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Malaria is a highly oxidative parasitic disease in which anemia is the most common clinical symptom. A major contributor to malarial anemia pathogenesis is the destruction of bystander, uninfected red blood cells. Metabolic fluctuations are known to occur in the plasma of individuals with acute malaria, emphasizing the role of metabolic changes in disease progression and severity. Here, we report that conditioned media fromPlasmodium falciparumculture induces oxidative stress in healthy uninfected RBCs. Additionally, we show the benefit of amino acid pre-exposure for RBCs and how this pre-treatment intrinsically prepares RBCs to mitigate oxidative stress.Key pointsIntracellular ROS is acquired in red blood cells incubated withPlasmodium falciparumconditioned mediaGlutamine, cysteine, and glycine amino acid supplementation increased glutathione biosynthesis and reduced ROS levels in stressed RBCs

The bone microenvironment cellular composition plays an essential role in bone health and is disrupted in bone pathologies, such as osteoporosis, osteoarthritis, and cancer. Flow cytometry protocols for hematopoietic stem cell lineages are well defined and well established. Additionally, a consensus for mesenchymal stem cell flow markers has been developed. However, flow cytometry markers for bone-residing cells-osteoblasts, osteoclasts, and osteocytes-have not been proposed. Here, we describe a novel partial digestion method to separate these cells from the bone matrix and present new markers for enumerating these cells by flow cytometry. We optimized bone digestion and analyzed markers across murine, nonhuman primate, and human bone. The isolation and staining protocols can be used with either cell sorting or flow cytometry. Our method allows for the enumeration and collection of hematopoietic and mesenchymal lineage cells in the bone microenvironment combined with bone-residing stromal cells. Thus, we have established a multi-fluorochrome bone marrow cell-typing methodology. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Partial digestion for murine long bone stromal cell isolation Alternate Protocol 1: Partial digestion for primate vertebrae stromal cell isolation Alternate Protocol 2: Murine vertebrae crushing for bone stromal cell isolation Basic Protocol 2: Staining of bone stromal cells Support Protocol 1: Fluorescence minus one control, isotype control, and antibody titration Basic Protocol 3: Cell sorting of bone stromal cells Alternate Protocol 3: Flow cytometry analysis of bone stromal cells Support Protocol 2: Preparing compensation beads.

Despite the wide availability of antiretroviral drugs, more than 250,000 infants are vertically infected with HIV-1 annually, emphasizing the need for additional interventions to eliminate pediatric HIV-1 infections. Here, we aimed to define humoral immune correlates of risk of mother-to-child transmission (MTCT) of HIV-1, including responses associated with protection in the RV144 vaccine trial. Eighty-three untreated, HIV-1–transmitting mothers and 165 propensity score–matched nontransmitting mothers were selected from the Women and Infants Transmission Study (WITS) of US nonbreastfeeding, HIV-1–infected mothers. In a multivariable logistic regression model, the magnitude of the maternal IgG responses specific for the third variable loop (V3) of the HIV-1 envelope was predictive of a reduced risk of MTCT. Neutralizing Ab responses against easy-to-neutralize (tier 1) HIV-1 strains also predicted a reduced risk of peripartum transmission in secondary analyses. Moreover, recombinant maternal V3–specific IgG mAbs mediated neutralization of autologous HIV-1 isolates. Thus, common V3-specific Ab responses in maternal plasma predicted a reduced risk of MTCT and mediated autologous virus neutralization, suggesting that boosting these maternal Ab responses may further reduce HIV-1 MTCT.

A successful HIV-1 vaccine must elicit immune responses that impede mucosal virus transmission, though functional roles of protective HIV-1 Envelope (Env)-specific mucosal antibodies remain unclear. Colostrum is a rich source of readily accessible mucosal B cells that may help define the mucosal antibody response contributing to prevention of postnatal HIV-1 transmission. To examine the HIV-1 Env-specific colostrum B-cell repertoire, single B cells were isolated from 17 chronically HIV-infected, lactating women, producing 51 blood and 39 colostrum HIV-1 Env-specific B-cell antibodies. All HIV-1 Env-specific colostrum-derived antibodies were immunoglobulin (Ig)G1 isotype and had mean heavy chain complementarity-determining region 3 (CDR3) lengths and mutation frequencies similar to those isolated from blood. However, variable heavy chain (VH) gene subfamily 1(∼)69 usage was higher among colostrum than blood HIV-1 Env-reactive antibodies (49% vs. 20%, P=0.006, Fisher's exact test). Additionally, more HIV-1 Env-specific colostrum antibodies were gp120 specific than those isolated from blood (44% vs. 16%, P=0.005, Fisher's exact test). One cross-compartment HIV-1 Env-specific clonal B-cell lineage was identified. These unique characteristics of colostrum B-cell antibodies suggest selective homing of HIV-1-specific IgG1-secreting memory B cells to the mammary gland and have implications for targeting mucosal B-cell populations by vaccination.

A strategy for HIV-1 vaccine development is to define envelope (Env) evolution of broadly neutralizing antibodies (bnAbs) in infection and to recreate those events by vaccination. Here, we report host tolerance mechanisms that limit the development of CD4-binding site (CD4bs), HCDR3-binder bnAbs via sequential HIV-1 Env vaccination. Vaccine-induced macaque CD4bs antibodies neutralize 7% of HIV-1 strains, recognize open Env trimers, and accumulate relatively modest somatic mutations. In naive CD4bs, unmutated common ancestor knock-in mice Env+B cell clones develop anergy and partial deletion at the transitional to mature B cell stage, but become Env− upon receptor editing. In comparison with repetitive Env immunizations, sequential Env administration rescue anergic Env+ (non-edited) precursor B cells. Thus, stepwise immunization initiates CD4bs-bnAb responses, but immune tolerance mechanisms restrict their development, suggesting that sequential immunogen-based vaccine regimens will likely need to incorporate strategies to expand bnAb precursor pools., An efficient HIV-1 vaccine will likely depend on eliciting broadly neutralizing antibodies (bnAb). Here the authors analyze the B cell repertoire in macaques and knock-in mice in response to sequential immunization with Env variants that induce a bnAb targeting the CD4-binding site of Env in a HIV-1 infected individual.

