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1.
mBio ; 13(2): e0375521, 2022 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-35227071

RESUMO

New treatment options against the widespread cancerogenic gastric pathogen Helicobacter pylori are urgently needed. We describe a novel screening procedure for inhibitors of H. pylori flagellar biosynthesis. The assay is based on a flaA flagellin gene-luciferase reporter fusion in H. pylori and was amenable to multi-well screening formats with an excellent Z factor. We screened various compound libraries to identify virulence blockers ("antimotilins") that inhibit H. pylori motility or the flagellar type III secretion apparatus. We identified compounds that either inhibit both motility and the bacterial viability, or the flagellar system only, without negatively affecting bacterial growth. Novel anti-virulence compounds which suppressed flagellar biosynthesis in H. pylori were active on pure H. pylori cultures in vitro and partially suppressed motility directly, reduced flagellin transcript and flagellin protein amounts. We performed a proof-of-principle treatment study in a mouse model of chronic H. pylori infection and demonstrated a significant effect on H. pylori colonization for one antimotilin termed Active2 even as a monotherapy. The diversity of the intestinal microbiota was not significantly affected by Active2. In conclusion, the novel antimotilins active against motility and flagellar assembly bear promise to complement commonly used antibiotic-based combination therapies for treating and eradicating H. pylori infections. IMPORTANCE Helicobacter pylori is one of the most prevalent bacterial pathogens, inflicting hundreds of thousands of peptic ulcers and gastric cancers to patients every year. Antibacterial treatment of H. pylori is complicated due to the need of combining multiple antibiotics, entailing serious side effects and increasing selection for antibiotic resistance. Here, we aimed to explore novel nonantibiotic approaches to H. pylori treatment. We selected an antimotility approach since flagellar motility is essential for H. pylori colonization. We developed a screening system for inhibitors of H. pylori motility and flagellar assembly, and identified numerous novel antibacterial and anti-motility compounds (antimotilins). Selected compounds were further characterized, and one was evaluated in a preclinical therapy study in mice. The antimotilin compound showed a good efficacy to reduce bacterial colonization in the model, such that the antimotilin approach bears promise to be further developed into a therapy against H. pylori infection in humans.


Assuntos
Infecções por Helicobacter , Helicobacter pylori , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Modelos Animais de Doenças , Flagelos/metabolismo , Flagelina/genética , Flagelina/metabolismo , Infecções por Helicobacter/tratamento farmacológico , Infecções por Helicobacter/microbiologia , Helicobacter pylori/metabolismo , Humanos , Camundongos , Estômago
2.
BMC Vet Res ; 18(1): 80, 2022 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-35219345

RESUMO

BACKGROUND: Primary photosensitization rarely occurs in horses and can easily be misinterpreted. Descriptions of the disease in horses after ingestion of parsnip are lacking. The aim of this case series was to describe the dermatological and ocular changes due to photosensitization and to raise awareness of parsnip being a possible aetiologic agent. CASE PRESENTATION: Nine horses from three different stables in Berlin and Brandenburg, Germany, presented variable degrees of erythema, scaling, crusting and necrosis of unpigmented skin at the head and prepuce. Horses were of different breeds with a median age of 15 ± 5.9 years. A mild leukocytosis was diagnosed in 1/9 horses at admission. Analyzed liver enzymes were within the reference ranges in all horses. Ocular changes were diagnosed as follows: blepharitis (3/9), conjunctivitis (7/9), corneal edema without additional signs of keratitis and/or uveitis (2/9), corneal edema with signs of uveitis (1/9) and photophobia (4/9). One horse developed a fluorescein positive corneal erosion. Skin biopsy (1/9) revealed a moderate to severe acute, eosinophilic and lymphocytic dermatitis with dermal edema and vasculitis. All stables housing these patients fed hay from the same distributer. Analyzed hay samples showed high contents of wild parsnip (plants, seeds, roots). Wild parsnip is widespread in Europe and contains furocoumarins, a family of photodynamic pigments, which may cause primary photodermatitis, keratoconjunctivitis and uveitis. Horses were treated according to severity of clinical symptoms systemically with flunixine meglumine (1.1 mg/kg BW 1-2x/day) or prednisolone (1 mg/kg BW 1x/day). Topically, either gentamicin (3x/day), dexamethasone (2-3x/day) and/or atropine (1x/day) were used. Skin care was provided with almond oil or dexpanthenol (2x/day). All horses were kept in a dark environment or were treated with sunscreen and facemasks. Duration of treatment varied from 6-30 days (median 11.3 days). CONCLUSION: Ingestion of wild parsnip (Pastinaca sativa) can induce primary photosensitization with dermatitis and ocular injury in horses. In times of extreme weather, hay may alter in botanical composition, resulting in high amounts of uncharacteristic plants causing novel problems.


