Acalabrutinib, venetoclax, and obinutuzumab as frontline treatment for chronic lymphocytic leukaemia: a single-arm, open-label, phase 2 study.

BACKGROUND: Both continuous therapy with acalabrutinib and fixed-duration therapy with venetoclax-obinutuzumab are effective for previously untreated chronic lymphocytic leukaemia. We hypothesised that frontline time-limited, minimal residual disease (MRD)-guided triplet therapy with acalabrutinib, venetoclax, and obinutuzumab would induce deep (ie, more patients with undetectable MRD) and durable remissions. METHODS: In this open-label, single-arm, investigator-sponsored, phase 2 study, patients with chronic lymphocytic leukaemia or small lymphocytic lymphoma were recruited from two academic hospitals in Boston, MA, USA. Eligible patients were aged 18 years or older, with an Eastern Cooperative Oncology Group performance status of 0-2, and were treatment naive. Patients were treated in 28 day cycles. Acalabrutinib monotherapy was given orally at 100 mg twice daily for cycle 1, then combined for six cycles with intravenous obinutuzumab (100 mg on cycle 2 day 1, 900 mg on day 2, 1000 mg on day 8, and 1000 mg on day 15 and on day 1 of cycles 3-7); and from the beginning of cycle 4, oral venetoclax was dosed daily using an accelerated ramp-up from 20 mg on day 1 to 400 mg by day 22 and continued at this dose thereafter. Patients continued on acalabrutinib 100 mg twice daily and venetoclax 400 mg once daily until day 1 of cycle 16 or day 1 of cycle 25. If the patient had undetectable MRD in the bone marrow they were given the option to discontinue therapy at the start of cycle 16 (if also in complete remission) or at the start of cycle 25 (if at least in partial remission). The primary endpoint was complete remission with undetectable MRD in the bone marrow (defined as <1 chronic lymphocytic leukaemia cell per 10 000 leucocytes as measured by four-colour flow cytometry), at cycle 16 day 1. Safety and activity endpoints were assessed in all patients who received at least one dose of any study drug. This study is registered with ClinicalTrials.gov, NCT03580928, and is ongoing. FINDINGS: Between Aug 2, 2018, and May 23, 2019, 37 patients with chronic lymphocytic leukaemia were enrolled and all received at least one dose of any study drug. The median age of patients was 63 years (IQR 57-70), and ten (27%) were female and 27 (73%) were male. Median follow-up was 27·6 months (IQR 25·1-28·2). At cycle 16 day 1, 14 (38% [95% CI 22-55]) of 37 participants had a complete remission with undetectable MRD in the bone marrow. The most common grade 3 or 4 haematological adverse event was neutropenia (16 [43%] of 37 patients). The most common grade 3-4 non-haematological adverse events were hyperglycaemia (three [8%]) and hypophosphataemia (three [8%]). Serious adverse events occurred in nine (24%) patients; the most common was neutropenia in three (8%) patients. There have been no deaths on study. INTERPRETATION: Acalabrutinib, venetoclax, and obinutuzumab is a highly active and well tolerated frontline therapy for chronic lymphocytic leukaemia. Although the primary endpoint of this study was not met, the high proportion of patients who had undetectable MRD in the bone marrow supports further investigation of this regimen, which is being tested against acalabrutinib-venetoclax and chemoimmunotherapy in an ongoing phase 3 study (NCT03836261). FUNDING: AstraZeneca and a Dana-Farber Cancer Institute Collaborative Award.

Left-right asymmetric heart jogging increases the robustness of dextral heart looping in zebrafish.

