Anticancer activity of Helicobacter pylori ribosomal protein (HPRP) with iRGD in treatment of colon cancer.

PURPOSE: As the conventional therapeutic approaches were not completely successful in the treatment of colon cancer, there is still a need for finding the most efficient therapeutic agents. Here we investigated the anticancer activity of HPRP-A1 that was derived from the N-terminal region of Helicobacter pylori ribosomal protein L1 (RpL1) alone or in combination with tumor-homing peptide iRGD and 5-Fluorouracil (5FU) on colon cancer cell lines (CT26 and HT29) and isograft models of colon cancer. METHOD: We assessed the tumor growth inhibitory activity of HPRP-A1 with or without iRGD and 5FU on colon cancer in vitro and in vivo. In the in vitro part, we investigate the effect of HPRP-A1 alone and in combination with iRGD/5FU. RESULTS: Our results demonstrated that co-administration of HPRP-A1 with iRGD increased the apoptosis, while these two peptides in combination with 5FU increased the intracellular level of p53 that upregulate the pro-apoptotic gene BAX and downregulate the anti-apoptotic gene BCL2. HPRP-A1 blocks the cell cycle progression in G0/G1. Co-administration of two peptides significantly reduced the size and weight of the tumors, while the group that received 5FU in combination with the peptides increased the necrotic and decrease the fibrotic area significantly in the tumor tissues, which also disrupt the oxidant/antioxidant balance. CONCLUSIONS: Our results indicated that HPRP-A1 could be considered an effective agent toward colon cancer in vitro and in vivo with the ability to enhance the effects of conventional chemotherapy agent 5FU.

Attenuated Salmonella typhimurium-mediated tumour targeting imaging based on peptides.

Attenuated bacteria-mediated tumor targeting diagnosis will provide a novel strategy for further cancer treatments owing to the intrinsic facultative anaerobic characteristic of bacteria and rapid proliferation in the tumor sites. In this work, we firstly investigate the in vivo behaviour of the attenuated Salmonella typhimurium (S. typhimuriumΔppGpp/lux) after intravenous injection. S. typhimurium exhibits rapid proliferation in tumor sites after three days of injection through bioluminescence imaging, the Luria-Bertani plate and the Gram-staining assay. In contrast, S. typhimurium does not proliferate in the normal tissues and could be excreted from the body of mice. Afterwards, a targeting peptide ubiquicidin (UBI) labeled with fluorescent dye Cy5.5 or radionuclide 125I was intravenously injected into the mice with or without S. typhimurium treatments for in vivo fluorescence imaging and single-photon emission computed tomography (SPECT/CT) imaging, respectively. The results show that the peptide UBI could specifically target the two independent bacteria-infected tumor models, the 4T1 murine breast cancer model and the CT26 mouse colon cancer model, realizing the sensitive multimodal imaging of tumors. Such a strategy (bacteria-mediated tumor targeting) may further improve the sensitivity to early diagnosis of tumors. We hope that our developed strategy could further be extended to cancer theranostics in the future, bringing good news for cancer patients.

Blocking Macrophage Migration Inhibitory Factor Protects Against Cisplatin-Induced Acute Kidney Injury in Mice.

Macrophage migration inhibitory factor (MIF) is elevated in patients with acute kidney injury (AKI) and is suggested as a potential predictor for renal replacement therapy in AKI. In this study, we found that MIF also plays a pathogenic role and is a therapeutic target for AKI. In a cisplatin-induced AKI mouse model, elevated plasma MIF correlated with increased serum creatinine and the severity of renal inflammation and tubular necrosis, whereas deletion of MIF protected the kidney from cisplatin-induced AKI by largely improving renal functional and histological injury, and suppressing renal inflammation including upregulation of cytokines such as interleukin (IL)-1ß, tumor necrosis factor-alpha (TNF-α), IL-6, inducible nitric oxide synthase (iNOS), MCP-1, IL-8, and infiltration of macrophages, neutrophils, and T cells. We next developed a novel therapeutic strategy for AKI by blocking the endogenous MIF with an MIF inhibitor, ribosomal protein S19 (RPS19). Similar to the MIF-knockout mice, treatment with RPS19, but not the mutant RPS19, suppressed cisplatin-induced AKI. Mechanistically, we found that both genetic knockout and pharmacological inhibition of MIF protected against AKI by inactivating the CD74-nuclear factor κB (NF-κB) signaling. In conclusion, MIF is pathogenic in cisplatin-induced AKI. Targeting MIF with an MIF inhibitor RPS19 could be a promising therapeutic potential for AKI.

