Enhanced immune reconstitution by sex steroid ablation following allogeneic hemopoietic stem cell transplantation

Delayed immune reconstitution in adult recipients of allogeneic hemopoietic stem cell transplantations (HSCT) is related to age-induced thymic atrophy. Overcoming this paucity of T cell function is a major goal of clinical research but in the context of allogeneic transplants, any strategy must not exacerbate graft-vs-host disease (GVHD) yet ideally retain graft-vs-tumor (GVT) effects. We have shown sex steroid ablation reverses thymic atrophy and enhances T cell recovery in aged animals and in congenic bone marrow (BM) transplant but the latter does not have the complications of allogeneic T cell reactivity. We have examined whether sex steroid ablation promoted hemopoietic and T cell recovery following allogeneic HSCT and whether this benefit was negated by enhanced GVHD. BM and thymic cell numbers were significantly increased at 14 and 28 days after HSCT in castrated mice compared with sham-castrated controls. In the thymus, the numbers of donor-derived thymocytes and dendritic cells were significantly increased after HSCT and castration; donor-derived BM precursors and developing B cells were also significantly increased. Importantly, despite restoring T cell function, sex steroid inhibition did not exacerbate the development of GVHD or ameliorate GVT activity. Finally, IL-7 treatment in combination with castration had an additive effect on thymic cellularity following HSCT. These results indicate that sex steroid ablation can profoundly enhance thymic and hemopoietic recovery following allogeneic HSCT without increasing GVHD and maintaining GVT.     

Parallel evolution of dwarf ecotypes in the forest tree Eucalyptus globulus

Three small populations of a dwarf ecotype of the forest tree Eucalyptus globulus are found on exposed granite headlands in south-eastern Australia. These populations are separated by at least 100 km. Here, we used 12 nuclear microsatellites and a chloroplast DNA marker to investigate the genetic affinities of the dwarf populations to one another and to their nearest populations of tall E. globulus. Cape Tourville was studied in greater detail to assess the processes enabling the maintenance of distinct ecotypes in close geographical proximity. The three dwarf populations were not related to one another and were more closely related to adjacent tall trees than to one another. At Cape Tourville the dwarf and tall ecotypes were significantly differentiated in microsatellites and in chloroplast DNA. The dwarf and tall populations differed in flowering time and no evidence of pollen dispersal from the more extensive tall to the dwarf population was found. The three dwarf populations have evolved in parallel from the local tall ecotypes. This study shows that small marginal populations of eucalypts are capable of developing reproductive isolation from nearby larger populations through differences in flowering time and/or minor spatial separation, making parapatric speciation possible.     

Rabies challenge of captive striped skunks (Mephitis mephitis) following oral administration of a live vaccinia-vectored rabies vaccine

Twenty-four adult striped skunks (Mephitis mephitis) were administered the raccoon product formulation of Rabies Vaccine, Live Vaccinia-Vectored (Raboral V-RG, Merial Limited, Athens, Georgia, USA), either by oral instillation or in vaccine-filled coated sachets either as single or multiple doses. A control group remained unvaccinated. Twenty-three of the skunks were challenged 116 days postvaccination with rabies virus (skunk isolate). Six of six naive skunks succumbed to challenge. Four of six skunks that received the vaccine by oral instillation survived challenge. The skunks that did not survive failed to seroconvert following vaccination. None of the skunks that accepted multiple doses of the vaccine offered in coated sachets survived challenge, nor were rabies virus-neutralizing antibodies (VNAs) detected in the sera. Likewise, none of the five skunks ingesting a single sachet developed VNA against rabies. However, in this group one skunk did survive rabies challenge. This preliminary study showed that the vaccinia-vectored oral rabies vaccine Raboral V-RG, as formulated for use in raccoons, is capable of protecting a percentage of skunks against rabies. However, although the fishmeal-coated sachets were readily consumed, subsequent challenge of these animals revealed poor vaccine delivery efficiency.     

