For the understanding of pathological states of bone tissues in oral surgery, it would be desirable to have the possibility to simulate these processes on bone cell models in vitro. These cultures, similarly to bone tissues, contain numerous proteins entrapped in the insoluble matrix. The major goal of this study was to verify whether a method based on direct in-matrix protein digestion could be suitable for the discrimination between different induced pathological states of bone cell models cultivated in vitro. Using in-sample specific protein digestion with trypsin followed by liquid chromatography-tandem mass spectrometry analysis of released peptides, 446 proteins (in average per sample) were identified in a bone cell in vitro model with induced cancer, 440 proteins were found in a model with induced inflammation, 451 proteins were detected in control in vitro culture, and 491 proteins were distinguished in samples of vestibular laminas of maxillary bone tissues originating from six different patients. Subsequent partial least squares - discrimination analysis of obtained liquid chromatography-tandem mass spectrometry data was able to discriminate among in vitro cultures with induced cancer, with induced inflammation, and control cultivation. Thus, the direct in-sample protein digestion by trypsin followed by liquid chromatography-tandem mass spectrometry analysis of released specific peptide fragments from the insoluble matrix and mathematical analysis of the mass spectrometry data seems to be a promising tool for the routine proteomic characterization of in vitro human bone models with induced different pathological states.

• MeSH
• Chromatography, Liquid methods   MeSH
• Humans MeSH
• Peptides analysis   MeSH
• Proteins chemistry   MeSH
• Proteolysis MeSH
• Proteomics methods   MeSH
• Oral Surgical Procedures * MeSH
• Tandem Mass Spectrometry * methods   MeSH
• Trypsin chemistry   MeSH
• Inflammation MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Chondroblastóm je vzácny benígny kostný nádor najčastejšie lokalizovaný v epifýze. Tvorí približne 1 % všetkých benígnych kostných tumorov. Vyskytuje sa hlavne u mladých pacientov s maximom výskytu v druhej dekáde života. Klinicky sa manifestuje bolesťou a obmedzením hybnosti postihnutého kĺbu. Jedná sa o histologicky benígnu afekciu, vyznačuje sa však agresívnym rastom, deštruuje okolie (spongióznu kosť, kortiku aj kĺbnu chrupavku) a môže expandovať intraartikulárne. Nádorové bunky produkujú cytokíny, ktoré sú dôvodom perilezionálnych inflamačných reakcií, ako je kostný edém, alebo výrazná synovitída v prípade intraartikulárnej propagácie. Približne v 20 % lokálne recidivuje aj napriek terapii. V práci publikujeme prípad 15-ročného chlapca s chondroblastómom proximálneho femuru. Po histologizácii bol ošetrený pomocou prístupu SHD (Surgical Hip Dislocation), ktorý umožňuje pomocou luxácie kĺbu ošetriť postihnutú hlavu stehennej kosti bez porušenia nutritívnych ciev. Po radikálnej exkochleácii a lokálnej chemoproteolýze bol defekt vyplnený parciálne resorbovateľným kostným cementom. Od operácie je pacient v sledovaní 1 rok s klinicky aj roentgenologicky dobrým výsledkom.

Chondroblastoma is a rare benign bone tumor, most often located in an epiphysis. It represents about 1 % of all benign bone tumors. Chondroblastoma is typical for young patients and occurs most often in the second decade of life. Clinical presentation consists of pain and restriction of movement in the affected joint. Histology shows a benign type of tumor, which can however grow aggressively and destroy its surroundings (spongiosa, cortical bone and joint cartilage) and expand into the joint cavity. Tumor cells produce cytokines, which are responsible for inflammatory processes occurring around the tumor such as bone edema or prominent synovitis in case of intraarticular growth. In 20 % of cases, we see a relapse despite treatment. In this article we present the case of a 15-year-old boy with a proximal femur chondroblastoma. After the initial biopsy, the tumor was treated surgically via surgical hip dislocation, which allows treatment of the femoral head without compromising nutritional blood vessels. After a radical excochleation and local chemoproteolysis, the defect was filled with partially resorbable bone cement. The patient has been monitored for a year now with a good clinical and radiological outcome.

