Systemic versus localized Bacillus Calmette Guérin immunotherapy of bladder cancer promotes an anti-tumoral microenvironment: Novel role of trained immunity.

Treatment for higher-risk non-muscle invasive bladder cancer (NMIBC) involves intravesical immunotherapy with Bacillus Calmette Guérin (BCG); however, disease recurrence and progression occur frequently. Systemic immunity is critical for successful cancer immunotherapy; thus, recurrence of NMIBC may be due to suboptimal systemic activation of anti-tumor immunity after local immunotherapy. We previously reported that systemically acquired trained immunity (a form of innate immune memory) in circulating monocytes is associated with increased time-to-recurrence in patients with NMIBC treated with BCG. Herein, we used a mouse model of NMIBC to compare the effects of intravesical versus intravenous (systemic) BCG immunotherapy on the local and peripheral immune microenvironments. We also assessed whether BCG-induced trained immunity modulates anti-tumor immune responses. Compared with intravesical BCG, which led to a tumor-promoting immune microenvironment, intravenous BCG resulted in an anti-tumoral bladder microenvironment characterized by increased proportions of cytotoxic T lymphocytes (CTLs), and decreased proportions of myeloid-derived suppressor cells. Polarization toward anti-tumoral immunity occurred in draining lymph nodes, spleen, and bone marrow following intravenous versus intravesical BCG treatment. Pre-treatment with intravesical BCG was associated with increased rate of tumor growth compared with intravenous BCG pre-treatment. Trained immunity contributed to remodeling of the tumor immune microenvironment, as co-instillation of BCG-trained macrophages with ovalbumin-expressing bladder tumor cells increased the proportion of tumor-specific CTLs. Furthermore, BCG-trained dendritic cells exhibited enhanced antigen uptake and presentation and promoted CTL proliferation. Our data support the concept that systemic immune activation promotes anti-tumor responses, and that BCG-induced trained immunity is important in driving anti-tumor adaptive immunity.

Tyrosinase-induced neuromelanin accumulation triggers rapid dysregulation and degeneration of the mouse locus coeruleus.

Motor symptoms in Parkinson's disease (PD) are caused by degeneration of dopamine (DA) neurons of the substantia nigra (SN), while early non-motor symptoms such as anxiety and sleep disturbances are likely mediated by dysfunction of locus coeruleus (LC) norepinephrine (NE) neurons. The LC develops α-synuclein pathology prior to SN DA neurons in PD, and later undergoes degeneration, but the mechanisms responsible for its vulnerability are unknown. The SN and LC are the only structures in the brain that produces appreciable amounts of neuromelanin (NM), a dark cytoplasmic pigment. It has been proposed that NM initially plays a protective role by sequestering toxic catecholamine metabolites and heavy metals, but may become harmful during aging and PD as they overwhelm cellular machinery and are released during neurodegeneration. Rodents do not naturally produce NM, limiting the study of causal relationships between NM and PD-associated LC pathology. Adapting a viral-mediated approach for expression of human tyrosinase, the enzyme responsible for peripheral melanin production, we successfully promoted pigmentation in mouse LC neurons that recapitulates key features of endogenous NM found in primates, including eumelanin and pheomelanin, lipid droplets, and a double-membrane encasement. Pigment expression results in mild neurodegeneration, reduced NE levels, transcriptional changes, and novelty-induced anxiety phenotypes as early as 1-week post-injection. By 6-weeks, NM accumulation is associated with severe LC neurodegeneration and a robust neuroinflammatory response. These phenotypes are reminiscent of LC dysfunction in PD, validating this model for studying the consequences of pigment accumulation in the LC as it relates to neurodegenerative disease.

RGS14 is neuroprotective against seizure-induced mitochondrial oxidative stress and pathology in hippocampus.

