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Human CD4+ T lymphocytes play an important role in inducing potent immune responses. T cells are activated and stimulated by peptides presented in human leucocyte antigen (HLA)-class II molecules. These HLA-class II molecules typically present peptides of between 12 and 20 amino acids in length. The region that interacts with the HLA molecule, designated as the peptide-binding core, is highly conserved in the residues which anchor the peptide to the molecule. In addition, as these peptides are the product of proteolytic cleavages, certain conserved residues may be expected at the N- and C-termini outside the binding core. To study whether similar conserved residues are present in different cell types, potentially harbouring different proteolytic enzymes, the ligandomes of HLA-DRB1*03:01/HLA-DRB > 1 derived from two different cell types (dendritic cells and EBV-transformed B cells) were identified with mass spectrometry and the binding core and N- and C-terminal residues of a total of 16,568 peptides were analysed using the frequencies of the amino acids in the human proteome. Similar binding motifs were found as well as comparable conservations in the N- and C-terminal residues. Furthermore, the terminal conservations of these ligandomes were compared to the N- and C-terminal conservations of the ligandome acquired from dendritic cells homozygous for HLA-DRB1*04:01. Again, comparable conservations were evident with only minor differences. Taken together, these data show that there are conservations in the terminal residues of peptides, presumably the result of the activity of proteases involved in antigen processing. Electronic supplementary material The online version of this article (10.1007/s00251-019-01129-6) contains supplementary material, which is available to authorized users.

The tuning fork tests have been under attack since their first use in clinical examination. However, the tuning fork is small and fits into every white coat, and tuning fork tests for hearing are easy, accurate and inexpensive. They should be used in patients with an acute unilateral hearing loss if an electric audiometer is not available. After more than 100 years, the tuning fork is not obsolete; tuning fork tests are very useful if used correctly and for the appropriate indication.

A new type of a large-area multi-touch tactile device operated by electrotactile stimulation is proposed. Being able to stimulate as many types of mechanoreceptors as possible, the use of electrical stimulation is widespread. However, the area of a device is usually limited because the more electrodes a device is composed of, the more difficult wires are placed if we want to deliver different signals to different regions of one touch panel simultaneously. To get closer to the possibility of integrating multi-touch tactile devices operated by electrotactile stimulation into smartphones or other flat-panel displays, this paper focuses on developing a large-area electrotactile display device driven by electrotactile stimulation that can fully cover smartphones, which has the advantages of larger touched area and better resolution comparing to traditional ones. It is also capable of delivering different stimulation simultaneously on the same surface. The new type of touch panel with cathodes-surrounding anodes taking responsibility for delivering signals and cathodes playing the role of switches to divide signals.

International audience; C-type lectins are a diverse group of proteins involved in many human physiological and pathological processes. Most C-type lectins are glycan-binding proteins, some of which are pivotal for innate immune responses against pathogens. Other C-type lectins, such as the macrophage galactose-type lectin (MGL), have been shown to induce immunosuppressive responses upon the recognition of aberrant glycosylation on cancer cells. MGL is known to recognize terminal N-acetylgalactosamine (GalNAc), such as the Tn antigen, which is commonly found on malignant cells. Even though this glycan specificity of MGL is well described, there is a lack of understanding of the actual glycoproteins that bind MGL. We present a glycoproteomic workflow for the identification of MGL-binding proteins, which we applied to study MGL ligands on the human Jurkat leukemia cell line. In addition to the known MGL ligands and Tn antigen-carrying proteins CD43 and CD45 on these cells, we have identified a set of novel cell-surface ligands for MGL. Importantly, for several of these, O-glycosylation has hitherto not been described. Altogether, our data provide new insight into the identification and structure of novel MGL ligands that presumably act as modulatory molecules in cancer immune responses.

