Pickering emulsions based on food byproducts: A comprehensive study of soluble and insoluble contents.

HYPOTHESIS: Pickering emulsions can be produced using raw particles obtained from uncracked vegetal food byproducts as sole stabilizers. The complexity brought by these non-purified ingredients will be their strength since insoluble particles and soluble compounds shall display good complementary properties, at the interface and the continuous phase. EXPERIMENTS: Emulsions were monitored over a one-month storage as regards to oil droplet diameter as main indicator of stability. Then, two main studies were carried out: 1) on the whole powders (water binding capacity, dry matter and insoluble content, size and morphology); 2) on the soluble content (size, charge, pH, Brix degree, surface tension measurements). FINDINGS: All byproducts stabilized-emulsions were stable during storage. They display various oil droplet sizes with sugar beet < apple < oat. Direct observation of the oil-water interfaces showed adsorption of the solid particles, and some voids corresponding to soluble elements from the byproducts' powders. The latter displayed surface-active properties. The insoluble particles are driving the oil droplet size and protecting against coalescence while soluble compounds can also adsorb at the interface, lowering droplet size, and also act as thickening agents from the continuous phase (pectins). Vegetal byproducts are thus meta ingredients, able to stabilize clean-label emulsions.

The immunosuppressant 15-deoxyspergualin [correction of 1,5-deoxyspergualin] reveals commonality between preT and preB cell differentiation.

15 [correction of 1,5] deoxyspergualin (DSG) is a potent immunosuppressant whose mechanism of action is still somewhat of a mystery. We have studied the generation of lymphocytes in mice treated with this drug. The differentiation of T cells in the thymus was blocked at an important early control point: the CD4-8- --> CD4+8+ transition, known to depend on the expression of a preTCR complex that includes the variable TCR-beta, but not TCR-alpha, chain. In clear contrast, a later control point, the CD4+8+ --> CD4+8- or CD4-8+ transition, dependent on the display of a conventional alpha:beta TCR complex, appeared unaffected, as did activation of mature T cells both in vitro and in vivo. Interestingly, preB cell differentiation in the bone marrow was blocked at a precisely equivalent point: the A-C --> C' transition, controlled by expression of a pre-receptor complex containing the Ig heavy, but not light, chain. Mature B cells seemed unperturbed. These findings have theoretical implications, suggesting common signaling pathways in early lymphocytes that are distinct from those employed by more mature cells, and are also of practical interest, to be considered in the design of DSG treatment protocols.

The V beta 17+ T cell repertoire: skewed J beta usage after thymic selection; dissimilar CDR3s in CD4+ versus CD8+ cells.

To ascertain how the actual repertoire of T cell receptors (TCRs) deviates from the theoretical, we have generated a large number of junctional region sequences from TCRs carrying the V beta 17 variable region. The greater than 600 sequences analyzed represent transcripts from nine different cell populations, permitting several comparisons: transcripts from an expressed vs. a non-expressed V beta 17 allele, those from E+ vs. E- mice, transcripts from immature vs. mature thymocytes, those from thymic vs. peripheral T cells, and those from CD4+ vs. CD8+ cells. These comparisons have allowed us to distinguish between the influence of molecular events involved in TCR gene rearrangement and that of various selection events that shape the T cell repertoire. Our most striking findings are: (a) that J beta usage is markedly skewed, partly due to recombination mechanics and partly due to selection forces: in particular, those mediated by the class II E molecule in the thymus; and (b) that TCRs on CD4+ and CD8+ cells show intriguing dissimilarities. In addition, we present evidence that N nucleotide additions occur with clear biases, probably due to idiosyncrasies of the recombination enzymes, and provide arguments that TCR and immunoglobulin CDR3s have distinct structures.

Major histocompatibility complex class II molecules can protect from diabetes by positively selecting T cells with additional specificities.

