MyD88 and not TRIF knockout is sufficient to abolish LPS-induced inflammatory responses in bone-derived macrophages.

Macrophages play an important role in the response to infection and/or repair of injury in tissues. To examine the NF-κB pathway in response to an inflammatory stimulus, we used wild-type bone-marrow-derived macrophages (BMDMs) or BMDMs with knockout (KO) of myeloid differentiation primary response 88 (MyD88) and/or Toll/interleukin-1 receptor domain-containing adapter-inducing interferon-ß (TRIF) via CRISPR/Cas9. Following treatment of BMDMs with lipopolysaccharide (LPS) to induce an inflammatory response, translational signalling of NF-κB was quantified via immunoblot and cytokines were measured. Our findings reveal that MyD88 KO, but not TRIF KO, decreased LPS-induced NF-κB signalling, and 10% expression of basal MyD88 expression was sufficient to partially rescue the abolished inflammatory cytokine secretion observed upon MyD88 KO.

Clinical and translational science award T32/TL1 training programs: program goals and mentorship practices.

INTRODUCTION: A national survey characterized training and career development for translational researchers through Clinical and Translational Science Award (CTSA) T32/TL1 programs. This report summarizes program goals, trainee characteristics, and mentorship practices. METHODS: A web link to a voluntary survey was emailed to 51 active TL1 program directors and administrators. Descriptive analyses were performed on aggregate data. Qualitative data analysis used open coding of text followed by an axial coding strategy based on the grounded theory approach. RESULTS: Fifty out of 51 (98%) invited CTSA hubs responded. Training program goals were aligned with the CTSA mission. The trainee population consisted of predoctoral students (50%), postdoctoral fellows (30%), and health professional students in short-term (11%) or year-out (9%) research training. Forty percent of TL1 programs support both predoctoral and postdoctoral trainees. Trainees are diverse by academic affiliation, mostly from medicine, engineering, public health, non-health sciences, pharmacy, and nursing. Mentor training is offered by most programs, but mandatory at less than one-third of them. Most mentoring teams consist of two or more mentors. CONCLUSIONS: CTSA TL1 programs are distinct from other NIH-funded training programs in their focus on clinical and translational research, cross-disciplinary approaches, emphasis on team science, and integration of multiple trainee types. Trainees in nearly all TL1 programs were engaged in all phases of translational research (preclinical, clinical, implementation, public health), suggesting that the CTSA TL1 program is meeting the mandate of NCATS to provide training to develop the clinical and translational research workforce.

LPS-stimulated NF-κB p65 dynamic response marks the initiation of TNF expression and transition to IL-10 expression in RAW 264.7 macrophages.

During injury and infection, inflammation is a response by macrophages to effect healing and repair. The kinetics of the responses of proinflammatory TNFα, anti-inflammatory IL-10, and inflammatory master regulator NF-κB elicited by lipopolysaccharide (LPS) may be critical determinants of the inflammatory response by macrophages; however, there is a lack of homogeneous kinetic data in this pathway. To address this gap, we used the RAW 264.7 macrophage cell line to define intracellular signaling kinetics and cytokine expression in cells treated with LPS for 15 min to 72 h. The abundance of IκBα was maximally reduced 45-min following LPS treatment, but expression increased at 10-h, reaching a maximum at 16 h. NF-κB phosphorylation was significantly increased 45-min following LPS treatment, maximal at 2-h, and decreased to basal levels by 6-h. Nuclear NF-κB expression was elevated 30-min following LPS treatment, maximal by 45-min, and returned to basal levels by 24-h. Binding of nuclear NF-κB to consensus oligonucleotide sequences followed a similar pattern to that observed for p-NF-κB, but lasted slightly longer. Following LPS treatment, TNFα mRNA expression began at 1-h, was maximal at 6-h, and decreased starting at 10-h. TNFα protein secretion in conditioned growth medium began at 4-h and was maximal by 16-h. IL-10 mRNA expression was induced by LPS at 10-h, and was maximal at 16-h. IL-10 protein secretion was induced at 16-h and was maximal at 24-h. Our data reveal the temporal kinetics of pro- and anti-inflammatory signaling events that may be important therapeutic targets for inflammatory diseases.

RNA transcript expression of IGF-I/PI3K pathway components in regenerating skeletal muscle is sensitive to initial injury intensity.

