Host-defence-related proteins in cows' milk.

Milk is a source of bioactive molecules with wide-ranging functions. Among these, the immune properties have been the best characterised. In recent years, it has become apparent that besides the immunoglobulins, milk also contains a range of minor immune-related proteins that collectively form a significant first line of defence against pathogens, acting both within the mammary gland itself as well as in the digestive tract of the suckling neonate. We have used proteomics technologies to characterise the repertoire of host-defence-related milk proteins in detail, revealing more than 100 distinct gene products in milk, of which at least 15 are known host-defence-related proteins. Those having intrinsic antimicrobial activity likely function as effector proteins of the local mucosal immune defence (e.g. defensins, cathelicidins and the calgranulins). Here, we focus on the activities and biological roles of the cathelicidins and mammary serum amyloid A. The function of the immune-related milk proteins that do not have intrinsic antimicrobial activity is also discussed, notably lipopolysaccharide-binding protein, RNase4, RNase5/angiogenin and cartilage-glycoprotein 39 kDa. Evidence is shown that at least some of these facilitate recognition of microbes, resulting in the activation of innate immune signalling pathways in cells associated with the mammary and/or gut mucosal surface. Finally, the contribution of the bacteria in milk to its functionality is discussed. These investigations are elucidating how an effective first line of defence is achieved in the bovine mammary gland and how milk contributes to optimal digestive function in the suckling calf. This study will contribute to a better understanding of the health benefits of milk, as well as to the development of high-value ingredients from milk.

Variation in consumption of cow milk proteins and lower incidence of Type 1 diabetes in Iceland vs the other 4 Nordic countries.

BACKGROUND: The incidence of Type 1 diabetes is lower in Iceland than in the other 4 Nordic Countries. Earlier studies have showed that the cow milk proteins A1 and B beta-casein, suggested to be diabetogenic, are in lower amount in Icelandic cow milk than in milk from the other 4 Nordic Countries, and the per capita consumption of these proteins correlates with the incidence of Type 1 diabetes. OBJECTIVE: To investigate whether lower consumption of the cow milk protein bovine serum albumin (BSA) (suggested to be diabetogenic) or higher consumption of immunoglobulin (Ig) or lactoferrin (LF) (suggested to be protective) is related to the lower incidence of Type 1 diabetes in Iceland. METHODS: The per capita consumption of milk proteins was calculated from an international database on consumption of milk and milk products and from the analysis of cow's milk samples. The samples were randomly collected from the largest consumption areas in Iceland and in the other 4 Nordic Countries. RESULTS: The per capita consumption of BSA was higher in Iceland (0.79 +/- 0.02 g/person per day) (mean +/- SEM) than in the other 4 Nordic Countries (0.43 +/- 0.05 g/person per day) (p = 0.025). The per capita consumption of Ig was also higher in Iceland than in the other 4 Nordic Countries (p = 0.025), while the consumption of LF was similar. Consumption of these 3 individual milk proteins did not correlate with the incidence of Type 1 diabetes in the 5 countries studied. CONCLUSION: Consumption of BSA, Ig or LF does not seem to explain the lower incidence of Type 1 diabetes in Iceland, compared with the other 4 Nordic Countries, while A1 and B beta-casein may contribute to varying diabetogenicity of cow's milk and explain the difference in incidence of Type 1 diabetes.

B cell immunodeficiency fails to develop in CD4-deficient mice infected with BM5: murine AIDS as a multistep disease.

The immunodeficiency syndrome murine AIDS (MAIDS), caused by the BM5 retrovirus preparation, involves the activation, division, and subsequent anergy of the entire CD4(+) T cell population as well as extensive B cell hyperproliferation and hypergammaglobulinemia, resulting in splenomegaly and lymphadenopathy, followed many weeks later by death. The development of MAIDS requires CD4(+) T cells and MHC class II expression by the infected host, supporting a role for T-B interaction in disease development or progression. To explore this possibility, we examined development of MAIDS in mice deficient in CD4 (CD4 knockout), in which T-B interactions are compromised. We find that in CD4 knockout hosts, BM5 causes T cell immunodeficiency in the remaining T cells but has only a limited ability to induce B cell phenotypic changes, hyperproliferation, hypergammaglobulinemia, or splenomegaly. There is also delayed death of infected mice. This implies that CD4 dependent T-B interaction is needed to induce the B cell aspects of disease and supports a multistep mechanism of disease in which B cell changes follow and are caused by CD4(+) T cell effects.

