GROWTH HORMONE ATTENUATES MYOCARDIAL FIBROSIS IN RATS WITH CHRONIC PRESSURE OVERLOAD-INDUCED LEFT VENTRICULAR HYPERTROPHY

1. The role of growth hormone (GH) in cardiac remodelling and function in chronic and persistent pressure overload-induced left ventricular hypertrophy has not been defined. The aim of the present study was to assess short-term GH treatment on left ventricular function and remodelling in rats with chronic pressure overload-induced hypertrophy.2. Twenty-six weeks after induction of ascending aortic stenosis (AAS), rats were treated with daily subcutaneous injections of recombinant human GH (1 mg/kg per day; AAS-GH group) or saline (AAS-P group) for 14 days. Sham-operated animals served as controls. Left ventricular function was assessed by echocardiography before and after GH treatment. Myocardial fibrosis was evaluated by histological analysis.3. Before GH treatment, AAS rats presented similar left ventricular function and structure. Treatment of rats with GH after the AAS procedure did not change bodyweight or heart weight, both of which were higher in the AAS groups than in the controls. After GH treatment, posterior wall shortening velocity (PWSV) was lower in the AAS-P group than in the control group. However, in the AAS-GH group, PWSV was between that in the control and AAS-P groups and did not differ significantly from either group. Fractional collagen (% of total area) was significantly higher in the AAS-P and AAS-GH groups compared with control (10.34 +/- 1.29, 4.44 +/- 1.37 and 1.88 +/- 0.88%, respectively; P < 0.05) and was higher still in the AAS-P group compared with the AAS-GH group.4. The present study has shown that short-term administration of GH to rats with chronic pressure overload-induced left ventricular hypertrophy induces cardioprotection by attenuating myocardial fibrosis.

Reduction of crotoxin-induced neuromuscular blockade by gamma radiation

A comparative study between crotoxin and gamma irradiated crotoxin was performed on the indirectly evoked twitches and tetani of sciatic nerve-extensor digitorum longus muscle of rats. Crotoxin (3 to 14 mu g/ml) decreased the amplitude of twitches and induced a slight tetanic fade, and irradiated crotoxin did not significantly affect either twitch amplitude or tetanic tension. Since gamma radiation reduced the neurotoxicity of crotoxin it may be useful for the production of anticrotalic serum. (C) 1998 Elsevier B.V. Ltd. All rights reserved.

Endogenous annexin A1 counter-regulates bleomycin-induced lung fibrosis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Hypervitaminosis A-induced hepatic fibrosis in a cat

Rationale: The excessive intake of vitamin A in the form of vitamin concentrate, supplement or vitamin-rich liver can result in hypervitaminosis A in man and animals. Although osteopathologies resulting from chronic vitamin A intoxication in cats are well characterized, no information is available concerning feline hypervitaminosis A-induced liver disease. Clinical summary: We report the first case of hepatic stellate cell lipidosis and hepatic fibrosis in a domestic cat that had been fed a diet based on raw beef liver. Radiographic examination revealed exostoses and ankylosis between vertebrae C1 and T7, compatible with deforming cervical spondylosis. Necropsy showed a slightly enlarged and light yellow to bronze liver. Microscopic and ultrastructural analyses of liver tissues revealed diffuse and severe liver fibrosis associated with hepatic stellate cell hyperplasia and hypertrophy. These cells showed immunopositive staining for α-smooth muscle actin and desmin markers. The necropsy findings of chronic liver disease coupled with osteopathology supported the diagnosis of hypervitaminosis A. Practical relevance: As in human hepatology, if there is dietary evidence to support increased intake of vitamin A, then hypervitaminosis A should be considered in the differential diagnosis of chronic liver disease in cats.

