Biodegradable Particles as Vaccine Delivery Systems: Size Matters

Poly(lactide-co-glycolide) (PLGA) particles have strong potential as antigen delivery systems. The size of PLGA particles used to vaccinate mice can affect the magnitude of the antigen-specific immune response stimulated. In this study, we fabricated and characterized 17 μm, 7 μm, 1 μm, and 300 nm PLGA particles coloaded with a model antigen ovalbumin (OVA) and CpG oligodeoxynucleotides (CpG ODN). PLGA particles demonstrated a size-dependent burst release followed by a more sustained release of encapsulated molecules. PLGA particles that were 300 nm in size showed the highest internalization by, and maximum activation of, dendritic cells. The systemic antigen-specific immune response to vaccination was measured after administration of two intraperitoneal injections, 7 days apart, of 100 μg OVA and 50 μg CpG ODN in C57BL/6 mice. In vivo studies showed that 300 nm sized PLGA particles generated the highest antigen-specific cytotoxic T cell responses by days  14 and 21. These mice also showed the highest IgG2a:IgG1 ratio of OVA-specific antibodies on day  28. This study suggests that the smaller the PLGA particle used to deliver antigen and adjuvants the stronger the antigen-specific cytotoxic T cell response generated.KEY WORDS: CpG ODN, cytotoxic T lymphocytes, dendritic cells, nanoparticles, poly (lactide-co-glycolide), vaccine     

Chitosan is a surprising negative modulator of cytotoxic CD8+ T cell responses elicited by adenovirus cancer vaccines

Adjuvants modulate protective CD8(+) T cell responses generated by cancer vaccines. We have previously shown that immunostimulatory cytosine-phosphodiester-guanine (CpG) oligodeoxynucleotide (ODN) significantly augments tumor protection in mice given adenovirus cancer vaccines. Here, we examined the impact of chitosan, another candidate vaccine adjuvant, on protection conferred by adenovirus cancer vaccines. Unexpectedly, immunization of mice with adenovirus cancer vaccines in combination with chitosan provided little protection against tumor challenge. This directly correlated with the reduced detection of Ag-specific CD8(+) T cells, interferon-γ (IFN-γ) production, and cytotoxic T cell activity. We ruled out immunosuppressive regulatory T cells since the frequency did not change regardless of whether chitosan was delivered. In mammalian cell lines, chitosan did not interfere with adenovirus transgene expression. However, infection of primary murine bone marrow-derived dendritic cells with adenovirus complexed with chitosan significantly reduced viability, transgene expression, and upregulation of major histocompatability (MHC) class I and CD86. Our in vitro observations indicate that chitosan dramatically inhibits adenovirus-mediated transgene expression and antigen presenting cell activation, which could prevent CD8(+) T cell activation from occurring in vivo. These surprising data demonstrate for the first time that chitosan vaccine formulations can negatively impact the induction of CD8(+) T cell responses via its effect on dendritic cells, which is clinically important since consideration of chitosan as an adjuvant for vaccine formulations is growing.     

Pharmacological, but not genetic, disruptions in 5-HT(2C) receptor function attenuate LPS anorexia in mice

Peripheral administration of bacterial lipopolysaccharide (LPS) elicits anorexia in several species, including rats and mice. There is strong evidence that antagonism of serotonergic activity at 2C receptors (5-HT(2C)R) attenuates LPS anorexia in rats. Here we used pharmacological and genetic approaches to examine the role of the 5-HT(2C)R in LPS anorexia in mice. In Experiment 1, SB 242084, a potent and selective 5-HT(2C) antagonist (0.3 mg/kg) was injected intraperitoneally 15 min before intraperitoneal LPS (2 microg/kg) injections just prior to dark onset in c57BL/6 mice. Food intake was recorded 1, 2 and 4 h after LPS administration. In Experiment 2, we recorded 2, 4 and 24 h food intake following dark onset intraperitoneal LPS (0.125, 0.25, 0.5, 1 and 2 microg/kg) injections in mice with a genetic deletion of 5-HT(2C)R and their WT controls. Our pharmacological results suggest that at least part of the anorexia following peripheral LPS administration is mediated by an increase in 5-HT-ergic activity at the 5-HT(2C)R. Our genetic data, in contrast, suggest that 5-HT(2C)R is not a necessary part of LPS anorexia.     

