Infusion of Recombinant Human Tissue Plasminogen Activator Through the Superior Mesenteric Artery in the Treatment of Acute Mesenteric Venous Thrombosis

Acute mesenteric venous thrombosis is an uncommon condition that is usually treated with systemic anticoagulation. Catheter-directed thrombolysis through the superior mesenteric artery may be a viable adjunct to treat this morbid condition. In the present article, we have described a case of superior mesenteric venous thrombosis treated with catheter-directed infusion of tissue plasminogen activator through the superior mesenteric artery.

Human Tissue Kallikrein Activity in Angiographically Documented Chronic Stable Coronary Artery Disease

AbstractBackground:Human tissue kallikrein (hK1) is a key enzyme in the kallikrein–kinin system (KKS). hK1-specific amidase activity is reduced in urine samples from hypertensive and heart failure (HF) patients. The pathophysiologic role of hK1 in coronary artery disease (CAD) remains unclear.Objective:To evaluate hK1-specific amidase activity in the urine of CAD patientsMethods:Sixty-five individuals (18–75 years) who underwent cardiac catheterism (CATH) were included. Random midstream urine samples were collected immediately before CATH. Patients were classified in two groups according to the presence of coronary lesions: CAD (43 patients) and non-CAD (22 patients). hK1 amidase activity was estimated using the chromogenic substrate D-Val-Leu-Arg-Nan. Creatinine was determined using Jaffé’s method. Urinary hK1-specific amidase activity was expressed as µM/(min · mg creatinine) to correct for differences in urine flow rates.Results:Urinary hK1-specific amidase activity levels were similar between CAD [0.146 µM/(min ·mg creatinine)] and non-CAD [0.189 µM/(min . mg creatinine)] patients (p = 0.803) and remained similar to values previously reported for hypertensive patients [0.210 µM/(min . mg creatinine)] and HF patients [0.104 µM/(min . mg creatinine)]. CAD severity and hypertension were not observed to significantly affect urinary hK1-specific amidase activity.Conclusion:CAD patients had low levels of urinary hK1-specific amidase activity, suggesting that renal KKS activity may be reduced in patients with this disease.

Pathology as the Cornerstone of Human Tissue Banking: European Consensus Expert Group Report

Aside from ethical considerations, the primary requirement for usage of human tissues in basic or translational research is the thorough characterization of tissues. The second, but equally essential, requirement is that tissues be collected, processed, annotated, and preserved in optimal conditions. These requirements put the pathologist at the center of tissue banking activities and of research aimed at discovering new biomarkers. Pathologists not only provide information identifying the specimen but also make decisions on what materials should be biobanked, on the preservation conditions, and on the timeline of events that precede preservation and storage. This central position calls for increased recognition of the role of the pathologist by the biomolecular community and places new demands on the pathologist's workload and scope of scientific activities. These questions were addressed by an Expert Group Meeting of the European Biological and Biomolecular Research Infrastructure (BBMRI). While detailed recommendations are published elsewhere (Bevilacqua et al., Virchows Archivs, 2010, in press), this article outlines the strategic and technological issues identified by the Expert Group and identifies ways forward for better integration of pathology in the current thrust for development of biomarker-based "personalized medicine.

Human Tissue Act 2004 (PDF 25 KB)

Draft Regulations for Consultation

Linear competitive inhibition of human tissue kallikrein by 4-aminobenzamidine and benzamidine and linear mixed inhibition by 4-nitroaniline and aniline

Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide (7.5-90.0 µM) by human tissue kallikrein (hK1) (4.58-5.27 nM) at pH 9.0 and 37ºC was studied in the absence and in the presence of increasing concentrations of 4-aminobenzamidine (96-576 µM), benzamidine (1.27-7.62 mM), 4-nitroaniline (16.5-66 µM) and aniline (20-50 mM). The kinetic parameters determined in the absence of inhibitors were: Km = 12.0 ± 0.8 µM and k cat = 48.4 ± 1.0 min-1. The data indicate that the inhibition of hK1 by 4-aminobenzamidine and benzamidine is linear competitive, while the inhibition by 4-nitroaniline and aniline is linear mixed, with the inhibitor being able to bind both to the free enzyme with a dissociation constant Ki yielding an EI complex, and to the ES complex with a dissociation constant Ki', yielding an ESI complex. The calculated Ki values for 4-aminobenzamidine, benzamidine, 4-nitroaniline and aniline were 146 ± 10, 1,098 ± 91, 38.6 ± 5.2 and 37,340 ± 5,400 µM, respectively. The calculated Ki' values for 4-nitroaniline and aniline were 289.3 ± 92.8 and 310,500 ± 38,600 µM, respectively. The fact that Ki'>Ki indicates that 4-nitroaniline and aniline bind to a second binding site in the enzyme with lower affinity than they bind to the active site. The data about the inhibition of hK1 by 4-aminobenzamidine and benzamidine help to explain previous observations that esters, anilides or chloromethyl ketone derivatives of Nalpha-substituted arginine are more sensitive substrates or inhibitors of hK1 than the corresponding lysine compounds.

