Expression pattern of the Hedgehog signaling pathway in pituitary adenomas.

Several studies have demonstrated the role of Wnt and Notch signaling in the pathogenesis of pituitary adenomas, but data are scarce regarding the role of Hedgehog signaling. In this study we investigated the differential expression of gene targets of the Hedgehog signaling pathway. Formalin-fixed, paraffin-embedded specimens from adult patients who underwent transphenoidal resection and normal human pituitary tissues that were obtained from autopsies were used. Clinical information and data from pre-operative MRI scan (extracellular tumor extension, tumor size, displacement of the optic chiasm) were retrieved from the Hospital's database. We used a customized RT(2) Profiler PCR Array, to investigate the expression of genes related to Notch and Hedgehog signaling pathways (PTCH1, PTCH2, GLI1, GLI3, NOTCH3, JAG1, HES1, and HIP). A total of 52 pituitary adenomas (32 non-functioning adenomas, 15 somatotropinomas and 5 prolactinomas) were used in the final analysis. In non-functioning pituitary adenomas there was a significant decrease (approximately 75%) in expression of all Hedgehog related genes that were tested, while Notch3 and Jagged-1 expression was found significantly increased, compared with normal pituitary tissue controls. In contrast, somatotropinomas demonstrated a significant increase in expression of all Hedgehog related genes and a decrease in the expression of Notch3 and Jagged-1. There was no significant difference in the expression of Hedgehog and Notch related genes between prolactinomas and healthy pituitary tissues. Hedgehog signalling appears to be activated in somatotropinomas but not in non-functioning pituitary adenomas in contrast to the expression pattern of Notch signalling pathway.

Effects of local application of simvastatin on bone regeneration in femoral bone defects in rabbit.

Simvastatin (SIM), which is widely used in hyperlipidemia treatment, has also attracted attention due to its anabolic effects on bones. This study is designed to investigate the effectiveness of 2 mg SIM combined with 3 different carriers as delivery systems. Bone defects were surgically created in the femoral bones of 14 New Zealand white rabbits. The carriers used were the inorganic bovine bone graft (BOS), the hydroxyapatite combined with calcium sulfate (HACS), and the collagen sponge (COS). The bone defects were divided for each time period into 7 groups, as follows: passive control group (CONT), active control groups (BOS), (HACS) and (COS) (no simvastatin), and groups (BOS + SIM), (HACS + SIM) (carrier and simvastatin combination). Animal were sacrificed at 4 and 8 weeks postoperatively, and bone defects areas were prepared for histological examination and histomorphometric evaluation. Analysis of variance demonstrated statistically significant differences between groups depending on the carrier used. At 4 weeks, the BOS + SIM group presented higher rates of new bone formation, whereas at 8 weeks more new bone formation was noted for the HACS + SIM group. This study suggests that local application of simvastatin, combined with an appropriate carrier, can promote new bone formation.

Manifestations of pilocytic astrocytoma: a pictorial review.

BACKGROUND: Pilocytic astrocytoma can be challenging to diagnose. METHODS: Its clinical presentations can differ, directly related to its size and location, and are relatively unreliable. Similarly, imaging findings also vary with the location of the pilocytic astrocytoma. RESULTS: This review provides an overview of the different imaging findings regarding pilocytic astrocytomas using both conventional and advanced magnetic resonance imaging sequences according to tumour location; the findings are strongly related to the tumour's tendency to infiltrate surrounding structures, being able to carry out gross total resection, and finally the prognosis. CONCLUSIONS: Knowledge of these imaging manifestations of pilocytic astrocytoma may be helpful to arrive at an accurate diagnosis. TEACHING POINTS: • To recognise the various imaging findings of pilocytic astrocytoma on both conventional and advanced magnetic resonance imaging sequences. • To identify the characteristic imaging findings according to tumour location. • To discuss the relevant differential diagnoses of pilocytic astrocytoma in each tumour location.

DARPP32, STAT5 and STAT3 mRNA expression ratios in glioblastomas are associated with patient outcome.

