A fast-degrading thiol-acrylate based hydrogel for cranial regeneration.

Successful regeneration of the cranium in patients suffering from cranial bone defects is an integral step to restore craniofacial function. However, restoration of craniofacial structure has been challenging due to its complex geometry, limited donor site availability, and poor graft integration. To address these problems, we investigated the use of a thiol-acrylate hydrogel as a cell carrier to facilitate cranial regeneration. Thiol-acrylate hydrogels were formulated with 5-15 wt% poly(ethylene glycol)-diacrylate (PEGDA) and 1-9 mm dithiothreitol (DTT). The degradation rate, swelling ratio, and shear modulus of the resulting hydrogel were first characterized. Then, pre-osteoblast-like cells (MC3T3-E1) were encapsulated in the hydrogel and cultured for up to 21 d. Our results demonstrate that compared to samples formulated from 15 wt% PEGDA, 5 wt% PEGDA samples showed lower storage modulus at day 10 (0.7 kPa versus 8.3 kPa), 62.7% higher in weight change after soaking for 10 d. While the 5 wt% PEGDA group showed an 85% weight loss between day 10 and 21, the 15 wt% PEGDA group showed a 5% weight gain in the same time period. Cell viability with 15 wt% PEGDA and 5 mm DTT hydrogel decreased by 41.3% compared to 5 wt% PEGDA and 5mM DTT gel at day 7. However, histological analysis of cells after 21 d in culture revealed that they had pericellular mineral deposition indicating that the cells were differentiating into osteoblasts lineage in all experimental groups. This study shows that thiol-acrylate hydrogels can be tailored to achieve different degradation rates, in order to enhance cell viability and differentiation. Thus, the findings of this study provide a fundamental understanding for the application of thiol-acrylate hydrogels in cranial bone regeneration.

Retroviral vector integration in post-transplant hematopoiesis in mice conditioned with either submyeloablative or ablative irradiation.

X-linked chronic granulomatous disease (X-CGD) is an inherited immunodeficiency with absent phagocyte NADPH-oxidase activity caused by defects in the gene-encoding gp91(phox). Here, we evaluated strategies for less intensive conditioning for gene therapy of genetic blood disorders without selective advantage for gene correction, such as might be used in a human X-CGD protocol. We compared submyeloablative with ablative irradiation as conditioning in murine X-CGD, examining engraftment, oxidase activity and vector integration in mice transplanted with marrow transduced with a gamma-retroviral vector for gp91(phox) expression. The frequency of oxidase-positive neutrophils in the donor population was unexpectedly higher in many 300 cGy-conditioned mice compared with lethally irradiated recipients, as was the fraction of vector-marked donor secondary CFU-S12. Vector integration sites in marrow, spleen and secondary CFU-S12 DNA from primary recipients were enriched for cancer-associated genes, including Evi1, and integrations in or near cancer-associated genes were more frequent in marrow and secondary CFU-S12 from 300 cGy-conditioned mice compared with fully ablated mice. These findings support the concept that vector integration can confer a selection bias, and suggest that the intensity of the conditioning regimen may further influence the effects of vector integration on clonal selection in post-transplant engraftment and hematopoiesis.

Granzyme B independently of perforin mediates noncytolytic intracellular inactivation of vesicular stomatitis virus.

Cytotoxic cells provide a crucial defense against DNA and RNA viral infections. Here we describe an in vitro model to study the fate of vesicular stomatitis virus (VSV) RNA in cells undergoing apoptosis. Using the [3H]uridine release assay, we show that human LAK cells induce the degradation of RNA in infected U937 cells in addition to inhibiting the production of infectious virions. LAK cell-mediated RNA degradation was blocked by the serine protease inhibitor, 3,4-dichloroisocoumarin. Purified human granzyme B but not inactivated granzyme B, granzyme A, or perforin rapidly induced degradation of RNA in VSV-infected U937 cells in a dose- and time-dependent manner without lysing the cells and suppressed viral production. Northern analysis of RNA extracted from infected cells with a VSV full-length cDNA probe confirmed that levels of viral transcripts were reduced by treatment with granzyme B. Nevertheless, the amount of host beta-actin mRNA was also reduced in infected cells, suggesting that treatment with granzyme B induced apoptosis. Consistent with this notion, infected cells exposed to granzyme B rapidly developed DNA strand breakage. Taken together, the data suggest that granzyme B in the absence of perforin reduced VSV production by activating a mechanism that degraded viral transcripts in infected U937 cells.

