Role of the inhibitor of serine peptidase 2 (ISP2) of Trypanosoma brucei rhodesiense in parasite virulence and modulation of the inflammatory responses of the host.

Trypanosoma brucei rhodesiense is one of the causative agents of Human African Trypanosomiasis (HAT), known as sleeping sickness. The parasite invades the central nervous system and causes severe encephalitis that is fatal if left untreated. We have previously identified ecotin-like inhibitors of serine peptidases, named ISPs, in trypanosomatid parasitic protozoa. Here, we investigated the role of ISP2 in bloodstream form T. b. rhodesiense. We generated gene-deficient mutants lacking ISP2 (Δisp2), which displayed a growth profile in vitro similar to that of wild-type (WT) parasites. C57BL/6 mice infected with Δisp2 displayed lower blood parasitemia, a delayed hind leg pathological phenotype and survived longer. The immune response was examined at two time-points that corresponded with two peaks of parasitemia. At 4 days, the spleens of Δisp2-infected mice had a greater percentage of NOS2+ myeloid cells, IFN-γ+-NK cells and increased TNF-α compared to those infected with WT and parasites re-expressing ISP2 (Δisp2:ISP2). By 13 days the increased NOS2+ population was sustained in Δisp2-infected mice, along with increased percentages of monocyte-derived dendritic cells, as well as CD19+ B lymphocytes, and CD8+ and CD4+ T lymphocytes. Taken together, these findings indicate that ISP2 contributes to T. b. rhodesiense virulence in mice and attenuates the inflammatory response during early infection.

Novel monoclonal antibody L2A5 specifically targeting sialyl-Tn and short glycans terminated by alpha-2-6 sialic acids.

Incomplete O-glycosylation is a feature associated with malignancy resulting in the expression of truncated glycans such as the sialyl-Tn (STn) antigen. Despite all the progress in the development of potential anti-cancer antibodies, their application is frequently hindered by low specificities and cross-reactivity. In this study, a novel anti-STn monoclonal antibody named L2A5 was developed by hybridoma technology. Flow cytometry analysis showed that L2A5 specifically binds to sialylated structures on the cell surface of STn-expressing breast and bladder cancer cell lines. Moreover, immunoblotting assays demonstrated reactivity to tumour-associated O-glycosylated proteins, such as MUC1. Tumour recognition was further observed using immunohistochemistry assays, which demonstrated a high sensitivity and specificity of L2A5 mAb towards cancer tissue, using bladder and colorectal cancer tissues. L2A5 staining was exclusively tumoural, with a remarkable reactivity in invasive and metastasis sites, not detectable by other anti-STn mAbs. Additionally, it stained 20% of cases of triple-negative breast cancers, suggesting application in diseases with unmet clinical needs. Finally, the fine specificity was assessed using glycan microarrays, demonstrating a highly specific binding of L2A5 to core STn antigens and additional ability to bind 2-6-linked sialyl core-1 probes. In conclusion, this study describes a novel anti-STn antibody with a unique binding specificity that can be applied for cancer diagnostic and future development of new antibody-based therapeutic applications.

Descripción cinemática de la marcha en pacientes con ataxia espinocerebelosa de tipo 2 / Biomechanical phenomenum description of gait in patients with type 2 spinocerebellar ataxia

Se realizó un estudio descriptivo y transversal de 23 pacientes con diagnóstico clínico, neurológico y genético de ataxia espinocerebelosa de tipo 2, atendidos en la consulta de Neurogenética del Hospital General Docente "Dr. Juan Bruno Zayas Alfonso" de Santiago de Cuba, desde enero de 2013 hasta diciembre de 2015, a fin de obtener un grupo reducido de parámetros espaciotemporales de la marcha en dichos pacientes, mediante la técnica videográfica. También se incluyó una muestra de 35 individuos sanos mayores de 18 años, escogidos al azar. Se observaron diferencias significativas entre las variables cinemáticas extraídas de la evaluación de la marcha de sujetos enfermos y sanos. El método utilizado permitió determinar que el tiempo de evolución de la enfermedad influye en la disminución de la velocidad de marcha y que aumenta el ancho de paso y las oscilaciones de la cadera

