Structural and physical features that distinguish tumor-controlling from inactive cancer neoepitopes.

Neoepitopes arising from amino acid substitutions due to single nucleotide polymorphisms are targets of T cell immune responses to cancer and are of significant interest in the development of cancer vaccines. However, understanding the characteristics of rare protective neoepitopes that truly control tumor growth has been a challenge, due to their scarcity as well as the challenge of verifying true, neoepitope-dependent tumor control in humans. Taking advantage of recent work in mouse models that circumvented these challenges, here, we compared the structural and physical properties of neoepitopes that range from fully protective to immunologically inactive. As neoepitopes are derived from self-peptides that can induce immune tolerance, we studied not only how the various neoepitopes differ from each other but also from their wild-type counterparts. We identified multiple features associated with protection, including features that describe how neoepitopes differ from self as well as features associated with recognition by diverse T cell receptor repertoires. We demonstrate both the promise and limitations of neoepitope structural analysis and predictive modeling and illustrate important aspects that can be incorporated into neoepitope prediction pipelines.

Structural dissimilarity from self drives neoepitope escape from immune tolerance.

T-cell recognition of peptides incorporating nonsynonymous mutations, or neoepitopes, is a cornerstone of tumor immunity and forms the basis of new immunotherapy approaches including personalized cancer vaccines. Yet as they are derived from self-peptides, the means through which immunogenic neoepitopes overcome immune self-tolerance are often unclear. Here we show that a point mutation in a non-major histocompatibility complex anchor position induces structural and dynamic changes in an immunologically active ovarian cancer neoepitope. The changes pre-organize the peptide into a conformation optimal for recognition by a neoepitope-specific T-cell receptor, allowing the receptor to bind the neoepitope with high affinity and deliver potent T-cell signals. Our results emphasize the importance of structural and physical changes relative to self in neoepitope immunogenicity. Considered broadly, these findings can help explain some of the difficulties in identifying immunogenic neoepitopes from sequence alone and provide guidance for developing novel, neoepitope-based personalized therapies.

Improving T Cell Receptor On-Target Specificity via Structure-Guided Design.

T cell receptors (TCRs) have emerged as a new class of immunological therapeutics. However, though antigen specificity is a hallmark of adaptive immunity, TCRs themselves do not possess the high specificity of monoclonal antibodies. Although a necessary function of T cell biology, the resulting cross-reactivity presents a significant challenge for TCR-based therapeutic development, as it creates the potential for off-target recognition and immune toxicity. Efforts to enhance TCR specificity by mimicking the antibody maturation process and enhancing affinity can inadvertently exacerbate TCR cross-reactivity. Here we demonstrate this concern by showing that even peptide-targeted mutations in the TCR can introduce new reactivities against peptides that bear similarity to the original target. To counteract this, we explored a novel structure-guided approach for enhancing TCR specificity independent of affinity. Tested with the MART-1-specific TCR DMF5, our approach had a small but discernible impact on cross-reactivity toward MART-1 homologs yet was able to eliminate DMF5 cross-recognition of more divergent, unrelated epitopes. Our study provides a proof of principle for the use of advanced structure-guided design techniques for improving TCR specificity, and it suggests new ways forward for enhancing TCRs for therapeutic use.

Accurate modeling of peptide-MHC structures with AlphaFold.

Major histocompatibility complex (MHC) proteins present peptides on the cell surface for T cell surveillance. Reliable in silico prediction of which peptides would be presented and which T cell receptors would recognize them is an important problem in structural immunology. Here, we introduce an AlphaFold-based pipeline for predicting the three-dimensional structures of peptide-MHC complexes for class I and class II MHC molecules. Our method demonstrates high accuracy, outperforming existing tools in class I modeling accuracy and class II peptide register prediction. We validate its performance and utility with new experimental data on a recently described cancer neoantigen/wild-type peptide pair and explore applications toward improving peptide-MHC binding prediction.

Physicochemical Heuristics for Identifying High Fidelity, Near-Native Structural Models of Peptide/MHC Complexes.

