Alpha alumina nanoparticle conjugation to cysteine peptidase A and B: an efficient method for autophagy induction

Background: autophagy as a cellular pathway facilitates several immune responses against infection. It also eliminates invading pathogens through transferring content between the cytosol and the lysosomal vesicles and contributes to the cross-presentation of exogenous antigens to T lymphocytes via MHC class I pathway. Autophagy induction is one of the main targets for new drugs and future vaccine formulations. Nanoparticles are one of the candidates for autophagy induction. Cysteine Peptidase A [CPA] and Cysteine Peptidase B [CPB] are two members of papain family [Clan CA, family C1] enzyme that have been considered as a virulence factor of Leishmania [L.] major, making them suitable vaccine candidates. In this research, Leishmania major cysteine peptidase A and B [CPA and CPB] conjugation to alpha alumina nanoparticle was the main focus and their entrance efficacy to macrophages was assessed

Methods: for this purpose, CPA and CPB genes were cloned in expression vectors. Related proteins were extracted from transformed Escherichia coli [E. coli] and purified using Ni affinity column. Alpha alumina nanoparticles were conjugated to CPA/CPB proteins using Aldehyde/Hydrazine Reaction. Autophagy induction in macrophages was assessed using acridine orange staining

Results: CPA/CPB protein loading to nanoparticles was confirmed by Fourier Transform Infrared Spectroscopy. alpha-alumina conjugated CPA/CPB antigen uptake by macrophages at different concentrations was confirmed using fluorescence microscope and flowcytometry. Highly efficient CPA/CPB protein loading to alpha-alumina nanoparticles and rapid internalization to macrophages introduced these nanocarriers as a delivery tool. Acridine orange staining demonstrated higher autophagy induction in CPA/CPB protein conjugated with alpha-alumina nanoparticles

Conclusion: alpha-alumina nanoparticles may be a promising adjuvant in the development of therapeutic leishmania vaccines through antigen delivery to intracellular compartments, induction of autophagy and cross presentation to CD[8] lymphocytes

Verification of ALDH activity as a biomarker in colon cancer stem cells-derived HT-29 cell line

Background: Recent evidence has suggested that epithelial cancers including colorectal cancer [CRC] have driven by a small population of self-renewing, multi-potent cells termed cancer stem cells [CSCs] which could be responsible for recurrence of cancer. Aldehyde dehydrogenase 1 [ALDH1] activity has used as a functional stem cell biomarker to isolate CSCs in different cancers such as colorectal cancer

Objectives: The main aim of this research was to determine the utility of ALDH1 activity along with CD44 and EPCAM in identifying stem cell-like cells in human HT-29 colonic adenocarcinoma cell line

Materials and Methods: In this experimental study, colon CSCs biomarkers including CD44, EPCAM and ALDH1 in colonospheres and parent cells have analyzed by flow cytometry. The expression levels of stemness genes in spheroid and parental cells have investigated using SYBR Green real-time PCR. In addition, in vivo xenografts assay has performed to determine tumorigenic potential of tumor spheroid cells in nude mice

Results: According to results, over 92% of spheroids were CD44+/EpCAM+, while parent cells only have expressed 38% of CD44/EpCAM biomarkers [P < 0.001]. Controversially, ALDH activity was about 2-fold higher in the parent cells than spheroid cells [P < 0.05]. In comparison with the parental cells, expression levels of ''stemness'' genes, like Sox2, Oct4, Nanog, C-myc, and Klf4 have significantly increased in colonosphere cells [P < 0.05]. Further, administration of 2500 spheroids could be sufficient to initiate tumor growth in nude mice, while 1x106 of parental cells has needed to form tumor

Conclusions: For the first time, we have shown that colonospheres with low ALDH1 activity has indicated increased tumorigenic potential and stemness properties. So, it hasn't seemed that ALDH1 could become a useful biomarker to identify CSCs population in HT-29 cell line

[Characterization of stemness properties of colonospheres in colon adenocarcinoma patients]