Added mass and wave radiation damping for flow-induced rotational vibrations of skinplates of hydraulic gates

One observed vibration mode for Tainter gate skinplates involves the bending of the skinplate about a horizontal nodal line. This vibration mode can be approximated as a streamwise rotational vibration about the horizontal nodal line. Such a streamwise rotational vibration of a Tainter gate skinplate must push away water from the portion of the skinplate rotating into the reservoir and draw water toward the gate over that portion of the skinplate receding from the reservoir. The induced pressure is termed the push-and-draw pressure. In the present paper, this push-and-draw pressure is analyzed using the potential theory developed for dissipative wave radiation problems. In the initial analysis, the usual circular-arc skinplate is replaced by a vertical, flat, rigid weir plate so that theoretical calculations can be undertaken. The theoretical push-and-draw pressure is used in the derivation of the non-dimensional equation of motion of the flow-induced rotational vibrations. Non-dimensionalization of the equation of motion permits the identification of the dimensionless equivalent added mass and the wave radiation damping coefficients. Free vibration tests of a vertical, flat, rigid weir plate model, both in air and in water, were performed to measure the equivalent added mass and the wave radiation damping coefficients. Experimental results compared favorably with the theoretical predictions, thus validating the theoretical analysis of the equivalent added mass and wave radiation damping coefficients as a prediction tool for flow-induced vibrations. Subsequently, the equation of motion of an inclined circular-arc skinplate was developed by incorporating a pressure correction coefficient, which permits empirical adaptation of the results from the hydrodynamic pressure analysis of the vertical, flat, rigid weir plate. Results from in-water free vibration tests on a 1/31-scale skinplate model of the Folsom Dam Tainter gate are used to demonstrate the utility of the equivalent added mass coefficient.

Gene transfer of hepatocyte growth factor by electroporation reduces bleomycin-induced lung fibrosis

Abnormal alveolar wound repair contributes to the development of pulmonary fibrosis after lung injury. Hepatocyte growth factor (HGF) is a potent mitogenic factor for alveolar epithelial cells and may therefore improve alveolar epithelial repair in vitro and in vivo. We hypothesized that HGF could increase alveolar epithelial repair in vitro and improve pulmonary fibrosis in vivo. Alveolar wound repair in vitro was determined using an epithelial wound repair model with HGF-transfected A549 alveolar epithelial cells. Electroporation-mediated, nonviral gene transfer of HGF in vivo was performed 7 days after bleomycin-induced lung injury in the rat. Alveolar epithelial repair in vitro was increased after transfection of wounded epithelial monolayers with a plasmid encoding human HGF, pCikhHGF [human HGF (hHGF) gene expressed from the cytomegalovirus (CMV) immediate-early promoter and enhancer] compared with medium control. Electroporation-mediated in vivo HGF gene transfer using pCikhHGF 7 days after intratracheal bleomycin reduced pulmonary fibrosis as assessed by histology and hydroxyproline determination 14 days after bleomycin compared with controls treated with the same vector not containing the HGF sequence (pCik). Lung epithelial cell proliferation was increased and apoptosis reduced in hHGF-treated lungs compared with controls, suggesting increased alveolar epithelial repair in vivo. In addition, profibrotic transforming growth factor-beta1 (TGF-beta1) was decreased in hHGF-treated lungs, indicating an involvement of TGF-beta1 in hHGF-induced reduction of lung fibrosis. In conclusion, electroporation-mediated gene transfer of hHGF decreases bleomycin-induced pulmonary fibrosis, possibly by increasing alveolar epithelial cell proliferation and reducing apoptosis, resulting in improved alveolar wound repair.

Effect of high-affinity anti-Pseudomonas aeruginosa lipopolysaccharide antibodies induced by immunization on the rate of Pseudomonas aeruginosa infection in patients with cystic fibrosis

Patients with cystic fibrosis (CF; N = 26) and with no prior history of infection with Pseudomonas aeruginosa were immunized with an octavalent O-polysaccharide-toxin A conjugate vaccine. During the next 4 years, 16 patients (61.5%) remained free of infection and 10 (38.5%) became infected. Total serum antilipopolysaccharide (LPS) antibody levels induced by immunization were comparable in infected and noninfected patients. In contrast, 12 of 16 noninfected versus 3 of 10 infected patients (p = 0.024) mounted and maintained a high-affinity anti-LPS antibody response. When compared retrospectively with the rate in a group of age- and gender-matched, nonimmunized, noncolonized patients with CF, the rate at which P. aeruginosa infections were acquired was significantly lower (p < or = 0.02) among all immunized versus nonimmunized patients during the first 2 years of observation. Subsequently, only those immunized patients who maintained a high-affinity anti-LPS antibody response had a significant reduction (p < or = 0.014) in the rate of infection during years 3 and 4. Smooth, typeable strains of P. aeruginosa predominated among immunized patients; rough, nontypeable strains were most frequently isolated from nonimmunized patients. Mucoid variants were isolated from one immunized patient versus six nonimmunized patients. These results indicate that the induction of a high-affinity P. aeruginosa anti-LPS antibody response can influence the rate of infection in patients with CF.

