Interleukin-6 release in the hepatic blood outflow during normothermic liver ischaemia in humans.

BACKGROUND: Liver surgery techniques have consistently improved and normothermic ischaemia of the liver is considered to be a safe procedure to reduce intraoperative haemorrhage. Hepatic failure, however, remains a significant complication. In liver ischaemia-reperfusion injury, cytokines play a key proinflammatory role. Cytokines may be part of the intercellular signalling that leads to recovery or to failure after major surgery. Moreover, they could be potential predictors of the outcome. Modulation of the pattern of cytokine response in the early postsurgery period could represent a new approach to minimise the impact of these procedures. AIMS: The aim of our study was to analyse the cytokine pattern in the hepatic blood outflow in patients undergoing surgical intervention of partial liver resection with clamping of the hepatic pedicle and liver ischaemia, and to correlate the cytokine behaviour with clinical parameters. PATIENTS: We studied eight patients (mean age 55 years) who underwent surgical intervention of liver resection during vascular exclusion of the hepatic pedicle. Patients were monitored for haemodynamic and haematological parameters during the pre-, infra- and postoperative period. METHODS: IL-I alpha, IL-6, TNF-alpha and IFN-gamma were assayed from peripheral and central vein blood at different times. Blood samples for cytokine assays were also drawn from the supra-hepatic veins after clamping of the porta hepatis. RESULTS: We found a significant increase of the IL-6 levels in the supra-hepatic samples during liver ischaemia, while the trend with IL-1alpha was less clear; IFN-gamma and TNF-alpha were undetectable with the methods used. IL-6 levels appeared to correlate positively with bilirubin and gamma-GT levels and negatively with the degree of acidosis. CONCLUSIONS: Our study confirms that during surgical ischaemic stress there is an increase of IL-6 serum levels more relevant in supra-hepatic vein blood. Cytokines could contribute to modulate the inflammatory response to liver ischaemia.

Expression and DNA binding activity of the Ku heterodimer in bladder carcinoma.

BACKGROUND: The Ku protein is a tightly associated heterodimer, comprised of 70-kilodalton (kD) and 86-kD subunits, that forms the DNA-dependent protein kinase (DNA-PK) complex together with the 470-kD DNA-PKcs catalytic subunit, and is involved mainly in DNA double-strand breaks (DSBs) repair. The objective of the current study was to investigate the expression and DNA-binding activity of the Ku protein in fresh tissues from patients with bladder carcinoma and to compare it with that in nontumor tissues obtained from the same organ. Moreover, the DNA-binding activity of Ku was assessed after exposure of the tumor cells to 1 or 2 grays (Gy) of X-rays. Furthermore, the level of phosphorylated Ku was analyzed in both the nuclear and cytoplasmic compartment of normal tissue after exposure to 2 Gy of X-rays. METHODS: The expression and DNA-binding activity of Ku protein were assessed in tumor samples from patients who all were diagnosed with transitional cell carcinoma (TCC) of the bladder using Western blot analysis and the electrophoretic mobility shift assay, respectively. RESULTS: Enhanced Ku activity and expression were found in tumor tissue compared with normal tissue for each patient. Moreover, variations in Ku activity were found in a dose-dependent manner after the tumor cells were exposed to 1 or 2 Gy of X-rays. A decrease in phosphorylated Ku in the cytoplasm and a parallel increase in the nucleus of normal tissue cells were observed after exposure to X-rays. CONCLUSIONS: The results of the current study suggest a possible role of Ku in regulating the DNA-PK activity of DSBs repair in bladder tumors.

Role of DNA-dependent protein kinase in recognition of radiation-induced DNA damage in human peripheral blood mononuclear cells.

The DNA-dependent protein kinase (DNA-PK) complex plays a crucial role in radiation-induced DNA damage recognition. The complex includes the ku heterodimer, which comprises ku 70 and ku 80 subunits, that binds DNA termini of breaks without sequence specificity, and the catalytic subunit DNA-PKCS: The activation of the DNA-PK complex was studied in X-irradiated peripheral blood mononuclear cells (PBMC) from subjects of different ages. Radiation-induced changes in the DNA-binding activity of the ku heterodimer, and in the concentrations of ku 70, ku 80, DNA-PKcs and phosphorylated ku 80 were determined in nuclear and cytoplasmic extracts. DNA-binding activity was increased by irradiation only in the nuclear extract of PBMC from young, but not from elderly subjects, whereas it was found unchanged in cytoplasmic extracts regardless of age. The radiation-induced activation of the DNA-PK complex may result from the increased concentrations of ku 80 and DNA-PKcs in the cytoplasm of PBMC from young, but not from elderly subjects, leading to a higher concentration of phosphorylated ku 80 which readily migrates to the nucleus where, after dimerization with ku 70, binds to DNA breaks. These findings suggest major steps involved in DNA-PK activation, and the intracellular and molecular changes that may account for the age-dependent impairment of DNA repair capacity in irradiated mammalian cells.

