Injection Of MDP Research Articles

A173 ELUCIDATING THE EFFECTS OF NOD2-MEDIATED SIGNALLING ON INTESTINAL RESIDENT-MEMORY T-CELL FORMATION AND FUNCTION

Abstract Background Aberrant resident memory T-cell (TRM) responses have been associated with increased intestinal inflammation and Crohn’s disease (CD) pathology in humans. Intestinal TRM cells are not only important for maintaining the integrity of the intestinal epithelial barrier, but also for the rapid clearance of pathogens in the intestine during infection. Understanding the signals received by the intestinal immune system to generate TRM responses is paramount to elucidating treatments for CD. Genetic mutations in NOD2 are associated with the highest risk of CD development. As a host intracellular sensor of bacterial peptidoglycan, NOD2 is critical for initiating both innate and adaptive immune responses. Furthermore, work from our lab as well as those of our collaborators suggest that NOD2 deficiency reduces systemic memory B and T-cell responses. However, the role of NOD2 in establishing memory T-cell responses in the intestine remains unclear. This work will therefore establish the role of NOD2 signaling in initiating and maintaining optimal TRM responses to achieve intestinal homeostasis and resilience to intestinal inflammation. Purpose It is the main objective of this project to determine whether NOD2-mediated signalling affects: 1. Antigen-specific T-cell priming in vivo 2. Bona fide intestinal TRM generation 3. Bona fide intestinal TRM function Method To address the effects of NOD2-signalling on intestinal T-cell priming in vivo, wildtype (WT) mice were adoptively transferred 50,000 naïve LCMV-specific (SMARTA) CD4+ T-cells. Mice were subsequently infected with LCMV-Armstrong in the presence or absence of the NOD2 agonist; MDP. 5-days following infection, the numbers and percentage of LCMV-specific T-cells in the mesenteric lymph nodes and spleen were examined. To examine the effects of NOD2 on intestinal TRM generation, littermate WT and NOD2 KO mice were infected with LCMV-Armstrong. Thirty-six-days following infection, the percentage and number of LCMV-specific CD4+ T-cells were profiled in the small and large intestinal lamina-propria by means of gp66-77 class-II MHC-tetramer staining. In another set of experiments, littermate WT and NOD2 KO mice were re-infected with LCMV-C13 30-days following LCMV-Armstrong immunization, and the interferon-response in the small intestine was profiled by quantitative PCR to assess the effect of NOD2-deficiency on antigen recall responses. Result(s) NOD2-stimulation by means of MDP injection increased the percentage and number of adoptively transferred SMARTA CD4+ T-cells in the mesenteric lymph nodes upon LCMV infection. Furthermore, NOD2-deficiency did not alter intestinal LCMV-specific CD4+ TRM seeding in the small and large intestinal lamina propria 36 days after infection. However, in vivo antigen recall experiments showed a decreased intestinal IFN response in NOD2 KO mice. Conclusion(s) Our findings reveal a potential role of NOD2 in the intestinal CD4+ T-cell priming and subsequent Ag-specific memory response. Disclosure of Interest None Declared

Normal Skeletal Standardized Uptake Values Obtained from Quantitative Single-Photon Emission Computed Tomography/Computed Tomography: Time-Dependent Study on Breast Cancer Patients.

Aim:To estimate the standard uptake values (SUVs) of Tc-99m methylene-diphosphonate (Tc-99m MDP) from normal skeletal sites in breast cancer patients using quantitative single-photon emission computed tomography (SPECT).Materials and Methods:A total of 60 breast cancer patients who underwent Tc-99m MDP SPECT/CT study at different postinjection acquisition times were included in this study. Based on postinjection acquisition time, patients were divided into four study groups (n_15 each), i.e. Ist (2 h), IInd (3 h), IIIrd (4 h), and IVth (5 h). Image quantification (SUVmax and SUVmean) was performed using Q.Metrix software. Delineation of volume of interest was shaped around different bones of the skeletal system.Results:The highest normal SUVmax and SUVmean values were observed in lumber and thoracic vertebra (8.89 ± 2.26 and 2.89 ± 0.58) for Group I and in pelvis and thoracic (9.6 ± 1.32 and 3.04 ± 0.64), (10.93 ± 3.91 and 3.65 ± 0.97), (11.33 ± 2.67 and 3.65 ± 0.22) for Group II, III and IV, respectively. Lowest normal SUVmax and SUVmean values were observed in humerus and ribs (3.22 ± 0.67 and 0.97 ± 0.18), (5.16 ± 1.82 and 1.18 ± 0.16) for Group I, IV, and in humerus (3.17 ± 0.58 and 0.85 ± 0.26), (3.98 ± 1.12 and 1.04 ± 0.28) for Group II and III, respectively. Significant difference (P < 0.05) noted in SUVmax for sternum, cervical, humerus, ribs, and pelvis with respect to time. However, significant difference (P < 0.05) noted in SUVmean for all skeletal sites with respect to time.Conclusions:Our study shows variability in normal SUV values for different skeletal sites in breast cancer patients. Vertebral bodies and pelvis contribute highest SUV values. Time dependency of SUVs emphasizes the usefulness of routinely acquired images at the same time after Tc-99m MDP injection, especially in follow-up studies.

