An Update on the Biologic Effects of Fenbendazole.

Fenbendazole remains the drug of choice to treat pinworm infection in laboratory rodents. When fenbendazole was last reviewed (15 y ago), the literature supported the drug's lack of toxic effects at therapeutic levels, yet various demonstrated physiologic effects have the potential to alter research outcomes. Although more recent reports continue to reflect an overall discordancy of results, several studies support the premise that fenbendazole affects the bone marrow and the immune system. No effects on reproduction were reported in an extensive study that assessed common treatment protocols in mice, and food intake was unchanged in rats. Behavioral studies are sparse, with only a single report of a subtle change in a rotarod performance in mice. Notably, unexpected results in tumor models during facility treatment with fenbendazole have prompted preclinical and clinical studies of the potential roles of benzimidazoles in cancer.

Impaired B Cell Apoptosis Results in Autoimmunity That Is Alleviated by Ablation of Btk.

While apoptosis plays a role in B-cell self-tolerance, its significance in preventing autoimmunity remains unclear. Here, we report that dysregulated B cell apoptosis leads to delayed onset autoimmune phenotype in mice. Our longitudinal studies revealed that mice with B cell-specific deletion of pro-apoptotic Bim (BBimfl/fl ) have an expanded B cell compartment with a notable increase in transitional, antibody secreting and recently described double negative (DN) B cells. They develop greater hypergammaglobulinemia than mice lacking Bim in all cells and accumulate several autoantibodies characteristic of Systemic Lupus Erythematosus (SLE) and related Sjögren's Syndrome (SS) including anti-nuclear, anti-Ro/SSA and anti-La/SSB at a level comparable to NODH2h4 autoimmune mouse model. Furthermore, lymphocytes infiltrated the tissues including submandibular glands and formed follicle-like structures populated with B cells, plasma cells and T follicular helper cells indicative of ongoing immune reaction. This autoimmunity was ameliorated upon deletion of Bruton's tyrosine kinase (Btk) gene, which encodes a key B cell signaling protein. These studies suggest that Bim-mediated apoptosis suppresses and B cell tyrosine kinase signaling promotes B cell-mediated autoimmunity.

Anti-inflammatory effects of α-MSH through p-CREB expression in sarcoidosis like granuloma model.

Lung inflammation due to sarcoidosis is characterized by a complex cascade of immunopathologic events, including leukocyte recruitment and granuloma formation. α-melanocyte stimulating hormone (α-MSH) is a melanocortin signaling peptide with anti-inflammatory properties. We aimed to evaluate the effects of α-MSH in a novel in vitro sarcoidosis model. An in vitro sarcoidosis-like granuloma model was developed by challenging peripheral blood mononuclear cells (PBMCs) derived from patients with confirmed treatment-naïve sarcoidosis with microparticles generated from Mycobacterium abscessus cell walls. Unchallenged PBMCsand developed granulomas were treated daily with 10 µM α-MSH or saline as control. Cytokine concentrations in supernatants of culture and in cell extracts were measured using Illumina multiplex Elisa and western blot, respectively. Gene expression was analyzed using RNA-Seq and RT-PCR. Protein secretion and gene expression of IL-7, IL-7R, IFN-γ, MC1R, NF-κB, phosphorylated NF-κB (p-NF-κB), MARCO, and p-CREB were measured with western blot and RNAseq. A significant increase in IL-7, IL-7R, and IFN-γ protein expression was found in developed granulomas comparing to microparticle unchallenged PBMCs. IL-7, IL-7R, and IFN-γ protein expression was significantly reduced in developed granulomas after exposure to α-MSH compared with saline treated granulomas. Compared with microparticle unchallenged PBMCs, total NF-κB and p-NF-κB were significantly increased in developed granulomas, while expression of p-CREB was not changed. Treatment with α-MSH promoted a significantly higher concentration of p-CREB in granulomas. The anti-inflammatory effects of α-MSH were blocked by specific p-CREB inhibition. α-MSH has anti-inflammatory properties in this in vitro granuloma model, which is an effect mediated by induction of phosphorylation of CREB.

