Optimality of Mutation and Selection in Germinal Centers

The population dynamics theory of B cells in a typical germinal center could play an important role in revealing how affinity maturation is achieved. However, the existing models encountered some conflicts with experiments. To resolve these conflicts, we present a coarse-grained model to calculate the B cell population development in affinity maturation, which allows a comprehensive analysis of its parameter space to look for optimal values of mutation rate, selection strength, and initial antibody-antigen binding level that maximize the affinity improvement. With these optimized parameters, the model is compatible with the experimental observations such as the ∼100-fold affinity improvements, the number of mutations, the hypermutation rate, and the “all or none” phenomenon. Moreover, we study the reasons behind the optimal parameters. The optimal mutation rate, in agreement with the hypermutation rate in vivo, results from a tradeoff between accumulating enough beneficial mutations and avoiding too many deleterious or lethal mutations. The optimal selection strength evolves as a balance between the need for affinity improvement and the requirement to pass the population bottleneck. These findings point to the conclusion that germinal centers have been optimized by evolution to generate strong affinity antibodies effectively and rapidly. In addition, we study the enhancement of affinity improvement due to B cell migration between germinal centers. These results could enhance our understanding of the functions of germinal centers.     

Clusterin Is a Potential Lymphotoxin Beta Receptor Target That Is Upregulated and Accumulates in Germinal Centers of Mouse Spleen during Immune Response

Clusterin is a multifunctional protein that participates in tissue remodeling, apoptosis, lipid transport, complement-mediated cell lysis and serves as an extracellular chaperone. The role of clusterin in cancer and neurodegeneration has been extensively studied, however little is known about its functions in the immune system. Using expression profiling we found that clusterin mRNA is considerably down-regulated in mouse spleen stroma upon knock-out of lymphotoxin β receptor which plays pivotal role in secondary lymphoid organ development, maintenance and function. Using immunohistochemistry and western blot we studied clusterin protein level and distribution in mouse spleen and mesenteric lymph nodes in steady state and upon immunization with sheep red blood cells. We showed that clusterin protein, represented mainly by the secreted heterodimeric form, is present in all stromal compartments of secondary lymphoid organs except for marginal reticular cells. Clusterin protein level rose after immunization and accumulated in light zones of germinal centers in spleen - the effect that was not observed in lymph nodes. Regulation of clusterin expression by the lymphotoxin beta signaling pathway and its protein dynamics during immune response suggest a specific role of this enigmatic protein in the immune system that needs further study.     

Determining the Binding Affinity of Therapeutic Monoclonal Antibodies towards Their Native Unpurified Antigens in Human Serum

Monoclonal antibodies (mAbs) are a growing segment of therapeutics, yet their in vitro characterization remains challenging. While it is essential that a therapeutic mAb recognizes the native, physiologically occurring epitope, the generation and selection of mAbs often rely on the use of purified recombinant versions of the antigen that may display non-native epitopes. Here, we present a method to measure both, the binding affinity of a therapeutic mAb towards its native unpurified antigen in human serum, and the antigen’s endogenous concentration, by combining the kinetic exclusion assay and Biacore’s calibration free concentration analysis. To illustrate the broad utility of our method, we studied a panel of mAbs raised against three disparate soluble antigens that are abundant in the serum of healthy donors: proprotein convertase subtilisin/kexin type 9 (PCSK9), progranulin (PGRN), and fatty acid binding protein (FABP4). We also determined the affinity of each mAb towards its purified recombinant antigen and assessed whether the interactions were pH-dependent. Of the six mAbs studied, three did not appear to discriminate between the serum and recombinant forms of the antigen; one mAb bound serum antigen with a higher affinity than recombinant antigen; and two mAbs displayed a different affinity for serum antigen that could be explained by a pH-dependent interaction. Our results highlight the importance of taking pH into account when measuring the affinities of mAbs towards their serum antigens, since the pH of serum samples becomes increasingly alkaline upon aerobic handling.     

