The anti-IRBP IgG1 and IgG2a response does not correlate with susceptibility to experimental autoimmune uveitis

Susceptibility to experimental autoimmune uveitis (EAU) in inbred mice has been associated with a dominant Th1 response. Elevated anti-inter-photoreceptor retinoid-binding protein (anti-IRBP) IgG2a/IgG1 antibody ratios have been implicated as candidate markers to predict disease severity. In the present study, both the anti-IRBP antibody isotype and severity of EAU phenotypes were examined in 4 non-isogenic genetically selected mouse lines to determine if they can be used as general markers of disease. Mice between 8 and 12 weeks old selected for high (H III) or low (L III) antibody response and for maximum (AIR MAX) or minimum (AIR MIN) acute inflammatory reaction (AIR) were immunized with IRBP. Each experiment was performed with at least 5 mice per group. EAU was evaluated by histopathology 21 days after immunization and the minimal criterion was inflammatory cell infiltration of the ciliary body, choroid and retina. Serum IgG1- and IgG2a-specific antibodies were determined by ELISA. EAU was graded by histological examination of the enucleated eyes. The incidence of EAU was lower in AIR MIN mice whereas in the other strains approximately 40 percent of the animals developed the disease. Low responder animals did not produce anti-IRBP IgG2a antibodies or interferon-gamma. No correlation was observed between susceptibility to EAU and anti-IRBP isotype profiles. Susceptibility to EAU is related to the intrinsic capacity to mount higher inflammatory reactions and increased production of anti-IRBP IgG2a isotype is not necessarily a marker of this immunologic profile.

The use of protein structure/activity relationships in the rational design of stable particulate delivery systems

The recombinant heat shock protein (18 kDa-hsp) from Mycobacterium leprae was studied as a T-epitope model for vaccine development. We present a structural analysis of the stability of recombinant 18 kDa-hsp during different processing steps. Circular dichroism and ELISA were used to monitor protein structure after thermal stress, lyophilization and chemical modification. We observed that the 18 kDa-hsp is extremely resistant to a wide range of temperatures (60 percent of activity is retained at 80ºC for 20 min). N-Acylation increased its ordered structure by 4 percent and decreased its ß-T1 structure by 2 percent. ELISA demonstrated that the native conformation of the 18 kDa-hsp was preserved after hydrophobic modification by acylation. The recombinant 18 kDa-hsp resists to a wide range of temperatures and chemical modifications without loss of its main characteristic, which is to be a source of T epitopes. This resistance is probably directly related to its lack of organization at the level of tertiary and secondary structures

Rabies infection and specific effect of vaccination in mice selected for high and low immunobiological parameters

Innate and acquired resistance to rabies infection was investigated in mice genetically selected for high(H) or low(L) antibody responsiveness from selections I, III and IV and in mice selected for maximal (AIRmax) or minimal (AIRmin) acute inflammatory reaction. These mouse lines were infected intramuscularly with different virus dilutions and the LD50 was determined. The HIII and HIV mouse lines were more susceptible than the LIII and LIV lines and the HI line showed a discrete but higher resistance than the LI line. Analysis of the interline (H x L) F1 hybrids from selections III and IV indicated different dominance effects on the "resistant" and "susceptible" phenotypes when the route of vaccination was changed. No differences were observed between the AIRmax and AIRmin mice, suggesting that inflammation plays a minor role in the resistance to rabies virus. The comparison of LD50 in mice vaccinated by distinct routes showed that the highest interline difference occurred after intramuscular vaccination (250-fold between H and L and 800-fold between F1 and L). These results indicate that different mechanisms may participate in acquired antirabies resistance.

