Bordetella pertussis Filamentous Hemagglutinin Enhances the Immunogenicity of Liposome-Delivered Antigen Administered Intranasally

In an attempt to increase the immunogenicity of mucosally delivered antigens, we incorporated the Bordetella pertussis filamentous hemagglutinin (FHA) adhesin into liposomes containing the glutathione S-transferase of Schistosoma mansoni (Sm28GST) as a model antigen. Outbred mice immunized twice intranasally with liposomes containing a constant suboptimal dose of Sm28GST and increasing doses of FHA produced anti-Sm28GST antibodies in a FHA dose-dependent manner. The addition of 3 μg of FHA to the liposomes induced more than 10-fold-higher anti-Sm28GST antibody titers, compared to those induced by liposomes without FHA. The presence of FHA did not alter the nature of the humoral immune response, and the sera contained anti-Sm28GST immunoglobulin G1 (IgG1), IgG2a, and IgG2b. However, anti-Sm28GST IgA was only detected when at least 3 μg of FHA was added to the preparation. These results show a promising potential for FHA to enhance the immunogenicity of mucosally administered antigens incorporated into liposomes.     

Retinoblastoma and p53 gene product expression in breast carcinoma: Immunohistochemical analysis and clinicopathologic correlation

We examined 100 breast cancers for retinoblastoma (Rb) and p53 protein expression by immunohistochemistry using the PMG3.245 and PAb 1801 antibodies. We assessed percentages of reactive cells and their intensity, as well as staining patterns. The results were correlated with neu protein reactivity and a panel of variables, including age, tumor size and type, nuclear grade, estrogen receptor/progesterone receptor content, and lymph node status. Retinoblastoma protein negativity, either partial or complete, was noted in 47% of cases. Surprisingly, a relatively stronger Rb reaction was seen in some high nuclear grade tumors. p53 positivity was found in 23% of cases and was a significant predictor of Rb loss. p53 also was correlated with poorly differentiated (nuclear grade III) neoplasms and neu expression but not with negative ER status. Tissue distribution profiles for Rb-negative and p53-positive cells were variable in this series, with both uniform and heterogeneous patterns observed. This suggests that Rb and p53 alterations may represent early or late events in transformation. Our findings further implicate Rb and p53 derangements in mammary oncogenesis.     

Comparison of the QUANTIPLEX HIV-1 RNA 2.0 Assay with the AMPLICOR HIV-1 MONITOR 1.0 Assay for Quantitation of Levels of Human Immunodeficiency Virus Type 1 RNA in Plasma of Patients Receiving Stavudine-Didanosine Combination Therapy

We compared the QUANTIPLEX HIV-1 RNA 2.0 assay with the AMPLICOR HIV-1 MONITOR 1.0 assay for quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in plasma in the Stadi trail, which evaluated a stavudine plus didanosine combination therapy in 52 patients. HIV-1 RNA baseline values measured with AMPLICOR HIV-1 MONITOR 1.0 were significantly higher than those measured with QUANTIPLEX HIV-1 RNA 2.0, and decreases in HIV-1 RNA levels from baseline were also found to be significantly higher when measured with the AMPLICOR HIV-1 MONITOR 1.0 assay. The frequency of HIV-1 RNA levels below the lower limit of quantitation was significantly higher with QUANTIPLEX HIV-1 RNA 2.0 than with AMPLICOR HIV-1 MONITOR 1.0. Reanalysis of these results by an ultrasensitive procedure of AMPLICOR HIV-1 MONITOR 1.0 or by a modified version of the test that included additional primers adapted for non-B HIV-1 clades yielded greater differences between the QUANTIPLEX HIV-1 RNA 2.0 assay and the AMPLICOR HIV-1 MONITOR 1.0 assay. Our results indicate that a valid comparison of the virological efficacies obtained with different antiretroviral drug regimens requires the use of the same viral load quantitation procedure; further standardization between the different HIV-1 RNA quantitation kits is therefore needed.     

