Intranasal Delivery of Pure Nanodrug Loaded Liposomes for Alzheimer's Disease Treatment by Efficiently Regulating Microglial Polarization.

The activated M1-like microglia induced neuroinflammation is the critical pathogenic event in Alzheimer's disease (AD). Microglial polarization from pro-inflammatory M1 toward anti-inflammatory M2 phenotype is a promising strategy. To efficiently accomplish this, amyloid-ß (Aß) aggregates as the culprit of M1 microglia activation should be uprooted. Interestingly, this study finds out that the self-reassembly of curcumin molecules into carrier-free curcumin nanoparticles (CNPs) exhibits multivalent binding with Aß to achieve higher inhibitory effect on Aß aggregation, compared to free curcumin with monovalent effect. Based on this, the CNPs loaded cardiolipin liposomes are developed for efficient microglial polarization. After intranasal administration, the liposomes decompose to release CNPs and cardiolipin in response to AD oxidative microenvironment. The CNPs inhibit Aß aggregation and promote Aß phagocytosis/clearance in microglia, removing roadblock to microglial polarization. Subsequently, CNPs are endocytosed by microglia and inhibit TLR4/NF-κB pathway for microglia polarization (M1→M2). Meanwhile, cardiolipin is identified as signaling molecule to normalize microglial dysfunction to prevent pro-inflammatory factors release. In AD transgenic mice, neuroinflammation, Aß burden, and memory deficits are relieved after treatment. Through combined attack by extracellularly eradicating roadblock of Aß aggregation and intracellularly inhibiting inflammation-related pathways, this nanotechnology assisted delivery system polarizes microglia efficiently, providing a reliable strategy in AD treatment.

High-throughput sequencing reveals differences in microbial community structure and diversity in the conjunctival tissue of healthy and type 2 diabetic mice.

BACKGROUND: To investigate the differences in bacterial and fungal community structure and diversity in conjunctival tissue of healthy and diabetic mice. METHODS: RNA-seq assays and high-throughput sequencing of bacterial 16 S rDNA and fungal internal transcribed spacer (ITS) gene sequences were used to identify differentially expressed host genes and fungal composition profiles in conjunctival tissues of diabetic BKS-db/db mice and BKS (control) mice. Functional enrichment analysis of differentially expressed genes and the correlation between the relative abundance of bacterial and fungal taxa in the intestinal mucosa were also performed. RESULTS: Totally, 449 differential up-regulated genes and 1,006 down-regulated genes were identified in the conjunctival tissues of diabetic mice. The differentially expressed genes were mainly enriched in metabolism-related functions and pathways. A decrease in conjunctival bacterial species diversity and abundance in diabetic mice compared to control mice. In contrast, fungal species richness and diversity were not affected by diabetes. The microbial colonies were mainly associated with cellular process pathways regulating carbohydrate and lipid metabolism, as well as cell growth and death. Additionally, some interactions between bacteria and fungi at different taxonomic levels were also observed. CONCLUSION: The present study revealed significant differences in the abundance and composition of bacterial and fungal communities in the conjunctival tissue of diabetic mice compared to control mice. The study also highlighted interactions between bacteria and fungi at different taxonomic levels. These findings may have implications for the diagnosis and treatment of diabetes.

De novo biosynthesis of anthocyanins in Saccharomyces cerevisiae using metabolic pathway synthases from blueberry.

