Partial characterization of a 29 kDa cysteine protease purified from Taenia solium metacestodes

A 29 kDa cysteine protease of Taenia solium metacestodes was purified by Mono Q anion-exchanger and Superose 6 HR gel filtration chromatography. The enzyme was effectively inhibited by cysteine protease inhibitors, such as iodoacetic acid (IAA) and trans-epoxy-succinyl-L-leucyl-amido (4-guanidino) butane (E-64) while inhibitors acting on serine- or metallo-proteases did not affect the enzyme activity. The purified enzyme degraded human immunoglobulin G (IgG), collagen and bovine serum albumin (BSA), but human IgG was more susceptible for proteolysis by the enzyme. To define the precise biological roles of the enzyme, more detailed biochemical and functional studies would be required.     

Regulation of fungal proteolysis on cyclic AMP-dependent protein kinase,cyclic AMP phosphodiesterase,glycogen synthase and histones

Limited proteolysis of catalytic and regulatory subunits of cyclic AMP-dependent protein kinase (A-pk), cyclic AMP phosphodiesterase, glycogen synthase, and histones by fungal protease (type XIX) was analyzed by the digested peptide bands in SDS polyacrylamide gel electrophoresis. The modulatory effects on proteolysis by nucleotides, polypeptides, and phospholipids may greatly depend on the intrinsic nature of substrates. The proteolysis of the regulatory subunit of A-pk and glycogen synthase was not regulated by nucleotides and nucleic acids. In comparison, phosphatidyl serine, cardiolipin, and pepstatin A stimulated the proteolysis of the catalytic subunit of A-pk. Whereas, DNA (Hind III digest), t-RNA, GTP, phosphatidyl serine, sphingosine inhibited the proteolysis of cyclic AMP phosphodiesterase. Moreover, MS2 RNA, DNA, t-RNA, dGTP, Phosphatidyl serine, phosphatidyl inositol, antipain, and chymostatin exerted inhibitory proteolytic effect on histone VIII-S. Some of these agents also had similar inhibitory effect on other types of histones (types III-S and VII-S). The inhibitory effect of phosphatidyl serine on proteolysis of histone may be due to their interaction which was monitored by the drastic increase of uv absorbance.     

Inhibition of immune precipitation by complement

Normal human complement serum (NHS) inhibited precipitin reactions between tetanus toxoid and human or rabbit anti-tetanus toxoid IgG antibody, between bovine serum albumin (BSA) and rabbit anti-BSA IgG antibody, and between hen egg albumin and rabbit anti-egg albumin IgG antibody. Ethylene-diaminetetraacetic acid (EDTA) prevented this inhibition. Mg-ethyleneglycol-bis(aminoethyl)-tetra-acetic acid-(EGTA) also prevented the inhibition except with lower concentrations of antibody and antigen. Therefore, the inhibition of immune precipitation seemed to occur mainly through the classical pathway of complement activation. The alternative pathway was usually dispensable, but it augmented the inhibition. Guinea pig complement serum (NGS) was less effective than NHS in inhibiting immune precipitation. Guinea pig serum deficient in C4 (C4DGS) did not inhibit the immune precipitation. Mouse complement serum was effective for inhibiting precipitation, and C5-deficient serum was as effective as normal serum. Therefore, the inhibition of immune precipitation is considered to occur by activation of complement up to the step of C3. The size of the soluble immune complexes formed in the presence of NHS varied depending on the concentrations of antibody and antigen, even when the ratio of antigen to antibody was constant. On incubation at 37 degrees C immune precipitation was inhibited by 1/2 dilution of NHS for 2 to 3 hr and then gradually increased to the level in the absence of complement. When the immune complexes were formed in the presence of serum containing complement, fragments of C4 and C3 were incorporated into the soluble immune complexes. The C3 fragments incorporated into the soluble complexes were C3b, iC3b, C3c, and C3d, some of which were bound covalently with heavy chains of IgG antibody molecules. Some of the covalent linkages between C3 fragments and IgG seemed to be destroyed by alkali treatment, but not by hydroxylamine treatment. The formation of covalent bonds between IgG and C3 and probably C4 was essential for inhibition of immune precipitation, because inhibitors of their formation, such as putrescine, cadaverine, and salicylhydroxamic acid, effectively prevented the inhibition of precipitation. When antigen and antibody reacted in the presence of mixtures of various combinations of isolated complement components, C1, C4, C2, and C3 showed maximal inhibition of immune precipitation, whereas factors I and H had little effect.     