Proliferation and expansion of cancer stem cells as spheroids were proved in previous studies. But, capability of primary tumor-derived stem cells to keep their unique properties in vitro is still disputed. So, the goal of this study was to isolate, expand and characterize of colon cancer-derived stem cells. In the present work, colon cancer stem cells markers including CD44 and EPCAM in spheroid and parental cells were analyzed by flow cytometry. The expression levels of stemness genes in both spheroid and parental cells were investigated using real-time PCR. Tumorigenic potential of spheroid cells was evaluated and used implantation of tumor xenografts into nude mice. Our data shows 79% of spheroids were CD44+/EpCAM+, while parental cells only expressed 20% of CD44/EpCAM markers [p< 0.01]. In compared with the parental cells, the expression levels of "stemness" genes, like Sox2, Oct4, Nanog, C-myc, and Klf4 were significantly increased in spheroid cells [p< 0.05]. Furthermore, as little as 1000 spheroid cells were sufficient to obtain tumor growth in nude mice, while 1x10[6] of parental cells was needed to generate tumor. Sphere formation assay is a useful method to enrich cancer stem cells. Spheroid cells showed increasing expression of stemness genes and tumorigenic activity in nude mice

Comparative study of the effect of LPS on the function of BALB/c and C57BL/6 peritoneal macrophages

Macrophages influence their environment and surrounding immune cells as soon as stimulators affect them. Different sources of macrophages induce different reactions in their neighboring immune cells,which result in non-uniform immunologic outcomes. In this experimental research, we compare the behavior of peritoneal macrophages to lipopolysaccharide [LPS] stimulation from BALB/cmice as an indicator of a type 2 immune response and from C57BL/6 mice as an indicator of a type 1 immune response. In this experimental study, peritoneal macrophages prepared from thioglycolate stimulated BALB/c and C57BL/6 micewere treated with 1microg/ml LPS. At different time points after LPS treatment, nitric oxide [NO], interferon gamma [IFN-gamma]. interleukin 4 [IL-4],transforming growth factor beta[1] [TGF-beta[1]], interleukin 17 [IL-17], and interleukin 10[IL-10] production were measured in the supernatants of all macrophage cultures.Indoleamine 2, 3 dioxygenase [IDO] and phagocytic activitywere analyzed in the different experimental groups. The supernatant effects of LPS-treated macrophages on splenocyte proliferation was assessed by the colorimetric method using a 3-[4,5-Dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide [MTT] reagent. According to cytokine analysis, different mouse strains show different cytokine patterns in response to LPS. C57BL/6 macrophages produced more IL-17, IL-10, and IFN-gamma while BALB/c macrophages produced more TGF-beta[1]., and IL-4. There was no significant difference in IDO activity between strains [p

Cloning and expression of leishmania infantum LPG3 gene by the lizard leishmania expression system

Various prokaryotic and eukaryotic expression systems have been developed for the production of recombinant proteins. In the present study, we used a new protein expression system based on the Iranian Lizard Leishmania, a trypanosomatid protozoan as a host, for the expression of LPG3 gene from Leishmania infantum [L.infantum]. The LPG3 gene was cloned in the expression cassette for integration into the small subunit of the ribosomal RNA locus of Lizard Leishmania genome by electroporation. Expression of the recombinant LPG3 protein was confirmed by western blotting and immunofluorescence staining. Western blotting confirmed the expression and production of rLPG3 protein. Immunofluoresence analysis also revealed the staining throughout the cytoplasm of transfected parasites, indicating that the protein has been expressed. These results demonstrate that Leishmania cells can be suggested an expression system for the production of recombinant LPG3 [rLPG3] to further research in vaccine designing against leishmaniasis

[Investigation of protective effects of Pseudomonas aeruginosa antiflagellar antibodies in burned-induced infection on BALB/c mice]