• Keywords
• exkochleace,
• MeSH
• Chondroblastoma * surgery   diagnostic imaging   pathology   MeSH
• Joint Dislocations MeSH
• Humans MeSH
• Adolescent MeSH
• Musculoskeletal Pain etiology   MeSH
• Femoral Neoplasms * surgery   diagnostic imaging   pathology   MeSH
• Orthopedic Procedures methods   MeSH
• Proteolysis MeSH
• Treatment Outcome MeSH
• Check Tag
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Publication type
• Case Reports MeSH

• MeSH
• Radiography methods   MeSH
• Publication type
• Monograph MeSH

• Conspectus
• Patologie. Klinická medicína
• NML Fields
• radiologie, nukleární medicína a zobrazovací metody
• patologie

BACKGROUND: Real life data regarding pharmacokinetics of vedolizumab in patients needing dose optimisation are scarce. We set to examine whether pre-optimisation vedolizumab levels associate with therapy outcomes and which mechanisms explain the associations. METHODS: A multicentre observational study assessed the outcome of dose increase in association with pre-escalation levels in vedolizumab-treated patients. SubsequentIy, α4β7 occupancy on peripheral blood [PB] and intestinal lamina propria [LP] tissues was investigated on various cellular subsets in patients undergoing lower endoscopy on infusion day. Cellular localisation of vedolizumab-bound α4β7 and effects on M1 and M2 macrophages were also explored. RESULTS: A total of 161 inflammatory bowel disease [IBD] patients were included. Among 129/161 patients intensified during maintenance [Week 14 onward], pre-intensification trough levels were comparable or higher among those subsequently attaining post-optimisation clinical, biomarker, and endoscopic remission, compared with non-remitting patients [p = 0.09, 0.25, 0.04, respectively]. Similar results were demonstrated for those dose-optimised during induction [Week 6, n = 32]. In the immune sub-study [n = 43], free α4β7 receptors at trough were similarly low among patients with/without mucosal healing, on PB T cells [p = 0.15], LP T cells [p = 0.88], and on PB eosinophils [p = 0.08]. Integrin receptors on M1 and M2 macrophages were also saturated by low levels of vedolizumab and anti-inflammatory cytokine secretion was not increased. Co-localisation and dissociation experiments demonstrated membranal α4β7 receptors of two origins: non-internalised and newly generated α4β7, but re-binding was still complete at very low concentrations. CONCLUSIONS: These results do not support pharmacokinetics as the mechanism responsible for loss of response to vedolizumab, nor do they support a need for higher drug concentration to enhance vedolizumab's immune effects. Higher pre-escalation levels may indicate less clearance [less severe disease] and higher likelihood of subsequent re-gained response, regardless of therapy escalation.

• MeSH
• Biomarkers analysis   MeSH
• C-Reactive Protein analysis   MeSH
• Adult MeSH
• Endoscopy, Gastrointestinal MeSH
• Gastrointestinal Agents administration & dosage   MeSH
• Antibodies, Monoclonal, Humanized administration & dosage   MeSH
• Inflammatory Bowel Diseases drug therapy   MeSH
• Middle Aged MeSH
• Humans MeSH
• Macrophages drug effects   MeSH
• Cell Adhesion Molecules analysis   MeSH
• Mucoproteins analysis   MeSH
• Serum Albumin analysis   MeSH
• Dose-Response Relationship, Drug MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Multicenter Study MeSH
• Observational Study MeSH

