Protective activity of preparations of blood plasma from donors with a high titer of natural antibodies to Re-glycolipid of gram-negative bacteria

The screening of the preparations of blood plasma obtained from 1,608 donors made it possible to establish the presence of high titers of natural antibodies to Re-glycolipid in 3% of the donors. Donor plasma containing antibodies to Re-glycolipid in a titer of 1:128 ensured a high level of protection for mice in experimental fecal peritonitis. The treatment of 10 patients having commonly occurring forms of peritonitis, caused by Gram-negative bacteria, with the use of such plasma yielded a positive clinical effect. The presence of correlation between the titers of antibodies to Re-glycolipid in blood plasma preparations and the content of high-density lipoproteids, expressed in per cent, was noted.     

The opsonizing activity of the sera of hamadryas baboons immunized with a preparation of the outer membranes of Francisella tularensis (based on data from the luminol-dependent luminescence method)]

The opsonizing properties of sera obtained from hamadryas baboons immunized with the preparation of F. tularensis outer membranes (OM) were studied with the use of luminol-dependent chemiluminescence (CL) of whole blood. The immunization of monkeys with the OM preparation was shown to lead to the formation of functionally active antibodies possessing opsonizing properties with respect to virulent F. tularensis. Immune sera obtained from the animals immunized with live vaccine and from those immunized with OM preparation had no essential differences in their opsonizing properties. The level of IgG antibodies in immune sera correlated with the CL parameters of whole blood in the presence of F. tularensis opsonized with these sera. Increased CL of phagocytes observed after addition of bacteria and immune sera under test to whole blood taken from a nonimmune donor made it possible to evaluate the functional activity of antibodies, thus permitting its use as a test for the evaluation of the effectiveness of new vaccine preparations.     

Lipid peroxidation and antioxidant defense of erythrocytes during bubbling air ionization of donor packed red blood cells

It is shown that erythrocytes in packed red blood cell preparations can be maintained in native state for a long time using bubbling air ionization (BAI). The BAI procedure proposed to prolong the storage period of donor erythrocytes makes it possible to reduce the level of destructive processes in packed cells stored until use, as indicated by a decrease in the lipid peroxidation intensity and an increase in the antioxidant activity in them.     

The standardization of the reverse indirect haemagglutination test for the assay of the viral antigen of Japanese encephalitis vaccine

The reverse indirect haemagglutination (RIHA) test has been standardized for the assay of the viral antigen of purified Japanese encephalitis (JE) vaccine. Glutaraldehyde fixed sheep erythrocytes were sensitized with ammonium sulphate purified antibodies to JE vaccine raised in mice. The sensitivity of the erythrocytes fell to about one hundredth of the initial sensitivity in the first two days after preparation. After initial loss in sensitivity the stability of the cells became stabilized and the cells retained their titre for one year at 4-8 degrees C. The initial loss in sensitivity was not reduced by storing the cells at -70 degrees C, but after freeze drying the sensitized cells with a stabilizer one day after their preparation the cells retained their sensitivity. The RIHA test has been found to be a highly reproducible and sensitive method for detecting viral antigen in 5-10 ng of protein nitrogen. The sensitivity of the test was affected by the origins of the erythrocytes, i.e. from the different sheep from which they were drawn. To obtain results more rapidly, goose erythrocytes were used in place of sheep erythrocytes and the sensitized goose erythrocytes gave RIHA results in only 40 min.     

Flow cytometric analysis of erythrocyte populations in Tn syndrome blood using monoclonal antibodies to glycophorin A and the Tn antigen.

Flow cytometric analysis employing monoclonal antibodies to the Tn antigen and glycophorin A was used to characterize the erythrocyte populations present in blood samples from individuals with Tn syndrome. Four monoclonal antibodies specific for the Tn antigen, Gal-NAc monosaccharide, on human erythrocytes were obtained from a fusion of splenocytes from a Biozzi mouse immunized with red cells from a Tn individual. These monoclonal antibodies specifically recognize GalNAc monosaccharide sites located on the erythrocyte cell surface sialoglycoproteins, glycophorin A and glycophorin B, and do not bind to fixed normal red cells presenting the Neu-NAc alpha 2-3Gal beta 1-3(NeuNAc alpha 2-6)GalNAc alpha 1-O-Ser(Thr) tetrasaccharide or to fixed neuraminidase-digested cells presenting the Gal-GalNAc disaccharide. The percentages of Tn-positive red cells in samples from six unrelated Tn donors ranged from 28 to 99%. Binding of the glycophorin A-specific monoclonal antibodies showed that the erythrocytes composing the Tn-negative fraction presented normal amounts of the M and N epitopes on glycophorin A. The presumed somatic mutational origin of Tn-positive cells was tested in blood samples from five normal donors; three possible Tn cells were observed after analysis of a total of 1.1 x 10(7) erythrocytes, suggesting that the frequency of such cells in normal individuals is less than 1 x 10(-6).     