BACKGROUND: Anthocyanins are water-soluble flavonoids in plants, which give plants bright colors and are widely used as food coloring agents, nutrients, and cosmetic additives. There are several limitations for traditional techniques of collecting anthocyanins from plant tissues, including species, origin, season, and technology. The benefits of using engineering microbial production of natural products include ease of use, controllability, and high efficiency. RESULTS: In this study, ten genes encoding enzymes involved in the anthocyanin biosynthetic pathway were successfully cloned from anthocyanin-rich plant materials blueberry fruit and purple round eggplant rind. The Yeast Fab Assembly technology was utilized to construct the transcriptional units of these genes under different promoters. The transcriptional units of PAL and C4H, 4CL and CHS were fused and inserted into Chr. XVI and IV of yeast strain JDY52 respectively using homologous recombination to gain Strain A. The fragments containing the transcriptional units of CHI and F3H, F3'H and DFR were inserted into Chr. III and XVI to gain Strain B1. Strain B2 has the transcriptional units of ANS and 3GT in Chr. IV. Several anthocyanidins, including cyanidin, peonidin, pelargonidin, petunidin, and malvidin, were detected by LC-MS/MS following the predicted outcomes of the de novo biosynthesis of anthocyanins in S. cerevisiae using a multi-strain co-culture technique. CONCLUSIONS: We propose a novel concept for advancing the heterologous de novo anthocyanin biosynthetic pathway, as well as fundamental information and a theoretical framework for the ensuing optimization of the microbial synthesis of anthocyanins.

Construction of Anthocyanin Biosynthesis System Using Chalcone as a Substrate in Lactococcus lactis NZ9000.

Anthocyanins are high-value natural compounds, but to date, their production still mainly relies on extraction from plants. A five-step metabolic pathway was constructed in probiotic Lactococcus lactis NZ9000 for rapid, stable, and glycosylated anthocyanin biosynthesis using chalcone as a substrate. The genes were cloned from anthocyanin-rich blueberry: chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanin synthase (ANS), and UDPG-flavonoid 3-O-glycosyltransferase (3GT). Using HR, the polysaccharide pellicle (PSP) segment of the cell wall polysaccharide synthesis (cwps) gene cluster from L. lactis NZ9000 was cloned into vector p15A-Cm-repDE. Then, CHI and F3H were placed sequentially under the control of NZProm 3 of this gene cluster in the vector, which was transformed into L. lactis NZ9000 to obtain Strain A. Furthermore, Strain B was constructed by placing F3H-DFR-ANS and 3GT under NZProm 2 and 3, respectively. Using LC-MS/MS analysis, several types of anthocyanins, including callistephin chloride, oenin chloride, malvidin O-hexoside, malvidin 3,5-diglucoside, and pelargonidin 3-O-malonyl-malonylhexoside, increased in the supernatant of the co-culture of Strains A and B compared to that of L. lactis NZ9000. This is the first time that a five-step metabolic pathway has been developed for anthocyanin biosynthesis in probiotic L. lactis NZ9000. This work lays the groundwork for novel anthocyanin production by a process involving the placement of several biosynthesis genes under the control of a gene cluster.

Self-Assembled Construction of Robust and Super Elastic Graphene Aerogel for High-Efficient Formaldehyde Removal and Multifunctional Application.

Simultaneously achieving exceptional mechanical strength and resilience of graphene aerogel (GA) remains a challenge, while GA is an ideal candidate for formaldehyde removal. Herein, flexible polyethyleneimine (PEI) is grafted chemically onto carbon nanotube (CNT) surface, and CNT-PEI@reduced GA (rGA) is fabricated via hydrothermal self-assembly, pre-frozen, and hydrazine reduction process. Introducing CNT-PEI contributes to well-interconnected/robust 3D network construction by connecting reduced graphene oxide (rGO) nanosheets through enhancing cross-linking, while entangled CNT-PEI is intercalated into rGO layers to avoid serious restacking of sheets, producing larger surface area and more formaldehyde adsorption sites. Ultralight CNT-PEI@rGA exhibits extreme high strength (276.37 kPa), reversible compressibility at 90% strain, and structural stability, while FA adsorption capacity reached 568.41 mg g-1 , ≈3.28 times of rGA, derivable from synergistic chemical-physical adsorption effect. Furthermore, CNT-PEI@rGA is ground into powder for first preparing polyoxymethylene (POM)/CNT-PEI@rGA composite, while formaldehyde emission amount is 69.63%/73.96% lower than that of POM at 60/230 °C. Moreover, CNT-PEI@rGA presents outstanding piezoresistive-sensing and thermal insulation properties, exhibiting high strain sensitivity, wide strain detection range, and long-term durability.

