A matrix metalloproteinase inhibitor to treat unresectable cholangiocarcinoma.

Matrix metalloproteinases (MMPs) are implicated in tumour invasion and metastasis. We report the first use of an MMP inhibitor to treat unresectable cholangiocarcinoma. Four men with stage IV cholangiocarcinoma received oral Marimastat (10 mg bd) indefinitely following relief of obstructive jaundice. Monthly measurements of the tumour marker CA 19-9 were used as an indicator of disease response and activity. CA 19-9 levels dropped sharply and stayed low in the two patients who appeared to respond. Mean survival of the four patients was 21.5 months (range 4-48 months). Side effects were well tolerated. A more extensive and detailed examination of MMP inhibitors to treat cholangiocarcinoma is indicated.

Human model of duodenogastro-oesophageal reflux in the development of Barrett's metaplasia.

BACKGROUND: Patients with an intrathoracic oesophagogastrostomy after subtotal oesophagectomy experience profound duodenogastro-oesophageal reflux (DGOR). This study investigated the degree of mucosal injury and histopathological changes in oesophageal squamous epithelium after subtotal oesophagectomy with gastric interposition in relation to the extent of postoperative DGOR. METHODS: Serial endoscopic assessment and systematic biopsy at the oesophagogastric anastomosis was undertaken in 40 patients following curative radical subtotal oesophagectomy and reconstruction with a gastric conduit subjected to a pyloroplasty. Thirty patients subsequently underwent combined 24-h ambulatory pH and bilirubin monitoring. RESULTS: Grade I-III oesophagitis was identified in 14 patients and oesophageal columnar epithelium in 19 patients. Biopsies from columnar regeneration revealed cardiac-type epithelium in ten patients and intestinal metaplasia in nine. Seven patients followed serially showed progression from cardiac-type epithelium to intestinal metaplasia. The incidence of Barrett's metaplasia was similar irrespective of the histological subtype of the resected tumour. Patients with oesophageal columnar epithelium had significantly higher acid (P = 0.015) and bilirubin (P = 0.011) reflux. CONCLUSION: Severe DGOR occurs following subtotal oesophagectomy and provides an environment for the acquisition of Barrett's metaplasia via a sequence of cardiac epithelium and eventual intestinal metaplasia.

Image analysis can be used to detect spatial changes in the histopathology of pancreatic tumours.

Pancreatic cancer is frequently associated with intense growth of fibrous tissue at the periphery of tumours, but the histopathological quantification of this stromal reaction has not yet been used as a prognostic factor because of the difficulty of obtaining quantitative measures using manual methods. Manual histological grading is a poor indicator of outcome in this type of cancer and there is a clinical need to establish a more sensitive indicator. Recent pancreatic tumour biology research has focused upon the stromal reaction and there is an indication that its histopathological quantification may lead to a new prognostic indicator. Histological samples from 21 cases of pancreatic carcinoma were stained using the sirius red, light-green method. Multiple images from the centre and periphery of each tumour were automatically segmented using colour cluster analysis to subdivide each image into representative colours. These were classified manually as stroma, cell cytoplasm or lumen in order to measure the area of each component in each image. Measured areas were analysed to determine whether the technique could detect spatial differences in the area of each tissue component over all samples, and within individual samples. Over all 21 cases, the area of stromal tissue at the periphery of the tumours exceeded that at the centre by an average of 10.0 percentage points (P < 0.001). Within individual tumours, the algorithm was able to detect significantly more stroma (P < 0.05) at the periphery than the centre in 11 cases, whilst none of the remaining cases had significantly more stromal tissue at the centre than the periphery. The results demonstrate that semi-automated analysis can be used to detect spatial differences in the area of fibrous tissue in routinely stained sections of pancreatic cancer.

Comparison of semi-automated image analysis and manual methods for tissue quantification in pancreatic carcinoma.