PLGA (85:15) nanoparticle based delivery of rL7/L12 ribosomal protein in mice protects against Brucella abortus 544 infection: A promising alternate to traditional adjuvants.

There is a compelling need for the development of suitable adjuvants for human use to enhance the efficacy of the upcoming vaccines for the prevention of life threatening infections. In the current study, we have tried to explore the immunogenic potential of nanoparticles (NPs) made of PLGA (poly lactic-co-glycolic acid), a biodegradable and biocompatible polymer approved by FDA for human use after entrapping rL7/L12 protein, an immunodominant antigen of Brucella. Adjuvant properties were exhibited by the formulation as it elicited high IgG antibody titers just after first immunization which increased significantly after the booster administration. A good elicitation of the Th1 cytokines especially IFN-γ was recorded. Amongst the IgG antibody subclasses, IgG1 remained the predominant subclass to be elicited in mice serum after immunization; however IgG1/2a ratio showed a mixed profile of Th1/Th2 response. Lymphocyte proliferation assay as a marker of amplification in cellular immunity demonstrated that the splenocytes of the immunized mice had a high proliferation index with reference to the control, revealing that L7/L12 entrapping PLGA nanoparticles are potent inducer of inflammatory cell response indispensable to combat Brucella infection. Enumeration of splenic CFU after 14 days of infection with Brucella abortus 544 showed a significant reduction in log CFU of splenic bacteria in the vaccinated mice as compared to the control group. Therefore it is evident that PLGA nano formulations delivering the entrapped vaccine candidate in mice elicit specific humoral as well as cellular responses specific to the entrapped Brucella antigen. So there is much promise in this approach and this work by highlighting the adjuvant properties of the PLGA nanospheres will accelerate the development of improved vaccines safe for human as well as veterinary use.

High efficacy of a 20 amino acid peptide of the acidic ribosomal protein P0 against the cattle tick, Rhipicephalus microplus.

Current strategies to control cattle ticks use integrated control programs (ICP) that include vaccination. Reduction in the use of chemicals and in the cost of tick control, the delay or elimination of acaricide resistance and the decreasing of environmental pollution are the advantages of using these programs. This integrated program is potentially applicable to all genotypes of chemical resistant ticks. However, the problem here is to improve the efficacy of anti-tick vaccines. The P0 protein is a structural component of the ribosome of all organisms. We have identified an immunogenic region of ribosomal protein P0 from Rhipicephalus spp. ticks that is not very conserved compared to the orthologous protein in their hosts. A synthetic 20 amino acid peptide from this sequence was effective as a vaccine against Rhipicephalus sanguineus infestations in an immunization and challenge experiment using rabbits. In this paper, the same peptide used as vaccine against the cattle tick Rhipicephalus Boophilus microplus shows a significant diminution in the number of engorged females recovered, in the weight of females and the weight of egg masses. The number of eggs hatched was also significantly reduced for the vaccinated group, with an overall effectivity for the antigen pP0 of 96%. These results, together with the conserved sequence of the P0 peptide among ticks, suggest that this antigen could be a good broad spectrum vaccine candidate. It would be expected to be active against many species of ticks and thus has promise in an ICP for effective control of ticks and thereby to improve the efficiency and productivity of the livestock industry.

Improved immunogenicity and protective efficacy of a divalent DNA vaccine encoding Brucella L7/L12-truncated Omp31 fusion protein by a DNA priming and protein boosting regimen.

