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This article describes the clinical application of gene therapy to a nonlethal disease, rheumatoid arthritis (RA). Intraarticular transfer of IL-1 receptor antagonist (IL-1Ra) cDNA reduces disease in animal models of RA. Whether this procedure is safe and feasible in humans was addressed in a phase I clinical study involving nine postmenopausal women with advanced RA who required unilateral sialastic implant arthroplasty of the 2nd-5th metacarpophalangeal (MCP) joints. Cultures of autologous synovial fibroblasts were established and divided into two. One was transduced with a retrovirus carrying IL-1Ra cDNA; the other provided untransduced, control cells. In a dose escalation, double-blinded fashion, two MCP joints were injected with transduced cells, and two MCP joints received control cells. One week later, injected joints were resected and examined for evidence of successful gene transfer and expression by using RT-PCR,ex vivoproduction of IL-1Ra,in situhybridization, and immunohistochemistry. All subjects tolerated the protocol well, without adverse events. Unlike control joints, those receiving transduced cells gave positive RT-PCR signals. Synovia that were recovered from the MCP joints of intermediate and high dose subjects produced elevated amounts of IL-1Ra (P= 0.01). Clusters of cells expressing high levels of IL-1Ra were present on synovia of transduced joints. No adverse events occurred. Thus, it is possible to transfer a potentially therapeutic gene safely to human rheumatoid joints and to obtain intraarticular, transgene expression. This conclusion justifies additional efficacy studies and encourages further development of genetic approaches to the treatment of arthritis and related disorders.

OBJECTIVE: Neurotransplantation has focused on disorders that involve subcortical brain targets. We evaluated the concepts of epileptic focus repair and changes in animal behavior through replacement of lost hippocampal neurons. The safety of hippocampal neurotransplantation was assessed in the rat kainic acid (KA) epilepsy model. METHODS: Sixty-three rats were studied and classified into six groups: KA plus 40,000 LBS-Neurons (Layton BioScience, Sunnyvale, CA; n = 13); KA plus 80,000 cells (n = 12); KA plus media (n = 9); no-KA plus 40,000 cells (n = 12); no-KA plus 80,000 cells (n = 12); and no-KA plus media (n = 5). Clinical observation (2 h daily) and electroencephalogram recording (3 h every other week) were performed to check for seizures until Week 11 after KA injection. On Week 12, the Morris water maze test was performed to assess spatial learning and memory. RESULTS: Four rats were excluded because of intracranial hematoma or abscess. In the clinical observation of seizures, the no-KA plus media group had significantly fewer seizures than rats that received KA followed by injection of 40,000 cells, 80,000 cells, or media (P = 0.001, 0.0004, and 0.004, respectively). On electroencephalographic analysis, there was no significant difference between any of the groups. Transplanted rats with KA-induced epilepsy did not have an increased number of seizures. In the Morris water maze test, the hidden platform task showed that the KA plus 80,000 cell group had significantly longer swim latencies than groups with no-KA plus 40,000 cells (P = 0.035) or no-KA plus 80,000 cells (P = 0.015), demonstrating the behavioral deficits caused by KA injection. The probe trial showed no significant difference for the percentage of time in the target quadrant between any of the groups. Histolugical studies showed that 26 (59%) of 44 transplanted rats had evidence of graft survival. CONCLUSION: The safety of cortical neurotransplantation was demonstrated, even in an animal model predisposed to epilepsy. We did not find evidence for cessation of seizures or improvement in behavior using this model.

Laboratory studies have established the potential for neuronal transplantation to be of benefit to patients. Experimental studies in normal animals indicate that brain implantation of neurons seems safe. Implanted neurons integrated with the host brain, sent out axonal processes to communicate with other nerve cells, released transmitters (the chemical messengers of nerve cell communication), and demonstrated typical neuronal proteins. This article discusses phase I and II trials of neuronal transplantation in humans with small strokes in critical brain locations such as the basal ganglia region. More work is needed to confirm safety and to identify optimal measures of efficacy in this setting.

