Denture Adhesives Associated with Silver Vanadate: Antimicrobial Approach Against Multi- Species Biofilms on Acrylic Resin Surfaces.

Alternatives have been sought to add an antimicrobial property to denture adhesives. This study evaluated the antimicrobial potential of adhesives associated with nanostructured silver vanadate decorated with silver nanoparticles (ß-AgVO3). Specimens in acrylic resin were treated with the adhesives associated with ß-AgVO3 (1%, 2.5%, 5% and 10%). As control, specimens treated only with Ultra Corega Cream (UCC) or Ultra Corega Powder (UCP) adhesive were used. Multispecies biofilm of Candida albicans, Candida glabrata, Streptococcus mutans and Staphylococcus aureus was evaluated by counting colony forming units per milliliter (CFU/mL), colorimetric assay and fluorescence microscopy. The data were analyzed using the two-way analysis of variance (ANOVA) and Bonferroni multiple comparisons test (α=0.05). For both adhesives, a small amount of ß-AgVO3 (1%) completely inhibited S. mutans (P⟨0.05). For the other microorganisms, there was a reduction in metabolic activity and complete inhibition in the groups with intermediate or greater amounts of nanomaterial (P⟨0.05), except for C. albicans, which was reduced (P⟨0.05) but not completely inhibited in UCP. Microscopy that showed less biofilm in the groups with ß-AgVO3 and in the UCC than UCP. Denture adhesives in powder and cream form with ß-AgVO3 showed potential antimicrobial activity against multispecies biofilm. Powder adhesive showed higher biofilm formation.

Early circulating tumour DNA kinetics measured by ultra-deep next-generation sequencing during radical radiotherapy for non-small cell lung cancer: a feasibility study.

BACKGROUND: The evaluation of circulating tumour DNA (ctDNA) from clinical blood samples, liquid biopsy, offers several diagnostic advantages compared with traditional tissue biopsy, such as shorter processing time, reduced patient risk and the opportunity to assess tumour heterogeneity. The historically poor sensitivity of ctDNA testing, has restricted its integration into routine clinical practice for non-metastatic disease. The early kinetics of ctDNA during radical radiotherapy for localised NSCLC have not been described with ultra-deep next generation sequencing previously. MATERIALS AND METHODS: Patients with CT/PET-staged locally advanced, NSCLC prospectively consented to undergo serial venepuncture during the first week of radical radiotherapy alone. All patients received 55Gy in 20 fractions. Plasma samples were processed using the commercially available Roche AVENIO Expanded kit (Roche Sequencing Solutions, Pleasanton, CA, US) which targets 77 genes. RESULTS: Tumour-specific mutations were found in all patients (1 in 3 patients; 2 in 1 patient, and 3 in 1 patient). The variant allele frequency of these mutations ranged from 0.05-3.35%. In 2 patients there was a transient increase in ctDNA levels at the 72 h timepoint compared to baseline. In all patients there was a non-significant decrease in ctDNA levels at the 7-day timepoint in comparison to baseline (p = 0.4627). CONCLUSION: This study demonstrates the feasibility of applying ctDNA-optimised NGS protocols through specified time-points in a small homogenous cohort of patients with localised lung cancer treated with radiotherapy. Studies are required to assess ctDNA kinetics as a predictive biomarker in radiotherapy. Priming tumours for liquid biopsy using radiation warrants further exploration.

Circulating tumour DNA is a potential biomarker for disease progression and response to targeted therapy in advanced thyroid cancer.