Essential and non-overlapping IL-2Rα-dependent processes for thymic development and peripheral homeostasis of regulatory T cells.

IL-2R signaling is essential for regulatory T cell (Treg) function. However, the precise contribution of IL-2 during Treg thymic development, peripheral homeostasis and lineage stability remains unclear. Here we show that IL-2R signaling is required by thymic Tregs at an early step for expansion and survival, and a later step for functional maturation. Using inducible, conditional deletion of CD25 in peripheral Tregs, we also find that IL-2R signaling is indispensable for Treg homeostasis, whereas Treg lineage stability is largely IL-2-independent. CD25 knockout peripheral Tregs have increased apoptosis, oxidative stress, signs of mitochondrial dysfunction, and reduced transcription of key enzymes of lipid and cholesterol biosynthetic pathways. A divergent IL-2R transcriptional signature is noted for thymic Tregs versus peripheral Tregs. These data indicate that IL-2R signaling in the thymus and the periphery leads to distinctive effects on Treg function, while peripheral Treg survival depends on a non-conventional mechanism of metabolic regulation.

Superior immune reconstitution using Treg-expanded donor cells versus PTCy treatment in preclinical HSCT models.

Posttransplant cyclophosphamide (PTCy) has been found to be effective in ameliorating acute graft-versus-host disease (GVHD) in patients following allogeneic hematopoietic stem cell transplantation (aHSCT). Adoptive transfer of high numbers of donor Tregs in experimental aHSCT has shown promise as a therapeutic modality for GVHD regulation. We recently described a strategy for in vivo Treg expansion targeting two receptors: TNFRSF25 and CD25. To date, there have been no direct comparisons between the use of PTCy and Tregs regarding outcome and immune reconstitution within identical groups of transplanted mice. Here, we assessed these two strategies and found both decreased clinical GVHD and improved survival long term. However, recipients transplanted with Treg-expanded donor cells (TrED) exhibited less weight loss early after HSCT. Additionally, TrED recipients demonstrated less thymic damage, significantly more recent thymic emigrants, and more rapid lymphoid engraftment. Three months after HSCT, PTCy-treated and TrED recipients showed tolerance to F1 skin allografts and comparable immune function. Overall, TrED was found superior to PTCy with regard to weight loss early after transplant and initial lymphoid engraftment. Based on these findings, we speculate that morbidity and mortality after transplant could be diminished following TrED transplant into aHSCT recipients, and, therefore, that TrED could provide a promising clinical strategy for GVHD prophylaxis.

Very Low Numbers of CD4+ FoxP3+ Tregs Expanded in Donors via TL1A-Ig and Low-Dose IL-2 Exhibit a Distinct Activation/Functional Profile and Suppress GVHD in a Preclinical Model.

Regulatory T cells (Tregs) are essential for the maintenance of tolerance and immune homeostasis. In allogeneic hematopoietic stem cell transplantation (aHSCT), transfer of appropriate Treg numbers is a promising therapy for the prevention of graft-versus-host disease (GVHD). We have recently reported a novel approach that induces the marked expansion and selective activation of Tregs in vivo by targeting tumor necrosis factor receptor superfamily 25 (TNFRSF25) and CD25. A potential advance to promote clinical application of Tregs to ameliorate GVHD and other disorders would be the generation of more potent Treg populations. Here we wanted to determine if very low doses of Tregs generated using the "2-pathway" stimulation protocol via TL1A-Ig fusion protein and low-dose IL-2 (targeting TNFRSF25 and CD25, respectively) could be used to regulate preclinical GVHD. Analysis of such 2-pathway expanded Tregs identified higher levels of activation and functional molecules (CD103, ICOS-1, Nrp-1, CD39, CD73, il-10, and tgfb1) versus unexpanded Tregs. Additionally, in vitro assessment of 2-pathway stimulated Tregs indicated enhanced suppressor activity. Notably, transplant of extremely low numbers of these Tregs (1:6 expanded Tregs/conventional T cells) suppressed GVHD after an MHC-mismatched aHSCT. Overall, these results demonstrate that 2-pathway stimulated CD4+ FoxP3+ Tregs were quantitatively and qualitatively more functionally effective than unexpanded Tregs. In total, the findings in this study support the notion that such 2-pathway stimulated Tregs may be useful for prevention of GVHD and ultimately promote more widespread application of aHSCT in the clinic.

