Photodynamic inactivation of Rhizopus oryzae – in vitro study

During the COVID-19 pandemic, several complications arose in infected patients, one of them being mucormycosis, which is an extremely aggressive fungal disease with a high mortality rate, especially in patients with compromised immune systems. Most cases of mucormycosis are caused by the fungus Rhizopus oryzae, also known as black fungus, with 90% of cases affecting the rhinocerebral site. The treatment tools used are based on high doses of amphotericin B and posaconazole, associated with surgical resections when possible. However, even with aggressive antifungal treatment, the estimated attributable mortality rate is high [1]. In the absence of surgical debridement of the infected tissue, antifungal treatment alone is not curative. So there is a need for development of adjuvant treatments. Antimicrobial Photodynamic Therapy (aPDT) may constitute an auxiliary therapeutic option for mucormycosis [2]. Due to the lack of reports on the photodynamic inactivation of R. oryzae, we investigated different protocols Photodithazine® (PDZ) as a photosensitizer. The response on the fungus growing rate under distinct treatment parameters as photosensitizer concentration, incubation time, and association with surfactant, will be presented for both white and black hyphal phases, and infective spore phase. Preliminary results show the potential use of photodynamic therapy for the inactivation and growth control of the R. oryzae.     

Cytotoxicity of fractionated paclitaxel (Taxol®) administration in vitro

Hintergrund

Paclitaxel (Taxol^®) ist eine neue Antikrebssubstanz mit einem neuartigen Wirkmechanismus. Es hat gegenüber zahlreichen malignen Erkrankungen klinische Aktivität demonstriert. Verschiedene Aspekte des Einsatzes von Paclitaxel wie das optimale Behandlungsschema sind noch ungeklärt. Darüber hinaus ist auch das Lösungsmittel von Paclitaxel, Cremophor EL/Ethanol, gegenüber Tumoren wirksam.

Material und Methode

Die In-vitro-Zytotoxizität von Paclitaxel (Taxol^®) wurde entsprechend der Einmalgabe (1mal 10 μM, Tag 1, Inkubationzeit: drei Stunden und 15 Stunden) und der fraktionierten Gabe (5mal 2 μM/d, Tag 1 bis 5, Inkubationzeit: drei Stunden pro Tag) anhand der Bestimmung der klonogenen Überlebensfraktion (Koloniebildungstest) und der DNA-Verteilung (Flußphotozytometrie) ermittelt. In den Kontrollpopulationen wurden das Lösungsmittel Cremophor EL/Ethanol sowie eine phosphatgepufferte Salzlösung (PBS) in identischer Dosierung und nach identischem Schema appliziert. Es wurde eine Fibroblasten-Säugetierzellinie (HyB14FAF28) verwendet.

Ergebnisse

Fraktionierte Paclitaxel-(Taxol^®-)Applikation ergab eine signifikant geringere klonogene Überlebensfraktion (0,63) im Vergleich mit der Einmalgabe über drei Stunden (0,84) und 15 Stunden (0,82). Die DNA-Analyse zeigte keinen Hinweis auf einen signifikanten Unterschied in der DNA-Verteilung der für Paclitaxel interessanten G₂/M-Phase während eines Zeitraums von zehn Tagen ab Applikation. In den Kontrollen ergaben sich für das Lösungsmittel Cremophor EL/Ethanol klonogene Überlebensfraktionen von 0,87 (Drei-Stunden-Gabe) and 0,88 (15-Stunden-Gabe) gegenüber 0.65 nach fraktionierter Gabe (5mal 2 μM/d, Tag 1 bis 5, Inkubationzeit: drei Stnden pro Tag). Die PBS-Kontrolle sowie die unbehandelte Kontrolle zeigten keinen signifikanten Effekt.

Schlußfolgerungen

Es scheint, daß sich bei dieser Fibroblasten-Säugertierzellinie die klonogene Überlebensfraktion nach Taxol^®-Gabe mit dem Behandlungsschema durch einen bis jetzt unbekannten Mechanismus, der keinen G₂/M-Block enthält, verändert. Die Ergebnisse zeigen einen Behandlungseffekt, der hauptsächlich auf der Kombination mit dem Lösungsmittel ohne einen zusätzlich induzierten Gewinn durch Paclitaxel basiert.     