Triggering of NOD2 Receptor Converts Inflammatory Ly6Chigh into Ly6Clow Monocytes with Patrolling Properties

The signals that regulate the fate of circulating monocytes remain unknown. In the present study, we demonstrate that triggering of the NOD2 receptor bymuramyl dipeptide (MDP) converts inflammatory Ly6Chigh monocytes into patrolling Ly6Clow monocytes. Administration of MDP to Nr4a1-/- mice, which lack Ly6Clow monocytes, or to Ly6Clow-depleted mice led to the emergence of blood-patrolling monocytes with a profile similar to that of Ly6Clow monocytes, including high expression of CX3CR1 and LFA1. Using intravital microscopy in animal models of inflammatory diseases, we also found that converted Ly6Chigh monocytes patrol the endothelium of blood vessels and that their presence contributes to a reduction in the inflammatory response following MDP injection. Our results demonstrate that NOD2 contributes to the regulation of blood monocytes and suggest that it could be therapeutically targeted to treat inflammatory diseases.

Increased Liver and Spleen Accumulation of Tc-99m Methylene Diphosphonate Associated With Intravenous Injection of MRI Contrast Gadolinium-Diethylenetriaminepentaacetic Acid

Several conditions that can cause diffuse hepatic or splenic uptake of Tc-99m methylene diphosphonate (Tc-99m MDP) have been previously reported. Nevertheless, diffuse abnormal liver and spleen uptake of Tc-99m MDP associated with intravenous injection of magnetic resonance imaging contrast gadolinium-diethylenetriaminepentaacetic acid is not previously known. In our series, we reported diffuse increased Tc-99m MDP activity in the liver and spleen in bone scans in patients who received Tc-99m MDP injection shortly after contrast-enhanced magnetic resonance imaging.

The acute phase response in Japanese quail ( Coturnix coturnix japonica)

Experiments were conducted to determine the effect of injection of lipopolysaccharide (LPS, from S. typhimurium) or muramyl dipeptide (MDP, N-acetylmuramyl- l-ala-isoglutamine) in Japanese quail. Doses of MDP between 0.3 and 10 mg/kg body wt. had no effect on body temperature. In contrast, doses of 1.0–22.5 mg LPS/kg body wt. caused significant increases in body temperature. None of the doses of LPS or MDP resulted in mortality. The febrile response to LPS was diminished following a second injection 48 h after the first, and was absent following a third injection. Plasma zinc, an indicator of the acute phase response, was significantly reduced by either LPS or MDP after the first injection ( P<0.001), but not after the second or third injection. Splenic interleukin 1-β (IL-1β) mRNA expression was increased after the first and last injection of LPS ( P<0.001), but only after the first injection of MDP ( P<0.005). Hepatic IL-1β mRNA expression was increased after the first, but not the third injection of LPS ( P<0.001), while MDP had no effect. These data indicate that Japanese quail are less sensitive to MDP than LPS, and that quail demonstrate tolerance to LPS following repeated injections.

Muramyl-dipeptide-induced fever in cats.

The characteristics of fever induced by intravenous injection of muramyl dipeptide (MDP; 25 micrograms/kg) were studied in cats implanted with electrodes for chronic recording of electromyograms, brain temperature (Tb) and subcutaneous temperature. Intravenous injection of MDP was followed by a bimodal fever response after a latent period of 19.4 +/- 1.3 min. Maximum Tb was reached 62.0 +/- 20.7 min after injection, with a second minor peak at about 4 h following injection. Tb returned to baseline after 6-8 h. A single intravenous injection of MDP induced a stable reproducible thermal response which allowed to study the properties of body temperature and effector mechanisms during fever in cats.