Building a left-right (L-R) asymmetric organ requires asymmetric information. This comes from various sources, including asymmetries in embryo-scale genetic cascades (including the left-sided Nodal cascade), organ-intrinsic mechanical forces, and cell-level chirality, but the relative influence of these sources and how they collaborate to drive asymmetric morphogenesis is not understood. During zebrafish heart development, the linear heart tube extends to the left of the midline in a process known as jogging. The jogged heart then undergoes dextral (i.e. rightward) looping to correctly position the heart chambers relative to one another. Left lateralized jogging is governed by the left-sided expression of Nodal in mesoderm tissue, while looping laterality is mainly controlled by heart-intrinsic cell-level asymmetries in the actomyosin cytoskeleton. The purpose of lateralized jogging is not known. Moreover, after jogging, the heart tube returns to an almost midline position and so it is not clear whether or how jogging may impact the dextral loop. Here, we characterize a novel loss-of-function mutant in the zebrafish Nodal homolog southpaw (spaw) that appears to be a true null. We then assess the relationship between jogging and looping laterality in embryos lacking asymmetric Spaw signals. We found that the probability of a dextral loop occurring, does not depend on asymmetric Spaw signals per se, but does depend on the laterality of jogging. Thus, we conclude that the role of leftward jogging is to spatially position the heart tube in a manner that promotes robust dextral looping. When jogging laterality is abnormal, the robustness of dextral looping decreases. This establishes a cooperation between embryo-scale Nodal-dependent L-R asymmetries and organ-intrinsic cellular chirality in the control of asymmetric heart morphogenesis and shows that the transient laterality of the early heart tube has consequences for later heart morphogenetic events.

Psychotic symptoms are associated with lower cortical folding in youth at risk for mental illness

Background: Cortical folding is essential for healthy brain development. Previous studies have found regional reductions in cortical folding in adult patients with psychotic illness. It is unknown whether these neuroanatomical markers are present in youth with subclinical psychotic symptoms. Methods: We collected MRIs and examined the local gyrification index in a sample of 110 youth (mean age ± standard deviation 14.0 ± 3.7 yr; range 9­25 yr) with a family history of severe mental illness: 48 with psychotic symptoms and 62 without. Images were processed using the Human Connectome Pipeline and FreeSurfer. We tested for group differences in local gyrification index using mixed-effects generalized linear models controlling for age, sex and familial clustering. Sensitivity analysis further controlled for intracranial volume, IQ, and stimulant and cannabis use. Results: Youth with psychotic symptoms displayed an overall trend toward lower cortical folding across all brain regions. After adjusting for multiple comparisons and confounders, regional reductions were localized to the frontal and occipital lobes. Specifically, the medial (B = ­0.42, pFDR = 0.04) and lateral (B = ­0.39, pFDR = 0.04) orbitofrontal cortices as well as the cuneus (B = ­0.47, pFDR = 0.03) and the pericalcarine (B = ­0.45, pFDR = 0.03) and lingual (B = ­0.38, pFDR = 0.04) gyri. Limitations: Inference about developmental trajectories was limited by the cross-sectional data. Conclusion: Psychotic symptoms in youth are associated with cortical folding deficits, even in the absence of psychotic illness. The current study helps clarify the neurodevelopmental basis of psychosis at an early stage, before medication, drug use and other confounds have had a persistent effect on the brain.

Affective lability in offspring of parents with major depressive disorder, bipolar disorder and schizophrenia.

Affective lability, defined as the propensity to experience excessive and unpredictable changes in mood, has been proposed as a potential transdiagnostic predictor of major mood and psychotic disorders. A parental diagnosis of bipolar disorder has been associated with increased affective lability in offspring. However, the association between affective lability and family history of other mood and psychotic disorders has not been examined. We measured affective lability using the self- and parent-reported Children's Affective Lability Scale in a cohort of 320 youth aged 6-17 years, including 137 offspring of a parent with major depressive disorder, 68 offspring of a parent with bipolar disorder, 24 offspring of a parent with schizophrenia, and 91 offspring of control parents. We tested differences in affective lability between groups using mixed-effects linear regression. Offspring of a parent with major depressive disorder (ß = 0.46, 95% CI 0.17-0.76, p = 0.002) or bipolar disorder (ß = 0.47, 95% CI 0.12-0.81, p = 0.008) had significantly higher affective lability scores than control offspring. Affective lability did not differ significantly between offspring of a parent with schizophrenia and offspring of control parents. Our results suggest that elevated affective lability during childhood is a marker of familial risk for mood disorders.

How activating mutations affect MEK1 regulation and function.

