Proteomic profiling of human dental enamel affected by molar incisor hypomineralisation of different clinical severity grades: an in vitro study.

PURPOSE: The aim of this study was to explore the potential to profile and distinguish varying clinical severity grades of MIH, compared to normal enamel, using proteomics. METHODS: Liquid chromatography-mass spectrometry analyses were conducted on enamel samples of extracted teeth, from 11 children and adolescents, spanning an age range of 6-18 years. Enamel powder samples were collected from extracted, third molars (n = 3) and first permanent molars diagnosed with MIH (n = 8). The MIH tooth samples were categorized into subgroups based on clinical severity grade. The data were statistically analyzed using ANOVA and Welch's t test. RESULTS: Teeth affected by MIH exhibited a diverse array of proteins, each with different functions related to dental enamel, distinguishing them from their normal enamel counterparts. The application of microdissection combined with LC-MS techniques has revealed the potential to discern unique proteomic profiles among MIH-affected teeth, characterized by varying clinical severity grades. Both analyzed MIH groups displayed consistent trends in the presentation of biological processes, including underabundance of proteins primarily associated with cell organization and biogenesis. Furthermore, proteins linked to cell death were overabundant in both MIH groups. CONCLUSION: Proteomics enabled the detection and differentiation of various proteins across different clinical severity grades of MIH.

BCG-induced cytokine release in bladder cancer cells is regulated by Ca2+ signaling.

Bacillus Calmette-Guérin (BCG) is widely used in the clinic to effectively treat superficial urinary bladder cancer. However, a significant proportion of patients who fail to respond to BCG risk cystectomy or death. Though more than 3 million cancer treatments with BCG occur annually, surprisingly little is known about the initial signaling cascades activated by BCG. Here, we report that BCG induces a rapid intracellular Ca2+ (calcium ion) signal in bladder cancer cells that is essential for activating the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and for synthesizing and secreting proinflammatory cytokines, including interleukin 8 (IL-8). A similar Ca2+ response was observed when cells were exposed to the supernatant of BCG. Studying cellular molecular mechanisms involved in the BCG signaling event, we found pivotal roles for phospholipase C and the Toll-like receptor 4. Further assessment revealed that this signaling pathway induces synthesis of IL-8, whereas exocytosis appeared to be controlled by global Ca2+ signaling. These results shed new light on the molecular mechanisms underlying BCG treatment of bladder cancer, which can help in improving therapeutic efficacy and reducing adverse side effects.

Ex Vivo Generation of Donor Antigen-Specific Immunomodulatory Cells: A Comparison Study of Anti-CD80/86 mAbs and CTLA4-lg Costimulatory Blockade.

Adoptive transfer of alloantigen-specific immunomodulatory cells generated ex vivo with anti-CD80/CD86 mAbs (2D10.4/IT2.2) holds promise for operational tolerance after transplantation. However, good manufacturing practice is required to allow widespread clinical application. Belatacept, a clinically approved cytotoxic T-lymphocyte antigen 4-immunoglobulin that also binds CD80/CD86, could be an alternative agent for 2D10.4/IT2.2. With the goal of generating an optimal cell treatment with clinically approved reagents, we evaluated the donor-specific immunomodulatory effects of belatacept- and 2D10.4/IT2.2-generated immunomodulatory cells. Immunomodulatory cells were generated by coculturing responder human peripheral blood mononuclear cells (PBMCs) (50 × 106 cells) with irradiated donor PBMCs (20 × 106 cells) from eight human leukocyte antigen-mismatched responder-donor pairs in the presence of either 2D10.4/IT2.2 (3 µg/106 cells) or belatacept (40 µg/106 cells). After 14 days of coculture, the frequencies of CD4+ T cells, CD8+ T cells, and natural killer cells as well as interferon gamma (IFN-γ) production in the 2D10.4/IT2.2- and belatacept-treated groups were lower than those in the control group. The percentage of CD19+ B cells was higher in the 2D10.4/IT2.2- and belatacept-treated groups than in the control group. The frequency of CD4+CD25+CD127lowFOXP3+ T cells increased from 4.1±1.0% (preculture) to 7.1±2.6% and 7.3±2.6% (day 14) in the 2D10.4/IT2.2- and belatacept-treated groups, respectively (p<0.05). Concurrently, delta-2 FOXP3 mRNA expression increased significantly. Compared with cells derived from the no-antibody treated control group, cells generated from both the 2D10.4/IT2.2- and belatacept-treated groups produced lower IFN-γ and higher interleukin-10 levels in response to donor-antigens, as detected by enzyme-linked immunospot. Most importantly, 2D10.4/IT2.2- and belatacept-generated cells effectively impeded the proliferative responses of freshly isolated responder PBMCs against donor-antigens. Our results indicate that belatacept-generated donor-specific immunomodulatory cells possess comparable phenotypes and immunomodulatory efficacies to those generated with 2D10.4/IT2.2. We suggest that belatacept could be used for ex vivo generation of clinical grade alloantigen-specific immunomodulatory cells for tolerance induction after transplantation.

