bluuebunny/crossref_metadata_2025 huggingface dataset:
A New Composite Biomarker for Cervical Cancer Diagnosis
Authors: Markovic Olivera , Markovic Nenad
Published: November 2023

In 2004, at the FASEB meeting, we introduced our new proprietary, biomarker‐based MarkPap® technology for detection of abnormal cells on cervical specimens.

At subsequent meetings, we followed with reports for further developments of this platform technology, e.g., semi automated processing, diagnosis at distance with digital (TelePap) and mobile (Mobile Pap) transmission of microscopic images from POC to medical centers. Empowering the methodology with IT technology (telecytopathology) it became infrastructure‐independent and a possibility for self‐specimen collection (MarkPap Self) opened a prospective for global cervical cancer screening.

On the most recent FASEB meetings we presented our results from building IT Telehealth Center for Telecytopathology with proprietary communication network to a global networking system enabling automatic exchange of secure digital image (high resolution, still, metadata) files between POCs and medical centers for the purpose of global cervical cancer screening.

This time, we are presenting a composite, meaningful biomarker MEDYKO, an acronym, composed of ME ‐ pre‐malignant changes (cervical acid phosphatase); DY‐dysplasia, dyskaryosis (malignancy); KO – koilocytes, HPV disease (poor prognosis). This composition of biomarkers provides instant visual information of the metabolic status of cell undergoing transformation, morphological status of the nucleus indicating DNA malignant alteration, and koilocytes indicating poor prognosis because of the chronic HPV disease. All three parameters were present in high percent of cases of advanced cervical cancer and hierarchically less expressed in HSIL, LSIL and ASCUS.

These results warrant further work on multiple biomarkers related to malignant disorders, which instant recognition on Pap smears could improve the clinical value of such new Pap test and return it to prior status of the “best anticancer screening test available”.

Support or Funding Information

BioSciCon, Inc.
Foxi3: A Sly Regulator of Bone Development, Promotes Prostate Cancer
Authors: Mukherjee Angana
Published: November 2023

Prostate cancer is the abnormal growth of cells in the prostate gland, and is the second leading cause of deaths among men in the United States. Studies have shown that 100% of men that die from prostate cancer have bone involvement. It is believed that tumor cells take on a bone phenotype, termed osteomimicry, thus permitting its growth at an ectopic location. Of the many cells resident in the bone, myeloid cells secrete various soluble factors that contribute to the high turnover rate of cells and molecular processes of bone development. Of particular interest, foxi‐3, a forkhead family transcription factor is critical in bone development and embryogenesis. However, its role in prostate cancer, has not been explored. Therefore, we hypothesized that foxi3 is a key factor in promoting prostate cancer progression to the bone. To investigate the clinical role of foxi3, we analyzed its expression and localization in human prostate cancer tissue and cell lines. Interestingly, there is a significant increase in foxi3 expression as cancer becomes more aggressive (p<0.01). Moreover, we see a 6000‐fold increase in foxi3 expression in bone metastatic cells than primary tumor cells. Furthermore, treatment with fibroblast growth factor, a regulator of bone development, resulted in differential expression and localization of foxi3 and bone markers in prostate cancer. Taken together, these data suggest a crucial role for foxi3 in prostate cancer progression to the bone.

Support or Funding Information

Troy University Faculty Development Grant ‐ 0409365
Breast Tumor Development and Cigarette Smoking: Accelerated Tumor Development in Calcium Independent Phospholipase A2γ Deficient Mice
Authors: Kispert Shannon E , McHowat Jane
Published: November 2023

The phospholipase A2 (PLA2) family of enzymes are responsible for cleavage of membrane phospholipids, releasing a free fatty acid and lysophospholipid. Various PLA2 dependent pathways are important in regulation of cell proliferation, differentiation, motility, and immune responses which can all be altered during tumor development and progression. Calcium independent PLA2 (iPLA2) enzymes are responsible for phospholipid remodeling as well as release of arachidonic acid or lysophosphatidic acid (LPA) which play major roles in downstream signaling pathways. The most commonly studied iPLA2 enzymes are iPLA2β and iPLA2γ, but their role in cancer development remains unknown. To determine what effects cigarette smoke exposure had on mammary tissue from wild type and iPLA2γ deficient mice, we exposed six month old females to cigarette smoke using the SCIREQ InExpose system for up to six months. Mammary tissue was removed and snap frozen for biochemistry or stored in formalin for immunohistochemical analysis. Following three and six months of cigarette smoke exposure, wild type mice showed atypical ductal hyperplasia. In comparison, the iPLA2γ knockout mice developed ductal carcinoma in situ following three months of cigarette smoke exposure. Recent studies from our lab have determined that iPLA2γ deletion in mice results in alterations of cytokine expression implicated in breast tumor progression when compared to wild type mice. Changes in cytokine expression may elucidate the mechanism by which iPLA2γ deficient mice develop tumors more rapidly than wild type mice in the presence of cigarette smoking.

