IL-2-like activity in lymph fluid following in vivo antigen challenge.

The majority of studies that characterize lymphokines utilize in vitro activation of lymphocytes. In an attempt to identify and characterize lymphokines released from tissue sites, we have cannulated sheep lymphatic vessels and collected lymph that drains a site of in vivo antigen challenge. Lymph draining directly from a site of intradermal antigen challenge (afferent lymph) and lymph draining an antigen-stimulated lymph node (efferent lymph) were assayed for lymphokine activity by the ability of cell-free lymph fluid to stimulate the proliferation of sheep Con A-blasts. Afferent and efferent lymph, both collected at 24 and 48 hr following in vivo antigen challenge, with either ovalbumin or PPD in primed animals, stimulates the proliferation of sheep Con A-blast cells. This in vivo-derived lymphokine activity and in vitro-generated sheep Con A supernatant has an active component with properties similar to interleukin-2 (IL-2) that has been described in several other species. The IL-2-like material is precipitated by 40-80% ammonium sulphate saturation, has a molecular weight (MW) of 20,000 MW as judged by gel filtration chromatography, and is eluted from an anion-exchange HPLC column with 125 mM NaCl. HPLC ion-exchange fractionation of the 20,000 MW material from lymph fluid shows differences between afferent and efferent lymph material. The fractionation of afferent material is similar to that of in vitro generated Con A supernatant material with a single peak of activity eluted by 125 mM NaCl. In contrast, the 20,000 MW material from efferent lymph elutes with peaks of activity at 125 and 300 mM NaCl.     

Genetic and molecular studies of plasmids coding for colonization factor antigen I and heat-stable enterotoxin in several escherichia coli serotypes.

Plasmids coding for colonization factor antigen I (CFA/I) and heat-stable enterotoxin (ST) were identified in 10 strains of human enterotoxigenic Escherichia coli. The strains, which belonged to serogroups O63, O114, O128, and O153, were isolated in Bangladesh, Latin America, Spain, and South Africa. Two strains produced heat-labile enterotoxin in addition to ST. CFA/I-ST plasmids were mobilized from two O128 strains into E. coli K-12 with the R factor R1-19K-. Like the prototype CFA/I-ST plasmid NTP113, mobilized previously from an E. coli O78 strain into K-12, these two plasmids were non-autotransferring. All 10 CFA/I-ST plasmids were incompatible with NTP113 and had molecular weights ranging from 59 X 10(6) to 72 X 10(6). The molecular properties of seven of these plasmids were compared with those of six CFA/I-ST plasmids previously mobilized from O78 strains from Ethiopia, South Africa, and Bangladesh and with those of one plasmid coding for CFA/I, ST and heat-labile enterotoxin from a South African strain of serogroup O63. Digestion with the restriction endonuclease HindIII showed that several plasmids had very similar fragment patterns and two were identical. Generally, a larger proportion of HindIII fragments were of common size in digests of plasmids identified in strains from related geographical areas, regardless of serogroup. However, all except one plasmid shared five or six HindIII fragments of the same size, one of which had been shown previously to be involved in CFA/I production. There was at least 90% DNA homology between CFA/I-ST plasmids with a molecular weight of about 58 X 10(6) from O78 strains from different sources. Most of the DNA sequences of these plasmids were present in a larger CFA/I-ST plasmid (72 X 10(6) from an O128 strain. The results of genetic and molecular studies suggest that CFA/I and ST production is determined by very similar plasmids in different serogroups of human enterotoxigenic E. coli from several sources.     

Patterns of major histocompatibility complex class II expression by T cell subsets in different immunological compartments. 2. Altered expression and cell function following activation in vivo

This study characterizes antigen-induced phenotypic and functional aspects of major histocompatibility complex (MHC) class II expression on recirculating T cells in efferent lymph. In vivo secondary, but not primary challenge is associated with both kinetic and phenotypic alterations in class II expression by T cells. All three major T cell subsets, CD4⁺, CD8⁺ and T19⁺ (γδ T cell receptor), show an approximate four fold increase in the level of MHC class II expression during secondary responses. No changes in B cell expression of class II were seen. Resting efferent lymph T cells are predominantly either class II^? or DR⁺DQ^? but this changes to DR⁺DQ⁺ after antigenic challenge. The antigen-presenting function of these class II⁺ T cells was investigated at daily intervals after in vivo antigenic challenge. T cells from non-activated lymph nodes could not induce proliferation of antigen-specific T cells with soluble antigen but were weakly stimulatory in allo-mixed lymphocyte reaction (MLR) at high (> 2:1) stimulator cell ratios. Activated T cells isolated during secondary in vivo responses, and expressing increased quantities of MHC class II, were positive stimulator cells in the MLR. In contrast these cells could not present soluble antigen or trypsin-digested antigen to the T cell lines. In the MLR assays, the relative stimulation by class II⁺ T cells correlates with the levels of class II expression. We conclude from these experiments that both quantitative and qualitative changes in MHC class II, induced on T cells under physiological conditions, play a role in the regulation of the immune response in vivo but that that role is not simply one of presentation of soluble antigen.     

Adhesion and ultrastructural properties of human enterotoxigenic Escherichia coli producing colonization factor antigens III and IV.

