Two distinct evolutionary conserved neural degeneration pathways characterized in a colonial chordate.

Colonial tunicates are marine organisms that possess multiple brains simultaneously during their colonial phase. While the cyclical processes of neurogenesis and neurodegeneration characterizing their life cycle have been documented previously, the cellular and molecular changes associated with such processes and their relationship with variation in brain morphology and individual (zooid) behavior throughout adult life remains unknown. Here, we introduce Botryllus schlosseri as an invertebrate model for neurogenesis, neural degeneration, and evolutionary neuroscience. Our analysis reveals that during the weekly colony budding (i.e., asexual reproduction), prior to programmed cell death and removal by phagocytes, decreases in the number of neurons in the adult brain are associated with reduced behavioral response and significant change in the expression of 73 mammalian homologous genes associated with neurodegenerative disease. Similarly, when comparing young colonies (1 to 2 y of age) to those reared in a laboratory for ∼20 y, we found that older colonies contained significantly fewer neurons and exhibited reduced behavioral response alongside changes in the expression of 148 such genes (35 of which were differentially expressed across both timescales). The existence of two distinct yet apparently related neurodegenerative pathways represents a novel platform to study the gene products governing the relationship between aging, neural regeneration and degeneration, and loss of nervous system function. Indeed, as a member of an evolutionary clade considered to be a sister group of vertebrates, this organism may be a fundamental resource in understanding how evolution has shaped these processes across phylogeny and obtaining mechanistic insight.

Complex mammalian-like haematopoietic system found in a colonial chordate.

Haematopoiesis is an essential process that evolved in multicellular animals. At the heart of this process are haematopoietic stem cells (HSCs), which are multipotent and self-renewing, and generate the entire repertoire of blood and immune cells throughout an animal's life1. Although there have been comprehensive studies on self-renewal, differentiation, physiological regulation and niche occupation in vertebrate HSCs, relatively little is known about the evolutionary origin and niches of these cells. Here we describe the haematopoietic system of Botryllus schlosseri, a colonial tunicate that has a vasculature and circulating blood cells, and interesting stem-cell biology and immunity characteristics2-8. Self-recognition between genetically compatible B. schlosseri colonies leads to the formation of natural parabionts with shared circulation, whereas incompatible colonies reject each other3,4,7. Using flow cytometry, whole-transcriptome sequencing of defined cell populations and diverse functional assays, we identify HSCs, progenitors, immune effector cells and an HSC niche, and demonstrate that self-recognition inhibits allospecific cytotoxic reactions. Our results show that HSC and myeloid lineage immune cells emerged in a common ancestor of tunicates and vertebrates, and also suggest that haematopoietic bone marrow and the B. schlosseri endostyle niche evolved from a common origin.

Combining CD47 blockade with trastuzumab eliminates HER2-positive breast cancer cells and overcomes trastuzumab tolerance.

Trastuzumab, a targeted anti-human epidermal-growth-factor receptor-2 (HER2) monoclonal antibody, represents a mainstay in the treatment of HER2-positive (HER2+) breast cancer. Although trastuzumab treatment is highly efficacious for early-stage HER2+ breast cancer, the majority of advanced-stage HER2+ breast cancer patients who initially respond to trastuzumab acquire resistance to treatment and relapse, despite persistence of HER2 gene amplification/overexpression. Here, we sought to leverage HER2 overexpression to engage antibody-dependent cellular phagocytosis (ADCP) through a combination of trastuzumab and anti-CD47 macrophage checkpoint immunotherapy. We have previously shown that blockade of CD47, a surface protein expressed by many malignancies (including HER2+ breast cancer), is an effective anticancer therapy. CD47 functions as a "don't eat me" signal through its interaction with signal regulatory protein-α (SIRPα) on macrophages to inhibit phagocytosis. Hu5F9-G4 (magrolimab), a humanized monoclonal antibody against CD47, blocks CD47's "don't eat me" signal, thereby facilitating macrophage-mediated phagocytosis. Preclinical studies have shown that combining Hu5F9-G4 with tumor-targeting antibodies, such as rituximab, further enhances Hu5F9-G4's anticancer effects via ADCP. Clinical trials have additionally demonstrated that Hu5F9-G4, in combination with rituximab, produced objective responses in patients whose diffuse large B cell lymphomas had developed resistance to rituximab and chemotherapy. These studies led us to hypothesize that combining Hu5F9-G4 with trastuzumab would produce an anticancer effect in antibody-dependent cellular cytotoxicity (ADCC)-tolerant HER2+ breast cancer. This combination significantly suppressed the growth of ADCC-tolerant HER2+ breast cancers via Fc-dependent ADCP. Our study demonstrates that combining trastuzumab and Hu5F9-G4 represents a potential new treatment option for HER2+ breast cancer patients, even for patients whose tumors have progressed after trastuzumab.

