The ZFP36 family of RNA binding proteins regulates homeostatic and autoreactive T cell responses.

RNA binding proteins are important regulators of T cell activation, proliferation, and cytokine production. The zinc finger protein 36 (ZFP36) family genes (Zfp36, Zfp36l1, and Zfp36l2) encode RNA binding proteins that promote the degradation of transcripts containing AU-rich elements. Numerous studies have demonstrated both individual and shared functions of the ZFP36 family in immune cells, but their collective function in T cells remains unclear. Here, we found a redundant and critical role for the ZFP36 proteins in regulating T cell quiescence. T cell-specific deletion of all three ZFP36 family members in mice resulted in early lethality, immune cell activation, and multiorgan pathology characterized by inflammation of the eyes, central nervous system, kidneys, and liver. Mice with T cell-specific deletion of any two Zfp36 genes were protected from this spontaneous syndrome. Triply deficient T cells overproduced proinflammatory cytokines, including IFN-γ, TNF, and GM-CSF, due to increased mRNA stability of these transcripts. Unexpectedly, T cell-specific deletion of both Zfp36l1 and Zfp36l2 rendered mice resistant to experimental autoimmune encephalomyelitits due to failed priming of antigen-specific CD4+ T cells. ZFP36L1 and ZFP36L2 double-deficient CD4+ T cells had poor proliferation during in vitro T helper cell polarization. Thus, the ZFP36 family redundantly regulates T cell quiescence at homeostasis, but ZFP36L1 and ZFP36L2 are specifically required for antigen-specific T cell clonal expansion.

Characterization of magnetic resonance-guided high-intensity focused ultrasound (MRgHIFU)-induced large-volume hyperthermia in deep and superficial targets in a porcine model.

PURPOSE: To characterize temperature fields and tissue damage profiles of large-volume hyperthermia (HT) induced by magnetic resonance-guided high-intensity focused ultrasound (MRgHIFU) in deep and superficial targets in vivo in a porcine model. METHODS: Nineteen HT sessions were performed in vivo with a commercial MRgHIFU system (Sonalleve® V2, Profound Medical Inc., Mississauga, ON, Canada) in hind leg muscles of eight pigs with temperature fields of cross-sectional diameter of 58-mm. Temperature statistics evaluated in the target region-of-interest (tROI) included accuracy, temporal variation, and uniformity. The impact of the number and location of imaging planes for feedback-based temperature control were investigated. Temperature fields were characterized by time-in-range (TIR, the duration each voxel stays within 40-45 °C) maps. Tissue damage was characterized by contrast-enhanced MRI, and macroscopic and histopathological analysis. The performance of the Sonalleve® system was benchmarked against a commercial phantom. RESULTS: Across all HT sessions, the mean difference between the average temperature (Tavg) and the desired temperature was -0.4 ± 0.5 °C; the standard deviation of temperature 1.2 ± 0.2 °C; the temporal variation of Tavg for 30-min HT was 0.6 ± 0.2 °C, and the temperature uniformity was 1.5 ± 0.2 °C. A difference of 2.2-cm (in pig) and 1.5-cm (in phantom) in TIR dimensions was observed when applying feedback-based plane(s) at different locations. Histopathology showed 62.5% of examined HT sessions presenting myofiber degeneration/necrosis within the target volume. CONCLUSION: Large-volume MRgHIFU-mediated HT was successfully implemented and characterized in a porcine model in deep and superficial targets in vivo with heating distributions modifiable by user-definable parameters.

Feasibility and safety assessment of magnetic resonance-guided high-intensity focused ultrasound (MRgHIFU)-mediated mild hyperthermia in pelvic targets evaluated using an in vivo porcine model.

