Background: Inflammatory cells contribute to the growth and spread of human malignancies by producing molecules that enhance tumour invasiveness. Aims: To characterise the inflammatory infiltrate in pancreatic ductal adenocarcinoma and to analyse its contribution to angiogenesis and its prognostic relevance. Methods: Immunohistochemistry was used to identify inflammatory cells and evaluate the expression of proangiogenic and prolymphangiogenic molecules (vascular endothelial growth factor A (VEGF-A), VEGF-C, and basic fibroblast growth factor (bFGF)) by inflammatory and cancer cells in 137 pancreatic cancers. Intratumorous microvessel density (IMD) was assessed using CD34 as an endothelial cell marker. Results: There were significantly more mast cells and macrophages in pancreatic cancers than in normal pancreas and the number of mast cells directly correlated with the presence of lymph node metastases. However, there was no relation between numbers of infiltrating inflammatory cells and the presence of chronic pancreatitis (CP)-like changes in the parenchyma surrounding the tumour. Double immunostaining revealed that both pancreatic mast cells and macrophages express VEGF-A, VEGF-C, and bFGF. These factors were also expressed in the tumour cells in many cases. The numbers of VEGF-A expressing tumour cells and bFGF expressing tumour and inflammatory cells significantly correlated with IMD. Moreover, tumours with higher IMD had higher numbers of infiltrating mast cells and macrophages. Conclusions: Mononuclear inflammatory cells of the non-specific immune response are recruited to pancreatic cancer tissues independent of the presence of CP-like changes, may influence the metastatic capacity of the cancer cells, and may contribute to the development of tumours with high angiogenic activity.

Background and objectives: Cancer stem cells (CSCs) have been implicated in tumor initiation, recurrence, metastatic spread and poor survival in multiple tumor types, breast cancers included. CSCs selectively overexpress key mitochondrial-related proteins and inhibition of mitochondrial function may represent a new potential approach for the eradication of CSCs. Because mitochondria evolved from bacteria, many classes of FDA-approved antibiotics, including doxycycline, actually target mitochondria. Our clinical pilot study aimed to determine whether short-term pre-operative treatment with oral doxycycline results in reduction of CSCs in early breast cancer patients.Methods: Doxycycline was administered orally for 14 days before surgery for a daily dose of 200 mg. Immuno-histochemical analysis of formalin-fixed paraffin-embedded (FFPE) samples from 15 patients, of which 9 were treated with doxycycline and 6 were controls (no treatment), was performed with known biomarkers of “stemness” (CD44, ALDH1), mitochondria (TOMM20), cell proliferation (Ki67, p27), apoptosis (cleaved caspase-3), and neo-angiogenesis (CD31). For each patient, the analysis was performed both on pre-operative specimens (core-biopsies) and surgical specimens. Changes from baseline to post-treatment were assessed with MedCalc 12 (unpaired t-test) and ANOVA.Results: Post-doxycycline tumor samples demonstrated a statistically significant decrease in the stemness marker CD44 (p-value < 0.005), when compared to pre-doxycycline tumor samples. More specifically, CD44 levels were reduced between 17.65 and 66.67%, in 8 out of 9 patients treated with doxycycline. In contrast, only one patient showed a rise in CD44, by 15%. Overall, this represents a positive response rate of nearly 90%. Similar results were also obtained with ALDH1, another marker of stemness. In contrast, markers of mitochondria, proliferation, apoptosis, and neo-angiogenesis, were all similar between the two groups.Conclusions: Quantitative decreases in CD44 and ALDH1 expression are consistent with pre-clinical experiments and suggest that doxycycline can selectively eradicate CSCs in breast cancer patients in vivo. Future studies (with larger numbers of patients) will be conducted to validate these promising pilot studies.

