Tissue Clearance Research Articles

Bystander Selection for Antimicrobial Resistance: Implications for Patient Health.

Antimicrobial therapy promotes resistance emergence in target infections and in off-target microbiota. Off-target resistance emergence threatens patient health when off-target populations are a source of future infections, as they are for many important drug-resistant pathogens. However, the health risks of antimicrobial exposure in off-target populations remain largely unquantified, making rational antibiotic stewardship challenging. Here, we discuss the contribution of bystander antimicrobial exposure to the resistance crisis, the implications for antimicrobial stewardship, and some novel opportunities to limit resistance evolution while treating target pathogens.

Detection of tumors with fluoromarker-releasing bacteria.

Combining the specificity of tumor-targeting bacteria with the sensitivity of biomarker detection would create a screening method able to detect small tumors and metastases. To create this system, we genetically modified an attenuated strain of Salmonella enterica to release a recombinant fluorescent biomarker (or fluoromarker). Salmonella expressing ZsGreen were intravenously administered to tumor-bearing mice and fluoromarker production was induced after 48 hr. The quantities and locations of bacteria and ZsGreen were measured in tumors, livers and spleens by immunofluorescence, and the plasma concentration of ZsGreen was measured using single-layer ELISA. In the plasma, the ZsGreen concentration was in the range of 0.5-1.5 ng/ml and was dependent on tumor mass (with a proportion of 0.81 ± 0.32 ng·ml-1 ·g-1 ). No adverse reaction to ZsGreen or bacteria was observed in any mice. ZsGreen was released at an average rate of 4.3 fg·CFU-1 ·hr-1 and cleared from the plasma with a rate constant of 0.259 hr-1 . ZsGreen production was highest in viable tissue (7.6 fg·CFU-1 ·hr-1 ) and lowest in necrotic tissue (0.47 fg·CFU-1 ·hr-1 ). The mass transfer rate constant from tumor to blood was 0.0125 hr-1 . Based on these measurements, this system has the capability to detect tumors as small as 0.12 g. These results demonstrate four essential mechanisms of this method: (i) preferential tumor colonization by bacteria, (ii) fluoromarker release in vivo, (iii) fluoromarker transport through tumor tissue and (iv) slow enough systemic clearance to enable measurement. This bacteria-based blood test would be minimally invasive and has the potential to identify previously undetectable microscopic tumors.

Measuring microdose ABY-029 fluorescence signal in a primary human soft-tissue sarcoma resection.

Microdose administration of ABY-029, an anti-epidermal growth factor receptor Affibody molecule conjugated to IRDye 800CW, is being studied in a Phase 0 trial for resection of soft-tissue sarcomas. The excised tissue of a single patient in the microdose administration group was imaged with both a wide-field fluorescence surgical system and a flat-bed scanning fluorescence imaging system. Here the resultant fluorescence from a breadloaf section of the primary tumor specimen and six region-specific tissue samples collected from that breadloaf are compared using these two imaging systems - a flatbed, black-box, fluorescence scanning system, the Odyssey CLx, and a open-air, wide-field, pre-clinical surgical imaging system, the Solaris. Florescence signal is compared using a variety of methods including: mean, standard deviation, variance, tumor-to-background ratio, biological-variance ratio, and contrast-to-noise ratio. The images produced from the Odyssey scanner have higher signal variance but more accurately represent the EGFR expression in small tissue sections. The Solaris system has higher depth sensitivity and volume averaging, and as such has lower signal variation and higher contrast-to-noise ratio.

