Maja Abram

Salivary levels of tumor necrosis factor-alpha in oral lichen planus.

OBJECTIVE: Oral lichen planus (OLP) is chronic inflammatory disease of the oral mucosa, presenting in various clinical forms. The etiology of OLP is still unknown but mounting evidence points to the immunologic basis of this disorder. AIM: Our study was undertaken to quantify the salivary levels of pro-inflammatory tumor necrosis factor-alpha (TNF-alpha) in the reticular and the erosive/atrophic forms of OLP, compared with age-matched healthy control volunteers. SUBJECTS AND METHODS: Whole saliva from 40 patients with active lesions of OLP, as well as from 20 healthy persons, was investigated for the presence of TNF-alpha by enzyme immunoassay. RESULTS: Salivary TNF-alpha levels were significantly increased in patients with OLP in comparison with healthy subjects. The presence of TNF-alpha showed positive correlation to clinical forms of OLP, being significantly higher in the erosive/atrophic type than in the reticular type of disease. CONCLUSION: Saliva provides an ideal medium for the detection of pro-inflammatory markers of the oral cavity. In patients with OLP, TNF-alpha levels in saliva are elevated, correlating with the severity of illness. Salivary TNF-alpha analysis may be a useful diagnostic tool and a potential prognostic marker in OLP.

Murine model of pregnancy‐associated Listeria monocytogenes infection

Listeria monocytogenes has been recognized as a significant pathogen, occurring worldwide, capable of causing animal and human infections. In its most severe form, listeriosis is an invasive disease that affects immunocompromised patients. Additionally, pregnant women represent a high-risk group for L. monocytogenes infection. Abortion, stillbirth or severe neonatal infection can be the serious outcome of such an infection. In an experimental murine model of pregnancy-associated listeriosis we studied the impact of L. monocytogenes on the maternal immune response and pregnancy outcome. In comparison to virgin animals, pregnant mice mounted lower levels of protective cytokines and were unable to eliminate the pathogen. The impaired maternal immune response that has been found both on the systemic and local level, facilitated bacterial multiplication in the liver, placenta and ultimately in the fetal tissues. This resulted in severe necrotizing hemorrhagic hepatitis and Listeria-induced placental necrosis, increasing the incidence of postimplantation loss and poor pregnancy outcome.

Stress response and pathogenic potential of Campylobacter jejuni cells exposed to starvation

Campylobacter jejuni is a Gram-negative, fragile, spiral bacterium, known worldwide to be a major cause of acute human enteritis. Like many other food-borne bacteria, campylobacters must be able to survive under diverse conditions both inside the host and in the environment. Understanding stress response mechanisms provides information necessary for improving food processing and strategies that enhance food safety as well as clarifying the pathogenesis of campylobacteriosis. We investigated the relation between stress response to starvation and pathogenic potential in C. jejuni. Starvation changed the morphology and physiology of C. jejuni cells. However, the lower metabolic activity of 5-h-starved culture was not a dormant state, but probably a viable but non-culturable (VBNC) form of the cells, since starved C. jejuni induced heat stress resistance. The health hazard potential of starved cells is still unclear. We showed that, in spite of starvation, C. jejuni survived in vitro within Caco-2 enterocites up to 4 days and caused systemic campylobacteriosis in vivo in a mouse model. However, bacterial numbers in investigated organs were significantly lower and the infection was resolved sooner. Our results show that nutrient insufficiency is responsible for C. jejuni transformation, influencing but not abolishing its survival and virulence properties while in the VBNC state.

