Humoral Immune Responses In Rats Research Articles

Lymphoid follicle antigen (Ag) delivery and enhanced rodent humoral immune responses mediated by Ag-containing PEGylated liposomes

Antigen (Ag) delivery to lymphoid follicles is important in achieving adaptive immunity. We recently developed a novel two-step Ag delivery system that efficiently induces cellular immune responses to Ags in mice by using priming intravenous (i.v.) injections of empty PEGylated liposomes (PEG-Lip) followed 3 days later by Ag-entrapped PEG-Lip (Ag-PEG-lip). In this study, we looked for humoral immune responses in rats and mice with IgG production specific to the encapsulated Ags. We observed that initial i.v. injections of empty PEG-Lip triggered accumulation of subsequent doses ovalbumin-PEG-Lip (OVA-PEG-lip) in splenic follicles and enhanced IgG production against OVA in both rats and mice. Anti-OVA IgG production was diminished by inhibition of splenic follicular accumulation of OVA-PEG-Lip by fingolimod (FTY720), which inhibits lymphocyte egress from lymphoid tissues. Thisindicates that the follicular accumulation of Ags that we observed is an indispensable and unique step in the production of anti-OVA IgG. Interestingly, in BALB/c nude mice, which are T cell deficient, a high follicular accumulation of OVA-PEG-Lip was observed, but anti-OVA IgG production was not observed. This suggests that T cells are also indispensable for the induction of cellular immune responses by our two-step immunization procedure. Our unique Ag delivery platform, which efficiently delivers Ags to splenic follicles, may be a useful technique for the enhancement of cellular immunity, as well as humoral immunity. Further experimental evaluation should be undertaken in relevant animal models in order for efficacy, safety and immunological correlates to be determined.

The effects of oat bran and soy flour on humoral immune response in rats fed hypercholesterolaemic diets

Previous studies have demonstrated the immunological and hypercholesterolaemic effects of soluble dietary fibre and soy protein in rats. In this study, we evaluated the effects of oat bran and soy flour on humoral immune response in Wistar rats fed hypercholesterolaemic diets. Animals (6 groups) fed with different diets for 6 wks were inoculated twice with antigen (IgY). Plasma samples were collected after each inoculation and anti-IgY antibody (IgM, IgG1 and IgG2a) levels were evaluated by ELISA. Animals receiving 1% cholesterol presented an increase in anti-IgY IgG1 and a reduction in anti-IgY IgM and IgG2a relative to control animals. These effects were abrogated in animals fed 1% cholesterol and oat bran or oat bran+soy protein, but not in animals fed 1% cholesterol and soy protein. Diets containing 1% cholesterol resulted in hepatic lesions and higher liver and spleen relative weights, but did not affect lipid profile, weight gain, food intake, or food conversion efficiency. In conclusion, a high-cholesterol diet influences classes of antibodies produced in response to an antigen in a way that can be reversed by oat bran.

Evaluation of primary and secondary responses to a T-cell-dependent antigen, keyhole limpet hemocyanin, in rats

To develop a rat T-cell-dependent antibody response (TDAR) model evaluating both primary and secondary antibody responses, keyhole limpet hemocyanin (KLH) was used to immunize rats twice during a 14-day course of study, a pattern closely linked to that of a short-term general toxicity study. Female rats of four representative strains (e.g., Sprague-Dawley, Wistar, Fischer, and Lewis) were immunized twice with intravenous administrations of KLH (300 µg/rat) on Days 5 and 9 during a 14-day treatment regimen with cyclophosphamide (CPA) at 1, 3, or 6 mg/kg/day. The primary and secondary immunizations of KLH markedly elevated serum anti-KLH IgM and IgG levels in all strains on Days 9 and 15. Remarkable higher levels of anti-KLH IgG (≈ 1000 µg/ml) were noted in all strains, which were more than 4-times compared with those of anti-KLH IgM levels at Day 9, indicating that predominant IgG reactions were induced by the dual immunizations. A large inter-individual variability in KLH-specific IgM and IgG production was observed in all strains. However, levels of the KLH-specific antibodies were considered sufficient for the evaluation, even in Sprague-Dawley and Wistar rats reported as strains with a wide range of variability since immunosuppression of CPA on responses in both anti-KLH IgM and IgG were observed in all strains to the same extent. In addition, the sensitivity of the KLH-ELISA assay system detecting the immunosuppressive effects of CPA was comparable to other assay systems with PFC assay or ELISA using SRBC. The results here demonstrated that these experimental designs could provide valuable information about the influence on both the primary and secondary humoral immune responses in rats when exposed to potential immunomodulatory drugs. Furthermore, the design of the presented TDAR study would support comprehensive evaluation together with the outcome of the conventional general toxicity study.