Support or Funding Information

Saint Louis University Presidents Research Fund
Anti‐invasive activity of capsaicin and non‐pungent capsaicin analogs in human SCLC
Authors: Akers Austin T , Hurley John D , Brown Kathleen C , Nolan Nicholas A , Dasgupta Piyali
Published: November 2023

Small cell lung cancer (SCLC) is the most aggressive lung malignancy. A majority of SCLC patients show extrapulmonary metastasis at the time of their presentation. The common sites of SCLC metastasis are bone and the brain. The invasion of malignant cells to the neighboring blood vessels and lymph nodes is a key step of metastasis. Recent data indicate that dietary compounds (by themselves or in combination with standard chemotherapy) reduce invasion and metastasis of several human cancers. Capsaicin is the pungent ingredient of chili peppers. Recent studies have shown that capsaicin inhibits the invasion and metastasis of several types of human cancers including melanoma, prostate cancer and cholangiosarcoma. We tested the anti‐invasive activity of capsaicin, as well as two non‐pungent capsaicin analogs, olvanil and arvanil, in human SCLC using the Boyden chamber assay and spherical invasion assay. We found that olvanil and arvanil possess greater anti‐invasive activity than capsaicin. The biological activity of capsaicin is mediated by transient receptor potential vanilloid (TRPV) receptors on target cells. However, arvanil and olvanil bind to both TRPV and cannabinoid receptors. Boyden chamber and spherical invasion assay revealed that the anti‐invasive effects of capsaicin, olvanil and arvanil were independent of both TRPV and cannabinoid receptors. The capsaicin analogs olvanil and arvanil may prove useful for the management and treatment of SCLC metastasis.

Support or Funding Information

Funding for our study was supported by the an NIH R15‐AREA Grant (1R15HL113681‐01A1), AICR Investigator Grant, and a NASA Undergraduate Fellowship to ATA.
Pharmacological Reversal of Adolescent Alcohol‐Induced Perturbation to Mesolimbic Dopamine Network Dynamics Rescues Maladaptive Decision Making in Adulthood
Authors: Schindler Abigail , Soden Marta , Weber Sophia , Zweifel Larry , Clark Jeremy
Published: November 2023

Alcohol is the most commonly used and abused substance among adolescents, promoting the development of substance use disorders and compromised decision‐making in adulthood. Using a preclinical model we have demonstrated that adolescent alcohol use results in maladaptive adult risk‐taking behavior that positively correlates with increased mesolimbic phasic dopamine transmission in response to risky options, but the underlying mechanisms remain unknown. Here we show that increased phasic dopamine after adolescent alcohol use is attributable to a selective perturbation of midbrain circuitry. Indeed, we demonstrate that VTA dopamine neurons exhibit enhanced IPSCs, but not EPSCs, leading to decreased basal dopamine levels in adulthood that negatively correlate with risk‐taking. We conclude that a novel disinhibitory mechanism in midbrain circuitry mediates the effects of alcohol in a model where increased inhibitory tone on dopamine neurons leads to a persistent decrease in basal dopamine and promotes a potentiation in stimulus‐evoked phasic dopamine release that drives maladaptive choice behavior. Based on these findings we employ a selective GABA(A) receptor agonist to pharmacologically reverse risk‐taking in adulthood in parallel with normalization of phasic dopamine. Together these results isolate the underlying circuitry involved in maladaptive decision‐making and identify a potential therapeutic target.

Support or Funding Information

R01AA021121 and T32AA07455
Neurochemical Profile of a Novel Allosteric Potentiator of the Dopamine D1 Receptor
Authors: Falcone Julie F , Mitchell Stephen N , Carter Guy , Maren Deanna L , Katner Jason , Chernet Eyassu , Wright Rebecca A , Wang Hong , Ryder John W , Morin Michelle , Gehlert Donald R , Johnson Michael P , Beck James P , Hao Junliang , Menezes Michelle M , Bruns Robert F , Svensson Kjell A
Published: November 2023

DETQ is a potent and selective dopamine D1 receptor potentiator that is active in several behavioral models (see abstracts by Heinz et al and Svensson et al at this meeting). We wished to explore the neurochemical basis for the behavioral effects of DETQ. Due to low affinity of DETQ in rodents, the current studies were carried out in transgenic mice in which both copies of the murine D1 receptor were replaced with its human counterpart (hD1 mice) (see Svensson et al). Receptor expression in hD1 mice was characterized using autoradiography of the D1‐selective antagonist 3H‐SCH23390. The pattern of D1 receptor distribution was very similar in wild‐type and hD1 mice, but the absolute level of expression was about 50% lower in hD1 mice. Using microdialysis in freely moving hD1 mice, DETQ and the D1 agonist SKF82958 increased extracellular levels of acetylcholine and histamine in cortical and subcortical areas. At a high dose we also observed enhanced extracellular levels of norepinephrine in the prefrontal cortex. Furthermore, a low dose of DETQ elevated hippocampal acetylcholine levels in an additive fashion with the acetylcholinesterase inhibitor rivastigmine. Brain levels of the histamine metabolites tele‐methylhistamine and tele‐methylimidazole acetic acid were also elevated in both microdialysate and post‐mortem tissue samples. The increases in brain histamine metabolites produced by DETQ were not present in wild‐type mice, indicating that the effects were mediated via the D1 receptor. Overall, these neurochemical changes correlated with brain exposure of DETQ and also with the behavioral effects (see Svensson et al.)

Effects on brain cyclic nucleotide levels were studied after microwave fixation of the forebran. Levels of cGMP were elevated in the striatum after both DETQ and SKF82958. Further changes in downstream signaling were explored measuring phosphorylation of the transcription factor CREB and the AMPA receptor GluR1. Both DETQ and SKF82958 increased pCREB and pGluR1 in the brain. Together, these data provide neurochemical evidence for enhanced synaptic plasticity of DETQ and SKF82958.

In conclusion, our in vivo neurochemical data support the proposal that the D1 potentiator DETQ selectively enhances D1 receptor function in the brain. Studies of neurotransmitter release, second messengers, and downstream signaling support a potential utility for D1 potentiators to enhance cognitive function in CNS disorders.

