Lesion Size In Mice Research Articles

Targeting NGF but not VEGFR1 or BDNF signaling reduces endometriosis-associated pain in mice

IntroductionEndometriosis is a chronic inflammatory disease that affects ∼ 10 % of women. A significant fraction of patients experience limited or no efficacy with current therapies. Tissue adjacent to endometriosis lesions often exhibits increased neurite and vascular density, suggesting that disease pathology involves neurotrophic activity and angiogenesis. ObjectivesWe aim to evaluate the potential for key tyrosine-kinase-receptor-coupled neurotrophic molecules to contribute to endometriosis-associated pain in mice. MethodsPeritoneal fluid was collected from endometriosis patients undergoing surgery and the levels of NGF and VEGFR1 regulators (VEGFA, VEGFB, PLGF, and sVEGFR1) were quantified by ELISA. VEGFR1 regulator concentrations were used to calculate VEGFR1 occupancy. We used genetic depletion, neutralizing antibodies, and pharmacological approaches to specifically block neurotrophic ligands (NGF or BDNF) or receptors (VEGFR1, TRKs) in a murine model of endometriosis-associated pain. Endometriosis-associated pain was measured using von Frey filaments, quantification of spontaneous abdominal pain-related behavior, and thermal discomfort. Disease parameters were evaluated by lesion size and prevalence. To evaluate potential toxicity, we measured the effect of entrectinib dose and schedule on body weight, liver and kidney function, and bone structure (via micro-CT). ResultsWe found that entrectinib (pan-Trk inhibitor) or anti-NGF treatments reduced evoked pain, spontaneous pain, and thermal discomfort. In contrast, even though calculated receptor occupancy revealed that VEGFR1 agonist levels are sufficient to support signaling, blocking VEGFR1 via antibody or tamoxifen-induced knockout did not reduce pain or lesion size in mice. Targeting BDNF-TrkB with an anti-BDNF antibody also proved ineffective. Notably, changing dosing schedule to once weekly eliminated entrectinib-induced bone-loss without decreasing efficacy against pain. ConclusionsThis suggests NGF-TrkA signaling, but not BDNF-TrkB or VEGF-VEGFR1, mediates endometriosis-associated pain. Moreover, entrectinib blocks endometriosis-associated pain and reduces lesion sizes. Our results also indicated that entrectinib-like molecules are promising candidates for endometriosis treatment.

Intraperitoneal Administration of 17-DMAG as an Effective Treatment against Leishmania braziliensis Infection in BALB/c Mice: A Preclinical Study.

Leishmaniasis is a significant global public health issue that is caused by parasites from Leishmania genus. With limited treatment options and rising drug resistance, there is a pressing need for new therapeutic approaches. Molecular chaperones, particularly Hsp90, play a crucial role in parasite biology and are emerging as promising targets for drug development. This study evaluates the efficacy of 17-DMAG in treating BALB/c mice from cutaneous leishmaniasis through in vitro and in vivo approaches. We assessed 17-DMAG's cytotoxic effect on bone marrow-derived macrophages (BMMΦ) and its effects against L. braziliensis promastigotes and intracellular amastigotes. Additionally, we tested the compound's efficacy in BALB/c mice infected with L. braziliensis via intraperitoneal administration to evaluate the reduction in lesion size and the decrease in parasite load in the ears and lymph nodes of infected animals. 17-DMAG showed selective toxicity [selective index = 432) towards Leishmania amastigotes, causing minimal damage to host cells. The treatment significantly reduced lesion sizes in mice and resulted in parasite clearance from ears and lymph nodes. It also diminished inflammatory responses and reduced the release of pro-inflammatory cytokines (IL-6, IFN-γ, TNF) and the regulatory cytokine IL-10, underscoring its dual leishmanicidal and anti-inflammatory properties. Our findings confirm the potential of 17-DMAG as a viable treatment for cutaneous leishmaniasis and support further research into its mechanisms and potential applications against other infectious diseases.