Quantification of PET infusion studies without true equilibrium: A tissue clearance correction

In some positron emission tomography (PET) studies, a reversibly binding radioligand is administered as a constant infusion to establish true equilibrium for quantification. This approach reduces scanning time and simplifies data analysis, but assumes similar behavior of the radioligand in plasma across the study population to establish true equilibrium in all subjects. Bias in outcome measurements can result if this assumption is not met. This work developed and validated a correction that reduces bias in total distribution volume (VT) estimates when true equilibrium is not present. This correction, termed tissue clearance correction (TCC), took the form , where β is the radioligand clearance rate in tissue, γ is a radiotracer-specific constant, and VT(A) is the apparent VT. Simulations characterized the robustness of TCC across imperfect values of γ and β and demonstrated reduction to false positive rates. This approach was validated with human infusion data for three radiotracers: [18F]FPEB, (−)-[18F]flubatine, and [11C]UCB-J. TCC reduced bias in VT estimates for all radiotracers and significantly reduced intersubject variance in VT for [18F]FPEB data in some brain regions. Thus, TCC improves quantification of data acquired from PET infusion studies.

Effects of Neurotrophic Factors in Glial Cells in the Central Nervous System: Expression and Properties in Neurodegeneration and Injury.

Astrocytes, oligodendrocytes, and microglia are abundant cell types found in the central nervous system and have been shown to play crucial roles in regulating both normal and disease states. An increasing amount of evidence points to the critical importance of glia in mediating neurodegeneration in Alzheimer’s and Parkinson’s diseases (AD, PD), and in ischemic stroke, where microglia are involved in initial tissue clearance, and astrocytes in the subsequent formation of a glial scar. The importance of these cells for neuronal survival has previously been studied in co-culture experiments and the search for neurotrophic factors (NTFs) initiated after finding that the addition of conditioned media from astrocyte cultures could support the survival of primary neurons in vitro. This led to the discovery of the potent dopamine neurotrophic factor, glial cell line-derived neurotrophic factor (GDNF). In this review, we focus on the relationship between glia and NTFs including neurotrophins, GDNF-family ligands, CNTF family, and CDNF/MANF-family proteins. We describe their expression in astrocytes, oligodendrocytes and their precursors (NG2-positive cells, OPCs), and microglia during development and in the adult brain. Furthermore, we review existing data on the glial phenotypes of NTF knockout mice and follow NTF expression patterns and their effects on glia in disease models such as AD, PD, stroke, and retinal degeneration.

Bacillus anthracis Protective Antigen Shows High Specificity for a UV Induced Mouse Model of Cutaneous Squamous Cell Carcinoma.

Squamous cell carcinoma (SCC) accounts for the majority of non-melanoma skin cancer related deaths, particularly in immunosuppressed persons. Identification of biomarkers that could be used to identify or treat SCC would be of significant benefit. The anthrax toxin receptors, Tumor Endothelial Marker 8 (TEM8) and Capillary Morphogenesis Gene 2 (CMG2), are endothelial receptors involved in extracellular matrix homeostasis and angiogenesis that are selectively upregulated on numerous tumors. One method of targeting these receptors is Protective Antigen (PA), a protein produced by B. anthracis that mediates binding and translocation of anthrax toxins into cells. PA targeted toxins have been demonstrated to selectively inhibit tumor growth and angiogenesis, but tumor selectivity of PA is currently unknown. In this work fluorescently labeled PA was shown to maintain receptor dependent binding and internalization in vitro. Utilizing a human papillomavirus transgenic mouse model that develops cutaneous SCC in response to ultraviolet irradiation we identified tumor uptake of PA in vivo. The intravenously administered PA resulted in tumor specific localization, with exclusive tumor detection 24 h post injection. Ex vivo analysis identified significantly higher fluorescence in the tumor compared to adjacent healthy tissue and major clearance organs, demonstrating low non-specific uptake and rapid clearance. While both TEM8 and CMG2 were observed to be overexpressed in SCC tumor sections compared to control skin, the intravenously administered PA was primarily co-localized with TEM8. These results suggest that PA could be systemically administered for rapid identification of cutaneous SCC, with potential for further therapeutic development.

