A Computational Tool for Quantitative Analysis of Vascular Networks

Angiogenesis is the generation of mature vascular networks from pre-existing vessels. Angiogenesis is crucial during the organism' development, for wound healing and for the female reproductive cycle. Several murine experimental systems are well suited for studying developmental and pathological angiogenesis. They include the embryonic hindbrain, the post-natal retina and allantois explants. In these systems vascular networks are visualised by appropriate staining procedures followed by microscopical analysis. Nevertheless, quantitative assessment of angiogenesis is hampered by the lack of readily available, standardized metrics and software analysis tools. Non-automated protocols are being used widely and they are, in general, time - and labour intensive, prone to human error and do not permit computation of complex spatial metrics. We have developed a light-weight, user friendly software, AngioTool, which allows for quick, hands-off and reproducible quantification of vascular networks in microscopic images. AngioTool computes several morphological and spatial parameters including the area covered by a vascular network, the number of vessels, vessel length, vascular density and lacunarity. In addition, AngioTool calculates the so-called “branching index” (branch points / unit area), providing a measurement of the sprouting activity of a specimen of interest. We have validated AngioTool using images of embryonic murine hindbrains, post-natal retinas and allantois explants. AngioTool is open source and can be downloaded free of charge

Effects of food nutrient content, insect age and stage in the feeding cycle on the FMRFamide immunoreactivity of diffuse endocrine cells in the locust gut

We have studied the influence of variations in dietary protein and digestible carbohydrate content, of insect age and of time during the feeding cycle on the endocrine cells of the ampullar region of the midgut in the African migratory locust Locusta migratoria L. Morphometric analysis of FMRFamide-like immunoreactivity was used as an indirect measure of the amount of FMRFamide-related peptides (FaRPs) stored in the gut endocrine cells. There was a highly significant correlation between FaRP content and the nutritional quality of the food, measured relative to the concentrations and ratio of protein to digestible carbohydrate in a nutritionally optimal diet. The direction of the relationship between FaRP content and diet quality varied with age during the fifth stadium. On day 1, FaRP levels increased with the nutritional quality of the food, while on day 4 the opposite relationship was observed. Release of peptide was triggered by the onset of a meal during ad libitum feeding, with cell FaRP levels returning to premeal values within 15 min of the meal ending. The results also suggested that cell contents were released during food deprivation beyond the normal intermeal interval. Locusts switched for a single meal during ad libitum feeding on day 4 from a low- to a high-carbohydrate food did not respond by reducing endocrine cell FaRP content. Our results show a relationship between the diffuse gut endocrine system and feeding and nutrition in locusts. The ampullar endocrine cells are in three-way contact with the midgut luminal contents, with the primary urine from the Malpighian tubules and with the haemolymph. They are thus ideally positioned to play an integrative receptor-secretory function in the regulation of a variety of post-ingestive processes, such as enzyme secretion, absorption, gut motility or nutrient metabolism

Dietary influences over proliferating cell nuclear antigen expression in the locust midgut

We have studied the influence of variations in dietary protein (P) and digestible carbohydrate (C), the quantity of food eaten, and insect age during the fifth instar on the expression of the proliferating cell nuclear antigen (PCNA) in the epithelial cells of the midgut (with special reference to the midgut caeca) in the African migratory locust, Locusta migratoria. Densitometric analysis of PCNA-immunostained cells was used as an indirect measure of the levels of expression of PCNA, and a PCNA cellular index (PCNA-I) was obtained. Measurements of the DNA content of the cells have also been carried out by means of microdensitometry of Feulgen-stained, thick sections of midgut. A comparison between the PCNA nuclear level and the DNA content was performed. The PCNA levels were significantly different among the cells of the five regions studied: caeca, anterior ventricle, medial ventricle, posterior ventricle and ampullae of the Malpighian tubules. We have studied in more detail the region with highest PCNA-I, i.e. the caeca. The quality and the quantity of food eaten under ad libitum conditions were highly correlated with both the PCNA and DNA levels in the caeca cells. Locusts fed a diet with a close to optimal P:C content (P 21%, C 21%) showed the highest PCNA and DNA content. In locusts fed a food that also contained a 1:1 ratio of P to C but was diluted three-fold by addition of indigestible cellulose (P 7%, C 7%), a compensatory increase in consumption was critical to maintaining PCNA levels. Our measurements also showed that the nuclear DNA content of the mature and differentiated epithelial cells was several-fold higher than the levels in the undifferentiated stem cells of the regenerative nests. These results, combined with the low number of mitotic figures found in the regenerative nests of the caeca and the marked variation in PCNA levels among groups, suggest that some type of DNA endoreduplication process may be taking place. Our data also indicate that the DNA synthetic activity in the midgut is related to feeding in locusts. The possible dietary and nutritional regulatory mechanisms and the significance of the differences found are discussed

