Leveraging binding pose metadynamics to optimise target fishing predictions for three diverse ligands and their true targets.

Computational target fishing plays an important role in target identification, particularly in drug discovery campaigns utilizing phenotypic screening. Numerous approaches exist to predict potential targets for a given ligand, but true targets may be inconsistently ranked. More advanced simulation methods may provide benefit in such cases by reranking these initial predictions. We evaluated the ability of binding pose metadynamics to improve the predicted rankings for three diverse ligands and their six true targets. Initial predictions using pharmacophore mapping showed no true targets ranked in the top 50 and two targets each ranked within the 50-100, 100-150, and 250-300 ranges respectively. Following binding pose metadynamics, ranking of true targets improved for four out of the six targets and included the highest ranked predictions overall, while rankings deteriorated for two targets. The revised rankings predicted two true targets ranked within the top 50, and one target each within the 50-100, 100-150, 150-200, and 200-250 ranges respectively. The findings of this study demonstrate that binding pose metadynamics may be of benefit in refining initial predictions from structure-based target fishing algorithms, thereby improving the efficiency of the target identification process in drug discovery efforts.

Automated Prediction of Malignant Melanoma using Two-Stage Convolutional Neural Network.

PURPOSE: A skin lesion refers to an area of the skin that exhibits anomalous growth or distinctive visual characteristics compared to the surrounding skin. Benign skin lesions are noncancerous and generally pose no threat. These irregular skin growths can vary in appearance. On the other hand, malignant skin lesions correspond to skin cancer, which happens to be the most prevalent form of cancer in the United States. Skin cancer involves the unusual proliferation of skin cells anywhere on the body. The conventional method for detecting skin cancer is relatively more painful. METHODS: This work involves the automated prediction of skin cancer and its types using two stage Convolutional Neural Network (CNN). The first stage of CNN extracts low level features and second stage extracts high level features. Feature selection is done using these two CNN and ABCD (Asymmetry, Border irregularity, Colour variation, and Diameter) technique. The features extracted from the two CNNs are fused with ABCD features and fed into classifiers for the final prediction. The classifiers employed in this work include ensemble learning methods such as gradient boosting and XG boost, as well as machine learning classifiers like decision trees and logistic regression. This methodology is evaluated using the International Skin Imaging Collaboration (ISIC) 2018 and 2019 dataset. RESULTS: As a result, the first stage CNN which is used for creation of new dataset achieved an accuracy of 97.92%. Second stage CNN which is used for feature selection achieved an accuracy of 98.86%. Classification results are obtained for both with and without fusion of features. CONCLUSION: Therefore, two stage prediction model achieved better results with feature fusion.

Social isolation postweaning alters reward-related dopamine dynamics in a region-specific manner in adolescent male rats.

Early development is characterized by dynamic transitions in brain maturation, which may be impacted by environmental factors. Here, we sought to determine the effects of social isolation from postweaning and during adolescence on reward behavior and dopaminergic signaling in male rats. Subjects were socially isolated or group housed at postnatal day 21. Three weeks later, extracellular dopamine concentrations were examined in the medial prefrontal cortex (mPFC) and nucleus accumbens shell (NAc) during a feeding bout. Surprisingly, opposing effects were found in which increased mPFC dopamine concentrations were observed in group housed, but not isolated, rats. In stark contrast, increased dopamine levels were found in the NAc of isolated, but not group housed, rats. Moreover, the absence of an effect in the mPFC of the isolated rats could not be reversed by subsequent group housing, demonstrating the remarkable long-term effects on dopamine signaling dynamics. When provided a highly palatable food, the isolated subjects exhibited a dramatic increase in mPFC dopamine levels when the chocolate was novel, but no effects following chronic chocolate consumption. In contrast, the group housed subjects showed significantly increased dopamine levels only with chronic chocolate consumption. The dopamine changes were correlated with differences in behavioral measures. Importantly, the deficit in reward-related behavior during isolation could be reversed by microinjection of either dopamine or cocaine into the mPFC. Together, these data provide evidence that social isolation from postweaning and during adolescence alters reward-induced dopamine levels in a brain region-specific manner, which has important functional implications for reward-related behavior.

