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- 2Materials and methods
The aim of the study was to evaluate intracellular interferon-γ (IFN-γ), and interleukin-4 (IL-4) levels in pre- and post-treatment periods of brucellosis patients and to determine the relationship between these parameters and patients’ clinical findings. Twenty-five patients diagnosed as brucellosis and 11 aged-matched healthy volunteers were included in the study. CD3+CD4+ T lymphocytes levels were significantly lower in patients with brucellosis as compared to the control group. CD3+CD8+ T lymphocytes and CD3+IFN-γ+ levels were increased in brucellosis patients compared with the control group. CD4+IFN-γ+ and CD4+IL-4+ levels were no different between patients and healthy individuals. CD3+IL-4+ levels decreased in patients compared with healthy controls. Pre-treatment CD3+IFN-γ+ levels dramatically increased in patients responsive to management compared with the unresponsive ones. In responsive cases, CD3+IFN-γ+ levels decreased statistically after the treatment while in unresponsive cases no meaningful change was observed with respect to treatment. Adding IFN-γ to the treatment for improving the depleted levels of IFN-γ can be beneficial in patients with brucellosis who shows a tendency to chronicity or patients who do not respond to the treatment.
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- 2Materials and methods
Brucellosis is one of the most important zoonosis that affects human welfare and livestock health worldwide. The disease is caused by bacteria of the genus Brucellae comprised of different species that vary in their affinity and virulence to several hosts [1,2]. Brucella is a facultative, intracellular pathogen that can reside within phagocytic cells of the host and is apparently resistant to the normal mechanisms of bacterial killing .
The response against Brucellae spp. involves the whole gamut of the immune system, from innate to adaptive immunity. In addition to the macrophage, which plays a major role in Brucella infections, other cells of the innate immune response are recruited and influenced by the interactions between bacteria and host [4–6]. The cellular immune response is a critical part of the defense of the host against intracellular bacterial infections [7,8]. Critical aspects in this response include secretion of IL-12 and IFN-γ involving antigen presenting cells APCs and Th1 cells [4–6]. IFN-γ reduces bacterial multiplication inside infected autologous macrophages [5,8,9]. Th1 cells, which produce interferon (IFN-γ), IL-2 and tumor necrosis factor (TNF)-β, evoke cell-mediated immunity and phagocyte-dependent inflammation.
Th2 cytokine, IL-4, evokes strong antibody responses and eosinophil accumulation; but inhibits several functions of phagocytic cells. Both environmental and genetic factors act in concert to determine the Th1 or Th2 polarization [8,10–12]. Both CD4+ and CD8+ T cell populations contribute to the immune response to B. abortus producing IFN-γ and IL-2. B. abortus can induce differentiation of Th0 into Th1-type cell .
Although there is substantial information about the role of cytokines and T cells in murine brucellosis, knowledge of cellular immune responses in humans is scarce. In order to characterize the cytokine profile of Th-cell-mediated effector responses, different methodological approaches have been attempted such as cloning RT-PCR, in situ hybridization, immunocytochemistry, ELIspot and intracellular staining by flow cytometry methods. Intracellular staining by flow cytometry methods used to detect polarized Th1 or Th2 responses is relatively simple and highly sensitive. It allows detection of cytokine synthesis at single cell level. In the present study, we evaluated the intracellular interferon-γ (IFN-γ), and interleukin-4 (IL-4) levels and their alterations with treatment in patients with brucellosis.
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- 2Materials and methods
The mean ages were 36.40 ± 18.23 years and 37.09 ± 19.31 years in the patients and healthy individuals, respectively. The most common complaints of the patients were fever and sweating (Table 1).
Table 1. Complaints of the patients with brucellosis
|Lack of appetite (anorexia)||12||48|
Brucella species were isolated from the blood cultures in 15 cases (60%). All the Brucella species were identified as Brucella melitensis. No response was observed to the treatment in six of the cases. Data taken by flow cytometric analysis of the groups before and after treatment are presented in Table 2.