RGS14 is a complex multifunctional scaffolding protein that is highly enriched within pyramidal cells (PCs) of hippocampal area CA2. There, RGS14 suppresses glutamate-induced calcium influx and related G protein and ERK signaling in dendritic spines to restrain postsynaptic signaling and plasticity. Previous findings show that, unlike PCs of hippocampal areas CA1 and CA3, CA2 PCs are resistant to a number of neurological insults, including degeneration caused by temporal lobe epilepsy (TLE). While RGS14 is protective against peripheral injury, similar roles for RGS14 during pathological injury in hippocampus remain unexplored. Recent studies show that area CA2 modulates hippocampal excitability, generates epileptiform activity and promotes hippocampal pathology in animal models and patients with TLE. Because RGS14 suppresses CA2 excitability and signaling, we hypothesized that RGS14 would moderate seizure behavior and early hippocampal pathology following seizure activity. Using kainic acid (KA) to induce status epilepticus (KA-SE) in mice, we show loss of RGS14 (RGS14 KO) accelerated onset of limbic motor seizures and mortality compared to wild type (WT) mice, and that KA-SE upregulated RGS14 protein expression in CA2 and CA1 PCs of WT. Utilizing proteomics, we saw loss of RGS14 impacted the expression of a number of proteins at baseline and after KA-SE, many of which associated unexpectedly with mitochondrial function and oxidative stress. RGS14 was shown to localize to the mitochondria in CA2 PCs of mice and reduce mitochondrial respiration in vitro . As a readout of oxidative stress, we found RGS14 KO dramatically increased 3-nitrotyrosine levels in CA2 PCs, which was greatly exacerbated following KA-SE and correlated with a lack of superoxide dismutase 2 (SOD2) induction. Assessing for hallmarks of seizure pathology in RGS14 KO, we observed worse neuronal injury in area CA3 (but none in CA2 or CA1), and a lack of microgliosis in CA1 and CA2 compared to WT. Together, our data demonstrates a newly appreciated neuroprotective role for RGS14 against intense seizure activity in hippocampus. Our findings are consistent with a model where, after seizure, RGS14 is upregulated to support mitochondrial function and prevent oxidative stress in CA2 PCs, limit seizure onset and hippocampal neuronal injury, and promote microglial activation in hippocampus.

Maternal Immune Cell and Cytokine Profiles to Predict Cardiovascular Risk Six Months after Preeclampsia.

Women who develop preeclampsia (PE) are at high risk for cardiovascular disease (CVD). Early identification of women with PE who may benefit the most from early cardiovascular risk screening and interventions remains challenging. Our objective was to assess whether cytokine and immune cell profiles after PE are helpful in distinguishing women at low and high CVD risk at 6-months postpartum. Individuals who developed PE were followed for immune cell phenotyping and plasma cytokine quantification at delivery, at 3-months, and at 6-months postpartum. Lifetime CVD risk was assessed at 6-months postpartum, and the immune cell and cytokine profiles were compared between risk groups at each time point. Among 31 participants, 18 (58.1%) exhibited high CVD-risk profiles at 6-months postpartum. The proportion of circulating NK-cells was significantly lower in high-risk participants at delivery (p = 0.04). At 3-months postpartum, high-risk participants exhibited a lower proportion of FoxP3+ regulatory T-cells (p = 0.01), a greater proportion of CD8+ T cells (p = 0.02) and a lower CD4+:CD8+ ratio (p = 0.02). There were no differences in immune cell populations at 6-months postpartum. There were no differences in plasma cytokines levels between risk groups at any time point. Subtle differences in immune cell profiles may help distinguish individuals at low and high CVD risk in the early postpartum period and warrants further investigation.

Enhancers of Host Immune Tolerance to Bacterial Infection Discovered Using Linked Computational and Experimental Approaches.

