Lymphatic and blood microvasculature organisation in pulmonary sarcoid granulomas.

Pulmonary sarcoid granulomas are characterised by their elective distribution along collecting lymphatics. However, relationships between granulomas and intralobular lymphatics or blood microvascularisation have not been investigated. Therefore, we undertook a specific analysis of blood capillaries and lymphatics supplying sarcoid granulomas to identify additional clues to understanding the pathophysiogenesis of these lesions. Six pulmonary samples were immunolabelled with D2-40, anti-CD34 and anti-CD31 antibodies, paying particular attention to the relationships between lymphatics and granulomas, and the pattern of blood microvessels supplying sarcoid lesions. A morphometric study of granulomas included their distance to lymphatics and a three-dimensional reconstruction of a granuloma in its lymphatic context. Intralobular granulomas were closely associated with lymphatics; apart from a few granulomas, blood capillaries stopped at the outer border of the fibrous ring surrounding granulomas, and perigranuloma capillaries were particularly scarce. Our observations of the lymphatic and blood microvascular environment of intralobular pulmonary sarcoid granulomas provide evidence for the critical role of lymphatics in the emergence of these lesions. Moreover, pulmonary sarcoid lesions could be considered avascular structures, thereby providing new insights into the understanding of the granuloma physiology and the distribution of blood-borne therapeutic agents.

Angiogenesis in the human thymoma assessed by subclassification of tumor-associated blood vessels and endothelial cells proliferation.

The prognostic value of tumor-associated angiogenesis is still a subject of debate. As microvascular density and the expression of different growth factors were not demonstrated to be good predictors of the response to antiangiogenic and antivascular therapy, there is a strong need to search for more sensitive markers. In the present study we evaluated by double immunohistochemical staining the profile of tumor-associated blood vessels and the rate of endothelial cell proliferation in patients with thymoma (n=38). Results were compared with specimens of normal thymus and from patients with myasthenia gravis. We found a significant increase in the number of immature and intermediate blood vessels in the tumor area of thymoma, regardless the histological type of the tumor. Proliferating endothelial cells were found in 15 cases, and co-expression of Ki67 and CD34 had the highest value in immature vessels. Both blood vessel type and endothelial cell proliferation significantly correlated with invasive thymoma. Based on these findings, it can be assumed that the type of tumor-associated vessel together with endothelial cell proliferation are useful predictors of invasion, immature and intermediate vessels can be targeted with antivascular drugs and endothelial cell proliferation could be used as a good predictor of the response to antiangiogenic therapy.

Diagnostic and clinical significance of D2-40 expression in the normal human thymus and thymoma.

Human thymus development and thymoma behavior remain elusive, in spite of many acquisitions in the field in last decades. In the present paper, we analyzed the immunohistochemical expression of D2-40 in the normal human thymus and thymoma. In both fetal and postnatal normal thymus, we found a strong expression of D2-40 in the subcapsular and cortico-medullary epithelial cells, and lack of expression in the thymus of involution. These findings support a role for podoplanin in the proliferation of some subtypes of epithelial cells of the normal thymus stroma. In thymoma, the expression of D2-40 was detected in neoplastic cells in 18 from 26 cases (69.23%). No correlation was found between D2-40 expression and histological types of thymoma, but strong correlation was noticed with tumor stage. Based on these results, it is suggested that D2-40 expression is a good predictor of invasion and can be considered as a potential target for therapy in selected cases.

Pancreatic metaplasia of gastric mucosa associated with gastroduodenal ulcer.

Metaplasia represents the process of transforming a well-differentiated adult tissue into another type of adult tissue. Pancreatic metaplasia of the gastric mucosa represents the process in which the normal mucosa of the stomach is replaced with pancreatic formations, which mimic the structure of pancreatic acini. We describe the case of a male patient aged 39 who was admitted for abdominal pain, vomiting, hematemesis, melena, pale teguments, intense perspiration and nausea. The patient underwent surgery for suturing a perforated duodenal ulcer five years prior to this episode (2002). A gastric ulcer complicated with superior digestive bleeding and a chronic duodenal ulcer complicated with partial stenosis and perivisceritis were found during surgery. Gastric wall fragments were harvested and underwent usual histological techniques and immunohistochemistry. We found an ulceration from the gastric mucosa to the submucosa, covered by fibrino-leukocytic detritus. In the mucosal chorion we found numerous round or oval shaped nested formations which occupied the lower two thirds of the chorion, to the muscularis mucosae. Some metaplasic acini contain cells variable in shape, color and immunophenotype. Surrounding the nested acini we found tubular formations, formed of cubic shaped cells, representing excretory canals which were continued by gastric glands or opened directly in the crypts of the gastric epithelium.