A descriptive and cross-sectional study of 23 patients with clinical, neurological and genetic diagnosis of type 2 spinocerebellar ataxia, assisted in the Neurogenetic Service of "Dr. Juan Bruno Zayas Alfonso" Teaching General Hospital in Santiago de Cuba, was carried out from January, 2013 to December, 2015, in order to obtain a reduced group of gait spacetemporal parameters in these patients, by means of the videographic technique. A sample of 35 healthy individuals over 18 years chosen at random, was also included. Significant differences between the biomechanical phenomenum variables extracted from the evaluation of gait in sick and healthy people were observed. The method used allowed to determine that the course of the disease influences in the decrease of gait speed and increases the step width and the hip oscillations

Towards the development of multifunctional chitosan-based iron oxide nanoparticles: Optimization and modelling of doxorubicin release.

In the present work composite nanoparticles with a magnetic core and a chitosan-based shell were produced as drug delivery systems for doxorubicin (DOX). The results show that composite nanoparticles with a hydrodynamic diameter within the nanometric range are able to encapsulate more DOX than polymeric nanoparticles alone corresponding also to a higher drug release. Moreover the synthesis method of the iron oxide nanoparticles influences the total amount of DOX released and a high content of iron oxide nanoparticles inhibits DOX release. The modelling of the experimental results revealed a release mechanism dominated by Fickian diffusion.

Thermal and magnetic properties of chitosan-iron oxide nanoparticles.

Chitosan is a biopolymer widely used for biomedical applications such as drug delivery systems, wound healing, and tissue engineering. Chitosan can be used as coating for other types of materials such as iron oxide nanoparticles, improving its biocompatibility while extending its range of applications. In this work iron oxide nanoparticles (Fe3O4 NPs) produced by chemical precipitation and thermal decomposition and coated with chitosan with different molecular weights were studied. Basic characterization on bare and chitosan-Fe3O4 NPs was performed demonstrating that chitosan does not affect the crystallinity, chemical composition, and superparamagnetic properties of the Fe3O4 NPs, and also the incorporation of Fe3O4 NPs into chitosan nanoparticles increases the later hydrodynamic diameter without compromising its physical and chemical properties. The nano-composite was tested for magnetic hyperthermia by applying an alternating current magnetic field to the samples demonstrating that the heating ability of the Fe3O4 NPs was not significantly affected by chitosan.

Chitosan-based nanoparticles as drug delivery systems for doxorubicin: Optimization and modelling.

In the present work, two drug delivery systems were produced by encapsulating doxorubicin into chitosan and O-HTCC (ammonium-quaternary derivative of chitosan) nanoparticles. The results show that doxorubicin release is independent of the molecular weight and is higher at acidic pH (4.5) than at physiological pH. NPs with an average hydrodynamic diameter bellow 200nm are able to encapsulate up to 70% and 50% of doxorubicin in the case of chitosan and O-HTCC nanoparticles, respectively. O-HTCC nanoparticles led to a higher amount of doxorubicin released than chitosan nanoparticles, for the same experimental conditions, although the release mechanism was not altered. A burst effect occurs within the first hours of release, reaching a plateau after 24h. Fitting mathematical models to the experimental data led to a concordant release mechanism between most samples, indicating an anomalous or mixed release, which is in agreement with the swelling behavior of chitosan described in the literature.

Thermal and magnetic properties of iron oxide colloids: influence of surfactants.

Iron oxide nanoparticles (NPs) have been extensively studied in the last few decades for several biomedical applications such as magnetic resonance imaging, magnetic drug delivery and hyperthermia. Hyperthermia is a technique used for cancer treatment which consists in inducing a temperature of about 41-45 °C in cancerous cells through magnetic NPs and an external magnetic field. Chemical precipitation was used to produce iron oxide NPs 9 nm in size coated with oleic acid and trisodium citrate. The influence of both stabilizers on the heating ability and in vitro cytotoxicity of the produced iron oxide NPs was assessed. Physicochemical characterization of the samples confirmed that the used surfactants do not change the particles' average size and that the presence of the surfactants has a strong effect on both the magnetic properties and the heating ability. The heating ability of Fe3O4 NPs shows a proportional increase with the increase of iron concentration, although when coated with trisodium citrate or oleic acid the heating ability decreases. Cytotoxicity assays demonstrated that both pristine and trisodium citrate Fe3O4 samples do not reduce cell viability. However, oleic acid Fe3O4 strongly reduces cell viability, more drastically in the SaOs-2 cell line. The produced iron oxide NPs are suitable for cancer hyperthermia treatment and the use of a surfactant brings great advantages concerning the dispersion of NPs, also allowing better control of the hyperthermia temperature.

