CLPFFD-PEG functionalized NIR-absorbing hollow gold nanospheres and gold nanorods inhibit ß-amyloid aggregation.

Gold nanoparticles with specific optical properties in combination with the CLPFFD peptide that exhibits selectivity for ß-amyloid (Aß) aggregates are promising photothermal absorbers for application in Alzheimer's disease therapy. We report on hollow gold nanospheres (HAuNS) and gold nanorods (AuNR), which exhibit strong plasmonic near infrared (NIR) absorbance in the optical window of biological tissue and which are functionalized with CLPFFD in two different ways. Therefore the peptide was either directly bound to the particle surface or indirectly to a particle-protecting polyethylene glycol (PEG) ligand shell, thereby reducing the CLPFFD density on the surfaces of both types of particles. Fully PEGylated particles were used for comparison. The effects on cell viability and the fundamental suitability of the HAuNS and AuNR conjugates as photothermal absorbers to inhibit Aß-fibrillation are analysed in vitro. The positive influence of the use of PEG ligands on the reduced cytotoxicity of the conjugates and on the Aß-disaggregation is discussed.

Unique transcriptional profile of liver-resident memory CD8+ T cells induced by immunization with malaria sporozoites.

Sterile immunity against live Plasmodium infection can be achieved by immunization with radiation-attenuated sporozoites. This protection is known to be mediated in part by antigen-specific memory CD8(+) T cells, presumably those residing in the liver. We characterized and compared the transcriptional profile of parasite-specific memory CD8(+) T cells residing in the liver and spleen after immunization of mice with irradiated sporozoites. Microarray-based expression analysis of these memory CD8(+) T cells indicated that liver-resident memory cells display a distinct gene expression profile. We found major differences in the expression of immune function genes as well as genes involved in the cell cycle, cell trafficking, transcription and intracellular signaling. Importantly, the malaria parasite-induced liver-resident CD8(+) T cells display a transcriptional profile different to that described for CD8(+) T cells following other microbial challenges.

Protective and pathogenic roles of CD8+ T cells during malaria infection.

CD8+ T cells play a key role in protection against pre-erythrocytic stages of malaria infection. Many vaccine strategies are based on the idea of inducing a strong infection-blocking CD8+ T cell response. Here, we summarize what is known about the development, specificity and protective effect of malaria-specific CD8+ T cells and report on recent developments in the field. Although work in mouse models continues to make progress in our understanding of the basic biology of these cells, many questions remain to be answered - particularly on the roles of these cells in human infections. Increasing evidence is also emerging of a harmful role for CD8+ T cells in the pathology of cerebral malaria in rodent systems. Once again, the relevance of these results to human disease is one of the primary questions facing workers in this field.

Peptide-based subunit vaccines against pre-erythrocytic stages of malaria parasites.

Malaria currently ranks among the most prevalent infections in tropical and sub-tropical areas throughout the world with relatively high morbidity and mortality particularly in young children. The widespread occurrence and the increased incidence of malaria in many countries, caused by drug-resistant parasites (Plasmodium falciparum and P. vivax) and insecticide-resistant vectors (Anopheles mosquitoes), indicate the need to develop new methods of controlling this disease. Experimental vaccination with radiation-attenuated sporozoites can protect animals and humans against the disease, demonstrating the feasibility of developing an effective malaria vaccine. However, vaccines based on radiation-attenuated sporozoites are not feasible for large scale application due to lack of in vitro culture system. Therefore, the development of peptide-based subunit vaccines has been undertaken as an alternative approach. Synthetic peptides containing defined B- and T-cell epitopes of different antigens expressed in sporozoites and/or liver stages have been used as subunit vaccines in experimental animal models. They have been shown to be highly immunogenic and capable of inducing protective immunity mediated by antibodies, as well as CD4+ and CD8+ T-cells.

Detection of malaria liver-stages in mice infected through the bite of a single Anopheles mosquito using a highly sensitive real-time PCR.

We describe a highly sensitive real-time PCR to detect and measure the development of the liver-stages of malaria parasites in mice infected with sporozoites ranging in number from 25 to more than 164,000, using the same reaction conditions. Furthermore, this assay detects and measures parasite loads in the livers of mice exposed to the bite of a single malaria-infected Anopheles mosquito. This unique method should greatly facilitate studies aimed at evaluating very precisely the efficacy of anti-malarial experimental drug treatments and vaccination regimens in conditions of infection resembling those found in the field.