Table 2. Pre-treatment and post-treatment intracellular cytokines and T cell counts in the cases (n= 25) and comparison with the control group (n= 11)
|(Means of %± SD)||Acute brucellosis cases (n= 25)||Control group (n= 11)|
|CD3+||52.72 ± 10.7||52.27 ± 15.79||48.38 ± 6.6|
|CD3+CD4+||44.62 ± 13.4a||50.85 ± 10.55||67.41 ± 9.9|
|CD3+CD8+||33.97 ± 10.4b||29.54 ± 7.04||23.03 ± 6.3|
|CD3+IFN-γ+||61.41 ± 13.8c||57.77 ± 11.60||45.23 ± 13.8|
|CD3+IL-4+||2.51 ± 1.0d||2.65 ± 1.01||3.40 ± 1.3|
|CD4+IFN-γ+||16.37 ± 5.8||15.23 ± 6.10||16.44 ± 6.8|
|CD4+IL-4+||2.20 ± 0.9||2.27 ± 0.93||2.62 ± 1.1|
CD3+CD4+ T lymphocytes levels remarkably decreased (p < 0.0001), but CD3+CD8+ T lymphocytes levels increased in patients in comparison with the control groups (p < 0.005). CD3+IFN-γ+ levels elevated in patients compared with the control groups (p < 0.01), on the contrary, CD3+IL-4+ levels decreased in patients (p < 0.05). CD4+IFN-γ+ levels did not differ between patients and healthy individuals. CD4+IL-4+ levels were not significantly higher in healthy individuals compared to the patients before treatment.
Intracellular cytokines levels and T cell counts were measured in the patients who responded to the treatment for 45 day and these results were compared with the patients who had no response to the treatment. Pre-treatment CD3+IFN-γ+ levels were higher in responsive patients than the unresponsive group (p < 0.05). Pre-treatment CD4+IFN-γ+ levels were also higher in patients responsive to the treatment than the patients unresponsive to the treatment but statistically significant differences were not observed between them (Table 3, Figs. 1 and 2). In responsive cases, while CD3+IFN-γ+ levels decreased significantly, no significant change was observed in CD3+IL-4+ and CD4+IL-4+ levels. In unresponsive cases, no significant changes were observed in pre- and post-treatment CD3+IFN-γ+ levels (Table 3). CD3+IL-4+ (pre-treatment period), CD4+IL-4+ (pre- and post-treatment periods) levels in the patients with positive blood culture were lower than the levels of the patients with negative blood culture. Patients received three different regimens: six of them received doxycycline + streptomycin, 16 of them doxycycline + rifampin, and three of them received doxycycline + streptomycin + rifampin; there were no significant differences in the levels of intracellular cytokines according to these regimens.
Table 3. Pre-treatment and post-treatment intracellular cytokines and T cell counts responder in the cases (n= 19) and comparison with the non-responder (n= 6)
|(Means of %± SD)||Responder (n= 19)||Non-responder (n= 6)|
|CD3+||57.09 ± 8.87||57.41 ± 9.55||56.13 ± 3.69||58.09 ± 6.29|
|CD3+CD4+||45.28 ± 14.25||51.16 ± 10.34||44.39 ± 9.68||50.25 ± 14.14|
|CD3+CD8+||34.37 ± 9.07||32.54 ± 7.56||34.15 ± 7.38||31.15 ± 8.04|
|CD3+IFN-γ+||69.11 ± 10.28a||59.90 ± 12.99||45.05 ± 4.24||43.64 ± 6.57|
|CD3+IL-4+||2.65 ± 0.88||2.89 ± 1.05||2.5 ± 0.49||2.39 ± 0.92|
|CD4+IFN-γ+||17.42 ± 5.74||17.11 ± 6.57||13.35 ± 4.12||12.11 ± 4.75|
|CD4+IL-4+||2.35 ± 0.85||2.54 ± 1.06||2.32 ± 0.54||2.08 ± 0.71|
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- 2Materials and methods
Even if intracellular cytokine mRNA level is high, its active extracellular levels can be found to be low [14,15]. In this report, in order to evaluate the changes in production of cytokines, we assessed the intracellular cytokine levels in the disease.
