Fully Automated Analysis of the Anatomic and Mechanical Axes From Pediatric Standing Lower Limb Radiographs Using Convolutional Neural Networks.

BACKGROUND: Lower limb alignment is the quantification of a set of parameters that are commonly measured radiographically to test for and track a wide range of skeletal pathologies. Determining limb alignment is a commonly performed yet laborious task in the pediatric orthopaedic setting and is therefore an interesting goal for automation. METHODS: We employ a machine learning approach using convolutional neural networks (CNNs) to segment pediatric weight-bearing lower limb radiographs. The results are then used with custom Matlab code to extract anatomic landmarks and to determine lower limb alignment parameters. RESULTS: Measurements obtained from the automated workflow proposed here were compared with manual measurements performed by orthopaedic surgery fellows. Mechanical axis deviation was determined within a mean of 2.02 mm. Lateral distal femoral angle and medial proximal tibial angle were determined with a mean deviation of 1.73 and 2.90 degrees, respectively. The calculation speed for the full set of mechanical and anatomic axis parameters was found to be ~2 seconds per radiograph. CONCLUSIONS: The CNN-based approach proposed in this work was shown to produce results comparable to orthopaedic surgery fellows at fast calculation speed. Although further work is needed to validate these results against radiographs and measurements from other centers, we see this as a promising start and a functional path that can be employed in further research. CLINICAL RELEVANCE: CNNs are a promising approach to automating commonly performed, repetitive tasks, especially those pertaining to image processing. The time savings are particularly important in clinical research applications where large sets of radiographs are routinely available and require analysis. With further development of these algorithms, we anticipate significantly improved agreement with expert-measured results and the calculation speed.

Early Investigation and Bracing in DDH Impacts Maternal Wellbeing and Breastfeeding.

BACKGROUND: The outcomes of Pavlik Harness (PH) management for Developmental Dysplasia of the Hip (DDH) are equivalent regardless of the initiation timing, if it is within the first 6 weeks of life. A PH may be a physical barrier to breastfeeding, which is important for nutrition, immunity, and normal child development. The diagnosis of DDH and early management with a PH may also negatively affect the maternal psychosocial wellbeing and the infant-maternal relationship. The purpose of this study is to investigate the impact of the diagnosis of DDH and the management with a PH has on maternal wellbeing and maintenance of breastfeeding, compared with being screened for but not diagnosed with DDH. METHODS: A retrospective cohort of the mothers of infants who were diagnosed with DDH and treated with a PH brace was compared with the mothers of infants who were screened for DDH only. The Hip Worries Inventory and Edinburgh Postnatal Depression Scale were completed by the mothers in both groups. The PH group also completed an in-house questionnaire specific to PH and breastfeeding. RESULTS: Eighty completed surveys were included, 50 from the treatment group. The mean age of the PH initiation was 6.2 weeks. The modified Hip Worries Inventory score was higher in the treatment group, with a mean difference (MD) of 9.7 out of 50 (95% confidence interval, CI, 6.8, 12.5). The MD of the Edinburgh Postnatal Depression Scale was 2.0 out of 30 (CI -0.5, 4.5). Although there was no difference in the breastfeeding ease before and after the PH initiation (MD-0.2, CI-0.7, 0.2), 83% of mothers found breastfeeding more difficult with a PH and 11% of mothers stopped breastfeeding earlier than planned because of the PH. CONCLUSIONS: Mothers of infants with DDH worry more about their child's hips and the PH. Screening alone may contribute to maternal psychological dejection and negative thoughts. The presence of a PH makes breastfeeding more difficult. LEVEL OF EVIDENCE: Retrospective comparative study, level III.

Multimodal Analgesia for Paediatric Patients Undergoing Lower Limb Reconstruction with External Fixators: A Prospective Case Series of Post-operative Pain and Functional Goals.

