Transmission of maternal antibody prenatally and from milk into serum of neonatal rabbits.

Rabbit dams fed 0.1% BSA for various periods before and during lactation produced anti-BSA of low avidity and IgG isotype in serum and milk. Milk anti-BSA and IgG concentrations were one-third to one-half of those in the serum. At birth, kits had IgG and anti-BSA serum concentrations approximately equal to their dams. Both fell rapidly for the first 10-20 days, levelling off at about 1 mg IgG/ml. Kits born to unimmunized dams and suckled by dams with anti-BSA in the milk showed increasing anti-BSA in serum for the first 12-16 days, falling by 20 days. Foster-suckling on immunized dams beginning at various times after birth showed antibody uptake from birth through 12 days of age. Thus immunoglobulins are among factors absorbed from milk that have potential for regulating the immune responses of rabbit neonates.     

Natural killer cell-dependent immunoglobulin G2a anti-bovine serum albumin (BSA) response elicited by high molecular weight dextran-BSA conjugates associated with dextran-mediated macrophage-natural killer cell interaction

The roles of the interferon-gamma (IFN-gamma) and interleukin-12 (IL-12) produced during natural killer (NK) cell interaction with macrophages (M phi) were investigated as the basis for the induction of immunoglobulin G2a (IgG2a) anti-bovine serum albumin (BSA) responses by high molecular weight dextran conjugated to BSA (HMW-DEX-BSA). BALB/c mice immunized with HMW-DEX-BSA produced significantly higher levels of both IgG1 and IgG2a anti-BSA than did mice immunized with BSA alone. Both IgG1 and IgG2a anti-BSA levels were higher in mice immunized with BSA conjugated to dextran of molecular weight (MW) 5 000 000-40 000 000 compared with dextran of MW 10,000-60,000. The enhancement of anti-BSA IgG2a levels but not of anti-BSA IgG1 levels was inhibited when free BSA was added to the HMW-DEX-BSA conjugate. NK cell depletion during HMW-DEX-BSA immunization of mice resulted in significantly lower anti-BSA IgG2a levels without affecting anti-BSA IgG1 levels. Naive splenocytes or M phi + NK cell co-cultures incubated with HMW-DEX or HMW-DEX-BSA produced higher IFN-gamma levels than splenocytes or co-cultures incubated with BSA alone. HMW-DEX stimulated both IFN-gamma and IL-12 production by M phi + NK cell co-cultures in a dose-dependent manner. DEX-induced IFN-gamma production by NK cells was dependent upon the presence of IL-12, and IL-12 production by M phi was dependent upon the presence of IFN-gamma in these co-cultures. Both M phi and NK cells bound DEX to their surfaces. These data demonstrate that BSA linked to HMW-DEX enhanced both T-helper-1- and T-helper-2-associated antibody responses to BSA. The results also indicate an IL-12-dependent positive feedback interaction between NK cells and M phi that supports a NK cell/IFN-gamma-dependent mechanism for enhancement of anti-BSA IgG2a antibody responses in mice immunized with HMW-DEX-BSA protein conjugates.     

Immune Response in the Bovine Mammary Gland After Intestinal, Local, and Systemic Immunization

The immune response in mammary glands of cattle was measured after intestinal, local, and systemic immunization with T4 bacteriophage. Nonlactating pregnant cows were immunized by infusions into the intestine or mammary gland and by subcutaneous injections in the region of the prescapular or external inguinal lymph nodes. Titers of antibodies of different isotypes were measured in serum and in lacteal secretions by enzyme-linked immunosorbent assay, and numbers of cells producing antibodies of each isotype were determined in lacteal secretions by the Jerne plaque assay. Substantial increases in immunoglobulin G subclass 1 (IgG1) and IgG2 antibody titers were detected in serum and lacteal secretions of animals immunized through an intestinal fistula. IgM and IgA antibody responses were low or undetectable. Low numbers of IgA and IgG1 plaque-forming cells were occasionally detected. It is proposed on the basis of these data that migration of antigen-stimulated IgG lymphoblasts, and perhaps of antigen, to spleen and peripheral lymph nodes may be dominant events after intestinal immunization of ruminants. This is consistent with the predominance of serum-derived IgG antibodies in colostrum and milk. Intramammary infusion of antigen gave rise to increases in antibody titers in all classes which were greater not only in lacteal secretions but also in blood serum than with either systemic route used. There was clear evidence from relative antibody titers for local synthesis of antibodies, principally IgA and IgG1, in the immunized glands. Comparison of IgA titers in secretions from the immunized glands with those in serum also suggested that locally synthesized IgA antibodies may have contributed in some measure to serum titers. Local synthesis in both immunized and nonimmunized glands was also reflected by the presence of increased numbers of IgA and IgG1 plaque-forming cells. It is hypothesized that antibody-forming cells responsible for local synthesis originated in lymphoid tissue within the mammary gland or from peripheral lymph nodes, depending upon the route of immunization.     