Plasma cytokine response in mice with bacterial infection

BACKGROUND: Exposure to microorganisms elicts the production of cytokines. These soluble factors enhance several innate immune functions and regulate the ensuing specific immune response aimed at limiting the spread of infection. AIM: This study was undertaken to quantify the plasma levels of pro-inflammatory cytokines during the course of primary Listeria monocytogenes and Campylobacter jejuni infection. Using an in vivo infection the relationship between endogenous cytokines and the bacterial number in the liver of infected animals was examined. METHODS: C57BL/6 mice were infected by the intraperitoneal route. At different time points we determined the number of colony-forming units of bacteria in the liver of infected animals and paralled these with the plasma levels of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) measured by enzyme immunoassays. RESULTS: L. monocytogenes infection lasted 10-11 days. IFN-gamma production occurred in the early phase but was more pronounced after day 4, following the appearance of specific immunity. The duration of experimental campylobacteriosis was 15 days. Early IFN-gamma production was not significant but a progressive rise of this cytokine in plasma was seen during the second week post infection. Mice produced measurable amounts of plasma TNF-alpha immediately after being given viable L. monocytogenes, peaking on day 2-3 when the greatest number of bacteria was present in the examined organs. During C. jejuni infection plasma TNF-alpha was produced in a similar manner, but the highest concentrations were found a few days later than in listeriosis, in correlation with the different course of campylobacteriosis. The quantity of IL-6 increased and decreased in concordance with clearance of L monocytogenes and the clinical status of the animals. C. jejuni did not promote the induction of this cytokine. This is to some extent an unusual finding. With respect to the role of IL-6 in Th2 responses and antibody production, the appearance of this cytokine in campylobacteriosis was more expected. DISCUSSION: During systemic bacterial infection, a network of pro-inflammatory cytokines is activated and blood levels of these cytokines are elevated, albeit inconsistently, with large individual variations and depending on microbial characteristics and structure.

Survival of stress exposed Campylobacter jejuni in the murine macrophage J774 cell line

Although campylobacters are relatively fragile and sensitive to environmental stresses, Campylobacter jejuni has evolved mechanisms for survival in diverse environments, both inside and outside the host. Their survival properties and pathogenic potential were assessed after subjecting food and clinical C. jejuni isolates to different stress conditions. After exposure to starvation (5 h and 15 h of nutrient depletion), a temperature shock (3 min at 55 degrees C) or oxidative stress (5 h and 15 h of atmospheric oxygen) we studied the culturability, viability and capability of adhesion, internalization and survival within the in vitro cell culture model using J774 murine macrophages. Starvation severely impaired C. jejuni culturability, particularly after 15 h of nutrient depletion. The number of viable cells decreased by 30-40%. Starved bacterial cells also showed a lower capability of adhesion, internalization and survival within macrophages. Despite the reduced culturability and viability of the heat treated cells, C. jejuni efficiently adhered to, and entered murine macrophages. However, the number of heat treated cells started to decrease more quickly than non-stressed cells. Within 24 h post infection all the cells were killed. The bacterial mechanisms involved in inactivating toxic oxygen products may enhance bacterial persistence through increased binding, entry and survival of both oxidatively stressed C. jejuni isolates inside the macrophages. Oxygen exposure increased the internalization and intracellular survival, although the cells cannot remain viable for extended periods within murine macrophages. However, any prolongation of survival in macrophages may increase the probability of transmission of bacteria in the host organism and have further implications in the pathogenesis of campylobacteriosis. This indicates that environmental stress conditions may be involved.

Primary Listeria monocytogenes infection in gestating mice.

The facultative intracellular Gram-positive bacteriumListeria monocytogenes is a food-borne pathogen of frequently underestimated importance. Pregnant women represent the high-risk group forL. monocytogenes infection. Abortion, stillbirth or neonatal infection can be the serious outcome of such an infection. Recovery from listeriosis, resistance mechanisms of the host and the effect ofL. monocytogenes on fetal development still remain to be fully understood. The results of our experiments showed an increased susceptibility of gestating BALB/c mice to primaryL. monocytogenes infection. The duration of listeriosis in gestating animals was almost twice longer than in the control group. Furthermore, it was clearly shown that the detrimental effect ofL. monocytogenes on fetal development was more pronounced if the infection was acquired earlier during gestation.

Superior induction and maintenance of protective CD8 T cells in mice infected with mouse cytomegalovirus vector expressing RAE-1γ

