Fructan, Rather Than Gluten, Induces Symptoms in Patients With Self-Reported Non-Celiac Gluten Sensitivity.

BACKGROUND & AIMS: Non-celiac gluten sensitivity is characterized by symptom improvement after gluten withdrawal in absence of celiac disease. The mechanisms of non-celiac gluten sensitivity are unclear, and there are no biomarkers for this disorder. Foods with gluten often contain fructans, a type of fermentable oligo-, di-, monosaccharides and polyols. We aimed to investigate the effect of gluten and fructans separately in individuals with self-reported gluten sensitivity. METHODS: We performed a double-blind crossover challenge of 59 individuals on a self-instituted gluten-free diet, for whom celiac disease had been excluded. The study was performed at Oslo University Hospital in Norway from October 2014 through May 2016. Participants were randomly assigned to groups placed on diets containing gluten (5.7 g), fructans (2.1 g), or placebo, concealed in muesli bars, for 7 days. Following a minimum 7-day washout period (until the symptoms induced by the previous challenge were resolved), participants crossed over into a different group, until they completed all 3 challenges (gluten, fructan, and placebo). Symptoms were measured by Gastrointestinal Symptom Rating Scale Irritable Bowel Syndrome (GSRS-IBS) version. A linear mixed model for analysis was used. RESULTS: Overall GSRS-IBS scores differed significantly during gluten, fructan, and placebo challenges; mean values were 33.1 ± 13.3, 38.6 ± 12.3, and 34.3 ± 13.9, respectively (P = .04). Mean scores for GSRS-IBS bloating were 9.3 ± 3.5, 11.6 ± 3.5, and 10.1 ± 3.7, respectively, during the gluten, fructan, and placebo challenges (P = .004). The overall GSRS-IBS score for participants consuming fructans was significantly higher than for participants consuming gluten (P = .049), as was the GSRS bloating score (P = .003). Thirteen participants had the highest overall GSRS-IBS score after consuming gluten, 24 had the highest score after consuming fructan, and 22 had the highest score after consuming placebo. There was no difference in GSRS-IBS scores between gluten and placebo groups. CONCLUSIONS: In a randomized, double-blind, placebo-controlled crossover study of individuals with self-reported non-celiac gluten sensitivity, we found fructans to induce symptoms, measured by the GSRS-IBS. Clinicaltrials.gov no: NCT02464150.

A Vaccine Approach for the Prevention of Infections by Multidrug-resistant Enterococcus faecium.

The incidence of multidrug-resistant Enterococcus faecium hospital infections has been steadily increasing. With the goal of discovering new vaccine antigens, we systematically fractionated and purified four distinct surface carbohydrates from E. faecium endocarditis isolate Tx16, shown previously to be resistant to phagocytosis in the presence of human serum. The two most abundant polysaccharides consist of novel branched heteroglycan repeating units that include signature sugars altruronic acid and legionaminic acid, respectively. A minor high molecular weight polysaccharide component was recognized as the fructose homopolymer levan, and a glucosylated lipoteichoic acid (LTA) was identified in a micellar fraction. The polysaccharides were conjugated to the CRM197 carrier protein, and the resulting glycoconjugates were used to immunize rabbits. Rabbit immune sera were evaluated for their ability to kill Tx16 in opsonophagocytic assays and in a mouse passive protection infection model. Although antibodies raised against levan failed to mediate opsonophagocytic killing, the other glycoconjugates induced effective opsonic antibodies, with the altruronic acid-containing polysaccharide antisera showing the greatest opsonophagocytic assay activity. Antibodies directed against either novel heteroglycan or the LTA reduced bacterial load in mouse liver or kidney tissue. To assess antigen prevalence, we screened a diverse collection of blood isolates (n = 101) with antibodies to the polysaccharides. LTA was detected on the surface of 80% of the strains, and antigens recognized by antibodies to the two major heteroglycans were co-expressed on 63% of these clinical isolates. Collectively, these results represent the first steps toward identifying components of a glycoconjugate vaccine to prevent E. faecium infection.

