Inside-out and outside-in organotypic normal human skin culture: JAK-STAT pathway is activated after pro-inflammatory psoriatic cytokine exposure.

The Janus kinases-signal transducers and activators of transcription (JAK-STAT) signalling pathway are a pleiotropic cascade that involves ligands such as cytokines, hormones, and growth factors. Among cytokines, interleukin (IL)-17, IL-22, IL-23, and tumour necrosis factor (TNF)-alpha play a pivotal role in psoriasis. We aimed at investigating in an organotypic experimental model of normal human skin (n = 7 women between 20-40 years old, non-smokers) the early, direct, and specific effects of IL-17, IL-22, IL-23, TNF-alpha and a combination of the four cytokines (Mix) on the JAK-STAT/pathway. The expression of the psoriatic marker keratin (K) 17 was analyzed by immunofluorescence and molecular techniques after exposure to IL-23 or Mix. The Mix elicited a strong K17 up-regulation in keratinocytes at 72 h, reinforcing the hypothesis of a synergistic effect of different cytokines. High levels of JAK1 and STAT3 activation were detected, suggesting the involvement of JAK1/STAT3 pathway in the upregulation of K17. As the present study in an organotypic model of human skin reports a variable expression of JAK-STAT upon different cytokine stimuli and most of the JAK inhibitors for the psoriasis treatment have proven to have a clinical efficacy, these observations have a relevance to better understand the mechanisms of JAK-inhibitors in the skin.

A therapeutic dose of FK506 does not affect collagen turnover pathways in healthy human gingival fibroblasts.

We previously demonstrated that a high dose of tacrolimus (1 mumol/L) induced expression of matrix metalloproteinase (MMP) proteins in human cultured gingival fibroblasts, suggesting a molecular mechanism maintaining gingival collagen homeostasis in tacrolimus-treated patients. Herein we have analyzed whether the effect on collagen turnover might be influenced by a therapeutic tacrolimus dose. Human gingival fibroblasts were incubated for 72 hours with 10 nmol/L, 100 nmol/L, and 1 mumol/L tacrolimus, or left untreated (CT). Collagen type I and III (COL-I, COL-III), lysyl hydroxylase 2b (LH2b), MMP-1 and -2, tissue inhibitor of MMP-1 and transforming growth factor-beta1 (TGF-beta1) mRNA levels were assayed by reverse-transcriptase polymerase chain reaction, collagen protein levels by dot blot, and MMP activity by sodium dodecyl sulfate zymography. Tacrolimus did not affect COL-I, COL-III, or MMP gene expression, while LH2b and TGF-beta1 tended to be down-regulated after 1 mumol/L FK506. Conversely, protein levels of MMP-1 (P = NS) and MMP-2 (P < .05 vs CT, 10 nmol/L, 100 nmol/L) were up-regulated after 1 mumol/L tacrolimus. Our findings confirmed that a high dose of tacrolimus does not induce interstitial collagen overexpression by gingival fibroblasts and induces up-regulation of MMPs protein levels. Interestingly, at doses corresponding to whole blood trough levels, tacrolimus did not exert any evident effect on collagen turnover pathways, suggesting that tacrolimus is likely to not affect collagen homeostasis in the gingival connective tissue compartment of FK506-immunosuppressed subjects. This effect did not seem to be dose-dependent.

Methionine oxidation as a major cause of the functional impairment of oxidized actin.

A significant specific increase in the actin carbonyl content has been recently demonstrated in human brain regions severely affected by the Alzheimer's disease pathology, in postischemic isolated rat hearts, and in human intestinal cell monolayers following incubation with hypochlorous acid (HOCl). We have very recently shown that exposure of actin to HOCl results in the immediate loss of Cys-374 thiol, oxidation of some methionine residues, and, at higher molar ratios of oxidant to protein, increase in protein carbonyl groups, associated with filament disruption and inhibition of filament formation. In the present work, we have studied the effect of methionine oxidation induced by chloramine-T (CT), which at neutral or slightly alkaline pH oxidizes preferentially Met and Cys residues, on actin filament formation and stability utilizing actin blocked at Cys-374. Methionines at positions 44, 47, and 355, which are the most solvent-exposed methionyl residues in the actin molecule, were found to be the most susceptible to oxidation to the sulfoxide derivative. Met-176, Met-190, Met-227, and Met-269 are the other sites of the oxidative modification. The increase in fluorescence associated with the binding of 8-anilino-1-naphtalene sulfonic acid to hydrophobic regions of the protein reveals that actin surface hydrophobicity increases with oxidation, indicating changes in protein conformation. Structural alterations were confirmed by the decreased susceptibility to proteolysis and by urea denaturation curves. Oxidation of some critical methionines (those at positions 176, 190, and 269) causes a complete inhibition of actin polymerization and severely affects the stability of actin filaments, which rapidly depolymerize. The present results would indicate that the oxidation of some critical methionines disrupts specific noncovalent interactions that normally stabilize the structure of actin filaments. We suggest that the process involving formation of actin carbonyl derivatives would occur at an extent of oxidative insult higher than that causing the oxidation of some critical methionine residues. Therefore, methionine oxidation could be a damaging event preceding the appearance of carbonyl groups on actin and a major cause for the functional impairment of the carbonylated protein recently observed both in vivo and in vitro.