OBJECTIVE: Skeletal muscle regeneration is a complex process involving the coordinated input from multiple stimuli. Of these processes, actions of the insulin-like growth factor-I (IGF-I) and phosphoinositide 3-kinase (PI3K) pathways are vital; however, whether IGF-I or PI3K expression is modified during regeneration relative to initial damage intensity is unknown. The objective of this study was to determine whether mRNA expression of IGF-I/PI3K pathway components was differentially regulated during muscle regeneration in mice in response to traumatic injury induced by freezing of two different durations. DESIGN: Traumatic injury was imposed by applying a 6-mm diameter cylindrical steel probe, cooled to the temperature of dry ice (-79°C), to the belly of the left tibialis anterior muscle of 12-week-old C57BL/6J mice for either 5s (5s) or 10s (10s). The right leg served as the uninjured control. RNA was obtained from injured and control muscles following 3, 7, and 21days recovery and examined by real-time PCR. Expression of transcripts within the IGF, PI3K, and Akt families, as well as for myogenic regulatory factors and micro-RNAs were studied. RESULTS: Three days following injury, there was significantly increased expression of Igf1, Igf2, Igf1r, Igf2r, Pik3cb, Pik3cd, Pik3cg, Pik3r1, Pik3r5, Akt1, and Akt3 in response to either 5s or 10s injury compared to uninjured control muscle. There was a significantly greater expression of Pik3cb, Pik3cd, Pik3cg, Pik3r5, Akt1, and Akt3 in 10s injured muscle compared to 5s injured muscle. Seven days following injury, we observed significantly increased expression of Igf1, Igf2, Pik3cd, and Pik3cg in injured muscle compared to control muscle in response to 10s freeze injury. We also observed significantly reduced expression of Igf1r and miR-133a in response to 5s freeze injury compared to control muscle, and significantly reduced expression of Ckm, miR-1 and miR-133a in response to 10s freeze injury as compared to control. Twenty-one days following injury, 5s freeze-injured muscle exhibited significantly increased expression of Igf2, Igf2r, Pik3cg, Akt3, Myod1, Myog, Myf5, and miR-206 compared to control muscle, while 10s freeze-injured muscles showed significantly increased expression of Igf2, Igf2r, Pik3cb, Pik3cd, Pik3r5, Akt1, Akt3, and Myog compared to control. Expression of miR-1 was significantly reduced in 10s freeze-injured muscle compared to control muscle at this time. There were no significant differences in RNA expression between 5s and 10s injury at either 7d or 21d recovery in any transcript examined. CONCLUSIONS: During early skeletal muscle regeneration in mice, transcript expressions for some components of the IGF-I/PI3K pathway are sensitive to initial injury intensity induced by freeze damage.

Induction of COX-2 expression by Helicobacter pylori is mediated by activation of epidermal growth factor receptor in gastric epithelial cells.

Chronic infection of the gastric mucosa by Helicobacter pylori is associated with an increased risk of developing gastric cancer; however, the vast majority of infected individuals never develop this disease. One H. pylori virulence factor that increases gastric cancer risk is the cag pathogenicity island, which encodes a bacterial type IV secretion system. Cyclooxygenase-2 (COX-2) expression is induced by proinflammatory stimuli, leading to increased prostaglandin E2 (PGE2) secretion by gastric epithelial cells. COX-2 expression is increased in gastric tissue from H. pylori-infected persons. H. pylori also activates the epidermal growth factor receptor (EGFR) in gastric epithelial cells. We now demonstrate that H. pylori-induced activation of COX-2 in gastric cells is dependent upon EGFR activation, and that a functional cag type IV secretion system and direct bacterial contact are necessary for full induction of COX-2 by gastric epithelial cells. PGE2 secretion is increased in cells infected with H. pylori, and this induction is dependent on a functional EGFR. Increased apoptosis in response to H. pylori occurs in cells treated with a COX-2 inhibitor, as well as COX-2-/- cells, indicating that COX-2 expression promotes cell survival. In vivo, COX-2 induction by H. pylori is significantly reduced in mice deficient for EGFR activation compared with wild-type mice with a fully functional receptor. Collectively, these findings indicate that aberrant activation of the EGFR-COX-2 axis may lower the threshold for carcinogenesis associated with chronic H. pylori infection.

Transactivation of EGFR by LPS induces COX-2 expression in enterocytes.

Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal morbidity and mortality in preterm infants. NEC is characterized by an exaggerated inflammatory response to bacterial flora leading to bowel necrosis. Bacterial lipopolysaccharide (LPS) mediates inflammation through TLR4 activation and is a key molecule in the pathogenesis of NEC. However, LPS also induces cyclooxygenase-2 (COX-2), which promotes intestinal barrier restitution through stimulation of intestinal cell survival, proliferation, and migration. Epidermal growth factor receptor (EGFR) activation prevents experimental NEC and may play a critical role in LPS-stimulated COX-2 production. We hypothesized that EGFR is required for LPS induction of COX-2 expression. Our data show that inhibiting EGFR kinase activity blocks LPS-induced COX-2 expression in small intestinal epithelial cells. LPS induction of COX-2 requires Src-family kinase signaling while LPS transactivation of EGFR requires matrix metalloprotease (MMP) activity. EGFR tyrosine kinase inhibitors block LPS stimulation of mitogen-activated protein kinase ERK, suggesting an important role of the MAPK/ERK pathway in EGFR-mediated COX-2 expression. LPS stimulates proliferation of IEC-6 cells, but this stimulation is inhibited with either the EGFR kinase inhibitor AG1478, or the selective COX-2 inhibitor Celecoxib. Taken together, these data show that EGFR plays an important role in LPS-induction of COX-2 expression in enterocytes, which may be one mechanism for EGF in inhibition of NEC.

TNF transactivation of EGFR stimulates cytoprotective COX-2 expression in gastrointestinal epithelial cells.

TNF and epidermal growth factor (EGF) are well-known stimuli of cyclooxygenase (COX)-2 expression, and TNF stimulates transactivation of EGF receptor (EGFR) signaling to promote survival in colon epithelial cells. We hypothesized that COX-2 induction and cell survival signaling downstream of TNF are mediated by EGFR transactivation. TNF treatment was more cytotoxic to COX-2(-/-) mouse colon epithelial (MCE) cells than wild-type (WT) young adult mouse colon (YAMC) epithelial cells or COX-1(-/-) cells. TNF also induced COX-2 protein and mRNA expression in YAMC cells, but blockade of EGFR kinase activity or expression inhibited COX-2 upregulation. TNF-induced COX-2 expression was reduced and absent in EGFR(-/-) and TNF receptor-1 (TNFR1) knockout MCE cells, respectively, but was restored upon expression of the WT receptors. Inhibition of mediators of EGFR transactivation, Src family kinases and p38 MAPK, blocked TNF-induced COX-2 protein and mRNA expression. Finally, TNF injection increased COX-2 expression in colon epithelium of WT, but not kinase-defective EGFR(wa2) and EGFR(wa5), mice. These data indicate that TNFR1-dependent transactivation of EGFR through a p38- and/or an Src-dependent mechanism stimulates COX-2 expression to promote cell survival. This highlights an EGFR-dependent cell signaling pathway and response that may be significant in colitis-associated carcinoma.

Learning from product labels and label changes: how to build pharmacogenomics into drug-development programs.

The 2010 US FDA-Drug Industry Association (DIA) Pharmacogenomics (PGx) Workshop follows a series that began in 2002 bringing together multidisciplinary experts spanning regulatory authorities, medical research, healthcare and industry. This report summarizes the 'Building PGx into Labels' sessions from the workshop, which discussed the critical elements in developing PGx outcomes leading to product labels that inform efficacy and/or safety. Examples were drawn from US prescribing information, which integrated PGx knowledge into medical decisions (e.g., panitumumab, warfarin and clopidogrel). Attendees indicated the need for broader dialog and for guidelines on evidentiary considerations for PGx to be included into product labels. Also discussed was the understanding of appropriate PGx placement on labels; how to encourage adoption by medical communities of label recommendations on PGx tests; and, given the global nature of drug development, worldwide considerations including European Summary of Product Characteristics.

Epidermal growth factor receptor activation protects gastric epithelial cells from Helicobacter pylori-induced apoptosis.

