In Vivo Polymer Mechanochemistry with Polynucleotides.

Polymer mechanochemistry utilizes mechanical force to activate latent functionalities in macromolecules and widely relies on ultrasonication techniques. Fundamental constraints of frequency and power intensity have prohibited the application of the polymer mechanochemistry principles in a biomedical context up to now, although medical ultrasound is a clinically established modality. Here, a universal polynucleotide framework is presented that allows the binding and release of therapeutic oligonucleotides, both DNA- and RNA-based, as cargo by biocompatible medical imaging ultrasound. It is shown that the high molar mass, colloidal assembly, and a distinct mechanochemical mechanism enable the force-induced release of cargo and subsequent activation of biological function in vitro and in vivo. Thereby, this work introduces a platform for the exploration of biological questions and therapeutics development steered by mechanical force.

The prostate-specific membrane antigen holds potential as a vascular target for endogenous radiotherapy with [177Lu]Lu-PSMA-I&T for triple-negative breast cancer.

INTRODUCTION: Overexpression of prostate-specific membrane antigen (PSMA) on the vasculature of triple-negative breast cancer (TNBC) presents a promising avenue for targeted endogenous radiotherapy with [177Lu]Lu-PSMA-I&T. This study aimed to assess and compare the therapeutic efficacy of a single dose with a fractionated dose of [177Lu]Lu-PSMA-I&T in an orthotopic model of TNBC. METHODS: Rj:NMRI-Foxn1nu/nu mice were used as recipients of MDA-MB-231 xenografts. The single dose group was treated with 1 × 60 ± 5 MBq dose of [177Lu]Lu-PSMA-I&T, while the fractionated dose group received 4 × a 15 ± 2 MBq dose of [177Lu]Lu-PSMA-I&T at 7 day intervals. The control group received 0.9% NaCl. Tumor progression was monitored using [18F]FDG-PET/CT. Ex vivo analysis encompassed immunostaining, TUNEL staining, H&E staining, microautoradiography, and autoradiography. RESULTS: Tumor volumes were significantly smaller in the single dose (p < 0.001) and fractionated dose (p < 0.001) groups. Tumor growth inhibition rates were 38% (single dose) and 30% (fractionated dose). Median survival was notably prolonged in the treated groups compared to the control groups (31d, 28d and 19d for single dose, fractionated dose and control, respectively). [177Lu]Lu-PSMA-I&T decreased the size of viable tumor areas. We further demonstrated, that [177Lu]Lu-PSMA-I&T binds specifically to the tumor-associated vasculature. CONCLUSION: This study highlights the potential of [177Lu]Lu-PSMA-I&T for endogenous radiotherapy of TNBC.

The Potential of PSMA as a Vascular Target in TNBC.

Recent studies proving prostate-specific membrane antigen (PSMA) expression on triple-negative breast cancer (TNBC) cells and adjacent endothelial cells suggest PSMA as a promising target for therapy of until now not-targetable cancer entities. In this study, PSMA and its isoform expression were analyzed in different TNBC cells, breast cancer stem cells (BCSCs), and tumor-associated endothelial cells. PSMA expression was detected in 91% of the investigated TNBC cell lines. The PSMA splice isoforms were predominantly found in the BCSCs. Tumor-conditioned media from two TNBC cell lines, BT-20 (high full-length PSMA expression, PSMAΔ18 expression) and Hs578T (low full-length PSMA expression, no isoform expression), showed significant pro-angiogenic effect with induction of tube formation in endothelial cells. All TNBC cell lines induced PSMA expression in human umbilical vein endothelial cells (HUVEC). Significant uptake of radiolabeled ligand [68Ga]Ga-PSMA was detected in BCSC1 (4.2%), corresponding to the high PSMA expression. Moreover, hypoxic conditions increased the uptake of radiolabeled ligand [177Lu]Lu-PSMA in MDA-MB-231 (0.4% vs. 3.4%, under hypoxia and normoxia, respectively) and MCF-10A (0.3% vs. 3.0%, under normoxia and hypoxia, respectively) significantly (p < 0.001). [177Lu]Lu-PSMA-induced apoptosis rates were highest in BT-20 and MDA-MB-231 associated endothelial cells. Together, these findings demonstrate the potential of PSMA-targeted therapy in TNBC.

[68Ga]Ga-Pentixafor and Sodium [18F]Fluoride PET Can Non-Invasively Identify and Monitor the Dynamics of Orthodontic Tooth Movement in Mouse Model.