Antibody responses induced by long-term vaccination with an octovalent conjugate Pseudomonas aeruginosa vaccine in children with cystic fibrosis

We assessed the serological responses over 10 years to repeated immunization of cystic fibrosis (CF) patients with an O-polysaccharide (OPS)-toxin A conjugate vaccine against Pseudomonas aeruginosa. A retrospective analysis was performed with sera from 25 vaccinated and 25 unvaccinated children treated at the same CF centre and matched for clinical management, age and gender. Yearly immunization led to sustained elevations of serum immunoglobulin G (IgG) antibody levels to all vaccine components. Eighteen unvaccinated patients but only eight vaccinated ones developed chronic pseudomonal lung infections. Infection rapidly caused further marked elevations of polysaccharide- but not toxin A-specific serum IgG in both immunized and nonimmunized patients, indicating that protection did not depend on the quantity of IgG present. However, qualitative analyses revealed that the protective capacity of specific serum IgG antibodies was linked to high affinity and to specificity for OPS serotypes rather than for lipopolysaccharide core epitopes.

Generation and characterization of antigenic variants induced by exposure of tumor cells to UV radiation in vitro

Antigenic changes present in nonantigenic tumor cells exposed to UV radiation (UV) in vitro were investigated by addressing the following questions: (1) Are antigenic variants (AV) produced that are rejected in normal but not immunosuppressed mice? (2) Does generation of AV depend upon intrinsic properties of the cells exposed or result from the action of UV? (3) Is antigenic modification induced by UV due to increased histocompatibility antigen expression? (4) Do AV crossreact immunologically with parental tumor or with other AV? and (5) Is the UV-associated common antigen expressed on UV-induced tumors present on UV-irradiated tumor cells? AV were generated at different frequencies following in vitro UV irradiation of a spontaneous murine fibrosarcoma (51% of cell lines tested), a murine melanoma (56%), and two melanoma clones (100% and 11%). This indicated that the percentage of AV produced is an intrinsic property of the cell line exposed. The increased antigenicity did not correlate with an increased expression of class I histocompatibility antigens. Immunological experiments demonstrated that the AV and parental cells shared a determinant that was susceptible to immune recognition, but incapable of inducing immunity. In contrast, the AV were noncrossreactive, suggesting that variant-specific antigens were also expressed. Finally, the AV were recognized by UV-induced suppressor cells, indicating that the UV-associated common antigen expressed by UV-induced tumors was also present. This investigation provides new information on the susceptibility of tumors to antigenic modification by UV and on the relationship between tumor antigens and neoplastic transformation. Furthermore, it suggests an immunological approach for cancer therapy. ^

Mechanisms of apoptosis in bleomycin-induced lung injury: Implications for pulmonary fibrosis