Study of vascular microdensity in areas of cerebral ischemia on experimental model.

Stroke is the third leading cause of death and the first cause of disability with an increasing incidence, especially of the ischemic type. There is no effective curative treatment for stroke and therefore the therapy for this disease currently relies on identifying patients at risk and instituting preventive measures. Since ischemic stroke in middle cerebral artery territory (and with preferential localization in the striated nuclei) is the main type of stroke in humans, animal models obtained by surgical ligation of the middle cerebral artery (MCAO) are valuable tools for the fundamental study of this disease. In this study, we investigated the morphological and immunohistochemical remodeling of the tissue after stroke and in particular the vascular component in a MCAO murine model. The analysis included the sequential sacrificing of animals at 1, 14, 28 and 60 days post-stroke and brain processing for paraffin embedding and sectioning. Our results show a gradual revascularization of the lesion as we move away from the time of the surgical intervention. This effect is accompanied by the development of an increasingly dense glial scar at the periphery of the lesion. The perilesional area itself, the penumbra, is characterized by minimal histological changes (such as eosinophilic neurons), but also by an increased expression of activated caspase 3 as a sign of apoptotic neurons and glial cells. Our study confirms the potential of the organism in its attempt to revascularize the injured area, and raises questions on the role of the glial scar in limiting the process of neovascularization.

Strategies to improve post-stroke behavioral recovery in aged subjects.

UNLABELLED: Old age is associated with an enhanced susceptibility to stroke and poor recovery from brain injury. Therefore, find therapeutic strategies aimed at improving functional recovery after brain ischemia in aged subjects is of considerable clinical interest. While environmental enrichment has been shown to improve the behavioral outcome of stroke in young animals, the effect of an enriched environment, hypothermia and Granulocyte-Colony Stimulating Factor (G-CSF) on behavioral and neuropathological recovery in aged animals is not known. Focal cerebral ischemia was produced by occlusion of the right middle cerebral artery in 3-month- and 20-month-old male Sprague-Dawley rats. The functional outcome was assessed in neurobehavioral tests conducted over a period of 14-28 days following surgery. Brain tissue then was immunostained for proliferating astrocytes and the infarct and scar tissue volumes were measured. Aged rats showed more severe behavioral impairments and diminished functional recovery compared to young rats. Most infarcted animals had disturbances of sensorimotor function, with recovery beginning later, progressing more slowly, and reaching a lower functional endpoint in aged animals. However, the enriched environment significantly improved the rate and extent of recovery in aged animals. Correlation analysis revealed that the beneficial effect of the enriched environment on recovery, both in young and aged rats, correlated highly with a reduction in infarct size, in the number of proliferating astrocytes, and in the volume of the glial scar. These results suggest that temporally modulating astrocytic proliferation and the ensuing scar formation might be a fruitful approach to improving functional recovery after stroke in aged rats. In aged humans, stroke is a major cause of disability for which no neuroprotective measures are available. A viable alternative to conventional drug-based neuroprotective therapies is brain/body cooling, or hypothermia. In animal studies of focal ischemia, short-term hypothermia consistently reduces infarct size. Nevertheless, efficient neuroprotection requires long-term, regulated lowering of whole body temperature. In this study, we show that two days post-stroke exposure of aged rats to a mixture of air and a mild inhibitor of oxidative phosphorylation, H2S, causes deep hypothermia (27.8+/-0.3 degrees C) and a 50% reduction in infarct size without obvious neurological deficits or physiological side effects. G-CSF treatment after stroke exerted a robust and sustained beneficial effect on survival rate and running function. Transient improvement after G-CSF treatment could be observed for coordinative motor function on the inclined plane test and for working memory in the radial maze test. At cellular level, G-CSF treatment increased the number of proliferating cells in the SVZ and the dentate gyrus and increased the number of newborn neurons in the SVZ, ipsilateral to the lesion. These results suggests that the G-CSF treatment in aged rats has a survival enhancing capacity and a beneficial effect on functional outcome most likely via supportive cellular processes such as neurogenesis. CONCLUSIONS: These findings are important for the further clinical development of the drug in elderly stroke patients. Future studies should focus on an optimization of treatment schedule to achieve a maximum of post-stroke recovery enhancement in aged subjects.

