HIV-1 Gag p24-Nef fusion peptide induces cellular and humoral immune response in a mouse model.

UNLABELLED: Many Human immunodeficiency virus (HIV) candidate vaccines have been tested in clinical trials, but none was sufficiently effective in the prevention of HIV infection. A HIV vaccine should induce humoral as well as cell-mediated response, the latter including the cytotoxic CD8+ T lymphocyte (CTL) response. In this study, we immunized BALB/c mice with a purified fusion peptide Gag p24-Nef and evaluated immune responses. As for the cellular responses, the adjuvanted fusion peptide induced lymphocyte proliferation, CTL response, and cytokines IFN-gamma and IL-4 in the Th1 pattern. Humoral immune response to the adjuvanted fusion peptide included an increase in IgG antibodies of more IgG2a than IgG1 subtype. These results indicate that the employed HIV-1 peptide construct can elicit both cellular and humoral immune responses in mice. Further studies aimed at memory T cells and other aspects of immune responses are needed before a comprehensive assessment of this candidate vaccine could be provided. KEYWORDS: epitopes; fusion peptide; HIV-1 p24-Nef; immune response.

Systemic infection with Candida albicans in breast tumor bearing mice: Cytokines dysregulation and induction of regulatory T cells.

OBJECTIVE: The effect of candidemia on immunologic parameters in breast tumor bearing patients is not well studied. Here, we hypothesised that candidemia in the tumor background may change the outcome of immunologic parameters and tumor condition. METHOD: Mice were divided into four groups, including normal, tumor, Candida infected (only Candidiasis) and tumor/Candidiasis groups. Tumor changes were recorded daily after tumor transplantation and induction of candidemia. Splenocytes of mice were harvested, cultured, and stimulated with PHA; afterwards, IL-4, IL-10, IFN-γ, TNF-α and TGF-ß cytokines were assessed using ELISA kits. We also evaluated the population of CD4+CD25+Foxp3+ regulatory T cells in the tumor infiltrated and splenocytes. RESULTS: The results showed that infection with C. albicans decreased the IFN-γ/IL-4 ratio in tumor/candidiasis and candidiasis groups versus their non-infected controls. IL-10, TGF-ß and TNF-α levels increased in the candidiasis group. In addition, Candidemia led to an increase in the Treg population in tumor microenvironment and splenocytes of experimental groups compared with non-infected controls. Finally, candidemia increased tumor growth of tumor/Candidiasis group compared with the tumor group. CONCLUSION: It seems that systemic infection with C. albicans could not only induce regulatory T cells but also result in dysregulation of cytokine network and thereby facilitate tumor growth.

Serologic and biochemical analysis of latent a1 IgG.

Immunization of rabbits with anti-allotype antibody (Ab) induces at least two populations of highly cross-reactive molecules. One of these populations bears the nominal VHa allotype of the producing rabbit and is designated the VHa-positive anti-IdX Ab. The other population lacks the expected nominal allotype and thus could represent an induced latent allotype-bearing Ig. To define better the putative latent allotypes, they were subjected to serologic, electron microscopic and biochemical analyses. The induced latent-like population was compared to nominal a1 and found to be indistinguishable in inhibition assays incorporating both rabbit anti-a1 Ab and mouse monoclonal anti-a1 Ab. In contrast, the latent-like Ig was less inhibitory than normal a1 Ig in assays with goat and guinea-pig anti-a1 Ab. The isolated anti-IdX population was less inhibitory than either nominal or latent a1 Ig in all assays. Immunoelectron microscopic analysis indicates that complexes composed of latent-like a1 molecules and Fab anti-a1 ab resemble allotype/anti-allotype (i.e. a1/anti-a1) complexes. Tryptic digests of the putative latent a1 H-chains reveals that these molecules share an a1-specific peptide with digests of nominal a1 H-chain. The peptides from both nominal and latent a1 IgG appear to have blocked N-terminal residues and have a similar though not identical amino acid composition. The composition of these peptides is correlated with the first nine amino acids of the nominal a1 H-chain. The data suggest that the induced latent a1-like molecules share the same major a1 epitope with nominal a1 but may differ in some subtle respects. The possible genetic bases for these observations are discussed.