Congenital infiltrating lipoma of the upper limb in a patient with von Willebrand disease

Infiltrating lipoma is a rare variety of lipoma, characterized by an infiltration of the adipose tissue of the muscles. Infiltrating lipomas are usually classified in two groups: intermuscular infiltrating lipoma and intramuscular infiltrating lipoma. Most are acquired, and they usually appear in middle-aged individuals. Exceptionally, they are congenital. In such cases they are not related to other diseases. We report an 8-year-old boy with a congenital infiltrating lipoma of the upper limb and von Willebrand disease. Both diseases are linked to an alteration in chromosome 12, but this clinical association seems to be random rather than causal

Adrenomedullin functions as an important tumor survival factor in human carcinogenesis

Adrenomedullin (AM) is a pluripotent regulatory peptide initially isolated from a human pheochromocytoma (adrenal tumor) and subsequently shown to play a critical role in cancer cell division, tumor neovascularization, and circumvention of programmed cell death, thus it is an important tumor cell survival factor underlying human carcinogenesis. A variety of neural and epithelial cancers have been shown to produce abundant amounts of AM. Recent findings have implicated elevation of serum AM with the onset of malignant expression. In addition, patients with tumors producing high levels of this peptide have a poor prognostic clinical outcome. Given that most human epithelial cancers display a microenvironment of reduced oxygen tension, it is interesting to note that AM and several of its receptors are upregulated during hypoxic insult. The existence of such a regulatory pathway has been implicated as the basis for the overexpression of AM/AM-R in human malignancies, thereby generating a subsequent autocrine/paracrine growth advantage for the tumor cell. Furthermore, AM has been implicated as a potential immune suppressor substance, inhibiting macrophage function and acting as a newly identified negative regulator of the complement cascade, protective properties which may help cancer cells to circumvent immune surveillance. Hence, AM's traditional participation in normal physiology (cited elsewhere in this issue) can be extended to a primary player in human carcinogenesis and may have clinical relevance as a biological target for the intervention of tumor progression

The aryl hydrocarbon receptor repressor is a putative tumor suppressor gene in multiple human cancers

The aryl hydrocarbon receptor repressor (AHRR) is a bHLH/Per-ARNT-Sim transcription factor located in a region of chromosome 5 (5p15.3) that has been proposed to contain one or more tumor suppressor genes. We report here consistent downregulation of AHRR mRNA in human malignant tissue from different anatomical origins, including colon, breast, lung, stomach, cervix, and ovary, and demonstrate DNA hypermethylation as the regulatory mechanism of AHRR gene silencing. Knockdown of AHRR gene expression in a human lung can- cer cell line using siRNA significantly enhanced in vitro anchorage-dependent and -independent cell growth as well as cell growth after transplantation into immunocompromised mice. In addition, knockdown of AHRR in non-clonable normal human mammary epithelial cells enabled them to grow in an anchorage-independent manner. Further, downregulation of AHRR expression in the human lung cancer cell line conferred resistance to apoptotic signals and enhanced motility and invasion in vitro and angiogenic potential in vivo. Ectopic expression of AHRR in tumor cells resulted in diminished anchorage-dependent and -independent cell growth and reduced angiogenic potential. These results therefore demonstrate that AHRR is a putative new tumor suppressor gene in multiple types of human cancers

¿Existe un intervalo de tiempo de isquemia fría seguro para el injerto renal?

