Publications

Multiplexed Immunofluorescent Characterization of T-Cell and Myeloid Cell Populations Reveals Histologic Subtype Differences Within the Immunosuppressive Landscape of Human Basal Cell Carcinoma

Published in Journal of Cutaneous Pathology, 2026

Basal cell carcinomas (BCCs) are thought to reside within an immunosuppressed tumor microenvironment, but a detailed characterization of BCC-associated immune cells was lacking. Utilizing multiplex immunohistochemistry (IHC) (Vectra Polaris, Akoya Biosciences), we designed two staining panels, targeting either T cells (CD3, CD4, CD8, FoxP3, PD1) or myeloid cells (neutrophil elastase, CD68, CD163, HLADR, CD1c). Formalin-fixed paraffin-embedded (FFPE) BCC tumor specimens (n = 30) were analyzed using the panels. The majority of immune cells reside at the peritumoral margin; therefore, a boundary zone of 150 μm around tumor nests was used to capture them. A CD8/T-reg ratio and M1/M2 macrophage ratio of < 0.5 was observed across all BCC specimens, confirming an overall immunosuppressive phenotype. Among different histologic subtypes, infiltrative BCC had significantly lower CD8 T cells (p = 0.038), T-regulatory cells (p = 0.039), and CD8/T-reg ratio (p = 0.048), as well as lower M1/M2 ratio (p = 0.034) compared to non-infiltrative BCC subtypes. The association of infiltrative BCC with a more immunosuppressed microenvironment may contribute to more aggressive biological behavior. Overall, these data demonstrate a method to determine T cell and myeloid profiles in fixed FFPE skin tumor tissues that can identify subtle immune profile differences.

Recommended citation: Shen AS, Heusinkveld LE, Zalavadia A, Branicky A, Anand S, Piliang M, Maytin EV. Multiplexed Immunofluorescent Characterization of T-Cell and Myeloid Cell Populations Reveals Histologic Subtype Differences Within the Immunosuppressive Landscape of Human Basal Cell Carcinoma. J Cutan Pathol. 2026 Mar 31. doi: 10.1111/cup.70098. Epub ahead of print. PMID: 41916617. https://onlinelibrary.wiley.com/doi/10.1111/cup.70098

Myosin IIA motor regulates attaching-effacing bacteria interactions with intestinal epithelium

Published in Gut Microbes, 2026

Attaching effacing (A/E) bacteria, such as enteropathogenic Escherichia coli (EPEC) and Citrobacter rodentium colonize intestinal epithelial cells (IECs) by inducing remodeling of the epithelial cytoskeleton and the formation of prominent actin pedestals at bacterial attachment sites. While non-muscle myosin II (NM II) is a key regulator of the actin cytoskeleton, whether it regulates IEC colonization by A/E pathogens is not known. To address this question, we targeted NM IIA and NM IIC, the NM II paralogs expressed in IECs. Our in vivo studies utilized mouse models with either intestinal epithelial-specific deletion of NM IIA (NM IIA cKO mice), expression of a NM IIA motor domain mutant, or total deletion of NM IIC (NM IIC tKO mice). In vitro experiments utilized IECs (HT-29cF8 and Caco-2BBE) with CRISPR-Cas9-mediated deletion of NM IIA or NM IIC. In addition, NM II activity in vitro was modulated pharmacologically, using either the pan-myosin inhibitor, blebbistatin, or a specific NM IIC activator, 4-hydroxyacetophenone (4-HAP). NM IIA cKO and NM IIA mutant mice demonstrated higher C. rodentium colonization, along with more severe mucosal inflammation and colonic crypt hyperplasia as compared to their controls. By contrast, NM IIC tKO mice was indistinguishable from their control with regard to C. rodentium colonization. Blebbistatin treatment increased EPEC attachment to IECs monolayers, whereas 4-HAP did not affect bacterial attachment. Genetic knockout of NM IIA, but not NM IIC, increased EPEC adhesion to IEC monolayers. Importantly, the increase in EPEC attachment exhibited by NM IIA-deficient IECs required an intact bacterial Type 3 secretion system and functional Tir effector, indicating that NM IIA functions in actin pedestal assembly. In summary, we describe a novel role for NM IIA in limiting intestinal epithelial colonization by A/E pathogens via the inhibition of pathogen-induced remodeling of the actin cytoskeleton.