Challenges in Antibody Development against Tn and Sialyl-Tn Antigens.

The carbohydrate antigens Tn and sialyl-Tn (STn) are expressed in most carcinomas and usually absent in healthy tissues. These antigens have been correlated with cancer progression and poor prognosis, and associated with immunosuppressive microenvironment. Presently they are used in clinical trials as therapeutic vaccination, but with limited success due to their low immunogenicity. Alternatively, anti-Tn and/or STn antibodies may be used to harness the immune system against tumor cells. Whilst the development of antibodies against these antigens had a boost two decades ago for diagnostic use, so far no such antibody entered into clinical trials. Possible limitations are the low specificity and efficiency of existing antibodies and that novel antibodies are still necessary. The vast array of methodologies available today will allow rapid antibody development and novel formats. Following the advent of hybridoma technology, the immortalization of human B cells became a methodology to obtain human monoclonal antibodies with better specificity. Advances in molecular biology including phage display technology for high throughput screening, transgenic mice and more recently molecularly engineered antibodies enhanced the field of antibody production. The development of novel antibodies against Tn and STn taking advantage of innovative technologies and engineering techniques may result in innovative therapeutic antibodies for cancer treatment.

Protein disulfide isomerases: Impact of thapsigargin treatment on their expression in melanoma cell lines.

Anti-cancer treatments usually elevate the content of unfolded or misfolded proteins in the endoplasmic reticulum (ER). Here we aimed to get insights into the relation between sensitivity of melanoma cell lines to the ER stress inducer thapsigargin (THG) and the genetic expression of protein disulfide isomerase family members (PDIs). The expression of PDIs was analysed by flow cytometry and real-time PCR. The results showed that SK-MEL-30, the less THG sensitive cell line, displays higher basal PDIs' expression levels and the sensitivity is increased by the PDIs inhibitor bacitracin. While SK-MEL-30 PDIs' expression is not THG dose-dependent, an increase in glucose related protein 78 (GRP78), PDIA5, PDIA6, and thioredoxin-related-transmembrane proteins' (TMX3 and TMX4) expression, in response to higher drug concentrations, was observed in MNT-1. The differences in PDIs' gene expression in MNT-1 suggest a different response to ER stress compared to the other cell lines and highlight the importance of understanding the diversity among cancer cells.

Cardiodesfibrilador implantável subcutâneo: um novo dispositivo para o tratamento da morte súbita / Subcutaneous implantable defibrillator: a new device for sudden death treatment

A morte súbita é um importante problema de saúde pública e o cardiodesfibrilador implantável (CDI) tem se mostrado mais efetivo que as drogas antiarrítmicas em seu tratamento. A despeito do grande benefício clínico dessa terapia, existem relatos de complicações relevantes relacionadas ao cabo-eletrodo transvenoso. Com o propósito de as minimizar, recentemente foi lançado nos mercados europeu, neozelandês e norte-americano um CDI com sistema totalmente subcutâneo (CDI-S) que dispensa a fluoroscopia. Os resultados iniciais de estudos clínicos publicados são promissores e mostram que o CDI-S converte com sucesso episódios de taquicardia ventricular (TV) e fibrilação ventricular (FV) induzidas ou espontâneas, com taxas de complicações ou de choques inapropriados semelhantes às do CDI convencional. Parece ser uma opção atraente para pacientes pediátricos com doença cardíaca congênita, pacientes com acesso venoso obstruído ou com alto risco de infecção, bem como para jovens com doença cardíaca elétrica, tais como miocardiopatia hipertrófica e síndromes de Brugada e do QT longo. Devido à impossibilidade de estimulação cardíaca permanente, não está indicado para os que necessitam de estimulação anti-bradicardia, terapia de ressincronização cardíaca (TRC) ou com história de taquicardia ventricular monomórfica repetitiva que se beneficiariam de estimulação anti-taquicardia (ATP). Estudos clínicos em andamento irão definir com maior precisão a segurança e a eficácia em longo prazo, o custo-efetividade e o perfil ideal dos pacientes que obterão os maiores benefícios dessa nova tecnologia...

