Phosphorylated KDR is expressed in the neoplastic and stromal elements of human renal tumours and shuttles from cell membrane to nucleus

Vascular endothelial growth factor (VEGF)-A is an important angiogenic factor in establishing the vasculature in renal cell carcinomas (RCCs). Since little is known about VEGF signalling in RCCs, the profile of phosphorylated KDR (pKDR) has been investigated and the intracellular location of the receptor has been examined in the present study. Using two monoclonal antibodies raised against the phosphorylated KDR epitopes (Y1059 and Y1214) known to mediate different VEGF functions, together with a commercial anti-KDR antibody and immunohistochemistry, the expression of pKDR was investigated in a series of normal (n = 25) and neoplastic kidneys (n = 54; clear cell n = 35; papillary n = 10; oncocytomas n = 8). pKDR was present in many tissue elements of both normal and neoplastic renal tissues, with strong expression in the cell membrane, cytoplasm, and nuclei of normal kidney and tumour cells, as well as endothelial cells in tumours of all histological types. Patterns and intensity were similar using both anti-pKDR antibodies. There was no significant correlation in clear cell carcinomas between pKDR expression and age (p = 0.57), tumour size (p = 0.2), gender (p = 0.59), grade (p = 0.2) or histological type (p = 0.36). To delineate further the intracellular processing that might account for the cellular distribution, confocal microscopy was also performed. Antibodies to the different phosphorylated epitopes demonstrated different intracellular staining patterns. This study shows that pKDR is present in a wide variety of renal tumours, suggesting that anti-VEGF therapy might have direct effects on tumour cells. It further suggests that cells traffic pKDR depending on the precise KDR tyrosines that are autophosphorylated in a manner that enables receptor activation to result in different functions.     

Cross-sectional analysis of clinical and environmental isolates of Pseudomonas aeruginosa: biofilm formation, virulence, and genome diversity

Chronic lung infections with Pseudomonas aeruginosa biofilms are associated with refractory and fatal pneumonia in cystic fibrosis (CF). In this study, a group of genomically diverse P. aeruginosa isolates were compared with the reference strain PAO1 to assess the roles of motility, twitching, growth rate, and overproduction of a capsular polysaccharide (alginate) in biofilm formation. In an in vitro biofilm assay system, P. aeruginosa displayed strain-specific biofilm formation that was not solely dependent on these parameters. Compared with non-CF isolates, CF isolates expressed two opposing growth modes: reduced planktonic growth versus efficient biofilm formation. Planktonic cells of CF isolates showed elevated sensitivity to hydrogen peroxide, a reactive oxygen intermediate, and decreased lung colonization in an aerosol infection mouse model. Despite having identical genomic profiles, CF sequential isolates produced different amounts of biofilm. While P. aeruginosa isolates exhibited genomic diversity, the genome size of these isolates was estimated to be 0.4 to 19% (27 to 1,184 kb) larger than that of PAO1. To identify these extra genetic materials, random amplification of polymorphic DNA was coupled with PAO1-subtractive hybridization. Three loci were found within the genomes of two CF isolates encoding one novel homolog involved in retaining a Shigella virulence plasmid (mvpTA) and two divergent genes that function in removing negative supercoiling (topA) and biosynthesis of pyoverdine (PA2402). Together, P. aeruginosa biodiversity could provide one cause for the variation of morbidity and mortality in CF. P. aeruginosa may possess undefined biofilm adhesins that are important to the development of an antibiofilm therapeutic target.     

A simple method for counting adherent cells: application to cultured human monocytes, macrophages and multinucleated giant cells

A simple method was devised for counting small numbers (10(4)-10(6)) of adherent mononuclear phagocytes, including populations containing multinucleated giant cells, which often arise during cultivation of human blood monocytes. Coverslips with adherent cells were transferred into small volumes (50-200 microliters) of 0.1 M citric acid, pH 2.2, containing 0.05% naphthol blue black and 1.0% of either Triton X-100 or Cetavlon. Triton X-100 was adequate for use with monocytes and macrophages from early cultures. However, Cetavlon was preferable for use with older cultures of adherent human mononuclear cells in order to prevent aggregation of the nuclei from giant cells. When multinucleated cells were present, separately stained coverslips were inspected to determine the mean number of nuclei per cell. This value, together with the number of nuclei per coverslip, permitted calculation of the number of cells per coverslip. The latter value is not readily derived from measurements of protein or DNA content in populations containing multinucleated giant cells. This counting method was simpler and more sensitive than several previously reported methods for enumerating adherent macrophages.     