Based on recent developments in glioblastoma subtyping, we examined DARPP32 (PPP1R1B), a neuronal marker against STAT5 and STAT3 that are pro-oncogenic in glioblastoma. mRNA ratios of DARPP32, STAT1, STAT3, STAT5A and STAT5B were assessed in routinely diagnosed gliomas s including a series of glioblastomas from patients (n = 67) treated with chemoradiotherapy (temozolomide), out of which 88 % had sequencing validated IDH-negative disease. DARPP32/STAT1 (p = 0.0007), DARPP32/STAT3 (p = 0.0004) and DARPP32/STAT5B (p = 0.0039) ratios were significantly higher in grade II and III as compared to grade IV tumours. The same high ratios were also associated with absence of immunohistochemically assessed AKT/PKB phosphorylation and survivin protein expression. High DARPP32/STAT3, DARPP32/STAT5B, and STAT5B/STAT3 ratios were associated with longer patient progression free (PFS) and overall survival (OS). Upon multivariate analysis, total/subtotal removal of the tumour (HR:0.431; 95%CI:0.241-0.771, Wald p = 0.005), high DARPP32/STAT5B (HR:0.341; 95%CI:0.169-0.690; Wald p = 0.003) and STAT5B/STAT3 mRNA ratios (HR:0.480; 95%CI:0.280-0.824; Wald p = 0.008) were independent favorable parameters for prolonged PFS. Extent of surgery (HR:0.198; 95%CI:0.101-0.390; p < 0.001) and high DARPP32/STAT5A ratios (HR:0.320; 95%CI:0.160-0.638, p = 0.001) were independently predictive for longer OS. The presented approach is applicable for prospective validation and appears promising towards an effective glioblastoma patient stratification in addition to IDH mutations. These data may contribute to understanding the biology of gliomas with respect to their potential neuronal characteristics and justify STAT-inhibiting therapeutic interventions in the same tumour system.

The effectiveness of intralesional injection of platelet-rich plasma in accelerating the healing of chronic ulcers: an experimental and clinical study.

The purpose of this prospective experimental and clinical study is to evaluate the effectiveness of the intralesional injection of platelet-rich plasma (PRP), in the management of non-healing chronic wounds. Skin defects were created in the ears of 20 white New Zealand rabbits. In the study group, autologous PRP was injected intralesionally. The control group was treated conservatively. Nineteen out of 20 cases of the study group healed within a mean time of 24·9 days. In the control group, seven defects healed within a mean period of 26·7 days, seven ulcers did not heal at day 28 and in six cases a full thickness ear defect was recorded. For a 3-year period, 26 patients with chronic ulcers underwent surgical debridement and intralesional injection of PRP. A histological study was performed before and 7 days after PRP injection. Ten patients healed within a mean period of 7 weeks. In 16 cases, PRP prepared the wound bed for the final and simpler reconstructive procedure. Intralesional injection is a newly described method for application of PRP and represents an effective therapeutic option when dealing with non-healing wounds.

The effects of chronically increased intra-abdominal pressure on the rabbit diaphragm.

BACKGROUND: Diaphragmatic muscular remodeling is caused by various conditions and was mainly studied in pulmonary pathologies and chronic alterations of intra-thoracic pressure. We investigate the effect of the chronically increased intra-abdominal pressure (IAP) on the diaphragm by morphological and biochemical analysis. METHODS: Thirty rabbits were divided into control and study groups. IAP was increased in group B to 12 mmHg for 2 months. The left hemidiaphragm underwent morphological, while the right underwent biochemical analysis. RESULTS: In H&E, all fibers were normal. ATPase analysis demonstrated that type I fibers show no differences between groups. Type ΙΙ(Α) were decreased (p = 0.016) while type ΙΙ(Β/X) fibers were increased (p = 0.025) in group B. Fibers with resistance to fatigue were decreased in group B (p = 0.024). In group B, biochemical activity for glutathione reductase (p = 0.004), glutathione peroxidase (p = 0.021), protein carbonylation (0.029), lipid peroxidation (p = 0.005), and balance of preoxidative-antioxidative factors (p = 0.006) was increased. CONCLUSIONS: Chronically increased IAP induces alterations to the rabbit diaphragm. Adaptation, equivalent to strenuous contraction, transforms the diaphragm to be functionally more efficient toward workload but makes it vulnerable against oxidative stress.

Diaphragmatic adaptation following intra-abdominal weight changing.