Human enterotoxigenic Escherichia coli (ETEC) producing colonization factor antigen III (CFA/III) and coli surface antigens 4, 5, and 6 (CS4, CS5, and CS6) of CFA/IV were examined ultrastructurally and for ability to adhere to human small intestinal enterocytes and to cultured human intestinal mucosa. Strains of serotypes O25:H-, O25:H42, and O167:H5 producing CFA/III plus CS6, CS4 plus CS6, and CS5 plus CS6, respectively, showed good adhesion to human enterocytes (1.8 to 4.2 bacteria per brush border) and cultured human intestinal mucosa, whereas variants lacking these antigens or producing only CS6 were nonadherent (0 to 0.03 bacterium per brush border). By electron microscopy, CFA/III, CS4, and CS5 appeared as morphologically distinct rodlike fimbriae: CFA/III was 7 to 8 nm in diameter, CS4 was 6 to 7 nm in diameter, and CS5 was 5 to 6 nm in diameter. CS5 was unusual in that it appeared to be composed of two fine fibrils arranged in a double-helical structure. CS6 was difficult to characterize morphologically but possibly has a very fine fibrillar structure. By specific fimbrial staining and immunoelectron microscopy. CS4 and CS5 were shown to promote mucosal adhesion of ETEC; a similar adhesion role for the CS6 antigen could not be confirmed. ETEC strains of serotypes O27:H7, O27:H20, O148:H28, and O159:H20 which produced CS6 showed good adhesion to human enterocytes (1.6 to 3.0 bacteria per brush border), whereas variants which lacked CS6 were nonadherent (0 to 0.01 bacterium per brush border). These strains, however, also produced fimbrial or fibrillar surface antigens, in addition to CS6, which probably represent additional coli surface antigens responsible for the observed adhesive properties of these ETEC serotypes.     

Characterization of the molecular chaperone calnexin in the channel catfish, Ictalurus punctatus, and its association with MHC class II molecules

Folding and assembly of MHC molecules in mammals occurs in the endoplasmic reticulum (ER), but has not been studied in teleosts. Calnexin (CNX) is an ER chaperone that associates with glycoproteins bearing a monoglucosylated N-linked oligosaccharide side chain. Here we report the first identification and characterization of a full-length CNX cDNA clone in a teleost, and the association of the CNX chaperone with MHC class II in a channel catfish T cell line. The 1.8 kb CNX clone encodes a protein of 607 amino acids that is 72% identical to the consensus sequence of mammalian CNXs. The association of CNX with class II is of particular interest because the native MHC class II alpha chain of Ictalurus punctatus does not bear any N-linked oligosaccharide consensus glycosylation sequences. Thus the assembly of class II molecules in the catfish probably proceeds via different steps than occurs in mammals.     

Sequence comparison of human and yeast telomeres identifies structurally distinct subtelomeric domains

We have sequenced and compared DNA from the ends of three human chromosomes: 4p, 16p and 22q. In all cases the pro-terminal regions are subdivided by degenerate (TTAGGG)n repeats into distal and proximal sub- domains with entirely different patterns of homology to other chromosome ends. The distal regions contain numerous, short (<2 kb) segments of interrupted homology to many other human telomeric regions. The proximal regions show much longer (approximately 10-40 kb) uninterrupted homology to a few chromosome ends. A comparison of all yeast subtelomeric regions indicates that they too are subdivided by degenerate TTAGGG repeats into distal and proximal sub-domains with similarly different patterns of identity to other non-homologous chromosome ends. Sequence comparisons indicate that the distal and proximal sub-domains do not interact with each other and that they interact quite differently with the corresponding regions on other, non- homologous, chromosomes. These findings suggest that the degenerate TTAGGG repeats identify a previously unrecognized, evolutionarily conserved boundary between remarkably different subtelomeric domains.      

Non-random migration of CD4 and CD8 T cells: changes in the CD4: CD8 ratio and interleukin 2 responsiveness of efferent lymph cells following in vivo antigen challenge

In this investigation we have examined some of the cellular and molecular changes in efferent lymph that drains from an antigen-stimulated peripheral lymph node. Resting efferent lymph is characterized by a higher percentage of CD4+ cells and consequently, a higher CD4/CD8 ratio than peripheral blood. Following antigen stimulation of a cannulated peripheral lymph node in antigen-primed sheep, the percentage of CD4+ cells in efferent lymph increases above the resting level during days 1, 2 and 3 post antigen stimulation. This is followed on days, 3, 4, and 5 after antigen stimulation by an increase in the percentage of CD8+ cells above the resting level which occurs as the percentage of CD4+ cells returns to the resting level. These changes cause the CD4/CD8 ratio to first increase above the resting value during the CD4 phase and then decrease below the resting value during the CD8 phase. During the CD4 phase a lymphokine activity is present in cell-free lymph fluid. Lymph fluid collected at this time supports the proliferation of activated T cells. Supernatants generated from efferent cells collected at a similar time and cultured in vivo for 24 h without any further stimulation are capable of releasing this material. During the CD8 phase cells expressing functional interleukin(IL)2 receptors appear in lymph fluid. The data suggests a sequential exit of T cell subsets from an antigen-stimulated lymph node and that the appearance of IL2-like activity and IL2-responsive cells in efferent lymph fluid are temporally distinct events.