Measure what matters: considerations for outcome measurement of care coordination for children with neurodevelopmental disabilities and medical complexity.

Introduction: Care Coordination (CC) is a significant intervention to enhance family's capacity in caring for children with neurodevelopmental disability and medical complexity (NDD-MC). CC assists with integration of medical and behavioral care and services, partnerships with medical and community-based supports, and access to medical, behavioral, and educational supports and services. Although there is some consensus on the principles that characterize optimal CC for children with NDD-MC, challenges remain in measuring and quantifying the impacts of CC related to these principles. Two key challenges include: (1) identification of measures that capture CC impacts from the medical system, care provider, and family perspectives; and (2) recognition of the important community context outside of a hospital or clinical setting. Methods: This study used a multilevel model variant of the triangulation mixed methods design to assess the impact of a CC project implemented in Alberta, Canada, on family quality of life, resource use, and care integration at the broader environmental and household levels. At the broader environmental level, we used linked administrative data. At the household level we used quantitative pre-post survey datasets, and aggregate findings from qualitative interviews to measure group-level impacts and an embedded multiple-case design to draw comparisons, capture the nuances of children with NDD-MC and their families, and expand on factors driving the high variability in outcome measures. Three theoretical propositions formed the basis of the analytical strategy for our case study evidence to explore factors affecting the high variability in outcome measures. Discussion: This study expanded on the factors used to measure the outcomes of CC and adds to our understanding of how CC as an intervention impacts resource use, quality of life, and care integration of children with NDD-MC and their families. Given the heterogeneous nature of this population, evaluation studies that account for the variable and multi-level impacts of CC interventions are critical to inform practice, implementation, and policy of CC for children with NDD-MC.

Hyperhydration to Improve Kidney Outcomes in Children with Shiga Toxin-Producing E. coli Infection: a multinational embedded cluster crossover randomized trial (the HIKO STEC trial).

BACKGROUND: Shiga toxin-producing E. coli (STEC) infections affect children and adults worldwide, and treatment remain solely supportive. Up to 15-20% of children infected by high-risk STEC (i.e., E. coli that produce Shiga toxin 2) develop hemolytic anemia, thrombocytopenia, and kidney failure (i.e., hemolytic uremic syndrome (HUS)), over half of whom require acute dialysis and 3% die. Although no therapy is widely accepted as being able to prevent the development of HUS and its complications, several observational studies suggest that intravascular volume expansion (hyperhydration) may prevent end organ damage. A randomized trial is needed to confirm or refute this hypothesis. METHODS: We will conduct a pragmatic, embedded, cluster-randomized, crossover trial in 26 pediatric institutions to determine if hyperhydration, compared to conservative fluid management, improves outcomes in 1040 children with high-risk STEC infections. The primary outcome is major adverse kidney events within 30 days (MAKE30), a composite measure that includes death, initiation of new renal replacement therapy, or persistent kidney dysfunction. Secondary outcomes include life-threatening, extrarenal complications, and development of HUS. Pathway eligible children will be treated per institutional allocation to each pathway. In the hyperhydration pathway, all eligible children are hospitalized and administered 200% maintenance balanced crystalloid fluids up to targets of 10% weight gain and 20% reduction in hematocrit. Sites in the conservative fluid management pathway manage children as in- or outpatients, based on clinician preference, with the pathway focused on close laboratory monitoring, and maintenance of euvolemia. Based on historical data, we estimate that 10% of children in our conservative fluid management pathway will experience the primary outcome. With 26 clusters enrolling a mean of 40 patients each with an intraclass correlation coefficient of 0.11, we will have 90% power to detect a 5% absolute risk reduction. DISCUSSION: HUS is a devastating illness with no treatment options. This pragmatic study will determine if hyperhydration can reduce morbidity associated with HUS in children with high-risk STEC infection. TRIAL REGISTRATION: ClinicalTrials.gov NCT05219110 . Registered on February 1, 2022.

Sequential perturbations to mouse corticogenesis following in utero maternal immune activation.

