Molecular characterization of the region 7q22.1 in splenic marginal zone lymphomas

Splenic marginal zone lymphomas (SMZL) are an uncommon type of B-cell non-Hodgkin's lymphoma (NHL-B) in which no specific chromosomal translocations have been described. In contrast, the most frequent cytogenetic abnormality is the loss of the long arm of chromosome 7 (7q). Previous reports have located this loss in the 7q32 region. In order to better characterize the genomic imbalances in SMZL, molecular studies were carried out in 73 patients with SMZL. To gain insight into the mapping at 7q a tiling array was also used. The results confirmed the loss of 7q as the most frequent change. In addition, several abnormalities, including 4q22.1, 1q21.3-q22, 6q25.3, 20q13.33, 3q28, 2q23.3-q24.1 and 17p13, were also present. A loss of 7q22.1 at 99925039-101348479 bp was observed in half of the cases. The region of 7q22.1 has not previously been characterised in SMZL. Our results confirmed the presence of a new region of loss on chromosome 7 in these NHL.     

Phylogenetic evidence for frequent positive selection and recombination in the meningococcal surface antigen PorB

Previous estimates of rates of synonymous (d(S)) and nonsynonymous (d(N)) substitution among Neisseria meningitidis gene sequences suggested that the surface loops of the variable outer membrane protein PorB were under only weak selection pressure from the host immune response. These findings were consistent with studies indicating that PorB variants were not always protective in immunological and microbiological assays and questioned the suitability of this protein as a vaccine component. PorB, which is expressed at high levels on the surface of the meningococcus, has been implicated in mechanisms of pathogenesis and has also been used as a typing target in epidemiological investigations. In this work, using more precise estimates of selection pressures and recombination rates, we have shown that some residues in the surface loops of PorB are under very strong positive selection, as great as that observed in human immunodeficiency virus-1 surface glycoproteins, whereas amino acids within the loops and the membrane-spanning regions of the protein are under purifying selection, presumably because of structural constraints. Congruence tests showed that recombination occurred at a rate that was not sufficient to erase all phylogenetic similarity and did not greatly bias selection analysis. Homology models of PorB structure indicated that many strongly selected sites encoded residues that were predicted to be exposed to host immune responses, implying that this protein is under strong immune selection and requires further examination as a potential vaccine candidate. These data show that phylogenetic inference can be used to complement immunological and biochemical data in the choice of vaccine candidates.     

IL-7 is required for the development of the intrinsic function of marginal zone B cells and the marginal zone microenvironment

The characteristic microarchitecture of the marginal zone (MZ), formed by locally interacting MZ-specific B cells, macrophages, and endothelial cells, is critical for productive marginal zone B cell (MZB cell) Ab responses. Reportedly, IL-7-deficient mice, although severely lymphopenic, retain small numbers of CD21(high)CD23(low) B cells consistent with MZB cell phenotype, suggesting that IL-7 signaling is not exclusively required for MZB cell lymphopoiesis. In this study, we investigated the function of IL-7(-/-) MZB cells and the IL-7(-/-) microenvironment using a model of hamster heart xenograft rejection, which depends exclusively on MZB cell-mediated production of T cell-independent IgM xenoantibodies (IgMXAb). C57BL/6-IL-7(-/-) mice accepted xenografts indefinitely and failed to produce IgMXAb, even after transfer of additional IL-7(-/-) or wild-type C57BL/6 MZB cells. Transfer of wild-type but not IL-7(-/-) B cells enabled SCID mice to produce IgMXAb. When transferred to SCID mice, wild-type but not IL-7(-/-) B cells formed B cell follicles with clearly defined IgM(+), MOMA-1(+), and MAdCAM-1(+) MZ structures. Conversely, adoptively transferred GFP(+) C57BL/6 B cells homed to the MZ area in a SCID but not an IL-7(-/-) environment. Naive IL-7(-/-) mice showed absent or aberrant splenic B cell structures. We provide evidence that IL-7 is critical for the development of the intrinsic function of MZB cells in producing rapidly induced IgM against T cell-independent type II Ags, for their homing potential, and for the development of a functional MZ microanatomy capable of attracting and lodging MZB cells.     

