HER2/HER3 dimerization resulting from overexpression of HER2 or neuregulin (NRG1) in cancer leads to HER3-mediated oncogenic activation of phosphoinositide 3-kinase (PI3K) signaling. Although ...ligand-blocking HER3 antibodies inhibit NRG1-driven tumor growth, they are ineffective against HER2-driven tumor growth because HER2 activates HER3 in a ligand-independent manner. In this study, we describe a novel HER3 monoclonal antibody (LJM716) that can neutralize multiple modes of HER3 activation, making it a superior candidate for clinical translation as a therapeutic candidate. LJM716 was a potent inhibitor of HER3/AKT phosphorylation and proliferation in HER2-amplified and NRG1-expressing cancer cells, and it displayed single-agent efficacy in tumor xenograft models. Combining LJM716 with agents that target HER2 or EGFR produced synergistic antitumor activity in vitro and in vivo. In particular, combining LJM716 with trastuzumab produced a more potent inhibition of signaling and cell proliferation than trastuzumab/pertuzumab combinations with similar activity in vivo. To elucidate its mechanism of action, we solved the structure of LJM716 bound to HER3, finding that LJM716 bound to an epitope, within domains 2 and 4, that traps HER3 in an inactive conformation. Taken together, our findings establish that LJM716 possesses a novel mechanism of action that, in combination with HER2- or EGFR-targeted agents, may leverage their clinical efficacy in ErbB-driven cancers.
Gold nanoparticles (AuNP) provide many opportunities in imaging, diagnostics, and therapy in nanomedicine. For the assessment of AuNP biokinetics, we intratracheally instilled into rats a suite of ...(198)Au-radio-labeled monodisperse, well-characterized, negatively charged AuNP of five different sizes (1.4, 2.8, 5, 18, 80, 200 nm) and 2.8 nm AuNP with positive surface charges. At 1, 3, and 24 h, the biodistribution of the AuNP was quantitatively measured by gamma-spectrometry to be used for comprehensive risk assessment. Our study shows that as AuNP get smaller, they are more likely to cross the air-blood barrier (ABB) depending strongly on the inverse diameter d(-1) of their gold core, i.e., their specific surface area (SSA). So, 1.4 nm AuNP (highest SSA) translocated most, while 80 nm AuNP (lowest SSA) translocated least, but 200 nm particles did not follow the d(-1) relation translocating significantly higher than 80 nm AuNP. However, relative to the AuNP that had crossed the ABB, their retention in most of the secondary organs and tissues was SSA-independent. Only renal filtration, retention in blood, and excretion via urine further declined with d(-1) of AuNP core. Translocation of 5, 18, and 80 nm AuNP is virtually complete after 1 h, while 1.4 nm AuNP continue to translocate until 3 h. Translocation of negatively charged 2.8 nm AuNP was significantly higher than for positively charged 2.8 nm AuNP. Our study shows that translocation across the ABB and accumulation and retention in secondary organs and tissues are two distinct processes, both depending specifically on particle characteristics such as SSA and surface charge.
The biokinetics of a size-selected fraction (70 nm median size) of commercially available and
48
V-radiolabeled
48
VTiO
2
nanoparticles has been investigated in female Wistar-Kyoto rats at retention ...timepoints 1 h, 4 h, 24 h and 7 days after oral application of a single dose of an aqueous
48
VTiO
2
-nanoparticle suspension by intra-esophageal instillation. A completely balanced quantitative body clearance and biokinetics in all organs and tissues was obtained by applying typical
48
VTiO
2
-nanoparticle doses in the range of 30-80 μg*kg
−1
bodyweight, making use of the high sensitivity of the radiotracer technique. The
48
VTiO
2
-nanoparticle content was corrected for nanoparticles in the residual blood retained in organs and tissue after exsanguination and for
48
V-ions not bound to TiO
2
-nanoparticles. Beyond predominant fecal excretion about 0.6% of the administered dose passed the gastro-intestinal-barrier after one hour and about 0.05% were still distributed in the body after 7 days, with quantifiable
48
VTiO
2
-nanoparticle organ concentrations present in liver (0.09 ng*g
−1
), lungs (0.10 ng*g
−1
), kidneys (0.29 ng*g
−1
), brain (0.36 ng*g
−1
), spleen (0.45 ng*g
−1
), uterus (0.55 ng*g
−1
) and skeleton (0.98 ng*g
−1
). Since chronic, oral uptake of TiO
2
particles (including a nano-fraction) by consumers has continuously increased in the past decades, the possibility of chronic accumulation of such biopersistent nanoparticles in secondary organs and the skeleton raises questions about the responsiveness of their defense capacities, and whether these could be leading to adverse health effects in the population at large. After normalizing the fractions of retained
48
VTiO
2
-nanoparticles to the fraction that passed the gastro-intestinal-barrier and reached systemic circulation, the biokinetics was compared to the biokinetics determined after IV-injection (Part 1). Since the biokinetics patterns differ largely, IV-injection is not an adequate surrogate for assessing the biokinetics after oral exposure to TiO
2
nanoparticles.
