Protective activity of secreted proteins of Streptococcus pneumoniae and Klebsiella pneumoniae

Protective efficacy of secreted proteins of Streptococcus pneumoniae and Klebsiella pneumoniae cultivated on cardiocerebral broth and semisynthetic growth medium respectively was studied in vivo. Fraction with molecular weight 30 - 50 kDa obtained by the method of membrane fractionation had high protective efficacy. Two-dose immunization of mice with this fraction provided 80 - 100% protection from infection by homologous strains of S. pneumoniae and K. pneumoniae. Cross-protective activity of the fraction was revealed when infecting immunized mice by different K-types of K. pneumoniae. Blood sera of mice immunized with 30 - 50 kDa fraction possessed preventive features protecting from infection 90% of animals while 100% of death in the control group. It was determined that protective efficacy of the mentioned fraction was determined by protein-containing antigens because proteolytic disruption of the protein component resulted in loss of protective properties of the preparation.     

Role of antibodies against outer-membrane proteins in murine resistance to infection with encapsulated Klebsiella pneumoniae

In the assessment of immunity to the encapsulated virulent strain of Klebsiella pneumoniae and its avirulent mutant defective for capsular polysaccharide (CPS), killed bacterial vaccine of both strains could protect mice equally against challenge with 100 x LD50 of encapsulated wild strain. Antisera to each strain conferred the same level of protection on naive mice upon transfer; the protective anti-mutant serum was highly capable of opsonizing the encapsulated bacteria. In addition to the common antigenic components shared by both strains, the wild strain had antigen(s) unrelated to the mutant since the protective capacity of the anti-wild serum was not affected by preabsorption with the mutant strain; the protection conferred by the anti-mutant serum was mediated by antibodies against non-capsular antigens since the antiserum did not contain antibodies against purified CPS detectable by ELISA. As possible candidates among the non-capsular antigens, outer-membrane proteins (OMPs) extracted from the mutant strain were examined for their immunogenicity. Immunoblotting of the protein-containing fraction and ELISA using LPS-free OMP suggested that a number of proteins were involved in the immune response evoked by K. pneumoniae. Furthermore, mice immunized with OMP or anti-OMP serum could overcome a lethal challenge with the wild strain. These results indicated that OMPs of K. pneumoniae are implicated as the protective antigens and may pave the way for the development of non-capsular, proteinaceous vaccines.     

Monoclonal antibodies to Mycoplasma pneumoniae antigens]

Approaches to obtaining stable mouse hybridomas, capable of producing monoclonal antibodies (McAb) to M. pneumoniae key antigens, were developed. As the result of hybridization experiments, 7 clones were obtained; of these, 4 clones stably synthesized IgG McAb. Clones H1/H9 and H9/B2 synthesized antibodies to thermolabile, proteinase-sensitive K protein, produced by cytoplasmic membranes of M. pneumoniae cells. The molecular weight of this protein was found to be 90 kD. McAb of clone H1/H9, labeled with horse-radish peroxidase and fluorescein isothiocyanate, specifically reacted with M. pneumoniae antigens in the immunofluorescence test and the enzyme immunoassay (EIA). The sensitivity of EIA was 0.25 ng/ml of antigen protein. These data are prerequisites for the development of diagnostic test systems for the detection of M. pneumoniae antigens in different biological substances obtained from patients with respiratory pathology.     

Isolation and partial characterisation of immunoglobulin from southern bluefin tuna Thunnus maccoyii Castelnau

Specific and total serum immunoglobulins were extracted by immunoaffinity, mannan-binding protein and Protein A affinity chromatography from southern bluefin tuna (Thunnus maccoyii Castelnau) immunised with rabbit IgG, and from non-immunised southern bluefin tuna. SDS-PAGE in 10% reducing gels revealed two heavy chains with molecular weights of approximately 74.6 +/- 1.3 kDa and 71.2 +/- 0.9 kDa, and two light chains with molecular weights of approximately 29 +/- 1.2 kDa and 28 +/- 1.0 kDa. Under non-reducing, but denaturing, conditions in 4% and 5% SDS-PAGE gels, a high molecular weight and a low molecular weight fraction were demonstrated. By gel filtration using Sephacryl HR 300 a molecular weight of 845 kDa, consistent with a tetramer, was obtained for the high molecular weight fraction, and a molecular weight of 168 kDa, consistent with a monomer, was obtained for the low molecular weight fraction. The extinction coefficient at A280 for the purified immunoglobulin (Ig) was determined to be 1.24. Tuna a-rabbit IgG Ig was reactive with all non-reduced mammalian IgG antigens tested, suggesting that common conformational antigenic determinants were recognised.     