The MEK1 kinase directly phosphorylates ERK2, after the activation loop of MEK1 is itself phosphorylated by Raf. Studies over the past decade have revealed a large number of disease-related mutations in the MEK1 gene that lead to tumorigenesis and abnormal development. Several of these mutations result in MEK1 constitutive activity, but how they affect MEK1 regulation and function remains largely unknown. Here, we address these questions focusing on two pathogenic variants of the Phe-53 residue, which maps to the well-characterized negative regulatory region of MEK1. We found that these variants are phosphorylated by Raf faster than the wild-type enzyme, and this phosphorylation further increases their enzymatic activity. However, the maximal activities of fully phosphorylated wild-type and mutant enzymes are indistinguishable. On the basis of available structural information, we propose that the activating substitutions destabilize the inactive conformation of MEK1, resulting in its constitutive activity and making it more prone to Raf-mediated phosphorylation. Experiments in zebrafish revealed that the effects of activating variants on embryonic development reflect the joint control of the negative regulatory region and activating phosphorylation. Our results underscore the complexity of the effects of activating mutations on signaling systems, even at the level of a single protein.

Disruptive mood dysregulation disorder in offspring of parents with depression and bipolar disorder.

BackgroundIt has been suggested that offspring of parents with bipolar disorder are at increased risk for disruptive mood dysregulation disorder (DMDD), but the specificity of this association has not been established.AimsWe examined the specificity of DMDD to family history by comparing offspring of parents with (a) bipolar disorder, (b) major depressive disorder and (c) a control group with no mood disorders.MethodWe established lifetime diagnosis of DMDD using the Schedule for Affective Disorders and Schizophrenia for School Aged Children for DSM-5 in 180 youth aged 6-18 years, including 58 offspring of parents with bipolar disorder, 82 offspring of parents with major depressive disorder and 40 control offspring.ResultsDiagnostic criteria for DMDD were met in none of the offspring of parents with bipolar disorder, 6 of the offspring of parents with major depressive disorder and none of the control offspring. DMDD diagnosis was significantly associated with family history of major depressive disorder.ConclusionsOur results suggest that DMDD is not specifically associated with a family history of bipolar disorder and may be associated with parental depression.

Brentuximab vedotin plus doxorubicin and dacarbazine in nonbulky limited-stage classical Hodgkin lymphoma.

ABVD (Adriamycin, bleomycin, vinblastine, dacarbazine) with or without radiation has been the standard treatment for limited-stage Hodgkin lymphoma (HL) but carries risks of bleomycin lung injury and radiation toxicity. Brentuximab vedotin (BV) is approved with AVD for stage III-IV HL, but carries increased risks of peripheral neuropathy (PN) and neutropenic fever, likely due to overlapping toxicity between BV and vinblastine. We therefore evaluated BV in combination with AD for 4 or 6 cycles based on interim positron emission tomography response. Thirty-four patients with nonbulky stage I-II HL were enrolled. Risk was early favorable in 53% and unfavorable in 47%. The overall and complete response rates (CRRs) were 100% and 97%, respectively, with a 5-year progression-free survival (PFS) of 91%. No differences in outcome were observed based on stage (I vs II) or risk status (early favorable vs unfavorable). The most common adverse events were nausea (85%), peripheral sensory neuropathy (59%), and fatigue (56%). There were no cases of grade-4 neutropenia or neutropenic fever, and no patient received granulocyte-colony stimulating factor. Most cases of PN were grade 1, and no patient experienced grade ≥3 PN. BV-AD produced a high CRR and durable PFS with most patients requiring 4 cycles of therapy. Compared with BV-AVD, the toxicity profile appeared improved, with predominantly grade 1 reversible PN and no case of grade 4 neutropenia or neutropenic fever. This regimen warrants further study in HL and may serve as a backbone for the addition of novel agents. This trial is registered on clinicaltrials.gov (NCT02505269).

Abrogation of MAP4K4 protein function causes congenital anomalies in humans and zebrafish.

