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Biomedical subjects

P B Bitterman

Publications and source records attributed to P B Bitterman.

At least 19 recordsLinked to original sources
PubMed2001

Inhibition of mesothelioma cell growth in vitro by doxycycline.

Malignant mesothelioma causes profound morbidity and nearly universal mortality that is often refractory to conventional treatment modalities of aggressive surgery, radiotherapy, or chemotherapy. Doxycycline, a commonly used antibiotic, has anti-tumor activity against several malignancies, but its anti-tumor effects on malignant mesothelioma have not been evaluated. We report here that concentrations of doxycycline achievable in serum with typical oral doses had cytostatic effects to varying extent on all eight of the mesothelioma cell lines studied but did not affect normal lung fibroblasts. Doxycycline inhibited the production of mitochondrial cytochrome c oxidase, especially in mesothelioma cells more sensitive to its cytostatic effects, and directly inhibited gelatinase A activity; both of these activities are putative mechanisms for the cytostatic activity of doxycycline in other tumor cells. Thus doxycycline may have a role as adjuvant therapy for malignant mesothelioma.

Anti-Bacterial Agents↗
PubMed2000

Translational control of the antiapoptotic function of Ras.

Activated Ras has been shown to provide powerful antiapoptotic signals to cells through well defined transcriptional and post- translational pathways, whereas translational control as a mechanism of Ras survival signaling remains unexplored. Here we show a direct relationship between assembly of the cap-dependent translation initiation apparatus and suppression of apoptosis by oncogenic Ras in vitro and in vivo. Decreasing protein synthesis with rapamycin, which is known to inhibit cap-dependent translation, increases the susceptibility of Ras-transformed fibroblasts to cytostatic drug-induced apoptosis. In contrast, suppressing global protein synthesis with equipotent concentrations of cycloheximide actually prevents apoptosis. Enforced expression of the cap-dependent translational repressor, the eukaryotic translation initiation factor (eIF) 4E-binding protein (4E-BPI), sensitizes fibroblasts to apoptosis in a manner strictly dependent on its ability to sequester eIF4E from a translationally active complex with eIF4GI and the co-expression of oncogenic Ras. Ectopic expression of 4E-BP1 also promotes apoptosis of Ras-transformed cells injected into immunodeficient mice and markedly diminishes their tumorigenicity. These results establish that eIF4E-dependent protein synthesis is essential for survival of fibroblasts bearing oncogenic Ras and support the concept that activation of cap-dependent translation by extracellular ligands or intrinsic survival signaling molecules suppresses apoptosis, whereas synthesis of proteins mediating apoptosis can occur independently of the cap.

Adaptor Proteins, Signal Transducing↗
PubMed2000

KGF pretreatment decreases B7 and granzyme B expression and hastens repair in lungs of mice after allogeneic BMT.

We investigated keratinocyte growth factor (KGF) as a pretreatment therapy for idiopathic pneumonia syndrome (IPS) generated as a result of lung damage and allogeneic T cell-dependent inflammatory events occurring in the early peri-bone marrow (BM) transplant (BMT) period. B10.BR (H2(k)) recipient mice were transplanted with C57BL/6 (H2(b)) BM with spleen cells after lethal irradiation with and without cyclophosphamide conditioning with and without subcutaneous KGF pretreatment. KGF-pretreated mice had fewer injured alveolar type II (ATII) cells at the time of BMT and exhibited ATII cell hyperplasia at day 3 post-BMT. The composition of infiltrating cells on day 7 post-BMT was not altered by KGF pretreatment, but the frequencies of cells expressing the T-cell costimulatory molecules B7.1 and B7.2 and mRNA for the cytolysin granzyme B (usually increased in IPS) were decreased by KGF. Sera from KGF-treated mice had increases in the Th2 cytokines interleukin (IL)-4, IL-6, and IL-13 4 days after cessation of KGF administration (i.e., at the time of BMT). These data suggest that KGF hinders IPS by two modes: 1) stimulation of alveolar epithelialization and 2) attenuation of immune-mediated injury as a consequence of failure to upregulate cytolytic molecules and B7 ligand expression and the induction of anti-inflammatory Th2 cytokines in situ.

