A novel microbead-based microfluidic device for rapid bacterial identification and antibiotic susceptibility testing

Effective treatment of infectious diseases depends on the ability to rapidly identify the infecting bacteria and the use of sensitive antibiotics. The currently used identification assays usually take more than 72 h to perform and have a low sensitivity. Herein, we present a microbead-based microfluidic platform that is highly sensitive and rapid for bacterial detection and antibiotic sensitivity testing. The platform includes four units, one of which is used for bacterial identification and the other three are used for susceptibility testing. Our results showed that Escherichia coli O157 at a cell density range of 10¹–10⁵ CFU/μL could be detected within 30 min. Additionally, the effects of three antibiotics on E. coli O157 were evaluated within 4–8 h. Overall, this integrated microbead-based microdevice provides a sensitive, rapid, reliable, and highly effective platform for the identification of bacteria, as well as antibiotic sensitivity testing.     

Quantitative activity-induced manganese-dependent MRI for characterizing cortical layers in the primary somatosensory cortex of the rat

The ability of Mn²⁺ to follow Ca²⁺ pathways upon stimulation transform them into remarkable surrogate markers of neuronal activity using activity-induced manganese-dependent MRI (AIM–MRI). In the present study, a precise follow-up of physiological parameters during MnCl₂ and mannitol infusions improved the reproducibility of AIM–MRI allowing in-depth evaluation of the technique. Pixel-by-pixel T₁ data were investigated using histogram distributions in the barrel cortex (BC) and the thalamus before and after Mn²⁺ infusion, after blood brain barrier opening and after BC activation. Mean BC T₁ values dropped significantly upon trigeminal nerve (TGN) stimulation (?38 %, P = 0.02) in accordance with previous literature findings. T₁ histogram distributions showed that 34 % of T_1s in the range 600–1500 ms after Mn²⁺ + mannitol infusions shifted to 50–350 ms after TGN stimulation corresponding to a twofold increase of the percentage of pixels with the lowest T_1s in BC. Moreover, T₁ changes in response to stimulation increased significantly from superficial cortical layers (I–III) to deeper layers (V–VI). Cortical cytoarchitecture detection during a functional paradigm was performed extending the potential of AIM–MRI. Quantitative AIM–MRI could thus offer a means to interpret local neural activity across cortical layers while identification of the role of calcium dynamics in vivo during brain activation could play a key role in resolving neurovascular coupling mechanisms.     

Walker 256 tumour cells increase substance P immunoreactivity locally and modify the properties of the blood–brain barrier during extravasation and brain invasion

It is not yet known how tumour cells traverse the blood–brain barrier (BBB) to form brain metastases. Substance P (SP) release is a key component of neurogenic inflammation which has been recently shown to increase the permeability of the BBB following CNS insults, making it a possible candidate as a mediator of tumour cell extravasation into the brain. This study investigated the properties of the BBB in the early stages of tumour cell invasion into the brain, and the possible involvement of SP. Male Wistar rats were injected with Walker 256 breast carcinoma cells via the internal carotid artery and euthanised at 1, 3, 6 and 9 days post tumour inoculation. Culture medium-injected animals served as controls at 1 and 9 days. Evidence of tumour cell extravasation across the BBB was first observed at 3 days post-inoculation, which corresponded with significantly increased albumin (p < 0.05) and SP immunoreactivity (p < 0.01) and significantly reduced endothelial barrier antigen labelling of microvessels when compared to culture medium control animals (p < 0.001). By day 9 after tumour cell inoculation, 100 % of animals developed large intracranial neoplasms that had significantly increased albumin in the peri-tumoral area (p < 0.001). The increased SP immunoreactivity and altered BBB properties at 3 days post-inoculation that coincided with early tumour invasion may be indicative of a mechanism for tumour cell extravasation into the brain. Thus, extravasation of tumour cells into the brain to form cerebral metastases may be a SP-mediated process.     