Pseudomonas aeruginosa is the major cause of septicemia and wound infection in burned patients. Immunotraphy is the best practical way for prevention and treatment of these infections. Flagella as one of the most important bacterial virulence factors has important role in attachment, motility, chemotaxis and TLR-5-dependent immune response so that it propounded as a vaccine candidate. Production of anti-flagellar antibodies and evaluation of its protective effects in burned induced infection of mice was the main aim of this study. In the first step, flagellar antigen prepared by ultra-centrifugation. Antiflagellar antibodies produced in rabbit and its impurity separated by absorption technique. Specification of the obtained antibodies for flagellar antigen was investigated via agglutination test. After determination of LD50 in a known strain, different dilutions of anti-flagellar antibodies injected in burned mice for passive immunization. The rate of bacterial spread from burn site was determined by quantification assay of bacteria in skin and liver. In this study, clinical isolate and PA103 in addition to ATCC 27853 strain were used for agglutination test. H-antiserum reduced mortality of burned mice challenged with ATCC 27853 strains about 80%. Counting of bacteria in the skin and liver showed that the number of bacteria in immunized mice, in contrast with control group, was significantly low. The results of this study showed that anti-flagellar antibodies of Pseudomonas can inhibit invasion of Pseudomonas and facilitate its opsonization, so these antibodies have protective effects in burned wound infections

[Analysis of HLA-G transcripts in PBMCs of normal and lupus individuals and effect of IFN-gamma on the expression of HLA-G]

HLA-G is a nonclassical major histocompatibility complex antigen and is expressed as seven isoforms, including four membrane bound [HLA-G1 to-G4] and three soluble [HLA-G5 to-G7] forms. The pattern of selective expression of HLA-G transcripts in tissues shows the existence of a tight transcriptional control on the gene expression. It has been revealed that cytokines including interfrons and IL-10 could cause stimulation of the HLA-G transcription. The purpose of this study was to examine the effects of IFN-gamma on the expression of HLA-G transcripts in both PBMCs of normal and SLE patients. Whole blood of 20 female SLE patients and 15 healthy donor candidates for Bone Marrow Transplantation were used. PBMCs were isolated from the whole blood by Ficoll gradient centrifugation and cultured with or without IFN-gamma /LPS for 48 hours. Total RNA was extracted from the cells by trizol method. After reverse transcription of RNA to cDNA and the performance of a multiplex PCR for beta actin and HLA-G, the PCR products were analyzed using electrophoresis on a 2% agarose gel and stained with ethidium bromide. The results showed that the transcription of HLA-G was higher in SLE patients compared to normal controls. Addition of IFN-gamma /LPS could influence the expression of this molecule by increasing the transcription of HLA-G in both normal and patient PBMCs [P?0.05]. Transcription of HLA-G gene could be increased by the cytokine IFN-gamma. This observation is in accordance with previous reports. This effect could be assigned to both normal and lupus Patients. The effects of the cytokine IFN-gamma /LPS in the induction of HLA-G transcription were higher in normal than lupus patients. Nevertheless, the total expression of HLA-G was higher in lupus patients

[ study of IgG immunoglobolin anti cryptosporidum parvum in infected neonatal BALB/C mice]

Cryptosporidiosis is a parasitic disease that is causing small protozoan genus Cryptosporidum and transmission take place through fecal-oral by direct contact or indirectly through food or drink. The aim of this study was detection of anti-Cryptosporidum parvum Immunoglobulin IgG, in newborn BALB/c infected with C. parvum. Oocysts of C. parvum were obtained from the feces of diarrheic lambs and following purification they were suspended in 2.5% aqueous potassium dichromate solution and stored at 4°C. Forty suckling BALB/c [3-4 days old] were divided in 8 groups [4 case groups and 4 control groups] each group consist of 5 suckling BALB/c. The mice in case groups were infected oraly with 105 C. parvum oocysts, and the mice in control groups served as non-infected. Blood samples were collected at 6, 9, 12 and 16 days post-infection [pi]. Immunoglobulin IgG were extracted by salting out method and confirmed with SDS-PAGE. Antibodies were analyzed by western blot and increased secretion of IgG was confirmed in neonatal mice infected with Cryptosporidum oocysts. Mean OD of Immunoglobulin IgG increased from 0.350 +/- 0.099 to 0.6776 +/- 0.099 in case groups but in control groups the increase was from 0.244 +/- 0.016 to 0.322 +/- 0.16 [P<0.05]. The type of antibody in neonetal mice infected with Cryptosporidum oocysts was IgG which is secreted against external memberane of oocysts. Significant differences in neonatal mice case groups as compared with the control groups were observed

