Activation of Intracellular Complement in Lungs of Patients With Severe COVID-19 Disease Decreases T-Cell Activity in the Lungs.

A novel coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), arose late in 2019, with disease pathology ranging from asymptomatic to severe respiratory distress with multi-organ failure requiring mechanical ventilator support. It has been found that SARS-CoV-2 infection drives intracellular complement activation in lung cells that tracks with disease severity. However, the cellular and molecular mechanisms responsible remain unclear. To shed light on the potential mechanisms, we examined publicly available RNA-Sequencing data using CIBERSORTx and conducted a Ingenuity Pathway Analysis to address this knowledge gap. In complement to these findings, we used bioinformatics tools to analyze publicly available RNA sequencing data and found that upregulation of complement may be leading to a downregulation of T-cell activity in lungs of severe COVID-19 patients. Thus, targeting treatments aimed at the modulation of classical complement and T-cell activity may help alleviate the proinflammatory effects of COVID-19, reduce lung pathology, and increase the survival of COVID-19 patients.

Combination Therapy of Mithramycin A and Immune Checkpoint Inhibitor for the Treatment of Colorectal Cancer in an Orthotopic Murine Model.

The axis of Programmed cell death-1 receptor (PD-1) with its ligand (PD-L1) plays a critical role in colorectal cancer (CRC) in escaping immune surveillance, and blocking this axis has been found to be effective in a subset of patients. Although blocking PD-L1 has been shown to be effective in 5-10% of patients, the majority of the cohorts show resistance to this checkpoint blockade (CB) therapy. Multiple factors assist in the growth of resistance to CB, among which T cell exhaustion and immunosuppressive effects of immune cells in the tumor microenvironment (TME) play a critical role along with other tumor intrinsic factors. We have previously shown the polyketide antibiotic, Mithramycin-A (Mit-A), an effective agent in killing cancer stem cells (CSCs) in vitro and in vivo in a subcutaneous murine model. Since TME plays a pivotal role in CB therapy, we tested the immunomodulatory efficacy of Mit-A with anti-PD-L1 mAb (αPD-L1) combination therapy in an immunocompetent MC38 syngeneic orthotopic CRC mouse model. Tumors and spleens were analyzed by flow cytometry for the distinct immune cell populations affected by the treatment, in addition to RT-PCR for tumor samples. We demonstrated the combination treatment decreases tumor growth, thus increasing the effectiveness of the CB. Mit-A in the presence of αPD-L1 significantly increased CD8+ T cell infiltration and decreased immunosuppressive granulocytic myeloid-derived suppressor cells and anti-inflammatory macrophages in the TME. Our results revealed Mit-A in combination with αPD-L1 has the potential for augmented CB therapy by turning an immunologically "cold" into "hot" TME in CRC.

SARS-CoV-2-Induced Gut Microbiome Dysbiosis: Implications for Colorectal Cancer.

The emergence of a novel coronavirus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), in December 2019 led to a worldwide pandemic with over 170 million confirmed infections and over 3.5 million deaths (as of May 2021). Early studies have shown higher mortality rates from SARS-CoV-2 infection in cancer patients than individuals without cancer. Herein, we review the evidence that the gut microbiota plays a crucial role in health and has been linked to the development of colorectal cancer (CRC). Investigations have shown that SARS-CoV-2 infection causes changes to the gut microbiota, including an overall decline in microbial diversity, enrichment of opportunistic pathogens such as Fusobacterium nucleatum bacteremia, and depletion of beneficial commensals, such as the butyrate-producing bacteria. Further, these changes lead to increased colonic inflammation, which leads to gut barrier disruption, expression of genes governing CRC tumorigenesis, and tumor immunosuppression, thus further exacerbating CRC progression. Additionally, a long-lasting impact of SARS-CoV-2 on gut dysbiosis might result in a greater possibility of new CRC diagnosis or aggravating the condition in those already afflicted. Herein, we review the evidence relating to the current understanding of how infection with SARS-CoV-2 impacts the gut microbiota and the effects this will have on CRC carcinogenesis and progression.