Antiproliferative effect of a prostatic cell-derived activity on the human androgen-dependent prostatic carcinoma cell line LNCaP.

We have identified a new antiproliferative activity from the conditioned medium of two androgen-independent prostatic cancer cell lines, PC3 and DU-145. This antiproliferative activity selectively inhibited cell proliferation of an androgen-dependent prostate cancer cell line LNCaP in a dose-dependent manner. No antiproliferative activity was observed against mouse fibroblast 3T3, normal human lymphocytes, human leukemic cells, including promyelocyte HL-60 or T cell HUT-78, or human adenocarcinoma cell lines, including prostatic cells JCA-1, ovary NIH:OVCAR-3, cervix C-33A, or breast MDA-MB-231. Cell cycle analysis revealed that the antiproliferative activity did not induce apoptosis in LNCaP cells, but it prevented some G1 LNCaP cells from entering into the S phase of the cell cycle. The antiproliferative activity was sensitive to high temperature (100 degrees C) and to proteinase digestion; however, it was resistant to 56 degrees C, pH 2.0, and reducing agent treatment, as well as to DNase and RNase digestion. The antiproliferative activity was partially purified by gel filtration, ion-exchange chromatography, and SDS-PAGE, with an apparent molecular weight of 50 kD. The antiproliferative activity was not affected by neutralizing antibody against TGF-beta 1,2,3, TNF-alpha, PDGF, EGF, IL-1, IL-2, IL-3, IL-4, or IL-6.

Identification of a co-stimulatory factor for human T cell proliferation.

A factor enhancing proliferation of human peripheral blood lymphocytes (PBL) was identified in the culture supernatant of a human myeloid cell line HL-60 after ion exchange separation. Partially purified lymphocyte growth enhancing factor (LGEF) was found to enhance mitogen-stimulated PBL or purified T cell proliferation in a dose dependent fashion. LGEF alone did not stimulate PBL or T cell proliferation and its activity was dependent on the presence of mitogen. LGEF mediated T cell proliferation was neither accompanied by an increase in the level of IL-2 receptor expression, nor was dependent on the presence of monocytes. In ELISA assay antibodies against IL-1, IL-2, IL-3 and IL-6 did not recognize the LGEF preparation. The comparison of LGEF with other lymphokines is discussed.

Characterization of the release of DNA by a human leukemia-cell line hl-60.

Release of DNA fragments into the culture supernatant of human promyelocytic cell line HL-60 was investigated. The released DNA has previously been shown to have a strong immunosuppressive effect on the proliferation of mitogen-stimulated human peripheral lymphocytes and some cancer cell lines. In this study, HL-60 cells were cultured for four days in serum-free medium and the amount of extracellular nucleic acid was monitored daily. Nucleic acids were isolated by phenol/chloroform extractions, followed by ethanol precipitations. A similar amount of DNA was identified in the HL-60 culture supernatant regardless of whether the cells were incubated for one day or up to four days. Also, the same amount of DNA was isolated from culture supernatants after washing the cells daily and reincubating into the fresh medium. During the first 8 hours of incubation no DNA material was detected in the culture supernatant, whereas at 24 h the concentration of the extracellular DNA reached a plateau. The data suggest the extracellular DNA is not accounted for totally by DNA from dying cells, and a regulatory mechanism may be involved which controls the release of DNA into the medium.

A murine IgG1 monoclonal antibody that binds specifically to outer surface protein A of Lyme disease spirochete Borrelia burgdorferi.

