Autosomal dominant ApoA4 mutations present as tubulointerstitial kidney disease with medullary amyloidosis.

Sporadic cases of apolipoprotein A-IV medullary amyloidosis have been reported. Here we describe five families found to have autosomal dominant medullary amyloidosis due to two different pathogenic APOA4 variants. A large family with autosomal dominant chronic kidney disease (CKD) and bland urinary sediment underwent whole genome sequencing with identification of a chr11:116692578 G>C (hg19) variant encoding the missense mutation p.L66V of the ApoA4 protein. We identified two other distantly related families from our registry with the same variant and two other distantly related families with a chr11:116693454 C>T (hg19) variant encoding the missense mutation p.D33N. Both mutations are unique to affected families, evolutionarily conserved and predicted to expand the amyloidogenic hotspot in the ApoA4 structure. Clinically affected individuals suffered from CKD with a bland urinary sediment and a mean age for kidney failure of 64.5 years. Genotyping identified 48 genetically affected individuals; 44 individuals had an estimated glomerular filtration rate (eGFR) under 60 ml/min/1.73 m2, including all 25 individuals with kidney failure. Significantly, 11 of 14 genetically unaffected individuals had an eGFR over 60 ml/min/1.73 m2. Fifteen genetically affected individuals presented with higher plasma ApoA4 concentrations. Kidney pathologic specimens from four individuals revealed amyloid deposits limited to the medulla, with the mutated ApoA4 identified by mass-spectrometry as the predominant amyloid constituent in all three available biopsies. Thus, ApoA4 mutations can cause autosomal dominant medullary amyloidosis, with marked amyloid deposition limited to the kidney medulla and presenting with autosomal dominant CKD with a bland urinary sediment. Diagnosis relies on a careful family history, APOA4 sequencing and pathologic studies.

In Vivo Cerebral Imaging of Mutant Huntingtin Aggregates Using 11C-CHDI-180R PET in a Nonhuman Primate Model of Huntington Disease.

Huntington disease (HD) is a neurodegenerative disorder caused by an expanded polyglutamine (CAG) trinucleotide expansion in the huntingtin (HTT) gene that encodes the mutant huntingtin protein (mHTT). Visualization and quantification of cerebral mHTT will provide a proxy for target engagement and a means to evaluate therapeutic interventions aimed at lowering mHTT in the brain. Here, we validated the novel radioligand 11C-labeled 6-(5-((5-methoxypyridin-2-yl)methoxy)benzo[d]oxazol-2-yl)-2-methylpyridazin-3(2H)-one (11C-CHDI-180R) using PET imaging to quantify cerebral mHTT aggregates in a macaque model of HD. Methods: Rhesus macaques received MRI-guided intrastriatal delivery of a mixture of AAV2 and AAV2.retro viral vectors expressing an HTT fragment bearing 85 CAG repeats (85Q, n = 5), a control HTT fragment bearing 10 CAG repeats (10Q, n = 4), or vector diluent only (phosphate-buffered saline, n = 5). Thirty months after surgery, 90-min dynamic PET/CT imaging was used to investigate 11C-CHDI-180R brain kinetics, along with serial blood sampling to measure input function and stability of the radioligand. The total volume of distribution was calculated using a 2-tissue-compartment model as well as Logan graphical analysis for regional quantification. Immunostaining for mHTT was performed to corroborate the in vivo findings. Results: 11C-CHDI-180R displayed good metabolic stability (51.4% ± 4.0% parent in plasma at 60 min after injection). Regional time-activity curves displayed rapid uptake and reversible binding, which were described by a 2-tissue-compartment model. Logan graphical analysis was associated with the 2-tissue-compartment model (r 2 = 0.96, P < 0.0001) and used to generate parametric volume of distribution maps. Compared with controls, animals administered the 85Q fragment exhibited significantly increased 11C-CHDI-180R binding in several cortical and subcortical brain regions (group effect, P < 0.0001). No difference in 11C-CHDI-180R binding was observed between buffer and 10Q animals. The presence of mHTT aggregates in the 85Q animals was confirmed histologically. Conclusion: We validated 11C-CHDI-180R as a radioligand to visualize and quantify mHTT aggregated species in a HD macaque model. These findings corroborate our previous work in rodent HD models and show that 11C-CHDI-180R is a promising tool to assess the mHTT aggregate load and the efficacy of therapeutic strategies.

