Characteristics of human neutrophils obtained by the chamber method of "skin window"

Viability, morphology and function of healthy human cells migrated into the chamber through the "skin window" were studied. After 18 to 20 hours there were 42.0 +/- 5.3 (x 10(6))/cm2 viable cells characterized by a high content of mature neutrophils (98.6 +/- 0.6%). Normal reactivity of migrated neutrophils was observed using the nitroblue tetrazolium reduction test. The chamber variant of the "skin window" method is recommended as a simple physiological technique for preparing pure populations of human neutrophils.     

The effect of local UVB skin irradiation on the rate of formazan deposition in the epidermis of hairless mice studied by means of a tetrazolium-reduction method

One-hundred-and-twenty hairless mice were irradiated with UVB (310 nm, exposure 60 mJ/cm2) on a limited area of the dorsal skin. At different time intervals after irradiation, the rate of endogenous dehydrogenase activity per mg dry epidermis was measured by the tetrazolium reduction method. The amount of formazan deposited remained normal for 18 h, and then increased, reaching a peak significantly higher than normal at 24 h, and thereafter returned to normal. At day 8 there was a new, probably significant peak. The reaction was followed for 14 days. It was concluded that UVB irradiation provokes a period of increased formazan deposition in the epidermis, similar to what has been observed after ionizing radiation and chemical carcinogens. The validity of the tetrazolium test for skin carcinogenic irritaments was thus also confirmed.     

The phagocytic activity of the neutrophilic granulocytes in patients with chronic candidiasis of the skin and mucous membranes]

24 patients with chronic candidiasis of the skin and mucous membranes have been examined. The following parameters have been evaluated: (1) ingestive capacity, (2) fungicidal capacity, (3) the state of the myeloperoxidase mechanism of respiratory explosion. In chronic candidiasis of the skin and mucous membranes the ingestive capacity of neutrophil granulocytes has been found to remain unchanged, constituting 70 +/- 3.5% in healthy persons and 60 +/- 4.2% in patients 60 minutes after interaction. The study has revealed the decrease of fungicidal activity in 50% of the patients which is probably linked with the constant activation of oxygen-dependent killing mechanisms (the index of the spontaneous nitro blue tetrazolium test has proved to be 8.9 +/- 0.8% in healthy persons and 15 +/- 3.4% in patients with candidiasis of the skin and mucous membranes) and the decreased reserve of the functional activity of neutrophil granulocytes. The activity of myeloperoxidase in patients with candidiasis of the skin and mucous membranes has been found to remain unchanged.     

The role of quality control in a skin bank: tissue viability determination.

New surgical procedures requiring viable skin have increased rapidly over the last few years. The cell viability assessment in allograft skin is a major step forward in burn treatment, since it is well-known that taking is correlated with grafted tissue viability. Various methods, both qualitative and quantitative, are currently used. Although qualitative assays (histomorphology, immunocytochemistry) are routinely performed in our laboratory, there arose a need to set up a standardised quantitative assay in an attempt to obtain a cut-off value so that the skin sample could be determined valid or not for grafting. Therefore, two different tetrazolium salt compounds MTT and WST-1, were compared in order to determine their efficacy in the evaluation of tissue viability. Several experimental conditions were analysed: 1- cellular cultures of keratinocytes and fibroblasts, 2- fresh skin tissue samples, 3- the same specimen tested daily for at least 2 weeks, 4- after cryopreservation and thawing. Viable cells were analysed by the cleavage of tetrazolium salts to formazan by cellular enzymes. The formazan dye produced by metabolically active cells was then quantified by measuring the absorbance of the dye solution at the appropriate wavelength. It was seen that WST-1 is easier to handle, more stable, has a wider line arrange, accelerated colour development and is more sensitive than MTT on fresh specimens and cell suspension. However, after 72 hours of storage at 4°C, most of the WST-1 tested specimens no longer gave any absorbance signal, whilst MTT specimens were seen to give a signal for more than two weeks. Moreover, after thawing WST-1 tested samples were almost negative,whilst MTT samples continued to give strong signals. In conclusion, WST-1 assay offers rapid and precise results as to the cell viability of fresh allografts and cell cultures, whilst the MTT method is much more useful in establishing viability after long conservation and cryopreservation. In our clinical experience, allografts transplanted at 72 hr post-harvesting or after cryopreservation showed a mean of take more than of 80%, demonstrating that the MTT system is more reliable for the determination of allograft viability. Studies are ongoing with larger clinical cohorts to establish the precise cut-off value for skin graft validation. This revised version was published online in July 2006 with corrections to the Cover Date.     

