Extracellular miR-6723-5p could serve as a biomarker of limbal epithelial stem/progenitor cell population.

BACKGROUND: Dysfunction or loss of limbal stem cells can result in limbal stem cell deficiency (LSCD), a disease that cause corneal opacity, pain, and loss of vision. Cultivated limbal epithelial transplantation (CLET) can be used to restore stem cell niche homeostasis and replenish the progenitor pool. Transplantation has been reported with high success rate, but there is an unmet need of prognostic markers that correlate with clinical outcomes. To date, the progenitor content in the graft is the only parameter that has been retrospectively linked to success. METHODS: In this study, we investigate extracellular micro RNAs (miRNAs) associated with stem/progenitor cells in cultivated limbal epithelial cells (cLECs). Using micro RNA sequencing and linear regression modelling, we identify a miRNA signature in cultures containing high proportion of stem/progenitor cells. We then develop a robust RNA extraction workflow from culture media to confirm a positive miRNA correlation with stem/progenitor cell proportion. RESULTS: miR-6723-5p is associated with cultures containing high proportion of stem/progenitor cells, and is detected in the basal layer of corneal epithelium. CONCLUSIONS: These results indicate that miR-6723-5p could potentially serve as a stem/progenitor cell marker in cLECs.

[Therapeutic effect of tonsillectomy on IgA nephropathy after kidney transplantation].

Objective: To observe the clinical effect of tonsillectomy on IgA nephropathy (IgAN) after renal transplantation. Methods: From March 2011 to July 2018, 201 kidney transplantation recipients who were diagnosed of IgAN by transplant renal biopsy in the Department of Organ Transplantation of the First Affiliated Hospital of Sun Yat-sen University were retrospectively reviewed, of which 18 patients underwent tonsillectomy after renal biopsy. The clinical data of the 18 patients were collected, patient and kidney survival time and function of the transplanted kidney were analyzed. Results: Of the 18 recipients, 13 were male and 5 were female, with an average age of (36.0±10.9) years. All 18 patients survived during follow-up. Two patients returned to dialysis treatment 10 months and 14 months after tonsillectomy, respectively. The creatinine was 94 (78, 133) µmol/L, 95 (74, 139) µmol/L, 106 (87, 158) µmol/L and 95(81, 147) µmol/L before tonsillectomy, 3 months, 1 year and 2 years after tonsillectomy, respectively (P=0.206). Urinary protein quantification was 0.31 (0.16, 1.38) g/24 h, 0.34 (0.10, 1.42) g/24 h, 0.33 (0.11, 0.56) g/24 h and 0.25 (0.10, 0.50) g/24 h at the same time points, respectively (P=0.104). The two patients who returned to dialysis were diagnosed of IgAN by transplant renal biopsy because of elevated creatinine, proteinuria and hematuria, 9 years and 4 years after kidney transplant respectively. Renal biopsy suggested that glomerular and segmental sclerosis were 7/24, 5/24 and 1/6, 2/6, respectively. Additionally, interstitial fibrosis and tubular atrophy (IF/TA) were both occupied 30% in the biopsies, and tonsillectomy was performed 461 days and 1 077 days after diagnosis of IgAN, respectively. Conclusions: Tonsillectomy can maintain the stability of renal function and prevent the aggravation of proteinuria in IgAN patients after renal transplantation. However, if pathology suggests obvious glomerulosclerosis or IF/TA, tonsillectomy may not be effective.

In vivo confocal microscopy of congenital aniridia-associated keratopathy.

PURPOSE: To explore the in vivo morphological changes of cornea and limbus in aniridia-associated keratopathy (AAK). METHODS: Three cases of AAK were examined with the application of in vivo confocal microscopy (IVCM). RESULTS: Abnormal structure of wing and basal layer of epithelium, the loss of subbasal nerves, and the presence of goblet cells at central cornea could be identified in the most severe case, along with the absence of Vogt palisades. The less extent of abnormalities in corneal epithelial cells, subbasal nerve, and Vogt palisades were visible in the moderate or mild cases. CONCLUSIONS: The morphological changes of cornea and limbus vary in AAK, and IVCM is a promising tool to determine the degree of limbal stem cell deficiency in patients with AAK.

Paediatric bacterial keratitis cases in Shanghai: microbiological profile, antibiotic susceptibility and visual outcomes.