Humoral immune response to the capsid components of recombinant adenoviruses: Routes of immunization modulate virus-induced Ig subclass shifts

This study examines in detail the capsid-specific humoral immune response of BALB/c mice after one single injection of a replication-defective adenovirus. Two routes of immunization, intravenous (i.v.) and intraperitoneal (i.p.), were compared for the response induced against the adenovirus particle and the three major components of the viral capsid, hexon, penton base, and fiber. A single immunization with the replication-defective adenovirus induces a long and persistent humoral response specific for the virus. However, the molecular components of the viral capsid are differentially recognized depending on the route of immunization. The sera from mice immunized i.p. recognized only the hexon protein and a preferential switch to the IgG2a subclass was obtained which remained stable 100 days post-immunization. The sera obtained from mice immunized i.v. gave a more complex response. At the beginning of the response, an isotype bias toward the IgG2a subclass was observed, but the isotype distribution changed during the whole period of the response. Neutralizing activity was maximum 45 days after immunization by both routes, and no activity was detectable after 3 months. However, the i.v. serum displayed a higher neutralizing activity than the i.p. serum. The IgM antiviral antibodies appeared to be an important component of the neutralizing activity, and the two routes of immunization do not induce the same IgG isotypes to neutralize viral infectivity. Extension of these findings to human gene therapy using recombinant adenoviruses may help to characterize the precise viral protein targets of neutralizing antibodies.     

The role of IgM rheumatoid factor in experimental immune vasculitis.

The effect of IgM rhematoid factor (RF) on reversepassive cutaneous Arthus reaction in rats was studied. The RF was obtained from the serum cryoglobulin of a patient with symptoms of purpura, arthralgia and digital gangrene. The cryoglobulins was of IgG-IgM type and when given i.v it induced a prompt hypocomplementaemia in experimental animals. The purified RF also induced low serum complement levels when injected i.v. along with complexes of non-complement-fixing, aggregated IgG. A reverse passive Arthus reaction was induced by intradermal injection of IgG anti-bovine serum albumin (BSA), followed by an i.v. dose of antigen (Ag). The cutaneous inflammatory reaction was aggravated by simultaneous administration of IgM RF intradermally, but not by IgM without antibody (Ab) properties. Intradermal injection of low concentrations of non-complement-fixing IgG anti-BSA, along with normal human IgM, followed by i.v. injection of BSA, resulted in a complete lack of cutaneous inflammation. At higher Ab concentrations there was only a mild inflammation. However, when IgM RF was substituted for normal IgM and injected with non-complement-fixing anti-BSA, an effective reverse passive cutaneous Arthus reaction and vasculitis was induced. The inflammatory response was greatly suppressed by decomplementation of animals by cobra venom factor. This study provides evidence favouring an inflammatory, complement-dependent role for RF in vasculitis.     

Distinctive humoral immune responses following anterior chamber and intravenous administration of soluble antigen. Evidence for active suppression of IgG2-secreting B lymphocytes.

