022). The GM‐CSF mRNA was similar (102·0 ± 6·0%versus 89·0 ± 5·0%; P = 0·475) in both groups. Stimulation of cells with PHA + PMA showed no IL‐12 expression but stimulation with lipopolysaccharide induced higher IL‐12 mRNA in patients than controls (83·0 ± 14·0%versus 33·0 ± 4·0%; P = 0·005). Our data suggest that an inflammatory cytokine signature exists in TA with a key role for TNF‐α, IL‐4, IL‐10 and IL‐12 in different pathological processes of the disease.

Expanded T cell populations in patients with wegener's granulomatosis: characteristics and correlates with disease activity

Patients with Wegeners granulomatosis have a high prevalence of expanded populations of CD4+ and CD8+ T cells bearing different α/β T cell receptors. To elucidate the role of these populations, we studied the phenotypic and functional characteristics of 13 expanded T cell populations in four patients for a period of 35–51 months. The expanded populations generally showed a persistently high expression of the activation markers HLA-DR and CD25. This expression was independent of the activity of the disease. The expanded populations also expressed CD45RO and/or CD45RA and most of them expressed CD57 but not CD28. Analysis of intracellular presence and secretion of IFN-γ, IL-2, and IL-4 showed that most of the expanded cell populations contained and/or secreted more of these cytokines than the nonexpanded populations, with an especially high expression/secretion of IFN-γ and IL-2. The expanded populations showed little proliferative response to Con A and OKT3. The proliferative response of the cells was partly restored after preincubation in medium alone. Some of the expanded populations were associated with disease activity, thus suggesting a link between expanded T cells and the disease. The activated status of the expanded populations and the tendency for certain populations to correlate in magnitude with disease activity suggest their involvement in the disease process. The relative stability of these cell populations indicates that the stimulus driving them is persistent, in agreement with the chronicity of the disease.

T cell receptor (TCR) V gene usage in patients with systemic necrotizing vasculitis

Wegeners granulomatosis (WG) and polyarteritis nodosa (PAN) are systemic necrotizing vasculitides of unknown etiology. These disorders run a fatal course if untreated. T lymphocytes are implicated in the pathogenesis of WG, since they have been found to infiltrate affected organs, and sIL‐2R correlates with disease activity. To elucidate further the role of T cells in necrotizing vasculitis, we have used a panel of 12 TCR V‐specific MoAbs to investigate the number of cells expressing certain Vα and Vβ gene segments in the CD4+ and CD8+ subsets of altogether 11 patients with WG or PAN. In the group of patients, we found abnormal expansions of T cells using particular TCR Vα or β gene products. These T cell expansions were more numerous, of a dramatically higher magnitude, and frequently more often found in the CD4 subset, compared with T cell expansions identified in healthy individuals. In long‐term studies of the T cell expansions for up to 18 months, a heterogeneous pattern was revealed, with no obvious correlation to clinical features such as disease activity or treatment. Studies of TCR V gene usage in this group of patients may help in understanding the pathogenesis of necrotizing vasculitis, and in the identification of unknown antigens, and may open the possibility to a highly selective immunotherapy by targeting disease‐mediating T cells.

Anti-annexin V antibodies in Takayasu's arteritis: prevalence and relationship with disease activity.

Annexin V has an important role in the regulation of apoptosis and antibodies directed against it have been shown to lead to apoptosis of vascular endothelial cells. To evaluate the role of anti‐annexin V antibodies (AA5A) in Takayasus arteritis (TA), we investigated these antibodies in the sera of 66 TA patients, 50 healthy controls and in the follow‐up sera of 12 active TA patients by enzyme‐linked immunosorbent assay. The AA5A‐positive patients were analysed further for the presence of anti‐endothelial cell antibodies (AECA) and anticardiolipin antibodies (ACLA) to determine the relationship of AA5A with these autoantibodies. AA5A were observed in 36% (24/66) of the patients versus 6% (3/50) of the controls (P < 0·001) and in 53% (19/36) of patients with active TA versus 17% (5/30) of those with inactive disease (P < 0·01). Levels of AA5A were also observed to be significantly higher in patients with TA compared to controls (0·557 ± 0·362 versus 0·259 ± 0·069; P < 0·0001) and in patients with active disease compared to those with inactive disease (0·700 ± 0·403 versus 0·385 ± 0·205; P < 0·0001). In the follow‐up study, 6/12 patients who became inactive during follow‐up also showed normalization of AA5A levels. AECA and ACLA were detected in 54% (13/24) and 12% (3/24) of the AA5A‐positive patients, respectively. Our results show that a significant proportion of TA patients have AA5A, which exhibit an association with AECA and because they have a correlation with disease activity thus appear to be involved in the disease pathogenesis.

