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BACKGROUND: This study examined the experiences of owners of dogs with leishmaniosis who treated their dogs with daily subcutaneous meglumine antimoniate injections. The owners' perceived ease of administering the injections, the occurrence of problems and the effects on the owners and on the dog‒owner bond were evaluated.METHODS: Dogs prescribed meglumine antimoniate as a treatment for leishmaniosis were identified using the database of the veterinary pharmacy of the Faculty of Veterinary Medicine, Utrecht University. An online questionnaire was sent to the owners of these dogs to evaluate the perceived ease of administering the injections, the occurrence of problems and the effects on the owner and the dog-owner bond.RESULTS: Responses were received from 64 dog owners. Most respondents (78%) reported that administering the injections was not difficult. Pain or the development of nodules at the injection site was reported in 50% and 40% of the dogs, respectively. Polyuria was reported in 44% of the dogs. Some owners reported that administering the injections had a negative impact on their psychological wellbeing (20%), and some would have liked more veterinary support (11%).LIMITATIONS: Some questions were answered by a limited number of people, and their responses may not be representative.CONCLUSION: Dog owners remain highly motivated to persevere with meglumine antimoniate treatment and are willing to administer the injections themselves. The availability of active support when needed during the therapy cycle may further improve their acceptance of and confidence in giving the injections.

Metabolism plays a crucial role in regulating the function of immune cells in both health and disease, with altered metabolism contributing to the pathogenesis of cancer and many inflammatory diseases. The local microenvironment has a profound impact on the metabolism of immune cells. Therefore, immunological and metabolic heterogeneity as well as the spatial organization of cells in tissues should be taken into account when studying immunometabolism. Here, we highlight challenges of investigating metabolic communication. Additionally, we review the capabilities and limitations of current technologies for studying metabolism in inflamed microenvironments, including single-cell omics techniques, flow cytometry-based methods (Met-Flow, single-cell energetic metabolism by profiling translation inhibition (SCENITH)), cytometry by time of flight (CyTOF), cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), and mass spectrometry imaging. Considering the importance of metabolism in regulating immune cells in diseased states, we also discuss the applications of metabolomics in clinical research, as well as some hurdles to overcome to implement these techniques in standard clinical practice. Finally, we provide a flowchart to assist scientists in designing effective strategies to unravel immunometabolism in disease-relevant contexts.

There is no curative treatment for chronic auto-inflammatory diseases including rheumatoid arthritis, and current treatments can induce off-target side effects due to systemic immune suppression. This work has previously shown that dexamethasone-pulsed tolerogenic dendritic cells loaded with the arthritis-specific antigen human proteoglycan can suppress arthritis development in a proteoglycan-induced arthritis mouse model. To circumvent ex vivo dendritic cell culture, and enhance antigen-specific effects, drug delivery vehicles, such as liposomes, provide an interesting approach. Here, this work uses anionic 1,2-distearoyl-sn-glycero-3-phosphoglycerol liposomes with enhanced loading of human proteoglycan-dexamethasone conjugates by cationic lysine tetramer addition. Antigen-pulsed tolerogenic dendritic cells induced by liposomal dexamethasone in vitro enhanced antigen-specific regulatory T cells to a similar extent as dexamethasone-induced tolerogenic dendritic cells. In an inflammatory adoptive transfer model, mice injected with antigen-dexamethasone liposomes have significantly higher antigen-specific type 1 regulatory T cells than mice injected with antigen only. The liposomes significantly inhibit the progression of arthritis compared to controls in preventative and therapeutic proteoglycan-induced arthritis mouse models. This coincides with systemic tolerance induction and an increase in IL10 expression in the paws of mice. In conclusion, a single administration of autoantigen and dexamethasone-loaded liposomes seems to be a promising antigen-specific treatment strategy for arthritis in mice.

In the Mediterranean area, a large reservoir of Leishmania infantum infected dogs is present. These dogs are imported into the Netherlands in considerable numbers, which leads to an increasing number of dogs suffering from leishmaniasis that present themselves to the Dutch veterinary practices. However, the seroprevalence of Leishmania in this imported population of dogs is still unknown. Therefore, Project Leishmania was founded to determine this seroprevalence. Part I of this paper describes the methodology of the database that was used to record the seroprevalence and other clinical findings in Project Leishmania participating dogs. Suggestions are offered to improve the database to minimise information bias and make future statistical analysis easier. Preliminary results show a Leishmania seroprevalence of 15.5% in imported dogs, which emphasises the need for awareness in Dutch veterinary practices for leishmaniasis in dogs with an importation history. Part II of this paper describes the research performed on the immunological aspect of Leishmania infections. Namely, an interesting characteristic of canine leishmaniasis is the fact that disease progression is extremely variable. Dogs can be infected and remain asymptomatic for long periods of time or they can develop mild to severe clinical symptoms. Research on leishmania infections in mammals show that the type 1 immune response, characterised by T-helper 1 cells and M1 macrophages, has been associated with parasite killing and asymptomatic infections, whereas the type 2 immune response, characterised by T-helper 2 cells and M2 macrophages, has been associated with parasite survival and symptomatic disease. Knowledge on these M1 and M2 macrophages in dogs is scarce and the specific role these macrophages play in relation to Leishmania infections has not been researched in dogs yet. Therefore, the polarisation of canine monocyte-derived macrophages into M1/M2 is investigated by the expression of cell surfaces m

