Optimized NGFR-derived hinges for rapid and efficient enrichment and detection of CAR T cells in vitro and in vivo.

Chimeric antigen receptor (CAR) T cell therapy has demonstrated unprecedented success with high remission rates for heavily pretreated patients with hematological malignancies. The hinge connecting the extracellular antigen recognition unit to the transmembrane domain provides the length and flexibility of the CAR constructs and ensures that the CAR can reach the target antigen and mediate recognition and killing of target cells. The hinge can also include specific amino acid sequences to improve CAR expression, influence T cell proliferation, and facilitate CAR T cell detection, enrichment, and even elimination. Here, we report the generation of two novel hinge domains derived from the low-affinity p75 chain of the human nerve growth factor receptor (NGFR), termed N3 and N4, which, when incorporated into the CAR backbone, allow detection as well as high-grade enrichment of CAR T cells with GMP-compatible immunomagnetic reagents. After optimizing the MACS protocol for excellent CAR T cell purity and yield, we demonstrated that N3- and N4-hinged CAR T cells are as efficacious as their CD8-hinged counterparts in vitro against hematological blasts and also in vivo in the control of acute monocytic leukemia in an immunodeficient mouse xenograft model. Thus, both hinges could potentially be an integral part of future CAR designs and universally applicable in clinical applications.

[Droplet exposure during tracheotomy : Case analysis and consequences with respect to COVID-19 patients]. / Tröpfchenexposition bei Tracheotomie : Fallanalyse und Konsequenzen in Bezug auf COVID-19-Patienten.

BACKGROUND: The COVID(coronavirus disease)-19 pandemic is characterized by high infectivity, droplet transmission, and high viral load in the upper respiratory tract. Severe disease courses are associated with interstitial pneumonia and ventilated patients, in whom tracheotomy (TT)-a droplet- and aerosol-producing medical intervention-is regularly necessary. TT as a potential infection risk for medical staff is scarcely found in the literature. Therefore, the aim of this study was to quantify droplet exposure of the surgical team during TT, to better define the requirements for personal protective equipment (PPE). MATERIALS AND METHODS: Surgical TT was performed in four non-infectious patients, during which the surgeon and his assistant both wore a surgical nasal mask with a transparent visor. After the procedure, the type, distribution, and number of droplets on the visor were determined macroscopically and microscopically. RESULTS: An average of 29 droplets were found on the middle third of the visor, 4 on the right third, and 13 on the left third, with an average droplet size of 571 µm (±â€¯381 µm). The smallest droplets were 55 µm, the largest 1431 µm. An increase in the number of droplets was found with increased ventilation during the procedure. Blood droplets were more common than secretion droplets. CONCLUSION: Contamination of the visor with droplets was demonstrated. Especially in the case of TT in highly infectious patients, e.g., COVID-19 patients, the use of hooded headgear in combination with breathing apparatus with air purification and power supply is recommended to ensure best protection from infection for the surgeon and the surgical assistant.

[Complications of anterior skull base surgery]. / Komplikationen bei Operationen an der Rhinobasis.

Anterior skull base operations are complex surgical procedures that are performed to treat serious and complicated diseases. Despite significant advances in surgical techniques, complications are relatively frequent and can be serious. Endoscopic skull base surgery seems to be associated with less complications than open techniques. Different classifications aimed at categorizing these complications have been suggested, the use of which can be recommended when reporting complication rates. The most relevant and frequent complications of anterior skull base surgery are hemorrhage, cerebrospinal fluid (CSF) leak, meningitis, and cranial nerve injury. Careful planning, close interdisciplinary cooperation, the competence of the skull base center and its members, and rigorous quality management are decisive for the avoidance of complications. With respect to the frequency and the seriousness of the complications, their meticulous and complete discussion with the patient before obtaining informed consent plays a central role.

Connexin40 controls endothelial activation by dampening NFκB activation.

