Transcriptional regulation of the claudin-16 gene by Mg2+ availability.

BACKGROUND/AIMS: Renal tubular Mg(2+) reabsorption is mediated predominantly by the tight junction channel protein claudin-16 which is encoded by the gene CLDN16. Hypermagnesemia decreases, whereas hypomagnesemia increases Mg(2+) reabsorption. This study examines the role of claudin-16 in the adaptive response of the kidney to Mg(2+) availability. METHODS/RESULTS: Mice received a low-, normal- or high Mg(2+) diet for up to 3 days. Mg(2+)-loaded animals displayed hypermagnesemia with increasing urine Mg(2+)/Ca(2+) levels paralleled by a decrease in claudin-16 protein and mRNA in the kidney. Mg(2+)- deprived animals developed hypomagnesemia with decreasing urine Mg(2+)/Ca(2+) levels associated with an increase in claudin-16 protein and mRNA abundance. Mg(2+) depletion markedly increased and Mg(2+) load decreased endogenous claudin-16 mRNA levels in calcium-sensing receptor-transfected HEK293 cells compared with native HEK293 cells. The effect of Mg(2+) availability on the human CLDN16 (hCLDN16) gene promoter was examined. Using a 2.5kb hCLDN16 5'-flanking DNA sequence, we show that magnesium depletion increases and Mg(2+) load decreases hCLDN16 promoter activity in transfected HEK293 cells. CONCLUSIONS: Changes in Mg(2+) availability may influence claudin-16 mediated Mg(2+) transport at the transcriptional level. The possible involvement of the cell membrane bound Ca(2+)/Mg(2+) sensing receptor or the potential role of a hypothetical Mg(2+) response element on the CLDN16 promoter in the Mg(2+)-induced response remains to be explored.

Physiological and catecholamine response to sympathetic stimulation in turner syndrome.

OBJECTIVE: Women with Turner syndrome have increased heart rate and high blood pressure (BP), and have been described as having high tolerance for emotional stress. We hypothesized that women with Turner syndrome have reduced catecholaminergic and physiological response to sympathetic stimulation, and that changes in BP and heart rate are related to their catecholamine response to sympathetic stimulation. DESIGN AND PATIENTS: Ten young women with Turner syndrome, age 17-34 years were the subjects of this study. Their response to sympathetic stimulation was compared to a group of 10 age-matched healthy women. MEASUREMENTS: After a period of 30 min resting, subjects and controls were subjected to an escalating series of sympathetic stimulation: orthostatic, cold pressor and exercise, and their plasma catecholamines and haemodynamic response were monitored and compared to resting levels. RESULTS: Resting heart rate was higher in Turner syndrome patients at 83 +/- 9 beats per min (bpm, mean +/- SD), as compared to controls (74 +/- 10 bpm, P < 0.05). Their supine BP was also higher at 122 +/- 9/84 +/- 6 vs. 106 +/- 11/70 +/- 9 mmHg (P < 0.02/< 0.02). The corresponding resting norepinephrine, but not epinephrine, was higher in Turner syndrome patients (2.54 +/- 1.09 nmol/l) as compared to controls (1.69 +/- 0.55 nmol/l, P < 0.02). In response to orthostatic stimulation and cold pressor test the systolic, but not the diastolic BP or heart rate, increased in Turner syndrome patients but not in the control group (P < 0.01). The change in blood catecholamine levels was comparable in both groups. Their physiological response to exercise was normal. Yet, the exercise-induced surge of norepinephrine and epinephrine in Turner syndrome patients was lower (P < 0.02). CONCLUSIONS: Turner syndrome is associated with dysregulation of the sympathetic nervous system (SNS), leading to tachycardia and high BP, increased resting norepinephrine levels, and a greater tolerance of the cathecholamine response to exercise.

Anti-cyclic citrullinated protein antibodies as a predictor of response to anti-tumor necrosis factor-alpha therapy in patients with rheumatoid arthritis.

OBJECTIVE: . The treatment of rheumatoid arthritis (RA) has changed dramatically with the introduction of anti-tumor necrosis factor (TNF) agents. Unfortunately, a subset of patients have partial or no response. No measurements were found to predict the efficacy of this therapy. Anti-cyclic citrullinated protein antibodies (anti-CCP) are highly specific and sensitive for RA, and their titer correlates with erosive disease. We investigated the correlation between the efficacy of infliximab therapy and the titer of anti-CCP. METHODS: Thirty consecutive seropositive patients with RA were treated with infusion of 3 mg/kg infliximab on Weeks 0, 2, 6, and 14. Clinical assessment and blood withdrawal were done before each treatment, i.e., at the minimal concentration of the drug. Disease activity was assessed by DAS28 score and by interleukin 6 (IL-6) level. Anti-CCP titer was measured by a commercial ELISA at Week 0 and Week 14. RESULTS: At baseline, 24 patients were positive for anti-CCP antibodies. In most patients there was a significant correlation between clinical response to therapy and anti-CCP titer. The results were especially noteworthy in those patients who showed a sustained and significant decrease in IL-6 levels through the entire period. CONCLUSION: Anti-CCP titer and IL-6 levels might be early predictors of the efficacy of anti-TNF therapy in patients with RA.

