LL-37_Renalexin hybrid peptide exhibits antimicrobial activity at lower MICs than its counterpart single peptides.

An alarming global public health and economic peril has been the emergence of antibiotic resistance resulting from clinically relevant bacteria pathogens, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species constantly exhibiting intrinsic and extrinsic resistance mechanisms against last-resort antibiotics like gentamycin, ciprofloxacin, tetracycline, colistin, and standard ampicillin prescription in clinical practices. The discovery and applications of antimicrobial peptides (AMPs) with antibacterial properties have been considered and proven as alternative antimicrobial agents to antibiotics. In this study, we have designed, produced, and purified a recombinant novel multifunctional hybrid antimicrobial peptide LL-37_Renalexin for the first time via the application of newly designed flexible GS peptide linker coupled with the use of our previously characterized small metal-binding proteins SmbP and CusF3H+ as carrier proteins that allow for an enhanced bacterial expression, using BL21(DE3) and SHuffle T7(DE3) Escherichia coli strains, and purification of the hybrid peptide via immobilized metal affinity chromatography. The purified tag-free LL-37_Renalexin hybrid peptide exhibited above 85% reduction in bacteria colony-forming units and broad-spectrum antimicrobial effects against Staphylococcus aureus, Escherichia coli, Methicillin-resistant Staphylococcus aureus (MRSA), and Klebsiella pneumoniae bacteria clinical isolates at a lower minimum inhibition concentration level (10-33 µM) as compared to its counterpart single-AMPs LL-37 and Renalexin (50-100 µM). KEY POINTS: • The hybrid antimicrobial peptide LL-37_Renalexin has been designed using a GS linker. • The peptide was expressed with the carrier proteins SmbP and CusF3H+. • The hybrid peptide shows antibacterial potency against clinical bacterial isolates.

In Silico Physicochemical Characterization of Fusion Proteins from Emerging Amazonian Arboviruses.

Mayaro (MAYV), Saint Louis encephalitis (SLEV), and Oropouche (OROV) viruses are neglected members of the three main families of arboviruses with medical relevance that circulate in the Amazon region as etiological agents of outbreaks of febrile illnesses in humans. As enveloped viruses, MAYV, SLEV, and OROV largely depend on their class II fusion proteins (E1, E, and Gc, respectively) for entry into the host cell. Since many aspects of the structural biology of such proteins remain unclear, the present study aimed at physicochemically characterizing them by an in silico approach. The complete amino acid sequences of MAYV E1, SLEV E, and OROV Gc proteins derived by conceptual translation from annotated coding regions in the reference sequence genome of the respective viruses were obtained from the NCBI Protein database in the FASTA format and then submitted to the ClustalO, Protcalc, Pepstats, Predator, Proscan, PCprof, Phyre2, and 3Drefine web servers for the determination of sequence identities, the estimation of residual properties, the prediction of secondary structures, the identification of potential post-translational modifications, the recognition of antigenic propensities, and the modeling/refinement of three-dimensional structures. Sequence identities were 20.44%, 18.82%, and 13.70% between MAYV/SLEV, SLEV/OROV, and MAYV/OROV fusion proteins, respectively. As for the residual properties, MAYV E1 and SLEV E proteins showed a predominance of the non-polar profile (56% and 55% of the residues, respectively), whereas the OROV Gc protein showed a predominance of the polar profile (52% of the residues). Regarding predicted secondary structures, MAYV E1 and SLEV E proteins showed fewer alpha-helices (16.51% and 15.17%, respectively) than beta-sheets (21.79% and 25.15%, respectively), while the opposite was observed in the OROV Gc protein (20.39% alpha-helices and 12.14% beta-sheets). Regarding post-translational modifications, MAYV E1, SLEV E, and OROV Gc proteins showed greater relative potential for protein kinase C phosphorylation, N-myristoylation, and casein kinase II phosphorylation, respectively. Finally, antigenic propensities were higher in the N-terminus half than in the C-terminus half of these three proteins, whose three-dimensional structures revealed three distinctive domains. In conclusion, MAYV E1 and SLEV E proteins were found to share more physicochemical characteristics with each other than the OROV Gc protein, although they are all grouped under the same class of viral fusion proteins.

Respiratory syncytial virus entry mechanism in host cells: A general overview.