Characterization of wild-type recombinant Bet v 1a as a candidate vaccine against birch pollen allergy

BACKGROUND: We describe the production in Escherichia coli as a recombinant protein of clinical grade wild-type Bet v 1a (rBet v 1a), to be used as a candidate vaccine against birch pollen allergy. METHODS: This recombinant protein was purified by hydrophobic interaction and ion exchange chromatography and characterized by SDS-PAGE, immunoprint and circular dichroism in parallel with natural Bet v 1 (nBet v 1) purified from a birch pollen extract. We also compared rBet v 1 and nBet v 1 for their capacity to induce histamine release from basophils and to stimulate T lymphocyte proliferation. RESULTS: rBet v 1a appears in SDS-PAGE as an 18-kDa monomeric protein, whereas purified nBet v 1 comprises a mixture of isoforms (resolving as three distinct bands and six spots after 1-dimensional and 2-dimensional electrophoresis, respectively). Both recombinant and natural purified Bet v 1 molecules are recognized by IgE from birch pollen-allergic patients as well as anti-Bet v 1 murine monoclonal antibodies, suggesting that the recombinant protein is correctly folded in a native configuration. Circular dichroism analysis confirmed that the two Bet v 1 molecules exhibit similar 3-dimensional structures, even if rBet v 1a appears more compact and stable in thermodenaturation/renaturation experiments. Both rBet v 1 and nBet v 1 induce the degranulation of sensitized basophils and proliferation of Bet v 1-specific T lymphocytes in a similar manner. CONCLUSIONS: On the basis of these structural and biological properties, rBet v 1a is a valid candidate vaccine against birch pollen allergy, currently evaluated in humans.     

NADPH oxidase priming and p47phox phosphorylation in neutrophils from synovial fluid of patients with rheumatoid arthritis and spondylarthropathy

Superoxide anion (O2°-)production by neutrophil NADPH oxidase participates in arthritic joint lesion formation. Proinflammatory cytokines such as tumor necrosis factor (TNF), interleukin 8 (IL-8) and granulocyte/macrophage-colony stimulating factor (GM-CSF) have a priming effect on neutrophil NADPH oxidase activity. NADPH oxidase activation is dependent on phosphorylation of p47phox, a cytosolic component of the enzyme. We studied O2°-production and p47phox phosphorylation in synovial fluid (SF) from patients with rheumatoid arthritis (RA) and spondylarthropathy (SpA) according to TNF, IL-8 and GM-CSF levels. O2°-production by neutrophils isolated from SF of all the arthritis patients (RA and SpA) was higher than that of circulating resting neutrophils and when stimulated with fMLP or PMA. In addition, p47phox was partially phosphorylated in SF neutrophils compared to circulating neutrophils. High levels of TNF and IL-8 (but not GM-CSF) are detected in patient's SF (compared to circulating blood levels). TNF levels were significantly higher in RA than in SpA SF. These results suggest that increased NADPH oxidase activity could be involved in arthritic joint inflammation through increased p47phox phosphorylation. This could be the result of the presence of high levels of priming agents such as TNF and IL-8 but not GM-CSF.     

Chemical constituents of diesel exhaust particles induce IL-4 production and histamine release by human basophils

BACKGROUND: An epidemiologic relationship between airway allergic diseases and exposure to atmospheric pollutants has been demonstrated and suggested to be one factor in the increasing prevalence of asthma. Diesel exhaust particles (DEPs) have been shown to participate in the development of allergic airway inflammation, in which the targets include macrophages, B and T cells, epithelial cells, and mast cells. In addition to the adjuvant effect of DEPs on total and allergen-specific IgE production, DEPs also act to induce chemokines and cytokines and may play a key role in primary sensitization. OBJECTIVE: DEPs have been shown to increase local IL-4-containing Kit(+) cells soon after in vivo nasal challenge. The aim of this study was to examine the effects of DEPs on human basophils, a key source of IL-4. METHODS: Peripheral blood leukocytes from allergic and control subjects were cultured in the presence of organic extracts of DEP (DEPex) with or without allergen. The cultures were analyzed for IL-4-containing cells by using multiparameter flow cytometry, IL-4 secretion with ELISA, and histamine release. RESULTS: Basophils, when exposed in vitro to DEPex, expressed IL-4 and released histamine significantly (P <.01) more than with antigen activation. DEPex did not synergize with allergen in cytokine production and histamine release. DEPex-induced basophil IL-4 expression peaked at 2 hours and persisted through 20 hours, in contrast to allergen-induced IL-4, which was transient. The effect of DEPex on basophil cytokine expression and histamine release was dose dependent and occurred with cells from both allergic and nonallergic subjects. DEPex induced IL-4 expression and histamine release in highly enriched basophil populations, suggesting it acts directly on basophils. Other peripheral blood leukocytes, including T cells, did not contribute to this cytokine expression. Preincubation with N-acetylcysteine completely abrogated DEPex-driven basophil IL-4 expression. CONCLUSIONS: Basophils are a direct target for DEPex, inducing IL-4 expression and histamine release in an IgE-allergen independent fashion. N-acetylcysteine inhibition of DEPex-driven IL-4 expression provides evidence that generation of reactive oxygen species is required for the effects observed. The capability of DEPex to activate basophils in both allergic and nonallergic subjects suggests a potential role of this pollutant in the increasing prevalence of allergic diseases.     

