Chromatin three-dimensional interactions mediate genetic effects on gene expression.

Studying the genetic basis of gene expression and chromatin organization is key to characterizing the effect of genetic variability on the function and structure of the human genome. Here we unravel how genetic variation perturbs gene regulation using a dataset combining activity of regulatory elements, gene expression, and genetic variants across 317 individuals and two cell types. We show that variability in regulatory activity is structured at the intra- and interchromosomal levels within 12,583 cis-regulatory domains and 30 trans-regulatory hubs that highly reflect the local (that is, topologically associating domains) and global (that is, open and closed chromatin compartments) nuclear chromatin organization. These structures delimit cell type-specific regulatory networks that control gene expression and coexpression and mediate the genetic effects of cis- and trans-acting regulatory variants on genes.

[Mononeuritis multiplex under the TNF-alpha inhibitor infliximab]. / Mononeuritis multiplex unter dem TNF-alpha-Hemmer Infliximab.

A 21-year-old patient with ankylosing spondylitis under treatment with the TNF-alpha inhibitor infliximab developed a multifocal, demyelinating axonal neuropathy affecting several peripheral nerves simultaneously (mononeuritis multiplex). This represents an additional rare peripheral nervous system side effect of infliximab therapy. The underlying cause is unknown. Intravenous immunoglobulin therapy (0.4 g/kg per day for five days) led to a complete regression of muscle paresis and sensory defects in this case.

Focal neuromyotonia: do I love you?

We present a rare case of focal neuromyotonia in a 73-year-old woman with a follow up of 5 years. The clinical picture showed a fixed contraction of the 3rd and 4th finger of the left hand. Similar to other published cases, our patient suffered from COPD and was treated with beta-2-sympathomimetics. This clinical picture shows a rare but rather salient differential diagnosis of Dupuytren's contracture. EMG of the affected muscles may yield a diagnosis and prevent the patient from a long and ineffective treatment "odyssey".

Presence and quantification of macrophages in squamous cell carcinoma of the cervix.

Cervical cancer is a major cause of morbidity and mortality in women. The presence of macrophages as well as other inflammatory cells has been noted in many of these tumors. Intratumoral macrophages/monocytes induce anergy to cytokine therapy and cause apoptosis in natural killer(NK) and T cells. The aim of this study was to better evaluate and quantify the presence of macrophages in these tumors. Twenty-four cases of squamous cell carcinoma of the cervix seen at our institution were evaluated. Sections were stained with CD68, a marker for macrophages. Staining was graded microscopically by two reviewers together on a scale of 0-4+, with 4+ representing the greatest number of positive cells. Image analysis was conducted to quantify the percent area stained in a given lesion. For each lesion, 10 fields were evaluated, and a mean percentage area stained was calculated. 4+ staining was observed in five cases, 3+ in zero cases, 2+ in three cases, 1+ in six cases, 1-2+ in one case, and nine cases were negative. Image analysis results correlated well with the light microscopic scoring. Presence of a prominent infiltrate of macrophages did not correlate with tumor grade or with histologic lymph node status, but showed a strong negative correlation with tumor stage. Some squamous cell carcinomas of the cervix show a prominent macrophage component in the tumor-associated inflammatory infiltrate. The presence of this prominent infiltration of macrophages did not correlate with tumor grade or lymph node status, but showed a strong negative correlation with tumor stage. The results suggest that immunotherapy may have a potential role in the treatment of cervical carcinoma. Computerized image analysis appears to be a valid measure to assess macrophage counts in such lesions.

Prophylactic hemodialysis after radiocontrast media in patients with renal insufficiency is potentially harmful.