Inhibition of host PARP1 contributes to the anti-inflammatory and antitubercular activity of pyrazinamide

The antibiotic pyrazinamide (PZA) is a cornerstone of tuberculosis (TB) therapy that shortens treatment durations by several months despite being only weakly bactericidal. Intriguingly, PZA is also an anti-inflammatory molecule shown to specifically reduce inflammatory cytokine signaling and lesion activity in TB patients. However, the target and clinical importance of PZA’s host-directed activity during TB therapy remain unclear. Here, we identify the host enzyme Poly(ADP-ribose) Polymerase 1 (PARP1), a pro-inflammatory master regulator strongly activated in TB, as a functionally relevant host target of PZA. We show that PZA inhibits PARP1 enzymatic activity in macrophages and in mice where it reverses TB-induced PARP1 activity in lungs to uninfected levels. Utilizing a PZA-resistant mutant, we demonstrate that PZA’s immune-modulatory effects are PARP1-dependent but independent of its bactericidal activity. Importantly, PZA’s bactericidal efficacy is impaired in PARP1-deficient mice, suggesting that immune modulation may be an integral component of PZA’s antitubercular activity. In addition, adjunctive PARP1 inhibition dramatically reduces inflammation and lesion size in mice and may be a means to reduce lung damage and shorten TB treatment duration. Together, these findings provide insight into PZA’s mechanism of action and the therapeutic potential of PARP1 inhibition in the treatment of TB.

Therapeutic effects of Lucilia sericata larval excretion/secretion products on Leishmania major under in vitro and in vivo conditions

BackgroundLeishmaniasis is a neglected infectious disease caused by protozoa of the genus Leishmania. The disease generally manifests as characteristic skin lesions which require lengthy treatment with antimonial drugs that are often associated with adverse side effects. Therefore, a number of studies have focused on natural compounds as promising drugs for its treatment. This study aimed to evaluate the effects of larval excretion/secretion products (ES) of Lucilia sericata in crude and fractionated forms on Leishmania major, by using in vitro and in vivo models.MethodsThe in vitro experiments involved evaluation of ES on both promastigotes and macrophage-engulfed amastigotes, whereas the in vivo experiments included comparative treatments of skin lesions in L. major-infected mice with Eucerin-formulated ES and Glucantime.ResultsThe half maximal inhibitory concentrations of the crude ES, > 10-kDa ES fraction, < 10-kDa ES fraction, and Glucantime were 38.7 μg/ml, 47.6 μg/ml, 63.3 μg/ml, and 29.1 μg/ml, respectively. Significant differences were observed between percentage viabilities of promastigotes treated with the crude ES and its fractions compared with the negative control (P < 0.0001). The crude ES was more effective on amastigotes than the two ES fractions at 300 μg/ml. The macroscopic measurements revealed that the reduction of lesion size in mice treated with the crude ES followed quicker cascades of healing than that of mice treated with Glucantime and the ES fractions.ConclusionsThe present study showed that the larval ES of L. sericata in both crude and fractionated forms are effective for both intracellular and extracellular forms of L. major. Also, the ES exert both topical and systemic effects on mice experimentally infected with L. major.Graphical abstract

NF-κB activation in retinal microglia is involved in the inflammatory and neovascularization signaling in laser-induced choroidal neovascularization in mice

PurposeTo evaluate Nuclear Factor NF-κB (NF-κB) signaling on microglia activation, migration, and angiogenesis in laser-induced choroidal neovascularization (CNV). MethodsNine-week-old C57BL/6 male mice were randomly assigned to IMD-0354 treated or untreated groups (5 mice, 10 eyes per group). CNV was induced with a 532-nm laser. Laser spots (power 250 mW, spot size 100 μm, time of exposure 50 ms) were created in each eye using a slit-lamp delivery system. Selective inhibitor of nuclear factor kappa-B kinase subunit beta (IKK2) inhibitor IMD-0354 (10 μg) was delivered subconjunctivally; vehicle-treated mice were the control. The treatment effect on CNV development was assessed at five days post-CNV induction in vivo in C57BL/6 and Cx3cr1gfp/wt mice by fluorescent angiography, fundus imaging, and ex vivo by retinal flatmounts immunostaining and Western blot analysis of RPE/Choroidal/Scleral complexes (RCSC) lysates. In vitro evaluations of IMD-0354 effects were performed in the BV-2 microglial cell line using lipopolysaccharide (LPS) stimulation. ResultsIMD-0354 caused a significant reduction in the fluorescein leakage and size of the laser spot, as well as a reduction in microglial cell migration and suppression of phospho-IκBα, Vascular endothelial growth factor (VEGF-A), and Prostaglandin-endoperoxide synthase 2 (COX-2). In vivo and ex vivo observations demonstrated reduced lesion size in mice, CD68, and Allograft inflammatory factor 1 (IBA-1) positive microglia cells migration to the laser injury site in IMD-0354 treated eyes. The data further corroborate with GFP-positive cells infiltration of the CNV site in Cx3cr1wt/gfp mice. In vitro IMD-0354 (10–25 ng/ml) treatment reduced NF-κB activation, expression of COX-2, caused decreased Actin-F presence and organization, resulting in reduced BV-2 cells migration capacity. ConclusionThe present data indicate that NF-κB activation in microglia and it's migration capacity is involved in the development of laser CNV in mice. Its suppression by NF-κB inhibition might be a promising therapeutic strategy for wet AMD.