Respiratory syncytial virus (RSV) is a virus that causes acute respiratory infections in neonates and older adults. To infect host cells, the attachment glycoprotein (G) interacts with a cell surface receptor. This interaction determines the specific cell types that are susceptible to infection. RSV possesses a type I fusion protein F. Type I fusion proteins are metastable when rearrangement of the prefusion F occurs; the fusion peptide is exposed transforming the protein into postfusion form. The transition between the prefusion form and its postfusion form facilitates the viral envelope and the host cell membrane to fuse, enabling the virus to enter the host cell. Understanding the entry mechanism employed by RSV is crucial for developing effective antiviral therapies. In this review, we will discuss the various types of viral fusion proteins and explore the potential entry mechanisms utilized by RSV. A deeper understanding of these mechanisms will provide valuable insights for the development of novel approaches to treat RSV infections.

In vitro and in vivo efficacies of Dectin-1-Fc(IgG)(s) fusion proteins against invasive fungal infections.

Fungal infections have increased in the last years, particularly associated to an increment in the number of immunocompromised individuals and the emergence of known or new resistant species, despite the difficulties in the often time-consuming diagnosis. The controversial efficacy of the currently available strategies for their clinical management, apart from their high toxicity and severe side effects, has renewed the interest in the research and development of new broad antifungal alternatives. These encompass vaccines and passive immunization strategies with monoclonal antibodies (mAbs), recognizing ubiquitous fungal targets, such as fungal cell wall ß-1,3-glucan polysaccharides, which could be used in early therapeutic intervention without the need for the diagnosis at species level. As additional alternatives, based on the Dectin-1 great affinity to ß-1,3-glucan, our group developed broad antibody-like Dectin1-Fc(IgG)(s) from distinct subclasses (IgG2a and IgG2b) and compared their antifungal in vitro and passive immunizations in vivo performances. Dectin1-Fc(IgG2a) and Dectin1-Fc(IgG2b) demonstrated high affinity to laminarin and the fungal cell wall by ELISA, flow cytometry, and microscopy. Both Dectin-1-Fc(IgG)(s) inhibited Histoplasma capsulatum and Cryptococcus neoformans growth in a dose-dependent fashion. For Candida albicans, such inhibitory effect was observed with concentrations as low as 0.098 and 0.049 µg/ml, respectively, which correlated with the impairment of the kinetics and lengths of germ tubes in comparison to controls. Previous opsonization with Dectin-1-Fc(IgG)(s) enhanced considerably the macrophage antifungal effector functions, increasing the fungi macrophages interactions and significantly reducing the intraphagosome fungal survival, as lower CFUs were observed. The administration of both Dectin1-Fc(IgG)(s) reduced the fungal burden and mortality in murine histoplasmosis and candidiasis models, in accordance with previous evaluations in aspergillosis model. These results altogether strongly suggested that therapeutic interventions with Dectin-1-Fc(IgG)(s) fusion proteins could directly impact the innate immunity and disease outcome in favor of the host, by direct neutralization, opsonization, phagocytosis, and fungal elimination, providing interesting information on the potential of these new strategies for the control of invasive fungal infections. LAY SUMMARY: Mycoses have increased worldwide, and new efficient therapeutics are needed. Passive immunizations targeting universally the fungal cell would allow early interventions without the species-level diagnosis. Lectins with affinity to carbohydrates could be used to engineer 'antibody-like' strategies.

Optimization of Expression and Purification of Schistosoma mansoni Antigens in Fusion with Rhizavidin.

Schistosomiasis causes significant morbidity and mortality. Vaccine efforts to date indicate the need to increase the immunogenicity of Schistosoma antigens. The multiple antigen-presenting system, whereby proteins are genetically fused to rhizavidin and affinity linked to biotinylated templates, enables the generation of robust immune responses. The objective of this work was to express and purify the S. mansoni antigens, SmTSP-2 and SmCD59.2, in fusion with rhizavidin. The fusion with rhizavidin greatly decreased the expression level of rSmTSP-2, but not rSmCD59.2, and both were expressed in the insoluble fraction, requiring optimization of culture conditions. Evaluation of different E. coli strains and media showed that BL21-DE3 cultured in Terrific Broth provided the highest expression levels of both proteins. Investigation of a range of time and temperature of induction showed that E. coli strains expressing rRzv:SmTSP-2 and rRzv:SmCD59.2 showed the highest protein production at 23 °C for 15 h. Recombinant proteins were purified by a single step of affinity chromatography allowing isolation of these proteins in high concentration and purity. The optimization process increased final soluble protein yield of rRzv:SmTSP-2 by fourfold and rRzv:SmCD59.2 by tenfold, providing ~ 20 mg/L of each protein. Optimized fusion protein production will allow antigen use in biotin-rhizavidin affinity platforms.

Bifunctional GM-CSF-derived peptides as tools for O-glycoengineering and protein tagging.