Dominant antibody responses in vaccinees who received the HIV-1 multiclade (A, B, and C) envelope (Env) DNA/recombinant adenovirus virus type 5 (rAd5) vaccine studied in HIV-1 Vaccine Trials Network (HVTN) efficacy trial 505 (HVTN 505) targeted Env gp41 and cross-reacted with microbial antigens. In this study, we asked if the DNA/rAd5 vaccine induced a similar antibody response in rhesus macaques (RMs), which are commonly used as an animal model for human HIV-1 infections and for testing candidate HIV-1 vaccines. We also asked if gp41 immunodominance could be avoided by immunization of neonatal RMs during the early stages of microbial colonization. We found that the DNA/rAd5 vaccine elicited a higher frequency of gp41-reactive memory B cells than gp120-memory B cells in adult and neonatal RMs. Analysis of the vaccine-induced Env-reactive B cell repertoire revealed that the majority of HIV-1 Env-reactive antibodies in both adult and neonatal RMs were targeted to gp41. Interestingly, a subset of gp41-reactive antibodies isolated from RMs cross-reacted with host antigens, including autologous intestinal microbiota. Thus, gp41-containing DNA/rAd5 vaccine induced dominant gp41-microbiota cross-reactive antibodies derived from blood memory B cells in RMs as observed in the HVTN 505 vaccine efficacy trial. These data demonstrated that RMs can be used to investigate gp41 immunodominance in candidate HIV-1 vaccines. Moreover, colonization of neonatal RMs occurred within the first week of life, and immunization of neonatal RMs during this time also induced a dominant gp41-reactive antibody response. IMPORTANCE Our results are critical to current work in the HIV-1 vaccine field evaluating the phenomenon of gp41 immunodominance induced by HIV-1 Env gp140 in RMs and humans. Our data demonstrate that RMs are an appropriate animal model to study this phenomenon and to determine the immunogenicity in new HIV-1 Env trimer vaccine designs. The demonstration of gp41 immunodominance in memory B cells of both adult and neonatal RMs indicated that early vaccination could not overcome gp41 dominant responses.

The RV144 ALVAC/AIDSVax HIV-1 vaccine clinical trial showed an estimated vaccine efficacy of 31.2%. Viral genetic analysis identified a vaccine-induced site of immune pressure in the HIV-1 envelope (Env) variable region 2 (V2) focused on residue 169, which is included in the epitope recognized by vaccinee-derived V2 monoclonal antibodies. The ALVAC/AIDSVax vaccine induced antibody-dependent cellular cytotoxicity (ADCC) against the Env V2 and constant 1 (C1) regions. In the presence of low IgA Env antibody levels, plasma levels of ADCC activity correlated with lower risk of infection. In this study, we demonstrate that C1 and V2 monoclonal antibodies isolated from RV144 vaccinees synergized for neutralization, infectious virus capture, and ADCC. Importantly, synergy increased the HIV-1 ADCC activity of V2 monoclonal antibody CH58 at concentrations similar to that observed in plasma of RV144 vaccinees. These findings raise the hypothesis that synergy among vaccine-induced antibodies with different epitope specificities contributes to HIV-1 antiviral antibody responses and is important to induce for reduction in the risk of HIV-1 transmission. IMPORTANCE The Thai RV144 ALVAC/AIDSVax prime-boost vaccine efficacy trial represents the only example of HIV-1 vaccine efficacy in humans to date. Studies aimed at identifying immune correlates involved in the modest vaccine-mediated protection identified HIV-1 envelope (Env) variable region 2-binding antibodies as inversely correlated with infection risk, and genetic analysis identified a site of immune pressure within the region recognized by these antibodies. Despite this evidence, the antiviral mechanisms by which variable region 2-specific antibodies may have contributed to lower rates of infection remain unclear. In this study, we demonstrate that vaccine-induced HIV-1 envelope variable region 2 and constant region 1 antibodies synergize for recognition of virus-infected cells, infectious virion capture, virus neutralization, and antibody-dependent cellular cytotoxicity. This is a major step in understanding how these types of antibodies may have cooperatively contributed to reducing infection risk and should be considered in the context of prospective vaccine design.

The ALVAC prime/ALVAC + AIDSVAX B/E boost RV144 vaccine trial induced an estimated 31% efficacy in a low-risk cohort where HIV‑1 exposures were likely at mucosal surfaces. An immune correlates study demonstrated that antibodies targeting the V2 region and in a secondary analysis antibody-dependent cellular cytotoxicity (ADCC), in the presence of low envelope-specific (Env-specific) IgA, correlated with decreased risk of infection. Thus, understanding the B cell repertoires induced by this vaccine in systemic and mucosal compartments are key to understanding the potential protective mechanisms of this vaccine regimen. We immunized rhesus macaques with the ALVAC/AIDSVAX B/E gp120 vaccine regimen given in RV144, and then gave a boost 6 months later, after which the animals were necropsied. We isolated systemic and intestinal vaccine Env-specific memory B cells. Whereas Env-specific B cell clonal lineages were shared between spleen, draining inguinal, anterior pelvic, posterior pelvic, and periaortic lymph nodes, members of Env‑specific B cell clonal lineages were absent in the terminal ileum. Env‑specific antibodies were detectable in rectal fluids, suggesting that IgG antibodies present at mucosal sites were likely systemically produced and transported to intestinal mucosal sites.

The ALVAC-HIV/AIDSVAX-B/E RV144 vaccine trial showed an estimated efficacy of 31%. RV144 secondary immune correlate analysis demonstrated that the combination of low plasma anti-HIV-1 Env IgA antibodies and high levels of antibody-dependent cellular cytotoxicity (ADCC) inversely correlate with infection risk. One hypothesis is that the observed protection in RV144 is partially due to ADCC-mediating antibodies. We found that the majority (73 to 90%) of a representative group of vaccinees displayed plasma ADCC activity, usually (96.2%) blocked by competition with the C1 region-specific A32 Fab fragment. Using memory B-cell cultures and antigen-specific B-cell sorting, we isolated 23 ADCC-mediating nonclonally related antibodies from 6 vaccine recipients. These antibodies targeted A32-blockable conformational epitopes ( n = 19), a non-A32-blockable conformational epitope ( n = 1), and the gp120 Env variable loops ( n = 3). Fourteen antibodies mediated cross-clade target cell killing. ADCC-mediating antibodies displayed modest levels of V-heavy (VH) chain somatic mutation (0.5 to 1.5%) and also displayed a disproportionate usage of VH1 family genes (74%), a phenomenon recently described for CD4-binding site broadly neutralizing antibodies (bNAbs). Maximal ADCC activity of VH1 antibodies correlated with mutation frequency. The polyclonality and low mutation frequency of these VH1 antibodies reveal fundamental differences in the regulation and maturation of these ADCC-mediating responses compared to VH1 bNAbs.