WstępKinesiotaping może być wykorzystywany jako samodzielna lub uzupełniająca metoda rehabilitacyjna. Jest przydatny w wielu urazach, zaburzeniach i schorzeniach. Celem badania jest zarejestrowanie częstości występowania alergii kontaktowych lub innych niekorzystnych zmian skórnych w grupie pacjentów z deficytem neurologicznym poddawanych terapii tą metodą.Materiał i metodyDo badania włączono 160 pacjentów z czterech krajów (Polska, Hiszpania, Wielka Brytania i Holandia), kobiet i mężczyzn, poddanych terapii z wykorzystaniem kinesiotapingu. Drugi autor (Polska) samodzielnie badał reakcje skórne podczas każdej nowej aplikacji, natomiast w pozostałych trzech krajach (Hiszpania, Wielka Brytania, Holandia) terapeuci zaczynali oklejanie, a rodzice/opiekunowie relacjonowali pierwszemu autorowi jego efekty za pomocą kwestionariusza internetowego.WynikiCzęstość występowania reakcji skórnych wyniosła 9,38%.WnioskiPomimo faktu, że wykorzystanie plastrów do kinesiotapingu jest uważane za efektywną, bezpieczną i przyjazną dla pacjenta formę terapii, ciągle istnieje potrzeba uważnej aplikacji plastra. Nabiera to szczególnego znaczenia u pacjentów neurologicznych, z możliwymi deficytami czucia, i/lub pacjentów z zaburzeniami przytomności. Zwiększona świadomość dotycząca możliwości wystąpienia reakcji alergicznych (alergii kontaktowej) powinna podwyższyć bezpieczeństwo omawianej metody.

In the last decade, a new surgical treatment modality was developed for frontal secondary headache, based on the assumption that the trigger of this pain entity is the entrapment of peripheral sensory nerves. The surgery entails a procedure, where an endoscopic approach is used to decompress the supraorbital and supratrochlear nerve branches, which are entrapped by the periosteum in the region of the corrugator supercilii muscle. Candidates for the surgery define their headache as moderate to severe persistent daily pressure or tension, localized in the frontal area, sometimes accompanied by symptoms of nausea and photophobia mimicking a primary headache-migraine. We created a step-by-step screening algorithm which is used to differentiate patients that have the highest chance for a successful surgical decompression. Up to now, published data regarding this type of surgery demonstrate long-lasting successful outcomes while adverse effects are minor. This article reviews and discusses from a surgeon's perspective decompression surgery for secondary headache attributed to supraorbital and supratrochlear nerve entrapment.

Item does not contain fulltext The spread of multidrug resistant Mycobacterium tuberculosis is one of the major challenges in tuberculosis control. In Eurasia, the spread of multidrug resistant tuberculosis is driven by the M. tuberculosis Beijing genotype. In this study, we examined whether selective advantages are present in the proteome of Beijing isolates that contribute to the emergence of this genotype. To this end, we compared the proteome of M. tuberculosis Beijing to that of M. tuberculosis H37Rv, both in the presence and absence of the first-line antibiotic rifampicin. During rifampicin exposure, both M. tuberculosis genotypes express proteins belonging to the DosR dormancy regulon, which induces a metabolically hypoactive-, drug tolerant phenotype. However, these markers of rifampicin tolerance were already more abundant in the M. tuberculosis Beijing isolate prior to drug exposure. To determine whether the a priori high abundance of specific proteins contribute to the formation of antibiotic resistance in M. tuberculosis Beijing, we quantified the abundance of 33 selected proteins in 27 clinical isolates from the five most common M. tuberculosis lineages using parallel reaction monitoring. The observed pre-existing high abundance of dormancy proteins in Beijing strains provides an evolutionary advantage that allows these strains to persist for prolonged periods during rifampicin treatment. SIGNIFICANCE: M. tuberculosis is the leading cause of death by a bacterial infection worldwide. Treatment-regimen to eradicate this pathogen make use of the first-line antibiotic rifampicin, which is considered to be the cornerstone of modern day anti-tuberculosis treatment. Despite the potency of rifampicin, there is an increasing occurrence of rifampicin resistant mutants in a specific cluster of M. tuberculosis, the Beijing genotype. Using both a data dependent acquisition and a targeted proteomic approach we identified markers of rifampicin tolerance to be high abundant in members of the M. tuberculosis Beijing genotype, already prior drug exposure. The identification of this M. tuberculosis Beijing specific trait will contribute to improved diagnostics and treatment of M. tuberculosis.

