A Novel Pan-RAS Inhibitor with a Unique Mechanism of Action Blocks Tumor Growth in Mouse Models of GI Cancer.

Here we characterize a novel pan-RAS inhibitor, ADT-007, that potently and selectively inhibited the growth of histologically diverse cancer cell lines with mutant or activated RAS irrespective of the RAS mutation or isozyme. Growth inhibition was dependent on activated RAS and associated with reduced GTP-RAS levels and MAPK/AKT signaling. ADT-007 bound RAS in lysates from sensitive cells with sub-nanomolar EC 50 values but did not bind RAS in lysates from insensitive cells with low activated RAS. Insensitivity to ADT-007 was attributed to metabolic deactivation by UGT-mediated glucuronidation, providing a detoxification mechanism to protect normal cells from pan-RAS inhibition. Molecular modeling and experiments using recombinant RAS revealed that ADT-007 binds RAS in a nucleotide-free conformation to block GTP activation. Local injection of ADT-007 strongly inhibited tumor growth in syngeneic immune competent and xenogeneic immune deficient mouse models of colorectal and pancreatic cancer and activated innate and adaptive immunity in the tumor microenvironment. SIGNIFICANCE: ADT-007 is a novel pan-RAS inhibitor with a unique mechanism of action having potential to circumvent resistance to mutant-specific KRAS inhibitors and activate antitumor immunity. The findings support further development of ADT-007 analogs and/or prodrugs with oral bioavailability as a generalizable monotherapy or combined with immunotherapy for RAS mutant cancers. BACKGROUND: It is projected that colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDA) will cause 52,580 and 49,830 deaths in the US in 2023, respectively (1). The 5-year survival rates for CRC and PDA are 65% and 12%, respectively (1). Over 50% of CRC and 90% of PDA patients harbor mutations in KRAS genes that are associated with poor prognosis, making the development of novel KRAS inhibitors an urgent unmet medical need (2).

A mouse model of Zhu-Tokita-Takenouchi-Kim syndrome reveals indispensable SON functions in organ development and hematopoiesis.

Rare diseases are underrepresented in biomedical research, leading to insufficient awareness. Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome is a rare disease caused by genetic alterations that result in heterozygous loss of function of SON. While patients with ZTTK syndrome live with numerous symptoms, the lack of model organisms hampers our understanding of SON and this complex syndrome. Here, we developed Son haploinsufficiency (Son+/-) mice as a model of ZTTK syndrome and identified the indispensable roles of Son in organ development and hematopoiesis. Son+/- mice recapitulated clinical symptoms of ZTTK syndrome, including growth retardation, cognitive impairment, skeletal abnormalities, and kidney agenesis. Furthermore, we identified hematopoietic abnormalities in Son+/- mice, including leukopenia and immunoglobulin deficiency, similar to those observed in human patients. Surface marker analyses and single-cell transcriptome profiling of hematopoietic stem and progenitor cells revealed that Son haploinsufficiency shifted cell fate more toward the myeloid lineage but compromised lymphoid lineage development by reducing genes required for lymphoid and B cell lineage specification. Additionally, Son haploinsufficiency caused inappropriate activation of erythroid genes and impaired erythropoiesis. These findings highlight the importance of the full gene expression of Son in multiple organs. Our model serves as an invaluable research tool for this rare disease and related disorders associated with SON dysfunction.

Breaking the stromal barrier in pancreatic cancer: Advances and challenges.

Pancreatic cancer (PC) remains a leading cause of mortality worldwide due to the absence of early detection methods and the low success rates of traditional therapeutic strategies. Drug resistance in PC is driven by its desmoplastic stroma, which creates a barrier that shields cancer niches and prevents the penetration of drugs. The PC stroma comprises heterogeneous cellular populations and non-cellular components involved in aberrant ECM deposition, immunosuppression, and drug resistance. These components can influence PC development through intricate and complex crosstalk with the PC cells. Understanding how stromal components and cells interact with and influence the invasiveness and refractoriness of PC cells is thus a prerequisite for developing successful stroma-modulating strategies capable of remodeling the PC stroma to alleviate drug resistance and enhance therapeutic outcomes. In this review, we explore how non-cellular and cellular stromal components, including cancer-associated fibroblasts and tumor-associated macrophages, contribute to the immunosuppressive and tumor-promoting effects of the stroma. We also examine the signaling pathways underlying their activation, tumorigenic effects, and interactions with PC cells. Finally, we discuss recent pre-clinical and clinical work aimed at developing and testing novel stroma-modulating agents to alleviate drug resistance and improve therapeutic outcomes in PC.

