Projections from the dorsomedial division of the bed nucleus of the stria terminalis to hypothalamic nuclei in the mouse.

As stressful environment is a potent modulator of feeding, we seek in the present work to decipher the neuroanatomical basis for an interplay between stress and feeding behaviors. For this, we combined anterograde and retrograde tracing with immunohistochemical approaches to investigate the patterns of projections between the dorsomedial division of the bed nucleus of the stria terminalis (BNST), well connected to the amygdala, and hypothalamic structures such as the paraventricular (PVH) and dorsomedial (DMH), the arcuate (ARH) nuclei and the lateral hypothalamic areas (LHA) known to control feeding and motivated behaviors. We particularly focused our study on afferences to proopiomelanocortin (POMC), agouti-related peptide (AgRP), melanin-concentrating-hormone (MCH) and orexin (ORX) neurons characteristics of the ARH and the LHA, respectively. We found light to intense innervation of all these hypothalamic nuclei. We particularly showed an innervation of POMC, AgRP, MCH and ORX neurons by the dorsomedial and dorsolateral divisions of the BNST. Therefore, these results lay the foundation for a better understanding of the neuroanatomical basis of the stress-related feeding behaviors.

[The ratio of tuberculosis-specific antigen to phytohemagglutinin in T-SPOT assay in the diagnosis of active tuberculosis].

Objective: The aim of this study was to determine the performance of the ratio of tuberculosis-specific antigen (TBAg) to phytohemagglutinin (PHA) (TBAg/PHA ratio) in T-SPOT assay in the diagnosis of active tuberculosis (ATB). Methods: Between January 2014 and January 2017, 378 Mycobacterium tuberculosis (MTB) culture positive patients (268 cases of pulmonary tuberculosis, 110 extra-pulmonary tuberculosis) and 824 healthy individuals were recruited from Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. T-SPOT assay was performed and TBAg/PHA ratio was calculated in all the participants. To validate the study, another group of 223 MTB culture positive TB patients with positive T-SPOT results were recruited from Guangzhou Chest Hospital between January 2017 and December 2017. This was a retrospective case-control study and differences between groups were analyzed using the Mann-Whitney U-test. Results: Of the 378 culture positive ATB patients, 344 patients had positive T-SPOT results. Of the 824 healthy individuals, 204 individuals had positive T-SPOT results. Using healthy individuals as the control group, the sensitivity and specificity of T-SPOT assay in the diagnosis of ATB were 91.0% (344/378) and 75.2% (620/824). Directly using T-SPOT results had a limited accuracy in distinguishing ATB from latent tuberculosis infection (LTBI). The area under the receiver operating characteristic (ROC) curve was between 0.7 and 0.8. However, a further calculation of the TBAg/PHA ratio showed a better performance than TBAg in distinguishing these two conditions, and the area under the ROC curve was 0.881 (95% CI: 0.853-0.909). If using the threshold value of 0.234, the sensitivity and specificity of the TBAg/PHA ratio in distinguishing ATB from LTBI were 69.5% (239/344) and 94.12% (192/204). The validation data showed that the performance of the TBAg/PHA ratio in distinguishing ATB from LTBI was also satisfactory, and the area under the ROC curve was 0.901 (95% CI: 0.872-0.931). Furthermore, the TBAg/PHA ratio had an important role in the diagnosis of extra-pulmonary tuberculosis. If using the threshold value of 0.234, the sensitivity and specificity of the TBAg/PHA ratio in the diagnosis of extra-pulmonary tuberculosis were 79.2% (76/96) and 94.1% (192/204). The area under the ROC curve was 0.932 (95% CI: 0.897-0.967). Conclusions: The TBAg/PHA ratio in T-SPOT assay was better than directly using T-SPOT results in distinguishing ATB from LTBI. This ratio also showed a potential use in the diagnosis of extra-pulmonary tuberculosis.