Phosphorylation and proteolytic degradation of nucleolin from 3T3-F442A cells

The effect of phosphorylation on the proteolysis of nucleolin has been investigated. Nucleolin is readily phosphorylated both in vitro and in vivo. Utilizing phosphorylation assays and immunoblotting with anti-nucleolin serum, we have observed that phosphorylation enhances nucleolin as a substrate for a protease. This protease activity cleaves the protein into a highly phosphorylated 30 kDa peptide and a 72 kDa peptide. The involvement of casein kinase II is suggested since this cleavage is promoted by spermine and inhibited by heparin, which are, respectively, a stimulator and an inhibitor of casein kinase II activity. The molecular identity of the protease and the physiologic significance of the proteolytic cleavage of nucleolin remain to be studied.     

The bactericidal action of human neutrophils on meningococci in vitro

32 Russian patients with late complement component deficiency (LCCD) were immunize with tetravalent meningococcal polysaccharide vaccine (A + C + W135 + Y). Their immune response and infectious morbidity rate were followed for 6 years and the partial protective efficacy of vaccination was demonstrated. As the antibody-mediated complement-induced bactericidal activity of plasma was completely absent in persons with LCCD, the bactericidal action of human neutrophils on meningococci of groups A, W135 and B was studied under the conditions of incubation with serum samples collected from persons with LCCD before and after vaccination. In LCCD serum alone the exponential growth of meningococci was observed, while the addition of human neutrophils resulted in the essential inhibition of the growth of meningococci (up to their complete elimination). The proportion of serum samples stimulating the elimination of group A and W 135 meningococci by neutrophils was almost 40% of the serum samples collected before vaccination and significantly increased among the serum samples collected after vaccination (up to 84%) or revaccination (up to 90%). At the same time the capacity of an individual serum sample to promote the bactericidal effect of neutrophils against meningococci correlated with its content of specific anti-polysaccharide IgG and IgM antibodies, as well as antibodies to the inner core of lipopolysaccharide. The interaction of neutrophils with meningococci was significantly inhibited after incubation in heat-inactivated serum, suggesting that this interaction was partly mediated along the following path: the binding of IgM and IgG antibodies with bacteria--the activation of complement and the deposition of C3 complement on bacteria--the binding of meningococci with CR3 receptors of neutrophils.     

Identification,characterization, and cloning of an immunoglobulin degrading enzyme in the cat flea,Ctenocephalides felis

The degradation of cat immunoglobulin G (IgG) in blood-fed adult C. felis midguts was examined. SDS-PAGE analysis of dissected midgut extracts obtained from C. felis that had been blood fed for various times between 0 to 44 h revealed that by 24 h most of the high molecular weight proteins, including the heavy chain of IgG, were digested. A 31-kDa serine protease with IgG degrading activity was purified from fed C. felis midguts by benzamidine affinity chromatography, hydrophobic interaction chromatography, and cation exchange chromatography. Three primary cleavage products between 30- and 40-kDa were observed when the purified protease was incubated with protein A purified cat IgG. N-terminal amino acid sequence analysis of the products revealed that the IgG degrading protease cleaves after specific cysteine and lysine residues within the hinge region of IgG. The enzyme is also capable of degrading other immunoglobulins, serum albumin, and hemoglobin, suggesting that it may have roles in both combating the host's immune system and providing nutrients for the flea. A cDNA clone encoding the 265 amino acid IgG degrading protease proenzyme was isolated. When expressed in a baculovirus/insect cell expression system, the recombinant protein had the same N-terminus as the processed 237 amino acid mature native protein and possessed IgG degrading activity indistinguishable from the native protein. Arch. Insect Biochem.     

Association of an albumin antigen with phosphatidylcholine liposomes alters the nature of immunoglobulins produced during the immune response against the antigen

Human serum albumin has been injected intravenously in rabbits either free in solution or associated with liposomes. Blood samples were obtained from the rabbits at various time intervals after injection, and two different antibody determinations were performed in each sample. Whereas a haemagglutination technique was applied for determination of predominantly IgM anti-human serum albumin antibodies, a second technique, using antigen-coated Sepharose beads and horseradish peroxidase-conjugated anti-rabbit IgG, was used to detect the IgG anti-human serum albumin antibodies. Liposomes appeared to enhance strongly the IgM response against human serum albumin. No such marked differences were found, however, between the IgG responses against liposome-associated or free human serum albumin. The conclusion is drawn that the immunoadjuvant effect of liposomes during the primary immune response against an albumin antigen is mainly due to an enhanced IgM antibody production.     

