Enhanced Antibody Production Research Articles

Cystine and theanine: amino acids as oral immunomodulative nutrients

The decreases in the glutathione (GSH) level in the mouse spleen and liver after immune stimulation are suppressed by the oral administration of cystine and theanine (CT). GSH is considered to be important for the control of immune responses. Antibody production in mice after infection is enhanced by the oral administration of CT. In humans, also, the oral administration of CT has been confirmed to enhance antibody production after vaccination against Flu and also reduce the incidence of cold. However, the GSH level is reduced by intense exercise and surgery. In clinical studies of body-builders and long-distance runners, the intake of CT suppressed excessive inflammatory reactions and a decline in immune functions after intense training. Surgery as well as intense exercise induces excessive inflammatory reactions. In mice, the preoperative administration of CT suppressed excessive inflammatory reactions associated with surgery and promoted the postoperative recovery. Moreover, in clinical studies of gastrectomized patients, CT intake suppressed excessive postoperative inflammatory reactions and induced early recovery. If infection is regarded as invasive stress, CT intake is considered to exhibit an immunomodulatory effect by suppressing the decrease in GSH due to invasive stress. The clarification of their detailed action mechanisms and their application as medical or function foods is anticipated.

Comparison of the immunogenicity of botulinum toxin type A and the efficacy of A1 and A2 neurotoxins in animals with A1 toxin antibodies

One issue with botulinum toxin type A products is a reduced therapeutic response in patients that have been injected with frequent dosing over a prolonged period. A possible cause of this is hemagglutinin, found in progenitor toxins, displaying adjuvant activity, enhancing antibody production against the toxin. We investigated whether there is any difference in immunogenicity between the LL toxin-derived subtype A1 (A1LL) and the neurotoxin-derived subtypes A1 and A2 (A1NTX and A2NTX, respectively), and investigated whether A2NTX is effective in animals which produce antibodies against A1LL.Neutralizing antibodies were detected in the A1LL-administered group; however, they were not detected in swine and rabbits administered multiple doses of A2NTX. These results indicate that A2NTX has a lower immunogenicity than A1LL. In rats with neutralizing antibodies, produced by the administration of A1LL, that were administered either A1NTX or A2NTX, A2NTX showed more potent inhibitory neuromuscular transmission than A1NTX. In human sera immunized with the botulinum toxoid vaccine (containing LL, L, and M toxoid derived subtype A1) reacted with either A1NTX or A2NTX, A2NTX showed more potent inhibitory neuromuscular transmission than A1NTX. This suggests that A2NTX has a greater therapeutic value in humans who have neutralizing antibodies against the A1 toxin.

Effect of culture pH on recombinant antibody production by a new human cell line, F2N78, grown in suspension at 33.0 °C and 37.0 °C

The human host cell line, F2N78, is a new somatic hybrid cell line designed for therapeutic antibody production. To verify its potential as a human host cell line, recombinant F2N78 cells that produce antibody against rabies virus (rF2N78) were cultivated at different culture pH (6.8, 7.0, 7.2, 7.4, and 7.6) and temperatures (33.0°C and 37.0°C). Regardless of the culture temperature, the highest specific growth rate was obtained at a pH of 7.0-7.4. Lowering the culture temperature from 37.0°C to 33.0°C suppressed cell growth while allowing maintenance of high cell viability for a longer period. However, it did not enhance antibody production because specific antibody productivity did not increase at 33.0°C. The highest maximum antibody concentration was obtained at 37.0°C and pH6.8. The N-linked glycosylation of the antibody was affected by the culture pH rather than the temperature. Nevertheless, G1F was dominant and G2F occupied a larger portion than G0F in all culture conditions. Compared to the same antibody produced from recombinant CHO cells, the antibody produced from rF2N78 cells has more galactose capping and was more similar to human plasma IgG. Taken together, the results obtained here demonstrate the potential of F2N78 as an alternative human host cell line for therapeutic antibody production.