In vivo tracking of macrophage activated killer cells to sites of metastatic ovarian carcinoma

Radio-labelling of blood cells is an established technique for evaluating in vivo migration of normal cells to sites of pathology such as infection and haemorrhage. A limitation of cellular immunotherapies to induce anti-tumour responses is in part due to the uncertain ability of cellular effectors to reach their intended target. We extended the approach of cell radiolabelling to accurately examine the in vivo distribution of cellular immunotherapy with ex-vivo macrophage activated killer (MAK) cells. We describe the use of two methods of cell labelling for tracking the destination of autologous-derived macrophage activated killer (MAK) cells linked to the bi-specific antibody MDX-H210 delivered either by intravenous (i.v.) or intraperitoneal (i.p.) injection in ten patients with peritoneal relapse of epithelial ovarian carcinoma. Our results demonstrate the feasibility of generating high numbers and purity of GMP quality MAK cells, which can be radiolabelled with (18)F-FDG or (111)In-oxime. MAK cell administration produced minimal infusional toxicity and demonstrated a reproducible pattern of in vivo distribution and active in vivo tracking to sites of known tumour following 8 of 16 i.v. infusions or 4 of 6 i.p. infusions. However, the leakage of (18)F-FDG limited the ability to confidently confirm the tracking of MAK cells to tumour in all cases and improved PET labels are required. The addition of MDX-H210 bispecific antibody did not alter the distribution of cells to tumour sites, but did accelerate the clearance of i.v. administered MAK cells from the pulmonary circulation. This data demonstrates that cellular cancer immunotherapies may be successfully delivered to the sites of active tumour following either i.v. or i.p. injection in a proportion of patients with metastatic cancer. Incorporation of tracking studies in early cycles of cellular immunotherapy may allow selection of patients who demonstrate successful targeting of the immunotherapy for ongoing treatment.     

GSK-3beta activity is increased in skeletal muscle after burn injury in rats

Previous reports suggest that burn-induced muscle proteolysis can be inhibited by treatment with GSK-3beta inhibitors, suggesting that burn injury may be associated with increased GSK-3beta activity. The influence of burn injury on muscle GSK-3beta activity, however, is not known. We determined the effect of a 30% total body surface full-thickness burn injury in rats on muscle GSK-3beta activity by measuring GSK-3beta activity and tissue levels of serine 9 phosphorylated GSK-3beta, p(Ser9)-GSK-3beta, by Western blot analysis and immunohistochemistry. Because burn-induced muscle wasting is, at least in part, mediated by glucocorticoids, we used dexamethasone-treated cultured muscle cells in which GSK-3beta expression was reduced with small interfering RNA (siRNA) to further assess the role of GSK-3beta in muscle atrophy. Burn injury resulted in a seven-fold increase in GSK-3beta activity in skeletal muscle. This effect of burn was accompanied by reduced tissue levels of p(Ser9)-GSK-3beta, suggesting that burn injury stimulates GSK-3beta in skeletal muscle secondary to inhibited phosphorylation of the enzyme. In addition, burn injury resulted in inhibited phosphorylation and activation of Akt, an upstream regulatory mechanism of GSK-3beta activity. Reducing the expression of GSK-3beta in cultured muscle cells with siRNA inhibited dexamethasone-induced protein degradation by approximately 50%. The results suggest that burn injury stimulates GSK-3beta activity in skeletal muscle and that GSK-3beta may, at least in part, regulate glucocorticoid-mediated muscle wasting.     

Role of vitronectin and fibronectin receptors in oral mucosal and dermal myofibroblast differentiation

Background information. The activation of fibroblasts into myofibroblasts is a crucial event in healing that is linked to remodelling and scar formation, therefore we determined whether regulation of myofibroblast differentiation via integrins might affect wound healing responses in populations of patient‐matched HOFs (human oral fibroblasts) compared with HDFs (human dermal fibroblasts). Results. Both the HOF and HDF cell types underwent TGF‐β1 (transforming growth factor‐β1)‐induced myofibroblastic differentiation [upregulation of the expression of α‐sma (α‐smooth muscle actin)], although analysis of unstimulated cells indicated that HOFs contained higher basal levels of α‐sma than HDFs (P<0.05). Functional blocking antibodies against the integrin subunits α5 (fibronectin) or αv (vitronectin) were used to determine whether the effects of TGF‐β1 were regulated via integrin signalling pathways. α‐sma expression in both HOFs and HDFs was down‐regulated by antibodies against both α5 and αv. Functionally, TGF‐β1 inhibited cell migration in an in vitro wound model and increased the contraction of collagen gels. Greater contraction was evident for HOFs compared with HDFs, both with and without stimulation by TGF‐β1 (P<0.05). When TGF‐β1‐stimulated cells were incubated with blocking antibodies against α5 and αv, gel contraction was decreased to that of non‐stimulated cells; however, blocking αv or α5 could not restore cellular migration in both HOFs and HDFs. Conclusions. Despite intrinsic differences in their basal state, the cellular events associated with TGF‐β1‐induced myofibroblastic differentiation are common to both HOFs and HDFs, and appear to require differential integrin usage; up‐regulation of α‐sma expression and increases in collagen gel contraction are vitronectin‐ and fibronectin‐receptor‐dependent processes, whereas wound re‐population is not.     

