Optimising the management of dysplastic lesions in the oesophagus with photodynamic therapy

The outcome of patients suffering from adeno and squamous carcinoma of the oesophagus remains poor. In the west, the incidence of adenocarcinoma has increased dramatically, with most cases occurring in association with Barrett's oesophagus (BE). Both adeno and squamous carcinoma are believed to progress through worsening degrees of dysplasia. This thesis assesses the role of Elastic Scattering Spectroscopy (ESS) as an objective diagnostic test for dysplasia and Photodynamic Therapy (PDT) with 5-aminolevulinic acid (ALA) as a less invasive treatment option. It also looks for a better understanding of the factors influencing mucosal healing after PDT. Using ESS, the sensitivity and specificity was 83% for distinguishing HGD/cancer from LGD/non dysplastic BE. Low dose ALA (30mg/kg) PDT eradicated 38% of HGD in BE compared with 67% eradication with a higher dose (60mg/kg). The higher dose also decreased the length of BE. In a study comparing red with green light (fixed light doses) for treating HGD, at 30 mg/kg ALA, 63% and 13 % of patients were clear of HGD with red and green laser respectively. At 60 mg/kg, the corresponding figures were 78% and 33% for the same light dose. 5 of 5 patients with LGD in BE and 4 of 5 patients with HGD in squamous mucosa had their dysplasia eradicated with ALA PDT. Successful PDT involves healing by regeneration of normal squamous mucosa. My in vitro studies created a PDT wound model using malignant oesophageal cell lines to assess the role of different cytokines in healing. Keratinocyte Growth Factor (KGF) was found to promote wound healing after PDT and significantly encouraged (p 0.001) the development of squamous cell lines. In conclusion: 1. ESS can differentiate dysplasia and early cancer from non-dysplastic and normal mucosa (sensitivity and specificity 83%). 2. PDT using high dose (60mg/kg) ALA (but not low dose) is effective in eradicating HGD in BE using red light. 3. The cytokine, KGF may promote healing with squamous mucosa after PDT. 4. Larger scale clinical trials are now required to confirm these results

TLR-related change in the chondrogenic differentiation potential of hTMSCs.

<p>Cells were cultured in chondrogenic induction medium (A). Cells demonstrated a sulphated extracellular matrix with toluidine blue staining over the 2-week period (B–D). B–D: toluidine blue staining on cultured hTMSCs for all groups. (B: unprimed, C: TLR3 primed, D: TLR4 primed). Magnification: ×400. Scale bars (A–D): 20 µm. No differences in visible color density were observed among the three groups. RT-PCR analysis of type II collagen and aggrecan mRNA expression in hTMSCs during 2 weeks of culture was performed. The expression levels of type II collagen did not differ among the three groups, but aggrecan expression was higher in the unexposed and TLR3 agonist exposure groups than in the TLR4 agonist exposure group.</p

TLR-related changes in cellular proliferation of hTMSCs.

<p>Cellular proliferation assay was performed for a period of 7 days. hTMSCs from all groups exhibit rapid proliferation from day 2 to 4, with the hTMSCs exposed to the TLR4 agonist expanding more rapidly compared to the others.</p

Gene expression assays used for real-time polymerase chain reaction for Multilineage differentiation.

<p>Gene expression assays used for real-time polymerase chain reaction for Multilineage differentiation.</p

Toll like receptor (TLR) expression (A), fluorescence Immunocytochemical staining (B) for TLR3, and change of TLR expression (C) of hTMSCs.

<p>(A): Protein expression was confirmed by flow cytometry analysis as positive for TLR3 (15.03%) and TLR4 (13.94%), with low positivity for TLR2 (3.53%) and TLR5 (5.75%). (B) Antibody staining of TLRs was performed following fixation and membrane permeabilization of the hTMSCs seeded on chamber slides. TLR3 expression was evaluated on hTMSCs by fluorescence microscope. TLR3 was stained diffusely in the cytoplasm (green) and nuclei were counterstained with DAPI (blue). Magnification: ×400. Scale bars: 20 µm. (C): When the cells were cultured in the absence (medium, unprimed) or presence of poly(I:C) (TLR3 primed), or LPS (TLR4 primed), the TLR4 agonist LPS significantly affects the expression of TLR2 and TLR4.</p