Rapid development of effective biotherapeutics has been a concern during the last couple decades. In our work we designed two novel peptide tags, GMOP and mGMOP, derived from the N-terminal region of human granulocyte and macrophage colony stimulating factor (hGM-CSF), which contain four and six potential O-glycosylation sites, respectively. These peptide tags were fused to the N-terminus of human interferon-α2b (hIFN-α2b), a therapeutic antiviral and antiproliferative protein rapidly cleared from circulation. Two new molecules were obtained which, consistently with the presence of O-glycans, showed higher molecular masses, more negatively charged isoforms, and higher sialic acid content compared to wild-type IFN. In vitro bioactivity of purified chimeras revealed a similar antiviral specific biological activity (SBA) compared to unmodified IFN. A reduction of antiproliferative SBA was only observed for mGMOP-IFN. Pharmacokinetic studies in rats showed a notable improvement in terminal half-life (t1/2elim) (3.3 and 2.8 times-longer) and a marked reduction of the apparent clearance (CLapp, 3.7 and 4.1-fold lower for GMOP-IFN and mGMOP-IFN in comparison with native IFN, respectively). Furthermore, the in vitro thermal and plasma stability of both proteins was improved. Finally, a monoclonal antibody (mAb) that recognizes an N-terminal GM-CSF epitope was able to bind both chimeras in western blots and ELISAs. This demonstrates the potential of both peptides to behave as bifunctional tags to create novel long-acting biotherapeutics and to facilitate detection and purification.

Optimization of expression and purification of schistosoma mansoni antigens in fusion with Rhizavidin

Schistosomiasis causes significant morbidity and mortality. Vaccine efforts to date indicate the need to increase the immunogenicity of Schistosoma antigens. The multiple antigen-presenting system, whereby proteins are genetically fused to rhizavidin and affinity linked to biotinylated templates, enables the generation of robust immune responses. The objective of this work was to express and purify the S. mansoni antigens, SmTSP-2 and SmCD59.2, in fusion with rhizavidin. The fusion with rhizavidin greatly decreased the expression level of rSmTSP-2, but not rSmCD59.2, and both were expressed in the insoluble fraction, requiring optimization of culture conditions. Evaluation of different E. coli strains and media showed that BL21-DE3 cultured in Terrific Broth provided the highest expression levels of both proteins. Investigation of a range of time and temperature of induction showed that E. coli strains expressing rRzv:SmTSP-2 and rRzv:SmCD59.2 showed the highest protein production at 23 °C for 15 h. Recombinant proteins were purified by a single step of affinity chromatography allowing isolation of these proteins in high concentration and purity. The optimization process increased final soluble protein yield of rRzv:SmTSP-2 by fourfold and rRzv:SmCD59.2 by tenfold, providing ~ 20 mg/L of each protein. Optimized fusion protein production will allow antigen use in biotin–rhizavidin affinity platforms.

Development of an Antigen Delivery Platform Using Lactobacillus acidophilus Decorated With Heterologous Proteins: A Sheep in Wolf's Clothing Story.

S-layers are bacterial structures present on the surface of several Gram-positive and Gram-negative bacteria that play a role in bacterial protection. In Lactobacillus acidophilus (L. acidophilus ATCC 4356), the S-layer is mainly composed of the protein SlpA. A tandem of two copies of the protein domain SLP-A (pfam: 03217) was identified at the C-terminal of SlpA, being this double SLP-A protein domain (in short dSLP-A) necessary and sufficient for the association of the protein to the L. acidophilus cell wall. A variety of proteins fused to the dSLP-A domain were able to spontaneously associate with high affinity to the cell wall of L. acidophilus and Bacillus subtilis var. natto, in a process that we termed decoration. Binding of dSLP-A-containing-proteins to L. acidophilus was stable at conditions that mimic the gastrointestinal transit in terms of pH, proteases, and bile salts. To evaluate if protein decoration of L. acidophilus can be adapted to generate an oral vaccine platform, a chimeric antigen derived from the bacterial pathogen Shiga-toxin-producing Escherichia coli (STEC) was constructed by fusing the sequences encoding the polypeptides EspA36-192, Intimin653-953, Tir240-378, and H7 flagellin352-374 (EITH7) to the dSLP-A domain (EITH7-dSLP-A). Recombinantly expressed EITH7-dSLP-A protein was affinity purified and combined with L. acidophilus cultures to allow the association of the chimeric antigen to the bacterial surface. EITH7-decorated L. acidophilus was orally administered to BALB/c mice and the induction of anti-EITH7 specific antibodies in sera and feces determined by ELISA. Mice presenting significantly higher anti-EITH7 antibodies titers were able to control more efficiently an experimental STEC infection than mice that received the non-decorated L. acidophilus carrier, indicating that antigen-decorated L. acidophilus can be adapted as a mucosal immunization delivery platform to elicit a protective immune response for vaccine purposes.