BACKGROUND & AIMS: Helicobacter pylori infection disrupts the balance between gastric epithelial cell proliferation and apoptosis, which is likely to lower the threshold for the development of gastric adenocarcinoma. H pylori infection is associated with epidermal growth factor (EGF) receptor (EGFR) activation through metalloproteinase-dependent release of EGFR ligands in gastric epithelial cells. Because EGFR signaling regulates cell survival, we investigated whether activation of EGFR following H pylori infection promotes gastric epithelial survival. METHODS: Mouse conditionally immortalized stomach epithelial cells (ImSt) and a human gastric epithelial cell line, AGS cells, as well as wild-type and kinase-defective EGFR (EGFRwa2) mice, were infected with the H pylori cag+ strain 7.13. Apoptosis, caspase activity, EGFR activation (phosphorylation), and EGFR downstream targets were analyzed. RESULTS: Inhibiting EGFR kinase activity or decreasing EGFR expression significantly increased H pylori-induced apoptosis in ImSt. Blocking H pylori-induced EGFR activation with a heparin-binding (HB)-EGF neutralizing antibody or abrogating a disintegrin and matrix metalloproteinase-17 (ADAM-17) expression increased apoptosis of H pylori-infected AGS and ImSt, respectively. Conversely, pretreatment of ImSt with HB-EGF completely blocked H pylori-induced apoptosis. H pylori infection stimulated gastric epithelial cell apoptosis in EGFRwa2 but not in wild-type mice. Furthermore, H pylori-induced EGFR phosphorylation stimulated phosphotidylinositol-3'-kinase-dependent activation of the antiapoptotic factor Akt, increased expression of the antiapoptotic factor Bcl-2, and decreased expression of the proapoptotic factor Bax. CONCLUSIONS: EGFR activation by H pylori infection has an antiapoptotic effect in gastric epithelial cells that appears to involve Akt signaling and Bcl family members. These findings provide important insights into the mechanisms of H pylori-associated tumorigenesis.

Fit-for-purpose pharmacogenomic biomarkers in drug development: a project team case study with 'what-ifs'.

Over the past four years, the annual US FDA-DIA pharmacogenomic workshops have brought together attendees with wide-ranging expertise spanning industry, regulatory authorities and academia. This special report summarizes a breakout session using a novel, interactive case format as a way to engage participants, raise awareness and share diverse learnings via 'real life' decisions that project teams might face in developing a new medicine. This case was situated just prior to approval by a Regulatory Authority as a project team is finalizing a new medicine label. To effectively integrate new biomarkers such as pharmacogenomics into developing new medicines, this session highlighted the importance in considering medical practice implications as relevant (or not) to information or actions by a prescriber; progressing validation beyond assay to clinical; and fitting pharmacogenomics into context with other evidence often built over decades during a drug's development. All converge onto a label that must communicate evidence-based use of a new medicine that is effective and safe.

Transactivation of EGF receptor and ErbB2 protects intestinal epithelial cells from TNF-induced apoptosis.

TNF is a pleiotropic cytokine that activates both anti- and proapoptotic signaling pathways, with cell fate determined by the balance between these two pathways. Activation of ErbB family members, including EGF receptor (EGFR/ErbB1), promotes cell survival and regulates several signals that overlap with those stimulated by TNF. This study was undertaken to determine the effects of TNF on EGFR and ErbB2 activation and intestinal epithelial cell survival. Mice, young adult mouse colon epithelial cells, and EGFR knockout mouse colon epithelial cells were treated with TNF. Activation of EGFR, ErbB2, Akt, Src, and apoptosis were determined in vivo and in vitro. TNF stimulated EGFR phosphorylation in young adult mouse colon epithelial cells, and loss of EGFR expression or inhibition of kinase activity increased TNF-induced apoptosis, which was prevented in WT but not by kinase-inactive EGFR expression. Similarly, TNF injection stimulated apoptosis in EGFR-kinase-defective mice (EGFR(wa2)) compared with WT mice. TNF also activated ErbB2, and loss of ErbB2 expression increased TNF-induced apoptosis. Furthermore, Src-kinase activity and the expression of both EGFR and ErbB2 were required for TNF-induced cell survival. Akt was shown to be a downstream target of TNF-activated EGFR and ErbB2. These findings demonstrate that EGFR and ErbB2 are critical mediators of TNF-regulated antiapoptotic signals in intestinal epithelial cells. Given evidence for TNF signaling in the development of colitis-associated carcinoma, this observation has significant implications for understanding the role of EGFR in maintaining intestinal epithelial cell homeostasis during cytokine-mediated inflammatory responses.

Inter-conversion of neuregulin2 full and partial agonists for ErbB4.