Immunization with a peptide mimicking Lipoteichoic acid protects mice against Staphylococcus aureus infection

Lipoteichoic acid (LTA), a major component of the cell wall of Staphylococcus aureus (S. aureus), is not generally considered as an ideal vaccine candidate since it is a thymus-independent antigen. In this study, we screened a 12-mer phage peptide library and identified a series of peptide sequences that can mimic the epitope of LTA. A tetra-branched multiple antigenic peptide, named MAP2-3, comprising one of the positive peptide sequences (GHKEDRQWCQHS), was synthesized. Immunization with MAP2-3 induced LTA-specific IgG antibodies, prolonged the survival time, and decreased the bacterial burden in organs of mice infected with S. aureus. Moreover, passive immunization with polyclonal anti-MAP2-3 sera reduced bacterial load in organs of mice with bacteremia, alleviated acute lung injury in mice with pneumonia, and decreased the size of lesions in mice with skin infection. The number of LTA-specific antibody-secreting cells in the spleen of MAP2-3 immunized mice were significantly higher than that in the control mice. In summary, as a surrogate of LTA, vaccination with MAP2-3 elicited humoral immune response and protected mice from S. aureus infection. This study provides a new option to design vaccines against S. aureus.

C-reactive protein is an atheroprotective molecule

Abstract The co-localization of plasma C-reactive protein (CRP) and atherogenic low-density lipoprotein (LDL) at the atherosclerotic lesions raises the possibility of a role of CRP in the disease process. In vitro, in its native pentameric structural form, CRP does not bind to oxidized LDL. However, in its non-native pentameric structural form, CRP is capable of binding to oxidized LDL. It has been proposed that CRP changes its structure at sites of inflammation to gain the oxidized LDL-binding activity. In vivo, native CRP is neither pro-atherosclerotic nor atheroprotective in animal models of atherosclerosis. We assumed that native CRP shows no effect because inflammatory microenvironment in the arterial wall in animal models of atherosclerosis is not appropriate and, therefore, the structure of CRP remains unchanged. Accordingly, we hypothesized that a CRP mutant, generated by site-directed mutagenesis, capable of binding to oxidized LDL without the requirement of any further structural change should show an effect on the disease. In the current study, we evaluated the impact of such a CRP mutant on the development of atherosclerosis employing LDL receptor knockout mouse model of atherosclerosis. We found that there was a two-week delay in the development of atherosclerotic lesions in the aortic root of mice treated with mutant CRP compared to mice that did not receive CRP. The development of atherosclerotic lesions in the whole aorta was also reduced; there was 30% reduction in the size of lesions in mice treated with mutant CRP. Overall, the data indicate that CRP is indeed an atheroprotective molecule.

Effect of hydroalcoholic extract of Echinacea purpurea in combination with meglumine antimoniate on treatment of Leishmania major-induced cutaneous leishmaniasis in BALB/c mice

Context:Progressive resistance of Leishmania parasite to available drugs including, meglumine antimoniate, has been reported from various regions of the world, especially Iran.Aims:This study was conducted to evaluate the effect of hydroalcoholic extract of Echinacea purpurea in a combination therapy with glucantime in the treatment of cutaneous leishmaniasis caused by Leishmania major.Materials and Methods:Hydroalcoholic extract of E. purpurea was prepared from the plant. Amastigote form of L. major was inoculated to the tail base of thirty mice. After their tails became wounded, mice were divided into six groups. The first group was used as control and the second group received 100 mg/kg of Echinacea extract (orally). The third group was treated by meglumine antimoniate with dose of 20 mg/kg. Combination therapy was used for group four, five, and six where the mice received a different concentration of extract (100–200 mg/kg) and glucantime (10–20 mg/kg). The size of the cutaneous lesion on tail base was measured regularly. Findings were analyzed by SPSS software and using Kruskal-Wallis test.Results:The sizes of the lesion were increased in all mice of control group by the time. The mean size of lesions in mice receiving the extract and/or receiving the extract along with meglumine antimoniate was lower than those of control mice, but the differences were not statistically significant (P > 0.05). On the other hand, the differences between the group of mice which received meglumine antimoniate alone, and the rest of groups were statistically significant (P < 0.05).Conclusion:E. purpurea extract in doses which have been used in this study and combination with meglumine antimoniate was not much effective against L. major in BALB/C mice.