To study the influence of colloidal stability on protein corona formation, gold nanoparticles are synthesized with five distinct surface modifications: coating with citric acid, ...bis(p‐sulfonatophenyl)phenylphosphine dihydrate dipotassium salt, thiol‐terminated methoxy‐polyethylene glycol, dodecylamine‐grafted poly(isobutylene‐alt‐maleic anhydride), and dodecylamine‐grafted poly(isobutylene‐alt‐maleic anhydride) conjugated with polyethylene glycol. The nanoparticles are incubated with serum or bronchoalveolar lavage fluid from C57BL/6 mice (15 min or 24 h) to assess the effect of differential nanoparticle surface presentation on protein corona formation in the air–blood barrier exposure pathway. Proteomic quantification and nanoparticle size measurements are used to assess protein corona formation. We show that surface modification has a clear effect on the size and the composition of the protein corona that is related to the colloidal stability of the studied nanoparticles. Additionally, differences in the composition and size of the protein corona are shown between biological media and duration of exposure, indicating evolution of the corona through this exposure pathway. Consequently, a major determinant of protein corona formation is the colloidal stability of nanoparticles in biological media and chemical or environmental modification of the nanoparticles alters the surface presentation of the functional epitope in vivo. Therefore, the colloidal stability of nanoparticles has a decisive influence on nano–bio interactions.
Colloidal stability effects on protein corona formation using 5 nm gold nanoparticles (Au NPs) with five distinct surface modifications are investigated. NPs are incubated with serum or bronchoalveolar lavage from C57BL/6 mice for 15 min or 24 h. Proteomic quantification and NP size measurements demonstrate that the colloidal stability of NPs is a major determinant of protein corona formation and evolution in biological media.
The biokinetics of a size-selected fraction (70 nm median size) of commercially available and
48
V-radiolabeled
48
VTiO
2
nanoparticles has been investigated in healthy adult female Wistar-Kyoto ...rats at retention time-points of 1 h, 4 h, 24 h, 7 d and 28 d after intratracheal instillation of a single dose of an aqueous
48
VTiO
2
-nanoparticle suspension. A completely balanced quantitative biodistribution in all organs and tissues was obtained by applying typical
48
VTiO
2
-nanoparticle doses in the range of 40-240 μg·kg
−1
bodyweight and making use of the high sensitivity of the radiotracer technique. The
48
VTiO
2
-nanoparticle content was corrected for residual blood retained in organs and tissues after exsanguination and for
48
V-ions not bound to TiO
2
-nanoparticles. About 4% of the initial peripheral lung dose passed through the air-blood-barrier after 1 h and were retained mainly in the carcass (4%); 0.3% after 28 d. Highest organ fractions of
48
VTiO
2
-nanoparticles present in liver and kidneys remained constant (0.03%).
48
VTiO
2
-nanoparticles which entered across the gut epithelium following fast and long-term clearance from the lungs via larynx increased from 5 to 20% of all translocated/absorbed
48
VTiO
2
-nanoparticles. This contribution may account for 1/5 of the nanoparticle retention in some organs. After normalizing the fractions of retained
48
VTiO
2
-nanoparticles to the fraction that reached systemic circulation, the biodistribution was compared with the biodistributions determined after IV-injection (Part 1) and gavage (GAV) (Part 2). The biokinetics patterns after IT-instillation and GAV were similar but both were distinctly different from the pattern after intravenous injection disproving the latter to be a suitable surrogate of the former applications. Considering that chronic occupational inhalation of relatively biopersistent TiO
2
-particles (including nanoparticles) and accumulation in secondary organs may pose long-term health risks, this issue should be scrutinized more comprehensively.