Histopathological changes in acute ischemic stroke.

We study here the histopathological changes in twenty-two cases of acute ischemic stroke. The average age of the patients was 62-year-old, and the interval from the onset of the disease to the death varied from 6 hours to 15 years. The brain lesions after acute stroke were observed in all regions. Their evolution allowed us to classify them in fourth stages. Phase one changes (1-2 days after onset) (n=2 patients) included red hypoxic and "ghost" neurons and other acute neuronal injury and spongiosis. The second phase (n=14 patients) was subdivided into: (a) a phase of acute inflammation (3-37 days after onset) (n=5 patients), where we observed especially features of acute inflammation together with coagulative necrosis, and (b) phase of chronic inflammation (10 days-53 years after the onset) (n=9 patients), in which prevail mononuclear and macrophage infiltrate, astrogliosis, spongiosis and neo-vascularization. In the third phase (26 days-23 years after the onset), we included six cases characterized by the absence of an inflammatory reaction, presence of cavitation, astrogliosis and macrophages. Our study describes the heterogeneity of brain injury after acute ischemic stroke with the participation of all brain components, and the chronology in which these lesions develop and evolve.

Fractal analysis of astrocytes in stroke and dementia.

The varied morphological forms in which astrocytes occur in brain of ischemic/hemorrhagic stroke and Alzheimer's disease (AD) patients are complex and the mechanisms that drive their formation are not yet properly understood. Subjective differences can be described between these pathologies in what it concerns astrocyte implication, but these have not been yet subjected to a morphometrical quantification. Here we apply a fractal dimension (FD) analysis algorithm to differentiate both between fibrous, protoplasmatic and activated astroglia; but also between the three pathological conditions studied. Analyzing more than 1000 astroglia, we show here first that FD can clearly differentiate between the three morphological subtypes. Second, we describe resemblances of the FD values for ischemic and hemorrhagic lesions, and significant differences when these are compared to AD patients. These results are further discussed and integrated in what it regards the preferential regions proved to be affected in these conditions, and which parallels our results. This work illustrates that fractal dimension analysis of astroglia is a useful method for quantitatively describing gliosis in different pathologies, and that it may offer more insight into the pathogenesis of brain diseases.

Hypothesis of microfractures by buckling theory of bone's trabeculas from vertebral bodies affected by osteoporosis.

Osteoporosis has become in recent years a public health problem considered a true "silent epidemic", by increasing the number of osteoporosis fractures in the world as a result of increased number of persons 3rd group of age by increasing life expectancy and reducing physical effort and the emergence of sedentary occupations, increasing incidence of obesity, diabetes, liver disease and kidney by applying widely corticosteroid therapy. Starting from the macroscopic and microscopic aspects of the bone spongy tissue affected by osteoporosis, from vertebral bodies, we try to explain the modality of damaging the bone micro-structure by buckling phenomenon, knowing that the bone tissue has at trabecular level, an elasticity degree and supports high levels of mechanical forces.

Immunohistochemical aspects of the evaluation of the inflammatory answer of the dental pulp.

The inflammatory answer of the dental pulp in front of an antigen presents a series of particularities caused by its special anatomical conditions. The evaluation of the inflammatory answer in the evaluation tests of the biocompatibility of the dental materials is made by histological methods without defining precise quantitative criteria to measure the pulp reaction. In this study, we followed the reaction of the pulp tissue to five antibodies (CD20, CD45Ro, CD4, CD8, and CD68) in order to evaluate the inflammatory aspects of the dental pulp. We did not find positive answers for the CD20 protein, specific for B-lymphocytes, for the fragments of normal pulp tissue taken into study. Even if they are in small number among the pulp cells, the T-lymphocytes that express the protein CD45Ro may be also found in the normal dental pulp. Of the two subsets of T-lymphocytes, we found positive answers on the studied preparations only for the CD8 protein. For the CD68 protein, strongly expressed by the macrophages, we obtained positive results both for the inflammated pulp tissue, and for the normal dental pulp, yet in a very small amount. The use of immunohistochemical techniques, with well-defined markers of the pulp inflammation, can offer better results for a highly accurate evaluation of the inflammatory answer of the dental pulp.