SARS-CoV-2 Immuno-Pathogenesis and Potential for Diverse Vaccines and Therapies: Opportunities and Challenges.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is a novel coronavirus that emerged from Wuhan, China in late 2019 causing coronavirus disease-19 (COVID-19). SARS-CoV-2 infection begins by attaching to angiotensin-converting enzyme 2 receptor (ACE2) via the spike glycoprotein, followed by cleavage by TMPRSS2, revealing the viral fusion domain. Other presumptive receptors for SARS-CoV-2 attachment include CD147, neuropilin-1 (NRP1), and Myeloid C-lectin like receptor (CLR), each of which might play a role in the systemic viral spread. The pathology of SARS-CoV-2 infection ranges from asymptomatic to severe acute respiratory distress syndrome, often displaying a cytokine storm syndrome, which can be life-threatening. Despite progress made, the detailed mechanisms underlying SARS-CoV-2 interaction with the host immune system remain unclear and are an area of very active research. The process's key players include viral non-structural proteins and open reading frame products, which have been implicated in immune antagonism. The dysregulation of the innate immune system results in reduced adaptive immune responses characterized by rapidly diminishing antibody titers. Several treatment options for COVID-19 are emerging, with immunotherapies, peptide therapies, and nucleic acid vaccines showing promise. This review discusses the advances in the immunopathology of SARS-CoV-2, vaccines and therapies under investigation to counter the effects of this virus, as well as viral variants.

The design and characterization of a gravitational microfluidic platform for drug sensitivity assay in colorectal perfused tumoroid cultures.

The tumor microenvironment plays a critical role in tumor initiation, progression, metastasis, and drug resistance. However, models recapitulating the complex 3D structure, heterogeneous cell environment, and cell-cell interactions found in vivo are lacking. Herein, we report on a gravitational microfluidic platform (GMP) retrofitted with MEMS sensors, which is integrated with 3D nanofiber scaffold-aided tumoroid culture. The results showed that this GMP for tumoroid growth mimics the tumor microenvironment more precisely than static culture models of colon cancer, including higher drug resistance, enhanced cancer stem cell properties, and increased secretion of pro-tumor cytokines. In addition, the GMP includes an integrated surface acoustic wave-based biosensing to monitor cell growth and pH changes to assess drug efficacy. Thus, this simple-to-use perfused GMP tumoroid culture system for in vitro and ex vivo studies may accelerate the drug development process and be a tool in personalized cancer treatment.

Advances in Translational Nanotechnology: Challenges and Opportunities.

The burgeoning field of nanotechnology aims to create and deploy nanoscale structures, devices, and systems with novel, size-dependent properties and functions. The nanotechnology revolution has sparked radically new technologies and strategies across all scientific disciplines, with nanotechnology now applied to virtually every area of research and development in the US and globally. NanoFlorida was founded to create a forum for scientific exchange, promote networking among nanoscientists, encourage collaborative research efforts across institutions, forge strong industry-academia partnerships in nanoscience, and showcase the contributions of students and trainees in nanotechnology fields. The 2019 NanoFlorida International Conference expanded this vision to emphasize national and international participation, with a focus on advances made in translating nanotechnology. This review highlights notable research in the areas of engineering especially in optics, photonics and plasmonics and electronics; biomedical devices, nano-biotechnology, nanotherapeutics including both experimental nanotherapies and nanovaccines; nano-diagnostics and -theranostics; nano-enabled drug discovery platforms; tissue engineering, bioprinting, and environmental nanotechnology, as well as challenges and directions for future research.

Withania Somnifera (Ashwagandha) and Withaferin A: Potential in Integrative Oncology.

Ashwagandha (Withania Somnifera, WS), belonging to the family Solanaceae, is an Ayurvedic herb known worldwide for its numerous beneficial health activities since ancient times. This medicinal plant provides benefits against many human illnesses such as epilepsy, depression, arthritis, diabetes, and palliative effects such as analgesic, rejuvenating, regenerating, and growth-promoting effects. Several clinical trials of the different parts of the herb have demonstrated safety in patients suffering from these diseases. In the last two decades, an active component of Withaferin A (WFA) has shown tremendous cytotoxic activity suggesting its potential as an anti-carcinogenic agent in treatment of several cancers. In spite of enormous progress, a thorough elaboration of the proposed mechanism and mode of action is absent. Herein, we provide a comprehensive review of the properties of WS extracts (WSE) containing complex mixtures of diverse components including WFA, which have shown inhibitory properties against many cancers, (breast, colon, prostate, colon, ovarian, lung, brain), along with their mechanism of actions and pathways involved.

