Microfluidic Immunoarray for Point-of-Care Detection of Cytokines in COVID-19 Patients.

The "cytokine storm" often induced in COVID-19 patients contributes to the onset of "acute respiratory distress syndrome" (ARDS) accompanied by lung infection and damage, multiorgan failure, and even death. This large increase in pro-inflammatory cytokines in blood may be related to severity. Rapid, on-demand cytokine analyses can thus be critical to inform treatment plans and improve survival rates. Here, we report a sensitive, low-cost, semiautomated 3D-printed microfluidic immunoarray to detect 2 cytokines and CRP simultaneously in a single 10 µL serum sample in 25 min. Accuracy was validated by analyzing 80 COVID-19 patient serum samples, with results well correlated to a commercial Meso Scale protein immunoassay. Capture antibodies immobilized in detection microwells in a flat well plate-type flow chamber facilitate the immunoassay, with a programmable syringe pump automatically delivering reagents. Chemiluminescence signals were captured in a dark box with a CCD camera integrated for 30 s. This system was optimized to detect inflammation biomarkers IL-6, IFN-γ, and CRP simultaneously in blood serum. Ultralow limits of detection (LODs) of 0.79 fg/mL for IL-6, 4.2 fg/mL for CRP, and 2.7 fg/mL for IFN-γ with dynamic ranges of up to 100 pg/mL were achieved. ROC statistical analyses showed a relatively good diagnostic value related to the samples assigned WHO COVID-19 scores for disease severity, with the best results for IL-6 and CRP. Monitoring these biomarkers for coronavirus severity may allow prediction of disease severity as a basis for critical treatment decisions and better survival rates.

Decreased progenitor TCF1 + T-cells correlate with COVID-19 disease severity.

COVID-19, caused by SARS-CoV-2, can lead to a severe inflammatory disease characterized by significant lymphopenia. However, the underlying cause for the depletion of T-cells in COVID-19 patients remains incompletely understood. In this study, we assessed the presence of different T-cell subsets in the progression of COVID-19 from mild to severe disease, with a focus on TCF1 expressing progenitor T-cells that are needed to replenish peripheral T-cells during infection. Our results showed a preferential decline in TCF1+ progenitor CD4 and CD8+ T-cells with disease severity. This decline was seen in various TCF1+ subsets including naive, memory and effector-memory cells, and surprisingly, was accompanied by a loss in cell division as seen by a marked decline in Ki67 expression. In addition, TCF1+ T-cells showed a reduction in pro-survival regulator, BcL2, and the appearance of a new population of TCF1 negative caspase-3 expressing cells in peripheral blood from patients with severe disease. The decline in TCF1+ T-cells was also seen in a subgroup of severe patients with vitamin D deficiency. Lastly, we found that sera from severe patients inhibited TCF1 transcription ex vivo which was attenuated by a blocking antibody against the cytokine, interleukin-12 (IL12). Collectively, our findings underscore the potential significance of TCF1+ progenitor T-cells in accounting for the loss of immunity in severe COVID-19 and outline an array of markers that could be used to identify disease progression.

Fat as a Friend or Foe of the Bone.

PURPOSE OF REVIEW: The objective of this review is to summarize the literature on the prevalence and diagnosis of obesity and its metabolic profile, including bone metabolism, focusing on the main inflammatory and turnover bone mediators that better characterize metabolically healthy obesity phenotype, and to summarize the therapeutic interventions for obesity with their effects on bone health. RECENT FINDINGS: Osteoporosis and fracture risk not only increase with age and menopause but also with metabolic diseases, such as diabetes mellitus. Thus, patients with high BMI may have a higher bone fragility and fracture risk. However, some obese individuals with healthy metabolic profiles seem to be less at risk of bone fracture. Obesity has become an alarming disease with growing prevalence and multiple metabolic comorbidities, resulting in a significant burden on healthcare and increased mortality. The imbalance between increased food ingestion and decreased energy expenditure leads to pathological adipose tissue distribution and function, with increased secretion of proinflammatory markers and harmful consequences for body tissues, including bone tissue. However, some obese individuals seem to have a healthy metabolic profile and may not develop cardiometabolic disease during their lives. This healthy metabolic profile also benefits bone turnover and is associated with lower fracture risk.

Levels of Angiotensin and Kinin Metabolite Peptides Related to COVID-19 Severity.