Synthesis of [18F]Favipiravir and Biodistribution in C3H/HeN Mice as Assessed by Positron Emission Tomography

Favipiravir (T705; 6-fluoro-3-hydroxypyrazine-2-carboxamide) is a pyrazine analog that has demonstrated potent antiviral activity against a broad spectrum of viruses in multiple in vivo disease models. To better understand the compounds anti-viral activity, assessment of the drug’s biodistribution and kinetics in vivo may lend insight into how best to evaluate the compound efficacy preclinically and to contribute to the design of clinical studies to take into account the compound’s pharmacokinetic distribution and kinetics. In the current study, a method for synthesis of [18F]favipiravir was developed and the biodistribution in mice naïve to and pre-dosed with favipiravir was assessed by PET and gamma counting of tissue samples. Fluorine-18 labeling of favipiravir was achieved in a one-pot, two-step synthesis using a commercially available precursor, methyl-5-chloroisoxazolo[4,5-b]pyrazine-3-carboxylate, with an overall radiochemical yield of 15–24%, a molar activity of 37–74 GBq/µmol in a 70 minute synthesis time. [18F]favipiravir tissue uptake and distribution was similar in naïve and pre-dosed mice; however, in the pre-dosed animals plasma clearance was more rapid and tissue clearance appeared to be prolonged. In conclusion, application of PET to the evaluation of favipiravir has demonstrated the importance of dosing regimen on the distribution and tissue uptake and clearance of the molecule. Favipiravir is cleared through the kidney as previously reported but the liver and intestinal excretion may also play an important role in compound elimination. Measurement of the tissue uptake of favipiravir as determined by PET may be a more important indicator of a compound’s potential efficacy than purely monitoring plasma parameters such as viremia and drug levels.

Phenotypic analysis of Myo10 knockout (Myo10tm2/tm2) mice lacking full-length (motorized) but not brain-specific headless myosin X

We investigated the physiological functions of Myo10 (myosin X) using Myo10 reporter knockout (Myo10tm2) mice. Full-length (motorized) Myo10 protein was deleted, but the brain-specific headless (Hdl) isoform (Hdl-Myo10) was still expressed in homozygous mutants. In vitro, we confirmed that Hdl-Myo10 does not induce filopodia, but it strongly localized to the plasma membrane independent of the MyTH4-FERM domain. Filopodia-inducing Myo10 is implicated in axon guidance and mice lacking the Myo10 cargo protein DCC (deleted in colorectal cancer) have severe commissural defects, whereas MRI (magnetic resonance imaging) of isolated brains revealed intact commissures in Myo10tm2/tm2 mice. However, reminiscent of Waardenburg syndrome, a neural crest disorder, Myo10tm2/tm2 mice exhibited pigmentation defects (white belly spots) and simple syndactyly with high penetrance (>95%), and 24% of mutant embryos developed exencephalus, a neural tube closure defect. Furthermore, Myo10tm2/tm2 mice consistently displayed bilateral persistence of the hyaloid vasculature, revealed by MRI and retinal whole-mount preparations. In principle, impaired tissue clearance could contribute to persistence of hyaloid vasculature and syndactyly. However, Myo10-deficient macrophages exhibited no defects in the phagocytosis of apoptotic or IgG-opsonized cells. RNA sequence analysis showed that Myo10 was the most strongly expressed unconventional myosin in retinal vascular endothelial cells and expression levels increased 4-fold between P6 and P15, when vertical sprouting angiogenesis gives rise to deeper layers. Nevertheless, imaging of isolated adult mutant retinas did not reveal vascularization defects. In summary, Myo10 is important for both prenatal (neural tube closure and digit formation) and postnatal development (hyaloid regression, but not retinal vascularization).

The mannose 6-phosphate/insulin-like growth factor 2 receptor mediates plasminogen-induced efferocytosis.