Botryllus schlosseri as a Unique Colonial Chordate Model for the Study and Modulation of Innate Immune Activity.

Understanding the mechanisms that sustain immunological nonreactivity is essential for maintaining tissue in syngeneic and allogeneic settings, such as transplantation and pregnancy tolerance. While most transplantation rejections occur due to the adaptive immune response, the proinflammatory response of innate immunity is necessary for the activation of adaptive immunity. Botryllus schlosseri, a colonial tunicate, which is the nearest invertebrate group to the vertebrates, is devoid of T- and B-cell-based adaptive immunity. It has unique characteristics that make it a valuable model system for studying innate immunity mechanisms: (i) a natural allogeneic transplantation phenomenon that results in either fusion or rejection; (ii) whole animal regeneration and noninflammatory resorption on a weekly basis; (iii) allogeneic resorption which is comparable to human chronic rejection. Recent studies in B. schlosseri have led to the recognition of a molecular and cellular framework underlying the innate immunity loss of tolerance to allogeneic tissues. Additionally, B. schlosseri was developed as a model for studying hematopoietic stem cell (HSC) transplantation, and it provides further insights into the similarities between the HSC niches of human and B. schlosseri. In this review, we discuss why studying the molecular and cellular pathways that direct successful innate immune tolerance in B. schlosseri can provide novel insights into and potential modulations of these immune processes in humans.

The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors.

CD47, a "don't eat me" signal for phagocytic cells, is expressed on the surface of all human solid tumor cells. Analysis of patient tumor and matched adjacent normal (nontumor) tissue revealed that CD47 is overexpressed on cancer cells. CD47 mRNA expression levels correlated with a decreased probability of survival for multiple types of cancer. CD47 is a ligand for SIRPα, a protein expressed on macrophages and dendritic cells. In vitro, blockade of CD47 signaling using targeted monoclonal antibodies enabled macrophage phagocytosis of tumor cells that were otherwise protected. Administration of anti-CD47 antibodies inhibited tumor growth in orthotopic immunodeficient mouse xenotransplantation models established with patient tumor cells and increased the survival of the mice over time. Anti-CD47 antibody therapy initiated on larger tumors inhibited tumor growth and prevented or treated metastasis, but initiation of the therapy on smaller tumors was potentially curative. The safety and efficacy of targeting CD47 was further tested and validated in immune competent hosts using an orthotopic mouse breast cancer model. These results suggest all human solid tumor cells require CD47 expression to suppress phagocytic innate immune surveillance and elimination. These data, taken together with similar findings with other human neoplasms, show that CD47 is a commonly expressed molecule on all cancers, its function to block phagocytosis is known, and blockade of its function leads to tumor cell phagocytosis and elimination. CD47 is therefore a validated target for cancer therapies.

