The treatment response of barrier membrane with amoxicillin-loaded nanofibers in experimental periodontitis.

BACKGROUND: Infection control is a major determinant of guided tissue regeneration (GTR). This study aims to develop an antibiotic-loaded membrane to assist periodontal repair. METHODS: Poly(D,L-lactic acid) (PDLLA) nanofibers encapsulating amoxicillin (PDLLA-AMX) were fabricated using the electrospinning technique, and their structures, drug encapsulation efficiency, and release characteristics were assessed. The viability and behaviors of periodontal ligament (PDL) cells on nanofibers, and antibacterial capabilities of nanofibers were evaluated in vitro. Early therapeutic efficiency of the antibiotic-loaded membranes was investigated in rats with ligature-induced experimental periodontitis, and the outcomes were evaluated by gene expression, microcomputed tomography imaging, and histology within 7 days of membrane placement. RESULTS: AMX was successfully encapsulated in the PDLLA nanofibers and released in a sustained manner. After initial attachment was achieved, cells stretched out along with the directions of nanofibers. The viability and expression of migration-associated gene of PDL cells were significantly improved, and the growth of Streptococcus sanguinis and Porphyromonas gingivalis was significantly reduced in the PDLLA-AMX group compared with the controls. On PDLLA-AMX-treated sites, wound dehiscence and sulcular inflammation were reduced. Collagen fiber matrix deposition was accelerated with upregulated type I collagen and interleukin-1ß, and downregulated matrix metalloproteinase-8, whereas periodontal bone level and the expressions of vascular endothelial growth factor and core-binding factor subunit alpha-1 were equivalent to conventional membrane treatment. CONCLUSIONS: PDLLA-AMX nanofibers inhibited bacterial growth and promoted the viability and mobility of PDL cells after initial cell attachment. Membranes with PDLLA-AMX nanofibers reduced inflammation and accelerated periodontal repair at an early stage, providing good prospects for the further development of GTR membranes.

Spatiotemporal segregation of human marginal zone and memory B cell populations in lymphoid tissue.

Human memory B cells and marginal zone (MZ) B cells share common features such as the expression of CD27 and somatic mutations in their IGHV and BCL6 genes, but the relationship between them is controversial. Here, we show phenotypic progression within lymphoid tissues as MZ B cells emerge from the mature naïve B cell pool via a precursor CD27-CD45RBMEM55+ population distant from memory cells. By imaging mass cytometry, we find that MZ B cells and memory B cells occupy different microanatomical niches in organised gut lymphoid tissues. Both populations disseminate widely between distant lymphoid tissues and blood, and both diversify their IGHV repertoire in gut germinal centres (GC), but nevertheless remain largely clonally separate. MZ B cells are therefore not developmentally contiguous with or analogous to classical memory B cells despite their shared ability to transit through GC, where somatic mutations are acquired.

IgE repertoire and immunological memory: compartmental regulation and antibody function.

It is now generally recognized that bone marrow is the survival niche for antigen-specific plasma cells with long-term immunological memory. These cells release antibodies into the circulation, needed to prime effector cells in the secondary immune response. These antibodies participate in the surveillance for antigen and afford immune defence against pathogens and toxins previously encountered in the primary immune response. IgE antibodies function together with their effector cells, mast cells, to exert 'immediate hypersensitivity' in mucosal tissues at the front line of immune defence. The constant supply of IgE antibodies from bone marrow plasma cells allows the rapid 'recall response' by mast cells upon re-exposure to antigen even after periods of antigen absence. The speed and sensitivity of the IgE recall response and potency of the effector cell functions are advantageous in the early detection and elimination of pathogens and toxins at the sites of attack. Local antigen provocation also stimulates de novo synthesis of IgE or its precursors of other isotypes that undergo IgE switching in the mucosa. This process, however, introduces a delay before mast cells can be sensitized and resume activity; this is terminated shortly after the antigen is eliminated. Recent results from adaptive immune receptor repertoire sequencing of immunoglobulin genes suggest that the mucosal IgE+ plasmablasts, which have undergone affinity maturation in the course of their evolution in vivo, are a source of long-lived IgE+ plasma cells in the bone marrow that are already fully functional.

Antibodies and superantibodies in patients with chronic rhinosinusitis with nasal polyps.

