Delayed Footpad Swelling Research Articles

Leishmania kinetoplast DNA contributes to parasite burden in infected macrophages: Critical role of the cGAS-STING-TBK1 signaling pathway in macrophage parasitemia.

Leishmania parasites harbor a unique network of circular DNA known as kinetoplast DNA (kDNA). The role of kDNA in leishmania infections is poorly understood. Herein, we show that kDNA delivery to the cytosol of Leishmania major infected THP-1 macrophages provoked increased parasite loads when compared to untreated cells, hinting at the involvement of cytosolic DNA sensors in facilitating parasite evasion from the immune system. Parasite proliferation was significantly hindered in cGAS- STING- and TBK-1 knockout THP-1 macrophages when compared to wild type cells. Nanostring nCounter gene expression analysis on L. major infected wild type versus knockout cells revealed that some of the most upregulated genes including, Granulysin (GNLY), Chitotriosidase-1 (CHIT1), Sialomucin core protein 24 (CD164), SLAM Family Member 7 (SLAMF7), insulin-like growth factor receptor 2 (IGF2R) and apolipoprotein E (APOE) were identical in infected cGAS and TBK1 knockout cells, implying their involvement in parasite control. Amlexanox treatment (a TBK1 inhibitor) of L. major infected wild type cells inhibited both the percentage and the parasite load of infected THP-1 cells and delayed footpad swelling in parasite infected mice. Collectively, these results suggest that leishmania parasites might hijack the cGAS-STING-TBK1 signaling pathway to their own advantage and the TBK1 inhibitor amlexanox could be of interest as a candidate drug in treatment of cutaneous leishmaniasis.

Immunization against Leishmania major Infection Using LACK- and IL-12-Expressing Lactococcus lactis Induces Delay in Footpad Swelling

Background Leishmania is a mammalian parasite affecting over 12 million individuals worldwide. Current treatments are expensive, cause severe side effects, and emerging drug resistance has been reported. Vaccination is the most cost-effective means to control infectious disease but currently there is no vaccine available against Leishmaniasis. Lactococcus lactis is a non-pathogenic, non-colonizing Gram-positive lactic acid bacterium commonly used in the dairy industry. Recently, L. lactis was used to express biologically active molecules including vaccine antigens and cytokines.Methodology/Principal findingsWe report the generation of L. lactis strains expressing the protective Leishmania antigen, LACK, in the cytoplasm, secreted or anchored to the bacterial cell wall. L. lactis was also engineered to secrete biologically active single chain mouse IL-12. Subcutaneous immunization with live L. lactis expressing LACK anchored to the cell wall and L. lactis secreting IL-12 significantly delayed footpad swelling in Leishmania major infected BALB/c mice. The delay in footpad swelling correlated with a significant reduction of parasite burden in immunized animals compared to control groups. Immunization with these two L. lactis strains induced antigen-specific multifunctional TH1 CD4+ and CD8+ T cells and a systemic LACK-specific TH1 immune response. Further, protection in immunized animals correlated with a Leishmania-specific TH1 immune response post-challenge. L. lactis secreting mouse IL-12 was essential for directing immune responses to LACK towards a protective TH1 response.Conclusions/SignificanceThis report demonstrates the use of L. lactis as a live vaccine against L. major infection in BALB/c mice. The strains generated in this study provide the basis for the development of an inexpensive and safe vaccine against the human parasite Leishmania.

Induction of Lymphatic Lesions by Brugia pahangi in Jirds with Large and Small Preexisting Homologous Intraperitoneal Infections

The effect of intraperitoneal (i.p.) infections induced by inoculations of 30 or 150 Brugia pahangi third-stage larvae (L3) on the development of infections and lymphatic lesions induced by subsequent homologous subcutaneous (s.c.) inoculations were compared in the present study. Lymphatic lesion severity, as judged by the numbers of lymph thrombi present, and lymphatic lesion scores were significantly reduced in both groups of jirds with existing i.p. infections. The numbers of adult worms that developed, locations of these worms, and the subsequent microfilaremias did not differ significantly between groups. All jirds with i.p. infections developed similar antibody titers to crude somatic adult antigen as measured by ELISA. These levels did not change following s.c. infections. Immediate and delayed footpad swelling responses were also similar in all groups. Results of these experiments support and extend previous studies indicating that i.p. infections of B. pahangi induce a hyporesponsive state in jirds to subsequent s.c. infections without significantly affecting the subsequent parasite burden. This effect appears to be independent of the numbers of L3 inoculated i.p. prior to lymphatic-induced infection. Circulating antibody titers and footpad swelling responses to B. pahangi antigen were not reduced in jirds with the hyporesponsive lymphatic inflammatory response and do not correlate with this condition.

