Dosimetric effect of tissue heterogeneity for 125I prostate implants

Aim - To use Monte Carlo (MC) together with voxel phantoms to analyze the tissue heterogeneity effect in the dose distributions and equivalent uniform dose (EUD) for (125)I prostate implants. Background - Dose distribution calculations in low dose-rate brachytherapy are based on the dose deposition around a single source in a water phantom. This formalism does not take into account tissue heterogeneities, interseed attenuation, or finite patient dimensions effects. Tissue composition is especially important due to the photoelectric effect. Materials and Methods - The computed tomographies (CT) of two patients with prostate cancer were used to create voxel phantoms for the MC simulations. An elemental composition and density were assigned to each structure. Densities of the prostate, vesicles, rectum and bladder were determined through the CT electronic densities of 100 patients. The same simulations were performed considering the same phantom as pure water. Results were compared via dose-volume histograms and EUD for the prostate and rectum. Results - The mean absorbed doses presented deviations of 3.3-4.0% for the prostate and of 2.3-4.9% for the rectum, when comparing calculations in water with calculations in the heterogeneous phantom. In the calculations in water, the prostate D 90 was overestimated by 2.8-3.9% and the rectum D 0.1cc resulted in dose differences of 6-8%. The EUD resulted in an overestimation of 3.5-3.7% for the prostate and of 7.7-8.3% for the rectum. Conclusions - The deposited dose was consistently overestimated for the simulation in water. In order to increase the accuracy in the determination of dose distributions, especially around the rectum, the introduction of the model-based algorithms is recommended.

Heterogeneous response of adipose tissue to cancer cachexia

Cancer cachexia causes disruption of lipid metabolism. Since it has been well established that the various adipose tissue depots demonstrate different responses to stimuli, we assessed the effect of cachexia on some biochemical and morphological parameters of adipocytes obtained from the mesenteric (MES), retroperitoneal (RPAT), and epididymal (EAT) adipose tissues of rats bearing Walker 256 carcinosarcoma, compared with controls. Relative weight and total fat content of tissues did not differ between tumor-bearing rats and controls, but fatty acid composition was modified by cachexia. Adipocyte dimensions were increased in MES and RPAT from tumor-bearing rats, but not in EAT, in relation to control. Ultrastructural alterations were observed in the adipocytes of tumor-bearing rat RPAT (membrane projections) and EAT (nuclear bodies).

Characterization of a monoclonal antibody recognizing mast cells

Immunohistochemical screening for monoclonal antibodies prepared by immunization of mice with a rat osteoblastic cell population led to identification of one antibody that reacted against a small population of cells present in the soft connective tissue compartment of 21 days fetal rat calvaria. The morphology of the cells and the immunohistochemical staining characteristics (a distinct intracellular granular pattern) suggested that the antibody might be reacting specifically against mast cells. We used combined histochemistry and immunohistochemistry to further characterize this antibody, designated RCJ102. Cryosections containing calvaria bone, soft connective tissues and skin were prepared from the top of the head of 21 days fetal rats, and from adult rats cryosections of lung, muscle, adipose tissue and small intestine were prepared. Some sections were labelled by indirect immunofluorescence with RCJ102; corresponding sections were labelled histochemically with toluidine blue. There was a direct correspondence between mast cells identified histochemically and cells labelling with RCJ102 in all tissues except intestine, in which the mast cell detectable by histochemistry were not labelled by RCJ102. These results suggest that the RCJ102 antibody will be a valuable new reagent for further elucidation of the heterogeneity described between connective tissue and intestinal mucosal mast cells.

An integrated nano-scale approach to profile miRNAs in limited clinical samples

Profiling miRNA expression in cells that directly contribute to human disease pathogenesis is likely to aid the discovery of novel drug targets and biomarkers. However, tissue heterogeneity and the limited amount of human diseased tissue available for research purposes present fundamental difficulties that often constrain the scope and potential of such studies. We established a flow cytometry-based method for isolating pure populations of pathogenic T cells from bronchial biopsy samples of asthma patients, and optimized a high-throughput nano-scale qRT-PCR method capable of accurately measuring 96 miRNAs in as little as 100 cells. Comparison of circulating and airway T cells from healthy and asthmatic subjects revealed asthma-associated and tissue-specific miRNA expression patterns. These results establish the feasibility and utility of investigating miRNA expression in small populations of cells involved in asthma pathogenesis, and set a precedent for application of our nano-scale approach in other human diseases. The microarray data from this study (Figure 7) has been submitted to the NCBI Gene Expression Omnibus (GEO; http://ncbi.nlm.nih.gov/geo) under accession no. GSE31030.