Quality of life improves early after gender reassignment surgery in transgender women.

BACKGROUND: Few studies have examined the long-term quality of life (QoL) of individuals with gender dysphoria, or how it is affected by treatment. Our aim was to examine the QoL of transgender women undergoing gender reassignment surgery (GRS). METHODS: We performed a prospective cohort study on 190 patients undergoing male-to-female GRS at Karolinska University Hospital between 2003 and 2015. We used the Swedish version of the Short Form-36 Health Survey (SF-36), which measures QoL across eight domains. The questionnaire was distributed to patients pre-operatively, as well as 1, 3, and 5 years post-operatively. The results were compared between the different measure points, as well as between the study group and the general population. RESULTS: On most dimensions of the SF-36 questionnaire, transgender women reported a lower QoL than the general population. The scores of SF-36 showed a non-significant trend to be lower 5 years post-GRS compared to pre-operatively, a decline consistent with that of the general population. Self-perceived health compared to 1 year previously rose in the first post-operative year, after which it declined. CONCLUSIONS: To our knowledge, this is the largest prospective study to follow a group of transgender patients with regards to QoL over continuous temporal measure points. Our results show that transgender women generally have a lower QoL compared to the general population. GRS leads to an improvement in general well-being as a trend but over the long-term, QoL decreases slightly in line with that of the comparison group. Level of evidence: Level III, therapeutic study.

Midface Osteotomies for Feminization of the Facial Skeleton.

Facial feminization surgery is a term to describe the surgical alteration of a masculine facial appearance to a more feminine appearance, which is most commonly performed for male-to-female transsexual individuals. To alter the midfacial relations, segmentalized osteotomies were performed in selected patients expanding on the established techniques for facial feminization surgery. All patients underwent a preoperative 3D computerized tomography scan and 3D photography before and after the surgery. The inclusion of the midface in surgery was determined based on the relative projection and angle of the zygomatic body compared with the supraorbital region (the supraorbital region was reduced in all patients). Patients were prospectively followed up by 3D surface photography and 3D computerized tomography scans. Rotation and advancement of the zygomatic region was found to be an effective way to further feminize the midfacial appearance in selected male-to-female transsexual patients. No major surgical complications occurred. Although somewhat technically challenging, we suggest that midface surgery should be considered for feminizing purposes in order for the patient to achieve a long-term favorable result compared with other alternative methods.

Long-Term Sensitivity and Patient-Reported Functionality of the Neoclitoris After Gender Reassignment Surgery.

INTRODUCTION: A cornerstone of treating gender dysphoria for transgender women is gender reassignment surgery (GRS) encompassing vaginoplasty and clitoroplasty. The neoclitoris is harvested as a flap with a neurovascular pedicle from the proximal dorsal part of the glans penis. Few long-term follow-ups exist on postoperative sensation and patient-reported sexual functionality of the neoclitoris. AIM: To examine the sensitivity of the neoclitoris and its relation to orgasm and sexual function at least 1 year after GRS. METHODS: Twenty-two patients were included, with a mean follow-up of 37 months (range = 12-63) after initial surgery. Tactile and vibratory sensitivities were measured with Semmes-Weinstein monofilaments and the Bio-Thesiometer vibratory measurement device, respectively. A questionnaire was provided to the patients, as were interview questions about body image, orgasm, pain, and general satisfaction with the surgery. MAIN OUTCOME MEASURES: Tactile and vibratory sensitivities of the neoclitoris and questionnaire on satisfaction with orgasm, sexual function, and general satisfaction. RESULTS: The average tactile threshold for the clitoris was 12.5 g/mm2 and the average vibratory threshold was 0.3 µm. Most participants (86%) experienced orgasm after surgery, had no or little pain, and were satisfied with the surgery. No statistical correlation was found between better or worse objective pressure and vibratory thresholds and patient answers to questions about the clitoris in the Body Image Scale for Transsexuals questionnaire. CONCLUSION: The neoclitoris derived from the glans penis in GRS provides long-term clitoral sensation that is erogenous. Overall, the vast majority of patients who undergo male-to female GRS experience orgasm and are satisfied with the surgery.

