Light-Induced Smooth Endoplasmic Reticulum Rearrangement in a Unique Interlaced Compartmental Pattern in Macaca mulatta RPE

PurposeTo investigate light-induced modifications of the smooth endoplasmic reticulum of the RPE in primates.MethodsEyes of three terminally anesthetized Rhesus monkeys were exposed to 5000 lux for 10 minutes or kept in the dark. Transmission electron microscopy and electron tomography were conducted on small fragments of retina sampled from different regions of the retina.ResultsRPE cells smooth endoplasmic reticulum shows a previously unknown arrangement characterized by an interlaced compartmental pattern (ICP). Electron tomograms and 3D-modelling demonstrated that the smooth endoplasmic reticulum with an ICP (ICPSER) consisted of four parallel, independent and interwoven networks of tubules arranged as interconnected coiled coils. Its architecture realized a compact labyrinthine structure of tightly packed tubules stabilized by intertubular filamentous tethers. On average, the ICPSER is present in about 14.6% of RPE cells. Although ICPSER was preferentially found in cells located in the peripheral and in the para/perifoveal retina, ICPSER cells significantly increased in number upon light exposure in the para/perifovea and in the fovea.ConclusionsAn ICPSER is apparently a unique feature to primate RPE. Its rapid appearance in the area centralis of the retina upon light exposure suggests a function related to the foveate structure of primate retina or to the diurnal habits of animals that may require additional protection from photo-oxidation or enhanced requests of visual pigments regeneration.     

Endogenous Interleukin-4 Does Not Suppress the Resistance Against a Primary or a Secondary Listeria monocytogenes Infection in Mice

Interleukin-4 (IL-4), a cytokine produced by T-helper 2 (Th2) cells, can inhibit the development of T-helper 1 (Th1) cells, which results in a decreased release of cytokines by the latter. As interferon-γ (IFN-γ), produced by Th1 cells, is involved in the resistance against a Listeria monocytogenes infection, the role of endogenously formed IL-4 during a Listeria infection in mice was investigated. Neutralization of endogenously formed cytokines by subcutaneously injected alginate-encapsulated monoclonal antibody (MoAb)-forming cells results in high antibody titres in the circulation over a long time period. The aim of the present study was to re-evaluate the effect of neutralization of IL-4 during a primary Listeria infection and to investigate the role of IL-4 during a secondary infection in mice using encapsulated MoAb-forming cells. During the course of a primary infection in mice given anti-IL-4 antibody-forming cells (anti-IL-4-FC), the number of Listeria found in the liver and spleen was comparable to that found in control mice given anti-β-galactosidase antibody-forming cells (anti-β-gal-FC). Activation of macrophages measured by inhibition of Toxoplasma gondii proliferation and the release of reactive nitrogen intermediates (RNI) was not affected by anti-IL-4-FC treatment during infection. Furthermore, during a secondary L. monocytogenes infection the number of bacteria in the liver and spleen of anti-IL-4-treated immune mice was comparable to anti-β-gal-FC-treated, control, immune mice. The concentration of IFN-γ in plasma of anti-IL-4-treated immune mice was similar to that of control immune mice. Taken together, these findings demonstrate that neutralization of endogenously formed IL-4 does not affect resistance to a primary or a secondary L. monocytogenes infection in mice.     

Elimination of resident macrophages from the livers and spleens of immune mice impairs acquired resistance against a secondary Listeria monocytogenes infection.

