Biomedical subjects
Publications and source records attributed to Y Kitano.
A 26-year-old woman developed a verrucous papule on the dorsum of the right hand during the treatment of housewive's hand dermatitis. Histological and ultrastructural study showed a typical appearance of Pagetoid reticulosis (Woringer-Kolopp disease).
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We report a case of eccrine sweat gland carcinoma arising from the occipital region, which later metastasized to the regional lymph nodes and spread widely to the viscera. After wide local excision of the primary tumor, it recurred locally, and then metastasized presumably via regional lymph nodes. Widespread metastasis to visceral organs occurred after a ten-year latent period from the appearance of the original tumor. Histochemical and enzymatic histochemical examinations of the specimens from the original tumor and metastasized lesions obtained at autopsy disclosed the characteristics of eccrine sweat gland tumor.
Dispase was used to obtain viable eccrine sweat glands from human skin in an intact shape. The full thickness of human skin was soaked in a solution of dispase in Eagle's minimum essential medium at a concentration of 500 units/ml and kept in a refrigerator at 4 degrees C for 24 h. The epidermal sheet with its appendages could then be easily separated from the dermis by lifting the epidermis with fine forceps. Electron-microscopic observation revealed that the eccrine sweat gland was completely separated froM the dermis at the basement membrane zone. The isolated epidermal sheet was scarcely dissociated by mechanical agitation in the presence of Ca2+ and Mg2+ ions. The eccrine sweat gland was cut away from the epidermis by using microscissors under a stereomicroscope. A cell suspension of the isolated eccrine sweat glands was obtained after trypsinization. The cells remained more than 90% viable up to 48 h in the culture medium. The obtained viable eccrine sweat glands will be useful for the study of the biology of sweating.
Dispase is a bacterial neutral protease which is obtained from the culture filtrate of Bacillus polymyxa. After 24-h treatment of the human skin with 500 and 1000 U/ml dispase, the epidermal sheet was easily peeled from the dermis, and its undersurface retained rete ridges. Electron microscopic observation showed that the basal surface was composed of cells with numerous slender villi and cytoplasmic projections. Although the intercellular spaces of the spinous as well as the basal layers were wide, all desmosomes were intact with their accompanying tonofilaments. An epidermal cell suspension was obtained by incubating the dispase-separated epidermal sheet in trypsin solution for 15 min and the viability of the cells was more than 95% as measured by the trypan blue dye exclusion test. A culture of keratinocytes was established by seeding the dissociated cells in the usual way.
The distribution of cytoplasmic microtubules in cultured guinea-pig keratinocytes was investigated using immunofluorescence (IF) microscopy with monospecific anti-tubulin antibodies and electron microscopy (EM). In culture, adherent cells displayed networks of thin fluorescent fibres, while a homogeneous and/or granular cytoplasmic IF was shown in the cells of upper layers as well as in trypsinized cells. By EM many microtubules were shown in adherent cells but there were fewer or none in the upper layers. An increase in calcium ion (Ca2+) concentration and the addition of an ionophore (X537A) to the culture medium caused disassembly of microtubules. This effect was cancelled by a calmodulin inhibitor. Cryostat sections of normal human and guinea-pig epidermis stained with anti-tubulin antibodies showed a homogeneous and/or granular cytoplasmic IF from basal to granular layers but no detectable IF was seen in the horny layer. These results suggest that keratinocytes contain a cellular pool of tubulin in various states of polymerization and that microtubule disassembly may occur during differentiation, probably being regulated by Ca2+-calmodulin complexes.
The stratified squamous epithelium of the skin, the epidermis, is a renewing cell population. In order for epidermis to remain the same size, each dividing basal cell must produce on an average, one daughter cell that will remain as a germinative cell and another that will perform terminal differentiation (differential mitosis). In order to investigate cell kinetics in the epidermis, the information obtained from in vivo study is limited, and offers only indirect evidence for the determination of cell cycle time and cell proliferation pattern. Keratinocytes in cell culture are unique in formation of a multilayered cellular sheet in which the keratinocytes form a structure resembling the epidermis in vivo, and keratinize at the top. In the early days of the primary culture of human keratinocytes, when the cells proliferate to form a monolayer sheet, direct access to cell proliferation pattern and measurement of interdivision time can be done using techniques of time-lapse cinemicrography. The primary cultures at 8-20 days of incubation were employed for cinemicrographic observation when small polygonal cells appeared in groups, when numerous mitotic figures were observed, and when stratification of the cells had not yet occurred. The appropriate field was marked and followed for up to 6 days. Photographic prints were made from the 16 mm cine film, and dendrograms were made and analyzed for pattern of cell proliferation and interdivision time. Most cells in the field divided two or three times during the period of observation. Sister-sister pairs of the second and third generations divided after approximately the same interdivision times. However, some cells have never divided. Some of the sister-sister pairs differed considerably in their interdivision times. In some cultures synchronous division was quite evident. The average interdivision time was about 26 hr in the majority of cultures, and it is suggested that the estimated long cell cycle time in vivo might be overestimated due to the existence of non-cycling cells in the germinative population.
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An aromatic retinoid Ro 10-9359 is metabolized after absorption from intestine to form Ro 10-1670 which is an active therapeutic compound. The epidermal keratinocytes, a main target tissue of retinoid therapy in dermatology, was examined in the capacity to metabolize the retinoid. The culture of human epidermal keratinocytes was treated with 10(-6) M Ro 10-9359 and the metabolites released in the medium was analyzed by HPLC. The HPLC profile showed a distinct peak of Ro 10-1670. The human skin fibroblasts, HeLa cells. Chang liver cells and 3T3 cells were less active in metabolizing Ro 10-9359, and only a small amount of Ro 10-1670 was detected in the culture of human skin fibroblasts treated with 10(-6) M Ro 10-9359 for 2 days. When these cells were disrupted by a glass homogenizer, and incubated with Ro 10-9359, no Ro 10-1670 formation was detected.