PURPOSE: The purpose of this study was to review the microbiological profile, in vitro antibiotic susceptibility and visual outcomes of paediatric microbial keratitis in Shanghai, China over the past 6 years. METHODS: Medical records of patients aged ≤16 years were reviewed, who were diagnosed as having bacterial keratitis between 1 January 2005 and 31 December 2010. Bacterial culture results and in vitro antibiotic susceptibility were analysed. A logistic regression analysis was conducted to evaluate the relationship between visual impairment and possible risk factors. RESULTS: Eighty consecutive cases of paediatric bacterial keratitis cases were included, among which 59 were identified as having positive culture. Staphylococcus epidermidis was the most commonly isolated organism (n=23; 39.0%), followed by Streptococcus pneumoniae (n=11; 18.6%) and Pseudomonas aeruginosa (n=6; 10.2%). Antibiotic sensitivities revealed that tested bacteria had low resistance rates to fluoroquinolones and aminoglycosides (8.3-18.4% and 12.5-24.4%, respectively). Multivariate logistic regression analysis proved that visual impairment was significantly associated with Gram-negative bacterial infection (odds ratio (OR)=7.626; P=0.043) and an increasing number of resistant antibiotics (OR=0.385; P=0.040). CONCLUSIONS: S. epidermidis was the most common isolated organism in Shanghai paediatric keratitis. The fluoroquinolones and aminoglycosides remained good choices for treating these patients. Gram-negative bacterial infection and an increasing number of resistant antibiotics were associated with worse visual prognoses in paediatric keratitis.

Quantitative studies of the distribution pattern for Salmonella Enteritidis in the internal organs of chicken after oral challenge by a real-time PCR.

This research was undertaken to identify and understand the regular distribution pattern for Salmonella Enteritidis (S. enteritidis) in the internal organs of chicken after oral challenge over a 3 wk period. We used a real-time, fluorescence-based quantitative polymerase chain reaction (FQ-PCR) to detect genomic DNA of S. enteritidis in the blood and the internal organs, including heart, liver, spleen, kidney, pancreas, and gallbladder, from chicken after oral challenge at different time points. The results showed that the spleen was positive at 12 h post inoculation (PI), and the blood was at 14 h PI. The organism was detected in the liver and heart at 16 h PI, pancrea was positive at 20 h PI, and the final organ to show a positive results were the kidney and gallbladder at 22 h PI. The copy number of S. enteritidis DNA in each tissue reached a peak at 24 h-36 h PI, with the liver and spleen containing high concentrations of S. enteritidis, whereas the blood, heart, kidney, pancreas, and gallbladder had low concentrations. S. enteritidis populations began to decrease and were not detectable at 3 d PI, but were still present up to 12 d PI in the gallbladder, 2 wk for the liver, and 3 wk for the spleen without causing apparent symptoms. The results showed that the liver and spleen may be the primary sites for S. enteritidis setting itself up as a commensa over a long time after oral challenge. Interestingly, it may be the first time reported that the gallbladder is a site of carriage for S. enteritidis over a 12 d period. This study will help to understand the mechanisms of action of S. enteritidis infection in vivo.

Simple and rapid detection of Salmonella serovar Enteritidis under field conditions by loop-mediated isothermal amplification.

AIM: The objective of this study is to develop a serovar-specific loop-mediated isothermal amplification (LAMP) method for sensitive, rapid, and inexpensive detection of Salmonella serovar Enteritidis under field conditions. METHODS: A set of six specific primers was designed with Salmonella Enteritidis DNA as the target. LAMP conditions were optimized by incubating the target DNA with the Bst DNA polymerase large fragment in a simple water bath. The sensitivity and specificity of LAMP was then compared with those of fluorescent quantitative real-time polymerase chain reaction (FQ-PCR). RESULTS: The results were as follows. (1) Serovar-specific Salmonella Enteritidis DNA was amplified at 65°C in as early as 20min in a water bath. (2) A colour change visible to the naked eye indicated a positive amplification reaction. (3) The detection limit of the LAMP assay was 4 copies µl(-1) ; thus, the sensitivity and specificity of this assay is similar to those of the FQ-PCR. CONCLUSIONS: LAMP is a high-throughput detection technique with high sensitivity, specificity, and simplicity; these factors make it suitable for specifically detecting Salmonella Enteritidis under field conditions and in laboratory settings. Thus, LAMP eliminates the need for complicated equipment and technical training in the detection of this specific serovar. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study involving the use of LAMP to detect Salmonella serovar-specific DNA sequences. It is also the first to report an ideal method of distinguishing between Salmonella Enteritidis and other Salmonella under field conditions.

Screening the Fusarium graminearum inhibitory mutant strain from Bacillus subtilis by atmospheric-pressure plasma jet.