Fabrication of two-layer poly(dimethyl siloxane) devices for hydrodynamic cell trapping and exocytosis measurement with integrated indium tin oxide microelectrodes arrays

The design, fabrication and test of a microfluidic cell trapping device to measure single cell exocytosis were reported. Procedures on the patterning of double layer template based on repetitive standard photolithography of AZ photoresist were investigated. The replicated poly(dimethyl siloxane) devices with 2.5 μm deep channels were proved to be efficient for stopping cells. Quantal exocytosis measurement can be achieved by targeting single or small clumps of chromaffin cells on top of the 10 μm?×?10 μm indium tin oxide microelectrodes arrays with the developed microdevice. And about 72 % of the trapping sites can be occupied by cells with hydrodynamic trapping method and the recorded amperometric signals are comparable to the results with traditional carbon fiber microelectrodes. The method of manufacturing the microdevices is simple, low-cost and easy to perform. The manufactured device offers a platform for the high throughput detection of quantal catecholamine exocytosis from chromaffin cells with sufficient sensitivity and broad application.     

In Vitro Biocompatibility and Antibacterial Efficacy of a Degradable Poly(l-lactide-co-epsilon-caprolactone) Copolymer Incorporated with Silver Nanoparticles

Silver nanoparticles (Ag-nps) are currently used as a natural biocide to prevent undesired bacterial growth in clothing, cosmetics and medical products. The objective of the study was to impart antibacterial properties through the incorporation of Ag-nps at increasing concentrations to electrospun degradable 50:50 poly(l-lactide-co-epsilon-caprolactone) scaffolds for skin tissue engineering applications. The biocompatibility of the scaffolds containing Ag-nps was evaluated with human epidermal keratinocytes (HEK); cell viability and proliferation were evaluated using Live/Dead and alamarBlue viability assays following 7 and 14 days of cell culture on the scaffolds. Significant decreases in cell viability and proliferation were noted for the 1.0 mg(Ag) g(scaffold)^?1 after 7 and 14 days on Ag-nps scaffolds. After 14 days, scanning electron microscopy revealed a confluent layer of HEK on the surface of the 0.0 and 0.1 mg(Ag) g(scaffold)^?1. Both 0.5 and 1.0 mg(Ag) g(scaffold)^?1 were capable of inhibiting both Gram positive and negative bacterial strains. Uniaxial tensile tests revealed a significant (p < 0.001) decrease in the modulus of elasticity following Ag-nps incorporation compared to control. These findings suggest that a scaffold containing between 0.5 and 1.0 mg(Ag) g(scaffold)^?1 is both biocompatible and antibacterial, and is suitable for skin tissue engineering graft scaffolds.     

Early administration of propofol protects against endotoxin-induced acute lung injury in rats by inhibiting the TGF-β1-Smad2 dependent pathway

Objective

To assess the effects of propofol treatments at different time points on acute lung injury and on the expression of transforming growth factor (TGF)-β1 and the downstream target of TGF-β1, Smad 2, in the lung tissues in the endotoxic rats.

Methods

Seventy-six Wistar rats were randomly assigned to five groups: control group (saline only), endotoxemic group [lipopolysaccharide (LPS) 8 mg kg^?1, i.v.], and three propofol-treated groups. For the propofol-treated groups, propofol (5 mg kg^?1, i.v. bolus) was administered either 1 h before LPS, simultaneously with LPS, and 1 h after LPS, and all were followed by infusion of 10 mg kg^?1 h^?1 of propofol for 5 h after LPS. Lung tissues were sampled to measure myeloperoxidase activity and expression of TGF-β1 and Smad2 and to assess pulmonary microvascular permeability and histopathological changes.

Results

The hemodynamics, arterial blood gases, 5 h survival rate, pulmonary microvascular permeability, and acute lung injury scores were significantly better, and expression of TGF-β1 and Smad2 and myeloperoxidase activity in lung tissues was significantly lower in the pretreatment and simultaneous treatment groups compared to the endotoxemic group. However, there were no significant differences in all observed variables between the endotoxemic and postreatment groups. Except for TGF-β1 expression in lung tissues, the other observed variables were also not significantly different between the pretreatment and simultaneous treatment groups.

Conclusions

In the endotoxic rat model, pretreatment and simultaneous treatment with propofol provided protection against acute lung injury by inhibiting the TGF-β1-Smad2 dependent pathway.     

