SARS-CoV-2 egress from Vero cells: a morphological approach.

Despite being extensively studied because of the current coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) interactions with mammalian cells are still poorly understood. Furthermore, little is known about this coronavirus cycle within the host cells, particularly the steps that lead to viral egress. This study aimed to shed light on the morphological features of SARS-CoV-2 egress by utilizing transmission and high-resolution scanning electron microscopy, along with serial electron tomography, to describe the route of nascent virions towards the extracellular medium. Electron microscopy revealed that the clusters of viruses in the paracellular space did not seem to result from collective virus release. Instead, virus accumulation was observed on incurved areas of the cell surface, with egress primarily occurring through individual vesicles. Additionally, our findings showed that the emission of long membrane projections, which could facilitate virus surfing in Vero cells infected with SARS-CoV-2, was also observed in non-infected cultures, suggesting that these are constitutive events in this cell lineage.

Cost-effective 3D lung tissue spheroid as a model for SARS-CoV-2 infection and drug screening.

BACKGROUND: The SARS-CoV-2 pandemic has spurred an unparalleled scientific endeavor to elucidate the virus' structure, infection mechanisms, and pathogenesis. Two-dimensional culture systems have been instrumental in shedding light on numerous aspects of COVID-19. However, these in vitro systems lack the physiological complexity to comprehend the infection process and explore treatment options. Three-dimensional (3D) models have been proposed to fill the gap between 2D cultures and in vivo studies. Specifically, spheroids, composed of lung cell types, have been suggested for studying SARS-CoV-2 infection and serving as a drug screening platform. METHODS: 3D lung spheroids were prepared by coculturing human alveolar or bronchial epithelial cells with human lung stromal cells. The morphology, size, and ultrastructure of spheroids before and after SARS-CoV-2 infection were analyzed using optical and electron microscopy. Immunohistochemistry was used to detect spike protein and, thus, the virus presence in the spheroids. Multiplex analysis elucidated the cytokine release after virus infection. RESULTS: The spheroids were stable and kept their size and morphology after SARS-CoV-2 infection despite the presence of multivesicular bodies, endoplasmic reticulum rearrangement, tubular compartment-enclosed vesicles, and the accumulation of viral particles. The spheroid responded to the infection releasing IL-6 and IL-8 cytokines. CONCLUSION: This study demonstrates that coculture spheroids of epithelial and stromal cells can serve as a cost-effective infection model for the SARS-CoV-2 virus. We suggest using this 3D spheroid as a drug screening platform to explore new treatments related to the cytokines released during virus infection, especially for long COVID treatment.

Low public awareness opens up new opportunities for highlighting milk as an iodine dietary source.

This international survey provides insights into public awareness of the importance of iodine as an essential trace mineral in human health along with knowledge of iodine dietary sources. The online questionnaire included sociodemographic aspects and dietary iodine consumer awareness on 7-point Likert-type questions. A total of 4,704 questionnaires from 16 countries were considered. Answers were analyzed through a multiple regression linear model including country, gender, age, education level, and employment status as fixed effects. Respondents were moderately aware of the importance of fish (4.86) and seafood (4.90) as dietary iodine sources, but less aware of milk as a primary iodine source (3.32). Respondent awareness varied considerably across countries. Age, education level, and employment status only modified their perception when asked about fish and seafood as a source of iodine, with elderly respondents, those highly educated and of working age being more aware of their relevance as dietary iodine sources. Respondent knowledge did not vary by age, education level, employment status, or gender when asked about cereals, vegetables and fruits, meat and milk as iodine-rich food sources. Consequently, labeling milk and dairy products as an iodine-rich food source should be considered. Public authorities can consider the results from this survey in promotional campaigns to improve the awareness of different iodine sources and their beneficial effect on health.

New azaaurone derivatives as potential multitarget agents in HIV-TB coinfection.