The Use of TB-Specific Antigen/Phytohemagglutinin Ratio for Diagnosis and Treatment Monitoring of Extrapulmonary Tuberculosis.

Extrapulmonary tuberculosis (EPTB) has become more common in recent years; however, the diagnosis of EPTB remains a challenge. In this study, we analyzed the performance of the ratio of TB-specific antigen (TBAg) to phytohemagglutinin (PHA) (TBAg/PHA ratio) in T-SPOT.TB (T-SPOT) assay for diagnosis and treatment monitoring of EPTB. Between 2012 and 2017, 734 EPTB patients were diagnosed and recruited from Tongji hospital, and 1,137 suspected EPTB patients who had other diagnoses were recruited as non-EPTB controls. To validate the study, another small group of EPTB patients and non-EPTB controls were recruited from Sino-French New City Branch of Tongji Hospital. The positive rate of peripheral blood T-SPOT in EPTB and non-EPTB were 88.15 and 32.28%. In T-SPOT positive patients, the direct T-SPOT results had limited value in distinguishing these two conditions. A further calculation of the TBAg/PHA ratio of T-SPOT showed improved performance in each form of EPTB. If using 0.20 as the threshold value of the TBAg/PHA ratio, the pooled sensitivity and specificity were 70.79 and 91.55% in distinguishing EPTB from non-EPTB. The validation results showed a better performance of the TBAg/PHA ratio in distinguishing these two conditions, with a sensitivity and specificity of 81.82 and 97.56%, respectively. Comparing with directly using T-SPOT results, the TBAg/PHA ratio was less affected by immunosuppression. Furthermore, PHA value reflected immunosuppression and could help to judge the credibility of T-SPOT results in EPTB patients with different immune status. The TBAg/PHA ratio was significantly decreased during anti-tuberculosis (TB) treatment, which suggests that it can also be used to monitor therapeutic efficacy. These data provide new insights into the role of T-SPOT assay in TB disease, and the TBAg/PHA ratio might be a useful tool for diagnosis and treatment monitoring of EPTB.

Diagnostic Performance of a 5-Marker Predictive Model for Differential Diagnosis Between Intestinal Tuberculosis and Crohn's Disease.

Background: The differentiation between intestinal tuberculosis (ITB) and Crohn's disease (CD) is a challenge. The aim of this study was to investigate a predictive model for differential diagnosis between ITB and CD. Methods: A total of 268 patients who were suspected of having ITB or CD were prospectively recruited between January 2013 and September 2016. The clinical, laboratory, radiological, endoscopic, and histological features were investigated and subjected to univariate and multivariate analyses. The final predictive model was developed based on the regression coefficients of multivariate logistic regression. To validate the model, the same regression equation was tested on the other group. Results: A total of 239 patients had a final diagnosis, including 86 ITB and 153 CD. Five variables (perianal disease, pulmonary involvement, longitudinal ulcer, left colon, and ratio of tuberculosis-specific antigen to phytohaemagglutinin) were selected for the predictive model to discriminate between ITB and CD. In the predictive model of the training data set, the area under the receiver operating characteristic (ROC) curve, sensitivity, specificity, and accuracy, with a cutoff level of 0.29, were 0.975 (95% confidence interval [CI], 0.939-0.993), 96.7%, 90.7%, and 92.8%, respectively. Application of the predictive model to the validation data set showed similar performance in distinguishing ITB from CD. The area under the ROC curve, sensitivity, specificity, and accuracy were 0.950 (95% CI, 0.871-0.987), 88.5%, 93.5%, and 91.7%, respectively. Conclusions: This 5-marker predictive model could be conveniently used by clinicians to draw a reliable differential diagnosis between ITB and CD in clinical practice. 10.1093/ibd/izy154_video1izy154.video15790725497001.

Use of TBAg/PHA ratio in distinguishing tuberculoma from cancer in solitary pulmonary nodule or mass.

