Improvement of insulin sensitivity in insulin resistant subjects during prolonged treatment with the anti-TNF-alpha antibody infliximab.
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Adult↗
Biomedical subjects
H P Brezinschek
Publications and source records attributed to H P Brezinschek.
The role of CD40-CD40 ligand (CD154) interactions in immunoglobulin light chain repertoire generation and somatic mutation.
To determine whether CD40 ligation influences the molecular and selective mechanisms that govern the development of the human Ig light chain repertoire, analysis of the Vkappa and Vlambda repertoires of CD19+ B cells obtained from a patient with X-linked hyper IgM syndrome (XHIM) and a nonfunctional CD154 was carried out. The nonproductive Vkappa and Vlambda repertoires were largely comparable to that of the normals with respect to V gene and J segment distribution as well as CDR3 length and VLJL joint complexity. Comparison of the nonproductive and productive repertoires indicated that a limited number of VL genes were positively and negatively selected in the XHIM patient. Although mutations were observed in the XHIM VL repertoires, the frequency of mutations was significantly lower than in normals. Typical targeting of these mutations into RGYW/WRCY motifs was significantly reduced and subsequent selection of RGYW/WRCY mutations, which is normally observed, was not found. These results indicate that CD40 ligation is not required for generation of the light chain repertoire, positive selection of some Vk rearrangements, negative selection of specific VL genes, and some degree of somatic mutation. Importantly, however, targeting of mutations to RGYW/WRCY motifs and subsequent selection of these mutated motifs does not occur in the absence of CD40 ligation.
Amino Acid Motifs↗
The influence of CD40-CD154 interactions on the expressed human V(H) repertoire: analysis of V(H) genes expressed by individual B cells of a patient with X-linked hyper-IgM syndrome.
Analysis of the V(H)DJ(H) repertoire of peripheral blood IgM(+) B cells from a patient with X-linked hyper-IgM syndrome (X-HIgM) was undertaken to determine whether the distribution of V(H) families in the productive repertoire might be regulated by in vivo CD40-CD154 interactions. The distribution of V(H) genes in the non-productive repertoire of IgM(+) B cells was comparable in X-HIgM and normals. Unlike the normal productive V(H) repertoire, however, in the X-HIgM patient the V(H)4 family was found at almost the same frequency as the V(H)3 family. This reflected a diminution in the positive selection of the V(H)3 family observed in normals and the imposition of positive selection of the V(H)4 family in the X-HIgM patient. Unique among the V(H)3 genes, V(H)3-23/DP-47 was positively selected in both normals and the X-HIgM patient. No major differences in the usage of J(H) or D segments or the complementarity-determining region (CDR) 3 were noted, although the foreshortening of the CDR3 noted in the mutated V(H) rearrangements of normals was absent in the X-HIgM patient. Finally, a minor degree of somatic hypermutation was noted in the X-HIgM patient. These results have suggested that specific influences on the composition of the V(H) repertoire in normals require CD40-CD154 interactions.
CD40 Antigens↗
Pairing of variable heavy and variable kappa chains in individual naive and memory B cells.
A functional Ig consists of two heterodimers each of which is composed of a heavy and a light chain. Although there is increasing knowledge about the events that govern the rearrangement of the genes encoding each individual chain, only very limited information is available about the mechanisms governing the pairing of variable heavy (V(H)) and variable light (V(L)) chains. Using a single cell PCR, we were able to obtain V(H) and Vkappa chains from 144 individual human CD19+/IgM+ B cells. Pairing of specific V(H) or Vkappa families was not observed, nor was the length or the amino acid composition of the CDR3s of V(H) and Vkappa chains in individual B cells similar. Comparison of V(H) and Vkappa genes in B cells in which one or both contained evidence of somatic hypermutation with those with no mutations revealed a significant decrease in the mean length of the V(H) CDR3. Moreover, there was a significant correlation between the frequencies of mutations in V(H) and Vkappa gene pairs in individual B cells. These results indicate that Ag-mediated selection as opposed to V(H)DJ(H) recombination or subsequent Ig chain pairing tended to approximate the CDR3 lengths and the frequency of mutations of V(H) and Vkappa in individual B cells.
Adult↗
Delineation of selective influences shaping the mutated expressed human Ig heavy chain repertoire.