The cellular and molecular mechanisms of orthodontic tooth movement (OTM) are not yet fully understood, partly due to the lack of dynamical datasets within the same subject. Inflammation and calcification are two main processes during OTM. Given the high sensitivity and specificity of [68Ga]Ga-Pentixafor and Sodium [18F]Fluoride (Na[18F]F) for inflammation and calcification, respectively, the aim of this study is to assess their ability to identify and monitor the dynamics of OTM in an established mouse model. To monitor the processes during OTM in real time, animals were scanned using a small animal PET/CT during week 1, 3, and 5 post-implantation, with [68Ga]Ga-Pentixafor and Na[18F]F. Both tracers showed an increased uptake in the region of interest compared to the control. For [68Ga]Ga-Pentixafor, an increased uptake was observed within the 5-week trial, suggesting the continuous presence of inflammatory markers. Na[18F]F showed an increased uptake during the trial, indicating an intensification of bone remodelling. Interim and end-of-experiment histological assessments visualised increased amounts of chemokine receptor CXCR4 and TRAP-positive cells in the periodontal ligament on the compression side. This approach establishes the first in vivo model for periodontal remodelling during OTM, which efficiently detects and monitors the intricate dynamics of periodontal ligament.

Effects of solution conductivity on macropore size dynamics in electroporated lipid vesicle membranes.

Using fast imaging microscopy, we investigate in detail the expansion of micron-sized pores occurring in individual electroporated giant unilamellar vesicles composed of the phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). To infer pore dynamics on the electrodeformed and electropermeabilized vesicles, we develop a computational approach and provide for the first time a direct evidence of quantitative agreement between experimental data and the well-established theoretical prediction of Smith, Neu and Krassowska (SNK). The analysis we describe also provides an extension to the current theoretical literature on how the conductivity ratio of the internal and the external vesicle solution plays a determinant role in the definition of the electrical force driving pore expansion kinetics.

Radiolabeled Nanocarriers as Theranostics-Advancement from Peptides to Nanocarriers.

Endogenous targeted radiotherapy is emerging as an integral modality to treat a variety of cancer entities. Nevertheless, despite the positive clinical outcome of the treatment using radiolabeled peptides, small molecules, antibodies, and nanobodies, a high degree of hepatotoxicity and nephrotoxicity still persist. This limits the amount of dose that can be injected. In an attempt to mitigate these side effects, the use of nanocarriers such as nanoparticles (NPs), dendrimers, micelles, liposomes, and nanogels (NGs) is currently being explored. Nanocarriers can prolong circulation time and tumor retention, maximize radiation dosage, and offer multifunctionality for different targeting strategies. In this review, the authors first provide a summary of radiation therapy and imaging and discuss the new radiotracers that are used preclinically and clinically. They then highlight and identify the advantages of radio-nanomedicine and its potential in overcoming the limitations of endogenous radiotherapy. Finally, the review points to the ongoing efforts to maximize the use of radio-nanomedicine for efficient clinical translation.

Tuning the Elasticity of Nanogels Improves Their Circulation Time by Evading Immune Cells.

Peptide receptor radionuclide therapy is used to treat solid tumors by locally delivering radiation. However, due to nephro- and hepato-toxicity, it is limited by its dosage. To amplify radiation damage to tumor cells, radiolabeled nanogels can be used. We show that by tuning the mechanical properties of nanogels significant enhancement in circulation half-life of the gel could be achieved. We demonstrate why and how small changes in the mechanical properties of the nanogels influence its cellular fate. Nanogels with a storage modulus of 37 kPa were minimally phagocytosed by monocytes and macrophages compared to nanogels with 93 kPa modulus. Using PET/CT a significant difference in the blood circulation time of the nanogels was shown. Computer simulations affirmed the results and predicted the mechanism of cellular uptake of the nanogels. Altogether, this work emphasizes the important role of elasticity even for particles that are inherently soft such as nano- or microgels.

Thin-layer approximation for the multi-physics and multiscale simulation of cell membrane electrodeformation.

Multi-physics simulation techniques provide a platform that is used to gain insights into complex biological problems with multiple length scales such as cell electrodeformation (ED) and electropermeabilization (EP). However, owing to the large degrees of freedom required to compute the electromechanical properties at very different length scales (membrane thickness, cell size, and customized tissue scaffold) finite element (FE) simulations can be computationally very expensive. Here, we report on a general method of analysis by which we can systematically simulate multiscale ED under direct-current electric fields. In the context of electromechanical continuum behavior, the key novelty of our work is the introduction of a specific Dirichlet boundary condition, i.e. thin-layer approximation (TLA), to represent the capacitive elastic cell membrane. To test the robustness of this newly proposed procedure, Maxwell stress tensor (MST) and cell displacement arising from ED forces obtained with the TLA are compared with a model using a physical thickness of the cell membrane. Furthermore, we present our results in terms of benchmark points for vesicle deformation induced by an electric field excitation and we confirm our approximate results are relevant to predict the aspect ratio characterizing the ellipsoidal deformation of an initially spherical vesicle.

