Inflammatory- and immune responses in relation to bacterial replication in mice following re-infections with Chlamydophila pneumoniae.

OBJECTIVE: Investigation of chronic infections with Chlamydophila pneumoniae. METHODS: BALB/c mice were repeatedly infected with C. pneumoniae and tested during a 1-year period. Production of histamine, IFN-gamma, IL-6 and antibodies was monitored by ELISA. Live bacteria were cultured and DNA was detected by PCR. Cellular immunity was tested by ELISPOT. RESULTS: After re-infections, culture positivity and persistence of DNA in lungs and blood were shorter. Detection of DNA at late time points indicated persistent infection in a few mice. Histamine was produced after primary and re-infections, and the level correlated with the number of viable bacteria in lung. IFN-gamma, IL-6 levels, IgG2/IgG1 ratio, IgA titres, and level of chlamydial heat-shock protein antibodies were higher after re-infections. IgM antibodies were demonstrated even after re-infections. High number of IFN-gamma-producing splenocytes was observed after the third inoculation. CONCLUSION: These results promote an understanding of the patho- and immune mechanisms after C. pneumoniae re-infections.

Chronic infections and histamine, CRP and IL-6 levels after percutaneous transluminal coronary angioplasty.

OBJECTIVE AND DESIGN: Our hypothesis was that percutaneous transluminal coronary angioplasty (PTCA) reactivates certain pathogens that contribute to inflammatory processes after the intervention. SUBJECTS: We determined the levels of antibodies to human Hsp60 and levels of histamine, CRP and IL-6 in sera from 28 patients of unstable angina prior to and on days 4 and 14 after PTCA. We compared the presence of Chlamydophila pneumoniae (Cpn) and human cytomegalovirus (HCMV) DNA in peripheral blood, and levels of antibodies to Cpn, HCMV, herpes simplex virus, Epstein-Barr virus and mycobacterial Hsp65 in the serum. RESULTS: Higher prevalence of Cpn and HCMV DNA was demonstrated after PTCA than before, but titers of antibodies to the pathogens did not increase. Levels of histamine, CRP and IL-6 were enhanced after PTCA. There was no association between the levels of histamine, CRP and IL-6 and the rate of pathogen DNA, or antibody titers to the pathogens, except an association between Cpn IgA and histamine levels before PTCA. CONCLUSIONS: Reactivation of Cpn and HCMV and inflammatory change characterized by increased levels of histamine, CRP and IL-6 following PTCA are suggested. An association might exist between Cpn IgA antibody and histamine levels in patients of unstable angina.

A soluble factor(s) released by MRC-5 cells early and late after human cytomegalovirus infection induces maturation of monocyte-derived dendritic cells.

A human cytomegalovirus (HCMV) strain passaged 10 times on MRC-5 human fibroblast cells failed to express immediate early (IE) antigens in immature dendritic cells (iDCs) after infection. However, both the early and the late HCMV conditioning medium, harvested from MRC-5 cells at 24 h or 7-9 days after infection, respectively, induced a higher ratio of DCs expressing maturation markers (CD40, CD83, CD86 and HLA-DR) on the surface of the cells. HCMV conditioning medium, ultracentrifuged to remove virus particles, exhibited a similarly enhanced expression of DC maturation markers. DCs treated with HCMV conditioning medium harvested late after infection increased the percentages of autologous CD4+ and CD8+ cells of seropositive donors to produce IFN-gamma and stimulated HCMV-specific lymphoproliferative responses. The early HCMC conditioning medium was also able to induce the functional maturation of DCs, as demonstrated by supplementing this medium with a Chlamydia pneumoniae antigen.

The interactions between human dendritic cells and microbes; possible clinical applications of dendritic cells.

The dendritic cells comprise several subsets that induce and regulate the immune responses against foreign and self-antigens, and that can therefore function as initiators of protective immunity and inducers of central or peripheral tolerance. The different subpopulations of dendritic cells interact with and also influence other cell populations of the immune system, such as T and B lymphocytes and natural killer cells. The factors that determine the given dendritic cell functions depend on the state of maturation and the local microenvironment. The interactions between dendritic cells and microorganisms are rather complex, but progress in the past few years has shed light on several aspects of these interactions. This review lays emphasis on the interactions between human dendritic cells, important components of the intima of arterial specimens at areas predisposed to atherosclerotic lesions, and Chlamydia pneumoniae and cytomegalovirus, the human pathogens most strongly implicated in the development of atherosclerosis. In addition, several examples of the potential clinical applications of dendritic cells are described.