In addition to crucial roles in normal human biology, peptide metabolites of the renin-angiotensin (RAS) and kallikrein-kinin systems (KKS) have been reported to be altered in COVID-19 patients. Here, we evaluate new data on RAS and KKS peptides in COVID-19 patient serum obtained from a recently developed, fully validated, and optimized stable isotope labeling LC-MS peptide assay. We found that the RAS peptides angiotensin (ANG) 1, 2, 1-5, and 1-7 were downregulated compared to COVID-free surrogate controls, while the KKS peptides Brad, Brad 1-8, and Brad 1-7 were upregulated. This paper focuses on uncovering the possible diagnostic value of these peptides using receiver operating characteristic (ROC) analyses of these data. ROC plots confirmed that all of the analyte peptides in 80 serum samples from COVID-19 patients were significantly altered from "normal" values of the control samples. The best diagnostic sensitivities and selectivities for COVID vs no COVID were found in ROC plots for Brad and Brad 1-7 (both 99% sensitivity, 100% selectivity). We then analyzed levels of all the peptides grouped according to preassigned values of the World Health Organization (WHO) COVID-19 Severity Index. ROC plots differentiated patients with a high WHO severity index from those with a low WHO severity index with moderate success, with BRAD (73% sensitivity, 79% selectivity) and Ang 1-7 (75% sensitivity, 65% selectivity) giving the best diagnostic performance. Results suggest the possible diagnostic value of these peptides as biomarkers to help identify moderate and serious COVID-19 cases at relatively early stages.

Searching for precision: Lorenz Eichstadt's Tabulae harmonicae coelestium motuum (Stetin 1644) and astronomical prediction after Kepler.

In the century between the creation of the first large, European astronomical observatory by Tycho Brahe in the 1580s and the national observatories of France and England in the 1660-1670s, astronomers constructed ever more sets of tables, derived from various geometrical and physical models, to compute planetary positions. But how were these tables to be evaluated? What level of precision or accuracy should be expected from mathematical astronomy? In 1644, the Stetin astronomer and calendar-maker Lorenz Eichstadt published a new set of tables, mostly cobbled together from earlier tables, which include a running commentary on how his tables might be expected to match 'observed' planetary positions. His earlier works also often display a rhetoric of 'exactitude' and 'error'. Eichstadt thus offers a case study of explicit discussions of 'precision' in mid-seventeenth astronomy. Although some tables could generate positions to arcseconds, Eichstadt argued that a regime of five arcminutes should be enough for most table users who were, presumably, computing horoscopes.

Quantitative detection of RAS and KKS peptides in COVID-19 patient serum by stable isotope dimethyl labeling LC-MS.

Angiotensin and kinin metabolic pathways are reported to be altered by many diseases, including COVID-19. Monitoring levels of these peptide metabolites is important for understanding mechanisms of disease processes. In this paper, we report dimethyl labeling of amines in peptides by addition of formaldehyde to samples and deutero-formaldehyde to internal standards to generate nearly identical isotopic standards with 4 m/z units larger per amine group than the corresponding analyte. We apply this approach to rapid, multiplexed, absolute LC-MS/MS quantitation of renin angiotensin system (RAS) and kallikrein-kinin system (KKS) peptides in human blood serum. Limits of detection (LODs) were obtained in the low pg mL-1 range with 3 orders of magnitude dynamic ranges, appropriate for determinations of normal and elevated levels of the target peptides in blood serum and plasma. Accuracy is within ±15% at concentrations above the limit of quantitation, as validated by spike-recovery in serum samples. Applicability was demonstrated by measuring RAS and KKS peptides in serum from COVID-19 patients, but is extendable to any class of peptides or other small molecules bearing reactive -NH2 groups.

PTHrP Regulates Fatty Acid Metabolism via Novel lncRNA in Breast Cancer Initiation and Progression Models.

