Tuning sterol extraction kinetics yields a renal-sparing polyene antifungal.

Decades of previous efforts to develop renal-sparing polyene antifungals were misguided by the classic membrane permeabilization model1. Recently, the clinically vital but also highly renal-toxic small-molecule natural product amphotericin B was instead found to kill fungi primarily by forming extramembraneous sponge-like aggregates that extract ergosterol from lipid bilayers2-6. Here we show that rapid and selective extraction of fungal ergosterol can yield potent and renal-sparing polyene antifungals. Cholesterol extraction was found to drive the toxicity of amphotericin B to human renal cells. Our examination of high-resolution structures of amphotericin B sponges in sterol-free and sterol-bound states guided us to a promising structural derivative that does not bind cholesterol and is thus renal sparing. This derivative was also less potent because it extracts ergosterol more slowly. Selective acceleration of ergosterol extraction with a second structural modification yielded a new polyene, AM-2-19, that is renal sparing in mice and primary human renal cells, potent against hundreds of pathogenic fungal strains, resistance evasive following serial passage in vitro and highly efficacious in animal models of invasive fungal infections. Thus, rational tuning of the dynamics of interactions between small molecules may lead to better treatments for fungal infections that still kill millions of people annually7,8 and potentially other resistance-evasive antimicrobials, including those that have recently been shown to operate through supramolecular structures that target specific lipids9.

Engineered ACE2 decoy mitigates lung injury and death induced by SARS-CoV-2 variants.

Vaccine hesitancy and emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) escaping vaccine-induced immune responses highlight the urgency for new COVID-19 therapeutics. Engineered angiotensin-converting enzyme 2 (ACE2) proteins with augmented binding affinities for SARS-CoV-2 spike (S) protein may prove to be especially efficacious against multiple variants. Using molecular dynamics simulations and functional assays, we show that three amino acid substitutions in an engineered soluble ACE2 protein markedly augmented the affinity for the S protein of the SARS-CoV-2 WA-1/2020 isolate and multiple VOCs: B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta). In humanized K18-hACE2 mice infected with the SARS-CoV-2 WA-1/2020 or P.1 variant, prophylactic and therapeutic injections of soluble ACE22.v2.4-IgG1 prevented lung vascular injury and edema formation, essential features of CoV-2-induced SARS, and above all improved survival. These studies demonstrate broad efficacy in vivo of an engineered ACE2 decoy against SARS-CoV-2 variants in mice and point to its therapeutic potential.

Yttrium-90 Radioembolization to the Prostate Gland: Proof of Concept in a Canine Model and Clinical Translation.

PURPOSE: To investigate the feasibility, safety, and absorbed-dose distribution of prostatic artery radioembolization (RE) in a canine model. MATERIALS AND METHODS: Fourteen male castrated beagles received dihydroandrosterone/estradiol to induce prostatic hyperplasia for the duration of the study. Each dog underwent fluoroscopic prostatic artery catheterization. Yttrium-90 (90Y) microspheres (TheraSphere; Boston Scientific, Marlborough, Massachusetts) were delivered to 1 prostatic hemigland (dose escalation from 60 to 200 Gy), with the contralateral side serving as a control. Assessments for adverse events were performed throughout the follow-up (Common Terminology Criteria for Adverse Events v5.0). Positron emission tomography/magnetic resonance (MR) imaging provided a confirmation after the delivery of absorbed-dose distribution. MR imaging was performed before and 3, 20, and 40 days after RE. Tissue harvest of the prostate, rectum, bladder, urethra, penis, and neurovascular bundles was performed 60 days after RE. RESULTS: All the animals successfully underwent RE. Positron emission tomography/MR imaging demonstrated localization to and good coverage of only the treated hemigland. No adverse events occurred. The MR imaging showed a significant dose-dependent decrease in the treated hemigland size at 40 days (25%-60%, P < .001). No extraprostatic radiographic changes were observed. Necropsy demonstrated no gross rectal, urethral, penile, or bladder changes. Histology revealed RE-induced changes in the treated prostatic tissues of the highest dose group, with gland atrophy and focal necrosis. No extraprostatic RE-related histologic findings were observed. CONCLUSIONS: Prostate 90Y RE is safe and feasible in a canine model and leads to focal dose-dependent changes in the gland without inducing unwanted extraprostatic effects. These results suggest that an investigation of nonoperative prostate cancer is warranted.