Hedgehog-responsive candidate cell of origin for diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine gliomas (DIPGs) are highly aggressive tumors of childhood that are almost universally fatal. Our understanding of this devastating cancer is limited by a dearth of available tissue for study and by the lack of a faithful animal model. Intriguingly, DIPGs are restricted to the ventral pons and occur during a narrow window of middle childhood, suggesting dysregulation of a postnatal neurodevelopmental process. Here, we report the identification of a previously undescribed population of immunophenotypic neural precursor cells in the human and murine brainstem whose temporal and spatial distributions correlate closely with the incidence of DIPG and highlight a candidate cell of origin. Using early postmortem DIPG tumor tissue, we have established in vitro and xenograft models and find that the Hedgehog (Hh) signaling pathway implicated in many developmental and oncogenic processes is active in DIPG tumor cells. Modulation of Hh pathway activity has functional consequences for DIPG self-renewal capacity in neurosphere culture. The Hh pathway also appears to be active in normal ventral pontine precursor-like cells of the mouse, and unregulated pathway activity results in hypertrophy of the ventral pons. Together, these findings provide a foundation for understanding the cellular and molecular origins of DIPG, and suggest that the Hh pathway represents a potential therapeutic target in this devastating pediatric tumor.

P66 is a bacterial mimic of CD47 that binds the anti-phagocytic receptor SIRPα and facilitates macrophage evasion by Borrelia burgdorferi.

Innate immunity, the first line of defense against pathogens, relies on efficient elimination of invading agents by phagocytes. In the co-evolution of host and pathogen, pathogens developed mechanisms to dampen and evade phagocytic clearance. Here, we report that bacterial pathogens can evade clearance by macrophages through mimicry at the mammalian anti-phagocytic "don't eat me" signaling axis between CD47 (ligand) and SIRPα (receptor). We identified a protein, P66, on the surface of Borrelia burgdorferi that, like CD47, is necessary and sufficient to bind the macrophage receptor SIRPα. Expression of the gene encoding the protein is required for bacteria to bind SIRPα or a high-affinity CD47 reagent. Genetic deletion of p66 increases phagocytosis by macrophages. Blockade of P66 during infection promotes clearance of the bacteria. This study demonstrates that mimicry of the mammalian anti-phagocytic protein CD47 by B. burgdorferi inhibits macrophage-mediated bacterial clearance. Such a mechanism has broad implications for understanding of host-pathogen interactions and expands the function of the established innate immune checkpoint receptor SIRPα. Moreover, this report reveals P66 as a novel therapeutic target in the treatment of Lyme Disease.

Multiple Forms of Neural Cell Death in the Cyclical Brain Degeneration of A Colonial Chordate.

Human neuronal loss occurs through different cellular mechanisms, mainly studied in vitro. Here, we characterized neuronal death in B. schlosseri, a marine colonial tunicate that shares substantial genomic homology with mammals and has a life history in which controlled neurodegeneration happens simultaneously in the brains of adult zooids during a cyclical phase named takeover. Using an ultrastructural and transcriptomic approach, we described neuronal death forms in adult zooids before and during the takeover phase while comparing adult zooids in takeover with their buds where brains are refining their structure. At takeover, we found in neurons clear morphologic signs of apoptosis (i.e., chromatin condensation, lobed nuclei), necrosis (swollen cytoplasm) and autophagy (autophagosomes, autolysosomes and degradative multilamellar bodies). These results were confirmed by transcriptomic analyses that highlighted the specific genes involved in these cell death pathways. Moreover, the presence of tubulovesicular structures in the brain medulla alongside the over-expression of prion disease genes in late cycle suggested a cell-to-cell, prion-like propagation recalling the conformational disorders typical of some human neurodegenerative diseases. We suggest that improved understanding of how neuronal alterations are regulated in the repeated degeneration-regeneration program of B. schlosseri may yield mechanistic insights relevant to the study of human neurodegenerative diseases.

SIRPα controls CD47-dependent platelet clearance in mice and humans.

