Vitamin D receptor-targeted treatment to prevent pathological dedifferentiation of pancreatic ß cells under hyperglycaemic stress.

Dedifferentiation has been identified as one of the causes of ß-cell failure resulting in type 2 diabetes (T2D). This study tested whether increasing vitamin D receptor (VDR) expression prevents dedifferentiation of ß cells in a high-glucose state in vitro. Culturing a mouse insulinoma cell line (MIN6) in a high-glucose environment decreased VDR expression. However, increased VDR following vitamin D3 (VD3) treatment improved insulin release of early-passage MIN6 and insulin index of db/- (heterozygous) islets to levels seen in normal functional islets. Treatment with VD3, its analogues and derivatives also increased the expression of essential transcription factors, such as Pdx1, MafA and VDR itself, ultimately increasing expression of Ins1 and Ins2, which might protect ß cells against dedifferentiation. VD3 agonist lithocholic acid (LCA) propionate was the most potent candidate molecule for protecting against dedifferentiation, and an e-pharmacophore mapping model confirmed that LCA propionate exhibits a stabilizing conformation within the VDR binding site. This study concluded that treating db/+ islets with a VD3 analogue and/or derivatives can increase VDR activity, preventing the pathological dedifferentiation of ß cells and the onset of T2D.

The Role of Positron Emission Tomography/Computed Tomography Imaging in Head and Neck Cancer after Radical Chemoradiotherapy: a Single Institution Experience.

AIMS: Positron emission tomography/computed tomography (PET/CT) is used to restage head and neck cancer 3 months after chemoradiotherapy. The purpose of this study was to determine the negative predictive value (NPV) of a scan reported as having no abnormal uptake and the positive predictive values (PPV) for different maximum standardised uptake value (SUVmax) thresholds. MATERIALS AND METHODS: Patients with squamous cell carcinoma of the oro-/hypopharynx/larynx (n = 206) were included. SUVmax and subsequent locoregional recurrence were documented. RESULTS: The median SUVmax was 11.2 (range 4-33)/4.6 (range 2-30), respectively, in patients with/without definite primary site recurrence (P = 0.004). The median SUVmax was 4.4 (range 2.6-15.6)/3.1 (range 2.1-4.6), respectively, in patients with/without definite nodal recurrence (P = 0.003). The NPV for a scan reported as having no abnormal uptake was 92%. The PPV for the SUVmax thresholds 4, 6 and 8, respectively, were 53, 65 and 92% (primary site) and 93, 100 and 100% (nodes). CONCLUSIONS: The NPV of PET/CT after chemoradiation is consistent with the literature and underlines the importance of PET/CT in restaging the primary site if salvage neck dissection is considered. The overall PPV of PET/CT remains low but is high for nodal SUVmax > 4. These data could be used to design risk-stratified follow-up schedules.

Diffusion-weighted magnetic resonance imaging for residual and recurrent cholesteatoma: a systematic review and meta-analysis.

BACKGROUND: Diagnosis and management of recurrent or residual cholesteatoma can be problematic. Diffusion-weighted imaging magnetic resonance imaging (MRI) sequences have been used for follow-up of such lesions. More recent non-echoplanar imaging (non-EPI) sequences are thought to be superior to older echoplanar imaging (EPI) sequences. OBJECTIVE OF REVIEW: Evaluate whether diffusion-weighted magnetic resonance imaging is useful in the diagnosis of recurrent or residual cholesteatoma. TYPE OF REVIEW: Systematic review and meta-analysis. SEARCH STRATEGY: MEDLINE, EMBASE, CINAHL, Web of Science and the Cochrane Database were searched, with no limits on date or language. STUDY SELECTION: Adults or children who had previously undergone tympanomastoid surgery by any method with confirmation of recurrence/residual disease by second-look/revision surgery. EVALUATION METHODS: Two reviewers independently reviewed studies. Data extracted on 11 domains and rechecked. DATA SYNTHESIS: Statistical analysis with SPSS. RESULTS: A total of 575 studies were identified of which 27 met the inclusion criteria. These covered 727 patient episodes. For EPI studies: sensitivity (sd) 71.82 (24.5), specificity (sd) 89.36 (13.4), PPV (sd) 93.36 (8.1) and NPV (sd) 73.36 (15.8). For non-EPI studies: sensitivity 89.79 (12.1), specificity (sd) 94.57 (5.8), PPV (sd) 96.50 (4.2) and NPV 80.46 (20.2). Improved sensitivity of non-EPI sequences reached significance (P = 0.02). CONCLUSIONS: Diffusion-weighted MRI is both sensitive and specific for the detection of recurrent or residual cholesteatoma following ear surgery. Non-EPI techniques are superior to EPI techniques.