Animals↗
PubMed1999

Lovastatin induces fibroblast apoptosis in vitro and in vivo. A possible therapy for fibroproliferative disorders.

Diseases associated with pathological fibroproliferation represent a major cause of morbidity and mortality. Despite the importance of this class of disorders, current therapy is of limited value, and no therapy is available to reduce the fibroblast population size within existing fibrotic lesions. In this regard, constitutive expression of growth-promoting genes can sensitize cells to undergo apoptosis. Studies in our laboratory have demonstrated that lovastatin potently induces apoptosis in fibroblasts constitutively expressing Myc, and that lung fibroblasts isolated from fibrotic lesions constitutively express growth-promoting genes. In this study, we sought to determine if nontransformed lung fibroblasts would manifest susceptibility to lovastatin-induced apoptosis similar to that observed in fibroblasts ectopically expressing Myc. Here we show that clinically achievable concentrations of lovastatin induce apoptosis in normal and fibrotic lung fibroblasts in vitro, as evidenced by acridine orange staining, terminal transferase nick end translation (TUNEL), and DNA laddering. Apoptosis of human lung fibroblasts was dose- and time-dependent, and blocked by exogenous mevalonic acid. Furthermore, apoptosis was associated with decreased levels of mature Ras, a molecule directly implicated in fibroblast rescue from apoptosis. The ability of lovastatin to induce fibroblast apoptosis in vivo was examined using a guinea pig wound chamber model. Lovastatin (5 microM, 8 d) reduced granulation tissue formation in the wound chambers by 64.7%, with associated ultrastructural evidence of fibroblast apoptosis. These findings support further study of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors as potential therapy for patients with fibroproliferative disorders.

Animals↗
PubMed1997

The critical early proinflammatory events associated with idiopathic pneumonia syndrome in irradiated murine allogeneic recipients are due to donor T cell infusion and potentiated by cyclophosphamide.

We have hypothesized that lung damage occurring in the peri-bone marrow transplant (BMT) period is critical for the subsequent generation of idiopathic pneumonia syndrome (IPS), a major complication following human BMT. The proinflammatory events induced by a common pre-BMT conditioning regimen, cyclophosphamide (Cytoxan(R)) (Cy) and total body irradiation, were analyzed in a murine BMT model. Electron microscopy indicated that Cy exacerbated irradiation-induced epithelial cell injury as early as day 3 after BMT. Allogenicity was an important contributing factor to lung injury as measured by lung wet and dry weights and decreased specific lung compliance. The most significant pulmonary dysfunction was seen in mice receiving both allogeneic T cells and Cy conditioning. IPS was associated with an influx of T cells, macrophages, and neutrophils early post-BMT. Hydroxyproline levels were not increased, indicating that the injury was not fibrotic early post-BMT. As early as 2 h after chemoradiation, host macrophages increased in number in the lung parenchyma. Continued increases in macrophages occurred if splenic T cells were administered with the donor graft. The expression of costimulatory B7 molecules correlated with macrophage numbers. Frequencies of cells expressing mRNA for the inflammatory proteins TNF-alpha, IL-1beta, and TGFbeta were increased. Cy accelerated the upregulation of TGFbeta and increase in host macrophages. The exacerbation of macrophage activation and severity of IPS was dependent on allogeneic T cells, implicating immune-mediated mechanisms as critical to the outcome of IPS. This demonstration of early injury after BMT indicates the need for very early therapeutic intervention before lung damage becomes profound and irreversible.

Animals↗
PubMed1996

Translational control of programmed cell death: eukaryotic translation initiation factor 4E blocks apoptosis in growth-factor-restricted fibroblasts with physiologically expressed or deregulated Myc.