Immunochemical identification of the growth hormone and prolactin in the hypophyseal polypeptide spectrum of chickens

Forty-eight fractions of polypeptides including 39 fractions with a molecular weight of 14-95 kD were identified in chick adenohypophysis by sodium dodecyl sulphate electrophoresis in 10-20% gradient polyacrylamide gel slabs. The immunochemical identification of the polypeptides was performed with the aid of the electroblotting of proteins and antisera to human STH, to bovine prolactin, and to the tissue-specific antigen A-1 of chick adenohypophysis. Antisera to human STH and to antigen A-1 reacted with the same major polypeptide fraction, m.w. 26 kD, characteristic of the caudal lobe of the adenohypophysis. Immunoreactive prolactin was present in chick adenohypophysis in the form of a polypeptide fraction with a molecular weight of 25 kD and in the form of two minor fractions of polypeptides with molecular weights of 27 and 28 kD. The data obtained indicate the identity of the adenohypophyseal tissue-specific antigen A-1 to chick STH.     

Immunobiological properties of Burkholderia pseudomallei and Burkholderia mallei capsular substances

The biopolymer composition, immunotropic and immunogenic properties of the fractions of B. pseudomallei and B. mallei were under study. The first two capsular fractions of these agents were found to be similar in their biopolymer composition that was indicative of their close relations. At the same time the causative agents of glanders proved to have decreased content of high molecular glycoproteids and LPS fragments. In the causative agents of melioidosis, capsular fractions K3 and K4 were characterized by the domination of proteins with a molecular weight of 42-25 kD. Fraction K4 in B. pseudomallei and fraction K1 in B. mallei had pronounced immunosuppressing properties ensuring the protection of encapsulated microbial cells in the body. The biopolymers forming fractions K1, K2, K3 in B. pseudomallei and fraction K2 in B. mallei were characterized by immunomodulating properties.     

Electrolyte and water transport in the newt renal tubule due to the action of low-molecular fractions isolated from the intestine of the hibernating ground squirrel

The micropuncture technique and X-ray microanalyses were used to evaluate Na, K, Ca, Mg, Cl and fluid transport in the early distal tubule mediated by low-molecular fractions isolated from the intestine of the hibernating ground squirrel (HSI). Perfusion of intertubular capillaries with a solution containing HSI with a molecular weight of 1-10 kD (0.1 mg.ml-1) decreased the relative reabsorption of fluid, Ca and Mg. The other low-molecular fraction 1-10 kD of m.v. (0.1 mg.ml-1) isolated from the intestine of alert ground squirrels (ASI 1-10) caused no significant changes of the ionic concentration and inulin concentration index in the distal tubule. Fraction 1 with molecular weight 1.2-4.5 kD (0.1 mg.ml-1) obtained by further division of HSI 1-10, decreased the reabsorption of fluid and ions under study. Fractions 2 (m.w. 0.98 kD) and 3 (m.w. 0.89 kD) had no effect on the ion and fluid transport in the same tubule. The addition of naloxone (0.4 mg.ml-1) blocked the action mediated by HSI 1-10 or fraction 1. Thus, the substance(s) capable of regulating Ca, Mg, and fluid transport in the distal tubule likely to be mediated by the opiate-dependent process has a molecular weight of 1.2-4.5 kD.     

Cell-surface associated proteins of corneal fibroblasts: dissection with monoclonal antibodies

It is now generally accepted that the cell surface is involved in the interaction of the cells with the extracellular matrix. To identify and characterize cell-surface-associated components of corneal fibroblasts, several monoclonal antibodies were developed. Hybridomas were developed by fusing mouse myeloma cells SP2/OAg14 with spleen cells from mice immunized with membrane fractions of corneal fibroblasts grown in culture. Twenty-five hybridomas secreting monoclonal antibodies to cell-surface components were selected by an enzyme-linked immunosorbent assay using corneal fibroblasts grown in microtiter plates as the substrate. Immunohistochemical staining demonstrated that the antigenic determinants recognized by these antibodies were not present on corneal epithelial cells, but were present on skin fibroblasts. The antigenic determinants recognized by two of these antibodies, designated 10D2 and 716, were matrix components of the corneal stroma. Immunochemical characterization of the antigens was carried out by indirect precipitation of the radioactively labeled cellular proteins with the monoclonal antibodies and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the precipitates. Four antibodies were able to precipitate antigens from cell extract in detectable amounts. Antibodies designated 5E2, 9G2, and 10D2 recognized antigens consisting of polypeptides of approximate molecular weights 105K and 110K, while antibody 716 recognized an antigen of 100K molecular weight. However, based on the tissue distribution and cell-surface distribution, these antibodies reacted with different antigenic determinants. The antigen recognized by 716 was also secreted by cells in culture but consisted of 220K and 200K polypeptide chains. It was tentatively identified as cellular fibronectin, based on the reaction of this antigen with polyclonal antibodies to plasma fibronectin.     