Immunoprophylactic Effects of Administering Honeybee (<i>Apis melifera</i>) Venom Spray against <i>Salmonella</i> Gallinarum in Broiler Chicks

ABSTRACTAntibiotics continue to be used as growth promoters in the poultry industry. Honeybee (Apis melifera) venom (HBV) possesses a number of beneficial biological activities, particularly for regulating the immune system. The aim of the present study was to evaluate the immunoprophylactic effects of HBV against Salmonella Gallinarum in broiler chicks as an initial step towards developing eco-friendly alternatives to reduce antibiotic use. HBV was administered using a spray technique. HBV improved body weight gain, particularly in the presence of infection. Moreover, HBV enhanced antibody production activity against formalin-killed S. Gallinarum. The CD4+:CD8+ T lymphocyte ratio, relative mRNA expression levels of interleukin-18 and interferon-γ, and serum lysozyme activity also increased following HBV administration before the infection period as well as during infection. HBV reinforced bacterial clearance and increased survivability against S. Gallinarum. Corresponding pathological analyses demonstrated that the HBV-sprayed group displayed mild and less severe abnormal changes compared with those in the control group. It was presumed that the prophylactic effects of HBV against S. Gallinarum were associated with its non-specific immune response stimulating activity. Thus, HBV may provide an alternative to reduce antibiotic use in the poultry industry.

Prolactin Levels Correlate with Abnormal B Cell Maturation in MRL and MRL/lpr Mouse Models of Systemic Lupus Erythematosus-Like Disease

Prolactin (PRL) plays an important role in modulating the immune response. In B cells, PRL enhances antibody production, including antibodies with self-specificity. In this study, our aims were to determine the level of PRL receptor expression during bone-marrow B-cell development and to assess whether the presence of high PRL serum concentrations influences absolute numbers of developing populations and disease outcome in lupus-prone murine models. We observed that the PRL-receptor is expressed in early bone-marrow B-cell; the expression in lupus-prone mice, which had the highest level of expression in pro-B cells and immature cells, differed from that in wild-type mice. These expression levels did not significantly change in response to hyperprolactinemia; however, populations of pro-B and immature cells from lupus-prone strains showed a decrease in the absolute numbers of cells with high PRL-receptor expression in response to PRL. Because immature self-reactive B cells are constantly being eliminated, we assessed the expression of survival factor BIRC5, which is more highly expressed in both pro-B and immature B-cells in response to PRL and correlates with the onset of disease. These results identify an important role of PRL in the early stages of the B-cell maturation process: PRL may promote the survival of self-reactive clones.

Ex Vivo Enzymatic Treatment of Aged CD4 T Cells Restores Cognate T Cell Helper Function and Enhances Antibody Production in Mice

Previous in vitro studies showed that CD4 T cells from old mice have defects in TCR signaling, immune synapse formation, activation, and proliferation. We reported that removing a specific set of surface glycoproteins by ex vivo treatment with O-sialoglycoprotein endopeptidase (OSGE) can reverse many aspects of the age-related decline in CD4 T cell function. However, the specific mechanism by which this process occurs remains unclear, and it is unknown whether this enzymatic treatment can also restore important aspects of adaptive immunity in vivo. By using an in vivo model of the immune response based on adoptive transfer of CD4 T cells from pigeon cytochrome C-specific transgenic H-2(k/k) TCR-Vα(11)Vβ(3) CD4(+) mice to syngeneic hosts, we demonstrate that aging diminishes CD28 costimulatory signals in CD4 T cells. These age-associated defects include changes in phosphorylation of AKT and expression of glucose transporter type I, inducible T cell costimulatory molecule, and CD40L, suggesting that the lack of CD28 costimulation contributes to age-dependent loss of CD4 function. All of these deficits can be reversed by ex vivo OSGE treatment. Blocking B7-CD28 interactions on T cells prevents OSGE-mediated restoration of T cell function, suggesting that changes in surface glycosylation, including CD28, may be responsible for the age-related costimulation decline. Finally, we show that the age-related decline in CD4 cognate helper function for IgG production and long-term humoral immunity can also be restored by OSGE treatment of CD4 T cells prior to adoptive transfer.

