Hepatocellular dysfunction after severe hypotension in the absence of blood loss is associated with the increased IL-6 and PGE2

Activation of ATP-sensitive K+ channels is involved in the coronary vascular response to decreases in perfusion pressure and ischemia. Since activation of ATP-sensitive K+ channels in collateral vessels may be important in determining flow to collateral-dependent myocardium, the ability of collaterals to respond to activation of the channel was tested. In the beating heart of dogs, we compared responses of non-collaterals less than 100 microns in diameter to collaterals of similar size using computer-controlled stroboscopic epi-illumination of the left ventricle coupled to a microscope-video system. Aprikalim, a selective activator of ATP-sensitive K+ channels (0.1-10 microM) produced similar dose-dependent dilation of non-collaterals and collaterals. Relaxation was decreased by inhibition of ATP-sensitive K+ channels with glibenclamide, but not by inhibition of nitric oxide synthase with nitro-L-arginine. Bradykinin (10-100 microM) produced similar dilation of non-collaterals and collaterals which was decreased by nitro-L-arginine but not glibenclamide. Thus, in microvascular collaterals, relaxation to both nitric oxide and activation of ATP-sensitive K+ channels is similar to non-collaterals.     

Sensitivity to Fas-mediated apoptosis in pediatric acute lymphoblastic leukemia is associated with a mutant p53 phenotype and absence of Bcl-2 expression

We investigated the antitumour effects of 1-(2,6-difluorophenyl)-1H,3H-thiazolo [3,4-a]benzimidazole (TBZ) a new anti-HIV-1 agent, on human promyelocytic HL60 leukaemia, both a parental and a multidrug resistant form (HL60R). HL60R overexpresses P-glycoprotein and, like HL60, lacks p53 protein expression. HL60 and HL60R show similar levels of Bcl-2 protein. In contrast to the conventional chemotherapeutic agents daunorubicin, etoposide and mitoxantrone, TBZ caused equal or even greater cytotoxicity in HL60R than in HL60, and this result was associated with a more marked induction of apoptosis in the drug resistant cells. The antitumour activity of TBZ occurred in the range of concentrations higher than those required to exert antiviral activity. TBZ seems to act in the presence of P-glycoprotein and Bcl-2 and in the absence of p53 and is able to circumvent the mechanisms of drug resistance and anti-apoptosis present in HL60R cells.     

IL-9 production of naive CD4+ T cells depends on IL-2, is synergistically enhanced by a combination of TGF-beta and IL-4, and is inhibited by IFN-gamma

Dense CD4+ T cells isolated from naive mice produce only trace amounts of IL-9 when stimulated by immobilized anti-CD3 in combination with anti-CD28 Abs. In this situation, IL-9 production is significantly stimulated by TGF-beta and further enhanced by the addition of IL-4, which, by itself, has only a minimal influence. IFN-gamma was found to inhibit the enhancing effect of IL-4. However, increasing amounts of IL-4 in the presence of a constant concentration of IFN-gamma could overcome the inhibitory activity of IFN-gamma. The application of CD4+ T cells isolated from IL-2 knockout mice unequivocally revealed that IL-2 is essential for the production of IL-9 by T cells. In addition, the use of T cells from IL-4 knockout mice elucidated that the basic (IL-2 + TGF-beta) mediated IL-9 production is independent of IL-4. Therefore, our results demonstrate that optimal IL-9 production of naive dense CD4+ T cells is positively regulated at different levels: 1) by IL-2, which is essential for IL-9 secretion; 2) followed by TGF-beta, which promotes a considerable increase in IL-9 production above the level induced by IL-2; and 3) finally, by IL-4, which requires the presence of IL-2 and TGF-beta to strongly enhance the production of IL-9. IFN-gamma inhibits the production of IL-9 mainly at the level of IL-4 by neutralizing the effect of this cytokine.     

Induction of tolerance with nondepleting anti-CD4 monoclonal antibodies is associated with down-regulation of TH2 cytokines