Most antibodies that broadly neutralize HIV-1 are highly somatically mutated in antibody clonal lineages that persist over time. Here, we describe the analysis of human antibodies induced during an HIV-1 vaccine trial (GSK PRO HIV-002) that used the clade B envelope (Env) gp120 of clone W6.1D (gp120 W6.1D ). Using dual-color antigen-specific sorting, we isolated Env-specific human monoclonal antibodies (MAbs) and studied the clonal persistence of antibodies in the setting of HIV-1 Env vaccination. We found evidence of V H somatic mutation induced by the vaccine but only to a modest level (3.8% ± 0.5%; range 0 to 8.2%). Analysis of 34 HIV-1-reactive MAbs recovered over four immunizations revealed evidence of both sequential recruitment of naïve B cells and restimulation of previously recruited memory B cells. These recombinant antibodies recapitulated the anti-HIV-1 activity of participant serum including pseudovirus neutralization and antibody-dependent cell-mediated cytotoxicity (ADCC). One antibody (3491) demonstrated a change in specificity following somatic mutation with binding of the inferred unmutated ancestor to a linear C2 peptide while the mutated antibody reacted only with a conformational epitope in gp120 Env. Thus, gp120 W6.1D was strongly immunogenic but over four immunizations induced levels of affinity maturation below that of broadly neutralizing MAbs. Improved vaccination strategies will be needed to drive persistent stimulation of antibody clonal lineages to induce affinity maturation that results in highly mutated HIV-1 Env-reactive antibodies.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the clonal expansion of CD5-expressing B lymphocytes that produce mAbs often reactive with microbial or autoantigens. Long-term culture of B-CLL clones would permit the collection and characterization of B-CLL mAbs to study antigen specificity and of B-CLL DNA to investigate molecular mechanisms promoting the disease. However, the derivation of long-term cell lines (eg, by EBV), has not been efficient. We have improved the efficiency of EBV B-CLL transformation of CpG oligonucleotide-stimulated cells by incubating patient peripheral blood mononuclear cells in the presence of an irradiated mouse macrophage cell line, J774A.1. Using this approach, peripheral blood mononuclear cells isolated from 13 of 21 B-CLL patients were transformed as documented by IGHV-D-J sequencing. Four clones grew and retained CD5 expression in culture for 2 to 4 months. However, despite documentation of EBV infection by expression of EBNA2 and LMP1, B-CLL cells died after removal of macrophage feeder cells. Nevertheless, using electrofusion technology, we generated 6 stable hetero-hybridoma cell lines from EBV-transformed B-CLL cells, and these hetero-hybridomas produced immunoglobulin. Thus, we have established enhanced methods of B-CLL culture that will enable broader interrogation of B-CLL cells at the genetic and protein levels.

Biome depletion, or loss of co-evolved constituents within the ecosystem of the human body, has become the leading suspect in epidemics of allergic, inflammatory and autoimmune diseases associated with post-industrial culture. Immunity in wild and laboratory rats has been used as a model for immunity in biomenormal and biome depleted environments, respectively. In this study, the ranges of numerous immune parameters (such as cytokine production and cell surface marker expression) in 8 wild rats overlapped with the ranges found in 7 laboratory rats. However, considering a number of parameters simultaneously as an “expression index” revealed a substantially greater range of immune activity in the wild compared with laboratory rats. These findings are consistent with the intuitive idea that the immune system is inherently malleable or flexible within environments of evolutionary adaptedness, and might suggest that biome reconstitution as an effective therapy could be achieved in a variety of ways.

Many HIV-1 envelope-reactive antibodies shortly after HIV-1 transmission may arise from crow-reactive memory B cells previously stimulated by non-HIV-1 host or microbial antigens, The initial antibody response to HIV-1 is targeted to envelope (Env) gp41, and is nonneutralizing and ineffective in controlling viremia. To understand the origins and characteristics of gp41-binding antibodies produced shortly after HIV-1 transmission, we isolated and studied gp41-reactive plasma cells from subjects acutely infected with HIV-1. The frequencies of somatic mutations were relatively high in these gp41-reactive antibodies. Reverted unmutated ancestors of gp41-reactive antibodies derived from subjects acutely infected with HIV-1 frequently did not react with autologous HIV-1 Env; however, these antibodies were polyreactive and frequently bound to host or bacterial antigens. In one large clonal lineage of gp41-reactive antibodies, reactivity to HIV-1 Env was acquired only after somatic mutations. Polyreactive gp41-binding antibodies were also isolated from uninfected individuals. These data suggest that the majority of gp41-binding antibodies produced after acute HIV-1 infection are cross-reactive responses generated by stimulating memory B cells that have previously been activated by non–HIV-1 antigens.

Microbiota can mislead antibodies Unlike the response to many viral infections, most people do not produce antibodies capable of clearing HIV-1. Non-neutralizing antibodies that target HIV-1's envelope glycoprotein (Env) typically dominate the response, which is generated by B cells that cross-react with Env and the intestinal microbiota. Williams et al. analyzed samples from individuals who had received a vaccine containing the Env protein, including the gp41 subunit. Most of the antibodies were non-neutralizing and targeted gp41. The antibodies also reacted to intestinal microbiota, suggesting that preexisting immunity to microbial communities skews vaccineinduced immune responses toward an unproductive target. Science , this issue 10.1126/science.aab1253 .

An HIV-1 DNA prime vaccine, with a recombinant adenovirus type 5 (rAd5) boost, failed to protect from HIV-1 acquisition. We studied the nature of the vaccine-induced antibody (Ab) response to HIV-1 envelope (Env). HIV-1-reactive plasma Ab titers were higher to Env gp41 than to gp120, and repertoire analysis demonstrated that 93% of HIV-1-reactive Abs from memory B cells responded to Env gp41. Vaccine-induced gp41-reactive monoclonal antibodies were non-neutralizing and frequently polyreactive with host and environmental antigens, including intestinal microbiota (IM). Next-generation sequencing of an immunoglobulin heavy chain variable region repertoire before vaccination revealed an Env-IM cross-reactive Ab that was clonally related to a subsequent vaccine-induced gp41-reactive Ab. Thus, HIV-1 Env DNA-rAd5 vaccine induced a dominant IM-polyreactive, non-neutralizing gp41-reactive Ab repertoire response that was associated with no vaccine efficacy.

