Treatment with the TGF-b superfamily cytokine MIC-1/GDF15 reduces the adiposity and corrects the metabolic dysfunction of mice with diet-induced obesity.

OBJECTIVES: To test the potential efficacy of recombinant macrophage inhibitory cytokine-1 (MIC-1/GDF15) as an obesity therapeutic. METHODS: Male C57BL/6 J mice, either fed on normal chow or high-fat diet for 16 weeks to induce diet-induced obesity, were infused with either recombinant MIC-1/GDF15 or vehicle for 34 days by osmotic minipump. During the experimental period metabolic parameters were measured. Blood and tissue were collected for analysis of inflammatory markers. RESULTS: MIC-1/GDF15 decreased food intake and body weight of high-fat-fed and chow-fed mice compared with their vehicle-treated control mice. MIC-1/GDF15 reduced body weight, accompanied by greater reduction in fat mass in high-fat-fed mice compared to its effect on chow-fed mice. Further, whilst MIC-1/GDF15-treated chow-fed mice lost lean as well as fat mass, MIC-1/GDF15-treated high-fat-fed mice lost fat mass alone. This reduction in body weight and adiposity was due largely to reduced food intake, but MIC-1/GDF15-treated high-fat-fed mice also displayed increased energy expenditure that may be due to increased thermogenesis. MIC-1/GDF15-treated high-fat-fed mice also had higher circulating level of adiponectin and lower tissue expression, and circulating levels of leptin and inflammatory mediators associated with insulin resistance. Peripheral insulin and glucose intolerance were improved in both MIC-1/GDF15-treated high-fat-fed and chow-fed mice compared to that of their vehicle-treated control mice. CONCLUSIONS: MIC-1/GDF15 is highly effective in reducing adiposity and correcting the metabolic dysfunction of mice with high-fat fed. These studies suggest that MIC-1/GDF15 may be a candidate anti-obesity therapeutic.

Macrophage inhibitory cytokine-1/growth differentiation factor-15 as a predictor of colonic neoplasia.

BACKGROUND: Serum macrophage inhibitory cytokine-1 (MIC-1/GDF15) concentration has been associated with colonic adenomas and carcinoma. AIMS: To determine whether circulating MIC-1/GDF15 serum concentrations are higher in the presence of adenomas and whether the level decreases after excision. METHODS: Patients were recruited prospectively from a single centre and stratified into five groups: no polyps (NP); hyperplastic polyps (HP); sessile serrated ademona (SSA); adenomas (AP); and colorectal carcinoma (CRC). Blood samples were collected immediately before and 4 weeks after colonoscopy. MIC-1/GDF15 serum levels were quantified using ELISA. RESULTS: Participants (n=301) were stratified as: NP; n=116 (52%), HP; n=37 (12%), SSA; n=19 (7%), AP; n=68 (23%); and CRC; n=3 (1%). Patients were excluded from the study due to nondiagnostic pathology (n=9, 3%) and exclusion criteria (n=20, 6%). In the 272 remaining subjects (M=149; F=123), age (P=.005), history of colonic polyps (P=.003) and family history of colonic polyps (P=.002) were associated with presence of adenomas. Baseline median MIC-1/GDF15 serum levels increased significantly from NP 609 (460-797) pg/mL, HP 582 (466-852) pg/mL, SSA 561 (446-837) pg/mL to AP 723 (602-1122) pg/mL and CRC 1107 (897-1107) pg/mL; (P<.001). In the pre- and postpolypectomy paired adenoma samples median MIC-1/GDF15 reduced significantly from 722 (603-1164) pg/mL to 685 (561-944) pg/mL (P=.002). A ROC analysis for serum MIC-1/GDF15 to identify adenomatous polyps indicated an area under the curve of 0.71. CONCLUSIONS: Our data suggest that serum MIC-1/GDF15 has the diagnostic characteristics to increase the detection of colonic neoplasia and improve screening.

Anorexia-cachexia and obesity treatment may be two sides of the same coin: role of the TGF-b superfamily cytokine MIC-1/GDF15.

