Ivana Mirkov

Acute cadmium administration to rats exerts both immunosuppressive and proinflammatory effects in spleen.

Conflicting data (both suppression and augmentation as well as lack of the effect) exist in respect to cadmium (Cd) and splenic T cell-based immune cell activity. Spleen is also the site of innate immune responses but impact of Cd on this type of immunity has been less explored. In the present study the effects of acute Cd administration on basic aspects of both T cell-based and innate immune spleen cell activity were examined in rats. Intraperitoneal injection of 1mg of Cd/kg resulted in decrease in concanavalin A (ConA) induced proliferation which seems to be more related to altered spleen cells responsiveness to IL-2 than to apoptosis. Differential effects on proinflammatory T cell derived cytokines were observed (decreases of IFN-γ gene expression and ConA-stimulated production, but increases in IL-17 mRNA levels with no effect on concentrations of protein product). Reduction of IFN-γ production seemed not to rely on IL-4 and IL-10, but at least partly on nitric oxide (NO). Increased activity relevant for innate immunity (granulocyte and CD11b(+) cell accumulation in the spleen, inducible nitric oxide synthase/iNOS expression and NO production by spleen cells) was observed, but there was a decrease in respiratory burst (dihydrorhodamine/DHR oxidation and nitroblue tetrazolium/NBT reduction). Increases of TNF-α and IL-1β gene expression and IL-1β protein product were noted as well. Administration of 0.5mg Cd/kg resulted in less pronounced (ConA-induced proliferation) or lack of the effect (IFN-γ production) on spleen T cell activities and on innate activities (granulocyte accumulation, NO production) as well. However, increases of spleen cell respiratory burst activity and IL-1β production were observed. Effects of lower cadmium doses (5ppm and 50ppm) on several aspects of spleen cell immune activity were observed in intermediate period of exposure (30 days, oral intake) as well. Differential effects of Cd on immune activities of spleen cells might contribute to our understanding of the complexity of immunomodulatory effects of this metal.

Toxicity of oral cadmium intake: Impact on gut immunity

Gastrointestinal tract is one of the main targets of cadmium (Cd), an important food and drinking water contaminant. In the present study, the effect of subchronic (30 days) oral (in water) intake of 5ppm and 50ppm of cadmium on immune responses in the gut was examined in rats. Cadmium consumption resulted in reduction of bacteria corresponding to Lactobacillus strain, tissue damage and intestinal inflammation [increases in high mobility group box 1 (HMGB1 molecules), superoxide dismutase (SOD) and catalase (CAT) activity and proinflammatory cytokine (TNF, IL-1β, IFN-γ, IL-17) content]. Draining (mesenteric) lymph node (MLN) stress response was observed [elevation of MLN glutathione (GSH) and metallothionein (MT) mRNA levels] and stimulation of both adaptive [cellularity, proliferation, proinflammatory (IFN-γ and IL-17) MLN cell cytokine responses] as well as innate immune activity (increases in numbers of NK and CD68(+) cells, oxidative activities, IL-1β). In contrast to proinflammatory milieu in MLN, decreased or unchanged antiinflammatory IL-10 response was observed. Stimulation of immune activities of MLN cells have, most probably, resulted from sensing of cadmium-induced tissue injury, but also from bacterial antigens that breached compromised intestinal barrier. These effects of cadmium should be taken into account when assessing dietary cadmium as health risk factor.

Effects of subacute oral warfarin administration on peripheral blood granulocytes in rats.

Warfarin affects mainly vitamin K dependent (VKD) processes, but the effects on some non-VKD-related activities such as tumor growth inhibition and mononuclear cell-mediated immune reactions were shown as well. In this study, the effect of subchronic (30 days) oral warfarin (0.35 mg/l and 3.5mg/l) intake on peripheral blood granulocytes in rats was investigated. Increase in prothrombin and partial thromboplastin time at high warfarin dose reflected its basic activity. Priming effect for respiratory burst was noted at both warfarin doses, while only high warfarin dose resulted in priming for adhesion, the rise in intracellular myeloperoxidase content/release and stimulation of nitric oxide production. Differential effects of high warfarin dose were noted on granulocyte cytokines IL-6 (lack of the effect), TNF-α (decreased release and mRNA expression) and IL-12 (increase in mRNA for IL-12 subunits p35 and p40). Changes in granulocytes seems not to rely on mitogen activated kinases p38 and ERK. Warfarin intake was associated with an increase in circulating IL-6, fibrinogen and haptoglobin and with changes in the activity of erythrocyte antioxidant enzymes superoxide dismutase and catalase. The effects of oral warfarin intake on peripheral blood granulocytes demonstrated in this study might be relevant for oral anticoagulant therapy strategies in humans.

