Stephen A. Wank

T Cells Targeting Carcinoembryonic Antigen Can Mediate Regression of Metastatic Colorectal Cancer but Induce Severe Transient Colitis

Autologous T lymphocytes genetically engineered to express a murine T cell receptor (TCR) against human carcinoembryonic antigen (CEA) were administered to three patients with metastatic colorectal cancer refractory to standard treatments. All patients experienced profound decreases in serum CEA levels (74-99%), and one patient had an objective regression of cancer metastatic to the lung and liver. However, a severe transient inflammatory colitis that represented a dose limiting toxicity was induced in all three patients. This report represents the first example of objective regression of metastatic colorectal cancer mediated by adoptive T cell transfer and illustrates the successful use of a TCR, raised in human leukocyte antigen (HLA) transgenic mice, against a human tumor associated antigen. It also emphasizes the destructive power of small numbers of highly avid T cells and the limitations of using CEA as a target for cancer immunotherapy.Autologous T lymphocytes genetically engineered to express a murine T cell receptor (TCR) against human carcinoembryonic antigen (CEA) were administered to three patients with metastatic colorectal cancer refractory to standard treatments. All patients experienced profound decreases in serum CEA levels (74-99%), and one patient had an objective regression of cancer metastatic to the lung and liver. However, a severe transient inflammatory colitis that represented a dose limiting toxicity was induced in all three patients. This report represents the first example of objective regression of metastatic colorectal cancer mediated by adoptive T cell transfer and illustrates the successful use of a TCR, raised in human leukocyte antigen (HLA) transgenic mice, against a human tumor associated antigen. It also emphasizes the destructive power of small numbers of highly avid T cells and the limitations of using CEA as a target for cancer immunotherapy.

Molecular cloning of the human brain and gastric cholecystokinin receptor: Structure, functional expression and chromosomal localization☆

The receptors for the brain and gastrointestinal peptide, cholecystokinin, can be classified into CCKA and CCKB subtypes. Having recently cloned the rat CCKB receptor, we used its cDNA to isolate the human CCKB receptor homologue from brain and stomach which encodes a 447 amino acid protein with 90% identity to both rat CCKB and canine gastrin receptors. Northern hybridization identifies transcripts from stomach, pancreas, brain and gallbladder. The CCKB receptor gene maps to chromosome 11. Expression of the receptor cDNA in COS-7 cells was characteristic of a CCKB receptor subtype pharmacology. These data confirm that we have cloned a novel gene for the human brain and stomach CCKB receptor.

The G-protein-coupled receptor GPR40 directly mediates long-chain fatty acid-induced secretion of cholecystokinin.

BACKGROUND & AIMS Long-chain fatty acid receptors G-protein-coupled receptor 40 (GPR40) (FFAR1) and GPR120 have been implicated in the chemosensation of dietary fats. I cells in the intestine secrete cholecystokinin (CCK), a peptide hormone that stimulates digestion of fat and protein, but these cells are rare and hard to identify. We sought to determine whether dietary fat-induced secretion of CCK is directly mediated by GPR40 expressed on I cells. METHODS We used fluorescence-activated cell sorting to isolate a pure population of I cells from duodenal mucosa in transgenic mice that expressed green fluorescent protein under the control of the CCK promoter (CCK-enhanced green fluorescent protein [eGFP] bacterial artificial chromosome mice). CCK-eGFP cells were evaluated for GPR40 expression by quantitative reverse transcription polymerase chain reaction and immunostaining. GPR40(-/-) mice were bred with CCK-eGFP mice to evaluate functional relevance of GPR40 on long-chain fatty acid-stimulated increases in [Ca(2+)]i and CCK secretion in isolated CCK-eGFP cells. Plasma levels of CCK after olive oil gavage were compared between GPR40(+/+) and GPR40(-/-) mice. RESULTS Cells that expressed eGFP also expressed GPR40; expression of GPR40 was 100-fold greater than that of cells that did not express eGFP. In vitro, linoleic, oleic, and linolenic acids increased [Ca(2+)]i; linolenic acid increased CCK secretion by 53% in isolated GPR40(+/+) cells that expressed eGFP. In contrast, in GPR40(-/-) that expressed eGFP, [Ca(2+)]i response to linoleic acid was reduced by 50% and there was no significant CCK secretion in response to linolenic acid. In mice, olive oil gavage significantly increased plasma levels of CCK compared with pregavage levels: 5.7-fold in GPR40(+/+) mice and 3.1-fold in GPR40(-/-) mice. CONCLUSIONS Long-chain fatty acid receptor GPR40 induces secretion of CCK by I cells in response to dietary fat.