The EGF family hormone NRG2beta potently stimulates ErbB4 tyrosine phosphorylation and coupling to IL3 independence. In contrast, the NRG2alpha splicing isoform has lower affinity for ErbB4, does not potently stimulate ErbB4 phosphorylation, and fails to stimulate ErbB4 coupling. Here we investigate these differences. The NRG2beta Q43L mutant potently stimulates ErbB4 phosphorylation but not ErbB4 coupling to IL3 independence. This failure to stimulate ErbB4 coupling is not due to differential ligand purity, glycosylation, or stability. The NRG2alpha K45F mutant potently stimulates ErbB4 phosphorylation but not ErbB4 coupling to IL3 independence. Thus, this failure to stimulate ErbB4 coupling is not due to inadequate affinity for ErbB4. In contrast, the NRG2alpha L43Q/K45F mutant stimulates ErbB4 coupling, even though it does not have greater affinity for ErbB4 than does NRG2alpha/K45F. Collectively, these data indicate that Gln43 of NRG2beta is both necessary and sufficient for NRG2 stimulation of ErbB4 coupling to IL3 independence.

The importance of dual 5alpha-reductase inhibition in the treatment of male pattern hair loss: results of a randomized placebo-controlled study of dutasteride versus finasteride.

BACKGROUND: Male pattern hair loss (MPHL) is a potentially reversible condition in which dihydrotestosterone is an important etiologic factor. OBJECTIVE: Our aim was to evaluate the efficacy of the type 1 and 2 5alpha-reductase inhibitor dutasteride in men with MPHL. METHODS: Four hundred sixteen men, 21 to 45 years old, were randomized to receive dutasteride 0.05, 0.1, 0.5 or 2.5 mg, finasteride 5 mg, or placebo daily for 24 weeks. RESULTS: Dutasteride increased target area hair count versus placebo in a dose-dependent fashion and dutasteride 2.5 mg was superior to finasteride at 12 and 24 weeks. Expert panel photographic review and investigator assessment of hair growth confirmed these results. Scalp and serum dihydrotestosterone levels decreased, and testosterone levels increased, in a dose-dependent fashion with dutasteride. LIMITATIONS: The study was limited to 24 weeks. CONCLUSION: Dutasteride increases scalp hair growth in men with MPHL. Type 1 and type 2 5alpha-reductase may be important in the pathogenesis and treatment of MPHL.

Phe45 of NRG2beta is critical for the affinity of NRG2beta for ErbB4 and for potent stimulation of ErbB4 signaling by NRG2beta*.

The Neuregulins (NRGs) are members of the epidermal growth factor (EGF) family of growth factors. EGF family members regulate the signaling of ErbB family receptor tyrosine kinases, including the epidermal growth factor receptor (EGFR/ErbB1), ErbB2/HER2/Neu, ErbB3/HER3 and ErbB4/HER4. We have previously demonstrated that the EGF family hormone NRG2beta is a potent ErbB4 agonist, whereas NRG2alpha is a weak ErbB4 agonist. We have also previously demonstrated that Phe45 of NRG2beta regulates the potency of NRG2beta. Here, we address the hypotheses that Phe45 regulates the potency of NRG2beta by regulating the affinity of NRG2beta for ErbB4. We demonstrate that Phe45 of NRG2beta indeed regulates the affinity of NRG2beta for ErbB4. Furthermore, a hydrophobic or uncharged amino acid side chain at residue 45 contributes to NRG2beta binding to ErbB4. These data indicate that Phe45 of NRG2beta may regulate the affinity of NRG2beta for ErbB4 by interacting with hydrophobic amino acids in ErbB4.

Marked suppression of dihydrotestosterone in men with benign prostatic hyperplasia by dutasteride, a dual 5alpha-reductase inhibitor.

Dihydrotestosterone (DHT) is the primary metabolite of testosterone in the prostate and skin. Testosterone is converted to DHT by 5alpha-reductase, which exists in two isoenzyme forms (types 1 and 2). DHT is associated with development of benign prostatic hyperplasia (BPH), and reduction in its level with 5alpha-reductase inhibitors improves the symptoms associated with BPH and reduces the risk of acute urinary retention and prostate surgery. A selective inhibitor of the type 2 isoenzyme (finasteride) has been shown to decrease serum DHT by about 70%. We hypothesized that inhibition of both isoenzymes with the dual inhibitor dutasteride would more effectively suppress serum DHT levels than selective inhibition of only the type 2 isoenzyme. A total of 399 patients with BPH were randomized to receive once-daily dosing for 24 wk of dutasteride (0.01, 0.05, 0.5, 2.5, or 5.0 mg), 5 mg finasteride, or placebo. The mean percent decrease in DHT was 98.4 +/- 1.2% with 5.0 mg dutasteride and 94.7 +/- 3.3% with 0.5 mg dutasteride, significantly lower (P < 0.001) and with less variability than the 70.8 +/- 18.3% suppression observed with 5 mg finasteride. Mean testosterone levels increased but remained in the normal range for all treatment groups. Dutasteride appeared to be well tolerated with an adverse event profile similar to placebo.

