Screening For Bone Marrow Cellularity Changes in Cynomolgus Macaques in Toxicology Safety Studies Using Artificial Intelligence Models.

Many compounds affect the cellularity of hematolymphoid organs including bone marrow. Toxicologic pathologists are tasked with their evaluation as part of safety studies. An artificial intelligence (AI) tool could provide diagnostic support for the pathologist. We looked at the ability of a deep-learning AI model to evaluate whole slide images of macaque sternebrae to identify and enumerate bone marrow hematopoietic cells. The AI model was trained and able to differentiate the hematopoietic cells from the other sternebrae tissues. We compared the model to severity scores in a study with decreased hematopoietic cellularity. The mean cells/mm2 from the model was lower for each increase in severity score. The AI model was trained by 1 pathologist, providing proof of concept that AI model generation can be fast and agile, without the need of a cross disciplinary team and significant effort. We see great potential for the role of AI-based bone marrow screening.

Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72.

Antibodies are a key resource in biomedical research yet there are no community-accepted standards to rigorously characterize their quality. Here we develop a procedure to validate pre-existing antibodies. Human cell lines with high expression of a target, determined through a proteomics database, are modified with CRISPR/Cas9 to knockout (KO) the corresponding gene. Commercial antibodies against the target are purchased and tested by immunoblot comparing parental and KO. Validated antibodies are used to definitively identify the most highly expressing cell lines, new KOs are generated if needed, and the lines are screened by immunoprecipitation and immunofluorescence. Selected antibodies are used for more intensive procedures such as immunohistochemistry. The pipeline is easy to implement and scalable. Application to the major ALS disease gene C9ORF72 identified high-quality antibodies revealing C9ORF72 localization to phagosomes/lysosomes. Antibodies that do not recognize C9ORF72 have been used in highly cited papers, raising concern over previously reported C9ORF72 properties.

Characterization of an evolutionarily conserved calcitonin signalling system in a lophotrochozoan, the Pacific oyster (Crassostrea gigas).

In Protostoma, the diuretic hormone 31 (DH31) signalling system was long considered as the orthologue of the chordate calcitonin (CT) signalling system. Using the Pacific oyster (Crassostrea gigas) transcriptomic database GigaTON, we characterized seven G-protein-coupled receptors (GPCRs) named Cragi-CTR1-7 and phylogenetically related to chordate CT receptors (CTRs) and to protostome DH31 receptors. Two CT precursors (Cragi-CTP1 and Cragi-CTP2) containing two CT-type peptides and encoded by two distinct genes with a similar organization were also characterized. These oyster neuropeptides (Cragi-CT1/2) exhibit the two N-terminal paired cysteine residues and, except CTP2-derived peptide (Cragi-CTP2dp), show the C-terminal proline-amide motif typical of deuterostome CT-type peptides. All mature Cragi-CTs except Cragi-CTP2dp were detected in visceral ganglion extracts using mass spectrometry. Cell-based assays revealed that the previously characterized oyster receptors Cg-CT-R and Cragi-CTR2 were specifically activated by Cragi-CT1b and Cragi-CT2, respectively. This activation does not require the co-expression of receptor activity-modifying proteins (RAMPs). Thus, oyster CT signalling appears functionally more closely related to vertebrate CT/CTR signalling than to calcitonin gene-related peptide/calcitonin receptor-like receptor (CGRP/CLR) signalling. Gene expression profiles in different adult tissues and in oysters acclimated to brackish water suggest the potential implication of both Cg-CT-R/Cragi-CT1b and Cragi-CTR2/Cragi-CT2 in water and ionic regulations, although with apparently opposite effects. The present study represents the first comprehensive characterization of a functional CT-type signalling system in a protostome and provides evidence for its evolutionarily ancient origin and its early role in osmotic homeostasis.

Experimental Models of Short Courses of Liposomal Amphotericin B for Induction Therapy for Cryptococcal Meningitis.