A Rational Design for the Nanoencapsulation of Poisonous Animal Venoms in Liposomes Prepared with Natural Phospholipids

Liposomes have been used since the 1970's to encapsulate drugs envisaging enhancement in efficacy and therapeutic index, avoidance of side effects and increase in the encapsulated agent stability. The major problem when encapsulating snake venoms is the liposomal membrane instability caused by venom phospholipases. Here the results obtained encapsulating Crotalus durissimus terrificus and a pool of Bothropic venoms within liposomes (LC and LB, respectively) used to produce anti-venom sera are presented. The strategy was to modify the immunization protocol to enhance antibody production and to minimize toxic effects by encapsulating inactivated venoms within stabilized liposomes. Chemically modified venoms were solubilized in a buffer containing an inhibitor and a chelating agent. The structures of the venoms were analyzed by UV, CD spectroscopy and ELISA. In spite of the differences in the helical content between natural and modified venoms, they were recognized by horse anti-sera. To maintain long-term stability, mannitol was used as a cryoprotectant. The encapsulation efficiencies were 59 % (LB) and 99 % (LC), as followed by filtration on Sephacryl S1000. Light scattering measurements led us to conclude that both, LB (119 ±47 nm) and LC (147±56 nm) were stable for 22 days at 4 °C, even after lyophilization. Genetically selected mice and mixed breed horses were immunized with these formulations. The animals did not show clinical symptoms of venom toxicity. Both, LB and LC enhanced by at least 30 % the antibody titers 25 days after injection and total IgG titers remained high 91 days after immunization. The liposomal formulation clearly exhibited adjuvant properties.

Cell-Penetrating Peptide-Linked Polymers as Carriers for Mucosal Vaccine Delivery

We evaluated the potential of poly(N-vinylacetamide-co-acrylic acid) modified with d-octaarginine, which is a typical cell-penetrating peptide, as a carrier for mucosal vaccine delivery. Mice were nasally inoculated four times every seventh day with PBS containing ovalbumin with or without the d-octaarginine-linked polymer. The polymer enhanced the production of ovalbumin-specific immunoglobulin G (IgG) and secreted immunoglobulin A (IgA) in the serum and the nasal cavity, respectively. Ovalbumin internalized into nasal epithelial cells appeared to stimulate IgA production. Ovalbumin transferred to systemic circulation possibly enhanced IgG production. An equivalent dose of the cholera toxin B subunit (CTB), which was used as a positive control, was superior to the polymer in enhancing antibody production; however, dose escalation of the polymer overcame this disadvantage. A similar immunization profile was also observed when ovalbumin was replaced with influenza virus HA vaccines. The polymer induced a vaccine-specific immune response identical to that induced by CTB, irrespective of the antibody type, when its dose was 10 times that of CTB. Our cell-penetrating peptide-linked polymer is a potential candidate for antigen carriers that induce humoral immunity on the mucosal surface and in systemic circulation when nasally coadministered with antigens.

Identification of antibody-interacting proteins that contribute to the production of recombinant antibody in mammalian cells

Protein folding and assembly processes are essential for antibody secretion; however, the endogenous proteins involved in these processes remain largely unknown. Therefore, except for some well-known endoplasmic reticulum (ER) chaperones such as GRP78/Bip and protein disulfide isomerase, enhancement of recombinant antibody expression by co-expression of interacting proteins has been largely elusive. Here, in addition to known ER chaperones, we identified additional endogenous proteins that interact with recombinant antibody in mammalian cells by immunoprecipitation coupled with liquid chromatography-tandem mass spectrometry. Most of our identified proteins enhanced antibody production, and furthermore, some of their combinations resulted in greater enhancement. In particular, eukaryotic initiation factor 4A combined with other proteins had approximately fourfold higher effect on antibody production. Identified proteins that could improve antibody expression contain not only ER-resident proteins like GRP78/Bip but also non-ER-resident proteins. These results suggest that this method could be effective in the investigation of novel proteins that are involved in enhancing recombinant antibody production because immunoprecipitation coupled with mass spectroscopy could identify proteins which directly interact with the antibody.