Assuntos
Furocumarinas , Doenças dos Cavalos , Pastinaca , Transtornos de Fotossensibilidade , Animais , Ingestão de Alimentos , Doenças dos Cavalos/induzido quimicamente , Doenças dos Cavalos/tratamento farmacológico , Cavalos , Transtornos de Fotossensibilidade/induzido quimicamente , Transtornos de Fotossensibilidade/veterinária , Melhoramento Vegetal
3.
Sci Rep ; 10(1): 16447, 2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-33020510

RESUMO

Manual count of mitotic figures, which is determined in the tumor region with the highest mitotic activity, is a key parameter of most tumor grading schemes. It can be, however, strongly dependent on the area selection due to uneven mitotic figure distribution in the tumor section. We aimed to assess the question, how significantly the area selection could impact the mitotic count, which has a known high inter-rater disagreement. On a data set of 32 whole slide images of H&E-stained canine cutaneous mast cell tumor, fully annotated for mitotic figures, we asked eight veterinary pathologists (five board-certified, three in training) to select a field of interest for the mitotic count. To assess the potential difference on the mitotic count, we compared the mitotic count of the selected regions to the overall distribution on the slide. Additionally, we evaluated three deep learning-based methods for the assessment of highest mitotic density: In one approach, the model would directly try to predict the mitotic count for the presented image patches as a regression task. The second method aims at deriving a segmentation mask for mitotic figures, which is then used to obtain a mitotic density. Finally, we evaluated a two-stage object-detection pipeline based on state-of-the-art architectures to identify individual mitotic figures. We found that the predictions by all models were, on average, better than those of the experts. The two-stage object detector performed best and outperformed most of the human pathologists on the majority of tumor cases. The correlation between the predicted and the ground truth mitotic count was also best for this approach (0.963-0.979). Further, we found considerable differences in position selection between pathologists, which could partially explain the high variance that has been reported for the manual mitotic count. To achieve better inter-rater agreement, we propose to use a computer-based area selection for support of the pathologist in the manual mitotic count.


Assuntos
Mastocitose Cutânea/patologia , Mitose/fisiologia , Algoritmos , Animais , Aprendizado Profundo , Cães , Processamento de Imagem Assistida por Computador/métodos , Mastócitos/patologia , Gradação de Tumores/métodos , Patologistas
4.
Artigo em Alemão | MEDLINE | ID: mdl-33086413

RESUMO

Lafora disease is an autosomal recessive lysosomal storage disorder leading to an accumulation of toxic glycogen bodies into the cells of the central nervous system and other tissues. In the progressive form of myoclonic epilepsy, clinical signs typically start around 7 years of age. Causal therapy is impossible, however, in the early stages the symptoms may at least be alleviated by modern antiepileptic drugs. In the case reported here, an approximately 7-year-old Beagle presented with daytime-dependent fasciculations, focal and generalized myoclonus ranging up to a brief tonic-clonic seizure. The signs could be triggered and augmented by stress, sounds and light. Histologic examination was performed on biopsy samples of skin, liver, muscle and nervous tissue to test for the clinical diagnosis of Lafora disease. Sarcoplasmic PAS-positive pla®ue deposits typical of Lafora bodies were detected in the muscle biopsies but not in any of the other specimens. Initial treatment with phenobarbital and imepitoin was unsuccessful. However, treatment with levetiracetam significantly alleviated the clinical signs. At time of writing this publication, 2 years following the diagnosis, the now 9-year-old dog shows occasional, stress-related increase in fokal myoclonic seizures. Episodes of collapse or tonic-clonic seizures did not occur to any further extent.


Assuntos
Anticonvulsivantes/uso terapêutico , Doenças do Cão , Doença de Lafora , Animais , Doenças do Cão/diagnóstico , Doenças do Cão/tratamento farmacológico , Cães , Doença de Lafora/diagnóstico , Doença de Lafora/tratamento farmacológico , Doença de Lafora/veterinária , Levetiracetam/uso terapêutico
5.
PLoS Pathog ; 16(3): e1008340, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32226027

RESUMO

Combination immunotherapy (CIT) is currently applied as a treatment for different cancers and is proposed as a cure strategy for chronic viral infections. Whether such therapies are efficient during an acute infection remains elusive. To address this, inhibitory receptors were blocked and regulatory T cells depleted in acutely Friend retrovirus-infected mice. CIT resulted in a dramatic expansion of cytotoxic CD4+ and CD8+ T cells and a subsequent reduction in viral loads. Despite limited viral replication, mice developed fatal immunopathology after CIT. The pathology was most severe in the gastrointestinal tract and was mediated by granzyme B producing CD4+ and CD8+ T cells. A similar post-CIT pathology during acute Influenza virus infection of mice was observed, which could be prevented by vaccination. Melanoma patients who developed immune-related adverse events under immune checkpoint CIT also presented with expanded granzyme-expressing CD4+ and CD8+ T cell populations. Our data suggest that acute infections may induce immunopathology in patients treated with CIT, and that effective measures for infection prevention should be applied.


