Gideon Zamir

Nucleotide deficiency promotes genomic instability in early stages of cancer development.

Chromosomal instability in early cancer stages is caused by stress on DNA replication. The molecular basis for replication perturbation in this context is currently unknown. We studied the replication dynamics in cells in which a regulator of S phase entry and cell proliferation, the Rb-E2F pathway, is aberrantly activated. Aberrant activation of this pathway by HPV-16 E6/E7 or cyclin E oncogenes significantly decreased the cellular nucleotide levels in the newly transformed cells. Exogenously supplied nucleosides rescued the replication stress and DNA damage and dramatically decreased oncogene-induced transformation. Increased transcription of nucleotide biosynthesis genes, mediated by expressing the transcription factor c-myc, increased the nucleotide pool and also rescued the replication-induced DNA damage. Our results suggest a model for early oncogenesis in which uncoordinated activation of factors regulating cell proliferation leads to insufficient nucleotides that fail to support normal replication and genome stability.

Identification of tissue-specific cell death using methylation patterns of circulating DNA

Significance We describe a blood test for detection of cell death in specific tissues based on two principles: (i) dying cells release fragmented DNA to the circulation, and (ii) each cell type has a unique DNA methylation pattern. We have identified tissue-specific DNA methylation markers and developed a method for sensitive detection of these markers in plasma or serum. We demonstrate the utility of the method for identification of pancreatic β-cell death in type 1 diabetes, oligodendrocyte death in relapsing multiple sclerosis, brain cell death in patients after traumatic or ischemic brain damage, and exocrine pancreas cell death in pancreatic cancer or pancreatitis. The approach allows minimally invasive monitoring of tissue dynamics in humans in multiple physiological and pathological conditions. Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic β-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics.

Providing more through less: current methods of retraction in SIMIS and NOTES cholecystectomy

BackgroundAs the field of minimally invasive surgery continues to develop, surgeons are confronted with the challenge of performing conventional laparoscopic surgeries through fewer incisions while maintaining the same degree of safety and surgical efficiency. Most of these methods involve elimination of the ports previously designated for retraction. As a result, minimally invasive surgeons have been forced to develop minimally invasive and ingenious methods for providing adequate retraction for these procedures. Herein we present our experience using endoloops and internal retractors to provide retraction during Single Incision Minimally Invasive Surgery (SIMIS) and Natural Orifice Transluminal Endoscopic Surgery (NOTES) cholecystectomy. We also present a review of the alternative retraction methods currently being employed for these surgeries.MethodsSIMIS was performed on 20 patients and NOTES was performed on 5 patients at our institution. Endoloops or internal retractors were used to provide retraction for all SIMIS procedures. Internal retractors provided retraction for all NOTES procedures.ResultsSuccessful cholecystectomy was accomplished in all cases. One SIMIS surgery required conversion to standard laparoscopy due to complex anatomy. There were no intraoperative complications. Although adequate retraction was accomplished in all cases, the internal retractors were found to provide superior and more versatile retraction compared to that of endoloops.ConclusionAdequate retraction greatly simplifies SIMIS and NOTES surgery. Endograb internal retractors were easy to use and were found to provide optimal retraction and exposure during these procedures without complications.

Phosphorylation of ribosomal protein S6 attenuates DNA damage and tumor suppression during development of pancreatic cancer

The signaling pathways that mediate the development of pancreatic ductal adenocarcinoma (PDAC) downstream of mutant Kras remain incompletely understood. Here, we focus on ribosomal protein S6 (rpS6), an mTOR effector not implicated previously in cancer. Phosphorylation of rpS6 was increased in pancreatic acinar cells upon implantation of the chemical carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) or transgenic expression of mutant Kras. To examine the functional significance of rpS6 phosphorylation, we used knockin mice lacking all five phosphorylatable sites in rpS6 (termed rpS6(P-/-) mice). Strikingly, the development of pancreatic cancer precursor lesions induced by either DMBA or mutant Kras was greatly reduced in rpS6(P-/-) mice. The rpS6 mutants expressing oncogenic Kras showed increased p53 along with increased staining of γ-H2AX and 53bp1 (Trp53bp1) in areas of acinar ductal metaplasia, suggesting that rpS6 phosphorylation attenuates Kras-induced DNA damage and p53-mediated tumor suppression. These results reveal that rpS6 phosphorylation is important for the initiation of pancreatic cancer.