Cloning and Characterization of the Signal Transduction of Four Splice Variants of the Human Pituitary Adenylate Cyclase Activating Polypeptide Receptor EVIDENCE FOR DUAL COUPLING TO ADENYLATE CYCLASE AND PHOSPHOLIPASE C

Alternative splicing of two exons of the rat pituitary adenylate cyclase activating polypeptide (PACAP) receptor gene generates four major splice variants that are differentially expressed in specific tissues and variably coupled to intracellular second messengers. To evaluate the potential implications of these findings in human physiology, the human PACAP receptor gene was cloned. Alternative splicing about two exons of the gene allowed for four major splice variants that were subsequently identified on cDNA cloning. Each of the four splice variant cDNAs (null, SV-1, SV-2, and SV-3) was stably expressed in NIH/3T3 cells at similar receptor densities. For each splice variant, PACAP (both PACAP-38 and PACAP-27) had similar affinity and potency for stimulating either adenylate cyclase or phospholipase C. However, each receptor splice variant differed in their ligand-stimulated maximal response (efficacy) for total inositol phosphate accumulation with the SV-2 showing the greatest efficacy, followed by the null, SV-1, and SV-3 splice variants. Therefore, unlike the rat, PACAP binds and stimulates signal transduction with nearly equal affinity and potency for each of the receptor splice variants although with varying efficacy for the stimulation of phospholipase C. These results suggest a novel and potentially important mechanism for a single hormone to not only couple to dual signal transduction cascades but also elicit tissue-specific differential activation of phospholipase C in humans.

The extracellular calcium sensing receptor is required for cholecystokinin secretion in response to L-phenylalanine in acutely isolated intestinal I cells

The extracellular calcium-sensing receptor (CaSR) has recently been recognized as an L-amino acid sensor and has been implicated in mediating cholecystokinin (CCK) secretion in response to aromatic amino acids. We investigated whether direct detection of L-phenylalanine (L-Phe) by CaSR results in CCK secretion in the native I cell. Fluorescence-activated cell sorting of duodenal I cells from CCK-enhanced green fluorescent protein (eGFP) transgenic mice demonstrated CaSR gene expression. Immunostaining of fixed and fresh duodenal tissue sections confirmed CaSR protein expression. Intracellular calcium fluxes were CaSR dependent, stereoselective for L-Phe over D-Phe, and responsive to type II calcimimetic cinacalcet in CCK-eGFP cells. Additionally, CCK secretion by an isolated I cell population was increased by 30 and 62% in response to L-Phe in the presence of physiological (1.26 mM) and superphysiological (2.5 mM) extracellular calcium concentrations, respectively. While the deletion of CaSR from CCK-eGFP cells did not affect basal CCK secretion, the effect of L-Phe or cinacalcet on intracellular calcium flux was lost. In fact, both secretagogues, as well as superphysiological Ca(2+), evoked an unexpected 20-30% decrease in CCK secretion compared with basal secretion in CaSR(-/-) CCK-eGFP cells. CCK secretion in response to KCl or tryptone was unaffected by the absence of CaSR. The present data suggest that CaSR is required for hormone secretion in the specific response to L-Phe by the native I cell, and that a receptor-mediated mechanism may inhibit hormone secretion in the absence of a fully functional CaSR.

