PLD3 and PLD4 are single-stranded acid exonucleases that regulate endosomal nucleic-acid sensing.

The sensing of microbial genetic material by leukocytes often elicits beneficial pro-inflammatory cytokines, but dysregulated responses can cause severe pathogenesis. Genome-wide association studies have linked the gene encoding phospholipase D3 (PLD3) to Alzheimer's disease and have linked PLD4 to rheumatoid arthritis and systemic sclerosis. PLD3 and PLD4 are endolysosomal proteins whose functions are obscure. Here, PLD4-deficient mice were found to have an inflammatory disease, marked by elevated levels of interferon-γ (IFN-γ) and splenomegaly. These phenotypes were traced to altered responsiveness of PLD4-deficient dendritic cells to ligands of the single-stranded DNA sensor TLR9. Macrophages from PLD3-deficient mice also had exaggerated TLR9 responses. Although PLD4 and PLD3 were presumed to be phospholipases, we found that they are 5' exonucleases, probably identical to spleen phosphodiesterase, that break down TLR9 ligands. Mice deficient in both PLD3 and PLD4 developed lethal liver inflammation in early life, which indicates that both enzymes are needed to regulate inflammatory cytokine responses via the degradation of nucleic acids.

Broadly neutralizing antibodies targeting HIV: Progress and challenges.

Anti-HIV broadly neutralizing antibodies (bNAbs) offer a novel approach to treating, preventing, or curing HIV. Pre-clinical models and clinical trials involving the passive transfer of bNAbs have demonstrated that they can control viremia and potentially serve as alternatives or complement antiretroviral therapy (ART). However, antibody decay, persistent latent reservoirs, and resistance impede bNAb treatment. This review discusses recent advancements and obstacles in applying bNAbs and proposes strategies to enhance their therapeutic potential. These strategies include multi-epitope targeting, antibody half-life extension, combining with current and newer antiretrovirals, and sustained antibody secretion.

Antiretrovirals to CCR5 CRISPR/Cas9 gene editing - A paradigm shift chasing an HIV cure.

The evolution of drug-resistant viral strains and anatomical and cellular reservoirs of HIV pose significant clinical challenges to antiretroviral therapy. CCR5 is a coreceptor critical for HIV host cell fusion, and a homozygous 32-bp gene deletion (∆32) leads to its loss of function. Interestingly, an allogeneic HSCT from an HIV-negative ∆32 donor to an HIV-1-infected recipient demonstrated a curative approach by rendering the recipient's blood cells resistant to viral entry. Ex vivo gene editing tools, such as CRISPR/Cas9, hold tremendous promise in generating allogeneic HSC grafts that can potentially replace allogeneic ∆32 HSCTs. Here, we review antiretroviral therapeutic challenges, clinical successes, and failures of allogeneic and allogeneic ∆32 HSCTs, and newer exciting developments within CCR5 editing using CRISPR/Cas9 in the search to cure HIV.

Automated Identification of Adults at Risk for In-Hospital Clinical Deterioration.

BACKGROUND: Hospitalized adults whose condition deteriorates while they are in wards (outside the intensive care unit [ICU]) have considerable morbidity and mortality. Early identification of patients at risk for clinical deterioration has relied on manually calculated scores. Outcomes after an automated detection of impending clinical deterioration have not been widely reported. METHODS: On the basis of a validated model that uses information from electronic medical records to identify hospitalized patients at high risk for clinical deterioration (which permits automated, real-time risk-score calculation), we developed an intervention program involving remote monitoring by nurses who reviewed records of patients who had been identified as being at high risk; results of this monitoring were then communicated to rapid-response teams at hospitals. We compared outcomes (including the primary outcome, mortality within 30 days after an alert) among hospitalized patients (excluding those in the ICU) whose condition reached the alert threshold at hospitals where the system was operational (intervention sites, where alerts led to a clinical response) with outcomes among patients at hospitals where the system had not yet been deployed (comparison sites, where a patient's condition would have triggered a clinical response after an alert had the system been operational). Multivariate analyses adjusted for demographic characteristics, severity of illness, and burden of coexisting conditions. RESULTS: The program was deployed in a staggered fashion at 19 hospitals between August 1, 2016, and February 28, 2019. We identified 548,838 non-ICU hospitalizations involving 326,816 patients. A total of 43,949 hospitalizations (involving 35,669 patients) involved a patient whose condition reached the alert threshold; 15,487 hospitalizations were included in the intervention cohort, and 28,462 hospitalizations in the comparison cohort. Mortality within 30 days after an alert was lower in the intervention cohort than in the comparison cohort (adjusted relative risk, 0.84, 95% confidence interval, 0.78 to 0.90; P<0.001). CONCLUSIONS: The use of an automated predictive model to identify high-risk patients for whom interventions by rapid-response teams could be implemented was associated with decreased mortality. (Funded by the Gordon and Betty Moore Foundation and others.).