Long-term sensory disturbances after orbitozygomatic fractures.

BACKGROUND: Orbitozygomatic fractures often lead to infraorbital nerve (ION) injury, and affected sensibility is a common long-term complaint within this patient group. We present a long-term follow-up study where the validated von Frey filament system was used for testing ION sensibility. Furthermore, we examined the incidence of persistent nerve injury and whether more complex fractures led to more pronounced ION sensibility disturbances. METHODS: Patients treated for facial fractures involving the orbitozygomatic complex were included and the follow-up time was 3 years or more. Depending on the location and severity of the fractures, the patients were divided into 4 groups. The patients answered a questionnaire before ION sensibility testing with von Frey filaments. RESULTS: Eighty-one patients were examined: 65 males (80%) and 16 females (20%). Examinations were conducted between 3.0 and 7.6 years (mean 4.9 years) after injury. Sixteen patients (20%) had affected and 6 patients (7.4%) had severely affected ION sensibility according to von Frey testing. No statistically significant differences were found in terms of questionnaire score between the groups. There was also no statistically significant correlation between questionnaire results and log von Frey values. Although the effect of groups could not be statistically verified using the log von Frey values, a larger proportion of patients with complex fractures had higher log von Frey values than the other groups. CONCLUSIONS: Patients with complex fractures report more permanent sensory disturbance of the ION after surgery than those with isolated orbitozygomatic fractures, although this could not be verified statistically with von Frey filament testing at several locations. Hence, a validated method for testing facial sensibility such as von Frey filaments, although sensitive, is inadequate to determine all aspects of sensory malfunction after orbitozygomatic fractures. This suggests that the patient's experience of long-term sensation after trauma may not be correlated with objective measures.

Solely Penile Skin for Neovaginal Construction in Sex Reassignment Surgery.

BACKGROUND: Gender reassignment surgery due to transsexualism (International Classification of Diseases, Tenth Revision: F64.0) is a procedure becoming increasingly common worldwide as a result of a significant increase in diagnostic incidence. Several methods have been described for this complex surgery, but no internationally agreed upon gold standard exists, in particular with regard to which methods allow for creating a sufficient neovaginal depth. METHODS: We use a 2-stage technique using solely penile skin for creating a neovaginal cavity and present the long-term outcome in terms of measured neovaginal depth. Eighty patients were included. Patients' neovaginal depth was measured in a standardized fashion 6 months or more after initial surgery. Results were compared with published data on female anatomy. RESULTS: The average neovaginal depth achieved was 10.2 cm. Having had a postoperative complication and noncompliance to neovaginal dilatation were both negatively correlated with neovaginal depth, whereas higher body mass index was not. Most patients received a neovaginal depth sufficient for penetrative intercourse and within the range for biological women. CONCLUSIONS: Using solely penile skin for the vaginal lining is a satisfactory surgical method to achieve adequate vaginal depth, provided that the postoperative dilatation regimen is followed. This holds true regardless of age or body mass index.

Composite orbital reconstruction using the vascularized segmentalized osteo-fascio-cutaneous fibula flap.

Reconstruction of composite orbital defects must address the orbit and an exposed skull base and/or maxillary region. The orbit should not only be covered but also reshaped to accommodate the orbital contents or an epithesis when warranted. This study presents a rationale for a near-anatomical reconstruction of the orbit, together with adjacent dead space obliteration, using the segmentalized osteo-fascia-cutaneous fibula flap. Before the flap transfer, a cutting template for the fibula is made according to the measures and requirements of the facial defect. The segmentalized bone is then osteosynthesized to the facial skeleton and revascularized. Thus, an orbital depth is created by the bony fibula, whereas the fascio-cutaneous part of the flap may be used for lining the orbit and obliteration of the skull base or the maxillary region, or resurface the palate and/or the nasal cavity.