BACKGROUND: Conventional biomarkers in thyroid cancer are not disease specific and fluctuate in advanced disease, making interpretation difficult. Circulating tumour DNA (ctDNA) has been shown to be a useful biomarker in other solid tumours. This is a multimutational study of ctDNA over multiple timepoints, designed to test the hypothesis that ctDNA is a potential biomarker in patients with advanced thyroid cancer. METHODS: Mutational analysis of archival tumour tissue was performed using NGS with a targeted gene panel. Custom TaqMan assays were designed for plasma ctDNA testing using digital droplet polymerase chain reaction. Concentrations of detected ctDNA were correlated with the conventional biomarker concentration and axial imaging status defined by the Response Evaluation Criteria in Solid Tumours criteria. RESULTS: Tumour tissue from 51 patients was obtained, with the following histologies: 32 differentiated (differentiated thyroid cancer [DTC]), 15 medullary (medullary thyroid cancer [MTC]), three poorly differentiated and one anaplastic. NGS analysis detected variants in 42 (82%) of cases. Plasma was assayed for these patients in 190 samples, and ctDNA was detected in 67% of patients. Earlier detection of disease progression was noted in three patients with MTC. In two cases (PTC and ATC), where conventional biomarkers were not detectable, ctDNA was detected before disease progression. Changes in ctDNA concentration occurred earlier than conventional markers in response to disease progression in multiple patients with DTC receiving targeted therapies. CONCLUSION: The majority of patients with advanced thyroid cancer had detectable ctDNA. ctDNA measurement may offer superiority over conventional markers in several scenarios: earlier detection of progression in MTC; as an alternative biomarker when conventional markers are not available; more rapid assessment of the disease status in response to targeted therapies, thereby potentially allowing prompter discontinuation of futile therapies. These early results support the hypothesis that ctDNA may be a clinically useful biomarker in thyroid cancer.

Spatial distribution of tuberculosis in Manaus and its social determinants, 2008-2013.

SETTING: Brazil ranks eighteenth worldwide in annual numbers of new tuberculosis (TB) cases. The municipality of Manaus, Amazonas State, has the highest incidence of TB in Brazil. OBJECTIVE: To evaluate the quality of TB epidemiological surveillance, and to describe the spatial distribution pattern of TB incidence in Manaus and its social determinants. DESIGN: An ecological study was performed based on secondary data from TB epidemiological surveillance reports. RESULTS: An index was developed to classify neighborhoods in terms of the quality of surveillance and suspected underreporting. Based on data from neighborhoods with better surveillance performance, we observed that the average number of residents per room, the unemployment rate and the proportion of households connected to a sewage system were significant predictors of TB incidence. Seven neighborhoods in the south and west of the city had clusters of high TB transmission. CONCLUSION: Our results suggest that the association between TB and social vulnerability is obscured by the poor quality of TB surveillance data. We identified priority areas that require immediate TB control interventions and those where local surveillance efforts should be improved, and generated information useful for formulating more effective actions.

Investigating the feasibility of tumour molecular profiling in gastrointestinal malignancies in routine clinical practice.

Background: Targeted capture sequencing can potentially facilitate precision medicine, but the feasibility of this approach in gastrointestinal (GI) malignancies is unknown. Patients and methods: The FOrMAT (Feasibility of a Molecular Characterisation Approach to Treatment) study was a feasibility study enrolling patients with advanced GI malignancies from February 2014 to November 2015. Targeted capture sequencing (mainly using archival formalin-fixed paraffin-embedded diagnostic/resection samples) was carried out to detect mutations, copy number variations and translocations in up to 46 genes which had prognostic/predictive significance or were targets in current/upcoming clinical trials. Results: Of the 222 patients recruited, 215 patients (96.8%) had available tissue samples, 125 patients (56.3%) had ≥16 genes successfully sequenced and 136 patients (61.2%) had ≥1 genes successfully sequenced. Sample characteristics influenced the proportion of successfully sequenced samples, e.g. tumour type (colorectal 70.9%, biliary 52.6%, oesophagogastric 50.7%, pancreas 27.3%, P = 0.002), tumour cellularity (high versus low: 78.3% versus 13.3%, P ≤ 0.001), tumour content (high versus low: 78.6% versus 27.3%, P = 0.001) and type of sample (resection versus biopsy: 82.4% versus 47.6%, P ≤ 0.001). Currently, actionable alterations were detected in 90 (40.5%) of the 222 patients recruited (66% of the 136 patients sequenced) and 2 patients subsequently received a targeted therapy. The most frequently detected currently actionable alterations were mutations in KRAS, BRAF, TP53 and PIK3CA. For the 205 patients with archival samples, the median time to obtain sequencing results was 18.9 weeks, including a median of 4.9 weeks for sample retrieval and 5.1 weeks for sequencing. Conclusions: Targeted sequencing detected actionable alterations in formalin-fixed paraffin-embedded samples, but tissue characteristics are of critical importance in determining sequencing success. Routine molecular profiling of GI tumours outside of clinical trials is not an effective use of healthcare resources unless more targeted drugs become available. ClinicalTrials.gov identifier: NCT02112357.

