Inhibition of C1r, C1s and generation of C1s by amidino compounds.

A series of amidino compounds has been investigated for their inhibitory effects on C1s, C1r, and generation of C1s. Diamidines consisting of two amidinophenyl residues linked in para position by a molecular bridge proved to be the strongest competitive inhibitors of C1s, whereas those linked in meta position were the strongest competitive inhibitors of C1r. They inhibited the overall generation of C1s when added to the system containing three subunits of C1 and Ca2+. Diphenylamidines were more active than single ring amidines. Of all the compounds tested, dibromopropamidine was the most effective inhibitor of C1s with Ki value 3 x 10(-5) M and deltaF' values 6.4 kcal x mole(-1), whereas amicarbalide and M and B 4596 were the strongest inhibitors of C1r with Ki values 3.5 x 10(-5) M and 3.25 x 10(-5) M and deltaF' values 6.3 and 6.34 kcal x mole(-1), respectively. Epsilon-Aminocaproic acid was also included in this study for comparison purposes and was found to be inert as to its effects on these reactions. The possibility that some of these amidino compounds might prove to be useful for treatment of hereditary angioneurotic edema is discussed.

Human plasma kallikreins and their inhibition by amidino compounds.

Human plasma kallikreins (EC 3.4.21.8) were purified as three distinct enzyme entities which hydrolyzed arginine esters and were active in releasing kinin from heated human plasma as measured by guinea pig ileum contraction bio-assay. The three enzymatically active fractions were termed as 19 S, 7 S-I and 7 S-II kallikreins. They represented purifications of 262- 2200- and 110-fold, respectively. These enzyme activities showed differences in physicochemical and biochemical properties as it appears from their elution profile on Sephadex G-200 and DEAE-cellulose columns, affinity for substrates and susceptibility of inhibition by various protease inhibitors such as trasylol and soya bean trypsin inhibitor. The data suggest that all these three enzyme preparations were most likely kallikreins. All these three enzymes (19 S, 7 S-I and 7 S-II) were inhibited by a series of amidino compounds competitively. Diamidines consisting of two amidinophenyl residues linked in para position by molecular bridge were comparatively stronger inhibitors of all of three enzymes than those linked in meta position and those having single ring structure. The possibility that some of these amidino compounds might prove to be useful for treatment of disease states where the kallikrein-kinin system plays a role, is discussed.

Some properties of proteolysis by polymorphonuclear leukocyte-granule extracts.

The extracts of granules of human polymorphonuclear leukocytes hydrolyzed a variety of proteins including human and bovine hemoglobin, human fibrinogen, human and bovine serum albumin, bovine elastin, and casein. The hydrolysis of all the proteins except fibrinogen and elastin was increased by addition of urea. Various inhibitors of trypsin, kallikrein, plasmin, Clr, Cls, and other proteolytic enzymes had no inhibitory effect. Slight inhibition was observed with polyanethol sulfonate and strong inhibition with normal human serum. Serum of patients with hereditary angioneurotic edema having no functional C1-esterase inhibitor was as effective in inhibiting the proteolysis as normal serum. The inhibitor was localized in 4S fractions of normal serum fractionated on Sephadex G-200. Fractionation of normal serum by ammonium sulfate precipitation, Sephadex G-200 filtration, and CM-Sephadex chromatography did not result in appearance of inhibitory activity in more than one protein peak, suggesting the possibility that only one inhibitor might be responsible. Since all fractions which contained the inhibitor of proteolysis also contained alpha1-antitrypsin, since sera of patients having low alpha1-antitrypsin levels contained less inhibitory activity, and since antibodies against alpha1-antitrypsin reversed the inhibition obtained from normal serum, the inhibition of proteolysis may be attributed to alpha1-antitrypsin.

Diagnostic procedures in immunodermatology.

