Investigation of Mitochondrial Related Variants in a Cerebral Small Vessel Disease Cohort.

Monogenic forms of cerebral small vessel disease (CSVD) can be caused by both variants in nuclear DNA and mitochondrial DNA (mtDNA). Mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is known to have a phenotype similar to Cerebral Autosomal Dominant Arteriopathy with Sub-cortical Infarcts and Leukoencephalopathy (CADASIL), and can be caused by variants in the mitochondrial genome and in several nuclear-encoded mitochondrial protein (NEMP) genes. The aim of this study was to screen for variants in the mitochondrial genome and NEMP genes in a NOTCH3-negative CADASIL cohort, to identify a potential link between mitochondrial dysfunction and CSVD pathology. Whole exome sequencing was performed for 50 patients with CADASIL-like symptomology on the Ion Torrent system. Mitochondrial sequencing was performed using an in-house designed protocol with sequencing run on the Ion GeneStudio S5 Plus (S5 +). NEMP genes and mitochondrial sequencing data were examined for rare (MAF < 0.001), non-synonymous variants that were predicted to have a deleterious effect on the protein. We identified 29 candidate NEMP variants that had links to either MELAS-, encephalopathy-, or Alzheimer's disease-related phenotypes. Based on these changes, variants affecting POLG, MTO1, LONP1, NDUFAF6, NDUFB3, and TCIRG1 were thought to play a potential role in CSVD pathology in this cohort. Overall, the exploration of the mitochondrial genome identified a potential role for mitochondrial related proteins and mtDNA variants contributing to CSVD pathologies.

Staged reconstruction of diaphyseal fractures with segmental defects: Surgical and patient-reported outcomes.

INTRODUCTION: Two-stage limb reconstruction is an option for patients with critical size segmental bone defects following acute trauma or non-union. Reconstruction is technically demanding and associated with a high complication rate. Current protocols for limb reconstruction have well-documented challenges, and no study has reported on patient outcomes using a validated questionnaire. In this study, we aimed to examine the clinical and patient-centered outcomes following our surgical protocol for two-stage limb reconstruction following critical size segmental defects. PATIENTS AND METHODS: A single surgeon performed reconstruction of long bone defects using antibiotic impregnated cement spacers and intramedullary cancellous bone autograft. A retrospective chart review was performed. Three reviewers independently measured time to union based on radiographs. The Lower Extremity Functional Scale (LEFS) survey was administered to patients after most recent follow-up. RESULTS: Ten limbs representing nine patients were included. All patients sustained a lower extremity injury, and one patient had bilateral lower extremity injuries. Average clinical follow-up was 18.3 months (range 7-33) from final surgical intervention, and follow-up to questionnaire administration was 28 months (range 24-37). The mean time between stages was 3.1 months. Average time to unrestricted weight-bearing was 7.9 months from Stage 1 (range 3.4-15.9) and 4.5 months from Stage 2 (range 1.1-11.6). Average time to full union was 16.7 months from Stage 1 (range 6.4-28.6) and 13.5 months from Stage 2 (range 1.8-27). Eight patients (nine limbs) participated in the LEFS survey, the average score was 53.1 (range 30-67), equating to 66% of full functionality (range 38%-84%). Complications included 5 infections, 3 non-unions, and one amputation. There was a moderate positive correlation between infection at any time point and non-union (R=0.65, p=0.03). DISCUSSION AND CONCLUSIONS: Outcomes in this small patient cohort were good despite risks of complication. There is an association between infection and non-union. Further studies addressing clinical and functional outcomes will help to guide expectations for future surgeons and patients.

Human gestation-associated tissues express functional cytosolic nucleic acid sensing pattern recognition receptors.

