Mucosal immunization with an unadjuvanted vaccine that targets Streptococcus pneumoniae PspA to human Fcγ receptor type I protects against pneumococcal infection through complement- and lactoferrin-mediated bactericidal activity.

Targeting an antigen to Fc receptors (FcR) can enhance the immune response to the antigen in the absence of adjuvant. Furthermore, we recently demonstrated that intranasal immunization with an FcγR-targeted antigen enhances protection against a category A intracellular mucosal pathogen, Francisella tularensis. To determine if a similar strategy could be applied to the important pathogen Streptococcus pneumoniae, we used an improved mucosal FcR-targeting strategy that specifically targets human FcγR type I (hFcγRI). A humanized single-chain antibody component in which the variable domain binds to hFcγRI [anti-hFcγRI (H22)] was linked in a fusion protein with the pneumococcal surface protein A (PspA). PspA is known to elicit protection against pneumococcal sepsis, carriage, and pneumonia in mouse models when administered with adjuvants. Anti-hFcγRI-PspA or recombinant PspA (rPspA) alone was used to intranasally immunize wild-type (WT) and hFcγRI transgenic (Tg) mice in the absence of adjuvant. The hFcγRI Tg mice receiving anti-hFcγRI-PspA exhibited elevated S. pneumoniae-specific IgA, IgG2c, and IgG1 antibodies in serum and bronchoalveolar lavage fluid. Neither immunogen was effective in protecting WT mice in the absence of adjuvant, but when PspA was targeted to hFcγRI as the anti-hFcγRI-PspA fusion, enhanced protection against lethal S. pneumoniae challenge was observed in the hFcγRI Tg mice compared to mice given nontargeted rPspA alone. Immune sera from the anti-hFcγRI-PspA-immunized Tg mice showed enhanced complement C3 deposition on bacterial surfaces, and protection was dependent upon an active complement system. Immune serum also showed an enhanced bactericidal activity directed against S. pneumoniae that appears to be lactoferrin mediated.

Natural killer and CD8 T cells dominate the response by human peripheral blood mononuclear cells to inactivated Francisella tularensis live vaccine strain.

Francisella tularensis is a category A biothreat agent, and as a result, it has recently generated much research interest. F. tularensis live vaccine strain (LVS) is an attenuated form of the virulent F. tularensis organism and has previously been used as a vaccine. However, because of safety concerns, it is no longer approved for this purpose. Thus, the use of inactivated organisms is preferable for vaccine purposes. Although many studies have been performed that examine human peripheral blood mononuclear cells (PBMC), and in particular CD4 T cells, responses to inactivated F. tularensis, there has been no study identifying the individual human cell populations within a mixed PBMC population that respond to this organism. We sought to address this deficit. Our results indicate that natural killer and CD8 T cells comprise the majority of cells responding to F. tularensis LVS. In addition, data suggest CD8 T cell responses are maximal when antibiotic-treated organisms are used and are minimal when formaldehyde-fixed organisms are used. Given the belief that CD8 T cells can play an important role in protection against F. tularensis infection, these studies have direct relevance to the development of F. tularensis vaccines that use inactivated organisms. In addition, important new knowledge is added to our understanding of the human immune response to F. tularensis LVS.

Modulation of epithelial sodium channel (ENaC) expression in mouse lung infected with Pseudomonas aeruginosa.