IL-10 and IL-12 production in response to mycobacterium tuberculosis total lipid antigens in multidrug-resistant tuberculosis

Mycobacterium tuberculosis lipid antigens take part in pathogenicity of the bacterium but the response of monocytes/macrophages to these antigens in tuberculosis is not well known. The aim of current investigation was to study the M. tuberculosis lipid antigens in tuberculosis pathogenesis. In the present study M. tuberculosis lipid antigens were extracted. Monocytes and macrophages from multidrug-resistant tuberculosis [MDR-TB], TB patients, asymptomatic healthy individuals with positive tuberculin skin test positive and healthy individuals with negative tuberculin skin test were collected using magnetic cell sorting. The cells were stimulated by M. tuberculosis total lipid antigens and IL-12 and IL-10 in their supernatants were measured by enzyme-linked immunosorbent assay. The IL-12 production by monocytes in response to M. tuberculosis total sonicate antigens in the MDR-TB patients did not show a considerable difference with the PPD positive healthy subjects, whereas in the active TB patients, IL-12 levels significantly decreased [p<0.05]. IL-10 production by monocytes in TB patients in response to total lipid antigens showed a significant increase in comparison to MDR-TB patients and healthy individuals. In the MDR-TB patients, IL-10 and IL-12 production by monocytes in response to M. tuberculosis lipid antigens are similar to the healthy subjects

Null allele frequencies at HLA-G locus in Iranian healthy subjects

HLA-G gene contains 15 alleles including a null allele, HLA-G*0105N. Previous studies have shown that HLA-G*0105N does not encode the complete HLA-G1 or HLA-G5 isoforms but encodes a functional HLA-G protein with the ability to inhibit NK cell cytolysis. Thus, although the biological functions of HLA-G1 and HLA-G5 proteins are abrogated, other isoforms such as HLA-G2 can replace their roles. Studies on the null allele of HLA-G gene could be useful in understanding the genetic variants of HLA-G alleles in ethnic groups. The goal of this research was to determine the frequency of HLA-G*0105N null allele in Iranian healthy subjects. The frequency of HLA-G*0105N null allele was evaluated in Iranian healthy subjects by PCR-RFLP method. Genomic DNA was isolated from the whole blood of 100 randomly selected, healthy, unrelated Iranian individuals using salting-out technique followed by PCR amplification of the exon 3 of HLA-G gene. PCR products were digested with PpUM-1 and the resulted fragments were analyzed using gel electrophoresis. In this study the restriction enzyme digestion confirmed homozygous HLA-G*0105N null allele for 9% of the population. Furthermore obtained results indicated that the total frequency of HLA-G*0105N null allele was 20% in the studied population of Iran. The final data analysis showed that the total frequency of this allele in Iranian people was higher than other ethnic groups that have been studied so far

Synergistic effect of LPS, IFN-gamma and iron on apoptosis of Balb/c mice macrophages following nitric oxide production

Previous studies have demonstrated that the nitric oxide [NO] dependent death of murine peritoneal macrophages activated in vitro with IFN- gamma and LPS is mediated through apoptosis. In the present study, we investigated the synergistic effect of LPS, IFN- gamma and iron on NO production and apoptosis. After determination of iron cytotoxicity, the peritoneal macrophages of Balb/c mice were cultured with iron, LPS, and IFN- gamma separately, or a mixture of these for 18 hr at 37 C, Then after 18 hr incubation, the level of NO in supernatant was measured by the Griess method. At the same time, after incubation with ethidium bromide and acridine orange dye, the apoptotic macrophages were detected by fluorescence microscopy. NO production was significantly greater than the control group in macrophages exposed to iron, LPS, or IFN- gamma alone [P=0.02], while no significant difference was detected in apoptosis rate in the presence of LPS [.P=0.08]. However, the differences were remarkable between NO production and apoptosis rate in the presence of iron, LPS and IFN- gamma [P

