Interleukin-10 of red nucleus plays anti-allodynia effect in neuropathic pain rats with spared nerve injury.

Our previous studies have shown that pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1ß) in red nucleus (RN) are involved in the development of neuropathic pain and play facilitated roles on the mechanical allodynia induced by peripheral nerve injury. The current study was designed to evaluate the expression and effect of IL-10, an anti-inflammatory cytokine, in the RN of rats with spared nerve injury (SNI). Immunohistochemical staining results demonstrated when 3 weeks after SNI, the expression level of IL-10 in the contralateral RN of SNI rats was apparently higher than those of sham-operated and normal rats. To further study the effect of IL-10 in the development of neuropathic pain, different doses of IL-10 (1.0, 0.5 and 0.1 µg/µl) were microinjected respectively into the RN contralateral to the nerve injury side of SNI rats. Results demonstrated that higher doses of IL-10 (1.0 and 0.5 µg/µl) significantly attenuated the mechanical allodynia of neuropathic rats, while 0.1 µg/µl of IL-10 did not show any analgesic effect. These results suggest that IL-10 of RN participates in the development of neuropathic pain and plays inhibitory roles on the mechanical allodynia induced by SNI.

A point mutation in the DNA-binding domain of HPV-2 E2 protein increases its DNA-binding capacity and reverses its transcriptional regulatory activity on the viral early promoter.

BACKGROUND: The human papillomavirus (HPV) E2 protein is a multifunctional DNA-binding protein. The transcriptional activity of HPV E2 is mediated by binding to its specific binding sites in the upstream regulatory region of the HPV genomes. Previously we reported a HPV-2 variant from a verrucae vulgaris patient with huge extensive clustered cutaneous, which have five point mutations in its E2 ORF, L118S, S235P, Y287H, S293R and A338V. Under the control of HPV-2 LCR, co-expression of the mutated HPV E2 induced an increased activity on the viral early promoter. In the present study, a series of mammalian expression plasmids encoding E2 proteins with one to five amino acid (aa) substitutions for these mutations were constructed and transfected into HeLa, C33A and SiHa cells. RESULTS: CAT expression assays indicated that the enhanced promoter activity was due to the co-expressions of the E2 constructs containing A338V mutation within the DNA-binding domain. Western blots analysis demonstrated that the transiently transfected E2 expressing plasmids, regardless of prototype or the A338V mutant, were continuously expressed in the cells. To study the effect of E2 mutations on its DNA-binding activity, a serial of recombinant E2 proteins with various lengths were expressed and purified. Electrophoresis mobility shift assays (EMSA) showed that the binding affinity of E2 protein with A338V mutation to both an artificial probe with two E2 binding sites or HPV-2 and HPV-16 promoter-proximal LCR sequences were significantly stronger than that of the HPV-2 prototype E2. Furthermore, co-expression of the construct containing A338V mutant exhibited increased activities on heterologous HPV-16 early promoter P97 than that of prototype E2. CONCLUSIONS: These results suggest that the mutation from Ala to Val at aa 338 is critical for E2 DNA-binding and its transcriptional regulation.

[The enhancement of DNA binding ability of a mutated E2 (A338V) protein of HPV-2].

HPV-2 is a very common type of HPV which causes common warts. The E2 protein of virus can repress the activity of the viral early promoter through binding to the specific binding sites in viral LCR. Previously we reported that the repression of a mutated E2 protein of HPV-2 isolated from a patient with huge common wart on the viral early promoter was obviously decreased, and A338V mutation located at the C terminal DNA binding region of E2 protein. In this study, we expressed and purified the recombinant mutated and prototype E2 fusion proteins, both in the contexts of the C terminal and the full length, by prokaryotic expression system. The electrophoretic mobility shift assay showed E2 protein could bind to double-stranded DNA oligos labeled with biotin that covered two E2 binding sites. The DNA binding abilities of both C terminal and full-length mutated E2 proteins were stronger than the prototype analogs. This result indicates that the enhancement of the mutated E2 DNA binding ability may be the molecular mechanism for its impact on the activity of viral promoter, which correlates with the phenotype of extensive common wart.