Incomplete dominance of the low antibody response to Cryptosporidium parvum antigens in mice selected for high and low antibody responsiveness

The humoral antibody response to Cryptosporidium was investigated in mice genetically selected for high (H) and low (L) antibody responsiveness. Groups of 4-5 mice from two different selections, general primary (GP) and general secondary (GS), were studied. Following immunization with Cryptosporidium parvum antigens, the maximum levels of IgG in the HGP, (X ñ SD = 1.13 ñ 0.35, N = 5) and in the HGS (0.42 ñ 0.15, N = 4) lines, and of IgM in the HGP line (0.86 ñ 0.53, N = 5) were significantly higher than those in their L counterparts (0.04 ñ 0.02, N = 5;0.05 ñ 0.02, N = 4 and 0.24 ñ 0.07, N = 5, respectively). These findings were similar to those reported for other immunogens. However, the IgG (0.22 ñ 0.05, N = 4) and the IgM (0.33 ñ 0.08, N = 4) responses to immunization of F1 (LGP x HGP) hybrids indicated an incomplete dominance of the low response, in contrast to the incomplete dominance of the high response described for many other antigens and representing an important exception. In addition, the H, L and F1 mice did not develop detectable infections when inoculated with live Cryptosporidium oocysts, supporting the view that a reduced or zero antibody production itself is not enough to permit the establishment of Cryptosporidium infection in adult mice.

Parasite and egg burden, hepatic collagen and histologic pattern of liver granulomas in selection III high and low antibody responder mice infected with Schistosoma mansoni

Selection III mice have particular immunological characteristics: they are high (H III) or low (L III) antibody producer animals, yet both lines display similar T cell responses and macrophage activities. We submittedthese mice to infection with Schistosoma mansoni to assess in vivo parasite and egg burden, hepatic collagen and cellular composition of granulomas in both lines. Titration of anti-Schistosoma IgG by ELISA showed remarkably higher values inH III line, at both studied periods (8th and 12th weeks post-infection). Nevertheless, the number of adult worms recovered from the portal system was similar inboth lines, being not associated with anti-Schistosoma antibody levels. There isan increase in hepatic collagen from the 8th to the 12th weeks post-infection, which is paralleled by an increase in the number of eggs in the liver. This association apparently occurs at the same radio in H III and L III animals. The most important difference found between the two lines was the outstanding contrast interms of volume and eosinophil counts in the granulomas, with lesions from H IIImice clearly being larger and containing more of these cells than LIII lesions

A comparative study of resistance to MHV3 infection in genetically homogeneous and heterogeneous mouse populations

Resistance to MHV3 infection was investigated in genetically homogeneous inbred (A/J, BALB/c) and genetically selected (High, Low) mouse lines. The A/J and L lines are resistant and the BALB/c and H mice are susceptible. The genetic analysis was performed on the F1 hybrids, as well as on the genetically heterogeneous F2 populations and backcrosses bred from HxL and A/JxBALB/c lines. The mortality rates of the F1 hybrids showed codominance of susceptibility and resistance characters. The results indicate that the same MHV3 susceptibility genes are present in isogenic and selected lines and corroborate previous results showing that at least two major genes are involved in the control of this response

Isotypic distribution of antibody responses in lines of mice selected for high or low immunoresponsiveness

The isotype distribution of antibody (Ab) responses to Salmonella antigens (Ag) was investigated in high (H) and low (L) Ab responder lines of mice from Selections III and carried out for responsiveness to flagellar (f) and somatic (s) Ag, respectively. Primary immuniztion resulted in higher Ab titers of all isotypes in response to both Ag in H mice fro m both selections and was confirmed after booster injections. The interline difference (H-L) in response to the distinct isotypes ranged from 3.0 to 7.0 log2 to Ag f in Selection III and from 2.0 to 5.1 log2 to Ag s in Selection IV. Comparison of isotype production to 3 Ag in Selections I,II,III and IV demonstrated that: 1) the highest responses in all mice are those against the selection Ag, 2) the isotypic pattern depends on both the Ag injected and the host's genetic constitution, and 3) the presence or lack of a multispecific effect is not due to isotype-restricted regulation

Preliminary results corroborating the polygenic control of immunological tolerance

Tolerance-susceptible (TS) and-resistant (TR) lines of mice are in the process of bidirectional genetic selection starting from a genetically hetrogeneous population achieved by the equilibrated intercrossing of eight inbred lines. Mice are intragastrically pretreated and then immunized with hen ovalbumin or bovine serum albunin and the extreme phenotypes are selected for assortative mating. The normal distribution of agglutinin titers in the F0 population and the significant interline difference already observed in the F2 and F3 generations indicate that oral tolerance is a character controlled by the additive effect of several independet loci. The mean heritability (h2) obtained thus far is 11% for the TS line and 19% for the TR line