From animal studies into clinical trials: the relevance of animal models to develop vaccines and therapies to reduce disease severity and prevent hRSV infection.

INTRODUCTION: Human respiratory syncytial virus (hRSV) is an important cause of lower respiratory tract infections in the pediatric and the geriatric population worldwide. There is a substantial economic burden resulting from hRSV disease during winter. Although no vaccines have been approved for human use, prophylactic therapies are available for high-risk populations. Choosing the proper animal models to evaluate different vaccine prototypes or pharmacological treatments is essential for developing efficient therapies against hRSV. AREAS COVERED: This article describes the relevance of using different animal models to evaluate the effect of antiviral drugs, pharmacological molecules, vaccine prototypes, and antibodies in the protection against hRSV. The animal models covered are rodents, mustelids, bovines, and nonhuman primates. Animals included were chosen based on the available literature and their role in the development of the drugs discussed in this manuscript. EXPERT OPINION: Choosing the correct animal model is critical for exploring and testing treatments that could decrease the impact of hRSV in high-risk populations. Mice will continue to be the most used preclinical model to evaluate this. However, researchers must also explore the use of other models such as nonhuman primates, as they are more similar to humans, prior to escalating into clinical trials.

Maternal hypothyroxinemia impairs spatial learning and synaptic nature and function in the offspring.

Neurological deficits in the offspring caused by human maternal hypothyroxinemia are thought to be irreversible. To understand the mechanism responsible for these neurological alterations, we induced maternal hypothyroxinemia in pregnant rats. Behavior and synapse function were evaluated in the offspring of thyroid hormone-deficient rats. Our data indicate that, when compared with controls, hypothyroxinemic mothers bear litters that, in adulthood, show prolonged latencies during the learning process in the water maze test. Impaired learning capacity caused by hypothyroxinemia was consistent with cellular and molecular alterations, including: 1) lack of increase of phosphorylated c-fos on the second day of the water maze test; 2) impaired induction of long-term potentiation in response to theta-burst stimulation to the Schaffer collateral pathway in the area 1 of the hippocampus Ammon's horn stratum radiatum, despite normal responses for input/output experiments; 3) increase of postsynaptic density protein 95 (PSD-95), N-methyl-D-aspartic acid receptor subunit 1, and tyrosine receptor kinase B levels in brain extracts; and 4) significant increase of PSD-95 at the PSDs and failure of this molecule to colocalize with N-methyl-D-aspartic acid receptor subunit 1, as it was shown by control rats. Our findings suggest that maternal hypothyroxinemia is a harmful condition for the offspring that can affect key molecular components for synaptic function and spatial learning.

In vitro removal of human IgG autoantibodies by affinity filtration using immobilized L-histidine onto PEVA hollow fiber membranes.

Histidine was immobilized onto PEVA membrane to obtain an affinity support for human IgG removal from serum with a view to clinical apheresis for the treatment of autoimmune diseases. These membranes were able to remove in vitro several autoantibodies from the serum of SLE patients.

Cytomegalovirus antigenemia in acquired immunodeficiency syndrome patients with untreated cytomegalovirus retinitis.

PURPOSE: To determine the frequency of cytomegalovirus (CMV) viremia in patients with acquired immunodeficiency syndrome (AIDS) and untreated CMV retinitis using conventional cell culture isolation and the sensitive CMV antigenemia assay. METHODS: We examined 24 AIDS patients with ophthalmologic diagnosis of untreated CMV retinitis and 24 AIDS patients without present or past retinitis (control patients) from three medical centers between September 1992 and March 1994. Cytomegalovirus antigenemia was detected by an indirect peroxidase staining in 300,000 cytocentrifuged neutrophils, using a mixture of murine monoclonal antibodies directed against the pp65 lower matrix protein of CMV. RESULTS: Positive antigenemia was demonstrated in eight (33.3%) of the 24 retinitis patients and in none of the 24 control patients (P < .001). Only two of the eight antigenemia-positive patients had a concurrent positive CMV isolation from blood leukocytes by conventional cell culture assay. CONCLUSIONS: These results emphasize the risk of extraocular disease in AIDS patients with CMV retinitis because the virus is often present in peripheral blood leukocytes. The CMV antigenemia assay may be a simple and rapid means of identifying those patients with unilateral retinitis at highest risk of developing CMV retinitis of the fellow eye or of visceral CMV disease if intravitreal injections or implants are used as sole treatment for CMV retinitis.

In vitro removal of human IgG by pseudobiospecific affinity membrane filtration on a large scale. A preliminary report.