Support or Funding Information

All authors are current or past employees of Eli Lilly & Co
Deficiency of macrophage migration inhibitory factor attenuates tau hyperphosphorylation in mouse models of Alzheimer's disease
Authors: Ye Richard D
Published: November 2023

Pathological features of Alzheimer's disease (AD) include aggregation of amyloid beta (Aβ) and tau protein. Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, has been implicated in the toxicity of aggregated Aβ. It remains unclear whether MIF affects hyperphosphorylation and aggregation of tau. The effects of MIF deficiency in tau hyperphosphorylation were examined in Mif‐/‐mice receiving intracerebroventricular (ICV) injection of Streptozotocin (STZ) and in APP/PS1 transgenic mice mated with Mif‐/‐ mice. MIF expression and astrocyte activation were evaluated in ICV‐STZ mice using immunofluorecence staining. Cultured primary astrocytes were treated with high glucose to mimic STZ function in vitro and the condition medium (CM) was collected. The level of tau hyperphosphorylation in neurons treated with the astrocyte CM was determined using Western blotting. MIF deficiency attenuated tau hyperphosphorylation in mice. ICV injection of STZ increased astrocyte activation and MIF expression in the hippocampus. MIF deficiency attenuated astrocyte activation in ICV‐STZ mice. CM from high glucose‐treated WT astrocytes increased tau hyperphosphorylation in cultured primary neurons, an effect absent from Mif‐/‐ astrocytes and WT astrocytes treated with the MIF inhibitor ISO‐1. ISO‐1 had no direct effect on tau phosphorylation in cultured primary neurons. These results suggest that MIF deficiency is associated with reduced astrocyte activation and tau hyperphosphorylation in the mouse AD models tested. Inhibition of MIF and MIF‐induced astrocyte activation may be useful in AD prevention and therapy.
Identification of potential PAR‐regulating proteinases in agricultural dust using an activity‐based serine proteinase probe
Authors: Hollenberg Morley D. , Polley Danny , Saifeddine Mahmoud , Gilmore Brendan , Romberger Debra J. , Heires Art J. , Nordgren Tara M , Toews Myron L
Published: November 2023

In previous work from our laboratories (PMIDs 26092994, 24152160, 21270400) we found that environmental allergens, including cockroach‐ and barn‐derived particulates, contain proteolytic enzymes that contribute to lung inflammation by cleaving and activating proteinase‐activated receptors (PARs). Preliminary work using in‐gel zymography demonstrated the presence of multiple serine proteinase inhibitor‐sensitive enzymes in hog barn dust extract (HDE: PMID 26092994). Because these HDE enzymes were blocked by alkylation of the active serine in many of the proteinases with PMSF and related agents, we reasoned that an activity‐based serine proteinase probe (ABP: PMID16554154) would also be able to alkylate the enzymes in the HDE. Using this novel approach (PMID 21270400), we ABP‐biotinylated the enzymes in HDE with ABPs selective for tryptic, chymotryptic, and elastase enzymes, respectively. Our data show differential reactivity of these ABP probes with enzymes in the HDE, identifying several enzymes in the mass range from 10 to 36 kDa, overlapping with the mass of ABP‐labeled porcine trypsin (28 kDa). Porcine trypsin itself is a major PAR‐regulating proteinase that is present in HDE, and this enzyme may play a prominent role in triggering lung inflammation in agricultural workers. Thus, HDE trypsin and other proteinases represent novel therapeutic targets both for preventing dust‐induced disease and for monitoring as environmental risk factors in many agricultural settings.

Support or Funding Information

Supported by the Canadian CIHR (MDH), The Lung Association of Alberta‐NWT (MDH), and NIOSH 2R01‐0H‐008539 (DJR)
Nephrotoxic Potential of 3,5‐Dichloroaniline Putative Metabolites in Isolated Renal Cortical Cells from Fischer 344 Rats
Authors: Rankin Gary , Racine Christopher , Ward Dakota , Tyree Conner , Pope Deborah , Anestis Dianne
Published: November 2023

Halogenated anilines are widely used as chemical intermediates in the manufacture of dyes, agricultural agents and as industrial intermediates. Previous studies in our laboratory have found that 3,5‐dichloroaniline (3,5‐DCA) is the most potent nephrotoxicant among mono‐ and dichloroanilines, both in vivo and in vitro. The potential role of renal biotransformation in 3,5‐DCA‐induced nephrotoxicity has also been examined. When isolated renal cortical cells (IRCC) from male Fischer 344 rats were pretreated with various biotransformation enzyme inhibitors, 3,5‐DCA –induced cytotoxicity was significantly attenuated. These studies indicated that 3,5‐DCA could be biotransformed via multiple pathways in the kidney to one or more toxic metabolite(s). The current study was designed to explore the nephrotoxic potential of three putative 3,5‐DCA metabolites [2‐amino‐4,6‐dichlorophenol (2A4,6DCP), 3,5‐dichlorophenylhydroxylamine (3,5‐DCPHA), and 3,5‐dichloroacetanilide (3,5‐DCAA)] in IRCC from male Fischer 344 rats. Briefly, IRCC were isolated using a collagenase perfusion technique, and IRCC (~4.1 million cells/ml; 3 ml) were exposed to various concentrations of a putative metabolite [2A4,6DCP or 3,5‐DCAA (0.5–1.5 mM); 3,5‐DCPHA (0.25–1.0 mM)]or vehicle (DMSO) for 60 or 90 min. Cytotoxicity was determined by lactate dehydrogenase (LDH) release assays. Results showed that there were no significant changes in LDH release following exposure to 3,5‐DCAA at any time point or concentration tested. However, 2A4,6DCP was a nephrotoxicant at all concentrations tested, but only after 90 min exposure. In contrast, 3,5‐DCPHA significantly increased LDH release at concentrations of 0.5 mM or greater after 60 min and at all concentrations tested after exposure for 90 min. These results suggest that at least two putative 3,5‐DCA metabolites [3,5‐DCPHA and/or 2A4,6DCP, but not 3,5‐DCAA] may contribute to 3,5‐DCA induced nephrotoxicity in vitro.