Role of the striatum in the humoral immune response to thymus-independent and thymus-dependent antigens in rats

Since the role of striatal GABAergic medium-sized spiny (MSP) neurons in the modulation of the immune responses is largely unknown, we evaluated the humoral immune response in rats with bilateral lesion of the striatum caused by quinolinic acid, which destroys MSP neurons. Sham-operated rats and those with striatal lesions were immunized either with TNP-LPS, a T-independent antigen type 1, or one of several T-dependent antigens: ovoalbumin, bovine serum albumin, lysozyme, sheep red blood cells (SRBC) or outer membrane proteins (OMP) of Salmonella enterica serovar Typhimurium. The specific levels of serum IgM and IgG, as well as intestinal IgA antibodies were determined either by enzyme-linked immunosorbent assay (ELISA) or a haemagglutination assay 5 or 7 days after immunization. Our results show that the lesion of striatal MSP neurons attenuated the primary antibody response to the T-independent antigen type 1 (TNP-LPS), but increased the antibody response to T-dependent antigens (proteins, SRBC and OMP), indicating that the striatal neurons modulate the humoral immune response in rats. The mechanisms involved are probably related to a reduction in both the number of B cells and the expression of caveolin-1 in the spleen, as well as an increase in the number of CD4 + T cells and in corticosterone levels of the serum.

Immunomodulatory role of Withania somnifera root powder on experimental induced inflammation: An in vivo and in vitro study

The aqueous suspension of Withania somnifera root powder was investigated for their in vivo and in vitro immunomodulatory properties. W. somnifera showed potent inhibitory activity towards the complement system, mitogen induced lymphocyte proliferation and delayed-type hypersensitivity reaction. Administration of W. somnifera root powder did not have a significant effect on humoral immune response in rats. Our results report immunosuppressive effect of W. somnifera root powder, thus it could be a candidate for developing as an immunosuppressive drug for the inflammatory diseases.

Adenoviral expression of a truncated S1 subunit of SARS-CoV spike protein results in specific humoral immune responses against SARS-CoV in rats

The causative agent of severe acute respiratory syndrome (SARS) has been identified as SARS-associated coronavirus (SARS-CoV), but the prophylactic treatment of SARS-CoV is still under investigation. We constructed a recombinant adenovirus containing a truncated N-terminal fragment of the SARS-CoV Spike (S) gene (from −45 to 1469, designated Ad-S N), which encoded a truncated S protein (490 amino-acid residues, a part of 672 amino-acid S1 subunit), and investigated whether this construct could induce effective immunity against SARS-CoV in Wistar rats. Rats were immunized either subcutaneously or intranasally with Ad-S N once a week for three consecutive weeks. Our results showed that all of the immunized animals generated humoral immunity against the SARS-CoV spike protein, and the sera of immunized rats showed strong capable of protecting from SARS-CoV infection in vitro. Histopathological examination did not find evident side effects in the immunized animals. These results indicate that an adenoviral-based vaccine carrying an N-terminal fragment of the Spike gene is able to elicit strong SARS-CoV-specific humoral immune responses in rats, and may be useful for the development of a protective vaccine against SARS-CoV infection.

HSP60, HSP70 and HSP90 from Trichinella spiralis as targets of humoral immune response in rats.

