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Rejection remains the main cause of premature graft loss after kidney transplantation, despite the use of potent immunosuppression. This highlights the need to better understand the composition and the cell-to-cell interactions of the alloreactive inflammatory infiltrate. Here, we performed droplet-based single-cell RNA sequencing of 35,152 transcriptomes from 16 kidney transplant biopsies with varying phenotypes and severities of rejection and without rejection, and identified cell-type specific gene expression signatures for deconvolution of bulk tissue. A specific association was identified between recipient-derived FCGR3A+ monocytes, FCGR3A +  NK cells and the severity of intragraft inflammation. Activated FCGR3A+ monocytes overexpressed CD47 and LILR genes and increased paracrine signaling pathways promoting T cell infiltration. FCGR3A +  NK cells overexpressed FCRL3 , suggesting that antibody-dependent cytotoxicity is a central mechanism of NK-cell mediated graft injury. Multiplexed immunofluorescence using 38 markers on 18 independent biopsy slides confirmed this role of FcγRIII+ NK and FcγRIII+ nonclassical monocytes in antibody-mediated rejection, with specificity to the glomerular area. These results highlight the central involvement of innate immune cells in the pathogenesis of allograft rejection and identify several potential therapeutic targets that might improve allograft longevity. Although long-term kidney allograft failure is broadly classified as T cell- or antibody-mediated, this dichotomy is not always apparent in all patients, highlighting the need for improved allograft tissue characterisation. Here, the authors use single-cell RNA sequencing and multiplex imaging for transcriptomic and spatial profiling of allograft tissue from patients experiencing different degrees of rejection severity.

Diabetic kidney disease (DKD) is a key microvascular complication of diabetes, with few therapies for targeting renal disease pathogenesis and progression. We performed transcriptional and protein studies on 103 unique blood and kidney tissue samples from patients with and without diabetes to understand the pathophysiology of DKD injury and its progression. The study was based on the use of 3 unique patient cohorts: peripheral blood mononuclear cell (PBMC) transcriptional studies were conducted on 30 patients with DKD with advancing kidney injury; Gene Expression Omnibus (GEO) data was downloaded, containing transcriptional measures from 51 microdissected glomerulous from patients with DKD. Additionally, 12 independent kidney tissue sections from patients with or without DKD were used for validation of target genes in diabetic kidney injury by kidney tissue immunohistochemistry and immunofluorescence. PBMC DKD transcriptional analysis, identified 853 genes (p < 0.05) with increasing expression with progression of albuminuria and kidney injury in patients with diabetes. GEO data was downloaded, normalized, and analyzed for significantly changed genes. Of the 325 significantly up regulated genes in DKD glomerulous (p < 0.05), 28 overlapped in PBMC and diabetic kidney, with perturbed FcER1 signaling as a significantly enriched canonical pathway. FcER1 was validated to be significantly increased in advanced DKD, where it was also seen to be specifically co-expressed in the kidney biopsy with tissue mast cells. In conclusion, we demonstrate how leveraging public and private human transcriptional datasets can discover and validate innate immunity and inflammation as key mechanistic pathways in DKD progression, and uncover FcER1 as a putative new DKD target for rational drug design.

