Hyperthermic Extracorporeal Applied Tumor Therapy for Six Cycles for Recurrent Metastatic Peritoneal Serous Papillary Carcinoma.

An elderly female with failed third-line peritoneal serous papillary carcinoma with metastasis (ovarian cancer) was treated by our proprietary method of whole-body hyperthermia-a recirculating extracorporeal circuit at 42°C for 120 minutes. She received six cycles, 28 days apart. Five index lesions were measured prior to and after each treatment. Results showed stable disease with reduced standard uptake volume. She then restarted six cycles of a previously failed chemotherapy, resulting in no evidence of disease for nine months; she survived for 27 months. Using our technology, the patient experienced an improvement in the quality of life and an increase in survival.

Synergistic interaction of hyperthermia and Gemcitabine in lung cancer.

Hyperthermia increases cytotoxicity of various antineoplastic agents. We investigated the cytotoxic effects of Gemcitabine and/or hyperthermia on BZR-T33 (human non-small-cell lung cancer cells) in vitro and in immune-suppressed athymic nude mice. Isobologram analysis of monolayer cell cultures for cytotoxicity demonstrates a synergistic interaction between hyperthermia and Gemcitabine. Clonogenic results show significant reductions in surviving fractions and colony size for both therapies; greatest reduction was for the combined therapy group. Using cell cycle analysis, hyperthermia enhanced Gemcitabine-induced G2-M arrest resulting in destruction of 3.5 log cells. Apoptotic studies (Annexin-V FITC staining) showed that hyperthermia augmented Gemcitabine-induced apoptosis. Transmission electron microscopy demonstrated pathology observed in cultures exposed to either therapy present in cultures exposed to both therapies. Studies in nude mice show that the combination therapy group had both an initial decrease in tumor size, and a significantly delayed rate of growth. Additionally, using tumor material harvested from nude mice two days after end to treatment reveals a significantly greater apoptotic index and significantly smaller mitotic index for the combined therapy group. Western blots of the same tumor material, showed that heat shock protein 70 was not significantly increased, however, caspase-3 activity of was significantly increased because of the combined therapy. In conclusion, the combined therapy is synergistic in effect because of hyperthermia enhancing Gemcitabine-induced apoptosis.

Smoke/burn injury-induced respiratory failure elicits apoptosis in ovine lungs and cultured lung cells, ameliorated with arteriovenous CO2 removal.

STUDY OBJECTIVE: s: The purpose of this study was to examine the effects of two supportive therapies, conventional mechanical ventilation (CMV) and arteriovenous CO(2) removal (AVCO(2)R), during treatment of severe smoke/burn injury-induced ARDS. DESIGN: Sheep were exposed to a smoke/burn injury (lethal dose causing death in 40% of animals); lung tissue and blood was collected prior to injury (control), when an ARDS criterion was met (PaO(2)/fraction of inspired oxygen ratio < 200), then after 72 h of either CMV (group 1) or AVCO(2)R (group 2). Lung tissue was studied by standard histopathologic techniques; cultured lung cells were studied in media supplemented with serum from all four groups. MEASUREMENTS AND RESULTS: In vivo assays demonstrate less apoptotic cell death, and in vitro assays show significantly greater (p < 0.05) cell survival in group 2 (AVCO(2)R) than in group 1 (CMV) or baseline. Differential gene expression demonstrates significantly higher messenger RNA levels of proapoptotic and tumor necrosis factor (TNF)-alpha in cells incubated in baseline media. After exposure of cultured lung cells to conditioned media, protein expression assay of the culture medium revealed no TNF-alpha, TNF receptor (TNFR)-1, or TNFR-2, however, cultured cell lysate reveals elevated levels of TNF-alpha, TNFR-1 and caspase-3 in all groups; most occurred in cells incubated in baseline media (p < 0.05). HOECHST stain, DNA fragmentation, and caspase-3 cleavage show that AVCO(2)R ameliorates apoptosis in this model. CONCLUSIONS: This in vitro work specifically examines cell death in lung cells as a result of smoke/burn injury and effects of therapeutic interventions. Our in vivo studies temporally correlate the clinical pathology to that studied in these lung cells and show that both in vivo and in vitro cell death is predominantly apoptotic and is significantly reduced by AVCO(2)R.

Percutaneous venovenous perfusion-induced systemic hyperthermia for lung cancer: a phase I safety study.