Small-molecule modulators of TRMT2A decrease PolyQ aggregation and PolyQ-induced cell death.

Polyglutamine (polyQ) diseases are characterized by an expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats encoding for an uninterrupted prolonged polyQ tract. We previously identified TRMT2A as a strong modifier of polyQ-induced toxicity in an unbiased large-scale screen in Drosophila melanogaster. This work aimed at identifying and validating pharmacological TRMT2A inhibitors as treatment opportunities for polyQ diseases in humans. Computer-aided drug discovery was implemented to identify human TRMT2A inhibitors. Additionally, the crystal structure of one protein domain, the RNA recognition motif (RRM), was determined, and Biacore experiments with the RRM were performed. The identified molecules were validated for their potency to reduce polyQ aggregation and polyQ-induced cell death in human HEK293T cells and patient derived fibroblasts. Our work provides a first step towards pharmacological inhibition of this enzyme and indicates TRMT2A as a viable drug target for polyQ diseases.

Modeling cell membrane electrodeformation by alternating electric fields.

With the aim of characterizing and gaining insight into the frequency response of cells suspended in a fluid medium and deformed with a controlled alternating electric field, a continuum-based analysis is presented for modeling electrodeformation (ED) via Maxwell stress tensor (MST) calculation. Our purpose here is to apply this approach to explain the fact that the electric field anisotropy and electrical conductivity ratio Λ of the cytoplasm and the extracellular medium significantly impact the MST exerted on the cytoplasm-membrane interface. One important finding is that the modulation of electrical cues and MST force by the frequency of the applied electric field provides an extremely rich tool kit for manipulating cells. We show the extreme sensitivity of proximity-induced capacitive coupling arising concomitantly when the magnitude of the MST increases as the distance between cells is decreased and the spatial anisotropy becomes important. Moreover, our model highlights the strongly localized character of the electrostatic field effect emanating from neighboring cells and suggests the possibility of exploiting cell distribution as a powerful tool to engineer the functional performance of cell assemblies by controlling ED and capacitive coupling. We furthermore show that frequency has a significant impact on the attenuation-amplification transition of MST, suggesting that shape anisotropy has a much weaker influence on ED of the cell membrane compared to the anisotropy induced by the orientation angle itself.

Proximity-induced electrodeformation and membrane capacitance coupling between cells.

Membrane capacitance and transmembrane potential are sensitive to the proximity of neighboring biological cells which eventually induces anisotropic perturbation of the local electric field distribution in a cell assembly and/or a tissue. The development of robust and reliable multiphysics approaches is essential to solve the challenge of analyzing proximity-induced capacitance coupling (CC) between cells. In this study, we ask to what extent this CC is a minor perturbation on the individual cells or whether it can fundamentally affect bio-electromechanical cues. A key component of our continuum electromechanical analysis is the consideration of elastic models of cells under steady state electric field excitation to characterize electrodeformation (ED). Analyzing the difference between the ED force for a pair of cells and its counterpart for a single reference cell allows us to determine a separation distance-orientation angle diagram providing evidence of a separation distance beyond which the electrostatic interactions between a pair of biological cells become inconsequential for the ED. An attenuation-amplification transition of ED force in this diagram suggests that anisotropy induced by the orientation angle of the cell pair relative to the applied electric field direction has a significant influence on ED and CC. We furthermore observe that the shape of this diagram changes when extracellular conductivity is varied. The results obtained are then contrasted with the corresponding diagrams of similar cell configurations under an oscillating electric field excitation below and above the α-dispersion frequency. This investigation may provide new opportunities for further assessment of electromechanical properties of engineered tissues.

A drug interaction study investigating the effect of Rifabutin on the pharmacokinetics of Maraviroc in healthy subjects.

Effects of steady-state rifabutin on the pharmacokinetics of steady-state maraviroc were investigated in fourteen healthy adult female and male volunteers. Maraviroc 300 mg twice daily (BID) was given orally with food for fifteen days. On day six, rifabutin 300 mg once daily (QD, P.O.) was added to the regimen. Formal pharmacokinetic (PK) sampling was performed on days five and fifteen. Individual plasma drug concentration-time data for maraviroc, and rifabutin on day fifteen, were obtained using validated High Performance Liquid Chromatography (HPLC) tandem Mass Spectrometry (MS/MS). Rifabutin steady state exposure was comparable to data in the literature. Maraviroc area under the curve (AUC) and minimum plasma concentration (Clast or Cmin) were reduced by 17% and 30% respectively when co-administered with rifabutin. No unexpected or serious adverse eventsoccurred. Based on the reduced exposure of maraviroc observed in this study, increasing the dose of maraviroc may be studied to normalize its moderately reduced exposure following rifabutin co-administration, a moderate inducer of CYP3A4.