Tumor-Localized Interleukin-2 and Interleukin-12 Combine with Radiation Therapy to Safely Potentiate Regression of Advanced Malignant Melanoma in Pet Dogs.

PURPOSE: Cytokines IL2 and IL12 exhibit potent anticancer activity but suffer a narrow therapeutic window due to off-tumor immune cell activation. Engineering cytokines with the ability to bind and associate with tumor collagen after intratumoral injection potentiated response without toxicity in mice and was previously safe in pet dogs with sarcoma. Here, we sought to test the efficacy of this approach in dogs with advanced melanoma. PATIENTS AND METHODS: This study examined 15 client-owned dogs with histologically or cytologically confirmed malignant melanoma that received a single 9-Gy fraction of radiotherapy, followed by six cycles of combined collagen-anchored IL2 and IL12 therapy every 2 weeks. Cytokine dosing followed a 3 + 3 dose escalation design, with the initial cytokine dose chosen from prior evaluation in canine sarcomas. No exclusion criteria for tumor stage or metastatic burden, age, weight, or neuter status were applied for this trial. RESULTS: Median survival regardless of the tumor stage or dose level was 256 days, and 10/13 (76.9%) dogs that completed treatment had CT-measured tumor regression at the treated lesion. In dogs with metastatic disease, 8/13 (61.5%) had partial responses across their combined lesions, which is evidence of locoregional response. Profiling by NanoString of treatment-resistant dogs revealed that B2m loss was predictive of poor response to this therapy. CONCLUSIONS: Collectively, these results confirm the ability of locally administered tumor-anchored cytokines to potentiate responses at regional disease sites when combined with radiation. This evidence supports the clinical translation of this approach and highlights the utility of comparative investigation in canine cancers.

Tumor-localized interleukin-2 and interleukin-12 combine with radiation therapy to safely potentiate regression of advanced malignant melanoma in pet dogs.

The clinical use of interleukin-2 and -12 cytokines against cancer is limited by their narrow therapeutic windows due to on-target, off-tumor activation of immune cells when delivered systemically. Engineering IL-2 and IL-12 to bind to extracellular matrix collagen allows these cytokines to be retained within tumors after intralesional injection, overcoming these clinical safety challenges. While this approach has potentiated responses in syngeneic mouse tumors without toxicity, the complex tumor-immune interactions in human cancers are difficult to recapitulate in mouse models of cancer. This has driven an increased role for comparative oncology clinical trials in companion (pet) dogs with spontaneous cancers that feature analogous tumor and immune biology to human cancers. Here, we report the results from a dose-escalation clinical trial of intratumoral collagen-binding IL-2 and IL-12 cytokines in pet dogs with malignant melanoma, observing encouraging local and regional responses to therapy that may suggest human clinical benefit with this approach.

Interpreting stress responses during routine toxicity studies: a review of the biology, impact, and assessment.

