Heat-killed Enterococcus faecalis Alters Nitric Oxide and CXCL12 Production but not CXCL8 and CCL3 Production by Cultured Human Dental Pulp Fibroblasts

Introduction: Fibroblasts are the most abundant cells in dental pulp. To investigate their capacity to produce the chemokines CCL3, CXCL8, and CXCL12 as well as nitric oxide (NO), we evaluated the production of these mediators in supernatants of cultured human dental pulp fibroblasts (HDPF) stimulated by heat-killed Enterococcus faecalis (HKEF). Methods: Primary cultures of HDPF were stimulated with medium alone or HKEF (1:1, 10:1, or 100:1 bacteria:fibroblast) for 1, 6, and 24 hours. Chemokines and NO were assessed through enzyme-linked immunosorbent assay and Griess reaction, respectively. Statistical analysis was performed by using analysis of variance and Tukey post test. Results: CCL3 was not detected, whereas constitutive CXCL8 was not affected. Production of CXCL12 was increased at 1 and 6 hours, and NO was increased at the concentration of 1:1 bacteria:fibroblast at 24 hours. Viability and proliferation assays did not reveal cell number differences. Conclusions: These findings demonstrate that heat-killed E. faecalis is able to increase production of CXCL12 and NO by HDPF. (J Endod 2010;36:91-94)

Genetic variation in the recognition of Porphyromonas gingivalis antigens in mice

T-cell cytokine profiles, anti Porphyromonas gingivalis antibodies and Western blot analysis of antibody responses were examined in BALB/c, CBA/CaH, C57BL6 and DBA/2J mice immunized intraperitoneally with different doses of P. gingivalis outer membrane antigens, Splenic CD4 and CD8 cells were examined for intracytoplasmic interleukin (IL)-4, interferon (IFN)-gamma and IL-LD by FAGS analysis and levels of anti-P. gingivalis antibodies in the serum samples determined by enzyme-linked immunosorbent assay. Western blot analysis was performed on the sera from mice immunized with 100 mug of P. gingivalis antigens. The four strains of mice demonstrated varying degrees of T-cell immunity although the T-cell cytokine profiles exhibited by each strain were not affected by different immunizing doses. While BALB/c and DBA/2J mice exhibited responses that peaked at immunizing doses of 100-200 mug of P. gingivalis antigens, CBA/CaH and C57BL6 demonstrated weak T-cell responsiveness compared with control mice. Like the T-cell responses, serum antibody levels were not dose dependent. DBA/23 exhibited the lowest levels of anti-P. gingivalis antibodies followed by BALB/c with CBA/CaH and C57BL6 mice demonstrating the highest levels. Western blot analysis showed that there were differences in reactivity between the strains to a group of 13 antigens ranging in molecular weight from 15 to 43 kDa. Antibody responses to a number of these bands in BALB/c mice were of low density, whereas CBA/CaH and C57BL6 mice demonstrated high-density bands and DBA/2J mice showed medium to high responses. In conclusion, different immunizing doses of P. gingivalis outer membrane antigens had little effect on the T-cell cytokine responses and serum anti-P. gingivalis antibody levels. Western blot analysis, however, indicated that the four strains of mice exhibited different reactivity to some lower-molecular-weight antigens. Future studies are required to determine the significance of these differences, which may affect the outcome of P. gingivalis infection.

Schistosomiasis in the People's Republic of China: Prospects and challenges for the 21st century

Schistosomiasis japonica is a serious communicable disease and a major disease risk for more than 30 million people living in the tropical and subtropical zones of China. Infection remains a major public health concern despite 45 years of intensive control efforts. It is estimated that 865, 000 people and 100,250 bovines are today infected in the provinces where the disease is endemic, and its transmission continues. Unlike tire other schistosome species known to infect humans, the oriental schistosome, Schistosoma japonicum, is a true zoonotic organism, with a range of mammalian reservoirs, making control efforts extremely difficult. Clinical features of schistosomiasis range from fever; headache, and lethargy to severe fibro-obstructive pathology leading to portal hypertension, ascites, and hepatosplenomegaly, which can cause premature death. Infected children ale stunted and have cognitive defects impairing memory and learning ability. Current control programs are heavily based on community chemotherapy with a single dose of the drug praziquantel, but vaccines (for use in bovines and humans) in combination with other control strategies ale needed to make elimination of the disease possible. In this article, we provide an overview of the biology, epidemiology clinical features, and prospects for cona ol of oriental schistosomiasis in the People's Republic of China.

