Elaine M. Elder

Transplantation of cultured human neuronal cells for patients with stroke

Article abstract Transplantation of cultured neuronal cells is safe in animal models and improves motor and cognitive deficits in rats with stroke. The authors studied the safety and feasibility of human neuronal cellular transplantation in patients with basal ganglia stroke and fixed motor deficits, including 12 patients (aged 44 to 75 years) with an infarct 6 months to 6 years previously (stable for at least 2 months). Serial evaluations (12 to 18 months) showed no adverse cell-related serologic or imaging-defined effects. The total European Stroke Scale score improved in six patients (3 to 10 points), with a mean improvement 2.9 points in all patients (p = 0.046). Six of 11 PET scans at 6 months showed improved fluorodeoxyglucose uptake at the implant site. Neuronal transplantation is feasible in patients with motor infarction.

Clonal Human (hNT) Neuron Grafts for Stroke Therapy : Neuropathology in a Patient 27 Months after Implantation

Although grafted cells may be promising therapy for stroke, survival of implanted neural cells in the brains of stroke patients has never been documented. Human NT2N (hNT) neurons derived from the NTera2 (NT2) teratocarcinoma cell line were shown to remain postmitotic, retain a neuronal phenotype, survive >1 year in host rodent brains and ameliorate motor and cognitive impairments in animal models of ischemic stroke. Here we report the first postmortem brain findings of a phase I clinical stroke trial patient implanted with human hNT neurons adjacent to a lacunar infarct 27 months after surgery. Neurofilament immunoreactive neurons were identified in the graft site, fluorescent in situ hybridization revealed polyploidy in groups of cells at this site just like polyploid hNT neurons in vitro, and there was no evidence of a neoplasm. These findings indicate that implanted hNT neurons survive for >2 years in the human brain without deleterious effects.

Serial [18F] fluorodeoxyglucose positron emission tomography after human neuronal implantation for stroke.

OBJECTIVEThere is no known effective treatment for chronic stroke. In this report, we used positron emission tomography (PET) with [18F]fluorodeoxyglucose (FDG) to map the metabolic brain response to neuronal cell implantation in the first human neuroimplantation trial for stroke. METHODSTwelve patients (nine men, three women; mean age ± standard deviation, 60.8 ± 8.3 yr) with chronic basal ganglia infarction and persistent motor deficit underwent FDG PET within 1 week before and 6 and 12 months after stereotactic implantation of human neuronal cells. Serial neurological evaluations during a 52-week postoperative period included the National Institutes of Health stroke scale and the European stroke scale. RESULTSAlterations in glucose metabolic activity in the stroke and surrounding tissue at 6 and 12 months after implantation correlated positively with motor performance measures. CONCLUSIONFDG PET performed as part of an initial open-label human trial of implanted LBS-Neurons (Layton BioScience, Sunnyvale, CA) for chronic stroke demonstrates a relationship between relative regional metabolic changes and clinical performance measures. These preliminary findings suggest improved local cellular function or engraftment of implanted cells in some patients.

Autologous lymphokine-activated killer cell therapy of Epstein-Barr virus-positive and -negative lymphoproliferative disorders arising in organ transplant recipients

Lymphoreticular malignancies, collectively called posttransplant lymphoproliferative disorders (PTLD), eventually develop in 2-5% of organ transplant recipients. They frequently undergo regression when immunosuppression is reduced or stopped. This feature has been associated with a previous or de novo Epstein-Barr virus (EBV) infection. We herein describe immunotherapy with autologous lymphokine-activated killer (LAK) cells in seven patients with PTLD (four EBV-positive patients and three EBV-negative patients). Autologous peripheral blood mononuclear cells were obtained by leukapheresis, depleted of monocytes, and cultured in the presence of interleukin 2 for 10 to 11 days. A single dose of 5.2 x 10(9) to 5.6 x 10(10) LAK cells was given intravenously. Systemic interleukin 2 was not administered. The four patients with EBV+ PTLD had complete tumor regression; two of them developed controllable rejection. Three patients are well 13-16 months after treatment; the fourth patient died of pneumonia 41 days after infusion. Three patients with EBV- lymphomas had no response despite prior evidence that their tumors also were subject to immune surveillance. Two of these three patients died after being given other treatment, and the third patient has persistent tumor. In conclusion, autologous LAK cell infusion was effective for treatment of four EBV+ organ transplant recipients. LAK cell efficacy for three patients with EBV- PTLD was not evaluable under the management circumstances in which this treatment was utilized.