Prolectin, a Glycan-binding Receptor on Dividing B Cells in Germinal Centers

Prolectin, a previously undescribed glycan-binding receptor, has been identified by re-screening of the human genome for genes encoding proteins containing potential C-type carbohydrate-recognition domains. Glycan array analysis revealed that the carbohydrate-recognition domain in the extracellular domain of the receptor binds glycans with terminal α-linked mannose or fucose residues. Prolectin expressed in fibroblasts is found at the cell surface, but unlike many glycan-binding receptors it does not mediate endocytosis of a neoglycoprotein ligand. However, compared with other known glycan-binding receptors, the receptor contains an unusually large intracellular domain that consists of multiple sequence motifs, including phosphorylated tyrosine residues, that allow it to interact with signaling molecules such as Grb2. Immunohistochemistry has been used to demonstrate that prolectin is expressed on a specialized population of proliferating B cells in germinal centers. Thus, this novel receptor has the potential to function in carbohydrate-mediated communication between cells in the germinal center.Membrane-bound mammalian glycan-binding receptors, often referred to as lectins, are believed to play multiple distinct roles in the immune system, decoding information in complex oligosaccharide structures on cell surfaces and soluble glycoproteins (1, 2). A host of glycan-binding receptors on dendritic cells and macrophages function in pathogen recognition, often resulting in uptake of microbes through endocytic mechanisms. Examples include the mannose receptor, DC-SIGN,³ langerin, and the macrophage galactose receptor. Glycan-binding receptors can also recognize glycans found on the surfaces of mammalian cells. Some of these receptors, such as the selectins, mediate adhesion between leukocytes and endothelia (3, 4). A small number of receptors, notably members of the siglec family, bind mammalian-type glycans and have been shown to have potential signaling functions (5). While multiple glycan-binding receptors have been described on cells of the myeloid lineage, the complement of such receptors on lymphocytes is much more restricted. The best characterized examples are the T-cell adhesion molecule L-selectin (4) and the B-cell receptor CD22, also designated siglec-2 (5).Genomic screening for potential glycan-binding receptors has usually been undertaken by initially searching for the presence of one of the several types of structural domains that are known to support sugar-binding activity (6). Knowledge of the structures of multiple families of modular carbohydrate-recognition domains (CRDs) has facilitated identification of proteins with potential sugar-binding activity and can lead to predictions of what types of ligands might be bound. Although the human genome has been extensively screened with profile-recognition algorithms that identify common sequence motifs associated with CRDs, refinements to the genome sequence and improvements in gene-recognition algorithms occasionally result in detection of novel proteins that contain putative CRDs.We describe a previously undetected glycan-binding receptor identified by re-screening of the human genome and provide characterization of its molecular and cellular properties. Based on its expression in a specialized population of proliferating B cells in germinal centers, we propose that it be designated prolectin. Our results suggest that prolectin functions in carbohydrate-mediated communication between cells in the germinal center.     

Lecithin-Agar Assay for Lecithinase Antibodies in Serum

A technique for assay of lecithinase antibodies in serum was developed in this laboratory by using a lecithin-agar plate diffusion procedure based on a combination of described plate assays. Egg yolk lipoprotein composed primarily of lecithin was used as a substrate for reaction with free or non-neutralized lecithinase C after incubation of known amounts of lecithinase C with various dilutions of control and test sera. It was found that the size of the reaction zone was a function of enzyme concentration and inversely proportional to the antibody concentration. Accuracy and precision of the assay were determined. In addition, lecithinase antibody levels in sera from experimentally inoculated rats and rabbits and sera from randomly selected human patients were studied.     

Prevalence of Serum IgG Antibodies to Cystic Echinococcus Antigen among Patients in an Uzbekistan Emergency Hospital

Cystic echinococcosis (CE) is one of the most widespread zoonotic helminthiases, which can last an asymptomatic infection for several years. The purpose of this study was to demonstrate serum antibody prevalence of CE among asymptomatic people in Uzbekistan using ELISA. A total of 2,547 serum samples were collected, 66 from confirmed CE patients and 2,481 of patients with other diseases than CE at a hospital in Tashkent, Uzbekistan. The serum samples were screened for CE specific IgG antibodies by ELISA using cystic fluid antigen obtained from sheep. The serum antibody positive rate was 89.4% (59/66) in CE and 3.6% (89/2,481) in other disease patients. The present ELISA recognized 89.4% sensitivity and 96.4% specificity. The ELISA absorbance of positive samples was distributed 0.271-0.971 for CE and 0.273-0.887 for other disease patients. The other disease patients with high absorbance over 0.3 were 50 (2.0%) who were presumed to be active CE patients. The patients in their 40s showed the highest positive rate of 5.2% (P=0.181), and women were 4.4% while men were 3.1% positive (P=0.136). The data confirmed that there are many asymptomatic patients of CE in Tashkent. It is indicated that CE is an endemic disease of public health importance in Uzbekistan.     