This study identifies three heat shock proteins (HSPs) using purified preparations from Trichinella spiralis larvae. The proteins: HSP60, HSP70 and HSP90 were found to be targets of the humoral immune response in rats. Three approaches were adopted to obtain T. spiralis HSP-enriched material and/or to purify HSPs to homogeneity. The former product was prepared using affinity chromatography on gelatin sepharose and elution with ATP. Pure 90 kDa-protein was isolated from parasite extract by sequential DEAE (A50) column chromatograpy and preparative electrophoresis. Immunoblot analysis using monoclonal antibodies to HSP60, HSP70 and HSP90 detected the HSP60 and HSP70 in the affinity-purified product and HSP90 in the product obtained by sequential anionic chromatography and preparative electrophoresis. Finally, the reactivy of preimmune, T. spiralis immune and irrelevant immune rat sera on immunoblots were also examined. Only sera taken from infected rats at time-points after day 7 following the first infection exhibited activity against 60, 70 and 90 kDa proteins on blots. The fact that the serum antibodies were anti-HSP was established by immunoadsorption of HSPs to microtiter plates coated with anti-HSP60, anti-HSP70, or anti-HSP90 and using rat sera, positive on blots, to also give positive scores by continued enzyme-linked immunosorbent assay.

Genetic control of the humoral responses to xenografts. III. Identification of the immunoglobulin V(H) genes responsible for encoding rat immunoglobin G xenoantibodies to hamster heart grafts.

We have previously reported that the early phases of the immune response of rats to hamster xenografts are characterized by the production of IgM xenoantibodies encoded by a restricted group of Ig germline V(H) genes (V(H)HAR family). In the later phases of the reaction, an IgM to IgG isotype switch occurs and our study examines the structure of the rearranged V(H)HAR genes used to encode IgG antibodies after this isotype switch. A quantitative polymerase chain reaction was used to investigate the changes in the levels of V(H)HAR+ IgG mRNA seen after xenotransplantation. cDNA libraries specific for V(H)HAR+ Iggamma chain were established from total RNA extracted from splenocytes of naive rats and xenograft recipients of hamster hearts at days 4, 8, 21, and 28 posttransplantation. Colony filter hybridization was used to estimate the relative frequency of the use of individual V(H)HAR+ IgG subclasses. Selected IgG clones from day 21 cDNA libraries were sequenced and analyzed for VH-D-J(H) gene usage and antibody combining site structure. The level of mRNA for V(H)HAR+ IgG increased 6-fold in xenograft recipients at day 21 post-transplantation when compared with naive animals. The relative frequency of isotype usage for V(H)HAR+ IgG1 antibodies alone increased from 22.3% at day 0 to 37.4% at day 21 PTx. Ten IgG clones from the day 21 cDNA libraries have been sequenced for the rearranged V(H)-D-J(H) genes. Thirty percent (3/10) of these IgG clones used V(H)HAR genes for the coding of heavy chain variable region with limited numbers of nucleic acid substitutions (>98% identity with their germline progenitors) although others demonstrated increased variation in nucleotide sequences (95-97% identity) when compared with germline V(H) genes. Analysis of the canonical binding site structure from the predicted amino acid sequences demonstrated that the majority of IgG clones (9/10) displayed a similar pattern of conserved configurations for their combining sites. The change in IgM to IgG antibody production in the later stages of the humoral immune response of rats to hamster xenografts is associated with an IgM to IgG isotype switch and an increased production of antibodies of the IgG1 isotype. Rat anti-hamster IgG xenoantibodies continue to express the V(H)HAR family of V(H) genes, many in their original germline configuration, to encode antibody recognition of the hamster target antigens. There are, however, a majority of antibodies for which the V(H) genes express evidence of increased nucleic acid sequence variation when compared to currently available germline sequences. The source of this variation is not known but may represent the expression of as yet unidentified germline genes and/or the introduction of T cell-driven somatic mutations. Despite the appearance of this variation, the unusual level of conservation in key antigen binding sites within the V(H) region suggests the variation, independent of its origin, may have a limited influence on the restricted nature of the host antibody response to xenografts.