PURPOSE: Acute renal failure induced by contrast media is an important cause of hospital-acquired renal insufficiency. Preexisting renal failure and the dose of contrast media are known risk factors for the development of radiocontrast nephropathy. We performed a randomized trial to test whether radiocontrast nephropathy can be avoided by prophylactic hemodialysis immediately after the administration of contrast media in patients with impaired renal function. SUBJECTS AND METHODS: Renal function and other parameters, hemodialysis requirement, and relevant clinical events were recorded before and during the 6 days after administration of contrast media in 113 patients with a baseline serum creatinine level >200 microm/L (>2.3 mg/dL). Patients were randomly assigned to either hemodialysis (n = 55) or nonhemodialysis (n = 58) treatment after parenteral low-osmolality contrast media. RESULTS: The characteristics of the patients in the two groups were similar. Compared with baseline levels, the mean [+/- SD] serum creatinine level decreased at day 1 (277 +/- 95 microm/L), peaked at day 4 (353 +/- 126 microm/L), and returned to baseline at day 6 (327 +/- 119 microm/L, P <0.05 by analysis of variance) after administration of contrast media in the hemodialysis group, whereas in the nonhemodialysis group, no significant changes in mean serum creatinine level were observed. Eleven patients required 1 or more hemodialyses (8 in the hemodialysis group and 3 in the nonhemodialysis group, P = 0.12), 6 of whom (4 vs. 2, P = 0.44) required 3 or more hemodialyses. Clinically relevant events included pulmonary edema (1 vs. 4 patients, P = 0.36), myocardial infarction (2 vs. 2), stroke (2 vs. 0, P = 0.24), and death (1 vs. 1). CONCLUSIONS: The strategy of performing hemodialysis immediately after the administration of low-osmolality contrast media in all patients with a reduced renal function did not diminish the rate of complications, including radiocontrast nephropathy.

Pigs expressing salivary phytase produce low-phosphorus manure.

To address the problem of manure-based environmental pollution in the pork industry, we have developed the phytase transgenic pig. The saliva of these pigs contains the enzyme phytase, which allows the pigs to digest the phosphorus in phytate, the most abundant source of phosphorus in the pig diet. Without this enzyme, phytate phosphorus passes undigested into manure to become the single most important manure pollutant of pork production. We show here that salivary phytase provides essentially complete digestion of dietary phytate phosphorus, relieves the requirement for inorganic phosphate supplements, and reduces fecal phosphorus output by up to 75%. These pigs offer a unique biological approach to the management of phosphorus nutrition and environmental pollution in the pork industry.

Metabolic and endocrine effects of metabolic acidosis in humans.

Metabolic acidosis is an important acid-base disturbance in humans. It is characterised by a primary decrease in body bicarbonate stores and is known to induce multiple endocrine and metabolic alterations. Metabolic acidosis induces nitrogen wasting and, in humans, depresses protein metabolism. The acidosis-induced alterations in various endocrine systems include decreases in IGF-1 levels due to peripheral growth hormone insensitivity, a mild form of primary hypothyroidism and hyperglucocorticoidism. Metabolic acidosis induces a negative calcium balance (resorption from bone) with hypercalciuria and a propensity to develop kidney stones. Metabolic acidosis also results in hypophosphataemia due to renal phosphate wasting. Negative calcium balance and phosphate depletion combine to induce a metabolic bone disease that exhibits features of both osteoporosis and osteomalacia. In humans at least, 1,25-(OH)2 vitamin D levels increase, probably through phosphate depletion-induced stimulation of 1-alpha hydroxylase. The production rate of 1,25-(OH)2 vitamin D is thus stimulated, and parathyroid hormone decreases secondarily. There is experimental evidence to support the notion that even mild degrees of acidosis, such as that occurring by ingestion of a high animal protein diet, induces some of these metabolic and endocrine effects. The possible role of diet-induced acid loads in nephrolithiasis, age-related loss of lean body mass and osteoporosis is discussed.

Characterization of acute inhibition of Na/H exchanger NHE-3 by dopamine in opossum kidney cells.