There is increasing evidence that cell cycle transit is potentially lethal, with survival depending on the activation of metabolic pathways which block apoptosis. However, the identities of those pathways coupling cell cycle transit to survival remain undefined. Here we show that the eukaryotic translation initiation factor 4E (eIF4E) can mediate both proliferative and survival signaling. Overexpression of eIF4E completely substituted for serum or individual growth factors in preserving the viability of established NIH 3T3 fibroblasts. An eIF4E mutant (Ser-53 changed to Ala) defective in mediating its growth-factor-regulated functions was also defective in its survival signaling. Survival signaling by enforced expression of eIF4E did not result from autocrine release of survival factors, nor did it lead to increased expression of the apoptosis antagonists Bcl-2 and Bcl-XL. In addition, the execution apparatus of the apoptotic response in eIF4E-overexpressing cells was found to be intact. Increased expression of eIF4E was sufficient to inhibit apoptosis in serum-restricted primary fibroblasts with enforced expression of Myc. In contrast, activation of Ha-Ras, which is required for eIF4E proliferative signaling, did not suppress Myc-induced apoptosis. These data suggest that the eIF4E-activated pathways leading to survival and cell cycle progression are distinct. This dual signaling of proliferation and survival might be the basis for the potency of eIF4E as an inducer of neoplastic transformation.

3T3 Cells↗
PubMed1996

Cell fusion to study nuclear-cytoplasmic interactions in endothelial cell apoptosis.

Studies examining the regulation of nuclear rearrangements during apoptosis have led to conflicting results. Cytoplasmic control of nuclear events has been strongly suggested by cell-free experimental systems. In contrast, strict cytoplasmic control cannot account for the results of fibroblast-thymocyte fusion experiments in which dexamethasone induction of polykaryons led only to thymocyte nuclear apoptosis. Unresolved by these fusion studies was whether fibroblast nuclei were indifferent to heterologous cytoplasmic signals. Our objective was to resolve this discrepancy using cell fusion in a homologous system. Our strategy was to fuse endothelial cells with high levels of susceptibility to the induction of apoptosis (log phase cells arrested in G1 for 48 hours by isoleucine deprivation) with those manifesting low levels of susceptibility (serum-deprived, G0). Resultant fused and unfused cells were induced to undergo apoptosis by incubation with tumor necrosis factor-alpha and cycloheximide. Depending on the parental cell of origin, between 14 and 30% of dikaryons contained one apoptotic and one intact nucleus, indicating that strict cytoplasmic control was not occurring. In accord with this, the total frequency of nuclear apoptosis was unchanged after fusion. However, the distribution of apoptotic nuclei revealed a pronounced cytoplasmic influence, with a two- to fivefold increase in coordinate nuclear behavior. This pattern of nuclear apoptosis was consistent with a model of control in which both the state of nuclear susceptibility to apoptosis and expression of cytoplasmic pro-apoptotic regulators determined whether nuclear apoptosis would eventuate.

Animals↗
PubMed1995

Cyclosporine A toxicity presenting with acute cerebellar edema and brainstem compression. Case report.

A 38-year-old man receiving cyclosporine A after bilateral lung transplantation for cystic fibrosis presented with cortical blindness, generalized seizures, and cerebellar edema. Progressive brainstem compression necessitated emergency posterior fossa decompression. Replacement of cyclosporine A with an alternative immunosuppressive agent, FK506, was followed by rapid neurological recovery and dramatic resolution of radiographic abnormalities. The etiology, clinical features, and radiographic findings of cyclosporine A neurotoxicity are discussed. The pertinent literature is reviewed.

Acute Disease↗
PubMed1994

Induction of endothelial cell apoptosis by TNF alpha: modulation by inhibitors of protein synthesis.