Support or Funding Information

Supported in part by NIH grant P20GM103434
Renoprotective Effects of 4‐Phenylbutyrate Against Palmitate‐Induced Renal Cell Injury
Authors: Munusamy Shankar , Alkhiyami Dania Ihssan , Berzou Souad , Gholamhussain Atefeh , Mohamed Hebat Alla Ahmed , Saleh Alaaeldin
Published: November 2023

Background

Increased dietary intake of fats rich in saturated fatty acids and dyslipidemia have been shown to significantly aggravate the risk of cardiovascular morbidity and mortality in patients with chronic kidney disease (CKD). Although multiple mechanisms including endoplasmic reticulum (ER) stress and oxidative stress are implicated in the pathophysiology of CKD, the effects of elevated free fatty acid (FFA) levels per se on renal cells is still unclear. Hence, we investigated the effects of palmitic acid (a saturated fatty acid) on rat renal proximal tubular cells in vitro, and tested the renoprotective effects of 4‐phenylbutyrate (4‐PBA), a chemical chaperone shown to inhibit ER stress in vitro and in vivo, in our model.

Methods

Subconfluent rat renal proximal tubular (NRK‐52E) cells were exposed to 100 mM palmitic acid in presence and absence of 5 mM 4‐PBA for 24 hours. Cells were then assessed for alterations in cell viability (using MTT assay), oxidative stress (via Dichlorofluorescein (DCF) staining) and the expression of ER stress markers (i.e., glucose‐regulated protein, GRP78 and C/EBP homologous protein, CHOP) and apoptosis (caspase‐3 and ‐12) using western blotting. Flow cytometry for cell cycle analysis and immunoblotting for p27 (an inhibitor of cell cycle) were performed to determine the effects of palmitate and 4‐PBA on the cell cycle in renal cells.

Results

Renal cells exposed to palmitate for 24 hours showed decreased viability (76.9% to that of control), increased DCF staining (1.3‐fold increase over control), and marked upregulation of GRP78 and CHOP. Similarly, the expression of active forms of caspase‐3 (1.8‐fold increase over control) and caspase‐12 (3‐fold increase over control) were also increased in renal cells treated with palmitate. These changes were significantly attenuated in cells co‐treated with 4‐PBA. Intriguingly, exposure to palmitate did not alter the cell cycle in renal cells, whereas treatment with 4‐PBA caused downregulation of p27 (54% to that of control) and increased the transition of cells from G1 to S‐phase and G2/M phase.

Conclusions

Our findings indicate that exposure to palmitate induces ER stress as well as oxidative stress in renal cells, and results in renal cell apoptosis. Co‐treatment with 4‐PBA protects renal cells against the perturbations induced by palmitate and improves renal cell viability, which underscores the renoprotective effects of 4‐PBA against fatty acid‐induced renal cell injury. Further studies are required to understand the mechanisms by which 4‐PBA prevents oxidative stress and alters cell cycle in renal cells.

Support or Funding Information

Supported by student research grants # QUST‐CPH‐FALL‐12/13‐6 and QUST‐CPH‐SPR‐12/13‐3 from the Office of Academic Research, Qatar University.
CaMKK2 activates oncogenic Akt in ovarian cancer cells and is a promising therapeutic target
Authors: Gocher Angela Marie , Azabdaftari Gissou , Karacosta Loukia , Franke Thomas F , Edelman Arthur M
Published: November 2023

Background

Ca2+/Calmodulin‐dependent protein kinase kinase 2 (CaMKK2) is highly expressed in prostate and breast cancer and has been shown to regulate cell proliferation and survival. In ovarian cancer (OvCa) cells CaMKK2 regulates G1/S phase cell cycle progression and inhibits apoptosis (preliminary results). Akt (PKB) is an oncogenic pro‐survival protein kinase that is commonly hyperactive in OvCa and contributes to resistance to first line platinum chemotherapy. Akt is activated by the canonical PI3K/PDK1 cascade resulting in Akt phosphorylation at its primary site Thr308. Inhibitors of the canonical Akt activation pathway are in clinical trials.

Results

We report here that RNAi‐mediated knockdown of CaMKK2 expression in OVCAR‐3 OvCa cells decreased phosphorylation of Akt at Thr308 (p‐Akt Thr308) (59.3%) to a similar extent as knockdown of PDK1 (50.4%) suggesting that in OvCa cells CaMKK2 may play a role comparable to that of PDK1 in maintaining Akt activity. Combined CaMKK2 and PDK1 knockdowns resulted in an additive (80.7%) decrease in p‐Akt Thr308 suggesting that CaMKK2 regulates Akt phosphorylation independently of the PDK1 pathway, a conclusion confirmed in another OvCa cell line SKOV‐3. Additive effects of CaMKK2 and PDK1 knockdowns on p‐Akt at the secondary activation site Ser473 were also achieved. The ability of recombinant purified Akt to phosphorylate an Akt specific peptide substrate was enhanced at least 9‐fold by purified CaMKK2, whereas kinase activity of Akt T308A/S473A was not increased by CaMKK2. In this assay direct phosphorylation of Akt Thr308 by CaMKK2 was confirmed by Western blotting. Pharmacological inhibition of CaMKK2 with STO‐609 resulted in decreased p‐Akt Thr308 and Ser473 as did the intracellular calcium chelator BAPTA‐AM and the CaM inhibitor W7 in OVCAR‐3 cells. STO‐609 treatment reduced the growth and viability of OVCAR‐3 cells. CaMKK2 knockdown in platinum‐resistant OVCAR‐3 cells significantly increased sensitization to the anti‐proliferative effects of carboplatin and of the PI3K inhibitor PX‐866, reducing the EC50 of PX‐866 more than 2‐fold.