Proteolysis activity of IgM antibodies from rheumatoid arthritis patients' sera: evidence of atypical catalytic site

The IgM antibodies from rheumatoid arthritis (RA) patients' sera were screened for peptide hydrolyzing activity. Recovery of structurally intact IgM antibodies (Abs), in a single step, was achieved using a weak anion-exchange methacrylate monolith disk. The IgM Abs from patients' sera hydrolyzed the Pro-Phe-Arg-4-methyl-coumaryl-7-amide (PFR-MCA) substrate appreciably compared to the healthy donors. The apparent K(m) values of IgM Abs from patients' sera were between 0.4 and 0.7 mM. Furthermore, IgM Abs displayed 5 to 10-folds greater proteolysis activity than IgG Abs, recovered from the same pathological serum. The proteolysis activity, as a function, was found to be independent of IgM-RF titer value. Affinity labeling approach targeted at the catalytic site histidine was studied, using a specific irreversible inhibitor, N-α-tosyl-L-lysine chloromethyl ketone (TLCK). Despite modification of catalytic His, observation of serine protease like activity suggest presence of an atypical catalytic framework in a few pathological IgM Abs.     

Serological and Genetic Evidence for Altered Complement System Functionality in Systemic Lupus Erythematosus: Findings of the GAPAID Consortium

Systemic lupus erythematosus is a chronic autoimmune disease with multifactorial ethiopathogenesis. The complement system is involved in both the early and late stages of disease development and organ damage. To better understand autoantibody mediated complement consumption we examined ex vivo immune complex formation on autoantigen arrays. We recruited patients with SLE (n = 211), with other systemic autoimmune diseases (n = 65) and non-autoimmune control subjects (n = 149). Standard clinical and laboratory data were collected and serum complement levels were determined. The genotype of SNP rs1143679 in the ITGAM gene was also determined. Ex vivo formation of immune complexes, with respect to IgM, IgG, complement C4 and C3 binding, was examined using a functional immunoassay on autoantigen microarray comprising nucleic acids, proteins and lipids. Complement consumption of nucleic acids increased upon binding of IgM and IgG even when serum complement levels were decreased due to consumption in SLE patients. A negative correlation between serum complement levels and ex vivo complement deposition on nucleic acid autoantigens is demonstrated. On the contrary, complement deposition on tested protein and lipid autoantigens showed positive correlation with C4 levels. Genetic analysis revealed that the non-synonymous variant rs1143679 in complement receptor type 3 is associated with an increased production of anti-dsDNA IgG antibodies. Notwithstanding, homozygous carriers of the previously reported susceptible allele (AA) had lower levels of dsDNA specific IgM among SLE patients. Both the non-synonymous variant rs1143679 and the high ratio of nucleic acid specific IgG/IgM were associated with multiple organ involvement. In summary, secondary complement deficiency in SLE does not impair opsonization of nucleic-acid-containing autoantigens but does affect other antigens and potentially other complement dependent processes. Dysfunction of the receptor recognizing complement opsonized immune complexes promotes the development of class-switched autoantibodies targeting nucleic acids.     

ATP-dependent degradation of 125I-bovine serum albumin by rabbit reticulocytes does not need repression of an endogenous inhibitor

The ubiquitin-dependent proteolysis of 125I-bovine serum albumin in rabbit reticulocytes has been investigated. Using various reticulocyte fractions (reticulocyte protease, inhibitor-free protease, "ubiquitin" and "inhibitor") in the presence or absence of ATP, we found that the repression of an endogenous inhibitor, as suggested by others for alpha-casein proteolysis, is unlikely for bovine serum albumin. Therefore, differences exist in the ATP-dependent proteolytic pathway of rabbit reticulocytes depending on the substrate. Fractionation of the reticulocyte ATP-dependent proteolytic system revealed at least two proteolytic and two inhibitory fractions involved in the proteolysis of bovine serum albumin.     

