Biomedical subjects
Publications and source records attributed to W L Epstein.
This in-depth review considers the known virological and molecular biological aspects of the molluscum contagiosum virus. Furthermore, the epidemiology of its infection of human skin is detailed, and the clinical, histological, and therapeutic information available at this time are cataloged. It is concluded that molluscum contagiosum has become an almost common skin disease that can prove very difficult to eradicate from an infected patient.
A soluble component that transfers granulomatous tissue reaction was fractionated from Schistosoma mansoni egg-induced hepatic granulomas (SMHG) by Sephacryl S-300 column chromatography. The fractions separately bound to inert, Affi-Gel agarose beads were inoculated subcutaneously in naive mice. The low molecular weight fraction, consisting of proteins 23 kd, 20 kd, and 16 kd, produced organized granulomas 6 to 7 weeks after inoculation. This fraction was further purified by high-pressure liquid chromatography (HPLC) gel filtration and gave three fractions eluting at retention times of 44, 46, and 48 minutes. Each fraction contained all low-molecular-weight proteins in varying amounts and induced skin granulomas when inoculated subcutaneously. Amino acid sequence of the major 20-kd protein showed 11 N-terminal residues identical to those of cyclophilin. Antisera raised to the protein with retention time of 46 minutes, reacted with cells in the granulomas but not surrounding liver tissue as detected by immunofluorescence microscopy. The findings indicate a low molecular weight soluble fraction of SMHG can induce new granuloma formation when injected in an immobilized form into skin of naive mice. The results suggest granuloma initiation factor is a homolog of the cyclophilin gene family.
Pathophysiological roles of macrophage chemotactic factor (MCF) in granulomatous inflammation were investigated. MCF was extracted in 10 mM phosphate-buffered saline, pH 7.4, from experimentally produced epithelioid cell granulomas in the liver and skin of mice. MCF activity reached a peak in the lesions prior to the time when granulomatous inflammation became maximal. MCF was then purified from 10-week-old hepatic granulomas and 2-week-old skin lesions by gel filtration, ion exchange column chromatography, and HPLC gel filtration. MCF from either liver or skin had a molecular weight about 650 kDa. MCF from hepatic granulomas was coupled to Affi-Gel beads and transplanted subcutaneously into naive mice. In vivo macrophage chemotaxis was observed around the beads and the cells formed a sheet, but organization of macrophages into granulomas did not occur with the MCF-active fractions. Macrophage chemotaxis alone is insufficient to elicit granulomatous inflammation.
An improved model of granulomatous inflammation in skin was developed by second passage skin grafting of isolated, lyophilized skin granulomas, originally elicited in naive mice by inoculations of lyophilized hepatic schistosome egg granulomas. The tissue reaction is caused by a single exposure to a noninfectious, acellular granulomagenic stimulus and occurs in healthy mice free of systemic disease. The model should prove useful for isolation of granuloma initiation factor(s). Furthermore, because there is a time lag before new granuloma formation begins, a window exists for analytical dissection of the initiation process. In this study we described the responses of host cells by autoradiography, and light and electron microscopy. The activity of angiotensin-converting enzyme and proline-specific endopeptidase showed a modulation during granuloma formation. In addition we found that severe immunosuppression with high dose cyclosporine therapy did not alter granuloma formation, supporting the idea that initiation of organized granulomas is T-cell independent.
Genetic influence on the development of granulomatous tissue reaction was investigated in C57BL/6 mice. Granulomas developed in the skin of euthymic C57BL/6 mice by transplantation of lyophilized hepatic granulomas were excised and lyophilized. The tissue mass free of parasite egg antigen and living cells was grafted into the skin of euthymic, athymic (nu/nu), and beige (bg/bg) C57BL/6 mice. Histological changes at the skin sites were studied weekly by light microscopy, and cells in newly developed granulomas at 6 weeks after grafting were examined by electron microscopy. Granulomatous inflammation occurred in all the variants but morphometric analysis showed that granulomatous inflammation was the most extensive in beige mice and least in athymic mice. The differences in the degree of tissue reaction were also quantified by measuring angiotensin converting enzyme and prolyl endopeptidase. Statistically significant differences among the animals with varying genetic background were confirmed by the marker enzyme activity. The findings confirm that initiation of a granulomatous response does not require T cells but T cell function is important for full expression of the reaction, while NK cell activity seems to suppress granuloma formation.