Most immunologic diseases are caused by the derailment of the humoral or cellular pathways of the immunologic defense system. This derailment results from numerous factors such as the inability of the patient to remove the pathogen; the consumption, defect, or deficiency in any component of these pathways, and the overproduction of any of the components. To diagnose these immunologic disorders one has to detect the pathogen and the reactions caused by it and to determine the cause of its nonclearance. The immunofluorescence techniques has been invaluable in detecting both the antigen that causes the disease and the reactions initiated by the antigen, such as the production of antibodies and the activation of the complement system. The immunoperoxidase technique has also been used for these purposes in certain instances. For detecting the circulating immune complexes which occur as intermediates in the chain of reactions initiated by the antigen, various physiochemical and biologic techniques have been used. However, none of these tests seems to be totally reliable for determining whether circulating immune complexes are present. The consumption of complement was detected by hemolytic estimations and radial immunodiffusion or rocket electrphoresis. These techniques were also useful in detecting the hereditary deficiencies in immunoglobulins and components of classical and alternative pathways of complement activation. Since these techniques cannot be used to estimate IgE, the radioallergosorbent test was used to measure such levels in the atopic patients. Cellular hypersensitivity was detected with skin tests together with methods which assess the ability of lymphocytes to produce mediators in response to antigen. Many of these mediator assays, however, are not suitable for this purpose. A satisfactory substitute appears to be to determine the factor in antigen-stimulated, lymphocyte culture supernatants which activates macrophages to take up radiolabeled colloidal gold or radiolabeled glucosamine. In contact allergic dermatitis, an increase in the IgD-bearing lymphocytes and granulocytes has also been correlated with cellular hypersensitivity. Lymphocytes and polymorphonuclear leukocytes coated with antibodies mainly directed against nuclear antigens of the basal layer cells of the noninvolved epidermis have invariably been encountered in psoriasis. The use of these findings for diagnostic purposes and for understanding the mechanisms of certain diseases is being explored.

CLq precipitin in the sera of patients with allergic vasculitis (Gougerot-Ruiter Syndrome).

The sera from two well-documented cases of allergic vasculitis were examined for the presence of C1q precipitins. Both sera contained material capable of precipitating C1q in agarose gel. The material from one of the sera was partially purified using ammonium sulfate precipitation. Sephadex G-200 filtration, and DEAE-cellulose chromatography. Attempts to identify the nature of C1q precipitin were unsuccessful, but it resembled the high-molecular-weight precipitins of sera from patients with systemic lupus erythematosus. In a lesion, deposition of fibrinogen, immunoglobulins, and complement were noted mainly in the vessel walls. No correlation between immunoglobulin and complement deposition in the skin and the presence of C1q precipitins in the blood could be established.

Antibodies eluted from lymphoid cell membrane. Occurrence in certain varieties of scleroderma.

By means of acid elution two antibodies could be removed successfully from the circulating lymphocytes of 11 patients with certain varieties of scleroderma. One was specifically directed against nuclear antigen(s) of endothelial cells (NEC) of the dermal blood vessels, and another against nuclear antigen(s) of epidermal basal cells (NBC) of the involved and uninvolved skin of the patients. In two cases of acroscleroderma, the eluates failed to react with either endothelial or basal cells of involved or uninvolved skin. In none of 20 healthy controls involved in this study could an antibody be eluted from the circulating lymphocytes. The aforementioned antibodies do not bind complement in vitro and do occur in the serum of four patients. Circulating antinuclear antibody (speckled type) was detectable in two cases of scleroderma.

Hereditary deficiency of C2 in association with linear scleroderma 'en coup de sabre'.