Impact of the COVID-19 Pandemic on Prenatal Care Utilization at a Public Hospital.

OBJECTIVE: The aim of the study is to compare rates of prenatal care utilization before and after implementation of a telehealth-supplemented prenatal care model due to the coronavirus disease 2019 (COVID-19) pandemic. STUDY DESIGN: Using electronic medical record data, we identified two cohorts of pregnant persons that initiated prenatal care prior to and during the COVID-19 pandemic following the implementation of telehealth (from March 1, 2019 through August 31, 2019, and from March 1, 2020, through August 31, 2020, respectively) at Grady Memorial Hospital. We used Pearson's Chi-square and two-tailed t-tests to compare rates of prenatal care utilization, antenatal screening and immunizations, emergency department and obstetric triage visits, and pregnancy complications for the prepandemic versus pandemic-exposed cohorts. RESULTS: We identified 1,758 pregnant patients; 965 entered prenatal care prior to the COVID-19 pandemic and 793 entered during the pandemic. Patients in the pandemic-exposed cohort were more likely to initiate prenatal care in the first trimester (46.1 vs. 39.0%, p = 0.01), be screened for gestational diabetes (74.4 vs. 67.0%, p <0.001), and receive dating and anatomy ultrasounds (17.8 vs. 13.0%, p = 0.006 and 56.9 vs. 47.3%, p <0.001, respectively) compared with patients in the prepandemic cohort. There was no difference in mean number of prenatal care visits between the two groups (6.9 vs. 7.1, p = 0.18). Approximately 41% of patients in the pandemic-exposed cohort had one or more telehealth visits. The proportion of patients with one or more emergency department visits was higher in the pandemic-exposed cohort than the prepandemic cohort (32.8 vs. 12.3%, p < 0.001). Increases in rates of labor induction were also observed among the pandemic-exposed cohort (47.1 vs. 38.2%, p <0.001). CONCLUSION: Rates of prenatal care utilization were similar before and during the COVID-19 pandemic. However, pregnant persons receiving prenatal care during the pandemic entered care earlier and had higher utilization of certain antenatal screening services than those receiving prenatal care prior to the pandemic. KEY POINTS: · Patients initiated prenatal care earlier during the COVID-19 pandemic.. · Uptake of telehealth services was low.. · Rates of diabetes screening and ultrasound use increased during the pandemic..

Factor Structure and Measurement Invariance of the Spanish Burnout Inventory Among Professionals Across 17 Countries and Regions.

Studies on the prevalence of burnout in professionals in service organizations who work in direct contact with the clients or users of the organization have concluded that burnout is a serious health disorder that has increased due to the COVID-19 pandemic. A significant advantage of the Spanish Burnout Inventory (SBI) over other instruments is that it provides a broader conceptualization of burnout by including feelings of guilt as a dimension of burnout to explain its development. However, the measurement invariance of the SBI across countries has not been investigated. The purpose of this study was to test the measurement invariance of the SBI among professionals across 17 countries and regions in Europe, Latin America, and Asia, and in different languages. All the countries showed a good fit to the four-factor model, except the Indian sample, which was excluded from the measurement invariance study. Using the alignment method, it was possible to verify the scalar measurement invariance of the four SBI factors across 15 countries and one Spanish region (16 samples). The comparison of estimated latent means indicates that France is the country with the lowest scores on the Enthusiasm factor and the highest scores on the negative factors (Exhaustion, Indolence, and Guilt). In contrast, the Andean countries, Colombia, Peru, and Ecuador, show the highest latent means on the Enthusiasm factor and the lowest means on the negative factors. These results support the validity of the SBI in the countries and regions in Europe and Latin America included in this study.