An evaluation of the polyacrylamide gel electrophoresis fractionation method for the production of Mycobacterium tuberculosis skin test preparations. II. Comparative skin testing on guinea-pigs

As part of a cooperative inter-laboratory WHO supported project for the fractionation of Mycobacterium tuberculosis skin test preparations, four fractions (designated 15, 7, 4.75 and 3.5%) were evaluated by comparative skin tests on sensitized guinea-pigs. The 7% fraction was the most potent in both homologously and heterologously sensitized animals, and the 4.75% and 3.5% gel fractions showed the lowest activity. Significant levels of cross-reactivity in guinea-pigs immunized with M. bovis BCG, M. kansasii, M. avium and M. intracellulare were demonstrated for all fractions examined, thus reflecting the antigenic relationships among these mycobacteria. These four fractions may qualify as starting material for further studies aiming at a reduction of skin test cross-reactivity.     

Gene expression in sheep skin and wool (hair)

We sequenced 2939 ESTs from fetal and adult sheep skin. Stages of gestation were picked to coincide with the major events in skin appendage (wool follicle) formation. Clustering analysis generated a nonredundant set of ESTs 2435 strong (83% nonredundant). Approximately 24% of these gave no hit to NCBI build 29 of the human genome, while 35% were tentatively classified by putative function based on BLASTX hits with a p(N) of <10(-4). In addition to bioinformatics analysis of our ESTs and gene mapping, we have generated a large EST spatial expression data set using in situ hybridization. One thousand one hundred forty-two ESTs have been used for in situ localization; about 31% are from adult sheep skin, 39% from late gestation fetal sheep skin, and 30% from midgestation fetal sheep skin. These probes have been used in over 3000 hybridization experiments. In this report, we summarize the results of in situs on adult sheep skin.     

Collagen from diamondback squid (Thysanoteuthis rhombus) outer skin

Collagens (acid-solubilized and pepsin-solubilized collagens) were prepared from diamondback squid outer skin and partially characterized. The yields of acid-solubilized and pepsin-solubilized collagens were about 1.3 and 35.6%, respectively, on a dry weight basis. Pepsin-solubilized collagen was heterotrimer with a chain composition of ala2a3. The patterns of peptide fragments were different from that of porcine skin collagen. Denaturation temperature was 27.5 degrees C, about 10 degrees C lower than that of porcine collagen. The amino acid composition of pepsin-solubilized collagen from diamondback squid outer skin was similar to that from cuttlefish outer skin. This squid is big among squid species, and its skin is thick. It is clear that diamondback squid outer skin has a potential as an alternative source of collagen to bovine skin and bone. At present, collagen using aquatic materials such as skin (cod and a deep-sea fish) and scale (sea bream and anchovy) is the development stage in the related industries. Unless the problem of BSE infection in land animals is resolved aquatic materials as an alternative source of collagen will attract much attention in the cosmetic and medical fields.     

Enhancement of nitroblue tetrazolium dye reduction by leucocytes exposed to a factor released from sensitised lymphocytes.

Leucocytes from purified protein derivative (PPD) skin test positive individuals, after exposure to PPD in vitro, show a significant increase in nitroblue tetrazolium (NBT) dye reduction when compared to leucocytes from PPD negative donors. The active principle was shown to be soluble, and was released from lymphocytes after a rapid, immunologically specific interaction with the sensitising antigen.     

DNA-synthesis with methylated poly(dA-dT) templates: possible role of O4-methylthymine as a pro-mutagenic base.

The alternating copolymer poly(dA-dT) has been methylated with either dimethyl sulphate (DMS) or N-methyl-N-nitrosourea (MNU) and the levels of the various methylation products determined. In addition to the methylated adenines formed by both methylating agents, MNU resulted also in the formation of 3-methylthymine, O4-methylthymine and phosphotriesters. The methylated polymers have been ution of complementary and non-complementary nucleotides determined. With the DMS methylated template no wrong nucleotide incorporation was detectable, but with the MNU methylated polymer the incorporation of dGMP was observed. The amount of dGMP incorporated correlated with the level of O4-methylthymine in the template over the range of methylation studied. The results indicate that O4-methylthymine is capable of miscoding on a one-to-one basis while the products of DMS methylation (1-, 3- and 7-methyladenines), and also possibly the phosphotriesters, do not lead to any misincorporation.     