Mutations in the K-ras gene are frequently found in lung tumours and are implicated in the development of lung cancer. In order to investigate the clinical usefulness of these mutations in lung cancer, we applied a sensitive method to compare mutations in codon 12 of the K-ras gene in DNA extracted from lung tumours and the matched sputum samples obtained from 22 lung cancer patients. K-ras mutations were identified in the lung tumours of 12 patients (54.5%) and in the sputum samples of 10 patients (45.5%). Nine patients showed an identical mutation in both the tumour and the matched sputum samples. There was a significant association between the presence of a K-ras mutation in a lung tumour and the detection of an identical mutation in the matched sputum sample of the lung cancer patient (kappa = 0.64, 95% confidence interval 0.32-0.95, p0.01). K-ras mutations were detected in sputum samples from cancer patients with all lung tumour grades, and both in the presence and the absence of lymph node metastasis. Therefore, K-ras mutations may provide useful diagnostic markers for lung cancer.

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.

OBJECTIVE There 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. METHODS Twelve 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. RESULTS Alterations in glucose metabolic activity in the stroke and surrounding tissue at 6 and 12 months after implantation correlated positively with motor performance measures. CONCLUSION FDG 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.

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.

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.

Background. Human lymphokine-activated cells (LAK cells) and interferon alpha (IFN-a) have been used clinically in the therapy of posttransplant lymphoproliferative disease (PTLD). However, the efficacy of such therapy has not been extensively tested under controlled experimental conditions. Methods. A B-cell line, derived from PTLD tissue and clonally related to the parent lesion, was tested for its response to IFN-a in vitro. The effects of LAK cells and IFN-a therapy were examined in a severe combined immunodeficiency disease (SCID) mouse model in vivo. Results. The PTLD cell line studied showed a 30% decrease in the rate of growth upon incubation with 500 U/ml of IFN-a. This in vitro response was also reproduced in vivo, in tumor therapy studies conducted in SCID mice. The magnitude of this inhibitory effect in vivo was a function of tumor burden and dose of IFN-a. In parallel experiments, LAK cells reduced the tumorigenicity of a lymphoblastoid cell line derived from the peripheral blood of a patient with PTLD, and prolonged the survival of SCID-beige mice with established lymphoproliferative disease. In contrast with two prior studies, in which the use of autologous cytotoxic T cells was found to be necessary, we found the administration of third-party non-HLAmatched LAK cells also to be effective in reducing tumor burden. Conclusions. These observations demonstrate the efficacy of immunotherapy for lymphoproliferative disease under controlled experimental conditions, and validate currently ongoing efforts exploring the utility of such therapy in the clinical setting. Posttransplant lymphoproliferative disease (PTLD*) is a complication of Epstein-Barr virus (EBV) infection in immu

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.

We tested a 105 amino acid synthetic mucin MUC-1 peptide that has 5 repeated immunodominant epitopes to evaluate toxicity and detect mucin-specific immune responses in patients with adenocarcinoma. We also studied the enhancement of these responses by vaccinating patients with the synthetic mucin peptide admixed with BCG. Mucins are glycoproteins present on the luminal surface of ductal epithelial cells and on tumors derived from them. The MUC-1 mucin is hypoglycosylated and nonpolarized on tumors and this exposes epitopes that can stimulate cytotoxic T-Cells (CTL). We vaccinated 63 patients with 100 μg of the 105aa mucin peptide mixed with BCG. Two additional vaccinations were given at 3-week intervals. All patients were able to tolerate vaccination, with most experiencing local ulceration at the vaccination site. All patients underwent hypersensitivity (DTH) testing with the 105aa and shorter mucin peptides, prior to vaccination. DTH responses were evaluated at 48 hr and the sites of highest peptide concentration were biopsied. Only 3 patients had a strong skin response to the long peptide. Examination of 55 biopsies showed intense T-Cell infiltration in 37 patients and lesser infiltration in 7. Seven of 22 patients tested had a 2- to 4-fold increase in mucin-specific CTLp. Serum levels of IL-6 were measured sequentially using the B9 hybridoma bioassay. Increasing serum levels of IL-6 correlated with constitutional symptoms (significance 0.001) and hypoalbuminemia (significance 0.007) but not with the extent of skin breakdown at vaccination sites. We conclude that mucin vaccination is safe and might serve to enhance specific responses to tumor antigens. IL-6 may be responsible for the constitution symptoms and hypoalbuminemia in these patients.