BACKGROUND: Induction of tolerance with anti-CD4 has mainly focused on monoclonal antibodies (mAbs) that deplete CD4+ T cells. In this study, the mechanisms by which nondepleting anti-CD4 mAbs induce tolerance in the Dark Agouti to PVG rat heart graft model were examined. METHODS: Five anti-CD4 mAbs were tested. Immunohistology and cytokine mRNA profiles were analyzed within grafts. Effects of combining anti-CD4 therapy with alloantibody (alloAb), interleukin (IL)-4, and anti-IL-4 mAb were also examined. RESULTS: All mAbs tested induced indefinite graft survival (>150 days), with blocking of alloAb production. Exogenous alloAb did not restore rejection. Similar T cell receptor alphabeta+, CD8+, IL-2 receptor+ T cell, macrophage, and natural killer cell infiltration and comparable MHC II and intercellular adhesion molecule-1 levels were seen in rejecting and tolerant grafts. mRNA for IL-2, interferon-gamma, lymphotoxin, tumor necrosis factor-alpha, transforming growth factor-beta, cytolysin, and granzyme-A/B was comparable, although inducible nitric oxide synthase was slightly reduced in tolerant grafts. IL-4 and IL-5 were significantly reduced in tolerant grafts, although IL-6, IL-10, and IL-13 levels were similar; this was consistent with partial T helper (Th)2 response inhibition, which was also manifested by inhibited alloAb. The combination of alloAb, IL-4, or anti-IL-4 mAb with anti-CD4 did not prevent tolerance induction. CONCLUSIONS: This study demonstrated that anti-CD4 mAb therapy did not inhibit activation and infiltration of Th1 and CD8+ effector T cells. Preferential induction of Th2 responses, especially IL-4, was not essential for the induction of tolerance. Our studies also found no evidence to support induction of anergy or transforming growth factor-beta as mechanisms of tolerance induction. These results question whether IL-4 is required for induction of transplantation tolerance.     

Bcl-2 but not Bax expression is associated with apoptosis in normal and transformed squamous epithelium

Aberrant regulation of apoptosis may contribute to tumorigenesis. Relative levels of apoptosis regulatory proteins, such as Bcl-2 and Bax as well as interactions of these proteins with other gene products, may contribute to the rate of apoptosis in neoplasia. We examined Bcl-2 expression in 104 squamous cell carcinomas of the head and neck, as well as histologically normal mucosa several centimeters away from the tumor, and in control normal mucosa from patients without cancer. Immunohistochemistry and immunoblotting demonstrated Bcl-2 expression in 30% (31 of 104) of squamous cell carcinoma, with an increase in Bcl-2 protein levels compared with control normal mucosa from noncancer patients. Bcl-2-positive tumors demonstrated a 5-fold decrease in the number of apoptotic cells compared with Bcl-2-negative tumors. Bcl-2 protein expression was associated with poorly differentiated tumor grade but was not correlated with Bax expression or patient survival. These findings demonstrate that Bcl-2 contributes to apoptosis in normal and transformed squamous epithelium.     

Igf2r and Igf2 gene expression in androgenetic, gynogenetic, and parthenogenetic preimplantation mouse embryos: absence of regulation by genomic imprinting

Genomic imprinting in mammals is believed to result from modifications to chromosomes during gametogenesis that inactivate the paternal or maternal allele. The genes encoding the insulin-like growth factor type 2 (Igf2) and its receptor (Igf2r) are reciprocally imprinted and expressed from the paternal and maternal genomes, respectively, in the fetal and adult mouse. We find that both genes are expressed in androgenetic, gynogenetic, and parthenogenetic preimplantation mouse embryos. These results indicate that inactivation of imprinted genes occurs postfertilization (most likely postimplantation) and that genomic imprinting and gene inactivation are separate processes. We propose that imprinting marks the chromosome so that regulatory factors expressed in cells at later times can recognize the imprint and selectively inactivate the maternal or paternal allele. For these genes, this finding invalidates models of genomic imprinting that require them to be inactive from the time of fertilization.     

Cisplatin resistance is associated with reduced interferon-gamma-sensitivity and increased HER-2 expression in cultured ovarian carcinoma cells

In ovarian carcinoma cells, the combination of interferon-gamma (IFN-gamma) and cisplatin (cDDP) has been reported to result in a synergistic amplification of antiproliferative activity. To assess whether IFN-gamma may also prevent the occurrence of cisplatin resistance, the human ovarian carcinoma cell line HTB-77 was treated repeatedly in an intermittent fashion with either cisplatin alone (HTB-77cDDP) or cisplatin plus IFN-gamma (HTB-77cDDP + IFN). After 8 months of treatment, both new lines (HTB-77cDDP or HTB-77cDDP + IFN) were found to be three times more resistant to cisplatin than the wild-type cells (HTB-77wt). IFN-gamma could not prevent the development of cisplatin resistance. Interestingly, both HTB-77cDDP and HTB-77cDDP + IFN cells were also less IFN-gamma sensitive than the parental line. Both cisplatin-resistant lines expressed p185HER-2 and HER-2 mRNA at a higher concentration than the HTB-77wt cells. IFN-gamma was in all three HTB-77 cell lines able to suppress the HER-2 message and its encoded protein. The expression of IFN-gamma-induced antigens, namely CA-125 and class II antigens of the major histocompatibility complex (HLA-DR), was markedly augmented by IFN-gamma in all three lines, whereby the most prominent effect was seen in HTB-77cDDP and HTB-77cDDP + IFN.     