Swift development of protective effector functions in naive CD8(+) T cells against malaria liver stages.

We generated T cell receptor transgenic mice specific for the liver stages of the rodent malaria parasite Plasmodium yoelii and studied the early events in the development of in vivo effector functions in antigen-specific CD8(+) T cells. Differently to activated/memory cells, naive CD8(+) T cells are not capable of exerting antiparasitic activity unless previously primed by parasite immunization. While naive cells need to differentiate before achieving effector status, the time required for this process is very short. Indeed, interferon (IFN)-gamma and perforin mRNA are detectable 24 h after immunization and IFN-gamma secretion and cytotoxic activity are detected ex vivo 24 and 48 h after immunization, respectively. In contrast, the proliferation of CD8(+) T cells begins after 24 h and an increase in the total number of antigen-specific cells is detected only after 48 h. Remarkably, a strong CD8(+) T cell-mediated inhibition of parasite development is observed in mice challenged with viable parasites only 24 h after immunization with attenuated parasites. These results indicate that differentiation of naive CD8(+) T cells does not begin only after extensive cell division, rather this process precedes or occurs simultaneously with proliferation.

Evaluation of a first aid and home safety program for Hispanic migrant adolescents.

BACKGROUND: Unintentional injuries are the major cause of death among children, adolescents, and young adults. This article presents an evaluation of an injury-prevention program for 11- to 16-year-old, Hispanic migrant youth. DESIGN: Randomized controlled trial with two conditions: first aid and home safety training and tobacco and alcohol prevention. Participants were assessed at baseline, at immediate post-intervention, and at 1-year follow-up. PARTICIPANTS: A total of 660 Hispanic adolescent and parent pairs participated in a program entitled Sembrando Salud (sowing the seeds of health). INTERVENTION: The intervention consisted of two conditions: first aid and home safety training and tobacco and alcohol prevention. Both groups were exposed to an eight-session, multimedia program presented by bilingual, bicultural college students. The sessions consisted of lectures, discussions, and skills development and practice. OUTCOME MEASURES: To examine the efficacy of the first aid and home safety intervention, adolescents were assessed for changes in first aid confidence, knowledge of items in a first aid kit, knowledge of how to respond in an emergency situation, acquisition of a first aid kit, and behavioral skills testing in response to two emergency scenarios. RESULTS: Similar changes in confidence were observed in both groups after the intervention. Participants in the first aid and home safety program were better able to identify items to include in a first aid kit, how to respond in an emergency situation, and reported fewer erroneous victim-caring procedures than the tobacco and alcohol prevention group. CONCLUSIONS: Sembrando Salud was successful at achieving and maintaining change in confidence and knowledge of first aid and emergency response skills over a yearlong period.

ELISPOT assay to measure antigen-specific murine CD8(+) T cell responses.

The enzyme-linked immunospot technique (ELISPOT) relies on the visualization of cytokine secretion by individual T cells following in vitro stimulation with antigen. This assay has been developed and standardized for the quantitative detection of antigen-specific CD8(+) T cells in mice subjected to different immunization protocols [J. Immunol. Methods 181 (1995) 45]. We have identified important variables that affect the efficacy of the ELISPOT assay and in this protocol we describe this methodology in detail. As a model, we used the production of interferon-gamma by CD8(+) T cells from peripheral blood, spleen and liver of mice immunized with malaria sporozoites expressing the H-2K(d)-restricted SYVPSAEQI. This protocol has also been used successfully to detect Th1 and Th2 epitope specific CD4(+) T cells.

Evaluation of a tobacco and alcohol use prevention program for Hispanic migrant adolescents: promoting the protective factor of parent-child communication.