Cytomegalovirus (CMV) infection, either de novo or as reactivation after allotransplantation and chronic immunosuppression, is recognized to cause detrimental alloimmune effects, inclusive of higher susceptibility to graft rejection and substantive impact on chronic graft injury and reduced transplant survival. To obtain further insights into the evolution and pathogenesis of CMV infection in an immunocompromised host we evaluated changes in the circulating host proteome serially, before and after transplantation, and during and after CMV DNA replication (DNAemia), as measured by quantitative polymerase chain reaction (QPCR). LC-MS-based proteomics was conducted on 168 serially banked plasma samples, from 62 propensity score-matched kidney transplant recipients. Patients were stratified by CMV replication status into 31 with CMV DNAemia and 31 without CMV DNAemia. Patients had blood samples drawn at protocol times of 3- and 12-months post-transplant. Additionally, blood samples were also drawn before and 1 week and 1 month after detection of CMV DNAemia. Plasma proteins were analyzed using an LCMS 8060 triple quadrupole mass spectrometer. Further, public transcriptomic data on time matched PBMCs samples from the same patients was utilized to evaluate integrative pathways. Data analysis was conducted using R and Limma. Samples were segregated based on their proteomic profiles with respect to their CMV Dnaemia status. A subset of 17 plasma proteins was observed to predict the onset of CMV at 3 months post-transplant enriching platelet degranulation (FDR, 4.83E-06), acute inflammatory response (FDR, 0.0018), blood coagulation (FDR, 0.0018) pathways. An increase in many immune complex proteins were observed at CMV infection. Prior to DNAemia the plasma proteome showed changes in the anti-inflammatory adipokine vaspin (SERPINA12), copper binding protein ceruloplasmin (CP), complement activation (FDR = 0.03), and proteins enriched in the humoral (FDR = 0.01) and innate immune responses (FDR = 0.01). Plasma proteomic and transcriptional perturbations impacting humoral and innate immune pathways are observed during CMV infection and provide biomarkers for CMV disease prediction and resolution. Further studies to understand the clinical impact of these pathways can help in the formulation of different types and duration of anti-viral therapies for the management of CMV infection in the immunocompromised host.

Advancement in proteomics methods for interrogating biological samples has helped identify disease biomarkers for early diagnostics and unravel underlying molecular mechanisms of disease. Herein, we examined the serum proteomes of 23 study participants presenting with one of two common arthropod-borne infections: Lyme disease (LD), an extracellular bacterial infection or West Nile virus infection (WNV), an intracellular viral infection. The LC/MS based serum proteomes of samples collected at the time of diagnosis and during convalescence were assessed using a depletion-based high-throughput shotgun proteomics (dHSP) pipeline as well as a non-depleting blotting-based low-throughput platform (MStern). The LC/MS integrated analyses identified host proteome responses in the acute and recovery phases shared by LD and WNV infections, as well as differentially abundant proteins that were unique to each infection. Notably, we also detected proteins that distinguished localized from disseminated LD and asymptomatic from symptomatic WNV infection. The proteins detected in both diseases with the dHSP pipeline identified unique and overlapping proteins detected with the non-depleting MStern platform, supporting the utility of both detection methods. Machine learning confirmed the use of the serum proteome to distinguish the infection from healthy control sera but could not develop discriminatory models between LD and WNV at current sample numbers. Our study is the first to compare the serum proteomes in two arthropod-borne infections and highlights the similarities in host responses even though the pathogens and the vectors themselves are different.

Cytomegalovirus (CMV) viremia continues to cause significant morbidity and mortality in kidney transplant patients with clinical complications including organ rejection and death. Whole blood gene expression dynamics in CMV viremic patients from onset of DNAemia through convalescence has not been well studied to date in humans. To evaluate how CMV infection impacts whole blood leukocyte gene expression over time, we evaluated a matched cohort of 62 kidney transplant recipients with and without CMV DNAemia using blood samples collected at multiple time points during the 12-month period after transplant. While transcriptomic differences were minimal at baseline between DNAemic and non-DNAemic patients, hundreds of genes were differentially expressed at the long-term timepoint, including genes enriching for pathways important for macrophages, interferon, and IL-8 signaling. Amongst patients with CMV DNAemia, the greatest amount of transcriptomic change occurred between baseline and 1-week post-DNAemia, with increase in pathways for interferon signaling and cytotoxic T cell function. Time-course gene set analysis of these differentially expressed genes revealed that most of the enriched pathways had a significant time-trend. While many pathways that were significantly down- or upregulated at 1 week returned to baseline-like levels, we noted that several pathways important in adaptive and innate cell function remained upregulated at the long-term timepoint after resolution of CMV DNAemia. Differential expression analysis and time-course gene set analysis revealed the dynamics of genes and pathways involved in the immune response to CMV DNAemia in kidney transplant patients. Understanding transcriptional changes caused by CMV DNAemia may identify the mechanism behind patient vulnerability to CMV reactivation and increased risk of rejection in transplant recipients and suggest protective strategies to counter the negative immunologic impact of CMV. These findings provide a framework to identify immune correlates for risk assessment and guiding need for extending antiviral prophylaxis.

