Enhancement of murine in vitro antibody formation by hyperthermia

The influence of hyperthermia on primary in vitro antibody formation by C57BL/6 splenocytes to a T-dependent antigen, sheep red blood cells (SRBC), and a T-independent antigen, trinitrophenyl-lipopolysaccharide (TNP-LPS), was evaluated. Following heat treatment (39 or 40 °C), spleen cells demonstrated two- to fivefold increases in antibody production to SRBC. This enhancement of humoral immunity is critically dependent on the timing of hyperthermia administration relative to antigenic exposure. Examination of the kinetics for the SRBC response revealed that heat significantly lengthens the time period of antibody production. Although a number of parameters were examined, antibody production to TNP-LPS from hyperthermically treated cultures were comparable to control (37 °C treated) cultures. Finally, we have determined that the heat-induced increases in antibody formation to SRBC are mediated through T lymphocytes.     

Inhibition of in vitro antibody synthesis by cyclophosphamide-induced suppressor cells

A population of suppressor lymphocytes appears in the spleens of mice 5 to 14 days after treatment with a high dose of cyclophosphamide (100–200 mg/kg body wt). Removal of carbonyl iron adherent cells or Ig^? cells from cyclophosphamide (CP)-treated spleen cells does not abolish suppressive activity. These suppressors are, however, sensitive to removal by treatment with anti-Thy-1.2 and rabbit complement. CP-treated spleen cells can suppress the in vitro primary response of normal spleen cells to the soluble hapten-protein conjugate DNP-MON or the particulate antigen HRBC when added at time of culture initiation or up to the second day of culture. CP-treated spleen cells can themselves respond in vitro to DNP-MON, as well as to HRBC, but with altered kinetics from that of normal spleen cells. Collectively, the data suggest that the CP-induced suppressors act late in the in vitro antibody response, possibly by prematurely shutting off antibody synthesis by B cells.     

Enhancement of a human antibody response in vitro mediated by interaction of interferon-alpha with T lymphocytes

This study describes the effects of human recombinant IFN-alpha 2 on antibody production in vitro. Whereas the inclusion of IFN-alpha 2 in cultures for 7 days had a relatively minor effect on pokeweed mitogen (PWM)-induced antibody production, it resulted in a dose-related enhancement of a hapten-specific primary antibody response. Comparison of PWM and IFN-induced [3H]thymidine uptake indicated that the observed IFN activation was not polyclonal. Pretreatment of T cells with IFN for 1 hr before recombination with untreated autologous B lymphocytes increased the anti-TNP response four-fold, whereas similar pretreatment of B lymphocytes had no effect. Furthermore, 2000 R x-irradiation of T cells before coculture with autologous B lymphocytes and IFN abrogated the TNP-specific response. These results indicate that IFN modulates TNP-specific antibody production via a radiosensitive T-helper function. Further subfractionation by panning suggests that the enhancement is mediated by the Leu-3a+ helper/inducer T cell subset. Evidence that a 1-hr exposure to IFN was sufficient to modulate antibody production prompted the examination of T cells for possible receptor mechanisms. Scatchard analysis of 125I-IFN-alpha 2 binding revealed approximately 65 high affinity IFN receptors per cell with an apparent dissociation constant (Kd) of 4.4 X 10(-10) M. This paper is the first demonstration of the role of T cells in mediating the effects of recombinant IFN-alpha 2 on human primary antibody responses in vitro. These data further suggest that the observed modulation of hapten-specific antibody production in vitro by IFN may involve the binding of IFN to specific cellular receptors expressed by T lymphocytes.     

Enhancement of antibody production by lysophosphatidylcholine and alkylglycerol

Inflammation products of normal and cancerous tissues, lysophosphatidylcholine and dodecylglycerol, were tested for their adjuvant effect on the antibody response. Mice treated with these agents and immunized with sheep erythrocytes simultaneously or at 3 days posttreatment developed a greatly enhanced antibody production as demonstrated by the Jerne plaque assay. Mice immunized at 3 days postadministration of agents did not significantly produce enhanced antibody-secreting cells as compared with those of mice simultaneously immunized. Since the mechanism of macrophage activation by lysophospholipids requires contribution of B and T cells, BALB/c-nu/nu mice treated with these agents and subsequently immunized with sheep erythrocytes did not produce antibodies. However, conditioned medium of in vitro-treated BALB/c-nu/nu B cells efficiently transmitted a signal to untreated BALB/c +/+ T cells for enhanced macrophage ingestion activity. This observation suggests that lysophospholipid-activated macrophages and T cells efficiently transmitted antigenic signal to the antibody-producing B cell population. Therefore, we conclude that these lipid metabolites have dual beneficial effects for the host by enhancing phagocytosis and antibody production. Thus, lysophosphatidylcholine and dodecylglycerol have potential practical application as adjuvants that could be administered separately or in combination with antigens.     

Enhancement of monoclonal antibody production by lysine-containing peptides

In the search for peptides that could effectively enhance the monoclonal antibody production of a model hybridoma, the performance of five lysine-containing peptides was compared. The capacity of the peptides to enhance the monoclonal antibody yield correlated with their growth-suppressing activity. No correlation of the production-enhancing activity with the character of the distribution of cell-cycle phases could be found. All of the tested peptides, including the negative control peptide Gly-Phe-Gly, altered the cell-cycle phases distribution in favor of the proportion of the S phase.The peptides added to the hybridoma culture were found to be gradually decomposed into dipeptides and free amino acids. Among the set of tested lysine-containing di- to pentapeptides, the best results were obtained with the tripeptide Gly-Lys-Gly. The growth-suppressing and production-enhancing capacity of this peptide supplement was obviously associated with the temporary presence of the intact peptide molecule in the culture media, because the addition of a mixture of free amino acids constituting this peptide, i.e., glycine and lysine, displayed a different effect-a slight promotion of cell growth.     

