Promoter methylation controls the expression of MAGE2, 3 and 4 genes in human cutaneous melanoma

Cancer-testis antigens expressed by different-histotype transformed cells are suitable targets for tumor immunotherapy. However, their heterogeneous expression in neoplastic lesions limits the eligibility of patients for cancer-testis antigen-directed vaccination, and low levels of cancer-testis antigens' expression may impair immune recognition of malignant cells. Because of the primary clinical relevance of cancer-testis antigens' expression in neoplastic tissues, 68 unrelated or sequential metastatic lesions from 56 patients were used to characterize the molecular mechanisms regulating the presence and levels of expression of different cancer-testis antigens of the MAGE family (i.e., MAGE2, 3 and 4) in cutaneous melanoma. Polymerase chain reaction-based methylation analyses showed that methylation status of specific cytosine-guanine dinucleotides in the promoters of investigated cancer-testis antigens correlated with their heterogeneous expression within unrelated metastatic melanoma lesions, and with their homogeneous expression among sequential metastases from three patients with melanoma. Unlike methylated promoters, unmethylated promoters of MAGE2, 3 and 4 genes drove the expression of reporter gene-enhanced green fluorescent protein after transient transfection of cancer-testis antigen-positive Mel 142 melanoma cells. Furthermore, de novo expression of MAGE3 gene induced by the treatment of Mel 195 melanoma cells with the DNA hypomethylating agent 5-aza-2'-deoxycytidine was associated with a 6%-12% demethylation of selected cytosine-guanine dinucleotides in its promoter. Finally, 5-aza-2'-deoxycytidine induced a 16-fold increase of MAGE3 expression in Mel 313 melanoma cells expressing constitutively low levels of the antigen, but did not affect that of Mel 275 melanoma cells expressing high baseline levels of MAGE3. Overall, these findings identify promoter methylation as a shared mechanism directly regulating the expression of therapeutic cancer-testis antigens in metastatic melanomas, and foresee the clinical use of 5-aza-2'-deoxycytidine to design new chemoimmunotherapeutic strategies in patients with melanoma.     

Uptake of alpha-tocopherol by the mammary gland but not by white adipose tissue is dependent on lipoprotein lipase activity around parturition and during lactation in the rat

This study was undertaken to test the potential role of changes in lipoprotein lipase (LPL) activity in the mammary gland and adipose tissue around parturition and lactation on the uptake of alpha-tocopherol in the rat. Plasma levels of alpha-tocopherol and triglycerides were higher in 20-day pregnant rats than in virgin rats, whereas its concentration was higher in the mammary gland of the former, and no differences were detected in adipose tissue between the groups. After an oral alpha-tocopherol and triglyceride load, both appeared in plasma faster in pregnant rats than in virgin rats, the change being even faster for alpha-tocopherol than for triglycerides. After 24 hours, both alpha-tocopherol and triglycerides in d < 1.006 lipoproteins were higher in pregnant rats than in virgin rats, LPL activity was higher in the mammary gland, and lower in adipose tissue in the former, whereas alpha-tocopherol concentration also appeared higher in the mammary gland of pregnant rats, and no differences were detected between the groups in adipose tissue. At day 13 of lactation, an oral load of alpha-tocopherol and triglycerides caused a higher increase of plasma alpha-tocopherol levels than triglycerides, and this effect decreased when rats had their litter removed 48 hours before analysis. In these litter-removed rats, the appearance of alpha-tocopherol and triglycerides in plasma was higher in d < 1.006 lipoproteins than in lactating rats. Also, both LPL activity and alpha-tocopherol concentration in the mammary gland plus milk was lower in litter-removed rats than in the lactating rats, whereas LPL in adipose tissue was higher in the former, although no difference in alpha-tocopherol was found. Thus, data are consistent with the role of LPL activity in the mammary gland modulating the uptake of alpha-tocopherol during pregnancy and lactation, although this is not true in adipose tissue.     

Corneal flap complications in refractive surgery: Part 1: development of an experimental animal model

PURPOSE: To report the outcome, learning curve, and complication rates of an experimental animal model for corneal flaps in refractive surgery. SETTING: Magill Research Center for Vision Correction, Storm Eye Institute, Charleston, South Carolina, USA. METHODS: Corneal flaps with a nasal or a temporal hinge were created in 190 eyes of 95 Dutch Belted rabbits using the Automated Corneal Shaper microkeratome (Bausch & Lomb Surgical). Diffuse lamellar keratitis (DLK) was induced by inoculating the corneal interfaces with 1 of 7 substances. Postoperatively, the eyes were examined with a slitlamp. Special emphasis was placed on corneal flap complications and the relationship between slipped flaps and hinge position and/or inoculation agent. RESULTS: A good corneal flap was achieved in 174 eyes (92%). The eyes with a nasal hinge had a lower incidence of slipped flaps (14%) than eyes with a temporal hinge (37%) (P =.02). CONCLUSION: With the animal model described, corneal flaps were created in a precise and reproducible way in more than 90% of eyes. Nasal hinged flaps showed less postoperative displacements than temporal hinged flaps and are adequate for further study.     