Resynchronization of blood pressure circadian rhythm after westward trans-7-meridian flight with and without melatonin treatment.

Blood pressure (BP) of a healthy 37-yr-old male traveling from Milan to Houston was monitored for 36 h before the flight and continued for 5 d after the arrival. The rhythmometric analysis of BP data was made to investigate the rate of adaptation to a rapid rest-activity cycle shift. Since two trips were evaluated, during the second one the subject took melatonin (3 mg) before the nocturnal rest. In the first trip the BP circadian rhythm synchronization occurred on the 5th day. In the second trip melatonin promoted an immediate but unstable adaptation to the new rest-activity cycle.

Glutathione circadian rhythm in duodenal mucosa of fasted rats.

Duodenal ulcer is a relevant clinical condition, but information about circadian rhythms of the duodenum is scarce. While the chronobiological model of gastric ulcerogenesis has been partly demonstrated, the time dependent variations of protective and aggressive factors of the duodenal mucosa have not yet been sufficiently documented. The aim of this study is to document the duodenal mucosa circadian dynamics of glutathione (GSH), a sulfhydryl compound, a well-known intracellular protective agent. Male Wistar rats, 8-9 weeks of age, were maintained at controlled temperature and humidity and 12L:12D day cycle since birth. Duodenal mucosa samples obtained by scraping were assayed with a spectrophotometric method. The data analysis with the Cosinor method demonstrated circadian rhythmicity for GSH content in the duodenal mucosa with higher levels during the dark period.

Modulation of X-ray-induced damage recognition and repair in ageing human peripheral blood mononuclear cells by an interleukin-6-type cytokine.

We have investigated the effects of an interleukin (IL)-6-type cytokine on the DNA-binding activity of ku and on unscheduled DNA repair in X-ray-treated peripheral blood mononuclear cells (PBMC) from human subjects of different ages. The cytokine used, called K-7/D-6, is an IL-6 variant with increased in vivo and in vitro biological activity compared to the wild type molecule. Ku is the DNA-binding component of the DNA-dependent protein kinase (DNA-PK). It binds the ends of various types of DNA discontinuity and is involved in the repair of DNA breaks caused by V(D)J recombination, isotype switching, physiological oxidation reactions, ionizing radiation and some chemotherapeutic drugs. The ku-dependent repair process, called non-homologous end joining, is the main DNA double strand break repair mechanism in irradiated mammalian cells. Results show that K-7/D-6 significantly increases DNA-binding activity of ku in irradiated PBMC from young but not from elderly subjects. However, K-7/D-6 is able to induce unscheduled DNA repair in irradiated PBMC from both young and elderly subjects. These effects of K-7/D-6 are relevant to the mechanisms of the cellular response to DNA damage.

Effect of age on DNA binding of the ku protein in irradiated human peripheral blood mononuclear cells (PBMC).

DNA binding of the ku protein was investigated in peripheral blood mononuclear cells (PBMC) from 24 subjects of different ages (20-89 years old) displaying age-related changes in DNA repair, mitotic responsiveness, and cytokine production. Ku is an heterodimeric protein composed of two subunits of 70 and 80 kDa, which is involved in the earliest steps of DNA damage recognition. DNA binding of ku 70/80 was found unchanged in normal PBMC from aging subjects but progressively declined in x-ray-irradiated PBMC from young to adult, and elderly subjects. This finding was concomitant with the age-related fall of DNA repair in the whole population.

Characteristics of gastric-vein lymphocytes with regard to the immune response to Helicobacter pylori.

BACKGROUND: Between peripheral blood and tissue-infiltrating lymphocytes there is an intermediate compartment, the blood of the organ-draining vessels, which could show unusual features. The aim of the present study was to analyse the characteristics of the lymphocytes from the stomach-draining vessels and the cytokine secretion by these lymphocytes. The CagA-mediated lymphocyte activation in Helicobacter pylori-infected subjects and the humoral response to this antigen were evaluated and correlated with clinical data. METHODS: We studied lymphocyte proliferation either with mitogens or with the CagA antigen and cytokine production and IgG anti-CagA by means of an enzyme-linked immunosorbent assay in peripheral blood and gastric-vein blood obtained during surgical intervention. RESULTS: We showed higher proliferative response and cytokine production in lymphocytes from the gastric vein. The mitogenic response to the CagA antigen was highly specific but poorly sensitive for the H. pylori infection in both the compartments. The overall cytokine profile in our patients affected by non-ulcer disease was of the Th0 type. CONCLUSIONS: Gastric-vein-derived lymphocytes seem to show unusual features, as they behave like peripheral blood lymphocytes but show higher responses to all the tested stimuli. It is possible that the interaction of the lymphocytes with the mucosal environment could activate the synthetic mechanisms, making the cells more 'responsive' to the stimulation. The CagA antigen is able to induce a specific T-lymphocyte response and is therefore a valid candidate antigen for the development of a vaccine.