Cryptococcal meningoencephalitis is a rapidly lethal infection in immunocompromised patients. Induction regimens are usually administered for 2 weeks. The shortest effective period of induction therapy with liposomal amphotericin B (LAMB) is unknown. The pharmacodynamics of LAMB were studied in murine and rabbit models of cryptococcal meningoencephalitis. The concentrations of LAMB in the plasma and brains of mice were measured using high-performance liquid chromatography (HPLC). Histopathological changes were determined. The penetration of LAMB into the brain was determined by immunohistochemistry using an antibody directed to amphotericin B. A dose-dependent decline in fungal burden was observed in the brains of mice, with near-maximal efficacy achieved with LAMB at 10 to 20 mg/kg/day. The terminal elimination half-life in the brain was 133 h. The pharmacodynamics of a single dose of 20 mg/kg was the same as that of 20 mg/kg/day administered for 2 weeks. Changes in quantitative counts were reflected by histopathological changes in the brain. Three doses of LAMB at 5 mg/kg/day in rabbits were required to achieve fungicidal activity in cerebrospinal fluid (cumulative area under the concentration-time curve, 2,500 mg · h/liter). Amphotericin B was visible in the intra- and perivascular spaces, the leptomeninges, and the choroid plexus. The prolonged mean residence time of amphotericin B in the brain suggests that abbreviated induction regimens of LAMB are possible for cryptococcal meningoencephalitis.

Nutritional Therapy.

This article provides the reader with steps needed to accurately assess patient nutrition behaviors that contribute to weight gain, inability to lose weight, or inability to sustain weight loss. Evidence-based approaches in nutrition therapy that can create the daily energy deficit needed to produce 1/2 to 2 pounds of weight loss per week, and the strategies to create the energy deficit, are presented. To optimize health, long-term weight loss maintenance is needed. The benefits of using a multidisciplinary team approach in treating obesity are highlighted.

Efficacy of an abbreviated induction regimen of amphotericin B deoxycholate for cryptococcal meningoencephalitis: 3 days of therapy is equivalent to 14 days.

UNLABELLED: Cryptococcal meningoencephalitis is an urgent global health problem. Induction regimens using 14 days of amphotericin B deoxycholate (dAmB) are considered the standard of care but may not be suitable for resource-poor settings. We investigated the efficacy of conventional and abbreviated regimens of dAmB for cryptococcal meningoencephalitis in both murine and rabbit models of cryptococcal meningoencephalitis. We examined the extent to which immunological effectors contribute to the antifungal effect. We bridged the results to humans as a first critical step to define regimens suitable for further study in clinical trials. There were significant differences in the murine plasma-versus-cerebrum dAmB concentration-time profiles. dAmB was detectable in the cerebrum throughout the experimental period, even following the administration of only three doses of 3 mg/kg. dAmB induced a fungistatic effect in the cerebrum with a 2- to 3-log10 CFU/g reduction compared with controls. The effect of 3 days of therapy was the same as that of daily therapy for 14 days. There was no evidence of increased numbers of CD3(+) CD4(+) or CD3(+) CD8(+) cells in treated mice to account for the persistent antifungal effect of an abbreviated regimen. The administration of dAmB at 1 mg/kg/day for 3 days was the same as daily therapy in rabbits. The bridging studies suggested that a human regimen of 0.7 mg/kg/day for 3 days resulted in nearly maximal antifungal activity in both the cerebrum and cerebrospinal fluid. An abbreviated regimen (3 days of therapy) of dAmB appears to be just as effective as conventional induction therapy for cryptococcal meningoencephalitis. IMPORTANCE: Cryptococcal meningitis is a significant and neglected infection that is associated with excessive morbidity and mortality. In well-resourced health care settings, induction therapy with at least 2 weeks of amphotericin B deoxycholate (dAmB) is advocated. Multiple clinical studies suggest that dAmB is the drug of choice for cryptococcal meningitis. In many parts of the world where the burden of cryptococcal meningitis is highest, it is infeasible to administer dAmB for prolonged periods. This paper provides the experimental basis for the efficacy of abbreviated regimens of dAmB for cryptococcal meningitis. The concept was explored in two well-validated laboratory animal models of cryptococcal meningitis, and the results were bridged to humans by using a range of mathematical modeling techniques. A 3-day regimen is as effective as the standard 14-day course. An abbreviated regimen is significantly more feasible and may enable better antifungal therapy to be administered to many patients with this frequently lethal disease.