The role of viral infections in adverse pregnancy outcomes has gained interest in recent years. Innate immune pattern recognition receptors (PRRs) and their signalling pathways, that yield a cytokine output in response to pathogenic stimuli, have been postulated to link infection at the maternal-fetal interface and adverse pregnancy outcomes. The objective of this study was to investigate the expression and functional response of nucleic acid ligand responsive Toll-like receptors (TLR-3, -7, -8 and -9), and retinoic acid-inducible gene 1 (RIG-I)-like receptors [RIG-I, melanoma differentiation-associated protein 5 (MDA5) and Laboratory of Genetics and Physiology 2(LGP2)] in human term gestation-associated tissues (placenta, choriodecidua and amnion) using an explant model. Immunohistochemistry revealed that these PRRs were expressed by the term placenta, choriodecidua and amnion. A statistically significant increase in interleukin (IL)-6 and/or IL-8 production in response to specific agonists for TLR-3 (Poly(I:C); low and high molecular weight), TLR-7 (imiquimod), TLR-8 (ssRNA40) and RIG-I/MDA5 (Poly(I:C)LyoVec) was observed; there was no response to a TLR-9 (ODN21798) agonist. A hierarchical clustering approach was used to compare the response of each tissue type to the ligands studied and revealed that the placenta and choriodecidua generate a more similar IL-8 response, while the choriodecidua and amnion generate a more similar IL-6 response to nucleic acid ligands. These findings demonstrate that responsiveness via TLR-3, TLR-7, TLR-8 and RIG-1/MDA5 is a broad feature of human term gestation-associated tissues with differential responses by tissue that might underpin adverse obstetric outcomes.

Thrombalexin: Use of a Cytotopic Anticoagulant to Reduce Thrombotic Microangiopathy in a Highly Sensitized Model of Kidney Transplantation.

Early activation of coagulation is an important factor in the initiation of innate immunity, as characterized by thrombotic microangiopathy (TMA). In transplantation, systemic anticoagulation is difficult due to bleeding. A novel "cytotopic" agent, thrombalexin (TLN), combines a cell-membrane-bound (myristoyl tail) anti-thrombin (hirudin-like peptide [HLL]), which can be perfused directly to the donor organ or cells. Thromboelastography was used to measure time to clot formation (r-time) in both rhesus and human blood, comparing TLN versus HLL (without cytotopic tail) versus negative control. Both TLN- and HLL-treated rhesus or human whole blood result in significantly prolonged r-time compared to kaolin controls. Only TLN-treated human endothelial cells and neonatal porcine islets prolonged time to clot formation. Detection of membrane-bound TLN was confirmed by immunohistochemistry and fluorescence activated cell sorter. In vivo, perfusion of a nonhuman primate kidney TLN-supplemented preservation solution in a sensitized model of transplantation demonstrated no evidence of TLN systemically. Histologically, TLN was shown to be present up to 4 days after transplantation. There was no platelet deposition, and TMA severity, as well as microvascular injury scores (glomerulitis + peritubular capillaritis), were less in the TLN-treated animals. Despite promising evidence of localized efficacy, no survival benefit was demonstrated.

Thrombalexins: Cell-Localized Inhibition of Thrombin and Its Effects in a Model of High-Risk Renal Transplantation.

Allograft transplantation into sensitized recipients with antidonor antibodies results in accelerated antibody-mediated rejection (AMR), complement activation, and graft thrombosis. We have developed a membrane-localizing technology of wide applicability that enables therapeutic agents, including anticoagulants, to bind to cell surfaces and protect the donor endothelium. We describe here how this technology has been applied to thrombin inhibitors to generate a novel class of drugs termed thrombalexins (TLNs). Using a rat model of hyperacute rejection, we investigated the potential of one such inhibitor (thrombalexin-1 [TLN-1]) to prevent acute antibody-mediated thrombosis in the donor organ. TLN-1 alone was able to reduce intragraft thrombosis and significantly delay rejection. The results confirm a pivotal role for thrombin in AMR in vivo. This approach targets donor organs rather than the recipient and is intended to be directly translatable to clinical use.

Immunotoxin Against a Donor MHC Class II Molecule Induces Indefinite Survival of Murine Kidney Allografts.

Rejection of donor organs depends on the trafficking of donor passenger leukocytes to the secondary lymphoid organs of the recipient to elicit an immune response via the direct antigen presentation pathway. Therefore, the depletion of passenger leukocytes may be clinically applicable as a strategy to improve graft survival. Because major histocompatibility complex (MHC) class II(+) cells are most efficient at inducing immune responses, selective depletion of this population from donor grafts may dampen the alloimmune response and prolong graft survival. In a fully MHC mismatched mouse kidney allograft model, we describe the synthesis of an immunotoxin, consisting of the F(ab')2 fragment of a monoclonal antibody against the donor MHC class II molecule I-A(k) conjugated with the plant-derived ribosomal inactivating protein gelonin. This anti-I-A(k) gelonin immunotoxin depletes I-A(k) expressing cells specifically in vitro and in vivo. When given to recipients of kidney allografts, it resulted in indefinite graft survival with normal graft function, presence of Foxp3(+) cells within donor grafts, diminished donor-specific antibody formation, and delayed rejection of subsequent donor-type skin grafts. Strategies aimed at the donor arm of the immune system using agents such as immunotoxins may be a useful adjuvant to existing recipient-orientated immunosuppression.