Tuberculosis (TB) disease, caused by Mycobacterium tuberculosis (Mtb) is the leading cause of death among people with human immunodeficiency virus (HIV) infection. No dual-target drug is currently being used to simultaneously treat both infections. This work aimed to obtain new multitarget HIV-TB agents, with the goal of optimizing treatments and preventing this coinfection. These compounds incorporate the structural features of azaaurones as anti-Mtb and zidovudine (AZT) as the antiretroviral moiety. The azaaurone scaffold displayed submicromolar activities against Mtb, and AZT is a potent antiretroviral drug. Six derivatives were synthetically generated, and five were evaluated against both infective agents. Evaluations of anti-HIV activity were carried out in HIV-1-infected MT-4 cells and on endogenous HIV-1 reverse transcriptase (RT) activity. The H37Rv strain was used for anti-Mtb assessments. Most compounds displayed potent antitubercular and moderate anti-HIV activity. (E)-12 exhibited a promising multitarget profile with an MIC90 of 2.82 µM and an IC50 of 1.98 µM in HIV-1-infected T lymphocyte cells, with an 84% inhibition of RT activity. Therefore, (E)-12 could be the first promising compound from a family of multitarget agents used to treat HIV-TB coinfection. In addition, the compound could offer a prototype for the development of new strategies in scientific research to treat this global health issue.

Evaluation of phenotypical and genotypical methods for the identification and typing of Stenotrophomonas maltophilia isolated from a pharmaceutical facility.

AIMS: Evaluate methods for identification and typing of Stenotrophomonas maltophilia isolated from a pharmaceutical facility. METHODS AND RESULTS: From 270 S. maltophilia strains identified by VITEK®2, 40 were selected and submitted to MALDI TOF-MS, 16S and 23S rRNA gene analysis, enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR), and an antimicrobial susceptibility profile. 16S rRNA sequencing was able to identify 39 (97.5%) strains as Stenotrophomonas spp. and one (2.5%) as Luteimonas huabeiensis. MALDI TOF-MS identified 37 (92.5%) strains as S. maltophilia, and three (7.5%) were not identified. PCR targeting 23S rRNA yielded a positive result for 39 (97.5%) strains. However, after sequencing, two strains were identified as Stenotrophomonas rhizophila, showing false-positive results. The confirmed S. maltophilia strains (n = 37) showed 35 distinct ERIC-PCR profiles and exhibited sensitivity to minocycline and levofloxacin, and six (16.3%) showed intermediate resistance to sulfamethoxazole-trimethoprim. CONCLUSION: Matrix-assisted laser desorption lonization-time of flight mass spectrometry (MALDI-TOF MS) was a satisfactory methodology for the identification of S. maltophilia, but expansion of the database is necessary for the identification of other species. 16S rDNA sequencing showed low resolution for Stenotrophomonas species differentiation. PCR targeting 23S rRNA could not differentiate S. maltophilia from S. rhizophila. ERIC-PCR was shown to be a useful tool for the microbial source tracking of S. maltophilia.

Phenotypical and molecular characterization of Acinetobacter spp. isolated from a pharmaceutical facility.

Characterizing microorganisms according to different criteria is useful when investigating sources of microbiological contamination in the pharmaceutical industry. The aim of this study was to characterize 38 Acinetobacter baumannii complex strains isolated from a biopharmaceutical industry by 16S rRNA sequencing, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS), multilocus sequence typing (MLST), antimicrobial susceptibility profile, biofilm formation, and sensibility to disinfectants. Thirty-three (86.9%) strains were identified by 16S rRNA gene sequencing as A. seifertii/pitti/nosocomialis/lactucae, four (10.5%) as A. baumannii, and one (2.6%) as A. vivianii/courvalini. MALDI-TOF/MS did not identify one strain, and incorrectly identified 30/37 (81.1%) strains as A. baumannii. Strains were assigned to 12 different STs, of which nine were newly defined in this study (STs 2091-2099). Twenty-six (68.4%) strains showed resistance to amikacin and gentamicin. Thirty-three (86.8%) strains were classified as moderately or strongly adherent on polystyrene. Alcohol 70%/15 min and quaternary ammonium 0.08%/20 min were not able to eliminate the biofilm formed, but sodium hypochlorite 0.1%/15 min was efficient. In conclusion, improved methods are needed to improve the identification of Acinetobacter strains in pharmaceutical industries. This organism is of particular concern as it forms recalcitrant biofilms, leading to persistence in the manufacturing environment and increased risk of product contamination.

Identification of Sutcliffiella horikoshii strains in an immunobiological pharmaceutical industry facility.