Insulin-dependent diabetes is heavily influenced by genes encoded within the major histocompatibility complex (MHC), positively by some class II alleles and negatively by others. We have explored the mechanism of MHC class II-mediated protection from diabetes using a mouse model carrying the rearranged T cell receptor (TCR) transgenes from a diabetogenic T cell clone derived from a nonobese diabetic mouse. BDC2.5 TCR transgenics with C57Bl/6 background genes and two doses of the H-2(g7) allele exhibited strong insulitis at approximately 3 wk of age and most developed diabetes a few weeks later. When one of the H-2(g7) alleles was replaced by H-2(b), insulitis was still severe and only slightly delayed, but diabetes was markedly inhibited in both its penetrance and time of onset. The protective effect was mediated by the Abetab gene, and did not merely reflect haplozygosity of the Abetag7 gene. The only differences we observed in the T cell compartments of g7/g7 and g7/b mice were a decrease in CD4(+) cells displaying the transgene-encoded TCR and an increase in cells expressing endogenously encoded TCR alpha-chains. When the synthesis of endogenously encoded alpha-chains was prevented, the g7/b animals were no longer protected from diabetes. g7/b mice did not have a general defect in the production of Ag7-restricted T cells, and antigen-presenting cells from g7/b animals were as effective as those from g7/g7 mice in stimulating Ag7-restricted T cell hybridomas. These results argue against mechanisms of protection involving clonal deletion or anergization of diabetogenic T cells, or one depending on capture of potentially pathogenic Ag7-restricted epitopes by Ab molecules. Rather, they support a mechanism based on MHC class II-mediated positive selection of T cells expressing additional specificities.

Evaluation of the functional equivalence of major histocompatibility complex class II A and E complexes.

Most mice display two conventional major histocompatibility complex class II isotypes, A and E. Several A+E- strains have been observed, but never any that are A-E+. Because of this and because of hints from several lines of functional analysis, it has been proposed that the two isotypes might not operate equivalently. This proposition has not been directly testable until now because of the lack of an E-only strain. We report the production of such mice, exploiting previously created class II-transgenic and class II-"knock-out" lines. A+E-, A-E-, and A-E+ littermates have been compared by a number of parameters. We find that E and A molecules are, for the most part, functionally equivalent. However, subtle differences are seen in their ability to engage CD4 molecules on immature thymocytes, and in the profile of receptors on T cells selected into the periphery.

Visualization of CD4/CD8 T cell commitment.

A system to innocuously visualize T cell lineage commitment is described. Using a "knock-in" approach, we have generated mice expressing a beta-galactosidase reporter in place of CD4; expression of beta-galactosidase in these animals appears to be an accurate and early indicator of CD4 gene transcription. We have exploited this knock-in line to trace CD4/CD8 lineage commitment in the thymus, avoiding important pitfalls of past experimental approaches. Our results argue in favor of a selective model of thymocyte commitment, demonstrating a fundamentally symmetrical process: engagement of either class of major histocompatibility complex (MHC) molecule by a differentiating CD4(+)CD8(+) cell can give rise to T cell antigen receptor (TCR)hi thymocytes of either lineage. Key findings include (a) direct demonstration of a substantial number of CD4-committed, receptor/coreceptor-mismatched cells in MHC class II- deficient mice, a critical prediction of the selective model; (b) highly efficient rescue of such "mismatched" intermediates by forced expression of CD8 in a TCR transgenic line, and an explanation of why previous experiments of this nature were less successful-a major past criticism of the selective model; (c) direct demonstration of an analogous, though smaller, population of CD8-committed mismatched intermediates in class I-deficient animals. Finally, we found no evidence of a CD4 default pathway.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) regulates the unfolding of autoimmune diabetes.

Evidence has been accumulating that shows that insulin-dependent diabetes is subject to immunoregulation. To determine whether cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is involved, we injected anti-CTLA-4 mAb into a TCR transgenic model of diabetes at different stages of disease. When injected into young mice, months before they would normally become diabetic, anti-CTLA-4 induced diabetes rapidly and essentially universally; this was not the result of a global activation of T lymphocytes, but did reflect a much more aggressive T cell infiltrate in the pancreatic islets. These effects were only observed if anti-CTLA-4 was injected during a narrow time window, before the initiation of insulitis. Thus, engagement of CTLA-4 at the time when potentially diabetogenic T cells are first activated is a pivotal event; if engagement is permitted, invasion of the islets occurs, but remains quite innocuous for months, if not, insulitis is much more aggressive, and diabetes quickly ensues.