Several groups, including our own, have independently demonstrated that effector memory T cells from non–alloantigen-primed donors do not cause graft-versus-host disease (GVHD). In the current study, we further investigated whether this approach could be extended to all memory T cells, and we studied the underlying mechanisms. Neither total memory T cells nor purified central memory T cells were able to induce GVHD. Memory T cells were at least 3-log less potent than bulk T cells in mediating GVHD. As expected, memory T cells failed to elicit cytotoxicity and proliferated poorly against alloantigens in standard 5-day mixed-lymphocyte cultures. However, the proliferative responses of memory T cells were more comparable with those of bulk and naive T cells when the culture time was shortened. Moreover, the frequencies of IL-2–secreting cells measured by 42-hour enzyme-linked immunosorbent spot (ELISPOT) assay were similar among naive, memory, and bulk T cells. These data indicated that memory T cells are able to respond to alloantigens initially but fail to develop to full potential. The abortive immune response, which was mediated by non–alloantigen-specific memory T cells in response to alloantigens, may explain why memory T cells from unprimed and non–alloantigen-primed donors could not induce GVHD.

Somatic mutations of immunoglobulin genes characterize mature memory B cells, and intraclonal B-cell diversification is typically associated with expansion of B-cell clones with greater affinity for antigen (antigen drive). Evidence for a role of antigen in progression of intraclonal chronic lymphocytic leukemia (CLL) cell diversification in patients with mutated immunoglobulin genes has not been previously presented. We performed a single-cell analysis of immunoglobulin heavy and light chains in 6 patients with somatically mutated CLL-cell immunoglobulin genes and identified 2 patients with multiple related (oligoclonal) subgroups of CLL cells. We constructed genealogic trees of these oligoclonal CLL-cell subgroups and assessed the effects of immunoglobulin somatic mutations on the ratios of replacement and silent amino acid changes in the framework and antigen-binding regions (CDRs) of the immunoglobulin heavy and light chains from each oligoclonal CLL-cell population. In one subject, the amino acid changes were consistent with an antigen-driven progression of clonally related CLL-cell populations. In the other subject, intraclonal diversification was associated with immunoglobulin amino acid changes that would have likely lessened antigen affinity. Taken together, these studies support the hypothesis that in some CLL cases intraclonal diversification is dependent on antigen interactions with immunoglobulin receptors.

SummaryMonoclonal antibodies derived from blood plasma cells of acute HIV-1-infected individuals are predominantly targeted to the HIV Env gp41 and cross-reactive with commensal bacteria. To understand this phenomenon, we examined anti-HIV responses in ileum B cells using recombinant antibody technology and probed their relationship to commensal bacteria. The dominant ileum B cell response was to Env gp41. Remarkably, a majority (82%) of the ileum anti-gp41 antibodies cross-reacted with commensal bacteria, and of those, 43% showed non-HIV-1 antigen polyreactivity. Pyrosequencing revealed shared HIV-1 antibody clonal lineages between ileum and blood. Mutated immunoglobulin G antibodies cross-reactive with both Env gp41 and microbiota could also be isolated from the ileum of HIV-1 uninfected individuals. Thus, the gp41 commensal bacterial antigen cross-reactive antibodies originate in the intestine, and the gp41 Env response in HIV-1 infection can be derived from a preinfection memory B cell pool triggered by commensal bacteria that cross-react with Env.

In vivo biocompatibility of soft-tissue implants is often hampered by development of capsules that eventually might contract and impair implant function. It has been shown that capsule formation can be significantly reduced by using materials with textured surface elements in the micron range. In this study the interaction of human fibroblasts with silicone surfaces was analyzed using cell cycle analysis. Silicone was textured with 2, 5, and 10 microns wide grooves (2MU, 5MU, 10MU, respectively) or kept smooth (SMT). Cell cycle analysis was performed after staining of cells with propidium iodide. Cells proliferated on the fibronectin-preadsorbed silicone, as demonstrated by increased coverage and occurrence of subpopulations in the S and G2/M phase of the cell cycle. Cells on SMT went faster into the S phase than cells on textured silicones. Cells on 10MU showed less proliferation than cells on 2MU and 5MU. Besides the basic percentages of cells in the different cycle phases, DNA profiles were also influenced by incubation time and texture, especially with respect to the presence of hypodiploid populations and asymmetry of the G0/G1 peak. Finally scatter characteristics were influenced. 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay data did not reveal significant differences among the different samples. Fibronectin preadsorption of silicone only resulted in slightly higher MTT conversion. Cell cycle analysis proved to be a sensitive screening method for proliferation on the silicone surfaces and provided information beyond the normal G0/G1, S and G2/M subpopulations.

Background The ALVAC-HIV/AIDSVAX-B/E RV144 vaccine efficacy trial showed an estimated efficacy of 31%. The immune correlates analysis raised the hypothesis that the observed protection in RV144 may be partially due to AntibodyDependent Cellular Cytotoxicity (ADCC)-mediating antibodies in the presence of low levels of Env IgA antibodies. In this study we analyzed the Ig VH family usage of vaccine-induced ADCC mAbs isolated from memory B cells of vaccinees. Methods From a total of 321,945 memory B-cells of 6 vaccinees we obtained 23 mAbs that mediated ADCC using IgG+ memory B-cell cultures (n=9) and Env-specific flow cytometric single memory B-cell sorting (n=14). ADCC activity was measured using both E.CM243 gp120-coated and E. CM235-infected target cells in a flow-based assay. Results ADCC-mediating mAbs displayed a disproportionate usage of VH1 family genes (17/23; 74%), in particular the VH1-2 gene segment (10/17; 59%), as recently observed for CD4bs broadly neutralizing antibodies (HAAD bNAbs). In contrast, only 17.1% of 111 heavy chains isolated from cultures that did not mediate ADCC used the VH1 gene. VH1 ADCC-mediating mAbs showed a high degree of V(D)J amino acid similarity to both the VH (68-84%) and VL (70-87%) HAAD motifs. V(D)J rearrangements displayed modest levels of affinity maturation (0.5-5.1% for heavy chains and 0.4-4.3% for light chains). While none of the VH1 ADCC-mediating mAbs was capable of mediating HIV-1 neutralization, the strength of their ADCC activity correlated with the levels of heavy chain somatic mutations (p=0.02). We produced the reverted unmutated ancestor antibodies of two VH1 ADCC-mediating mAbs: one bound to B.MN Env and both reacted against autoantigens. Conclusion ADCC-mediating antibodies induced by the ALVAC-HIV/ AIDSVAX-B/E vaccine underwent limited affinity maturation, and preferentially used VH1 gene segments which share the HAAD motif with CD4bs bNAbs. These observations raise the hypothesis that HIV-1 Env preferentially selects VH1 family usage for distinct subsets of antibodies with different functions.

