Changes in histone deacetylase (HDAC) expression patterns and activity of HDAC inhibitors in urothelial cancers

ObjectiveTo determine histone deacetylase (HDAC) isoenzyme expression patterns in urothelial cancer tissues and cell lines and investigate their potential to predict the efficacy of the HDAC inhibitor vorinostat.Materials and methodsExpression of HDAC mRNAs was determined by quantitative RT-PCR in 18 urothelial cancer cell lines (UCC), normal uroepithelial controls (NUC), 24 urothelial cancer tissues, and 12 benign controls. Results were compared with published microarray data. Effects of pan-HDAC inhibitor vorinostat and on UCCs were determined by viability and apoptosis assays, cell cycle analysis, and measurements of p21^CIP1, thymidylate synthase (TS), and EZH2. In addition, protein expression levels of HDACs were investigated in UCCs.ResultsProminent changes in UCCs were HDAC2 and/or HDAC8 up-regulation in 11 of 18 cell lines and decreased expression of HDAC4, HDAC5, and/or HDAC7 mRNA in 15 of 18 cell lines. In cancer tissues, HDAC8 was likewise significantly up-regulated (P = 0.002), whereas HDAC2 up-regulation was detected only in a subset of tumors (9/24, P = 0.085). Overexpression of HDAC2 and HDAC8 mRNA did not correspond with the protein level. Vorinostat induced G2/M arrest, an increase in the sub-G1 fraction, up-regulation of p21, and down-regulation of TS in all UCC. Effects on EZH2 and PARP cleavage as well as activation of caspase 3/7 differed between cell lines. Associations between the overall sensitivity to the pan-HDACi vorinostat and overexpression of HDAC2 and HDAC8 mRNA were not observed.ConclusionsIn urothelial cancer, up-regulation of HDAC2 and HDAC8 and down-regulation of HDAC4, HDAC5, and HDAC7 mRNA are common findings. The treatment effect of the pan-HDAC inhibitor vorinostat was variable in UCCs and up-regulation of HDAC2 and HDAC8 was not predictive for treatment response. Whether selective targeting of HDAC2, HDAC8, or other HDACs deregulated in urothelial cancer (e.g., HDAC4, HDAC5, and HDAC7) result in a more consistent treatment response needs further investigation.     

Immunohistochemistry in the classification of systemic forms of amyloidosis: a systematic investigation of 117 patients

Amyloidoses are characterized by organ deposition of misfolded proteins. This study evaluated immunohistochemistry as a diagnostic tool for the differentiation of amyloid subentities, which is warranted for accurate treatment. A total of 117 patients were systematically investigated by clinical examination, laboratory tests, genotyping, and immunohistochemistry on biopsy specimens. Immunohistochemistry enabled the classification in 94% of the cases. For subsequent analysis, the patient population was divided into 2 groups. The first group included all patients whose diagnosis could be verified by typical clinical signs or an inherited amyloidogenic mutation. In this group, immunohistochemical subtyping was successful in 49 of 51 cases and proved accurate in each of the 49 cases, corresponding to a sensitivity of 96% and a specificity of 100%. The second group included patients with systemic light chain amyloidosis without typical signs, senile transthyretin, or hereditary amyloidosis with a concomitant monoclonal gammopathy. Immunohistochemistry allowed to define the subentities in 61 of 66 (92%) of these cases. Immunohistochemistry performed by a highly specialized pathologist combined with clinical examination and genotyping leads to a high accuracy of amyloidosis classification and is the standard in our center. However, new techniques, such as mass spectroscopy-based proteomics, were recently developed to classify inconclusive cases.     