We have developed a pseudobiospecific affinity membrane device for selective removal of human IgG from plasma or serum in vitro for clinical apheresis application. The pseudobiospecific affinity ligand L-histidine was immobilized through an ether linkage onto poly(ethylenevinyl alcohol) hollow fiber cartridge. The obtained affinity membranes showed high selectivity for IgG adsorption from untreated human serum. These membranes are able to adsorb IgG1, IgG2, IgG3 if Mops buffer is used, and more selectively IgG1, and IgG3 in Tris-HCl buffer. With respect to the binding capacity, the pseudobiospecific affinity membrane used showed a higher capacity as compared to protein A-membranes described in the literature. Due to the high capacity, specificity and stability of the histidine affinity membranes, in addition to their lower cost, the approach proposed in this paper may offer a useful alternative to protein A based devices in the treatment of immune-related diseases.

[Sexually transmitted diseases: concepts, attitudes and perceptions among dustmen]. / Doenças sexualmente transmissíveis: conceitos, atitudes e percepções entre coletores de lixo.

The concepts regarding sexually transmitted diseases (STD) of 41 (63.07%) dustmen of a country town in S. Paulo State, Brazil, are presented in order to provide support for the preparation of health education programmes on STD for this and similar populational groups. The data collected from interviews with these workers show that a considerable number of them have inadequate concepts about STD. These results demonstrate the lack of information and education on this subject, and the need to implement educational activities.

[Educational communication between nurses and students about sexual health promotion]. / Comunicação educativa do enfermeiro na promoção da saúde sexual do escolar.

As school has been a crucial space for the development of knowledge and abilities in order to assure changes of behavior and considering the lack of reports about sexuality and STD/AIDS to the students, the present study aims to search scholars from three classes of high school, from a town surrounding the city of Ribeirão Preto-São Paulo. Authors identified students' problems by carrying out and assessing joint educative actions on the problems that have been found. Results have showed that these students relate AIDS to fatality and temerity and perhaps they are influenced by the message issued at the first decade of the history of this disease while now, the tendency is to work with up to date knowledge and abilities. They form an opinion about AIDS as a sex and preventible disease. A it however, authors detected misinformation in another basic aspects. Therefore, we suggest nurses to work hard with this question.

[Leisure--a way to alleviate tension in the work environment of an intensive care unit: a concept of the nursing team]. / Lazer--um caminho para aliviar as tensões no ambiente de trabalho em UTI: uma concepção da equipe de enfermagem.

In the present study, we aimed at verifying close to ICU nursing team the service representation and this unit meaning, looking at finding the sense of leisure and its implication in the professional environment. We worked with DUMAZEDIER framework, considering the application of leisure in health promotion, through a quali-quantitative survey. We have investigated 10 members of the nursing team, among different categories, most of them were women, half single, from 28 to 45 years old, with predominantly 10 years in service. They showed pleasure in their job (90%), although they consider it stressing (50%). The job was identified as assistance (80%) and requiring vocation/donation (50%). They also showed relationship and communication. They have the concept of leisure as entertainment/deconcentration (80%), relaxation (20%) etc., emphasizing the importance of discussing the theme. They admit tension/stress caused in the location, considering the need of leisure in the service, to help communication and alleviate the tension. We suggest to nurses special attention on this.

Secretion of IL-16 through TNFR1 and calpain-caspase signaling contributes to MRSA pneumonia.

Staphylococcus aureus is a major cause of severe pneumonia. Multiple mechanisms of proinflammatory signaling are activated to recruit immune cells into the airway in response to S. aureus. We found that interleukin-16 (IL-16), a T cell cytokine that binds CD4, is potently activated by S. aureus, specifically by protein A (SpA), and to a much greater extent than by Gram-negative pathogens or lipopolysaccharide. IL-16 production involved multiple signals including ligation of tumor necrosis factor receptor (TNFR) family members or epidermal growth factor receptor, both receptors for SpA and generation of Ca(2+) fluxes to activate calpains and caspase-3. Although human airway epithelial cells, vascular endothelial cells, THP-1 and Jurkat T cells released IL-16 in response to S. aureus in vitro, in a murine model of pneumonia, CD4(+) cells were the major source of IL-16 suggesting the involvement of an autocrine signaling pathway. The production of IL-16 contributed to lung damage as neutralization of IL-16 enhanced S. aureus clearance and resulted in diminished lung pathology in S. aureus pneumonia. Our results suggest that the ability of S. aureus to activate TNFR1 and Ca(2+)/calpain signaling contribute to T cell activation and excessive inflammation in the setting of acute pneumonia.