A very sensitive enzyme-linked immunoabsorbent assay to Staphylococcal protein A in the presence of immunoglobulins

The production of recombinant hepatitis B virus surface antigen (rHBsAg) purified by immunoaffinity chromatography with monoclonal antibodies is used to obtain a vaccine against this virus. Monoclonal antibodies to rHBsAg from mouse ascites have been purified by Staphylococcal Protein A (SpA)--prior coupling to Sepharose CL-4B (Amersham-Bioscences, Uppsala, Sweden). A high sensitivity immunoassay has been developed for the quantification of part-per-million of SpA contaminants likely to co-purify with monoclonal antibodies obtained by Protein A affinity chromatography, in the presence of immunoglobulins. Specific sheep polyclonal Abs against SpA (SpAc1) were used as plate coating and the SpA detection was possible thanks to the conjugates of sheep Ab fragments F(ab)(2) (fSpAc1) and horseradish peroxidase (fSpAc1-peroxidase), reducing the possible unspecific interaction between SpA and Fc fragments. The immunoassay was shown to be specific for SpA contaminants. The quantification limit of the assay was 0.39 ng/ml spreading to the measurement of contamination levels less than 2 ppm of SpA in final preparations of monoclonal antibodies used for the immunopurification of pharmaceutical products, which is quite low for this application.     

Immunological studies on the localization of phosphatidylglycerol in the membranes of Mycoplasma hominis.

Phosphatidylglycerol is the main component (87%) of the membrane phospholipids of Mycoplasma hominis. It is immunologically active. Antibodies directed against phosphatidylglycerol were detected in rabbits intravenously immunised with native M. hominis or isolated M. hominis membranes. The intravenous method of immunisation was chosen in order to select for a response to surface antigenic determinants. Anti-phosphatidylglycerol antibodies were induced in rabbits by intravenously injecting the flocculated complexes of methylated bovine serum albumin and a phosphatidylglycerol/phosphatidylcholine/cholesterol mixture. These antibodies were specifically bound to intact M. hominis, as shown by complement fixation and Coombs tests. Native M. hominis were not agglutinated by anti-phosphatidylglycerol antibodies; but after partial digestion of the membrane proteins with Pronase, the mycoplasmas were heavily agglutinated by the anti-phosphatidylglycerol antibodies. The same amount of anti-phosphatidylglycerol antibodies was bound to intact M. hominis, containing 600 mug of phosphatidylglycerol as to 6 mug of phosphatidylglycerol in the optimal configurational arrangement of a mixed phosphatidylglycerol/phosphatidylcholine/cholesterol micelle. It is concluded that the major part of the phosphatidylglycerol in native M. hominis membranes is masked, probably by membrane proteins, and is not accessible to the anti-phosphatidylglycerol antibodies.     

Detection of poliovirus antigens and antibodies: microindirect haemagglutination and haemagglutination inhibition tests for poliovirus types I, II, and III.

Microtitre indirect haemagglutination (IHA) and IHA-inhibition (IHAI) procedures were adapted to determine the reactivities of type I, II, and III poliovirus antibodies and antigens. Glutaraldehyde-fixed sheep erythrocytes were sensitized for these tests with concentrated, partially purified preparations of type I, II, and III poliovirus. Antibody titres by IHA were generally 10 to 100 times greater than serum microneutralization (SN) titres. The SN and IHA reactivities of three kinds of sera were compared. Of these sera, virus type specific antibodies, in monospecific guinea pig sera one week after immunization and in sera from hyperimmunized horses, could be readily differentiated and measured; antibodies in human diagnostic specimens, however, showed some intertypic cross reactivity. Monovalent one-week immune guinea pig sera reacted specifically in the IHAI test to differentiate viruses, and could be used for virus typing and differentiating strains of poliovirus type III.     