Brucellosis is one of the most common zoonotic diseases caused by species of Brucella. At present, there is no commercially available vaccine for the human brucellosis. Brucella melitensis and Brucella abortus are the main causes of human brucellosis, worldwide. The outer membrane protein 31 (Omp31) and L7/L12 are immunodominant and protective antigens conserved among human Brucella pathogens. The purpose of the current study was to evaluate and compare the immunogenicity and protective efficacy of the L7/L12-TOmp31 construct administered as DNA/DNA and DNA/Pro vaccine regimens. Vaccination of BALB/c mice with the DNA/Pro regimen provided more protection levels against B. melitenisis and B. abortus challenge than did the DNA/DNA regimen. IgG1 and IgG2a titers were higher in the sera from DNA/Pro-immunized mice than in those from mice immunized with DNA alone. Moreover, splenocytes from DNA/Pro-immunized mice produced significantly higher levels of IFN-γ than did those from mice given DNA alone. The pcDNA-L7/L12-TOmp31 priming followed by rL7/L12-TOmp31 boosting led to improved protection against B. abortus or B. melitensis infection.

Plasmodium riboprotein PfP0 induces a deviant humoral immune response in Balb/c mice.

Passive immunization with antibodies to recombinant Plasmodium falciparum P0 riboprotein (rPfP0, 61-316 amino acids) provides protection against malaria. Carboxy-terminal 16 amino acids of the protein (PfP0C0) are conserved and show 69% identity to human and mouse P0. Antibodies to this domain are found in 10-15% of systemic lupus erythematosus patients. We probed the nature of humoral response to PfP0C0 by repeatedly immunizing mice with rPfP0. We failed to raise stable anti-PfP0C0 hybridomas from any of the 21 mice. The average serum anti-PfP0C0 titer remained low (5.1 ± 1.3 × 104). Pathological changes were observed in the mice after seven boosts. Adsorption with dinitrophenyl hapten revealed that the anti-PfP0C0 response was largely polyreactive. This polyreactivity was distributed across all isotypes. Similar polyreactive responses to PfP0 and PfP0C0 were observed in sera from malaria patients. Our data suggests that PfP0 induces a deviant humoral response, and this may contribute to immune evasion mechanisms of the parasite.

Protection of BALB/C mice against Brucella abortus 544 challenge by vaccination with combination of recombinant human serum albumin-l7/l12 (Brucella abortus ribosomal protein) and lipopolysaccharide.

BACKGROUND: The immunogenic Brucella abortus ribosomal protein L7/L12 and Lipopolysaccharide (LPS) are promising candidate antigens for the development of subunit vaccines against brucellosis. OBJECTIVE: This study was aimed to evaluate the protection of combination of recombinant HSA-L7/L12 fusion protein with LPS in Balb/c mouse. MATERIALS AND METHODS: The recombinant HSA-L7/L12 fusion protein in Saccharomyces cerevisiae was expressed and purified by affinity chromatography column. LPS was extracted by n-butanol, purified by ultracentrifugation. BALB/c mouses were immunized in 9 groups with PBS, HSA, tHSA-L7/L12, L7/L12, LPS, LPS+ HSA, LPS+ tHSA-L7/L12, LPS+ L7/L12, B. abortus S19. ELISA, LTT tests and challenging two weeks after last injection were carried out. Bacterial count of spleen of immunized BALB/c mouse was done four weeks after challenging with virulent strain B. abortus 544. RESULTS: In ELISA test the specific antibodies of tHSA-L7/L12 exhibited a dominance of immunoglobulin IgG1 over IgG2a. LPS-HSA and tHSA-L7/L12 + LPS produced a significantly higher antibody titer than LPS alone and L7/L12+LPS (P < 0.05). The predominant IgG subtype for LPS and L7/L12+LPS were IgG3. However, tHSA-L7/L12+ LPS and LPS+ HAS elicited predominantly IgG1 and IgG3 subtypes. In addition, the tHSA-L7/L12 fusion protein and L7/L12 elicited a strong T-cell proliferative response upon restimulation in vitro with recombinant tHSA-L7/L12 and L7/L12, suggesting the induction of a cellular immunity response in vivo. However, there was no significant difference proliferative response in L7/L12 and tHSA-L7/L12 fusion protein (P > 0.05). The combination of tHSA-L7/L12 fusion protein with LPS and B. abortus S19 induce higher level of protection against challenge with the virulent strain B. abortus 544 in BALB/c mice than other groups (P = 0.005). CONCLUSIONS: The combination of tHSA-L7/L12 fusion protein with LPS had higher protective ability than LPS and fusion protein distinctly.