A meta-analysis on morphological, physiological and biochemical responses of plants with PGPR inoculation under drought stress.

Plant growth-promoting rhizobacteria (PGPR) can help plants to resist drought stress. However, the mechanisms of how PGPR inoculation affect plant status under drought remain incompletely understood. We performed a meta-analysis of plant response to PGPR inoculation by compiling data from 57 PGPR-inoculation studies, including 2, 387 paired observations on morphological, physiological and biochemical parameters under drought and well-watered conditions. We compare the PGPR effect on plants performances among different groups of controls and treatments. Our results reveal that PGPR enables plants to restore themselves from drought-stressed to near a well-watered state, and that C4 plants recover better from drought stress than C3 plants. Furthermore, PGPR is more effective underdrought than well-watered conditions in increasing plant biomass, enhancing photosynthesis and inhibiting oxidant damage, and the responses of C4 plants to the PGPR effect was stronger than that of C3 plants under drought conditions. Additionally, PGPR belonging to different taxa and PGPR with different functional traits have varying degrees of drought-resistance effects on plants. These results are important to improve our understanding of the PGPR beneficial effects on enhanced drought-resistance of plants.

The role of neutrophils in corneal nerve regeneration.

BACKGROUND: To investigate the role of neutrophils in corneal nerve regeneration. METHODS: A mouse model simulating corneal nerve injury was established and samples from corneal scraping with and without neutrophil closure were collected. These samples were used for corneal nerve staining, ribonucleic acid sequencing, and bioinformatics. Differential expression analysis was used to perform enrichment analysis to identify any significant differences between these two groups. The differential genes were then intersected with neutrophil-associated genes and a protein-protein interaction network was constructed using the intersected genes. The immune infiltration between the two groups was examined along with the immune cell variation between the high and low gene expression groups. RESULTS: Neutrophil removal delays corneal epithelial and nerve regeneration. A total of 546 differential genes and 980 neutrophil-associated genes, with 27 genes common to both sets were obtained. Molecular Complex Detection analysis yielded five key genes, namely integrin subunit beta 2 (ITGB2), matrix metallopeptidase 9 (MMP9), epidermal growth factor (EGF), serpin family E member 1 (SERPINE1), and plasminogen activator urokinase receptor (PLAUR). Among these genes, ITGB2, SERPINE1, and PLAUR exhibited increased expression in the neutrophil-confined group, while MMP9 and EGF showed decreased expression, with MMP9 and EGF displaying a more significant difference. Immune infiltration was also observed between the two groups, revealing significant differences in the infiltration of M0 macrophages, activated mast cells, and neutrophils. Moreover, the neutrophil levels were lower in the groups with low MMP9 and EGF expressions and higher in the high-expression group. CONCLUSION: Neutrophil confinement might significantly affect the MMP9 and EGF expression levels. Strategies to inhibit MMP9 could potentially yield therapeutic benefits.

Renal Yolk Sac Tumor Clinically Misdiagnosed as Nephroblastoma: A Case Report.

Background: Yolk sac tumor is a germ cell tumor (GCT) that occurs in infants and adolescents and affects various sites. There is a trend to treat pediatric renal tumors before a tissue diagnosis. We report a renal yolk sac tumor clinically misdiagnosed as Wilms tumor, based on ultrasound (US) and MRI.Case Report: This 21-month-old male infant was discovered to have a space occupying lesion in the right kidney. Because the tumor was large, initial radiotherapy preceded surgical resection. Histologically, the tumor was a yolk sac tumor.Conclusion: Imaging examination of renal yolk sac tumor can easily be misdiagnosed as Wilms tumor. SIOP treatment plan for Wilms tumor requires preoperative chemotherapy, which is different from the treatment regimen for yolk sac tumor. Preoperative alpha-fetoprotein could have been helpful in avoiding this clinical misdiagnosis.