A mouse model of ZTTK syndrome reveals indispensable SON functions in organ development and hematopoiesis.

Rare diseases are underrepresented in biomedical research, leading to insufficient awareness. Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome is a rare disease caused by genetic alterations that result in heterozygous loss-of-function of SON. While ZTTK syndrome patients suffer from numerous symptoms, the lack of model organisms hamper our understanding of both SON and this complex syndrome. Here, we developed Son haploinsufficiency (Son+/-) mice as a model of ZTTK syndrome and identified the indispensable roles of Son in organ development and hematopoiesis. Son+/- mice recapitulated clinical symptoms of ZTTK syndrome, including growth retardation, cognitive impairment, skeletal abnormalities, and kidney agenesis. Furthermore, we identified hematopoietic abnormalities in Son+/- mice, similar to those observed in human patients. Surface marker analyses and single-cell transcriptome profiling of hematopoietic stem and progenitor cells revealed that Son haploinsufficiency inclines cell fate toward the myeloid lineage but compromises lymphoid lineage development by reducing key genes required for lymphoid and B cell lineage specification. Additionally, Son haploinsufficiency causes inappropriate activation of erythroid genes and impaired erythroid maturation. These findings highlight the importance of the full gene dosage of Son in organ development and hematopoiesis. Our model serves as an invaluable research tool for this rare disease and related disorders associated with SON dysfunction.

Obesity associated pancreatic ductal adenocarcinoma: Therapeutic challenges.

Obesity is a prominent health issue worldwide and directly impacts pancreatic health, with obese individuals exhibiting a significant risk for increasing pancreatic ductal adenocarcinoma (PDAC). Several factors potentially explain the increased risk for the development of PDAC, including obesity-induced chronic inflammation within and outside of the pancreas, development of insulin resistance and metabolic dysfunction, promotion of immune suppression within the pancreas during inflammation, pre- and malignant stages, variations in hormones levels (adiponectin, ghrelin, and leptin) produced from the adipose tissue, and acquisition of somatic mutations in tumor once- and suppressor proteins critical for pancreatic tumorigenesis. In this manuscript, we will explore the broad impact of these obesity-induced risk factors on the development and progression of PDAC, focusing on changes within the tumor microenvironment (TME) as they pertain to prevention, current therapeutic strategies, and future directions for targeting obesity management as they relate to the prevention of pancreatic tumorigenesis.

In vitro and in vivo immune assessments of genetically-engineered pig skin grafts in New World (squirrel) monkeys.

Half a million patients in the USA alone require treatment for burns annually. Following an extensive burn, it may not be possible to provide sufficient autografts in a single setting. Genetic manipulations (GM) of pigs offer the possibility of reducing primate humoral and cellular rejection of pig skin xenografts and thus extending graft survival. We compared the survival of skin grafts from pigs with 9-GM with that of autografts and allografts in squirrel monkeys. Monitoring for rejection was by (1) macroscopic examination, (2) histopathological examination of skin biopsies, and (3) measurement of anti-monkey and anti-pig IgM and IgG antibodies. Autografts (n = 5) survived throughout the 28 days of follow-up without histopathological features of rejection. Median survival of allografts (n = 6) was 14 days and of pig xenografts (n = 12) 21 days. Allotransplantation was associated with an increase in anti-monkey IgM, but the anticipated subsequent rise in IgG had not yet occurred at the time of euthanasia. Pig grafts were associated with increases in anti-pig IgM and IgG. In all cases, histopathologic features of rejection were similar. 9-GM pig skin xenografts survive at least as long as monkey skin allografts (and trended to survive longer), suggesting that they are a realistic clinical option for the temporary treatment of burns. Although monkeys with pig skin grafts developed anti-pig IgM and IgG antibodies, these did not cross-react with monkey antigens, indicating that a primary 9-GM pig skin graft would not be detrimental to a subsequent monkey skin allograft.