Analysis of marginal zone B cell development in the mouse with limited B cell diversity: role of the antigen receptor signals in the recruitment of B cells to the marginal zone

The quasimonoclonal (QM) mouse provides an intelligible model to analyze the B cell selection as the competition between two major 4-hydroxy-3-nitrophenylacetyl-specific B cell populations whose BCR are comprised of the knockin V(H)17.2.25 (V(H)T)-encoded H chain and the lambda1 or lambda2 L chain. In this study, we show the QM system is useful to examine how BCR signals guide a subset of B cells to the marginal zone (MZ). Compared with the control C57BL/6 mice, the QM mice had approximately 2.7-fold increased number of B cells exhibiting the MZ B cell phenotype and a larger MZ area in the spleen. Interestingly, V(H)T/lambda2 B cells significantly predominated over V(H)T/lambda1 B cells in MZ-(V(H)T/lambda1:V(H)T/lambda2 approximately 3:7) and transitional 2-B cell subsets, while these two populations were comparable in immature, transitional 1, and mature counterparts. Thus, the biased use of lambda2 in the MZ B cells may be the result of selection in the periphery. The enlargement of MZ B cell compartment and the preferred recruitment of the V(H)T/lambda2 B cells were further augmented by doubling the V(H)T gene, but dampened by the dysfunction of Bruton's tyrosine kinase, suggesting a positive role of BCR signaling in this selection. Comparison of Ag specificity between V(H)T/lambda1 and V(H)T/lambda2 IgM mAbs revealed a polyreactive nature of the V(H)T/lambda2 BCR, including the reactivity with ssDNA. Taken together, it is suggested that polyreactivity (including self-reactivity) of BCR is crucial in driving B cells to differentiate into the MZ phenotype.     

Distortion of symmetrical introgression in a hybrid zone: evidence for locus-specific selection and uni-directional range expansion

The fate of species integrity upon natural hybridization depends on the interaction between selection and dispersal. The relative significance of these processes may be studied in the initial phase of contact before selection and gene flow reach equilibrium. Here we study a hybrid zone of two salamander species, Lyciasalamandra antalyana and Lyciasalamandra billae, at the initial phase of hybridization. We quantify the degree and mode of introgression using nuclear and mtDNA markers. The hybrid zone can be characterized as an abrupt transition zone, the central hybrid zone being only c. 400 m, but introgressed genes were traced up to 3 km. Introgression was traced in both sexes but gene flow may be slightly male-biased. Indirect evidence suggests that hybrid males are less viable than females. Introgression occurred at two levels: (1) locus-specific selection led to different allelic introgression patterns independent of species, while (2) asymmetrical species-level introgression occurred predominately from L. antalyana to L. billae due to range expansion of the former. This indicates that foreign genes can be incorporated into novel genomic environments, which in turn may contribute to the great diversity of morphological variants in Lyciasalamandra.     

Purifying selection and birth-and-death evolution in the histone H4 gene family

Histones are small basic proteins encoded by a multigene family and are responsible for the nucleosomal organization of chromatin in eukaryotes. Because of the high degree of protein sequence conservation, it is generally believed that histone genes are subject to concerted evolution. However, purifying selection can also generate a high degree of sequence homogeneity. In this study, we examined the long-term evolution of histone H4 genes to determine whether concerted evolution or purifying selection was the major factor for maintaining sequence homogeneity. We analyzed the proportion (p(S)) of synonymous nucleotide differences between the H4 genes from 59 species of fungi, plants, animals, and protists and found that p(S) is generally very high and often close to the saturation level (p(S) ranging from 0.3 to 0.6) even though protein sequences are virtually identical for all H4 genes. A small proportion of genes showed a low level of p(S) values, but this appeared to be caused by recent gene duplication. Our findings suggest that the members of this gene family evolve according to the birth-and-death model of evolution under strong purifying selection. Using histone-like genes in archaebacteria as outgroups, we also showed that H1, H2A, H2B, H3, and H4 histone genes in eukaryotes form separate clusters and that these classes of genes diverged nearly at the same time, before the eukaryotic kingdoms diverged.     

Macrophages of the splenic marginal zone are essential for trapping of blood-borne particulate antigen but dispensable for induction of specific T cell responses

Rapid removal of pathogens from the circulation by secondary lymphoid organs is prerequisite for successful control of infection. Blood-borne Ags are trapped mainly in the splenic marginal zone. To identify the cell populations responsible for Ag trapping in the marginal zone, mice were selectively depleted of marginal zone macrophages and marginal metallophilic macrophages. In the absence of these cells, trapping of microspheres and Listeria monocytogenes organisms was lost, and early control of infection was impaired. Depletion of marginal zone macrophages and marginal metallophilic macrophages, however, did not limit Ag presentation because Listeria-specific protective T cell immunity was induced. Therefore, marginal zone macrophages and marginal metallophilic macrophages are crucial for trapping of particulate Ag but dispensable for Ag presentation.     