Prevention of Th2-like cell responses by coadministration of IL-12 and IL-18 is associated with inhibition of antigen-induced airway hyperresponsiveness, eosinophilia, and serum IgE levels

Allergic asthma is thought to be regulated by Th2 cells, and inhibiting this response is a promising mode of intervention. Many studies have focused on differentiation of Th cells to the Th1 or Th2 subset in vitro. IL-4 is essential for Th2 development, while IL-12 induces Th1 development, which can be enhanced by IL-18. In the present study, we investigated whether IL-12 and IL-18 were able to interfere in Th2 development and the associated airway symptoms in a mouse model of allergic asthma. Mice were sensitized with OVA using a protocol that induces IgE production. Repeated challenges by OVA inhalation induced elevated serum levels of IgE, airway hyperresponsiveness, and a predominantly eosinophilic infiltrate in the bronchoalveolar lavage concomitant with the appearance of Ag-specific Th2-like cells in lung tissue and lung-draining lymph nodes. Whereas treatments with neither IL-12 nor IL-18 during the challenge period were effective, combined treatment of IL-12 and IL-18 inhibited Ag-specific Th2-like cell development. This inhibition was associated with an absence of IgE up-regulation, airway hyperresponsiveness, and cellular infiltration in the lavage. These data show that, in vivo, the synergistic action of IL-12 and IL-18 is necessary to prevent Th2-like cell differentiation, and consequently inhibits the development of airway symptoms in a mouse model of allergic asthma.     

Increased ratio of bcl-2/bax expression is associated with bovine leukemia virus-induced leukemogenesis in cattle

To further investigate the molecular basis underlying the dysregulation of B cell homeostasis associated with bovine leukemia virus disease progression in cattle, bovine bax was cDNA cloned and sequenced. The predicted amino acid sequence of bovine Bax revealed a 192-amino-acid protein having extensive identity with the human (97%), murine (93%), and rat (94%) homologues. Because the ratio of Bcl-2 to Bax is believed to predetermine the susceptibility to a given apoptotic stimulus, the relative expression of the genes encoding these oncoproteins was evaluated in cattle naturally infected with BLV. In BLV-infected cattle an increase in the ratios of bcl-2/bax mRNA and protein expression correlated with advancing stages of disease. These findings suggest that in addition to the maintenance of BLV-associated hematopoietic malignancies, the reciprocal expression of Bcl-2/Bax may modulate the induction of B cell expansion typical of BLV disease progression.     

Increased matrix metalloproteinase-2 activity induced by TGF-beta 1 in duct cells of human salivary gland is associated with the development of cyst formation in vivo

Proteolytic enzyme activity has been shown to be important for cyst formation. In this study, we constructed a cyst-like structure in vivo and analyzed molecular mechanisms involved in the development of the lesion. When SV40-immortalized duct cells of normal human salivary gland (NS-SV-DC) were treated with TGF-beta 1 at a concentration of 1 ng/ml or 5 ng/ml followed by co-inoculation with Matrigel into the backs of nude mice, they formed large cysts containing fluid when 5 ng/ml of TGF-beta 1 was used. Analysis of the fluid demonstrated high MMP activity. Immunohistochemical staining exhibited strong reactivity with anti-MMP-2 antibody in TGF-beta 1 (5 ng/ml)-treated NS-SV-DC. Northern blot analysis indicated that the expression of TGF-beta 1 and MMP-2 mRNAs in cells was greatly enhanced by treatment with 5 ng/ml TGF-beta 1. These findings suggest that the in vivo cyst formation by TGF-beta 1-treated cells is associated with continuous induction of MMP-2 activity.     

