Nutrient supply and nucleus pulposus cell function: effects of the transport properties of the cartilage endplate and potential implications for intradiscal biologic therapy.

OBJECTIVE: Intradiscal biologic therapy is a promising strategy for managing intervertebral disc degeneration. However, these therapies require a rich nutrient supply, which may be limited by the transport properties of the cartilage endplate (CEP). This study investigated how fluctuations in CEP transport properties impact nutrient diffusion and disc cell survival and function. DESIGN: Human CEP tissues harvested from six fresh cadaveric lumbar spines (38-66 years old) were placed at the open sides of diffusion chambers. Bovine nucleus pulposus (NP) cells cultured inside the chambers were nourished exclusively by nutrients diffusing through the CEP tissues. After 72 h in culture, depth-dependent NP cell viability and gene expression were measured, and related to CEP transport properties and biochemical composition determined using fluorescence recovery after photobleaching and Fourier transform infrared (FTIR) spectroscopy. RESULTS: Solute diffusivity varied nearly 4-fold amongst the CEPs studied, and chambers with the least permeable CEPs appeared to have lower aggrecan, collagen-2, and matrix metalloproteinase-2 gene expression, as well as a significantly shorter viable distance from the CEP/nutrient interface. Increasing chamber cell density shortened the viable distance; however, this effect was lost for low-diffusivity CEPs, which suggests that these CEPs may not provide enough nutrient diffusion to satisfy cell demands. Solute diffusivity in the CEP was associated with biochemical composition: low-diffusivity CEPs had greater amounts of collagen and aggrecan, more mineral, and lower cross-link maturity. CONCLUSIONS: CEP transport properties dramatically affect NP cell survival/function. Degeneration-related CEP matrix changes could hinder the success of biologic therapies that require increased nutrient supply.

Agitation in adults in the post-anaesthesia care unit after general anaesthesia.

BACKGROUND: Agitation after general anaesthesia can lead to self-harm, violence against staff, and increased resource utilisation. We aimed to assess patient and procedural characteristics associated with this complication in adults. METHODS: We identified cases of agitation (Richmond Agitation-Sedation Scale score +3 or +4, or administration of haloperidol) in patients after general anaesthesia in the PACU from July 1, 2010 to September 30, 2016. The cases were matched 1:1 with control patients without agitation by age, sex, and procedure. Potential clinical associations were assessed with a multivariable analysis. RESULTS: We identified agitation in 510 patients [incidence: 2.5 cases/1000 patients; 95% confidence interval (CI): 2.3-2.7]. Variables associated with agitation were substance misuse [odds ratio (OR): 6.77; 95% CI: 1.23-37.2; P=0.03], cognitive impairment (OR: 4.66; 95% CI: 1.79-12.1; P=0.002), obesity (OR: 2.49; 95% CI: 1.66-3.73; P<0.001), psychiatric problems (OR: 2.05; 95% CI: 1.32-3.19; P=0.002), fall risk (OR: 1.66; 95% CI: 1.02-2.70; P=0.04), postoperative presence of a tracheal tube (OR: 16.6; 95% CI: 7.25-38.2; P<0.001), urine catheter (OR: 7.25; 95% CI: 4.31-12.2; P<0.001), nasogastric tube (OR: 4.06; 95% CI: 1.51-10.9; P=0.006), or chest tube (OR: 3.46; 95% CI: 1.07-11.2; P=0.006). Compared with control patients, more agitated patients had postoperative delirium (16.1% vs 6.3%; P<0.001) and pulmonary complications (9.8% vs 4.7%; P=0.002). CONCLUSIONS: Agitation after general anaesthesia was associated with postoperative indwelling catheters, tracheal intubation and patient features suggestive of pre-existing mental health problems. Anticipation of high-risk patients could allow allocation of staffing resources to provide a safe environment for anaesthetic recovery.

Does bowel preparation for inflammatory bowel disease surgery matter?