In-Depth Characterisation of Retinal Pigment Epithelium (RPE) Cells Derived from Human Induced Pluripotent Stem Cells (hiPSC)

Induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) has widely been appreciated as a promising tool to model human ocular disease emanating from primary RPE pathology. Here, we describe the successful reprogramming of adult human dermal fibroblasts to iPSCs and their differentiation to pure expandable RPE cells with structural and functional features characteristic for native RPE. Fibroblast cultures were established from skin biopsy material and subsequently reprogrammed following polycistronic lentiviral transduction with OCT4, SOX2, KLF4 and L-Myc. Fibroblast-derived iPSCs showed typical morphology, chromosomal integrity and a distinctive stem cell marker profile. Subsequent differentiation resulted in expandable pigmented hexagonal RPE cells. The cells revealed stable RNA expression of mature RPE markers RPE65, RLBP and BEST1. Immunolabelling verified localisation of BEST1 at the basolateral plasma membrane, and scanning electron microscopy showed typical microvilli at the apical side of iPSC-derived RPE cells. Transepithelial resistance was maintained at high levels during cell culture indicating functional formation of tight junctions. Secretion capacity was demonstrated for VEGF-A. Feeding of porcine photoreceptor outer segments revealed the proper ability of these cells for phagocytosis. IPSC-derived RPE cells largely maintained these properties after cryopreservation. Together, our study underlines that adult dermal fibroblasts can serve as a valuable resource for iPSC-derived RPE with characteristics highly reminiscent of true RPE cells. This will allow its broad application to establish cellular models for RPE-related human diseases.     

LCMV-specific CD4 T cell dependent polyclonal B-cell activation upon persistent viral infection is short lived and extrafollicular

Persistent virus infections with non- or poorly cytopathic viruses are commonly associated with B cell dysregulations. These include the induction of hypergammaglobulinemia and the emergence of virus-unspecific antibodies. These seemingly unspecific antibody responses interfere with the virus-specific humoral immunity and contribute to delayed virus control. Whether these virus-unspecific antibodies are induced in the B cell follicle or at extrafollicular sites and whether one specific CD4 T cell subset is involved in the polyclonal B cell activation is unclear. Here we studied virus-unrelated IgG antibody responses against self or foreign antigens in the context of persistent lymphocytic choriomeningitis virus (LCMV) infection. We found that the LCMV-unspecific antibody response is short-lived and induced predominantly at extrafollicular sites and depends on the presence of LCMV-specific CD4 T cells. Our data support a scenario in which activated, virus-specific CD4 T cells provide help to non-specific B cells at extrafollicular sites, supporting the production of virus unspecific IgG antibodies during persistent viral infection.     

Intraocular pressure changes following 20G pars‐plana vitrectomy

Purpose: To observe the excursions of short‐term intraocular pressure (IOP) after 20‐G pars‐plana vitrectomy (ppV). Material and methods: In a prospective study, 851 patients (age: mean 63 ± 15 years) underwent unilateral ppV for various vitreoretinal diseases using different endotamponades [Balanced Salt Solution (BSS) 33.1%, Air 7.2%, SF₆ 33.6%, silicon oil 5000 cst 26.1%]. Intraocular pressure was measured in all patients before and at 3, 6, 24 and 48 hr after surgery. Survival analysis was performed to determine the cumulative hazard of IOP changes depending on endotamponade and time point after ppV (Log‐Rang ‐ Mantel Cox; p < 0.0001). Results: At baseline, IOP ranged from 0 to 50 mmHg (mean IOP: 15.3 ± 5.3 mmHg). Mean IOP after surgery revealed a slight elevation (3 hr: 16.5 ± 11.0 mmHg; 6 hr 16.9 ± 9.8 mmHg; 24 hr 19.7 ± 8.0 mmHg; 48 hr 17.3 ± 6.2 mmHg; range: 0–64 mmHg). Silicon oil filling revealed highest mean values at already 3 hr after surgery (21.8 mmHg). Also, BSS filling showed a peak after 3 hr; however, mean values were lower. Equivalent high IOP values as for silicon oil tamponade were found for gas filling; however, maximal peak was reached after 24 hr but not after 3 hr post‐treatment. The cumulative hazard in all patients to reach IOP ≥ 30 mmHg after 24 hr was 23.9%; (IOP ≥ 40 mmHg = 8.2%). Herein, oil filling revealed highest risk at all time points after surgery. The risk of suffering from IOP < 5 mmHg lasting longer than 6 hr was only 1.2% after 20 G vitrectomy. Conclusion: Intraocular pressure measurements after ppV are important to prevent unintentional high IOP, especially within the early phase (3 hr post‐treatment) in eyes with silicon oil filling. Gas filling leads to prolonged IOP increase (24 hr post‐treatment). Long‐lasting hypotony (≥6 hr) is very rare after 20G vitrectomy.     