Ruthenium-Clotrimazole complex has significant efficacy in the murine model of cutaneous leishmaniasis

In previous studies we reported a novel series of organometallic compounds, RuII complexed with clotrimazole, displaying potent trypanosomatid activity with unnoticeable toxicity toward normal mammalian cells. In view of the promising activity of Ru-clotrimazole complexes against Leishmania major (L. major), the present work sought to investigate the anti-leishmanial activity of the AM162 complex in the murine model of cutaneous leishmaniasis. In addition, to facilitate the design of new therapeutic strategies against this disease, we investigated the mode of action of two Ru-clotrimazole complexes in L. major promastigotes. Overall, we demonstrate that AM162 significantly reduced the lesion size in mice exposed to L. major infection. In addition, Ru-clotrimazole compounds are able to induce a mitochondrial dependent apoptotic-like death in the extracellular form of the parasite based on labeling of DNA fragments, mitochondrial depolarization, cell cycle alteration profile and plasma membrane phospholipid externalization. Our findings reveal a promising efficacy of the Ru-clotrimazole AM162 complex for the treatment of cutaneous leishmaniasis, as well as pro-apoptotic activity and thus guarantees further evaluation in pre-clinical studies.

Anti-Leishmanial Activity (In Vitro and In Vivo) of Allicin and Allicin Cream Using Leishmania major (Sub-strain Zymowme LON4) and Balb/c Mice

BackgroundLeishmania is a unicellular protozoan parasite that produces several human diseases, ranging from localized self-healing cutaneous lesions to deadly visceral infections.ObjectiveThe effect of allicin on the growth of Leishmania major (L. major) promastigotes was evaluated under in vitro conditions. Moreover, the efficacy of a topical allicin cream was examined in BALB/c (Bagg albino, laboratory-bred strain of the House Mouse) mice with cutaneous leishmanial lesions compared to the currently used drug, sodiumstibogluconate (pentostam).MethodsCytotoxiciy and promastigote proliferation were measured. Different concentrations (50, 100, 150, and 200 μM) of liquid allicin were tested on L. major promastigotes twice: after 24 and 48 hours using an MTT colorimetric assay. In the in vivo condition, the efficacies of allicin cream and liquid allicin at two concentrations (0.15 μM/mouse and 0.30 μM/mouse) were evaluated. Serum factors of the control and treated groups were tested to evaluate the toxic effects of allicin on the liver and kidney.ResultsAllicin at a concentration of 50 μM inhibited the growth of Leishmania promastigotes. Topical application of allicin cream reduced lesion sizes in mice. No significant differences in biochemical analysis were observed between the control and treated groups.ConclusionsAllicin has antileishmanial effects under in vitro and in vivo conditions and may be used in clinical applications.

Choroidal Neovascularization Is Inhibited in Splenic-Denervated or Splenectomized Mice with a Concomitant Decrease in Intraocular Macrophage.