Target cell-induced perforin mRNA turnover in NK3.3 cells is mediated by multiple elements within the mRNA coding region.

We have previously shown that in cytolytic cells exposed to sensitive targets the mRNA of the cytolytic protein perforin undergoes rapid downregulation. We now demonstrate that perforin message undergoes accelerated turnover in NK3.3 cells exposed to sensitive TC. This inducible mRNA decay phenomenon is specific for cytolytic protein messages, as levels of the constitutive message beta-actin are unchanged. This TC-induced perforin mRNA turnover cannot be attributed to a blockage of RNA synthesis, or to a rapid half-life (t 1/2). To determine the region(s) within the perforin transcript responsible for governing this TC-mediated turnover event, various segments of the perforin cDNA were cloned and inserted into the 3' UTR of rabbit beta globin (RG). Constructs containing perforin coding region cDNA, but not 3' UTR cDNA, mediated TC-induced mRNA turnover. These data indicate that multiple elements governing perforin mRNA stability reside within the coding region, a novel type of mRNA regulation not previously described.

Fas-mediated cytotoxicity remains intact in perforin and granzyme B antisense transfectants of a human NK-like cell line.

Cell-mediated cytotoxicity (CMC) has traditionally been thought to involve the release of granule components, including perforin and granzymes, from the effector cell (EC) onto the target cell (TC) membrane. Recently, a granule-independent cytolytic mechanism involving the interaction of Fas antigen (CD95) with Fas ligand has been described. We have generated antisense perforin (YT-xP1) and granzyme B (YT-xGrB) transfectants of the human NK-like cell line YT-INDY. These transfectants have greatly reduced cytolytic ability when compared to the vector-transfected control cell line (YT-neo). In this study, however, we demonstrate that the antisense transfectants retain the ability to lyse Fas+ TC. Fas-mediated lysis is Ca(2+)-independent and is inhibited by a monoclonal anti-Fas blocking Ab, M3. By RT-PCR, we detect message for FasL in unstimulated YT-xP1 and YT-xGrB transfectants, as well as in unstimulated YT-neo. By flow cytometry, we show that YT-neo, YT-xGrB, and YT-xP1 constitutively express surface FasL. These data indicate that in a human NK-like cell line, similar to the murine system, the granule and Fas pathways of cytotoxicity function independently of one another. At least with the TC tested, our data also indicate that the granule and Fas pathways together account for nearly 100% of the cytolytic ability of YT-INDY.

Stably transfected antisense granzyme B and perforin constructs inhibit human granule-mediated lytic ability.

In human NK cells and CTL it has been shown that release of lytic molecules is, at least in part, responsible for the lysis of target cells (TC). Of the various types of molecules thought to be involved in cell-mediated cytotoxicity (CMC), perforin and the serine proteases (granzymes A and B) are the best described. Using mammalian expression vectors (pRSV-neo and pSV2-neo), antisense constructs for perforin and granzyme B were independently electroporated into YT-INDY, a human non-MHC-restricted, IL-2-independent, cytotoxic lymphocyte. Transfected YT-INDY was then selected for expression of the plasmid by antibiotic G418 resistance. The presence of plasmid was confirmed by detection of the integrated plasmid G418 resistance gene using PCR. The presence of antisense perforin in YT-INDY (YT-xPFP) inhibited lytic ability by > 95% compared to YT-INDY transfected with plasmid alone or plasmid with unrelated antisense (YT-neo, YT-ctrl, respectively). Likewise, the presence of antisense GrB (YT-xGrB) inhibited the lytic ability of YT-INDY by > 95%. Western analysis revealed a 30% decrease in the level of perforin and a 55% decrease in granzyme B protein levels compared to YT-neo. Northern analysis using oligo probes complementary to perforin and granzyme B mRNA showed a decrease in their respective message levels. In conclusion, stably transfected antisense constructs for perforin and granzyme B essentially eliminated the lytic ability of YT-INDY. These results strongly indicate that both perforin and granzyme B are required by this human cytotoxic lymphocyte for effective TC lysis.