Reassessment of SST4 Somatostatin Receptor Expression Using SST4-eGFP Knockin Mice and the Novel Rabbit Monoclonal Anti-Human SST4 Antibody 7H49L61.

Among the five somatostatin receptors (SST1-SST5), SST4 is the least characterized, which is in part due to the lack of specific monoclonal antibodies. We generated a knockin mouse model that expresses a carboxyl-terminal SST4-eGFP fusion protein. In addition, we extensively characterized the novel rabbit monoclonal anti-human SST4 antibody 7H49L61 using transfected cells and receptor-expressing tissues. 7H49L61 was then subjected to immunohistochemical staining of a series of formalin-fixed, paraffin-embedded normal and neoplastic human tissues. Characterization of SST4-eGFP mice revealed prominent SST4 expression in cortical pyramidal cells and trigeminal ganglion cells. In the human cortex, 7H49L61 disclosed a virtually identical staining pattern. Specificity of 7H49L61 was demonstrated by detection of a broad band migrating at 50-60 kDa in immunoblots. Tissue immunostaining was abolished by preadsorption of 7H49L61 with its immunizing peptide. In the subsequent immunohistochemical study, 7H49L61 yielded a predominant plasma membrane staining in adrenal cortex, exocrine pancreas, and placenta. SST4 was also found in glioblastomas, parathyroid adenomas, gastric and pancreatic adenocarcinomas, pheochromocytomas, and lymphomas. Altogether, we provide the first unequivocal localization of SST4 in normal and neoplastic human tissues. The monoclonal antibody 7H49L61 may also prove of great value for identifying SST4-expressing tumors during routine histopathological examinations.

PNL2: A Useful Adjunct Biomarker to HMB45 in the Diagnosis of Uterine Perivascular Epithelioid Cell Tumor (PEComa).

Perivascular epithelioid cell tumors (PEComa) are rare neoplasms characterized by co-expression of melanocytic and muscle markers. HMB45 and Melan-A are used to confirm a PEComa diagnosis; however, both are often focally expressed and sensitivity for Melan-A is low. PNL2 is a reliable biomarker for epithelioid melanoma and renal angiomyolipoma/PEComa. The objective of this study was to determine PNL2 utility in diagnosing uterine PEComas as well as distinguishing PEComas from uterine smooth muscle tumors (SMTs). Twenty-one uterine PEComas and 45 SMTs were analyzed for PNL2; a subset was also stained for HMB45, Melan-A, Cathepsin-K, Desmin, and h-Caldesmon. Cases were scored as negative (0), focal (<10% of tumor cells), or patchy to diffusely positive (>10% of tumor cells). PEComas were positive for PNL2, HMB45, and Melan-A in 86%, 100%, and 57% of cases, respectively. In PEComas, PNL2 was patchy to diffusely positive more frequently (10/18, 56%) than Melan-A (4/12, 33%). In contrast, 2 of 45 (4%) SMTs were focally PNL2 positive; HMB45 was focally positive in 4 SMTs (11%) and all were negative for Melan-A. Desmin and h-Caldesmon were positive in 90% and 57% of PEComas, and 91% and 82% of SMTs. Cathepsin-K was positive in 100% of PEComas and 93% of SMTs. PNL2 is a useful biomarker for the diagnosis of uterine PEComa, with comparable sensitivity and specificity to HMB45. In contrast, PNL2 stains more PEComas when compared with Melan-A. Cathepsin-K, Desmin, and h-Caldesmon are of little utility for distinguishing PEComas and SMTs; however, lack of Cathepsin-K argues against PEComa. These results suggest that PNL2 should be used in conjunction with HMB45 in the diagnosis of PEComa of the uterine corpus.

Somatostatin Type 2 Receptor Antibody Enhances Mechanical Hyperalgesia in the Dorsal Root Ganglion Neurons after Sciatic Nerve-pinch Injury: Evidence of Behavioral Studies and Bax Protein Expression.