Evaluation of protective effect of multi-epitope DNA vaccine encoding six antigen segments of Toxoplasma gondii in mice.

To investigate the vaccine potential of multi-epitope vaccines against toxoplasmosis, a multi-epitope DNA vaccine, eukaryotic plasmid pcDNA3.1/T-ME expressing six antigen segments (SAG1(238-256), SAG1(281-320), GRA1(170-193), GRA4(331-345), GRA4(229-245), and GRA2(171-185)) of Toxoplasma gondii was constructed. We investigated the efficacy of pcDNA3.1/T-ME with or without co-administration of a CpG-oligodeoxynucleotide (CpG-ODN) as an adjuvant to protect mice (BALB/c and C57BL/6) against toxoplasmosis. High survival rates were observed in mice immunized with pcDNA3.1/T-ME when challenged with T. gondii RH strain. Lymphocyte proliferation assays, cytokine, and antibody determinations show that mice immunized with pcDNA3.1/T-ME produced stronger humoral and Th1-type cellular immune responses compared to untreated mice or those immunized with empty plasmids. However, co-immunization with CpG-ODN resulted in impaired immune responses. Our data demonstrates that multi-epitope DNA vaccination is a potential strategy for the control of toxoplasmosis and paves the way for further investigations into producing a multi-epitope anti-T. gondii DNA vaccine.

Nerve growth factor of red nucleus involvement in pain induced by spared nerve injury of the rat sciatic nerve.

Nerve growth factor (NGF), a member of the neurotrophin family, is essential for the development and maintenance of sensory neurons and for the formation of central pain circuitry. The current study was designed to evaluate the expression of NGF in the brain of rats with spared nerve injury (SNI), using immunohistochemical technique. The results showed that the level of NGF in the Red nucleus (RN) of SNI rats was apparently higher than that of sham-operated rats. To further study the effect of NGF in the development of neuropathic pain, different doses of anti-NGF antibody (20, 2.0 and 0.2 microg/ml) were microinjected into the RN contralateral to the nerve injury side of SNI rats. The data suggested that the higher doses of anti-NGF antibody (20 and 2.0 microg/ml) significantly attenuated the mechanical allodynia of neuropathic rats, while the 0.2 microg/ml antibody showed no analgesic effect. These results suggest that the NGF of RN is involved in the development of neuropathic allodynia in SNI rats.

[Construction of multi-epitope DNA vaccine for Toxoplasma gondii and the study on protective immunity response in mice].

AIM: To construct multi-epitope DNA vaccine for Toxoplasma gondii and study its protective immunity response. METHODS: The gene encoding six polypeptides of T. gondii, which consists of plenty of T and B epitopes, was cloned into the eucaryotic expression vector pcDNA3.1(+). BALB/c mice were vaccinated by this multi-epitope based DNA vaccine (intramuscular needle injection). The specific antibody and T cell proliferation were determined. Meanwhile, the DNA-vaccinated mice were challenged with a lethal dose of T. gondii tachyzoites for further observation. RESULTS: The eukaryotic expression plasmid (pcDNA3.1/T-ME) encoding plenty of T. gondii epitopes was constructed successfully. pcDNA3.1/T-ME immunization induced T. gondii specific humoral and cellular immunity in mice. The mice immunized with pcDNA3.1/T-ME survived significantly longer than the mice in control after challenged by T. gondii RH strain infection. CONCLUSION: The multi-epitope DNA vaccine can induce the protective immunity against T. gondii infection effectively in vivo, which is a potential strategy to control T. gondii infection.

[Bactericidal activity of GLL-37, a novel derivative of the human antimicrobial peptide LL-37].