The pharmaceutical industry must comply with the requirements for good manufacturing practices to reduce inherent contamination risks in the production process. Bacillus and related genera are among the main bacterial isolated from clean areas, raw material, and products in the pharmaceutical industries, but the correct identification of these species is still a challenge. The aim of this study was to characterize by phenotyping, protein profiling, and 16S rRNA gene sequencing Sutcliffiellahorikoshii strains (n = 6) isolated from an immunobiological pharmaceutical facility, and to propose the reclassification of Bacillus tianshenii to the genus Sutcliffiella, and Sutcliffiella tianshenii sp. nov. The strains were characterized by VITEK®2, matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) using VITEK®MS, and 16S rRNA gene sequencing analysis. MALDI-TOF/MS did not identify any strains that were identified by 16S rRNA as S. horikoshii. VITEK®2 showed false-positive results, with misidentification as B. sporothermodurans (reclassified as Heyndrickxia sporothermodurans) and Geobacillus thermoleovorans. After MALDI-TOF/MS database expansion, with the creation of SuperSpectrum, the strains were correctly identified as S. horikoshii. This study is the first report of isolation of S. horikoshii strains from a pharmaceutical industry. More studies are necessary to better understand the ability of S. horikoshii to contaminate the environment and products.

Identification and characterisation of SARS-CoV-2 and Human alphaherpesvirus 1 from a productive coinfection in a fatal COVID-19 case.

BACKGROUND: During routine Coronavirus disease 2019 (COVID-19) diagnosis, an unusually high viral load was detected by reverse transcription real-time polymerase chain reaction (RT-qPCR) in a nasopharyngeal swab sample collected from a patient with respiratory and neurological symptoms who rapidly succumbed to the disease. Therefore we sought to characterise the infection. OBJECTIVES: We aimed to determine and characterise the etiological agent responsible for the poor outcome. METHODS: Classical virological methods, such as plaque assay and plaque reduction neutralisation test combined with amplicon-based sequencing, as well as a viral metagenomic approach, were performed to characterise the etiological agents of the infection. FINDINGS: Plaque assay revealed two distinct plaque phenotypes, suggesting either the presence of two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains or a productive coinfection of two different species of virus. Amplicon-based sequencing did not support the presence of any SARS-CoV-2 genetic variants that would explain the high viral load and suggested the presence of a single SARS-CoV-2 strain. Nonetheless, the viral metagenomic analysis revealed that Coronaviridae and Herpesviridae were the predominant virus families within the sample. This finding was confirmed by a plaque reduction neutralisation test and PCR. MAIN CONCLUSIONS: We characterised a productive coinfection of SARS-CoV-2 and Herpes simplex virus 1 (HSV-1) in a patient with severe symptoms that succumbed to the disease. Although we cannot establish the causal relationship between the coinfection and the severity of the clinical case, this work serves as a warning for future studies focused on the interplay between SARS-CoV-2 and HSV-1 coinfection and COVID-19 severity.

Samanea tubulosa Benth. (Fabaceae): Antinociceptive effect on acute pain in mice: K+ATP channel and opioid activity.

Samanea tubulosa Benth. it has been widely used in traditional medicine to treat inflammatory processes. The present study aimed to investigate the antinociceptive effect and mechanism of action of the fractions obtained from the Samanea tubulosa pods in mice. The antinociceptive activity was evaluated in formalin, capsaicin and glutamate tests and the. The possible mechanisms of action involved in the antinociceptive effect of the hexane and ethyl acetate fraction in the opioid system, also the the K + ATP channels and the L-arigine pathways of nitric oxide were evaluated. The chemical characterization analysis revealed in the hexane fraction the presence of triterpenes such as lupenone and lupeol. In the glutamate test, the hexane and ethyl acetate fractions showed antinociceptive activity at the dose of 12.5 and 25 mg kg-1. The antinociception produced by the hexane and ethyl acetate fractions was significantly reversed by naloxone, indicating that the fractions act through the opioid pathway. Antinociceptive response of the ethyl acetate fraction was blocked by glibenclamide, indicating that this fraction acts via the K + ATP channels activation. It is concluded that the fractions under study exert antinociceptive activity possibly related to the opioid route and through K+ ATP channels activation.

