Amal Halabi

TCR ζ Down-Regulation under Chronic Inflammation Is Mediated by Myeloid Suppressor Cells Differentially Distributed between Various Lymphatic Organs

T cell AgR ζ chain down-regulation associated with T cell dysfunction has been described in cancer, infectious, and autoimmune diseases. We have previously shown that chronic inflammation is mandatory for the induction of an immunosuppressive environment leading to this phenomenon. To identify the key immunosuppressive components, we used an in vivo mouse model exhibiting chronic inflammation-induced immunosuppression. Herein, we demonstrate that: 1) under chronic inflammation secondary lymphatic organs display various immunological milieus; ζ chain down-regulation and T cell dysfunction are induced in the spleen, peripheral blood, and bone marrow, but not in lymph nodes, correlating with elevated levels of Gr1+Mac-1+ myeloid suppressor cells (MSC); 2) MSC are responsible for the induction of such an immunosuppression under both normal and inflammatory conditions; and 3) normal T cells administered into mice exhibiting an immunosuppressive environment down-regulate their ζ expression. Such an environment is anticipated to limit the success of immunotherapeutic strategies based on vaccination and T cell transfer, which are currently under investigation for immunotherapy of cancer.

Mouse model of experimental periodontitis induced by Porphyromonas gingivalis/Fusobacterium nucleatum infection: bone loss and host response

AIM To compare the effect of oral infection with Porphyromonas gingivalis or Fusobacterium nucleatum versus infection with both bacteria on mouse periodontal tissues, and to characterize the inflammatory response. MATERIALS AND METHODS Mice were orally infected with P. gingivalis, F. nucleatum or both. At 42 days post-infection, alveolar bone loss was quantified using micro-computerized tomography. Tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta levels induced by the infection were quantified using the subcutaneous chamber model. RESULTS Mice orally infected with F. nucleatum/P. gingivalis exhibited significantly more bone loss compared with that of mono-infected and sham-infected mice. F. nucleatum/P. gingivalis infection also increased the levels of TNF-alpha and IL1beta compared with the levels found in the mono-infected groups. CONCLUSIONS Polymicrobial infection with P. gingivalis/F. nucleatum aggravates alveolar bone loss and induces a stronger inflammatory response compared with that observed upon infection with either bacterium alone. The results suggest that oral infection of mice with a mixture of P. gingivalis and F. nucleatum may be superior to mono-infection models of experimental periodontitis.

Eimeria maxima: identification of gametocyte protein antigens.

The antigenicity of Eimeria maxima gametocyte proteins during the course of an infection and when injected into mice and rabbits was demonstrated using the Western blotting technique. Serum taken from chickens at various times postinfection reacted to a few gametocyte proteins, with the strongest reactivity seen with serum taken 14-days postinfection. Two major antigens having molecular weights of 56,000 and 82,000 were consistently detected by these sera. Using immune rabbit or mouse sera to whole gametocyte detergent extracts, the 56,000 and 82,000 molecular weight proteins were again the immunodominant antigens, despite their representing only a small proportion of the extract which was used to immunize the animals. These results, together with those obtained by Rose (1971) using recovered chicken serum to passively immunize chickens, indicate that these two gametocyte antigens may play a role in protective immunity to E. maxima.

Behavior of two osteoblast-like cell lines cultured on machined or rough titanium surfaces.

BACKGROUND Two osteosarcoma-derived cell lines have been extensively used to investigate the biological events occurring on titanium surfaces: MG63 and Saos-2. However, the behavior of the two lines on different titanium surfaces has never been compared. AIM The aim of the present study was to compare the behavior of MG63 and Saos-2 cells on two different titanium surfaces, machined and rough (sandblasting and acid-etched). We compared cell proliferation and morphology, alkaline phosphatase (ALP) activity and secretion of osteocalcin (OC). RESULTS The most pronounced difference between the two cell lines was that ALP activity in the Saos-2 cells was 10-fold higher than in the MG63 cells. The proliferation rate of the MG63 cells was much higher than that of the Saos-2 cells at all the tested cell concentrations. MG-63 cells, but not Saos-2 cells, grown on rough surface titanium proliferated more rapidly than cells grown on machined surfaces. Morphological analysis revealed that Saos-2 cells and cells grown on the rougher surface, displayed a more mature phenotype. The level of OC secreted by the Saos-2 cells, but not the MG63 cells, were higher on the rough surface than on the machined surface. CONCLUSIONS This study shows that Saos-2 cells exhibit a more mature osteoblast phenotype, compared with that of MG63 cells, rendering them a good candidate for an in vitro model of osseointegration.