Immunological properties of inulin-type fructans.

Beneficial effects of inulin-type fructans are discussed in view of studies that applied the oligosaccharides in colon cancer, chronic inflammatory diseases, vaccination efficacy, and prevention of infection and allergy. In the present paper, we discuss their immunomodulating effects. It is suggested that immunomodulation is elicited through indirect and direct mechanisms. Indirect mechanisms encompass stimulation of growth and activity of lactic acid bacteria, but can also be caused by fermentation products of these bacteria, i.e., short chain fatty acids. Evidence for direct effects on the immune system generally remains to be confirmed. It is suggested that inulin-type fructans can be detected by gut dendritic cells (DCs), through receptor ligation of pathogen recognition receptors (PRRs) such as Toll-like receptors, nucleotide oligomerization domain containing proteins (NODs), C-type lectin receptors, and galectins, eventually inducing pro- and anti-inflammatory cytokines. DCs may also exert antigen presenting capacity toward effector cells, such as B cells, T cells, and natural killer cells locally, or in the spleen. Inulin-type fructans may also ligate PRRs expressed on gut epithelium, which could influence its barrier function. Inulin-type fructans are potent immunomodulating food components that hold many promises for prevention of disease. However, more studies into the mechanisms, dose-effect relations, and structure-function studies are required.

ß(2→6)-Type fructans attenuate proinflammatory responses in a structure dependent fashion via Toll-like receptors.

Graminan-type fructans (GTFs) have demonstrated immune benefits. However, mechanisms underlying these benefits are unknown. We studied GTFs interaction with Toll-like receptors (TLRs), performed molecular docking and determined their impact on dendritic cells (DCs). Effects of GTFs were compared with those of inulin-type fructans (ITFs). Whereas ITFs only contained ß(2→1)-linked fructans, GTFs showed higher complexity as it contains additional ß(2→6)-linkages. GTFs activated NF-κB/AP-1 through MyD88 and TRIF pathways. GTFs stimulated TLR3, 7 and 9 while ITFs activated TLR2 and TLR4. GTFs strongly inhibited TLR2 and TLR4, while ITFs did not inhibit any TLR. Molecular docking demonstrated interactions of fructans with TLR2, 3, and 4 in a structure dependent fashion. Moreover, ITFs and GTFs attenuated pro-inflammatory cytokine production of stimulated DCs. These findings demonstrate immunomodulatory effects of GTFs via TLRs and attenuation of cytokine production in dendritic cells by GTFs and long-chain ITF.

Neonatal treatment with low doses of anti-idiotypic antibody leads to the expression of a silent clone.

BALB/c mice immunized with bacterial levan (BL) produce an immune response that fails to generate antibody expressing the idiotype (Id) of the beta (2 leads to 6) fructosan-binding myeloma protein ABPC 48 (A48). Pretreatment of newborn BALB/c mice (at 1 d of age) with 0.01-10 microgram of affinity purified BALB/c anti-A48 Id antibody followed by immunization with BL 1-2 mo later produces an anti-BL response that expresses the A48 Id. This shows that A48 Id+ anti-BL clones belong to a normally silent fraction of the anti-BL repertoire. The activation of A48 Id+ anti-BL clones anti-A48 Id antibody is specific because the pretreatment of newborn mice with anti-MOPC 384 Id antibody, followed by immunization with BL, does not lead to its activation. Moreover, pretreatment of mice with anti-A48 Id antibody does not alter the MOPC 460 Id+ component of the anti-TNP response. It is also important to note that the activation of the A48 Id+ clone in pretreated mice requires subsequent immunization with BL.

T cell regulation of IgG subclass antibody production in response to T-independent antigens.