INTRODUCTION: Differentiation of tuberculoma from cancer in solitary pulmonary nodule or mass still remains a major challenge in diagnostic laboratories. OBJECTIVES: The objective of this study is to determine the performance of T-SPOT.TB assay in discriminating these 2 diseases. METHODS: We prospectively enrolled 331 patients with a solitary pulmonary nodule or mass on computed tomography scans. Conventional tests and T-SPOT.TB assay were simultaneously performed in all participants. RESULTS: Our results showed that the performance of directly using T-SPOT.TB results in distinguishing tuberculoma from cancer in solitary pulmonary nodule or mass was not satisfactory because of moderate sensitivity and specificity. However, a further calculation of the ratio of TB-specific antigen (TBAg) to phytohemagglutinin (PHA) (TBAg/PHA ratio) of T-SPOT.TB assay may lead to improvement in distinguishing these 2 diseases. If using the threshold value of 0.236, the sensitivity and specificity of the TBAg/PHA ratio in distinguishing tuberculoma from cancer in solitary pulmonary nodule or mass were, respectively, 80.6% and 93.3%. The area under the curve (AUC) of the receiver operating characteristic curve was 0.921 (95% confidence interval, 0.875-0.967). Furthermore, the TBAg/PHA ratio may also be used to distinguish tuberculoma from other benign diseases (AUC: 0.909, sensitivity: 85.07%, specificity: 90%). CONCLUSIONS: Calculation of the TBAg/PHA ratio might provide a useful non-invasive tool for distinguishing tuberculoma from cancer in patients with a solitary pulmonary nodule or mass in TB-endemic countries.

The performance of the TBAg/PHA ratio in the diagnosis of active TB disease in immunocompromised patients.

OBJECTIVES: The results of the T-SPOT.TB (T-SPOT) assay are reduced in immunocompromised patients with active tuberculosis (ATB), and it is difficult using T-SPOT results to distinguish ATB from latent tuberculosis infection (LTBI) in this condition. The aim of this study was to determine the performance of the TBAg/PHA ratio in T-SPOT assay in the diagnosis of ATB in immunocompromised patients. METHODS: One hundred and forty three immunocompromised ATB patients and 124 LTBI individuals were diagnosed according to conventional tests and T-SPOT assay. RESULTS: The results of T-SPOT assay are of no value in the diagnosis of ATB in immunocompromised patients. However, the number of phytohaemagglutinin (PHA) spot-forming cells (sfc) in T-SPOT assay was substantially decreased in immunocompromised ATB patients compared with that in LTBI individuals. Receiver operating characteristic (ROC) analysis revealed that a further calculation of the TBAg/PHA ratio (the larger of the ESAT-6/PHA and CFP-10/PHA) showed a better performance in distinguishing these two diseases. Using the threshold value of 0.316, the sensitivity and specificity for distinguishing immunocompromised ATB patients from LTBI individuals were respectively 79.21 and 94.05%. CONCLUSIONS: Our findings suggest that the TBAg/PHA ratio might have some significance for the diagnosis of TB disease in immunocompromised patients.

Using the TBAg/PHA ratio in the T-SPOT(®).TB assay to distinguish TB disease from LTBI in an endemic area.

SETTING: An important limitation of the T-SPOT(®).TB assay is its inability to distinguish active tuberculosis (TB) from latent tuberculous infection (LTBI). OBJECTIVE: We proposed a new calculation method for the T-SPOT assay and assessed its effect on distinguishing active TB from LTBI. DESIGN: A total of 162 active TB patients and 97 LTBI individuals were diagnosed according to conventional tests and the T-SPOT assay. RESULTS: The results of early secreted antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) in T-SPOT cannot be recommended for distinguishing TB from LTBI. The number of phytohaemagglutinin (PHA) spot-forming cells (sfc) in the T-SPOT assay was reduced in active TB patients. The ESAT-6/PHA or CFP-10/PHA ratios in active TB patients were significantly higher than in individuals with LTBI. Using 0.295 as the threshold ratio of Mycobacterium tuberculosis-specific antigen (TBAg) sfc to PHA sfc (TBAg/PHA ratio, the larger of ESAT-6/PHA and CFP-10/PHA), the sensitivity and specificity were 82.1% and 90.7% in distinguishing active TB from LTBI. The TBAg/PHA ratio might also be used to monitor the effect of anti-tuberculosis treatment. CONCLUSIONS: Calculating the TBAg/PHA ratio might have the potential to diagnose active TB and distinguish TB disease from LTBI.

