Corneal regeneration by transplantation of corneal epithelial cell sheets fabricated with automated cell culture system in rabbit model

We have performed clinical applications of cell sheet-based regenerative medicine with human patients in several fields. In order to achieve the mass production of transplantable cell sheets, we have developed automated cell culture systems. Here, we report an automated robotic system utilizing a cell culture vessel, cell cartridge. The cell cartridge had two rooms for epithelial cells and feeder layer cells separating by porous membrane on which a temperature-responsive polymer was covalently immobilized. After pouring cells into this robotic system, cell seeding, medium change, and microscopic examination during culture were automatically performed according to the computer program. Transplantable corneal epithelial cell sheets were successfully fabricated in cell cartridges with this robotic system. Then, fabricated cell sheets were transplanted onto ocular surfaces of rabbit limbal epithelial stem cell deficiency model after 6-h transportation using a portable homothermal container to keep inner temperature at 36 °C. Within one week after transplantation, normal corneal epithelium was successfully regenerated. This automatic cell culture system would be useful for industrialization of tissue-engineered products for regenerative medicine.     

Evaluation of laboratory methods routinely used to detect the effect of aspirin against new reference methods

Background

Aspirin, a commonly used antiplatelet agent, blocks platelet thromboxane A₂ (TXA₂) formation from arachidonic acid (AA) by acetylating platelet cyclooxygenase-1 (COX-1). Laboratory methods currently used to detect this antiplatelet effect of aspirin provide variable results. We have reported three methods that assess platelet COX-1 acetylation (inactivation) by aspirin and its direct consequences. The first and second assays use monoclonal anti-human-COX-1 antibodies that only detect acetylated (inactivated) COX-1 and active (non-acetylated) COX-1, respectively. The third method measures platelet production of TXB₂ (the stable metabolite of TXA₂) in vitro in response to AA. We compared the results of these three reference methods with other routinely used methods for assessing the functional consequences aspirin treatment.

Methods

108 healthy volunteers were treated with low-dose aspirin for 7 days. On day 7 following aspirin treatment COX-1 in the platelets was fully acetylated whereas only non-acetylated COX-1 was present in the day 0 platelets. Further, TXB₂ production by day 7 platelets was completely blocked. The following tests were performed on the samples obtained from study participants before and after seven days of aspirin treatment: PFA-100 closure time with collagen/epinephrine cartridge, VerifyNow® (VN) Aspirin Assay, platelet aggregation and ATP secretion using AA, ADP, epinephrine and collagen as agonists.

Results

Comparing the pre-treatment and day 7 values, methods that use AA as platelet agonist (AA-induced platelet aggregation/secretion and VN Aspirin Assay) showed high discriminative power. In contrast, results of the other tests showed considerable overlap between day 7 and day 0 values.

Conclusions

Only assays that clearly distinguish between acetylated and non-acetylated platelet COX-1 are useful for establishing the antiplatelet effect of aspirin. The other tests are not suitable for this purpose.     

Antidepressants differentially modify the extinction of an aversive memory task in female rats

Treatment of major depression, posttraumatic stress disorder and other psychopathologies with antidepressants can be associated with improvement of the cognitive deficits related to these disorders. Although the mechanisms of these effects are not completely elucidated, alterations in the extinction of aversive memories are believed to play a role in these psychopathologies. We have recently verified that female rats present low levels of extinction when submitted to the plus-maze discriminative avoidance task. In the present study, female rats were treated long term with clinically used antidepressants (fluoxetine, nortriptyline or mirtazapine) and subjected to the plus-maze discriminative avoidance task to evaluate learning, memory, extinction and anxiety-related behaviors as well as behavioral despair in the forced swimming test. All groups learned the task and exhibited retrieval. Chronic treatment with fluoxetine (but not with the other antidepressants tested) increased extinction of the discriminative task. In the forced swimming test, the animals treated with fluoxetine and mirtazapine showed decreased immobility duration. In conclusion, fluoxetine potentiated extinction, while both fluoxetine and mirtazapine were effective in ameliorating depressive-like behavior in the forced swimming test, suggesting a possible dissociation between the effects on mood and the extinction of aversive memories in female rats.     

Effect of hemoperfusion cartridge on different internal environmental indicators

BACKGROUND:

This study aimed to observe the effect of hemoperfusion (HP) cartridge on different internal environment indicators at different time points in patients with acute blood poisoning and to find alternative indicators for the detection of blood poisoning.

METHODS:

The levels of internal environment indicators (blood pH, P_vCO₂, P_vO₂, blood lactate, potassium, free calcium, bicarbonate, and blood glucose) before and after HP treatment were recorded for patients with acute poisoning at time points of 30 minutes and 120 minutes. After calculating the difference value δ, the statistical software was used to analyze the statistical difference of the influence caused by HP cartridge at two time points. According to the formula, adsorption rate % = ×100, the adsorption rate of each indicator was calculated respectively.