Recommended citation: Naydenov NG, Zafar A, Lechuga S, Zalavadia A, Marino-Melendez A, Hammer JA, Fowler VM, McDonald C, Campellone KG, Ivanov AI. Myosin IIA motor regulates attaching-effacing bacteria interactions with intestinal epithelium. Gut Microbes. 2026 Dec 31;18(1):2638002. doi: 10.1080/19490976.2026.2638002. Epub 2026 Feb 28. PMID: 41761878; PMCID: PMC12959189. https://www.tandfonline.com/doi/full/10.1080/19490976.2026.2638002

Microbial Metabolite 4EPS Inhibits AT1R to Reduce Blood Pressure and Aortic Aneurysm Outcome

Published in Hypertension, 2026

Plasma accumulation of the gut microbial metabolite 4-ethylphenylsulfate (4EPS), derived from dietary amino acid, tyrosine, has been associated with cardiovascular, renal, metabolic, and neurological disorders. AngII (angiotensin II) infusion increases circulating 4EPS in mice, suggesting a potential mechanistic role. We hypothesized that 4EPS modulates AngII-regulated pathophysiology and disease progression by directly inhibiting AT1R (angiotensin II type 1 receptor). This hypothesis was tested by combining AT1R pharmacology, cell signaling assays, ex vivo vascular studies, an AngII-induced aortic aneurysm growth model, and plasma proteomics analysis. in vitro, 4EPS reduced the binding of both AngII and the antagonist candesartan to AT1R and suppressed AngII-induced calcium signaling. Ex vivo, 4EPS attenuated AngII-mediated vasoconstriction. In vivo, high-fat diet-fed ApoE-null mice coinfused with AngII and 4EPS showed significant blunting of blood pressure elevation and a marked reduction in aortic aneurysm-related mortality compared with mice infused with AngII alone. Analysis of aortic remodeling revealed increased elastin preservation and decreased thickening of the intimal and medial layers in 4EPS-treated animals. Plasma proteomics indicated alterations in actin-cytoskeletal signaling pathways consistent with reduced activation of ERK (extracellular-regulated kinase) 1/2, filamin-A, and proteins involved in vascular smooth muscle cell motility. These findings identify 4EPS as a benign, endogenous AT1R antagonist that diminishes AngII-mediated hemodynamic and vascular pathology. By suppressing cytoskeletal signaling associated with vascular remodeling, 4EPS provides significant protection against hypertension and aortic aneurysm progression in mice, revealing a previously unrecognized protective role for a gut microbial metabolite in modulating renin-angiotensin system activity.

Recommended citation: Harford TJ, Singh KD, Pardhi TR, Desnoyer R, Ravi T, Jara ZP, Zalavadia A, Stenson K, Naga Prasad SV, Karnik SS. Microbial Metabolite 4EPS Inhibits AT1R to Reduce Blood Pressure and Aortic Aneurysm Outcome. Hypertension. 2026 Jan 22. doi: 10.1161/HYPERTENSIONAHA.125.25364. PMID: 41568432. https://www.ahajournals.org/doi/epub/10.1161/HYPERTENSIONAHA.125.25364

Leveraging dysregulated tumor metabolism for targeting anticancer bacteria

Published in Science Advances, 2025

Widespread application of bacterial-based cancer therapy is limited because of the need to increase therapeutic bacteria specificity to the tumor to improve treatment safety and efficacy. Here, we harness the altered tumor metabolism and specifically elevated kynurenine accumulation to target engineered bacteria to the cancer site. We cloned and leveraged kynurenine-responsive transcriptional regulator (KynR) with its cognate promoter in Escherichia coli. Optimizing KynR expression coupled with overexpressing kynurenine transporter and amplifying the response through plasmid copy number–based signal amplification enabled the response to kynurenine at the low micromolar levels. Knocking out genes essential for cell wall synthesis and supplying these genes via kynurenine-controlled circuits allowed tuning Salmonella enterica growth in response to kynurenine. Our kynurenine-controlled S. enterica (hereafter named AD95+) showed superior tumor specificity in breast and ovarian cancer murine models compared to S. enterica VNP20009, one of the best characterized tumor-specific strains. Last, AD95+ showed anticancer properties compared to vehicle controls, demonstrating the potential as an anticancer therapeutic.