Pompe Disease and Autophagy: Partners in Crime, or Cause and Consequence?

Pompe disease or glycogen storage disease type II (OMIM: 232300) is a lysosomal storage disorder resulting from a partial or total lack of acid alphaglucosidase, which may produce muscle weakness, gait abnormalities, or even death by respiratory failure. In the last decade, autophagy has been proposed as a mechanism involved in the severity of symptoms related to this disorder and as a potential therapeutic target to alleviate disease progression. This review summarizes the relationship between autophagy and Pompe disease, including what information has been recently discovered and what remains unclear.

Prognostic role of the LCS6 KRAS variant in locally advanced rectal cancer: results of the EXPERT-C trial.

BACKGROUND: Lethal-7 (let-7) is a tumour suppressor miRNA which acts by down-regulating several oncogenes including KRAS. A single-nucleotide polymorphism (rs61764370, T > G base substitution) in the let-7 complementary site 6 (LCS-6) of KRAS mRNA has been shown to predict prognosis in early-stage colorectal cancer (CRC) and benefit from anti-epidermal growth factor receptor monoclonal antibodies in metastatic CRC. PATIENTS AND METHODS: We analysed rs61764370 in EXPERT-C, a randomised phase II trial of neoadjuvant CAPOX followed by chemoradiotherapy, surgery and adjuvant CAPOX plus or minus cetuximab in locally advanced rectal cancer. DNA was isolated from formalin-fixed paraffin-embedded tumour tissue and genotyped using a PCR-based commercially available assay. Kaplan-Meier method and Cox regression analysis were used to calculate survival estimates and compare treatment arms. RESULTS: A total of 155/164 (94.5%) patients were successfully analysed, of whom 123 (79.4%) and 32 (20.6%) had the LCS-6 TT and LCS-6 TG genotype, respectively. Carriers of the G allele were found to have a statistically significantly higher rate of complete response (CR) after neoadjuvant therapy (28.1% versus 10.6%; P = 0.020) and a trend for better 5-year progression-free survival (PFS) [77.4% versus 64.5%: hazard ratio (HR) 0.56; P = 0.152] and overall survival (OS) rates (80.3% versus 71.9%: HR 0.59; P = 0.234). Both CR and survival outcomes were independent of the use of cetuximab. The negative prognostic effect associated with KRAS mutation appeared to be stronger in patients with the LCS-6 TT genotype (HR PFS 1.70, P = 0.078; HR OS 1.79, P = 0.082) compared with those with the LCS-6 TG genotype (HR PFS 1.33, P = 0.713; HR OS 1.01, P = 0.995). CONCLUSION: This analysis suggests that rs61764370 may be a biomarker of response to neoadjuvant treatment and an indicator of favourable outcome in locally advanced rectal cancer possibly by mitigating the poor prognosis of KRAS mutation. In this setting, however, this polymorphism does not appear to predict cetuximab benefit.

HER2 in high-risk rectal cancer patients treated in EXPERT-C, a randomized phase II trial of neoadjuvant capecitabine and oxaliplatin (CAPOX) and chemoradiotherapy (CRT) with or without cetuximab.