BACKGROUND: The intratracheal instillation of Pseudomonas aeruginosa entrapped in agar beads in the mouse lung leads to chronic lung infection in susceptible mouse strains. As the infection generates a strong inflammatory response with some lung edema, we tested if it could modulate the expression of genes involved in lung liquid clearance, such as the alpha, beta and gamma subunits of the epithelial sodium channel (ENaC) and the catalytic subunit of Na+-K+-ATPase. METHODS: Pseudomonas aeruginosa entrapped in agar beads were instilled in the lung of resistant (BalB/c) and susceptible (DBA/2, C57BL/6 and A/J) mouse strains. The mRNA expression of ENaC and Na+-K+-ATPase subunits was tested in the lung by Northern blot following a 3 hours to 14 days infection. RESULTS: The infection of the different mouse strains evoked regulation of alpha and beta ENaC mRNA. Following Pseudomonas instillation, the expression of alphaENaC mRNA decreased to a median of 43% on days 3 and 7 after infection and was still decreased to a median of 45% 14 days after infection (p < 0.05). The relative expression of betaENaC mRNA was transiently increased to a median of 241%, 24 h post-infection before decreasing to a median of 43% and 54% of control on days 3 and 7 post-infection (p < 0.05). No significant modulation of gammaENaC mRNA was detected although the general pattern of expression of the subunit was similar to alpha and beta subunits. No modulation of alpha1Na+-K+-ATPase mRNA, the catalytic subunit of the sodium pump, was recorded. The distinctive expression profiles of the three subunits were not different, between the susceptible and resistant mouse strains. CONCLUSIONS: These results show that Pseudomonas infection, by modulating ENaC subunit expression, could influence edema formation and clearance in infected lungs.

Evaluation of risk factors associated with endometriosis.

OBJECTIVE: To investigate the association between several factors such as demographics, personal habits, reproductive factors, menstrual characteristics, contraception, and clinical profile and the probability of having endometriosis. SETTING: Ten clinical institutions in the Montreal area. PATIENT(S): A total of 2777 subjects who underwent surgery for diagnostic laparoscopy, tubal ligation, or hysterectomy between January 1998 and July 2002. Eight hundred ninety subjects were classified as cases on the basis of the presence of endometriotic lesions found at the time of surgery, whereas 1881 controls had no surgical evidence of endometriosis. MAIN OUTCOME MEASURE(S): The association between potential risk factors and endometriosis was estimated by Pearson chi(2) and by crude and adjusted odds ratios. RESULT(S): Results indicated a positive association between endometriosis and education level for subjects who underwent hysterectomy. An inverse relation between gravidity and endometriosis was also found within a subgroup of subjects who had diagnostic laparoscopy. Furthermore, the proportion of uterine leiomyoma was significantly higher in cases compared with in controls, among subjects who underwent surgery for diagnostic laparoscopy or hysterectomy but not for tubal ligation. CONCLUSION(S): These observations underline the importance of taking into account the clinical profile of subjects when investigating risk factors for endometriosis.

Blood leukocyte subsets are modulated in patients with endometriosis.

OBJECTIVE: To determine whether blood leukocyte populations could be modulated in endometriosis. DESIGN: Case-control study. SETTING: Eight clinical institutions of the Montreal area. PATIENT(S): Women with regular menstrual cycles who underwent laparoscopy or laparotomy between 1997 and 2001 and who were not under hormonal treatment for at least 3 months were selected. This study includes 175 cases and 131 controls. MAIN OUTCOME MEASURE(S): The proportion of blood leukocytes expressing markers for T, B lymphocytes, monocytes, or natural killer (NK) cells were compared by flow cytometric analysis between cases and controls. RESULT(S): Age and parity were identified as important confounders. Given that smoking, history of acute infection, and previous use of oral contraceptives strongly correlate with the level of some blood leukocyte populations, these parameters were taken into account in addition with age and parity when the level of blood leukocyte subsets were evaluated in cases and controls. Blood monocytes expressing CD14 and CD44 molecules were increased in patients with endometriosis. Alternatively, B lymphocytes were shown to be significantly decreased in cases compared with controls. CONCLUSION(S): Although these results suggest that endometriosis is associated with some systemic manifestations, the exact role of these modulations remains unclear.

Development of a nonsurgical diagnostic tool for endometriosis based on the detection of endometrial leukocyte subsets and serum CA-125 levels.