Purpose: To evaluate the feasibility and assess safety parameters of magnetic resonance-guided high-intensity focused ultrasound (MRgHIFU)-mediated hyperthermia (HT; heating to 40-45 °C) in various pelvic targets in a porcine model in vivo.Methods: Thirteen HT treatments were performed in six pigs with a commercial MRgHIFU system (Sonalleve V2, Profound Medical Inc., Mississauga, Canada) to muscle adjacent to the ventral/dorsal bladder wall and uterus to administer 42 °C (±1°) for 30 min (±5%) using an 18-mm target diameter and 100 W power. Feasibility was assessed using accuracy, uniformity, and MR-thermometry performance-based metrics. Safety parameters were assessed for tissues in the targets and beam-path by contrast-enhanced MRI, gross-pathology and histopathology.Results: Across all HT sessions, the mean difference between average temperature (Tavg) and the target temperature within the target region-of-interest (tROI, the cross-section of the heated volume at focal depth) was 0.51 ± 0.33 °C. Within the tROI, the temperature standard deviation averaged 1.55 ± 0.31 °C, the average 30-min Tavg variation was 0.80 ± 0.17 °C, and the maximum difference between Tavg and the 10th- or 90th-percentile temperature averaged 2.01 ± 0.44 °C. The average time to reach ≥41 °C and cool to ≤40 °C within the tROI at the beginning and end of treatment was 47.25 ± 27.47 s and 66.37 ± 62.68 s, respectively. Compared to unheated controls, no abnormally-perfused tissue or permanent damage was evident in the MR images, gross pathology or histological analysis.Conclusions: MRgHIFU-mediated HT is feasible and safety assessment is satisfactory for treating an array of clinically-mimicking pelvic geometries in a porcine model in vivo, implying the technique may have utility in treating pelvic targets in human patients.

Phylogenetic and functional analysis of ADAMTS13 identifies highly conserved domains essential for allosteric regulation.

The metalloprotease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats member 13) prevents microvascular thrombosis by cleaving von Willebrand factor (VWF) within platelet-rich thrombi, and cleavage depends on allosteric activation of ADAMTS13 by the substrate VWF. Human ADAMTS13 has a short propeptide, metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains (proximal domains), followed by 7 T and 2 CUB (complement components C1r and C1s, sea urchin protein Uegf, and bone morphogenetic protein-1) domains (distal domains). Distal domains inhibit the catalytic proximal domains; binding of distal T8-CUB domains to the VWF D4 domain relieves autoinhibition and promotes cleavage of the nearby VWF A2 domain. However, the role of specific ADAMTS13 distal domains in this allosteric mechanism is not established. Assays of plasma ADAMTS13 from 20 placental mammals, birds, and amphibians show that allosteric regulation is broadly conserved, and phylogenetic analysis of 264 vertebrates shows the long propeptide, T3, T4, T6, and T6a domains have been deleted several times in placental mammals, birds, and fish. Notably, pigeon ADAMTS13 has only 3 distal T domains but was activated normally by human VWF D4 and cleaved VWF multimers, preferentially under fluid shear stress. Human ADAMTS13 constructed to resemble pigeon ADAMTS13 retained normal allosteric regulation and shear-dependent cleavage of VWF. Thus, the T3-T6 domains of human ADAMTS13 are dispensable. Conversely, deletion of T7 or T8 abolished allosteric activation. For most species, some sequence changes in the VWF substrate can markedly increase the rate of cleavage, suggesting that ADAMTS13 and VWF have not evolved to be optimal enzyme-substrate pairs. These properties may reflect evolutionary pressure to balance the risk for VWF-dependent bleeding and thrombosis.

Identification and taxonomic characterization of Bordetella pseudohinzii sp. nov. isolated from laboratory-raised mice.