Immunoregulatory role of neurotransmitters

The nervous and endocrine systems modulate the immune system functions through releasing neurotransmitters, neuropeptides and endocrine hormones as they regulate the other physiological functions. The immune system in turn communicates with the nervous and endocrine systems through secreting immunocompetent substances. In this report we review our concepts and evidence concerning the immunoregulatory role of acetylcholine (ACh) and monoamine neurotransmitters which include noradrenaline (NA), 5-hydroxytryptamine (5-HT) and dopamine (DA). The immunoregulatory role comprises two aspects, the modulation of immune functions by neurotransmitters and the effect of the immune system on nervous system functions. The inhibition of ACh biosynthesis in the central nervous system (CNS) caused the enhancement of the humoral immune response of rats to sheep red blood cells (SRBC); by contrast, the inhibition of acetylcholinesterase (AChE) activity in the CNS resulted in the suppression of the immune response. It seems that ACh in the brain plays an immunoinhibitory role. The role can be blocked by atropine, a muscarinic antagonist, but not by hexamethonium, a nicotinic antagonist. During the humoral immune response (days 3–6 after SRBC injection), activity of AChE in the hypothalamus and hippocampus was strikingly lower. It is suggested that a functional connection is present in the ACh of the brain and the immune system. In vitro, ACh at 10 −9 to 10 −4 mol/l dose range significantly strengthened the spleen cell proliferation induced by concanavalin (Con A). The action of ACh only occurred either before or just after T lymphocytes were activated through muscarinic cholinergic receptors. In vivo, the depletion of monoamine neurotransmitters or only NA in the CNS caused the impairment of the anti-SRBC response of rats. During the phases of days 2–7 post-immunization, the metabolic alterations of NA, 5-HT and DA emerged in the CNS and the lymphoid organs of rats, which mainly exhibited that in the peak periods of the antibody response, the metabolism of the monoamine neurotransmitters in the hypothalamus and hippocampus was markedly increased, but NA content in the spleen and thymus was significantly decreased. These results provide evidence for the bidirectional information exchange network between the monoamine neurotransmitters and the immune system. Exposure to NA (at 10 −8–10 −5 mol/l concentration range) in vitro was shown to inhibit the Con A-induced proliferation of the rat spleen cells. This effect of NA was related to the early events involved in the initiation of T cell proliferation and was mediated by either alpha- or beta- adrenergic receptors. The evidence that altering 5-HT level in the central or peripheral nervous systems through various ways of administering the drugs to regulate 5-HT biosynthesis led to the variations of the antibody response, and that cyproheptadine, an antagonist of serotoninergic receptors, can block the action of 5-HT show that 5-HT may exert an immunoinhibitory effect, which appears to be mediated via the peripheral mechanism to relate to the 5-HT receptors. However, the antibody response can cause changes in 5-HT metabolism in the CNS. The possible reasons for these results are discussed. Collectively, the antibody response arouses the metabolic variations of ACh, NA, 5-HT and DA in the central and peripheral nervous systems and then, these alterations can in turn influence immune function through neurotransmitter relevant receptors present on the immunocytes. The purpose of this interaction is most likely to maintain the homeostasis of the immune and other physiological functions.

Lesions of the hippocampus enhance or depress humoral immunity in rats

The effect on the humoral immune response in rats by bilaterally destroying cell bodies in the hippocampus with kainic acid or by electrolytic lesion was studied. After 3 days a humoral immune response was evoked by injecting 1 ml of 10% sheep red blood cells. Five days later, the haemolysin content (MC50) was measured. Chemical destruction of pyramidal cell bodies in CA2 and CA3 significantly increased humoral immunity as measured by HC50, but larger chemical lesions that also included CA1 did not. Electrical destruction of cell bodies and axonal pathways in CA2 and CA3 did not significantly affect HC50. These results suggest that different areas of the hippocampus can stimulate or inhibit humoral immunity.