Methylnitrosourea as challenge mutagen in assessment of the DNA mismatch repair (MMR) activity: Association with some types of cancer

Total repair capability is a widely used phenotypic marker of predisposition to cancer. Evaluation of this parameter implies using a challenge mutagen in an in vitro system to unmask latent genetic instability and repair insufficiency in the target cells. Traditionally, these investigations involve two tests, evaluation of mutagenic susceptibility (chromosomal aberrations) and genotoxic effect (DNA comet assay). The present study was focused on analysis of the effect of methylnitrosourea (MNU) on resting and PHA-stimulated lymphocytes from healthy donors and patients with gynecological cancer. Cytotoxic effect of MNU (apoptotic lymphocyte death) was estimated using two parameters, interaction of the cells with the annexin V-FITC complex, and morphological changes of the nuclei after their staining with the mixture of two DNA-tropic dyes. The genotoxic effect of MNU, namely, secondary double-strand DNA breaks, was scored using the neutral comet assay, modified for the calculation of the comets produced exclusively by BrUdr-labeled proliferating lymphocytes. The proportion of these comets was represented as the proliferative cell index. It was shown that resting lymphocytes were resistant to genotoxic and cytotoxic effects of MNU. The response of proliferating cells to the action of MNU was expressed as the development of secondary DNA breaks (P < 0.01), along with the increased frequency of apoptosis (P < 0.05). The genotoxic effect of MNU on stimulated lymphocytes of gynecological cancer patients was fourfold lower compared to healthy donor lymphocytes. In response to the MNU action, patient lymphocytes did not change their proliferative index, while in healthy donor lymphocytes proliferative index was two times decreased in response to the MNU action. The data obtained pointed to the association between the cytotoxic response of the lymphocytes to the action of MNU and gynecological cancer. Since only proliferating lymphocytes response to the genotoxic effect of MNU, and the effect is revealed a day after the mutagen action, it is suggested that this phenomenon is associated with postreplicative repair, MMR, the substrate of which is O⁶-methylguanin. The MMR deficiency in patient lymphocytes determines their tolerance to the action of MNU. Genotoxic response of lymphocytes to the action of MNU can serve as a marker of MMR, as well as of the MMR deficiency-associated gynecological cancer.     

Disruption of DNA synthesis and structure of mouse L1210 leukemia cells, sensitive and resistant to 1-methyl-1 nitrosourea and 1,3-bis(2-chloroethyl)-1-nitrosourea in vivo

Leukemia L1210 cells with acquired resistance to 1-methyl-1-nitrosourea (MNU) (L1210/MNU) and 1.3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (L1210/BCNU) were developed from leukemia L1210 cells sensitive to these drugs (L1210/0). The modal chromosome number of leukemia L1210/MNU and L1210/BCNU cells increases from 40 (L1210/0) to 41. It was shown that in leukemia L1210/MNU cells the inhibition of DNA synthesis after MNU administration in a therapeutic dose (80 mg/kg) is lasted within 24 hours, while that in leukemia L1210/0 cell--within 96 hours. After administration of BCNU (20 mg/kg) inhibition of DNA synthesis in leukemia L1210/BCNU cells reached of 50% of control in comparison with practically complete inhibition of DNA synthesis in leukemia L1210/0 cells. Centrifugation on alkaline sucrose density gradients revealed no differences in the rate of sedimentation of leukemia L1210/0, L1210/MNU and L1210/BCNU cell lysates. After 1 hour treatment with MNU of mice bearing L1210/MNU and L1210/0 leukemia cells single-strand breaks in DNA were determined. After 4 hours these strand-breaks retained in leukemia L1210/0 cells, but were eliminated in leukemia L1210/MNU cells. Administration of BCNU to mice with leukemia L1210/0 and L1210/BCNU cells resulted in both cases in the production of DNA aggregates. There is no complete cross-resistance between MNU and BCNU which allows a substitution of these drugs providing for the increase in their therapeutic efficiency.     

