Mathematical analysis of antigen selection in somatically mutated immunoglobulin genes associated with autoimmunity.

Affinity maturation is a process by which low-affinity antibodies are transformed into highly specific antibodies in germinal centres. This process occurs by hypermutation of immunoglobulin heavy chain variable (IgH V) region genes followed by selection for high-affinity variants. It has been proposed that statistical tests can identify affinity maturation and antigen selection by analysing the frequency of replacement and silent mutations in the complementarity determining regions (CDRs) that contact antigen and the framework regions (FRs) that encode structural integrity. In this study three different methods that have been proposed for detecting selection: the binomial test, the multinomial test and the focused binomial test, have been assessed for their reliability and ability to detect selection in human IgH V genes. We observe first that no statistical test is able to identify selection in the CDR antigen-binding sites, second that tests can reliably detect selection in the FR and third that antibodies from nasal biopsies from patients with Wegener's granulomatosis and pathogenic antibodies from systemic lupus erythematosus do not appear to be as stringently selected for structural integrity as other groups of functional sequences.

Related IgA1 and IgG producing cells in blood and diseased mucosa in ulcerative colitis.

BACKGROUND: Ulcerative colitis (UC) is a chronic inflammatory bowel disease in which the colonic mucosa is infiltrated with plasma cells producing IgG autoantibodies. It is not known whether this represents a local mucosal response which has switched to IgG or a peripheral response which may have been initiated by peripheral antigen which homed to the colonic mucosa. The clonal distribution of IgG secreting cells and isotype switched variants in UC is not known. AIMS: To investigate the clonal distribution of mucosal IgG in UC and to search for related IgG and IgA secreting cells in normal and diseased mucosa and blood in UC. To investigate characteristics which may discriminate between the mucosal and peripheral repertoire in the normal mucosa and in UC. PATIENTS: Blood and normal and diseased mucosa from two patients with UC were studied. METHODS: Immunoglobulin gene analysis and clone specific polymerase chain reaction were used to study the clonal distribution and characteristics of IgG and related IgA in the mucosa and blood of patients with UC. RESULTS: The IgG response in the mucosa of UC patients included widespread clones of cells that were present in both the diseased mucosa and blood but that were scarce in normal mucosa. Clonally related IgA class switch variants, all IgA1, were detected but also only in the diseased mucosa and blood. This suggests that these clones home preferentially to the diseased mucosa. We showed that J(H)1 usage was characteristic of the peripheral repertoire, and that examples of J(H)1 usage were observed in mucosal IgG in UC. CONCLUSIONS: Overall, these data are consistent with a model of UC in which a peripheral response is expressed and expanded in the colonic mucosa.

Sequence analysis of light chain genes from human intestinal plasma cells demonstrates that lambda genes are almost all in-frame and highly mutated and most kappa genes are highly mutated when in-frame and minimally mutated when out-of-frame.

Around 80 % of immunoglobulin (Ig)-producing cells in man are located in the gut, with a preponderance of IgA- and IgM-producing cells that express heavily mutated IgVH genes. Here we describe the characteristics of Ig light chain genes isolated from human ileal and colonic lamina propria plasma cells. We focused on the properties of the two most commonly used light chain families, Vkappa1 and Vlambda2. Out-of-frame lambda rearrangements were very rare, suggesting that these lambda light chains may have undergone sequential rearrangements until successful conformation was achieved. This has not been observed in the human peripheral B cell population. The in-frame lambda gene rearrangements were highly mutated, with a frequency of mutation that was indistinguishable from that observed in many groups of heavy chain variable regions used by intestinal plasma cells. The in-frame kappac chain rearrangements were also highly mutated, but contained a subgroup of genes (27.3 %) that showed over 98 % homology with the germ-line gene. The majority of unused kappa chain genes were unmutated. A strong tendency for preferential mutation of G over C nucleotides was observed. Detailed analysis of the sequences in which the biases were observed suggested that this was likely to be due to selection, rather than a characteristic of the mechanism introducing the mutations.

Effect of somatic hypermutation on potential N-glycosylation sites in human immunoglobulin heavy chain variable regions.

