Genetic variation in the humoral immune responses of mice to the nematode Trichuris muris

Genetically based differences in the antibody responses to the large intestinal nematode Trichuris muris were studied in two groups of H-2 congenic strains of mice that differed in their relative resistance to infection with this parasite. The primary antibody response to parasite excretory/secretory (E/S) antigen was predominantly an IgG response with the strains forming two distinct groups, defined by their genetic background. The more susceptible B10 genetic background mice had strikingly higher antibody levels than mice of the BALB genetic background. Superimposed upon these background effects were clearly defined influences attributable to H-2-linked genes, strains which differed genetically only at H-2 loci exhibiting differences in the kinetics of the antibody response. Only B10.G and B10.BR mice showed any great increase in IgM levels post-infection. No IgA specific to E/S antigen was detected in the peripheral circulation of any strain at any time post-infection. Antibody responses to a 40-43 kD antigen revealed clear H-2-linked gene effects, with mice sharing the H-2k haplotype (B10.BR, BALB/K) exhibiting considerably higher total antibody levels than strains expressing other haplotypes; mice of the H-2d haplotype (BALB/c, B10.D2/n) responded very weakly to this antigen. A Western blot analysis of antigen recognition by antibody revealed similarities between the mouse strains in their total antibody responses to T. muris E/S antigen. However, immunoprecipitation studies showed that in general the more susceptible B10 congenic strains had wider spectra of antigen recognition than the BALB congenics. Strains sharing the same H-2 haplotype had dissimilar antigen recognition profiles, but strains sharing the H-2b haplotype (B10, BALB/B) recognized a low mol. wt antigen (20-23 kD) not recognized by any other strain, suggesting an exclusively H-2b restriction in the recognition of this antigen. These results support the conclusion that both H-2-linked and background genes play important roles in controlling the humoral immune response to T. muris infection.     

Method for studying the development, structure and microflora of denture plaque

Abstract – Pieces of sandblasted, self-adhesive tape were stuck to the fitring surface of maxillary dentures in 17 edentulous subjects with climically normal palatal mucosa. After 1 week, during which the subjects abstained from denture hygience, the tape samples were removed for cultural and microscopical examinations. The microorganisms were suspended in RTF by ultrasonic treatment and total viable anaerobic counts and viable counts of yeasts per cm² of tape were calculated after inoculation of serial dilutions on nonselective medium and Sabouraud agar. Total anaerobic viable counts ranged from 4×10⁴ to 5×10⁸. By repeated sampling the largest intra-individual variation was 10-fold, only. Yeast counts constituted less than 1% of the total viable counts in all but two of the subjects. Light and electron microscopy showed a bacterial plaque o predominantly cocci and rods.     

Increased hepatic microsomal activity after halothane anaesthesia in children

The effect of anaesthesia and surgery on microsomal enzyme activity was studied in 19 children aged 4-9 years, scheduled for tonsillectomy. The children were randomly allocated to either halothane or ketamine anaesthesia. Antipyrine clearance was measured before and 4 days after surgery by a salivary one-sample technique. Statistically significant (p less than 0.001) increases in antipyrine clearance was found in children who received halothane anaesthesia. The antipyrine clearance was increased by a mean of 26% 4 days after surgery, compared with a pre-operative control measurement. No significant change in antipyrine clearance was observed in children who received ketamine anaesthesia. There was also a significant difference in antipyrine clearance changes after surgery between the two groups (p less than 0.05). Halothane has enzyme-inducing properties after a single exposure in children, while a single dose of ketamine does not.     

In vitro antigen presenting cell-derived IL-10 and IL-6 correlate with Trichuris muris isolate-specific survival

Trichuris muris , the mouse whipworm, is used as a laboratory model of the human parasite T. trichiura . Three laboratory isolates of T. muris exist — the E, J and S isolates. Previous data have shown that the S isolate survives to chronicity in C57BL/6 mice unlike the E and J isolates, which are expelled. The ability of the S isolate to persist is thought to be due to it secreting unique excretory/secretory antigens, which interact with APCs such that protective T cell responses do not develop. To determine whether APCs respond differently to E/S antigens from the three isolates we cultured isolate-specific E/S with bone marrow-derived macrophages (BMMΦ) and dendritic cells (BMDCs) in vitro . Markers of co-stimulation and levels of MHC-II were analysed by FACS and cytokine levels in supernatants quantified. E/S antigens from the S isolate consistently stimulated significantly higher levels of IL-10 and IL-6 from both macrophages (F4/80⁺CD11b⁺CD11c⁻) and dendritic cells (CD11c⁺CD11b⁺F4/80⁻) compared to J and E isolate E/S. If these in vitro differences in APC-derived cytokines, particularly IL-10, are biologically significant in vivo , they may contribute to the S isolate survival, by creating a regulatory cytokine environment in which protective immune responses are less effective.     

