Aldo Arturo Reséndiz-Albor

Intranasal Coadministration of the Cry1Ac Protoxin with Amoebal Lysates Increases Protection against Naegleria fowleri Meningoencephalitis

ABSTRACT Cry1Ac protoxin has potent mucosal and systemic adjuvant effects on antibody responses to proteins or polysaccharides. In this work, we examined whether Cry1Ac increased protective immunity against fatal Naegleria fowleri infection in mice, which resembles human primary amoebic meningoencephalitis. Higher immunoglobulin G (IgG) than IgA anti-N. fowleri responses were elicited in the serum and tracheopulmonary fluids of mice immunized by the intranasal or intraperitoneal route with N. fowleri lysates either alone or with Cry1Ac or cholera toxin. Superior protection against a lethal challenge with 5 × 104 live N. fowleri trophozoites was achieved for immunization by the intranasal route. Intranasal immunization of N. fowleri lysates coadministered with Cry1Ac increased survival to 100%; interestingly, immunization with Cry1Ac alone conferred similar protection to that achieved with amoebal lysates alone (60%). When mice intranasally immunized with Cry1Ac plus lysates were challenged with amoebae, both IgG and IgA mucosal responses were rapidly increased, but only the increased IgG response persisted until day 60 in surviving mice. The brief rise in the level of specific mucosal IgA does not exclude the role that this isotype may play in the early defense against this parasite, since higher IgA responses were detected in nasal fluids of mice intranasally immunized with lysates plus either Cry1Ac or cholera toxin, which, indeed, were the treatments that provided the major protection levels. In contrast, serum antibody responses do not seem to be related to the protection level achieved. Both acquired and innate immune systems seem to play a role in host defense against N. fowleri infection, but further studies are required to elucidate the mechanisms involved in protective effects conferred by Cry1Ac, which may be a valuable tool to improve mucosal vaccines.

Heterologous Prime-Boost Strategy to Immunize Very Young Infants against Measles: Pre-clinical Studies in Rhesus Macaques

Infants in developing countries are at high risk of developing severe clinical measles if they become infected during the “window of vulnerability” (age 4–9 months), when declining maternal antibodies do not protect against wild virus, yet impede successful immunization by attenuated measles vaccine. We developed two Sindbis replicon‐based DNA vaccines expressing measles virus hemagglutinin and fusion protein with the goal of priming young infants to respond safely and effectively to subsequent boosting with attenuated measles vaccine. Intradermal prime with DNA vaccines by needle‐free injection followed by aerosol or parenteral boost with licensed measles vaccine was well tolerated by juvenile and young infant rhesus macaques, and protected against clinical measles and viremia on wild‐type virus challenge. A proteosome‐measles vaccine administered alone (three doses) or as a boost following DNA vaccine priming was also safe and protective. These promising results pave the way for clinical trials to assess this prime–boost strategy.

Lactoferrin increases both resistance to Salmonella typhimurium infection and the production of antibodies in mice.

Lactoferrin (Lf) is a multifunctional iron-binding glycoprotein with antibacterial and immunomodulatory activities. The antibacterial influence of orally administered bovine Lf (bLf) against murine infection caused by Salmonella typhimurium (S. typhimurium) has scarcely been explored. In the current study, Balb/c mice were treated orally for 7 days with either 5 or 100mg of bovine lactoferrin (bLf). On day 7 of treatment, mice were intragastrically infected with a lethal or sublethal dose of colony forming units (CFU) of S. typhimurium. During treatment with bLf, feces from mice sublethally infected were harvested daily to prepare fecal suspensions, which were serially diluted and plated onto Salmonella Shigella agar to estimate CFU/g of feces. After sacrificing the animals on day 7, 14 or 21 post-infection, samples of intestinal fluid, Peyers patches (PP), liver and spleen were collected to count the number of CFU by plate dilution. Intestinal secretions were also employed, along with serum samples, to evaluate total IgA, IgG and IgM antibodies, and those against Salmonella surface proteins and bLf by ELISA assay. In lethally infected mice both bLf doses decreased mortality. In sublethally infected mice, both bLf doses decreased bacterial shedding in feces and intestinal fluid, and also reduced bacterial colonization at PP and bacterial translocation in the liver and spleen. Levels of total and those IgG and IgM in serum and IgA in intestinal secretions against Salmonella surface proteins and bLf were enhanced with both doses of bLf. These findings suggest that the effect of bLf against the infection by S. typhimurium in mice may be the result of an antimicrobial activity linked with its modulatory effect on immunocompetent cells (from intestinal and peripheral organs) involved in antibody production.

Regionalization of pIgR expression in the mucosa of mouse small intestine.

