Clinical benefits of single vs repeated courses of mesenchymal stem cell therapy in epilepsy patients.

PURPOSE: Epilepsy is defined as "drug-resistant" when existing anti-epileptic drugs (AED) are found to have minimal to no effect on patient's condition. Therefore the search and testing of new treatment strategies is warranted. This study focuses on the effects of autologous mesenchymal stem cells (MSC) in drug-resistant epilepsy patients within a Phase I/II open-label registered clinical trial NCT02497443. MATERIALS/METHODS: A total of 67 patients was included (29 males, 38 females, mean age 33 ± 1.3 yo). The patients received either standard treatment with AEDs, or AEDs supplemented with one or two courses of therapy with autologous bone marrow-derived MSCs expanded in vitro. MSC therapy courses were 6 months apart, and each course consisted of two cell injections: an intravenous infusion of MSCs, followed within 1 week by an intrathecal injection. Primary outcome of the study was safety, secondary outcome was efficacy in terms of seizure frequency reduction and response to treatment. RESULTS: MSC injections proved safe and did not cause any severe side effects. In MSC group (n = 34), 61.7% patients responded to therapy at 6 months timepoint (p < 0.01 vs control, n = 33), and the number rose to 76.5% by 12 months timepoint. Decrease in anxiety and depression scores and paroxysmal epileptiform activity was observed in MSC group based on HADS and EEG, respectively, and MMSE score has also improved. Another observation was that concomitant administration of levetiracetam, but not other AEDs, correlated significantly with the success of MSC therapy. Second course of MSC therapy facilitated further reduction in seizure count and epileptiform EEG activity (p < 0.05 vs single course). CONCLUSIONS: Application of autologous mesenchymal stem cell-based therapy in patients with pharmacoresistant epilepsy demonstrated significant anticonvulsant potential. This effect lasted for at least 1 year, with repeated administration of MSCs conveying additional clinical benefit.

Treatment of refractory epilepsy patients with autologous mesenchymal stem cells reduces seizure frequency: An open label study.

PURPOSE: Existing anti-epileptic drugs (AED) have limited efficiency in many patients, necessitating the search for alternative approaches such as stem cell therapy. We report the use of autologous patient-derived mesenchymal stem cells (MSC) as a therapeutic agent in symptomatic drug-resistant epilepsy in a Phase I open label clinical trial (registered as NCT02497443). PATIENTS AND METHODS: The patients received either standard treatment with AED (control group), or AED supplemented with single intravenous administration of undifferentiated autologous MSC (target dose of 1×106cells/kg), followed by a single intrathecal injection of neurally induced autologous MSC (target dose of 0.1×106cells/kg). RESULTS: MSC injections were well tolerated and did not cause any severe adverse effects. Seizure frequency was designated as the main outcome and evaluated at 1 year time point. 3 out of 10 patients in MSC therapy group achieved remission (no seizures for one year and more), and 5 additional patients became responders to AEDs, while only 2 out of 12 patients became responders in control group (difference significant, P=0.0135). CONCLUSIONS: MSC possess unique immunomodulatory properties and are a safe and promising candidate for cell therapy in AED resistant epilepsy patients.

Autologous mesenchymal stromal cells as a therapeutic in ALS and epilepsy patients: Treatment modalities and ex vivo neural differentiation.

Stem cell therapy for incurable central nervous system disorders has long been viewed as a promising therapeutic option. In this review, we discuss the existing data and approaches on cell transplantation in the context of the neural differentiation potential of adult autologous stem cells, focusing on those of mesenchymal origin as easily accessible and well studied. Mesenchymal stromal cells (MSCs) are a heterogeneous cell population with a remarkable therapeutic plasticity, demonstrated by their ability to dampen inflammation, inhibit pathogenic immune responses and secrete neuroprotective factors. To demonstrate and discuss the broad therapeutic potential of MSCs, this review focuses on two examples of neurological conditions: amyotrophic lateral sclerosis and epilepsy. We review the lessons from animal models and clinical trials, and consider encouraging newly published clinical data on therapeutic applications of neurally induced MSCs.