Mithramycin A Inhibits Colorectal Cancer Growth by Targeting Cancer Stem Cells.

The pivotal role of cancer initiating stem cells (CSCs) in tumor initiation, growth, metastasis and drug resistance has led to the postulation of a 'total cancer therapy' paradigm, which involves targeting both cancer cells and CSCs for effective therapy. However, the progress in identifying drugs for total cancer therapy has been limited. Herein, we show for the first time that mithramycin A (Mit-A) can successfully inhibit CSC proliferation, in addition to inhibiting bulk cancer cells in a model of colorectal cancer (CRC), the second leading cause of death among men and women in the United States. To this end, a polymeric nanofiber scaffold culture system was established to develop 3D tumor organoids (tumoroids) from CRC cell lines such as HT29, HCT116, KM12, CT26 and MC38 as well as ex vivo mouse tumors. These tumoroids possessed increased expression of CSC markers and transcription factors, expanded the number of CSCs in culture and increased CSC functional properties measured by aldehyde dehydrogenase activity. Screening of an NCI library of FDA approved drugs led to the identification of Mit-A as a potential total cancer therapy drug. In both sphere and tumoroid culture, Mit-A inhibits cancer growth by reducing the expression of cancer stemness markers. In addition, Mit-A inhibits the expression of SP1, a previously known target in CRCs. Moreover, Mit-A significantly reduces growth of tumoroids in ex vivo cultures and CRC tumor growth in vivo. Finally, a dose-dependent treatment on CRC cells indicate that Mit-A significantly induces the cell death and PARP-cleavage of both CSC and non-CSC cells. Taken together the results of these in vitro, ex vivo and in vivo studies lead to the inference that Mit-A is a promising drug candidate for total cancer therapy of CRCs.

Multimodal Theranostic Nanoformulations Permit Magnetic Resonance Bioimaging of Antiretroviral Drug Particle Tissue-Cell Biodistribution.

RATIONALE: Long-acting slow effective release antiretroviral therapy (LASER ART) was developed to improve patient regimen adherence, prevent new infections, and facilitate drug delivery to human immunodeficiency virus cell and tissue reservoirs. In an effort to facilitate LASER ART development, "multimodal imaging theranostic nanoprobes" were created. These allow combined bioimaging, drug pharmacokinetics and tissue biodistribution tests in animal models. METHODS: Europium (Eu3+)- doped cobalt ferrite (CF) dolutegravir (DTG)- loaded (EuCF-DTG) nanoparticles were synthesized then fully characterized based on their size, shape and stability. These were then used as platforms for nanoformulated drug biodistribution. RESULTS: Folic acid (FA) decoration of EuCF-DTG (FA-EuCF-DTG) nanoparticles facilitated macrophage targeting and sped drug entry across cell barriers. Macrophage uptake was higher for FA-EuCF-DTG than EuCF-DTG nanoparticles with relaxivities of r2 = 546 mM-1s-1 and r2 = 564 mM-1s-1 in saline, and r2 = 850 mM-1s-1 and r2 = 876 mM-1s-1 in cells, respectively. The values were ten or more times higher than what was observed for ultrasmall superparamagnetic iron oxide particles (r2 = 31.15 mM-1s-1 in saline) using identical iron concentrations. Drug particles were detected in macrophage Rab compartments by dual fluorescence labeling. Replicate particles elicited sustained antiretroviral responses. After parenteral injection of FA-EuCF-DTG and EuCF-DTG into rats and rhesus macaques, drug, iron and cobalt levels, measured by LC-MS/MS, magnetic resonance imaging, and ICP-MS were coordinate. CONCLUSION: We posit that these theranostic nanoprobes can assess LASER ART drug delivery and be used as part of a precision nanomedicine therapeutic strategy.