An ultrasensitive impedimetric glycan biosensor with controlled glycan density for detection of lectins and influenza hemagglutinins.

An impedimetric glycan biosensor with optimised glycan density was applied for the detection of lectins and influenza hemagglutinins down to attomolar concentrations (aM).

2-DE-based proteomic analysis of common bean (Phaseolus vulgaris L.) seeds.

Common bean (Phaseolus vulgaris L.) is the most important grain legume for direct human consumption. Proteomic studies in legumes have increased significantly in the last years but few studies have been performed to date in P. vulgaris. We report here a proteomic analysis of bean seeds by two-dimensional electrophoresis (2-DE). Three different protein extraction methods (TCA-acetone, phenol and the commercial clean-up kit) were used taking into account that the extractome can have a determinant impact on the level of quality of downstream protein separation and identification. To demonstrate the quality of the 2-DE analysis, a selection of 50 gel spots was used in protein identification by mass spectrometry (MALDI-TOF MS and MALDI-TOF/TOF). The results showed that a considerable proportion of spots (70%) were identified in spite of incomplete genome/protein databases for bean and other legume species. Most identified proteins corresponded to storage protein, carbohydrate metabolism, defense and stress response, including proteins highly abundant in the seed of P. vulgaris such as the phaseolin, the phytohemagglutinin and the lectin-related α-amylase inhibitor.

Optimización de técnicas proteómicas para el estudio de la judía común Phaseolus vulgaris L / Optimization of proteomic techniques for the study of the common bean Phaseolus vulgaris L

La finalidad del presente trabajo es la optimización de la técnica de electroforesis bidimensional (2-DE) y la evaluación de los diferentes métodos de extracción de proteínas para la obtención y estudio del proteoma de la judía común Phaseolus vulgaris. Como material biológico se han usado tejidos de semilla y hoja de la línea Calima perteneciente al acervo genético andino y de la línea ICA-Pijao perteneciente al acervo mesoamericano de esta especie, respectivamente. Se han evaluado tres métodos de extracción de proteínas: TCA-acetona, fenol y el kit comercial clean up. La electroforesis bidimensional se ha optimizado para geles con un gradiente de pH 4-7 en la primera dimensión. Los resultados obtenidos ponen de manifiesto que el método óptimo de extracción de semilla es el fenol y para la hoja, el idóneo es el basado en el kit de precipitación. Con este estudio se puede ver cuál es el protocolo de precipitación más adecuado para cada tejido de Phaseolus por lo que con estos resultados se intenta dar solución a uno de los temas más controvertidos de la técnica (AU)

The purpose of this study is the optimization of two-dimensional electrophoresis (2-DE) technique and the evaluation of different protein extraction methods to obtain and study the proteome of the common bean Phaseolus vulgaris. As biological material, seed and leaf tissues have been used. These tissues belong to the Calima line of the Andean gene pool and to the Ica-Pijao line of the Mesoamerican pool of this species, respectively. Three protein extraction methods have been evaluated: TCA-acetone, phenol and the commercial kit «clean up». Two-dimensional electrophoresis gels have been optimized for a gradient of pH 4-7 in the first dimension. The results show that phenol is the optimal method of seed protein extraction while the optimal method of leaf protein extraction is based on the precipitation kit. This study determines the most appropriate protocol of precipitation for each tissue of Phaseolus, and settles one of the most controversial topics of the technique (AU)

Nutritional and anti-nutritional potential of three accessions of itching bean (Mucuna pruriens (L.) DC var. pruriens): an under-utilized tribal pulse.