The incidence of T-cell receptor gene rearrangements in childhood B-lineage acute lymphoblastic leukemia is related to immunophenotype and fusion oncogene expression.

Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangement is conventionally used for assessment of lymphoid malignant cells. TCR genes rearrangements were reported to occur at high frequency in B-lineage acute lymphoblastic leukemia (ALL). Therefore, we have analyzed 83 children with acute B-lineage ALL (67 de novo patients and 19 relapses) by PCR analysis for clonal IgH, incomplete TCRD (Vdelta2-Ddelta3 and Ddelta2-Ddelta3) and TCRG rearrangements. It was shown that clonal cross-lineage TCR rearrangements were associated with more immature immunophenotype (CD34+, CD117+, CyIgM-) of leukemic cells from patients' bone marrow (BM) samples as compared to cell samples without cross-lineage TCR rearrangements. That was equally detected both in de novo and relapsed cases of disease. Low frequency of clonal TCRG rearrangements was associated with expression of E2A/PBX chimeric oncogene. We suggest that TCRG and TCRD clonal rearrangements in leukemic B-cells are associated with early stages of their differentiation.

Aberrant expression of tumor suppressor genes and their association with chimeric oncogenes in pediatric acute lymphoblastic leukemia.

Aberrant expression of tumor suppressor genes WT 1, RB 1, p53, homozygous deletion of p16 gene and their relationship with expression of oncogenes BCR-ABL, TEL-AML 1, MLL-AF 4, E2A-PBX 1, SIL-TAL 1 were determined in bone marrow samples of children with de novo B-lineage (n=170) and T-lineage (n=25) acute lymphoblastic leukemia (ALL). In contrast to expression of chimeric oncogenes alterations in p16, WT 1, RB 1 and p53 expression were T/B-lineage-unrestricted. Significant association between expression of MLL-AF 4 and WT 1, E2A-PBX 1 and p53; SIL-TAL 1 and homozygous deletion of p16 has been demonstrated.

Accumulation of chlorine e6 derivatives in cells with different level of expression and function activity of multidrug resistance protein P-gp 170.

AIM: This work deals with studying the influence of overexpression and function activity of multidrug transporter protein P-gp 170 on the intracellular accumulation of several porphyrin sensitizers, including chlorin e6 (Chl e6), di- (DME) and trimethyl esters (TME) of Chl e6. METHODS: A parental IM9 cell line and two IM9 drug-resistant IM9 sublines were used. With flow cytometry technique the rates of chlorines accumulation in different IM9 cells and values were related to the extent of multidrug resistance (MDR) phenotype. RESULTS: Using P-gp 170-specific antibodies and fluorescent probe JC-1 an increased expression and function activity of P-gp 170 was detected for drug-resistant cells. It was obtained that drug-resistant IM9 cells accumulated chlorines to a lesser extent than the respective wild type, however the differences did not exceed 30%. Verapamil, cyclosporine, known to reverse the MDR phenotype affects equally IM9-Vinc IM9-Tax and Im9 cells. CONCLUSION: Our results demonstrate that the P-gp 170 does not appear to play a role in the intracellular accumulation of chlorines. Since the enhanced activity of P-gp 170 in tumor cells is a factor of their resistance to the action of various antitumor drugs, we conclude that photodynamic therapy could be useful in killing cells exhibiting the MDR.

Whole body hyperthermia in adjuvant therapy of children with renal cell carcinoma.

Whole body hyperthermia (WBH) in combination with chemotherapy has been proven to be effective in some patients with advanced malignancies. However, only limited experience exists regarding the application of WBH with chemotherapy in children. We present the results of applying WBH and chemotherapy in five children with advanced renal cell carcinoma (RCC). WBH (3 hr, 41.8-42.5 degrees C) combined with doxorubicin (50 mg/m2) and interferon-alpha (3 MU/m2) were applied to patients after nephrectomy and lymph node dissection. Each patient received three to eight courses of treatment three times weekly. All children tolerated the combined therapy well without complications. Follow-up of 7-68 months (median: 22 months) showed no tumor progression in patients with locoregional (n = 3) and metastatic (n = 2) disease. WBH with moderate dose doxorubicin and INF-alpha might be a feasible treatment option in childhood RCC.