Adults hospitalized with breakthrough COVID-19 have lower mortality than matched unvaccinated adults.

BACKGROUND: Coronavirus disease 2019 (COVID-19) breakthrough infections are common. OBJECTIVE: Evaluate in-hospital mortality of patients with COVID-19 by vaccination status using retrospective cohort study. METHODS: We generated propensity scores for receipt of full vaccination in adults requiring supplemental oxygen hospitalized at Kaiser Permanente Northern California (1 April 2021 to 30 November 2021) with positive severe acute respiratory syndrome coronavirus 2 polymerase chain reaction tests. Optimal matching of fully vaccinated/unvaccinated patients was performed comparing in-hospital mortality. RESULTS: Of 7305 patients, 1463 (20.0%) were full, 138 (1.9%) were partial, and 5704 (78.1%) were unvaccinated. Fully vaccinated were older than partial or unvaccinated (71.0, 63.0, and 54.0 years, respectively, p < 0.001) with more comorbidities (Comorbidity Point Scores 33.0, 22.0, and 10.0, p < 0.001) and immunosuppressant (11.5%, 8.7%, and 3.0%, p < 0.001) or chemotherapy exposure (2.8%, 0.7%, and 0.4%, p < 0.001). Fewer fully vaccinated patients died compared to matched unvaccinated (9.0% vs. 16.3%, p < 0.0001). CONCLUSION: Fully vaccinated patients are less likely to die compared to matched unvaccinated patients.

An Slfn2 mutation causes lymphoid and myeloid immunodeficiency due to loss of immune cell quiescence.

Here we describe a previously unknown form of inherited immunodeficiency revealed by an N-ethyl-N-nitrosourea-induced mutation called elektra. Mice homozygous for this mutation showed enhanced susceptibility to bacterial and viral infection and diminished numbers of T cells and inflammatory monocytes that failed to proliferate after infection and died via the intrinsic apoptotic pathway in response to diverse proliferative stimuli. They also had a greater proportion of T cells poised to replicate DNA, and their T cells expressed a subset of activation markers, suggestive of a semi-activated state. We positionally ascribe the elektra phenotype to a mutation in the gene encoding Schlafen-2 (Slfn2). Our findings identify a physiological role for Slfn2 in the defense against pathogens through the regulation of quiescence in T cells and monocytes.

Model Based Design of a Low Cost and Compliant Low Profile Prosthetic Foot.

This paper describes the design of a simple and low-cost compliant low-profile prosthetic foot based on a cantilevered beam of uniform strength. The prosthetic foot is developed such that the maximum stress experienced by the beam is distributed approximately evenly across the length of the beam. Due to this stress distribution, the prosthetic foot exhibits compliant behavior not achievable through standard design approaches (e.g., designs based on simple cantilevered beams). Additionally, due to its simplicity and use of flat structural members, the foot can be manufactured at low cost. An analytical model of the compliant behavior of the beam is developed that facilitates rapid design changes to vary foot size and stiffness. A characteristic prototype was designed and constructed to be used in both a benchtop quasi-static loading test as well as a dynamic walking test for validation. The model predicted the rotational stiffness of the prototype with 5% error. Furthermore, the prototype foot was tested alongside two commercially available prosthetic feet (a low profile foot and an energy storage and release foot) in level walking experiments with a single study participant. The prototype foot displayed the lowest stiffness of the three feet (6.0, 7.1, and 10.4 Nm/deg for the prototype foot, the commercial low profile foot, and the energy storage and release foot, respectively). This foot design approach and accompanying model may allow for compliant feet to be developed for individuals with long residual limbs.