The commonly used histological assessment of pathological states of alveolar bone tissues in oral surgery needs laborious and time-consuming processing by an experienced histologist. Therefore, a simpler and faster methodology is required in this field. Following this demand, this paper reports a straightforward approach using the tryptic cleavage of proteins directly in bone without its demineralization, followed by the capillary electrophoresis-ultraviolet detection profiling of the yielded protein digest. Cleavage-derived peptides were separated by capillary electrophoresis in acidic background electrolytes, pH 2.01-2.54. The best resolution of peptide fragments with the highest peak capacity was achieved in the background electrolyte composed of 55 mM H3 PO4 , 14 mM tris(hydroxymethyl)aminomethan, pH 2.01. The differences in the obtained capillary electrophoresis-ultraviolet detection profiles with characteristic patterns for particular bone samples were subsequently discriminated by linear discriminant analysis over principal components. This approach was first verified on porcine bone tissues as model samples; jawbone and calf bone tissues could be discriminated with an accuracy of 100%. Subsequently, the method was capable of differentiating unequivocally between human healthy and inflammatory alveolar bone tissues obtained from oral surgery. This procedure seems to be promising as complement or even an alternative to the traditional histological discrimination between healthy and inflammatory bone tissues in oral surgery.

• MeSH
• Electrophoresis, Capillary MeSH
• Bone and Bones chemistry   metabolism   MeSH
• Humans MeSH
• Swine MeSH
• Oral Surgical Procedures * MeSH
• Trypsin chemistry   metabolism   MeSH
• Inflammation diagnosis   metabolism   surgery   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Wnt and BMP signaling pathways are two key molecular machineries regulating development and homeostasis. The efficient coordination of Wnt and BMP is essential in many developmental processes such as establishment of antero-posterior and dorso-ventral body axis, regulation of convergent extension, or development of various organ systems. SMAD ubiquitination regulatory factor (Smurf) family of E3 ubiquitin ligases are important and evolutionary conserved regulators of TGF-β/BMP signaling pathways. Smurf2 has been previously shown to regulate Wnt/planar cell polarity (PCP) signaling pathway by ubiquitinating Prickle1, one of the key components of PCP. We explored the role of Smurf2 in Wnt pathways in further detail and identified that Smurf2 is also a ubiquitin ligase of Dishevelled (DVL), the key cytoplasmic signal transducer in the Wnt pathway. Interestingly, the Smurf2 and DVL relationship expands beyond substrate-E3 ligase. We can show that DVL activates Smurf2, which allows Smurf2 to ubiquitinate its substrates from Wnt/PCP (Prickle1) as well as TGF-β/BMP (Smad2) pathways more efficiently. Using SMAD7 as an example of Smurf2 activator we show that DVL and SMAD7 both activates Smurf2 activity. In HEK293 cells the deficiency of DVL phenocopies absence of Smurf2 and leads to the increased phosphorylation of R-Smads. Smurf2-DVL connection provides a novel and intriguing point of crosstalk for Wnt and BMP pathways.

• MeSH
• Models, Biological MeSH
• HEK293 Cells MeSH
• Bone Morphogenetic Proteins metabolism   MeSH
• Humans MeSH
• Tumor Suppressor Proteins metabolism   MeSH
• Dishevelled Proteins metabolism   MeSH
• LIM Domain Proteins metabolism   MeSH
• Proteolysis MeSH
• Wnt Signaling Pathway * MeSH
• Signal Transduction MeSH
• Transforming Growth Factor beta metabolism   MeSH
• Ubiquitination MeSH
• Ubiquitin-Protein Ligases metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Pyknodysostóza je vzácné genetické onemocnění patřící ke sklerotizujícím kostním dysplaziím. Pyknodysostóza je způsobena ztrátovou mutací v genu pro katepsin K(CTSK), který hraje důležitou roli v odbourávání kostních proteinů, osteoklasty tak nemohou efektivně resorbovat organickou kostní matrix. Důsledkem nedostatku katepsinu K je porucha růstu, dysplazie lebečních a obličejových kostí a článků prstů, osteoskleróza a zvýšená kostní lomivost. Dědičnost je autosomálně recesivní. Prezentovaný pacient byl poslán na antropologii pro poruchu růstu a orofaciální abnormity. Antropometrické vyšetření prokázalo malý vzrůst se zkrácenými končetinami, úzký hrudník a ramena, relativní makrocefalii, opožděný uzávěr velké fontanely a hypoplazii dolní čelisti. Měl krátké distální články prstů a onychodystrofii. Radiologické vyšetření prokázalo difusní osteosklerózu skeletu a akroosteolýzu distálních článků prstů. Na základě těchto nálezů bylo vysloveno podezření na pyknodysostózu. Molekulárně genetické vyšetření pak tuto diagnózu potvrdilo. Terapie tohoto onemocnění je symptomatická a multidisciplinární, včetně neinvazivní dechové podpory pro spánkovou apnoe, ortopedické léčby zlomenin, dentomaxilární rekonstrukce atd. Prognóza je příznivá, onemocnění není progresivní. Znalosti klíčových znaků a časná radiologická diagnóza této vrozené vady umožní prevenci a léčbu obvyklých komplikací.