AIM: The purpose of this study was to determine if bowel preparation influences outcomes in patients with inflammatory bowel disease undergoing surgery. METHODS: The database of the American College of Surgeons National Surgical Quality Improvement Program, Procedure Targeted Colectomy, from 2012 to 2014 was analyzed. Inflammatory bowel disease patients undergoing colorectal resection with or without bowel preparation were included in the study. RESULTS: In all, 3679 patients with inflammatory bowel disease were identified. 42.5% had no bowel preparation, 21.5% had mechanical bowel preparation only, 8.8% had oral antibiotic bowel preparation only and 27.2% had combined mechanical and oral antibiotic preparation. Combined mechanical and oral antibiotic preparation is associated with lower rates of anastomotic leak, ileus, surgical site infection, organ space infection, wound dehiscence and sepsis/septic shock. CONCLUSION: Combined mechanical and oral antibiotic preparation for inflammatory bowel disease patients undergoing colectomy is associated with decreased rates of surgical site infection, anastomotic leak, ileus. Combined bowel preparation should be the standard of care for inflammatory bowel disease patients undergoing colorectal resection.

Multistate outbreak of listeriosis caused by imported cheese and evidence of cross-contamination of other cheeses, USA, 2012.

Listeria monocytogenes is a foodborne pathogen that can cause bacteraemia, meningitis, and complications during pregnancy. In July 2012, molecular subtyping identified indistinguishable L. monocytogenes isolates from six patients and two samples of different cut and repackaged cheeses. A multistate outbreak investigation was initiated. Initial analyses identified an association between eating soft cheese and outbreak-related illness (odds ratio 17·3, 95% confidence interval 2·0-825·7) but no common brand. Cheese inventory data from locations where patients bought cheese and an additional location where repackaged cheese yielded the outbreak strain were compared to identify cheeses for microbiological sampling. Intact packages of imported ricotta salata yielded the outbreak strain. Fourteen jurisdictions reported 22 cases from March-October 2012, including four deaths and a fetal loss. Six patients ultimately reported eating ricotta salata; another reported eating cheese likely cut with equipment also used for contaminated ricotta salata, and nine more reported eating other cheeses that might also have been cross-contaminated. An FDA import alert and US and international recalls followed. Epidemiology-directed microbiological testing of suspect cheeses helped identify the outbreak source. Cross-contamination of cheese highlights the importance of using validated disinfectant protocols and routine cleaning and sanitizing after cutting each block or wheel.

Population structure and cryptic speciation in bonnethead sharks Sphyrna tiburo in the south-eastern U.S.A. and Caribbean.

Population structure and lineage diversification within a small, non-dispersive hammerhead shark species, the bonnethead shark Sphyrna tiburo, was assessed. Sphyrna tiburo is currently described as one continuously distributed species along the Atlantic continental margins of North, Central and South America, but recent genetic analysis of an insular population (Trinidad) suggests the possibility of cryptic speciation. To address this issue S. tiburo were sampled at six sites along c. 6200 km of continuous, continental coastline and from one island location (Grand Bahama) across a discontinuity in their distribution (the Straits of Florida), in order to test if they constitute a single lineage over this distribution. A total of 1030 bp of the mitochondrial control region (CR) was obtained for 239 S. tiburo, revealing 73 distinct haplotypes, high nucleotide diversity (0·01089) and a pair of highly divergent lineages estimated to have separated 3·61-5·62 million years ago. Mitochondrial cytochrome oxidase I and nuclear internal transcribed spacer loci show the same pattern. Divergence is similar within S. tiburo to that observed between established elasmobranch sister species, providing further evidence of cryptic speciation. A global AMOVA based on CR confirms that genetic diversity is primarily partitioned among populations (ΦST = 0·828, P < 0·001) because the divergent lineages are almost perfectly segregated between Belize and North America-The Bahamas. An AMOVA consisting solely of the North American and Bahamian samples is also significantly different from zero (ΦST = 0·088, P < 0·001) and pairwise FST is significantly different between all sites. These findings suggest that S. tiburo comprises a species complex and supports previous research indicating fine population structure, which has implications for fisheries management and biodiversity conservation.

