Whole-genome sequencing reveals activation-induced cytidine deaminase signatures during indolent chronic lymphocytic leukaemia evolution.

Patients with chromosome 13q deletion or normal cytogenetics represent the majority of chronic lymphocytic leukaemia (CLL) cases, yet have relatively few driver mutations. To better understand their genomic landscape, here we perform whole-genome sequencing on a cohort of patients enriched with these cytogenetic characteristics. Mutations in known CLL drivers are seen in only 33% of this cohort, and associated with normal cytogenetics and unmutated IGHV. The most commonly mutated gene in our cohort, IGLL5, shows a mutational pattern suggestive of activation-induced cytidine deaminase (AID) activity. Unsupervised analysis of mutational signatures demonstrates the activities of canonical AID (c-AID), leading to clustered mutations near active transcriptional start sites; non-canonical AID (nc-AID), leading to genome-wide non-clustered mutations, and an ageing signature responsible for most mutations. Using mutation clonality to infer time of onset, we find that while ageing and c-AID activities are ongoing, nc-AID-associated mutations likely occur earlier in tumour evolution.

Cardiac and great vessel injuries in children after blunt trauma: an institutional review.

PURPOSE: The purpose of this study was to review the incidence of cardiac and great vessel injury after blunt trauma in children. METHOD: A retrospective review of 2,744 patients with injuries from blunt mechanisms was performed. RESULTS: Eleven patients sustained cardiac injury. Four patients had clinically evident cardiac contusions. All recovered. Four patients who died from central nervous system injury were found to have cardiac contusions at autopsy. None had clinical evidence of contusion before demise. One patient had a traumatic ventricular septal defect (VSD) that required operative repair. Autopsy findings showed a VSD in another patient, and a third patient was found to have a ventricular septal aneurysm that was treated medically. Two patients had great vessel injuries. One patient had a contained disruption of the superior vena cava that was managed nonoperatively. Another patient had a midthoracic periaortic hematoma without intimal disruption found at autopsy. One patient had cardiac and great vessel injuries. Discrete aneurysms of 2 coronary artery branches and the pulmonary outflow tract were identified by cardiac catheterization. This patient was treated nonoperatively. CONCLUSIONS: Cardiac and great vessel injury after blunt trauma are uncommon in children. Cardiac contusion was the most common injury encountered but had minimal clinical significance. Noncontusion cardiac injury is rare. No patient with aortic transection was identified.

Zone I retroperitoneal hematoma identified by computed tomography scan as an indicator of significant abdominal injury.

OBJECTIVE: All zone I retroperitoneal hematomas (Z1RPHs) identified at laparotomy for blunt trauma traditionally require exploration. The purpose of this study was to correlate patient outcome after blunt abdominal trauma with the presence of Z1RPH diagnosed on admission computed tomography (CT) scan. METHODS: This is a retrospective review of patients with blunt trauma who were admitted to a Level 1 trauma center and who underwent CT scan during a 40-month period. All scans with a traumatic injury were reviewed to identify and grade Z1RPH as mild, moderate, or severe. Patients requiring operative treatment were compared with those who were observed. Statistical analysis was performed with Student's t test and chi-square test, with P < .05 considered significant. RESULTS: Eighty-five (15.5%) of the CT scans were positive for Z1RPH. None of the 50 patients with a mild Z1RPH had their treatment altered. Of the 29 patients with a moderate or severe Z1RPH, 8 required celiotomy. The patients requiring celiotomy had significant elevations of solid viscus score (SVS) (4.9 +/- 1.6 versus 1.8 +/- 0.3), abdominal Abbreviated Injury Scale (3.8 +/- 0.3 versus 2.6 +/- 0.3), and transfusion requirements (13 +/- 4 versus 2 +/- 1). All patients (N = 4) with an SVS >4 required operative treatment. Seventy-two percent of patients with more than 1 intra-abdominal injury required abdominal exploration. CONCLUSIONS: The presence of a moderate or severe Z1RPH and more than 1 intra-abdominal injury or an SVS >4 on admission CT scan is an important radiographic finding. This injury pattern should be considered a contraindication for nonoperative treatment of the associated solid organ injury.

Addisonian crisis in a liver transplant patient due to fluconazole withdrawal.