Immunoglobulins are known to be variably glycosylated. Although most carbohydrate is likely to be associated with the constant region, the variable region may also be glycosylated. Variable region glycosylation is known to be associated with antigen binding; in one model, the presence of carbohydrate on the external surface of CDR2 was shown to increase the affinity by up to ten-fold. In this study we have studied the effect of somatic hypermutation on potential sites of N-glycosylation in IgV(H) genes used by mucosal plasma cells secreting IgM, IgA and IgG in adults and children. Mucosal plasma cells secreting IgM, IgA and IgG are all heavily mutated from childhood. We have observed a tendency to lose the germline encoded N-glycosylation sites in all populations studied (a range of 43-67% of genes showing loss of the site). The tendency to lose the site was associated with a higher frequency of somatic hypermutation. We have also analysed the tendency to create potential N-glycosylation sites, and observed that this was greater in IgA and IgG than IgM and was again associated with the high frequency of somatic hypermutation. We observed no evidence of selection for either loss or gain of potential N-glycosylation sites. The changes in potential glycosylation status associated with a high frequency of somatic hypermutation is likely to increase the diversity of mucosal immunoglobulins, but is not as likely to affect the peripheral immune system where the frequency of somatic hypermutation is generally lower.

Characteristics of human IgA and IgM genes used by plasma cells in the salivary gland resemble those used in duodenum but not those used in the spleen.

Immunologically, the parotid salivary gland is an effector site that secretes large quantities of polyspecific Abs into the saliva, mainly of the IgA isotype. It is considered to be part of the common mucosal immune system but the inductive site for the Ab-producing cells of the salivary gland has not yet been clearly identified. The origin and diversity of cells of B lineage can be investigated by analyzing their Ig heavy chain genes (IgH). We have obtained sequences of IgM and IgA VH4-34 genes from plasma cells in human salivary gland, duodenal lamina propria, and splenic red pulp. Related sequences were found in different areas sampled within each tissue studied, indicating that the plasma cells carrying these genes are widespread with limited diversity. Examples of related IgH genes that are isotype switched were also seen in the salivary gland. The genes from plasma cells of the salivary gland were highly mutated, as were duodenal plasma cell sequences. The level of mutation was significantly higher than that seen in splenic plasma cell sequences. Analysis of CDR3 regions showed that the sequences from salivary gland had significantly smaller CDR3 regions than sequences from spleen, due to differences in number and type of DH regions used. Sequences from duodenum also had smaller CDR3 regions. Therefore, plasma cells from human duodenum and salivary gland showed characteristics that differed from those of human splenic plasma cells.

Characteristics of IgVH genes used by human intestinal plasma cells from childhood.

Plasma cells secreting immunoglobulin M (IgM) and IgA in human intestinal mucosa are the largest antibody-producing population in the human body. Despite this there have been relatively few studies of the characteristics and maturation of the genes which encode the mucosal immunoglobulins. We have previously demonstrated that intestinal plasma cells use highly mutated IgVH genes, likely to reflect germinal centre origin. Here we show that IgVH genes used by intestinal lamina propria plasma cells secreting IgM and IgA are highly mutated from childhood, with no change in the frequency of mutation through to adulthood, though IgVH genes used by IgM are significantly less mutated than those used by IgA. There was no difference between the IgA subclasses in either the frequency or distribution of mutations. The frequency of mutation in IgVH4-34 genes used by IgG was also studied in the adult biopsies, and was found to be of the same order as that observed in IgA and was significantly higher than that observed in IgM. We have identified IgM and IgA sequences which share identical CDR3 and distribution of mutations. Isotype switching may therefore occur after extensive mutation of IgM sequences, and IgM- and IgA-secreting plasma cells with the same specificity may occur within the same microenvironment. IgM should therefore be considered to be a component of secondary immune responses in the gut.

B-cell lymphoma associated with chronic lymphatic leukaemia: two cases with contrasting aggressive and indolent behaviour.

The term Richter's syndrome is used to describe the transformation of chronic lymphatic leukaemia (CLL) into a high-grade systemic lymphoma and is associated with a poor prognosis. We have undertaken detailed molecular studies in two patients with cutaneous B-cell lymphoma (CBCL) and CLL. Patient 1 exhibited a low-grade CBCL with different immunoglobulin gene rearrangements in blood and skin. By contrast, patient 2 showed identical gene rearrangements, confirmed by gene sequencing, and died within 4 months of presentation. The latter patient fulfilled the criteria for a diagnosis of cutaneous Richter's syndrome, whereas the former patient demonstrated the coincidence of CLL with a primary CBCL. Our results highlight the importance of gene rearrangement studies with sequencing for the accurate diagnosis of cutaneous Richter's syndrome.