2013 multistate outbreaks of Cyclospora cayetanensis infections associated with fresh produce: focus on the Texas investigations.

The 2013 multistate outbreaks contributed to the largest annual number of reported US cases of cyclosporiasis since 1997. In this paper we focus on investigations in Texas. We defined an outbreak-associated case as laboratory-confirmed cyclosporiasis in a person with illness onset between 1 June and 31 August 2013, with no history of international travel in the previous 14 days. Epidemiological, environmental, and traceback investigations were conducted. Of the 631 cases reported in the multistate outbreaks, Texas reported the greatest number of cases, 270 (43%). More than 70 clusters were identified in Texas, four of which were further investigated. One restaurant-associated cluster of 25 case-patients was selected for a case-control study. Consumption of cilantro was most strongly associated with illness on meal date-matched analysis (matched odds ratio 19·8, 95% confidence interval 4·0-∞). All case-patients in the other three clusters investigated also ate cilantro. Traceback investigations converged on three suppliers in Puebla, Mexico. Cilantro was the vehicle of infection in the four clusters investigated; the temporal association of these clusters with the large overall increase in cyclosporiasis cases in Texas suggests cilantro was the vehicle of infection for many other cases. However, the paucity of epidemiological and traceback information does not allow for a conclusive determination; moreover, molecular epidemiological tools for cyclosporiasis that could provide more definitive linkage between case clusters are needed.

Comparison of vertebral and intervertebral disc lesions in aging humans and rhesus monkeys.

OBJECTIVE: To compare gross and histologic patterns of age-related degeneration within the intervertebral disc and adjacent vertebra between rhesus monkeys and humans. MATERIALS AND METHODS: We examined age-related patterns of disc degeneration from mid-sagittal sections of the intervertebral disc and adjacent vertebral bodies (VB) among six rhesus monkey thoracolumbar and seven human lumbar spines. Gross morphology and histopathology were assessed via the Thompson grading scheme and other degenerative features of the disc and adjacent bone. RESULTS: Thompson grades ranged from 3 through 5 for rhesus monkey discs (T9-L1) and 2 through 5 for the human discs (T12-S1). In both rhesus monkey and human discs, presence of distinct lesions was positively associated with Thompson grade of the overall segment. Degenerative patterns differed for radial tears, which were more prevalent with advanced disc degeneration in humans only. Additionally, compared to the more uniform anteroposterior disc degeneration patterns of humans, rhesus monkeys showed more severe osteophytosis and degeneration on the anterior border of the vertebral column. CONCLUSIONS: Rhesus monkey spines evaluated in the present study appear to develop age-related patterns of disc degeneration similar to humans. One exception is the absence of an association between radial tears and disc degeneration, which could reflect species-specific differences in posture and spinal curvature. Considering rhesus monkeys demonstrate similar patterns of disc degeneration, and age at a faster rate than humans, these findings suggest longitudinal studies of rhesus monkeys may be a valuable model for better understanding the progression of human age-related spinal osteoarthritis (OA) and disc degeneration.

Low back pain patients with Modic type 1 changes exhibit distinct bacterial and non-bacterial subtypes.

Objectives: Modic type 1 changes (MC1) are vertebral endplate bone marrow (BM) lesions observed on magnetic resonance images in sub-populations of chronic low back pain (CLBP) patients. The etiopathogenesis remains unknown and treatments that modify the underlying pathomechanisms do not exist. We hypothesized that two biological MC1 subtypes exist: a bacterial and a non-bacterial. This would have important implications for developing treatments targeting the underlying pathomechanisms. Methods: Intervertebral disc (IVD) samples adjacent to MC1 (n â€‹= â€‹34) and control (n â€‹= â€‹11) vertebrae were collected from patients undergoing spinal fusion. Cutibacterium acnes (C.acnes) genome copy numbers (GCNs) were quantified in IVD tissues with 16S qPCR, transcriptomic signatures and cytokine profiles were determined in MC1 and control BM by RNA sequencing and immunoassay. Finally, we assessed if C.acnes GCNs are associated with blood plasma cytokines. Results: IVD tissues from control levels had <870 â€‹C.acnes GCNs/gram IVD. MC1-adjacent IVDs had either "low" (<870) or "high" (>870) C.acnes GCNs. MC1 patients with "high" C.acnes GCNs had upregulated innate immune cell signatures (neutrophil, macrophage/monocyte) and pro-inflammatory cytokines related to neutrophil and macrophage/monocyte function in the BM, consistent with a host defense against bacterium. MC1 patients with "low" C.acnes GCNs had increased adaptive immune cell signatures (T-and B-cell) in the BM and elevated IL-13 blood plasma levels. Conclusion: Our study provides the first evidence for the existence of bacterial (C.acnes "high") and non-bacterial (C.acnes "low") subtypes in MC1 patients with CLBP. This supports the need for different treatment strategies.

