Pandemic Response Requires Research Samples: A U.S. Safety-Net Hospital's Experience and Call for National Action.

Biorepositories provide a critical resource for gaining knowledge of emerging infectious diseases and offer a mechanism to rapidly respond to outbreaks; the emergence of the novel coronavirus, SARS-CoV-2, has proved their importance. During the COVID-19 pandemic, the absence of centralized, national biorepository efforts meant that the onus fell on individual institutions to establish sample repositories. As a safety-net hospital, Boston Medical Center (BMC) recognized the importance of creating a COVID-19 biorepository to both support critical science at BMC and ensure representation in research for its urban patient population, most of whom are from underserved communities. This article offers a realistic overview of the authors' experience in establishing this biorepository at the onset of the COVID-19 pandemic during the height of the first surge of cases in Boston, Massachusetts, with the hope that the challenges and solutions described are useful to other institutions. Going forward, funders, policymakers, and infectious disease and public health communities must support biorepository implementation as an essential element of future pandemic preparedness.

On-Site, On-Demand 3D-Printed Nasopharyngeal Swabs to Improve the Access of Coronavirus Disease-19 Testing.

Diagnostic testing that facilitates containment, surveillance, and treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or future respiratory viruses, depends on a sample collection device that efficiently collects nasopharyngeal tissue and that can be manufactured on site when an outbreak or public health emergency is declared by a government. Here two novel stereolithography-based three-dimensional (3D)-printed nasopharyngeal swabs are reported which are made using a biocompatible and sterilizable photoresist. Such swabs are readily manufactured on-site and on-demand to ensure availability, if supply chain shortages emerge. Additionally, the 3D-printed swabs easily adapt to current workflow and testing procedures in hospital clinical laboratories to allow for effortless scaling up of test kits. Finally, the 3D-printed nasopharyngeal swabs demonstrate concordant SARS-CoV-2 testing results between the 3D-printed swabs and the COPAN commercial swabs, and enable detection of SARS-CoV-2 in clinical samples obtained from autopsies.

qRT-PCR Platforms for Diagnosing and Reporting SARS-CoV-2 Infection in Human Samples.

The protocols herein outline the use of qRT-PCR to detect the presence of SARS-CoV-2 genomic RNA in patient samples. In order to cope with potential fluctuations in supply chain and testing demands and to enable expedient adaptation of reagents and assays on hand, we include details for three parallel methodologies (one- and two-step singleplex and one-step multiplex assays). The diagnostic platforms described can be easily adapted by basic science research laboratories for SARS-CoV-2 diagnostic testing with relatively short turnaround time. For complete details on the use and execution of this protocol, please refer to Vanuytsel et al. (2020).

Impact of specific preclinical variables on coagulation biomarkers in cancer-associated thrombosis.

Coagulation biomarkers are being actively studied for their diagnostic and prognostic value in patients with venous thromboembolism and cancer, as well as in the study of pathogenic mechanisms between cancer and thrombosis. For the results of such studies to be accurate and reproducible, attention must be paid to minimize sources of error in all phases of testing. The pre-analytical phase of laboratory testing is known to be fraught with the majority of errors. Coagulation testing is particularly susceptible to conditions during collection, processing, transport and storage of specimens which can lead to clinically significant errors in results. In addition, changes in pre-analytical conditions can impact different biomarkers differently. Therefore, research studies investigating coagulation biomarkers must carefully standardize not just the analytical phase, but also the pre-analytical phase of testing to ensure accuracy and reliability. We briefly review the impact of pre-analytical conditions on coagulation testing in general, and on specific biomarkers in cancer and thrombosis. In addition, we provide recommendations to reduce pre-analytical errors by developing and sharing standard operating procedures that specifically target standardization of methodologies for collecting specimens and measuring current and emerging coagulation biomarkers in cancer studies.

Rapid Implementation of a SARS-CoV-2 Diagnostic Quantitative Real-Time PCR Test with Emergency Use Authorization at a Large Academic Safety Net Hospital.