Significance There are a number of pathogens for which the immunity acquired postinfection does not fully protect against reinfection and disease. Therefore, vaccines offering superior protection compared with the protection following natural infection are needed. Due to a unique pattern of immune response induced by cytomegaloviruses (CMVs), live attenuated CMVs are attractive candidates for vaccine vectors. Here we have demonstrated that a recombinant CMV vector expressing RAE-1γ, a cellular ligand for activating NKG2D receptor expressed on several types of immune cells, has tremendous potential for subverting viral immunoevasion and enhancing the efficiency of the CD8 T-cell response against vectored antigens. This study demonstrates a significant new approach in designing T-cell–based vaccine vectors. Due to a unique pattern of CD8 T-cell response induced by cytomegaloviruses (CMVs), live attenuated CMVs are attractive candidates for vaccine vectors for a number of clinically relevant infections and tumors. NKG2D is one of the most important activating NK cell receptors that plays a role in costimulation of CD8 T cells. Here we demonstrate that the expression of CD8 T-cell epitope of Listeria monocytogenes by a recombinant mouse CMV (MCMV) expressing the NKG2D ligand retinoic acid early-inducible protein 1-gamma (RAE-1γ) dramatically enhanced the effectiveness and longevity of epitope-specific CD8 T-cell response and conferred protection against a subsequent challenge infection with Listeria monocytogenes. Unexpectedly, the attenuated growth in vivo of the CMV vector expressing RAE-1γ and its capacity to enhance specific CD8 T-cell response were preserved even in mice lacking NKG2D, implying additional immune function for RAE-1γ beyond engagement of NKG2D. Thus, vectors expressing RAE-1γ represent a promising approach in the development of CD8 T-cell–based vaccines.

Sequence Types 235, 111, and 132 Predominate among Multidrug-Resistant Pseudomonas aeruginosa Clinical Isolates in Croatia

ABSTRACT A population analysis of 103 multidrug-resistant Pseudomonas aeruginosa isolates from Croatian hospitals was performed. Twelve sequence types (STs) were identified, with a predominance of international clones ST235 (serotype O11 [41%]), ST111 (serotype O12 [15%]), and ST132 (serotype O6 [11%]). Overexpression of the natural AmpC cephalosporinase was common (42%), but only a few ST235 or ST111 isolates produced VIM-1 or VIM-2 metallo-β-lactamases or PER-1 or GES-7 extended-spectrum β-lactamases.

In Vivo Modulation of Campylobacter jejuni Virulence in Response to Environmental Stress

Campylobacters have developed a number of mechanisms for responding to environmental conditions, although the different virulence properties of these cells following exposure to stress are still poorly understood. We analyzed in vitro stress responses and the consequent in vivo modulation of Campylobacter jejuni pathogenicity in BALB/c mice, as a result of the exposure of the C. jejuni to environmental stress (starvation, oxidative stress, heat shock). In vitro, the influence of starvation and oxidative stress was milder than that of heat shock, although the majority of the stress conditions influenced the survival of C. jejuni. During starvation, C. jejuni viability was maintained longer than its culturability. Additionally, starvation elicited transformation of stressed bacteria to coccoid forms. In contrast, bacteria exposed to oxygen remained culturable, but their viability decreased. Pre-starvation did not contribute to improved survival of C. jejuni cells during oxygen exposure. Changes in bacteria numbers and the levels of several cytokines (interleukins 6 and 10, tumor necrosis factor-α, interferon-γ) were followed in vivo, in liver homogenates from the mice intravenously infected with either control (untreated) or stressed C. jejuni. The systemic infection with the control or stressed C. jejuni occurred with different production dynamics of the cytokines investigated. Starvation was the most powerful stress factor, which significantly decreased infectious potential of C. jejuni during the first 3 days postinfection. The most pronounced differences in cytokine production were found in interferon-γ and interleukin-10 production, which indicates that these have roles in the immune response to C. jejuni infection. These in vivo studies of environmental impact on bacterial virulence reveal that microbial adaptation during stress challenge is crucial not just for pathogen survival out of the host, but also during host-pathogen interactions, and thus for the bacterial pathogenicity.

Systemic and local CC chemokines production in a murine model of Listeria monocytogenes infection.

Repeated intragastric inoculation of Listeria monocytogenes into BALB/c mice resulted in prolonged bacteraemia and severe hepatic infection. Bacteria could also be isolated from the brain tissue of all experimental mice. During the inflammatory process, chemokine concentrations typically increased at the local site in comparison to the systemic level. The liver-to-serum ratio was more pronounced in the case of macrophage inflammatory protein 1α (MIP-1α), suggesting its role in the inflammatory response in the liver. The ratio of brain-to-serum concentration of monocyte chemoattractant protein 1 (MCP-1) remained the same as in the control animals, while it was lower in the infected mice, both in the case MIP-1α and in the case of regulated on activation, normal T cell expressed and secreted (RANTES). This is in correlation with slight inflammatory infiltrates found in the brain tissue early in infection.