Objective measurements of tissue area during histological examination of carcinoma can yield valuable prognostic information. However, such measurements are not made routinely because the current manual approach is time consuming and subject to large statistical sampling error. In this paper, a semi-automated image analysis method for measuring tissue area in histological samples is applied to the measurement of stromal tissue, cell cytoplasm and lumen in samples of pancreatic carcinoma and compared with the standard manual point counting method. Histological samples from 26 cases of pancreatic carcinoma were stained using the sirius red, light-green method. Images from each sample were captured using two magnifications. Image segmentation based on colour cluster analysis was used to subdivide each image into representative colours which were classified manually into one of three tissue components. Area measurements made using this technique were compared to corresponding manual measurements and used to establish the comparative accuracy of the semi-automated image analysis technique, with a quality assurance study to measure the repeatability of the new technique. For both magnifications and for each tissue component, the quality assurance study showed that the semi-automated image analysis algorithm had better repeatability than its manual equivalent. No significant bias was detected between the measurement techniques for any of the comparisons made using the 26 cases of pancreatic carcinoma. The ratio of manual to semi-automatic repeatability errors varied from 2.0 to 3.6. Point counting would need to be increased to be between 400 and 1400 points to achieve the same repeatability as for the semi-automated technique. The results demonstrate that semi-automated image analysis is suitable for measuring tissue fractions in histological samples prepared with coloured stains and is a practical alternative to manual point counting.

The pattern of recurrence of adenocarcinoma of the oesophago-gastric junction.

Knowledge of the pattern of recurrence of surgically treated cases of adenocarcinoma of the oesophago-gastric junction is important both for better understanding of their biological nature and for future strategic planning of therapy. The aim of this study is to demonstrate and compare the pattern of dissemination and recurrence in patients with Type I and Type II adenocarcinoma of oesophago-gastric junction. A prospective audit of the clinico-pathological features of patients who had undergone surgery with curative intent for adenocarcinoma of oesophago-gastric junction between 1991 and 1996 was undertaken. Patients were followed up by regular clinical examination. Clinical evaluation was supported by ultrasound, computerised tomography, radio-isotope bone scan, endoscopy and laparotomy each with biopsy and histology where appropriate. One hundred and sixty-nine patients with oesophago-gastric junction tumours (94 Type I and 75 Type II) have been followed up for a median of 75.3 (57-133) months. One hundred and three patients developed proven recurrent disease. The median time to recurrence was 23.3 (14.2-32.4) months for Type I and 20.5 (11.6-29.4) for Type II cancers. The most frequent type of recurrence was haematogenous (56% of Type I recurrences and 54% of Type II) of which 56% were detected within 1 year of surgery. The most frequent sites were to liver (27%), bone (18%) brain (11%) and lung (11%). Local recurrence occurred in 33% of Type I cancer and 29% of Type II recurrences. Nodal recurrence occurred in 18 and 25% of Type I and Type II cancer recurrences, most frequently to coeliac or porta hepatis nodes (64%). Only 7% of Type I and 15% of Type II cancer recurrences were by peritoneal dissemination. Type I and Type II adenocarcinoma of the oesophago-gastric junction have a predominantly early, haematogenous pattern of recurrence. There is a need to better identify the group of patients with small metastases at the time of diagnosis who are destined to develop recurrent disease in order that they may be spared surgery and those with micro metastases in order that they can be offered multi-modality therapy including early post operative or neo-adjuvant chemotherapy.

Functional cloning of Src-like adapter protein-2 (SLAP-2), a novel inhibitor of antigen receptor signaling.

In an effort to identify novel therapeutic targets for autoimmunity and transplant rejection, we developed and performed a large-scale retroviral-based functional screen to select for proteins that inhibit antigen receptor-mediated activation of lymphocytes. In addition to known regulators of antigen receptor signaling, we identified a novel adaptor protein, SLAP-2 which shares 36% sequence similarity with the known Src-like adaptor protein, SLAP. Similar to SLAP, SLAP-2 is predominantly expressed in hematopoietic cells. Overexpression of SLAP-2 in B and T cell lines specifically impaired antigen receptor-mediated signaling events, including CD69 surface marker upregulation, nuclear factor of activated T cells (NFAT) promoter activation and calcium influx. Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade. The SLAP-2 protein contains an NH2-terminal myristoylation consensus sequence and SH3 and SH2 Src homology domains, but lacks a tyrosine kinase domain. In antigen receptor-stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl. Deletion of the COOH terminus of SLAP-2 blocked function and abrogated its association with Cbl. Mutation of the putative myristoylation site of SLAP-2 compromised its inhibitory activity and impaired its localization to the membrane compartment. Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems.