Gait analysis has been used for the assessment of gait-related disabilities and to provide detailed information. The gait characteristics of a healthy adult population have often been utilised to evaluate and monitor a subject's walk and patterns. This paper summarises a study that aims to compare the Functional Ambulation Performance (FAP) scores in male subjects wearing safety shoes versus regular walking shoes (RWS). Participants walked with RWS along the GAITRite® electronic walkway 30 times in self-selected speed and were analysed by the FAP; the subjects then repeated the same procedure with safety footwear. The data were then gathered in test groups containing 15 measured walks enabling the research team to establish an accurate and average FAP score for each test group. A descriptive statistic for the collected data was performed to determine the gait variables of the FAP score. Walking velocity, step and stride length, step and stride time, and step width among other measures were collect...

Objective:Sudden sensorineural hearing loss in divers may be caused by either inner-ear barotrauma or inner-ear decompression sickness. There is no consensus on the best treatment option. This study aimed to evaluate the therapeutic value of hyperbaric oxygen therapy for sudden sensorineural hearing loss in divers.Method:A literature review and three cases of divers with sudden sensorineural hearing loss treated with hyperbaric oxygen therapy are presented.Results:Hyperbaric oxygen therapy resulted in hearing improvement in 80 per cent of patients: 39 per cent had hearing improvement and 41 per cent had full recovery.Conclusion:Hyperbaric oxygen therapy improved hearing in divers with sudden sensorineural hearing loss.

Precise knowledge of the nerve supply of the corrugator supercilii muscle (CSM) is a prerequisite for performing a selective denervation of the CSM. The authors' goal was to determine the course and variability of the motor nerves of the CSM in relation to fixed orbital landmarks. The facial nerve branches toward the CSM were identified during microscopic dissection of 9 Caucasian formaldehyde-fixed cadaver half-heads. The distances between the branches and defined landmarks were measured. All branches to the transverse head of the CSM ran between 15 and 32 mm superior to the lateral orbital margin. Medially the CSM was supplied by a superficial zygomatic, buccal, or bucco-zygomatic branch, which was much smaller than the temporal branches. This branch ran 4 to 7 mm medial to the medial canthus. This anatomical knowledge can be applied for surgical denervation of the corrugator supercilii muscle. The authors suggest a surgical procedure for corrugator denervation through a blepharoplasty incision.

Summary The initiation of type 1 diabetes (T1D) requires a break in peripheral tolerance. New insights into neoepitope formation indicate that post-translational modification of islet autoantigens, for example via deamidation, may be an important component of disease initiation or exacerbation. Indeed, deamidation of islet autoantigens increases their binding affinity to the T1D highest-risk human leucocyte antigen (HLA) haplotypes HLA-DR3/DQ2 and -DR4/DQ8, increasing the chance that T cells reactive to deamidated autoantigens can be activated upon T cell receptor ligation. Here we investigated human pancreatic islets and inflammatory and tolerogenic human dendritic cells (DC and tolDC) as potential sources of deamidated islet autoantigens and examined whether deamidation is altered in an inflammatory environment. Islets, DC and tolDC contained tissue transglutaminase, the key enzyme responsible for peptide deamidation, and enzyme activity increased following an inflammatory insult. Islets treated with inflammatory cytokines were found to contain deamidated insulin C-peptide. DC, heterozygous for the T1D highest-risk DQ2/8, pulsed with native islet autoantigens could present naturally processed deamidated neoepitopes. HLA-DQ2 or -DQ8 homozygous DC did not present deamidated islet peptides. This study identifies both human islets and DC as sources of deamidated islet autoantigens and implicates inflammatory activation of tissue transglutaminase as a potential mechanism for islet and DC deamidation.

