Photodynamic purging of alloreactive T cells for adoptive immunotherapy after haploidentical stem cell transplantation

After haploidentical stem cell transplantation immune recovery is inevitably slow and infectious related mortality is about 30-40%. Immune reconstitution could be improved by infusing donor T cells, but the obstacle is graft-versus-host disease. In a mixed lymphocyte reaction, alloantigen-stimulated T cells uptake 4,5-dibromorhodamine methyl ester (TH9402), a compound that is structurally similar to rhodamine. TH9402 preferentially localizes in mitochondria and when exposed to 500- to 600-nm wavelength visible light delivered through the Theralux device (Kiadis Pharma, Amsterdam, The Netherlands), it becomes highly cytotoxic through oxidative damage. This study investigated a range of parameters, and combinations thereof, with the aim of achieving optimal T cell allodepletion and preservation of pathogen-specific responses. We report on 11 clinical scale dry runs which reproducibly yielded the following results. Blood mononuclear cells were stimulated with haploidentical irradiated (20 Gy). Blood mononuclear cells in a mixed lymphocyte reaction. Cells were then incubated with TH9402 and exposed to light delivered through the Theralux device. Optimal conditions for T cell allodepletion emerged as (1) duration of mixed lymphocyte reaction: 24 h; (2) responder cell concentration: 3-5x10(6)/ml; (3) TH9402 concentration: 5 microM; (4) quantity of internalized TH9402, as measured by mean fluorescence intensity (MFI): 20,000-25,000 MFI; (5) energy delivered: 0.1 J/cm(2). Only under these conditions were the frequencies (by limiting dilution analyses) of alloantigen-specific T cells maximally reduced, i.e., 2467+/-639 (mean+/-SD) times, when compared with non-TH9402-treated cells. Pathogen-specific responses to pathogen antigens such as Cytomegalovirus, Adenovirus, Varicella Zoster Virus, Herpes Simplex Virus, Aspergillus fumigatus, Candida albicans, Toxoplasma gondii were retained, although with a 19+/-9.7 times reduction in frequency. This remarkable drop in frequency of alloreactive T cells is expected to allow safe infusion of relatively large numbers of T cells across histocompatibility barriers for adoptive transfer of donor immunity. Consequently, a clinical trial is planned to incorporate infusion of photo-allodepleted donor T cells after haploidentical stem cell transplantation with the aim of decreasing infection-related mortality.     

Transforming growth factor-beta 1 expression in the peri-implant soft tissues of healthy and failing dental implants

BACKGROUND: Transforming growth factor-beta (TGF-beta) is composed of a family of multifunctional polypeptide growth factors involved in embryogenesis, inflammation, regulation of immune response, angiogenesis, wound healing, and extracellular matrix formation. TGF-beta1 is the most common isoform found in human tissues. A role of TGF-beta in the pathogenesis of periodontal disease has been suggested. The aim of the present study was a comparative immunohistochemical evaluation of TGF-beta1 in normal keratinized gingiva and in the peri-implant soft tissues surrounding failing non-submerged implants. METHODS: Twenty patients participated in this study. Ten biopsies from healthy keratinized mucosa and 10 biopsies from peri-implant soft tissues surrounding failing implants were obtained (one biopsy per patient). The biopsies were obtained from different patients. RESULTS: In 5 cases of healthy mucosa, the stromal cells were positive between 1 to 5. In 7 cases, the epithelial layers were positive, between 1 and 18 cells. The superficial epithelial layer was negative in all cases. In 9 cases, there was a positivity of the vascular component, between 2 and 16 vessels. In failing implants, the stromal cells were positive in 6 cases, between 1 and 4. In all cases, cells of the epithelial layers were positive, between 15 and 40. The vascular component was positive in all cases, between 12 and 30 vessels. The differences between TGF-beta1 expression in the epithelium around healthy and failing implants were statistically significant (P < 0.0001). The differences between TGF-beta1 expression in the blood vessels in the soft tissues around healthy and failing implants were also statistically significant (P < 0.0001). No statistically significant difference was observed between the 2 groups in the TGF-beta1 expression in the stromal cells (P = 0.88). CONCLUSION: TGF-beta1 may be one of the most important factors in the regulation of the infiltrate, and in the production of tissue repair with a stimulation of fibroblasts and endothelial cells.     

Plasma adiponectin for prediction of cardiovascular events and mortality in high-risk patients

Context: The prognostic value of plasma levels of adiponectin, an adipocytokine with antiatherogenic, antiinflammatory, and insulin-sensitizing effects, is contentious. Objective: The objective of the study was to investigate whether plasma adiponectin levels predict cardiovascular (CV) events and mortality in high-risk coronary artery disease (CAD) patients. Design, Setting, Participants, and Main Outcome Measure: We measured plasma adiponectin and examined its impact on the incidence of CV deaths and events at follow-up in the context of all potentially relevant background covariates in 712 high-risk patients of the Genetic and ENvironmental factors in Coronary Atherosclerosis study who underwent coronary angiography for suspected CAD. Based on the population plasma adiponectin median (6.38 mug/ml, interquartile range 4.2-10.2), we split the patients in a high- and a low-plasma adiponectin subgroup. After a median follow-up of 3.8 years (interquartile range 3.3-4.3 yr), outcome data were obtained in 100% of the patients and 45 CV deaths (6.4%) were recorded. Kaplan-Meier analysis unexpectedly showed a higher CV death rate in high-plasma adiponectin than low-plasma adiponectin patients. By contrast, multivariate Cox regression analysis, in which potential confounders, including ongoing medical treatment, were considered, showed no impact of plasma adiponectin on CV death. Similar negative results were obtained using the propensity score that considered all relevant covariables and medical treatment rate, which differed between the high- and low-plasma adiponectin group. Conclusions: In high-risk CAD patients, plasma adiponectin above the median (6.38 mug/ml) implies a paradoxical higher risk of CV death. However, when relevant covariates that differ between high- and low-plasma adiponectin groups are considered, this association wanes, indicating that the clustering of plasma adiponectin with other covariates can abolish its impact on CV prognosis.     