The role of CD4 cells in protective immunity to Brugia pahangi

The BALB/c mouse immunized sub-cutaneously (s.c.) with 45 kRad attenuated third stage larvae (L₃) of the lymphatic filarial nematode Brugia pahangi is strongly immune to a challenge infection (75–100% reduction in recovery at day six post challenge). Analysis of spleen cell supernatants from immunized mice re-stimulated in vitro, with parasite antigen or the non specific T cell mitogen Con-A reveals a cytokine profile (IL-4, IL-5 and IL-9) which indicates that the Th2 subset of CD4 cells has been expanded. In an attempt to formally prove a critical role for CD4 cells in immunity in this model system, immunized mice were given either anti-CD4 or anti-CD8 neutralizing antibodies. Administration of anti-CD4 antibody had a significant and detrimental effect on the immune response whereas anti-CD8 antibody had a negligible effect on immunity. The efficacy of antibody in neutralizing their target cells was determined by fluorescence activated cell sorting analysis (FACS). Spleen cells from anti-CD4 treated immunized mice, when re-stimulated with parasite antigen had a significantly reduced potential to secrete IL-4, IL-5 and IL-9 in vitro and serum from these mice had reduced levels of parasite specific IgG and IgE. These results demonstrate a critical role for CD4 T cells in host protective immunity to B. pahangi in vivo and strongly suggest that some component of the Th2 response plays an important role in the immune response elicited in this model system.     

MHC-restricted antibody responses to Trichuris muris excretory/secretory (E/S) antigen

Summary Two panels of H-2 recombinant mice were used in a detailed serological study to analyse the role of H-2-linked genes in the control of the antibody response to excretory/secretory (E/S) antigens of Trichuris muris. An apparent H-2 ^q ( 1-A ^q) restriction on the early development of high levels of IgGl antibody to E/S antigen was revealed by ELISA. No such restriction was demonstrated for the specific IgG2a response patterns. Recognition of two high molecular weight antigens (90–95 kDa, 105–110 kDa) by IgG antibodies was also shown to be almost exclusively H-2 ^q restricted and may be related at least in part to the high antibody levels seen for H-2 ^q strains of mice. Immune serum from resistant (B10.BR × B10.G) Fl hybrid mice ( H-2 ^qk) containing high levels of IgGl antibodies specific for T. muris E/S and IgG antibodies which recognized the 90–95 kDa and 105–110 kDa E/S antigens was effective in transferring protection to the non-responsive B10.BR mouse strain as seen on day 35 post-infection (p.i.). It is suggested that the IgG responses described for the generally very resistant H-2 ^q mouse strains may contribute to, but not be an absolute requirement for. protective immunity, antibody-mediated damage facilitating a subsequent cellular attack in certain strains of mice.     

Microbiologic study on developing plaque in human fissures

abstract — Occlusal fissures from unerupted third molars were implanted in occlusal fillings in molars of six dental students. Two experiments were performed consecutively in each tooth. After 7 d in the mouth the fissure was removed, opened and dropped into a prereduced suspending medium and the contents suspended by ultrasound. In phase contrast microscopy the microflora consisted of 81–93% cocci and 7–19% rods. No fusiforms, filaments, spirilla, spirochetes or yeasts were seen. In Gram-stained smears 81–96% of the bacteria were Gram-positive. The anaerobic total viable counts (AnVC) were 1–33×10⁶ per fissure. Streptococcal counts on Mitis Salivarius Agar constituted 29–60% of AnVC. In differential counts, S. mutans constituted 0–40%, S. sanguis 0–78% and S. salivarius 0–63% of the colonies present. Lactobacilli grew on Rogosa SL Agar from five of the subjects. In four of these, lactobacilli constituted 0.002–4% of the total viable counts, whereas in one sample they made up 65%. Hemophili isolated on chocolate agar containing bacitracin constituted 0–15% (median 0.7%) of the AnVC. Although the large variation indicates each fissure to be a separate ecologic system, it may be concluded that the flora differs from that of 7-d dento-gingival plaque at least by the absence of fusiforms, filaments, spirilla and spirochetes. The proportional distributions of Streptococcal species also differ from those reported for approximal and buccal plaque as well as for saliva.     

Immunity to coccidiosis: genetic influences on lymphocyte and cytokine responses to infection with Eimeria vermiformis in inbred mice

Cellular and cytokine responses to infection with Eimeria vermiformis were compared in BALB/c (resistant) and C57BL/6 (B6-susceptible) inbred mice. Cellular responses in the mesenteric lymph node (MLN) occurred sooner after primary infection in the resistant BALB/c strain. In contrast, proliferative responses occurred earlier after challenge in B6 mice. Resting levels of CD4 + ve and CD8 + ve T-lymphocytes in the MLN differed between the two strains but the relative numbers of each subset remained relatively constant throughout primary infection. MLN cells taken at intervals after infection were assayed for release of the cytokines IFN-gamma, IL-5 and IL-10 after culture in vitro with the mitogen Concanavalin A (Con-A) or with parasite antigen. With either stimulus cells from resistant BALB/c mice released IFN-gamma and IL-5 earlier after infection than did B6 cells. The strains had a comparable absolute ability to produce IFN-gamma but BALB/c cells released more IL-5 than did B6, levels declining, rather than increasing, during primary infection in the latter. Only cells from BALB/c mice released IL-10 during infection. Cells taken after a secondary infection released relatively little cytokine after pulsing in vitro. These data suggest that the difference in response phenotype between the two strains when infected with E. vermiformis reflect a kinetic, rather than a qualitative, difference in ability to mount protective T-helper (Th) cell subset responses. No evidence was found for a Th2-mediated interference with ability to release IFN-gamma, the cytokine most closely associated with protective immunity.