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J G Clark

Publications and source records attributed to J G Clark.

At least 19 recordsLinked to original sources
PubMed2005

Pulmonary function testing prior to hematopoietic stem cell transplantation.

The pretransplant pulmonary function test plays an important role in the management of noninfectious pulmonary complications after hematopoietic stem cell transplantation (HCT). Although these tests are widely used as standard preoperative assessments in the nontransplant population, common conditions associated with the HCT patient requires that particular attention be given to interpretation of pulmonary function testing (PFT) results, such as comparison of serial pulmonary function tests and evaluation of the diffusion capacity. Although their utility in helping to predict the likelihood of developing post transplant pulmonary complications and mortality is not well established, current data indicate that pretransplant PFTs are important as a reference for the interpretation of post transplant PFTs and for identifying patients at high risk for developing pulmonary complications and/or mortality after HCT. Future studies of pretransplant pulmonary function should consider the advances in HCT, so that pretransplant PFTs will become a useful tool in pretransplant risk assessment and help the transplant oncologist to determine the most appropriate conditioning regimen for a patient with compromised lung function.

Hematopoietic Stem Cell Transplantation↗
PubMed2004

Implications of early airflow decline after myeloablative allogeneic stem cell transplantation.

The clinical significance of early airflow decline after myeloablative allogeneic hematopoietic SCT is uncertain. We performed a retrospective cohort analysis to determine if airflow decline by day 100 is associated with later development of transplant-related airflow obstruction (AFO) and increased mortality risk. Overall, 750 (40%) patients had airflow decline by day 100. Development of airflow decline by day 100 was associated with an increased risk for AFO at 1 year (relative risk 2.6, 95% confidence interval 2.1-3.1) but not with an increase in mortality risk (hazard ratio (HR) 0.86, P=0.05). However, patients with the fastest rate of decline between day 100 and 1 year (12.5% per year +/-24) had the highest mortality risk (HR 3.2, P<0.001). In conclusion, airflow measurements made on day 100 do not predict the rate of airflow decline between day 100 and 1 year, and therefore are not useful as a single measurement for determining mortality risk associated with development of AFO. Closer monitoring of the rate of airflow decline during the first year may facilitate the timely detection and treatment of early airflow decline and prevent the development of fixed AFO and increased mortality risk after hematopoietic stem cell transplant.

Adolescent↗
PubMed2003

Incidence, risk factors, and mortality from pneumonia developing late after hematopoietic stem cell transplantation.

The incidence, etiology, outcome, and risk factors for developing pneumonia late after hematopoietic stem cell transplantation (SCT) were investigated in 1359 patients transplanted in Seattle. A total of 341 patients (25% of the cohort) developed at least one pneumonic episode. No microbial or tissue diagnosis (ie clinical pneumonia) was established in 197 patients (58% of first pneumonia cases). Among the remaining 144 patients, established etiologies included 33 viral (10%), 31 bacterial (9%), 25 idiopathic pneumonia syndrome (IPS, 7%), 20 multiple organisms (6%), 19 fungal (6%), and 16 Pneumocystis carinii pneumonia (PCP) (5%). The overall cumulative incidence of first pneumonia at 4 years after discharge home was 31%. The cumulative incidences of pneumonia according to donor type at 1 and 4 years after discharge home were 13 and 18% (autologous/syngeneic), 22 and 34% (HLA-matched related), and 26 and 39% (mismatched related/unrelated), respectively. Multivariate analysis of factors associated with development of late pneumonia after allografting were increasing patient age (RR 0.5 for <20 years, 1.2 for >40 years, P=0.009), donor HLA-mismatch (RR 1.6 for unrelated/mismatched related, P=0.01), and chronic graft-versus-host disease (GVHD; RR 1.5, P=0.007). Our data suggest that extension of PCP prophylaxis may be beneficial in high-risk autograft recipients. Further study of long-term anti-infective prophylaxis based on patient risk factors after SCT appear warranted.

Adult↗
PubMed2001

Association of TLR4 mutations and the risk for acute GVHD after HLA-matched-sibling hematopoietic stem cell transplantation.

