Biomedical subjects

Basil Golding

Publications and source records attributed to Basil Golding.

13 recordsLinked to original sources

PubMed2006

Brucella abortus bacA mutant induces greater pro-inflammatory cytokines than the wild-type parent strain.

The inner-membrane protein BacA affects Brucella LPS structure. A bacA deletion mutant of Brucella abortus, known as KL7 (bacA(mut)-KL7), is attenuated in BALB/c mice and protects against challenge. Thus, bacA mutation was a candidate for incorporation into live attenuated vaccines. We assessed bacA(mut)-KL7 in 2 additional mouse strains: the more resistant C57BL/6 that produces interferon-gamma throughout the infection and the highly susceptible interferon-gamma-deficient C57BL/6 in which brucellae exhibit continual exponential growth. While it was hypothesized that bacA(mut)-KL7 would exhibit even greater attenuation relative to its parent strain B. abortus 2308 in C57BL/6 mice than it did in BALB/c mice, this was not the case. Moreover, it was more pathogenic in C57BL/6 interferon-gamma-deficient mice than 2308 causing abscesses and wasting even though the splenic loads of bacA(mut)-KL7 were significantly lower. These 2 observations were correlated, respectively, with an ability of IFNgamma-activated macrophages to equivalently control strains 2308 and bacA(mut)-KL7 and the ability of bacA(mut)-KL7 organism and its LPS to induce greater amounts of pro-inflammatory cytokines than 2308. We conclude that attenuation properties of bacA mutation are dependent upon the nature of the host but more importantly that bacterial gene deletion can result in increased host pathology without an increase in bacterial load, crucial considerations for vaccine design.

Animals↗

PubMed2006

Inhibitory effects of B cells on antitumor immunity.

B-cell functions in antitumor immunity are not well understood. In this study, we evaluated the role of B cells in the development of antitumor immunity using Friend murine leukemia virus gag-expressing mouse EL-4 (EL-4 gag), D5 mouse melanoma, or MCA304 mouse sarcoma cells. To screen tumors for susceptibility to B-cell-deficient immune environments, spleen cells from naive C57BL/6 [wild-type (WT)] and B-cell knockout (BKO) mice were cultured with irradiated tumor cells in vitro. When cells were stimulated with EL-4 gag or D5 (but not MCA304 tumors), IFN-gamma production from CD8 T cells and natural killer cells was markedly decreased in WT compared with BKO cultures. IFN-gamma production was correlated with CD40 ligand expression on the tumor and inversely with interleukin-10 (IL-10) production by B cells. Sorted WT B cells produced more IL-10 than CD40 knockout (CD40KO) B cells when cocultured with EL-4 gag or D5 (but not MCA304). IFN-gamma production by BKO cells was reduced by the addition of sorted naive WT B cells (partially by CD40KO B cells) or recombinant mouse IL-10. In vivo tumor progression mirrored in vitro studies in that WT mice were unable to control tumor growth whereas EL-4 gag and D5 tumors (but not MCA304) were eliminated in BKO mice. Robust in vivo antitumor CTLs developed only in BKO tumor-challenged mice. Our studies provide the first mechanistic basis for the concept that B-cell depletion could therapeutically enhance antitumor immune responses to certain tumors by decreasing IL-10 production from B cells.

Animals↗

PubMed2006

Passive immunotherapy of Bacillus anthracis pulmonary infection in mice with antisera produced by DNA immunization.

Because of the high failure rate of antibiotic treatment in patients with anthrax there is a need for additional therapies such as passive immunization with therapeutic antibodies. In this study, we used codon-optimized plasmid DNAs (DNA vaccines) encoding Bacillus anthracis protective antigen (PA) to immunize rabbits for producing anti-anthrax antibodies for use in passive immunotherapy. The antisera generated with these DNA vaccines were of high titer as measured by ELISA. The antisera were also able to protect J774 macrophage cells by neutralizing the cytotoxic effect of exogenously added anthrax lethal toxin, and of the toxin released by B. anthracis (Sterne strain) spores following infection. In addition, the antisera passively protected mice against pulmonary challenge with an approximate 50 LD50 dose of B. anthracis (Sterne strain) spores. The protection in mice was obtained when the antiserum was given 1h before or 1h after challenge. We further demonstrated that IgG and F(ab')2 components purified from anti-PA rabbit hyperimmune sera retained similar levels of neutralizing activities against both exogenously added B. anthracis lethal toxin and toxin produced by B. anthracis (Sterne strain) spores. The high titer antisera we produced will enable an immunization strategy to supplement antibiotic therapy for improving the survival of patients with anthrax.

