Programmed Cell Death-1 (PD-1) anchoring to the GPI-linked co-receptor CD48 reveals a novel mechanism to modulate PD-1-dependent inhibition of human T cells.

Activation of PD-1 by anchoring it to Antigen Receptor (AR) components or associated co-receptors represents an attractive approach to treat autoimmune conditions. In this study, we provide evidence that CD48, a common lipid raft and Src kinase-associated coreceptor, induces significant Src kinase-dependent activation of PD-1 upon crosslinking, while CD71, a receptor excluded from these compartments, does not. Functionally, using bead-conjugated antibodies we demonstrate that CD48-dependent activation of PD-1 inhibits proliferation of AR-induced primary human T cells, and similarly, PD-1 activation using PD-1/CD48 bispecific antibodies inhibits IL-2, enhances IL-10 secretion, and reduces NFAT activation in primary human and Jurkat T cells, respectively. As a whole, CD48-dependent activation of PD-1 represents a novel mechanism to fine tune T cell activation, and by functionally anchoring PD-1 with receptors other than AR, this study provides a conceptual framework for rational development of novel therapies that activate inhibitory checkpoint receptors for treatment of immune-mediated diseases.

Review of the Role of Intraoperative Neurophysiological Monitoring in Spinal Surgery With a Focus on the True and False Positives and a Clinical Correlation.

BACKGROUND: Spinal surgery is a technically challenging endeavor with potentially devastating complications. Intraoperative neurophysiological monitoring (IONM) is a method of preventing and identifying damage to the spinal cord. OBJECTIVE: The aim of our study was to examine the clinical utility of IONM in spinal surgeries performed at our institution and what effect, if any, subsequent interventions had on postoperative patient outcomes. METHODS: This is a retrospective cohort study of 169 patients who underwent spinal surgery with IONM at 2 institutions between 2013 and 2018. Signal changes detected were recorded as well as the surgeon's response to these changes. Neurological status was recorded using a standard neurological examination and characterized as per the McCormick Neurological Scale. Patients were followed up for 12 months after surgery. RESULTS: A total of 169 spinal surgery cases with concurrent use of spinal cord monitoring were carried out in our institution between 2013 and 2018. The youngest patient was 14 years old, and the oldest was 92 years old (mean, 51.9 ± 19.6 years). There were 100 female patients and 69 male patients. Most patients (n = 124) had no signal changes. Signal changes were observed in 26.6% of the cases (n = 45). Most of these signal changes were rectified through repositioning of the patient (n = 24). The other 21 patients saw no improvement in their signals before the end of their procedures; however, these 21 patients had no postoperative deficits (grade I). This brought the false positive rate to 38% (21/55); the false negative rate was 1.8% (3/169). CONCLUSION: This study showed similar outcomes in patients whether IONM signals were recovered or not. The false positive and false negative rates were high. Our study helps to raise awareness about IONM's strengths and weaknesses to inform future clinical practice. We recommend prioritizing clinical judgment in spinal surgery cases and using IONM with caution.

Spinal metastasis: The rise of minimally invasive surgery.

INTRODUCTION: Bone is the third most common site of metastatic cancer, of which the spine is the most frequently involved. As metastatic cancer prevalence rises and surgical techniques advance, operative intervention for spinal metastases is expected to rise. In the first operative cohort of spinal metastasis in Ireland, we describe the move towards less invasive surgery, the causative primary types and post-operative survival. METHODS: This is a retrospective cohort study of all operative interventions for spinal metastasis in a tertiary referral centre over eight years. Primary spinal tumours and local invasion to the spine were excluded. Median follow up was 1895 days. RESULTS: 225 operative procedures in 196 patients with spinal metastasis were performed over eight years. Average cases per year increased form 20 per year to 29 per year. Percutaneous procedures became more common, accounting for the majority (53%) in the final two years. The most common primary types were breast, myeloma, lung, prostate and renal. Overall survival at 1 year was 51%. Primary type was a major determinant of survival, with breast and the haematological cancers demonstrating good survival, while lung had the worst prognosis. CONCLUSION: This is the first descriptive cohort of operative interventions for spinal metastasis in an Irish context. Surgery for spinal metastasis is performed at an increasing rate, especially through minimally-invasive means. The majority of patients survive for at least one year post-operatively. Prudent resource planning is necessary to prepare for this growing need.