Teleost IL-6 promotes antibody production through STAT3 signaling via IL-6R and gp130

Teleost IL-6 is upregulated after antigen stimulation; therefore, we hypothesized that fish IL-6 contributes to antibody production during immune responses against infections. To verify this hypothesis, we first cloned IL-6R and gp130 in fugu (Takifugu rubripes) in the present study. The membrane and soluble forms of IL-6R were identified by the identification of cDNA clones of IL-6R homologues. Three STAT3-docking sites were found in the intracellular region of fugu gp130. Expression analysis showed that fugu IL-6R and gp130 were expressed in mIgM+ B cells, suggesting that fugu B cells are stimulated by IL-6. Recombinant fugu IL-6 (rfIL-6) increased the gene expression of secretory antibodies by mIgM+ B cells in vitro. The rfIL-6 and soluble form of rfIL-6R activated STAT3 phosphorylation in the B cells and a cultured cell line transfected with fugu gp130. These results indicate that fugu IL-6 enhances antibody production in the B-cell lineage via gp130 and STAT3 signaling.

Identification of Immunopotentiating Lactic Acid Bacteria that Induce Antibody Production by <i>in vitro</i> Stimulated Human Peripheral Blood Mononuclear Cells

L-leucyl-L-leucine methyl ester (LLME) is known to remove lysosome-rich cells from human peripheral blood mononuclear cells (PBMCs). To evaluate the immunopotentiating ability of lactic acid bacteria (LAB), we adopted the in vitro stimulation protocol of LLME-treated PBMCs as a model assay system and monitored the level of antibody produced by stimulated PBMCs. The results indicated that several LAB strains have immunopotentiating ability against PBMCs, as evidenced by the enhanced antibody production and increased number of antigen-specific B cells. Next, we identified T cells as the direct target cells of the immunopotentiating LAB strain L32, suggesting that L32 induced antibody production by PBMCs through T-cell activation. Finally, we tested the immunopotentiating ability of ligands for Toll-like receptor 2 (TLR2), which is known to mediate the LAB signal, and observed that both L32 and one of the TLR2 ligands, LTA-BS, induced antigen-specific antibody production by in vitro stimulated PBMC. This suggests that L32 and LTA-BS can be used as an adjuvant for stimulating immune reaction in PBMCs.

Antibody responses initiated by Clec9A-bearing dendritic cells in normal and Batf3−/− mice

Injection of antigens coupled to antibodies against the dendritic cell (DC) surface molecule Clec9A has been shown to produce strongly enhanced antibody responses even without co-administration of adjuvants, via antigen presentation by DC on MHC class II and consequent production of follicular helper T cells. A series of mutant mice were tested to determine the DC subtypes responsible for this MHC II presentation of targeted antigen, compared to presentation of antigen on MHC I. A new clec9A null mouse was developed; these mice did not give enhanced antibody production, confirming the response was dependent on Clec9A-expressing DC. However targeting of antigen to Clec9A in batf3 null mice produced enhanced antibody responses despite the marked reduction in CD8+ DC, the major Clec9A-expressing DC subtype. This was shown to be dependent on efficient MHC II presentation by minor Clec9A-expressing DC subtypes in the environment of the Batf3−/− mice, namely early cells of the CD8 DC lineage and the plasmacytoid-related CD8+ DC subset, but not by plasmacytoid cells themselves. However in normal mice most MHC II presentation of the Clec9A-targeted antigen was by the major CD8+ DC population, the DC also responsible for presentation on MHC I.

Chemotypical variations in Withania somnifera lead to differentially modulated immune response in BALB/c mice

Withania somnifera (Ashwagandha) is a plant with known ethnomedicinal properties and its use in Ayurvedic medicine in India is well documented. The present investigation reports on immunomodulatory efficacy of aqueous-ethanol extracts of roots of three selected Withania somnifera chemotypes designated as NMITLI 101R, NMITLI 118R and NMITLI 128R. Each chemotype was administered 10-100 mg/kg orally to BALB/c mice once daily for 14 days. The immunomodulatory consequences were recorded by determining the humoral immune response with the help of hemagglutination, plaque forming cell assay and cellular response by measuring delayed type hypersensitivity reaction. Additionally, other immune parameters such as proliferation of T and B cells, intracellular and secreted Th1 and Th2 cytokines along with modulation in ROS production by peritoneal macrophages were monitored after feeding with lower doses (3-30 mg/kg/day) of these three chemotypes individually. NMITLI 101R incited both humoral and cellular immune response in terms of higher number of antibody producing cells and enhanced foot pad swelling at the 10mg dose as also dose dependent B and T cell proliferations. Levels of intracellular and secreted cytokines post-NMITLI 101R treatment illustrated generation of mixed Th1/Th2 response that remained more polarized towards Th1. This chemotype also generated maximum reactive oxygen species. NMITLI 118R provoked comparatively reduced immune response in all humoral and cellular parameters at lower doses but induced highly polarized Th1 cytokine response. In contrast, NMITLI 128R led to enhanced antibody production with minimal cellular response demonstrating marginally Th2 dominance at a lower dose. Taken together, it may therefore be concluded that there were distinct modulation in the immune response exhibited by the three chemotypes of Withania somnifera and NMITLI 101R appeared to possess a better immunostimulatory activity than the other chemotypes at lower doses.