Animals↗

PubMed2005

The cytotoxic T lymphocyte response against a protein antigen does not decrease the antibody response to that antigen although antigen-pulsed B cells can be targets.

The role of activated CD8+ T cells in shaping the dynamics of in vivo antigen presentation and immune responses is a subject receiving more attention. We studied whether cytotoxic T lymphocyte (CTL) would limit antibody responses by targeting antigen-specific B cells. A modified in vivo CTL assay was developed and used herein to demonstrate cytotoxicity in vivo, and to show that antigen-specific B cells that process exogenous antigen and present peptide in association with MHC class I can be the targets of CD8+ T cells. B cells from C57BL/6 mice immunized with ovalbumin (OVA)/alum were pulsed with OVA in vitro, and transferred into C57BL/6 recipient mice that had been immunized with vaccinia virus expressing SIINFEKL minigene to generate CD8+ CTL against K(b)/SIINFEKL. OVA-pulsed B220+ B cells from OVA-immunized mice were killed to a greater extent than B220+ B cells from naïve mice (28+/-20% versus 12+/-16%, p=0.0042). However, mice receiving vaccinia-SIINFEKL and generating CTL, did not appear to target endogenous B cells, since both primary and secondary antibody responses to OVA were unaffected. Our findings indicate that CTL responses to the protein antigen do not interfere with endogenous B cell responses, even though exogenous B cells expressing the CTL epitope can be efficiently lysed.

Animals↗

PubMed2005

Th1-like cytokine induction by heat-killed Brucella abortus is dependent on triggering of TLR9.

In this report we provide evidence, for the first time, that bacterial DNA in the context of heat-killed Brucella abortus (HKBA) engages TLR9 in dendritic cells (DC), resulting in a Th1-like cytokine response. This is based on the findings that HKBA induction of IL-12p40 is: 1) abolished in DC from TLR9(-/-) mice; 2) blocked by suppressive oligodeoxynucleotides; 3) simulated by bacterial DNA derived from HKBA; and 4) abrogated by DNase or methylation of the DNA from HKBA. Furthermore, the effect of HKBA can be inhibited by chloroquine, indicating that endosomal acidification is required and supporting the notion that DNA from HKBA is interacting with TLR9 at the level of the endosome, as is the case with CpG oligodeoxynucleotides. In addition to DC, HKBA can elicit IL-12p40 secretion from macrophages, in which case the effect is wholly MyD88 dependent but only partially TLR9 dependent. This probably explains why HKBA effects in vivo are only partially reduced in TLR9(-/-), but absent in MyD88(-/-) mice. Because of their intimate interactions with T cells, the DC response is most likely to be critical for linking innate and adaptive immune responses, whereas the macrophage reaction may play a role in enhancing NK cell and bystander immune responses. In addition to IL-12p40, HKBA induces other Th1-like cytokines, namely, IFN-alpha and IFN-gamma, in a TLR9-dependent manner. These cytokines are important in protection against viruses and bacteria, and their induction enhances HKBA as a potential carrier for vaccines.

Animals↗

PubMed2005

Programming of CTL with heat-killed Brucella abortus and antigen allows soluble antigen alone to generate effective secondary CTL.

Optimal generation of cytotoxic T cell (CTL) responses continues to be a challenge in the production of vaccines against pathogens such as HIV-1, in part because it is difficult to introduce soluble protein antigens (Ag) into the MHC class I pathway. Using heat-killed Brucella abortus (HKBA) as an adjuvant and ovalbumin (ova) protein as an Ag, we demonstrated that a high dose of Ag was required for systemic and effective CTL. In an adoptive transfer model, primary and secondary ova-specific OT-1 CD8 cell expansion by HKBA plus high dose of ova were partially CD4 T cell-dependent. Interestingly, primary stimulation with HKBA plus ova allowed effective secondary stimulation with ova alone that was equivalent to HKBA plus ova in terms of IFN-gamma production from Ag-specific CD8 cells. Thus a combination of adequate Ag dose, and selection of appropriate adjuvants can meet the threshold not only for primary effective CTL responses to soluble protein Ags but for secondary CTL responses following stimulation with protein Ag alone.

Adoptive Transfer↗

PubMed2005

Identification of a novel immunodominant cytotoxic T lymphocyte epitope derived from human factor VIII in a murine model of hemophilia A.