Recent advances in hepatocellular carcinoma therapy.

Hepatocellular carcinoma (HCC), also called malignant hepatoma, is one of the deadliest cancers due to its complexities, reoccurrence after surgical resection, metastasis and heterogeneity. Incidence and mortality of HCC are increasing in Western countries and are expected to rise as a consequence of the obesity epidemic. Multiple factors trigger the initiation and progression of HCC including chronic alcohol consumption, viral hepatitis B and C infection, metabolic disorders and age. Although Sorafenib is the only FDA approved drug for the treatment of HCC, numerous treatment modalities such as transcatheter arterial chemoembolization/transarterial chemoembolization (TACE), radiotherapy, locoregional therapy and chemotherapy have been tested in the clinics. Polymeric nanoparticles, liposomes, and micelles carrying small molecules, proteins, peptides and nucleic acids have attracted great attention for the treatment of various cancers including HCC. Herein, we discuss the pathogenesis of HCC in relation to its various recent treatment methodologies using nanodelivery of monoclonal antibodies (mAbs), small molecules, miRNAs and peptides. Synopsis of recent clinical trials of mAbs and peptide drugs has been presented with a broad overview of the pathogenesis of the disease and treatment efficacy.

Pharmacokinetics and Biodistribution of GDC-0449 Loaded Micelles in Normal and Liver Fibrotic Mice.

PURPOSES: To determine the pharmacokinetic parameters and biodistribution of GDC-0449 loaded polymeric micelles after systemic administration into common bile duct ligation (CBDL) induced liver fibrotic mice. METHODS: We used GDC-0449 encapsulated methoxy poly (ethylene glycol)-block-poly (2-methyl-2-carboxyl-propylene carbonate)-graft-dodecanol (PEG-PCD) non-targeted polymeric micelles for GDC-0449 delivery to normal and liver fibrotic mice. To maximize GDC-0449 delivery to hepatic stellate cells (HSCs), mixed micelles formulations with 10, 20 and 30% w/w mannose-6-phosphate (M6P)-conjugated micelles were administered to normal and liver fibrotic mice for targeting M6P/IGF-IIR overexpressed on activated HSCs and biodistribution of GDC-0449 was determined at 30 and 120 min post systemic administration. RESULTS: GDC-0449 distributed to all major organs after systemic administration of drug loaded micelles, with higher accumulation in the liver of both normal and fibrotic mice. The plasma concentration versus time profiles suggest rapid clearance of GDC-0449 after systemic administration of drug loaded micelles in both normal and fibrotic mice, with similar plasma clearance (CL), area under the curve (AUCint) and volume of distribution at steady state (Vss). However, there is significant increase in GDC-0449 accumulation in the liver when M6P-conjugated mixed micelles were injected, with the highest GDC-0449 concentration in the liver with mixed micelles carrying 30% M6P-conjugated polymer. HSCs accounted for 14.19% of GDC-0449 accumulation for M6P-targeted micelles in fibrotic mice compared to 5.62% of non-targeted micelles in the liver uptake study. CONCLUSION: M6P-conjugated GDC-0449 loaded mixed micelles may be used as a potential drug delivery vehicle for treating liver fibrosis.

Micelle Mixtures for Coadministration of Gemcitabine and GDC-0449 To Treat Pancreatic Cancer.