Conclusions

These data show that CaMKK2 regulates cell growth and viability of OvCa cells and directly phosphorylates Akt Thr308 to promote its activity via a non‐PDK1‐dependent pathway. CaMKK2 silencing/inhibition potentiates the anti‐proliferative effects of compounds used to treat OvCa (carboplatin) or in development (PX‐866). CaMKK2 may be a novel therapeutic target for OvCa in combination with the PI3K inhibitors currently in clinical trials and/or with carboplatin.

Support or Funding Information

UBF
In Vitro Validation of Cancer Immunotherapy Reagents Using an Impedance‐Based Platform
Authors: Cerignoli Fabio , Zhu Jenny , Guenther Garret , Radecke Chris , Xi Biao , Zhao Leyna , Abassi Yama
Published: November 2023

Immunotherapy is emerging as one of the most promising approaches in cancer treatment, allowing specific targeting of cancer cells from immune cells, mediated by surface marker expression. In vitro characterization of the efficacy and potency of cancer immunotherapy reagents is a necessary step before moving to animal models and Phase I and II clinical studies. In this study we report the validation and optimization of protocols for analyzing the efficacy of reagents designed to stimulate the immune response against cancer cells using an impedance‐based and label‐free platform. The platform allows detection of cell adhesion and cell death trough monitoring the change in conductance of microelectrodes embedded in the bottom of 96‐wells cell culture plates. Because cell adhesion alters the conductivity of the electrodes, any change in cell morphology, cell adhesion or cell number is detected as variation of an impedance‐related parameter named Cell index (Ci). The platform avoids the use of any chemical label or sample processing, allowing continuous monitoring of cell activity over prolonged time periods. Furthermore, the untouched cells are still available for secondary assays that can better clarify the biological mechanism involved in the response.

To mimic tumors that are target of immunotherapy efforts we utilized several carcinoma cell lines whit different level of expression of the EpCAM membrane protein. Such antigen is overexpressed in many tumors and is currently under evaluation in clinical trials. In PC3 prostate cancer cells we measured cell cytotoxicity of Primary Blood Mononuclear Cells (PBMCs) mediated by an EpCAM/CD3 Bispecific T cell Engager (BiTE) antibody. The use of the impedance‐based platform allowed measuring the full kinetic of the cytotoxic activity under different effector:target ratios and BiTE concentrations. Apoptosis of tumor cells was confirmed trough flow cytometer analysis. We also demonstrated the relation between amount of PBMCs cytotoxic activity and EpCAM surface expression trough testing MCF7 (high EpCAM expressor) and HeLa (low EpCAM expressor).

To evaluate cancer immunotherapy protocols in lymphomas and leukemia, we developed a protocol where lymphomas cells are immobilized on electrodes‐coated plates and the T‐Lymphoblast cell line TALL‐104 is used as cytotoxic effector.

Taken together our results demonstrate high sensitivity of the impedance‐based approach in measuring cytotoxic activity in sophisticated high throughput set up. Furthermore, the availability of continuous recording of the Cell Index over the temporal scale allows better characterization, comparing to alternative end point assays, of construct efficacy.
Sex Differences in Sympathetic Neurotransmission in Normotensive Rat Mesenteric Arteries
Authors: Gebremedhin Kibrom , Galligan James J , Xu Hui
Published: November 2023

Hypertension is less common in premenopausal women than in men possibly due to high estrogen levels in females. The diameter of mesenteric arteries (MA) is an important determinant of system blood pressure and sympathetic nerve activity is a major determinant of MA diameter. Sympathetic nerve supplying MA uses norepinephrine (NE) and ATP as vasoconstrictor transmitters in male rats. We tested the hypothesis that noradrenergic/purinergic neuroeffector transmission to MA also occurs in female rats. Young (10–14 week), normotensive Sprague Dawley male and female rats were used. The estrus cycle stage in female was determined cytologically by vaginal lavage before euthanasia. MA (200 – 250 μm inner diameter) were harvested in a physiological buffer solution and mounted in a pressure (60 mmHg) myograph and inner diameter was measured continuously. Electrical field stimulation (30 stimuli at 0.2–30Hz) and drug addition to the chamber were used to constrict MA. Neurogenic constriction was greater in male compared to female arteries. Neurogenic constriction did not vary with the estrus cycle in female rats. Neurogenic constrictions were blocked by combined application of prazosin (0.1 μM, α1‐adrenergic receptor antagonist) and PPADS (10 μM, P2X‐purinergic receptor antagonist) as well as suramin (100 μM, P2X and P2Y‐purinergic receptor antagonist) in arteries from male and female rats. PPADS/Suramin inhibited ATP and α,β‐methylene ATP, but not KCl, ‐induced MA contraction. Constrictions caused by exogenous NE, ATP,α,β‐methylene ATP or elevated KCl were similar in MA from male and female rats. Immunostaining for tyrosine hydroxylase (a sympathetic nerve marker) revealed no observable differences in nerve density and distribution in MA from male and female rats. Our data indicate that there are fundamental differences in the mechanisms of neurogenic vasoconstriction in MA of male and female rats. Differences in neurotransmission and/or nerve fiber distribution in male and in female rats might contribute to and explain the sex differences in hypertension prevalence between human males and females.

Support or Funding Information

Supported by NHLBI 2P01HL07687
Burn Injury Alters Cardiac β‐Adrenergic Receptor Signaling in Interleukin 6 Knockout Mice
Authors: Guillory Ashley N , El Ayadi Amina , Porter Craig , Herndon David N , Finnerty Celeste C
Published: November 2023

Introduction

Burn injury disrupts many signaling pathways including those that are β‐adrenergic receptor (β1‐AR) dependent. Studies performed in various animal models have shown that burn injury causes cardiac dysfunction primarily attributed to altered β‐AR and pro‐inflammatory signaling. Interleukin 6 is involved in the regulation of cardiac hypertrophy as well as the development of heart failure, both of which are also regulated by β‐ARs.