Assessing the clinical impact of CYP2C9 pharmacogenetic variation on phenytoin prescribing practice and patient response in an integrated health system.

OBJECTIVE: To assess the impact of CYP2C9 variation on phenytoin patient response and clinician prescribing practice where genotype was unknown during treatment. METHODS: A retrospective analysis of Resource on Genetic Epidemiology Research on Adult Health and Aging cohort participants who filled a phenytoin prescription between 1996 and 2017. We used laboratory test results, medication dispensing records, and medical notes to identify associations of CYP2C9 genotype with phenytoin blood concentration, neurologic side effects, and medication dispensing patterns reflecting clinician prescribing practice and patient response. RESULTS: Among 993 participants, we identified 69% extensive, 20% high-intermediate, 10% low-intermediate, and 2% poor metabolizers based on CYP2C9 genotypes. Compared with extensive metabolizer genotype, low-intermediate/poor metabolizer genotype was associated with increased dose-adjusted phenytoin blood concentration [21.3 pg/mL, 95% confidence interval (CI): 13.6-29.0 pg/mL; P < 0.01] and increased risk of neurologic side effects (hazard ratio: 2.40, 95% CI: 1.24-4.64; P < 0.01). Decreased function CYP2C9 genotypes were associated with medication dispensing patterns indicating dose decrease, use of alternative anticonvulsants, and worse adherence, although these associations varied by treatment indication for phenytoin. CONCLUSION: CYP2C9 variation was associated with clinically meaningful differences in clinician prescribing practice and patient response, with potential implications for healthcare utilization and treatment efficacy.

The Impact of Pharmacy-specific Predictors on the Performance of 30-Day Readmission Risk Prediction Models.

RESEARCH OBJECTIVE: Pharmacists are an expensive and limited resource in the hospital and outpatient setting. A pharmacist can spend up to 25% of their day planning. Time spent planning is time not spent delivering an intervention. A readmission risk adjustment model has potential to be used as a universal outcome-based prioritization tool to help pharmacists plan their interventions more efficiently. Pharmacy-specific predictors have not been used in the constructs of current readmission risk models. We assessed the impact of adding pharmacy-specific predictors on performance of readmission risk prediction models. STUDY DESIGN: We used an observational retrospective cohort study design to assess whether pharmacy-specific predictors such as an aggregate pharmacy score and drug classes would improve the prediction of 30-day readmission. A model of age, sex, length of stay, and admission category predictors was used as the reference model. We added predictor variables in sequential models to evaluate the incremental effect of additional predictors on the performance of the reference. We used logistic regression to regress the outcomes on predictors in our derivation dataset. We derived and internally validated our models through a 50:50 split validation of our dataset. POPULATION STUDIED: Our study population (n=350,810) was of adult admissions at hospitals in a large integrated health care delivery system. PRINCIPAL FINDINGS: Individually, the aggregate pharmacy score and drug classes caused a nearly identical but moderate increase in model performance over the reference. As a single predictor, the comorbidity burden score caused the greatest increase in model performance when added to the reference. Adding the severity of illness score, comorbidity burden score and the aggregate pharmacy score to the reference caused a cumulative increase in model performance with good discrimination (c statistic, 0.712; Nagelkerke R, 0.112). The best performing model included all predictors: severity of illness score, comorbidity burden score, aggregate pharmacy score, diagnosis groupings, and drug subgroups. CONCLUSIONS: Adding the aggregate pharmacy score to the reference model significantly increased the c statistic but was out-performed by the comorbidity burden score model in predicting readmission. The need for a universal prioritization tool for pharmacists may therefore be potentially met with the comorbidity burden score model. However, the aggregate pharmacy score and drug class models still out-performed current Medicare readmission risk adjustment models. IMPLICATIONS FOR POLICY OR PRACTICE: Pharmacists have a great role in preventing readmission, and therefore can potentially use one of our models: comorbidity burden score model, aggregate pharmacy score model, drug class model or complex model (a combination of all 5 major predictors) to prioritize their interventions while exceeding Medicare performance measures on readmission. The choice of model to use should be based on the availability of these predictors in the health care system.