Tumor-infiltrating lymphocytes (TIL) freshly obtained from human malignant melanomas as well as the same TIL grown in the presence of interleukin 2 (IL2) were studied for gene expression of the T-cell receptor (TCR) variable beta regions (V beta). To perform the TCR-V beta analysis, total RNA was isolated from TIL and reverse-transcribed into cDNA, which was then amplified by PCR using 22 different 5' primers specifically recognizing the sequences of 20 V beta gene families and a 3' primer annealing to the constant region of the beta chain. The TCR-alpha constant region (C alpha) gene was co-amplified as a standard for the calculation of the percentage of each TCR-V beta gene expressed. The frequency of individual V beta regions expressed on TIL was computed from the ratio of cpm V beta to cpm C alpha for each V beta region in relation to the total of all 22 ratios. With fresh TIL obtained from 8 different melanomas, oligoclonal distribution of V beta genes expressed on TIL was observed, in comparison with a broader and unrestricted distribution seen with peripheral-blood T cells of 8 normal individuals. The oligoclonal patterns of V beta-gene expression in fresh melanoma TIL were distinct in every tumor. Several of the V beta-genes usually expressed in normal PBL were not expressed in fresh TIL in melanoma TIL cultured in the presence of IL2 and IL4 and in the absence of autologous tumor (AuTu) or antigen-presenting cells for 23 to 65 days, selection of T-cell lines expressing a restricted number of V beta genes occurred. Although in 4/5 TIL cultures this selection involved the V beta 7 gene, no relationship could be established between V beta gene expression in fresh TIL and that in T-cell lines outgrowing in long-term cultures. Selection in culture of CD3+CD8+ T-cell lines with V beta-gene expression restricted to 1 or 2 V beta families did not correlate with the presence or level of AuTu cytotoxicity mediated by these T cells. The results indicate that in TIL cultures random selection of T-cell lines with reactivity not relevant to AuTu may account for poor expression or loss of AuTu cytotoxicity by most TIL cultured long-term in the presence of cytokines and in the absence of specific antigenic stimulation.

Cytolytic T lymphocytes play an important role in host defense against viral infections, including human immunodeficiency virus (HIV). In a phase I clinical trial (protocol 080 of the AIDS Clinical Trials Group), generation of CD8+ effector cells from peripheral blood of patients with acquired immunodeficiency syndrome (AIDS)-related complex (ARC) or AIDS and safety of autologous adoptive transfer of these cells were evaluated. For therapeutic infusions, CD8+ T cells were purified by positive selection on anti-CD8 monoclonal antibody-coated flasks from leukapheresed peripheral blood of seven patients. These CD8+ T cells were cultured in the presence of interleukin-2 and phytohemagglutinin for up to 3 weeks to obtain cells sufficient for therapeutic infusions (10(8) to 10(10)). All 31 cell cultures established from the seven patients and used for therapy were highly enriched in CD8+ (mean, 97%), CD8+HLA-DR+ (50%), cytotoxic CD8+CD11b- (82%), and memory CD29+ (78%) T lymphocytes. In vitro expanded CD8+ cells had excellent cytotoxic function at the time they were used for therapy, including HIV-specific activity against autologous targets infected with vaccinia vectors expressing HIV-IIIb antigens, gag, pol, and env. Anti-HIV activity of cultured CD8+ cells was significantly higher than that of autologous fresh peripheral blood lymphocytes. Our results show that CD8+ T lymphocytes obtained from peripheral blood of symptomatic HIV-infected patients can be purified, cultured to obtain large numbers of cells with enhanced anti-HIV activity, and safely infused into patients with AIDS as a form of immunotherapy.