Determinant capture as a possible mechanism of protection afforded by major histocompatibility complex class II molecules in autoimmune disease.

How peptide-major histocompatibility complex (MHC) class II complexes are naturally generated is still unknown, but accumulating evidence suggests that unfolding proteins or long peptides can become bound to class II molecules at the dominant determinant before proteolytic cleavage. We have compared the immunogenicity of hen egg-white lysozyme (HEL) in nonobese diabetic (NOD), (NOD x BALB/c)F1, and E(d) alpha transgenic NOD mice. We find that a response to the subdominant ANOD-restricted determinant disappears upon introduction of an E(d) molecule, and is restored when scission of HEL separates this determinant from its adjoining, competitively dominant, E(d)-restricted determinant. This suggests that the E(d) molecule binds and protects its dominant determinant on a long peptide while captured neighboring determinants are lost during proteolysis. These results provide clear evidence for "determinant capture" as a mechanism of determinant selection during antigen processing and a possible explanation for MHC-protective effects in insulin-dependent diabetes mellitus.

I-A alpha polymorphic residues that determine alloreactive T cell recognition.

An individual's T lymphocytes are highly reactive to allogeneic MHC molecules. As a step in deciphering the mechanism of allorecognition by T lymphocytes, we have attempted to identify the TCR's target on MHC class II molecules, in particular the polymorphic residues that determine the specificity of recognition. We have generated a panel of Ak-reactive, Ab-nonreactive T cell hybridomas, and sets of L cell transfectants displaying A alpha A beta molecules with wild-type, chimeric or single site-mutated A alpha chains, with reciprocal interchanges between Ak and Ab. We then measured the stimulation of the T hybridomas in response to the transfectants. The results indicate that the hybridomas recognize diverse and complex determinants, with contributions from both A alpha and A beta chains, and from several regions or amino acids of the A alpha chain. The data are most consistent with a model in which alloreactivity results from the presentation of peptides to the T cell by an allogeneic MHC molecule, peptides that cannot be presented by the responder's own MHC complexes. The specificity of allorecognition seems to be imparted mainly by peptide/MHC molecule rather than TCR/MHC molecule contacts.

CD1-restricted CD4+ T cells in major histocompatibility complex class II-deficient mice.

Rather unexpectedly, major histocompatibility complex class II-deficient mice have a significant population of peripheral CD4+ T lymphocytes. We have investigated these cells at the population and clonal levels. CD4+ T lymphocytes from class II-deficient animals are thymically derived, appear early in ontogeny, exhibit the phenotype of resting memory cells, are potentially functional by several criteria, and have a diverse T cell receptor repertoire. They do not include substantially elevated numbers of NK1.1+ cells. Hybridomas derived after polyclonal stimulation of the CD4+ lymphocytes from class II-deficient animals include a subset with an unusual reactivity pattern, responding to splenocytes from many mouse strains including the strain of origin. Most members of this subset recognize the major histocompatibility complex class Ib molecule CD1; their heterogeneous reactivities and T cell receptor usage further suggest the involvement of peptides and/or highly variable posttranslational modifications.

An enhancer factor defect in a mutant Burkitt lymphoma cell line.

RJ 2.2.5 is an immunoselected mutant of the Burkitt lymphoma line Raji. It fails to display MHC class II antigens at the cell surface due to a transcriptional defect. We have identified the function of a regulatory factor that is defective in RJ 2.2.5 cells; this factor is absolutely required for the activity of an MHC class II gene enhancer.

Tetracycline-controllable selection of CD4(+) T cells: half-life and survival signals in the absence of major histocompatibility complex class II molecules.