Parathyroid hormone-related peptide (PTHrP) is the primary cause of malignancy-associated hypercalcemia (MAH). We previously showed that PTHrP ablation, in the MMTV-PyMT murine model of breast cancer (BC) progression, can dramatically prolong tumor latency, slow tumor growth, and prevent metastatic spread. However, the signaling mechanisms using lineage tracing have not yet been carefully analyzed. Here, we generated Pthrpflox/flox; Cre+ mT/mG mice (KO) and Pthrpwt/wt; Cre+ mT/mG tumor mice (WT) to examine the signaling pathways under the control of PTHrP from the early to late stages of tumorigenesis. GFP+ mammary epithelial cells were further enriched for subsequent RNA sequencing (RNAseq) analyses. We observed significant upregulation of cell cycle signaling and fatty acid metabolism in PTHrP WT tumors, which are linked to tumor initiation and progression. Next, we observed that the expression levels of a novel lncRNA, GM50337, along with stearoyl-Coenzyme A desaturase 1 (Scd1) are significantly upregulated in PTHrP WT but not in KO tumors. We further validated a potential human orthologue lncRNA, OLMALINC, together with SCD1 that can be regulated via PTHrP in human BC cell lines. In conclusion, these novel findings could be used to develop targeted strategies for the treatment of BC and its metastatic complications.

Very Low and High Levels of Vitamin D Are Associated with Shorter Leukocyte Telomere Length in 148,321 UK Biobank Participants.

Background: Shorter leukocyte telomere length (LTL) is observed in multiple age-related diseases, which are also associated with vitamin D deficiency (i.e., osteosarcopenia, neurocognitive disorders, cancer, osteoarthritis, etc.), suggesting a close association between vitamin D and LTL. In this study, we examined the relationship between vitamin D levels and LTL in older participants of the UK Biobank. Methods: Data were collected from the UK Biobank. Participants aged 60 and older (n = 148,321) were included. Baseline LTL was measured using a multiplex qPCR technique and expressed as the ratio of the telomere amplification product (T) to that of a single-copy gene (S) (T/S ratio). Serum 25-hydroxyvitamin D (25OHD) was stratified by z score and linked to LTL in a linear regression model adjusting for covariates. Results: Compared to the medium level, a low (in the range of 16.6 nmol/L, 29.7 nmol/L) or extremely low (≤16.6 nmol/L) level of serum 25OHD was associated with shorter LTL: 0.018 SD (standardized ß = -0.018, 95% CI -0.033 to -0.003, p = 0.022) and 0.048 SD (standardized ß = -0.048, 95% CI -0.083 to -0.014, p = 0.006), respectively. Additionally, the high serum 25OHD groups (>95.9 nmol/L) had 0.038 SD (standardized ß = -0.038, 95% CI -0.072 to -0.004, p = 0.030) shorter mean LTL than the group with medium 25OHD levels. The associations above were adjusted for multiple variables. Conclusions: In this population-based study, we identified an inverted U-shape relationship between LTL and vitamin D status. Our findings could be affected by unmeasured confounders. Whether high or low vitamin D-associated shorter LTL is mechanistically related to age-related conditions remains to be elucidated.

Improved platform for breast cancer circulating tumor cell enrichment and characterization with next-generation sequencing technology.

Circulating tumor cells (CTCs) represent cells shed from the primary tumor or metastatic sites and can be used to monitor treatment response and tumor recurrence. However, CTCs circulate in extremely low numbers making in-depth analysis beyond simple enumeration challenging when collected from peripheral blood. Furthermore, tumor heterogeneity, a hallmark of many tumors, especially breast cancer, further complicates CTC characterization. To overcome this limitation, we developed a platform based on the large-scale isolation of CTCs by apheresis, allowing us to collect CTCs in large numbers, which were preserved live in liquid nitrogen for further characterization. Flow cytometry followed by cell sorting (FACS) was performed using a combination of antibodies directed against cell surface markers of white blood cells (CD45) and epithelial tumor cells (CK8). Analysis of subpopulations CD45+/- and CK8+/- by bulk RNA sequencing (RNAseq) and the CD45-/CK8 positive population by single-cell RNAseq was performed. The CD45- population was enriched using CD45 magnetic beads separation and examined by IHC for pan-cytokeratin and immunofluorescence (IF) for specific markers, including the elusive circulating cancer stem cells (CSCs). CSC-rich mammospheres were grown in vitro for further analysis and treated to examine their response to chemotherapeutic agents. Finally, mammospheres were transplanted into the mammary fat pad and bone of immunodeficient mice to examine tumor growth in vivo. This platform enables the detection and collection of CTCs in early and late-stage breast cancer patients of every subtype. Markers including CD44/24, ALDH1 and CXCR4 were identified by IF and showed high expression following mammosphere culture, which responded predictably to chemotherapeutic agents. Mammospheres were also transplanted into nude mice and induced tumors in the mammary fat pad and bone following intra-tibial transplantation. Finally, bulk RNA analysis of the FACS isolated CD45+/- and CK8+/- cells showed a clear separation of CD45- away from CD45+ populations. Single-cell RNAseq of the FACS isolated CD45-/CK8+ cells showed the presence of 4-5 clusters, confirming the high degree of heterogeneity of CTCs. Our platform for large-scale isolation of CTCs using apheresis is suitable for an in-depth analysis of the cancer phenotype and may eventually allow evaluation in real-time of the disease process to optimize cancer regimens.

