Lake, Douglas F.

The diagnosis of coccidioidomycosis is often achieved serologically by the detection of antibodies against fungal antigens. While several serologic tests are available for coccidioidomycosis, all of them are performed in a laboratory setting causing delays in diagnosis and therapeutic intervention. Point-of-care testing offers the ability to provide a shorter time to result by avoiding specimen send-out, minimizing processing steps, and employing expeditious immunochemical techniques. A preliminary trial of a rapid anti-coccidioidal antibody lateral flow assay (LFA) using fingerstick blood was performed on 22 patients with coccidioidomycosis at the point of care during outpatient clinic visits. Patients were tested longitudinally over the course of one year. An LFA reader was implemented to provide an objective result by quantifying the intensity of the test line band. There was close qualitative concordance observed between positive LFA results with send-out immunodiffusion (89.5%) and complement fixation (78.4%) standard of care clinical laboratory assays. Additionally, the relationship between LFA test line density values and traditional complement fixation antibody titers was assessed.

Abstract Description Coccidiomycosis is an endemic fungal infection caused by Coccidioides spp., and diagnosis is often made with serology. Coccidiomycosis is especially problematic for immunocompromised patients, with greater risk for disseminated infection and mortality. This study evaluated current strategies for detecting antibodies to Coccidioides, including enzyme immunoassay (EIA), immunodiffusion (ID) and complement fixation (CF) and applied a new ID assay for improved sensitivity and specificity. Clinical sensitivity and specificity studies were done using 1544 unique serum specimens for which Coccidioides EIA testing was clinically requested. A full panel of EIA IgG, EIA IgM, ID and CF was performed on each, comparing our in-house immunodiffusion assay using reagents from Cactus Bio, LLC to our current reference laboratory. Full method validation was completed, as prescribed by CLIA. Using existing reference lab testing as the standard, a sensitivity of 96.9%, specificity 85.9%, PPV 54.9%, NPV 99.4% was achieved. Additional chart review demonstrated the in-house ID improved to 98.0% sensitivity, 94.6 specificity, 84.2% PPV, 99.4% NPV. Such high NPV supports using reflex testing, which allowed our lab to eliminate hundreds of unnecessary CF referrals each month, saving diagnostic time, financial waste and lab resources. A reflexive testing algorithm paired with this enhanced ID assay is especially useful for the immunosuppressed, for which Coccidioides infections carry a greater risk. Topic Categories Translational and Interventional Immunology (TI)

Abstract Glutamine dependence of cancer cells reduces local glutamine availability, which hinders antitumor T-cell functionality and facilitates immune evasion. We thus speculated that glutamine deprivation might be limiting efficacy of chimeric antigen receptor (CAR) T-cell therapies in patients with cancer. We have seen that antigen-specific T cells are unable to proliferate or produce interferon gamma (IFN-γ) in response to antigen stimulation when glutamine concentration is limited. Using multiple myeloma (MM) as a glutamine-dependent disease model, we found that murine CAR T cells selectively targeting B-cell maturation antigen (Bcma) in MM cells were sensitive to glutamine deprivation. However, CAR T cells engineered to increase glutamine uptake by expression of the glutamine transporter Asct2 exhibited enhanced proliferation and responsiveness to antigen stimulation, increased production of IFN-γ, and heightened cytotoxic activity, even under conditions of low glutamine concentration. Mechanistically, Asct2 overexpression reprogrammed the metabolic fitness of CAR T cells by upregulating the mechanistic target of rapamycin complex 1 gene signature, modifying the solute carrier transporter repertoire, and improving both basal oxygen consumption rate and glycolytic function, thereby enhancing CAR T-cell persistence in vivo. Accordingly, expression of Asct2 increased the efficacy of Bcma-CAR T cells in syngeneic and genetically engineered mouse models of MM, which prolonged mouse survival. In patients, higher-level expression of ASCT2 by MM cells predicted poor outcome to combined immunotherapy and BCMA-CAR T-cell therapy. Our results indicate that reprogramming glutamine metabolism may enhance antitumor CAR T-cell functionality in MM. This approach may also be effective for other cancers that depend on glutamine as a key energy source and metabolic hallmark.