BACKGROUND: HER2 is an established therapeutic target in breast and gastric cancers. The role of HER2 in rectal cancer is unclear, as conflicting data on the prevalence of HER2 expression in this disease have been reported. We evaluated the prevalence of HER2 and its impact on the outcome of high-risk rectal cancer patients treated with neoadjuvant CAPOX and CRT±cetuximab in the EXPERT-C trial. PATIENTS AND METHODS: Eligible patients with available tumour tissue for HER2 analysis were included. HER2 expression was determined by immunohistochemistry (IHC) in pre-treatment biopsies and/or surgical specimens (score 0-3+). Immunostaining was scored according to the consensus panel recommendations on HER2 scoring for gastric cancer. Tumours with equivocal IHC result (2+) were tested for HER2 amplification by D-ISH. Tumours with IHC 3+ or D-ISH ratio ≥2.0 were classified as HER2+. The impact of HER2 on primary and secondary end points of the study was analysed. RESULTS: Of 164 eligible study patients, 104 (63%) biopsy and 114 (69%) surgical specimens were available for analysis. Only 3 of 104 (2.9%) and 3 of 114 (2.6%) were HER2+, respectively. In 77 patients with paired specimens, concordance for HER2 status was found in 74 (96%). Overall, 141 patients were assessable for HER2 and 6 out of 141 (4.3%) had HER2 overexpression and/or amplification. The median follow-up was 58.6 months. HER2 was not associated with a difference in the outcome for any of the study end points, including in the subset of 90 KRAS/BRAF wild-type patients treated±cetuximab. CONCLUSIONS: Based on the low prevalence of expression as recorded in the EXPERT-C trial, HER2 does not appear to represent a useful therapeutic target in high-risk rectal cancer. However, the role of HER2 as a potential predictive biomarker of resistance to anti-EGFR-based treatments and a therapeutic target in anti-EGFR refractory metastatic colorectal cancer (CRC) warrants further investigation. TRIAL REGISTRATION: ISRCTN Register: 99828560.

Biomarker analysis in oesophagogastric cancer: Results from the REAL3 and TransMAGIC trials.

BACKGROUND: REAL3 (Randomised ECF for Advanced or Locally advanced oesophagogastric cancer 3) was a phase II/III trial designed to evaluate the addition of panitumumab (P) to epirubicin, oxaliplatin and capecitabine (EOC) in untreated advanced oesophagogastric adenocarcinoma, or undifferentiated carcinoma. MAGIC (MRC Adjuvant Gastric Infusional Chemotherapy) was a phase III study which demonstrated that peri-operative epirubicin, cisplatin and infused 5-fluorouracil (ECF) improved survival in early oesophagogastric adenocarcinoma. PATIENTS AND METHODS: Analysis of response rate (RR; the primary end-point of phase II) and biomarkers in the first 200 patients randomised to EOC or modified dose (m) EOC+P in REAL3 was pre-planned to determine if molecular selection for the on-going study was indicated. KRAS, BRAF and PIK3CA mutations and PTEN expression were assessed in pre-treatment biopsies and results correlated with response to mEOC+P. Association between these biomarkers and overall survival (OS) was assessed in MAGIC patients to determine any prognostic effect. RESULTS: RR was 52% to mEOC+P, 48% to EOC. Results from 175 assessable biopsies: mutations in KRAS (5.7%), BRAF (0%), PIK3CA (2.5%) and loss of PTEN expression (15.0%). None of the biomarkers evaluated predicted resistance to mEOC+P. In MAGIC, mutations in KRAS, BRAF and PIK3CA and loss of PTEN (phosphatase and tensin homolog) were found in 6.3%, 1.0%, 5.0% and 10.9%, respectively, and were not associated with survival. CONCLUSIONS: The RR of 52% in REAL3 with mEOC+P met pre-defined criteria to continue accrual to phase III. The frequency of the mutations was too low to exclude any prognostic or predictive effect.