Background During the recent H1N1 influenza pandemic, excess morbidity and mortality was seen in young but not older adults suggesting that prior infection with influenza strains may have protected older subjects. In contrast, a history of recent seasonal trivalent vaccine in younger adults was not associated with protection. Methods and Findings To study hemagglutinin (HA) antibody responses in influenza immunization and infection, we have studied the day 7 plasma cell repertoires of subjects immunized with seasonal trivalent inactivated influenza vaccine (TIV) and compared them to the plasma cell repertoires of subjects experimentally infected (EI) with influenza H3N2 A/Wisconsin/67/2005. The majority of circulating plasma cells after TIV produced influenza-specific antibodies, while most plasma cells after EI produced antibodies that did not react with influenza HA. While anti-HA antibodies from TIV subjects were primarily reactive with single or few HA strains, anti-HA antibodies from EI subjects were isolated that reacted with multiple HA strains. Plasma cell-derived anti-HA antibodies from TIV subjects showed more evidence of clonal expansion compared with antibodies from EI subjects. From an H3N2-infected subject, we isolated a 4-member clonal lineage of broadly cross-reactive antibodies that bound to multiple HA subtypes and neutralized both H1N1 and H3N2 viruses. This broad reactivity was not detected in post-infection plasma suggesting this broadly reactive clonal lineage was not immunodominant in this subject. Conclusion The presence of broadly reactive subdominant antibody responses in some EI subjects suggests that improved vaccine designs that make broadly reactive antibody responses immunodominant could protect against novel influenza strains.

V2/V3 conformational epitope antibodies that broadly neutralize HIV-1 (PG9 and PG16) have been recently described. Since an elicitation of previously known broadly neutralizing antibodies has proven elusive, the induction of antibodies with such specificity is an important goal for HIV-1 vaccine development. A critical question is which immunogens and vaccine formulations might be used to trigger and drive the development of memory B cell precursors with V2/V3 conformational epitope specificity. In this paper we identified a clonal lineage of four V2/V3 conformational epitope broadly neutralizing antibodies (CH01 to CH04) from an African HIV-1-infected broad neutralizer and inferred their common reverted unmutated ancestor (RUA) antibodies. While conformational epitope antibodies rarely bind recombinant Env monomers, a screen of 32 recombinant envelopes for binding to the CH01 to CH04 antibodies showed monoclonal antibody (MAb) binding to the E.A244 gp120 Env and to chronic Env AE.CM243; MAbs CH01 and CH02 also bound to transmitted/founder Env B.9021. CH01 to CH04 neutralized 38% to 49% of a panel of 91 HIV-1 tier 2 pseudoviruses, while the RUAs neutralized only 16% of HIV-1 isolates. Although the reverted unmutated ancestors showed restricted neutralizing activity, they retained the ability to bind to the E.A244 gp120 HIV-1 envelope with an affinity predicted to trigger B cell development. Thus, E.A244, B.9021, and AE.CM243 Envs are three potential immunogen candidates for studies aimed at defining strategies to induce V2/V3 conformational epitope-specific antibodies.

Telomeres are noncoding DNA regions at the end of the chromosomes that are crucial for genome stability. Since telomere length decreases with cell division, they can be used as a signature of cell proliferation history. T-cell reconstitution in severe combined immunodeficiency (SCID) subjects, recipients of T-cell-depleted, allogeneic-related bone marrow cells, is due to the development and maturation of donor T-cell precursors in the infant’s vestigial thymus and to homeostatic proliferation of mature T cells in the peripheral organs. Since T-cell function, thymic output, and T-cell clonal diversity are maintained long term in these patients, we investigated whether donor T-cell engraftment resulted in increased telomere shortening. Our study of seven SCID patients, following successful bone marrow transplantation, demonstrates that the patients’ peripheral T cells did not exhibit greater than normal telomere shortening.

Although mice associated with a single bacterial species have been used to provide a simple model for analysis of host-bacteria relationships, bacteria have been shown to display adaptability when grown in a variety of novel environments. In this study, changes associated with the host-bacterium relationship in mice monoassociated with Escherichia coli K-12 over a period of 1,031 days were evaluated. After 80 days, phenotypic diversification of E. coli was observed, with the colonizing bacteria having a broader distribution of growth rates in the laboratory than the parent E. coli . After 1,031 days, which included three generations of mice and an estimated 20,000 generations of E. coli , the initially homogeneous bacteria colonizing the mice had evolved to have widely different growth rates on agar, a potential decrease in tendency for spontaneous lysis in vivo , and an increased tendency for spontaneous lysis in vitro . Importantly, mice at the end of the experiment were colonized at an average density of bacteria that was more than 3-fold greater than mice colonized on day 80. Evaluation of selected isolates on day 1,031 revealed unique restriction endonuclease patterns and differences between isolates in expression of more than 10% of the proteins identified by two-dimensional electrophoresis, suggesting complex changes underlying the evolution of diversity during the experiment. These results suggest that monoassociated mice might be used as a tool for characterizing niches occupied by the intestinal flora and potentially as a method of targeting the evolution of bacteria for applications in biotechnology.

Monoclonal B-cell lymphocytosis (MBL) is a preclinical hematologic syndrome characterized by small accumulations of CD5(+) B lymphocytes. Most MBL share phenotypic characteristics with chronic lymphocytic leukemia (CLL). Although some MBL progress to CLL, most MBL have apparently limited potential for progression to CLL, particularly those MBL with normal absolute B-cell counts ('low-count' MBL). Most CLL are monoclonal and it is not known whether MBL are monoclonal or oligoclonal; this is important because it is unclear whether MBL represent indolent CLL or represent a distinct premalignant precursor before the development of CLL. We used flow cytometry analysis and sorting to determine immunophenotypic characteristics, clonality and molecular features of MBL from familial CLL kindreds. Single-cell analysis indicated four of six low-count MBL consisted of two or more unrelated clones; the other two MBL were monoclonal. 87% of low-count MBL clones had mutated immunoglobulin genes, and no immunoglobulin heavy-chain rearrangements of V(H) family 1 were observed. Some MBL were diversified, clonally related populations with evidence of antigen drive. We conclude that although low-count MBL share many phenotypic characteristics with CLL, many MBL are oligoclonal. This supports a model for step-wise development of MBL into CLL.

