Establishing Human Lacrimal Gland Cultures with Secretory Function

PURPOSE: Dry eye syndrome is a multifactorial chronic disabling disease mainly caused by the functional disruptions in the lacrimal gland. The treatment involves palliation like ocular surface lubrication and rehydration. Cell therapy involving replacement of the gland is a promising alternative for providing long-term relief to patients. This study aimed to establish functionally competent lacrimal gland cultures in-vitro and explore the presence of stem cells in the native gland and the established in-vitro cultures. METHODS: Fresh human lacrimal gland from patients undergoing exenteration was harvested for cultures after IRB approval. The freshly isolated cells were evaluated by flow cytometry for expression of stem cell markers ABCG2, high ALDH1 levels and c-kit. Cultures were established on Matrigel, collagen and HAM and the cultured cells evaluated for the presence of stem cell markers and differentiating markers of epithelial (E-cadherin, EpCAM), mesenchymal (Vimentin, CD90) and myofibroblastic (α-SMA, S-100) origin by flow cytometry and immunocytochemistry. The conditioned media was tested for secretory proteins (scIgA, lactoferrin, lysozyme) post carbachol (100 µM) stimulation by ELISA. RESULTS: Native human lacrimal gland expressed ABCG2 (mean±SEM: 3.1±0.61%), high ALDH1 (3.8±1.26%) and c-kit (6.7±2.0%). Lacrimal gland cultures formed a monolayer, in order of preference on Matrigel, collagen and HAM within 15-20 days, containing a heterogeneous population of stem-like and differentiated cells. The epithelial cells formed 'spherules' with duct like connections, suggestive of ductal origin. The levels of scIgA (47.43 to 61.56 ng/ml), lysozyme (24.36 to 144.74 ng/ml) and lactoferrin (32.45 to 40.31 ng/ml) in the conditioned media were significantly higher than the negative controls (p<0.05 for all comparisons). CONCLUSION: The study reports the novel finding of establishing functionally competent human lacrimal gland cultures in-vitro. It also provides preliminary data on the presence of stem cells and duct-like cells in the fresh and in-vitro cultured human lacrimal gland. These significant findings could pave way for cell therapy in future

Biomarkers in chronic obstructive pulmonary disease patients for prediction of lung cancer development

According to the World Health Organization (2016), chronic obstructive pulmonary disease (COPD) and lung cancer (along with trachea and bronchial cancers) are third and sixth among 10 top causes of death globally. The association between lung cancer and COPD has been widely established owing to their common endogenous and exogenous risk factors. Mechanistically, lung cancer and COPD are interlinked diseases in many ways such as oxidative stress-associated DNA damage, inflammation, and telomere shortening. An increase in lung cancer has been well correlated with smoking, which is likely to occur up to five folds higher in smokers with COPD than normal lung function subjects. In majority of cases, lung cancer development, especially in COPD patients, is asymptomatic and only diagnosed at advanced stages with poor prognosis. The development of biomarkers for early prediction of lung cancer in both high- and low-risk COPD patients will help clinicians for their better follow-up, early diagnosis, and improved therapeutic management

Molecular Understanding of Growth Inhibitory Effect from Irradiated to Bystander Tumor Cells in Mouse Fibrosarcoma Tumor Model