Altered migration, recruitment, and somatic hypermutation in the early response of marginal zone B cells to T cell-dependent antigen

The early responses of follicular (Fo) and marginal zone (MZ) B cells to T cell-dependent Ag were compared using anti-hen egg lysozyme (HEL+) B cells capable of class switch recombination and somatic hypermutation (SHM). Purified CD21/(35int)CD23high Fo and CD21/35(high)CD23low MZ splenic B cells from SW(HEL) Ig-transgenic mice were transferred into wild-type recipients and challenged with HEL-sheep RBC. Responding HEL+ B cells from both populations switched efficiently to IgG1, generated syndecan-1+ Ab-secreting cells, and exhibited equivalent rates of proliferation. However, the expansion of HEL+ MZ B cells lagged significantly behind that of HEL+ Fo B cells due to less efficient homing to the outer periarteriolar lymphatic sheath and reduced recruitment into the proliferative response. Despite the equivalent rates of class switch recombination, the onset of SHM was delayed in the MZ subset, indicating that these two activation-induced cytidine deaminase-dependent events are uncoupled in the early response of MZ B cells. Migration of HEL+ B cells into germinal centers coincided with the onset of SHM, occurring more rapidly with Fo vs MZ responders. These results are consistent with the concept that Fo and MZ B cells have evolved to specialize in T cell-dependent and T-independent responses respectively.     

The marginal zone and marginal sinus in the spleen of the gerbil. A light and electron microscopy study

The spleen of the adult gerbil (M. unguiculatus) is characterized by the absence of venous sinuses and ellipsoid sheaths. The follicle (white pulp) is separated from the surrounding red pulp by a distinct marginal zone. The cell types in the marginal zone are common to both the follicle and red pulp. Separating the marginal zone from the follicle is a vascular channel of capillary dimension, the marginal sinus. A number of terminal segments of the arterial vessels within the follicle were observed to form a direct connection with the marginal sinus. Ultrastructurally, discontinuities were evident within the walls of the marginal sinus that would permit passage of both cellular and plasma components from the marginal sinus to either marginal zone or the follicle.     

B cells are crucial for both development and maintenance of the splenic marginal zone

The splenic marginal zone is a unique compartment that separates the lymphoid white pulp from the surrounding red pulp. Due to the orchestration of specialized macrophages and B cells flanking a marginal sinus, this compartment plays an important role in uptake of blood-borne Ags and it gives the spleen its specialized function in antibacterial immunity. In this study, we demonstrate that both development and maintenance of this marginal zone is highly dependent on the presence of B cells. Spleens from B cell-deficient mice were found to lack both metallophilic and marginal zone macrophages as well as mucosal addressin cellular adhesion molecule-1+ sinus lining cells. Using an inducible Cre/loxP-driven mouse model in which mature B cells could be partially depleted by removal of the B cell receptor subunit Igalpha, we could show that the integrity and function of an established marginal zone was also dependent on the presence of B cells. This was confirmed in a transgenic model in which all B cells were gradually depleted due to overexpression of the TNF family member CD70. The loss of all cellular subsets from the marginal zone in these CD70 transgenic mice was effectively prevented by crossing these mice on a CD27(-/-) or TCRalpha(-/-) background, because this prohibited the ongoing B cell depletion. Therefore, we conclude that B cells are not only important for the development, but also for maintenance, of the marginal zone. This direct correlation between circulating B cells and the function of the spleen implies an increased risk for B cell lymphopenic patients with bacterial infections.     

Defective microarchitecture of the spleen marginal zone and impaired response to a thymus-independent type 2 antigen in mice lacking scavenger receptors MARCO and SR-A

The macrophage scavenger receptor macrophage receptor with a collagenous structure (MARCO) is expressed in mice by the marginal zone macrophages of the spleen and by macrophages of the medullary cords of lymph nodes, as well as the peritoneal macrophages. MARCO is a relative of scavenger receptor A (SR-A), the more widely expressed prototypic member of the scavenger receptor family. In the present study, we found that genetic ablation of MARCO leads to changes in the organization of the splenic marginal zone, and causes a significant reduction in the size of the resident peritoneal macrophage population, possibly due to changes in adhesion and migration capacity. In mice lacking both MARCO and SR-A these effects are even more apparent. During ontogeny, the appearance and organization of the MARCO-expressing cells in the spleen precedes the appearance of other receptors on macrophages in the marginal zone, such as SIGNR1 and Siglec-1. In the absence of MARCO, a clear delay in the organization of the marginal zone was observed. Similar findings were seen when the reappearance of the various subsets from precursors was studied after depleting macrophages from the adult spleen by a liposome treatment. When challenged with a pneumococcal polysaccharide vaccine, a T-independent type 2 Ag for which an intact marginal zone is crucial, the knockout mice exhibited a clearly impaired response. These findings suggest that both MARCO and SR-A, in addition to being important scavenger receptors, could be involved in the positioning and differentiation of macrophages, possibly through interaction with endogenous ligands.     