We report 21 families displaying neurodevelopmental differences and multiple congenital anomalies while bearing a series of rare variants in mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4). MAP4K4 has been implicated in many signaling pathways including c-Jun N-terminal and RAS kinases and is currently under investigation as a druggable target for multiple disorders. Using several zebrafish models, we demonstrate that these human variants are either loss-of-function or dominant-negative alleles and show that decreasing Map4k4 activity causes developmental defects. Furthermore, MAP4K4 can restrain hyperactive RAS signaling in early embryonic stages. Together, our data demonstrate that MAP4K4 negatively regulates RAS signaling in the early embryo and that variants identified in affected humans abrogate its function, establishing MAP4K4 as a causal locus for individuals with syndromic neurodevelopmental differences.

An Internet-Based Cognitive Behavioral Therapy Program for Anxiety and Depression (Tranquility): Adaptation Co-design and Fidelity Evaluation Study.

BACKGROUND: Internet-based cognitive behavioral therapy (iCBT) is a necessary step toward increasing the accessibility of mental health services. Yet, few iCBT programs have been evaluated for their fidelity to the therapeutic principles of cognitive behavioral therapy (CBT) or usability standards. In addition, many existing iCBT programs do not include treatments targeting both anxiety and depression, which are commonly co-occurring conditions. OBJECTIVE: This study aims to evaluate the usability of Tranquility-a novel iCBT program for anxiety-and its fidelity to CBT principles. This study also aims to engage in a co-design process to adapt Tranquility to include treatment elements for depression. METHODS: CBT experts (n=6) and mental health-informed peers (n=6) reviewed the iCBT program Tranquility. CBT experts assessed Tranquility's fidelity to CBT principles and were asked to identify necessary interventions for depression by using 2 simulated client case examples. Mental health-informed peers engaged in 2 co-design focus groups to discuss adaptations to the existing anxiety program and the integration of interventions for depression. Both groups completed web-based surveys assessing the usability of Tranquility and the likelihood that they would recommend the program. RESULTS: The CBT experts' mean rating of Tranquility's fidelity to CBT principles was 91%, indicating a high fidelity to CBT. Further, 5 out of 6 CBT experts and all mental health-informed peers (all participants: 11/12, 88%) rated Tranquility as satisfactory, indicating that they may recommend Tranquility to others, and they rated its usability highly (mean 76.56, SD 14.07). Mental health-informed peers provided suggestions on how to leverage engagement with Tranquility (eg, adding incentives and notification control). CONCLUSIONS: This preliminary study demonstrated the strong fidelity of Tranquility to CBT and usability standards. The results highlight the importance of involving stakeholders in the co-design process and future opportunities to increase engagement.

A multiple baseline trial of adapted prolonged exposure psychotherapy for individuals with early phase psychosis, comorbid substance misuse, and a history of adversity: A study protocol.

Background: Adversity is prevalent among people with psychotic disorders, especially those within the first 5 years of a psychotic disorder, called early phase psychosis. Although adversity can lead to many negative outcomes (e.g., posttraumatic stress symptoms), very few treatments for adversity-related sequelae have been tested with individuals with psychotic disorders, and even fewer studies have specifically tested interventions for people in early phase psychosis. Furthermore, people who misuse substances are commonly excluded from adversity treatment trials, which is problematic given that individuals with early phase psychosis have high rates of substance misuse. For the first time, this trial will examine the outcomes of an adapted 15-session prolonged exposure protocol (i.e., PE+) to observe whether reductions in adversity-related psychopathology occurs among people with early phase psychosis and comorbid substance misuse. Methods: This study will use a multiple-baseline design with randomization of participants to treatment start time. Participants will complete baseline appointments prior to therapy, engage in assessments between each of the five therapy modules, and complete a series of follow-up appointments 2 months after the completion of therapy. Primary hypothesized outcomes include clinically significant reductions in (1) negative psychotic symptoms measured using the Positive and Negative Syndrome Scale, (2) adversity-related sequelae measured using the Trauma Symptom Checklist-40, and (3) substance use frequency and overall risk score measured with the Alcohol, Smoking, and Substance Involvement Screening Test. We also anticipate that clinically significant reductions in hopelessness and experiential avoidance, measured with the Beck Hopelessness Scale and Brief Experiential Avoidance Questionnaire, the theorized mechanisms of change of PE+, will also be observed. A secondary outcome is a hypothesized improvement in functioning, measured using the Clinical Global Impression and Social and Occupational Functioning Assessment scales. Discussion: The results of this treatment trial will contribute to the advancement of treatment research for individuals in early phase psychosis who have current substance misuse and a history of adversity, and the findings may provide evidence supporting the use of hopelessness and experiential avoidance as mechanisms of change for this treatment. Clinical trial registration: Clinicaltrials.gov, NCT04546178; registered August 28, 2020, https://clinicaltrials.gov/ct2/show/NCT04546178?term=NCT04546178&draw=2&rank=1.