Specialized Proresolving Mediators Rescue Infant Mice from Lethal Citrobacter rodentium Infection and Promote Immunity against Reinfection.

Infants are generally highly susceptible to oral pathogens. Intestinal infection and the associated diarrhea are significant global causes of morbidity and mortality in infants. Among the enteric pathogens, enteropathogenic Escherichia coli (EPEC) stands out as showing the highest risk for infection-induced death in infants ≤12 months old. We have developed an experimental model of infant infection with EPEC, using the mouse-specific pathogen Citrobacter rodentium Our murine infant model is similar to EPEC infection in human infants since infant mice are much more susceptible to C. rodentium infection than adult mice; infants infected with 50-fold fewer bacteria than the standard adult dose uniformly succumbed to the infection. Infant infection is characterized by high early and sustained bacterial titers and profound intestinal inflammation associated with extensive necrosis and systemic dissemination of the bacteria. Therefore, it seems likely that infant deaths result from sepsis secondary to intestinal damage. Recently, specialized proresolving mediators (SPM) have been found to exert profound beneficial effects in adult models of infection. Thus, we investigated the actions of two proresolving lipid mediators, resolvin D1 (RvD1) and resolvin D5 (RvD5), on the course of infection in infants. Strikingly, postinfection treatment with RvD1 and RvD5 reduced bacterial loads, mitigated inflammation, and rescued the infants from death. Furthermore, postinfection treatment with RvD1 and RvD5 led to protection from reinfection associated with C. rodentium-specific IgG responses comparable to those in adults. These results indicate that SPM may provide novel therapeutic tools for the treatment of pathological intestinal infections in infants.

The Lower Limit of Regulatory CD4+ Foxp3+ TCRß Repertoire Diversity Required To Control Autoimmunity.

The TCR repertoire of regulatory T cells (Tregs) is highly diverse. The relevance of this diversity to maintain self-tolerance remains unknown. We established a model where the TCR repertoire of normal polyclonal Tregs was limited by serial transfers into IL-2Rß-/- mice, which lack functional Tregs. After a primary transfer, the donor Treg TCR repertoire was substantially narrowed, yet the recipients remained autoimmune-free. Importantly, upon purification and transfer of donor-derived Tregs from an individual primary recipient into neonatal IL-2Rß-/- mice, the secondary recipients developed autoimmunity. In this study, the Treg TCRß repertoire was reshaped and further narrowed. In contrast, secondary IL-2Rß recipients showed fewer symptoms of autoimmunity when they received donor Tregs that were premixed from several primary recipients to increase their TCRß repertoire diversity. About 8-11% of the Treg TCRß repertoire was estimated to be the minimum required to establish and maintain tolerance in primary IL-2Rß-/- recipients. Collectively, these data quantify where limitations imposed on the Treg TCRß repertoire results in a population of Tregs that cannot fully suppress polyclonal autoreactive T cells. Our data favor a model where the high diversity of the Treg TCR provides a mechanism for Tregs to actively adapt and effectively suppress autoreactive T cells, which are not fixed, but are evolving as they encounter self-antigens.

Marked in Vivo Donor Regulatory T Cell Expansion via Interleukin-2 and TL1A-Ig Stimulation Ameliorates Graft-versus-Host Disease but Preserves Graft-versus-Leukemia in Recipients after Hematopoietic Stem Cell Transplantation.