Infectious diseases are a significant burden in global healthcare. Pathogens engage with different host defense mechanisms. However, it is currently unknown if there are disease-specific immune signatures and/or if different pathogens elicit common immune-associated molecular entities to common therapeutic interventions. We studied patients enrolled through the Human Immunology Project Consortium (HIPC), which focuses on immune responses to various infections. Blood samples were collected and analyzed from patients during infection and follow-up time points at the convalescent stage. The study included samples from patients with Lyme disease (LD), tuberculosis (TB), malaria (MLA), dengue virus (DENV), and West Nile virus (WNV), as well as kidney transplant patients with cytomegalovirus (CMV) and polyomavirus (BKV) infections. Using an antibody-based assay, we quantified ~ 350 cell surface markers, cytokines, and chemokines involved in inflammation and immunity. Unique protein signatures were identified specific to the acute phase of infection irrespective of the pathogen type, with significant changes during convalescence. In addition, tumor necrosis factor receptor superfamily member 6 (TNR6), C–C Motif Chemokine Receptor 7 (CCR7), and C–C motif chemokine ligand-1 (CCL1) were increased in the acute and convalescent phases across all viral, bacterial, and protozoan compared to blood from healthy donors. Furthermore, despite the differences between pathogens, proteins were enriched in common biological pathways such as cell surface receptor signaling pathway and response to external stimulus. In conclusion, we demonstrated that irrespective of the pathogen type, there are common immunoregulatory and proinflammatory signals.

Cytomegalovirus (CMV) infection, either de novo or as reactivation after allotransplantation and chronic immunosuppression, is recognized to cause detrimental alloimmune effects, inclusive of higher susceptibility to graft rejection and substantive impact on chronic graft injury and reduced transplant survival. To obtain further insights into the evolution and pathogenesis of CMV infection in an immunocompromised host we evaluated changes in the circulating host proteome serially, before and after transplantation, and during and after CMV DNA replication (DNAemia), as measured by quantitative polymerase chain reaction (QPCR). LC-MS-based proteomics was conducted on 168 serially banked plasma samples, from 62 propensity score-matched kidney transplant recipients. Patients were stratified by CMV replication status into 31 with CMV DNAemia and 31 without CMV DNAemia. Patients had blood samples drawn at protocol times of 3- and 12-months post-transplant. Additionally, blood samples were also drawn before and 1 week and 1 month after detection of CMV DNAemia. Plasma proteins were analyzed using an LCMS 8060 triple quadrupole mass spectrometer. Further, public transcriptomic data on time matched PBMCs samples from the same patients was utilized to evaluate integrative pathways. Data analysis was conducted using R and Limma. Samples were segregated based on their proteomic profiles with respect to their CMV Dnaemia status. A subset of 17 plasma proteins was observed to predict the onset of CMV at 3 months post-transplant enriching platelet degranulation (FDR, 4.83E-06), acute inflammatory response (FDR, 0.0018), blood coagulation (FDR, 0.0018) pathways. An increase in many immune complex proteins were observed at CMV infection. Prior to DNAemia the plasma proteome showed changes in the anti-inflammatory adipokine vaspin (SERPINA12), copper binding protein ceruloplasmin (CP), complement activation (FDR=0.03), and proteins enriched in the humoral (FDR=0.01) and innate immune responses (FDR=0.01). Plasma proteomic and transcriptional perturbations impacting humoral and innate immune pathways are observed during CMV infection and provide biomarkers for CMV disease prediction and resolution. Further studies to understand the clinical impact of these pathways can help in the formulation of different types and duration of anti-viral therapies for management of CMV infection in the immunocompromised host.