A facile strategy to construct biocompatible poly(vinyl alcohol)-based self-healing hydrogels.

Endowing polymer hydrogels with good self-healing ability can autonomously repair damage with improved reliability. In this work, the benzaldehyde group was first grafted onto a biocompatible poly(vinyl alcohol) (PVA) molecular chain by esterification to obtain aldehyde-functionalized PVA (APVA), and the reversible imine bonds were further formed by reacting with amine groups on a quaternized chitosan (HTCC) chain. And thus, the self-healing APVA/HTCC hydrogel was fabricated with such imine bonds as crosslinking points together with hydrogen bonds. Many more imine bonds of hydrogels formed with increasing aldehyde content, resulting in increasing crosslinking density, decreasing average pore diameter and formation of a compact dynamic network, imparting certain mechanical strength and toughness with hydrogels. Furthermore, the healing efficiency of the hydrogel reached as high as 91.7% by self-healing without any external stimulus and its microstructure could be reconstructed after damage, exhibiting rapid recovery and dynamic features. Biocompatible self-healing PVA-based hydrogels exhibited great potential application in biomedical fields, like smart infill biomaterials, tissue engineering scaffolds, etc.

YKL-39 is an independent prognostic factor in gastric adenocarcinoma and is associated with tumor-associated macrophage infiltration and angiogenesis.

BACKGROUND: Gastric cancer has a high incidence and mortality rate. Angiogenesis is necessary for tumor infiltration and metastasis and affects patient prognosis. YKL-39 has monocyte chemotactic activity and pro-angiogenic activity in some tumors. In this study, we investigated the relationship between YKL-39 and tumor-associated macrophages and microangiogenesis in gastric cancer to determine its potential as a prognostic biomarker. MATERIALS AND METHODS: A total of 119 patients with gastric cancer who had undergone gastrectomy at the 940th Hospital of the Joint Security Force between 2014 and 2018 were included in this study. We assayed the protein expression of YKL-39, CD68, and CD34 by immunohistochemistry in tissues of 119 patients with gastric cancer, as well as the intracellular expression of YKL-39 and CD68 by immunofluorescence. Data were analyzed with SPSS Statistics 25.0 to explore the impact of expression of YKL-39, CD68, and CD34 in gastric cancer patients and the relationship among them. RESULTS: Our results show that YKL-39 was expressed in both the nucleus and cytoplasm of gastric cancer cells and tumor mesenchyme. YKL-39 protein expression was associated with the depth of tumor infiltration, lymph node metastasis, and TNM stage; CD68 protein expression was associated with lymph node metastasis and TNM stage; CD34 protein expression was not associated with clinicopathological characteristics. Expression of YKL-39 was positively correlated with CD68 and CD34 (p < 0.001), and high expression of YKL-39 was associated with poor prognosis (p < 0.05). CONCLUSION: In gastric cancer, YKL-39 expression is positively correlated with the degree of tumor-associated macrophage infiltration and angiogenesis, and is a potential prognostic marker for gastric cancer.

Cation/Anion Codoped and Cobalt-Free Li-Rich Layered Cathode for High-Performance Li-Ion Batteries.

Lithium-rich layered oxides have received great attention due to their high energy density as cathode material. However, the progressive structural transformation from layered to spinel phase triggered by transition-metal migration and the irreversible release of lattice oxygen leads to voltage fade and capacity decay. Here, we report a Fe, Cl codoped and Co-free Li-rich layered cathode with significantly improved structural stability. It is revealed that the Fe and Cl codoping can facilitate the Li-ion diffusion and improve the rate performance of the materials. Moreover, the calculations show that the structural stability is enhanced by Fe and Cl codoping. As a result, the Fe and Cl codopant reduces the irreversible release of lattice oxygen, mitigates voltage fade, and improves the first-cycle Coulombic efficiency. This work provides a low-cost, environmentally friendly, practical strategy for high-performance cathode materials.