Class-specific effects of selenium on PWM-driven human antibody synthesis in vitro

The influence of inorganic and organic forms of selenium (Se) on human antibody production was studied in a Pokeweed Mitogen (PWM)-driven in vitro system. Mitogen-stimulated peripheral blood mononuclear cells (PBMC) of eight healthy donors were cultured with different Se compounds at concentrations between 10(-3) and 10(-9) M. At high Se levels (10(-3)-10(-4) M), IgM and IgG production of all donors were strongly inhibited owing to reduced cell viability. However, in five of eight donors, low levels of Se enhanced IgG secretion. This was most effective in the presence of inorganic Se, whereas selenomethionine and selenocystine were less effective. In contrast to IgG, IgM synthesis was significantly reduced by low Se levels in five donors. No significant correlation between donor serum Se levels and antibody production in vitro was found. The addition of low levels of Se to PBMC, stimulated with PHA or PWM, showed no effect on proliferation, whereas a high concentration (5 x 10(-3) M) of sodium selenite and selenocystine suppressed proliferation owing to reduced cell viability. Thus, the present results show that Se supplementation can enhance human antibody production and, moreover, suggest some selectivity of Se action on human immune responses that may result in increased switching from IgM to IgG production.     

Enhancement of carotenoid synthesis by fungal metabolites

Carotenoids, mainly the all-trans--carotene, may be produced by the fungus Phycomyces blakesleeanus under normal fermentation conditions. A number of chemical compounds that stimulate or inhibit fungal carotenogenesis have been reported earlier. A procedure for the screening of organisms that enhance carotenogenesis on co-culturing with P. blakesleeanus is described. A water-soluble stimulator of carotenogenesis was obtained from the fermentation broth of an unidentified Aspergillus sp. isolated from soil. The crude preparation appears to be ten times more potent than dimethylphthalate.     

Myogenesis in vitro. Enhancement by dibutyryl cAMP

Cholera enterotoxin (CT) increased the concentration of adenosine 3′-5′-cyclic monophosphate (cAMP) in monolayer cultures of adrenal tumor cells after a 60 min lag phase in contrast to the rapid effect of adrenocorticotropin (ACTH). The change in intracellular cAMP was accompanied by the release of steroids into the culture medium and a reversible alteration of monolayer morphology.     

Enhancement of histamine synthesis in mouse skin by epinephrine

Intravenous administration of 10-40 micrograms/Kg epinephrine to mice leads to a transient 50% increase in skin histamine, followed by an increase in blood histamine. Delayed inhibition by alpha-fluorometyl histidine (alpha FMH), suggests that these changes follow stimulation of pre-formed tissue histidine decarboxylase.     

Role of mercaptoethanol and endotoxin in stimulating B lymphocyte colony formation in vitro.

Mercaptoethanol is necessary to permit B lymphocyte colony formation in semi-solid agar cultures of cells from normal mouse lymphoid organs. Transfer studies on developing colonies showed that, in part, this was a direct action on B lymphocyte colony cells but evidence was produced that in the presence of mercaptoethanol lymphoid organ cells release a factor promoting colony growth. Endotoxin strongly potentiated B lymphocyte colony formation in vitro by a direct action on colony cells but in the absence of mercaptoethanol did not allow cell survival or proliferation.     

Therapeutic anti-methamphetamine antibody fragment-nanoparticle conjugates: synthesis and in vitro characterization

Treatments specific to the medical problems caused by methamphetamine (METH) abuse are greatly needed. Toward this goal, we are developing new multivalent anti-METH antibody fragment-nanoparticle conjugates with customizable pharmacokinetic properties. We have designed a novel anti-METH single chain antibody fragment with an engineered terminal cysteine (scFv6H4Cys). Generation 3 (G3) polyamidoamine dendrimer nanoparticles were chosen for conjugation due to their monodisperse properties and multiple amine functional groups. ScFv6H4Cys was conjugated to G3 dendrimers via a heterobifunctional PEG cross-linker that is reactive to a free amine on one end and a thiol group on the other. PEG modified dendrimers were synthesized by reacting the PEG cross-linker with dendrimers in a stoichiometric ratio of 11:1, which were further reacted with 3-fold molar excess of anti-METH scFv6H4Cys. This reaction resulted in a heterogeneous mix of G3-PEG-scFv6H4Cys conjugates (dendribodies) with three to six scFv6H4Cys conjugated to each dendrimer. The dendribodies were separated from the unreacted PEG modified dendrimers and scFv6H4Cys using affinity chromatography. A detailed in vitro characterization of the PEG modified dendrimers and the dendribodies was performed to determine size, purity, and METH binding function. The dendribodies were found to have affinity for METH identical to that of the unconjugated scFv6H4Cys in saturation binding assays, whereas the PEG modified dendrimers had no affinity for METH. These data suggest that an anti-METH scFv can be successfully conjugated to a PEG modified dendrimer nanoparticle with no adverse effects on METH binding properties. This study is a critical step toward preclinical characterization and development of a novel nanomedicine for the treatment of METH abuse.