Fluconazole is an antifungal agent commonly used in liver transplant patients. In addition to its antifungal activity, it is a potent inhibitor of the liver cytochrome P450 enzymes. These enzymes degrade a wide range of metabolically active compounds including glucocorticoids. In this report, we identify an episode of Addisonian crisis that occurred in a liver transplant patient receiving prednisone immunosuppression after fluconazole was discontinued. We postulate the mechanism for the crisis was a reversal of the fluconazole-induced suppression of the P450 enzymes. The resulting increased activity altered the patient's glucocorticoid metabolism leading to an Addisonian crisis.

The molecular regulation of protein breakdown following burn injury is different in fast- and slow-twitch skeletal muscle.

We compared the effect of burn injury on the energy-ubiquitin-dependent proteolytic pathway in the fast-twitch extensor digitorum longus (EDL) and the slow-twitch soleus muscle in rats. Rats were subjected to a 30% total body surface area full-thickness burn or sham procedure. At various time points after injury, total and myofibrillar protein breakdown rates were determined in incubated EDL and soleus muscles. The energy-dependent component of protein break-down was determined by incubating muscles in energy-depleting medium. Messenger RNA levels for ubiquitin and RC3, a 20S proteasome subunit, were measured by Northern blot analysis. Burn injury resulted in an approximately 50% increase in total protein breakdown and a 3-4 fold increase in myofibrillar protein breakdown in EDL muscles, and this response reflected increased energy-dependent protein breakdown. In contrast, protein breakdown rates were not significantly influenced by the burn injury in soleus muscles. Ubiquitin mRNA levels were increased almost 10-fold in EDL and approximately 4.5-fold in soleus muscles following burn injury. Burn injury resulted in a 2-fold increase in RC3 mRNA in EDL with no significant changes noted in soleus muscles. The results suggest that the more pronounced effect of burn injury on protein breakdown in fast-twitch than in slow-twitch muscle may reflect different regulation of proteolysis at the molecular level.

Endotoxemia in mice stimulates production of complement C3 and serum amyloid A in mucosa of small intestine.

We examined the effect of endotoxemia in mice on protein and mRNA levels for the acute phase proteins complement C3 and serum amyloid A (SAA) in jejunal mucosa. Endotoxemia was induced in mice by the subcutaneous injection of 250 microg lipopolysaccharide per mouse. Control mice were injected with saline. C3 and SAA were measured by ELISA. Messenger RNA levels were determined by Northern blot analysis or competitive PCR. Immunohistochemistry was performed to determine in which cell type(s) C3 and SAA were present. Mucosal C3 and SAA protein and mRNA levels were increased in endotoxemic mice. Immunohistochemistry showed that C3 was present in both enterocytes and cells of the lamina propria, whereas SAA was seen mainly in lamina propria cells. Results suggest that endotoxemia stimulates production of C3 and SAA in small intestinal mucosa. The response may be regulated at the transcriptional level and probably reflects increased synthesis of the acute phase proteins in both enterocytes and cells of the lamina propria.

Sepsis-induced increase in muscle proteolysis is blocked by specific proteasome inhibitors.

Recent studies suggest that sepsis stimulates ubiquitin-dependent protein breakdown in skeletal muscle. The 20S proteasome is the catalytic core of the ubiquitin-dependent proteolytic pathway. We tested the effects in vitro of the proteasome inhibitors N-acetyl-L-leucinyl-L-leucinal-L-norleucinal (LLnL) and lactacystin on protein breakdown in incubated muscles from septic rats. LLnL resulted in a dose- and time-dependent inhibition of protein breakdown in muscles from septic rats. Lactacystin blocked both total and myofibrillar muscle protein breakdown. In addition to inhibiting protein breakdown, LLnL reduced muscle protein synthesis and increased ubiquitin mRNA levels, probably reflecting inhibited proteasome-associated ribonuclease activity. Inhibited muscle protein breakdown caused by LLnL or lactacystin supports the concept that the ubiquitin-proteasome pathway plays a central role in sepsis-induced muscle proteolysis. The results suggest that muscle catabolism during sepsis may be inhibited by targeting specific molecular mechanisms of muscle proteolysis.

Intraoperative events common to videoscopic preperitoneal mesh inguinal herniorrhaphy.

