Experiments were conducted to optimize triploid induction parameters, and assess triploid sterility, in burbot. Duration and timing of shocks were based on degree minutes, temperature multiplied by ...time, denoted as °C min. Hydrostatic shock experiments investigated the duration of shock using 7500 or 8500 psi at 180°C min post‐fertilization. Thermal shocks investigated duration of shock and post‐fertilization shock timing using a shock of 16°C. Sterility experiments investigated egg survival when diploids were crossed with triploids. Hydrostatic shock of 7500 psi for 10 or 20°C min can induce triploidy ≥90% and exhibits survival that is statistically similar, p ≤ 0.05, to controls. Hydrostatic shock of 8500 psi for 5 or 10°C min yielded triploid induction of 93% and 100%, respectively, with survival that is statistically similar to controls, p ≤ 0.05. Thermal induction experiments indicated shocks at 120°C min post‐fertilization, for durations between 350 and 450°C min, have potential to induce triploidy ≥90% while facilitating survival statistically similar to controls, p ≤ 0.05. Induction of tetraploidy was observed. Sterility experiments determined that triploid burbot are functionally sterile. These results may allow production of burbot where sterility is required.
Intensive commercial production of burbot fingerlings will depend on consistent supply of eggs and larvae at multiple times during the year; however, no information on out-of-season spawning of ...burbot exists. The current study was designed to shift sexual maturation of burbot broodstock by six months from the natural spawning season through temperature and photoperiod manipulation. Results demonstrated that out-of-season spawning of burbot was successful. Broodstock, egg quality, and larval condition data were compared between out-of-season and in-season groups. For both production regimes, broodstock were sampled for condition factor and egg production, and embryos were monitored for development, viability, and survival. Larvae were reared to 35 days post hatch (dph) for growth and survival comparison. There was no significant difference in pre-spawn condition factor between in-season and out-of-season males or females. Post-spawn condition factor for out-of-season females was not statistically different from in-season females, 0.83 (±0.06) and 0.66 (±0.07) respectively. However, post-spawn condition factor for out-of-season male broodstock was significantly higher, 0.82 (±0.04), than in-season fish, 0.64 (±0.02). Eggs produced relative to weight of pre-spawn female did not differ significantly between in-season and out-of-season production, 328.5 (±22.95) and 213 (±38.04) eggs/g, respectively. Fertilization did not differ significantly between treatments, averaging 90% for both in and out-of-season production. Egg survival was higher, 61.18% (±9.3), in the final week of incubation for out-of-season groups compare to eggs from in-season broodstock, 20.59% (±10.6). However, no difference in larval growth or survival was observed out to the 35dph termination point. Overall, egg quality and larval survival was not negatively impacted for broodstock spawned out-of-season. This is the first report demonstrating that burbot eggs can be produced outside the natural spawning season by altering photoperiod and temperature. As commercial production of this species develops, this will be an important tool to mitigate the risk of relying on a single annual spawning season and can ensure a consistent supply of burbot fingerlings throughout the year.
•Sexual maturation and spawning of burbot was shifted six months from natural spawning season with photothermal manipulation•Egg and larval quality did not differ between out of season and natural in-season broodstock•As commercial production of burbot develops this will be an important tool to alleviate risks of an annual spawning season
The feasibility of triploid induction for burbot (Lota lota) was determined following a series of hydrostatic (pressure) and thermal (heat) shock treatments. Hydrostatic shock treatments were ...designed to test a range of variables including 1) duration of shock; 2) timing of shock (post-fertilization); and 3) shock pressure. Shock times post-fertilization and shock duration were varied by degree minutes (°C minutes). A hydrostatic shock of 8,500psi at 180 °C minutes post-fertilization for 10 °C minutes yielded the highest percent triploid induction (100%) and survival (95%) relative to the controls. Duration of pressure-shock longer than 10 °C min at 8,500psi and higher, resulted in 100% pre-hatch mortality. A reduced shock pressure (7,500psi) resulted in a high percent triploidy (100%), but pre-hatch larval survival was 65.5% at a shock duration of 30 °C minutes. Thermal shock treatments included: duration of shock, timing of shock, and shock temperature. Triploid induction and survival were greatest following a thermal shock of 16 °C at 120 °C minutes post-fertilization for 500 °C minutes. This resulted in 96.6% triploidy and 57.4% survival relative to control groups. Shock temperatures above 16 °C generally resulted in a higher percent triploid induction but lower survival. Results presented here confirm that triploid induction in burbot is possible. Further work is needed to confirm scale up potential, survival dynamics, sterility of triploid burbot, and changes in growth performance. Production of sterile burbot may increase opportunities for culturing burbot in areas where escapement may be a concern or when growth is inhibited due to reproductive maturation.
•Heat shock of 16 °C at 120 °C minutes post-fertilization for 500 °C minutes yielded 96.6% triploidy relative to control groups.•Hydrostatic shock of 8,500psi at 180 °C minutes post-fertilization for 10 °C minutes produced 100% triploid induction.•Triploid burbot production may allow for culture in areas were escapement is a concern or increase growth potential.