BACKGROUND: Chronic rhinosinusitis with nasal polyps is associated with local immunoglobulin hyperproduction and the presence of IgE antibodies against Staphylococcus aureus enterotoxins (SAEs). Aspirin-exacerbated respiratory disease is a severe form of chronic rhinosinusitis with nasal polyps in which nearly all patients express anti-SAEs. OBJECTIVES: We aimed to understand antibodies reactive to SAEs and determine whether they recognize SAEs through their complementarity-determining regions (CDRs) or framework regions. METHODS: Labeled staphylococcal enterotoxin (SE) A, SED, and SEE were used to isolate single SAE-specific B cells from the nasal polyps of 3 patients with aspirin-exacerbated respiratory disease by using fluorescence-activated cell sorting. Recombinant antibodies with "matched" heavy and light chains were cloned as IgG1, and those of high affinity for specific SAEs, assayed by means of ELISA and surface plasmon resonance, were recloned as IgE and antigen-binding fragments. IgE activities were tested in basophil degranulation assays. RESULTS: Thirty-seven SAE-specific, IgG- or IgA-expressing B cells were isolated and yielded 6 anti-SAE clones, 2 each for SEA, SED, and SEE. Competition binding assays revealed that the anti-SEE antibodies recognize nonoverlapping epitopes in SEE. Unexpectedly, each anti-SEE mediated SEE-induced basophil degranulation, and IgG1 or antigen-binding fragments of each anti-SEE enhanced degranulation by the other anti-SEE. CONCLUSIONS: SEEs can activate basophils by simultaneously binding as antigens in the conventional manner to CDRs and as superantigens to framework regions of anti-SEE IgE in anti-SEE IgE-FcεRI complexes. Anti-SEE IgG1s can enhance the activity of anti-SEE IgEs as conventional antibodies through CDRs or simultaneously as conventional antibodies and as "superantibodies" through CDRs and framework regions to SEEs in SEE-anti-SEE IgE-FcεRI complexes.

Transitional B Cells in Early Human B Cell Development - Time to Revisit the Paradigm?

The B cell repertoire is generated in the adult bone marrow by an ordered series of gene rearrangement processes that result in massive diversity of immunoglobulin (Ig) genes and consequently an equally large number of potential specificities for antigen. As the process is essentially random, the cells exhibiting excess reactivity with self-antigens are generated and need to be removed from the repertoire before the cells are fully mature. Some of the cells are deleted, and some will undergo receptor editing to see if changing the light chain can rescue an autoreactive antibody. As a consequence, the binding properties of the B cell receptor are changed as development progresses through pre-B ≫ immature ≫ transitional ≫ naïve phenotypes. Using long-read, high-throughput, sequencing we have produced a unique set of sequences from these four cell types in human bone marrow and matched peripheral blood, and our results describe the effects of tolerance selection on the B cell repertoire at the Ig gene level. Most strong effects of selection are seen within the heavy chain repertoire and can be seen both in gene usage and in CDRH3 characteristics. Age-related changes are small, and only the size of the CDRH3 shows constant and significant change in these data. The paucity of significant changes in either kappa or lambda light chain repertoires implies that either the heavy chain has more influence over autoreactivity than light chain and/or that switching between kappa and lambda light chains, as opposed to switching within the light chain loci, may effect a more successful autoreactive rescue by receptor editing. Our results show that the transitional cell population contains cells other than those that are part of the pre-B ≫ immature ≫ transitional ≫ naïve development pathway, since the population often shows a repertoire that is outside the trajectory of gene loss/gain between pre-B and naïve stages.

Significant Differences in Physicochemical Properties of Human Immunoglobulin Kappa and Lambda CDR3 Regions.

