Molecular genetic characterization of XRCC4 function

XRCC4 is a generally expressed protein of 334 amino acids that is involved in the repair of DNA double-strand breaks and in V(D)J recombination, but its function is unknown. In this study, we have used a mutational approach and the yeast two-hybrid method to perform an initial characterization of this protein. We show that the XRCC4 protein is located in the nucleus. We also demonstrate that several potential phosphorylation sites are not required for XRCC4 function in a transient V(D)J recombination assay. In addition, we show that XRCC4 forms a homodimer in vivo with the homodimerization domain being located within amino acids 115-204. Finally, we define a core domain of XRCC4 that functions in V(D)J recombination and comprises amino acids 18-204. Potential functions of XRCC4 are discussed.      

A gene signature of inhibitory MHC receptors identifies a BDCA3(+) subset of IL-10-induced dendritic cells with reduced allostimulatory capacity in vitro

Dendritic cells (DC) are crucial gatekeepers in regulating immunity. Whereas mature immunostimulatory myeloid DC (DC(ims)) potently promote immune responses, IL-10-induced myeloid DC (DC-IL-10) counteract T cell activation. To elucidate the molecular repertoire by which DC-IL-10 secure reduced T cell activation, comparative gene expression profiling was done using Affymetrix U133A microarrays. Among the genes overexpressed in DC-IL-10, eight immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing inhibitory molecules (ILT2, ILT3, ILT4, ILT5, DCIR, PILRA, FcgammaRIIB, SLAM) were found. Phenotypic analysis of DC-IL-10 defined an ILT(high) DC subset further characterized by expression of CD14, TLR2, DC-SIGN, and CD123 and the lack of lymphocyte, monocyte/macrophage, and plasmacytoid DC markers such as CD3, CD8, and CD68. A unique feature of the ILT(high) DC subset was expression of the novel DC marker BDCA3, which was not detected on monocytes, immature DC, DC(ims), or ILT(low) DC-IL-10. While the allogeneic T cell proliferation induced by the entire DC-IL-10 population was approximately 30% of that stimulated by DC(ims), allogeneic MLR responses driven by the ILT(high) DC subset were reduced to 8% of the allostimulatory capacity of DC(ims), although secretion of the inhibitory cytokine IL-10 and other Th1/Th2-associated cytokines was similar in these cells.     

Rflp analysis of the L-myc oncogene in head and neck-cancer - relationship study with susceptibility and disease progression

The L-myc DNA restriction fragment length polymorphism (RFLF), revealed by EcoRI digestion, has been evaluated in a case-control study including 161 head and neck cancer (HNSCC) patients and 160 normal healthy individuals with similar smoking and alcohol habits. No significant difference in the distribution of L-myc genotypes (LL, LS or SS) was found between the two populations implying thus no predisposition to head and neck tumour by either allele. There was no significant association between L-myc genotypes and the usual clinicopathological features such as T staging, differentiation status and lymph node involvement. Moreover, follow-up data from 154 patients was obtained and correlated with the L-myc pattern. No significant difference was observed in metastasis occurrence, multiple cancer incidence and survival data in the patients classified according to the L-myc genotypes; only a trend to preferentially develop metastasis in lung for patients with S allele was noted. In conclusion, our data shows that the L-myc typing does not contribute to HNSCC risk or prognosis assessment. A review of L-myc RFLP published studies shows contradictory results even on the same type of tumour and emphasizes the lacunae in understanding the biological role of L-myc for valid interpretation of L-myc allelic associations with cancer susceptibility or prognosis.     

A randomized controlled trial testing the effectiveness of a universal school-based depression prevention program 'Op Volle Kracht' in the Netherlands

Background  

The incidence of depressive symptoms increases during adolescence, from 10.0% to 24.5% at age 11 to 15, respectively. Experiencing elevated levels of depressive symptoms increases the risk of a depressive disorder in adulthood. A universal school-based depression prevention program Op Volle Kracht (OVK) was developed, based on the Penn Resiliency Program, aimed at preventing the increase of depressive symptoms during adolescence and enhancing positive development. In this study the effectiveness of OVK will be tested and possible mediators of program effects will be focus of study as well.     