Hemolysis in several animal species after rapid freezing of blood

The effect of various freezing rates on the extent of hemolysis in human, bovine and ovine erythrocytes, which are known to have different cell volumes, water contents and permeabilities, was investigated. Blood in stainless steel capillary tubes was frozen at various rates by abrupt immersion of the capillaries into cooling baths at temperatures ranging from ?20° to ?130°C. Minimum lysis values were obtained at freezing temperatures of ?40°, ?50° and ?70°C with, respectively, human, bovine and ovine blood. The smallest, highly permeable sheep erythrocytes were the least damaged at the highest freezing rates; the largest human cells with the highest water content, suffered the greatest damage; intermediate values were obtained with ox blood. At the lower freezing rates, the largest, human cells were the least damaged; the highest hemolysis values were obtained with the smallest, highly permeable sheep erythrocytes; ox blood again gave intermediate values. These results are in agreement with current views that, (1) very rapid freezing results in the formation of damaging intracellular ice; (2) injury associated with slow freezing is related to the extent of dehydration or to the increase in electrolyte concentration which accompanies ice formation; (3) minimum hemolysis is obtained under those freezing conditions in which osmotic dehydration has been sufficient to prevent the formation of intracellular ice, but has left enough water in the cells to prevent the damaging effects of dehydration and high electrolyte concentrations.     

Purification and chemical characterization of antigens from sheep erythrocytes

A mixture of glycoproteins and glycolipids was solubilized from sheep erythrocytes membranes under the effect of high ionic strength (2 M NaCl, 0.5 M Tris). Several antigenic fractions could be purified from the mixture using gel filtration on Sephadex G-200 and block electrophoresis on Pevikon C870; two fractions were found to raise antibodies in a primary reaction and these antibodies effectively sensitized erythrocytes to lysis by complement. The majority of other fractions elicited a weaker primary reaction which was detectable by both agglutination and haemolysis. The fraction, migrating fastest towards the cathode, elicited after immunization a formation of antibodies that could be detected almost exclusively by haemagglutination. The fraction, which elicited in the primary reaction a high titre of haemolytic antibodies, is composed of 72% proteins, 11% lipids and 15% saccharides.     

Mycoplasma hominis septicaemia.

Mycoplasma hominis septicaemia occurred in a patient with a malignant lymphoma of lymphoblastic type in leukaemic phase. M hominis was isolated several times from blood cultures with antibody titres against the micro-organism rising to a high level despite severe immunosuppression. M hominis was detected in the blood after subculture of the blood culture bottles despite their macroscopically normal appearance. The patient''s pyrexia resolved without treatment with antibiotics effective against M hominis.     

Immunologic parameters of monkeys infected by urogenital mycoplasmas

In monkeys contained in captivity conditions in open-air cages or in group cages human mycoplasmas are often detected: antigens of Mycoplasma hominis in blood serum were revealed in 33.3% of cases, and antibodies to it--in 15.6% of cases. IgM to M. hominis were detected more often than IgG. In 8 monkeys both types of immunoglobulins were detected. Rates of detection of Ureaplasma urealyticum antigens and specific antibodies were 43.1% and 31.1% respectively, and IgG were found more frequently than IgM (in 22 cases both types of immunoglobulins were revealed). High rates of M. hominis and U. urealyticum antigens and antibodies detection in blood serum of both healthy monkeys and monkeys with urogenital tract diseases show prevalence of human mycoplasmas carriage among monkeys contained in captivity conditions.     

Expression and purification of a novel mannose-binding lectin from Pinellia ternata

Pinellia ternata agglutinin (PTA) from the tubers of P. ternata is a monocot mannose-binding lectin that catalytically agglutinated rabbit erythrocytes. The potential effect of PTA has gained considerable interest in recent years owing to clinical use of native PTA as the preparation against cancer and for plant protection against insect pests. Here we report a successful strategy to allow high-level expression of PTA as inclusion bodies in Escherichia coli M15. Purification of refolded recombinant protein from solubilized inclusion bodies by Ni-NTA agarose affinity chromatography yielded biological activity recombinant PTA (final yield of about 10 mg/L). The recombinant PTA agglutinated rabbit erythrocytes to a dilution similar to that determined for "native" lectin purified from P. ternata. The expression and purification system makes it possible to obtain sufficient quantities of biologically active and homogenous recombinant PTA sufficient to carry out advanced clinical trials. This is the first report on the large-scale expression and purification of biologically active recombinant PTA from E. coli.     