68Ga-DOTATOC PET/CT and somatostatin receptor (sst1-sst5) expression in normal human tissue: correlation of sst2 mRNA and SUVmax

By targeting somatostatin receptors (sst) radiopeptides have been established for both diagnosis and therapy. For physiologically normal human tissues the study provides a normative database of maximum standardized uptake value (SUV(max)) and sst mRNA.

Characterization of the human tissue transglutaminase gene promoter

Transglutaminases are a family of calcium-dependent enzymes, that catalyze the covalent cross-linking of proteins by forming $\varepsilon(\gamma$-glutamyl)lysine isopeptide bonds. In order to investigate the molecular mechanisms regulating the expression of the tissue transglutaminase gene and to determine its biological functions, the goal of this research has been to clone and characterize the human tissue transglutaminase promoter. Thirteen clones of the tissue transglutaminase gene were obtained from the screening of a human placental genomic DNA library. A 1.74 Kb fragment derived from DNA located immediately upstream of the translation start site was subcloned and sequenced. Sequence analysis of this DNA fragment revealed that it contains a TATA box (TATAA), a CAAT box (GGACAAT), and a series of potential transcription factor binding sites and hormone response elements. Four regions of significant homology, a GC-rich region, a TG-rich region, an AG-rich region, and HR1, were identified by aligning 1.8 Kb of DNA flanking the human, mouse, and guinea pig tissue transglutaminase genes.^ To measure promoter activity, we subcloned the 1.74 Kb fragment of the tissue transglutaminase gene into a luciferase reporter vector to generate transglutaminase promoter/luciferase reporter constructs. Transfection experiments showed that this DNA segment includes a functional promoter with high constitutive activity. Deletion analysis revealed that the SP1 sites or corresponding sequences contribute to this activity. We investigated the role of DNA methylation in regulating the activity of the promoter and found that in vitro methylation of tissue transglutaminase promoter/luciferase reporter constructs suppressed their basal activity. Methylation of the promoter is inversely correlated with the expression of the tissue transglutaminase gene in vivo. These results suggest that DNA methylation may be one of the mechanisms regulating the expression of the gene. The tumor suppressor gene product p53 was also shown to inhibit the activity of the promoter, suggesting that induction of the tissue transglutaminase gene is not involved in the p53-dependent programmed cell death pathway. Although retinoids regulate the expression of the tissue transglutaminase gene in vivo, retinoid-inducible activity can not be identified in 3.7 Kb of DNA 5$\sp\prime$ to the tissue transglutaminase gene.^ The structure of the 5$\sp\prime$ end of the tissue transglutaminase gene was mapped. Alignment analysis of the human tissue transglutaminase gene with other human transglutaminases showed that tissue transglutaminase is the simplest member of transglutaminase superfamily. Transglutaminase genes show a conserved core of exons and introns but diverse N-terminuses and promoters. These observations suggest that key regulatory sequences and promoter elements have been appended upstream of the core transglutaminase gene to generate the diversity of regulated expression and regulated activity characteristic of the transglutaminase gene family. ^

Reliable LC3 and p62 autophagy marker detection in formalin fixed paraffin embedded human tissue by immunohistochemistry