The binding of Host Defense Peptides (HDPs) to the endotoxin of Gram-negative bacteria has important unsolved aspects. For most HDPs, it is unclear if binding is part of the antibacterial mechanism or whether LPS actually provides a protective layer against HDP killing. In addition, HDP binding to LPS can block the subsequent TLR4-mediated activation of the immune system. This dual activity is important, considering that HDPs are thought of as an alternative to conventional antibiotics, which do not provide this dual activity. In this study, we systematically determine, for the first time, the influence of the O-antigen and Lipid A composition on both the antibacterial and anti-endotoxin activity of four HDPs (CATH-2, PR-39, PMAP-23, and PMAP36). The presence of the O-antigen did not affect the antibacterial activity of any of the tested HDPs. Similarly, modification of the lipid A phosphate (MCR-1 phenotype) also did not affect the activity of the HDPs. Furthermore, assessment of inner and outer membrane damage revealed that CATH-2 and PMAP-36 are profoundly membrane-active and disrupt the inner and outer membrane of Escherichia coli simultaneously, suggesting that crossing the outer membrane is the rate-limiting step in the bactericidal activity of these HDPs but is independent of the presence of an O-antigen. In contrast to killing, larger differences were observed for the anti-endotoxin properties of HDPs. CATH-2 and PMAP-36 were much stronger at suppressing LPS-induced activation of macrophages compared to PR-39 and PMAP-23. In addition, the presence of only one phosphate group in the lipid A moiety reduced the immunomodulating activity of these HDPs. Overall, the data strongly suggest that LPS composition has little effect on bacterial killing but that Lipid A modification can affect the immunomodulatory role of HDPs. This dual activity should be considered when HDPs are considered for application purposes in the treatment of infectious diseases.

Several reports demonstrated the susceptibility of domestic cats to SARS-CoV-2 infection. Here, we describe a thorough investigation of the immune responses in cats after experimental SARS-CoV-2 inoculation, along with the characterization of infection kinetics and pathological lesions. Specific pathogen-free domestic cats ( n = 12) were intranasally inoculated with SARS-CoV-2 and subsequently sacrificed on DPI (days post-inoculation) 2, 4, 7 and 14. None of the infected cats developed clinical signs. Only mild histopathologic lung changes associated with virus antigen expression were observed mainly on DPI 4 and 7. Viral RNA was present until DPI 7, predominantly in nasal and throat swabs. The infectious virus could be isolated from the nose, trachea and lungs until DPI 7. In the swab samples, no biologically relevant SARS-CoV-2 mutations were observed over time. From DPI 7 onwards, all cats developed a humoral immune response. The cellular immune responses were limited to DPI 7. Cats showed an increase in CD8+ cells, and the subsequent RNA sequence analysis of CD4+ and CD8+ subsets revealed a prominent upregulation of antiviral and inflammatory genes on DPI 2. In conclusion, infected domestic cats developed a strong antiviral response and cleared the virus within the first week after infection without overt clinical signs and relevant virus mutations.

In the Mediterranean area, a large reservoir of Leishmania infantum infected dogs is present. These dogs are imported into the Netherlands in considerable numbers, which leads to an increasing number of dogs suffering from leishmaniasis that present themselves to the Dutch veterinary practices. However, the seroprevalence of Leishmania in this imported population of dogs is still unknown. Therefore, Project Leishmania was founded to determine this seroprevalence. Part I of this paper describes the methodology of the database that was used to record the seroprevalence and other clinical findings in Project Leishmania participating dogs. Suggestions are offered to improve the database to minimise information bias and make future statistical analysis easier. Preliminary results show a Leishmania seroprevalence of 15.5% in imported dogs, which emphasises the need for awareness in Dutch veterinary practices for leishmaniasis in dogs with an importation history. Part II of this paper describes the research performed on the immunological aspect of Leishmania infections. Namely, an interesting characteristic of canine leishmaniasis is the fact that disease progression is extremely variable. Dogs can be infected and remain asymptomatic for long periods of time or they can develop mild to severe clinical symptoms. Research on leishmania infections in mammals show that the type 1 immune response, characterised by T-helper 1 cells and M1 macrophages, has been associated with parasite killing and asymptomatic infections, whereas the type 2 immune response, characterised by T-helper 2 cells and M2 macrophages, has been associated with parasite survival and symptomatic disease. Knowledge on these M1 and M2 macrophages in dogs is scarce and the specific role these macrophages play in relation to Leishmania infections has not been researched in dogs yet. Therefore, the polarisation of canine monocyte-derived macrophages into M1/M2 is investigated by the expression of cell surfaces m

Current treatments for Rheumatoid Arthritis aim at remission, often rendering patients immunocompromised. Novel treatments options would therefore be beneficial. It is hypothesized that a dysfunction of Regulatory T cells plays a role in the pathology of Rheumatoid Arthritis. Thus, ex vivo polyclonal culturing of Regulatory T cells, followed by adoptive cell therapy would be a potential new therapy. Hereby, the number and function of Regulatory T cells can be restored. Although this approach shows potential, it comes with limitations, such as a lack of potency and specificity together with a higher risk of generalized immunosuppression. To overcome these, the use of antigen-specific Regulatory T cells would be more beneficial. However, antigen-specific expansion is challenging due to, among others, low available cell numbers. A method in which the high cell yields of polyclonal expansion are combined with the specificity of antigen-specific Regulatory T cells is therefore needed. In our research we move towards a method by which Regulatory T cells can be expanded in an antigen-specific manner. To this end, a protocol for the polyclonal expansion of Regulatory T cells is established using low concentrations of TransAct, high concentrations of rhIL-2 and addition of a TNFR2-agonist, leading to high expansion numbers while retaining a stable phenotype. We show that cells expanded under these conditions can potentially be restimulated in an antigen-specific manner with mammalian B29a, a heat shock protein hypothesized to be involved in Rheumatoid Arthritis. This enables us to obtain a high number of B29a-specific Regulatory T cells from which potentially, in future research, TCRs can be isolated.