Development of a cytomegalovirus vaccine: lessons from recent clinical trials.

Cytomegalovirus-caused diseases are preventable. We believe that both neutralising antibodies and cell-mediated immunity are necessary for prevention. Of the CMV proteins, gB and pp65 are the minimum requirements in a vaccine to induced neutralising antibodies and cytotoxic T-lymphocyte (CTL) responses. Immunisation with additional proteins, e.g., gH, gN for neutralising antibodies and IE1exon 4 and pp150 for CTL responses, would strengthen protective immune responses. Approaches to development of a safe and effective cytomegalovirus (CMV) vaccine for the prevention of CMV diseases include: a) a live attenuated vaccine (Towne strain); b) recombinant constructs of the attenuated Towne and the virulent Toledo CMV strains; c) subunit glycoprotein B (gB) adjuvanted with MF59 to induce neutralising antibodies; d) phosphoprotein 65 (pp65) peptide-based vaccines to induce (CTL) for use in therapeutic vaccination; e) canarypox-CMV recombinants, e.g., ALVAC-CMV(gB) and ALVAC-CMV (pp65) to induce neutralising antibodies and CTL responses, respectively; f) DNA plasmids containing the genes for gB and pp65; g) dense bodies containing the key antigens. The attenuated Towne strain, gB/MF59, ALVAC-CMV(gB) and ALVAC-CMV(pp65) approaches have already been tested in clinical trials. The Towne vaccine induced neutralising antibodies and cell-mediated immunity (including CTLs) mitigated CMV disease in seronegative renal transplant recipients and protected against a low-dose virulent CMV challenge in normal volunteers but did not prevent infection in mothers of children excreting CMV. Immunisation with gB/MF59 resulted in high levels of neutralising antibodies in seronegative subjects. ALVAC-CMV(gB) did not induce neutralising antibodies but primed the immune system to a Towne strain challenge, while ALVAC-CMV(pp65) induced long-lasting CTL responses in all originally seronegative volunteers, with CTL precursor frequency similar to naturally seropositive individuals. These results suggest that CMV diseases can be prevented or attenuated and that a vaccine combining ALVAC-CMV(pp65) with gB/MF59 may induce sufficient CTLs and neutralising antibodies to protect against CMV diseases. Meanwhile, other approaches such as DNA peptide and dense body vaccines, should enter Phase I trials. All candidate vaccines will have to demonstrate that immunogenicity provides protection. Combined vaccines containing canarypox (ALVAC) vectors to express CMV-pp65 to induce CTLs and of subunit gB, given together with an appropriate adjuvant to induce neutralising antibodies, should be tested in a target population for the prevention of CMV infection and disease.

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.

Chlamydia pneumoniae in atherosclerotic middle cerebral artery.

BACKGROUND AND PURPOSE: Atherosclerotic middle cerebral arteries are frequent sites of thrombosis, leading to stroke. Previous studies have suggested a role for Chlamydia pneumoniae in the pathogenesis of atherosclerosis. However, the presence of this pathogen in atherosclerotic middle cerebral arteries has heretofore not been documented. In the present study, we analyzed atheromatous plaques from middle cerebral arteries for the presence of C pneumoniae. METHODS: Atherosclerotic middle cerebral arteries from 15 cadavers who died of natural causes and corresponding nonatherosclerotic arteries from 4 otherwise healthy trauma victims were examined. Assays for C pneumoniae DNA were carried out by nested polymerase chain reaction (nPCR) specific for the C pneumoniae ompA gene. The presence of the bacterium was assessed by transmission electron microscopy. RESULTS: Five of the 15 atherosclerotic arterial samples and none of the control tissues were positive for C pneumoniae by nPCR. Particles similar in morphology and size to C pneumoniae elementary bodies were detected by transmission electron microscopy in 4 of the 5 nPCR-positive atherosclerotic samples. CONCLUSIONS: The demonstration of C pneumoniae in atherosclerotic middle cerebral arteries is consistent with the hypothesis that this bacterium is involved in acute and chronic cerebrovascular diseases.

