Plasmodium knowlesi, a zoonotic malaria, is now considered the fifth species of Plasmodium causing malaria in humans. With its 24-hour erythrocytic stage of development, it has raised concern regarding its high potential in replicating and leading to severe illness. Spleen is an important site for removal of parasitized red blood cells and generating immunity. We reported a case of knowlesi malaria in a non-immune, splenectomized patient. We observed the delay in parasite clearance, high parasitic counts, and severe illness at presentation. A thorough search through literature revealed several case reports on falciparum and vivax malaria in splenectomized patients. However, literature available for knowlesi malaria in splenectomized patient is limited. Further studies need to be carried out to clarify the role of spleen in host defense against human malaria especially P. knowlesi.
Mice were chronically infected with cysts of ME49 strain of Toxoplasma gondii. At different periods post-infection, their spleens were removed and single cell suspensions were made. Lymphocyte transformation experiments were performed on the lymphocyte suspensions using three different kinds of antigens of ME49 strain of T. gondii, namely soluble, excretory/secretory and cystic forms. The results showed that the pattern of lymphocyte responsiveness was dependent on the kind of antigen employed for induction of the blastogenesis. Using soluble and cystic forms of the antigen, different periods of lymphocyte suppression and lymphocyte proliferation were demonstrated. However, with the use of excretory/secretory antigen, no significant suppression of lymphocyte stimulation was noted throughout the course of infection. Thus excretory/secretory antigen may be the best form of antigen for stimulation of the cell-mediated immune response and hence it appears to be a good candidate for vaccine in toxoplasmosis.
Spleen cells from saline- and Porphyromonas gingivalis-primed mice were cultured and stimulated with or without P. gingivalis and added with or without various concentration of sodium fluoride (NaF). Cell proliferation, antigen-specific IgG antibodies and both IFN-gamma and IL-10 levels were determined by a colorimetric assay, ELISA and commercial ELISA kits respectively. The results showed that NaF at concentration of 1 x 10(-6) M enhanced but at concentration of 1 x 10(-1) M abolished the immune response to P. gingivalis, suggesting that NaF at low concentration may act as an adjuvant but at high concentration may be toxic to the P. gingivalis-induced murine splenic immune response in vitro.
The aim of the present study was to investigate the effects of betulinic acid (BetA) on the expression and distribution pattern of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH‑d), an indirect indicator of nitric oxide (NO) synthase in the thymus and spleen of mice. Mice were randomly assigned to four main groups (n=48 per group): Experimental group (BetA), positive control group (goniothalamin), vehicle control group (dimethyl sulfoxide) and control group (without vehicle). Each group was further divided into three equal subgroups according to the treatment length (4, 8 and 12 days). BetA treatment induced the expression of NADPH‑d activity in the thymus and spleen without any significant changes in the morphology of the organs. Furthermore, the expression pattern of NADPH‑d in BetA‑treated animals was significantly increased compared with that in the control animals. NADPH‑d expression in the thymus and spleen suggests that NO signaling may be a potential mechanism underlying the BetA‑induced immunomodulation in these organs. These findings are of direct clinical relevance and may contribute to the further development of BetA as a therapeutic drug.
The first stage in Peyer's patch development in the fetal lamb is characterized by the colonization of the rudimentary Peyer's patches by precursor cells expressing the IgM surface receptor. In the fetal lamb, the spleen has been implicated as the source of gene-rearranged IgM(+) B lymphocytes. This study was intended to quantitate IgM(+) lymphocytes in the spleen, lymph nodes and liver of fetal lambs at various gestational ages between 63 and 110 days using flow cytometry. Flow-cytometric analysis revealed that IgM(+) lymphocytes were rare in the liver being consistently less than 1% at every gestational age examined. IgM(+) lymphocytes were detected in the spleen (mean 9.18%) and prescapular lymph nodes (mean 11.89%) as early as 63 days. In both spleen and lymph nodes, the highest representation of IgM(+) lymphocytes occurred between 70 and 86 days gestation. The highest mean percentage of IgM(+) lymphocytes was observed in the spleen (22.63%) and lymph nodes (17.02%) at 75 days gestation. From 98 days onwards, B-lymphocyte density gradually decreased in both spleen and prescapular lymph nodes. This study indicates that substantial populations of IgM(+) lymphocytes were present in both the spleen and prescapular lymph nodes from 70 days gestation and implies that both of these locations could be potential sources for the normal colonization of the ileal Peyer's patches.