Connexins are proteins forming gap junction channels for intercellular communication. Connexin40 (Cx40) is highly expressed by endothelial cells (ECs) of healthy arteries but this expression is lost in ECs overlying atherosclerotic plaques. Low/oscillatory shear stress observed in bends and bifurcations of arteries is atherogenic partly through activation of the pro-inflammatory NFκB pathway in ECs. In this study, we investigated the relation between shear stress, Cx40 and NFκB. Shear stress-modifying casts were placed around carotid arteries of mice expressing eGFP under the Cx40 promoter (Cx40+/eGFP ). We found that Cx40 expression is decreased in carotid regions of oscillatory shear stress but conserved in high and low laminar shear stress regions. These results were confirmed in vitro. Using phage display, we retrieved a binding motif for the intracellular regulatory Cx40 C-terminus (Cx40CT), i.e. HS[I, L, V][K, R]. One of the retrieved peptides (HSLRPEWRMPGP) showed a 58.3% homology with amino acids 5-to-16 of IκBα, a member of the protein complex inhibiting NFκB activation. Binding of IκBα (peptide) and Cx40 was confirmed by crosslinking and en face proximity ligation assay on carotid arteries. TNFα-induced nuclear translocation of NFκB in ECs was enhanced after reducing Cx40 with siRNA. Transfection of HeLa cells with either full-length Cx40 or Cx40CT demonstrated that Cx40CT was sufficient for inhibition of TNFα-induced NFκB phosphorylation. Finally, Tie2CreTgCx40fl/flApoe-/- mice showed exaggerated shear stress-induced atherosclerosis and enhanced NFκB nuclear translocation. Our data show a novel functional IκBα-Cx40 interaction that may be relevant for the control of NFκB activation by shear stress in atherogenesis.

Optimized human CYP4B1 in combination with the alkylator prodrug 4-ipomeanol serves as a novel suicide gene system for adoptive T-cell therapies.

Engineering autologous or allogeneic T cells to express a suicide gene can control potential toxicity in adoptive T-cell therapies. We recently reported the development of a novel human suicide gene system that is based on an orphan human cytochrome P450 enzyme, CYP4B1, and the naturally occurring alkylator prodrug 4-ipomeanol. The goal of this study was to systematically develop a clinically applicable self-inactivating lentiviral vector for efficient co-expression of CYP4B1 as an ER-located protein with two distinct types of cell surface proteins, either MACS selection genes for donor lymphocyte infusions after allogeneic stem cell transplantation or chimeric antigen receptors for retargeting primary T cells. The U3 region of the myeloproliferative sarcoma virus in combination with the T2A site was found to drive high-level expression of our CYP4B1 mutant with truncated CD34 or CD271 as MACS suitable selection markers. This lentiviral vector backbone was also well suited for co-expression of CYP4B1 with a codon-optimized CD19 chimeric antigen receptor (CAR) construct. Finally, 4-ipomeanol efficiently induced apoptosis in primary T cells that co-express mutant CYP4B1 and the divergently located MACS selection and CAR genes. In conclusion, we here developed a clinically suited lentiviral vector that supports high-level co-expression of cell surface proteins with a potent novel human suicide gene.

Nasal levels of soluble IL-33R ST2 and IL-16 in allergic rhinitis: inverse correlation trends with disease severity.

BACKGROUND: Serum levels of IL-16, IL-33 and the decoy receptor of IL-33, soluble ST2, are elevated in allergic rhinitis. Recent studies show that IL-16, soluble ST2 or anti-IL-33 reduce type 2 cytokines (such as IL-5) and eosinophilia in murine models of allergic asthma or allergic rhinitis respectively. OBJECTIVE: In this study, we studied the release of IL-5, IL-16, IL-33 and soluble ST2 in allergic rhinitis patients after nasal allergen challenge and natural pollen exposure. METHODS: The nasal lavages of 15 allergic and 14 non-allergic volunteers were collected during the pollen allergy season. In addition, six allergic volunteers underwent unilateral nasal allergen and control challenge out of season and nasal secretions and sera were collected. IL-5, IL-16, IL-33 and soluble ST2 in nasal secretions and sera were measured by electrochemiluminescent assay or ELISA, respectively. RESULTS: Nasal IL-5, IL-16 and soluble ST2 levels were significantly increased in seasonally pollen exposed allergic volunteers compared to control subjects (P < 0.001, P = 0.018 and P = 0.002 respectively), whereas IL-33 remained undetectable. Nasal IL-16 showed a weak inverse correlation trend with nasal symptoms (r = -0.48, P = 0.07). Nasal soluble ST2 concentrations were inversely correlated with nasal symptoms (r = -0.61, P = 0.02) and positively correlated with IL-16 (r = 0.56, P = 0.03). Significant increases of nasal IL-5, IL-16 and ST2 but not IL-33 were observed after nasal allergen challenge. At 24 h after allergen challenge, local ST2 and IL-5 concentrations showed an inverse correlation trend (r = -0.83, P = 0.04). Serum levels of IL-5, IL-16 and soluble ST2 rose in at least five of six volunteers tested at 5 or 24 h post-challenge. CONCLUSIONS AND CLINICAL RELEVANCE: The observed upregulation of soluble ST2 and IL-16 after nasal allergen challenge and during peak pollination season suggests potential regulatory roles of these cytokines in the inflammatory reaction in allergic rhinitis.