Carrier Frequency of the Bloom SyndromeblmMutation in the Ashkenazi Jewish Population

Bloom syndrome is more common in individuals of Ashkenazi Jewish descent than in any other population, and one particular mutation in the Bloom syndrome gene,blm^Ash,is homozygous in nearly all Ashkenazi Jewish persons with Bloom syndrome. We have determined the frequency ofblm^Ashin 1491 Ashkenazi Jewish persons with no known history of Bloom syndrome and found that 1 in 107 persons was heterozygous. Although not common, genetic screening for Bloom syndrome is feasible in this population.     

Relationship between extracellular stimulation of intracellular killing and oxygen-dependent microbicidal systems of monocytes.

Human monocytes require serum components immunoglobulin G, C3/C3b, and B/Bb to exert optimal microbicidal action against ingested microorganisms. The present study was performed to find out whether these factors act by enhancing oxygen-dependent antimicrobial mechanisms. Serum enhanced oxygen consumption and superoxide production by monocytes before phagocytosis, but did not further increase these processes in monocytes that had recently ingested bacteria. Furthermore, serum did not boost iodination during intracellular killing by monocytes. Phorbol myristate acetate, N-formyl-methyonyl-leucyl-phenylalanine, concanavalin A, and concanavalin A-Sephadex all stimulated the conversion of O2 to H2O2 by monocytes, but only concanavalin A augmented intracellular killing. Reactive oxygen intermediates generated by cell-free enzymes (xanthine oxidase or glucose oxidase) in concentrations comparable to those accumulating extracellularly during incubation of monocytes containing bacteria with phorbol myristate acetate did not promote intracellular killing. The presence of catalase during phagocytosis inhibited killing, but had no effect on killing in the postphagocytic state. Monocytes deprived of glucose for 24 h showed markedly impaired O2 consumption, O2- generation, and bacterial killing; all of these effects were rapidly reversed by restoration of glucose. It is concluded that both an intact respiratory burst and extracellular serum factors are necessary for optimal killing of intracellular Staphylococcus aureus by human monocytes. Serum does not appear to act by enhancing the respiratory burst, but rather to have a separate, synergistic role, the biochemical basis of which is unknown.     

Autocrine growth of human blymphocytes: Maintained response to autostimulatory factors is the special feature of immortalization by Epstein-Barr virus—A hypothesis

The autocrine growth profile of human B lymphocytes transformed with Epstein-Barr virus (EBV) was found to comprise three distinct components: a B-cell growth factor (BCGF); an interleukin-1 (IL-1)-like activity; an activity requiring cell-to-cell contact for its action. Observations on the inhibition of the EBV-carrying Daudi lymphoma line by α-interferon indicated that loss of response to these autostimulatory factors was underlying growth cessation. Furthermore, a putative for BCGF was found to be down-regulated on B cells stimulated with non-transforming mitogens but constitutively expressed following EBV-transformation. Taken together with recent evidence that normal B cells produce autostimulatory factors, these findings suggest that the special feature of autocrine growth by EBV-immortalized cells is a maintenance of what should normally be a transient phenotype, possibly through deregulation of receptor expression. This hypothesis is discussed.     

Beyond empathy decline: Do the barriers to compassion change across medical training?

Advances in Health Sciences Education - Background: Despite being a mandated, foundational value in healthcare, research on compassion remains limited. Studying the individual, patient, clinical,...     

Human Milk Exosomal MicroRNA: Associations with Maternal Overweight/Obesity and Infant Body Composition at 1 Month of Life

Among all the body fluids, breast milk is one of the richest sources of microRNAs (miRNAs). MiRNAs packaged within the milk exosomes are bioavailable to breastfeeding infants. The role of miRNAs in determining infant growth and the impact of maternal overweight/obesity on human milk (HM) miRNAs is poorly understood. The objectives of this study were to examine the impact of maternal overweight/obesity on select miRNAs (miR-148a, miR-30b, miR-29a, miR-29b, miR-let-7a and miR-32) involved in adipogenesis and glucose metabolism and to examine the relationship of these miRNAs with measures of infant body composition in the first 6 months of life. Milk samples were collected from a cohort of 60 mothers (30 normal-weight [NW] and 30 overweight [OW]/obese [OB]) at 1-month and a subset of 48 of these at 3 months of lactation. Relative abundance of miRNA was determined using real-time PCR. The associations between the miRNAs of interest and infant weight and body composition at one, three, and six months were examined after adjusting for infant gestational age, birth weight, and sex. The abundance of miR-148a and miR-30b was lower by 30% and 42%, respectively, in the OW/OB group than in the NW group at 1 month. miR-148a was negatively associated with infant weight, fat mass, and fat free mass, while miR-30b was positively associated with infant weight, percent body fat, and fat mass at 1 month. Maternal obesity is negatively associated with the content of select miRNAs in human milk. An association of specific miRNAs with infant body composition was observed during the first month of life, suggesting a potential role in the infant’s adaptation to enteral nutrition.