Human susceptibility to Schistosoma japonicum in China correlates with antibody isotypes to native antigens

Antibody isotypic responses (IgE, IgA, IgG1, IgG2, IgG3 and IgG4) to Schistosoma japonicum antigens-adult worm (AWA), soluble egg (SEA) and the recombinant proteins TEG (22.6-kDa tegumental antigen, Sj22) and PMY (paramyosin, Sj97)-were measured (in 1998) in a cohort of 179 Chinese subjects 2 years post-treatment. Subjects in the highest intensity re-infection group (> 100 eggs per gram faeces) had significantly higher levels of IgG1 and IgG4 against AWA. Analysis of IgG4/IgE ratios for AWA and SEA linked IgG4 excess to re-infection and IgE excess to non-re-infection. Two years after chemotherapeutic cure, 29 subjects, who were re-infected or never infected but highly water-exposed, were classified as epidemiologically susceptible (n = 15) or epidemiologically insusceptible to infection (n = 14). IgG4 levels against native antigens (AWA and SEA) were higher in susceptibles and IgE levels were higher in insusceptibles but antibody responses to the recombinant proteins (PMY and TEG) showed no clear pattern or difference between susceptibility groups. These and earlier findings provide evidence that immunity develops against schistosomiasis japonica in China and that susceptibility/resistance correlates with antibody isotypes against native schistosome antigens.

Up-regulation of p21(WAF1)/(CIP1) by histone deacetylase inhibitors reduces their cytotoxicity

Histone deacetylase inhibitors show promise as chemotherapeutic agents and have been demonstrated to block proliferation in a wide range of tumor cell lines. Much of this antiproliferative effect has been ascribed to the up-regulated expression of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1). In this article, we report that p21 expression was up-regulated by relatively low doses of the histone deacetylase inhibitor azelaic bishydroxamic acid (ABHA) and correlated with a proliferative arrest. Higher doses of ABHA were cytotoxic. Cells that did not up-regulate p21 expression were hypersensitive to killing by ABHA and died via apoptosis, whereas up-regulation of p21 correlated with reduced sensitivity and a block in the apoptotic mechanism, and these cells seemed to die by necrosis. Using isogenic p21(+/+) and p21(-/-) cell lines and direct inhibition of caspase activity, we demonstrate that the reduced sensitivity to killing by ABHA is a consequence of inhibition of apoptosis by up-regulated p21 expression. These data indicate the enormous potential of therapeutic strategies that bypass the cytoprotective effect of p21 and act on the same molecular targets as the histone deacetylase inhibitors.

Experimental infections of pigs with Japanese encephalitis virus and closely related Australian flaviviruses

The flavivirus Japanese encephalitis (JE) virus has recently emerged in the Australasian region. To investigate the involvement of infections with related enzootic flaviviruses, namely Murray Valley encephalitis (MVE) virus and Kunjin (KUN) virus, on immunity of pigs to JE virus and to provide a basis for interpretation of serologic data, experimental infections were conducted with combinations of these viruses. Antibody responses to primary and secondary infections were evaluated using panels of monoclonal antibody-based blocking enzyme-link-ed immuno-sorbent assays and microtiter scrum neutralization tests (mSNTs). Identification of the primary infecting virus was possible only using the mSNTs. Following challenge, unequivocal diagnosis was impossible due to variation in immune responses between animals and broadened and/or anamnestic responses. Viremia for JE virus was readily detected in pigs following primary infection, but was not detected following prior exposure to MVE or KUN viruses. Boosted levels of existing cross-neutralizing antibodies to JE virus suggested a role for this response in suppressing JE viremia.

Adoptive transfer of autologous Epstein-Barr virus-specific cytotoxic T cells for nasopharyngeal carcinoma