Methods

Male wild‐type and interleukin 6 knockout mice received a 30% total body surface area full thickness burn. Animals were sacrificed and hearts were isolated 10 days post injury. We examined the expression of β‐ARs and downstream proteins via Western blotting. Protein kinase A activity was measured in cytosolic fractions using an enzyme immunoassay kit.

Results

β1‐AR protein expression was significantly higher in the knockout animals after burn injury (p=0.01). Protein kinase A activity and Troponin I phosphorylation were also significantly higher in the interleukin 6 knockout mice post burn (p=0.003; 0.03). Conversely, β2‐AR protein expression was significantly lower in the burned knockout animals (p=0.03). Sarcoplasmic reticulum calcium ATPase protein expression was significantly lower in both the wild‐type and interleukin 6 knockout mice after burn injury (p=0.003).

Conclusion

These data indicate that interleukin 6 may play a role in the disruption of cardiac β‐AR signaling including proteins involved in calcium handling after burn injury. Burn induced cardiac dysfunction has been well documented but is not clearly understood. Understanding how these signaling pathways are altered post injury may provide insight to the appropriate method of treatment.

Support or Funding Information

External Funding: Shriners Hospitals for Children (84291,80500, 84202,71008); National Institutes of Health (R01GM056687, R01HD049471‐07, R01AR065270, R01GM112936)
Superoxide via Sp3 but Not NF‐Kb Regulates Renal AT1 Receptor Expression and Function in Human Kidney Cells
Authors: Saleem Mohammad , Asghar Mohammad
Published: November 2023

Oxidative stress plays a causal role in higher Angiotensin II type 1 receptor (AT1R) function and hypertension. Both redox‐sensitive transcription factors have been implicated in regulation of AT1R. Here, we tested a hypothesis that superoxide but hydrogen peroxide (H2O2) via Sp3 but not NF‐kB p‐65 transcription factor regulates renal AT1R. We tested our hypothesis in human kidney (HK2) cells by undertaking following studies. Diethyldithiocarbamate (DDC), a superoxide prodrug, but not H2O2 increased nuclear accumulation of both Sp3 and NF‐kB in HK2 cells as measured by immunoblotting. Importantly, this effect was attenuated with Tempol, a superoxide mimetic. Further, DDC increased AT1R mRNA and protein levels that were attenuated with Tempol treatment, measured respectively by RT‐qPCR and immunoblotting. However, H2O2 did not show any significant effect on AT1R mRNA and protein levels. Moreover, Sp3 plasmid increased while Sp3 siRNA decreased AT1R protein levels in HK2 cells. Interestingly, NF‐kB plasmid did not show any significant effect on AT1R protein levels. Moreover, NF‐kB shRNA and PDTC, an NF‐kB inhibitor, both increased AT1R mRNA, suggesting an inhibitory effect of NF‐kB on AT1R. In addition, DDC treatment showed an exaggerated response of Ang II on protein kinase C (PKC) activity, an indicator of AT1R function, which was attenuated with Tempol treatment. Collectively, our results suggest that superoxide but not H2O2 via Sp3 but not NF‐kB up‐regulate AT1 receptor expression and function that may contribute to hypertension.

Support or Funding Information

NIH/NIA: AG039836
Gamma Linoleic Acid and Trans Linoleic Acid Effects on Atherogenic Dieted Rabbit Tissue Sensitivity to Insulin
Authors: Beard Elizabeth Letitia , Swafford Albert Nicholas
Published: November 2023

The effects of adding 5% Primrose oil‐(PO) [ containing 9% Gamma Linolenic Acid (GLA)] and/or 5% Soy Oil (SO) [containing 35% Trans Linoleic acid (TLA)] ‐to the Atherogenic Diet (AD) fed to rabbits for a 30 day interval, were studied regarding their ability to alter their tissue sensitivity to insulin. New Zealand strain male rabbits were divided into diet groups with four animals/group. Group I‐(AD Control ) rabbits were maintained on AD containing 92% Purina Rabbit Chow, 6% Corn Oil and 2% Cholesterol. Group II (AD+SO) animals were fed AD + 5% SO, Group III (AD+PO) animals received AD+ 5%.PO, and Group IV (AD+SO+PO), rabbits were fed AD + 2.5% PO and 2.5% SO. Blood platelet samples were isolated from the blood plasma samples drawn from each group. These were incubated in media with exogenous insulin and glucose then washed, homogenized and assayed for their glucose content. The mean glucose values determined in the platelet homogenates were: I. (AD Control ) : 0.41mg/dl, II. (AD+SO ): 0.51mg/dl, III. (AD+ PO): 1.09 mg/dl and IV. (AD + {2.5%PO + 2.5% SO): 0.96 mg/dl. The mean cholesterol levels found in the blood plasma samples of these groups were: I. (AD Controls): 72.6mg/dl. II. (AD+ SO): 73.5 mg ‘dl, III. (AD+PO): 52mg/dl, and in IV. (AD+ (2.5%SO and 2.5%PO), the level rose to 59 mg/dl. Blood platelet mean cholesterol levels were : I. (AD Control) :75.6mg/dl, II. (AD +SO): 56.3mg/dl, III. (AD+PO) values fell to 33.3 mg/dl, and IV. (AD+2.5%PO+2.5%SO): 33.9mg/dl. Rabbits fed AD with PO/GLA supplement, were found to have reduced plaque development in their aortae at autopsy compared to the those lacking GLA supplement in the diet. GLA supplementation to an atherogenic diet appears in these findings to work against the development of hypercholesterolemia and decresased sensitivity to insulin that develops in rabbits with prolonged feeding of an atherogenic diet containing 2% cholesterol as well as atherogenic plaque build up in the aorta..