During a secondary Listeria monocytogenes infection in mice, the bacteria are eliminated more rapidly from the liver and spleen than during a primary infection. This acquired resistance against a secondary infection is dependent on T lymphocytes, which induce enhanced elimination of bacteria via stimulation of effector cells such as neutrophils, resident macrophages, exudate macrophages, and hepatocytes. The aim of the present study was to determine the role of the resident macrophages in acquired resistance against a secondary L. monocytogenes infection in mice. Mice which had recovered from a sublethal primary infection with 0.1 50% lethal dose (LD50) of L. monocytogenes intravenously (i.v.), i.e., immune mice, received a challenge of 1 LD50 of L. monocytogenes i.v. to induce a secondary infection. At 2 days prior to challenge, immune mice were given an i.v. injection of liposomes containing dichloromethylene-diphosphonate (L-Cl2MDP) to selectively eliminate resident macrophages from the liver and spleen. Control immune mice received either phosphate-buffered saline (PBS) or liposomes containing PBS (L-PBS). Treatment of mice with L-Cl2MDP effectively eliminated resident macrophages from the liver and spleen but did not affect the number of granulocytes, monocytes, or lymphocytes in peripheral blood or their migration to a site of inflammation. Phagocytosis and killing of L. monocytogenes by peritoneal exudate cells elicited with heat-killed L. monocytogenes were similar in all groups of immune mice. On day 3 of a secondary infection, the number of L. monocytogenes organisms in the livers and spleens of L-Cl2MDP-treated immune mice was 4 log10 units higher than in immune mice treated with PBS or L-PBS. The concentration of reactive nitrogen intermediates in plasma, a measure of the severity of infection, was 70-fold higher for L-Cl2MDP-treated immune mice than for PBS- or L-PBS-treated immune mice. Treatment with L-Cl2MDP significantly increased the number of inflammatory foci in the liver and spleen, decreased their size, and affected their structure. From these results, we conclude that resident macrophages are required for the expression of acquired resistance against a secondary L. monocytogenes infection in mice.     

Transfected Plasmodium knowlesi produces bioactive host gamma interferon: a new perspective for modulating immune responses to malaria parasites

Transgenic pathogenic microorganisms expressing host cytokines such as gamma interferon (IFN-gamma) have been shown to manipulate host-pathogen interaction, leading to immunomodulation and enhanced protection. Expression of host cytokines in malaria parasites offers the opportunity to investigate the potential of an immunomodulatory approach by generating immunopotentiated parasites. Using the primate malaria parasite Plasmodium knowlesi, we explored the conditions for expressing host cytokines in malaria parasites. P. knowlesi parasites transfected with DNA constructs for expressing rhesus monkey (Macaca mulatta) IFN-gamma under the control of the heterologous P. berghei apical membrane antigen 1 promoter, produced bioactive IFN-gamma in a developmentally regulated manner. IFN-gamma expression had no marked effect on in vitro parasite development. Bioactivity of the parasite-produced IFN-gamma was shown through inhibition of virus cytopathic effect and confirmed by using M. mulatta peripheral blood cells in vitro. These data indicate for the first time that it is feasible to generate malaria parasites expressing bioactive host immunomodulatory cytokines. Furthermore, cytokine-expressing malaria parasites offer the opportunity to analyze cytokine-mediated modulation of malaria during the blood and liver stages of the infection.     

Granulocyte-macrophage colony-stimulating factor is not involved in production of reactive nitrogen intermediates by or toxoplasmastatic activity of gamma interferon-activated murine macrophages.

The induction of reactive nitrogen intermediates (RNI) and toxoplasmastatic activity of murine macrophages by recombinant gamma interferon (rIFN-gamma) is mediated by an autocrine pathway involving tumor necrosis factor alpha (TNF-alpha). To investigate whether cytokines other than TNF-alpha play a role in the activation of these effector functions, granulocyte-macrophage colony-stimulating factor (GM-CSF) was studied. Recombinant GM-CSF (rGM-CSF) could stimulate peritoneal macrophages, since this cytokine stimulated the production of prostaglandin E2 by these cells. However, rGM-CSF did not induce either the release of RNI by or the toxoplasmastatic activity of macrophages. rGM-CSF in combination with various concentrations of rIFN-gamma did not enhance these effector functions more than rIFN-gamma alone. Furthermore, neutralization of endogenously produced GM-CSF by monoclonal antibodies did not affect the release of RNI by or the toxoplasmastatic activity of rIFN-gamma-activated macrophages. Together these results indicate that GM-CSF is not involved in RNI production by and toxoplasmastatic activity of IFN-gamma-activated murine macrophages.     