Persistent diplopia after fractures involving the orbit related to nerve injury.

BACKGROUND: Fractures in the facial skeleton are common and may lead to orbital sequelae caused by the injury and/or the surgery. In this long-term follow-up, we examined the nature of sequelae after facial fractures involving the orbit and whether a higher complexity of the fractures produced more sequelae compared to simpler fracture patterns, and if so, to what extent. METHODS: Patients surgically treated for facial fractures involving the orbit at the Karolinska University Hospital with a follow-up duration of ≥3 years were included in this retrospective study and were examined by a neuro-ophthalmologist. Based on the location and severity of the fractures, the patients were divided into four groups according to fracture complexity: 1) isolated zygomatic fracture, 2) isolated orbital floor blowout fracture, 3) zygomatic fracture combined with blowout fracture and 4) bilateral or multiple fracture patterns. RESULTS: Out of 154 patients, 81 patients (53%) attended follow-up examinations, 65 male (80%) and 16 female (20%). The duration of follow-up was 3.0-7.6 years (mean of 4.9 years). The incidence of diplopia was 3.7%, visual loss 2.5%, dystopia 4.9% and visible enophthalmos (>2 mm) 8.6%. Severe diplopia (2.5%) was due to nerve injuries. Visual loss was encountered only in group 4 with complex fractures. Fracture complexity had an effect on the presence of any sequelae, with group 4 presenting a higher percentage of patients with sequelae than the other three groups. However, no statistically significant effect of group could be found on the individual, quantitative output values of dystopia and enophthalmos. CONCLUSIONS: In this study, severe persistent diplopia in patients was due to nerve injuries, which emphasizes the need for preoperative ophthalmologic examinations, in all patients with fractures involving the orbit. A higher fracture complexity was found to lead to a higher percentage of patients presenting sequelae.

Development of a bioactive implant for repair and potential healing of cranial defects.

The repair of complex craniofacial bone defects is challenging and a successful result is dependent on the size of the defect, quality of the soft tissue covering the defect, and choice of reconstruction method. The objective of this study was to develop a bioactive cranial implant that could provide a permanent reconstructive solution to the patient by stimulating bone healing of the defect. In this paper the authors report on the feasibility and clinical results of using such a newly developed device for the repair of a large traumatic and therapy-resistant cranial bone defect. The patient had undergone numerous attempts at repair, in which established methods had been tried without success. A mosaic-designed device was manufactured and implanted, comprising interconnected ceramic tiles with a defined calcium phosphate composition. The clinical outcome 30 months after surgery revealed a restored cranial vault without postoperative complications. Computed tomography demonstrated signs of bone ingrowth. Examination with combined (18)F-fluoride PET and CT provided further evidence of bone healing of the cranial defect.

Oversized design of the anterolateral thigh flap for head and neck reconstruction.

Most surgical protocols for head and neck cancer extirpation require concurrent neck lymph node dissection. Hence, the defect involves not only the cancerous anatomical structures but also the neck. The tissue deficiency in the latter region imposes potential problems such as carotid and jugular vein exposure risking blowout, infections and orocutaneous fistulae in the neck, and a cosmetically untoward sunken appearance. The free anterolateral thigh flap (ALT) provides abundant tissue and is often the flap of choice in head and neck reconstruction. To replace the proper amount of postoperative soft tissue deficit in the neck area, we use an oversized ALT flap design. This allows reconstruction of the specific anatomical defects, protection of the important neck structures, and prevention of a sunken appearance. An oversized flap may also provide additional coverage for fixation hardware to prevent its exposure, especially after radiotherapy. In the event of partial flap loss, some viable parts of the oversized flap may be possible to advance or rotate to replace the nonviable part to avoid a repeated free flap procedure or other more complicated reconstructive procedures.

Quiescence and γH2AX in neuroblastoma are regulated by ouabain/Na,K-ATPase.