Prospective Study of the Ability of Computed Axial Tomography to Localize Gastrinomas in Patients With Zollinger-Ellison Syndrome

The ability of routine computed tomography (CT) performed with oral and intravenous contrast to localize gastrinomas in 61 consecutive patients with Zollinger-Ellison syndrome was evaluated prospectively. The results of CT scanning were subsequently evaluated in all patients by either surgery, autopsy, or percutaneous biopsy. Thirteen of 14 patients with CT scans positive for hepatic metastases and 5 of 13 patients with CT scans negative for hepatic metastases were found to have gastrinoma in the liver. For gastrinoma metastatic to the liver, CT scanning had a specificity of 98%, a sensitivity of 72%, a positive predictive value of 93%, and a negative predictive value of 90%. Twenty-two of 23 patients with positive extrahepatic CT scans and 15 of 33 patients with negative extrahepatic CT scans were found to have extrahepatic gastrinomas. For extrahepatic gastrinoma, CT scanning had a specificity of 95%, a sensitivity of 59%, a positive predictive value of 96%, and a negative predictive value of 54%. The ability of CT scan to detect gastrinomas both in the liver and extrahepatically was directly related to tumor size, detecting 0% of tumors less than 1 cm and 83%-95% of tumors greater than 3 cm. The location of the extrahepatic gastrinoma was also an important determinant in that approximately 80% of pancreatic gastrinomas but only 35% of extrapancreatic gastrinomas were detected. The present results indicate that because of its convenience and accuracy, CT scanning with oral and intravenous contrast material should be the initial procedure to evaluate the extent of gastrinoma. A positive CT scan is almost always correct; therefore, a CT scan detecting metastatic gastrinoma to the liver would avoid unnecessary surgery and, if positive for extrahepatic gastrinoma, would assist the surgeon in finding the gastrinoma. A negative CT is less reliable; therefore, patients should undergo other localizing studies before exploratory laparotomy.

Prospective Study of Chemotherapy in Patients With Metastatic Gastrinoma

Ten consecutive patients with metastatic gastrinoma that increased in size over time were studied prospectively during treatment with monthly cycles of streptozotocin (3 g/m2), 5-fluorouracil (1.2 g/m2), and adriamycin (40 mg/m2) to determine the response rate and time-courses of changes during chemotherapy and to assess various methods of evaluating the effect of chemotherapy. Forty percent of patients demonstrated an initial objective response (greater than or equal to 25% decrease in tumor size with no new lesions) and 60% failed chemotherapy (greater than or equal to 25% increase in tumor size or appearance of new lesions). The mean dose of streptozotocin was 27 g/m2 with objective responses occurring at 3.7 +/- 0.7 mo and failures at 4.5 +/- 0.7 mo. Responses lasted 9.7 +/- 2.8 cycles and no complete responses occurred. Survival was not significantly different in responders versus nonresponders (26 +/- 11 vs. 15 +/- 4.8 mo, p greater than 0.1). Changes in serum gastrin concentration, basal acid output, or sensitivity to a given dose of histamine H2-receptor antagonist did not reflect changes in tumor size. Computed tomography and angiography were the best methods to assess changes in tumor size during chemotherapy, whereas liver-spleen scan and ultrasound were relatively insensitive. All patients developed side effects with chemotherapy: 100% had vomiting, 80% alopecia, 40% transient proteinuria, and 20% leukopenia. The present results indicate that chemotherapy with streptozotocin, 5-fluorouracil, and adriamycin is much less effective in patients with extensive metastatic gastrinoma than previously reported. Computed tomography scanning is the method of choice to assess changes in tumor size. Changes in serum gastrin concentration, acid secretion, or tumor size assessed by liver-spleen scan or ultrasound are not sensitive indicators of the tumor response during chemotherapy.

