Concomitant Treatment with Doxycycline and Rifampicin in Balb/c Mice Infected with Brucella abortus 2308 Fails to Reduce Inflammation and Motor Disability.

Brucellosis is an infection widely distributed around the world, and in some countries it is considered a public health problem. Brucellosis causes insidious symptoms that make it difficult to diagnose. Infection can also trigger chronic pain and neuropsychiatric complications. Antibiotics are not always effective to eradicate infection, contributing to chronicity. We aimed to investigate the effects of antibiotic treatment on proinflammatory cytokines, neurotransmitters, corticosterone, and behavior in a murine model of infecrion of B. abortus strain 2308. Four study groups were created: (a) control; (b) antibiotic control; (c) infected with B. abortus 2308; and (d) infected and treated with rifampicin and doxycycline. We determined B. abortus 2308 colony-forming units (CFUs), the count of dendritic cells, and macrophages in the spleen; serum levels of cytokines and corticosterone; levels of serotonin, dopamine, epinephrine, and norepinephrine in the brain; and equilibrium, physical strength, anxiety, and hopelessness tests. The infected and treated mice group was compared with the control and infected mice to assess whether treatment is sufficient to recover neuroimmunoendocrine parameters. Our results showed that despite the treatment of brucellosis with rifampicin and doxycycline, antibiotic-treated mice showed a persistence of B. abortus 2308 CFUs, an increased count in macrophage number, and higher circulating levels of corticosterone. Furthermore, the levels of IL-12, IL-6, and TNF-α remained higher. We found a decrease in muscular strength and equilibrium concomitant to changes in neurotransmitters in the hippocampus, cerebellum, and frontal cortex. Our data suggest that the remaining bacterial load after antibiotic administration favors inflammatory, neurochemical, and behavioral alterations, partly explaining the widespread and paradoxical symptomatology experienced by patients with chronic brucellosis.

Imipramine Administration in Brucella abortus 2308-Infected Mice Restores Hippocampal Serotonin Levels, Muscle Strength, and Mood, and Decreases Spleen CFU Count.

Brucellosis infection causes non-specific symptoms such as fever, chills, sweating, headaches, myalgia, arthralgia, anorexia, fatigue, and mood disorders. In mouse models, it has been associated with increased levels of IL-6, TNF-α, and IFN-γ, a decrease in serotonin and dopamine levels within the hippocampus, induced loss of muscle strength and equilibrium, and increased anxiety and hopelessness. Imipramine (ImiP), a tricyclic antidepressant, is used to alleviate neuropathic pain. This study evaluated the effects of ImiP on Balb/c mice infected with Brucella abortus 2308 (Ba) at 14- and 28-days post-infection. Serum levels of six cytokines (IFN-γ, IL-6, TNF-α, IL-12, MCP-1. and IL-10) were assessed by FACS, while the number of bacteria in the spleen was measured via CFU. Serotonin levels in the hippocampus were analyzed via HPLC, and behavioral tests were conducted to assess strength, equilibrium, and mood. Our results showed that mice infected with Brucella abortus 2308 and treated with ImiP for six days (Im6Ba14) had significantly different outcomes compared to infected mice (Ba14) at day 14 post-infection. The mood was enhanced in the forced swimming test (FST) (p < 0.01), tail suspension test (TST) (p < 0.0001), and open-field test (p < 0.0001). Additionally, there was an increase in serotonin levels in the hippocampus (p < 0.001). Furthermore, there was an improvement in equilibrium (p < 0.0001) and muscle strength (p < 0.01). Lastly, there was a decrease in IL-6 levels (p < 0.05) and CFU count in the spleen (p < 0.0001). At 28 days, infected mice that received ImiP for 20 days (Im20Ba28) showed preservation of positive effects compared to infected mice (Ba28). These effects include the following: (1) improved FST (p < 0.0001) and TST (p < 0.0001); (2) better equilibrium (p < 0.0001) and muscle strength (p < 0.0001); (3) decreased IL-6 levels (p < 0.05); and (4) reduced CFU count in the spleen (p < 0.0001). These findings suggest the potential for ImiP to be used as an adjuvant treatment for the symptoms of brucellosis, which requires future studies.