Lipopolysaccharide (LPS) has been implicated in the pathogenesis of graft-versus-host disease (GVHD). The toll-like receptor (TLR)-4 has been recently identified as a major receptor for LPS. Mutations of TLR4 have been associated with LPS hyporesponsiveness. We hypothesized that TLR4 mutations reduce the risk of acute GVHD in allogeneic marrow transplant recipients. In a preliminary study to determine the frequency of TLR4 mutations and their possible association with GVHD, we tested 237 patients and their HLA-identical sibling donors for 2 TLR4 polymorphisms. All patients received methotrexate and cyclosporine for GVHD prophylaxis. One or more mutants were detected in 10.8% of patients and 10.6% of donors. Multivariable logistic regression models were used to analyze the association between TLR4 mutations and probability (1-sided) of GVHD. The odds ratio (adjusted for advanced disease, total body irradiation dose, and patient age) for development of grades II to IV GVHD when a mutation was present in the recipient was 0.63 (95% confidence interval [CI], 0.25-1.60; P = .16). When a mutation was present in the donor, the adjusted odds ratio was 0.88 (95% CI, 0.36-2.17; P = .40). When a mutation was present in both recipient and donor, the odds ratio was 0.72 (95% CI, 0.22-2.32; P = .29). Among 24 patients with TLR4 mutations in either donor or recipient, 4 (16.7%) developed gram-negative bacteremia. Among 213 patients without mutations, 14 (6.6%) developed gram-negative bacteremia (P = .09). The data indicate that a reduced risk of acute GVHD is associated with TLR4 mutations and that TLR4 mutations may increase the risk for gram-negative bacteremia. However, these associations are not statistically significant in recipients of HLA-matched sibling marrow transplants who are prophylactically treated for infections and GVHD. A much larger study population would be needed to confirm the role of LPS in the pathogenesis of GVHD in humans.

Bacteremia↗
PubMed2001

Selective induction of tissue inhibitor of metalloproteinase-1 in bleomycin-induced pulmonary fibrosis.

Tissue inhibitors of metalloproteinases (TIMPs) are multifunctional proteins that have the capacity to modify cellular activities and to modulate matrix turnover. We demonstrate that TIMP-1 messenger RNA (mRNA) and protein expression are selectively and markedly increased in a murine model of bleomycin-induced pulmonary fibrosis. Northern analysis showed that lung steady-state TIMP-1 mRNA levels increased 14-fold after bleomycin administration compared with control mice. Expression of the genes for TIMP-2, TIMP-3, and interstitial collagenase (matrix metalloproteinase-13) was unaltered in the injured lung. In situ hybridization demonstrated that TIMP-1 gene induction was spatially restricted to areas of lung injury. Metalloproteinase inhibitory activity of relative molecular mass of ~ 21 to 28 kD, corresponding to the molecular weights for TIMP-1 and TIMP-2, was identified in lung extracts of bleomycin-injured mice by reverse zymography. Western analysis demonstrated that TIMP-1 protein levels in bronchoalveolar lavage fluid (BALF) of bleomycin-treated mice increased 220- and 151-fold at Days 4 and 28, respectively, compared with control mice. TIMP-2 immunoreactive protein in the BALF increased 20- and 103-fold relative to controls at Days 4 and 28, respectively. These results demonstrate that TIMP-1 gene expression is selectively increased, and that the expression of TIMP-1 and TIMP-2 is differentially regulated in bleomycin-induced pulmonary fibrosis. The profound and durable increase in TIMP-1 and TIMP-2 proteins suggests an important regulatory role for these antiproteases in the inflammatory and fibrotic responses to bleomycin-induced lung injury.

Animals↗
PubMed2000

Neutralization of tumor necrosis factor-alpha action delays but does not prevent lung injury induced by alloreactive T helper 1 cells.

BACKGROUND: Lung injury occurs frequently after allogeneic bone marrow transplantation in association with graft-versus-host disease, an immune response that involves both cellular and cytokine components. In a murine model, we recently showed that cloned alloreactive T helper (Th)1 cells can cause lung injury associated with increased production of tumor necrosis factor (TNF)-alpha by alveolar macrophages (J Immunol 1998; 161: 1913). METHODS: To evaluate the role of TNF-alpha in this model, we injected in vitro-activated Th1 cells into the following: (1) recipients deficient in receptors for TNF; (2) C57BL/6 control mice; (3) C57BL/6 mice, pretreated with soluble TNFRIIFc (a dimorphic high-affinity TNF antagonist); (4) mice expressing TNFRIIFc transgene under control of the surfactant apoprotein C promoter (SPCTNFRIIFc); and (5) wild-type littermate controls (C57BL/6) (n=3-6 mice/group). RESULTS: At 1 and 3 days after i.v. Th1 cell transfer, recipients were killed for analysis of lung histology, bronchoalveolar lavage (BAL) protein, and BAL cell counts. Control mice (wild type) at day 1 after injection had a mild to moderate mononuclear perivasculitis and increased interstitial cellularity. At day 3, lesions were more severe and perivasculitis also involved larger veins. TNFR-deficient mice had normal lung or minimal lung inflammation at day 1. At day 3, perivasculitis of medium-sized vessels was present, but there was no apparent involvement of larger veins. Results in mice treated with soluble TNFRIIFc and transgenic mice (SPCsTNFRIIFc) were similar to controls. BAL protein and BAL cell counts did not differ between any of the experimental groups. CONCLUSIONS: We conclude that lung inflammation induced by Th1 cells may be only delayed when TNF-alpha action is blocked. The persistence of abnormalities indicates that other proinflammatory pathways are involved in injury caused by these cells.