VEGF and VEGFRs expression in oral squamous cell carcinoma.

Oral cancer is an important cause of worldwide morbidity and mortality, with substantial economic, physiological, and psychosocial impacts due to its treatment modality and a great risk for recurrences and second primary oral squamous cell carcinomas (OSCC) development. Therefore, it is very important to understand the underlying cell biology of such tumors. It is now a well-accepted fact that angiogenesis is essential for the growth and metastasis of solid tumors, including head and neck squamous cell carcinoma. The main factor responsible for angiogenesis is VEGF and its receptors. It has been demonstrated that VEGFRs are also present on tumor cells themselves and other cells from the tumor microenvironment, in addition to tumoral endothelial cells (ECs). Therefore between these cells take place numerous and different interactions mediated via paracrine/autocrine pathways that promote angiogenesis, uncontrolled tumor proliferation and metastasation. In consequence, estimation of VEGF expression and its receptors became a reliable prognostic tool in OSCCS, predicting the poor disease-free survival, poor overall survival, and metastatic disease. Understanding the distribution and role of VEGF and its receptors in the progression of OSCC will be essential to the development and design of new therapeutic strategies.

The Treatment of Arterial Hypertension.

Arterial hypertension is the leading cause of death worldwide and is one of the most important public health problems. Arterial hypertension is a major cardiovascular risk factor with an increasing incidence. In this paper we set out to analyze a group of 3050 patients hospitalized between January 2013 and December 2017 in terms of drug therapy. We found that the majority of patients received drug treatment with a converting-enzyme inhibitor as a monotherapy, and the most common drug association was the association between conversion enzyme inhibitor and calcium channel blocker.

Colorectal Cancer: An Update on Treatment Options and Future Perspectives.

Throughout the years, colorectal cancer has steadily become a global health problem. While other types of cancers have seen a decline in cases because of screening and vaccination programs, colorectal cancer has risen become the third most diagnosed cancer worldwide and, more worryingly, the second leading cancer-related cause of death. The introduction of targeted therapy has been widely considered a major paradigm shift in the treatment of colorectal cancer, which agents such as bevacizumab and cetuximab quickly becoming mainstay options in the treatment of locally advanced or metastatic disease. However, this type of treatment has also shown its limitations, with limited or no benefit for a large portion of the patients. With more and more knowledge being gathered on the molecular mechanisms which govern the malignant phenotype presented by colorectal cancer, scientists are engaged in a continuous effort to develop new therapies based on these discoveries.

A Statistical Analysis of Risk Groups in Colorectal Cancer Patients.

Colorectal cancer (CRC) is considered a major global health concern due to an increasing number of new cases and cancer-related deaths each year, strong link to dietary habits prevalent in middle and high-income countries and limited therapeutic options especially in locally-advanced and metastatic settings. To counter this growing problem, the scientific community has strived to underpin the major molecular mechanisms behind the aggressive phenotype displayed by CRC and also develop new agents to selectively target and inhibit these core drivers. This evolution has allowed the separation of patients according to different risk groups in concordance with epidemiological parameters alongside novel biomarkers such as gene alterations, protein overexpression and aberrant signaling pathways. In this study we included 20 patients who underwent colonoscopy and were later received histopathologic confirmation of CRC. The statistical anamnestic data obtained from the patients (age, gender, home distribution, signs and symptoms) was corroborated with the results obtained from the histopathologic and immunohistochemical analysis of the samples obtained via colonoscopy. The average age was 63.8 years, the male: female ratio was 2.33 and the origin of 2/3 of the patients was urban and the most encountered symptoms were transit disorders (75%). In terms of colonoscopy results, the majority of tumors were found on the rectum (85%), 90% of tumors were adenocarcinomas, having a vegetant aspect in 60% of the cases and a moderate degree of differentiation in 50% of situations.