BACKGROUND: The diaphragm, the major respiratory muscle, contains three types of muscular fibers in dynamic balance between them. The fiber ratios vary in time in function of conditions, such as aging, hypoproteinemia, exercise, and chronic respiratory load. The diaphragmatic adaptation following abdominal conditions remains an unexplored field. This experimental study aims to identify the changes of the diaphragm due to chronic abdominal weight load. This may find application in conditions such as pregnancy, ascites, visceromegaly, large masses, and morbid obesity. METHODS: Thirty rabbits were divided into control (A) and study (B) groups. Group B was loaded with weight for 2 months. The left costal hemidiaphragm were stained with H&E and ATPase (fiber typing), while the right underwent biochemical analysis (prooxidative-antioxidative balance, lipid peroxidation, superoxide dismutase, glutathione reductase and peroxidase activities, total glutathione, and protein carbonylation). RESULTS: In H&E, all fibers were within normal range. ATPase analysis demonstrated reduction of type I (p = 0.019) and an increase of the type ΙΙ(Α) fibers ratio (p < 0.001) in group B, while the type ΙΙ(Β/X) fibers ratio remained stable. The above suggest remodeling of type I fibers into type II(A). Concerning biochemical analysis, difference was observed in glutathione peroxidase activity (p < 0.001). CONCLUSIONS: Chronically loaded abdomen leads to morphological adaptations of the costal diaphragm, but with minor oxidative stress. These diaphragmatic morphological changes are equivalent to heart failure or severe COPD, showing that this remodeling makes the muscle more efficient towards work load, but more vulnerable to fatigue.

The effect of chronically increased intra-abdominal pressure on rectus abdominis muscle histology an experimental study on rabbits.

BACKGROUND: The aim of this study was to specify the histologic response of the rectus abdominis muscle of the rabbit, to the chronically increased intra-abdominal pressure. MATERIALS AND METHODS: Forty-five New Zealand white rabbits were divided into three groups. In all groups, a rubber bag was implanted into the peritoneal cavity. In group A (n=15) the bags were kept empty. In group B (n=15) the bags were filled with normal saline in order to achieve an intra-abdominal pressure of over 12 mmHg. This pressure was kept at this level for 8 wk. In group C (n=15) the intra-abdominal rubber bags were filled with lead covered by silicone, equiponderant to the mean weight of the normal saline insufflated in group B. After 8 wk we took biopsies of the rectus abdominis muscle and counted the proportion of the different types of muscular fibers (type I, IIA, and IIB/X). RESULTS: Significant difference was found in the proportion of the three types of muscle fibers. Intra-abdominal hypertension led to an increase in type I fibers (P=0.008). No difference was noticed between groups A and C. CONCLUSIONS: The histologic response to the increased intra-abdominal pressure was an increase in type I muscle fibers. Charging with lead did not cause any significant change in the proportion of muscular fibers.

Targeted KRAS mutation assessment on patient tumor histologic material in real time diagnostics.

BACKGROUND: Testing for tumor specific mutations on routine formalin-fixed paraffin-embedded (FFPE) tissues may predict response to treatment in Medical Oncology and has already entered diagnostics, with KRAS mutation assessment as a paradigm. The highly sensitive real time PCR (Q-PCR) methods developed for this purpose are usually standardized under optimal template conditions. In routine diagnostics, however, suboptimal templates pose the challenge. Herein, we addressed the applicability of sequencing and two Q-PCR methods on prospectively assessed diagnostic cases for KRAS mutations. METHODOLOGY/PRINCIPAL FINDINGS: Tumor FFPE-DNA from 135 diagnostic and 75 low-quality control samples was obtained upon macrodissection, tested for fragmentation and assessed for KRAS mutations with dideoxy-sequencing and with two Q-PCR methods (Taqman-minor-groove-binder [TMGB] probes and DxS-KRAS-IVD). Samples with relatively well preserved DNA could be accurately analyzed with sequencing, while Q-PCR methods yielded informative results even in cases with very fragmented DNA (p<0.0001) with 100% sensitivity and specificity vs each other. However, Q-PCR efficiency (Ct values) also depended on DNA-fragmentation (p<0.0001). Q-PCR methods were sensitive to detect99%) could accurately be analyzed at a sensitivity level of 10% (external validation of TMGB results). DNA quality and tumor cell content were the main reasons for discrepant sequencing/Q-PCR results (1.5%). CONCLUSIONS/SIGNIFICANCE: Diagnostic targeted mutation assessment on FFPE-DNA is very efficient with Q-PCR methods in comparison to dideoxy-sequencing. However, DNA fragmentation/amplification capacity and tumor DNA content must be considered for the interpretation of Q-PCR results in order to provide accurate information for clinical decision making.