Noninvasive Imaging of Activated Complement in Ischemia-Reperfusion Injury Post-Cardiac Transplant.

Ischemia-reperfusion injury (IRI) is inevitable in solid organ transplantation, due to the transplanted organ being ischemic for prolonged periods prior to transplantation followed by reperfusion. The complement molecule C3 is present in the circulation and is also synthesized by tissue parenchyma in early response to IRI and the final stable fragment of activated C3, C3d, can be detected on injured tissue for several days post-IRI. Complement activation post-IRI was monitored noninvasively by single photon emission computed tomography (SPECT) and CT using (99m) Tc-recombinant complement receptor 2 ((99m) Tc-rCR2) in murine models of cardiac transplantation following the induction of IRI and compared to (99m) Tc-rCR2 in C3(-/-) mice or with the irrelevant protein (99m) Tc-prostate-specific membrane antigen antibody fragment (PSMA). Significant uptake with (99m) Tc-rCR2 was observed as compared to C3(-/-) or (99m) Tc-PSMA. In addition, the transplanted heart to muscle ratio of (99m) Tc-rCR2 was significantly higher than (99m) Tc-PSMA or C3(-/-) . The results were confirmed by histology and autoradiography. (99m) Tc-rCR2 can be used for noninvasive detection of activated complement and in future may be used to quantify the severity of transplant damage due to complement activation postreperfusion.

BRAF inhibitors: From the laboratory to clinical trials.

BRAF is one of the most commonly mutated proto-oncogenes and plays a significant role in the development of numerous cancers of high clinical impact. Due to the commonality of BRAF mutations, a number of BRAF inhibitors have been developed as tools in the management of patients with cancers dependent on the action of mutant BRAF to drive cellular proliferation. In this review, we examine the current state of clinical trials and laboratory research concerning BRAF inhibitors in development and available for clinical use. We contrast the effectiveness of type-I and type-II BRAF inhibitors, the former typically showing much more restricted inhibitory selectivity and greater patient response rates.

B-Raf mutation: a key player in molecular biology of cancer.

B-Raf is one of the more commonly mutated proto-oncogenes implicated in the development of cancers. In this review, we consider the mechanisms and clinical impacts of B-Raf mutations in cancer and discuss the implications for the patient in melanoma, thyroid cancer and colorectal cancer, where B-Raf mutations are particularly common.

Balancing mechanical strength with bioactivity in chitosan-calcium phosphate 3D microsphere scaffolds for bone tissue engineering: air- vs. freeze-drying processes.

The objective of this study was to evaluate the potential benefit of 3D composite scaffolds composed of chitosan and calcium phosphate for bone tissue engineering. Additionally, incorporation of mechanically weak lyophilized microspheres within those air-dried (AD) was considered for enhanced bioactivity. AD microsphere, alone, and air- and freeze-dried microsphere (FDAD) 3D scaffolds were evaluated in vitro using a 28-day osteogenic culture model with the Saos-2 cell line. Mechanical testing, quantitative microscopy, and lysozyme-driven enzymatic degradation of the scaffolds were also studied. FDAD scaffold showed a higher concentration (p < 0.01) in cells per scaffold mass vs. AD constructs. Collagen was ∼31% greater (p < 0.01) on FDAD compared to AD scaffolds not evident in microscopy of microsphere surfaces. Alternatively, AD scaffolds demonstrated a superior threefold increase in compressive strength over FDAD (12 vs. 4 MPa) with minimal degradation. Inclusion of FD spheres within the FDAD scaffolds allowed increased cellular activity through improved seeding, proliferation, and extracellular matrix production (as collagen), although mechanical strength was sacrificed through introduction of the less stiff, porous FD spheres.

