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Citrullinemia type 1 (CTLN1) is an autosomal recessive disorder of metabolism caused by a deficiency of argininosuccinate synthetase. Despite optimal management, CTLN1 patients still suffer from lethal metabolic instability and experience life-threatening episodes of acute hyperammonemia. A murine model of CTLN1 (fold/fold) that displays lethality within the first 21 days of life was used to determine the efficacy of adeno-associated viral (AAV) gene transfer as a potential therapy. An AAV serotype 8 (AAV8) vector was engineered to express the human ASS1 cDNA under the control of a liver-specific promoter (thyroxine-binding globulin, TBG), AAV8-TBG-hASS1, and delivered to 7-10 days old mice via intraperitoneal injection. Greater than 95% of the mice were rescued from lethality and survival was extended beyond 100 days after receiving a single dose of vector. AAV8-TBG-hASS1 treatment resulted in liver-specific expression of hASS1, increased ASS1 enzyme activity, reduction in plasma ammonia and citrulline concentrations and significant phenotypic improvement of the fold/fold growth and skin phenotypes. These experiments highlight a gene transfer approach using AAV8 vector for liver-targeted gene therapy that could serve as a treatment for CTLN1. [ABSTRACT FROM AUTHOR]

Human GraB (hGraB) preferentially induces apoptosis via Bcl-2-regulated mitochondrial damage but can also directly cleave caspases and caspase substrates in cell-free systems. How hGraB kills cells when it is delivered by cytotoxic lymphocytes (CL) and the contribution of hGraB to CL-induced death is still not clear. We show that primary human natural killer (hNK) cells, which specifically used hGraB to induce target cell death, were able to induce apoptosis of cells whose mitochondria were protected by Bcl-2. Purified hGraB also induced apoptosis of Bcl-2-overexpressing targets but only when delivered at 5- to 10-fold the concentration required to kill cells expressing endogenous Bcl-2. Caspases were critical in this process as inhibition of caspase activity permitted clonogenic survival of Bcl-2-overexpressing cells treated with hGraB or hNK cells but did not protect cells that only expressed endogenous Bcl-2. Our data therefore show that hGraB triggers caspase activation via mitochondria-dependent and mitochondria-independent mechanisms that are activated in a hierarchical manner, and that the combined effects of Bcl-2 and direct caspase inhibition can block cell death induced by hGraB and primary hNK cells.Cell Death and Differentiation (2008) 15, 708–717; doi:10.1038/sj.cdd.4402300; published online 18 January 2008 [ABSTRACT FROM AUTHOR]

Loss of Bid confers clonogenic survival to granzyme B-treated cells, however the exact role of Bid-induced mitochondrial damage – upstream or downstream of caspases – remains controversial. Here we show that direct cleavage of Bid by granzyme B, but not caspases, was required for granzyme B-induced apoptosis. Release of cytochrome c and SMAC, but not AIF or endonuclease G, occurred in the absence of caspase activity and correlated with the onset of apoptosis and loss of clonogenic potential. Loss of mitochondrial trans-membrane potential (ΔΨm) was also caspase independent, however if caspase activity was blocked the mitochondria regenerated their ΔΨm. Loss of ΔΨm was not required for rapid granzyme B-induced apoptosis and regeneration of ΔΨm following cytochrome c release did not confer clonogenic survival. This functional dissociation of cytochrome c and SMAC release from loss of ΔΨm demonstrates the essential contribution of Bid upstream of caspase activation during granzyme B-induced apoptosis.Cell Death and Differentiation (2006) 13, 607–618. doi:10.1038/sj.cdd.4401772; published online 16 September 2005 [ABSTRACT FROM AUTHOR]

Apoptosis of malignant cells has been suggested as an important mechanism of the action of bexarotene in the treatment of cutaneous T-cell lymphoma (CTCL).Our purpose was to examine thein vivoandin vitroresponses of patients with Sézary syndrome treated with oral bexarotene and assess them for apoptosis of the Sézary cells.Six patients with CTCL with circulating Sézary cells, participating in a clinical trial of oral bexarotene (300 mg m−2 daily) were included in the study. Peripheral blood from the patients was analysed forin vivoandin vitroapoptosis.None of the six patients demonstratedin vivoapoptosis.In vitroapoptosis of Sézary cells was demonstrated in one patient following exogenous bexarotene.Apoptosis is not detectable in the circulation of patients with Sézary syndrome treated with bexarotene. [ABSTRACT FROM AUTHOR]

The molecular pathways responsible for apoptosis in response to granzyme B have remained unresolved. Here we present data supporting the notion that granzyme Bmediated cell death is largely dependent on a pathway that is inhibitable by Bcl-2 or its viral analog BHRF1. We used a panel of stably transfected FDC-P1 mouse myeloid cell lines to show that overexpression of functional, wild-type Bcl-2 or BHRF1 rescued cells from granzyme B-mediated apoptosis, whereas mutated (Gly[sup 145] → Glu) Bcl-2, or wild-type Bcl-2 directed to the plasma membrane conferred no protection. Overexpression of Bcl-2 resulted in inhibition of multiple parameters of apoptosis in response to purified perforin and granzyme B, including DNA fragmentation, changes in light scatter profile indicating cell shrinkage and increased refractivity, loss of mitochondrial membrane potential and inhibited colony formation in clonogenic assays. Nevertheless, when exposed to cytotoxic lymphocytes, FDC-P1 and YAC-1 cells overexpressing Bcl-2 remained susceptible to death imparted by cytolytic granules, irrespective of whether the granules contained granzyme B. Thus, alternative granzyme Bindependent pathways can be activated by intact lymphocytes to overcome Bcl-2-like inhibitors of apoptosis, enabling CTLs to overcome potential viral blocks to granzyme B-mediated cell death. [ABSTRACT FROM AUTHOR]