Anorexia-cachexia associated with cancer and other diseases is a common and often fatal condition representing a large area of unmet medical need. It occurs most commonly in advanced cancer and is probably a consequence of molecules released by tumour cells, or tumour-associated interstitial or immune cells. These may then act directly on muscle to cause atrophy and/or may cause anorexia, which then leads to loss of both fat and lean mass. Although the aetiological triggers for this syndrome are not well characterized, recent data suggest that MIC-1/GDF15, a transforming growth factor-beta superfamily cytokine produced in large amounts by cancer cells and as a part of other disease processes, may be an important trigger. This cytokine acts on feeding centres in the hypothalamus and brainstem to cause anorexia leading to loss of lean and fat mass and eventually cachexia. In animal studies, the circulating concentrations of MIC-1/GDF15 required to cause this syndrome are similar to those seen in patients with advanced cancer, and at least some epidemiological studies support an association between MIC-1/GDF15 serum levels and measures of nutrition. This article will discuss its mechanisms of central appetite regulation, and the available data linking this action to anorexia-cachexia syndromes that suggest it is a potential target for therapy of cancer anorexia-cachexia and conversely may also be useful for the treatment of severe obesity.

Modeling of subglacial hydrological development following rapid supraglacial lake drainage.

The rapid drainage of supraglacial lakes injects substantial volumes of water to the bed of the Greenland ice sheet over short timescales. The effect of these water pulses on the development of basal hydrological systems is largely unknown. To address this, we develop a lake drainage model incorporating both (1) a subglacial radial flux element driven by elastic hydraulic jacking and (2) downstream drainage through a linked channelized and distributed system. Here we present the model and examine whether substantial, efficient subglacial channels can form during or following lake drainage events and their effect on the water pressure in the surrounding distributed system. We force the model with field data from a lake drainage site, 70 km from the terminus of Russell Glacier in West Greenland. The model outputs suggest that efficient subglacial channels do not readily form in the vicinity of the lake during rapid drainage and instead water is evacuated primarily by a transient turbulent sheet and the distributed system. Following lake drainage, channels grow but are not large enough to reduce the water pressure in the surrounding distributed system, unless preexisting channels are present throughout the domain. Our results have implications for the analysis of subglacial hydrological systems in regions where rapid lake drainage provides the primary mechanism for surface-to-bed connections. KEY POINTS: Model for subglacial hydrological analysis of rapid lake drainage eventsLimited subglacial channel growth during and following rapid lake drainagePersistence of distributed drainage in inland areas where channel growth is limited.

The interaction of serum testosterone levels and androgen receptor CAG repeat polymorphism on the risk of erectile dysfunction in aging Taiwanese men.

Testosterone has been found to play important roles in men's sexual function. However, the effects of testosterone can be modulated by androgen receptor (AR) CAG repeat polymorphism. It could also contribute to the risk of erectile dysfunction (ED). The aim of this study is to evaluate the interaction of serum testosterone levels and AR CAG repeat polymorphism on the risk of ED in aging Taiwanese men. This cross-sectional data of Taiwanese men older than 40 years were collected from a free health screening held between August 2010 and August 2011 in Kaohsiung city, Taiwan. All participants completed a health questionnaires included five-item version of the International Index of Erectile Function (IIEF-5) and the International Prostate Symptoms Score, received a detailed physical examination and provided 20 cm3 whole blood samples for biochemical and genetic evaluation. The IIEF-5 was used to evaluate ED. Serum albumin, total testosterone (TT), and sex hormone-binding globulin levels were measured. Free testosterone level was calculated. AR gene CAG repeat polymorphism was determined by direct sequencing. Finally, 478 men with the mean age of 55.7 ± 4.8 years were included. When TT levels were above 330 ng/dL, the effect of testosterone level on erectile function seemed to reach a plateau and a significantly negative correlation between AR CAG repeat length and the score of IIEF-5 was found (r = -0.119, p = 0.034). After adjusting for other covariates, the longer AR CAG repeat length was still an independent risk factor for ED in subjects with TT above 330 ng/dL (p = 0.006), but not in TT of 330 ng/dL or below. In conclusion, both serum testosterone levels and AR CAG repeat polymorphism can influence erectile function concomitantly. In subjects with normal TT concentration, those with longer AR CAG repeat lengths have a higher risk of developing ED.