A leukocyte chemotactic factor with a molecular weight of around 12,500 daltons is found in large amounts in psoriatic scales. Except for complement-activated serum none of the extracts prepared from individual contaminants found in psoriatic scales showed an elution pattern similar to that of the psoriatic leukotactic factor (PLF) by Sephadex G-75 chromatography. Checkerboard experiments showed that PLF was highly chemotactic and mildly chemokinetic for polymorphonuclear leukocytes (PMNs). Purification of PLF yielded a cationic peptide fraction similar to that of anaphylatoxin. Intradermal injection of a potent PLF solution into guinea pigs produced degenerative changes in the epidermis within 24 h, particularly in the differentiated cells of the upper epidermis. It increased epidermopoiesis as assessed by incorporation of 3H-thymidine. Planimetric analysis of the epidermis revealed a marked increase in epidermal thickness after 5 days. Since PLF per se did not influence the proliferative capabilities of isolated basal cells or exert any damaging effect on differentiated epidermal cells in human epidermal cell culture, we speculated that PLF induced the advent of PMNs abound with hydrolytic enzymes and toxic substances, which were responsible for the damage of the upper epidermis resulting in the formation of parakeratosis and of acanthosis.
In the culture of epidermal keratinocytes, the cellular growth rate is reported to be accelerated by cholera toxin. The mechanism by which cholera toxin exerts biological effects is thought to result from changes in intracellular cyclic AMP concentrations. But in many reports cyclic AMP elevating agents appeared to inhibit growth of keratinocytes in culture. This study was done to clarify the discrepancy of this problem. Determination of cyclic AMP revealed that cholera toxin over a range of 10-14-10-8 M increased the intracellular concentration of cyclic AMP of cultured keratinocytes about 100-fold over the controls after incubation for 6 hr. When a small number (10(5)) of cells were inoculated in a 60 X 15 mm culture dish, cholera toxin strongly stimulated colony growth. When a relatively larger number (8 X 10(5)) of cells were inoculated in a dish, cholera toxin moderately accelerated cell division, and increased DNA and protein levels of the culture during early days of cultivation. But after about 20 days, of cultivation when the culture reached confluence, cholera toxin decreased both DNA and protein content in a culture dish. The cultures were pulse labeled with 3H-thyrmidine at 12 and 24 hr after the addition of 10-10 M cholera toxin, and its uptake into DNA was determined. In the early days of cultivation the uptake of 3H-thymidine increased after treatment with cholera toxin. But in the late days of cultivation, cholera toxin decreased the rate of 3H-thymidine incorporation into DNA. These results indicated that cholera toxin-cyclic AMP has effects on the proliferation of keratinocytes in culture biphasically according to cellular concentrations in culture.
The normal senescent fibroblasts in culture accumulate a significantly high proportion of altered enzymes, and the alterations are considered to be the manifestation of ageing in molecular terms. To detect the possible molecular alterations in patients with De Sanctis-Cacchione syndrome, the severest form of xeroderma pigmentosum, in which repair processes to UV light-damaged DNA are defective and the neurologic abnormalities are considered to reflect accelerated ageing, we studied the heat stability of glucose-6-phosphate dehydrogenase (G6PD) in crude extracts of cultured skin fibroblasts. Three patients with the syndrome were the center of our investigation. Even at early passage in culture the heat-labile portion of G6PD was increased in the cells from patients in comparison to normal controls. The life span of the cells in culture from patients was not reduced below normal age-matched controls, and no appreciable senescent appearance was observed. The increase in the heat-labile portion of G6PD from cells of DeSanctis-Cacchione syndrome patients to reflect that defective repair of DNA damage occurs, rather than being a direct result of ageing of cultured cells.
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Human epidermal keratinocytes in cell culture are unique in reorganizing the formation of a stratified cellular sheet and performing terminal differentiation. The course of keratinocytes in culture was delineated over 50 days in the primary culture. The rapid increase in total DNA content commencing 2 weeks after plating corresponded to the appearance of small polygonal keratinocytes in groups. The DNA content reached its maximum after 25 days of cultivation, decreasing gradually thereafter. The protein content increase slowly until 16 to 18 days of cultivation, and then increased rapidly, though slightly behind the rapid increase in DNA. The maximum value of protein content was reached at 32 days, after which the value decreased when the keratinized cells of the uppermost layer began to detach. The heat-labile fraction of glucose-6-phosphate dehydrogenase was increased in 33- and 43-day cultures, when the DNA content was decreasing.
In 35 out of 38 cases of patients suffering from intrahepatic stones, complete removal of the stones has been carried out with repeated postoperative choledochoscopies, the average being 14.5. A follow-up study revealed that 34 cases remain asymptomatic and are leading a normal social life. The remaining one case died from pancreas carcinoma 22 months after successfully removing the entire stones. With the successful results using postoperative choledochoscopy, we believe that the endoscopy is the preferred approach insofar as it can relieve symptoms, prolong life and render cure in some cases, although it may be only palliative in others. This safer and more reliable procedure as opposed to the blind technique which is carried out under fluoroscopic control should be routinely used for management of intrahepatic stones.