Diagnosis of medullary thyroid cancer

Medullary thyroid cancer (MTC) accounts for 5-10% of all thyroid cancers. The majority of medullary thyroid cancers are sporadic, but 25% of cases are inherited as a result of germline mutations in the RET proto-oncogene. In sporadic cases MTC presents as a thyroid nodule discovered at palpation or at thyroid ultrasonography, and is indistinguishable from thyroid nodules of different histology. Since effective treatment of MTC is only possible when the tumour is limited to the thyroid gland, early discovery has a decisive impact on how radical initial surgical treatment needs to be. Recent data suggest that in sporadic cases, early discovery of thyroid nodular disease is possible when screening serum calcitonin measurement, while screening for germline RET proto-oncogene mutations is fundamental in first degree relatives of patients with hereditary MTC

A New Methodology of Viewing Extra-Axial Fluid and Cortical Abnormalities in Children with Autism via Transcranial Ultrasonography

Background: Autism spectrum disorders (ASDs) are developmental conditions of uncertain etiology which have now affected more than 1% of the school-age population of children in many developed nations. Transcranial ultrasonography (TUS) via the temporal bone appeared to be a potential window of investigation to determine the presence of both cortical abnormalities and increased extra-axial fluid (EAF). Methods: TUS was accomplished using a linear probe (10–5 MHz). Parents volunteered ASD subjects (N = 23; males 18, females 5) for evaluations (mean = 7.46 years ± 3.97 years), and 15 neurotypical siblings were also examined (mean = 7.15 years ± 4.49 years). Childhood Autism Rating Scale (CARS2(®)) scores were obtained and the ASD score mean was 48.08 + 6.79 (Severe). Results: Comparisons of the extra-axial spaces indicated increases in the ASD subjects. For EAF we scored based on the gyral summit distances between the arachnoid membrane and the cortical pia layer (subarachnoid space): (1) <0.05 cm, (2) 0.05–0.07 cm, (3) 0.08–0.10 cm, (4) >0.10 cm. All of the neurotypical siblings scored 1, whereas the ASD mean score was 3.41 ± 0.67. We also defined cortical dysplasia as the following: hypoechoic lesions within the substance of the cortex, or disturbed layering within the gray matter. For cortical dysplasia we scored: (1) none observed, (2) rare hypoechogenic lesions and/or mildly atypical cortical layering patterns, (3) more common, but separated areas of cortical hypoechogenic lesions, (4) very common or confluent areas of cortical hypoechogenicity. Again all of the neurotypical siblings scored 1, while the ASD subjects’ mean score was 2.79 ± 0.93. Conclusion: TUS may be a useful screening technique for children at potential risk of ASDs which, if confirmed with repeated studies and high resolution MRI, provides rapid, non-invasive qualification of EAF, and cortical lesions

Friend erythroleukemia cells induce angiogenesis in chick embryo chorioallantoic membrane and in human umbilical vein endothelial cells

The effects of Friend erythroleukemia cells on angiogenesis were studied in chick embryo chorioallantoic membrane assay and in human umbilical vein endothelial cells. In chorioallantoic membrane assay, the conditioned medium of Friend cells stimulated in vivo angiogenesis to an extent comparable to that observed with Prostaglandin El, used as positive control. Prostaglandin El added to conditioned medium of Friend cells did not further increase angiogenesis. Conditioned medium of Friend erythroleukemia cells also stimulated proliferation of human umbilical vein endothelial cells to an extent comparable to that observed with fetal bovine serum, used as positive control. Conditioned medium and fetal bovine serum together did not affect human umbilical vein endothelial cells proliferation, as compared to that observed when tested separately. These results seem to indicate that Friend erythroleukemia cells produce and secrete factors stimulating angiogenesis. These findings extend and confirm the hypothesis that successful angiogenesis is necessary for development of leukemia

Telomeres and Thyroid Cancer

Telomeres are specialized structures at the ends of chromosomes, consisting of hundreds of repeated hexanucleotides (TTAGGG)n. Genetic integrity is partly maintained by the architecture of telomeres and it is gradually lost as telomeres progressively shorten with each cell replication, due to incomplete lagging DNA strand synthesis and oxidative damage. Telomerase is a reverse transcriptase enzyme that counteracts telomere shortening by adding telomeric repeats to the G-rich strand. It is composed of a telomerase RNA component and a protein component, telomerase reverse transcriptase. In the absence of telomerase or when the activity of the enzyme is low compared to the replicative erosion, apoptosis is triggered. Patients who have inherited genetic defects in telomere maintenance seem to have an increased risk of developing familial benign diseases or malignant diseases. At the somatic level, telomerase is reactivated in the majority of human carcinomas, suggesting that telomerase reactivation is a critical step for cancerogenesis

