Changes in phenotype or connectivity of primary afferent neurons following peripheral nerve injury may contribute to the hyperalgesia and allodynia associated with neuropathic pain conditions. Although earlier studies using partial nerve injury models have focused on the role of damaged fibres in the generation of ectopic discharges and pain, it is now thought that remaining undamaged fibres may be equally important. We have examined the expression of the sensory neuron-specific cation channel Vanilloid Receptor 1 (VR1), an important transducer of noxious stimuli, in three models of nerve injury in the rat, using anatomical separation or fluorescent retrograde tracers to identify damaged or undamaged sensory neurons. After total or partial sciatic nerve transection, or spinal nerve ligation, VR1-immunoreactivity (IR) was significantly reduced in the somata of all damaged dorsal root ganglion (DRG) neuronal profiles, compared to controls. However, after partial transection or spinal nerve ligation, VR1 expression was greater in the undamaged DRG somata than in controls. Unexpectedly, after L5 spinal nerve ligation, VR1-IR of the A-fibre somata increased approximately 3-fold in the uninjured L4 DRG compared to controls; a much greater increase than seen in the somata with C-fibres. Furthermore, we found that VR1-IR persisted in the transected sciatic nerve proximal to the lesion, despite its down-regulation in the damaged neuronal somata. This persistence in the nerve proximal to the lesion after nerve section, together with increased VR1 in DRG neurons left undamaged after partial nerve injury, may be crucial to the development or maintenance of neuropathic pain.

Proteins associated with cancer cell plasma membranes are rich in known drug and antibody targets as well as other proteins known to play key roles in the abnormal signal transduction processes required for carcinogenesis. We describe here a proteomics process that comprehensively annotates the protein content of breast tumor cell membranes and defines the clinical relevance of such proteins. Tumor-derived cell lines were used to ensure an enrichment for cancer cell-specific plasma membrane proteins because it is difficult to purify cancer cells and then obtain good membrane preparations from clinical material. Multiple cell lines with different molecular pathologies were used to represent the clinical heterogeneity of breast cancer. Peptide tandem mass spectra were searched against a comprehensive data base containing known and conceptual proteins derived from many public data bases including the draft human genome sequences. This plasma membrane-enriched proteome analysis created a data base of more than 500 breast cancer cell line proteins, 27% of which were of unknown function. The value of our approach is demonstrated by further detailed analyses of three previously uncharacterized proteins whose clinical relevance has been defined by their unique cancer expression profiles and the identification of proteinbinding partners that elucidate potential functionality in cancer.

Activation of either B1 or B2 bradykinin receptors by kinins released from damaged tissues contributes to the development and maintenance of inflammatory hyperalgesia. Whereas B2 agonists activate sensory neurones directly, B1 agonists were thought only to have indirect actions on sensory neurones. The recent discovery of constitutive B1 receptor expression in the rat nervous system lead us to re-investigate the role of neuronal B1 receptors in inflammatory hyperalgesia. Therefore we have examined B1 bradykinin receptor regulation in rat dorsal root ganglia in a model of inflammatory hyperalgesia, and correlated it with hyperalgesic behaviour. Twenty-four hours after injection of Freund's complete adjuvant into one hindpaw, there was a significant increase in B1 protein expression (measured by immunohistochemistry) in both ipsilateral and contralateral dorsal root ganglion neurones, whereas axotomy resulted in reduction of B1 protein in ipsilateral dorsal root ganglia. In behavioural experiments, the B1 antagonist desArg10HOE140, administered by either intrathecal or systemic routes, attenuated Freund's complete adjuvant-induced mechanical hyperalgesia in the inflamed paw, but did not affect mechanical allodynia. The B1 agonist, desArg9BK, did not affect paw withdrawal thresholds in nai;ve rats following intraplantar administration into the paw, whilst intrathecal administration elicited mechanical hyperalgesia. However, after Freund's complete adjuvant-induced inflammation, desArg9BK caused a marked mechanical hyperalgesia, by either route, of the contralateral, uninflamed hindpaw, correlating with the observed contralateral and ipsilateral increases in receptor levels. Our results suggest a functional role for B1 receptors expressed both in the periphery and in the spinal cord, in mechanical hyperalgesia during inflammation.