Pulmonary pathogenesis in a murine model of inhaled arsenical exposure.

Arsenic trioxide (ATO), an inorganic arsenical, is a toxic environmental contaminant. It is also a widely used chemical with industrial and medicinal uses. Significant public health risk exists from its intentional or accidental exposure. The pulmonary pathology of acute high dose exposure is not well defined. We developed and characterized a murine model of a single inhaled exposure to ATO, which was evaluated 24 h post-exposure. ATO caused hypoxemia as demonstrated by arterial blood-gas measurements. ATO administration caused disruption of alveolar-capillary membrane as shown by increase in total protein and IgM in the bronchoalveolar lavage fluid (BALF) supernatant and an onset of pulmonary edema. BALF of ATO-exposed mice had increased HMGB1, a damage-associated molecular pattern (DAMP) molecule, and differential cell counts revealed increased neutrophils. BALF supernatant also showed an increase in protein levels of eotaxin/CCL-11 and MCP-3/CCL-7 and a reduction in IL-10, IL-19, IFN-γ, and IL-2. In the lung of ATO-exposed mice, increased protein levels of G-CSF, CXCL-5, and CCL-11 were noted. Increased mRNA levels of TNF-a, and CCL2 in ATO-challenged lungs further supported an inflammatory pathogenesis. Neutrophils were increased in the blood of ATO-exposed animals. Pulmonary function was also evaluated using flexiVent. Consistent with an acute lung injury phenotype, respiratory and lung elastance showed significant increase in ATO-exposed mice. PV loops showed a downward shift and a decrease in inspiratory capacity in the ATO mice. Flow-volume curves showed a decrease in FEV0.1 and FEF50. These results demonstrate that inhaled ATO leads to pulmonary damage and characteristic dysfunctions resembling ARDS in humans.

Early Life Stress in Mice Leads to Impaired Colonic Corticosterone Production and Prolonged Inflammation Following Induction of Colitis.

BACKGROUND: Early life stress (ELS) is an environmental trigger believed to promote increased risk of IBD. Our goal was to identify mechanisms whereby ELS in mice affects susceptibility to and/or severity of gut inflammation. METHODS: We utilized 2 published animal models of ELS. In the first model, newborn mice were separated from the dam daily for 4 to 8 hours starting on postnatal day 2 and then weaned early on postnatal day 17. Control mice were left undisturbed with the dams until weaning on postnatal day 21. In the second model, dams were fed dexamethasone or vehicle ad libitum in drinking water on postpartum days 1 to 14. Plasma and colonic corticosterone were measured in juvenile and adult mice. Colitis was induced in 4-week-old mice via intraperitoneal injection of interleukin (IL)-10 receptor blocking antibody every 5 days for 15 days. Five or 15 days later, colitis scores and transcripts for Tnf, glucocorticoid receptors, and steroidogenic enzymes were measured. RESULTS: Mice exposed to ELS displayed reduced plasma and colonic corticosterone. Control animals showed improvements in indices of inflammation following cessation of interleukin-10 receptor blockade, whereas ELS-exposed animals maintained high levels of Tnf and histological signs of colitis. In colitic animals, prior exposure to ELS was associated with significantly lower expression of genes associated with corticosterone synthesis and responsiveness. Finally, TNF stimulation of colonic crypt cells from ELS mice led to increased inhibition of corticosterone synthesis. CONCLUSIONS: Our study identifies impaired local glucocorticoid production and responsiveness as a potential mechanism whereby ELS predisposes to chronic colitis in susceptible hosts.

Using 2 distinct animal models, this study shows that in mice, early life stress leads to reduced colonic corticosterone and that induction of colitis after stress removal results in reduced transcription of glucocorticoid synthesis genes, increased Tnf, and enhanced chronicity of intestinal inflammation.

Mucociliary transport deficiency and disease progression in Syrian hamsters with SARS-CoV-2 infection.