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.

Adherent lymphokine-activated killer (A-LAK) cells, selected from peripheral blood lymphocytes (PBL) of normal human donors by adherence to plastic, and cultured in the presence of interleukin 2 (IL-2), are highly enriched in CD3-CD56+ natural killer (NK) cells. These IL-2-activated NK cells proliferate extensively upon further culture in conditioned medium containing IL-2. In contrast, we previously found that with PBL of some patients with advanced cancer, the same procedure often failed to yield high enrichment of NK cells or substantial expansion in the numbers of these effector cells. To obtain sufficient numbers of A-LAK cells for adoptive immunotherapy in cancer patients, an improved method for generation of human A-LAK cells with irradiated mitogen-stimulated allogeneic PBL- or Epstein-Barr virus-transformed lymphoblastoid cell lines was introduced. In paired experiments, A-LAK cultures with feeder cells showed significantly enhanced IL-2-driven proliferation of A-LAK cells obtained from normal donors or patients with metastatic melanoma, renal cell carcinoma, and other types of solid cancers. The growth-promoting effect of feeders for A-LAK cells resulted in significantly improved expansion of CD3-CD56+ (NK) effector cells in A-LAK cultures established from normal donors. Cells in these cultures also had significantly higher levels of antitumor cytotoxicity against K562 and Daudi targets than did A-LAK cells grown in the absence of feeder cells. Enrichment in CD3-CD56+ cells and antitumor activity also occurred in patient A-LAK cultures supplemented with mitogen-stimulated feeder cells, but was not statistically significant. Overall, despite improved proliferation and CD3-CD56+ cell content of A-LAK cultures established in the presence of mitogen-activated feeder cells, only 39% (21/54) of patients tested generated A-LAK cells that would be judged acceptable for large-scale therapeutic use by criteria based on fold expansion and purity of A-LAK cells. These results suggest that in comparison to normal individuals, NK cells of many patients with advanced solid tumors are defective in their ability to respond by proliferation to IL-2 even in the presence of exogenously supplied growth factors.

Considerable evidence has accumulated indicating that cultured human or rodent tumor cells can be successfully transduced with cytokine genes and selected in the appropriate antibiotic-containing culture media. The selected transductants are generally able to secrete the cytokine coded for by the transduced gene, and in many cases, substantial levels (e.g., ng quantities) of the cytokine are produced. Using retroviral vectors, it has been possible to obtain stably transduced tumor cells with a variety of cytokine genes (1-4). These tumor cells have been used for immunotherapy of cancer in numerous animal models of tumor growth or metastasis, and more recently, in vaccination protocols in patients with cancer. One possible criticism that can be leveled at this type of vaccination approach is that cultured, genetically modified, and selected tumor cells might have phenotypic characteristics that are substantially different from those of unmodified tumor cells. Since retroviral vectors are often used for transduction, it is also possible that viral antigens expressed on transduced tumor cells contribute to the immune response generated as a result of vaccination. Also, primary cultures of human tumor cells are often difficult to establish and maintain.