Sustained Antibody Responses Depend on CD28 Function in Bone Marrow Resident Plasma Cells

Abstract 182Protective immunity against infection requires sustained antibody production by long-lived plasma cells (LLPC) that survive for years/decades within specialized niches. What regulates/supports this survival remains largely unknown. However, it has been shown that normal and transformed (human multiple myeloma) LLPC are critically dependent on the bone marrow microenvironment, including cell-to-cell interactions. This lead us to hypothesize that modulating these interactions could either enhance antibody production for vaccine development or, conversely, compromise the survival of transformed/normal LLPC in the bone marrow microenvironment. We have shown that the T cell costimulatory receptor CD28 expressed on both normal and transformed LLPC, plays an essential role in survival. While LLPC and short-lived plasma cells (SLPC) both express CD28, its activation in vitro only significantly increases survival and IgG production in LLPC. Consistent with these findings, we show in vivo, vaccinated bone marrow CD28−/−:μMT chimeras had significantly reduced long-term antibody titers and decreased LLPC (but not SLPC) t1/2 from 426 to 63 days. These findings demonstrate the existence of a distinct bone marrow (BM) LLPC subset necessary to sustain antibody titers, and establish a central role for CD28 function in the maintenance of plasma cells and humoral immunity.While CD28 signaling has been shown to play an important role in maintaining long-term humoral immune responses, the mechanism by which CD28 signaling affects PC function has not yet been determined. To further elucidate CD28 signaling in BM PC, we utilized CD28 conditional knock-in mice. In these mice, the CD28 cytoplasmic tail is mutated at either the YMNM or proline-rich motifs, resulting in an inhibition of PI3K or vav signaling, respectively. We found that CD28-vav signaling deficient BM PC were selectively depleted in vivo and could not be rescued by CD28 activation in in vitro serum starvation conditions. Furthermore, anti-CD28 mAb drove a 1.5 fold increase in Blimp-1 expression in BM PC, compared to control. This increase was regulated through the CD28-vav signaling pathway, as CD28 activation in CD28-vav signaling deficient BM PC did not increase Blimp-1 expression. To further determine if CD28 is acting directly on the Blimp-1 promoter, we examined in silico for a CD28RE composite element, previously reported to transcriptionally regulate IL-2 production in T cells and IL-8 production in myeloma cells. To our surprise, we found a CD28RE “like” site 4712bp upstream of the Blimp-1 start site. To confirm CD28 transcriptionally regulates Blimp-1 promoter activity, we transfected the CD28+ plasmacytoma cell line J558 with full-length or truncated Blimp-1 promoter constructs (i.e. 7000bp, 4500bp, 1500bp). We found CD28 activation enhances Blimp-1 activity in J558 cells transfected with full-length-Blimp-1, and this activity was lost when the promoter was truncated. Using site-directed mutagenesis, we confirmed the CD28RE is required for induction of Blimp-1 in PC. Furthermore, we show CD28 activation of Blimp-1 increases the BCMA receptor in BM PC. Taken together, our data suggests the CD28-vav signaling pathway in PC induces a CD28RE composite element, which is necessary for the induction of the key PC transcriptional regulator Blimp-1, required to maintain LLPC and humoral immunity. Disclosures:No relevant conflicts of interest to declare.