A 32-year-old man suffering from linear frontoparietal scleroderma was found to have low (less than 10% normal) serum classical pathway activity although C1q, C3, C4, C5, and total alternative pathway activity was normal. Addition of purified C2 led to complete restoration of the total hemolytic activity of the classical pathway. The C2 hemolytic assays showed that the patient was not totally deficient in C2. He had about 30% of the normal C2 level. Studies on his available nucleus family members in the Netherlands also showed that the deficiency was inherited; one of the patient's brothers and one of his daughters had half of the normal C2 levels. The C2 deficiency could not be corrected by a three-week regimen of danazol. To the best of our knowledge, this is the first documented case concerning an association of linear frontoparietal scleroderma with C2 deficiency.

Hereditary congenital hypopigmented and hyperpigmented macules.

Congenital hypomelanotic and hypermelanotic macules traced in three generations of a family suggested autosomal dominant inheritance. Some affected membbers also showed retarded growth and mental deficiency. Light microscopic findings of "splitdopa" preparations of lesional and normal skin were comparable, except that background staining of keratinocytes in dark macules was higher than in control skin. In light macules it was lower. Ultrastructurally, hypomelanotic skin showed small melanosomes (0.3 mu) that occurred in keratinocytes in melanosome complexes. Hypermelanotic skin revealed large melanosomes (0.6 mu) that were singly distributed in keratinocytes. Melanosome size in normal skin averaged 0.4 mu; distribution pattern was mixed. Melanin granules inside keratinocytes were fully melanized. Hyperpigmented, normal and hypopigmented skin of one person had histological features of black oriental and white skin. This clinical picture could well represent a new neurocutaneous syndrome different from tuberous sclerosis.

Immunopathological studies on alopecia areata.

Anti-endothelial cell antibodies could be removed from circulating lymphocytes by means of acid elution techniques in eight patients with different degrees of alopecia areata. These antibodies were specifically directed against the endothelial cells in the capillary network of the hair bulb, indicating the existence of an antigen, which is unique to these particular endothelial cells. These antibodies do not bind complement "in vitro" and are species-specific. Circulating ANA (speckled type) were only noticed in case with alopecia areata in spots. A significant decrease in circulating T cells was noticed in six of eight patients with a certain degree of alopecia.

Studies on the immunologic aspects of keloids and hypertrophic scars.

A significant difference between keloids and hypertrophic scars could be demonstrated by means of acid elution of lymphoid blood cells and immunofluorescence studies. A total of 20 patients (13 patients with keloids and seven with hypertrophic scars) were investigated. All the 13 patients with keloids revealed in the eluates antinuclear antibodies belonging to one or more of the five main classes and directed mainly against fibroblasts. On the other hand, there were no antinuclear antibodies detectable in the eluates of the seven patients with hypertrophic scars and in over 40 healthy controls.

The recruitment of inflammatory cells using the skin-window technique.

The chemotaxis of inflammatory cells induced by the skin-window technique using IgD as cytotaxigen or cytotaxinogen was studied in 16 patients with allergic contact dermatitis. Six patients with leg ulcer served as controls. By means of this method the recruitment of inflammatory cells with receptors for IgD could be shown.

Allotransplantation of cultured human skin.

Human skin allografts cultured for approximately 6 weeks on a solid medium were transplanted to non-related human recipients with skin defects of various etiology. Although based on this study no answer could be given to the question whether these grafts survived or were replaced by cells of the recipient, it was observed that in most cases the grafts were not rejected and remained in situ several months up to over a year.

Specific Y chromosome fluorescence in interphase nuclei of epithelial cells in human buccal smears.

A simplified method is described to demonstrate Y chromosome fluorescence in interphase nuclei of epithelial cells in human buccal smears using Quinacrine Mustard. This technique turned out to be efficient in our hands and would be of value in determining the sex chromosomes in the epithelial cells of the skin in cases of allogeneic transplantations from females to males and vice versa to determine the peroid during which the grafts survived.

Immunofluorescence studies in reactional leprosy with relevance to treatment.

