Biology subjects

Wandinger-Ness, A.

Publications and source records attributed to Wandinger-Ness, A..

4 recordsLinked to original sources

bioRxiv2022

Humanized NBSGW PDX Models of Disseminated Ovarian Cancer Recapitulate Key Aspects of the Tumor Immune Environment within the Peritoneal Cavity

The importance of the immune microenvironment in ovarian cancer progression, metastasis, and response to therapies has become increasingly clear, especially with the new emphasis on immunotherapies. In order to leverage the power of patient-derived xenograft (PDX) models within a humanized immune microenvironment, three ovarian cancer PDX were grown in humanized NBSGW mice engrafted with human CD34+ cord blood-derived hematopoietic stem cells. Analysis of cytokine levels in the ascites fluid and infiltrating immune cells in the tumors demonstrated that these humanized PDX (huPDX) established an immune tumor microenvironment similar to what has been reported for ovarian cancer patients. The lack of human myeloid cell differentiation has been a major setback for humanized mouse models, but our analysis shows that PDX engraftment increases the human myeloid population in the peripheral blood. Analysis of cytokines within the ascites fluid of huPDX revealed high levels of human M-CSF, a key myeloid differentiation factor as well as other elevated cytokines that have previously been identified in ovarian cancer patient ascites fluid including those involved in immune cell differentiation and recruitment. Human tumor-associated macrophages and tumor-infiltrating lymphocytes were detected within the tumors of humanized mice, demonstrating immune cell recruitment to tumors. Comparison of the three huPDX revealed certain differences in cytokine signatures and in the extent of immune cell recruitment. Our studies show that huNBSGW PDX models reconstitute important aspects of the ovarian cancer immune tumor microenvironment making this a superior approach for therapeutic trials.

cancer biology↗

bioRxiv2022

G-Trap Assay I: Small GTPases as sensitive immune response biomarkers for active bacteremia

The annual toll of sepsis is a 33% mortality rate for hospitalized patients with a cost of greater than 60 billion dollars in the U.S. There is a correlation between sepsis mortality rates and time to treatment with broad-spectrum antibiotics. Consequently, antibiotics are prescribed to nearly every patient suspected of bacteremia. However, once determined that broad-spectrum antibiotics are not required, it is unclear how to optimize the de-escalation of the antibiotics. There is an urgent need for methods to distinguish bacteremia from sterile inflammation and to assess antibiotic efficacy. Rho (Rac1 and RhoA) and Ras (Rap1) family GTPases are dynamic nodes of signaling convergence used by immune-activated leukocytes migrating to sites of infection. This study targeted the onset of GTPase activation as a biomarker of infection-induced immune activation in trauma patients. GTP binding assays were performed using a novel GTPase effector trap flow cytometry assay (G-Trap). Here, we demonstrate increased GTP binding to small GTPases (Rac1, Rap1, and RhoA) of resting cells serially exposed to plasma samples from bacteremic trauma patients. Responses to the serial samples showed that GTPase activation was influenced by the concentration of circulating pro- and anti-inflammatory mediators, in tandem with synergy or antagonism from the cytokines and the antibiotic treatment.

immunology↗

bioRxiv2022

G-Trap Assay II: Characterization of blood Leukocyte Functionality differentiates immune activation and immune suppression in bacteremia patient samples

Sepsis is a severe organ dysfunction syndrome caused by a dysregulation of the immune systems response to infection. Unfortunately, most infection-causing pathogens arent routinely detectable in real-time to enable targeted and lifesaving treatment. Thus, clinicians frequently have limited data on which to base treatment decisions. A complete blood count with differential is available within 24 h, and positive culture is only available in ~30% of cases. Furthermore, a blood culture, the traditional gold standard for accurate diagnosis of bacteremia, may take up to five days for results, long after a clinical decision for sepsis management is required. Circulating leukocytes can sense chemotactic signals released by bloodborne pathogens or focal infections not in the bloodstream. Our earlier study showed that pathogen and host immune factors released in the bloodstream stimulated GTP binding of Ras homology (Rho) GTPases (guanosine triphosphatase) such as Rac1 in quiescent endothelial and human leukocytes after exposure to blood plasma from infected patients.[1] In this study, we measured Rac1*GTP as a biomarker of immune functionality of peripheral blood monocytes and polymorphonuclear cells extracted from blood samples drawn for diagnostic use in blood culture assays; from 120 non-infected control patients and serial blood test samples from 28 patients with a confirmed diagnosis of bloodstream infection. 18 cases presented with Rac1*GTP elevation of [≥]3 fold above that of control samples. Ten patients with normal or below-normal GTPase activity, accompanied by neutrophilia or pancytopenia. We used Principal Component Analysis to differentiate the 2D spatial distribution of infected patients and negative controls. Measuring differential leukocyte functionality in infected and control patients blood samples with the G-Trap assay may provide an innovative process for a real-time distinction between infection and non-infectious etiologies.

immunology↗

bioRxiv2022

Reconstitution of renal cyst formation in 3D culture reveals a role for AMOT and Yap1 in determining cyst size.

Despite substantial progress in identifying and understanding causative mutations in autosomal dominant polycystic kidney disease (ADPKD), little is known about subsequent cellular events leading to cyst formation. In prior studies we reported that Cadherin 8, a type II Cadherin, expression is sufficient to induce cyst emergence from HK-2 cells grown as tubule arrays in collagen matrix (1). However, emergent cysts did not exhibit the luminal enlargement observed in ADPKD. In this communication, we reconstitute cyst emergence with consequent cyst lumen expansion in 3D culture by stable co-expression of Cadherin 8 in combination with a constitutively active mutant of YAP1, the key effector of the HIPPO pathway. Specifically, immortalized cells derived from ADPKD cyst epithelia formed cysts with substantially larger lumen sizes when transduced with YAP1-5SA. Conversely, expression of the YAP1 inhibitor, AMOTL1, in these cells resulted in their forming cysts with smaller lumens than control cells. Our data show that cyst formation results from a sequential two-step process consisting of cyst initiation and subsequent cyst expansion. Taken together, cyst initiation induced by Cadherin 8 expression is proposed to result from decreased cell-cell adhesion while cyst expansion is driven by increased Yap1 activity.

cell biology↗