Current therapeutic strategies against bacterial infections focus on reduction of pathogen load using antibiotics; however, stimulation of host tolerance to infection in the presence of pathogens might offer an alternative approach. Computational transcriptomics and Xenopus laevis embryos are used to discover infection response pathways, identify potential tolerance inducer drugs, and validate their ability to induce broad tolerance. Xenopus exhibits natural tolerance to Acinetobacter baumanii, Klebsiella pneumoniae, Staphylococcus aureus, and Streptococcus pneumoniae bacteria, whereas Aeromonas hydrophila and Pseudomonas aeruginosa produce lethal infections. Transcriptional profiling leads to definition of a 20-gene signature that discriminates between tolerant and susceptible states, as well as identification of a more active tolerance response to gram negative compared to gram positive bacteria. Gene pathways associated with active tolerance in Xenopus, including some involved in metal ion binding and hypoxia, are found to be conserved across species, including mammals, and administration of a metal chelator (deferoxamine) or a HIF-1α agonist (1,4-DPCA) in embryos infected with lethal A. hydrophila increased survival despite high pathogen load. These data demonstrate the value of combining the Xenopus embryo infection model with computational multiomics analyses for mechanistic discovery and drug repurposing to induce host tolerance to bacterial infections.

Surgical stabilization of traumatic elbow joint luxation and proximal ulnar fracture in a silvery langur (Trachypithecus cristatus).

CASE DESCRIPTION: A 3-year-old 5-kg sexually intact female silvery langur housed in a single-species group at a zoological institution was presented because of acute trauma to the left forelimb. CLINICAL FINDINGS: Radiography of the left forelimb revealed a type II Monteggia fracture (proximal ulnar fracture with cranial displacement and caudal luxation of the radial head). During surgery, disruption of the annular ligament and rupture of the lateral collateral ligament were noted. TREATMENT AND OUTCOME: The langur underwent open reduction and internal fixation of the ulnar fracture and placement of a radioulnar positional screw, a prosthetic lateral collateral ligament, and a temporary hinged type 1A external skeletal fixator. The langur was returned to group housing, underwent behavioral training, and was periodically anesthetized for physical therapy sessions to improve range of motion of the left elbow joint. The external skeletal fixator was removed 4 weeks after surgery, and the radioulnar positional screw was removed 6 weeks after surgery. Three months after surgery, the range of motion of the langur's left elbow joint was considered normal, and the animal returned to normal activity. CLINICAL RELEVANCE: For the captive silvery langur of the present report, surgical stabilization and postoperative management of a type II Monteggia fracture of the left forelimb were successful with recovery of elbow joint function. These techniques may be applied to other captive nonhuman primates, including those that brachiate or are members of social species that must be housed with conspecifics in the postoperative period to maintain group dynamics.

PHARMACOKINETICS OF A SINGLE ORAL DOSE OF PONAZURIL IN THE INDIAN PEAFOWL (PAVO CRISTATUS).

Ponazuril, a novel coccidiocidal triazinetrione, has shown promise in addressing apicomplexan diseases in mammals and birds. This study describes the pharmacokinetics of ponazuril in healthy adult Indian peafowl (Pavo cristatus) following a single oral dose administered at two different dosages. Peafowl (four males and four females) were administered compounded ponazuril at 20 or 40 mg/kg orally in a double crossover design, with a 2-wk washout period. Blood was collected from each bird at 2, 4, 8, 24, 48, 72, 96, and 120 h after administration for plasma concentration of ponazuril using high-pressure liquid chromatography. Fecals were evaluated for coccidial shedding for 3 consecutive d prior to the ponazuril trial, 1 wk after the first dose of ponazuril, and 1 wk after the second dose of the trial. After the first trial, one peafowl administered 20 mg/kg ponazuril was shedding coccidia, but no coccidia were detected by the end of the second trial. Ponazuril reached peak concentrations (Tmax) at 21.38 h + 5.25 and 22.04 h + 7.39, and peak concentration (Cmax) were 11.82 µg/ml + 3.01 and 18.42 µg/ml + 4.13, for 20 and 40 mg/kg doses, respectively. Ponazuril was detected at 120 h with a concentration of 9.48 µg/ml + 2.59 and 12.25 µg/ml + 2.89 and a half-life of 219.4 + 58.7 h and 186.7 + 58.7 h, for and 40 mg/kg doses, respectively. Ponazuril in peafowl was well absorbed orally, plasma concentrations increased with dose, and elimination was slower than current dosages for birds would suggest. No obvious adverse effects were observed at either dosage.