The neuronal t-SNARE complex is a parallel four-helix bundle.

Assembly of the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complex is an essential step for neurotransmitter release in synapses. The presynaptic plasma membrane associated proteins (t-SNAREs), SNAP-25 (synaptosome-associated protein of 25,000 Da) and syntaxin 1A may form an intermediate complex that later binds to vesicle-associated membrane protein 2 (VAMP2). Using spin labeling electron paramagnetic resonance (EPR), we found that the two t-SNARE proteins assemble into a parallel four-helix bundle that consists of two identical syntaxin 1A components and the N-terminal and C-terminal domains of SNAP-25. Although the structure is generally similar to that of the final SNARE complex, the middle region of the helical bundle appears more flexible in the t-SNARE complex. Such flexibility might facilitate interactions between VAMP2 and the t-SNARE complex.

The pattern of metastatic lymph node dissemination from adenocarcinoma of the esophagogastric junction.

BACKGROUND: The incidence of adenocarcinoma of the esophagogastric junction is rapidly increasing, and the extent of lymphadenectomy for such tumors remains controversial. The aim of this study was to identify the pattern of dissemination by examination of all lymph nodes retrieved from resected tumors of the esophagogastric junction. METHODS: The endoscopic and pathologic reports of patients who underwent RO resection for adenocarcinoma of the esophagogastric junction between January 1996 and November 1999 were examined. Patients with type 1 tumors (distal esophagus) underwent subtotal esophagectomy with 2-field lymphadenectomy. Patients with type 2 (gastric cardia) tumors underwent transhiatal D2 total gastro-esophagectomy. Lymph node groups were dissected from the main specimens and examined separately. RESULTS: One hundred and four type 1 and 48 type 2 tumors were studied. Median nodal recovery was 23 lymph nodes (type 1, 22 lymph nodes; type 2, 23 lymph nodes). Seventy-eight percent of the type 1 tumors with nodal metastases had dissemination in both the abdomen and mediastinum. The common abdominal sites were the paracardiac and the left gastric stations. Within the mediastinum, paraesophageal, paraaortic and tracheobronchial metastases were more often encountered. Type 2 tumors had positive lymph nodes most frequently in the left and right paracardiac, lesser curve (N1 group), and left gastric (N2 group) territories. Nodal status correlated with increasing depth of tumor invasion (P =.002). CONCLUSIONS: The pattern of nodal dissemination for cardia tumors concurs with that described by other studies. The current definition of nodal fields in the abdomen and mediastinum for esophageal tumors relates to experience with squamous carcinomas. Our results demonstrate a different pattern of dissemination for junctional esophageal adenocarcinomas. The nodal stations to be resected in radical lymphadenectomies for such tumors should be redefined.

The use of retroviruses as pharmaceutical tools for target discovery and validation in the field of functional genomics.

Retrovirally mediated functional genomics enables identification of physiologically relevant cellular therapeutic targets. Unique properties of retroviruses make them ideal tools for the introduction of large and diverse libraries of potential genetic effectors to a variety of cell types. The identification and recovery of intracellular library elements responsible for altered disease responses establishes a direct basis for pharmaceutical development. Recent innovations in retroviral infection efficiency and expression control have broadened application of the methodology to include libraries of mutagenized cDNAs, peptides and ribozyme genetic effectors.

Mucin gene expression in human urothelium and in intestinal segments transposed into the urinary tract.