A new generation of single-cell assays for measuring the frequency of peptide-specific T lymphocytes in cellular populations has become widely available. These assays, enzyme-linked immunospot (ELISPOT), cytokine flow cytometry, and tetramer binding, are used frequently for monitoring of cancer patient responses to vaccination therapies. We concomitantly used these three assays to determine the frequency of CD8(+) T cells in the circulation of 8 patients with metastatic melanoma who had received a multiepitope peptide/dendritic cell-based vaccine. Using peripheral blood mononuclear cells harvested before and a week after the last of four vaccines, we observed that the three assays detected a substantially different frequency of peptide-specific CD8(+) T cells. The tetramer assay consistently detected the highest numbers of peptide-specific CD8(+) T cells, followed by cytokine flow cytometry and then ELISPOT. There was no significant concordance among the assays in measuring the numbers of CD8(+) T cells specific for each of the peptides in the peripheral circulation of the patient. No significant pre- to postvaccine changes in the number of CD8(+) T cells specific for any of the peptides were observed. Thus, the dendritic cell-based vaccine was observed not to augment immune responses to the peptides in the patients. Because of a low frequency of the peptide-specific T cells in the peripheral circulation of the patients, these sensitive single-cell assays were used to measure values at the lower limit of detection. For this and other reasons, including the issues of tetramer specificity and ELISPOT sensitivity, caution in interpretation and serious attention to quality control are needed in monitoring of immune responses to anticancer vaccines.

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.

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.

Fifty samples of lung tissue from patients with non-small cell lung cancer were analyzed for the expression and localization of biomarkers related to squamous differentiation and programmed cell death. These markers include tissue transglutaminase (tTG), keratinocyte transglutaminase (kTG), involucrin, loricrin, and Bcl-2. We found that all of these markers are overexpressed in tumors as compared with histologically normal lung epithelium, where expression is minimal. Expression of the oncoprotein, Bcl-2, increased starting in squamous metaplasia and remained elevated in all lesions, including frank carcinoma. In contrast, expression of the other markers was elevated in the histologically abnormal noninvasive lesions but was decreased somewhat in invasive malignancy. In addition, we found that tTG, kTG, and Bcl-2, when expressed, were detected in mutually exclusive areas. These findings suggest that (1) these markers may prove useful, with more extensive testing and clinical correlation, in predicting risk for the development of lung cancer; and (2) pulmonary carcinogenesis may result from the failure of differentiation and programmed cell death mechanisms in the presence of oncogene overexpression rather than through oncogene/tumor suppressor gene abnormalities alone.

A sensitive method was developed and applied to examine the distribution of K- ras gene mutations in histologically differing areas of lung tissues obtained from lung cancer patients. This method, which combines polymerase chain reaction (PCR), mutation allele enrichment (MAE), and denaturing gradient gel electrophoresis (DGGE), allows detection of one K- ras mutant allele present in 10 4 to 10 5 wild-type alleles. It was applied to analyze mutations in codon 12 of the K- ras gene in 43 tissue sites microdissected from paraffin-embedded sections obtained from 8 archival cases of lung cancer, all previously shown to have codon 12 K- ras mutations by direct sequencing. In four cases, mutations were detected only in the tumor, while in the other four cases, the same mutations were also found in tissues adjacent to tumors, using the MAE + DGGE method. No mutations were detected among normal-appearing cells in areas distant from the tumors in any of the cases studied. These findings demonstrate that K- ras mutations can be detected at low frequencies in normal-appearing cells from tissues adjacent to the tumor in some lung cancer cases. In addition, this approach also allowed detection of multiple mutations in colorectal tissues obtained from colorectal cancer patients. Thus, the MAE + DGGE method may be applicable to study of K- ras mutations in premalignant or morphologically suspicious lesions in bronchial mucosa or other types of human cancer.

