Expression and localisation of insulin receptor substrate 2 in normal intestine and colorectal tumours. Regulation by intestine-specific transcription factor CDX2.

BACKGROUND AND AIMS: Self-renewal and differentiation of intestinal epithelium is a tightly regulated process, whose perturbations are implicated in human colorectal tumourigenesis. The insulin/insulin-like growth factor (IGF) signalling pathway may play an important role in intestinal epithelium homeostasis. Insulin receptor substrate 2 (IRS2) is a poorly characterised component in this pathway. METHODS: Using complementary in vitro and in vivo human and murine models, expression (mRNA and protein levels), localisation (immunohistochemistry) and regulation of IRS2 were investigated in the normal intestine and colorectal tumours. In silico analysis of the human IRS2 promoter was performed together with reporter and chromatin immunoprecipitation assays. RESULTS: Significant IRS2 expression was detected in the intestine, with specific protein localisation in the villus region of the ileum and in the surface epithelium of the colon. In human HT29 and Caco2 cells, IRS2 mRNA levels increased with spontaneous and induced differentiation, together with CDX2 (caudal-related homeobox protein 2), P21 and KLF4 (Krüppel-like factor 4). Adenoviral infection with human CDX2 induced IRS2 expression in APC- (adenomatous polyposis coli) and beta-catenin-mutated cells. On the other hand, IRS2 downregulation was observed in differentiated enterocytes after adenoviral infection with short hairpin CDX2 (shCDX2), in the intestine of CDX2 heterozygous mice and in colorectal tumours of Apc(Min/+) and patients with familial adenomatous polyposis (FAP). The human IRS2 promoter region presents several CDX2-binding sites where CDX2 immunoprecipitated in vivo. IRS2 reporters were functionally activated via CDX2 and blocked via a dominant-negative CDX2 protein. CONCLUSIONS: Combining gain- and loss-of-function approaches, an intriguing scenario is presented whereby IRS2 is significantly expressed in the apical intestinal compartment and is directly controlled by CDX2 in normal intestine and tumours.

Circadian variations of autologous mixed lymphocyte reactions and endogenous cortisol.

The degree of proliferation of human T cells stimulated with autologous PHA-T cells and with autologous non-T cells displays circadian variations. The highest proliferation occurs with cells isolated from blood drawn at 8 a.m. in mixed lymphocyte reactions (MLR) with autologous PHA-T cells and from blood drawn at 8 p.m. in MLR with autologous non-T cells. The circadian variations of autologous MLRs appear to reflect changes in the proliferative response of T cells. In autologous MLRs with non-T cells as stimulators the extent of proliferation was inversely correlated with the level of endogenous cortisol. The circadian variations of autologous MLRs do not reflect non-specific changes in the proliferative and stimulatory properties of T and non-T cells, since circadian variations were not observed in the proliferative response of T cells to mitogens and in allogeneic MLRs. Circadian variations of autologous MLRs must be taken into account when analyzing abnormalities of these reactions in pathological conditions.

Differential modulation by tumor necrosis factor and immune interferon of HLA class-II antigens expressed by melanoma cells.

Tumor necrosis factor (TNF-alpha) was compared to immune interferon (IFN-gamma) for its ability to modulate the expression and shedding of HLA antigens, of intercellular adhesion molecule I (ICAM I) and of high-molecular-weight melanoma-associated antigen (HMW MAA) by a panel of melanoma cell lines. The latter included the melanoma cell line MeWo and its metastatic variant MeM 50-10, which display differential susceptibility to modulation of HLA class-II antigens by IFN-gamma and the cell lines SK-MEL-93-DX-2 and SK-MEL-93-DX-3, which originated from anatomically distinct metastases in patient DX. TNF-alpha enhanced the expression of HLA class-I antigens on all 7 melanoma cell lines tested, although to a lower extent than IFN-gamma and the combination of IFN-gamma and TNF-alpha. TNF-alpha displayed a differential effect on the expression of HLA class-II antigens by the 7 melanoma cell lines: it enhanced it on 3 out of the 4 cell lines with constitutive expression of HLA class-II antigens and induced them on 1 of 3 cell lines without detectable expression of these antigens. The effects of IFN-gamma were different since it enhanced HLA class-II antigens on the 4 cell lines with constitutive expression of these antigens and induced them on 2 out of the remaining 3 lines. Interestingly, both TNF-alpha and IFN-gamma enhanced the expression of HLA class-II antigens by SK-MEL-93-DX-3 cells. On the other hand only TNF-alpha induced the expression of HLA class-II antigens by MeWo cells and only IFN-gamma induced such expression by MeM 50-10 cells and by SK-MEL-93-DX-2 cells. The effect of the combination of TNF-alpha and IFN-gamma was similar to that of the individual cytokines. Both TNF-alpha and IFN-gamma displayed a differential effect on the expression of the gene products of the HLA-D region by the melanoma cell lines. Northern blot analysis with HLA-DR beta-, DQ beta- and DP beta-specific probes suggests that the modulation of HLA class-II antigens by both cytokines reflects transcriptional and post-transcriptional events. TNF-alpha enhanced the expression of ICAM-I on all the melanoma cell lines, although to a lower extent than IFN-gamma and the combination of IFN-gamma and TNF-alpha. Lastly, neither TNF-alpha nor IFN-gamma displayed a marked effect on the expression of HMW-MAA by the melanoma cell lines tested.