Pycnodysostosis is arare genetic disorder belonging to sclerosing skeletal dysplasias. Pycnodysostosis is caused by loss of function mutations in the cathepsin K(CTSK) gene which plays an important role in enzymatic digestion of the bone proteins, therefore osteoclasts cannot effectively break down the organic matrix of bone. Deficiency of cathepsin Kresults in dwarfism, dysplasia of the skull and facial bones and phalanges, osteosclerosis, and fragility. It is inherited in an autosomal recessive trait. The presented patient was referred to our department due to growth failure and orofacial abnormality. Anthropometric examination proved short stature with short limbs, narrow chest and shoulder, relative macrocephaly, delayed closure of the anterior fontanelle and mandibular hypoplasia. He has short tips of fingers and onychodystrophy. Radiological examination ascertained diffuse osteosclerosis of the skeleton and acro-osteolysis of the distal phalanges. On the basis of these findings the suspicion on Pycnodysostosis was pronounced. Molecular genetic testing proved this diagnosis. The therapy of this disease is symptomatic and multidisciplinary, including assisted non-invasive ventilation due to sleep apnoea, orthopaedic treatment of fractures, dento-maxillar reconstructions, etc. The prognosis is quite promising, the disease is not progressive. Knowledge of the key signs and early X-ray diagnosis of this inherited disorder will facilitate the prevention and treatment of common complications.

Despite the increasing number of studies concerning insect immunity, Lutzomyia longipalpis immune responses in the presence of Leishmania infantum chagasi infection has not been widely investigated. The few available studies analyzed the role of the Toll and IMD pathways involved in response against Leishmania and microbial infections. Nevertheless, effector molecules responsible for controlling sand fly infections have not been identified. In the present study we investigated the role a signal transduction pathway, the Transforming Growth Factor-beta (TGF-β) pathway, on the interrelation between L. longipalpis and L. i. chagasi. We identified an L. longipalpis homolog belonging to the multifunctional cytokine TGF-β gene family (LlTGF-β), which is closely related to the activin/inhibin subfamily and potentially involved in responses to infections. We investigated this gene expression through the insect development and in adult flies infected with L. i. chagasi. Our results showed that LlTGF-β was expressed in all L. longipalpis developmental stages and was upregulated at the third day post L. i. chagasi infection, when protein levels were also higher as compared to uninfected insects. At this point blood digestion is finished and parasites are in close contact with the insect gut. In addition, we investigated the role of LlTGF-β on L. longipalpis infection by L. i. chagasi using either gene silencing by RNAi or pathway inactivation by addition of the TGF-β receptor inhibitor SB431542. The blockage of the LlTGF-β pathway increased significantly antimicrobial peptides expression and nitric oxide levels in the insect gut, as expected. Both methods led to a decreased L. i. chagasi infection. Our results show that inactivation of the L. longipalpis TGF-β signal transduction pathway reduce L. i. chagasi survival, therefore suggesting that under natural conditions the parasite benefits from the insect LlTGF-β pathway, as already seen in Plamodium infection of mosquitoes.