Serum of normal subjects contains IgG with antibody-like specificity for phospholipids

Using counterimmunoelectrophoresis, sera from normal subjects display precipitin activity against liposomes made by phosphatidic acid, phosphatidyl ethanolamine, phosphatidyl serine and cardiolipin. No activity is detected by using liposomes made with phosphatidyl choline, sphingomyelin and phosphatidyl inositol. The same pattern of precipitation is obtained when pure IgG from normal subjects is tested against phospholipids. No precipitin activity is found by using pure IgM. The precipitin reaction is prevented by preincubating liposomes with Fab obtained from normal IgG.     

Purification and Characterization of Canine Serum Ferritin-binding Proteins

Ferritin-binding protein (FBP) is known to interact with circulating ferritins in mammals. Canine FBPs were purified from canine serum by affinity chromatography and were identified as IgM, IgG, and IgA by immunoblotting with alkaline phosphatase-labeled antibodies to canine IgM, IgG, and IgA heavy chains. Following further purification by application to a Sephacryl S-300 column, canine FBPs were separated into 81.3- and 27.7-kDa bands by sodium dodecyl sulfate-polyacryamide gel electrophoresis, and the 81.3-kDa band reacted with the anti-canine IgM heavy chain antibody. Purified canine FBP bound to canine liver ferritin, but not to canine albumin and transferrin. FBP showed greater binding to the expressed bovine ferritin H-chain homopolymer than to the expressed bovine ferritin L-chain homopolymer. The binding of FBP with canine liver ferritin was dose-dependently inhibited by anti-rat liver ferritin antibody, and the anti-ferritin antibody dissociated the bound FBP in a dose-dependent manner, even after binding FBP with liver ferritin. The canine ferritin H subunit peptide fragment with amino acid residues 148–155 (NH₂-GDHVTNLR-COOH) in its C-terminal region was recognized by FBP. These results indicate that canine serum FBPs are autoantibodies to ferritin (IgM, IgG, and IgA) and that anti-ferritin autoantibody (IgM) recognizes the C-terminal region of ferritin H subunit.     

Effect of Serum Heat-Inactivation and Dilution on Detection of Anti-WNV Antibodies in Mice by West Nile Virus E-protein Microsphere Immunoassay

Immunopathogenesis studies employing West Nile virus (WNV) mice model are important for the development of antivirals and vaccines against WNV. Since antibodies produced in mice early during WNV infection are essential for clearing virus from the periphery, it is important to detect early and persistent anti-WNV antibodies. ELISA and plaque reduction neutralization tests are traditionally used for detection of anti-WNV antibodies and WNV-neutralizing antibodies, respectively. Although these assays are sensitive and specific, they are expensive and time consuming. Microsphere immunoassays (MIA) are sensitive, specific, allow for high throughput, are cost effective, require less time to perform than other methods, and require low serum volumes. Several assay parameters such as serum heat-inactivation (HI) and dilution can alter WNV MIA sensitivity. We examined the effect of these parameters on WNV E-protein MIA (WNV E-MIA) for the enhanced detection of anti-WNV IgM and IgG antibodies. WNV E-MIA was conducted using serial dilutions of HI and non-HI (NHI) serum collected at various time points from mice inoculated with WNV. HI significantly enhanced detection of IgM and IgG antibodies as compared to NHI serum. WNV IgM and IgG antibodies in HI sera were detected earlier at day 3 and IgM antibodies persisted up to day 24 after infection. HI serum at 1∶20 dilution was found to be optimal for detection of both IgM and IgG antibodies as compared to higher-serum dilutions. Further, addition of exogenous complement to the HI serum decreased the WNV E-MIA sensitivity. These results suggest that serum-HI and optimal dilution enhance WNV E-MIA sensitivity by eliminating the complement interference, thereby detecting low-titer anti-WNV antibodies during early and late phases of infection. This improved MIA can also be readily employed for detection of low-titer antibodies for detection of other infectious agents and host proteins.     

Growth hormone alters metabolic effects and proteolysis of insulin in adipose tissue during lactation.