The role of sympathetic and parasympathetic nerve systems on the smooth muscle of rat seminal vesicles - experimental results and speculation for physiological implication on ejaculation.

Ejaculation is a process involving sympathetic and parasympathetic effects during different stages - emission and ejection. Some conditions of ejaculation dysfunction are associated with autonomic nerves. However, the exact effects of autonomic nerves on ejaculation are not well defined. Autonomic agonists induce different recorded trace patterns of seminal vesicular contraction. The different traces contain different components of phasic and tonic contraction, which may have physiological implications. In this study, we examined isolated rat seminal vesicle (SV) contraction by phenylephrine (PE), acetylcholine, and their respective antagonists and then speculated upon physiological roles of sympathetic and parasympathetic nerves on SV during ejaculation. We found that PE and Ach both achieved good contraction of rat SV. Compared to α1b for sympathetic and M1, M2 for parasympathetic receptors, α1a and M3 are the relatively dominant subtypes on rat SV. Adrenergic and cholinergic agonists cause different trace patterns of SV contraction. We speculated that the sympathetic effect is dominant during emission to squeeze seminal fluid out and that the parasympathetic effect is dominant during ejection to provide an anti-reflux effect on the ejaculatory duct.

Earthquake in a maze: compressional rupture branching during the 2012 M(w) 8.6 Sumatra earthquake.

Seismological observations of the 2012 moment magnitude 8.6 Sumatra earthquake reveal unprecedented complexity of dynamic rupture. The surprisingly large magnitude results from the combination of deep extent, high stress drop, and rupture of multiple faults. Back-projection source imaging indicates that the rupture occurred on distinct planes in an orthogonal conjugate fault system, with relatively slow rupture speed. The east-southeast-west-northwest ruptures add a new dimension to the seismotectonics of the Wharton Basin, which was previously thought to be controlled by north-south strike-slip faulting. The rupture turned twice into the compressive quadrant, against the preferred branching direction predicted by dynamic Coulomb stress calculations. Orthogonal faulting and compressional branching indicate that rupture was controlled by a pressure-insensitive strength of the deep oceanic lithosphere.

Evaluation of cost effectiveness for conservative and active management strategies for acoustic neuroma.

OBJECTIVE: To determine whether economic costs associated with a conservative management strategy for unilateral acoustic neuroma offer an economic advantage over active management options. DESIGN: Cost and sensitivity analysis on a prospective cohort. SETTING: Single centre study within a tertiary referral centre in Toronto, Canada. PARTICIPANTS: 72 patients (32 males, 40 females) aged 36 to 78 years with unilateral acoustic neuroma were assigned initially to a conservative management strategy. Entry criteria were small tumour size (less than 15 mm in the cerebellopontine angle), patient preference and/or significant co-morbidity. INTERVENTIONS: MRI scanning was performed every 6 months for the first year, annually subsequently and then every 2-3 years as required. Clinical review occurred every 6 months. Failure of conservative management led to active treatment. MAIN OUTCOME MEASURES: Cost analysis was performed to determine the mean total cost per patient for continued conservative management over the follow up period compared to the mean upfront total cost per patient undergoing active intervention. RESULTS: Cost analysis within the Canadian health care system determined the mean total cost per patient for microsurgical removal at CAD$22,402 (12,545 pounds; 14,561 euros), for gamma knife radiotherapy at CAD$27659 (15,489 pounds; 17,978 euros), for LINAC radiotherapy at CAD$9,003(5,041 pounds; 5,852 euros) and for conservative management at CAD$9,651 (5,405 pounds;6,273 euros) over the follow-up timeframe. CONCLUSION: An economic advantage can be demonstrated for the conservative management of acoustic neuromas compared to microsurgical removal and gamma knife radiotherapy on the proviso that no increase in active treatment complications arose from continued tumour growth during the period of observation.

The effects of alanine-substituted conantokin-G and ifenprodil on the human spermine-activated N-methyl-D-aspartate receptor.