AIMS: The aim of this study was to improve the antagonistic activity of Bacillus subtilis JA towards Fusarium graminearum by screening high-yielding mutant using the atmospheric-pressure plasma jet (APPJ). METHODS AND RESULTS: Atmospheric-pressure plasma jet was applied as mutagenic source for breeding high-yielding mutant strain. Helium was used as APPJ operating gas. The mutation effects of different treatment times of APPJ were studied. The mutant strain designated as B. subtilis B06 was successfully screened out, which showed higher antagonistic activity against F. graminearum in vitro. Its inhibition zone against the indicator fungus increased by 23% compared to the original one. HPLC and ESI (electrospray ionization) mass spectrometry analysis indicated that antifungal compounds produced by the mutant and original strain belonged to the lipopeptide, surfactin and iturin families. The mutant strain showed favourable properties of faster growth in the fermentation process and higher production of antibiotics. The lipopeptide production of the mutant was 2.3-fold as that of the original strain. CONCLUSIONS: A mutant strain with strong antagonistic activity and high yielding of antibiotics was obtained by APPJ in this study. The mutant could be used as a promising biocontrol agent in agriculture. SIGNIFICANCE AND IMPACT OF STUDY: This study provides a novel mutagenic source for breeding high-yielding microbial mutant, which would be very useful in the application of some valuable metabolites from micro-organism.

Quantitative analysis of Salmonella Enteritidis loads in ducklings after nasal inoculation.

The distribution patterns of Salmonella Enteritidis in systemic organs, which have not been described previously, should be studied to better understand its pathogenesis in vivo. We inoculated the ducklings with Salmonella Enteritidis via the nasal route and performed a real-time PCR assay for determining the concentration of Salmonella Enteritidis DNA and studied the histopathology of various tissues postinoculation. The results show that the Salmonella Enteritidis load in systemic organs has a close correlation with the progression of disease. Further, rapid dissemination and active replication of Salmonella Enteritidis in multiple systemic organs accelerated the progression of disease.

Replication kinetics of Salmonella enteritidis in internal organs of ducklings after oral challenge: a quantitative time-course study using real-time PCR.

This research was undertaken to understand the replication kinetics of Salmonella enteritidis (S. enteritidis) in the internal organs of ducklings after oral challenge over a 2 wk period. A serovar-specific real-time, fluorescence-based quantitative polymerase chain reaction (FQ-PCR) assay was used to detect genomic DNA of S. enteritidis in the blood and the internal organs at different time points respectively. The results showed that the spleen was positive at 12 h post inoculation (PI) and the blood was at 14 h PI. The organism was detected in the liver and heart at 16 h PI, the pancreas and kidney were positive at 20 h PI, and the final organ to show a positive results was the gallbladder at 22 h PI. The copy number of S. enteritidis DNA in each tissue reached a peak at 24 h-36 h PI, with the liver and spleen containing the highest concentration of S. enteritidis. The blood, heart, kidney, pancreas, and gallbladder had low concentrations. S. enteritidis populations began to decrease and were not detectable at 3 d PI, but were still present up to 2 wk for the spleen without causing apparent symptoms. To make the results meaningful, a side-by-side bacteriology method (IFA) was performed. The results of IFA were similar to the FQ-PCR assay. This research provided a significant data for understanding the life cycle of S. enteritidis in the internal organs, and may help to understand the pathogenesis of S.entertidis in the future.

The pathogenesis of Salmonella enteritidis in experimentally infected ducks: a quantitative time-course study using taqman polymerase chain reaction.

Ducks were subcutaneously infected with a high-virulence strain of Salmonella enterica ssp. enterica serovar Enteritidis (Salmonella Enteritidis). The kinetics of the Salmonella Enteritidis genomic DNA loads, the immunohistochemical localization of the bacterial antigens, and the histopathological examination in various tissues were investigated. The results showed that the time course of the appearance of the Salmonella Enteritidis bacterial antigens and the lesions in various tissues was coincident with the bacterial load of the organism in various infected tissues. This suggests that Salmonella Enteritidis loads in systemic organs are closely correlated with the progression of the infection.

Population pharmacokinetics of mycophenolic acid in senile Chinese kidney transplant recipients.

