Engineering Pancreatic Islets to Transiently Codisplay on Their Surface Thrombomodulin and CD47 Immunomodulatory Proteins as a Means of Mitigating Instant Blood-Mediated Inflammatory Reaction following Intraportal Transplantation.

Most pancreatic islets are destroyed immediately after intraportal transplantation by an instant blood-mediated inflammatory reaction (IBMIR) generated through activation of coagulation, complement, and proinflammatory pathways. Thus, effective mitigation of IBMIR may be contingent on the combined use of agents targeting these pathways for modulation. CD47 and thrombomodulin (TM) are two molecules with distinct functions in regulating coagulation and proinflammatory responses. We previously reported that the islet surface can be modified with biotin for transient display of novel forms of these two molecules chimeric with streptavidin (SA), that is, thrombomodulin chimeric with SA (SA-TM) and CD47 chimeric with SA (SA-CD47), as single agents with improved engraftment following intraportal transplantation. This study aimed to test whether islets can be coengineered with SA-TM and SA-CD47 molecules as a combinatorial approach to improve engraftment by inhibiting IBMIR. Mouse islets were effectively coengineered with both molecules without a detectable negative impact on their viability and metabolic function. Coengineered islets were refractory to destruction by IBMIR ex vivo and showed enhanced engraftment and sustained function in a marginal mass syngeneic intraportal transplantation model. Improved engraftment correlated with a reduction in intragraft innate immune infiltrates, particularly neutrophils and M1 macrophages. Moreover, transcripts for various intragraft procoagulatory and proinflammatory agents, including tissue factor, HMGB1 (high-mobility group box-1), IL-1ß, IL-6, TNF-α, IFN-γ, and MIP-1α, were significantly reduced in coengineered islets. These data demonstrate that the transient codisplay of SA-TM and SA-CD47 proteins on the islet surface is a facile and effective platform to modulate procoagulatory and inflammatory responses with implications for both autologous and allogeneic islet transplantation.

Protocol for transplanting pancreatic islets into the parametrial fat pad of female mice.

Although the male epididymal fat pad is an effective site for islet transplantation, females lack this tissue. Here, we present a protocol to assess the parametrial fat pad (PFP) adjacent to the uterine horn in females as an alternative site for islet transplantation. We describe steps for islet isolation from the pancreas, counting, transplantation into PFP, and monitoring for engraftment. Transplantation into PFP is minimally invasive, time efficient, and supports long-term engraftment of syngeneic islets and rejection of allogeneic islets. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022).1.

The impact of armed conflict on animal well-being and welfare, and analyzing damage assessment on the veterinary sector: The case of Ethiopia's Tigray region.

Tigray war broke out on November 4, 2020, and lasted until November 3, 2022. The war has caused a significant loss of human life and a catastrophic economic and humanitarian crisis. The war affected the food and water supplies to farmers to care their livestock and this led to animal death, malnutrition, and suffering. In addition, a significant number of animals have been subjected to flee the region and killed during the warfare. The veterinary sector is significantly damaged and animals became a victim. Veterinarians and animal health workers have fled the region because of the war, and this adds an extra burden to the sector. Although the impact of this war on animal life, welfare and overall, on the livestock infrastructure is significant, no study has been conducted so far. We analyzed the level of damage to the veterinary sector and number of animal loss following the war. Our analytical study showed the war has claimed a total of 2,487,047 cattle, 1,690,096 sheep, 3,803,860 goat, 610,976 donkey, 4,280,815 poultry, and 231,985 beehives. Such loss led to an estimated total financial loss of 53.56 billion Ethiopian birr (∼1.01 billion USD). We also analyzed the destruction level of the veterinary infrastructures in percentage, and the associated financial loss due to facility damage and animal disappearances from the region. A complete destruction (100 %) of veterinary facilities was reported in 10 districts and this accounts to an estimated financial loss of 68.59 million Ethiopian birrs (1.3 million USD). In conclusion, Tigray war has caused an immense impact to animal welfare and veterinary sector and a collaborative effort between governmental and nongovernmental organizations, and professional bodies is required to restore to restor the sector. This study also highlighted how the war jeopardize animal right and wellbeing. Thus, we believe this study will be an input for national and international policy makers working on international convention for animal protection and rights.

Measuring the Manipulation of T Helper Immune Responses by Schistosoma mansoni.