Sudden death is an important public health problem; the implantable cardioverter defibrillator (ICD) has proven more effective than antiarrhythmic drugs in the treatment of sudden death. Despite the great clinical benefit of this therapy, there are reports of significant complications related to transvenous lead-electrode. In order to minimize these complications, recently was launched on the market in Europe, New Zealand and the United States a subcutaneous system (S-ICD) that even eliminates the need for fluoroscopy. The initial results of published clinical studies have been promising and show that ICD-S successfully converts the ventricular tachycardia episodes (VT)/ventricular fibrillation(VF), induced or spontaneous, with rates of complications or inappropriate shocks similar to conventional ICD. It seems to be an attractive option for pediatric patients with congenital heart disease that limits the implantation of transvenous leads, in cases of obstructed venous access, in patients at high risk of infection, as well as young people with electrical heart disease, such as hypertrophic cardiomyopathy, Brugada syndrome and long QT syndrome. Due to the impossibility of permanent cardiac pacing, the S-ICD is not indicated for patients that requiring anti-bradycardia pacing, or cardiac resynchronization therapy (CRT), or with a history of repetitive monomorphic ventricular tachycardia that would benefit from anti-tachycardia pacing (ATP). Ongoing clinical studies will better define its safety and long-term efficacy, cost effectiveness and better patient profile that this new technology will benefit...

Effects of surfactants on the magnetic properties of iron oxide colloids.

Iron oxide nanoparticles are having been extensively investigated for several biomedical applications such as hyperthermia and magnetic resonance imaging. However, one of the biggest problems of these nanoparticles is their aggregation. Taking this into account, in this study the influence of three different surfactants (oleic acid, sodium citrate and Triton X-100) each one with various concentrations in the colloidal solutions stability was analyzed by using a rapid and facile method, the variation in the optical absorbance along time. The synthesized nanoparticles through chemical precipitation showed an average size of 9 nm and a narrow size distribution. X-ray diffraction pattern and Fourier Transform Infrared analysis confirmed the presence of pure magnetite. SQUID measurements showed superparamagnetic properties with a blocking temperature around 155 K. In addition it was observed that neither sodium citrate nor Triton X-100 influences the magnetic properties of the nanoparticles. On the other hand, oleic acid in a concentration of 64 mM decreases the saturation magnetization from 67 to 45 emu/g. Oleic acid exhibits a good performance as stabilizer of the iron oxide nanoparticles in an aqueous solution for 24h, for concentrations that lead to the formation of the double layer.

Identification and Functional Characterisation of CRK12:CYC9, a Novel Cyclin-Dependent Kinase (CDK)-Cyclin Complex in Trypanosoma brucei.

The protozoan parasite, Trypanosoma brucei, is spread by the tsetse fly and causes trypanosomiasis in humans and animals. Both the life cycle and cell cycle of the parasite are complex. Trypanosomes have eleven cdc2-related kinases (CRKs) and ten cyclins, an unusually large number for a single celled organism. To date, relatively little is known about the function of many of the CRKs and cyclins, and only CRK3 has previously been shown to be cyclin-dependent in vivo. Here we report the identification of a previously uncharacterised CRK:cyclin complex between CRK12 and the putative transcriptional cyclin, CYC9. CRK12:CYC9 interact to form an active protein kinase complex in procyclic and bloodstream T. brucei. Both CRK12 and CYC9 are essential for the proliferation of bloodstream trypanosomes in vitro, and we show that CRK12 is also essential for survival of T. brucei in a mouse model, providing genetic validation of CRK12:CYC9 as a novel drug target for trypanosomiasis. Further, functional characterisation of CRK12 and CYC9 using RNA interference reveals roles for these proteins in endocytosis and cytokinesis, respectively.

Cell phenotypic change due to Cryptosporidium parvum infection in immunocompetent mice.

