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

SUMMARY: 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 muns 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.

Usefulness of animal models of aspergillosis in studying immunity against Aspergillus infections.

Aspergillosis represents a spectrum of fungal diseases which are caused by fungi of the genus Aspergillus. Animal models have been developed and used to address immune-based mechanisms of defense against these fungi. Invertebrate models enabled mass screening of virulence attributes of Aspergillus species as well as mechanisms of acquired resistance to antifungal agents. This review represents a concise view of cellular and humoral participants in an immune response to Aspergillus gained mostly from rodent models of aspergillosis. The survey of immune defense mechanisms was given, including the role of innate immune cells (macrophages, neutrophils, monocytes, eosinophils, innate-like lymphocytes) and receptors in antifungal response, the significance of dendritic cells in activation of specific adaptive T cell-mediated immune responses and the regulatory mechanisms of excessive response. Insight into innate immune defense mechanisms gained using non-vertebrate models of infections with Aspergillus sp. was given as well. The contribution of animal models to the current knowledge of immune mechanisms of resistance or susceptibility to these fungi was stressed and the significance of data gained from these models in forming the basis for the design of therapeutic strategies in prevention and/or treatment of aspergillosis was pointed out.

Stimulator of proliferation of spleen colony-forming cells in T-cell deprived mice treated with cyclophosphamide or irradiation.

A role for T-cells in the regulation of CFU-S proliferation was investigated by determining the presence and activity of CFU-S proliferation stimulator (CFU-S stimulator) in adult mouse bone marrow after irradiation or cyclophosphamide (Cy) treatment. CBA mice previously deprived of T-cells by thymectomy, irradiation and bone marrow reconstitution (TIR) were thereafter treated with 4.5 Gy irradiation or 200 mg/kg Cy. Regenerating bone marrow cells of TIR and corresponding control mice after irradiation or Cy treatment produced CFU-S stimulator. The dose dependent increase in cytosine arabinoside cell death of normal bone marrow day 8 CFU-S was found when both CFU-S stimulators obtained after irradiation of TIR or corresponding control animals were tested. CFU-S stimulator activity in the bone marrow of TIR-Cy treated mice was also detected, but the effect was not dose-dependent. This was not related to the presence of an inhibitor of CFU-S proliferation. It appears that the CFU-S stimulator activity is not related to IL-6, IL-1 or IL-2, or to an inhibitor of IL-6 or IL-1 activity. The results demonstrate the existence of CFU-S proliferation stimulator unrelated to the two major monokines in the bone marrow of immunosuppressed mice.

Stimulator of proliferation of spleen colony-forming cells in acute sterile inflammation.

The presence and activity of a spleen colony-forming cell (CFU-S) proliferation stimulator was investigated in rat bone marrow after induction of sterile inflammation. Wistar rats were treated intraperitoneally with two 15 ml injections of 3.5% polyvinylpyrrolidone (PVP) at 18 h intervals, and the presence of CFU-S proliferation stimulator determined in bone marrow 6, 20 and 24 h after the first injection. The marrow of these mice was used to condition medium which was then fractionated using Amicon Diaflo ultrafiltration membranes. The 30-50 kDa fraction, taken from 20h post-PVP-bone marrow extract, was found to induce cycling of d8-CFU-S in normal mouse bone marrow. This activity was not related to the presence of interleukins-1, -2 or -6 like activities in the material tested. The results demonstrate the existence of CFU-S proliferation stimulator in the bone marrow of rats with sterile inflammation (i.e. in an in vivo tissue response to non-specific cell stimulation), similar to that originally described as a macrophage product in mouse bone marrow after treatment with a variety of cytotoxic agents.

Aberrant levels of cytokines within the healing wound after burn injury.