INTRODUCTION The approved amyloid antibodies for early Alzheimer's disease (AD) carry a boxed warning about the risk of amyloid‐related imaging abnormalities (ARIAs) that are highest in apolipoprotein E (APOE) ε4/ε4 homozygotes. ALZ‐801/valiltramiprosate, an oral brain‐penetrant amyloid beta oligomer inhibitor is being evaluated in APOE ε4/ε4 homozygotes with early AD. METHODS This Phase 3 randomized, double‐blind, placebo‐controlled, 78‐week study of ALZ‐801 administered as 265 mg twice per day tablets, enrolled 50‐ to 80‐year‐old homozygotes with Mini‐Mental State Examination (MMSE) ≥ 22 and Clinical Dementia Rating–Global Score 0.5 or 1.0. The study is powered to detect a 2.0 to 2.5 drug–placebo difference on the Alzheimer's Disease Assessment Scale 13‐item Cognitive subscale primary outcome with 150 subjects/arm. The key secondary outcomes are Clinical Dementia Rating–Sum of Boxes and Instrumental Activities of Daily Living; volumetric magnetic resonance imaging and fluid biomarkers are additional outcomes. RESULTS The APOLLOE4 Phase 3 trial enrolled 325 subjects with a mean age of 69 years, 51% female, MMSE 25.6, and 65% mild cognitive impairment. Topline results are expected in 2024. DISCUSSION APOLLOE4 is the first disease‐modification AD trial focused on APOE ε4/ε4 homozygotes. Oral ALZ‐801 has the potential to be the first effective and safe anti‐amyloid treatment for the high‐risk APOE ε4/ε4 population. Highlights The APOLLOE4 Phase 3, placebo‐controlled, 78‐week study is designed to evaluate the efficacy and safety of ALZ‐801 265 mg twice per day in early Alzheimer's disease (AD) subjects with the apolipoprotein E (APOE) ε4/ε4 genotype. The enrolled early AD population (N = 325) has 51% females, a mean age = 69 years, and a mean Mini‐Mental State Examination = 25.6, with the majority being mild cognitive impairment subjects, a similar disease stage to the lecanemab Phase 3 AD trial (Clarity AD). The primary outcome is the cognitive Alzheimer's Disease Assessment Scale 13‐item Cognitive subscale, with two functional measures as key secondary outcomes (Clinical Dementia Rating–Sum of Boxes, Amsterdam‐Instrumental Activities of Daily Living), and with hippocampal volume and fluid biomarkers as additional outcomes. The study is unique in allowing a large number of microhemorrhages or siderosis at baseline magnetic resonance imaging, lesions that indicate concomitant cerebral amyloid angiopathy (CAA). At baseline, 32% of the enrolled population had at least 1 microhemorrhage, 24% had 1 to 4, and 8% had > 4 microhemorrhages; 10% had at least 1 siderosis lesion; with more males than females having microhemorrhages (63% vs. 37%) and siderosis (68% vs. 32%). Study results will become available in the second half of 2024 and, if positive, ALZ‐801 may become the first oral drug to demonstrate a favorable benefit/risk profile in APOE ε4/ε4 AD subjects.