Allogeneic immunity clears latent virus following allogeneic stem cell transplantation in SIV-infected ART-suppressed macaques.

Allogeneic hematopoietic stem cell transplantation (alloHSCT) from donors lacking C-C chemokine receptor 5 (CCR5Δ32/Δ32) can cure HIV, yet mechanisms remain speculative. To define how alloHSCT mediates HIV cure, we performed MHC-matched alloHSCT in SIV+, anti-retroviral therapy (ART)-suppressed Mauritian cynomolgus macaques (MCMs) and demonstrated that allogeneic immunity was the major driver of reservoir clearance, occurring first in peripheral blood, then peripheral lymph nodes, and finally in mesenteric lymph nodes draining the gastrointestinal tract. While allogeneic immunity could extirpate the latent viral reservoir and did so in two alloHSCT-recipient MCMs that remained aviremic >2.5 years after stopping ART, in other cases, it was insufficient without protection of engrafting cells afforded by CCR5-deficiency, as CCR5-tropic virus spread to donor CD4+ T cells despite full ART suppression. These data demonstrate the individual contributions of allogeneic immunity and CCR5 deficiency to HIV cure and support defining targets of alloimmunity for curative strategies independent of HSCT.

The combined impact of testosterone and Western-style diet on endometriosis severity and progression in rhesus macaques†.

Polycystic ovary syndrome (PCOS) is associated with irregular menstrual cycles, hyperandrogenemia, and obesity. It is currently accepted that women with PCOS are also at risk for endometriosis, but the effect of androgen and obesity on endometriosis has been underexplored. The goal of this study was to determine how testosterone (T) and an obesogenic diet impact the progression of endometriosis in a nonhuman primate (NHP) model. Female rhesus macaques were treated with T (serum levels approximately 1.35 ng/ml), Western-style diet (WSD; 36% of calories from fat compared to 16% in standard monkey chow) or the combination (T + WSD) at the time of menarche as part of a longitudinal study for ~7 years. Severity of endometriosis was determined based on American Society for Reproductive Medicine (ASRM) revised criteria, and staged 1-4. Stages 1 and 2 were associated with extent of abdominal adhesions, while stages 3 and 4 were associated with presence of chocolate cysts. The combined treatment of T + WSD resulted in earlier onset of endometriosis and more severe types associated with large chocolate cysts compared to all other treatments. There was a strong correlation between glucose clearance, homeostatic model assessment for insulin resistance (HOMA-IR), and total percentage of body fat with presence of cysts, indicating possible indirect contribution of hyperandrogenemia via metabolic dysfunction. An RNA-seq analysis of omental adipose tissue revealed significant impacts on a number of inflammatory signaling pathways. The interactions between obesity, hyperandrogenemia, and abdominal inflammation deserve additional investigation in NHP model species.

Cost of pre-surgical evaluation for epilepsy surgery: A single-center experience.

OBJECTIVE: To estimate the cost and time taken to evaluate adults with drug-resistant focal epilepsy for potentially curative surgery. METHODS: We reviewed data on 100 consecutive individuals at a tertiary referral center evaluated for epilepsy surgery in 2017. The time elapsed between referral and either surgery or a definitive decision not to progress was measured. National Health Service tariffs applicable to our setting were used to estimate the total cost of evaluation for individuals following different routes through the pre-surgical pathway. After surgery, self-reported seizure freedom rates were obtained from each individual to assess the approximate cost of pre-surgical evaluation per additional person seizure-free. RESULTS: Of 100 individuals evaluated, 27 had surgery, 63 had a definitive decision not to have surgery, and ten were awaiting further investigations. The median duration of the pre-surgical evaluation was 29.7 months (IQR 18.6-44.1 months), with a median cost per person of £9138 (IQR £6984-£14,868). Those who proceeded to Stage Two investigations (including fluorodeoxyglucose positron emission tomography, ictal single-photon emission computerized tomography and intracranial electroencephalography) had a higher cost and extended evaluation length. After a median of 3.1 (IQR 2.3-3.7) years, 15/27 people who had surgery were seizure-free. This equated to an approximate cost of £123,500 spent per additional person seizure-free. CONCLUSION: Pre-surgical evaluation is long and costly, particularly for those who require icEEG. For those with drug-resistant focal epilepsy, surgery is, however, associated with a greater chance of seizure freedom. The suitability and risk-benefit ratio of surgery should be considered at each step of the pre-surgical pathway.

