Rapid and effective generation of nanobody based CARs using PCR and Gibson Assembly

Recent approval of chimeric antigen receptor (CAR) T cell therapy by the European Medicines Agency (EMA)/Federal and Drug Administration (FDA) and the remarkable results of CAR T clinical trials illustrate the curative potential of this therapy. While CARs against a multitude of different antigens are being developed and tested (pre)clinically, there is still a need for optimization. The use of single -chain variable fragments (scFvs) as targeting moieties hampers the quick generation of functional CARs and could potentially limit the efficacy. Instead, nanobodies may largely circumvent these difficulties. We used an available nanobody library generated after immunization of llamas against Cluster of Differentiation (CD) 20 through DNA vaccination or against the ectodomain of CD33 using soluble protein. The nanobody specific sequences were amplified by PCR and cloned by Gibson Assembly into a retroviral vector containing two different second -generation CAR constructs. After transduction in T cells, we observed high cell membrane nanoCAR expression in all cases. Following stimulation of nanoCAR-expressing T cells with antigen-positive cell lines, robust T cell activation, cytokine production and tumor cell lysis both in vitro and in vivo was observed. The use of nanobody technology in combination with PCR and Gibson Assembly allows for the rapid and effective generation of compact CARs

Crosstalk between Virulence Loci: Regulation of Salmonella enterica Pathogenicity Island 1 (SPI-1) by Products of the std Fimbrial Operon

Invasion of intestinal epithelial cells is a critical step in Salmonella infection and requires the expression of genes located in Salmonella pathogenicity island 1 (SPI-1). A key factor for SPI-1 expression is DNA adenine (Dam) methylation, which activates synthesis of the SPI-1 transcriptional activator HilD. Dam-dependent regulation of hilD is postranscriptional (and therefore indirect), indicating the involvement of unknown cell functions under Dam methylation control. A genetic screen has identified the std fimbrial operon as the missing link between Dam methylation and SPI-1. We show that all genes in the std operon are part of a single transcriptional unit, and describe three previously uncharacterized ORFs (renamed stdD, stdE, and stdF). We present evidence that two such loci (stdE and stdF) are involved in Dam-dependent control of Salmonella SPI-1: in a Dam− background, deletion of stdE or stdF suppresses SPI-1 repression; in a Dam+ background, constitutive expression of StdE and/or StdF represses SPI-1. Repression of SPI-1 by products of std operon explains the invasion defect of Salmonella Dam− mutants, which constitutively express the std operon. Dam-dependent repression of std in the ileum may be required to permit invasion, as indicated by two observations: constitutive expression of StdE and StdF reduces invasion of epithelial cells in vitro (1,000 fold) and attenuates Salmonella virulence in the mouse model (>60 fold). In turn, crosstalk between std and SPI-1 may play a role in intestinal infections by preventing expression of SPI-1 in the caecum, an intestinal compartment in which the std operon is known to be expressed

Regional and experiential differences in surgeon preference for the treatment of cervical facet injuries: a case study survey with the AO Spine Cervical Classification Validation Group

Purpose: The management of cervical facet dislocation injuries remains controversial. The main purpose of this investigation was to identify whether a surgeon’s geographic location or years in practice influences their preferred management of traumatic cervical facet dislocation injuries. Methods: A survey was sent to 272 AO Spine members across all geographic regions and with a variety of practice experience. The survey included clinical case scenarios of cervical facet dislocation injuries and asked responders to select preferences among various diagnostic and management options. Results: A total of 189 complete responses were received. Over 50% of responding surgeons in each region elected to initiate management of cervical facet dislocation injuries with an MRI, with 6 case exceptions. Overall, there was considerable agreement between American and European responders regarding management of these injuries, with only 3 cases exhibiting a significant difference. Additionally, results also exhibited considerable management agreement between those with ≤ 10 and > 10 years of practice experience, with only 2 case exceptions noted. Conclusion: More than half of responders, regardless of geographical location or practice experience, identified MRI as a screening imaging modality when managing cervical facet dislocation injuries, regardless of the status of the spinal cord and prior to any additional intervention. Additionally, a majority of surgeons would elect an anterior approach for the surgical management of these injuries. The study found overall agreement in management preferences of cervical facet dislocation injuries around the globe

Factors Associated with Revision Surgery after Internal Fixation of Hip Fractures

Background: Femoral neck fractures are associated with high rates of revision surgery after management with internal fixation. Using data from the Fixation using Alternative Implants for the Treatment of Hip fractures (FAITH) trial evaluating methods of internal fixation in patients with femoral neck fractures, we investigated associations between baseline and surgical factors and the need for revision surgery to promote healing, relieve pain, treat infection or improve function over 24 months postsurgery. Additionally, we investigated factors associated with (1) hardware removal and (2) implant exchange from cancellous screws (CS) or sliding hip screw (SHS) to total hip arthroplasty, hemiarthroplasty, or another internal fixation device. Methods: We identified 15 potential factors a priori that may be associated with revision surgery, 7 with hardware removal, and 14 with implant exchange. We used multivariable Cox proportional hazards analyses in our investigation. Results: Factors associated with increased risk of revision surgery included: female sex, [hazard ratio (HR) 1.79, 95% confidence interval (CI) 1.25-2.50; P = 0.001], higher body mass index (fo

Low level of evidence for all treatment modalities for irreparable posterosuperior rotator cuff tears