RESULTS:

The difference of indicators at different time points in inlet and outlet such as blood glucose, free-calcium, and lactate was statistically significant (P<0.05), but the difference in indicators such as pH, P_vCO₂, P_vO₂, potassium, sodium, and bicarbonate was not statistically significant (P>0.05).

CONCLUSION:

During HP treatment, the indicators of blood glucose, free-calcium and lactate were significantly affected by HP cartridge, and the effect varies with time.KEY WORDS: Hemoperfusion cartridge, Internal environment indicators, Blood glucose, Adsorption rate     

Use of Fast Transfer Analysis Cartridges for Cervical Sampling and Real Time PCR Based High Risk HPV Testing in Cervical Cancer Prevention - a Feasibility Study from South India

Background: Molecular testing for human papillomavirus (HPV) is the most objective and reproducible ofall cervical cancer screening tests and also less demanding in terms of training and quality assurance. However,there is an impending need for cost effective molecular HPV testing methods with sampling ease, easy storagemeasures and minimum turn around times suitable for a low resource setting. Objective : Our aim was toevaluate the feasibility of using a fast transfer analysis (FTA) mini elute cartridge for cervical sampling toidentify high risk HPV by real time PCR and to compare molecular HPV testing and Pap cytology testing topredict histologically confirmed cervical precancer (CIN 2+ lesions) in a cervical cancer prevention program.Materials and Methods: This was conducted as a pilot study (n=200) on women sampled using FTA mini elutecartridges, genotyped by two different real time PCR assays, detecting 13 high risk HPV (HR HPV) species,including HPV16 along with its physical DNA status. Results obtained from each of the tests were comparedand analysed using suitable statistical tests. Results: With FTA mini elute cartridge samples HR HPV positivitywas seen in 48/200 (24%). Of these, presence of HPV 16 DNA was observed in 28/48 (58.3%) women. High riskHPV was positive in 20% (37/185) of women with benign cytology and 73.3% (11/15) of women with abnormalcytology findings. A very significant correlation (χ2 = 22.090 ; p=0.000) was observed between cytology and HRHPV findings showing an increasing trend of HR HPV prevalence in 50% (1/2) of LSIL, 75% (3/4) of HSIL and100% (3/3) of SCC. Of the CIN 2+ lesions identified by histopathology, 88.9% (8/9) had HR HPV. A significantassociation (χ2=11.223 ; p=0.001) of HR HPV and histopathologically confirmed CIN 2+ lesions was found.Sensitivity of the two tests were comparable but specificity of Pap testing was better (90.7% vs 70.4%) to predicthistopathologically diagnosed cervical precancers. Conclusions: The current study explored the feasibility ofusing a FTA mini elute cartridge for cervical sampling for the first time in India as a part of a community basedcervical cancer prevention program. We suggest that FTA based sampling is suitable and feasible for real timebased HPV testing. Molecular HR HPV testing can be more sensitive and useful to identify high risk womenrequiring Pap testing which is more specific to detect histologically confirmed cervical precancer.     

Optimizing recovery of peptides from an octadecylsilyl (ODS) cartridge

An analytical procedure has been studied in detail to optimize the recovery of the radiolabeled and unlabeled synthetic peptides substance P and methionine-enkephalin from a reversed phase octadecylsilyl disposable cartridge, which is one of the first steps to preferentially enrich peptides from a biologic matrix. Our previous research on biologic peptides indicated the need for the present study. The column flow-rate was found to be one of the most important experimental parameters; and its effect on the recovery of those two synthetic peptides was determined by the measurement of recovered radioactivity and by high performance liquid chromatography U.V. detection. The accuracy and reproducibility of the flow-rate affected recovery, and it was necessary to regulate the flow-rate with a syringe infusion pump. Recovery was optimum using a slow rate for both sample application and elution. Furthermore, a slow elution was more important than a slow rate of sample application. Such basic recovery studies on individual synthetic peptides are required before undertaking peptide extractions from biologic matrices.     

Highly efficient solid phase supported radiosynthesis of [11C]PiB using tC18 cartridge as a “3‐in‐1” production entity

Pittsburgh compound B ([¹¹C]PiB) is the gold standard positron emission tomography (PET) tracer for the in vivo imaging of amyloid plaques. Currently, it is synthesized by either solution chemistry or using a “dry loop” approach followed by HPLC purification within 30 minutes starting from [¹¹C]CO₂. Here, we report a novel, highly efficient solid phase supported carbon‐11 radiolabeling procedure using commercially available disposable tC18 cartridge as a “3‐in‐1” entity: reactor, purifier, and solvent replacement system. [¹¹C]PiB is synthesized by passing gaseous [¹¹C]CH₃OTf through a tC18 cartridge preloaded with a solution of precursor. Successive elution with aqueous ethanol solutions allows for nearly quantitative separation of the reaction mixture to provide chemically and radiochemically pure PET tracer. [¹¹C]PiB suitable for human injection is produced within 10 minutes starting from [¹¹C]CH₃OTf (20 min from [¹¹C]CO₂) in 22% isolated yield not corrected for decay and molar activity of 190 GBq/μmol using 0.2 mg of precursor. This technique reduces the amount of precursor and other supplies, avoids use of preparative HPLC and toxic solvents, and decreases the time between consecutive production batches. Solid phase supported technique can facilitate [¹¹C]PiB production compliant with Good Manufacturing Practice (GMP) and improve synthesis reliability.     