BACKGROUND: Our previous study has indicated that somatostatin potently inhibits neuropathic pain through the activation of its type 2 receptor (SSTR2) in mouse dorsal root ganglion and spinal cord. However, the underlying mechanism of this activation has not been elucidated clearly. OBJECTIVE: The aim of this study is to perform the pharmacological studies on the basis of sciatic nerve-pinch mice model and explore the underlying mechanism involving SSTR2. METHODS: On the basis of a sciatic nerve-pinch injury model, we aimed at comparing the painful behavior and dorsal root ganglion neurons neurochemical changes after the SSTR2 antibody (anti- SSTR2;5µl,1µg/ml) administration in the mouse. RESULTS: After pinch nerve injury, we found that the mechanical hyperalgesia and severely painful behavior (autotomy) were detected after the application of SSTR2 antibody (anti-SSTR2; 5µl, 1µg/ml) on the pinch-injured nerve. The up-regulated phosphorylated ERK (p-ERK) expression and the apoptotic marker (i.e., Bax) were significantly decreased in DRGs after anti-SSTR2 treatment. CONCLUSION: The current data suggested that inhibitory changes in proteins from the apoptotic pathway in anti-SSTR2-treated groups might be taking place to overcome the protein deficits caused by SSTR2 antibody and supported the new therapeutic intervention with SSTR2 antagonist for neuronal degeneration following nerve injury.

Immunohistochemical Expression of Somatostatin Receptor Subtypes in a Panel of Neuroendocrine Neoplasias.

Neuroendocrine neoplasias (NENs) are known to express somatostatin receptors (SSTRs) 1-5, which are G-protein-coupled cell membrane receptors. Somatostatin receptor imaging and therapy utilizes the SSTR expression. Synthetic somatostatin analogs with radioligands are used to detect primary tumors, metastases, and recurrent disease. Receptor analogs are also used for treating NENs. Furthermore, commercially available SSTR antibodies can be used for the immunohistochemical (IHC) detection of SSTRs. We investigated different SSTR antibody clones applying diverse IHC protocol settings to identify reliable clones and feasible protocols for NENs. A tissue microarray including NENs from 12 different primary sites were stained. Only UMB clones were able to localize SSTR on the cell membranes of NENs. SSTR2 (UMB1) emerged as the most common subtype followed by SSTR5 (UMB4) and SSTR1 (UMB7). SSTR3 (UMB5) expression was mainly cytoplasmic. Yet, SSTR4 expression was weak and located primarily in the cytoplasm. Thus, appropriate IHC protocols, including proper positive and negative controls, represent requirements for high-quality NEN diagnostics and for planning personalized therapy.

Characterization of Functional Primary Cilia in Human Induced Pluripotent Stem Cell-Derived Neurons.

Recent advances in human induced pluripotent stem cells (hiPSCs) offer new possibilities for biomedical research and clinical applications. Neurons differentiated from hiPSCs may be promising tools to develop novel treatment methods for various neurological diseases. However, the detailed process underlying functional maturation of hiPSC-derived neurons remains poorly understood. Here, we analyze the developmental architecture of hiPSC-derived cortical neurons, iCell GlutaNeurons, focusing on the primary cilium, a single sensory organelle that protrudes from the surface of most growth-arrested vertebrate cells. To characterize the neuronal cilia, cells were cultured for various periods and evaluated immunohistochemically by co-staining with antibodies against ciliary markers Arl13b and MAP2. Primary cilia were detected in neurons within days, and their prevalence and length increased with increasing days in culture. Treatment with the mood stabilizer lithium led to primary cilia length elongation, while treatment with the orexigenic neuropeptide melanin-concentrating hormone caused cilia length shortening in iCell GlutaNeurons. The present findings suggest that iCell GlutaNeurons develop neuronal primary cilia together with the signaling machinery for regulation of cilia length. Our approach to the primary cilium as a cellular antenna can be useful for both assessment of neuronal maturation and validation of pharmaceutical agents in hiPSC-derived neurons.

Immunochemical strategy for quantification of G-coupled olfactory receptor proteins on natural nanovesicles.

Cell membrane proteins are involved in a variety of biochemical pathways and therefore constitute important targets for therapy and development of new drugs. Bioanalytical platforms and binding assays using these membrane protein receptors for drug screening or diagnostic require the construction of well-characterized liposome and lipid bilayer arrays that act as support to prevent protein denaturation during biochip processing. Quantification of the protein receptors in the lipid membrane arrays is a key issue in order to produce reproducible and well-characterized chips. Herein, we report a novel immunochemical analytical approach for the quantification of membrane proteins (i.e., G-protein-coupled receptor, GPCR) in nanovesicles (NVs). The procedure allows direct determination of tagged receptors (i.e., c-myc tag) without any previous protein purification or extraction steps. The immunochemical method is based on a microplate ELISA format and quantifies this tag on proteins embedded in NVs with detectability in the picomolar range, using protein bioconjugates as reference standards. The applicability of the method is demonstrated through the quantification of the c-myc-olfactory receptor (OR, c-myc-OR1740) in the cell membrane NVs. The reported method opens the possibility to develop well-characterized drug-screening platforms based on G-coupled proteins embedded on membranes.