Assuntos
Anticorpos/administração & dosagem , Melanoma/imunologia , Melanoma/terapia , Infecções por Retroviridae/imunologia , Linfócitos T Reguladores/imunologia , Infecções Tumorais por Vírus/imunologia , Animais , Antígeno B7-H1/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Vírus da Leucemia Murina de Friend/fisiologia , Humanos , Imunoterapia/efeitos adversos , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Retroviridae/patologia , Infecções por Retroviridae/virologia , Infecções Tumorais por Vírus/patologia , Infecções Tumorais por Vírus/virologia
6.
Vet Pathol ; 57(2): 214-226, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31808382

RESUMO

Mitotic count (MC) is an important element for grading canine cutaneous mast cell tumors (ccMCTs) and is determined in 10 consecutive high-power fields with the highest mitotic activity. However, there is variability in area selection between pathologists. In this study, the MC distribution and the effect of area selection on the MC were analyzed in ccMCTs. Two pathologists independently annotated all mitotic figures in whole-slide images of 28 ccMCTs (ground truth). Automated image analysis was used to examine the ground truth distribution of the MC throughout the tumor section area, which was compared with the manual MCs of 11 pathologists. Computerized analysis demonstrated high variability of the MC within different tumor areas. There were 6 MCTs with consistently low MCs (MC<7 in all tumor areas), 13 cases with mostly high MCs (MC ≥7 in ≥75% of 10 high-power field areas), and 9 borderline cases with variable MCs around 7, which is a cutoff value for ccMCT grading. There was inconsistency among pathologists in identifying the areas with the highest density of mitotic figures throughout the 3 ccMCT groups; only 51.9% of the counts were consistent with the highest 25% of the ground truth MC distribution. Regardless, there was substantial agreement between pathologists in detecting tumors with MC ≥7. Falsely low MCs below 7 mainly occurred in 4 of 9 borderline cases that had very few ground truth areas with MC ≥7. The findings of this study highlight the need to further standardize how to select the region of the tumor in which to determine the MC.


Assuntos
Doenças do Cão/patologia , Técnicas Histológicas/veterinária , Neoplasias Cutâneas/veterinária , Animais , Contagem de Células/veterinária , Cães , Processamento de Imagem Assistida por Computador , Mastócitos/patologia , Índice Mitótico/veterinária , Gradação de Tumores/veterinária , Variações Dependentes do Observador , Patologistas , Neoplasias Cutâneas/patologia , Software
7.
Front Immunol ; 10: 1864, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31474978

RESUMO

An overt pro-inflammatory immune response is a key factor contributing to lethal pneumococcal infection in an influenza pre-infected host and represents a potential target for therapeutic intervention. However, there is a paucity of knowledge about the level of contribution of individual cytokines. Based on the predictions of our previous mathematical modeling approach, the potential benefit of IFN-γ- and/or IL-6-specific antibody-mediated cytokine neutralization was explored in C57BL/6 mice infected with the influenza A/PR/8/34 strain, which were subsequently infected with the Streptococcus pneumoniae strain TIGR4 on day 7 post influenza. While single IL-6 neutralization had no effect on respiratory bacterial clearance, single IFN-γ neutralization enhanced local bacterial clearance in the lungs. Concomitant neutralization of IFN-γ and IL-6 significantly reduced the degree of pneumonia as well as bacteremia compared to the control group, indicating a positive effect for the host during secondary bacterial infection. The results of our model-driven experimental study reveal that the predicted therapeutic value of IFN-γ and IL-6 neutralization in secondary pneumococcal infection following influenza infection is tightly dependent on the experimental protocol while at the same time paving the way toward the development of effective immune therapies.