BACKGROUND: Videoscopic preperitoneal mesh (VPM) inguinal herniorrhaphy avoids the entry into the abdominal cavity, which is necessary with other videoscopic techniques. Despite this advantage, surgeons have been slow to adopt this technique. We reviewed our experience with VPM inguinal herniorrhaphy, specifically investigating the technical aspects of this approach. METHODS: Data were collected prospectively. Operative notes were reviewed retrospectively detailing intraoperative events not "typical" with the VPM technique. RESULTS: One hundred consecutive patients undergoing VPM repair of 127 hernias were studied. The repair was completed in all but 2 patients. Mean operating time was 120 minutes (60 to 146). In 36 repairs there were 59 intraoperative "events" requiring specific maneuvers to correct. Events identified were the need for transection of the hernia sac, creation and repair of a peritoneal tear, and need to divide the inferior epigastric vessels. No complications related to these events occurred. When events occurred, operative times were significantly longer (146+/-45 versus 83+/-23 minutes; P <0.05). CONCLUSION: Intraoperative events are common with VPM herniorrhaphy. These events significantly prolong operating time. A surgeon's lack of familiarity with such events and how to deal with them may in part explain the reluctance to widely apply the VPM technique.

Sepsis and endotoxaemia in mice stimulate the expression of interleukin-I and interleukin-6 in the central nervous system.

1. In previous studies, experimental endotoxaemia was found to stimulate cytokine production in the central nervous system. The effect of sepsis on brain cytokines is not fully known. We compared the effect of endotoxaemia and sepsis on brain interleukin-1 and interleukin-6 expression. 2. Male A/J mice were injected subcutaneously with lipopolysaccharide (10 mg/kg) or an equal volume of saline as control. Sepsis was induced by caecal ligation and puncture (CLP); control mice underwent sham-operation. Brain tissue was assayed for interleukin-1 and interleukin-6 by ELISA. Northern blotting or the polymerase chain reaction was used to determine cytokine mRNA levels. 3. Administration of endotoxin induced a greater than fourfold increase in brain interleukin-1, a greater than threefold increase in interleukin-6 and an increase in mRNA for both cytokines. Caecal ligation and puncture resulted in increased brain interleukin-1 and interleukin-6 levels, but the changes were less pronounced and occurred later than after injection of endotoxin. There was no detectable difference in brain interleukin-1 mRNA between septic and sham-operated mice, whereas interleukin-6 mRNA was increased in brains of septic animals. 4. Sepsis and endotoxaemia resulted in similar, although not identical, changes in brain interleukin-1 and interleukin-6 concentrations and mRNA levels, suggesting that increased cytokine production in the central nervous system is part of the systemic response to sepsis and may be mediated by endotoxin.

Intracellular regulation of protein degradation during sepsis is different in fast- and slow-twitch muscle.

We tested the hypothesis that the difference in the response to sepsis of protein breakdown between fast- and slow-twitch skeletal muscle reflects differential activation of the energy-ubiquitin-dependent proteolytic pathway. In addition, we defined the time course and the tissue specificity of sepsis-induced changes in the expression of the ubiquitin pathway. Sepsis was induced in rats by cecal ligation and puncture; control rats were sham operated. Energy-dependent protein breakdown was measured in incubated extensor digitorum longus (EDL) and soleus muscles. Ubiquitin mRNA levels were determined by Northern blot analysis. Sepsis resulted in increased energy-dependent protein breakdown and upregulated expression of ubiquitin mRNA in the fast-twitch EDL but not in the slow-twitch soleus muscle. The sepsis-induced increase in ubiquitin mRNA levels in the EDL muscle was noticeable before the increase in energy-dependent protein breakdown. Sepsis increased ubiquitin mRNA levels in the diaphragm (a mixed fiber-type muscle) but not in heart, liver, kidney, or intestine, consistent with a tissue-specific regulation of the ubiquitin system during sepsis. The results suggest that the difference in protein breakdown during sepsis between fast- and slow-twitch muscles reflects differential activation of the energy-ubiquitin-dependent proteolytic pathway. The data also suggest that the expression of the ubiquitin pathway is upregulated in a time-dependent fashion during sepsis and that this response is not a generalized phenomenon but is tissue specific.