In utero exposure to maternal immune activation (MIA) is an environmental risk factor for neurodevelopmental and neuropsychiatric disorders. Animal models provide an opportunity to identify mechanisms driving neuropathology associated with MIA. We performed time-course transcriptional profiling of mouse cortical development following induced MIA via poly(I:C) injection at E12.5. MIA-driven transcriptional changes were validated via protein analysis, and parallel perturbations to cortical neuroanatomy were identified via imaging. MIA-induced acute upregulation of genes associated with hypoxia, immune signaling, and angiogenesis, by 6 hr following exposure. This acute response was followed by changes in proliferation, neuronal and glial specification, and cortical lamination that emerged at E14.5 and peaked at E17.5. Decreased numbers of proliferative cells in germinal zones and alterations in neuronal and glial populations were identified in the MIA-exposed cortex. Overall, paired transcriptomic and neuroanatomical characterization revealed a sequence of perturbations to corticogenesis driven by mid-gestational MIA.

Alterations in Retrotransposition, Synaptic Connectivity, and Myelination Implicated by Transcriptomic Changes Following Maternal Immune Activation in Nonhuman Primates.

BACKGROUND: Maternal immune activation (MIA) is a proposed risk factor for multiple neuropsychiatric disorders, including schizophrenia. However, the molecular mechanisms through which MIA imparts risk remain poorly understood. A recently developed nonhuman primate model of exposure to the viral mimic poly:ICLC during pregnancy shows abnormal social and repetitive behaviors and elevated striatal dopamine, a molecular hallmark of human psychosis, providing an unprecedented opportunity for studying underlying molecular correlates. METHODS: We performed RNA sequencing across psychiatrically relevant brain regions (prefrontal cortex, anterior cingulate, hippocampus) and primary visual cortex for comparison from 3.5- to 4-year-old male MIA-exposed and control offspring-an age comparable to mid adolescence in humans. RESULTS: We identify 266 unique genes differentially expressed in at least one brain region, with the greatest number observed in hippocampus. Co-expression networks identified region-specific alterations in synaptic signaling and oligodendrocytes. Although we observed temporal and regional differences, transcriptomic changes were shared across first- and second-trimester exposures, including for the top differentially expressed genes-PIWIL2 and MGARP. In addition to PIWIL2, several other regulators of retrotransposition and endogenous transposable elements were dysregulated following MIA, potentially connecting MIA to retrotransposition. CONCLUSIONS: Together, these results begin to elucidate the brain-level molecular processes through which MIA may impart risk for psychiatric disease.

Baseline immunoreactivity before pregnancy and poly(I:C) dose combine to dictate susceptibility and resilience of offspring to maternal immune activation.

Despite the potential of rodent models of maternal immune activation (MIA) to identify new biomarkers and therapeutic interventions for a range of psychiatric disorders, current approaches using these models ignore two of the most important aspects of this risk factor for human disease: (i) most pregnancies are resilient to maternal viral infection and (ii) susceptible pregnancies can lead to different combinations of phenotypes in offspring. Here, we report two new sources of variability-the baseline immunoreactivity (BIR) of isogenic females prior to pregnancy and differences in immune responses in C57BL/6 dams across vendors-that contribute to resilience and susceptibility to distinct combinations of behavioral and biological outcomes in offspring. Similar to the variable effects of human maternal infection, MIA in mice does not cause disease-related phenotypes in all pregnancies and a combination of poly(I:C) dose and BIR predicts susceptibility and resilience of pregnancies to aberrant repetitive behaviors and alterations in striatal protein levels in offspring. Even more surprising is that the intermediate levels of BIR and poly(I:C) dose are most detrimental to offspring, with higher BIR and poly(I:C) doses conferring resilience to measured phenotypes in offspring. Importantly, we identify the BIR of female mice as a biomarker before pregnancy that predicts which dams will be most at risk as well as biomarkers in the brains of newborn offspring that correlate with changes in repetitive behaviors. Together, our results highlight considerations for optimizing MIA protocols to enhance rigor and reproducibility and reveal new factors that drive susceptibility of some pregnancies and resilience of others to MIA-induced abnormalities in offspring.