A rational approach to “in vitro” thyroid function testing

A system of in vitro thyroid function testing is proposed whereby laboratory staff select the most appropriate screening test depending on the information supplied by the clinician. Total serum triiodothyronine (T3) or thyroxine (T4) are used for screening as appropriate. In borderline cases, secondary tests are performed automatically according to a flow chart.This system improves efficiency and is cost effective, saving approximately £1,800 annually in a laboratory handling about 5,000 requests for thyroid function tests each year.     

CKMB和cTnI在缺氧诱导in vitro心肌细胞中的表达

目的:研究in vitro心肌细胞在长时间缺氧刺激后,CKMB和cTnI的表达特点,为探讨缺氧诱发心脏高表达CKMB和cTnI的机制提供参考。方法:采用体外培养新生Wister大鼠心肌细胞模型,两步法RT-PCR和Western Blotting分别检测上清和胞内HIF-1α和CKMB、cTnI在缺氧培养40分钟后连续时间点的表达变化。结果:缺氧后15min,上清CKMB表达显著升高,后随时间推移缓慢升高;胞内表达在0min～24h均显著高于上清(P<0.05),1h后达峰值。上清cTnI浓度在缺氧15min后急剧升高,24h后达峰值;胞内cTnI浓度在缺氧45min后达峰值;缺氧心肌细胞内、上清cTnI表达在15min～48h均有显著性变化(P<0.05),且呈近似同步性。胞内cTnI浓度在24h后低于上清。结论:单纯长时间缺氧刺激可引起invitro心肌细胞内高表达CKMB和cTnI,并向胞外释放,推测缺氧导致胞膜渗漏是主要释放机制。     

The Hounsfield value for cortical bone geometry in the proximal humerus—an in vitro study

Introduction  

Fractures of the proximal humerus represent a major osteoporotic burden. Recent developments in CT imaging have emphasized the importance of cortical bone thickness distribution in the prevention and management of fragility fractures. We aimed to experimentally define the CT density of cortical bone in the proximal humerus for building cortical geometry maps.     

mBAND analysis of chromosome aberrations in lymphocytes exposed in vitro to α-particles and γ-rays

Purpose:?To investigate the profiles of chromosome damage induced in vitro by exposure to α-particles and γ-rays.

Materials and methods:?Human peripheral blood lymphocytes were exposed to three dose regimes: α-particle doses of 0.2 and 0.5 Gy and a γ-ray dose of 1.5 Gy. After culturing for 47 hours, chromosome aberrations involving the number 5 chromosomes were identified using a multi-coloured banding (mBAND) technique.

Results:?Analysis of the frequencies of chromosome 5 breaks within aberrant cells and within aberrant number 5 chromosomes demonstrated that α-particle irradiation is more likely to result in multiple breaks in a chromosome than γ-irradiation. Additionally, overdispersion was observed for all doses for the distribution of breaks amongst all cells analysed and breaks amongst total number 5 chromosomes, with this being greatest for the 0.2 Gy α-particle dose. The ratio of interchanges to intrachanges (F ratio) was 1.4 and 2.4 for 0.2 and 0.5 Gy α-particles respectively and 5.5 for 1.5 Gy γ-rays. Evaluation of simple versus complex exchanges indicated ratios of 1.9 and 2.7 for 0.2 and 0.5 Gy α-particles respectively and 10.6 for 1.5 Gy γ-rays. The majority of the intrachanges involving chromosomes 5 induced by α-particle radiation were associated with more complex exchanges.

Conclusions:?This study has confirmed that exchanges induced by exposure to high linear energy transfer (LET) α-particle radiation comprise a greater proportion of intrachanges than those induced by exposure to low LET γ-rays. However, since the majority of these are associated with complex rearrangements and likely to be non-transmissible, this limits their applicability as a marker of past in vivo exposure.     

