Differential sensitivity to apoptosis between the human small and large intestinal mucosae: linkage with segment-specific regulation of BCL-2 homologs and involvement of signaling pathways.

The small and large intestines differ in their expression profiles of Bcl-2 homologs. Intestinal segment-specific Bcl-2 homolog expression profiles are acquired as early as by mid-gestation (18-20 weeks) in man. In the present study, we examined the question whether such distinctions underlie segment-specific control mechanisms of intestinal cell survival. Using mid-gestation human jejunum and colon organotypic cultures, we analyzed the impact of growth factors (namely insulin; 10 microg/ml) and pharmacological compounds that inhibit signal transduction molecules/pathways (namely tyrosine kinases, Fak, P13-K/Akt, and MEK/Erk) on cell survival and Bcl-2 homolog expression (anti-apoptotic: Bcl-2, Bcl-X(L), Mcl-1; pro-apoptotic: Bax, Bak, Bad). The relative activation levels of p125Fak, p42Erk-2, and p57Akt were analyzed as well. Herein, we report that (1) the inhibition of signal transduction molecules/pathways revealed striking differences in their impact on cell survival in the jejunum and colon (e.g., the inhibition of p125Fak induced apoptosis with a significantly greater extent in the jejunum [approximately 43%] than in the colon [approximately 24%]); (2) sharp distinctions between the two segments were noted in the modulatory effects of the various treatments on Bcl-2 homolog steady-state levels (e.g., inhibition of tyrosine kinase activities in the jejunum down-regulated all anti-apoptotics analyzed while increasing Bax, whereas the same treatment in the colon down-regulated Bcl-X(L) only and increased all pro-apoptotics); and (3) in addition to their differential impact on cell survival and Bcl-2 homolog expression, the MEK/Erk and P13-K/Akt pathways were found to be distinctively regulated in the jejunum and colon mucosae (e.g., insulin in the jejunum increased p42Erk-2 activation without affecting that of p57Akt, whereas the same treatment in the colon decreased p42Erk-2 activation while increasing that of p57Akt). Altogether, these data show that intestinal cell survival is characterized by segment-specific susceptibilities to apoptosis, which are in turn linked with segmental distinctions in the involvement of signaling pathways and the regulation of Bcl-2 homolog steady-state levels. Therefore, these indicate that cell survival is subject to segment-specific control mechanisms along the proximal-distal axis of the intestine.

Early acquisition of bowel segment-specific Bcl-2 homolog expression profiles during development of the human ileum and colon.

The adult small and large intestines display distinct expression profiles of Bcl-2 homologs, known regulators of apoptosis. This is thought to indicate that control mechanisms of intestinal apoptosis are gut segment-specific. Little is known on the expression of Bcl-2 homologs during gut development. In man, intestinal features and functions are acquired largely by mid-gestation (18-20 wks); the question whether segment-specific controls of intestinal apoptosis are also acquired early during development remains open. In the present study, we approached this by investigating the expression of six Bcl-2 homologs (Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, Bad), and one nonhomologous associated molecule (Bag-1), during development of the human ileum and colon (12-20 wks of gestation). Beginning at 18 wks, we found that the epithelial localization of Bcl-2 homologs displayed differential patterns (or gradients) in both the ileum and colon; however, the patterns of some of the homologs differed between the two segments. For instance, Bag-1 and Bcl-2 exhibited crypt-villus decreasing gradients of expression in the ileum but not in the colon, whereas Mcl-1 displayed differing compartimentalizations between the two segments. Further analyses indicated that the steady-state expression levels of Bcl-2 homologs underwent modulations between 12 and 20 wks; however, the observed developmental profiles contrasted significantly between the two segments. For example, Bcl-2, Bag-1 and Bak levels increased in the colon, but the levels of these same homologs decreased in the ileum. Furthermore, by 18-20 wks, we found that the expression levels of each Bcl-2 homolog analyzed differed greatly between the ileum and colon. Altogether, these data indicate that the expression of Bcl-2 homologs is modulated differentially during human gut development in order to establish, by mid-gestation, distinct expression profiles for the small and large intestines. This in turn suggests that gut segment-specific control mechanisms of human intestinal apoptosis are acquired early during fetal life.

Early establishment of epithelial apoptosis in the developing human small intestine.