COVID-19 Detection Using a 3D-Printed Micropipette Tip and a Smartphone.

The COVID-19 pandemic has caused over 7 million deaths worldwide and over 1 million deaths in the US as of October 15, 2022. Virus testing lags behind the level or availability necessary for pandemic events like COVID-19, especially in resource-limited settings. Here, we report a low cost, mix-and-read COVID-19 assay using a synthetic SARS-CoV-2 sensor, imaged and processed using a smartphone. The assay was optimized for saliva and employs 3D-printed micropipette tips with a layer of monoclonal anti-SARS-CoV-2 inside the tip. A polymeric sensor for SARS-CoV-2 spike (S) protein (COVRs) synthesized as a thin film on silica nanoparticles provides 3,3',5-5'-tetramethylbenzidine responsive color detection using streptavidin-poly-horseradish peroxidase (ST-poly-HRP) with 400 HRP labels per molecule. COVRs were engineered with an NHS-PEG4-biotin coating to reduce nonspecific binding and provide affinity for ST-poly-HRP labels. COVRs binds to S-proteins with binding strengths and capacities much larger than salivary proteins in 10% artificial saliva-0.01%-Triton X-100 (as virus deactivator). A limit of detection (LOD) of 200 TCID50/mL (TCID50 = tissue culture infectious dose 50%) in artificial saliva was obtained using the Color Grab smartphone app and verified using ImageJ. Viral load values obtained in 10% pooled human saliva spiked with inactivated SARS-COV-2 virus gave excellent correlation with viral loads obtained from qPCR (p = 0.0003, r = 0.99).

Wnt/ß-catenin pathway inhibitors, bone metabolism and vascular health in kidney transplant patients.

BACKGROUND AND OBJECTIVES: Sclerostin, dickkopf-related protein 1 (DKK1), fibroblast growth factor-23 (FGF23) and α-klotho have been shown to play an important role in bone and vascular disease of chronic kidney disease. We aimed to evaluate the evolution of these bone markers in newly kidney transplanted patients, and whether they are associated with bone metabolism and vascular stiffness. DESIGN, SETTING, PARTICIPANTS AND MEASUREMENTS: This is a longitudinal single-center observational cohort study. Circulating levels of Wnt/ß-catenin pathway inhibitors (sclerostin, DKK1, FGF23 and α-klotho), arterial stiffness (carotid-femoral pulse-wave velocity (PWV), carotid-radial PWV, PWV ratio, augmented index) and bone parameters were assessed before (M0), and at 3 (M3) and 6 months (M6) after transplantation. Generalized estimating equations were conducted for comparative analyses between the three time points. We used a marginal structural model for repeated measures for the impact of changes in bone markers on the evolution of arterial stiffness. Multivariate linear regression analyses were performed for the associations between Wnt/ß-catenin pathway inhibitors and mineral metabolism parameters. RESULTS: We included 79 patients (70% male; median age of 53 (44-60) years old). The levels of sclerostin (2.06 ± 1.18 ng/mL at M0 to 0.88 ± 0.29 ng/mL at M6, p ≤ 0.001), DKK1 (364.0 ± 266.7 pg/mL at M0 to 246.7 ± 149.1 pg/mL at M6, p ≤ 0.001), FGF23 (5595 ± 9603 RU/mL at M0 to 137 ± 215 RU/mL at M6, p ≤ 0.001) and α-klotho (457.6 ± 148.6 pg/mL at M0 to 109.8 ± 120.7 pg/mL at M6, p < 0.05) decreased significantly after kidney transplant. Sclerostin and FGF23 were positively associated with carotid-femoral (standardized ß = 0.432, p = 0.037 and standardized ß = 0.592, p = 0.005) and carotid-radial PWV (standardized ß = 0.259, p = 0.029 and standardized ß = 0.242, p = 0.006) throughout the 6 months of follow-up. The nature of the associations between bone markers and bone metabolism parameters varies after kidney transplant. CONCLUSIONS: The circulating levels of Wnt/ß-catenin pathway inhibitors and α-klotho significantly decrease after kidney transplantation, while sclerostin and FGF23 levels might be associated with improvement of vascular stiffness and blood pressure.