Abstract OBJECTIVE To compare 2 point-of-care lateral flow assays (LFAs) with immunodiffusion (ID) IgG results for anti-coccidioidal antibody detection in dogs with coccidioidomycosis. A further aim was to compare the quantifiable output of 1 of the LFAs to ID antibody titers. SAMPLE Serum banked from 73 client-owned dogs diagnosed with pulmonary or disseminated coccidioidomycosis. METHODS ID was used to determine antibody presence and titer against a coccidioidal antigen preparation. All sera were subsequently tested on an LFA based on recombinant chitinase 1 (CTS1) and the commercially available sōna LFA. LFA results were analyzed and compared to ID IgG results and clinical diagnosis. RESULTS All assays showed similar sensitivities in detecting anti-coccidioidal antibodies (83.6% to 89.0%). When compared with ID IgG, the CTS1 LFA had a positive percent agreement of 100%, while the sōna LFA had a positive percent agreement of 91.4%. Since the CTS1 LFA is semiquantitative, we were able to compare test line densities with ID titers and found a strong correlation between the 2 assays (Spearman ρ = 0.82). CLINICAL RELEVANCE This is the first side-by-side evaluation of a commercially available LFA (sōna) and a newer more rapid anti-CTS1 antibody LFA using serum from dogs with coccidioidomycosis. Both LFAs tested have similar sensitivity to ID IgG results. The CTS1 LFA can be read after 10 minutes and is semiquantitative, while the sōna LFA is read after 30 minutes, and the results are subject to interpretation. Accurate and fast detection of anti-coccidioidal antibodies allows clinicians to initiate appropriate treatment without diagnostic delay.

ABSTRACTCoccidioidesspp. are dimorphic fungi that are capable of infecting human and non-human mammals and can cause diverse manifestations of coccidioidomycosis or Valley fever (VF). In combination with clinical symptoms and radiographic findings, antibody-based diagnostic tests are often used to diagnose and monitor patients with VF. Chitinase 1 (CTS1) has previously been identified as the seroreactive antigen used in these diagnostic assays to detect anticoccidial IgG. Here, an indirect enzyme-linked immunosorbent assay to detect IgG to CTS1 demonstrated 165 of 178 (92.7%) patients with a positive result by immunodiffusion (ID) and/or complement fixation (CF) had antibodies to the single antigen CTS1. We then developed a rapid antibody lateral flow assay (LFA) to detect anti-CTS1 antibodies. Out of 143 samples tested, the LFA showed 92.9% positive percent agreement [95% confidence interval (CI), 84.3%–96.9%] and 97.7% negative percent agreement (95% CI, 87.9%–99.6%) with ID and CF assays. Serum or plasma from canines, macaques, and dolphins was also tested by the CTS1 LFA. Test line densities of the CTS1 LFA correlated in a linear manner with the reported CF and ID titers for human and non-human samples, respectively. This 10-min point-of-care test for the rapid detection of anti-coccidioidal antibodies could help to inform healthcare providers in real-time, potentially improving the efficiency of healthcare delivery.

Histone‐lysine N‐methyltransferase SETD2 (SETD2), the sole histone methyltransferase that catalyzes trimethylation of lysine 36 on histone H3 (H3K36me3), is often mutated in clear cell renal cell carcinoma (ccRCC). SETD2 mutation and/or loss of H3K36me3 is linked to metastasis and poor outcome in ccRCC patients. Epithelial‐to‐mesenchymal transition (EMT) is a major pathway that drives invasion and metastasis in various cancer types. Here, using novel kidney epithelial cell lines isogenic for SETD2, we discovered that SETD2 inactivation drives EMT and promotes migration, invasion, and stemness in a transforming growth factor‐beta‐independent manner. This newly identified EMT program is triggered in part through secreted factors, including cytokines and growth factors, and through transcriptional reprogramming. RNA‐seq and assay for transposase‐accessible chromatin sequencing uncovered key transcription factors upregulated upon SETD2 loss, including SOX2, POU2F2 (OCT2), and PRRX1, that could individually drive EMT and stemness phenotypes in SETD2 wild‐type (WT) cells. Public expression data from SETD2 WT/mutant ccRCC support the EMT transcriptional signatures derived from cell line models. In summary, our studies reveal that SETD2 is a key regulator of EMT phenotypes through cell‐intrinsic and cell‐extrinsic mechanisms that help explain the association between SETD2 loss and ccRCC metastasis.