Stress and dexfenfluramine: Effects on the immune response and energy balance in the rat

The effects of stress, dexfenfluramine ( d-Fen), and a combination of both were investigated on ingestive behavior, body weight, and the humoral immune response in the rat. Three-hundred and 84 male Sprague-Dawley rats were split into four groups of 96 animals. In a balanced design, each group was submitted or not to repeated intense stress for 20 consecutive days. Animals were also treated with 5 mg/kg/day d-Fen (IP, 1 ml/kg) or an equal volume of placebo (saline) for 28 days. The humoral immune response of rats to sheep red blood cells (50% solution, 1 ml IP at day 0) was assessed from the antibody titer on days 4, 8, 12, 16, 20, and 28. Antibodies were assayed by direct hemagglutination and by the Coombs' test. Plasma corticosterone was also measured on days 0 and 12. The effects of stress and d-Fen on ingestive behavior and body weight were consistent with previously published results. In addition, rats treated with d-Fen had a significantly reduced body weight (−20 g) 5 weeks after the end of the treatment, whereas the loss in body weight induced by stress had totally disappeared. Stress did not decrease animals' immune response despite a massive corticosterone secretion on day 0, with a marked response lasting for at least 12 days. d-Fen reduced the corticosterone levels determined on day 12. Antibody production was slightly but significantly reduced in rats receiving d-Fen. These results indicate that the humoral immune response may be independent of corticosterone secretion, explaining why the d-Fen-induced reduction in the corticosterone response to stress did not compensate for the effect of this drug on antibody production.

Use of Immunoresponsiveness to Demonstrate That the Dietary Requirement for Copper in Young Rats Is Greater with Dietary Fructose than Dietary Starch

Weaned male Lewis rats were pair-fed diets containing 62.7% fructose or starch and either 6-7 mg Cu/kg diet (adequate) or 0.7 mg Cu/kg diet (deficient) for 33 d. Antibody titers after primary immunization with sheep erythrocytes were significantly lower in rats fed copper-deficient diets. Compared to starch, fructose markedly attenuated antibody production in copper-deficient rats. Dietary carbohydrate did not affect the humoral immune response of rats fed diets with adequate copper. Concentrations of copper in thymus, spleen, liver and heart were also significantly lower in rats fed fructose with deficient copper (F-Cu) than in the group fed starch with deficient copper. Thymic hypogenesis was observed only in the F-Cu group. Tissue concentrations of copper were reduced before antibody production was impaired. Repletion of previously copper-deficient rats rapidly restored immunocompetence and stimulated thymic growth. Immunoresponsiveness and tissue concentrations of copper reached control levels in rats fed diets containing starch and fructose at 1.2-1.6 and 2.2-2.9 mg Cu/kg diet, respectively. The results demonstrate that the amount of dietary copper required for optimal function of the humoral immune system, thymic growth and maintenance of normal tissue levels of this essential micronutrient is greater when young rats are fed diets with fructose than with starch.

Evaluation of ELISA for detecting in vivo chemical immunomodulation.

Immunotoxicology is a relatively new field of investigation and is becoming recognized and used by toxicologists involved in drug and chemical testing. The purpose of this study was to evaluate the enzyme-linked immunosorbent assay (ELISA) as a means of quantitating humoral immune responses in rats exposed to immunomodulating chemicals. The ELISA proved to be highly sensitive and quantitative, simple to perform, and reliable. The assay is also economically feasible and automated so that large numbers of samples can be analyzed at one time. The antigen used in the study was bovine serum albumin, and the immunosuppressive chemicals used to validate the system were lead, polychlorinated biphenyl, cyclophosphamide, and dexamethasone. Practical application of the procedure to immunotoxicology testing is discussed.

Enhanced humoral immune response in rats by hyaluronic acid

The effect of hyaluronic acid (HA) on antibody responses to egg albumin, dog albumin and birch pollen proteins, was studied in rats. The highest antibody responses to dog and egg albumin were obtained in rats immunized with a coprecipitate of the antigen with HA. However, pretreatment of rats with a subcutaneous injection of HA up to three (3) days prior to the priming injection of dog albumin also significantly enhanced the secondary antibody responses to that antigen. Possible explanations of this stimulatory effect on the humoral immune response are discussed.