Geriatric Syndromes in Older Adults Hospitalized with COVID-19 in Montreal, Canada.

Background: Older adults are more vulnerable to severe infection and mortality due to COVID-19. They have atypical presentations of the disease without respiratory symptoms, making early diagnosis clinically challenging. We aimed to compare the baseline characteristics, presentation, and disease course of older (≥70 yrs & ≥90 yrs) and younger (<70 yrs) patients hospitalized with COVID-19. Methods: Retrospective review of 429 consecutive patients hospitalized at two tertiary care hospitals in Montreal, Canada, with PCR-confirmed COVID-19. Baseline characteristics, presentation, in-hospital complications, and outcomes were recorded. Desegregation by age was performed to compare older versus younger individuals. Additional subgroup analyses were performed amongst patients ≥70 stratifying by sex, living situation, and those presenting with geriatric syndromes compared to those without. Results: Patients ≥70 (n=260) presented less frequently with respiratory symptoms compared to patients <70 (n=169) (52% vs. 32%). 11% of patients ≥70 and 24% of patients ≥90 presented with a geriatric syndrome as their sole symptom compared to 3% of those <70. Older adults were more likely to develop disease complications (including delirium, acute kidney injury, and hypernatremia) and had higher in-hospital mortality (32% vs. 13%). Subgroup analyses revealed heightened vulnerability to complications in older men, those from long-term care, and those with at least one geriatric syndrome upon presentation. Conclusions: Older adults presenting to hospital with COVID-19 often have no respiratory symptoms and can present with only a geriatric syndrome. New geriatric syndromes in older individuals should trigger evaluation for COVID-19 and consideration for early initiation of multidisciplinary care to prevent complications.

Immunoarray Measurements of Parathyroid Hormone-Related Peptides Combined with Other Biomarkers to Diagnose Aggressive Prostate Cancer.

Parathyroid hormone-related peptide (PTHrP) is related to bone metastasis and hypercalcemia in prostate and breast cancers and should be an excellent biomarker for aggressive forms of these cancers. Current clinical detection protocols for PTHrP are immunoradiometric assay and radioimmunoassay but are not sensitive enough to detect PTHrPs at early stages. We recently evaluated a prostate cancer biomarker panel, including serum monocyte differentiation antigen (CD-14), ETS-related gene protein, pigment epithelial-derived factor, and insulin-like growth factor-1, with promise for identifying aggressive prostate cancers. This panel predicted the need for patient biopsy better than PSA alone. In the present paper, we report an ultrasensitive microfluidic assay for PTHrPs and evaluate their diagnostic value and the value of including them with our prior biomarker panel to diagnose aggressive forms of prostate cancer. The immunoarray features screen-printed carbon sensor electrodes coated with 5 nm glutathione gold nanoparticles with capture antibodies attached. PTHrPs are bound to a secondary antibody attached to a polyhorseradish peroxidase label and delivered to the sensors to provide high sensitivity when activated by H2O2 and a mediator. We obtained an unprecedented 0.3 fg mL-1 limit of detection for PTHrP bioactive moieties PTHrP 1-173 and PTHrP 1-86. We also report the first study of PTHrPs in a large serum pool to identify aggressive malignancies. In assays of 130 human patient serum samples, PTHrP levels distinguished between aggressive and indolent prostate cancers with 83-91% clinical sensitivity and 78-96% specificity. Logistic regression identified the best predictive model as a combination of PTHrP 1-86 and vascular endothelial growth factor-D. PTHrP 1-173 alone also showed a high ability to differentiate aggressive and indolent cancers.

Parathyroid hormone-related protein (PTHrP) and malignancy.

PTHrP (parathyroid hormone related protein) is an important mediator of malignancy-related tumor progression and hypercalcemia that shares considerable homology and functionality with parathyroid hormone. In this chapter, we review what has been elucidated to date regarding PTHrP's role in malignancies. Starting with a review of calcium metabolism and regulation, we then summarize the discovery and structure of PTHrP and development of sensitive immunoassays for specific measurement. Subsequently, we explore its role in tumor progression, with emphasis on the primary tumor as well as skeletal and non-osseus metastases. We then consider the clinical implications of PTHrP in cancer before concluding with a discussion of both established and potential treatments for malignancy associated hypercalcemia and bone metastases.

