Douglas F. Lake's research while affiliated with Arizona State University and other places

Valley Fever (VF), caused by fungi in the genus Coccidioides, is a prevalent disease in southwestern and western parts of the United States that affects both humans and animals, such as dogs. Although the immune responses to infection with Coccidioides spp. are not fully characterized, antibody-detection assays are used in conjunction with clinical presentation and radiologic findings to aid in the diagnosis of VF. These assays often use Complement Fixation (CF) and Tube Precipitin (TP) antigens as the main targets of IgG and IgM reactivity, respectively. Our group previously reported evidence of over 800 genes expressed at the protein level in C. posadasii. However, antibody reactivity to the majority of these proteins has never been explored. Using a new, high-throughput screening technology, the Nucleic Acid Programmable Protein Array (NAPPA), we screened serum specimens from dogs against 708 of these previously identified proteins for IgG reactivity. Serum from three separate groups of dogs was analyzed and revealed a small panel of proteins to be further characterized for immuno-reactivity. In addition to CF/CTS1 antigen, sera from most infected dogs showed antibody reactivity to endo-1,3-betaglucanase, peroxisomal matrix protein, and another novel reactive protein, CPSG_05795. These antigens may provide additional targets to aid in antibody-based diagnostics.

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.

Early reports showed that patients with COVID-19 who had a previous diagnosis of Coccidioidomycosis (Valley fever, VF) who had cleared VF months and years prior had re-emergence of VF after COVID-19. However, total numbers of annual cases reported to CDC did not appear to rise dramatically. We therefore investigated serial infection of Coccidioides posadasii (C.p.) and variants of SARS-CoV2 in an K18-hACE2 transgenic mouse to assess the impact on disease outcomes. We intranasally challenged mice sequentially with sub-lethal doses of 100 plaque forming units (PFUs) per mouse of SARS-CoV-2 (variants WA-1, Delta, Omicron BA1) and 24 hours later with 100 arthroconidia per mouse of a low virulence Coccidioides posadasii strain 1038 and vice versa. Lungs, brain, and spleen were extracted and cultured to assess fungal burden and half of the lung was saved for histopathology. To assess pathogenesis, we repeated experiment but sacrificed a subset of mice on days 1, 3, 5, and 6 post-infections in order to assess viral/fungal burden in the lungs. Serum was collected at each time point to measure differences in systemic cytokine/chemokine responses. Experiments were conducted under IACUC protocol # 21-025 at Northern Arizona University. We examined differences in disease outcome between the co-infected groups and groups that only received a primary infection. Co-infected groups had a more severe disease progression as well as decreased survival. Interestingly, results differed depending on SARS-CoV-2 variant (WA-1, Delta, Omicron), infection timing (CoV2 first, C.p. second or vice versa). The groups that were infected with the virus first had decreased survival, increased morbidity (significant weight loss), and increased fungal and viral burdens. There were also differences in immune responses and number/size of spherules, the virus first groups had an increase in circulating pro-inflammatory cytokines and larger more abundant spherules. This is the first in vivo investigation of a co-infection of SARS-CoV-2 and Coccidioides to date. Because of the increased severity of disease, we contemplate the serious implications to the populations that live in areas of high fungal burden and how the SARS-CoV-2 virus can complicate disease progression.

Coccidioides spp. 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.

As amniote vertebrates, lizards are the most closely related organisms to humans capable of appendage regeneration. Lizards can autotomize, or release their tails as a means of predator evasion, and subsequently regenerate a functional replacement. Green anoles ( Anolis carolinensis ) can regenerate their tails through a process that involves differential expression of hundreds of genes, which has previously been analyzed by transcriptomic and microRNA analysis. To investigate protein expression in regenerating tissue, we performed a whole proteomic analysis of regenerating tail tip and base. This is the first proteomic data set available for any anole lizard. We identified a total of 2646 proteins—976 proteins only in the regenerating tail base, 796 only in the tail tip, and 874 in both tip and base. For over 90% of these proteins in these tissues, we were able to assign a clear orthology to gene models in either the Ensembl or NCBI databases. For 13 proteins in the tail base, 9 proteins in the tail tip, and 10 proteins in both regions, the gene model in Ensembl and NCBI matched an uncharacterized protein, confirming that these predictions are present in the proteome. Ontology and pathways analysis of proteins expressed in the regenerating tail base identified categories, including actin filament‐based process, ncRNA metabolism, regulation of phosphatase activity, small GTPase‐mediated signal transduction, and cellular component organization or biogenesis. Analysis of proteins expressed in the tail tip identified categories, including regulation of organelle organization, regulation of protein localization, ubiquitin‐dependent protein catabolism, small GTPase‐mediated signal transduction, morphogenesis of epithelium, and regulation of biological quality. These proteomic findings confirm pathways and gene families activated in tail regeneration in the green anole as well as identify uncharacterized proteins whose role in regrowth remains to be revealed. This study demonstrates the insights that are possible from the integration of proteomic and transcriptomic data in tail regrowth in the green anole, with potentially broader application to studies in other regenerative models. KEY POINTS This research is highly interdisciplinary, combining our previous analyses with these most recent findings: Appendage regeneration is a conserved trait among vertebrates and has been characterized in animals ranging from teleost fish (zebrafish), urodele amphibians (axolotl), anuran amphibians ( Xenopus frog), squamate reptiles (various species of lizards), and even crocodilians (American alligator). Comparative genomic and proteomic analysis of this process allows us to identify the genetic pathways and cellular processes under evolutionary selection for this regrowth capacity. Activating these conserved genetic pathways and cellular processes will be critical to developing regenerative medical therapies in humans. The identification of proteins expressed in regeneration extends analyses based only on predicted proteins from transcriptomic analysis, and permits integration with protein‐expression studies of regrowing nervous and musculoskeletal structures.