The potent and selective RIPK2 inhibitor BI 706039 improves intestinal inflammation in the TRUC mouse model of inflammatory bowel disease.

Mouse and human data implicate the NOD1 and NOD2 sensors of the intestinal microbiome and the associated signal transduction via the receptor interacting protein kinase 2 (RIPK2) as a potential key signaling node for the development of inflammatory bowel disease (IBD) and an attractive target for pharmacological intervention. The TRUC mouse model of IBD was strongly indicated for evaluating RIPK2 antagonism for its effect on intestinal inflammation based on previous knockout studies with NOD1, NOD2, and RIPK2. We identified and profiled the BI 706039 molecule as a potent and specific functional inhibitor of both human and mouse RIPK2 and with favorable pharmacokinetic properties. We dosed BI 706039 in the spontaneous TRUC mouse model from age 28 to 56 days. Oral, daily administration of BI 706039 caused dose-responsive and significant improvement in colonic histopathological inflammation, colon weight, and terminal levels of protein-normalized fecal lipocalin (all P values <0.001). These observations correlated with dose responsively increasing systemic levels of the BI 706039 compound, splenic molecular target engagement of RIPK2, and modulation of inflammatory genes in the colon. This demonstrates that a relatively low oral dose of a potent and selective RIPK2 inhibitor can modulate signaling in the intestinal immune system and significantly improve disease associated intestinal inflammation.NEW & NOTEWORTHY The RIPK2 kinase at the apex of microbiome immunosensing is an attractive target for pharmacological intervention. A low oral dose of a RIPK2 inhibitor leads to significantly improved intestinal inflammation in the murine TRUC model of colitis. A selective and potent inhibitor of the RIPK2 kinase may represent a new class of therapeutics that target microbiome-driven signaling for the treatment of IBD.

Cervical spine hangman fracture secondary to a gelastic seizure.

A 42-year-old man reported to our service with a 1-week history of vague cervical neck pain on a background history of a gelastic seizure disorder. Radiological imaging confirmed a type II hangman's fracture through the C2 pedicle. A CT angiogram of carotid and vertebral arteries was normal, and the patient was managed with an occipitocervical fusion. The patient had no neurological insult and was discharged on day 5 with a Miami-J collar for 6 weeks. Although there are case report evidence of lumbar and thoracic vertebral fractures secondary to seizures, this is the first report of a spine injury resulting from a gelastic seizure.

Erythema Ab Igne and Its Relation to Spinal Pathology.

Erythema ab igne is a skin condition usually caused by abnormal exposure to heat in a variety of forms. While common in the past, its incidence has declined significantly to become a rare occurrence today. Today, we still see an odd case once in a while, although they have become a rarity. We describe a case of a young man who presented with erythema ab igne with a unique cause.

Signs of Nature in Spine Radiology.

As medical science developed over time, we have relied on natural imagery to help us recognise and remember things. In this review article, we will be discussing some radiological signs named because of their resemblance to the occurrences in the natural world.

An Orthopaedic Fracture Clinic Service Audit: A Complete Loop.

