Early separation of B and T lymphocyte precursors in chick embryo.

Embryonic chimeras were used to demonstrate an early separation of chicken T and B cell precursors. Genetically polymorphic cell surface antigens, Bu-1 and Ov, which are expressed on cells of the B and T lineage, respectively, are useful markers in adoptive cell transfer studies. Allelic products Bu-1a and Bu-1b can be detected with monoclonal antibodies (mAbs) L22 and 11G2, respectively, and the Ov antigen with mAb 11A9. Chimeric chickens were constructed by reconstituting irradiated 14-d Ov- H.B19 embryos with the sorted Bu-1+ or Bu-1- fractions of spleen cells from age-matched H.B19 Ov+ embryos. Chimeras were analyzed, 3-4 wk after hatching, for the presence of Ov+ cells in the bursa, thymus, spleen, and peripheral blood lymphocytes. T cell precursors giving rise to thymocytes and peripheral T cells were present only in the Bu-1-, but not in the Bu-1+, fraction. We previously demonstrated that, in contrast, all B cell precursors in spleen from 14-d embryos are exclusively present in the Bu-1+ fraction. We also analyzed the immunoglobulin light chain gene rearrangement in these populations by polymerase chain reaction. We show here that VJ recombination occurs in the Bu-1+, but not in the Bu-1-, fraction of spleen. These data demonstrate an early commitment to the B cell lineage, which occurs before the colonization of the bursa of Fabricius. Segregation of B cell precursors from the other hemopoietic precursors, and consequently separation of T and B cell precursors, occurs before the colonization of the primary lymphoid organs.

Are serum inflammatory markers age dependent in acute appendicitis?

BACKGROUND: Preoperative measurement of body inflammatory agents reduces unnecessary appendectomies by up to 30 percent. A decline in the formation of blood leukocytes and C-reactive protein with aging may hinder the correct diagnosis of appendicitis. STUDY DESIGN: White cell count and C-reactive protein were determined before appendectomy in 600 patients aged 0 to 5 years, 6 to 19 years, 20 to 39 years, 40 to 59 years, 60 to 79 years, and older than 80 years. Their records were analyzed. The sensitivity, specificity, diagnostic accuracy, and receiver-operating characteristic curves for C-reactive protein and white cell count to predict appendicitis were calculated separately for each age group. RESULTS: The rates of negative explorations and perforations were highest at both extremes of age. In uncomplicated appendicitis, the diagnostic potential of white cell count was better than C-reactive protein in all age groups except infants. The C-reactive protein was elevated similarly throughout human life, but only in those with perforated appendicitis. The receiver-operating characteristic curves confirmed that the performance of white cell count was better than C-reactive protein in the correct diagnosis in every age group except infants and octogenarians. CONCLUSIONS: The leukocyte response declines in 0- to 5-year-old children with appendicitis, but the C-reactive protein response is well preserved in all other age groups.

Chicken IgA H chains. Implications concerning the evolution of H chain genes.

A cDNA clone (A1) encoding for a novel chicken Ig H chain isotype was isolated. In sequence comparison to mammalian H chains, A1 C region was most closely related to the alpha isotype. For example, identities of 35%, 32%, and 31% at the amino acid level to rabbit C alpha, C mu, and C gamma were observed, respectively. Distribution of the glucosamine acceptor sites (Asn-X-Ser/Thr) in A1 C region was typical of alpha H chains. Moreover, A1 C region probe hybridized to a 2.2-kb RNA species expressed in the epithelial lymphoid tissues. Thus, A1 was identified as the avian homologue for mammalian alpha H chains. Interestingly, the chicken C alpha was structurally consistent with four complete CH domains, whereas only three domains are present in the mammalian C alpha genes. In addition, interdomain sequence alignments suggested that the homologue for the chicken C alpha 2 domain is missing from the mammalian alpha H chains. Thus, the present data suggest evolution of the IgA isotype before the segregation of avian and mammalian species. Also, the first C alpha gene may have consisted of four CH domains, whereas reorganization of the C alpha 2 region led to the generation of hinge region in the mammalian alpha H chains.

D-D recombination diversifies the CDR 3 region of chicken immunoglobulin heavy chains.