Two novel mutations and a previously unreported intronic polymorphism in the NOTCH3 gene.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary disease of small vessel caused by mutations in the NOTCH3 gene (NCBI Gene ID: 4854) located on chromosome 19p13.1. NOTCH3 consists of 33 exons which encode a protein of 2321 amino acids. Exons 3 and 4 were found to be mutation hotspots, containing more than 65% of all CADASIL mutations. We performed direct sequencing on an ABI 3130 Genetic Analyser to screen for mutations and polymorphisms on 300 patients who were clinically suspected to have CADASIL. First, exons 3 and 4 were screened in NOTCH3 and if there were no variations found, then extended CADASIL testing (exons 2, 11, 18 and 19) was offered to patients. Here we report two novel non-synonymous mutations identified in the NOTCH3 gene. The first mutation, located in exon 4 was found in a 49-year-old female and causes an alanine to valine amino acid change at position 202 (605C>T). The second mutation, located in exon 11, was found in a 66-year-old female and causes a cysteine to arginine amino acid change at position 579 (1735T>C). We also report a 46-year-old male with a known polymorphism Thr101Thr (rs3815188) and an unreported polymorphism NM_000435.2:c.679+60G>A observed in intron 4 of the NOTCH3 gene. Although Ala202Ala (rs1043994) is a common polymorphism in the NOTCH3 gene, our reported novel mutation (Ala202Val) causes an amino acid change at the same locus. Our other reported mutation (Cys579Arg) correlates well with other known mutations in NOTCH3, as the majority of the CADASIL-associated mutations in NOTCH3 generally occur in the EGF-like (epidermal growth factor-like) repeat domain, causing a change in the number of cysteine residues. The intronic polymorphism NM_000435.2:c.679+60G>A lies close to the intron-exon boundary and may affect the splicing mechanism in the NOTCH3 gene.

Development of an eight gene expression profile implicating human breast tumours of all grade.

The goal of improving systemic treatment of breast cancers is to evolve from treating every patient with non-specific cytotoxic chemotherapy/hormonal therapy, to a more individually-tailored direct treatment. Although anatomic staging and histological grade are important prognostic factors, they often fail to predict the clinical course of this disease. This study aimed to develop a gene expression profile associated with breast cancers of differing grades. We extracted mRNA from FFPE archival breast IDC tissue samples (Grades I-III), including benign tumours. Affymetrix GeneChip(®) Human Genome U133 Plus 2.0 Arrays were used to determine gene expression profiles and validated by Q-PCR. IHC was used to detect the AXIN2 protein in all tissues. From the array data, an independent group t-test revealed that 178 genes were significantly (P ≤ 0.01) differentially expressed between three grades of malignant breast tumours when compared to benign tissues. From these results, eight genes were significantly differentially expressed in more than one comparison group and are involved in processes implicated in breast cancer development and/or progression. The two most implicated candidates genes were CLD10 and ESPTI1 as their gene expression profile from the microarray analysis was replicated in Q-PCR analyses of the original tumour samples as well as in an extended population. The IHC revealed a significant association between AXIN2 protein expression and ER status. It is readily acknowledged and established that significant differences exist in gene expression between different cancer grades. Expansion of this approach may lead to an improved ability to discriminate between cancer grade and other pathological factors.

Semaphorin-plexin signalling genes associated with human breast tumourigenesis.