BACKGROUND: Interventions designed to prevent tobacco and alcohol use targeting high-risk adolescents are limited. In addition, few studies have attempted to improve parent-child communication skills as a way of improving and maintaining healthy youth decision-making. METHODS: A total of 660 Hispanic migrant families participated in a randomized pre-post control group study that was utilized to determine the impact of the intervention on parent-child communication. Both treatment and attention-control groups of youth were exposed to an eight-session culturally sensitive program presented by bilingual/bicultural college students. Parents jointly attended three of the eight sessions and participated in helping their child complete homework assignments supporting the content of each session. The content of the treatment intervention included (1) information about tobacco and alcohol effects, (2) social skills training (i.e., refusal skills), and (3) the specific development of parent-child communication skills to support healthy youth decisions. RESULTS: Significant intervention by household size interactions for both parent and youth perceptions of communication were found indicating that the treatment was effective in increasing communication in families with fewer children. Based on the effect size and the previously established relationship between communication and susceptibility to tobacco and alcohol use, it was determined that the intervention effect could be translated into a future 5 to 10% decrease in susceptibility for these smaller families. CONCLUSIONS: A culturally sensitive family-based intervention for migrant Hispanic youth was found to be effective in increasing perceived parent-child communication in families with fewer children. It is expected that increases in this important protective factor will lead to later observed decreases in tobacco and alcohol use.

Immunogenicity of Ty-VLP bearing a CD8(+) T cell epitope of the CS protein of P. yoelii: enhanced memory response by boosting with recombinant vaccinia virus.

We characterized the immunogenicity of the hybrid Ty-virus-like carrying the CD8(+) T cell epitope (SYVPSAEQI) of the circumsporozoite (CS) protein of Plasmodium yoelii (TyCS-VLP), a rodent malaria parasite. Balb/c mice were immunized with hybrid TyCS-VLP, and their CS-specific CD8(+) T cell response was quantitatively evaluated with the ELISPOT assay, based on the enumeration of epitope specific gamma-interferon secreting CD8(+) T cell. A single immunization with the TyCS-VLP by a variety of routes and doses indicated that the maximal response occurred in mice, which were immunized with 50 micrograms of these particles, administered via intramuscular. Combined immunization of mice with this TyCS-VLP followed by recombinant vaccinia virus expressing the entire P. yoelii CS protein (VacPyCS) or irradiated sporozoites, induced high levels of IFN-gamma-producing cells. The immunization regime, priming with TyCS-VLP and boosting with VacPyCS generated a potent protective immune response, which strongly inhibited P. yoelii liver stages development and protected 62% of the mice against a subsequent live P. yoelii sporozoite challenge.

Granulocyte-colony stimulating factor treatment of lupus autoimmune disease in MRL-lpr/lpr mice.

G-CSF not only functions as an endogenous hemopoietic growth factor for neutrophils, but also displays pro-Th2 and antiinflammatory properties that could be of therapeutic benefit in autoimmune settings. We evaluated the effect of treatment with G-CSF in a murine model of spontaneous systemic lupus erythematosus, a disease in which G-CSF is already administered to patients to alleviate neutropenia, a common complication. Chronic treatment of lupus-prone MRL-lpr/lpr mice with low doses (10 microg/kg) of recombinant human G-CSF, despite the induction of a shift toward the Th2 phenotype of the autoimmune response, increased glomerular deposition of Igs and accelerated lupus disease. Conversely, high-dose (200 microg/kg) treatment with G-CSF induced substantial protection, prolonging survival by >2 mo. In the animals treated with these high doses of G-CSF, neither the Th1/Th2 profile nor the serum levels of TNF-alpha and IL-10 were modified. Despite the presence of immune complexes in their kidney glomeruli, no inflammation ensued, and serum IL-12 and soluble TNF receptors remained at pre-disease levels. This uncoupling of immune complex deposition and kidney damage resulted from a local down-modulation of FcgammaRIII (CD16) expression within the glomeruli by G-CSF. Our results demonstrate a beneficial effect of high doses of G-CSF in the prevention of lupus nephritis that may hold promise for future clinical applications, provided caution is taken in dose adjustment.

IL-12 delivery from recombinant vaccinia virus attenuates the vector and enhances the cellular immune response against HIV-1 Env in a dose-dependent manner.