Formation Tracking Control for Multi-Agent Networks with Fixed Time Convergence via Terminal Sliding Mode Control Approach.

This paper investigates formation tracking control for multi-agent networks with fixed time convergence. The control task is that the follower agents are required to form a prescribed formation within a fixed time and the geometric center of the formation moves in sync with the leader. First, an error system is designed by using the information of adjacent agents and a new control protocol is designed based on the error system and terminal sliding mode control (TSMC). Then, via employing the Lyapunov stability theorem and the fixed time stability theorem, the control task is proved to be possible within a fixed time and the convergence time can be calculated by parameters. Finally, numerical results illustrate the feasibility of the proposed control protocol.

DNase I improves corneal epithelial and nerve regeneration in diabetic mice.

DNase I has been reported to improve diabetic wound healing through the clearance of neutrophils extracellular traps (NETs) caused by neutrophil aggregation. However, the function of DNase I on diabetic corneal wound healing remains unclear. Here, we investigated the effect and mechanism of topical DNase I application on diabetic mouse corneal epithelial and nerve regeneration. Corneal epithelial defects, inflammatory response, regeneration-related signalling pathways, oxidative stress, corneal innervation and sensation were examined and compared between the diabetic and normal mice. The results confirmed firstly the increased NETs production during the delayed corneal epithelial wound healing of diabetic mice, which was significantly improved through either DNase I or Cl-amidine administration. Mechanistically, DNase I improved inflammation resolution, reactivated epithelial regeneration-related signalling pathways and attenuated the accumulation of reactive oxygen species (ROS). Moreover, DNase I application also promoted corneal nerve regeneration and restored the impaired corneal sensitivity in diabetic mice. Therefore, these results indicate that topical DNase I application promotes corneal epithelial wound healing and mechanical sensation restoration in diabetic mice, representing the potential therapeutic approach for diabetic keratopathy.

Enhancing the Gas Separation Selectivity of Mixed-Matrix Membranes Using a Dual-Interfacial Engineering Approach.

We report a dual-interfacial engineering approach that uses a sub-20 nm polycrystalline MOF-74 shell as a transition phase to engineer the MOF-polymer interface. The application of a shell MOF layer divides the original single interface problem into two interfaces: MOF-MOF and MOF-polymer, which can be individually addressed. The greater external surface area created by the uneven MOF-74 shell containing high-density open metal sites allows the MOF to interact with 300% polymer at the interface compared to traditional MOF, thereby ensuring good interfacial compatibility. When applied on UiO-66-NH2, its respective mixed-matrix membranes exhibit a simultaneous increase of CO2/CH4 separation selectivity and CO2 permeability with increasing MOF loading, implying a defect-free interface. When applied on MOF-801, the mixed-matrix membranes exhibit an ethylene/ethane separation selectivity up to 5.91, a drastic 76% increase compared to that of the neat polymer owing to a "gas focusing" mechanism promoted by the preferred pore orientation in the MOF-74 layer. This represents one of the most selective ethylene/ethane separation membranes reported to date.

TIM-3 blockade combined with bispecific antibody MT110 enhances the anti-tumor effect of γδ T cells.