Recommended citation: Santos A, Wang Z, Bharti R, Dey G, Sangwan N, Baldwin W, Zalavadia A, Myers A, Huffman OG, Lathia JD, Hazen SL, Reizes O, Dwidar M. Leveraging dysregulated tumor metabolism for targeting anticancer bacteria. Sci Adv. 2025 Jun 13;11(24):eads1630. doi: 10.1126/sciadv.ads1630. Epub 2025 Jun 13. PMID: 40512849; PMCID: PMC12164975. https://www.science.org/doi/full/10.1126/sciadv.ads1630

Coactosin-like protein 1 regulates integrity and repair of model intestinal epithelial barriers via actin binding dependent and independent mechanisms

Published in Frontiers in Cell and Developmental Biology, 2024

The actin cytoskeleton regulates the integrity and repair of epithelial barriers by mediating the assembly of tight junctions (TJs), and adherens junctions (AJs), and driving epithelial wound healing. Actin filaments undergo a constant turnover guided by numerous actin-binding proteins, however, the roles of actin filament dynamics in regulating intestinal epithelial barrier integrity and repair remain poorly understood. Coactosin-like protein 1 (COTL1) is a member of the ADF/cofilin homology domain protein superfamily that binds and stabilizes actin filaments. COTL1 is essential for neuronal and cancer cell migration, however, its functions in epithelia remain unknown. The goal of this study is to investigate the roles of COTL1 in regulating the structure, permeability, and repair of the epithelial barrier in human intestinal epithelial cells (IEC). COTL1 was found to be enriched at apical junctions in polarized IEC monolayers in vitro. The knockdown of COTL1 in IEC significantly increased paracellular permeability, impaired the steady state TJ and AJ integrity, and attenuated junctional reassembly in a calcium-switch model. Consistently, downregulation of COTL1 expression in Drosophila melanogaster increased gut permeability. Loss of COTL1 attenuated collective IEC migration and decreased cell-matrix attachment. The observed junctional abnormalities in COTL1-depleted IEC were accompanied by the impaired assembly of the cortical actomyosin cytoskeleton. Overexpression of either wild-type COTL1 or its actin-binding deficient mutant tightened the paracellular barrier and activated junction-associated myosin II. Furthermore, the actin-uncoupled COTL1 mutant inhibited epithelial migration and matrix attachment. These findings highlight COTL1 as a novel regulator of the intestinal epithelial barrier integrity and repair.

Recommended citation: Lechuga, S., Marino-Melendez, A., Davis, A., Zalavadia, A., Khan, A., Longworth, M. S., & Ivanov, A. I. (2024). Coactosin-like protein 1 regulates integrity and repair of model intestinal epithelial barriers via actin binding dependent and independent mechanisms. Frontiers in Cell and Developmental Biology, 12, 1405454. https://www.frontiersin.org/journals/cell-and-developmental-biology/articles/10.3389/fcell.2024.1405454/full

VISTA promotes the metabolism and differentiation of myeloid-derived suppressor cells by STAT3 and polyamine-dependent mechanisms

Published in Cell Reports, 2024

Myeloid-derived suppressor cells (MDSCs) impair antitumor immune responses. Identifying regulatory circuits during MDSC development may bring new opportunities for therapeutic interventions. We report that the V-domain suppressor of T cell activation (VISTA) functions as a key enabler of MDSC differentiation. VISTA deficiency reduced STAT3 activation and STAT3-dependent production of polyamines, which causally impaired mitochondrial respiration and MDSC expansion. In both mixed bone marrow (BM) chimera mice and myeloid-specific VISTA conditional knockout mice, VISTA deficiency significantly reduced tumor-associated MDSCs but expanded monocyte-derived dendritic cells (DCs) and enhanced T cell-mediated tumor control. Correlated expression of VISTA and arginase-1 (ARG1), a key enzyme supporting polyamine biosynthesis, was observed in multiple human cancer types. In human endometrial cancer, co-expression of VISTA and ARG1 on tumor-associated myeloid cells is associated with poor survival. Taken together, these findings unveil the VISTA/polyamine axis as a central regulator of MDSC differentiation and warrant therapeutically targeting this axis for cancer immunotherapy.