Increasing access to microfluidics for studying fungi and other branched biological structures.

BACKGROUND: Microfluidic systems are well-suited for studying mixed biological communities for improving industrial processes of fermentation, biofuel production, and pharmaceutical production. The results of which have the potential to resolve the underlying mechanisms of growth and transport in these complex branched living systems. Microfluidics provide controlled environments and improved optical access for real-time and high-resolution imaging studies that allow high-content and quantitative analyses. Studying growing branched structures and the dynamics of cellular interactions with both biotic and abiotic cues provides context for molecule production and genetic manipulations. To make progress in this arena, technical and logistical barriers must be overcome to more effectively deploy microfluidics in biological disciplines. A principle technical barrier is the process of assembling, sterilizing, and hydrating the microfluidic system; the lack of the necessary equipment for the preparatory process is a contributing factor to this barrier. To improve access to microfluidic systems, we present the development, characterization, and implementation of a microfluidics assembly and packaging process that builds on self-priming point-of-care principles to achieve "ready-to-use microfluidics." RESULTS: We present results from domestic and international collaborations using novel microfluidic architectures prepared with a unique packaging protocol. We implement this approach by focusing primarily on filamentous fungi; we also demonstrate the utility of this approach for collaborations on plants and neurons. In this work we (1) determine the shelf-life of ready-to-use microfluidics, (2) demonstrate biofilm-like colonization on fungi, (3) describe bacterial motility on fungal hyphae (fungal highway), (4) report material-dependent bacterial-fungal colonization, (5) demonstrate germination of vacuum-sealed Arabidopsis seeds in microfluidics stored for up to 2 weeks, and (6) observe bidirectional cytoplasmic streaming in fungi. CONCLUSIONS: This pre-packaging approach provides a simple, one step process to initiate microfluidics in any setting for fungal studies, bacteria-fungal interactions, and other biological inquiries. This process improves access to microfluidics for controlling biological microenvironments, and further enabling visual and quantitative analysis of fungal cultures.

Brain, Immunity, Gut: "BIG" Links between Pregnancy and Autism.

Although dysregulation of brain, immune, and gut physiology during pregnancy have each been implicated in neuropsychiatric disorders, whether and how these presumably distinct systems are linked to cause disease is unclear. Kim et al. (2017) and Shin Yim et al. (2017) identify a pathway to explain how these aspects of our physiology are deeply and inextricably connected.

Maternal immune activation: Implications for neuropsychiatric disorders.

Epidemiological evidence implicates maternal infection as a risk factor for autism spectrum disorder and schizophrenia. Animal models corroborate this link and demonstrate that maternal immune activation (MIA) alone is sufficient to impart lifelong neuropathology and altered behaviors in offspring. This Review describes common principles revealed by these models, highlighting recent findings that strengthen their relevance for schizophrenia and autism and are starting to reveal the molecular mechanisms underlying the effects of MIA on offspring. The role of MIA as a primer for a much wider range of psychiatric and neurologic disorders is also discussed. Finally, the need for more research in this nascent field and the implications for identifying and developing new treatments for individuals at heightened risk for neuroimmune disorders are considered.

Immune mediators in the brain and peripheral tissues in autism spectrum disorder.

Increasing evidence points to a central role for immune dysregulation in autism spectrum disorder (ASD). Several ASD risk genes encode components of the immune system and many maternal immune system-related risk factors--including autoimmunity, infection and fetal reactive antibodies--are associated with ASD. In addition, there is evidence of ongoing immune dysregulation in individuals with ASD and in animal models of this disorder. Recently, several molecular signalling pathways--including pathways downstream of cytokines, the receptor MET, major histocompatibility complex class I molecules, microglia and complement factors--have been identified that link immune activation to ASD phenotypes. Together, these findings indicate that the immune system is a point of convergence for multiple ASD-related genetic and environmental risk factors.

Alterations in immune cells and mediators in the brain: it's not always neuroinflammation!