Mechanism of the lethal effect of Riparin E against bacterial and yeast strains.

Riparins are alkamides naturally found in the fruits of Aniba riparia (Nees) Mez, but currently synthetic molecules as Riparin E (Rip-E) can be obtained. Potential biological of Rip-E as schistosomicidal agent against Schistosoma mansoni worms, as well as against Staphylococcus aureus strains has already been described. However, the mechanism of action related to antimicrobial activity of Rip-E against bacterial or fungi species has not yet been reported. This study had as objective to evaluate the Rip-E antimicrobial activity against Gram-positive and Gram-negative bacteria, as well as against yeast species of clinical importance. Minimal inhibitory concentrations of the compound against bacterial and yeast strains were determined by microdilution method. To verify if a possible lethal effect caused by Rip-E were related to plasma membrane damage, microbial cells treated with Rip-E were stained with 7-aminoactinomycin D (7-AAD) and analyzed by flow cytometry. Rip-E showed a bactericide effect against Gram-positive species S. aureus and S. epidermidis, as well as, against Gram-negative species Escherichia coli and Salmonella enterica Typhimurium, but was inactive against Pseudomonas aeruginosa. Moreover, Rip-E showed activity against fungi species Candida albicans and C. tropicalis. S. aureus, E. coli and C. albicans cells treated with Rip-E were marked with 7-aminoactinomycin D (7-AAD) indicating that Rip-E can cause plasma membrane damage, acting as a potential microbicide agent for prevention or treatment of infectious diseases.

Influence of laboratory procedures on postthawing cell viability and hematopoietic engraftment after autologous peripheral blood stem cell transplantation.

BACKGROUND: The kinetics of hematopoietic recovery after autologous stem cell transplantation (ASCT) may be affected by laboratory procedures. The aim of this study was to evaluate the influence of characteristics of the cryopreserved units of peripheral blood stem cells (PBSC) on postthawing cell viability and engraftment outcomes after ASCT. STUDY DESIGN AND METHODS: This was a retrospective cohort study including individuals referred for ASCT. Cryopreservation was conducted at a single processing facility between 2014 and 2019, and patients received clinical care at six transplant centers. Covariates and outcome data were retrieved from participants' records. RESULTS: The study population comprised 619 patients (345 [55.7%] male). Median age was 53 years. Multiple myeloma was the most common diagnosis (62.7%). Higher preapheresis CD34+ cell count, lower nucleated cell (NC) concentration per cryobag, and composition of the cryoprotectant solution (5% dimethyl sulfoxide [DMSO] and 6% hydroxyethyl starch) were statistically significantly associated with higher postthawing cell viability. The linear regression model for time to neutrophil and platelet engraftment included the infused CD34+ cell dose and the composition of the cryoprotectant solution. Patients who had PBSC cryopreserved using 10% DMSO solution presented six times higher odds (odds ratio [OR] = 6.9; 95% confidence interval [CI]: 2.2-21.1; p = .001) of delayed neutrophil engraftment (>14 days) and two times higher odds (OR = 2.3, 95%CI: 1.4-3.7; p = .001) of prolonged hospitalization (>18 days). DISCUSSION: The study showed that mobilization efficacy, NC concentration, and the composition of the cryoprotectant solution significantly affected postthawing cell viability. In addition, the composition of the cryoprotectant solution significantly impacted engraftment outcomes and time of hospitalization after ASCT.

Magnetic resonance imaging and neurological findings in dogs with disc-associated cervical spondylomyelopathy: a case series.