Protective Efficacy of Lectin-Fc(IgG) Fusion Proteins In Vitro and in a Pulmonary Aspergillosis In Vivo Model.

Aspergillosis cases by Aspergillus fumigatus have increased, along with fungal resistance to antifungals, urging the development of new therapies. Passive immunization targeting common fungal antigens, such as chitin and ß-glucans, are promising and would eliminate the need of species-level diagnosis, thereby expediting the therapeutic intervention. However, these polysaccharides are poorly immunogenic. To overcome this drawback, we developed the lectin-Fc(IgG) fusion proteins, Dectin1-Fc(IgG2a), Dectin1-Fc(IgG2b) and wheat germ agglutinin (WGA)-Fc(IgG2a), based on their affinity to ß-1,3-glucan and chitooligomers, respectively. The WGA-Fc(IgG2a) previously demonstrated antifungal activity against Histoplasma capsulatum, Cryptococcus neoformans and Candida albicans. In the present work, we evaluated the antifungal properties of these lectin-Fc(s) against A. fumigatus. Lectin-Fc(IgG)(s) bound in a dose-dependent manner to germinating conidia and this binding increased upon conidia germination. Both lectin-Fc(IgG)(s) displayed in vitro antifungal effects, such as inhibition of conidia germination, a reduced length of germ tubes and a diminished biofilm formation. Lectin-Fc(IgG)(s) also enhanced complement deposition on conidia and macrophage effector functions, such as increased phagocytosis and killing of fungi. Finally, administration of the Dectin-1-Fc(IgG2b) and WGA-Fc(IgG2a) protected mice infected with A. fumigatus, with a 20% survival and a doubled life-span of the infected mice, which was correlated to a fungal burden reduction in lungs and brains of treated animals. These results confirm the potential of lectin-Fc(IgGs)(s) as a broad-spectrum antifungal therapeutic.

Synthetic Monopartite Peptide That Enables the Nuclear Import of Genes Delivered by the Neurotensin-Polyplex Vector.

Neurotensin (NTS)-polyplex is a multicomponent nonviral vector that enables gene delivery via internalization of the neurotensin type 1 receptor (NTSR1) to dopaminergic neurons and cancer cells. An approach to improving its therapeutic safety is replacing the viral karyophilic component (peptide KPSV40; MAPTKRKGSCPGAAPNKPK), which performs the nuclear import activity, by a shorter synthetic peptide (KPRa; KMAPKKRK). We explored this issue and the mechanism of plasmid DNA translocation through the expression of the green fluorescent protein or red fluorescent protein fused with KPRa and internalization assays and whole-cell patch-clamp configuration experiments in a single cell together with importin α/ß pathway blockers. We showed that KPRa electrostatically bound to plasmid DNA increased the transgene expression compared with KPSV40 and enabled nuclear translocation of KPRa-fused red fluorescent proteins and plasmid DNA. Such translocation was blocked with ivermectin or mifepristone, suggesting importin α/ß pathway mediation. KPRa also enabled NTS-polyplex-mediated expression of reporter or physiological genes such as human mesencephalic-derived neurotrophic factor (hMANF) in dopaminergic neurons in vivo. KPRa is a synthetic monopartite peptide that showed nuclear import activity in NTS-polyplex vector-mediated gene delivery. KPRa could also improve the transfection of other nonviral vectors used in gene therapy.

Guia prático de atualização: medicamentos biológicos no tratamento da asma, doenças alérgicas e imunodeficiências / Practical update guide: biological drugs in the treatment of asthma, allergic diseases and immunodeficiencies

O presente guia apresenta revisão extensa sobre imunobiológicos utilizados, liberados e ainda sob estudo, para o tratamento da asma, doenças alérgicas e imunodeficiências. Além das características físico-químicas de alguns desses fármacos, são revisadas as indicações e os resultados de estudos clínicos realizados para avaliar eficácia e segurança. Separados por doença específica, são apresentados os principais agentes disponíveis e aprovados para utilização segundo as normas regulatórias nacionais.

This guide presents an extensive review of immunobiological drugs used, approved and/or under investigation for the treatment of asthma, allergic diseases and immunodeficiencies. In addition to the physicochemical characteristics of some of these drugs, their indications and results of clinical studies evaluating efficacy and safety are reviewed. The main agents available and approved for use in each specific disease according to national regulatory standards are presented.