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.

Independent and joint effects of antibodies to human heat-shock protein 60 and Chlamydia pneumoniae infection in the development of coronary atherosclerosis.

BACKGROUND: Studies have suggested that the prevalence of antibodies against heat-shock proteins (HSPs), Chlamydia pneumoniae (CPN), and cytomegalovirus (CMV) is associated with coronary artery disease (CAD), but the independent or joint effects of human (h) HSP60 antibodies and these pathogens in patients have not been fully elucidated. METHODS AND RESULTS: A total of 405 subjects (276 patients with CAD and 129 control individuals) were tested for serum antibodies to hHSP60, CPN, and CMV immediate-early-1 (IE1) antigens. Patients were also assessed for serum cholesterol, triglyceride levels, and smoking habit. Significantly elevated levels of antibodies to hHSP60 and CPN but not to CMV-IE1 antigens were documented in CAD patients. Multiple logistic regression analysis and subanalyses of selected subjects showed that these associations were independent of age, sex, smoking, and serum lipid levels. Antibodies to hHSP60 and CPN did not correlate quantitatively; however, the relative risk of disease development was substantially increased in subjects with high antibody levels to both hHSP60 and CPN:, reaching an odds ratio of 82.0 (95% CI 10.6 to 625.0). CONCLUSIONS: High levels of antibodies to hHSP60 and CPN: are independent risk factors for coronary atherosclerosis, but their simultaneous presence substantially increases the risk for disease development.

Chronic infections and atherosclerosis.

The inability of traditional risk factors such as hypercholesterolemia, hypertension, and smoking to explain the incidence of atherosclerosis (AT) in about 50% of the cases prompted a search for additional putative risk factors involved in the development of the disease. Infectious agents have long been suspected to initiate/contribute to the process of AT. It has also been suggested that inflammation, either related to infectious agents or independent from infection, may mediate the atherogenic process [1, 2].

Chlamydia pneumoniae inhibits apoptosis in human peripheral blood mononuclear cells through induction of IL-10.

Chlamydia pneumoniae is a common cause of pulmonary infection, with serum positivity in at least 50% of the general population. In this study, we report that human PBMCs exposed to C. pneumoniae are resistant to apoptosis induced by the potent photoactivated chemotherapeutic agents 8-methoxypsoralen and hypericin. In contrast, PBMCs treated with a heat-inactivated inoculum exhibit normal susceptibility to apoptosis. We also observed that human PBMCs are responsive to C. pneumoniae infection by secretion of key immune regulatory cytokines, including IL-12 and IL-10. While IL-12 may play an important role in limiting C. pneumoniae proliferation within cells, IL-10 serves an anti-inflammatory function by down-regulating proinflammatory cytokines such as IL-12 and TNF-alpha. Depletion of endogenous IL-10, but not of IL-12, abolished the apoptosis resistance of C. pneumoniae-infected PBMCs. Furthermore, addition of exogenous IL-10, but not IL-12, significantly increased the resistance of control inoculum-treated PBMCs to photoactivated 8-methoxypsoralen- and hypericin-induced apoptosis. Therefore, we conclude that C. pneumoniae possesses an antiapoptotic mechanism. The resistance to apoptosis observed in PBMCs exposed to C. pneumoniae is due, at least partially, to the IL-10 induced during C. pneumoniae infection.

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.

Involvement of a p53-dependent pathway in rubella virus-induced apoptosis.

In light of the important role of apoptotic cell death in the pathogenesis of several viral infections, we asked whether the cytopathogenicity evoked by rubella virus (RV) might also involve apoptotic mechanisms. The To-336 strain of RV induced apoptosis in Vero and RK-13 cells, but not in fibroblast cell lines. UV-inactivated RV virions did not elicit the apoptotic response, indicating that productive infection is required for the induction of cell death. Both p53 and p21 protein levels were highly elevated in RV-infected Vero cells. The level of p21 mRNA was increased, while expression of the p53 gene was unaffected by RV infection. A dominant-negative p53 mutant (p53(W248)) conferred partial protection from RV-induced apoptosis. These data implicate a p53-dependent apoptotic pathway in the cytopathogenicity of RV, thereby suggesting a mechanism by which RV exerts its teratogenic effects.

