Morphological changes and serological reactions in cultured Leishmania donovani promastigotes.

Promastigotes of Leishmania donovani cultured in liquid media gradually changed from a majority of elongated forms through a predominance of intermediate spindle-shaped parasites to a majority of round forms, often aflagellate. Quantitative and qualitative differences among these subpopulations, separated on Percoll gradients, could be demonstrated when they were run on SDS-PAGE gels and stained from proteins or carbohydrates. When cultures containing a majority of one or other form were used as antigenic substrate in serological tests (ELISA and immunofluorescence) the older, round forms were more highly reactive with patients' sera. These forms may represent different phases of the parasite, related to function, and the density variations would provide a convenient method to separate them.

Serodiagnosis of Sudanese visceral and mucosal leishmaniasis: comparison of ELISA--immunofluorescence and indirect haemagglutination.

Two established immunodiagnostic techniques, immunofluorescence and indirect haemagglutination, were compared with ELISA (enzyme-linked immunosorbent assay) using intact promastigotes as antigen for the detection of specific antibodies against Leishmania in the serum of patients with visceral or mucosal leishmaniasis from the Sudan. The ELISA was found to be more sensitive and more specific than either of the other two tests.

ELISA using intact promastigotes for immunodiagnosis of kala-azar.

The commonly used diagnostic methods for visceral leishmaniasis (kala-azar) have many disadvantages. ELISA appears to be a more easily applied test for diagnosis, follow-up of treatment and epidemiology. The preparation of the ELISA antigen has given rise to difficulties in standardization since these are usually soluble. Intact cultured promastigotes have been used to develop an ELISA which is sensitive and specific while avoiding the extra handling which is a source of variability and lack of reproducibility. When tested on sera from kala-azar patients and patients with other tropical diseases, this ELISA was able to detect specific antibodies at very high serum dilutions. It is thus promising as a screening method applicable in the field.

The binding of activated C3 to polysaccharides and immunoglobulins.

The nature of the acceptors for activated C3 present on immunoglobulins was studied by using the fixation of C3 to Sepharose beads after activation with trypsin as a model system. C3 fixation to Sepharose is a property exclusively linked to the short active state of C3. This binding could be inhibited by various carbohydrates and their affinity for C3 was calculated from the extent of the inhibition of the binding of C3 to Sepharose. Mono-, di-tri- and tetrasaccharides showed on a molar basis an increasing but still weak affinity for active C3. The C3 fixation of Sepharose could be inhibited by immunoglobulins and the degree of inhibition was proportional to the hexose content of the immunoglobulin preparations. The isolated polysaccharide moiety of IgG gave the same inhibition as the intact IgG. In addition cell wall polysaccharides of Salmonella abortus equi were found to have a high affinity for active C3. Thus, the more complex polysaccharides such as those present on immunoglobulins and cell walls might function as acceptor for activated C3.

C-1 inactivator and cold-promoted activation of factor VII.

When exposed to temperatures between -5 degree and +5 degree C, plasma from pregnant women and from certain blood donors show shortening of the Thrombotest clotting time, kallikrein formation, and activation of blood-clotting factor VII. This phenomenon has been called cold-promoted activation of factor VII (CPA). In this study, it was found that CPA-positive plasma or serum samples which had been exposed to low temepratures showed spontaneous disappearance of C-1-inactivator activity in parallel to the shortening of the Thrombotest clotting time. C-1-inactivator antigen was not affected by storage at 4 degree C. In these CPA-possitive samples the loss of C-1-inactivator activity is caused partially by the formation of kallikrein at this temperature because when kallikrelin was added to C-1 inactivator, the latter was inactive when tested in the esterolytic assay. The formation of Hageman factor fragments may add to further loss. Purified C-1 inactivator effectively inhibited the CPA phenomenon, whereas alpha2-macroglobulin did so only weakly. This finding indicates that during exposure of CPA-positive plasma samples to low temperatures, Hageman factor fragments, which are inhibited only by C-1 inactivator, induce the activation of the kallikrein system and blood clotting factor VII. The reported lowered activity of C-1 inactivator in pregnancy is probably an artifact caused by generation of CPA during storage, since in fresh samples the levels were compeletely normal. Similarly, various subjects classified as belonging to the variant type of HANE (low C-1-inactivator activity with a normal antigen content) were found to have normal C-1-inactivator activity when determinations were made on fresh instead of frozen samples. It is recommended that plasma or serum samples should not be exposed to temperatures between -5degree and +5degree C prior to the determination of C-1-inactivator activity. Moreover, during purification procedures of kallikrein-binding antiproteases such as C-1 inactivator and also alpha2-macroglobulin, the occurrence of CPA should be avoided by the use of CPA-negative plasma as starting material.

