Mechanisms of carcinogenicity/chemotherapy by O6-methylguanine

Alkylating agents are a structurally diverse group of compounds that cause a wide range of biological effects, including cell death, mutation and cancer. DNA damaged by these agents contains widely different amounts of 12 alkylated purines/pyrimidines and two phosphotriester isomers. The biological effects appear to be mediated predominantly by attack at the O(6) position of guanine. DNA extracted from various normal human tissues contains detectable levels of O(6)-alkylguanine, the source of which has not been defined. Given that, following DNA replication, this lesion cannot only generate point mutations but can also initiate mismatch repair-mediated DNA recombination and cell death, it seems worthwhile to consider the possible contribution of these events and cell killing to the aetiology of human cancer. There is increasing evidence that point mutations are not the only mechanism involved in malignant transformation by alkylating agents. Some cancer chemotherapeutic agents exploit the cytotoxic effects of O(6)-alkylguanine and an understanding of the processing of this lesion has allowed strategies to be developed that should increase the effectiveness of such agents.     

Interleukin-7-engineered mesenchymal cells: in vitro effects on naive T-cell population.

T-cell homeostasis is regulated by several molecules; among these, interleukin (IL)-7 plays an essential role in the survival and homeostatic proliferation of peripheral naive T cells. In a previous study, we investigated whether human mesenchymal stromal cells (MSCs) could be engineered with the IL-7 gene to produce functional level of this cytokine. In the present study, we analyzed the impact of different quantities of IL-7 produced by MSCs on the survival and proliferation of a negative immunoselected naive (CD3(+)/CD45RA(+)) T-cell population. Co-cultivation of peripheral naive T cells with MSCs producing low (16 pg/mL) or high (1000 pg/mL) IL-7 levels or in the presence of exogenous IL-7 (0.01 ng/mL and 100 ng/mL) maintained the CD3(+)/CD45RA(+) naive T-cell phenotype. Chemokine receptor CCR7(+) expression was also maintained among this T-cell population. Naive T-cell molecular characteristics were maintained as assessed by the Vbeta spectratyping complexity score, which showed the maintenance of a broad T-cell repertoire. No Th1 or Th2 differentiation was observed, as assessed by interferon-gamma or IL-4 accumulation. In contrast, only MSCs producing high amounts of IL-7 caused increased activation (CD25 31.2% +/- 12% vs 10% +/- 3.5%; P < .05), proliferation (CD71 17.8+/-7% vs 9.3%+/-3, P < .05), apoptosis (assessed by annexin V: 18.6% +/- 5% vs 14.9% +/- 2.6%; P > .05), and the phase S cell cycle (15% vs 6.9%, P > .05). Exogenous IL-7 exhibited no significant effect. In conclusion, we demonstrated that IL-7 produced by MSCs has a dose-independent effect on naive T-cell survival while exerting a dose-dependent effect on activation/proliferation. Due to the continuous production of IL-7 by engineered cells, our system is more efficacious than exogenous IL-7.     

Treg-Cell Control of a CXCL5-IL-17 Inflammatory Axis Promotes Hair-Follicle-Stem-Cell Differentiation During Skin-Barrier Repair

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Good Safety Profile and Efficacy of Leucocyte Interferon-α in Combination with Oral Ribavirin in Treatment-Naive Patients with Chronic Hepatitis C

Background: The differential tolerability profile of various interferon (IFN)-α preparations used in combination with ribavirin for the treatment of chronic hepatitis C needs to be elucidated. Approximately 8% of patients receiving recombinant IFNα-2b plus ribavirin discontinue treatment because of adverse events. Human leucocyte IFNα is deemed to have a better safety profile than recombinant IFNα. We therefore compared the safety profile and efficacy of ribavirin combined with leucocyte IFNα or with recombinant IFNα-2b in treatment-naive patients with chronic hepatitis C. Study design: We randomised 423 patients to either leucocyte IFNα 3MU three times weekly plus ribavirin (210 patients) or the same dose of recombinant IFNα-2b plus ribavirin (213 patients). Patients were treated for 24 weeks and followed-up for a further 48 weeks. The primary endpoint was the safety profile of the two therapies; the secondary endpoint was the rate of sustained response. Results: In patients receiving leucocyte IFNα, the total number of adverse events was lower than in the group receiving recombinant IFNα (259 vs 441 patients), and the percentage of patients discontinuing treatment because of adverse events or laboratory abnormalities was significantly reduced (4% vs 11%; p = 0.013). Sustained response was observed in 47% of patients receiving leucocyte IFNα plus ribavirin and in 44% of patients receiving IFNα-2b plus ribavirin. Conclusions: Both therapeutic regimens were effective in inducing a sustained response in naive patients. However, the safety profile of leucocyte IFNα plus ribavirin was more favourable than that observed with the administration of recombinant IFNα-2b plus ribavirin, suggesting that leucocyte IFNα may be an alternative option in patients with reduced tolerability to other IFNs.     

