Dietary restriction impacts health and lifespan of genetically diverse mice.

Caloric restriction extends healthy lifespan in multiple species1. Intermittent fasting, an alternative form of dietary restriction, is potentially more sustainable in humans, but its effectiveness remains largely unexplored2-8. Identifying the most efficacious forms of dietary restriction is key for developing interventions to improve human health and longevity9. Here we performed an extensive assessment of graded levels of caloric restriction (20% and 40%) and intermittent fasting (1 and 2 days fasting per week) on the health and survival of 960 genetically diverse female mice. We show that caloric restriction and intermittent fasting both resulted in lifespan extension in proportion to the degree of restriction. Lifespan was heritable and genetics had a larger influence on lifespan than dietary restriction. The strongest trait associations with lifespan included retention of body weight through periods of handling-an indicator of stress resilience, high lymphocyte proportion, low red blood cell distribution width and high adiposity in late life. Health effects differed between interventions and exhibited inconsistent relationships with lifespan extension. 40% caloric restriction had the strongest lifespan extension effect but led to a loss of lean mass and changes in the immune repertoire that could confer susceptibility to infections. Intermittent fasting did not extend the lifespan of mice with high pre-intervention body weight, and two-day intermittent fasting was associated with disruption of erythroid cell populations. Metabolic responses to dietary restriction, including reduced adiposity and lower fasting glucose, were not associated with increased lifespan, suggesting that dietary restriction does more than just counteract the negative effects of obesity. Our findings indicate that improving health and extending lifespan are not synonymous and raise questions about which end points are the most relevant for evaluating aging interventions in preclinical models and clinical trials.

Novel PE and APC tandems: Additional near-infrared fluorochromes for use in spectral flow cytometry.

Recent advances in flow cytometry instrumentation and fluorochrome chemistries have greatly increased fluorescent conjugated antibody combinations that can be used reliably and easily in routine experiments. The Cytek Aurora flow cytometer was first released with three excitation lasers (405, 488, and 640 nm) and incorporated the latest Avalanche Photodiode (APD) technology, demonstrating significant improvement in sensitivity for fluorescent emission signals longer than 800 nm. However, there are limited commercially available fluorochromes capable of excitation with peak emission signals beyond 800 nm. To address this gap, we engineered six new fluorochromes: PE-750, PE-800, PE-830 for the 488 nm laser and APC-750, APC-800, APC-830 for the 640 nm laser. Utilizing the principal of fluorescence resonance energy transfer (FRET), these novel structures were created by covalently linking a protein donor dye with an organic small molecule acceptor dye. Additionally, each of these fluorochrome conjugates were shown to be compatible with fixation/permeabilization buffer reagents, and demonstrated acceptable brightness and stability when conjugated to antigen-specific monoclonal antibodies. These six novel fluorochrome reagents can increase the numbers of fluorochromes that can be used on a spectral flow cytometer.

Perspectives on psychedelic treatment and research in eating disorders: a web-based questionnaire study of people with eating disorders.

Approximately 1.25 million people in the UK suffer from an eating disorder, yet the treatment options show limited efficacy, warranting the need for novel approaches. This study aimed to investigate the perspectives of people with eating disorders on the use of complementary therapies and psychedelic research and treatment. Two hundred participants with eating disorders took part in this web survey study. The majority of participants (70%) had used a complementary treatment to manage their eating disorder. Participants believed that psychedelic research was worthwhile in the context of a moderate level of concern. The most popular solutions to meet these concerns included providing education around psychedelics and their effects and use in psychiatry and experiencing endorsement from professionals in the area. Moreover, participant responses emphasized the need for a safe, monitored environment and the patient-therapist rapport in the context of psychedelic treatment. The findings are explored concerning future trials of psychedelics as a treatment for eating disorders.

Murinization and H Chain Isotype Matching of the Anti-GITR Antibody DTA-1 Reduces Immunogenicity-Mediated Anaphylaxis in C57BL/6 Mice.