Assuntos
Coinfecção/imunologia , Citocinas/imunologia , Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Algoritmos , Animais , Anticorpos Neutralizantes/imunologia , Coinfecção/microbiologia , Coinfecção/virologia , Citocinas/metabolismo , Feminino , Humanos , Vírus da Influenza A/fisiologia , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-6/imunologia , Interleucina-6/metabolismo , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/virologia , Camundongos Endogâmicos C57BL , Modelos Imunológicos , Testes de Neutralização , Infecções por Orthomyxoviridae/virologia , Infecções Pneumocócicas/microbiologia , Pneumonia/imunologia , Pneumonia/microbiologia , Pneumonia/virologia , Streptococcus pneumoniae/fisiologia
8.
J Equine Vet Sci ; 78: 6-9, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31203985

RESUMO

Recently, a syndrome called "equine idiopathic hemorrhagic cystitis" was described and clinical features compared with bladder neoplasia. In this case report, we describe a case of hemorrhagic cystitis with a favorable outcome in a high-performance dressage horse, in which exercise intensity might be the etiologic factor for the development of bladder-wall hyperplasia and hematuria. A 14-year-old Warmblood gelding was presented with a history of hematuria of 2-day duration. The high-level dressage horse had performed on the previous 3 weekends and was trained at least three times a week at performance intensity level. Sonographically, the dorsal bladder wall was about 1.5 cm thick and the different layers of the bladder wall could not be differentiated. Endoscopy revealed that the bladder was highly edematous and showed diffuse submucosal bleeding. Histopathological differential diagnoses were severe reactive hyperplasia or a low-grade transitional cell carcinoma. Four months later, bladder wall thickness had decreased to 1.0 cm and the different layers of the bladder wall were easily visible sonographically. Endoscopy showed a normal bladder mucosa. On histopathology, hyperplasia of the epithelium was significantly decreased. A diet low in calcium was recommended after the checkup, and the owners started working the horse very lightly for 2 days a week. Over the following 2 months, hematuria had not recurred. In conclusion, it seems likely that hemorrhagic cystitis in this horse was exercise-associated, but as repeated provocation by high exercise intensity was not performed in this case, this remains an assumption.


Assuntos
Cistite/veterinária , Condicionamento Físico Animal , Animais , Hematúria/veterinária , Hemorragia/veterinária , Cavalos , Masculino , Recidiva Local de Neoplasia/veterinária
9.
Front Neurol ; 9: 937, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30510535

RESUMO

Background and Purpose: The gut communicates with the brain bidirectionally via neural, humoral and immune pathways. All these pathways are affected by acute brain lesions, such as stroke. Brain-gut communication may therefore impact on the overall outcome after CNS-injury. Until now, contradictory reports on intestinal function and translocation of gut bacteria after experimental stroke have been published. Accordingly, we aimed to specifically investigate the effects of transient focal cerebral ischemia on intestinal permeability, gut associated lymphoid tissue and bacterial translocation in an exploratory study using a well-characterized murine stroke model. Methods: After 60 min of middle cerebral artery occlusion (MCAO) we assessed intestinal morphology (time points after surgery day 0, 3, 5, 14, 21) and tight junction protein expression (occludin and claudin-1 at day 1 and 3) in 12-week-old male C57Bl/6J mice. Lactulose/mannitol/sucralose test was performed to assess intestinal permeability 24-72 h after surgery. To investigate the influence of cerebral ischemia on the local immune system of the gut, main immune cell populations in Peyer's patches (PP) were quantified by flow cytometry. Finally, we evaluated bacterial translocation to extraintestinal organs 24 and 72 h after MCAO by microbiological culture and fluorescence in situ hybridization targeting bacterial 16S rRNA. Results: Transient MCAO decreased claudin-1 expression in the ileum but not in the colon. Intestinal morphology (assessed by light microscopy) and permeability did not change measurably after MCAO. After MCAO, animals had significantly fewer B cells in PP compared to naïve mice. Conclusions: In a murine model of stroke, which leads to large brain infarctions in the middle cerebral artery territory, we did not find evidence for overt alterations neither in gut morphology, barrier proteins and permeability nor presence of intestinal bacterial translocation.

10.
Artigo em Alemão | MEDLINE | ID: mdl-29727875

RESUMO

In this report, the case of a tumourous enlargement of the mammary gland of a 10-year-old, female, Göttingen minipig is described. Noticeable clinical signs were an inflammatory enlargement of one complex of the mammary gland with a teat lesion and a foul-odour, liquid secretion. Further symptoms included reduced general health, with expression of pain, loss of appetite and dehydration. Mastectomy with histopathological examination of the tissue led to the diagnosis of an adenocarcinoma. The pig recovered without complications and was free of symptoms 6 months later.


