Elicitation of broadly protective sarbecovirus immunity by receptor-binding domain nanoparticle vaccines.

Understanding vaccine-elicited protection against SARS-CoV-2 variants and other sarbecoviruses is key for guiding public health policies. We show that a clinical stage multivalent SARS-CoV-2 spike receptor-binding domain nanoparticle (RBD-NP) vaccine protects mice from SARS-CoV-2 challenge after a single immunization, indicating a potential dose-sparing strategy. We benchmarked serum neutralizing activity elicited by RBD-NPs in non-human primates against a lead prefusion-stabilized SARS-CoV-2 spike (HexaPro) using a panel of circulating mutants. Polyclonal antibodies elicited by both vaccines are similarly resilient to many RBD residue substitutions tested, although mutations at and surrounding position 484 have negative consequences for neutralization. Mosaic and cocktail nanoparticle immunogens displaying multiple sarbecovirus RBDs elicit broad neutralizing activity in mice and protect mice against SARS-CoV challenge even in the absence of SARS-CoV RBD in the vaccine. This study provides proof of principle that multivalent sarbecovirus RBD-NPs induce heterotypic protection and motivates advancing such broadly protective sarbecovirus vaccines to the clinic.

Flagellin-modulated inflammasome pathways characterize the human alveolar macrophage response to Burkholderia pseudomallei, a lung-tropic pathogen.

Melioidosis is an emerging tropical infection caused by inhalation, inoculation, or ingestion of the flagellated, facultatively intracellular pathogen Burkholderia pseudomallei. The melioidosis case fatality rate is often high, and pneumonia, the most common presentation, doubles the risk of death. The alveolar macrophage is a sentinel pulmonary host defense cell, but the human alveolar macrophage in B. pseudomallei infection has never been studied. The objective of this study was to investigate the host-pathogen interaction of B. pseudomallei infection with the human alveolar macrophage and to determine the role of flagellin in modulating inflammasome-mediated pathways. We found that B. pseudomallei infects primary human alveolar macrophages but is gradually restricted in the setting of concurrent cell death. Electron microscopy revealed cytosolic bacteria undergoing division, indicating that B. pseudomallei likely escapes the alveolar macrophage phagosome and may replicate in the cytosol, where it triggers immune responses. In paired human blood monocytes, uptake and intracellular restriction of B. pseudomallei are similar to those observed in alveolar macrophages, but cell death is reduced. The alveolar macrophage cytokine response to B. pseudomallei is characterized by marked interleukin (IL)-18 secretion compared to monocytes. Both cytotoxicity and IL-18 secretion in alveolar macrophages are partially flagellin dependent. However, the proportion of IL-18 release that is driven by flagellin is greater in alveolar macrophages than in monocytes. These findings suggest differential flagellin-mediated inflammasome pathway activation in the human alveolar macrophage response to B. pseudomallei infection and expand our understanding of intracellular pathogen recognition by this unique innate immune lung cell.

FlgM is required to evade NLRC4-mediated host protection against flagellated Salmonella.

Salmonella enterica serovar Typhimurium is a leading cause of gastroenteritis worldwide and a deadly pathogen in children, immunocompromised patients, and the elderly. Salmonella induces innate immune responses through the NLRC4 inflammasome, which has been demonstrated to have distinct roles during systemic and mucosal detections of flagellin and non-flagellin molecules. We hypothesized that NLRC4 recognition of Salmonella flagellin is the dominant protective pathway during infection. To test this hypothesis, we used wild-type, flagellin-deficient, and flagellin-overproducing Salmonella to establish the role of flagellin in mediating NLRC4-dependent host resistance during systemic and mucosal infections in mice. We observed that during the systemic phase of infection, Salmonella efficiently evades NLRC4-mediated innate immunity. During mucosal Salmonella infection, flagellin recognition by the NLRC4 inflammasome pathway is the dominant mediator of protective innate immunity. Deletion of flgM results in constitutive expression of flagellin and severely limits systemic and mucosal Salmonella infections in an NLRC4 inflammasome-dependent manner. These data establish that recognition of Salmonella's flagellin by the NLRC4 inflammasome during mucosal infection is the dominant innate protective pathway for host resistance against the enteric pathogen and that FlgM-mediated evasion of the NLRC4 inflammasome enhances virulence and intestinal tissue destruction.

Collapsing glomerulopathy: unraveling varied pathogeneses.