BACKGROUND: Canine cervical spondylomyelopathy can be separated into osseous and disc-associated (DA-CSM) forms. Our aim was to describe the magnetic resonance imaging (using a high-field scanner) and neurological findings in dogs with DA-CSM and investigate a relationship between these findings. RESULTS: Sixty-three dogs were included: 60/63 (95 %) were large breeds, with Doberman Pinschers and males over-represented (70 %). Mean and median age at the time of diagnosis was 7.25 and 7.2 years (range 0.41-12 years). Chronic signs were noted in 52/63 (83 %) dogs, with proprioceptive ataxia the most common. Main site of spinal cord compression was commonly C6-7 or C5-6. Thirty-six (57 %) dogs had various sites of spinal cord compression. Most dogs younger than 6 years of age had a single affected site. Foraminal stenosis was present in 51/63 dogs (81 %). T2-weighted hyperintensity was present in 40/63 dogs (63 %). 88 % of the articular processes showed degenerative changes, which correlated strongly with intervertebral disc degeneration. Ligamentum flavum hypertrophy was seen in 38 % of dogs. No correlation was observed between neurologic signs and number of affected sites. A moderate positive correlation was observed between severity of spinal cord compression and neurologic grade (r 0.48; p < 0.001). CONCLUSIONS: DA-CSM was predominantly observed in older, male Dobermans, with lesions located in the caudal cervical vertebral region. It was also seen in dogs 3 years of age or even younger (8 %). Single compressive lesions were more common in dogs younger than 6 years of age. Many dogs had concomitant changes (e.g.: ligamentum flavum hypertrophy and foraminal stenosis). Most dogs with ligamentum flavum hypertrophy were 6 years or older. A positive correlation was observed between severity of spinal cord compression and neurologic grade, but multilevel compression was not associated with more severe neurologic signs. A very high percentage of dogs had articular process degenerative changes. Possible biomechanical or genetic relationships between degenerative changes in articular processes, ligamentum flavum, and intervertebral discs warrants further investigation.

Milk lymphocyte profile and macrophage functions: new insights into the immunity of the mammary gland in quarters infected with Corynebacterium bovis.

BACKGROUNDS: The present study explored the viability of bovine milk macrophages, their intracellular production of reactive oxygen and nitrogen species (RONS), and their phagocytosis of Staphylococcus aureus, as well as the profile of lymphocytes, from healthy udder quarters and udder quarters infected by Corynebacterium bovis. The study included 28 healthy udder quarters from 12 dairy cows and 20 udder quarters infected by C. bovis from 10 dairy cows. The percentages of macrophages and lymphocytes were identified by flow cytometry using monoclonal antibodies. Macrophage viability, RONS production, and S. aureus phagocytosis were evaluated by flow cytometry. RESULTS: Milk samples from quarters infected with C. bovis showed a lower percentage of macrophages but an increased number of milk macrophages per mL and a higher percentage of macrophages that produced intracellular RONS and phagocytosed S. aureus. No effect of C. bovis infection on macrophage viability was found. Udder quarters infected by C. bovis showed a higher percentage of T cells and CD4+ T lymphocytes, but no effect was found on the percentage of CD8+ CD4- T, CD8- CD4- T, or B lymphocytes. CONCLUSIONS: Thus, our results corroborate, at least in part, the finding that intramammary infections by C. bovis may offer protection against intramammary infections by major pathogens.

Association of neurologic signs with high-field MRI findings in 100 dogs with osseous-associated cervical spondylomyelopathy.

Although osseous-associated cervical spondylomyelopathy (OA-CSM) findings have been well described using magnetic resonance imaging (MRI), there are no large-scale published studies on the associations between dog size, age, high-field MRI and neurologic findings. Using a retrospective, observational study design, we aimed to investigate an association between neurologic and high-field MRI characteristics in OA-CSM. Records were reviewed for dogs diagnosed with OA-CSM using high-field MRI. One-hundred dogs were included: 73/100 (73%) were giant breeds, 27/100 (27%) large breeds. Mean and median ages were, respectively, 3.1 and 2 years (0.3-9.75 years), with 2.6 and 2 years for giant-breed; and 4.4 and 4 years for large-breed dogs. The majority of dogs were male (75%) with chronic presentation (89%), more than one site of spinal cord compression (78%) and foraminal stenosis (91%). Dogs with multiples sites of spinal cord compression were more likely to have severe spinal cord compression (p < 0.001), severe foraminal stenosis (p < 0.001) and ligamentum flavum/soft tissue proliferation (p = 0.03) than those with a single compressive site. There was a weak correlation between neurologic grade and severity of spinal cord compression (r = 0.27; p = 0.007), number of affected sites (r = 0.24; p = 0.0183) and spinal cord T2W hyperintensity (r = 0.24; p = 0.0152). Intervertebral disc degeneration was seen in 80% of dogs. Age did not appear to have a prominent role in the manifestation of OA-CSM. This study showed that OA-CSM affects a sizeable proportion of young large-breed, in addition to giant-breed dogs.

