Using an Aluminum Hydroxide-Chitosan Matrix Increased the Vaccine Potential and Immune Response of Mice against Multi-Drug-Resistant Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative, immobile, aerobic nosocomial opportunistic coccobacillus that causes pneumonia, septicemia, and urinary tract infections in immunosuppressed patients. There are no commercially available alternative antimicrobials, and multi-drug resistance is an urgent concern that requires emergency measures and new therapeutic strategies. This study evaluated a multi-drug-resistant A. baumannii whole-cell vaccine, inactivated and adsorbed on an aluminum hydroxide-chitosan (mAhC) matrix, in an A. baumannii sepsis model in immunosuppressed mice by cyclophosphamide (CY). CY-treated mice were divided into immunized, non-immunized, and adjuvant-inoculated groups. Three vaccine doses were given at 0D, 14D, and 28D, followed by a lethal dose of 4.0 × 108 CFU/mL of A. baumannii. Immunized CY-treated mice underwent a significant humoral response, with the highest IgG levels and a higher survival rate (85%); this differed from the non-immunized CY-treated mice, none of whom survived (p < 0.001), and from the adjuvant group, with 45% survival (p < 0.05). Histological data revealed the evident expansion of white spleen pulp from immunized CY-treated mice, whereas, in non-immunized and adjuvanted CY-treated mice, there was more significant organ tissue damage. Our results confirmed the proof-of-concept of the immune response and vaccine protection in a sepsis model in CY-treated mice, contributing to the advancement of new alternatives for protection against A. baumannii infections.

Duffy blood system and G6PD genetic variants in vivax malaria patients from Manaus, Amazonas, Brazil.

BACKGROUND: Over a third of the world's population is at risk of Plasmodium vivax-induced malaria. The unique aspect of the parasite's biology and interactions with the human host make it harder to control and eliminate the disease. Glucose-6-phosphate dehydrogenase (G6PD) deficiency and Duffy-negative blood groups are two red blood cell (RBC) variations that can confer protection against malaria. METHODS: Molecular genotyping of G6PD and Duffy variants was performed in 225 unrelated patients (97 with uncomplicated and 128 with severe vivax malaria) recruited at a Reference Centre for Infectious Diseases in Manaus. G6PD and Duffy variants characterizations were performed using Real Time PCR (qPCR) and PCR-RFLP, respectively. RESULTS: The Duffy blood group system showed a phenotypic distribution Fy(a + b-) of 70 (31.1%), Fy(a + b +) 96 (42.7%), Fy(a-b +) 56 (24.9%) and Fy(a-b-) 1 (0.44%.) The genotype FY*A/FY*B was predominant in both uncomplicated (45.3%) and severe malaria (39.2%). Only one Duffy phenotype Fy(a-b) was found and this involved uncomplicated vivax malaria. The G6PD c.202G > A variant was found in 11 (4.88%) females and 18 (8.0%) males, while c.376A > G was found in 20 females (8.88%) and 23 (10.22%) male patients. When combined GATA mutated and c.202G > A and c.376A > G mutated, was observed at a lower frequency in uncomplicated (3.7%) in comparison to severe malaria (37.9%). The phenotype Fy(a-b +) (p = 0.022) with FY*B/FY*B (p = 0.015) genotype correlated with higher parasitaemia. CONCLUSIONS: A high prevalence of G6PD c202G > A and c.376A > G and Duffy variants is observed in Manaus, an endemic area for vivax malaria. In addition, this study reports for the first time the Duffy null phenotype Fy(a-b-) in the population of the Amazonas state. Moreover, it is understood that the relationship between G6PD and Duffy variants can modify clinical symptoms in malaria caused by P. vivax and this deserves to be further investigated and explored among this population.

Inactivated alpha toxin from Clostridium novyi type B in nano-emulsion protect partially protects Swiss mice from lethal alpha toxin challenge.

Nano-emulsions are promising carriers for antigen delivery. Here, we evaluated the efficacy of a water-oil nano-emulsion containing concentrated, inactivated Clostridium novyi (C. novyi) type B supernatant culture (nano-iCnB) in protecting Swiss mice against a lethal dose of alpha toxin concentrated extract. Proteins were confirmed in the nano-iCnB and their stabilities were determined according physical parameters such as Zeta Potential (ZP). Biochemical, hematological parameters and morphological appearance of liver, spleen and thigh muscle alterations were examined to determine the safety of the compound. Partial protection against lethal doses was achieved in immunized mice despite low IgG titers. These data suggest that our nano-emulsion is a simple and efficient method of promoting antigen delivery for toxin-related diseases.