Cloning and characterization of the acidic ribosomal protein P2 of Cryptosporidium parvum, a new 17-kilodalton antigen.

Cryptosporidium infection is commonly observed among children and immunocompromised individuals in developing countries, but large-scale outbreaks of disease among adults have not been reported. In contrast, outbreaks of cryptosporidiosis in the United States and Canada are increasingly common among patients of all ages. Thus, it seems likely that residents of regions where Cryptosporidium is highly endemic acquire some level of immunity, while residents of the developed world do not. A new immunodominant Cryptosporidium parvum antigen in the 15- to 17-kDa size range was identified as the Cryptosporidium parvum 60S acidic ribosomal protein P2 (CpP2). We developed a recombinant protein-based enzyme-linked immunosorbent assay for serologic population surveillance for antibodies that was 89% sensitive and 92% specific relative to the results of the large-format Western blot assay. The human IgG response is directed almost exclusively toward the highly conserved, carboxy-terminal 15 amino acids of the protein. Although IgG antibody cross-reactivity was documented with sera from patients with acute babesiosis, the development of an anti-CpP2 antibody response in our Peru study population correlated better with Cryptosporidium infection than with infection by any other parasitic protozoan. In Haiti, the prevalence of antibodies to CpP2 plateaus at 11 to 20 years of age. Because anti-CpP2 IgG antibodies were found only among residents of countries in the developing world where Cryptosporidium infection occurs early and often, we propose that this response may be a proxy for the intensity of infection and for acquired immunity.

Abnormal olfactory function demonstrated by manganese-enhanced MRI in mice with experimental neuropsychiatric lupus.

Mice with experimental neuropsychiatric lupus (NPSLE), induced by anti-ribosomal-P antibodies, developed depression-like behavior and a diminished sense of smell. Manganese-enhanced MRI (MEMRI) allows in vivo mapping of functional neuronal connections in the brain, including the olfactory tract. The aim of this study was to analyze and describe, via the MEMRI technique, the effect of the anti-ribosomal-P injection on the olfactory pathway. Twenty mice were intra-cerebra-ventricular injected to the right hemisphere: 10 with human anti-ribosomal-P antibodies and 10 with human IgG antibodies (control). Depression was addressed by forced swimming test and smell function was evaluated by smelling different concentrations of menthol. MEMRI was used to investigate the olfactory system in these mice. Passive transfer of anti-ribosomal-P to mice resulted in a depression-like behavior, accompanied with a significant deficit in olfactory function. MEMRI of these mice demonstrated significant reduction (P < 0.001) in normalized manganese enhancement ratios of olfactory structures, compared to control mice. We concluded that an impaired olfactory neuronal function in mice with experimental depression, mediated by passive transfer of human-anti-ribosomal-P, can be demonstrated by MEMRI.

Liposomised recombinant ribosomal L7/L12 protein protects BALB/c mice against Brucella abortus 544 infection.

Brucella abortus, a facultative intracellular pathogen, is of tremendous zoonotic importance because of its ability to induce spontaneous abortion in cattle and other livestock. It is also known to cause persistent undulant fever, endocarditis, arthritis, osteomyelitis and meningitis in humans. The available vaccines against this dreadful infection suffer from limitations like short-term immunity, increased risk of hypersensitivity and low prophylactic index in the recipients. In the present study, we have demonstrated that liposomal form of a recombinant ribosomal L7/L12 protein, a B-T cell antigen of B. abortus, activates strong immune response in the host. In contrast, free antigen generates moderate immune response in the immunised animals. The liposomisation of rL7/L12 protein causes tremendous increase in cell-mediated immune response in terms of delayed type hypersensitivity, T-cell proliferation and up-regulation in type I cytokine expression, etc. Moreover, the liposome encapsulated antigen elicited stronger humoral immune response as compared to standard vaccine (S-19) or IFA-L7/L12 combination in the immunised animals. The effectiveness of liposome-based vaccine was also substantiated by better systemic clearance of bacterial load after challenging the animals with B. abortus 544 pathogen. The results of the present study suggest the potential of liposome-based rL7/L12 antigen as prospective and efficient candidate vaccine capable of eliciting both cell mediated as well as humoral immune responses against experimental murine brucellosis.

