Indeterminate QuantiFERON Gold Plus Results Reveal Deficient Interferon Gamma Responses in Severely Ill COVID-19 Patients.

SARS-CoV-2 is a novel positive-sense single-stranded RNA virus that has caused a recent pandemic. Most patients have a mild disease course, while approximately 20% have moderate to severe disease, often requiring hospitalization and, in some cases, care in the intensive care unit. By investigating a perceived increased rate of indeterminate QuantiFERON-TB Gold Plus results in hospitalized COVID patients, we demonstrate that severely ill COVID-19 patients have at least a 6-fold reduction of interferon gamma (IFN-γ) levels compared to control patients. What is more, over 60% of these severely ill COVID-19 patients' peripheral T cells were found to be unable to produce measurable IFN-γ when stimulated with phytohemagglutinin (PHA), a potent IFN-γ mitogen, reflected by an indeterminate QuantiFERON-TB Gold Plus result. This defect of IFN-γ production was independent of absolute lymphocyte counts and immunosuppressive therapy. It was associated with increased levels of interleukin-6 (IL-6), which was a predictor of patient outcomes for our cohort when measured early in the course of disease. Finally, in a subset of COVID-19 patients, we found elevated IL-10 levels in addition to IL-6 elevation. In addition to finding a significant limitation of interferon-gamma release assay (IGRA) testing in severely ill COVID-19 patients, these data provide evidence that many of these patients demonstrate a focused Th2 immune response with inhibition of IFN-γ signaling and, in many cases, significant elevations of IL-6.

PD-L1 expression in pancreaticobiliary adenosquamous carcinoma: a single-institution case series.

Background: The programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway is a potent negative regulator of T-cell-mediated immune response that is upregulated in many neoplasms. Pancreaticobiliary adenosquamous carcinoma (PB-ASC) is an aggressive cancer that carries a poorer prognosis compared with pure pancreaticobiliary adenocarcinoma (PB-AC). To date, there is little published information regarding PD-L1 expression in PB-ASC. The aim of the study was to examine the relationship between PD-L1 expression and tumor-infiltrating lymphocytes in PB-ASC and PB-AC. Methods: We evaluated 15 PB-ASCs (10 pancreatic, 5 gallbladder) and 34 control PB-ACs (22 pancreatic ductal, and 12 gallbladder) for tumor expression of PD-L1 using anti-PD-L1 (E1L3N) antibody. All tumors were classified into three immune phenotypes: immune inflamed (II), immune excluded (IE), and immune desert (ID) according to the distribution of tumor-infiltrating lymphocytes in tumor tissues. Results: The frequency of PD-L1 expression was significantly higher in PB-ASC (10/15; 66.7%) than in PB-AC (3/34; 8.8%). In PB-ASC, PD-L1 expression occurred exclusively in the squamous component in six cases, exclusively in the glandular component in one case, and in both the squamous and the glandular components in three cases. PD-L1 expression in PB-ASC was irrespective of the tumor immune status, whereas its expression in PB-AC was observed only in tumors with the II or IE phenotype. The ID phenotype was relatively rare (4/15; 26.7%) in PB-ASC compared with PB-AC (22/34; 65%; P=0.02). Conclusions: PB-ASCs are notably enriched in inflammatory response and showed significantly higher PD-L1 expression than PB-AC (P<0.001), suggesting a potential therapeutic role for immune checkpoint inhibitors in managing patients with PB-ASC.

Beyond the Syndrome: Extensive Congenital Abnormalities in an Infant With Trisomy 21.

Herein we discuss the clinical course and subsequent autopsy of a female infant with trisomy 21 with balanced Rastelli Type "C" complete atrioventricular septal defect (AVSD), tetralogy of Fallot and right aortic arch with mirror image branching pattern who underwent a palliative right modified Blalock-Taussig-Thomas shunt (mBTTS) for hypoxemia from progressive right ventricular outflow tract obstruction. The baby was found to have multiple concomitant pathologic findings not typically seen with this constellation of cardiac anatomy. Autopsy revealed significant abdominal adhesions with near-complete stenosis of the transverse colon. In addition, the infant was found to have significantly elongated villi within the small and large bowel and a relatively large collagenous polyp in the small bowel. The decedent also had an abnormal tracheal bronchus, characterized by an additional superior right-sided bronchus, which is an extremely rare abnormality. Her clinical course was complicated by severe pulmonary hypertensive arteriolar changes out of proportion to what would be typical for her age, trisomy 21 status, and degree of left to right intracardiac shunting. Furthermore, she had refractory anasarca and recurrent chylous pleural effusions without gross lymphatic abnormalities that may have been secondary to systemic capillary leak syndrome (SCLS) versus severe pulmonary hypertension. Due to the aforementioned findings, the family elected for comfort care and the baby expired shortly after extubation. Overall, the infant had multiple, rare coexisting congenital abnormalities that likely represents an extreme phenotype of trisomy 21 that has not been described in the literature to date.