BACKGROUND: Dopamine (DA) is a principal natriuretic hormone that defends extracellular fluid volume from a Na load. Natriuresis is effected partly through inhibiting the proximal tubule Na/H exchanger NHE-3. Changes in NHE-3 phosphorylation is one mechanism by which NHE-3 activity is regulated. METHODS: We used opossum kidney (OK) cells to characterize the differential and synergistic effects of DA receptor subtype-1 (DA1) and -2 (DA2) agonists and the effect of blockade of protein kinase A (PKA) or protein kinase C (PKC) on NHE-3 activity and phosphorylation. RESULTS: DA and DA1 agonists inhibited NHE-3 activity, and DA1 antagonist blocked the effect of either DA or DA1 agonist. DA2 agonist alone had no effect, but DA2 antagonist reduced the DA effect on NHE-3 activity. DA1 and DA2 agonists together were more potent than DA1 alone. PKA inhibition eliminated the effect of DA1 agonist and partially blocked the effect of DA on NHE-3 activity. PKC inhibition did not block the DA effect. DA1 agonist and PKA activation phosphorylated NHE-3 on identical sites. Despite lack of effect on NHE-3 activity, DA2 agonists increased NHE-3 phosphorylation. DA-induced NHE-3 phosphorylation was distinct from DA1 and PKA but closely resembled DA2. CONCLUSION: We postulate the following: (1) DA modifies NHE-3 phosphorylation by activating PKA through DA1 and by other kinases/phosphatases via DA2. (2) DA1 is sufficient to inhibit NHE-3, while DA2 is insufficient but plays a synergistic role by altering NHE-3 phosphorylation.

Factitious hyperkalemia.

Pseudohyperkalemia, or factitious hyperkalemia, constitutes an artificially high plasma potassium level (P(K)) from a variety of possible causes. Occasionally, the cause cannot be elucidated. Three patients who showed unusually large differences between free-flowing and tourniquet (stasis) potassium levels prompted us to investigate the influence of tourniquets in routine phlebotomy in eight healthy volunteers. P(K) showed a consistent but rather small average increase of 0.2 mEq/L (P < 0.001) during tourniquet use; however, the range was 10-fold, from 0.05 to 0.5 mEq/L in our subjects. We suggest there may be large variability leading to an excessive increase in P(K) in some individuals. In the three patients presented, average excessive increases in P(K) of 1.6, 1.3, and 1.7 mEq/L were seen. Although diagnosing and treating true hyperkalemia remains paramount, recognizing factitious hyperkalemia is important to preclude unnecessary investigations and potentially hazardous intervention.

Acute regulation of Na+/H+ exchanger NHE3 by parathyroid hormone via NHE3 phosphorylation and dynamin-dependent endocytosis.

Parathyroid hormone (PTH) is a potent inhibitor of mammalian renal proximal tubule Na(+) transport via its action on the apical membrane Na(+)/H(+) exchanger NHE3. In the opossum kidney cell line, inhibition of NHE3 activity was detected from 5 to 45 min after PTH addition. Increase in NHE3 phosphorylation on multiple serines was evident after 5 min of PTH, but decrease in surface NHE3 antigen was not detectable until after 30 min of PTH. The decrease in surface NHE3 antigen was due to increased NHE3 endocytosis. When endocytic trafficking was arrested with a dominant negative dynamin mutant (K44A), the early inhibition (5 min) of NHE3 activity by PTH was not affected, whereas the late inhibition (30 min) and decreased surface NHE3 antigen induced by PTH were abrogated. We conclude that PTH acutely inhibits NHE3 activity in a biphasic fashion by NHE3 phosphorylation followed by dynamin-dependent endocytosis.

Acute regulation of Na/H exchanger NHE3 activity by protein kinase C: role of NHE3 phosphorylation.

Acute hormonal modulation of NHE3 activity is partly mediated by kinases, including protein kinase C (PKC). We examined the role of NHE3 phosphorylation in regulating its activity in response to PKC activation by phorbol 12-myristate 13-acetate (PMA). In pooled NHE-deficient fibroblasts transfected with NHE3, PMA increased NHE3 activity and phosphorylation. When six potential PKC target serines were mutated, NHE3 phosphorylation was drastically reduced and PMA failed to regulate NHE3 phosphorylation or function. To examine whether NHE3 phosphorylation is sufficient for functional regulation by PKC, we exploited the heterogeneous response of NHE3 activity to PMA in individual clones of transfectants. Clones with stimulatory, inhibitory, or null responses to PMA were observed. Despite the diverse functional response, changes in NHE3 phosphorylation as revealed by tryptic phosphopeptide maps were similar in all clones. We conclude that although phosphorylation appears to be necessary, it is insufficient to mediate PKC regulation of NHE3 function and factors extrinsic to the NHE3 protein must be involved.