We evaluated the effects of Ala-7-conantokin-G (Con-G(A7)) and ifenprodil on the modulation by spermine of [(3)H]MK801 binding to human cortical membranes. Human cortical tissue was obtained at autopsy and stored at -80 degrees C until assay. Both Con-G(A7) and ifenprodil inhibited [(3)H]MK801 binding, but spermine affected these inhibitions differently. Con-G(A7) IC(50) changed little with spermine concentration, indicative of a non-competitive interaction, whereas the rightward shift in ifenprodil IC(50) with increasing spermine concentration suggested partial competition. When the two agents were tested against the biphasic activation of [(3)H]MK801 binding by spermine, they again differed in their effects. In the activation phase Con-G(A7) was a non-competitive inhibitor of spermine activation, and may even enhance the spermine EC(50), while the ifenprodil data indicated a partially competitive interaction. Both agents were non-competitive in the inhibitory phase. Overall, the data suggest that Con-G(A7) and ifenprodil interact differently with the polyamine modulation of the glutamate-N-methyl-D-aspartate receptor.

Regulation of epithelial transport and barrier function by distinct protein kinase C isoforms.

The phorbol ester phorbol 12-myristate 13-acetate (PMA) inhibits Cl(-) secretion (short-circuit current, I(sc)) and decreases barrier function (transepithelial resistance, TER) in T84 epithelia. To elucidate the role of specific protein kinase C (PKC) isoenzymes in this response, we compared PMA with two non-phorbol activators of PKC (bryostatin-1 and carbachol) and utilized three PKC inhibitors (Gö-6850, Gö-6976, and rottlerin) with different isozyme selectivity profiles. PMA sequentially inhibited cAMP-stimulated I(sc) and decreased TER, as measured by voltage-current clamp. By subcellular fractionation and Western blot, PMA (100 nM) induced sequential membrane translocation of the novel PKC epsilon followed by the conventional PKC alpha and activated both isozymes by in vitro kinase assay. PKC delta was activated by PMA but did not translocate. By immunofluorescence, PKC epsilon redistributed to the basolateral domain in response to PMA, whereas PKC alpha moved apically. Inhibition of I(sc) by PMA was prevented by the conventional and novel PKC inhibitor Gö-6850 (5 microM) but not the conventional isoform inhibitor Gö-6976 (5 microM) or the PKC delta inhibitor rottlerin (10 microM), implicating PKC epsilon in inhibition of Cl(-) secretion. In contrast, both Gö-6976 and Gö-6850 prevented the decline of TER, suggesting involvement of PKC alpha. Bryostatin-1 (100 nM) translocated PKC epsilon and PKC alpha and inhibited cAMP-elicited I(sc). However, unlike PMA, bryostatin-1 downregulated PKC alpha protein, and the decrease in TER was only transient. Carbachol (100 microM) translocated only PKC epsilon and inhibited I(sc) with no effect on TER. Gö-6850 but not Gö-6976 or rottlerin blocked bryostatin-1 and carbachol inhibition of I(sc). We conclude that basolateral translocation of PKC epsilon inhibits Cl(-) secretion, while apical translocation of PKC alpha decreases TER. These data suggest that epithelial transport and barrier function can be modulated by distinct PKC isoforms.

Cystic prostate metastases to the brain parenchyma: report of two cases and review of the literature.

Prostate carcinoma is among the leading causes of cancer death for American men. Intracranial metastases from prostatic adenocarcinoma are unusual, and cystic metastases are rare. Two cases of cystic intraparenchymal metastases from prostatic adenocarcinoma are reported. Our comprehensive literature review revealed that prostatic adenocarcinoma metastases are rarely diagnosed antemortem. If these lesions are detected early and treated with surgery and radiation therapy, survival time for patients with prostatic adenocarcinoma may be increased; however, given the rarity of these cases, routine CNS imaging of men with metastatic prostate cancer does not seem warranted.

Specific depletion of human anti-adenovirus antibodies facilitates transduction in an in vivo model for systemic gene therapy.