Evaluation of somatostatin receptor subtype expression in human neuroendocrine tumors using two sets of new monoclonal antibodies.

INTRODUCTION: The expression and reliable detection of somatostatin receptor subtypes (SSTR1-5) is a prerequisite for the successful use of somatostatin analogs in neuroendocrine tumors (NETs). Two sets of monoclonal antibodies (mAbs) against human SSTR1, 2A, 3 and 5 have recently been developed by two independent laboratories using rabbit and mouse hybridomas. Our aim was to evaluate the usefulness of both sets of mAbs for detection of SSTRs in NET samples as they are routinely collected in clinical practice. METHODS: Mouse and rabbit mAbs were characterized in SSTR1, 2A, 3 and 5-transfected HEK293 cells and human archival samples of pancreatic tissue and NET. Comparative analysis of mAbs was also conducted by immunostaining of a tissue microarray composed of 75 cores of NET. RESULTS: Immunohistochemical analysis of HEK293 cells showed that both rabbit and mouse mAbs specifically detect their cognate receptor subtype, with mild cytoplasmic cross-reactivity observed for rabbit mAbs. Both sets of mAbs labeled normal pancreatic islets and showed similar patterns of immunoreactivity in NET controls. Direct comparison of mAb sets using a NET tissue microarray revealed strong correlation between rabbit and mouse mAbs against SSTR1 and 5, and moderate correlation for SSTR3. The rabbit mAb against SSTR2A showed higher affinity for its cognate receptor than the corresponding mouse mAb, resulting in a more reliable detection of this SSTR. CONCLUSIONS: mAbs from both sets are reliable tools for the detection of SSTR1, 3 and 5, whereas the rabbit mAb against SSTR2A is recommended for use in routine clinical testing due to its superior binding affinity.

Expression of SSTR2a, but not of SSTRs 1, 3, or 5 in somatotroph adenomas assessed by monoclonal antibodies was reduced by octreotide and correlated with the acute and long-term effects of octreotide.

CONTEXT: Reduced expression of somatostatin receptors (SSTRs) in somatotroph adenomas and their potential down-regulation after medical treatment may explain the unsatisfactory response to octreotide in particular acromegalic patients. The expression of SSTRs other than SSTR2a has not been studied in large, unselected cohorts using novel rabbit monoclonal antibodies. OBJECTIVE: We aimed to determine the expression of SSTRs 1, 2a, 3, and 5 in somatotroph adenomas, to correlate expression with clinical characteristics and the response to octreotide, and to ascertain whether preoperative octreotide treatment affected SSTR expression. DESIGN, SETTING, PATIENTS: The study included 78 adenomas from patients operated on consecutively during 2000 to 2010. After exclusion of 13 patients, immunohistochemical analysis with rabbit monoclonal antibodies against SSTRs 1, 2a, 3, and 5 (clones UMB-7, -1, -5, and -4) was performed on 65 adenomas. INTERVENTION: Twenty-eight patients received preoperative octreotide, and 37 patients were operated on without pretreatment. Twenty-six patients were randomized to direct surgery (n = 13) or to octreotide pretreatment (n = 13). MAIN OUTCOME MEASURE: SSTR expression was evaluated using a 12-grade scoring system. The responses to the octreotide test dose (GH reduction) and to 6 months of octreotide (IGF-I reduction) were measured. RESULTS: The majority of adenomas showed membranous expression of SSTRs 2a and 5. SSTR2a expression was reduced in the pretreated group and correlated with the acute octreotide test results and the effect of octreotide treatment. In a linear regression model with SSTR2a expression as the determinant, the correlation with the acute test response improved after adjustment for medical pretreatment. CONCLUSION: Rabbit monoclonal antibodies are reliable markers of SSTRs in somatotroph adenomas. SSTR2a expression correlated with the response to octreotide and was reduced after octreotide treatment, indicating the need for adjustment when SSTR2a expression is correlated with baseline characteristics. Evaluation of SSTR subtypes may be an important aspect of improving the medical treatment for acromegaly.

Correlation of immunohistopathological expression of somatostatin receptor-2 in breast cancer and tumor detection with 68Ga-DOTATOC and 18F-FDG PET imaging in an animal model.