Chronic inflammation leading to pulmonary fibrosis develops in response to environmental pollutants, radiotherapy, or certain cancer chemotherapeutic agents. Studies have shown that several cell types accumulate during the inflammatory process, but little information is known about what actually triggers and stimulates persistent inflammation culminating in fibrosis. As a first step in defining the events that precipitate inflammation in the lung, the biological mechanism(s) mediating apoptosis and cellular targets must be identified. The purpose of this study was to determine the molecular mechanism(s) of bleomycin-induced apoptosis in the lung using mice deficient in genes that we hypothesized to play a key role in apoptosis. Intratracheal administration of bleomycin led to caspase-mediated DNA fragmentation characteristic of apoptosis. The effects of bleomycin were associated with translocation of p53 from the cytosol to the nucleus only in alveolar macrophages that had been exposed to the drug in vivo, suggesting that the lung microenvironment regulated p53 activation. Experiments with a thiol antioxidant (N-acetylcysteine) in vivo and nitric oxide donors in vitro confirmed that reactive oxygen species were required for p53 activation. A specific role for NO was demonstrated in experiments with iNOS−/− macrophages, which failed to demonstrate nuclear p53 localization after in vivo bleomycin exposure. Strikingly, rates of bleomycin-induced apoptosis were at least two-fold higher in iNOS−/− and p53−/− C57BL/6 mice compared to wild-type controls. Laser Scanning Cytometry (LSC) analysis revealed that bleomycin exposure resulted in a 2-fold induction in Fas and FasL expression in wild-type mice but not iNOS−/− or p53−/− mice. Experiments using gld mice confirmed that the Fas/FasL pathway was the primary mechanism of bleomycin-induced apoptosis in the lung. LSC-mediated analysis indicated that bleomycin exposure resulted in a 2-fold induction in Bax expression in iNOS−/− and P53−/− mice but not wild-type mice. Furthermore, LSC analysis revealed that bleomycin exposure induced a 3-fold increase in thrombospondin expression in wild-type mice. However, thrombospondin was not expressed in either the iNOS−/− or p53−/− mice, implicating a thrombospondin-mediated apoptotic cell clearance mechanism in the lung. Together, these results demonstrate that iNOS and p53 positively regulate apoptosis via the Fas/FasL pathway and mediate a novel apoptosis-suppressing pathway in the lung. ^

Adenosine induced pulmonary fibrosis and the contribution of the adenosine A2B receptor to the induction of osteopontin in a mouse model of pulmonary fibrosis

Pulmonary fibrosis is a devastating and lethal lung disease with no current cure. Research into cellular signaling pathways able to modulate aspects of pulmonary inflammation and fibrosis will aid in the development of effective therapies for its treatment. Our laboratory has generated a transgenic/knockout mouse with systemic elevations in adenosine due to the partial lack of its metabolic enzyme, adenosine deaminase (ADA). These mice spontaneously develop progressive lung inflammation and severe pulmonary fibrosis suggesting that aberrant adenosine signaling is influencing the development and/or progression of the disease in these animals. These mice also show marked increases in the pro-fibrotic mediator, osteopontin (OPN), which are reversed through ADA therapy that serves to lower lung adenosine levels and ameliorate aspects of the disease. OPN is known to be regulated by intracellular signaling pathways that can be accessed through adenosine receptors, particularly the low affinity A2BR receptor, suggesting that adenosine receptor signaling may be responsible for the induction of OPN in our model. In-vitro, adenosine and the broad spectrum adenosine receptor agonist, NECA, were able to induce a 2.5-fold increase in OPN transcripts in primary alveolar macrophages. This induction was blocked through antagonism of the A2BR receptor pharmacologically, and through the deletion of the receptor subtype in these cells genetically, supporting the hypothesis that the A2BR receptor was responsible for the induction of OPN in our model. These findings demonstrate for the first time that adenosine signaling is an important modulator of pulmonary fibrosis in ADA-deficient mice and that this is in part due to signaling through the A2BR receptor which leads to the induction of the pro-fibrotic molecule, otseopontin. ^

Immunologic characteristics of immunogenic variants generated by {\it in vitro\/} treatment of a methylcholanthrene-induced murine fibrosarcoma MCA-F with 1 methyl-3-nitro-1-nitrosoguanidine, 5 -aza-2$\sp\prime$-deoxycytidine, or ultraviolet radiation