Ultrastructural Features of Neuroendocrine Differentiated Carcinomas of the Breast

The ultrastructural patterns of neuroendocrine (NE) differentiated breast carcinomas are analyzed and discussed. Reports in the literature describe wide variations in the size of observed dense-core membrane-bound granules and discrepancies in their interpretation. In the present study 24 cases of breast carcinoma with recognized morphologic, histochemical, and immunocytochemical features of NE tumors were investigated. Five different types of dense-core granules of neurosecretory (NS) type (confirmed by the ultrastructural localization of chromogranin A) and five different cell types were recognized. Some amphicrine cells were found to contain both mucin and NS granules. Another notable ultrastructural feature of breast NE carcinomas was the presence of clear vesicles of presynaptic type, which correlated with expression of synaptophysin.     

Regulation of human erythrocyte metabolism by insulin: cellular distribution of 6-phosphofructo-1-kinase and its implication for red blood cell function

Human erythrocytes are highly specialized cells whose function is oxygen transport. These cells' sole metabolic source of energy is the fermentation of glucose via glycolysis. They contain an active insulin receptor and respond to insulin by increasing phosphorylation of tyrosine residues in several proteins. However, no metabolic effects have yet been associated with activation of this receptor in human erythrocytes. Here, we show that insulin increases the rate of glycolysis in human erythrocytes. Lactate production increased 56 and 173% in the presence of 10 and 100 nM insulin, respectively. A higher insulin concentration (1000 nM) partially reversed the stimulation of glycolysis. These effects occur through activation of the key glycolytic enzyme 6-phosphofructo-1-kinase, which exhibits the same pattern of modulation by insulin as seen for glycolytic flux. This modulation also occurs physiologically since ex vivo experiments revealed 50% stimulation of 6-phosphofructo-1-kinase (PFK) activity following a high carbohydrate meal. Insulin increases phosphorylation of PFK and redistributes the enzyme in red blood cells, causing it to detach from the erythrocyte membrane: upon insulin stimulation, the amount of enzyme associated with the plasma decreases by 86%. Detachment is a common mechanism of enzyme activation. As a consequence, insulin prevents up to 68% of red cells hemolysis. These results show that insulin regulates erythrocyte glycolysis and viability and suggest that this regulation is associated to other erythrocyte functions such as oxygen transport. Finally, we suggest that this regulatory mechanism might be compromised in patients with diabetes mellitus.     

Fibrin-polyurethane composites for articular cartilage tissue engineering: a preliminary analysis

In this study we investigated the use of a fibrin hydrogel to improve the potential of a polyurethane (PU) scaffold-based system for articular cartilage tissue engineering. PU-only ("no-fibrin") and PU-fibrin ("fibrin") composites were cultured for up to 28 days and analyzed for DNA content, glycosaminoglycan (GAG) content, type II collagen content, GAG release, and gene expression of aggrecan, collagen I, and collagen II. The use of fibrin allowed for higher viable cell-seeding efficiency (10% higher DNA content on day 2 in fibrin versus no-fibrin composites) and more even cell distribution on seeding, a more than 3-fold increase in the percentage of newly synthesized GAG retained in the constructs, and 2- to 6-fold higher levels of type II collagen and aggrecan gene expression through day 14. Addition of aprotinin to the medium inhibited fibrin degradation, most noticeably in the center of the constructs, but had little effect on biochemical composition or gene expression. Short-term mechanical compression (0-10% sinusoidal strain at 0.1 Hz for 1 h, applied twice daily for 3 days) doubled the rate of GAG release from the constructs, but had little effect on gene expression, regardless of the presence of fibrin. Although further work is needed to optimize this system, the addition of fibrin hydrogel to encapsulate cells in the stiff, macroporous PU scaffold is a step forward in our approach to articular cartilage tissue engineering.     

Chemokines and pro-inflammatory cytokines in Down's syndrome: an early marker for Alzheimer-type dementia?

BACKGROUND: People with Down's syndrome (DS) show early Alzheimer-like dementia. It has been suggested that the pro-inflammatory cytokine class plays a role in Alzheimer's disease (AD). The study aims at verifying whether pro-inflammatory cytokines in DS are correlated with age, affective symptoms and intellectual decline to a different degree than in subjects with non-DS learning disabilities. METHODS: Cases: 19 subjects with DS; controls: sex- and age-matched individuals with learning disabilities caused by perinatal ischaemic damage. The level of mental retardation was assessed according to DSM-IV; psychopathological symptoms were measured by the Assessment and Information Rating Profile. Serum levels of cytokines were determined with ELISA. RESULTS: DS patients showed higher levels of cytokines and chemokines, with the exception of RANTES; but the only significant difference detected was for MIP-1alpha. A correlation between the degree of mental retardation and IL-6, and between MIP-lalpha and age was found in patients with DS, but not in controls. CONCLUSIONS: The data obtained suggest a possible involvement of chemokines in the inflammatory and degenerative processes similar to AD in DS. Further longitudinal research is required to confirm these findings.