Differences Between Brucella Antigens Involved in Indirect Hemagglutination Tests with Normal and Tanned Red Blood Cells

Brucella antigens capable of sensitizing normal and tanned sheep red blood cells for indirect hemagglutination were compared with antigens involved in agglutination, gel diffusion, and immunoelectrophoresis. Hyperimmune rabbit sera, before and after absorption with various antigenic preparations from smooth and rough B. abortus, were used in the tests. Normal erythrocytes could be sensitized with an NaOH-treated ether-water extract (EW-T) of smooth Brucella. Tanned erythrocytes could be sensitized with a water-soluble extract from ultrasonically disrupted smooth or rough Brucella. The EW-T produced a single precipitation band and the water-soluble antigens produce 6 to 23 bands in immunoelectrophoresis with unabsorbed sera. After absorption of antisera with water-soluble extracts from smooth or rough Brucella cells or from smooth or rough cell walls, the hemagglutinins for sensitized tanned erythrocytes and the precipitins for water-soluble antigens were removed. Absorption with living smooth or rough Brucella cells or with EW-T did not remove these antibodies. The precipitins and hemagglutinins for the antigen EW-T, and agglutinins for smooth cells, were absorbed by smooth antigens but not by rough antigens. It appears that the antigens which sensitize tanned erythrocytes and diffuse through agar gels are present in both smooth and rough forms and may be situated in the cytoplasm or in the internal part of the cell wall, whereas the agglutinogen and the antigen which attaches to normal erythrocytes are surface antigens found only on the smooth Brucella cell.     

Immunomodulation by myxospores of Myxococcus xanthus

Glycerol-induced myxospores of Myxococcus xanthus caused non-specific modulation of humoral and cellular immune responses in laboratory animals. The number of cells which formed specific haemolysins in spleens of mice immunized with sheep erythrocytes was increased when 0.5 X 10(8) myxospores were inoculated 2 d after the erythrocytes, and decreased when myxospores were injected 2 d before or at the same time as the erythrocytes. Both the IgG primary response and the secondary response to erythrocytes were decreased in rabbits after pretreatment with 2 X 10(8) myxospores per rabbit. Delayed-type hypersensitivity to sheep erythrocytes was also suppressed in mice after intraperitoneal (i.p.) injection of 0.3 X 10(8) myxospores. One day after i.p. injection of myxospores, neither an inflammatory response nor bone marrow cell depletion was observed in mice. These results support the idea that M. xanthus myxospores possess diverse immunomodulation properties apparently due to factors different from the classical LPS of Gram-negative bacteria.     

Importance of spontaneous micronucleated erythrocytes in bottlenose dolphin (Tursiops truncatus) to marine toxicology studies

The objective of the work was to characterize the presence of spontaneous micronucleated erythrocytes (MNES) from peripheral blood of bottlenose dolphins (Tursiops truncatus) to evaluate the possibility to use this species as potential bioindicator of genotoxic compounds. Forty-eight blood samples from 12 bottlenose dolphins were obtain from three Mexican dolphinariums, and from 10 dolphins was possible to obtain more than one sample at different sampling times. Smears were processed and observed with an epifluorescence microscope. The average of MNES and polychromatic erythrocytes (PCE) from the 48 samples was 24.3 +/- 6.1 MNES/10,000 total erythrocytes (TE), and 9.1 +/- 5.5 PCE/1,000 TE. MNES and PCE number did not show differences between gender and age. No variations in the MNES values of the bottlenose dolphins that were sampled more than one occasion were found. Comparisons among dolphinariums revealed differences in MNES frequency, with the highest significant frequency observed in dolphins from dolphinarium "A" (26.0 +/- 5.9 MNES/10,000 TE) than dolphinarium "B" (19.5 +/- 3.1 MNES/10,000 TE) (p < 0.05) and dolphinarium "C" (18.6 +/- 3.5 MNES/10,000 TE) (p < 0.007). The presence of MNES and PCE in the bottlenose dolphin may provide a useful marine mammal model to detect DNA damage by means of micronuclei test in peripheral blood erythrocytes to evaluate genotoxicity and cytotoxicity expositions.     