Neuroinflammation was once a clearly defined term denoting pathological immune processes within the central nervous system (CNS). Historically, this term was used to indicate the four hallmarks of peripheral inflammaton that occur following severe CNS injuries, such as stroke, injury or infection. Recently, however, the definition of neuroinflammation has relaxed to the point that it is often now assumed to be present when even only a single classical hallmark of inflammation is measured. As a result, a wide range of disorders, from psychiatric to degenerative diseases, are now assumed to have an integral inflammatory component. Ironically, at the same time, research has revealed unexpected nonclassical immune actions of immune mediators and cells in the CNS in the absence of pathology, increasing the likelihood that homeostatic and adaptive immune processes in the CNS will be mistaken for neuroinflammation. Thus, we suggest reserving the term neuroinflammation for contexts where multiple signs of inflammation are present to avoid erroneously classifying disorders as inflammatory when they may instead be caused by nonimmune etiologies or secondary immune processes that serve adaptive roles.

MHCI requires MEF2 transcription factors to negatively regulate synapse density during development and in disease.

Major histocompatibility complex class I (MHCI) molecules negatively regulate cortical connections and are implicated in neurodevelopmental disorders, including autism spectrum disorders and schizophrenia. However, the mechanisms that mediate these effects are unknown. Here, we report a novel MHCI signaling pathway that requires the myocyte enhancer factor 2 (MEF2) transcription factors. In young rat cortical neurons, MHCI regulates MEF2 in an activity-dependent manner and requires calcineurin-mediated activation of MEF2 to limit synapse density. Manipulating MEF2 alone alters synaptic strength and GluA1 content, but not synapse density, implicating activity-dependent MEF2 activation as critical for MHCI signaling. The MHCI-MEF2 pathway identified here also mediates the effects of a mouse model of maternal immune activation (MIA) on connectivity in offspring. MHCI and MEF2 levels are higher, and synapse density is lower, on neurons from MIA offspring. Most important, dysregulation of MHCI and MEF2 is required for the MIA-induced reduction in neural connectivity. These results identify a previously unknown MHCI-calcineurin-MEF2 signaling pathway that regulates the establishment of cortical connections and mediates synaptic defects caused by MIA, a risk factor for autism spectrum disorders and schizophrenia.

Flow cytometric identification and functional characterization of immature and mature circulating endothelial cells.

OBJECTIVE: We sought to identify and characterize 2 distinct populations of bona fide circulating endothelial cells, including the endothelial colony-forming cell (ECFC), by polychromatic flow cytometry (PFC), colony assays, immunomagnetic selection, and electron microscopy. METHODS AND RESULTS: Mononuclear cells from human umbilical cord blood and peripheral blood were analyzed using our recently published PFC protocol. A population of cells containing both ECFCs and mature circulating endothelial cells was determined by varying expressions of CD34, CD31, and CD146 but not AC133 and CD45. After immunomagnetic separation, these cells failed to form hematopoietic colonies, yet clonogenic endothelial colonies with proliferative potential were obtained, thus verifying their identity as ECFCs. The frequency of ECFCs were increased in cord blood and were extremely rare in the peripheral blood of healthy adults. We also detected another mature endothelial cell population in the circulation that was apoptotic. Finally, when comparing this new protocol with a prior method, we determined that the present protocol identifies circulating endothelial cells, whereas the earlier protocol identified extracellular vesicles. CONCLUSIONS: Two populations of circulating endothelial cells, including the functionally characterized ECFC, are now identifiable in human cord blood and peripheral blood by PFC.

Application of polychromatic flow cytometry to identify novel subsets of circulating cells with angiogenic potential.

Defining whether human circulating proangiogenic cells represent a subset of the hematopoietic system and express CD45 or are hematopoietic derivatives that do not express CD45 (and are called endothelial progenitor cells) remains controversial. We have previously developed a polychromatic flow cytometry (PFC) protocol to isolate subsets of hematopoietic cells and we now identify the circulating pool of CD34(+)CD45(dim) cells representing functional circulating hematopoietic stem and progenitor cells (CHSPCs) that can be separated on the basis of AC133 expression and report that the AC133(+) subset of the CHSPCs enhances the growth of tumor blood vessels in vivo in immunodeficient mice. In addition, the ratio of AC133(+) proangiogenic CHSPCs to AC133(-) nonangiogenic CHSPCs unambiguously correlates with the severity of the clinical state of patients with peripheral arterial disease. In sum, a PFC protocol validated via in vitro and in vivo analyses, can be used to interrogate the roles of human hematopoietic elements in the growth and maintenance of the vasculature.