Schistosoma mansoni uses different mechanisms to escape its host's immunity. Understanding the ability of memory T cells to withstand this pathogen's manipulation is important for the development of effective vaccines against this immunomodulatory pathogen. In this study, ovalbumin (OVA) transgenic S. mansoni is used as a tool to investigate whether fully differentiated Th1, Th2 and Th17 cells are able to withstand pathogen manipulation. Naïve T cells from OT-II T cell receptor transgenic mice with a specificity for OVA were differentiated into Th1, Th2, and Th17 polarised memory cells in vitro. These cells were adoptively transferred into recipient mice to investigate whether these polarised immune memory T cells are resilient in the face of pathogen-mediated manipulation. After transferring memory cells, mice were challenged with OVA-transduced S. mansoni eggs as well as wild-type controls. The in vitro differentiated Th1, Th2 and Th17 memory cells continued to produce the same cytokines when challenged by OVA-expressing S. mansoni eggs as to these they produced when transferred in vivo, suggesting that the Th phenotypes of the memory T cells remains unaltered in the face of stimulation by S. mansoni. The ability of memory T cells to remain resilient to manipulation by the parasite suggests that vaccines might be able to produce immune memory responses able to withstand S. mansoni immune manipulation and hence protect the host from infection.

Validating Immunomodulatory Responses of r-LdODC Protein and Its Derived HLA-DRB1 Restricted Epitopes against Visceral Leishmaniasis in BALB/c Mice.

Vaccination is considered the most appropriate way to control visceral leishmaniasis (VL). With this background, the r-LdODC protein as well as its derived HLA-DRB1-restricted synthetic peptides (P1: RLMPSAHAI, P2: LLDQYQIHL, P3: GLYHSFNCI, P4: AVLEVLSAL, and P5: RLPASPAAL) were validated in BALB/c mice against visceral leishmaniasis. The study was initiated by immunization of the r-LdODC protein as well as its derived peptides cocktail with adjuvants (r-CD2 and MPL-A) in different mice groups, separately. Splenocytes isolated from the challenged and differentially immunized mice group exhibited significantly higher IFN-γ secretion, which was evidenced by the increase in the expression profile of intracellular CD4+IFN-γ T cells. However, the IL-10 secretion did not show a significant increase against the protein and peptide cocktail. Subsequently, the study confirmed the ability of peptides as immunoprophylactic agents, as the IE-I/AD-I molecule overexpressed on monocytes and macrophages of the challenged mice group. The parasitic load in macrophages of the protein and peptides cocktail immunized mice groups, and T cell proliferation rate, further established immunoprophylactic efficacy of the r-LdODC protein and peptide cocktail. This study suggests that the r-LdODC protein, as well as its derived HLA-DRB1-restricted synthetic peptides, have immunoprophylactic potential and can activate other immune cells' functions towards protection against visceral leishmaniasis. However, a detailed study in a humanized mice model can explore its potential as a vaccine candidate.

Recognition of Schistosoma mansoni egg-expressed ovalbumin by T cell receptor transgenic mice.

Schistosoma mansoni eggs can influence immune responses directed at them, and the mechanisms by which this is achieved are being unravelled. Going towards, developing effective tools for the study of how S. mansoni influences naïve T cells, we have developed S. mansoni eggs expressing chicken ovalbumin (OVA), using a lentiviral transduction system. Indeed, such a parasite could be used in conjunction with cells from OT-II transgenic mice as a source of naïve, antigen-specific T cells. The expression of the transgenic protein was confirmed by real-time RT-PCR of OVA-specific mRNA and western blotting using polyclonal antibodies specific for OVA. T cells from OT-II transgenic mice expressing a T cell receptor specific for the OVA323-339 peptide recognised the OVA-transduced S. mansoni eggs. Using flow cytometry on CFSE-labelled OT-II splenocytes, we demonstrated that OVA-transduced eggs elicit higher OT-II proliferative responses than untransduced eggs. The OT-II T cells also produced TNF-α and IFN-γ following exposure to OVA-transduced eggs. In addition, moderate amounts of IL-6 and IL-17A were also detected. In contrast, no IL-10, IL-4 and IL-2 were detected in cultures, whether the cells were stimulated with transduced or untransduced eggs. Thus, the cytokine signatures showed the transfected eggs induced predominantly a Th1 response, with a small amount of IL-6 and IL-17.

Investigating immune responses to parasites using transgenesis.

Parasites comprise diverse and complex organisms, which substantially impact human and animal health. Most parasites have complex life-cycles, and by virtue of co-evolution have developed multifaceted, often life-cycle stage-specific relationships with the immune system of their hosts. The complexity in the biology of many parasites often limits our knowledge of parasite-specific immune responses, to in vitro studies only. The relatively recent development of methods to stably manipulate the genetic make-up of many parasites has allowed a better understanding of host-parasite interactions, particularly in vivo. In this regard, the use of transgenic parasites can facilitate the study of immunomodulatory mechanisms under in vivo conditions. Therefore, in this review, we specifically highlighted the current developments in the use of transgenic parasites to unravel the host's immune response to different life-cycle stages of some key parasite species such as Leishmania, Schistosoma, Toxoplasma, Plasmodium and Trypanosome and to some degree, the use of transgenic nematode parasites is also briefly discussed.