Langerhans cells in hypospadias : an analysis of Langerin (CD207) and HLA-DR on epidermal sheets and full thickness skin sections

Background: Hypospadias are among the most common genital malformations. Langerhans Cells (LCs) play a pivotal role in HIV and HPV infection. The migration of LC precursors to skin coincides with the embryonic period of hypospadias development and genetic alterations leading to the formation of hypospadias impact the development of ectodermally derived tissues. We hypothesized that this might be associated with a difference in frequency or morphology of epidermal and dermal LCs in hypospadias patients. Methods: A total of 43 patients from two centers were prospectively included into this study after parental consent and ethics approval. Epidermal and dermal sheets were prepared from skin samples of 26 patients with hypospadias, 13 patients without penile malformations and 4 patients with penile malformations other than hypospadias. Immunofluorescence staining of sheets was performed with anti-HLA-DR-FITC and anti-CD207/Langerin-A594 antibodies. Skin sections from 11 patients without penile malformation and 11 patients with hypospadias were stained for Langerin. Frequencies as well as morphology and distribution of epidermal and dermal LCs on sheets and sections were microscopically evaluated. Cell counts were compared by unpaired t-tests. Results: There was no difference in frequency of epidermal LCs, Neither on sheets (87361 vs. 940 +/- 84LCs/mm(2), p=0.522) nor on sections (32 +/- 3 vs. 30 +/- 2LCs/mm(2), p=0.697). Likewise, the frequency of dermal LCs (5,9 +/- 0,9 vs. 7.5 +/- 1.3LCs/mm(2), p=0.329) was comparable between patients with hypospadias and without penile malformation. No differences became apparent in subgroup analyses, comparing distal to proximal hypospadias (p=0.949), younger and older boys (p=0.818) or considering topical dihydrotestosterone treatment prior to surgery (p=0.08). The morphology of the LCs was not different comparing hypospadias patients with boys without penile malformations. Conclusions: p id=Par Cs are present in similar frequencies and with a comparable morphology and distribution in patients with hypospadias as compared to children without penile malformations. This suggests that patients with hypospadias are not different from patients with normal penile development considering this particular compartment of their skin immunity

Characterization of DLK1(PREF1)+/CD34+ cells in vascular stroma of human white adipose tissue

AbstractSorting of native (unpermeabilized) SVF-cells from human subcutaneous (s)WAT for cell surface staining (cs) of DLK1 and CD34 identified three main populations: ~10% stained cs-DLK1+/cs-CD34−, ~20% cs-DLK1+/cs-CD34+dim and ~45% cs-DLK1−/cs-CD34+. FACS analysis after permeabilization showed that all these cells stained positive for intracellular DLK1, while CD34 was undetectable in cs-DLK1+/cs-CD34− cells. Permeabilized cs-DLK1−/cs-CD34+ cells were positive for the pericyte marker α-SMA and the mesenchymal markers CD90 and CD105, albeit CD105 staining was dim (cs-DLK1−/cs-CD34+/CD90+/CD105+dim/α-SMA+/CD45−/CD31−). Only these cells showed proliferative and adipogenic capacity. Cs-DLK1+/cs-CD34− and cs-DLK1+/cs-CD34+dim cells were also α-SMA+ but expressed CD31, had a mixed hematopoietic and mesenchymal phenotype, and could neither proliferate nor differentiate into adipocytes. Histological analysis of sWAT detected DLK1+/CD34+ and DLK1+/CD90+ cells mainly in the outer ring of vessel-associated stroma and at capillaries. DLK1+/α-SMA+ cells were localized in the CD34− perivascular ring and in adventitial vascular stroma. All these DLK1+ cells possess a spindle-shaped morphology with extremely long processes. DLK1+/CD34+ cells were also detected in vessel endothelium. Additionally, we show that sWAT contains significantly more DLK1+ cells than visceral (v)WAT. We conclude that sWAT has more DKL1+ cells than vWAT and contains different DLK1/CD34 populations, and only cs-DLK1−/cs-CD34+/CD90+/CD105+dim/α-SMA+/CD45−/CD31− cells in the adventitial vascular stroma exhibit proliferative and adipogenic capacity

