Distribution of immune deposits in the skin of reversed passive arthus reaction using horseradish peroxidase as antigen.

The distribution of immune deposits in the skin of reversed passive Arthus reaction was investigated using horseradish peroxidase (HRP) as antigen. Free and non-reacted HRP, after leaking from small vessels, spreads widely in connective tissue, in epidermis, and in hair follicles in a diffuse and homogeneous pattern. When a specific antibody is administered, HRP appears as granular deposits, which can be considered to be immune deposits, and they adhere to the tissues. When sufficient amounts of antibody is intracutaneously administered, these granular deposits are seen at the wall of venules or in the vicinity of the venules, but when a small amount of antibody is injected, the deposits are seen widely in the connective tissue and are apt to be accumulated at the basement membrane of the dermo-epidermal junction and of hair follicles. Free HRP passes the basement membrane easily, but the immune complexes are considered unable to pass it and are deposited under it.     

Serotonin metabolism in the arthus reaction

To better characterize the role of serotonin in the Arthus reaction, we examined the concentration of the amine and the activities of serotonin-metabolizing enzymes, monoamine oxidase (MAO) and serotonin-N-acetyltransferase (NAT), in reaction sites induced in guinea pig skin. The specific activity of total MAO in the intact skin was 108.0 +/- 15.9 pmol/min/mg protein, and consisted of about 92% of type A activity and 8% of type B. The activity of total MAO was about 10 times greater than that of NAT. Total MAO activity increased to 130%-150% of control levels at 2 h after initiation of the reaction and approximated the control level at 3 to 6 h. Subsequently, the activity decreased linearly to 50% at 12 h and to 20% at 24 h. Although the time-dependent changes of MAO type A activity were similar to those of total MAO activity, MAO type B activity increased to 42% at 30 min, remained at 30%-40% until 6 h, and then decreased to 20% at 12 h and to 5% at 24 h. NAT activity in the reaction sites decreased with time to 50% of the control at 30 min and to 35% at 4 h and was stationary until 24 h. The serotonin concentration decreased linearly with time to 16% of the control level at 1 h, increased sharply to 240% at 6 h, and remained at more than 200% until 24 h. This biphasic change in serotonin concentration seems to be related to the dynamic changes in the activities of serotonin-degrading enzymes. In addition, the accumulation of platelets in the reaction sites may increase serotonin concentration and MAO activity subsequent to 1 h after the initiation.     

A study of the permeability barrier in epidermis and oral epithelium using horseradish peroxidase as a tracer in vitro

Small biopsies of skin, and of keratinized and non-keratinized oral mucosa were incubated with horseradish peroxidase, fixed and reacted to demonstrate peroxidase activity at the light and electron microscope level. In all tissues peroxidase extended through the intercellular spaces of the epithelium up to, but not into, the stratum corneum of keratinized, or the superficial layer of non-keratinized, tissue. This distribution corresponds to that seen in studies using the tracer in vivo where the presence of an intercellular permeability barrier has been demonstrated in the upper cell layers. It is suggested that the method is a useful way of investigating the permeability of normal and pathological human epithelial tissues.     

Impaired histamine metabolism in the arthus reaction induced in guinea-pig skin

Summary The activities of two histamine-metabolizing enzymes, histamine-N-methyltransferase (HMT) and diamine oxidase (DAO), were examined in various types of experimentally induced cutaneous inflammations in guinea pigs. In intact guinea-pig skin, the specific activities of HMT and DAO were 24.8±1.7 pmol/min per milligram of protein or 0.930±0.097 pmol/min per milligram of the wet weight of skin specimen, and 6.0±0.7 pmol/min per milligram of protein or 0.189±0.011 pmol/min per milligram of the wet weight, respectively. Both enzyme activities were markedly reduced in skin lesions of the Arthus reaction (P<0.005), while those in dinitro-chlorobenzene allergic dermatitis, croton-oil dermatitis, and the intact areas in Arthus-reaction-induced aminals were almost within the normal limits. The activity of HMT decreased linearly with time from the onset of the Arthus reaction, reaching about 20% of the control activity at 48 h; the activity of DAO decreased even from the early stages of the reaction, and this decrease continued throughout first 48 h of the reaction. These results suggest that impaired histamine metabolism in the skin lesions of the reaction plays a distinct role in the formation and development of the Arthus reaction.     