A major 62-kD intranuclear matrix polypeptide is a component of metaphase chromosomes.

We have isolated and partially characterized a major intranuclear matrix polypeptide from rat liver. This polypeptide, which is reversibly stabilized into the intranuclear matrix under conditions which promote intermolecular disulfide bond formation, has a Mr of 62,000 and pI of 6.8-7.2 as determined by two-dimensional IEF/SDS-PAGE. A chicken polyclonal antiserum was raised against the polypeptide purified from two-dimensional polyacrylamide gels. Affinity-purified anti-62-kD IgG was prepared and used to immunolocalize this polypeptide in rat liver tissue hepatocytes. In interphase hepatocytes the 62-kD antigen is localized in small, discrete patches within the nucleus consistent with the distribution of chromatin. The staining is most prominent at the nuclear periphery and somewhat less dense in the nuclear interior. Nucleoli and cytoplasm are devoid of staining. During mitosis the 62-kD antigen localizes to the condensed chromosomes with no apparent staining of cytoplasmic areas. The chromosomal staining during mitosis is uniform with no suggestion of the patching seen in interphase nuclei. Fractionation and immunoblotting studies using rat hepatoma tissue culture cells blocked in metaphase with colcemid confirm the chromosomal localization of this 62-kD intranuclear protein during mitosis. The 62-kD polypeptide fractionates completely with metaphase chromosome scaffolds generated by sequential treatment of isolated chromosomes with DNAse I and 1.6 M NaCl, suggesting that this major 62-kD intranuclear protein may be involved in maintaining metaphase chromosomal architecture.

Overexpression of protein kinase C betaII induces colonic hyperproliferation and increased sensitivity to colon carcinogenesis.

Protein kinase C betaII (PKC betaII) has been implicated in proliferation of the intestinal epithelium. To investigate PKC betaII function in vivo, we generated transgenic mice that overexpress PKC betaII in the intestinal epithelium. Transgenic PKC betaII mice exhibit hyperproliferation of the colonic epithelium and an increased susceptibility to azoxymethane-induced aberrant crypt foci, preneoplastic lesions in the colon. Furthermore, transgenic PKC betaII mice exhibit elevated colonic beta-catenin levels and decreased glycogen synthase kinase 3beta activity, indicating that PKC betaII stimulates the Wnt/adenomatous polyposis coli (APC)/beta-catenin proliferative signaling pathway in vivo. These data demonstrate a direct role for PKC betaII in colonic epithelial cell proliferation and colon carcinogenesis, possibly through activation of the APC/beta-catenin signaling pathway.

Morphine, opioids, and the feline pulmonary vascular bed.