Our objective was to examine the induction of endothelial cell apoptosis by the proinflammatory ligand, TNF alpha. We hypothesized that TNF alpha influences endothelial cell viability by altering the balance of regulatory molecules that either induce or suppress apoptosis. Since the identity of these regulators is unknown, our approach was to examine induction of endothelial cell apoptosis by TNF alpha alone and in the context of an inhibitor of transcription or translation. TNF alpha was able to induce bovine pulmonary artery endothelial cell apoptosis in a dose-dependent fashion. Inhibition of transcription with actinomycin D or translation with cycloheximide also resulted in apoptosis, which reached a maximum value of approximately 35 to 40% of cells after 24 h. TNF alpha induction of apoptosis was either potentiated or abrogated by cycloheximide, depending on the timing of TNF alpha exposure in relation to inhibition of protein synthesis. When cycloheximide was added concomitantly with midrange concentrations of TNF alpha, there was both a dramatic acceleration and a synergistic increase in the observed apoptotic response, with all endothelial cells dying within 24 h. When cycloheximide was added as a function of time after termination of TNF alpha exposure, the synergistic induction of apoptosis was maintained at > 70% of its maximum value for 1 h, declining monotonically in a time-dependent fashion to baseline values after 6 h. In contrast, when TNF alpha was added after protein synthesis was inhibited, no additional increase in apoptosis above that observed with inhibition of protein synthesis alone was observed. Our results are consistent with the concept that endothelial cell viability depends on an interaction of inducers and suppressors of apoptosis, which are susceptible to modulation by TNF alpha.

Animals↗
PubMed1994

Alveolar epithelial cells regulate the induction of endothelial cell apoptosis.

Mesenchymal cell apoptosis is important during development, tissue homeostasis, and repair. We sought to determine whether type II alveolar epithelial cells influence mesenchymal cell apoptosis, using the model of tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis of endothelial cells. Apoptosis was quantified by morphology and confirmed by electrophoretic DNA size analysis. Endothelial cells exposed to 20 ng/ml of TNF-alpha for 16 h exhibited apoptosis in 14.4 +/- 1.4% (SE) of the cells, whereas serum-free conditioned media (CM) from primary cultures of rat type II cells reduced TNF-alpha-induced apoptosis by 52% to 7.5 +/- 0.9% (P < 0.01). Flow cytometric analysis of subdiploid DNA content per cell also showed that CM reduced the percentage of cells with TNF-alpha-induced DNA degradation by 48 +/- 1.7%. The protective effect of CM was concentration dependent and also was effective across a range of TNF concentrations. This CM factor was trypsin sensitive and stable at 65 degrees C for 1 h. It bound to a Mono-Q anion-exchange resin, eluting in a discrete peak at 1.18 M NaCl and pH 8.5. Therefore alveolar type II cells release a heat-stable peptide(s) that protects endothelial cells against apoptosis induced by TNF. Our results suggest that alveolar epithelial cells regulate the response of mesenchymal cells to factors that induce apoptosis during injury and repair.

Animals↗
PubMed1994

Pathogenesis of pulmonary fibrosis: platelet-derived growth factor precedes structural alterations in the Hermansky-Pudlak syndrome.

Peptides that modulate mesenchymal cell function have been detected in the fibrotic lung disorders once physiologic dysfunction is present. Despite this close association with manifest disease, their role in initiating alveolar remodeling remains unknown. We examined the hypothesis that one potent peptide, platelet-derived growth factor (PDGF), would be present at the alveolar surface before the onset of physiologic dysfunction in patients in whom pulmonary fibrosis subsequently develops. Bronchoalveolar lavage and physiologic assessment were performed in asymptomatic patients with the Hermansky-Pudlak syndrome (n = 30), obligate heterozygous (n = 9), and normal volunteers (control group). Lavage cell number and profile were normal, but alveolar macrophages demonstrated characteristic autofluorescence and ultrastructural features of ceroid. Lavage fluid from physiologically normal patients with Hermansky-Pudlak syndrome and from those with occult restrictive disease demonstrated two PDGF-related peptides (14 kd and 38 kd). Radioligand binding and fibroblast proliferation assay demonstrated that the peptides were functional. By immunoassay the concentration of PDGF in lavage fluid was six times greater than control values (p < 0.01). In situ hybridization together with bioassay indicated that alveolar macrophages were one cellular source of PDGF. Similar results were obtained for heterozygotes. These data identify macrophage-derived PDGF peptides as important candidate molecules in the initiation of alveolar remodeling in the fibrotic lung disorders.