Biased JH usage in plasma cell immunoglobulin gene sequences from colonic mucosa in ulcerative colitis but not in Crohn's disease.

BACKGROUND: Ulcerative colitis is an inflammatory disease of the colonic and rectal mucosa. Autoantibodies have been observed in ulcerative colitis which may have a role in the pathogenesis of the disease. Evidence also suggests that there is an hereditary predisposition towards the disease, although no individual genes have been identified. AIMS: This is a pilot study of immunoglobulin heavy chain genes (IgH) in ulcerative colitis to determine whether they have any particular genetic characteristics which may lead to a better understanding of the disease aetiology. SUBJECTS: Colonic or rectal tissue was obtained from five children with ulcerative colitis. Tissue was also obtained from five children with Crohn's disease and five children who did not have inflammatory bowel disease as controls. METHODS: B cells and IgD+ B cells were identified by immunohistochemistry on frozen sections. Areas of lamina propria containing plasma cells, and areas of IgD+ B cells were microdissected. The immunoglobulin genes were PCR amplified, cloned, and sequenced. Sequences were analysed for content of somatic mutations and composition of heavy chain. RESULTS: An increase in the use of JH6 and DXP'1, and a decrease in the use of JH4, gene segments in immunoglobulin genes from lamina propria plasma cells, and from virgin IgD+ B cells, was found in patients with ulcerative colitis. These biases were not present in the control groups. CONCLUSIONS: There is a fundamental difference in the immunoglobulin genes from patients with ulcerative colitis. Whether this is caused by a difference in content of immunoglobulin gene segments in the germline or a difference in the recombination mechanism is not known.

Analysis of immunoglobulin genes in splenic marginal zone lymphoma suggests ongoing mutation.

Splenic marginal zone lymphoma (SMZL) is a low-grade primary splenic B cell lymphoma, originally thought to be related to splenic marginal zone B cells. Later studies showed that SMZL sometimes may be accompanied by villous lymphocytes in the peripheral blood, a condition previously characterized as splenic lymphoma with villous lymphocytes (SLVL). The relationship between SMZL and splenic marginal zone B cells has recently been called into question. We report four further cases of SMZL, two of which were associated with villous lymphocytes in the peripheral blood. In addition to immunophenotypical analysis, we have studied the IgV(H) genes in each case, because the extent and patterns of their mutation can indicate the normal B cell counterpart of lymphomas. The IgV(H) genes in the four cases of SMZL studied are mutated, which is consistent with their origin from postfollicular marginal zone B cells. Evidence of ongoing mutation was also observed. This contrasts with a study showing that blood-borne tumor cells in SLVL show no sign of ongoing mutation. It is possible that the ongoing mutations in the cases studied here are acquired in a splenic microenvironment, such as that found in the follicle center.

Base-specific sequences that bias somatic hypermutation deduced by analysis of out-of-frame human IgVH genes.

Somatic hypermutation introduces mutations into IgV genes during affinity maturation of the B cell response. Mutations are introduced nonrandomly, and are generally targeted to the complementarity determining regions (CDRs). Subsequent selection against mutations that result in lower affinity or nonfunctional Ig increases the relative number of mutations in the CDRs. Investigation of somatic hypermutation is hampered by the effects of selection. We have avoided this by studying out-of-frame human IgVH4.21 and 251 genes, which, being unused alleles, are unselected. By comparison of the frequency of A, C, G, and T nucleotides at positions -3 to +3 around mutated or unmutated A, C, and G nucleotides, we have identified flanking sequences that most commonly surround mutated bases. Distinct trends in flanking sequences that were unique for each base were observed. Statistically significant trends that were common to both IgVH4.21 and 251 were used to deduce motifs that bias somatic hypermutation. The motifs deduced from this data, with targeted bases in regular type, are AANB, WDCH, and DGHD (where W = A/T, B = C/G/T, D = A/G/T, H = A/C/T, and N = any base). Mutations from C and G in two further groups of out-of-frame human IgVH genes, not used in the deduction of the motifs, occurred significantly within the motifs for C and G. The proposed target sequence for G is within the reverse complement of the target sequence for C, suggesting that the hypermutation mechanism may target only G or C. The mutation in the complementary base would appear on the other strand following replication.