Twenty-three biopsies of skin lesions of patients with various types of leprosy, showing a recent reaction, were examined by means of immunofluorescence (IF) methods. The patients were divided into two groups according to the number of inflammatory cells, staining with various FTC-labelled anti-immunoglobulin antisera, in representative areas of the biopsies. It was found that the presence of these cells was correlated with a good response to thalidomide treatment.

Phenylalanine and UVA light for the treatment of vitiligo.

The administration of phenylalanine (Phe) combined with UVA exposure was found to be effective in vitiligo. Phe is an amino acid which constitutes part of the daily dietary protein, and when orally administered in a dose of 50 mg/kg body weight, it results in an elevated plasma level. Since peak concentrations of Phe in the blood are reached between 30 and 45 min after ingestion, UVA exposure was administered at this time. After 4 months (32 treatments) reasonable repigmentation preferentially occurred in the skin area of subcutaneous fat (adipose tissue). Apart from the repigmentation of hypo-pigmented macules, vitiligo patients can tolerate more sun than usual, especially at the vitiliginous lesion, and they experience no sunburn as a result of Phe-UVA therapy. Normal skin also tans very well.

Immunocompetent cells in psoriasis. In situ immunophenotyping by monoclonal antibodies.

Immunocompetent cells in exacerbating untreated psoriasis vulgaris skin lesions were immunophenotypically studied by the application of a selection of monoclonal antibodies in a two-stage immunoperoxidase technique. Epidermal changes include: focal accumulation of immunoglobulins in the stratum corneum, as demonstrated by a mixture of monoclonal anti-kappa and anti-lambda antibodies; focal accumulation of OKM-1 positive but Mo-2 negative cells high in the epidermis, reflecting granulocytes in Munro's abscesses; a marked decrease in epidermal Langerhans cells with focal abnormal clumping and smaller dendrites, as demonstrated by monoclonal anti-HLA-DR and anti-T6 (OKT-6) antibodies; and, sporadic exocytosis of mainly T1 (Leu-1), T8 (Leu-2a) positive suppressor/cytotoxic T lymphocytes. The dermal infiltrates were found to consist mainly of partically activated T1 (Leu-1), T4 (Leu-3a) positive T-helper/inducer cells with a smaller compartment of T1 (Leu-1), T8 (Leu-2a) positive suppressor/cytotoxic lymphocytes. These cells were found in close apposition to T6 (OKT-6), HLA-DR positive Langerhans cells and further accompanied by a minor compartment of OKM-1, Mo-2 positive monocytes. No B-cells or plasma cells could be demonstrated in the dermis. Natural killer cells were observed only incidentally. These results fit best with the hypothesis that psoriasis is a chronic inflammatory condition as a result of persistent stimulation of T cells by immunogen(s) of epidermal origin.

Microenema of 8-methoxypsoralen in photochemotherapy of psoriasis.

Reasonably high plasma levels were obtained 1/2 h after 8-methoxypsoralen (8-MOP) administration by microenema. This method was used in photochemotherapy of psoriasis and reasonably good clinical results with no serious side effects were obtained. The advantages of this modality include noninvolvement of the upper gastrointestinal tract, high bioavailability of psoralen, peak levels at a predictable time, and rapid elimination of the drug.

Psoriasis specific chromosomal proteins, antibodies against them and disease activity.

It has been demonstrated recently that the lymphoid cells of patients with psoriasis have antibodies directed against the psoriasis specific non-histone proteins. A conceptual hypothesis for the role of these antibodies in the pathogenesis of psoriasis presented in this communication, is as follows. Perhaps the psoriasis specific non-histone proteins following phosphorylation bind to histone which keeps psoriasis gene(s) repressed. This may result in the displacement of histone from the DNA. Antibodies against psoriasis specific non-histone proteins may facilitate the displacement of histone. DNA set free, possibly containing psoriasis gene(s), can then be transcribed into RNA causing a shift of the resting pool of keratinocytes in the symptom-free skin of psoriasis patients to the proliferating pool of keratinocytes in the psoriasis lesions.