Characterization of Non-hormone Expressing Endocrine Cells in Fetal and Infant Human Pancreas

Context: Previously, we identified chromograninA positive hormone-negative (CPHN) cells in high frequency in human fetal and neonatal pancreas, likely representing nascent endocrine precursor cells. Here, we characterize the putative endocrine fate and replicative status of these newly formed cells.Objective: To establish the replicative frequency and transcriptional identity of CPHN cells, extending our observation on CPHN cell frequency to a larger cohort of fetal and infant pancreas.Design, Setting, and Participants: 8 fetal, 19 infant autopsy pancreata were evaluated for CPHN cell frequency; 12 fetal, 24 infant/child pancreata were evaluated for CPHN replication and identity.Results: CPHN cell frequency decreased 84% (islets) and 42% (clusters) from fetal to infant life. Unlike the beta-cells at this stage, CPHN cells were rarely observed to replicate (0.2 ± 0.1 vs. 4.7 ± 1.0%, CPHN vs. islet hormone positive cell replication, p < 0.001), indicated by the lack of Ki67 expression in CPHN cells whether located in the islets or in small clusters, and with no detectable difference between fetal and infant groups. While the majority of CPHN cells express (in overall compartments of pancreas) the pan-endocrine transcription factor NKX2.2 and beta-cell specific NKX6.1 in comparable frequency in fetal and infant/child cases (81.9 ± 6.3 vs. 82.8 ± 3.8% NKX6.1+-CPHN cells of total CPHN cells, fetal vs. infant/child, p = 0.9; 88.0 ± 4.7 vs. 82.1 ± 5.3% NKX2.2+-CPHN cells of total CPHN cells, fetal vs. infant/child, p = 0.4), the frequency of clustered CPHN cells expressing NKX6.1 or NKX2.2 is lower in infant/child vs. fetal cases (1.2 ± 0.3 vs. 16.7 ± 4.7 clustered NKX6.1+-CPHN cells/mm2, infant/child vs. fetal, p < 0.01; 2.7 ± 1.0 vs. 16.0 ± 4.0 clustered NKX2.2+-CPHN cells/mm2, infant/child vs. fetal, p < 0.01).Conclusions: The frequency of CPHN cells declines steeply from fetal to infant life, presumably as they differentiate to hormone-expressing cells. CPHN cells represent a non-replicative pool of endocrine precursor cells, a proportion of which are likely fated to become beta-cells.Precis: CPHN cell frequency declines steeply from fetal to infant life, as they mature to hormone expression. CPHN cells represent a non-replicative pool of endocrine precursor cells, a proportion of which are likely fated to become beta-cells

Breaking the seals: Efficient mRNA detection from human archival paraffin-embedded tissue

During our study on HOXA13, HOXD12, and HOXD13 mRNA expression in human adult and embryonic tissues, we were confronted with the fact that, within our specimen collection, as in other University Departments in Europe, <20% of all samples yielded reliable labeling, while most samples were resistant to hybridization by standard protocols due to over-fixation. Fixation is essential for specimen stability, especially when samples are stored at room temperature and used for histology, and people tend to be more worried about under- than over-fixation. On the other hand fixation inhibits penetration by the probe and may also trap mRNA within ribosomes. Therefore, we developed a nonradioactive in situ hybridization technique, which allows detection of mRNA expressed on low levels from a variety of differentially fixed tissues while maintaining tissue integrity. This was achieved by improving target retrieval and probe detection. In contrast with others, our method allows reliable staining from tissues that are fixed in paraformaldehyde from four hours to over one week, and archived samples that were stored at room temperature for several years (17–19 yr in some cases) and exceeds detection limits of purely fluorescent methods. Our protocol is highly suitable for detecting CDX-2 mRNA in carcinoma specimens, but especially designed to investigate mRNAs in nonpathological adult and embryonic tissues. Due to the use of standardized probes, we do not expect problems in detecting other mRNAs expressed in suitable amounts

Efficient mRNA detection from human archival paraffin-embedded tissue: An update

We recently published an in situ hybridization protocol for archival tissue using a commercial hybridization buffer. This buffer is no more available. Therefore, we have developed an improved protocol with a defined hybridization buffer