Podoplanin expression in advanced-stage gastric carcinoma and prognostic value of lymphatic microvessel density.

In gastric cancer, lymph node metastasis is a major prognostic factor. Tumor lymphangiogenesis promotes metastasis in experimental models, but in human tumors data about the presence and clinical significance of lymphatic vessels in the tumor area are controversial. We investigated 70 patients with advanced-stage gastric carcinoma and the pathological examination showed 40 cases with intestinal subtype and 30 cases with diffuse subtype. Forty three from 70 cases had regional lymph node metastasis. Additional slides were stained with an antibody against podoplanin, and lymphatic microvessel density (LMVD) was evaluated in the tumoral and peritumoral areas. Lymphatic vessels were identified in tumor area in all cases and LMVD was higher in the peritumoral than in the tumor area. Podoplanin-positive vessels in tumor area were usually small, with narrow lumen. A significant correlation was found between LMVD and stage of the tumor (p<0.002) and lymph node metastasis (p<0.031), but not with the pathological subtype and grade of the tumor. We found tumor cells in the lumen of lymphatic vessels in 11 cases, whereas tumor cells expressing podoplanin were found in 4 cases of less differentiated diffuse subtype gastric carcinoma. In conclusion, our results suggest that LMVD predicts tumor stage and lymph node metastasis, and podoplanin-positive tumor cells select a subgroup of tumors with high potential of invasion and metastasis.

Immunohistochemical characterization of tumoral vessels in oral squamous cell carcinoma.

Tumors require a blood supply for growth and hematogenous dissemination. Angiogenesis is one of the mechanism by which tumors acquire their microcirculation. Structurally and functionally, these newborn vessels are abnormal, showing increased permeability, delayed maturation, and potential for rapid proliferation. Such vascular defects could be an explanation for the aggressivity of oral squamous cell carcinoma (OSCC). For these reason we studied the morphology of tumoral vessels in such tumors by using immunohistochemistry and immunofluorescence. Forty formalin-fixed, paraffin-embedded tissue blocks of OSCC were processed for double enzymatic and fluorescence immunohistochemistry. We were interested in analyzing the tumor vessel architecture, and their maturity and activity in such tumors. The tumor vessel architecture had a chaotic pattern, mostly of different sizes, aberrant morphology, tortuous, without clear lumen, and irregularly branches. Regarding pericytes recruitment, the immature and intermediate vessel types (both negative to smooth muscle actin-SMA) were the most numerous type of tumoral vessels. The mature ones (positive to SMA) were readily more numerous at the invasive front of OSCC (85.4 vessels/4 mm(2) +/- 38.3), especially in poor differentiated tumoral type. Investigation of the tumor vessel basal membrane, as reactivity for collagen IV, revealed variability in thickness (2.59 microm +/- 0.48), small surface projections, discontinuities and loose associations with endothelial cells; these abnormalities being more obviously at the tumor-host interface and in poor differentiated OSCC. The most active angiogenesis was noticed in poor differentiated OSCC (0.23 +/- 0.04), at the tumor-host interface with the immature and intermediated vessel as the most active tumor vessel types. In conclusion, our study revealed some peculiar structurally and functionally defects of tumor vessels in OSCC, changes that could be selective targets for the new developing antiangiogenic drugs.

Endoglin (CD105) and microvessel density in oral squamous cell carcinoma.

Recent studies revealed that CD105 is intensively expressed in tumor vasculature, and may be an important prognostic indicator for the outcome in a number of malignancies. The aim of our study was to evaluate the CD105 expression and microvessel density in oral squamous cell carcinoma (OSCC). Nineteen surgical specimens with OSCC were immunohistochemical analyzed with CD105 (Endoglin). We determined the microvessel density (MVD) by "hot spot method". Endoglin was intensively expressed in vessels from the inner and invading front of all investigated OSCC. The highest value of MVD, about 30.89 +/- 22.4, were record in peritumoral area of OSCC, since intratumoral MVD average was about 10.18 +/- 4.7. We did not observe any significant association of MVD with age, sex, primary tumor's location, clinical stage or differentiation grade. In conclusion, CD105 expression is up-regulated in OSCC, and has a significant role in the development of such malignancies.