EVALUATION OF PASSIVE TRANSFER IN NINE SPECIES OF CERVIDAE.

The reliability of packed cell volumes (PCV), total solids (TS), blood glucose (BG), γ-glutamyl transferase (GGT), and glutaraldehyde test in determining passive transfer of colostral immunoglobulins was investigated in nine species of cervids: axis deer (Axis axis), hog deer (Axis porcinus), sika deer (Cervus nippon), tufted deer (Elaphodus cephalophus), Père David's deer (Elaphurus davidianus), pudu (Pudu puda), sambar deer (Rusa unicolor), barasinga deer (Rucervus duvaucelli), and Eld's deer (Rucervus eldii). Individually the parameters evaluated were significant though imperfect predictors of passive transfer status in cervids. Interpreted collectively as a panel along with neonate condition, these tests were clinically helpful in diagnosing failure of passive transfer (FPT). Collectively interpreting test results as a panel along with clinical assessment of the animal is recommended. Some species-specific variations in TS, GGT, and glutaraldehyde test results were identified.

SARCOCYSTOSIS IN A CAPTIVE FLOCK OF THICK-BILLED PARROTS (RHYNCHOPSITTA PACHYRHYNCHA) FROM 2005 TO 2016: MORBIDITY, MORTALITY, DIAGNOSTICS, AND MANAGEMENT STRATEGIES.

Sarcocystosis was diagnosed in a captive flock of thick-billed parrots (Rhynchopsitta pachyrhyncha) at the Wildlife Conservation Society's Queens Zoo. Since the index case in 2005, 45% of mortalities in birds over 30 days of age were due to sarcocystosis. Sarcocystis falcatula was repeatedly identified as the causative agent. The disease predominantly affected younger adult parrots. Administration of antiparasitic medications prior to development of respiratory signs prolonged life in infected birds, but disease was fatal until utilization of a three-drug combination (pyrimethamine, trimethoprim-sulfamethoxazole, and ponazuril). This protocol may require in excess of 6 mo of therapy to achieve clinical resolution of active disease. Plasma creatine kinase activity was found to be the most useful test in diagnosing infection and monitoring response to therapy. Polymerase chain reaction (PCR) for apicomplexan organisms on antemortem whole blood, blood smears, or dried blood spots helped confirm suspected cases, but due to the poor sensitivity was sometimes misleading when assessing response to therapy or resolution of clinical disease. Preventive measures, focusing on exclusion and removal of Virginia opossums (Didelphis virginiana) from zoo grounds failed to curtail the occurrence of sarcocystosis in the flock. Other preventative steps, such as modification of feeding stations to exclude potential arthropod paratenic hosts and prophylaxis trials with diclazuril, appeared to successfully mitigate new infections. Given the diagnostic and therapeutic challenges, prevention of exposure to S. falcatula is essential to ex-situ conservation efforts for thick-billed parrots.

DIFFUSIBLE IODINE-BASED CONTRAST-ENHANCED COMPUTED TOMOGRAPHY AS A NECROPSY AID: A CASE REPORT EVALUATING RESPIRATORY DISEASE IN MACROCEPHALON MALEO.

This study describes the novel use of diffusible iodine-based contrast-enhanced computed tomography (diceCT) as a digital necropsy aid. DiceCT was used postmortem to evaluate the cause of progressive respiratory disease in a juvenile maleo (Macrocephalon maleo). The technique facilitated soft-tissue contrast and a three-dimensional investigation of sinus and choanal anatomy as a means to identify normal and pathologic morphologies. Results showed right-sided narial occlusion by mucoid debris, along with left-sided choanal stenosis caused by osteomyelitis and reactive bone formation. The high spatial resolution afforded by diceCT enabled targeted histology and quantification of the clinical impact of pathologies, which contributed to an effective 60% loss in nasal airway aperture for this individual. This study demonstrates how adding diceCT to traditional necropsy can proffer additional understanding of an individual's pathology, and the resulting data can enhance research programs in vertebrate anatomy, evolution, and health.