OBJECTIVE: To study the effect of a burn injury on the course of cellular and cytokine changes in a wound and the relationship of these cytokines to the amounts of protein and collagen deposited at the site of the wound. DESIGN: A randomized control trial was done in which one group of rats were subjected to a severe burn injury. With the use of a sponge matrix model, the wound-healing parameters were evaluated. MATERIALS: A random sample of eight inbred albino Oxford rats per group were used in all experiments. INTERVENTIONS: Rats were subjected to a severe scald injury. Polyvinyl sponges were used as the wound-healing model. MAIN OUTCOME MEASURE: The obtained results implied that the wound-healing process is impaired after a severe burn injury. RESULTS: The wounds in these animals with burn injuries contained a lower number and an altered type of infiltrating cells with aberrant levels of cytokines, higher levels of interleukin-6, and lower levels of tumor necrosis factor and interleukin-1 in the fluids of the wounds. The parameters of healing (amounts of protein and collagen deposited at the site of the wound) were significantly lower in animals with burn injuries on days 7 and 14. CONCLUSION: The underlying mechanism of the impaired healing of a wound after burn injury could lie in the altered migration of inflammatory cells to the site of the wound and in the aberrant cytokine levels within the wound.

Peripheral blood granulocyte activity following contact sensitization of rats with dinitrochlorobenzene.

Contact hypersensitivity (CHS) reaction is a classic example of a cell-mediated reaction. As the afferent phase of the reaction includes inflammation, CHS is a suitable model for investigating non-specific immunity. Some aspects of granulocyte activity in the afferent phase of experimentally induced CHS to dinitrochlorobenzene (DNCB) in two genetically different rat strains, AO and DA were examined in this study. A shift in the ratio of granulocytes to lymphocytes in favour of granulocytes and an increase in granulocyte survival were noted in DA rats. Granulocytes from both strains demonstrated increased levels of NBT reduction and an increase in their adhesion to plastic. Decreased granulocyte adhesion in the presence of monoclonal antibodies to beta2 integrins (anti-CD11b/c and anti-CD18) points to the contribution of these molecules to granulocyte adhesiveness during the sensitization phase of CHS. Stimulation of adhesion in the presence of anti-CD11a antibody, points to a differential modulation of adhesion molecule activity during the afferent phase of CHS. Changes in functional activity of granulocytes demonstrated in this study might contribute to the development of CHS in rats.

Early inflammatory cytokine and acute phase protein response under the stress of thermal injury in rats.

The acute inflammatory response associated with thermal injury was examined in rats. The appearance of mediators of inflammation in the systemic circulation, including cytokines interleukin-1 (IL-1), tumor necrosis factor (TNF) and interleukin-6 (IL-6) and acute phase proteins were assessed during initial 72 h following thermal injury. Increased levels of activity were noted for all three cytokines, but with a different time-course. While serum IL-1 activity was elevated throughout the 3-day period of observation, the levels of serum TNF activity were enhanced after 12 h and on days 1 and 3 following scalding injury. The values of IL-6 were already increased one hour after thermal injury and increased progressively up to day 1 following scalding. Alpha2-macroglobulin and haptoglobin levels were increased 12 h after thermal injury, rising further on days 1 and 3. Positive correlation was found between the time-course of increased serum IL-6 activity and alpha2-macroglobulin, as well as between TNF and haptoglobin in the serum.

Anticardiolipin antibodies in women with unexplained infertility.

Concept of autoimmune basis of infertility is still controversial, particularly regarding the presence of non-organ specific autoantibodies. Non-organ specific anticardiolipin (aCL) and antithyroglobulin (TgAt) antibodies were detected in infertile women. Both partners were evaluated according to the criteria of The American Society for Reproductive Medicine. All the results were normal in cases of unexplained infertility. Antisperm antibodies (ASA) were determined by a mixed antiglobulin reaction (MAR) and the Kibrick agglutination assay, aCL by commercial ELISA, TgAt by commercial RIA. Fertile women had children. Subjects were grouped in fertile (n=27), infertile (n=65), and cases of unexplained infertility (n=42). In fertile women, aCL was below the negative cut-off value (100 %), while women with unexplained infertility were positive in 23.8 %. Anticardiolipin (aCL) antibodies were detected in 21.5 % of infertile patients, most of them with unexplained infertility (15.4 %). Other positive women had partners with ASA (4.6 %), or exhibited a negative postcoital test (1.5 %). In this study aCL were not detected in women with ASA. TgAt incidence was increased in infertile (20 %) and unexplained infertility group (21.4 %) compared to the fertile controls (18.5 %). Increased incidence of aCL and TgAt in infertile women supports the contention that these autoantibodies contribute to the infertility

Granulocyte and plasma cytokine activity in acute cadmium intoxication in rats.