Influence of epidemiology, immunosuppressive regimens, clinical presentation, and treatment on kidney transplant outcomes of patients diagnosed with tuberculosis: A retrospective cohort analysis.

Tuberculosis (TB) mortality is high among kidney transplant (KT) recipients. Although local epidemiology is an important factor, diagnostic/therapeutic challenges and immunosuppressive therapy (ISS) may influence outcomes. We analyzed the cumulative incidence (CumI) of TB in KT recipients receiving a variety of ISS with long-term follow-up. Our retrospective single-center cohort study included all KT procedures performed between January 1, 1998, and August 31, 2014, with follow-up until August 31, 2014. Induction therapy was based on perceived immunological risk; maintenance ISS included prednisone and calcineurin inhibitor (CNI) plus azathioprine (AZA), and mycophenolic acid (MPA) or mechanistic target of rapamycin inhibitor (mTORi). Thirty-four patients received belatacept/MPA. KT was performed on 11 453 patients and followed for 1989 (IQR 932 to 3632) days. Among these, 152 patients were diagnosed with TB (CumI 1.32%). Median time from KT to TB was 18.8 (IQR 7.2 to 60) months, with 59% of patients diagnosed after the first year. Unadjusted analysis revealed an increasing confidence interval (CI) of TB (0.94% CNI/AZA vs 1.6% CNI/MPA [HR = 1.62, 95% CI = 1.13 to 2.34, P = .009] vs 2.85% CNI/mTORi [HR = 2.45, 95% CI = 1.49 to 4.32, P < .001] vs 14.7% belatacept/MPA [HR = 13.14, 95% CI = 5.27 to 32.79, P < .001]). Thirty-seven (24%) patients died, and 39 (25.6%) patients experienced graft loss. Cytomegalovirus infection (P = .02) and definitive ISS discontinuation (P < .001) were associated with death. Rejection (P = .018) and ISS discontinuation (P = .005) occurred with graft loss. TB occurred at any time after KT and was influenced by ISS.

Urinary tract infections, immunity, and vaccination. / Infecciones del tracto urinario, inmunidad y vacunación

Urinary tract infections (UTI) are considered one of the main causes of morbidity worldwide, and uropathogenic Escherichia coli (UPEC) is the etiological agent associated with these infections. The high morbidity produced by the UTI and the limitation of antibiotic treatments promotes the search for new alternatives against these infections. The knowledge that has been generated regarding the immune response in the urinary tract is important for the development of effective strategies in the UTI prevention, treatment, and control. Molecular biology and bioinformatic tools have allowed the construction of fusion proteins as biomolecules for the development of a viable vaccine against UTI. The fimbrial adhesins (FimH, CsgA, and PapG) of UPEC are virulence factors that contribute to the adhesion, invasion, and formation of intracellular bacterial communities. The generation of recombinant proteins from fimbrial adhesins as a single molecule is obtained by fusion technology. A few in vivo and in vitro studies have shown that fusion proteins provide an efficient immune response and protection against UTI produced by UPEC. Intranasal immunization of immunogenic molecules has generated a response in the urinary tract mucosa compared with other routes of immunization. The objective of this review was to propose a vaccine designed against UTI caused by UPEC, describing the general scenario of the infection, the mechanism of pathogenicity of bacteria, and the immune response of the host.

Las infecciones del tracto urinario (ITU) se consideran como una de las principales causas de morbilidad en el mundo, y Escherichia coli uropatogénica (UPEC, por sus siglas en inglés) es el agente causal asociado a estas infecciones. La alta morbilidad generada por las ITU y la limitación de tratamientos debido al aumento de la resistencia bacteriana a los diversos antibióticos inducen la búsqueda de nuevas alternativas contra estas infecciones. El conocimiento que se ha generado acerca de la respuesta inmunitaria en el tracto urinario (TU) es importante para el desarrollo de estrategias efectivas en la prevención, el tratamiento y el control de las ITU. Los avances en las herramientas de biología molecular y bioinformática han permitido generar proteínas de fusión consideradas como biomoléculas potenciales para el desarrollo de una vacuna viable contra las ITU. Las adhesinas fimbriales (FimH, CsgA y PapG) de UPEC son factores de virulencia que contribuyen a la adherencia, la invasión y la formación de comunidades bacterianas intracelulares. Pocos estudios in vivo e in vitro han mostrado que las proteínas de fusión promueven una respuesta inmunitaria eficiente y de protección contra las ITU causadas por UPEC. Adicionalmente, la vía de inmunización intranasal con moléculas inmunogénicas ha generado una respuesta en la mucosa del TU en comparación contra otras vías de inmunización. El objetivo de esta revisión fue proponer un diseño de vacuna contra las ITU causadas por UPEC, describiendo el panorama general de la infección, el mecanismo de patogenicidad de la bacteria y la respuesta inmunitaria del huésped.