Estrogen blunts neuroendocrine and metabolic responses to hypoglycemia

This study tested the hypothesis that estrogen is the mechanism responsible for the sexual dimorphism present in the neuroendocrine and metabolic responses to hypoglycemia. Postmenopausal women receiving (E2; n = 8) or not receiving (NO E2; n = 9) estrogen replacement were compared with age- and BMI-matched male subjects (n = 8) during a single-step 2-h hyperinsulinemic-hypoglycemic clamp. Plasma insulin (599 +/- 28 pmol/l) and glucose (2.9 +/- 0.03 mmol/l) levels were similar among all groups during the glucose clamp. In response to hypoglycemia, epinephrine (2.8 +/- 0.6 vs. 5.8 +/- 0.8 and 4.4 +/- 0.5 nmol/l), glucagon (57 +/- 8 vs. 77 +/- 8 and 126 +/- 18 ng/l), and endogenous glucose production (2 +/- 2 vs. 10 +/- 2 and 6 +/- 3 micro mol x kg(-1) x min(-1)) were significantly lower in E2 vs. both NO E2 and male subjects (P < 0.05). These reduced counterregulatory responses resulted in significantly greater glucose infusion rates (16 +/- 2 vs. 6 +/- 2 and 6 +/- 3 micro mol x kg(-1) x min(-1); P < 0.01) in E2 vs. both NO E2 and male subjects. Pancreatic polypeptide was significantly lower (P < 0.05) in both the E2 and NO E2 groups compared with the male subjects (136 +/- 20 and 136 +/- 23 vs. 194 +/- 16 pmol/l). Last, glycerol (36 +/- 3 vs. 47 +/- 5 micro mol/l; P < 0.05), lactate (1.4 +/- 0.1 vs. 1.8 +/- 0.2 mmol/l; P < 0.05), and muscle sympathetic nerve activity (19 +/- 4 to 27 +/- 4 vs. 27 +/- 5 to 42 +/- 6 bursts/min; P < 0.05) responses to hypoglycemia were all significantly lower in E2 vs. NO E2 subjects. We conclude that estrogen appears to play a major role in the sexual dimorphism present in counterregulatory responses to hypoglycemia in healthy humans.     

Cortisol acts through central mechanisms to blunt counterregulatory responses to hypoglycemia in conscious rats

Physiological levels of cortisol have been found to blunt neuroendocrine and metabolic responses to subsequent hypoglycemia in humans. The aim of this study was to determine whether cortisol acts directly on the brain to elicit this effect. A total of 41 conscious unrestrained Sprague-Dawley rats were studied during 2-day experiments. Day 1 consisted of two episodes of clamped 2-h hyperinsulinemic (30 pmol. kg(-1) x min(-1)) hypoglycemia (2.8 +/- 0.1 mmol/l; n = 12; ANTE HYPO), euglycemia (6.2 +/- 0.1 mmol/l; n = 12; ANTE EUG), or euglycemia (6.2 +/- 0.1 mmol/l) plus simultaneous intracerebroventricular (ICV) infusion of cortisol (25 microg/h; n = 9; ANTE EUG+Cort) or saline (24 microl/h; n = 8; ANTE EUG+Sal). For all groups, day 2 consisted of a 2-h hyperinsulinemic (30 pmol x kg(-1) x min(-1)) hypoglycemic (2.9 +/- 0.2 mmol/l) clamp. Plasma epinephrine and glucagon incremental area under the curve (Delta AUC) responses were significantly less in ANTE EUG+Cort and ANTE HYPO versus both ANTE EUG and ANTE EUG+Sal (P < 0.05). The Delta AUC responses of plasma norepinephrine were significantly lower in ANTE EUG+Cort versus both ANTE EUG and ANTE EUG+Sal (P < 0.05). Endogenous glucose production was significantly less in ANTE HYPO and ANTE EUG+Cort versus the other groups (P < 0.05). Lastly, the glucose infusion rate to maintain the desired hypoglycemia was significantly greater in ANTE EUG+Cort and ANTE HYPO versus the other two groups (P < 0.05). In summary, ICV infusion of cortisol significantly blunted norepinephrine, epinephrine, glucagon, and endogenous glucose production responses to next-day hypoglycemia. We conclude that cortisol can act directly on the central nervous system to blunt counterregulatory responses to subsequent hypoglycemia in the conscious rat.     

Retroperitoneal abscesses--analysis of a series of 66 cases

OBJECTIVE: To analyze our experience with the management of retroperitoneal abscesses. PATIENTS AND METHODS: A retrospective study was made of 66 patients with retroperitoneal abscesses treated at our hospital from January 1975 to July 2001 for the purpose of analyzing the diagnosis and treatment of these rare infections. In each case, we analyzed patient characteristics, abscess location and origin, predisposing factors, clinical presentation, microbiology, radiographic findings, treatment, and outcome. RESULTS: In our series, the most frequent type of abscess was perinephric (45.4%), and the most frequent origin was the kidney (72.7%), generally renal lithiasis or previous urological surgery. Gram-negative bacilli were the microorganisms most often involved as causal agents of abscesses. CT had the best diagnostic performance (95%). Percutaneous drainage resolved the abscess in 86.3% of the patients in which it was used, compared with 87.5% for traditional surgical drainage. In 4 cases, the only treatment was administration of antibiotics. In all these cases the abscesses were smaller than 3 cm and patients were in good general condition. The mortality rate was excellent (1.5%), probably due to the low rate of comorbidity in our patients. CONCLUSIONS: Gram-negative bacilli were the most frequent microorganisms in our retroperitoneal abscesses. CT was the imaging technique that produced the most reliable and rapid diagnosis. Radiographically-guided percutaneous drainage was a safe and effective therapeutic alternative when used as definitive treatment or preoperatively.     

Radiodermatitis. Effectiveness of a collagen dust healing product

Injuries caused by radio-therapy prove difficult to cure. Catrix is a healing product composed of a collagen particle base having a proven effectiveness in the treatment of various kinds of ulcers. In a study on 33 patients who received radio-therapy and who suffered from injuries caused by roentgen dermatitis, this product succeeded in curing 100% of these ulcers, demonstrating great speed in producing an effect, while the average treatment duration was 5 days. There were no adverse reactions. Therefore, this product received positive marks from both professionals and patients.