Induction of protective immunity against Schistosoma mansoni by a nonliving vaccine. IV. Fractionation and antigenic properties of a soluble adult worm immunoprophylactic activity.

An aqueous buffer-soluble, nonparticulate fraction of adult Schistosoma mansoni worms (SWAP) was separated by gel filtration on Ultragel AcA-34, and portions of the eluate were tested for their capacity to induce protective immunity against cercarial challenge when administered intradermally to mice in combination with the adjuvant BCG. All of the immunogenic activity was found in a single peak of protein excluded in the void volume of the column. This same fraction was determined by SDS-PAGE and Western immunoblotting to be unique in that it contained a component of Mr (X 10(-3) 97 (97,000) recognized monospecifically by antibodies from mice vaccinated with unseparated SWAP plus BCG. Similarly, the protective fraction was unique in possessing the capacity to elicit 24 hr delayed footpad swelling responses, as well as lymphokine production, in SWAP-BCG-immunized mice. These results suggest that the immunogenic activity of SWAP resides in a restricted population of molecules, and possibly in the 97,000 antigen detected with antibodies from vaccinated animals. Because both the protective capacity of unfractionated SWAP and the serologic reactivity of the 97,000 antigen are sensitive to digestion with protease, it is likely that the immunologic activity of these molecules is dependent on peptide-bonded structural elements.

Glycopeptides in soluble egg antigen of Schistosoma mansoni: isolation, characterization, and elucidation of their immunochemical and immunopathological relation to the major egg glycoprotein (MEG).

The major egg glycoprotein (MEG) of Schistosoma mansoni was purified by ion-exchange chromatography of glycoprotein fraction obtained from soluble egg antigen (SEA) by lectin affinity chromatography. Small carbohydrate-rich fragments (CRF) contained in the glycoprotein fraction of SEA were isolated by ultrafiltration followed by dialysis (10 to 13 kd). Comparison of MEG and CRF yielded the following results: purified MEG (70 kd) contains about 77% carbohydrate, and CRF contains 92.5% carbohydrate. When radioiodinated and run by SDS-PAGE, each yielded a single band with respective Rf values of around 0.33 and 1.0 CRF is capable of inhibiting, in a Farr-type RIA, the binding of 125I-MEG to serum from chronically infected mice. Furthermore, CRF and MEG exhibit a single and continuous line of radioimmunodiffusion. CRF, unlike SEA, SEA glycoproteins, or purified MEG, is incapable of eliciting delayed footpad swelling in egg-sensitized mice or of inducing granulomatous hypersensitivity, when given at amounts equivalent to or higher than MEG by protein or carbohydrate content. Thus, whereas SEA, SEA glycoproteins, or MEG elicited in a representative test net swelling of 0.28 mm, 0.34 mm, and 0.29 mm, respectively, CRF gave net swellings of 0.06 mm, similar to the control value (0.07 mm) in unsensitized mice. Also, mice sensitized to viable eggs, SEA, or purified MEG exhibited, after i.v. challenge with viable eggs, a mean area of granulomas in the lungs of 12,389 micron2, 16,412 micron2, and 12,354 micron2, respectively, as compared with 7940 micron2 in CRF-sensitized mice and 8428 micron2 in unsensitized control mice. Thus, CRF appears to contain fragments of MEG that are serologically active but immunopathologically inactive at the concentrations used.

Suppression of cell-mediated immune responses in vivo and in vitro by 1-thiocarbamoyl-2-imidazolidinone

Both in vivo and in vitro, the niridazole metabolite, 1-thiocarbamoyl-2-imidazolidinone (TCI), was found to suppress various manifestations of cell-mediated immunity. In vivo, doses of 10−6 g/kg every other day delayed rejection of BALB/cJ (H−2d) skin allografts by C57BL/6J (H−2b) mice. Much lower single doses (∼10−11g/kg) markedly reduced both pulmonary granuloma formation around, Schistosoma mansoni eggs in C57BL/6J mice and delayed footpad swelling in sensitized CF1 mice responding to S. mansoni egg antigen (SEA). Reduction of footpad swelling occured when TCI was given 24 hr before, but not 4 hr after, antigenic challenge. In vitro, SEA-dependent production of eosinophil stimulation promoter (ESP) activity by sensitized mouse spleen cells was decreased > 90% by 10−10gTCI/ml. TCI failed, however, to inhibit the action of preformed ESP lymphokine on eosinophils. The blastogenic response of sensitized lymph node cells to SEA was reduced by > 70% by 10−11to −10−8gTCI/ml. The drug produced no effect on either antigen-dependent lymphokine production or blastogenesis if it was added 5 min after, instead of before, stimulating antigen.