Dual mechanisms of regulation of Na/H exchanger NHE-3 by parathyroid hormone in rat kidney.

Parathyroid hormone (PTH) is a potent inhibitor of mammalian renal proximal tubule sodium absorption via suppression of the apical membrane Na/H exchanger (NHE-3). We examined the mechanisms by which PTH inhibits NHE-3 activity by giving an acute intravenous PTH bolus to parathyroidectomized rats. Parathyroidectomy per se increased apical membrane NHE-3 activity and antigen. Acute infusion of PTH caused a time-dependent decrease in NHE-3 activity as early as 30 min. Decrease in NHE-3 activity at 30 and 60 min was accompanied by increased NHE-3 phosphorylation. In contrast to the rapid changes in NHE-3 activity and phosphorylation, decrease in apical membrane NHE-3 antigen was not detectable until 4-12 h after the PTH bolus. The decrease in apical membrane NHE-3 occurred in the absence of changes in total renal cortical NHE-3 antigen. Pretreatment of the animals with the microtubule-disrupting agent colchicine blocked the PTH-induced decrease in apical NHE-3 antigen. We propose that PTH acutely cause a decrease in NHE-3 intrinsic transport activity possibly via a phosphorylation-dependent mechanism followed by a decrease in apical membrane NHE-3 antigen via changes in protein trafficking.

Acute inhibition of Na/H exchanger NHE-3 by cAMP. Role of protein kinase a and NHE-3 phosphoserines 552 and 605.

Regulation of the renal Na/H exchanger NHE-3 by protein kinase A (PKA) is a key intermediate step in the hormonal regulation of acid-base and salt balance. We studied the role of NHE-3 phosphorylation in this process in NHE-deficient AP-1 cells transfected with NHE-3 and in OKP cells expressing native NHE-3. A dominant-negative PKA-regulatory subunit completely abolished the effect of cAMP on NHE-3 activity demonstrating a role of PKA in the functional regulation of NHE-3 by cAMP. NHE-3 isolated from cAMP-treated cells showed lower phosphorylation by purified PKA in vitro suggesting that NHE-3 is a PKA substrate in vivo. Although changes in NHE-3 whole protein phosphorylation is difficult to detect in response to cAMP addition, the tryptic phosphopeptide map of in vivo phosphorylated NHE-3 showed a complex pattern of constitutive and cAMP-induced phosphopeptides. To test the causal relationship between phosphorylation and activity, we mutated eight serines in the cytoplasmic domain to glycine or alanine. Single or multiple mutants harboring S552A or S605G showed no PKA activation or reduced regulation by PKA activation. Ser-552 and Ser-605 were phosphorylated in vivo. However, multiple mutations of serines other than Ser-552 or Ser-605 also reduced the functional PKA regulation. We conclude that regulation of NHE-3 by PKA in vivo involves complex mechanisms, which include phosphorylation of Ser-552 and Ser-605.

Interferon-alpha activates signal transducers and activators of transcription 5 and 6 in Daudi cells.

In most cells studied so far, interferon-alpha (IFN-alpha) activates signal transducer and activator of transcription (Stat1), Stat2 and Stat3, whereas interferon-gamma (IFN-gamma) induces Stat1 only. In general, each of the several dozens of cytokines, growth factors and hormones that signal through the Janus kinases-signal transducers and activators of transcription (Jak-STAT) pathway activates a distinct subset of STATs, and this selectivity is thought to be essential for the specificity of the cellular responses toward these ligands. Here, we have studied the pattern of STAT activation in the human lymphoblastoid cell line Daudi in response to IFN-alpha. In addition to Stat1, Stat2 and Stat3 activation, IFN-alpha was found to directly induce activation of Stat5 and Stat6. Cell-type-specific activation of additional STATs could be responsible for cell-type-specific responses to IFN-alpha.

Central hypogonadism: distinguishing idiopathic low testosterone from pituitary tumors.