Actin carbonylation: from a simple marker of protein oxidation to relevant signs of severe functional impairment.

The number of protein-bound carbonyl groups is an established marker of protein oxidation. Recent evidence indicates a significant increase in actin carbonyl content in both Alzheimer's disease brains and ischemic hearts. The enhancement of actin carbonylation, causing the disruption of the actin cytoskeleton and the loss of the barrier function, has also been found in human colonic cells after exposure to hypochlorous acid (HOCl). Here, the effects of oxidation induced by HOCl on purified actin are presented. Results show that HOCl causes a rapidly increasing yield of carbonyl groups. However, when carbonylation becomes evident, some Cys and Met residues have been already oxidized. Covalent intermolecular cross-linking as well as some noncovalent aggregation of carbonylated actin have been found. The covalent cross-linking, unaffected by reducing and denaturing agents, parallels an increase in dityrosine fluorescence. Moreover, HOCl-mediated oxidation induces the progressive disruption of actin filaments and the inhibition of F-actin formation. The molar ratios of HOCl to actin that lead to inhibition of actin polymerization seem to have effect only on cysteines and methionines. The process that involves oxidation of amino acid side chains with formation of a carbonyl group would occur at an extent of oxidative insult higher than that causing the oxidation of some critical amino acid residues. Therefore, the increase in actin content of carbonyl groups found in vivo would indicate drastic oxidative modification leading to drastic functional impairments.

Age-dependent expression of fibrosis-related genes and collagen deposition in the rat myocardium.

OBJECTIVES: We sought to characterize the evolution, during maturational growth and early ageing, of the messenger abundance of four genes involved in cardiac fibrosis regulation (procollagens alpha2(I) and alpha1(III), transforming growth factors beta1, and beta3) and corroborate it with the alterations in collagen deposition in cardiac interstitium and around coronary arteries. METHODS: Messenger RNA was quantified in LV and RV of 2-, 6-, 12- and 19-month-old male Sprague-Dawley rats (n = 5 per group) with Northern blot analysis. Collagen deposition was quantified with a semi-automated image analyser on Sirius red-stained sections of LV tissue. RESULTS: There was an age-related monotonous decrease of procollagen type I (COL-I) transcript abundance in LV (p < 0.001) but not in RV. Procollagen type III (COL-III) expression decreased rapidly during maturational growth, both in LV and RV. On the other hand, collagen deposition in myocardial interstitium and around coronary arteries was slightly augmented during the maturational period of life (2-12 months), but with a higher rate during early ageing (up to 19 months). This was not accompanied by a significant thickening of the wall of coronary arteries. Transforming growth factor beta1, (TGF-beta1) and transforming growth factor beta3 (TGF-beta3) transcript abundance showed no major variations during ageing. CONCLUSIONS: These results reflect a striking ventricular difference regarding the age-dependent expression of COL-I. The expression of TGF-beta(s), pleiotropic factors known to influence collagen pathway at different levels, does not seem to be profoundly altered during ageing. The discrepancy between protein and COL-I and COL-III mRNA levels indicates differences in age-related mRNA stability and/or regulation of collagen translation.

Left ventricular response to beta-adrenergic stimulation in aging rats.