Gene therapy of hemophilia A could be complicated by the development of immune responses against the vector as well as the Factor VIII (FVIII) transgene. Previous efforts have been focused on identifying FVIII inhibitor antibody epitopes, whereas the cytotoxic T lymphocyte (CTL) epitopes have not been characterized. CTL would kill cells expressing such epitopes and thus limit the efficacy of gene therapy. To investigate CTL responses against human FVIII in a mouse model of hemophilia A, a computer algorithm program (BIMAS) was employed to predict CTL epitopes of human FVIII. The potential binding of these predicted peptides to MHC class I K(b) was evaluated in a TAP-deficient cell line. When recombinant vaccinia virus expressing B domain-deleted human FVIII (vv-FVIII) was used to immunize E16 hemophilia A mice, a specific CTL response against FVIII152-159 was generated. In contrast, a CTL response to four other FVIII peptides was not detected. Therefore, FVIII152-159 represents a dominant CTL epitope. Identification of this epitope raises the possibility that CTL response to FVIII gene-transduced cells can be diminished by deliberatively mutating the dominant CTL epitope while retaining the biologic function of FVIII for hemophilia A gene therapy.

Algorithms↗

PubMed2004

Treatment of anthrax infection with combination of ciprofloxacin and antibodies to protective antigen of Bacillus anthracis.

Currently there is no effective treatment for inhalational anthrax beyond administration of antibiotics shortly after exposure. There is need for new, safe and effective treatments to supplement traditional antibiotic therapy. Our study was based on the premise that simultaneous inhibition of lethal toxin action with antibodies and blocking of bacterial growth by antibiotics will be beneficial for the treatment of anthrax. In this study, we tested the effects of a combination treatment using purified rabbit or sheep anti-protective antigen (PA) antibodies and the antibiotic ciprofloxacin in a rodent anthrax model. In mice infected with a dose of Bacillus anthracis Sterne strain corresponding to 10 LD(50), antibiotic treatment with ciprofloxacin alone only cured 50% of infected animals. Administration of anti-PA IgG in combination with ciprofloxacin produced 90-100% survival. These data indicate that a combination of antibiotic/immunoglobulin therapy is more effective than antibiotic treatment alone in a rodent anthrax model.

Animals↗

PubMed2004

Systemic and mucosal immunity in rhesus macaques immunized with HIV-1 peptide and gp120 conjugated to Brucella abortus.

HIV vaccine testing in primates is an important method for determining the possibility of vaccine benefit in humans. Goals of HIV-1 vaccination include establishing neutralizing antibodies and a strong CD8(+) T-cell response. We tested a novel vaccine conjugate for its ability to elicit relevant immune responses to HIV proteins and peptides in rhesus macaques. A neutralizing epitope, V3 loop peptide from HIV-1 envelope, was coupled to heat-inactivated Brucella abortus (V3-HKBA). Rhesus macaques were immunized with this conjugate in the anterior thigh. After two immunizations V3-specific antibodies were found in the sera and at mucosal sites. Neutralizing activity of these antibodies was demonstrated by syncytia inhibition assays. Cellular immune recall responses were demonstrated by antigen-specific induction of interferon-gamma and Regulation on Activation Noraml T Cell Expressed and Secreted (RANTES) secretion in vitro. These results confirm and extend preliminary studies in mice that suggest HKBA is an effective carrier that promotes neutralizing antibody secretion at relevant mucosal sites, as well as cellular immune responses that are correlated with viral protection.

AIDS Vaccines↗

PubMed2004

Characterization of antibodies to capsular polysaccharide antigens of Haemophilus influenzae type b and Streptococcus pneumoniae in human immune globulin intravenous preparations.

The most common infections in primary immune deficiency disease (PIDD) patients involve encapsulated bacteria, mainly Haemophilus influenzae type b (Hib) and Streptococcus pneumoniae (pneumococcus). Thus, it is important to know the titers of Hib- and pneumococcus-specific antibodies that are present in immune globulin (Ig) intravenous (IGIV) preparations used to treat PIDD. In this study, seven IGIV preparations were tested by enzyme-linked immunosorbent assay and opsonophagocytic activity for antibody titers to the capsular polysaccharides of Hib and five pneumococcal serotypes. Differences in Hib- and pneumococcus-specific antibody titer were observed among various IGIV preparations, with some products having higher- or lower-than-average titers. Opsonic activity also varied among preparations. As expected, IgG2 was the most active subclass of both binding and opsonic activity except against pneumococcal serotype 6B where IgG3 was the most active. This study determines antibody titers against capsular polysaccharides of Hib and pneumococcus in seven IGIV products that have been shown to be effective in reducing infections in PIDD patients. As donor antibody levels and manufacturing methods continue to change, it may prove useful from a regulatory point of view to reassess IGIV products periodically, to ensure that products maintain antibody levels that are important for the health of IGIV recipients.