Antibody variable regions are composed of a heavy and a light chain, and in humans, there are two light chain isotypes: kappa and lambda. Despite their importance in receptor editing, the light chain is often overlooked in the antibody literature, with the focus being on the heavy chain complementarity-determining region (CDR)-H3 region. In this paper, we set out to investigate the physicochemical and structural differences between human kappa and lambda light chain CDR regions. We constructed a dataset containing over 29,000 light chain variable region sequences from IgM-transcribing, newly formed B cells isolated from human bone marrow and peripheral blood. We also used a published human naïve dataset to investigate the CDR-H3 properties of heavy chains paired with kappa and lambda light chains and probed the Protein Data Bank to investigate the structural differences between kappa and lambda antibody CDR regions. We found that kappa and lambda light chains have very different CDR physicochemical and structural properties, whereas the heavy chains with which they are paired do not differ significantly. We also observed that the mean CDR3 N nucleotide addition in the kappa, lambda, and heavy chain gene rearrangements are correlated within donors but can differ between donors. This indicates that terminal deoxynucleotidyl transferase may work with differing efficiencies between different people but the same efficiency in the different classes of immunoglobulin chain within one person. We have observed large differences in the physicochemical and structural properties of kappa and lambda light chain CDR regions. This may reflect different roles in the humoral immune response.

Assessment of B Cell Repertoire in Humans.

The B cell receptor (BCR) repertoire is highly diverse. Repertoire diversity is achieved centrally by somatic recombination of immunoglobulin (Ig) genes and peripherally by somatic hypermutation and Ig heavy chain class-switching. Throughout these processes, there is selection for functional gene rearrangements, selection against gene combinations resulting in self-reactive BCRs, and selection for BCRs with high affinity for exogenous antigens after challenge. Hence, investigation of BCR repertoires from different groups of B cells can provide information on stages of B cell development and shed light on the etiology of B cell pathologies. In most instances, the third complementarity determining region of the Ig heavy chain (CDR-H3) contributes the majority of amino acids to the antibody/antigen binding interface. Although CDR-H3 spectratype analysis provides information on the overall diversity of BCR repertoires, this fairly simple technique analyzes the relative quantities of CDR-H3 regions of each size, within a range of approximately 10-80 bp, without sequence detail and thus is limited in scope. High-throughput sequencing (HTS) techniques on the Roche 454 GS FLX Titanium system, however, can generate a wide coverage of Ig sequences to provide more qualitative data such as V, D, and J usage as well as detailed CDR3 sequence information. Here we present protocols in detail for CDR-H3 spectratype analysis and HTS of human BCR repertoires.

Age-related aspects of human IgM(+) B cell heterogeneity.

The CD27(+) IgD(+) B cell population, known as IgM memory, reduces with age. It is thought that this population is responsible for pneumococcal polysaccharide T-independent responses, and that the age-related reduction might be partially responsible for the increased susceptibility of older people to bacterial pathogens. There are other IgM(+) B cell populations that do not express IgD. We compared the different IgM populations using high-throughput sequencing of the immunoglobulin (Ig) gene repertoire and multidimensional cell phenotyping and found that the different populations of IgM cells, defined by CD27 and IgD expression, have repertoire differences. Some of these differences are likely indicative of different selection pressures in an immune response, although the older individuals were found to have a changed repertoire in naive B cells, which may contribute to some of the changes seen in memory cells. In addition, even within the CD27(+) IgD(+) IgM memory population there are multiple cell types. We show that the level of IgM expression varies substantially and hypothesize that this distinguishes between T-dependent and T-independent types of IgM memory cells. Significant age-related changes in the relative proportions of these populations may exacerbate the reduction in T-independent responders in old age.

Ageing of the B-cell repertoire.

Older people are more susceptible to infection, less responsive to vaccination and have a more inflammatory immune environment. Using spectratype analysis, we have previously shown that the B-cell repertoire of older people shows evidence of inappropriate clonal expansions in the absence of challenge, and that this loss of B-cell diversity correlates with poor health. Studies on response to vaccination, using both spectratyping and high-throughput sequencing of the repertoire, indicate that older responses to challenge are lacking in magnitude and/or delayed significantly. Also that some of the biologically significant differences may be in different classes of antibody. We have also previously shown that normal young B-cell repertoires can vary between different phenotypic subsets of B cells. In this paper, we present an analysis of immunoglobulin repertoire in different subclasses of antibody in five different populations of B cell, and show how the repertoire in these different groups changes with age. Although some age-related repertoire differences occur in naive cells, before exogenous antigen exposure, we see indications that there is a general dysregulation of the selective forces that shape memory B-cell populations in older people.

Immunoglobulin kappa variable region gene selection during early human B cell development in health and systemic lupus erythematosus.