A regenerative approach to the treatment of multiple sclerosis.

Progressive phases of multiple sclerosis are associated with inhibited differentiation of the progenitor cell population that generates the mature oligodendrocytes required for remyelination and disease remission. To identify selective inducers of oligodendrocyte differentiation, we performed an image-based screen for myelin basic protein (MBP) expression using primary rat optic-nerve-derived progenitor cells. Here we show that among the most effective compounds identifed was benztropine, which significantly decreases clinical severity in the experimental autoimmune encephalomyelitis (EAE) model of relapsing-remitting multiple sclerosis when administered alone or in combination with approved immunosuppressive treatments for multiple sclerosis. Evidence from a cuprizone-induced model of demyelination, in vitro and in vivo T-cell assays and EAE adoptive transfer experiments indicated that the observed efficacy of this drug results directly from an enhancement of remyelination rather than immune suppression. Pharmacological studies indicate that benztropine functions by a mechanism that involves direct antagonism of M1 and/or M3 muscarinic receptors. These studies should facilitate the development of effective new therapies for the treatment of multiple sclerosis that complement established immunosuppressive approaches.

Bispecific small molecule-antibody conjugate targeting prostate cancer.

Bispecific antibodies, which simultaneously target CD3 on T cells and tumor-associated antigens to recruit cytotoxic T cells to cancer cells, are a promising new approach to the treatment of hormone-refractory prostate cancer. Here we report a site-specific, semisynthetic method for the production of bispecific antibody-like therapeutics in which a derivative of the prostate-specific membrane antigen-binding small molecule DUPA was selectively conjugated to a mutant αCD3 Fab containing the unnatural amino acid, p-acetylphenylalanine, at a defined site. Homogeneous conjugates were generated in excellent yields and had good solubility. The efficacy of the conjugate was optimized by modifying the linker structure, relative binding orientation, and stoichiometry of the ligand. The optimized conjugate showed potent and selective in vitro activity (EC50 ~ 100 pM), good serum half-life, and potent in vivo activity in prophylactic and treatment xenograft mouse models. This semisynthetic approach is likely to be applicable to the generation of additional bispecific agents using drug-like ligands selective for other cell-surface receptors.

An anti-B cell maturation antigen bispecific antibody for multiple myeloma.

The development of immunotherapies for multiple myeloma is critical to provide new treatment strategies to combat drug resistance. We report a bispecific antibody against B cell maturation antigen (BiFab-BCMA), which potently and specifically redirects T cells to lyse malignant multiple myeloma cells. BiFab-BCMA lysed target BCMA-positive cell lines up to 20-fold more potently than a CS1-targeting bispecific antibody (BiFab-CS1) developed in an analogous fashion. Further, BiFab-BCMA robustly activated T cells in vitro and mediated rapid tumor regression in an orthotopic xenograft model of multiple myeloma. The in vitro and in vivo activities of BiFab-BCMA are comparable to those of anti-BCMA chimeric antigen receptor T cell therapy (CAR-T-BCMA), for which two clinical trials have recently been initiated. A BCMA-targeted bispecific antibody presents a promising treatment option for multiple myeloma.

An immunosuppressive antibody-drug conjugate.

We have developed a novel antibody-drug conjugate (ADC) that can selectively deliver the Lck inhibitor dasatinib to human T lymphocytes. This ADC is based on a humanized antibody that selectively binds with high affinity to CXCR4, an antigen that is selectively expressed on hematopoietic cells. The resulting dasatinib-antibody conjugate suppresses T-cell-receptor (TCR)-mediated T-cell activation and cytokine expression with low nM EC50 and has minimal effects on cell viability. This ADC may lead to a new class of selective immunosuppressive drugs with improved safety and extend the ADC strategy to the targeted delivery of kinase inhibitors for indications beyond oncology.

Interleukin-7 is required for CD4(+) T cell activation and autoimmune neuroinflammation.