Probiotics are live microorganisms that confer health benefits through the gastrointestinal microbiota. This nutritional supplement may benefit athletes who undergo rigorous training by maintaining their gastrointestinal functions and overall health. In this study the influence of moderate physical exercise using a graded treadmill exercise, alone or in combination with the consumption of a soy product fermented with Lactobacillus plantarum LAB12 (LAB12), on tumour necrosis factor alpha (TNF-α) responses was investigated in a murine model. Male BALB/c mice were randomly divided into four groups of six mice each (control, exercise alone, LAB12 and LAB12 + exercise). Mice treated with the potential probiotic LAB12 were orally gavaged for 42 days. At autopsy, blood and spleen from the animals were collected. The splenocytes were cultured in the presence of a mitogen, concanavalin A (Con A). The amount of TNF-α produced by the Con A-stimulated splenocytes was quantified using ELISA, while their proliferation was determined using the [(3)H]-thymidine incorporation method. This study shows that LAB12-supplemented and exercise-induced mice showed marked increase (P<0.05) in cell proliferation compared to the control animals. TNF-α production was suppressed (P<0.05) in the LAB12 group compared to the untreated mice. These results demonstrate that supplementation with LAB12 has immunomodulatory effects, under conditions of moderate physical exercise, which may have implications for human athletes. Further investigation in human trials is warranted to confirm and extrapolate these findings.
Toxoplasmosis in humans and other animals is caused by the protozoan parasite Toxoplasma gondii. During the process of host cell invasion and parasitophorous vacuole formation by the tachyzoites, the parasite secretes Rhoptry protein 8 (ROP8), an apical secretory organelle. Thus, ROP8 is an important protein for the pathogenesis of T. gondii. The ROP8 DNA was constructed into a pVAX-1 vaccine vector and used for immunizing BALB/c mice. Immunized mice developed immune response characterized by significant antibody responses, antigen-specific proliferation of spleen cells, and production of high levels of IFN-γ (816 ± 26.3 pg/mL). Challenge experiments showed significant levels of increase in the survival period (29 days compared with 9 days in control) in ROP8 DNA vaccinated mice after a lethal challenge with T. gondii. Results presented in this study suggest that ROP8 DNA is a promising and potential vaccine candidate against toxoplasmosis.
Diabetes mellitus is a predisposing factor of melioidosis, contributing to higher mortality rates in diabetics infected with Burkholderia pseudomallei. To investigate how diabetes alters the inflammatory response, we established a streptozotocin (STZ) -induced diabetic murine acute-phase melioidosis model. Viable B. pseudomallei cells were consistently detected in the blood, liver and spleen during the 42-hr course of infection but the hyperglycaemic environment did not increase the bacterial burden. However, after 24 hr, granulocyte counts increased in response to infection, whereas blood glucose concentrations decreased over the course of infection. A genome-wide expression analysis of the STZ-diabetic murine acute melioidosis liver identified ~1000 genes whose expression was altered in the STZ-diabetic mice. The STZ-diabetic host transcriptional response was compared with the normoglycaemic host transcriptional response recently reported by our group. The microarray data suggest that the presence of elevated glucose levels impairs the host innate immune system by delaying the identification and recognition of B. pseudomallei surface structures. Consequently, the host is unable to activate the appropriate innate immune response over time, which may explain the increased susceptibility to melioidosis in the STZ-diabetic host. Nevertheless, a general 'alarm signal' of infection as well as defence programmes are still triggered by the STZ-diabetic host, although only 24 hr after infection. In summary, this study demonstrates that in the face of a B. pseudomallei acute infection, poor glycaemic control impaired innate responses during the early stages of B. pseudomallei infection, contributing to the increased susceptibility of STZ-induced diabetics to this fatal disease.