The plasminogen system is harnessed in a wide variety of physiological processes, such as fibrinolysis, cell migration, or efferocytosis; and accordingly, it is essential upon inflammation, tissue remodeling, wound healing, and for homeostatic maintenance in general. Previously, we identified a plasminogen receptor in the mannose 6‐phosphate/insulin‐like growth factor 2 receptor (M6P/IGF2R, CD222). Here, we demonstrate by means of genetic knockdown, knockout, and rescue approaches combined with functional studies that M6P/IGF2R is up‐regulated on the surface of macrophages, recognizes plasminogen exposed on the surface of apoptotic cells, and mediates plasminogen‐induced efferocytosis. The level of uptake of plasminogen‐coated apoptotic cells inversely correlates with the TNF‐α production by phagocytes indicating tissue clearance without inflammation by this mechanism. Our results reveal an up‐to‐now undetermined function of M6P/IGF2R in clearance of apoptotic cells, which is crucial for tissue homeostasis.

Treatment of postoperative chyle leak after pancreatic surgery – A single center experience

Background: Postoperative chyle leak (CL) is a common complication in pancreatic surgery, where extensive soft tissue clearance is indispensable. Currently, the efficacy of different treatment options for CL is unclear. The aim of the present study was to evaluate treatment options of patients with CL after pancreatic surgery. Methods: CL was defined according to the recently published definition of the International Study Group of Pancreatic Surgery. 228 patients with CL or serous drainage after pancreatic surgery between 01/2014 and 03/2016 were included in this retrospective analysis. Early (≤5 days) and late drain removal were compared regarding the risk for CL and morbidity. A subgroup analysis on those patients who had drain removal despite of persistent CL with respect to the need of subsequent CT-guided drainage within three months postoperatively was performed. Results: At total of 60 patients with CL was identified. 41 patients with CL were treated with medium-chain triglyceride-diet, with a median duration of therapy of 12 days. Early drain removal is associated with a decreased risk or detection rate of CL (p = 0.008) and is not associated with an increased morbidity. After drain removal despite of persistent CL, none of the patients had CT-drainage with proof of CL within three months postoperatively. Conclusion: Early drain removal and removal of the surgical drains despite of persistent CL may be as safe as continuous drainage until complete resolution of CL.

Increased erythrocyte aggregation following an acute bout of eccentric isokinetic exercise does not exceed two days.

Isokinetic exercise is used to reduce strength imbalance and to enhance performance. The aim of this study was to investigate the acute effects of a single bout of eccentric isokinetic exercise on hemorheology (erythrocyte deformability and aggregation), total oxidant/antioxidant status (TOS/TAS) and oxidative stress index (OSI) in active individuals. The study comprises 11 active, healthy, male subjects (mean age of 19.45 ± 0.31 years, BMI 22.05 ± 0.51 kg/m2). They performed single, unilateral eccentric contractions of knee flexors and extensors with dominant leg on a dynamometer. Isokinetic hamstring, quadriceps strength were recorded at eccentric (30, 120° s-1) angular velocities. Eight active age-matched healthy male subjects were included as a control group, who did not receive exercise. Blood samples were obtained before, immediately after and two days after the exercise session. Hemorheological parameters were measured by an ektacytometer. TOS/TAS were determined using a commercial kit. A session of eccentric isokinetic exercise did not affect erythrocyte deformability and oxidative stress indices, whereas red blood cell (RBC) aggregation was increased initially and returned to pre-exercise levels after two days following exercise. Our results suggest that, increased RBC aggregation following an acute bout of isokinetic exercise may result in increased plasma skimming that augments tissue perfusion and clearance of metabolites within a period of two days following exercise.

Failure rates for stainless steel versus titanium alloy infrazygomatic crest bone screws: A single-center, randomized double-blind clinical trial.