Infecciones del tracto urinario, inmunidad y vacunación / Urinary tract infections, immunity, and vaccination

Resumen Las infecciones del tracto urinario (ITU) se consideran como una de las principales causas de morbilidad en el mundo, y Escherichia coli uropatogénica (UPEC, por sus siglas en inglés) es el agente causal asociado a estas infecciones. La alta morbilidad generada por las ITU y la limitación de tratamientos debido al aumento de la resistencia bacteriana a los diversos antibióticos inducen la búsqueda de nuevas alternativas contra estas infecciones. El conocimiento que se ha generado acerca de la respuesta inmunitaria en el tracto urinario (TU) es importante para el desarrollo de estrategias efectivas en la prevención, el tratamiento y el control de las ITU. Los avances en las herramientas de biología molecular y bioinformática han permitido generar proteínas de fusión consideradas como biomoléculas potenciales para el desarrollo de una vacuna viable contra las ITU. Las adhesinas fimbriales (FimH, CsgA y PapG) de UPEC son factores de virulencia que contribuyen a la adherencia, la invasión y la formación de comunidades bacterianas intracelulares. Pocos estudios in vivo e in vitro han mostrado que las proteínas de fusión promueven una respuesta inmunitaria eficiente y de protección contra las ITU causadas por UPEC. Adicionalmente, la vía de inmunización intranasal con moléculas inmunogénicas ha generado una respuesta en la mucosa del TU en comparación contra otras vías de inmunización. El objetivo de esta revisión fue proponer un diseño de vacuna contra las ITU causadas por UPEC, describiendo el panorama general de la infección, el mecanismo de patogenicidad de la bacteria y la respuesta inmunitaria del huésped.

Abstract Urinary tract infections (UTI) are considered one of the main causes of morbidity worldwide, and uropathogenic Escherichia coli (UPEC) is the etiological agent associated with these infections. The high morbidity produced by the UTI and the limitation of antibiotic treatments promotes the search for new alternatives against these infections. The knowledge that has been generated regarding the immune response in the urinary tract is important for the development of effective strategies in the UTI prevention, treatment, and control. Molecular biology and bioinformatic tools have allowed the construction of fusion proteins as biomolecules for the development of a viable vaccine against UTI. The fimbrial adhesins (FimH, CsgA, and PapG) of UPEC are virulence factors that contribute to the adhesion, invasion, and formation of intracellular bacterial communities. The generation of recombinant proteins from fimbrial adhesins as a single molecule is obtained by fusion technology. A few in vivo and in vitro studies have shown that fusion proteins provide an efficient immune response and protection against UTI produced by UPEC. Intranasal immunization of immunogenic molecules has generated a response in the urinary tract mucosa compared with other routes of immunization. The objective of this review was to propose a vaccine designed against UTI caused by UPEC, describing the general scenario of the infection, the mechanism of pathogenicity of bacteria, and the immune response of the host.

Renal Allograft Survival in Nonhuman Primates Infused With Donor Antigen-Pulsed Autologous Regulatory Dendritic Cells.

Systemic administration of autologous regulatory dendritic cells (DCreg; unpulsed or pulsed with donor antigen [Ag]), prolongs allograft survival and promotes transplant tolerance in rodents. Here, we demonstrate that nonhuman primate (NHP) monocyte-derived DCreg preloaded with cell membrane vesicles from allogeneic peripheral blood mononuclear cells induce T cell hyporesponsiveness to donor alloantigen (alloAg) in vitro. These donor alloAg-pulsed autologous DCreg (1.4-3.6 × 106 /kg) were administered intravenously, 1 day before MHC-mismatched renal transplantation to rhesus monkeys treated with costimulation blockade (cytotoxic T lymphocyte Ag 4 immunoglobulin [CTLA4] Ig) and tapered rapamycin. Prolongation of graft median survival time from 39.5 days (no DCreg infusion; n = 6 historical controls) and 29 days with control unpulsed DCreg (n = 2), to 56 days with donor Ag-pulsed DCreg (n = 5) was associated with evidence of modulated host CD4+ and CD8+ T cell responses to donor Ag and attenuation of systemic IL-17 production. Circulating anti-donor antibody (Ab) was not detected until CTLA4 Ig withdrawal. One monkey treated with donor Ag-pulsed DCreg rejected its graft in association with progressively elevated anti-donor Ab, 525 days posttransplant (160 days after withdrawal of immunosuppression). These findings indicate a modest but not statistically significant beneficial effect of donor Ag-pulsed autologous DCreg infusion on NHP graft survival when administered with a minimal immunosuppressive drug regimen.