The apoptosis modulator and tumour suppressor protein RBM5 is a phosphoprotein

RBM5/LUCA-15/H37 is a nuclear SR-related RNA binding protein with the ability to modulate both apoptosis and the cell cycle, and retard tumour formation. How RBM5 functions to carry out these, potentially interrelated, biological activities is unknown. Since reversible phosphorylation has been shown to play an important role in the regulation of SR protein function, apoptosis and cell cycle control, in an attempt to elucidate the underlying mechanisms regulating RBM5 function, the phosphorylation status of RBM5 was investigated. Whole cell lysate from growing cell cultures was treated with the broad phosphatase spectrum of CIP, resulting in a decrease in the molecular mass of RBM5. A similar decrease in molecular mass, of a subset of RBM5 proteins, was observed during growth factor deprivation, in a manner consistent with partial dephosphorylation of RBM5. Molecular mass increased upon growth factor addition, demonstrating that this apoptosis-associated alteration in molecular mass was a reversible process. Immunoprecipitation and mutagenesis experiments strongly suggested that phosphotyrosines are not present in RBM5 under normal growth conditions, and that serine 69 is phosphorylated, but not by Akt kinase. Taken together, these results suggest that reversible phosphorylation of RBM5 is a mechanism capable of regulating RBM5 participation in modulating apoptosis, and perhaps tumour suppression.     

Telomere rapid deletion regulates telomere length in Arabidopsis thaliana

Telomere length is maintained in species-specific equilibrium primarily through a competition between telomerase-mediated elongation and the loss of terminal DNA through the end-replication problem. Recombinational activities are also capable of both lengthening and shortening telomeres. Here we demonstrate that elongated telomeres in Arabidopsis Ku70 mutants reach a new length set point after three generations. Restoration of wild-type Ku70 in these mutants leads to discrete telomere-shortening events consistent with telomere rapid deletion (TRD). These findings imply that the longer telomere length set point is achieved through competition between overactive telomerase and TRD. Surprisingly, in the absence of telomerase, a subset of elongated telomeres was further lengthened, suggesting that in this background a mechanism of telomerase-independent lengthening of telomeres operates. Unexpectedly, we also found that plants possessing wild-type-length telomeres exhibit TRD when telomerase is inactivated. TRD is stochastic, and all chromosome ends appear to be equally susceptible. The frequency of TRD decreases as telomeres shorten; telomeres less than 2 kb in length are rarely subject to TRD. We conclude that TRD functions as a potent force to regulate telomere length in Arabidopsis.     

Targeted disruption of the glutaredoxin 1 gene does not sensitize adult mice to tissue injury induced by ischemia/reperfusion and hyperoxia

To understand the physiological function of glutaredoxin, a thiotransferase catalyzing the reduction of mixed disulfides of protein and glutathione, we generated a line of knockout mice deficient in the cytosolic glutaredoxin 1 (Grx1). To our surprise, mice deficient in Grx1 were not more susceptible to acute oxidative insults in models of heart and lung injury induced by ischemia/reperfusion and hyperoxia, respectively, suggesting that either changes in S-glutathionylation status of cytosolic proteins are not the major cause of such tissue injury or developmental adaptation in the Glrx1-knockout animals alters the response to oxidative insult. In contrast, mouse embryonic fibroblasts (MEFs) isolated from Grx1-deficient mice displayed an increased vulnerability to diquat and paraquat, but they were not more susceptible to cell death induced by hydrogen peroxide (H(2)O(2)) and diamide. A deficiency in Grx1 also sensitized MEFs to protein S-glutathionylation in response to H(2)O(2) treatment and retarded deglutathionylation of the S-glutathionylated proteins, especially for a single prominent protein band. Additional experiments showed that MEFs lacking Grx1 were more tolerant to apoptosis induced by tumor necrosis factor alphaplus actinomycin D. These findings suggest that various oxidants may damage the cells via distinct mechanisms in which the action of Grx1 may or may not be protective and Grx1 may exert its function on specific target proteins.     

Identification of a novel class of succinyl-nitrile-based Cathepsin S inhibitors

The synthesis and in vitro activities of a series of succinyl-nitrile-based inhibitors of Cathepsin S are described. Several members of this class show nanomolar inhibition of the target enzyme as well as cellular potency. The inhibitors displaying the greatest potency contain N-alkyl substituted piperidine and pyrrolidine rings spiro-fused to the alpha-carbon of the P1 residue.