In the present study, we describe the development of a DNA vaccine against chicken anemia virus. The VP1 and VP2 genes of CAV were amplified and cloned into pBudCE4.1 to construct two DNA vaccines, namely, pBudVP1 and pBudVP2-VP1. In vitro and in vivo studies showed that co-expression of VP1 with VP2 are required to induce significant levels of antibody against CAV. Subsequently, the vaccines were tested in 2-week-old SPF chickens. Chickens immunized with the DNA-plasmid pBudVP2-VP1 showed positive neutralizing antibody titer against CAV. Furthermore, VP1-specific proliferation induction of splenocytes and also high serum levels of Th1 cytokines, IL-2 and IFN-γ were detected in the pBudVP2-VP1-vaccinated chickens. These results suggest that the recombinant DNA plasmid co-expressing VP1 and VP2 can be used as a potential DNA vaccine against CAV.
The peroxisome proliferator-activated receptors (PPARs) have been implicated in regulating the immune response. We determined the relative changes in the transcriptional expression of PPAR isoforms (α, γ1 and γ2) and cytokines involved in the pathogenesis of type 1 diabetes (T1D) in the immune cells of 5 weeks, 10 weeks and diabetic male non-obese diabetic (NOD) mice compared to those of female NOD mice from our previous studies, "normalized" against their respective non-obese diabetic resistant (NOR) mice controls. Overall PPARα was significantly more elevated in the macrophages of female NOD mice of all age groups whereas PPARγ, particularly the PPARγ2 isoform was more depressed in the macrophages and CD4(+) lymphocytes of female NOD mice compared to their male counterparts. The pro-inflammatory cytokines, IL-1 and TNFα, as well as the Th1 cytokines, IL-2 and IFNγ were more elevated in female NOD mice whereas the Th2 cytokine, IL-4, was more depressed in these mice compared to their male counterparts. These findings suggest that the preponderance of T1D in female NOD mice may be influenced by the more pronounced changes in the expression of PPAR isoforms and pathogenic cytokines compared to those in male NOD mice.
The aim of this study was to determine the effect of exogenous nitric oxide (NO) on the induction of murine splenic immune response to Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS) in vitro. BALB/c mice were immunized with A. actinomycetemcomitans LPS and a control group was sham-immunized. Spleen cells were obtained, cultured and stimulated with A. actinomycetemcomitans LPS with or without the presence of S-nitroso acetyl-penicillamine (SNAP), a NO donor, and carboxy-PTIO, an NO scavenger. Culture supernatants were assessed for inducible nitric oxide synthase (iNOS) activity, specific IgG subclass levels, and both IFN-gamma and IL-4 levels. The results showed that in A. actinomycetemcomitans LPS-stimulated cells, SNAP enhances iNOS activity but inhibits the levels of specific IgG2a and IFN-gamma suggesting a Th1 response. The effect of SNAP on these immune parameters was ablated by carboxy-PTIO. These results suggest that exogenous NO may suppress the Th1-like immune response of A. actinomycetemcomitans LPS-stimulated murine spleen cells.
The aim of the present study was to test the hypothesis that colchicine may alter Aggregatibacter actinomycetemcomitans-induced immune response and abscess formation in mice. BALB/c mice were either sham-immunized or immunized with heat-killed A. actinomycetemcomitans. Spleen cells were stimulated with heat-killed A. actinomycetemcomitans in the presence or absence of colchicine. Specific IgG subclass antibodies, interferon-gamma (IFN-gamma), interleukin-4 (IL-4) and cell proliferation were determined. The animals were sham-immunized (group I) or immunized with heat-killed A. actinomycetemcomitans (groups II-VII). Colchicine was administered intraperitoneally before (group III), on the same day of (group IV), or after (group V) the primary immunization and on the same day of (group VI) or after (group VII) the secondary immunization. All groups were challenged with viable A. actinomycetemcomitans. The levels of serum-specific IgG subclasses and both IFN-gamma and IL-4 before and after bacterial challenge were assessed. The diameter of skin lesions was assessed. The results showed that colchicine augmented splenic-specific IgG1 and IL-4 as well as cell proliferation but suppressed specific IgG2a and IFN-gamma levels. Enhancement of serum-specific IgG1 and IL-4 levels, suppression of specific IgG2a and IFN-gamma levels as well as DTH response, and delayed healing of the lesions were observed in groups IV and VI, but not in the remaining groups of animals. Therefore, these results suggest that colchicine may induce a T helper 2 (Th2)-like immunity specific to A. actinomycetemcomitans in vitro and that colchicine administered on the same day as the immunization may stimulate a non-protective Th2-like immunity in A. actinomycetemcomitans-induced infections in mice.