A reliable animal model that captures the key features of human cancer is essential to bring new insights for oncology research. In this context, spontaneously occurring canine cancers are excellent models to study tumor development and progression, since dogs share the same environment as humans and present many similarities regarding anatomic, histological, molecular and clinical features. Here, we aimed to identify and characterize intratumor heterogeneity using mass spectrometry imaging (MSI) of proteolytic peptides in order to simultaneously detect morphological and molecular alterations whilst preserving tissue integrity, directing the search for cancer biomarkers. Samples were obtained from five dogs diagnosed with metastatic disease, and MALDI-FT-ICR-MSI data of 17 primary tumors were co-registered to hematoxylin and eosin stained slides. Unsupervised clustering of tumor spectra was performed using the bisecting k-means method in SCiLS Lab (Bruker Daltonics, DE) and two molecularly distinct clusters (CL1 and CL2) were selected regardless morphological presentation. We also performed a supervised analysis, by annotating (FlexImagingTM, Bruker Daltonics, DE) and classifying tumor regions of interest into low- and high-grade populations according to previously established morphological criteria. LC/MS-MS of extracted peptides was used for protein identity assignment based on mass matching, and functional enrichment was performed using a human protein-coding database. Discriminative peaks were identified using ROC and Wilcoxon methods. M/z signals presenting AUC > 0.7 and FDR < 0.05, and biological functions with FDR < 0.05 were considered significant. We found 266 features discriminating low- and high-grade tumor populations, resulting in 44 protein IDs, and 185 features significantly different between CL1 and CL2, with 35 IDs retrieved. CANX, RSPA5 and PDIA3, found down-regulated in high-grade compared to low-grade regions, enriched for protein folding in endoplasmic reticulum process, suggesting that morphology changes may have arisen from protein misfolding due to reticular stress. Both protein sets enriched for cell adhesion molecule binding, cadherin binding and structural molecule activity. HMGN3, HNRNPC and histones H2AFY and HIST1H1C, resulting from the comparison of clusters CL1 and CL2, also enriched for nucleosome binding and chromatin DNA binding, alterations that could have not been detected only by conventional histopathological evaluation. Ongoing analyses aim to further characterize the intratumor molecular heterogeneity of microscopically indistinct populations and its association to cancer progression and metastasis. Citation Format: Yonara G. Cordeiro, Bram P.A.M Heijs, Leandra M. Mulder, René J. van Zeijl, Peter van Veelen, Arnoud de Ru, Ricardo F. Strefezzi, Heidge Fukumasu. Mass spectrometry imaging for the assessment of intratumor molecular heterogeneity in canine metastatic mammary carcinomas: A comparative approach for biomarker discovery [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2709.

The rate of spontaneous complete recovery from sudden deafness is only 39%, so additional therapy is warranted.1 Fishman and Cullen do not mention hyperbaric oxygen therapy in their paragraph on how patients with sudden hearing loss should be managed.2 The Cochrane systematic review finds a significant positive effect for this treatment, which is more than …

The most frequent subtype of acute myeloid leukemia (AML) is defined by mutations in the nucleophosmin 1 (NPM1) gene. Mutated NPM1 (ΔNPM1) is an attractive target for immunotherapy, since it is an essential driver gene and 4 bp frameshift insertions occur in the same hotspot in 30%-35% of AMLs, resulting in a C-terminal alternative reading frame of 11 aa. By searching the HLA class I ligandome of primary AMLs, we identified multiple ΔNPM1-derived peptides. For one of these peptides, HLA-A*02:01-binding CLAVEEVSL, we searched for specific T cells in healthy individuals using peptide-HLA tetramers. Tetramer-positive CD8+ T cells were isolated and analyzed for reactivity against primary AMLs. From one clone with superior antitumor reactivity, we isolated the T cell receptor (TCR) and demonstrated specific recognition and lysis of HLA-A*02:01-positive ΔNPM1 AML after retroviral transfer to CD8+ and CD4+ T cells. Antitumor efficacy of TCR-transduced T cells was confirmed in immunodeficient mice engrafted with a human AML cell line expressing ΔNPM1. In conclusion, the data show that ΔNPM1-derived peptides are presented on AML and that CLAVEEVSL is a neoantigen that can be efficiently targeted on AML by ΔNPM1 TCR gene transfer. Immunotherapy targeting ΔNPM1 may therefore contribute to treatment of AML.