PurposeTo determine the involvement of sympathetic activity in choroidal neovascularization (CNV) using laser-induced CNV in a mouse model.MethodsWe investigated changes in the proportions of intraocular lymphocytes, granulocytes, and three macrophage subtypes (Ly6Chi, Ly6Cint, and Ly6Clo) after laser injury in mice using flow cytometry, and evaluated CNV lesion size in mice lacking inflammatory cells. Further, we evaluated the lesion size in mice administered the β3 receptor antagonist, splenic-denervated and splenectomized mice. We also assessed changes in the proportions of intraocular macrophages and peripheral blood monocytes in splenic-denervated and splenectomized mice. Lastly, lesion size was compared between splenic-denervated mice with or without adoptive transfer of macrophages following laser injury. After Ly5.1 mice spleen-derived Ly6Chi cells were transferred into Ly5.2 mice, the proportions of intraocular Ly5.1+Ly6Chi cells were compared.ResultsIn WT mice, the proportion of CD4+ T cells recruited into the eye increased progressively from day 3 to day 7 after laser injury, whereas, intraocular CD8+ T cells did not change significantly. Proportions of B220+ cells, granulocytes, and two subtypes of intraocular macrophages (Ly6Chi and Ly6Clo) peaked at day 3 following laser injury. In contrast, Ly6Cint/loCD64+ subtype showed a significantly higher percentage at day 7 after laser injury. There were no differences in lesion size between CD4–/–or Rag2–/–mice and controls, whereas lesion size was significantly reduced in CCR2−/− mice and clodronate liposome-treated mice. CNV lesion area was significantly reduced in mice with β3 blocker treatment, splenic-denervated and splenectomized mice compared with controls. Intraocular Ly6Chi macrophages were also reduced by splenic denervation or splenectomy. Adoptive transfer of spleen-derived Ly6Chi cells increased the lesion size in splenic-denervated mice. Compared with controls, intraocular donor-derived Ly6Chi cells recruited into the eye were reduced in splenic-denervated and splenectomized mice.ConclusionsAlthough lymphocytes had little effect on CNV formation, Ly6Chi macrophages/monocytes exacerbated CNV in mice. Sympathetic activity might contribute to CNV via the recruitment of macrophages to the eye.

Abstract 676: 14q32 MicroRNA Inhibition Reduces Atherosclerotic Lesion Formation and Increases Plaque Stability

Introduction: Atherosclerosis is a multifactorial disease involving inflammatory, metabolic and lipid-related processes. As microRNAs (miRs) regulate expression of up to hundreds of target genes, we aimed to identify miRs that target genes involved in all processes of atherosclerosis. Using www.targetscan.org, we performed a Reverse Target Prediction on a set of 150 atherosclerosis-related genes. We found enrichment of binding sites for several miRs from a single gene cluster on human chromosome 14. Methods: ApoE-/- mice fed a Western type diet received semi-constrictive collars around both carotid arteries to induce atherosclerotic lesion formation. Gene Silencing Oligonucleotides (GSOs) were used to inhibit 3 14q32 miRs: miR-329, miR-494 and miR-495. Mice were injected with 1 mg GSO at day 4 after collar placement and again with 0.5 mg GSO at day 18. Mice were sacrificed at day 7 and day 28 for analysis of carotid arteries, liver, spleen, bone marrow and blood samples. Results: Atherosclerotic plaque analysis revealed a striking decrease of lesion size in mice treated with GSO-494 and GSO-495 (GSO-Control: 47 ± 11*103 μm2; GSO-494: 16 ± 3*103 μm2; GSO-495: 22 ± 5*103 μm2), whereas a trend towards reduced plaque size was observed in mice treated with GSO-329 (27 ± 6*103 μm2). Moreover, plaque stability was increased in mice treated with GSO-494 and GSO-495, determined by both a decrease in necrotic core size (GSO-494: 80% decrease; GSO-495: 60% decrease compared to GSO-Control) and an increase in plaque collagen content (GSO-Control: 6.6 ± 1.6%; GSO-494: 12.7 ± 2.1%; GSO-495: 15.8 ± 3.1%). Furthermore, inhibition of miR-494 and miR-495 resulted in decreased plasma cholesterol levels (GSO-Control: 30.4 ± 1.1 mM; GSO-494: 26.4 ± 0.7 mM; GSO-495: 26.4 ± 1.6) and decreased VLDL fractions. Conclusions: Inhibition of miR-494, miR-495 and, to a lesser extent, miR-329, leads to reduced plaque size, increased plaque stability and reduced plasma cholesterol levels. This makes 14q32 miRs promising novel therapeutic targets in atherosclerosis.

Slit2 Overexpression Results in Increased Microvessel Density and Lesion Size in Mice With Induced Endometriosis