Personalized cancer medicine: molecular diagnostics, predictive biomarkers, and drug resistance.

The progressive elucidation of the molecular pathogenesis of cancer has fueled the rational development of targeted drugs for patient populations stratified by genetic characteristics. Here we discuss general challenges relating to molecular diagnostics and describe predictive biomarkers for personalized cancer medicine. We also highlight resistance mechanisms for epidermal growth factor receptor (EGFR) kinase inhibitors in lung cancer. We envisage a future requiring the use of longitudinal genome sequencing and other omics technologies alongside combinatorial treatment to overcome cellular and molecular heterogeneity and prevent resistance caused by clonal evolution.

A comparison of three methods for detecting KRAS mutations in formalin-fixed colorectal cancer specimens.

BACKGROUND: KRAS mutation testing is required to select patients with metastatic colorectal cancer (CRC) to receive anti-epidermal growth factor receptor antibodies, but the optimal KRAS mutation test method is uncertain. METHODS: We conducted a two-site comparison of two commercial KRAS mutation kits - the cobas KRAS Mutation Test and the Qiagen therascreen KRAS Kit - and Sanger sequencing. A panel of 120 CRC specimens was tested with all three methods. The agreement between the cobas test and each of the other methods was assessed. Specimens with discordant results were subjected to quantitative massively parallel pyrosequencing (MPP). DNA blends were tested to determine detection rates at 5% mutant alleles. RESULTS: Reproducibility of the cobas test between sites was 98%. Six mutations were detected by cobas that were not detected by Sanger, and five were confirmed by MPP. The cobas test detected eight mutations which were not detected by the therascreen test, and seven were confirmed by MPP. Detection rates with 5% mutant DNA blends were 100% for the cobas and therascreen tests and 19% for Sanger. CONCLUSION: The cobas test was reproducible between sites, and detected several mutations that were not detected by the therascreen test or Sanger. Sanger sequencing had poor sensitivity for low levels of mutation.

Selective FLT3 inhibition of FLT3-ITD+ acute myeloid leukaemia resulting in secondary D835Y mutation: a model for emerging clinical resistance patterns.

Acquired resistance to selective FLT3 inhibitors is an emerging clinical problem in the treatment of FLT3-ITD(+) acute myeloid leukaemia (AML). The paucity of valid pre-clinical models has restricted investigations to determine the mechanism of acquired therapeutic resistance, thereby limiting the development of effective treatments. We generated selective FLT3 inhibitor-resistant cells by treating the FLT3-ITD(+) human AML cell line MOLM-13 in vitro with the FLT3-selective inhibitor MLN518, and validated the resistant phenotype in vivo and in vitro. The resistant cells, MOLM-13-RES, harboured a new D835Y tyrosine kinase domain (TKD) mutation on the FLT3-ITD(+) allele. Acquired TKD mutations, including D835Y, have recently been identified in FLT3-ITD(+) patients relapsing after treatment with the novel FLT3 inhibitor, AC220. Consistent with this clinical pattern of resistance, MOLM-13-RES cells displayed high relative resistance to AC220 and Sorafenib. Furthermore, treatment of MOLM-13-RES cells with AC220 lead to loss of the FLT3 wild-type allele and the duplication of the FLT3-ITD-D835Y allele. Our FLT3-Aurora kinase inhibitor, CCT137690, successfully inhibited growth of FLT3-ITD-D835Y cells in vitro and in vivo, suggesting that dual FLT3-Aurora inhibition may overcome selective FLT3 inhibitor resistance, in part due to inhibition of Aurora kinase, and may benefit patients with FLT3-mutated AML.

Low-energy laser therapy for prevention of oral mucositis in hematopoietic stem cell transplantation.