Inoculation of soluble antigen into the anterior chamber (AC) of the eyes of mice and rats induces a distinctive form of immune deviation known as anterior chamber-associated immune deviation (ACAID). Similarly, intravenous injections of soluble antigen induce immune deviation. In both instances, a selective impairment of delayed hypersensitivity (DH) is observed, whereas humoral immunity is said to be preserved. Recently, we noted that radiolabelled bovine serum albumin (BSA) was not eliminated in an immune fashion from the blood of animals pretreated with this antigen via AC and intravenous (i.v.) routes of inoculation. This was puzzling because the sera of these animals contained easily measurable anti-BSA antibodies. We have examined the characteristics of the anti-BSA humoral responses of mice following i.v. and AC inoculation of BSA in order to understand the reason for the lack of immune elimination. The results indicate that AC and i.v. recipients fail to eliminate antigen in an immune fashion because they produce insufficient amounts of complement-fixing (IgG2) antibodies, even though the other isotypes of immunoglobulins are well represented in the humoral anti-BSA response. The pattern of antibody isotype production, especially after boosting with BSA in complete Freund's adjuvant (CFA), implies that activation of IgG2-secreting, BSA-specific B cells is suppressed. Evidence is presented demonstrating this suppression to be antigen-specific and mediated by CD8+ T lymphocytes. These data are compatible with the hypothesis that interleukin-4 (IL-4)-secreting T helper (Th) cells are selectively activated in ACAID, whereas interferon-gamma (IFN-gamma)IL-2-secreting Th cells are actively suppressed.     

Origin and Kinetics of IgA, IgG and IgM Milk Antibodies in Primary and Secondary Responses of Rats

IgA and IgM antibodies were detected in rat milk after immunization with ferritin in Peyer's patches (Pp) 1 day after parturition but not after intramammary gland or intravenous immunization. The antibody levels decreased from day 9 to day 17 of the nursing period and were undetectable during a second lactation period. Despite the absence of milk IgM antibodies after intramammary gland or intravenous immunization, the serum levels of the IgM antibodies were similar after all three immunization methods. IgA antibodies were not found in serum after any of the immunization methods.IgG antibodies appeared in serum and milk after P. intramammary gland, and intravenous immunization. Milk and serum IgG antibodies from all the Pp-immunized animals decreased from day 9 to day 17 of the lactation period. After intramammary gland immunization, however, the IgG antibody levels increased in all the milk samples, but only in four of seven sera. The milk and serum IgG antibody levels were lower but still detectable during a second lactation period. Re-injection of ferritin in the Pp during a third lactation period resulted in higher levels of milk IgA, IgG and IgM antibodies than after the first injection. Rats with serum IgG antibodies against Escherichia coli 08 naturally present in their gut flora had no corresponding milk antibodies of any isotype. The results suggest tht milk antibodies of all three isotypes stem from local production in the mammary gland and that blood IgG and IgM antibodies originate at least partly from stimulation in Pp.     

Bacillus thuringiensis Cry1Ac Protoxin is a Potent Systemic and Mucosal Adjuvant

Recently we demonstrated that recombinant Cry1Ac protoxin from Bacillus thuringiensis is a potent systemic and mucosal immunogen. In this study we compared the adjuvant effects of Cry1Ac and cholera toxin (CT) for the hepatitis B surface antigen (HBsAg) and bovine serum albumin (BSA). The antibody responses of intestinal secretions and serum were determined by ELISA in Balb/c mice immunized through the intragastric (IG) or intraperitoneal (IP) routes. When HBsAg was administered via IG, the anti-HBsAg intestinal response was not enhanced by either Cry1Ac or CT, whereas via IP Cry1Ac increased the anti-HBsAg intestinal immunoglobulin (Ig)G response and CT increased the intestinal IgA and IgM responses. Serum anti-BSA antibodies increased when BSA was co-administered with CT or Cry1Ac by both routes. Cholera toxin and Cry1Ac co-administered via IP increased the IgG anti-BSA response in fluid of the large intestine and CT also increased the IgA and IgM responses slightly. When co-administered via IP, CT and Cry1Ac did not affect the IgG anti-BSA response of the small intestine significantly. We conclude that Cry1Ac is a mucosal and systemic adjuvant as potent as CT which enhances mostly serum and intestinal IgG antibody responses, especially at the large intestine, and its effects depend on the route and antigen used. These features make Cry1Ac of potential use as carrier and/or adjuvant in mucosal and parenteral vaccines.     

Effect of vitamin A on the systemic and local antibody responses to intragastrically administered bovine serum albumin.

The effects of vitamin A on the immune response to bovine serum albumin (BSA) were studied in adult mice. Treatment with vitamin A by the intragastric or parenteral routes markedly increased the local as well as the systemic antibody response to different concentrations of the antigen (BSA). In contrast, in animals given BSA alone, antigen concentrations above a certain dose resulted in a decreased or even absent anti-BSA response. These studies suggest that vitamin A may be an appropriate adjuvant in oral immunization.     