The occurrence of D-D recombination during the embryonic differentiation of chicken B cells was studied. Ig heavy (H) chains were amplified by polymerase chain reaction from day-12 bursal cDNA, and 30 random V-D-J regions were analysed by DNA sequencing. No gene conversion events were observed in any of the V regions, indicating that diversification of the H chains by gene conversion is not yet activated at this stage of embryonic B-cell development. In contrast, the V-D-J joint regions were extremely heterogeneous. Most of the sequenced V-D-J joints were formed by direct joining of the single-germline V mu 1 gene, one of the multiple-germline D elements, and the single J gene. However, three V-D-J regions were clearly longer in size, and their D-region structure indicated recombination between two or three different germline D elements. Thus, the present data suggest that D-D recombination may have a role in diversification of the Ig H-chain repertoire.

The evolutionarily conserved avian Aiolos gene encodes alternative isoforms.

Aiolos is an Ikaros-related lymphoid regulatory protein involved in B cell development and function. To evaluate the role of Aiolos in avian B lymphopoiesis, we have cloned and characterized the first non-mammalian Aiolos ortholog in the avian. In sharp contrast to the avian Ikaros, expressed already at the multipotential stage and prior to the colonization of the lymphoid rudiments, Aiolos transcripts were not expressed in early ontogeny and were first detected in cells isolated from the embryonic bursa of Fabricius and thymus. In accordance with Ikaros, the avian Aiolos is also highly related to the mammalian homolog, thus suggesting an evolutionarily conserved function in lymphocyte development. Interestingly, in contrast to the mammalian Aiolos, at least one alternatively spliced form of avian Aiolos is detected in addition to the primary full-length transcript. Both of these alternate transcripts are expressed in bursa, thymus and peripheral lymphoid cells, and no major differences in the expression were detected during lymphocyte development. Furthermore, avian Aiolos is clearly expressed at various stages of B cell development, thus supporting recent evidence for the importance of Aiolos in B cell development and function.

B-cell differentiation in the chicken: expression of immunoglobulin genes in the bursal and peripheral lymphocytes.

We have studied the expression of immunoglobulin genes in the chicken B-cell precursors, and of a B-cell surface marker (Bu-1) on the bursal and peripheral B cells during normal ontogeny. Since there is no way of distinguishing the precursor cells from the more mature bursal lymphocytes on the basis of surface markers, we chose to study the total bursal lymphocyte population at ages when the numbers of the various precursor cells (bursal, early post-bursal, and post-bursal stem cells) in the bursa are estimated to be at their highest. Thereafter, comparisons with the more mature lymphocytes in the peripheral organs were made. As a result, levels of the lambda and mu transcripts and expression of Bu-1 antigen in the chicken B-cell precursors were found to be unchanged during the post-hatching period. In the light of these experiments, the later events of B-cell differentiation, i.e. the development from the bursal to post-bursal B lymphocytes, occurs without the lambda, mu, and Bu-1 gene loci involved. On the other hand, the higher level of lambda and mu expression in the splenic B lymphocytes indicates that the post-bursal stem cells mature into highly active plasma cells after seeding to the peripheral organs.

Rearrangement of immunoglobulin light chain genes in the chicken occurs prior to colonization of the embryonic bursa of Fabricius.

We have applied polymerase-chain-reaction-directed immunoglobulin gene analysis to study the embryonic differentiation of chicken B cells. Immunoglobulin light chain DNA segments in the rearranged configuration were amplified from cells of the intraembryonic mesenchyme as early as day 7 of incubation. We showed by sequencing that the rearranged variable region genes in these early B-cell progenitors were not different from the germ-line V lambda 1 gene (the single functional light chain variable region gene in chickens). In the bursal B lymphocytes, on the other hand, clear gene conversion events were first observed at day 15 of embryonic development. The present data indicate that rearrangement of light chain genes in the chicken occurs independently of the bursa of Fabricius and that diversification of the variable region begins only later, when the surface immunoglobulin-positive B cells are proliferating in the bursal follicles.

Novel serum inflammatory markers in acute appendicitis.

BACKGROUND: Cytokines and leukocyte adhesion molecules are activated and found in increased concentrations in bacterial infection. The purpose of this study was to investigate whether some of these new serum markers could be feasible as a single on-admission test to predict acute appendicitis (AA). METHODS: In an open prospective study the diagnostic potentials of two cytokine measurements (interleukin-6 and interleukin-8), soluble leukocyte adhesion molecule (CD44), C-reactive protein (CRP) and white blood cell (WBC) count were compared in 80 consecutive patients who had undergone surgery for suspected AA. The diagnostic performance of each parameter was tested by using receiver operating characteristic (ROC) curves. RESULTS: Phlegmonous AA was found in 34%, gangrenous AA in 40% and perforated AA in 5% of the patients. The proportion of negative explorations was 21%. Preoperative serum concentrations of IL-6 and CRP were elevated only in gangrenous and perforated AA. The concentrations of IL-8 and CD44 remained unchanged in AA. The sensitivity (84%), specificity (79%) and diagnostic accuracy (82%) of IL-6 were higher than the values for CRP, WBC, IL-8 and CD44 in predicting AA. CONCLUSION: ROC analysis confirmed that IL-6 showed the best trend in the diagnosis of AA. However, the diagnosis of AA was not greatly improved by any of the new serum markers as single on-admission tests.