IL-7 is known to be vital for T cell homeostasis but has previously been presumed to be dispensable for TCR-induced activation. Here, we show that IL-7 is critical for the initial activation of CD4(+) T cells in that it provides some of the necessary early signaling components, such as activated STAT5 and Akt. Accordingly, short-term in vivo IL-7Rα blockade inhibited the activation and expansion of autoantigen-specific CD4(+) T cells and, when used to treat experimental autoimmune encephalomyelitis (EAE), prevented and ameliorated disease. Our studies demonstrate that IL-7 signaling is a prerequisite for optimal CD4(+) T cell activation and that IL-7R antagonism may be effective in treating CD4(+) T cell-mediated neuroinflammation and other autoimmune inflammatory conditions.

Role of nucleic acid-sensing TLRs in diverse autoantibody specificities and anti-nuclear antibody-producing B cells.

Nucleic acid (NA)-sensing TLRs (NA-TLRs) promote the induction of anti-nuclear Abs in systemic lupus erythematosus. However, the extent to which other nonnuclear pathogenic autoantibody specificities that occur in lupus and independently in other autoimmune diseases depend on NA-TLRs, and which immune cells require NA-TLRs in systemic autoimmunity, remains to be determined. Using Unc93b1(3d) lupus-prone mice that lack NA-TLR signaling, we found that all pathogenic nonnuclear autoantibody specificities examined, even anti-RBC, required NA-TLRs. Furthermore, we document that NA-TLRs in B cells were required for the development of antichromatin and rheumatoid factor. These findings support a unifying NA-TLR-mediated mechanism of autoantibody production that has both pathophysiological and therapeutic implications for systemic lupus erythematosus and several other humoral-mediated autoimmune diseases. In particular, our findings suggest that targeting of NA-TLR signaling in B cells alone would be sufficient to specifically block production of a broad diversity of autoantibodies.

Multiformat T-cell-engaging bispecific antibodies targeting human breast cancers.

Four different formats of bispecific antibodies (bsAbs) were generated that consist of anti-Her2 IgG or Fab site-specifically conjugated to anti-CD3 Fab using the genetically encoded noncanonical amino acid. These bsAbs varied in valency or in the presence or absence of an Fc domain. Different valencies did not significantly affect antitumor efficacy, whereas the presence of an Fc domain enhanced cytotoxic activity, but triggered antigen-independent T-cell activation. We show that the bsAbs can efficiently redirect T cells to kill all Her2 expressing cancer cells, including Her2 1+ cancers, both in vitro and in rodent xenograft models. This work increases our understanding of the structural features that affect bsAb activity, and underscores the potential of bsAbs as a promising therapeutic option for breast cancer patients with low or heterogeneous Her2 expression.

Anti-IFN-α/ß receptor antibody treatment ameliorates disease in lupus-predisposed mice.

The demonstration in humans and mice that nucleic acid-sensing TLRs and type I IFNs are essential disease mediators is a milestone in delineating the mechanisms of lupus pathogenesis. In this study, we show that Ifnb gene deletion does not modify disease progression in NZB mice, thereby strongly implicating IFN-α subtypes as the principal pathogenic effectors. We further document that long-term treatment of male BXSB mice with an anti-IFN-α/ß receptor Ab of mouse origin reduced serologic, cellular, and histologic disease manifestations and extended survival, suggesting that disease acceleration by the Tlr7 gene duplication in this model is mediated by type I IFN signaling. The efficacy of this treatment in BXSB mice was clearly evident when applied early in the disease process, but only partial reductions in some disease characteristics were observed when treatment was initiated at later stages. A transient therapeutic effect was also noted in the MRL-Fas(lpr) model, although overall mortality was unaffected. The combined findings suggest that IFN-α/ß receptor blockade, particularly when started at early disease stages, may be a useful treatment approach for human systemic lupus erythematosus and other autoimmune syndromes.

Targeting human C-type lectin-like molecule-1 (CLL1) with a bispecific antibody for immunotherapy of acute myeloid leukemia.

