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

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

Comparison of the osteogenic differentiation potentials of hTMSCs cultured under serum-free medium versus those of hTMSCs cultured under serum-containing medium.

<p>Under osteogenic conditions, alkaline phosphatase staining (x 400) of hTMSCs cultured in serum-free medium (left) and serum-containing medium (right) demonstrated similar levels of alkaline phosphatase expression, as assessed visually. The mRNA expression levels of osteocalcin, Runt-related transcription factor 2, and type I collagen in hTMSCs (B) were perceived by RT-PCR. hTMSCs cultured under serum-free medium showed higher expression of osteogenic differentiation markers versus hTMSCs cultured under serum-containing medium.</p

Karyotype analysis of hTMSCs cultured in serum-free medium.

<p>To check whether cells derived from serum-free cultivation showed the chromosomal stability or not, the cytogenetic karyotypes of cells at passage 3 (left) and 6 (right) were analyzed. All sex chromosomes were XY. No chromosome eliminations, displacements, or imbalances were observed.</p

Comparison of the adipogenic differentiation potentials of hTMSCs cultured in serum-free medium versus those of hTMSCs cultured in serum-containing medium.

<p>Under adipogenic conditions, adipogenesis was perceived after 2 weeks of culture by staining of intracytoplasmic microvacuole with Oil Red O. Visual assessment revealed similar levels of intracytoplasmic Oil Red O staining (x 400) in hTMSCs cultured under serum-free medium (left) and in hTMSCs cultured under serum-containing medium (right). The mRNA expression levels of peroxisome proliferator activated receptor r (PPARr) and AcylCoA synthetase (ACS) in hTMSCs (B) were detected by RT-PCR. hTMSCs cultured under serum-free medium exhibited higher expression of adipogenic differentiation markers compared with hTMSCs cultured under serum-containing medium.</p

Comparison of proliferation of hTMSCs cultured in serum-free medium versus that of hTMSCs cultured under serum-containing medium.

<p>A cellular proliferation assay was conducted for 7 days. hTMSCs cultured in serum-free medium showed rapid proliferation from 3 to 4 days. The proliferation patterns resembled those observed in MSCs cultured under serum-containing medium. Additionally, culture under serum-free medium did not affect the proliferation of hTMSCs.</p

Comparison of the chondrogenic differentiation potentials of hTMSCs cultured in serum-free medium versus those of hTMSCs cultured in serum-containing medium.

<p>Under chondrogenic conditions (A), visual assessment revealed similar levels of toluidine blue staining (x 400) in hTMSCs cultured under serum-free medium (left) and hTMSCs cultured under serum-containing medium (right). However, RT-PCR analysis of type II collagen and aggrecan mRNA expression in hTMSCs cultured under serum-free medium showed consistently increased expression, but much lower expression of chondrogenic differentiation markers compared with the levels in hTMSCs cultured under serum-containing medium.</p

Effects of serum-free cultivation on cytokine and chemokine release by hTMSCs.

<p>Release of cytokines and chemokines [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] from hTMSCs cultured under serum-free medium compared to serum-containing medium was evaluated with an enzyme-linked immunosorbent assay. hTMSCs cultured in serum-free medium exhibited upregulation of IL-8 and GM-CSF, but downregulation of IL-1β, IL-10, IL-12, RANTES, and TNF-α versus the control group. These patterns of cytokine and chemokine secretion were different from those of hTMSCs cultured under serum-containing medium.</p

Morphology (A) after primary explant culture and fluorescence-activated cell sorting analysis of human nasal inferior turbinate derived mesenchymal stem cells (hTMSCs) cultured under serum-free medium (B) and serum-containing medium (C).

<p>Cells in both groups attached to the culture dish and displayed a similar spindle-shaped, fibroblast-like shape (magnification x 100) (A). Flow cytometry analysis revealed that hTMSCs from both groups were positive for MSC markers (CD73, CD90, and CD105) and negative for hematopoietic cell markers (CD14, CD19, CD34, and HLA-DR) (B and C).</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