Metabotropic glutamate receptor 5 (mGluR5) protein increased after sciatic nerve section in ipsilateral L4 and L5 DRG neuronal profiles, with most of the increase occurring in myelinated A-fiber somata. mGluR5 also increased in lamina II of the ipsilateral spinal cord and the proximal sciatic nerve stump in this model. After L5 spinal nerve ligation, mGluR5 immunoreactivity increased dramatically not only in damaged L5 but also in the neighboring undamaged L4. Interestingly, after partial sciatic nerve section, mGluR5 expression did not change in either L4 or L5 DRG neuronal profiles. Both spinal nerve ligation and sciatic nerve partial section produced significant mechanical and thermal hyperalgesia and tactile allodynia. After partial sciatic nerve section, the mGluR5-specific antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) had no effect on any of these behaviors. However, after L5 spinal nerve ligation, although MPEP failed to alter the induced tactile allodynia or mechanical hyperalgesia, it dose dependently reversed the developed thermal hyperalgesia. Therefore, reversal of thermal hyperalgesia by MPEP correlates with increased mGluR5 in lumbar DRG A-fiber somata after nerve injury. Furthermore, A-fibers in the uninjured L4 DRG after L5 spinal nerve ligation that have increased mGluR5 are the same A-fibers that newly express vanilloid receptor 1 after such injury. Together, these results suggest that, after L5 spinal nerve injury, mGluR5 expression on A-fibers is essential to the development of thermal hyperalgesia. After partial nerve section, however, it is unlikely that thermal responses are mediated through mGluR5 because no such increase in mGluR5 is detected in this model and MPEP is ineffective.

B-cell-specific plasma-membrane proteins are potential targets for either small molecule or antibody-based therapies. We have sought to annotate proteins expressed at the cell surface membrane in patients with chronic lymphocytic leukemia (CLL) using plasma-membrane-based proteomic analysis to identify previously uncharacterized and potentially B-cell-specific proteins. Proteins from plasma-membrane fractions were separated on one-dimensional gels and trypsinized fractions subjected to high-throughput MALDI-TOF mass spectrometry. Using this method, many known B-cell surface antigens were detected, but also known proteins not previously described in this disease or in this cellular compartment, including cell surface receptors, membrane-associated enzymes and secreted proteins, and completely unknown proteins. To validate the method, we show that BLK, a B-cell-specific kinase, is located in the CLL-plasma-membrane fraction. We also describe two novel proteins (MIG2B and B-cell novel protein #1, BCNP1), which are expressed preferentially in B cells. MIG2B is in a highly conserved and defined gene family containing two plasma-membrane-binding ezrin/radixin/moesin domains and a pleckstrin homology domain; the Caenorhabditis elegans homolog (UNC-112) is a membrane-associated protein that colocalizes with integrin at cell-matrix adhesion complexes. BCNP1 is a completely unknown protein with three predicted transmembrane domains, with three alternatively spliced final exons. Proteomic analysis may thus define new potential therapeutic targets.