Sepsis is associated with increased mRNAs of the ubiquitin-proteasome proteolytic pathway in human skeletal muscle.

Previous studies provided evidence that sepsis-induced muscle proteolysis in experimental animals is caused by increased ubiquitin-proteasome-dependent protein breakdown. It is not known if a similar mechanism accounts for muscle proteolysis in patients with sepsis. We determined mRNA levels for ubiquitin and the 20 S proteasome subunit HC3 by Northern blot analysis in muscle tissue from septic (n = 7) and non-septic (n = 11) patients. Plasma and muscle amino acid concentrations and concentrations in urine of 3-methylhistidine (3-MH), creatinine, and cortisol were measured at the time of surgery to assess the catabolic state of the patients. A three- to fourfold increase in mRNA levels for ubiquitin and HC3 was noted in muscle tissue from the septic patients concomitant with increased muscle levels of phenylalanine and 3-MH and reduced levels of glutamine. Total plasma amino acids were decreased by approximately 30% in the septic patients. The 3-MH/creatinine ratio in urine was almost doubled in septic patients. The cortisol levels in urine were higher in septic than in control patients but this difference did not reach statistical significance. The results suggest that sepsis is associated with increased mRNAs of the ubiquitin-proteasome pathway in human skeletal muscle.

Sepsis increases oxidatively damaged proteins in skeletal muscle.

BACKGROUND: Muscle wasting and negative nitrogen balance are common findings in septic patients. It is not clear what signals this loss of body protein. Proteins modified by reactive oxygen species have been shown to be rapidly degraded. OBJECTIVE: To test the hypothesis that sepsis results in an increased amount of oxidatively damaged proteins in skeletal muscle. METHODS: Exposure of proteins to reactive oxygen species results in the incorporation of carbonyl groups into amino acids with metal binding sites. The formation of carbonyl group derivatives in sarcoplasmic and myofibrillar proteins was measured in the extensor digitorum longus and soleus muscles of septic rats 4 to 48 hours after cecal ligation and puncture and in control rats that underwent sham operation. RESULTS: Protein carbonyl content was increased 8 and 16 hours after cecal ligation and puncture in the extensor digitorum longus and soleus muscles, respectively. When muscles were incubated in vitro, the carbonyl content in protein decreased in muscles from septic rats but not in muscles from rats that had the sham operation. The loss of carbonyl groups in incubated septic muscles occurred also in energy-depleted muscles. CONCLUSIONS: Muscle proteins are oxidatively damaged during sepsis and an energy-independent proteolytic pathway participates in the degradation of these proteins. Damage to muscle proteins by reactive oxygen species may signal the selective removal of postsynthetically modified proteins, contributing to accelerated muscle protein degradation in sepsis.

Identification of altered gene expression in skeletal muscle during sepsis using differential display.

Different aspects of muscle metabolism are altered during sepsis and there is evidence that some of these changes may be regulated at the gene level. Differential display is a recently described technique to identify genes whose expression has changed during a biological process. This technique utilizes reverse transcriptase-polymerase chain reaction (RT-PCR) to compare mRNA signals in tissues during two different conditions. We used differential display to test the hypothesis that gene expression is altered in skeletal muscle during sepsis. Sepsis was induced in rats by cecal ligation and puncture (CLP). Control rats were sham-operated. Sixteen hours after CLP or sham operation, extensor digitorum longus muscles were harvested and RNA was extracted. Following differential display, 30 fragments (F1-F30) were identified that appeared to be uniquely expressed in muscles from sham-operated or septic rats. These fragments were reamplified by PCR and used as probes in Northern blot analysis. Messenger RNA levels corresponding to 2 of the 30 fragments (F5 and F24) were confirmed to be increased by Northern blot analysis in septic muscle. Following cloning and sequencing, F5 was found to display significant homology to the gene sequence of the guanine nucleotide releasing protein MSS4. The sequence of F24 did not match any reported gene sequence and may therefore represent a previously unidentified gene. The results support the hypothesis that gene expression is altered in skeletal muscle during sepsis.

Sepsis increases putrescine concentration and protein synthesis in mucosa of small intestine in rats.