Recent advances in immuno-oncology have shown that the immune system can be activated to induce long-term, durable antitumor responses. For immuno-oncology drug development, immune activation is often explored using rat Abs in immunocompetent mouse models. Although these models can be used to show efficacy, antidrug immune responses to experimental protein-based therapeutics can arise. Immunogenicity of surrogate Abs may therefore represent an important obstacle to the evaluation of the antitumor efficacy of immunomodulator Abs in syngeneic models. A recent publication has shown that anti-glucocorticoid-induced TNFR family-related protein agonistic Ab DTA-1 (rat or murinized IgG2a) can induce the development of anaphylaxis in C57BL/6 mice upon repeated i.p. dosing because of an anti-idiotypic anti-drug Ab immune response. This study was undertaken to address the impact of the immunogenicity derived from the Fc and variable domains. To this end, chimerized (rat V domains/mouse constant regions) and murinized (95% mouse sequence) DTA-1-based surrogate Abs with a murine IgG2c H chain isotype were created. Chimerization and murinization of DTA-1 did not affect receptor binding and glucocorticoid-induced TNFR family-related protein-induced T cell agonistic properties. Similar in vivo antitumor efficacy and intratumoral CD8+/regulatory T cells were also observed. Finally, treatment of C57BL/6 mice with the chimerized and murinized DTA-1 Abs on a C57BL/6-matched IgG2c isotype resulted in reduced development and severity of anaphylaxis as measured by decline of body temperature, behavioral effects, serum IL-4, IgE, and anti-drug Ab levels. These results suggest that careful murinization and selection of a strain-matched H chain isotype are critical to generate ideal surrogate Abs for testing immuno-oncology mechanisms in vivo.

Identifying predictors of adverse outcomes after termination of seclusion in psychiatric intensive care units.

BACKGROUND: Seclusion is a restrictive practice that many healthcare services are trying to reduce. Previous studies have sought to identify predictors of seclusion initiation, but few have investigated factors associated with adverse outcomes after seclusion termination. AIMS: To assess the factors that predict an adverse outcome within 24 h of seclusion termination. METHOD: In a cohort study of individuals secluded in psychiatric intensive care units, we investigated factors associated with any of the following outcomes: actual violence, attempted violence, or reinitiation of seclusion within 24 h of seclusion termination. Among the seclusion episodes that were initiated between 29 March 2018 and 4 March 2019, we investigated the exposures of medication cooperation, seclusion duration, termination out of working hours, involvement of medical staff in the final seclusion review, lack of insight, and agitation or irritability. In a mixed-effects logistic regression model, associations between each exposure and the outcome were calculated. Odds ratios were calculated unadjusted and adjusted for demographic and clinical variables. RESULTS: We identified 254 seclusion episodes from 122 individuals (40 female, 82 male), of which 106 (41.7%) had an adverse outcome within 24 h of seclusion termination. Agitation or irritability was associated with an adverse outcome, odds ratio 1.92 (95% CI 1.03 to 3.56, P = 0.04), but there was no statistically significant association with any of the other exposures, although confidence intervals were broad. CONCLUSIONS: Agitation or irritability in the hours preceding termination of seclusion may predict an adverse outcome. The study was not powered to detect other potentially clinically significant factors.

Origin of trisomy: no evidence to support the ovarian mosaicism theory.

OBJECTIVE: Trisomy is the most common type of chromosome abnormality, affecting 4% of clinically recognised pregnancies, of which, trisomies 16, 21 and 22 are the most prevalent. It has been suggested that a large proportion of maternally derived trisomic pregnancies, specifically trisomy 21, are the result of low-level ovarian mosaicism. In this study, we aimed to reproduce these previously published results on trisomy 21 and investigate the other common maternally derived trisomies (i.e. trisomies 16 and 22) by determining chromosome copy number in fetal ovarian and control skin cells. METHODS: Ovarian and control skin tissue was collected from eight karyotypically normal female fetuses of between 10 and 14 weeks gestation, which were terminated for social reasons. Tissues were dissociated and fluorescence in situ hybridisation was performed with break-apart probes: CBFß (16q22), RUNX1 (21q22) and EWSR1 (22q12). RESULTS: A small number of trisomic cells, 13 out of 51,146 cells examined (0.025%), were identified in both ovarian and control skin samples. Only three of these trisomic cells were present in the fetal ovarian tissue. CONCLUSION: This study found no evidence of fetal ovarian mosaicism for trisomies 16, 21 and 22.

Genomic abnormalities of TP53 define distinct risk groups of paediatric B-cell non-Hodgkin lymphoma.