Generation of T Cells Specific for the Wild-Type Sequence p53 264–272 Peptide in Cancer Patients: Implications for Immunoselection of Epitope Loss Variants

Alterations in the p53 gene occur frequently and can lead to accumulation of p53 protein in squamous cell carcinomas of the head and neck (SCCHN). Since accumulation of p53 is associated with enhanced presentation of wild-type sequence (wt) p53 peptides to immune cells, the development of pan vaccines against SCCHN has focused on wt p53 epitopes. We used the HLA-A2.1-restricted wt p53264–272 epitope to generate CTL from circulating precursor T cells of HLA-A2.1+ healthy donors and patients with SCCHN. Autologous peptide-pulsed dendritic cells were used for in vitro sensitization. CTL specific for the wt p53264–272 peptide were generated from PBMC obtained from two of seven normal donors and three of seven patients with SCCHN. These CTL were HLA class I restricted and responded to T2 cells pulsed with p53264–272 peptide as well as HLA-A2-matched SCCHN cell lines naturally presenting the epitope. Paradoxically, none of the tumors in the three patients who generated CTL could adequately present the epitope; two had a wt p53 genotype and no p53 protein accumulation, while the third tumor expressed a point mutation (R to H) in codon 273 that prevents presentation of the p53264–272 epitope. In contrast, patients who did not generate CTL had tumors that accumulated altered p53 and potentially could present the p53264–272 epitope. These findings suggest that in vivo, CTL specific for the wt p53264–272 peptide might play a role in the elimination of tumor cells expressing this epitope and in immunoselection of epitope-loss tumor cells. Immunoselection of tumors that become resistant to anti-p53 immune responses has important implications for future p53-based vaccination strategies.

Host immune response in renal cell cancer: Interleukin-4 (IL-4) and IL-10 mRNA are frequently detected in freshly collected tumor-infiltrating lymphocytes

Human renal cell cancer (RCC) is clearly responsive to immunotherapy. Clinical responses may be mediated by “non-specific” (e. g. natural killer, NK, cells) or “specific” MHC-class-I-restricted tumor-specific CD8+ T lymphocytes. Typically RCC progresses, however, despite significant infiltration of various lymphoid cells. We examined freshly isolated RCC tumor-infiltrating lymphocytes (TIL) derived from seven RCC patients for cytokine expression by the polymerase chain reaction (PCR). Established RCC tumor cell lines derived from these RCC patients were negative for interleukin-2 (IL-2), IL-4, IL-10, and interferon γ and found to be positive for tumor necrosis factor α (TNFα), IL-6, IL-1β, granulocyte/macrophage-colony-stimulating factor (GM-CSF), and transforming growth factor β1 (TGFβ1) message as detected by PCR. An identical pattern of cytokine mRNA expression was identified in other long-term RCC lines and in normal human kidney cells upon culture, but not in two Wilms tumor cell lines tested. Short-term-, and long-term-established RCC lines, but not Wilms tumor lines, secreted substantial levels of GM-CSF, TNFα, IL-1β, and IL-6 as detected by enzyme-linked immunosorbent assay. Both RCC lines and Wilms tumor lines secreted TGFβ1. In comparison, normal kidney cells secreted IL-6 and GM-CSF, but not IL-1β, or TFGβ1 under identical in vitro cell culture conditions. We applied PCR-based methods to characterize the cytokine mRNA expression pattern in immune cells infiltrating into renal cell cancer without the need for expansion of such effector cells in vitro. Examining freshly collected RCC TIL by PCR from patients with primary cell cell cancer, we could demonstrate that such cells, but not lympho-mononuclear cells harvested from normal human kidney tissue, typically exhibit IL-4 and IL-10 mRNA expression.

Posttransplant adoptive immunotherapy with activated natural killer cells in patients with metastatic breast cancer.

Relapse after high-dose chemotherapy is the main cause of therapeutic failure in patients with metastatic breast cancer. Adoptive immunotherapy with interleukin-2 (IL-2) plus activated natural killer cells may eliminate residual disease without excessive toxicity. The authors sought to determine if immunotherapy immediately after transplantation would affect engraftment and the toxicity associated with transplantation. Fifteen consecutive patients with metastatic breast cancer were allocated to three cohorts. Cohort 1 (five patients) received high-dose cyclophosphamide, thiotepa, and carboplatin (CTCb) followed by peripheral blood stem cell infusion and granulocyte colony-stimulating factor at 10 micrograms/kg. Cohort 2 (five patients) received in addition rhIL-2 (2 x 10(6) IU/m2/day) for 4 days intravenously via continuous infusion after peripheral blood stem cell infusion. In cohort 3 (five patients), peripheral blood stem cell transplant was followed by infusion of autologous activated NK cells and rhIL-2 (2 x 10(6) IU/m2/day) for 4 days (via continuous intravenous infusion). Generation of activated NK cells was possible in all patients in cohort 3. All patients has successful engraftment. Median time to absolute neutrophil count more than 0.5 x 10(9)/L was 8 days (range, 8 to 11 days) in cohort 1, 9 days (range, 7 to 11 days) in cohort 2, and 9 days (range, 8 to 9 days) in cohort 3. Median time until the platelet count was more than 20 x 10(9)/L was 14 days (range, 9 to 22 days) in cohort 1, 11 days (range, 6 to 14 days) in cohort 2, and 12 days (range, 11 to 21 days) in cohort 3. All patients developed neutropenic fevers, but the overall toxicity associated with the infusion of IL-2 (cohort 2) or IL-2 plus activated NK cells (cohort 3) did not differ from that observed in cohort 1. Complete responses were achieved in one patient in cohort 1, in two patients in cohort 2, and in one patient in cohort 3. In conclusion, post-transplant adoptive immunotherapy with activated NK cells plus IL-2 is feasible, well tolerated, and does not adversely affect engraftment.