The unique specificity of the B cell receptor is generated by an ordered sequence of gene rearrangement events. Once IGH genes have rearranged, rearrangement at the IGK locus is initiated followed by the IGL locus if functional IGK rearrangement is not achieved. Receptor specificity can subsequently be altered by secondary light chain editing based on the features of the heavy and light chain combination. The final profile of expressed genes is not random and biases in this profile are associated with several autoimmune diseases. However, how and when biases are created is not known. To increase our understanding of the processes of selection and editing of IGK rearrangements, we compared four groups of rearrangements of IGK acquired by next generation sequencing. First, expressed rearrangements of IGK from cDNA of IGK expressing B cells. Second, productive rearrangements of IGK from DNA of the same kappa expressing B cells. Third, non-productive rearrangements of IGK from DNA of IGK and IGL expressing B cells, and fourth productively rearranged IGK from DNA of IGL expressing B cells. The latter group would have been rejected during B cell development in favour of rearrangement at the IGL locus and are therefore selected against. We saw evidence that rearranged IGK segments can be selected at a checkpoint where the decision to rearrange the IGL locus is made. In addition, our data suggest that mechanisms regulating the expression or not of IGK rearrangements may also contribute to repertoire development and also that this latter component of the selection process is defective in SLE.

Influence of seasonal exposure to grass pollen on local and peripheral blood IgE repertoires in patients with allergic rhinitis.

BACKGROUND: Previous studies of immunoglobulin gene sequences in patients with allergic diseases using low-throughput Sanger sequencing have limited the analytic depth for characterization of IgE repertoires. OBJECTIVES: We used a high-throughput, next-generation sequencing approach to characterize immunoglobulin heavy-chain gene (IGH) repertoires in patients with seasonal allergic rhinitis (AR) with the aim of better understanding the underlying disease mechanisms. METHODS: IGH sequences in matched peripheral blood and nasal biopsy specimens from nonallergic healthy control subjects (n = 3) and patients with grass pollen-related AR taken in season (n = 3) or out of season (n = 4) were amplified and pyrosequenced on the 454 GS FLX+ System. RESULTS: A total of 97,610 IGH (including 8,135 IgE) sequences were analyzed. Use of immunoglobulin heavy-chain variable region gene families 1 (IGHV1) and 5 (IGHV5) was higher in IgE clonotypic repertoires compared with other antibody classes independent of atopic status. IgE repertoires measured inside the grass pollen season were more diverse and more mutated (particularly in the biopsy specimens) and had more evidence of antigen-driven selection compared with those taken outside of the pollen season or from healthy control subjects. Clonal relatedness was observed for IgE between the blood and nasal biopsy specimens. Furthermore in patients with AR, but not healthy control subjects, we found clonal relatedness between IgE and IgG classes. CONCLUSION: This is the first report that exploits next-generation sequencing to determine local and peripheral blood IGH repertoires in patients with respiratory allergic disease. We demonstrate that natural pollen exposure was associated with changes in IgE repertoires that were suggestive of ongoing germinal center reactions. Furthermore, these changes were more often apparent in nasal biopsy specimens compared with peripheral blood and in patients with AR compared with healthy control subjects.

Tunable microwave generation of a monolithic dual-wavelength distributed feedback laser.

The dynamic behavior of a monolithic dual-wavelength distributed feedback laser was fully investigated and mapped. The combination of different driving currents for master and slave lasers can generate a wide range of different operational modes, from single mode, period 1 to chaos. Both the optical and microwave spectrum were recorded and analyzed. The detected single mode signal can continuously cover from 15GHz to 50GHz, limited by photodetector bandwidth. The measured optical four-wave-mixing pattern indicates that a 70GHz signal can be generated by this device. By applying rate equation analysis, the important laser parameters can be extracted from the spectrum. The extracted relaxation resonant frequency is found to be 8.96GHz. With the full operational map at hand, the suitable current combination can be applied to the device for proper applications.

Age-Related Changes in Human Peripheral Blood IGH Repertoire Following Vaccination.