Assuntos
Adenocarcinoma/veterinária , Neoplasias Mamárias Animais/diagnóstico , Neoplasias Mamárias Animais/cirurgia , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/cirurgia , Adenocarcinoma/diagnóstico , Adenocarcinoma/patologia , Adenocarcinoma/cirurgia , Animais , Feminino , Neoplasias Mamárias Animais/patologia , Mastectomia/veterinária , Suínos , Doenças dos Suínos/patologia , Porco Miniatura
11.
J Zoo Wildl Med ; 49(1): 247-251, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29517425

RESUMO

Between June and November 2015, 25 woodpeckers (Picidae) with neurologic signs or unknown cause of death were admitted to a veterinary clinic. Alive birds were clinically examined. Birds that were found dead or died despite intensive care treatment were forwarded to a pathologic examination. Necropsy and subsequent tests included screening for several infectious agents and toxins. Three birds tested positive for Sarcocystis calchasi. Toxoplasma gondii was detected in one bird demonstrating intracerebral cysts. Mycoplasma gypis was detected in one woodpecker in the absence of respiratory signs. Several microbial pathogens (eg, Aspergillus fumigatus, Clostridium perfringens, and Escherichia coli) were isolated from single individuals. However, there was no consistent finding in all birds that could explain nervous signs and mortality of the woodpeckers examined. To the authors' knowledge, M. gypis and S. calchasi were detected in a woodpecker for the first time in this study.


Assuntos
Doenças das Aves/diagnóstico , Aves , Doenças do Sistema Nervoso Central/veterinária , Sarcocystis/isolamento & purificação , Sarcocistose/veterinária , Animais , Doenças das Aves/epidemiologia , Doenças das Aves/patologia , Doenças do Sistema Nervoso Central/diagnóstico , Doenças do Sistema Nervoso Central/epidemiologia , Doenças do Sistema Nervoso Central/patologia , Alemanha/epidemiologia , Sarcocistose/diagnóstico , Sarcocistose/epidemiologia , Sarcocistose/patologia
12.
Vet Pathol ; 55(4): 490-500, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29402206

RESUMO

Integration of new technologies, such as digital microscopy, into a highly standardized laboratory routine requires the validation of its performance in terms of reliability, specificity, and sensitivity. However, a validation study of digital microscopy is currently lacking in veterinary pathology. The aim of the current study was to validate the usability of digital microscopy in terms of diagnostic accuracy, speed, and confidence for diagnosing and differentiating common canine cutaneous tumor types and to compare it to classical light microscopy. Therefore, 80 histologic sections including 17 different skin tumor types were examined twice as glass slides and twice as digital whole-slide images by 6 pathologists with different levels of experience at 4 time points. Comparison of both methods found digital microscopy to be noninferior for differentiating individual tumor types within the category epithelial and mesenchymal tumors, but diagnostic concordance was slightly lower for differentiating individual round cell tumor types by digital microscopy. In addition, digital microscopy was associated with significantly shorter diagnostic time, but diagnostic confidence was lower and technical quality was considered inferior for whole-slide images compared with glass slides. Of note, diagnostic performance for whole-slide images scanned at 200× magnification was noninferior in diagnostic performance for slides scanned at 400×. In conclusion, digital microscopy differs only minimally from light microscopy in few aspects of diagnostic performance and overall appears adequate for the diagnosis of individual canine cutaneous tumors with minor limitations for differentiating individual round cell tumor types and grading of mast cell tumors.


Assuntos
Doenças do Cão/diagnóstico , Interpretação de Imagem Assistida por Computador , Processamento de Imagem Assistida por Computador , Neoplasias Cutâneas/veterinária , Animais , Doenças do Cão/patologia , Cães , Ensaio de Proficiência Laboratorial , Microscopia/veterinária , Reprodutibilidade dos Testes , Neoplasias Cutâneas/diagnóstico por imagem , Neoplasias Cutâneas/patologia
13.
Eur J Microbiol Immunol (Bp) ; 8(4): 128-134, 2018 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-30719329

RESUMO

In chronic obstructive pulmonary disease (COPD), acute exacerbations and emphysema development are characteristics for disease pathology. COPD is complicated by infectious exacerbations with acute worsening of respiratory symptoms with Moraxella catarrhalis as one of the most frequent pathogens. Although cigarette smoke (CS) is the primary risk factor, additional molecular mechanisms for emphysema development induced by bacterial infections are incompletely understood. We investigated the impact of M. catarrhalis on emphysema development in CS exposed mice and asked whether an additional infection would induce a solubilization of pro-apoptotic and pro-inflammatory endothelial monocyte-activating-protein-2 (EMAPII) to exert its activities in the pulmonary microvas-culature and other parts of the lungs not exposed directly to CS. Mice were exposed to smoke (6 or 9 months) and/or infected with M. catarrhalis. Lungs, bronchoalveolar lavage fluid (BALF), and plasma were analyzed. CS exposure reduced ciliated area, caused rarefaction of the lungs, and induced apoptosis. EMAPII was increased independent of prior smoke exposure in BALF of infected mice. Importantly, acute M. catarrhalis infection increased release of matrixmetalloproteases-9 and -12, which are involved in emphysema development and comprise a mechanism of EMAPII release. Our data suggest that acute M. catarrhalis infection represents an independent risk factor for emphysema development in smoke-exposed mice.