Strain‐dependent activation of the mouse immune response is correlated with Porphyromonas gingivalis‐induced experimental periodontitis

AIMS To evaluate the effect of oral infection with three Porphyromonas gingivalis strains on alveolar bone loss (ABL) and its correlation with the mouse immune response. MATERIALS AND METHODS Mice were orally infected with P. gingivalis strains 381, 33277 and 53977. After 42 days, maxillae were analysed for ABL using micro-computed tomography and the serum for anti-P.gingivalis IgG1 and IgG2a levels. The cytokine response to P. gingivalis was tested using the subcutaneous chamber model. RESULTS The P. gingivalis 53977-infected group showed the highest ABL, which was significantly different from all other groups (p<0.001). In addition, the humoral response to P. gingivalis 53977 was significantly lower than the response to P. gingivalis 381 and 33277 (p< or =0.01). The IgG2a/IgG1 ratio was higher in the P. gingivalis 33277-infected group (1.6) compared with the P. gingivalis 381-infected group (0.51). Four days post-infection, interleukin (IL)-1beta levels remained significantly higher in the P. gingivalis 53977-infected group only (1198.2+/-260.0, p<0.05), while IL-4 levels remained significantly higher in the P. gingivalis 381-infected group (265.8+/-131.6, p<0.05). CONCLUSIONS The high levels of ABL induced by P. gingivalis 53977 were inversely correlated with the humoral response to this bacterium. In addition, ABL was correlated with an elevated pro-inflammatory response.

Repeat bacterial challenge in a subcutaneous chamber model results in augmented tumour necrosis factor‐α and interferon‐γ response, and suppression of interleukin‐10

The present study compared the effect of a single or a repeat challenge with the Gram‐negative pathogen Porphyromonas gingivalis on the local inflammatory response within subcutaneous chamber model in mice. Subcutaneous chambers were implanted 2 weeks prior to the final challenge. The repeat‐challenge (REP) group received two intrachamber bacterial injections 14 days apart, while the single‐injection group (SIN) received only a single bacterial challenge. Injection of saline was used as the control. The cellular contents of the chamber exudates were used for differential cell counts, and the supernatants were analysed for tumour necrosis factor‐α (TNF‐α), interferon‐γ (IFN‐γ), and interleukin (IL)‐10 levels. Immunoglobulin G1 (IgG1) and IgG2a levels to P. gingivalis in the exudates were also determined. The results showed that the leucocyte counts increased significantly post‐challenge, and the REP group showed the highest number of lymphocytes and neutrophils. Both P. gingivalis‐challenged groups exhibited significant increase in TNF‐α and IL‐10 levels at day 1 post‐challenge. TNF‐α levels in the chamber exudate were threefold higher in the REP group compared with the SIN group on day 1 post‐challenge (P < 0·05). In contrast, IL‐10 levels were significantly lower in the REP group 1 day post‐challenge compared with the SIN group. The REP group had significantly higher levels of IFN‐γ at baseline, and this difference remained significant 1 day post‐challenge. Analysis of antibody levels to P. gingivalis showed that while the control and the SIN groups had no anti‐P. gingivalis IgG in the chamber exudate during the 7‐day study period, the REP group showed high anti‐P. gingivalis IgG levels. In addition, the titres of IgG2a were fivefold higher than the IgG1 titres. The results showed that a repeat local challenge with P. gingivalis augmented the proinflammatory cytokines TNF‐α and IFN‐γ, while inhibiting the accumulation of the anti‐inflammatory cytokine IL‐10. This shift towards a T helper 1 (Th1)‐dominant response was reflected in the relatively high anti‐P. gingivalis IgG2a titres in the local inflammatory environment 7 days post‐challenge.

EFFECTS OF TETRACYCLINES ON THE PATHOLOGIC ACTIVITY OF ENDOTOXIN: IN VITRO AND IN VIVO STUDIES

Lipopolysaccharide (LPS) is considered to be one of the major virulence factors of Gram-negative bacteria. Recently, tetracyclines (TTCs) were found to prevent the patho-physiological changes associated with LPS in vivo and the secretion of inflammatory mediators in vitro. However, the mechanism by which TTCs prevents LPS-induced pathology in vivo is still unclear. In order to shed light on that problem, we carried out in vitro and in vivo experiments. TTC inhibited the secretion of nitric oxide (NO) and TNFa from LPSstimulated macrophages and inhibited macrophage-induced thymocyte proliferation. However, TTC inhibited NO secretion with use of concentrations five-fold lower than those that inhibited TNFα secretion and thymocyte proliferation. The secretion of NO was inhibited by the addition of TTC to the cultures up to 6 hrs post-LPS stimulation. TTC inhibition of LPS-induced NO secretion was not reversed by the addition of recombinant TNFa, and TTC inhibition of LPS-induced TNFa secretion was not reversed by the addition of NO donor. These results suggest that the inhibition of TNFa by TTC is not the result of the inhibition of LPS-induced NO secretion or vice versa. In vivo experiments had shown that TTC prevented mortality in LPS-treated mice, but not in mice pre-sensitized with galactosamine prior to the LPS challenge. These results suggest that TTC activity in vivo is due not to the suppression of synthesis of inflammatory mediators but rather to the induction of acute phase-like response, which antagonizes the LPS-induced activity.