To develop vaccination strategies against HIV-1 infection aimed to specifically enhance the cell-mediated immunity (CMI), we have engineered vaccinia virus (VV) recombinants expressing HIV-1 Env (rVVenv) and murine IL-12 (rVVlucIL-12) genes or coexpressing both genes (rVVenvIL-12). In mice inoculated with rVVlucIL-12 there is a rapid clearance of the virus, and this correlates with the induction of high levels of IL-12 and IFN-gamma in serum and spleen early after infection. Enzyme-linked immunospot analysis of mice inoculated with rVVlucIL-12, revealed a nearly 2-fold increase in the number of specific anti-VV CD8+ T cells compared with that in mice given control rVV, and the serum Ab response was biased in favor of a Th1 response. An enhancement of about 2-fold in the number of anti-gp160 IFN-gamma-secreting CD8+ T cells was observed in mice inoculated with rVVenvIL-12, when a dose of 1 x 107 PFU/mouse was used, but this enhancement was not observed when mice were given 5 x 107 PFU. This variation with virus dosage was confirmed in mice immunized simultaneously with different multiplicities of rVV expressing singly the env or IL-12 genes. The highest specific CMI was obtained in mice coadministered a low dose (2 x 104 PFU) of rVVlucIL-12 and 1 x 107 PFU of rVVenv. Our findings provide evidence for specific enhancement of the CMI to HIV-1 Env by the differential expression of IL-12 and env genes delivered from VV recombinants. This approach can be of wide vaccination interest as a means to improve immune responses to other Ags.

Soluble tumor necrosis factor receptors in chronic hepatitis C: a correlation with histological fibrosis and activity.

BACKGROUND/AIMS: Tumor necrosis factor-alpha (TNF) is a mediator of inflammation and cellular immune response. Soluble TNF receptors (sTNFR) sTNF-R55 and sTNF-R75, which compete with cellular receptors for the binding of TNF, have been detected at high levels in infectious diseases including human immunodeficiency virus and HBV infection. In order to investigate the activation of the TNF system in HCV infection, we have analyzed the balance between TNF and sTNF-R in 60 HCV-infected subjects according to their clinical, biological, virological and histological characteristics. METHODS: Serum TNF, sTNF-R55 and sTNF-R75 levels were determined by ELISA before any therapy and were compared to a control group of 60 healthy subjects and a group of 34 HBV-infected patients. RESULTS: Mean TNF levels were 50.5+/-4.5 pg/ml in HCV patients, and undetectable (<5 pg/ml) in the control subjects. sTNF-R55 and sTNF-R75 levels were significantly higher in HCV-infected patients than in the controls: 2.88+/-0.14 ng/ml vs. 1.30+/-0.05, (p = 0.0001), and 9.54+/-0.58 ng/ml vs. 4.19+/-016, (p = 0.0001), respectively. sTNF-R55 and TNF-alpha levels in HCV patients were not significantly different from levels in HBV patients. sTNF-R75 levels were slightly lower than in HBV patients (9.54+/-0.58 vs. 11.4+/-0.79 ng/ml, p = 0.03). In contrast to other infectious diseases, there was no correlation between levels of sTNF-R and TNF. sTNF-R75 but not TNF levels were correlated with aminotransferases levels (p = 0.0001 and p = 0.0015 for aspartate and alanine aminotransferase, respectively), while sTNF-R55 levels were significantly correlated only with aspartate aminotransferase levels (p = 0.003). sTNF-R75 levels were significantly correlated with the Metavir activity index (p = 0.01), and sTNF-R55 and sTNF-R75 levels were significantly higher in patients with vs. without cirrhosis (3.22+/-0.21 vs. 2.54+/-0.17 ng/ml (p<0.02) and 11.6+/-0.86 vs. 7.5+/-0.53 ng/ml (p<0.001), respectively). sTNF-R55, sTNF-R75 and TNF levels were not correlated with viral load, genotype or response to interferon therapy. CONCLUSIONS: Levels of soluble TNF receptors, and particularly sTNF-R75, are significantly correlated with the severity of the disease but not with virological parameters such as quantitative viremia and genotype. High TNF-R production could thus suggest that HCV-related liver disease involves immunological mechanisms, including activation of the TNF system.

Phenotypic and functional characterization of CD8(+) T cell clones specific for a mouse cytomegalovirus epitope.