Bronchiolar stem cells have been functionally defined in vivo on the basis of their resistance to chemical (naphthalene) injury, their infrequent proliferation relative to other progenitor cell types, and their coexpression of the airway and alveolar secretory cell markers Clara cell secretory protein and pro-surfactant protein C, respectively. Cell surface markers that have previously been used for their prospective isolation included Sca-1 and CD34. Using transgenic animal models associated with stem cell expansion, ablation, and lineage tracing, we demonstrate that CD34pos cells do not belong to the airway epithelial lineage and that cell surface Sca-1 immunoreactivity does not distinguish between bronchiolar stem and facultative transit-amplifying (Clara) cell populations. Furthermore, we show that high autofluorescence (AFhigh) is a distinguishing characteristic of Clara cells allowing for the fractionation of AFlow bronchiolar stem cells. On the basis of these data we show that the defining phenotype of the bronchiolar stem cell is CD45neg CD31neg CD34neg Sca-llow AFlow. This refinement in the definition of bronchiolar stem cells provides a critical tool by which to assess functional and molecular distinctions between bronchiolar stem cells and the more abundant pool of facultative transit-amplifying (Clara) cells.

The death of CD4 + CCR5 + T cells is a hallmark of human immunodeficiency virus (HIV) infection. We studied the plasma levels of cell death mediators and products—tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), Fas ligand, TNF receptor type 2 (TNFR-2), and plasma microparticles—during the earliest stages of infection following HIV type 1 (HIV-1) transmission in plasma samples from U.S. plasma donors. Significant plasma TRAIL level elevations occurred a mean of 7.2 days before the peak of plasma viral load (VL), while TNFR-2, Fas ligand, and microparticle level elevations occurred concurrently with maximum VL. Microparticles had been previously shown to mediate immunosuppressive effects on T cells and macrophages. We found that T-cell apoptotic microparticles also potently suppressed in vitro immunoglobulin G (IgG) and IgA antibody production by memory B cells. Thus, release of TRAIL during the onset of plasma viremia (i.e., the eclipse phase) in HIV-1 transmission may initiate or amplify early HIV-1-induced cell death. The window of opportunity for a HIV-1 vaccine is from the time of HIV-1 transmission until establishment of the latently infected CD4 + T cells. Release of products of cell death and subsequent immunosuppression following HIV-1 transmission could potentially narrow the window of opportunity during which a vaccine is able to extinguish HIV-1 infection and could place severe constraints on the amount of time available for the immune system to respond to the transmitted virus.

IRG proteins, or immunity-related GTPases (also known as p47 GTPases), are a group of IFN-regulated proteins that are highly expressed in response to infection. The proteins localize to intracellular membranes including vacuoles that contain pathogens in infected macrophages and other host cells. Current data indicate that the IRG protein Irgm1 (LRG-47) is critical for resistance to intracellular bacteria. This function is thought to be a consequence of regulating the survival of vacuolar bacteria in host cells. In the current work, the role of Irgm1 in controlling resistance to Salmonella typhimurium was explored to further define the mechanism through which the protein regulates host resistance. Irgm1-deficient mice displayed increased susceptibility to this bacterium that was reflected in increased bacterial loads in spleen and liver and decreased maturation of S. typhimurium granulomas. The mice also displayed an inability to concentrate macrophages at sites of bacterial deposition. In vitro, the ability of Irgm1-deficient macrophages to suppress intracellular growth of S. typhimurium was impaired. Furthermore, adhesion and motility of Irgm1-deficient macrophages after activation with IFN-γ was markedly decreased. Altered adhesion/motility of those cells was accompanied by changes in cell morphology, density of adhesion-associated proteins, and actin staining. Together, these data suggest that in addition to regulating the maturation of pathogen-containing vacuoles, Irgm1 plays a key role in regulating the adhesion and motility of activated macrophages.

Chronic lymphocytic leukemia (CLL) results in the accumulation of mature immunologically defective lymphocytes in GO phase. Lymphocytes from CLL patients were exposed to UVC radiation to determine whether these cells are capable of undergoing apoptosis, as a response to DNA damage. Lymphocytes from CLL patients were found to be readily killed by ultraviolet light-C (UVC) radiation. Cells from healthy donors were minimally affected by doses of UVC ten times higher then those which caused dramatic drops in the metabolism of CLL cells. At four hours after irradiation, the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) had dropped by 50% for CLL cells exposed to a dose of 10 J/m2. In contrast, there was no significant drop for healthy cells exposed to 100 J/m2. Cell death was measured by trypan blue staining, flow cytometry of Annexin V-PI stained cells, and Wright staining. By 24 hours after irradiation, significant amounts of cell death were observed in CLL cells at doses which had no significant effects on viability of healthy lymphocytes. The extreme sensitivity of CLL lymphocytes to UVC indicates that phototherapy should be explored as a potential treatment for this neoplasm.

Background Antibody Dependent Cellular Cytotoxicity (ADCC) may be a contributing factor of immune responses controlling HIV-1 replication. Understanding the epitopes recognized by ADCC-mediating antibodies is likely to be important for the development of an effective AIDS vaccine. We characterized the epitope specificity and breadth of the ADCC-mediating antibody response elicited by the RV144 vaccine regimen.

