Protein kinase D interaction with TLR5 is required for inflammatory signaling in response to bacterial flagellin

Protein kinase D (PKD), also called protein kinase C (PKC)mu, is a serine-threonine kinase that is involved in diverse areas of cellular function such as lymphocyte signaling, oxidative stress, and protein secretion. After identifying a putative PKD phosphorylation site in the Toll/IL-1R domain of TLR5, we explored the role of this kinase in the interaction between human TLR5 and enteroaggregative Escherichia coli flagellin in human epithelial cell lines. We report several lines of evidence that implicate PKD in TLR5 signaling. First, PKD phosphorylated the TLR5-derived target peptide in vitro, and phosphorylation of the putative target serine 805 in HEK 293T cell-derived TLR5 was identified by mass spectrometry. Furthermore, mutation of serine 805 to alanine abrogated responses of transfected HEK 293T cells to flagellin. Second, TLR5 interacted with PKD in coimmunoprecipitation experiments, and this association was rapidly enhanced by flagellin treatment. Third, pharmacologic inhibition of PKC or PKD with G?6976 resulted in reduced expression and secretion of IL-8 and prevented the flagellin-induced activation of p38 MAPK, but treatment with the PKC inhibitor G?6983 had no significant effects on these phenotypes. Finally, involvement of PKD in the p38-mediated IL-8 response to flagellin was confirmed by small hairpin RNA-mediated gene silencing. Together, these results suggest that phosphorylation of TLR5 by PKD may be one of the proximal elements in the cellular response to flagellin, and that this event contributes to p38 MAPK activation and production of inflammatory cytokines in epithelial cells.     

IgE-antibody-dependent immunotherapy of solid tumors: cytotoxic and phagocytic mechanisms of eradication of ovarian cancer cells

Abs have a paramount place in the treatment of certain, mainly lymphoid, malignancies, although tumors of nonhemopoietic origin have proved more refractory ones. We have previously shown that the efficacy of immunotherapy of solid tumors, in particular ovarian carcinoma, may be improved by the use of IgE Abs in place of the conventional IgG. An IgE Ab (MOv18 IgE) against an ovarian-tumor-specific Ag (folate binding protein), in combination with human PBMC, introduced into ovarian cancer xenograft-bearing mice, greatly exceeded the analogous IgG1 in promoting survival. In this study, we analyzed the mechanisms by which MOv18 IgE may exert its antitumor activities. Monocytes were essential IgE receptor-expressing effector cells that mediated the enhanced survival of tumor-bearing mice by MOv18 IgE and human PBMC. Monocytes mediated MOv18 IgE-dependent ovarian tumor cell killing in vitro by two distinct pathways, cytotoxicity and phagocytosis, acting respectively through the IgE receptors FcepsilonRI and CD23. We also show that human eosinophils were potent effector cells in MOv18 IgE Ab-dependent ovarian tumor cell cytotoxicity in vitro. These results demonstrate that IgE Abs can engage cell surface IgE receptors and activate effector cells against ovarian tumor cells. Our findings offer a framework for an improved immunotherapeutic strategy for combating solid tumors.     

Multiple and additive functions of ALDH3A1 and ALDH1A1: cataract phenotype and ocular oxidative damage in Aldh3a1(-/-)/Aldh1a1(-/-) knock-out mice