Using a voxel-based krogh cylinder array to simulate microvascular contrast enhancement

The goal of this study is to better simulate microscopic and voxel-based dynamic contrast enhancement in magnetic resonance imaging. Specifically, errors imposed by the traditional two-compartment model are reduced by introducing a novel Krogh cylinder network. The two-compartment model was developed for macroscopic pharmacokinetic analysis of dynamic contrast enhancement and generalizing it to voxel dimensions, due to the significant decrease in scale, imposes physiologically unrealistic assumptions. In the project, a system of microscopic exchange between plasma and extravascular-extracellular space is built while numerically simulating the local contrast agent flow between and inside image elements. To do this, tissue parameter maps were created, contrast agent was introduced to the tissue via a flow lattice, and various data sets were simulated. The effects of sources, tissue heterogeneity, and the contribution of individual tissue parameters to an image are modeled. Further, the study attempts to demonstrate the effects of a priori flow maps on image contrast, indicating that flow data is as important as permeability data when analyzing tumor contrast enhancement. In addition, the simulations indicate that it may be possible to obtain tumor-type diagnostic information by acquiring both flow and permeability data.

The 434(G > C) polymorphism in the eosinophil cationic protein gene and its association with tissue eosinophilia in oral squamous cell carcinomas

Objective: The aim of this study was to investigate the prevalence of the Eosinophil cationic protein (ECP)-gene polymorphism 434(G > C) in oral squamous cell carcinoma (OSCC) patients and its association with tumor-associated tissue eosinophilia (TATE), demographic, clinical, and microscopic variables. Methods: The ECP genotypes of 165 healthy individuals and 157 OSCC patients were detected by PCR-RFLP analysis after cleavage of the amplified DNA sequence with enzyme PstI. TATE was obtained by morphometric analysis. Chi-square test or Fisher`s exact test was used to analyze the association of ECP-gene polymorphism 434(G > C) with TATE, demographic, clinical, and microscopic variables in OSCC patients. Disease-free survival and overall survival were calculated by the Kaplan-Meier product-limit actuarial method and the comparison of the survival curves were performed using log rank test. Results: Most of healthy individuals (53.33%) and OSCC patients (57.97%) were heterozygous for the ECP 434(G > C) polymorphism. Based on numerical differences, our results showed that OSCC patients with intense TATE and at least one C allele had a higher frequency of bilateral neck dissection, local recurrence, vascular embolization, involved resection margins, and postoperative radiotherapy. No statistically significant differences on survival rates were found in OSCC patients presenting different ECP 434(G > C) genotypes. Conclusions: These results suggest a tendency towards a poor clinical outcome in OSCC patients with intense TATE and 434GC/CC genotypes, probably due to an ECP genetic variant with altered cytotoxic activity.

Heterogeneity in olfactory neurons in mouse revealed by differential expression of glycoconjugates

Cell surface glycoconjugates have been implicated in the growth and guidance of subpopulations of primary olfactory axons. While subpopulations of primary olfactory neurons have been identified by differential expression of carbohydrates in the rat there are few reports of similar subpopulations in the mouse. We have examined the spatiotemporal expression pattern of glycoconjugates recognized by the lectin from Wisteria floribunda (WFA) in the mouse olfactory system. In the developing olfactory neuroepithelium lining the nasal cavity, WFA stained a subpopulation of primary olfactory neurons and the fascicles of axons projecting to the target tissue, the olfactory bulb. Within the developing olfactory bulb, WFA stained the synaptic neuropil of the glomerular and external plexiform layers. In adults, strong expression of WFA ligands was observed in second-order olfactory neurons as well as in neurons in several higher order olfactory processing centres in the brain. Similar, although distinct, staining of neurons in the olfactory pathway was detected with Dolichos biflorus agglutinin. These results demonstrate that unique subpopulations of olfactory neurons are chemically coded by the expression of glycoconjugates. The conserved expression of these carbohydrates across species suggests they play an important role in the functional organization of this region of the nervous system.