Brucella abortus induces mast cell activation through TLR-2 and TLR-4.

The Gram-negative bacteria Brucella abortus is a major cause of brucellosis in animals and humans. The host innate immune response to B. abortus is mainly associated with phagocytic cells such as dendritic cells, neutrophils, and macrophages. However, as mast cells naturally reside in the main bacterial entry sites they may be involved in bacterial recognition. At present, little is known about the role of mast cells during B. abortus infection. The role of the innate immune receptors TLR2 and TLR4 in activation of mast cells by B. abortus (strain RB51) infection was analyzed in this study. The results showed that B. abortus did not induce mast cell degranulation, but did induce the synthesis of the cytokines IL-1ß, IL-6, TNF-α, CCL3, CCL4, and CCL5. Furthermore, B. abortus stimulated key cell signaling molecules involved in mast cell activation such as p38 and NF-κB. Blockade of the receptors TLR2 and TLR4 decreased TNF-α and IL-6 release by mast cells in response to B. abortus. Taken together, our results demonstrate that mast cells are activated by B. abortus and may play a role in inducing an inflammatory response during the initial phase of the infection.

Behavioral and Neurochemical Shifts at the Hippocampus and Frontal Cortex Are Associated to Peripheral Inflammation in Balb/c Mice Infected with Brucella abortus 2308.

Brucellosis is a zoonosis affecting 50,000,000 people annually. Most patients progress to a chronic phase of the disease in which neuropsychiatric symptoms upsurge. The biological processes underlying the progression of these symptoms are yet unclear. Peripheral inflammation mounted against Brucella may condition neurochemical shifts and hence unchained neuropsychiatric disorders. Our work aimed at establishing whether neurological, behavioral, and neurochemical disarrays are circumstantially linked to peripheral inflammation uprise secondary to Brucella abortus 2308 infections. We then evaluated, in control and Brucella-infected mice, skeletal muscle strength, movement coordination, and balance and motivation, as well as dopamine, epinephrine, norepinephrine, and serotonin availability in the cerebellum, frontal cortex, and hippocampus. Serum levels of proinflammatory cytokines and corticosterone in vehicle-injected and -infected mice were also estimated. All estimates were gathered at the infection acute and chronic phases. Our results showed that infected mice displayed motor disabilities, muscular weakness, and reduced motivation correlated with neurochemical and peripheral immunological disturbances that tended to decrease after 21 days of infection. The present observations support that disturbed peripheral inflammation and the related neurochemical disruption might lead to mood disorders in infected mice. Future experiments must be aimed at establishing causal links and to explore whether similar concepts might explain neurological and mood disorders in humans affected by brucellosis.

The histone deacetylase inhibitor valproic acid attenuates phospholipase Cγ2 and IgE-mediated mast cell activation.

Mast cell activation through the high-affinity IgE receptor (FcεRI) plays a central role in allergic reactions. FcεRI-mediated activation triggers multiple signaling pathways leading to degranulation and synthesis of different inflammatory mediators. IgE-mediated mast cell activation can be modulated by different molecules, including several drugs. Herein, we investigated the immunomodulatory activity of the histone deacetylase inhibitor valproic acid (VPA) on IgE-mediated mast cell activation. To this end, bone marrow-derived mast cells (BMMC) were sensitized with IgE and treated with VPA followed by FcεRI cross-linking. The results indicated that VPA reduced mast cell IgE-dependent degranulation and cytokine release. VPA also induced a significant reduction in the cell surface expression of FcεRI and CD117, but not other mast cell surface molecules. Interestingly, VPA treatment inhibited the phosphorylation of PLCγ2, a key signaling molecule involved in IgE-mediated degranulation and cytokine secretion. However, VPA did not affect the phosphorylation of other key components of the FcεRI signaling pathway, such as Syk, Akt, ERK1/2, or p38. Altogether, our data demonstrate that VPA affects PLCγ2 phosphorylation, which in turn decreases IgE-mediated mast cell activation. These results suggest that VPA might be a key modulator of allergic reactions and might be a promising therapeutic candidate.