Aim: Limb reconstruction with external fixators requires appropriate pain management to promote effective analgesia and healing while minimising adverse events of the analgesic technique used. The objective of this prospective case series was to evaluate a multimodal analgesia regimen designed to reduce opioid requirements and hence reduce the opioid-related side effect profile. Materials and methods: A prospective cohort of patients undergoing lower limb reconstruction surgery (LRS) were managed through an evidence-informed multimodal analgesia guideline (MMAG), including acetaminophen, pregabalin, dexmedetomidine, IV lidocaine, and opioids. Outcome measures included intraoperative and post-operative opioid administration, post-operative pain scores, time to achieve mobilisation milestones, and post-operative complications. Surveys were conducted to obtain patient reported experiences. Results: 26 patients were included in this prospective case series. 110.59 (84.29, 162.13) (median, interquartile range) µg/kg/hr intraoperative IV morphine equivalent opioids were administered. In the first 48 hours post-operatively, patients received 11.49 (6.41, 19.35) µg/kg/hr of IV morphine equivalent dose. Median level of pain (0-10) in the first 48 post-operative hours was 2 (1, 2). Patients achieved mobilisation. And 19/20 patients surveyed reported 'yes' to having effective pain management; 17/20 patients had no unwanted side effects associated with analgesia medications. There were no cases of compartment syndrome. Conclusion: This multimodal analgesia regime applied to patients undergoing lower LRS with external fixators demonstrates the feasibility of this analgesic regimen which revealed effective pain control, early mobilisation, with minimal side effects, but warrants further study. Clinical significance: This study provides valuable evidence that this standardised multimodal anaesthesia and analgesia regimen is feasible, offers adequate post-operative comfort and encourages early mobilization while minimising opioid use and adverse events in a paediatric LRS population at our institution. How to cite this article: Wang AWT, Chhina H, Cooper A. Multimodal Analgesia for Paediatric Patients Undergoing Lower Limb Reconstruction with External Fixators: A Prospective Case Series of Post-operative Pain and Functional Goals. Strategies Trauma Limb Reconstr 2023;18(3):140-147.

Human complete NFAT1 deficiency causes a triad of joint contractures, osteochondromas, and B-cell malignancy.

The discovery of humans with monogenic disorders has a rich history of generating new insights into biology. Here we report the first human identified with complete deficiency of nuclear factor of activated T cells 1 (NFAT1). NFAT1, encoded by NFATC2, mediates calcium-calcineurin signals that drive cell activation, proliferation, and survival. The patient is homozygous for a damaging germline NFATC2 variant (c.2023_2026delTACC; p.Tyr675Thrfs∗18) and presented with joint contractures, osteochondromas, and recurrent B-cell lymphoma. Absence of NFAT1 protein in chondrocytes caused enrichment in prosurvival and inflammatory genes. Systematic single-cell-omic analyses in PBMCs revealed an environment that promotes lymphomagenesis with accumulation of naïve B cells (enriched for oncogenic signatures MYC and JAK1), exhausted CD4+ T cells, impaired T follicular helper cells, and aberrant CD8+ T cells. This work highlights the pleiotropic role of human NFAT1, will empower the diagnosis of additional patients with NFAT1 deficiency, and further defines the detrimental effects associated with long-term use of calcineurin inhibitors.

Paediatric elbow fractures and public play spaces: adherence to standards for children's playground equipment and surfacing.

Background: Supracondylar humerus fractures (SCHF) are the most common fractures sustained following a fall onto an outstretched hand among healthy children, and one of the leading causes of hospital admission and surgical intervention. The aim of this study was to examine SCHF occurring at public play spaces-particularly to determine whether or not the playground equipment implicated in injurious falls aligned with Canadian playground safety standards. Methods: Cases of children who attended the provincial paediatric orthopaedic clinic following SCHF at a public playground between April 2017 and October 2019 were included in the study. A research assistant visited each playground to measure the play structure type and dimensions, height of the equipment at the point from which the child fell and the type and depth of the surface material, and compare measurements to the 2016 safety standards. Child demographics and injury classification were also noted. Descriptive statistics were calculated and a scatterplot of fall height and surface depth was generated. Results: Forty-three sites, representing 47 SCHF cases (18 female, 29 male), were included in the final analysis. Fourteen children sustained type 1 fracture, 23 had type 2 fracture and the remaining 10 had type 3 fracture. Five children with type 2 fracture and all 10 children with type 3 fracture required surgery. The majority of sites had engineered wood fibre surfacing, with surfacing at 35 sites being less than 300 mm deep. Twenty-six play structures were upper body equipment (ie, monkey bars or similar), seven were track rides, five were rotating structures and the rest comprised a variety of classified and unclassified structures. Twenty-seven children fell from a height exceeding 2 m. Conclusions: The majority of SCHF cases occurred at playgrounds with insufficient surface depth and/or non-compliant equipment. Upper body equipment, track rides and rotating play structures were of particular concern, as the children fell from heights exceeding the recommended standard, likely reflecting the degradation and compaction of the surfacing material over time.

