Clinical Evaluation of IDH Mutation Status in Formalin-Fixed Paraffin-Embedded Tissue in Gliomas.

BACKGROUND AND OBJECTIVE: Determination of isocitrate dehydrogenase (IDH) 1/2 mutational status is crucial for a glioma diagnosis. It is common for IDH mutational status to be determined via a two-step algorithm that utilizes immunohistochemistry studies for IDH1 R132H, the most frequent variant, followed by next-generation sequencing studies for immunohistochemistry-negative or immunohistochemistry-equivocal cases. The objective of this study was to evaluate adding a rapid real-time polymerase chain reaction (RT-PCR) assay to the testing algorithm.  METHODS: We validated a modified, commercial, qualitative, RT-PCR assay with the ability to detect 14 variants in IDH1/2 in formalin-fixed paraffin-embedded glioma tumor specimens. The assay was validated using 51 tumor formalin-fixed paraffin-embedded specimens. During clinical implementation of this assay, 48 brain tumor specimens were assessed for IDH result concordance and turnaround time to result. RESULTS: Concordance between the RT-PCR and sequencing and IHC studies was 100%. This RT-PCR assay also showed concordant results with IHC for IDH1 R132H for 11 of the 12 (92%) tumor specimens with IDH mutations. The RT-PCR assay yielded faster results (average 2.6 days turnaround time) in comparison to sequencing studies (17.9 days), with complete concordance. CONCLUSIONS: In summary, we report that this RT-PCR assay can reliably be performed on formalin-fixed paraffin-embedded specimens and has a faster turnaround time than sequencing assays and can be clinically implemented for determination of IDH mutation status for patients with glioma.

Saliva versus Upper Respiratory Swabs: Equivalent for Severe Acute Respiratory Syndrome Coronavirus 2 University Screening while Saliva Positivity Is Prolonged After Symptom Onset in Coronavirus Disease 2019 Hospitalized Patients.

Reopening of schools and workplaces during the ongoing coronavirus disease 2019 (COVID-19) pandemic requires affordable and convenient population-wide screening methods. Although upper respiratory swab is considered the preferable specimen for testing, saliva offers several advantages, such as easier collection and lower cost. In this study, we compared the performance of saliva with upper respiratory swab for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. Paired saliva and anterior nares specimens were collected from a largely asymptomatic cohort of students, faculty, and staff from the University of Pennsylvania. Paired saliva and combined nasopharyngeal/oropharyngeal (NP/OP) specimens were also collected from hospitalized patients with symptomatic COVID-19 following confirmatory testing. All study samples were tested by real-time PCR in the Hospital of the University of Pennsylvania. In the university cohort, positivity rates were 37 of 2500 for saliva (sensitivity, 86.1%) and 36 of 2500 for anterior nares (sensitivity, 83.7%), with an overall agreement of 99.6%. In the hospital study cohort, positivity rates were 35 of 49 for saliva (sensitivity, 89.3%) and 28 of 49 for NP/OP (sensitivity, 75.8%), with an overall agreement of 75.6%. A larger proportion of saliva than NP/OP samples tested positive after 4 days of symptom onset in hospitalized patients. Our results show that saliva has an acceptable sensitivity and is comparable to upper respiratory swab, supporting the use of saliva for SARS-CoV-2 detection in both symptomatic and asymptomatic populations.

An Autocrine Proliferation Repressor Regulates Dictyostelium discoideum Proliferation and Chemorepulsion Using the G Protein-Coupled Receptor GrlH.

In eukaryotic microbes, little is known about signals that inhibit the proliferation of the cells that secrete the signal, and little is known about signals (chemorepellents) that cause cells to move away from the source of the signal. Autocrine proliferation repressor protein A (AprA) is a protein secreted by the eukaryotic microbe Dictyostelium discoideum AprA is a chemorepellent for and inhibits the proliferation of D. discoideum We previously found that cells sense AprA using G proteins, suggesting the existence of a G protein-coupled AprA receptor. To identify the AprA receptor, we screened mutants lacking putative G protein-coupled receptors. We found that, compared to the wild-type strain, cells lacking putative receptor GrlH (grlH¯ cells) show rapid proliferation, do not have large numbers of cells moving away from the edges of colonies, are insensitive to AprA-induced proliferation inhibition and chemorepulsion, and have decreased AprA binding. Expression of GrlH in grlH¯ cells (grlH¯/grlHOE ) rescues the phenotypes described above. These data indicate that AprA signaling may be mediated by GrlH in D. discoideumIMPORTANCE Little is known about how eukaryotic cells can count themselves and thus regulate the size of a tissue or density of cells. In addition, little is known about how eukaryotic cells can sense a repellant signal and move away from the source of the repellant, for instance, to organize the movement of cells in a developing embryo or to move immune cells out of a tissue. In this study, we found that a eukaryotic microbe uses G protein-coupled receptors to mediate both cell density sensing and chemorepulsion.