Vaccination with the Leishmania infantum acidic ribosomal P0 protein plus CpG oligodeoxynucleotides induces protection against cutaneous leishmaniasis in C57BL/6 mice but does not prevent progressive disease in BALB/c mice.

We have examined the efficacy of the administration in mice of a molecularly defined vaccine based on the Leishmania infantum acidic ribosomal protein P0 (rLiP0). Two different challenge models of murine cutaneous leishmaniasis were used: (i) subcutaneous inoculation of L. major parasites in susceptible BALB/c mice (a model widely used for vaccination analysis) and (ii) the intradermal inoculation of a low infective dose in resistant C57BL/6 mice (a model that more accurately reproduces the L. major infection in natural reservoirs and in human hosts). First, we demonstrated that C57BL/6 mice vaccinated with LiP0-DNA or rLiP0 protein plus CpG oligodeoxynucleotides (ODN) were protected against the development of dermal pathology and showed a reduction in the parasite load. This protection was associated with production of gamma interferon (IFN-gamma) in the dermal site. Secondly, we showed that immunization with rLiP0 plus CpG ODN is able to induce only partial protection in BALB/c, since these mice finally developed a progressive disease. Further, we demonstrated that LiP0 vaccination induces a Th1 immunological response in both strains of mice. In both cases, the antibodies against LiP0 were predominantly of the immunoglobulin G2a isotype, which was correlated with an rLiP0-stimulated production of IFN-gamma in draining lymph nodes. Finally, we demonstrated that LiP0 vaccination does not prevent the Th2 response induced by L. major infection in BALB/c mice. Taken together, these data indicate that the BALB/c model of cutaneous leishmaniasis may undervalue the potential efficacy of some vaccines based on defined proteins, making C57BL/6 a suitable alternative model to test vaccine candidates.

The P domain of the P0 protein of Plasmodium falciparum protects against challenge with malaria parasites.

Monoclonal antibodies (MAbs) specific for the P domain of the Plasmodium falciparum P0 phosphoriboprotein (PfP0) blocked the invasion of RBCs by P. falciparum. Vaccination with this P-domain peptide protected mice upon malaria parasite challenge. The absolute specificity of the MAbs and the PfP0 P peptide makes them potential protective malaria reagents.

T cell reactivity to MHC class II-bound self peptides in systemic lupus erythematosus-prone MRL/lpr mice.

The epitopes recognized by pathogenic T cells in systemic autoimmune disease remain poorly defined. Certain MHC class II-bound self peptides from autoimmune MRL/lpr mice are not found in eluates from class II molecules of MHC-identical C3H mice. Eleven of 16 such peptides elicited lymph node cell and spleen cell T cell proliferation in both MRL/lpr (stimulation index = 2.03-5.01) and C3H mice (stimulation index = 2.03-3.75). IL-2 and IFN-gamma production were detected, but not IL-4. In contrast to what was seen after immunization, four self peptides induced spleen cell proliferation of T cells from naive MRL/lpr, but not from C3H and C57BL/6.H2(k), mice. These peptides were derived from RNA splicing factor SRp20, histone H2A, beta(2)-microglobulin, and MHC class II I-A(k)beta. The first three peptides were isolated from I-E(k) molecules and the last peptide was bound to I-A(k). T cell responses, evident as early as 1 mo of age, depended on MHC class II binding motifs and were inhibited by anti-MHC class II Abs. Thus, although immunization can evoke peripheral self-reactive T cells in normal mice, the presence in MRL/lpr mice of spontaneous T cells reactive to certain MHC-bound self peptides suggests that these T cells actively participate in systemic autoimmunity. Peptides eluted from self MHC class II molecules may yield important clues to T cell epitopes in systemic autoimmunity.

Characterization of autoantibodies generated in mice by immunization with the C-terminal region of Trypanosoma cruzi ribosomal P1 and P2 proteins.