PURPOSE OF REVIEW: Collapsing glomerulopathy presents clinically with nephrotic syndrome and rapid progressive loss of kidney function. Animal models and patient studies have uncovered numerous clinical and genetic conditions associated with collapsing glomerulopathy, as well as putative mechanisms, which will be reviewed here. RECENT FINDINGS: Collapsing glomerulopathy is classified pathologically as a variant of focal and segmental glomerulosclerosis (FSGS). As such, most research efforts have focused on the causative role of podocyte injury in driving the disease. However, studies have also shown that injury to the glomerular endothelium or interruption of the podocyte-glomerular endothelial cell signaling axis can also cause collapsing glomerulopathy. Furthermore, emerging technologies are now enabling exploration of diverse molecular pathways that can precipitate collapsing glomerulopathy using biopsies from patients with the disease. SUMMARY: Since its original description in the 1980s, collapsing glomerulopathy has been the subject of intense study, and these efforts have uncovered numerous insights into potential disease mechanisms. Newer technologies will enable profiling of the intra-patient and inter-patient variability in collapsing glomerulopathy mechanisms directly in patient biopsies, which will improve the diagnosis and classification of collapsing glomerulopathy.

Digital spatial profiling of collapsing glomerulopathy.

Collapsing glomerulopathy is a histologically distinct variant of focal and segmental glomerulosclerosis that presents with heavy proteinuria and portends a poor prognosis. Collapsing glomerulopathy can be triggered by viral infections such as HIV or SARS-CoV-2. Transcriptional profiling of collapsing glomerulopathy lesions is difficult since only a few glomeruli may exhibit this histology within a kidney biopsy and the mechanisms driving this heterogeneity are unknown. Therefore, we used recently developed digital spatial profiling (DSP) technology which permits quantification of mRNA at the level of individual glomeruli. Using DSP, we profiled 1,852 transcripts in glomeruli isolated from formalin fixed paraffin embedded sections from HIV or SARS-CoV-2-infected patients with biopsy-confirmed collapsing glomerulopathy and used normal biopsy sections as controls. Even though glomeruli with collapsing features appeared histologically similar across both groups of patients by light microscopy, the increased resolution of DSP uncovered intra- and inter-patient heterogeneity in glomerular transcriptional profiles that were missed in early laser capture microdissection studies of pooled glomeruli. Focused validation using immunohistochemistry and RNA in situ hybridization showed good concordance with DSP results. Thus, DSP represents a powerful method to dissect transcriptional programs of pathologically discernible kidney lesions.

Characterizing Viral Infection by Electron Microscopy: Lessons from the Coronavirus Disease 2019 Pandemic.

The severe acute respiratory syndrome coronavirus 2 pandemic has infected millions of individuals in the United States and caused hundreds of thousands of deaths. Direct infection of extrapulmonary tissues has been postulated, and using sensitive techniques, viral RNA has been detected in multiple organs in the body, including the kidney. However, direct infection of tissues outside of the lung has been more challenging to demonstrate. This has been in part due to misinterpretation of electron microscopy studies. In this perspective, we will discuss what is known about coronavirus infection, some of the basic ultrastructural cell biology that has been confused for coronavirus infection of cells, and rigorous criteria that should be used when identifying pathogens by electron microscopy.

Pathogenesis of coronavirus disease 2019-associated kidney injury.

PURPOSE OF REVIEW: The current review summarizes the pathologic findings in kidneys from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients who have had autopsies or undergone biopsy, and the pathogenic mechanisms implicated in coronavirus disease 2019 (COVID-19)-associated kidney diseases. RECENT FINDINGS: Direct infection of the kidney by SARS-CoV-2 is not common, and convincing morphologic evidence of substantive kidney infection by SARS-CoV-2 is lacking. Severe COVID-19-associated acute kidney injury is likely multifactorial and results from the physiologic disturbances and therapies used to treat this illness. COVID-19-associated collapsing glomerulopathy (COVAN) is seen almost exclusively in patients with apolipoprotein L1 high-risk genotypes with no evidence of direct infection of the kidney by SARS-CoV-2. SUMMARY: The prevailing evidence does not support substantive or persistent infection of kidneys in COVID-19 and indirect means of tissue injury are favored, although a 'hit and run' model cannot be excluded. COVAN frequently occurs in patients with mild respiratory systems, suggesting that innate and adaptive immune responses to SARS-CoV-2 infection may provide the second hit needed for the development of collapsing glomerulopathy in susceptible individuals.

Multicenter Clinicopathologic Correlation of Kidney Biopsies Performed in COVID-19 Patients Presenting With Acute Kidney Injury or Proteinuria.