Imaging in head and neck cancer: United Kingdom National Multidisciplinary Guidelines.

This guideline is endorsed by the specialty associations involved in the care of head and neck cancer patients in the UK. This paper summarises the current imaging modalities in use for head and neck cancer evaluation. It highlights their role in the management with recommendations on modality choice for each cancer subsite. Recommendations • Offer appropriate radiological imaging, based on tumour extent, site and local expertise, to stage tumours and plan treatment for patients diagnosed with head and neck cancer. (G) • Consider positron emission tomography combined with computed tomography (PET-CT) imaging if conventional cross-sectional imaging identifies no primary site. (R) • Offer PET-CT imaging 12 weeks after non-surgical treatment to detect residual disease. (R).

Notch-induced transcription factors are predictive of survival and 5-fluorouracil response in colorectal cancer patients.

BACKGROUND: The purpose of this study was to evaluate the expression of Notch-induced transcription factors (NTFs) HEY1, HES1 and SOX9 in colorectal cancer (CRC) patients to determine their clinicopathologic and prognostic significance. METHODS: Levels of HEY1, HES1 and SOX9 protein were measured by immunohistochemistry in a nonmalignant and malignant tissue microarray of 441 CRC patients, and the findings correlated with pathologic, molecular and clinical variables. RESULTS: The NTFs HEY1, HES1 and SOX9 were overexpressed in tumours relative to colonic mucosa (OR=3.44, P<0.0001; OR=7.40, P<0.0001; OR=4.08 P<0.0001, respectively). HEY1 overexpression was a negative prognostic factor for all CRC patients (HR=1.29, P=0.023) and strongly correlated with perineural and vascular invasion and lymph node (LN) metastasis. In 5-fluorouracil (5-FU)-treated patients, the tumour overexpression of SOX9 correlated with markedly poorer survival (HR=8.72, P=0.034), but had no predictive effect in untreated patients (HR=0.70, P=0.29). When HEY1, HES1 and SOX9 expression were combined to predict survival with chemotherapy, in treated patients there was an additive increase in the risk of death with each NTF overexpressed (HR=2.09, P=0.01), but no prognostic import in the untreated patient group (HR=0.74, P=0.19). CONCLUSION: The present study is the first to discover that HEY1 overexpression correlates with poorer outcome in CRC, and NTF expression is predictive of CRC patient survival with 5-FU chemotherapy. If confirmed in future studies, testing of NTF expression has the potential to enter routine pathological practice for the selection of patients to undergo chemotherapy alone or in combination with Notch inhibitors.

The role of positron emission tomography/CT imaging in head and neck cancer patients after radical chemoradiotherapy.

OBJECTIVES: Positron emission tomography with CT (PET/CT) scanning is increasingly being used in head and neck cancer to assess response after radical concomitant chemoradiotherapy. The purpose of this study was to assess the use of PET/CT following chemoradiotherapy at our institution. METHODS: All patients receiving radical chemoradiotherapy for head and neck cancer over a 9-year period were retrospectively identified. Outcome data including local control and overall survival were collected for all patients. The negative predictive value of PET/CT for local recurrence was calculated. Of those with a reported positive PET/CT scan the maximum standardised uptake values were compared with the incidence of local recurrence. RESULTS: 92 patients were identified having a post-treatment PET/CT from a total of 301 patients receiving radical concomitant chemoradiotherapy. Median time from completion of chemoradiotherapy to PET/CT scan was 3 (range 2-8) months. Median follow-up in surviving patients was 19 and 25 months in the PET/CT and non-PET/CT groups, respectively. The negative predictive value for local recurrence was 91.8%. The median maximum standardised uptake values were 10.2 (range 3.1-33) and 6.89 (range 3.1-30) in those with local recurrence and with no local recurrence, respectively. CONCLUSIONS: Post-chemoradiotherapy PET/CT may aid subsequent management decisions. Patients with a negative PET/CT scan after radical chemoradiotherapy have a 91.8% chance of remaining free of local recurrence 19 months post-treatment. A higher maximum standardised uptake value on the post-chemoradiotherapy PET/CT may predict subsequent local recurrence and warrants further investigation. Advances in knowledge Post-chemoradiotherapy PET/CT imaging aids subsequent management decisions.