INTRODUCTION: Gene expression profiling has enabled us to demonstrate the heterogeneity of breast cancers. The potential of a tumour to grow and metastasise is partly dependant on its ability to initiate angiogenesis or growth and remodelling of new blood vessels, usually from a pre-existing vascular network, to ensure delivery of oxygen, nutrients, and growth factors to rapidly dividing transformed cells along with access to the systemic circulation. Cell-cell signalling of semaphorin ligands through interaction with their plexin receptors is important for the homeostasis and morphogenesis of many tissues and has been widely studied for a role in neural connectivity, cancer, cell migration and immune responses. This study investigated the role of four semaphorin/plexin signalling genes in human breast cancers in vivo and in vitro. MATERIALS AND METHODS: mRNA was extracted from formalin fixed paraffin embedded archival breast invasive ductal carcinoma tissue samples of progressive grades (grades I-III) and compared to tissue from benign tumours. Gene expression profiles were determined by microarray using the Affymetrix GeneChip® Human Genome U133 Plus 2.0 Arrays and validated by Q-PCR using a Corbett RotorGene 6000. Following validation, the gene expression profile of the identified targets was correlated with those of the human breast cancer cell lines MCF-7 and MDA-MD-231. RESULTS: The array data revealed that 888 genes were found to be significantly (p≤0.05) differentially expressed between grades I and II tumours and 563 genes between grade III and benign tumours. From these genes, we identified four genes involved in semaphorin-plexin signalling including SEMA4D which has previously been identified as being involved in increased angiogenesis in breast cancers, and three other genes, SEMA4F, PLXNA2 and PLXNA3, which in the literature were associated with tumourigenesis, but not directly in breast tumourigenesis. The microarray analysis revealed that SEMA4D was significantly (P=0.0347) down-regulated in the grade III tumours compared to benign tumours; SEMA4F, was significantly (P=0.0159) down-regulated between grades I and II tumours; PLXNA2 was significantly (P=0.036) down-regulated between grade III and benign tumours and PLXNA3 significantly (P=0.042) up-regulated between grades I and II tumours. Gene expression of SEMA4D was validated using Q-PCR, demonstrating the same expression profile in both data sets. When the sample set was increased to incorporate more cases, SEMA4D continued to follow the same expression profile, including statistical significance for the differences observed and small standard deviations. In vitro the same pattern was present where expression for SEMA4D was significantly higher in MDA-MB-231 cells when compared to MCF-7 cells. The expression of SEMA4F, PLXNA2 and PLXNA3 could not be validated using Q-PCR, however in vitro analysis of these three genes revealed that both SEMA4F and PLXNA3 followed the microarray trend in expression, although they did not reach significance. In contrast, PLXNA2 demonstrated statistical significance and was in concordance with the literature. DISCUSSION: We, and others, have proposed SEMA4D to be a gene with a potentially protective effect in benign tumours that contributes to tumour growth and metastatic suppression. Previous data supports a role for SEMA4F as a tumour suppressor in the peripheral nervous system but our data seems to indicate that the gene is involved in tumour progression in breast cancer. Our in vitro analysis of PLXNA2 revealed that the gene has higher expression in more aggressive breast cancer cell types. Finally, our in vitro analysis on PLXNA3 also suggest that this gene may have some form of growth suppressive role in breast cancer, in addition to a similar role for the gene previously reported in ovarian cancer. From the data obtained in this study, SEMA4D may have a role in more aggressive and potentially metastatic breast tumours. CONCLUSIONS: Semaphorins and their receptors, the plexins, have been implicated in numerous aspects of neural development, however their expression in many other epithelial tissues suggests that the semaphorin-plexin signalling system also contributes to blood vessel growth and development. These findings warrant further investigation of the role of semaphorins and plexins and their role in normal and tumour-induced angiogenesis in vivo and in vitro. This may represent a new front of attack in anti-angiogenic therapies of breast and other cancers.

Investigation of the 1758G>C and 2880A>G variants within the NCOA3 gene in a breast cancer affected Australian population.

NCOA3 is a known low to moderate-risk breast cancer susceptibility gene, amplified in 5-10% and over expressed in about 60% of breast tumours. Additionally, this over expression is associated with Tamoxifen resistance and poor prognosis. Previously, two variants of NCOA3, 1758G>C and 2880A>G have been associated with breast cancer in two independent populations. Here we assessed the influence of the two NCOA3 variants on breast cancer risk by genotyping an Australian case-control study population. 172 cases and 178 controls were successfully genotyped for the 1758G>C variant and 186 cases and 182 controls were successfully genotyped for the 2880A>G variant using high-resolution melt analysis (HRM). The genotypes of the 1758G>C variant were validated by sequencing. χ(2) tests were performed to determine if significant differences exist in the genotype and allele frequencies between the cases and controls. χ(2) analysis returned no statistically significant difference (p>0.05) for genotype frequencies between cases and controls for 1758G>C (χ(2)=0.97, p=0.6158) or 2880A>G (χ(2)=2.09, p=0.3516). Similarly, no statistical difference was observed for allele frequencies for 1758G>C (χ(2)=0.07, p=0.7867) or 2880A>G (χ(2)=0.04, p=0.8365). Haplotype analysis of the two SNPs also showed no difference between the cases and the controls (p=0.9585). Our findings in an Australian Caucasian population composed of breast cancer sufferers and an age matched control population did not support the findings of previous studies demonstrating that these markers play a significant role in breast cancer susceptibility. Here, no significant difference was detected between breast cancer patients and healthy matched controls by either the genotype or allele frequencies for the investigated variants (all p ≥ 0.05). While an association of the two variants and breast cancer was not detected in our case-control study population, exploring these variants in a larger population of the same kind may obtain results in concordance with previous studies. Given the importance of NCOA3 and its involvement in biological processes involved in breast cancer and the possible implications variants of the gene could have on the response to Tamoxifen therapy, NCOA3 remains a candidate for further investigations.

