DNA fusion vaccines enter the clinic

Induction of effective immune attack on cancer cells in patients requires conversion of weak tumor antigens into strong immunogens. Our strategy employs genetic technology to create DNA vaccines containing tumor antigen sequences fused to microbial genes. The fused microbial protein engages local CD4+ T cells to provide help for anti-tumor immunity, and to reverse potential regulation. In this review, we focus on induction of CD8+ T cells able to kill target tumor cells. The DNA vaccines incorporate tumor-derived peptide sequences fused to an engineered domain of tetanus toxin. In multiple models, this design induces strong CD8+ T-cell responses, able to suppress tumor growth. For clinical relevance, we have used “humanized” mice expressing HLA-A2, successfully inducing cytolytic T-cell responses against a range of candidate human peptides. To overcome physical restriction in translating to patients, we have used electroporation. Clinical trials of patients with cancer are showing induction of responses, with preliminary indications of suppression of tumor growth and evidence for clinically manageable concomitant autoimmunity.     

Arginine deficiency causes runting in the suckling period by selectively activating the stress kinase GCN2

Suckling "F/A2" mice, which overexpress arginase-I in their enterocytes, develop a syndrome (hypoargininemia, reduced hair and muscle growth, impaired B-cell maturation) that resembles IGF1 deficiency. The syndrome may result from an impaired function of the GH-IGF1 axis, activation of the stress-kinase GCN2, and/or blocking of the mTORC1-signaling pathway. Arginine deficiency inhibited GH secretion and decreased liver Igf1 mRNA and plasma IGF1 concentration, but did not change muscle IGF1 concentration. GH supplementation induced Igf1 mRNA synthesis, but did not restore growth, ruling out direct involvement of the GH-IGF1 axis. In C2C12 muscle cells, arginine withdrawal activated GCN2 signaling, without impacting mTORC1 signaling. In F/A2 mice, the reduction of plasma and tissue arginine concentrations to ～25% of wild-type values activated GCN2 signaling, but mTORC1-mediated signaling remained unaffected. Gcn2-deficient F/A2 mice suffered from hypoglycemia and died shortly after birth. Because common targets of all stress kinases (eIF2α phosphorylation, Chop mRNA expression) were not increased in these mice, the effects of arginine deficiency were solely mediated by GCN2.     

Bacterial-epithelial contact is a key determinant of host innate immune responses to enteropathogenic and enteroaggregative Escherichia coli

Background

Enteropathogenic (EPEC) and Enteroaggregative (EAEC) E. coli have similar, but distinct clinical symptoms and modes of pathogenesis. Nevertheless when they infect the gastrointestinal tract, it is thought that their flagellin causes IL-8 release leading to neutrophil recruitment and gastroenteritis. However, this may not be the whole story as the effect of bacterial adherence to IEC innate response(s) remains unclear. Therefore, we have characterized which bacterial motifs contribute to the innate epithelial response to EPEC and EAEC, using a range of EPEC and EAEC isogenic mutant strains.

Methodology

Caco-2 and HEp-2 cell lines were exposed to prototypical EPEC strain E2348/69 or EAEC strain O42, in addition to a range of isogenic mutant strains. E69 [LPS, non-motile, non-adherent, type three secretion system (TTSS) negative, signalling negative] or O42 [non-motile, non-adherent]. IL-8 and CCL20 protein secretion was measured. Bacterial surface structures were assessed by negative staining Transmission Electron Microscopy. The Fluorescent-actin staining test was carried out to determine bacterial adherence.

Results

Previous studies have reported a balance between the host pro-inflammatory response and microbial suppression of this response. In our system an overall balance towards the host pro-inflammatory response is seen with the E69 WT and to a greater extent O42 WT, which is in fit with clinical symptoms. On removal of the external EPEC structures flagella, LPS, BFP, EspA and EspC; and EAEC flagella and AAF, the host inflammatory response is reduced. However, removal of E69 lymphostatin increases the host inflammatory response suggesting involvement in the bacterial mediated anti-inflammatory response.

Conclusion

Epithelial responses were due to combinations of bacterial agonists, with host-bacterial contact a key determinant of these innate responses. Host epithelial recognition was offset by the microbe''s ability to down-regulate the inflammatory response. Understanding the complexity of this host-microbial balance will contribute to improved vaccine design for infectious gastroenteritis.     

Immunolocalization of influenza A virus and markers of inflammation in the human Parkinson's disease brain

Although much is known regarding the molecular mechanisms leading to neuronal cell loss in Parkinson's disease (PD), the initiating event has not been identified. Prevailing theories including a chemical insult or infectious agent have been postulated as possible triggers, leading to neuroinflammation. We present immunohistochemical data indicating the presence of influenza A virus within the substantia nigra pars compacta (SNpc) from postmortem PD brain sections. Influenza A virus labeling was identified within neuromelanin granules as well as on tissue macrophages in the SNpc. Further supporting a role for neuroinflammation in PD was the identification of T-lymphocytes that colocalized with an antibody to caspase-cleaved Beclin-1 within the SNpc. The presence of influenza A virus together with macrophages and T-lymphocytes may contribute to the neuroinflammation associated with this disease.     