To compare failure rates for stainless steel (SS) and titanium alloy (TiA) bone screws (BSs) placed in the infrazygomatic crest (IZC). A total of 386 consecutive patients (76 male, 310 female; mean age 24.3 years, range 10.3-59.4 years) received IZC BSs (SS or TiA) via a double-blind, split-mouth design. BSs penetrated attached gingiva (AG) or moveable mucosa (MM) with 5 mm of soft tissue clearance. All BSs were immediately loaded and reactivated monthly with ≤14 oz (397 g or 389 cN) applied directly to the upper archwire bilaterally for 6 months to retract the maxilla to correct Class II or bimaxillary protrusion. Of the 772 devices, there were 49 (6.3%) failures: 27 SS (7.0%) and 22 TiA (5.7%). The 1.3% difference was not statistically significant ( P = .07). There was no significant relationship between SS or TiA failures relative to (1) right vs left side, (2) unilateral vs bilateral, or (3) age at failure. Significantly ( P < .05) increased failure rates were noted for SS screws in only two subgroups: AG site (7.4%) and right side (7.8%). Unilateral failure occurred in 21 patients (5.4%), and bilateral failures occurred in 14 of the total 772 patients (1.8%). The overall success rate of 93.7% indicates that both SS and TiA are clinically acceptable for IZC BSs.

SIV/SHIV-Zika co-infection does not alter disease pathogenesis in adult non-pregnant rhesus macaque model.

Due to the large geographical overlap of populations exposed to Zika virus (ZIKV) and human immunodeficiency virus (HIV), understanding the disease pathogenesis of co-infection is urgently needed. This warrants the development of an animal model for HIV-ZIKV co-infection. In this study, we used adult non-pregnant macaques that were chronically infected with simian immunodeficiency virus/chimeric simian human immunodeficiency virus (SIV/SHIV) and then inoculated with ZIKV. Plasma viral loads of both SIV/SHIV and ZIKV co-infected animals revealed no significant changes as compared to animals that were infected with ZIKV alone or as compared to SIV/SHIV infected animals prior to ZIKV inoculation. ZIKV tissue clearance of co-infected animals was similar to animals that were infected with ZIKV alone. Furthermore, in co-infected macaques, there was no statistically significant difference in plasma cytokines/chemokines levels as compared to prior to ZIKV inoculation. Collectively, these findings suggest that co-infection may not alter disease pathogenesis, thus warranting larger HIV-ZIKV epidemiological studies in order to validate these findings.

Granulocyte-macrophage colony-stimulating factor improves mouse peripheral nerve regeneration following sciatic nerve crush.

Peripheral nerve injuries severely impair patients' quality of life as full recovery is seldom achieved. Upon axonal disruption, the distal nerve stump undergoes fragmentation, and myelin breaks down; the subsequent regeneration progression is dependent on cell debris removal. In addition to tissue clearance, macrophages release angiogenic and neurotrophic factors that contribute to axon growth. Based on the importance of macrophages for nerve regeneration, especially during the initial response to injury, we treated mice with granulocyte-macrophage colony-stimulating factor (GM-CSF) at various intervals after sciatic nerve crushing. Sciatic nerves were histologically analyzed at different time intervals after injury for the presence of macrophages and indicators of regeneration. Functional recovery was followed by an automated walking track test. We found that GM-CSF potentiated early axon growth, as indicated by the enhanced expression of growth-associated protein at 7days postinjury. Inducible nitric oxide synthase expression increased at the beginning and at the end of the regenerative process, suggesting that nitric oxide is involved in axon growth and pruning. As expected, GM-CSF treatment stimulated macrophage infiltration, which increased at 7 and 14days; however, it did not improve myelin clearance. Instead, GM-CSF stimulated early brain-derived neurotrophic factor (BDNF) production, which peaked at 7days. Locomotor recovery pattern was not improved by GM-CSF treatment. The present results suggest that GM-CSF may have beneficial effects on early axonal regeneration.