Regulatory T cells (Tregs) are critical for self-tolerance. Although adoptive transfer of expanded Tregs limits graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation (HSCT), ex vivo generation of large numbers of functional Tregs remains difficult. Here, we demonstrate that in vivo targeting of the TNF superfamily receptor TNFRSF25 using the TL1A-Ig fusion protein, along with IL-2, resulted in transient but massive Treg expansion in donor mice, which peaked within days and was nontoxic. Tregs increased in multiple compartments, including blood, lymph nodes, spleen, and colon (GVHD target tissue). Tregs did not expand in bone marrow, a critical site for graft-versus-malignancy responses. Adoptive transfer of in vivo-expanded Tregs in the setting of MHC-mismatched or MHC-matched allogeneic HSCT significantly ameliorated GVHD. Critically, transplantation of Treg-expanded donor cells facilitated transplant tolerance without GVHD, with complete sparing of graft-versus-malignancy. This approach may prove valuable as a therapeutic strategy promoting transplantation tolerance.

IL-2Rß-dependent signaling and CD103 functionally cooperate to maintain tolerance in the gut mucosa.

A network of mechanisms operates to maintain tolerance in the gut mucosa. Although CD103 marks many lymphoid cells within the gut, its direct functional role in intestinal tolerance is poorly understood. CD103 may be part of a redundant pathway, as CD103(-/-) mice do not exhibit autoimmunity. To reduce such redundancy, CD103(-/-) mice were crossed to mice (designated Y3) whose T cells expressed a mutant IL-2Rß-chain that lowers IL-2R signaling. Unlike overtly healthy Y3 mice, all Y3/CD103(-/-) mice rapidly developed severe colitis. The large intestine of these mice contained an increase in CD4(+) Th1 and Th17 effector cells and a reduced ratio of regulatory T cells (Tregs). Importantly, colitis was effectively prevented by the transfer of wild-type Tregs into Y3/CD103(-/-) mice. Impaired intestinal tolerance was not attributed to an obvious lack of CD103-dependent gene regulation or intestinal homing/retention by Tregs nor a lack of functional activities typically associated with CD103(+) dendritic cells, such as peripherally induced Treg development or imprinting CCR9 and α4ß7 homing molecules on Tregs and T effector cells. Transcriptome analysis of Tregs was consistent with altered homeostasis due to impaired IL-2Rß-dependent signaling with minimal dysregulation added by the absence of CD103. Rather, the absence of CD103 functioned to alter the localization of the cells within the gut microenvironment that may alter Treg homeostasis. Thus, IL-2Rß-dependent signaling and CD103 normally cooperate through distinctive processes to promote Treg homeostasis and immune tolerance.

Monoclonal hyperglobulinemia in lymphosarcoma in a cockatiel (Nymphicus hollandicus) and a blue and gold macaw (Ara ararauna).

A 15-yr-old cockatiel (Nymphicus hollandicus) presented with decreased activity and appetite. A presumptive diagnosis of lymphoproliferative disease was made because of a monoclonal band observed on plasma protein electrophoresis. The owner declined further diagnostics or treatment, and the bird died 2 yr later. In a second case, a 4-yr-old blue and gold macaw (Ara ararauna) presented with lethargy and anorexia. Despite supportive treatment, the bird died 7 days later. Blood studies also demonstrated a monoclonal band on plasma protein electrophoresis. The histologic review of tissues from both birds found extensive neoplastic lymphoplasmocytic cells in the liver and supported a diagnosis of lymphosarcoma.

Effect of fenbendazole on an autoimmune mouse model.