ALDH3A1 (aldehyde dehydrogenase 3A1) is abundant in the mouse cornea but undetectable in the lens, and ALDH1A1 is present at lower (catalytic) levels in the cornea and lens. To test the hypothesis that ALDH3A1 and ALDH1A1 protect the anterior segment of the eye against environmentally induced oxidative damage, Aldh1a1(-/-)/Aldh3a1(-/-) double knock-out and Aldh1a1(-/-) and Aldh3a1(-/-) single knock-out mice were evaluated for biochemical changes and cataract formation (lens opacification). The Aldh1a1/Aldh3a1- and Aldh3a1-null mice develop cataracts in the anterior and posterior subcapsular regions as well as punctate opacities in the cortex by 1 month of age. The Aldh1a1-null mice also develop cataracts later in life (6-9 months of age). One- to three-month-old Aldh-null mice exposed to UVB exhibited accelerated anterior lens subcapsular opacification, which was more pronounced in Aldh3a1(-/-) and Aldh3a1(-/-)/Aldh1a1(-/-) mice compared with Aldh1a1(-/-) and wild type animals. Cataract formation was associated with decreased proteasomal activity, increased protein oxidation, increased GSH levels, and increased levels of 4-hydroxy-2-nonenal- and malondialdehyde-protein adducts. In conclusion, these findings support the hypothesis that corneal ALDH3A1 and lens ALDH1A1 protect the eye against cataract formation via nonenzymatic (light filtering) and enzymatic (detoxification) functions.     

A comprehensive evaluation of cattle introgression into US federal bison herds

Genetic introgression, especially from interspecies hybridization, is a significant threat to species conservation worldwide. In this study, 11 US federal bison populations were comprehensively examined for evidence of both mitochondrial and nuclear domestic cattle (Bos taurus) introgression. Mitochondrial introgression was examined using established polymerase chain reaction methods and confirmed through analysis of D-loop sequences. Nuclear introgression was assessed in 14 chromosomal regions through examination of microsatellite electromorph and sequence differences between bison and domestic cattle. Only one population was identified with domestic cattle mitochondrial DNA introgression. In contrast, evidence of nuclear introgression was found in 7 (63.6%) of the examined populations. Historic accounts of bison transfers among populations were corroborated with evidence of introgressed DNA transmission. While neither nuclear nor mitochondrial domestic cattle introgression was detected in bison from Grand Teton National Park, Sully's Hill National Game Preserve, Wind Cave National Park, or Yellowstone National Park, adequate sample sizes were available only from the last 2 populations to allow for statistical confidence (>90%) in nuclear introgression detection limits. The identification of genetically unique and undisturbed populations is critical to species conservation efforts, and this study serves as a model for the genetic evaluation of interspecies introgression.     

Functional analysis of KSRP interaction with the AU-rich element of interleukin-8 and identification of inflammatory mRNA targets

mRNA stability is a major determinant of inflammatory gene expression. Rapid degradation of interleukin-8 (IL-8) mRNA is imposed by a bipartite AU-rich element (ARE) in the 3′ untranslated region (R. Winzen et al., Mol. Cell. Biol. 24:4835-4847, 2004). Small interfering RNA-mediated knockdown of the ARE-binding protein KSRP resulted in stabilization of IL-8 mRNA or of a β-globin reporter mRNA containing the IL-8 ARE. Rapid deadenylation was impaired, indicating a crucial role for KSRP in this step of mRNA degradation. The two IL-8 ARE domains both contribute to interaction with KSRP, corresponding to the importance of both domains for rapid degradation. Exposure to the inflammatory cytokine IL-1 has been shown to stabilize IL-8 mRNA through p38 mitogen-activated protein (MAP) kinase and MK2. IL-1 treatment impaired the interaction of KSRP with the IL-8 ARE in a manner dependent on p38 MAP kinase but apparently independent of MK2. Instead, evidence that TTP, a target of MK2, can also destabilize the IL-8 ARE reporter mRNA is presented. In a comprehensive approach to identify mRNAs controlled by KSRP, two criteria were evaluated by microarray analysis of (i) association of mRNAs with KSRP in pulldown assays and (ii) increased amounts in KSRP knockdown cells. According to both criteria, a group of 100 mRNAs is controlled by KSRP, many of which are unstable and encode proteins involved in inflammation. These results indicate that KSRP functions as a limiting factor in inflammatory gene expression.     