Flow-directed microcatheters for cerebral embolizations using Ethibloc – an in vitro study

We conducted an in vitro performance study to test the flow-directed Spinnaker microcatheter for potential endovascular embolizations with Ethibloc as embolic agent. We performed in vitro testing using 50 Spinnaker 1.8F hydrophilic infusion microcatheters of three different types. As embolic agent, different mixtures of Ethibloc and Lipiodol (E/L 1:1, 1:2, 1:3) were used. In an in vitro setting, each catheter was injected to a maximum of ten times (E/L 1:1, n = 20 catheters; E/L 1:2, n = 20; E/L 1:3, n = 10). In addition, we evaluated the thinner Spinnaker 1.5F microcatheter (165/20, n = 10). Five Spinnaker 1.5F catheters were each injected with a mixture of Ethibloc and Lipiodol (E/L 1:2 and 1:3). When a mixture of E/L 1:1 was used, 9 of 20 (45%) Spinnaker 1.8F microcatheters ruptured distally near the tip of the microcatheter, each during injection nos. 5-9. One Spinnaker 1.8F was distally occluded after the fifth injection. With a mixture of E/L 1:2, 4 of 20 (20%) Spinnaker 1.8F microcatheters ruptured distally, during injection no. 6 (n = 2), 7 (n = 1), and 8 (n = 1). With a mixture of E/L 1:3, all ten Spinnaker 1.8F microcatheters could be injected without any problems. All ten microcatheters of the thinner Spinnaker 1.5F ruptured (10-18 cm proximally to the catheter tip). This in vitro study using the flow-directed Spinnaker microcatheter revealed that microcatheters can rupture. The Spinnaker 1.8F can rupture when a mixture of Ethibloc and Lipiodol 1:1 or 1:2 and more than four injections are used. The Spinnaker 1.8F does not rupture when a mixture of E/L 1:3 is used, or with a mixture of E/L 1:1 or 1:2 and injecting to a maximum of four times. The Spinnaker 1.5F microcatheter is not suitable for embolizations using Ethibloc. For embolizations with Ethibloc we therefore recommend the use of a guidewire-directed microcatheter.     

Ionizing radiation induces up-regulation of functional β 1-integrin in human lung tumour cell lines in vitro

Purpose : Cell-matrix interactions are in part mediated through the β 1-integrin pathway regulating cell survival, proliferation, adhesion and migration. This study was performed to elucidate alterations of expression of the β 1-integrin and its co-localized protein kinase, integrin-linked kinase (ILK), after exposure to ionizing radiation in two lung carcinoma cell lines in the presence or absence of different β 1-integrin-dependent matrix proteins. Materials and methods : Exponentially growing A549 and SKMES1 cells grown on fibronectin, laminin, BSA or plastic were exposed to 2 Gy or 6 Gy. Besides colony formation assays (0.5-8 Gy) and immediate plating experiments, flow cytometry (for β 1-integrin) and immunoblotting (for β 1-integrin and ILK) were carried out to analyze the protein expression. The localization of both proteins plus filamentous (f-) actin was further examined by immunofluorescence staining and laser confocal scanning microscopy. Functionality of the β 1 receptor subunit after irradiation was investigated in adhesion assays. Results : A549 and SKMES1 cells grown on fibronectin or laminin demonstrated a significant increase in cell survival after irradiation compared to cells grown on BSA or plastic. Immediate plating of cells after irradiation on fibronectin did not show an improved survival. Flow cytometric and Western blot data showed a dose- and matrix-dependent induction of β 1-integrin and ILK expression after irradiation within 48 h. Adhesion to fibronectin or laminin compared to BSA or plastic was increased by 10-fold after irradiation, demonstrating these specific cell surface receptors to be functional. The staining of β 1-integrin and ILK in A549 cells confirmed the radiation-induced up-regulation of both proteins. Additionally, β 1-integrin and ILK co-localized with accumulated actin fibers at the cytoplasmic face of the cell membrane at confined areas. Conclusions : Ionizing radiation strongly induced the expression of functional β 1-integrin and ILK in the two lung cancer cell lines, A549 and SKMES1, dependent on different matrices used. Additionally, the subcellular localization of both proteins was altered by irradiation, and the individual cellular radiosensitivity was reduced in the presence of an extracellular matrix. On the one hand, this may result in aggravated therapeutic approaches and on the other hand, cells could adhere more strongly in their environment by the increase in functional surface receptor density preventing metastasis. Concerning intravascular located tumour cells, β 1-integrin up-regulation might enable these cells to adhere to the endothelium, which represents a prerequisite for metastatic disease. Identification of such mechanisms will provide considerable insights into the understanding of tumorigenicity and metastatic phenotypes, possibly leading to new, optimized radiochemotherapeutic regimens.     