New Hepatitis B Vaccine Schedule for Children with Celiac Disease

Dear Editor, The article by Ertekin et al. published in Hepatitis Monthly indicates that a new hepatitis B virus (HBV) vaccine schedule should be created for children with celiac disease (CD), and the response of all children with CD to the HBV vaccine should be routinely investigated [1]. The authors investigated a group of children with CD vaccinated against HBV and found that the anti-HBs titer was not detected in a significant percentage (38.5%) of children. In addition, the clinical presentation of the disease and dietary compliance of the patients seemed to play a role in nonresponsiveness to the HBV vaccine. Therefore, the authors suggest a different immunization schedule. Most studies indicate that major histocompatibility complex (MHC) and human leukocyte II antigens (HLA-II) are responsible for nonresponsiveness to HBV vaccine [2][3][4][5]. However, Nemes et al. [6] found that the percentage of seroconversion (95.5%) in a group of patients vaccinated after initiating dietary treatment was similar to that in healthy individuals, and overall, 36 out of 37 nonresponders consuming a controlled gluten-free diet showed seroconversion after administering a booster dose of vaccine. This finding shows that HLA DQ alleles do not have a primary role in nonresponsiveness to HBV vaccine. The nature of protection afforded by vaccination is complex and, although both humoral and cytotoxic T-cell responses play a role, long-term protection is dependent upon immunological memory. The general belief is that immunological memory persists for a long period after natural infection and administration of live vaccines. For many years, the response to HBV vaccination is determined by measuring the anti-HBs titer, and a patient with an anti-HBs level < 10 mIU/mL is considered to be a nonresponder. However, responsiveness to HBV vaccination is usually determined by testing within 2-6 months after receiving the third dose of the vaccine. Thus, the nonresponsiveness to HBV vaccine might be secondary to the tendency of older individuals having a weaker response to the vaccine or not developing immunity. In patients with anti-HBs level < 10 IU/L, a schedule for booster vaccinations should be proposed, but no consensus has been achieved on the management of nonresponders to date. A high dose of HB vaccine is administered in healthy individuals, but this procedure is not uniformly successful [7]. Recently, 90% seroconversion was observed in a pilot study in patients after administration of a booster dose of vaccine by the intradermal (ID) route [8]. Large prospective randomized studies are required to validate this procedure to improve the efficacy and safety of the current standard protocol. The mechanism of induction of immunogenicity via intradermal administration is not clearly established but may be related to the sequestration of the antigens in the dermis for a long time after intradermal injection. The dermis contains large numbers of dendritic cells (the most potent antigen-presenting cells) that are important for the ID route of immunization, and these cells are thought to induce cell-mediated immune responses, particularly CD4+ and CD8+ T-cell responses. In addition, dendritic cells enhance antibody production by B cells through efficient induction of CD4+ T-cell modulation of B cells and thus a lower antigen dose is required to produce an immune response.

Lactobacillus rhamnosus GG secreting an antigen and Interleukin‐2 translocates across the gastrointestinal tract and induces an antigen specific immune response

Lactobacillus rhamnosus strain GG (LGG) is a probiotic organism. In this present study, LGG that express the green fluorescence protein (LGG-GFP) and IL-2 and GFP as a fusion protein (LGG-IL-2-GFP) were used to examine bacterial uptake and the immune response induced by oral immunization. Using TEM to examine the intestinal tissue, the Lactobacilli were localized in M cells and in venules. After oral immunization, most of the bacteria were excreted in feces only a small fraction (0.15%) was retained in the intestine at 48 hr. However, more LGG-IL-2-GFP was found in the MLN and spleen than LGG-GFP. The loop ligation method was used to evaluate LGG uptake and both LGG-GFP and LGG-IL-2-GFP were found to translocate at the same rate. Analysis of LGG internalization in J774 macrophage cells indicated that IL-2 increased survival of LGG and this may explain the increased presence of these bacteria in the MLN for a longer period. After oral immunization, specific mucosal antibody production as well as GFP specific CTL activity was demonstrated. IL-2 co-expression with GFP further enhanced antibody production and CTL activity. In conclusion, Lactobacillus rhamnosus GG expressing an antigen could generate an effective immune response to the antigen and IL-2 improved the response generated probably by increasing LGG expressing antigen survival in immune cells.