Endothelial abnormalities in adolescents with type 1 diabetes: a biomarker for vascular sequelae?

OBJECTIVE: To evaluate whether counts of circulating colony forming unit-endothelial cells (CFU-ECs), cells co-expressing CD34, CD133, and CD31 (CD34+CD133+CD31+), and CD34+CD45- cells are altered in adolescents with type 1 diabetes and if the changes in counts correlate with endothelial dysfunction. STUDY DESIGN: Adolescents with diabetes (ages 18 to 22 years) and race- and sex-matched control subjects were studied. We assessed circulating CFU-ECs, using colony assays, and CD34+CD133+CD31+ and CD34+CD45- cells, using poly-chromatic flow cytometry. CFU-ECs and CD34+CD133+CD31+ are hematopoietic-derived progenitors that inversely correlate with cardiovascular risk in adults. CD34+CD45- cells are enriched for endothelial cells with robust vasculogenic potential. Vascular reactivity was tested by laser Doppler iontophoresis. RESULTS: Subjects with diabetes had lower CD34+CD133+CD31+ cells, a trend toward reduced CFU-ECs, and increased CD34+CD45- cells compared with control subjects. Endothelium-dependent vasodilation was impaired in subjects with diabetes, which correlated with reductions in circulating CD34+CD133+CD31+ cells. CONCLUSIONS: Long-term sequelae of type 1 diabetes include vasculopathies. Endothelial progenitor cells promote vascular health by facilitating endothelial integrity and function. Lower CD34+CD133+CD31+ cells may be a harbinger of future macrovascular disease risk. Higher circulating CD34+CD45- cells may reflect ongoing endothelial damage. These cells are potential biomarkers to guide therapeutic interventions to enhance endothelial function and to prevent progression to overt vascular disease.

Identification of endothelial cells and progenitor cell subsets in human peripheral blood.

An assay for circulating cell subsets in human peripheral blood by flow cytometry is used as a biomarker to determine cardiovascular disease risk and tumor responsiveness to chemotherapy since endothelial progenitor cells (EPCs) function in vasculogenesis and angiogenesis. Despite analytical advances in polychromatic flow cytometry (PFC), conventional approaches are routinely utilized to enumerate and isolate EPCs, which has led to varied results in clinical studies, potential cellular misidentification, and thus a lack of a plausible biological explanation for how purported EPCs function. Herein, a reproducible PFC protocol is provided to identify a rare circulating endothelial colony-forming cell (ECFC) with proliferative potential, along with a population of circulating progenitor cells (CPCs) in which the ratio analysis distinguishes between healthy and disease populations. In sum, a reliable PFC protocol, which can be used to investigate the roles of human hematopoietic and endothelial elements in the growth and maintenance of the vasculature, is described.

Genetic and cellular evidence of vascular inflammation in neurofibromin-deficient mice and humans.

Neurofibromatosis type 1 (NF1) results from mutations in the NF1 tumor suppressor gene, which encodes the protein neurofibromin. NF1 patients display diverse clinical manifestations, including vascular disease, which results from neointima formation and vessel occlusion. However, the pathogenesis of NF1 vascular disease remains unclear. Vessel wall homeostasis is maintained by complex interactions between vascular and bone marrow-derived cells (BMDCs), and neurofibromin regulates the function of each cell type. Therefore, utilizing cre/lox techniques and hematopoietic stem cell transplantation to delete 1 allele of Nf1 in endothelial cells, vascular smooth muscle cells, and BMDCs alone, we determined which cell lineage is critical for neointima formation in vivo in mice. Here we demonstrate that heterozygous inactivation of Nf1 in BMDCs alone was necessary and sufficient for neointima formation after vascular injury and provide evidence of vascular inflammation in Nf1+/- mice. Further, analysis of peripheral blood from NF1 patients without overt vascular disease revealed increased concentrations of inflammatory cells and cytokines previously linked to vascular inflammation and vasoocclusive disease. These data provide genetic and cellular evidence of vascular inflammation in NF1 patients and Nf1+/- mice and provide a framework for understanding the pathogenesis of NF1 vasculopathy and potential therapeutic and diagnostic interventions.