Understanding respiratory syncytial virus infection to improve treatment and immunity.

Despite significant research since it was discovered more than 50 years ago, respiratory syncytial virus (RSV) continues to be the leading agent causing infant hospitalization and respiratory distress worldwide. Although RSV normally does not cause mortality, this virus is recognized as a major public health and economic burden around the globe. RSV can modulate host immunity leading to an inflammatory response that produces lung damage and virus dissemination in the host airways. Remarkably, infection with the virus elicits poor immunity that in most cases fails to protect against subsequent exposures. Here, we review advances made on the understanding of the lifecycle of the virus, some of the molecular mechanisms it has evolved to cause pathology and ineffective immunity during infection. Hopefully, ongoing research will contribute to developing new drugs and candidate vaccines that will decrease the health burden caused by this virus.

Evasion of host immunity by virulent Salmonella: implications for vaccine design.

Dendritic cells (DCs) are professional antigen presenting cells (APCs) capable of linking innate and adaptive immunity during infection. After recognition of pathogen-associated molecular patterns (PAMPs), DCs can engulf, process and present bacteria-derived antigens on MHC molecules to T cells. Because of the key role that DCs play on the initiation of innate and adaptive immunity, alterations in their function could render the host susceptible to bacterial dissemination. Consistent with this notion, is the observation that several pathogenic bacteria have evolved mechanisms to impair the DC capacity to prime naïve T cells. One of such bacteria is Salmonella enterica serovar Typhimurium, which causes a typhoid-like disease in mice and gastroenteritis in humans. Recent studies have shown that virulent Salmonella can use intestinal DCs to spread inside the host, evading T cell priming. The avoidance of T cell recognition by Salmonella is in large part achieved by the activity of gene products encoded on Salmonella Pathogenicity Islands -1 and - 2. The understanding of some of the remarkable molecular virulence mechanisms displayed by Salmonella has contributed to the design of new vaccines capable of inducing protective immunity against this pathogen in mouse models. Here we describe recent data underscoring the virulence mechanisms used by Salmonella to exploit DC function and discuss strategies based on this new knowledge aimed at the design of new efficient and safe vaccines against this pathogen.

Virulence mechanisms displayed by Salmonella to impair dendritic cell function.

Dendritic cells (DCs) link innate and adaptive immunity by directly recognizing pathogen-associated molecular patterns (PAMPs) on bacteria. DCs can capture and degrade bacteria and present their antigens on MHC molecules to T cells. PAMP recognition promotes DC maturation, a phenotypic change that empowers them to prime naïve T cells. As a result, an adaptive immune response that specifically targets bacteria-derived antigens is initiated. Consequently, any impairment of DC function might contribute to bacterial survival and dissemination in the host. Therefore, the characterization of DC-bacteria interactions is required to understand the mechanisms used by virulent bacteria to avoid adaptive immunity. An example of a bacterial pathogen capable of interfering with DC function is Salmonella enterica serovar Typhimurium (S. Typhimurium), which causes a typhoid-like disease in mice. Virulent strains of S. Typhimurium are able to differentially modulate the entrance to DCs and avoid lysosomal degradation, to prevent antigen presentation on MHC molecules. These features of virulent S. Typhimurium are controlled by virulence factors encoded by Salmonella Pathogenicity Islands 1 and 2. Modulation of DC functions by the activity of these gene products is supported by several recent studies, which have shown that pathogenesis might depend on this attribute of virulent S. Typhimurium. Here we discuss recent data showing that several virulence factors from Salmonella are required to differentially modulate DC function and adaptive immunity in the host.

Evaluation of immobilized metal-ion affinity chromatography (IMAC) as a technique for IgG(1) monoclonal antibodies purification: the effect of chelating ligand and support.

Monoclonal antibodies (MAbs) have been used for therapies and some analytical procedures as highly purified molecules. Many techniques have been applied and studied, focusing on monoclonal antibodies purification. In this study, an immobilized metal affinity chromatography membrane was developed and evaluated for the purification of anti-TNP IgG(1) mouse MAbs from cell culture supernatant after precipitation with a 50% saturated ammonium sulfate solution. The chelating ligands iminodiacetic acid, carboxymethylated aspartic acid (CM-Asp), nitrilotriacetic acid, and tris (carboxymethyl) ethylenediamine in agarose gels with immobilized Ni(II) and Zn(II) ions were compared for the adsorption and desorption of MAbs. The most promising chelating ligand--CM-Asp--was then coupled to poly(ethylene vinyl alcohol) (PEVA) hollow fiber membranes. According to SDS-PAGE and ELISA analyses, a higher selectivity and a purification factor of 85.9 (fraction eluted at 500 mM Tris) were obtained for IgG(1) using PEVA-CM-Asp-Zn(II). The anti-TNP MAb could be eluted under mild pH conditions causing no loss of antigen binding capacity.