Substantial clinical evidence supports the notion that ciliary function in the airways is important in COVID-19 pathogenesis. Although ciliary damage has been observed in both in vitro and in vivo models, the extent or nature of impairment of mucociliary transport (MCT) in in vivo models remains unknown. We hypothesize that SARS-CoV-2 infection results in MCT deficiency in the airways of golden Syrian hamsters that precedes pathological injury in lung parenchyma. Micro-optical coherence tomography was used to quantitate functional changes in the MCT apparatus. Both genomic and subgenomic viral RNA pathological and physiological changes were monitored in parallel. We show that SARS-CoV-2 infection caused a 67% decrease in MCT rate as early as 2 days postinfection (dpi) in hamsters, principally due to 79% diminished airway coverage of motile cilia. Correlating quantitation of physiological, virological, and pathological changes reveals steadily descending infection from the upper airways to lower airways to lung parenchyma within 7 dpi. Our results indicate that functional deficits of the MCT apparatus are a key aspect of COVID-19 pathogenesis, may extend viral retention, and could pose a risk factor for secondary infection. Clinically, monitoring abnormal ciliated cell function may indicate disease progression. Therapies directed toward the MCT apparatus deserve further investigation.

A single intranasal administration of AdCOVID protects against SARS-CoV-2 infection in the upper and lower respiratory tracts.

The coronavirus disease 2019 (COVID-19) pandemic has illustrated the critical need for effective prophylactic vaccination to prevent the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Intranasal vaccination is an attractive approach for preventing COVID-19 as the nasal mucosa is the site of initial SARS-CoV-2 entry and viral replication prior to aspiration into the lungs. We previously demonstrated that a single intranasal administration of a candidate adenovirus type 5-vectored vaccine encoding the receptor-binding domain of the SARS-CoV-2 spike protein (AdCOVID) induced robust immunity in both the airway mucosa and periphery, and completely protected K18-hACE2 mice from lethal SARS-CoV-2 challenge. Here we show that a single intranasal administration of AdCOVID limits viral replication in the nasal cavity of K18-hACE2 mice. AdCOVID also induces sterilizing immunity in the lungs of mice as reflected by the absence of infectious virus. Finally, AdCOVID prevents SARS-CoV-2 induced pathological damage in the lungs of mice. These data show that AdCOVID not only limits viral replication in the respiratory tract, but it also prevents virus-induced inflammation and immunopathology following SARS-CoV-2 infection.

Ferret Systemic Coronavirus in Alpha-1 Antitrypsin Knockout Ferrets.

Ferret systemic coronavirus (FRSCV) causes a highly fatal disease of ferrets (Mustela putorius furo). It is believed to be a mutated variant of ferret enteric coronavirus (FRECV) and has a clinical presentation similar to that of feline infectious peritonitis virus (FIPV) in cats. The interplay of infectious diseases and host genetics will become a greater issue in the research environment as genetically modified species other than rodents become available due to advances in gene editing technology. In this case series, we present the clinical and histopathologic features of a FRSCV outbreak that affected 5 out of 10 ferrets with α-1 antitrypsin knockout (AAT KO) over an approximately 1-y period. Clinical features varied, with the affected ferrets presenting with some combination of wasting, hind limb paralysis, incontinence or sudden death. Multiple ferrets had gross pathologic lesions consistent with FRSCV, but the lesions were typically mild. Microscopic pyogranulomatous inflammation was present in 4 ferrets. Immunohistochemistry using an anti-feline coronavirus antibody that cross reacts with ferret coronavirus confirmed infection of intralesional macrophages in 4 out of 5 animals with suspected FRSCV infection. PCR testing of formalin fixed tissue was negative for all ferrets. PCR testing of feces from healthy wild-type ferrets indicated that the endemic presence of FRECV genotype 2, while PCR surveillance testing of other in-house AAT KO ferrets revealed both enteric coronavirus genotypes 1 and 2. This case series highlights the potential for greater disease incidence in the future as genetically modified ferrets are used more often, and may support exclusion of FRECV and similar viruses from highly susceptible ferret genotypes.

Observations on hydronephrosis after pig kidney transplantation in baboons.