Recommended citation: Zhang, K., Zakeri, A., Alban, T., Dong, J., Ta, H. M., Zalavadia, A. H., Branicky, A., Zhao, H., Juric, I., Husich, H., Parthasarathy, P. B., Rupani, A., Drazba, J. A., Chakraborty, A. A., Ching-Cheng Huang, S., Chan, T., Avril, S., & Wang, L. L. (2024). VISTA promotes the metabolism and differentiation of myeloid-derived suppressor cells by STAT3 and polyamine-dependent mechanisms. Cell reports, 43(1), 113661. https://www.sciencedirect.com/science/article/pii/S2211124723016728

Ifit2 restricts murine coronavirus spread to the spinal cord white matter and its associated myelin pathology

Published in Journal of Virology, 2023

Interferon-induced protein with tetratricopeptide repeats 2, Ifit2, is critical in restricting neurotropic murine-β-coronavirus, RSA59 infection. RSA59 intracranial injection of Ifit2-deficient (-/-) compared to wild-type (WT) mice results in impaired acute microglial activation, reduced CX3CR1 expression, limited migration of peripheral lymphocytes into the brain, and impaired virus control followed by severe morbidity and mortality. While the protective role of Ifit2 is established for acute viral encephalitis, less is known about its influence during the chronic demyelinating phase of RSA59 infection. To understand this, RSA59 infected Ifit2-/- and Ifit2+/+ (WT) were observed for neuropathological outcomes at day 5 (acute phase) and 30 post-infection (chronic phase). Our study demonstrates that Ifit2 deficiency causes extensive RSA59 spread throughout the spinal cord gray and white matter, associated with impaired CD4+ T and CD8+ T cell infiltration. Further, the cervical lymph nodes of RSA59 infected Ifit2-/- mice showed reduced activation of CD4+ T cells and impaired IFNγ expression during acute encephalomyelitis. Interestingly, BBB integrity was better preserved in Ifit2-/- mice, as evidenced by tight junction protein Claudin-5 and adapter protein ZO-1 expression surrounding the meninges and blood vessels and decreased Texas red dye uptake, which may be responsible for reduced leukocyte infiltration. In contrast to sparse myelin loss in WT mice, the chronic disease phase in Ifit2-/- mice was associated with severe demyelination and persistent viral load, even at low inoculation doses. Overall, our study highlights that Ifit2 provides antiviral functions by promoting acute neuroinflammation and thereby aiding virus control and limiting severe chronic demyelination.

Recommended citation: Madhav Sharma, Debanjana Chakravarty, Ajay Zalavadia, Amy Burrows, Patricia Rayman, Nikhil Sharma, Lawrence C. Kenyon, Cornelia Bergmann, Ganes C. Sen, Jayasri Das Sarma. Ifit2 restricts murine coronavirus spread to the spinal cord white matter and its associated myelin pathology. Journal of Virology. e00749-23. doi:10.1128/jvi.00749-23 https://doi.org/10.1128/jvi.00749-23

The MIF promoter SNP rs755622 is associated with immune activation in glioblastoma

Published in JCI Insight, 2023

Intratumoral heterogeneity is a defining hallmark of glioblastoma, driving drug resistance and ultimately recurrence. Many somatic drivers of microenvironmental change have been shown to affect this heterogeneity and, ultimately, the treatment response. However, little is known about how germline mutations affect the tumoral microenvironment. Here, we find that the single-nucleotide polymorphism (SNP) rs755622 in the promoter of the cytokine macrophage migration inhibitory factor (MIF) is associated with increased leukocyte infiltration in glioblastoma. Furthermore, we identified an association between rs755622 and lactotransferrin expression, which could also be used as a biomarker for immune-infiltrated tumors. These findings demonstrate that a germline SNP in the promoter region of MIF may affect the immune microenvironment and further reveal a link between lactotransferrin and immune activation.