Prediction of Structure and Molecular Interaction with DNA of BvrR, a Virulence-Associated Regulatory Protein of Brucella.

Brucellosis, also known as "undulant fever" is a zoonotic disease caused by Brucella, which is a facultative intracellular bacterium. Despite efforts to eradicate this disease, infection in uncontrolled domestic animals persists in several countries and therefore transmission to humans is common. Brucella evasion of the innate immune system depends on its ability to evade the mechanisms of intracellular death in phagocytic cells. The BvrR-BvrS two-component system allows the bacterium to detect adverse conditions in the environment. The BvrS protein has been associated with genes of virulence factors, metabolism, and membrane transport. In this study, we predicted the DNA sequence recognized by BvrR with Gibbs Recursive Sampling and identified the three-dimensional structure of BvrR using I-TASSER suite, and the interaction mechanism between BvrR and DNA with Protein-DNA docking and molecular dynamics (MD) simulation. Based on the Gibbs recursive Sampling analysis, we found the motif AAHTGC (H represents A, C, and T nucleotides) as a possible sequence recognized by BvrR. The docking and EMD simulation results showed that C-terminal effector domain of BvrR protein is likely to interact with AAHTGC sequence. In conclusion, we predicted the structure, recognition motif, and interaction of BvrR with DNA.

Immune Response to Mucosal Brucella Infection.

Brucellosis is one of the most prevalent bacterial zoonosis of worldwide distribution. The disease is caused by Brucella spp., facultative intracellular pathogens. Brucellosis in animals results in abortion of fetuses, while in humans, it frequently manifests flu-like symptoms and a typical undulant fever, being osteoarthritis a common complication of the chronic infection. The two most common ways to acquire the infection in humans are through the ingestion of contaminated dairy products or by inhalation of contaminated aerosols. Brucella spp. enter the body mainly through the gastrointestinal and respiratory mucosa; however, most studies of immune response to Brucella spp. are performed analyzing models of systemic immunity. It is necessary to better understand the mucosal immune response induced by Brucella infection since this is the main entry site for the bacterium. In this review, some virulence factors and the mechanisms needed for pathogen invasion and persistence are discussed. Furthermore, some aspects of local immune responses induced during Brucella infection will be reviewed. With this knowledge, better vaccines can be designed focused on inducing protective mucosal immune response.

Cervical Cancer Cells Express Markers Associated with Immunosurveillance.

Cervical cancer is the second most frequent cancer in women in Mexico, and its development depends on the presence of human papillomaviruses in the uterine cervix. These oncogenic viruses transform cells where the control over cell cycle disappears, and the capacity to induce apoptosis is absent. On the other hand, some mutations confer to the transformed cells the ability to evade recognition by the immune system. The expression of markers of the immune system such as CD95, MICA/B, CD39, CD73, NKp30, NKp46, CD44, CD24, NKG2A, and CTLA-4 was analysed by flow cytometry on cervical cancer cells INBL (HPV 18, stage IVB), HeLa (HPV 18), CaSki (HPV 16), and C33A (HPV-). Our results showed the presence of atypical markers on cervical cancer cells; some of them are molecules involved in tumour cell recognition such as MICA/B and CD95. Other markers associated with immune system escape, such as CD39, CD73, and CTLA-4, were also present. Furthermore, we found that some cervical cancer cells expressed typical markers of NK cells like NKp30, NKp46, NKG2A, and KIR3DL1. It is not clear whether these molecules confer any gain to the tumour cells or if they represent a disadvantage, but we hypothesise that these molecules that are present in cervical cancer cells allow them to mimic in front of the immune system.