Epidermal cell proliferation following an active arthus reaction in the guinea pig

Active Arthus reactions were provoked by injections of 100 micrograms horseradish peroxidase (HRP), 10 micrograms HRP and 100 micrograms bovine serum albumin (BSA) into the skin of sensitized guinea pigs. Labeling indices (LI) of epidermal basal cells were measured 1, 4, 8, 24, 48 and 72 h later by the in vivo 3H-thymidine labeling technique, and compared with those obtained with injections of antigens into the skin of non-sensitized guinea pigs. From 1-8 h after the induction of an active Arthus reaction, the LI of epidermal basal cells of the skin injected with 100 micrograms HRP decreased to a remarkably low value. On the other hand, those obtained with the reaction against 10 micrograms HRP were significantly high. At 24 h after the reaction, LI were as high as those obtained in non-sensitized guinea pigs with control intradermal injections, though the former persisted high until 48 h after the injection. In addition, decreased LI of the epidermal basal cells were observed in the skin 4 h after intradermal injections of immune complexes. It was suggested that DNA synthetic activity of the epidermis increases in a mild active Arthus reaction, while the activity may be suppressed in a severe active Arthus reaction up to 8 h after provocation.     

Studies on the role of polymorphonuclear leukocyte granule elastase in arthus reaction

Summary In the present study, attempts were made to clarify the role of an elastase-like enzyme in Arthus reaction by using rabbit-peritoneal polymorphonuclear leukocytes (PMNs). It was first confirmed that the injection of large amounts of PMNs with antibody can increase tissue damage in reversed Arthus reaction. The five various fractions, which were intact PMNs, whole homogenate, granule fraction, nuclei and cell debris, and membrane extract, were prepared from rabbit-peritoneal exudate. Each fraction was intracutaneously injected into the abdominal wall of rabbit. It was of particular interest that the tissue damage induced by membrane extract occurred earlier and was more severe than that caused by the others. Furthermore, membrane extract possessed remarkably higher caseinolytic and elastinolytic activities than the others. The membrane extract was partially purified by Sephadex G-100 column chromatography. The protein concentration of the elastase-like enzyme solution was 3.64 mg/ml, and its specific activity was 195 caseinolytic units and 0.45 elastinolytic units per milligram of protein. The cutaneous reaction by membrane extract was dependent on the concentration of this elastase-like enzyme and could be strongly inhibited by Elastatinal. From these results, the authors suggest that an elastase-like enzyme which originates in PMN granules is the key enzyme that promotes the severity of tissue damage in Arthus reaction.     

Cytomembrane-derived Birbeck granules transport horseradish peroxidase to the endosomal compartment in the human Langerhans cells.

It has been previously shown that the cytomembrane of human Langerhans cells (LC) has the capacity to fold upon itself, thereby forming Birbeck granules (BG), which then internalize. I confirmed this by exposing LC in vitro to horseradish peroxidase (HRP) at +8 degrees C and +37 degrees C. On incubation at +37 degrees C the label appeared not only in BG, but also in tubular structures and vesicles of different shapes and sizes. Interconnections between these labeled endosomal structures were common. The LC cytomembrane could form BG at +8 degrees C and, moreover, the BG was the only organelle that was labeled and internalized at this temperature. Thus, cytomembrane-derived BG are endocytotic in nature and link the exterior of the cell and the endosomal compartment. The membrane interlinking of the BG may eventually dissolve and the BG then transform into an endosomal vesicle.     

Immunoblot analyses of Brazilian pemphigus foliaceus antigen using different antigen sources

Summary We investigated the Brazilian pemphigus foliaceus (BPf) antigen applying the immunoblotting method to two different antigen sources using 27 patients' sera. Twelve BPf sera reacted specifically with a 150 kD protein in extract of dispase separated human epidermis, while 18 sera yielded a similar protein band in bovine muzzle desmosomal preparation. The diversity of staining intensities between the two samples suggested the heterogeneity of BPf antigens in terms of epitopes. Japanese sporadic pemphigus foliaceus (Pf) sera showed similar results but Japanese pemphigus vulgaris (Pv) sera recognized different antigens of 130 kD or 135 kD, suggesting that BPf is similar to Japanese Pf but is distinct from Pv in respect to the antigenic substance. Furthermore, the present study showed that immunoblot analysis using different antigen sources should be a valuable tool to determine clinical types of pemphigus.This work was supported in part by grant from the Ministry of Health and Welfare of Japan. Dr. Marilia M. Ogawa was an awardee of the Uehara Memorial Foundation Research Fellowship Program (1988)     