Comparative Ultrastructural Analysis of Thalamocortical Innervation of the Primary Motor Cortex and Supplementary Motor Area in Control and MPTP-Treated Parkinsonian Monkeys.

The synaptic organization of thalamic inputs to motor cortices remains poorly understood in primates. Thus, we compared the regional and synaptic connections of vGluT2-positive thalamocortical glutamatergic terminals in the supplementary motor area (SMA) and the primary motor cortex (M1) between control and MPTP-treated parkinsonian monkeys. In controls, vGluT2-containing fibers and terminal-like profiles invaded layer II-III and Vb of M1 and SMA. A significant reduction of vGluT2 labeling was found in layer Vb, but not in layer II-III, of parkinsonian animals, suggesting a potential thalamic denervation of deep cortical layers in parkinsonism. There was a significant difference in the pattern of synaptic connectivity in layers II-III, but not in layer Vb, between M1 and SMA of control monkeys. However, this difference was abolished in parkinsonian animals. No major difference was found in the proportion of perforated versus macular post-synaptic densities at thalamocortical synapses between control and parkinsonian monkeys in both cortical regions, except for a slight increase in the prevalence of perforated axo-dendritic synapses in the SMA of parkinsonian monkeys. Our findings suggest that disruption of the thalamic innervation of M1 and SMA may underlie pathophysiological changes of the motor thalamocortical loop in the state of parkinsonism.

Innate immune memory is associated with increased disease-free survival in bladder cancer patients treated with bacillus Calmette-Guérin.

INTRODUCTION: While studies suggest that innate immune memory acquired by circulating monocytes may mediate the benefit of bacillus Calmette-Guérin (BCG) in the treatment of patients with high-risk non-muscle-invasive bladder cancer (NMIBC), prospective studies are lacking. Innate immune memory is defined by enhanced release of pro-inflammatory cytokines by innate immune cells following a secondary challenge with pattern recognition receptor (PRR) ligands. METHODS: Peripheral blood monocytes isolated from 33 patients with intermediate- or high-risk NMIBC before and after two or five induction BCG instillations were stimulated with the PRR ligand lipopolysaccharide (LPS). Inflammatory cytokine levels in the culture medium were measured. Extent of innate immune memory acquisition was determined by dividing the levels of cytokines released after BCG instillation by the levels released prior to BCG therapy. RESULTS: Monocytes secreted variable levels of TNFα, IL-1ß, IL-6, IFNγ, IL-12, and IL-10. Compared with patients with recurrences, the post-BCG:pre-BCG ratio of IL-12 in monocyte cultures from patients without recurrences after five BCG instillations was significantly increased. Patients with no innate immune memory (based on IL-12 ratios) had significantly shorter time to recurrence than patients with innate immune memory (p<0.001). Eighty-four percent (16/19) of patients with innate immune memory vs. only 22% (2/9) of patients without memory had disease-free survival of over 500 days. CONCLUSIONS: Results demonstrate a potential link between BCG-induced innate immune memory peripherally and local anti-tumor responses. Further validation will increase our understanding of the mode of action of BCG and, therefore, will be used to enhance its effectiveness.

CEFOVECIN PROTEIN BINDING AS A PREDICTOR FOR EXTENDED DURATION OF ACTION: A REVIEW OF CURRENT LITERATURE AND IN VITRO ANALYSIS IN MULTIPLE ZOOLOGICAL SPECIES.