14.
Int J Cancer ; 141(12): 2551-2561, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-28833076

RESUMO

Targeted oncogene inactivation by small molecule inhibitors can be very effective but tumor recurrence is a frequent problem in the clinic. Therapy by inactivation of the cancer-driving oncogene in transplanted tumors was shown to be augmented in the presence of T cells. However, these experiments did not take into account the long-term, usually tolerogenic, interaction of de novo malignancies with the immune system. Here, we employed mice, in which SV40 large T (Tag) and firefly luciferase (Luc) as fusion protein (TagLuc) could be regulated with the Tet-on system and upon activation resulted in tumors after a long latency. TagLuc inactivation induced profound tumor regression, demonstrating sustained oncogene addiction. While tumor relapse after TagLuc inactivation was prevented in immunocompetent mice bearing transplanted tumors, autochthonous tumors relapsed or recurred after therapy discontinuation indicating that the immune system that coevolved with the malignancy over an extended period of time lost the potency to mount an efficient anti-tumor immune response. By contrast, adoptively transferred CD8+ T cells targeting the cancer-driving oncogene eradicated recurrent autochthonous tumors, highlighting a suitable therapy option in a clinically relevant model.


Assuntos
Linfócitos T CD8-Positivos/transplante , Doxiciclina/administração & dosagem , Inativação Gênica , Sistema Imunitário/metabolismo , Neoplasias Experimentais/terapia , Transferência Adotiva , Animais , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Doxiciclina/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transplante de Neoplasias , Neoplasias Experimentais/imunologia , Proteínas Recombinantes de Fusão/genética
15.
Histochem Cell Biol ; 147(6): 707-719, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28247028

RESUMO

Loss of alveolar barrier function with subsequent respiratory failure is a hallmark of severe pneumonia. Although junctions between endo- and epithelial cells regulate paracellular fluid flux, little is known about their composition and regulation in the human alveolar compartment. High autofluorescence of human lung tissue in particular complicates the determination of subcellular protein localization. By comparing conventional channel mode confocal imaging with spectral imaging and linear unmixing, we demonstrate that background fluorescent spectra and fluorophore signals could be rigorously separated resulting in complete recovery of the specific signal at a high signal-to-noise ratio. Using this technique and Western blotting, we show the expression patterns of tight junction proteins occludin, ZO-1 as well as claudin-3, -4, -5 and -18 and adherence junction protein VE-cadherin in naive or Streptococcus pneumoniae-infected human lung tissue. In uninfected tissues, occludin and ZO-1 formed band-like structures in alveolar epithelial cells type I (AEC I), alveolar epithelial cells type II (AEC II) and lung capillaries, whereas claudin-3, -4 and -18 were visualised in AEC II. Claudin-5 was detected in the endothelium only. Claudin-3, -5, -18 displayed continuous band-like structures, while claudin-4 showed a dot-like expression. Pneumococcal infection reduced alveolar occludin, ZO-1, claudin-5 and VE-cadherin but did not change the presence of claudin-3, -4 and -18. Spectral confocal microscopy allows for the subcellular structural analysis of proteins in highly autofluorescent human lung tissue. The thereby observed deterioration of lung alveolar junctional organisation gives a structural explanation for alveolar barrier disruption in severe pneumococcal pneumonia.


Assuntos
Caderinas/metabolismo , Síndrome da Persistência do Padrão de Circulação Fetal/metabolismo , Infecções Pneumocócicas/metabolismo , Alvéolos Pulmonares/anormalidades , Humanos , Síndrome da Persistência do Padrão de Circulação Fetal/microbiologia , Infecções Pneumocócicas/microbiologia , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/microbiologia , Streptococcus pneumoniae
16.
Stroke ; 47(5): 1354-63, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27056982