A system that allows the study, in a gentle fashion, of the role of MHC molecules in naive T cell survival is described. Major histocompatibility complex class II-deficient mice were engineered to express Ealpha chains only in thymic epithelial cells in a tetracycline (tet)-controllable manner. This resulted in tet-responsive display of cell surface E complexes, positive selection of CD4(+)8(-) thymocytes, and generation of a CD4(+) T cell compartment in a class II-barren periphery. Using this system, we have addressed two unresolved issues: the half-life of naive CD4(+) T cells in the absence of class II molecules (3-4 wk) and the early signaling events associated with class II molecule engagement by naive CD4(+) T cells (partial CD3 zeta chain phosphorylation and ZAP-70 association).

Initiation of autoimmune diabetes by developmentally regulated presentation of islet cell antigens in the pancreatic lymph nodes.

Little is known about the events triggering lymphocyte invasion of the pancreatic islets in prelude to autoimmune diabetes. For example, where islet-reactive T cells first encounter antigen has not been identified. We addressed this issue using BDC2.5 T cell receptor transgenic mice, which express a receptor recognizing a natural islet beta cell antigen. In BDC2.5 animals, activated T cells were found only in the islets and the lymph nodes draining them, and there was a close temporal correlation between lymph node T cell activation and islet infiltration. When naive BDC2.5 T cells were transferred into nontransgenic recipients, proliferating cells were observed only in pancreatic lymph nodes, and this occurred significantly before insulitis was detectable. Surprisingly, proliferation was not seen in 10-day-old recipients. This age-dependent dichotomy was reproduced in a second transfer system based on an unrelated antigen artificially expressed on beta cells. We conclude that beta cell antigens are transported specifically to pancreatic lymph nodes, where they trigger reactive T cells to invade the islets. Systemic or extrapancreatic T cell priming, indicative of activation via molecular mimicry or superantigens, was not seen. Compromised presentation of beta cell antigens in the pancreatic lymph nodes of juvenile animals may be the root of a first "checkpoint" in diabetes progression.

Genetic influences on the end-stage effector phase of arthritis.

K/BxN T cell receptor transgenic mice are a model of inflammatory arthritis, most similar to rheumatoid arthritis, that is critically dependent on both T and B lymphocytes. Transfer of serum, or just immunoglobulins, from arthritic K/BxN animals into healthy recipients provokes arthritis efficiently, rapidly, and with high penetrance. We have explored the genetic heterogeneity in the response to serum transfer, thereby focussing on the end-stage effector phase of arthritis, leap-frogging the initiating events. Inbred mouse strains showed clear variability in their responses. A few were entirely refractory to disease induction, and those which did develop disease exhibited a range of severities. F1 analyses suggested that in most cases susceptibility was controlled in a polygenic additive fashion. One responder/nonresponder pair (C57Bl/6 x NOD) was studied in detail via a genome scan of F2 mice; supplementary information was provided by the examination of knock-out and congenic strains. Two genomic regions that are major, additive determinants of the rapidity and severity of K/BxN serum-transferred arthritis were highlighted. Concerning the first region, on proximal chromosome (chr)2, candidate assignment to the complement gene C5 was confirmed by both strain segregation analysis and functional data. Concerning the second, on distal chr1, coinciding with the Sle1 locus implicated in susceptibility to lupus-like autoimmune disease, a contribution by the fcgr2 candidate gene was excluded. Two other regions, on chr12 and chr18 may also contribute to susceptibility to serum-transferred arthritis, albeit to a more limited degree. The contributions of these loci are additive, but gene dosage effects at the C5 locus are such that it largely dominates. The clarity of these results argues that our focus on the terminal effector phase of arthritis in the K/BxN model will bear fruit.

The Type 1 Diabetes PhysioLab Platform: a validated physiologically based mathematical model of pathogenesis in the non-obese diabetic mouse.