In organized granulomatous inflammation, activated macrophages terminally differentiate into epithelioid cells (ECs). By light microscopy, individual ECs appear similar, irrespective of the inciting agent. Ultrastructural analysis of a number of clinical and experimental granulomas in man and mice reveals, however, three distinct varieties of these cells. They are classified as follows: (1) EC-I cells containing a mixture of lysosomal-dense bodies and phagolysosomes and arrays of rough endoplasmic reticulum in the cytoplasm; (2) EC-II cells showing primarily a secretory mode with prominent Golgi bodies and rich arrangement of dilated rough endoplasmic reticulum with no phagocytic vacuoles; and (3) EC-III cells containing large multisized vesicles crowded throughout the cytoplasm and presently of unknown function. Although the unique appearance of these cells offers little diagnostic help, the cells do distribute differentially in different granulomas. The EC-I cells tend to predominate in infectious granulomas and where an excess of foreign material and debris is present. The EC-II cells are most characteristic of active sarcoidal granulomas, and the EC-III cells appear in chronic, long-standing granulomas. The heterogeneic nature of these cells seems to suggest their functions in granulomatous inflammation are not always the same.
Changes in the expression of glycoconjugates in cells of the inner root sheath (IRS) and outer root sheath (ORS) of human anagen hair follicles were investigated by lectin histochemistry. Concanavalin A (Con A) and Ricinus communis (RCA-I) stained hair follicle cells regardless of their differentiation stages. In IRS, Ulex europeaus-I (UEA-I) bound to the surface of the cells as soon as they were morphologically defined, and Glycine max (SBA) stained as their differentiation progressed. Innermost (IM) cells of ORS layers were reactive with UEA-I at the stage where Henle's cells were keratinized, while the reactivity of UEA-I was lost at the site of the completion of IRS keratinization where SBA reaction was detected. Staining of both UEA-I and SBA was prominent in other ORS cells at the levels where SBA binding in IM cells became strong. The staining intensity increased up to the position of the follicular isthmus. In addition, a sugar residue recognized by Dolichos biflorus (DBA) was detected in differentiated cells of ORS. In contrast, the DBA reaction was not found at all in cells of IRS, infundibulum, and epidermis. These findings identified a complexity of carbohydrate metabolism in the cells of different layers at various stages of keratinization. IM cells differentiate independently from other ORS cells but seem responsive to the degree of IRS keratinization. All ORS cells possess a unique sugar moiety not found in other keratinocytes either in the hair or epidermis.
1. Angiotensin I hydrolases, Mr 140,000 and Mr 70,000 were separated by gel filtration from Tris-HCl buffer extract of hepatic granulomas developed in mice with schistosomiasis. Two enzymes had different substrate specificity. 2. Mr 140,000 hydrolase activity was inhibited by captopril as reported for angiotensin converting enzyme (ACE), while that of Mr 70,000 hydrolase activity was inhibited by potato carboxypeptidase inhibitor. 3. An intermediary, des-Leu10-angiotensin I and then angiotensin II were formed from angiotensin I by Mr 70,000 hydrolase. 4. The findings suggest that Mr 70,000 enzyme is tissue carboxypeptidase A, and it generates angiotensin II in granulomatous inflammation as does ACE.
The relationship between natural killer (NK) cells and granulomatous inflammation was investigated using two experimental granuloma models in C57BL/6 mice. The hepatic granuloma model was produced by infection with Schistosoma mansoni (S. mansoni), and the skin granuloma model was established by subcutaneous inoculation of the isolated hepatic granulomas. NK cell activity in lymph nodes and spleen, measured by 51Cr-release assay against YAC-1 cells, was compared to that in age-matched control mice. The activity decreased progressively as granulomas developed without changing the number of NK-1.1+ cells. The reduced NK cell activity was not reversed by administration of indomethacin. In order to further substantiate whether NK cells contribute to granulomatous inflammation, NK cells were depleted in the mice by injection of anti-NK-1.1 mAb. Reduction of NK cell activity (70-90%) was achieved during granuloma formation. An increase of about 20% in the mean granuloma diameter was detected in the mAb-treated mice in both models. Moreover, the percentage of granuloma takes in the skin model was enhanced (65% increase). The mAb treatment did not alter T cell counts in granulomas, T cell subset numbers or proliferative response in spleen. These findings indicate that NK cells directly play a regulatory role in granulomatous inflammation.
We investigated the effect of granulomatous inflammation in skin on lymphocyte maturation in athymic (nu/nu) mice. Hepatic egg granulomas developed in euthymic (nu/+) mice with schistosomiasis were transplanted into skin of nu/nu mice. During skin granuloma development the rate of DNA synthesis and interleukin 2 activity of lymphocytes from lymph nodes, with and without concanavalin A stimulation, showed that the nu/nu cells were activated to levels of untreated nu/+ lymph node cells. Activation of splenic lymphocytes was not detected in the grafted nu/nu mice. Also, immunohistochemical staining demonstrated an increase in cells expressing Thy 1.2, Lyt-1 or L3T4 surface markers in the skin and lymph nodes, but not in spleen. The findings indicate that a granulomatous reaction in nu/nu mouse skin induces local, but not systemic, proliferation and differentiation of lymphocytes, to a low degree compatible with resting nu/+ mice.