In southwestern and western regions of the United States, at least half a million diagnostic tests for coccidioidomycosis (valley fever) are run annually. Enzyme immunoassays (EIAs) are blood tests which require precise dilution of patient serum prior to testing.

Abstract Background While evaluating COVID-19 vaccine responses using a rapid neutralizing antibody (NAb) test, we observed that 25% of RNA vaccine recipients did not neutralize >50%. We termed this group “vaccine poor responders” (VPRs). The objective of this study was to determine if individuals who neutralized <50% would remain VPRs, or if a third dose would elicit high levels of NAbs. Methods 269 healthy individuals ranging in age from 19 to 80 (Average age = 51; 165 females and 104 males) who received either BNT162b2 (Pfizer) or mRNA1273 (Moderna) vaccines were evaluated. NAb levels were measured: i) 2-4 weeks after a second vaccine dose, ii) 2-4 months after the second dose, iii) within 1-2 weeks prior to a third dose and iv) 2-4 weeks after a third RNA vaccine dose. Results Analysis of vaccine recipients revealed that 25% did not neutralize above 50% (Median neutralization = 21%, titers <1:80) within a month after their second dose. Twenty-three of these VPRs obtained a third dose of either BNT162b2 or mRNA-1273 vaccine 1-8 months (average = 5 months) after their second dose. Within a month after their third dose, VPRs showed an average 20-fold increase in NAb levels (range: 46%-99%). Conclusions The results suggest that VPRs are not permanently poor responders; they can generate high NAb levels with an additional vaccine dose. Although it is not known what levels of NAbs protect from infection or disease, those in high-risk professions may wish to keep peripheral NAb levels high, limiting infection, and potential transmission.

Longitudinal studies assessing durability of the anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) humoral immune response have generated conflicting results. This has been proposed to be due to differences in patient populations, the lack of standardized methodologies, and the use of assays that measure distinct aspects of the humoral response.

Abstract Background Coccidioidomycosis is often diagnosed with a collection of tests that measure a patient’s ability to mount an immune response to the fungus (antibody-based diagnostics) utilizing fungal protein preparations. Here we present an antigen-based assay that detects and quantifies coccidioidal chitinase-1 (CTS1) in diagnostic antigen preparations with potential for use in human serum. Methods An inhibition-based enzyme-linked immunoassay (ELISA) was developed that utilizes a monoclonal antibody specific for coccidioidal CTS1. CTS1 was quantified in commercial antigen preparations using recombinant CTS1 as a standard. Sera from 192 individuals from an endemic area were tested which included 78 patients (40.6%) with proven or probable coccidioidomycosis. Results The quantity of CTS1 in diagnostic commercial antigen preparations from different suppliers varied. Temporal constraints of availability of different lots of commercial antigens does not allow for immediate comparison of lot-to-lot variability. Assay results from patient serum samples correlated with low- and high-titer serology from patients with a coccidioidomycosis diagnosis. Further analysis suggested that patient derived anti-CTS1 antibodies may overlap with the mouse monoclonal antibody used in the assay. This unexpected overlap in CTS1 binding suggests the assay can detect antigen, antibody, or both, which contributes to its high level of clinical sensitivity of 89.74% and specificity of 94.90%. Conclusions The CTS1 inhibition ELISA described in this report is a promising tool to aid in quality control of antigens used in the diagnosis of coccidioidomycosis. Further optimization is needed to harness its utility as a diagnostic tool to aid in diagnosis and disease monitoring of coccidioidomycosis.