Targeting an alternate Wilms' tumor antigen 1 peptide bypasses immunoproteasome dependency.

Designing effective antileukemic immunotherapy will require understanding mechanisms underlying tumor control or resistance. Here, we report a mechanism of escape from immunologic targeting in an acute myeloid leukemia (AML) patient, who relapsed 1 year after immunotherapy with engineered T cells expressing a human leukocyte antigen A*02 (HLA-A2)-restricted T cell receptor (TCR) specific for a Wilms' tumor antigen 1 epitope, WT1126-134 (TTCR-C4). Resistance occurred despite persistence of functional therapeutic T cells and continuous expression of WT1 and HLA-A2 by the patient's AML cells. Analysis of the recurrent AML revealed expression of the standard proteasome, but limited expression of the immunoproteasome, specifically the beta subunit 1i (ß1i), which is required for presentation of WT1126-134. An analysis of a second patient treated with TTCR-C4 demonstrated specific loss of AML cells coexpressing ß1i and WT1. To determine whether the WT1 protein continued to be processed and presented in the absence of immunoproteasome processing, we identified and tested a TCR targeting an alternative, HLA-A2-restricted WT137-45 epitope that was generated by immunoproteasome-deficient cells, including WT1-expressing solid tumor lines. T cells expressing this TCR (TTCR37-45) killed the first patients' relapsed AML resistant to WT1126-134 targeting, as well as other primary AML, in vitro. TTCR37-45 controlled solid tumor lines lacking immunoproteasome subunits both in vitro and in an NSG mouse model. As proteasome composition can vary in AML, defining and preferentially targeting these proteasome-independent epitopes may maximize therapeutic efficacy and potentially circumvent AML immune evasion by proteasome-related immunoediting.

Plasma Mucin-1 (CA15-3) Levels in Autosomal Dominant Tubulointerstitial Kidney Disease due to MUC1 Mutations.

INTRODUCTION: Patients with ADTKD-MUC1 have one allele producing normal mucin-1 (MUC1) and one allele producing mutant MUC1, which remains intracellular. We hypothesized that ADTKD-MUC1 patients, who have only 1 secretory-competent wild-type MUC1 allele, should exhibit decreased plasma mucin-1 (MUC1) levels. To test this hypothesis, we repurposed the serum CA15-3 assay used to measure MUC1 in breast cancer to measure plasma MUC1 levels in ADTKD-MUC1. METHODS: This cross-sectional study analyzed CA15-3 levels in a reference population of 6,850 individuals, in 85 individuals with ADTKD-MUC1, and in a control population including 135 individuals with ADTKD-UMOD and 114 healthy individuals. RESULTS: Plasma CA15-3 levels (mean ± standard deviation) were 8.6 ± 4.3 U/mL in individuals with ADTKD-MUC1 and 14.6 ± 5.6 U/mL in controls (p < 0.001). While there was a significant difference in mean CA15-3 levels, there was substantial overlap between the 2 groups. Plasma CA15-3 levels were <5 U/mL in 22% of ADTKD-MUC1 patients, in 0/249 controls, and in 1% of the reference population. Plasma CA15-3 levels were >20 U/mL in 1/85 ADTKD-MUC1 patients, in 18% of control individuals, and in 25% of the reference population. Segregation of plasma CA15-3 levels by the rs4072037 genotype did not significantly improve differentiation between affected and unaffected individuals. CA15-3 levels were minimally affected by gender and estimated glomerular filtration rate. DISCUSSION/CONCLUSIONS: Plasma CA15-3 levels in ADTKD-MUC1 patients are approximately 40% lower than levels in healthy individuals, though there is significant overlap between groups. Further investigations need to be performed to see if plasma CA15-3 levels would be useful in diagnosis, prognosis, or assessing response to new therapies in this disorder.

Terumo spectra optia leukapheresis of cynomolgus macaques for hematopoietic stem cell and T cell collection.

Macaques are physiologically relevant animal models of human immunology and infectious disease that have provided key insights and advanced clinical treatment in transplantation, vaccinology, and HIV/AIDS. However, the small size of macaques is a stumbling block for studies requiring large numbers of cells, such as hematopoietic stem cells (HSCs) for transplantation, antigen-specific lymphocytes for in-depth immunological analysis, and latently-infected CD4+ T-cells for HIV cure studies. Here, we provide a detailed protocol for collection of large numbers of HSCs and T-cells from cynomolgus macaques as small as 3 kg using the Terumo Spectra Optia apheresis system, yielding an average of 5.0 × 109 total nucleated cells from mobilized animals and 1.2 × 109 total nucleated cells from nonmobilized animals per procedure. This report provides sufficient detail to adapt this apheresis technique at other institutions, which will facilitate more efficient and detailed analysis of HSCs and their progeny blood cells.