Phase II study of neoadjuvant imatinib in glioblastoma: evaluation of clinical and molecular effects of the treatment.

PURPOSE: Phase I-II studies indicate that imatinib is active in glioblastoma multiforme. To better understand the molecular and clinical effects of imatinib in glioblastoma multiforme, we conducted a neoadjuvant study of imatinib with pretreatment and posttreatment biopsies. EXPERIMENTAL DESIGN: Patients underwent a computerized tomography-guided biopsy of their brain tumors. If diagnosed with glioblastoma multiforme, they were immediately treated with 7 days of imatinib 400 mg orally twice daily followed by either definitive surgery or re-biopsy. Pretreatment and posttreatment tissue specimens were tested by immunohistochemistry for Ki67 and microvessel destiny, and posttreatment specimens were analyzed for the presence of intact imatinib in tissue. Furthermore, pretreatment and posttreatment pairs were analyzed by Western blotting for activation of platelet-derived growth factor receptor, epidermal growth factor receptor (EGFR), phosphoinositide 3-kinase/AKT, and mitogen-activated protein kinase signaling pathways. Pharmacokinetic studies were also done. RESULTS: Twenty patients were enrolled. Median survival was 6.2 months. Intact imatinib was detected in the posttreatment tissue specimens using mass spectrometry. There was no evidence of a drug effect on proliferation, as evidenced by a change in Ki67 expression. Biochemical evidence of response, as shown by decreased activation of AKT and mitogen-activated protein kinase or increased p27 level, was detected in 4 of 11 patients with evaluable, matched pre- and post-imatinib biopsies. Two patients showed high-level EGFR activation and homozygous EGFR mutations, whereas one patient had high-level platelet-derived growth factor receptor-B activation. CONCLUSIONS: Intact imatinib was detected in glioblastoma multiforme tissue. However, the histologic and immunoblotting evaluations suggest that glioblastoma multiforme proliferation and survival mechanisms are not substantially reduced by imatinib therapy in most patients.

Neuroprotective effect of long-term MgSO4 administration after cerebral hypoxia-ischemia in newborn rats is related to the severity of brain damage.

Previous studies have shown contradictory results regarding magnesium-mediated neuroprotection in animal models of perinatal asphyxia. The aim of this study is to investigate the effects of MgSO(4) postasphyxial treatment on hypoxia-ischemia (HI)-induced brain injury in neonatal rats and the possibility that this effect is related to the severity of brain damage. Seven-day-old rats underwent unilateral carotid artery ligation followed by 1 or 2 hours of hypoxia (8% O(2)) and MgSO(4) administration. Adenosine triphosphate/phosphocreatine and glutamate/glutamine measurements and neuropathological evaluation of the hippocampus were used to assess the effects of HI and MgSO(4). HI caused time-dependent changes in energy stores, amino acid concentrations, and brain damage. Administration of MgSO(4) after 1 hour but not after 2 hours of hypoxia resulted in significant prevention of HI-induced brain injury. MgSO(4) administration results in a significant protection against moderate HI-induced brain damage, whereas it fails to offer a similar effect against severe brain damage.

hTERT immunopositivity patterns in the normal brain and in astrocytic tumors.