OBJECTIVE: To develop and investigate GLL-37, a substitution analogue of the human antimicrobial peptide LL-37 with anti-enzymatic degradation activity and improved efficacy. METHODS: The bactericidal activities of LL-37 and newly developed GLL-37 against 6 Gram-negative and -positive bacteria were determined by Broth microdilution assays. The minimum inhibitory concentrations of LL-37 and GLL-37 against E.coli ATCC 25922 in different NaCl concentration medium were also detected. Both peptides were co-incubated with elastase, and then analyzed by PAGE electrophoresis and bactericidal activity determination. RESULT: GLL-37 showed a stronger elastase resistance ability than LL-37, and was significantly more effective than LL-37 under high-salt condition. CONCLUSION: The antimicrobial peptide GLL-37 derived form LL-37 has the potential as a new therapeutic agent for bacterial infections.

[Analgesic effect of interleukin-2 in mouse models of spared nerve injury].

OBJECTIVE: To investigate the analgesic effect of interleukin-2 (IL-2) in mice with spared nerve injury (SNI). METHOD: IL-2 was intraperitoneally injected in mice with induced SNI, and von Frey Filaments test and cold plate test were carried out to accesses the analgesic effects of IL-2 and the effect of naloxone in antagonizing the effects of IL-2. RESULTS: IL-2 produced analgesic effects against hyperalgesia and allodynia in mouse models of SNI, and the effect of IL-2 lasted for more than 24 h, showing a double-peak pattern in its action with the two peaks occurring at 30 and 105 min, respectively. The effect of IL-2 could be significantly antagonized by naloxone. CONCLUSIONS: IL-2 has long-lasting analgesic effects in mouse models of SNI model, showing a double-peak pattern of its action. The analgesic effect of IL-2 is probably mediated by opiate receptor.

Resveratrol induces apoptosis, influences IL-6 and exerts immunomodulatory effect on mouse lymphocytic leukemia both in vitro and in vivo.

Resveratrol, a dietary polyphenol found in grapes, has been proposed to act as a chemopreventive or anti-tumor agent in numerous epidemiologic studies. In this study, we investigate the antitumor and immunomodulation effects of resveratrol on mouse lymphocytic leukemia cells L1210 both in vitro and in vivo. Our finding indicates that resveratrol inhibits proliferation, induces apoptosis, and influences cell cycle of L1210 cells in a dose- and time-dependent manner in vitro. Furthermore, resveratrol can exert a dose-related regulatory effect on both innate and specific immune function to L1210-bearing mice. A normalization of CD4/CD8 ratios is noted as well as an enhancement of lymphocyte proliferation, NK cell activity and anti-SRBC titers. Interleukin-6 cellular content and release are suppressed by resveratrol as well as mRNA expression. In conclusion, the data provide new findings with respect to resveratrol mechanism of action to mouse lymphocytic leukemia.

[Inducement of cytotoxic T cell responses in mice by constructing a multi-CTL epitope-based DNA vaccine].

AIM: To construct a multi-CTL epitope-based DNA vaccine to induce specific CTL responses. METHODS: Multi-CTL epitope gene which encoded two HCV epitopes(H-2(d)) was cloned into the eucaryotic expression vector pcDNA3.1 to construct a multi-CTL epitope-based DNA vaccine. BALB/c mice were vaccinated with the DNA vaccine and the specific CTL responses to target cells (P815, H-2(d)) pulsed by different CTL epitope peptide were detected. RESULTS: The multi-CTL epitope-based DNA vaccine which directed against two HCV CTL epitopes induced specific CTL responses to each of the two CTL epitopes independently and enhanced the total specific CTL response. CONCLUSION: The multi-CTL epitope-based DNA vaccine is constructed by using multi-CTL epitopes linked as encoding sequence through natural flanking amino acid residues. It can not only induce specific CTL responses to each CTL epitope independently but also enhance the total specific CTL response.

mu- but not delta- and kappa-opioid receptor mediates the nucleus submedius interferon-alpha-evoked antinociception in the rat.