Children with B-cell non-Hodgkin lymphoma (B-NHL) have an excellent chance of survival, however, current clinical risk stratification places as many as half of patients in a high-risk group receiving very intensive chemo-immunotherapy. TP53 alterations are associated with adverse outcome in many malignancies; however, whilst common in paediatric B-NHL, their utility as a risk classifier is unknown. We evaluated the clinical significance of TP53 abnormalities (mutations, deletion and/or copy number neutral loss of heterozygosity) in a large UK paediatric B-NHL cohort and determined their impact on survival. TP53 abnormalities were present in 54.7% of cases and were independently associated with a significantly inferior survival compared to those without a TP53 abnormality (PFS 70.0% vs 100%, p < 0.001, OS 78.0% vs 100%, p = 0.002). Moreover, amongst patients clinically defined as high-risk (stage III with high LDH or stage IV), those without a TP53 abnormality have superior survival compared to those with TP53 abnormalities (PFS 100% vs 55.6%, p = 0.005, OS 100% vs 66.7%, p = 0.019). Biallelic TP53 abnormalities were either maintained from the presentation or acquired at progression in all paired diagnosis/progression Burkitt lymphoma cases. TP53 abnormalities thus define clinical risk groups within paediatric B-NHL and offer a novel molecular risk stratifier, allowing more personalised treatment protocols.

An anti-PD-1-GITR-L bispecific agonist induces GITR clustering-mediated T cell activation for cancer immunotherapy.

Costimulatory receptors such as glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) play key roles in regulating the effector functions of T cells. In human clinical trials, however, GITR agonist antibodies have shown limited therapeutic effect, which may be due to suboptimal receptor clustering-mediated signaling. To overcome this potential limitation, a rational protein engineering approach is needed to optimize GITR agonist-based immunotherapies. Here we show a bispecific molecule consisting of an anti-PD-1 antibody fused with a multimeric GITR ligand (GITR-L) that induces PD-1-dependent and FcγR-independent GITR clustering, resulting in enhanced activation, proliferation and memory differentiation of primed antigen-specific GITR+PD-1+ T cells. The anti-PD-1-GITR-L bispecific is a PD-1-directed GITR-L construct that demonstrated dose-dependent, immunologically driven tumor growth inhibition in syngeneic, genetically engineered and xenograft humanized mouse tumor models, with a dose-dependent correlation between target saturation and Ki67 and TIGIT upregulation on memory T cells. Anti-PD-1-GITR-L thus represents a bispecific approach to directing GITR agonism for cancer immunotherapy.

Design, creation and in vitro testing of a reduced immunogenicity humanized anti-CD25 monoclonal antibody that retains functional activity.

Humanized and fully human sequence-derived therapeutic antibodies retain the capacity to induce anti-drug antibodies. Daclizumab (humanized version of the murine anti-Tac antibody; E.HAT) was selected for a proof of concept application of engineering approaches to reduce potential immunogenicity due to its demonstrated immunogenicity in the clinic. Reduced immunogenicity variants of E.HAT were created by identifying and modifying a CD4+ T cell epitope region in the VH region. Variant epitope region peptides were selected for their reduced capacity to induce CD4+ T cell proliferative responses in vitro. Variant antibody molecules were created, and CD25 affinity and potency were similar to the unmodified parent antibody. Fab fragments from the variant antibodies induced a lower frequency and magnitude of responses in human peripheral blood mononuclear cells proliferation tests. By the empirical selection of two amino acid mutations, fully functional humanized E.HAT antibodies with reduced potential to induce immune responses in vitro were created.

A beta-lactamase with reduced immunogenicity for the targeted delivery of chemotherapeutics using antibody-directed enzyme prodrug therapy.

Antibody-directed enzyme prodrug therapy (ADEPT) delivers chemotherapeutic agents in high concentration to tumor tissue while minimizing systemic drug exposure. beta-Lactamases are particularly useful enzymes for ADEPT systems due to their unique substrate specificity that allows the activation of a variety of lactam-based prodrugs with minimal interference from mammalian enzymes. We evaluated the amino acid sequence of beta-lactamase from Enterobacter cloacae for the presence of human T-cell epitopes using a cell-based proliferation assay using samples from 65 community donors. We observed a low background response that is consistent with a lack of preexposure to this enzyme. beta-Lactamase was found to contain four CD4+ T-cell epitopes. For two of these epitopes, we identified single amino acid changes that result in significantly reduced proliferative responses while retaining stability and activity of the enzyme. The beta-lactamase variant containing both changes induces significantly less proliferation in human and mouse cell assays, and 5-fold lower levels of IgG1 in mice were observed after repeat administration of beta-lactamase variant with adjuvant. The beta-lactamase variant should be very suitable for the construction of ADEPT fusion proteins, as it combines high activity toward lactam prodrugs, high plasma stability, a monomeric architecture, and a relatively low risk of eliciting an immune response in patients.