Studying the effects of early HIV infection on human antibody responses, M. Anthony Moody and colleagues find rapid polyclonal B cell differentiation and structural damage to gut-associated lymphoid tissue., 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, Editors' Summary Background Acquired immunodeficiency syndrome (AIDS) has killed more than 25 million people since 1981 and more than 30 million people are now infected with the human immunodeficiency virus (HIV), which causes AIDS. HIV infects and kills a type of immune system cell called CD4+ T lymphocytes. These cells are needed to maintain a vigorous immune response, so people infected with HIV eventually become susceptible to other infections and develop full-blown AIDS. However, early during HIV infection, other parts of the immune system attempt to fight off the virus. Soon after infection, immune system cells called B lymphocytes begin to produce HIV-specific antibodies (proteins that recognize viral molecules called antigens). The first antibodies to HIV usually appear two to seven weeks after infection; from about 12 weeks after infection, antibodies are made that can kill the specific HIV type responsible for the infection (neutralizing antibodies). Why Was This Study Done? Unfortunately, by this time, it is too late for the antibody (“humoral”) immune response to clear HIV from the body. Indeed, the humoral immune response to HIV is very slow; for most viruses, neutralizing antibodies appear within days of infection. To help them design an effective HIV vaccine, scientists need to understand how the virus delays humoral responses to HIV infection (and how it later causes the production of HIV-specific antibodies to decline). Little is known, however, about the early effects of HIV infection on B lymphocytes. These cells are born and mature in the bone marrow. “Naïve” B lymphocytes, each of which carries an antigen-specific receptor (a protein that binds to a specific antigen), then enter the blood and circulate around the body, passing through the “peripheral lymphoid organs”. Exposure to antigens in these organs, which include lymph nodes and gut-associated lymphoid tissues, activates the subset of B lymphocytes that recognize the specific antigens that are present. Finally, with the help of activated T lymphocytes, the activated B lymphocytes proliferate and change (differentiate) into antibody-secreting cells and memory B lymphocytes (which respond more quickly to antigen than naïve B lymphocytes). In this study, the researchers investigate the effects of early HIV-1 infection on B lymphocytes in blood and in gut-associated lymphoid tissues. What Did the Researchers Do and Find? The researchers collected blood from patients as early as 17 days after HIV-1 infection and tissue samples from the lower portion of the small intestine (a region rich in gut-associated lymphoid structures called Peyer's patches) from 47 days after infection onward. When they analyzed the B lymphocytes in these samples (which were collected during two trials organized by the US Center for HIV/AIDS Vaccine Immunology [CHAVI]), they found that HIV-1 infection rapidly induced the activation of many different B cells that recognized a variety of antigens (polyclonal activation), as well as the appearance of differentiated B cells in blood and in gut-associated lymphoid tissue. The B lymphocytes that were activated in the gut made HIV-specific antibodies but also antibodies against unrelated antigens (such as flu virus proteins). Finally, the structure of Peyer's patches was altered early in HIV-1 infection. More specifically, most of the lymphoid follicles (organized collections of lymphocytes and antigen-presenting cells) in the Peyer's patches showed signs of damage and T- and B-lymphocyte death and the number of germinal centers (regions in lymphoid follicles in which B lymphocytes proliferate) was reduced. What Do These Findings Mean? Although the depletion of gut-associated CD4+ T lymphocytes in early HIV-1 infection is well known, these new results demonstrate the effects of early HIV-1 infection on gut-associated and circulating B lymphocytes. The results of this study are limited by the methods used to analyze the antibodies induced by HIV infection and by only taking tissue samples from one region of the gut. Nevertheless, the findings of polyclonal B-cell activation and damage to gut-associated lymphoid follicles soon after HIV-1 infection may have implications for HIV-1 vaccine design. Specifically, these findings suggest that an effective HIV-1 vaccine will need to ensure that significant levels of neutralizing antibodies are present in people before HIV-1 infection and that other protective immune defenses are fully primed so that, in the event of HIV-1 infection, the virus can be dealt with effectively before it disables any part of the immune system. Additional Information Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000107. Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS HIV InSite has comprehensive information on all aspects of HIV/AIDS, including an article about how HIV-1 infection affects the immune system Information is available from Avert, an international AIDS charity on many aspects of HIV/AIDS, including information on the stages of HIV infection, and on AIDS vaccines (in English and Spanish) The US Center for HIV/AIDS Vaccine Immunology (CHAVI) Web site provides information on research designed to solve major problems in HIV vaccine development and design

The effect of in ovo bursectomy on hatchability, mortality rate, and antibody response to keyhole limpet hemocyanin (KLH) was studied in hypertensive and hypotensive lines of turkeys. Experiments were conducted to assess the optimal time to perform surgical in ovo bursectomy in turkey embryos for the lowest mortality rate. Factors such as the ease of operation, hatchability, and least deaths were considered. Surgical bursectomies at 25, 24, 23, and 22 day embryonation resulted in 75, 20, 0, and 10% hatchability, respectively. The surgical procedure performed at 25-day embryonation resulted in superior hatchability and ease of operation, and it was less traumatic as evidenced by fewer deaths. The hatchability of embryos injected with testosterone (TE) at 15 days incubation was 55%. Birds subjected to surgery or chemical treatment had increased mortality rates. Cyclophosphamide (CY) treatment at 1 and 2 days posthatch caused a significant increase in deaths over groups not treated with CY. Both primary and secondary KLH responses were significantly lower in bursectomized and TE-injected groups, than in shamoperated or nonoperated control groups.

Background: Monoclonal B cell lymphocytosis (MBL) is a pre-clinical syndrome characterized by small accumulations of monoclonal B lymphocytes in the peripheral blood. MBL have an immunophenotype similar to CLL: CD5+, CD19+, CD20lo, CD23+, CD79blo, sIglo. The biologic characteristics and clinical implications of MBL remain unclear. We hypothesized that MBL have a biology similar to CLL and represent a pre-neoplastic state of CLL. Characterization and longitudinal evaluation of MBL may provide insight into mechanisms of CLL leukemogenesis. Methods: We defined MBL as a population of CD5+, CD20lo cells that were kappa/lambda restricted and comprised at least 2% of the CD19+ peripheral B cell compartment. Persons with MBL were ascertained by flow cytometric screening of peripheral blood from unaffected members of CLL kindreds. Flow cytometric analysis and fluorescence-activated cell sorting (FACS) were used to determine the surface immunophenotype and molecular characteristics of MBL. MBL cells were bulk-sorted and analyzed using FISH for loci associated with clinical CLL [13q14.3 (D13S319), 17p13.1 (TP53), 11q22.3 (ATM), and 12p11.1-q11 (enumeration probe)]. Single MBL cells were sorted and genomic DNA analyzed by PCR to determine immunoglobulin heavy and light chain sequences. Results: Twelve out of 113 (11%) unaffected family members were found to have MBL. Seven of these individuals provided additional blood for MBL characterization. All 7 MBL subjects had normal complete blood counts and leukocyte differentials. Six of the 7 MBL subjects had MBL counts less than 150 cells/μL. All MBL samples had immunophenotypes similar to CLL. One MBL sample was CD38+/Zap70+, one was CD38+/Zap70−, one was CD38−/Zap70+, and 4 samples were CD38−/Zap70−. The ratio of median fluorescence intensity of CD69:CD71 was low ( Conclusions: This cohort of familial MBL recapitulated much of the biologic diversity of clinical CLL. By several different phenotypic measures, the MBL phenotype was similar to typical CLL. To our knowledge, this is the first report of CD38+, Zap70+, and IgVH unmutated MBL. In contrast to most CLL, we found that many MBL consisted of more than one clonal population. Likewise, we identified evidence of ongoing antigen-driven intraclonal diversification in MBL.