A hybrid protein containing MSP1a repeats and Omp7, Omp8 and Omp9 epitopes protect immunized BALB/c mice against anaplasmosis.

Anaplasma marginale (A. marginale) has a remarkable impact on livestock production, and an effective vaccine is not currently available due to the inexistence of a small animal model. Recently, BALB/c mice were successfully infected with A. marginale, resulting in an acute and persistent anaplasmosis infection. Here, we designed a hybrid protein containing repeats of polypeptide 1a from major surface protein-1 complex (MSP1a) repeats and common epitopes of outer membrane proteins (OMPs) OMP7, OMP8 and OMP9 expressed in Escherichia coli. Our proof-of-concept assessed vaccinal effectiveness against a challenge with live bacteria. The MSP1a/OMP7/8/9 immunized BALB/C mice exhibited a strong reduction in rickettsemia and had no signs of anaplasmosis or hepatic lesions. In contrast, the non-immunized mice exhibited signs of anaplasmosis and a body weight loss associated with increases in monocyte and neutrophil counts. Furthermore, the non-immunized mice displayed atrophies with chronic inflammatory infiltrates in the spleen and increased binucleation and hydropic degeneration in the hepatocytes. Our findings demonstrated that immunization with our hybrid protein induced a strong reduction in rickettsemia and conferred protection against anaplasmosis. Therefore, given the strong evidence of the protective effect against anaplasmosis, hybrid protein designs are potential candidates for the rational design of vaccinal subunits.

Immunogenetic markers associated with a naturally acquired humoral immune response against an N-terminal antigen of Plasmodium vivax merozoite surface protein 1 (PvMSP-1).

BACKGROUND: Humoral immune responses against proteins of asexual blood-stage malaria parasites have been associated with clinical immunity. However, variations in the antibody-driven responses may be associated with a genetic component of the human host. The objective of the present study was to evaluate the influence of co-stimulatory molecule gene polymorphisms of the immune system on the magnitude of the humoral immune response against a Plasmodium vivax vaccine candidate antigen. METHODS: Polymorphisms in the CD28, CTLA4, ICOS, CD40, CD86 and BLYS genes of 178 subjects infected with P. vivax in an endemic area of the Brazilian Amazon were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The levels of IgM, total IgG and IgG subclasses specific for ICB2-5, i.e., the N-terminal portion of P. vivax merozoite surface protein 1 (PvMSP-1), were determined by enzyme-linked immuno assay. The associations between the polymorphisms and the antibody response were assessed by means of logistic regression models. RESULTS: After correcting for multiple testing, the IgG1 levels were significantly higher in individuals recessive for the single nucleotide polymorphism rs3116496 in CD28 (p = 0.00004). Furthermore, the interaction between CD28 rs35593994 and BLYS rs9514828 had an influence on the IgM levels (p = 0.0009). CONCLUSIONS: The results of the present study support the hypothesis that polymorphisms in the genes of co-stimulatory components of the immune system can contribute to a natural antibody-driven response against P. vivax antigens.

Phenotypic and Molecular Aspects of Staphylococcus spp. Isolated from Hospitalized Patients and Beef in the Brazilian Amazon.

The aim of this study was to characterize and compare Staphylococcus spp. isolated from hospitalized patients and beef marketed in the city of Porto Velho-RO, Brazil. The isolates were subjected to antibiogram tests, adherence capacity tests, detection of the mecA gene, and epidemiological investigation by the random amplified polymorphic DNA (RAPD) technique, using the primers M13 and H12. Among the 123 Staphylococcus spp. isolates, 50 were identified as S. aureus and 73 as coagulase-negative Staphylococcus; among the latter, 7 species were identified. It was observed that the coagulase-negative Staphylococcus isolates showed greater adhesion ability than S. aureus. The profile of antimicrobial susceptibility was different among isolates, all of which were susceptible to vancomycin and linezolid, and had high penicillin resistance rates, varying according to the bacterial class and the source. In this study, all strains were negative for mecA gene detection; however, 36% of S. aureus and 17% of coagulase-negative Staphylococcus were resistant to oxacillin. The genetic relationship of these bacteria, analyzed by RAPD, was able to discriminate the species of coagulase-negative Staphylococcus strains of S. aureus along its origin. It was concluded that the isolates of Staphylococcus spp. derived from beef and human infections differ genetically. Thus, it is suggested that isolates from beef, which were grouped within hospital isolates, were probably carried via contact with beef in hospital professionals or patients.