Growth factor withdrawal in combination with sodium butyrate addition extends culture longevity and enhances antibody production in CHO cells

The effect of growth factor (GF) and sodium butyrate (NaBu) on Chinese hamster ovary (CHO) cell growth, cell viability and antibody production was investigated using shaking flasks in GF-containing and GF-deficient medium containing 0, 1 and 3mM NaBu. The withdrawal of GF and the addition of NaBu suppressed cell growth, but they significantly increased specific antibody productivity, q(Ab). Interestingly, the withdrawal of GF in combination with the addition of NaBu markedly retarded cell death, leading to extended culture longevity. For instance, at 3mM NaBu, cell viability fell below 80% after day 4 in GF-containing medium, but it remained over 80% until day 18 in GF-deficient medium. Due to the enhanced q(Ab) and the extended culture longevity, approximately 2-fold increase in total antibody production was achieved in pseudo-perfusion culture with 1mM NaBu in GF-deficient medium, compared to the culture in GF-containing medium. The effect of GF and NaBu on the change in the expression and activity of cellular proteins, c-Myc, Bcl-2 and pyruvate dehydrogenase (PDH), was also investigated. Both the withdrawal of GF and the addition of NaBu decreased the expression of c-Myc. The expression of Bcl-2 was enhanced by the addition of NaBu in a dose-dependent manner while it was not affected by the withdrawal of GF. In addition, both the withdrawal of GF and the addition of NaBu reduced metabolic rates, q(Glc), q(Lac) and Y(Lac/Glc), and increased PDH activity while not affecting PDH expression, suggesting that they may reduce the glycolytic rates, but enhance the conversion rates of pyruvate to TCA intermediates. Taken together, the withdrawal of GF in combination with the addition of NaBu can be considered as a relevant strategy for alleviating NaBu-induced cell apoptosis and enhancing antibody production since it can be easily implemented as well as enhance q(Ab) and extend culture longevity.

Autophagy and apoptosis of recombinant Chinese hamster ovary cells during fed‐batch culture: Effect of nutrient supplementation

Upon nutrient depletion during recombinant Chinese hamster ovary (rCHO) cell batch culture, cells are subjected to apoptosis, type I programmed cell death (PCD), and autophagy which can be type II PCD or a cell survival mechanism. To investigate the effect of nutrient supplementation on the two PCDs and protein production in rCHO cells, an antibody-producing rCHO cell line was cultivated in batch and fed-batch modes. The feed medium containing glucose, amino acids, and vitamins was determined through flask culture tests and used in bioreactor cultures. In the bioreactor cultures, the nutrient feedings extended the culture longevity and enhanced antibody production. In addition, cells in the fed-batch culture showed delayed onset of both apoptosis and autophagy, compared with those in the batch culture. The inhibition of apoptosis was demonstrated by a decreased amount of cleaved caspase-7 protein and less fragmentation of chromosomal DNA. Concurrently, reduced LC3 conversion, from LC3-I to LC3-II, was observed in cells that received the feeds. Cultivation with pharmacological autophagy inducer (rapamycin) or inhibitor (bafilomycin A1) indicated that autophagy is necessary for the cells to survive under nutrient depletion. Taken together, the delayed and relieved cell death by nutrient supplementation could improve antibody production.