Introduction The British Orthopaedic Association Standards for Trauma (BOAST) Guideline 7 informs the standard of care patients should expect when they come to orthopaedic fracture clinics in the United Kingdom (UK). Objectives We compared our fracture clinic's practice against the standards set by BOAST Guideline 7 to make changes for aligning with the standard of care. We aimed to then re-audit our practice for further evaluation against the guidelines. Material and methods We prospectively collected data from 100 patients presenting to the fracture clinics of different orthopaedic consultants working in our hospital, using the Royal College of Surgeons in Ireland's (RCSI's) satisfaction with outpatients services (SWOPS) questionnaire. We made some improvements, recommended changes to the hospital management, and conducted a re-audit, collecting data from another 100 patients. Results With reference to improvements, we were only able to make them on behalf of the doctors and clinical auxiliary staff. We were able to decrease the waiting time from a patient's initial presentation in the accident and emergency (A&E) department to an appointment at the fracture clinic. A few improvements were made to the waiting area facilities. However, the cumulative changes resulted in a positive attitude in patient satisfaction levels. Conclusion Considering our complete audit loop, we found gaps and enabled improvements, but areas of concern remain, which will need to be addressed in the future.

A review of the application of vancomycin powder to posterior spinal fusion wounds with a focus on side effects and infection. A prospective study.

INTRODUCTION: Deep wound infection after spinal surgery is a potentially devastating complication and is associated with higher morbidity, mortality and healthcare costs. Different measures including intraoperative application of vancomycin powder to wounds have been employed previously to decrease the infection rate. OBJECTIVES: The primary objective of this ongoing clinical study is to evaluate the systemic uptake of prophylactically applied vancomycin in instrumented spinal fusion surgery. Secondary outcomes are to show any side effects including nephrotoxicity related to its local application and record superficial and deep wound infections. METHODS: A prospective study has been designed to recruit consecutive patients, between September 2013 and September 2014, operated by a single surgeon. All patients undergoing instrumented spinal fusion surgery (elective and trauma) in a single institution over a 12-month period were included. One gram of vancomycin powder was applied to the subfascial layer, and serum levels were measured at 6, 12 and 24 h post-administration. All patients routinely had renal functions checked postoperatively to evaluate nephrotoxic effects. A second cohort of patients was then recruited to apply 2 g of vancomycin subfascially. The patients were followed up for a 2-year period. RESULTS: Twenty-four patients, both trauma and elective, had 1 g of vancomycin powder applied to the subfascial layer. Twenty-eight patients had 2 g of vancomycin powder applied to the subfascial layer. Four patients reached systemic levels in the 2-g group; however, only one patient had clinically detectable but nonsignificant levels in the 1-g group. There were no adverse effects detected. CONCLUSION: This study demonstrates that systemic uptake of vancomycin after local application to the wound is negligible for the vast majority of patients. However, it has shown clinical and biochemical safety for its use and remains a cost-effective and low-risk strategy to combat surgical site and deep wound infections.

Prospective study using anterior approach did not show association between Modic 1 changes and low grade infection in lumbar spine.

INTRODUCTION: The modern literature is producing a rapidly growing number of articles which highlight the relationship between infection and lumbar disc degeneration. However, the means by which samples are collected is questionable. Posterior approach surgery is not free from skin contamination. The possibility of intraoperative contamination of disc biopsies cannot be excluded. OBJECTIVE: The objective of this study was to determine if an association existed between lumbar disc degeneration and chronic infection of the intervertebral disc. MATERIALS AND METHODS: 313 patients (186/127, F/M) with chronic low back pain secondary to degenerative disc disease which was resistant to medical treatment were included in a single-centre prospective study. All underwent a lumbar anterior video-assisted minimally invasive fusion or disc prosthesis in L4-L5 and/or L5-S1 via an anterior retroperitoneal approach. The patients MRI scans demonstrated in Pfirrmann's classification grade IV or V disc degeneration; 385 disc drives were taken. In terms of Modic changes, 303 Modic 1, 58 Modic II and 24 absence of Modic change, respectively. All underwent intraoperative biopsy, performed according to a strict aseptic protocol. The biopsies were then cultured for 4 weeks with specialised enrichment cultures and subjected to histopathological analysis. RESULTS: The mean age was 47 ± 8.6 years sterile cultures were obtained in 379 samples (98.4%) and 6 were positive (1.6%). The cultured bacteria were: Propionibacterium acnes (n:2), Staphylococcus epidermidis (n:2), Citrobacter freundii (n:1), and Saccharopolyspora hirsuta (n:1). Histopathological analysis did not demonstrate any evidence of a neutrophilia. There were no delayed or secondary infections. DISCUSSION AND CONCLUSION: Unlike the posterior approach where contamination is common, the anterior video-assisted approach allows a biopsy without skin contact. This approach to the spine is the most effective way to eliminate the risk of contamination. Our results confirm the absence of any relationship between infection and disc degeneration. We suggest that the 6 positive samples in our study may be related to contamination. The absence of infection at 1-year followup is an additional argument in favour of our results. In conclusion, our study shows no association between infection and disc degeneration. The pathophysiology of disc degeneration is complex, but the current literature opens new perspectives.