Meta-analysis of immunohistochemical prognostic markers in resected pancreatic cancer.

BACKGROUND: The potential prognostic value of several commonly investigated immunohistochemical markers in resected pancreatic cancer is variably reported. The objective of this study was to conduct a systematic review of literature evaluating p53, p16, smad4, bcl-2, bax, vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) expression as prognostic factors in resected pancreatic adenocarcinoma and to conduct a subsequent meta-analysis to quantify the overall prognostic effect. METHODS: Relevant literature was identified using Medline, EMBASE and ISI Web of Science. The primary end point was overall survival assessed on univariate analysis. Only studies analysing resected pancreatic adenocarcinoma were eligible for inclusion and the summary log(e) hazard ratio (logHR) and variance were pooled using an inverse variance approach. Evidence of heterogeneity was evaluated using the χ(2) test for heterogeneity and its impact on the meta-analysis was assessed by the I(2) statisic. Hazard ratios greater than one reflect adverse survival associated with positive immunostaining. RESULTS: Vascular endothelial growth factor emerged as the most potentially informative prognostic marker (11 eligible studies, n=767, HR=1.51 (95% confidence interval, CI=1.18-1.92)) with no evidence of any significant publication bias (Egger's test, P=0.269). Bcl-2 (5 eligible studies, n=314, HR=0.51 (95% CI=0.38-0.68)), bax (5 studies, n=274, HR=0.63 (95% CI=0.48-0.83)) and p16 (3 studies, n=229, HR=0.63 (95% CI=0.43-0.92)) also returned significant overall survival differences, but in smaller patient series due to a lack of evaluable literature. Neither p53 (17 studies, n=925, HR=1.22 (95% CI=0.96-1.56)), smad4 (5 studies, n=540, HR=0.88 (95% CI=0.61-1.27)) nor EGFR (4 studies, n=250, HR=1.35 (95% CI=0.80-2.27)) was found to represent significant prognostic factors when analysing the pooled patient data. There was evidence of significant heterogeneity in four of the seven study groups. CONCLUSION: These results support the case for immunohistochemical expression of VEGF representing a significant and reproducible marker of adverse prognosis in resected pancreatic cancer.

Single nucleotide polymorphisms and mRNA expression of VEGF-A in papillary thyroid carcinoma: potential markers for aggressive phenotypes.

BACKGROUND AND OBJECTIVES: Polymorphisms of the VEGF gene are known to affect the biological behaviour of cancers but have seldom been studied in thyroid cancer. The aim of the current study is to evaluate the prevalence and relevance of VEGF-A polymorphisms and mRNA expression in papillary thyroid carcinoma (PTC). MATERIALS AND METHODS: Genomic DNA and total RNA were isolated from paraffin-embedded tissue from 91 PTC (51 conventional PTC and 40 follicular variant) and 78 control thyroid tissues. Three DNA polymorphisms (+936C > T, +405C > G and -141A > C) in the 3' and 5' untranslated region (3'-UTR, 5'-UTR) of VEGF-A were studied using PCR and RFLP. Also, the mRNA expression of VEGF-A in these tissues was studied by real-time PCR. RESULTS: Distribution of polymorphisms in the 5'-UTR (VEGF-VEGF -141A > C and +405C > G) and 3'-UTR (VEGF +936C > T) were all significantly different in PTC and benign thyroid tissue (p = 0.0001, 0.001 and 0.028 respectively). The VEGF -141 C allele was more common in PTC with lymph node metastases (p = 0.026). VEGF + 405 Galleles andVEGF +936 CC genotype were more common in PTC of advanced pathological staging (p = 0.018 and 0.017 respectively). Also, increased expression of VEGF-A mRNA was noted in PTC compared to control (p = 0.009). Within the group of patients with conventional PTC, those with lymph nodal metastases had a higher level of VEGF-A mRNA expression than other patients (p = 0.0003). CONCLUSION: These findings suggest that VEGF polymorphisms and mRNA expression may predict the aggressiveness behaviour of thyroid cancer.

Considerations for the use of plasma cytokeratin 18 as a biomarker in pancreatic cancer.

