Adoptive Transfer Immunization of Mice

This procedure is designed to enrich and expand antibody-forming cells for use in generating monoclonal antibodies. Gamma-irradiation is used to wipe out the immune system in a recipient animal, after which spleen cells that have reverted to memory cells are obtained from syngeneic donor animals and transferred to the irradiated animal, allowing the implanted immune cells to take over. This method can produce an 80-fold enrichment of antibody-producing cells over that obtained in the original immunized animal.

Protective activity of specific transfer factor against Salmonella typhimurium infection

Specific transfer factor (TF) extracted from spleens of sensitized and non-sensitized guinea pigs to study the efficacy of transfer of cellular immunity specific for salmonella . Two groups each of five guinea pigs were used for in vivo TF preparation. The First group was inoculated with 1 ml of aromatic dependent Salmonella typhimurium SL 1479 vaccine at a dose of 10' cfu/ml intramuscularly twice at two weeks intervals. The second group was injected with trypticase soy broth similarly . These two groups used as a donor for TF, and TFn respectively. Twenty one recipient guinea pigs were divided into three groups, the first group was TF at a dose of 1 ml equivalent to 5X10 cell intramuscularly three times/2 days intervals, Similarly the second group was given TF, where as the third group was given PBS- Cell mediated immunity in recipient animal was evaluated by delayed type hypersensitivity - skin test, Macrophage migration inhibition test (MIF) and then challenged with virulent Salmonella typhimurium. The TFt recipient group induced skin test and showed migration indices less than 0.8 and overcome the challenge organism. Contrary to TFn & PBS recipient groups which did not show any response for skin test and given migration indices more than 0.8 and did not show resistance for virulent Salmonella typhimurium .

Sensory neurons expressing the atypical olfactory receptor guanylyl cyclase D are required for the acquisition of odor preferences by mice in diverse social contexts

ABSTRACTAnimals use social communication to learn important information from conspecifics that can guide appropriate behavioral choices. For example, during the social transmission of food preference (STFP), conspecific semiochemicals detected by mouse olfactory sensory neurons (OSNs) expressing the atypical olfactory receptor guanylyl cyclase D (GC-D+ OSNs) promote the acquisition of food preferences in the recipient animal, mitigating the risk of ingesting food contaminated with toxins or pathogens. However, it is unclear if GC-D+ OSNs mediate preference learning outside this specific context. Here, we report that GC-D+ OSNs are required for the acquisition of odor preferences by both adult and juvenile mice, and that GC-D-dependent preference could be formed for conditionally aversive odors. We used a two-choice olfactory behavioral test to assess odor preferences in adult Gucy2d +/+, +/- and -/- mice that encountered novel odors together with GC-D+ OSN stimuli (guanylin family peptides), during social investigation of a live conspecific, or during suckling as pups. Gucy2d +/+ and +/-mice (which express functional GC-D), but not Gucy2d -/- littermates, successfully acquire a preference for the demonstrated odor in any of these behavioral paradigms. Mice could even acquire a GC-D-dependent preference for odors to which they had recently formed a conditioned aversion. Together, these results demonstrate that GC-D+ OSNs mediate the acquisition of socially-transmitted odor preferences in different social and experiential contexts and at different life stages.

Development of light-weight video-tracking technology for use in wildlife research: A case study on kangaroos

There have been significant advances in the development of animal-borne sensor technologies, or biologgers, in recent years. This has resulted in tremendous capacity for wildlife researchers to remotely collect physiological, behavioural and social data from wildlife in circumstances that were unthinkable just decades ago. While this technology can provide us with a unique insight into the “secret lives” of wild animals, there is a need to evaluate the utility of these new sensors versus traditional wildlife research methodologies, and to critically evaluate the integrity of the data collected by ensuring that these devices themselves do not alter the physiology or behaviour of the recipient animal. This paper reports on the development of a light weight “animal borne video and environmental data collection system” (AVED), which can be deployed on animals as small as 11 kg, whilst still meeting the desired 3% body weight threshold. This AVED (referred to as the “Kangaroo-cam”) simultaneously collects video footage and GPS location data for an average of 19 h. Kangaroo-cams were deployed on seven kangaroos as a proof of concept of their potential utility for the study of location specific behaviour and diet in a medium-sized terrestrial herbivore. Following device recovery and data processing, we were able to successfully score 83 foraging events which allowed us to determine diet based on visual identification (to the family level) of plants consumed. This approach could be further broadened to include a comparison of plant species consumed versus plant species encountered to provide a novel approach to diet selection analysis. When combined with GPS mapping of foraging locations, this approach would allow researchers to address questions on diet selection at both fine (within patch) and broad (habitat) spatial scales, overcoming some of the limitations of traditional diet selection methodologies. However, animal capture and collar deployment caused a significant elevation in stress hormone concentrations within the first 24 h post-capture, which highlighted the need to incorporate a time-delay capacity into these devices. We conclude the paper by reviewing recent advances in the development of AVED technology and providing suggestions for the improvement of this Kangaroo-cam device.