Expression of interferon-γ and tumor necrosis factor-α in bone marrow T cells and their levels in bone marrow plasma in patients with aplastic anemia

Immune-mediated stem cell damage has been postulated to be responsible for disease initiation and progression in aplastic anemia (AA). It is hypothesized that T lymphocytes play a major role in destroying the bone marrow (BM) stem cells of AA patients by infiltrating the BM and secreting excessive levels of anti-hematopoietic cytokines, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α). We undertook this study to assess the pathogenic significance of anti-hematopoietic cytokines such as IFN-γ and TNF-α in BM T cells and plasma of AA patients. Significantly elevated levels of IFN-γ and TNF-α were found in the BM plasma of AA patients compared to controls (p=0.05 and 0.006, respectively). Intracellular IFN-γ and not TNF-α in BM CD3+ T cells of AA patients was significantly higher compared to controls (p=0.04 and p=0.2, respectively). A follow-up analysis of expression of these cytokines in BM T cells and their levels in BM plasma in five AA patients before and 180 days (6 months) after antithymocyte globulin (ATG) and cyclosporin A (CsA) therapy showed a decline 180 days after therapy compared to pre-therapy. We thus conclude that increased production of both IFN-γ and TNF-α in the BM may contribute to disease pathogenesis in AA and ATG therapy may induce hematological remission by suppressing the elevated levels of IFN-γ and TNF-α in AA BM.

Mesenchymal Stem Cells from Fetal Heart Attenuate Myocardial Injury after Infarction: An In Vivo Serial Pinhole Gated SPECT-CT Study in Rats

Mesenchymal stem cells (MSC) have emerged as a potential stem cell type for cardiac regeneration after myocardial infarction (MI). Recently, we isolated and characterized mesenchymal stem cells derived from rat fetal heart (fC-MSC), which exhibited potential to differentiate into cardiomyocytes, endothelial cells and smooth muscle cells in vitro. In the present study, we investigated the therapeutic efficacy of intravenously injected fC-MSC in a rat model of MI using multi-pinhole gated SPECT-CT system. fC-MSC were isolated from the hearts of Sprague Dawley (SD) rat fetuses at gestation day 16 and expanded ex vivo. One week after induction of MI, 2×106 fC-MSC labeled with PKH26 dye (n = 6) or saline alone (n = 6) were injected through the tail vein of the rats. Initial in vivo tracking of 99mTc-labeled fC-MSC revealed a focal uptake of cells in the anterior mid-ventricular region of the heart. At 4 weeks of fC-MSC administration, the cells labeled with PKH26 were located in abundance in infarct/peri-infarct region and the fC-MSC treated hearts showed a significant increase in left ventricular ejection fraction and a significant decrease in the end diastolic volume, end systolic volume and left ventricular myo-mass in comparison to the saline treated group. In addition, fC-MSC treated hearts had a significantly better myocardial perfusion and attenuation in the infarct size, in comparison to the saline treated hearts. The engrafted PKH26-fC-MSC expressed cardiac troponin T, endothelial CD31 and smooth muscle sm-MHC, suggesting their differentiation into all major cells of cardiovascular lineage. The fC-MSC treated hearts demonstrated an up-regulation of cardio-protective growth factors, anti-fibrotic and anti-apoptotic molecules, highlighting that the observed left ventricular functional recovery may be due to secretion of paracrine factors by fC-MSC. Taken together, our results suggest that fC-MSC therapy may be a new therapeutic strategy for MI and multi-pinhole gated SPECT-CT system may be a useful tool to evaluate cardiac perfusion, function and cell tracking after stem cell therapy in acute myocardial injury setting.

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.

Controversies About the Chromosomal Stability of Cultivated Mesenchymal Stem Cells: Their Clinical Use is it Safe?

The usefulness of adult stem cells in research and therapeutic applications highly relies on their genomic integrity and stability. Many laboratories including ours have addressed this concern using methods such as karyotyping, Qbanding, fluorescent in situ hybridization, array CGH, flow cytometry and Pap test to evaluate number and structure of chromosomes and cellular phenotype. This review attempts to summarize the findings reported so far for the studies on chromosomal aberrations in adult stem cells and warrant to perform certain basic tests before transplantation to avoid any adverse reactions, which will thus aid in better therapeutic output after cellular transplantation in the treatment of various diseases.