PURPOSE: The repertoire of mucin (MUC) gene expression in the normal human urothelium is poorly defined and the alterations in MUC gene expression following transposition of intestinal segments into the urinary tract has not previously been studied. The aims of this study were to define MUC gene expression in the normal human urothelium; and in transposed intestinal segments. MATERIALS AND METHODS: Non-isotopic in-situ hybridization was carried out using eight digoxigenin labeled oligonucleotide mucin gene probes (MUC 1 - 7). Immunohistochemistry using NCL-MUC1 and NCL-MUC2 monoclonal antibodies was performed on sections of paraffin-embedded tissues. Twenty-seven patients were investigated (normal human urothelium, n = 6; transposed ileal segments, n = 14 and normal ileal controls, n = 7). RESULTS: MUC1 and MUC4 were the predominant mucin genes expressed in the normal urothelium with MUC3 being expressed in a third of cases studied; MUC2, 5AC, 5B, 6 and 7 were not expressed. Despite the morphological changes seen in transposed ileal segments, MUC2 and MUC3 continued to be expressed in these segments albeit in a disorganised fashion. Both MUC1 and MUC4 were up-regulated in transposed ileal segments, genes expressed by the normal human urothelium. All eight mucin genes were expressed in an area of pyloric-type metaplasia found in one transposed ileal segment. In patients with clam enterocystoplasty there was evidence of increasing up-regulation of MUC2, 3, 4 and 5AC expression in the urothelium toward the anastomotic site. CONCLUSION: Transposition of ileal segments into the urinary tract results in up-regulation of MUC1 and MUC4, the predominant MUC genes expressed in the human bladder. The clinical implication of the up-regulation of some MUC genes toward the anastomotic site in patients with an enteroplasty and the aberrant expression of MUC5AC - MUC7 by transposed segments is at present unclear.

The tissue effect of argon plasma coagulation on esophageal and gastric mucosa.

BACKGROUND: Argon plasma coagulation is a diathermy-based non-contact therapeutic endoscopic modality that may have a lower risk of perforation than other tissue ablation techniques. METHODS: Its effect was studied on three fresh esophageal and three fresh gastric resection specimens using power settings from 40 to 99 Watts at 90 degrees, with 1 mm separation using pulse durations of 1 and 3 seconds. A scoring system for depth of tissue damage was created and samples were analyzed blindly by a gastrointestinal histopathologist. RESULTS: There was significantly greater damage to gastric tissue using a 3-second (compared with 1-second) pulse (p = 0.003) and marginally significantly greater damage to esophageal tissue using the 3-second pulse (p = 0.053). Tissue damage was related to power setting for gastric (p = 0.031) but not for esophageal tissue (p = 0. 065). Only 1 of 42 esophageal samples and 2 of 42 gastric samples examined showed damage extending into the muscularis propria. CONCLUSIONS: Deep tissue damage that could lead to perforation was rare with argon plasma coagulation. The depth of gastric mucosal damage increased with increased pulse duration and increasing power settings, and, although the depth of esophageal mucosal damage was marginally related to pulse duration, it was not related to the power setting. (Gastrointest Endosc 2000;52:342-5).

Retroviral delivery of peptide modulators of cellular functions.

Stable transduction of genetic material, in combination with sensitive methodologies for in vivo study of cell physiology, provides an opportunity to efficiently evaluate the functions of regulatory proteins. To dissect the minimal therapeutic function of such proteins, we have stably expressed protein microdomains as fusions, composed of short peptides, and detected specific subfunctions distinct from holoprotein function, using flow cytometry and other techniques. We demonstrate that retroviral delivery of the 24-amino-acid proliferating cell nuclear antigen-binding motif (p21C), derived from the C-terminus of the cell cycle inhibitor protein, p21, is sufficient to induce cell cycle arrest. Cells expressing this peptide motif reversibly execute both G1- and G2-checkpoint controls that are normally activated subsequent to interference with DNA synthesis. The p21C effect is distinct from results obtained with an intact p21 protein that also binds cyclin-CDK complexes and arrested cells exclusively at the G1/S transition. Thus, microdomains can exert unique biological effects compared to the parental molecules from which they were derived. To further evaluate the peptide delivery strategy, we analyzed the role of various kinases in IgE-mediated stimulation of mast cell exocytosis. Primary bone marrow-derived mast cells were transduced with retroviral constructs encoding short-kinase inhibitor motifs and analyzed by flow cytometry for effects on exocytosis. We found that a specific protein kinase A (PKA) inhibitor peptide suppressed IgE-mediated stimulation of mast cell exocytosis. This anti-exocytotic effect was mimicked by a small molecule inhibitor of PKA (KT5720). Thus, the ability to express protein microdomains can be a powerful means to subtly perturb cellular physiology in manners that reveal new paths for therapeutic intervention. We believe that such approaches might allow for new forms of gene therapy to become available.