Cardiac senescence is associated with enhanced expression of angiotensin II receptor subtypes

Recent studies have pointed out the differential role of angiotensin II (Ang II) receptor subtypes, AT1 and AT2, in cardiac hypertrophy and fibrosis during pathological cardiac growth. Because senescence is characterized by an important cardiovascular remodeling, we examined the age-related expression of cardiac Ang II receptors in rats. AT1 and AT2 receptor subtype messenger RNA (mRNA) levels were quantitated by RT-PCR. In parallel, specific Ang II densities were determined in competition binding experiments using specific antagonists. AT1a and AT1b mRNA levels were markedly up-regulated (5.6-fold) in the left ventricle of 24-month-old rats compared with 3-month-old rats, but not in the right ventricle. In contrast, AT2 gene expression was increased in both ventricles of senescent rats (4.2- and 2.8-fold in the left and right ventricles, respectively). Similarly, AT1 and AT2 gene expression was increased 2.3- and 2-fold, respectively, in freshly isolated cardiomyocytes from aged rats. Furthermore, AT1 and AT2 specific binding was increased in the aged left ventricular myocardium. Even though the mechanistic pathway of this up-regulation of Ang II receptor subtype gene expression might be intrinsic to developmental gene reprogramming, the up-regulation of AT1 mRNA accumulation in the left ventricle during aging could also be secondary to age-related hemodynamic changes, whereas increased AT2 gene expression in both ventricles may depend upon hormonal and humoral factors.     

IGFBP-2 expression in a human cell line is associated with increased IGFBP-3 proteolysis, decreased IGFBP-1 expression and increased tumorigenicity

Insulin-like growth factors (IGF-I and -II) play an active role in cell proliferation. In biological fluids, they are non-covalently bound to high-affinity binding proteins (IGFBPs), at least 6 species of which have been identified to date, but with poorly defined functions. One of these IGFBPs, IGFBP-2, is secreted by most cell lines and appears to be involved in cell proliferation. A human epidermoid carcinoma cell line, KB 3.1, which produces IGFBP-1 and -3 and small amounts of IGFBP-4, but no IGFBP-2, was stably transfected with an expression vector comprising IGFBP-2 complementary DNA (cDNA), whose expression was placed under the control of the constitutive and ubiquitous cytomegalovirus promoter. After an s.c. injection of these IGFBP-2-expressing KB 3.1 cells into nude mice, tumours developed more quickly than in controls, they were 3 to 4 times larger and grew about 3 times as fast. Concomitant with IGFBP-2 expression in these tumours, were a decrease in IGFBP-1 expression and an increase in IGFBP-3 proteolysis, both of which increase the bioavailability of the IGF-II produced by the cells. The increased IGFBP-3 proteolysis most probably resulted from amplified expression of tissue-type plasminogen activator (t-PA) and depression of its inhibitor (PAI-I) observed in IGFBP-2-expressing xenografts. Our findings suggest that IGFBP-2 plays a role in this model of experimental tumorigenesis via a mechanism that remains unclear, but appears to involve increased protease activity and IGF-II bioavailability.     

Interstitial pneumonitis in bone marrow transplant recipients is associated with local production of TH2-type cytokines and lack of T cell-mediated cytotoxicity

BACKGROUND: Interstitial pneumonitis, especially associated with cytomegalovirus (CMV) infection, is a serious complication after bone marrow transplantation (BMT), with a high fatality rate despite adequate antiviral treatment. The aim of this study was to elucidate the local immunopathogenesis of interstitial pneumonitis caused by CMV or other agents in BMT recipients. METHODS: Cryopreserved lung tissue obtained from 12 patients with interstitial pneumonitis following BMT was analyzed for cytokine production at the single-cell level using a cytokine-specific monoclonal antibody and immunohistochemical technique. Cytokine production in individual cells was analyzed using monoclonal antibodies to 23 different human cytokines: interleukin (IL)-1 to IL-13, tumor necrosis factor (TNF)-alpha, TNF-beta, interferon-gamma (IFNgamma), granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and transforming growth factor (TGF)-beta1 to 3. RESULTS: Marrow transplant patients with interstitial pneumonia had increased numbers of infiltrating alveolar macrophages, CD3+, CD4+ T cells, and CD40+ B cells and significantly increased numbers of IL-4-, IL-10-, IL-1-, TGF-beta1-, TGF-beta2-, and TGF-beta3-producing cells than controls. IL-2-, IFN-gamma-, and TNF-beta-producing cells were undetectable in most patients with CMV pneumonitis (n=7). Neither perforin-positive CD8+ T lymphocytes nor up-regulation of the apoptotic pathway was detected in lung tissue from patients with interstitial pneumonia. In contrast, extensive local production of IgA, IgG, and IgM was demonstrated in all patients. Intracellular and extensive extracellular deposition of CD68, the L-1 antigen synthesized in CD14+ macrophages, was found. CONCLUSIONS: The cytokine profile suggested that Th1-type cytokine production was absent, whereas production of Th2-type cytokines was significantly up-regulated. Interstitial pneumonitis in BMT recipients with fatal outcome (11/12 patients) was associated with dysregulation in the local cytokine network notable for a predominant Th2 immune response with minimal or absent T cell-mediated cytotoxicity.