Direct hemoperfusion with a polymyxin B-immobilized cartridge in intestinal warm ischemia reperfusion

AIM： To investigate the effectiveness of direct hemoperfusion with polymyxin B-immobilized fibers （DHPPMX therapy） on warm ischemia-reperfusion （I/R） injury of the small intestine.
METHODS： The proximal jejunum and distal ileum of mongrel dogs were resected. Warm ischemia was performed by clamping the superior mesenteric artery （SMA） and vein （SMV） for 2 h. Blood flow to the proximal small intestine was restored 1 h after reperfusion, and the distal small intestine was used as a stoma. The experiment was discontinued 6 h after reperfusion. The dogs were divided into two groups： the DHP-PMX group （n = 6, DHP-PMX was performed for 180 min; from 10 min prior to reperfusion to 170 rain after reperfusion） and the control group （n = 5）. The rate pressure product （RPP）, SMA blood flow, mucosal tissue blood flow, and intramucosal pH （pHi） were compared between the two groups. The serum interleukin （IL）-10 levels measured 170 min after reperfusion were also compared.
RESULTS： The RPP at 6 h after reperfusion was significantly higher in the PMX group than in the control group （12174 ± 1832 mmHg/min vs 8929 ± 1797 mmHg/min, P 〈 0.05）. The recovery rates of the SMA blood flow at I and 6 h after reperfusion were significantly better in the PMX group than in the control group （61%±7% vs 44% ±4%, P 〈 0.05, and 59%±5% vs 35%±5%, P 〈 0.05, respectively）. The recovery rate of the mucosal tissue blood flow and the pHi levels at 6 h after reperfusion were significantly higher in the PMX group （61%±8% vs 31%±3%, P 〈 0.05 and 7.91±0.06 vs 7.69±0.08, P 〈 0.05, respectively）. In addition, the serum IL-IO levels just before DHP-PMX removal were significantly higher in the PMX group than in the control group （1 569 ± 253 pg/mL vs 211± 40 pg/mL, P 〈 0.05）.
CONCLUSION： DHP-PMX therapy reduced warm I/R injury of the small intestine. IL-10 may play a role in inhibiting I/R injury during DHP-PMX therapy.     

树脂血液灌流治疗毒鼠强中毒的疗效观察

目的评价HA型血液灌流器清除人体内毒鼠强(tetramine)的效果。方法(1)12例毒鼠强中毒患者入院后,选用HA型血液灌流器(由苯乙烯／二乙烯苯聚合而成的高分子大孔树脂作为吸附剂)进行血液灌流治疗,用气相色谱法分别测定毒鼠强中毒患者入院、灌流即刻、灌流结束时及结束后不同时间点血液毒鼠强浓度。(2)气相色谱法测定HA型血液灌流器中毒鼠强的吸附量。结果(1)随入院时间的推移患者血中毒鼠强浓度呈逐渐下降趋势,其降低幅度与HP治疗次数及每次HP的间隔时间有关,即在HP集中治疗阶段(每日或隔日一次),血中毒鼠强浓度呈较大幅度下降。(2)HA-330ml灌流器在开始治疗时血毒鼠强浓度为219～54μg／L之间(平均值为117．4μg／L),吸附毒鼠强的含量范围在4．385～0．836mg之间(平均值为2．03mg);HA-230ml灌流器在毒鼠强血药浓度在192～55μg／L范围(平均115μg／L)时,吸附毒鼠强的含量在2．375～0．328mg之间(平均值：1．508mg)。结论(1)血液灌流能有效清除人体内毒鼠强;(2)在血TETS浓度100μg／L左右时,树脂灌流器对毒鼠强的吸附量可能大于活性炭吸附剂。     

某化工厂全封闭车间职业病危害防护措施效果分析

目的对某化工厂全封闭车间存在的职业病危害因素及防护措施效果进行分析。方法依据《中华人民共和国职业病防治法》《工业企业设计卫生标准》（GBZ1—2002）,采用现场职业卫生学调查和职业病危害因素检测等方法对防护措施效果综合分析。结果该车间主要职业病危害因素为丙酮、噪声。丙酮检测点STEL、TWA合格率分别为77．8％、90．0％,卷曲机单元丙酮STEL最高为1146．2mg／m^3。卷曲机单元噪声强度合格率为66．7％,其余单元均合格。结论在该车间的主要职业病危害防护措施中,空调通风系统需改善,公司职业卫生管理有待加强。