Viral opportunistic infections in Mauritian cynomolgus macaques undergoing allogeneic stem cell transplantation mirror human transplant infectious disease complications.

Allogeneic hematopoietic stem cell transplantation (HSCT) and xenotransplantation are accompanied by viral reactivations and virus-associated complications resulting from immune deficiency. Here, in a Mauritian cynomolgus macaque model of fully MHC-matched allogeneic HSCT, we report reactivations of cynomolgus polyomavirus, lymphocryptovirus, and cytomegalovirus, macaque viruses analogous to HSCT-associated human counterparts BK virus, Epstein-Barr virus, and human cytomegalovirus. Viral replication in recipient macaques resulted in characteristic disease manifestations observed in HSCT patients, such as polyomavirus-associated hemorrhagic cystitis and tubulointerstitial nephritis or lymphocryptovirus-associated post-transplant lymphoproliferative disorder. However, in most cases, the reconstituted immune system, alone or in combination with short-term pharmacological intervention, exerted control over viral replication, suggesting engraftment of functional donor-derived immunity. Indeed, the donor-derived reconstituted immune systems of two long-term engrafted HSCT recipient macaques responded to live attenuated yellow fever 17D vaccine (YFV 17D) indistinguishably from untransplanted controls, mounting 17D-targeted neutralizing antibody responses and clearing YFV 17D within 14 days. Together, these data demonstrate that this macaque model of allogeneic HSCT recapitulates clinical situations of opportunistic viral infections in transplant patients and provides a pre-clinical model to test novel prophylactic and therapeutic modalities.

Clonal Decomposition and DNA Replication States Defined by Scaled Single-Cell Genome Sequencing.

Accurate measurement of clonal genotypes, mutational processes, and replication states from individual tumor-cell genomes will facilitate improved understanding of tumor evolution. We have developed DLP+, a scalable single-cell whole-genome sequencing platform implemented using commodity instruments, image-based object recognition, and open source computational methods. Using DLP+, we have generated a resource of 51,926 single-cell genomes and matched cell images from diverse cell types including cell lines, xenografts, and diagnostic samples with limited material. From this resource we have defined variation in mitotic mis-segregation rates across tissue types and genotypes. Analysis of matched genomic and image measurements revealed correlations between cellular morphology and genome ploidy states. Aggregation of cells sharing copy number profiles allowed for calculation of single-nucleotide resolution clonal genotypes and inference of clonal phylogenies and avoided the limitations of bulk deconvolution. Finally, joint analysis over the above features defined clone-specific chromosomal aneuploidy in polyclonal populations.

The human IL-15 superagonist ALT-803 directs SIV-specific CD8+ T cells into B-cell follicles.

Sequestering of latent HIV in follicular helper T cells within B-cell follicles that largely exclude cytotoxic T cells is a major barrier to cellular immune-based approaches to eradicate HIV. Here, we show that the clinical-grade human interleukin-15 (IL-15) superagonist ALT-803 activates and redirects simian immunodeficiency virus (SIV)-specific CD8+ T cells from the peripheral blood into B-cell follicles. In agreement with the increased trafficking of SIV-specific cytotoxic T cells to sites of cryptic viral replication, lymph nodes of elite controlling macaques contained fewer cells expressing SIV RNA or harboring SIV DNA post-ALT-803 treatment. These data establish ALT-803 as an immunotherapeutic for HIV and other chronic viral pathogens that evade host immunity by persisting in B-cell follicles.