RATIONALE & OBJECTIVE: Kidney biopsy data inform us about pathologic processes associated with infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We conducted a multicenter evaluation of kidney biopsy findings in living patients to identify various kidney disease pathology findings in patients with coronavirus disease 2019 (COVID-19) and their association with SARS-CoV-2 infection. STUDY DESIGN: Case series. SETTING & PARTICIPANTS: We identified 14 native and 3 transplant kidney biopsies performed for cause in patients with documented recent or concurrent SARS-CoV-2 infection treated at 7 large hospital systems in the United States. OBSERVATIONS: Men and women were equally represented in this case series, with a higher proportion of Black (n=8) and Hispanic (n=5) patients. All 17 patients had SARS-CoV-2 infection confirmed by reverse transcriptase-polymerase chain reaction, but only 3 presented with severe COVID-19 symptoms. Acute kidney injury (n=15) and proteinuria (n=11) were the most common indications for biopsy and these symptoms developed concurrently or within 1 week of COVID-19 symptoms in all patients. Acute tubular injury (n=14), collapsing glomerulopathy (n=7), and endothelial injury/thrombotic microangiopathy (n=6) were the most common histologic findings. 2 of the 3 transplant recipients developed active antibody-mediated rejection weeks after COVID-19. 8 patients required dialysis, but others improved with conservative management. LIMITATIONS: Small study size and short clinical follow-up. CONCLUSIONS: Cases of even symptomatically mild COVID-19 were accompanied by acute kidney injury and/or heavy proteinuria that prompted a diagnostic kidney biopsy. Although acute tubular injury was seen among most of them, uncommon pathology such as collapsing glomerulopathy and acute endothelial injury were detected, and most of these patients progressed to irreversible kidney injury and dialysis.

Utility of abdominal skin punch biopsy for detecting systemic amyloidosis.

BACKGROUND: Early and accurate diagnosis of systemic amyloidosis (SA) is critical for optimal patient outcomes. Biopsy of clinically uninvolved skin and subcutaneous tissue including abdominal skin punch biopsy (ASPB) is often used as a surrogate for affected organ sampling. There is a lack of published data on the sensitivity and specificity of ASPB for diagnosing SA. METHODS: Retrospective chart review between 2000 and 2020 of all ASPB was performed to diagnose SA. Amyloid deposition was confirmed by Congo red stain. Study group includes patients with histopathologically and clinically confirmed diagnosis of SA. Control group includes patients without histopathology of amyloid deposition and no clinical SA. RESULTS: Forty-one patients meeting inclusion criteria were analyzed; 23 study group and 18 control group patients. The overall diagnostic sensitivity of ASPB was 43% (95% CI 23%-66%) and the specificity 100% (95% CI 81%-100%). The AL amyloidosis diagnostic sensitivity was 64% (95% CI 35%-87%). ASPB >10 mm in depth had 100% (95% CI 54%-100%) sensitivity compared to 24% for depth ≤10 mm (P = .002). CONCLUSIONS: ASPB is a minimally invasive and highly specific method of diagnosing SA. It is particularly sensitive for diagnosing AL amyloidosis and the diagnostic sensitivity can be significantly improved with adequate biopsy depth and diameter.

Collapsing glomerulopathy in older adults.

Collapsing glomerulopathy has been described in settings of viral infections, drug, genetic, ischemic, renal transplant, and idiopathic conditions. It has a worse prognosis than other morphologic variants of focal segmental glomerulosclerosis, and may be treated with aggressive immunosuppression. In this study, we sought to characterize the clinical and morphologic findings in older adults with collapsing glomerulopathy. Renal biopsies and associated clinical data from patients aged 65 or older with a diagnosis of collapsing glomerulopathy were retrospectively reviewed at 3 academic institutions. Patients (n = 41, 61% male, median age 71) usually had hypertension (88%), nephrotic range proteinuria (91%), and renal insufficiency (median serum creatinine 2.5 mg/dL). A likely precipitating drug (5%) or vascular procedure (5%) was identified in a minority of cases; viral infections were infrequent. Renal biopsies contained a median of 40% globally and 16% segmentally sclerotic glomeruli. Approximately 60% of cases had moderate or severe arteriosclerosis, arteriolar hyalinosis, and/or tubular atrophy and interstitial fibrosis; 7% had atheroembolic disease and 5% had thrombotic microangiopathy. In 28 patients with available follow-up information, eight (19%) were treated with immunosuppressives, which were not tolerated by 2. At a median interval of 14 months, 5 (18%) patients had died, 12 (43%) had end stage renal disease, and 12 were alive with renal insufficiency and proteinuria. Treatment with immunosuppressive therapy did not have a significant benefit with regard to the primary outcome of overall or renal survival. One steroid-treated patient with diabetes died 6 weeks after biopsy, with invasive rhinoorbital Rhizopus infection. In conclusion, collapsing glomerulopathy in older patients is usually not associated with viral infections, and is accompanied by significant chronic injury in glomeruli, vasculature, and tubulointerstitium. Aggressive immunosuppression likely contributed to one death in a patient with diabetes, and did not yield an overall or renal survival advantage in this cohort.