Activated natural killer (A-NK) cells, a subset of CD56 dim CD3 - lymphocytes, are obtained from PBMC of normal donors by adherence to plastic and culture in the presence of IL2. In this study we tested the feasibility of generating A-NK cells in patients with Ph + chronic myeloid leukaemia (CML). Cultures obtained from patients with early chronic phase (ECP ; n=7) contained a mean (±SD) of 83 ± 7%) of CD56 + CD3 - cells, and those from patients with advanced chronic phase (ACP ; n = 7) contained 27±13% CD56 + CD3 - cells. In three patients with leukaemia in a blastic phase (BP) it was only possible to obtain one culture enriched in CD56 + CD3 - cells (81%). Cellular aggregates of myeloid cells and large granular lymphocytes were observed in early A-NK cell cultures. Paired freshly-adherent and cultured A-NK cells were tested for the presence of BCR/abl mRNA by RT-PCR. The BCR/abl - cells were detected in all 12 preparations of the freshly adherent A-NK cells tested. In 6/12 the BCR/abl + cells were no longer detectable by RT-PCR on day 14 of culture. Both proliferation and antileukaemic cytotoxicity were significantly higher (P = 0.002 and P = 0.029, respectively) in the BCR/abl - cultures than those in the six BCR/abl + cultures. 5/6 BCR/abl - cultures were highly enriched in A-NK cells on day 14, and 1/6 contained predominantly CD56 + CD3 + cells. Only 2/6 BCR/abl + cultures were enriched in A-NK cells on day 14, but they had poor cytotoxicity and a low proliferative index. Myeloid cells (CD33 + ) were more frequently detected in the BCR/abl + than BCR/abl - A-NK cell cultures (P = 0.028). These observations suggest that : (1) populations of benign A-NK cells can be generated from the peripheral blood of CML patients : (2) the ability to generate A-NK cells is impaired in patients with advanced CML ; and (3) the ability to generate A-NK cells with antileukaemic activity correlates with the disappearance of BCR/abl + cells from these cultures.

Human autologous dermal fibroblasts have been cultured, transduced with the interleukin-4 (IL-4) gene and used as a vaccine together with irradiated autologous tumor cells in patients with cancer participating in a phase I/II clinical trial at the University of Pittsburgh Cancer Institute. In support of this clinical trial, methods have been devised to facilitate isolation of fibroblasts from freshly harvested skin specimens, to enhance their outgrowth in large-scale cultures, and to assay cytokine (IL-4) production following transduction with the cytokine gene +/- irradiation. Fibroblasts were isolated from skin specimens by enzymatic digestion, grown in primary cultures, and transduced with a retroviral vector containing the gene for human IL-4 and the NeoR gene as a selectable marker. Following selection in G418, the irradiated, IL-4-producing fibroblasts were administered to patients in a vaccine containing irradiated autologous tumor cells. Seventy-eight specimens of human skin were processed to obtain fibroblast suspensions. Cultures of fibroblasts were established from 68 of the 78 specimens (87%). Of 33 transduced and selected fibroblast cultures, 21 produced at least 1,000 units of IL-4/24 hours per 10(6) cells, as determined by ELISA, and 17/33 or 51% were used for therapy. The primary cultures were typically maintained for up to seven or eight passages. The mean +/- SD overall time for obtaining a required number of transduced, selected cells was 53 +/- 4 days. The fibroblasts continued to produce IL-4 in culture for 3 weeks even after irradiation. Similar results have been obtained with a retroviral vector encoding IL-12. This study shows that human dermal fibroblasts can be consistently and reproducibly expanded and genetically modified to serve as a source of cytokines or other gene products for gene therapy trials.

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 gamma and found to be positive for tumor necrosis factor alpha (TNF alpha), IL-6, IL-1 beta, granulocyte/macrophage-colony-stimulating factor (GM-CSF), and transforming growth factor beta 1 (TGF beta 1) 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, TNF alpha, IL-1 beta, and IL-6 as detected by enzyme-linked immunosorbent assay. Both RCC lines and Wilms tumor lines secreted TGF beta 1. In comparison, normal kidney cells secreted IL-6 and GM-CSF, but not IL-1 beta, or TFG beta 1 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.