Few reports exist on the differences in cell populations or immunological functions between the proximal and distal segments of the small intestine (SI). In the current contribution we analyzed the expression of the polymeric immunoglobulin receptor (pIgR) and alpha chains as well as the density of IgA-producing cells from the proximal and distal intestinal segments from Balb/c mice. Furthermore, by using real-time RT-PCR we quantified the expression of cytokines (TNF-alpha, IFN-gamma, IL-4 and TGF-beta), Toll-like receptor-4 (TLR-4), and the glucocorticoid receptor (GR) involved in pIgR expression in intestinal epithelial cells (IEC). In this study, for the first time it has been demonstrated that the expression of the pIgR as well as alpha chain was greater in the proximal than the distal segment of the small intestine of normal mice. Moreover, we found striking differences in the expression of cytokines at the different intestinal compartments. Whereas the expression of TNF-alpha, IFN-gamma and TGF-beta was higher in lamina propria lymphocytes (LPL) of the distal than proximal segment, it was higher in IEC of the proximal than distal segment. In contrast, the expression of the gene for IL-4 was higher in the LPL of the proximal segment and the IEC of the distal segment. Although the overall expression of TNF-alpha, IL-4, IFN-gamma and TGF-beta was higher in the whole mucosa of the distal than proximal segment, we propose that cytokines produced by epithelial cells (TNF-alpha, IFN-gamma and TGF-beta) autocrinally up-regulate the expression of mRNA for the pIgR. Finally the expression of the GR was higher in the proximal segment, while the expression of the gene for TLR-4 was significantly higher in the IEC of the distal than proximal segment. The higher expression of pIgR found in the proximal segment is probably related to the effect on epithelial cells of the higher production of TNF-alpha, IFN-gamma and TGF-beta, as well as the higher expression of the glucocorticoid receptors. The increased expression of pIgR in the proximal segment appears primarily responsible for the increased secretory IgA levels in the small intestine of mice. These results confirm and extend previous findings supporting the compartmentalization of the intestinal immune system.

Sindbis Virus-Based Measles DNA Vaccines Protect Cotton Rats against Respiratory Measles: Relevance of Antibodies, Mucosal and Systemic Antibody-Secreting Cells, Memory B Cells, and Th1-Type Cytokines as Correlates of Immunity

ABSTRACT Measles remains an important cause of pediatric morbidity and mortality in developing countries, especially among infants who are too young to receive the current licensed live attenuated measles vaccine. We developed two Sindbis virus DNA vaccines encoding the measles virus hemagglutinin (pMSIN-H) and fusion proteins (pMSINH-FdU) and examined their immunogenicities and protective efficacies when administered alone or followed by the live measles virus vaccine in cotton rats. Neutralizing antibodies, mucosal and systemic antibody-secreting cells, memory B cells, and gamma interferon-secreting T cells developed after priming and increased after boosting. pMSIN-H priming conferred 100% protection against pulmonary measles, whereas pMSINH-FdU protected only in conjunction with the live measles virus vaccine boost.

Effects on secretory IgA levels in small intestine of mice that underwent moderate exercise training followed by a bout of strenuous swimming exercise

Intestinal homeostasis effectors, secretory IgA (SIgA) and polymeric immunoglobulin receptor (pIgR), have been evaluated in proximal and distal small intestine with moderate-exercise training but not with strenuous exercise or a combination of these two protocols. Therefore, two groups of mice (n=6-8) were submitted to strenuous exercise, one with and one without previous training. The control group had no exercise protocol. Assessment was made of intestinal SIgA and plasma adrenal hormones (by immunoenzymatic assay), alpha-chain and pIgR proteins in intestinal mucosa (by Western blot), lamina propria IgA plasma-cells (by cytofluorometry), mRNA expression (by real-time PCR) for pIgR, alpha- and J-chains in liver and intestinal mucosa, and pro- and anti-inflammatory cytokines in mucosa samples. Compared to other exercise protocols, training plus strenuous exercise elicited: (1) higher levels of SIgA and pIgR in the proximal intestine (probably by hepatobiliary contribution); (2) higher levels of SIgA in the distal segment; (3) lower mRNA expression of some SIgA- and most pro-inflammatory pIgR-producing cytokines. SIgA and pIgR in both segments were derived from an existing pool of their corresponding producing cells. The apparent decreased translation of mRNA transcripts underlies lower levels of SIgA and pIgR in distal than proximal small intestine. There was no significant difference in the relatively high adrenal hormone levels found in both exercised groups. Further study is required about the effects of training plus strenuous exercise on pool-derived SIgA levels and mRNA expression of pro-inflammatory pIgR-producing cytokines. These results could have important implications for intestinal disorders involving inflammation and infection.

Different antiamebic antibody isotype patterns in the large and small intestine after local and systemic immunization of mice with glutaraldehyde fixed Entamoeba histolytica trophozoites.

