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Background Flat epithelial atypia (FEA) of the breast is characterised by a few layers of mildly atypical luminal epithelial cells. Genetic changes found in ductal carcinoma in situ (DCIS) and invasive ductal breast cancer (IDC) are also found in FEA, albeit at a lower concentration. So far, miRNA expression changes associated with invasive breast cancer, like miR-21, have not been studied in FEA. Methods We performed miRNA in-situ hybridization (ISH) on 15 cases with simultaneous presence of normal breast tissue, FEA and/or DCIS and 17 additional cases with IDC. Expression of the miR-21 targets PDCD4, TM1 and PTEN was investigated by immunohistochemistry. Results Two out of fifteen cases showed positive staining for miR-21 in normal breast ductal epithelium, seven out of fifteen cases were positive in the FEA component and nine out of twelve cases were positive in the DCIS component. A positive staining of miR-21 was observed in 15 of 17 IDC cases. In 12 cases all three components were present in one tissue block and an increase of miR-21 from normal breast to FEA and to DCIS was observed in five cases. In three cases the FEA component was negative, whereas the DCIS component was positive for miR-21. In three other cases, normal, FEA and DCIS components were negative for miR-21 and in the last case all three components were positive. Overall we observed a gradual increase in percentage of miR-21 positive cases from normal, to FEA, DCIS and IDC. Immunohistochemical staining for PTEN revealed no obvious changes in staining intensities in normal, FEA, DCIS and IDC. Cytoplasmic staining of PDCD4 increased from normal to IDC, whereas, the nuclear staining decreased. TM1 staining decreased from positive in normal breast to negative in most DCIS and IDC cases. In FEA, the staining pattern for TM1 was similar to normal breast tissue. Conclusion Upregulation of miR-21 from normal ductal epithelial cells of the breast to FEA, DCIS and IDC parallels morphologically defined carcinogenesis. No clear relation was observed between the staining pattern of miR-21 and its previously reported target genes.

IntroductionSerum calcification propensity is emerging as an independent predictor for cardiovascular outcomes in high-risk populations. Calcification propensity can be monitored by the maturation time of calciprotein particles in serum (T50 test). A low T50 value is an independent determinant of cardiovascular morbidity and mortality in various populations. Aim was to investigate the T50 and its relationship to type 2 diabetes mellitus.Research design and methodsUsing nephelometry, serum T50 was cross-sectionally measured in 932 stable patients with type 2 diabetes mellitus (55% male) with a median age of 66 (62–75) years, diabetes duration of 6.5 (3.0–10.2) years and hemoglobin A1c (HbA1c) of 49 (44–54) mmol/mol.ResultsSerum T50 was normally distributed with a mean value of 261±66 min. In linear regression, serum T50 was lower in women and current smokers. A lower T50 value was found in patients with a higher HbA1c or higher systolic blood pressure, insulin users and patients with a longer history of diabetes. The association with HbA1c was independent of other determinants in multivariable analysis. There was no association between T50 and previous macrovascular events or the presence of microvascular disease.ConclusionsSerum calcification propensity is independently associated with glycemic control, suggesting that a lower HbA1c may be associated with better cardiovascular outcomes. Retrospective analysis could not establish an association between a history of macrovascular events and T50, and prospective studies will have to be performed to address this hypothesis.Trial registration numberNCT01570140.

Recent studies have shown that certain non-coding short RNAs, called miRNAs, play an important role in diffuse large B-cell lymphomas. Patients with diffuse large B-cell lymphoma have great diversity in both clinical characteristics, site of presentation and outcome. The aim of our study is to validate the differential expression in germinal center and non-germinal center diffuse large B-cell lymphoma,s and to study to the extent to which the primary site of differentiation is associated with the miRNA expression profile. We studied 50 cases of de novo diffuse large B-cell lymphoma for the expression of 15 miRNAs (miR-15a, miR-15b, miR-16, miR-17-3p, miR-17-5p, miR-18a, miR-19a, miR-19b, miR-20a, miR-21, miR-92, miR-127, miR-155, miR-181a and miR-221). Apart from 19 nodal cases without extranodal dissemination (stages I and II), we selected two groups with unambiguous stages I and II extranodal presentation; 9 cases of primary central nervous system, 11 cases of primary testicular and 11 cases of other primary extranodal diffuse large B-cell lymphomas. All cases were analyzed with qRT-PCR. In situ hybridization for the most differentially expressed miRNAs was performed to show miRNA expression in tumor cells, but not in background cells. MiR-21 and miR-19b showed the highest expression levels. No significant differences were seen between germinal center and non-germinal center diffuse large B-cell lymphomas in either the total or the nodal group for any of the 15 miRNAs. Two miRNAs showed significant differences in expression levels for diffuse large B-cell lymphoma subgroups according to the site of presentation. MiR-17-5p showed a significant higher expression level in the central nervous system compared with testicular and nodal diffuse large B-cell lymphomas ( P <0.05). MiR-127 levels were significantly higher in testicular than in central nervous system and in nodal diffuse large B-cell lymphomas ( P <0.05). We conclude that the location of diffuse large B-cell lymphoma is an important factor in determining the differential expression of miRNAs.