Characteristics of Human Turbinate-Derived Mesenchymal Stem Cells Are Not Affected by Allergic Condition of Donor

<div><p>The characteristics of mesenchymal stem cells (MSCs) derived from human turbinates (hTMSCs) have not been investigated in allergic rhinitis. We evaluated the influence of allergic state of the donor on the characteristics, proliferation, and differentiation potential of hTMSCs, compared with hTMSCs derived from non-allergic patients. hTMSCs were isolated from five non-allergic and five allergic patients. The expression of toll-like receptors (TLRs) in hTMSCs was measured by FACS, and cell proliferation was measured using a cell counting kit. Cytokine secretion was analyzed using multiplex immunoassays. The osteogenic, chondrogenic, and adipogenic differentiation potentials of hTMSCs were evaluated by histology and gene expression analysis. In allergic patients, FACS analysis showed that TLR3 and TLR4 were more highly expressed on the surface of hTMSCs than TLR2 and TLR5. The proliferation of hTMSCs was not influenced by the presence of TLR priming. The expression of IL-6, IL-8, IL-12, IP-10, and RANTES was upregulated after the TLR4 priming. The differentiation potential of hTMSCs was not influenced by TLR priming. These characteristics of hTMSCs were similar to those of hTMSCs from non-allergic patients. We conclude that the allergic condition of the donor does not influence TLR expression, proliferation, or immunomodulatory potential of hTMSCs.</p></div

Effects of growth suppressive effect of hTMSCs on human peripheral blood mononuclear cells (PBMC).

<p>(A) During hTMSC/PBMC coculture, the proliferation of cocultured PBMCs were suppressed significantly on day 2 and 3, while single-cultured PBMCs showed steady expansion for a period of 3days. (B) The cell counts of PBMCs on day 3 were similar with the pattern of growth inhibition analysis.</p

Isolation (A) and fluorescence-activated cell sorting analysis (B) of human turbinate mesenchymal stromal cells (hTMSCs).

<p>Adherent cells with fibroblastic morphology were analyzed. Flow cytometry analysis after three passages was used to confirm hTMSCs as positive for CD29, CD73, and CD90, with negative for CD14, CD19, CD34, and HLA-DR. (A) Magnification: ×400. Scale bars: 20 µm.</p

Effects of TLR3 and TLR4 on cytokine and chemokine secretion by hTMSCs.

<p>Secretion of cytokines and chemokines such as IL-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-12, IP-10 (CXCL10), RANTES (CCL5), TNF-α, GM-CSF, and IFN-γ of hTMSCs after stimulation with TLR3 and TLR4 were measured with enzyme-linked immunosorbent assay. Among them, IL-4, IL-6, IL-8, IL-10, IP-10 (CXCL10), and RANTES (CCL5) were detectable with mean values of more than 1 ng/ml under standard culture conditions. LPS strongly induced expression of IL-6, IL-8, IL-12, IP-10 (CXCL10), RANTES (CCL5), TNF-α, and GM-CSF, while poly(I:C) did not affect the secretion of cytokines and chemokines.</p

Evaluation of characteristic of human turbinate derived mesenchymal stem cells cultured in the serum free media

<div><p>We evaluated the effect of serum-free and xeno-cultivation (SFXFM) on the characterization, proliferation, and differentiation properties of human nasal stem cells (airway tissue; hTMSCs). hTMSCs were isolated from 10 patients, after which patient samples were separated into two groups, an SFXFM group and a control group. The control group was treated with bovine serum-containing medium. FACS analysis revealed that SFXFM-cultured hTMSCs maintained a characteristic mesenchymal stem cell phenotype. hTMSC proliferation was not influenced by SFXFM. In addition, upregulation of IL-8 and GM-CSF and downregulation of RANTES expression were shown in response to SFXFM. Moreover, two-lineage differentiation properties (osteocyte and adipocyte) of hTMSCs were enhanced under SFXFM. Finally, the genetic stability of SFXFM-cultured hTMSCs was demonstrated by normal karyotype results. SFXFM enables good expansion, multipotentiality, and normal genotype maintenance of MSCs. Moreover, this approach serves as a substitute to conventional media for the cultivation of capable MSCs for upcoming medical applications.</p></div