Previous studies have indicated that interferon-alpha (IFN-alpha) can bind to opioid receptors and exerts an antinociceptive effect in both peripheral and central nervous systems. The current study investigated the antinociceptive effect of IFN-alpha unilaterally microinjected into the thalamic nucleus submedius (Sm) of rats on noxious thermal stimulus, and the roles of different subtypes of opioid receptors in mediating the Sm IFN-alpha-evoked antinociception. The results indicated that unilateral microinjection of IFN-alpha (4, 8, 16 pmol) into the Sm dose-dependently increased the hind paw withdrawal latency from the noxious heat stimulus, and this effect was reversed by pretreatment with non-selective opioid receptor antagonist naloxone (200 pmol) and specific mu-opioid receptor antagonist beta-FNA (1 nmol) into the same sites, whereas delta-opioid receptor antagonist ICI174,864 (1 nmol) and kappa-opioid receptor antagonist nor-BNI (1 nmol) failed to alter the effect of IFN-alpha. These results suggest that Sm is involved in IFN-alpha-evoked antinociception and mu- but not delta- and kappa-opioid receptor mediates the Sm IFN-alpha-evoked antinociception.

Effective induction of type 1 cytotoxic T cell responses in mice with DNA vaccine encoding two hepatitis C virus cytotoxic T lymphocyte epitopes.

The aims of this study were to explain whether a multiple cytotoxic T lymphocyte (CTL) epitope-based anti-hepatitis C virus (HCV) DNA vaccine can induce specific CTL responses to each HCV CTL epitope independently and long-term CD8(+) T cell memory responses, and to determine the cytokine secretion pattern and subtype of epitope-specific cytotoxic T cells. A multi-CTL epitope gene, which consists of two epitopes of HCV (H-2(d)-restricted HCV core(133142) and E1(315322)), was cloned into the eukaryotic expression vector pcDNA3.1. BALB/c mice (H-2(d) restricted) were vaccinated intramuscularly with this multi-CTL epitope-based DNA vaccine. The epitope-specific CTLs against target cells (P815,H-2(d) restricted) pulsed with various CTL epitope peptides were detected by lactate dehydrogenase release assay, and the precursor frequency of epitope-specific CTLs was determined by limiting dilution analysis. Cytokines (interleukin [IL]-2, IL-4, and interferon-) in culture supernatants were determined by enzyme-linked immunosorbent assay. The multi-CTL epitope-based DNA vaccine directed against two HCV CTL epitopes could induce specific CTL responses to each of the two CTL epitopes independently and long-term CD8(+) T cell memory responses. The epitope-specific cytotoxic T cells produced helper T cell type 1 cytokines. This work demonstrated that multiepitope DNA vaccination is a potential strategy to control HCV infection.

[Preliminary study on anti-tumor function of resveratrol and its immunological mechanism].

AIM: To explore the immunological mechanism of the anti-tumor function of resveratrol (Res) through in vitro or vivo experiments. METHODS: The tumor cell growth repression rate was measured by MTT colorimetry. Flow cytometry was used to analyze the cell cycle of Hep2 cells (laryngeal squamous cancer cell line). The immune function and cytokine levels in the serum of the tumor-bearing mice were determined by MTT colorimetry, hemolysis spectrophotography, improved Mayer's method and ELISA. RESULTS: Res inhibited the tumor cell growth and enhanced the apoptosis of tumor cells in time-concentration-dependent manners showing the phenomenon of obvious G(0)/G(1) blocking and apoptotic peak. The maximal tumor inhibition rate came up to 42.76%. Furthermore, Res improved function of T, B lymphocytes, killing activity of NK cells, release of antibodies, and the total complement activity in serum. It also increased contents of IL-2 and TGF-beta1 but reduced that of IL-8 and VEGF. CONCLUSION: Res can not only affect tumor cells directly but also exert anti-tumor efficiency through reinforcing cell-mediated, humoral immune response and accommodating lymphocytes to secrete cytokines.