A murine monoclonal antibody, designated MA-2G9, directed against outer surface protein A (OspA) of the Lyme disease spirochete, Borrelia burgdorferi, has been produced. Antibody MA-2G9, IgG1 subclass, was purified by affinity chromatography on protein G Sepharose column and used for purification of OspA antigen from Borrelia burgdorferi cell lysate. Epitope specificity was studied by Western immunoblotting, using several strains of B. burgdorferi and non-Lyme disease bacteria such as Treponema pallidum and B. hermsii. The MA-2G9 monoclonal antibody reacted specifically with recombinant OspA as well as with native OspA in sonicated B. burgdorferi strains. No reaction was observed with T. pallidum, Escherichia coli, Staphylococcus aureus and B. hermsii lysates. The MA-2G9 antibody also recognized the denatured form of OspA indicating that it is directed against sequential epitope and not conformational epitope.

Antigens of Lyme disease of spirochaete Borrelia burgdorferi inhibits antigen or mitogen-induced lymphocyte proliferation.

Modulation of cellular immune responses by the spirochaete Borrelia burgdorferi, the bacteria that causes Lyme disease, was demonstrated. When cultured in the presence of sonicated Borrelia preparation (Bb), the mitogen- or antigen-stimulated proliferative responses of normal lymphocytes were consistently lowered. Bb caused the greatest reduction in Concanavalin A (ConA) or antigen-stimulated proliferation, where almost 100% reduction in proliferation could be achieved. Bb also reduced phytohemagglutinin-M (PHA) or pokeweed mitogen (PWM)-stimulated peripheral blood lymphocyte (PBL) proliferation, with the PWM proliferation being the least affected. This regulatory activity was not due to toxicity and was determined to be caused by Bb protein antigens. The degree of the proliferation reduction was directly proportional to both Bb quantity and length of exposure to lymphocytes. IL-2 production was significantly reduced from Bb-exposed lymphocytes. The entry of lymphocytes into the proliferating phases of the cell cycle was also shown to be blocked. These results have demonstrated an immune suppressive mechanism of B. burgdorferi. The magnitude of host immune responses may be dependent on the degree of suppression which is related to the spirochaete quantity and their length of presence in the host.

Human prostatic-carcinoma cells modulate lymphocyte-proliferation by an immunosuppressive activity.

We have identified an immunosuppressive activity from the conditioned medium of an androgen-independent human prostatic carcinoma cell line, JCA-1. This activity is constitutively produced by JCA-1 cells and is capable of suppressing normal human peripheral blood lymphocyte proliferation irreversibly in a dose-dependent manner. Immunosuppressive activity was semi-purified by a combination of ion-exchange chromatography and gel filtration with an apparent molecular weight of 40-55 kDa. The immunosuppressive activity was not cytolytic to lymphocytes and was sensitive to 56 degrees C, reducing agent as well as to protease digestion. Cell cycle analysis revealed that the suppressive activity did not induce apoptosis, but it prevented some G(1) lymphocytes from entering into the S phase of the cell cycle.

Purification of an acid-stable bovine leukocyte interferon.

Bovine leukocyte interferon (BoL-IFN), produced in bovine peripheral blood leukocytes after priming and induction with Sendai virus, was concentrated by precipitation with KSCN (pH 3.5) and purified by gel column chromatography. Recovery of BoL-IFN from precipitation was higher when crude BoL-IFN containing more fetal bovine serum (FBS) was used. However, purity of BoL-IFN recovered from the gel filtration column was highest when crude BoL-IFN with no FBS was used. The use of 25% ethylene glycol in the column elution buffer resulted in over 93% recovery of the applied IFN activity, versus only 25% when buffer contained no ethylene glycol. Column-purified BoL-IFN was further concentrated by ultrafiltration and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in denaturing buffer. When crude BoL-IFN containing no FBS was used for purification, BoL-IFN from a selected column fraction applied to SDS-PAGE resulted in a single narrow band with an apparent molecular weight (MW) of 19,000 Da. Extraction of the SDS-PAGE gel resulted in a single peak of IFN activity indicating identity of the activity and the polypeptide. This proved to be a practical method for obtaining sufficient quantities of purified natural BoL-IFN for use in the production of monoclonal antibodies to BoL-IFN and other biological experiments.