Valiltramiprosate (ALZ-801) is an oral inhibitor of amyloid oligomer formation. Tramiprosate (active agent in ALZ-801) had shown promising efficacy signals with favorable safety in ∼1300 APOE4 carriers with AD, with no observed ARIA-E. This trial evaluated Valiltramiprosate in APOE4/4 homozygotes with Early AD. This 78-week double-blind, placebo-controlled, two-arm trial that randomized 325 homozygotes (162 to placebo, 163 to 265 mg BID), stratified by MCI (MMSE 27-30) or Mild AD (MMSE 22-26). MRI (1.5/3 Tesla) were conducted every 26 weeks and analyzed by Clario Inc. The clinical outcomes were ADAS-Cog13 (primary), CDR-SB (key secondary) and DAD (disability assessment for dementia, secondary). Hippocampal volume (HV) was the main imaging outcome. MMRM was the primary analysis model. Safety population (N =325) was 51% females, 90% Caucasian, mean age 68 years, MMSE 25.6, 39% with MCI (MMSE 27-30) and 30% with baseline microhemorrhages and/or siderosis. In the full analysis set (N =320), the placebo-adjusted least-square mean (LSM) difference in change from baseline (CBL) on ADAS-Cog13 favored drug non-significantly (delta=-0.50; p =0.61, 11% vs placebo CBL); CDR-SB and DAD numerically favored drug by 23% and 29%, but HV favored drug significantly (74 mm , nominal p =0.017, 18% less atrophy). Prespecified Mild AD showed small nonsignificant clinical effects favoring placebo, but showed numerical benefit on HV (51mm , 12% p = 0.115). In prespecified MCI, all outcomes favored drug (nominal p -values): ADAS-Cog13= -2.14 (p =0.041; 52%); CDR-SB= -0.65 (p =0.053, 104%); DAD= 6.09 (p =0.016, 96%); and HV= 108 mm (p = 0.004, 26% less atrophy). Nausea (mostly mild) was the most common adverse event; incidence of ARIA-E was the same as placebo. In the overall population, ADAS-Cog13 did not achieve significance, but the hippocampus showed significant 18% slowing of atrophy. The pre-specified Mild group showed trends to HV atrophy slowing that did not translate to clinical benefits. The pre-specified MCI group showed significant 28% HV atrophy slowing with meaningful cognitive and functional benefits and positive trends on several secondary clinical outcomes. Overall safety was favorable with no increased ARIA. In the high-risk APOE4/4 population, this positive benefit-risk profile supports valiltramiprosate's potential as an oral disease-modifying treatment for APOE4/4 homozygotes with MCI.

Valiltramiprosate/ALZ-801, an oral inhibitor of amyloid oligomer formation, was evaluated in a Phase 3 trial in APOE4/4 homozygotes with Early AD, with main results presented at this meeting (Power et al, 2025). APOE4 is a risk factor for cerebral amlyloid angiopathy (CAA) and ARIA. Tramiprosate (active agent in ALZ-801) had shown no ARIA-E in prior studies, allowing the APOLLOE4 trial to include subjects with any number of microhemorrhages (MH) and several siderosis lesions at screening MRI. This 78-week placebo-controlled trial enrolled 325 homozygotes who received placebo or 265 mg BID. Anti-coagulants were exclusionary. In addition to adverse event (AE) assessments, MRIs were performed every 26 weeks and evaluated by Clario Inc. for occurrence of ARIA with edema and/or microhemorrhage/siderosis (ARIA-E, ARIA-H). Safety population (N =325) was 51% females, 89% Caucasian, mean age 68 years, MMSE 25.6, with MCI vs. Mild AD (39% vs 61%); 298 had screening MRI (149/arm provided ≥1 post-dose MRI). At baseline, 32% had >1 MH (placebo, 32%, active, 31%); 11% in placebo and 8% in active arm had siderosis (maximum 4 and 5 lesions, respectively); and 5% in each arm had ≥ 10 MH. New ARIA-E occurred during the study in 5 subjects in each arm (3%) and new macrohemorrhages in 2 subjects/arm. Increase in MH from low to high category (0, 1-4, 5-9, ≥10 MH) was seen in 14% placebo and 9% active arm. Radiographic ARIA were all mild except 1 severe (placebo) and 1 moderate (active); all were without symptoms. AEs with incidence >5% and >2x placebo rate were nausea (26%), weight decrease (14%), decreased appetite and vomiting (10% each); with lower incidence of headache, falls and dizziness in active arm. APOLLOE4 enrolled homozygotes with a high CAA burden who would be ineligibile for amyloid antibody treatments. Valiltramiprosate overall safety was favorable and consistent with prior studies. ARIA-E incidence was similar to placebo, and ARIA-H was lower than placebo with no symptomatic ARIA events over 78 weeks of treatment. Coupled with promising efficacy in MCI subjects, valiltramiprosate can provide a favorable benefit-risk profile for the high-risk APOE4/4 patients with unmet treatment need.