BACKGROUND: Opioid-induced vasodepressor responses have been reported in a variety of species and laboratory models. The aim of this study was to ascertain the relative potencies of different clinically relevant opioids compared with traditional vasodepressor agents in the feline pulmonary vascular bed. A second aim was to study the effects of morphine and to identify the receptors involved in the mediation or the modulation of these effects. METHODS: This was a prospective vehicle-controlled study involving an intact chest preparation of adult mongrel cats. The effects of various opioids, morphine, fentanyl, remifentanil, sufentanil, and meperidine were compared with other vasodepressor agents. Additionally, the effects of L-N(5)-(1-iminoethyl) ornithine hydrochloride (L-NIO) (nitric oxide synthase inhibitor), nimesulide [selective cyclooxygenase (COX)-2 inhibitor], glibenclamide (ATP-sensitive K+ channel blocker), naloxone (non-selective opioid receptor antagonist), and diphenhydramine (histamine H(1)-receptor antagonist) were investigated on pulmonary arterial responses to morphine and other selected agonists in the feline pulmonary vascular bed. The systemic pressure and lobar arterial perfusion pressure were continuously monitored, electronically averaged, and recorded. RESULTS: In the cat pulmonary vascular bed of the isolated left lower lobe, morphine, remifentanil, fentanyl, sufentanil, and meperidine induced a dose-dependent moderate vasodepressor response and it appeared that sufentanil was the most potent on a nanomolar basis. The effects of morphine were not significantly altered after administration of L-NIO, nimesulide, and glibenclamide. However, the vascular responses to morphine were significantly attenuated following administration of naloxone and diphenhydramine. CONCLUSION: The results of the present study suggest that sufentanil appears to have slightly more potency and morphine the least of the five opioid agonists studied on a nanomolar basis. Morphine-induced vasodilatory responses appeared to be mediated or modulated by both opioid receptor and histamine-receptor-sensitive pathways.

Larger vertebral endplate concavities cause higher failure load and work at failure under high-rate impact loading of rabbit spinal explants.

Vertebral fractures are among the most common of all osteoporosis related fracture types and its risk assessment is largely based on bone quality measures. Morphometric parameters are not yet considered, although endplate thickness and concavity shape were found to be important in fracture prediction in low-rate tests. We hypothesized that, under high-rate impact loading, the shape and size of the central endplate concavity are of key importance for fracture prediction. Therefore, we tested rabbit spinal segment explants in vitro under high-rate impact loading. With a combination of microCT to describe endplate morphometry, high-speed video imaging, and impact force measurement, endplate morphometry was correlated to the mechanical response. We found that endplate concavity shape and volume were important in describing the mechanical response: larger concavities caused higher failure load. We suggest a model for the fracture mechanism under high-rate impact loading, considering the morphometry of the endplates: wider and more voluminous concavities are protective whereas steeper slopes of the concavity edges and increasing bone volume fraction of the central endplate moiety are disadvantageous. Therefore, the shape and size of endplate morphometry are important in vertebral fracture prediction and should be considered included in vertebral fracture risk assessment.

Evi1 is a survival factor which conveys resistance to both TGFbeta- and taxol-mediated cell death via PI3K/AKT.

In hematopoietic cells the transforming potential of the ecotropic viral integration site 1 (Evi1) oncogene is thought to be dependent upon the ability to inhibit TGFbeta signaling. Although Evi1 has recently been implicated in certain epithelial cancers, the effects of Evi1 on transformation and TGFbeta signaling in epithelial cells are not completely understood. Herein, we have determined the effects of Evi1 on TGFbeta signaling in intestinal epithelial cells. Stable expression of Evi1 in non-transformed intestinal epithelial cells inhibited induction of some Smad3-dependent TGFbeta target genes, such as PAI1. However, TGFbeta-mediated induction of cellular adhesion signaling components such as integrin1 and paxillin was not inhibited by Evi1; nor did Evi1 inhibit TGFbeta-mediated epithelial to mesenchymal transition. Likewise, Evi1 did not inhibit TGFbeta-mediated downregulation of cyclin D1 or block TGFbeta-mediated growth inhibition. However, Evi1 did inhibit TGFbeta-mediated apoptosis by a process that involves phosphoinositide-3-kinase (PI3K) and its downstream effector AKT. The ability of Evi1 to suppress apoptosis is not restricted to TGFbeta-mediated cell death, since Evi1 also protects intestinal epithelial cells from taxol-mediated apoptosis. Evi1 is overexpressed in some human colon cancer cell lines, and overexpression is associated with amplification of the Evi1 gene. Knockdown of Evi1 by siRNA inhibited AKT phosphorylation in HT-29 human colon cancer cells and increased their sensitivity to taxol-mediated apoptosis. These data indicate that Evi1 functions as a survival gene in intestinal epithelial cells and colon cancer cells, activating PI3K/AKT and conveying resistance to both physiological and therapeutic apoptotic stimuli.