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.

Early atherosclerotic plaques in the aorta following cytomegalovirus infection of mice.

We show here that BALB/c mice inoculated with murine cytomegalovirus (MCMV) express viral antigens in the endothelial and smooth muscle cells of the aortic wall, and that accumulation of inflammatory cells in the aortic lumen, similar to that seen in early atherosclerotic lesions in humans, colocalizes with the site of virus antigen expression. Immunosuppression of the mice at the time of virus infection increased the expression of viral antigens and the size of early atherosclerotic lesions in the intima. The percentage of the low-density lipoprotein cholesterol (LDL-C), the major lipid contributor to atherosclerotic plaques, was significantly increased in the serum of MCMV-infected mice, whether or not the mice were fed a high cholesterol diet. Human cytomegalovirus (HCMV) significantly increased the esterified cholesterol component of the total cholesterol in a human arterial smooth muscle cell line infected in vitro with HCMV. These results suggest that CMV infection is involved in two of the major mechanisms that lead to development of atherosclerosis, i.e., immune injury and high LDL-C.

Application of near infrared spectroscopy to the determination of haemoglobin.

Seventy-two whole blood samples were investigated to determine the relationship between their spectral data measured in the near infrared (NIR) wavelength region and haemoglobin content based on laboratory data determined by a routine standard method as reference. Blood samples were obtained from the 1st Department of Medicine, Imre Haynal University of Health Sciences. Donors were selected randomly without respect to age, sex, state of health or medical treatment, from apparently healthy volunteers as well as from ambulatory and hospitalized patients. NIR spectra were measured with a SPECTRALYZER 1025 (PMC) computerized spectrophotometer in the 1000-2500 nm wavelength region. The relationship between laboratory data and values of the second derivative (i.e. second order finite difference) of the log(1/TF) spectra measured at different wavelengths was determined by multiple linear regression (MLR) using three- and four-term linear summation equations. The cross-validated standard error of performance (SEP) for haemoglobin was 1.348 g dL-1 with a three term model and 1.251 g dL-1 with a four term model over the range from 5.9 to 20 g dL-1. This preliminary study indicates that NIR measurements can be directly related to haemoglobin content and can be used to determine haemoglobin content in human whole blood.

Human colorectal cancer (CRC) antigen CO17-1A/GA733 encoded by adenovirus inhibits growth of established CRC cells in mice.

The human colorectal carcinoma (CRC)-associated Ag CO17-1A/GA733, originally defined by mAbs CO17-1A and GA733, has been a useful target in passive immunotherapy of CRC patients with mAb and in active immunotherapy with anti-idiotypic Abs mimicking the CO17-1A or GA733 epitope. Both approaches have targeted single epitopes. We investigated the capacity of full-length CO17-1A/GA733 Ag expressing multiple potentially immunogenic epitopes and encoded by recombinant adenovirus 5 (Ad5 GA733-2) to induce humoral, cellular, and/or protective immunity in mice. Ad5 GA733-2 induced Ag-specific Abs that reacted predominantly to CO17-1A- and GA733-unrelated epitopes on the Ag and lysed Ag-positive CRC targets in conjunction with effector cells. Ad5 GA733-2-immune mice developed Ag-specific, proliferative lymphocytes of Th1 type and cytolytic lymphocytes. The use of Ad5 GA733-2 to immunize mice bearing established syngeneic CRC cells transfected with the human Ag induced significant and specific tumor regression. Cured mice resisted rechallenge with human CO17-1A/GA733 Ag-negative parental CRC cells, suggesting that targeting the human Ag on the murine transfectants induced protective immunity to other Ag expressed by the parental tumor. These results may explain the high potency of the recombinant vaccine. Thus, rAd5 GA733-2 may have potential as a vaccine for CRC patients.