Animals↗
PubMed2000

Adherence of adoptively transferred alloreactive Th1 cells in lung: partial dependence on LFA-1 and ICAM-1.

T helper type 1 (Th1) cells are important effectors in a number of immune-mediated lung diseases. We recently described a murine model of lung injury induced by adoptive transfer of cloned alloreactive Th1 cells. To investigate mechanisms that result in injury to the lung, we studied the in vivo distribution of (51)Cr-labeled Th1 cells. One hour after intravenous administration, >85% of injected radioactivity was left in the lung, and at 24 h, 40% of radioactivity was left in the lung. Adherence of Th1 cells in the lung was significantly inhibited by neutralizing antibody to lymphocyte function-associated antigen-1. Th1 cell adherence also was decreased in lungs of mice deficient in intercellular adhesion molecule-1 (ICAM-1). Th1 cell transfer further induced expression of ICAM-1 and vascular cell adhesion molecule-1 in the lung. Vascular cell adhesion molecule-1-immunoreactive protein was markedly induced in lung endothelium by alloreactive Th1 cells. These findings indicate that Th1 cells localize in normal lung by a mechanism involving lymphocyte function-associated antigen-1 and ICAM-1. Alloreactive cells further induce endothelial adhesion molecules that may facilitate recruitment of inflammatory cells to the lung and amplify Th1 cell-induced lung injury.

Adoptive Transfer↗
PubMed2000

Chemokine expression in Th1 cell-induced lung injury: prominence of IFN-gamma-inducible chemokines.

Proinflammatory responses generated by T helper type 1 (Th1) cells may contribute significantly to immune-mediated lung injury. We describe a murine model of Th1 cell-induced lung injury in which adoptive transfer of alloreactive Th1 cells produces pulmonary inflammation characterized by mononuclear cell vasculitis, alveolitis, and interstitial pneumonitis. To investigate the link between activation of Th1 cells in the lung and inflammatory cell recruitment, we characterized cytokine and chemokine mRNA expression in Th1 cells activated in vitro and in lung tissue after adoptive transfer of Th1 cells. Activated Th1 cells per se express mRNA for interferon (IFN)-gamma and several members of the tumor necrosis factor family as well as the C-C chemokine receptor-5 ligands regulated on activation normal T cells expressed and secreted and macrophage inflammatory protein-1alpha and -1beta. Additional chemokine genes were induced in the lung after Th1 cell administration, most notably IFN-gamma-inducible protein (IP-10) and monokine induced by IFN-gamma (MIG). Remarkable increases in IP-10- and MIG-immunoreactive proteins were present in inflammatory foci lung and identified in macrophages, endothelium, bronchial epithelium, and alveolar structures. The findings suggest that IFN-gamma-inducible chemokines are an important mechanism for amplifying inflammation initiated by Th1 cells in the lung.

Animals↗
PubMed1999

Perflubron enhances adenovirus-mediated gene expression in lungs of transgenic mice with chronic alveolar filling.

Perfluorochemical (PFC) liquids have both low surface tension and a high capacity to dissolve O2 and CO2, and have been shown to improve gas exchange and lung compliance in animal models of lung injury. We have previously demonstrated that perflubron and other PFC liquids enhance transgene expression in lungs of spontaneously breathing normal rodents after intratracheal instillation of either adenoviral or liposomal vectors followed by a single instillation of PFC liquid. We reasoned that PFC liquids may also be useful for enhancing transgene expression in abnormal lungs. GM-CSF knockout mice develop chronic accumulation of surfactant lipids and proteinaceous material in alveolar spaces and serve as a useful model of chronic alveolar filling. Intratracheal instillation of the adenoviral vector Adlac-Z resulted in patchy in situ distribution of beta-Gal activity, predominantly in larger proximal airways. In contrast, in mice instilled with Adlac-Z followed by instillation of a single dose of perflubron (10 ml/kg body weight), increased expression was observed in distal airway and alveolar epithelial cells. In particular, expression was observed in epithelial cells of debris-filled alveoli. Spectrophotometric measure of quantitative beta-Gal activity in lung homogenates demonstrated increased activity in lungs of mice receiving Adlac-Z plus perflubron compared with lungs of animals receiving Adlac-Z alone. These studies demonstrate that use of perflubron enhances transgene expression in lungs of animals with a chronic alveolar filling process. This approach may be applicable for gene delivery in diseases marked by chronic airway or alveolar filling such as cystic fibrosis.