Nutrient regulation of gene expression by the sterol regulatory element binding proteins: increased recruitment of gene-specific coregulatory factors and selective hyperacetylation of histone H3 in vivo.

We have evaluated the mechanism for sterol-regulated gene expression by the sterol regulatory element binding proteins (SREBPs) in intact cells. We show that activation of SREBPs by sterol depletion results in the increased binding of Sp1 to a site adjacent to SREBP in the promoter for the low density lipoprotein (LDL) receptor gene in vivo. Similarly, sterol depletion resulted in the increased recruitment of two distinct SREBP coregulatory factors, NF-Y and CREB, to the promoter for hydroxymethyl glutaryl CoA reductase, another key gene of intracellular cholesterol homeostasis. Furthermore, increased acetylation of histone H3 but not H4 was also detected in chromatin from both promoters on SREBP activation. Thus, SREBP activation results in the similar selective recruitment of different coregulatory generic transcription factors to two separate cholesterol-regulated promoters. These studies demonstrate the utility of the chromatin immunoprecipitation technique for analyzing the differential action of low-abundance transcription factors in fundamental regulatory events in intact cells. Our results also provide key in vivo support for the mechanism proposed from cell-free experiments, where SREBP increased the binding of Sp1 to the LDL receptor promoter. Finally, our findings also indicate that subtle differences in the pattern of core histone acetylation play a role in selective gene activation.

Rab37 is a novel mast cell specific GTPase localized to secretory granules.

GTPases regulate a myriad of cellular functions including signal transduction, cytoskeletal organization and membrane trafficking. Rab GTPases act to coordinate the membrane dynamics of cells by organizing and regulating the activity of effector proteins important in vesicle trafficking. Rab37 is a novel Rab GTPase specifically expressed in the MC-9 mast cell line and bone marrow mast cells. Rab37 is 74% identical to Rab26 and 47% identical to Rab8, a GTPase important in Golgi to plasma membrane vesicle trafficking in mammalian cells. When green fluorescent protein tagged Rab37 is expressed in bone marrow mast cells, the secretory granules are labeled. These data suggest that Rab37 may play an important role in mast cell degranulation making this protein a potentially important target for therapeutic intervention in the treatment of allergy.

Syntaxin 2 splice variants exhibit differential expression patterns, biochemical properties and subcellular localizations.

The syntaxins are a large protein family implicated in the targeting and fusion of intracellular transport vesicles. A subset of proteins of this family are the four syntaxin 2 splice variants, syntaxins 2A (2), 2B (2'), 2C (2") and 2D. Each syntaxin 2 variant contains an identical, or nearly identical, amino-terminal cytoplasmic domain followed by a distinct hydrophobic (syntaxins 2A and 2B) or hydrophilic (syntaxins 2C and 2D) carboxyl-terminal domain. To investigate whether the difference among the syntaxin 2 variants is functionally important, we have examined comparatively their RNA transcript and protein expression patterns, membrane associations, protein-protein interactions and intracellular localizations. Analysis of the RNA transcript and protein expression patterns demonstrated that syntaxins 2A, 2B and 2C are broadly, but not uniformly, expressed while syntaxin 2D expression is restricted to the brain. Subcellular fractionation studies showed that syntaxins 2A and 2B behave as integral membrane proteins while syntaxin 2C is only partially associated with membranes. In vitro biochemical assays demonstrated that the syntaxin 2 variants exhibit similar yet distinct interactions with other proteins implicated in vesicular trafficking, including SNAP-25, SNAP-23, VAMP-2 and n-sec1. In a variety of nonpolarized cell types, syntaxins 2A and 2B localized to both the plasma membrane and endosomal membranes. However, in two polarized epithelial cell lines, MDCK and Caco-2, syntaxin 2A localized predominantly to the apical plasma membrane while syntaxin 2B was associated with both the apical and the basolateral membranes. These observations indicate that the distinct carboxyl-terminal domains of the syntaxin 2 variants influence their biochemical and localization properties and may therefore confer upon these variants different functional roles in the regulation of intracellular membrane trafficking.

Systemic and mucosal immunity is elicited after both intramuscular and intravaginal delivery of human immunodeficiency virus type 1 DNA plasmid vaccines to pregnant chimpanzees.