BACKGROUND: Significant delays in the rapid development and distribution of diagnostic testing for SARS-CoV-2 (COVID-19) infection have prevented adequate public health management of the disease, impacting the timely mapping of viral spread and the conservation of personal protective equipment. Furthermore, vulnerable populations, such as those served by the Boston Medical Center (BMC), the largest safety net hospital in New England, represent a high-risk group across multiple dimensions, including a higher prevalence of pre-existing conditions and substance use disorders, lower health maintenance, unstable housing, and a propensity for rapid community spread, highlighting the urgent need for expedient and reliable in-house testing. METHODS: We developed a SARS-CoV-2 diagnostic medium-throughput qRT-PCR assay with rapid turnaround time and utilized this Clinical Laboratory Improvement Amendments (CLIA)-certified assay for testing nasopharyngeal swab samples from BMC patients, with emergency authorization from the Food and Drug Administration (FDA) and the Massachusetts Department of Public Health. FINDINGS: The in-house testing platform displayed robust accuracy and reliability in validation studies and reduced institutional sample turnaround time from 5-7 days to less than 24 h. Of over 1,000 unique patient samples tested, 44.1% were positive for SARS-CoV-2 infection. CONCLUSIONS: This work provides a blueprint for academic centers and community hospitals lacking automated laboratory machinery to implement rapid in-house testing. FUNDING: This study was supported by funding from the Boston University School of Medicine, the National Institutes of Health, Boston Medical Center, and the Massachusetts Consortium on Pathogen Readiness (MASS CPR).

Metabolites in a mouse cancer model enhance venous thrombogenicity through the aryl hydrocarbon receptor-tissue factor axis.

Patients with malignancy are at 4- to 7-fold higher risk of venous thromboembolism (VTE), a potentially fatal, yet preventable complication. Although general mechanisms of thrombosis are enhanced in these patients, malignancy-specific triggers and their therapeutic implication remain poorly understood. Here we examined a colon cancer-specific VTE model and probed a set of metabolites with prothrombotic propensity in the inferior vena cava (IVC) ligation model. Athymic mice injected with human colon adenocarcinoma cells exhibited significantly higher IVC clot weights, a biological readout of venous thrombogenicity, compared with the control mice. Targeted metabolomics analysis of plasma of mice revealed an increase in the blood levels of kynurenine and indoxyl sulfate (tryptophan metabolites) in xenograft-bearing mice, which correlated positively with the increase in the IVC clot size. These metabolites are ligands of aryl hydrocarbon receptor (AHR) signaling. Accordingly, plasma from the xenograft-bearing mice activated the AHR pathway and augmented tissue factor (TF) and plasminogen activator inhibitor 1 (PAI-1) levels in venous endothelial cells in an AHR-dependent manner. Consistent with these findings, the endothelium from the IVC of xenograft-bearing animals revealed nuclear AHR and upregulated TF and PAI-1 expression, telltale signs of an activated AHR-TF/PAI-1 axis. Importantly, pharmacological inhibition of AHR activity suppressed TF and PAI-1 expression in endothelial cells of the IVC and reduced clot weights in both kynurenine-injected and xenograft-bearing mice. Together, these data show dysregulated tryptophan metabolites in a mouse cancer model, and they reveal a novel link between these metabolites and the control of the AHR-TF/PAI-1 axis and VTE in cancer.

Broad Consent for Research on Biospecimens: The Views of Actual Donors at Four U.S. Medical Centers.

Commentators are concerned that broad consent may not provide biospecimen donors with sufficient information regarding possible future research uses of their tissue. We surveyed with interviews 302 cancer patients who had recently provided broad consent at four diverse academic medical centers. The majority of donors believed that the consent form provided them with sufficient information regarding future possible uses of their biospecimens. Donors expressed very positive views regarding tissue donation in general and endorsed the use of their biospecimens in future research across a wide range of contexts. Concerns regarding future uses were limited to for-profit research and research by investigators in other countries. These results support the use of broad consent to store and use biological samples in future research.