MicroRNAs (miRNAs) are an important class of small RNAs that regulate gene expression at the post-transcriptional level. It has become evident that miRNAs are involved in hematopoiesis, and that deregulation of miRNAs may give rise to hematopoietic malignancies. The aim of our study was to establish miRNA profiles of naïve, germinal center (GC) and memory B cells, and validate their expression patterns in normal lymphoid tissues. Quantitative (q) RT-PCR profiling revealed that several miRNAs were elevated in GC B cells, including miR-17-5p, miR-106a and miR-181b. One of the most abundant miRNAs in all three B-cell subsets analyzed was miR-150, with a more than 10-fold lower level in GC B cell as compared with the other two subsets. miRNA in situ hybridization (ISH) in tonsil tissue sections confirmed the findings from the profiling work. Interestingly, gradual decrease of miR-17-5p, miR-106a and miR-181b staining intensity from the dark to the light zone was observed in GC. A strong cytoplasmic staining of miR-150 was observed in a minority of the centroblasts in the dark zone of the GC. Inverse staining pattern of miR-150 against c-Myb and Survivin was observed in tonsil tissue sections, suggesting possible targeting of these genes by miR-150. In line with this, the experimental induction of miR-150 lead to reduced c-Myb, Survivin and Foxp1 expression levels in the Burkitt's lymphoma cell line, DG75. In conclusion, miRNA profiles of naïve, GC and memory B cells were established and validated by miRNA ISH. Within the GC cells, a marked difference was observed between the light and the dark zone.

Little is known about the gene expression profile and significance of the rosetting CD4+CD26− T cells in classical Hodgkin's lymphoma (cHL). To characterize these T cells, CD4+CD26− and CD4+CD26+ T-cell populations were sorted from lymph node (LN) cell suspensions from nodular sclerosis HL (NSHL) and reactive LNs. mRNA profiles of stimulated and resting cell subsets were evaluated with quantitative RT-PCR for 46 genes. We observed a higher percentage of CD4+CD26− T cells in NSHL than in reactive LNs. The resting CD4+CD26− T cells in NSHL showed higher mRNA levels of CD25, CTLA4, OX40 and CCR4 compared with in LNs, supporting a regulatory T-cell (Treg) type, and this was validated by immunohistochemistry. Moreover, these cells showed low or no expression of the Th1- or Th2-related cytokines IL-2, IFN- γ , IL-13, IL-12B, IL-4, and IL-5, and the chemoattractant receptor CRTH2. Besides Tregs, Th17 cells may exist in NSHL based on the significantly higher IL-17 mRNA level for both T-cell populations in NSHL. Upon stimulation in vitro , lack of upregulation of mRNA levels of most cytokine genes indicated an anergic character for the CD4+CD26− T-cell subset. Anergy fits with the Treg profile of these cells, probably explaining the immunosuppressive mechanism involved in NSHL.

The majority of disease modifying therapies for multiple sclerosis (MS) reduce inflammation, but do no’t target remyelination. Development of remyelinating therapies will benefit from a method to quantify myelin kinetics in patients with MS. We labeled myelin in vivo with deuterium, and modeled kinetics of myelin breakdown products β‐galactosylceramide (β‐GalC) and N‐Octadecanoyl‐sulfatide (NO‐Sulf). Five patients with MS received 120 ml 70% D2O daily for 70 days and were compared with six healthy subjects who previously received the same procedure. Mass spectrometry and compartmental modeling were used to quantify the turnover rate of β‐GalC and NO‐Sulf in cerebrospinal fluid (CSF). Turnover rate constants of the fractions of β‐GalC and NO‐Sulf with non‐negligible turnover were 0.00186 and 0.00714, respectively, in both healthy subjects and patients with MS. The turnover half‐life of β‐GalC and NO‐Sulf was calculated as 373 days and 96.5 days, respectively. The effect of MS on the NO‐Sulf (49.4% lower fraction with non‐negligible turnover) was more pronounced compared to the effect on β‐GalC turnover (18.3% lower fraction with non‐negligible turnover). Kinetics of myelin breakdown products in the CSF are different in patients with MS compared with healthy subjects. This may be caused by slower myelin production in these patients, by a higher level of degradation of a more stable component of myelin, or, most likely, by a combination of these two processes. Labeling myelin breakdown products is a useful method that can be used to quantify myelin turnover in patients with progressive MS and can therefore be used in proof‐of‐concept studies with remyelination therapies.

Objective ITPR3, encoding inositol 1,4,5‐trisphosphate receptor type 3, was previously reported as a potential candidate disease gene for Charcot‐Marie‐Tooth neuropathy. Here, we present genetic and functional evidence that ITPR3 is a Charcot‐Marie‐Tooth disease gene. Methods Whole‐exome sequencing of four affected individuals in an autosomal dominant family and one individual who was the only affected individual in his family was used to identify disease‐causing variants. Skin fibroblasts from two individuals of the autosomal dominant family were analyzed functionally by western blotting, quantitative reverse transcription PCR, and Ca2+ imaging. Results Affected individuals in the autosomal dominant family had onset of symmetrical neuropathy with demyelinating and secondary axonal features at around age 30, showing signs of gradual progression with severe distal leg weakness and hand involvement in the proband at age 64. Exome sequencing identified a heterozygous ITPR3 p.Val615Met variant segregating with the disease. The individual who was the only affected in his family had disease onset at age 4 with demyelinating neuropathy. His condition was progressive, leading to severe muscle atrophy below knees and atrophy of proximal leg and hand muscles by age 16. Trio exome sequencing identified a de novo ITPR3 variant p.Arg2524Cys. Altered Ca2+‐transients in p.Val615Met patient fibroblasts suggested that the variant has a dominant‐negative effect on inositol 1,4,5‐trisphosphate receptor type 3 function. Interpretation Together with two previously identified variants, our report adds further evidence that ITPR3 is a disease‐causing gene for CMT and indicates altered Ca2+ homeostasis in disease pathogenesis.