Effect of Sendai inducing virus in cytopathic effect inhibition assay of bovine leukocyte interferon.

Various methods for removal or inactivation of Sendai inducing virus from natural bovine leukocyte interferon (IFN) preparations were compared and the effects of Sendai virus in microtiter cytopathic effect (CPE) inhibition assay were investigated. Ultraviolet light and ultracentrifugation at either 108,000 X g or 145,000 X g for 2 h proved to be inadequate methods for complete removal of inducing virus from IFN preparations, as judged by the effects of residual virus in CPE inhibition assay. Optimal methods for inducer virus removal/inactivation were ultracentrifugation at 175,000 X g or dialysis against pH 2.0 buffer. Sendai virus itself was capable of inducing IFN production in assay cells at concentrations commonly found in virus-induced IFN preparations.

Compositional changes of PBL population in patients with chronic hepatitis B virus infection.

In this report we have analysed the peripheral blood lymphocyte of several patients with chronic hepatitis B virus infection with flow cytometry. Based on the presence and absence of the HBeAb, patients were divided into two groups. In both, all the patients were HBsAg positive with normal range of serum alanine aminotranferase (23.9 +/- 17.8). We have found that the immunophenotypic profiles of patients were different from healthy donors with significant decrease in CD(3)(+) T cells, specially CD(8)(+) T cells and a significant increase in the CD(19)(+) B cells. The differences were seen in other subset of T cells (CD(4)(+)) or NK cells (CD(56)(+)/CD(16)(+)) and HLA-DR markers were not significant. When the phenotypic profiles of both groups were compared with each other, such changes were more dominant in group II, with HBeAb positive than in group I, with HBeAb negative. Also, we have seen a correlation between the increase of CD(19)(+) B cells and the decrease of CD CD(3)(+) T cells. No such correlation was observed with other cells.

Kinetics of large-scale production of bovine leukocyte interferon, using three viral inducers.

Kinetics of large-scale production of naturally derived bovine leukocyte interferon (IFN) was investigated using Sendai virus, Newcastle disease virus, and infectious bovine rhinotracheitis virus inducers. Cultures were tested for IFN production every 6 hours for 66 hours. The effect of varying the priming dose of Sendai virus from 0 to 50% of total virus dose and the effect of varying the priming time from 0 to 4 hours before induction also were investigated. Other factors explored were effects of varying the fetal bovine serum concentration (from 0 to 8%) and individual cow donors on bovine IFN titers. Highest bovine leukocyte IFN titers (15,314 U/ml) were obtained using Sendai virus (priming dose, 60 hemagglutinating units/ml; inducing dose, 240 hemagglutinating units/ml) and incubating for 12 hours. Up to 24 L (over 360 million U) of naturally derived leukocyte IFN were produced at one time.

Dynamics in the one-dimensional extended ionic Hubbard model.

We investigate single-particle spectral densities and dynamical charge and spin structure factors of the one-dimensional extended ionic Hubbard model in the band insulator regime by using the perturbative continuous unitary transformations method. The one-body staggered potential is considered as the unperturbed part and the hopping term, on-site electron-electron interaction, and the nearest-neighbor repulsive interaction are treated as the perturbations. The excitation spectrum of this model was determined in a previous work (Hafez and Jafari 2010 Eur. Phys. J. B 78 323). It was shown that when the intersite interaction is off, there are two antibound state modes and one bound state mode in the singlet channel and two bound state modes in the triplet channel, while for finite values of intersite interaction two bound state modes were found in each channel. Our results for dynamical charge and spin structure factors indicate that only one of two bound/antibound state modes can be probed by electron-energy-loss spectroscopy and inelastic neutron scattering experiments.