In vivo suppression of delayed hypersensitivity by thiabendazole and diethylcarbamazine.

Several anthelminthic agents, such as niridazole and metronidazole, have been demonstrated to have striking effects on the immune system, apparently independent of their antiparasitic activities. In the present study, we have examined the effect of thiabendazole and diethylcarbamazine on two parameters of delayed hypersensitivity, lung granuloma formation around Schistosoma mansoni eggs and delayed footpad edema in response to schistosome egg antigens. Both drugs caused significant reduction of lung granuloma size in unsensitized animals when given as daily doses for 8 days. Thiabendazole was the more potent suppressant, affecting granuloma size in unsensitized animals when given as single dose and in sensitized animals using a multiple dose regimen. Diethylcarbamazine was without effect on granuloma size in sensitized animals, but inhibited delayed footpad swelling when given daily for 6 days. Neither drug affected granulomas induced by non-antigenic plastic beads. These data support the hypothesis that some of the clinical activities of these drugs may be mediated by interference with host response to antigenic stimuli.

Immune competence and diabetes mellitus: II. Experimental mouse studies

An experimental model utilizing congenitally diabetic mice was established to answer the question whether such animals have altered immune responses and ability to ward off infections. Cellular immunity was suppressed in diabetic mice when the parameters of granuloma formation, allograft rejection, and delayed footpad swelling were tested. Spleen cells from diabetic mice grown in tissue culture had an impaired mitogenic response compared to those from control animals. In addition, when diabetic mice were inoculated with the common human intraoral organisms, B-hemolytic streptococcus and Staphylococcus aureus, they could not contain the infection as well as normal littermates. Thus, immune function and infectious control mechanisms appear to be deficient in diabetic mice.

Delayed-type hypersensitivity in mice immunized with Trypanosoma rhodesiense antigens.

When mice were immunized intravenously, subcutaneously, or by the footpad route with formaldehyde-killed Trypanosoma rhodesiense, delayed-type hypersensitivity was elicited by the use of frozen-thawed trypanosomal antigen. The delayed footpad swelling technique was used to measure delayed hypersensitivity. Hypersensitivity induction was dose dependent (greater than or equal to 10(6) formaldehyde-treated T. rhodesiense) and was affected by the route of immunization. The footpad route induced higher levels of hypersensitivity than other routes of immunization. Mice immunized with a single dose of formaldehyde-treated antigen and challenged with live T. rhodesiense did not survive. Yet, mice immunized subcutaneously with formaldehyde-treated antigen and then injected with frozen-thawed antigen and challenged 28 days after immunization survived. The results suggest that T-cell activation, manifested by delayed hypersensitivity responses, was a necessary component in the protective response, perhaps functioning in a helper cell capacity.

Impairment of Cell-Mediated Immunity in Mutation Diabetic Mice (db/db)

Mutation diabetes in the mouse occurs in the C57BL/Ks strain. All homozygous animals (db/db) develop obesity, elevated blood sugar levels and increased or normal blood insulin concentration. The defects in cellular immunity in db/db mice and their littermate controls were examined both in vivo and in vitro. Significant suppression of delayed footpad swelling and first and second set skin allograft rejection time were observed. In addition, DNA synthesis in spleen cells after nonspecific mitogen stimulation was markedly inhibited. Diabetic animals with a mean blood sugar of 512 +/- 101 mg/100 ml did not respond to exogenous insulin therapy by lowering their blood sugar levels or reversing the defect in DNA synthesis. Adding insulin to spleen cell culture in vitro had no demonstrable effect on their response to mitogens. Thus, mutation diabetic mice with their known defect in the peripheral utilization of insulin have markedly suppressed cell-mediated immune mechanisms.

Delayed Immune Reactions in Mice Immunized with Malarial Antigen

Mice were immunized subcutaneously, intravenously or in a footpad with antigens prepared from a lethal strain of Plasmodium berghei yoelii. A delayed footpad swelling (DFS) reaction was observed 4 days after immunization, and was detectable at least 42 days after immunization. However, IV immunization was the least efficacious is producing hypersensitivity. Regardless of the type of antigen used for immunization, mice responded similarly when live parasites were used to elicit DFS. This study revealed that hypersensitivity depended on route of immunization and type of antigen.