Accumulating data about the impact of hTERT in astrocytic tumor carcinogenesis and recent evidence about its association with disease outcome prompt the evaluation of this molecule with methods applicable in routine pathology practice. In this study, we investigated hTERT protein expression with immunohistochemistry (IHC) and the NCL-hTERT antibody in 49 astrocytic tumors. Results were validated with the assessment of hTERT mRNA (relative quantification, identification of splice variants, in situ hybridization). Specific nuclear hTERT immunostaining patterns (IPs) were characterized as patterns As (single large dot) and Am (multiple dots) without nucleoplasm staining and pattern B (nucleoplasm staining with or without dots), corresponding to low and high relative hTERT expression values (P<0.0001). Low- and high-grade astrocytic tumors were found positive for hTERT in 74 and 85% of cases, respectively. Heterogeneity in the distribution of hTERT-positive cells was observed in all tumors. The prevailing nuclear IPs differed significantly between pilocytic astrocytomas (pattern As) and the rest of histologic types up to glioblastoma (patterns Am and B) (P<0.0001). The described nuclear IPs were also observed in non-neoplastic cells. Positive endothelial cells were found in astrocytic tumors of all grades, even when tumor cells showed no hTERT immunoreactivity. A subset of mature normal neurons was positive for hTERT (pattern As), suggesting a role for this molecule in neuronal maintenance in the adult brain. The nuclear hTERT IPs described here may reflect the functional status of non-neoplastic brain and neoplastic astrocytic cells and support the model of a continuum in the development of glioblastomas from diffuse fibrillary astrocytomas.

Post-operative combined radiation and chemotherapy with temozolomide and irinotecan in patients with high-grade astrocytic tumors. A phase II study with biomarker evaluation.

BACKGROUND: Clinical studies have shown that temozolomide (TMZ) and irinotecan demonstrate activity in high grade astrocytic tumors (HGAT). However, the optimal schedule of administration is unknown. PATIENTS AND METHODS: In the present study, a total of 45 HGAT patients, 38 with glioblastoma multiforme (GBM) and 7 with anaplastic astrocytoma (AA), were treated with TMZ, 150 mg/m(2) on days 1-5, followed by irinotecan, 150 mg/m(2) on days 6 and 17, every 4 weeks for 6 cycles or until the occurrence of unacceptable toxicity or disease progression. Radiation therapy (60 Gy) was initiated on the first day of treatment. RESULTS: Twenty-two patients completed six cycles of treatment. Most frequently recorded side-effects included neutropenia (37%), nausea/vomiting (66%), diarrhea (31%) and infection (44%). Five episodes of vaso-occlusive disease, all of them fatal, were observed. After a median follow-up of 49.8 months, median progression-free survival for patients with GBM was 7.7 months, while median overall survival was 12.8 months. There were six long-term survivors, three of them with GBM. Two out of the five biomarkers studied, epidermal growth factor receptor (EGFR) and vascular endothelial growth factor-C (VEGF-C), were found to be overexpressed in 74% of the tumors, however they had no predictive value for progression-free or overall survival. CONCLUSION: The combination of TMZ and irinotecan, as administered in this study, was accompanied by high rates of toxicity, especially myelotoxicity and infection. Further development of this regimen in the treatment of HGAT is not recommended.

Hypoxia-ischemia affects erythropoietin and erythropoietin receptor expression pattern in the neonatal rat brain.

Erythropoietin (EPO), known for its role in erythroid differentiation, has been suggested to have non-hematopoietic functions in the brain, especially during development. In the present study, we investigated the expression of erythropoietin and erythropoietin receptor (EPOR) in the developing rat brain following hypoxia-ischemia. Seven-day-old rats underwent unilateral, permanent carotid artery ligation followed by 1 h of hypoxia, and their brains were examined immediately, 24 h or 4 days after hypoxia-ischemia. RT-PCR and Western blot analysis revealed that hypoxia-ischemia only marginally affected EPO expression. Immunohistochemical study of brains 4 days after hypoxia showed that 60 min of hypoxia (resulting in cortical infarction and severe neuronal loss in other regions) led to the increased EPO immunoreactivity, especially in the boundaries of the damaged cerebral cortex, associated with astrocytosis. In contrast, EPOR was dramatically upregulated within 24 h after hypoxia-ischemia. These results suggest that there is a rapid response of EPOR to the hypoxic-ischemic stimulus, which seems to precede that of EPO, leading to the hypothesis that the EPO/EPOR system is implicated in the processes of neuroprotection from hypoxia-ischemia.

A rare case of juxtaarticular osteoid osteoma of the calcaneus initially misdiagnosed as juvenile chronic arthritis.

Juxtaarticular osteoid osteoma is frequently misdiagnosed because the symptoms may mimic arthritis, and radiographs may not be characteristic. A rare case of subtalar pain lasting 5 years in a female teenager is presented here. The initial diagnosis was monarticular juvenile chronic arthritis. Family history was misleading because her mother had rheumatoid arthritis (RA).