Mouse hitchhiker mutants have spina bifida, dorso-ventral patterning defects and polydactyly: identification of Tulp3 as a novel negative regulator of the Sonic hedgehog pathway.

The mammalian Sonic hedgehog (Shh) signalling pathway is essential for embryonic development and the patterning of multiple organs. Disruption or activation of Shh signalling leads to multiple birth defects, including holoprosencephaly, neural tube defects and polydactyly, and in adults results in tumours of the skin or central nervous system. Genetic approaches with model organisms continue to identify novel components of the pathway, including key molecules that function as positive or negative regulators of Shh signalling. Data presented here define Tulp3 as a novel negative regulator of the Shh pathway. We have identified a new mouse mutant that is a strongly hypomorphic allele of Tulp3 and which exhibits expansion of ventral markers in the caudal spinal cord, as well as neural tube defects and preaxial polydactyly, consistent with increased Shh signalling. We demonstrate that Tulp3 acts genetically downstream of Shh and Smoothened (Smo) in neural tube patterning and exhibits a genetic interaction with Gli3 in limb development. We show that Tulp3 does not appear to alter expression or processing of Gli3, and we demonstrate that transcriptional regulation of other negative regulators (Rab23, Fkbp8, Thm1, Sufu and PKA) is not affected. We discuss the possible mechanism of action of Tulp3 in Shh-mediated signalling in light of these new data.

Youth Experience Tracker Instrument: A self-report measure of developmental antecedents to severe mental illness.

AIM: We sought to examine the structure, internal consistency, convergent and criterion validity of the Youth Experience Tracker Instrument (YETI), a new brief self-report measure designed to facilitate early identification of risk for severe forms of mental illness, including major depressive disorder, bipolar disorder, and schizophrenia. METHODS: We collected 716 YETIs from 315 individuals aged 8 to 27 with and without familial risk of severe mental illness. The YETI measures six developmental antecedents that precede and predict serious forms of mental illness: affective lability, anxiety, basic symptoms, depressive symptoms, psychotic-like experiences, and sleep. A battery of concurrent questionnaires and interviews measured the same constructs. RESULTS: The best-fitting bifactor model supported the validity of both total score and antecedent-specific subscales. Internal consistency was high for the total score (ω = 0.94) and subscales (ω = 0.80-0.92; ρ = 0.72). The total score captured the majority of information from the 26 YETI items (hierarchical omega ωh = 0.74). Correlations of YETI subscales with established measures of the same constructs (r = 0.45-0.80) suggested adequate convergent validity. We propose cut-offs with high negative predictive values to facilitate efficient risk screening. CONCLUSION: The YETI, a brief self-report measure of antecedents, provides an alternative to using multiple longer instruments. Future research may examine the predictive validity of the YETI for the onset of major mood and psychotic disorders.

The novel mouse mutant, chuzhoi, has disruption of Ptk7 protein and exhibits defects in neural tube, heart and lung development and abnormal planar cell polarity in the ear.