We have determined the major immunoglobulin isotypes (IgG, IgA, IgM) of antiamebic antibodies induced in the serum and in the large and small intestine after local (oral and rectal) or systemic (intraperitoneal and intramuscular) immunization of mice with glutaraldehyde-fixed Entamoeba histolytica trophozoites (GFT). IgA predominated in the small intestine after immunization through all routes, whereas in the large intestine similar antibody levels of the major isotypes were induced by rectal, intraperitoneal and intramuscular immunization. The intramuscular route elicited intestinal responses lower than those induced by the rectal and intraperitoneal routes, but higher than the slight IgA antibody increase observed after oral immunization. The differences in antiamebic antibody response patterns at the large and small intestine suggest that there are different mucosal effector compartments. They also indicate that isotype analysis of mucosal antibodies from the sites where an infectious agent resides is needed to evaluate whether a vaccine candidate induces responses of higher protective value in the appropriate site, and that the study of antibody responses must not be limited to sampling the serum or mucosal sites distant to the relevant one.

In silico and in vitro studies to elucidate the role of Cu2+ and galanthamine as the limiting step in the amyloid beta (1–42) fibrillation process

The formation of fibrils and oligomers of amyloid beta (Aβ) with 42 amino acid residues (Aβ1–42) is the most important pathophysiological event associated with Alzheimers disease (AD). The formation of Aβ fibrils and oligomers requires a conformational change from an α‐helix to a β‐sheet conformation, which is encouraged by the formation of a salt bridge between Asp 23 or Glu 22 and Lys 28. Recently, Cu2+ and various drugs used for AD treatment, such as galanthamine (Reminyl®), have been reported to inhibit the formation of Aβ fibrils. However, the mechanism of this inhibition remains unclear. Therefore, the aim of this work was to explore how Cu2+ and galanthamine prevent the formation of Aβ1–42 fibrils using molecular dynamics (MD) simulations (20 ns) and in vitro studies using fluorescence and circular dichroism (CD) spectroscopies. The MD simulations revealed that Aβ1–42 acquires a characteristic U‐shape before the α‐helix to β‐sheet conformational change. The formation of a salt bridge between Asp 23 and Lys 28 was also observed beginning at 5 ns. However, the MD simulations of Aβ1−42 in the presence of Cu2+ or galanthamine demonstrated that both ligands prevent the formation of the salt bridge by either binding to Glu 22 and Asp 23 (Cu2+) or to Lys 28 (galanthamine), which prevents Aβ1−42 from adopting the U‐characteristic conformation that allows the amino acids to transition to a β‐sheet conformation. The docking results revealed that the conformation obtained by the MD simulation of a monomer from the 1Z0Q structure can form similar interactions to those obtained from the 2BGE structure in the oligomers. The in vitro studies demonstrated that Aβ remains in an unfolded conformation when Cu2+ and galanthamine are used. Then, ligands that bind Asp 23 or Glu 22 and Lys 28 could therefore be used to prevent β turn formation and, consequently, the formation of Aβ fibrils.

Changes in protein and gene expression of angiotensin II receptors (AT1 and AT2) in aorta of diabetic and hypertensive rats.

Abstract Diabetes and hypertension have been associated with cardiovascular diseases and stroke. Some reports have related the coexistence of hypertension and diabetes with increase in the risk of developing vascular complications. Recently some studies have shown results suggesting that in the early stages of diabetes and hypertension exist a reduced functional response to vasopressor agents like angiotensin II (Ang II), which plays an important role in blood pressure regulation mechanism through the activation of its AT1 and AT2 receptors. For that reason, the aim of this work was to study the gene and protein expression of AT1 and AT2 receptors in aorta of diabetic SHR and WKY rats. Diabetes was induced by the administration of streptozotocin (60 mg/kg i.p.). After 4 weeks of the onset of diabetes, the protein expression was obtained by western blot and the mRNA expression by RT-PCR. Our results showed that the hypertensive rats have a higher mRNA and protein expression of AT1 receptors than normotensive rats while the AT2 expression remained unchanged. On the other hand, the combination of diabetes and hypertension increased the mRNA and protein expression of AT1 and AT2 receptors significantly. In conclusion, our results suggest that diabetes with hypertension modifies the mRNA and protein expression of AT1 and AT2 receptors. However, the overexpression of AT2 could be associated with the reduction in the response to Ang II in the early stage of diabetes.

Infections of Diabetic Foot Ulcers With Methicillin-Resistant Staphylococcus aureus

Infected diabetic foot is the most common reason for hospitalization and complications in patients with type 2 diabetes mellitus (DM2). Methicillin-resistant Staphylococcus aureus (MRSA) is frequently isolated from such lesions, and its presence is growing, seriously deteriorating the infected patient’s quality of life. The aim of this study was to assess the prevalence of MRSA as well as other microbiota in 100 patients diagnosed with (DM2) and with infected foot ulcers at the Hospital General de Mexico. The main results obtained show a prevalence of Staphylococcus aureus (42%), followed by Escherichia coli (36%) and, in lower percentages, other bacteria. MRSA was predominant (34%), and we conclude that the use of cefoxitin instead of oxacillin as the first-choice antibiotic has an advantage because it is a better inducer of methicillin-resistance expression.