DnaJ Homolog Subfamily B Member 9 Is a Putative Autoantigen in Fibrillary GN.

Fibrillary GN is a rare form of GN of uncertain pathogenesis that is characterized by the glomerular accumulation of randomly arranged, nonbranching fibrils (12-24 nm) composed of Ig and complement proteins. In this study, we used mass spectrometry to comprehensively define the glomerular proteome in fibrillary GN compared with that in controls and nonfibrillary GN renal diseases. We isolated glomeruli from formalin-fixed and paraffin-embedded biopsy specimens using laser capture microdissection and analyzed them with liquid chromatography and data-dependent tandem mass spectrometry. These studies identified DnaJ homolog subfamily B member 9 (DNAJB9) as a highly sampled protein detected only in fibrillary GN cases. The glomerular proteome of fibrillary GN cases also contained IgG1 as the dominant Ig and proteins of the classic complement pathway. In fibrillary GN specimens only, immunofluorescence and immunohistochemistry with an anti-DNAJB9 antibody showed strong and specific staining of the glomerular tufts in a distribution that mimicked that of the immune deposits. Our results identify DNAJB9 as a putative autoantigen in fibrillary GN and suggest IgG1 and classic complement effector pathways as likely mediators of the destructive glomerular injury in this disease.

LMX1B-Associated Nephropathy With Type III Collagen Deposition in the Glomerular and Tubular Basement Membranes.

Variants in the LMX1B gene cause nail-patella syndrome, a rare autosomal dominant disorder characterized by dysplasia of nails, patella and elbow abnormalities, iliac "horns," and glaucoma. We describe an adult man with nephrotic syndrome and no systemic manifestations of nail-patella syndrome at the time of his initial kidney biopsy. His kidney biopsy was initially interpreted as a form of segmental sclerosis with unusual fibrillar deposits. At the time of consideration for kidney transplantation, a family history was notable for end-stage renal disease in 3 generations. Subsequent reanalysis of the initial biopsy showed infiltration of the lamina densa by type III collagen fibrils, and molecular studies identified a pathogenic variant in one allele of LMX1B (a guanine to adenine substitution at nucleoide 737 of the coding sequence [c.737G>A], predicted to result in an arginine to glutamine substitution at amino acid 246 [p.Arg246Gln]). This variant has been described previously in multiple unrelated families who presented with autosomal dominant nephropathy without nail and patellar abnormalities.

Flagellin induces antibody responses through a TLR5- and inflammasome-independent pathway.

Flagellin is a potent immunogen that activates the innate immune system via TLR5 and Naip5/6, and generates strong T and B cell responses. The adaptor protein MyD88 is critical for signaling by TLR5, as well as IL-1Rs and IL-18Rs, major downstream mediators of the Naip5/6 Nlrc4-inflammasome. In this study, we define roles of known flagellin receptors and MyD88 in Ab responses generated toward flagellin. We used mice genetically deficient in flagellin recognition pathways to characterize innate immune components that regulate isotype-specific Ab responses. Using purified flagellin from Salmonella, we dissected the contribution of innate flagellin recognition pathways to promote Ab responses toward flagellin and coadministered OVA in C57BL/6 mice. We demonstrate IgG2c responses toward flagellin were TLR5 and inflammasome dependent; IgG1 was the dominant isotype and partially TLR5 and inflammasome dependent. Our data indicate a substantial flagellin-specific IgG1 response was induced through a TLR5-, inflammasome-, and MyD88-independent pathway. IgA anti-FliC responses were TLR5 and MyD88 dependent and caspase-1 independent. Unlike C57BL/6 mice, flagellin-immunized A/J mice induced codominant IgG1 and IgG2a responses. Furthermore, MyD88-independent, flagellin-induced Ab responses were even more pronounced in A/J MyD88(-/-) mice, and IgA anti-FliC responses were suppressed by MyD88. Flagellin also worked as an adjuvant toward coadministered OVA, but it only promoted IgG1 anti-OVA responses. Our results demonstrate that a novel pathway for flagellin recognition contributes to Ab production. Characterization of this pathway will be useful for understanding immunity to flagellin and the rationale design of flagellin-based vaccines.