OBJECTIVE: To attempt to determine clinical or hormonal characteristics that could help distinguish benign idiopathic low testosterone (ILT) from pituitary tumor. METHODS: On retrospective review of medical records of patients encountered by Johns Hopkins endocrine staff between 1985 and July 1995, 64 patients who fulfilled our enrollment criteria--27 men with ILT and 37 patients with imaging-proven pituitary tumor--were identified. Men 21 years of age or older needed to have had serum testosterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin levels measured before hormonal replacement therapy or pituitary tumor extirpation (or both) and a high-quality imaging scan (computed tomography or magnetic resonance imaging) done and interpreted by the Johns Hopkins radiology staff. RESULTS: In comparison with men who had ILT, men with pituitary tumors had similar serum testosterone levels and significantly higher serum levels of LH, FSH, and prolactin. In addition, significantly more men with pituitary tumors had visual field abnormalities, headaches, and symptoms of hypothyroidism in comparison with the men with ILT. In contrast, the group with ILT complained significantly more of impotence, erectile dysfunction, and depression than did the group with pituitary tumors. The age at initial assessment was comparable in both study groups. CONCLUSION: Although age at initial manifestation did not predict the presence of pituitary tumor, the group of men with tumors were more likely than those with ILT to have serum testosterone levels <150 ng/dL, higher serum gonadotropin and prolactin levels, and visual field abnormalities and less likely to have sexual dysfunction. Therefore, on the basis of our data, we recommend that men with these findings should be referred for a magnetic resonance image to exclude the presence of a tumor.

[Electromyography as a diagnostic aid in tetanus]. / Elektromyogramm als diagnostische Hilfe beim Tetanus.

In developing countries tetanus is still a real problem because of its high incidence and mortality. In all countries with high medical standards it has become rare. For this reason many clinicians are no longer familiar with the disease and its diagnosis. Until now no laboratory test has been readily available to confirm or rule out tetanus. However, the diagnosis can be performed by a simple and readily available electromyogram (EMG). We performed EMGs in 13 patients in whom tetanus was suspected but whose case history or clinical findings left some doubt. In 7 cases the EMG was typical for tetanus, showing spontaneous activity of motor units which could not be suppressed voluntarily and with shortening or absence of the silent period after a stretch reflex or after electrical stimulation of the nerve. In 2 cases we found one of the two diagnostic features. In all these 9 cases the diagnosis of tetanus was confirmed by the further development of the disease. In the remaining 4 patients the EMG was normal and in the course it was confirmed that they were not suffering from tetanus. Therefore, we consider electromyography a very useful and reliable tool either to confirm or rule out the diagnosis of tetanus.

Chronic renal allograft rejection: immunologic and nonimmunologic risk factors.

The pathogenesis of chronic renal allograft rejection is unknown. It is also unclear why cyclosporine has failed to prevent chronic rejection. We examined possible risk factors for graft loss to chronic rejection among 706 renal transplants using the Cox proportional hazards model with fixed and time-dependent covariates. Both the number and the severity of acute rejection episodes were independent risk factors for chronic rejection [relative risk (95% confidence interval) 2.31 (2.04 to 2.60) and 1.53 (1.27 to 1.84), respectively]. Cyclosporine and cyclosporine withdrawal had no effect on chronic rejection. Acute rejections occurring within the first three months after transplantation, when cyclosporine most effectively prevented acute rejection, also had no effect on chronic rejection. Risk factors that were independent of acute rejection and not clearly attributable to immune mechanisms included serum albumin [0.20 (0.10 to 0.38) for each g/dl], proteinuria [1.42 (1.29 to 1.57) for each g/24 hr], and serum triglycerides -1.09 (1.03 to 1.16) for each 100 mg/dl-. These results suggest that the reduction in acute rejection episodes from cyclosporine has failed to reduce graft failure from chronic rejection, possibly because the early (within the first 3 months) and mild acute rejection episodes that are most effectively prevented by cyclosporine do not cause chronic rejection. In addition, the results suggest that there may be a number of nonimmunologic risk factors for chronic rejection.

Serum albumin and mortality after renal transplantation.