The characterization of the spectrum of the antibody response to Mycobacterium tuberculosis antigenic determinants by immunoblotting]

The spectrum of antibody response to M. tuberculosis antigenic determinants H37Rv and M. bovis antigenic determinants BCG was studied in serum samples from 33 healthy donors and 31 patients with infiltrative pulmonary tuberculosis by the method of immunoblotting. The study revealed that most frequently tuberculosis patients showed response to Ag-H37Rv with molecular weights of 52, 39, 35, 21, 31, 68 kD (44.4-22.2%) and Ag-BCG with molecular weights 60, 58, 50, 25, 54, 70 kD. (33.3-22.2%). By month 9 of effective chemotherapy binding predominantly with Ag-H37Rv determinants of 31, 62, 35, 75, 56, 28, 19, 5, 13 kD (75-37.5%) and Ag-BCG determinants of 13, 34, 38, 44, 19, 36, 45, 52, 58, 60 70 kD (37.5-25%) were registered. Some differences in the spectra of antibody response to Ag-H37Rv and Ag-BCG determinants were noted.     

Study of the fractions and fibrinolytic activity of fungal mannan

Characteristics of the fraction composition of extracellular mannan produced by Rhodotorula rubra are presented. Various lots of the polysaccharide mainly contained two fractions similar by their chemical structures and differing in the solution relative viscosity. HPLC was used for determining the molecular weight of the samples. In the isolated fractions it differed 3-5 fold. Relationship between the fibrinolytic activity of the polysaccharide and its molecular weight was revealed. The samples of mannan with the molecular weight of 400-500 kD had the highest capacity for lowering the fibrinogen blood levels in rats. The polysaccharide with the molecular weight of less than 100 kD had practically no fibrinolytic activity.     

Fractionation and quantitation of egg antigens from Schistosoma japonicum by the single-tube kinetic-dependent enzyme-linked immunosorbent assay (k-ELISA): higher antigenic activity in urea-soluble than in aqueous-soluble fractions

To identify sources of high potency antigens for use in serodiagnosis, aqueous-soluble egg antigens from Schistosoma japonicum were extracted with Dulbecco's phosphate-buffered saline. Residual particulates were solubilized with Tris-buffered 8 M urea, yielding a urea-soluble egg antigen fraction. The urea-soluble fraction was further fractionated with Bio Gel A50m and QAE-Sephadex. All fractions were quantitatively assayed for their specific antigenic activities against serum specimens from infected rabbits by the single-tube enzyme-linked immunosorbent assay (k-ELISA). In antigen rate-limiting conditions, the urea-soluble particulate fractions were more antigenically active than the aqueous-soluble fraction. In antigen-excess and antibody-limiting assay conditions, the ideal conditions for serologic assays, the urea-derived antigens also showed superior activities against sera from infected humans. Analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on gradient gels revealed numerous low molecular weight protein bands in the aqueous-soluble fraction, whereas the urea-soluble fractions appeared to be much simpler with the majority of their proteins concentrated in one or two high molecular weight bands (greater than or equal to 200 kdaltons). Electro-transfer blots of the SDS-PAGE onto nitrocellulose papers and subsequent visualization of antigens by enzyme-linked immunoabsorbence confirmed these findings. The above data suggest that the urea-soluble fraction of S. japonicum eggs is antigenically active and has potential use in the development of a diagnostic reagent.     

IGM and IGG response to 29-35-kDa Toxoplasma gondii protein fractions in experimentally infected goats

The evolution of the humoral responses of IgG and IgM against 29-35-kDa Toxoplasma gondii fractions from experimentally infected goats were studied and compared by ELISA with the use of whole T. gondii soluble extracts and 29-35-kDa electroeluted proteins as antigens. The IgM response to electroeluted proteins was detected from wk 1 to wk 3 postinfection, showing a similar evolution to that observed when T. gondii crude extracts were used as antigens. These results suggest that this group of proteins could be used for a more specific detection of anti-T. gondii IgM. In the same way, the IgG response was equivalent in both cases, although when 29-35-kDa T. gondii fractions were used as antigens, the level of specific IgGs reached a peak 2 wk before than when T. gondii crude extract was used. The detection by ELISA of anti-T. gondii IgM in goats does not seem to be affected by the presence of specific IgG in serum samples when 29-35-kDa protein fractions were used as antigens.     