Differential Roles for Interleukin-23 and Interleukin-17 in Intestinal Immunoregulation.

Interleukin-23 (IL-23) and IL-17 are cytokines currently being targeted in clinical trials. Although inhibition of both of these cytokines is effective for treating psoriasis, IL-12 and IL-23 p40 inhibition attenuates Crohn's disease, whereas IL-17A or IL-17 receptor A (IL-17RA) inhibition exacerbates Crohn's disease. This dichotomy between IL-23 and IL-17 was effectively modeled in the multidrug resistance-1a-ablated (Abcb1a(-/-)) mouse model of colitis. IL-23 inhibition attenuated disease by decreasing colonic inflammation while enhancing regulatory T (Treg) cell accumulation. Exacerbation of colitis by IL-17A or IL-17RA inhibition was associated with severe weakening of the intestinal epithelial barrier, culminating in increased colonic inflammation and accelerated mortality. These data show that IL-17A acts on intestinal epithelium to promote barrier function and provide insight into mechanisms underlying exacerbation of Crohn's disease when IL-17A or IL-17RA is inhibited.

Optimization of experimental conditions for functional in vitro characterization of humanized antibodies specific for staphylococcal enterotoxin B.

Staphylococcus aureus is a common nosocomial infection and its resistance to penicillin and methicillin antibiotics is a growing clinical problem. We previously described the development of a humanized anti-Staphylococcus enterotoxin B (SEB) antibody derived from the mouse antibody made by the 20B1 hybridoma. This antibody was humanized and characterized kinetically by surface plasmon resonance demonstrating that the humanized clones retained binding to SEB. Clones were then functionally characterized in an in vitro assay demonstrating that the murine 20B1, chimeric and humanized antibodies potently inhibited SEB-induced murine splenocyte proliferation assay. Here, we describe a human cell-based screening assay, optimized by varying multiple experimental parameters that resulted in an assay that was used to demonstrate full and potent neutralization by the parental, chimeric and humanized antibodies. The replacement of fetal bovine serum (FBS) with normal human serum (NHS) was found to be a crucial factor in the performance of the human cell based screening assay that enabled the calculation of mAb efficacy and potency. In addition, we found that anti-SEB antibodies showed similar efficacy and potency with a triple mutant Fc region (designed to be effector function null) or a wild-type Fc region, which is in contrast to previously described studies.

Chemokine receptor CCR7 regulates the intestinal TH1/TH17/Treg balance during Crohn's-like murine ileitis.

The regulation of T cell and DC retention and lymphatic egress within and from the intestine is critical for intestinal immunosurveillance; however, the cellular processes that orchestrate this balance during IBD remain poorly defined. With the use of a mouse model of TNF-driven Crohn's-like ileitis (TNF(Δ) (ARE)), we examined the role of CCR7 in the control of intestinal T cell and DC retention/egress during experimental CD. We observed that the frequency of CCR7-expressing TH1/TH17 effector lymphocytes increased during active disease in TNF(Δ) (ARE) mice and that ΔARE/CCR7(-/-) mice developed exacerbated ileitis and multiorgan inflammation, with a marked polarization and ileal retention of TH1 effector CD4(+) T cells. Furthermore, adoptive transfer of ΔARE/CCR7(-/-) effector CD4(+) into lymphopenic hosts resulted in ileo-colitis, whereas those transferred with ΔARE/CCR7(+/+) CD4(+) T cells developed ileitis. ΔARE/CCR7(-/-) mice had an acellular draining MLN, decreased CD103(+) DC, and decreased expression of RALDH enzymes and of CD4(+)CD25(+)FoxP3(+) Tregs. Lastly, a mAb against CCR7 exacerbated ileitis in TNF(Δ) (ARE) mice, phenocopying the effects of congenital CCR7 deficiency. Our data underscore a critical role for the lymphoid chemokine receptor CCR7 in orchestrating immune cell traffic and TH1 versus TH17 bias during chronic murine ileitis.