There is long-standing interest in accurately modeling the structural features of peptides bound and presented by class I MHC proteins. This interest has grown with the advent of rapid genome sequencing and the prospect of personalized, peptide-based cancer vaccines, as well as the development of molecular and cellular therapeutics based on T cell receptor recognition of peptide-MHC. However, while the speed and accessibility of peptide-MHC modeling has improved substantially over the years, improvements in accuracy have been modest. Accuracy is crucial in peptide-MHC modeling, as T cell receptors are highly sensitive to peptide conformation and capturing fine details is therefore necessary for useful models. Studying nonameric peptides presented by the common class I MHC protein HLA-A*02:01, here we addressed a key question common to modern modeling efforts: from a set of models (or decoys) generated through conformational sampling, which is best? We found that the common strategy of decoy selection by lowest energy can lead to substantial errors in predicted structures. We therefore adopted a data-driven approach and trained functions capable of predicting near native decoys with exceptionally high accuracy. Although our implementation is limited to nonamer/HLA-A*02:01 complexes, our results serve as an important proof of concept from which improvements can be made and, given the significance of HLA-A*02:01 and its preference for nonameric peptides, should have immediate utility in select immunotherapeutic and other efforts for which structural information would be advantageous.

A previously unappreciated polymorphism in the beta chain of I-As expressed in autoimmunity-prone SJL mice: Combined impact on antibody, CD4 T cell recognition and MHC class II dimer structural stability.

In the process of structure-function studies on the MHC class II molecule expressed in autoimmunity prone SJL mice, I-As, we discovered a disparity from the reported sequence of the MHC class II beta chain. The variant is localized at a highly conserved site of the beta chain, at residue 58. Our studies revealed that this single amino acid substitution of Pro for Ala at this residue, found in I-As, changes the structure of the MHC class II molecule, as evidenced by a loss of recognition by two monoclonal antibodies, and elements of MHC class II conformational stability identified through molecular dynamics simulation. Two other rare polymorphisms in I-As involved in hydrogen bonding potential between the alpha chain and the peptide main chain are located at the same end of the MHC class II binding pocket, studied in parallel may impact the consequences of the ß chain variant. Despite striking changes in MHC class II structure, CD4 T cell recognition of influenza-derived peptides was preserved. These disparate findings were reconciled by discovering, through monoclonal antibody blocking approaches, that CD4 T cell recognition by I-As restricted CD4 T cells focused more on the region of MHC class II at the peptide's amino terminus. These studies argue that the conformational variability or flexibility of the MHC class II molecule in that region of I-As select a CD4 T cell repertoire that deviates from the prototypical docking mode onto MHC class II peptide complexes. Overall, our results are consistent with the view that naturally occurring MHC class II molecules can possess polymorphisms that destabilize prototypical features of the MHC class II molecule but that can maintain T cell recognition of the MHC class II:peptide ligand via alternate docking modes.

A class-mismatched TCR bypasses MHC restriction via an unorthodox but fully functional binding geometry.

MHC restriction, which describes the binding of TCRs from CD4+ T cells to class II MHC proteins and TCRs from CD8+ T cells to class I MHC proteins, is a hallmark of immunology. Seemingly rare TCRs that break this paradigm exist, but mechanistic insight into their behavior is lacking. TIL1383I is a prototypical class-mismatched TCR, cloned from a CD4+ T cell but recognizing the tyrosinase tumor antigen presented by the class I MHC HLA-A2 in a fully functional manner. Here we find that TIL1383I binds this class I target with a highly atypical geometry. Despite unorthodox binding, TCR signaling, antigen specificity, and the ability to use CD8 are maintained. Structurally, a key feature of TIL1383I is an exceptionally long CDR3ß loop that mediates functions that are traditionally performed separately by hypervariable and germline loops in canonical TCR structures. Our findings thus expand the range of known TCR binding geometries compatible with normal function and specificity, provide insight into the determinants of MHC restriction, and may help guide TCR selection and engineering for immunotherapy.

Reversion analysis reveals the in vivo immunogenicity of a poorly MHC I-binding cancer neoepitope.

High-affinity MHC I-peptide interactions are considered essential for immunogenicity. However, some neo-epitopes with low affinity for MHC I have been reported to elicit CD8 T cell dependent tumor rejection in immunization-challenge studies. Here we show in a mouse model that a neo-epitope that poorly binds to MHC I is able to enhance the immunogenicity of a tumor in the absence of immunization. Fibrosarcoma cells with a naturally occurring mutation are edited to their wild type counterpart; the mutation is then re-introduced in order to obtain a cell line that is genetically identical to the wild type except for the neo-epitope-encoding mutation. Upon transplantation into syngeneic mice, all three cell lines form tumors that are infiltrated with activated T cells. However, lymphocytes from the two tumors that harbor the mutation show significantly stronger transcriptional signatures of cytotoxicity and TCR engagement, and induce greater breadth of TCR reactivity than those of the wild type tumors. Structural modeling of the neo-epitope peptide/MHC I pairs suggests increased hydrophobicity of the neo-epitope surface, consistent with higher TCR reactivity. These results confirm the in vivo immunogenicity of low affinity or 'non-binding' epitopes that do not follow the canonical concept of MHC I-peptide recognition.