Wellness coaching for obesity: a case report.

D.S. presented to a medical and surgical weight-loss program to initiate bariatric surgery. He had made numerous attempts at weight loss to no avail and was taking steps toward bariatric surgery as a last viable option. D.S.'s health insurance provider required 3 months of supervised weight loss prior to approval for surgery, and this was initiated with a board-certified bariatrician (MD) and a registered dietitian nutritionist (RDN)/wellness coach. D.S. presented with a body mass index (BMI) >40 and was classified as morbidly obese with comorbidities of high cholesterol and hyperglycemia and degenerative joint disease (DJD) of the knees. D.S. began the process outlined by his insurance company, meeting with the MD and RDN/wellness coach monthly. A plan was developed by D.S. and his RDN/wellness coach that alligned with his wellness vision, values, and lifestyle. D.S. ate meals and snacks at regular intervals throughout the day, consumed little to no red meat, increased his consumption of fruits and vegetables, and spent 1 hour daily in a swimming pool-walking, swimming, or both. By the end of the 3-month period required by the insurance provider, D.S. had lost more than 30 lbs, improved his exercise capacity, no longer used a cane, and chose to continue with coaching rather than undergo bariatric surgery. D.S. continued to meet with the MD and RDN monthly for 1 year and averaged a 10-lb weight loss per month for a total of 120 lbs, normalizing his blood panels and improving his joint mobility. D.S. continued to meet with the RDN/wellness coach for a total of 10 visits during year 2 and quarterly visits through year 3. D.S. lost a total of 240 lbs, maintained the weight loss over the 3-year period, and achieved these results solely through lifestyle interventions. Although bariatric surgery is a viable treatment option for class 2 and 3 obesity, many patients pursue this treatment option without the help of medical and commercial weight loss personnel to improve the likelihood of weight loss sustainability. The investment of lifestyle intervention in this circumstance was less than $5000 (exculsive of blood panels) compared with the $20 000 cost of bariatric surgery at the time of intervention.

D. S. acudió a un programa médico y quirúrgico de pérdida de peso para someterse a una cirugía bariátrica. Había realizado numerosos intentos infructuosos para perder peso y se dirigió hacia la cirugía bariátrica como última opción viable. El proveedor del seguro médico de D. S. le exigía que perdiera peso de forma supervisada durante 3 meses antes de acceder a la intervención quirúrgica, para lo cual se puso en manos de un médico bariatra titulado y de un nutricionista dietista titulado/monitor de bienestar.D. S. presentaba un índice de masa corporal (IMC) > 40 y su estado se clasificó como de obesidad mórbida con las comorbilidades de colesterol elevado, hiperglucemia y artropatía degenerativa de las rodillas. D. S. inició el proceso establecido por su compañía de seguros, durante el cual se reunió cada mes con el médico y con el nutricionista/ monitor de bienestar. D. S. y su nutricionista/monitor de bienestar elaboraron un plan de acuerdo con su visión del bienestar, sus valores y su estilo de vida. D. S. ingirió alimentos y tentempiés a intervalos regulares a lo largo del día, apenas consumió carnes rojas, incrementó su consumo de frutas y verduras, y pasó una hora diaria en la piscina caminando, nadando o haciendo ambas cosas.Al final de los 3 meses establecidos por su compañía de seguros, D. S. había perdido más de 13,5 kg, mejoró su capacidad para hacer ejercicio, dejó de usar un bastón y decidió continuar con la formación en lugar de someterse a la cirugía bariátrica. D. S. siguió manteniendo reuniones mensuales con el médico y con el nutricionista durante 1 año y perdió por término medio 4,5 kg al mes hasta un total de 55 kg, con una normalización de sus valores hematológicos y una mejoría de su movilidad articular. D. S. acudió a un total de 10 visitas con el nutricionista/ monitor de bienestar durante el segundo año, y a lo largo del tercer año se reunió con él trimestralmente. D. S. perdió un total de 109 kg, mantuvo la pérdida de peso a lo largo de los 3 años y consiguió estos resultados exclusivamente mediante intervenciones en el estilo de vida.Aunque la cirugía bariátrica constituye una opción de tratamiento viable para las obesidades de clase 2 y 3, muchos pacientes recurren a esta opción sin ayuda del personal médico y comercial dedicado a la pérdida de peso para incrementar la probabilidad de que esta se mantenga. En este caso, la suma invertida en la intervención sobre el estilo de vida fue inferior a 5.000 dólares (sin contar los análisis de sangre), en comparación con los 20.000 dólares que costaba la cirugía bariátrica en el momento de la intervención.