Serum Antibodies to Bovine Coronavirus in Swedish Sheep

Altogether 218 sheep sera from 40 flocks in different parts of Sweden were screened for antibodies to bovine Coronavirus (BCV). Nineteen per cent of the sera were positive and there was a significantly higher frequency (p<0.05) of at least one positive sample in flocks with more than 100 adult sheep than in smaller flocks. There was also a significantly higher frequency (p<0.001) of positive samples from sheep older than 4 years than from younger ones. Only a weak relationship between BCV positivity (2 or more positive samples, p<0.05) and cattle contact was demonstrated in this study. Possible transmission routes and other factors that could have affected the result are discussed. In light of our finding that all 5 sheep experimentally exposed to BCV through contact with infectious cow faeces seroconverted, we conclude that the antibodies found in Swedish sheep are probably the result of BCV infections directly or indirectly transmitted from cattle.     

Systemic Antibodies Can Inhibit Mouse Mammary Tumor Virus-Driven Superantigen Response in Mucosa-Associated Lymphoid Tissues

Many mucosal pathogens invade the host by initially infecting the organized mucosa-associated lymphoid tissue (o-MALT) such as Peyer’s patches or nasal cavity-associated lymphoid tissue (NALT) before spreading systemically. There is no clear demonstration that serum antibodies can prevent infections in o-MALT. We have tested this possibility by using the mouse mammary tumor virus (MMTV) as a model system. In peripheral lymph nodes or in Peyer’s patches or NALT, MMTV initially infects B lymphocytes, which as a consequence express a superantigen (SAg) activity. The SAg molecule induces the local activation of a subset of T cells within 6 days after MMTV infection. We report that similar levels of anti-SAg antibody (immunoglobulin G) in serum were potent inhibitors of the SAg-induced T-cell response both in peripheral lymph nodes and in Peyer’s patches or NALT. This result clearly demonstrates that systemic antibodies can gain access to Peyer’s patches or NALT.     

Polyclonal B Cell Differentiation and Loss of Gastrointestinal Tract Germinal Centers in the Earliest Stages of HIV-1 Infection

Background

The antibody response to HIV-1 does not appear in the plasma until approximately 2–5 weeks after transmission, and neutralizing antibodies to autologous HIV-1 generally do not become detectable until 12 weeks or more after transmission. Moreover, levels of HIV-1–specific antibodies decline on antiretroviral treatment. The mechanisms of this delay in the appearance of anti-HIV-1 antibodies and of their subsequent rapid decline are not known. While the effect of HIV-1 on depletion of gut CD4⁺ T cells in acute HIV-1 infection is well described, we studied blood and tissue B cells soon after infection to determine the effect of early HIV-1 on these cells.

Methods and Findings

In human participants, we analyzed B cells in blood as early as 17 days after HIV-1 infection, and in terminal ileum inductive and effector microenvironments beginning at 47 days after infection. We found that HIV-1 infection rapidly induced polyclonal activation and terminal differentiation of B cells in blood and in gut-associated lymphoid tissue (GALT) B cells. The specificities of antibodies produced by GALT memory B cells in acute HIV-1 infection (AHI) included not only HIV-1–specific antibodies, but also influenza-specific and autoreactive antibodies, indicating very early onset of HIV-1–induced polyclonal B cell activation. Follicular damage or germinal center loss in terminal ileum Peyer''s patches was seen with 88% of follicles exhibiting B or T cell apoptosis and follicular lysis.

Conclusions

Early induction of polyclonal B cell differentiation, coupled with follicular damage and germinal center loss soon after HIV-1 infection, may explain both the high rate of decline in HIV-1–induced antibody responses and the delay in plasma antibody responses to HIV-1. Please see later in the article for Editors'' Summary     

Affinity Distance Values among Somatic Metaphase Chromosomes in Maize

In maize root-tip metaphase preparations, all distances between two chromosomes were measured in 50 cells from each of seven stocks and in 30 from one stock; four were arrested with cold, two with 8-hydroxyquinoline, one with colchicine and one with monobromonaphthalene. Standardized, affinity-distance values were calculated for all pairs of homologues and pairs of nonhomologues from each preparation. The homologues of pair X were the least separated, those of pair I the most separated in the cold-arrested stocks. All but pairs I and VIII were shown to be significantly different from the observed mean. The observed mean was less than but not significantly different from the theoretical value for a random distribution. The use of chemical agents for metaphase arrest increased the separation of homologues, except for pair I.—Eleven percent of the comparisons of nonhomologues from cold-arrested, as contrasted to none of the comparisons from the c-metaphase treatments, were significantly different from the theoretical value for a random distribution. This was considered evidence for limited primary nonhomologue association in maize. Although there were specific, differential responses to the two arrest agents, the population of homologous pairs approached a random distribution only in chemically arrested stocks.—Primary homologue association was considered to be maintained by two mechanisms, the more common involving the microtubules and the second involving the nucleolus.—Interpretations are offered regarding the claims of somatic association in other species, especially man. The opportunity in maize for experimentally modifying distance values by cytogenetic techniques is discussed.     