Five carboxyl-terminal residues of neuregulin2 are critical for stimulation of signaling by the ErbB4 receptor tyrosine kinase.

The neuregulins (NRGs) are members of the epidermal growth factor (EGF) family of peptide growth factors. These hormones are agonists for the ErbB family of receptor tyrosine kinases, a family that includes the epidermal growth factor receptor (EGFR/ErbB1), ErbB2/Neu/HER2, ErbB3/HER3, and ErbB4/HER4. We recently observed that the EGF family hormone NRG2beta is a potent agonist for ErbB4. In contrast, NRG2alpha, a splicing isoform of the same gene that encodes NRG2beta, is a poor ErbB4 agonist. We hypothesized that carboxyl-terminal residues of NRG2beta are critical for stimulation of ErbB4 tyrosine phosphorylation and coupling to downstream signaling events. Here, we demonstrate that the substitution of a lysine residue for Phe45 in NRG2beta results in reduced ligand potency. We also demonstrate that substitution of a phenylalanine for Lys45 in NRG2alpha results in increased ligand potency. Finally, analyses of the gain-of-function NRG2alpha Chg5 mutant demonstrate that Gln43, Met47, Asn49, and Phe50 regulate ligand efficacy. Thus, these data indicate that carboxyl-terminal residues of NRG2beta are critical for activation of ErbB4 signaling. Moreover, these NRG2alpha and NRG2beta mutants reveal new insights into models for ligand-induced ErbB family receptor tyrosine phosphorylation and coupling to downstream signaling events.

Neuregulin isoforms exhibit distinct patterns of ErbB family receptor activation.

During the last decade, several novel members of the Epidermal Growth Factor family of peptide growth factors have been identified. Most prominent among these are the Neuregulins or Heregulins. To date, four different Neuregulin genes have been identified (Neuregulin1-4) and several different splicing isoforms have been identified for at least two of these genes (Neuregulin1 and Neuregulin2). While Neuregulin1 isoforms have been extensively studied, comparatively little is known about Neuregulin3, Neuregulin4, or the Neuregulin2 isoforms. Indeed, there has been no systematic comparison of the activities of these molecules. Here we demonstrate that Neuregulin2alpha and Neuregulin2beta stimulate ErbB3 tyrosine phosphorylation and coupling to biological responses. In contrast, Neuregulin3 and Neuregulin4 fail to activate ErbB3 signaling. Furthermore, Neuregulin2beta, but not Neuregulin2alpha, stimulates ErbB4 tyrosine phosphorylation and coupling to biological responses. Finally, both Neuregulin3 and Neuregulin4 stimulate modest amounts of ErbB4 tyrosine phosphorylation. However, whereas Neuregulin3 stimulates a modest amount of ErbB4 coupling to biological responses, Neuregulin4 fails to stimulate ErbB4 coupling to biological responses. This suggests that there are qualitative as well as quantitative differences in ErbB family receptor activation by Neuregulin isoforms.

Effects of vagal afferent stimulation on cervical spinothalamic tract neurons in monkeys.

The purpose of this study was to determine if electrical stimulation of vagal afferents inhibited activity of primate spinothalamic tract (STT) neurons located in cervical segments of the spinal cord. Previous studies show vagal inhibition of STT neurons in more caudal segments of the cord, which receive visceral spinal inputs and somatic inputs from proximal body regions. We hypothesized that activation of vagal afferents would inhibit cervical STT neurons that were excited by cardiopulmonary sympathetic afferents and not inhibit those cells inhibited or unaffected by this visceral input. Because visceral pain is referred to proximal somatic fields, we also hypothesized that STT neurons with excitatory somatic fields confined to distal areas would not be inhibited by vagal stimulation. In 42 cervical STT neurons, we found no difference in effects of vagal stimulation between cells excited or not excited by stimulation of cardiopulmonary sympathetic afferents. Responses to vagal stimulation also were the same for cervical STT cells with proximal or distal somatic fields. Furthermore, there was no difference in the inhibitory effects of vagal stimulation in cervical as compared to thoracic STT neurons. We concluded that vagal afferent stimulation causes a general inhibitory effect at all levels of the spinal cord on neurons which transmit nociceptive information.