After Ag exposure, somatic hypermutation and subsequent selection play significant roles in shaping the peripheral B cell repertoire. However, the disparate impact of each process has not been completely delineated. To address this, the mutational patterns of a large panel of productive V(H)DJ(H) rearrangements of individual human B cells were analyzed and compared with those of a previously reported panel of nonproductive V(H)DJ(H) rearrangements. The productive V(H) rearrangements exhibited a significantly lower mutational frequency and a significantly smaller number of replacement mutations than the nonproductively rearranged genes, suggesting that structural constraints of the Ig molecule and selective influences both impacted the repertoire, militating against replacement mutations. Positive selection favored a mean of four to six replacements in complementarity-determining region 1 (CDR1) and CDR2, and less than two replacements in the framework regions (FRs). In contrast, the negative impact of replacement mutations generated an increased number of silent mutations within both the CDRs and FRs of the productive repertoire accompanied by a net increase in the ratio of replacement to silent mutations in the CDRs compared with that in the FRs. Moreover, there was a negative influence on the distribution of amino acid changes resulting from mutations of highly mutable codons, such as AGY, TAY, GTA, and GCT, preferentially leading to conservative changes in the expressed Ig repertoire. The results are consistent with the conclusion that the expressed repertoire is limited, compared with the potential generated by the mutational machinery, by the dual requirements of avoiding autoreactivity and satisfying structural constraints of an intact Ig molecule.
Codon↗
Comparable impact of mutational and selective influences in shaping the expressed repertoire of peripheral IgM+/CD5- and IgM+/CD5+ B cells.
Somatic hypermutation and subsequent selection play a significant role in shaping the peripheral B cell repertoire. This repertoire is composed of CD5+ (5%) and CD5- B cells (95%) which are known to traffic through different lymphoid compartments. Previous studies have shown that V(H) gene usage by CD5+ and CD5- B cells is similar, although mutations are more frequent in the latter. However, the effect of mutation and subsequent selection on the expressed V(H) repertoire of CD5+ and CD5- B cells has not been delineated in detail. This study, therefore, analyzed the mutational pattern of individual IgM+/CD5+ and IgM+/CD5- B cells. In both populations, mutations can occur without heavy chain isotype switching. Despite the differences in mutational frequency, the patterns of mutation and subsequent selection were comparable in CD5+ and CD5- B cells. These results imply that although mutations are more frequent in CD5- B cells, the overall mechanisms governing somatic hypermutation and subsequent positive and negative selection are similar in CD5+ and CD5- B cells.
Adenine↗
Similar characteristics of the CDR3 of V(H)1-69/DP-10 rearrangements in normal human peripheral blood and chronic lymphocytic leukaemia B cells.
The variable heavy chain (V(H)) gene segment V(H)1-69/DP-10 has been shown to be over-represented in B-cell chronic lymphocytic leukaemia (CLL). Because of certain similar characteristics of their complementarity determining region 3 (CDR3), including preferential utilization of J(H)6 elements and an extended length, it has been suggested that antigenic stimulation might be involved in leukaemogenesis. Utilizing single-cell PCR to amplify and sequence genomic DNA from individual normal human peripheral blood B cells, we have obtained 7/421 productively and 1/69 nonproductively rearranged V(H) genes that used V(H)1-69/DP-10. All productive rearrangements were unmutated, used J(H)6 and had an average CDR3 length similar to that previously found in V(H)1-69/DP-10-expressing CLL cells. These results suggest that CLL may arise from B cells commonly found in the peripheral B-cell repertoire and do not represent expansion of a unique subset of specific antigen-reactive B cells.
Adult↗
Analysis of the targeting of the hypermutational machinery and the impact of subsequent selection on the distribution of nucleotide changes in human VHDJH rearrangements.
B cells are unique in that they generate and tolerate a high rate of mutations in their antigen receptor genes and employ these mutations as a basis of avidity maturation. The precise role of the mutational machinery versus subsequent selection in determining the frequency and distribution of mutations has not been fully analyzed. To address these issues, the influence of the intrinsic mutational machinery and subsequent selection on the frequency and distribution of mutations in the expressed human immunoglobulin repertoire was analyzed. Analysis of non-productively rearranged VH genes from individual human B cells provided an opportunity to examine the immediate impact of somatic hypermutation without superimposed selective influences. Comparison with the frequency and distribution of mutations in the productively rearranged human VH genes permitted an estimate of the influences of subsequent selection.
B-Lymphocytes↗
Analysis of the human VH gene repertoire. Differential effects of selection and somatic hypermutation on human peripheral CD5(+)/IgM+ and CD5(-)/IgM+ B cells.
