Stability of avian leukosis virus type-specific antigen in the cell after fixation in periodate-lysine-paraformaldehyde

Stability of avian leukosis virus type-specific antigen in chicken embryo fibroblasts after fixation in periodate-lysine-paraformaldehyde was tested by the indirect immunoperoxidase method. Antigen that had been steeped in 0.015M Tris-HCl saline buffer solution containing 20% bovine serum and 30% glycerin was preserved at -20 degrees C, 4 degrees C and room temperature without loss of its antigenicity as long as 16 weeks. It was also stable against heat treatment at 56 degrees C, acid treatment and ethyl ether treatment.     

Use of skin biopsies for assessing levels of organochlorines in walruses (Odobenus rosmarus)

Skin and blubber samples of ten adult male Pacific walruses (Odobenus rosmarus divergens) from Alaska were used to investigate the relationship between organochlorine (OC) levels in skin and blubber of individuals. For analyses we selected 11 components that were quantified in the blubber of all individuals: hexachlorocyclohexanes (αHCH and βHCH), the DDT (dichlorodiphenyltrichloroethane) metabolite p,p′DDE, oxychlordane, and 7 individual PCB congeners, 28, 99, 105, 118, 138, 153 and 180. The correlation between the levels in the two types of tissues was significant and the relation was isometric for all components. The regression coefficient between levels in blubber (dependent variable) and levels in skin (independent variable) was different from 1 for only four of the components. The mean levels in the two types of tissues were significantly different for 3 of the 11 chemical components (βHCH, oxychlordane, and PCB28). Although this analysis is based on only ten individuals, we propose that skin samples taken by biopsy darts can be used to monitor OC levels in walruses. In August 1993 skin biopsies were collected from 25 adult male Atlantic walruses (O. r. rosmarus) at haul-out sites in southeastern Svalbard in the Norwegian Arctic and from 28 walruses of different sex and age at haul-out sites at Franz Josef Land in the Russian Arctic. The mean levels of OCs were 2–10 times higher at Svalbard than at Franz Josef Land. The dominant OC component was PCB153 in both areas. A principal component analysis detected differences between areas in OC levels but not in patterns. Since the Franz Josef Land samples were mainly taken from females and young individuals and the Svalbard samples were taken largely from adult males, we believe the differences in tissue OC levels observed from these areas can be explained by differences in sex and age of the walrus sampled. Comparable organochlorine levels in skin samples from walruses from other areas are not available. However, compared to the corresponding OC levels found in walrus blubber in other areas, the OC levels from Svalbard and Franz Josef Land are higher. The high levels of OCs in walruses from Svalbard and Franz Josef Land may be a combined effect of high pollution level in the environment and seal-eating habits. In the present study we show that it is possible to use skin biopsies taken by a non-destructive method to assess OC levels in walruses. Accepted: 24 October 1999     

The extraction of polymeric collagen from biopsies of human skin

1. Medium sized biopsies (100 mm2) of human skin from 14 subjects yielded sufficient polymeric collagen for depolymerisation and ultrastructural investigations. 2. The yields obtained from one skin specimen by the alpha-amylase, EDTA and lyotropic relaxation (water) methods of extracting polymeric collagen are similar. 3. The responses to depolymerisation treatments of the three polymeric collagen samples extracted by each of the three methods from one skin specimen are cross-correlated. There are however electron microscopical differences between the three polymeric collagen samples. 4. The results show that it feasible to study the polymeric collagen of normal and diseased human skin from medium sized biopsies.     

The complement fixation test in vaccinia with antigens from the brain,the testis and the skin

Summary Eight antigens, prepared from the brain, the testis and the skin of rabbits after the tissues had been inoculated with vaccinia virus, have been compared in the complement fixation test with vaccinia-immune-sera. It was rare that a serviceable antigen could be prepared from the brain. Antigens from the testis and the skin were always active, but the activity of the former surpassed that of the latter. The best results were obtained with a saline extract 1 : 100 of freshly removed testis; when frozen this extract may be kept for some weeks. In the sera of vaccinated rabbits and of a monkey complement fixing antibodies could be detected regularly, even 3 months after vaccination. In sera of men, who had been revaccinated more than 3 months earlier, this was only rarely the case.     