Leber Congenital Amaurosis: Comprehensive Survey of the Genetic Heterogeneity, Refinement of the Clinical Definition, and Genotype-Phenotype Correlations as a Strategy for Molecular Diagnosis

Leber congenital amaurosis (LCA) is the earliest and most severe form of all inherited retinal dystrophies, responsible for congenital blindness. Disease-associated mutations have been hitherto reported in seven genes. These genes are all expressed preferentially in the photoreceptor cells or the retinal pigment epithelium but they are involved in strikingly different physiologic pathways resulting in an unforeseeable physiopathologic variety. This wide genetic and physiologic heterogeneity that could largely increase in the coming years, hinders the molecular diagnosis in LCA patients. The genotyping is, however, required to establish genetically defined subgroups of patients ready for therapy. Here, we report a comprehensive mutational analysis of the all known genes in 179 unrelated LCA patients, including 52 familial and 127 sporadic (27/127 consanguineous) cases. Mutations were identified in 47.5% patients. GUCY2D appeared to account for most LCA cases of our series (21.2%), followed by CRB1 (10%), RPE65 (6.1%), RPGRIP1 (4.5%), AIPL1 (3.4%), TULP1 (1.7%), and CRX (0.6%). The clinical history of all patients with mutations was carefully revisited to search for phenotype variations. Sound genotype-phenotype correlations were found that allowed us to divide patients into two main groups. The first one includes patients whose symptoms fit the traditional definition of LCA, i.e., congenital or very early cone-rod dystrophy, while the second group gathers patients affected with severe yet progressive rod-cone dystrophy. Besides, objective ophthalmologic data allowed us to subdivide each group into two subtypes. Based on these findings, we have drawn decisional flowcharts directing the molecular analysis of LCA genes in a given case. These flowcharts will hopefully lighten the heavy task of genotyping new patients but only if one has access to the most precise clinical history since birth.

Regional myocardial function in healthy adults: assessment through tissue Doppler echocardiography

OBJECTIVE: To characterize left ventricular regional myocardial function through tissue Doppler echocardiography in healthy adults and to assess the influence of aging in this function. METHODS: In 45 healthy volunteers divided in two groups (< 45 and > 45 years old) we assessed longitudinal and radial regional function (velocities, times intervals and velocity-time integrals). Data were compared in each group and between groups. RESULTS: Systolic function: a) longitudinal: higher velocities and integrals in lateral and inferior walls and in basal segments, with a trend to reduction of these parameters with aging; b) radial: higher basal velocities, no significant change with aging. Diastolic function: a) longitudinal: higher velocities in lateral and inferior walls and in basal segments. With aging e and e/a velocities and integrals decreased, a increased and older individuals showed lower percentage of segments with e/a >1; b) radial: aging was associated with lower e and higher a velocities. CONCLUSION: 1) Tissue Doppler echocardiography detects physiological differences between regional myocardial function of different ventricular segments, in velocities, times intervals and integrals, with physiological heterogeneity and asynchrony; 2) Many of these data are age dependent; 3) Our data contribute to define normal values, and may become useful when compared with data from populations with heart diseases.

Heterogeneity of human monocytes: an optimized four-color flow cytometry protocol for analysis of monocyte subsets.

Monocytes are central mediators in the development of atherosclerotic plaques. They circulate in blood and eventually migrate into tissue including the vessel wall where they give rise to macrophages and dendritic cells. The existence of monocyte subsets with distinct roles in homeostasis and inflammation suggests specialization of function. These subsets are identified based on expression of the CD14 and CD16 markers. Routinely applicable protocols remain elusive, however. Here, we present an optimized four-color flow cytometry protocol for analysis of human blood monocyte subsets using a specific PE-Cy5-conjugated monoclonal antibody (mAb) to HLA-DR, a PE-Cy7-conjugated mAb to CD14, a FITC-conjugated mAb to CD16, and PE-conjugated mAbs to additional markers relevant to monocyte function. Classical CD14(+)CD16(-) monocytes (here termed "Mo1" subset) expressed high CCR2, CD36, CD64, and CD62L, but low CX(3)CR1, whereas "nonclassical" CD14(lo)CD16(+) monocytes (Mo3) essentially showed the inverse expression pattern. CD14(+)CD16(+) monocytes (Mo2) expressed high HLA-DR, CD36, and CD64. In patients with stable coronary artery disease (n = 13), classical monocytes were decreased, whereas "nonclassical" monocytes were increased 90% compared with healthy subjects with angiographically normal coronary arteries (n = 14). Classical monocytes from CAD patients expressed higher CX(3)CR1 and CCR2 than controls. Thus, stable CAD is associated with expansion of the nonclassical monocyte subset and increased expression of inflammatory markers on monocytes. Flow cytometric analysis of monocyte subsets and marker expression may provide valuable information on vascular inflammation. This may translate into the identification of monocyte subsets as selective therapeutic targets, thus avoiding adverse events associated with indiscriminate monocyte inhibition.