Identification of cytoplasmic p53 protein in corneal epithelium of vertebrates.

The tumour suppressor gene p53 encodes a phosphoprotein involved in the control of cell growth. It's expression and function have been documented in malignancy, apoptosis and the aging processes. Recently, p53 expression has been demonstrated in normal murine tissues, including whole eye. Currently, we intend to map and to characterize p53 expression in the normal cornea across different species. To do this, eyes of animals were enucleated after sacrifice by CO(2) narcosis and then p53 expression in whole eyes (cornea) was mapped by indirect immunohistochemical staining techniques using the anti-p53 monoclonal antibodies PAb 248, PAb 421 and PAb 240 (alternatively called mAb 248, mAb 421 and mAb 240, respectively). Additionally, eyes were freshly dissected to separate the corneas, for quantitating p53 expression, using Western blot analysis. We found strong cytoplasmic p53 expression in the corneal epithelium of various vertebrate species by immunohistochemistry and by Western analysis. High levels of cytoplasmic p53 protein were normally found in normal corneal epithelium of various vertebrate species. Hence, these data may indicate that p53 may have a new evolutionary significant function in the eye.

Fast bedside measurement of blood count and C-reactive protein in neonates with suspected late-onset sepsis.

UNLABELLED: Conventional and fast complete blood count (CBC) and C-reactive protein (CRP) measurements were compared in 56 neonates with suspected sepsis. Mean differences between pairs (conventional minus fast) of the corresponding CBC and CRP values were significant for the six CBC parameters tested (p<0.02), but not for CRP (p=0.18). CONCLUSION: In neonatal sepsis, conventional and fast methods are comparable only for CRP but not for CBC variables.

Association between elevated liver enzymes and C-reactive protein: possible hepatic contribution to systemic inflammation in the metabolic syndrome.

OBJECTIVE: The objective of this study was to test whether the frequent association between liver enzyme elevations and various components of the metabolic syndrome is associated with higher C-reactive protein (CRP) levels. METHODS AND RESULTS: Alanine aminotransferase (ALT), alkaline phosphatase (Alk-P), and high-sensitivity CRP were measured in 1740 subjects. Adjusted geometric mean CRP was calculated for subjects with normal and elevated ALT and for subjects with normal and elevated Alk-P, adjusting for age, sex, smoking, physical activity, body mass index, fasting glucose, triglycerides, the presence of hypertension and low HDL cholesterol, and use of aspirin or hormone replacement therapy. Adjusted CRP levels were higher in subjects with elevated ALT (2.21 versus 1.94 mg/L, P=0.028) or elevated Alk-P (2.58 versus 1.66 mg/L, P<0.0001). Logistic regression showed that compared with subjects with normal liver function tests, the adjusted odds for high-risk CRP (>3 mg/L) were significantly higher in subjects with elevated ALT (OR, 1.5; 95% CI, 1.2 to 1.9, P=0.002) or elevated Alk-P (OR, 2.1; 95% CI, 1.7 to 2.6, P<0.0001). CONCLUSIONS: Elevations of liver enzymes are associated with higher CRP concentrations. Hepatic inflammation secondary to liver steatosis is a potential contributor to the low-grade inflammation associated with the metabolic syndrome.

Plasma endothelin-1 levels in patients with complex regional pain syndrome.

The clinical characteristics of complex regional pain syndrome (CRPS)--spontaneous and stimulus-evoked pain, autonomic abnormalities, motor dysfunction, and trophic changes in the affected limb--are well known. However, its pathogenesis is unclear, and the diagnosis is often delayed, in part due to lack of objective laboratory tests. Endothelin-1 (ET-1) is a potent vasoconstrictor that has recently been shown to produce pain, allodynia, edema, and muscle weakness, as well as to exert a direct excitatory effect on nociceptive afferents. Furthermore, new evidence indicates that ET-1 is involved in various cancer- and non-cancer-related painful conditions. The aim of the present explorative study was to determine the ET-1 plasma levels in patients with CRPS in an attempt to identify a 'laboratory marker' for CRPS and to search for evidence suggesting that ET-1 may be involved in the pathogenesis of CRPS. ET-1 plasma levels were determined in 20 severely affected CRPS patients, in eight patients with non-CRPS chronic painful conditions, and in 10 healthy volunteers. The results showed that there were no significant differences in ET-1 plasma levels between the three groups. We conclude that the plasma level of ET-1 cannot be regarded as a 'marker' for CRPS. Yet, the possibility that ET-1 is involved in the pathophysiology of CRPS has not been excluded and deserves further investigation.