Recombinant adenoviral (rAd) vectors are capable of mediating high-efficiency gene transfer in vivo. Under conditions requiring systemic administration, however, the use of rAd vectors can be problematic due to the presence of circulating anti-adenovirus antibodies developed either through natural infection or during the course of treatment. We developed a passive immunization model in SCID/Beige mice to assess the effect of human and mouse anti-adenovirus antibodies on systemic administration of a rAd vector expressing beta-galactosidase (rAd-betagal). In this model, the in vitro neutralizing activity of human or mouse antibodies used for passive immunization correlated well with inhibition of transduction of the liver following i.v. administration of rAd-betagal. Depletion of antibodies to individual adenovirus structural proteins (hexon, penton, fiber) by affinity chromatography demonstrated that antibodies to each of the three virion components contributed to neutralization of infectivity in vitro and to inhibition of transduction in vivo. Depletion of antibodies against all three structural proteins from human or mouse immune serum prior to passive immunization restored in vivo transduction activity to levels comparable to those obtained with nonimmune serum. Our data suggest that depletion of both murine and human anti-adenoviral antibodies can restore transduction in vivo during systemic rAd gene therapy in hosts previously exposed to adenovirus.

Extended lung expression and increased tissue localization of viral IL-10 with adenoviral gene therapy.

IL-10 is a pleiotropic cytokine that acts as an important regulator of macrophage, T cell, and natural killer cell functions. Human IL-10 (hIL-10) has both stimulatory and inhibitory effects on a wide variety of cell types. Viral IL-10 (vIL-10) possesses only a subset of hIL-10's activities, predominantly its suppression of cytokine synthesis by T helper type 1 clones. In the present report, we evaluated tissue accumulation and biological activity of hIL-10 and vIL-10 in vivo in individual organs by using a first-generation adenoviral (Ad) vector administered intratracheally and intravenously. We report the observation that Ad vectors delivering vIL-10, but not hIL-10, are associated with prolonged expression in the lung (>42 days) when delivered intratracheally. In contrast, there was no prolongation in vIL-10 expression when Ad vectors were intravenously administered, although vIL-10 levels in the tissue, but not serum, were markedly increased relative to hIL-10. Moreover, we report an augmented capacity of expressed vIL-10 versus hIL-10 to suppress the acute inflammatory responses in the lung to intratracheal administration of Ad. These findings confirm fundamental differences in Ad-induced expression of vIL-10 and hIL-10 when administered to the lungs. The results further suggest that Ad vectors expressing vIL-10 may have a role as anti-inflammatory agents in the treatment of acute and chronic lung inflammation.

Evaluation of endostatin antiangiogenesis gene therapy in vitro and in vivo.

Progressive growth and metastasis of solid tumors require angiogenesis, or the formation of new blood vessels. Endostatin is a 20-kDa carboxy-terminal fragment of collagen XVIII that has been shown to inhibit endothelial cell proliferation and tumor angiogenesis. Replication-deficient recombinant adenovirus (rAd) vectors were constructed, which encoded secreted forms of human and mouse endostatin (HECB and MECB, respectively), and, as a control, human alkaline phosphatase (APCB). Accumulation of endostatin was demonstrated in supernatants of cultured cells infected with the endostatin rAds. These supernatants disrupted tubule formation, inhibited migration and proliferation, and induced apoptosis in human dermal vascular endothelial cells or human vascular endothelial cells. Endostatin-containing supernatants had no effect on the proliferation of MidT2-1 mouse mammary tumor cells in vitro. A pharmacokinetic study of MECB in immunocompetent FVB mice demonstrated a 10-fold increase of serum endostatin concentrations 3 days after intravenous administration of 1x10(10) particles of this rAd (215-257 ng/mL compared to 12-38 ng/mL in control rAd-treated mice). Intravenous administration of MECB reduced b-FGF stimulated angiogenesis into Matrigel plugs by 38%. Intratumoral MECB inhibited growth of MidT2-1 syngeneic mammary tumors in FVB mice, but had minimal impact on the growth of MDA-MB-231 human breast tumors in SCID mice. Intravenous therapy with MECB also initially inhibited growth of MidT2-1 tumors, but this activity was subsequently blocked by induced anti-rAd antibodies. In summary, endostatin gene therapy effectively suppressed angiogenic processes in vitro and in vivo in several model systems.