Clinical Responses to Gene Therapy in Joints of Two Subjects with Rheumatoid Arthritis

This paper provides the first evidence of a clinical response to gene therapy in human arthritis. Two subjects with rheumatoid arthritis received ex vivo, intraarticular delivery of human interleukin-1 receptor antagonist (IL-1Ra) cDNA. To achieve this, autologous synovial fibroblasts were transduced with a retrovirus, MFG-IRAP, carrying IL-1Ra as the transgene, or remained as untransduced controls. Symptomatic metacarpophalangeal (MCP) joints were injected with control or transduced cells. Joints were clinically evaluated on the basis of pain; the circumference of MCP joint 1 was also measured. After 4 weeks, joints underwent surgical synovectomy. There were no adverse events in either subject. The first subject responded dramatically to gene transfer, with a marked and rapid reduction in pain and swelling that lasted for the entire 4 weeks of the study. Remarkably, joints receiving IL-1Ra cDNA were protected from flares that occurred during the study period. Analysis of RNA recovered after synovectomy revealed enhanced expression of IL-1Ra and reduced expression of matrix metalloproteinase-3 and IL-1beta. The second subject also responded with reduced pain and swelling. Thus, gene transfer to human, rheumatoid joints can be accomplished safely to produce clinical benefit, at least in the short term. Using this ex vivo procedure, the transgene persisted within the joint for at least 1 month. Further clinical studies are warranted.

Gene therapy of malignant gliomas: a pilot study of vaccination with irradiated autologous glioma and dendritic cells admixed with IL-4 transduced fibroblasts to elicit an immune response.

IND 8918 Principal Investigator: Hideho Okada, M.D., Ph.D. Co-investigators: Ian F. Pollack, M.D. Frank Lieberman, M.D. Associate Investigators: Neurological Surgery L. Dade Lunsford, M.D. Douglas Kondziolka, M.D. David Schiff, M.D. Jason Attanucci, B.S. Biologic Therapy University of Pittsburgh Cancer Institute Howard Edington, M.D. William Chambers, Ph.D. Pawel Kalinski, M.D., Ph.D. Donna Kinzler, R.N. IMCPL/CT Theresa Whiteside, Ph.D. Elaine Elder, Sc.D. Statistics Douglas Potter, Ph.D.

Modulation of the cAMP relay in Dictyostelium discoideum by ammonia and other metabolites: possible morphogenetic consequences.

Using a perfusion technique (P.N. Devreotes, P.L. Derstine, and T.L. Steck, 1979, J. Cell Biol. 80, 291-299), it has been shown that cAMP secretion by aggregation-competent cells in response to an exogenous cAMP signal is significantly reduced by exposure to NH4Cl or any of a set of carboxylic acids that includes propionate, succinate, pyruvate, and acetate. The effects of NH4Cl and any of the carboxylic acids are additive and the combinations restrict cAMP secretion to barely detectable or insignificant levels. The inhibitions are rapidly expressed, and are reversible. The activity of NH4Cl is marked at pH 7.2 and undetectable at pH 6.2. Hence, NH3 is presumably the active molecular species. Propionate activity is significantly greater at pH 6.2 than 7.2, indicating that the un-ionized acid is the active species. The data presented herein indicate that these effects are exerted via two separate and independent routes. During exposure of cAMP-stimulated cells to NH4Cl, the decrease in intracellular cAMP accumulation was even greater than the decrease in extracellular accumulation. Hence, NH3 appears to act as a cAMP accumulation inhibitor (CAI). In contrast, exposure to carboxylic acid concentrations that drastically reduce extracellular cAMP accumulation can actually enhance or, at worst, only slightly reduce intracellular accumulation. Hence, the carboxylic acids appear to act as cAMP release inhibitors (CRI). Stationary phase cells incubated on solid substratum in the presence of NH4Cl plus succinate (or propionate) for 18 hr failed to exhibit even the earliest signs of aggregation. If then harvested and redeposited in the absence of the metabolites, they proceeded through the morphogenetic sequence with approximately normal kinetics, suggesting that no significant morphogenetic competence had been achieved during their previous tenure. The morphogenetic implications of cAMP relay modulation are discussed.