Immune protection against pulmonary infections, such as seasonal flu and invasive pneumonia, is severely attenuated with age, and vaccination regimes for the elderly people often fail to elicit effective immune response. We have previously shown that influenza and pneumococcal vaccine responses in the older population are significantly impaired in terms of serum antibody production, and have shown repertoire differences by CDR-H3 spectratype analysis. Here we report a detailed analysis of the B cell repertoire in response to vaccine, including a breakdown of sequences by class and subclass. Clustering analysis of high-throughput sequencing data enables us to visualize the response in terms of expansions of clonotypes, changes in CDR-H3 characteristics, and somatic hypermutation as well as identifying the commonly used IGH genes. We have highlighted a number of significant age-related changes in the B cell repertoire. Interestingly, in light of the fact that IgG is the most prevalent serum antibody and the most widely used as a correlate of protection, the most striking age-related differences are in the IgA response, with defects also seen in the IgM repertoire. In addition there is a skewing toward IgG2 in the IgG sequences of the older samples at all time points. This analysis illustrates the importance of antibody classes other than IgG and has highlighted a number of areas for future consideration in vaccine studies of the elderly.

B cell immunosenescence: different features of naive and memory B cells in elderly.

Elderly people show a reduced protection against new infections and a decreased response to vaccines as a consequence of impairment of both cellular and humoral immunity. In this paper we have studied memory/naïve B cells in the elderly, evaluating surface immunoglobulin expression, production of the pro- and anti-inflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-10, and presence of somatic hypermutation, focusing on the IgG(+)IgD(-)CD27(-) double negative (DN) B cells that are expanded in the elderly. Our results show that naïve B cells from young donors need a sufficiently strong stimulus to be activated "in vitro", while naïve B cells from old subjects are able to produce IL-10 and TNF-α when stimulated "physiologically" (α-CD40/IL-4), suggesting that these cells might play a role in the control of the immuno-inflammatory environment in the elderly. In addition, in the elderly there is an accumulation of DN B cells with a reduced rate of somatic hypermutation. Thus, DN B lymphocytes may be exhausted cells that are expanded and accumulate as a by-product of persistent stimulation or impaired germinal center formation.

Vaccination-induced changes in human B-cell repertoire and pneumococcal IgM and IgA antibody at different ages.

It is well known that older people are more susceptible to morbidity and mortality from infectious diseases, particularly from pulmonary diseases such as pneumococcal pneumonia where vaccines do not provide efficient protection as in younger populations. We have previously shown that the B-cell repertoire in the old is reduced and hypothesise that this may contribute to the impaired humoral responses of the elderly. Here, we investigated the repertoire and antibody responses to winter vaccination in two age groups, aged 18-49 and 65-89. We found that the serum IgM and IgA pneumococcal responses were significantly impaired in the older group, with no difference in IgG levels. IGHM spectratype analysis seems to be the most promising in terms of its predictive ability for vaccine responses. Spectratypes showed a clear change in the repertoire at day 7 after vaccination, with a return to the baseline levels at day 28. The changes at day 7 reflected expansion of IGH sequences that have smaller, more hydrophilic, CDR3 regions, and these changes were attenuated in the older group. The older group was more likely to have spectratypes indicative of a reduced diversity at day 0 and day 28. On average, the baseline repertoire in the older group was comprised of larger CDR3 regions than in the younger group. In conclusion, IgA and IgM responses are significantly impaired in the elderly pneumococcal response and are likely key mediators of protection. Hydrophilicity and/or small size of the IGH CDR3 appear to be important in these responses.

B cells and immunosenescence: a focus on IgG+IgD-CD27- (DN) B cells in aged humans.

Immunosenescence contributes to the decreased ability of the elderly to control infectious diseases, which is also reflected in their generally poor response to new antigens and vaccination. It is known that the T cell branch of the immune system is impaired in the elderly mainly due to expansion of memory/effector cells that renders the immune system less able to respond to new antigens. B lymphocytes are also impaired in the elderly in terms of their response to new antigens. In this paper we review recent work on B cell immunosenescence focusing our attention on memory B cells and a subset of memory B cells (namely IgG(+)IgD(-)CD27(-)) that we have demonstrated is increased in healthy elderly.

The relationship between CD27 negative and positive B cell populations in human peripheral blood.