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 (TGF-β)-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 (ATAC-seq) 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 cells. Public expression data from SETD2 wild-type/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.

Galectin-1 is a β-galactoside-binding lectin that has been implicated as a suppressive molecule in cancer and autoimmune diseases. Gal-1 has known immunomodulatory activity and was found to be expressed on regulatory T cells, leading to the potential for targeted immunotherapies. Anti-Gal-1 monoclonal antibodies were generated in this study using classical hybridoma techniques. MAb 6F3 was found to bind to Gal-1 by Western blot and ELISA. Flow cytometry was used to determine cell surface and intracellular binding of mAb 6F3 to Gal-1 in PBMC-derived Tregs and tumor cells, including Treg-like cell lines. These results suggest mAb 6F3 may be used to further study Gal-1 protein expression and function.

Coccidioides immitis and Coccidioides posadasii are soil-dwelling fungi of arid regions in North and South America that are responsible for Valley fever (coccidioidomycosis). Forty percent of patients with Valley fever exhibit symptoms ranging from mild, self-limiting respiratory infections to severe, life-threatening pneumonia that requires treatment. Misdiagnosis as bacterial pneumonia commonly occurs in symptomatic Valley fever cases, resulting in inappropriate treatment with antibiotics, increased medical costs, and delay in diagnosis. In this proof-of-concept study, we explored the feasibility of developing breath-based diagnostics for Valley fever using a murine lung infection model. To investigate potential volatile biomarkers of Valley fever that arise from host–pathogen interactions, we infected C57BL/6J mice with C. immitis RS (n = 6), C. posadasii Silveira (n = 6), or phosphate-buffered saline (n = 4) via intranasal inoculation. We measured fungal dissemination and collected bronchoalveolar lavage fluid (BALF) for cytokine profiling and for untargeted volatile metabolomics via solid-phase microextraction (SPME) and two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC×GC-TOFMS). We identified 36 volatile organic compounds (VOCs) that were significantly correlated (p < 0.05) with cytokine abundance. These 36 VOCs clustered mice by their cytokine production and were also able to separate mice with moderate-to-high cytokine production by infection strain. The data presented here show that Coccidioides and/or the host produce volatile metabolites that may yield biomarkers for a Valley fever breath test that can detect coccidioidal infection and provide clinically relevant information on primary pulmonary disease severity.

Coccidioides immitis and Coccidioides posadasii are soil-dwelling fungi of arid regions in North and South America that are responsible for Valley fever (coccidioidomycosis). Forty percent of patients with Valley fever exhibit symptoms ranging from mild, self-limiting respiratory infections, to severe, life-threatening pneumonia that requires treatment. Misdiagnosis as bacterial pneumonia commonly occurs in symptomatic Valley fever cases, resulting in inappropriate treatment with antibiotics, increased medical costs, and delay in diagnosis. In this study, we explored the feasibility of developing breath-based diagnostics for Valley fever using a murine lung infection model. To investigate potential volatile biomarkers of Valley fever that arise from host-pathogen interactions, we infected C57BL/6J mice with C. immitis RS and C. posadasii Silveira via intranasal inoculation. We measured fungal dissemination and collected bronchoalveolar lavage fluid (BALF) for cytokine profiling and for untargeted volatile metabolomics via solid phase microextraction (SPME) and two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-TOFMS). We identified 36 volatile organic compounds (VOCs) that were significantly correlated to cytokine abundances and clustered mice by disease severity. These 36 VOCs were also able to separate mice with a moderate to high disease severity by infection strain. The data presented here show that Coccidioides and/or the host produce volatile metabolites that may yield biomarkers for a Valley fever breath test that can detect Coccidioidal infection and provide clinically relevant information on disease severity. IMPORTANCE Coccidioidomycosis, or Valley fever, is a fungal disease endemic to the North and South American arid regions. Forty percent of individuals infected with Valley fever will exhibit symptoms consistent with community-acquired pneumonia. However, misdiagnosis frequently occurs in these cases, resulting in inappropriate treatment with antibiotics, increased medical costs, and delay in receiving an accurate diagnosis. Herein, we used a murine lung infection model as a step towards developing a breath-based diagnostic for Valley fever. We infected C57BL/6J mice with C. immitis RS and C. posadasii Silveira and collected bronchoalveolar lavage fluid for untargeted volatile metabolomics. We observed that volatile metabolites in the bronchoalveolar lavage fluid of Cocci-inoculated mice were significantly correlated to disease severity, as measured by immune response. The data presented here show that Coccidioides and/or the host produce volatile metabolites that may yield biomarkers for a Valley fever breath test.