This study addresses the questions of whether the frequency of generation and in vivo cross-reactivity of highly immunogenic tumor clones induced in a single parental murine fibrosarcoma cell line MCA-F is more closely related to the agent used to induce the Imm$\sp{+}$ clone or whether these characteristics are independent of the agents used. These questions were addressed by treating the parental tumor cell line MCA-F with UV-B radiation (UV-B), 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), or 5-aza-2$\sp\prime$-deoxycytidine (5-azaCdR). The frequency of Imm$\sp{+}$ variant generation was similarly high for the three different agents, suggesting that the frequency of Imm$\sp{+}$ generation was related more closely to the cell line than to the inducing agent used. Cross-reactivity was tested with two Imm$\sp{+}$ clones from each treatment group in a modified immunoprotection assay that selectively engendered antivariant, but not antiparental immunity. Under these conditions each clone, except one, immunized against itself. The MNNG-induced clones engendered stronger antivariant immunity but a weaker variant cross-reactive immunity could also be detected.^ This study also characterized the lymphocyte populations responsible for antivariant and antiparental immunity in vivo. Using the local adoptive transfer assay (LATA) and antibody plus complement depletion of T-cell subsets, we showed that immunity induced by the Imm$\sp{+}$ variants against the parent MCA-F was transferred by the Thy1.2$\sp{+}$, L3T4a$\sp{+}$, Lyt2.1$\sp{-}$ (CD4$\sp{+}$) population, without an apparent contribution by Thy1.2$\sp{+}$, L3T4a$\sp{-}$, Lyt2.1$\sp{+}$ (CD8$\sp{+}$) cells. A role for Lyt2.1$\sp{+}$T lymphocytes in antivariant, but not antiparent immunity was supported by the results of LATA and CTL assays. Immunization with low numbers of viable Imm$\sp{+}$ cells, or with high numbers of non viable Imm$\sp{+}$ cells engendered only antivariant immunity without parental cross-protection. The associative recognition of parental antigens and variant neoantigens resulting in strong antiparent immunity was investigated using somatic cells hybrids of Imm$\sp{+}$ variants of MCA-F and an antigenically distinct tumor MCA-D. An unexpected result of these latter experiments was the expression of a unique tumor-specific antigen by the hybrid cells. These studies demonstrate that the parental tumor-specific antigen and the variant neoantigen must be coexpressed on the cell surface to engender parental cross-protective immunity. (Abstract shortened with permission of author.) ^

The role of p53 in skin cancer induction by UV radiation and as a potential tumor antigen in UV-induced murine skin tumors

Carcinoma of the skin is the most common type of human cancer in the United States. Ultraviolet radiation (UVR) present in the sunlight is thought to be the major carcinogen responsible for induction of skin cancer. In UV-associated skin carcinogenesis, mutations in p53 are not only present with very high frequency, but occur early in the course of tumor development. In addition, UV-induced skin tumors in mice exhibit unique immunological characteristics. They are highly antigenic and express both individually-specific tumor transplantation antigens recognized by effector T cells and the UV-associated common antigen recognized by UV-induced suppressor T cells. ^ To examine the hypothesis that p53 plays a critical role in preventing skin cancer induction by UVR, mice constitutively lacking one or two functional p53 alleles were compared to wild-type mice for their susceptibility to UV carcinogenesis. Both p53 +/– and –/– mice showed greater susceptibility to skin cancer induction than wild-type mice, and –/– mice were the most susceptible, Accelerated tumor development in the p53 +/– mice was not associated with loss of the remaining wild-type allele of p53 , but in many cases was associated with UV-induced mutations in p53. Our studies clearly demonstrate the essential role of p53 in protection against UV carcinogenesis, particularly in the eye and epidermis. ^ The role of p53 in the antigenicity of UV-induced murine skin tumors was also addressed. Primary UV-induced tumors from p53 –/–, +/– and +/+ mice were transplanted into both normal and immunosuppressed mice, and rates of tumor rejection were compared. Tumors from mice with only one or no functional p53 alleles were less antigenic than those from mice with two functional p53 alleles. Moreover, tumors with no functional p53 also failed to grow well in chronically UV-irradiated mice. These results indicate that p53 contributes to the strong antigenicity of UV-induced murine skin tumors, and suggest that it may play a critical role in expression of the UV-associated common antigen recognized by suppressor T cells. ^ In this study we also monitored the effect of UVR on the development of lymphoid malignancies in p53 deficient mice. The incidence of lymphoid malignancies in UV-irradiated p53 +/– mice was drastically enhanced compared to that in unirradiated counterparts. The immune responses of the mice were identical and were suppressed to the same extent by UV irradiation regardless of the p53 genotype. These data provide the first experimental evidence that exposure to UVR can contribute to the development of lymphoid neoplasms in genetically susceptible hosts. ^