Stress often occurs during toxicity studies. The perception of sensory stimuli as stressful primarily results in catecholamine release and activation of the hypothalamic-pituitary-adrenal (HPA) axis to increase serum glucocorticoid concentrations. Downstream effects of these neuroendocrine signals may include decreased total body weights or body weight gain; food consumption and activity; altered organ weights (e.g., thymus, spleen, adrenal); lymphocyte depletion in thymus and spleen; altered circulating leukocyte counts (e.g., increased neutrophils with decreased lymphocytes and eosinophils); and altered reproductive functions. Typically, only some of these findings occur in a given study. Stress responses should be interpreted as secondary (indirect) rather than primary (direct) test article-related findings. Determining whether effects are the result of stress requires a weight-of-evidence approach. The evaluation and interpretation of routinely collected data (standard in-life, clinical pathology, and anatomic pathology endpoints) are appropriate and generally sufficient to assess whether or not changes are secondary to stress. The impact of possible stress-induced effects on data interpretation can partially be mitigated by toxicity study designs that use appropriate control groups (e.g., cohorts treated with vehicle and subjected to the same procedures as those dosed with test article), housing that minimizes isolation and offers environmental enrichment, and experimental procedures that minimize stress and sampling and analytical bias. This article is a comprehensive overview of the biological aspects of the stress response, beginning with a Summary (Section 1) and an Introduction (Section 2) that describes the historical and conventional methods used to characterize acute and chronic stress responses. These sections are followed by reviews of the primary systems and parameters that regulate and/or are influenced by stress, with an emphasis on parameters evaluated in toxicity studies: In-life Procedures (Section 3), Nervous System (Section 4), Endocrine System (Section 5), Reproductive System (Section 6), Clinical Pathology (Section 7), and Immune System (Section 8). The paper concludes (Section 9) with a brief discussion on Minimizing Stress-Related Effects (9.1.), and a final section explaining why Parameters routinely measured are appropriate for assessing the role of stress in toxicology studies (9.2.).

Diagnostic accuracy of seven radiographic views, alone and in combination, for diagnosis of pectoral girdle fractures in wild passerines after window collisions.

OBJECTIVE: To determine the prevalence of pectoral girdle fractures in wild passerines found dead following presumed window collision and evaluate the diagnostic accuracy of various radiographic views for diagnosis of pectoral girdle fractures. SAMPLE: Cadavers of 103 wild passerines that presumptively died as a result of window collisions. PROCEDURES: Seven radiographic projections (ventrodorsal, dorsoventral, lateral, and 4 oblique views) were obtained for each cadaver. A necropsy was then performed, and each bone of the pectoral girdle (coracoid, clavicle, and scapula) was evaluated for fractures. Radiographs were evaluated in a randomized order by a blinded observer, and results were compared with results of necropsy. RESULTS: Fifty-six of the 103 (54%) cadavers had ≥ 1 pectoral girdle fracture. Overall accuracy of using individual radiographic projections to diagnose pectoral girdle fractures ranged from 63.1% to 72.8%, sensitivity ranged from 21.3% to 51.1%, and specificity ranged from 85.7% to 100.0%. The sensitivity of using various combinations of radiographic projections to diagnose pectoral girdle fractures ranged from 51.1% to 66.0%; specificity ranged from 76.8% to 96.4%. CLINICAL RELEVANCE: Radiography alone appeared to have limited accuracy for diagnosing fractures of the bones of the pectoral girdle in wild passerines after collision with a window. Both individual radiographic projections and combinations of projections resulted in numerous false negative but few false positive results.

Safety and efficacy of cold atmospheric plasma for the sterilization of a Pasteurella multocida-contaminated subcutaneously implanted foreign body in rabbits.

OBJECTIVE: To determine whether a stainless steel implant sterilized with a novel cold atmospheric plasma sterilization (CAPS) device adversely affects local tissues in rabbits and whether CAPS was as effective as steam sterilization with an autoclave to inactivate Pasteurella multocida. ANIMALS: 31 healthy New Zealand White rabbits. PROCEDURES: Steam-autoclaved stainless steel implants inoculated with P multocida underwent a second steam autoclave sterilization (AIA) or CAPS (AICAPS). One AIA implant and 3 AICAPS implants were randomly placed subcutaneously at 4 sites in 21 rabbits (84 implants). These rabbits were monitored daily for 5 days for evidence of systemic illness and local tissue reactions at the implantation sites and then euthanized. Samples were taken from each implant site for bacterial culture and histologic examination. RESULTS: Cultures of samples obtained from all sites were negative for bacterial growth. No significant difference was observed in mean skin thickness or erythema between AIA and AICAPS implant sites on any observed day. Also, individual histologic grades for the epidermis, dermis, subcutis, and muscle and total histologic grade were not significantly different between AIA and AICAPS implant sites. CONCLUSIONS AND CLINICAL RELEVANCE: Cold atmospheric plasma sterilization was noninferior to steam sterilization of P multocida-contaminated stainless steel implants in the rabbits in the present study. However, studies of the efficacy of CAPS for inactivation of other important bacteria are needed.