BACKGROUND: Fludeoxyglucose positron emission topography ((18)F-FDG PET) is insufficiently sensitive at detecting small or low-grade breast tumors. The characterization of somatostatin receptors (SSTR) in tumors and the development of (68)Ga-DOTATOC PET for imaging could be of interest. The aim of this study was to validate an animal model expressing SSTR2 and to correlate the immunohistochemical (IHC) analysis with (18)F-FDG and (68)Ga-DOTATOC uptake in vivo. MATERIALS AND METHODS: Ten nude mice were xenografted with the ZR-75-1 breast tumor cell line. Imaging was performed with (68)Ga-DOTATOC and (18)F-FDG and correlated to IHC analysis of SSTR2. RESULTS: IHC analyses showed that the tumors expressed SSTR2. On PET imaging, the tumors were barely visible with (18)F-FDG, whereas with (68)Ga-DOTATOC, specific two-fold higher uptake was observed (p<0.005). CONCLUSION: Our results suggest that (68)Ga-DOTATOC PET could be used for detection of breast tumors not detected with (18)F-FDG. SSTR2 status should be assessed to allow for individual treatment.

Reevaluation of sst1 somatostatin receptor expression in human normal and neoplastic tissues using the novel rabbit monoclonal antibody UMB-7.

BACKGROUND: The somatostatin receptor 1 (sst1) is widely distributed throughout the body and is also present in neoplastic tissues. However, little is known about its precise tissue distribution, regulation and function, which may in part be due to the lack of specific monoclonal anti-sst1 antibodies. METHODS: We have characterized the novel rabbit monoclonal anti-human sst1 antibody UMB-7 using sst1-expressing cells and human pituitary samples. The antibody was then used for immunohistochemical staining of a large panel of formalin-fixed, paraffin-embedded human tissues. RESULTS: Western blot analyses of BON-1 cells and human pituitary revealed a broad band migrating at a molecular weight of 45,000-60,000. After enzymatic deglycosylation the size of this band decreased to a molecular weight of 45,000. UMB-7 yielded an efficient immunostaining of distinct cell populations in the human tissue samples with a predominance of plasma membrane staining, which was completely abolished by preadsorption of UMB-7 with its immunizing peptide. The sst1 receptor was detected in anterior pituitary, pancreatic islets, distal tubules, enteric ganglion cells and nerve fibers, chief cells of the gastric mucosa, macrophages and mast cells. In addition, sst1 was observed in pituitary adenomas, gastrointestinal neuroendocrine tumors and pheochromocytoma as well as in pancreatic adenocarcinomas, gastric carcinomas, urinary bladder carcinomas and sarcomas. CONCLUSIONS: UMB-7 may prove of great value in the identification of sst1-expressing tumors during routine histopathological examinations. This may open up new routes for diagnostic and therapeutic intervention.

Immunoreactivity score using an anti-sst2A receptor monoclonal antibody strongly predicts the biochemical response to adjuvant treatment with somatostatin analogs in acromegaly.

CONTEXT: Somatostatin receptor subtype 2 (sst2A) protein expression has been demonstrated to positively correlate with somatostatin analog treatment outcome in GH-secreting adenomas. Recently, a new rabbit monoclonal anti-sst2A antibody (clone UMB-1) has been validated as a reliable method to selectively detect sst2A protein levels in formalin-fixed tissues. OBJECTIVE: The aim of the study was to establish whether the evaluation of sst2A protein levels, assessed with a routine reproducible immunohistochemistry protocol using UMB-1 antibody, may predict the successful adjuvant therapy with somatostatin analogs in acromegalic patients. DESIGN, SETTING, AND PATIENTS: Thirty-six acromegalic patients from our referral hospital were evaluated retrospectively. Sst2A expression analysis was performed by immunohistochemistry in 25 patients and by quantitative RT-PCR in 26 patients. Sst2A immunoreactivity was evaluated using an immunoreactivity score (IRS), which takes into account both the percentage of positive cells and staining intensity. INTERVENTIONS: Patients with persistent disease after surgery (n = 26) were treated with somatostatin analogs for a median duration of 6 months. MAIN OUTCOME MEASURE: GH and IGF-I levels were measured before and after postoperative treatment. RESULTS: Sst2A IRS showed a significant positive correlation with both GH (P = 0.039) and IGF-I (P = 0.001) suppression by octreotide. Sst2A IRS was negatively associated with IGF-I levels reached after treatment (P = 0.001), and patients that achieved IGF-I normalization showed significantly higher sst2A IRS compared to the group that was not normalized (P = 0.002). A sst2A IRS of at least 5 showed a sensitivity of 86% and a specificity of 91% in predicting IGF-I normalization during adjuvant octreotide treatment. CONCLUSION: Sst2A IRS with the anti-sst2A antibody UMB-1 represents a valid tool in the clinical practice to identify acromegalic patients likely to be responders to adjuvant therapy with the currently available somatostatin analogs.