Acute myeloid leukemia (AML), which is the most common acute adult leukemia and the second most common pediatric leukemia, still has a poor prognosis. Human C-type lectin-like molecule-1 (CLL1) is a recently identified myeloid lineage restricted cell surface marker, which is overexpressed in over 90% of AML patient myeloid blasts and in leukemic stem cells. Here, we describe the synthesis of a novel bispecific antibody, αCLL1-αCD3, using the genetically encoded unnatural amino acid, p-acetylphenylalanine. The resulting αCLL1-αCD3 recruits cytotoxic T cells to CLL1 positive cells, and demonstrates potent and selective cytotoxicity against several human AML cell lines and primary AML patient derived cells in vitro. Moreover, αCLL1-αCD3 treatment completely eliminates established tumors in an U937 AML cell line xenograft model. These results validate the clinical potential of CLL1 as an AML-specific antigen for the generation of a novel immunotherapeutic for AML.

A CXCR4-targeted site-specific antibody-drug conjugate.

A chemically defined anti-CXCR4-auristatin antibody-drug conjugate (ADC) was synthesized that selectively eliminates tumor cells overexpressing the CXCR4 receptor. The unnatural amino acid p-acetylphenylalanine (pAcF) was site-specifically incorporated into an anti-CXCR4 immunoglobulin G (IgG) and conjugated to an auristatin through a stable, non-cleavable oxime linkage to afford a chemically homogeneous ADC. The full-length anti-CXCR4 ADC was selectively cytotoxic to CXCR4(+) cancer cells in vitro (half maximal effective concentration (EC50 )≈80-100 pM). Moreover, the anti-CXCR4 ADC eliminated pulmonary lesions from human osteosarcoma cells in a lung-seeding tumor model in mice. No significant overt toxicity was observed but there was a modest decrease in the bone-marrow-derived CXCR4(+) cell population. Because CXCR4 is highly expressed in a majority of metastatic cancers, a CXCR4-auristatin ADC may be useful for the treatment of a variety of metastatic malignancies.

Evaluation of an outreach programme for patients with COVID-19 in an integrated healthcare delivery system: a retrospective cohort study.

OBJECTIVES: In the first year of the COVID-19 pandemic, health systems implemented programmes to manage outpatients with COVID-19. The goal was to expedite patients' referral to acute care and prevent overcrowding of medical centres. We sought to evaluate the impact of such a programme, the COVID-19 Home Care Team (CHCT) programme. DESIGN: Retrospective cohort. SETTING: Kaiser Permanente Northern California. PARTICIPANTS: Adult members before COVID-19 vaccine availability (1 February 2020-31 January 2021) with positive SARS-CoV-2 tests. INTERVENTION: Virtual programme to track and treat patients with 'CHCT programme'. OUTCOMES: The outcomes were (1) COVID-19-related emergency department visit, (2) COVID-19-related hospitalisation and (3) inpatient mortality or 30-day hospice referral. MEASURES: We estimated the average effect comparing patients who were and were not treated by CHCT. We estimated propensity scores using an ensemble super learner (random forest, XGBoost, generalised additive model and multivariate adaptive regression splines) and augmented inverse probability weighting. RESULTS: There were 98 585 patients with COVID-19. The majority were followed by CHCT (n=80 067, 81.2%). Patients followed by CHCT were older (mean age 43.9 vs 41.6 years, p<0.001) and more comorbid with COmorbidity Point Score, V.2, score ≥65 (1.7% vs 1.1%, p<0.001). Unadjusted analyses showed more COVID-19-related emergency department visits (9.5% vs 8.5%, p<0.001) and hospitalisations (3.9% vs 3.2%, p<0.001) in patients followed by CHCT but lower inpatient death or 30-day hospice referral (0.3% vs 0.5%, p<0.001). After weighting, there were higher rates of COVID-19-related emergency department visits (estimated intervention effect -0.8%, 95% CI -1.4% to -0.3%) and hospitalisation (-0.5%, 95% CI -0.9% to -0.1%) but lower inpatient mortality or 30-day hospice referral (-0.5%, 95% CI -0.7% to -0.3%) in patients followed by CHCT. CONCLUSIONS: Despite CHCT following older patients with higher comorbidity burden, there appeared to be a protective effect. Patients followed by CHCT were more likely to present to acute care and less likely to die inpatient.