Purpose: Advanced stage gastrointestinal cancers represent a high unmet need requiring new effective therapies. We investigated the anti-tumor activity of a novel T-cell engaging antibody (B7-H6/CD3 ITE) targeting B7-H6, a tumor-associated antigen that is expressed in gastrointestinal tumors. Experimental Design: Membrane proteomics and immunohistochemistry analysis identified B7-H6 as tumor- associated antigen in gastrointestinal tumor tissues with no to very little expression in normal tissues. The anti-tumor activity and mode of action of B7-H6/CD3 ITE was evaluated in in vitro co-culture assays, in humanized mouse tumor models, and in Colorectal Cancer Precision Cut Tumor Slice Cultures. Results: B7-H6 expression was detected in 98 % of colorectal, 77 % of gastric cancer, and 63 % of pancreatic cancer tissue samples. B7-H6/CD3 ITE-mediated redirection of T cells towards B7-H6-positive tumor cells resulted in B7-H6-dependent lysis of tumor cells, activation and proliferation of T cells, and cytokine secretion in in vitro co-culture assays, and infiltration of T cells into tumor tissues associated with tumor regression in in vivo CRC models. In primary patient-derived Colorectal Cancer Precision Cut Tumor Slice Cultures, treatment with B7-H6/CD3 ITE elicited cytokine secretion by endogenous tumor infiltrating immune cells. Combination with anti-PD-1 further enhanced the activity of the B7-H6/CD3 ITE. Conclusions: These data highlight the potential of the B7-H6/CD3 ITE to induce T-cell re-directed lysis of tumor cells and recruitment of T cells into non-inflamed tumor tissues leading to anti-tumor activity in in vitro, in vivo, and human tumor slice cultures which supports further evaluation in a clinical study.

Aims To measure the milk to plasma ratio (M/P) of quinapril and its active metabolite quinaprilat in lactating mothers and to assess likely infant exposure. Methods A single dose of quinapril 20 mg was administered to six healthy mothers who had been breastfeeding their infants for at least 2 weeks. Blood was sampled for the measurement of quinapril and quinaprilat at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 16 and 24 h. Milk was collected for measurement of quinapril and quinaprilat concentrations over the periods x4±0, 0±4, 4±8, 8±12, 12±18, 18±24 h. The areas under the plasma and milk concentration-time curves were estimated and an M/P ratio derived for both quinapril and quinaprilat. Results The M/P ratio for quinapril was 0.12 (95% CI 0.09,0.14). No quinapril was detected in milk after 4 h. No quinaprilat was detected in any of the milk samples. The estimated`dose' of quinapril that would be received by the infant was 1.6% (95% CI 1.0,2.2) of the maternal dose, adjusted for respective weights. Conclusions Quinapril appears to be`safe' during breastfeeding according to conventional criteria, although as always, the risk:bene®t ratio should be considered when it is to be given to a nursing mother.

Antibody-drug conjugates (ADCs) are a recent and exciting development for targeted therapy of cancer. Their efficacy is governed by ADC-intrinsic characteristics such as avidity, drug load and linker chemistry, and mechanisms of activation and action, which can be controlled or clarified in the early stages of ADC development. In contrast, the properties that define a promising ADC target are still somewhat unclear. OGAP is a unique proteomic database that integrates information at the tissue, disease and protein isoform level across diseases, indications, and normal tissues to clarify protein expression levels and profiles. Specifically, it currently holds information on ∼2,000,000 human protein peptide sequences, ∼16,000 human proteins sequenced, ∼7,000 cancer membrane proteins, ∼50 tissues/organs, and ∼60 diseases. Building on OGAP and a proprietary sample preparation and processing workflow that relies on state-of-the-art high-throughput mass spectrometry and data processing to provide quantitative information on over 4,000 membrane-enriched proteins from ∼ 15,000 unique peptide sequences per analysis, we have established a novel predictive tool to establish each protein's potential to serve as a target for ADC development. The tool considers proteomic and target-specific information on antigenicity, structure, function, expression level, regulation, and tissue distribution in order to highlight the most suitable candidates for ADC development. We will demonstrate the utility of this process for the protein family of G-protein coupled receptors (GPCRs), which according to a recent bioinformatics prediction encompasses 899 distinct members in the human genome. These cell surface receptors are the target of more than one third of conventional drugs, yet their potential for ADCs is largely unexplored. Here we show that proteomics in the context of the OGAP database can highlight which of this large family of receptors have the potential to become true ADC targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3869. doi:1538-7445.AM2012-3869