BACKGROUND: Veno-venous perfusion-induced systemic hyperthermia (VV-PISH) homogeneously raises core body temperature potentially improving outcomes from metastatic lung cancer. METHODS: Patients (n = 10) with stage IV lung cancer, received VV-PISH (>or= 42 degrees C to or= 70. Time to target temperature was 47 +/- 2 minutes, as electrolytes remained normal, without patient or circuit complications. Extubation occurred between 6 and 18 hours. Hospital stay was 4.6 +/- 1.1 days; median length-of-survival after hyperthermia was 271 days. For concurrent controls (n = 16, stage IV lung cancer), median length-of-survival from time of diagnosis to death was 96 days, but for the VV-PISH patients it was significantly longer at 450 days (p < 0.05). All patients returned to pretreatment status following treatment and died from progression of lung cancer. CONCLUSIONS: Venovenous perfusion-induced systemic hyperthermia is safe, technically feasible, and achieves target temperature. Survival may be enhanced in stage IV lung cancer.

Development of a three-dimensional model of lung cancer using cultured transformed lung cells.

Despite great strides in understanding cancer biology, the role cellular differentiation and three-dimensional (3-D) structural organization play in metastasis and malignancy remains unclear. Development of 3-D cultures may ultimately provide a model facilitating discovery and interpretation of more relevant information for the expression and role of antibodies in lung cellular pathobiology. The purpose was to develop traditional monolayer (ML) and 3-D cultures of a known transformed metastatic lung cell line and then determine similarities and differences between cultures in terms of differentiation, molecular marker expression and metastasis. A transformed lung cell line (BZR-T33) was initially transfected with green fluorescent protein (GFP) in ML culture. Nude mice were inoculated with BZR-T33 and observed for metastasis. BZR-T33 was grown as ML and 3-D cultures under identical conditions. Immunohistochemical comparison for degree of antibody expression between cultures and control tissue were studied. Electron microscopy (EM) for identification of ultra structures was done and compared between cultures. A 3-D co-culture containing GFP-transformed cells over an immortalized lung-cell line was developed. The GFP-transfected cell line formed tumors and metastasized in mice. EM identified significant mitochondrial and granular endoplasmic reticular pathology in ML not seen in 3-D. Degree of differentiation shows ultra structures and antibody expressions were more representative of control tissue in 3-D than ML. The co-culture experiment in 3-D demonstrates the ability of transformed cells to penetrate the sub-layer of immortalized cells. Development of 3-D cultures will provide a new and powerful tool to study lung biology and pathobiology.

Peri-operative alemtuzumab (Campath-1H) and plasmapheresis for high-PRA positive lymphocyte crossmatch heart transplant: a strategy to shorten left ventricular assist device support.

Patients on a left ventricular assist device (LVAD) often have a high level of panel-reactive antibodies (PRA). Conventional therapy is to await a heart from a negative prospective-crossmatch donor. We transplanted three high-PRA patients with non-crossmatched hearts, using intra- and post-operative plasmapheresis and long-term T-/B-/plasma-cell therapy with alemtuzumab. Three highly sensitized patients (70%, 94% and 96% T-PRA; 63%, 24% and 73% B-PRA) were transplanted after 29, 187 and 94 days LVAD support. The first patient (Case 1) had an erroneous prospective negative crossmatch (due to an outside laboratory's use of the wrong patient's serum) with immediate allograft dysfunction. The correct serum showed a strongly positive crossmatch; plasmapheresis followed by alemtuzumab (20 mg intravenously) shortly after arrival in the ICU resulted in rapid hemodynamic improvement. Encouraged by this success, the next two patients (Cases 2 and 3) underwent LVAD explant and heart transplant with the next available ABO-identical, non-crossmatched donors, using plasmapheresis on bypass immediately before heart implant and alemtuzumab 20 mg intravenously upon ICU arrival, with uneventful courses. All three patients had positive retrospective T- and B-cell crossmatches. Maintenance immunosuppression consisted of cyclosporine and routine prednisone taper, with plasmapheresis as needed (Patient 1, x10; Patient 2, x5) based on diastolic dysfunction. Mycophenolate mofetil was started as a third agent several months post-transplant. Patients are presently New York Heart Association (NYHA) Class I at 26, 16 and 13 months post-transplant. In this small series with follow-up, immediate antibody removal with plasmapheresis, combined with alemtuzumab, a long-acting antibody to CD52 (expressed on T, B and some plasma cells), appears effective in allowing transplantation in sensitized, positive crossmatch recipients. Expanded use of this strategy could shorten LVAD support in many sensitized patients.

Cellular differentiation in three-dimensional lung cell cultures.