Bone marrow myeloid cells in regulation of multiple myeloma progression.

Survival, growth, and response to chemotherapy of cancer cells depends strongly on the interaction of cancer cells with the tumor microenvironment. In multiple myeloma, a cancer of plasma cells that localizes preferentially in the bone marrow, the microenvironment is highly enriched with myeloid cells. The majority of myeloid cells are represented by mature and immature neutrophils. The contribution of the different myeloid cell populations to tumor progression and chemoresistance in multiple myeloma is discussed.

Functional similarities between the dictyostelium protein AprA and the human protein dipeptidyl-peptidase IV.

Autocrine proliferation repressor protein A (AprA) is a protein secreted by Dictyostelium discoideum cells. Although there is very little sequence similarity between AprA and any human protein, AprA has a predicted structural similarity to the human protein dipeptidyl peptidase IV (DPPIV). AprA is a chemorepellent for Dictyostelium cells, and DPPIV is a chemorepellent for neutrophils. This led us to investigate if AprA and DPPIV have additional functional similarities. We find that like AprA, DPPIV is a chemorepellent for, and inhibits the proliferation of, D. discoideum cells, and that AprA binds some DPPIV binding partners such as fibronectin. Conversely, rAprA has DPPIV-like protease activity. These results indicate a functional similarity between two eukaryotic chemorepellent proteins with very little sequence similarity, and emphasize the usefulness of using a predicted protein structure to search a protein structure database, in addition to searching for proteins with similar sequences.

Peritoneal Dialysis Fluid and Some of Its Components Potentiate Fibrocyte Differentiation.

Long-term peritoneal dialysis (PD) often results in the development of peritoneal fibrosis. In many other fibrosing diseases, monocytes enter the fibrotic lesion and differentiate into fibroblast-like cells called fibrocytes. We find that peritoneal tissue from short-term PD patients contains few fibrocytes, while fibrocytes are readily observed in the peritoneal membrane of long-term PD patients. The PD fluid Dianeal (Baxter Healthcare Corporation, Deerfield, IL, USA) contains dextrose, a number of electrolytes including sodium chloride, and sodium lactate. We find that PD fluid potentiates human fibrocyte differentiation in vitro and implicates sodium lactate in this potentiation. The plasma protein serum amyloid P (SAP) inhibits fibrocyte differentiation. Peritoneal dialysis fluid and sodium chloride decrease the ability of human SAP to inhibit human fibrocyte differentiation in vitro Together, these results suggest that PD fluid contributes to the development of peritoneal fibrosis by potentiating fibrocyte differentiation.

Bone marrow PMN-MDSCs and neutrophils are functionally similar in protection of multiple myeloma from chemotherapy.

Multiple myeloma (MM) is an incurable cancer of plasma cells localized preferentially in the bone marrow (BM). Resistance to chemotherapy represents one of the main challenges in MM management. BM microenvironment is known to play a critical role in protection of MM cells from chemotherapeutics; however, mechanisms responsible for this effect are largely unknown. Development of MM is associated with accumulation of myeloid-derived suppressor cells (MDSCs) mostly represented by pathologically activated relatively immature polymorphonuclear neutrophils (PMN-MDSCs). Here, we investigated whether PMN-MDSCs are responsible for BM microenvironment-mediated MM chemoresistance. Using in vivo mouse models allowing manipulation of myeloid cell number, we demonstrated a critical role for myeloid cells in MM growth and chemoresistance. PMN-MDSCs isolated from MM-bearing host are immunosuppressive and thus, functionally distinct from their counterpart in tumor-free host neutrophils. We found, however, that both PMN-MDSCs and neutrophils equally promote MM survival from doxorubicin and melphalan and that this effect is mediated by soluble factors rather than direct cell-cell contact. Our data indicate that targeting PMN-MDSCs would enhance chemotherapy efficacy in MM.

Role of the neutrophil chemorepellent soluble dipeptidyl peptidase IV in decreasing inflammation in a murine model of arthritis.