A tissue carboxypeptidase-A-like enzyme was purified to apparent homogeneity from terminally differentiated epidermal cells of 2-day-old rats by potato inhibitor affinity chromatography followed by FPLC Mono Q column chromatography. The enzyme has an Mr of 35,000 as determined by SDS-polyacrylamide gel electrophoresis and HPLC gel filtration. It has a pH optimum of 8.5 for hydrolysis of benzyloxycarbonyl-Phe-Leu (Km = 0.22 mM, kcat = 57.9 s-1). The enzyme does not hydrolyze substrates with Arg, Lys and Pro at the C-terminal and Pro at the penultimate position. Angiotensin I was effectively hydrolyzed (Km = 0.06 mM, kcat = 6.48 s-1) and produced both des-Leu10-angiotensin I and angiotensin II. The enzyme activity, relatively stable at 4 degrees C and pH 8.0-10.5, was inactivated at pH values higher than 12.0 and lower than 5.0 or at 65 degrees C for 10 min. Inhibitor profiles of the epidermal enzyme also differed slightly from those of tissue carboxypeptidase A of pancreatic or mast cell origin.
Damage and repair of cell-surface glycoconjugates were examined in human palmar skin following friction-blister injury, using biotinylated lectins and the avidin-biotin complex method. In normal skin, concanavalin A, Ricinus communis, and Triticum vulgaris bound to the surface of cells from the basal layer to the granular layer. After injury, binding of concanavalin A was absent in the plasma membrane, but appeared in the cytoplasm at perinuclear sites. The surface reaction was recovered in basal and spinous cells, but not in granular cells, when cell maturation began at 5 days after injury. In contrast, binding of Ricinus communis and Triticum vulgaris was, in general, much more resistant to tissue damage. Even in some cells, where the surface staining became obscure at an early period, a normal staining pattern reappeared by 6 h after injury. Staining of Ulex europeus I and Glycine max, detected on the surface of upper spinous and granular cells in normal skin, disappeared immediately after the injury, but recovered quickly on the surfaces of the differentiated cells. These findings suggest that at least 2 oligosaccharide sequences, one binding with concanavalin A, and the other with Ricinus communis and Triticum vulgaris, may exist on epidermal cells. Addition of terminal carbohydrates, detectable with binding of Ulex europeus I and Glycine max, appears to occur on the Ricinus communis I and Triticum vulgaris-bound oligosaccharide chain.
Proteins with approximate molecular weights of greater than 300,000 (EP greater than 300K) and 49,000 (EP 49K) were partially purified from terminally differentiated cells of 2-day-old rat epidermis. They were extracted in 0.34 M sucrose containing 0.01 M citric acid and purified by Sephacryl S-300 chromatography followed by reverse-phase column chromatography. The major constituents of EP greater than 300K and EP 49K were focused around pH 10 to 11 by sucrose gradient isoelectric focusing. Both proteins were effective at inhibiting colony formation of Candida albicans and C. tropicalis, but neither inhibited the growth of C. parapsilosis. The effect was maximum below pH 5.0 and reduced considerably above pH 5.0. The activity of EP greater than 300K on C. albicans TIMM 1623 (group A) was much stronger than that of EP 49K, whereas both proteins similarly inhibited C. albicans TIMM 1604 (group B). Their effects against C. albicans TIMM 1623 were dose dependent and were activated after a longer preincubation time, and NaCl concentration influenced their potency. At a low salt concentration and a 60-min preincubation at pH 4.5, the 50% effective dose for EP greater than 300K was calculated to be 1.7 x 10(-9) M, whereas that for EP 49K was 1.8 x 10(-7) M.
Subcutaneous grafts of fresh or freeze-dried hepatic schistosome egg granulomas in inbred mice caused increased numbers of total cells, T cell subsets, and elevated DNA synthesis in regional lymph nodes. This in vivo T cell activation was not apparent by in vitro tests. Spleen cells were unaffected both in vivo and in vitro. Immunosuppressive therapy with ciclosporin or monoclonal antibodies to T cell subsets profoundly depressed the T cell numbers and function, even when primed by a prior graft. Subsequent grafting partly reversed these effects, and T cells became activated. The findings indicate that in vitro assessment of activation and suppression of T cell function does not necessarily correspond to the events occurring in vivo.