Recommended citation: Alban, T.J., Grabowski, M.M., Otvos, B., Bayik, D., Wang, W., Zalavadia, A., Makarov, V., Troike, K., McGraw, M., Rabljenovic, A. and Lauko, A., 2023. The MIF promoter SNP rs755622 is associated with immune activation in glioblastoma. JCI insight, 8(13). https://doi.org/10.1172/jci.insight.160024

Adaptation to chronic ER stress enforces pancreatic β-cell plasticity

Published in Nature Communications, 2022

Pancreatic β-cells are prone to endoplasmic reticulum (ER) stress due to their role in insulin secretion. They require sustainable and efficient adaptive stress responses to cope with this stress. Whether episodes of chronic stress directly compromise β-cell identity is unknown. We show here under reversible, chronic stress conditions β-cells undergo transcriptional and translational reprogramming associated with impaired expression of regulators of β-cell function and identity. Upon recovery from stress, β-cells regain their identity and function, indicating a high degree of adaptive plasticity. Remarkably, while β-cells show resilience to episodic ER stress, when episodes exceed a threshold, β-cell identity is gradually lost. Single cell RNA-sequencing analysis of islets from type 1 diabetes patients indicates severe deregulation of the chronic stress-adaptation program and reveals novel biomarkers of diabetes progression. Our results suggest β-cell adaptive exhaustion contributes to diabetes pathogenesis.

Recommended citation: Chien-Wen Chen, Bo-Jhih Guan, Mohammed Alzahrani, Zhaofeng Gao, Long Gao, Syrena Bracey, Jing Wu, Cheikh Mbow, Raul Jobava, Leena Haataja, Ajay Zalavadia, Ashleigh Schaffer, Hugo Hugo Lee, Thomas Laframboise, Ilya Bederman, Peter Arvan, Clayton Mathews, Ivan Gerling, Klaus Kaestner, Boaz Tirosh, Feyza Engin, Maria Hatzoglou. Adaptation to chronic ER stress enforces pancreatic β-cell plasticity. Nature Communications, 13, 4621 (2022). https://doi.org/10.1038/s41467-022-32425-7

P-Cadherin Regulates Intestinal Epithelial Cell Migration and Mucosal Repair, but is Dispensable for Colitis Associated Colon Cancer

Published in Cells, 2022

Recurrent chronic mucosal inflammation, a characteristic of inflammatory bowel diseases (IBD), perturbs the intestinal epithelial homeostasis resulting in formation of mucosal wounds and, in most severe cases, leads to colitis-associated colon cancer (CAC). The altered structure of epithelial cell-cell adhesions is a hallmark of intestinal inflammation contributing to epithelial injury, repair, and tumorigenesis. P-cadherin is an important adhesion protein, poorly expressed in normal intestinal epithelial cells (IEC) but upregulated in inflamed and injured mucosa. The goal of this study was to investigate the roles of P-cadherin in regulating intestinal inflammation and CAC. P-cadherin expression was markedly induced in the colonic epithelium of human IBD patients and CAC tissues. The roles of P-cadherin were investigated in P-cadherin null mice using dextran sulfate sodium (DSS)-induced colitis and an azoxymethane (AOM)/DSS induced CAC. Although P-cadherin knockout did not affect the severity of acute DSS colitis, P-cadherin null mice exhibited faster recovery after colitis. No significant differences in the number of colonic tumors were observed in P-cadherin null and control mice. Consistently, the CRISPR/Cas9-mediated knockout of P-cadherin in human IEC accelerated epithelial wound healing without affecting cell proliferation. The accelerated migration of P-cadherin depleted IEC was driven by activation of Src kinases, Rac1 GTPase and myosin II motors and was accompanied by transcriptional reprogramming of the cells. Our findings highlight P-cadherin as a negative regulator of IEC motility in vitro and mucosal repair in vivo. In contrast, this protein is dispensable for IEC proliferation and CAC development.