Parathyroid Hormone-Related Protein Inhibition Blocks Triple-Negative Breast Cancer Expansion in Bone Through Epithelial to Mesenchymal Transition Reversal.

Parathyroid hormone-related protein (PTHrP) plays a major role in skeletal metastasis but its action mechanism has not been fully defined. We previously demonstrated the crucial importance of PTHrP in promoting mammary tumor initiation, growth, and metastasis in a mouse model with a mammary epithelium-targeted Pthlh gene ablation. We demonstrate here a novel mechanism for bone invasion involving PTHrP induction of epithelial to mesenchymal transition (EMT) and cancer stem cells (CSCs) regulation. Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated Pthlh gene ablation was used to study EMT markers, phenotype, and invasiveness in two triple-negative breast cancer (TNBC) cell types (established MDA-MB-231 and patient-derived PT-TNBC cells). In vitro, Pthlh ablation in TNBC cells reduced EMT markers, mammosphere-forming ability, and CD44high/CD24low cells ratio. In vivo, cells were injected intratibially into athymic nude mice, and therapeutic treatment with our anti-PTHrP blocking antibody was started 2 weeks after skeletal tumors were established. In vivo, compared to control, lytic bone lesion from Pthlh -ablated cells decreased significantly over 2 weeks by 27% for MDA-MB-231 and by 75% for PT-TNBC-injected mice (p < 0.001). Micro-CT (µCT) analyses also showed that antibody therapy reduced bone lytic volume loss by 52% and 48% for non-ablated MDA-MB-231 and PT-TNBC, respectively (p < 0.05). Antibody therapy reduced skeletal tumor burden by 45% and 87% for non-ablated MDA-MB-231 and PT-TNBC, respectively (p < 0.002) and caused a significant decrease of CSC/EMT markers ALDH1, vimentin, and Slug, and an increase in E-cadherin in bone lesions. We conclude that PTHrP is a targetable EMT molecular driver and suggest that its pharmacological blockade can provide a potential therapeutic approach against established TNBC-derived skeletal lesions. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Hydroxychloroquine (HCQ) decreases the benefit of anti-PD-1 immune checkpoint blockade in tumor immunotherapy.

Immunotherapy using checkpoint blockade (ICB) with antibodies such as anti-PD-1 has revolutionised the treatment of many cancers. Despite its use to treat COVID-19 patients and autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, the effect of hydroxychloroquine (HCQ) on cancer immunotherapy has not been examined. In this study, remarkably, we find that HCQ alone, or in combination with azithromycin (AZ), at doses used to treat patients, decreased the therapeutic benefit of anti-PD-1 in cancer immunotherapy. No deleterious effect was seen on untreated tumors. Mechanistically, HCQ and HCQ/AZ inhibited PD-L1 expression on tumor cells, while specifically targeting the anti-PD-1 induced increase in progenitor CD8+CD44+PD-1+TCF1+ tumor infiltrating T cells (TILs) and the generation of CD8+CD44+PD-1+ effectors. Surprisingly, it also impaired the appearance of a subset of terminally exhausted CD8+ TILs. No effect was seen on the presence of CD4+ T cells, FoxP3+ regulatory T cells (Tregs), thymic subsets, B cells, antibody production, myeloid cells, or the vasculature of mice. This study indicates for the first time that HCQ and HCQ/AZ negatively impact the ability of anti-PD-1 checkpoint blockade to promote tumor rejection.

Vitamin D Regulates CXCL12/CXCR4 and Epithelial-to-Mesenchymal Transition in a Model of Breast Cancer Metastasis to Lung.