Adenoviridae↗
PubMed1999

Histochemical discrimination of endogenous mammalian beta-galactosidase activity from that resulting from lac-Z gene expression.

Minces of several organs from the transgenic mouse ROSAbeta-gal 26 (ROSA-26), which robustly expresses bacterial lac-Z in most tissues, were exposed to 4-bromo-5-chloro-3-indoyl-beta-D-galactopyrosanide (X-gal) at pH ranging from 7.5 to 9.5 to determine the optimal pH for in situ demonstration of bacterial beta-galactosidase activity (neutral pH optimum) while minimizing detection of potentially confounding endogenous mammalian beta-galactosidase (acidic pH optimum). Similar studies were performed with organ minces from C57BL/6 mice, Sprague-Dawley rats, New Zealand white rabbits, and macaques to confirm the effect of pH on minimizing detection of endogenous mammalian beta-galactosidase. In all organs evaluated; heart, liver, spleen, kidney, brain, and skeletal muscle, endogenous beta-galactosidase activity was rarely detected following incubation at pH greater than 7.5. In contrast, bacterial beta-galactosidase activity in the ROSA-26 mice was strongly detected in organ minces following incubation at pH 8.0-9.0. These findings are similar to previous observations we have made in lung minces and confirm that a simple alteration of a commonly used histochemical technique for detecting in situ beta-galactosidase activity, raising the reaction buffer pH to weakly alkaline range, can reliably distinguish between endogenous activity and that resulting from exogenous bacterial gene expression.

Animals↗
PubMed1999

Perfluorochemical liquid-enhanced adenoviral vector distribution and expression in lungs of spontaneously breathing rodents.

Perfluorochemical (PFC) liquids have been shown to improve gas exchange and lung compliance in models of lung injury. We reasoned they may also be useful as a vehicle for gene transfer by improving transgene distribution throughout the lung as well as increasing total transgene expression. We have developed a model for PFC liquid use in spontaneously breathing rodents that obviates the need for intubation and ventilation. Intratracheal instillation of the adenoviral vector Adlac-Z resulted in patchy distribution of beta-galactosidase (beta-gal) activity as demonstrated using X-gal histochemistry. In contrast, in rats instilled with Adlac-Z followed by instillation of PFC liquid, more uniformly distributed and increased beta-gal activity was observed. Activity in distal airway and alveolar epithelium was particularly increased. Quantitative measure of beta-gal activity in lung homogenates demonstrated a 3- to 6-fold increase in total activity in lungs of rats receiving Adlac-Z and PFC liquid compared to animals receiving Adlac-Z alone. These studies show that PFC liquids can enhance both the distribution and the total amount of transgene expressed following adenoviral-mediated vector transfer to lungs during spontaneous breathing. Use of PFC liquids may increase the efficacy of gene transfer strategies for treatment of cystic fibrosis and other lung diseases.

Adenoviridae↗
PubMed1999

Idiopathic pneumonia after bone marrow transplantation: cytokine activation and lipopolysaccharide amplification in the bronchoalveolar compartment.

OBJECTIVE: To determine whether idiopathic pneumonia syndrome (IPS), a form of noninfectious lung injury that follows bone marrow transplantation, is associated with cytokine activation and increased susceptibility to lipopolysaccharide (LPS). DESIGN: Case series. SETTING: Tertiary referral center for marrow transplantation. PATIENTS: Recipients with biopsy-confirmed IPS; normal volunteers and marrow transplant recipients without IPS were analyzed as controls. MEASUREMENTS AND MAIN RESULTS: Levels of lymphocyte and macrophage-derived cytokines as well as components of the LPS, LPS-binding protein (LBP), and CD14 system in bronchoalveolar lavage (BAL) fluid were determined. We found evidence of increased vascular permeability (BAL protein) and inflammatory cytokine activation (interleukin-1, interleukin-2, interleukin-6, and tumor necrosis factor-alpha) in patients with IPS. Patients without IPS had BAL fluid cytokine and protein levels that were similar to levels in BAL fluid from normal volunteers. Moreover, components of the LPS amplification system (LBP and soluble CD14) were increased in patients with IPS but not in patients without IPS. CONCLUSIONS: These results provide direct evidence for proinflammatory cytokine activation in IPS and suggest that these patients might be at increased risk for LPS-mediated injury through the LBP amplification pathway.