Submicrometer TiO
2
particles, including nanoparticulate fractions, are used in an increasing variety of consumer products, as food additives and also drug delivery applications are envisaged. Beyond ...exposure of occupational groups, this entails an exposure risk to the public. However, nanoparticle translocation from the organ of intake and potential accumulation in secondary organs are poorly understood and in many investigations excessive doses are applied. The present study investigates the biokinetics and clearance of a low single dose (typically 40-400 μg/kg BW) of
48
V-radiolabeled, pure TiO
2
anatase nanoparticles (
48
VTiO
2
NP) with a median aggregate/agglomerate size of 70 nm in aqueous suspension after intravenous (IV) injection into female Wistar rats. Biokinetics and clearance were followed from one-hour to 4-weeks. The use of radiolabeled nanoparticles allowed a quantitative
48
VTiO
2
NP balancing of all organs, tissues, carcass and excretions of each rat without having to account for chemical background levels possibly caused by dietary or environmental titanium exposure. Highest
48
VTiO
2
NP accumulations were found in liver (95.5%ID after one day), followed by spleen (2.5%), carcass (1%), skeleton (0.7%) and blood (0.4%). Detectable nanoparticle levels were found in all other organs. The
48
VTiO
2
NP content in blood decreased rapidly after 24 h while the distribution in other organs and tissues remained rather constant until day-28. The present biokinetics study is part 1 of a series of studies comparing biokinetics after three classical routes of intake (IV injection (part 1), ingestion (part 2), intratracheal instillation (part 3)) under identical laboratory conditions, in order to test the common hypothesis that IV-injection is a suitable predictor for the biokinetics fate of nanoparticles administered by different routes. This hypothesis is disproved by this series of studies.
Industrially produced quantities of TiO
nanoparticles are steadily rising, leading to an increasing risk of inhalation exposure for both professionals and consumers. Particle inhalation can result in ...inflammatory and allergic responses, and there are concerns about other negative health effects from either acute or chronic low-dose exposure.
To study the fate of inhaled TiO
-NP, adult rats were exposed to 2-h intra-tracheal inhalations of
V-radiolabeled, 20 nm TiO
-NP aerosols (deposited NP-mass 1.4 ± 0.5 μg). At five time points (1 h, 4 h, 24 h, 7d, 28d) post-exposure, a complete balance of the
VTiO
-NP fate was quantified in organs, tissues, carcass, lavage and body fluids, including excretions. After fast mucociliary airway clearance (fractional range 0.16-0.31), long-term macrophage-mediated clearance (LT-MC) from the alveolar region is 2.6-fold higher after 28d (integral fraction 0.40 ± 0.04) than translocation across the air-blood-barrier (integral fraction 0.15 ± 0.01). A high NP fraction remains in the alveoli (0.44 ± 0.05 after 28d), half of these on the alveolar epithelium and half in interstitial spaces. There is clearance from both retention sites at fractional rates (0.02-0.03 d
) by LT-MC. Prior to LT-MC,
VTiO
-NP are re-entrained to the epithelium as reported earlier for 20 nm inhaled gold-NP (AuNP) and iridium-NP (IrNP).
Comparing the 28-day biokinetics patterns of three different inhaled NP materials TiO
-NP, AuNP and IrNP, the long-term kinetics of interstitial relocation and subsequent re-entrainment onto the lung-epithelium is similar for AuNP and Ir-NP but slower than for TiO
-NP. We discuss mechanisms and pathways of NP relocation and re-entrainment versus translocation. Additionally, after 28 days the integral translocated fractions of TiO
-NP and IrNP across the air-blood-barrier (ABB) are similar and become 0.15 while the translocated AuNP fraction is only 0.04. While NP dissolution proved negligible, translocated TiO
-NP and IrNP are predominantly excreted in urine (~ 0.1) while the urinary AuNP excretion amounts to a fraction of only 0.01. Urinary AuNP excretion is below 0.0001 during the first week but rises tenfold thereafter suggesting delayed disagglomeration. Of note, all three NP dissolve minimally, since no ionic radio-label release was detectable. These biokinetics data of inhaled, same-sized NP suggest significant time-dependent differences of the ABB translocation and subsequent fate in the organism.
When particles incorporated within a mammalian organism come into contact with body fluids they will bind to soluble proteins or those within cellular membranes forming what is called a protein ...corona. This binding process is very complex and highly dynamic due to the plethora of proteins with different affinities and fractions in different body fluids and the large variation of compounds and structures of the particle surface. Interestingly, in the case of nanoparticles (NP) this protein corona is well suited to provide a guiding vehicle of translocation within body fluids and across membranes. This NP translocation may subsequently lead to accumulation in various organs and tissues and their respective cell types that are not expected to accumulate such tiny foreign bodies. Because of this unprecedented NP accumulation, potentially adverse biological responses in tissues and cells cannot be neglected a priori but require thorough investigations. Therefore, we studied the interactions and protein binding kinetics of blood serum proteins with a number of engineered NP as a function of their physicochemical properties. Here we show by in vitro incubation tests that the binding capacity of different engineered NP (polystyrene, elemental carbon) for selected serum proteins depends strongly on the NP size and the properties of engineered surface modifications. In the following attempt, we studied systematically the effect of the size (5, 15, 80 nm) of gold spheres (AuNP), surface-modified with the same ionic ligand; as well as 5 nm AuNP with five different surface modifications on the binding to serum proteins by using proteomics analyses. We found that the binding of numerous serum proteins depended strongly on the physicochemical properties of the AuNP. These in vitro results helped us substantially in the interpretation of our numerous in vivo biokinetics studies performed in rodents using the same NP. These had shown that not only the physicochemical properties determined the AuNP translocation from the organ of intake towards blood circulation and subsequent accumulation in secondary organs and tissues but also the the transport across organ membranes depended on the route of AuNP application. Our in vitro protein binding studies support the notion that the observed differences in in vivo biokinetics are mediated by the NP protein corona and its dynamical change during AuNP translocation in fluids and across membranes within the organism.