Patients with advanced malignancies, participating in our ongoing phase I interleukin-4 (IL-4) gene therapy protocol at the Pittsburgh Cancer Institute, were vaccinated with irradiated autologous tumor cells together with IL-4 gene-transduced irradiated autologous fibroblasts. The level of expression of the IL-4 gene in cultured transduced and selected fibroblasts and in biopsies obtained from vaccination sites was evaluated using quantitative reverse transcription-polymerase chain reaction (RT-PCR). The number of copies of IL-4 mRNA/ng of total cellular RNA was determined in the transduced fibroblasts. Good agreement was observed between IL-4 message expression, as determined by RT-PCR, and IL-4 production, as determined by enzyme-linked immunosorbent assay (ELISA) in the fibroblast supernatants. Tissue biopsies of multiple vaccination sites were obtained from the patients to determine the level of gene expression in situ for IL-4 and Neo-r. The Neo-r gene was used as a marker for transduced fibroblasts. Two weeks after the first vaccination, mRNA for the IL-4 gene was still detectable in all tissue biopsies. The Neo-r gene was also detectable, indicating the presence of transduced fibroblasts in the biopsy. After the second vaccination, expression of the IL-4 and Neo-r genes was generally the highest on day 1 after vaccine administration and was considerably lower but still detectable on day 14 in all biopsies tested. These data indicate that autologous dermal fibroblasts transduced with the IL-4 and Neo-r genes and used as a source of IL-4 in tumor vaccine are able to express the IL-4 gene in vivo.

Melanoma represents the single best example of a human tumor that has been shown to elicit specific T-cell reactivity. The responsiveness of some patients with metastatic melanoma to treatment with the prototypic T-cell growth factor (TCGF), interleukin-2 (IL-2), indicates that T cells play a role in antitumor immunity. Interleukin-4 (IL-4), another TCGF that has been administered clinically to humans, was not associated with tumor response in our trials conducted at the Surgery Branch of the National Cancer Institute. Combination trials of IL-2 with IL-4 have shown no increase in responsiveness of melanoma or other tumors when compared to IL-2 alone. However, enhanced expansion of tumor-infiltrating lymphocytes (TILs) in vitro has been observed with combinations of low-dose IL-2 and IL-4. We have begun a study evaluating the trafficking of such expanded lymphocytes following their adoptive transfer in association with systemic administration of IL-2 and IL-4. We have established several TIL cultures from fresh tumor samples, maintained them in long-term culture, and marked them with the neomycin phosphotransferase gene using the LNL6 retroviral vector. Such TILs appear to demonstrate no notable alterations in phenotype or cytolytic activity when compared to their nontransduced counterparts. In addition to IL-2 and IL-4, there are a variety of other novel TCGFs that are now available for evaluation in preclinical and clinical trials. IL-7 induces proliferation and lymphokine-activated killer (LAK) cell activity from human peripheral blood mononuclear cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Two patients in whom pneumonia due to Legionella pneumophila developed while they were receiving immunosuppressive therapy had serologic evidence of prior infection with the same serogroup of L. pneumophila two and eight months prior to their clinical pneumonia. This suggests that the pneumonia in these patients may have been due to the reactivation of a latent infection, possibly due to their immunosuppressed state. A new enzyme-linked immunosorbent assay (ELISA) was developed to detect IgG and IgM antibodies to L. pneumophila, and the kinetics of these antibody responses were useful diagnostically.

Five strains of bacteria (strains 684, 687, U7W, U8W, and MICU-B) that were biochemically and morphologically indistinguishable from Legionella pneumophila were recovered from the environment. Strains U7W, U8W, and MICU-B were antigenically identical to L. pneumophila strain Dallas 1E (serogroup 5), as determined by direct fluorescent antibody staining and immunodiffusion. Although strains 694 and 687 shared antigenic determinants with L. pneumophila Dallas 1E, they could be distinguished by immunodiffusion and differential absorption studies. However, as determined by DNA hybridization, the antigenically distinct strains 684 belong to the same DNA homology cluster as previously described authentic strains of L. pneumophila, whereas strain U7W, U8W, and MICU-B belong to a separate homology group. Therefore, two groups could be identified among these environmental isolates; one represents an antigenic subclass of serogroup 5 L. pneumophila (strains 684, and 687), and the other, although antigenically indistinguishable from serogroup 5 L. pneumophila, probably represents a new Legionella species (strains U7W, U8W, and MICU-B).