Partial purification and characterization of Gigantocotyle explanatum somatic antigens

Soluble extracts of Gigantocotyle explanatum, isolated from the liver of buffalo Bubalus bubalis were fractionated on Sephadex G-200 columns. Nine major fractions referred to as F1, F2, F3, F4, F5, F6, F7, F8 and F9 were separated. Each fraction was tested by ELISA for antigenicity using sera from G. explanatum-infected field buffaloes. Fractions F1 and F2 were highly antigenic, F3, F4, F6 and F7 were moderately antigenic and F5, F8 and F9 were poorly antigenic. Analyses by SDS-PAGE revealed that each fraction comprised several polypeptide(s) in the molecular weight range of <29 to >205 kDa. Results of Western blotting indicated that not all polypeptides which appeared in the SDS-PAGE were antigenic. The antigenic molecules of each fraction were mostly in the low molecular weight range of <14 to >94 kDa with the polypeptides in the range of >14, 14, 18, 21-25 and 34-36 kDa.     

Stimulation of Tubulin Synthesis by Lactate in Isolated Spermatogenic Cells

The effect of lactate on synthesis of new proteins in isolated spermatids and spermatocytes of rats was examined. Lactate stimulated[³⁵S]methionine ([³⁵S]met) incorporation into both spermatids and spermatocytes. The rate of protein synthesis was positively correlated with the intracellular level of ATP. The [³⁵S]met-labeled proteins in the two types of cells were compared by one and two dimensional polyacrylamide gel electrophoresis (1D and 2D-PAGE) and autoradiography. The syntheses of several stagespecific and non-specific proteins were observed. When spermatids and spermatocytes were cultured in medium without lactate, two major proteins of molecular weight (Mr) 43 kD and 55 kD were detected in the water-soluble fraction (105,000 g supernatant), and one major protein of Mr 24 kD was observed in the membrane-rich fraction. Addition of lactate to the incubation medium dramatically increased the synthesis of six proteins (Mr 14 kD, 16 kD, 43 kD, 55 kD, 84 kD and 135 kD) in the water-soluble fractions of spermatids and spermatocytes, but did not stimulate the synthesis of the Mr 24 kD protein in the membrane-rich fraction. In addition, after 1D and 2D-PAGE and electrophoretic transfer to nitrocellulose, two proteins of Mr 43 kD and 55 kD were identified as actin and tubulin, respectively, on the basis of their reactivities with specific antisera. Tubulin was also produced by in vitro translation using a spermatid lysate. These results suggest that lactate may play an important role in changing the cell structure and shape during spermatogenesis by regulating the syntheses of actin and tubulin.     

The antigenic and allergenic properties of an extract from the house dust mite Dermatophagoides farinae]

To evaluate the antigenic composition and allergenic activity of D. farinae extracts, the methods of rocket and cross immunoelectrophoresis, gel chromatography and the inhibition of the radioallergosorbent test have been used. The presence of 9-20 antigenic components has been established. The fractions of the extract with a molecular weight of 10-200 kD possessed the highest capacity of inhibiting the binding of specific IgE antibodies from the pool of sera from patients sensitized to D. farinae.     

Secretion of noncomplexed 'Big' (10-18 kD) forms of insulin-like growth factor-II by 12 soft tissue sarcoma cell lines

The paraneoplastic production of pro-insulin-like growth factor-II (IGF-II) forms causes tumour hypoglycaemias and presumably also has an effect on tumour cell growth. We investigated the molecular weights of IGF-II forms and their ability to form complexes with IGF binding proteins (IGFBPs) in conditioned culture media (CM) from 12 paediatric soft tissue sarcoma (STS) cell lines and from two healthy fibroblast lines. Untreated CM were separated by size exclusion chromatography using biocompatible HPLC. Subsequently, IGF-II, IGFBP-2 and IGFBP-3 were determined in the HPLC fractions by specific RIAs. In the CM, IGF-II concentrations between 0.5 and 8.6 ng/10(6) cells were measured but no IGF-I was detectable. Parallel to this investigation, a high IGF-II mRNA level averaging 44.4 +/- 29.7% was measured by semi-quantitative RT-PCR. The STS cell lines secreted a higher proportion of big-IGF-II forms reaching 10-18 kD (10-33% of the total IGF-II secreted) compared to the healthy fibroblasts (2.5-5%). At the same time, the proportion of IGF-II bound with IGFBP in complexes of 35- 70 kD and 150 kD was reduced by up to 85% in CM from tumour cells. The tumour cell lines apparently secrete a different spectrum of IGF-II forms than healthy fibroblasts. The reduced ability to form complexes with IGFBP and the higher molecular weight of the IGF-II forms produced by the tumour cells indicate that these forms could in fact be the known tumour-associated pro-IGF-II forms. Due to these characteristics, the big-IGF-II forms probably have an altered biological effect on the tumour cells when compared to IGF-II.     