Effects of vitamin D-binding protein-derived macrophage-activating factor on human breast cancer cells

BACKGROUND: Searching for additional therapeutic tools to fight breast cancer, we investigated the effects of vitamin D-binding protein-derived macrophage activating factor (DBP-MAF, also known as GcMAF) on a human breast cancer cell line (MCF-7). MATERIALS AND METHODS: The effects of DBP-MAF on proliferation, morphology, vimentin expression and angiogenesis were studied by cell proliferation assay, phase-contrast microscopy, immunohistochemistry and western blotting, and chorioallantoic membrane (CAM) assay. RESULTS: DBP-MAF inhibited human breast cancer cell proliferation and cancer cell-stimulated angiogenesis. MCF-7 cells treated with DBP-MAF predominantly grew in monolayer and appeared to be well adherent to each other and to the well surface. Exposure to DBP-MAF significantly reduced vimentin expression, indicating a reversal of the epithelial/mesenchymal transition, a hallmark of human breast cancer progression. CONCLUSION: These results are consistent with the hypothesis that the known anticancer efficacy of DBP-MAF can be ascribed to different biological properties of the molecule that include inhibition of tumour-induced angiogenesis and direct inhibition of cancer cell proliferation, migration and metastatic potential

Role of angiotensin-converting enzyme and vitamin D receptor gene polymorphisms in cancer anorexia-cachexia syndrome

The ubiquitin-proteasome pathway is a crucial connection between aberrant immune system activation, systemic inflammation and Cancer Anorexia-Cachexia Syndrome (CACS), a syndrome that culminates in hyper-activation of the ubiquitin-proteasome pathway. Angiotensin directly up-regulates this pathway, while vitamin D down-regulates it indirectly through the insulin-like growth factor-1 pathway. We investigated the genetic predisposition towards CACS in a cancer population, examining Insertion/Deletion (I/D) polymorphism of angiotensin-converting enzyme gene and FokI and BsmI polymorphisms of vitamin D receptor gene. Sixty-two cancer patients were recruited and divided into three groups: primary cachectic (C1, n = 14; dysmetabolic body weight loss ≥5% in 6 months); secondary cachectic (C2, n = 34; similar weight loss, mechanic or iatrogenic origin); and non-cachectic (NC, n = 16). C2+NC were merged in the control group. The three groups showed significant differences in average prognostic inflammatory nutritional index (C1: 26.4±23.4; C2: 5.4±5.6; NC: 0.37±0.5), C-reactive protein serum levels (C1: 6.6±2.1; C2: 2.4±2.2; NC: 1.0±2.0 mg/dL), albumin serum levels (C1: 3.1±0.6; C2: 3.5±0.4; NC 3.7±0.6 g/dL), weight loss (C1: 22±8; C2: 15±6.7; NC 5±6%) and life expectancy (C1: 6.4±3.3; C2: 25±28; NC: 45±25 months). However, none of the chosen polymorphisms showed any statistically significant correlation with CACS. The complexity of the changes of the immune system in the chronic inflammation state associated with CACS is far greater than expected and further studies are required to identify genetic independent markers of progression toward CACS.