RESUMO

BACKGROUND AND PURPOSE: Antibiotics disturbing microbiota are often used in treatment of poststroke infections. A bidirectional brain-gut microbiota axis was recently suggested as a modulator of nervous system diseases. We hypothesized that gut microbiota may be an important player in the course of stroke. METHODS: We investigated the outcome of focal cerebral ischemia in C57BL/6J mice after an 8-week decontamination with quintuple broad-spectrum antibiotic cocktail. These microbiota-depleted animals were subjected to 60 minutes middle cerebral artery occlusion or sham operation. Infarct volume was measured using magnetic resonance imaging, and mice were monitored clinically throughout the whole experiment. At the end point, tissues were preserved for further analysis, comprising histology and immunologic investigations using flow cytometry. RESULTS: We found significantly decreased survival in the middle cerebral artery occlusion microbiota-depleted mice when the antibiotic cocktail was stopped 3 days before surgery (compared with middle cerebral artery occlusion specific pathogen-free and sham-operated microbiota-depleted mice). Moreover, all microbiota-depleted animals in which antibiotic treatment was terminated developed severe acute colitis. This phenotype was rescued by continuous antibiotic treatment or colonization with specific pathogen-free microbiota before surgery. Further, infarct volumes on day one did not differ between any of the experimental groups. CONCLUSIONS: Conventional microbiota ensures intestinal protection in the mouse model of experimental stroke and prevents development of acute and severe colitis in microbiota-depleted mice not given antibiotic protection after cerebral ischemia. Our experiments raise the clinically important question as to whether microbial colonization or specific microbiota are crucial for stroke outcome.


Assuntos
Antibacterianos/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Acidente Vascular Cerebral/microbiologia , Animais , Feminino , Infarto da Artéria Cerebral Média/microbiologia , Camundongos , Camundongos Endogâmicos C57BL
17.
Brain Res ; 1642: 364-375, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27067188

RESUMO

Although injectable anesthetics are still widely used in laboratory rodents, scientific data concerning pain and distress during and after stereotactic surgery are rare. However, optimal anesthesia protocols have a high impact on the quality of the derived data. We therefore investigated the suitability of recommended injectable anesthesia with a traditionally used monoanesthesia for stereotactic surgery in view of optimization and refinement in rats. The influence of the recommended complete reversal anesthesia (MMF; 0.15mg/kg medetomidine, 2mg/kg midazolam, 0.005mg/kg fentanyl; i.m.) with or without reversal and of chloral hydrate (430mg/kg, 3.6%, i.p.) on various physiological, biochemical and behavioral parameters (before, during, after surgery) was analyzed. Isoflurane was also included in stress parameter analysis. In all groups, depth of anesthesia was sufficient for stereotactic surgery with no animal losses. MMF caused transient exophthalmos, myositis at the injection site and increased early postoperative pain scores. Reversal induced agitation, restlessness and hypothermia. Even the low concentrated chloral hydrate led to peritonitis and multifocal liver necrosis, corresponding to increased stress hormone levels and loss in body weight. Increased stress response was also exerted by isoflurane anesthesia. Pronounced systemic toxicity of chloral hydrate strongly questions its further use in rodent anesthesia. In view of undesired effects of MMF and isoflurane, thorough consideration of anesthesia protocols for particular research projects is indispensable. Reversal should be restricted to emergency situations. Our data support further refinement of the current protocols and the importance of sham operated controls.


Assuntos
Anestesia/métodos , Modelos Animais , Ratos , Técnicas Estereotáxicas , Anestesia/efeitos adversos , Anestésicos/administração & dosagem , Anestésicos/toxicidade , Animais , Hidrato de Cloral/administração & dosagem , Hidrato de Cloral/toxicidade , Feminino , Fentanila/administração & dosagem , Injeções/efeitos adversos , Isoflurano/administração & dosagem , Masculino , Medetomidina/administração & dosagem , Midazolam/administração & dosagem , Dor Pós-Operatória/patologia , Dor Pós-Operatória/fisiopatologia , Dor Pós-Operatória/prevenção & controle , Estresse Psicológico/etiologia , Estresse Psicológico/patologia , Estresse Psicológico/fisiopatologia , Estresse Psicológico/prevenção & controle
18.
Arch Toxicol ; 90(5): 1093-102, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26070365

RESUMO

Colorectal cancer is one of the most frequent cancers in Western countries. Chronic intestinal diseases such as Crohn's disease and ulcerative colitis, in which the intestinal barrier is massively disturbed, significantly raise the risk of developing a colorectal tumour. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a genotoxic heterocyclic aromatic amine that is formed after strongly heating fish and meat. In this study, the hypothesis that PhIP uptake in the gut is increased during chronic colitis was tested. Chronic colitis was induced by oral administration of dextran sulphate sodium (DSS) to Fischer 344 rats. The transport of PhIP in eight different rat intestinal segments was examined in Ussing chambers. The tissues were incubated with 10 µM PhIP for 90 min, and the concentration of PhIP was determined in the mucosal and serosal compartments of the Ussing chambers as well as in the clamped tissues by LC-MS. Although chronic colitis was clearly induced in the rats, no differences in the intestinal transport of PhIP were observed between control and DSS-treated animals. The hypothesis that in the course of chronic colitis more PhIP is taken up by the intestinal epithelium, thereby increasing the risk of developing colorectal cancer, could not be confirmed in the present report.