Comparison of five elastin histochemical stains to identify pulmonary small vasculature*

Optimization of an elastic tissue histochemical stain to enable clear, crisp visualization and quantification of pulmonary small vasculature is central to the histomorphologic quantitation of pulmonary vasculature wall thickness. To accomplish elastic tissue histochemical stain optimization, five histochemical elastin stains were compared to identify the internal and external elastic laminae of small arteries (50–100 μm in external diameter) to very small intra-acinar vessels (10–50 μm in external diameter) in rat lung tissue sections. The five elastin stains included: a modified Verhoeff’s elastin stain, Miller’s elastic van Gieson, and three modifications of the Miller’s stain. The Miller elastin stain is a progressive procedure that does not require a differentiation step, thus enabling consistency and reliability of staining from slide to slide. A modified Miller’s histochemical staining methodology successfully highlighted the pulmonary small caliber vasculature wall thickness. The modified method was technically easier and less time consuming to perform than regressive methods. To improve elastin-to-background contrast, modifications to the Miller’s stain included bypassing the nuclear staining and using a neutral red counterstain in place of the van Gieson counterstain, both of which greatly facilitated observer-assisted pulmonary vascular structure identification for histomorphometric quantitation.     

Similarity of permeabilities for Ficoll,pullulan, charge‐modified albumin and native albumin across the rat peritoneal membrane

Aim: Compared to neutral globular proteins, neutral polysaccharides, such as dextran, pullulan and Ficoll, appear hyperpermeable across the glomerular filtration barrier. This has been attributed to an increased flexibility and/or asymmetry of polysaccharides. The present study investigates whether polysaccharides are hyperpermeable also across the continuous capillaries in the rat peritoneum. Methods: In anaesthetized Wistar rats, FITC–Ficoll or FITC–pullulan together with ¹²⁵I‐human serum albumin (RISA) or neutralized ¹²⁵I‐bovine serum albumin (nBSA) were given intravenously, after which peritoneal dialysis (PD) using conventional PD fluid (Gambrosol 1.5%) was performed for 120 min. Concentrations of FITC‐polysaccharides and radioactive albumin species in plasma and dialysis fluid were analysed with high‐performance size exclusion chromatography and a gamma counter respectively. Transperitoneal clearance values were calculated for polysaccharides in the molecular radius range 36–150 Å, and for RISA and nBSA. Results: Ficoll and pullulan showed more or less identical permeabilities, compared to RISA and nBSA, across the peritoneal membrane. Although RISA‐clearance, 5.50 ± 0.28 (μL min^?1; ±SEM), tended to be lower than the clearances of Ficoll_36Å (6.55 ± 0.25), pullulan_36Å (6.08 ± 0.22) and nBSA (6.56 ± 0.23), the difference was not statistically significant. This is in contrast to the hyperpermeability exhibited by polysaccharides across the glomerular filtration barrier and also contrasts with the charge selectivity of the latter. Conclusion: The phenomenon of molecular flexibility is more important for a macromolecule’s permeability through the glomerular filter than across the continuous peritoneal capillary endothelium. Furthermore, it seems that charge plays a subordinate role in the steady‐state transport across the combined peritoneal capillary–interstitial barrier.     

The microwell-mesh: A novel device and protocol for the high throughput manufacturing of cartilage microtissues

Microwell platforms are frequently described for the efficient and uniform manufacture of 3-dimensional (3D) multicellular microtissues. Multiple partial or complete medium exchanges can displace microtissues from discrete microwells, and this can result in either the loss of microtissues from culture, or microtissue amalgamation when displaced microtissues fall into common microwells. Herein we describe the first microwell platform that incorporates a mesh to retain microtissues within discrete microwells; the microwell-mesh. We show that bonding a nylon mesh with an appropriate pore size over the microwell openings allows single cells to pass through the mesh into the microwells during the seeding process, but subsequently retains assembled microtissues within discrete microwells. To demonstrate the utility of this platform, we used the microwell-mesh to manufacture hundreds of cartilage microtissues, each formed from 5 × 10³ bone marrow-derived mesenchymal stem/stromal cells (MSC). The microwell-mesh enabled reliable microtissue retention over 21-day cultures that included multiple full medium exchanges. Cartilage-like matrix formation was more rapid and homogeneous in microtissues than in conventional large diameter control cartilage pellets formed from 2 × 10⁵ MSC each. The microwell-mesh platform offers an elegant mechanism to retain microtissues in microwells, and we believe that this improvement will make this platform useful in 3D culture protocols that require multiple medium exchanges, such as those that mimic specific developmental processes or complex sequential drug exposures.     