Different beta-casein fractions in Icelandic versus Scandinavian cow's milk may influence diabetogenicity of cow's milk in infancy and explain low incidence of insulin-dependent diabetes mellitus in Iceland.

OBJECTIVES: To compare children with insulin-dependent diabetes mellitus (IDDM) with controls in Iceland regarding their consumption of cow's milk in infancy, and to investigate the beta-casein fractions in Scandinavian and Icelandic cow's milk. The A1 variant of beta-casein has been shown to be diabetogenic in animal studies, and suggestions have been made that the B variant of beta-casein acts similarly. Differences in the relative proportions of beta-casein fractions might explain the lower incidence of IDDM in Iceland than in Scandinavia. METHODS: A retrospective case-control study on IDDM patients and matching controls was performed in Iceland to compare their diets in infancy. Fifty-five children with IDDM born in Iceland over a 16-year period and randomly collected controls (n = 165) were recruited to the study. Mothers of the children answered questions on breastfeeding habits and on when cow's milk products were introduced. Samples of cow's milk from randomly selected milk batches from the largest consumption areas in Iceland and Scandinavia were collected. The milk samples were freeze-dried and their beta-casein fractions were analyzed using capillary electrophoresis. RESULTS: No significant difference was found between IDDM patients and controls in the frequency and duration of breastfeeding or the first introduction of cow's milk products. The analyses of milk samples showed that the percentage of the A1 and B variants of beta-casein in Icelandic milk was significantly lower than in the milk from the Scandinavian countries. CONCLUSIONS: Cow's milk consumption in infancy is not related to IDDM in Iceland. The lower fraction of A1 and B beta-caseins in Icelandic cow's milk may explain why there is a lower incidence of IDDM in Iceland than in Scandinavia.

Reciprocal regulation of polarized cytokine production by effector B and T cells.

Although B cells produce cytokines it is not known whether B cells can differentiate into effector subsets that secrete polarized arrays of cytokines. We have identified two populations of "effector" B cells (Be1 and Be2) that produce distinct patterns of cytokines depending on the cytokine environment in which the cells were stimulated during their primary encounter with antigen and T cells. These effector B cell subsets subsequently regulate the differentiation of naïve CD4+ T cells to TH1 and TH2 cells through production of polarizing cytokines such as interleukin 4 and interferon gamma. In addition, Be1 and Be2 cells could be identified in animals that were infected with pathogens that preferentially induce a Type 1 and Type 2 immune response. Together these results suggest that, in addition to their well defined role in antibody production, B cells may regulate immune responses to infectious pathogens through their production of cytokines.

E2A activity is induced during B-cell activation to promote immunoglobulin class switch recombination.

The basic helix-loop-helix protein, E2A, is required for proper early B lymphopoiesis. Specifically, in E2A-deficient mice, B-cell development is blocked at the progenitor stage prior to the onset of immunoglobulin (Ig) V(D)J recombination. Here, we demonstrate that E2A plays an additional role during peripheral B lymphopoiesis. Upon activation of primary mature B lymphocytes, both E2A protein levels and DNA-binding activity are induced. Furthermore, we show that mature B cells, expressing a dominant-negative E2A antagonist, proliferate normally in response to mitogenic signaling and appropriately express the early and late activation markers CD69, CD44, IgD and B220. However, in the absence of E2A activity, B lymphocytes are blocked in their ability to express secondary Ig isotypes. We demonstrate that the defect lies at the level of DNA rearrangements between the Ig switch regions. These data suggest that E2A is an essential target during B-cell activation and its induction is required to promote Ig class switch recombination.

Type I (insulin-dependent) diabetes mellitus and cow milk: casein variant consumption.