The effect of Gcmaf complexed with oleic acid on multiple myeloma cultures

Abstract: Deglycosylated vitamin D-binding protein-derived macrophage-activating factor (GcMAF) is known to be a strong immune stimulatory natural molecule. Data in literature demonstrate that GcMAF has a direct role in decreasing cell proliferation of different cancer cell lines. In this study we evaluate the direct effect of GcMAF complexed with oleic acid (OA-GcMAF) on human multiple myeloma cells (KMS-12- BM), as well as the effect on the same cell line of human macrophages (CRL9853) previously activated by OA-GcMAF. Cell viability and living cell number were evaluated respectively by tetrazolium dye cell viability assay and by Trypan blue staining. Interactions between activated macrophages and myeloma cells were studied by time lapse photography. Our results show that OA-GcMAF decreases the cell viability of KMS-12-BM with a dose-dependent pathway. Furthermore OA-GcMAF activates human macrophages, which in turn phagocytise myeloma cancer cells. OA-GcMAF confirms its double effect on cancer cells: a direct inhibition of their viability and, at the same time, an efficient macrophage activation leading to a significant depletion of cancer cell population. Introduction: In recent years the interest of vitamin D-binding protein-derived macrophage-activating factor (GcMAF) as a potent immunotherapeutic agent has increased. The GcMAF has been shown to be effective in stimulating murine macrophages in vitro to phagocytose human breast carcinoma cultures (1, 2), as well as inhibiting the growth of prostate cancer cells (3). It has also been the agent that has been referenced as reducing tumour burden in several clinical approaches (4, 5). In previous studies GcMAF has been used to stimulate Raw 264.7 cells (murine macrophage cell line) that were observed in vitro to phagacytose MCF-7 cells (human breast carcinoma). In this study we demonstrate the effect of GcMAF stabilized with oleic acid (OA-GcMAF) directly on KMS-12-BM multiple myeloma cells and on co-culture of stimulated human macrophages (CRL9853) and KMS-12-BM. Materials and Methods: Cell lines: KMS-12-BM: human multiple myeloma cell line was purchased from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) and cultured in RPMI 1640 supplemented with 10% FBS and 2mM L-Glutamine (Life Technologies, Paisley, UK). Cultures were passaged every 3-4 days. CRL9853: human spleen macrophage was purchased from ATCC (American Type Culture Collection, Teddington, UK) and routinely cultured in IMDM supplemented with 10% FBS and 2mM L-Glutamine (Life Technologies). Cultures were passaged every 3-4 days. Prior to assay, CRL9853 cells were activated culturing them for 72h in the presence of OAGcMAF at a concentration 100ng/ml in complete medium. Stimuli: OA-GcMAF, commercially available, was prepared at Immuno Biotech Ltd. (Guernsey, Channel Island) with a proprietary procedure previously described (6). Cell viability assay: Cell viability was evaluated by the reduction of a tetrazolium salt (WST-8) as an index of cell dehydrogenases’ activity. KMS-12-BM cells were seeded into a 96-well plate at a density of 3x104 cells/well in their appropriate starvation medium (without FBS). After incubation for 24h the cell line was treated for 24h with the following different concentrations of OA-GcMAF ([8-80-800 pM). At the end of the treatment, the medium was replaced with 100μl of fresh starvation medium plus 10μl of WST-8. The 96-well plate was incubated for 3h at 37°C and the optical density (O.D.) was directly measured at A450nm by Multiscan FC photometer (ThermoScientific, Milano, Italy). Cell counting – Trypan blue assay: To corroborate the results obtained by cell viability assay, a viable cell count was performed. Briefly, KMS-12-BM cells were plated into a 6-well plate at a density of 2x105 cells/well in starvation medium. After 24h incubation, human multiple myeloma cells were treated with OA-GcMAF at the increasing concentrations (8-80-800 pM) for 24h. At the end of the treatment, a volume of cell suspension was collected and the viable cell number was counted by Trypan Blue staining. Video-time lapse photography: KMS-12-BM cells were seeded into a 24-well plate at a density of 1x106 cells/well with a 1ml volume. The cells were allowed to settle for a minimum of 2h prior to the addition of the OA-GcMAF-activated CRL9853 macrophages. The staging mat was set at a temperature of 37°C and allowed to equilibrate prior to placement of the 24-well plate. The OA-GcMAF-activated CRL9853 macrophages were added to a final concentration of 5x105 cells per well in 1ml. HEPES (Fisher Scientific, Loughborough, UK) was added to each well to provide a final concentration of 25mM to stabilize the culture pH. Once activated macrophages and the HEPES were added, the 24-well plate was observed microscopically and an image selected. An initial frame was taken and a timelapse film initiated. A frame was taken every 3 minutes until filming was stopped. Results: Cell viability assay: Cell viability (Figure 1), evaluated both by tetrazolium dye cell assay (A) and by Trypan blue staining (B), decreased when KMS-12-BM cells were treated with increasing concentrations of OAGcMAF. In particular, when cells were treated with OAGcMAF (800 pM) a significant reduction (p<0.01) in cell viability was observed in comparison to the untreated control cells. Discussion: It has been shown that Gc-MAF activates Raw 264.7 murine macrophages to phagocytose and destroy MCF-7 human breast carcinoma cells (1). An identical effect has been recorded for the first time with the stimulation of human macrophages CRL9853 on KMS-12-BM cell line . The OA-GcMAF-stimulated human macrophages seek out surround and phagocytose the myeloma cell lines destroying them. This demonstrates that the CRL9853 behave as postulated against KMS-12- BM cell line. In addition the effect of increased cell death in the presence of OA-GcMAF alone as indicated by direct evaluation of viable cell counts and viability assay also provides supports to the previous data generated (1). This study provides some inferred evidence to support the in vivo clinical data that has recently been published (6) providing some insight into the method of potential tumour removal by stimulated macrophages. In conclusion OA-GcMAF has demonstrated two major effects: a direct decrease of KMS-12- BM cell viability and an efficient activation of human macrophages, which become able to phagocytose and destroy the myeloma cells. The studies will be expanded further to encompass additional cancer cell lines