Intestinal helminths infection of rats (Ratus norvegicus) in the Belgrade area (Serbia): the effect of sex, age and habitat

Gastrointestinal helminths of Norway rat (Rattus norvegicus) from the Belgrade area were studied as a part of a wider ecological research of rats in Serbia (data on the distribution, population ecology, economic and epizoothiological-epidemiological importance, and density control). Rats were captured from May 2005 to July 2009 at both urban and suburban-rural sites. Of a total of 302 trapped rats 48% were males and 52% females, with 36.5% and 38.8% of juvenile-subadult individuals, per sex respectively. Intestinal helminth infection was noted in 68.5% of rats, with a higher prevalence in male hosts and in adult individuals. Higher numbers of infected juveniles-subadults were noted in suburban-rural habitats, while an opposite tendency was noted in adult rats. Seven helminth species were recovered, of which five were nematode (Heterakis spumosa, Nippostrongylus brasiliensis, Capillaria sp., Trichuris muris and Syphacia muris) and two cestode species (Hymenolepis diminuta and Rodentolepis fraterna). The most prevalent parasites were Heterakis spumosa (36.7%) and Hymenolepis diminuta (30.5%). Sex and habitat-related differences were noted in the prevalence of infection with Capillaria sp. and Trichuris muris, while there were no age-related differences in the prevalence of infection with any individual helminth species. Significantly higher prevalence of infection was noted in summer as compared to spring or winter, with a tendency to be higher in autumn as compared to spring. The only significant difference in the prevalence of infection between habitat-related was noted during spring. H. spumosa was most prevalent in summer, while H. diminuta and N. brasiliensis in autumn. The mean intensity of infection with H. spumosa, R. fraterna, S. muris and T. muris was higher in autumn than in the other seasons, while N. brasiliensis and Capillaria sp. occured in winter. No more than four helminth species were found in one host.

Gender Differences in Pulmonary Inflammation Following Systemic Cadmium Administration in Rats

OBJECTIVE To examine the presence of gender differences in pulmonary inflammation evoked by acute systemic cadmium administration in rats. METHODS Presence of basic indicators of lung inflammation (inflammatory cytokine lung content, leukocyte infiltration and activity of cells recovered from lungs by enzyme digestion) was analyzed and compared in animals of the two sexes. RESULTS Intraperitoneal administration of cadmium (1.0 mg/kg) resulted in higher cadmium content in lungs of female rats. Higher tumor necrosis factor (TNF) content was noted in lung homogenates of male rats, while interleukin-6 (IL-6) content was slightly, but significantly greater in lungs of female rats. Increased leukocyte infiltration was observed in lungs of male rats, mainly due to neutrophils. Increased responsiveness to phorbol myristate acetate (PMA) stimulation was noted in cells recovered from lungs of male rats. Rise in intracellular content of myeloperoxidase (MPO) was noted in lung cells from cadmium-treated rats of both sexes, but higher in cells from male rats. CONCLUSIONS Presented data documented a more intense pulmonary inflammatory response to systemic cadmium administration in males, with higher IL-6 levels in lungs of female individuals. These sex differences in proinflamatory activity of cadmium in lungs should be taken into consideration in studying the remote toxicity of this heavy metal.

Contact allergic response to dinitrochlorobenzene (DNCB) in rats: Insight from sensitization phase

Contact hypersensitivity (CHS) is a T-cell-mediated skin inflammatory reaction to cutaneous exposure to small sensitizing chemicals, haptens. Majority of CHS studies were conducted in mice and there is paucity of data in other experimental animals. In the present study, characteristics of contact hypersensitivity reaction to dinitrochlorobenzene (DNCB) were determined in Th1-prone Dark Agouti (DA) rats by evaluating sensitization phase as a function of time-dependent changes in draining lymph nodes (DLN). Apart from basic indices of DLN activity (cellularity and proliferation), the production of cytokines relevant for CHS induction, interleukin-6 (IL-6), interferon-γ (IFN-γ), interleukin-17 (IL-17) and interleukin-4 (IL-4) was analyzed. Anti-inflammatory cytokine interleukin-10 (IL-10) production by DLN cells was determined as well. Highest production of IL-6, IFN-γ and IL-17 in sensitized animals was observed at day 3 after DNCB application, with a decrease at day 5. Increased messages for IFN-γ and IL-17 were noted at this time point. In contrast to inflammatory cytokines, anti-inflammatory cytokine interleukin-4 (IL-4) was undetectable during the entire sensitization phase. Differential pattern (IL-6 and IFN-γ) and level (IFN-γ and IL-17) of inflammatory cytokine production was noted in sensitized Th2-prone Albino Oxford (AO) rats. Similarly to DA rats, no changes in IL-4 were noted in AO rats. Strain-dependent differences in inflammatory cytokine production seem to be based on anti-inflammatory cytokine interleukin-10 (IL-10). Production of IFN-γ concomitantly with undetectable IL-4 in both strains classify rat CHS to DNCB as Th1/type 1 reaction. Detection of IL-17 in sensitized DLN cells points to the involvement of T(IL-17) cells in rat contact hypersensitivity.