Squamous cell carcinomas of the head and neck in Fanconi anemia: risk, prevention, therapy, and the need for guidelines.

Fanconi anemia (FA) is a rare recessive DNA repair disorder that is clinically characterized by congenital malformations, progressive bone marrow failure, and increased incidence of malignancies, especially acute myeloid leukemia and squamous cell carcinomas of the head and neck (HNSCCs) and the anogenital regions. On a cellular level, typical features of the disorder are a high degree of genomic instability and an increased sensitivity to bi-functionally alkylating agents. So far, germ-line defects in 15 different FA genes have been identified. Some of these FA genes are also established as tumor susceptibility genes for familiar cancers.In recent years, the prevention and therapy of HNSCCs in FA patients has become more important as the percentage of patients surviving into adulthood is rising. HNSCCs appear in very young FA patients without common risk factors. Since cisplatin-based chemotherapy in combination with radiotherapy, essential parts of the standard treatment approach for sporadic HNSCCs, cannot be used in FA patients due to therapy-associated toxicities and mortalities even with reduced dosing, surgery is the most important treatment option for HNSCCs, in FA patients and requires an early and efficient detection of malignant lesions. So far, no uniform treatment protocol for the management of HNSCCs in FA patients exists. Therefore, we propose that the information on affected FA patients should be collected worldwide, practical therapeutic guidelines developed and national treatment centers established.

Connexin channel-dependent signaling pathways in inflammation.

Inflammation is a highly regulated process with common but also specific characteristics in each tissue affected. Recruitment of leukocytes from the blood to the injured tissue is an important early step in the inflammatory cascade. This review highlights the role of connexins (Cxs) in the regulation of both acute and chronic inflammatory processes. Cxs form gap junction channels that provide a cytoplasmic continuity between adjacent cells allowing the intercellular exchange of ions and metabolites. Their structural halves form connexons or hemichannels. Each of them consists of 6 Cx proteins and hemichannels not taking part in gap junction formation but facilitating the release of small molecules such as ATP. Based on the differential distribution of various Cxs in different tissues such as the brain, lung capillaries and large blood vessels, our aim was to analyze the specific roles of Cxs in the inflammatory process in these tissues. Three typical sites of inflammation were chosen to shed light on similarities and differences in several types of responses: (1) atherosclerosis as a model for chronic inflammation, (2) the lung as an example of acute inflammation and (3) the 'immune-privileged' environment of the brain to highlight specific reactions of the vasculature to ischemic damage and inflammation at this site.

Prostaglandin E2regulation of cystic fibrosis transmembrane conductance regulator activity and airway surface liquid volume requires gap junctional communication.

Stimulation of the cystic fibrosis transmembrane conductance regulator (CFTR) by protease-activated receptors (PARs) at the basolateral membranes and by adenosine receptors (ADO-Rs) at the apical membrane maintain airway surface liquid (ASL) volume, which is required to ensure hydrated and clearable mucus. Both pathways involve the release of prostaglandin E2 (PGE2) and the stimulation of their basolateral receptors (EP-Rs). We sought to determine whether gap junctions contribute to the coordination of these pathways for modulating CFTR activity and mucus hydration. We used RT-PCR and Western blotting to determine connexin (Cx), CD73, and EP-R expression in a Calu-3 airway epithelial cell line grown on Transwell (Corning Costar, Cambridge, MA) inserts. We used dye coupling to evaluate gap junctional intercellular communication (GJIC). We used Ussing chamber studies and X-Z confocal microscopy to monitor Cl(-) secretion and ASL volume regulation. We found that connexin 43 (Cx43)-mediated GJIC was increased either by endogenous ADO after the hydrolysis of purine nucleotides by CD73 or by the direct activation of ADO-Rs. Inhibition of phospholipase A2 and cyclooxygenase prevented ADO-dependent increases in GJIC, suggesting the involvement of PGE2. PGE2 was found to increase GJIC markedly by stimulating EP4-Rs. The modulation of ADO signaling also affected the PAR-dependent activation of CFTR. The reduction of GJIC by CD73 or Cx43 inhibition prevented PAR-evoked CFTR currents in Ussing chambers. The inhibition of GJIC resulted in a failure of PGE2 to increase ASL volume in Calu-3 cells and in primary cultures of well-differentiated human airway epithelial cells. Thus, gap junctions coordinate a signaling network comprising CFTR, ADO-Rs, PARs, and EP-Rs, and are required for ASL volume homeostasis.