Vitamin D deficiency is associated with poor cancer outcome in humans, and administration of vitamin D or its analogs decreases tumor progression and metastasis in animal models. Using the mouse mammary tumor virus-polyoma middle T antigen (MMTV-PyMT) model of mammary cancer, we previously demonstrated a significant acceleration of carcinogenesis in animals on a low vitamin D diet and a reduction in spontaneous lung metastases when mice received vitamin D through perfusion. We investigate here the action mechanism for vitamin D in lung metastasis in the same non-immunodeficient model and demonstrate that it involves the control of epithelial to mesenchymal transition as well as interactions between chemokine C-X-C motif chemokine 12 (CXCL12) and its receptor C-X-C chemokine receptor type 4 (CXCR4). In vitro, 10-9M vitamin D treatment modifies the phenotype of MMTV-PyMT primary mammary tumor cells and significantly decreases their invasiveness and mammosphere formation capacity by 40% and 50%, respectively. Vitamin D treatment also inhibits phospho-signal transducer and activator of transcription 3 (p-STAT3), zinc finger E-box-binding homeobox 1 (Zeb1), and vimentin by 52%, 75%, and 77%, respectively, and increases E-cadherin by 87%. In vivo, dietary vitamin D deficiency maintains high levels of Zeb1 and p-STAT3 in cells from primary mammary tumors and increases CXCL12 expression in lung stroma by 64%. In lung metastases, vitamin D deficiency increases CXCL12/CXCR4 co-localization by a factor of 2.5. These findings indicate an involvement of vitamin D in mammary cancer "seed" (primary tumor cell) and "soil" (metastatic site) and link vitamin D deficiency to epithelial-to-mesenchymal transition (EMT), CXCL12/CXCR4 signaling, and accelerated metastasis, suggesting vitamin D repleteness in breast cancer patients could enhance the efficacy of co-administered therapies in preventing spread to distant organs.

Safety and effectiveness concerns of lopinavir/ritonavir in COVID-19 affected patients: a retrospective series.

CONTEXT: Originally developed for treatment of human immunodeficiency virus (HIV), the antiviral combination lopinavir/ritonavir (LPV/r) is being repurposed for treating the novel coronavirus disease (COVID-19) despite minimal experience in this markedly different population and an in-vitro derived EC50 against SARS-CoV-2 several hundred-fold greater than for HIV. We present a case series including a case of severe hyponatremia and a 32-fold overdose raising safety and effectiveness concerns in COVID-19 patients. METHODS: We measured LPV trough concentrations in 12 patients and reviewed their clinical charts for side effects known to occur in HIV patients. FINDINGS: Compared to established LPV trough concentrations in HIV patients, concentrations in COVID-19 patients were 3-fold greater (19.37 ± 10.12 mcg/mL versus 6.25 mcg/mL). In addition, cholestasis and dyslipidemia toxicity thresholds were exceeded in 12/12 and 11/12 patients respectively. No patients achieved the presumed therapeutic concentration. Side effects included gastrointestinal symptoms (5/12), electrolyte imbalances (4/12), liver enzyme disturbances (5/12) and triglyceride elevations (2/12). CONCLUSION: No patients reached presumed therapeutic LPV concentrations despite experiencing side effects and exceeding cholestasis and dyslipidemia toxicity thresholds. This raises concerns for the safety and effectiveness of LPV/r. Clinicians should consider closely monitoring for side effects and not necessarily attribute them to COVID-19.

PTHrP Drives Pancreatic Cancer Growth and Metastasis and Reveals a New Therapeutic Vulnerability.

Pancreatic cancer metastasis is a leading cause of cancer-related deaths, yet very little is understood regarding the underlying biology. As a result, targeted therapies to inhibit metastasis are lacking. Here, we report that the parathyroid hormone-related protein (PTHrP encoded by PTHLH) is frequently amplified as part of the KRAS amplicon in patients with pancreatic cancer. PTHrP upregulation drives the growth of both primary and metastatic tumors in mice and is highly enriched in pancreatic ductal adenocarcinoma metastases. Loss of PTHrP-either genetically or pharmacologically-dramatically reduces tumor burden, eliminates metastasis, and enhances overall survival. These effects are mediated in part through a reduction in epithelial-to-mesenchymal transition, which reduces the ability of tumor cells to initiate metastatic cascade. Spp1, which encodes osteopontin, is revealed to be a downstream effector of PTHrP. Our results establish a new paradigm in pancreatic cancer whereby PTHrP is a driver of disease progression and emerges as a novel therapeutic vulnerability. SIGNIFICANCE: Pancreatic cancer often presents with metastases, yet no strategies exist to pharmacologically inhibit this process. Herein, we establish the oncogenic and prometastatic roles of PTHLH, a novel amplified gene in pancreatic ductal adenocarcinoma. We demonstrate that blocking PTHrP activity reduces primary tumor growth, prevents metastasis, and prolongs survival in mice.This article is highlighted in the In This Issue feature, p. 1601.