PKCι regulates nuclear YAP1 localization and ovarian cancer tumorigenesis.

Atypical protein kinase Cι (PKCι) is an oncogene in lung and ovarian cancer. The PKCι gene PRKCI is targeted for frequent tumor-specific copy number gain (CNG) in both lung squamous cell carcinoma (LSCC) and ovarian serous carcinoma (OSC). We recently demonstrated that in LSCC cells PRKCI CNG functions to drive transformed growth and tumorigenicity by activating PKCι-dependent cell autonomous Hedgehog (Hh) signaling. Here, we assessed whether OSC cells harboring PRKCI CNG exhibit similar PKCι-dependent Hh signaling. Surprisingly, we find that whereas PKCι is required for the transformed growth of OSC cells harboring PRKCI CNG, these cells do not exhibit PKCι-dependent Hh signaling or Hh-dependent proliferation. Rather, transformed growth of OSC cells is regulated by PKCι-dependent nuclear localization of the oncogenic transcription factor, YAP1. Lentiviral shRNA-mediated knockdown (KD) of PKCι leads to decreased nuclear YAP1 and increased YAP1 binding to angiomotin (AMOT), which sequesters YAP1 in the cytoplasm. Biochemical analysis reveals that PKCι directly phosphorylates AMOT at a unique site, Thr750, whose phosphorylation inhibits YAP1 binding. Pharmacologic inhibition of PKCι decreases YAP1 nuclear localization and blocks OSC tumor growth in vitro and in vivo. Immunohistochemical analysis reveals a strong positive correlation between tumor PKCι expression and nuclear YAP1 in primary OSC tumor samples, indicating the clinical relevance of PKCι-YAP1 signaling. Our results uncover a novel PKCι-AMOT-YAP1 signaling axis that promotes OSC tumor growth, and provide a rationale for therapeutic targeting of this pathway for treatment of OSC.

Elevated protein kinase C betaII is an early promotive event in colon carcinogenesis.

Protein kinase C (PKC) has been implicated in colon carcinogenesis in humans and in rodent models. However, little is known about the specific role of individual PKC isozymes in this process. We recently demonstrated that elevated expression of PKC betaII in the colonic epithelium induces hyperproliferation in vivo (N. R. Murray et al., J. Cell Biol., 145: 699-711, 1999). Because hyperproliferation is a major risk factor for colon cancer, we assessed whether specific alterations in PKC betaII expression occur during azoxymethane-induced colon carcinogenesis in mice. An increase in PKC betaII expression was observed in preneoplastic lesions (aberrant crypt foci, 3.7-fold) compared with saline-treated animals, and in colon tumors (7.8-fold; P = 0.011) compared with uninvolved colonic epithelium. In contrast, PKC alpha and PKC betaI (a splicing variant of PKC betaII) expression was slightly decreased in aberrant crypt foci and dramatically reduced in colon tumors. Quantitative reverse transcription-PCR analysis revealed that PKC mRNA levels do not directly correlate with PKC protein levels, indicating that PKC isozyme expression is likely regulated at the posttranscriptional/translational level. Finally, transgenic mice expressing elevated PKC betaII in the colonic epithelium exhibit a trend toward increased colon tumor formation after exposure to azoxymethane. Taken together, our results demonstrate that elevated expression of PKC betaII is an important early, promotive event that plays a role in colon cancer development.

Regulation of energy metabolism in Morris hepatoma 7777 and 7800.