Previously published Type I (insulin-dependent) diabetes mellitus incidence in 0 to 14-year-old children from 10 countries or areas was compared with the national annual cow milk protein consumption. Countries which were selected for study had appropriate milk protein polymorphism studies, herd breed composition information and low dairy imports from other countries. Total protein consumption did not correlate with diabetes incidence (r = +0.402), but consumption of the beta-casein A1 variant did (r = +0.726). Even more pronounced was the relation between beta-casein (A1+B) consumption and diabetes (r = +0.982). These latter two cow caseins yield a bioactive peptide beta-casomorphin-7 after in vitro digestion with intestinal enzymes whereas the common A2 variant or the corresponding human or goat caseins do not. beta-casomorphin-7 has opioid properties including immunosuppression, which could account for the specificity of the relation between the consumption of some but not all beta-casein variants and diabetes incidence.

Induction of CD8+ CTL recognizing mycobacterial peptides.

Mycobacterium tuberculosis is the single, most important cause of morbidity attributable to a single infectious organism. CD8+ T cells play an important role in anti-tuberculous immune responses in both mice and humans. Data concerning the identity of mycobacterial antigens recognized by CD8+ T cells is limited; consequently, few CTL epitopes have been characterized. The authors identified allele-specific (H-2b and d) MHC class I binding motifs in six prominent M. tuberculosis protein antigens (the 19 and 38 kDa lipoglycoproteins and the 10, 16, 65 and 70 kDa stress proteins). These predicted epitopes were tested for MHC binding as well as their ability to elicit peptide-specific CTL following in vivo priming. The authors were able to identify eight previously undescribed mycobacterial CTL epitopes by using spleen cells from peptide-immunized mice. In addition, CTL specific for at least one of these epitopes also recognized the naturally processed epitope presented on transfected EL4 target cells. These mycobacteria-derived CTL epitopes could be important for future analysis of the involvement of CD8+ T cells in M. tuberculosis infection, pathogenesis and vaccine development.

Mutagenesis of an immunodominant T cell epitope can affect recognition of different T and B determinants within the same antigen.

The effects of mutagenesis of residues of a major T cell epitope were investigated in order to expand knowledge from synthetic peptides to the naturally processed antigen. The impact of substitutions within the core of the immunodominant p61-80/PT19 mycobacterial epitope was ascertained in respect of this epitope per se, or of a C-terminal (140-159) overlapping T/B epitope and of a conformational B epitope. The core substitution A71L impaired T immunogenicity of the target epitope within the protein, but not in the peptide, whereas the N73A substitution impaired the responses in both instances. Notably, each of these single amino acid mutations abrogated the T but not the B immunogenicity of the C-terminal epitope. Furthermore, mutation of five core residues (71-76) also ablated expression of a monoclonal antibody defined conformational B epitope. In conclusion, immunological analysis of mutated proteins revealed functional associations between topographically distinct antigenic determinants which may account for the previously observed differences in the specificity of immune responses between immunised and infected hosts.

MRI of pulmonary embolism using Gd-DTPA-polyethylene glycol polymer enhanced 3D fast gradient echo technique in a canine model.

This study was to evaluate the accuracy of MR angiography (MRA) using a Gd-DTPA-polyethylene glycol polymer (Gd-DTPA-PEG) with a 3D fast gradient echo (3D fgre) technique in diagnosing pulmonary embolism in a canine model. Pulmonary emboli were created in six mongrel dogs (20-30 kg) by injecting tantalum oxide-doped autologous blood clots into the femoral veins via cutdowns. MRI was performed with a 1.5 T GE Signa imager using a 3D fgre sequence (11.9/2.3/15 degrees) following intravenous injection of 0.06 mmol Gd/kg of Gd-DTPA-PEG. The dogs were euthanized and spiral CT of the lungs were then obtained on the deceased dogs. The MRI images were reviewed independently and receiver-operating-characteristic (ROC) curves were used for statistical analysis using spiral CT results as the gold standard. The pulmonary emboli were well visualized on spiral CT. Out of 108 pulmonary segments in the six dogs, 24 contained emboli >2 mm and 27 contained emboli < or = 2 mm. With unblinded review, MRI detected 79% of emboli >2 mm and only 48% of emboli < or = 2 mm. The blinded review results were significantly worse. Gd-DTPA-PEG enhanced 3D fgre MRI is potentially able to demonstrate pulmonary embolism with fairly high degree of accuracy, but specialized training for the interpretations will be required.