OBJECTIVE: To determine whether the proportion of several leukocyte subsets is modulated in the endometrium of patients with endometriosis and, if yes, whether it can be used for diagnostic purposes. DESIGN: Case-control study. SETTING: Eight clinical institutions of the Montreal area. PATIENT(S): Women who underwent laparoscopy or laparotomy between 1997 and 2001, who had regular menstrual cycles and were not under hormone treatment for the previous 3 months were selected. This study included 368 women, 173 with surgically confirmed endometriosis and 195 controls with no surgical evidence of endometriosis. MAIN OUTCOME MEASURE(S): Cytometry analysis was used to measure the proportion of several leukocyte subsets among CD45(+) endometrial cells. RESULT(S): The proportion of CD3(+), CD16(+), CD3(-)HLADR(-), CD3(-)CD45RA(-), CD3(+)CD16(-), CD3(+)CD56(-), CD56(-)CD16(+), and CD16b(+) leukocytes was significantly altered in the endometrium of cases compared with controls. A multiple logistic regression model was adjusted with these endometrial leukocytes, serum CA-125 levels, risk factors, and confounders. The diagnostic performance of this predictive model was defined by a specificity of 95% and a sensitivity of 61%. Furthermore, the positive and negative predictive values were 91% and 75%, respectively. CONCLUSION(S): This predictive model represents a novel diagnostic tool to identify women with a high likelihood of suffering from endometriosis.

Concentrations of alpha-fetoprotein, insulin-like growth factor binding protein-3, c-erbB-2, and epidermal growth factor in serum of patients with endometriosis.

OBJECTIVE: Endometriosis, although it is a benign disorder, shares many similarities with cancer. There is increasing levels of evidence suggesting that some circulating factors involved in gynecologic cancers, such as alpha-fetoprotein (AFP), insulin-like growth factor binding protein-3 (IGFBP-3), c-erbB-2 (HER-2/neu), and epidermal growth factor (EGF), could also play a role in endometriosis. Hence, the present study was aimed at evaluating whether the levels of these molecules are modulated in the serum of patients with endometriosis. METHODS: Levels of AFP, IGFBP-3, c-erbB-2, and EGF were determined by enzyme-linked immunosorbent assay in serum from 36 subjects with surgically confirmed endometriosis and 36 controls with no surgical evidence of the disease. In addition, information such as demographic characteristics, personal habits, menstrual characteristics, and clinical profile was collected from each participating subject. RESULTS: No significant difference was found between serum levels of AFP, IGFBP-3, c-erbB-2, and EGF in patients with endometriosis and controls, even when we adjusted for potential confounders and took into account the menstrual cycle. Moreover, no correlation was observed between the serum concentrations of these molecules and the stage of the disease. However, a correlation was detected between soluble levels of IGFBP-3 and presence of uterine leiomyoma. CONCLUSION: Although AFP, IGFBP-3, c-erbB-2, and EGF are not altered in the circulation of patients with endometriosis, their involvement in the development of endometriotic lesions cannot be excluded.

Utilization of Fc receptors as a mucosal vaccine strategy against an intracellular bacterium, Francisella tularensis.

Numerous studies have demonstrated that targeting Ag to Fc receptors (FcR) on APCs can enhance humoral and cellular immunity. However, studies are lacking that examine both the use of FcR-targeting in generating immune protection against infectious agents and the use of FcRs in the induction of mucosal immunity. Francisella tularensis is a category A intracellular mucosal pathogen. Thus, intense efforts are underway to develop a vaccine against this organism. We hypothesized that protection against mucosal infection with F. tularensis would be significantly enhanced by targeting inactivated F. tularensis live vaccine strain (iFt) to FcRs at mucosal sites, via intranasal immunization with mAb-iFt complexes. These studies demonstrate for the first time that: 1) FcR-targeted immunogen enhances immunogen-specific IgA production and protection against subsequent infection in an IgA-dependent manner, 2) FcgammaR and neonatal FcR are crucial to this protection, and 3) inactivated F. tularensis, when targeted to FcRs, enhances protection against the highly virulent SchuS4 strain of F. tularensis, a category A biothreat agent. In summary, these studies show for the first time the use of FcRs as a highly effective vaccination strategy against a highly virulent mucosal intracellular pathogen.

Enhanced antigen-specific antibody and cytokine responses when targeting antigen to human FcGAMMA receptor type I using an anti-human FcGAMMA receptor type I-streptavidin fusion protein in an adjuvant-free system.