Haemopoietic reconstitution after sublethal irradiation: comparison of the effects of different haemopoietic cytokines on murine lymphocytes and bone marrow cells.

Sublethally irradiated mice were injected with recombinant cytokines to stimulate haemopoietic reconstitution. Interleukin (IL)-11 and IL-6 were able to significantly accelerate the recovery of thymus, spleen and bone marrow cells when used in combination with IL-3, but not alone. Stem cell factor (SCF) also displayed detectable effects when used with IL-3. Conversely, the IL-6 superagonist K-7/D-6 was able, even when used alone, to induce recovery of thymus, spleen and bone marrow cells up to the level of unirradiated controls. Together, these results indicate that it is possible to attain complete recovery of lymphoid organs and tissues as early as 7 d after irradiation by use of haemopoietic cytokines.

Activation of gp130 signaling in vivo by the IL-6 super-agonist K-7/D-6 accelerates repopulation of lymphoid organs after irradiation.

Stimulation of the gp130 signaling pathway by IL-6 is known to contribute significantly to hematopoietic expansion in vitro, mostly in combination with other cytokines. In the present study we have investigated whether a similar effect can be observed also in vivo using short-term assays in which irradiated mice were analyzed for repopulation of lymphoid organs. Mice were injected with a combination of soluble IL-6Ralpha either with wild-type (wt) human IL-6 or with an IL-6 variant, called K-7/D-6, that shows a 70-fold higher IL-6Ralpha affinity. We observed that while wt IL-6 was able to induce a partial effect only in combination with IL-3, K-7/D-6 bypassed the need for IL-3 and yielded complete recovery. In lethally irradiated mice reconstituted with syngeneic bone marrow cells K-7/D-6 strongly accelerated the repopulation of thymus and spleen and hastened blood neutrophil recovery. These results underscore the potential of the gp130 signaling pathway in hematopoietic reconstitution after myeloablative regimens and open the possibility to fully exploit it with a super-active IL-6 variant.

Cell proliferation and ku protein expression in ageing humans.

Previous studies on DNA repair in ageing have demonstrated increased frequencies of single and double strand breaks in lymphocytes from elderly subjects and, as a consequence, decreased efficiency in DNA replication. We have investigated the relationship between cell proliferation and the nuclear expression of ku protein in a human population of 43 subjects of different ages. Ku is an heterodimeric protein composed of two subunits of 70 and 80 kDa, which is involved in the early steps of DNA damage recognition. In the present study, PBL from subjects of different ages were PHA-activated to evaluate the stimulation index and the production of Th1- and Th2-type cytokines. Moreover, nuclear extracts were obtained from activated lymphocytes to evaluate by a gel retardation assay the presence and the functional activity of the heterodimer ku 70/80. Our results indicate that ageing affects the mitotic responsiveness and cytokine production to a significant extent, but only marginally the expression of ku 70/80. These findings suggest that the age-related impairment in DNA repair mechanisms are only in part related to the reduced expression of ku protein able to recognize DNA damage.

Regulation of cytokine production in aging: use of recombinant cytokines to upregulate mitogen-stimulated spleen cells.

We investigated the production of IL-2 and IFN-gamma (Th1 type) and IL-4 (Th2 type) cytokines by mitogen-activated spleen cells from young, adult and old mice. Cytokine production was evaluated in culture supernatants by CTLL proliferation (IL-2), ELISA (IFN-gamma), CT4.S proliferation (IL-4) and in mRNA extracted from activated CD4+ cells by RT-PCR (IL-2, IFN-gamma and IL-4). Results show that the production of IL-2, as protein and mRNA, is profoundly depressed by aging, whereas that of IFN-gamma, as protein and mRNA, firstly declines and then increases with age. The production of IL-4, as protein, monotonically declines with aging whereas, as mRNA, firstly decreases and then increases above the level in young mice. Spleen cells in culture were also incubated with mitogens and with a recombinant cytokine (IL-1 beta, IL-2, IL-3, IL-4, IL-12 or IFN-gamma) at various concentrations. It was found that recombinant cytokines by and large enhance cytokine production when the level induced by mitogens only is low. This conclusion applies to IL-2 and IFN-gamma production as protein and mRNA. The addition of recombinant cytokines also increases the production of IL-4 at the protein level in spleen cells from old mice but, at the mRNA level, only in spleen cells from young mice. This finding suggests age-related changes in IL-4-specific mRNA transcription rate and post-transcriptional half-life as well as translation kinetics.