The effect of T lymphocytes on the IgM, IgG3, IgG1, IgG2b, and IgG2a responses of B lymphocytes to the type-2 T-independent antigens, trinitrophenylated (TNP)-Ficoll, and TNP-Levan, was investigated. T cell-bearing nu/+ mice were found to produce substantially higher IgG2 serum anti-TNP antibody than their athymic counterparts, and nu/nu and nu/+ IgG2a titers exhibiting more disparity than nu/nu and nu/+ IgG2b titers. The Igm, IgG3, and IgG1 anti-TNP levels in nu/nu and nu/+ mice were indistinguishable. By cell transfer experiments, it was determined that this variance in nude and heterozygote IgG2 responses could not be explained by B cell differences between the two strains or by suppressive effects on IgG2 production within nu/nu mice. Rather, the difference was shown to be the result of the absence of T cells at the time B cells were responding to antigen. In the absence of T cells, the strength of the nu/nu anti-TNP antibody response was found to be in the following order: IgM > IgG3 > IgG1 > IgG2b > IgG2a, a heirarchy identical with the recently proposed heavy chain gene order. The possibilities that T cells influence IgG2 production via their specific recognition of IgG2-bearing B cells or via signals to increase heavy chain switching of responding B cell clones are discussed.

Regulation of the anti-inulin antibody response by a nonallotype-linked gene.

The antibody response to the inulin [(In), beta-(2 leads to 1) fructosan] determinant of bacterial levan [(BL), a beta-(2 leads to 6) polyfructosan that contains beta-(2 leads to 1) branch points] requires the presence of the a haplotype of the Igh gene complex. BALB/c (Igh a) mice immunized with BL produce IgG anti-In antibodies of a single spectrotype by isoelectric focusing analysis. C57BL/6 mice, which possess the b haplotype of the Igh gene complex and which fail to produce anti-In antibodies, nevertheless possess a gene, spectrotype regulation gene 1 (Sr-1), that regulates the isoelectric focusing (IEF) pattern of anti-In antibodies in mice of the a haplotype. Thus, the IEF patterns of anti-In antibodies of (BALB/c x C57BL/6)F1 mice and of B.C8 mice (C57BL/Ka . Igh-Ca) are considerably more complex than those of BALB/c. Backcross analysis indicates that Sr-1 is not linked to the Igh complex, the major histocompatibility complex, or to the genes that code for coat color. Studies of the heterogeneity of anti-In antibodies in recombinant inbred lines and their progeny from matings to BALB/c and C.B20 (BALB/c . Igh-Cb) suggest the existence of other regulatory genes.

Immunochemical and molecular characterization of regulatory idiotopes expressed by monoclonal antibodies exhibiting or lacking beta 2-6 fructosan binding activity.

Hybridomas secreting antibodies bearing the ABPC48 (A48) regulatory idiotype (Id) were generated from BALB/c mice treated at birth or as adults with minute amounts of anti-A48-Id antibodies. The majority of these antibodies were recognized by the syngeneic monoclonal anti-A48-Id and anti-UPC-10-Id antibodies, IDA10 and 10-1, respectively. In Northern blotting experiments, most of these hybridomas were shown to use VH (heavy chain variable region) genes related to the 441-4 germline VH gene that encodes the A48 VH region. Hybridization was detected between polyadenylated H chain mRNA, isolated from the majority of the hybridomas, and the VH probe. Southern blots confirmed these results by showing a rearrangement of VH-related sequences to the JH (H chain joining segment) clusters on these same hybridomas. The antibodies from all of the hybridomas that derived from neonatal mice and half of those derived from adult mice showed specificity for fructosan determinants that, in most cases, was different from the beta 2-6 fructosan linkage specificity of A48. Surprisingly, several of the non-fructosan-binding hybridomas generated from the adult mice and the MOPC-173 myeloma demonstrated a clear specificity for the beta 1-6-D-galactan determinant. Of four galactan-binding myeloma proteins studied. XRPC 44 alone shared idiotypy with the UPC-10 myeloma. These findings suggest a possible clonal crossreactive regulation mediated by regulatory idiotopes. The crossreactive regulation concept is discussed.

ABPC 48 cross-reactive idiotopes in BALB/c mice. Natural and levan-induced expression.