To study the regulation of lipogenesis in adipose tissue by insulin and growth hormone during lactation, tissue was biopsied from primiparous bovines at 30 days antepartum and 60 days postpartum. Tissue was cultured for 24 hr or 48 hr in M199 with acetate and glucose, with a change of medium at 24 hr. The three in vitro treatments were: insulin and hydrocortisone at 10 and 50 ng/ml, respectively (IH); IH + 10 ng/ml of growth hormone (G10); and IH + 100 ng/ml of growth hormone (G100). IH allowed lipogenesis rates from 50% to 85% of those in fresh tissue. Addition of 10 ng/ml of growth hormone reduced (P less than 0.05) lipogenesis; at 100 ng/ml, the effect was only slightly greater. The hypothesis that insulin and growth hormone could be degraded by bovine adipose tissue was tested. Adipose tissue cell-free extracts degraded 125I-labeled insulin, but did not degrade labeled growth hormone. The insulin protease activity was further characterized and had a pH optimum of 7.1, a maximum hydrolysis of approximately 70%, and a hydrated molecular mass of approximately 23,000 daltons. Insulin proteolysis was inhibited by specific insulin protease inhibitors and stimulated by disulfide reducing agents. Bovine growth hormone, prolactin, and histone inhibited (P less than 0.05) the proteolysis of insulin, while bovine serum albumin, egg albumin, trypsin inhibitor, and lysozyme did not. Adipose tissue from pregnant and lactating bovines was sensitive to insulin and growth hormone, and growth hormone may modulate activity of an insulin-specific protease.     

Studies on the interaction between protein A and immunoglobulin G. I. Effect of protein A on the functional activity of IgG.

Staphylococcal protein (A (PA) and IgG anti-Forssman immunoglobulin formed complexes that behaved functionally like IgM in their ability to lyse sheep erythrocytes (E) in the presence of whole guinea pig complement (GPC) and to fix purified guinea pig C1. Concanavalin A, a plant lectin that inhibited IgM but not IgG hemolytic activity, inhibited the hemolytic activity of IgG-protein A complexes that behaved like IgM but had no effect on complexes that behaved functionally like IgG. Since Con A is known to bind specifically to glucose and mannose residues, our results suggested that the interaction of protein A with the Fc region of IgG led to exposure of sugar moieties that may participate in complement (C) binding. The production of IgM-like complexes depended on the ratio of protein A to IgG and the empirical formula of these IgM-like complexes was found to be [(IgG)2PA]n. As the ratio of PA to IgG was increased, the resulting complexes tended to behave functionally like IgG but with reduced hemolytic activity and C1 fixing ability. Furthermore, the binding of C1 to EIgG was inhibited by PA and the binding of PA to EIgG was inhibited by C1 indicating that the binding sites for C1 and PA were located near each other or were identical. Our results offer a reasonable explanation for the reported effects of PA or mixtures of PA and IgG in vitro and in vivo.     

Humoral components of immunological response in nephrotic syndrome

Serum and urine were collected from 58 patients with nephrotic syndrome. Immunoglobulins (IgA, IgG and IgM), complement (C3) and transferrin levels were measured by single radial immunodiffusion. The extent of glomerular injury was estimated by determining the selectivity of proteinuria. The relationship between the severity of glomerular damage and serum concentrations of immunoglobulins and complement was assessed. Higher IgM and lower IgG serum concentrations were found in nephrotic patients than in normal controls (157 +/- 108 mg+ vs 127 +/- 38 mg% for IgM, 929 +/- 537 mg% for IgG). The difference was statistically significant (p less than 0.05 for IgM, p less than 0.001 for IgG). No correlation was present between the selectivity of proteinuria and serum levels of IgA, IgM, IgG or C3. The results indicate that abnormalities in humoral components of the immune system are present in nephrotic patients and are probably related to a basic immunological defect in the patients rather than to the severity of glomerular damage.     

Echinococcus multilocularis: distribution and persistence of specific host immunoglobulins on cysts membranes

Cyst vesicles were obtained from larval cyst masses of Echinococcus multilocularis grown in Medium 858 in vitro or isolated from the intraperitoneal or subcutaneous larval cyst mass of C57BL/6J mice infected 12 weeks previously. Antigenic determinants were present on the outermost section of the laminated layer and throughout the germinal layer of the cysts. Specific host antibodies of the IgG1, IgG2a, IgG2b, and IgM classes and complement component C3 but not host IgA or C-reactive protein were detected on the intact cysts by the indirect immunofluorescent technique. Specific antibodies were bound to the epitopes on the laminated layer but were not detected on the germinal layer. Host albumin, however, was found on the laminated layer, germinal layer, and within the intact cyst. IgG2a and IgG2b were high-affinity antibodies but IgG1 and IgM were low-affinity antibodies as they were eluted easily from the laminated layer with two washes of sodium phosphate buffer (0.01 M, pH 7.2) containing 0.15 M NaCl. The significance of bound antibody in complement activation and antibody-dependent cell-mediated cytotoxicity of the proliferative phase (cyst) of alveolar hydatid cyst is discussed.     