Increased susceptibility of melanin-concentrating hormone-deficient mice to infection with Salmonella enterica serovar Typhimurium.

Melanin-concentrating hormone (MCH) was initially identified in mammals as a hypothalamic neuropeptide regulating appetite and energy balance. However, the wide distribution of MCH receptors in peripheral tissues suggests additional functions for MCH which remain largely unknown. We have previously reported that mice lacking MCH develop attenuated intestinal inflammation when exposed to Clostridium difficile toxin A. To further characterize the role of MCH in host defense mechanisms against intestinal pathogens, Salmonella enterocolitis (using Salmonella enterica serovar Typhimurium) was induced in MCH-deficient mice and their wild-type littermates. In the absence of MCH, infected mice had increased mortality associated with higher bacterial loads in blood, liver, and spleen. Moreover, the knockout mice developed more-severe intestinal inflammation, based on epithelial damage, immune cell infiltrates, and local and systemic cytokine levels. Paradoxically, these enhanced inflammatory responses in the MCH knockout mice were associated with disproportionally lower levels of macrophages infiltrating the intestine. Hence, we investigated potential direct effects of MCH on monocyte/macrophage functions critical for defense against intestinal pathogens. Using RAW 264.7 mouse monocytic cells, which express endogenous MCH receptor, we found that treatment with MCH enhanced the phagocytic capacity of these cells. Taken together, these findings reveal a previously unappreciated role for MCH in host-bacterial interactions.

Distribution of somatostatin receptor 5 in mouse and bullfrog retinas.

Somatostatin (SRIF), as a neuroactive peptide in the CNS, may act as a neuromodulator through activation of five specific receptor subtypes (sst(1)-sst(5)). In this work we conducted a comparative study of the expression of sst(5) in mouse and bullfrog retinas by immunofluorescence double labeling. Basically, the expression profiles of sst(5) in the retinas of the two species were similar. That is, in the inner retina sst(5) was localized to dopaminergic and cholinergic amacrine cells, stained by tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) respectively, and cells in the ganglion cell layer, whereas in the outer retina immunostaining for sst(5) was observed in horizontal cells. However, a more widespread, abundant distribution of labeling for sst(5), as compared to mouse retina, was seen in bullfrog retina: strong labeling for sst(5) was diffusely distributed in both outer and inner plexiform layers (OPL and IPL) in the bullfrog retina, but the labeling was only observed in the IPL of the mouse retina. In addition, bullfrog photoreceptors, both rods and cones, but not mouse ones, were labeled by sst(5). In combination with the experiments showing that SRIF-immunoreactivity was mainly found in the inner retina, our results suggest that SRIF, released from SRIF-containing cells in the inner retina, may play a neuromodulatory role in both outer and inner retina mediated by volume transmission via sst(5) in bullfrog retina, while the SRIF action may be largely restricted to the mouse inner retina.

Somatostatin receptor subtype 2A immunohistochemistry using a new monoclonal antibody selects tumors suitable for in vivo somatostatin receptor targeting.

High overexpression of somatostatin receptors in neuroendocrine tumors allows imaging and radiotherapy with radiolabeled somatostatin analogues. To ascertain whether a tumor is suitable for in vivo somatostatin receptor targeting, its somatostatin receptor expression has to be determined. There are specific indications for use of immunohistochemistry for the somatostatin receptor subtype 2A, but this has up to now been limited by the lack of an adequate reliable antibody. The aim of this study was to correlate immunohistochemistry using the new monoclonal anti-somatostatin receptor subtype 2A antibody UMB-1 with the gold standard in vitro method quantifying somatostatin receptor levels in tumor tissues. A UMB-1 immunohistochemistry protocol was developed, and tumoral UMB-1 staining levels were compared with somatostatin receptor binding site levels quantified with in vitro I-[Tyr]-octreotide autoradiography in 89 tumors. This allowed defining an immunohistochemical staining threshold permitting to distinguish tumors with somatostatin receptor levels high enough for clinical applications from those with low receptor expression. The presence of >10% positive tumor cells correctly predicted high receptor levels in 95% of cases. In contrast, absence of UMB-1 staining truly reflected low or undetectable somatostatin receptor expression in 96% of tumors. If 1% to 10% of tumor cells were stained, a weak staining intensity was suggestive of low somatostatin receptor levels. This study allows for the first time a reliable recommendation for eligibility of an individual patient for in vivo somatostatin receptor targeting based on somatostatin receptor immunohistochemistry. Under optimal methodological conditions, UMB-1 immunohistochemistry may be equivalent to in vitro receptor autoradiography.