Longitudinal SARS-CoV-2 Testing among the Unvaccinated Is Punctuated by Intermittent Positivity and Variable Rates of Increasing Cycle Threshold Values.

The coronavirus disease 2019 (COVID-19) pandemic is complicated by cases of vaccine breakthrough and reinfection and widespread transmission of variants of concern (VOCs). Consequently, the need to interpret longitudinal positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) tests is crucial in guiding clinical decisions regarding infection control precautions and treatment. Although diagnostic real-time reverse transcription (RT)-PCR tests yield CT values that are inversely correlated with RNA quantity, these tests are only approved for qualitative interpretation. In this study, we performed a retrospective review of 72,217 SARS-CoV-2 positive tests and identified 264 patients with longitudinal positivity prior to vaccination and VOC circulation. Patients with longitudinal positivity fell into two categories: short-term (207, 78%) or prolonged (57, 22%) positivity, defined as ≤28 (range, 1 to 28; median, 16) days and >28 (range, 29 to 152; median, 41) days, respectively. In general, CT values increased over time in both groups; however, 11 short-term-positive patients had greater amounts of RNA detected at their terminal test than at the first positive test, and 6 patients had RNA detected at CT values of <35 at least 40 days after initial infection. Oscillating positive and negative results occurred in both groups, although oscillation was seen three times more frequently in prolonged-positive patients. Patients with prolonged positivity had diverse clinical characteristics but were often critically ill and were discharged to high-level care or deceased (22%). Overall, this study demonstrates that caution must be emphasized when interpreting CT values as a proxy for infectivity, a predictor of severity, or a guide for patient care decisions in the absence of additional clinical context, particularly among the unvaccinated population. IMPORTANCE We describe the duration of positivity and the COVID-19 treatment and outcome characteristics of an unvaccinated population of patients with prolonged SARS-CoV-2 positivity. This investigation serves to highlight challenges in using CT values to guide clinical decisions among unvaccinated individuals.

LPA Induces Metabolic Reprogramming in Ovarian Cancer via a Pseudohypoxic Response.

Although hypoxia has been shown to reprogram cancer cells toward glycolytic shift, the identity of extrinsic stimuli that induce metabolic reprogramming independent of hypoxia, especially in ovarian cancer, is largely unknown. In this study, we use patient-derived ovarian cancer cells and high-grade serous ovarian cancer cell lines to demonstrate that lysophosphatidic acid (LPA), a lipid growth factor and GPCR ligand whose levels are substantially increased in ovarian cancer patients, triggers glycolytic shift in ovarian cancer cells. Inhibition of the G protein α-subunit Gαi2 disrupted LPA-stimulated aerobic glycolysis. LPA stimulated a pseudohypoxic response via Rac-mediated activation of NADPH oxidase and generation of reactive oxygen species, resulting in activation of HIF1α. HIF1α in turn induced expression of glucose transporter-1 and the glycolytic enzyme hexokinase-2 (HKII). Treatment of mice bearing ovarian cancer xenografts with an HKII inhibitor, 3-bromopyruvate, attenuated tumor growth and conferred a concomitant survival advantage. These studies reveal a critical role for LPA in metabolic reprogramming of ovarian cancer cells and identify this node as a promising therapeutic target in ovarian cancer.Significance: These findings establish LPA as a potential therapeutic target in ovarian cancer, revealing its role in the activation of HIF1α-mediated metabolic reprogramming in this disease. Cancer Res; 78(8); 1923-34. ©2018 AACR.

Lysophosphatidic acid stimulates epithelial to mesenchymal transition marker Slug/Snail2 in ovarian cancer cells via Gαi2, Src, and HIF1α signaling nexus.

Recent studies have identified a critical role for lysophosphatidic acid (LPA) in the progression of ovarian cancer. Using a transcription factor activation reporter array, which analyzes 45 distinct transcription factors, it has been observed that LPA observed robustly activates the transcription factor hypoxia-induced factor-1α (HIF1α) in SKOV3.ip ovarian cancer cells. HIF1α showed 150-fold increase in its activation profile compared to the untreated control. Validation of the array analysis indicated that LPA stimulates a rapid increase in the levels of HIF1α in ovarian cancer cells, with an observed maximum level of HIF1α-induction by 4 hours. Our report demonstrates that LPA stimulates the increase in HIF1α levels via Gαi2. Consistent with the role of HIF1α in epithelial to mesenchymal transition (EMT) of cancer cells, LPA stimulates EMT and associated invasive cell migration along with an increase in the expression levels N-cadherin and Slug/Snail2. Using the expression of Slug/Snail2 as a marker for EMT, we demonstrate that the inhibition of Gαi2, HIF1α or Src attenuates this response. In line with the established role of EMT in promoting invasive cell migration, our data demonstrates that the inhibition of HIF1α with the clinically used HIF1α inhibitor, PX-478, drastically attenuates LPA-stimulates invasive migration of SKOV3.ip cells. Thus, our present study demonstrates that LPA utilizes a Gαi2-mediated signaling pathway via Src kinase to stimulate an increase in HIF1α levels and downstream EMT-specific factors such as Slug, leading to invasive migration of ovarian cancer cells.