Reticulocyte lipoxygenase, ingensin, and ATP-dependent proteolysis

Lipoxygenase purified from rabbit reticulocyte lysate has a molecular mass of 68 kDa on SDS gel and a pI of 5.97. Lipoxygenase is inhibited by nordihydroguaiaretic acid (NDGA), 3-amino-1-(m-(trifluoromethyl)phenyl)-2-pyrazoline (BW755C), 5,8,11,14-eicosatetraynoic acid (ETYA), salicylhydroxamate (SHAM) or hemin. Metal ions or nucleotides do not affect its activity. The addition of certain of these inhibitors to the reticulocyte extract also inhibited the ATP-dependent proteolysis of casein, one of the distinct characteristics of reticulocytes. No clear correlation between lipoxygenase activity and ATP-dependent proteolysis could be detected. Hemin and NDGA inhibited both processes, but the concentrations necessary for inhibition were quite different. SHAM completely inhibited lipoxygenase, but not proteolysis. o-Phenanthroline inhibited ATP-dependent proteolysis, but had no effect on lipoxygenase activity. We have also purified a high-molecular-mass protease, ingensin, from reticulocyte extract. This protease accounted for more than 90% of the casein-degrading activity in reticulocyte extract. NDGA inhibited ingensin at the same concentrations required for inhibition of ATP-dependent proteolysis. These results suggest that lipoxygenase is not indispensable for the ATP-dependent proteolysis and the novel high-molecular-mass protease, ingensin, may be involved in the process.     

Activation of serum complement leads to inhibition of ascorbic acid transport

Ascorbic acid is transported into 3T6 fibroblasts by a carrier-mediated, energy-dependent saturable active process with a Km of 112 microM and Vmax of 158 pmole/min/mg protein. The transport is dependent on extracellular Na+ concentration which reduces the Km. It was recently observed in this laboratory that bovine serum contained a heat-labile factor which, after interaction with bacterial endotoxin (lipopolysaccharides), inhibited ascorbic acid transport (J.J. Alleo and H. Padh, Proc Soc Exp Biol Med 179:128-131, 1985). We report here that the inhibition of ascorbic acid transport by endotoxin is mediated by the activation of serum complement. This was done by examining the activation of complement by other activators like zymosan and immunocomplexes (e.g., albumin and antibodies to albumin). Ascorbate transport was inhibited by the mixture of unheated serum and the activators. No inhibition was observed with serum devoid of C3 (component 3 of the complement). When C3-deficient serum was reconstituted by the addition of purified C3, the endotoxin-induced inhibition of ascorbate transport was restored. The implication of these findings is that in spite of a normal intake and blood level of the vitamin, tissues may not be getting adequate vitamin C during disease states when the complement in serum is activated. In other words, what may be considered an adequate intake of vitamin C under health conditions may not be adequate under disease conditions.     

Estimation and Identification of Streptococcus dysgalactiae Protective Antibody in Sera of Rabbits and Cattle

Rabbit and cow anti-Streptococcus dysgalactiae sera were tested by bacterial agglutination, complement fixation, hemagglutination, and immunodiffusion for the presence of antibody. The results of these tests were compared with mouse-protection studies on the same serum to estimate which in vitro test would best reflect the in vivo protective capacity of serum. Identification of the antibody constituents responsible for the mouse protection, hemagglutination, and complement fixation titers were established by reacting whole and diluted antisera with mercaptoethanol before and after testing. Results indicate that the complement fixation test may be a more accurate indicator of IgG protective bovine and rabbit antibody, whereas the hemagglutination test may more readily reflect a wider range of protective antibody levels and IgM. The complement fixation test showed some shared responses to IgG and IgM in both the rabbit and cow, whereas the IgM components seemed to be the predominant factor influencing hemagglutination titers in the rabbit and more so in the bovine. Mouse protection tests with mercaptoethanol-treated cow and rabbit sera indicate that the protective capacity of these antisera is shared between IgM and IgG components.