Determining the complex cytokine profile during an infectious disease is important in the understanding of the pathogenesis, prognosis and treatment of disease . Facultative intracellular bacteria, including Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium leprae, Brucella abortus and Salmonella spp., survive within normal resident macrophages and other non-professional phagocytes. Th1-dominated immune responses predominantly produce a phagocyte-dependent inflammation. Th2 cells evoke strong antibody responses, including IgE.
The passive transfer of immune cells in BALB/c mice has shown that the protection against Brucella abortus is mediated by CD4+ T cells, and more recently, it has also been demonstrated that CD8+ T cells play a role in the resistance to Brucella infection . In this study, CD3+CD4+ T lymphocytes levels in patients were decreased but CD3+CD8+ T lymphocytes levels were increased.
Moreno-Lafont et al.  demonstrated that peripheral blood mononuclear cells from chronically ill patients with brucellosis proliferated in response to a sonicated bacterial suspension rich in internal antigens, whereas cells from patients with acute brucellosis did not. Patients with untreated acute brucellosis have a diminished proliferative and IFN-γ response to the polyclonal mitogens PHA [20,21]. However, Giambartolomei et al.  found that this response was specific to cytoplasmic protein (CP). In this study, it was shown that IFN-γ levels in patients with brucellosis were enhanced to PMA, despite some contradictory results in the literature [20,21]. The Th1-like immune response showed an increase of CD3+IFN-γ+ levels with reduced CD3+IL-4+ and CD4+IL-4+ levels. In a study, both CD3+CD4+ and CD3+CD8+ T cells produced IFN-γ in mice infected with Brucella abortus but CD3+CD8+ cells produced more IFN-γ than CD3+CD4+ cells . In our study, CD3+IFN-γ+ levels increased in patients with brucellosis in comparison with the control group. However, CD4+IFN-γ+ levels were not significantly different between patients and healthy individuals. We suggest that elevated levels of CD3+IFN-γ+ resulted from the increased numbers of CD3+CD8+ T lymphocytes in patients with brucellosis.
Gamma interferon (IFN-γ) produced by CD4+ and CD8+ T lymphocytes plays an important role in recovery from infection by these organisms [17,24]. Giambartolomei et al.  demonstrated that patients with acute brucellosis display a Th1 type response with cell proliferation and production of IFN-γ and IL-2, whereas the patients with the chronic form of the disease (7 of 11 non-responder patients) do not. Likewise, in this study, pre-treatment CD3+IFN-γ+ levels were statistically higher in persons who responded to the treatment than in non-responders. However, pre-treatment CD4+IFN-γ+ levels showed higher levels in responders than non-responding patients were but not statistically significant. The low-level synthesis of IFN-γ in non-responders could be responsible for a reduced cellular immune response. It has been reported that serum levels of IFN-γ dropped as a result of treatment . We observed that levels of intracellular CD3+IFN-γ+ significantly decreased at the end of the treatment in responder patients, whereas the levels of CD3+IL-4 and CD4+IL-4+ did not show any significant alteration.
During Brucella infection various cytokines such as IFN-γ, TNF-α, IL-2, IL-10, IL-12 control intracellular multiplication of Brucella in macrophages. IFN-γ is the most relevant for generating macrophages with strong activity for killing intracellular Brucella. Furthermore, cytokines such as IL-2, IL-10 and IL-12 influence the acquired cellular resistance and specifically contribute to the control of Brucella multiplication. These cytokines seem to act via the IFN-γ-dependent pathway . The Th1/Th2 paradigm not only allows a better understanding of the fundamental mechanisms involved in the protection against infectious agents and in the pathogenesis of several immunopathological disorders, but also provides the basis for the development of novel therapeutic strategies.