The incidence of heart failure in the population increases steeply among older people. Overactivation of the sympathetic nervous system is associated to and responsible for worsening of heart failure. This study describes the influence of aging on short-term left ventricular (LV) adaptation to b-adrenergic stimulation in Wistar rats. In controls at 18 mo, interstitial fibrosis was increased with respect to 3-mo-old rats, whereas myocytes dimension and the messenger RNA (mRNA) abundance of atrial natriuretic peptide (ANP), a2(I) procollagen, transforming growth factor (TGF-b1, TGF-b3), and secreted protein, acidic and rich in cysteine (SPARC) were not different. To determine how aging affects LV adaptation to adrenergic stimulation, two groups of animals received isoproterenol (ISO, 1 mg/kg/d) for 3 days. There was no significant difference between young and older rats with respect to increase in LV weight, myocytes dimension, and mRNA abundance of all the genes considered, except a1(III) procollagen. These findings indicate that despite limited compensatory hypertrophy and higher fibrosis, LV from aged nonsenescent rats preserves the capacity to adapt to b-adrenergic stimulation through the upregulation of several genes encoding extracellular matrix-related proteins.

Age-dependent expression of fibrosis-related genes and collagen deposition in rat kidney cortex.

Because progressive fibrosis is a histological hallmark of the aging kidney, we sought to characterize the course of some fibrosis-related genes [pro-alpha2(I)collagen (COL-I), pro-alpha1(III)collagen (COL-III), and transforming growth factors beta1 and beta3 (TGF-beta1 and TGF-beta3)] of interstitial collagen accumulation [COL-I and COL-III proteins, hydroxyproline (PRO-OH), histology] and its degradation (matrix metalloproteinase MMP-1 and -2) during maturation and early aging in rats. During the lifespan considered we observed no changes in the mRNA, except that COL-I mRNA tended to be up-regulated from 2 to 19 months of age. However, progressive fibrosis was histologically detectable, with COL-I accumulation (p < .05 and p < .01 in 12-month- and 19-month-old rats vs the youngest), and confirmed by the PRO-OH tissue levels (p = .0001); COL-III seemed to be less involved. The MMP-1 protein level decreased significantly in the cortex of 12-month- and 19-month-old rats (p < .05), whereas MMP-2 protein level and activity remained essentially unchanged. These results show that, during aging of the kidney, (i) renal cortex fibrosis is explained by COL-I accumulation as a consequence of an altered balance between its synthesis and degradation, and (ii) the expression of the pleiotropic factor TGF-beta in the renal cortex is not modified.

Extracellular signal-regulated kinases 1 and 2 phosphorylated neurons in the tele- and diencephalon of rat after visceral pain stimulation: an immunocytochemical study.

We aimed at verifying whether extracellular signal-regulated kinases (erks) 1 and 2 are activated, i.e. phosphorylated, in forebrain neurons after visceral pain stimulation (VPS). Ether and urethane anaesthetized rats received an intraperitoneal injection of acetic acid or were left untreated (ECT, UCT). After 2 h the animals were perfused. Paraffin embedded brain sections immunoreacted with an antibody selective for the phosphorylated erks. The light microscope analysis revealed only a few labelled neurons in ECT, while in UCT, positive cells were detected. In VPS rats (VPSR) positive cells were mainly distributed in regions, such as the hypothalamic anterior and thalamic paraventricular midline nuclei, amygdala, hippocampal and parahippocampal, insular and perirhinal cortex, involved in nociception and/or visceral activities. Our data suggest an association of erks activation with the emotional component of nociception; moreover, they show that erks activation is not suppressed by anaesthesia.

Megaesophagus in an asthmatic patient and beta2 stimulant treatment by inhalation.

Megaesophagus is a severe esophageal malformation. We report a case of megaesophagus in an asthmatic patient affected by congenital non-haemolytic anaemia and undergoing beta2 stimulant treatment by inhalation. Our case could be due to chronic beta2 receptor stimulation with imbalance of alpha and beta receptor, without any implication of favism.

Cholesterol screening in the adolescent.

