While human prostate cancers and cell lines express Fas, most of these cell lines are resistant to Fas-mediated death. In the present studies we addressed the ability of IFN-gamma to influence Fas-mediated cell death in prostate cancer cells. In vitro exposure of the human cell lines LNCaP and PC3 and the mouse cell line RM-1 to agonist anti-Fas antibody and/or soluble Fas ligand resulted in killing of only PC3 cells. However, preincubation with IFN-gamma resulted in synergistic killing in all three cell lines. In vitro treatment of RM-1 with a replication-incompetent adenovirus expressing mouse FasL (Ad.FasL) resulted in maximal cell kill near 40%, which correlated with baseline Fas expression. The addition of IFN-gamma enhanced cell kill to a degree consistent with the resulting higher levels of Fas and maintained synergistic killing at very low doses of vector. Co-inoculation of orthotopic RM-1 primary tumors with Ad.mFasL and an adenovirus expressing mouse IL-12 (Ad.mIL-12) to drive host production of IFN-gamma negated the survival advantage of Ad.mIL-12 alone. However, the staggered injection of Ad.mIL-12 and Ad.FasL achieved almost threefold higher levels of apoptosis in primary tumor tissue and doubled median survival. Therefore, IFN-gamma is capable of bestowing increased sensitivity to Fas-mediated cell death in prostate cancer cells and, in a gene therapy approach, may define a powerful tool to treat prostate cancers.

Immune responses against E1-deleted adenovirus vectors and/or their transgene products result in the rapid elimination of vector-transduced cells and the generation of neutralizing antibodies. Different strategies of immunomodulation to stabilize transgene expression at therapeutic levels and to permit productive vector readministration have been examined. Our previous studies have shown that depletion of macrophages from spleen and liver decreases hepatic inflammation, significantly prolongs transgene expression, and delays the onset of humoral immune responses after systemic administration of an E1-deleted adenovirus vector. In the present study, we have examined the effects of macrophage depletion in combination with temporary blockade of CD40 ligation on E1-deleted adenovirus vector-mediated gene transfer. Alone, each of these treatments significantly inhibited the humoral immune response against the transgene product and prolonged its expression. Together, these treatments completely stabilized transgene expression and inhibited the production of neutralizing anti-adenovirus antibodies, permitting successful vector readministration. Animals rendered immunologically unresponsive to vector and transgene antigens regained their ability to mount productive immune responses against the vector after recovery of immune function, but remained unresponsive to the transgene product. These experiments demonstrate that this treatment is transient and antigen-specific.

We studied 133 California phenylketonuria (PKU) patients and one obligate heterozygote to delineate the molecular basis of PKU in a population with greater ethnic diversity than in previous studies, and to determine whether a correlation exists between genotype and clinical phenotype, with the latter defined by both the diagnostic pretreatment blood phenylalanine (PHE) level and cognitive (IQ) test scores. To determine PAH genotypes, we used PCR-mediated amplification, denaturing gradient gel electrophoresis, and direct sequencing on dried whole blood samples. Where possible, mutation severity was defined according to predicted in vitro PAH enzyme activity estimated by using Cos cell expression analysis for a given mutation. We then asked whether mutation severity, as defined by such expression analysis, correlated with pretreatment PHE levels or with IQ test results. A mutation was identified in 236 (88%) of 267 mutant alleles. Seventeen new mutant alleles were found; A47E, T81P, I102T, E182G, T328D, Y343P, K371R, Y387H, A389E, E422K, IVS9nt5, IVS11nt20, delS70, del364-368/del198-220, delF299, delT323, and -1C/T. In striking contrast to a number of studies in other populations, in this study, based on predicted PAH activity, we observed no correlation between mutation severity and pretreatment PHE levels. There was also no correlation between genotype and IQ. We conclude that in samples collected from an ethnically heterogeneous population, there is no correlation of mutation severity with either pretreatment PHE levels or IQ measurement in treated patients. We caution that genetic counseling in PKU should incorporate the notion that prognosis may not be predicted with precision based on mutation analysis in a given patient.

A high degree of molecular heterogeneneity at the phenylalanine hydroxylase (PAH) locus was established by examining RFLP haplotypes and PAH mutations in the families of 13 Egyptians with phenylketenouria (PKU). Thirteen different haplotypes were unequivocally determined in these kindreds. Haplotypes 1.8, 3.9, 4.3, 7.8, 22.11, 27.6, and 52.8 were found segregating with normal chromosomes, whilst haplotypes 1.8, 5.9, 23.8, 32.8, the newly assigned 73.9, and two as yet incomplete but novel haplotypes were found segregating with the mutant chromosomes. There was no particular preference for a single haplotype among normal or mutant chromosomes. Nine different mutations were also identified among the 26 alleles.

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