To explore the pharmacokinetic characteristics of mycophenolic acid (MPA) among elderly Chinese kidney transplant recipients, we enrolled 24 patients over 60 years old (65.6 +/- 3.6) as the (Gs) group and 24 patients of 39.6 +/- 14.3 years old as a control group (Ga). Venous blood samples were taken at 0 (predose), 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, and 12 hours after the morning dose of mycophenolate mofetil at 10 to 12 weeks posttransplant. Plasma MPA concentrations were measured by a validated high-performance liquid chromatography method. Within 6-month posttransplant follow-up, there had not been an acute rejection episode when five elderly and one other adult experienced severe adverse events (SAEs), such as pneumonia and leukocytopenia. MPA area under the curve (AUC) in Gs was significantly lower than that among Ga (P < .05), while there was no significant difference in predose, peak concentrations, or peak times (P > .05). The concentration-time curve of Gs showed a bipeak pattern in five patients (20.8%) during the early stage (2 to 4 hours postdose). AUC in the subgroup of Gs with SAEs (n = 5) was significantly higher than that of elderly subjects without SAEs (n = 19) (P = .042). When Gs were subdivided at a cutting AUC point of 25 mug/mL, the SAE incidence was significantly higher in the subgroup with a higher AUC than than those with the lower AUC (P = .047). Through multiple stepwise regression, we obtained a minimal model to estimate MPA AUC of elderly recipients: AUC = 3.0410 + 9.8588 x C(0) + 0.5963 x C(0.5) + 2.5612 x C(3) (R(2) = .893).

Characteristics of neoplasm occurrence and the therapeutic effect of sirolimus in South Chinese kidney transplant recipients.

Kidney transplantation (KTx) recipients are at a higher risk of oncogenesis when compared to the general population. Sirolimus (SRL), a potent immunosuppressant, has shown promising antineoplastic effects in vitro and in vivo. This study retrospectively analyzed the neoplasm occurrence and the efficiency of SRL on unresectable malignancies in South Chinese KTx recipients. Thirty-three (1.64%) of 2017 patients who received KTx from January 1984 to December 2004 developed neoplasms at 4 to 117 months posttransplant, mostly in digestive organs (33.3%), the hematologic system (15.2%), or the skin (12.1%). The most common type was liver cancer (24.2%), followed by skin cancer, lymphoma, and thyroid cancer (9.1%). The median survival times were 41.5 and 6.0 months for those who did (n = 10) receive radical surgery or did not (n = 23), respectively. The 20-month survival rates were 70.0% versus 13.0% (P < .01). For unresectable patients, the median survival time of those treated with SRL (n = 8) was 14.5 months compared to 3.0 months for those who did not (n = 15). The survival rates at 12(th) and 20(th) months were 75.0% and 37.5% in the SRL group and 6.7% and 0% in the non-SRL group (P < .05). In conclusion, when compared with Western studies, a lower incidence and unique location pattern (liver cancer-dominant) are characteristics of de novo posttransplant neoplasms in South Chinese KTx recipients. Early diagnosis and feasible radical surgery are favorable for prognosis, and SRL is a treatment of choice for KTx recipients with neoplasms.

Crystallographic and biochemical analysis of cocaine-degrading antibody 15A10.

Catalytic antibody 15A10 hydrolyzes the benzoyl ester of cocaine to form the nonpsychoactive metabolites benzoic acid and ecgonine methylester. Here, we report biochemical and structural studies that characterize the catalytic mechanism. The crystal structure of the cocaine-hydrolyzing monoclonal antibody (mAb) 15A10 has been determined at 2.35 A resolution. The binding pocket is fairly shallow and mainly hydrophobic but with a cluster of three hydrogen-bond donating residues (TrpL96, AsnH33, and TyrH35). Computational docking of the transition state analogue (TSA) indicates that these residues are appropriately positioned to coordinate the phosphonate moiety of the TSA and, hence, form an oxyanion hole. Tyrosine modification of the antibody with tetranitromethane reduced hydrolytic activity to background level. The contribution from these and other residues to catalysis and TSA binding was explored by site-directed mutagenesis of 15A10 expressed in a single chain fragment variable (scFv) format. The TyrH35Phe mutant had 4-fold reduced activity, and TrpL96Ala, TrpL96His, and AsnH33Ala mutants were all inactive. Comparison with an esterolytic antibody D2.3 revealed a similar arrangement of tryptophan, asparagine, and tyrosine residues in the oxyanion hole that stabilizes the transition state for ester hydrolysis. Furthermore, the crystal structure of the bacterial cocaine esterase (cocE) also showed that the cocE employs a tyrosine hydroxyl in the oxyanion hole. Thus, the biochemical and structural data are consistent with the catalytic antibody providing oxyanion stabilization as its major contribution to catalysis.

Natural and artificial enzymes against cocaine. I. Monoclonal antibody 15A10 and the reinforcing effects of cocaine in rats.