Cyclophosphamide pretreatment and protection against malaria.

Mice pretreated with cyclophosphamide were able to overcome infection from a lethal malarial strain. The development of resistance was preceded by increased hypersensitivity to malarial antigens. Hypersensitivity was demonstrable by a delayed footpad swelling technique.

Induced and spontaneous diabetes mellitus and suppression of cell-mediated immunologic responses. Granuloma formation, delayed dermal reactivity and allograft rejection.

These investigations delineate the recently described suppression of a form of cellular hypersensitivity in mice with streptozotocin-induced diabetes mellitus using a variety of cell-mediated immunologic responses in animals with several different forms of diabetes. Streptozotocin- and alloxan-induced diabetic mice and db/db genetically determined diabetic mice showed reductions in the areas of inflammation around Schistosoma mansoni eggs injected into the pulmonary vasculature of 68, 70, 77%, respectively. In contrast, streptozotocin-induced diabetes had no effect on the nonimmunologic foreign body granuloma around divinyl benzene copolymer beads injected into the pulmonary arterioles. Animals protected from diabetes by treatment with nicotinamide before streptozotocin administration did not develop hyperglycemia and had normal areas of immunologic granuloma formation around schistosome eggs. Treatment with insulin reversed the suppression of schistosome egg granuloma formation in both streptozotocin- and alloxan-diabetic animals. Two additional in vivo parameters of cellular immunologic reactivity were examined in streptozotocin-induced diabetes: delayed footpad swelling was essentially eliminated; skin graft survival across the H-2 area was significantly prolonged from 10.2 days in the controls to 14.4 days in moderately diabetic A/J mice. These observations suggest that diabetes mellitus is associated with suppression of cell-mediated reactions in vivo and that the defect is reversible with insulin treatment.

Niridazole

Abstract Niridazole, a human anthelminthic drug, is shown to be a potent long-acting suppressor of delayed hypersensitivity. Much lower doses than used in the therapy of helminth infections suppressed granuloma formation around Schistosoma mansoni eggs for periods up to 32 days and inhibited delayed footpad swelling in mice previously sensitized with eggs. Marked prolongation of skin allograft survival in mice across the H-2 area was produced. The drug is administered orally, has been used clinically on a massive scale, and appears to be relatively free of the cytotoxic effects of most immunosuppressive agents.

Schistosoma mansoni: Antigenic secretions and the etiology of egg granulomas in mice

In order to determine the exact role of antigenic secretions in the formation of the granuloma around S. Mansoni eggs, the following three experiments were performed: 1. (1) The early stages of granuloma formation around eggs injected intravenously into the pulmonary microvasculature of mice were studied at 12-hr intervals and correlated with the onset of delayed footpad swelling. No reaction was seen around the eggs until 72 hr, at which time delayed footpad swelling was also first seen. 2. (2) Immature eggs (without secretory glands) obtained from worms maintained in tissue culture resulted in virtually no granuloma formation at 8 days in un-sensitized animals. Soluble egg antigens extracted from immature eggs and injected intraperitoneally did not sensitize mice to granuloma formation around eggs subsequently injected into the pulmonary veins. 3. (3) Eggs maintained in tissue culture over a period of 14 days gradually lost their ability to elicit granuloma formation when injected into the pulmonary vessels. Tissue culture medium obtained at similar time intervals and injected intraperitoneally into mice, concurrently lost its ability to sensitize the animals to granuloma formation around fresh eggs later injected intravenously. On the basis of the above results it is concluded that granuloma formation around S. mansoni eggs is due wholly to their output of antigenic secretions.

Delayed hypersensitivity-type granuloma formation and dermal reaction induced and elicited by a soluble factor isolated from Schistosoma mansoni eggs.

A soluble material has been isolated from schistosome eggs which in minute quantities without the addition of adjuvant induces sensitization to a delayed hypersensitivity type of granuloma forming around intact schistosome eggs. This material is secreted by intact eggs and is found in high concentration in the fluid released during the hatching process. When adsorbed to bentonite particles this substance elicits hypersensitivity type granuloma formation in specifically sensitized animals. The granuloma sensitizing factor also both induces and elicits delayed footpad swelling in mice. Quantities which sensitize with respect to these delayed type reactions do not induce antibody formation detectable by a sensitive hemagglutination. technique within the duration of the above experiments.