Postmortem fetal MR imaging: comparison with findings at autopsy.

OBJECTIVE: The purpose of this study was to prospectively compare findings from postmortem fetal MR imaging with findings at autopsy. SUBJECTS AND METHODS: Twenty-six fetuses were imaged on a 1.5-T MR scanner using two-dimensional and high-resolution three-dimensional fast spin-echo techniques immediately before autopsy. The MR images were reviewed independently by three radiologists who evaluated then for major and minor malformations. These findings were then compared with those at autopsy. RESULTS: The 26 subjects had 47 major and 11 minor malformations. All three radiologists correctly identified 37 of the major malformations on the MR images (detection rate, 79%), and at least one of the three reviewers correctly identified 43 of the abnormalities (detection rate, 91%). Only one of the 11 minor anomalies was identified by any reviewer. Reviewers made six false-positive diagnoses. In two cases, both with major CNS malformations, MR imaging was superior to autopsy in defining in situ relationships. CONCLUSION: Although autopsy remains the study of choice for evaluating causes of fetal death, MR imaging is an excellent alternative when autopsy is refused. Additionally, MR imaging may be a valuable adjunct to autopsy for fetuses with CNS anomalies.

The application of aqueous two-phase systems to the purification of pharmaceutical proteins from transgenic sheep milk.

Transgenic sheep milk containing the protein human alpha 1-Antitrypsin (AAT) was partitioned in Poly(ethylene glycol) (PEG)-Sulphate and PEG-Phosphate biphasic systems. Individual partition coefficients for AAT and some of the milk proteins were determined in these systems. The effects of PEG molecular weight, pH and the inclusion of NaCl on the partitioning of the proteins were also studied. It was found that increasing the concentration of NaCl and decreasing the molecular weight of the PEG resulted in an increase of the partition coefficients of the proteins to the upper (PEG) phase. This partitioning effect was greater for the more hydrophobic proteins and particularly in systems having a pH close to the isoelectric point of the protein. Solubilities of the proteins in increasing concentrations of ammonium sulphate were measured in order to investigate the effects of hydrophobic and electrostatic interactions on the partitioning of these proteins in aqueous two-phase systems. Those proteins that precipitated at low levels of ammonium sulphate showed an increase in partition coefficient at low concentrations of NaCl, or they were precipitated at the interface of the phase at low concentrations of NaCl. Proteins that had low salting out constants in ammonium sulphate solutions were relatively unaffected by NaCl in ATPS. It is probable however that conformational changes and the state of aggregation of proteins are also important and should be invoked in describing the partitioning behavior observed for beta-Lg for example. Comparison of theoretical and experimental values for AAT yield and purity showed clearly that partition coefficients are influenced by the degree of purity and values obtained with purified standards are not necessarily the same as for the same protein present in a complex mixture. Under the most favourable conditions using a 4% w/w loading of transgenic ovine milk, we obtained a 91% yield of AAT in the PEG phase with a purity of 73%.

Increase of tuberculous infection in the organs of B cell-deficient mice.

Protective immunity against infection with Mycobacterium tuberculosis is imparted by T cells rather than antibodies, but B cells can play a role as antigen-presenting cells and in granuloma formation. We re-evaluated the role of B cells in the course of tuberculous infection in mu-chain knock-out (Ig-) mice. Surprisingly, the organs of M. tuberculosis-infected Ig- mice were found to have three- to eight-fold elevated counts of viable bacilli compared with normal littermates at 3-6 weeks post-infection. Splenic interferon-gamma responses to whole antigen were unimpaired, whilst proliferation to certain mycobacterial peptides was found to be diminished. However, bacille Calmette-Guérin (BCG) vaccination significantly reduced the infection in Ig- mice. The mechanisms by which B cells can influence primary tuberculous infection need further study.