Bacterial Capsules↗

PubMed2003

Heat-killed Brucella abortus induces TNF and IL-12p40 by distinct MyD88-dependent pathways: TNF, unlike IL-12p40 secretion, is Toll-like receptor 2 dependent.

Cattle and humans are susceptible to infection with the Gram-negative intracellular bacterium Brucella abortus. Heat-killed B. abortus (HKBA) is a strong Th1 adjuvant and carrier. Previously, we have demonstrated that dendritic cells produce IL-12 in response to HKBA stimulation. In the present study, we use knockout mice and in vitro reconstitution assays to examine the contribution of signaling by Toll-like receptors (TLRs) and their immediate downstream signaling initiator, myeloid differentiation protein MyD88, in the activation following stimulation by HKBA. Our results show that HKBA-mediated induction of IL-12p40 and TNF is dependent on the adapter molecule MyD88. To identify the TLR involved in HKBA recognition, we examined HKBA responses in TLR2- and TLR4-deficient animals. TNF responses to HKBA were TLR4 independent; however, the response in TLR2-deficient mice was significantly delayed and reduced, although not completely abolished. Studies using Chinese hamster ovary/CD14 reporter cell lines stably transfected with either human TLR2 or human TLR4 confirmed the results seen with knockout mice, namely TLR2, but not TLR4, can mediate cellular activation by HKBA. In addition, human embryonic kidney 293 cells, which do not respond to HKBA, were made responsive by transfecting TLR2, but not TLR4 or TLR9. Taken together, our data demonstrate that MyD88-dependent pathways are crucial for activation by HKBA and that TLR2 plays a role in TNF, but not IL-12p40 pathways activated by this microbial product.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Mucosal immunity in mice immunized with HIV-1 peptide conjugated to Brucella abortus.

We have previously shown that a V3-loop peptide from HIV-1 envelope conjugated to heat-inactivated Brucella abortus (Ba) (V3-Ba) is capable of inducing antibodies that neutralize HIV-1 and cytotoxic T cells (CTL) that kill HIV-1-infected targets, even in mice that lack CD4(+) T cells. In this paper we show that intranasal (i.n.) immunization elicits neutralizing antibodies and IFNgamma-secreting T cells at mucosal surfaces. This approach may protect individuals from HIV-1 infection and reduce transmission from infected individuals to their sexual partners and offspring.

AIDS Vaccines↗

PubMed2002

Recombinant adeno-associated virus serotype 2 vectors mediate stable interleukin 10 secretion from salivary glands into the bloodstream.

We have constructed a recombinant adeno-associated virus serotype 2 vector encoding human interleukin 10 (rAAVhIL10). IL-10 is a potent antiinflammatory/immune cytokine, which has received growing attention for its therapeutic potential. Human IL-10 (hIL-10) production was virus dose dependent after in vitro infection of HSG cells, a human submandibular gland cell line. The vector-derived hIL-10 produced was biologically active, as the medium from rAAVhIL10-infected HSG cells caused a dose-dependent blockade of IL-12 secretion from spleen cells of IL-10 knockout mice challenged with heat-killed Brucella abortus. Administration of rAAVhIL10 (10(10) genomes per gland) to both mouse submandibular glands led to hIL-10 secretion into the bloodstream (approximately 1-5 pg/ml), that is, in an endocrine manner, which was stable for approximately 2 months. Salivary gland administration of rAAVhIL10 under experimental conditions was more efficacious than intravenous administration (approximately 0.5-0.7 pg/ml). Also, hIL-10 was readily secreted in vitro from organ cultures of minced submandibular glands infected with rAAVhIL10, 6 or 8 weeks earlier. Consistent with these results, hIL-10 mRNA was detected by reverse transcription-polymerase chain reaction in submandibular glands of mice infected with rAAVhIL10 but not from control mice. At these doses, little to no hIL-10 was detected in mouse saliva. Using a rAAV serotype 2 vector encoding beta-galactosidase, we observed that the primary parenchymal target cells were ductal. These findings represent the first report of rAAV use to target exocrine glands for systemic secretion of a therapeutic protein, and support the notion that rAAV serotype 2 vectors may be useful in salivary glands for local (periglandular) and systemic gene-based protein therapeutics.

Animals↗