It has previously been suggested that CD4+ T cells play a pivotal role in regulating the immune response to periodontal pathogens. The aim of the present study therefore was to determine delayed type hypersensitivity (DTH), spleen cell proliferation, serum and splenic anti-Porphyromonas gingivalis antibody levels, and lesion sizes following challenge with viable P. gingivalis in CD4-depleted BALB/c mice immunized with P. gingivalis outer membrane proteins (OMP).
Animal studies suggest that inducible nitric oxide synthase (iNOS) may be associated with destructive periodontal disease. l-N(6)-(1-Iminoethyl)-lysine (L-NIL) has been shown to inhibit iNOS in a selective manner, and hence the aim of the present study was to test the hypothesis that treatment with l-NIL may induce a T-cell helper 1 (Th1)-like immune response by Aggregatibacter (Actinobacillus) actinomycetemcomitans lipopolysaccharide (LPS)-stimulated murine spleen cells in vitro. BALB/c mice were either sham-immunized or immunized with A. actinomycetemcomitans LPS. Spleen cells were stimulated with A. actinomycetemcomitans LPS in the presence or absence of L-NIL. Nitric oxide (NO), iNOS activity, specific IgG subclass antibodies, interferon-gamma (IFN-gamma), and interleukin-4 (IL-4) levels and cell proliferation were determined. The results showed that treatment with L-NIL suppressed both NO production and iNOS activity but enhanced specific IgG2a, IFN-gamma levels, and increased cell proliferation following stimulation with A. actinomycetemcomitans LPS-stimulated cells. The results of the present study suggest that inhibition of iNOS activity by L-NIL may skew the A. actinomycetemcomitans LPS-stimulated murine splenic immune response towards the Th1-like immune profile in vitro.
Studies have shown that infectious bursal disease virus (IBDV) infects lymphoid cells, mainly B cells and macrophages. This study was aimed to examine the involvement of chicken splenic-derived dendritic cells (ch-sDCs) in specific-pathogen-free chickens following inoculation with IBDV vaccine strain (D78) and a very virulent (vv) strain (UPM0081). Following IBDV infection, enriched activated ch-sDCs were collected by using the negative selection method and were examined based on morphology and immunophenotyping to confirm the isolation method for dendritic cells (DCs). The presence of IBDV on enriched activated ch-sDCs was analyzed based on the immunofluorescence antibody test (IFAT), flow cytometry, and quantitative real-time PCR (RT-qPCR) while the mRNAs of several cytokines were detected using RT-qPCR. The isolated ch-sDCs resembled typical DC morphologies found in mammals by having a veiled shape and they grew in clusters. Meanwhile, the expression of DC maturation markers, namely CD86 and MHCII, were increased at day 2 and day 3 following vvIBDV and vaccine strain inoculation, respectively, ranging from 10% to 40% compared to the control at 2.55% (P < 0.05). At day 3 postinfection, IBDV VP3 proteins colocalized with CD86 were readily detected via IFAT and flow cytometry in both vaccine and vvIBDV strains. In addition, enriched activated ch-sDCs were also detected as positive based on the VP4 gene by RT-qPCR; however, a higher viral load was detected on vvIBDV compared to the vaccine group. Infection with vaccine and vvIBDV strains induced the enriched activated ch-sDCs to produce proinflammatory cytokines and Th1-like cytokines from day 3 onward; however, the expressions were higher in the vvIBDV group (P < 0.05). These data collectively suggest that enriched activated ch-sDCs were permissive to IBDV infection and produced a strong inflammatory and Th1-like cytokine response following vvIBDV infection as compared to the vaccine strain.