Suppression of autophagy and HCK signaling promotes PTGS2high FCGR3− NK cell differentiation triggered by ectopic endometrial stromal cells

ABSTRACTImpaired NK cell cytotoxic activity contributes to the local dysfunctional immune environment in endometriosis (EMS), which is an estrogen-dependent gynecological disease that affects the function of ectopic endometrial tissue clearance. The reason for the impaired cytotoxic activity of NK cells in an ectopic lesion microenvironment (ELM) is largely unknown. In this study, we show that the macroautophagy/autophagy level of endometrial stromal cells (ESCs) from EMS decreased under negative regulation of estrogen. The ratio of peritoneal FCGR3− NK to FCGR3+ NK cells increases as EMS progresses. Moreover, the autophagy suppression results in the downregulation of HCK (hematopoietic cellular kinase) by inactivating STAT3 (signal transducer and activator of transcription 3), as well as the increased secretion of the downstream molecules CXCL8/IL8 and IL23A by ESCs, and this increase induced the upregulation of FCGR3− NK cells and decline of cytotoxic activity in ELM. This process is mediated through the depression of microRNA MIR1185-1-3p, which is associated with the activation of the target gene PTGS2 in NK cells. FCGR3− NK with a phenotype of PTGS2/COX2high IFNGlow PRF1low GZMBlow induced by hck knockout (hck−/-) or 3-methyladenine (3-MA, an autophagy inhibitor)-stimulated ESCs accelerates ESC’s growth both in vitro and in vivo. These results suggest that the estrogen-autophagy-STAT3-HCK axis participates in the differentiation of PTGS2high IFNGlow PRF1low GZMBlow FCGR3− NK cells in ELM and contributes to the development of EMS. This result provides a scientific basis for potential therapeutic strategies to treat diseases related to impaired NK cell cytotoxic activity.Abbreviations: anti-FCGR3: anti-FCGR3 with neutralizing antibody; Ctrl-ESC: untreated ESCs; CXCL8: C-X-C motif chemokine ligand 8; ectoESC: ESCs from ectopic lesion; ELM: ectopic lesion microenvironment; EMS: endometriosis; ESCs: endometrial stromal cells; eutoESC:eutopic ESCs; HCK: hematopoietic cellular kinase; HCK(OE): overexpression of HCK; IFNG: interferon gamma; IL23A (OE): overexpression of IL23A; KLRK1: Killer cell lectin like receptor K1; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; 3 -MA: 3-methyladenine; 3-MA-ESC: 3-MA-treated ESCs; MIR1185-1-3p+: overexpression of HsMIR1185-1-3p; NK: natural killer; normESCs: normal ESCs; Rap-ESC:rapamycin-treated ESCs; PCNA: proliferating cell nuclear antigen; PF: peritoneal fluid; SFKs: SRC family of cytoplasmic tyrosine kinases; si-HCK: silencing of HCK; siIL23A: silencing of IL23A; USCs: uterus stromal cells.

Intravenously-injected gold nanoparticles (AuNPs) access intracerebral F98 rat gliomas better than AuNPs infused directly into the tumor site by convection enhanced delivery.

BackgroundIntravenously (IV)-injected gold nanoparticles (AuNPs) powerfully enhance the efficacy of X-ray therapy of tumors including advanced gliomas. However, pharmacokinetic issues, such as slow tissue clearance and skin discoloration, may impede clinical translation. The direct infusion of AuNPs into the tumor might be an alternative mode of delivery.Materials and methodsUsing the advanced, invasive, and difficult-to-treat F98 rat glioma model, we have studied the biodistribution of the AuNPs in the tumor and surrounding brain after either IV injection or direct intratumoral infusion by convection-enhanced delivery using light microscopy immunofluorescence and direct gold visualization.ResultsIV-injected AuNPs localize more specifically to intracerebral tumor cells, both in the main tumor mass and in the migrated tumor cells as well as the tumor edema, than do the directly infused AuNPs. Although some of the directly infused AuNPs do access the main tumor region, such access is largely restricted.ConclusionThese data suggest that IV-injected AuNPs are likely to have a greater therapeutic benefit when combined with radiation therapy than after the direct infusion of AuNPs.