The interaction of Streptococcus pneumoniae with plasmin mediates transmigration across endothelial and epithelial monolayers by intercellular junction cleavage

The precise mechanisms by which Streptococcus pneumoniae overcomes epithelial and endothelial barriers to access underlying human tissues remain to be determined. The plasminogen system is highly important for the tissue barrier degradation which allows cell migration. Plasminogen is known to interact with pneumococci via enolase, glyceraldehyde-3-phosphate dehydrogenase, and choline-binding protein E. These observations prompted us to evaluate the role of this proteolytic system in the pneumococcal invasion process. We observed that coating of S. pneumoniae R6 strain with plasminogen or inactivated plasmin increased adherence to pulmonary epithelial A549 and vascular endothelial EaHy cells in vitro. This indicates that plasminogen-mediated adherence is independent of the protease activity and involves plasminogen binding to receptors on eukaryotic cell surfaces. Conversely, decreased adherence of bacterial cells coated with active plasmin was observed, indicating that the protease activity limits bacterial attachment on the cell surface. We were then interested in investigating the role of the proteolytic plasmin activity in the traversal of tissue barriers. We observed that adherence of plasmin-coated D39 (encapsulated) or R6 (unencapsulated) pneumococci induced sporadic disruptions of EaHy and A549 monolayer cell junctions. This was not observed when plasmin was inhibited by aprotinin. Endothelial junction disorganization may proceed by proteolysis of the cell junction components. This is supported by our observation of the in vitro cleavage by plasmin bound to pneumococci of recombinant vascular endothelial cadherin, the main component of endothelial adherens junctions. Finally, junction damage induced by plasmin may be related to tissue barrier traversal, as we measured an increase of S. pneumoniae transmigration across epithelial A549 and endothelial EaHy layers when active plasmin was present on the bacterial surface. Our results highlight a novel function for the plasminogen recruitment at the bacterial surface in facilitating adherence of pneumococci to endothelial and epithelial cells, while active plasmin degrades intercellular junctions. This process promotes migration of pneumococci through cell barriers by a pericellular route, a prerequisite for dissemination of S. pneumoniae in the host organism.     

人血脑屏障体外实验模型的建立及缺氧-复氧对其通透性的影响

目的建立人血脑屏障体外实验模型，并探讨缺氧-复氧对血脑屏障模型通透性的影响。 方法将分离、纯化的人脑微血管内皮细胞（HB-MVEC）在细胞插入器上培养至汇合状态，以液面试漏试验确定血脑屏障模型的形成，并通过形态学检查、跨内皮细胞电阻（TEER）测定和辣根过氧化物酶（HRP）通透率对血脑屏障模型进行鉴定；以未达汇合状态的HB-MVEC及培养至汇合状态的人脐静脉内皮细胞（HUVEC）作为对照。观察缺氧-复氧处理（缺氧2h和复氧1、2、4、8、24h）和缺氧-复氧时存在白细胞激活产物（缺氧2h后在有白细胞激活产物存在情况下复氧1h）对血脑屏障模型通透性的影响，以及前列腺素E、α1抗胰蛋白酶和丹参单体764-3对血脑屏障模型的保护作用。各项实验均观察3孔细胞。 结果HB-MVEC在细胞插入器培养至汇合状态后，液面试漏试验呈阳性；扫描电镜观察显示细胞间无间隙，透射电镜检查证实细胞间存在紧密连接。血脑屏障模型、未达汇合状态HB-MVEC和HUVEC的TEER分别为（46.0±1.3）、（30.8±1.4）、（7.5±2.1）Ω/cm^2；向细胞插入器内加入含HRP的培养基培养1h后，HRP通透率分别为0.17%±0.03%、0.26%±0.04%和0.94%±0.07%；缺氧2h和复氧1、2、4、8、24h HRP通透率分别为3.97%±0.94%、6.06%±0.75%、7.17%±0.18%、7.96%±0.47%、8.57%±0.62%、10.37%±0.78%。血脑屏障模型缺氧2h后在有白细胞激活产物的情况下复氧1h，其HRP通透率为8.87%±0.76%，明显高于无白细胞激活产物组（7.20%±0.87%）；而前列腺素E、α1抗胰蛋白酶和丹参单体764-3能够减弱这种情况下的血脑屏障模型通透性增加，3组的HRP通透率分别为7.08%±0.89%，6.01%±0.57%和5.53%±0.62%。 结论应用HB-MVEC可以构建血脑屏障体外模型，缺氧-复氧明显增加血脑屏障模型的通透性，前列腺素E、α1抗胰蛋白酶和丹参单体764-3具有保护血脑屏障     

BBB ON CHIP: microfluidic platform to mechanically and biochemically modulate blood-brain barrier function