Tumor cells from NPC patients are regularly and latently infected with EBV. To examine whether the virus serves as target for immune intervention of the cancer, we determined levels of EBV-specific CTLp in peripheral blood from NPC patients, long-term survivors of the cancer and healthy subjects. CTLp levels of test subjects varied between 3-3,000/10(6) PBMCs. The plasma EBV burden increased when the CTLp level fell below 150, whereas the EBV burden of PBMCs was not correlated with CTLp level. Compared with healthy carriers, CTLp levels of patients were lower and varied over a wider range, between 3-1,500/10(6) PBMCs. The quantitative immune deficit was probably attributed to the tumor because, first, CTLp in survivors was restored to levels similar to those in healthy carriers after the tumor had been successfully treated. Second, the CTLp level changed as disease progressed, being lower in local disease, increased in locoregional disease and decreased again when the tumor metastasized. Based on these findings, 4 patients with advanced disease were infused with 5 x 10(7)-3 x 10(8) autologous EBV CTLs. The treatment was safe and unaccompanied by inflammatory or other complications, but whether it improved tumor control could not be discerned from the large tumor bulk. Nevertheless, the treatment regularly increased CTLp levels of patients, maintained it at higher levels for protracted periods and, in 3 patients, restored host surveillance of EBV replication, reducing the plasma EBV burden. Taken together, these results provided a rationale to further explore EBV as a target of immune intervention of NPC. (C) 2001 Wiley-Liss, Inc.

Metastatic lesions in the joint associated with acute inflammatory arthritis after dendritic cell immunotherapy for metastatic melanoma

A 47 year old man undergoing immunotherapy for metastatic melanoma with autologous dendritic cells pulsed with autologous tumour peptide and hepatitis a surface antigen developed acute left ankle arthritis. Gout and acute infection were excluded, and an autoimmune aetiology or occult metastasis were considered. The arthritis initially subsided with indomethacin, but the symptoms recurred 2 months later, and magnetic resonance imaging demonstrated metastatic melanoma of the left talus. Immunohistochemical staining of a cerebral metastatic deposit biopsied 1 week after the onset of arthritis demonstrated T-cell and macrophage infiltration of the tumour. In addition, the patient developed melanoma-specific delayed type hypersensitivity and cytotoxic T-cell responses after vaccination. Thus, the monoarthritis represented an 'appropriate' inflammatory response directed against metastatic melanoma. (C) 2001 Lippincott Williams & Wilkins.

Histone hyperacetylation induced by histone deacetylase inhibitors is not sufficient to cause growth inhibition in human dermal fibroblasts

Use of specific histone deacetylase inhibitors has revealed critical roles for the histone deacetylases (HDAC) in controlling proliferation. Although many studies have correlated the function of HDAC inhibitors with the hyperacetylation of histones, few studies have specifically addressed whether the accumulation of acetylated histones, caused by HDAC inhibitor treatment, is responsible for growth inhibition. In the present study we show that HDAC inhibitors cause growth inhibition in normal and transformed keratinocytes but not in normal dermal fibroblasts, This was despite the observation that the HDAC inhibitor, suberic bishydroxamate (SBHA), caused a kinetically similar accumulation of hyperacetylated histones, This cell type-specific response to SBHA was not due to the inactivation of SBHA by fibroblasts, nor was it due to differences in the expression of specific HDAC family members. Remarkably, overexpression of HDACs 1, 4, and 6 in normal human fibroblasts resulted in cells that could be growth-inhibited by SBHA. These data suggest that, although histone acetylation is a major target for HDAC inhibitors, the accumulation of hyperacetylated histones is not sufficient to cause growth inhibition in all cell types, This suggests that growth inhibition, caused by HDAC inhibitors, may be the culmination of histone hyperacetylation acting in concert with other growth regulatory pathways.

Structural effects of lipophilic methotrexate conjugates on model phospholipid biomembranes

Lipophilic conjugates of the antitumor drug methotrexate (MTX) with lipoamino acids (LAAs) have been previously described as a tool to enhance MTX passive entrance into cells, overcoming a form of transport resistance which makes tumour cells insensitive to the antimetabolite. A knowledge of the mechanisms of interaction of such lipophilic derivatives with cell membranes could be useful for planning further lipophilic MTX derivatives with an optimal antitumour activity. To this aim, a calorimetric study was undertaken using a biomembrane model made from synthetic 1,2-dipalmitoyl-glycero-3-phosphocholine (DPPC) multilamellar liposomes. The effects of MTX and conjugates on the phase transition of liposomes were investigated using differential scanning calorimetry. The interaction of pure MTX with the liposomes was limited to the outer part of the phospholipid bilayers, due to the polar nature of the drug. Conversely, its lipophilic conjugates showed a hydrophobic kind of interaction, perturbing the packing order of DPPC bilayers. In particular, a reduction of the enthalpy of transition from the gel to the liquid crystal phase of DPPC membranes was observed. Such an effect was related to the structure and mole fraction of the conjugates in the liposomes. The antitumour activity of MTX conjugates was evaluated against cultures of a CCRF-CEM human leukemic T-cell line and a related MTX resistant sub-line. The in vitro cell growth inhibitory activity was higher for bis(tetradecyl) conjugates than for both the other shorter- and longer-chain derivatives. The biological effectiveness of the various MTX derivatives correlated very well with the thermotropic effects observed on the phase transition of DPPC biomembranes. (C), 2001 Elsevier Science B.V All rights reserved.

Vaccine efficacy of recombinant cathepsin D aspartic protease from Schistosoma japonicum

Mice were vaccinated with recombinant Schistosoma japonicum cathepsin D aspartic protease, expressed in both insect cells and bacteria, in order to evaluate the vaccine efficacy of the schistosome protease. Mean total worm burdens were significantly reduced in vaccinated mice by 21-38%, and significant reductions in female worm burdens were also recorded (22-40%). Vaccination did not reduce fecundity; rather, we recorded increased egg output per female worm in vaccinated animals, suggesting a crowding effect. Vaccinated mice developed high levels of antibodies (predominantly IgG1, IgG2a and IgG2b isotypes), but there was no correlation between antibody levels and protective efficacy. Immune sera from vaccinated mice did not inhibit the in vitro degradation of human haemoglobin by the recombinant protease, and passive transfer of serum or antibodies from vaccinated animals, before and after parasite challenge, did not significantly reduce worm or egg burdens in recipient animals. These results suggest that antibodies may not play a key role in the protective effect elicited, and that protection may be due to a combination of humoral and cell-mediated responses.

Further studies on an intermediate host murine model showing that a primary Echinococcus granulosus infection is protective against subsequent oncospheral challenge

We describe the use of a murine model to evaluate resistance against subsequent challenge following a primary infection with oncospheres of Echinococcus granulosus. Mice (Kunming strain) were infected with hatched oncospheres of Echinococcus granulosus; 21 days later a second challenge was given by a different route of infection. A primary infection by intraperitoneal (i.p.) injection stimulated 100 and 90.5% protection in terms of reduced cyst numbers against a secondary infection given subcutaneously (s.c.) or intravenously (i.v.) respectively. A primary infection given s.c. followed by i.p. or i.v. challenge resulted in 84.0 and 100% protection, respectively. Intravenous infection followed by i.p. or s.c. challenge resulted in 98.5 and 69.4% protection, respectively. With the i.v. route of infection, almost all resultant cysts were present in the lungs. The data show that a primary infection with oncospheres can induce total or a high degree of protection against a subsequent challenge and confirms that natural (concomitant) immunity can be stimulated in the intermediate host as the result of a primary infection. This may explain the decline in hydatid infection in sheep older than 2 years in hyper-endemic areas such as those found in Xingjiang, China. These older sheep may have been earlier infected and have subsequently self-cured, with the primary infection stimulating an immune response that protects the intermediate host animals from further infection. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

CD40 and CD86 upregulation with divergent CMRF44 expression on blood dendritic cells in inflammatory bowel diseases

OBJECTIVE: Dendritic cells (DC) are the only antigen-presenting cells that can activate naive T lymphocytes and initiate a primary immune response. They are also thought to have a role in immune tolerance. DC traffic from the blood to peripheral tissue where they become activated. They then present antigen and the costimulating signals necessary to initiate an immune response. In this study, we investigated the number, subsets, and activation pattern of circulating and intestinal DC from patients with clinically mild ulcerative colitis (UC) or Crohn's disease. METHODS: Patients were recruited, if they were not taking immunosuppressive therapy, and were assessed for clinical severity of their disease using for UC, the Clinical Activity Index, and for Crohn's disease, the Crohn's Disease Activity Index. Blood CD11c(+) and CD11c(-) DC subsets, expression of costimulatory antigens, CD86 and CD40, and the early differentiation/activation antigen, CMRF44, were enumerated by multicolor flow cytometry of lineage negative (lin(-) = CD3(-), CD19(-), CD14(-), CD16(-)) HLA-DR+ DC. These data were compared with age-matched healthy and the disease control groups of chronic noninflammatory GI diseases (cGI), acute noninflammatory GI diseases (aGI), and chronic non-GI inflammation (non-GI). In addition, cryostat sections of colonoscopic biopsies from healthy control patients and inflamed versus noninflamed gut mucosa of inflammatory bowel disease (IBD) patients were examined for CD86(+) and CD40(+)lin(-) cells. RESULTS: Twenty-one Crohn's disease and 25 UC patients, with mean Crohn's Disease Activity Index of 98 and Clinical Activity Index of 3.1, and 56 healthy controls, five cGI, five aGI, and six non-GI were studied. CD11c(+) and CD11c(-) DC subsets did not differ significantly between Crohn's, UC, and healthy control groups. Expression of CD86 and CD40 on freshly isolated blood DC from Crohn's patients appeared higher (16.6%, 31%) and was significantly higher in UC (26.6%, 46.3%) versus healthy controls (5.5%, 25%) (p = 0.004, p = 0.012) and non-GI controls (10.2%, 22.8%) (p = 0.012, p = 0.008), but not versus cGI or aGI controls. CD86(+) and CD40(+) DC were also present in inflamed colonic and ileal mucosa from UC and Crohn's patients but not in noninflamed IBD mucosa or normal mucosa. Expression of the CMRF44 antigen was low on freshly isolated DC, but it was upregulated after 24-h culture on DC from all groups, although significantly less so on DC from UC versus Crohn's or healthy controls (p = 0.024). The CMRF44(+) antigen was mainly associated with CD11c(+) DC, and in UC was inversely related to the Clinical Activity Index (r = -0.69, p = 0.0002). CONCLUSIONS: There is upregulation of costimulatory molecules on blood DC even in very mild IBD but surprisingly, there is divergent expression of the differentiation/activation CMRF44 antigen. Upregulation of costimulatory molecules and divergent expression of CMRF44 in blood DC was also apparent in cGI and aGI but not in non-GI or healthy controls, whereas intestinal CD86(+) and CD40(+) DC were found only in inflamed mucosa from IBD patients. Persistent or distorted activation of blood DC or divergent regulation of costimulatory and activation antigens may have important implications for gut mucosal immunity and inflammation. (Am J Gastroenterol 2001;96:2946-2956. (C) 2001 by Am. Coll. of Gastroenterology).

Irradiation-induced oral candidiasis in an experimental murine model

The aim of this experiment was to establish a mouse model of irradiation-induced oral candidiasis and to explore the cellular populations and mechanisms by which the infection is cleared from the oral mucosa. BALB/c mice received irradiation to the head and neck equivalent to 800 Rad using a Cobalt 60 gamma source. Both irradiated and non-irradiated mice were infected orally with 1 X 10(8) Candida albicans yeasts. Compared with untreated controls, irradiated animals developed a more severe infection of longer duration, with hyphae penetrating the oral mucosa. Monoclonal antibody depletion of CD4(+) but not CD8(+) T cells from the systemic circulation prolonged the infection in irradiated mice, but not in controls. Supernatants of submandibular and superficial cervical lymph node cultures from irradiated animals demonstrated significantly higher titers of interleukin-12, but similar levels of interferon-gamma compared with controls. Screening for cytokine production by an RNase protection assay detected only macrophage migration inhibition factor in irradiated and non-irradiated oral tissues from day 8 onwards. The results of this study demonstrate a requirement for CD4(+) T cells in the recovery from oral candidiasis induced by head and neck irradiation in mice, and are consistent with a role for Th-1-type cytokines in host resistance.

Low level expression of human papillomavirus type 16 (HPV16) E6 in squamous epithelium does not elicit E6 specific B- or T-helper immunological responses, or influence the outcome of immunisation with E6 protein

Mice transgenic for E6/E7 oncogenes of Human Papillomavirus type 16 display life-long expression of E6 in lens and skin epithelium, and develop inflammatory skin disease late in life, which progresses to papillomata and squamous carcinoma in some mice. We asked whether endogenous expression of E6 induced a specific immunological outcome, i.e. immunity or tolerance, or whether the mice remained immunologically naive to E6. We show that prior to the onset of skin disease, E6 transgenic mice did not develop a spontaneous E6-directed antibody response, nor did they display T-cell proliferative responses to dominant T-helper epitope peptides within E6. In contrast, old mice in which skin disease had arisen, developed antibodies to E6. We also show that following immunisation with E6, specific antibody responses did not differ significantly among groups of EB-transgenic mice of different ages (and therefore of different durations and amounts of exposure to endogenous E6), and non-transgenic controls. Additionally, E6 immunisation-induced T-cell proliferative responses were similar in E6-transgenic and non-transgenic mice. These data are consistent with the interpretation that unimmunised Eb-transgenic mice that have not developed inflammatory skin disease remain immunologically naive to E6 at the B- and Th levels. There are implications for E6-mediated tumorigenesis in humans, and for the development of putative E6 therapeutic vaccines. (C) 2001 Elsevier Science B.V. All rights reserved.