Introduction
Objectives of this study, as part of a nation-wide HIV pre-exposure prophylaxis (PrEP) evaluation project, were to determine the incidence of infections with HIV, chlamydia, gonorrhea, ...syphilis, hepatitis A/B/C in persons using PrEP, and to describe the health care funded PrEP use in Germany. Additionally, factors associated with chlamydia/gonorrhea and syphilis infections were assessed.
Methods
Anonymous data of PrEP users were collected at 47 HIV-specialty centers from 09/2019–12/2020. Incidence rates were calculated per 100 person years (py). Using longitudinal mixed models, we analyzed risk factors associated with sexually transmitted infections (STIs).
Results
4620 PrEP users were included: 99.2% male, median age 38 years (IQR 32–45), 98.6% men who have sex with men (MSM). The median duration of PrEP exposure was 451 days (IQR 357–488), totaling 5132 py. Four HIV infections were diagnosed, incidence rate 0,078/100py (95% CI 0.029–0.208). For two, suboptimal adherence was reported and in the third case, suboptimal adherence and resistance to emtricitabine were observed. One infection was likely acquired before PrEP start. Incidence rates were 21.6/100py for chlamydia, 23.7/100py for gonorrhea, 10.1/100py for syphilis and 55.4/100py for any STI and decreased significantly during the observation period. 65.5% of syphilis, 55.6% of chlamydia and 50.1% of gonorrhea cases were detected by screening of asymptomatic individuals. In a multivariable analysis among MSM younger age, PrEP start before health insurance coverage and daily PrEP were associated with greater risk for chlamydia/gonorrhea. Symptom triggered testing and a history of STI were associated with a higher risk for chlamydia/gonorrhea and syphilis. A significantly lower risk for chlamydia/gonorrhea and syphilis was found for observations during the COVID-19 pandemic period.
Conclusions
We found that HIV-PrEP is almost exclusively used by MSM in Germany. A very low incidence of HIV infection and decreasing incidence rates of STIs were found in this cohort of PrEP users. The results were influenced by the SARS-CoV-2 pandemic. Rollout of PrEP covered by health insurance should be continued to prevent HIV infections. Increased PrEP availability to people at risk of HIV infection through the elimination of barriers requires further attention. Investigation and monitoring with a longer follow-up would be of value.
Glioblastomas are neuroepithelial tumors with lost cellular differentiation and tenfold increased growth rates compared to low-grade gliomas. Despite of very aggressive treatment options based on ...surgery, irradiation, and chemotherapy, the prognosis of affected patients has remained poor and showed only slight improvements during the last 30 years. Research on glioblastoma border zone was hindered by the tumor's intense invasion into the brain parenchyma and the lack of suitable tumor cell markers. Nevertheless, the compact tumor mass and tumor invasion zone are composed of distinct cell types that need to be distinguished from each other to be addressed selectively. As the isoform 140 of the neural cell adhesion molecule (NCAM-140) was recently demonstrated to be lost in human gliomas with rising WHO grade, human multiform glioblastomas were characterized as a NCAM-140 negative entity displaying three main distinct invasion patterns. Evaluation of putative therapy targets within the tumor tissue and tumor invasion zone has been made possible through NCAM-140 negativity. In the present study, brain tissue controls and human glioblastoma samples with compact tumor mass and invasion areas were analyzed for their vascularization at the tumor border and the expression of thrombin receptor protease-activated receptor type 1 (PAR-1) within tumor tissue and vascular vessel walls. Use of NCAM-140 enabled the identification of the tumor invasion zone and its experimental investigation. Tissue vascularization was found to be significantly increased in the compact tumor mass of glioblastomas compared to their invasion zone and tumor-free controls with a significantly high and specific overexpression of PAR-1 within tumor cells and within tumor blood vessels depending upon the tumor area. This suggests thereby a functional role of the thrombin receptor PAR-1 in glioma cell malignancy and glioblastoma neoangiogenesis.
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