The blood-brain barrier (BBB) is a unique feature of the human body, preserving brain homeostasis and preventing toxic substances to enter the brain. However, in various neurodegenerative diseases, the function of the BBB is disturbed. Mechanisms of the breakdown of the BBB are incompletely understood and therefore a realistic model of the BBB is essential. We present here the smallest model of the BBB yet, using a microfluidic chip, and the immortalized human brain endothelial cell line hCMEC/D3. Barrier function is modulated both mechanically, by exposure to fluid shear stress, and biochemically, by stimulation with tumor necrosis factor alpha (TNF-α), in one single device. The device has integrated electrodes to analyze barrier tightness by measuring the transendothelial electrical resistance (TEER). We demonstrate that hCMEC/D3 cells could be cultured in the microfluidic device up to 7 days, and that these cultures showed comparable TEER values with the well-established Transwell assay, with an average (± SEM) of 36.9 Ω.cm² (± 0.9 Ω.cm²) and 28.2 Ω.cm² (± 1.3 Ω.cm²) respectively. Moreover, hCMEC/D3 cells on chip expressed the tight junction protein Zonula Occludens-1 (ZO-1) at day 4. Furthermore, shear stress positively influenced barrier tightness and increased TEER values with a factor 3, up to 120 Ω.cm². Subsequent addition of TNF-α decreased the TEER with a factor of 10, down to 12 Ω.cm². This realistic microfluidic platform of the BBB is very well suited to study barrier function in detail and evaluate drug passage to finally gain more insight into the treatment of neurodegenerative diseases.     

Water permeability of the mammalian cochlea: functional features of an aquaporin-facilitated water shunt at the perilymph–endolymph barrier

The cochlear duct epithelium (CDE) constitutes a tight barrier that effectively separates the inner ear fluids, endolymph and perilymph, thereby maintaining distinct ionic and osmotic gradients that are essential for auditory function. However, in vivo experiments have demonstrated that the CDE allows for rapid water exchange between fluid compartments. The molecular mechanism governing water permeation across the CDE remains elusive. We computationally determined the diffusional (P _D) and osmotic (P _f) water permeability coefficients for the mammalian CDE based on in silico simulations of cochlear water dynamics integrating previously derived in vivo experimental data on fluid flow with expression sites of molecular water channels (aquaporins, AQPs). The P _D of the entire CDE (P _D?=?8.18?×?10^?5 cm s^?1) and its individual partitions including Reissner's membrane (P _D?=?12.06?×?10^?5 cm s^?1) and the organ of Corti (P _D?=?10.2?×?10^?5 cm s^?1) were similar to other epithelia with AQP-facilitated water permeation. The P _f of the CDE (P _f?=?6.15?×?10^?4 cm s^?1) was also in the range of other epithelia while an exceptionally high P _f was determined for an epithelial subdomain of outer sulcus cells in the cochlear apex co-expressing AQP4 and AQP5 (OSCs; P _f?=?156.90?×?10^?3 cm s^?1). The P _f/P _D ratios of the CDE (P _f/P _D?=?7.52) and OSCs (P _f/P _D?=?242.02) indicate an aqueous pore-facilitated water exchange and reveal a high-transfer region or “water shunt” in the cochlear apex. This “water shunt” explains experimentally determined phenomena of endolymphatic longitudinal flow towards the cochlear apex. The water permeability coefficients of the CDE emphasise the physiological and pathophysiological relevance of water dynamics in the cochlea in particular for endolymphatic hydrops and Ménière's disease.     

The Interstitial Space of Adipose Tissue as Determined by Single Injection and Equilibration Techniques

The interstitial ¹⁴C-sucrose space was determined in the subcutaneous adipose tissue of the dog using both a single injection-indicator dilution technique and an equilibration-tissue sampling method. ¹³¹I-albumin and ⁵¹Cr-labelled erythrocytes served as intravascular indicators. The conventional extrapolation method for single injection curves yielded space values several times lower than those measured by the equilibration technique, although sampling was continued until the venous outflow samples contained less than 1 % of the peak ¹⁴C-sucrose activity. This discrepancy may be due to long transit times, for which the conventional extrapolation procedure does not account. An extrapolation procedure is proposed based on the assumption that the area under the normalized extra- and intravascular curves should be equal. By this procedure as well as by the equilibration technique the interstitial ¹⁴C-sucrose space in adipose tissue measures approximately 10 ml × 100 g^-1. The single injection technique does not appear applicable for space determinations in adipose tissue during sympathetic nerve stimulation (3–7 Hz), possibly due to “trapping” of the tracers within the tissue.     