Structure Based Prediction of Neoantigen Immunogenicity.

The development of immunological therapies that incorporate peptide antigens presented to T cells by MHC proteins is a long sought-after goal, particularly for cancer, where mutated neoantigens are being explored as personalized cancer vaccines. Although neoantigens can be identified through sequencing, bioinformatics and mass spectrometry, identifying those which are immunogenic and able to promote tumor rejection remains a significant challenge. Here we examined the potential of high-resolution structural modeling followed by energetic scoring of structural features for predicting neoantigen immunogenicity. After developing a strategy to rapidly and accurately model nonameric peptides bound to the common class I MHC protein HLA-A2, we trained a neural network on structural features that influence T cell receptor (TCR) and peptide binding energies. The resulting structurally-parameterized neural network outperformed methods that do not incorporate explicit structural or energetic properties in predicting CD8+ T cell responses of HLA-A2 presented nonameric peptides, while also providing insight into the underlying structural and biophysical mechanisms governing immunogenicity. Our proof-of-concept study demonstrates the potential for structure-based immunogenicity predictions in the development of personalized peptide-based vaccines.

Potentiating and inhibitory effects of periodate-oxidized ATP analogs on contractions of vas deferens to ATP.

Previous studies have shown that treatment of guinea-pig isolated vas deferens with the affinity label periodate-oxidized ATP (2',3'-dialdehyde ATP), results in two irreversible effects on biphasic contractile responses to ATP, i.e., potentiation of the P2X purinoceptor-mediated first phase and inhibition of the ecto-kinase-mediated second phase. The present experiments were designed to evaluate whether periodate-oxidized ADP, periodate-oxidized AMP, and periodate-oxidized adenosine, produce similar effects. Periodate-oxidized ATP and periodate-oxidized ADP (10(-2) M) elicited contraction of the vas deferens (periodate-oxidized ATP > periodate-oxidized ADP; periodate-oxidized AMP and periodate-oxidized adenosine had no agonist activity. After incubation of the preparations for 5 min with 10(-2) M periodate-oxidized ATP, periodate-oxidized ADP, periodate-oxidized AMP or periodate-oxidized adenosine, the first phase of contraction to submaximal ATP concentrations was potentiated. Simultaneously, periodate-oxidized ATP, periodate-oxidized ADP and periodate-oxidized AMP inhibited the second contractile phase, whereas periodate-oxidized adenosine did not. The results indicate that the requirement for 5'-phosphate to produce potentiation and inhibition is different: 5'-phosphate is not needed to potentiate the first phase of contraction to ATP, but at least one 5'-phosphate is required to inhibit the second phase of contraction.

Effect of parity on pituitary prolactin response to metoclopramide and domperidone: implications for the enhancement of lactation.

OBJECTIVE: The gastrointestinal motility agents metoclopramide and domperidone are known to increase pituitary prolactin (PRL) secretion and breast milk production. This study compared the effect of single doses of two strengths of metoclopramide and a single dose of domperidone on PRL secretion. METHODS: Ten nonpregnant women had baseline evaluation of serum PRL concentrations. The PRL concentrations were then determined after random oral administration of metoclopramide 10 mg, metoclopramide 5 mg, and domperidone 10 mg. Blood samples were drawn in the first 7 days of the menstrual cycle, at 13 time points over a 6-hour period (0, 15, 30, 45, 60, 75, 90, 120, 150, 180, 240, 300, and 360 minutes), with the zero time point beginning at 0800 hours. Variables such as weight, height, age, gravidity, parity, and oral contraceptive use were recorded. RESULTS: Baseline PRL concentrations showed the natural circadian rhythm. Metoclopramide and domperidone both caused a significant increase in PRL. However, PRL secretion was most influenced by parity. Nulliparous women had the quickest and highest PRL secretion with metoclopramide 10 mg, compared with the PRL response with metoclopramide 5 mg and domperidone 10 mg. Conversely, multiparous women had PRL secretion patterns that were equivalent between the medications. CONCLUSIONS: The PRL response to the medications was most influenced by parity. Therefore, we suggest that the medication therapy of choice for enhancing lactation may not be the same in all women, but may instead be determined by parity.