Interstitial eosinophilic aggregates in diabetic nephropathy: allergy or not?

BACKGROUND: Interstitial eosinophilic aggregates (IEA) in renal biopsies often suggest allergic tubulointerstitial nephritis, yet clear associations with drug reactions are often difficult to establish. IEA are also encountered in diabetic nephropathy (DN) and thought to be attributed to medication exposure. METHODS: Native medical kidney biopsies performed at the University of Washington Medical Center were reviewed, including DN (n = 64), IgA nephropathy (IgAN, n = 28), membranous nephropathy (MN, n = 14), focal and segmental glomerulosclerosis (FSGS, n = 27) and membranoproliferative glomerulonephritis (MPGN, n = 28). IEA were defined as ≥5 eosinophils per high power field. The severity of interstitial fibrosis and tubular atrophy (IFTA) was scored semi-quantitatively as minimal, mild, moderate or severe. RESULTS: IEA were remarkably more prevalent in DN (41%), when compared with IgAN (7%, P = 0.001), MN (8%, P = 0.017) or MPGN (14%, P = 0.013), but not FSGS (26%, P = 0.18). In DN cases, univariate analysis revealed that IEA were associated with greater IFTA severity, but not with the percentage of glomerulosclerosis, mesangial expansion, history of drug allergy, number of prescribed medications or particular class of medications (antibiotics, NSAIDs, aspirin, thiazide, loop diuretics, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, calcium channel blockers, beta blockers, insulin, sulfonylurea, metformin or allopurinol). Multivariate analysis showed that the severity of IFTA was the only significant predictor for IEA (P < 0.01) after stepwise adjustment for age, number of medications, drug allergy, diabetes type, % global glomerulosclerosis and mesangial expansion. CONCLUSIONS: Our study shows that IEA are more common in DN, when compared with other types of glomerulopathy. In DN, IEA are associated with the severity of IFTA but not with prescribed medications or clinical history of allergy. This suggests that in DN IEA are often associated with chronic tubulointerstitial injury and are not diagnostic of an allergic interstitial nephritis.

Acyl-CoA synthetase 1 is induced by Gram-negative bacteria and lipopolysaccharide and is required for phospholipid turnover in stimulated macrophages.

The enzyme acyl-CoA synthetase 1 (ACSL1) is induced by peroxisome proliferator-activated receptor α (PPARα) and PPARγ in insulin target tissues, such as skeletal muscle and adipose tissue, and plays an important role in ß-oxidation in these tissues. In macrophages, however, ACSL1 mediates inflammatory effects without significant effects on ß-oxidation. Thus, the function of ACSL1 varies in different tissues. We therefore investigated the signals and signal transduction pathways resulting in ACSL1 induction in macrophages as well as the consequences of ACSL1 deficiency for phospholipid turnover in LPS-activated macrophages. LPS, Gram-negative bacteria, IFN-γ, and TNFα all induce ACSL1 expression in macrophages, whereas PPAR agonists do not. LPS-induced ACSL1 expression is dependent on Toll-like receptor 4 (TLR4) and its adaptor protein TRIF (Toll-like receptor adaptor molecule 1) but does not require the MyD88 (myeloid differentiation primary response gene 88) arm of TLR4 signaling; nor does it require STAT1 (signal transducer and activator of transcription 1) for maximal induction. Furthermore, ACSL1 deletion attenuates phospholipid turnover in LPS-stimulated macrophages. Thus, the regulation and biological function of ACSL1 in macrophages differ markedly from that in insulin target tissues. These results suggest that ACSL1 may have an important role in the innate immune response. Further, these findings illustrate an interesting paradigm in which the same enzyme, ACSL1, confers distinct biological effects in different cell types, and these disparate functions are paralleled by differences in the pathways that regulate its expression.

Chronic Ifosfamide toxicity: kidney pathology and pathophysiology.

Ifosfamide is a nitrogen mustard alkylating agent used as both a first-line and a salvage chemotherapeutic agent in the treatment of testicular germ cell tumors, various sarcomas, carcinomas, and some lymphomas. A well-known complication of ifosfamide therapy is transient acute kidney injury. However, in a minority of patients, the reduction in kidney function is progressive and permanent, sometimes occurring long after exposure to ifosfamide. Scattered reports have described the pathologic findings in kidneys permanently affected by ifosfamide toxicity. We present the findings of an illustrative case and review the pathology and molecular mechanisms of long-term ifosfamide toxicity with implications for personalized medicine.