Improved assessment of viability and germination of Cattleya (Orchidaceae) seeds following storage

Effective seed storage methods for orchid species provide a unique opportunity to preserve large amounts of germplasm. Early evaluation of the physiological status of seeds is useful for selecting seed lots for conservation. To score seed quality, seeds from nine Cattleya species (Cattleya granulosa, Cattleya hegeriana, Cattleya intermedia, Cattleya mossiae, Cattleya purpurata, Cattleya sanguiloba, Cattleya tenuis, Cattleya tigrina and Cattleya walkeriana) were germinated on Knudson C medium and viability was measured using a tetrazolium (TZ) test following pre-conditioning in a 10% sucrose solution, both before and after storage at −18°C for 3 mo. Tetrazolium test results were quantified using digital imaging of germinating seeds on a flatbed scanner. Seed viability (with or without pre-conditioning in sucrose), germination, Germination Velocity Index (GVI) and the correlation between viability measurements and germination were also evaluated. Both the tetrazolium test and the germination results showed that there was no loss of viability after storage, although in five seed lots, the GVI decreased. Pre-conditioning improved the correlation between viability exhibited in the tetrazolium test and seed germination. Image analysis proved to be a powerful tool for analysing these small seeds.     

Surface secretions of the skin of Blennius (Lipophrys) pholis L.

Mucus is secreted to the surface of the body and fin webs of Blennius pholis by superficial epithelial cells and by goblet cells. Some goblet cells secrete sulphated acid glycoproteins, others produce a mucus which is neutral or mixed in its reactions. The superficial epithelial cells of these areas secrete sulphated acid glycoproteins, seen by electron microscopy as electron-lucent or moderately lucent vesicles; this secretion is not normally visible external to the skin in transmission electron microscope (TEM) sections. These cells do not react to the bromphenol blue test for proteins. Over part of the surface of the pelvic fins and the distal parts of the rays of the pectoral fins, the skin contains no goblet cells and bears a thick external secretion, or cuticle, containing protein and glycoprotein which is mainly neutral in reaction, although some cells at the edges of the region secrete weakly sulphated or non-sulphated acidic glycoprotein. The protein content of the columnar superficial epithelial cells of these regions correlates with the fibrous nature of the secreted cuticular layer as seen by TEM; the columnar cells are characterized by extensive ribosomal endoplasmic reticulum and vesicles which stain darkly with phosphotungstic acid, less so with uranyl acetate. The distal part of the cell, containing these vesicles, reacts positively to the PAS stain. In some places the borders of the zones with fibrous cuticle are characterized by cuboidal superficial epithelial cells which give a strong positive reaction to alcian blue at pH 1.0, indicating the presence of sulphated acid glycoproteins, but also react positively to the bromphenol blue test for proteins.     

The skin of primates. XXX. The skin of the woolly monkey. (Lagothrix lagotricha)

The anatomical and histochemical features of the skin of the woolly monkey are intermediate between those of the Cercopithecoidea and the Pithecoidea. The animal has a prehensile tail, the glabrous, friction surface of which is similar to that of the fingers. The epidermis is heavily pigmented. The dermal vascularization is relatively well-developed and similar to that of the skin of the Cercopithecoidea. Hair follicles grow in groups of 4 to 15, as in the skin of the Pithecoidea. In the hairy skin, eccrine sweat glands occur only in the tail and genitalia. The woolly monkey, like the green monkey, possesses only acetylcholinesterase-containing nerve fibers around its eccrine sweat glands.     

Factitious (self-inflicted) skin wounds.

We represent 4 of our recent cases of factitious skin wounds. The general aspects of these wounds--including etiology, diagnosis, detection, and treatment--are discussed.     

Microflora associated with the skin of the bowhead whale (Balaena mysticetus)

A study of the microbiological flora isolated from cultures of normal and lesional skin tissue samples collected from 19 bowhead whales (Balaena mysticetus) over a 4 yr period is presented. These cultures were obtained from 30 tissue samples (17 normal, 13 lesion) and 248 swab samples (157 normal, 91 lesion). Seven hundred-thirty bacterial and yeast isolations were made (285 normal, 445 lesion). Distribution revealed that 56% of the gram positive bacterial isolates, 75% of the gram negative bacterial isolates and 64% of the yeast isolates recovered were associated with lesional skin. It was found that 80% of one group of Corynebacterium sp. isolates, 90% of the Acinetobacter sp. isolates and 94% of the Moraxella sp. isolates were associated with lesional skin. Although the primary yeasts recovered were Candida spp., they were found on both normal and lesional skin. Enzymatic assays of isolates from normal and lesional skin demonstrated production of enzymes capable of causing necrosis. The majority of the microorganisms recovered were facultative anaerobes and many of them could be considered potential pathogens of mammalian hosts.     