[Breast cancer metastases in the head and neck region]. / Mammakarzinommetastasen im Kopf-Hals-Bereich.

Breast cancer metastases to the head and neck region are very rare and therefore represent a challenge for the clinician in terms of diagnosis and therapy. Recent advances in breast cancer treatment have achieved longer median survival times in affected patients. However, at the same time, the risk of a clinical manifestation of metastasis increases. Here we present the cases of two breast cancer patients who developed filiae into the petrous portion of the temporal bone and one very rare case of metastasis to the larynx. Diagnosis, therapy and distinctive features of metastasis to the head and neck region are discussed.Secondary to long-term endocrine hormone therapy, a reduction in estrogen receptor expression occurred in all three cases. We believe that the loss of steroid receptor expression contributed to tumor resistance to endocrine therapy. Moreover, this receptor loss hindered the pathologist from confirming the diagnosis of metastases at very unusual sites.

Endothelial-specific deletion of connexin40 promotes atherosclerosis by increasing CD73-dependent leukocyte adhesion.

BACKGROUND: Endothelial dysfunction is the initiating event of atherosclerosis. The expression of connexin40 (Cx40), an endothelial gap junction protein, is decreased during atherogenesis. In the present report, we sought to determine whether Cx40 contributes to the development of the disease. METHODS AND RESULTS: Mice with ubiquitous deletion of Cx40 are hypertensive, a risk factor for atherosclerosis. Consequently, we generated atherosclerosis-susceptible mice with endothelial-specific deletion of Cx40 (Cx40del mice). Cx40del mice were indeed not hypertensive. The progression of atherosclerosis was increased in Cx40del mice after 5 and 10 weeks of a high-cholesterol diet, and spontaneous lesions were observed in the aortic sinuses of young mice without such a diet. These lesions showed monocyte infiltration into the intima, increased expression of vascular cell adhesion molecule-1, and decreased expression of the ecto-enzyme CD73 in the endothelium. The proinflammatory phenotype of Cx40del mice was confirmed in another model of induced leukocyte recruitment from the lung microcirculation. Endothelial CD73 is known to induce antiadhesion signaling via the production of adenosine. We found that reducing Cx40 expression in vitro with small interfering RNA or antisense decreased CD73 expression and activity and increased leukocyte adhesion to mouse endothelial cells. These effects were reversed by an adenosine receptor agonist. CONCLUSIONS: Cx40-mediated gap junctional communication contributes to a quiescent nonactivated endothelium by propagating adenosine-evoked antiinflammatory signals between endothelial cells. Alteration in this mechanism by targeting Cx40 promotes leukocyte adhesion to the endothelium, thus accelerating atherosclerosis.

Surgical management of retropharyngeal abscesses.

CONCLUSION: Retropharyngeal abscess (RPA) is a rare, potentially life-threatening disease, requiring appropriate otorhinolaryngologic as well as radiologic diagnostics, and medical and surgical intervention by a transoral, transcervical or transnasal approach in a multidisciplinary setting. OBJECTIVES: The risks and benefits of surgical intervention in patients with RPA were assessed. The main outcome measure was the clinical resolution of the abscess. PATIENTS AND METHODS: A retrospective chart review was performed at a tertiary care university hospital over a period of 28 months. Eleven patients aged 1 to 68 years with the diagnosis of RPA were included. RESULTS: All patients presented with restricted cervical mobility and all patients had CT and/or MRI scan on admission. The mean abscess volume was 9.4 cm(3). Surgical intervention was performed in all cases, including transoral (n=5), transcervical (n=3) or combined transoral and transcervical (n=2) drainage. In one patient RPA close to the skull base was drained by an MRI-guided transnasal approach. All patients recovered; however, there was one recurrence and in one case surgical tracheotomy was unavoidable during the course of disease. Growth of streptococcal species was verified in six of the examined abscesses. Abscessing lymphadenitis, infection of a cervical cyst, and previous ganglionar local opioid analgesia treatment were identified as causative factors.

Connexin43 modulates neutrophil recruitment to the lung.