A year in review: Are diversity, equity, and inclusion initiatives fixing systemic barriers?

Are current diversity, equity, and inclusion initiatives addressing systemic issues? This article highlights the progress thus far and emphasizes the systemic and cultural shifts needed to support and retain historically excluded scientists.

α3* Nicotinic Acetylcholine Receptors in the Habenula-Interpeduncular Nucleus Circuit Regulate Nicotine Intake.

Allelic variation in CHRNA3, the gene encoding the α3 nicotinic acetylcholine receptor (nAChR) subunit, increases vulnerability to tobacco dependence and smoking-related diseases, but little is known about the role for α3-containing (α3*) nAChRs in regulating the addiction-related behavioral or physiological actions of nicotine. α3* nAChRs are densely expressed by medial habenula (mHb) neurons, which project almost exclusively to the interpeduncular nucleus (IPn) and are known to regulate nicotine avoidance behaviors. We found that Chrna3tm1.1Hwrt hypomorphic mice, which express constitutively low levels of α3* nAChRs, self-administer greater quantities of nicotine (0.4 mg kg-1 per infusion) than their wild-type littermates. Microinfusion of a lentivirus vector to express a short-hairpin RNA into the mHb or IPn to knock-down Chrna3 transcripts markedly increased nicotine self-administration behavior in rats (0.01-0.18 mg kg-1 per infusion). Using whole-cell recordings, we found that the α3ß4* nAChR-selective antagonist α-conotoxin AuIB almost completely abolished nicotine-evoked currents in mHb neurons. By contrast, the α3ß2* nAChR-selective antagonist α-conotoxin MII only partially attenuated these currents. Finally, micro-infusion of α-conotoxin AuIB (10 µm) but not α-conotoxin MII (10 µm) into the IPn in rats increased nicotine self-administration behavior. Together, these data suggest that α3ß4* nAChRs regulate the stimulatory effects of nicotine on the mHb-IPn circuit and thereby regulate nicotine avoidance behaviors. These findings provide mechanistic insights into how CHRNA3 risk alleles can increase the risk of tobacco dependence and smoking-related diseases in human smokers.SIGNIFICANCE STATEMENT Allelic variation in CHRNA3, which encodes the α3 nicotinic acetylcholine receptor (nAChR) subunit gene, increases risk of tobacco dependence but underlying mechanisms are unclear. We report that Chrna3 hypomorphic mice consume greater quantities of nicotine than wild-type mice and that knock-down of Chrna3 gene transcripts in the habenula or interpeduncular nucleus (IPn) increases nicotine intake in rats. α-Conotoxin AuIB, a potent antagonist of the α3ß4 nAChR subtype, reduced the stimulatory effects of nicotine on habenular neurons, and its infusion into the IPn increased nicotine intake in rats. These data suggest that α3ß4 nAChRs in the habenula-IPn circuit regulate the motivational properties of nicotine.

Adolescent Cannabinoid and Nicotine Exposure Differentially Alters Adult Nicotine Self-Administration in Males and Females.