Biases in sperm use in the mallard: no evidence for selection by females based on sperm genotype

If we are to understand fully the factors influencing fertilization success, it is essential to untangle male and female effects on sperm use. In many species, differences in fertilizing ability have been found between males or male genotypes, but the impact of female effects is less clear and may vary between taxa. Here, we examine sperm use in the mallard (Anas platyrhynchos), a species of bird in which forced copulation forms a major component of the mating system, to investigate whether there is any evidence for post-insemination female choice or rejection of particular sperm genotypes. Current models of sperm use in birds suggest observed patterns of paternity are a result of passive sperm loss from the reproductive tract and the relative timing of inseminations. Although this type of model successfully predicted average values of last male precedence observed in this species, there was considerable variation between females in their pattern of sperm use, with a tendency for females to use sperm of a single genotype. However, females did not consistently prefer one genotype over another in repeated inseminations with identical sperm mixtures, suggesting that post-insemination female preference based on sperm genotype did not account for this variation.     

DNA sequence analysis of the C4 antigen WH: evidence for two mechanisms of expression

 Amino acid and protein analyses have allowed the construction of a model for the C4-based Rodgers and Chido blood group antigens. The single low-frequency allele (WH) in this blood group system, however, has not been characterized at the molecular level. Two WH⁺ donors were studied by C4 agarose gel electrophoreses, immunoblot studies using monoclonal anti-Rg: 1 or anti-Ch: 1, serological phenotyping, polymerase chain reaction-restriction fragment length polymorphism of their C4 genes, and DNA sequencing of the WH allele. The first donor had the C4A1, A3 phenotype; the C4A1 carried Ch: 1, 3, 6 (thus exhibiting reversed antigenicity) and the C4A3 carried the WH antigen. The amino acid sequence of the WH allele was PCPVLD at positions 1101 – 1106, S at position 1157, and VDLL at positions 1188 – 1191. A second donor typed as C4A2, A4, B1 and was also WH⁺. Immunoblot analysis showed that a C4B1 protein expressed Rg: 1. Sequence analysis of the C4B genes showed the amino acids LSPVIH at positions 1101 – 1106, S at position 1157, and ADLR at positions 1188 – 1191. Thus, the WH antigen is a conformational epitope that can arise through different mechanisms on either a C4A or C4B gene. Received: 22 November 1995 / Revised: 19 February 1996     

Expression of the CD31 antigen in normal B-cells and non Hodgkin's lymphomas

The role cell adhesion molecules play in the biological and clinical behaviour of non Hodgkin's lymphomas (NHL) has been reported in several studies. This study reports the findings on B-cells taken from various healthy control tissues and compared them to B-cells from 83 malignant B-lymphomas, that had been classified according to the WHO classification. Flow cytometry was used to investigate the surface expression of CD31, an adhesion molecule involved in B-cell development and vascular adhesion mechanisms. Quantification of the fluorescence signals showed specific patterns of CD31 expression on normal B-cell subpopulations and different NHL groups. Our results demonstrate that CD31 expression is modulated during the differentiation process in normal B-cells, high in pre-B-I cells, low in pre-B-II precursors, intermediate in the mature B-cell subpopulations or, depending on the functional state absent in activated follicular centre cells, present in pre- and post- germinal centre cells. When the CD31 expression is evaluated as fluorescence intensity in NHL, it reveals a heterogeneous pattern related to histogenetic derivation (high in small lymphocytic lymphoma, low in follicular lymphoma, intermediate in marginal zone and large cell lymphomas). These observations suggest that CD31 might well play a critical role in the ontogeny and physiology of B-lymphocytes. Therefore, on the basis of these observations we propose the CD31 molecule as an interesting additional useful parameter to be used for the differential diagnosis of NHL and hypothese that it has a pathophysiologic role in NHL evolution.     