Engineered High-Affinity ACE2 Peptide Mitigates ARDS and Death Induced by Multiple SARS-CoV-2 Variants.

Vaccine hesitancy and continuing emergence of SARS-CoV-2 variants of concern that may escape vaccine-induced immune responses highlight the urgent need for effective COVID-19 therapeutics. Monoclonal antibodies used in the clinic have varying efficacies against distinct SARS-CoV-2 variants; thus, there is considerable interest in engineered ACE2 peptides with augmented binding affinities for SARS-CoV-2 Spike protein. These could have therapeutic benefit against multiple viral variants. Using molecular dynamics simulations, we show how three amino acid substitutions in an engineered soluble ACE2 peptide (sACE2 2 .v2.4-IgG1) markedly increase affinity for the SARS-CoV-2 Spike (S) protein. We demonstrate high binding affinity to S protein of the early SARS-CoV-2 WA-1/2020 isolate and also to multiple variants of concern: B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), and B.1.617.2 (Delta) SARS-CoV-2 variants. In humanized K18-hACE2 mice, prophylactic and therapeutic administration of sACE2 2 .v2.4-IgG1 peptide prevented acute lung vascular endothelial injury and lung edema (essential features of ARDS) and significantly improved survival after infection by SARS-CoV-2 WA-1/2020 as well as P.1 variant of concern. These studies demonstrate for the first time broad efficacy in vivo of an ACE2 decoy peptide against multiple SARS-CoV-2 variants and point to its therapeutic potential.

N-Methyl-N-Nitrosourea (MNU): A positive control chemical for p53+/- mouse carcinogenicity studies.

This study evaluated the effects of a single intraperitoneal injection of N-methyl-N-nitrosourea (MNU) in citrate buffer (pH 4.5) at a dose of 75 mg/kg in thirty male and thirty female p53+/- mice followed by a six-month observation period. Fifteen control mice per sex received a single intraperitoneal injection of citrate buffer. Fifty-six of sixty mice treated with MNU died or were sacrificed before the end of the observation period. Twenty-four males and twenty-seven females treated with MNU developed malignant lymphoma of the thymus; of these, twenty-three males and twenty-seven females had corresponding enlargement or masses in the thymus at necropsy. Lymphoblasts in thymic lymphomas stained positively for mouse CD3 antigen, indicating a T-cell lineage. One control female mouse had malignant lymphoma of the spleen that did not involve the thymus. Nine males and five females treated with MNU had adenomas or adenocarcinomas of the small intestine, whereas no intestinal neoplasms were observed in control mice. These findings support the use of a single dose of MNU as a positive control chemical in six-month p53+/- mouse carcinogenicity studies and suggest that examination of the thymus alone is sufficient to evaluate the validity of the model system.

Comparison of tumor histology to dynamic contrast enhanced magnetic resonance imaging-based physiological estimates.