Acute-Phase Proteins↗
PubMed1999

Transforming growth factor-alpha deficiency reduces pulmonary fibrosis in transgenic mice.

Despite evidence that implicates transforming growth factor-alpha (TGF-alpha) in the pathogenesis of acute lung injury, the contribution of TGF-alpha to the fibroproliferative response is unknown. To determine whether the development of pulmonary fibrosis depends on TGF-alpha, we induced lung injury with bleomycin in TGF-alpha null-mutation transgenic mice and wild-type mice. Lung hydroxyproline content was 1.3, 1.2, and 1.6 times greater in wild-genotype mice than in TGF-alpha-deficient animals at Days 10, 21, and 28, respectively, after a single intratracheal injection of bleomycin. At Days 7 and 10 after bleomycin treatment, lung total RNA content was 1.5 times greater in wild-genotype mice than in TGF-alpha-deficient animals. There was no significant difference between mice of the two genotypes in lung total DNA content or nuclear labeling indices after bleomycin administration. Wild-genotype mice had significantly higher lung fibrosis scores at Days 7 and 14 after bleomycin treatment than did TGF-alpha-deficient animals. There was no significant difference between TGF-alpha-deficient mice and wild-genotype mice in lung inflammation scores after bleomycin administration. To determine whether expression of other members of the epidermal growth factor (EGF) family is increased after bleomycin-induced injury, we measured lung EGF and heparin-binding- epidermal growth factor (HB-EGF) mRNA levels. Steady-state HB-EGF mRNA levels were 321% and 478% of control values in bleomycin-treated lungs at Days 7 and 10, respectively, but were not significantly different in TGF-alpha-deficient and in wild-genotype mice. EGF mRNA was not detected in normal or bleomycin-treated lungs of mice of either genotype. These results show that TGF-alpha contributes significantly to the pathogenesis of pulmonary fibrosis after bleomycin-induced injury, and that compensatory increases in other EGF family members do not occur in TGF-alpha-deficient mice.

Animals↗
PubMed1998

Lung injury induced by alloreactive Th1 cells is characterized by host-derived mononuclear cell inflammation and activation of alveolar macrophages.

We have investigated a murine model of acute lung injury caused by i.v. administration of a T cell clone (CD4+, Th1 phenotype) that recognizes Ly5, a polymorphic cell surface glycoprotein expressed on hemopoietic cells. Alloreactive cloned T cells, specific for host Ly5 Ag, cause a mononuclear cell pulmonary vasculitis and interstitial pneumonitis. In further studies of the cellular mechanisms involved in this model, we found that mature host T cells or B cells are not required, since lung injury was comparable in transgenic host mice that lack these cells (RAG-1 knockout). Cloned T cells labeled in vitro with bromodeoxyuridine were localized in inflammation foci in lung, but the majority of cells in the foci were not labeled. Using transgenic mice that constitutively express lacZ, we determined that the mononuclear cell vasculitis is of host cell origin. Alveolar macrophages (AM) from T cell-treated mice spontaneously secreted TNF-alpha in culture, whereas TNF-alpha was not detected in AM cultures from control mice. TNF-alpha production in response to LPS stimulation was significantly higher in AM cultures derived from T cell-treated mice than in those from control mice. Challenge with sublethal doses of LPS resulted in 50% mortality in T cell-treated mice and was associated with augmented AM TNF-alpha production and protein in bronchoalveolar lavage fluid. We conclude that immune activation of T cells of the Th1 phenotype can initiate lung injury characterized by a host-derived mononuclear cell inflammation and activation of AM.

Adoptive Transfer↗
PubMed1998

T cells specific for a polymorphic segment of CD45 induce graft-versus-host disease with predominant pulmonary vasculitis.