Fenbendazole is an anthelmintic drug widely used to treat and prevent pinworm infection in laboratory rodents. Data regarding possible side effects of fenbendazole on the immune system are conflicting, potentially due to the design of treatment protocols. The purpose of the current study was to determine the effects of 2 fenbendazole therapeutic regimens (continuous for 5 wk and alternating weeks [that is, 1 wk on, 1 wk off] for 9 wk) on the development of autoimmune disease in (NZB × NZW)F1 mice. No significant differences in survival curves or weight were observed between the treatment groups and cohort mice receiving nonmedicated feed. At the termination of the experiment, there were no differences in tissue pathology. Hematocrit decreased and BUN increased over time in all groups, but no significant differences were present between groups. After the cessation of treatment, mice fed the medicated diet continuously for 5 wk showed an increase in antiDNA antibody. Although this difference was significant, it did not affect survival curves or disease-related tissue or blood changes. These data indicate that common protocols of fenbendazole treatment do not alter the progression of autoimmune disease in (NZB × NZW)F1 mice.

Quantitation of acute phase proteins and protein electrophoresis in monitoring the acute inflammatory process in experimentally and naturally infected mice.

Serologic screening for infectious disease in sentinel mice from rodent colonies is expensive and labor-intensive, often involving multiple assays for several different infectious agents. Previously, we established normal reference ranges for the protein fractions of several laboratory strains of mice by using a commercially available agarose system of protein electrophoresis. In the current study, we address protein fractionation and quantitation of acute phase proteins (APP) in mice experimentally infected with Sendai virus or mouse parvovirus. We further investigate this methodology by using samples from sentinel mice from colonies with endemic infection. All study groups showed significant increases in gamma globulins. Various other protein fractions showed mild variable changes; significant differences were not detected for individual APP. These results contrast the significant changes observed in APP and protein electrophoresis by using the standard methods of inducing inflammatory responses through injection of complete Freund adjuvant or LPS. These present data suggest that although quantitation of individual APP may not be helpful, gamma globulin levels may reflect infection in laboratory mice and provide a possible adjunct to traditional screening methods.

CD4(+) CD25(+) Foxp3(+) T regulatory cells with limited TCR diversity in control of autoimmunity.

The importance of high TCR diversity of T regulatory (Treg) cells for self-tolerance is poorly understood. To address this issue, TCR diversity was measured for Treg cells after transfer into IL-2Rbeta(-/-) mice, which develop lethal autoimmunity because of failed production of Treg cells. In this study, we show that high TCR diversity of pretransferred Treg cells led to selection of therapeutic Treg cells with lower TCR diversity that prevented autoimmunity. Pretransferred Treg cells with lower diversity led to selection of Treg cells through substantial peripheral reshaping with even more restricted TCR diversity that also suppressed autoimmune symptoms. Thus, in a setting of severe breakdown of immune tolerance because of failed production of Treg cells, control of autoimmunity is achieved by only a fraction of the Treg TCR repertoire, but the risk for disease increased. These data support a model in which high Treg TCR diversity is a mechanism to ensure establishing and maintaining self-tolerance.

Acute phase response in animals: a review.

The acute phase response is a complex systemic early-defense system activated by trauma, infection, stress, neoplasia, and inflammation. Although nonspecific, it serves as a core of the innate immune response involving physical and molecular barriers and responses that serve to prevent infection, clear potential pathogens, initiate inflammatory processes, and contribute to resolution and the healing process. Acute phase proteins, an integral part of the acute phase response, have been a focus of many applications in human diagnostic medicine and recently have been identified in common animal species. Potential applications to diagnosis, prognosis, assessment of animal health, and laboratory animal welfare are readily apparent.

Reference values for serum proteins of common laboratory rodent strains.

Protein electrophoresis is a common proven technique to determine the protein components of plasma or serum in human, veterinary, and laboratory animal medicine. Changes in albumin and globulin protein levels can provide early and valuable diagnostic and prognostic information. Here we describe a preliminary analysis of the distribution of serum protein fractions in adult BALB/c, C57BL/6, and CD1 mice and Sprague-Dawley rats and describe the changes in protein values from birth to maturity in BALB/c mice and Sprague-Dawley rats. Quantifiable changes in the electrophoretic profile were apparent in mice with chronic-active dermatitis.