Modification of delayed-type hypersensitivity reactions by muramyldipeptide in guinea pigs

N-Acetylmuramyl- l-alanyl- d-isoglutamine (muramyldipeptide, MDP) modulated delayed-type hypersensitivity (DTH) reactions and induced severe inflammatory lesions in guinea pigs. The animals immunized with heat-killed Mycobacterium tuberculosis were challenged with the purified protein derivative (PPD) at the flanks and the corneas to prepare DTH reactions at 2 weeks after the immunization, thereafter 24 h the animals received subcutaneous injections of MDP at the flanks of the opposite side. At the skin with the DTH reaction, increase of swelling and redness accompanied with hemorrhage and necrosis were observed. As corneal reactions in the animals that had received MDP, increase of cornea thickness, opaque and grayish-white and the projection of eyes accompanied with severe iritis were observed. Modification of the skin reaction occurred from 2 h after the MDP injection, rapidly increased to the maximum level around 10 h, maintained the level until 24 h, then slowly decreased. The polymorphonuclear leukocyte infiltration was observed from 15 min after the MDP injection, and tumor necrosis factor alpha, interleukin (IL)-1, and IL-6 levels in the serum and skin lesions increased after the MDP injection. Synthetic muramyltripeptide ( N-acetylmuramyl- l-alanyl- d-isoglutaminyl- l-lysine) also provoked definite skin reactions, while the larger peptidoglycan fragments and various inflammatory agents including cytokines so far examined were inactive in this respect. Cortisone and heparin inhibited definitely and slightly the reaction, respectively. A comparison was made with the modified DTH reaction and the necrotic reactions which we reported previously.

Modification of delayed-type hypersensitivity reactions by muramyldipeptide in guinea pigs

N-Acetylmuramyl-L-alanyl-D-isoglutamine (muramyldipeptide, MDP) modulated delayed-type hypersensitivity (DTH) reactions and induced severe inflammatory lesions in guinea pigs. The animals immunized with heat-killed Mycobacterium tuberculosis were challenged with the purified protein derivative (PPD) at the flanks and the corneas to prepare DTH reactions at 2 weeks after the immunization, thereafter 24 h the animals received subcutaneous injections of MDP at the flanks of the opposite side. At the skin with the DTH reaction, increase of swelling and redness accompanied with hemorrhage and necrosis were observed. As corneal reactions in the animals that had received MDP, increase of cornea thickness, opaque and grayish-white and the projection of eyes accompanied with severe iritis were observed. Modification of the skin reaction occurred from 2 h after the MDP injection, rapidly increased to the maximum level around 10 h, maintained the level until 24 h, then slowly decreased. The polymorphonuclear leukocyte infiltration was observed from 15 min after the MDP injection, and tumour necrosis factor alpha, interleukin (IL)-1, and IL-6 levels in the serum and skin lesions increased after the MDP injection. Synthetic muramyltripeptide (N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-lysine) also provoked definite skin reactions, while the larger peptidoglycan fragments and various inflammatory agents including cytokines so far examined were inactive in this respect. Cortisone and heparin inhibited definitely and slightly the reaction, respectively. A comparison was made with the modified DTH reaction and the necrotic reactions which we reported previously.

Role of non-parenchymal liver cells in ischaemia-reperfusion liver injury: protective effects of muramyl dipeptide.

It has been suggested that non-parenchymal liver cells play a central role after ischaemia and reperfusion of the liver. Male Lewis rats were subjected to 90 min of warm liver ischaemia. Four groups were constituted: group 1, no treatment; group 2, muramyl dipeptide treatment, activation of Kupffer cells; group 3, dextran sulphate injection, Kupffer cell blockade; and group 4, gadolinium chloride administration, Kupffer cell blockade. Dextran sulphate (4 mg/100 g) and gadolinium chloride (GdCl2, 0.7 mg/100 g) were given intravenously on day 2. MDP was injected intravenously (500 mg/250 g) 24 h before and 10 min after the intervention. Mortality rates were assessed and serum transaminases, histology of the liver and Kupffer cell phagocytic activity were evaluated 6 h after the end of ischaemia. MDP treatment significantly (P < 0.001) reduced mortality (30%) in comparison with the non-treated group (60%). The mortality rate was significantly higher in the dextran sulphate-treated (80%) and gadolinium chloride-treated (90%) groups in comparison with group 1. A significant reduction in transaminase levels was observed after MDP treatment, while blockade of Kupffer cells resulted in higher serum transaminase levels. The extent of necrosis and congestion was improved by MDP administration, while disruption of the vascular and sinusoidal integrity of the liver and extensive areas of necrosis were observed in dextran sulphate and gadolinium chloride-treated rats. Sheep red blood cell 51Cr liver uptake was deeply depressed 6 h after the end of ischaemia in group 1 (10 +/- 1.2%/g tissue). MDP injection restored the Kupffer cell activity (30.6 +/- 3.22%/g tissue) while dextran sulphate and gadolinium chloride administration markedly decreased SRBC 51Cr liver uptake. Our findings demonstrate that MDP in able to protect the liver from ischaemic insult while blockade of Kupffer cells was deleterious in rats subjected to liver ischaemia.