Cefovecin is a third-generation cephalosporin antibiotic with an efficacy of 2 wk following a single injection in domestic dogs and cats. A high degree of plasma protein binding to cefovecin has been proposed as one of the mechanisms responsible for the long elimination half-life, but protein binding has not been evaluated extensively in nondomestic species. In this study, a review of the current literature was conducted, and pharmacokinetic data were compiled for species in which cefovecin has been evaluated thus far. Additionally, in vitro cefovecin protein binding was evaluated in plasma from 22 nondomestic species representing a broad range of taxa. Animals of the order Carnivora demonstrated protein-binding levels of >98%, which is supportive of the long elimination half-life seen in related species. Protein binding was highly variable in Artiodactyl and Perissodactyl species, with dolphins (Tursiops truncatus) and barasingha (Rucervus duvaucelii) displaying high protein binding (99.12% to >99%), but not gazelles (Eudorcas thomsonii) or equids (91.76-92.70%). Cefovecin was not highly bound in any reptiles or birds, corresponding to short half-lives reported for these taxa. These results suggest that a high percentage of plasma protein binding in vitro may predict in which species cefovecin may exhibit a long half-life and duration of action in vivo. These findings may aid in selecting species for cefovecin pharmacokinetic research and for empirical treatment of infections caused by susceptible bacteria.

Glutamatergic inputs to GABAergic interneurons in the motor thalamus of control and parkinsonian monkeys.

The primate ventral motor thalamus contains a large number of GABAergic interneurons of poorly understood function and anatomical connectivity. Glutamatergic inputs to these cells arise predominantly from corticothalamic (in both basal ganglia- and cerebellar-receiving ventral motor thalamic territories; BGMT and CBMT, respectively) and cerebellothalamic terminals (in CBMT). In Parkinson's disease patients and animal models, neuronal activity is abnormal within both BGMT and CBMT. Historically, such motor thalamic dysregulation has been largely attributed to changes in inhibitory tone from the basal ganglia output nuclei, ignoring the potential role of other thalamic inputs in such processes, particularly within the CBMT, which is largely devoid of direct basal ganglia afferents. We have recently reported changes in the abundance and structural morphology of corticothalamic terminals in BGMT of parkinsonian monkeys. In this study, we assessed potential changes in the prevalence of cortical (vesicular glutamate transporter 1-positive, vGluT1-positive) and subcortical (vGluT2-positive) glutamatergic inputs in contact with GABAergic interneurons in BGMT and CBMT of MPTP-treated parkinsonian monkeys. Our findings revealed that interneurons represent a major target of both sets of glutamatergic terminals. In both BGMT and CBMT of control and parkinsonian monkeys, 29%-38% of total asymmetric axodendritic synapses (putative glutamatergic) were formed by vGluT1-positive terminals and 11%-17% of total vGluT1-positive terminals targeted dendrites of GABAergic interneurons. In CBMT, 16%-18% of asymmetric synaptic inputs on interneurons involved vGluT2-containing terminals. No major differences in the extent of glutamatergic innervation of thalamic GABAergic interneurons were found between control and parkinsonian monkeys.

An in vivo brain-bacteria interface: the developing brain as a key regulator of innate immunity.

Infections have numerous effects on the brain. However, possible roles of the brain in protecting against infection, and the developmental origin and role of brain signaling in immune response, are largely unknown. We exploited a unique Xenopus embryonic model to reveal control of innate immune response to pathogenic E. coli by the developing brain. Using survival assays, morphological analysis of innate immune cells and apoptosis, and RNA-seq, we analyzed combinations of infection, brain removal, and tail-regenerative response. Without a brain, survival of embryos injected with bacteria decreased significantly. The protective effect of the developing brain was mediated by decrease of the infection-induced damage and of apoptosis, and increase of macrophage migration, as well as suppression of the transcriptional consequences of the infection, all of which decrease susceptibility to pathogen. Functional and pharmacological assays implicated dopamine signaling in the bacteria-brain-immune crosstalk. Our data establish a model that reveals the very early brain to be a central player in innate immunity, identify the developmental origins of brain-immune interactions, and suggest several targets for immune therapies.