In the adult small intestine, the dynamic renewal of the epithelium is characterized by a sequence of cell production in the crypts, cell maturation and cell migration to the tip of villi, where apoptosis is undertaken. Little is known about enterocytic apoptosis during development. In man, intestinal architectural features and functions are acquired largely by mid-gestation (18-20 wks); the question whether the establishment of enterocytic apoptotic processes parallels or not the acquisition of other intestinal functional features remains open. In the present study, we approached this question by examining enterocytic apoptosis during development of the human jejunum (9-20 wks gestation), using the ISEL (in situ terminal uridine deoxynucleotidyl nick-end labelling) method. Between 9 and 17 wks, apoptotic enterocytes were not evidenced. However, beginning at the 18 wks stage, ISEL-positive enterocytes were regularly observed at the tip of villi. Since the Bcl-2 family of proteins constitutes a critical checkpoint in apoptosis, acting upstream of the apoptotic machinery, we investigated the expression of six Bcl-2 homologs (Bcl-2, Bcl-X(L), Mcl-1, Bax, Bak, Bad) and one non-homologous associated molecule (Bag-1). By immunofluorescence, we found that all homologs analyzed were expressed by enterocytes between 9 and 20 wks. However, Bcl-2 homologs underwent a gradual compartmentalization of epithelial expression along the maturing crypt-villus axis, to establish gradients of expression by 18-20 wks. Western blot analyses indicated that the expression levels of Bcl-2 homologs were modulated during morphogenesis of the crypt-villus axis, in parallel to their gradual compartmentalization of expression. Altogether, these data suggest that regulatory mechanisms of human enterocytic apoptosis become established by mid-gestation (18-20 wks) and coincide with the maturation of the crypt-villus axis of cell proliferation, differentiation and renewal.

[Lipid levels in children with psoriasis]. / Studio dei livelli lipidemici in bambini psoriasici.

Elevated blood cholesterol (above the 95 degrees percentile) was found in three of thirty-one children with psoriasis, as in adult subjects with psoriasis. Therefore a steadily evaluation of lipids pattern is suggested for patients with psoriasis, even children, in order to start an appropriate management.

Chromosomal alterations in peripheral blood lymphocytes, urinary mutagenicity and excretion of polycyclic aromatic hydrocarbons in six psoriatic patients undergoing coal tar therapy.

Six male non-smoking subjects treated for psoriasis with topical applications of pure coal tar or 4% coal tar-containing ointment were examined in order to assess the genotoxic risk associated with this type of therapy. Mutagenicity in urine samples collected before and during the coal tar therapy was evaluated in the plate incorporation assay on Salmonella typhimurium strain TA98 in the presence of S9 mix and beta-glucuronidase. Total urinary polycyclic aromatic hydrocarbon (PAH) levels were evaluated in parallel by high resolution gas chromatography/mass spectrometry. In addition, sister chromatid exchanges and chromosomal aberrations were also analysed in peripheral blood lymphocytes collected before, during and after the end of the coal tar applications. The results suggest that urinary mutagenicity levels as well as the frequencies of chromosome aberrations and sister chromatid exchanges in lymphocytes are related to the levels of exposure to coal tar. Moreover the kinetics of repair of chromosome damage in relation to different exposure levels and the capacity of the urinary mutagenicity assay to correctly identify the exposure to significant levels of PAH are discussed.

Mutagenic activity and polycyclic aromatic hydrocarbon levels in urine of humans exposed to therapeutical coal tar.

The urinary mutagenicity and the excretion of polycyclic aromatic hydrocarbons (PAH) in three non-smoking male patients, treated for psoriasis with cutaneous applications of crude coal tar, were analysed. Mutagenicity of the urinary extracts was measured by the plate incorporation assay using Salmonella typhimurium strains TA 98 and TA 100 in the presence of liver S9 fraction from Aroclor-induced rats with or without beta-glucuronidase. After concentration, hydrolysis and reduction of the urine sample, PAH levels were measured by high resolution gas chromatography/mass spectrometry. Following cutaneous treatment with coal tar, the urine of all three subjects showed noticeable levels of PAH and/or metabolites and marked mutagenicity both on strain TA 98 and TA 100 in the presence of S9 fraction. The addition of beta-glucuronidase increased the mutagenicity of the urinary extracts, the maximum values being attained on strain TA 100 in the presence of both microsomal fraction and deconjugating enzymes. The mutagenicity of urinary extracts from subjects treated therapeutically with crude coal tar was correlated (r = 0.788, P less than 0.01) with the total PAH levels in their urine. The PAH excreted in urine were mainly low molecular weight compounds, while benzo[a]anthracene was present in scarce amounts and the excretion of benzo[a]pyrene did not increase following the cutaneous exposure to the crude coal tar.

[Tryptophan metabolism in patients with vitiligo]. / Studi sul metabolismo del triptofano in pazienti con vitiligine.

Our previous research showed that tryptophan is an important precursor in the biogenesis of melanins. Therefore, with the purpose of observing whether a relationship exists between tryptophan metabolism and diseases characterized by an altered process of skin pigmentation in man, we studied the metabolism of this aminoacid along the kynurenine pathway in 29 vitiliginous patients (11 males and 18 females) and in 21 control subjects (11 males and 10 females) by determining 10 urinary metabolites after an oral loading of 50 mg/kg body weight L-tryptophan. The mean total excretion of the metabolites in patients resulted similar to that of the controls. However, considering the individual metabolites one can observe a decreased excretion of 3-hydroxykynurenine, o-aminohippuric acid and 3-hydroxyanthranilic acid and an increased excretion of xanthurenic acid and of its 8-methyl ether in the group of vitiliginous patients in respect to the controls. These results seem to indicate a decreased formation of nicotinic acid from tryptophan. Moreover, in relation to the fact that 3-hydroxykynurenine could be the metabolite through which tryptophan is involved in melanin biosynthesis, this study supports the hypothesis of a connection of tryptophan metabolism with the lack of pigmentation in vitiligo.