BACKGROUND: The planar cell polarity (PCP) signalling pathway is fundamental to a number of key developmental events, including initiation of neural tube closure. Disruption of the PCP pathway causes the severe neural tube defect of craniorachischisis, in which almost the entire brain and spinal cord fails to close. Identification of mouse mutants with craniorachischisis has proven a powerful way of identifying molecules that are components or regulators of the PCP pathway. In addition, identification of an allelic series of mutants, including hypomorphs and neomorphs in addition to complete nulls, can provide novel genetic tools to help elucidate the function of the PCP proteins. RESULTS: We report the identification of a new N-ethyl-N-nitrosourea (ENU)-induced mutant with craniorachischisis, which we have named chuzhoi (chz). We demonstrate that chuzhoi mutant embryos fail to undergo initiation of neural tube closure, and have characteristics consistent with defective convergent extension. These characteristics include a broadened midline and reduced rate of increase of their length-to-width ratio. In addition, we demonstrate disruption in the orientation of outer hair cells in the inner ear, and defects in heart and lung development in chuzhoi mutants. We demonstrate a genetic interaction between chuzhoi mutants and both Vangl2Lp and Celsr1Crsh mutants, strengthening the hypothesis that chuzhoi is involved in regulating the PCP pathway. We demonstrate that chuzhoi maps to Chromosome 17 and carries a splice site mutation in Ptk7. This mutation results in the insertion of three amino acids into the Ptk7 protein and causes disruption of Ptk7 protein expression in chuzhoi mutants. CONCLUSIONS: The chuzhoi mutant provides an additional genetic resource to help investigate the developmental basis of several congenital abnormalities including neural tube, heart and lung defects and their relationship to disruption of PCP. The chuzhoi mutation differentially affects the expression levels of the two Ptk7 protein isoforms and, while some Ptk7 protein can still be detected at the membrane, chuzhoi mutants demonstrate a significant reduction in membrane localization of Ptk7 protein. This mutant provides a useful tool to allow future studies aimed at understanding the molecular function of Ptk7.

Swimming toward solutions: Using fish and frogs as models for understanding RASopathies.

The RAS signaling pathway regulates cell growth, survival, and differentiation, and its inappropriate activation is associated with disease in humans. The RASopathies, a set of developmental syndromes, arise when the pathway is overactive during development. Patients share a core set of symptoms, including congenital heart disease, craniofacial anomalies, and neurocognitive delay. Due to the conserved nature of the pathway, animal models are highly informative for understanding disease etiology, and zebrafish and Xenopus are emerging as advantageous model systems. Here we discuss these aquatic models of RASopathies, which recapitulate many of the core symptoms observed in patients. Craniofacial structures become dysmorphic upon expression of disease-associated mutations, resulting in wider heads. Heart defects manifest as delays in cardiac development and changes in heart size, and behavioral deficits are beginning to be explored. Furthermore, early convergence and extension defects cause elongation of developing embryos: this phenotype can be quantitatively assayed as a readout of mutation strength, raising interesting questions regarding the relationship between pathway activation and disease. Additionally, the observation that RAS signaling may be simultaneously hyperactive and attenuated suggests that downregulation of signaling may also contribute to etiology. We propose that models should be characterized using a standardized approach to allow easier comparison between models, and a better understanding of the interplay between mutation and disease presentation.

Basic symptoms in offspring of parents with mood and psychotic disorders.

BACKGROUND: Basic symptoms, defined as subjectively perceived disturbances in thought, perception and other essential mental processes, have been established as a predictor of psychotic disorders. However, the relationship between basic symptoms and family history of a transdiagnostic range of severe mental illness, including major depressive disorder, bipolar disorder and schizophrenia, has not been examined. AIMS: We sought to test whether non-severe mood disorders and severe mood and psychotic disorders in parents is associated with increased basic symptoms in their biological offspring. METHOD: We measured basic symptoms using the Schizophrenia Proneness Instrument - Child and Youth Version in 332 youth aged 8-26 years, including 93 offspring of control parents, 92 offspring of a parent with non-severe mood disorders, and 147 offspring of a parent with severe mood and psychotic disorders. We tested the relationships between parent mental illness and offspring basic symptoms in mixed-effects linear regression models. RESULTS: Offspring of a parent with severe mood and psychotic disorders (B = 0.69, 95% CI 0.22-1.16, P = 0.004) or illness with psychotic features (B = 0.68, 95% CI 0.09-1.27, P = 0.023) had significantly higher basic symptom scores than control offspring. Offspring of a parent with non-severe mood disorders reported intermediate levels of basic symptoms, that did not significantly differ from control offspring. CONCLUSIONS: Basic symptoms during childhood are a marker of familial risk of psychopathology that is related to severity and is not specific to psychotic illness. DECLARATION OF INTEREST: None.