INTRODUCTION: During adolescence, exposure to nicotine or cannabis independently induces effects on neuromaturation and later cognitive function. However, the potential effect of both drugs under co-use conditions has become of increasing concern given the prevalence of e-cigarettes, legalization of cannabis, and availability of synthetic "spice" cannabinoid agonists. AIMS AND METHODS: The current studies investigated the effects of exposure to a cannabinoid receptor agonist (WIN55,212-2) and/or nicotine over a discrete time period in mid-adolescence on later intravenous nicotine self-administration in adult male and female mice. We further examined whether cannabinoid agonist administration in adulthood would alter nicotine reinforcement, with either acute or chronic pairing across 7 days. RESULTS: We found that adult males exhibited increased nicotine self-administration at a lower, rewarding nicotine dose following adolescent cannabinoid exposure, either alone or with nicotine coadministration. In contrast, adult females demonstrated an opposing effect in which adolescent cannabinoid and nicotine coexposure resulted in decreased nicotine intake compared with the nicotine only and control groups. Furthermore, after maintaining nicotine self-administration across sessions, pretreatment with a low dose of the cannabinoid agonist decreased nicotine intake in both male and female control mice, and this lowering effect was evidenced after both acute and chronic treatment. However, the cannabinoid agonist was ineffective in altering nicotine intake in mice previously exposed to nicotine, cannabinoid agonist, or both during adolescence. CONCLUSIONS: These data provide evidence that adolescent drug exposure can alter later nicotine reinforcement in a sex-specific manner and can further modulate the effectiveness of interventions in reducing nicotine intake during adulthood. IMPLICATIONS: These studies demonstrate a significant impact of nicotine, cannabinoids, or coexposure on developmental processes during adolescence. Differential effects were observed within each sex, with opposing results found for cannabinoid exposure on nicotine intake in males and females. Intriguingly, we also evidenced resistance to the lowering effects of a cannabinoid agonist on nicotine intake in adulthood based on adolescent drug exposure. Thus, these findings have important implications for our understanding of the impact of nicotine and cannabinoids (eg, Δ9-tetrahydrocannabinol (THC) and synthetic "spice" cannabinoids) during development, with further implications for the effectiveness of therapeutic interventions based on prior drug exposure in youth.

Evaluation of a Ultraviolet B Light Emitting Diode (LED) for Producing Vitamin D3 in Human Skin.

AIM: A commercially available light emitting diode (LED) that transmitted narrow band ultraviolet B (UVB) radiation was evaluated for its efficacy and efficiency to produce vitamin D3 in human skin. MATERIALS AND METHODS: Human skin samples were obtained from surgical procedures. The LED had peak emission wavelength of 295 nm. Skin samples were exposed to the UVB-LED for varying times and then were analyzed by high-pressure liquid chromatography (HPLC) to determine the vitamin D3 content. RESULTS: There was a statistically significant time- and dose-dependent increase in the percent of 7-dehydrocholesterol that was converted to vitamin D3 in the skin type II samples; 1.3%±0.5, 2.3%±0.6 and 4.5%±1.67 after exposure to 0.75 (11.7 mJ/cm2), 1.5 (23.4 mJ/cm2) and 3 (46.8 mJ/cm2) minimal erythemal doses (MEDs), respectively. CONCLUSION: The UVB-LED was effective and efficient in generating vitamin D3 in human skin, in vitro. The amount of vitamin D3 production increased in a dose-dependent fashion with increased UVB energy. UVB-LEDs can be developed for devices that can efficiently produce vitamin D3 in human skin.

Effectiveness and safety of extending intrauterine device duration: a systematic review.

OBJECTIVE: To systematically review the literature to determine if extended use of intrauterine devices, including the copper or levonorgestrel intrauterine device, beyond approved durations is effective and safe for preventing pregnancy. STUDY DESIGN: We searched PubMed, Embase, Cumulative Index to Nursing and Allied Health Literature, and Global Index Medicus from inception through June 2019. We considered primary studies of women using the T380A copper or currently available levonorgestrel intrauterine devices past their approved duration (extended use). The primary outcome was pregnancy. Safety outcomes included adverse events and side effects that led to discontinuation. We abstracted study data into evidence tables and assessed study quality as related to the risk of bias, precision, and external validity. For each intrauterine device type, we calculated a pooled pregnancy rate during the first 2 years of extended use using the Poisson inverse variance method. RESULTS: Of 4068 studies identified by our search, 4 good-to-poor-quality studies of the 52 mg levonorgestrel intrauterine device (approved for 5 years), with a total of 2098 women starting extended use, and 2 good-to-fair-quality studies of the T380A copper intrauterine device (approved for 10 years), with 245 women starting extended use, met inclusion criteria. For the levonorgestrel intrauterine device, the pooled pregnancy rate was 0.02 per 100 person-years (95% confidence interval, 0.00-0.45) in year 6, 0.03 per 100 person-years (95% confidence interval, 0.00-0.71) in year 7, and 0.02 per 100 person-years (95% confidence interval, 0.00-0.29) in years 6 and 7 combined. During this same time, annual rates of adverse events or discontinuation owing to side effects ranged from 0 to 3.7 per 100 participants. For the copper intrauterine device, the pooled pregnancy rate for years 11 and 12 was 0.0 per 100 person-years (95% confidence interval, 0.0-0.8), and annual rates of adverse events and discontinuation owing to side effects during extended use ranged from 0 to 4.6 per 100 participants. CONCLUSION: The available evidence suggests that rates of pregnancy, adverse events, and discontinuation owing to side effects during the first 2 years of extended use of the 52 mg levonorgestrel intrauterine device and the T380A copper intrauterine device are low and comparable to rates during approved duration of use. Data were limited in quantity and quality, and may not be generalizable to all intrauterine device users.