Impact of Human Mutant TGFß1/Fc Protein on Memory and Regulatory T Cell Homeostasis Following Lymphodepletion in Nonhuman Primates.

Transforming growth factor ß1 (TGFß1) plays a key role in T cell homeostasis and peripheral tolerance. We evaluated the influence of a novel human mutant TGFß1/Fc (human IgG4 Fc) fusion protein on memory CD4+ and CD8+ T cell (Tmem) responses in vitro and their recovery following antithymocyte globulin (ATG)-mediated lymphodepletion in monkeys. TGFß1/Fc induced Smad2/3 protein phosphorylation in rhesus and human peripheral blood mononuclear cells and augmented the suppressive effect of rapamycin on rhesus Tmem proliferation after either alloactivation or anti-CD3/CD28 stimulation. In combination with IL-2, the incidence of CD4+ CD25hi Foxp3hi regulatory T cells (Treg) and Treg:Th17 ratios were increased. In lymphodepleted monkeys, whole blood trough levels of infused TGFß1/Fc were maintained between 2 and 7 µg/mL for 35 days. Following ATG administration, total T cell numbers were reduced markedly. In those given TGFß1/Fc infusion, CD8+ T cell recovery to predepletion levels was delayed compared to controls. Additionally, numbers of CD4+ CD25hi CD127lo Treg increased at 4-6 weeks after depletion but subsequently declined to predepletion levels by 12 weeks. In all monkeys, CD4+ CD25hi Foxp3hi Treg/CD4+ IL-17+ cell ratios were reduced, particularly after stopping TGFß1/Fc infusion. Thus, human TGFß1/Fc infusion may delay Tmem recovery following lymphodepletion in nonhuman primates. Combined (low-dose) IL-2 infusion may be required to improve the Treg:Th17 ratio following lymphodepletion.

Spontaneous resolution of acute rejection and tolerance induction with IL-2 fusion protein in vascularized composite allotransplantation.

Vascularized composite allotransplantation (VCA) has emerged as a treatment option for treating nonlife-threatening conditions. Therefore, in order to make VCA a safe reconstruction option, there is a need to minimize immunosuppression, develop tolerance-inducing strategies and elucidate the mechanisms of VCA rejection and tolerance. In this study we explored the effects of hIL-2/Fc (a long-lasting human IL-2 fusion protein), in combination with antilymphocyte serum (ALS) and short-term cyclosporine A (CsA), on graft survival, regulatory T cell (Treg) proliferation and tolerance induction in a rat hind-limb transplant model. We demonstrate that hIL-2/Fc therapy tips the immune balance, increasing Treg proliferation and suppressing effector T cells, and permits VCA tolerance as demonstrated by long-term allograft survival and donor-antigen acceptance. Moreover, we observe two distinct types of acute rejection (AR), progressive and reversible, within hIL-2/Fc plus ALS and CsA treated recipients. Our study shows differential gene expression profiles of FoxP3 versus GzmB, Prf1 or interferon-γ in these two types of AR, with reversible rejection demonstrating higher Treg to Teff gene expression. This correlation of gene expression profile at the first clinical sign of AR with VCA outcomes can provide the basis for further inquiry into the mechanistic aspects of VCA rejection and future drug targets.

Transgenic plants expressing ω-ACTX-Hv1a and snowdrop lectin (GNA) fusion protein show enhanced resistance to aphids.

Recombinant fusion proteins containing arthropod toxins have been developed as a new class of biopesticides. The recombinant fusion protein Hv1a/GNA, containing the spider venom toxin ω-ACTX-Hv1a linked to snowdrop lectin (GNA) was shown to reduce survival of the peach-potato aphid Myzus persicae when delivered in artificial diet, with survival <10% after 8 days exposure to fusion protein at 1 mg/ml. Although the fusion protein was rapidly degraded by proteases in the insect, Hv1a/GNA oral toxicity to M. persicae was significantly greater than GNA alone. A construct encoding the fusion protein, including the GNA leader sequence, under control of the constitutive CaMV 35S promoter was transformed into Arabidopsis; the resulting plants contained intact fusion protein in leaf tissues at an estimated level of 25.6 ± 4.1 ng/mg FW. Transgenic Arabidopsis expressing Hv1a/GNA induced up to 40% mortality of M. persicae after 7 days exposure in detached leaf bioassays, demonstrating that transgenic plants can deliver fusion proteins to aphids. Grain aphids (Sitobion avenae) were more susceptible than M. persicae to the Hv1a/GNA fusion protein in artificial diet bioassays (LC50 = 0.73 mg/ml after 2 days against LC50 = 1.81 mg/ml for M. persicae), as they were not able to hydrolyze the fusion protein as readily as M. persicae. Expression of this fusion protein in suitable host plants for the grain aphid is likely to confer higher levels of resistance than that shown with the M. persicae/Arabidopsis model system.