Humanized staphylococcal enterotoxin B (SEB)-specific monoclonal antibodies protect from SEB intoxication and Staphylococcus aureus infections alone or as adjunctive therapy with vancomycin.

BACKGROUND: Staphylococcal enterotoxin B (SEB), a potential biological warfare agent, is a potent superantigen that contributes to the virulence of methicillin-resistant Staphylococcus aureus (MRSA), which is a major health threat in the United States. Efforts to develop toxin-neutralizing antibodies as adjunctive therapies are justified, given the high mortality and frequent failure of therapy despite available antibiotics. METHODS: Murine SEB-specific mAb 20B1 was humanized, and treatment benefits of Hu-1.6/1.1 and Hu-1.4/1.1 variants were investigated in mice in an SEB intoxication model, as well as in sepsis and deep-tissue infection models. RESULTS: Hu-1.6/1.1 and Hu-1.4/1.1 protected mice against SEB-induced lethal shock. Hu-1.6/1.1 also enhanced survival of mice that developed fatal sepsis after challenge with a SEB-producing MRSA strain. Combined treatment of Hu-1.6/1.1 with vancomycin further increased survival and altered cytokine responses, compared with monotherapy with either monoclonal antibody or vancomycin alone. Efficacy was also demonstrated in the deep-tissue infection model, where Hu-1.4/1.1 bound to SEB in vivo and decreased abscess formation, as well as proinflammatory cytokine levels. CONCLUSIONS: SEB-neutralizing mAb 20B1 was successfully humanized. The mAb affects outcome by modulating the proinflammatory host response in both the sepsis and the intoxication models, which justifies further development.

Staphylococcal Enterotoxin B-specific monoclonal antibody 20B1 successfully treats diverse Staphylococcus aureus infections.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) has become a major health threat in the United States. Staphylococcal enterotoxin B (SEB) is a potent superantigen that contributes to its virulence. High mortality and frequent failure of therapy despite available antibiotics have stimulated research efforts to develop adjunctive therapies. METHODS: Treatment benefits of SEB-specific monoclonal antibody (mAb) 20B1 were investigated in mice in sepsis, superficial skin, and deep-tissue infection models. RESULTS: Mice challenged with a SEB-producing MRSA strain developed fatal sepsis, extensive tissue skin infection, and abscess-forming deep-seeded thigh muscle infection. Animals preimmunized against SEB or treated passively with mAb 20B1 exhibited enhanced survival in the sepsis model, whereas decrease of bacterial burden was observed in the superficial skin and deep-tissue models. mAb 20B1 bound to SEB in the infected tissue and decreased abscess formation and proinflammatory cytokine levels, lymphocyte proliferation, and neutrophil recruitment. CONCLUSIONS: mAb 20B1, an SEB-neutralizing mAb, is effective against MRSA infection. mAb 20B1 protects against lethal sepsis and reduces skin tissue invasion and deep-abscess formation. The mAb penetrates well into the abscess and binds to SEB. It affects the outcome of S. aureus infection by modulating the host's proinflammatory immune response.

Anterior cruciate ligament graft tensioning. Is the maximal sustained one-handed pull technique reproducible?