Habitat selection by dispersing yellow-headed blackbirds: evidence of prospecting and the use of public information

In migratory birds individuals may prospect for potential breeding sites months before they attempt to breed and should use the cues most predictive of future reproductive success when selecting a breeding site. However, what cues individuals use when prospecting and which cues are used in selecting a breeding site are unknown for most species. I investigated whether yellow-headed blackbirds (Xanthocephalus xanthocephalus) prospect for future breeding sites and whether they select breeding habitats based on food availability, male or female density, or the average number of young produced per female in the previous year. Although it is often assumed that migratory birds prospect for potential breeding sites at the end of the breeding season, I investigated this by recording all visits to sites early and late in the breeding season. I found that males and females who visited sites other than the site at which they bred were more likely to disperse than individuals only observed at the site where they bred, and that males and females were more likely to prospect late in the breeding season. Both food availability and density in year ^x were not predictive of the number of young per female in year ^x+1; however, the number of young produced per female at a site in year ^x was predictive of the number of young per female in year ^x+1. As expected, dispersers used the most informative cue, the number of young per female and moved to sites with a relatively high number of young per female. This study suggests that individuals prospect for potential breeding sites late in the breeding season when they can use information gathered from the reproductive success of other individuals (i.e., public information) to select a breeding site.     

No evidence for strong recent positive selection favoring the 7 repeat allele of VNTR in the DRD4 gene

The human dopamine receptor D4 (DRD4) gene contains a 48-bp variable number of tandem repeat (VNTR) in exon 3, encoding the third intracellular loop of this dopamine receptor. The DRD4 7R allele, which seems to have a single origin, is commonly observed in various human populations and the nucleotide diversity of the DRD4 7R haplotype at the DRD4 locus is reduced compared to the most common DRD4 4R haplotype. Based on these observations, previous studies have hypothesized that positive selection has acted on the DRD4 7R allele. However, the degrees of linkage disequilibrium (LD) of the DRD4 7R allele with single nucleotide polymorphisms (SNPs) outside the DRD4 locus have not been evaluated. In this study, to re-examine the possibility of recent positive selection favoring the DRD4 7R allele, we genotyped HapMap subjects for DRD4 VNTR, and conducted several neutrality tests including long range haplotype test and iHS test based on the extended haplotype homozygosity. Our results indicated that LD of the DRD4 7R allele was not extended compared to SNP alleles with the similar frequency. Thus, we conclude that the DRD4 7R allele has not been subjected to strong recent positive selection.     

The Rac2 guanosine triphosphatase regulates B lymphocyte antigen receptor responses and chemotaxis and is required for establishment of B-1a and marginal zone B lymphocytes

We have defined roles for the hemopoietic-specific Rho guanosine triphosphatase, Rac2, in B lymphocyte development and function through examination of rac2(-/-) mice. Rac2-deficient mice displayed peripheral blood B lymphocytosis and marked reductions in peritoneal cavity B-1a lymphocytes, marginal zone B lymphocytes, and IgM-secreting plasma cells as well as reduced concentrations of serum IgM and IgA. The rac2(-/-) B lymphocytes exhibited reduced calcium flux following coligation of B cell AgR and CD19 and reduced chemotaxis in chemokine gradients. T cell-independent responses to DNP-dextran were of reduced magnitude, but normal kinetics, in rac2(-/-) mice, while T-dependent responses to nitrophenyl-keyhole limpet hemocyanin were subtly abnormal. Rac2 is therefore an essential element in regulating B lymphocyte functions and maintaining B lymphocyte populations in vivo.     

Diversity in and evidence for selection on the mitochondrial genome of Phytophthora infestans

Two extant nomenclature systems were reconciled to relate six mitochondrial DNA (mtDNA) haplotypes of Phytophthora infestans, the oomycete pathogen causing late blight disease on potato and tomato. Carter's haplotypes I-a and I-b were included in Goodwin's haplotype A, while Carter's haplotypes II-a and II-b were included in Goodwin's haplotype B. In addition, haplotypes E and F were included in Carter's haplotype I-b. The mutational differences separating the various haplotypes were determined, and we propose that either haplotype I-b(A) or haplotype I-a(A) is the putative ancestral mtDNA of P. infestans, because either can center all the other haplotypes in a logical stepwise network of mutational changes. The occurrence of the six haplotypes in 548 isolates worldwide was determined. Haplotypes I-a and II-a were associated with diverse genotypes worldwide. As previously suggested, haplotype I-b was found only in the US-1 clonal lineage and its variants (n = 99 isolates from 16 countries on 5 continents), and haplotype II-b was limited to the US-6 clonal lineage and its derivatives (n = 36). In a confirmation of a previous suggestion, the randomly mating population in the Toluca Valley of central Mexico (n = 78) was monomorphic for mtDNA haplotype I-a(A). We hypothesize that selection there may be driving the dominance of that single mtDNA haplotype.