Mass rearing of Lucilia sericata Meigen (Diptera: Calliphoridae).

OBJECTIVE: To carry out an experimental study with the main objective of mass rearing of sheep flies (Lucilia sericata). METHODS: Hand collection and beef- or cattle liver-baited net traps were used for field fly sampling from April, 2010 to November, 2010. The samples collected from different places were placed in properly labeled tubes and sent to the Entomology Laboratory. Since maggot identification is important in inducing mortality, they were kept under insectary condition to develop to adult stage and identified using systematic keys. RESULTS: A total of 218 flies were collected in three rounds of sampling from the field of Tehran and Karaj Counties. In the first generation, 433 flies including 135 (31.17%) male, and 298 (68.82%) female were yielded. The female/male of parent ratio was calculated as 1.72 in Tehran and in Karaj areas, whereas it was 2.20% and 1.81%, respectively in F1 and F2 generations, respectively. CONCLUSIONS: During this study, the mass rearing of sheep blow fly has been established at the School of Public Health, Tehran University of Medical Sciences and can be used for producing flies for maggot therapy.

Preparation and evaluation of chitosan-DNA-FAP-B nanoparticles as a novel non-viral vector for gene delivery to the lung epithelial cells.

Gene delivery using cationic polymers such as chitosan shows good biocompatibility, but reveals low transfection efficiency. Fibronectin Attachment Protein of Mycobacterium bovis (FAP-B) which is responsible for the attachment of many Mycobacteria on the Fibronectin molecule of epithelial cell membrane can be considered as a new targeting ligand and can improve transfection rates in epithelial cells. In this study, chitosan-DNA nanoparticles were prepared using coacervation process. The effect of stirring speed and charge ratio (N/P) on the size and zeta potential of nanoparticles were evaluated. FAP-B ligand was added to nanoparticles at the specific condition to form chitosan-DNA-FAP-B nanoparticles via electrostatic attraction. Transfection efficiency of the final nanoparticles was investigated in A549 (alveolar epithelial cells). Cell viability was investigated using MTT assay. The optimum speed of stirring which was yielded the smallest chitosan-DNA nanoparticles with a narrow distribution (227±43 nm), was 500 rpm with the corresponding N/P ratio of 20. Chitosan-DNA-FAP-B nanoparticles presented the size of 279±27 nm with transfection efficiency about 10-fold higher than chitosan-DNA nanoparticles and resulted in 97.3% cell viability compared to 71.7% using Turbofect controls. Chitosan-DNA-FAP-B nanoparticles showed good transfection efficiency without cell toxicity. They have small particle size around 279 nm which make them a promising candidate as a novel non-viral gene vector for gene delivery to lung epithelial cells.

Evaluation of silica gel-immobilized phosphorylcholine columns for size exclusion chromatography and their application in the analysis of the subunit structures of fish-egg lectins.

Columns of phosphorylcholine (PC) immobilized on silica gel were shown to be useful for size exclusion chromatography (SEC) of proteins. The columns provided good separation of proteins in 50mM sodium phosphate buffer (pH 6.9) containing 0.25 M NaCl, and there was a linear relationship between the retention times and the logarithmic values of the molecular weights with a correlation coefficient (R(2)) of 0.978-0.992. The columns were used in analyzing the subunit structures of the rhamnose-binding lectins CSL1, CSL2, and CSL3, isolated from chum salmon (Oncorhynchus keta) eggs. Although the lectins, which are a group of carbohydrate-binding and hydrophobic proteins, behaved anomalously in SEC with conventional matrices, they could be eluted from the immobilized PC columns without non-size-related retention, thereby allowing their molecular weights to be reliably estimated.

Hyperosmolarity causes inflammation through the methylation of protein phosphatase 2A.