Background: Segregation analyses suggest that CLL has one of the highest inherited risks among all types of cancer. However, no common germline mutations that confer an inherited predisposition to CLL have been found. Identification of a CLL predisposition gene should lead to improved prevention and treatment strategies. Gene identification studies to date have been limited by few affected patients per family, late age of disease onset, and probable genetic heterogeneity. Identification of a phenotypic marker for carriers of a CLL predisposition gene would dramatically improve the likelihood of finding a causative gene. Monoclonal B-cells with typical CLL immunophenotype (monoclonal B-cell lymphocytosis, MBL) can be identified in normal relatives of patients with overt CLL. We hypothesize that MBL identify unaffected carriers of a CLL predisposition gene. Methods: We used 6-color flow cytometric methods to analyze peripheral blood samples for MBL. We defined MBL as a population of CD5+, CD20lo cells that were kappa/lambda restricted and comprised at least 2% of the CD19+ peripheral B cell compartment. We identified extended families segregating 2 or more members with CLL from patients receiving care at Duke and the Durham VA. We collected peripheral blood from both affected and unaffected family members for complete blood count, flow cytometry for MBL detection, and DNA purification. Genotyping studies were performed using PCR to amplify highly polymorphic short tandem repeat sequences (STR) at candidate loci. Results: We ascertained 16 extended families with CLL and obtained samples from 138 family members. Twelve of the 113 (11%) unaffected participating family members had MBL. In one family, evaluation of 32 members revealed 3 with overt CLL and 5 with MBL. Two distinct inheritance patterns emerge upon review of the families ascertained and analyzed to date. Five families suggested a dominant mode of inheritance of the combined CLL/MBL phenotype: multiple family members with MBL, vertical transmission, and an approximate 50% affection rate for among persons over age 40. In these 5 families, 11 of 64 (17%) unaffected family members had MBL while in the other 11 families no unaffected family members had MBL (0 of 48). In some of the latter 11 families, analysis of obligate carriers of a CLL predisposition gene showed no MBL. We pooled the 5 families with MBL for screening of candidate loci associated with an inherited predisposition to CLL from previously published linkage studies. Using markers at these candidate loci, we excluded linkage to 5q22.1, 6p22, 9q21.33, 10q24.1, 11q13.1, 13q22.2, and 14q32.2. Conclusions: A combined MBL/CLL phenotype appears to be inherited as a dominant trait in some cases of familial CLL. This supports the hypothesis of genetic heterogeneity in the development of CLL. A familial analysis of both CLL and MBL greatly increases the statistical power for genetic analysis, and may allow for families to be pooled for genetic analysis based on the MBL phenotype. Gene identification studies are ongoing.

Background Rheumatoid arthritis (RA) and its accepted animal model, murine collagen-induced arthritis (CIA), are classic autoimmune inflammatory diseases which require proinflammatory cytokine production for pathogenesis. We and others have previously used N, N-dimethylglycine (DMG) and extracts from the New Zealand green-lipped mussel Perna canaliculus (Perna) as potent immunomodulators to modify ongoing immune and/or inflammatory responses. Methods In our initial studies, we treated lipopolysaccahride (LPS) stimulated THP-1 monocytes in vitro with increasing concentrations of Perna extract or DMG. Additionally, we treated rat peripheral blood neutrophils with increasing concentrations of Perna extract and measured superoxide burst. In subsequent in vivo experiments, CIA was induced by administration of type II collagen; rats were prophylactically treated with either Perna or DMG, and then followed for disease severity. Finally, to test whether Perna and/or DMG could block or inhibit an ongoing pathologic disease process, we induced CIA in mice and treated them therapeutically with either of the two immunomodulators. Results Following LPS stimulation of THP-1 monocytes, we observed dose-dependent reductions in TNF-α and IL-12p40 production in Perna treated cultures. DMG treatment, however, showed significant increases in both of these cytokines in the range of 0.001–1 μM. We also demonstrate that in vitro neutrophil superoxide burst activity is dose-dependently reduced in the presence of Perna. Significant reductions in disease incidence, onset, and severity of CIA in rats were noted following prophylactic treatment with either of the two immunomodulators. More importantly, amelioration of mouse CIA was observed following therapeutic administration of Perna. In contrast, DMG appeared to have little effect in mice and may act in a species-specific manner. Conclusion These data suggest that Perna, and perhaps DMG, may be useful supplements to the treatment of RA in humans.

Background In acute HIV-1 infection (AHI) there are infectioninduced polyclonal shifts in blood and bone marrow Bcell subsets from naïve to memory cells and plasmablasts/ plasma cells (PCs) coupled with decreased numbers of naive B cells. To study the initial antibody response to HIV, we have used recombinant technology to create a database of PC antibody sequences derived from 3 early stage AHI subjects.

Methods Blood PCs from three AHI subjects obtained approximately 17, 20 and 30 days after HIV-1 transmission were sorted into 96-well plates for amplification of VH and VL genes by RT/PCR. The isolated VH and VL genes were expressed as recombinant IgG1 mAbs in 293T cells by transfection using linear Ig expression cassettes, The specificity of produced mAbs was determined by ELISA and luminex bead immunoassays against a panel of HIV-1 and non-HIV-1 antigens.

Background HIV-1 vaccines must induce protective antibodies at mucosal surfaces; the role of IgA in protection remains unknown. The HIV-1 Env antibody response begins ~day 17 after transmission, and derives from a polyreactive memory B cell pool of gut flora-reactive IgG1 and IgA B cells. Whereas the IgG Env antibody response persists years after acute HIV-1 infection, the initial IgA response decreases over the first month. There is also selective destruction of terminal ileum germinal centers in early HIV-1 infection (EHI). To determine HIV-1 IgA responses in gut, we isolated Env-reactive antibodies from ileum from patients in EHI and chronic HIV-1 infection (CHI).