Autophagy assures cellular homeostasis, and gains increasing importance in cancer, where it impacts on carcinogenesis, propagation of the malignant phenotype and development of resistance. To date, its tissue-based analysis by immunohistochemistry remains poorly standardized. Here we show the feasibility of specifically and reliably assessing the autophagy markers LC3B and p62 (SQSTM1) in formalin fixed and paraffin embedded human tissue by immunohistochemistry. Preceding functional experiments consisted of depleting LC3B and p62 in H1299 lung cancer cells with subsequent induction of autophagy. Western blot and immunofluorescence validated antibody specificity, knockdown efficiency and autophagy induction prior to fixation in formalin and embedding in paraffin. LC3B and p62 antibodies were validated on formalin fixed and paraffin embedded cell pellets of treated and control cells and finally applied on a tissue microarray with 80 human malignant and non-neoplastic lung and stomach formalin fixed and paraffin embedded tissue samples. Dot-like staining of various degrees was observed in cell pellets and 18/40 (LC3B) and 22/40 (p62) tumors, respectively. Seventeen tumors were double positive for LC3B and p62. P62 displayed additional significant cytoplasmic and nuclear staining of unknown significance. Interobserver-agreement for grading of staining intensities and patterns was substantial to excellent (kappa values 0.60 - 0.83). In summary, we present a specific and reliable IHC staining of LC3B and p62 on formalin fixed and paraffin embedded human tissue. Our presented protocol is designed to aid reliable investigation of dysregulated autophagy in solid tumors and may be used on large tissue collectives.

Species-specific metastasis of human tumor cells in the severe combined immunodeficiency mouse engrafted with human tissue.

We have attempted to model human metastatic disease by implanting human target organs into the immunodeficient C.B-17 scid/scid (severe combined immunodeficiency; SCID) mouse, creating SCID-hu mice. Preferential metastasis to implants of human fetal lung and human fetal bone marrow occurred after i.v. injection of human small cell lung cancer (SCLC) cells into SCID-hu mice; the homologous mouse organs were spared. Clinically more aggressive variant SCLC cells metastasized more efficiently to human fetal lung implants than did cells from classic SCLC. Metastasis of variant SCLC to human fetal bone marrow was enhanced in SCID-hu mice exposed to gamma-irradiation or to interleukin 1 alpha. These data indicate that the SCID-hu mice may provide a model in which to study species- and tissue-specific steps of the human metastatic process.

Human vascular to cardiac tissue selectivity of L- and T-type calcium channel antagonists

1 Voltage-operated calcium channel (VOCC) antagonists are effective antihypertensive and antianginal agents but they also depress myocardial contractility. 2 We compared four L-type calcium channel antagonists, felodipine, nifedipine, amlodipine and verapamil and a relatively T-type selective calcium channel antagonist, mibefradil, on human and rat isolated tissue assays to determine their functional vascular to cardiac tissue selectivity (V/C) ratio. 3 The V/C ratio was calculated as the ratio of the IC50 value of the antagonist that reduced (by 50%) submaximally contracted (K+ 62 mM) human small arteries from the aortic vasa vasorum (vascular, V) mounted in a myograph and the IC50 value of the antagonist that reduced (-)-isoprenaline (6 nM) submaximally stimulated human right atrial trabeculae muscle (cardiac, C) mounted in organ chambers. 4 The average pIC(50) Values (-log IC50 M) for the human vascular preparations were felodipine 8.30, nifedipine 7.78, amlodipine 6.64, verapamil 6.26 and mibefradil 6.22. The average pIC(50) values for the cardiac muscle were felodipine 7.21, nifedipine 6.95, verapamil 6.91, amlodipine 5.94, and mibefradil 4.61. 5 The V/C ratio calculated as antilog [pIC(50)V-pIC(50)C] is thus mibefradil 41, felodipine 12, nifedipine 7, amlodipine 5 and verapamil 0.2. 6 In rat small mesenteric arteries the pIC(50) values for the five drugs were similar to the values for human vasa vasorum arteries contracted by K+ 62 mM. However for methoxamine (10 mu M) contraction in the rat arteries the pIC(50) values were lower for felodipine 7.24 and nifedipine 6.23, but similar for verapamil 6.13, amlodipine 6.28 and mibefradil 5.91. 7 In conclusion in the human tissue assays, the putative T-channel antagonist mibefradil shows the highest vascular to cardiac selectivity ratio; some 3 fold higher than the dihydropyridine, felodipine, and some 200 fold more vascular selective than the phenylalkylamine, verapamil. This favourable vascular to cardiac selectivity for mibefradil, from a new chemical class of VOCC antagonist, may be explained by its putative T-channel selectivity.