The phagocytosis and toxicity of amorphous silica

Background

Inhalation of crystalline silica is known to cause an inflammatory reaction and chronic exposure leads to lung fibrosis and can progress into the disease, silicosis. Cultured macrophages bind crystalline silica particles, phagocytose them, and rapidly undergo apoptotic and necrotic death. The mechanism by which particles are bound and internalized and the reason particles are toxic is unclear. Amorphous silica has been considered to be a less toxic form, but this view is controversial. We compared the uptake and toxicity of amorphous silica to crystalline silica.

Methodology/Principal Findings

Amorphous silica particles are phagocytosed by macrophage cells and a single internalized particle is capable of killing a cell. Fluorescent dextran is released from endo-lysosomes within two hours after silica treatment and Caspase-3 activation occurs within 4 hours. Interestingly, toxicity is specific to macrophage cell lines. Other cell types are resistant to silica particle toxicity even though they internalize the particles.The large and uniform size of the spherical, amorphous silica particles allowed us to monitor them during the uptake process. In mCherry-actin transfected macrophages, actin rings began to form 1-3 minutes after silica binding and the actin coat disassembled rapidly following particle internalization. Pre-loading cells with fluorescent dextran allowed us to visualize the fusion of phagosomes with endosomes during internalization. These markers provided two new ways to visualize and quantify particle internalization. At 37°C the rate of amorphous silica internalization was very rapid regardless of particle coating. However, at room temperature, opsonized silica is internalized much faster than non-opsonized silica.

Conclusions/Significance

Our results indicate that amorphous and crystalline silica are both phagocytosed and both toxic to mouse alveolar macrophage (MH-S) cells. The pathway leading to apoptosis appears to be similar in both cases. However, the result suggests a mechanistic difference between FcγRIIA receptor-mediated and non-opsonized silica particle phagocytosis.     

Differential regulation of circulating and renal ACE2 and ACE in hypertensive mRen2.Lewis rats with early-onset diabetes

We examined the impact of early diabetes on the circulating and kidney renin-angiotensin system (RAS) in male and female mRen2.Lewis (mRen2) hypertensive rats. Diabetes (DB) was induced by streptozotocin (STZ; 65 mg/kg) at 11 wk of age for 4 wk without insulin replacement. Systolic blood pressures were not increased in DB males or females compared with controls (CON). Circulating angiotensin-converting enzyme 2 (ACE2) increased ninefold (P < 0.05) in DB females and threefold (P < 0.05) in DB males, but circulating ACE and ANG II were higher in the DB groups. Serum C-reactive protein was elevated in DB females but not DB males, and the vascular responses to acetylcholine and estradiol were attenuated in the DB females. Proteinuria, albuminuria, and angiotensinogen excretion increased to a similar extent in both DB females and males. Glomerular VEGF expression also increased to a similar extent in both DB groups. Renal inflammation (CD68(+)cells) increased only in DB females although males exhibited greater inflammation that was not different with DB. Cortical ACE2 did not change in DB females but was reduced (30%) in DB males. Renal neprilysin activity (>75%, P < 0.05) was markedly reduced in the DB females to that in the DB and CON males. ACE activity was significantly lower in both female (75%, P < 0.05) and male (50%; P < 0.05) DB groups, while cortical ANG II and Ang-(1-7) levels were unchanged. In conclusion, female mRen2 rats are not protected from vascular damage, renal inflammation, and kidney injury in early STZ-induced diabetes despite a marked increase in circulating ACE2 and significantly reduced ACE within the kidney.     

A logistic delay differential equation model for Chagas disease with interrupted spraying schedules

This work studies a mathematical model for the dynamics of Chagas disease, a parasitic disease that affects humans and domestic mammals throughout rural areas in Central and South America. It presents a modified version of the model found in Spagnuolo et al. [A model for Chagas disease with controlled spraying, J. Biol. Dyn. 5 (2011), pp. 299-317] with a delayed logistic growth term, which captures an overshoot, beyond the vector carrying capacity, in the total vector population when the blood meal supply is large. It studies the steady states of the system in the case of constant coefficients without spraying, and the analysis shows that for given-averaged parameters, the endemic equilibrium is stable and attracting. The numerical simulations of the model dynamics with time-dependent coefficients are shown when interruptions in the annual insecticide spraying cycles are taken into account. Simulations show that when there are spraying schedule interruptions, spraying may become ineffective when the blood meal supply is large.     

Speech spectrum's correlation with speakers' Eysenck personality traits

The current study explored the correlation between speakers' Eysenck personality traits and speech spectrum parameters. Forty-six subjects completed the Eysenck Personality Questionnaire. They were instructed to verbally answer the questions shown on a computer screen and their responses were recorded by the computer. Spectrum parameters of /sh/ and /i/ were analyzed by Praat voice software. Formant frequencies of the consonant /sh/ in lying responses were significantly lower than that in truthful responses, whereas no difference existed on the vowel /i/ speech spectrum. The second formant bandwidth of the consonant /sh/ speech spectrum was significantly correlated with the personality traits of Psychoticism, Extraversion, and Neuroticism, and the correlation differed between truthful and lying responses, whereas the first formant frequency of the vowel /i/ speech spectrum was negatively correlated with Neuroticism in both response types. The results suggest that personality characteristics may be conveyed through the human voice, although the extent to which these effects are due to physiological differences in the organs associated with speech or to a general Pygmalion effect is yet unknown.