Monoclonal antibodies against the human somatostatin receptor subtypes 1-5: development and immunohistochemical application in neuroendocrine tumors.

BACKGROUND: Activation of somatostatin receptors (sstr1-5) by somatostatin and its analogues exerts an inhibitory effect on hormone secretion and provides the basis for the treatment of a range of endocrine diseases such as acromegaly, Cushing's disease and neuroendocrine tumors (NET). The lack of well-characterized commercially available sstr subtype-specific antibodies prevents routine identification of the sstr expression profile in patients. METHODS: We generated and characterized new mouse monoclonal antibodies (mAbs) targeting the five human sstr subtypes using ELISA and immunohistochemistry, and tested their suitability in formalin-fixed and paraffin-embedded (FFPE) human tissues and archival samples of normal pancreatic tissue and NET. RESULTS: All mAbs were highly specific with no cross-reactivity. The sstr1-5 immunoreactivity in gastrointestinal NET (n=67) was correlated with clinicopathologic data. With the exception of sstr3, NET were highly positive for all receptor subtypes (42, 63, 6, 32 and 65% of tumors were positive for sstr1, sstr2a, sstr3, sstr4 and sstr5, respectively). sstr1, sstr2a and sstr5 were present at the plasma membrane and in the cytoplasm of tumor cells, whereas sstr3 and sstr4 were almost exclusively cytoplasmic. Immunoreactivity of sstr1, sstr2a and sstr4 tended to decrease as tumor aggressiveness increased. sstr5 showed an opposite pattern, with higher staining in well-differentiated carcinomas compared with well-differentiated tumors. sstr5 immunoreactivity was correlated with the presence of metastases and angioinvasion, suggesting a possible association with more aggressive behavior. CONCLUSION: Determination of the sstr1-5 by immunohistochemistry using subtype-specific mAbs is feasible in FFPE tissue and may provide a tool for routine clinical practice.

SSTR5 P335L monoclonal antibody differentiates pancreatic neuroendocrine neuroplasms with different SSTR5 genotypes.

BACKGROUND: Somatostatin receptor type 5 (SSTR5) P335L is a hypofunctional, single nucleotide polymorphism of SSTR5 with implications in the diagnostics and therapy of pancreatic neuroendocrine neoplasms. The purpose of this study is to determine whether a SSTR5 P335L-specific monoclonal antibody could sufficiently differentiate pancreatic neuroendocrine neoplasms (PNENs) with different SSTR5 genotypes. METHODS: Cellular proliferation rate, SSTR5 mRNA level, and SSTR5 protein level were measured by performing MTS assay, a quantitative reverse transcription polymerase chain reaction study, Western blot analysis, and immunohistochemistry, respectively. SSTR5 genotype was determined with the TaqMan SNP Genotyping assay (Applied Biosystems, Foster City, CA). RESULTS: We found that the SSTR5 analogue RPL-1980 inhibited cellular proliferation of CAPAN-1 cells more than that of PANC-1 cells. Only PANC-1 (TT) cells, but not CAPAN-1 (CC) cells expressed SSTR5 P335L. In 29 white patients with PNENs, 38% had a TT genotype for SSTR5 P335L, 24% had a CC genotype for WT SSTR5, and 38% hada CT genotype for both SSTR5 P335L and WT SSTR5. Immunohistochemistry using SSTR5 P335L monoclonal antibody detected immunostaining signals only from the neuroendocrine specimens with TT and CT genotypes, but not those with CC genotypes. CONCLUSION: A SSTR5 P335L monoclonal antibody that specifically recognizes SSTR5 P335L but not WT SSTR5 could differentiate PNENs with different SSTR5 genotypes, thereby providing a potential tool for the clinical diagnosis of PNEN.

Reassessment of sst(5) somatostatin receptor expression in normal and neoplastic human tissues using the novel rabbit monoclonal antibody UMB-4.