BACKGROUND: Enzyme-linked immunoassays of full-length (M65) and/or caspase-cleaved (M30) cytokeratin 18 (CK18) released from epithelial cells undergoing necrosis and/or apoptosis, respectively, may have prognostic or predictive biomarker utility in a range of solid tumour types. Characterisation of baseline levels of circulating full length and cleaved CK18 specifically in patients with pancreatic cancer. METHODS: Plasma samples from 103 patients with pancreatic cancer stored at -80 degrees C were assayed for M65 and M30 levels. The median (inter-quartile range (IQR)) duration of plasma storage was 34 (23-57) months. Patients with metastatic disease (n=19) were found to have greater median (IQR) M65 levels (1145 (739-1698) U l(-1)) compared with the locally advanced (n=20; 748 (406-1150) U l(-1)) and resected (n=64; 612 (331-987) U l(-1)) patients (P=0.002). Elevated M65 levels were associated with poorer overall survival on univariate (P<0.001) but not multivariate (P=0.202) analysis. M65 concentrations also exhibited significant associations with concurrent serum-bilirubin levels (P<0.001) and the duration of plasma storage (P<0.001). CONCLUSIONS: Baseline plasma CK18 levels in pancreatic cancer are affected by the presence of obstructive jaundice and prolonged plasma storage. Clinical biomarker studies utilising serial CK18 levels are warranted in pancreatic cancer, provided consideration is given to these potentially confounding factors.

Quantitative measurement of multifunctional quantum dot binding to cellular targets using flow cytometry.

Semiconductor nanocrystals such as quantum dots (QDs) are a potentially powerful resource in the fields of flow cytometry and fluorescence microscopy. QD size and fluorescence characteristics offer attractive features for use in targeted delivery systems and detection by flow cytometry. While quantitative measurements of a variety of fluorescent molecules are routinely performed, fluorophores for which no calibration standards exist, such as QDs, pose a problem for quantitation in flow cytometry. Our goal was to develop a targeted nanoparticle delivery platform as well as a corresponding method to accurately and quantitatively assess the performance of this system. We synthesized surface-modified QD probes targeted to cellular surface receptors and measured the MFI of the resulting cell-probe conjugates by flow cytometry. MFI was converted to mean equivalent R-PE intensity (MEPE) using standard calibration microspheres. Known concentrations of both R-PE and QD probes were measured by fluorometry to relate R-PE and QD fluorescence. Fluorometry results were then used to translate MEPE measurements to the number of bound QD probes. The targeted probes exhibited superior binding characteristics over unmodified and untargeted particles. This binding interaction was shown to be specific and mediated by the NGR targeting peptide tethered to the QD surface. The calibration method developed to assess this system proved successful at converting raw fluorescence data to quantitative probe binding values. We demonstrate the synthesis and performance of a highly modular nanoparticle system capable of targeted binding and fluorescent imaging. The calibration method implemented to quantify the performance of this system represents a potentially powerful tool to utilize truly quantitative flow cytometry measurements with an array of fluorescent molecules, including QDs.

Are periods of low atmospheric pressure associated with an increased risk of abdominal aortic aneurysm rupture?

INTRODUCTION: Seasonal variation in rates of abdominal aortic aneurysm (AAA) rupture has previously been described. However, only two studies, to date, have suggested that periods of low atmospheric pressure may account for this observation and both returned apparently contradictory findings. The objective of this study was to demonstrate whether periods of low atmospheric pressure are indeed associated with an increased likelihood of AAA rupture presentation. PATIENTS AND METHODS: A total of 182 cases of ruptured AAA were identified retrospectively from two centres over a 6-year period from January 2000 to December 2005. Local meteorological data for the corresponding period was obtained from the UK Meteorological Office which was recorded daily at a local weather station. Statistical analysis using Student's t-test, ANOVA (Kruskal-Wallis), linear regression and multiple logistic regression was conducted to identify significant relationships from the data. RESULTS: Days of rupture presentation were associated with a significantly lower daily mean atmospheric pressure when compared with days when no rupture occurred (P = 0.025). Multiple logistic regression demonstrated a significant association between low daily atmospheric pressure and rupture incidence (P = 0.033) which appeared to be independent of temperature. A significant seasonal trough in atmospheric pressure was observed in October and November (Kruskal-Wallis, P < 0.001); however, there was no corresponding autumnal peak in rupture incidence (P = 0.232). CONCLUSIONS: The findings of this study appear to confirm the assertion that low atmospheric pressure is associated with an increased risk of AAA rupture on a day-to-day basis; however, no evidence was identified to support a change in policy regarding prioritising elective surgery based on seasonal risk.