The pathway of fat oxidation in two experimental hepatomas was studied in order to demonstrate that a specific deficit in the energy metabolism of a tumor might contribute to the cachexia of the host. Forty-eight male Buffalo rats were divided into four groups of 12 each. One group was implanted s.c. with Morris hepatoma 7777 and one group was implanted with Morris hepatoma 7800, whereas the other two groups served as controls. All groups were fed standard rat chow diet ad libitum until the tumors reached 2 cm in diameter. The animals were then fasted for 24 hr prior to sacrifice and excision of tumor and liver for assays. During the period of tumor growth, the animals bearing the 7777 hepatoma lost weight, but the weight of the 7800 hepatoma-bearing rats did not differ significantly from that of the control animals. The livers of both groups of animals showed evidence of fatty acid oxidation in vivo and in vitro, and, as expected, during fasting, pyruvate dehydrogenase was inactivated and the rate of fatty acid synthesis was low. A qualitatively similar picture was seen with the better-differentiated 7800 hepatoma. In contrast, the 7777 hepatoma exhibited low levels of fatty acyl coenzyme CoA, no appreciable activity of carnitine palmitoyl transferase and fortified homogenates of the tumor were unable to oxidize palmitate. In keeping with these observations, pyruvate dehydrogenase remained in the active form, and fatty acid synthesis continued unabated in the fasted state in these tumors. Ketone bodies could not be oxidized by fortified homogenates of the liver or by either tumor, probably due to the lack of 3-ketoacid thiotransferase, which was undetectable in these tissues. We hypothesize that flow-through pyruvate dehydrogenase during fasting in Morris hepatoma 7777, occurring as a result of the defect in fat oxidation, contributes to the weight loss of these animals.

No association of the I1307K APC allele with ovarian cancer risk in Ashkenazi Jews.

Familial adenomatous polyposis is a dominantly inherited colon cancer syndrome associated with germ-line mutations in the APC tumor suppressor gene. An APC gene sequence alteration, the I1307K allele, occurs in 6% of the Ashkenazi Jewish population and is reported to double the risk for colorectal cancer. We screened a population of 190 Ashkenazi women who were diagnosed with epithelial ovarian carcinoma for the I1307K variant and measured the effect of this allele on the risk for cancer development in their first-degree relatives. We identified the I1307K allele in 7.9% (15 of 190) of our ovarian cancer cases. The average age of ovarian cancer diagnosis in carriers of the I1307K allele (57.5 years) was not statistically different than the age for noncarriers (56.4 years; P = 0.70). Among the 1087 first-degree relatives, there were 23 cases of colorectal cancer; 3 of 100 relatives of probands with the I1307K allele (3.0%) had a history of colorectal cancer versus 20 of 987 relatives of probands without the I1307K allele (2.1%; relative risk, 1.48; 95% confidence interval, 0.45-4.88; P = 0.462). Relatives of the I1307K carriers had a risk of 38.0% for developing any cancer to age 80, similar to the risk for relatives of noncarriers of the I1307K allele (42.1%; P = 0.86). The average age of diagnosis of cancer of any type was not different between relatives of carriers (59.0 years) and noncarriers (60.4 years). In the Ashkenazi Jewish population, the I1307K allele is unlikely to increase the risk of ovarian cancer or of cancer in general.

NF-kappaB/RelA transactivation is required for atypical protein kinase C iota-mediated cell survival.

In chronic myelogenous leukemia (CML), the oncogene bcr-abl encodes a dysregulated tyrosine kinase that inhibits apoptosis. We showed previously that human erythroleukemia K562 cells are resistant to antineoplastic drug (taxol)-induced apoptosis through the atypical protein kinase C iota isozyme (PKC iota), a kinase downstream of Bcr-Abl. The mechanism(s) by which PKC iota mediates cell survival to taxol is unknown. Here we demonstrate that PKC iota requires the transcription factor nuclear factor-kappaB (NF-kappaB) to confer cell survival. At apoptosis-inducing concentrations, taxol weakly induces IkappaB(alpha) proteolysis and NF-kappaB translocation in K562 cells, but potently induces its transcriptional activity. Inhibition of NF-kappaB activity (by blocking IkappaB(alpha) degradation) significantly sensitizes cells to taxol-induced apoptosis. Likewise, K562 cells expressing antisense PKC iota mRNA or kinase dead PKC iota (PKC iota-KD) are sensitized to taxol; these cells are rescued from apoptosis by NF-kappaB overexpression. Expression of constitutively active PKC iota (PKC iota-CA) upregulates NF-kappaB transactivation and rescues cells from apoptosis in the absence of Bcr-Abl tyrosine kinase activity. Using a chimeric GAL4-RelA transactivator, we find that taxol potently activates GAL4-RelA-dependent transcription. This activation was further upregulated by expression of PKC iota-CA and inhibited by expression of PKC iota-KD. Our results indicate that RelA transactivation is an important downstream target of the PKC iota-mediated Bcr-Abl signaling pathway and is required for resistance to taxol-induced apoptosis.