AIM: To evaluate the clinical effects of laser therapy on the prevention and reduction of oral mucositis in patients who underwent hematopoietic stem cell transplantation (HSCT). PATIENTS AND METHODS: From January 2003 to September 2004, 24 patients received prophylactic laser therapy (L+ group). The applications started from the beginning of the conditioning regimen up to day +2. The oral assessment was performed daily until day +30. This group was compared with historical controls, namely 25 patients, who did not receive laser therapy (L- group). RESULTS: All patients developed some grade of mucositis. However, the L- group presented initial mucositis by 4.36 days, whereas the L+ group presented it in 6.12 days (P = 0.01). The maximum mucositis occurred between day +2 and day +6 with healing by day +25 in the L- group and between day +2 and day +7 with healing by day +14 for the L+ group (P = 0.84). Laser therapy also reduced the time of oral pain from 5.64 to 2.45 days (P = 0.04), and decreased the consumption of morphine (P = 0.07). CONCLUSION: This study suggests that laser therapy can be useful in oral mucositis to HSCT patients and improve the patient's quality of life. However, controlled randomized trials should be performed to confirm the real efficacy of laser therapy.

Fixed tibial subluxation after successful anterior cruciate ligament reconstruction.

Anterior cruciate ligament reconstruction results in improved function and stability in many patients. However, it is not known whether the improved stability is associated with an improved tibiofemoral relationship. We used stress radiographs to determine not only stability but also the tibiofemoral relationship in 15 patients who had a clinically successful anterior cruciate ligament reconstruction. Their results were compared with those of 14 volunteers with normal knees. The average Lysholm score for the patients was 94. None of the patients had more than 3 mm of side-to-side difference on KT-1000 arthrometer testing. Maximal anteroposterior tibial translation as measured by stress radiography was slightly increased in the reconstructed knees but was not statistically significantly different (6.6 +/- 3.2 mm versus 5.0 +/- 3.3 mm). However, with a posteriorly directed stress the tibia in the reconstructed knees did not translate posteriorly to the same extent as did the control knees, resulting in a significant difference in tibial position (-1.2 +/- 3.0 mm versus -4.0 +/- 3.3 mm). Surgical anterior cruciate ligament reconstruction may result in reduced anteroposterior tibial translation, accomplished, in part, through restraining posterior translation, leaving the tibia with persistent subluxation. Fibrosis and contracture of the posterior structures may explain this phenomenon.

A study on the acute anti-ulcer effects of narra (Pterocarpus Indicus Will.) on indomethacin-induced gastric ulcers in male albino rats (Rattus rattus)

Gastric, duodenal and peptic ulcers are collectively ranked as one of the causes of deaths in the country. Management of these diseases comes at a high cost. The researchers explored the use of indigenous narra (Pterocarpus indicus Will.) as a low cost alternative to other expensive medications. This study aimed to determine the presence and degree of ulcerations in Indomethacin-induced gastric ulcers in male albino rats after treatment with either narra leaf decoction or sucralfate. It also aimed to compare the anti-ulcer effects of different dosages of narra leaf decoction with that of sucralfateTwenty-five male rats weighting 115-200 grams were randomly divided into five groups. Gastric ulcers were induced by orally administering 30 mg/kg body weight (BW) of Indomethacin in all rats. Treatments were divided as follows: Negative control (NSS)-10 mg/kg BW normal saline solution; positive control (SUC)-1 g/kg BW sucralfate; Narra group 1 (N1)-3.94 g/kg BW narra leaf decoction; Narra group 2 (N2)-9.89 g/kg BW narra leaf decoction; and, Narra group 3 (N3)-24.84 g/kg BW narra leaf decoction. All treatments were administered using oral gavage and were repeated at intervals of 24 hours for three days. Six hours after the last administration of treatment, the rats were sacrificed and their stomachs excised. Gross analysis was done using the Bests Ulcer Staging Index while histopathological analysis was performed according to the presence and degree of ulcers and hemorrhage. Results were analyzed using Kruskal- Wallis Test for one-way ANOVAOn gross analysis, ulcers and hemorrhages were seen in some of the rat stomachs but the difference in the effects of the treatments on the different groups was not statistically significant. On histopathological analysis, ulcers and hemorrhages were evident in the NSS group but were not noted in the SUC group. The difference between the SUC and NSS groups was statistically significant. Histopathologic studies also showed the following: 2/5 rats had ulcers in Narra group 1; 2/5 in Narra group 2; and, none in Narra group 3 (but all of these rats died before the end of the experiment). However, histopathological differences among the treatment groups were not statistically significantThese findings suggest that narra may have anti-ulcer effects. It is recommended that a dosage higher than 24.84 g/kg BW (the highest narra dosage administered for three days in this experiment) be used in further experiments. The duration of exposure to the drug should also be lengthened. (Author)