Abciximab for treatment of thromboembolic complications during endovascular coiling of intracranial aneurysms.

BACKGROUND AND PURPOSE: Thromboembolism is a recognized complication occurring during endovascular coil embolization of intracranial aneurysms. Recently, there has been much interest in glycoprotein IIb/IIIa inhibitors to treat such complications, but the evidence is limited. We reviewed our use of one such agent, abciximab, which we commonly administer and believe to be a safe and suitable rescue agent in this setting. MATERIALS AND METHODS: We retrospectively reviewed cases in which abciximab was administered in our institution between 2001 and 2007. Clinical outcome was assessed by the modified Rankin Scale (mRS) at 6 months. Good outcome was defined as no significant clinical sequelae compared with baseline status or clinical improvement (mRS < 2). Poor outcome was defined as no resolution of a new clinical deficit that developed postprocedure at 6 months (mRS > 2). Angiographic appearance of thromboembolic phenomena and posttreatment outcome was assessed with the Thrombolysis in Myocardial Infarction (TIMI) scale. RESULTS: Thirty-eight patients were included, with good outcome observed in 30 (79%) and poor outcome in 8 (21%) patients. Angiographic improvement based on TIMI scoring was seen in 24 (63%) patients, and no improvement was seen in 14 (37%). In 4 patients (11%), good outcome was obtained at 6 months despite no angiographic improvement on TIMI. No cases of intracranial rebleed or additional neurologic deficit following administration of abciximab were encountered. CONCLUSION: In this small retrospective series, abciximab was safe and effective when used as a rescue agent for thromboembolic complications encountered during coiling of intracerebral aneurysms.

Peripheral nerve injury induces cannabinoid receptor 2 protein expression in rat sensory neurons.

We have localized cannabinoid receptor 2 protein in rat and mouse somatic sensory nervous system, using an antibody that recognizes mouse cannabinoid receptor 2. Little or no cannabinoid receptor 2 immunoreactivity was found in sections of naive rat or mouse dorsal root ganglia or spinal cord. This was in accord with the lack of detectable cannabinoid receptor 2 mRNA in (dorsal root ganglion) neurons by in situ hybridization experiments described in the literature. However, we could detect cannabinoid receptor 2 immunoreactivity following unilateral nerve damage-either by sciatic nerve section, or by spinal nerve ligation. It was localized to the superficial laminae of the dorsal horn of the spinal cord, ipsilateral to the nerve damage, coincident with the area of termination of damaged afferents which was marked by loss of isolectin B4 binding. This upregulation was not seen in cannabinoid receptor 2 null mice. The cannabinoid receptor 2 protein in spinal cord appeared to be expressed on sensory neuron afferent terminals as it colocalized with two markers of damaged afferents, namely growth associated protein-43 and the neuropeptide galanin. Moreover, it did not colocalize with markers of activated microglial cells (OX-42) or astroglial cells (glial fibrillary acidic protein) in rat spinal cord. In the peripheral nerve, accumulation of cannabinoid receptor 2 immunoreactivity was seen in nerve sections proximal, but not distal, to the ligation site, suggesting transport down the nerve from the cell bodies. Although convincing cannabinoid receptor 2 immunoreactivity was seen in neither uninjured nor injured dorsal root ganglion neuron cell bodies in tissue sections, expression was detectable in isolated, cultured neurons that had received a prior axotomy in vivo. This clear demonstration of CB(2) receptors on sensory neurons suggests an additional cellular target for CB(2) agonist induced analgesia, at least in neuropathic models.

Estrogen-induced osteogenesis in intact female mice lacking ERbeta.

We recently found that estrogen receptor (ER) antagonists prevent high-dose estrogen from inducing the formation of new cancellous bone within the medullary cavity of mouse long bones. In the present investigation, we studied the role of specific ER subtypes in this response by examining whether this is impaired in female ERbeta(-/-) mice previously generated by targeted gene deletion. Vehicle or 17beta-estradiol (E(2)) (range 4-4,000 microg. kg(-1). day(-1)) was administered to intact female ERbeta(-/-) mice and wild-type littermates by subcutaneous injection for 28 days. The osteogenic response was subsequently assessed by histomorphometry performed on longitudinal and cross sections of the tibia. E(2) was found to cause an equivalent increase in cancellous bone formation in ERbeta(-/-) mice and littermate controls, as assessed at the proximal and distal regions of the proximal tibial metaphysis. E(2) also resulted in a similar increase in endosteal mineral apposition rate in these two genotypes, as assessed at the tibial diaphysis. In contrast, cortical area in ERbeta(-/-) mice was found to be greater than that in wild types irrespective of E(2) treatment, as was tibial bone mineral density as measured by dual-energy X-ray absorptiometry, consistent with previous reports of increased cortical bone mass in these animals. We conclude that, although ERbeta acts as a negative modulator of cortical modeling, this isoform does not appear to contribute to high-dose estrogen's ability to induce new cancellous bone formation in mouse long bones.