BACKGROUND: Tensioning of anterior cruciate ligament (ACL) reconstruction grafts affects the clinical outcome of the procedure. As yet, no consensus has been reached regarding the optimum initial tension in an ACL graft. Most surgeons rely on the maximal sustained one-handed pull technique for graft tension. We aim to determine if this technique is reproducible from patient to patient. FINDINGS: We created a device to simulate ACL reconstruction surgery using Ilizarov components and porcine flexor tendons. Six experienced ACL reconstruction surgeons volunteered to tension porcine grafts using the device to see if they could produce a consistent tension. None of the surgeons involved were able to accurately reproduce graft tension over a series of repeat trials. CONCLUSIONS: We conclude that the maximal sustained one-handed pull technique of ACL graft tensioning is not reproducible from trial to trial. We also conclude that the initial tension placed on an ACL graft varies from surgeon to surgeon.

An antibody to IP-10 is a potent antagonist of cell migration in vitro and in vivo and does not affect disease in several animal models of inflammation.

IP-10 secretion is induced by pro-inflammatory cytokines and mediates the migration of CXCR3+ cells. Its elevation in clinical samples has been associated with multiple inflammatory diseases and its antagonism has been reported to be effective in several animal models of inflammatory disease. We generated a mouse anti-mouse IP-10 monoclonal antibody (mAb; Clone 20A9) that specifically bound murine IP-10 with high affinity and inhibited in vitro IP-10 induced BaF3/mCXCR3 cell migration with an IC(50) of approximately 4 nM. The 20A9 mAb was completely absorbed in vivo and had dose proportional pharmacokinetic exposure with a serum half life of 2.4-6 days. The 20A9 mAb inhibited IP-10 mediated T-cell recruitment to the airways, indicating that it is effective in vivo. However, administration of the 20A9 mAb had no significant effect on disease in mouse models of delayed type hypersensitivity, collagen induced arthritis, cardiac allograft transplantation tolerance, EAE or CD4+ CD45RBHi T-cell transfer-induced IBD. These data suggest that the 20A9 mAb can antagonize IP-10 mediated chemotaxis in vitro and in vivo and that this is insufficient to cause a therapeutic benefit in multiple mouse models of inflammatory disease.

Methods of inducing inflammatory bowel disease in mice.

Animal models of experimentally induced inflammatory bowel disease (IBD) are useful for understanding more about the mechanistic basis of disease, identifying new targets for therapeutic intervention, and testing novel therapeutic agents. This unit provides detailed protocols for four of the most commonly used mouse models of experimentally induced intestinal inflammation: chemical induction of colitis by dextran sodium sulfate (DSS), hapten-induced colitis via 2,4,6-trinitrobenzene sulfonic acid (TNBS), Helicobacter-induced colitis in mdr1a(-/-) mice, and the CD4(+) CD45RB(hi) SCID transfer colitis model.

Spontaneous colitis occurrence in transgenic mice with altered B7-mediated costimulation.

The B7 costimulatory molecules govern many aspects of T cell immune responses by interacting with CD28 for costimulation, but also with CTLA-4 for immune suppression. Although blockade of CTLA-4 with Ab in humans undergoing cancer immune therapy has led to some cases of inflammatory bowel disease, spontaneous animal models of colitis that depend upon modulation of B7 interactions have not been previously described. In this study, we demonstrate that mice expressing a soluble B7-2 Ig Fc chimeric protein spontaneously develop colitis that is dependent on CD28-mediated costimulation of CD4(+) T cells. We show that the chimeric protein has mixed agonistic/antagonist properties, and that it acts in part by blocking the cell intrinsic effects on T cell activation of engagement of CTLA-4. Disease occurred in transgenic mice that lack expression of the endogenous B7 molecules (B7 double knock-out mice), because of the relatively weak costimulatory delivered by the chimeric protein. Surprisingly, colitis was more severe in this context, which was associated with the decreased number of Foxp3(+) regulatory T cells in transgenic B7 double knock-out mice. This model provides an important tool for examining how B7 molecules and their effects on CTLA-4 modulate T cell function and the development of inflammatory diseases.