Decreased lymphocyte blastogenesis, IL2 production and NK activity following nifedipine administration to healthy humans.

The effects of a single oral dose of nifedipine on part of the immune response in healthy humans has been investigated in terms of two different immune functions: T lymphocyte proliferation and NK activity. Both functions are known to require calcium ions. Ten healthy subjects were bled before and 30 min, and 4 and 24 h after receiving 10 mg nifedipine. Lymphocyte proliferation, both in mitogen-activated lymphocyte cultures, and in autologous and allogeneic mixed lymphocyte reactions, was significantly reduced (up to 48%) 30 min after drug administration and reverted to normal 4 h later. The inhibition could be attributed to reduction in IL2 production by the T cells isolated 30 min following the administration of nifedipine, since they normally express IL2-receptors. The addition of recombinant IL2 of 200 U.ml-1 to the cell cultures restored their responsiveness. NK activity was significantly reduced 30 min and 4 h after drug administration and returned to normal at the 24th h. This function was also restored by the addition of IL2. The data suggest that calcium channel blockers may inhibit, at least transiently, lymphocyte functions in vivo.

Expression of HLA-class II antigens and proliferative capacity in autologous mixed lymphocyte reactions of human T lymphocytes exposed in vitro to alpha-endorphin.

alpha-Endorphin (aEP) inhibited the expression of HLA-Class II antigens by PHA-primed T lymphocytes and reduced mitogen-induced T-cell proliferation up to 35%. This action was time related and not naloxone sensitive. When aEP was added to autologous and allogeneic lymphocyte cultures (both of non-T/T and T/T type), it inhibited lymphocyte blastogenesis up to 40%. These findings, indicating that aEP can influence some functions of immunocompetent cells, provide evidence for the functional interrelationship between the neuroendocrine and the immune systems.

Morphine-induced TSH release in normal and hypothyroid subjects.

The effects of morphine (10 mg i.v.), an opioid agonist, and of naloxone (10 mg i.v.), an opioid antagonist, on serum levels of TSH and PRL were studied in 7 hypothyroid patients and in 5 normal volunteers. Morphine administration induced a prompt, significant increase in serum TSH and PRL in all subjects. The degree of PRL release after morphine was similar in the two groups, while, as regards TSH, the increase was more evident in hypothyroid subjects. Pretreatment with naloxone (4 mg i.v. 5 min before morphine administration) blocked these effects in all subjects. In contrast, naloxone alone was not able to affect significantly TSH and PRL secretion. Moreover, in 5 other euthyroid volunteers, morphine significantly enhanced the response of TSH and PRL to TRH stimulation (200 micrograms i.v.). These data demonstrate that morphine exerts a stimulatory action on TSH and PRL secretion: the possible mode of action of this drug and the physiologic significance of these findings are discussed.

Pulmonary fat embolism in the traumatised patient: development of a prophylactic programme in a city accident hospital.

Low molecular weight plasma expanders, proteinase inhibitor and phosphatidilcholine have been used in a city accident hospital prophylactically against fat pulmonary embolism (F.P.E.) in traumatised patients. Their routine use in patients at risk has shown a decrease in the frequency of this syndrome, suggesting the validity of such treatment.