The candidate vaccine strain Brucella ovis ∆abcBA is protective against Brucella melitensis infection in mice.

Brucellosis is a major zoonotic disease, and Brucella melitensis is the species most often associated with human infection. Vaccination is the most efficient tool for controlling animal brucellosis, with a consequent decrease of incidence of human infections. Commercially available live attenuated vaccines provide some degree of protection, but retain residual pathogenicity to human and animals. In this study, Brucella ovis ∆abcBA (Bo∆abcBA), a live attenuated candidate vaccine strain, was tested in two formulations (encapsulated with alginate and alginate plus vitelline protein B [VpB]) to immunize mice against experimental challenge with B. melitensis strain 16M. One week after infection, livers and spleens of immunized mice had reduced numbers of the challenge strain B. melitensis 16M when compared with those of nonimmunized mice, with a reduction of approximately 1-log10 of B. melitensis 16M count in the spleens from immunized mice. Moreover, splenocytes stimulated with B. melitensis antigens in vitro secreted IFN-γ when mice had been immunized with Bo∆abcBA encapsulated with alginate plus VpB, but not with alginate alone. Body and liver weights were similar among groups, although spleens from mice immunized with Bo∆abcBA encapsulated with alginate were larger than those immunized with Bo∆abcBA encapsulated with alginate plus VpB or nonimmunized mice. This study demonstrated that two vaccine formulations containing Bo∆abcBA protected mice against experimental challenge with B. melitensis.

Antimicrobial activity of Phyllanthus amarus Schumach. & Thonn and inhibition of the NorA efflux pump of Staphylococcus aureus by Phyllanthin.

The aim of this study was to evaluate the antimicrobial activity of ethanoic extract of P. amarus (PAEE) and its compound Phyllanthin, as well as, investigate if these natural products could modulate the fluoroquinolone-resistance in S. aureus SA1199-B by way of overexpression of the NorA efflux pump. Microdilution tests were carried out to determine the minimal inhibitory concentration (MIC) of the PAEE or Phyllanthin against several bacterial and yeast strains. To evaluate if PAEE or Phyllanthin were able to act as modulators of the fluoroquinolone-resistance, MICs for Norfloxacin and ethidium bromide were determined in the presence or absence of PAEE or Phyllanthin against S. aureus SA1199-B. PAEE showed antimicrobial activity against Gram-negative strains, meanwhile Phyllanthin was inactive against all strains tested. Addition of PAEE or Phyllanthin, to the growth media at sub-inhibitory concentrations enhanced the activity of the Norfloxacin as well as, Ethidium Bromide, against S. aureus SA1199-B. These results indicate that Phyllanthin is able to modulate the fluoroquinolone-resistance possibly by inhibition of NorA. This hypothesis was supported by in silico docking analysis which confirmed that Phyllantin is a NorA ligand. Thus, this compound could be used as a potentiating agent of the Norfloxacin activity in the treatment of infections caused by fluoroquinolone-resistant S. aureus.

Long-term computed tomography follow-up in great Danes with or without signs of osseous- associated cervical Spondylomyelopathy.

BACKGROUND: Osseous- associated cervical spondylomyelopathy (OA-CSM) has a high prevalence in Great Danes. In order to understand the progression of osseous changes, we aimed to perform a long-term computed tomographic (CT) follow-up study of Great Dane dogs with and without OA-CSM. Canine CSM is comparable to a common neurologic disease often diagnosed in older people termed cervical spondylotic myelopathy or degenerative cervical myelopathy, which is progressive in nature. The natural history of cervical spondylotic myelopathy in people has been well described, whereas there is scarce information on the natural history of canine OA-CSM. Our first goal was to evaluate if follow-up CT studies showed any changes compared to initial CT studies in Great Dane dogs with a diagnosis of OA-CSM. Our second goal was to establish whether clinically normal Great Danes went on to develop any vertebral changes or clinical signs consistent with OA-CSM. We enrolled Great Danes diagnosed with OA-CSM and clinically normal Great Danes who had previously participated in a prospective study. All dogs had clinical and CT follow-up evaluations. RESULTS: Twelve Great Dane dogs were investigated: six OA-CSM affected and six clinically normal dogs. The median time between CT studies was 28 months (OA-CSM dogs) and 25 months (normal dogs). On follow-up CT, two OA-CSM-affected dogs developed new sites of stenosis, and two clinically normal dogs developed new sites of stenosis (one each). Disc spaces most commonly affected were C4-C5, C5-C6 and C6-C7. New sites of foraminal stenosis were noted in two of the CSM-affected and four of the clinically normal dogs. Morphometric evaluation showed no statistically significant differences between the initial and follow-up CT studies in the OA-CSM affected or normal groups. CONCLUSION: Our long-term CT follow-up study documented progression of vertebral canal stenosis in four out of twelve dogs. The majority of dogs did not develop new sites of stenosis or show progression of vertebral lesions.