Langerhans Cell Histiocytosis (LCH) is a neoplastic disorder of hematopoietic origin characterized by inflammatory lesions containing clonal histiocytes (LCH-cells) intermixed with various immune cells, including T cells. In 50-60% of LCH-patients, the somatic BRAF(V600E) driver mutation, which is common in many cancers, is detected in these LCH-cells in an otherwise quiet genomic landscape. Non-synonymous mutations like BRAF(V600E) can be a source of neoantigens capable of eliciting effective antitumor CD8(+) T cell responses. This requires neopeptides to be stably presented by Human Leukocyte Antigen (HLA) class I molecules and sufficient numbers of CD8(+) T cells at tumor sites. Here, we demonstrate substantial heterogeneity in CD8(+) T cell density in n = 101 LCH-lesions, with BRAF(V600E) mutated lesions displaying significantly lower CD8(+) T cell:CD1a(+) LCH-cell ratios (p = 0.01) than BRAF wildtype lesions. Because LCH-lesional CD8(+) T cell density had no significant impact on event-free survival, we investigated whether the intracellularly expressed BRAF(V600E) protein is degraded into neopeptides that are naturally processed and presented by cell surface HLA class I molecules. Epitope prediction tools revealed a single HLA class I binding BRAF(V600E) derived neopeptide (KIGDFGLATEK), which indeed displayed strong to intermediate binding capacity to HLA-A*03:01 and HLA-A*11:01 in an in vitro peptide-HLA binding assay. Mass spectrometry-based targeted peptidomics was used to investigate the presence of this neopeptide in HLA class I presented peptides isolated from several BRAF(V600E) expressing cell lines with various HLA genotypes. While the HLA-A*02:01 binding BRAF wildtype peptide KIGDFGLATV was traced in peptides isolated from all five cell lines expressing this HLA subtype, KIGDFGLATEK was not detected in the HLA class I peptidomes of two distinct BRAF(V600E) transduced cell lines with confirmed expression of HLA-A*03:01 or HLA-A*11:01. These data indicate that the in silico predicted HLA class I binding and proteasome-generated neopeptides derived from the BRAF(V600E) protein are not presented by HLA class I molecules. Given that the BRAF(V600E) mutation is highly prevalent in chemotherapy refractory LCH-patients who may qualify for immunotherapy, this study therefore questions the efficacy of immune checkpoint inhibitor therapy in LCH.

IMPORTANCE: Endoscopic surgical decompression of the supratrochlear nerve (STN) and supraorbital nerve (SON) is a new treatment for patients with frontal chronic headache who are refractory to standard treatment options. OBJECTIVE: To evaluate and compare treatment outcomes of oral medication, botulinum toxin type A (BoNT/A) injections, and endoscopic decompression surgery in frontal secondary headache attributed to STN and supraorbital SON entrapment. DESIGN, SETTING, AND PARTICIPANTS: Prospective cohort study of 22 patients from a single institution (Diakonessen Hospital Utrecht) with frontal headache of moderate-to-severe intensity (visual analog scale [VAS] score, 7-10), frontally located, experienced more than 15 days per month, and described as pressure or tension that intensifies with pressure on the area of STN and SON. A screening algorithm was used that included examination, questionnaire, computed tomography of the sinus, injections of local anesthetic, and BoNT/A in the corrugator muscle. INTERVENTIONS: Different oral medication therapy for headache encountered in the study cohort, as well as BoNT/A injections (15 IU) into the corrugator muscle. Surgical procedures were performed by a single surgeon using an endoscopic surgical approach to release the supraorbital ridge periosteum and to bluntly dissect the glabellar muscle group. MAIN OUTCOMES AND MEASURES: Headache VAS intensity after oral medication and BoNT/A injections. Additionally, early postoperative follow-up consisted of a daily headache questionnaire that was evaluated after 1 year. RESULTS: In total, 22 patients (mean [SD] age, 42.0 [15.3] years; 7 men and 15 women) were included in this cohort study. Oral medication therapy reduced the headache intensity significantly (mean [standard error of the mean {SEM}] VAS score, 6.45 [0.20] [95% CI, 0.34-3.02; P