Diagnostic coronary angiography induces a systemic inflammatory response in patients with stable angina.

BACKGROUND: Systemic markers of inflammation increase after percutaneous coronary intervention (PCI). The rise in inflammatory markers after PCI is frequently attributed to the inflammatory stimulus associated with coronary artery injury during balloon inflation and coronary stent implantation. The aim of this study was the determine whether diagnostic coronary angiography performed in patients with stable angina triggers a systemic inflammatory response. METHODS: We prospectively studied patients with chronic stable angina undergoing either coronary angiography (n = 13) or coronary angiography followed by PCI (n = 13). Peripheral blood samples were obtained before and 24 hours, 48 hours, and 4 weeks after the procedure and analyzed for C-reactive protein (CRP), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha). Patients with periprocedural myocardial necrosis were excluded. RESULTS: There was a significant increase in CRP levels at 24 and 48 hours in both the coronary angiography (P <.05) and PCI (P <.01) groups. IL-6 levels peaked at 24 hours in both the coronary angiography (median, 2.5-9.5 pg/mL; P =.01) and PCI (median, 3.0-8.2 pg/mL; P <.005) groups. At 4 weeks, both CRP and IL-6 returned to baseline levels. TNF-alpha levels were unchanged with either coronary angiography or PCI. The magnitude of the rise of CRP and IL-6 levels was not significantly different between the groups. There was a fair correlation between baseline and peak postprocedural levels of CRP (r = 0.67, P =.008) and IL-6 (r = 0.48, P =.016). CONCLUSION: Uncomplicated diagnostic coronary angiography triggers a systemic inflammatory response in patients with stable angina. The contribution of coronary angiography should be considered in interpreting the significance of the systemic inflammatory response observed after PCI.

Catecholaminergic pathways, chromaffin cells, and human disease.

Recent studies demonstrate major effects of adrenal medullary and catecholaminergic pathways on a wide variety of normal physiologic and regulatory events. Alterations in these pathways, involving changes in catecholamines or in proteins and peptides costored and coreleased with catecholamines, may lead to profound changes in autonomic, cardiovascular, neuroendocrine, metabolic, nociceptive, and immune function. These findings have important implications for a variety of human disease states. In addition, molecules associated with catecholaminergic function may provide novel diagnostic and therapeutic strategies for human disease and suggest specific genetic loci as important and fruitful targets for further genetic and pharmacogenetic studies.

Hyponatremia as a complication of cardiac catheterization: a prospective study.

BACKGROUND: A decrease in plasma sodium (P(Na)) concentration is common after surgery and attributed to the secretion of antidiuretic hormone in response to such nonosmotic stimuli as pain or nausea. In this setting, acute hyponatremia may lead to seizures, coma, and permanent neurological damage. Sporadic case reports have described severe neurological symptoms caused by hyponatremia occurring within hours after cardiac catheterization. We evaluated the prevalence, contributing clinical circumstances, and course of hyponatremia in patients undergoing cardiac catheterization. METHODS: We prospectively studied 309 consecutive patients scheduled for an elective cardiac catheterization. Plasma and urine electrolytes and urine osmolarity were measured at baseline and again 1 to 4 hours and 24 hours after the procedure. RESULTS: P(Na) level was 139.4 +/- 2.3 mEq/L at baseline. At 1 to 4 hours, P(Na) level decreased to 134.2 +/- 3.6 mEq/L (P < 0.0001). Mild (> or =5 to 10 mEq/L), moderate (11 to 14 mEq/L), and severe (> or =15 mEq/L) reductions in P(Na) levels occurred in 50%, 5%, and 0.3% of patients, respectively. At the 24-hour point, P(Na) level increased to 137.3 +/- 2.4 mEq/L, but was significantly lower compared with baseline (P < 0.0001). In hyponatremic patients at the 1- to 4- and 24-hour points, mean urine osmolarity values were 428 +/- 139 and 420 +/- 204 mOsm/kg, respectively; almost every urine sample was inappropriately concentrated. Multivariate logistic regression identified the amount of electrolyte-free water administered to be a predictor for the development of hyponatremia (3.7-fold incremental risk for every 1 L administered to a 70-kg patient). CONCLUSION: An acute reduction in P(Na) level commonly occurs shortly after cardiac catheterization. The cause of hyponatremia appears to be related to the administration of hypotonic fluids, together with impaired urinary dilutional capacity. Although symptomatic hyponatremia is rare, the diagnosis should be entertained when neurological symptoms develop in this setting.

Increased emotional distress in daughters of breast cancer patients is associated with decreased natural cytotoxic activity, elevated levels of stress hormones and decreased secretion of Th1 cytokines.