Asynchrony in circadian rhythms of gastric function in the rat. A model for gastric mucosal injury.

A model for gastric mucosal injury is proposed in which a key pathogenetic event is the disruption in the normal relationships among several circadian rhythms of gastric function. In the rat a circadian rhythm in acid secretion was found to be out of phase with a circadian rhythm in gastric pepsin secretion, another aggressive factor, and several mucosal defensive factors (mucus and bicarbonate efflux and tissue prostacyclin content). Gastric corpus mucosal blood flow circadian patterns paralleled the the rhythmicity in acid secretion and, therefore, was out of phase with the other measured mucosal defensive factors. Thus, gastric mucosal defense was maintained by different mechanisms over the 24-hr cycle. During the dark phase, when this species was active and when acid secretion was highest, enhanced damage by topical acidified aspirin was documented, despite increased mucosal blood flow. Natural asynchrony in circadian rhythms of gastric function can be protective of gastric mucosal integrity but disruption of this circadian interplay of gastric aggressive and defensive factors could theoretically lead to greater vulnerability to damage. In the human, a circadian rhythm in basal gastric acidity has been described but no information exists as to the possibility of similar rhythmic variation in other gastric factors (aggressive and defensive) and possible disruption of these rhythms in disease.

Effect of constant light on rhythmic gastric functions in fasting rats.

The effects of four weeks of continuous illumination (LL), a subacute stress, on gastric mucosal endogenous aggressive and defensive factors were studied. Young male Sprague-Dawley rats were used with two different illumination regimens: LL and 12 hr light/12 hr dark (LD). At the end of three to four weeks of either regimen of illumination, gastric acid secretion, pepsin secretion, mucus secretion, and potential difference (PD) were studied. All gastric parameters, except mucus secretion, were significantly reduced by LL. The reduction in acid secretion (13.3%) was not significant after Bonferroni correction for the four t tests Pepsin secretion and PD were 27.9% and 24.6% less, respectively. These differences were significant after Bonferroni correction. The LD rats showed significant circadian rhythms for acid, mucus, and pepsin secretion. The LL rats showed significant rhythmicity for these same parameters with period lengths different from 24 hr. Gross inspection of the gastric mucosa indicated that 69.8% of the LL rats had lesion scores of 1.0 or higher, while none of the LD rats had scores above 0.5.

Circadian rhythm of pepsin efflux in the fasting rat stomach.

Gastric pepsin efflux, a putative aggressive factor because of its proteolytic activity, was examined to determine if it displays circadian rhythmicity as has been shown for other factors such as acid, bicarbonate, mucus, blood flow, potential difference, and tissue prostacyclin activity. Ninety-six fasted Sprague-Dawley male rats, 6-7 weeks of age were acclimated in sound-attenuating, light-proof chambers on a 12/12 light/dark schedule. They were studied in groups of 12 at 3-h intervals. After anesthesia and minor surgery, the stomach was cannulated and filled with 2 ml of saline for two sequential periods of 30 min. The samples were tested for pepsin according to the modified hemoglobin substrate colorimetric method. The data were analyzed with cosinor rhythmometric techniques. Pepsin efflux displayed significant (p < 0.05) circadian rhythmicity with an acrophase value or peak time at 06:49 h after lights on, during the lights-on resting phase. In contrast, the acrophase for acid secretion in the same model occurs during the dark period, when the rats are normally active. We postulate that differences in the circadian patterns of acid and pepsin may be protective.

Dynamics of nonprotein and protein sulfhydryl compounds in rat gastric mucosa: possible implication for cytoprotection and ulcerogenesis.

The hypothesis that nonprotein and protein sulfhydryls in gastric mucosa may play some role in the defensive and offensive processes of gastric epithelium was tested in this study in the intact rat gastric mucosa. Both sulfhydryl compounds presented statistically significant changes during the 24-hour day. The content of nonprotein sulfhydryls was less during the dark span than during the light span, while the circadian acrophase of protein sulfhydryls occurred during dark span. These results may offer a new interpretation of the greater vulnerability to ulcerogenic agents of the gastric mucosa of rats during their usual activity span.