A role for macrophage migration inhibitory factor in protective immunity against Aspergillus fumigatus.

Inflammation plays an important role in protective immunity against fungi, including the opportunistic pathogen, Aspergillus fumigatus. The balance between pro-inflammatory and anti-inflammatory cytokines is a key determinant of infection outcome. Since macrophage migration inhibitory factor (MIF) is an upstream regulator of many cytokines, we analyzed herein the role of endogenous MIF in the host control of hematogenously disseminated aspergillosis using MIF⁻/⁻ mice. As revealed by their mortality rate, MIF⁻/⁻ mice were more susceptible to disseminated infection than WT mice. Moreover, pharmacologic inhibition of MIF with (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester, (ISO-1) increased the susceptibility of WT mice to lethal infection. The higher tissue fungal burden early in sublethal infection indicated increased susceptibility of MIF⁻/⁻ mice to sublethal infection as well. Substantial down-regulation of innate and acquired antifungal responses, characterized by decreased production of IL-1β, IL-6, TNF-α, IFN-γ and IL-17 in the spleen was noted in sublethally infected MIF⁻/⁻ mice. In contrast, IL-4 was higher in MIF⁻/⁻ than in WT mice. Taken together, our findings show that MIF contributes to host resistance against progressive invasive A. fumigatus infection by controlling downstream pro-inflammatory versus anti-inflammatory cytokine production thus determining the outcome of infection.

Lungs: Remote inflammatory target of systemic cadmium administration in rats

Pulmonary inflammation is a biological response to cadmium entering the body via the respiratory route. Systemic administration of this metal revealed the lungs as a significant site of its disposition. In this study, the presence of basic indicators of lung inflammation (leukocyte infiltration and activity of cells recovered from lungs by enzyme digestion) was analyzed in the rat model of acute systemic cadmium intoxication. Intraperitoneal administration of both cadmium doses (0.5mg/kg and 1.0mg/kg) resulted in increased numbers of neutrophils. Signs of spontaneous activation of lung cells including the capacity of reduction of nitroblue tetrazolium (NBT), increase in myeloperoxidase (MPO) intracellular content and increase in interleukin-6 (IL-6) production were noted at both cadmium doses. Increased lung cell responsiveness to stimulation in vitro was noted at the higher cadmium dose. The presence of pulmonary inflammatory parameters in rats administered intraperitoneally with cadmium revealed the lungs as remote inflammatory targets of this metal.

Differential effects of cadmium administration on peripheral blood granulocytes in rats

Infiltration of circulatory inflammatory cells is a common histopathological finding in target organs following cadmium administration, but there is paucity of data concerning their activity. In this study, the effects of sublethal (1 mg/kg) cadmium on peripheral blood polymorphonuclear (PMN) cells were examined 48 h following administration in rats, when tissue (liver and lung) infiltration of these cells was observed. Cadmium administration resulted in systemic inflammatory cytokine and acute phase response with an increase in circulatory neutrophil numbers and cells that express CD11b molecules. Rise in basic aspects of oxidative activity including intracellular myeloperoxidase (MPO), reactive oxygen (nitroblue tetrazolium/NBT cytochemical assay) and nitrogen (Griess assay) species production was observed in PMNs from cadmium-administered rats. A decrease in levels of mRNA for IL-1β, TNF-α and IL-6 was noted, but production of these cytokines was affected differentially. Described effects of cadmium on PMNs add further to the understanding of inflammatory potential of this environmental contaminant.

Oral warfarin affects peripheral blood leukocyte IL-6 and TNFα production in rats

Warfarin is a Vitamin K (VK) antagonist that affects Vitamin K-dependent (VKD) processes, including blood coagulation, as well as processes unrelated to hemostasis such as bone growth, calcification, and growth of some cell types. In addition, warfarin exerts influence on some non-VKD-related activities, including anti-tumor and immunomodulating activity. With respect to the latter, both immune stimulating and suppressive effects have been noted in different experimental systems. To explore the in vivo immunomodulatory potential of warfarin on one type of activity (i.e., cytokine production) in two different immune cell populations (i.e., mononuclear or polymorphonuclear cells), effects of subchronic oral warfarin intake in rats on pro-inflammatory cytokine (i.e., TNFα, IL-6) production by peripheral blood mononuclear and polymorphonuclear cells (granulocytes) was examined. Differential effects of warfarin intake on TNFα and IL-6 were noted, depending on the type of peripheral blood leukocytes and on the cytokine examined. Specifically, a lack of effect on TNFα and a priming of IL-6 production by mononuclear cells along with a decrease in TNFα and a lack of effect on IL-6 in polymorphonuclear cells were seen in warfarin-exposed hosts. The cell- and cytokine-dependent effects from subchronic oral warfarin intake on peripheral blood leukocytes demonstrated in this study could, possibly, differentially affect reactions mediated by these cells. Ultimately, the observed effects in rats might have implications for those humans who are on long-term/prolonged warfarin therapy.