Erythropoietin-stimulated Raf-1 tyrosine phosphorylation is associated with the tyrosine kinase Lyn in J2E erythroleukemic cells.

The serine/threonine kinase Raf-1 is crucial for transducing intracellular signals emanating from numerous growth factors. Here we used the J2E erythroid cell line transformed by the nu-raf/nu-myc oncogenes to examine the effects of erythropoietin on endogenous Raf-1 activity. Despite the presence of constitutively active v-raf in these cells, Raf-1 exokinase activity increased after erythropoietin stimulation. This increase in enzymatic activity coincided with tyrosine phosphorylation of Raf-1 on residue Y341. Significantly, the tyrosine kinase Lyn coimmunoprecipitated with Raf-1, and Raf-1 was not tyrosine-phosphorylated in a J2E subclone lacking Lyn. Therefore, it was concluded that Lyn may be the kinase responsible for tyrosine phosphorylating Raf-1 and increasing its exokinase activity in response to erythropoietin.

Role of endothelial nitric oxide synthase in estrogen-induced osteogenesis.

It is well recognized that high-dose estrogen induces a marked osteogenic response in long bones of female mice. In light of evidence which suggests that nitric oxide synthase (NOS) plays a role in regulation of osteoblast activity, we analyzed whether NOS is involved in mediating this response. Intact female mice were administered 17beta-estradiol (E(2)) either alone or in combination with N(G)-nitro-L-arginine methylester (L-NAME) or aminoguanidine (AG), over 24 days. The former inhibits both constitutive and inducible isoforms of NOS, whereas the latter is a selective inhibitor of inducible NOS. Bone mineral density (BMD) of the femur was subsequently measured by dual-energy X-ray absorptiometry (DXA), and histomorphometry performed at the proximal metaphysis on longitudinal tibial sections. As expected, E(2) given alone led to a marked accumulation of cancellous bone at the proximal tibial metaphysis, associated with a significant gain in femoral BMD, and an increase in cancellous mineralizing surfaces as assessed by histomorphometry. Neither L-NAME nor AG affected cancellous histomorphometric indices when given alone. However, when administered in combination with L-NAME, the magnitude of the skeletal response to E(2) was significantly reduced. The tendency for L-NAME to reduce estrogen-induced bone formation within the proximal tibial metaphysis was more marked distally compared with proximally. In contrast, AG showed no tendency to suppress the osteogenic response to E(2). Subsequently, we examined the effect of E(2) administration on expression within mouse femoral bone marrow of endothelial NOS (eNOS), which is the predominant constitutive isoform of NOS within bone. No change in eNOS mRNA levels was observed following E(2) administration, as assessed by reverse transcription-polymerase chain reaction (RT-PCR). Taken together, our results suggest that eNOS plays a role in mediating estrogen-induced bone formation in intact female mice, possibly as a consequence of posttranscriptional regulation of eNOS activity by estrogen.

Karyotypic abnormalities associated with haemopoietic lineage switching are not linked with mutations to p53.

Leukemic cells can undergo lineage switching to display the phenotypic features of another haemopoietic pathway, as exemplified by B lymphoma and erythroleukemic cell lines generating variants with a monocytic appearance. Unlike the diploid parental lines, the vast majority of myeloid derivative lines examined (12 of 13 lines) were aneuploid. As p53 is involved in the maintenance of chromosomal stability, we investigated the role of p53 in the emergence of abnormal karyotypes in cells which had undergone lineage switching. Single strand conformation polymorphism and sequence analysis of cDNA, together with protein immunoprecipitations, were used to assess the p53 status of parental and variant cell lines. Unexpectedly, four or five monocytic lines with chromosomal alterations contained wild type p53. Conversely, a p53 point mutation found in one aneuploid monocytic line was also present in the diploid parental pre-B cell. These results provide strong evidence that mechanisms other than p53 mutations are responsible for karyotypic abnormalities seen in cells that have undergone lineage switching.