Cryptosporidium parvum is an intracellular parasite causing enteritis which can become life-threatening in immunocompromised host. Immunoregulatory T cells play a central role in the regulatory network of the host. Here, we proposed to characterize the populations of immune cells during infection and reinfection with C. parvum. Four-week-old BALB/C mice were inoculated with oocysts of C. parvum at days 0 and 22. Fecal and blood samples, spleens, and small intestines were collected for analysis. Peripheral blood and spleen cell populations were characterized by flow cytometry. After infection (days 0 to 21), mice presented higher values of neutrophils, eosinophils, NK cells and CD4(+)CD25(high) T cells in peripheral blood. After reinfection, this upward trend continued in the following days for all four populations in infected mice. At day 35, infected mice presented similar values to the control group, except for CD4(+)CD25(high) T cells, which remained higher in infected mice. A possible correlation between alterations in blood and spleen cell populations was also studied, but no consistent association could be established. Small intestine sections were screened for intracellular stages of the parasite but no evidence of pathology was observed. Here, we report information which may be important for the understanding of the specific cell-mediated response in immunocompetent mice to C. parvum infection. Although some questions remain unanswered and complementary studies are needed, our results are expected to contribute to a better understanding of innate and Treg cells role in the clearance process of this parasite.

Dynamics of cytokines and immunoglobulins serum profiles in primary and secondary Cryptosporidium parvum infection: usefulness of Luminex® xMAP technology.

Infection by Cryptosporidium parvum triggers a complex array of innate and adaptive cell mediated immune response, playing an important role in controlling the infection. To date, there are no studies applying the Luminex® xMAP technology to determine profiles of cytokines and immunoglobulins in the context of an infection by C. parvum. In this study, we analyzed these immune mediators in the serum of immunocompetent mice inoculated with C. parvum oocysts, using Luminex, to understand how the immune system responds to an infection by this parasite. Animal sera were also analyzed by ELISA to determine the expressed immunoglobulin isotype profile, and compare the obtained trend with data obtained by Luminex. Specific-pathogen-free BALB/C mice were inoculated with oocysts of C. parvum at days 0 and 22. Peripheral blood was aseptically collected from sacrificed mice on several time points, and immune mediators were evaluated in serum samples. Infection was confirmed by the presence of C. parvum DNA in feces by a nested-PCR assay (60-kDa glycoprotein). Luminex results showed predominance in the secretion of IgG1 and IgG2a, confirmed by ELISA, which also showed that IgG1 is the major immunoglobulin isotype produced during the infection. The analysis of cytokines suggests a preferential Th(1) over the Th(2) response, with increased production of TNF-α, IFN-γ and GM-CSF. This work contributed to a better understanding of the immune response to the infection by C. parvum, as well as demonstrated the advantage of Luminex® xMAP technology to study immune mediators, using small sample volumes.

Application of hyperthermia for cancer treatment: recent patents review.

Cancer is one of the main causes of death in the world and its incidence increases every day. Current treatments are insufficient and present many breaches. Hyperthermia is an old concept and since early it was established as a cancer treatment option, mainly in superficial cancers. More recently the concept of intracellular hyperthermia emerged wherein magnetic particles are concentrated at the tumor site and remotely heated using an applied magnetic field to achieve hyperthermic temperatures (42-45°C). Many patents have been registered in this area since the year 2000. This review presents the most relevant information, organizing them according to the hyperthermic method used: 1) external Radio-Frequency devices; 2) hyperthermic perfusion; 3) frequency enhancers; 4) apply heating to the target site using a catheter; 5) injection of magnetic and ferroelectric particles; 6) injection of magnetic nanoparticles that may carry a pharmacological active drug. The use of magnetic nanoparticles is a very promising treatment approach since it may be used for diagnostic and treatment. An ideal magnetic nanoparticle would be able to detect and diagnose the tumor, carry a pharmacological active drug to be delivered in the tumor site, apply hyperthermia through an external magnetic field and allow treatment monitoring by magnetic resonance imaging.