OBJECTIVE: The frequent overexpression of somatostatin receptors (sst) in neuroendocrine tumors provides the molecular basis for the diagnostic and therapeutic application of stable somatostatin analogs. Whereas octreotide acts mainly via the sst(2) receptor, the novel pan-somatostatin analog pasireotide exhibits particular high affinity for the sst(5) receptor. To determine whether a patient is a candidate for octreotide or pasireotide therapy, it is important to evaluate the somatostatin receptor status. However, so far highly specific rabbit monoclonal antibodies have been developed for the sst(2) receptor only (clone UMB-1). METHODS: Here, we have extensively characterized a novel rabbit monoclonal antibody for the human sst(5) receptor (clone UMB-4). In a comparative immunohistochemical study, the expression of sst(5) and sst(2) receptors was assessed using UMB-4 and UMB-1, respectively. RESULTS: Western blot experiments unequivocally demonstrated that UMB-4 selectively detected its cognate sst(5) receptor and did not cross-react with other proteins present in crude tissue homogenates. UMB-4 yielded a highly effective immunostaining of distinct cell populations in formalin-fixed, paraffin-embedded human tissues with a predominance of plasma membrane staining. In the pituitary, sst(5) was present on all growth hormone (GH)- and adrenocorticotropin hormone (ACTH)-producing cells whereas sst(2) was only observed on a subpopulation of GH-positive cells. Consequently, sst(5) was detectable on the majority of GH and ACTH adenomas. In contrast, sst(2) was only seen on GH but not on ACTH adenomas. CONCLUSIONS: The rabbit monoclonal antibodies UMB-4 and UMB-1 will facilitate the assessment of the somatostatin receptor status of human tumors during routine histopathological examinations.

Molecular imaging with 68Ga-SSTR PET/CT and correlation to immunohistochemistry of somatostatin receptors in neuroendocrine tumours.

PURPOSE: Somatostatin receptors (SSTR) are known for an overexpression in gastroenteropancreatic neuroendocrine tumours (GEP-NET). The aim of the present study was to find out if the receptor density predicted by the semi-quantitative parameters generated from the static positron emission tomography (PET/CT) correlated with the in vitro immunohistochemistry using a novel rabbit monoclonal anti-SSTR2A antibody (clone UMB-1) for specific SSTR2A immunohistochemistry and polyclonal antibodies for SSTR1 and 3-5. METHODS: Overall 14 surgical specimens generated from 34 histologically documented GEP-NET patients were correlated with the preoperative (68)Ga-DOTA-NOC PET/CT. Quantitative assessment of the receptor density was done using the immunoreactive score (IRS) of Remmele and Stegner; the additional 4-point IRS classification for immunohistochemistry and standardized uptake values (SUV(max) and SUV(mean)) were used for PET/CT. RESULTS: The IRS for SSTR2A and SSTR5 correlated highly significant with the SUV(max) on the PET/CT (p < 0.001; p < 0.05) and the IRS for SSTR2A with the SUV(mean) (p < 0.013). The level of SSTR2A score correlated significantly with chromogranin A staining and indirectly to the tumour grading. CONCLUSION: The highly significant correlation between SSTR2A and SSTR5 and the SUV(max) on the (68)Ga-DOTA-NOC PET/CT scans is concordant with the affinity profile of (68)Ga-DOTA-NOC to the SSTR subtypes and demonstrates the excellent qualification of somatostatin analogues in the diagnostics of NET. This study correlating somatostatin receptor imaging using (68)Ga-DOTA-NOC PET/CT with immunohistochemically analysed SSTR also underlines the approval of therapy using somatostatin analogues, follow-up imaging as well as radionuclide therapy.

Immunodetection of the MCHR1 antibody in vitiligo patient sera.