To analyze the immunoglobulin repertoire of human IgM+ B cells and the CD5(+) and CD5(-) subsets, individual CD19(+)/ IgM+/CD5(+) or CD5(-) B cells were sorted and non-productive as well as productive VH gene rearrangements were amplified from genomic DNA and sequenced. In both subsets, the VH3 family was overrepresented largely as a result of preferential usage of a small number of specific individual family members. In the CD5(+) B cell subset, all other VH families were found at a frequency expected from random usage, whereas in the CD5(-) population, VH4 appeared to be overrepresented in the nonproductive repertoire, and also negatively selected since it was found significantly less often in the productive compared to the nonproductive repertoire; the VH1 family was significantly diminished in the productive rearrangements of CD5(-) B cells. 3-23/DP-47 was the most frequently used VH gene segment and was found significantly more often than expected from random usage in productive rearrangements of both CD5(+) and CD5(-) B cells. Evidence for selection based on the D segment and the JH gene usage was noted in CD5(+) B cells. No differences were found between the B cell subsets in CDR3 length, the number of N-nucleotides or evidence of exonuclease activity. Somatically hypermutated VHDJH rearrangements were significantly more frequent and extensive in CD5(-) compared to CD5(+) IgM+ B cells, indicating that IgM+ memory B cells were more frequent in the CD5(-) B cell population. Of note, the frequency of specific VH genes in the mutated population differed from that in the nonmutated population, suggesting that antigen stimulation imposed additional biases on the repertoire of IgM+ B cells. These results indicate that the expressed repertoire of IgM+ B cell subsets is shaped by recombinational bias, as well as selection before and after antigen exposure. Moreover, the influences on the repertoires of CD5(+) and CD5(-) B cells are significantly different, suggesting that human peripheral blood CD5(+) and CD5(-) B cells represent different B cell lineages, with similarities to murine B-1a and B-2 subsets, respectively.
Adult↗
Molecular mechanisms and selective influences that shape the kappa gene repertoire of IgM+ B cells.
To analyze the human kappa chain repertoire and the influences that shape it, a single cell PCR technique was used that amplified Vkappa Jkappa rearrangements from genomic DNA of individual human B cells. More than 350 productive and 250 nonproductive Vkappa Jkappa rearrangements were sequenced. Nearly every functional Vkappa gene segment was used in rearrangements, although six Vkappa gene segments, A27, L2, L6, L12a, A17, and O12/O2 were used preferentially. Of these, A27, L2, L6, and L12a showed evidence of positive selection based on the variable region and not CDR3, whereas A17 was overrepresented because of a rearrangement bias based on molecular mechanisms. Utilization of Jkappa segments was also nonrandom, with Jkappa1 and Jkappa2 being overrepresented and Jkappa3 and Jkappa5 underrepresented in the nonproductive repertoire, implying a molecular basis for the bias. In B cells with two Vkappa Jkappa rearrangements, marked differences were noted in the Vkappa segments used for the initial and subsequent rearrangements, whereas Jkappa segments were used comparably. Junctional diversity was generated by n-nucleotide addition in 60% and by exonuclease trimming in 75% of the Vkappa Jkappa rearrangements analyzed. Despite this large degree of diversity, a strict CDR3 length was maintained in both productive and nonproductive rearrangements. More than 23% of the productive rearrangements, but only 7% of the nonproductive rearrangements contained somatic hypermutations. Mutations were significantly more frequent in Vkappa sequences derived from CD5- as compared with CD5+ B cells. These results document that the gene segment utilization within the Vkappa repertoire is biased by both intrinsic molecular processes as well as selection after light chain expression. Moreover, IgM+ memory cells with highly mutated kappa genes reside within the CD5- but not the CD5+ B cell compartment.
B-Lymphocytes↗
Analysis of the frequency and pattern of somatic mutations within nonproductively rearranged human variable heavy chain genes.
Somatic hypermutation plays an essential role in avidity maturation of Ab. To characterize the effects of hypermutation without the imposed bias of Ag-mediated selection, the mutational pattern of 37 nonproductively rearranged VH genes amplified from individual human B cells was analyzed. A high frequency of mutations as well as frequent replacement mutations were observed in the complementarity-determining regions (CDR) and in the framework regions of nonproductive VHDJH rearrangements. Comparison with 57 productive VH rearrangements indicated that replacement mutations, especially those occurring in the framework regions, were less frequent in productively rearranged VH genes, suggesting that they were deleted from the expressed repertoire. A number of factors contributed to the nonrandom localization of mutations, including: the targeting of specific motifs, such as AGY, GCY, GTA, TAY, and RGYW; an increased frequency of some commonly mutated motifs in the CDRs; and an apparent increased likelihood of mutations of CDR nucleotides. Each of these appeared to bias the mutational machinery, resulting in an increased frequency of replacement mutations in the CDRs of nonproductive VH rearrangements.
Base Composition↗
Staphylococcal enterotoxin D functions as a human B cell superantigen by rescuing VH4-expressing B cells from apoptosis.