Spontaneous release of Fc receptor-like material from human lymphoblastoid cell lines.

In the culture medium of some human lymphoblastoid cell lines material is released with the following properties: (a) hemagglutination reaction of IgG-sensitized erythrocytes; (b) enhancement of precipitation of DNA-anti-DNA complexes; (c) inhibition of binding of C1q to immune complexes; (d) inhibition of immune complex binding to lymphocytes; (e) inhibition of antibody-dependent lymphocytotoxicity. The material is not identical with C1q or rheumatoid factor, it is heat resistant (30 min at 56 degrees C); the molecular weight is about 100 000 daltons and it is capable of inhibiting antibody production in vitro. It is suggested that this material consists of Fc receptors spontaneously shed from lymphocyte membranes.

In vitro activation of C1s in plasma of patients with hereditary angioneurotic oedema.

The serum of patients with hereditary angioneurotic oedema contains small amounts of inhibitor of Cl-esterase (Cl-s) which is usually present in appreciable amounts in the serum of healthy individuals. In the citrated plasma of these patients in remission Cl-s activity was not detectable, but prolonged incubation with various alcohols, detergents, acetone, phenols and metal chelating agents generated the enzyme. Normal plasma did not respond to these reagents. The generation of Cl-s by alcohol and EDTA was inhibited by addition of natural inhibitor of Cl-s but not by soyabean trypsin inhibitor, trasylol and hexadimethrine bromide. Hexadimethrine bromide did not block the generation when experiments were carried out in silicon coated glassware. Incubation with kaolin and kallikrein generated Cl-s in remission plasma. Density gradient centrifugation studies showed that alcohol caused at least partial dissociation of Cl in remission plasma but not in normal plasma. This effect was similar to that of EDTA described in earlier reports. The possible explanations for the findings are 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.

Interaction of human lymphocytes with fluid phase human C3b detected by immunofluorescence.

Human lymphocytes obtained from tonsils and peripheral blood were found to bind human fluid phase C3b, obtained by trypsin treatment. This binding was detected by indirect immunofluorescence (IIF) using specific anti-C3 antisera. Lymphocytes isolated from thymus tissue scored low percentages in IIF, indicating that the main population of thymus-derived lymphocytes are T cells. The distribution pattern of C3b-binding cells was compared with that of cells forming rosettes with sheep erythrocytes coated with antibody and complement (EAC) and with sheep erythrocytes (E) only, as well as with that of Ig-bearing lymphocytes, as detected by direct immunofluorescence. It appeared that the distribution pattern of lymphocytes which can bind fluid phase C3b is similar to that of EAC rosette-forming and of Ig-bearing lymphocytes. Pre-incubation of the lymphocytes with C3b and pretreatment of the cells with trypsin decreased the capacity to form rosettes and to bind C3b to their surface. Human monocytes granulocytes and erythrocytes did not bind fluid phase C3b, as judged by IIF. Therefore, the selective binding of fluid phase C3b to lymphocytes provides a specific method for the detection of complement-reactive lymphocytes in lymphoid cell preparations.

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.