Frecuencia de los transcriptos p190BCR-ABL y p210BCR-ABL en una población colombiana con leucemia mieloide crónica (LMC) usando RT-PCR cualitativa / Detection of p190BCR-ABL AND p210BCR-ABL fusion transcripts in patients with chronic myeloid leukemia (CML) using qualitative RT-PCR / Frequência dos transcritos p190BCR-ABL e p210BCR- ABL numa população colombiana com leucemia mielóide crônica (LMC) usando RT-PCR qualitativa

Introducción: la leucemia mieloide crónica (LMC) se caracteriza por la presencia del cromosoma Filadelfia (Ph) que resulta de la translocación recíproca balanceada t(9;22)(q34;q11); este marcador cromosómico se encuentra con menor frecuencia en pacientes con leucemia linfoide aguda (LLA). Objetivo: determinar la frecuencia de las fusiones génicas BCR-ABL, que codifican para los transcriptos p210BCR-ABL y p190 BCR-ABL en pacientes colombianos con diagnóstico de LMC, en diferentes fases de la enfermedad o de su tratamiento. Materiales y métodos: estudio descriptivo de corte transversal de 31 pacientes con LMC (15-78 años). El análisis se hizo a partir de muestras de sangre periférica con la técnica PCR anidada cualitativa para las isoformas P210 BCR-ABL (b3a2 e b2a2) y P190 BCR-ABL (e1a2). Resultados: se detectó el transcripto p210BCR-ABL en 29 de los 31 casos (93,6%). En ellos se identificaron las fusiones génicas b2a2 (16/29; 55,2%), b3a2 (10/29; 34,5%) y la coexpresión b3a2 y b2a2 (3/29; 10,3%). Conclusión: la fusión génica b2a2 fue la más frecuente en esta población con LMC.

Introduction: Chronic myelogenous leukemia (CML) is characterized by the presence of the Philadelphia chromosome (Ph), resulting from the balanced reciprocal translocation t(9;22)(q34;q11). This marker chromosome is found less frequently in patients suffering from acute lymphoblastic leukemia. Objective: To determine the frequency of BCR-ABL gene fusions encoding the p210BCR-ABL y p190 BCR-ABL transcripts in Colombian patients diagnosed with CML in different stages of the disease and/or its treatment. Materials and methods: Cross sectional, descriptive study of thirty one CML patients (aged 15-78). Analysis was carried out through qualitative nested PCR for the isoforms P210 BCR-ABL (b3a2 e b2a2) and P190 BCR-ABL (e1a2), and based on peripheral blood samples. Results: In 29 of the 31 patients (93.6%) transcript p210BCR-ABL was detected; b2a2 and b3a2 gene fusions and the coexpression b3a2 y b2a2 were identified in 55.2% (16/29), 34.5% (10/29) and 10.3% (3/29) of the cases, respectively. Conclusion: b2a2 gene fusion was the most frequent in this CML population.

Introdução: a leucemia mielóide crônica (LMC) caracteriza- se pela presença do cromossomo Filadélfia (Ph) que resulta da translocação recíproca balanceada t(9;22)(q34;q11); este marcador cromossômico se encontra com menor frequência em pacientes com leucemia linfoide aguda (LLA). Objetivo: determinar a frequência das fusões genéticas BCR-ABL, que codificam para os transcritos p210BCR-ABL e p190 BCR-ABL em pacientes colombianos com diagnóstico de LMC, em diferentes fases da doença ou de seu tratamento. Materiais e métodos: estudo descritivo de corte transversal de 31 pacientes com LMC (15-78 anos). A análise se fez a partir de mostras de sangue periférico com a técnica PCR aninhada qualitativa para as isoformas P210 BCR-ABL (b3a2 e b2a2) e P190 BCR-ABL (e1a2). Resultados: detectou-se o transcrito p210BCR-ABL em 29 dos 31 casos (93,6%). Neles se identificaram as fusões genéticas b2a2 (16/29; 55,2%), b3a2 (10/29; 34,5%) e a co-expressão b3a2 e b2a2 (3/29; 10,3%). Conclusão: a fusão genética b2a2 foi a mais frequente nesta população com LMC.