Inhibition of hypoxia-inducible factor 1α accumulation by glyceryl trinitrate and cyclic guanosine monophosphate.

A key mechanism mediating cellular adaptive responses to hypoxia involves the activity of hypoxia-inducible factor 1 (HIF-1), a transcription factor composed of HIF-1α, and HIF-1ß subunits. The classical mechanism of regulation of HIF-1 activity involves destabilisation of HIF-1α via oxygen-dependent hydroxylation of proline residues and subsequent proteasomal degradation. Studies from our laboratory revealed that nitric oxide (NO)-mediated activation of cyclic guanosine monophosphate (cGMP) signalling inhibits the acquisition of hypoxia-induced malignant phenotypes in tumour cells. The present study aimed to elucidate a mechanism of HIF-1 regulation involving NO/cGMP signalling. Using human DU145 prostate cancer cells, we assessed the effect of the NO mimetic glyceryl trinitrate (GTN) and the cGMP analogue 8-Bromo-cGMP on hypoxic accumulation of HIF-1α. Concentrations of GTN known to primarily activate the NO/cGMP pathway (100 nM-1 µM) inhibited hypoxia-induced HIF-1α protein accumulation in a time-dependent manner. Incubation with 8-Bromo-cGMP (1 nM-10 µM) also attenuated HIF-1α accumulation, while levels of HIF-1α mRNA remained unaltered by exposure to GTN or 8-Bromo-cGMP. Furthermore, treatment of cells with the calpain (Ca2+-activated proteinase) inhibitor calpastatin attenuated the effects of GTN and 8-Bromo-cGMP on HIF-1α accumulation. However, since calpain activity was not affected by incubation of DU145 cells with various concentrations of GTN or 8-Bromo-cGMP (10 nM or 1 µM) under hypoxic or well-oxygenated conditions, it is unlikely that NO/cGMP signalling inhibits HIF-1α accumulation via regulation of calpain activity. These findings provide evidence for a role of NO/cGMP signalling in the regulation of HIF-1α, and hence HIF-1-mediated hypoxic responses, via a mechanism dependent on calpain.

Thalamic degeneration in MPTP-treated Parkinsonian monkeys: impact upon glutamatergic innervation of striatal cholinergic interneurons.

In both Parkinson's disease (PD) patients and MPTP-treated non-human primates, there is a profound neuronal degeneration of the intralaminar centromedian/parafascicular (CM/Pf) thalamic complex. Although this thalamic pathology has long been established in PD (and other neurodegenerative disorders), the impact of CM/Pf cell loss on the integrity of the thalamo-striatal glutamatergic system and its regulatory functions upon striatal neurons remain unknown. In the striatum, cholinergic interneurons (ChIs) are important constituents of the striatal microcircuitry and represent one of the main targets of CM/Pf-striatal projections. Using light and electron microscopy approaches, we have analyzed the potential impact of CM/Pf neuronal loss on the anatomy of the synaptic connections between thalamic terminals (vGluT2-positive) and ChIs neurons in the striatum of parkinsonian monkeys treated chronically with MPTP. The following conclusions can be drawn from our observations: (1) as reported in PD patients, and in our previous monkey study, CM/Pf neurons undergo profound degeneration in monkeys chronically treated with low doses of MPTP. (2) In the caudate (head and body) nucleus of parkinsonian monkeys, there is an increased density of ChIs. (3) Despite the robust loss of CM/Pf neurons, no significant change was found in the density of thalamostriatal (vGluT2-positive) terminals, and in the prevalence of vGluT2-positive terminals in contact with ChIs in parkinsonian monkeys. These findings provide new information about the state of thalamic innervation of the striatum in parkinsonian monkeys with CM/Pf degeneration, and bring up an additional level of intricacy to the consequences of thalamic pathology upon the functional microcircuitry of the thalamostriatal system in parkinsonism. Future studies are needed to assess the importance of CM/Pf neuronal loss, and its potential consequences on the neuroplastic changes induced in the synaptic organization of the thalamostriatal system, in the development of early cognitive impairments in PD.