Der Einfluss ionisierender Strahlen auf die Entwicklung der Sekundärkatarakt in vitro

BACKGROUND AND PURPOSE: Histologically, the posterior capsule opacification (PCO) corresponds to regenerative tissue of transformed lens epithelial cells (LECs) with extracellular matrix production. In this study, the influence of ionizing radiation on proliferating LECs and the development of PCO was investigated in vitro. MATERIAL AND METHODS: Each four and 14 pork lenses, respectively, were irradiated with 6 MeV electrons with single doses of 8, 10, 12, and 20 Gy. 1-2 h after irradiation the lens was removed by capsulorrhexis and hydrodissection. After fixation of the capsular bag in a special device the proliferation of residual LECs was examined daily. The experiment was considered to be finished when the capsular bag was completely opacified by confluent cell proliferates. RESULTS: Single dose irradiation with electrons in a dose range from 8 to 12 Gy significantly protracted the development of PCO with complete inhibition of PCO after application of 20 Gy. CONCLUSION: To inhibit PCO in vitro, a single dose of 20 Gy is necessary. The actual in vitro model allows an optimal investigation of PCO formation under different external influences and is therefore very suitable for radiobiological questions.     

Goldvalve detachable balloon: “in vitro” assessment of safety and imaging artifacts in a 3-T MR system

The Goldvalve balloon is the only currently available detachable balloon. We undertook a study to determine safety and imaging artifacts in a MR environment. We found no evidence for heating or deflection of the balloon in a comprehensive series of in vitro experiments at 3 T. MR imaging at field strengths up to 3 T of patients with implanted Goldvalve balloons is safe. Imaging artifacts are minimal.     

Cell death induced by 131 I in a differentiated thyroid carcinoma cell line in vitro: necrosis or apoptosis?

AIM: The apoptotic and necrotic dose-response of thyroid carcinoma cells following irradiation with I was evaluated. METHODS: In our in-vitro model, cells of well-differentiated papillary thyroid carcinoma (B-CPAP) were incubated with increasing activity concentrations of I for 2 days. Changes in cell viability and the extents of necrosis and apoptosis were evaluated both immediately and 2 days after irradiation. RESULTS: Viability of B-CPAP cells diminished with increasing I activity concentration. No apoptosis was detectable immediately after irradiation. Two days after irradiation significant apoptosis was found. The lowest I activity concentration at which apoptosis was detectable corresponds to about 1 MBq . ml. At higher activity concentrations a larger percentage of cells became apoptotic but the proportion decreased again at activity concentrations >10 MBq . ml. Likewise, necrosis was minimal at low activity concentrations and showed an exponential increase with rising I activity concentrations (>5-10 MBq . ml). Necrosis was already detectable immediately after irradiation and was the predominant form of cell death at high activity concentrations. CONCLUSION: The data suggest that the nature of the cytotoxic effect of I and whether it leads to apoptotic or necrotic cell death is dose-dependent. High I doses seem to produce mainly necrotic phenomena, whereas at low I activity concentrations apoptotic phenomena prevail. The predominance of delayed apoptosis could explain why radioiodine therapy at lower doses is often linked to delayed onset and possible continuation of thyroid volume reduction over some months and even up to a year.     

MRI with intraoral orthodontic appliance—a comparative in vitro and in vivo study of image artefacts at 1.5?T

Objectives:

We investigated artefacts caused from orthodontic appliances at 1.5-T MRI of the head and neck region and whether the image quality can be improved utilizing the artefact-minimizing sequence WARP.

Methods:

In vitro tests were performed by phantom measurements of different orthodontic devices applying different types of MR sequences [echoplanar imaging (EPI), turbo spin echo (TSE) and TSE-WARP, gradient echo (GRE)]. Two independent readers determined after calibration the level of artefacts. Subsequently, the interobserver agreement was calculated. The measurement of artefacts was based on the American Society for Testing Materials Standard F 2119-07. For in vivo imaging, one test person was scanned with an inserted multibracket appliance. The level of artefacts for 27 target regions was evaluated.