OBJECTIVE: To determine whether an intraarticular injection of the neutrophil chemorepellent dipeptidyl peptidase IV (DPPIV; CD26) can attenuate inflammation and decrease the severity of arthritis in a murine model. METHODS: DBA/1 mice were immunized with type II collagen/Freund's complete adjuvant to produce collagen-induced arthritis (CIA). On day 25 postimmunization, recombinant human DPPIV (rhDPPIV) or phosphate buffered saline was injected intraarticularly, and arthritis severity scores were recorded 3 times per week. The hind legs of mice in both groups were fixed, decalcified, paraffin embedded, and sectioned. Pathologic scores for inflammation and neutrophil infiltration were recorded on a scale of 1-8, and the number of neutrophils was determined by morphometric cell counts. In addition, Mac-2-positive macrophages and articular damage were assessed using anti-Mac-2 antibodies and histologic staining, respectively. RESULTS: Injection of rhDPPIV reduced the mean score of arthritis severity in mice with CIA. DPPIV treatment reduced the overall extent of inflammation and articular damage around the arthritic joint and periarticular tissue, and also decreased neutrophil and macrophage infiltration. CONCLUSION: A localized injection of the neutrophil chemorepellent DPPIV reduces inflammation and the severity of the disease in a murine model of arthritis.

A retinoblastoma orthologue is required for the sensing of a chalone in Dictyostelium discoideum.

Retinoblastoma-like proteins regulate cell differentiation and inhibit cell proliferation. The Dictyostelium discoideum retinoblastoma orthologue RblA affects the differentiation of cells during multicellular development, but it is unclear whether RblA has a significant effect on Dictyostelium cell proliferation, which is inhibited by the secreted proteins AprA and CfaD. We found that rblA⁻ cells in shaking culture proliferate to a higher density, die faster after reaching stationary density, and, after starvation, have a lower spore viability than wild-type cells, possibly because in shaking culture, rblA⁻ cells have both increased cytokinesis and lower extracellular accumulation of CfaD. However, rblA⁻ cells have abnormally slow proliferation on bacterial lawns. Recombinant AprA inhibits the proliferation of wild-type cells but not that of rblA⁻ cells, whereas CfaD inhibits the proliferation of both wild-type cells and rblA⁻ cells. Similar to aprA⁻ cells, rblA⁻ cells have a normal mass and protein accumulation rate on a per-nucleus basis, indicating that RblA affects cell proliferation but not cell growth. AprA also functions as a chemorepellent, and RblA is required for proper AprA chemorepellent activity despite the fact that RblA does not affect cell speed. Together, our data indicate that an autocrine proliferation-inhibiting factor acts through RblA to regulate cell density in Dictyostelium, suggesting that such factors may signal through retinoblastoma-like proteins to control the sizes of structures such as developing organs or tumors.

Dipeptidyl peptidase IV is a human and murine neutrophil chemorepellent.

In Dictyostelium discoideum, AprA is a secreted protein that inhibits proliferation and causes chemorepulsion of Dictyostelium cells, yet AprA has little sequence similarity to any human proteins. We found that a predicted structure of AprA has similarity to human dipeptidyl peptidase IV (DPPIV). DPPIV is a serine protease present in extracellular fluids that cleaves peptides with a proline or alanine in the second position. In Insall chambers, DPPIV gradients below, similar to, and above the human serum DPPIV concentration cause movement of human neutrophils away from the higher concentration of DPPIV. A 1% DPPIV concentration difference between the front and back of the cell is sufficient to cause chemorepulsion. Neutrophil speed and viability are unaffected by DPPIV. DPPIV inhibitors block DPPIV-mediated chemorepulsion. In a murine model of acute respiratory distress syndrome, aspirated bleomycin induces a significant increase in the number of neutrophils in the lungs after 3 d. Oropharyngeal aspiration of DPPIV inhibits the bleomycin-induced accumulation of mouse neutrophils. These results indicate that DPPIV functions as a chemorepellent of human and mouse neutrophils, and they suggest new mechanisms to inhibit neutrophil accumulation in acute respiratory distress syndrome.

A Dictyostelium secreted factor requires a PTEN-like phosphatase to slow proliferation and induce chemorepulsion.

In Dictyostelium discoideum, AprA and CfaD are secreted proteins that inhibit cell proliferation. We found that the proliferation of cells lacking CnrN, a phosphatase and tensin homolog (PTEN)-like phosphatase, is not inhibited by exogenous AprA and is increased by exogenous CfaD. The expression of CnrN in cnrN cells partially rescues these altered sensitivities, suggesting that CnrN is necessary for the ability of AprA and CfaD to inhibit proliferation. Cells lacking CnrN accumulate normal levels of AprA and CfaD. Like cells lacking AprA and CfaD, cnrN cells proliferate faster and reach a higher maximum cell density than wild type cells, tend to be multinucleate, accumulate normal levels of mass and protein per nucleus, and form less viable spores. When cnrN cells expressing myc-tagged CnrN are stimulated with a mixture of rAprA and rCfaD, levels of membrane-associated myc-CnrN increase. AprA also causes chemorepulsion of Dictyostelium cells, and CnrN is required for this process. Combined, these results suggest that CnrN functions in a signal transduction pathway downstream of AprA and CfaD mediating some, but not all, of the effects of AprA and CfaD.