Enhanced immunogenicity of a functional enzyme by T cell epitope modification.

BACKGROUND: T helper epitopes are necessary for the induction of high titers of antigen-specific IgG antibodies. We are interested in the epitope modification of intact proteins as a method to enhance their immunogenicity for the generation of recombinant protein-based vaccines. RESULTS: Hartley strain guinea pig T cell epitopes were mapped for two related bacterial proteases. Two T cell epitopes were found in one of the proteases, while a comparatively reduced immunogenicity protease had no detectable T cell epitopes. A T cell epitope sequence homologous to the immunogenic protease was created in the less immunogenic protease by changing a single amino acid. Proliferative responses to the whole protein parent enzyme were two-fold higher in splenocyte cultures from variant-immunized animals. We found that the single amino acid change in the variant resulted in a protein immunogen that induced higher titers of antigen-specific IgG antibody at low doses and at early time points during the immunization protocol. The serum from parent- and variant-immunized guinea pigs cross-reacted at both the protein and the peptide level. Finally, animals primed to the variant but boosted with the parent enzyme had higher levels of antigen-specific IgG than animals immunized with the parent enzyme alone. CONCLUSIONS: With a single amino acid change we have introduced a T cell epitope into a comparatively low-immunogenic enzyme and have increased its immunogenicity while retaining the enzyme's original proteolytic function. The ability to immunomodulate proteins while leaving their function intact has important implication for the development of recombinant vaccines and protein-based therapeutics.

Human population-based identification of CD4(+) T-cell peptide epitope determinants.

A human cell-based method to identify functional CD4(+) T-cell epitopes in any protein has been developed. Proteins are tested as synthetic 15-mer peptides offset by three amino acids. Percent responses within a large donor population are tabulated for each peptide in the set. Peptide epitope regions are designated by difference in response frequency from the overall background response rate for the compiled dataset. Epitope peptide responses are reproducible, with a median coefficient of variance of 21% when tested on multiple random-donor sets. The overall average response rate within the dataset increases with increasing putative human population antigenic exposure to a given protein. The background rate was high for HPV16 E6, and was low for human-derived cytokine proteins. The assay identified recall epitope regions within the donor population for the protein staphylokinase. For an industrial protease with minimal presumed population exposure, immunodominant epitope peptides were identified that were found to bind promiscuously to many HLA class II molecules in vitro. The peptide epitope regions identified in presumably unexposed donors represent a subset of the total recall epitopes. Finally, as a negative control, the assay found no peptide epitope regions in human beta2-microglobulin. This method identifies functional CD4(+) T-cell epitopes in any protein without pre-selection for HLA class II, suggests whether a donor population is pre-exposed to a protein of interest, and does not require sensitized donors for in vitro testing.

A human dendritic cell-based method to identify CD4+ T-cell epitopes in potential protein allergens.

We developed an assay to determine the location of immunodominant CD4(+) T-cell epitopes in any protein. The method uses CD4(+) T cells from community donors in conjunction with dendritic cells derived in vitro. Synthetic peptides constructed to describe the sequence of the protein of interest are cocultured with dendritic cells and CD4(+) T cells, and T-cell proliferation is measured. Data are compiled over a large replicate of human donors to pinpoint immunodominant, usually promiscuous epitope regions. We have applied this technique to a known food allergen, the Brazil nut 2S storage globulin protein, and to two potential food allergens, the Cry1Ab and Cry3Aa proteins. We show epitope data for these three proteins. This assay can be used as a tool to guide the selection and qualification of future potential food transgenes.

An in vitro human cell-based assay to rank the relative immunogenicity of proteins.

A method to rank proteins based on their relative immunogenicity has been devised. A statistical analysis of peptide-specific responses in large human donor pools provides a structure index value metric that ranked four industrial enzymes in the order determined by both mouse and guinea pig exposure models. The ranking method also compared favorably with human sensitization rates measured in occupationally exposed workers. Structure index values for other proteins known to cause immune responses in humans were also determined and found to be higher than the value determined for human beta2-microglobulin. Using values from known immunogenic and putative nonimmunogenic proteins, a cut-off value was established. The structure index value calculation provides a comparative method to predict subsequent immunogenicity on a human population basis without the need to use animal models. Information provided by this assay can be used in the early development of protein therapies and other protein-based applications to select or create reduced immunogenicity variants.