Immunogenicity of peptides for B cells is not impaired by overlapping T-cell epitope topology.

The epitope specificity of T-cell help to B cells and of surface immunoglobulin-mediated B-cell-binding of antigens usually involves topographically distinct antigenic determinants. The possibility of cross-recognition of the same peptide sequence by both T cells and antibodies has been a matter of conflicting opinions. We investigated this subject by detailed mapping of T- and B-cell epitopes within four immunogenic mycobacterial peptides. The identified core sequences of T- and B-cell epitopes showed different topology within each peptide: they were partially overlapping or adjacent in two P38-derived peptides, but entirely overlapping in two P19-derived peptides. The critically important result using the two truncated peptides (P19/67-78 and P19/146-155) containing only the fully overlapping epitope cores was, that they retained full potency for inducing antibody responses. However, despite this desirable overlap of determinants, antipeptide sera failed to block the proliferation of corresponding T-cell hybridomas. We conclude, that our study, in contrast to previous findings, suggests that overlapping topology of T- and B-cell epitopes within synthetic peptides does not necessarily impair B-cell immunogenicity.

Secretion of the mycobacterial 19-kilodalton protein by Escherichia coli, a novel method for the purification of recombinant mycobacterial antigens.

We have reported previously (R.G. Hewinson and W.P. Russell, J. Gen. Microbiol. 139:1253-1259, 1993) the secretion by Escherichia coli of a recombinant form of the immunogenic protein MPB70 of Mycobacterium bovis when the protein is translated from its native initiation codon. N-terminal sequence analysis of the purified protein revealed that the signal peptide of MPB70 was cleaved by an endopeptidase of E. coli at the same cleavage site as reported for the protein in M. bovis. Since both the B- and T-cell antigenicities of the purified recombinant protein were similar to that of the native protein, the 19-kDa antigen of M. bovis was used as a model to test whether the signal peptide of MPB70 could direct the secretion of heterologous proteins in E. coli and whether antigen produced in this way retained antigenicity superior to that of recombinant protein produced as a fusion to glutathione-S-transferase. A chimeric protein was produced in which the signal peptide of MPB70 was fused to the 19-kDa antigen of M. bovis at amino acid residue 23. This chimeric protein was found to be secreted into the periplasm and culture medium of recombinant E. coli, and the signal peptide was cleaved by an endopeptidase of E. coli during secretion. Secretion of the 19-kDa antigen facilitated purification of the antigen by two-stage preparative electrophoresis which gave yields of 2.5 mg of purified, soluble 19-kDa antigen from 2.5 g (wet weight) of E. coli. Antigen purified in this way retained both B- and T-cell antigenicities. Moreover, the nonspecific mitogenic activity of the purified 19-kDa antigen was low, while the magnitude of the T-cell response induced by the purified antigen was considerably higher than that observed with purified antigen produced as a fusion protein with glutathione-S-transferase.

Cross-recognition by T cells of an epitope shared by two unrelated mycobacterial antigens.

The molecular mimicry represented by cross-recognition of determinants shared by unrelated antigens by antibodies or T cells is of broad immunological interest. In this study, we analyzed the cross-recognition by CD4+ T cells of a peptide epitope shared by two mycobacterial proteins of diverse sequence, represented by the 19-kDa antigen of Mycobacterium tuberculosis and the 28-kDa antigen of Mycobacterium leprae. This epitope was immunodominant with respect to the 19-kDa antigen, but cryptic in relation to the 28-kDa antigen. The cross-reactive epitope cores were identified by Pepscan window analysis and found to be eight residues long in both antigens (residues 69-76 and 127-134). Alignment of these octameric sequences revealed two identical and five conservatively related amino acids. Within the epitope core, two residues (73Asn and 76Ile) were identified as critical for recognition on the basis of inhibition of the cross-reactive T cell proliferative response using singly substituted analog peptides. These results suggest that T cell cross-reactive epitopes can exist in proteins with apparently not more than random levels of sequence homology. Their potential for unsuspected cross-sensitization may play a role in the maintenance of T cell memory, in the pathogenesis of autoimmune diseases and possibly in a wide range of host immune responses to infectious pathogens.