Factors that affect human hemopoiesis are produced by T-cell growth factor dependent and independent cultured T-cell leukemia-lymphoma cells

Some laboratory results and clinical situations suggest that human T cells may be important in the regulation of growth of hematopoietic cells. Since the discovery of T-cell growth factor (TCGF), systems are now available for the long-term specific in vitro propagation of mature normal or neoplastic human T cells, providing an opportunity to study the influence of T cells on hematopoiesis. Recently, 24 cell lines from patients with cutaneous T-cell lymphoma (CTCL) and T-cell acute lymphoblastic leukemia (T-ALL) were grown with TCGF and then assessed for release of humoral factors that affect hematopoiesis. Conditioned media (CM) from these cell lines were tested for erythroid burst- promoting activity (BPA) and granulocyte colony-stimulating activity (CSA). BPA was detected in CM from 3/6 cultures of T-ALL patients and 4/6 CTCL cultures. CSA was found in the CM from 6/8 cultures of T-ALL patients, 7/12 CTCL cultures, and 3/4 CTCL cell lines that become independent of exogenous TCGF for growth. The CSA from several of the neoplastic T-cell cultures stimulated high levels of eosinophil colonies, a possible source of the eosinophilia seen in these patients. The ability of continuously proliferating human T lymphocytes, which retain functional specificity and responsiveness to normal humoral regulation, to produce factors that directly or indirectly stimulate myeloid and erythroid colony formation lends further credence to the role of T lymphocytes in regulating hematopoiesis.     

Evaluation of erythropoiesis in long-term hamster bone marrow suspension cultures: absence of a requirement for adherent monolayer cells

In long-term hamster bone marrow cultures, proliferation and differentiation of hemopoietic stem cells occurs for several months without need for hydrocortisone or adherent stromal elements, which are requirements for bone marrow growth in all other species studied. Only the most primitive erythroid progenitors (BFU-E) are produced in the cultures. Following treatment of the cells with erythropoietin, these progenitor cells undergo differentiation into mature hemoglobinized red blood cells. Concomitant addition of erythropoietin (Epo) and prostaglandin-E1 (PGE1) results in the production of large numbers of maturing red blood cells. In cultures stimulated with Epo and PGE1, as many as 70% of the cells are benzidine-positive, while Epo alone stimulated as many as 45% of the cells to become erythroid. Epo and PGE1 do not have any apparent deleterious effect on the continuous hemopoiesis occurring in these cultures. Under identical conditions, syngeneic adherent cell cultures do not produce any erythroid elements. The development of mature red blood cells from primitive erythroid precursors occurs in the presence of Epo alone and without any apparent need for adherent stromal elements. These cultures provide a useful in vitro model for dissecting the positive and negative signals that regulate erythropoiesis.     

Heparin-binding epidermal growth factor–like growth factor attenuates acute lung injury and multiorgan dysfunction after scald burn

Background

Impaired gut barrier function and acute lung injury (ALI) are significant components of the multiorgan dysfunction syndrome that accompanies severe burns. Heparin-binding epidermal growth factor–like growth factor (HB-EGF) has been shown to reduce inflammation, preserve gut barrier function, and protect the lungs from acute injury in several models of intestinal injury; however, comparable effects of HB-EGF after burn injury have never been investigated. The present studies were based on the hypothesis that HB-EGF would reduce the severity of ALI and multiorgan dysfunction after scald burns in mice.

Materials and methods

Mice were randomized to sham, burn (25% of total body surface area with full thickness dorsal scald), and burn + HB-EGF groups. The HB-EGF group was pretreated with two enteral doses of HB-EGF (1200 μg/kg/dose). Mice were resuscitated after injury and sacrificed at 8 h later. Their lungs were harvested for determination of pulmonary myeloperoxidase activity, wet:dry ratios, and terminal deoxynucleotidyl transferase dUTP nick end label and cleaved caspase 3 immunohistochemistry. Lung function was assessed using the SCIREQ Flexivent. Splenic apoptosis was quantified by Western blot for cleaved caspase 3, and intestinal permeability was measured using the everted gut sac method.