Evaluation of school-based life skills programmes in a high-risk sample: a controlled longitudinal multi-centre study

Aim  

Previous studies have demonstrated a positive effect of school-based life skills programmes on the prevention of substance abuse and other health-risk behaviours in children and adolescents. However, the comparison and interpretation of study results is difficult due to methodological problems. In particular, the effectiveness of such programmes within high-risk groups remains uncertain. In this study, we investigated the effects of two school-based life skills programmes on substance abuse and subjective health in a sample with a high proportion of socially disadvantaged pupils.     

Localized immunoglobulin light chain amyloidosis: Novel insights including prognostic factors for local progression

In localized light chain amyloidosis (locAL), amyloidogenic light chains (aLC) are produced and deposited locally by a B-cell clone. We present 293 patients with immunohistochemically confirmed locAL. Lung (nodular pulmonary) with 63 patients was the most involved organ. The aLC was λ in 217 cases (κ:λ ratio 1:3). A local B-cell clone was identified in 30% of cases. Sixty-one (21%) had a concomitant autoimmune disorder (cAD). A monoclonal component (MC) were present in 101 (34%) patients and were more frequent in subjects with cAD (51% vs 34%; P = .03). Cigarette smoking was more prevalent in lung locAL (54% vs 37%; P = .018). After a median follow-up of 44 months, 16 patients died and 5- and 10-years locAL progression-free survival (PFS) were 62% and 44%. Interestingly, locAL-PFS was shorter among patients with an identified clonal infiltrate at amyloid deposition site (40 vs 109 months; P = .02) and multinuclear giant cells and/or an inflammatory infiltrate resulted in longer locAL-PFS in lung involvement (65 vs 42 months; P = .01). However, no differences in locAL PFS were observed in patients with cAD, a MC and involved organ site. Treatment was administered in 163 (54%) patients and was surgical in 135 (46%). Median locAL-PFS after first treatment was 56 months. Responders had longer locAL-PFS (78 vs 17 months; P < .001). Three patients with lung locAL and a MC were diagnosed as systemic AL amyloidosis at follow-up. In summary, locAL pathogenesis seems to be heterogeneous and the clonal infiltrate leads local progression.     

Health-related quality of life in children with disorders of sex development (DSD)

Disorders of sex development (DSD) are rare genetic conditions resulting in atypical development of the sex organs. While some evidence is available on psychosexual outcomes, much less is known about the quality of life in this population, especially in children. Health-related quality of life (HRQOL) is a widely accepted endpoint for assessment and evaluation of interventions and medical care. Within the German DSD Network study, 86 children aged 8–12 years with several subtypes of DSD were recruited from Germany, Austria and Switzerland. Demographic, medical and psychosocial variables were collected through interviews of the attending physicians, the children and the parents. HRQOL was the primary outcome. It was assessed by the KINDL-R Questionnaire [2001]. Psychosexual determinants included gender identity/gender dysphoria, gender role behaviour, the child’s knowledge about the condition and number/timing of genital surgery. A significant reduction of HRQOL was reported in children’s self-report (p?<?0.001), in particular in the area of self-esteem (p?<?0.001), physical well-being (p?<?0.01) and school functioning (p?<?0.05). Girls with congenital adrenal hyperplasia who experienced gender dysphoria reported lower HRQOL scores compared to the study group at large. Atypical gender role behaviour was not associated with HRQOL. Conclusion: Psychosocial support of children with DSD and their families appears to be necessary in at least some cases and must be accessible for all patients.