DBCP who are aware of their increased risk of developing breast cancer may suffer from high emotional distress. Chronic stress may interfere with NCA and low NCA is associated with increased cancer risk. We studied 80 DBCP and 47 age- and education-matched healthy females (controls). Heparinized venous blood (30 ml) was drawn from all subjects between 8 and 9 A.M., and each participant answered a set of psychologic questionnaires. In addition, the first-morning urine sample was collected. DBCP scored significantly higher in emotional distress compared to controls. Levels of stress hormones in DBCP were higher and in vitro secretion of IL-2, IL-12 and IFN-gamma lower compared to controls. NCA against NK-resistant (MCF-7, COLO-205, U937) and NK-sensitive (K562) cell lines was significantly lower in DBCP and much less augmented by in vitro preincubation with IL-2 or IL-12 compared to controls. NCA and in vitro Th1 cytokine secretion were inversely correlated with the degree of emotional distress and the level of stress hormones in blood or urine. High emotional distress and elevated levels of stress hormones are associated with impaired immune surveillance functions in DBCP. This may contribute to the increased risk of DBCP to develop breast cancer. An interventional trial to enhance coping and reduce stress levels may help to decrease the risk for breast cancer onset in DBCP.

p53 expression in the normal murine eye.

PURPOSE: The tumor-suppressor gene p53 encodes a phosphoprotein involved in the control of cell growth. Its expression and function have been documented in malignancy, apoptosis, and other abnormal cell proliferation processes. Recently, expression of p53 has been demonstrated in certain normal tissues, including whole eye. The purpose of the study was to map and to characterize expression of p53 in the normal murine eye. METHODS: Eyes of adult C57BL/6 mice were enucleated after death by CO2 narcosis. Expression of p53 in frozen sections of whole cryoprotected eyes was mapped by indirect immunofluorescence microscopy using the anti-p53 monoclonal antibodies 248 and 421 and the polyclonal antibody FL-393. Additionally, eyes were freshly dissected to separate the various ocular tissues. In these ocular tissues, expression of p53 was quantitated with ELISA and Western blot analyses. RESULTS: Strong expression of p53 was observed in various normal ocular tissues. The corneal and conjunctival epithelium exhibited very high cytoplasmic p53 protein levels. High nuclear p53 protein staining was seen in the lens epithelial cells of the central and pre-equatorial zones and in the lens fiber nuclear bow, situated posterior to the epithelial germinative zone. Cells of the actual lens germinative zone did not stain for p53 protein. Low levels of p53 protein were expressed in retinal tissue. CONCLUSIONS: High levels of p53 protein are found in various normal murine ocular tissues, especially the corneal and conjunctival structures and the lens epithelium. Each of these tissues demonstrate unique patterns of staining.

Educating a new generation of clinical laboratory scientists.

In many countries the new generation of laboratory scientists comes from the graduates of the biological sciences. Their training in the sciences is usually of high quality, but is almost totally lacking in the clinical application of their scientific knowledge. They obtain this clinical knowledge most often by on-the-job training and experience. This paper describes a new undergraduate academic program in Laboratory Medicine developed at the Faculty of Medicine of the Technion, the Israel Institute of Technology. The program is carried out with the collaboration of the Faculty of Biology and the Faculty of Medicine, and upon completion of the classroom studies there is a period of internship for practical experience. The first students of the program are now in their internship period and will graduate this year.

Effect of ilioinguinal nerve block on the catecholamine plasma levels in orchidopexy: comparison with caudal epidural block.

BACKGROUND: Both caudal epidural and ilioinguinal/iliohypogastric nerve blocks have been used to provide effective intra- and postoperative analgesia. Stress response hormone levels can be used as an objective method to assess the analgesic efficacy of the anaesthetic techniques used in infraumbilical surgery in children. In this study, we compared catecholamine blood levels in children undergoing these two different supplementary analgesic/anaesthetic techniques. METHODS: Thirty male paediatric patients undergoing orchidopexy, ASA I, received inhalation general anaesthesia, and were randomly allocated to one of two groups: a caudal group (n = 15) and an ilioinguinal/iliohypogastric nerve block group (n = 15). Plasma epinephrine and norepinephrine concentrations were measured at the induction time, at the end of surgery, and in the postanaesthesia care unit. Postoperative pain score was also assessed in the postanaesthesia care unit. RESULTS: In both groups, there was a substantial decrease in the catecholamine blood levels; however, there were significantly higher levels of epinephrine in the ilioinguinal group at the end of surgery (P = 0.008) and in the recovery room (P = 0.02) and a significant higher level of norpinephrine in the recovery room (P = 0.008). CONCLUSIONS: The result of this study revealed that caudal epidural block was more effective than ilioinguinal block in suppressing the stress response as reflected in epinephrine and norepinephrine blood levels in orchidopexy patients.