<div><p>Even though bystander effects pertaining to radiation risk assessment has been extensively studied, the molecular players of radiation induced bystander effect (RIBE) in the context of cancer radiotherapy are poorly known. In this regard, the present study is aimed to investigate the effect of irradiated tumor cells on the bystander counterparts in mouse fibrosarcoma (WEHI 164 cells) tumor model. Mice co-implanted with WEHI 164 cells γ-irradiated with a lethal dose of 15 Gy and unirradiated (bystander) WEHI 164 cells showed inhibited tumor growth, which was measured in terms of tumor volume and Luc⁺WEHI 164 cells based bioluminescence <i>in vivo</i> imaging. Histopathological analysis and other assays revealed decreased mitotic index, increased apoptosis and senescence in these tumor tissues. In addition, poor angiogenesis was observed in these tumor tissues, which was further confirmed by fluorescence imaging of tumor vascularisation and CD31 expression by immuno-histochemistry. Interestingly, the growth inhibitory bystander effect was exerted more prominently by soluble factors obtained from the irradiated tumor cells than the cellular fraction. Cytokine profiling of the supernatants obtained from the irradiated tumor cells showed increased levels of VEGF, Rantes, PDGF, GMCSF and IL-2 and decreased levels of IL-6 and SCF. Comparative proteomic analysis of the supernatants from the irradiated tumor cells showed differential expression of total 24 protein spots (21 up- and 3 down-regulated) when compared with the supernatant from the unirradiated control cells. The proteins which showed substantially higher level in the supernatant from the irradiated cells included diphosphate kinase B, heat shock cognate, annexin A1, angiopoietin-2, actin (cytoplasmic 1/2) and stress induced phosphoprotein 1. However, the levels of proteins like annexin A2, protein S100 A4 and cofilin was found to be lower in this supernatant. In conclusion, our results provided deeper insight about the damaging RIBE in an <i>in vivo</i> tumor model, which may have significant implication in improvement of cancer radiotherapy.</p></div

RT-PCR showing appropriate product bands for scIgA, lactoferrin and lysozyme in day 14 <i>in-vitro</i> cultures on all HAM (H), collagen I (C) and Matrigel™(M).

<p>Negative (N) panel shows no amplification product.</p

Flow cytometry data: Percentage of various marker in freshly isolated, day 14–18 and day 21–25 of in-vitro culture of human lacrimal gland.

<p>Flow cytometry data: Percentage of various marker in freshly isolated, day 14–18 and day 21–25 of in-vitro culture of human lacrimal gland.</p

Immunohistochemistry on normal human lacrimal gland.

<p>H&E staining shows the normal histology of the lacrimal gland. Marker staining pattern shows localization of pan-cytokeratin (AE1/AE3) and lysozyme (Lzy) in the cytoplasm of the acinar cells while c-kit is seen in the plasma membrane of acinar cells. p63, glial fibrillary acidic protein (GFAP), S-100 protein and ∝-SMA localize in the myoepithelial cells enveloping the acinar cells. Vimentin is seen in the myoepithelial cells and also in some of the acinar cells. All images are at 40× magnification except H&E which is at 10×.</p

Secretome Assessment by ELISA.

<p>a–c): Standard calibration curve for lysozyme, scIgA and lactoferrin. d–f): Plot of mean optical density values for secreted proteins lysozyme, scIgA and lactoferrin on HAM, collagen and Matrigel™ pre and post carbachol stimulation.</p

Establishment of human lacrimal gland primary cultures.

<p>a) Heterogenous cell population isolated after enzymatic digestion of the lacrimal gland. b) Cell clumps adhere to the substrate as discrete islands and show initiation of proliferation within 1–3 days. c) The islands proliferate and form a confluent monolayer within 15–20 days. d) The cells in the monolayer show thin cytoplasmic border, vesicular nucleus and granularity in the cytoplasm. e–f) Spherules are formed by day 16–18 (arrow head). g–i) Cord-like connections (arrow head) are seen to develop between the spherules.</p

Immunocytochemistry on in-vitro cultured human lacrimal gland cells.

<p>Cells with epithelial morphology stain positively with E-cadherin, CK3/12, lysozyme and p63; oval and plump cells stain positive for myoepithelial markers GFAP and S100 protein while the spindle shaped cells are seen to be positive for mesenchymal markers CD90 and vimentin. Some cells also show immunopositivity for ABCG2. Secondary antibody uses is fluoresceine isothiocyanate (green) and the counter-stain is propidium iodide (red).</p