Insights Into Thrombopoiesis From Infused Human Megakaryocytes Into Mice

Thrombopoiesis is the process by which megakaryocytes (Megs) release platelets (Plts), but issues remain as to the detailed in vivo mechanisms underlying this process. We now report new insights into this process by studying infused human Megs into immunocompromized NOD/SCID, gamma-interferon deleted (NSG) mice. Prior in situ microscopy has suggested that Megs release varied-size cytoplasmic fragments up to whole Megs in size into the medullary vascular space. Other studies have suggested that at least a portion of thrombopoiesis occurs by Megs lodged in the lungs. We previously infused ex vivo-generated murine Megs into mice and found that these Megs become entrapped in the animals’ lungs, and in <1.5 hrs, release functional Plts (termed here “Meg-Plts”) that have a similar half-life as infused mouse donor-derived Plts (termed here “Donor-Plts”). To better understand the biology of thrombopoiesis, we have infused ex vivo-generated human Megs into NSG mice. These studies replicated many of the observations seen with infused murine Megs: Human Megs were entrapped in the lungs with delayed release of human Meg-Plts, and these Meg-Plts had the same half-life as infused human Donor-Plts. Human Plts differ from murine Plts in size so this parameter was analyzed following infusion of human Megs using forward cell scatter analysis. We noted that 10 mins post-infusion, the Meg-Plt size range was wide and displayed a non-bell-shaped distribution. This distribution was in contrast to the tight bell-shaped curves seen for the endogenous murine Plts and for infused human Donor-Plts. However, by 3 hrs post-human Meg infusion - at the time of peak Meg-Plt counts - the human Meg-Plts now displayed an identical bell-shaped distribution curve as infused human Donor-Plt. The smaller, human Meg-Plts had disappeared. The size and distribution of these Meg-Plts then remained near identical to Donor-Plts for the remaining portion of the 48 hr post-infusion study. However, after impairing macrophage clearance in NSG recipient mice with clodronate-ladened liposome infusion, the small Meg-Plts did not disappear and were present at 48 hrs. Using thiazole orange (TO) to stain platelets for RNA content, we noted that ∼70% of all Meg-Plts were initially TO+ compared to the steady-state of ∼10% for mouse endogenous platelets. This high TO+ state decreased to near 10% by 24 hrs post-infusion. Up to ∼6 hrs, all of the large Meg-Plts were TO+, while the smaller-sized Meg-Plts were predominantly TO-. Unless the mice were treated with clodronate-ladened liposomes, these TO-, small Meg-Plts disappeared before 6 hrs. In conclusion, these data support that ex vivo-generated human Megs release physiologic platelets in the pulmonary vascular bed of NSG mice with the same size range/distribution and survival as infused human Donor-Plts. Mean Meg-Plt size depends on the species of origin of the infused Megs rather than on the species of the recipient animal. We did not detect large Meg cytoplasmic fragments that underwent further size reduction although our technique may not be capable of detecting small numbers of such fragments or the small size changes that would accompany platelet maturation from preplatelets. Our data also suggest that Megs generated in culture release a wide size range of non-physiologic Plt-like particles that when infused are cleared rapidly by macrophages. Disclosures: No relevant conflicts of interest to declare.