CD27 expression has been used to distinguish between memory and naive B cells in humans. However, low levels of mutated and isotype-switched CD27-IgD- cells are seen in healthy adults, and these are increased in some autoimmune diseases and in the elderly. Thus CD27 is not a universal marker of memory B cells in humans. Various hypotheses have been put forward as to the function of the CD27- memory population. Since we have previously found high-throughput IGHV repertoire analysis useful to distinguish "innate-like" memory B cells (CD27+IgD+), we have employed similar analyses to elucidate the relationship between CD27- and CD27+ memory B cells. IgM+IgD- memory cells in both the CD27+ and CD27- compartments share the unique characteristics of the "innate-like" IgM+IgD+CD27+ cells. The switched CD27+ and CD27- memory cells share a similar IGHV repertoire, having more in common with each other than with "innate-like" memory cells, although it is interesting that IgG2 and IgA2 subclasses of antibody in both switched memory populations have a more "innate-like" repertoire. Clonality analysis shows evidence of a close clonal relationship between the two populations in that both CD27- and CD27+ switched memory cells can be found in the same genealogical tree. The expression of CD27 does not appear to occur in a linear developmental fashion, since we see CD27- cells as precursors of CD27+ cells and vice versa. Despite the similarities, the CDR-H3 repertoire of the CD27- cells is significantly different from both the CD27+IgD+ and CD27+IgD- populations, indicating that perhaps the lack of CD27 might be related to binding properties of the Ig CDR-H3 region.

High-throughput immunoglobulin repertoire analysis distinguishes between human IgM memory and switched memory B-cell populations.

B-cell receptor (BCR) diversity is achieved centrally by rearrangement of Variable, Diversity, and Joining genes, and peripherally by somatic hypermutation and class-switching of the rearranged genes. Peripheral B-cell populations are subject to both negative and positive selection events in the course of their development that have the potential to shape the BCR repertoire. The origin of IgM(+)IgD(+)CD27(+) (IgM memory) cells is controversial. It has been suggested that they may be a prediversified, antigen-independent, population of cells or that they are a population of cells that develop in response to T-independent antigens. Most recently, it was suggested that the majority of IgM memory cells are directly related to switched memory cells and are early emigrants from the germinal center reaction. Advances in sequencing technology have enabled us to undertake large scale IGH repertoire analysis of transitional, naive, IgM memory and switched memory B-cell populations. We find that the memory B-cell repertoires differ from the transitional and naive repertoires, and that the IgM memory repertoire is distinct from that of class-switched memory. Thus we conclude that a large proportion of IgM memory cells develop in response to different stimuli than for class-switched memory cell development.

The ageing B cell population: composition and function.

Age-related changes in the structure and function of the immune system, collectively termed immunosenescence, result in poor responses to infections, increased susceptibility to cancers and increased incidence of autoimmune diseases. The humoral immune response, maintained by the B cell compartment, has a key role in an effective immune system-not only in producing high affinity antibodies that are crucial for vaccination strategies, but in assisting other components of the immune system in their function. Hence an understanding of B cell immunosenescence in particular is vital in designing strategies to combat the effects of age on immune function. Numerous studies have been undertaken using small animal models in order to understand immunosenescence, and these have contributed greatly to our understanding of the events that underpin impaired immune responses. However, there are key differences between the human and the mouse and a clear understanding of these differences is required when extrapolating from one species to the other. In this article we present an overview of B cell development and summarise current data on age-related B cell changes, at both the population level and at the individual mechanistic level. Areas of similarity and difference between human and mouse models are highlighted.

B-cell diversity decreases in old age and is correlated with poor health status.

Older people suffer from a decline in immune system, which affects their ability to respond to infections and to raise efficient responses to vaccines. Effective and specific antibodies in responses from older individuals are decreased in favour of non-specific antibody production. We investigated the B-cell repertoire in DNA samples from peripheral blood of individuals aged 86-94 years, and a control group aged 19-54 years, using spectratype analysis of the IGHV complementarity determining region (CDR)3. We found that a proportion of older individuals had a dramatic collapse in their B-cell repertoire diversity. Sequencing of polymerase chain reaction products from a selection of samples indicated that this loss of diversity was characterized by clonal expansions of B cells in vivo. Statistical analysis of the spectratypes enabled objective comparisons and showed that loss of diversity correlated very strongly with the general health status of the individuals; a distorted spectratype can be used to predict frailty. Correlations with survival and vitamin B12 status were also seen. We conclude that B-cell diversity can decrease dramatically with age and may have important implications for the immune health of older people. B-cell immune frailty is also a marker of general frailty.