We have seen hydronephrosis (obstructive nephropathy) at necropsy in 3 of 11 (21%) genetically-engineered pig kidneys that functioned in baboons for >36 days, even when the clinical and histopathological features of rejection were minimal. We briefly report one such case and illustrate the macroscopic and microscopic appearances of such a kidney and ureter. The causes of the observed changes remain uncertain. In our small experience, there seems to be no correlation between the development of hydronephrosis and (i) the surgical technique, (ii) the genotype of the pig, (iii) the length of the pig ureter, or (iv) the immunosuppressive and anti-inflammatory therapy administered. We suggest that the distal ureteric thickening may be the result of an inflammatory response. In two cases, we resolved the problem by carrying out a secondary side-to-side anastomosis between the proximal pig ureter and the baboon bladder.

Impaired Mitochondria Promote Aging-Associated Sebaceous Gland Dysfunction and Pathology.

Mitochondrial dysfunction is one of the hallmarks of aging. Changes in sebaceous gland (SG) function and sebum production have been reported during aging. This study shows the direct effects of mitochondrial dysfunction on SG morphology and function. A mitochondrial DNA (mtDNA) depleter mouse was used as a model for introducing mitochondrial dysfunction in the whole animal. The effects on skin SGs and modified SGs of the eyelid, lip, clitoral, and preputial glands were characterized. The mtDNA depleter mice showed gross morphologic and histopathologic changes in SGs associated with increased infiltration by mast cells, neutrophils, and polarized macrophages. Consistently, there was increased expression of proinflammatory cytokines. The inflammatory changes were associated with abnormal sebocyte accumulation of lipid, defective sebum delivery at the skin surface, and the up-regulation of key lipogenesis-regulating genes and androgen receptor. The mtDNA depleter mice expressed aging-associated senescent marker. Increased sebocyte proliferation and aberrant expression of stem cell markers were observed. These studies provide, for the first time, a causal link between mitochondrial dysfunction and abnormal sebocyte function within sebaceous and modified SGs throughout the whole body of the animal. They suggest that mtDNA depleter mouse may serve as a novel tool to develop targeted therapeutics to address SG disorders in aging humans.

Cardiac and Pulmonary Histopathology in Baboons Following Genetically-Engineered Pig Orthotopic Heart Transplantation.

BACKGROUND Although improving, survival after pig orthotopic heart transplantation (OHTx) in baboons has been mixed and largely poor. The causes for the high incidence of early failure remain uncertain. MATERIAL AND METHODS We have carried out pig OHTx in 4 baboons. Two died or were euthanized within hours, and 2 survived for 3 and 8 months, respectively. There was evidence of a significant 'cytokine storm' in the immediate post-OHTx period with the elevations in IL-6 correlating closely with the final outcome. RESULTS All 4 baboons demonstrated features suggestive of respiratory dysfunction, including increased airway resistance, hypoxia, and tachypnea. Histopathological observations of pulmonary infiltration by neutrophils and, notably, eosinophils within vessels and in the perivascular and peribronchiolar space, with minimal cardiac pathology, suggested a role for early lung acute inflammation. In one, features suggestive of transfusion-related acute lung injury were present. The 2 longer-term survivors died of (i) a cardiac dysrhythmia with cellular infiltration around the conducting tissue (at 3 months), and (ii) mixed cellular and antibody-mediated rejection (at 8 months). CONCLUSIONS These initial findings indicate a potential role of acute lung injury early after OHTx. If this response can be prevented, increased survival may result, providing an opportunity to evaluate the factors affecting long-term survival.

Pan-RAS inhibitors: Hitting multiple RAS isozymes with one stone.