Enhancement of antitumor response to sarcoma 45 in rats by combination of whole-body hyperthermia and interleukin-2.

AIM: To evaluate the summarized effect of hyperthermia and interleukin-2 (IL-2) administration on antitumor defense in tumor-bearing rats. METHODS: Nonbred rats after subcutaneous inoculation of sarcoma 45 cells were treated with whole-body hyperthermia (WBH, +42.5 degrees C, 60 min) and interleukin-2 (IL-2, 10,000 U/kg of body weight). Parameters of tumor growth and survival of animals were monitored till day 33 after tumor cell inoculation. RESULTS: Combined application of WBH and IL-2 at 5th day after tumor cell transplantation resulted in a delay of tumor growth and improvement of survival parameters in comparison with control group or animals that received separate treatment. Therapeutic efficacy of WBH combined with IL-2 was analogous to a single-dose chemotherapy with cyclophosphamide. CONCLUSION: Combined application of WBH and IL-2 is the useful approach for cancer immunotherapy.

Significance of serum immunoglobulin G for leukocytosis and prognosis in childhood B-lineage acute lymphoblastic leukemia.

BACKGROUND: This study was conducted to evaluate the significance of serum level of immunoglobulins (Igs) and particularly IgG for leukemic cell persistence in peripheral blood (PB) and prognosis for childhood acute lymphoblastic leukemia (ALL). PROCEDURE: Human sera were obtained from 68 children with primary B-lineage ALL at diagnosis and 46 healthy children (control). Serum level of IgM, IgG, IgA, IgG1, IgG2, IgG3, IgG4, antitumor antibody, homogeneous IgG were quantified by turbidimetric or enzyme-linked immunosorbent assays. RESULTS: The mean values of serum IgM, IgG, IgA at diagnosis were not differed significantly in ALL patients and control children. The level of IgM and IgG1 inversely correlated with white blood cell (WBC) count in PB of patients. Normal range of serum IgG, separated by 25th and 75th percentiles of IgG variables, was associated in patients with decreased WBC count in PB but not in bone marrow (BM) versus patients with low concentration of IgG. Normal range of IgG also favors low frequency of homogeneous IgG and antitumor antibodies. Patients with high level of IgG, besides increased frequency of homogeneous IgG and antitumor antibodies, had worse 3-year overall survival (OS) rate as compared to patients with normal level of IgG (58.8 vs. 91.2%, P = 0.014). CONCLUSIONS: The normal level of serum IgG at diagnosis is a beneficial prognostic factor associated with lower rate of leukemic cell persistence in PB and better outcome of childhood B-lineage ALL.

Comparative measurement of spontaneous apoptosis in pediatric acute leukemia by different techniques.

BACKGROUND: To distinguish between subgroups of patients with acute leukemia, the rate of spontaneous (culture-induced) apoptosis of leukemic cells was evaluated using five methods. METHODS: Leukemic cells (cells) from the bone marrow of children with acute lymphoblastic leukemia (ALL, n = 112) and acute myeloid leukemia (AML, n = 30) were cultured for 20 h in vitro. The level of apoptosis was detected by fluorescent microscopy after staining with acridine orange (AO) or by flow cytometry after staining using PI, JC-1, the APO-BRDU kit, or the AnnexinV-FITC kit. RESULTS: ALL cells were significantly more sensitive to spontaneous apoptosis versus AML cells, as was detected by all methods. The least sensitive technique was apoptosis detection by sub-G1-peak/PI-staining. No difference in the rate of apoptosis in cells was determined between T- and B-lineage ALL patients. In patients with B-lineage ALL, strong positive correlation existed between the level of cells with loss of mitochondrial membrane potential (JC-1), chromatin condensation (AO), and externalization of phosphatidylserine (AnnexinV+PI+). The proportion of AnnexinV+PI- cells had no correlative link with any other apoptotic cell subpopulation. CONCLUSIONS: We found different sensitivities of ALL and AML cells to undergoing spontaneous apoptosis in vitro. Detection of the early/intermediate, but not the late stage of apoptosis is of preferable for correct assignment of spontaneous apoptosis in pediatric acute leukemia.