The R13 peptide sequence (EEEDDDMGFGLFD) that corresponds to the C-terminal region of Trypanosoma cruzi ribosomal P1 and P2 proteins differs from the eukariotic P consensus sequence EESDDDMGFGLFD (H13) only in a nonconservative amino acid substitution. Since the immunization with R13 peptide coupled to a carrier protein like OVA would break the tolerance to a self-sequence and generate autoantibodies, we characterized the antibodies induced in mice by R13 immunization, analyzing by ELISA their capacity to bind to R13 and the self-sequence H13. Besides, we studied the course of these reactivities a long time after immunization. It was found that all R13-immunized mice had antibodies against H13 and this reactivity was always lower than R13 reactivity. The anti-H13 reactivity evaluated by competitive ELISA demonstrated that the H13 peptide is able to inhibit the binding of immune sera to R13 at high doses. When the levels and the avidity of anti-R13 and anti-H13 were evaluated at 10 and 80 days post third immunization, it was observed that anti-R13 levels were higher than anti-H13 levels in all sera from 10 days after the third immunization. However, avidity of both antibodies was high. In sera from 80 days post third immunization, anti-R13 and anti-H13 levels and avidity either remained elevated or showed a rise, whereas anti-OVA levels declined. Moreover, when ECG traces were obtained from immunized mice, the heart functional alterations observed at 10 days continued at 80 days after the third immunization, showing an association with the levels of the antibodies. In addition, the isotype pattern that recognizes R13 and the self-sequence H13 is different. For anti-R13 response, IgG1 reactivity was higher than IgG2; meanwhile, for anti-H13 response IgG2 reactivity was higher than IgG1. These results indicate that sera from R13-immunized mice bind the H13 sequence and this autoreactivity may be self-perpetuating.

[Isotypic characteristics of serum and secretory O antibodies and local immunological memory in the parenteral immunization of guinea pigs with a ribosomal Shigella vaccine]. / Izotipicheskaia kharakteristika syvorotochnykh i sekretornykh O-antitel i mestnaia immunologicheskaia pamiat' pri parenteral'noi immunizatsii morskikh svinok ribosomal'noi shigelleznoi vaktsinoi.

Guinea pigs were immunized subcutaneously with ribosomal vaccine prepared from S. sonnei and their systemic and local humoral response was studied by means of ELISA techniques with the use of monospecific antisera to guinea pig IgA and IgG. Injection of the ribosomal vaccine leads to a significant rise in the levels of IgA O-antibodies in tears, IgG and IgA O-antibodies in the serum. The presence of IgA O-antibodies in tears was seemingly the result of their local synthesis rather than the seepage of serum IgA. The stimulation of the local and systemic anti-O response was more pronounced after parenteral immunization with the ribosomal vaccine than after immunization with the corresponding dose of lipopolysaccharide (LPS). Parenteral immunization with the ribosomal vaccine induced the development of both systemic and local memory. The priming effect produced by relatively small doses of this vaccine (40 micrograms), administered parenterally, was similar to the effect of prolonged and intensive stimulation ensured by 10-day feeding with LPS (the total dose being 5,000 micrograms).

Characterization of the immune response in subcutaneous chambers of guinea pigs immunized with a ribosomal preparation from Neisseria gonorrhoeae.

Immunization of guinea pigs with ribosomal preparations has been previously shown to be protective against chamber infection with Neisseria gonorrhoeae, and this protection could be correlated with the presence of serum bactericidal antibody. Analysis of the chamber fluids from ribosome-immunized and nonimmunized guinea pigs has demonstrated that the chamber fluid may accurately reflect serum antibody levels and proteins. At least one major population of antiribosomal antibodies is present in both immune serum and chamber fluid as revealed by lines of identity between these components. Antibody- and complement-mediated bactericidal assays revealed that matched chamber fluids and sera from immune animals had comparable bactericidal titers. These results suggest that the antibody-complement-mediated bactericidal activity plays a major role in the protection against N. gonorrhoeae infection. Protection could not be explained on the basis of the cellular components of the inflammatory response since both immune and nonimmune chambers had comparable increases in polymorphonuclear neutrophils, monocytes, and lymphocytes after challenge.