Advantages and Risks of Husbandry and Housing Changes to Improve Animal Wellbeing in a Breeding Colony of Common Marmosets (Callithrix jacchus)

Between 1975 and 2014, housing conditions for laboratory-housed marmosets changed dramatically after the introduction of new guidelines designed to improve their care and wellbeing. According to these guidelines, our facility provided marmosets with outside enclosures, switched to deep litter as bedding material, and discontinued the use of disinfectant agents in animal enclosures. However, both deep litter and access to outside enclosures hypothetically increase the risk of potential exposure to pathogenic microorganisms. We evaluated whether these housing and husbandry modifications constituted an increased veterinary risk for laboratory-housed common marmosets (Callithrix jacchus). After the animals had been exposed to these new housing conditions for 2.5 y, we examined their intestinal bacterial flora and feces, the deep litter, and insects present in the housing. In addition, we assessed the marmosets’ general health and the effect of outdoor housing on, for example, vitamin D levels. Although numerous bacterial strains—from nonpathogenic to potentially pathogenic—were cultured, we noted no increase in illness, mortality, or breeding problems related to this environmental microflora. Housing laboratory marmosets in large enriched cages, with both indoor and outdoor enclosures, providing them with deep litter, and eliminating the use of disinfectants present an increased veterinary risk. However, after evaluating all of the collected data, we estimate that the veterinary risk of the new housing conditions is minimal to none in terms of clinical disease, disease outbreaks, abnormal behavior, and negative effects on reproduction.The Biomedical Primate Research Centre (Rijswijk, The Netherlands) houses a self-sustaining breeding colony of common marmosets (Callithrix jacchus) for the purpose of conducting biomedical research on life-threatening human diseases. The marmoset colony was formed in 1975 and has been used mainly for research on autoimmune diseases, neurodegenerative disorders, and comparative genetics.^{3,5,6,14,21,29}After the introduction of new European and Dutch guidelines regarding animal care and welfare, animal housing conditions changed markedly between 1975 and 2014. Our facility responded promptly to these new guidelines, by providing larger and more complex cages comprising outdoor enclosures, each with an attached heated indoor enclosure, where the animals are housed in family groups to improve animal wellbeing.^22,35 The concept of environmental enrichment continued to be developed and optimized over the years.Potential benefits of outdoor enclosures are exposure to seasonal fluctuations in light and climate and increased sensory stimulation. These enclosures provide opportunities for exploration and manipulation that are considered to contribute positively to the animals’ wellbeing. Furthermore, marmosets housed indoors with no access to UV light are susceptible to metabolic bone diseases. Marmosets cannot synthesize vitamin D3 (cholecalciferol) from the plant form of the vitamin (ergocalciferol, vitamin D2). Without access to UVB radiation, they cannot form vitamin D3 from 7-dehydroxycholesterol in the skin.³¹ In addition to dietary supplementation with vitamin D3, we surmised that access to unfiltered sunlight in outside enclosures would limit or prevent vitamin D deficiency.Another change initiated in response to BPRC''s new housing guidelines was the cleaning of the housing facilities. Scent marking is an important aspect of the natural behavior of marmosets. In laboratory settings, marmosets scent-mark their cages constantly.¹¹ To minimize the removal of scents, disinfectants are no longer used to clean the enclosures. In addition to effects on scent, limiting disinfectant use could have other beneficial effects. For example, the chemical disinfection of their environment was suggested to be one cause of chromosomal disorders in marmosets.¹³A third important housing-related change was the provision of deep litter in the outdoor and indoor enclosures. Deep litter is a floor covering, preferably of organic origin, that promotes activities including locomotion, foraging, and playing. In general, the changes associated with providing deep litter typically involved a shift in the animals’ behavioral profiles toward those that might be observed in their wild counterparts; therefore, the provision of deep litter is seen as environmental enrichment.^{7,9,10,24,30,32}Although some of these changes have been implemented in zoos, primate centers that breed marmosets for research purposes have been more reticent because of potential health issues. To evaluate whether the new housing conditions enhance the animals’ wellbeing, we studied their benefits and potential threats to the animals, the practical consequences for personnel and management, and the influence on experimental results. In particular, the health risks for the marmosets due to increased microbiologic exposure because of the new housing conditions were examined. The aim of the study was to determine whether these changes in their housing constituted not only an improvement in their wellbeing but also a possible increased veterinary risk for laboratory-housed common marmosets.     

Effect of granulocyte-macrophage colony-stimulating factor on the number of leucocytes and course of Listeria monocytogenes infection in naive and leucocytopenic mice.