Results:

In vitro: ceramic brackets and ferromagnetic steel brackets produced artefact radii up to 1.12 and 7.40 cm, respectively. WARP reduced these artefacts by an average of 32.7%. The Bland–Altman-Plot indicated that maximum measurement differences of 3 mm have to be expected with two calibrated observers. In vivo: the EPI sequence for brain imaging was not analysable. The TSE sequence of the brain did not demonstrate artefacts except for the nasal cavity. Conversely, the TSE sequence of the cervical spine revealed severe artefacts in the midface region. The GRE sequence appeared to be more susceptible to artefacts than did the TSE sequence.

Conclusions:

In vitro measurements allow an estimation of the in vivo artefact size. Orthodontic appliances may often remain intraorally when performing MRI. WARP showed a more significant effect in vitro than in vivo.     

Dose-effect of chromosome aberration in lymphocytes of human peripheral blood irradiated with high dose of 60Co γ rays in vitro

Objective To explore the dose-effect of chromosome aberration,and establish a dose-effect curve of dicentrie and ring (die+r) in human peripheral blood lymphocytes irradiated with high dose of γ-rays.Methods Human peripheral blood was obtained from three healthy individuals,and exposed to 60Co γ-rays with doses between 0 and 50 Gy.Colchicine was added immediately and the cells were incubated at 37℃ for 52,72 and 96 h,respectively.Metaphase index (MI) and dic+r were counted,and a dose-effect curve was fitted.Dose estimation were performed according to the curve for the two patients who had accidentally received high doses of irradiation.Results MI was reduced with the dose increasing.Frequencies of dic+r were increased until 23 Gy,and then saturation was observed.A dose-effect curve was fitted during 5-23 Gy,and the doseeffect was as follows:γ=-1.608(±0.300)+0.830(±0.051) D-0.013(±0.002) D2(R2=0.998),where γ is the yield of dic+r,and D is the dose.The dose estimated for the two patients were in accordance with those by physical method,ESR method and clinical symptoms.Conclusions The highest dose that could be estimated is 23 Gy with the established dic+r dose-effect curve by using the eolchicine black method and prolonged culture time,which suggests the potential of chromosome aberration analysis as biological dosemeter.     

Synthesis, radioiodination and in vitro and in vivo sigma receptor studies of N-1-allyl-N´-4-phenethylpiperazine analogs

IntroductionSigma-1 (σ₁) receptor radioligands are useful for basic pharmacology studies and for imaging studies in neurology, psychiatry and oncology. We derived a hybrid structure, N-1-allyl-N´-4-phenethylpiperazine, from known ligands TPCNE and SA4503 for use as a scaffold for development of radioiodinated σ₁ receptor ligands.MethodsE-and Z-N-1-(3′-iodoallyl)-N´-4-(3″,4″-dimethoxyphenethyl)-piperazine (E-1 and Z-1), N-1-allyl-N´-4-(3′,4′-dimethoxyphenethyl)-piperazine (2) and E-N-1-(3′-iodoallyl)-N´-4-(3″-methoxy-4′´-hydroxyphenethyl)-piperazine (3) were synthesized. Affinities for σ₁ and σ₂ receptors were determined. [¹²⁵I]E-1 and [¹²⁵I]Z-1 were prepared and evaluated in vivo in mice. [¹²⁵I]E-1 was further evaluated in σ₁ receptor binding assays in vitro.ResultsE-1 displayed moderately high apparent affinity (15 nM) for σ₁ sites and 84-fold selectivity against σ₂ sites. Z-1 showed similar σ₁ affinity, but only 23-fold selectivity. In contrast, 2 exhibited poor binding to both subtypes, while 3 had good affinities but poor selectivity. E-1 profiled as a probable antagonist in the phenytoin shift assay. [¹²⁵I]E-1 and [¹²⁵I]Z-1 were prepared in good yields and with high specific radioactivities. Log D_7.4 values (2.25 and 2.27) fall within the optimal range for in vivo studies. Both radioligands selectively labeled σ₁ receptors in mouse brain and peripheral organs in vivo. [¹²⁵I]E-1 showed a higher level of specific binding than [¹²⁵I]Z-1 and displayed good metabolic stability. Further, [¹²⁵I]E-1 selectively labeled σ₁ receptors in mouse brain homogenates (K_d 3.79 nM; B_max=599 fmol/mg protein).Conclusions[¹²⁵I]E-1 is a selective σ₁ receptor radioligand that exhibits properties amenable to in vitro and in vivo studies, with possible extension to single photon emission computed tomography using iodine-123.     