Measurement of chemopreventive efficacy in skin biopsies

OBJECTIVE: To explore methods suitable for quantitative assessment of the efficacy of chemopreventive intervention. STUDY DESIGN: High-resolution imagery of nuclei from the suprabasal and basal cell layers of sun-damaged skin were recorded. There were 10 cases. A shave biopsy was taken from an area of clearly evident solar keratosis before and after treatment with 2-difluoromethyl-dlornithine (DFMO) and from the colateral forearm, treated with a placebo. A number of karyometric variables were computed and combined to derive marker features that provided a numeric measure of the degree of nuclear deviation from normal. RESULTS: DFMO treatment was effective overall in reducing the degree of nuclear abnormality seen in the biopsies; in 8 of the 10 cases there was a significant improvement. The placebo-treated arm did not show a statistically different abnormality from the untreated arm. CONCLUSION: Karyometric analysis can provide numeric measures that allow documentation of statistically significant regression of actinic keratotic lesions following treatment with DFMO.     

Influence of fixation and decalcification on the immunohistochemical staining of cell-specific markers in paraffin-embedded human bone biopsies

A number of fixation and decalcification procedures were evaluated to determine their suitability for immunohistochemistry on trephine samples of bone marrow after paraffin embedding. In particular, the immunoreactivity of antigens characteristic for various hematopoietic cell lines (immunoglobulin heavy and light chains for plasmacytoid cells; elastase for neutrophil myeloid cells; lysozyme, alpha-1-antitrypsin and alpha-1-antichymotrypsin for hystiocytic cells; leukocyte common antigen for lymphocytes; hemoglobin and glycophorin A for erythroid cells; Factor VIII-related antigen for thrombocytoid cells) as well as some antigens specific for epithelial tumors (CEA, 115D8, and keratin) were investigated. Fixation in a mercuric chloride-formaldehyde mixture followed by decalcification in acetic acid-formaldehyde-saline proved to be the best procedure for antigen preservation and retention of morphologic detail. Moreover, there is no need of trypsinization when using this procedure. The only exception was Factor VIII-related antigen in megakaryocytes, which was best demonstrated in trypsin-digested sections of formalin-fixed and acetic acid-decalcified biopsies.     

Assessment of chemokine profiles in human skin biopsies by an immunoaffinity capillary electrophoresis chip

Atopic dermatitis is a skin condition resulting in a skin rash from exposure to environmental factors. Skin biopsies taken from patients suffering from atopic dermatitis were micro-dissected and analyzed using a microchip-based immunoaffinity CE system for the presence of CXCL1, CXCL5 and CXCL8 and CCL1, CCL3 and CCL5 chemokines. Disposable immunoaffinity disks with immobilized antibodies were used to capture the CXC and CC chemokines from the homogenized skin samples. The captured analytes were then labeled with AlexaFluor 633, eluted from the disk and separated by CE. The labeled chemokines were identified and quantified by laser induced fluorescence. The total analysis time was less than 40min, including the biopsy microdissection, pre-analysis preparation of the sample and the ICE-CHIP analysis, which took less than 10min with inter- and intra-assay CV's below 6.4%. Microchip-based immunoaffinity CE could distinguish between normal skin biopsies and those with inflammation. Patients with neutrophil cellular infiltrates by histopathology showed increased concentrations of CXCL1, CXCL5 and CXCL8 while increases of CCL1, CCL3 and CCL5 corresponded to the patient group demonstrating monocytic and T-lymphocyte infiltration by histopathology. This system demonstrates the ability to identify and quantify immunochemical analytes in frozen sections taken from clinical histopathology samples.     

Use of a gelatin substrate for culturing diploid human skin fibroblasts

Gelatin coating of the growth surface of commercially available polystyrene Petri dishes is proposed for successful cultivation of human skin fibroblasts. A comparison of culture growth dynamics on gelatin, glass and polystyrene allowed to recommend gelatin as the substrate in order to receive an abundant similar material with a low population density viability. The absence of changes in carbohydrate metabolism changes during cell culturing on gelatin provides an opportunity to use these cells in studying regulation of carbohydrate metabolism.     