As ideal cells that can be used for adoptive cell therapy, γδ T cells are a group of homogeneous cells with high proliferative and tumor killing ability. However, γδ T cells are apt to apoptosis and show decreased cytotoxicity under persistent stimulation in vitro and cannot aggregate at tumor sites efficiently in vivo, both of which are two main obstacles to tumor adoptive immunotherapy. In this study, we found that the immune checkpoint T-cell immunoglobulin domain and mucin domain 3 (TIM-3) were up-regulated significantly on γδ T cells during their ex vivo expansion and this up-regulation contributed to the dysfunction of γδ T cells. Although the killing ability of γδ T cells against breast cancer cells which exhibited a high level of epithelial cell adhesion molecule (EpCAM) was enhanced, the level of TIM-3 on γδ T cells was also further up-regulated under the application of the bispecific antibody MT110 (anti-CD3 × anti-EpCAM) which can redirect T cells to target cells. Besides, these γδ T cells with up-regulated TIM-3 exhibited an increased susceptibility to apoptosis. By reinvigorating dysfunctional γδ T cells and promoting them to accumulate at tumor sites, the combined use of TIM-3 inhibitor and MT110 could further enhance the anti-tumor effect of the adoptively transfused γδ T cells. These results may have clinical implications for the design of new translational anti-tumor regimens aimed at combining checkpoint blockade and immune cell redirection.

Practical Synthesis of Phosphinic Amides/Phosphoramidates through Catalytic Oxidative Coupling of Amines and P(O)-H Compounds.

Herein, we report a highly efficient ZnI2 -triggered oxidative cross-coupling reaction of P(O)-H compounds and amines. This operationally simple protocol provides unprecedented generic access to phosphinic amides/phosphoramidate derivatives in good yields and short reaction time. Besides, the reaction proceeds under mild conditions, which avoids the use of hazardous reagents, and is applicable to scale-up syntheses as well as late-stage functionalization of drug molecules. The stereospecific coupling is also achieved from readily available optically enriched P(O)-H compounds.

METHIONINE ADENOSYLTRANSFERASE4 Mediates DNA and Histone Methylation.

DNA and histone methylation coregulate heterochromatin formation and gene silencing in animals and plants. To identify factors involved in maintaining gene silencing, we conducted a forward genetic screen for mutants that release the silenced transgene Pro35S::NEOMYCIN PHOSPHOTRANSFERASE II in the transgenic Arabidopsis (Arabidopsis thaliana) line L119 We identified MAT4/SAMS3/MTO3/AT3G17390, which encodes methionine (Met) adenosyltransferase 4 (MAT4)/S-adenosyl-Met synthetase 3 that catalyzes the synthesis of S-adenosyl-Met (SAM) in the one-carbon metabolism cycle. mat4 mostly decreases CHG and CHH DNA methylation and histone H3K9me2 and reactivates certain silenced transposons. The exogenous addition of SAM partially rescues the epigenetic defects of mat4 SAM content and DNA methylation were reduced more in mat4 than in three other mat mutants. MAT4 knockout mutations generated by CRISPR/Cas9 were lethal, indicating that MAT4 is an essential gene in Arabidopsis. MAT1, 2, and 4 proteins exhibited nearly equal activity in an in vitro assay, whereas MAT3 exhibited higher activity. The native MAT4 promoter driving MAT1, 2, and 3 cDNA complemented the mat4 mutant. However, most mat4 transgenic lines carrying native MAT1, 2, and 3 promoters driving MAT4 cDNA did not complement the mat4 mutant because of their lower expression in seedlings. Genetic analyses indicated that the mat1mat4 double mutant is dwarfed and the mat2mat4 double mutant was nonviable, while mat1mat2 showed normal growth and fertility. These results indicate that MAT4 plays a predominant role in SAM production, plant growth, and development. Our findings provide direct evidence of the cooperative actions between metabolism and epigenetic regulation.

Epithelium-derived miR-204 inhibits corneal neovascularization.

