Colitis induced by proteinase-activated receptor-2 agonists is mediated by a neurogenic mechanism

Proteinase-activated receptor-2 (PAR2) activation induces colonic inflammation by an unknown mechanism. We hypothesized that PAR2 agonists administered intracolonically in mice induce inflammation via a neurogenic mechanism. Pretreatment of mice with neurokinin-1 and calcitonin-gene-related peptide (CGRP) receptor antagonists or with capsaicin showed attenuated PAR2-agonist-induced colitis. Immunohistochemistry demonstrated a differential expression of a marker for the type-1 CGRP receptor during the time course of PAR2-agonist-induced colitis, further suggesting a role for CGRP. We conclude that PAR2-agonist-induced intestinal inflammation involves the release of neuropeptides, which by acting on their receptors cause inflammation. These results implicate PAR2 as an important mediator of intestinal neurogenic inflammation.     

TPX2 Impacts Acetylation of Histone H4 at Lysine 16: Implications for DNA Damage Response

During interphase, the spindle assembly factor TPX2 is compartmentalized in the nucleus where its roles remain largely uncharacterized. Recently, we found that TPX2 regulates the levels of serine 139-phosphoryated H2AX (γ-H2AX) at chromosomal breaks induced by ionizing radiation. Here, we report that TPX2 readily associates with the chromatin in the absence of ionizing radiation. Overexpression of TPX2 alters the DAPI staining pattern of interphase cells and depletion of TPX2 constitutively decreases the levels of histone H4 acetylated at lysine16 (H4K16ac) during G1-phase. Upon ionizing irradiation, this constitutive TPX2 depletion-dependent decrease in H4K16ac levels correlates with increased levels of γ-H2AX. The inversely correlated levels of H4K16ac and γ-H2AX can also be modified by altering the levels of SIRT1, herein identified as a novel protein complex partner of TPX2. Furthermore, we find that TPX2 depletion also interferes with formation of 53BP1 ionizing radiation-induced foci, known to depend on γ-H2AX and the acetylation status of H4K16. In brief, our study is the first indication of a constitutive control of TPX2 on H4K16ac levels, with potential implications for DNA damage response.     

Conditional deletion of Shp2 tyrosine phosphatase in thymocytes suppresses both pre-TCR and TCR signals

It is well known that T cell differentiation and maturation in the thymus is tightly controlled at multiple checkpoints. However, the molecular mechanism for the control of this developmental program is not fully understood. A number of protein tyrosine kinases, such as Zap-70, Lck, and Fyn, have been shown to promote signals required for thymocyte development, whereas a tyrosine phosphatase Src homology domain-containing tyrosine phosphatase (Shp)1 has a negative effect in pre-TCR and TCR signaling. We show in this study that Shp2, a close relative of Shp1, plays a positive role in T cell development and functions. Lck-Cre-mediated deletion of Shp2 in the thymus resulted in a significant block in thymocyte differentiation/proliferation instructed by the pre-TCR at the beta selection step, and reduced expansion of CD4(+) T cells. Furthermore, mature Shp2(-/-) T cells showed decreased TCR signaling in vitro. Mechanistically, Shp2 acts to promote TCR signaling through the ERK pathway, with impaired activation of ERK kinase observed in Shp2(-/-) T cells. Thus, our results provide physiological evidence that Shp2 is a common signal transducer for pre-TCR and TCR in promoting T cell maturation and proliferation.     

Temporal and Spatial Analysis of Clinical and Molecular Parameters in Dextran Sodium Sulfate Induced Colitis

Background

Inflammatory bowel diseases (IBD), including mainly ulcerative colitis (UC) and Crohn''s disease (CD), are inflammatory disorders of the gastrointestinal tract caused by an interplay of genetic and environmental factors. Murine colitis model induced by Dextran Sulfate Sodium (DSS) is an animal model of IBD that is commonly used to address the pathogenesis of IBD as well as to test efficacy of therapies. In this study we systematically analyzed clinical parameters, histological changes, intestinal barrier properties and cytokine profile during the colitic and recovery phase.

Methods

C57BL/6 mice were administered with 3.5% of DSS in drinking water for various times. Clinical and histological features were determined using standard criteria. Myeloperoxidase (MPO) activity, transepithelial permeability and proinflammatory mediators were determined in whole colon or proximal and distal parts of colon.

Results

As expected after administration of DSS, mice manifest loss of body weight, shortening of colon length and bloody feces. Histological manifestations included shortening and loss of crypts, infiltration of lymphocytes and neutrophil, symptoms attenuated after DSS withdrawal. The MPO value, as inflammation indicator, also increases significantly at all periods of DSS treatment, and even after DSS withdrawal, it still held at very high levels. Trans-mucosal permeability increased during DSS treatment, but recovered to almost control level after DSS withdrawal. The production of proinflammatory mediators by colonic mucosa were enhanced during DSS treatment, and then recovered to pre-treated level after DSS withdrawal. Finally, enhanced expression of proinflammatory mediators also revealed a different profile feature in proximal and distal parts of the colon.

Conclusion

Experimental colitis induced by DSS is a good animal model to study the mechanisms underlying the pathogenesis and intervention against IBD, especially UC.     