Investigating the gut-brain axis in a neurodevelopmental rodent model of schizophrenia.

BACKGROUND: Although the aetiology of schizophrenia remains unknown, it has been suggested that it might occur in response to alterations in the gut-brain axis (GBA), the bi-directional communication system between the gut and the brain. The current study aimed to determine whether the "two-hit" animal model of neuropsychopathology (maternal immune activation combined with adolescent cannabinoid exposure), produced abnormalities in the GBA. METHOD: Pregnant Wistar rats were administered the viral mimetic polyI:C on gestational day 19 and offspring were administered the synthetic cannabinoid HU210 from postnatal days 35-48. Evidence of GBA activation was assessed in the hypothalamus, colon and fecal samples from male and female offspring at adolescence and adulthood. RESULTS: Findings were sex-specific with adolescent female offspring exhibiting an increased hypothalamic inflammatory profile, increased hypothalamic CRHR1 mRNA, and decreased fecal expression of Bifidobacterium longum, however, no changes were detected in colonic inflammation or integrity. CONCLUSION: These results indicate that the rat two-hit model, documented to produce behavioural and neuroanatomical abnormalities, also produces hypothalamic and microbiota abnormalities. The results also demonstrate significant sex differences, suggesting that this model may be useful for investigating the role of the GBA in the aetiology of neurodevelopmental disorders such as schizophrenia.

Cannabinoid and nicotine exposure during adolescence induces sex-specific effects on anxiety- and reward-related behaviors during adulthood.

Nicotine and cannabis use during adolescence has the potential to induce long lasting changes on affective and cognitive function. Here, we examined whether adolescent exposure to nicotine, the cannabinoid agonist WIN55-212,2 (WIN), or co-exposure to both would alter operant learning, locomotion, and anxiety- and reward-related behaviors in male and female mice during adulthood. Males exposed to a moderate dose of WIN (2 mg/kg) or co-exposed to nicotine and the moderate dose of WIN exhibited decreased anxiety-associated behaviors and increased cognitive flexibility, but did not differ in operant learning or generalized locomotion. In contrast, differences were not found among the females in these measures at the moderate WIN dose or in both sexes with exposure to a low WIN dose (0.2 mg/kg). Furthermore, a sex-dependent dissociative effect was found in natural reward consumption. Males exposed to the moderate dose of WIN or co-exposed to nicotine and the moderate dose of WIN demonstrated increased sucrose consumption. In contrast, females exposed to the moderate dose of WIN exhibited a decrease in sucrose consumption, which was ameliorated with co-administration of nicotine. Together, these novel findings demonstrate that adolescent exposure to cannabinoids in the presence or absence of nicotine results in altered affective and reward-related behaviors during adulthood.

Altered glutamate clearance in ascorbate deficient mice increases seizure susceptibility and contributes to cognitive impairment in APP/PSEN1 mice.