PURPOSE: The purpose of this study was to compare histologically determined cellularity and extracellular space to dynamic contrast-enhanced magnetic resonance imaging (DCE MRI)-based maps of a two-compartment model's parameters describing tumor contrast agent extravasation, specifically tumor extravascular extracellular space (EES) volume fraction (ve), tumor plasma volume fraction (vp) and volume-normalized contrast agent transfer rate between tumor plasma and interstitium (KTRANS/VT). MATERIALS AND METHODS: Obtained ve, vp and KTRANS/VT maps were estimated from gadolinium diethylenetriamine penta-acetic acid DCE T1-weighted gradient-echo images at resolutions of 469, 938 and 2500 microm. These parameter maps were compared at each resolution to histologically determined tumor type, and the high-resolution 469-microm maps were compared with automated cell counting using Otsu's method and a color-thresholding method for estimated intracellular (Vintracellular) and extracellular (Vextracellular) space fractions. RESULTS: The top five KTRANS/VT values obtained from each tumor at 469 and 938 microm resolutions are significantly different from those obtained at 2500 microm (P<.0001) and from one another (P=.0014). Using these top five KTRANS/VT values and the corresponding tumor EES volume fractions ve, we can statistically differentiate invasive ductal carcinomas from noninvasive papillary carcinomas for the 469- and 938-microm resolutions (P=.0017 and P=.0047, respectively), but not for the 2500-microm resolution (P=.9008). The color-thresholding method demonstrated that ve measured by DCE MRI is statistically similar to histologically determined EES. The Vextracellular obtained from the color-thresholding method was statistically similar to the ve measured with DCE MRI for the top 10 KTRANS/VT values (P>.05). DCE MRI-based KTRANS/VT estimates are not statistically correlated with histologically determined cellularity. CONCLUSION: DCE MRI estimates of tumor physiology are a limited representation of tumor histological features. Extracellular spaces measured by both DCE MRI and microscopic analysis are statistically similar. Tumor typing by DCE MRI is spatial resolution dependent, as lower resolutions average out contributions to voxel-based estimates of KTRANS/VT. Thus, an appropriate resolution window is essential for DCE MRI tumor diagnosis. Within this resolution window, the top KTRANS/VT values with corresponding ve are diagnostic for the tumor types analyzed in this study.

Thallium toxicosis in a Pit Bull Terrier.

Thallotoxicosis is described in an adult Pit Bull Terrier. The dog exhibited anorexia, emesis, weakness, conscious proprioceptive deficits, and a hemorrhagic diarrhea before death. A severe, acute necrotizing enterocolitis was evident upon histological examination, as was a multifocal to coalescing pulmonary edema. Liver and kidney thallium concentrations were 18 and 26 ppm, respectively. The source of the thallium was determined to be thallium sulfate obtained by a person with the intent to harm family members. Although thallium has not been produced in the United States for 20 years, this report demonstrates the need to consider thallium toxicosis as a differential diagnosis for animals presenting with vague and mixed gastrointestinal and neurological signs.

Moxidectin steady state prior to inoculation protects cats from subsequent, repeated infection with Dirofilaria immitis.

BACKGROUND: Infection of cats with Dirofilaria immitis causes seroconversion on antibody tests and pulmonary pathology, often without subsequent development of adult heartworms. Consistent administration of topical 10% imidacloprid-1% moxidectin has been shown to result in sustained plasma levels of moxidectin in cats after three to five treatments, a pharmacokinetic behavior known as "steady state". METHODS: To evaluate the ability of moxidectin at "steady state" to protect cats from subsequent infection with D. immitis, cats (n = 10) were treated with the labeled dose of topical 10% imidacloprid-1% moxidectin for four monthly treatments. Each cat was inoculated with 25 third-stage larvae of D. immitis 7, 14, 21, and 28 days after the last treatment; non-treated cats (n = 9) were inoculated on the same days, serving as infection controls. Blood samples were collected from each cat from 1 month prior to treatment until 7 months after the final inoculation and tested for antibody to, and antigen and microfilaria of, D. immitis. RESULTS: Measurement of serum levels of moxidectin confirmed steady state in treated cats. Cats treated with topical 10% imidacloprid-1% moxidectin prior to trickle inoculation of D. immitis L3 larvae throughout the 28 day post-treatment period remained negative on antibody and antigen tests throughout the study and did not develop gross or histologic lesions characteristic of heartworm infection. A majority of non-treated cats tested antibody positive by 3-4 months post infection (6/9) and, after heat treatment, tested antigen positive by 6-7 months post-infection (5/9). Histologic lesions characteristic of D. immitis infection, including intimal and medial thickening of the pulmonary artery, were present in every cat with D. immitis antibodies (6/6), although adult D. immitis were confirmed in only 5/6 antibody-positive cats at necropsy. Microfilariae were not detected at any time. CONCLUSIONS: Taken together, these data indicate that prior treatment with 10% imidacloprid-1% moxidectin protected cats from subsequent infection with D. immitis for 28 days, preventing both formation of a detectable antibody response and development of pulmonary lesions by either immature stages of D. immitis or young adult heartworms.