To study the character of graft-vs-host disease (GVHD) induced by T cells specific for hemopoietic cells, T cells specific for a polymorphic segment of CD45 were transferred into CD45 congenic mice. C57BL/6 mice that express the CD45b allele were immunized with a 13 mer peptide representing the polymorphic segment (p257-268) of CD45a protein. Conversely, C57BL/6 mice congenic for CD45a were immunized with the CD45b peptide. CD4+ T cells specific for allelic CD45 peptides were elicited. Importantly, T cells specific for CD45 peptides proliferated specifically and vigorously in response to spleen cells expressing the appropriate polymorphic CD45 protein. T cells specific for CD45 induced a substantial graft-vs-host response (GVHR) with predominant early pulmonary vasculitis and later more widespread interstitial mononuclear cell infiltration and alveolitis. No GVHR was induced in bone marrow chimeras expressing only donor hemopoietic cells. Thus, donor T cell recognition of host hemopoietic cells is sufficient to elicit GVHR, but the classical skin, liver, and gut manifestations of GVHD were not observed. The CD45-specific T cells used secreted Th1 cytokines, but without detectable soluble IL-2. Studies using CD45-specific T cells with different effector functions might allow further dissection of donor cell requirements for GVHD syndromes.

Animals↗
PubMed1998

Elevated transforming growth factor-alpha levels in bronchoalveolar lavage fluid of patients with acute respiratory distress syndrome.

The acute respiratory distress syndrome (ARDS) frequently results in a fibroproliferative response that precludes effective alveolar repair. Transforming growth factor-alpha (TGF-alpha), a potent epithelial and mesenchymal cell mitogen, may modulate the response to lung injury. In this study, we determined whether bronchoalveolar lavage fluid (BALF) concentrations of TGF-alpha are increased during the first 2 wk after the onset of ARDS and, if so, whether increased TGF-alpha levels in lavage fluid are associated with increased levels of procollagen peptide III (PCP III), a biological marker of fibroproliferation, and with increased fatality rates. We enrolled 74 consecutive patients with ARDS prospectively identified on admission to the intensive care unit of a tertiary care hospital, and 11 patients with chronic interstitial lung disease. Thirteen healthy volunteers served as control subjects. TGF-alpha concentrations were measured in BALF recovered on Days 3, 7, and 14 after the onset of ARDS (total of 130 lavage samples). TGF-alpha was detected in the lavage fluid of 90% of patients with ARDS (67 of 74), and in 100% of patients with idiopathic pulmonary fibrosis (IPF) (10 of 10), but in none of 13 normal volunteers. At each day tested, the median lavage TGF-alpha level of patients with ARDS was significantly higher than that of normals. The overall fatality rate was 45% (33 of 74 patients). In a univariate analysis, the median TGF-alpha levels in nonsurvivors were 1.5-fold higher at Day 7 (p = 0.06) and 1.8-fold higher at Day 14 (p = 0.048). The fatality rate was 4 times higher (CI 1.6, 17.5) for patients with both increased lavage TGF-alpha and PCP III concentrations at Day 7 than for patients with low TGF-alpha and PCP III values, indicating a synergistic relationship between TGF-alpha and PCP III. We conclude that increased levels of TGF-alpha in BALF are common in patients with ARDS and that lavage TGF-alpha is associated with a marker of the fibroproliferative response in sustained ARDS.

Adolescent↗
PubMed1997

In situ histochemical detection of beta-galactosidase activity in lung: assessment of X-Gal reagent in distinguishing lacZ gene expression and endogenous beta-galactosidase activity.

Bacterial lacZ is one of the most commonly used reporter genes for assessing gene transfer to lung. However, lung contains endogenous beta-galactosidase (beta-Gal), which can confound estimation of exogenous lacZ expression by histochemical techniques (i.e., X-Gal) for in situ demonstration of enzyme activity. We investigated several parameters of the X-Gal reaction, including time and temperature of X-Gal exposure as well as lung tissue processing and fixation techniques, and found that none of these could be used to distinguish between endogenous and exogenous beta-Gal activities. The mammalian and bacterial beta-Gal enzymes, however, have pH optima in the acidic and neutral ranges, respectively. Exposing whole lung, lung minces, or mounted frozen sections of lung to X-Gal at mildly alkaline pH (pH 8.0-8.5), minimized detection of endogenous activity in lungs from a variety of species while preserving that resulting from bacterial enzyme activity in a transgenic mouse expressing lacZ. This technique was also useful in distinguishing endogenous activity from that resulting from adenovirus-mediated lacZ gene transfer to diploid lung fibroblasts in primary culture. An appropriate buffer that maintains the desired pH throughout the duration of X-Gal exposure must be used.

Animals↗