Three accessions of the under-utilized legume itching bean (Mucuna pruriens var. pruriens) were analysed for proximate composition, mineral profiles, vitamins (niacin and ascorbic acid), fatty acid profiles, amino acid profiles of total seed protein, in vitro protein digestibility and certain anti-nutritional factors. All three accessions of M. pruriens var. pruriens contained higher amounts of crude protein and crude lipid when compared with most of the commonly consumed pulses. The fatty acid profiles revealed that the seed lipids contained a higher concentration of palmitic acid and linoleic acids. Amino acid profiles of M. pruriens var. pruriens revealed that the seed protein contained relatively higher levels of certain essential amino acids compared with the FAO/WHO requirement pattern. The investigated seeds are rich in minerals such as potassium, calcium, magnesium, phosphorus, iron and manganese. Anti nutritional substances such as total free phenolics, tannins, 3,4-dihydroxyphenylalanine, phytic acid, hydrogen cyanide, trypsin inhibitor activity, oligosaccharides and phytohaemagglutinating activity were investigated. The anti-nutritional fatty acid, behenic acid, also was detected in the present study.

Age-dependent changes in the pigeon bursa of Fabricius vasculature: a comparative study using light microscopy and scanning electron microscopy of vessel casts.

The present study was carried out to analyse the vascularization of the pigeon bursa cloacalis of Fabricius and to determine whether it undergoes age-dependent changes during its functionally most important growth period after hatching of the pigeon. Morphological assessment of vascular corrosion casts, studied qualitatively and quantitatively, was applied for the first time to investigate the vascularization of the pigeon pigeon bursa of Fabricius. This also allowed us to analyse the microvasculature and morphological aspects of the vessel interrelationships as occurring in the natural state. The casts were compared with histological sections stained by haematoxylin-eosin and by binding of the lectin e-PHA (Phaseolus vulgaris, erythroagglutinin) to blood vessels. The vascular architecture of the bursa of Fabricius of the pigeon revealed that the organ is irrigated via two pathways, first through the terminal capillary system of lymphoid follicles arising from the internal pudendal artery, and secondly through arteries originating from the cloacal vasculature of the collum of the organ supplying the periluminal capillary system of the pigeon bursa of Fabricius. Both systems are drained by a venous system which is collateral to the system of the internal pudendal artery and clearly functions as a direct link between the lumen and vasculature of the cloaca or gut, respectively, and the bursa fabricii. This could allow the lymphocytes to be confronted with antigens from the contents of the gut, and their subsequent transport into the secondary lymphoid organs of the organism. Our results demonstrate that the blood vessels, as major and supplying part of the lymphoid system of the bursa Fabricii, clearly reflect three different phases of development: the evolution phase from about day 20 until day 50 post-hatching, the mature phase from days 50 to 90, and the involution phase after day 90. During the evolution phase the density of the vessel system rapidly increases, while in the mature phase the vascular architecture is maintained. The involution phase is dominated by vascular degeneration combined with shrinkage of the whole organ. Therefore, the morphology of the vasculature distinctly reflects the functional status of this primary lymphoid organ during its lifespan.

Analysis of unconventional approaches for the rapid detection of surface lectin binding ligands on human cell lines.