Thymic generation and regeneration: a new paradigm for establishing clinical tolerance of stem cell-based therapies

Tolerance to tissue-engineering products is a major obstacle hindering the clinical application of this rapidly advancing technology. Manipulation of central tolerance, by establishing thymus chimerism of both donor and host-derived haemopoietic cells (haemopoietic stem cell transplant--HSCT), should purge any T cells reactive to potential donor organ or tissue transplant. A functional thymus, however, is required to induce chimerism and repopulate the peripheral T cell pool, but age-related thymic atrophy and damage caused by ablative conditioning regimes significantly reduce thymic function and increase incident of infection-dependent morbidity and mortality. Thus rejuvenation of the thymus alongside HSCT may potentiate the use of this strategy in the clinic. In addition, the use of thymic epithelial progenitor cell technology may allow growth of ex vivo thymic tissue for use in clinical situations of immunodeficiency as well as in establishing tolerance to tissue/organ products derived from the same source.     

Abiotic influences on embryo growth: statoliths as experimental tools in the squid early life history

Statolith size and growth was used to determine the influence of abiotic factors on the growth of Loligo vulgaris and Sepioteuthis australis embryos. Recently spawned egg masses collected from the field were incubated in the laboratory under different levels of light intensity, photoperiod, or short periods of low salinity (30‰). Double tetracycline staining was used to follow statolith growth. In L. vulgaris constant light conditions produced significantly slower growth in the embryonic statoliths and embryos held at summer photoperiod had slower statolith growth than those held at winter photoperiods. However once they hatched out there was no evidence that photoperiod affected statolith growth. After hatching, in all photoperiods statolith growth rates decreased in comparison with late embryonic rates. In S. australis embryos, differences between the high and medium light intensities for summer and intermediate photoperiods were found, suggesting that under summer incubation temperature, longer daylengths at medium light intensity favoured higher statolith growth for this species. In comparison to controls, slower statolith growth in S. australis embryos due to low salinity only occurred when exposed for 72 h. Comparison with previous studies indicates that temperature seems to be the main abiotic factor influencing statolith growth during early stages, however, interactions among all abiotic factors needs to be determined as well as the unknown influence of other isolated factors, e.g., oxygen concentration within the egg mass.     

Pim-1 regulates cardiomyocyte survival downstream of Akt

The serine-threonine kinases Pim-1 and Akt regulate cellular proliferation and survival. Although Akt is known to be a crucial signaling protein in the myocardium, the role of Pim-1 has been overlooked. Pim-1 expression in the myocardium of mice decreased during postnatal development, re-emerged after acute pathological injury in mice and was increased in failing hearts of both mice and humans. Cardioprotective stimuli associated with Akt activation induced Pim-1 expression, but compensatory increases in Akt abundance and phosphorylation after pathological injury by infarction or pressure overload did not protect the myocardium in Pim-1-deficient mice. Transgenic expression of Pim-1 in the myocardium protected mice from infarction injury, and Pim-1 expression inhibited cardiomyocyte apoptosis with concomitant increases in Bcl-2 and Bcl-X(L) protein levels, as well as in Bad phosphorylation levels. Relative to nontransgenic controls, calcium dynamics were significantly enhanced in Pim-1-overexpressing transgenic hearts, associated with increased expression of SERCA2a, and were depressed in Pim-1-deficient hearts. Collectively, these data suggest that Pim-1 is a crucial facet of cardioprotection downstream of Akt.     

An anti-infective peptide that selectively modulates the innate immune response

We show that an innate defense-regulator peptide (IDR-1) was protective in mouse models of infection with important Gram-positive and Gram-negative pathogens, including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus and Salmonella enterica serovar Typhimurium. When given from 48 h before to 6 h after infection, the peptide was effective by both local and systemic administration. Because protection by IDR-1 was prevented by in vivo depletion of monocytes and macrophages, but not neutrophils or B- and T-lymphocytes, we conclude that monocytes and macrophages are key effector cells. IDR-1 was not directly antimicrobial: gene and protein expression analysis in human and mouse monocytes and macrophages indicated that IDR-1, acting through mitogen-activated protein kinase and other signaling pathways, enhanced the levels of monocyte chemokines while reducing pro-inflammatory cytokine responses. To our knowledge, an innate defense regulator that counters infection by selective modulation of innate immunity without obvious toxicities has not been reported previously.