Detection of Brucella abortus by a platform functionalized with protein A and specific antibodies IgG.

Oriented immobilization of antibodies on a sensor surface is critical for enhancing both the antigen-binding capacity and the sensitivity of immunosensors. In this study, we describe a strategy to adsorb immunoglobulin G (IgG) anti-Brucella antibodies onto a silicon surface, oriented by protein A obtained from Staphylococcus aureus (SpA). X-ray photoelectron spectroscopy and atomic force microscopy were used to characterize topographically, morphologically, and chemical changes of the sensor functionalization. The activity of the biosensor was assessed by confocal microscopy, scanning electronic microscopy, and bacteria capture assays (BCA). According to the BCA, the efficiency of Brucella abortus detection with the SpA-IgG anti Brucella biosensor was three-fold higher than that of the random orientated IgG anti Brucella biosensor. The limit of detection was 1 × 106 CFU/ml. These data show that the orientation of antibodies immobilization is crucial to developing immunosensors for bacterial antigen detection as Brucella spp and improve its sensibility level. Functionalization with protein A increases Brucella detection by an antibody-coated surface. Functionalized silicon surface for Brucella detection was characterized by atomic force microscopy, X-ray photoelectron spectroscopy and confocal microscopy.

Description of CD8+ Regulatory T Lymphocytes and Their Specific Intervention in Graft-versus-Host and Infectious Diseases, Autoimmunity, and Cancer.

Gershon and Kondo described CD8+ Treg lymphocytes as the first ones with regulating activity due to their tolerance ability to foreign antigens and their capacity to inhibit the proliferation of other lymphocytes. Regardless, CD8+ Treg lymphocytes have not been fully described-unlike CD4+ Treg lymphocytes-because of their low numbers in blood and the lack of specific and accurate population markers. Still, these lymphocytes have been studied for the past 30 years, even after finding difficulties during investigations. As a result, studies have identified markers that define their subpopulations. This review is focused on the expression of cell membrane markers as CD25, CD122, CD103, CTLA-4, CD39, CD73, LAG-3, and FasL as well as soluble molecules such as FoxP3, IFN-γ, IL-10, TGF-ß, IL-34, and IL-35, in addition to the lack of expression of cell activation markers such as CD28, CD127 CD45RC, and CD49d. This work also underlines the importance of identifying some of these markers in infections with several pathogens, autoimmunity, cancer, and graft-versus-host disease as a strategy in their prevention, monitoring, and cure.

Chronic Intake of Commercial Sweeteners Induces Changes in Feeding Behavior and Signaling Pathways Related to the Control of Appetite in BALB/c Mice.

Nonnutritive sweetener use is a common practice worldwide. Although considered safe for human consumption, accumulating evidence suggests these compounds may affect metabolic homeostasis; however, there is no consensus on the role of frequent sweetener intake in appetite and weight loss. We sought to determine whether frequent intake of commercial sweeteners induces changes in the JAK2/STAT3 signaling pathway in the brain of mice, as it is involved in the regulation of appetite and body composition. We supplemented adult BALB/c mice with sucrose, steviol glycosides (SG), or sucralose, daily, for 6 weeks. After supplementation, we evaluated body composition and expression of total and phosphorylated JAK2, STAT3, and Akt, as well as SOCS3 and ObRb, in brain tissue. Our results show that frequent intake of commercial SG decreases energy intake, adiposity, and weight gain in male animals, while increasing the expression of pJAK2 and pSTAT3 in the brain, whereas sucralose increases weight gain and pJAK2 expression in females. Our results suggest that chronic intake of commercial sweeteners elicits changes in signaling pathways that have been related to the control of appetite and energy balance in vivo, which may have relevant consequences for the nutritional state and long term health of the organism.

CD38 expression in early B-cell precursors contributes to extracellular signal-regulated kinase-mediated apoptosis.