B cell maturation in the chicken Harderian gland.

We have characterized maturation of B lymphocytes in the chicken Harderian gland. Expression of Ig genes was studied by using lambda L and mu H chain-specific DNA probes. In unstimulated chickens, the concentration of mu H chain and lambda L chain mRNA in the Harderian gland was observed to be greater than 8 times higher than in the bursa of Fabricius or spleen. By using in situ hybridization, the plasma cells expressing mu mRNA were located in central area of the gland packed around the tubules. Antibodies produced by the Harderian plasma cells were measured from the tears before and after antigenic stimulation. In unstimulated chickens high levels of total IgM, IgA, and IgG were observed. After ocular stimulation with tetanus toxoid, specific antitetanus IgG and IgA antibodies appeared in the tears but IgM antibodies were barely detectable. These results indicate that after antigenic stimulation the Harderian B cells rapidly mature through IgM secretion to the production of IgG or IgA. Southern blot analysis of the Harderian total genomic DNA showed strong rearrangement in the lambda L chain locus. In contrast, the band indicating major rearrangement in the mu H chain locus gave a very poor hybridization signal, indicating deletion of C mu genes in the Harderian gland DNA. As a conclusion, our present data indicate for the Harderian gland a role in terminal B cell differentiation and Ig class switch.

Bursectomy of chicken embryos at 60 hours of incubation leads to an oligoclonal B cell compartment and restricted Ig diversity.

Chickens that have been surgically bursectomized at 60 h of embryonic development usually generate Ig producing B cells; however, the bursectomized chickens are incapable of specific antibody responses, even after repeated immunization. In the present work, we analyzed the molecular basis of this immunodeficiency. In the bursectomized chickens, DNA sequencing revealed a repertoire of Ig L and H chains with a low number of different V-J and V-D-J joints, indicating an oligoclonal B cell compartment. In addition, the L and H chains belonging to each B cell clone had similar gene conversion events in the V region. In situ hybridization to Harderian gland tissue sections showed, that B cells of the bursectomized chickens were, however, capable of terminal plasma cell maturation. Thus, in chickens that were lacking the bursal microenvironment, 1) only a few B cell precursors differentiated into mature Ig-producing B cells, 2) low rate of gene conversion resulted in restricted Ig diversity. Regarding the chicken B cell differentiation, the present data support a model that the induction of B cell differentiation is a bursa-independent event, whereas the bursa of Fabricius has a crucial role in the amplification and diversification of the embryonic B cell repertoire.

Diagnosis of Helicobacter pylori infection by using pyloriset EIA-G and EIA-A for detection of serum immunoglobulin G (IgG) and IgA antibodies.

We evaluated the performance of new enzyme immunoassay (EIA) kits (Pyloriset; Orion Corporation, Orion Diagnostica, Espoo, Finland) for the detection of immunoglobulin G (IgG) and IgA antibodies to Helicobacter pylori in serum. Serum samples from 195 patients with upper abdominal complaints were collected. Biopsy specimens of the gastric mucosae were taken for histological analysis and bacterial culture. The sensitivity, specificity, and positive and negative predictive values, and efficacy of the Pyloriset EIA-G in detecting IgG antibodies to H. pylori were 92, 84, 88, 90, and 89%, respectively, when compared with those of the reference methods used. The corresponding data for detection of IgA antibodies were 80, 89, 89, 79, and 84%, respectively. The overall prevalence of defined H. pylori positivity was 54%. Moreover, the antibody tests showed a very good correlation with the biopsy findings. IgG antibodies were found in 93% of sera from patients with documented gastritis and H. pylori positivity, whereas only 4% of the sera from patients with documented gastritis and H. pylori-negative patients was positive. The results obtained for IgA antibodies were 81 and 6%, respectively. We conclude that the Pyloriset EIA-G, the test for IgG antibodies, is a good and reliable test for the detection of antibodies to H. pylori and as an indication of H. pylori infection. The determination of IgA antibodies may be used as a test that complements the IgG antibody assay.