Bordetella hinzii is known to cause respiratory disease in poultry and has been associated with a variety of infections in immunocompromised humans. In addition, there are several reports of B. hinzii infections in laboratory-raised mice. Here we sequenced and analysed the complete genome sequences of multiple B. hinzii-like isolates, obtained from vendor-supplied C57BL/6 mice in animal research facilities on different continents, and we determined their taxonomic relationship to other Bordetella species. The whole-genome based and 16S rRNA gene based phylogenies each identified two separate clades in B. hinzii, one was composed of strains isolated from poultry, humans and a rabbit whereas the other clade was restricted to isolates from mice. Distinctly different estimated DNA-DNA hybridization values, average nucleotide identity scores, gene content, metabolic profiles and host specificity all provide compelling evidence for delineation of the two species, B. hinzii - from poultry, humans and rabbit - and Bordetella pseudohinzii sp. nov. type strain 8-296-03T (=NRRL B-59942T=NCTC 13808T) that infect mice.

Metabotropic glutamate receptor 5 antagonism with fenobam: examination of analgesic tolerance and side effect profile in mice.

BACKGROUND: The metabotropic glutamate receptor 5 noncompetitive antagonist fenobam is analgesic in rodents. Future development of fenobam as an analgesic in humans will require a favorable long-term treatment profile and a lack of significant deleterious side effects. This study aimed to determine whether tolerance to fenobam's analgesic effects developed over 14 days and to assess for side effects in mice. METHODS: Mouse models of pain, locomotor behavior, and coordination were used. Fenobam or vehicle (n = 8 or 11 per group) was administered for 14 days, and analgesic tolerance to fenobam was assessed using the formalin test. Histopathologic examination, hematology, and clinical chemistry analysis after 14-day fenobam administration were also assessed (n = 12 or 9). The effects of fenobam on locomotor activity were assessed in the open field and elevated zero maze (n = 8 or 7). Coordination was assessed using ledge crossing and vertical pole descent tasks (n = 11 or 10). RESULTS: Tolerance to fenobam's analgesic effect did not develop after 14 days. Chronic fenobam administration resulted in statistically significantly less weight gain compared with vehicle control subjects, but did not cause any physiologically or statistically significant hematologic abnormalities, altered organ function, or abnormal histopathology of the liver, brain, or testes. Fenobam administration resulted in a metabotropic glutamate receptor 5-dependent increase in exploratory behavior but does not impair motor coordination at analgesic doses. CONCLUSIONS: Analgesic tolerance to repeat fenobam dosing does not develop. Chronic dosing of up to 14 days is well tolerated. Fenobam represents a promising candidate for the treatment of human pain conditions.

Laparoscopic fixation of biologic mesh at the hiatus with fibrin or polyethylene glycol sealant in a porcine model.

BACKGROUND: The objective of this study was to determine the acute and chronic fixation strengths achieved by fibrin or polyethylene glycol (PEG) sealants to secure biologic mesh at the esophageal hiatus in a porcine model. METHODS: For this study, 32 female domestic pigs were divided into four groups of 8 each. The four groups respectively received acute fibrin sealant, acute PEG sealant, chronic fibrin sealant, and chronic PEG sealant. Laparoscopically, a 5.5 × 8.5-cm piece of Biodesign Surgisis Hiatal Hernia Graft (porcine small intestine submucosa) was oriented with the U-shaped cutout around the gastroesophageal junction and the short axis in the craniocaudal direction to simulate hiatal reinforcement with a biologic mesh. The mesh then was secured with 2 ml of either fibrin sealant or PEG sealant. The pigs in the acute groups were maintained alive for 2 h to allow for complete polymerization of the sealants, and the pigs in the chronic group were maintained alive for 14 days. After the pigs were euthanized, specimens of the mesh-tissue interface were subjected to lap shear testing to determine fixation strength, and hematoxylin and eosin (H&E) stained slides were evaluated for evidence of remodeling. RESULTS: No significant differences were observed between the acute and chronic fixation strengths or the remodeling characteristics of the two sealants. However, fixation strength increased significantly over time for both types of sealant. Evidence of remodeling also was significantly more pronounced in the chronic specimens than in the acute specimens. CONCLUSIONS: This study demonstrated the feasibility of using fibrin or PEG sealants to secure biologic mesh at the hiatus in a porcine model.