The effect of mycophenolate mofetil on disease development in the gld.apoE (-/-) mouse model of accelerated atherosclerosis and systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that is characterized by autoantibody production and inflammatory disease involving multiple organs. Premature atherosclerosis is a common complication of SLE and results in substantial morbidity and mortality from cardiovascular disease (CVD). The reasons for the premature atherosclerosis in SLE are incompletely understood, although chronic inflammation is thought to play an important role. There is currently no known preventative treatment of premature atherosclerosis in SLE. Mycophenolate mofetil (MMF) is an immunosuppressive agent that is commonly used for treatment of patients with SLE. In order to study the impact of this drug on murine lupus disease including premature atherosclerosis development, we treated gld.apoE(-/-) mice, a model of SLE and accelerated atherosclerosis, with MMF. We maintained seven-week old gld.apoE(-/-) mice on a high cholesterol Western diet with or without MMF. After 12 weeks on diet, mice receiving MMF showed decreased atherosclerotic lesion area compared to the control group. MMF treatment also improved the lupus phenotype, indicated by a significant decrease circulating autoantibody levels and ameliorating lupus nephritis associated with this model. This data suggests that the effects of MMF on the immune system may not only be beneficial for lupus, but also for inflammation driving lupus-associated atherosclerosis.

Rapamycin delays but does not prevent recovery from acute renal failure: role of acquired tubular resistance.

BACKGROUND: We reported that rapamycin impairs recovery after acute renal failure (ARF) in rats. The objective of this study was to determine if recovery will eventually occur after ARF despite continued rapamycin treatment. METHODS: ARF was induced in rats by renal artery occlusion. Glomerular filtration rate (GFR), morphology, and tubular cell proliferation were assessed either 2, 4, 6, or 7 days later. Rats were treated daily with rapamycin or vehicle throughout the study. Cultured mouse proximal tubular (MPT) cells were used to compare the antiproliferative effects of rapamycin after exposure for 1 and 7 days. RESULTS: Two days after ARF, GFR was reduced severely but comparably in vehicle and rapamycin rats. In controls, GFR began to increase after day 2 and was normal by day 6. In rapamycin rats, GFR did begin to improve until after day 4 and reached normal values by day 7. In controls, many proliferating tubular cells were present in outer medulla on day 2, after which proliferation progressively decreased. By contrast, in rapamycin rats, proliferating cells were sparse on day 2, but then increased substantially through days 4 and 6. Cultured MPT cells exposed to rapamycin for 7 days were approximately 10-fold more resistant to the antiproliferative effects of rapamycin than cells exposed for 1 day. CONCLUSIONS: Rapamycin delays but does not prevent renal recovery after ARF. MPT cells become resistant to rapamycin after prolonged exposure. We speculate that the ultimate recovery of renal function after ARF is due to the development of acquired tubular cell resistance to rapamycin.

Rapamycin ameliorates proteinuria-associated tubulointerstitial inflammation and fibrosis in experimental membranous nephropathy.