Purpose: This systematic review assesses evidence for improvements in outcome for all reported types of treatment modalities [physical therapy, tenotomy or tenodesis of the long head of the biceps, debridement, partial repair, subacromial spacer, deltoid flap, muscle transfer, rotator cuff advancement, graft interposition, superior capsular reconstruction (SCR), and reversed shoulder arthroplasty (RSA)] used for irreparable posterosuperior rotator cuff tears without glenohumeral osteoarthritis. The primary aim was to be able to inform patients about expectations of the amount of clinical improvement after these treatments. Methods: A systematic search was conducted in MEDLINE, EMBASE, CINAHL, and Cochrane databases for studies on irreparable posterosuperior rotator cuff lesions without glenohumeral osteoarthritis, published from January 2007 until January 2019, with minimum 2-year follow-up. Studies with pre-operative and/or intra-operative determination of cuff tear irreparability were included. We defined the non-adjusted Constant Score as the primary outcome. Results: Sixty studies (2000 patients) were included with a fair mean quality score, according to the Modified Coleman Methodology Score. The employed definitions of ‘irreparable’ were mainly based on MRI criteria and were highly variable among studies. The smallest weighted mean preoperative to post-operative improvements in Constant Score were reported for biceps tenotomy/tenodesis (10.7 points) and physical therapy (13.0). These were followed by debridement (21.8) and muscle transfer (27.8), whereas the largest increases were reported for partial repair (32.0), subacromial spacer (32.5), rotator cuff advancement (33.2), RSA (34.4), graft reconstruction (35.0), deltoid flap (39.8), and SCR (47.4). Treatment using deltoid flap showed highest mean weighted improvement in Constant Score among studies with available medium-term (4–5-year) follow-up. Treatments deltoid flap, muscle transfer, and debridement were the only treatments with available long-term (8–10-year) follow-up and showed similar improvements in Constant Score at this time point. Conclusion: The variability in patient characteristics, co-interventions, outcome reporting, and length of follow-up in studies on irreparable rotator cuff tears without osteoarthritis complicates sound comparison of treatments. Clinically important treatment effects were seen for all 11 different treatment modalities. Level of evidence: IV

Chyluria and chylothorax after posterior selective fusion for adolescent idiopathic scoliosis

PURPOSE: To describe and discuss the diagnostic and treatment complexity of lymphatic system complications after scoliosis surgery. METHODS: Surgery for adolescent idiopathic scoliosis is very commonly performed with posterior pedicle screw instrumentation. Complications of the anteriorly based lymphatic system are, therefore, rare. We present a case with complications related to the lymphatic system, which have not been reported before after this type of surgery. RESULTS: After standard Th3 to Th12 posterior spinal reduction and fusion of a moderate thoracic curve, chyluria and a chylothorax developed in an adolescent girl. This appeared to be caused by an obstruction of the thoracic duct. Thorax drainage and finally thoracoscopic intervention prevented further pulmonal impairment. The exact cause could not be identified and the persistent lymph drainage problems had to be treated with a medium chain triglyceride diet. CONCLUSION: With this report, we aim to create awareness of the lymphatic system in general and the possibility of severe complications, even after a posterior only approach of the vertebral column

Shoulder kinematics and muscle activity following latissimus dorsi transfer for massive irreparable posterosuperior rotator cuff tears in shoulders with pseudoparalysis

Background: The aim of this study was to evaluate the thoracohumeral (TH) and glenohumeral (GH) motion with muscle activity after latissimus dorsi transfer (LDT) in a shoulder with a massive irreparable posterosuperior rotator cuff tear (MIRT) and pseudoparalysis compared with the asymptomatic contralateral shoulder (ACS). Methods: We recruited and evaluated 13 patients after LDT in a shoulder with preoperative clinical pseudoparalysis and an MIRT on magnetic resonance imaging, with a minimum follow-up period of 1 year, and with a Hamada stage of 3 or less. Three-dimensional electromagnetic tracking was used to assess shoulder active range of motion in both the LDT shoulder and the ACS. The maximal active elevation of the shoulder (MAES) was assessed and consisted of forward flexion, scapular abduction, and abduction in the coronal plane. Maximal active internal rotation and external rotation were assessed separately. Surface electromyography (EMG) was performed to track activation of the latissimus dorsi (LD) and deltoid muscles during shoulder motion. EMG was scaled to its maximal isometric voluntary contraction recorded in specified strength tests. Results: In MAES, TH motion of the LDT shoulder was not significantly different from that of the ACS (F1,12 = 1.174, P = .300) but the GH contribution was significantly lower in the LDT shoulder for all motions (F1,12 = 11.230, P = .006). External rotation was significantly greater in the ACS (26° ± 10° in LDT shoulder vs. 42° ± 11° in ACS, P < .001). The LD percentage EMG maximum showed no significant difference between the LDT shoulder and ACS during MAES (F1,11 = 0.005, P = .946). During maximal active external rotation of the shoulder, the LDT shoulder showed a higher percentage EMG maximum than the ACS (3.0% ± 2.9% for LDT shoulder vs. 1.2% ± 2.0% for ACS, P = .006). Conclusions: TH motion improved after LDT in an MIRT with pseudoparalysis and was not different from the ACS except for external rotation. However, GH motion was significantly lower after LDT than in the ACS in active-elevation range of motion. The LD was active after LDT but not more than in the ACS except for active external rotation, which we did not consider relevant as the activity did not rise above 3% EMG maximum. The favorable clinical results of LDT do not seem to be related to a change in LD activation and might be explained by its effect in preventing proximal migration of the humeral head in active elevation

Physical rehabilitation for older people in long-term care.

The worldwide population is progressively ageing, with an expected increase in morbidity and demand for long-term care. Physical rehabilitation is beneficial in older people, but relatively little is known about effects on long-term care residents. This is an update of a Cochrane review first published in 2009