A series of CD8(+) T cell clones, specific for the IE1 epitope YPHFMPTNL, of the immediate-early protein 1 of the murine cytomegalovirus (MCMV) were generated in order to determine their protective activity against this infection and correlate their phenotypic markers with antiviral activity. We found that the adoptive transfer of three of these anti-MCMV CD8(+) T cell clones into irradiated naive mice resulted in protection against challenge, while another CD8(+) T cell clone, of the same specificity, failed to confer protection. The clones that conferred protection against lethal challenge reduced greatly viral replication in the lung and other organs of the mice. Using one of the protective anti-MCMV CD8(+) T cell clones we found that in order to be fully protective the cells had to be transferred to recipient mice no later than 1 day after MCMV challenge. The adoptive transfer of these CD8(+) T cell clones also protected CD4(+) T-cell-depleted mice. Phenotypic characterization of the anti-MCMV clones revealed that the nonprotective clone expressed very low levels of CD8 molecules and produced only small amounts of TNF-alpha upon antigenic stimulation. Most importantly, our current study demonstrates that this MHC class I-restricted IE1 epitope of MCMV is efficiently presented to CD8(+) T cell clones in vivo and further strengthens the possibility of the potential use of CD8(+) T cell clones as immunotherapeutic tools against cytomegalovirus-induced disease.

Pre-erythrocytic malaria vaccine: mechanisms of protective immunity and human vaccine trials.

In order to provide a rational basis for the development of a pre-erythrocytic malaria vaccine we have aimed at: (a) elucidating the mechanisms of protection, and (b) identifying vaccine formulations that best elicit protection in experimental animals and humans. Based on earlier successful immunization of experimental animals with irradiated sporozoites, human volunteers were exposed to the bites of large numbers of Plasmodium falciparum or P. vivax infected irradiated mosquitoes. The result of this vaccine trial demonstrated for the first time that a pre-erythrocytic vaccine, administered to humans, can result in their complete resistance to malaria infection. However, since infected irradiated mosquitoes are unavailable for large scale vaccination, the alternative is to develop subunit vaccines. The human trials using irradiated sporozoites provided valuable information on the human immune responses to pre-erythrocytic stages and studies on mice an excellent experimental model to characterize protective immune mechanisms. The circumsporozoite protein, the first pre-erythrocytic antigen identified, is present in all malaria species, displaying a similar structure, with a central region of repeats, and two conserved regions, essential for parasite development. Most pre-erythrocytic vaccine candidates are based on the CS protein, expressed in various cell lines, microorganisms, and recently the corresponding DNA. We and others have identified CS-specific B and T cell epitopes, recognized by the rodent and human immune systems, and used them for the development of synthetic vaccines. We used synthetic peptide vaccines, multiple antigen peptides and polyoximes, for immunization, first in experimental animals, and recently in two human safety and immunogenicity trials. We also report here on our work on T cell mediated immunity, particularly the protection of mice immunized with viral vectors expressing CS-specific cytotoxic CD8+ T cell epitopes, and the striking booster effect of recombinant vaccinia virus. To what degree CD8+ T cells, and/or other T cells specific for sporozoites and/or liver stage epitopes, contribute to pre-erythrocytic protective immunity in humans, remains to be determined.

Induction and inhibition of CD40-CD40 ligand interactions: a new strategy underlying host-virus relationships.

Interaction between CD40 and the CD40 ligand (CD40L) is required for mouse mammary tumor virus (MMTV) propagation. We found that Fas was expressed on B cells and CD40L on a small subset of viral superantigen-cognate T cells 12 h after MMTV(SW) infection. CD40L and Fas were down-regulated after 24 h. All CD4 T cells then became resistant to anti-CD3-induced CD40L induction in vitro for 2 wk. Initiation of CD40L expression and its rapid shut-off was associated with IL-12 production and was controlled by IFN-gamma and shedding of soluble CD40. These results suggest that a rapid, transient CD40-CD40L interaction involving a small number of cells is sufficient for MMTV propagation. Modulation of CD40L expression may be a major mechanism regulating the balance between viral propagation and host defenses, allowing mutual survival.

Efficient induction of protective anti-malaria immunity by recombinant adenovirus.

The immunogenicity of a previously constructed replication-defective recombinant adenovirus expressing the CS protein of Plasmodium yoelii was compared with that of irradiated sporozoites. We found that immunization of BALB/c mice with a single dose of this recombinant adenovirus induced a much greater CS-specific T-cell response compared with immunization with irradiated sporozoites. More importantly, we found that this recombinant adenovirus induces similar or higher levels of protective immunity than those induced by irradiated sporozoites, eliciting an appreciable resistance to malaria infection.