We recently reported that Slit/Roundabout (ROBO) 1 pathway may be a constituent biomarker for recurrence of endometriosis, likely through promoting angiogenesis. In this study, we sought to determine as whether Slit2 overexpression can facilitate angiogenesis, increase lesion size, and induce hyperalgesia in mice with induced endometriosis. We used 30 Slit2 transgenic (S) and 29 wild-type (W) mice and cross-transplanted endometrial fragments from S to W (group SW) and vice versa (group WS), and also within the S and W (groups SS and WW, respectively), into the peritoneal cavity, inducing endometriosis. We also performed a sham surgery within both S and W mice (groups Sm and Wm, respectively). The size of the ectopic implants, microvessel density (MVD) and immunoreactivity to ROBO1, and vascular endothelial cell growth factor (VEGF) in ectopic and eutopic endometrium, along with hotplate and tail-flick tests in all mice, were then evaluated. We found that the induction of endometriosis resulted in generalized hyperalgesia, which was unaffected by Slit2 overexpression. Slit2 overexpression did increase the lesion size significantly and correlated positively with the MVD in ectopic and eutopic endometrium. Slit2 expression levels appear to correlate with the MVD, but not with VEGF immunoreactivity, in ectopic endometrium. Consequently, we conclude that Slit2 may play an important role in angiogenesis in endometriosis. The increased angiogenesis, as measured by MVD, but not VEGF immunoreactivity, likely resulted in increased lesion size in induced endometriosis. Thus, SLIT2/ROBO1 pathway may be a potential therapeutic target for treating endometriosis.

Abstract 3872: Transgenic HuR in Astrocytes Increases Ischemic Lesion Size in Mice

Introduction: Stroke is a serious neurological disorder that affects more than 800,000 Americans each year. HuR, an mRNA binding protein, regulates gene expression by modulating the stability and translational efficiency of mRNAs containing adenine-uridine rich elements in the 3’ untranslated region. Genes implicated in neuroprotection and post-ischemic repair may be regulated by HuR. The current investigation utilizes transgenic mice engineered to express Flag-HuR in astrocytes using the glial fibrillary acidic protein (GFAP) promoter. We hypothesized that modulation of mRNA stability in astrocytes would change the outcome from transient cerebral ischemia. Methods: Four mouse cohorts were studied: male or female and transgenic (TG) or wild-type (WT). Mice underwent 30-minute transient right middle cerebral artery occlusion (MCAO) and survived for 24 or 72h. Neurological scores were recorded after ischemia and prior to euthanasia, and brain tissue was sectioned for histology and immunolabeling. In a blinded fashion, Flag-HuR expressing nuclei were counted in peri-lesional regions of interest (ROI) and contralateral mirror ROIs, and ischemic/contralateral indices were calculated. Representative lesion size was determined in hematoxylin-eosin sections at the level of Bregma 1.10 mm. Results:HuR-Flag expression: The transgene was expressed in peri-lesional astrocytes. 24h after MCAO, hormonally intact female mice had higher levels of Flag-HuR expression: Flag-HuR index was 5.9 ± 1.9 in females (n = 7) vs. 2.0 ± 0.7 in males (n = 6), p = 0.002, Mann-Whitney rank sum test. 72h after MCAO, TG expression declined in females to levels comparable to those of males. Lesion size: There was no difference in lesion size between sex-matched TG and WT mice 24h post-MCAO, but at 72h lesion size was larger in TG females than WT females: 47% ± 12 (n = 10) vs. 22% ± 11 (n = 5), respectively, p = 0.002, t-test. Analysis of neurological scores, sex hormone levels, and long term survival is ongoing. Discussion: We hypothesized that altering mRNA stability in ischemic astrocytes would change outcome from cerebral ischemia. Initial characterization of TG mice in which HuR was expressed under control of the GFAP promoter showed that ischemia results in peri-lesional upregulation of the transgene, which is more robust in females than males 24h after MCAO and comparable to males 72h after MCAO. Transgene expression correlated with increased lesion size in females 72h after MCAO. While the mechanism of this effect requires elucidation, our results suggest that HuR is a viable novel target for further investigation in cerebral ischemia.

Electroacupuncture at the ST36 acupoint increases interleukin-4 responsiveness in macrophages, generation of alternatively activated macrophages and susceptibility to Leishmania major infection