CD38 is a 45,000 molecular weight transmembrane protein that is expressed in immature and mature lymphocytes. However, the expression and function of CD38 during B-cell differentiation in mice is poorly understood. Here, we report that CD38 is expressed from the earliest stages of B-cell development. Pre-pro-B, pro-B, pre-B and immature B cells from murine bone marrow all stained positive for CD38. Interestingly, CD38 expression increases with B-cell maturation. To assess the role of CD38 during B-cell maturation, CD38-deficient mice were analysed. CD38(-/-) mice showed a significant increase in both the frequency of B-lineage cells and the absolute numbers of pre-pro-B cells in bone marrow; however, no other differences were observed at later stages. CD38 cross-linking in Ba/F3 cells promoted apoptosis and marked extracellular signal-regulated kinase (ERK) phosphorylation, and these effects were reduced by treatment with the mitogen-activated protein kinase/ERK kinase inhibitor PD98059, and similar effects were observed in B-cell precursors from bone marrow. These data demonstrate that B-cell precursors in mouse bone marrow express functional CD38 and implicate the early ligation of CD38 in the ERK-associated regulation of the B-lineage differentiation pathway.

Salmonella Typhi OmpS1 and OmpS2 porins are potent protective immunogens with adjuvant properties.

Salmonella enterica serovar Typhi (S. Typhi) is the causal agent of typhoid fever, a disease that primarily affects developing countries. Various antigens from this bacterium have been reported to be targets of the immune response. Recently, the S. Typhi genome has been shown to encode two porins--OmpS1 and OmpS2--which are expressed at low levels under in vitro culture conditions. In this study, we demonstrate that immunizing mice with either OmpS1 or OmpS2 induced production of specific, long-term antibody titres and conferred protection against S. Typhi challenge; in particular, OmpS1 was more immunogenic and conferred greater protective effects than OmpS2. We also found that OmpS1 is a Toll-like receptor 4 (TLR4) agonist, whereas OmpS2 is a TLR2 and TLR4 agonist. Both porins induced the production of tumour necrosis factor and interleukin-6, and OmpS2 was also able to induce interleukin-10 production. Furthermore, OmpS1 induced the over-expression of MHC II molecules in dendritic cells and OmpS2 induced the over-expression of CD40 molecules in macrophages and dendritic cells. Co-immunization of OmpS1 or OmpS2 with ovalbumin (OVA) increased anti-OVA antibody titres, the duration and isotype diversity of the OVA-specific antibody response, and the proliferation of T lymphocytes. These porins also had adjuvant effects on the antibody response when co-immunized with either the Vi capsular antigen from S. Typhi or inactivated 2009 pandemic influenza A(H1N1) virus [A(H1N1)pdm09]. Taken together, the data indicate that OmpS1 and OmpS2, despite being expressed at low levels under in vitro culture conditions, are potent protective immunogens with intrinsic adjuvant properties.

Comparative proteome analysis of Brucella abortus 2308 and its virB type IV secretion system mutant reveals new T4SS-related candidate proteins.

Brucella abortus is an alpha-2 proteobacteria with a type IV secretion system (T4SS) known as virB, which is necessary to gain virulence by building up a replicative vacuole associated with the endoplasmic reticulum of the host cell. A virB T4SS mutant of the B. abortus 2308 strain and its wild-type strain were grown in acid medium in order to obtain and analyze their proteomes, looking for putative proteins that may serve as T4SS substrates and those that may be subjected to T4SS regulation. A total of 47 overexpressed and 22 underexpressed proteins from the virB T4SS mutant strain were selected and sequenced. Some of the 69 analyzed proteins have not been described before either as over or under-expressed in relation to a virB T4SS mutation, whereas some of them have been already described by other groups as potentially important secretory proteins in other Brucella species. An important number of the proteins identified are outer membrane and periplasmic space protein, which makes them become particularly important new T4SS-related candidate proteins.

Activation and proliferation of T lymphocyte subpopulations in patients with brucellosis.