Transmigration of neutrophils through the microvascular endothelium is a cardinal event of acute inflammation. It has been suggested that gap junctions made of connexin43 (Cx43) may serve as a conducting pathway to spread inflammatory signals within the lung capillary network. To determine whether Cx43 contributes to neutrophil transmigration in vivo, the number of transmigrated neutrophils was monitored in lungs of Cx43 mouse models subjected to inflammation by intratracheal instillations of Pseudomonas aeruginosa lipopolysaccharide (LPS). Cx43 was detected in inflamed lungs independently of neutrophil recruitment, whereas Cx43 up-regulation was not detected in mice genetically protected from inflammation. Mice heterozygous for the Cx43 gene (gja1) showed a 56% (P < 0.01) reduction in airway neutrophil count. In contrast, increased (P < 0.05) neutrophil recruitment in response to LPS was observed in a mouse model expressing a mutant Cx43 with enhanced channel conductivity. In vitro adhesion assays showed that reduced conductivity of Cx43 channels with (43)Gap26, a Cx43 blocking peptide, decreased adhesion of neutrophils to endothelial cells. Finally, we found that instillation of (43)Gap26 in inflamed lungs reduced neutrophil transmigration by 65% (P < 0.05). These results indicate that inflammatory mediators up-regulate alveolar Cx43 to promote neutrophil recruitment to the airspace. Cx43 may therefore represent a pharmacological target in lung diseases characterized by excessive neutrophil recruitment to the airways.

[Risk of hemorrhage after adenoidectomy and tonsillectomy. Value of the preoperative determination of partial thromboplastin time, prothrombin time and platelet count]. / Risiko von Blutungen nach Adenotomie und Tonsillektomie. Aussagekraft der präoperativen Bestimmung von PTT, Quick und Thrombozytenzahl.

BACKGROUND: Hemorrhage after tonsillectomy and adenoidectomy remains a serious complication. Therefore, routine preoperative coagulation screening, including activated partial thromboplastin time (aPTT), prothrombin time (PT) and platelet count (PLC), are regularly performed, also for medicolegal reasons. In the recently published statement of the German Society of Otorhinolaryngology, Head and Neck Surgery the need for routine preoperative coagulation screening is discussed, but so far no standardized procedure had been established. According to this statement - at least for children - routine preoperative coagulation screening is not mandatory as long as the thorough medical history provides no evidence for a coagulation disorder ( http://www.hno.org/kollegen/gerinnung_te_ae.html ). The present study was undertaken to determine the occurrence of postoperative hemorrhage on the one hand, and the incidence of abnormal preoperative routine coagulation parameters or pathological anamnesis findings on the other. PATIENTS AND METHODS: In 688 patients, a standardized clinical history was obtained using a questionnaire. Coagulation screening included aPTT, PT, and PLC was also carried out. Bleeding complications were then correlated with anamnesis features and abnormalities in coagulation screening. RESULTS: In 39 (5.7%) of the 688 patients we found abnormal coagulation values, which were confirmed in repeated analyses. In six of these a detailed analysis revealed occult coagulation disorders requiring correction only in the case of bleeding complications who were previously unknown. Fifteen patients were already known to have a coagulation disorder, and the anamnesis identified no additional patient at risk. Thus, 21 patients with coagulation disorders requiring correction in the case of a bleeding complication underwent surgery. However, only eight (38%) of these showed abnormal routine coagulation parameters. Surgical treatment of postoperative hemorrhage was required in 12 patients, all of whom had normal values for aPTT, PT and PLC. CONCLUSION: The frequently performed determination of routine coagulation parameters (aPTT, PT, PLC) is not able to reliably identify relevant coagulation disorders or to predict the risk for postoperative hemorrhagic complications after adenoidectomy or tonsillectomy.

[MALT lymphoma of the orbit]. / MALT-Lymphom der Orbita.

MALT lymphomas of the orbit are rare orbital tumors; the differential diagnosis needs to exclude inflammatory pseudotumors of the orbit, but also benign lymphoproliferations, pseudolymphomas, and other orbital neoplasms. After histological confirmation of the diagnosis staging is necessary, and - as long as the disease is localized exclusively in the orbit - radiation therapy should be started. The clinical picture of MALT lymphoma and its differential diagnosis and treatment are discussed with reference to an actual case.

[Assurance-associated aspects of head and neck carcinomas: disposition, exposition, prognosis]. / Versicherungsrelevante Aspekte bei Karzinomen der Kopf-hals-Region: Disposition, Exposition, Prognose.

After a brief introduction into the basics of head and neck oncology, the current review focusses on potential predictive markers and predisposing factors for these kinds of cancers. Furthermore, prognosis and degree of disability of cancer patients are commented on. With a survival rate of approximately 50%, the prognosis of head and neck carcinomas seems rather good, but it is in fact unsatisfactory. Since the treatment of advanced head and neck cancers is often accompanied by profound functional (articulation, phonation, respiration, and deglutition) and aesthetic impairment, it repeatedly leads to a high degree of disability and occupational invalidity. Furthermore, special limitations of head and neck cancer patients are discussed as well as the consequences they may have on the qualification for certain jobs.