Electrophysiological study of dorsal respiratory neurons in the medulla oblongata of the rat.

There has been controversy whether the dorsal respiratory group (DRG), identified in the cat and several other species as a concentration of mainly inspiratory neurons located in the ventrolateral subnucleus of the solitary tract, also exists in the rat. The aim of this study was to re-examine this question by systematically exploring this region with extracellular microelectrodes, in anesthetized and artificially ventilated rats. One-hundred and forty-two units were recorded which fired in phase with central respiratory cycles (determined by recording from the phrenic nerve) and/or lung inflations. One-hundred and nineteen recordings were thought to be from neuronal cell bodies (confirmed in some cases by excitatory responses to microelectrophoretic administration of DL-homocysteic acid), while the remaining 23 were from lung vagal afferents. Most neurons in the former group (87/119) were inspiratory. Out of 96 neurons tested for spinal projections only 14 (12 inspiratory, 2 expiratory) responded antidromically following stimulation at C3 segment. These results confirm the existence of the DRG in the rat and demonstrate that neurons located in this region have firing patterns generally similar to those previously described in the cat. The main difference is the relative paucity in the rat of neurons projecting spinally below the C2 level, which indicates that most DRG neurons in this species do not project directly to phrenic and intercostal motoneurons, but to other, as yet unidentified, neuronal groups within the brainstem or upper cervical segments.

Expiratory neurons of the Bötzinger Complex in the rat: a morphological study following intracellular labeling with biocytin.

The term "Bötzinger Complex" (BOT) refers to a distinct group of neurons, located near the rostral portion of the nucleus ambiguus, which are known to play an important role in the control of respiratory movements. Previous studies conducted in cats have demonstrated that most of these neurons are active during expiration, exerting a monosynaptic inhibitory action on several subpopulations of inspiratory neurons in the medulla and spinal cord. The aim of this study was to examine morphological properties and possible synaptic targets of BOT neurons in the rat. Forty-one expiratory neurons were labeled intracellularly with biocytin; 12 were interneurons (BOT neurons) and 29 were motoneurons. The latter could not be antidromically activated following stimulation of the superior laryngeal or vagal nerves. BOT neurons showed extensive axonal arborisations in the ipsilateral medulla, with some projections to the contralateral side. Bouton-like axon varicosities mainly clustered in two areas: near the parent cell bodies, and in the area corresponding to the rostral part of the ventral respiratory group (VRG). In five pairs of labeled neurons, each consisting of one BOT neuron and one inspiratory neuron in the rostral VRG, no appositions were identified at the light microscopic level between axons of BOT neurons and dendrites or cell bodies of inspiratory neurons. These results demonstrate that some features of BOT expiratory neurons in the rat are similar to those previously described in cats. The differences include their more ventral location in relation to the compact formation of nucleus ambiguus (retrofacial nucleus), and the relative paucity in the rat of neurons displaying an augmenting pattern of activity and of neurons with spinally projecting axons. In addition, we were unable to find morphological evidence for contacts between labeled BOT neurons and ipsilateral inspiratory neurons near the obex level, a finding not consistent with previous electrophysiological studies in the cat in which such synaptic connections have been identified.