Hedgehog (Hh) signaling plays an important role in the development and metastasis of pancreatic ductal adenocarcinoma (PDAC). Although gemcitabine (GEM) has been used as a first-line therapy for PDAC, its rapid metabolism and short plasma half-life restrict its use as a single chemotherapy. Combination therapy with more than one drug is a promising approach for treating cancer. Herein, we report the use of methoxy poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate)-graft-dodecanol (mPEG-b-PCC-g-DC) copolymer for conjugating GEM and encapsulating a Hh inhibitor, vismodegib (GDC-0449), into its hydrophobic core for treating PDAC. Our objective was to determine whether the micelle mixtures of these two drugs could show better response in inhibiting Hh signaling pathway and restraining the proliferation and metastasis of pancreatic cancer. The in vivo stability of GEM significantly increased after conjugation, which resulted in its increased antitumor efficacy. Almost 80% of encapsulated GDC-0449 and 19% conjugated GEM were released in vitro at pH 5.5 in 48 h in a sustained manner. The invasion, migration, and colony forming features of MIA PaCa-2 cells were significantly inhibited by micelle mixture carrying GEM and GDC-0449. Remarkable increase in PARP cleavage and Bax proved increased apoptosis by this combination formulation compared to individual micelles. This combination therapy efficiently inhibited tumor growth, increased apoptosis, reduced Hh ligands PTCH-1 and Gli-1, and lowered EMT-activator ZEB-1 when injected to athymic nude mice bearing subcutaneous tumor generated using MIA PaCa-2 cells compared to monotherapy as observed from immunohistochemical analysis. In conclusion, micelle mixtures carrying GEM and GDC-0449 have the potential to treat pancreatic cancer.

Co-delivery of small molecule hedgehog inhibitor and miRNA for treating liver fibrosis.

In liver fibrosis, secretion of growth factors and hedgehog (Hh) ligands by hepatic parenchyma upon repeated insults results in transdifferentiation of quiescent hepatic stellate cells (HSCs) into active myofibroblasts which secrete excessive amounts of extracellular matrix (ECM) proteins. An Hh inhibitor GDC-0449 and miR-29b1 can play an important role in treating liver fibrosis by inhibiting several pro-fibrotic genes. Our in-silico analysis indicate that miR-29b1 targets several profibrotic genes like collagen type I & IV, c-MYC, PDGF-ß and PI3K/AKT which are upregulated in liver fibrosis. Common bile duct ligation (CBDL) resulted in an increase in Ptch-1, Shh and Gli-1 expression. miR-29b1 and GDC-0449 were co-formulated into micelles using methoxy poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft-dodecanol-graft-tetraethylenepentamine) (mPEG-b-PCC-g-DC-g-TEPA) copolymer, and injected systemically into CBDL mice. High concentrations of GDC-0449 and miR-29b1 were delivered to liver cells as determined by in situ liver perfusion at 30 min post systemic administration of their micelle formulation. There was a significant decrease in collagen deposition in the liver and serum injury markers, leading to improvement in liver morphology. Combination therapy was more effective in providing hepatoprotection, lowering liver injury related serum enzyme levels, reducing fibrotic protein markers such as collagen, α-SMA, FN-1 and p-AKT compared to monotherapy. In conclusion, inhibition of Hh pathway and restoration of miR-29b1 have the potential to act synergistically in treating CBDL-induced liver fibrosis in mice.

Fluorescent polymer-based post-translational differentiation and subtyping of breast cancer cells.

Herein, we report the application of synthesized fluorescent, water soluble polymers for post-translational subtyping and differentiation of breast cancer cells in vitro. The fluorescence emission spectra from these polymers were modulated differently in the presence of conditioned cell culture media from various breast cancer cells. These polymers differentiate at a post-translation level possibly due to their ability to interact with extracellular enzymes that are over-expressed in cancerous conditions.

Differentiation of prostate cancer cells using flexible fluorescent polymers.

Using water-soluble, fluorescent, flexible polymers, we have devised a novel methodology for identification and differentiation of prostate cancer cells. Using a stepwise linear discriminant analysis, we demonstrate that the differential modulations of the polymer emission intensities in the presence of conditioned cell culture media can be used to distinguish between prostate cancer subtypes and between cancerous and noncancer cells. The differences in the compositions of the conditioned cell culture media are likely contributing to different fluorescence spectral patterns of the polymers. This in vitro approach may provide a novel platform for the development of an alternative prostate cancer diagnostic and subtyping technique.

Fluorescent water soluble polymers for isozyme-selective interactions with matrix metalloproteinase-9.

Matrix metalloproteinases (MMPs) are overexpressed in various pathological conditions, including cancers. Although these isozymes have similar active sites, the patterns of exposed amino acids on their surfaces are different. Herein, we report the synthesis and molecular interactions of two water soluble, fluorescent polymers which demonstrate selective interactions with MMP-9 compared to MMP-7 and -10.