A model to rule out smear-negative tuberculosis among symptomatic HIV patients using C-reactive protein.

SETTING: Improved diagnostic algorithms for sputum smear-negative tuberculosis (SNTB) are needed to address the dramatic increase in SNTB in regions with high human immunodeficiency virus (HIV) prevalence. OBJECTIVE: To determine whether the addition of C-reactive protein (CRP) to a prediction model using simple clinical criteria improves the diagnosis of SNTB among mostly antiretroviral-naïve adult HIV TB suspects in an out-patient setting. DESIGN: A multiple logistic regression model was derived from a database of 228 HIV patients to predict the risk of SNTB using data from a previous prospective study. RESULTS: The derived model demonstrated that male sex, night sweats, fever, low body mass index and anemia increased the probability of having SNTB. CRP improved the accuracy of the model (without CRP, area under the curve [AUC] 0.75, 95%CI 0.68-0.81 vs. model with CRP, AUC 0.81, 95%CI 0.76-0.87, P = 0.0014) to predict SNTB. Using reclassification tables, CRP correctly reclassified 27.9% of the patients (net reclassification improvement, P = 0.0005) into higher or lower risk categories. The strongest effect was seen in the reclassification improvement among patients with no TB, which was 20.6% (P = 0.0023). CONCLUSION: CRP improved the performance of the prediction model in the diagnosis of SNTB in HIV patients, and may play a role in ruling out SNTB in this population. Prospective validation of this model is needed.

Performing allogeneic and autologous hematopoietic SCT in the outpatient setting: effects on infectious complications and early transplant outcomes.

This retrospective single-center study compared the incidence, spectrum and effect of infections in 1045 consecutive allogeneic (allo) and autologous (auto) hematopoietic SCT (HSCT) performed between 1995 and 2006 in the inpatient (IP) or outpatient (OP) setting. We analyzed 374 allo-HSCT (196 IP and 178 OP) and 671 auto-HSCT (163 IP and 508 OP). The incidence of infection was lower both in auto-OP (25% OP vs 33% IP, P=0.042) and allo-OP cohorts (42.7% OP vs 55.6% IP, P=0.012). The mean number of infections per transplant was lower in both auto-OP (0.39 OP vs 0.57 IP, P=0.05) and in allo-OP cohorts (0.78 OP vs 1.09 IP, P=0.018). The 100-day non-relapse mortality (NRM) for OP auto-HSCT was 4.72% and for IP 3.95% (P=0.68). The 100-day NRM for OP allo-HSCT was lower at 14.1% than it was for IP at 22.6% (P=0.041). Time to onset of first infection and spectrum of infections was similar in all groups. We conclude that performing allo- and auto-HSCT in the OP setting results in short-term outcomes, including infections complications that are comparable to the standard IP setting.

Monoclonal B cells detected in autologous PBSC grafts from patients with classical Hodgkin lymphoma: impact on relapse and survival following transplantation.

Autologous peripheral blood stem cell transplantation (PBSCT) for Hodgkin lymphoma (HL) is curative for many patients with relapsed or refractory disease. Relapsing disease, however, remains a major problem. Neoplastic transformation of B-lymphocytes probably underlies the development of classical HL. Whether clonal B cells are critical for disease evolution and response to therapy in HL remains uncertain. We investigated the impact of clonal B cells detected in peripheral blood stem cell (PBSC) collections on the outcome of patients with HL undergoing transplant. Qualitative semi-nested PCR was carried out on genomic DNA from mononuclear cells from PBSCs to determine the presence of clonal immunoglobulin heavy chain (IgH) complementary-determining region 3 (CDR3) gene rearrangements. Clinical factors were assessed for their association with relapse, overall survival (OS) and progression-free survival (PFS). Among 39 patients undergoing PBSCT, 12 grafts (31%) were PCR positive for clonal IgH rearrangements. OS was better in the PCR-negative group (logrank test, P=0.041). The OS at 5 years was 81% in PCR-negative versus 39% in PCR-positive patients; hazard ratio was 3.23 (95% confidence interval: 0.98-10.63). There was a trend towards better PFS (logrank test, P=0.12), estimated as 71% at 5 years in PCR-negative versus 41% in PCR-positive patients. Clonal B-lymphocytes in PBSC collections of patients with HL identify patients at risk of poor outcome. Larger series are needed to confirm our observations. Insight regarding the role of monoclonal B cells may lead to improved therapies.