Fibronectin and laminin increase resistance to ionizing radiation and the cytotoxic drug Ukrain® in human tumour and normal cells in vitro

Purpose: Cell–extracellular matrix (ECM) interactions are thought to mediate drug and radiation resistance. Dependence of cell survival, β1‐integrin expression and cell cycling on the ECM proteins and β1‐integrin ligands fibronectin (FN) and laminin (LA) were examined in malignant and normal cells exposed to the cytotoxic drug Ukrain plus/minus irradiation.

Materials and methods: Human A549 lung cancer and MDAMB231 (MDA231) breast cancer cells and normal fibroblasts (HSF1) grown on FN, LA, bovine serum albumin (BSA) or polystyrene were treated with Ukrain (1?µg?ml^?1, 24?h) plus/minus irradiation (2–8?Gy) and the effects studied using colony formation assays, flow cytometry (β1‐integrin, DNA analysis) and adhesion assays.

Results: FN and LA reduced the cytotoxic effect of single Ukrain treatment compared with polystyrene and BSA. FN and LA also abolished Ukrain‐dependent radiosensitization in A549 cells and decreased the radiosensitivity of MDA231 and HSF1 cells. Single Ukrain exposure on polystyrene significantly reduced β1‐integrin expression and promoted G2‐phase accumulation of A549 cells. In contrast, Ukrain‐treated MDA231 and HSF1 cells showed elevated β1‐integrin expression and no Ukrain‐specific cell cycle effect. Under Ukrain‐radiation exposure, irradiation, FN or LA abolished Ukrain‐mediated reduction of β1‐integrin expression and G2‐phase accumulation in A549 cells, whereas in MDA231 cells and fibroblasts β1‐integrin expression and cell cycle distribution were stabilized. Cell adhesion to FN or LA was significantly impaired (A549) or improved (MDA231, HSF1) upon Ukrain treatment.

Conclusions: The data corroborate the findings of other groups that cell adhesion‐mediated resistance to either single or combined drug and radiation exposure is tightly correlated to specific ECM proteins. By demonstrating a strong modulatory impact of FN and LA on the radiosensitivity‐modifying activity of the drug Ukrain, these findings are also highly important for the assessment of drug and radiation effects within in vitro cytotoxicity studies. The data give the first mechanistic insights into specific FN‐ and LA‐modulated cellular resistance mechanisms as well as into the important role for β1‐integrins using the unique cytotoxic and radiosensitivity‐modifying drug Ukrain.     

Angiographic CT: in vitro comparison of different carotid artery stents—does stent orientation matter?

Introduction

Our aim was to evaluate the in vitro visualization of different carotid artery stents on angiographic CT (ACT). Of particular interest was the influence of stent orientation to the angiography system by measurement of artificial lumen narrowing (ALN) caused by the stent material within the stented vessel segment to determine whether ACT can be used to detect restenosis within the stent.

Methods

ACT appearances of 17 carotid artery stents of different designs and sizes (4.0 to 11.0 mm) were investigated in vitro. Stents were placed in different orientations to the angiography system. Standard algorithm image reconstruction and stent-optimized algorithm image reconstruction was performed. For each stent, ALN was calculated.

Results

With standard algorithm image reconstruction, ALN ranged from 19.0 to 43.6 %. With stent-optimized algorithm image reconstruction, ALN was significantly lower and ranged from 8.2 to 18.7 %. Stent struts could be visualized in all stents. Differences in ALN between the different stent orientations to the angiography system were not significant.

Conclusion

ACT evaluation of vessel patency after stent placement is possible but is impaired by ALN. Stent orientation of the stents to the angiography system did not significantly influence ALN. Stent-optimized algorithm image reconstruction decreases ALN but further research is required to define the visibility of in-stent stenosis depending on image reconstruction.