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The neuromuscular junction has retained through evolution the capacity to regenerate after damage, but little is known on the inter‐cellular signals involved in its functional recovery from trauma, autoimmune attacks, or neurotoxins. We report here that CXCL12α, also abbreviated as stromal‐derived factor‐1 (SDF‐1), is produced specifically by perisynaptic Schwann cells following motor axon terminal degeneration induced by α‐latrotoxin. CXCL12α acts via binding to the neuronal CXCR4 receptor. A CXCL12α‐neutralizing antibody or a specific CXCR4 inhibitor strongly delays recovery from motor neuron degeneration in vivo. Recombinant CXCL12α in vivo accelerates neurotransmission rescue upon damage and very effectively stimulates the axon growth of spinal cord motor neurons in vitro. These findings indicate that the CXCL12α‐CXCR4 axis plays an important role in the regeneration of the neuromuscular junction after motor axon injury. The present results have important implications in the effort to find therapeutics and protocols to improve recovery of function after different forms of motor axon terminal damage.

Ductal pancreatic carcinoma (DPC) is a deadly disease with an incidence of 9 cases in 100,000 people per year and a mortality rate close to 100%. Allelic losses in the long arm of chromosome 9 are commonly encountered in many human malignancies but no data are yet available about DPC. We screened 40 lasermicrodissected DPC samples and 6 pre-invasive lesions for 9 microsatellite mapping markers of region 9q21.3 through 9q34.2. A small overlapping region of deletion, spanning 8 million base pairs, was identified between D9S127 and D9S105. Two genes, RSG3 and KLF4, mapped to 9q31.1 through 9q32, were further investigated. A highly significant association was found between KLF4 gene expression levels and genomic status. Similarly, absence of immunohistochemical expression of KLF4 protein was found in 86.8% cases of DPC (33/ 38). Overexpression of KLF4 in a human pancreatic carcinoma cell line induced a significant decrease in the proliferation associated with up-regulation of p21 and the down-regulation of cyclin D1. In conclusion, we identified a novel oncosuppressor region located at the 9q 31.1-3 locus that is lost in DPC at high frequency. Loss of KLF4 expression is closely related to the genomic loss, and its restoration inhibits cancer cell proliferation, suggesting a key suppressor role in pancreatic tumorigenesis. Pancreatic cancer is the fifth leading cause of cancerrelated death in both men and women in the Western world, being responsible for 5% of all cancer-related deaths.1 The lack of reliable early diagnostic methods and effective therapeutic regimens makes the mortality rates in persons with pancreatic carcinoma virtually the same as the incidence rates. A radical surgical approach is possible in only 10% of cases, and adjuvant therapies are virtually ineffective.2 A better understanding of the molecular mechanisms leading to pancreatic tumorigenesis may provide new markers for early diagnosis and potential targets for therapeutic intervention.Ductal pancreatic carcinoma (DPC) is by far the most common pancreatic tumor type, accounting for about 90% of all pancreatic malignancies. At present, a molecular model of DPC development has been proposed; it involves key genes, such K-ras, HER2neu, p16, p53, and DPC4 (smad4). Activating mutations in the K-ras oncogene and the overexpression of Her-2/neu gene are considered "early" genetic events because they occur in pre-invasive lesions (pancreatic intra-epithelial neoplasias, or PanINs) 3,4 and are followed by homozygous deletions in the p16 tumor suppressor gene locus. 5,6 Later in the tumor's progression, inactivations of p53 and DPC4 tumor suppressor genes are thought to be key events that lead to fully transformed phenotypes (carcinoma in situ, or PanIN-3). 7-11Wide genomic instability characterizes DPC, as has been documented by the large collection of cytogenetic abnormalities reported in the literature. [12][13][14][15][16][17] Among these, 9p (p16 locus), 17p (p53 locus), and 18q (DPC4 locus) have been reported to have been deleted in more than 60%...