The involvement of interleukin-18 (IL-18) and the effects of modulating its release on the course of malaria infection were investigated using Plasmodium berghei ANKA infection in ICR mice as a model. Results demonstrated that plasma IL-18 concentrations in malarial mice were significantly elevated and positively correlated with the percentage parasitaemia development. Significant expressions of IL-18 were also observed in the brain, spleen and liver tissues. Slower development of parasitaemia was observed significantly upon inhibition and neutralization of IL-18, whereas faster development of parasitaemia was recorded when the circulating levels of IL-18 were further augmented during the infection. Inhibition and neutralization of IL-18 production also resulted in a significant decrease of plasma concentrations of pro-inflammatory cytokines (TNFα, IFNγ, IL-1α and IL-6), whereas the anti-inflammatory cytokine, IL-10, was significantly increased. Augmenting the release of IL- 18 during the infection on the other hand resulted in the opposite. Early mortality in malarial mice was also observed when the circulating levels of IL-18 were further augmented. Results proved the important role of IL-18 in immune response against malaria and suggest that IL-8 is pro-inflammatory in nature and may involve in mediating the severity of the infection through a pathway of elevating the pro-inflammatory cytokine and limiting the release of anti-inflammatory cytokine.
Polycyclic aromatic hydrocarbons (PAHs) are persistent pollutants and chemically a class of structurally similar chemical compounds characterized by the presence of fused aromatic rings. This research was undertaken to find out immunotoxic effects produced by pyrene, phenanthrene and fluoranthene. These chemicals were injected into developing chicks at three dose levels (0.2, 2 and 20 mg per kg) through allantioc route to rule out possible mechanisms involved in immunotoxicity. DNA adduct produced by PAHs in immune organs were analyzed by DNA adduct enzyme-linked immunosorbent assay (ELISA) kit and DNA damage was assessed by comet assay. A significant increase in the DNA adduct levels was found in thymus and bursa in 2 mg and 20 mg dose levels of pyrene, fluoranthene and phenanthrene treated groups, whereas those in spleen simulated the value of controls. Comet assay indicated that PAHs especially pyrene, fluoranthene and phenanthrene were capable of inducing increased level of comet parameters in thymus at all the dose levels. Bursa of Fabricius and spleen also showed a gradual rise in comet parameters corresponding to all dose levels, but the increase was more marked as in thymus. Thus, it can be concluded that DNA adducts produced by PAHs lead to single-strand breaks and reduced DNA repair, which ultimately begin a carcinogenic process. Hence, this experiment can be considered as a strong evidence of genotoxic potential of PAHs like pyrene, phenanthrene and fluoranthene in developing chicks.
Type 1 diabetes is an autoimmune disease that results from the destruction of the insulin-producing pancreatic beta islet cells, probably via the influence of cytokines. However, direct correlation between the expression of selected cytokines by various immune cells at different time points during the progression of the disease has not yet been clearly demonstrated. In this study, we showed that the mRNA expression of the pro-inflammatory cytokines, TNF-alpha, IL-1 beta, IL-6, and GM-CSF, were increased while the anti-inflammatory cytokine, TGF-beta, decreased in the peritoneal macrophages of nonobese diabetic (NOD) mice. IL-6 expression however decreased when the mice became diabetic. Surprisingly the expression of IFN-gamma and IL-2 by splenic CD4+ cells were lower in 5-week-old NOD mice as compared to the nonobese diabetic resistant (NOR) control mice, but their expression was higher in older NOD mice. The expression of IL-4 and IL-10 decreased in splenic CD4-positive lymphocytes. Splenic CD8-positive lymphocytes expressed increased levels of IFN-gamma and IL-10 but the latter decreased sharply when diabetes occurred. The relevance of these findings to the pathogenesis of type 1 diabetes is discussed.