The incidence, causes, and consequences of hypoalbuminemia after renal transplantation are not well defined. We examined clinical correlates of serum albumin measured at 3 months, 6 months, 1 year, and annually thereafter in 706 renal transplant recipients who survived at least 6 months with a functioning allograft. Follow-up was 7.0 +/- 4.2 years. Hypoalbuminemia (< or = 3.5 g/dL) was most common at 3 months (31%, n = 692), least common at 1 year (12%, n = 656), and then became increasingly common among survivors, for example, 14% (n = 466) at 4 years, 20% (n = 204) at 8 years, and 29% (n = 77) at 12 years after transplantation. By multiple linear regression, variables that correlated (P < 0.05) with lower serum albumin at 3, 6, 12, and 24 months included age, diabetes, proteinuria, and cytomegalovirus infection. Other independent correlates on at least one of these occasions included renal function and chronic disease (malignancy, liver disease, and cardiovascular disease). Serum albumin, as a time-averaged and time-dependent covariate, was a strong independent risk factor for death using Cox proportional hazards analysis (relative risk for each g/dL increment, 0.26; 95% confidence interval, 0.16 to 0.44 [1.00 = no risk]). The effects of albumin on mortality were independent of age, diabetes, serum lipids, renal function, chronic liver disease, malignancies, and cardiovascular disease. The effects of albumin on mortality were evident even when the analysis was restricted to patients dying several years after albumin was measured. Thus, hypoalbuminemia is common and serum albumin is a strong independent risk factor for all-cause mortality after renal transplantation.

Cardiovascular disease after renal transplantation.

Although cardiovascular disease is a major cause of morbidity and mortality after renal transplantation, its pathogenesis and treatment are poorly understood. We conducted separate analyses of risk factors for ischemic heart disease, cerebral, and peripheral vascular disease after 706 renal transplants, all of which functioned for at least 6 months. We used Cox proportional hazards analysis to examine the effects of multiple pretransplant and posttransplant risk factors and included time-dependent variables measured at 3, 6, and 12 months, and annually to last follow-up at 7.0 +/- 4.2 yr. The independent relative risk (RR) of diabetes was 3.25 for ischemic heart disease, 3.21 for cerebral vascular disease, and 28.18 peripheral vascular disease (P < 0.05). The RR of each acute rejection episode was 1.40 for ischemic heart disease and 1.24 for cerebral vascular disease. Among serum lipid levels, high-density lipoprotein cholesterol was the best predictor of ischemic heart disease (RR = 0.80 for each 10 mg/dL). Posttransplant ischemic heart disease was strongly predictive of cerebral (5.80) and peripheral vascular disease (5.22), whereas ischemic heart disease was predicted by posttransplant cerebral (8.25) and peripheral vascular disease (4.58). Other risk factors for vascular disease included age, gender, cigarette smoking, pretransplant splenectomy, and serum albumin. Hypertension and low-density lipoprotein cholesterol had no effect, perhaps because of aggressive pharmacologic treatment. Thus, the incidence of cardiovascular disease continues to be high after renal transplantation, and multiple risk factors suggest a number of possible strategies for more effective treatment and prevention.

[The status of the dentition in 2 Swiss population groups before the introduction of refined sugar]. / Gebisszustand bei zwei Schweizer Bevölkerungs-gruppen vor Einführung des raffinierten Zuckers.

Caries and parodontitis have been one of the most spread diseases of mankind. In an archaeological study, 53 graves of Bernese patricians were exhumed. 22 skulls of adults with 392 teeth were examined. They lived between the 16th and the 18th century in the community of Worb BE. In some cases identification could be accomplished by comparison of oil paintings with skulls, using a computerized technique. The aim of this study was to evaluate the prevalence of caries, the loss of periodontal bone, the amount of calculus and of abrasion. To compare the dental situation with another population segment sex- and age-matched dates were used. This second population segment lived between the 12th and 18th century and belonged to a lower social class than the patricians from Worb. The comparison of these two groups showed significant differences only in the abrasion pattern. We conclude that the social class did not significantly influence most of the parameters studied here. The different abrasion pattern could be due to different dietary habits.