Optimizing Staining Protocols for Laser Microdissection of Specific Cell Types from the Testis Including Carcinoma In Situ

Microarray and RT-PCR based methods are important tools for analysis of gene expression; however, in tissues containing many different cells types, such as the testis, characterization of gene expression in specific cell types can be severely hampered by noise from other cells. The laser microdissection technology allows for enrichment of specific cell types. However, when the cells are not morphologically distinguishable, it is necessary to use a specific staining method for the target cells. In this study we have tested different fixatives, storage conditions for frozen sections and staining protocols, and present two staining protocols for frozen sections, one for fast and specific staining of fetal germ cells, testicular carcinoma in situ cells, and other cells with embryonic stem cell-like properties that express the alkaline phosphatase, and one for specific staining of lipid droplet-containing cells, which is useful for isolation of the androgen-producing Leydig cells. Both protocols retain a morphology that is compatible with laser microdissection and yield RNA of a quality suitable for PCR and microarray analysis

Surface chemistry mediated albumin adsorption, conformational changes and influence on innate immune responses

Available online 10 May 2022The surface chemistry of biomaterials plays a pivotal role in regulating the type, amount, and conformational changes of adsorbed proteins, which then modulates the subsequent innate immune responses. Albumin is the most abundant protein in serum and a major constituent of the protein corona that forms on biomaterial surfaces. Therefore, it is important to understand the role of surface chemistry in albumin adsorption, surface induced conformational changes, and how this affects the subsequent immune responses. To interrogate these events, we generated model substrata with four tailored surface chemistries rich in amines, oxazolines, carboxylic acid groups and pure hydrocarbons via plasma polymerization. The positively charged amine and oxazoline rich surface chemistries caused greatest albumin adsorption and protein conformational changes. Our data demonstrated that macrophages (differentiated THP-1 cells) were able to interact with unfolded albumin via their scavenger receptors. The innate inflammatory responses were studied by measuring the expression of pro- and anti- inflammatory markers. As an overall trend, the pre-adsorption of albumin resulted in a reduction in the level of expression of pro-inflammatory cytokines while the secretion of anti-inflammatory markers was stimulated. This study provides valuable information, which could aid in the design of future biomaterials that elicit predictable immune response.Panthihage Ruvini L Dabare, Akash Bachhuka, Dennis Palms, Emma Parkinson- Lawrence, John D Hayball, Agnieszka Mierczynska, Krasimir Vasile

Outcomes in third and fourth kidney transplants based on the type of donor

Background. An increasing number of patients are requiring multiple retransplants. We assessed outcomes of third and fourth kidney transplants, to aid decision making on the most suitable donor type. Methods. Data were collected retrospectively for 2561 transplants, including 69 third and 8 fourth, performed from 2000 to 2017. Demographics and outcomes for the combined third/fourth group were compared to first and second transplants. Within the third/fourth kidney transplant group, comparisons were made between deceased donors (n = 39), live donor HLA-compatible (n = 23) and -incompatible (n = 13) transplants, as well as between standard (n = 25) and extended-criteria (n = 14) deceased donor transplants. Results. Patient survival did not differ significantly by transplant number (P = 0.532), whereas death-censored graft survival declined progressively, from 89% at 5 years in first, 85% in second and 74% in the third/fourth transplant group (P < 0.001). Within the combined third/fourth transplant subgroup, 5-year graft survival was found to be 100% in recipients of HLA-compatible live donors, compared to 75% in deceased donors and 53% in HLA-incompatible live donors, although this difference did not reach statistical significance (P = 0.083). No significant difference in patient survival (P = 0.356) or complication rates (P = 0.757) were detected between these groups. For recipients of deceased donors in the third/fourth transplant group, there were no significant differences between standard versus extended-criteria donors for any of the outcomes considered. Conclusions. Despite variable functional outcomes, third and fourth kidney transplant recipients experience comparable patient survival rates to first and second transplants, regardless of the donor type. In selected patients, HLA-incompatible live donors and extended-criteria deceased donors should be considered

Availability and Affordability of Essential Medicines: Implications for Global Diabetes Treatment

The purpose of this review is to highlight the key issues with regard to the value, affordability, and availability of diabetes treatments