Results

Mice subjected to scald burn injury had increased lung myeloperoxidase levels, increased pulmonary and splenic apoptosis, elevated airway resistance and bronchial reactivity, and increased intestinal permeability compared with sham mice. These abnormalities were significantly attenuated in mice that were subjected to scald burn injury but treated with enteral HB-EGF.

Conclusions

These data suggest that HB-EGF protects mice from ALI after scald burn and attenuates the severity of postburn multiorgan dysfunction.     

Arginase and α‐smooth muscle actin induction after hyperoxic exposure in a mouse model of bronchopulmonary dysplasia

The L‐arginine/NO pathway is an important regulator of pulmonary hypertension, the leading cause of mortality in patients with the chronic lung disease of prematurity, bronchopulmonary dysplasia. L‐arginine can be metabolized by NO synthase (NOS) to form L‐citrulline and NO, a potent vasodilator. Alternatively, L‐arginine can be metabolized by arginase to form urea and L‐ornithine, a precursor to collagen and proline formation important in vascular remodelling. In the current study, we hypothesized that C3H/HeN mice exposed to prolonged hyperoxia would have increased arginase expression and pulmonary vascular wall cell proliferation. C3H/HeN mice were exposed to 14 days of 85% O₂ or room air and lung homogenates analyzed by western blot for protein levels of arginase I, arginase II, endothelial NOS (eNOS), ornithine decarboxylase (ODC), ornithine aminotransferase (OAT), and α‐smooth muscle actin (α‐SMA). Hyperoxia did not change arginase I or eNOS protein levels. However, arginase II protein levels were 15‐fold greater after hyperoxia exposure than in lungs exposed to room air. Greater protein levels of ODC and OAT were found in lungs following hyperoxic exposure than in room air animals. α‐SMA protein levels were found to be 7‐fold greater in the hyperoxia exposed lungs than in room air lungs. In the hyperoxia exposed lungs there was evidence of greater pulmonary vascular wall cell proliferation by α‐SMA immunohistochemistry than in room air lungs. Taken together, these data are consistent with a more proliferative vascular phenotype, and may explain the propensity of patients with bronchopulmonary dysplasia to develop pulmonary hypertension.     

Use of a case-mix approach to study the trends in the incidence of second primary cancers

Purpose

To analyze trends in second primary cancer (SPC) incidence by using a case-mix approach to standardize on first cancer site distribution.

Transforming growth factor-beta trans-modulates the expression of colony stimulating factor receptors on murine hematopoietic progenitor cell lines

Transforming growth factor beta (TGF-beta) is a potent and selective growth inhibitor of early hematopoietic progenitors and leukemic cells. The cellular mechanism(s) underlying this antiproliferative effect is, however, currently unknown. In the present study, we demonstrate that TGF-beta inhibits the expression of granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL-3), and granulocyte-CSF (G-CSF) receptors on murine factor-dependent and independent hematopoietic progenitor cell lines without a significant change in receptor affinity. A maximum reduction in GM-CSF receptor numbers of 65% to 77% was observed by 96-hour incubation with TGF-beta. The TGF- beta induced trans-down-modulation of GM-CSF receptors was prolonged, noncytotoxic but reversible, and not due to endogenous production of GM- CSF. The TGF-beta induced reduction in CSF receptor numbers preceded TGF-beta's growth inhibitory action. In addition, the ED50 (1 to 10 pmol/L) for TGF-beta's CSF receptor modulatory and antiproliferative effect was similar. The effect of TGF-beta on cell surface CSF receptor expression was specific, because the expression of other cell surface proteins (Ly 5 and Ly 17) was not affected by TGF-beta treatment, and because other growth inhibitors (tumor necrosis factor and interferon) did not affect CSF receptor expression. These data suggest that the downregulation of the growth of hematopoietic progenitor cells by TGF- beta involves reducing the cell surface expression on growth factor receptors.