Hypermutation, diversity and dissemination of human intestinal lamina propria plasma cells.

In this work we have microdissected lamina propria plasma cells and used polymerase chain reaction and sequencing to investigate immunoglobulin (Ig) gene rearrangements and mutations in human intestine. In addition, specific primers were designed for individual Ig gene rearrangements to analyze the distribution of related B cell and plasma cell clones at different sites along the bowel. Confirming our earlier work, intestinal IgVH genes were highly mutated in plasma cells from older individuals (> 30 years). IgVH genes were significantly less mutated in samples taken from patients aged 11-30 years, and there were fewer mutations again in samples from young children (< 11 years). In age-matched specimens the number of mutations was equivalent in the duodenum and colon. Using complementarity-determining region 3 primers to amplify specific Ig gene rearrangements, evidence was also found for the existence of related lamina propria plasma cells along the small bowel and colon, although these were quite scarce. In addition, analysis of the numbers of related clones in a random sampling from discrete areas of lamina propria indicates that the local population is diverse. These results suggest that the highly mutated IgVH genes in adult intestinal plasma cells are a consequence of chronic antigen exposure with age. Duodenal plasma cells are as highly mutated as colonic plasma cells, despite the fact that the upper bowel has no indigenous microbial flora (the stimulus for intestinal plasma cells). They also show that the plasma cell population is diverse and can be widely disseminated along the bowel.

The complete genome sequence of the gram-positive bacterium Bacillus subtilis.

Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.

The Bacillus subtilis genome from gerBC (311 degrees) to licR (334 degrees).

As part of the international project to sequence the Bacillus subtilis genome, the DNA region located between gerBC (311 degrees) and licR (334 degrees) was assigned to the institut Pasteur. In this paper, the cloning and sequencing of 176 kb of DNA and the analysis of the sequence of the entire 271 kb region (6.5% of the B. subtilis chromosome) is described; 273 putative coding sequences were identified. Although the complete genome sequences of seven other organisms (five bacteria, one archaeon and the yeast Saccharomyces cerevisiae) are available in public database, 65 genes from this region of the B. subtilis chromosome encode proteins without significant similarities to other known protein sequences. Among the 208 other genes, 115 have paralogues in the currently known B. subtilis DNA sequences and the products of 178 genes were found to display similarities to protein sequences from public databases for which a function is known. Classification of these genes shows a high proportion of them to be involved in the adaptation to various growth conditions (non-essential cell wall constituents, catabolic and bioenergetic pathways); a small number of the genes are essential or encode anabolic enzymes.

Anaerobic transcription activation in Bacillus subtilis: identification of distinct FNR-dependent and -independent regulatory mechanisms.

Bacillus subtilis is able to grow anaerobically using alternative electron acceptors, including nitrate or fumarate. We characterized an operon encoding the dissimilatory nitrate reductase subunits homologous to the Escherichia coli narGHJI operon and the narK gene encoding a protein with nitrite extrusion activity. Downstream from narK and co-transcribed with it a gene (fnr) encoding a protein homologous to E.coli FNR was found. Disruption of fnr abolished both nitrate and fumarate utilization as electron acceptors and anaerobic induction of narK. Four putative FNR binding sites were found in B.subtilis sequences. The consensus sequence, centred at position -41.5, is identical to the consensus for the DNA site for E.coli CAP. Bs-FNR contained a four cysteine residue cluster at its C-terminal end. This is in contrast to Ec-FNR, where a similar cluster is present at the N-terminal end. It is possible that oxygen modulates the activity of both activators by a similar mechanism involving iron. Unlike in E.coli, where fnr expression is weakly repressed by anaerobiosis, fnr gene expression in B.subtilis is strongly activated by anaerobiosis. We have identified in the narK-fnr intergenic region a promotor activated by anaerobiosis independently of FNR. Thus induction of genes involved in anaerobic respiration requires in B.subtilis at least two levels of regulation: activation of fnr transcription and activation of FNR to induce transcription of FNR-dependent promoters.