The heterozygous DQ2/8 (DQA1*05:01-DQB1*02:01/DQA1*03:01-DQB1*03:02) genotype confers the highest risk in type 1 diabetes (T1D), whereas the DQ6/8 (DQA1*02:01-DQB1*06:02/DQA1*03:01-DQB1*03:02) genotype is protective. The mechanism of dominant protection by DQ6 (DQB1*06:02) is unknown. We tested the hypothesis that DQ6 interferes with peptide binding to DQ8 by competition for islet epitope (“epitope stealing”) by analysis of the islet ligandome presented by HLA-DQ6/8 and -DQ8/8 on dendritic cells pulsed with islet autoantigens preproinsulin (PPI), GAD65, and IA-2, followed by competition assays using a newly established “epitope-stealing” HLA/peptide-binding assay. HLA-DQ ligandome analysis revealed a distinct DQ6 peptide-binding motif compared with the susceptible DQ2/8 molecules. PPI and IA-2 peptides were identified from DQ6, of DQ6/8 heterozygous dendritic cells, but no DQ8 islet peptides were retrieved. Insulin B6-23, a highly immunogenic CD4 T-cell epitope in patients with T1D, bound to both DQ6 and DQ8. Yet, binding of InsB6-23 to DQ8 was prevented by DQ6. We obtained first functional evidence of a mechanism of dominant protection from disease, in which HLA molecules associated with protection bind islet epitopes in a different, competing, HLA-binding register, leading to “epitope stealing” and conceivably diverting the immune response from islet epitopes presented by disease-susceptible HLA molecules in the absence of protective HLA.

The signal peptide region of preproinsulin (PPI) contains epitopes targeted by human leucocyte antigen-A (HLA-A)-restricted (HLA-A0201, A2402) cytotoxic T-cells as part of the pathogenesis of β-cell destruction in type 1 diabetes. We extended PPI epitope discovery to disease-associated HLA-B*1801 and HLA-B*3906 (risk) and HLA-A*1101 and HLA-B*3801 (protective) alleles revealing that 4/6 alleles present epitopes derived from the signal peptide region. During co-translational translocation of PPI, its signal peptide is cleaved and retained within the endoplasmic reticulum (ER) membrane, implying it is processed for immune recognition outside of the canonical, proteasome-directed pathway. Using in vitro translocation assays with specific inhibitors and gene knockout in PPI-expressing target cells we show that PPI signal peptide antigen processing requires signal peptide peptidase (SPP). The intramembrane protease SPP generates cytoplasm-proximal epitopes, which are transporter-associated-with-antigen-processing (TAP)-dependent, and ER-luminal (TAP-independent) epitopes, each presented by different HLA class I molecules, and N-terminal trimmed by ER aminopeptidase 1 (ERAP1) for optimal presentation. In vivo, TAP expression is significantly up-regulated and correlated with HLA class I hyper-expression in insulin-containing islets of patients with type 1 diabetes. Thus, PPI signal peptide epitopes are processed by SPP and loaded for HLA-guided immune recognition via pathways that are enhanced during disease pathogenesis.