Using monoclonal antiidiotypic antibodies, we developed a sensitive binding assay that detects molecules with one or with two idiotopes of the ABPC48 idiotype. ABPC48 cross-reactive idiotypes were thus shown to be present in substantial amounts in sera of nonimmunized mice. Levan binding sites are found on these idiotypes. During the life time of the mice, the natural anti-levan titer increases while ABPC48 idiotypic expression remains constant, suggesting different controls for these two activities. On the other hand, ABPC48 cross-reactive idiotypes participate--as minor components--in the response that follows a deliberate immunization with bacterial levan. This induction process is likely to reflect the selection of idiotopes expressed by the B cell clones preactivated in sera of nonimmunized mice rather than the activation of silent clones. We suggest that a similar situation might explain the reported emergence of ABPC48 idiotypes in animals primed with antiidiotypic antibodies and subsequently stimulated with levan.

Allosuppressor and allohelper T cells in acute and chronic graft-vs-host disease. I. Alloreactive suppressor cells rather than killer T cells appear to be the decisive effector cells in lethal graft-vs.-host disease.

Splenic T cells from B10 donors were injected into irradiated (B10 x DBA/2)F1 mice. Either 5 or 6 d later, activated donor T cells were recovered from the spleens of these primary F1 (1 degree F1) recipients and transferred to groups of nonirradiated syngeneic F1 (2 degrees F1) recipients. Whereas day-5-activated parental T cells induced the characteristic symptoms of acute graft-vs.-host disease (GVHD) and eventually lethal GVHD, day-6-activated B10 T cells failed to induce acute GVHD but induced symptoms of chronic GVHD. Interestingly, the inability of day-6-activated T cells to induce lethal GVHD could not be ascribed to a lack in anti-F1 T killer cells. The combined results of functional studies indicated that day-6 cells were enriched for alloreactive helper T cells, whereas day-5 cells were enriched for alloreactive suppressor cells. Hence, our findings indicate that acute GVHD and lethal GVHD are caused by alloreactive donor T suppressor but not T killer cells, and that symptoms of chronic GVHD are caused by alloreactive donor T helper cells.

Prebiotics, immune function, infection and inflammation: a review of the evidence.

Beta2-1 fructans are carbohydrate molecules with prebiotic properties. Through resistance to digestion in the upper gastrointestinal tract, they reach the colon intact, where they selectively stimulate the growth and/or activity of beneficial members of the gut microbiota. Through this modification of the intestinal microbiota, and by additional mechanisms, beta2-1 fructans may have beneficial effects upon immune function, ability to combat infection, and inflammatory processes and conditions. In this paper, we have collated, summarised and evaluated studies investigating these areas. Twenty-one studies in laboratory animals suggest that some aspects of innate and adaptive immunity of the gut and the systemic immune systems are modified by beta2-1 fructans. In man, two studies in children and nine studies in adults indicate that the adaptive immune system may be modified by beta2-1 fructans. Thirteen studies in animal models of intestinal infections conclude a beneficial effect of beta2-1 fructans. Ten trials involving infants and children have mostly reported benefits on infectious outcomes; in fifteen adult trials, little effect was generally seen, although in specific situations, certain beta2-1 fructans may be beneficial. Ten studies in animal models show benefit of beta2-1 fructans with regard to intestinal inflammation. Human studies report some benefits regarding inflammatory bowel disease (four positive studies) and atopic dermatitis (one positive study), but findings in irritable bowel syndrome are inconsistent. Therefore, overall the results indicate that beta2-1 fructans are able to modulate some aspects of immune function, to improve the host's ability to respond successfully to certain intestinal infections, and to modify some inflammatory conditions.

Intestinal IgA Regulates Expression of a Fructan Polysaccharide Utilization Locus in Colonizing Gut Commensal Bacteroides thetaiotaomicron.