Guidelines for screening populations for hypercholesterolemia are controversial. In pediatric patients, screening has been targeted to those with a family history of hypercholesterolemia or early myocardial infarction. Because specific guidelines for adolescents have not been developed, we undertook a study of cholesterol screening in 224 consecutive patients (mean age, 14.8 +/- 2.2 years; range, 11-20 years). In this study, 33 (14.7%) adolescents had levels above 185 mg/dL; 71 (32.7%) of the patients reported a positive family history for early myocardial infarction or elevated lipids. A positive family history obtained from the adolescent patient had a 36% sensitivity, 69% specificity, 17% positive predictive value, and an 87% negative predictive value compared with elevated cholesterol level. A positive history reported by the parent had a 65% sensitivity, 46% specificity, 16% positive predictive value, and an 89% negative predictive value. A positive family history obtained from either parent or adolescent had a 45% sensitivity, 69% specificity, 19% positive predicative value, and 89% negative predictive value. Family history by the adolescent was significantly different from parental history (p < 0.001). Family history is a poor predictor for cholesterol level in adolescents.

Polyamine levels and ornithine decarboxylase activity in blood and erythrocytes in human diseases.

Serum and erythrocyte levels of the polyamines spermine, spermidine and putrescine, as well as ornithine decarboxylase in erythrocytes, were studied in patients with different neoplasms (breast, lung and colon cancer) and in those with a nonmalignant proliferative disease (familial polyposis). The blood levels of polyamines and the spermine/putrescine ratio were significantly higher in all tumors and in nonmalignant colon polyposis. In erythrocyte ornithine decarboxylase activity, spermine and spermidine levels, as well as spermidine/putrescine and spermine/putrescine ratios showed a significant decrease after surgery and chemotherapy. Our data suggest that high levels of blood polyamines and erythrocyte ornithine decarboxylase activity are related to cell proliferation and cancer treatment, but that levels of polyamines in serum and erythrocytes are still significantly high after cancer treatment and are similar to those in polyposis disease. Polyamines are related to nuclear activity during differentiation; therefore, the altered turnover of polyamines could be a sign of abnormal nuclear function. Since polyamines stimulate protooncogene expression, their high levels could be considered an important cofactor in malignant cell transformation.

D.D.S.H.: developmental dysplasia of the spastic hip: strategies of management in cerebral palsy. A new suggestive algorithm.

Hip problems in cerebral palsy are relatively frequent (25-75%). Subluxation and dislocation of the hip is proportional to the neuromuscular involvement and is often due to alteration caused by spastic muscle forces acting on the femoral head in the acetabular cavity. The EMMA approach (Early Multilevel Minimally-invasive Approach) has been designed to restore muscle balance, decrease hip migration and prevent bone deformities thereby avoiding future pain with minimal biological cost to the patient. EMMA is suitable for most patients, especially those with increased tone, poor muscle control and selectivity, Reimer Index (R.I.) 20%. We consider age and R.I crucial prerequisites for treatment steps. EMMA 1) age 2-4 years, RI 20%: multilevel injection of botulinum toxin in case of muscular hyperactivity without morphological alterations of the couple muscle-tendon (contractures). EMMA 2) age 4-6, RI 20%: multilevel aponeurectomies in case of muscular hyperactivity with morphological alterations of the couple muscle-tendon (retraction). EMMA 3) early bone surgery (growth plates). This approach has been adopted in the last 4 years to prevent bone deformities and give early mobilisation and early control of the pain. EMMA is simple to apply even in infants, both for hip containment and to decrease spasticity.

Effects of aging and cyclosporin a on collagen turnover in human gingiva.

BACKGROUND: WE AIMED AT CHARACTERIZING THE AGING GINGIVA ANALYZING: i) collagen content and turnover in human gingival tissues and fibroblasts obtained from healthy young and aging subjects. ii) the effect of cyclosporin A administration in human cultured gingival fibroblasts obtained from aging compared to young subjects. METHODS: Morphological analysis was performed on haematoxylin-eosin and Sirius red stained paraffin-embedded gingival biopsies from young and aging healthy subjects. The expression of the main genes and proteins involved in collagen turnover were determined by real time PCR, dot blot and SDS-zymography on cultured young and aging gingival fibroblasts, and after cyclosporin A administration. RESULTS: Our results suggest that in healthy aged people, gingival connective tissue is characterized by a similar collagen content and turnover. Collagen turnover pathways are similarly affected by cyclosporin A treatment in young and aging gingival fibroblasts. CONCLUSIONS: Cyclosporin A administration affects gingival collagen turnover pathways in young and aging fibroblasts at the same extent, suggesting that during aging cyclosporin A administration is not related to relevant collagen turnover modifications.

Early epidermal response after a single dose of gamma-rays in organotypic culture of human breast skin.