Biomechanical and histologic evaluation of fenestrated and nonfenestrated biologic mesh in a porcine model of ventral hernia repair.

BACKGROUND: The purpose of this study was to compare tissue incorporation and adhesion characteristics of a novel fenestrated versus nonfenestrated crosslinked porcine dermal matrix (CPDM) (Bard CollaMend) in a porcine model of ventral hernia repair. STUDY DESIGN: Bilateral abdominal wall defects were created in 24 Yucatan minipigs, resulting in 48 defects, which were allowed to mature for 21 days. Twelve defects were repaired with fenestrated CPDM using a preperitoneal technique, 12 with fenestrated CPDM using an intraperitoneal technique, 12 with nonfenestrated CPDM using a preperitoneal technique, and 12 with nonfenestrated CPDM using an intraperitoneal technique. Half of the animals in the intraperitoneal group were euthanized after 1 month, and the other half after 3 months. Similarly, half of the animals in the preperitoneal group were euthanized after 1 month, and the other half after 6 months. Biomechanical testing and histologic evaluation were performed. RESULTS: Intraperitoneal placement of the CPDM products resulted in significantly greater adhesed area compared with preperitoneal placement (p < 0.05). Tissue ingrowth into preperitoneal fenestrated and nonfenestrated CPDM resulted in significantly greater incorporation strengths after 6 months compared with 1 month (p = 0.03 and p < 0.0001). Histologic analysis showed significantly greater cellular infiltration, extracellular matrix deposition, and neovascularization, with less fibrous encapsulation through the center of the fenestrations compared with all other sites evaluated, including nonfenestrated grafts. CONCLUSIONS: Histologic findings revealed increased tissue incorporation at fenestration sites compared with nonfenestrated grafts regardless of implant location or time in vivo. However, preperitoneal placement resulted in greater incorporation strength, less adhesed area, and lower adhesion scores compared with intraperitoneal placement for both fenestrated and nonfenestrated CPDM.

Histologic and biomechanical evaluation of crosslinked and non-crosslinked biologic meshes in a porcine model of ventral incisional hernia repair.

BACKGROUND: The objective of this study was to evaluate the biomechanical characteristics and histologic remodeling of crosslinked (Peri-Guard, Permacol) and non-crosslinked (AlloDerm, Veritas) biologic meshes over a 12 month period using a porcine model of incisional hernia repair. STUDY DESIGN: Bilateral incisional hernias were created in 48 Yucatan minipigs and repaired after 21 days using an underlay technique. Samples were harvested at 1, 6, and 12 months and analyzed for biomechanical and histologic properties. The same biomechanical tests were conducted with de novo (time 0) meshes as well as samples of native abdominal wall. Statistical significance (p < 0.05) was determined using 1-way analysis of variance with a Fisher's least significant difference post-test. RESULTS: All repair sites demonstrated similar tensile strengths at 1, 6, and 12 months and no significant differences were observed between mesh materials (p > 0.05 in all cases). The strength of the native porcine abdominal wall was not augmented by the presence of the mesh at any of the time points, regardless of de novo tensile strength of the mesh. Histologically, non-crosslinked materials showed earlier cell infiltration (p < 0.01), extracellular matrix deposition (p < 0.02), scaffold degradation (p < 0.05), and neovascularization (p < 0.02) compared with crosslinked materials. However, by 12 months, crosslinked materials showed similar results compared with the non-crosslinked materials for many of the features evaluated. CONCLUSIONS: The tensile strengths of sites repaired with biologic mesh were not impacted by very high de novo tensile strength/stiffness or mesh-specific variables such as crosslinking. Although crosslinking distinguishes biologic meshes in the short-term for histologic features, such as cellular infiltration and neovascularization, many differences diminish during longer periods of time. Characteristics other than crosslinking, such as tissue type and processing conditions, are likely responsible for these differences.