TNF-alpha receptor signaling and IL-10 gene therapy regulate the innate and humoral immune responses to recombinant adenovirus in the lung.

Recombinant adenovirus-mediated gene therapy has demonstrated great promise for the delivery of genes to the pulmonary epithelium. However, dose-dependent inflammation and local immune responses abbreviate transgene expression. The purpose of these studies was to determine the role of TNF-alpha and individual TNF receptor signaling to adenovirus clearance and immune responses, and whether coexpression of human IL-10 could reduce inflammation and extend the duration of transgene expression in the lung. beta-Galactosidase expression in mice receiving intratracheal instillation of Adv/beta-gal (adenovirus construct expressing beta-galactosidase) was transient (less than 14 days), but a significant early increase of beta-galactosidase expression was seen in mice lacking either or both TNF-alpha receptors. Absence of TNF-alpha or the p55 receptor significantly attenuated the Ab response to both adenovirus and beta-galactosidase. Human IL-10 expression in the lung suppressed local TNF-alpha production following AdV/hIL-10 (adenovirus construct expressing human IL-10) delivery, but did not lead to increased or prolonged transgene expression when coexpressed with beta-galactosidase. Expression of human IL-10 following AdV/hIL-10 instillation extended at least 14 days, was nonimmunogenic, and suppressed the development of neutralizing Abs against adenoviral proteins as well as against human IL-10. We conclude that TNF-alpha signaling through both the p55 and p75 receptor plays important roles in the clearance of adenoviral vectors and the magnitude of the humoral immune response. Additionally, although coexpression of human IL-10 with beta-galactosidase had only modest effects on transgene expression, we demonstrate that AdV/hIL-10 is well tolerated, has extended expression compared with beta-galactosidase, and is nonimmunogenic in the lung.

Identification of HLA-A3-restricted cytotoxic T lymphocyte epitopes from carcinoembryonic antigen and HER-2/neu by primary in vitro immunization with peptide-pulsed dendritic cells.

The human carcinoembryonic antigen (CEA) and HER-2/neu are potential target antigens for CTL specific immunotherapy for common malignancies such as breast, lung, colon, and gastric carcinomas. Several CTL epitopes restricted by HLA-A2, the most common human histocompatibility molecule, have been previously reported. However, to develop CTL-based immunotherapies for the general population, it is necessary to identify epitopes restricted by other common histocompatibility alleles. Here, we describe two HLA-A3-restricted CTL epitopes from the CEA and HER-2/neu antigens. HLA-A3 binding synthetic peptides from CEA and HER-2/neu were tested for immunogenicity by in vitro primary CTL induction protocol using peripheral blood mononuclear cells from normal healthy volunteers. One peptide from CEA (CEA[9(61)]: HLFGYSWYK) and one peptide from HER-2/neu (HER2[9(754)]: VLRENTSPK) were shown to induce CTL that was capable of killing a tumor cell line expressing HLA-A3 and the corresponding tumor-associated antigen. Additional MHC binding studies with the most common HLA molecules belonging to the HLA-A3 superfamily (HLA-A*1101, -A*3101, -A*3301, and -A*6801), demonstrated that CEA[9(61)] binds five of five A3 supertype molecules with high affinity, and the HER2[9(754)] epitope was able to bind to four of the same five alleles. These results indicate that these two new CTL epitopes should be immunogenic in individuals expressing either HLA-A3, or other members of the HLA-A3 superfamily.

Identification of gp100-derived, melanoma-specific cytotoxic T-lymphocyte epitopes restricted by HLA-A3 supertype molecules by primary in vitro immunization with peptide-pulsed dendritic cells.