Complications of high grade liver injuries: management and outcomewith focus on bile leaks.

BackgroundAlthough liver injury scale does not predict need for surgical intervention, a high-grade complex liver injury should alert the physician to expect an increased risk of hepatic complications following trauma. The aim of the current study was to define hepatic related morbidity in patients sustaining high-grade hepatic injuries that could be safely managed non-operatively.Patients and methodsThis is a retrospective study of patients with liver injury admitted to Hadassah-Hebrew University Medical Centre over a 10-year period. Grade 3-5 injuries were considered to be high grade. Collected data included the number and types of liver-related complications. Interventions which were required for these complications in patients who survived longer than 24 hours were analysed.ResultsOf 398 patients with liver trauma, 64 (16%) were found to have high-grade liver injuries. Mechanism of injury was blunt trauma in 43 cases, and penetrating in 21. Forty patients (62%) required operative treatment. Among survivors 22 patients (47.8%) developed liver-related complications which required additional interventional treatment. Bilomas and bile leaks were diagnosed in 16 cases post-injury. The diagnosis of bile leaks was suspected with abdominal CT scan, which revealed intraabdominal collections (n = 6), and ascites (n = 2). Three patients had continuous biliary leak from intraabdominal drains left after laparotomy. Nine patients required ERCP with biliary stent placement, and 2 required percutaneous transhepatic biliary drainage. ERCP failed in one case. Four angioembolizations (AE) were performed in 3 patients for rebleeding. Surgical treatment was found to be associated with higher complication rate. AE at admission was associated with a significantly higher rate of biliary complications. There were 24 deaths (37%), the majority from uncontrolled haemorrhage (18 patients). There were only 2 hepatic-related mortalities due to liver failure.ConclusionsA high complication rate following high-grade liver injuries should be anticipated. In patients with clinical evidence of biliary complications, CT scan is a useful diagnostic and therapeutic tool. AE, ERCP and temporary internal stenting, together with percutaneous drainage of intra-abdominal or intrahepatic bile collections, represents a safe and effective strategy for the management of complications following both blunt and penetrating hepatic trauma.

Efficiency of Cell-Free and Cell-Associated Virus in Mucosal Transmission of Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus

ABSTRACT Effective strategies are needed to block mucosal transmission of human immunodeficiency virus type 1 (HIV-1). Here, we address a crucial question in HIV-1 pathogenesis: whether infected donor mononuclear cells or cell-free virus plays the more important role in initiating mucosal infection by HIV-1. This distinction is critical, as effective strategies for blocking cell-free and cell-associated virus transmission may be different. We describe a novel ex vivo model system that utilizes sealed human colonic mucosa explants and demonstrate in both the ex vivo model and in vivo using the rectal challenge model in rhesus monkeys that HIV-1-infected lymphocytes can transmit infection across the mucosa more efficiently than cell-free virus. These findings may have significant implications for our understanding of the pathogenesis of mucosal transmission of HIV-1 and for the development of strategies to prevent HIV-1 transmission.

Results of reconstruction in major pelvic and extremity venous injuries.