Interaction of human immunoglobulin G with l-histidine immobilized onto poly(ethylene vinyl alcohol) hollow-fiber membranes.

L-Histidine as pseudobiospecific ligand was immobilized onto poly(ethylene vinyl alcohol) hollow-fiber membranes to obtain an affinity support for immunoglobulin G (IgG) purification. The interaction of human IgG with the affinity membranes was studied by chromatography and equilibrium binding analysis. Adsorption was possible over a broad pH range and was found to depend strongly on the nature of the buffer ions rather than on ionic strength. With zwitterionic buffers like morpholinopropanesulfonic acid (Mops) and hydroxyethylpiperazineethanesulfonic acid (Hepes), much higher adsorption capacities were obtained than with other buffers like Tris-HCl and phosphate buffers. An inhibition analysis revealed that non-zwitterionic buffers competitively inhibit IgG binding, whereas Mops and Hepes in their zwitterionic form do not. By choosing the appropriate buffer system, it was possible to adsorb specifically different IgG subsets. The IgG molecules were found to adsorb on membrane immobilized histidine via their Fab part. Determination of dissociation constants at different temperatures allowed calculation of thermodynamic adsorption parameters. Decrease in KD with increasing temperature and a positive entropy value between 20 and 35 degrees C (in Mops buffer) indicated that adsorption is partially governed by hydrophobic forces in that temperature range, whereas at lower temperatures, electrostatic forces are more important for adsorption.

Separation of immunoglobulin G from human serum by pseudobioaffinity chromatography using immobilized L-histidine in hollow fibre membranes.

L-Histidine, intended as a pseudobiospecific ligand, was immobilized on poly(ethylenevinyl alcohol) hollow fibre membranes after their activation with epichlorohydrin or butanediol diglycidyl ether. The affinity membranes obtained allowed the one-step separation of immunoglobulin G (IgG) from untreated human serum. Elution was possible under mild conditions with discontinuous pH or salt gradients. IgM was also adsorbed to a certain extent and partially separated from IgG by pH gradient elution. The bound IgG fractions showed pI values between 8 and 9.5 and contained IgG1 and IgG3. The dissociation constants for IgG on the bisoxirane- and epichlorohydrin-activated membranes coupled with histidine were determined by equilibrium binding analysis to be 2.5 x 10(-5) and 2.0 x 10(-5) M, respectively. The maximum binding capacity of the affinity hollow fibre membranes was 80 and 70 mg of IgG per gram of support, respectively. With a cartridge of surface area 1 m2 (about 19 g of fibres), during a 60-min run, theoretically up to 1.5 g of IgG can be removed from human serum. The histidine affinity membranes are very stable owing to the simple nature of the ligand and the coupling via an ether linkage. Reproducible results were obtained over more than 1 year even with untreated human serum being used regularly.

Strategies for the depyrogenation of contaminated immunoglobulin G solutions by histidine-immobilized hollow fiber membrane.

The depyrogenation of different IgG solutions using the histidine-linked hollow fiber membrane developed in our laboratory is presented here. Three strategies for endotoxin (ET) removal were investigated according to the immobilized histidine's ability to bind different immunoglobulins: (1) ET removal from 1 mg/ml non histidine-binding mouse monoclonal IgG1 (MabCD4) solution was achieved in the presence of acetate buffer (pH 5.0) without any protein loss. (2) For contaminated human IgG, combined adsorption of ET and IgG in the presence of MOPS of Tris buffer was tested, followed by differential elution using increasing salt concentrations. This attempt was not successful since ET were quantitatively found in the IgG elution fraction. (3) Alternatively, it was proposed to adsorb selectively ET in the presence of acetate buffer (pH 5.0) under non binding conditions for human IgG. Human IgG could then be purified if necessary with the same membrane in the presence of MOPS buffer (pH 6.5). With a 1 m2 histidine-PEVA module under these operating conditions, it is estimated that the depyrogenation of 3 l of 1 mg/ml IgG (human or murine) solution containing 80 EU/ml of ET should be possible.