The immunogenicity of humanized and fully human antibodies: residual immunogenicity resides in the CDR regions.

Monoclonal antibodies represent an attractive therapeutic tool as they are highly specific for their targets, convey effector functions and enjoy robust manufacturing procedures. Humanization of murine monoclonal antibodies has vastly improved their in vivo tolerability. Humanization, the replacement of mouse constant regions and V framework regions for human sequences, results in a significantly less immunogenic product. However, some humanized and even fully human sequence-derived antibody molecules still carry immunological risk. To more fully understand the immunologic potential of humanized and human antibodies, we analyzed CD4(+) helper T cell epitopes in a set of eight humanized antibodies. The antibodies studied represented a number of different VH and VL family members carrying unique CDR regions. In spite of these differences, CD4(+) T cell epitopes were found only in CDR-sequence containing regions. We were able to incorporate up to two amino acid modifications in a single epitope that reduced the immunogenic potential while retaining full biologic function. We propose that immunogenicity will always be present in some antibody molecules due to the nature of the antigen-specific combining sites. A consequence of this result is modifications to reduce immunogenicity will be centered on the affinity-determining regions. Modifications to CDR regions can be designed that reduce the immunogenic potential while maintaining the bioactivity of the antibody molecule.

Elimination of an immunodominant CD4+ T cell epitope in human IFN-beta does not result in an in vivo response directed at the subdominant epitope.

The BALB/cByJ mouse strain displays an immunodominant T cell response directed at the same CD4(+) T cell epitope peptide region in human IFN-beta, as detected in a human population-based assay. BALB/cByJ mice also recognize a second region of the protein with a lesser magnitude proliferative response. Critical residue testing of the immunodominant peptide showed that both BALB/cByJ mice and the human population response were dependent on an isoleucine residue at position 129. A variant IFN-beta molecule was constructed containing the single amino acid modification, I129V, in the immunodominant epitope. The variant displayed 100% of control antiproliferation activity. Mice immunized with unmodified IFN-beta responded weakly in vitro to the I129V variant. However, BALB/cByJ mice immunized with the I129V variant were unable to respond to either the I129V variant or the unmodified IFN-beta molecule by either T cell proliferation or Ag-specific IgG1 Ab production. This demonstrates that a single amino acid change in an immunodominant epitope can eliminate an immune response to an otherwise intact therapeutic protein. The elimination of the immunodominant epitope response also eliminated the response to the subdominant epitope in the protein. Modifying functionally immunodominant T cell epitopes within proteins may obviate the need for additional subdominant epitope modifications.

Characterization of HLA DR3/DQ2 transgenic mice: a potential humanized animal model for autoimmune disease studies.

Linkage studies indicate close associations of certain HLA alleles with autoimmune diseases. To better understand how specific HLA alleles are related to disease pathogenesis, we have generated an HLA DR3/DQ2 transgenic mouse utilizing a 550-kb yeast artificial chromosome (YAC) construct containing the complete DRalpha, DRbeta1, DRbeta3, DQalpha, and DQbeta regions. The transgenic mouse (4D1/C2D) in an I-Abeta(o) background appears healthy with no signs of autoimmune diseases. Lymphoid tissues as well as CD4(+) T cells develop normally. Characterization of the transgene expression demonstrates that approximately 90% of B cells express high levels of DR3 and 50-70% of B cells express DQ2. CD11c(+) dendritic cells express high levels of DR and DQ. Approximately 12-18% of resting T cells are positive for DR expression, and further up-regulation to 40-50% expression is seen upon activation with anti-CD3/anti-CD28 mAb. These results suggest that the transgenic construct confers a high fidelity to the normal human temporal and spatial expression profile. Analysis of T cell receptor repertoire in transgenic mice confirms that DR3/DQ2 are able to mediate thymic selection. Furthermore, transgenic mice respond to a DR3-restricted antigen, demonstrating antigen processing and presentation by antigen-presenting cells (APC). Purified T cells from ovalbumin (OVA)-immunized 4D1 mice respond to human APC co-cultured with OVA, suggesting appropriate antigen/DR3 or DQ2 recognition by murine T cells. Immunoglobulin isotype switching is also observed, indicating functional T-B cognate interactions. Thus, the DR3/DQ2 transgenic mouse has normal lymphoid development and functionality that are mediated by HLA transgenes and can be used to investigate HLA-associated immunological questions.