Inhibition of p38alpha MAPK disrupts the pathological loop of proinflammatory factor production in the myelodysplastic syndrome bone marrow microenvironment.

Myelodysplastic syndromes (MDS) are common causes of ineffective hematopoiesis and cytopenias in the elderly. Various myelosuppressive and proinflammatory cytokines have been implicated in the high rates of apoptosis and hematopoietic suppression seen in MDS. We have previously shown that p38 MAPK is overactivated in MDS hematopoietic progenitors, which led to current clinical studies of the selective p38alpha inhibitor, SCIO-469, in this disease. We now demonstrate that the myelosuppressive cytokines TNFalpha and IL-1beta are secreted by bone marrow (BM) cells in a p38 MAPK-dependent manner. Their secretion is stimulated by paracrine interactions between BM stromal and mononuclear cells and cytokine induction correlates with CD34+ stem cell apoptosis in an inflammation-simulated in vitro bone marrow microenvironment. Treatment with SCIO-469 inhibits TNF secretion in primary MDS bone marrow cells and protects cytogenetically normal progenitors from apoptosis ex vivo. Furthermore, p38 inhibition diminishes the expression of TNFalpha or IL-1beta-induced proinflammatory chemokines in BM stromal cells. These data indicate that p38 inhibition has anti-inflammatory effects on the bone marrow microenvironment that complements its cytoprotective effect on progenitor survival. These findings support clinical investigation of p38alpha as a potential therapeutic target in MDS and other related diseases characterised by inflammatory bone marrow failure.

Src family kinases promote AML cell survival through activation of signal transducers and activators of transcription (STAT).

We investigated the role of Src family kinases (SFKs) in the regulation of STAT activation in myeloid leukemia cells. Two of 6 AML cell lines displayed constitutive STAT5 activation, whereas four cell lines had constitutive SFK activity. Treatment with the SFK inhibitors suppressed STAT5 activation and decreased viability. Akt phosphorylation and Mcl-1 expression decreased after SFK inhibition accompanied by apoptosis induction. In primary AML specimens, SFK inhibitors suppressed proliferation in 5 of 14 specimens. These data indicate that Src-STAT5 and Src-Akt pathways are integral survival signal pathways in AML cells. Src inhibition may represent a novel treatment strategy for investigation in AML.

In vitro hyperglycemia or a diabetic intrauterine environment reduces neonatal endothelial colony-forming cell numbers and function.

OBJECTIVE: Emerging data demonstrate that maternal diabetes has long-term health consequences for offspring, including the development of hypertension. In adults, circulating endothelial progenitor cells (EPCs) participate in vascular repair, and EPC numbers and function inversely correlate with the risk of developing vascular disease. Therefore, our objectives were to determine whether hyperglycemia or exposure to a diabetic intrauterine environment alters EPC function. RESEARCH DESIGN AND METHODS: We used well-established clonogenic endothelial colony-forming cell (ECFC) assays and murine transplantation experiments to examine human vasculogenesis. RESULTS: Both in vitro hyperglycemia and a diabetic intrauterine environment reduced ECFC colony formation, self-renewal capacity, and capillary-like tube formation in matrigel. This cellular phenotype was linked to premature senescence and reduced proliferation. Further, cord blood ECFCs from diabetic pregnancies formed fewer chimeric vessels de novo after transplantation into immunodeficient mice compared with neonatal ECFCs harvested from uncomplicated pregnancies. CONCLUSIONS; Collectively, these data demonstrate that hyperglycemia or exposure to a diabetic intrauterine environment diminishes neonatal ECFC function both in vitro and in vivo, providing potential mechanistic insights into the long-term cardiovascular complications observed in newborns of diabetic pregnancies.