Role of Selective Angiography in the Management of Patients With Zollinger-Ellison Syndrome

To determine the ability of selective abdominal angiography to localize gastrinoma in patients with Zollinger-Ellison syndrome, selective angiography was performed in 70 consecutive patients and the results were assessed prospectively by either surgery, autopsy, or percutaneous biopsy. In addition, to define the role of angiography in the management of patients with gastrinoma, we compared the results of angiography with those of computed tomography (CT) scanning in 58 patients who underwent both tests. For gastrinoma in the liver, angiography had a specificity of 100% and a sensitivity of 86% with a positive predictive value of 100% and a negative predictive value of 94%. For extrahepatic gastrinoma, angiography had a specificity of 94% and a sensitivity of 68%, a positive predictive value of 97% and a negative predictive value of 53%. Comparison of CT scanning and angiography demonstrated that for hepatic tumor CT demonstrated 72% and angiography 89% of tumors, and the combination detected all tumors with no false-positive results. Outside the liver, CT scanning detected 57%, angiography 70%, and the combination 73% of tumors with a false-positive rate of 7%. These results indicate that if a CT scan is performed first, then the addition of selective angiography will detect a further 28% of hepatic tumors and a further 16% of extrahepatic tumors, but that 24% of extrahepatic tumors will still be missed. Angiography is a useful adjunct to CT particularly in patients in whom surgery is contemplated.

Medical management of patients with Zollinger-Ellison syndrome who have had previous gastric surgery: A prospective study

We examined prospectively the criteria for medical management in 16 patients with Zollinger-Ellison syndrome who had had previous gastric surgery. Each patient received sufficient antisecretory medication to lower gastric acid output to less than 10 mEq/h during the last hour before the next dose of drug. The 7 patients with a vagotomy but no gastric resection were symptom-free and had no mucosal disease. Of 9 patients with a partial gastrectomy, 7 had mucosal disease, with or without symptoms, and 6 of the 7 patients had acid outputs of 5-10 mEq/h. In these patients, antisecretory medication was increased to reduce output to less than 5 mEq/h and symptoms and mucosal abnormalities resolved in each patient. Patients with Zollinger-Ellison syndrome and a vagotomy can be treated safely by reducing acid secretion to less than 10 mEq/h, but in patients with a partial gastrectomy, acid secretion must be reduced to less than 5 mEq/h, and adequacy of therapy must be checked further by endoscopy.

Calcium-sensing receptor is a physiologic multimodal chemosensor regulating gastric G-cell growth and gastrin secretion

The calcium-sensing receptor (CaR) is the major sensor and regulator of extracellular Ca2+, whose activity is allosterically regulated by amino acids and pH. Recently, CaR has been identified in the stomach and intestinal tract, where it has been proposed to function in a non-Ca2+ homeostatic capacity. Luminal nutrients, such as Ca2+ and amino acids, have been recognized for decades as potent stimulants for gastrin and acid secretion, although the molecular basis for their recognition remains unknown. The expression of CaR on gastrin-secreting G cells in the stomach and their shared activation by Ca2+, amino acids, and elevated pH suggest that CaR may function as the elusive physiologic sensor regulating gastrin and acid secretion. The genetic and pharmacologic studies presented here comparing CaR-null mice and wild-type littermates support this hypothesis. Gavage of Ca2+, peptone, phenylalanine, Hepes buffer (pH 7.4), and CaR-specific calcimimetic, cinacalcet, stimulated gastrin and acid secretion, whereas the calcilytic, NPS 2143, inhibited secretion only in the wild-type mouse. Consistent with known growth and developmental functions of CaR, G-cell number was progressively reduced between 30 and 90 d of age by more than 65% in CaR-null mice. These studies of nutrient-regulated G-cell gastrin secretion and growth provide definitive evidence that CaR functions as a physiologically relevant multimodal sensor. Medicinals targeting diseases of Ca2+ homeostasis should be reviewed for effects outside traditional Ca2+-regulating tissues in view of the broader distribution and function of CaR.