INTRODUCTION: Aspects of human biology that are not sufficiently addressed by current cell culture models are cellular differentiation and three-dimensional (3-D) structural organization. A model that more closely associates the presence and biology of organelles to molecular expressions relevant to these organelles may provide evidence of cellular differentiation and the beginning steps in the construction of a 3-D architecture. The development of a new model--3-D cell cultures--may ultimately provide a better understanding of lung biology and pathobiology. PURPOSE: The purpose of this study was to develop both traditional monolayer and 3-D cell cultures of a known and well documented normal lung cell line and then to determine similarities and differences between these cultures in terms of differentiation and molecular marker expression. RESULTS: Electron microscopy identified presence of lipid inclusion, microvilli, extra cellular matrix, and tight junctions in the 3-D cultures; differentiation not seen in ML cultures. The degree of differentiation determined by immunohistochemistry when the cell line was grown as ML or 3-D cultures shows that ultra-structure and marker expressions were more representative of control tissue than when cells were grown in 3-D than as MLs. METHODS: An immortalized cell line was grown as a traditional monolayer (ML) in culture flasks and as 3-D cultures in rotating walled vessels and incubated under identical conditions. Comparison for presence of differentiation and marker expression between these cultures and control tissue collected from surgical patient specimens was studied. Electron microscopy for identification of ultra structures, and immunohistochemistry (ZO-1, EMA, ICAM-1, villin, tubulin, CK 18, VWF, Collagen IV and human mucin) for phenotypic comparisons between cells in ML and 3-D cell cultures was conducted. SUMMARY: The development of 3-D cell cultures will provide for a new and more powerful tool in the study of lung biology and pathobiology.

A mechanism of hyperthermia-induced apoptosis in ras-transformed lung cells.

Lung cancer, the leading cause of cancer-related deaths in both men and women, is the consequence of disordered apoptosis, induction of which may have therapeutic utility. Hyperthermia has been identified as a stimulus for apoptosis. We investigated the mechanism of hyperthermia-induced cell death in ras-transformed lung cells. Effect of hyperthermia (43 degrees C for 180 min) was compared between two cell lines, an immortalized (sv-40) normal human bronchial epithelial (BEAS2-B) and its malignant transformed (H-ras transfected) counterpart (BZR-T33). Survival after hyperthermia: 7-d growth culture BEAS2-B, 1.03 +/- 0.007 and BZR-T33, 0.39 +/- 0.008 (P < 0.05); clonogenic assays BEAS2-B, 0.76 +/- 0.003 and BZR-T33, 0.41 +/- 0.004 (P < 0.05). Hoechst positive (apoptotic) cells: BEAS2-B, 11 +/- 3% and BZR-T33, 78 +/- 5% (P < 0.05). TUNEL, DNA fragmentation, and Annexin-V all corroborate this result. Western blot comparing the effect of hyperthermia in BZR-T33 cells to BEAS2-B cells revealed: TRAIL and FAS-L displayed significant increases (threefold and twofold, respectively); caspase-3 showed a decrease in uncleaved form and an increase in cleaved form, and a 50-fold increase in activity effectively blocked with the caspase-3 inhibitor DEVD-fmk; caspase-9 showed near depletion of uncleaved; poly (ADP-ribose) polymerase (PARP) degradation was clearly visible during heating. After hyperthermia, gene expression demonstrates a 5.7-fold increase in TRAIL and insignificant changes in tumor necrosis factor-alpha (TNF-alpha), FAS-L, and caspases 3, 8, 9 in transformed cells. Data demonstrated that hyperthermia induces apoptosis in transformed cells, and that apoptosis is mediated by caspase-3 as a result of activation of cell-death membrane receptors of the tumor-necrosis-factor family. In summary, these data suggest that hyperthermia could become an additional modality in the multidisciplinary approach to the treatment of lung cancer.

Leukocyte filtration in lung transplantation.

Controlled reperfusion of the transplanted lung has been used in nine consecutive patients to decrease manifestations of lung reperfusion injury. An extracorporeal circuit containing a roller pump, heat exchanger and leukodepleting filter is primed with substrate-enhanced reperfusion solution mixed with approximately 2000 ml of the patient's blood. This solution is slowly recirculated to remove leukocytes prior to reperfusion. When the pulmonary anastomoses are completed, the pulmonary artery is cannulated through the untied anastomosis using a catheter containing a pressure lumen for measurement of infusion pressure. An atrial clamp is left in place on the patient's native atrial cuff to decrease the risk of systemic air embolism during the brief period of reperfusion from the extracorporeal reservoir. During reperfusion, the water bath to the heat exchanger is kept at 35 degrees C and the flow rate for reperfusion solution is between 150 and 200 m/min, keeping the pulmonary artery pressure <14 mmHg. Eight of nine patients were ventilated on 40% inspired oxygen within a few hours of operation and 7/9 were extubated on or before postoperative day 1. Six of nine patients are long-term survivors.