Proteomic analysis of Chromobacterium violaceum and its adaptability to stress.

BACKGROUND: Chromobacterium violaceum (C. violaceum) occurs abundantly in a variety of ecosystems, including ecosystems that place the bacterium under stress. This study assessed the adaptability of C. violaceum by submitting it to nutritional and pH stresses and then analyzing protein expression using bi-dimensional electrophoresis (2-DE) and Maldi mass spectrometry. RESULTS: Chromobacterium violaceum grew best in pH neutral, nutrient-rich medium (reference conditions); however, the total protein mass recovered from stressed bacteria cultures was always higher than the total protein mass recovered from our reference culture. The diversity of proteins expressed (repressed by the number of identifiable 2-DE spots) was seen to be highest in the reference cultures, suggesting that stress reduces the overall range of proteins expressed by C. violaceum. Database comparisons allowed 43 of the 55 spots subjected to Maldi mass spectrometry to be characterized as containing a single identifiable protein. Stress-related expression changes were noted for C. violaceum proteins related to the previously characterized bacterial proteins: DnaK, GroEL-2, Rhs, EF-Tu, EF-P; MCP, homogentisate 1,2-dioxygenase, Arginine deiminase and the ATP synthase ß-subunit protein as well as for the ribosomal protein subunits L1, L3, L5 and L6. The ability of C. violaceum to adapt its cellular mechanics to sub-optimal growth and protein production conditions was well illustrated by its regulation of ribosomal protein subunits. With the exception of the ribosomal subunit L3, which plays a role in protein folding and maybe therefore be more useful in stressful conditions, all the other ribosomal subunit proteins were seen to have reduced expression in stressed cultures. Curiously, C. violeaceum cultures were also observed to lose their violet color under stress, which suggests that the violacein pigment biosynthetic pathway is affected by stress. CONCLUSIONS: Analysis of the proteomic signatures of stressed C. violaceum indicates that nutrient-starvation and pH stress can cause changes in the expression of the C. violaceum receptors, transporters, and proteins involved with biosynthetic pathways, molecule recycling, energy production. Our findings complement the recent publication of the C. violeaceum genome sequence and could help with the future commercial exploitation of C. violeaceum.

Duffy antigen receptor for chemokine (DARC) polymorphisms and its involvement in acquisition of inhibitory anti-duffy binding protein II (DBPII) immunity.

The Plasmodium vivax Duffy binding protein (PvDBP) and its erythrocytic receptor, the Duffy antigen receptor for chemokines (DARC), are involved in the major P. vivax erythrocyte invasion pathway. An open cohort study to analyze DARC genotypes and their relationship to PvDBP immune responses was carried out in 620 volunteers in an agricultural settlement of the Brazilian Amazon. Three cross-sectional surveys were conducted at 6-month intervals, comprising 395, 410, and 407 subjects, respectively. The incidence rates of P. vivax infection was 2.32 malaria episodes per 100 person-months under survey (95% confidence interval [CI] of 1.92-2.80/100 person-month) and, of P. falciparum, 0.04 per 100 person-months (95% CI of 0.007-0.14/100 person-month). The distribution of DARC genotypes was consistent with the heterogeneous ethnic origins of the Amazon population, with a predominance of non-silent DARC alleles: FY*A > FY*B. The 12-month follow-up study demonstrated no association between DARC genotypes and total IgG antibodies as measured by ELISA targeting PvDBP (region II, DBPII or regions II-IV, DBPII-IV). The naturally acquired DBPII specific binding inhibitory antibodies (BIAbs) tended to be more frequent in heterozygous individuals carrying a DARC-silent allele (FY*BES). These results provide evidence that DARC polymorphisms may influence the naturally acquired inhibitory anti-Duffy binding protein II immunity.