For over a decade our laboratory has developed and used a novel histochemical assay using derivatized agarose beads to examine the surface properties of various cell types. Most recently, we have used this assay to examine lectin binding ligands on two human cell types, CCL-220, a colon cancer cell line, and CRL-1459, a non-cancer colon cell line. We found that CCL-220 cells bound specific lectins better than CRL-1459, and this information was used to test for possible differential toxicity of these lectins in culture, as a possible approach in the design of more specific anti-cancer drugs. Although we have examined the validity of the bead-binding assay in sea urchin cell systems, we have not previously validated this technique for mammalian cells. Here the binding results of the bead assay are compared with conventional fluorescence assays, using lectins from three species (Triticum vulgaris, Phaseolus vulgaris, and Lens culinaris) on the two colon cell lines. These lectins were chosen because they seemed to interact with the two cell lines differently. Binding results obtained using both assays were compared for frozen, thawed and fixed; cultured and fixed; and live cells. Both qualitative and quantitative fluorescence results generally correlated with those using the bead assay. Similar results were also obtained with all of the three different cell preparation protocols. The fluorescence assay was able to detect lower lectin binding ligand levels than the bead assay, while the bead assay, because it can so rapidly detect cells with large numbers of lectin binding ligands, is ideal for initial screening studies that seek to identify cells that are rich in surface binders for specific molecules. The direct use of frozen, thawed and fixed cells allows rapid mass screening for surface molecules, without the requirement for costly and time consuming cell culture.

Enterally but not parenterally administered Phaseolus vulgaris lectin induces growth and precocious maturation of the gut in suckling rats.

BACKGROUND: The lectin, phytohemagglutinin (PHA) has been shown to induce growth and functional maturation of the gastrointestinal (GI) tract in suckling rats. OBJECTIVES: To investigate the effect of the administration route, and whether enteral exposure to PHA was necessary to induce functional maturation. METHODS: Fourteen-day-old rats were daily administered PHA via orogastric feeding (0.05 mg PHA/g BW) or via subcutaneous injection (0.05 or 0.005 mg PHA/g BW) for 3 days, while the controls received saline orogastrically. At 17 days of age, organ weight, intestinal and pancreatic function, and plasma corticosterone levels were analyzed. Moreover, 14-days old pups receiving a single dose of PHA, enterally or parenterally, were sacrificed after 12 h and examined for organ PHA binding using immunohistochemistry. RESULTS: Enteral PHA exposure resulted in PHA binding in the epithelial lining of the small intestine, increased gastrointestinal growth, reduced intestinal macromolecular absorption, altered the disaccharidase expression towards an adult-like pattern, and increased the pancreatic protein and trypsin contents. In contrast, parenteral PHA exposure (high dose) resulted in PHA-binding in extra-intestinal organs, increased liver and spleen weight, and decreased thymus weight. Moreover, the intestinal maltase activity increased moderately, and the transfer of BSA to blood plasma was partially reduced. Both PHA treatments led to elevated plasma corticosterone levels. CONCLUSION: These results demonstrated that enteral exposure to PHA was necessary to induce the precocious maturation of the GI tract and the pancreas, while parenteral administration affects the extra-intestinal organs. Furthermore, the enteral effects were probably not mediated via a corticosteroid dependent pathway.

Immobilized zinc affinity chromatography of pectin hydroxamic acids for purification of trypsin inhibitors from soybean and sweet potato.

Commercial pectin (with a 94% degree of esterification, DE94) suspended in methanol was reacted with methanolic alkaline hydroxylamine at room temperature for 20 h to prepare pectin hydroxamic acids (PHAs). The prepared PHA was coupled to the epoxy-activated Sepharose 6B gel to get immobilized PHA resins. The immobilized PHA resin was then balanced in column with 2 mM ZnCl2 in 50 mM Tris-HCl buffer (pH 7.9) to test the immobilized Zn-PHA gel as solid phase for immobilized metal affinity chromatography for the purification of trypsin inhibitors (TIs) from soybean and sweet potato. Using TI activity staining, it was found that purified TIs from the commercial soybean and sweet potato after trypsin affinity column purification could be adsorbed onto an immobilized Zn-PHA affinity column and eluted by 100 mM EDTA in 10 mM Tris-HCl buffer (pH 7.9). The immobilized Zn-PHA affinity column was used for TI purifications from crude extracts of sweet potato. The recovery of TI activity for one step was 90%, with 19.74-fold purification increase.