This study concerns the effect of recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) on the number of circulating leucocytes, activation of peritoneal macrophages and proliferation of Listeria monocytogenes in various organs of naive and leucocytopenic mice. Mice were rendered leucocytopenic by sublethal total body irradiation or cyclophosphamide treatment. GM-CSF treatment enhanced the number of granulocytes and monocytes in peripheral blood during L. monocytogenes infection in naive mice, but not in irradiated or cyclophosphamide-treated mice. In naive mice, irradiated and cyclophosphamide-treated mice, GM-CSF did not affect the course of L. monocytogenes infection in thigh muscle, spleen and liver. However, GM-CSF treatment significantly increased the number of macrophages in the peritoneal cavity of naive mice during infection; these macrophages were more enlarged and showed a higher frequency of binucleated and multinucleated cells relative to non-GM-CSF-treated mice. Together, these results demonstrated that GM-CSF increased the number of circulating granulocytes and monocytes, and the number of peritoneal macrophages during infection with L. monocytogenes in naive mice, but did not affect the course of the infection in thigh muscle, spleen or liver of these mice. In leucocytopenic mice, however, GM-CSF did not affect the number of circulating phagocytes, which explains that this factor had no effect on the proliferation of the bacteria in the various organs.     

A prime-boost immunisation regimen using DNA followed by recombinant modified vaccinia virus Ankara induces strong cellular immune responses against the Plasmodium falciparum TRAP antigen in chimpanzees

Two chimpanzees were vaccinated intramuscularly against malaria using plasmid DNA expressing the pre-erythrocytic antigens thrombospondin related adhesion protein (PfTRAP) and liver stage specific antigen-1 (PfLSA-1) of Plasmodium falciparum together with GM-CSF protein. A recombinant modified vaccinia virus Ankara (MVA) expressing PfTRAP was injected intramuscularly 6 weeks later to boost the immune response. This sequence of antigen delivery induced a specific and long-lasting T cell and antibody response to PfTRAP as detected by ELISPOT assay and ELISA. Antibody responses were detected after four DNA injections, and were boosted by injection of recombinant MVA expressing PfTRAP. Interferon-gamma secreting antigen-specific T cells were detected in both animals, but only after boosting with recombinant MVA. By screening a panel of PfTRAP-derived peptides, an epitope was identified that was recognized by cytotoxic T lymphocytes in one of the chimpanzees studied. T cells specific for this epitope were present in PBMCs and liver-infiltrating lymphocytes at a frequency of between 1 in 200 and 1 in 500. The high immunogenicity of this prime-boost regimen in chimpanzees supports further assessment of this delivery strategy for the induction of protection against P. falciparum malaria in humans.     

Activation of murine peritoneal macrophages by Salmonella typhimurium

The results of these studies indicate that the interaction between activated macrophages and S. typhimurium depends on the characteristics of the micro-organism and the kind of activation.     

Tumour necrosis factor, but not interferon-gamma, is essential for acquired resistance to Listeria monocytogenes during a secondary infection in mice.

Mice with a secondary Listeria monocytogenes infection eliminate the bacteria much faster and more efficiently from their organs than mice with a primary infection. During the course of a secondary infection, serum concentrations of interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF) are higher than during a primary infection. The aim of the present study was to determine whether these cytokines are involved in the acquired resistance to L. monocytogenes during a secondary infection in mice. In order to neutralize cytokines, alginate-encapsulated cells, which form anti-cytokine monoclonal antibodies, were injected into the nuchal region of mice during a Listeria infection. Mice recovered from a sublethal primary Listeria infection, which acquired cell-mediated immunity, received a subcutaneous injection of anti-IFN-gamma-forming cells, or anti-TNF-forming cells, and 4 days later received an intravenous injection with 10 50% lethal dose (LD50) L. monocytogenes. The number of bacteria recovered from the liver and spleen of immune mice treated with anti-IFN-gamma-forming cells was slightly larger (approximately 1 log10) than that found for immune mice treated with anti-beta-galactosidase-forming cells, called immune control mice. The organs of immune mice treated with anti-TNF-forming cells yielded significantly more (approximately 4 log10) bacteria than those of immune control mice, more than those of immune mice treated with anti-IFN-gamma-forming cells, and comparable numbers to those of non-immune mice. Taken together, these results demonstrate that TNF is essential in acquired resistance to L. monocytogenes during a secondary infection in mice, while IFN-gamma plays a minor role.