Respiratory syncytial virus recombinant F protein (residues 255-278) induces a helper T cell type 1 immune response in mice

We have developed and evaluated an immunodominant respiratory syncytial virus (RSV) F antigen in a mouse model. The antigenic region corresponding to amino acids 255-278 of the RSV F protein was cloned into a vector containing the ctxA(2)B gene of cholera toxin (CT). The recombinant protein was expressed in Escherichia coli and analyzed on sodium dodecyl sulfate-polyacrylamide gels. The purified protein was evaluated by immunoblot and ganglioside GM(1) enzyme-linked immunosorbent assay to confirm the expression of the RSV F protein and to correct association of the recombinant protein to form a holotoxin-like chimera, respectively. We hypothesized that genetic fusion of modified CT-based adjuvant with RSV F immunodominant epitopes (rRF-255) would induce protective humoral and cellular immune responses in mice. Intranasal immunization of mice with rRF-255 overall induced higher concentrations of anti-RSV F-specific antibodies in both serum and saliva as compared with mice immunized intranasally with RSV or phosphate-buffered saline (PBS). Antibody isotype analysis (IgA, IgG1, IgG2a, and IgG2b) was also performed. The predominant IgG2a antibody isotype response in combination with cytokine analysis of helper T cell type 1 (interferon-gamma, interleukin [IL]-2, IL-12 p70, and tumor necrosis factor-alpha) and helper T cell type 2 (IL-4 and IL-10) responses revealed that rRF-255 antigen induces a prominent helper T cell type 1 immune response in mice. The rRF-255 antigen also induced serum neutralizing antibodies in immunized mice. Analysis of RSV load in lungs showed that rRF-255 immunization provided significant protection compared with PBS control animals.     

The effect of dose in tolerance induction on the subsequent response to a cross-reactive antigen

The amount of antibody produced by BSA-tolerant rabbits as a result of immunization with DNP—BSA is dependent upon the amount of BSA used to induce tolerance. Tolerance was induced by initial injection of 100 μg of antigen followed by progressively higher doses. Rabbits rendered tolerant with a maximum BSA dose of 1 mg had a mean serum anti-BSA antibody concentration of 0.39 mg/ml after immunization with DNP—BSA, whereas rabbits rendered tolerant with a maximum BSA dose of 100 mg had a mean serum anti-BSA concentration of 0.02 mg after the same course of immunization. This compares with a mean of 1.08 mg of anti-BSA antibody in normal rabbits immunized with DNP—BSA. There was a similar reduction in the concentration of anti-DNP antibodies and of conjugate-specific antibodies in the tolerant groups.

The results are discussed in terms of a thermodynamically-controlled induction of tolerance in individual precursor cells.

     

The effect of intraperitoneal and intramammary immunization of sheep on the numbers of antibody-containing cells in the mammary gland,and antibody titres in blood serum and mammary secretions.

The contribution of gut-associated lymphoid tissue (GALT) to the local response in the mammary gland is well documented in laboratory animals and has been evaluated in this study in ruminants. Ewes were immunized intraperitoneally (IP) with antigen in Freund''s complete adjuvant (FCA), a procedure which stimulates the production of antibodies of the IgA class in the intestine, and challenged intramammarily (IMam) either during colostrum formation or mammary gland involution. Despite a substantial IgA antibody-containing cell (ACC) response in the intestine in IP immunized sheep, there was no evidence to suggest a relocation of IgA-specific ACC to the mammary gland. There was, however, an IgA antibody response in mammary secretion of IP immunized animals, regardless of whether the mammary gland was locally immunized, but the origin of this antibody is unclear. IP/IMam immunized sheep did have an enhanced antigen-specific ACC response of the IgG1 isotype in locally immunized glands, but whether these cells were of GALT or systemic origin is also unclear.     

The humoral immune response of mice to intra-mammary immunization with ovalbumin.