Role of trabectedin in the treatment of soft tissue sarcoma.

Interest in marine natural products has allowed the discovery of new drugs and trabectedin (ET-743, Yondelis), derived from the marine tunicate Ecteinascidia turbinata, was approved for clinical use in 2007. It binds to the DNA minor groove leading to interferences with the intracellular transcription pathways and DNA-repair proteins. In vitro antitumor activity was demonstrated against various cancer cell lines and soft tissue sarcoma cell lines. In phase I studies tumor responses were observed also in osteosarcomas and different soft tissue sarcoma subtypes. The most common toxicities were myelosuppression and transient elevation of liver function tests, which could be reduced by dexamethasone premedication. The efficacy of trabectedin was established in three phase II studies where it was administered at 1.5 mg/m2 as a 24 h intravenous infusion repeated every three weeks, in previously treated patients. The objective response rate was 3.7%-8.3% and the tumor control rate (which included complete response, partial response and stable disease) was obtained in half of patients for a median overall survival reaching 12 months. In nonpretreated patients the overall response rate was 17%. Twenty-four percent of patients were without progression at six months. The median overall survival was almost 16 months with 72% surviving at one year. Predictive factors of response are being explored to identify patients who are most likely to respond to trabectedin. Combination with other agents are currently studied with promising results. In summary trabectedin is an active new chemotherapeutic agents that has demonstrated its role in the armamentarium of treatments for patients with sarcomas.

Candidate gene linkage analysis indicates genetic heterogeneity in Marfan syndrome

Marfan syndrome (MFS) is an autosomal dominant disease of the connective tissue that affects the ocular, skeletal and cardiovascular systems, with a wide clinical variability. Although mutations in the FBN1 gene have been recognized as the cause of the disease, more recently other loci have been associated with MFS, indicating the genetic heterogeneity of this disease. We addressed the issue of genetic heterogeneity in MFS by performing linkage analysis of the FBN1 and TGFBR2 genes in 34 families (345 subjects) who met the clinical diagnostic criteria for the disease according to Ghent. Using a total of six microsatellite markers, we found that linkage with the FBN1 gene was observed or not excluded in 70.6% (24/34) of the families, and in 1 family the MFS phenotype segregated with the TGFBR2 gene. Moreover, in 4 families linkage with the FBN1 and TGFBR2 genes was excluded, and no mutations were identified in the coding region of TGFBR1, indicating the existence of other genes involved in MFS. Our results suggest that the genetic heterogeneity of MFS may be greater that previously reported.

Heterogeneity correction in the construction of optimized planning in radiotherapy using linear programming

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Pharmacological modulation of allergic inflammation in the rat airways and association with mast cell heterogeneity

Administration of ovalbumin by aerosol to sensitised rats produced a rapid (15 min) protein exudation in different airway tissues, as determined by Evans blue staining. This was associated with marked mast cell degranulation determined by histological examination, with there being no difference between mucosal and connective tissue mast cells. A 5-day administration regimen with compound 48/80 selectively depleted connective tissue mast cell (Positive to berberine staining) without modifying ovalbumin-induced plasma protein extravasation. Treatment of rats with dexamethasone (1 mg/kg, - 12 h) or nor-dihydroguaiaretic acid (30 mg/kg i.p., - 30 min) significantly reduced ovalbumin-induced protein extravasation and preserved mucosal mast cell morphology. Indomethacin (4 mg/kg i.v., - 30 min) exerted no effect on either parameter. In conclusion, we propose the mucosal mast cell as a target cell responsible at least partly for the inhibitory actions of known anti-inflammatory drugs. We suggest an involvement of endogenous leukotriene(s), but not prostanoid(s), in mucosal mast cell activation/degranulation. (C) 2001 Published by Elsevier B.V. B.V.