Adolescent↗
PubMed1993

Role of mesenchymal cell death in lung remodeling after injury.

Repair after acute lung injury requires elimination of granulation tissue from the alveolar airspace. We hypothesized that during lung repair, signals capable of inducing the death of the two principal cellular elements of granulation tissue, fibroblasts and endothelial cells, would be present at the air-lung interface. Bronchoalveolar lavage fluid obtained from patients during lung repair induced both fibroblast and endothelial cell death, while fluid obtained at the time of injury or from patient controls did not. The mode of cell death for endothelial cells was apoptosis. Fibroblast death, while morphologically distinct from necrosis, also differed from typical apoptosis. Only proliferating cells were susceptible to the bioactivities in lavage fluid, which were trypsin sensitive and lipid insoluble. Histological examination of lung tissue from patients after lung injury revealed evidence of apoptotic cells within airspace granulation tissue. Our results suggest that cell death induced by peptide(s) present at the air-lung interface may participate in the remodeling process that accompanies tissue repair after injury.

Acute Disease↗
PubMed1992

Obliterative bronchiolitis after lung transplantation: a fibroproliferative disorder associated with platelet-derived growth factor.

Fibroproliferative disorders are characterized by accumulations of mesenchymal cells and connective tissue in critical locations, leading to organ dysfunction. We examined the role of platelet-derived growth factor (PDGF) in the pathogenesis of obliterative bronchiolitis, a fibroproliferative process that occurs after lung transplantation and results in small airway occlusion. Bronchoalveolar lavage fluid from obliterative bronchiolitis patients significantly stimulated fibroblast migration, whereas fluid from patient controls did not. Quantitation by radioligand binding assay demonstrated increased concentrations of PDGF in lavage fluid from obliterative bronchiolitis patients (patients, 104 +/- 26.9 pM; controls, 8.4 +/- 6.9 pM; P < 0.01). Heparin affinity, gel filtration, and Western blot analysis confirmed the presence of PDGF in lavage fluid. Immunohistochemical and in situ hybridization studies of histologic sections and bronchoalveolar lavage cells suggest that alveolar macrophages are one cellular source. Prospective evaluation of sequential bronchoalveolar lavage samples from a patient who developed obliterative bronchiolitis demonstrated markedly increased PDGF concentrations before the onset of irreversible airflow obstruction. These findings are consistent with a role for PDGF in the fibroproliferative changes observed in obliterative bronchiolitis.

Adult↗
PubMed1992

Pathogenesis of fibrosis in acute lung injury.

The anatomic changes that occur in response to acute lung injury significantly impair gas exchange. As is the case with skin wounds, a fibroproliferative response follows lung injury. In the lungs, however, this can result in life-threatening obliteration of alveolar air spaces. A better understanding of the mechanisms involved in lung repair may allow the development of therapies that regulate the fibroproliferative response. Studies from our laboratory have identified a peptide in bronchoalveolar lavage fluid from patients with acute lung injury that promotes the migration and replication of lung fibroblasts. This peptide is related to platelet-derived growth factor (PDGF) antigenically as well as by receptor-binding criteria; its molecular weight is 14 kilodaltons (kDa) as compared to 29 kDa for PDGF. Despite the potent activity of the 14 kDa peptide, however, such a growth signal may not be absolutely required for tissue granulation. The possibility that lung fibroblasts from patients with acute lung injury might be capable of dividing without exogenous stimulation will be examined. Another theoretical consideration is the signals that regulate termination of the fibroproliferative response. Insights into the molecular mechanisms involved in lung repair may result in therapies that modulate the sometimes maladaptive fibroproliferative response following acute lung injury.