Gut-derived immunoglobulin A (IgA) is the most abundant antibody secreted in the gut that shapes gut microbiota composition and functionality. However, most of the microbial antigens targeted by gut IgA remain unknown, and the functional effects of IgA targeting these antigens are currently understudied. This study provides a framework for identifying and characterizing gut microbiota antigens targeted by gut IgA. We developed a small intestinal ex vivo culture assay to harvest lamina propria IgA from gnotobiotic mice, with the aim of identifying antigenic targets in a model human gut commensal, Bacteroides thetaiotaomicron VPI-5482. Colonization by B. thetaiotaomicron induced a microbe-specific IgA response that was reactive against diverse antigens, including capsular polysaccharides, lipopolysaccharides, and proteins. IgA against microbial protein antigens targeted membrane and secreted proteins with diverse functionalities, including an IgA specific against proteins of the polysaccharide utilization locus (PUL) that are necessary for utilization of fructan, which is an important dietary polysaccharide. Further analyses demonstrated that the presence of dietary fructan increased the production of fructan PUL-specific IgA, which then downregulated the expression of fructan PUL in B. thetaiotaomicron, both in vivo and in vitro Since the expression of fructan PUL has been associated with the ability of B. thetaiotaomicron to colonize the gut in the presence of dietary fructans, our work suggests a novel role for gut IgA in regulating microbial colonization by modulating their metabolism.IMPORTANCE Given the significant impact that gut microbes have on our health, it is essential to identify key host and environmental factors that shape this diverse community. While many studies have highlighted the impact of diet on gut microbiota, little is known about how the host regulates this critical diet-microbiota interaction. In our present study, we discovered that gut IgA targeted a protein complex involved in the utilization of an important dietary polysaccharide: fructan. While the presence of dietary fructans was previously thought to allow unrestricted growth of fructan-utilizing bacteria, our work shows that gut IgA, by targeting proteins responsible for fructan utilization, provides the host with tools that can restrict the microbial utilization of such polysaccharides, thereby controlling their growth.

Specific inulin-type fructan fibers protect against autoimmune diabetes by modulating gut immunity, barrier function, and microbiota homeostasis.

SCOPE: Dietary fibers capable of modifying gut barrier and microbiota homeostasis affect the progression of type 1 diabetes (T1D). Here, we aim to compare modulatory effects of inulin-type fructans (ITFs), natural soluble dietary fibers with different degrees of fermentability from chicory root, on T1D development in nonobese diabetic mice. METHODS AND RESULTS: Female nonobese diabetic mice were weaned to long- and short-chain ITFs [ITF(l) and ITF(s), 5%] supplemented diet up to 24 weeks. T1D incidence, pancreatic-gut immune responses, gut barrier function, and microbiota composition were analyzed. ITF(l) but not ITF(s) supplementation dampened the incidence of T1D. ITF(l) promoted modulatory T-cell responses, as evidenced by increased CD25+ Foxp3+ CD4+ regulatory T cells, decreased IL17A+ CD4+ Th17 cells, and modulated cytokine production profile in the pancreas, spleen, and colon. Furthermore, ITF(l) suppressed NOD like receptor protein 3 caspase-1-p20-IL-1ß inflammasome in the colon. Expression of barrier reinforcing tight junction proteins occludin and claudin-2, antimicrobial peptides ß-defensin-1, and cathelicidin-related antimicrobial peptide as well as short-chain fatty acid production were enhanced by ITF(l). Next-generation sequencing analysis revealed that ITF(l) enhanced Firmicutes/Bacteroidetes ratio to an antidiabetogenic balance and enriched modulatory Ruminococcaceae and Lactobacilli. CONCLUSION: Our data demonstrate that ITF(l) but not ITF(s) delays the development of T1D via modulation of gut-pancreatic immunity, barrier function, and microbiota homeostasis.

Immune response induced by a single or several syngeneic monoclonal antiABPC48 antiidiotypic antibodies: no predominant coexpression of ABPC48 idiotopes.