A phosphoprotein phosphatase (PPase: EC 3.1.3.2) was recently purified from rat epidermis. The enzyme dephosphorylates phosphoprotein, and its properties, such as pH optimum, inhibitor spectrum, and Fe2+ activation, differ from those of other soluble phosphatases. We investigated in 2-day-old rat skin the distribution of immunologically detectable PPase and intracellular localization of PPase activity. The reaction of rabbit monospecific anti-PPase IgG was identified in granular and cornified cells by the avidin-biotin complex method. For activity staining, basic principles of the Gomori lead-salt method and azo dye technique with the substrates p-nitrophenylphosphate (p-NPP) and alpha-naphthyl phosphate (NP), respectively, were modified according to the biochemical properties of PPase activity which is resistant to formalin, Na tartrate, and NaF. Activity was detectable in granular cells including keratohyalin granules and the lower strata of cornified cells. The activity was inhibited by 1 mM CuSO4 and enhanced by a mixture of 0.5 mM FeSO4 and 1 mM ascorbic acid. We consider that PPase may be involved in dephosphorylation of histidine-rich proteins in granular and cornified cells and may play a key role in intracellular catabolism associated with epidermal cell differentiation.
Plasminogen activator (PA) and PA inhibitor were partially purified from 2-d-old rat epidermis and characterized in order to elucidate the enzyme-inhibitor interaction in epidermis. PA extracted with buffer containing KSCN was first purified by Blue-Sepharose affinity chromatography. Separation of two PAs, with relative molecular mass (Mr) of 66000 and 44000, was accomplished by Con A-Sepharose column chromatography. The Mr 66000 enzyme had the properties of tissue-type PA (t-PA), while the Mr 44000 enzyme showed those of urokinase-type PA (u-PA) as determined by immunological and fibrin-binding studies. PA inhibitor was extracted in 1,4-piperazinediethanesulphonic acid buffer and purified by (NH4)2SO4 precipitation, gel filtration followed by a Mono Q column in an FPLC system. This inhibitor showed Mr 60000 and inhibited human u-PA activity in a dose- and time-dependent manner, but did not inhibit the activity of t-PA from human and murine melanoma cells or plasmin. It inhibited epidermal PA, Mr 44000, more effectively than it did the Mr 66000 epidermal PA. It was stable at 60 degrees C for 60 min or between pH 5 and 11. This study indicates that both u-PA and t-PA function in normal rat epidermis. On the other hand, an inhibitor which preferentially acts against u-PA exists, but inhibitor to t-PA does not appear to operate under normal epidermal functions.
The mononuclear phagocyte system is the central body defense called upon to deal with persistent foreign objects or irritants. Depending on circumstances, the macrophages function as phagocytes or secretory cells or both. Granulomas may form in the absence of immunologic modulation (foreign body granuloma) or they may develop under immunologic control (hypersensitivity granulomas). In either case, the secretory products of macrophages are qualitatively similar and differ mainly in the total amounts produced. If cell-mediated immunity dictates hypersensitivity granulomas as commonly taught, one has to explain what aberrent signals are sent from T cells or what abnormal message is perceived in the macrophages which causes them to differentiate to purely secretory epithelioid cells or produce such abnormal pathologic patterns as necrobiosis. Alternatively, some granulomatous hypersensitivity reactions such as organized epithelioid cell granulomas may be initiated primarily by a macrophage secretion product with cellular immunity acting to amplify or promote granuloma formation and organization. The known heterogeneity of macrophages is insufficient to account for the varied observations. Future experimentation will uncover the different differentiation programs of monocytes required for the varied and various granulomas seen in skin.
Natural killer (NK) cell activity is down-regulated in patients with active chronic inflammatory processes, and appears abnormal in sarcoidosis. We investigated NK cell activity in lymph nodes (LN) and spleen cells of C57BL/6 mice by using a 51Cr-release assay against Yac-1 tumor target cells. Hepatic granulomas were caused by infection with 75 cercariae of Schistsoma mansoni ans skin granulomas formed by transplanting isolated liver granulomas into skin of another group of naive mice. Also, anti-NK-1.1 MoAb was injected intraperitoneally into some mice (1 mg IgG/mouse) 1 wk before and weekly for 4 wks after grafting; control mice received saline. Well-organized granulomas appeared in both liver and skin. NK activity (%) was markedly reduce in LN and spleen: (table; see text) The results showed a relationship between granuloma formation and reduction of NK cell activity; both systemically and locally. MoAb did not alter total LN and spleen cell numbers, but reduced NK cell activity by 70-90%. Mean diameter of skin granulomas developed in treated mice was greater (p less than 0.05). NK cells may act as suppressor cells in granulomatous inflammation.