Effect of the antitumor antibiotic chromomycin A3 on the humoral immune response in rats.

Chromomycin A3 (250 mug/kg) suppressed the humoral immune response in rats against sheep erythrocytes when administered 48 h or later after antigenic stimulus. The antibiotic at this dose enhanced immunity when given along with or before antigen administration. The natural heterohemagglutinin levels in rabbits and guinea pigs were not affected by the antibiotic (10 mug/kg per day x 7).

Enhancement of immune response by the proteinaceous crystal of [formula omitted] var [formula omitted

The proteinaceous crystal of Bacillus thuringiensis var thuringiensis was found to enhance humoral immune response in rats and guinea pigs immunised with sheep red blood cells. The enhancement was due to the increased levels of both 19S and 7S antibodies in the sera of the treated animals. A novel synthesis of 7S haemolytic antibodies was observed in case of crystal treated animals.

The effect of N-nitroso-N-methylurea and N-dimethylnitrosamine on cell mediated and humoral immune responses in rats and mice.

N-Nitroso-N-methylurea (NMU) induced a marked dose dependent leucopoenia which was associated with an increased survival of skin allografts in adult rats and in 2 strains of mice. The humoral immune response to NMU as assessed by haemolytic plaque formation and haemagglutination was also much reduced. Dimethylnitrosamine (DMN) which, like NMU is a powerful carcinogen and an alkylating agent, showed no immunosuppressive activity after a single dose in rats on either a normal diet or fed a protein-free diet which enhances kidney tumourigenesis. In mice DMN at a near LD(50) dose (14 mg/kg) had no effect on skin graft survival but did reduce the humoral response. At half this dose level, however, no immunosuppressive effect was seen. The results support the conclusion that the immunosuppressive activity of a chemical carcinogen is not necessarily associated with the expression of its carcinogenicity.

A STUDY OF THE DEVELOPMENT OF CYTOTOXIC ANTIBODIES PRODUCED IN RECIPIENTS OF XENOGRAFTS (HETEROGRAFTS) OF ILIAC BONE

1. The humoral immune response of rats against sheep iliac bone grafts has been examined. 2. Fresh, marrow-containing grafts elicited a brisk and sustained antibody response. Attempts to wash out the marrow were not uniformly successful in removing cellular antigens from the grafts. 3. Decalcifying and freezing bone grafts at —20 degrees Celsius do not impair immunogenicity to any significant extent. Immunogenicity was found to be reduced in grafts subjected to freeze-drying. 4. Deproteinised Oswestry bone grafts and Kiel grafts gave rise to antibody production in a few recipients, and in the case of the former this response did not occur until after six or seven weeks from grafting. 5. The highest degree of osteogenesis in composite bone xenograft-autografts was found by Salama and colleagues (1973) to occur in Oswestry bone grafts. It is suggested that osteogenesis in xenografts may be impaired by an immune response.

Effect of Anti-Kupffer Cell Serum on Phagocytosis and Humoral Antibody Formation

To ascertain the effects of selective impairment of the fixed macrophage compartment on host defense, rabbit anti-rat Kupffer cell serum was raised and tested for its ability to depress phagocytosis and to serve as an immunosuppressant. The ability of the anti-Kupffer cell serum (AKS) to depress intravascular clearance rates of gelatinized RE test lipid emulsion indicated that the antiserum can functionally impair overt phagocytic activity. The phagocytic impairment was manifested by a significantly decreased liver phagocytosis of the lipid emulsion, whereas only a slight decrease in phagocytosis by the spleen was noted. When titered in vitro, AKS was cytotoxic to hepatic and splenic macrophages. Although AKS induced some degree of immunosuppressive activity, it was not as effective as antilymphocytic serum in suppressing the humoral immune response of rats to sheep red blood cells. Selectivity in the cytotoxic activity of AKS was manifested by toxicity for macrophages but not toxicity for thymocytes or splenic lymphocytes. It is suggested that AKS as a specific antimacrophage serum may be useful for assessing the contribution of the reticuloendothelial system to host defense physiology and metabolism.