BALB/c mice were immunized with monoclonal BALB/c antibodies IDA10, IDA16 and IDA17 raised against the BALB/c ABPC48 myeloma protein. Several procedures of immunization--copolymers with lipopolysaccharide or keyhole limpet hemocyanin, simultaneous or sequential injections of different IDAs--were performed in an attempt to orient the immune response towards the production of ABPC48-like idiotypes. We used a binding assay which identifies two idiotopes on the same molecule to measure the population of antibodies induced in these responses. The expression of ABPC48 cross-reactive idiotypes in immune sera was analyzed. The results show that, with all immunization protocols, immune responses to different monoclonal antiidiotypic antibodies are mostly independent of each other: the coexpression of ABPC48 idiotopes, either private or recurrent on the induced antibodies, is rarely found; it makes it difficult to discriminate by a serological approach between cross-reactive idiotypes and anti-antiidiotypic antibodies. We discuss the interest of combining molecular and serological approaches to identify these two populations of antibodies.

The expression of a private idiotope requires pretreatment with noncomplementary anti-idiotypic antibodies.

The levan-binding ABPC48 myeloma protein is characterized by 3 idiotopes, (Ids), defined by 3 syngeneic monoclonal anti-idiotypic antibodies (IDA 10, IDA 16 and IDA 17). When BALB/c mice are immunized with levan, they produce anti-levan antibodies, some of which carry the Id 10 and Id 16 but not the Id 17 determinants. In the present study, we attempted to induce the synthesis of Id 17 positive anti-levan molecules. We found that immunization with IDA 17 antibodies alone was ineffective in inducing an Id 17 positive anti-levan response. By contrast, successive immunizations with IDA 10, IDA 16 and IDA 17 antibodies resulted in the synthesis of Id 17 positive anti-levan immunoglobulins. The synthesis of these molecules was concommitant with the induction of Id 10 and Id 16 positive anti-levan antibodies. Thus our data suggest that the Id 17 determinant on anti-levan antibodies is coexpressed with Id 10 and Id 16, and that successive anti-idiotypic treatment may result in the selective expansion of rare ABPC48 cross-reactive idiotype B-cell clone precursors.

V lambda-light chain genes reconstitute immune responses to defined carbohydrate antigens or haptens by utilizing different VH genes.

The contribution of the lambda-light chain to the development of peripheral B cell repertoire and generation of specific antibodies to haptens and polysaccharide antigens was studied in genetically manipulated kappa-deficient and lambda 2-transgenic mice. The results clearly demonstrate a non-stoichiometric VH gene family expression in the absence of k-light chain and suggest a non-stochastic pairing between VH and V lambda genes, expressed in the peripheral B cell repertoire. A shift in VH gene utilization in the case of VI lambda + antibodies was evident in response to beta 2-6 fructosan and TNP hapten. These observations demonstrate the availability of compensatory mechanisms in the absence of VK genes and are consistent with the hypothesis that VH gene family expression is controlled by genetic factors from inside the VH locus. Furthermore, genetic factors from outside the VH locus, namely restricted available light chain diversity, may lead to a shift in VH gene utilization in the peripheral B cell repertoire.

Functional antibody single-chain fragments from the cytoplasm of Escherichia coli: influence of thioredoxin reductase (TrxB).

The cytoplasmic expression of a functional antibody (Ab) fragment, containing the correct intradomain disulfide bonds, was investigated in E. coli. We used a single-chain Fv (scFv) fragment of the levan-binding Ab ABPC48, which was shown to be functional only in the presence of the disulfide bonds. Significant amounts of functional, disulfide-containing scFv could be produced in the cytoplasm of E. coli in the absence of thioredoxin reductase (TrxB) activity. The amount of soluble protein remained largely unchanged by this null mutation. A stronger promoter did not result in further improved yields of functional Ab fragment, despite much higher protein production, suggesting that inefficient disulfide formation was still limiting the yield of active scFv. This method of expressing functional Ab fragments in the cytoplasm of E. coli may be important for screening and selection systems.

The molecular origin of regulatory idiotypes.