Recent reports have indicated the potential usefulness of anticocaine catalytic monoclonal antibodies in reducing cocaine's toxic and reinforcing effects by altering its pharmacokinetics to favor increased metabolism to the systemically inert products ecgonine methylester and benzoic acid. The present study was designed to further these findings by evaluating the hypothesis that administration of the anticocaine catalytic monoclonal antibody mAb 15A10 would dose and time dependently reduce behavior maintained by a range of doses of i.v. cocaine. Male Sprague-Dawley rats were trained in daily 8-h sessions to self-administer i.v. cocaine. A within-session multiple-dose protocol was used wherein rats were allowed access to saline or one of six doses of cocaine [0 (saline), 0.015, 0.03, 0.06, 0 (saline), 0.125, 0.25, or 0.5 mg/kg/injection] each hour in the order stated. After demonstrating stable dose-response curves over 3 consecutive days, rats were given 30-min pretreatments of saline or mAb 15A10, (10, 30, or 100 mg/kg i.v.). Antibody, but not saline, pretreatments significantly altered dose-response curves for cocaine self-administration in a dose- and time-dependent manner, resulting in downward and rightward shifts in rates of responding across the cocaine dose range. These effects were apparently not attributable to general behavioral suppression, because operant behavior for an alternative reinforcer was not likewise affected. The present data extend previous work indicating that pharmacokinetic approaches may be of worth in the search for clinically effective cocaine antagonists.

In vivo cell penetration and intracellular transport of anti-Sm and anti-La autoantibodies.

Anti-nuclear autoantibodies (ANA) are the hallmark of systemic autoimmune diseases. Yet, the in vivo function of ANA remains controversial to a large extent due to the intracellular nature of their antigenic targets. It has been reported that a subset of autoantibodies can penetrate live cells and translocate into the subcellular compartments containing the corresponding antigens. The studies presented herein show that murine anti-Sm and anti-La monoclonal autoantibodies can also enter a variety of cell types from different animal species and that the cell penetration activity is not isotype-restricted. Interestingly, only mAb with cross-reactivity against double-stranded DNA did enter cells. Both these autoantibodies rapidly accumulate in the nucleus of viable cells but display different penetration kinetics. In co-localization experiments, monoclonal autoantibodies did not accumulate significantly within endocytic vesicles containing dextran, suggesting that they are internalized by mechanisms distinct from conventional receptor-mediated endocytosis. This report represents the first evidence that anti-La and anti-Sm autoantibodies are capable of entering live cells. Our observations support the notion that the phenomenon of intracellular autoantibodies may have a larger scope than previously reported and are consistent with a potential pathogenic role for ANA.

Luciferase: a sensitive and quantitative probe for blood-brain barrier disruption.

A novel method for quantitative analysis of blood-brain barrier (BBB) disruption is described, using luciferase as a probe in a murine model system. Purified luciferase was delivered to mouse brain by osmotic BBB disruption with hypertonic mannitol; control animals received an intracarotid inoculation of saline prior to infusion of luciferase. Delivery of luciferase to brain tissue was then assessed by enzyme assay of tissue extracts, and by immunohistochemical staining. Luciferase activity in the brain of mannitol-treated animals was found to be significantly elevated (approx. sevenfold), when compared to activity in control (saline-treated) mice. This finding was confirmed by quantitative immunohistochemical staining of tissue sections, using a luciferase-specific antibody. These studies showed that there was an eight-fold elevation in the level of extravascular luciferase particles within the brain of mannitol-treated animals, as compared to controls. Taken together these data show that purified recombinant luciferase can be used as a sensitive probe, with which to study the integrity of the BBB.

New experiments on shear modulus of elasticity of arteries.

Although the mechanical properties of blood vessels have been studied extensively, the shear modulus of the blood vessel wall is still unknown. New data on the shear modulus of elasticity of rat arteries and its variation with axial stretch and blood pressure are presented. The data were obtained from a new instrument designed and constructed by us to perform simultaneous torsion, inflation, and longitudinal stretching tests. It was found under physiological conditions (pressure = 120 mmHg or 16 kPa; longitudinal stretch = 1.2 relative to zero-stress state), the shear modulus of normal rat thoracic aorta is G = 137 +/- 18 kPa. The difference of shear modulus at body temperature (37 degrees C) and room temperature (25 degrees C) is within 10%. The shear modulus varies significantly with changing longitudinal and circumferential strains in proportion to the strain energy due to these strains. A constitutive equation based on a pseudo strain energy function is proposed. The vessel wall is not transversely isotropic in the incremental sense. When the rat was subjected to high blood pressure due to constriction of its aorta, the shear modulus does not vary significantly with the length of time the animal was subjected to hypertension.