High Affinity Antibodies against Influenza Characterize the Plasmablast Response in SLE Patients After Vaccination

Breakdown of B cell tolerance is a cardinal feature of systemic lupus erythematosus (SLE). Increased numbers of autoreactive mature naïve B cells have been described in SLE patients and autoantibodies have been shown to arise from autoreactive and non-autoreactive precursors. How these defects, in the regulation of B cell tolerance and selection, influence germinal center (GC) reactions that are directed towards foreign antigens has yet to be investigated. Here, we examined the characteristics of post-GC foreign antigen-specific B cells from SLE patients and healthy controls by analyzing monoclonal antibodies generated from plasmablasts induced specifically by influenza vaccination. We report that many of the SLE patients had anti-influenza antibodies with higher binding affinity and neutralization capacity than those from controls. Although overall frequencies of autoreactivity in the influenza-specific plasmablasts were similar for SLE patients and controls, the variable gene repertoire of influenza-specific plasmablasts from SLE patients was altered, with increased usage of JH6 and long heavy chain CDR3 segments. We found that high affinity anti-influenza antibodies generally characterize the plasmablast responses of SLE patients with low levels of autoreactivity; however, certain exceptions were noted. The high-avidity antibody responses in SLE patients may also be correlated with cytokines that are abnormally expressed in lupus. These findings provide insights into the effects of dysregulated immunity on the quality of antibody responses following influenza vaccination and further our understanding of the underlying abnormalities of lupus.     

Affinity among mountain ranges in Megamexico: A phytogeographical scenario

The concept of Megamexico as a phytogeographic unit has been introduced by Rzedowski (1991) on the basis of distribution patterns of genera. Until now precise information on the resemblance between plant communities in the proposed Megamexico was scarce. This phytosociological study documents the affinities between plant communities within Megamexico, based on studies from mountain ranges in Mexico (Sierra Chichinautzin, Sierra Nevada) and Guatemalan mountain ranges (Sierra de los Cuchumatanes, Cadena Volcánica). Data, collected in the style of Braun-Blanquet during extensive fieldwork, were pooled into a single data set, and analyzed by using (1) TWINSPAN, for plant community classification, (2) ordination (DCA), (3) alpha log series biodiversity-index to measure intergroup diversity, and (4) information on the altitudinal distribution of the vegetation belts. Classification showed that four communities were common to the two groups of mountain ranges, namely, alpine bunchgrass-land, pine forest, fir forest and mixed forest. Along the altitudinal gradient Mexican communities are distributed 200 m higher than their Guatemalan equivalents. This appears to be an illustration of the Massenerhebungseffekt. DCA showed that the first axis represents a set of minor differences of closely related ecological factors (e.g., temperature, precipitation) and the second a humidity gradient. The biodiversity index showed that the Mexican mixed forest was significantly more diverse than the Guatemalan mixed forest. From the present results, ecological conditions among mountain ranges in Megamexico differed significantly. To conclude, phytogeographical units in Megamexico can better be defined on basis of both historical and ecological characteristics of the communities.Abbreviations: community refers to plant community throughout the paper.     

Affinity groups among plant pathogenic species of phytophthora in culture

Cluster analysis of the plant-pathogenic species ofPhytophthora in culture was conducted based on the morphological, physiological and pathological characters. The dendrograms of similarity coefficients produced five distinct affinity groups broadly in agreement with those established by Waterhouse (1963). The merging of Group V and Group VI of Waterhouse was proposed.     

Serum Antibodies to the Catalase Antigen of Aspergillus fumigatus in Cattle

Aspergillus fumigatus is an important agent of mycotic infection in cattle and a potent source of antigens. However, the efficacy of serological diagnosis of aspergillosis in cattle remains controversial. Corbel (1972) and Knudtson et al. (1974) considered a precipitin assay useful as a supplementary test in the diagnosis of mycotic abortion, whereas Wiseman et al. (1984) found the specificity too low to justify its routine use. We have studied 1) the antibody response to the catalase antigen of A. fumigatus in experimentally infected cattle and 2) the prevalence of catalase antibodies and A. fumigatus precipitins in healthy and diseased cattle. The aim was to ascertain how far detection of antibodies to a defined fungal antigen can contribute to the often difficult diagnosis of mycosis.     