Long Term Humoral Immunity Is Dependent on CD28 Expression In Plasma Cells

AbstractAbstract 1737Protective immunity against infection requires sustained antibody production by long-lived plasma cells (LLPC) that survive for years/decades within specialized niches. What regulates/supports this survival remains largely unknown. However, is has been shown normal and transformed (human multiple myeloma) LLPC are critically dependent on the bone marrow microenvironment including cell-to-cell interactions. Leading us to rationalize, modulating this interaction could either enhance antibody production for cancer vaccine development or conversely compromise the survival of transformed/normal LLPC in the bone marrow microenvironment. We have shown the T cell costimulatory receptor CD28 expressed on both normal and transformed LLPC plays an essential role. While LLPC and short-lived plasma cells (SLPC) both express CD28, its activation in vitro only significantly increases the survival and IgG production of LLPC. These observations led us to directly investigate the role of CD28 in LLPC survival as well as cell-cell interactions with CD80/CD86+ bone marrow derived dendritic cells (BMDC). Utilizing normal murine bone marrow and splenic PC as our model system we further investigated the role of CD28 in LLPC function and survival. We have previously shown, in vitro serum starvation experiments, direct activation of CD28 increased survival of LLPC by 12-fold (p<0.05), whereas CD28 activation of SLPC did not induce survival. Addition of BMDC improved the survival of LLPC 2-fold over that seen with media alone, and resulted in a significant increase in IgG production (p<0.001). In contrast, CD28-/- PC had no increase in survival when cocultured with BMDC, suggesting a direct role for CD28 in PC-DC interaction. Consistent with these findings we now show that in vivo, vaccinated bone marrow CD28-/-:μMT chimeras had significantly reduces long-term antibody titers and LLPC (but not SLPC) survival from t1/2 of 426 to 63 days. Additionally, LLPC CD28 modulates the microenvironment by inducing CD80/CD86+ stromal cell production of the supportive cytokine IL-6 (p<0.001 vs. BMDC/PC alone), which was abrogated by blocking CD80 and CD86 (p<0.05). From the above experiments we hypothesized IL-6 was playing a significant role in the survival of LLPC, however to our surprise LLPC cocultured with WT or IL-6-/- BMDC maintained equivalent LLPC numbers, interestingly however LLPC cocultures with BMDC showed a 3-fold increase of IgG compared to LLPC cocultured with IL-6-/- BMDC (p<0.001). These data suggest CD28 is a key molecular component in LLPC survival, whereas IL-6 contributes to Ig production. Our data demonstrates that signaling through CD28 directly supports the survival of LLPC, sustaining long term protective antibody titers. These findings suggest CD28 plays an important role in maintaining the quality of protective durable humoral immunity. Strategies to augment CD28 signaling may lead to greater LLPC survival and persistent antibody titers in cancer vaccine development. Conversely, blocking CD28 signaling could compromise the survival of transformed myeloma cells which are critically dependent on the bone marrow microenvironment. Disclosures:Boise:University of Chicago: Patents & Royalties.

Modulation of in vivo immunoglobulin production by endogenous histamine and H1R and H2R agonists and antagonists

The present study was designed to delineate the immunomodulatory role of histamine receptors (H1R and H2R) and their antibody generation in a rabbit model. Six groups containing 18 rabbits each received either vehicle (sterile distilled water, 1ml/kg×b.i.d), histamine (100µg/kg×b.i.d.), H1R agonist (HTMT, 10µg/kg×b.i.d.), H2R agonist (amthamine, 10µg/kg×b.i.d.), H1R antagonist (pheniramine, 10mg/kg×b.i.d.) or H2R antagonist (ranitidine, 10mg/kg×b.i.d.). All animals were subsequently immunized with an intravenous injection of sheep red bood cells (SRBC). Estimations of total serum immunoglobulins (Igs), immunoglobulin M (IgM) and immunoglobulin G (IgG) were performed by ELISA and hemagglutination assay (HA) at days 0 (pre-immunization), 7, 14, 21, 28 and 58 (post-immunization). Both the ELISA and the HA showed similar production of Igs, IgM and IgG but the results were found comparatively more significant by ELISA as opposed to HA. Results showed that histamine could influence a detectable antibody response to SRBC early (i.e., at day 7), which lasted until day 58. Immunomodulatory processes showed suppression of an Ig generation in the H1R-antagonist group with enhancement in the H2R-antagonist group. The H1R-agonist group showed an increased Ig production in comparison to the H2R-agonist group. The IgM production was inhibited in the H1R-antagonist group as compared to the H2R-antagonist group, and it was also suppressed in H1R-agonist group as compared to H2R-agonist group. IgG production was inhibited in the H1R-antagonist group as opposed to the H2R-antagonist group. In contrast, the H1R-agonist group increased IgG production as compared to the H2R-agonist group. All the results were found to be statistically significant (p<0.05 or p<0.01). In conclusion, histamine and its receptor (H1R and H2R) agonists enhance antibody production by triggering the histamine receptors (H1R and H2R), and both the H1R antagonist and the H2R antagonist positively or negatively regulate the antibody production. The findings of this study may have clinical significance.