BACKGROUND: Skin reaction is the most common side-effect of radiation therapy. Radiation-induced dermal fibrosis has been characterized histologically, but little is known about the epidermis overlying fibronecrotic lesions. OBJECTIVES: To characterize the epidermal response 24 h after a single clinically relevant dose of gamma-rays in cultured human breast skin. METHODS: Biopsies obtained from cosmetic surgery (n = 7) were placed epidermis upwards in a Transwell system, and were exposed to a single dose of gamma-irradiation (2 Gy). A parallel set of nonirradiated skin fragments was incubated under the same conditions. Both irradiated and nonirradiated fragments were harvested 24 h after irradiation and processed for light microscopy and molecular biology analysis. A quantitative analysis of cell proliferation was performed after 5-bromo-2'-deoxyuridine incorporation. Cytokeratin 10 (CK10) and desmocollin 1 (Dsc1) expression was evaluated by immunofluorescence. Dsc1 and transforming growth factor (TGF)-beta1 gene expression was measured by reverse transcriptase-polymerase chain reaction analysis. RESULTS: The mean percentage inhibition of epidermal proliferation in irradiated samples was 53.7% (P < 0.01, paired Student's t-test). The inhibition of cell proliferation was significant in five of seven samples (P < 0.05, unpaired Student's t-test). Normal cell architecture was found in irradiated samples. Throughout the epithelial compartment, the distribution patterns of CK10 and Dsc1 were comparable in nonirradiated and irradiated fragments. Condensation of CK10 filaments suggested a cytoskeletal rearrangement in irradiated samples. Dsc1 and TGF-beta1 mRNA levels were, respectively, reduced and unmodified 24 h after irradiation. CONCLUSIONS: A perturbation of epidermal homeostasis occurs as early as 24 h after a single dose of gamma-rays. Our immunofluorescence observations indicate that keratinocyte terminal differentiation is not yet affected at the protein level 24 h after exposure to gamma-rays. The lack of an inverse relationship between TGF-beta1 gene expression and epidermal proliferation, together with decreased Dsc1 gene expression, may represent the early molecular basis for the development of the late effects of radiotherapy observed many months/years after radiotherapy. Our findings set the stage for further investigation of the best time to begin topical treatment at the start of radiation therapy.

Appendicitis in children.

A study of 406 consecutive children operated upon for appendicitis from July 1982 to July 1987 was compared with a previously published study of 657 children with the same diagnosis operated upon between 1972 and 1982. This was done to determine if the methods of therapy continue to yield low complication rates and zero mortality rates. The routine use of antibiotic coverage for both aerobic and anaerobic bacteria in perforated appendicitis resulted in low complication rates, 3.2 per cent for major and 2.5 per cent for minor complications. Major complications included small intestinal obstruction and intra-abdominal abscess. Minor complications included wound infection and prolonged ileus. These rates are similar to those of the first investigation. The mortality rate continued to be zero. Complete peritoneal lavage was used in patients with generalized peritonitis or extensive localized peritonitis. Operative lysis of adhesions for small intestinal obstruction was required in four of these patients. This did not occur in patients with perforated appendicitis with abscess formation or more localized peritonitis who had no lavage. The technique rather than the disease process may be responsible for the complication.

Changes in expression of the albumin, fibronectin and type I procollagen genes in CCl4-induced liver fibrosis: effect of pyridoxol L,2-pyrrolidon-5 carboxylate.

The protective activity of pyridoxol L,2-pyrrolidon-5 carboxylate (metadoxine) was investigated in a rat model of carbon tetrachloride (CCL4)-induced hepatic fibrosis. After 6 weeks of CCl4 treatment, the animals developed fibrosis and inflammation of the liver while those treated with CCl4 + metadoxine had less severe lesions (P < 0.05). Since in liver fibroplasia there are quantitative changes of the extracellular matrix components and almost invariably a decrease in albumin synthesis, we have also investigated by Northern blot analysis the expression of the cellular fibronectin, pro-alpha 2(I)collagen and albumin genes. There were striking increases in fibronectin and pro-alpha 2(I)collagen mRNA contents in the livers of CCL4-treated animals and these enhancements were less evident in the metadoxine-treated rats. In contrast, albumin mRNA levels, almost identical in control and metadoxine-treated rats, were lower in the CCl4-treated animals. These data suggest that metadoxine might slow the development of CCl4-mediated liver fibrosis.