Groups of lactating mice were immunized intra-mammarily on the second day of lactation with 20 micrograms, 150 micrograms or 400 micrograms of ovalbumin (OVA). This resulted in the appearance of IgG in serum, and IgA and IgG in milk. In serum, no IgA antibodies were detected 16 days after immunization in any of the groups. The serum response of IgG was variable and not related directly to the immunizing dose. Both IgA and IgG antibodies were absent in milk 5 days after immunization and IgG antibody level in milk increased significantly throughout lactation as measured 10 and 15 days after inoculation. No IgA antibodies appeared in the milk of the 20 micrograms and 150 micrograms group; however, responses appeared in milk with the highest dose (400 micrograms), but the number of responders for IgG increased in milk but not in blood. The results suggest that intra-mammary immunization can provoke a local IgA response in milk, and that serum is not a major source of IgG in that fluid. Moreover, the kinetics of the IgA and IgG responses differ.     

Isolation of porcine immunoglobulins and determination of the immunoglobulin classes of transmissible gastroenteritis viral antibodies

The porcine immunoglobulins M (IgM), A (IgA), and G (IgG) were isolated and purified and some of the properties of the porcine milk IgA were examined. Monospecific antisera which were prepared against these immunoglobulins in rabbits were then used to absorb a particular class of immunoglobulin from sow serum, colostrum, and milk in an attempt to identify the immunoglobulin classes of neutralizing antibodies to the porcine enteric virus, transmissible gastroenteritis (TGE). The results of these absorption studies suggest that in colostrum and milk from sows experimentally (orally) or naturally infected with live virulent TGE virus, IgA is the predominant immunoglobulin class of TGE antibodies. Both IgA and IgG TGE antibodies appeared to be present in the serum from these sows, but with IgG TGE antibodies predominating. In contrast, in the serum, colostrum and milk from sows vaccinated intramuscularly or intramammarily with live attenuated TGE virus, the TGE antibody activity was associated mainly with the IgG class of immunoglobulins. These results provide additional data indicating that the route of infection or vaccination markedly influences the immunoglobulin class of antibodies in colostrum and milk. Secondly, IgA antibodies in mammary secretions are probably essential for providing optimal passive immunity of nursing pigs against infection with TGE virus.     

Detection of antibody specificity of raw bovine and human milk to bacterial lipopolysaccharides using PCFIA

Raw milk from non‐immunized cows and raw human milk from lactating mothers were examined for specificity and antibody activity against lipopolysaccharides (LPS) of five pathogenic bacteria, i.e. Escherichia coli O111:B4, E. coli O128:B12, Salmonella enteritidis, S. typhimurium and Shigella flexneri 1A in a particle concentration fluorescence immunoassay (PCFIA). Bacterial LPS was covalently coated on submicron polystyrene particles and used in an antibody sandwich technique with commercially available rabbit anti‐bovine fluorescein‐labeled IgG or goat anti‐human fluorescein‐labeled IgA. Comparison was made to a skim milk sample obtained from vaccinated cows. In general, specific activity of milk IgG against bacterial LPS did not parallel the trend of total non‐specific activity. Although the specific anti‐LPS activities were in general high in milk from vaccinated cows, milk from non‐immunized cows also showed significant activity against the bacterial LPS, sometimes higher than the vaccinated cows. IgA in human milk samples showed a wider range of antibody activity against the LPS fractions than bovine milk samples. The specificity of antibody from milk obtained from non‐immunized cows against pathogenic bacteria demonstrated that immunization of cows in order to obtain milk with high titer antibody may not be necessary as milk from non‐immunized cows can provide IgG with sufficient antibody activity against the LPS of bacterial pathogens. This can translate into significant savings and reduce stress imposed on animals for immunization.     

Intraperitoneal immunization of human subjects with tetanus toxoid induces specific antibody-secreting cells in the peritoneal cavity and in the circulation, but fails to elicit a secretory IgA response.

Five patients on continuous ambulatory peritoneal dialysis (CAPD) were immunized intraperitoneally with tetanus toxoid (TT) through an indwelling catheter. Four control patients on CAPD received the same dose of TT intramuscularly. Before immunization, virtually no anti-TT antibody-secreting cells (AbSC) were detected by the enzyme-linked immunospot (ELISPOT) assay in peripheral blood or peritoneal fluid from patients of either group. One to 2 weeks after immunization, high frequencies of TT-specific AbSC were detected in the circulation and peritoneal cavity. More than 80% of those cells were of the IgG isotype, with IgA accounting for most of the remainder. Patients receiving TT by the i.p. route showed significantly higher frequencies of specific IgG and IgA AbSC in the peritoneal cavity than patients immunized intramuscularly. Frequencies of AbSC in peripheral blood did not significantly differ between the two groups. Immunization with TT by both routes resulted in a significant increase of IgG anti-TT antibodies in serum, saliva and peritoneal fluid. A significant IgA antibody response was seen only in serum and peritoneal effluents. Therefore, i.p. immunization of human subjects with TT elicited both a localized response in the peritoneal cavity as well as a systemic response in serum, but did not induce a salivary IgA response.     