Objective: It is aimed to characterize the true relationship of the cold ischemia time (CIT) with graft survival and with the principal post-transplantation events.aterial and methods: We analyzed 378 kidney transplants, studying the relationship of the CIT with graft survival using a univariate analysis according to the COX model and seeking the optimum cutoff according to the Kaplan-Meier method and log-rank test. The relationship between CIT and the principal events of the post-transplant was studied using the binary logistic regression. Results: The mean follow-up of all the group was 77.8 months (± 51 SD) and the mean CIT was 14.8 hours (± 5.1 SD). The univariate analysis revealed that the CIT was not related with the graft survival as a continuous variable (OR = 1.04; 95% CI: 0.9-1.08; p > 0.05). On establishing the cutoff at 18 hours, we found differences in the actuarial survival. Survival at 5 years was 91% with CIT 18 h. Each hour of cold ischemia increased risk of delay in the graft function by 10% (OR = 1.1; 95% CI: 1.05-1.15; p < 0.001) and also conditioned a greater incidence of acute rejection (41.5% vs. 55.3%; p = 0.02) and less time to the first rejection episode (72.6 days ± 137 vs. 272.2 days ± 614.8; p = 0.023) after 18 hours. The CIT did not seem to be related (p < 0.05) with the rest of the post-transplantation events, such as surgical complications or hospital admissions. Conclusions: In our experience, cold ischemia under 18 hours does not seem to negatively affect graft survival

Expression of complement factor H by lung cancer cells: effects on the activation of the alternative pathway of complement

The complement system is important in immunosurveillance against tumors. However, malignant cells are usually resistant to complement-mediated lysis. In this study, we examine the expression of factor H, an inhibitor of complement activation, and factor H-like protein 1 (FHL-1), its alternatively spliced form, in lung cancer. We also evaluate the potential effect of factor H/FHL-1 in the protection of lung cancer cells against the activation of the complement cascade. By Northern blot analysis we demonstrate a high expression of factor H and FHL-1 in most non-small cell lung cancer cell lines, although neuroendocrine pulmonary tumors (small cell lung carcinoma and carcinoid cell lines) had undetectable levels. Western blot analysis of conditioned medium showed the active secretion of factor H and FHL-1 by cells that were positive by Northern blot. Expression of factor H/FHL-1 mRNA was also shown in a series of non-small cell lung cancer biopsies by in situ hybridization. Interestingly, many cultured lung cancer cells were able to bind fluorescence-labeled factor H to their surfaces. Deposition of C3 fragments from normal human serum on H1264, a lung adenocarcinoma cell line, was more efficient when factor H/FHL-1 activity was blocked by specific antibodies. Blocking factor H/FHL-1 activity also enhanced the release of anaphylatoxin C5a and moderately increased the susceptibility of these cells to complement-mediated cytotoxicity. In summary, we demonstrate the expression of factor H and FHL-1 by some lung cancer cells and analyze the contribution of these proteins to the protection against complement activation

Edad del donante y su influencia en la supervivencia del injerto

INTRODUCTION: In 2007 in Spain 43% of donors were older than 60 years. This produces a worse graft quality and probably a worse survival. OBJECTIVE: Our objective is to analyze the influence of donor age on graft survival. MATERIAL AND METHODS: We analyze retrospectively 216 renal consecutive transplants realized between 2000 and 2008. A univaried and multivaried study (Cox regression) was performed and Kaplan-Meyer test with log rank for graft survival. RESULTS: Follow-up mean of 40 months (+/-33,4 SD). The univaried analysis of graft survival showed that donor age had a significative influence on graft survival. (OR=1,03; 95% CI 1,01-1,05) (p: 0,009). Studying the relation between donor and recipient age we find an inverse correlation (Pearson's Correlation: 0,55. p<0,0001), but there are significative differences after the adjustment for recipient age. (OR: 1,02; 95% CI 1,01-1,04) (p: 0,04). Optimal cut-point value determined by the ROC analysis was 60 years. The graft survival of donors over 60 years is 79% (95% CI; 74-84%) and 71% (95% CI; 65-77%) at 3 and 5 years in contrast with 94% (95% CI; 94-96%) and 90% (95% CI; 88-92 in donors under 60. (p: 0,002). The multivaried study of the influential factors on graft survival reveals that donor age dichotomized in older or younger than 60, the presence of a surgical immediate reintervention and a delayed graft function were independent influence factors. CONCLUSIONS: Donor age over 60 years has a negative and independent prognostic influence on graft survival