Staphylococcal enterotoxins are potent superantigens, in that they activate T cells bearing specific V beta-chain gene segments. In this study, we analyzed the capacity of staphylococcal enterotoxin D (SED) to function as a B cell superantigen. SED induced T cell-dependent polyclonal proliferation and differentiation of B cells. In the absence of T cells, SED induced survival of B cells uniquely expressing VH4 containing IgM. The mechanism of survival of VH4-expressing B cells appeared to relate to the countering of apoptosis initiated by the engagement of HLA-DR by SED. Analysis of the VH4 gene products expressed by SED-stimulated B cells revealed the usage of six of the known functional VH4 genes with a variety of different CDR3 regions, employing different DH and JH gene segments. Moreover, the sequence analysis identified a possible site for SED binding of VH4 that includes the solvent-exposed surfaces of 3' CDR2/FR3 and/or FR1. Thus, SED appears to function as a unique B cell superantigen by inducing survival of VH4-expressing B cells.
Adult↗
Expression of the beta 7 integrin by human endothelial cells.
Integrin adhesion receptors mediate fundamental intercellular interactions of many cell types as well as cellular interactions with specific extracellular matrix molecules. To date, the beta 7 integrin has been shown to be expressed by leukocyte subsets and to mediate interactions of these cells with extracellular matrix molecules as well as with endothelial and epithelial cells. The data presented here indicate that human endothelial cells also express the beta 7 integrin both in vitro and in situ. Analysis of cDNA indicated that endothelial beta 7 was identical to that expressed by leukocytes. Cell surface expression of beta 7 was increased by exposure of the endothelium to the pro-inflammatory cytokines, tumor necrosis factor-alpha and interleukin-1 beta. In leukocytes, beta 7 complexes with alpha 4 or alpha E integrin chains. Endothelial cells also expressed a number of alpha-integrin chains, including alpha 4, but not alpha E. The expression and utilization of beta 7, presumably complexed with alpha 4, by endothelial cells may be instrumental in the maintenance of the function or phenotype of endothelial cells.
Antigens, CD↗
Analysis of the heavy chain repertoire of human peripheral B cells using single-cell polymerase chain reaction.
The VH gene repertoire of human peripheral B cells was analyzed using PCR analysis of individual blood B cells. Because genomic DNA of single B cells was analyzed, data from both productive and nonproductive VDJ rearrangements were obtained. Nine out of 75 B cells contained both functional and nonfunctional rearrangement products, whereas 62/75 had a single productive VDJ rearrangement. The distribution of VH families was ordered in accordance with the germline complexity, although a bias toward VH3 and some of its members was found. This bias was noted in both the productively and nonproductively rearranged repertoires, indicating that it resulted from molecular and not selective processes. Evidence for negative selection of certain VH3 and VH4 family members was noted in that they were found less often as productive than nonproductive VDJ rearrangements. In addition, evidence for positive selection based on CDR3 was obtained, in that JH6 and DXP'1 were found at a higher frequency in the productively compared with the nonproductively rearranged repertoire. The CDR3 segments were markedly heterogenous, with a much greater degree of variability noted in the nonproductive VDJ rearrangements. Finally, a much greater frequency of mutations was noted in the nonproductively rearranged VH genes within individual B cells. These results have begun to delineate the human peripheral B cell repertoire and identify processes that shape it both positively and negatively.
B-Lymphocytes↗
Cell-type specific response of peripheral blood lymphocytes to methotrexate in the treatment of rheumatoid arthritis.
The mode of action of methotrexate in the treatment of rheumatoid arthritis is still questionable. Although in vitro results suggest an immunosuppressive effect of methotrexate, several clinical studies have failed to confirm these effects in patients treated with oral low-dose methotrexate. With respect to the highly variable bioavailability of methotrexate, we investigated the effects of an intravenous administration of 15 mg methotrexate per week on peripheral blood lymphocyte subsets in eight patients with rheumatoid arthritis. Methotrexate after 12 weeks significantly (P < 0.01) reduced total peripheral blood lymphocytes and led to a pronounced redistribution of lymphocyte subsets with a preferred reductive effect on B-lymphocytes (P < 0.005) and T-lymphocytes (P < 0.05). Natural killer cells and killer cell-like T cells, on the other hand, were unaffected by the treatment. Our results suggest a cell-type specific effect of intravenously administered low-dose methotrexate on peripheral blood lymphocytes. This effect, in our opinion, may contribute to the mode of action of methotrexate as an immunosuppressive drug in the treatment of rheumatoid arthritis.
