Human skin biopsies

Frienkel in 1960 (F19) studied normal human skin biopsy specimens in vitro using radioactive glucose as a precursor. Her main findings were that (1) most of the glucose was converted to lactate (at least 75%) ... 

Figure 2. Indirect immunofluorescence staining of human skin biopsies from a patient treated topically with coal tar and a control. Slides were treated with RNase, proteinase K and 4N HCl before staining with antisera 29 (1 50 dilution) and goat anti-rabbit IgG antibody conjugated with fluorescein (IGO). Magnification was 220fold. A.) section from patient B) section from control...

Torma, H., Lontz, W., Liu, W, Rollman, O, and Vahlquist, A (1994) Expression of cytosolic retinoid-binding protein genes in human skin biopsies and cultured keratinocytes and fibroblasts Br. J Derm. 131,243-249. 

Kroll et al. used X-band spectral/spatial EPRI together with pH-sensitive nitroxide probes to study the microacidity of rat and human skin biopsies. A dose of nitroxide solution was placed on the skin surface and allowed to penetrate into the sample prior to the commencement of EPRI measurements. Magnetic field gradients were applied perpendicular to the skin surface, in order to allow spatial discrimination within the various layers of the skin. Local pH was deduced from the spectral axis of the spectral/spatial data, by determination of the hyperfine coupling constant, which is pH-sensitive in these compounds. Plots of pH versus depth into the skin could be produced, and the shape of the curves was found to change under the influence of topical drug treatment. This preliminary study showed the potential for one-dimensional pH imaging in the skin, a tool that is likely to be of use to the pharmaceutical industry. 

Kadotanl, T. Sato, H. Ohama, K. and Takahara, H. "A Technical Note on the Antenatal Chromosome Analysis by Transabdominal Fetal Skin Biopsy". Jap. Jour. Human Genet., (1972), 16, 42-47. 

Human skin fibroblasts are cultured from skin biopsy samples. The dermis is cut into small pieces (0.5 mm on each side) and placed in a dish containing DMEM, 10% (v/v) FCS, and 1% (v/v) antibiotic-antimycotic solution. When these primary cultures are confluent they are split into novel dishes. Cells between passages three and six are used for experiments. 

Human skin fibroblasts are cultured from skin biopsy samples. The dermis is cut into small pieces (0.5 mm on each side) and placed into a dish in DMEM containing 10% (v/v) FCS and 1% (v/v) antibiotic-antimycotic solution. When these primary cultures are confluent they are split and cells between passage three and six are used for experiments. For the cholesterol efflux assay, cells are grown in 24-well plates to 60-80% confluence and are labeled with [1,2-3H]-cholesterol (1 pCi/well) for 24 h. Cells are then washed with DMEM and incubated for 4 h at 37°C with DMEM containing BSA (0.2%, v/v) and either 0 (negative control) or 5-30 pg/ml apoA-I. The efflux medium is collected and centrifuged to remove cell debris. Cells are solubilized in 0.1 mol/1 NaOH and the radioactivity in the efflux media and in the cell lysates is determined by scintillation counting [11, 30, 75]. 

An 84-year-old woman with polymyalgia rheumatica and a 79-year-old woman with undifferentiated connective tissue disease and leukocytoclastic vasculitis were given prednisolone 20 mg/day with subsequent dosage reductions. The first patient developed a raised purpuric rash and lymphedema of the left leg within 5 months and the second developed large purple nodules on the soles of her feet and the backs of her hands accompanied by periorbital and peripheral edema. Skin biopsies showed Kaposi s sarcoma, and both patients had raised IgG antibody titers to human herpesvirus-8. 

Human skin specimens were obtained from reduction abdominoplasty (tummy tuck) and used to generate acute wounds as described elsewhere [42]. A 3 mm biopsy punch was used to create an acute wound. Skin specimens were maintained at the air-liquid interface at 37°C, 5% CO2, and 95% relative humidity for 6-7 days. 

Figure 7.3 A. Human psoriatic tissue (200x). B. Normal human skin (200x). Both samples were immunostained with caspase 14 antibody. Punch biopsies (4 mm) of normal and psoriatic skin samples were obtained from different patients who provided written, informed consent under an IRB-approved protocol. Samples were fixed in 10% neutral-buffered formalin, paraffin embedded, cut to 5-pm sections, and stained with caspase 14 antibody by immunohistochemistry.

Miyamoto et al. have also demonstrated in the dry skin and itch mouse model (water + acetone ether treated) that the scratching response can be inhibited by the use of atropine, a nonspecific muscarinic acetylcholine receptor (mAChR) antagonist, and 4-diphenyl-acetoxy-N-methyl-piperidine (4-DAMP), an M3 mAChR antagonist.32 They further showed that Mi and M2 mAChR antagonist were not able to inhibit the scratch response. This report suggests the role of acetylcholine, and the M3 specific receptor as a potential player in dry-skin-associated pruritus. In addition, skin biopsies in human subjects with atopic dermatitis were found to have increased levels of acetylcholine compared with normal controls, which suggests that abnormal concentrations of neurotransmitters may also be involved in itch secondary to xeroderma.33... 