3-D Ultrasound Imaging Reliability of Measuring Dysplasia Metrics in Infants.

Developmental dysplasia of the hip is a hip abnormality that ranges from mild acetabular dysplasia to irreducible femoral head dislocations. While 2-D B-mode ultrasound (US)-based dysplasia metrics or disease metrics are currently used clinically to diagnose developmental dysplasia of the hip, such estimates suffer from high inter-exam variability. In this work, we propose and evaluate 3-D US-derived dysplasia metrics that are automatically computed and demonstrate that these automatically derived dysplasia metrics are considerably more reproducible. The key features of our automatic method are (i) a random forest-based learning technique to remove regions across the coronal axis that do not contain bone structures necessary for dysplasia-metric extraction, thereby reducing outliers; (ii) a bone segmentation method that uses rotation-invariant and intensity-invariant filters, thus remaining robust to signal dropout and varying bone morphology; (iii) a novel slice-based learning and 3-D reconstruction strategy to estimate a probability map of the hypoechoic femoral head in the US volume; and (iv) formulae for calculating the 3-D US-derived dysplasia metrics. We validate our proposed method on real clinical data acquired from 40 infant hip examinations. Results show a considerable (around 70%) reduction in variability in two key 3-D US-derived dysplasia metrics compared with their 2-D counterparts.

Discovery of a Bromodomain and Extraterminal Inhibitor with a Low Predicted Human Dose through Synergistic Use of Encoded Library Technology and Fragment Screening.

The bromodomain and extraterminal (BET) family of bromodomain-containing proteins are important regulators of the epigenome through their ability to recognize N-acetyl lysine (KAc) post-translational modifications on histone tails. These interactions have been implicated in various disease states and, consequently, disruption of BET-KAc binding has emerged as an attractive therapeutic strategy with a number of small molecule inhibitors now under investigation in the clinic. However, until the utility of these advanced candidates is fully assessed by these trials, there remains scope for the discovery of inhibitors from new chemotypes with alternative physicochemical, pharmacokinetic, and pharmacodynamic profiles. Herein, we describe the discovery of a candidate-quality dimethylpyridone benzimidazole compound which originated from the hybridization of a dimethylphenol benzimidazole series, identified using encoded library technology, with an N-methyl pyridone series identified through fragment screening. Optimization via structure- and property-based design led to I-BET469, which possesses favorable oral pharmacokinetic properties, displays activity in vivo, and is projected to have a low human efficacious dose.

Discovery of Potent, Efficient, and Selective Inhibitors of Phosphoinositide 3-Kinase δ through a Deconstruction and Regrowth Approach.

A deconstruction of previously reported phosphoinositide 3-kinase δ (PI3Kδ) inhibitors and subsequent regrowth led to the identification of a privileged fragment for PI3Kδ, which was exploited to deliver a potent, efficient, and selective lead series with a novel binding mode observed in the PI3Kδ crystal structure.

Treating Tissue Factor-Positive Cancers with Antibody-Drug Conjugates That Do Not Affect Blood Clotting.

The primary function of tissue factor (TF) resides in the vasculature as a cofactor of blood clotting; however, multiple solid tumors aberrantly express this transmembrane receptor on the cell surface. Here, we developed anti-TF antibody-drug conjugates (ADC) that did not interfere with the coagulation cascade and benchmarked them against previously developed anti-TF ADCs. After screening an affinity-matured antibody panel of diverse paratopes and affinities, we identified one primary paratope family that did not inhibit conversion of Factor X (FX) to activated Factor X (FXa) and did not affect conversion of prothrombin to thrombin. The rest of the antibody panel and previously developed anti-TF antibodies were found to perturb coagulation to varying degrees. To compare the anticancer activity of coagulation-inert and -inhibitory antibodies as ADCs, a selection of antibodies was conjugated to the prototypic cytotoxic agent monomethyl auristatin E (MMAE) through a protease-cleavable linker. The coagulation-inert and -inhibitory anti-TF ADCs both killed cancer cells effectively. Importantly, the coagulation-inert ADCs were as efficacious as tisotumab vedotin, a clinical stage ADC that affected blood clotting, including in patient-derived xenografts from three solid tumor indications with a need for new therapeutic treatments-squamous cell carcinoma of the head and neck (SCCHN), ovarian, and gastric adenocarcinoma. Furthermore, a subset of the anti-TF antibodies could also be considered for the treatment of other diseases associated with upregulation of membranous TF expression, such as macular degeneration. Mol Cancer Ther; 17(11); 2412-26. ©2018 AACR.