BACKGROUND: T-cell proliferation is a standard method to evaluate cellular immune responses against intracellular infectious agents. The present study was undertaken to look for expression of an early activation marker (CD69) and proliferation using a nonradioactive method to evaluate cellular immune response against a salt-extractable antigen from Brucella melitensis 16M (RCM-BM) in patients suffering from brucellosis. METHODS: Expression of CD69 on membrane of CD4+ and CD8+ T-cells was determined by flow cytometry. Lymphoproliferation was determined by tritiated thymidine and 5-bromo-2'-deoxyuridine (BrdU) incorporation using liquid scintillation counter or flow cytometry, respectively, to evaluate DNA synthesis. RESULTS: Thirty healthy donors and 24 patients suffering from brucellosis were included in this study. In all cases, incubation with mitogen induced expression of CD69 and proliferation of both CD4+ and CD8+ T-cells. In contrast, only brucellosis patients responded with expression of CD69 and proliferation against RCM-BM antigen from Brucella melitensis (p < 0.001). CONCLUSIONS: Methods used in this study were useful to evaluate immune response against specific antigen or polyclonal stimulation. CD4+ and CD8+ T cells from patients became equally activated and proliferated in response to RCM-BM antigen. Our data suggest that both T-cell subpopulations play an important role in immune response against Brucella.

Antigen-specific activation and proliferation of CD4+ and CD8+ T lymphocytes from brucellosis patients.

Salt-extractable antigen from Brucella melitensis 16M (RCM-BM) was used to evaluate the immune response from acute and chronic patients suffering from Brucella infections (in Mexico); their responses were compared with those of healthy controls. As a readout we used upregulation of CD69 (a well-established early activation marker for lymphocytes), lymphocyte proliferation by 3[H]thymidine or 5-bromo-2-deoxyuridine (BrdU) incorporation measured by liquid scintillation or flow cytometry, respectively, and production of gamma interferon (IFN gamma). We compared the antigen-specific response with the response induced by phytohaemagglutinin (PHA) as a positive control. There was no difference between acute patients and the healthy controls in the percentages of CD3+, CD4+ or CD8+ lymphocytes. However, we found that chronic patients had a significant (P < 0.05) increase in the CD8+ T cells, in line with previous studies. Antigen-specific responses to RCM-BM showed a significant (P < 0.05) upregulation of CD69 in both CD4+ and CD8+ T lymphocytes in acute brucellosis patients and in CD8+ T lymphocytes in chronic patients, indicating that both populations became activated by this antigen preparation. Moreover, lymphocyte proliferation from both acute and chronic patients in response to RCM-BM was highly significant (P < 0.001) when compared with healthy controls. However, there were no apparent differences between acute and chronic patients. Although the incorporation of BrdU showed similar results it provided additional information, since we demonstrated that both CD4+ and CD8+ T lymphocytes from acute and chronic patients proliferated equally well in response to RCM-BM. Similar results were observed with intracellular IFN gamma determination. As a whole, our data suggest an important role for both CD4+ and CD8+ T lymphocytes in Brucella infection in humans. As has been reported in mice, it is feasible that activated CD8+ T cells participate in protection against Brucella in humans through cytotoxicity or/and by the production of factors such as interferon and granulysin. The role of these cells should be carefully analysed to understand better their participation in human infection by Brucella.

Aspectos de la relación huésped-parásito en brucelosis / Aspects of host-parasite relation in brucellosis

La brucelosis es una zoonosis causada por bacterias del género Brucella, las cuales son gérmenes intracelulares facultativos. Se sabe que en este tipo de infecciones la respuesta inmunitaria celular desempeña un papel relevante para conferir protección al huésped; sin embargo, los mecanismos exactos a través de los cuales se establece la inmunidad celular no han sido bien comprendidos. Se analizan algunas de las probables interacciones celulares involucradas en la respuesta protectora y la función de las poblaciones celulares participantes, así como algunos mecanismos que permiten a la bacteria evadir la respuesta inmunitaria del huésped