Vena cava filters and inferior vena cava thrombosis

OBJECTIVE: Retrievable vena cava filters (R-VCF) are a recent addition to the therapeutic armamentarium for the prevention of pulmonary embolism. However, unlike permanent vena cava filters (P-VCF), outcomes data are limited regarding complication rates. METHODS: This was a retrospective comparative analysis of consecutive patients undergoing placement of R-VCF vs P-VCF at Wake Forest University School of Medicine from January 2000 to December 2004. Data collected included demographics, procedural specifics, filter type, indications, and complications. Summary data are expressed as number (percentage) or mean +/- SD. Continuous and categorical variables were analyzed by using t and Fisher exact testing, as appropriate. Four additional patients with vena cava thrombosis were also referred to our institution for treatment during the study period, all with opposed biconical VCFs (OptEase and TrapEase filters) recently placed at other facilities. This last group of patients is described but not included in the analysis. RESULTS: A total of 189 VCF (165 P-VCF and 24 R-VCF) cases were examined. No significant differences in VCF groups were observed according to age, documented hypercoagulability, or concomitant anticoagulation. Significant differences were observed according to sex (30.3% of P-VCF vs 62.5% of R-VCF patients were female), morbid obesity (4.2% of P-VCF vs 25% of R-VCF patients), active malignancy (20% of P-VCF vs 41.7% of R-VCF patients), and indication for VCF placement. Over a median follow-up of 8.5 months, no case of significant hemorrhage, no VCF migration, and four cases of vena cava thrombosis were observed. Vena cava thrombosis was observed more frequently in the presence of R-VCF when compared with P-VCF (12.5% vs 0.6%; P = .007). All observed vena cava thromboses were associated with severe clinical symptoms and occurred in patients who received opposed biconical VCF designs. CONCLUSIONS: In our experience, both P-VCF and R-VCF can be placed safely. Among both permanent and retrievable devices, however, opposed biconical designs seem to be associated with an increased risk for vena cava thrombosis. Although causative factors remain unclear, filter design and resultant flow dynamics may play an important role, because all episodes of vena cava thrombosis occurred in patients with a single-filter design.     

Identification of a virulence-associated determinant, dihydrolipoamide dehydrogenase (lpd), in Mycoplasma gallisepticum through in vivo screening of transposon mutants

To effectively analyze Mycoplasma gallisepticum for virulence-associated determinants, the ability to create stable genetic mutations is essential. Global M. gallisepticum mutagenesis is currently limited to the use of transposons. Using the gram-positive transposon Tn4001mod, a mutant library of 110 transformants was constructed and all insertion sites were mapped. To identify transposon insertion points, a unique primer directed outward from the end of Tn4001mod was used to sequence flanking genomic regions. By comparing sequences obtained in this manner to the annotated M. gallisepticum genome, the precise locations of transposon insertions were discerned. After determining the transposon insertion site for each mutant, unique reverse primers were synthesized based on the specific sequences, and PCR was performed. The resultant amplicons were used as unique Tn4001mod mutant identifiers. This procedure is referred to as signature sequence mutagenesis (SSM). SSM permits the comprehensive screening of the M. gallisepticum genome for the identification of novel virulence-associated determinants from a mixed mutant population. To this end, chickens were challenged with a pool of 27 unique Tn4001mod mutants. Two weeks postinfection, the birds were sacrificed, and organisms were recovered from respiratory tract tissues and screened for the presence or absence of various mutants. SSM is a negative-selection screening technique whereby those mutants possessing transposon insertions in genes essential for in vivo survival are not recovered from the host. We have identified a virulence-associated gene encoding dihydrolipoamide dehydrogenase (lpd). A transposon insertion in the middle of the coding sequence resulted in diminished biologic function and reduced virulence of the mutant designated Mg 7.     

Long-term parental and family adaptation following pediatric brain injury

OBJECTIVE: To determine whether parents of children with traumatic brain injuries (TBI) report increased injury-related burden, distress, and family dysfunction and to examine the effects of attrition on the results. METHODS: Children with severe TBI, moderate TBI, and orthopedic injuries were followed at six time points from baseline to 6 years after injury. Parents completed measures of injury-related burden, psychological distress, and family functioning at each assessment. Mixed model analysis was used to examine long-term changes. RESULTS: Attrition was higher among families in the severe TBI group with lower burden thereby amplifying group differences. The severe TBI group reported higher injury-related burden over time after injury than the other groups. Family functioning was moderated by social resources. Families of children with severe TBI and low resources reporting deteriorating functioning over the follow-up interval. CONCLUSIONS: Although environmental advantages moderate long-term effects on family functioning, families of children with severe TBI experience long-standing injury-related burden.     

The Oryza bacterial artificial chromosome library resource: construction and analysis of 12 deep-coverage large-insert BAC libraries that represent the 10 genome types of the genus Oryza

Rice (Oryza sativa L.) is the most important food crop in the world and a model system for plant biology. With the completion of a finished genome sequence we must now functionally characterize the rice genome by a variety of methods, including comparative genomic analysis between cereal species and within the genus Oryza. Oryza contains two cultivated and 22 wild species that represent 10 distinct genome types. The wild species contain an essentially untapped reservoir of agriculturally important genes that must be harnessed if we are to maintain a safe and secure food supply for the 21st century. As a first step to functionally characterize the rice genome from a comparative standpoint, we report the construction and analysis of a comprehensive set of 12 BAC libraries that represent the 10 genome types of Oryza. To estimate the number of clones required to generate 10 genome equivalent BAC libraries we determined the genome sizes of nine of the 12 species using flow cytometry. Each library represents a minimum of 10 genome equivalents, has an average insert size range between 123 and 161 kb, an average organellar content of 0.4%-4.1% and nonrecombinant content between 0% and 5%. Genome coverage was estimated mathematically and empirically by hybridization and extensive contig and BAC end sequence analysis. A preliminary analysis of BAC end sequences of clones from these libraries indicated that LTR retrotransposons are the predominant class of repeat elements in Oryza and a roughly linear relationship of these elements with genome size was observed.