Ascorbate (vitamin C) is critical as a first line of defense antioxidant within the brain, and specifically within the synapse. Ascorbate is released by astrocytes during glutamate clearance and disruption of this exchange mechanism may be critical in mediating glutamate toxicity within the synapse. This is likely even more critical in neurodegenerative disorders with associated excitotoxicity and seizures, in particular Alzheimer's disease, in which ascorbate levels are often low. Using Gulo-/- mice that are dependent on dietary ascorbate, we established that low brain ascorbate increased sensitivity to kainic acid as measured via behavioral observations, electroencephalography (EEG) measurements, and altered regulation of several glutamatergic system genes. Kainic acid-induced immobility was improved in wild-type mice following treatment with ceftriaxone, which upregulates glutamate transporter GLT-1. The same effect was not observed in ascorbate-deficient mice in which sufficient ascorbate is not available for release. A single, mild seizure event was sufficient to disrupt performance in the water maze in low-ascorbate mice and in APPSWE/PSEN1dE9 mice. Together, the data support the critical role of brain ascorbate in maintaining protection during glutamatergic hyperexcitation events, including seizures. The study further supports a role for mild, subclinical seizures in cognitive decline in Alzheimer's disease.

Association between transmission rate and disease severity for Actinobacillus pleuropneumoniae infection in pigs.

A better understanding of the variation in infectivity and its relation with clinical signs may help to improve measures to control and prevent (clinical) outbreaks of diseases. Here we investigated the role of disease severity on infectivity and transmission of Actinobacillus pleuropneumoniae, a bacterium causing respiratory problems in pig farms. We carried out transmission experiments with 10 pairs of caesarean-derived, colostrum-deprived pigs. In each pair, one pig was inoculated intranasally with 5×10(6) CFUs of A. pleuropneumoniae strain 1536 and housed together with a contact pig. Clinical signs were scored and the course of infection was observed by bacterial examination and qPCR analysis of tonsillar brush and nasal swab samples. In 6 out of 10 pairs transmission to contact pigs was observed, but disease scores in contact infected pigs were low compared to the score in inoculated pigs. Whereas disease score was positively associated with bacterial load in inoculated pigs and bacterial load with the transmission rate, the disease score had a negative association with transmission. These findings indicate that in pigs with equal bacterial load, those with higher clinical scores transmit A. pleuropneumoniae less efficiently. Finally, the correlation between disease score in inoculated pigs and in positive contact pigs was low. Although translation of experimental work towards farm level has limitations, our results suggest that clinical outbreaks of A. pleuropneumoniae are unlikely to be caused only by spread of the pathogen by clinically diseased pigs, but may rather be the result of development of clinical signs in already infected pigs.

Autophagy is involved in starvation response and cell death in Blastocystis.

Previous studies have demonstrated that colony forms of Blastocystis undergo cell death with numerous membrane-bound vesicles containing organelles located within the central vacuole, resembling morphological features of autophagy. In this study, we investigated whether Blastocystis underwent autophagy upon amino acid starvation and rapamycin treatment. Concurrently, we provide new insight into a possible function of the central vacuole. The use of the autophagy marker monodansylcadaverine, and the autophagy inhibitors 3-methyladenine and wortmannin, showed the existence of autophagy in amino-acid-starved and rapamycin-treated Blastocystis. Confocal microscopy and transmission electron microscopy studies also showed morphological changes that were suggestive of autophagy. The unusually large size of the autophagic compartments within the parasite central vacuole was found to be unique in Blastocystis. In addition, autophagy was found to be triggered when cells were exposed to the cytotoxic antibody mAb 1D5, and autophagy was intensified in the presence of the caspase inhibitor zVAD.fmk. Taken together, our results suggest that the core machinery for autophagy is conserved in Blastocystis, and that it plays an important role in the starvation response and cell death of the parasite.