Bacteroides Lipopolysaccharides (LPS) Induce Anaphylactoid and Lethal Reactions in LPS-Responsive and -Nonresponsive Mice Primed with Muramyl Dipeptide

Lipopolysaccharides (LPS) (100 micrograms/mouse) from Bacteroides gingivalis elicited anaphylactoid reactions in N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) (100 micrograms)-primed C3H/HeN mice 0-48 h after MDP injection. The reaction resulted in death within 1 h when the LPS was injected 2-10 h after the MDP injection. LPS prepared from other Bacteroides and Salmonella species but not from others, including Escherichia coli, could induce the reaction. LPS-nonresponsive C3H/HeJ and C57BL/6 and C57BL/10 strains were also responsive to the reaction, while AKR, BALB/c, DBA/2, and ICR mice were not. By contrast, Bacteroides LPS exhibited weak lethal toxicity compared with E. coli LPS in galactosamine-sensitized C3H/HeN mice, while neither LPS had activity in C3H/HeJ mice. LPS from E. coli and Bacteroides intermedius induced serum tumor necrosis factor (TNF) activity in MDP-primed mice, except for C3H/HeJ mice; B. gingivalis LPS rarely induced TNF in C3H/HeN or C3H/HeJ mice, indicating no involvement of TNF in the anaphylactoid reaction. Serotonin could substitute for MDP for the reaction, and methysergide, a serotonin antagonist, inhibited the activity.

The use of radionuclide bone scanning in the diagnosis of tibial 'stress' fractures in the horse: a review of five cases.

Five horses, all two year old Thoroughbreds, presented with acute onset hindlimb lameness following moderate to fast exercise. The use of a hand-held scintillation detector after Tc99 MDP injection greatly aided the diagnosis of tibial stress fractures in these horses. Radiological findings were subtle, involving only periosteal new bone and callus at the fracture site. In two cases this took several weeks to develop but in three others was present at the onset of lameness, indicating insidious pre-fracture bone pathology. The site of the most obvious radiological signs was constant in all five cases, being the proximal lateral tibia some 8 to 9 cm from the femoro-tibial joint surface. The diagnosis of five cases of tibial fracture in one racing season suggests that this condition has been underdiagnosed in the past in this clinic, prior to the availability of bone scanning.

Curative properties of muramyl dipeptide in experimental Naegleria meningoencephalitis

Naegleria fowleri is a free-living amoeba which causes a fatal meningoencephalitis in man. Mice injected with the immunostimulant MDP or an attenuated 11RX strain of Salmonella enteritidis showed some resistance to an intranasal challenge with N. fowleri. In addition it was observed that some of the mice infected with N. fowleri and showing symptoms of naegleria meningoencephalitis, given a single injection of MDP were cured of this disease. Our findings suggest that the use of immunostimulants could be a new approach in the quest for therapeutic agents for this disease.

Adjuvants for secretory immune responses.

Salivary IgA responses of rats receiving MDP were elevated after oral GTF administration and after infection with S. mutans bearing surface GTF. Intragastric administration (but not injection) of MDP enhanced salivary IgA responses to i.g. GTF. Subcutaneous injection (but not i.g. administration) of MDP enhanced IgA responses to GTF injected in the salivary gland vicinity. Conjugation of MDP to GTF did not enhance the secretory response to the antigen. Nevertheless, s.g.v. administration of OVA in IFA dramatically enhanced the secretory and serum responses. Injection of MDP may further enhance the secretory response. The combination of routes of adjuvant and antigen administration were critical in selectively enhancing a secretory immune response.

Analysis of the antigenic relationship of various derivatives of [formula omitted] (MDP), using anti-MDP antibodies

Antibodies to N- acetyl-muramyl- l-alanyl- d-isoglutamine (MDP) were produced in rabbits by injection of MDP conjugated to various carriers [bovine gamma globulin (BGG), methylated BSA or sheep erythrocytes]. The anti-MDP was assayed by a direct enzyme-linked immunosorbent assay (ELISA) using horse radish peroxidase linked to MDP-Lys. Various derivatives of MDP were employed in an inhibition of ELISA for analysis of specificity of antibodies and study of the relationship of configuration to biological activity. The results confirmed previous findings that MDP alone is not immunogenic but can act as a hapten when conjugated to carriers. The antibodies were shown to be primarily directed against the muramyl residue. Modifications of this region of MDP yielded derivatives with weak reactivity against anti-MDP, while some changes of other regions had no effect on its antigenicity. Optical isomers of MDP had reduced activity as compared to MDP and polymeric MDP was a strong inhibitor. The structure and function relationship is discussed for some derivatives.