Proteinuria is a risk factor for progression of chronic renal failure. A model of proteinuria-associated tubulointerstitial injury was developed and was used to examine the therapeutic effect of rapamycin. Two studies were performed. In study A, proteinuric rats were given sheep anti-Fx1A to induce experimental membranous nephropathy; control rats received normal sheep serum. Four weeks later, groups were subdivided and underwent laparotomy alone (two kidneys), nephrectomy alone (one kidney), or nephrectomy with polectomy (0.6 kidney). Renal function and morphology were evaluated 4 wk later. Whereas control rats never developed proteinuria, anti-Fx1A induced severe proteinuria. Proteinuria was unaffected by renal mass reduction. Proteinuric rats developed tubulointerstitial disease that was most severe in rats with 0.6 kidneys. Renal function (GFR) was reduced by loss of renal mass and was reduced further in proteinuric rats with 0.6 kidneys. In study B, the effect of rapamycin on the expression of candidate proinflammatory and profibrotic genes and the progression of proteinuria-associated renal disease were examined. All rats received an injection of anti-Fx1A and were nephrectomized and then divided into groups to receive rapamycin or vehicle. Gene expression, renal morphology, and GFR were evaluated after 4, 8, and 12 wk. Rapamycin reduced expression of the proinflammatory and profibrotic genes (monocyte chemotactic protein-1, vascular endothelial growth factor, PDGF, TGF-beta(1), and type 1 collagen). Tubulointerstitial inflammation and progression of interstitial fibrosis that were present in vehicle-treated rats were ameliorated by rapamycin. Rapamycin also completely inhibited compensatory renal hypertrophy. In summary, rapamycin ameliorates the tubulointerstitial disease associated with chronic proteinuria and loss of renal mass.

NF-kappaB activation precedes increases in mRNA encoding neurokinin-1 receptor, proinflammatory cytokines, and adhesion molecules in dextran sulfate sodium-induced colitis in rats.

Nuclear factor kappa B (NF-kappa B) plays a key role in initiating inflammation associated with colitis. A systematic study was conducted in the rat DSS colitis model to determine the temporal relationship between NF-kappa B activation and expression of substance P (SP), neurokinin-1 receptor (NK-1R), proinflammatory cytokines, and adhesion molecules. Rats were given 5% DSS in their water and sacrificed daily for 6 days. Colon tissue was collected for assessment of histological changes, NF-kappa B activation, myeloperoxidase (MPO) activity, and expression of NK-1R, SP, TNFalpha, IL-1beta, VCAM-1, ICAM-1, E-selectin, CINC-1, MIP-1alpha, and iNOS. NF-kappa B activation increased, biphasically, on Day 1 and again on Days 4-6. The mRNA levels for ICAM-1, CINC-1, IL-1beta, TNFalpha, VCAM-1, and NK-1R rose significantly (P < 0.05) by 2-4 days. Increased iNOS mRNA levels, MPO activity, and mucosal damage occurred on Day 6. These data demonstrate that NF-kappa B activation substantially precedes the onset of physical disease signs and active inflammation.

A neurokinin 1 receptor antagonist reduces an ongoing ileal pouch inflammation and the response to a subsequent inflammatory stimulus.

Ileal pouch-anal anastomosis (IPAA) is an excellent surgical option for patients with chronic ulcerative colitis (CUC) requiring colectomy; however, persistent episodes of ileal pouch inflammation, or pouchitis, may result in debilitating postoperative complications. Because considerable evidence implicates substance P (SP) as an inflammatory mediator of CUC, we investigated whether SP participates in the pathophysiology of pouchitis. With the use of a rat model of IPAA that we developed, we showed that ileal pouch MPO levels and neurokinin 1 receptor (NK-1R) protein expression by Western blot analysis were significantly elevated 28 days after IPAA surgery. In situ hybridization and immunohistochemistry showed that the increase in NK-1R protein expression was localized to the lamina propria and epithelia of pouch ileum. The intraperitoneal administration of the NK-1R antagonist (NK-1RA) CJ-12,255 for 4 days, starting on day 28, was effective in reducing MPO levels. Starting on day 28, animals with IPAA were given 5% dextran sulfate sodium (DSS) in their drinking water for 4 days, which caused histological and physical signs of clinical pouchitis concomitant with significant increases in ileal pouch MPO concentrations as well as NK-1R protein expression by Western blot analysis. In situ hybridization and immunohistochemistry showed that the increase in NK-1R protein expression was especially evident in crypt epithelia of pouch ileum. When the NK-1RA was administered 1 day before starting DSS and continued for the duration of DSS administration, the physical signs of clinical pouchitis and the rise in MPO were prevented. These data implicate SP in the pathophysiology of pouchitis and suggest that NK-1RA may be of therapeutic value in the management of clinical pouchitis.