Tannins, trypsin inhibitors and lectin cytotoxicity in tepary (Phaseolus acutifolius) and common (Phaseolus vulgaris) beans.

This study compared the levels of antinutritional components and cytotoxic effect of extracts, from tepary (Phaseolus acutifolius) and common (Phaseolus vulgaris) beans. Antinutritional factors were evaluated by determining their effect on the viability of epithelial cells isolated from rat small intestine. The protein and carbohydrates content were similar in all the genotypes studied (20 and 60%, respectively). Common beans presented higher content of trypsin inhibitors, tannins and lectins than tepary beans. There was not a significant correlation between tannins and cooking time. However, water absorption and cooking time correlated significantly (p < 0.05). Considerable variation was observed in lectin activity (1302-18161 Ul/mg) of extracts from different beans. Tannins, lectins, trypsin inhibitors and fat content differed between bean varieties whereas protein content was similar. The percent cellularity on rat epithelial cells was significantly different among protein extracts from different bean cultivars and ranged between 53.5% and 87.4% (p < 0.05). These results suggest that the incorporation of tepary beans in the diet would not alter the current nutritional contribution of common beans or introduce adverse toxic effects. The agronomic characteristics of tepary beans make them attractive for cultivation. However, the harder to cook phenomenon may be a limiting factor that needs further consideration.

Does immune activation continue during an attack-free period in familial Mediterranean fever?

Although some information is available regarding immune activation in familial Mediterranean fever (FMF), little is known about either peripheral blood T cell activation marker expression or the T cell proliferative response to phytohaemagglutinin (PHA). In the present study, we aimed to investigate the percentages of peripheral blood lymphocyte subsets, T cell expression of cellular activation markers (CD25, CD69, HLA-DR), the T cell response to PHA and serum levels of soluble interleukin-2 receptor (sIL-2R) and interleukin (IL)-10 in patients with FMF. Forty patients with FMF were enrolled into the study. Control groups were sex- and age-matched and consisted of 20 healthy blood donors and 15 patients with inactive Behcet's disease. The patients with FMF in an attack period had higher levels of sIL-2R than those in an attack-free period, and also in comparison with both control groups. The levels of sIL-2R were also found to be higher in patients with FMF in an attack-free period than those in both control groups. The mean levels of IL-10 were found to be lower in patients with FMF in an attack-free period than those in an attack period and were also lower than those in the healthy controls. In an acute attack period, the absolute counts of CD3+HLA-DR+, CD4+CD69+, CD8+CD25+ and CD8+CD69+ T cells in peripheral blood samples were also higher than those in both control groups. Both the percentages and absolute counts of CD4+CD69+ T cells in peripheral blood samples of patients with FMF in an attack-free period were slightly but significantly higher than those in the healthy controls. In conclusion, our study indicates that the T cell system is abnormally activated in patients with FMF in both the attack and attack-free period and that decreased IL-10 levels may create a tendency to perpetuate subclinical immune activation in the attack-free period.

Combination antiangiogenesis therapy with marimastat, captopril and fragmin in patients with advanced cancer.

Marimastat, low molecular weight heparins and captopril have antiangiogenic activity in vitro and in animal models. We studied the safety and efficacy of the combination of these drugs in patients with advanced cancer. In all, 50 patients were enrolled. Captopril was given orally at a dose of 50 mg bd daily. Fragmin was administered as a daily subcutaneous injection of 200 units kg(-1) for the first 28 days and 5000 units thereafter. Marimastat was given at 10 mg bd orally. Serum, plasma and urinary angiogenic factors were measured at baseline and after 1 month of treatment. Inhibition of release of tumour necrosis factor alpha (TNF-alpha) from peripheral lymphocytes was used as a surrogate pharmacodynamic end point. There was one case of haemorrhagic stroke and one upper gastrointestinal haemorrhage. The commonest toxicity was myalgia. One of 10 patients with renal cancer had a partial response, and three patients had a prolonged period of stable disease. The treatment significantly inhibited phytohaemagglutinin (PHA)-stimulated TNF-alpha release from patient's lymphocytes. The combination of marimastat, fragmin and captopril is well tolerated and has in vivo activity. Inhibition of PHA-stimulated TNF-alpha release from lymphocytes is a surrogate pharmacodynamic marker of metalloprotease inhibition.