Type 1 diabetes is an autoimmune disease whose clinical onset signifies a lifelong requirement for insulin therapy and increased risk of medical complications. To increase the efficiency and confidence with which drug candidates advance to human type 1 diabetes clinical trials, we have generated and validated a mathematical model of type 1 diabetes pathophysiology in a well-characterized animal model of spontaneous type 1 diabetes, the non-obese diabetic (NOD) mouse. The model is based on an extensive survey of the public literature and input from an independent scientific advisory board. It reproduces key disease features including activation and expansion of autoreactive lymphocytes in the pancreatic lymph nodes (PLNs), islet infiltration and beta cell loss leading to hyperglycaemia. The model uses ordinary differential and algebraic equations to represent the pancreas and PLN as well as dynamic interactions of multiple cell types (e.g. dendritic cells, macrophages, CD4+ T lymphocytes, CD8+ T lymphocytes, regulatory T cells, beta cells). The simulated features of untreated pathogenesis and disease outcomes for multiple interventions compare favourably with published experimental data. Thus, a mathematical model reproducing type 1 diabetes pathophysiology in the NOD mouse, validated based on accurate reproduction of results from multiple published interventions, is available for in silico hypothesis testing. Predictive biosimulation research evaluating therapeutic strategies and underlying biological mechanisms is intended to deprioritize hypotheses that impact disease outcome weakly and focus experimental research on hypotheses likely to provide insight into the disease and its treatment.

T helper cell subsets in insulin-dependent diabetes.

It has been proposed that the development of insulin-dependent diabetes is controlled by the T helper 1 (TH1) versus TH2 phenotype of autoreactive TH cells: TH1 cells would promote diabetes, whereas TH2 cells would actually protect from disease. This proposition was tested by establishing cultures of TH1 and TH2 cells that express an identical diabetogenic T cell receptor and comparing their ability to initiate disease in neonatal nonobese diabetic mice. TH1-like cells actively promoted diabetes; TH2-like cells invaded the islets but did not provoke disease--neither did they provide substantial protection.

Diversity of endogenous epitopes bound to MHC class II molecules limited by invariant chain.

The invariant chain (Ii) binds nascent major histocompatibility complex (MHC) class II molecules, blocking peptide binding until the complex dissociates in the endosomes. This may serve to differentiate the MHC class I and II antigen presentation pathways and enable class II molecules to efficiently bind peptides in the endosomes. This hypothesis was addressed by probing spleen cells from a combination of knock-out and transgenic mice with a large panel of T cell hybridomas. The Ii molecule blocked the presentation of a range of endogenously synthesized epitopes, but some epitopes actually required Ii. Thus, the influence of Ii on presentation does not follow simple rules. In addition, mice expressing Ii were not tolerant to epitopes unmasked in its absence, a finding with possible implications for autoimmunity.

Positive selection of T cells induced by viral delivery of neopeptides to the thymus.

The relation between an antigenic peptide that can stimulate a mature T cell and the natural peptide that promoted selection of this cell in the thymus is still unknown. An experimental system was devised to address this issue in vivo-mice expressing neopeptides in thymic stromal cells after adenovirus-mediated delivery of invariant chain-peptide fusion proteins. In this system, selection of T cells capable of responding to a given antigenic peptide could be promoted by the peptide itself, by closely related analogs lacking agonist and antagonist activity, or by ostensibly unrelated peptides. However, the precise repertoire of T cells selected was dictated by the particular neopeptide expressed.

Independent modes of natural killing distinguished in mice lacking Lag3.

The LAG3 protein has several features in common with CD4, suggesting that it may be important in controlling T cell reactivity. However, mice with a Lag3 null mutation have now been shown to exhibit a defect in the natural killer cell, rather than the T cell, compartment. Killing of certain tumor targets by natural killer cells from these mice was inhibited or even abolished, whereas lysis of cells displaying major histocompatibility complex class I disparities remained intact. It appears that LAG3 is a receptor or coreceptor that defines different modes of natural killing.

Arthritis provoked by linked T and B cell recognition of a glycolytic enzyme.

The hallmark of rheumatoid arthritis (RA) is specific destruction of the synovial joints. In a mouse line that spontaneously develops a disorder with many of the features of human RA, disease is initiated by T cell recognition of a ubiquitously expressed self-antigen; once initiated, pathology is driven almost entirely by immunoglobulins. In this study, the target of both the initiating T cells and pathogenic immunoglobulins was identified as glucose-6-phosphate isomerase, a glycolytic enzyme. Thus, some forms of RA or related arthritides may develop by a mechanism fundamentally different from the currently popular paradigm of a joint-specific T cell response.