Assuntos
Carcinógenos/metabolismo , Colite/metabolismo , Sulfato de Dextrana , Imidazóis/metabolismo , Absorção Intestinal , Mucosa Intestinal/metabolismo , Animais , Carcinógenos/toxicidade , Cromatografia Líquida , Doença Crônica , Colite/induzido quimicamente , Colite/patologia , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/metabolismo , Modelos Animais de Doenças , Imidazóis/toxicidade , Intestinos/patologia , Cinética , Masculino , Ratos Endogâmicos F344 , Fatores de Risco , Espectrometria de Massas por Ionização por Electrospray
19.
J Avian Med Surg ; 29(3): 216-23, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26378668

RESUMO

A 6-week-old, parent-reared peregrine falcon ( Falco peregrinus ) was presented with spastic hypertonus of its hind limbs of unknown origin and duration. Radiologic examination revealed smooth periosteal reactions ventrally at thoracic vertebrae 5 to 7. Contrast-enhanced computed tomography identified the swelling as inflammation; antibiotic, antimycotic, anti-inflammatory, and analgesic treatments were initiated, and vitamins and minerals were supplemented. Because the bird's condition did not improve after 10 days, it was euthanatized and submitted for postmortem examination. On histopathologic examination, chronic, active osteomyelitis was diagnosed in thoracic vertebrae 5 to 7, and chronic, active arthritis was present in both the right shoulder and left elbow joints. Staphylococcus hyicus was isolated from these 3 locations, as well as from lungs and liver, indicating a chronic septic staphylococcosis. Although infections with Staphylococcus species are occasional causes of vertebral osteomyelitis in juvenile poultry with active growth plates, it is only sporadically reported in raptors and companion birds. This case report is the first description of the clinical features and diagnostic and pathologic findings in a juvenile peregrine falcon with hematogenous osteomyelitis and arthritis associated with septicemia caused by S hyicus.


Assuntos
Artrite Infecciosa/veterinária , Doenças das Aves/microbiologia , Falconiformes , Osteomielite/veterinária , Coluna Vertebral/patologia , Infecções Estafilocócicas/veterinária , Staphylococcus hyicus/isolamento & purificação , Animais , Antibacterianos/uso terapêutico , Anti-Inflamatórios não Esteroides/uso terapêutico , Artrite Infecciosa/tratamento farmacológico , Artrite Infecciosa/microbiologia , Artrite Infecciosa/patologia , Doenças das Aves/patologia , Fluoroquinolonas/uso terapêutico , Masculino , Meloxicam , Osteomielite/tratamento farmacológico , Osteomielite/microbiologia , Osteomielite/patologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/patologia , Tiazinas/uso terapêutico , Tiazóis/uso terapêutico
20.
PLoS One ; 10(4): e0124927, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25928296

RESUMO

In our previous work we could identify defects in human regulatory T cells (Tregs) likely favoring the development of graft-versus-host disease (GvHD) following allogeneic stem cell transplantation (SCT). Treg transcriptome analyses comparing GvHD and immune tolerant patients uncovered regulated gene transcripts highly relevant for Treg cell function. Moreover, granzyme A (GZMA) also showed a significant lower expression at the protein level in Tregs of GvHD patients. GZMA induces cytolysis in a perforin-dependent, FAS-FASL independent manner and represents a cell-contact dependent mechanism for Tregs to control immune responses. We therefore analyzed the functional role of GZMA in a murine standard model for GvHD. For this purpose, adoptively transferred CD4+CD25+ Tregs from gzmA-/- mice were analyzed in comparison to their wild type counterparts for their capability to prevent murine GvHD. GzmA-/- Tregs home efficiently to secondary lymphoid organs and do not show phenotypic alterations with respect to activation and migration properties to inflammatory sites. Whereas gzmA-/- Tregs are highly suppressive in vitro, Tregs require GZMA to rescue hosts from murine GvHD, especially regarding gastrointestinal target organ damage. We herewith identify GZMA as critical effector molecule of human Treg function for gastrointestinal immune response in an experimental GvHD model.


Assuntos
Gastroenteropatias/imunologia , Gastroenteropatias/prevenção & controle , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Granzimas/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Movimento Celular/imunologia , Modelos Animais de Doenças , Gastroenteropatias/genética , Expressão Gênica , Doença Enxerto-Hospedeiro/genética , Granzimas/genética , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Imunofenotipagem , Imunoterapia Adotiva , Tecido Linfoide/imunologia , Camundongos , Camundongos Knockout , Fenótipo
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