Comparison of gemcitabine versus the matrix metalloproteinase inhibitor BAY 12-9566 in patients with advanced or metastatic adenocarcinoma of the pancreas: a phase III trial of the National Cancer Institute of Canada Clinical Trials Group.

PURPOSE: To compare the selective matrix metalloproteinase inhibitor BAY 12-9566 with the nucleoside analog gemcitabine in the treatment of advanced pancreatic cancer. METHODS: Patients with advanced pancreatic adenocarcinoma who had not previously received chemotherapy were randomly assigned to receive BAY 12-9566 800 mg orally bid continuously or gemcitabine 1,000 mg/m2 administered intravenously on days 1, 8, 15, 22, 29, 36, and 43 for the first 8 weeks, and then days 1, 8, and 15 of each subsequent 28-day cycle. The primary end point was overall survival; secondary end points were progression-free survival, tumor response, quality of life, and clinical benefit. The planned sample size of the study was 350 patients. Two formal interim analyses were planned. RESULTS: The study was closed to accrual after the second interim analysis on the basis of the recommendation of the National Cancer Institute of Canada Clinical Trials Group Data Safety Monitoring Committee. There were 277 patients enrolled onto the study, 138 in the BAY 12-9566 arm and 139 in the gemcitabine arm. The rates of serious toxicity were low in both arms. The median survival for the BAY 12-9566 arm and the gemcitabine arm was 3.74 months and 6.59 months, respectively (P <.001; stratified log-rank test). The median progression-free survival for the BAY 12-9566 and gemcitabine arms was 1.68 and 3.5 months, respectively (P <.001). Quality-of-life analysis also favored gemcitabine. CONCLUSION: Gemcitabine is significantly superior to BAY 12-9566 in advanced pancreatic cancer.

Analysis of MDR1 expression in normal and malignant endometrium by reverse transcription-polymerase chain reaction and immunohistochemistry.

The purpose of this study was to quantitate the expression of human MDR1 mRNA levels in normal endometrium and in endometrial carcinoma and to determine the association of MDR1 levels with prognostic indicators. Endometrial samples from 43 postmenopausal patients with endometrial carcinoma and 38 patients (controls) with benign disease undergoing hysterectomy were snap-frozen. MDR1 levels were determined by quantitative reverse transcription-PCR (RT-PCR) and compared to sensitive and resistant cell lines. Immunohistochemistry was done with MM4.17, an anti-MDR1 antibody, on paraffin sections, and the results were compared to those obtained from RT-PCR. Data was analyzed using the Kruskal-Wallis and Bonferroni tests, setting the P value at 0.05. In both postmenopausal endometrial tissue and tumors, MDR1 expression was localized to the epithelial cell layer. Comparison of immunohistochemistry and RT-PCR results demonstrated a correlation of 80%. In control patients, MDR1 expression was significantly higher in postmenopausal endometrium (n = 15) than in the proliferative premenopausal endometrium (n = 15; P = 0.0024). MDR1 expression in all tumors was lower than that measured in the postmenopausal controls. Between each tumor group, there was no significant difference in the MDR1 levels observed. MDR1 expression was significantly lower in patients with high nuclear grade (n = 18) tumors when compared to patients with low nuclear grade (n = 14; P = 0.04) tumors. Comparison of MDR1 levels with multiple prognostic indicators for endometrial cancer was only significant for nuclear grade. The data indicate that MDR1 expression is not a major component of the drug resistance observed in primary endometrial tumors.