Lectin-binding pattern in extramammary Paget's disease by horseradish peroxidase (HRP)-labeling method--specific staining with Dolichos biflorus agglutinin (DBA)

Lectin-binding pattern in extramammary Paget's disease was studied using seven different lectins (Con A, WGA, RCA-I, PNA, SBA, DBA, and UEA-I) by means of the horseradish peroxidase (HRP)-labeling method. By light microscopy it was observed that Con A, WGA, RCA-I, and DBA stained almost all the extramammary Paget cells, while PNA, SBA, and UEA-I stained only some of them. Normal keratinocytes and tumor cells from other diseases such as mammary Paget's disease, malignant melanoma, squamous cell carcinoma, basal cell epithelioma, Bowen's disease, and seborrheic keratosis were positively stained with Con A, WGA, and RCA-I, but not with DBA except in some of the mammary Paget's cells. By electron microscopy it was observed that DBA stained the cell membrane and the Golgi apparatus of the extramammary Paget cells. The present results suggest that DBA is a specific lectin for glycoconjugates in extramammary Paget cells.     

Enzyme-linked immunosorbent assay for detecting gonococcal antibodies using two antigenically different gonococcal pili as antigen.

Antibodies to pilar antigens of two gonococcal strains isolated in Rotterdam (6650 and 1443) were detected using enzyme-linked immunosorbent assays (ELISA). Paired sera (the first sample taken at the first examination (D1) and the second 11-22 days later (D2)) from women with and without gonorrhoea attending a sexually transmitted disease (STD) clinic were studied. The sensitivity of the ELISA using gonococcal pili 6650 as antigen (ELISA 6650) was significantly higher than that using gonococcal pili 1443 as antigen (ELISA 1443). The specificity of the two tests differed little. On D1 the sensitivity in women with uncomplicated gonorrhoea was 69% in the ELISA 6650 and 45% in the ELISA 1443; the corresponding values in asymptomatic infected women were 75% and 57% respectively. The agreement (both in positive and in negative results) between the two tests was less than might have been expected (kappa = 0.41).     

间接血凝法和对流免疫电泳法测定抗ENA抗体

本文分析和比较了被动间接血凝法(PHA)和对流免疫电泳(CIE)检测抗可提取性抗原(ENA)抗体的结果.发现SLE患者中35,700(60/68)抗RNP抗体阳性,39.9(67/168)抗Sm抗体阳性.15例MCTD中抗RNP抗体均阳性10000 PHA法检测抗Sm抗体敏感,而CIE法检测抗RNP抗体敏感.在PHA法中56℃加热一小时处理ENA和RNase消化ENA结果相似.     

Effects of dapsone on passive Arthus reaction and chemotaxis and phagocytosis of polymorphonuclear leukocytes

Summary The effects of dapsone (diaminodiphenyl sulfone) on the polymorphonuclear leukocyte (PMNL) function and on the elicitation of the passive Arthus reaction were investigated in rabbits and guinea-pigs. NBT-reduction was markedly enhanced in PMNL of animal receiving 5 mg dapsone/kg for 3 days, whereas chemotaxis and phagocytosis were not influenced. Passive Arthus reaction was clearly suppressed by the same treatment regimen. 2 mg/kg of the drug had no effect on the parameters examined.     

Lectin-binding pattern in extramammary Paget's disease by horseradish peroxidase (HRP)-labeling method —Specific staining with Dolichos biflorus agglutinin (DBA)

Summary Lectin-binding pattern in extramammary Paget's disease was studied using seven different lectins (Con A, WGA, RCA-I, PNA, SBA, DBA, and UEA-I) by means of the horseradish peroxidase (HRP)-labeling method. By light microscopy it was observed that Con A, WGA, RCA-I, and DBA stained almost all the extramammary Paget cells, while PNA, SBA, and UEA-I stained only some of them. Normal keratinocytes and tumor cells from other diseases such as mammary Paget's disease, malignant melanoma, squamous cell carcinoma, basal cell epithelioma, Bowen's disease, and seborrheic keratosis were positively stained with Con A, WGA, and RCA-I, but not with DBA except in some of the mammary Paget's cells. By electron microscopy it was observed that DBA stained the cell membrane and the Golgi apparatus of the extramammary Paget cells. The present results suggest that DBA is a specific lectin for glycoconjugates in extramammary Paget cells.Supported in part by grants-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan, and Japan Private School Promotion Foundations