PURPOSE Outcome and venous patency after reconstruction in major pelvic and extremity venous injuries was studied. METHODS We retrospectively reviewed 46 patients with 47 venous injuries. RESULTS Injuries were caused by penetrating trauma in 37 extremities, blunt trauma in 6 patients, and were iatrogenic in 4 patients. Pelvic veins were injured in 4 patients, lower-extremity veins were injured in 39 limbs in 38 patients, and upper-extremity veins were injured in 4 patients. Concomitant arterial injuries occurred in 37 patients. Venous repairs were mostly of the complex type and included spiral or panel grafts in 15 (32%) reconstructions, interposition grafts or patch venoplasty in 19 (40%) reconstructions, end-to-end and lateral repair in 11 patients, and ligation in 2 patients. Two patients underwent early amputation. Early transient limb edema occurred in 2 patients, and postoperative venous occlusions were documented in 4 patients. Full function was regained in 39 (81%) extremities. No variable, including 4 retrospectively applied extremity injury scores (mangled extremity severity score [MESS], limb salvage index [LSI], mangled extremity syndrome index [MESI], predictive salvage index [PSI]), correlated with outcome. High values on all 4 scores were significantly associated with reexplorations (P <.02), which were done in 8 patients for debridement (5), arrest of bleeding (2), and repair of a missed arterial injury (1). Follow-up of 28 +/- 6 months on 27 patients (57%; duplex scan in 18, continuous-wave Doppler and plethysmography in 9, and venography in 3) showed 1 occlusion 6 weeks after the injury and patency of all other venous reconstructions. CONCLUSION Reconstructions of major venous injuries with a high rate of complex repairs result in a large proportion of fully functional limbs and a high patency rate. A high extremity injury score predicts the need for reexploration of the extremity. Mostocclusions occur within weeks of injury, and the subsequent delayed occlusion rate is very low.

Vascular complications post orthotopic liver transplantation.

From October 1991 to May 1998, 63 OLT in 57 patients were performed at Hadassah Hebrew University Medical Center. These included 58 whole liver transplantations, four reduced size, and one living-related liver transplantation. There were 25 females and 32 males with a mean age of 40.7 year (range 3.5–68). The follow-up was a mean of 21 months (range of 0.1–78). In 52 OLT donor liver arterial inflow was based on recipient hepatic artery (HA), and in 10 OLT on recipient aorta (Ao). In one OLT, this information was not available. Portal vein (PV) reconstruction was by end-to-end anastomosis in all transplants except in one OLT with recipient PV thrombosis (PVT), in which an interposition donor iliac vein was used. Inferior vena cava (IVC) reconstruction was performed in a standard manner in 53 OLT, and piggy-back in 10 OLT. Vascular complications were diagnosed based on clinical, ultrasonographic, angiographic, and operative findings. Statistical analysis was performed using Fisher’s exact test with significance accepted at P , .05.

Intra-arterial thrombolysis and stent placement for traumatic carotid dissection with subsequent stroke: a combined, simultaneous endovascular approach.

Traumatic carotid dissection is a well-known cause of ischemic stroke and although the treatment of the dissection itself has received some attention in recent years, the treatment of the concomitant stroke has been less investigated. We present a 43-year-old patient with blunt traumatic internal carotid artery dissection associated with subocclusive stenosis and major cerebral thromboembolic complication. Combined, simultaneous intra-arterial fibrinolysis and carotid stenting through a bilateral approach was successfully performed allowing the complete clinical recovery of the patient. Contralateral carotid artery approach allowed the beginning of intra-arterial thrombolysis without delay, while stent-assisted angioplasty of the injured carotid was simultaneously performed. This approach was proved to be safe and effective and may deserve further evaluation.

Torture rhabdomyorhexis--a pseudo-crush syndrome.

Two patients who were systematically tortured and deprived of any oral intake presented with acute renal failure several days later. Unlike the classical crush syndrome, we describe a clinical entity wherein repeated direct muscle injury from blunt trauma, in addition to forced dehydration, led to myoglobinuria and renal failure. The literature is reviewed, and biochemical indices predicting severity of injury, pathophysiology, and management protocol are described. This pseudo-crush syndrome caused by rhabdomyorhexis in addition to rhabdomyolysis is an unusual entity, in part related to extreme sociopolitical factors.