Use of SMART-generated cDNA for gene expression studies in multiple human tumors

We demonstrate here that SMART PCR-amplified cDNAs arrayed on a nylon membrane are suitable for high-throughput tissue expression profiling when starting biological materials are limited. We show that SMART cDNA accurately reflects gene expression patterns found in total RNA by comparing the expression level of several target genes in SMART PCR-amplified cDNAs and their corresponding total RNAs. We also arrayed cDNAs from 68 matched tumor and normal samples on a nylon membrane to determine whether SMART PCR-amplified cDNA could be used for detecting differentially expressed genes in these tissues. These arrays containing normalized tumor and normal cDNAs were hybridized with probes for glutathione peroxidase and gelsolin. The hybridization results revealed cancer-related and patient-specific gene expression differences between tumor and normal tissues for these genes. These studies show that SMART PCR-amplified cDNAs maintain the complexity of the original mRNA population and are thus suitable for high-throughput studies to compare the relative abundance of target genes and to detect differentially expressed genes in a wide variety of tissues simultaneously.     

Stimulation of skin's energy metabolism provides multiple benefits for mature human skin

As an organism ages, there is a decline in mitochondrial function and cellular energy balance. This decline is both accelerated by and can cause the formation of reactive oxygen species (ROS) that damage nuclear and mitochondrial DNA, lipid membranes as well as structural and catalytic proteins, especially those involved in energetic pathways of cells. Further, ROS have also been linked to some of the detrimental skin changes that occur as a result of photoaging. We have previously shown that levels of Coenzyme Q10 (CoQ10), a component of the respiratory chain in mitochondria, are reduced in skin cells from aging donors, and that topical supplementation can ameliorate processes involved in skin aging. Creatine is another important component of the cellular energy system and phosphocreatine, its phosphorylated form, functions as a reservoir for high energy phosphates. Unfortunately the creatine system and thus the energy storage mechanism in skin are negatively affected by aging and conditions of oxidative stress. This article reviews some of our in vivo data about the synergistic effects of combining a stabilized form of Creatine with CoQ10 and clearly depicts their beneficial effects as active ingredients in topical formulations.     

An improved method for the demonstration of fibrin in biopsies of rabbit skin homografts

Summary Tissues embedded in resin are convenient for routine use when the presence or absence of fibrin in them is to be confirmed using the electron microscope. To visualize fibrin using the light microscope, sections (1.0–2.0 m) from such specimens should be stained with Methylene Blue-Azure II-Basic Fuchsin (MBBF). Staining with MBBF is more controllable than with other methods and it requires only two short staining steps. Compared with Giemsa, MBBF provides a polychromatic, as opposed to a monochromatic end-result, sharply contrasting fibrin (blue) against collagen (pink-violet).     

Use of monoclonal antibodies to follow the phylogenic distribution of human renal antigens

Nine human differentiation antigens have been defined by monoclonal antibodies (M. Abs) developed from mice immunized with embryonic kidney cells (mesonephros or metanephros of 7 week-developmental ages). Their spatial and temporal distributions during human kidney organization were previously studied [3]. In this paper we have attempted to follow by immunofluorescence their phylogenic location, from fish to mammals. Six of them recognized the same structures as in humans: proximal convoluted tubules (PCT) (EG9.11, EG19.6, E116.1), glomerular basement membrane (GBM) (EG14.1) and extracellular matrix (EK8.1, EK17.1). However, staining was limited to certain mammals. EK17.1 has been characterized as an anti-fibronectin. These antibodies revealed the same histological structures in the human mesonephros and metanephros. The three other antibodies revealed epitopes appearing earlier in evolution and whose histological distribution varied according to species. These antibodies stained different structures in the mesonephros and metanephros. Thus, the staining particularities observed during human renal ontogenesis were found again in the phylogenetical study.