Differentially conserved staphylococcal SH3b_5 cell wall binding domains confer increased staphylolytic and streptolytic activity to a streptococcal prophage endolysin domain.

Staphylococcal peptidoglycan hydrolases are a potential new source of antimicrobials. A large subset harbors C-terminal SH3b_5 cell wall binding domains. These C-terminal domains have been shown to be necessary for accurate cell wall recognition and subsequent staphylolytic activity for some endolysins. Over fifty proteins of staphylococcal or phage origin containing SH3b domains were aligned, yielding five highly repetitive groups of proteins. Representative C-termini from these five groups, and six staphylococcal proteins for which no homologues have been identified, were aligned, revealing two distinct SH3b_5 subgroups with overlapping but differentially conserved residues. A premise behind this research is that there may be unique cell wall binding properties conferred by these staphylococcal domains that could be exploited to specifically enhance anti-staphylococcal efficacy in heterologous protein fusion constructs. To identify functional differences between the two subgroups, the native Cpl-7 cell wall binding domains of the streptococcal LambdaSa2 endolysin were replaced by staphylococcal SH3b domains from both subgroups. SH3b domains from either lysostaphin (bacteriocin) or LysK (phage endolysin) resulted in a approximately 5x increase in staphylolytic activity conferred on the streptococcal endopeptidase domain, and surprisingly these same fusions maintained significant streptolytic activity suggesting that the staphylococcal SH3b domains are not always staphylococcal-specific. A comparison of the differences in lytic activity conferred on the LambdaSa2 endopeptidase domain by either LysK or lysostaphin SH3b domain differed by no more than a factor of two. Through the collection of peptidoglycan hydrolase sequences, three new putative intron-containing phage endolysin genes were identified in public data sets for the phages G1, X2 and 85.

Progression of lesions in the respiratory tract of broilers after single infection with Escherichia coli compared to superinfection with E. coli after infection with infectious bronchitis virus.

The progression of Escherichia coli lesions was studied in the respiratory tract of 4-week-old commercial broilers. Lesions were induced after a single intratracheal E. coli infection, and after an infection with E. coli preceded 5 days earlier by an oculo-nasal and intratracheal infectious bronchitis virus (IBV) infection of either the virulent M41 strain or the H120 vaccine strain. Trachea, lung and thoracic airsac lesions were examined macroscopically and microscopically. Tissue samples were taken at 3h post-inoculation (hpi), and 1, 2, 4 and 7 days post-inoculation (dpi) with E. coli. The location of both pathogens was assessed by immunohistochemistry. Single E. coli inoculation induced pneumonia and airsacculitis; in case it was preceded by IBV infection, the same macroscopical lesions and also viral tracheitis were found. No clear difference existed between the single and dual infected birds with respect to inflammatory reactions in the lung, which had disappeared within 7 days, except for the presence of more follicles in dual infected birds. IBV antigen was detected in secondary bronchi and airsacs up to 2 dpi and in the trachea up to 4 dpi. E. coli bacteria were found in the tracheal lumen included in purulent material, the parabronchi and airsacs. In lung tissue E. coli antigen was found up to 4 dpi. No clear difference existed between single and dual inoculated birds regarding the presence of E. coli in the lung. In the airsacs, a few bacteria were found from 0.5 hpi up to 4 dpi in E. coli and IBV-E. coli inoculated birds. Although both pathogens were cleared beyond detection at 7 dpi, in IBV-E. coli inoculated birds lesions in the airsac persisted, in contrast to broilers inoculated with E. coli only. In the present study it is shown that 4-week-old broilers are not resistant to intratracheal E. coli inoculation, however, these birds can overcome the induced E. coli infection within a short time span. Moreover, a preceding infection with vaccine or virulent IBV does not seem to impair the clearance of E. coli in the respiratory tract of broilers, but rather induces an exaggerated inflammatory response in the airsacs only, which seems to be the mechanism behind the pattern of airsacculitis in commercial poultry in the field.