Application of different methods in the diagnosis of respiratory mycoplasmosis

A complex of methods for the detection of Mycoplasma pneumoniae and Mycoplasma hominis in children and adults with respiratory diseases (acute, chronic and obstructive bronchitis, pneumonia, recurring croup, bronchial asthma), as well as in children frequently having acute respiratory diseases, has been worked out and tested. Both infective agents are frequently detected in the above mentioned pathological processes as monoinfection or mixed infection. Mycoplasma antigens are capable of prolonged (up to 1 year) persistence in the patient body in spite of etiotropic therapy and the presence of specific antibodies. The method of the preliminary treatment of specimens for the polymerase chain reaction is proposed: the specimens are subjected to prolonged deproteinization, which makes it possible to detect M. pneumoniae in some cases of chronic infection when it cannot be detected by routine methods.     

Use of protein A in the immunosorbent analysis of antibodies to the tick-borne encephalitis virus

The results of the studies made with a view to developing the method for the determination of specific antibodies to the antigen of tick-borne encephalitis virus in human blood serum and liquor are presented. The method is based on the capacity of Staphylococcus aureus protein A to bind with Fc-region of immunoglobulins, which makes it possible to use this protein as the "second" system of antibodies. The conditions for the sorption of the antigen on polystyrene test tubes and for binding 125I-or horse radish peroxidase-labeled protein A preparations with antibodies have been determined, and the method has been approved in tests made on sera and liquor obtained from donors and tick-borne encephalitis patients.     

Immunoglobulin-positive mononuclear cells in human peripheral blood: detection by mixed anti-globulin and direct anti-globulin-rosetting reactions.

Ig-bearing mononuclear cells were identified in Ficoll-Hypaque preparations of human peripheral blood by using mixed anti-globulin (MAG) and direct anti-globulin rosettes; indicator cells consisted of sheep erythrocytes coated with human F(ab')2 or anti-F(ab')2 antibody, respectively. Of the cell population isolated from 10 normal subjects, a mean of 68% was lymphocytes. However, fewer than 50% of the cells with detectable surface Ig were lymphocytes. On viable cell preparations using chromic chloride-treated sheep erythrocytes (CrCl3SRBC) coated with anti-F(ab')2 antibody, a mean of 20.1% of the lymphocytes formed rosettes, i.e., were B. Up to 6% of peripheral blood lymphocytes formed mixed Ig-rosettes and E-rosettes. On viable lymphocytes using F(ab')2-coated CrCl3SRBC, MAG rosettes were insensitive in detection of B lymphocytes. Formaldehyde treatment of lymphocytes increased the number of B cells detectable to 25.5% of the lymphocyte population. Study of T-enriched and B-enriched populations showed that the observed increase in B cell reactivity was real and not due to MAG-rosetting T cells. A one-stage procedure for T and B lymphocyte separation is described.     

Relationship of hydrogen peroxide production by Mycoplasma pulmonis to virulence for catalase-deficient mice

Mycoplasma pulmonis, an etiological agent of murine pneumonia, produced about 0.065 mumoles of hydrogen peroxide (H(2)O(2)) per hr per 10(10) colony-forming units. When glucose was present at a concentration of 0.01 m, H(2)O(2) production was increased by 50%. To determine if H(2)O(2) production by M. pulmonis could be correlated with virulence, normal, acatalasemic, and acatalatic mice were infected with the organism. Three days after infection with M. pulmonis significantly more acatalatic mice had pneumonia than did normal or acatalasemic mice. The pneumonia in acatalatic mice was also more severe than in the other two groups. Five days after infection, pneumonia in the acatalatic mice was resolved, whereas normal mice were severely affected. The presence of pneumonia and the severity were correlated with the recovery of M. pulmonis from the lesions. In vitro studies of the effect of catalase on M. pulmonis showed that exogenously supplied catalase stimulated the growth of M. pulmonis at 37 C and prolonged its survival at 25 C. Hemolysis of sheep blood, guinea pig blood, rabbit blood, and normal and acatalasemic mouse blood by M. pulmonis was inversely related to the catalase activity of the erythrocytes. These findings suggest that H(2)O(2) secretion contributes to the virulence of M. pulmonis and to the death of the microorganism in the absence of host catalase.