The human melanocyte lineage-specific antigen gp100 contains several epitopes recognized by cytotoxic T lymphocytes (CTL). However, most of the epitopes reported to date are HLA-A2.1-restricted. Despite the high frequency of HLA-A2.1 in melanoma patients, effective population coverage requires the identification of epitopes restricted by other frequent HLA alleles. Herein, HLA-A3 binding, gp100-derived synthetic peptides were tested for their capacity to elicit anti-melanoma CTL in vitro using CD8+ T cells from healthy donors as responders and peptide-pulsed autologous dendritic cells as antigen-presenting cells. Of 7 peptides tested, 2 (gp100[9(87)] and gp100[10(86)]) induced CTLs that killed melanoma cell lines expressing HLA-A3 and gp100. Additional MHC-binding studies to various HLA molecules belonging to the HLA-A3 superfamily (HLA-A*1101, -A*3101, -A*3301 and -A*6801) were performed to determine whether these CTL epitopes could further increase potential population coverage. Further experiments indicated that the peptide gp100[9(87)], which bound to HLA-A11 with high affinity, was capable of inducing specific CTLs that killed melanoma cells expressing gp100 and HLA-A11 molecules. Our results indicate that the gp100[9(87)] peptide corresponds to a CTL epitope which may be restricted by either the HLA-A3 or HLA-A11 allele, emphasizing its utility for the design and development of epitope-based therapies for melanoma.

The multi-epitope approach for immunotherapy for cancer: identification of several CTL epitopes from various tumor-associated antigens expressed on solid epithelial tumors.

One approach to development of specific cancer immunotherapy relies on the induction of cytotoxic T lymphocytes (CTL) specific for tumor-associated antigens (TAA). Induction of TAA-specific CTL could be used towards the eradication of established tumors, or to prevent their dissemination or recurrence after primary treatment. The present study identifies a set of CTL epitopes from TAA frequently found on solid epithelial tumors such as breast, lung and gastro-intestinal tumors. Specifically, HLA-A2.1 binding peptides from the MAGE2, MAGE3, HER-2/neu and CEA antigens were tested for their capacity to elicit in vitro anti-tumor CTL using lymphocytes from normal volunteers and autologous dendritic cells as antigen-presenting cells. A total of 6 new epitopes (MAGE2[10(157)], MAGE3[9(112)], CEA[9(691)], CEA[9(24)], HER2[9(435)] and HER2[9(5)]) were identified which were capable of specifically recognizing tumor cell lines lines expressing HLA-A2.1 and the corresponding TAA. In one case (CEA[9(24)]), induction of vigorous anti-tumor CTL responses required epitope engineering to increase HLA-A2.1 binding affinity. Finally, most of the newly identified epitopes (5 out of 6) were found to be highly crossreactive with other common HLA alleles of the A2 supertype (A2.2, A2.3, A2.6 and A6802), thus demonstrating their potential in providing broad and non-ethnically biased population coverage. The results are discussed in the context of the development of multi-epitope-based therapies with broad applicability for patients suffering from commonly found tumors.

Specific T cell recognition of peptides derived from prostate-specific antigen in patients with prostate cancer.

OBJECTIVES: To determine if proteins known to be expressed by both benign and malignant prostate epithelium can be recognized by T cells from patients with prostate cancer. We examined 7 HLA-A2 patients with prostate cancer for evidence of T cell reactivity with prostate-specific antigen (PSA). METHODS: Four peptides derived from PSA were chemically synthesized and shown to bind to HLA-A2. As a control, we also examined the immunogenic influenza matrix peptide Flu58-66 that binds to HLA-A2. These peptides were used to stimulate peripheral blood lymphocytes by in vitro stimulation. RESULTS: In 1 patient, specific recognition of peptide PSA141-150 was observed. The remaining 6 patients had no reactivity with any PSA-derived peptide. The T cell line with specific recognition of peptide PSA141-150 failed to recognize an autologous B cell blast line expressing endogenous PSA following infection with a recombinant PSA vaccinia virus construct. Three of the 7 patients demonstrated specific reactivity with Flu58-66. CONCLUSIONS: We found specific recognition of one PSA-derived peptide in 1 patient of 7 with prostate cancer. The peptide-specific lymphocyte cell line did not recognize endogenous PSA, suggesting that the peptide may not be produced by prostate cancer cells producing PSA. Specific recognition of PSA peptides was not common in our patients with prostate cancer. Whether such activity can be induced by vaccination strategies or can be therapeutic in men with established prostate cancer remains to be demonstrated.