Mutations in the three RAS oncogenes are present in approximately 30% of all human cancers that drive tumor growth and metastasis by aberrant activation of RAS-mediated signaling. Despite the well-established role of RAS in tumorigenesis, past efforts to develop small molecule inhibitors have failed for various reasons leading many to consider RAS as "undruggable." Advances over the past decade with KRAS(G12C) mutation-specific inhibitors have culminated in the first FDA-approved RAS drug, sotorasib. However, the patient population that stands to benefit from KRAS(G12C) inhibitors is inherently limited to those patients harboring KRAS(G12C) mutations. Additionally, both intrinsic and acquired mechanisms of resistance have been reported that indicate allele-specificity may afford disadvantages. For example, the compensatory activation of uninhibited wild-type (WT) NRAS and HRAS isozymes can rescue cancer cells harboring KRAS(G12C) mutations from allele-specific inhibition or the occurrence of other mutations in KRAS. It is therefore prudent to consider alternative drug discovery strategies that may overcome these potential limitations. One such approach is pan-RAS inhibition, whereby all RAS isozymes co-expressed in the tumor cell population are targeted by a single inhibitor to block constitutively activated RAS regardless of the underlying mutation. This chapter provides a review of past and ongoing strategies to develop pan-RAS inhibitors in detail and seeks to outline the trajectory of this promising strategy of RAS inhibition.

T and B lymphocyte dynamics after genetically-modified pig-to-baboon kidney xenotransplantation with an anti-CD40mAb-based immunosuppressive regimen.

BACKGROUND: The aim was to monitor recovery of T/B lymphocytes in baboons after depletion by anti-thymocyte globulin (ATG) and anti-CD20mAb (Rituximab), followed by pig kidney transplantation and maintenance therapy with an anti-CD40mAb-based regimen. METHODS: In baboons (n = 14), induction was with ATG and anti-CD20mAb, and maintenance with (i) anti-CD40mAb, (ii) rapamycin, and (iii) methylprednisolone. Follow-up was for 6 months, or until rejection or other complication developed. Baboon blood was collected at intervals to measure T/B cells and subsets by flow cytometry. In a separate study in baboons receiving the same immunosuppressive regimen (n = 10), the populations of T/B lymphocytes in PBMCs, lymph nodes, and spleen were examined. RESULTS: After induction therapy, the total lymphocyte count and the absolute numbers of CD3+, CD4+, and CD8+T cells fell by >80%, and no CD22+B cells remained (all p < 0.001). T cell numbers began to recover early, but no CD22+B cells were present in the blood for 2 months. Recovery of both T and B cells remained at <30% of baseline (p < 0.001), even if rejection developed. At 6 months, effector memory CD8+T cells had increased more than other T cell subsets, but a greater percentage of B cells were naïve. In contrast to blood and spleen, T and B cells were not depleted in lymph nodes. CONCLUSIONS: ATG and anti-CD20mAb effectively decreased T and B lymphocytes in the blood and, in the presence of anti-CD40mAb maintenance therapy, recovery of these cells was inhibited. The recovery of effector memory CD8+T cells may be detrimental to long-term graft survival.

Profound thrombocytopenia associated with administration of multiple anti-inflammatory agents in baboons.

Congestion, granular platelet debris both within macrophage and extracellularly, and neutrophil infiltration in the spleen of a baboon that was euthanized with profound thrombocytopenia.

Poly (acetyl, arginyl) glucosamine disrupts Pseudomonas aeruginosa biofilms and enhances bacterial clearance in a rat lung infection model.

Pseudomonas aeruginosa is a common opportunistic pathogen that can cause chronic infections in multiple disease states, including respiratory infections in patients with cystic fibrosis (CF) and non-CF bronchiectasis. Like many opportunists, P. aeruginosa forms multicellular biofilm communities that are widely thought to be an important determinant of bacterial persistence and resistance to antimicrobials and host immune effectors during chronic/recurrent infections. Poly (acetyl, arginyl) glucosamine (PAAG) is a glycopolymer that has antimicrobial activity against a broad range of bacterial species, and also has mucolytic activity, which can normalize the rheological properties of cystic fibrosis mucus. In this study, we sought to evaluate the effect of PAAG on P. aeruginosa bacteria within biofilms in vitro, and in the context of experimental pulmonary infection in a rodent infection model. PAAG treatment caused significant bactericidal activity against P. aeruginosa biofilms, and a reduction in the total biomass of preformed P. aeruginosa biofilms on abiotic surfaces, as well as on the surface of immortalized cystic fibrosis human bronchial epithelial cells. Studies of membrane integrity indicated that PAAG causes changes to P. aeruginosa cell morphology and dysregulates membrane polarity. PAAG treatment reduced infection and consequent tissue inflammation in experimental P. aeruginosa rat infections. Based on these findings we conclude that PAAG represents a novel means to combat P. aeruginosa infection, and may warrant further evaluation as a therapeutic.