Recommended citation: Naydenov, N.G.; Lechuga, S.; Zalavadia, A.; Mukherjee, P.K.; Gordon, I.O.; Skvasik, D.; Vidovic, P.; Huang, E.; Rieder, F.; Ivanov, A.I. P-Cadherin Regulates Intestinal Epithelial Cell Migration and Mucosal Repair, but Is Dispensable for Colitis Associated Colon Cancer. Cells 2022, 11, 1467. https://doi.org/10.3390/cells11091467

Cardiac expression of microRNA-7 is associated with adverse cardiac remodeling

Published in Nature - Scientific Reports, 2021

Although microRNA-7 (miRNA-7) is known to regulate proliferation of cancer cells by targeting Epidermal growth factor receptor (EGFR/ERBB) family, less is known about its role in cardiac physiology. Transgenic (Tg) mouse with cardiomyocyte-specific overexpression of miRNA-7 was generated to determine its role in cardiac physiology and pathology. Echocardiography on the miRNA-7 Tg mice showed cardiac dilation instead of age-associated physiological cardiac hypertrophy observed in non-Tg control mice. Subjecting miRNA-7 Tg mice to transverse aortic constriction (TAC) resulted in cardiac dilation associated with increased fibrosis bypassing the adaptive cardiac hypertrophic response to TAC. miRNA-7 expression in cardiomyocytes resulted in significant loss of ERBB2 expression with no changes in ERBB1 (EGFR). Cardiac proteomics in the miRNA-7 Tg mice showed significant reduction in mitochondrial membrane structural proteins compared to NTg reflecting role of miRNA-7 beyond the regulation of EGFR/ERRB in mediating cardiac dilation. Consistently, electron microscopy showed that miRNA-7 Tg hearts had disorganized rounded mitochondria that was associated with mitochondrial dysfunction. These findings show that expression of miRNA-7 in the cardiomyocytes results in cardiac dilation instead of adaptive hypertrophic response during aging or to TAC providing insights on yet to be understood role of miRNA-7 in cardiac function.

Recommended citation: Gupta, M.K., Sahu, A., Sun, Y. et al. Cardiac expression of microRNA-7 is associated with adverse cardiac remodeling. Sci Rep 11, 22018 (2021). https://doi.org/10.1038/s41598-021-00778-6

A Broadly Tunable Surface Plasmon-Coupled Wavelength Filter for Visible and Near Infrared Hyperspectral Imaging

Published in Cleveland State University, College of Sciences and Health Professions., 2018

The development of ultra-compact handheld hyperspectral imagers has been impeded by the scarcity of small widefield tunable wavelength filters. The widefield modality is preferred for handheld imaging applications in which image registration can be performed to counter scene shift caused by irregular user motions that would thwart scanning approaches. Conventional widefield tunable filters like the liquid crystal tunable filter and acousto-optic tunable filter achieve narrow passbands across a wide spectral range by utilizing large interaction lengths, thereby increasing the thickness of the device along the optical path. In addition, these technologies rely on rather bulky external control circuitry and, in the case of acousto-optic filters, high power requirements. In the work presented here, we introduce a novel widefield tunable filter for visible and near infrared imaging based on surface plasmon coupling that can be miniaturized without sacrificing performance. The surface plasmon coupled tunable filter (SPCTF) provides diffraction limited spatial resolution with a <10nm nominal passband and a spurious free spectral range of more than 300nm. Acting on the π-polarized component, the device is limited to transmitting 50 percent of unpolarized incident light. This is higher than the throughput of comparable Lyot-based liquid crystal tunable filters that employ a series of linear polarizers. The design of the SPTF is presented along with a comparison of its performance to calculated estimates of transmittance, spectral resolution, and spectral range.

Recommended citation: Zalavadia, Ajaykumar. "A Broadly Tunable Surface Plasmon-Coupled Wavelength Filter for Visible and Near Infrared Hyperspectral Imaging." Electronic Thesis or Dissertation. Cleveland State University, 2018. OhioLINK Electronic Theses and Dissertations Center. 16 Jun 2020. http://rave.ohiolink.edu/etdc/view?acc_num=csu1522253688346498

A novel surface plasmon coupled tunable wavelength filter for hyperspectral imaging

Published in Proceedings of SPIE, Novel Optical Systems Design and Optimization XX., 2017

Recommended citation: John F. Turner II, and Ajaykumar H. Zalavadia, "A novel surface plasmon coupled tunable wavelength filter for hyperspectral imaging", Proc. SPIE 10376, Novel Optical Systems Design and Optimization XX, 103760A (24 August 2017). https://doi.org/10.1117/12.2274671

Ajay Zalavadia, Ph.D.

Publications