Effects of fenbendazole on the murine humoral immune system.

Pinworms are highly contagious parasites that have been effectively treated in laboratory rodents with fenbendazole (FBZ). Whether FBZ has any detrimental side effects that may compromise experimental results is unknown. Here we asked whether the immune systems from young and aged mice are altered under FBZ treatment. We compared control and FBZ-treated groups of young (age, 2 to 4 mo) and old (age, 22 to 24 mo) BALB/cN mice. The treated mice received a total of 4 wk (alternating-week treatment regimen) of FBZ-medicated feed. Spleen and bone marrow were collected for immunologic assays, and heart, stomach, intestines, kidneys, and liver were evaluated by histopathology. Our results indicate that FBZ treatment has significant effects on the immune systems of mice; these effects are greater in aged mice. FBZ treatment adversely affected mRNA and protein expression of E2A (a transcription factor crucial for B lymphocytes) in activated precursor B lymphocytes obtained from the bone marrow of young and old mice. These effects were reversed by 6 wk on regular feed after the end of treatment. Activated B lymphocytes from the spleens of young and old mice showed decreased function (cell proliferation, E2A mRNA and protein expression) through the last time point of FBZ treatment but recovered by 2 to 4 wk after treatment. Our findings suggest that FBZ treatment may alter sensitive immune and molecular measures as presented here, and postponing the experimental use of mice until at least 6 wk after treatment should be considered.

Downregulation of interleukin-7 and hepatocyte growth factor in the thymic microenvironment is associated with thymus involution in tumor-bearing mice.

During mammary tumorigenesis, there is a profound thymic involution associated with severe depletion of the most abundant subset of thymocytes, CD4(+)CD8(+) immature cells, and an early arrest in at least two steps of T cell differentiation. Thymic atrophy that is normally related with aging has been observed in other model systems, including graft-vs-host disease (GVHD) and tumor development. However, the mechanisms involved in this phenomenon remain to be elucidated. Vascular endothelial growth factor (VEGF) has been associated with thymic involution, when expressed at high levels systemically. In thymuses of D1-DMBA-3 tumor-bearing mice, this growth factor is diminished relative to the level of normal thymuses. Interestingly, the expression of hepatocyte growth factor (HGF), which has been associated with proliferation, cell survival, angiogenesis and B-cell differentiation, is profoundly down-regulated in thymuses of tumor bearers. In parallel, IL-7 and IL-15 mRNA, crucial cytokines involved in thymocytes development and cellular homeostasis, respectively, are also down-regulated in the thymuses of tumor hosts as compared to those of normal mice. Injection of HGF into mice implanted with mammary tumors resulted in normalization of thymic volume and levels of VEGF, IL-7 and IL-15. While, injections of IL-7 partially restored the thymic involution observed in the thymuses of tumor-bearing mice, injection of IL-15 did not have any significant effects. Our data suggest that the downregulation of HGF and IL-7 may play an important role in the thymic involution observed in tumor-bearing hosts.

Effects of storage temperature and time on clinical biochemical parameters from rat serum.

Serum is often frozen and banked for analysis at a later date. This study assessed the stability of 17 analytes in rat serum during refrigeration at 4 degrees C and extended storage at -20 degrees C (frost-free and nonfrost-free freezers) and -70 degrees C. Samples were analyzed by using an automated dry-slide chemistry analyzer at time 0 and then stored as aliquots for analysis at time points including day 7, 30, 90, and 360. After 7 d of refrigeration, only creatine kinase activity had varied by more than 10% of the starting value. Freezing at -70 degrees C was clearly superior to -20 degrees C where changes were observed in CO(2) as early as day 30 and alanine aminotransferase as early as day 90. Samples stored in frost-free and nonfrost-free -20 degrees C freezers did not differ significantly through day 90. Factors such as storage time and temperature should be considered when designing any retrospective study.