Multiple cutaneous leiomyomas in the perineum of a horse.

Multiple cutaneous masses developed in the perineum of a 14-year-old Saddlebred stallion over a period of approximately 5 years. Clinically, the masses ranged in size from 3- to 9-mm diameter and were not ulcerated, painful, or pruritic. Three of the masses were surgically excised and submitted for microscopic evaluation. The masses were dome shaped to nodular, located in the superficial dermis, and composed of haphazardly arranged bundles of plump spindle-shaped cells. The tumor cells immunoreacted with monoclonal antibodies directed against desmin, muscle-specific actin, and smooth muscle actin, confirming a smooth muscle origin. Multiple cutaneous leiomyomas have not been reported previously in horses.

Primary epitheliotropic intestinal T-cell lymphoma in a horse.

A 17-year-old Quarterhorse gelding with a clinical diagnosis of protein-losing enteropathy was submitted for necropsy following a 4-5-month duration of weight loss, decreased appetite, and hypoproteinemia. Gross findings included multiple 1-2-cm diameter ulcers on the luminal surfaces of the duodenum and ileum. Histologic examination revealed individual large, round cells infiltrating much of the mucosal epithelium of the duodenum, jejunum, ileum, and colon in addition to multifocal areas of ulceration. Similar round cells infiltrated Brunner's glands and expanded the submucosa beneath the foci of ulceration. Immunohistochemical staining indicated the round cell population was of T-lymphocyte origin. Several features of this equine neoplasm bear similarities to enteropathy-associated T-cell lymphoma in humans.

Paranasal meningioma in a horse.

Paranasal meningioma was diagnosed in a 5-year-old Appaloosa gelding. The mass occupied the right maxillary, frontal, and sphenopalatine sinuses but did not invade the calvarium. The diagnosis was based on histologic evaluation, positive immunohistochemical staining for vimentin and cytokeratin, and ultrastructural features including the presence of interdigitating spindle cells with numerous desmosomes.

Characterization of cardiac lesions in calves after ingestion of Japanese yew (Taxus cuspidata).

Plants of the genus Taxus are common ornamental shrubs that contain cardiotoxic alkaloids. Gross lesions consistent with heart failure are frequently reported in fatal cases; however, microscopic lesions in the heart have not been well characterized. The current report describes 2 related outbreaks in which 7 of 30, 250-kg calves died after confirmed exposure to clippings of Japanese yew (Taxus cuspidata). Three calves died 24 hr after initial exposure, with no significant gross or histologic lesions. Leaves of the yew plant were identified within the rumen contents, and Taxus alkaloids were confirmed by gas chromatography-mass spectrometry. Following the initial diagnosis, the yew clippings were burned. Two days later, the remaining calves were reintroduced to the enclosure. Within 24 hr, 3 additional calves began to show clinical signs of depression (3/3) or labored breathing (1/3), and by the fourth day, these 3 calves and an additional calf were found dead. Partially burnt yew leaves were found during close inspection of the enclosure. Two of 3 calves submitted for necropsy were severely autolyzed; the third had pulmonary edema and mild fibrinous pleural effusion. Histologic lesions in the latter included multifocal cardiac myocyte hypereosinophilia, sarcolemma fragmentation, pyknosis, karyolysis, myocyte loss, and a mild interstitial lymphoplasmacytic infiltrate with edema. Moderate fibrinosuppurative interstitial pneumonia was the only other significant finding. Cardiac changes were attributed to damage from the initial exposure to Taxus 6 days prior to death.