BackgroundElectroacupuncture (EA) has been used to treat inflammatory diseases. Alternatively activated macrophages (AAMo) stimulated by cytokines such as interleukin (IL)-4, IL-10 and IL-13 are anti-inflammatory and mildly microbicidal. This study aimed to evaluate whether EA at the Zusanli acupoint (ST36) would change the profile of healthy murine macrophages, particularly the generation of AAMo and susceptibility to Leishmania major infection.MethodsBALB/c mice were treated with EA (15/30 Hz) at the ST36 acupoint for 20 min/d for 5 d. After the final EA session, the mice were euthanized and their peritoneal cells were harvested and counted for determination of arginase activity, nitric oxide (NO) production and microbicidal activity after culture in the presence or absence of IL-4, interferon-γ (IFNγ) or lipopolysaccharide (LPS) or both IFNγ and LPS. Twelve mice were infected with L. major promastigotes into the footpads after the final EA session and the infection course was monitored.ResultsPeritoneal cells freshly obtained from EA-treated mice had similar arginase and microbicidal activities to cells from sham-treated mice. After culture with IL-4, cells from EA-treated mice exhibited significant increases in the arginase activity (sham: 58 ± 11.3 vs. EA: 80.7 ± 4.6%, P = 0.025) and number of parasites/infected cell (sham: 2.5 ± 0.4 vs. EA: 4.3 ± 0.8 cells, P = 0.007). The NO production was lower in cells from EA-treated mice cultured in the presence of a combination of IFNγ and LPS (sham: 31.6 ± 6.5 vs. EA: 22.3 ± 2.1 μM, P = 0.025). The lesion size in mice infected with L. major promastigotes was larger in EA-treated mice (sham: 3.26 ± 0.29 vs. EA: 2.23 ± 0.4 mm, P = 0.039).ConclusionEA at the ST36 acupoint increases IL-4 responsiveness in macrophages, Generation of AAMo and susceptibility to L. major infection

Genotypically distinct strains of Leishmania major display diverse clinical and immunological patterns in BALB/c mice

Infection with Leishmania major species is endemic in many provinces of Iran. Isolates from four endemic areas located in north (Damghan), center (Kashan), west (Dehloran), and south (Shiraz) of country which showed major distinctive polymorphism by RAPD-PCR method were evaluated. Isolates were inoculated to different groups of BALB/c mice and their clinical and immunological status was compared. Lesion size, parasite burden and T cell phenotype in lymph node (LN), and cytokine secretion in the culture of LN mononuclear cells were determined. The results showed the lowest and highest lesion sizes in mice infected by Shiraz strain (3.02 ± 0.52 mm) and Kashan strain (5.20 ± 0.45), respectively, 8 weeks after inoculation. No significant difference was observed between other strains. The parasite burden was significantly lower in lymph node of mice infected with strain of Damghan (1.51 × 10 7) than Kashan (3.60 × 10 9) and Shiraz (7.08 × 10 9) strains, 8 weeks post-infection. However, Dehloran strain showed intermediate load of viable parasites (1.51 × 10 9) in LN, 8 weeks post-infection. High ratios of IFN-γ/IL-4 were shown in mice inoculated by strain of Dehloran (3.17) and Damghan (2.66), but not in mice infected by other strains, 8 weeks post-infection. The highest and lowest ratios of CD4 +/CD8 + T cells were found in LN cells of mice infected with Kashan (1.82) and Dehloran (1.00) strains, respectively. Results indicate that the lowest and intermediate loads of parasites induced by Damghan and Dehloran strains along with higher ratio of IFN-γ/IL-4 produced by both strains in LN of inoculated mice suggest that these strains have the capacity to shift the immune responses to a predominant Th1 response after 8 weeks infection in BALB/c mice and might be the ideal strains for vaccine studies and development of candidate vaccine against leishmaniasis.

Confocal scanning laser microscopy measurments of atherosclerotic lesions in mice aorta: A fast evaluation method for volume determinations

Atherosclerotic lesion size in mice is routinely evaluated by morphometrical measurements on serial sections in order to obtain volume measurements. The technique of confocal scanning laser microscopy (CSLM) makes it possible to optically scan and thereby evaluate a tissue sample. We here describe a method for measuring lesion volume in ApoE/LDLr deficient mice at 20 and 30 weeks of age using the non-destructive procedure of CSLM. Whole mount preparations of opened aorta with the lumen side facing the cover slip were analysed under 10× magnification in a CSLM (Leica). The autofluorescence of the elastic fibres of the lamina interna as opposed to the non-fluorescing lesion was used to define the bottom and top of the lesion during scanning. Ten to forty images were collected 2.4 μm apart, depending on the size of the lesion, and the stack of images was then analysed using Imaris (Bitplane). After the CSLM evaluation, the aortas were de-mounted, embedded in paraffin, sectioned, stained in hematoxylin and eosin and the volume re-evaluated with conventional morphometry. Statistical evaluation showed that the results obtained with CSLM and the results of morphometry were positively correlated. Area measurements of the plaques using en face preparations of aorta showed that the plaque area was generally larger at the left side and a significant increase of plaque area along the length of the thoracic aorta. Our results showed that atherosclerotic lesions in mice can be quantitatively evaluated by CSLM.