Paucity of Plasmodium vivax mature schizonts in peripheral blood is associated with their increased cytoadhesive potential.

There is now a growing body of evidence that challenges the current view that Plasmodium vivax-infected erythrocyte (Pv-iE) are unable to sequester. Here we used ex vivo adhesion assays with Pv-iE before and after maturation to demonstrate a higher binding potential of schizonts compared to other asexual stages. These experimental results are correlated with our observations in a panel of 50 vivax malaria patients where schizonts were completely absent in 27 isolates, and few schizonts were observed in the remaining patients. These observations prompt a paradigm shift in P. vivax biology and open avenues to investigate the role of Pv-iE sequestration.

N-terminal Plasmodium vivax merozoite surface protein-1, a potential subunit for malaria vivax vaccine.

The human malaria is widely distributed in the Middle East, Asia, the western Pacific, and Central and South America. Plasmodium vivax started to have the attention of many researchers since it is causing diseases to millions of people and several reports of severe malaria cases have been noticed in the last few years. The lack of in vitro cultures for P. vivax represents a major delay in developing a functional malaria vaccine. One of the major candidates to antimalarial vaccine is the merozoite surface protein-1 (MSP1), which is expressed abundantly on the merozoite surface and capable of activating the host protective immunity. Studies have shown that MSP-1 possesses highly immunogenic fragments, capable of generating immune response and protection in natural infection in endemic regions. This paper shows humoral immune response to different proteins of PvMSP1 and the statement of N-terminal to be added to the list of potential candidates for malaria vivax vaccine.

High levels of IgG3 anti ICB2-5 in Plasmodium vivax-infected individuals who did not develop symptoms.

BACKGROUND: Plasmodium vivax has the potential to infect 2.85 billion individuals worldwide. Nevertheless, the limited number of studies investigating the immune status of individuals living in malaria-endemic areas, as well as the lack of reports investigating serological markers associated with clinical protection, has hampered development of vaccines for P. vivax. It was previously demonstrated that naturally total IgG against the N-terminus of P. vivax merozoite surface protein 1 (Pv-MSP1) was associated with reduced risk of malarial infection. METHODS: Immune response against Pv-MSP1 (N-terminus) of 313 residents of the Rio Pardo rural settlement (Amazonas State, Brazil) was evaluated in a cross-sectional and longitudinal follow up over two months (on site) wherein gold standard diagnosis by thick blood smear and rRNA gene-based nested real-time PCR were used to discriminate symptomless Plasmodium vivax-infected individuals who did not develop clinical symptoms during a 2-months from those uninfected ones or who have had acute malaria. The acquisition of antibodies against Pv-MSP1 was also evaluated as survival analysis by prospective study over a year collecting information of new malaria infections in surveillance database. RESULTS: The majority of P. vivax-infected individuals (52-67%) showed immune recognition of the N-terminus of Pv-MSP1. Interesting data on infected individuals who have not developed symptoms, total IgG levels against the N-terminus Pv-MSP1 were age-dependent and the IgG3 levels were significantly higher than levels of subjects had acute malaria or those uninfected ones. The total IgG anti ICB2-5 was detected to be an important factor of protection against new malaria vivax attacks in survival analysis in a prospective survey (p = 0.029). CONCLUSIONS: The study findings illustrate the importance of IgG3 associated to 2-months of symptomless in P. vivax infected individuals and open perspectives for the rationale of malaria vaccine designs capable to sustain high levels of IgG3 against polymorphic malaria antigens.

Thrombocytopenia in Plasmodium vivax malaria is related to platelets phagocytosis.