The microenzyme-linked immunosorbent assay (ELISA) for the detection of immunoglobulin M and G (IgM, IgG) antibodies to Legionella pneumophila serogroup 1 antigens was evaluated. IgM antibodies were measured by both double-sandwich and single-sandwich techniques. These assays were compared with the previously standardized indirect immunofluorescence test in four groups of subjects: (i) pneumonia patients with culture-proven Legionnaires disease with serogroup 1 isolates, (ii) pneumonia patients with serogroup 1 organisms detected by direct immunofluorescence testing of respiratory secretions but without culture confirmation, (iii) pneumonia patients with negative culture and direct immunofluorescence tests, and (iv) healthy hospital employees. In addition, the sensitivity and specificity of the IgG ELISA were evaluated with larger groups of controls and Legionnaires disease patients. The ELISA was more sensitive than the indirect immunofluorescence test. However, it detected antibody rises in pneumonia patients without culture or direct immunofluorescence evidence of L. pneumophila serogroup 1 infection, thereby suggesting that the specificity of the ELISA was slightly lower than that of the indirect immunofluorescence test. The double-sandwich ELISA was a sensitive method for detecting IgM antibodies and, as previously reported, appeared to be free from interference by rheumatoid factor. IgM anti- Legionella antibodies detected by the ELISA appeared earlier and were less persistent than IgG antibodies. In addition, the IgM ELISA was useful in detecting antibodies in necropsy serum samples obtained from patients dying acutely of Legionnaires disease. The data presented show that the ELISA is a reliable method for the detection of specific anti- Legionella antibodies.

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.

Despite increasing survival rates for patients with colorectal cancer, additional treatment options are required, including active or passive immunotherapy for patients with metastatic disease. Freshly harvested colorectal cancer specimens and in vitro cultured colorectal cancer cell lines were examined for IL-7 protein secretion in order to examine the potential role of this cytokine in the interaction between tumour cells and the host immune system. Freshly harvested colorectal cancer specimens (21/21), or normal adjacent mucosa (3/3), as well as long-term established colorectal cancer cell lines (3/4) exhibited IL-7 mRNA expression as detected by RT-PCR and confirmed by Southern Blot analysis. Freshly harvested colorectal cancer tissue (16/18), or long-term established colorectal cancer cell lines (2/4) secreted in vitro IL-7 as detected by ELISA. In contrast, breast, pancreatic, or lung cancer cell lines, as well as several haematopoietic cancer cells lines, tested negative for IL-7 mRNA and protein. The authors tested different cytokines (IL-1 beta, IL-2, IL-7, or a combination of IL-1 beta/IL-7) in vitro for the ability to expand tumour-infiltrating T lymphocytes (TIL) from individual patients (n = 9) with colorectal cancer. TIL populations were tested at day 14 after in vitro propagation for phenotypic analysis by FACS and for reactivity directed against NK and LAK sensitive target cells and autologous cancer cells as measured by cytotoxicity and cytokine release. TIL obtained from colorectal cancer lesions can be efficiently expanded in the presence of IL-7, some(3/9) of which appear to exhibit autologous tumour recognition as measured by cytolytic effector functions and by detection of IFN gamma and TNF alpha release. Detection of IL-7 mRNA expression in colorectal cancer, in normal mucosa adjacent to tumour, as well as the ability of colorectal cancer tissue to secrete IL-7, raises new questions about the biology of the host/tumour interactions in colorectal cancer.

Excerpt The pittsburgh pneumonia agent is a newly recognized cause of pneumonia (1, 2). Although most patients have been renal transplant recipients (1-4), others have had malignant disease or rece...