Pharmacodynamics of liposomal amphotericin B and flucytosine for cryptococcal meningoencephalitis: safe and effective regimens for immunocompromised patients.

BACKGROUND: Cryptococcal meningoencephalitis is a lethal infection with relatively few therapeutic options. The optimal dosage of liposomal amphotericin B (LAmB) alone or in combination with flucytosine is not known. METHODS: A murine model of cryptococcal meningoencephalitis was used. The fungal density in the brain was determined using quantitative cultures. Pharmacokinetic-pharmacodynamic relationships were determined for LAmB and flucytosine administered alone. The effect of the combination was described using the Greco model and a mathematical model. The results were bridged to humans. RESULTS: Inoculation resulted in hematogenous dissemination and logarithmic growth within the central nervous system. There was histological evidence of multifocal infection throughout the brain. Both LAmB and flucytosine produced a dose-dependent reduction in fungal burden. The effect of the combination of agents in the brain was additive. Bridging studies suggested that a human dosage of LAmB 3 mg/kg/d resulted in a submaximal antifungal effect. Regimens of LAmB 6 mg/kg/d alone, LAmB 3 mg/kg/d plus flucytosine 50 mg/kg/d, and LAmB 3 mg/kg/d plus flucytosine 100 mg/kg/d all resulted in near-maximal antifungal activity. CONCLUSIONS: Potential regimens for further study in clinical trials include LAmB 6 mg/kg/d alone, LAmB 3 mg/kg/d plus flucytosine 50 mg/kg/d, and LAmB 3 mg/kg/d plus flucytosine 100 mg/kg/d.

Safety and efficacy of activated transfected killer cells for neutropenic fungal infections.

BACKGROUND: Invasive fungal infections cause considerable morbidity and mortality in neutropenic patients. White blood cell transfusions are a promising treatment for such infections, but technical barriers have prevented their widespread use. METHODS: To recapitulate white blood cell transfusions, we are developing a cell-based immunotherapy using a phagocytic cell line, HL-60. We sought to stably transfect HL-60 cells with a suicide trap (herpes simplex virus thymidine kinase), to enable purging of the cells when desired, and a bioluminescence marker, to track the cells in vivo in mice. RESULTS: Transfection was stable despite 20 months of continuous culture or storage in liquid nitrogen. Activation of these transfected cells with retinoic acid and dimethyl sulfamethoxazole enhanced their microbicidal effects. Activated transfected killer (ATAK) cells were completely eliminated after exposure to ganciclovir, confirming function of the suicide trap. ATAK cells improved the survival of neutropenic mice with lethal disseminated candidiasis and inhalational aspergillosis. Bioluminescence and histopathologic analysis confirmed that the cells were purged from surviving mice after ganciclovir treatment. Comprehensive necropsy, histopathology, and metabolomic analysis revealed no toxicity of the cells. CONCLUSIONS: These results lay the groundwork for continued translational development of this promising, novel technology for the treatment of refractory infections in neutropenic hosts.

