Optimization of immunohistochemical detection of collagen type II in osteochondral sections by comparing decalcification and antigen retrieval agent combinations

Bone containing tissues such as osteochondral joint are resistant to routine tissue processing, therefore require decalcification. This technique causes removal of mineral salts, but in the process may macerate the organic tissue, hence the need for tissue fixation. Such severe processing demands careful antigen retrieval to necessitate optimal staining. The aim of our study was to compare five different antigen retrieval protocols (heat retrieval and protein digestion) following decalcification of rabbit knee joints using two different techniques (20% formic acid and 10% ethylenediamine-tetra acetic acid: EDTA). Osteochondral sections were compared based on time required for decalcification, ease of sectioning, morphological integrity using HE staining and antigen preservation (Collagen type II) using immunohistochemistry. The two decalcification solutions did not impair the tissue morphology and ease of sectioning. Joints processed with formic acid decalcified four times faster than EDTA. Among the five antigen retrieval approaches, maximal collagen II uptake with minimal nonspecific staining was found with protein digestion (pronase and hyaluronidase) in both formic acid and EDTA sections. For osteo-chondral sections, we recommend using 10% EDTA for decalcification and pronase plus hyaluronidase for antigen retrieval if maintaining tissue morphology is crucial, whereas if time is of the essence, 20% FA with pronase plus hyaluronidase is the faster option while still preserving structural integrity. Clin. Anat. 33:343–349, 2020. © 2019 Wiley Periodicals, Inc.     

Influence of TAT-peptide polymerization on properties and transfection activity of TAT/DNA polyplexes.

Use of bioactive cationic peptides as gene carriers is limited by instability of their DNA complexes in vivo and by the loss of their biological activity due to undesired interactions of their bioactive parts with the DNA. To overcome the two major limitations, biodegradable high-molecular-weight form of TAT peptide (POLYTAT) sensitive to cellular redox-potential gradients was synthesized in this study by oxidative polycondensation. Physicochemical and transfection properties of DNA polyplexes based on POLYTAT were investigated and compared with polyplexes based on TAT polymer prepared by in situ template-assisted polymerization. Physicochemical properties of TAT-based polyplexes were affected by the molecular weight and method of polymerization of the TAT peptide. All TAT-based DNA polyplexes exhibited reduced cytotoxicity when compared with control polyethylenimine (PEI) polyplexes. Polyplexes based on both high-molecular-weight TAT polypeptides exhibited increased transfection efficiency compared to control TAT peptide but lower than that of PEI polyplexes. The evidence shows that transfection activity of TAT-based polyplexes is strongly dependent on the presence of chloroquine and therefore suggests that TAT polyplexes are internalized by an endocytosis. Overall, high-molecular-weight reducible polycations based on bioactive peptides has the potential as versatile carriers of nucleic acids that display low cytotoxicity and can prove to be especially beneficial in cases that require surface presentation of membrane-active or cell-specific targeting peptides.     

Cytokines and growth factors in keratinocytes and sweat glands in chronic venous leg ulcers. An immunohistochemical study

The role of growth factors and cytokines in the impaired healing of chronic leg ulcers remains uncertain. The aim of this study was to determine whether changes in the amount and location of cytokines and growth factors may be associated with impaired healing in chronic leg ulcers. Biopsies from leg ulcers of 21 patients and from normal skin of nine healthy volunteers were examined immunohistochemically for selected growth factors and cytokines. Greater staining intensity was found in keratinocytes at the edges of ulcers compared to normal skin, or skin adjacent to the ulcers. Staining at the ulcer edge was more intense in nonhealing ulcers for only vascular endothelial growth factor and platelet-derived growth factor, whereas staining in the adjacent skin was more intense for all factors in the nonhealing phase. For all factors staining was cytoplasmic, suggesting production in these areas. This study shows up-regulation of the production of cytokines and growth factors in keratinocytes of chronic leg ulcers that is greater when the ulcers are nonhealing.     

Picotamide inhibition of excess in vitro thromboxane B2 release by colorectal mucosa in inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease is associated with increased mucosal release of eicosanoids. Among these, thromboxane A2 has been proposed as a possible inflammatory mediator; its suppression may be a useful therapeutic option. METHODS: Using a tissue incubation technique, we compared release of immunoreactive thromboxane B2 by colonic biopsies from patients with ulcerative colitis, Crohn's disease and controls, and assessed the inhibitory effect of picotamide, a thromboxane synthesis inhibitor-receptor antagonist, which has been widely used in Italy for management of ischaemic heart and cerebrovascular disease. RESULTS: Increased amounts of thromboxane B2 were released from biopsies from patients with active ulcerative colitis (median 238 pg/20 min/mg wet weight (interquartile range 147- 325), n = 12) and active Crohn's disease (252 (174-450), 6) compared with those from patients with quiescent ulcerative colitis (95 (61- 140), 12) or Crohn's disease (105 (57-201), 13), or controls (136 (64- 206), 8). Incubation with picotamide at concentrations between 100 microM and 1 mM reduced thromboxane B2 release (IC50 890 microM). CONCLUSION: Since increased thromboxane A2 production may have pathogenetic importance, thromboxane synthesis inhibitor-receptor antagonists such as picotamide merit therapeutic trial in the management of inflammatory bowel disease.     

Follistatin and activin A production by the male reproductive tract

Follistatin is a binding protein for the activin and inhibin family of hormones, regulating their biological activity. In the male reproductive tract, the interaction of these factors is likely to be involved in the regulation of the proliferation of several cell types. We have investigated the presence of follistatin and activin A in seminal plasma using specific immunoassays and have localized follistatin and activin/inhibin subunits in the adult human testis, prostate and seminal vesicle to establish their likely sources. High concentrations of immunoreactive follistatin were present in seminal plasma in normal men (mean 97.9 ng/ml; 1.43 ng/ml in peripheral plasma) and were similar in men with oligo/azoospermia and following vasectomy. Follistatin immunoreactivity was localized to both Leydig and Sertoli cells of the testis, and to epithelial cells of the prostate gland and seminal vesicle, which are likely to be the predominant sources of the hormone in seminal plasma. Activin A was also present in seminal plasma in normal men but was undetectable following vasectomy, thus deriving from the testis. Consistent with this finding, the betaA-subunit was immunolocalized in Sertoli and Leydig cells but was not present in seminal vesicle or prostate gland. The functional significance of the high concentrations of follistatin secreted into seminal plasma by the prostate gland and/or seminal vesicle is uncertain, but they may regulate the biological activity of testis-derived activin A and inhibin B.