Perceived Sodium Reduction Barriers Among Patients with Chronic Kidney Disease: Which Barriers Are Important and Which Patients Experience Barriers?

Purpose

The purposes of this study were to assess the importance of perceived sodium reduction barriers among patients with chronic kidney disease (CKD) and identify associated sociodemographic, clinical, and psychosocial factors.

Method

A total of 156 patients with CKD completed a questionnaire assessing sodium reduction barriers (18 self-formulated items), depressive symptoms (Beck Depression Inventory), perceived autonomy support (Modified Health Care Climate Questionnaire), and self-efficacy (Partners in Health Questionnaire). Factor analysis was used to identify barrier domains. Correlation coefficients were computed to examine relationships between barrier domains and patient characteristics.

Results

Nine barrier domains were identified. Barriers perceived as important were as follows: high sodium content in products, lack of sodium feedback, lack of goal setting and discussing strategies for sodium reduction, and not experiencing CKD-related symptoms (mean scores > 3.0 on 5-point scales, ranging from 1 ‘no barrier’ to 5 ‘very important barrier’). Other barriers (knowledge, attitude, coping skills when eating out, and professional support) were rated as moderately important (rated around midpoint), and the barrier ‘intrinsic motivation’ was rated as somewhat important (mean score = 1.9). Sodium reduction barrier domains were not associated with gender and kidney function, but were associated with age, level of education, number of comorbidities, perceived autonomy support, depressive symptoms, and self-efficacy (range r = 0.17–0.35). Patients with lower self-efficacy and perceived autonomy support scores experienced most sodium reduction barriers.

Conclusion

Patients with CKD experience multiple important sodium reduction barriers and could benefit from support strategies that target various sodium reduction barriers and strengthen beliefs regarding self-efficacy and autonomy support. Additionally, environmental interventions should be implemented to reduce sodium levels in processed foods.

     

Suppression by isoproterenol of endothelial cell morphology and barrier function changes induced by platelet-activating factor

Using a model to study vascular permeability on hydrostatically perfused bovine pulmonary artery endothelial cell (EC) monolayers and software to analyze cell morphological parameters automatically in a computer image workstation, we studied the effects of isoproterenol (IPN) on platelet-activating factor (PAF)-induced changes in EC monolayer permeability and cell morphological parameters. Albumin has fortifying effects on endothelial barrier function. As albumin concentration in the perfusate increased (0, 1, 5, 10, 20 mg/ml), EC monolayer hydraulic conductivity (Lp) decreased gradually while Lp of the filter membranes did not change. After treatment of the EC monolayer with PAF 10^–8 mol/liter for 30 min, transmonolayer fluid flow, protein clearance rate, and Lp value increased noticeably. At the same time, cell area decreased and intercellular distance and percentage of intercellular space area in total cell monolayer increased. Pretreatment with 10^–4 mol/liter IPN blocked PAF-induced EC permeability and morphological changes, suggesting that EC contraction and intercellular gap formation are important mechanisms for PAF-induced high vascular permeability. IPN inhibits the effects of PAF via stabilization of EC morphology, protection of intercellular junction, and blockade of intercellular gap formation.Grant support: National Natural Sciences Foundation 39200144.     

Human epidermal growth factor receptor signaling in acute lung injury

Acute lung injury (ALI) is a syndrome marked by increased permeability across the pulmonary epithelium resulting in pulmonary edema. Recent evidence suggests that members of the human epidermal growth factor receptor (HER) family are activated in alveolar epithelial cells during ALI and regulate alveolar epithelial barrier function. These tyrosine kinase receptors, which also participate in the pathophysiology of pulmonary epithelial malignancies, regulate cell growth, differentiation, and migration as well as cell-cell adhesion, all processes that influence epithelial injury and repair. In this review we outline mechanisms of epithelial injury and repair in ALI, activation patterns of this receptor family in pulmonary epithelial cells as a consequence injury, how receptor activation alters alveolar permeability, and the possible intracellular signaling pathways involved. Finally, we propose a theoretical model for how HER-mediated modulation of alveolar permeability might affect lung injury and repair. Understanding how these receptors signal has direct therapeutic implications in lung injury and other diseases characterized by altered epithelial barrier function.     