There is a continuing need for alternatives to current human adjuvants. Recombinant protein vaccines, which target antigen to human Fc gamma receptor type I (hFcgammaRI) on hFcgammaRI-expressing antigen presenting cells, provide one potential alternative. Using a recombinant anti-hFcgammaRI-antigen fusion protein and adjuvant independent mouse model, we demonstrate enhanced antigen-specific antibody responses to low doses of antigen, when targeting antigen to hFcgammaRI in vivo. Enhanced antibody production to hFcyRI-targeted antigen is evident in both primary and secondary immune responses, as compared to that of non-targeted antigen. Furthermore, antibody isotype and cytokine responses following immunization with hFcgammaRI-targeted antigen, suggest enhancement of both Th1 and Th2 responses.

A pilot feasibility study of an extension of the acquaintanceship recruitment procedure in recent-onset psychosis.

The acquaintanceship recruitment procedure is an appealing yet infrequently used method to recruit controls, allowing a very close match between patients and controls. We used an extension of the acquaintanceship procedure to investigate the feasibility of this method to recruit controls in a neuropsychological study of recent-onset psychotic patients. Twenty-five recent-onset psychotic patients attending a multidisciplinary program devoted to recent-onset psychoses were contacted, among whom 13 agreed to participate to the study. At the end of the process, only four control participants were assessed. This pilot study suggests that several obstacles prevent the use of this procedure to recruit controls in this research focusing on recent-onset psychotic disorders.

Serum concentrations of insulin-like growth factor-1, soluble tumor necrosis factor receptor-1 and angiogenin in endometriosis patients.

PROBLEM: Many soluble factors contributing to the pathophysiology of endometriosis are found at abnormal levels in patients suffering from the disease. We postulated that levels of these factors could also be altered in the serum of patients. We compared levels of insulin-like growth factor-1 (IGF-1), soluble form of tumor necrosis factor-alpha (TNF-alpha) receptor-1 (sTNFR-1) and angiogenin in the serum of patients with endometriosis and controls. METHOD OF STUDY: Levels of IGF-1, sTNFR-1 and angiogenin were measured by enzyme-linked immunosorbent assay in samples from 148 patients (77 cases and 71 controls) with diagnostic confirmed by laparoscopy. Correlations with demographic data and stage of the disease were evaluated and potential confounders in the study population were controlled. RESULTS: A significant increase in sTNFR-1 and angiogenin serum levels was observed in cases in comparison with controls, but only for patients in the follicular phase of the cycle. No significant difference was found in serum levels of IGF-1, sTNFR-1 and angiogenin between cases and controls in the luteal phase of the cycle. Correlations between levels of angiogenin and stage of the disease could also be observed. CONCLUSION: sTNFR-1 and angiogenin represent potential blood markers for endometriosis.

Quantitative assessment of human endometriotic tissue maintenance and regression in a noninvasive mouse model of endometriosis.

Endometriosis is a prevalent disease characterized by the estrogen-dependent ectopic growth of endometrial tissue. Most of the current medical therapies consist in inducing a hypoestrogenic state in patients, but these treatments are associated with severe side effects and high recurrence rates. The development of convenient and reliable endometriosis animal models would be instrumental to accelerate the emergence of new therapeutic alternatives. Recently, we developed an improved experimental model for endometriosis, relying on the infection of human endometrial fragments by an adenovirus carrying the green fluorescent protein. Following injection of fluorescent fragments into nude mice, the implantation and growth of endometriotic-like lesions could be followed noninvasively. In the present work, we demonstrate that this model can be used to quantify the size of fluorescent endometriotic lesions by in vivo imaging. To this end, we repeatedly measured lesion size over a 4-week period in mice supplemented or not with estradiol. The model was adequate to confirm previous results showing that estrogen is dispensable for the implantation phase of endometrial tissue, whereas it is required for lesion maintenance. As a proof of concept for inducing regression of established lesions, ganciclovir was used to treat animals implanted with human fluorescent endometrial fragments expressing thymidine kinase. A significant decrease in lesion size was observed by in vivo imaging in ganciclovir-treated mice. Together, the data indicate that the noninvasive animal model described here provides a tool for drug testing and/or gene target validation in endometriosis.