An in vitro evaluation of a prototype Delrin carrier for the Thermafil obturation system.

OBJECTIVE: To produce and test a prototype carrier for the Thermafil obturation system. METHOD AND MATERIALS: A prototype carrier was created in a new plastic material (Delrin) with a new profile design made up of 15 intersecting cones of decreasing diameter and a 0.06 taper. Thirty extracted premolar teeth were root filled, 15 with obturators with the prototype carriers and 15 with the traditional Thermafil obturators. The samples were made transparent and examined with an optical microscope equipped with a millimeter scale in order to observe the aspect of gutta-percha stripping from the carrier and evaluate its degree by measuring the length of carrier deprived of gutta-percha on the buccal, mesial, distal, and lingual root surface. The analysis of variance (ANOVA) test was carried out for statistical analysis. RESULTS: The prototype carriers made contact with the dentinal walls (mean length of stripping: 5.80 mm for size 25, 3.42 for 30, and 1.40 for 35) but less than the traditional Thermafil obturators (mean length of stripping: 6.68 mm for size 25, 7.98 for 30, and 6.86 for 35). The ANOVA test revealed no statistically significant difference between groups for P <.01. The prototype carriers contacted the walls in short horizontal band-shaped areas around the circumference, while the Thermafil obturators had contact in long vertical strip-shaped areas. CONCLUSION: The prototype carrier, contacting the dentinal walls in short areas all around the circumference, allows the centering of the obturator into the root canal.     

CD1a-positive cells in odontogenic cysts

Langerhans cells (LC) are bone marrow-derived cells that have a CD1a-positive immunophenotype and are an important portion of the cell-mediated immune response. The aim of this study was an immunohistochemical evaluation of CD1a positive cells in different types of oral cysts. Fifty-five cysts were studied: 18 odontogenic keratocysts (OKC), of which five were orthokeratotic and 13 parakeratotic; 19 radicular cysts; and 18 dentigerous cysts. Positive LC was 80% for orthokeratotic OKC, 33% for parakeratotic OKC, approximately 35% for radicular cysts, and approximately 20% for dentigerous cysts. The results show that OKC with well-differentiated epithelial linings presented a greater number of LC than the other cysts. However, when the cyst wall was inflamed there were no differences in LC expression in the different types of cysts. The data confirm that LC distribution seems to be associated with the degree of differentiation of the epithelia.     

Phenotypic and molecular dissection of metaplastic breast cancer and the prognostic implications

Metaplastic breast carcinoma (MBC) is relatively rare but accounts for a significant proportion of global breast cancer mortality. This group is extremely heterogeneous and by definition exhibits metaplastic change to squamous and/or mesenchymal elements, including spindle, squamous, chondroid, osseous, and rhabdomyoid features. Clinically, patients are more likely to present with large primary tumours (higher stage), distant metastases, and overall, have shorter 5-year survival compared to invasive carcinomas of no special type. The current World Health Organisation (WHO) diagnostic classification for this cancer type is based purely on morphology – the biological basis and clinical relevance of its seven sub-categories are currently unclear. By establishing the Asia-Pacific MBC (AP-MBC) Consortium, we amassed a large series of MBCs (n = 347) and analysed the mutation profile of a subset, expression of 14 breast cancer biomarkers, and clinicopathological correlates, contextualising our findings within the WHO guidelines. The most significant indicators of poor prognosis were large tumour size (T3; p = 0.004), loss of cytokeratin expression (lack of staining with pan-cytokeratin AE1/3 antibody; p = 0.007), EGFR overexpression (p = 0.01), and for ‘mixed’ MBC, the presence of more than three distinct morphological entities (p = 0.007). Conversely, fewer morphological components and EGFR negativity were favourable indicators. Exome sequencing of 30 cases confirmed enrichment of TP53 and PTEN mutations, and intriguingly, concurrent mutations of TP53, PTEN, and PIK3CA. Mutations in neurofibromatosis-1 (NF1) were also overrepresented [16.7% MBCs compared to ∼5% of breast cancers overall; enrichment p = 0.028; mutation significance p = 0.006 (OncodriveFM)], consistent with published case reports implicating germline NF1 mutations in MBC risk. Taken together, we propose a practically minor but clinically significant modification to the guidelines: all WHO_1 mixed-type tumours should have the number of morphologies present recorded, as a mechanism for refining prognosis, and that EGFR and pan-cytokeratin expression are important prognostic markers. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.