Annexin A3 expression after stroke in the aged rat brain.

In an effort to identify new proteins involved in functional recovery after cerebral ischemia, young (3 months) and aged (18 months) male rats were subjected to middle cerebral artery (MCA) occlusion. Brains were harvested at 3- and 14-days post ischemia and proteins from the peri-infarcted and the corresponding contralateral area and total proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis followed by mass spectrometry analysis. Annexin A3 (ANXA3) was identified as one upregulated protein in the post-ischemic rat brain. Using western blotting, real-time PCR and immunohistochemistry, we confirmed that at 3-14 days post-stroke, ANXA3 expression in the peri-infarct area was consistently increased over the corresponding area of control rats. Double staining revealed that ANXA3 is produced by activated microglial cells. We found that aged rats also had more newly proliferating cells expressing ANXA3 than young rats do. Occasionally, ANXA3-immunopositive cells wraped around neurons, suggesting that annexin A3 may be involved in the removal of dying neurons after stroke.

Immunohistochemical expression of vascular endothelial growth factor (VEGF) in intestinal type gastric carcinoma.

BACKGROUND: Gastric cancer still represents a difficult problem in the field of oncology, in terms of morbidity and mortality. The local progression and systemic spread is significantly influenced by tumor angiogenesis and lymphangiogenesis. In spite of many studies on the topic, data about the significance of growth factors in gastric cancer is controversial. AIM: to investigate the immunohistochemical expression of VEGF and to evaluate the relationships with the tumors stage and grade. MATERIAL AND METHODS: The immunohistochemical expression of VEGF was investigated on 80 patients with intestinal type gastric carcinoma. Specimens were fixed in buffer formalin, embedded in paraffin, and sections were stained with Hematoxylin-Eosin and immunohistochemistry was performed for VEGF (clone VG-1). Evaluation was performed using the VEGF score, based on the intensity of reaction and percent of positive cells. RESULTS: The reaction for VEGF was positive in 52 from 80 cases (70%). The final product of reaction was found in the cytoplasm of tumor cells, with granular pattern. Positive reaction was also found in eight from 28 cases with associated intestinal metaplasia, and in six from nine cases with gastric dysplasia. In the adjacent apparently normal mucosa, the reaction was positive in hyperplastic gastric pits and parietal cells. A strong correlation was found between VEGF expression and lymph node status and grade of the primary, but not with the stage of the tumor. CONCLUSIONS: The investigation of the immunohistochemical expression of VEGF in the intestinal type of gastric carcinoma showed positive reaction in 70% of the cases. It was demonstrated the expression of VEGF in intestinal metaplasia and gastric dysplasia, which could signify an early angiogenic switch during tumorigenesis.

The study of E-cadherine and CD44 immunoexpression in oral squamous cell carcinoma.

In spite of the progresses achieved in surgical treatment, radiotherapy and chemotherapy, the survival rate in oral squamous cell carcinoma (OSCC) remain unchanged among past three decades, which made this neoplasia a major problem of health in the entire world. There were investigated 42 cases of OSCC with different sites and various grades of differentiation, by histological and immunohistochemical techniques (by LSAB/HRP method), using E-cadherine and CD44. Considering the differentiation grade, the cases were histopathologically classified as 9 cases of well-differentiated squamous carcinoma, 14 cases of moderately differentiated squamous carcinoma, and 19 cases of poorly differentiated squamous carcinoma. The E-cadherine immunoexpression study indicated an immunostaining degree 3 in well-differentiated squamous carcinoma, 2 in moderately differentiated squamous carcinoma, and 1 in poorly differentiated squamous carcinoma. The results of CD44 immunostaining indicated in most of the cases an immunostaining degree 2, especially in moderately differentiated OSCC. The immunostaining degree 3 corresponded to nine cases of well-differentiated OSCC, and other two cases of moderately differentiated OSCC. Immunostaining degree 1 corresponded to poorly differentiated OSCC. The results indicate the possibility of using the two-immunohistochemical markers as prognostic factors in OSCC.