The angiogenic phenotype of 13 normal adrenal glands (N), 13 aldosterone-producing adenomas (APA), 12 cortisol-producing adenomas (CPA), 13 nonfunctioning adrenal cortical adenomas (NFA), and 13 adrenal cortical carcinomas (CA) was investigated. Intratumoral vascular density was explored by CD34, a marker of endothelial cells, and the angiogenic status was investigated by vascular endothelial growth factor (VEGF) expression, an important angiogenic factor expressed by tumoral cells. Vascular density, quantified as the number of vessels per square millimeter, was significantly lower (P < 0.0001) in CA (110.3 +/- 27.8) than in N (336.6 +/- 14.5), APA (322.8 +/- 19.1), CPA (288.5 +/- 14.3), and NFA (274.2 +/- 19.8). VEGF expression, calculated as the percentage of positive cells, was significantly greater (P < 0.0001) in CA (85.3 +/- 2.1) than in APA (56.5 +/- 7.5), CPA (38.5 +/- 7.0), N (33.1 +/- 6.1), and NFA (0.76 +/- 0.6). In APA, a negative relation between CD34 and plasma renin activity (P < 0.0002) and a positive association between CD34 and aldosterone levels (P < 0.05) was found. In conclusion, the angiogenic phenotype of CA is characterized by VEGF overexpression but low vascularization, a finding suggesting a dissociation between angiogenic potential and neoangiogenic capabilities of these tumors. The lack of VEGF expression in NFA and the close association between angiogenesis and functional status in APA also suggest a possible influence of the angiogenic phenotype on hormonal secretion of these endocrine tumors.

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Transglutaminase 2 (TG2 or TGM2) is a multi-functional enzyme which catalyzes transamidation reactions or acts as a G-protein in intracellular signalling. Tgm2 -/-Mice lacking TG2 activity are glucose intolerant and show impairment of insulin secretion, suggesting an important physiological role for TG2 in the pancreatic β cell. We have previously described a TGM2 heterozygous missense mutation ((c.998A>G, p.N333S) in a 14 year-old patient with insulin-treated diabetes and in his diabetic father. The aim of this study was to further investigate the role of TG2 in early-onset type 2 diabetes. We analysed the TGM2 gene in 205 patients with clinically defined Maturity Onset Diabetes of the Young (MODY) or early-onset type 2 diabetes. We found two novel heterozygous mutations (c.989T>G, p.M330R; c.992T>A, p.I331N), which were not detected in 300 normoglycemic controls. All mutations were in residues which are located close to the catalytic site and impaired transamidating activity in vitro. Gene expression of TGM family genes and localization of TG2 in normal human pancreas indicated that TG2 is the only transglutaminase significantly expressed in human pancreatic islet cells. We conclude that reduced TG2 activity can contribute to disorders of glucose metabolism possibly via an impairment of insulin secretion. © 2007 Wiley-Liss, Inc. INTRODUCTIONTransglutaminase 2 (TG2; EC 2.3.2.13) is an ubiquitous enzyme with two well established, reciprocally regulated, activities: it cross-links proteins by virtue of its transamidating activity and acts as a G-protein in intracellular signalling (Griffin et al., 2002;Lorand and Graham, 2003). TG2 has been involved in several cellular processes such as cell adhesion and cell death, but its physiological role is still a matter of debate (Griffin et al., 2002;Lorand and Graham, 2003). In order to shed light on the function of TG2 in mammals, TG2 knock-out mice (Tgm2 -/-) have been generated (Bernassola et al., 2002;Nanda et al., 2001). Surprisingly, mice lacking TG2 activity do not show any major defects in apoptosis, but rather are glucose intolerant and show impairment of insulin secretion, suggesting a physiological role for TG2 in the pancreatic β cell (Bernassola et al., 2002). This is in keeping with previous studies showing that agents known to inhibit transglutaminase activity (e.g. monodansylcadaverine) can decrease glucose-stimulated insulin secretion from rat pancreatic islets in a dose-related manner (Sener et al., 1985, Bungay et al., 1986. In addition, we have described a TG2 heterozygous missense mutation p.N333S (c.998A>G) located in the TG2 catalytic site in a family clinically classified as maturity onset diabetes of the young (MODY; MIM# 606391) (Fig. 1A) (Bernassola et al., 2002). Thus, the aim of this study was to further investigate whether additional mutations in TG2 were also associated with early-onset type 2 diabetes (MIM# 125853). SUBJECTS AND METHODS Families with maturity onset diabetes of the young (MODY) or early onset type 2 diabetesWe studied the ...