STANDING COMPUTED TOMOGRAPHY IN NONANESTHETIZED LITTLE PENGUINS (EUDYPTULA MINOR) TO ASSESS RESPIRATORY SYSTEM ANATOMY AND MONITOR DISEASE.

Multidetector computed tomography (MDCT) scans were performed in clinically healthy, nonanesthetized, standing little penguins (Eudyptula minor) to determine reference ranges for air-sac and lung volumes, as well as lung density. Five of 15 clinically healthy birds were diagnosed with pulmonary granulomas on initial MDCT scans. Granulomas were not readily apparent on radiographs, even in cases where the entire normal pulmonary parenchymal architecture was effaced on the MDCT scan. Serial MDCT scans after antifungal and antimycobacterial therapies demonstrated a response to treatment. MDCT scanning in nonanesthetized little penguins proved to be a well-tolerated, non-invasive imaging modality for respiratory diseases that are otherwise difficult to diagnose, including aspergillosis and mycobacteriosis.

DIAGNOSIS AND MANAGEMENT OF MYCOBACTERIOSIS IN A COLONY OF LITTLE PENGUINS (EUDYPTULA MINOR).

A group of zoo-housed little penguins (Eudyptula minor) was diagnosed with mycobacteriosis. While undergoing multidetector computed tomography (MDCT) imaging for an unrelated research project, pulmonary lesions were detected in multiple individuals. In general, birds appeared healthy and free of outward signs of disease. After the loss of three individuals, polyclonal mycobacterial disease due to Mycobacterium avium-intracellulare complex was confirmed. Surviving birds were treated with rifampin (45 mg/kg), ethambutol (30 mg/kg), clarithromycin (10 mg/kg), and enrofloxacin (30 mg/kg) compounded into a single capsule administered once a day in food. After 3 mo of therapy, MDCT imaging documented a decrease in nodule size and number in all remaining birds, with further improvement documented after 13 mo of treatment. MDCT imaging was invaluable for diagnosing disease, documenting disease progression over time, and assessing response to therapy. Early initiation of therapy before the development of outward signs of disease led to resolution of mycobacterial pulmonary lesions in multiple penguins. Mycobacterial disease in this group of little penguins, as well as previously published reports, suggests that the species is at increased risk for developing mycobacteriosis.

Ultrastructural localization of DREADDs in monkeys.

Designer receptors exclusively activated by designer drugs (DREADDs) are extensively used to modulate neuronal activity in rodents, but their use in primates remains limited. An essential need that remains is the demonstration that DREADDs are efficiently expressed on the plasma membrane of primate neurons. To address this issue, electron microscopy immunogold was used to determine the subcellular localization of the AAV vector-induced DREADDs hM4Di and hM3Dq fused to different tags in various brain areas of rhesus monkeys and mice. When hM4Di was fused to mCherry, the immunogold labelling was mostly confined to the intracellular space, and poorly expressed at the plasma membrane in monkey dendrites. In contrast, the hM4Di-mCherry labelling was mostly localized to the dendritic plasma membrane in mouse neurons, suggesting species differences in the plasma membrane expression of these exogenous proteins. The lack of hM4Di plasma membrane expression may limit the functional effects of systemic administration of DREADD-actuators in monkey neurons. Removing the mCherry and fusing of hM4Di with the haemagglutinin (HA) tag resulted in strong neuronal plasma membrane immunogold labelling in both monkeys and mice neurons. Finally, hM3Dq-mCherry was expressed mostly at the plasma membrane in monkey neurons, indicating that the fusion of mCherry with hM3Dq does not hamper membrane incorporation of this specific DREADD. Our results suggest that the pattern of ultrastructural expression of DREADDs in monkey neurons depends on the DREADD/tag combination. Therefore, a preliminary characterization of plasma membrane expression of specific DREADD/tag combinations is recommended when using chemogenetic approaches in primates.