Performance of Two Commercial ELISAs for Detecting IgA Anti-Human and Anti-Guinea Pig Tissue Transglutaminase Antibodies

Background: Sensitivity and specificity of anti-human tissue transglutaminase antibodies (anti-htTGA) seem to be superior to those of anti-tissue transglutaminase of guinea pig (anti-gptTGA) for screening patients with celiac disease (CD), but there are still controversies. The aim of this study was to evaluate the performance of two INOVA ELISA kits to detect IgA anti-htTGA and anti-gptTGA in patients with and without CD. Methods: The study groups were comprised of 49 anti-endomysial antibody (EMA)-positive untreated-CD, and 123 controls (EMA-negative treated CD, EMA-negative chronic diarrhea, autoimmune hepatitis, inflammatory bowel disease and healthy people). Results: The agreement between the two ELISAs was statistically significant in all study groups and there was no significant difference between them (92.7% agreement; kappa=0.70; kappa p=0.001; McNemar p=1). All patients with serum reactivity of more than 100 units had histologic diagnosis of CD. In seven of 10 patients with treated-CD who had control biopsies, villous atrophy was still present in four who tested positive by both kits. Two of three celiacs with histologic remission tested positive for both anti-tTGA. Conclusions: the anti-gptTGA and anti-htTGA determination were equally efficient in identifying patients with untreated-CD with high titers of EMA. Whatever the anti-tTGA ELISA used, the reactivity above 100 units was always related to active CD diagnosed by histologic alterations in intestinal biopsies. The anti-tTGA reactivity by both kits was not only similar in determining histologic activity in the follow-up of CD after a gluten free diet, but also in identifying positive sera from the control groups, regardless if CD has been confirmed by duodenal biopsies. (Clin. Lab. 2010;56:29-35)

Tamoxifen down-regulates CaMKII messenger RNA levels in normal human breast tissue

Tamoxifen was proven to reduce the incidence of breast cancer by 49% in women at increased risk of the disease in the Breast Cancer Prevention Trial. In order to identify potential candidates to explain the preventive effect induced by tamoxifen on breast cancer, normal breast tissue obtained from 42 fibroadenoma patients, randomly assigned to receive placebo or tamoxifen, was analyzed by the reverse Northern blot and RT-PCR techniques. The cDNA fragments used on Northern blot membranes were generated by the Human Cancer Genome Project funded by the Ludwig Institute for Cancer Research and FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil). Total RNA was obtained from normal breast tissue from patients with clinical, cytological and ultrasound diagnosis of fibroadenoma. After a 50-day treatment with tamoxifen (10 or 20 mg/day) or placebo, normal breast tissue adjacent to the tumor was collected during lumpectomy with local anesthesia. One differentially expressed gene, Calcium/calmodulin-dependent protein kinase II (CaMKII), was found to be down-regulated during TAM treatment. CaMKII is an ubiquitous serine/threonine protein kinase that has been implicated in the diverse effects of hormones utilizing Ca2+ as a second messenger as well as in c-fos activation. These results indicate that the down-regulation of CaMKII induced by TAM might represent alternative or additional mechanisms of the action of this drug on cell cycle control and response to hormones in normal human breast tissue.

Expression and Regulation of Human Cytochrome P450 4F Isoforms in Tissue Samples and Under TNF-Alpha Challenges

CYP4F (Cytochrome P4504F) enzymes metabolize endogenous molecules including leukotrienes, prostaglandins and arachidonic acid. The involvement of these endogenous compounds in inflammation has led to the hypothesis that changes in the inflamed tissue environment may affect the expression of CYP4Fs during the pro-inflammatory state, which in turn may modulate inflammatory conditions during the anti-inflammatory state. We demonstrated that inflamed tissues have different levels of CYP4F isoform expression profiles in a number of human samples when compared to the average population. The CYP4F isoform expression levels change with the degree of inflammation present in tissue. Further investigation in cell culture studies revealed that inflammatory cytokines, in particular TNF-α, play a role in regulating the expression of the CYP4F family. One of the isoforms, CYP4F11, had different characteristics than that of the other five CYP4F family members. CYP4F11 metabolizes xenobiotics while the other isoforms metabolize endogenous compounds with higher affinity. CYP4F11 also was expressed at high quantities in the brain, and was up-regulated by TNF-α, while the other isoforms were not expressed at high quantities in the brain and were down-regulated by TNF-α. We identified the AP-1 protein of the JNK pathway as the signaling protein that causes significant increase in CYP4F11 expression. Since TNF-α stimulation causes a simultaneous activation of both JNK pathway and NF-κB signaling, we investigated further the role that NF-κB plays on expression of the CYP4F11 gene. We concluded that although there is a significant increase in CYP4F11 expression in the presence of TNF-α, the activation of NF-κB signaling inhibits CYP4F11 expression in a time dependent manner. The expression of CYP4F11 is only significantly increased after 24 hours of treatment with TNF-α; at shorter time points NF-κB signaling overpowers the JNK pathway activation. We believe that these findings may in the future lead to improved drug design for modulating inflammation.