Changes in the number and ex vivo function of peripheral blood neutrophils were investigated following intraperitoneal administration of cadmium-chloride in rats. Besides a dose-dependent increase in the number of peripheral blood neutrophils, changes were found in the functional state of isolated polymorphonuclear leukocytes (PMNs). Increased spontaneous adhesion and activation, and TNF activity in a conditioned medium were observed in cultures of granulocytes in comparison to granulocytes from control (saline-treated) animals. Increased levels of plasma activity of inflammatory cytokines, tumor necrosis factor (TNF) and interleukin-6 (IL-6) were noted following cadmium administration. Cytological signs of pulmonary inflammation were revealed histologically and the majority of neutrophils recovered from the lungs by enzyme digestion exhibited a capacity of nitroblue tetrazolium (NBT) reduction. Our data demonstrate that acute cadmium intoxication leads to a systemic inflammatory response characterized by numerical and functional changes in the granulocyte compartment and to increased levels of inflammation-related cytokine activity in the circulation. Correlations between the increased number of peripheral blood neutrophils and IL-6 plasma activity (r=0.776, p<0.00001) and the number of neutrophils recovered from the lung tissue (r=0.893, p<0.00001) suggested that systemic cadmium-induced inflammation might be involved in the pulmonary toxicity of cadmium.

Effects of treatment with interleukin-1 receptor antagonist on endogenous interleukin-1 levels in normal and irradiated mice.

The in vivo effects of recombinant human interleukin-1 receptor antagonist (rhIL-1Ra) administration on endogenous IL-1 levels in the circulation and conditioned media (CM) from different immunohematopoietic organ/tissues were studied in CBA mice under steady state and postirradiation conditions. In normal mice, constitutive IL-1 levels were demonstrated in the plasma, CM of peritoneal exudate cells and full-thickness skin explants with low or undetectable levels in CM of splenic and bone marrow cell suspensions. In irradiated mice (2 Gy, X rays) on day 3 post exposure a significant increase of IL-1 levels was seen in the circulation and CM of peritoneal exudate cells, with no significantly different levels in postirradiation bone marrow, spleen and skin. After rhIL-1Ra treatment of the animals (2 x 50 microg/mouse, i.p.), significantly elevated IL-1 levels were observed in the skin and CM of peritoneal exudate cells in normal mice, whereas slightly increased levels were detected in CM of splenic cells. The rhIL-1Ra administration in irradiated mice led to decreased IL-1 concentrations in the circulation, and CM of peritoneal exudate cells and skin. The results pointed out the importance of IL-1 secretion and receptor expression in the maintenance of homeostasis in steady state, as well as during recovery after irradiation. Modulatory effects of IL-1Ra on IL-1 production were dependent on basic endogenous IL-1 concentration.

Increased activity of lymph node cells in experimental thermal injury: changes in accessory cells in injured area-draining lymph nodes.

Accessory cell content and some of their functional characteristics were determined in regional lymph nodes which drain burn injury (DLN) in rats. Increase in percentages of non-specific esterase-positive cells and NBT+ macrophages and in numbers of dendritic cells were noted in cytospin preparations of draining lymph node cells (DLC) 24 and 72 h following thermal injury. An accumulation of B cells was also noted in the DLN paracortex region at these time points. Enrichment of ED1+ (rat macrophage marker) cells was noted in the adherent DLC population. Increased activity of interleukin-1 (IL-1) in conditioned medium from adherent DLC population and the increased stimulatory capacity of whole DLC or dendritic cell enriched-DLC fraction were noted in functional assays. Enrichment in accessory cells and an increase in their functional activity could contribute to the endogenous activity of regional lymph nodes which drain burned areas.

Post-traumatic activation of draining lymph node cells. II. Proliferative and phenotypic characteristics.