Heterotopic uterine transplantation by vascular anastomosis in the mouse

A method of heterotopic uterine transplantation was developed in the mouse as a model system for studies of uterine function and transplant immunology of the uterus. The model involved transplantation of the right uterine horn and the cervix by vascular anastomosis to a donor animal with the intact native uterus remaining in situ. F1-hybrids of inbred C57BL/6 x CBA/ca (B6 CBAF1) mice of 6-8 weeks of age (n=42) were used. The specific pelvic vascular anatomy of these mice was first examined by intra-aortal injection of a two-component silicon-rubber curing agent. The surgery of the donor animal involved microsurgical isolation of the right uterine horn and the cervix, with preserved vascular supply from the aorta through the right uterine artery. After isolation of the uterine horn with vascular supply and venous drainage, including approximately 3 mm of the inferior vena cava and aorta, the organ was put on ice. The recipient animal was prepared by exposing and mobilizing the infrarenal part of the aorta and the vena cava. The grafted uterus was placed in the abdomen on the left side and the aorta and vena cava of the graft were anastomosed end-to-side to the aorta and vena cava of the recipient animal with 11-0 sutures. The total time for these procedures declined with time and was 125+/-4 min for the last 28 operations. Viability of the uterus was confirmed, several days later, by demonstrating a blood flow similar to that of the native uterus, and histology of the grafted uterus demonstrated normal morphology, including intact ultrastructure of the endothelial cells. The overall survival rate of the recipient animals increased with learning from approximately 40% in animals 1-21 to 71% in animals 22-42. The proportion of viable grafts, as judged by normal blood flow and histology among the surviving mice was 25% in animals 1-21 and 87% in animals 22-42. An undisturbed function of the transplanted uterus horn was finally demonstrated by its ability to implant inserted blastocysts and to carry pregnancy with fetal weight being similar to that of fetuses in the native uterus and controls. In conclusion, this is the first report of successful transplantation of the uterus with proven functionality in the mouse. The model should be useful for many aspects of research in uterine physiology and pathophysiology.

The significance of transferrin for intestinal iron absorption

A mechanism is proposed by which apotransferrin is secreted from mucosal cells, loaded with iron in the intestinal lumen, and then the intact complex is taken into the cell. Within the cell, iron is released and transferred to the blood stream, whereas iron-free transferrin returns to the brush border to be recycled. We have investigated this hypothesis by measuring intestinal absorption of radioiron and 125I-labeled plasma transferrin using tied-off gut segments in normal and iron-deficient rats. There was no absorption of diferric transferrin from the ileum, but high absorption from the duodenum and jejunum segments. Jejunal absorption occurred as a function of the dose offered and showed saturation kinetics. In normal animals, 4 micrograms of the 50 micrograms of transferrin iron was absorbed over 1 hr. In iron-deficient animals, mean values as high as 13 micrograms were observed. Radioiron content of the jejunal mucosa bore a linear relationship to the dose administered and was inversely proportional to the amount of iron entering the plasma. Recycling of transferrin was indicated by the presence of labeled apotransferrin in the lumen, first observed between 15 and 60 min after the injection of diferric transferrin. A high resistance of diferric and apotransferrin to proteolytic degradation within the gut lumen was demonstrated. Comparative studies with lactoferrin and ferritin disclosed poor availability of their iron for absorption. The small amount that was absorbed did not relate to the iron status of the recipient animal. These studies support the role of mucosal transferrin as a shuttle protein for iron absorption.

The significance of transferrin for intestinal iron absorption

A mechanism is proposed by which apotransferrin is secreted from mucosal cells, loaded with iron in the intestinal lumen, and then the intact complex is taken into the cell. Within the cell, iron is released and transferred to the blood stream, whereas iron-free transferrin returns to the brush border to be recycled. We have investigated this hypothesis by measuring intestinal absorption of radioiron and 125I-labeled plasma transferrin using tied-off gut segments in normal and iron-deficient rats. There was no absorption of diferric transferrin from the ileum, but high absorption from the duodenum and jejunum segments. Jejunal absorption occurred as a function of the dose offered and showed saturation kinetics. In normal animals, 4 micrograms of the 50 micrograms of transferrin iron was absorbed over 1 hr. In iron-deficient animals, mean values as high as 13 micrograms were observed. Radioiron content of the jejunal mucosa bore a linear relationship to the dose administered and was inversely proportional to the amount of iron entering the plasma. Recycling of transferrin was indicated by the presence of labeled apotransferrin in the lumen, first observed between 15 and 60 min after the injection of diferric transferrin. A high resistance of diferric and apotransferrin to proteolytic degradation within the gut lumen was demonstrated. Comparative studies with lactoferrin and ferritin disclosed poor availability of their iron for absorption. The small amount that was absorbed did not relate to the iron status of the recipient animal. These studies support the role of mucosal transferrin as a shuttle protein for iron absorption.