Abstract 3716: Tumor multicomponent targeting nano-micelles with synergistic combination to overcome drug resistance and reprogramming of macrophages in renal cell carcinoma

Abstract Renal Cell Carcinoma (RCC) contributes &amp;gt;90% of the most common form of kidney tumor and remains one of the ten leading causes of cancer death in the US. Tyrosine kinase inhibitors (TKIs) and mammalian target of rapamycin (mTOR) inhibitors have increased therapeutic possibilities for treating RCC. Although the impact on disease progression is encouraging, a substantial proportion of patients do not respond adequately, and therapy resistance almost inevitably occurs. Eventually, new strategies have emerged that include immunotherapy such as PD-1 inhibitor (Nivolumab) and the combination of chemo-immune therapy. Possibly, combination treatment aimed at different, non-related pathways may be advantageous. In this regard, we would like to come up with synergistic therapeutic strategies for nonresponsive, highly aggressive tumor types to tackle the current clinical challenges. We have worked on different RCCs tumor models that in general have a poor prognosis with conventional therapies. We also pursued different combination regimens, including drugs that work on the mTOR inhibition (everolimus), inhibit RTK-inhibitor or VEGFR (cabozantinib or sorafenib). Also, there is a critical need to develop safe and effective delivery vehicles that can carry the payload to the right target tissue and cell. Thus, different types of nanoparticles have been designed to deliver a variety of therapeutic agents to target hypoxic tumor microenvironment by using carbonic anhydrase-9 (CA9); FRα for targeting cancer epithelial cells; and FRβ for targeting tumor-associated macrophages (TAM) and combination delivery of RTK-inhibitor Cabozantinib (CB) and sorafenib with our own apoptosis inducer/CARP-1 protein activator CFM-4.16 (C4.16) for overcoming drug resistance and reprograming TAM for RCC therapy. The current work also focused on multimodal approaches, including (a) Optimization of hypoxia marker conjugated targeted polymer-lipid nano-formulation (PLNP) using copper-free click chemistry; (b) In vitro and in vivo pre-clinical testing of targeted -PLNP loaded with polypharmacy in inhibiting RCCs using mice bearing resistant RCCs and patient-derived xenografts (PDX). The results of efficacy and biodistribution of targeted PLNPs in animals bearing RCC xenografts and PDX model showed higher accumulation of drugs at tumor sites with higher tumor growth inhibition. Also, the targeted formulation showed high binding affinity and specific tumor uptake, faster normal tissue clearance, and less non-target organ uptake. These findings portent promising therapeutic potential of hypoxia-targeted -PLNPs for treating RCCs. Citation Format: Hashem O. Alsaab, Samaresh Sau, Vino T. Cheriyan, Rami Alzhrani, Ulka Vaishampayan, Arun K. Rishi, Arun K. Iyer. Tumor multicomponent targeting nano-micelles with synergistic combination to overcome drug resistance and reprogramming of macrophages in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3716.

Abstract 4107: Tumor multifunctional targeting polymeric nanomicelles with polypharmacy payload for effective therapy and imaging of resistant renal cell carcinoma