Successful hematopoietic stem cell mobilization with vinorelbine and filgrastim in germ cell tumor

Germ cell tumor (GCT) is the most frequent cancer in young men and is highly curable. Almost 80 % of patients with the disease in an advanced stage achieve a reliable response to cisplatin combination chemotherapy. For relapsing or refractory disease, autologous hematopoietic stem cell transplantation (HSCT) is an effective therapy. The two most used mobilization strategies for HSC collection are filgrastim alone or filgrastim after chemotherapy (chemomobilization). HSC collection with filgrastim mobilization can be difficult, especially in highly treated patients. While the addition of chemotherapy improves mobilization and reduces the number of apheresis sessions, it can increase morbidity rate as well. We describe a case of a 45-year-old male with classical seminoma who was submitted to orchiectomy. Two months after, he presented progression of the tumor. He received four cycles of cisplatin, etoposide and bleomycin, with residual retroperitoneal mass and cervical lymphadenopathy. Further, he was submitted to three more cycles of cisplatin, ifosfamide and paclitaxel. Thereupon, he showed partial response. At that moment, autologous HSC transplantation was considered. In the first mobilization, filgrastim alone was used without success in harvesting. The second mobilization consisted of vinorelbine at day 1 (35 mg/m²) and filgrastim (16 µg/kg) started at day 5. The peak of CD34+ cells in peripheral blood was 32.6 × 10⁶ cells/L on day 8, with 4.73 × 10⁶ cells/kg CD34+ collected on days 8 and 9. The benefits of this scheme include: (a) outpatient administration, (b) fewer doses of filgrastim, (c) minimal risk of febrile neutropenia and (d) reliable prediction of collection day. For these reasons, we conclude that vinorelbine chemomobilization is a great option for GCT, particularly in patients with high risk of mobilization failure. Furthermore, it requires less resource usage, hospitalizations and transfusions than conventional chemomobilization.     

Refinement and verification of test conditions for colony-forming unit assay in rat bone marrow cells with preservable colony specimens

We established test conditions for colony-forming unit assay using rat bone marrow cells in which the colony specimens can be preserved. In our test system, all colonies can be transferred from a petri dish to a slide glass with whole agar medium because the agar medium volume is small and movable, enabling the agar medium to be dried and then fixed on the slide glass. Appropriate conditions for the colony-forming unit granulocyte–macrophage assay were as follows: 10 to 30 ng/ml recombinant murine granulocyte and macrophage colony-stimulating factor, 1?×?10⁵ cells/dish, and culture periods of 3 days for neutrophil colonies and 8 days for macrophage colonies. Appropriate conditions for the colony-forming unit erythroid assay were as follows: 2 IU/ml recombinant human erythropoietin, 0.25 to 1?×?10⁵ cells/dish, and culture period of 2–3 days. We also examined responses of bone marrow toxicants 5-fluorouracil and doxorubicin in these test systems, finding that both compounds decreased the numbers of colonies in a concentration-dependent manner in both assays. These findings suggest that these test systems are useful tools in evaluating bone marrow toxicity of these particular compounds.     

A primary culture system of mouse thick ascending limb cells with preserved function and uromodulin processing

The epithelial cells lining the thick ascending limb (TAL) of the loop of Henle perform essential transport processes and secrete uromodulin, the most abundant protein in normal urine. The lack of differentiated cell culture systems has hampered studies of TAL functions. Here, we report a method to generate differentiated primary cultures of TAL cells, developed from microdissected tubules obtained in mouse kidneys. The TAL tubules cultured on permeable filters formed polarized confluent monolayers in ～12 days. The TAL cells remain differentiated and express functional markers such as uromodulin, NKCC2, and ROMK at the apical membrane. Electrophysiological measurements on primary TAL monolayers showed a lumen-positive transepithelial potential (+9.4?±?0.8 mV/cm²) and transepithelial resistance similar to that recorded in vivo. The transepithelial potential is abolished by apical bumetanide and in primary cultures obtained from ROMK knockout mice. The processing, maturation and apical secretion of uromodulin by primary TAL cells is identical to that observed in vivo. The primary TAL cells respond appropriately to hypoxia, hypertonicity, and stimulation by desmopressin, and they can be transfected. The establishment of this primary culture system will allow the investigation of TAL cells obtained from genetically modified mouse models, providing a critical tool for understanding the role of that segment in health and disease.