BACKGROUND: Cellular quiescence is a state of reversible proliferation arrest that is induced by anti-mitogenic signals. The endogenous cardiac glycoside ouabain is a specific ligand of the ubiquitous sodium pump, Na,K-ATPase, also known to regulate cell growth through unknown signalling pathways. METHODS: To investigate the role of ouabain/Na,K-ATPase in uncontrolled neuroblastoma growth we used xenografts, flow cytometry, immunostaining, comet assay, real-time PCR, and electrophysiology after various treatment strategies. RESULTS: The ouabain/Na,K-ATPase complex induced quiescence in malignant neuroblastoma. Tumour growth was reduced by >50% when neuroblastoma cells were xenografted into immune-deficient mice that were fed with ouabain. Ouabain-induced S-G2 phase arrest, activated the DNA-damage response (DDR) pathway marker γH2AX, increased the cell cycle regulator p21(Waf1/Cip1) and upregulated the quiescence-specific transcription factor hairy and enhancer of split1 (HES1), causing neuroblastoma cells to ultimately enter G0. Cells re-entered the cell cycle and resumed proliferation, without showing DNA damage, when ouabain was removed. CONCLUSION: These findings demonstrate a novel action of ouabain/Na,K-ATPase as a regulator of quiescence in neuroblastoma, suggesting that ouabain can be used in chemotherapies to suppress tumour growth and/or arrest cells to increase the therapeutic index in combination therapies.

Oral immunosuppressive medication for growing pigs in transplantation studies.

Immunosuppressive (IS) medication is needed to avoid graft rejection in porcine transplantation models. An ideal IS therapy should have no side-effects, but increased susceptibility to infections, disturbed intestinal microflora and toxic effects on organs and tissues are commonly reported. The aim of the present study was to design an IS protocol with tacrolimus and mycophenolic acid to be used for maintenance therapy in the post-transplant period. An eligible whole blood trough value for tacrolimus was 5-15 µg/L. Conventional specific pathogen-free pigs were fitted with an indwelling catheter under general anaesthesia, and after the acclimatization period three groups were formed: group A (n= 4) received 0.15 mg/kg body weight (BW) twice daily tacrolimus and 500 mg twice daily mycophenolic acid; group B (n= 4) received 0.3 mg/kg BW twice daily tacrolimus and 500 mg twice daily mycophenolic acid; group C (n= 2) did not receive any medication. Daily clinical examinations and analyses of blood concentrations of tacrolimus and glucose were performed. Total and differential white blood cell counts, enzyme activities, bilirubin and electrolyte concentrations were measured every fourth day. At the end of the experiment, the pigs were killed with an overdose of pentobarbital intravenously and a necropsy was performed immediately. All animals seemed to tolerate the IS treatment well. No alterations in their clinical state of health were observed throughout the study and daily weight gain was similar for the three groups. The necropsy did not reveal any pathological findings related to medication. The study showed that 0.25 mg/kg BW twice daily tacrolimus and 500 mg twice daily mycophenolic acid would be an appropriate maintenance dosage for conventional pigs.

RET PLCγ phosphotyrosine binding domain regulates Ca2+ signaling and neocortical neuronal migration.

The receptor tyrosine kinase RET plays an essential role during embryogenesis in regulating cell proliferation, differentiation, and migration. Upon glial cell line-derived neurotrophic factor (GDNF) stimulation, RET can trigger multiple intracellular signaling pathways that in concert activate various downstream effectors. Here we report that the RET receptor induces calcium (Ca(2+)) signaling and regulates neocortical neuronal progenitor migration through the Phospholipase-C gamma (PLCγ) binding domain Tyr1015. This signaling cascade releases Ca(2+) from the endoplasmic reticulum through the inositol 1,4,5-trisphosphate receptor and stimulates phosphorylation of ERK1/2 and CaMKII. A point mutation at Tyr1015 on RET or small interfering RNA gene silencing of PLCγ block the GDNF-induced signaling cascade. Delivery of the RET mutation to neuronal progenitors in the embryonic ventricular zone using in utero electroporation reveal that Tyr1015 is necessary for GDNF-stimulated migration of neurons to the cortical plate. These findings demonstrate a novel RET mediated signaling pathway that elevates cytosolic Ca(2+) and modulates neuronal migration in the developing neocortex through the PLCγ binding domain Tyr1015.

Molecular and immunological characterization of Can f 4: a dog dander allergen cross-reactive with a 23 kDa odorant-binding protein in cow dander.