Reduced Expression of Gamma Interferon in Serum and Marked Lymphoid Depletion Induced by Porphyromonas gingivalis Increase Murine Morbidity and Mortality due to Cytomegalovirus Infection

ABSTRACT Porphyromonas gingivalis, a gram-negative anaerobe, is a major etiological agent of severe forms of periodontal disease. Although periodontal disease is considered a localized disease, accumulating evidence indicates that it may lead to a predisposition to a decline in immunocompetence. Human cytomegalovirus (CMV) commonly infects all human populations without producing significant clinical symptoms. Immunocompromised patients usually develop a primary or reactivated CMV infection, which is associated with high rates of morbidity and mortality. The aim of this study was to determine whether P. gingivalis increases animal susceptibility to CMV infection. Mice were inoculated with CMV and infected locally with P. gingivalis 3 days after the virus inoculation. Mortality rates were monitored, and traces of viral DNA and bacterial infection were detected systemically by using real-time PCR. Local and systemic cytokine secretion was measured, and histological sections were used to assess the pathological state of infected organs. P. gingivalis- and CMV-coinfected mice showed dramatically higher mortality rates than mice infected with P. gingivalis or CMV only. Although the organs of coinfected mice exhibited decreased viral titers, distinct necrosis and tissue damage were more evident in the livers and spleens of these mice than in those of mice infected with CMV only. Furthermore, systemic gamma interferon levels were decreased in coinfected mice, and marked lymphoid depletion was observed in their necrotic organs. In parallel control Escherichia coli-CMV coinfection experiments, the mortality and pathological results were the same as those found in mice infected with CMV only. Our results suggest a specific influence of P. gingivalis on the mouse immune response, causing increased susceptibility to CMV infection.

Inflammatory response to chlorhexidine, minocycline HCl and doxycycline HCl in an in vivo mouse model

AIM To examine the effect of locally delivered antimicrobial drugs on the inflammatory response in an in vivo mouse chamber model. MATERIAL AND METHODS Two weeks following chamber implantation, 24 BALB/c mice, in the experimental group, were given an intra-chamber challenge of heat-killed Porphyromonas gingivalis, followed immediately by injection of the specific antimicrobial drug: 2000 microg/ml chlorhexidine (CHX); 1500 microg/ml minocycline HCl;and 1500 microg/ml doxycycline HCl (concentrations achieved in the periodontal pocket with commercial controlled-release delivery systems). A second group of 24 animals received only the antimicrobial treatment without P. gingivalis challenge. Intra-chamber exudates were sampled at 2 and 24 h following the challenge, and leucocytes, TNFalpha, IFNgamma and IL-10 were evaluated. RESULTS At 2 h, minocycline HCl induced high levels of IL-10, TNFalpha and IFNgamma, while CHX reduced the levels of TNFalpha and IFNgamma. By 24 h, these responses were attenuated. Following bacterial challenge, the antibacterial agents attenuated the inflammatory process, each in its own fashion. CONCLUSIONS Antibacterial agents applied locally have the ability to induce an inflammatory response. They also modify the inflammatory response to P. gingivalis independent of their antimicrobial effect. CHX and doxycycline HCl appear to have the most marked anti-inflammatory effect.

Induction of tumor necrosis factor α and interleukin-1β in subcutaneously implanted chamber by lipopolysaccharide

Lipopolysaccharide (LPS) is the major component of the outermost membrane of Gram-negative bacteria and is considered to be one of the major virulence factors of these bacteria. While the effect of systemic injection of LPS is well characterized, the characterization of cytokine secretion in response to local injection of LPS is lacking. The present study was designed to determine the local production of tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) over a 4 day period following injection of LPS into subcutaneous implanted chambers in mice. Mice were challenged by a single or repeated injection of Salmonella typhosa LPS into the chambers. Chamber fluids were aspirated at different time intervals and were used for assessment of leukocyte and cytokine levels. A single injection of LPS was found to induce cell influx into the chamber which peaked after 4 h. TNFα and IL-1β levels increased rapidly, reaching their maximum levels within 4 h. After 24 h, TNFα levels declined markedly and were undetectable at 48 and 96 h. TNFα mRNA levels in the sedimented cells followed a similar pattern. In contrast, IL-1β showed a more gradual decrease with levels significantly different from baseline still being present 96 h post-LPS challenge. Four consecutive daily injections of LPS into the chambers resulted in undetectable levels of TNFα in the chamber fluid, while significant levels of IL-1β were detected. These levels were significantly higher than the levels of IL-1β in the chamber fluid 96 h after a single injection and approximately 60% of the levels measured 24 h after a single intra-chamber injection of LPS. The results emphasize the difference between single and repeated exposure to LPS in vivo, and suggest a role for TNFα in the initial phase of the local inflammatory response and for IL-1β in the later phase.