MicroRNA-204 (miR-204) is highly expressed in cornea, here we explored the role and mechanism of miR-204 in corneal neovascularization (CNV). Mouse CNV was induced by intrastromal placement of suture in BALB/c mice with the subconjunctival injection of miR-204 agomir or negative control. Human primary limbal epithelial cells (LECs) and immortalized microvascular endothelial cells (HMECs) were used to evaluate the expression changes and anti-angiogenic effects of miR-204 under biomechanical stress (BS). The expression and localization of miR-204, vascular endothelial growth factor (VEGF) and their receptors were detected by quantitative real-time PCR, in situ hybridization, immunohistochemistry and Western blot. The results showed that miR-204 expression was mainly localized in epithelium and down-expressed in vascularized cornea. Subconjunctival injection of miR-204 agomir inhibited CNV and reduced the expression of VEGF and VEGF receptor 2. Similarly, miR-204 overexpression attenuated the increased expression of VEGF by biomechanical stress in LECs, and suppressed the proliferation, migration, and tube formation of HMECs. These novel findings indicate that epithelium-derived miR-204 inhibits suture-induced CNV through regulating VEGF and VEGF receptor 2.

The Value of Serum Complement C1q in the Diagnosis of Acute Ischemic Stroke.

BACKGROUND: To investigate the relationship between the levels of serum complement C1q and the risk and severity of acute ischemic stroke, a total of 154 patients with acute ischemic stroke and 42 healthy volunteers as normal controls were enrolled in the present study. METHODS: According to the onset time of stroke, patients were divided into three groups. Using an immune transmission turbidity method, the levels of serum complement C1q were detected to investigate the relationship between the level of serum complement C1q and the incidence and severity of acute ischemic stroke. The risk factors of these groups were calculated using a conditional logistic regression model. The assessment of neurological function impairment was carried out according to the National Institute of Health Stroke Scale. Then correlation anal- ysis was carried out between the level of serum complement C1q among patients with acute ischemic stroke and the degree of neurological function impairment. RESULTS: The results showed that the level of serum complement C1q was higher in the ischemic stroke group than in the control group. Using a conditional logistic regression model it was discovered that serum complement C1q was the independent pathogenic factor of cerebral infarction. There also was a decreasing trend in the level of serum complement C1q with the extension of the onset time and an increasing trend in the level of serum complement C1q with the increase in the maximum diameter of infarction volume. CONCLUSIONS: Serum complement C1q is an independent risk factor for acute outbreak of ischemic stroke, whose level is closely related to the outbreak and infarct size and neurological function impairment.

Characterization of the corneal surface in limbal stem cell deficiency and after transplantation of cultured allogeneic limbal epithelial cells.

PURPOSE: The objective of this study was to characterize the changes that occur in the cornea during Limbal Stem Cell Deficiency (LSCD) and on the corneal surface after transplantation of ex vivo cultured allogeneic limbal epithelial transplantation (CALET). METHODS: Forty-one pannus were analyzed to characterize the changes found in the cornea in LSCD. Nineteen impression cytology samples, including 14 pannus and five corneal buttons, obtained during subsequent procedures from patients who had undergone CALET were examined to assess the effect of CALET and to determine the long-term fate of donor cells. The presence of donor and recipient epithelial cells in each sample was determined by short tandem repeat (STR) amplification and fluorescent-multiplex polymerase chain reaction (PCR). Phenotypic analysis of the epithelium was performed by immunohistochemistry and real-time PCR. RESULTS: The expression of lineage markers was similar between pannus and conjunctivae, but not to corneas. Objective long-term benefits from the transplantation were recorded in most cases. After CALET, the lineage markers in the excised corneal buttons and pannus showed a limbus phenotype. DNA analysis of the 19 cases showed no donor cells present on the ocular surface beyond three months after CALET. CONCLUSIONS: LSCD was characterized by ingrowth of abnormal, inflamed tissue with a conjunctival phenotype. CALET was a useful technique for restoring the ocular surface in LSCD. However, such benefits did not necessarily correlate with survival of measurable numbers of donor cells on the ocular surface. The absence of donor DNA beyond three months raises questions regarding the period of ongoing immunosuppression and the origin of the regenerated corneal epithelium.