BACKGROUND: Dog dander is an important cause of respiratory allergy but its content of allergenic components is still incompletely known. While Can f 1, 2, 3 and 5 have been studied in detail, only fragmentary information is available on the lipocalin Can f 4. OBJECTIVE: To purify, clone and characterize dog dander allergen Can f 4. METHODS: Can f 4 was purified from dog dander extract by size exclusion, ion exchange and reverse phase chromatography. A cDNA encoding Can f 4 was cloned and used to produce recombinant Can f 4 in Escherichia coli. A 23 kDa protein from cow dander, displaying cross-reactivity with Can f 4, was purified and identified by amino acid sequencing and mass spectrometry. IgE antibody binding to dog and cow dander extract and to individual dog allergens among 37 dog allergic subjects and 44 pollen allergic controls was studied using ImmunoCAP. RESULTS: A dog genome segment containing the Can f 4 gene was bioinformatically identified and enabled the cloning of Can f 4 cDNA. Recombinant Can f 4 displayed close immunological and biochemical similarity to purified natural Can f 4 and bound IgE antibodies from 13/37 (35%) sera of dog allergic subjects. Can f 4 reactive sera showed IgE binding to a 23 kDa protein present in cow dander extract, related to a family of odorant-binding proteins. The dog and cow proteins shared 37% sequence identity and their cross-reactivity was demonstrated by IgE inhibition experiments. CONCLUSION: Recombinant Can f 4 brings the panel of available dog allergens closer to completion and will be important in component-resolved diagnostics in allergy to animal epithelial allergens.

Positron emission tomography in clinical islet transplantation.

The fate of islets in clinical transplantation is unclear. To elude on this positron emission tomography combined with computed tomography (PET/CT) was performed for 60 min during islet transplantation in five patients receiving six transplants. A fraction of the islets (23%) were labeled with 18F-fluorodeoxyglucose ([(18)F]FDG) and carefully mixed with unlabeled islets just prior to intraportal transplantation. The peak radioactivity concentration in the liver was found at 19 min after start of islet infusion and corresponded to only 75% of what was expected, indicating that islets are lost during the transplantation procedure. No accumulation of radioactivity was found in the lungs. A nonphysiological peak of C-peptide was found in plasma during and immediately after transplantation in all subjects. Distribution in the liver was heterogeneous with wide variations in location and concentration. Islets found in areas with concentrations of >400 IEQ/cc liver tissue varied between 1% and 32% of the graft in different subjects. No side effects attributed to the PET/CT procedure were found. Clinical outcome in all patients was comparable to that previously observed indicating that the [(18)F]FDG labeling procedure did not harm the islets. The technique has potential to be used to assess approaches to enhance islet survival and engraftment in clinical transplantation.

Differential membrane compartmentalization of Ret by PTB-adaptor engagement.

Glial cell line-derived neurotrophic factor family ligands act through the receptor tyrosine kinase Ret, which plays important roles during embryonic development for cell differentiation, survival, and migration. Ret signaling is markedly affected by compartmentalization of receptor complexes into membrane subdomains. Ret can propagate biochemical signaling from within concentrates in cholesterol-rich membrane microdomains or lipid rafts, or outside such regions, but the mechanisms for, and consequences of, Ret translocation between these membrane compartments remain largely unclear. Here we investigate the interaction of Shc and Frs2 phosphotyrosine-binding domain-containing adaptor molecules with Ret and their function in redistributing Ret to specialized membrane compartments. We found that engagement of Ret with the Frs2 adaptor results in an enrichment of Ret in lipid rafts and that signal transduction pathways and chemotaxis responses depend on the integrity of such rafts. The competing Shc adaptor did not promote Ret translocation to equivalent domains, and Shc-mediated effects were less affected by disruption of lipid rafts. However, by expressing a chimeric Shc protein that localizes to lipid rafts, we showed that biochemical signaling downstream of Ret resembled that of Ret signaling via Frs2. We have identified a previously unknown mechanism in which phosphotyrosine-binding domain-containing adaptors, by means of relocating Ret receptor complexes to lipid rafts, segregate diverse signaling and cellular functions mediated by Ret. These results reveal the existence of a novel mechanism that could, by subcellular relocation of Ret, work to amplify ligand gradients during chemotaxis.