DNA vaccines encoding human immunodeficiency virus type 1 (HIV-1) env/rev and gag/pol were delivered intravaginally (IVAG) and intramuscularly (IM) to 2 pregnant chimpanzees. Vaccination was well tolerated and each chimpanzee developed antibodies (up to 1 year later) to both vaccines. Placental transfer of anti-Env and anti-Gag IgG was demonstrated in both maternal/infant pairs. Specific IgG was also demonstrated in saliva, vaginal, and rectal washes after IVAG immunization. Predominantly anti-HIV-1 IgA was detected in the milk of both mothers after both IM and IVAG immunization. Cellular responses included Gag-specific proliferation of lymphocytes and cytotoxic T lymphocytes against both antigens. These data suggest a strategy for induction of mucosal and systemic responses after both IM and IVAG delivery of DNA vaccines in a primate model and could ultimately be useful in lowering maternal-to-fetal transmission of HIV-1, perinatally and through breastfeeding.

Co-stimulation of promoter for low density lipoprotein receptor gene by sterol regulatory element-binding protein and Sp1 is specifically disrupted by the yin yang 1 protein.

Sterol regulation of gene expression in mammalian cells is mediated by an interaction between the cholesterol-sensitive sterol regulatory element-binding proteins (SREBPs) and promoter-specific but generic co-regulatory transcription factors such as Sp1 and NF-Y/CBF. Thus, sterol-regulated promoters that require different co-regulatory factors could be regulated independently through targeting the specific interaction between the SREBPs and the individual co-regulatory proteins. In the present studies we demonstrate that transiently expressed yin yang 1 protein (YY1) inhibits the SREBP-mediated activation of the low density lipoprotein (LDL) receptor in a sensitive and dose-dependent manner. The inhibition is independent of YY1 binding directly to the LDL receptor promoter, and we show that the same region of YY1 that interacts in solution with Sp1 also interacts with SREBP. Furthermore, other SREBP-regulated genes that are not co-regulated by Sp1 are either not affected at all or are not as sensitive to the repression. Thus, the specific interaction that occurs between SREBPs and Sp1 to stimulate the LDL receptor promoter is a specific target for inhibition by the YY1 protein, and we provide evidence that the mechanism can be at least partially explained by the ability of YY1 to inhibit the interaction between SREBP and Sp1 in solution in vitro. The LDL receptor is the key gene of cholesterol uptake, and the rate-controlling genes of cholesterol synthesis are stimulated by the concerted action of SREBPs along with coregulators that are distinct from Sp1. Therefore, repression of gene expression through specifically targeting the interaction between SREBP and Sp1 would provide a molecular mechanism to explain how cholesterol uptake can be regulated independently from cholesterol biosynthesis in mammalian cells.

Correlation between spiral computed tomography, endoscopic ultrasonography and findings at operation in pancreatic and ampullary tumours.

BACKGROUND: Spiral computed tomography (CT) allows high-resolution examination of the pancreas, surrounding vascular structures, lymph nodes and liver. Endoscopic ultrasonography (EUS) also allows high-resolution imaging of the pancreas and adjacent structures but is an invasive procedure. With the availability of spiral CT, the role of EUS in the investigation of patients with suspected pancreatic or ampullary tumours is unclear. METHODS: Forty-eight patients with clinical suspicion of a pancreatic or ampullary tumour underwent both spiral CT and EUS. Thirty-four patients had surgical exploration, of whom 17 underwent pancreatic resection and 17 had biliary and gastric bypass. The results of spiral CT and EUS were compared with the operative findings. RESULTS: The final histological diagnosis was ductal adenocarcinoma (24 patients), ampullary carcinoma (six), serous cystadenoma (two) and chronic pancreatitis (two). EUS demonstrated 33 and spiral CT 26 of the 34 primary lesions. EUS was particularly useful in the assessment of small resectable tumours missed by spiral CT. The sensitivity and specificity of EUS and spiral CT for detecting involvement by the tumour of the superior mesenteric vein, portal vein and lymph nodes were similar, but EUS was less effective at evaluating the superior mesenteric artery. CONCLUSION: EUS is an important additional investigation after spiral CT in patients with a suspected pancreatic or ampullary tumour.