Modeling Syndromic Congenital Heart Defects in Zebrafish.

Cardiac development is a dynamic process regulated by spatial and temporal cues that are integrated to effect molecular, cellular, and tissue-level events that form the adult heart. Disruption of these highly orchestrated events can be devastating for cardiac form and function. Aberrations in heart development result in congenital heart defects (CHDs), which affect 1 in 100 infants in the United States each year. Zebrafish have proven informative as a model organism to understand both heart development and the mechanisms associated with CHDs due to the similarities in heart morphogenesis among vertebrates, as well as their genetic tractability and amenability to live imaging. In this review, we discuss the mechanisms of zebrafish heart development and the utility of zebrafish for understanding syndromic CHDs, those cardiac abnormalities that occur in the context of multisystem disorders. We conclude with avenues of zebrafish research that will potentially inform future therapeutic approaches for the treatment of CHDs.

A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging.

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to lessen the impact of debilitating conditions but remain elusive in many cases. Only by understanding the causes and pathology of diseases associated with aging, can scientists begin to identify potential therapeutic targets and develop strategies for intervention. The most common age-related conditions are neurodegenerative disorders such as Parkinson's disease and blindness. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Genome wide association studies have previously identified loci that are associated with increased susceptibility to this disease and identified two regions of interest: complement factor H (CFH) and the 10q26 locus, where the age-related maculopathy susceptibility 2 (ARMS2) and high-temperature requirement factor A1 (HtrA1) genes are located. CFH acts as a negative regulator of the alternative pathway (AP) of the complement system while HtrA1 is an extracellular serine protease. ARMS2 is located upstream of HtrA1 in the primate genome, although the gene is absent in mice. To study the effects of these genes, humanized knock-in mouse lines of Cfh and ARMS2, knockouts of Cfh, HtrA1, HtrA2, HtrA3 and HtrA4 as well as a conditional neural deletion of HtrA2 were generated. Of all the genetically engineered mice produced only mice lacking HtrA2, either systemically or in neural tissues, displayed clear phenotypes. In order to examine these mice thoroughly and systematically, an initial phenotyping schedule was established, consisting of a series of tests related to two main diseases of interest: AMD and Parkinson's. Genetically modified mice can be subjected to appropriate experiments to identify phenotypes that may be related to the associated diseases in humans. A phenotyping regimen with a mitochondrial focus is presented here alongside representative results from the tests of interest.

Protoporphyrins enhance oligomerization and enzymatic activity of HtrA1 serine protease.

High temperature requirement protein A1 (HtrA1), a secreted serine protease of the HtrA family, is associated with a multitude of human diseases. However, the exact functions of HtrA1 in these diseases remain poorly understood. We seek to unravel the mechanisms of HtrA1 by elucidating its interactions with chemical or biological modulators. To this end, we screened a small molecule library of 500 bioactive compounds to identify those that alter the formation of extracellular HtrA1 complexes in the cell culture medium. An initial characterization of two novel hits from this screen showed that protoporphyrin IX (PPP-IX), a precursor in the heme biosynthetic pathway, and its metalloporphyrin (MPP) derivatives fostered the oligomerization of HtrA1 by binding to the protease domain. As a result of the interaction with MPPs, the proteolytic activity of HtrA1 against Fibulin-5, a specific HtrA1 substrate in age-related macular degeneration (AMD), was increased. This physical interaction could be abolished by the missense mutations of HtrA1 found in patients with cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). Furthermore, knockdown of HtrA1 attenuated apoptosis induced by PPP-IX. These results suggest that PPP-IX, or its derivatives, and HtrA1 may function as co-factors whereby porphyrins enhance oligomerization and the protease activity of HtrA1, while active HtrA1 elevates the pro-apoptotic actions of porphyrin derivatives. Further analysis of this interplay may shed insights into the pathogenesis of diseases such as AMD, CARASIL and protoporphyria, as well as effective therapeutic development.