Direct Detection of Disease-Associated Prions in Brain and Lymphoid Tissue Using Antibodies Recognizing the Extreme N-terminus of PrPC

A simple diagnostic test is described for the detection of TSE in bovine, ovine and human brain and lymphoid tissue that obviates the use of proteinase K as a discriminating reagent. The immunoassay utilises high affinity anti-peptide antibodies that appear blind to the normal isoform of prion protein (PrP^C). These reagents have been produced with novel N-terminal chimeric peptides and we hypothesise that the retention and stability of the extreme N-terminus of PrP in the disease-associated aggregate makes it an operationally specific marker for TSE. Accordingly, the assay involves homogenisation of the tissue directly in 8M guanidine hydrochloride, a simple one-step capture of PrP^Sc followed by detection with a europium-labelled anti-PrP^C antibody. This rapid assay clearly differentiates between levels of disease-associated PrP extracted from brain and lymphoid tissues taken from confirmed TSE positive and negative cattle and sheep. The assay can also be used to detect PrP^Sc in cases of vCJD.     

Acute SIV Infection in Sooty Mangabey Monkeys Is Characterized by Rapid Virus Clearance from Lymph Nodes and Absence of Productive Infection in Germinal Centers

Lymphoid tissue immunopathology is a characteristic feature of chronic HIV/SIV infection in AIDS-susceptible species, but is absent in SIV-infected natural hosts. To investigate factors contributing to this difference, we compared germinal center development and SIV RNA distribution in peripheral lymph nodes during primary SIV infection of the natural host sooty mangabey and the non-natural host pig-tailed macaque. Although SIV-infected cells were detected in the lymph node of both species at two weeks post infection, they were confined to the lymph node paracortex in immune-competent mangabeys but were seen in both the paracortex and the germinal center of SIV-infected macaques. By six weeks post infection, SIV-infected cells were no longer detected in the lymph node of sooty mangabeys. The difference in localization and rate of disappearance of SIV-infected cells between the two species was associated with trapping of cell-free virus on follicular dendritic cells and higher numbers of germinal center CD4⁺ T lymphocytes in macaques post SIV infection. Our data suggests that fundamental differences in the germinal center microenvironment prevent productive SIV infection within the lymph node germinal centers of natural hosts contributing to sustained immune competency.     

Side-by-Side Comparison of Commonly Used Biomolecules That Differ in Size and Affinity on Tumor Uptake and Internalization

The ability to use a systemically injected agent to image tumor is influenced by tumor characteristics such as permeability and vascularity, and the size, shape, and affinity of the imaging agent. In this study, six different imaging biomolecules, with or without specificity to tumor, were examined for tumor uptake and internalization at the whole body, ex-vivo tissue, and cellular levels: antibodies, antibody fragments (Fab), serum albumin, and streptavidin. The time of peak tumor uptake was dependent solely on the size of molecules, suggesting that molecular size is the major factor that influences tumor uptake by its effect on systemic clearance and diffusion into tumor. Affinity to tumor antigen failed to augment tumor uptake of Fab above non-specific accumulation, which suggests that Fab fragments of typical monoclonal antibodies may fall below an affinity threshold for use as molecular imaging agents. Despite abundant localization into the tumor, albumin and streptavidin were not found on cell surface or inside cells. By comparing biomolecules differing in size and affinity, our study highlights that while pharmacokinetics are a dominant factor in tumor uptake for biomolecules, affinity to tumor antigen is required for tumor binding and internalization.     

Direct Detection of Disease Associated Prions in Brain and Lymphoid Tissue Using Antibodies Recognizing the Extreme N Terminus of PrPC

A simple diagnostic test is described for the detection of TSE in bovine, ovine and human brain and lymphoid tissue that obviates the use of proteinase K as a discriminating reagent. The immunoassay utilises high affinity anti-peptide antibodies that appear blind to the normal isoform of prion protein (PrP^C). These reagents have been produced with novel N-terminal chimeric peptides and we hypothesise that the retention and stability of the extreme N-terminus of PrP in the disease-associated aggregate makes it an operationally specific marker for TSE. Accordingly, the assay involves homogenisation of the tissue directly in 8M guanidine hydrochloride, a simple one-step capture of PrP^Sc followed by detection with a europium-labelled anti-PrP^C antibody. This rapid assay clearly differentiates between levels of disease-associated PrP extracted from brain and lymphoid tissues taken from confirmed TSE positive and negative cattle and sheep. The assay can also be used to detect PrP^Sc in cases of vCJD.Key Words: BSE, scrapie and vCJD Diagnosis, DELFIA®, novel N terminal antibodies