Antigenic profile and localization of Clonorchis sinensis proteins in the course of infection.

In the course of Clonorchis sinensis infection, antigens presented to the hosts may be in a close relation to growth of the fluke. The antigenic proteins stimulating IgG antibody production were chronologically identified by immunoblot and localized by immunohistochemical staining. In the early stage of infection until 12 weeks post-infection (PI), antigens were proteins with molecular mass larger than 34 kDa which were derived from the tegument, testes and intrauterine eggs. After 20 weeks PI, antigens recognized were 29, 27 and 26 kDa proteins from the intestine, excretory bladder and reproductive organs. It is suggested that the tegumental proteins are the most potent antigens and the excretory-secretory proteins with middle molecular mass of 26-45 kDa contribute to the high level production of antibodies after 20 weeks of the C. sinensis infection.     

Expression of differentiation and age-related antigens on chicken erythroleukemia cells transformed by avian erythroblastosis virus (AEV)

Immature circulating chicken red cells express on their surface two antigenic molecules referred to as Im 48 kD and Im 140 kD antigens. The Im 140 kD antigen is not present beyond the erythroblast stage while the expression of Im 48 kD antigenic molecule remains detectable on circulating erythrocytes of embryos and young chickens, but not on erythrocytes of adult animals. In addition to Im 48 kD and Im 140 kD antigens, the avian erythroblastosis virus (AEV)-transformed erythroid cells express two novel high molecular weight (MW) immature antigens referred to as Im 150 kD and Im 160 kD. Since the transformed erythroid cells are apparently blocked at a stage close to the colony-forming units erythrocytic (CFU-E), these molecules might be expressed on these progenitor cells. The age-related antigenic molecules referred to as E1 48 kD and A 40 kD/A 85 kD antigens are detected on erythrocytes of embryos (and young chickens) and adult animals respectively. The E1 48 kD antigen as well as an antigen related to the A 40 kD were also detected on AEV-transformed erythroid cells deriving from both young chicken bone marrow and yolk sac. The presence of an adult antigen on the embryonic cells might well be related to the transformation by AEV, since the yolk sac CFU-E progenitor cells do not bear the adult antigenicity.     

Monoclonal antibodies specific for members of the genus Endotrypanum

Twenty-six monoclonal antibodies were produced against membrane-enriched preparations of Endotrypanum schaudinni or Endotrypanum sp. promastigotes. Fifteen of these monoclonal antibodies (E1-E15) reacted only with the standard strain of E. schaudinni, M6159. Monoclonal antibodies E16-E26 were considered Endotrypanum specific; no cross reactivity was detected with any other genus of the family Trypanosomatidae (Leishmania, Trypanosoma, Leptomonas, Herpetomonas or Crithidia) by dot-blot radioimmune assay. By indirect immunofluorescence assay, the antigens recognized by Endotrypanum specific monoclonal antibodies appear to be associated with the surface of the parasite. Based on Western blot analysis, 4 antigenic molecules ranging in molecular weight from 24 kD to 160 kD were identified by monoclonal antibodies specific for the strain of E. schaudinni, M6159. Monoclonal antibodies specific for the genus Endotrypanum identified an antigen of molecular weight 48 kD as well as a diffuse component migrating with an apparent molecular weight of 64-200 kD.     

Immune response to an allergenic fraction ofSetaria digitata in human filariasis

A low molecular weight antigenic fraction has been isolated from saline-soluble extracts of cattle filarial parasiteSetaria digitata. This glycoprotein fraction (Fr III) which appears to have low phosphorylcholine content cross-reacted with infective larval (L₃) antigens ofWuchereria bancrofti. Binding of human chronic filarial serum with L₃ antigens could be inhibited partially by Fr III. The fraction elicited immediate cutaneous hypersensitivity reaction in people living inWuchereria bancrofti endemic regions. The proportion of skin test positive cases was found to be highest in endemic normals in contrast to infected cases. IgE levels were however not different in chronic filariasis and in endemic normals (or in asymptomatic microfilaraemic carriers). On the other hand, specific IgG level was considerably enhanced only in chronic filariasis