Neural-specific deletion of Htra2 causes cerebellar neurodegeneration and defective processing of mitochondrial OPA1.

HTRA2, a serine protease in the intermembrane space, has important functions in mitochondrial stress signaling while its abnormal activity may contribute to the development of Parkinson's disease. Mice with a missense or null mutation of Htra2 fail to thrive, suffer striatal neuronal loss, and a parkinsonian phenotype that leads to death at 30-40 days of age. While informative, these mouse models cannot separate neural contributions from systemic effects due to the complex phenotypes of HTRA2 deficiency. Hence, we developed mice carrying a Htra2-floxed allele to query the consequences of tissue-specific HTRA2 deficiency. We found that mice with neural-specific deletion of Htra2 exhibited atrophy of the thymus and spleen, cessation to gain weight past postnatal (P) day 18, neurological symptoms including ataxia and complete penetrance of premature death by P40. Histologically, increased apoptosis was detected in the cerebellum, and to a lesser degree in the striatum and the entorhinal cortex, from P25. Even earlier at P20, mitochondria in the cerebella already exhibited abnormal morphology, including swelling, vesiculation, and fragmentation of the cristae. Furthermore, the onset of these structural anomalies was accompanied by defective processing of OPA1, a key molecule for mitochondrial fusion and cristae remodeling, leading to depletion of the L-isoform. Together, these findings suggest that HTRA2 is essential for maintenance of the mitochondrial integrity in neurons. Without functional HTRA2, a lifespan as short as 40 days accumulates a large quantity of dysfunctional mitochondria that contributes to the demise of mutant mice.

Survival of the anterior cruciate ligament graft and the contralateral ACL at a minimum of 15 years.

BACKGROUND: The risks for primary anterior cruciate ligament (ACL) rupture have been established. What is less well known is the risk of graft rupture after reconstruction and also the risk of a primary ACL rupture in the contralateral knee. PURPOSE: To determine the long-term survival of the ACL graft and the contralateral ACL (CACL) after reconstruction and to identify factors that increase the odds of subsequent ACL injury. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: All patients having undergone primary ACL reconstruction in 1993 or 1994 by a single surgeon in a single unit were considered. Patients were contacted to complete a subjective interview by telephone or e-mail questionnaire at a minimum of 15 years after surgery. RESULTS: A total of 755 patients met the inclusion criteria, and ACL reconstruction was performed using a single-incision endoscopic technique with either autologous bone-patellar tendon-bone graft (BPTB; n = 314) or hamstring tendon graft (HT; n = 359) and metal interference screw fixation. Of these patients, 673 (89%) completed the questionnaire; 23% had sustained either a graft rupture or CACL rupture. Expected survival of the ACL graft was 95%, 93%, 91%, and 89% at a respective 2, 5, 10, and 15 years after reconstruction. Expected survival of the CACL was 97%, 93%, 90%, and 87%, respectively. Survival of the ACL graft was less favorable in men than in women (P = .007); ACL graft survival was not significantly different between the HT (88%) or BPTB (91%) groups (P = .149). Rupture of the CACL occurred twice as frequently as graft rupture in the BPTB group (graft survival, 84% vs 89%; P = .003). A positive family history of ACL rupture doubled the odds of both ACL graft and CACL rupture. The mean International Knee Documentation Committee subjective score at 15 years was 85. Return to preinjury sport levels was reported in 73% of patients, and 51% were still participating in strenuous or very strenuous activities at 15 years. CONCLUSION: Fifteen years after ACL reconstruction, expected survival of the ACL graft was 89% and expected survival of the CACL was 86%. Graft choice did not affect ACL graft rupture, but using BPTB increased the risk of CACL rupture compared with HT. Men had a less favorable survival rate of the ACL graft than did women, and a family history of ACL rupture increased the risk of both ACL graft and CACL rupture.