LPA-mediated migration of ovarian cancer cells involves translocalization of Gαi2 to invadopodia and association with Src and ß-pix.

Lysophosphatidic acid (LPA) plays a critical role in the migration and invasion of ovarian cancer cells. However, the downstream spatiotemporal signaling events involving specific G protein(s) underlying this process are largely unknown. In this report, we demonstrate that LPA signaling causes the translocation of Gαi2 into the invadopodia leading to its interaction with the tyrosine kinase Src and the Rac/CDC42-specific guanine nucleotide exchange factor, ß-pix. Our results establish that Gαi2 activates Rac1 through a p130Cas-dependent pathway in ovarian cancer cells. Moreover, our report reveals that knockdown of Gαi2 leads to loss of ß-pix and active-Rac association in the invadopodia. We also show that knockdown of Gαi2 leads to the complete loss of translocation to p130Cas to focal adhesions. Finally, when Gαi2 is knocked down, this led to the total distribution of Src being shifted primarily from invadopodia and the leading edge of the cells to the perinuclear region, suggesting that Src is inactive in the absence of Gαi2. Overall, our report provides tantalizing evidence that Gαi2 is a critical signaling component of a large signaling complex in the invadopodia that if disrupted could serve as an excellent target for therapy in ovarian and potentially other cancers.

The gep proto-oncogene Gα12 mediates LPA-stimulated activation of CREB in ovarian cancer cells.

Lysophosphatidic acid (LPA) plays a critical role in the pathophysiology of ovarian cancers. Previous studies have shown that LPA stimulates the proliferation of ovarian cancer cells via Gα12. The present study utilizing Protein/DNA array analyses of LPA-stimulated HeyA8 cells in which the expression of Gα12 was silenced, demonstrates for the first time that Gα12-dependent mitogenic signaling by LPA involves the atypical activation cAMP-response element binding protein (CREB). Results indicate that the robust activation of CREB by LPA is an early event that can be monitored by the phosphorylation of SER133 of CREB as early as 3min. The findings that the expression of the constitutively activated mutant of Gα12 stimulates CREB even in the absence of LPA in multiple ovarian cancer cell lines confirm the direct role of Gα12 in the activation of CREB. This is further substantiated by the observation that the silencing of Gα12 drastically attenuates LPA-stimulated phosphorylation of CREB. Our results also establish that LPA-Gα12-dependent activation of CREB is through a cAMP-independent, but Ras-ERK-dependent mechanism. More significantly, our findings indicate that the expression of the dominant negative S133A mutant of CREB leads to a reduction in LPA-stimulated proliferation of HeyA8 ovarian cancer cells. Thus, results presented here demonstrate for the first time that CREB is a critical signaling node in LPA-LPAR and Gα12/gep proto-oncogene stimulated oncogenic signaling in ovarian cancer cells.

LPA Stimulates the Phosphorylation of p130Cas via Gαi2 in Ovarian Cancer Cells.

Ovarian cancer is the most deadly gynecological cancer, with previous studies implicating lysophosphatidic acid (LPA) in the progression of approximately 90% of all ovarian cancers. LPA potently stimulates the tyrosine phosphorylation of p130Cas, a scaffolding protein, which, upon phosphorylation, recruits an array of signaling molecules to promote tumor cell migration. Our work presented here identifies Gαi2 as the major G protein involved in tyrosine phosphorylation of p130Cas in a panel of ovarian cancer cells consisting of HeyA8, SKOV3, and OVCA429. Our results also indicate that the G12 family of G proteins that are also involved in LPA-mediated migration inhibits tyrosine phosphorylation of p130Cas. Using p130Cas siRNA, we demonstrate that p130Cas is a necessary downstream component of LPA Gαi2-induced migration and collagen-1 invasion of ovarian cancer cells. Considering the fact that LPA stimulates invasive migration through the coordination of multiple downstream signaling pathways, our current study identifies a separate unique signaling node involving p130Cas and Gαi2 in mediating LPA-mediated invasive migration of ovarian cancer cells.