BACKGROUND: Although thrombocytopenia is a hematological disorder commonly reported in malarial patients, its mechanisms are still poorly understood, with only a few studies focusing on the role of platelets phagocytosis. METHODS AND FINDINGS: Thirty-five malaria vivax patients and eight healthy volunteers (HV) were enrolled in the study. Among vivax malaria patients, thrombocytopenia (<150,000 platelets/µL) was found in 62.9% (22/35). Mean platelet volume (MPV) was higher in thrombocytopenic patients as compared to non-thrombocytopenic patients (p = 0.017) and a negative correlation was found between platelet count and MPV (r = -0.483; p = 0.003). Platelets from HV or patients were labeled with 5-chloromethyl fluorescein diacetate (CMFDA), incubated with human monocytic cell line (THP-1) and platelet phagocytosis index was analyzed by flow cytometry. The phagocytosis index was higher in thrombocytopenic patients compared to non-thrombocytopenic patients (p = 0.042) and HV (p = 0.048). A negative correlation was observed between platelet count and phagocytosis index (r = -0.402; p = 0.016). Platelet activation was assessed measuring the expression of P-selectin (CD62-P) in platelets' surface by flow cytometry. No significant difference was found in the expression of P-selectin between thrombocytopenic patients and HV (p = 0.092). After evaluating the cytokine profile (IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and IL-17) in the patients' sera, levels of IL-6, IL-10 and IFN-γ were elevated in malaria patients compared to HV. Moreover, IL-6 and IL-10 values were higher in thrombocytopenic patients than non-thrombocytopenic ones (p = 0.044 and p = 0.017, respectively. In contrast, TNF-α levels were not different between the three groups, but a positive correlation was found between TNF-α and phagocytosis index (r = -0.305; p = 0.037). CONCLUSION/SIGNIFICANCE: Collectively, our findings indicate that platelet phagocytosis may contribute to thrombocytopenia found in vivax malaria. Finally, we believe that this study opens new avenues to explore the mechanisms involved in platelet dysfunction, commonly found in vivax malaria patients.

Plasmodium vivax Duffy binding protein: baseline antibody responses and parasite polymorphisms in a well-consolidated settlement of the Amazon Region.

OBJECTIVE: To investigate risk factors associated with the acquisition of antibodies against Plasmodium vivax Duffy binding protein (PvDBP) - a leading malaria vaccine candidate - in a well-consolidated agricultural settlement of the Brazilian Amazon Region and to determine the sequence diversity of the PvDBP ligand domain (DBP(II)) within the local malaria parasite population. METHODS: Demographic, epidemiological and clinical data were collected from 541 volunteers using a structured questionnaire. Malaria parasites were detected by conventional microscopy and PCR, and blood collection was used for antibody assays and molecular characterisation of DBP(II). RESULTS: The frequency of malaria infection was 7% (6% for P. vivax and 1% for P. falciparum), with malaria cases clustered near mosquito breeding sites. Nearly 50% of settlers had anti-PvDBP IgG antibodies, as detected by enzyme-linked immunosorbent assay (ELISA) with subject's age being the only strong predictor of seropositivity to PvDBP. Unexpectedly, low levels of DBP(II) diversity were found within the local malaria parasites, suggesting the existence of low gene flow between P. vivax populations, probably due to the relative isolation of the studied settlement. CONCLUSION: The recognition of PvDBP by a significant proportion of the community, associated with low levels of DBP(II) diversity among local P. vivax, reinforces the variety of malaria transmission patterns in communities from frontier settlements. Such studies should provide baseline information for antimalarial vaccines now in development.

On cytoadhesion of Plasmodium vivax: raison d'être?

It is generally accepted that Plasmodium vivax, the most widely distributed human malaria parasite, causes mild disease and that this species does not sequester in the deep capillaries of internal organs. Recent evidence, however, has demonstrated that there is severe disease, sometimes resulting in death, exclusively associated with P. vivax and that P. vivax-infected reticulocytes are able to cytoadhere in vitro to different endothelial cells and placental cryosections. Here, we review the scarce and preliminary data on cytoadherence in P. vivax, reinforcing the importance of this phenomenon in this species and highlighting the avenues that it opens for our understanding of the pathology of this neglected human malaria parasite.

On cytoadhesion of Plasmodium vivax: raison d'être?

It is generally accepted that Plasmodium vivax, the most widely distributed human malaria parasite, causes mild disease and that this species does not sequester in the deep capillaries of internal organs. Recent evidence, however, has demonstrated that there is severe disease, sometimes resulting in death, exclusively associated with P. vivax and that P. vivax-infected reticulocytes are able to cytoadhere in vitro to different endothelial cells and placental cryosections. Here, we review the scarce and preliminary data on cytoadherence in P. vivax, reinforcing the importance of this phenomenon in this species and highlighting the avenues that it opens for our understanding of the pathology of this neglected human malaria parasite.