Over the last decade, more data has revealed that increased surface expression of the "don't eat me" CD47 protein on cancer cells plays a role in immune evasion and tumor progression, with CD47 blockade emerging as a new therapy in immuno-oncology. CD47 is critical in regulating cell homeostasis and clearance, as binding of CD47 to the inhibitory receptor SIRPα can prevent phagocytosis and macrophage-mediated cell clearance. The purpose of this study was to examine the role of the CD47-SIRPα signal in platelet homeostasis and clearance. Therapeutic reagents targeting the CD47-SIRPα axis are very promising for treatment of hematologic malignancies and solid tumors, but lead to transient anemia or thrombocytopenia in a subset of patients. We found that platelet homeostatic clearance is regulated through the CD47-SIRPα axis and that therapeutic blockade to disrupt this interaction in mice and in humans has a significant impact on platelet levels. Furthermore, we identified genetic variations at the SIRPA locus that impact platelet levels in humans such that higher SIRPA gene expression is associated with higher platelet levels. SIRPA expression at either end of the normal range may affect clinical outcomes of treatment with anti-CD47 therapy.

Is hypoimmunogenic stem cell therapy safe in times of pandemics?

The manipulation of human leukocyte antigens (HLAs) and immune modulatory factors in "universal" human pluripotent stem cells (PSCs) holds promise for immunological tolerance without HLA matching. This paradigm raises concerns should "universal" grafts become virally infected. Furthermore, immunological manipulation might functionally impair certain progeny, such as hematopoietic stem cells. We discuss the risks and benefits of hypoimmunogenic PSCs, and the need to further advance HLA matching and autologous strategies.

The efficacy of single pharmacist medication review among type II diabetic patients who take six chronic medications or more: a case-control study.

BACKGROUND: Pharmacist medication review has been implemented in many health organizations throughout the world in an attempt to alleviate the underlying risk of polypharmacy in elderly patients. These consultations are often frequent and prolonged, and are thus associated with increased costs. To date, data regarding the most effective way to utilize pharmacist consultations for the improvement of health status is scant. AIM: To evaluate the effectiveness of a single pharmacist consultation on changes in chronic medication regimes and on selected outcomes of diabetes 1-year after the consultation. METHODS: A case-control study included an intervention group of 740 patients who had pharmacist consultations and a reference group of 1476 matched patients who did not have a pharmacist consultation. 1-year outcome measures were compared including changes in medications, improved safety, and objective variables such as Hba1c, blood pressure, and lipid profile. RESULTS: In the pharmacist consultation group, there were significantly more treatment changes ([mean 1.5 vs. 0.7, p < 0.001 medications were stopped], and [mean 1.3 vs. 0.4, p < 0.05 medications were started]). Patient safety improved with a general reduction in opiates and benzodiazepines ([50.0% vs. 31.6%, p < 0.05 opioids were stopped] and [58.8% vs 43.8%, p < 0.001 benzodiazepines were stopped]). Sulfonylurea treatment reduced (10.7% vs. 3.6%, p < 0.05 patients who stopped Sulfonylurea) and Glucagon-like peptide-1 receptor agonists (GLP-1) increased (16.4% vs. 11.2%, p < 0.001 patients who started GLP-1). Additionally, HbA1c levels showed a small decrease in the pharmacist consultation group ([- 0.18 ± 1.11] vs. [- 0.051 ± 0.80], p = 0.0058) but no significant differences were found regarding blood pressure or lipids profile. CONCLUSION: A single pharmacist consultation beneficially impacted specific clinical and patient safety outcomes. Pharmacist consultations may thus help resolve polypharmacy complexities in primary care.

Evolutionary perspective on the hematopoietic system through a colonial chordate: allogeneic immunity and hematopoiesis.