Do Immediate Postoperative Radiographs Change Patient Management After Fracture Fixation? A Systematic Review.

OBJECTIVES: To evaluate whether immediate (0-3 days) postoperative radiography leads to alterations in the management of patients postfracture fixation. DATA SOURCES: Systematic review of English-language articles in the MEDLINE (1946-2016), EMBASE (1974-2016), CDSR (2005-2016), CENTRAL (1948-2016), and Google Scholar databases using Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. STUDY SELECTION: Randomized or non-randomized controlled trials and prospective or retrospective cohort studies that addressed surgical management of the upper extremity, lower extremity or hip fractures were eligible for review. All included studies needed to have performed radiography within 0-3 days of surgery and reported any directly resulting management changes. DATA EXTRACTION: Data were independently extracted by 2 reviewers using a standardized data collection form with predefined data fields for demographics, interventions, study methods, complications, and management outcomes. DATA SYNTHESIS: A random-effects model was applied, and pooled effects for absolute benefit increase (ABI) and number needed to treat (NNT) were calculated. CONCLUSIONS: Combining the 11/12 articles that reported by patient numbers, the ABI of immediate postoperative radiography for management change was 0.13% [95% confidence interval (CI), 0.00078%-0.60%; NNT = 753]. The ABI for identification of complications was 0.22% (95% CI, 0.0015%-1.24%; NNT = 453). Current literature suggests that immediate postoperative radiography does not lead to management change in most patients after fracture fixation. More comprehensive reporting, along with further prospective comparative research, is encouraged. LEVEL OF EVIDENCE: Diagnostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Computational and experimental analysis of short peptide motifs for enzyme inhibition.

The metabolism of living systems involves many enzymes that play key roles as catalysts and are essential to biological function. Searching ligands with the ability to modulate enzyme activities is central to diagnosis and therapeutics. Peptides represent a promising class of potential enzyme modulators due to the large chemical diversity, and well-established methods for library synthesis. Peptides and their derivatives are found to play critical roles in modulating enzymes and mediating cellular uptakes, which are increasingly valuable in therapeutics. We present a methodology that uses molecular dynamics (MD) and point-variant screening to identify short peptide motifs that are critical for inhibiting ß-galactosidase (ß-Gal). MD was used to simulate the conformations of peptides and to suggest short motifs that were most populated in simulated conformations. The function of the simulated motifs was further validated by the experimental point-variant screening as critical segments for inhibiting the enzyme. Based on the validated motifs, we eventually identified a 7-mer short peptide for inhibiting an enzyme with low µM IC50. The advantage of our methodology is the relatively simplified simulation that is informative enough to identify the critical sequence of a peptide inhibitor, with a precision comparable to truncation and alanine scanning experiments. Our combined experimental and computational approach does not rely on a detailed understanding of mechanistic and structural details. The MD simulation suggests the populated motifs that are consistent with the results of the experimental alanine and truncation scanning. This approach appears to be applicable to both natural and artificial peptides. With more discovered short motifs in the future, they could be exploited for modulating biocatalysis, and developing new medicine.

Relationships Between Severity of Deformity and Impingement in Slipped Capital Femoral Epiphysis.