Allogeneic stem cell transplantation in fully MHC-matched Mauritian cynomolgus macaques recapitulates diverse human clinical outcomes.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a critically important therapy for hematological malignancies, inborn errors of metabolism, and immunodeficiency disorders, yet complications such as graft-vs.-host disease (GvHD) limit survival. Development of anti-GvHD therapies that do not adversely affect susceptibility to infection or graft-vs.-tumor immunity are hampered by the lack of a physiologically relevant, preclinical model of allogeneic HSCT. Here we show a spectrum of diverse clinical HSCT outcomes including primary and secondary graft failure, lethal GvHD, and stable, disease-free full donor engraftment using reduced intensity conditioning and mobilized peripheral blood HSCT in unrelated, fully MHC-matched Mauritian-origin cynomolgus macaques. Anti-GvHD prophylaxis of tacrolimus, post-transplant cyclophosphamide, and CD28 blockade induces multi-lineage, full donor chimerism and recipient-specific tolerance while maintaining pathogen-specific immunity. These results establish a new preclinical allogeneic HSCT model for evaluation of GvHD prophylaxis and next-generation HSCT-mediated therapies for solid organ tolerance, cure of non-malignant hematological disease, and HIV reservoir clearance.

Uterus-like Masses in a Rhesus Macaque (Macaca mulatta).

Endometriosis is a relatively common condition in women and some populations of adult female rhesus macaques. However, endometriosis with extensive smooth muscle proliferation, as occurs in endomyometrioma and uterus-like mass (ULM), is rare in women. This report describes a case of endometriosis with extensive smooth muscle metaplasia resembling multiple ULM in a 20-y-old female rhesus macaque. During a protocol-related procedure, a large, smooth, globoid, freely moveable mass was palpated in the midabdomen. Ultrasonography revealed a cystic structure from which dark brown fluid was aspirated. During exploratory laparotomy, an 8-cm spherical mass in the greater omentum and 3 additional masses (diameter, 2 to 5 cm) attached to the omentum were excised. Microscopic examination of the masses revealed numerous foci of ectopic endometrial glands and stroma frequently surrounded by bundles of smooth muscle and fibrous connective tissue. The gross and histologic lesions in this macaque bore many similarities to ULM in women. To our knowledge, this case represents the first report of endometriosis resembling a uteruslike mass in a NHP.

Lithium Protects Against Anaesthesia Neurotoxicity In The Infant Primate Brain.

Exposure of infant animals, including non-human primates (NHPs), to anaesthetic drugs causes apoptotic death of neurons and oligodendrocytes (oligos) and results in long-term neurodevelopmental impairment (NDI). Moreover, retrospective clinical studies document an association between anaesthesia exposure of human infants and significant increase in NDI. These findings pose a potentially serious dilemma because millions of human infants are exposed to anaesthetic drugs every year as part of routine medical care. Lithium (Li) at clinically established doses is neuroprotective in various cerebral injury models. We therefore investigated whether Li also protects against anaesthesia neurotoxicity in infant NHPs. On postnatal day 6 NHPs were anaesthetized with the widely used anaesthetic isoflurane (ISO) for 5 h employing the same standards as in a human pediatric surgery setting. Co-administration of Li completely prevented the acute ISO-induced neuroapoptosis and significantly reduced ISO-induced apoptosis of oligodendroglia. Our findings are highly encouraging as they suggest that a relatively simple pharmacological manipulation might protect the developing primate brain against the neurotoxic action of anaesthetic drugs while not interfering with the beneficial actions of these drugs. Further research is needed to determine Li's potential to prevent long-term NDI resulting from ISO anaesthesia, and to establish its safety in human infants.

Activation of innate immune-response genes in little brown bats (Myotis lucifugus) infected with the fungus Pseudogymnoascus destructans.

Recently bats have been associated with the emergence of diseases, both as reservoirs for several new viral diseases in humans and other animals and, in the northern Americas, as hosts for a devastating fungal disease that threatens to drive several bat species to regional extinction. However, despite these catastrophic events little Information is available on bat defences or how they interact with their pathogens. Even less is known about the response of bats to infection during torpor or long-term hibernation. Using tissue samples collected at the termination of an experiment to explore the pathogenesis of White Nose Syndrome in Little Brown Bats, we determined if hibernating bats infected with the fungus Pseudogymnoascus destructans could respond to infection by activating genes responsible for innate immune and stress responses. Lesions due to fungal infection and, in some cases, secondary bacterial infections, were restricted to the skin. However, we were unable to obtain sufficient amounts of RNA from these sites. We therefore examined lungs for response at an epithelial surface not linked to the primary site of infection. We found that bats responded to infection with a significant increase in lungs of transcripts for Cathelicidin (an anti-microbial peptide) as well as the immune modulators tumor necrosis factor alpha and interleukins 10 and 23. In conclusion, hibernating bats can respond to experimental P. destructans infection by activating expression of innate immune response genes.