Validation of a new experimental model of extrusive luxation on maxillary molars of rats: a histological study.

BACKGROUND/AIM: The aim of this study was to test and validate a new model of extrusive luxation trauma on maxillary first molars of rats. MATERIAL AND METHODS: Forty adult male rats (Rattus norvegicus albinus, Wistar; weight = 230-250 g), 45 days old, were divided into eight groups (n = 5): control groups, in which animals were not subjected to any procedure and waited 1 day (GC1D) or 3 days (GC3D) for euthanasia, and experimental groups, in which animals were subjected to forces of 1100cN, 1300cN, or 1500cN and waited 1 or 3 days for euthanasia (GT1100/1D, GT1100/3D, GT1300/1D, GT1300/3D, GT1500/1D, GT1500/3D). In animals of the experimental groups, trauma was produced by an extrusive force in maxillary first right molars. Four-micrometer serial cuts stained with hematoxylin and eosin (HE) were made. Descriptive microscopic analysis of first upper right molar and semi-quantitative analysis (scores 1 to 4) of intensity of acute and chronic inflammation and vascular changes in the periodontal ligament and active and inactive external root resorption were conducted. The distribution of scores in the groups was compared using the Freeman-Halton extension of Fisher's exact test. The significance level was 5%. RESULTS: It was observed that vascular disorders (bleeding) on the periodontal ligament became more evident with increasing extrusive force. CONCLUSIONS: This new method was capable of generating histological changes, proving its secure application in this research area. The 1500cN force produced more damage on the periodontal ligament. CLINICAL RELEVANCE: The validation of a new experimental method can produce more reliable evidence in further research.

Cigarette Smoke During Breastfeeding in Rats Changes Glucocorticoid and Vitamin D Status in Obese Adult Offspring.

Maternal smoking increases obesogenesis in the progeny. Obesity is associated with several hormonal dysfunctions. In a rat model of postnatal tobacco smoke exposure, we previously reported increased central fat depot and disruption of some hormonal systems in the adult offspring. As both glucocorticoids and vitamin D alter lipogenesis and adipogenesis, here we evaluated the metabolism of these two hormones in visceral adipose tissue (VAT) and liver by Western blotting, and possible associations with lipogenesis biomarkers in adult rats that were exposed to tobacco smoke during their suckling period. At postnatal day (PN) 3, dams and offspring of both sexes were exposed (S group) or not (C group) to tobacco smoke, 4 × 1 h/day. At PN180, corticosteronemia was lower in S male and higher in S female offspring, without alterations in peripheral glucocorticoid metabolism and receptor. Adrenal ACTH receptor (MC2R) was higher in both sexes of S group. Despite unchanged serum vitamin D, liver 25-hydroxylase was higher in both sexes of S group. Male S offspring had higher 1α-hydroxylase, acetyl-CoA carboxylase (ACC), and fatty acid synthase (FAS) in VAT. Both sexes showed increased ACC protein content and reduced sirtuin mRNA in liver. Male S offspring had lower liver peroxisome proliferator-activated receptor-α. Tobacco exposure during lactation induced abdominal obesity in both sexes via distinct mechanisms. Males and females seem to develop HPA-axis dysfunction instead of changes in glucocorticoid metabolism and action. Lipogenesis in VAT and liver, as well as vitamin D status, are more influenced by postnatal smoke exposure in male than in female adult rat offspring.