Antibody-induced foot oedema,hyperalgesia and monoarticular arthritis in rats,guinea-pigs and rabbits for testing anti-inflammatory,antiarthritic agents

To develop a simple and convenient test based on immunologic events that can be used for testing anti-inflammatory antiarthritic drugs, experiments were performed on rabbits, guinea-pigs and rats.A monoarticular arthritis was provoked injecting a knee joint with: (a) ovalbumin or bovin serum albumin (BSA) in immunized rabbits (active immunization); (b) rabbit anti-BSA serum in rabbits previously treated i.v. with BSA (passive immunization); (c) rabbit anti-guinea-pig or anti-rat serum in normal guinea-pigs and rats.The diameter of the joint was measured with a caliper. The development of the joint swelling was found to be favourably influenced by anti-inflammatory drugs given orally or intra-articularly. The test was considered suitable but not practical for testing a large number of compounds.Foot oedema and hyperalgesia were provoked by injecting rabbit anti-rat serum into the foot pad of normal rats. Volume and sensitivity to a nociceptive stimulation (compression) were determined. Steroidal and non-steroidal anti-inflammatory drugs and miscellaneous drugs were tested orally in a preventive-type schedule of treatment. The test was found suitable for testing large number of compounds. The results were compared to the results obtained testing the same compounds against foot oedema and hyperalgesia provoked by carrageenan.Some of the drugs tested modified foot swelling and hyperalgesia differently in the two experimental models of inflammation.     

The transfer of immunoglobulins IgG, IgA and IgM from serum to colostrum and milk in the sow

The proportion of IgA, IgG and IgM in sow colostrum and milk that is derived from serum has been determined by measuring the transfer of ¹²⁵I-labelled immunoglobulin from serum. All colostral IgG and a high proportion of IgM are derived from serum as is 40 per cent of IgA. Thus it would appear that colostrum is not a true secretion since 90 per cent of its immunoglobulin content is of serum origin.

In milk, however, 90 per cent of IgA and IgM and 70 per cent of IgG would appear to be locally produced in mammary tissue.

     

Specific antibody production against a soluble antigen in the Harderian gland of the domestic fowl.

Three groups of chickens were immunized with bovine serum albumen (BSA). One group received the antigen i.v., the second had BSA dropped into the orbit and the third group was immunized both systemically and by topical application of BSA. Agar-gel diffusion studies showed antibodies to BSA to be present in the tears and sera of the first and third groups but only in the tears of the second group. Immunofluorescence revealed positive anti-BSA staining in lymphoid cells of the spleens of the first and third groups and in plasma cells of Harderian glands of all three groups. It is concluded that systemic and/or topical applications of BSA affect the tears and Harderian gland whereas only systemic injections produce an effect in the spleen and serum. The Harderian gland may have an important part to play in the local immunologic mechanism of the eye and upper respiratory tract of the fowl.     

FcRn is not the receptor mediating the transfer of serum IgG to colostrum in pigs

In contrast to humans or rabbits, in which maternal IgG is transmitted to offspring prenatally via the placenta or the yolk sac, large domestic animals such as pigs, cows and sheep transmit IgG exclusively through colostrum feeding after delivery. The extremely high IgG content in colostrum is absorbed by newborns via the small intestine. Although it is widely accepted that the neonatal Fc receptor, FcRn, is the receptor mediating IgG transfer across both the placenta and small intestine, it remains unclear whether FcRn also mediates serum IgG transfer across the mammary barrier to colostrum/milk, especially in large domestic animals. In this study, using a FcRn knockout pig model generated with a CRISPR‐Cas9‐based approach, we clearly demonstrate that FcRn is not responsible for the IgG transfer from serum to colostrum in pigs, although like in other mammals, it is involved in IgG homeostasis and mediates IgG absorption in the small intestine of newborns.