These cells have all been grown under conditions which vary the number of PDs per passage, and at 5% or 10% CO2 in the incubator, and samples frozen at each passage up to senescence. Standard protocols are also used for the initial isolation of the cells from human skin, cartiledge and limbal biopsies. 

Evaluation must distinguish between primary irritation responses, which may disappear within a couple of days, and sensitization responses which may develop more slowly, persist longer, and are characterized by pruritis, erythema, edema, papules, vesicles, bullae, or a combination of these. Further identification of sensitization reactions may involve microscopic examination of skin biopsy samples. Some issues which may be encountered in human studies include reactions early in the induction phase which may be indicative of preexisting sensitization to the test material, or a delayed response at 192 h instead of at 48 or 96 h. Follow-up of subjects not completing the study may yield valuable information on the adverse effects of a preparation. 

Experiments by Bickers and Kappas (1978), Li et al. (1995) and Genevois et al. (1998) have examined the role of AHH and cytochrome P450 in the metabolism of coal tar products. A coal tar solution (crude coal tar diluted to 20% with ethanol and polysorbate 80) was applied to clinically unaffected skin of three patients with severe atopic dermatitis and six patients with generalized psoriasis (Bickers and Kappas 1978). Another skin area at least 10 cm away was not treated or was treated with 100 mL of the vehicle alone. Twenty-four hours later, a 6-mm punch biopsy was obtained from coal tar treated and control areas and the effect on AHH activity was determined. Application of coal tar to the skin caused induction of cutaneous AHH activity that varied from 2.4-5.4-fold over the enzyme activity in untreated skin areas. There were no sex differences in inducibility between patients with psoriasis and patients with atopic dermatitis. Relative inducibility of human skin AHH by coal tar did not appear to be a function of the basal level of the enzyme. 

By combining swab and scrub test methods on the same site, differential identification of both surface and subsurface microorganisms can be obtained [60]. A study by Chevalier [18] compared results of the scrub method to results of aerobic counts obtained from biopsies with remarkable similarity in data sets. From detailed studies using the scrub test method, the percentage of different species and types can be calculated for various parts of the body [107]. Due to the aggressiveness of this method, skin damage inevitably occurs and some studies of the human skin, under dynamic conditions, are precluded. 

A 68-year-old man with type 2 diabetes treated with insulin and oral hypoglycemic agents developed pruritic plaques of more than 15 cm diameter at the site of his insulin injections. Skin biopsy showed an Arthus type reaction. Various insulin therapies, including insulin glargine, insulin detemir, and human insulin, produced the same response. Intra-dermal tests were positive to a variety of insulins and protamine. He was desensitized using subcutaneous human insulin and orally fexofenadine 180 mg bd and was then successfully treated with insulin glargine. The fexofenadine was stopped 6 months later. 

FIGURE 47.1 Histological comparison of native skin and cultured skin substitute prepared for treatment of pediatric bum patient, (a) Native human skin, (b) Cultured skin substitute in vitro, shown after 6 days of incubation. Graft was transplanted to patient after 10 days of in vitro incubation. (c),(d) Healed autograft (c), and healed cultured skin substitute (d), biopsied 3 weeks after grafting to excised full-thickness burn wounds. Scale bars = 0.1 mm. 

NIR-FT-Raman spectra of skin from pig ear, guinea pig and mouse were recorded with excitation at 1064 nm and compared to spectra of human skin. The R(V )-representation was used to eliminate the intense Rayleigh band. The total water content in each sample was estimated from the intensities of the OH-stretching vibrations at about 3200 cm. A low-wavenumber band around 180 cm (- 5.5 terahertz) was characteristic of a bulk-like liquid water structure. Water content and structure in skin from pig ear, guinea pig and human were similar and different from mouse skin. Differences in loss of bulk water were observed for skin samples after freezing and thawing. Skin biopsies of human skin with various skin tumors showed an increase of water with a bulk-like structure in skin with malignant skin tumors. 

It is difficult to explain why the loss of water differs by freezing and thawing for different skin samples. Further experiments are necessary. However, knowledge of what happens by freezing and thawing of skin samples is very important, because skin biopsies often are frozen before pathological examination. The water content of animal skin may also be important for selection of animal skin in pharmaceutical skin penetration studies. In laboratory studies animal skin with water content similar to human skin should be chosen. The low-wavenumber Raman spectrum in the R( v )-representation is a way to follow similarities and changes in the content of free water in skin samples. 

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