Support or Funding Information

American Heart Association‐ Louisiana
Sigma Receptor Agonism by Afobazole Attenuates Lymphatic Pumping in Juvenile Rat Mesenteric Collecting Vessels
Authors: Trujillo Andrea Nichole , Adderley Shaquria P , Katnik Christopher P , Cuevas Javier , Breslin Jerome W
Published: November 2023

The mechanisms that govern tone and phasic contractions of collecting lymphatics are known to involve transient increases in intracellular free Ca2+ but are still poorly understood. The role of sigma (σ) receptors, chaperone proteins recently identified to modulate intracellular free Ca2+ has not been previously explored. We studied mesenteric collecting lymphatics isolated from 5–9 week old male Sprague Dawley rats, investigating whether: 1) sigma receptors, are expressed in lymphatic collecting vessels; and 2) Afobazole modulates tone and pumping. Lymphatics were mounted onto resistance‐matched glass micropipettes, in a 37°C albumin physiological salt solution (APSS) bath with 1–2 cm H2O luminal pressure. Concentrations of 50, 100 and 150 μM Afobazole were assessed. Video recordings were used to obtain contraction frequency (CF), end diastolic diameter (EDD), end systolic diameter (ESD), phasic contraction amplitude (AMP), tone, and ejection fraction (EF). Ca2+‐free APSS was used to obtain the maximal passive diameter (MaxD) used to normalize EDD, ESD and AMP. Expression of the sigma 1 (σ1) receptor in lymphatics was verified by q‐PCR and western blot. Afobazole (50–150 μM) decreased CF, AMP/MaxD, tone and EF, and increased ESD/MaxD compared to baseline. A 15‐min pre‐treatment with either BD 1047 dihydrobromide or BD 1063 dihydrochloride, both σ1 receptor antagonists, blocked the effects produced by 50 and 100 μM Afobazole. Similar pre‐treatment with SM‐21, a specific antagonist of the σ2 receptor, did not block the responses to Afobazole (50–100 μM). Lastly, using multi‐photon laser‐scanning microscopy, the σ1 receptor was detected in lymphatic collecting vessels, closely associated with the endothelium. This study shows, for the first time, that agonists of the σ1 receptor can negatively modulate juvenile mesenteric lymphatic vessel contractile function.

Support or Funding Information

Supported by NIH grant L40HL097863
Impact of Dynamic Transmural Pressure on Lymph Propulsion in Lymphatic Vessels
Authors: Jamalian Samira , Davis Michael J , Zawieja David C , Moore James E
Published: November 2023

Transmural pressure is an important determinant of pumping in collecting lymphatic vessels. Here we studied the impact of dynamic transmural pressure in lymphatic vessels using our physiology‐based computational model [1]. In parallel, we conducted in vitro studies in isolated lymphatic vessels from rat mesentery to examine the effect of dynamic transmural pressure. Vessel segments containing multiple valves were isolated, cannulated, and pressurized at varying levels of inlet and outlet pressures (Pin = Pout, zero transaxial pressure difference). Dynamic pressure waves at different physiologic frequencies (0 – 0.5 Hz) and amplitudes (0 – Pin) were introduced at the inlet and outlet reservoirs. Intraluminal pressure (measured with a servo‐null system) and inner diameter were recorded throughout the experiment. At all pressure amplitudes and frequencies (both higher and lower than normal contraction frequency of the vessel), the dynamic pressure wave was superimposed on the physiologic contraction of the vessel and was detectable in both the intraluminal pressure and diameter traces. The results of our computational model demonstrated that dynamic transmural pressure increased the flow rate at low baseline transmural pressures. Under these circumstances increasing the amplitude and/or frequency of oscillations further facilitated the pumping. Additionally, our computational model enables us to control the transmural pressure by varying external pressure (independent of inlet and outlet pressures). This allows us to study conditions with non‐zero transaxial pressure differences. For example, for an adverse pressure difference of 3 cmH2O and average transmural pressure of 3 cmH2O, average flow rate increased 10% when transmural pressure oscillated with an amplitude of 1 cmH2O; doubling the contraction frequency resulted in a further 15% higher flow rate. These results suggest that dynamic transmural pressure can play an important role in maintaining lymph propulsion in areas of weaker intrinsic pumping activity.

Support or Funding Information

Grant support: NIH R01 HL094269, U01 HL123420 (JEM), NIH R01 HL‐120867 (MJD). JEM wishes to acknowledge the support of The Royal Society, The Royal Academy of Engineering, and The Sir Leon Bagrit Trust.
A Novel Mouse Model of Inducible Lymphangiogenesis Through Controlled Local Expression of VEGF‐D
Authors: Rutkowski Joseph M
Published: November 2023