BACKGROUND: In situ pinning, a low-risk treatment for slipped capital femoral epiphysis (SCFE), leaves the slipped femoral head in place and may reduce range of motion (ROM) and cause impingement. It is unclear when a more complex surgery should be considered, because the relationships between severity, slip stability, remodeling, impingement, and ROM are unknown. RESEARCH QUESTIONS: (1) Do more severe acute SCFE deformities (no bony remodeling) result in a greater loss of flexion ROM?(2) Does the presence or location of impingement on the pelvis vary with severity of acute SCFE deformity? METHODS: We developed a 3D geometric model of acute SCFE deformity from 1 computed tomography scan of a normal adolescent hip. Ethics board approval was obtained from our institution. Bone models were created from the segmented pelvis, epiphysis, and subphyseal femur.In total, 3721 SCFE deformities were simulated by combining posterior and inferior slips in the axial and coronal planes, respectively. Southwick angles were estimated from a frog-leg lateral projection. Deformities were divided into mild (0 to 30 degrees), moderate (30 to 60 degrees), and severe (≥60 degrees) Southwick groups. Each joint was flexed in combination with internal/external rotation until contact occurred. A total of 121 ROM trials, with different degrees of internal/external rotation (0 to 90 degrees at 1.5-degree steps) were performed for each deformity. RESULTS: In total, 3355 simulated SCFE deformities (363 could not be rotated out of impingement) were analyzed.Increasing slip severity reduced flexion ROM across the range of internal/external rotation. Contact occurred for most mild deformities, and for all moderate and severe deformities in at least 1 ROM trial. Impingement was observed mainly on the anterosuperior aspect of the acetabulum. CONCLUSIONS: Increasing slip severity in acute SCFE reduced flexion and increased incidence of impingement, primarily occurring on the anterosuperior aspect of the acetabulum. The impingement patterns observed are consistent with damaged cartilage locations seen in clinical literature. CLINICAL RELEVANCE: In this experimental model, moderate and severe acute slips in SCFE lead to reduced ROM and impingement with the acetabulum. This suggests that in situ pinning may result in impingement of moderate and severe acute SCFE slips.

Regulation of B-cell development and tolerance by different members of the miR-17∼92 family microRNAs.

The molecular mechanisms that regulate B-cell development and tolerance remain incompletely understood. In this study, we identify a critical role for the miR-17∼92 microRNA cluster in regulating B-cell central tolerance and demonstrate that these miRNAs control early B-cell development in a cell-intrinsic manner. While the cluster member miR-19 suppresses the expression of Pten and plays a key role in regulating B-cell tolerance, miR-17 controls early B-cell development through other molecular pathways. These findings demonstrate differential control of two closely linked B-cell developmental stages by different members of a single microRNA cluster through distinct molecular pathways.

Evolution of a Novel, Orally Bioavailable Series of PI3Kδ Inhibitors from an Inhaled Lead for the Treatment of Respiratory Disease.

A four-step process of high-quality modeling of existing data, deconstruction, identification of replacement cores, and an innovative synthetic regrowth strategy led to the rapid discovery of a novel oral series of PI3Kδ inhibitors with promising selectivity and excellent in vivo characteristics.

Robotic Left Ventricular Assist Device Implantation Using Left Thoracotomy Approach in Patients with Previous Sternotomies.

Left ventricular assist devices (LVADs) are commonly used as either a bridge-to-transplant or a destination therapy. The traditional approach for LVAD implantation is via median sternotomy, but many candidates for this procedure have a history of failed cardiac surgeries and previous sternotomy. Redo sternotomy increases the risk of heart surgery, particularly in the setting of advanced heart failure. Robotics facilitates a less invasive approach to LVAD implantation that circumvents some of the morbidity associated with a redo sternotomy. We compared the outcomes of all patients at our institution who underwent LVAD implantation via either a traditional sternotomy or using robotic assistance. The robotic cohort showed reduced resource utilization including length of hospital stay and use of blood products. As the appropriate candidates become elucidated, robotic assistance may improve the safety and cost-effectiveness of reoperative LVAD surgery.

A novel method for assessing postoperative femoral head reduction in developmental dysplasia of the hip.

PURPOSE: Developmental dysplasia of the hip (DDH) affects approximately 1 % of live births. Dislocated hips require reduction and stabilisation in a spica cast, and reduction efficacy is assessed radiologically. Numerous measurements are used to ascertain the adequacy of reduction but can be inconsistent in evaluating femoral head position. This study describes the morphology of the developing acetabulum in DDH and validates a novel method to assess adequate reduction of the dysplastic hip following closed or open reduction. METHODS: A retrospective review was performed of 66 consecutive patients undergoing reduction of hip dislocation over a 2-year period. Three independent reviewers evaluated postoperative CT scans to assess anterior-posterior (AP) displacement and modified Shenton's line. Acetabular morphology was also assessed along with hip congruency using a described novel 'posterior neck line'. RESULTS: Dislocated hips were successfully identified using the posterior neck line with a sensitivity of 0.71 and specificity of 0.88 giving a negative predictive value of 0.97. The interobserver reliability of this technique was higher in comparison against both (AP) displacement and modified Shenton's line. CONCLUSIONS: We have shown a novel approach in assessing the acetabular morphology of DDH and a novel technique to accurately confirm the reduction of dislocated hips following open or closed reduction.