Proliferative and phenotypic characteristics of cells in regional lymph nodes that drain burn injury were examined in rats on day 3 postburn, i.e. at the time of maximal spontaneous proliferation and of interleukin-2 and accessory cytokine (IL-1 and IL-6) production. The importance of IL-1 in spontaneous proliferation of draining lymph node cells was demonstrated by stimulation of IL-2-driven proliferation by recombinant IL-1 in vitro and by susceptibility of unstimulated proliferation to anti-IL-1 antibodies, while requirements for IL-6 in draining lymph node cell proliferation were less pronounced. Cell surface phenotyping revealed a slightly increased percentage of CD25+ cells in the blast cell population of freshly isolated draining lymph node cells after injury, which increased further during cultivation. Enrichment in CD8+ cells on day 3 following burn injury was demonstrated, while no changes in total cell population and CD4+ cells was noted. This was however preceded by pronounced percentual decrease of total T cells and CD4+ cells and by increases of B cells and MHC class II+ cells on day 1 postburn. Inhibition of draining lymph node cell proliferation by anti-MHC class II antibodies suggested that this proliferation was class II MHC dependent. The contribution of cell proliferation and/or cell influx to day 3 postburn draining lymph node cell activity is discussed.

Inhibitory action of cyclosporin A on the early events of T cell activation: effects on T cell-independent production of IL1 and on IL1-dependent expression of reactivity to IL2.

Cyclosporin A (CsA) has a selective immunosuppressive effect on T cell functions, but its mode of action on IL1 activities and production remain unclear and controversial. While it has been documented that CsA has a profound effect on the ability of IL1 to promote IL2 production, its effects on the ability of IL1 to promote IL2-responsiveness are not known. Macrophage-depleted nylon wool non-adherent thymocytes (NATs) were preincubated for 6 h with or without IL1 in the presence of a wide range of concentrations of CsA (1 ng/ml-1 microgram/ml), washed and then pulsed with Con A. After washings in medium with alpha-MM, the capacity of NATs to proliferate in response to IL2 was determined. Both background and IL1-dependent acquisition of IL2-reactivity were inhibited by concentrations of CsA as low as 5 ng/ml. The effects of CsA (1 ng/ml-1 microgram/ml) on T cell-independent production of IL1 by irradiated, carrageenan-stimulated peritoneal macrophages were also examined. Up to 50 ng/ml CsA had no effect on IL1 production but higher concentrations produced a dose-dependent inhibition that was complete at 1 microgram/ml. These results show that CsA inhibits both T cell-independent production of IL1 and IL1-dependent expression of reactivity to IL2.

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-1ß, 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.

Host immune defense against Aspergillus fumigatus: insight from experimental systemic (disseminated) infection.

Model of systemic Aspergillus fumigatus infection induced by intravenous application of conidia is suitable for studying important aspects of invasive aspergillosis including relationship between infection and mortality, dissemination of infection and immune mechanisms involved in host resistance to this fungus. Use of this model allows the investigation of both innate and adaptive immune response characteristics in resistant/susceptible host, and investigating the contribution of genetic background and cytokine gene deficiency improves the knowledge of the diversity of mechanisms of immune response to Aspergillus infection. Studying of various aspects of systemic aspergillosis contributes to development of antifungal drugs.

Inflammatory and immune mechanisms in contact hypersensitivity (CHS) in rats.

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 this review, after a brief survey of murine CHS, hitherto known characteristics of CHS in rats were presented including inflammatory and immune mechanisms of both sensitization and elicitation phases. Survey of literature of rat CHS is presented, with our data concerning the importance of genetic background both in the induction and in the expression of reaction to dinitrochlorobenzene. The knowledge of CHS in rats, preferred animal in immunopharmacological studies, might help development of immunomodulatory intervention in contact allergy.

Differential effects of in vivo cadmium administration on lymphocytes and granulocytes in rats.

Effect of in vivo administration of 0.5, 1 or 2 mg cadmium/kg body mass on peripheral blood polymorphonuclear leukocyte and splenic lymphocyte functional state and viability was examined in rats. Decreased proliferative capacity in vitro of splenic lymphocytes to T-cell mitogen concanavalin A was noted at all administered cadmium dosages. No changes in cell viability, except at the highest cadmium dose, were noted in lymphocyte cultures. Increase in some of the aspects of granulocyte function including spontaneous adhesion and activation was seen and was accompanied by increase in granulocyte survival ex vivo. Rise in cytokine inflammatory mediators which might influence lymphocyte and neutrophil functions was noted also. Differential effects of cadmium on these 2 cell types might be considered when studying in vivo cadmium toxicity.