West Nile 25A virus infection of B-cell-deficient ((micro)MT) mice: characterization of neuroinvasiveness and pseudoreversion of the viral envelope protein.

The attenuated West Nile virus 25A strain (WN25A) was investigated for its neuroinvasive properties in B-cell-deficient (microMT) mice. After peripheral inoculation, WN25A caused fatal encephalitis in the majority of 6-8-week-old mice, characterized by a systemic infection with viraemia, moderate virus burdens in peripheral tissues and a high titre of brain-associated virus. Mice generally succumbed to infection within a few weeks of infection. However, others survived for as long as 10 weeks, and some for even longer. Normal age-matched C57BL/6 mice showed no signs of illness after inoculation with WN25A virus. Nucleotide sequencing of WN25A viruses recovered from the brains of B-cell-deficient mice revealed that the conserved N-linked glycosylation site in the viral envelope protein was abolished by substitution of a serine residue at position 155. This was found to be a pseudoreversion relative to the wild-type WN-Israel strain, based on virulence testing of one such brain-associated virus in both B-cell-deficient and normal C57BL/6 mice. This study provides further characterization of the mouse virulence properties of the attenuated WN25A virus in the context of B-cell deficiency. Replication in these mice does not involve rapid neuroadaptation or reversion of WN25A virus to a neuroinvasive phenotype. Molecular modelling studies suggest a difference in local structure of the E protein associated with either an asparagine or serine residue at position 155 compared with the tyrosine found in the virulent parental WN-Israel virus.

The iron chelator deferasirox protects mice from mucormycosis through iron starvation.

Mucormycosis causes mortality in at least 50% of cases despite current first-line therapies. Clinical and animal data indicate that the presence of elevated available serum iron predisposes the host to mucormycosis. Here we demonstrate that deferasirox, an iron chelator recently approved for use in humans by the US FDA, is a highly effective treatment for mucormycosis. Deferasirox effectively chelated iron from Rhizopus oryzae and demonstrated cidal activity in vitro against 28 of 29 clinical isolates of Mucorales at concentrations well below clinically achievable serum levels. When administered to diabetic ketoacidotic or neutropenic mice with mucormycosis, deferasirox significantly improved survival and decreased tissue fungal burden, with an efficacy similar to that of liposomal amphotericin B. Deferasirox treatment also enhanced the host inflammatory response to mucormycosis. Most importantly, deferasirox synergistically improved survival and reduced tissue fungal burden when combined with liposomal amphotericin B. These data support clinical investigation of adjunctive deferasirox therapy to improve the poor outcomes of mucormycosis with current therapy. As iron availability is integral to the pathogenesis of other infections (e.g., tuberculosis, malaria), broader investigation of deferasirox as an antiinfective treatment is warranted.

The development of methods for immunophenotypic and lymphocyte function analyzes for assessment of Southern sea otter (Enhydra lutris nereis) health.

The Southern sea otter (Enhydra lutris nereis) is listed as threatened under the Endangered Species Act. The population began a pattern of slow decline in 1995. The decline was attributed to high adult mortality rates with infectious disease being the major cause of death. Multiple pathogens were implicated in these deaths including opportunistic pathogens such as Coccidiodes immitis and Toxoplasma sp. These findings suggested that the immunological health of mature animals in this population might be compromised. The primary goal of this study was to establish techniques for assessing phenotypic and functional baseline data for peripheral blood mononuclear cells (PBMC) in free-ranging sea otters. Standard total and differential white blood cell counts were augmented by emumeration of T and B lymphocyte subsets. Lymphocyte function was determined by both mitogen-induced proliferation and expression of IL-2 receptors. In addition to establishing normal ranges for adult animals, age-related changes were identified in B lymphocyte numbers and cell-surface density of major histocompatability complex class II (MHC II) proteins. The predominant lymphocyte subpopulation in Southern sea otters is the T lymphocyte. Substantial variation among individual animals was observed within the B lymphocyte population both in cell number and density of MHC II expression. Pups had greater numbers of T and B lymphocyte, as well as, greater MHC II expression on B lymphocytes than adults. Mitogen-induced proliferation of peripheral blood mononuclear cells (PBMC) was variable among individual animals with no significant difference in cell response between age class and gender. Concanavalin (ConA) was a more effective mitogen in stimulating proliferation and interleukin (IL)-2 receptor expression than pokeweed. This data can be used to augment routine hematology profiles and aid in the identification of animals with immunologic perturbations.