Abundance of H-2 promiscuous T cells specific for mycobacterial determinants in H-2b/d F1 hybrid mice.

A majority of immunodominant epitopes of mycobacterial antigens are known to be recognized by murine T cells in the context of several H-2 haplotypes. In this study, we established the frequency of T cells able to recognize these peptides promiscuously, i.e. in the context of allogeneic antigen-presenting cells, using hybridomas from peptide-immunized H-2 homologous and heterologous mice. The degree of promiscuity in homozygous mice varied between 4-27% between different specificities and genetic backgrounds. In particular, the results showed that promiscuity between Ab and Ad in respect to a peptide from the Mycobacterium tuberculosis 38-kDa protein (residues 350-369) was displayed by 22% of BALB/c and 4% of C57BL/10-derived hybrids, but by 42% of [BALB/c x C57BL/10] F1-derived clones. This represents a significant increase (p < 0.001) of T cell promiscuity compared to the parental haplotypes. It is noteworthy that considerably lower peptide concentrations were able to stimulate the promiscuous hybridomas compared to the H-2-restricted hybrids. This finding suggests a functional advantage of promiscuous T cells which enables them to expand preferentially in the initial stages of infections with M. tuberculosis and thus enables the host to mount a rapid protective immune response.

Human T cell responses to peptide epitopes of the 16-kD antigen in tuberculosis.

The 16-kD protein constituent of the Mycobacterium tuberculosis complex has been known mainly for its prominent serological immunogenicity and species specificity in tuberculous infection. In this study, we evaluated the T cell immune repertoire in 27 sensitized healthy subjects and 46 patients with active tuberculosis using 14 overlapping 20mer peptides spanning the entire sequence of this protein. Four of the tested peptides individually stimulated proliferation of blood mononuclear cells from more than 50% of healthy controls. Tuberculosis patients reacted to a narrower peptide range and with a 17-27% lower rate of responses to the four most immunogenic peptides, but these differences do not distinguish in any simple way between the T cell repertoire of patients and sensitized healthy subjects. The most immunogenic peptide (91-110) was recognized by 67% of healthy subjects and by 50% of tuberculosis patients. Importantly, several non-responders to this peptide were stimulated with the other three most permissive peptides with sequences of 111-130, 71-91 and 21-40, resulting in an overall response rate to at least one of these four peptides of 93% in healthy controls and 74% in tuberculosis patients. In view of this additive effect between the most immunogenic peptides, their combined use may achieve sufficient sensitivity in a test aimed at the specific discrimination between infected and non-infected healthy subjects. The major interest in testing with these peptides rests in their species specificity, which is not achieved using purified protein derivative (PPD).

H-2-associated effects of flanking residues on the recognition of a permissive mycobacterial T-cell epitope.

Previously we have identified an immunodominant, eight-residue, epitope core sequence (TAAGNVNI) from the 19,000 MW protein of Mycobacterium tuberculosis, which is recognized in the context of multiple H-2 I-A molecules. In this study, the role of residues flanking this T-cell epitope core was examined, using a series of 20 mer analogue peptides in which the native flanking residues were progressively replaced with L-alanine. Analogue peptides were tested for their capacity to stimulate a CD4+ 19,000 MW protein-specific T-cell line, revealing that all but one N-terminal flanking residue could be replaced collectively by alanine without significant loss of stimulatory activity. However, clear H-2-associated differences in the requirement for flanking residues were demonstrated with peptide-specific T-cell hybridomas. In particular, H-2d-derived hybridomas were much more stringent in their requirement for flanking residues than were H-2b hybridomas. All polyalanine-substituted peptides bound I-Ab molecules, with affinities similar to the native unsubstituted peptide. In contrast, significantly reduced binding to I-Ad was observed with several analogue peptides, although without a clear relationship to the degree of substitution. Furthermore, in H-2b mice, neither immunogenicity nor cross-reactivity with the native peptide showed a clear inverse relationship with respect to the degree of alanine substitution. The results presented in this paper indicate that flanking residues can influence T-cell specificity and that these effects may be controlled by major histocompatibility complex (MHC) haplotype.