A complete immunochemical and molecular profile was generated for a group of hybridoma and myeloma antibodies bearing the A48 regulatory idiotype (RI). These A48 RI+ antibodies were derived from normal or idiotypically manipulated mice and were selected either for utilization of a VHX24 VH gene or expression of the A48 RI. Among the hybridomas selected for VHX24 VH utilization a variety of antibody specificities were seen with the fructosan specificity occurring least frequently and the N-acetylglucosamine specificity occurring most frequently. A variety of Vk families were used with a bias for the Vk1 family by the antibodies deriving from untreated mice. The A48RI was expressed by only 3 of these antibodies, none of which were fructan specific. Two used the canonical VHX24-Vk10 combination utilized by the A48 and UPC 10 prototypes, and one used the VHX24-Vkl combination. This demonstration of A48 RI expression ny non-fructan specific, non-VHX24+Vk10+ antibodies was extended by showing expression of this Id by two monoclonal antibodies specific for the Sm self-antigen, one rheumatoid factor and two monoclonal antibodies specific for influenza virus hemagglutinin molecule. They used different VH-VL combinations. Among the monoclonal antibodies selected for A48 RI expression all exhibited fructan binding activity and the vast majority used the VHX24-Vk10 association. A collective analysis of the VH and VL sequences of all these A48RI+ antibodies showed idiotype expression was not associated with any particular germline VH or VL gene. D, Jk or JH sequence. Three positions on the light chain and one on the heavy chain were identified which could represent the structural correlates for the A48 regulatory idiotype.

Transfer of T or CD8+ cells from hemorrhaged mice produce alterations in bacterial antigen specific plasma cell repertoires in normal syngeneic recipients.

Hemorrhage has multiple effects on immunologic response, including alteration of B cell repertoires and T cell function. This study examined possible relationships between these two phenomena by determining the effects of T cells and T cell subsets transferred from hemorrhaged donors into normal, unhemorrhaged syngeneic recipients on B cell repertoires. Mice given total T or CD8+ cells from hemorrhaged animals then immunized with the bacterial polysaccharide antigen levan had a decreased percentage of plasma cells producing antibody to levan compared to that in mice given T or CD8+ cells from unhemorrhaged animals. These effects of post hemorrhage CD8+ cells were not seen after transfer into nu/nu mice, indicating that these cells did not directly affect B cell function, but rather required other T cell populations in order to alter the B cell repertoire. These results demonstrate that hemorrhage-induced alterations in bacterial antigen specific B cell repertoires may result from T and CD8+ cell mediated changes in T-B interactions.

Immunochemical studies on dextran-specific and levan-specific myeloma proteins from NZB mice.

Two dextran-specific (PC 3858 and PC 3936) and one levan-specific (PC 3660) NZB myeloma proteins were studied by quantitative precipitin and precipitin-inhibition assays. Both myeloma antidextrans were alphaD-(1 leads to 6) specific and precipitated strongly with a synthetic, linear dextran, molecular weight 35,500, and with other dextrans. The two myeloma antidextrans differed with respect to their relative reactivities with dextrans containing various proportions of alpha-D-(1 leads to 6), alpha-D-(1 leads to 4)-like, and alpha-D-(1 leads to 3)-like linkages. In inhibition assays, the two antidextran myeloma proteins behaved differently from each other, from alpha-D-(1 leads to 6)-specific BALB/c myeloma antidextrans, and from the human antidextrans previously studied. Isomalto-oligosaccharides IM3, IM4, and IM5 were all equal in inhibitory power but were only about 60% as potent as IM6 and IM7, which also inhibited equally on a molar basis. Although precipitation with linear dextran suggests that both may have groove-type sites, as previously inferred for QUPC 52, the size of their combining sites is uncertain. It is not clear whether the sites are only as big as three glucose residues with the increased inhibition by six and seven glucose residues being attributable to partial bivalence and to their ability to combine in several ways along the chain, or whether the site is as big as six glucose residues with the increment in binding by the fourth and fifth glucose residues being minimal and the sixth contributing considerable additional binding-energy. The fructan-specific myeloma protein did not react with inulin, but reacted with many levans and with perennial rye-grass levan containing only beta-D-(2 leads to 6) links. The levan-antilevan reaction was not inhibited by beta-D-(2 leads to 1)- linked oligosaccharides. The findings suggest that PC 3660 has a specificity for (2 leads to 6)-linked chains.