• MeSH
• Survival Analysis MeSH
• Insect Vectors immunology   parasitology   MeSH
• Host-Pathogen Interactions * MeSH
• Leishmania infantum growth & development   MeSH
• Immunity, Innate MeSH
• Psychodidae immunology   parasitology   MeSH
• Signal Transduction MeSH
• Gene Expression Profiling MeSH
• Transforming Growth Factor beta metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

To successfully feed, ticks inject pharmacoactive molecules into the vertebrate host including cystatin cysteine protease inhibitors. However, the molecular and cellular events modulated by tick saliva remain largely unknown. Here, we describe and characterize a novel immunomodulatory cystatin, Iristatin, which is upregulated in the salivary glands of feeding Ixodes ricinus ticks. We present the crystal structure of Iristatin at 1.76 Å resolution. Purified recombinant Iristatin inhibited the proteolytic activity of cathepsins L and C and diminished IL-2, IL-4, IL-9, and IFN-γ production by different T-cell populations, IL-6 and IL-9 production by mast cells, and nitric oxide production by macrophages. Furthermore, Iristatin inhibited OVA antigen-induced CD4+ T-cell proliferation and leukocyte recruitment in vivo and in vitro. Our results indicate that Iristatin affects wide range of anti-tick immune responses in the vertebrate host and may be exploitable as an immunotherapeutic.

• MeSH
• Cystatins classification   genetics   pharmacology   MeSH
• Cytokines metabolism   MeSH
• Epoxy Compounds metabolism   MeSH
• Phylogeny MeSH
• Immunosuppressive Agents chemistry   metabolism   pharmacology   MeSH
• Ixodes chemistry   genetics   metabolism   MeSH
• Crystallography, X-Ray MeSH
• Macrophages drug effects   metabolism   MeSH
• Nitric Oxide metabolism   MeSH
• Arthropod Proteins chemistry   genetics   pharmacology   MeSH
• Proteolysis drug effects   MeSH
• Amino Acid Sequence MeSH
• Sequence Homology, Amino Acid MeSH
• Salivary Cystatins chemistry   genetics   pharmacology   MeSH
• T-Lymphocytes drug effects   metabolism   MeSH
• Tyrosine analogs & derivatives   metabolism   MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Studies have suggested a role for the mammalian (or mechanistic) target of rapamycin (mTOR) in skeletal development and homeostasis, yet there is no evidence connecting mTOR with the key signaling pathways that regulate skeletogenesis. We identified a parathyroid hormone (PTH)/PTH-related peptide (PTHrP)-salt-inducible kinase 3 (SIK3)-mTOR signaling cascade essential for skeletogenesis. While investigating a new skeletal dysplasia caused by a homozygous mutation in the catalytic domain of SIK3, we observed decreased activity of mTOR complex 1 (mTORC1) and mTORC2 due to accumulation of DEPTOR, a negative regulator of both mTOR complexes. This SIK3 syndrome shared skeletal features with Jansen metaphyseal chondrodysplasia (JMC), a disorder caused by constitutive activation of the PTH/PTHrP receptor. JMC-derived chondrocytes showed reduced SIK3 activity, elevated DEPTOR, and decreased mTORC1 and mTORC2 activity, indicating a common mechanism of disease. The data demonstrate that SIK3 is an essential positive regulator of mTOR signaling that functions by triggering DEPTOR degradation in response to PTH/PTHrP signaling during skeletogenesis.

• MeSH
• HEK293 Cells MeSH
• Homozygote MeSH
• Intracellular Signaling Peptides and Proteins metabolism   MeSH
• Humans MeSH
• Mutation, Missense genetics   MeSH
• Mechanistic Target of Rapamycin Complex 1 metabolism   MeSH
• Mechanistic Target of Rapamycin Complex 2 metabolism   MeSH
• Mutant Proteins chemistry   metabolism   MeSH
• Osteogenesis * MeSH
• Parathyroid Hormone metabolism   MeSH
• Parathyroid Hormone-Related Protein metabolism   MeSH
• Protein Kinases chemistry   deficiency   genetics   metabolism   MeSH
• Proteolysis MeSH
• Growth Plate metabolism   MeSH
• Amino Acid Sequence MeSH
• Signal Transduction * MeSH
• TOR Serine-Threonine Kinases metabolism   MeSH
• Inheritance Patterns genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH