Polymorphism in the IL-8 gene, but not in the TLR4 gene, increases the severity of acute pancreatitis.

BACKGROUND/AIM: Activated granulocytes and inflammatory mediators of the innate immune response play fundamental roles in the pathogenesis of acute pancreatitis. We studied whether polymorphisms of interleukin-8 (IL-8) and Toll-like receptor 4 (TLR4) genes correlate with the severity of acute pancreatitis. METHODS: Patients with acute pancreatitis (n = 92) were grouped according to the severity of the disease on the basis of the Ranson scores. Healthy blood donors (n = 200) served as controls. The IL-8 -251 gene polymorphism was analyzed by amplification-refractory mutation system; the single-nucleotide polymorphisms (Asp299Gly and Thr399Ile) of TLR4 were investigated by using a real-time polymerase chain reaction method with melting point analysis. RESULTS: The IL-8 A/T heterozygote mutant variants were detected with a significantly higher frequency among the patients with severe pancreatitis than among the healthy blood donors (60 vs. 42%; p = 0.0264, odds ratio = 2.071, 95% confidence interval = 1.101-3.896), while the frequency of the normal allelic genotype (TT) was higher among the patients with mild pancreatitis than in the group with severe pancreatitis (35 vs. 16%; p = 0.051, odds ratio = 2.917, 95% confidence interval = 1.089-7.811). There was no significant correlation between TLR4 polymorphisms and the acute pancreatitis itself, but nonsignificantly increased frequencies of Asp299Gly and Thr399Ile heterozygotes among patients with severe infected pancreatic necrosis could be observed relative to the patients with mild pancreatitis. CONCLUSIONS: Determination of the frequency of IL-8 polymorphism in acute pancreatitis may be informative and may provide further evidence concerning the role of IL-8 in the severe form of this disease. The possible role of TLR4 polymorphism in the outcome of severe acute pancreatitis requires further investigations in a larger series of patients.

Chlamydia pneumoniae exacerbates aortic inflammatory foci caused by murine cytomegalovirus infection in normocholesterolemic mice.

Inflammatory foci induced by murine cytomegalovirus infection in normocholesterolemic mice were present temporarily in the aortic wall, but some of these foci developed into advanced lesions that persisted late after infection. The early foci induced by virus infection were significantly exacerbated following a single inoculation with Chlamydia pneumoniae.

Optimization of DNA immunization against human cytomegalovirus.

The immune responses of mice injected with plasmids VR-gB and VR-gB Delta tm expressing the full-length membrane-anchored, or secreted forms of human cytomegalovirus (HCMV)-glycoprotein B (gB), respectively, and VR-pp65 expressing the HCMV-phosphoprotein 65 (pp65) were analyzed. Pretreatment of mice with the local anesthetic bupivacaine did not enhance antibody production, and IFN-alpha co-expressed with the immunizing plasmids induced a moderate increase in the antibody response. However, antibody response was higher in mice inoculated at three sites in the musculus quadriceps than in mice inoculated at one site with the same dose and in the same muscle. pVR-gB Delta tm induced significantly higher antibody titers than the construct expressing the membrane-anchored form of gB, and priming with pVR-gB Delta tm followed by boosting with the gB subunit resulted in high-titer antibody responses. Immunization with VR-pp65 induced dose-dependent CTL responses in about 50% of the mice at a dose of 50 microg. Co-expression of IFN-alpha did not affect the number of responding mice. These findings might be important for optimization of humoral and cellular immune responses to HCMV after DNA vaccination.

A canarypox vector-expressing cytomegalovirus (CMV) phosphoprotein 65 induces long-lasting cytotoxic T cell responses in human CMV-seronegative subjects.

The major matrix phosphoprotein 65 (pp65) of cytomegalovirus (CMV) is an important target of HLA-restricted cytotoxic T cells (CTL) after natural infection. A canarypox-CMV pp65 recombinant was studied for its ability to induce CMV pp65-specific CTL, helper T lymphocytes, and antibodies in a phase I clinical trial. Twenty-one CMV-seronegative adult volunteers were randomized to receive immunizations at months 0, 1, 3, and 6 with either canarypox-CMV pp65 or placebo. In canarypox-CMV pp65-immunized subjects, pp65-specific CTL were elicited after only 2 vaccinations and were present at months 12 and 26 in all subjects tested. Cell-depletion studies indicated that the CTL were phenotype CD8(+). Peripheral blood mononuclear cells proliferated in response to stimulation with purified pp65, and antibodies specific for pp65 also were detected. Canarypox-CMV pp65 is the first recombinant vaccine to elicit CMV-specific CTL responses, which suggests the potential usefulness of this approach in preventing disease caused by CMV.

Cytotoxic T lymphocyte (CTL) responses to human cytomegalovirus pp65, IE1-Exon4, gB, pp150, and pp28 in healthy individuals: reevaluation of prevalence of IE1-specific CTLs.

The prevalence of human cytomegalovirus (HCMV) pp65-, pp150-, IE1-exon4-, gB- and pp28-specific cytotoxic T lymphocyte (CTL) responses was compared among 34 healthy individuals, grouped by neutralizing antibody titers. Moderately and highly seropositive donors showed predominantly pp65- and IE1-exon4-specific CTL responses (92% and 76% of the donors, respectively), with similar precursor frequencies in the 2 donors tested. In addition, highly seropositive and a few moderately seropositive donors showed CTL responses to gB and pp150 (33% and 30% of the donors, respectively). No individual recognized pp28 as a target in the CTL assay. Phenotypic analysis revealed a mixed effector population of CD4+ and CD8+ (1 donor) or only CD8+ cells for pp65-specific effectors (2 donors). IE1-exon4- and pp150-specific effectors were CD8+ (2 donors and 1 donor, respectively), whereas gB-specific CTLs were CD4+ (1 donor). These data may help to design a cellular immunity-based vaccine effective against HCMV diseases.

Roles of interleukin-12 and gamma interferon in murine Chlamydia pneumoniae infection.

BALB/c and strain 129 mice infected intranasally with Chlamydia pneumoniae displayed a moderate-to-severe inflammation in the lungs and produced interleukin-12 (IL-12), gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and IL-10, with peak levels on days 1 to 3 postinfection (p.i.), returning to basal levels by day 16 p.i. Anti-IL-12 treatment resulted in less-severe pathological changes but higher bacterial titers on days 3 and 7 p.i. By day 16 p.i., the inflammatory responses of control antibody-treated mice subsided. The bacterial titers of both anti-IL-12- and control antibody-treated mice decreased within 3 weeks to marginally detectable levels. Anti-IL-12 treatment significantly reduced lung IFN-gamma production and in vitro spleen cell IFN-gamma production in response to either C. pneumoniae or concanavalin A. In gamma-irradiated infected mice, cytokine production was delayed, and this delay correlated with high bacterial titers in the lungs. Following C. pneumoniae infection, 129 mice lacking the IFN-gamma receptor alpha chain gene (G129 mice) produced similar IL-12 levels and exhibited similarly severe pathological changes but had higher bacterial titers than 129 mice. However, by day 45 p.i., bacterial titers became undetectable in both wild-type 129 and G129 mice. Thus, during C. pneumoniae lung infection, IL-12, more than IFN-gamma, plays a role in pulmonary-cell infiltration. IFN-gamma and IL-12, acting mostly through its induction of IFN-gamma and Th1 responses, play an important role in controlling acute C. pneumoniae infection in the lungs, but eventually all mice control the infection to undetectable levels by IL-12- and IFN-gamma-independent mechanisms.

A canarypox vector expressing cytomegalovirus (CMV) glycoprotein B primes for antibody responses to a live attenuated CMV vaccine (Towne).

To develop a vaccine against cytomegalovirus (CMV), a canarypox virus (ALVAC) expressing CMV glycoprotein (gB) was evaluated alone or in combination with a live, attenuated CMV vaccine (Towne). Three doses of 106.5 TCID50 of ALVAC-CMV(gB) induced very low neutralizing or ELISA antibodies in most seronegative adults. However, to determine whether ALVAC-CMV(gB) could prime for antibody responses, 20 seronegative adults randomly received either 106.8 TCID50 of ALVAC-CMV(gB) or 106.8 TCID50 of ALVAC-RG, expressing the rabies glycoprotein, administered at 0 and 1 month, with all subjects receiving a dose of 103.5 pfu of the Towne vaccine at 90 days. For subjects primed with ALVAC-CMV(gB), neutralizing titers and ELISA antibodies to CMV(gB) developed sooner, were much higher, and persisted longer than for subjects primed with ALVAC-RG. All vaccines were well tolerated. These results demonstrate that ALVAC-CMV(gB) primes the immune system and suggest a combined-vaccine strategy to induce potentially protective levels of neutralizing antibodies.

Induction of human cytomegalovirus (HCMV)-glycoprotein B (gB)-specific neutralizing antibody and phosphoprotein 65 (pp65)-specific cytotoxic T lymphocyte responses by naked DNA immunization.

Plasmids expressing the human cytomegalovirus (HCMV) glycoprotein B (gB) (UL55) or phosphoprotein 65 (pp65) (UL83) were constructed and evaluated for their ability to induce immune responses in mice. The full-length gB as well as a truncated form expressing amino acids 1-680 of gB, and lacking the fragment encoding amino acids 681 907 including the transmembrane domain of gB (gB680) were evaluated. Immunization of mice with plasmids coding for gB or gB680 induced ELISA and neutralizing antibodies, with the highest titres in mice immunized with the gB680 plasmid. Mice immunized with the gB plasmid predominantly produced IgG2a gB-specific antibody, while the gB680 plasmid raised mostly IgG1 anti-gB antibody. Mice immunized with the pp65 plasmid developed pp65-specific cytotoxic T lymphocytes (CTL) and ELISA antibodies. Immunization with a mixture of both gB and pp65 plasmids raised antibodies to both proteins and pp65-specific CTL, indicating a lack of interference between these two plasmids. These results suggest that DNA immunization is a useful approach for vaccination against HCMV disease.

Combined effects of flavonoids and acyclovir against herpesviruses in cell cultures.

The combined antiviral effects of some flavonoid compounds and acycloguanosine (acyclovir, Zovirax) were studied on the multiplication of herpes simplex virus types 1 and 2 in HEp-2 cells and on pseudorabies (Aujeszky) virus in chick embryo fibroblast cells by the yield reduction method. The flavonoids quercetin, quercitrin (quercetin-3-L-rhamnoside) and apigenin exhibit antiviral activity against these herpesviruses, and acyclovir is currently one of the most effective antiherpetic agents. In these studies, the simultaneous application of flavonoids with acyclovir resulted in an enhanced antiviral activity. A mathematical formula was used to interpret the drug interaction, resulting in FIC (fractional inhibitory concentration) indices. Meaning a synergic interaction, all combinations exhibited synergy, FIC values of 0.6-0.8 being commonly observed.