Gestational malaria associated to Plasmodium vivax and Plasmodium falciparum placental mixed-infection followed by foetal loss: a case report from an unstable transmission area in Brazil.

Gestational malaria is a multi-factorial syndrome leading to poor outcomes for both the mother and foetus. Although an unusual increasing in the number of hospitalizations caused by Plasmodium vivax has been reported in Brazil, mortality is rarely observed. This is a report of a gestational malaria case that occurred in the city of Manaus (Amazonas State, Brazil) and resulted in foetal loss. The patient presented placental mixed-infection by Plasmodium vivax and Plasmodium falciparum after diagnosis by nested-PCR, however microscopic analysis failed to detect P. falciparum in the peripheral blood. Furthermore, as the patient did not receive proper treatment for P. falciparum and hospitalization occurred soon after drug treatment, it seems that P. falciparum pathology was modulated by the concurrent presence of P. vivax. Collectively, this case confirms the tropism towards the placenta by both of these species of parasites, reinforces the notion that co-existence of distinct malaria parasites interferes on diseases' outcomes, and opens discussions regarding diagnostic methods, malaria treatment during pregnancy and prenatal care for women living in unstable transmission areas of malaria, such as the Brazilian Amazon.

On the cytoadhesion of Plasmodium vivax-infected erythrocytes.

BACKGROUND: Plasmodium falciparum and Plasmodium vivax are responsible for most of the global burden of malaria. Although the accentuated pathogenicity of P. falciparum occurs because of sequestration of the mature erythrocytic forms in the microvasculature, this phenomenon has not yet been noted in P. vivax. The increasing number of severe manifestations of P. vivax infections, similar to those observed for severe falciparum malaria, suggests that key pathogenic mechanisms (eg, cytoadherence) might be shared by the 2 parasites. METHODS: Mature P. vivax-infected erythrocytes (Pv-iEs) were isolated from blood samples collected from 34 infected patients. Pv-iEs enriched on Percoll gradients were used in cytoadhesion assays with human lung endothelial cells, Saimiri brain endothelial cells, and placental cryosections. RESULTS: Pv-iEs were able to cytoadhere under static and flow conditions to cells expressing endothelial receptors known to mediate the cytoadhesion of P. falciparum. Although Pv-iE cytoadhesion levels were 10-fold lower than those observed for P. falciparum-infected erythrocytes, the strength of the interaction was similar. Cytoadhesion of Pv-iEs was in part mediated by VIR proteins, encoded by P. vivax variant genes (vir), given that specific antisera inhibited the Pv-iE-endothelial cell interaction. CONCLUSIONS: These observations prompt a modification of the current paradigms of the pathogenesis of malaria and clear the way to investigate the pathophysiology of P. vivax infections.

Violacein extracted from Chromobacterium violaceum inhibits Plasmodium growth in vitro and in vivo.

Violacein is a violet pigment extracted from the gram-negative bacterium Chromobacterium violaceum. It presents bactericidal, tumoricidal, trypanocidal, and antileishmanial activities. We show that micromolar concentrations efficiently killed chloroquine-sensitive and -resistant Plasmodium falciparum strains in vitro; inhibited parasitemia in vivo, even after parasite establishment; and protected Plasmodium chabaudi chabaudi-infected mice from a lethal challenge.

Time evolution of the activation energy in a batch chemical oscillator.

The batch-operated bromate/phosphate/acetone/dual catalyst system was studied at four temperatures between 5 and 35 degrees C. The dynamics was simultaneously followed by potential measurements with platinum and bromide selective electrodes, and spectroscopically at two different wavelengths. By simultaneously recording these four time series it was possible to characterize the dynamics of the sequential oscillations that evolve in time. The existence of three sequential oscillatory patterns at each temperature allowed estimating the activation energies in each case. Along with the activation energy of the induction period, it was possible to trace the time evolution of the overall activation energy at four different stages as the reaction proceeds. The study was carried out for two different sets of initial concentrations and it was observed that the overall activation energy increases as reactants turn into products. This finding was propounded as a result of the decrease in the driving force, or the system's affinity, of the catalytic oxidative bromination of acetone with acidic bromate, as the closed system evolves toward the thermodynamic equilibrium.