Interferon gamma alters the phenotype of rat thymic epithelial cells in culture and increases interleukin-6 production.

Rat thymic epithelial cells (TEC) in long-term culture were characterized by anticytokeratin monoclonal antibodies (mAbs) and electron microscopy. Phenotypic analysis performed by a large panel of mAbs showed that the highest percentage of these cells was of the subcapsular/medullary type. Recombinant rat interferon (IFN)-gamma up-regulated class-I and class-II MHC expression by TEC in culture as confirmed by immunohistochemistry and flow cytometry, but did not significantly alter other cell markers. TEC supernatants of IFN-gamma-treated cultures showed higher interleukin-6 (IL-6) activity, compared to the control, as determined by proliferation of the IL-6-sensitive B9-cell line. Increased IL-6 activity was probably not a consequence of increased TEC number in IFN-gamma-treated cultures because IFN did not significantly stimulate TEC proliferation in vitro. In contrast, IL-6 significantly stimulated TEC proliferation, indicating that this cytokine is not only a regulatory molecule for T-cell proliferation, but could also be an autocrine growth factor for thymic epithelium.

Rat thymic epithelial cells in culture constitutively secrete IL-1 and IL-6.

To study the in vitro interactions between rat thymic non-lymphoid cells and thymocytes, we established a system for long-term cultivation of thymic epithelial cells (TEC). TEC were cultivated and successfully propagated for over 8 months in RPMI 1640 medium containing 15% FCS, dexamethasone, insulin, epidermal growth factor, and poly-L-lysin as an adhesive matrix. Their epithelial nature has been confirmed using monoclonal anti-cytokeratin (CK) antibodies. More than 95% of these cells were reactive with K 8.13 and CK 8 mAbs, which are pan-epithelial markers for rat TEC in situ. An epithelial cell clone (TE-R 2.5) established from a long-term TEC culture was 100% reactive with these anti-CK antibodies. Phenotypic analysis of TEC cultures was performed by a large panel of mAbs reactive with a subset of rat TEC or CK polypeptides as well as UIex europaeus agglutinin I using a streptavidin-biotin immunofluorescence assay. Although the results obtained demonstrated phenotypic heterogeneity among these cells, most cultures, including the TE-R 2.5 clone, were of subcapsular/medullary phenotype. Medium conditioned by TEC cultures exhibited IL-1 and IL-6 activities when tested on D10S and B9 sensitive cell lines, respectively. Cytokine activities were neutralized (IL-1) or significantly inhibited (IL-6) by specific polyclonal antibodies. In addition, both anti-IL-1 and anti-IL-6 antibodies reacted with TEC in culture and epithelial (CK-positive) cells on thymic cryostat sections, indicating that thymic epithelium provides an important intrathymic source for molecules contributing to T cell activation.

Acute sterile inflammation--correlation between cellular changes and extramedullary-produced regulators in vivo.

Rats with polyvinylpyrrolidone (PVP)-induced sterile inflammation were used as a model in vivo for investigation of granulopoiesis and extramedullary-produced regulators. The data obtained demonstrated the invasion of massive numbers of granulocytes at the site of inflammation (peritoneal cavity) during the first 24 h of the acute phase of inflammation. To meet the organism's needs for granulocytes the activation of granulopoiesis in bone marrow occurred simultaneously. Accelerated production of granulocytic cells is manifested by involvement of granulocytic proliferative compartment in various stages of differentiation (CFU-GM and morphologically recognizable proliferative granulocytes--PG). Together with cellular changes within the granulocytic cells line, the changes in the content of investigated regulators influencing granulopoiesis were observed. At different time intervals the levels of interleukin-6 (IL-6), colony-stimulating activity (CSA), and granulocytic stimulating activity (GSA) were increased locally at the site of inflammation as well as in serum. The data obtained provide evidence that inducible granulopoiesis during the acute phase of inflammation is under the control of extramedullary-produced regulators, thus confirming their role in the regulation of granulocytic production in vivo.