Human vitiligo is an acquired depigmenting skin disorder characterized by milk-white skin macules resulting from a chronic and progressive loss of melanocytes. It has been suggested that autoimmune mechanisms are involved in this disorder. We undertook this project to obtain an MCHR1-C linear peptide containing four main MCHR1 B-cell epitopes in an attempt to detect the IgG antibody against MCHR1 in vitiligo. The target gene encoding the MCHR1-C peptide was cloned into a pGEX-4T-2 expression vector, expressed in Escherichia coli BL21, and purified using a GST column. The molecular weight was analyzed by SDS-PAGE and western blotting. The IgG antibody response to MCHR1 was detected by ELISA with MCHR1-C as a coated antigen, and the absorption experiment was used to assess the binding ability of the Ab detected by MCHR1-C with a membrane protein in human epidermal melanocytes. The purified peptide MCHR1-C with a molecular weight of 33 kDa was obtained, and could bind to the MCHR1 antibody in human sera. Of the vitiligo patient sera examined, 24 of 145 (16.55%) tested positive for the MCHR1 antibody, and the frequency in the vitiligo patient group was significantly greater compared to the normal control. However, no significant difference between gender, disease duration, clinical subtype or family history between the two groups was observed. In addition, the Ab detected by MCHR1-C in sera was specifically absorbed by the membrane protein obtained from human epidermal melanocytes. Collectively, our data suggest that the MCHR1-C peptide can be used to detect the MCHR1 antibody in vitiligo patients as a coated antigen instead of MCHR1 protein. We conclude that the level of MCHR1 antibody in active vitiligo patients is significantly higher than that in healthy individuals, while gender, disease duration, clinical subtype and family history have no association with the level of MCHR1 antibody in vitiligo patients.

The hypofunctional effect of P335L single nucleotide polymorphism on SSTR5 function.

BACKGROUND: Somatostatin receptor subtype 5 (SSTR5) mediates the inhibitory effect of somatostatin on insulin expression/secretion and cell proliferation. A number of single nucleotide polymorphisms (SNPs) of SSTR5 have been identified, including P335L, a nonsynonymous SNP located in the protein C-terminal region and encrypted by the codon CCG (proline) or the codon CTG (leucine). In the present study we sought to determine the distribution of the SSTR5 P335L SNP in a cohort of pancreatic cancer patients and whether the P335L SNP affected cellular function of SSTR5 in human pancreatic cancer. METHODS: The P335L germline genotype of 246 patients with pancreatic cancer (213 Caucasians, 16 Hispanics, and 17 African Americans) and 17 human pancreatic cell lines was determined with the TaqMan SNP Genotyping assay. Human SSTR5 leucine variant (L335) was generated by performing site-directed mutagenesis using SSTR5 proline variant (P335) as a template. Transient transfections were performed in HEK293, Mia PaCa-2, and ß-TC-6 cells using Lipofectamine 2000. The expression of SSTR5 L335 was determined with a mouse monoclonal anti-SSTR5 L335 antibody generated in our laboratory. The cell proliferation rate was measured by performing MTS assays. Insulin concentration was measured by performing ELISA assays. RESULTS: Genotyping of the patients' blood indicated that the frequency of the T allele (CT and TT genotypes) in codon 335 of SSTR5 in Caucasians, Hispanics, and African Americans was 52, 69, and 35%, respectively, which was race-dependent. Statistical analysis indicated that association between the frequency of the T allele and the existence of pancreatic cancer in each race missed significance perhaps due to limited sample size. In 17 tested human pancreatic cancer cell lines, 5 (Capan-2, HPAF-II, Panc03.27, Panc-1, and -3) were homozygous (TT genotype) and 9, including Mia PaCa-2, were heterozygous (CT genotype). Overexpression of SSTR5 L335 in Mia PaCa-2 cells enhanced cell proliferation compared to overexpression of SSTR5 P335. Overexpression of SSTR5 P335 enhanced the inhibitory effect of SSTR5 agonist RPL-1980 on cell proliferation of Mia PaCa-2 cells and glucose-stimulated insulin secretion from mouse insulinoma cells, while overexpression of SSTR5 L335 blocked the inhibitory effect of RPL-1980. Overexpression of SSTR5 L335 enhanced PDX-1 expression in Mia PaCa-2 cells. A specific monoclonal antibody was generated to detect SSTR5 P335L. CONCLUSION: SSTR5 P335L SNP widely exists in the human population, in patients with pancreatic cancer, and is race-dependent. The SNP is also present in selected human pancreatic cancer cell lines. In contrast to SSTR5 P335, overexpression of the SSTR5 L335 variant resulted in cellular proliferation and PDX-1 overexpression in human pancreatic cancer cells. Its overexpression blocked the inhibitory effect of an SSTR5-specific analog on human pancreatic cancer cell proliferation and on glucose-stimulated insulin secretion from mouse insulinoma cells. These data suggest that SSTR5 P335L is a hypofunctional protein with a potentially harmful effect on function, as well as potential latent effect, and therefore it could affect the clinical response to somatostatin analog therapy for patients with pancreatic cancer.