Abstract Renal cell carcinoma (RCC) is very difficult to treat as the cells are mostly resistant to many current therapies. Therefore, newer treatments including better ways of drug delivery are urgently needed to fight this malignant disease efficiently. The ongoing work was done by generating and characterizing RCC cells that are resistant to everolimus, a front-line mTOR-targeted therapy, and tested whether a class of CARP-1 functional mimetic (CFM) compounds inhibits parental and everolimus-resistant RCCs in combination with sorafenib. CFMs inhibited RCC cell viability in a dose-dependent manner that was comparable to everolimus treatments as well as the combination with sorafenib. CFM-4.16 inhibited viabilities of everolimus-resistant RCC cells, albeit CFM-4.16 combined with sorafenib was more effective than CFM-4.16 alone. CFM-dependent loss of RCC cell viabilities was due in part to reduced cyclin B1 levels, activation of pro-apoptotic, stress-activated protein kinases (SAPKs), and apoptosis. However, CFMs and sorafenib are hydrophobic, and their dose escalation for in vivo studies remains challenging. We addressed the solubility and delivery concerns of CFM-4.16 and sorafenib by utilizing a nanotechnology-based approach. Our data thus far revealed that we have been successful in overcoming the solubility concerns of CFM-4.16 and sorafenib by encapsulating it in water-soluble vitamin E TPGS-based micellar nanoformulation that enables high drug loading and affords its intravenous administration for animal testing. We also use our polymeric NPs formulation for in vitro efficacy in parental, wild-type and everolimus-resistant RCC. After confirming the anticancer activity of CFM-4.16-loaded nanoformulations in vitro, we finally examined the intravenous (i.v.) administration of the CFM-4.16-loaded polymeric NPs in a clinically relevant RCC mouse model. The CFM-4.16 formulation inhibited the viability of parental and everolimus-resistant RCC cells in vitro and suppressed the growth of parental A498 RCC-cell-derived xenografts (RCC animal model), in part by stimulating apoptosis. After optimizing its potency and biologic stability, we performed imaging of animal inoculated with RCC tumor and PDX model by conjugation with NIR dye. The results showed high binding affinity and specific tumor uptake, faster normal tissue clearance and less non-target organ uptake. These findings make CFM-4.16 an important potential therapeutic in combination with sorafenib using nanoformulation in the treatment of RCCs. Citation Format: Hashem O. Alsaab, Samaresh Sau, Vino T. Cheriyan, Ulka Vaishampayan, Arun K. Rishi, Arun K. Iyer. Tumor multifunctional targeting polymeric nanomicelles with polypharmacy payload for effective therapy and imaging of resistant renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4107.

Testosterone Retention Mechanism in Sertoli Cells: A Biochemical Perspective

Mechanism(s) involved in regulating Intratesticular Testosterone levels (iT) have assumed importance in recent years, from the point of view of hormonal contraception. Contraceptives using Testosterone (T) in combination with Progestins (P), for more effective suppression of pituitary gonadotropins thereby iT, are not 100% effective in suppressing spermatogenesis in human males, likely due to pesrsistence of Intratesticular Dihydrotestosterone (iD) in poor-responders. Several lacunae pertaining to the mechanism of action of principal male hormone T during spermatogenesis remain to be resolved. Notably, the mechanism through which T brings about the stage-specific differentiation of germ cells lacking Androgen Receptors (AR). Testosterone is a highly anabolic steroid with a rapid tissue clearance rate. T is intratesticular substrate for synthesis of Dihydrotestosterone (DHT) and Estradiol (E2) involved in spermtaogenesis. Therefore, it is important to delineate the mechanism(s) for retention of iT, in order to understand regulation of its bioavailability in testis. In depth studies, pertaining to the role of androgen-binding protein(s) in sequestration, retention and bioavailability of T/DHT are required to understand male fertility regulation. The appropriate approach to overcome this lacuna would be development of mice lacking functional testicular Androgen-Binding Protein (ABPKO), but not deficient T/DHT, Luteinizing Hormone (LH) and Follicle-Stimulating Hormone (FSH), in order to understand its physiological functions. Insights gained about androgen retention mechanism(s) from the ABPKO murine model will be of immense help in improving the efficacy of male hormonal contraceptives and infertility management.

ITC Commentary on Metformin Clinical Drug-Drug Interaction Study Design That Enables an Efficacy- and Safety-Based Dose Adjustment Decision.

Metformin drug-drug interaction (DDI) studies are conducted during development of drugs that inhibit organic cation transporters and/or multidrug and toxin extrusion proteins (OCTs/MATEs). Monitoring solely changes in systemic exposure, the typical DDI study endpoint appears inadequate for metformin, which is metabolically stable, has poor passive membrane permeability, and undergoes transporter-mediated tissue distribution and clearance. Evaluation of renal clearance, antihyperglycemic effects, and potentially lactate as an exploratory safety marker, can support rational metformin dose adjustment. The proposed DDI study design aims to adequately inform metformin dosing during comedication.