Recent studies suggest that sepsis stimulates mucosal polyamine and protein synthesis. It is not known in which cell type polyamine biosynthesis is increased during sepsis and if polyamines regulate mucosal protein synthesis. We examined the effect of sepsis in rats on polyamine biosynthesis in isolated jejunal enterocytes and measured mucosal protein synthesis following inhibition of ornithine decarboxylase (ODC) activity with difluoromethylornithine. ODC and S-adenosylmethionine decarboxylase (SAMDC) activities and putrescine concentrations were increased in isolated jejunal enterocytes 16 h after induction of sepsis by cecal ligation and puncture. Enterocyte spermidine and spermine levels were not influenced by sepsis. Mucosal ODC and SAMDC activities and polyamine levels were increased following treatment of rats with interleukin-1 but not tumor necrosis factor. Treatment of rats with difluoromethylornithine prevented the sepsis-induced increase in mucosal ODC activity, putrescine concentration, and protein synthesis rate. The results suggest that sepsis increases ODC and SAMDC activities and putrescine concentrations in enterocytes of the small intestine. This metabolic response to sepsis may be regulated by interleukin-1 although other mechanisms may also be involved. Increased mucosal protein synthesis during sepsis may at least in part be regulated by increased putrescine levels.

Influence of sepsis and endotoxemia on polyamine metabolism in mucosa of small intestine in rats.

We examined the influence of sepsis and endotoxemia in rats on the biosynthesis of polyamines in small-intestinal mucosa. Sepsis was induced by cecal ligation and puncture (CLP); control rats were sham-operated. In other experiments, rats were treated with two subcutaneous injections of endotoxin (1 mg/kg) or corresponding injections of sterile saline. Ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) activities and concentrations of putrescine, spermidine, and spermine were measured in jejunal mucosa at intervals during 16 hours. Sepsis stimulated ODC and SAMDC activities and increased putrescine and spermidine concentrations in jejunal mucosa. Injection of endotoxin resulted in metabolic changes similar to those observed following CLP. The results suggest that sepsis and endotoxemia stimulate polyamine biosynthesis in mucosa of small intestine. The role of polyamines in the regulation of cell proliferation and metabolic changes in the intestinal mucosa during sepsis remains to be determined.

Energy-ubiquitin-dependent muscle proteolysis during sepsis in rats is regulated by glucocorticoids.

Recent studies suggest that sepsis-induced increase in muscle proteolysis mainly reflects energy-ubiquitin-dependent protein breakdown. We tested the hypothesis that glucocorticoids activate the energy-ubiquitin-dependent proteolytic pathway in skeletal muscle during sepsis. Rats underwent induction of sepsis by cecal ligation and puncture or were sham-operated and muscle protein breakdown rates were measured 16 h later. The glucocorticoid receptor antagonist RU 38486 or vehicle was administered to groups of septic and sham-operated rats. In other experiments, dexamethasone (2.5 or 10 mg/kg) was injected subcutaneously in normal rats. Total and myofibrillar proteolysis was determined in incubated extensor digitorum longus muscles as release of tyrosine and 3-methylhistidine, respectively. Energy-dependent proteolysis was determined in incubated muscles depleted of energy with 2-deoxyglucose and 2,4-dinitrophenol. Levels of muscle ubiquitin mRNA and free and conjugated ubiquitin were determined by Northern and Western blot, respectively. RU 38486 inhibited the sepsis-induced increase in total and myofibrillar energy-dependent protein breakdown rates and blunted the increase in ubiquitin mRNA levels and free ubiquitin. Some, but not all, sepsis-induced changes in ubiquitin protein conjugates were inhibited by RU 38486. Injection of dexamethasone in normal rats increased energy-dependent proteolysis and ubiquitin mRNA levels. The results suggest that glucocorticoids regulate the energy-ubiquitin-dependent proteolytic pathway in skeletal muscle during sepsis.

Sepsis stimulates polyamine biosynthesis in the liver and increases tissue levels of ornithine decarboxylase mRNA.