Evolution and selection have shaped diverse immune systems throughout phylogeny, the vast majority of which remain unexplored. Botryllus schlosseri is a colonial tunicate, a sister group to vertebrates, that develops as a chordate, then metamorphoses to an asexually reproductive invertebrate that every week makes the same body plan from budded stem cells. Genetically distinct B. schlosseri colonies can fuse to form a chimera, or reject each other based on allogeneic recognition. In chimeras, circulating germline and somatic stem cells participate in development; stem cells compete in all individuals in the fused colonies, with rejection preventing germline parasitism. Here we review the isolation and characterization of B. schlosseri hematopoietic stem cells (HSC) and their niches, and the role of the immune effector cells in allorecognition.

Normal and Neoplastic Stem Cells.

A stem cell is broadly defined as a cell that retains the capacity to self-renew, a feature that confers the ability to continuously make identical daughter cells or additional cells that will differentiate into downstream progeny. This highly regulated genetic program to retain "stemness" is under active investigation. Research in our laboratory has explored similarities and differences in embryonic, tissue-specific, and neoplastic stem cells and their terminally differentiated counterparts. In this review, we will focus on the contributions of our laboratory, in particular on the studies that identified the mouse hematopoietic stem cell (HSC) and the human leukemic stem cell. These studies have led to significant improvements in both preclinical and clinical research, including improved clinical bone marrow transplantation protocols, isolation of nonleukemic HSCs, a cancer immunotherapy currently in clinical trials, and development of a HSC reporter mouse. These studies and the current follow-up research by us and others will continue to identify the properties, function, and regulation of both normal and neoplastic stem cells.

Calreticulin is the dominant pro-phagocytic signal on multiple human cancers and is counterbalanced by CD47.

Under normal physiological conditions, cellular homeostasis is partly regulated by a balance of pro- and anti-phagocytic signals. CD47, which prevents cancer cell phagocytosis by the innate immune system, is highly expressed on several human cancers including acute myeloid leukemia, non-Hodgkin's lymphoma, and bladder cancer. Blocking CD47 with a monoclonal antibody results in phagocytosis of cancer cells and leads to in vivo tumor elimination, yet normal cells remain mostly unaffected. Thus, we postulated that cancer cells must also display a potent pro-phagocytic signal. Here, we identified calreticulin as a pro-phagocytic signal that was highly expressed on the surface of several human cancers, but was minimally expressed on most normal cells. Increased CD47 expression correlated with high amounts of calreticulin on cancer cells and was necessary for protection from calreticulin-mediated phagocytosis. Blocking the interaction of target cell calreticulin with its receptor, low-density lipoprotein receptor-related protein, on phagocytic cells prevented anti-CD47 antibody-mediated phagocytosis. Furthermore, increased calreticulin expression was an adverse prognostic factor in diverse tumors including neuroblastoma, bladder cancer, and non-Hodgkin's lymphoma. These findings identify calreticulin as the dominant pro-phagocytic signal on several human cancers, provide an explanation for the selective targeting of tumor cells by anti-CD47 antibody, and highlight the balance between pro- and anti-phagocytic signals in the immune evasion of cancer.

Insulin-like growth factor 2 is required for progression to advanced medulloblastoma in patched1 heterozygous mice.

Medulloblastoma (MB) can arise in the cerebellum due to genetic activation of the Sonic Hedgehog (Shh) signaling pathway. During normal cerebellum development, Shh spurs the proliferation of granule neuron precursors (GNP), the precursor cells of MB. Mutations in the Shh receptor gene patched1 (ptc1+/-) lead to increased MB incidence in humans and mice. MB tumorigenesis in mice heterozygous for ptc1+/- shows distinct steps of progression. Most ptc1+/- mice form clusters of preneoplastic cells on the surface of the mature cerebellum that actively transcribe Shh target genes. In approximately 15% of mice, these preneoplastic cells will become fast-growing, lethal tumors. It was previously shown that the loss of function of insulin-like growth factor 2 (igf2) suppresses MB formation in ptc1+/- mice. We found that igf2 is not expressed in preneoplastic lesions but is induced as these lesions progress to more advanced MB tumors. Igf2 is not required for formation of preneoplastic lesions but is necessary for progression to advanced tumors. Exogenous Igf2 protein promoted proliferation of MB precursor cells (GNP) and a MB cell line, PZp53(MED). Blocking igf2 signaling inhibited growth of PZp53(MED) cells, implicating igf2 as a potential clinical target.