Antithrombotic properties of trillium coated connectors

Trillium coating (Medtronic Inc., Minneapolis, MN) offers, in addition to the presence of heparin, endothelium-like properties of its negatively charged surface. Its thromboresistant properties on coated connectors are tested here and compared with uncoated standard connectors, as well as with the Carmeda BioActive surface (CBAS) heparin surface coating. A partial cardiopulmonary bypass bovine model (body weight 68 +/- 5 kg) was selected, and the surfaces were exposed to the blood stream (pump flow 3.5 L/min) for up to 350 minutes without systemic heparinization. Thereafter, another set of samples was exposed to stagnant blood for 20 minutes. Besides hemodynamic, hematologic, and biochemical analyses, the macroscopic appearance of 45 blood exposed surface samples were graded semiquantitatively on a scale of 0 to 10: no macroscopic deposits = grade 0, one spot (1 mm diameter) = grade 1, two spots = grade 2, five or more spots = grade 5, 10% of the surface covered with clots = grade 6, 100% covered = grade 10. When exposed to blood flow, Trillium and CBAS coatings showed a statistically significant (p = 0.03) better thromboresistance (score: 0 +/- 0 for both) than uncoated connectors (score: 0.8 +/- 1.5) in this nonheparinized model. The same holds true when the connectors were exposed to stagnant blood (score: 0 +/- 0 for both coatings vs 4.3 +/- 2.8 for controls; p = 0.03). Therefore, Trillium coating exhibits significant antithrombotic properties that outperform standards for connectors used in clinical perfusion.     

The relative contribution of direct and indirect antigen recognition pathways to the alloresponse and graft rejection depends upon the nature of the transplant

In this study, we measured direct and indirect T-cell alloresponses mediated by CD4(+) and CD8(+) T cells in three mouse transplantation models: skin, cornea, and retina. We show that the contribution of direct and indirect antigen recognition pathways to the alloresponse to fully allogeneic grafts varies depending upon the nature of the tissue/organ transplanted. The implications of this finding for understanding the cellular mechanisms by which rejection is mediated in different transplant models are discussed.     

Prevalence of transmitted HIV-1 drug resistance and the role of resistance algorithms: data from seroconverters in the CASCADE collaboration from 1987 to 2003

OBJECTIVES: To examine factors influencing the rate of transmitted drug resistance (TDR) among seroconverters, with particular emphasis on 3 widely used genotypic drug resistance algorithms. METHODS: The study used data from CASCADE (Concerted Action on Seroconversion to AIDS and Death in Europe), a collaboration of seroconverter cohorts in Europe and Canada. Genotypic resistance data were derived within 18 months of the last seronegative test or date of laboratory evidence of acute infection and before the initiation of antiretroviral therapy. The Stanford algorithm was used to analyze each individual's nucleotide sequence. A multivariate logistic model was used to assess independent relationships between the presence of TDR and exposure category, sex, age at seroconversion, and year of seroconversion. The paper also describes 3 alternative definitions of resistance: the Stanford algorithm, the key resistance mutations defined by the International AIDS Society, and the Agence Nationale de Recherches sur le Sida (ANRS) algorithm. RESULTS: Forty-five of 438 patients (10.3%) seroconverting between 1987 and 2003 were infected with a drug-resistant HIV-1 variant. Forty patients (9.1%) showed resistance mutations to only 1 class of antiretroviral drugs, 2 (0.5%) to 2 classes, and 3 (0.7%) to 3 classes of antiretroviral therapy. It was suggested that individuals seroconverting later in calendar time were more likely to have TDR (relative risk 3.89 and 95% CI: 0.84 to 18.02, and relative risk 4.69 and 95% CI: 1.03 to 21.31, for 1996-1999 and 2000-2003, respectively, compared with pre-1996; P trend = 0.08). This trend was apparent regardless of the definition of TDR used. The total estimated proportion of individuals with TDR varied between 10.3% and 15.5% according to which definition was used. CONCLUSIONS: Evidence was found for the rise of TDR over time. A specific definition of what constitutes TDR rather than a simple list of mutations is needed.