Laparoscopic resection for colon cancer: would all patients benefit?

PURPOSE: This study was designed to assess whether the exclusion criteria used in the Clinical Outcomes of Surgical Therapy and Colon Cancer Laparoscopic or Open Resection trials affected the generalizability of their findings. METHODS: A prospective database of consecutive laparoscopic resections performed for colon cancer was reviewed. Patients were categorized into two groups: inclusion group and exclusion group, based on the selection criteria used in the Clinical Outcomes of Surgical Therapy and Colon Cancer Laparoscopic or Open Resection trials. Baseline and perioperative data were analyzed by using t-tests, Wilcoxon's rank-sum, chi-squared, and Fisher's exact test. Kaplan-Meier survival curves, followed by adjustment for tumor nodes metastasis stage and age utilizing a Cox proportional hazard model, were performed. RESULTS: The inclusion group had 221 patients and the exclusion group had 166 (median age and gender distribution were similar). The exclusion group had a higher conversion rate (23 vs. 11.3 percent; P=0.0023). There was no difference in intraoperative complications (9 percent for exclusion group vs. 8.6 percent for inclusion group; P=0.8), operative time (180 minutes for exclusion group vs.172 minutes for inclusion group; P=0.24), or postoperative complication rates (33.7 percent for exclusion group vs. 26 percent for inclusion group; P=0.13). No difference was detected in perioperative mortality rates, length of stay, days to diet as tolerated, and adjusted two-year survival. CONCLUSIONS: No differences were found in outcomes between the two groups in terms of operative/postoperative complications, length of stay, perioperative mortality, and two-year survival. It seems that all patients with colon cancer can potentially benefit from a laparoscopic approach.

Effects of cross-linkage on fatigue life and failure modes of stainless steel posterior spinal constructs.

This study tested the effects of cross-linkage on the fatigue performance of posterior spinal constructs (i.e., AcroMed stainless steel Isola systems). The failure modes encountered during fatigue were also examined. The results of this study confirmed earlier findings that the use of cross-linkage does not significantly affect the stability of posterior constructs during axial loading. Their influence in torsion loading is much more pronounced. During the fatigue tests, posterior stainless steel spinal implants instrumented without cross-linkage reached 1 million cycles at 500- and 750-N loads. When the load was increased to 1,000 N, the number of cycles to failure dropped by two-thirds. These findings demonstrate that the endurance limit was between 750 N and 1,000 N for spinal constructs without cross-linkage, with the limit being closer to 750 N. Devices equipped with one or two cross-linkages reached 1 million cycles at 500 N. The number of cycles to failure dropped dramatically as the load was increased to 750 and 1,000 N. It appears that the endurance limits for spinal devices using cross-linkage should be 500 and 750 N, with the limit closer to the 500-N load. All rod fractures occurred near the junction between the longitudinal and transverse rods. Stress concentration was greatly in the vicinity of that contact point. These results should provide a basis for future improvement in endurance limits of spinal implants equipped with cross-linkage. Higher endurance limits will reduce the toxic effects encountered during fracture modes. The implants will also be better able to withstand the high physiologic loads experienced by obese individuals.

Biomechanical assessment of titanium and stainless steel posterior spinal constructs: effects of absolute/relative loading and frequency on fatigue life and determination of failure modes.

The goal of this study was to examine the effects of absolute/relative loads and frequency on the fatigue life of titanium and stainless steel posterior spinal constructs, and to determine the failure fracture modes. The stainless steel constructs had higher stiffness and yield strength than the titanium constructs, but the ultimate static strength was almost equal for both types of constructs. Titanium constructs, however, exhibited higher variability than the stainless steel constructs. In fatigue tests, the stainless steel constructs were significantly affected by the external load and were frequency independent. It appears from fatigue curves that 500 N can be approximated as the endurance limit for the stainless steel constructs. Titanium constructs were load-frequency dependent, and their endurance limit was somewhere between the 500 and 750 N load levels. There were no differences in performance between the stainless steel and titanium constructs at 16 Hz. At 4 Hz, titanium constructs performed as well or better than stainless steel constructs. Also, the titanium constructs resulted in better performance than the stainless steel constructs in the elastic region, and with smaller differences in the plastic region. Most of the failure modes for stainless steel constructs were in screw bending at 16 Hz with a smaller percentage of rod fractures at high loads, with a higher percentage of rod fractures observed for the stainless steel constructs at 4 Hz. Most of the failure modes for titanium constructs occurred in screw bending or fracture.