Dynamic proteomics in individual human cells uncovers widespread cell-cycle dependence of nuclear proteins.

We examined cell cycle-dependent changes in the proteome of human cells by systematically measuring protein dynamics in individual living cells. We used time-lapse microscopy to measure the dynamics of a random subset of 20 nuclear proteins, each tagged with yellow fluorescent protein (YFP) at its endogenous chromosomal location. We synchronized the cells in silico by aligning protein dynamics in each cell between consecutive divisions. We observed widespread (40%) cell-cycle dependence of nuclear protein levels and detected previously unknown cell cycle-dependent localization changes. This approach to dynamic proteomics can aid in discovery and accurate quantification of the extensive regulation of protein concentration and localization in individual living cells.

Immune-related mechanisms participating in resistance and susceptibility to glutamate toxicity.

Glutamate is an essential neurotransmitter in the CNS. However, at abnormally high concentrations it becomes cytotoxic. Recent studies in our laboratory showed that glutamate evokes T cell-mediated protective mechanisms. The aim of the present study was to examine the nature of the glutamate receptors and signalling pathways that participate in immune protection against glutamate toxicity. We show, using the mouse visual system, that glutamate-induced toxicity is strain dependent, not only with respect to the amount of neuronal loss it causes, but also in the pathways it activates. In strains that are genetically endowed with the ability to manifest a T cell-dependent neuroprotective response to glutamate insult, neuronal losses due to glutamate toxicity were relatively small, and treatment with NMDA-receptor antagonist worsened the outcome of exposure to glutamate. In contrast, in mice devoid of T cell-dependent endogenous protection, NMDA receptor antagonist reduced the glutamate-induced neuronal loss. In all strains, blockage of the AMPA/KA receptor was beneficial. Pharmacological (with alpha2-adrenoceptor agonist) or molecular intervention (using either mice overexpressing Bcl-2, or DAP-kinase knockout mice) protected retinal ganglion cells from glutamate toxicity but not from the toxicity of NMDA. The results suggest that glutamate-induced neuronal toxicity involves multiple glutamate receptors, the types and relative contributions of which, vary among strains. We suggest that a multifactorial protection, based on an immune mechanism independent of the specific pathway through which glutamate exerts its toxicity, is likely to be a safer, more comprehensive, and hence more effective strategy for neuroprotection. It might suggest that, because of individual differences, the pharmacological use of NMDA-antagonist for neuroprotective purposes might have an adverse effect, even if the affinity is low.

Death-associated protein (DAP) kinase plays a central role in ceramide-induced apoptosis in cultured hippocampal neurons.

Treatment of cultured hippocampal neurons with high concentrations of short-chain acyl ceramide derivatives, such as N-hexanoyl-D-sphingosine (C(6)-Cer), results in apoptotic cell death. We now show that death-associated protein (DAP) kinase plays an important role in mediating this effect. Upon incubation with C(6)-Cer, DAP kinase levels are elevated as early as 1 h after treatment, reaching levels 2-3-fold higher than untreated cells after 4 h. Neurons cultured from DAP kinase-deficient mice were significantly less sensitive to apoptosis induced by C(6)-Cer or by ceramide generated by high concentrations of nerve growth factor. A peptide corresponding to the 17 amino acids at the C terminus of DAP kinase protected wild type neurons from C(6)-Cer-induced death and from death induced by the addition of exogenous bacterial neutral sphingomyelinase, whereas a scrambled peptide had no protective effect, implying that the DAP kinase C-terminal tail inhibits the function of DAP kinase. Together, these data demonstrate that DAP kinase plays a central role in ceramide-induced cell death in neurons, but the pathway in which DAP kinase is involved is not the only one via which ceramide can induce apoptosis.