Análisis de la funcionabilidad de prótesis ortopédicas transfemorales / Analysis of the functional qualities of trans-femoral orthopedic prostheses

Introducción: la funcionabilidad de los artificios protésicos de miembros inferiores suele expresarse en términos de relacionar sus beneficios operacionales con aquellas necesidades funcionales y de bienestar del amputado. Este bienestar se relaciona fundamentalmente con un mínimo gasto energético del paciente y con la ausencia de enfermedades en las articulaciones residuales y que se inducen por la prótesis durante el régimen de marcha. Objetivo: evaluar la funcionabilidad de prótesis transfemorales utilizadas en Cuba mediante la integración de los resultados del análisis cinemático de la marcha del amputado, combinados con análisis de mecánica de sólidos rígidos aplicando simulaciones numéricas. Métodos: el análisis de la marcha de 8 pacientes con amputaciones unilaterales transfemorales y 5 sujetos normales (control) se realizó mediante técnicas videográficas. Se efectuó la estimación dinámica de las fuerzas que actúan en los segmentos articulares y se determinaron las tensiones y deformaciones que sufren las zonas del muñón y de la articulación del tobillo mediante el método de los elementos finitos. Resultados: cambios significativos en la dinámica de los segmentos articulados del tobillo-rodilla y cadera se reflejan como el resultado de la asimetría de la marcha del miembro protésico y el sano. Tal desequilibrio sugiere ser una de las principales causas de las enfermedades degenerativas de la cadera observadas en este tipo de paciente. Conclusiones: el análisis de la funcionabilidad de los artificios protésicos transfemorales solo es posible mediante la integración de los métodos cinemáticos de caracterización de la marcha, métodos dinámicos que establezcan la posibilidad de replicar la actividad del miembro sano. Mediante este estudio fue posible además el estudio de la resistencia mecánica y se vincularon todos los resultados a la aparición de enfermedades tanto en el miembro intacto como en el residual

Introduction: the functional quality of the prosthetic devices of lower extremities is usually expressed in terms to relate the functional benefits to those functional needs and the wellbeing of the amputee. This wellbeing is related mainly to a minimal energetic output of patient and to the lack of diseases of residual joints induced by the prosthesis during the gait. Objective: to assess the functional qualities of trans-femoral prostheses used in Cuba by the integration of results of kinematics analysis of the gait of amputee, combined with mechanics analysis of rigid solids applying numerical simulations. Methods: the gait's analysis of 8 patients with trans-femoral unilateral amputations and 5 normal subjects (controls) was made by videographic techniques. The dynamics estimation of strengths acting on the articular segments was carried out and determination of strain and deformations suffering the stump zones and of the ankle joint by finite elements method. Results: the significant changes in the dynamics of ankle-knee-hip articulation segments are present as the result of gait asymmetry of the prosthetic limb and the healthy one. This imbalance may be one of the leading causes of degenerative diseases of hip observed in this type of patient. Conclusions: the analysis of functional qualities of trans-femoral prosthetic devices only is possible by means of the integration of cinematic methods of gait characterization, dynamic methods establishing the possibility to replicate to the activity of healthy limb. Using this study also it was possible the mechanical resistance study linking all results to the appearance of diseases of intact limb as well as the residual one

Besnoitia besnoiti protein disulfide isomerase (BbPDI): molecular characterization, expression and in silico modelling.

Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a disease with a high prevalence in tropical and subtropical regions and re-emerging in Europe. Despite the great economical losses associated with besnoitiosis, this disease has been underestimated and poorly studied, and neither an effective therapy nor an efficacious vaccine is available. Protein disulfide isomerase (PDI) is an essential enzyme for the acquisition of the correct three-dimensional structure of proteins. Current evidence suggests that in Neosporacaninum and Toxoplasmagondii, which are closely related to B. besnoiti, PDI play an important role in host cell invasion, is a relevant target for the host immune response, and represents a promising drug target and/or vaccine candidate. In this work, we present the nucleotide sequence of the B. besnoiti PDI gene. BbPDI belongs to the thioredoxin-like superfamily (cluster 00388) and is included in the PDI_a family (cluster defined cd02961) and the PDI_a_PDI_a'_c subfamily (cd02995). A 3D theoretical model was built by comparative homology using Swiss-Model server, using as a template the crystallographic deduced model of Tapasin-ERp57 (PDB code 3F8U chain C). Analysis of the phylogenetic tree for PDI within the phylum apicomplexa reinforces the close relationship among B. besnoiti, N. caninum and T. gondii. When subjected to a PDI-assay based on the polymerisation of reduced insulin, recombinant BbPDI expressed in E. coli exhibited enzymatic activity, which was inhibited by bacitracin. Antiserum directed against recombinant BbPDI reacted with PDI in Western blots and by immunofluorescence with B. besnoiti tachyzoites and bradyzoites.