Limited chloroplast gene transfer via recombination overcomes plastomegenome incompatibility between Nicotiana tabacum and Solanum tuberosum

Green cybrids with a new nucleus-chloroplast combination cannot be selected after protoplast fusion in the intersubfamilial Nicotiana-Solanum combination. As an approach to overcome the supposed plastomegenome incompatibility, a partial plastome transfer by genetic recombination has been considered. After fusions of protoplasts of a light-sensitive Nicotiana tabacum (tobacco) plastome mutant and lethally irradiated protoplasts of wild-type Solanum tuberosum (potato), a single green colony was recovered among 2.5×10⁴ colonies. The regenerated plants had tobacco-like (although abnormal) morphology, but were normally green, and sensitive to tentoxin, demonstrating chloroplast markers of the potato parent. Restriction enzyme analysis of the chloroplast DNA (cpDNA) revealed recombinant, nonparental patterns. A comparison with physical maps of the parental cpDNA demonstrated the presence of a considerable part of the potato plastome flanked by tobacco-specific regions. This potacco plastome proved to be stable in backcross and backfusion experiments, and normally functional in the presence solely of N. tabacum nucleus.     

Partial phase diagram of aqueous bovine carbonic anhydrase: analyses of the pressure-dependent temperatures of the low- to physiological-temperature nondenaturational conformational change and of unfolding to the molten globule state

At 1.0 atm pressure and in 150 mM sodium phosphate (pH = 7.0), bovine carbonic anhydrase undergoes a nondenaturational conformational change at 30.3 degrees C and an unfolding transition from the physiological conformer to the molten globule state at 67.4 degrees C. The pressure dependences of the temperatures of these transitions have been studied under reversible conditions for the purpose of understanding DeltaH degrees , DeltaS degrees , and DeltaV for each conformational change. Temperatures for the low-temperature to physiological-temperature conformational change T(L-->P) are obtained from physiologically relevant conditions using slow-scan-rate differential scanning calorimetry. Temperatures for the physiological-temperature conformation to molten globule state conversion T(P-->MG) are obtained from differential scanning calorimetry measurements of the apparent transition temperature in the presence of guanidine hydrochloride extrapolated to zero molar denaturant. The use of slow-scan-rate differential scanning calorimetry permits the calculation of the activation volume for the conversion of the low-temperature conformer to the physiological-temperature conformer DeltaV(double dagger)(L-->P). At 1.0 atm pressure, the transition from the low-temperature conformer to the physiological-temperature conformer involves a volume change DeltaV(L-->P) = 15 +/- 2 L/mole, which contrasts with the partial unfolding of the physiological-temperature conformer to the molten globule state (DeltaV(P-->MG) = 26 +/- 9 L/mole). The activation volume for this process DeltaV(double dagger)(L-->P) = 51 +/- 9 L/mole and is consistent with a prior thermodynamic analysis that suggests the conformational transition from the low-temperature conformation to the physiological-temperature conformation possesses a substantial unfolding quality. These results provide further evidence the structure of the enzyme obtained from crystals grown below 30 degrees C should not be regarded as the physiological structure (the normal bovine body temperature is 38.3 degrees C). These results should therefore have implications in any area that seeks to correlate the crystal structure of bovine carbonic anhydrase to physiological function.     

The related adaptors, adaptor in lymphocytes of unknown function X and Rlk/Itk-binding protein, have nonredundant functions in lymphocytes

Adaptors play a critical role in regulating signaling pathways that control lymphocyte development and activation. Adaptor in lymphocytes of unknown function X (ALX) and Rlk/Itk-binding protein (RIBP) are adaptors related by structure and sequence, coexpressed in T cells. Mice deficient for each adaptor demonstrated that ALX and RIBP, respectively, negatively and positively regulate T cell activation in response to TCR/CD28 stimulation. However, these results did not preclude that they may function redundantly in other cell populations, or in response to other stimuli. Therefore, to understand the relationship between these related adaptors, ALX/RIBP-deficient mice were generated. We demonstrate that although ALX and RIBP are expressed throughout T cell development, T cell development occurs normally in these mice. Using the H-Y TCR transgenic model, positive and negative selection were found to proceed unimpeded in the absence of ALX and RIBP. We demonstrate that RIBP is also expressed in B cells; however, RIBP- and ALX/RIBP-deficient mice had normal B cell development, and responded equivalently to wild type in response to IgM, CD40, B cell-activating factor/B lymphocyte stimulator, CpG, and LPS. Interestingly, T cells deficient in both ALX and RIBP behaved similarly to those deficient in ALX alone during T cell activation in response to TCR/CD28, exhibiting increased IL-2 production, CD25 expression, and proliferation, thus showing that ALX deficiency masked the effect of RIBP deficiency. ALX/RIBP-deficient T cells did not have any alterations in either activation-induced cell death or Th1/2 polarization. Therefore, we did not find any functional redundancy or synergy during lymphocyte development, selection, activation, or survival in ALX/RIBP-deficient mice, demonstrating that these molecules function independently.     

Contrasting effects of T cell growth factors on T cell responses to melanoma in vitro

 Determinants of T cell responses to tumor cells remain largely unknown. In the present study we have used long-term cultures of human melanoma cells and autologous peripheral blood lymphocytes to examine the influence of cytokines with T cell growth activity on the phenotype and cytotoxic and proliferative response of T cells to melanoma. It was found that addition of interleukin-4 (IL-4) inhibited the response of CD8⁺ T cells and promoted the response of the CD4 subset. IL-2 or IL-7 was effective in increasing melanoma-specific cytotoxic T lymphocyte (CTL) activity in cultures where CD8 T cells were predominant, whereas IL-4 followed by IL-2 was most effective in cultures where CD4 T cells predominated. IL-10 or IL-12 inhibited proliferation and CTL activity against melanoma in long-term cultures. The effects of IL-12 were reproduced in long-term cultures of T cells stimulated with mAb against CD3 and were shown to depend on prior exposure of T cells to IL-12 before IL-2. As yet unidentified factors, such as co-factor expression on melanoma, appear to be as important as exogenous cytokines in determining the nature of T cell responses to melanoma. These results suggest that analysis of responses in long-term culture may assist in defining the role of key cytokines and other determinants of immune responses to melanoma. Received: 4 June 1996 / Accepted: 12 November 1996