Expression of HLA-G in the skin of patients with pemphighus vulgaris

Studies on HLA-G, a nonpolymorphic antigen of non-classical HLA class I, is of basic and clinical significance. In the present study, the expression of HLA-G proteins in the human skin tissue sections of normal and autoimmune pemphigus vulgaris [PV] individuals were investigated using monoclonal antibodies. The antibodies recognized both membrane-bound and soluble isoforms of HLA-G. RT-PCR was performed to assess the patterns of HLA-G mRNA transcripts in the epidermal cells of PV and normal subjects. HLA-G expression could only be detected at transcriptional level in normal skin tissues. However cells derived from PV subjects expressed detectable HLA-G molecules at both transcriptional and translational levels. In addition, the RT-PCR patterns of HLA-G amplification revealed a reduction in HLA-G2 and an increase in HLA-G1 transcripts in epidermal cells of PV patients as compared to normal cells. These observations further support suggestions in the literature regarding the role of HLA-G in induction of tolerance in autoimmune individuals

Immune Responses of CD4 [+] T-cells of MDR-TB patients to M. tuberculosis total lipid antigens

CD4 [+]T-cell have a central role in protective immune responses to Mycobacterium tuberculosis [M. tuberculosis] protein antigens, but function of these cells in response to M. tuberculosis total lipid antigens has remained unclear. The present study was undertaken to determine role of CD4[+] T cells in the MDR-TB patients against M. tuberculosis total lipid antigens. CD4[+]T- cells were isolated from MDR-TB patients and stimulated with M. tuberculosis total lipid antigens. Proliferative responses and cytokine secretion were assessed by MTT and ELISA, respectively. Our study results showed that proliferative responses of stimulated CD4[+]T- cells to M. tuberculosis total lipid antigens and IFN-gamma production in MDR-TB patients were significantly lower than those of the PPD-positive subjects [P < 0.05] whereas, IL-4 production in the MDR-TB patients was elevated[P < 0.05]. Responses of CD4[+]T -cells of MDR-TB patients to total lipid antigens was significantly lower than that of PPDpositive healthy subjects. Therefore, it seems that M. tuberculosis lipid antigens, as protein antigens, have an important role in specific immune response

Protective role of antigens from peritoneal exudates of infected mice against toxoplasmosis

Toxoplasma gondii is an obligate intracellular parasite that infects all mammalian cells. Several antigens such as excreted/secreted antigens have been identified as a potential vaccine candidate. To determine how excreted/ secreted antigens from peritoneal exudates of infected mice [mESA] stimulate cell-mediated immune responses and induce protective immunity against toxoplasmosis in the murine model. The supernatants produced from the peritoneal fluids, were fractionated by precipitation in ammonium sulphate solution [30-80% saturated]. For induction of cell-mediated immune responses, delayed type hypersensitivity was measured, in injected footpad. Response to purified antigen was measured by lymphocyte proliferation assay. Nitric oxide was measured by Griess method. For immunization, Balb/c mice were immunized 2 times with mESA, mESA-40% and Toxoplasma Lysate Antigen [TLA]. The virulent RH strain of Toxoplasma gondii was used for challenging. The pattern of lymphocyte responsiveness was dependent on the antigen employed. In sensitized mice, those received mESA-40% displayed higher lymphocyte response than mice stimulated by mESA [p<0.05]. The highest amounts of nitric oxide were observed in macrophages, which received mESA-40% and mESA [p<0.05]. Mice immunized with mESA-40% survived longer than those immunized with mESA and other antigens [p<0.05]. As fraction 40% [mESA-40%] showed a good result in induction of cellmediated responses in the murine model, the purification and isolation of the mESA 40% is highly recommended for future study

Detection of lactoferrin in the neutrophils and plasma of the patients suffering from hepatitis C

Lactoferrin [LF] has antimicrobial properties against bacteria, fungi and several viruses including herpes virus, HIV and hepatitis C virus. The aim of this study was to detect LF in PMNs and plasma of the patients suffering from hepatitis C and the healthy persons. The sonicated solutions of PMNs of two groups were evaluated by SDS-PAGE [10%], isoelectric focusing [30%] and dot blotting .The level of LF in plasma was measured by ELISA. The results confirmed the presence of LF in PMNs of the two groups. ELISA showed that the level of LF in plasma of patients was higher than normal persons. Based on these findings we conclude that not only the production of LF was not reduced in the patients but also its level was significantly increased compared with the normal persons [P<0.0001]