Potent and broadly reactive HIV-2 neutralizing antibodies elicited by a vaccinia virus vector prime-C2V3C3 polypeptide boost immunization strategy.

Human immunodeficiency virus type 2 (HIV-2) infection affects about 1 to 2 million individuals, the majority living in West Africa, Europe, and India. As for HIV-1, new strategies for the prevention of HIV-2 infection are needed. Our aim was to produce new vaccine immunogens that elicit the production of broadly reactive HIV-2 neutralizing antibodies (NAbs). Native and truncated envelope proteins from the reference HIV-2ALI isolate were expressed in vaccinia virus or in bacteria. This source isolate was used due to its unique phenotype combining CD4 independence and CCR5 usage. NAbs were not elicited in BALB/c mice by single immunization with a truncated and fully glycosylated envelope gp125 (gp125t) or a recombinant polypeptide comprising the C2, V3, and C3 envelope regions (rpC2-C3). A strong and broad NAb response was, however, elicited in mice primed with gp125t expressed in vaccinia virus and boosted with rpC2-C3. Serum from these animals potently neutralized (median 50% neutralizing titer, 3,200) six of six highly divergent primary HIV-2 isolates. Coreceptor usage and the V3 sequence of NAb-sensitive isolates were similar to that of the vaccinating immunogen (HIV-2ALI). In contrast, NAbs were not reactive on three X4 isolates that displayed major changes in V3 loop sequence and structure. Collectively, our findings demonstrate that broadly reactive HIV-2 NAbs can be elicited by using a vaccinia virus vector-prime/rpC2-C3-boost immunization strategy and suggest a potential relationship between escape to neutralization and cell tropism.

Gene sequencing, modelling and immunolocalization of the protein disulfide isomerase from Plasmodium chabaudi.

Malaria remains one of the major human parasitic diseases, particularly in subtropical regions. Most of the fatal cases are caused by Plasmodium falciparum. The rodent parasite Plasmodium chabaudi has been the model of choice in research due to its similarities to human malaria, including developmental cycle, preferential invasion of mature erythrocytes, synchrony of asexual development, antigenic variation, gene sinteny as well as similar resistance mechanisms. Protein disulfide isomerase (PDI) is an essential catalyst of the endoplasmic reticulum in different biological systems with folding and chaperone activities. Most of the proteins exported by parasites have to pass through the endoplasmic reticulum before reaching their final destination and their correct folding is critical for parasite survival. PDI constitutes a potential target for the development of alternative therapy strategies based on the inhibition of folding and chaperoning of exported proteins. We here describe the sequencing of the gene coding for the PDI from P. chabaudi and analyse the relationship to its counterpart enzymes, particularly with the PDI from other Plasmodium species. The model constructed, based on the recent model deduced from the crystallographic structure 2B5E, was compared with the previous theoretical model for the whole PDI molecule constructed by threading. A recombinant PDI from P. chabaudi was also produced and used as an antigen for monoclonal antibody production for application in PDI immunolocalization.

Plasmodium chabaudi: expression of active recombinant chabaupain-1 and localization studies in Anopheles sp.

Plasmodium cysteine proteases have been shown to be immunogenic and are being used as malaria potential serodiagnostic markers and vaccine targets. Genes encoding two Plasmodium chabaudi cysteine proteases chabaupain-1 (CP-1) and chabaupain-2 (CP-2) were identified and further expressed in Escherichia coli. Solubilisation of recombinant CP-1 and CP-2 was achieved by decreasing the temperature of induction. Anopheles gambiae tissues infected with Plasmodium were analyzed by Western blotting using the anti-CP-1 antibody showing that CP-1 is only present in the A. gambiae midguts being absent from other infected mosquito biological material. Anti-CP-1 anti-serum recognized a 30 kDa band in P. chabaudi, Plasmodium berghei and Plasmodium yoelii lysates but does not recognize the recombinant CP-2 extracts suggesting high antibody specificity.