Mucociliary Transport Deficiency and Disease Progression in Syrian Hamsters with SARS-CoV-2 Infection.

Substantial clinical evidence supports the notion that ciliary function in the airways plays an important role in COVID-19 pathogenesis. Although ciliary damage has been observed in both in vitro and in vivo models, consequent impaired mucociliary transport (MCT) remains unknown for the intact MCT apparatus from an in vivo model of disease. Using golden Syrian hamsters, a common animal model that recapitulates human COVID-19, we quantitatively followed the time course of physiological, virological, and pathological changes upon SARS-CoV-2 infection, as well as the deficiency of the MCT apparatus using micro-optical coherence tomography, a novel method to visualize and simultaneously quantitate multiple aspects of the functional microanatomy of intact airways. Corresponding to progressive weight loss up to 7 days post-infection (dpi), viral detection and histopathological analysis in both the trachea and lung revealed steadily descending infection from the upper airways, as the main target of viral invasion, to lower airways and parenchymal lung, which are likely injured through indirect mechanisms. SARS-CoV-2 infection caused a 67% decrease in MCT rate as early as 2 dpi, largely due to diminished motile ciliation coverage, but not airway surface liquid depth, periciliary liquid depth, or cilia beat frequency of residual motile cilia. Further analysis indicated that the fewer motile cilia combined with abnormal ciliary motion of residual cilia contributed to the delayed MCT. The time course of physiological, virological, and pathological progression suggest that functional deficits of the MCT apparatus predispose to COVID-19 pathogenesis by extending viral retention and may be a risk factor for secondary infection. As a consequence, therapies directed towards the MCT apparatus deserve further investigation as a treatment modality.

Host Genetics But Not Commensal Microbiota Determines the Initial Development of Systemic Autoimmune Disease in BXD2 Mice.

OBJECTIVE: To determine the extent to which the gut microbiome influences systemic autoimmunity in a mouse model of lupus. METHODS: We generated germ-free (GF) lupus-prone BXD2 mice, which under normal conditions develop spontaneous germinal centers (GCs) and high titers of serum autoantibodies. GF status was confirmed by gut bacterial culture. The autoimmune phenotypes of 6- and 12-month-old gnotobiotic GF BXD2 mice and specific pathogen-free (SPF) BXD2 mice were compared. Serum levels of autoantibodies were measured by enzyme-linked immunosorbent assay. Histologic sections of the mouse kidney and joints were evaluated. Flow cytometry was used to analyze GCs and age-associated B cells. CD4+ T cells were analyzed for PD-1+ICOS+ activated T cells, T follicular regulatory (Tfr) cells (Foxp3+CD25+ PD-1+CXCR5+), and PD-1+ICOS+ T cells expressing interleukin-17A (IL-17A) or interferon-γ (IFNγ) after stimulation with phorbol myristate acetate (PMA)/ionomycin. RESULTS: In 6-month-old mice, GF status did not affect splenomegaly, GC B cells, age-associated B cells, or serum autoantibody levels, except for IgG antihistone. GF BXD2 mice exhibited a significantly higher percentage of Tfr cells compared to their SPF counterparts (P < 0.05). At 12 months of age, however, GF BXD2 mice had significantly diminished IgG autoantibody levels and a lower percentage of GC B cells and age-associated B cells (P < 0.05). Following stimulation with PMA/ionomycin, PD-1+ICOS+ CD4+ T cells expressed significantly lower IL-17A, but not IFNγ, levels in GF BXD2 mice compared to SPF BXD2 mice (P < 0.01). SPF BXD2 mice and GF BXD2 mice developed equivalent renal and joint disease with no significant differences in severity. CONCLUSION: Our results suggest a model in which genetics plays a dominant role in determining the initial development of autoimmunity. In contrast, gut microbiomes may regulate the persistence of certain aspects of systemic autoimmunity.