Adult↗
Serum levels of interleukin-6 and tumour-necrosis-factor-alpha are not correlated to disease activity in patients with rheumatoid arthritis after treatment with low-dose methotrexate.
Cytokines are major mediators of inflammatory responses in rheumatoid arthritis. Some of them have been shown to correlate with the disease activity and thus are proposed to be used for monitoring patients. Therefore the effects of a low-dose therapy with methotrexate on serum concentrations of interleukin-6 (IL-6) and tumour-necrosis-factor-alpha (TNF-alpha) were examined in eight patients with seropositive rheumatoid arthritis. Serum levels of IL-6 and TNF-alpha were significantly elevated in patients compared to healthy controls. Before the onset of MTX treatment IL-6 concentrations were correlated to the c-reactive protein (P < 0.05) but the correlation was abolished after treatment. For TNF-alpha no correlations neither before nor after treatment were observed. Both cytokines remained substantially elevated after MTX treatment despite a clear reduction in disease activity. Thus we suggest that one of the effects of MTX might be the inhibition of some of the actions of IL-6 and TNF-alpha.
Arthritis, Rheumatoid↗
Effects of cytokine application on glucocorticoid secretion in an animal model for systemic scleroderma.
We previously reported on an altered immune-endocrine feedback loop via the hypothalamo-pituitary-adrenal (HPA) axis in Obese strain (OS) chickens afflicted with spontaneous autoimmune thyroiditis. These animals are deficient in plasma corticosterone increase after antigenic challenge or application of cytokine-containing conditioned medium of mitogen-stimulated spleen cells (CM). To investigate whether the impaired ability to respond to cytokines with glucocorticoid-increasing factor (GIF) activity, e.g. interleukin 1 (IL 1), is restricted to OS chickens as a model for an organ-specific autoimmune disease, we extended our experiments to another autoimmune-prone animal strain, the chickens of the University of California at Davis line 200 (UCD-200). These animals develop an inherited inflammatory fibrotic disease that closely resembles human progressive systemic sclerosis (scleroderma). Application of GIF-containing CM to UCD-200 chickens leads to a transient increase in glucocorticoid serum levels within 1-2 hours comparable to that of controls. But, while corticosterone levels in the latter returned to normal baseline levels after 4 hours, they were still elevated in autoimmune chickens. Although the peak of the glucocorticoid hormone serum concentrations was equal to that of controls, UCD-200 had to secrete twice as much adrenocorticotropic hormone to achieve this corticosterone serum level due to an apparent hyporesponsiveness of the adrenal gland to this secretagogue. The altered cytokine-induced glucocorticoid secretion is found in early as well as in chronic, sclerotic stages of the disease. Cellular alterations in the peripheral blood of UCD-200 chickens during the prolonged elevated corticosterone section, i.e. between 2-4 hours after CM application, are characterized by a significant decrease in the percentage of CD4+ and CD8+ cells. Furthermore, a significant increase in B cells up to 24 hours with a maximum after 1 hour was found. The proliferative response to the mitogen concanavalin A of peripheral mononuclear cells was inversely correlated to the serum corticosterone level, showing a permanent decrease of 80-90% after 1-4 hours in autoimmune animals. This functional alteration in UCD-200 was accompanied by an 80% decrease in serum interleukin 2 (sIL 2) activity 4 hours after CM application. Twenty-four hours later an eight-fold increase in sIL 2 rebound activity was found, indicating that the inhibitory effect of corticosterone in UCD-200 chickens is not long-lasting.
Adrenocorticotropic Hormone↗
Increased immune activation during and after physical exercise.
The present study has been performed to examine the pattern of immune response during and following a long-duration of physical exercise. We have measured plasma concentrations of serum soluble immune activation markers namely soluble interleukin-2 receptor (sIL-2R), soluble CD8 (sCD8), soluble intercellular adhesion molecule 1 (sICAM-1), soluble CD23 (sCD23), soluble tumor necrosis factor receptor (sTNF-R) and neopterin in 18 individuals before, during (ascent: 3 h, descent: 2 h) and after an alpine tour. Compared to baseline levels, all the parameters were significantly increased on top of the mountain and/or after descent. Within 36 hours after the tour sIL-2R, sCD8 and sICAM-1 decreased. In contrast, sTNF-R and neopterin levels remained higher than baseline throughout the study, only partially decreasing 24 and 36 hours from start. These data show immune system activation induced by physical exercise. The increase of parameters sTNF-R and neopterin, reflecting activation of macrophages, was sustained. The data suggest that immune activation phenomena may be involved in the pathogenesis of impaired immune function after exercise and the exercise-induced asthma.
Adult↗