Expression of N-acetylglucosaminyltransferase v is associated with prognosis and histology in non-small cell lung cancers.

PURPOSE: N-Acetylglucosaminyltransferase V (GnT-V), a key enzyme in the formation of branching of asparagine-linked oligosaccharides, is strongly linked to tumor invasion and metastasis of colon and breast cancers. However, GnT-V is expressed in many tissues, including normal lung. GnT-V expression has not been examined previously in human lung cancers. The objective of this study is to examine GnT-V expression in non-small cell lung cancers (NSCLCs) and to determine its relationship to biological and clinicopathological characteristics and prognosis. EXPERIMENTAL DESIGN: GnT-V expression was studied by immunohistochemistry in 217 surgically resected NSCLCs and analyzed statistically in relation to various characteristics. RESULTS: High GnT-V expression was found in 113 (52.1%) NSCLCs, and low GnT-V expression was found in 104 (47.9%) NSCLCs. Multivariate logistic regression analysis revealed a significant association between low GnT-V expression and squamous cell carcinomas, as compared with nonsquamous cell carcinomas (P = 0.02). Among biological characteristics of tumors, Ki-67 labeling index was higher in tumors with low GnT-V expression than in those with high GnT-V expression, although this difference was not statistically significant (P = 0.09). Patients with tumors having low GnT-V expression had significantly shorter survival time than patients with tumors having high GnT-V expression in 103 patients with pStage I NSCLCs (5-year survival rates, 49% and 86%, respectively; P = 0.0009), as well as in 59 patients with pStage I non-squamous cell carcinomas (5-year survival rates, 54% and 89%, respectively; P = 0.007). Low GnT-V expression was a significant unfavorable prognostic factor in pStage I NSCLCs (hazard ratio, 2.86; P = 0.002) and in pStage I nonsquamous cell carcinomas (hazard ratio, 3.02; P = 0.02). Furthermore, beta1-6 branching of asparagine-linked oligosaccharides, which are products of GnT-V, were increased highly or moderately in 8 of 10 tumors with high GnT-V expression, as judged by leukoagglutinating phytohemagglutinin staining. CONCLUSIONS: GnT-V expression is associated with histology in NSCLCs. Low GnT-V expression is associated with shorter survival and poor prognosis in pStage I overall NSCLCs and non-squamous cell carcinomas.

Organization of parabrachial projections from the spinal trigeminal nucleus oralis: an anterograde tracing study in the rat.

In recent years, we have accumulated data showing that the spinal trigeminal nucleus oralis (Sp5O) contributes to the processing of somatosensory inputs from the orofacial region. Although the parabrachial area (PB) represents the main brainstem relay for autonomic, nociceptive, and gustatory afferents, few data are available regarding the topographical distribution of the efferent projections from the Sp5O to the PB. We have addressed this question with the rat, by using the anterograde tracer Phaseolus vulgaris leucoagglutinin. A dense trigeminoparabrachial pathway from the Sp5O toward, predominantly, the ipsilateral PB was revealed. Projections come mainly from the dorsal part of the Sp5O that was found to innervate densely the medial, external medial, and ventral lateral subnuclei. In contrast, the ventral part of the Sp5O projected almost exclusively to an as yet not formally described region, located dorsally and laterally to the lateral tip of the brachium conjunctivum, close to the Kölliker-Fuse nucleus. These results suggest that distinct regions within the Sp5O may be involved in the processing of gustatory and nociceptive information.