The influence of sepsis on polyamine metabolism in the liver was studied in rats. Sepsis was induced by cecal ligation and puncture; control rats were sham-operated. Sepsis resulted in increased concentrations in liver tissue of putrescine and spermidine and stimulated activity of the enzymes ornithine decarboxylase (ODC) and s-adenosylmethionine decarboxylase. A similar metabolic response was seen following the subcutaneous injection of 1 mg/kg of endotoxin or following the e intraperitoneal injection of 100 micrograms/kg of human recombinant tumor necrosis factor (TNF)-alpha or interleukin-1 alpha (IL-1 alpha). ODC mRNA levels determined by Northern blots were increased in liver tissue of septic rats, suggesting that the increase in ODC activity may be regulated at the transcriptional level although increased stability of the messenger could give rise to similar results. Treatment of rats with either TNF antiserum, recombinant IL-1 receptor antagonist, or the glucocorticoid receptor antagonist RU 38486, did not prevent the sepsis-induced increase in hepatic ODC activity. The data suggest that sepsis stimulates the biosynthesis of polyamines in liver tissue and that this response to sepsis may not primarily be mediated by TNF, IL-1, or glucocorticoids. The biological role of increased liver polyamines during sepsis, in particular their relationship with the synthesis of acute phase proteins, remains to be determined.

Sepsis and endotoxemia stimulate intestinal interleukin-6 production.

BACKGROUND: Endotoxemia stimulates tumor necrosis factor (TNF) and interleukin-1 (IL-1) production in mucosa of the small intestine, but the effect on IL-6 production is not known. Intestinal IL-6 may be especially important, considering its role in the acute phase response. We tested the influence of endotoxemia and sepsis in mice on intestinal IL-6 and IL-6 messenger RNA (mRNA) levels. METHODS: Mice were injected with lipopolysaccharide (LPS 10 mg/kg) or saline solution. In some experiments animals were pretreated with indomethacin (5 mg/kg) or N-nitro-L-arginine (NNA, 100 mg/kg) before LPS injection. In other experiments, sepsis was induced by cecal ligation and puncture (CLP); controls were sham operated. Serum and jejunal mucosa were harvested at intervals during 16 hours, and IL-6 levels were determined by enzyme-linked immunosorbent assay. IL-6 mRNA was detected by polymerase chain reaction. RESULTS: Endotoxemia and sepsis increased serum and mucosal IL-6 and IL-6 mRNA, with maximum levels noted at 1 and 4 hours after LPS and at 8 hours after CLP. Pretreatment of endotoxemic mice with indomethacin or NNA blunted the increase in mucosal IL-6. CONCLUSIONS: Results suggest that sepsis and endotoxemia stimulate IL-6 production in small intestinal mucosa and that this response may be transcriptionally regulated. The effect of endotoxemia may be partly mediated by prostaglandins and nitric oxide. The results also suggest that the intestinal mucosa may be a participant in the cytokine response, rather than just a passive bystander.

Nitric oxide inhibits LPS-induced IL-6 production in enterocytes.

In recent studies, production of interleukin-6 (IL-6) in cultured enterocytes was stimulated by lipolysaccharide (LPS). In other cell types, IL-6 production was inhibited by nitric oxide (NO). We tested the hypothesis that LPS-induced IL-6 production in the enterocyte is regulated, at least in part, by NO. IEC-6 cells (a rat intestinal epithelial cell line) were cultured for 3 days with different combinations of LPS (1-10 micrograms/ml), the NO synthase inhibitor N-omega-nitro-L-arginine (NNA, 3-300 microM), L-arginine (10 mM), the NO donor sodium nitroprusside (SNP, 0.5-1 microM), or medium alone as control. IL-6 levels in the culture medium were determined by the B9 murine hybridoma bioassay. Nitrite, a stable end product of NO metabolism, was measured by HPLC. PCR was performed to determine inducible NO synthase (iNOS) mRNA expression in the IEC-6 cells. Treatment of IEC-6 cells with LPS stimulated IL-6 production. LPS-induced IL-6 production was further increased by NNA in a dose-dependent fashion. This effect of NNA was abolished by the addition of L-arginine. SNP caused a dose-dependent decrease in IL-6 production. Nitrite production was increased in a dose-dependent fashion after LPS treatment. PCR revealed an increase in iNOS mRNA expression in IEC-6 cells after administration of 1 microgram/ml LPS. The results suggest that NO inhibits LPS-induced IL-6 production in the enterocyte. NO may be an important regulator of intestinal cytokine response during sepsis and endotoxemia.