Lymphatic vessels are an integral part of the body's circulatory system. Lymphatic circulation modulates tissue fluid balance and inflammation and provides a conduit for endocrine and lipid transport as well as a pathway for cancer cell metastasis. The growth of new lymphatic vessels in the adult – lymphangiogenesis – is predominantly mediated through vascular endothelial growth factor receptor‐3 (VEGFR‐3) signaling upon binding of its ligands VEGF‐C and VEGF‐D. Lymphangiogenesis may be therapeutic in conditions of lymphatic deficiency (e.g., post‐lymphadenectomy), but may also be a negative driver of lymphatic proliferative pathologies such as GLA, Gorham‐Stout disease, and lymphangioleiomyomatosis. While human VEGF‐C and –D both can bind VEGFR‐2 as well, mouse VEGF‐D only binds VEGFR‐3. We took advantage of this unique interaction and generated mice capable of inducible, tissue‐specific expression of VEGF‐D under a tightly‐controlled tetracycline response element (TRE) promoter. This limits VEGF‐D expression to be cell‐specific and only upon doxycycline (administered in the water or chow) activation, thus providing a novel model of local lymphangiogenesis. We confirmed tight, dose‐responsive VEGF‐D expression with the TRE‐VEGF‐D mouse crossed to mice with tissue‐specific promoters for the lung (CCSP‐rtTA), kidney (KSP‐rtTA), and adipose tissue (adiponectin‐rtTA). Induction of VEGF‐D expression in these tissues resulted in marked lymphatic endothelial cell massive lymphatic vessel hyperplasia and hypertrophy. The inducible TRE‐VEGF‐D mouse thus provides a novel murine platform to study the adult mechanisms and therapies of an array of disease‐ and tissue‐specific models of lymphangiogenesis.

Support or Funding Information

AHA 12SDG12050287
Morphometric Analyses of Longitudinal and Circular Smooth Muscle and Lacteals of the Small Intestine of Zucker Lean and Obese Rats
Authors: Burgess Andrea R , Trujillo Andrea N , Barter Micaela , Breslin Jerome W
Published: November 2023

Lymph lacteals are the entry point of absorbed fats and cholesterol in the villi of the small intestine. In a recent study, lacteals and the leakiness of the submucosal lympahtic network was implicated as a source for adult‐onset obesity (Harvey et al. 2005). The purpose of this study was to determine what structural differences may exist in the lacteals, submucosa, and other layers of the ileum between lean and obese Zucker rats using histochemistry (H&E) and immunofluorescence microscopy. We hypothesized that due to increased food intake and levels of digested fats in the obese Zucker rat, the lacteals and the smooth muscle layers of the ileum will modify from their normal appearance to accommodate the increased intake of fats and volume of food respectively. Ileum from lean and obese Zucker rats was harvested, fixed in buffered formalin overnight, processed and embedded in paraffin. Cross sections (2–3 μm) of the ileum were mounted on slides and stained with H&E to measure longitudinal and circular smooth muscle layer thickness. Similar sections were incubated with a LYVE‐1 antibody to identify lymphatic endothelial cells of the lacteals and a smooth muscle actin (SMA) antibody to label smooth muscle cells extending into the villi. Layers of longitudinal and circular smooth muscle, identified in the ileum with H&E staining, were significantly thicker in obese Zucker rats compared to lean rats. Preliminary immunolabeling with LYVE‐1 identified positive endothelial cells in crypts of the sub mucosae and SMA‐positive smooth muscle cells that extended into the villi (lamina propria). SMA was also detected in the circular and longitudinal muscle layers and the muscularis muscosae as predicted. Histological data obtained from the ileum support the hypothesis that increased food intake increases intestinal smooth muscle layer thickness in obese Zucker rats.

Support or Funding Information

This work was supported by an APS/NHLBI STRIDE Undergraduate Summer Research Fellowship (NIH R25HL115473) and NIH grant L40HL097863.
IGF‐1 deficiency exacerbates hypertension‐induced cerebromicrovascular rarefaction in mice: implications for cognitive decline
Authors: Tarantini Stefano , Giles Cory , Toth Peter , Ashpole Nicole M , Tucsek Zsuzsanna , Valcarcel‐Ares Marta Noa , Gautam Tripti , Sonntag William E , Wren Jonathan D , Csiszar Anna , Ungvari Zoltan
Published: November 2023

Aging is associated with marked deficiency in circulating insulin‐like growth factor (IGF‐1), which has been shown to contribute to age‐related cognitive decline. The mechanisms by which IGF‐1 deficiency impairs brain function are multifaceted and likely involve impairment of cerebral microcirculation. Accordingly, previously we have shown that IGF‐1 deficiency impairs cerebromicrovascular endothelial function, neurovascular coupling and adaptation of myogenic autoregulatory mechanisms to hypertension, contributing to blood‐brain barrier disruption and neuroinflammation. The present study was designed to characterize the effects of IGF‐1 deficiency on microvascular density in the brain and its pathogenic role in hypertension‐induced microvascular injury. Circulating IGF‐1 deficiency was induced in mice by AAV‐mediated knockdown of IGF‐1 in the liver (Igf1f/f+TBG‐Cre‐AAV8). Hypertension was induced by chronic infusion of angiotensin‐II. We found that circulating IGF‐1 deficiency is associated with decreased microvascular density and exacerbates hypertension‐induced microvascular rarefaction in the cortex and the hippocampus. Hippocampal microvascular rarefaction positively correlated with impairment of hippocampal‐dependent cognitive function. To elucidate the mechanisms underlying the synergistic effects of hypertension and IGF‐1 deficiency on microvascular density hippocampal mRNA expression of genes involved in regulation of angiogenesis and microvascular regression was assessed using a targeted qPCR array. Both hypertension and IGF‐1 deficiency appear to decrease the expression of a number of genes involved in the maintenance of the structural integrity of the cerebral microcirculation (e.g. Vegfb, Pdgfrb), while increasing the expression of genes promoting endothelial apoptosis and/or microvascular regression (e.g. Tnfa, Angpt2). Gene set enrichment analysis scores calculated for positive and negative regulators of angiogenesis suggest that interaction of hypertension in IGF‐1 deficiency results in an exacerbated negative angiogenesis signature. Collectively, IGF‐1 deficiency promotes cerebromicrovascular rarefaction and exacerbates hypertension‐induced structural damage to the cerebral microcirculation, which likely contribute to the deleterious effects of age‐related IGF‐1 deficiency on higher brain function in the elderly.

Support or Funding Information

This work was supported by grants from the American Heart Association and the National Institute on Aging