Bronchoalveolar Lavage Fluid↗
PubMed1992

Hyperplasia of type II pneumocytes in acute lung injury. Cytologic findings of sequential bronchoalveolar lavage.

The organizing stage of acute lung injury syndrome is characterized, in part, by a reactive hyperplasia of type II pneumocytes. These cells may be recovered in large numbers and in an excellent state of preservation by bronchoalveolar lavage. It was noted recently that such cells in lavage samples may mimic adenocarcinoma. To examine the dynamics of type II pneumocyte shedding in acute lung injury, we studied 62 bronchoalveolar lavage samples from 38 patients with acute onset of the adult respiratory distress syndrome (median age, 54 years). A single lavage was performed in 25 patients, whereas from two to five studies were performed in 13 individuals. The timing of lavage ranged from 1 to 435 days after the onset of respiratory distress. Type II pneumocytes were present in 12 specimens as follows: 6 of 24 (25%) samples from days 1 to 3, 4 of 9 (44%) samples from days 4 to 10, and 2 of 11 (18%) samples from days 21 to 32. Specimens obtained after day 32 never contained such cells. Cytologically, type II pneumocytes may resemble the cells of acute radiation injury. Alternatively, they may be smaller, with an increased nuclear-cytoplasmic ratio, nuclear membrane irregularities, and prominent nucleoli, thus resembling the cells of adenocarcinoma. They may shed singly or in groups. A spectrum of changes from small cells to large, fully hyperplastic type II cells may be seen. Recognition of the morphologic features of type II pneumocytes and careful clinical correlation usually will suffice to prevent a false diagnosis of malignancy. Sequential lavage provides a means to study pulmonary epithelial changes after lung injury.

Acute Disease↗
PubMed1991

Safety of bronchoalveolar lavage in the critically ill, mechanically ventilated patient.

OBJECTIVE: To assess complications of bronchoalveolar lavage in the intubated, mechanically ventilated patient. DESIGN: A retrospective, consecutive case series. SETTING: Medical, surgical, and bone marrow transplant critical care units at a university teaching hospital. PATIENTS: Ninety-nine consecutive critically ill, mechanically ventilated patients undergoing bronchoalveolar lavage were included in the study. INTERVENTIONS: All patients underwent bronchoalveolar lavage using a standard method designed to maximize the safety of the procedure. MEASUREMENTS: Each patient's hospital chart was reviewed for immediate and delayed medical complications of the procedure, including cardiac arrhythmias, bleeding, and hemodynamic disturbances. Specific indices of lung mechanics (peak inspiratory airway pressure and static compliance) and oxygenation (alveolar to arterial oxygen tension gradient [P(A-a)O2] and the ratio of FIO2/PaO2) were measured before and 4 hrs after bronchoalveolar lavage to assess durable physiologic consequences of the procedure. RESULTS: No complications occurred that required premature termination of bronchoalveolar lavage. Three patients exhibited adverse effects (hypotension in two and wheezing in one) immediately after the procedure, all of which resolved promptly with treatment. No statistically significant changes were observed in the variables of arterial oxygenation or pulmonary mechanics. Although the sample mean did not change significantly for any of the oxygen variables, 19% of the patients experienced widening of the P(A-a)O2 by greater than 100 torr (greater than 13.3 kPa). A systematic analysis indicated that there was no statistically significant relationship between readily available clinical variables (including duration of mechanical ventilation before bronchoalveolar lavage and prebronchoalveolar lavage P[A-a]O2), and deterioration in oxygenation after the procedure. CONCLUSIONS: We conclude that bronchoalveolar lavage is a well-tolerated procedure in critically ill, mechanically ventilated patients, provided that risk factors for complications are corrected before the procedure and one adheres to procedural guidelines focused on patient safety. Clinically important complications are uncommon. Some patients exhibit deterioration in oxygen after bronchoalveolar lavage; this occurrence cannot be predicted before the procedure.

Adolescent↗