An evaluation of the characteristics and performance of neonatal phototherapy equipment.

This paper reviews the current state of knowledge and practice in neonatal phototherapy, and assesses methods of evaluating the characteristics and performance of different equipment. Artificial lighting (usually fluorescent) has been used for the past 30 years in the treatment of neonatal jaundice. Widely differing light outputs and spectra are used, making comparison and evaluation difficult for clinicians. Manufacturers of neonatal phototherapy equipment have no standard for assessing the performance of their equipment, and information that is supplied is at best confusing or deceptive to the users. Best practice is usually based on empirical data from equipment in use, but there is wide agreement that present phototherapy is sub-optimal, i.e. does not achieve maximum rate of bilirubin clearance for minimum therapeutic dose. Several studies in the last ten years have emphasized the importance of both the wavelength and intensity of light for optimal phototherapy. These are discussed and a technique is proposed for normalizing the output of different systems to make comparison easier and to enable optimal treatments to be designed.

Two miniature monitors for long-term ambulatory studies.

Two very low-power monitors are described. One typically limb-worn to record the level of physical activity of a person; the other, used with surface electrodes, to record three parameters of an EMG signal. The monitoring period can extend to days with a very accurate 'time of event' log. A novel method has been devised to double the resolution of the data gathered. A PC is used to store and process the collected data, which can be displayed or printed as one-, or four-channel, time-related histogram. The gathered data can also be analysed for trends and other statistical analysis. The hardware is relatively simple and made from low-cost components. Neither monitor uses a microprocessor for data gathering. The system is very easy to use requiring minimal operator training. Where 'no-frills' ambulatory monitoring is called for, the systems presented would compare very favourably with other commercially available monitors, either on cost, complexity of setting up, or on weight.

Stray RF field strength during radiofrequency endometrial ablation.

A study has been carried out of the RF field strength around a total of 17 patients undergoing radio-frequency endometrial ablation in three hospital centres. The mean equivalent power density at the position of the surgeon exceeded 10 W m-2, which is the derived investigation level for exposure to electromagnetic fields. This value is derived from a basic restriction in the average specific absorption rate in the body to 0.4 W kg-1. This limit applies to general population as well as occupational exposure and is well below that which has been shown to be hazardous to health. Also, the working conditions did not place physical stress on the surgeon. The maximum value of the specific energy absorption rate in the head or trunk should not exceed 10 W kg-1. Measured values on the field strength suggest that this organ dose limit was not exceeded. It is concluded that staff exposure to stray RF radiation during radio-frequency endometrial ablation is not considered to be hazardous to health.

An adolescent services plan to incorporate status offenders into the child welfare system.

A model plan is presented for adolescent services that would facilitate the current trend to shift troubled but nondelinquent youngsters from the juvenile justice system into the child welfare system.

Assessment of child sexual abuse: eighteen months' experience at the Child Protection Center.

One hundred fifty-seven cases of alleged sexual abuse were assessed. Analysis of these data with respect to age, sex, relationship to assailant, and type of assault corroborate other published data. A 9.5% incidence of gonorrhea and 1.4% pregnancy rate were detected. Referrals were usually made through a parent. Concomitant social work and police investigation are necessary, especially in complex cases. Initial screening enables the scheduling of appointments for medical assessment in many cases.

Product liability aspects of bioengineering.

The introduction of the Consumer Protection Act 1987 brought the problems of product liability directly into hospital bioengineering and medical physics departments. This law makes great demands upon manufacturers, but also on suppliers and maintainers of medical equipment. Departments which manage medical equipment carry a responsibility for ensuring the requirements of product liability are known by all those whose actions may affect the potential of a piece of equipment to injure or damage a patient. This paper addresses these problems from a bioengineering, rather than a legal, viewpoint. The problems of design, manufacture, modification, maintenance and use are discussed and examples of potential problems are identified. The paper concludes that as long as good standards of quality control and professionalism are applied in bioengineering and medical physics departments then there is little to be feared from this piece of legislation.

A microprocessor based instrument for the spectral analysis of the EEG in anaesthesia.

An instrument for monitoring the frequency components contained in the electroencephalogram is described. The technique uses the fast Fourier transform which is implemented in a microprocessor controlled 'black box'. The control and display of the data are organised by a conventional microcomputer. The system is designed for use by practising anaesthetists in a district general hospital environment and is, therefore, inexpensive and simple to operate. The operation of the equipment is described and some limited results from early clinical trials are presented.