A novel ADP-ribosylation like factor (ARL-6), interacts with the protein-conducting channel SEC61beta subunit.

We report here the isolation of a new member of the ADP-ribosylation factor (ARF)-like family (ARL-6) present in the J2E erythroleukemic cell line, but not its myeloid variants. Consistent with this lineage-restricted expression, ARL-6 mRNA increased with erythropoietin-induced maturation of J2E cells, and decreased with interleukin 6-induced differentiation of M1 monoblastoid cells. In tissues, ARL-6 mRNA was most abundant in brain and kidney. While ARL-6 protein was predominantly cytosolic, its membrane association increased following exposure to GTP-gammaS, like many members of the ARF/ARL family. Using the yeast two-hybrid system, six molecules which interact with ARL-6 were identified including SEC61beta, a subunit of the heterotrimeric protein conducting channel SEC61p. Co-immunoprecipitation of ARL-6 confirmed a stable association between ARL-6 and SEC61beta in COS cells. These results demonstrate that ARL-6, a novel member of the ADP-ribosylation factor-like family, interacts with the SEC61beta subunit.

Increased transcription of the E mu-myc transgene and mRNA stabilisation produce only a modest elevation in Myc protein.

Mice bearing the E mu-myc transgene, which links the immunoglobulin heavy chain enhancer (E mu) with c-myc, are predisposed to developing B cell lymphomas. Several B lineage cell lines have been isolated from these animals, and some have been converted to macrophages following infection with v-raf. In this study we compared the regulation of myc expression in E mu-myc B lymphoma lines, their macrophage counterparts and other non-myc transformed B cell lines. Nuclear run-on analyses demonstrated that transcription of the transgene was elevated in E mu-myc B cell lines. Moreover, the presence of a 600 bp phiX174 marker in the 3' end of the transgene produced a marked stabilisation of this RNA species. Consequently, steady state myc mRNA levels in the E mu-myc B lymphoma cells were tenfold higher than the macrophage derivatives and non-myc transformed B lineage lines. Despite the considerable difference in myc RNA levels, the E mu-myc B cell lines contained only 30-50% more Myc protein than the other cell lines. This discrepancy between RNA and protein content was not due to increased degradation of the protein as the half life was normal in the transgenic cell lines. These results indicate that both E mu and phiX174 sequences influence transgenic myc expression and that protein levels do not correlate with RNA content in E mu-myc cell lines.

Prevention of apoptosis in J2E erythroid cells by erythropoietin: involvement of JAK2 but not MAP kinases.

The J2E erythroid cell line, transformed by retroviral v-raf/v-myc oncogenes, proliferates and differentiates in response to erythropoietin. Here we show that J2E cells undergo apoptosis rapidly after serum withdrawal and that only erythropoietin of seven growth factors tested, could protect the cells from death. The role of JAK2 and MAP kinases in transmitting viability signals initiated by erythropoietin was examined in these cells. Despite constitutive raf kinase activity, phosphorylation of MAP kinases fell after serum withdrawal. However, an antisense oligonucleotide strategy revealed that JAK2, but not the MAP kinases, was involved in transmitting signals to maintain the viability of J2E cells. Several cell cycle proteins and transcription factors were also studied; although c-jun rose sharply during apoptosis, erythropoietin could not suppress this increase. It was concluded that erythropoietin-induced protection from apoptosis involved JAK2, but not MAP kinases or c-jun.

Four outbreaks of Salmonella enteritidis phage type 4 food poisoning linked to a single baker.

An outbreak of gastrointestinal illness occurred among a party of diners at a hotel in South Yorkshire. A case control study identified a gateau, from an outside supplier, as the likeliest vehicle of infection. Further gateaux from the same baker's premises and other outlets were examined microbiologically. Three other outbreaks of food poisoning in neighbouring districts were recognised and found to be associated with gateaux supplied by the same baker. A total of 32 cases were identified. Stool specimens from 24 cases grew Salmonella enteritidis phage type (PT) 4. The same organism was also grown from gateau in the domestic refrigerator of the chef of one hotel, cheesecake made by the same baker, and a gateau and ingredients from the baker's premises. The isolates of S. enteritidis PT4 were all fully sensitive to antibiotics, and had the same plasmid and pulsed field gel electrophoresis profiles. It is most likely that cross contamination occurred from infected raw eggs on the baker's premises. The control measures instituted probably prevented two further outbreaks, and the baker now uses only pasteurised eggs. The benefits of close cooperation between different local and health authority districts in the investigation of the outbreaks are discussed.