Cryopreservation of skin using a murine model: validation of a prognostic viability assay

In order to evaluate the many variables that can affect cryopreservation success, a simple, highly reproducible model system is required. We have evaluated the use of tetrazolium reductase activity as a prognostic indicator of skin viability in an inbred murine model. Two inbred hairless mouse strains were characterized in studies on autografting and allografting following different skin-storage protocols. Skin tetrazolium reductase (TR) activity correlated well with oxygen consumption, and with graft success--the ultimate performance criterion--following varying degrees of cryogenic injury. The assay was shown to be highly reproducible. In a series of factorial experiments the only factors affecting TR activity were those concerning the mouse donors, i.e., mouse strain, age, sex, and body area. The effects of these factors on TR activity were fully characterized.     

Tetrazolium [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction by mycoplasmas.

We investigated 22 mycoplasma and acholeplasma species for their ability to reduce tetrazolium salts by using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The test results were evaluated visually, as well as spectrophotometrically, by using an enzyme-linked immunosorbent assay reader. Our results were very similar to the results obtained when the tetrazolium salt reduction assay described by Aluotto et al. was used. However, the MTT reduction assay appeared to be better because it is faster, more objective and sensitive, easier to evaluate, and less expensive; in addition, it allows quantitative determinations. By using regression analysis a linear correlation between formazan production and the number of colony-forming units was demonstrated for all of the species investigated, indicating that the MTT assay can also be used for growth, toxicity, or chemosensitivity tests for the mycoplasma species that are capable of reducing tetrazolium salts.     

Climate change and human skin cancer.

As part of an inventory of potential interactions between effects of ozone depletion and climate change, a possible effect of ambient temperature on sun-induced skin cancers was suggested. Mouse experiments had shown that increased room temperature enhanced ultraviolet (UV) radiation-induced carcinogenesis; the effective UV dose was increased by 3-7% per degrees C. The present investigation was aimed at studying a possible temperature effect on human skin cancer. Existing data on the incidence of human skin cancer were analyzed, as available from two special surveys of non-melanoma skin cancer in the United States. The incidence of non-melanoma skin cancer in the ten regions surveyed not only correlated significantly with the ambient UV dose but also with the average daily maximum temperature in summer. For squamous cell carcinoma the incidence was higher by 5.5% (SE 1.6%) per degrees C and for basal cell carcinoma by 2.9% (SE 1.4%) per degrees C. These values correspond to an increase of the effective UV dose by about 2% per degrees C. Although the precise nature of this correlation with temperature requires further studies, it can be concluded that the temperature rises coming with climate change can indeed amplify the induction of non-melanoma skin cancers by UV radiation in human populations.     

Ellagic acid protects rat embryos in culture from the embryotoxic effects of N-methyl-N-nitrosourea.

Ellagic acid is a naturally occurring plant phenol that has demonstrated anticarcinogenic and antimutagenic activity in several test systems. Given the common proposed etiopathogenic processes of mutagenesis, carcinogenesis, and teratogenesis induced by genotoxic chemicals, the present study was initiated to determine whether ellagic acid would protect rat embryos in culture from the teratogenic effects of N-methyl-N-nitrosourea (MNU). Ellagic acid alone (as used in these experiments; 50 microM in DMSO) was not embryotoxic. Ellagic acid (50 microM) significantly (P less than 0.01) prevented MNU (75 microM)-induced effects including mortality (absence of heart beat), abnormal formation of the cephalic neural tube derivatives, and delayed differentiation as assessed by a morphological scoring system. These embryoprotective effects were dose responsive. Sequential treatment of embryos with ellagic acid followed by MNU in fresh media also was embryoprotective with no diminution of effect. The site at which ellagic acid interrupts the critical teratogenic events induced by MNU is apparently within the embryo and/or placenta. This model of chemical embryoprotection may be useful in determining the role of cell death and/or mutation in the teratogenic mechanism of action of methylating agents.