Chronic fuel oil toxicity in American mink (Mustela vison): systemic and hematological effects of ingestion of a low-concentration of bunker C fuel oil.

Petroleum oil enters the coastal marine environment through various sources; marine mammals such as sea otters that inhabit this environment may be exposed to low concentrations of petroleum hydrocarbons through ingestion of contaminated prey. The inability to perform controlled studies in free-ranging animals hinders investigations of the effects of chronic petroleum oil exposure on sea otter morbidity and mortality, necessitating the development of a reliable laboratory model. We examined the effects of oral exposure to 500 ppm bunker C fuel oil over 113-118 days on American mink, a species phylogenetically related to the sea otter. Hematological parameters and organs were examined for fuel oil-associated changes. Hepatic cytochrome P4501A1 mRNA expression and fecal cortisol concentrations were also measured. Ingestion of fuel oil was associated with a decrease in erythrocyte count, hemoglobin concentration (Hgb), hematocrit (HCT), and an increase in mean corpuscular volume (MCV). Total leukocytes were elevated in the fuel oil group from increases in neutrophils, lymphocytes, and monocytes. Significant interactions between fuel oil and antigen challenge were found for erythrocyte parameters, monocyte and lymphocyte counts. Liver and adrenal weights were increased although mesenteric lymph node weights were decreased in the fuel oil group. Hepatic cytochrome P4501A1 mRNA was elevated in the fuel oil group. Fecal cortisol concentration did not vary between the two groups. Our findings show that fuel oil exposure alters circulating leukocyte numbers, erythrocyte homeostasis, hepatic metabolism and adrenal physiology and establish a framework to use mink as a model for sea otters in studying the systemic effects of marine contaminants.

Immunophenotypic and functional effects of bunker C fuel oil on the immune system of American mink (Mustela vison).

The relationship between exposure to environmental contaminants and immunotoxicity in vulnerable marine species is unknown. In this study, we used American mink (Mustela vision) as a surrogate species for the sea otter to examine the immunotoxic effects of chronic exposure to a low concentration of bunker C fuel oil (500 ppm admixed in the feed for 113-118 days). The mink immune system was monitored over time by flow cytometric analysis for alterations in the immunophenotype of blood lymphocytes and monocytes and by mitogen-stimulated proliferation assays for changes in peripheral blood mononuclear cell function. Fuel oil exposure caused a mild, yet significant (P < 0.05) increase in the absolute numbers of specific peripheral blood lymphocyte subsets (CD3+T cells) and monocytes, an increase in the level of expression of functionally significant cell surface proteins (MHC II, CD18), and an increase in mitogen-induced mononuclear cell proliferative responses. This heightened state of cellular activation along with the increase in specific cell surface protein expression on both the innate and adaptive immune cells is similar to the pro-inflammatory or "adjuvant-like" effect described in laboratory models of polycyclic aromatic hydrocarbon exposure in other species. These results show the benefits of using a controlled laboratory model for detecting and characterizing subtle petroleum oil-induced perturbations in immune responses. In addition this study establishes a framework for studying the effects of environmental petroleum oil exposure on the immune system of free-ranging marine mammals. Expansion of these studies to address biolgical significance is warranted.