Transforming growth factor ß neutralization ameliorates pre-existing hepatic fibrosis and reduces cholangiocarcinoma in thioacetamide-treated rats.

Considerable evidence has demonstrated that transforming growth factor ß (TGF-ß) plays a key role in hepatic fibrosis, the final common pathway for a variety of chronic liver diseases leading to liver insufficiency. Although a few studies have reported that blocking TGF-ß with soluble receptors or siRNA can prevent the progression of hepatic fibrosis, as yet no evidence has been provided that TGF-ß antagonism can improve pre-existing hepatic fibrosis. The aim of this study was to examine the effects of a murine neutralizing TGF-ß monoclonal antibody (1D11), in a rat model of thioacetamide (TAA)-induced hepatic fibrosis. TAA administration for 8 weeks induced extensive hepatic fibrosis, whereupon 1D11 dosing was initiated and maintained for 8 additional weeks. Comparing the extent of fibrosis at two time points, pre- and post-1D11 dosing, we observed a profound regression of tissue injury and fibrosis upon treatment, as reflected by a reduction of collagen deposition to a level significantly less than that observed before 1D11 dosing. Hepatic TGF-ß1 mRNA, tissue hydroxyproline, and plasminogen activator inhibitor 1 (PAI-1) levels were significantly elevated at the end of the 8 week TAA treatment. Vehicle and antibody control groups demonstrated progressive injury through 16 weeks, whereas those animals treated for 8 weeks with 1D11 showed striking improvement in histologic and molecular endpoints. During the course of tissue injury, TAA also induced cholangiocarcinomas. At the end of study, the number and area of cholangiocarcinomas were significantly diminished in rats receiving 1D11 as compared to control groups, presumably by the marked reduction of supporting fibrosis/stroma. The present study demonstrates that 1D11 can reverse pre-existing hepatic fibrosis induced by extended dosing of TAA. The regression of fibrosis was accompanied by a marked reduction in concomitantly developed cholangiocarcinomas. These data provide evidence that therapeutic dosing of a TGF-ß antagonist can diminish and potentially reverse hepatic fibrosis and also reduce the number and size of attendant cholangiocarcinomas.

Enhanced efficacy of melanoma vaccines in the absence of B lymphocytes.

Provoking a specific cellular immune response against tumor-associated antigens is a promising therapeutic strategy to treat cancers with defined antigens such as melanoma. In recent clinical trials, however, immune responses against melanoma antigens have been elicited without consistent clinical responses, suggesting the need for approaches that potentiate the specific cellular immune response. Since B lymphocytes have been reported to exert a negative effect on the cellular arm of the immune response in certain model systems, the authors compared the protective immunity elicited by melanoma antigens in B cell-deficient microMT mice to that obtained in fully immunocompetent C57BL/6 mice. Immunization with melanoma-associated antigens was accomplished using recombinant adenovirus (Ad) vectors encoding human gp100 (Ad2/gp100) or murine TRP-2 (Ad2/mTRP-2). A single dose of Ad2/gp100 or Ad2/mTRP-2 inhibited the growth of established subcutaneous B16 melanoma tumors in B cell-deficient but not wild-type C57BL/6 mice. The enhanced tumor protection observed in B cell-deficient mice appeared to be associated with potentiation of the magnitude and longevity of the specific cellular immune response. Natural killer (NK) cells were also found to be essential to the protective immune response in microMT mice because NK cell depletion with anti-asialo-GM1 antibody resulted in both the loss of tumor growth suppression and attenuation of the specific cellular immune response. The authors conclude that the protective cell-mediated immunity provoked by Ad-based cancer vaccines is enhanced in the absence of B cells, suggesting that a therapeutic regimen that includes depletion of B lymphocytes may be beneficial to cancer vaccine therapy.

Correction of the nonlinear dose response improves the viability of adenoviral vectors for gene therapy of Fabry disease.

Systemic administration of recombinant adenoviral vectors for gene therapy of chronic diseases such as Fabry disease can be limited by dose-dependent toxicity. Because administration of a high dose of Ad2/CMVHI-alpha gal encoding human alpha-galactosidase A results in expression of supraphysiological levels of the enzyme, we sought to determine whether lower doses would suffice to correct the enzyme deficiency and lysosomal storage abnormality observed in Fabry mice. Reducing the dose of Ad2/CMVHI-alpha gal by 10-fold (from 10(11) to 10(10) particles/mouse) resulted in a greater than 200-fold loss in transgene expression. In Fabry mice, the reduced expression of alpha-galactosidase A, using the lower dose of Ad2/CMVHI-alpha gal, was associated with less than optimal clearance of the accumulated glycosphingolipid (GL-3) from the affected lysosomes. It was determined that this lack of linearity in dose response was not due to an inability to deliver the recombinant viral vectors to the liver but rather to sequestration, at least in part, of the viral vectors by the Kupffer cells. This lack of correlation between dose and expression levels could be obviated by supplementing the low dose of Ad2/CMVHI-alpha gal with an unrelated adenoviral vector or by depleting the Kupffer cells before administration of Ad2/CMVHI-alpha gal. Prior removal of the Kupffer cells, using clodronate liposomes, facilitated the use of a 100-fold lower dose of Ad2/CMVHI-alpha gal (10(9) particles/mouse) to effect the nearly complete clearance of GL-3 from the affected organs of Fabry mice. These results suggest that practical strategies that minimize the interaction between the recombinant adenoviral vectors and the reticuloendothelial system (RES) may improve the therapeutic window of this vector system. In this regard, we showed that pretreatment of mice with gamma globulins also resulted in significantly enhanced adenovirus-mediated transduction and expression of alpha-galactosidase A in the liver.

Analysis of cellular immune responses in the peripheral blood of mice using real-time RT-PCR.

Murine cancer models are commonly used in the evaluation of immunotherapeutic strategies. However, one of the major limitations in the monitoring of cellular immune responses induced by various vaccination approaches is that existing immunoassays require sacrifice of the animals for collection of the spleen or lymph nodes for analysis. We report here the development of an assay to quantitate antigen-specific T cell responses in murine blood, without euthanasia, using real-time RT-PCR for measurement of interferon-gamma mRNA levels. C57BL/6 mice were immunized with an adenoviral vector encoding the melanoma antigen gp100 (Ad2/gp100) or were left untreated. Small samples of whole blood were collected by retro-orbital puncture for analysis of T cell reactivity. The mice were then euthanized and spleen cells were isolated for comparative analyses. Blood and spleen cells were restimulated with either a peptide containing the dominant gp100 MHC Class I-restricted epitope, gp100(25-33), or a negative control peptide containing an irrelevant Class I-restricted epitope from ovalbumin. IFN-gamma mRNA was detected in gp100 peptide-pulsed whole blood as well as in spleen cells recovered from Ad2/gp100-treated mice, but not in untreated mice. In addition, there was a strong correlation in the magnitude of the gp100-specific response of spleen cells from an individual animal when measured by real-time RT-PCR with the more conventional enzyme-linked immunospot (ELISPOT) method (P<0.001). Finally, the gp100-specific immune response measured in the peripheral blood of individual animals by real-time RT-PCR or ELISPOT showed a significant correlation with the response measured in the spleen (P=0.001). We conclude that real-time RT-PCR measurement of IFN-gamma mRNA induced by antigenic stimulation is an attractive method to measure an antigen-specific cellular immune response in small samples of whole blood as it does not require euthanasia, mirrors the response observed in the spleen and correlates with the response measured using the conventional ELISPOT method.