Evidence-based management of developmental dysplasia of the hip.

Developmental Dysplasia of the Hip (DDH) refers to a spectrum of abnormalities involving the developing hip. These abnormalities range from mild instability to frank dislocation of the joint. It is important to treat the condition effectively in order to encourage the hip to develop normally and produce good long-term results. This article reviews the evidence related to the treatment of DDH. The quality of evidence for DDH management remains low, with little uniformity in terminology and most studies being retrospective in nature. Given this, it is not possible to recommend or reject most treatment modalities based on existing studies.

Robotic-assisted implantation of ventricular assist device after sternectomy and pectoralis muscle flap.

Left ventricular assist devices are increasingly important in the management of advanced heart failure. Most patients who benefit from these devices have had some prior cardiac surgery, making implantation of higher risk. This is especially true in patients who have had prior pectoralis flap reconstruction after sternectomy for mediastinitis. We outline the course of such a patient, in whom the use of robotic assistance allowed for a less invasive device implantation approach with preservation of the flap for transplantation.

Non-viral adeno-associated virus-based platform for stable expression of antibody combination therapeutics.

Antibody combination therapeutics (ACTs) are polyvalent biopharmaceuticals that are uniquely suited for the control of complex diseases, including antibiotic resistant infectious diseases, autoimmune disorders and cancers. However, ACTs also represent a distinct manufacturing challenge because the independent manufacture and subsequent mixing of monoclonal antibodies quickly becomes cost prohibitive as more complex mixtures are envisioned. We have developed a virus-free recombinant protein expression platform based on adeno-associated viral (AAV) elements that is capable of rapid and consistent production of complex antibody mixtures in a single batch format. Using both multiplexed immunoassays and cation exchange (CIEX) chromatography, cell culture supernatants generated using our system were assessed for stability of expression and ratios of the component antibodies over time. Cultures expressing combinations of three to ten antibodies maintained consistent expression levels and stable ratios of component antibodies for at least 60 days. Cultures showed remarkable reproducibility following cell banking, and AAV-based cultures showed higher stability and productivity than non-AAV based cultures. Therefore, this non-viral AAV-based expression platform represents a predictable, reproducible, quick and cost effective method to manufacture or quickly produce for preclinical testing recombinant antibody combination therapies and other recombinant protein mixtures.

PD-1 coinhibitory signals: the link between pathogenesis and protection.

In the majority of HIV-1 infected individuals, the adaptive immune response drives virus escape resulting in persistent viremia and a lack of immune-mediated control. The expression of negative regulatory molecules such as PD-1 during chronic HIV infection provides a useful marker to differentiate functional memory T cell subsets and the frequency of T cells with an exhausted phenotype. In addition, cell-based measurements of virus persistence equate with activation markers and the frequency of CD4 T cells expressing PD-1. High-level expression of PD-1 and its ligands PD-L1 and PD-L2 are found on hematopoietic and non-hematopoietic cells, and are upregulated by chronic antigen stimulation, Type 1 and Type II interferons (IFNs), and homeostatic cytokines. In HIV infected subjects, PD-1 levels on CD4 and CD8 T cells continue to remain high following combination anti-retroviral therapy (cART). System biology approaches have begun to elucidate signal transduction pathways regulated by PD-1 expression in CD4 and CD8 T cell subsets that become dysfunctional through chronic TCR activation and PD-1 signaling. In this review, we summarize our current understanding of transcriptional signatures and signal transduction pathways associated with immune exhaustion with a focus on recent work in our laboratory characterizing the role of PD-1 in T cell dysfunction and HIV pathogenesis. We also highlight the therapeutic potential of blocking PD-1-PD-L1 and other immune checkpoints for activating potent cellular immune responses against chronic viral infections and cancer.