Role of leukotriene B4 receptors in the development of atherosclerosis: potential mechanisms.

Leukotriene B4 (LTB4), a potent leukocyte chemoattractant, is known to promote several inflammatory diseases, including atherosclerosis. We sought to determine mechanisms through which LTB4 modulates atherosclerosis in cell lines expressing LTB4 receptors, BLT-1, and in mice deficient in BLT-1 as well as macrophage cell lines derived from BLT-1+/+ and BLT-1-/- mice. Analysis of global changes in gene expression induced by LTB4 in rat basophilic leukemia cells (RBL-2H3) expressing the human BLT-1 showed highest-fold increase in expression of fatty acid translocase/CD36 and the chemokine MCP1/JE/CCL2, which are critical in atherogenesis. To determine the importance of BLT-1 in atherogenesis, we crossed BLT-1-null mice with apolipoprotein (apo)-E-deficient mice, which develop severe atherosclerosis. Deletion of BLT-1 significantly reduced the lesion formation in apo-E-/- mice only during initiating stages (4 and 8 weeks) but had no effect on the lesion size in mice fed atherogenic diet for 19 weeks. Macrophage cell lines from BLT-1-deficient mice expressed the low-affinity LTB4 receptor, BLT-2, and exhibited chemotaxis to LTB4. The effects of LTB4 in atherosclerosis are likely mediated through the high-affinity BLT-1 and the low-affinity BLT-2 receptors. LTB4 promotes atherosclerosis by chemo-attracting monocytes, by providing an amplification loop of monocyte chemotaxis via CCL2 production, and by converting monocytes to foam cells by enhanced expression of CD36 and fatty acid accumulation.

Macrophage-specific p53 expression plays a crucial role in atherosclerosis development and plaque remodeling.

We first showed that absence of p53 accelerates atherosclerosis development in apoE-deficient mice. In this study, we investigated how macrophage-specific loss of p53 function might modulate atherosclerosis development in LDL receptor-deficient mice, a model for familial hypercholesterolemia. We transferred bone marrow cells isolated from p53+/+ and p53-/- mice to lethally irradiated LDL receptor-/- mice and evaluated the aortic atherosclerotic lesion areas in the recipients at different times afterward. At 15 weeks and again at 20 weeks, we found larger aortic lesion size in mice receiving p53-/- cells compared with those that received p53+/+ cells. By measuring the rate of bromodeoxyuridine incorporation, we found that the absence of p53 in macrophages stimulates cellular proliferation. In contrast, the rate of apoptosis in the atheromatous lesion was similar in the two groups of mice. Furthermore, we found that the absence of macrophage-specific p53 expression was associated with vulnerable-appearing lesions marked by increased tissue necrosis and reduced collagen deposition. p53 plays a crucial role in atherosclerosis lesion development and remodeling, and macrophage-specific p53 deficiency stimulates cellular proliferation leading to a vulnerable-appearing phenotype of lesions in a mouse model of familial hypercholesterolemia.

In vitro environmental regulation of Porphyromonas gingivalis growth and virulence.

Porphyromonas gingivalis appears to be a major contributor to periodontal disease, especially soft tissue destruction, which is reflected by the ability to cause invasive, spreading lesions, and tissue inflammation in a murine abscess model. This study investigated the role of hemin on the regulation of growth and virulence of P. gingivalis strains. P. gingivalis strains W50, A7A1-28, 3079, 381, W50/BEI, and NG4B19 were grown in broth and on blood agar plates. P. gingivalis cells grown under iron-depleted conditions for multiple passages showed significantly decreased lesion size in mice, in contrast to cells grown under iron-normal (5 microg/ml) and iron-elevated conditions. Statistically significant (P < 0.01) decreases in gingipain enzyme activity were found among the strains grown under iron-depleted conditions. P. gingivalis grown in the presence of blood induced significantly different lesion type, lesion size, lesion onset, and mortality. Elevated hemin resulted in increased cell-associated iron in P. gingivalis, which increased the capacity of the microorganism to survive at times of iron deprivation. These results indicate that hemin or iron availability regulates multiple aspects related to P. gingivalis virulence, including growth, survival, gingipain levels, and iron accumulation.