OBJECTIVE AND DESIGN: We evaluated the role of the osmolarity in the pro-inflammatory responses of epithelial cells. MATERIAL: Twenty-five female Wistar rats and colorectal (HT-29) and bladder (T24) cell lines were used. TREATMENTS: Rats and cells were exposed for 48 hours to hyperosmotic solutions. METHODS: Interleukin-8 (IL-8) production was measured by Enzyme Linked ImmunoSorbent Assay, mRNA transcription of pro-inflammatory cytokines by microarrays or RNase Protection Assay. Nuclear factor-kappa B (NF-kappaB) pathway and Protein Phosphatase 2A (PP2A) activations were measured. Myeloperoxydase (MPO) activation and Macrophage-Inflammatory Protein-2 (MIP-2) transcription were monitored. RESULTS: The exposure to hyperosmotic solutions enhanced the production of IL-8 and induced pro-inflammatory cytokines transcription. In vivo, MPO enhanced activity accompanied by an increased MIP-2 transcription was observed. In vitro, NF-kappaB activation is accompanied by an inhibitor of kappa B-alpha degradation and inhibitor of kappa B kinase (IKK gamma) activation. We demonstrated the induction of IKK gamma after methylation and activation of PP2A. Cytokine induction was inhibited by okadaic acid and calyculin A and stimulated by xylitol. CONCLUSION: Hyperosmolarity can induce pro-inflammatory cytokine responses in colorectal and bladder epithelial cells. Inflammation appears to be the simple consequence of a shift of methylation of PP2A which in turn activates NF-kappaB.

Auditory wavelet transform based on auditory wavelet families.

The auditory periphery system receives a one dimensional acoustical signal that describes how the local pressure varies with time. However, this one dimensional signal information is then somehow unfolded into a two dimensional time-frequency plane, that tells us when which frequency occurs. The hearing process is based on compromise between time localization and frequency localization. A kind of time-frequency or wavelet type transformation is done in auditory signal processing. In this study the similarities between auditory transform based on the auditory physiological process and wavelet transform are introduced. Specially, band pass filter bank properties and variable time and frequency resolutions with the signal frequency are considered. The main goal is to find the scaling function while the numerical values of the wavelet function were measured. If the wavelet function and the scaling function from the measured data are estimated, then the wavelet coefficients and the scaling coefficients could be calculated. Therefore, the multiresolution implementation of auditory based wavelet transform is possible.

Nitric oxide induction as a novel immunoepidemiological target in malaria-infected patients from endemic areas of the Islamic Republic of Iran.

OBJECTIVE: Malaria has been prevalent for a long time in Iran and continues to be a health problem despite substantial control programs. In addition to numerous cytokines, nitric oxide (NO) is thought to be a key molecule and a novel target of malaria immunopathology. MATERIAL AND METHODS: The objective of this research was to measure reactive nitrogen intermediates (RNI) as stable metabolites of NO induction in plasma of malaria-infected patients in Iran. In this study, 235 blood samples from malaria patients and 80 blood samples from healthy controls were randomly collected from different malarial endemic provinces of Iran, located in southeastern (Sistan & Balouchestan, Hormozgan, Kerman) and northwestern (Ardabil) areas. The involvement of NO in malaria patients has been investigated by statistical analysis of RNI values. Griess micro assay (GMA) was used during Plasmodium vivax, P. falciparum and mixed infections, in order to evaluate whether RNI changes are related to the provincial areas, parasite strains, clinical symptoms and age and gender parameters. RESULTS: The results showed a significant increase of RNI level in malaria patients compared with the control groups of Ardabil (p<0.01), Sistan & Balouchestan, Hormozgan and Kerman (p<0.001) provinces. The level of RNI was higher in mixed plasmodial infection than in single infection. CONCLUSIONS: The high level of RNI was dependent on the type of infection, the plasmodia strain, the clinical symptoms, the age groups and the endemic provinces. Although, this study did not clarify the pathogenic and/or protective role of NO in malaria, our findings provide a novel immunoepidemiological aspect of basal NO production in patients with malaria in endemic areas in Iran.