T cell affinity for antigen initiates adaptive immunity. However, the contribution of low affinity cells to a response is unknown as it has not been possible to assess the entire affinity range of a ...polyclonal T cell repertoire. In this study, we used a highly sensitive two-dimensional binding assay to identify low affinity cells in polyclonal autoreactive and pathogen-reactive CD4(+) T cell populations specific for myelin oligodendrocyte glycoprotein (MOG) and lymphocytic choriomeningitis virus (LCMV) antigens, respectively. Low affinity CD4(+) T cells, below detection with peptide-major histocompatibility complex class II tetramers, were at least as frequent as high affinity responders and contributed significant effector cytokines in both primary antigen-specific responses. We further demonstrated that MOG- and LCMV-specific CD4(+) T cells possessed similarly broad ranges in their affinities (>100-fold wide), only differing in the frequencies of low and high affinity cells. Thus, low as well as high affinity CD4(+) T cells are critical effectors in autoimmune and pathogen-specific responses.
In order to recognize and combat a diverse array of pathogens the immune system has a large repertoire of T cells having unique T cell receptors (TCRs) with only a few clones specific for any given ...antigen. We discuss how the number of different possible TCRs encoded in the genome (the potential repertoire) and the number of different TCRs present in an individual (the realized repertoire) can be measured. One puzzle is that the potential repertoire greatly exceeds the realized diversity of naïve T cells within any individual. We show that the existing hypotheses fail to explain why the immune system has the potential to generate far more diversity than is used in an individual, and propose an alternative hypothesis of "evolutionary sloppiness." Another immunological puzzle is why mice and humans have similar repertoires even though humans have over 1000-fold more T cells. We discuss how the idea of the "protecton," the smallest unit of protection, might explain this discrepancy and estimate the size of "protecton" based on available precursor frequencies data. We then consider T cell cross-reactivity - the ability of a T cell clone to respond to more than one epitope. We extend existing calculations to estimate the extent of expected cross-reactivity between the responses to different pathogens. Our results are consistent with two observations: a low probability of observing cross-reactivity between the immune responses to two randomly chosen pathogens; and the ensemble of memory cells being sufficiently diverse to generate cross-reactive responses to new pathogens.
T cells recognizing self-peptides that mediate autoimmune disease and those that are responsible for efficacious immunity against pathogens may differ in affinity for antigen due to central and ...peripheral tolerance mechanisms. Here we utilize prototypical self-reactive (myelin) and viral-specific (LCMV) T cells from T cell receptor (TCR) transgenic mice (2D2 and SMARTA, respectively) to explore affinity differences. The T cells responsive to virus possessed >10,000 fold higher 2D affinity as compared to the self-reactive T cells. Despite their dramatically lower affinity for their cognate ligand, 2D2 T cells respond with complete, albeit delayed, activation (proliferation and cytokine production). SMARTA activation occurs rapidly, achieving peak phosphorylation of p38 (1 minute), Erk (30 minutes), and Jun (3 hours) as well as CD69 and CD25 upregulation (3 and 6 hours, respectively), with a corresponding early initiation of proliferation. 2D2 stimulation with MOG results in altered signaling--no phospho-Erk or phospho-p38 accumulation, significantly delayed activation kinetics of Jun (12 hours), and delayed but sustained SHP-1 activity--as well as delayed CD69 and CD25 expression (12-24 hours), and slow initiation of proliferation. This delay was not intrinsic to the 2D2 T cells, as a more potent antigen with >100-fold increased 2D affinity restored rapid response kinetics in line with those identified for the viral antigen. Taken together, these data demonstrate that time can offset low TCR affinity to attain full activation and suggest a mechanism by which low affinity T cells participate in autoimmune disease.
Of interest to the etiology of demyelinating autoimmune disease is the potential to aberrantly activate CD4
T cells due to cross-recognition of multiple self-epitopes such as has been suggested for ...myelin oligodendrocyte glycoprotein epitope 35-55 (MOG
) and neurofilament medium protein epitope 15-35 (NFM
). NFM
is immunogenic in C57BL/6 mice but fails to induce demyelinating disease by polyclonal T cells despite having the same TCR contact residues as MOG
, a known encephalitogenic Ag. Despite reported cross-reactivity with MOG-specific T cells, the polyclonal response to NFM
did not expand threshold numbers of MOG
tetramer-positive T cells. Furthermore, NFM lacked functional synergy with MOG to promote experimental autoimmune encephalomyelitis because NFM-deficient synonymous with knockout mice developed an identical disease course to wild-type mice after challenge with MOG
Single-cell analysis of encephalitogenic T cells using the peptide:MHC monomer-based two-dimensional micropipette adhesion frequency assay confirmed that NFM was not a critical Ag driving demyelinating disease because NFM
-specific T cells in the CNS were predominantly reactive to MOG
The absence of NFM contribution to disease allowed mapping of the amino acids required for encephalitogenicity and expansion of high-affinity, MOG-specific T cells that defined the polyclonal response. Alterations of N-terminal residues outside of the NFM
core nonamer promoted expansion of high-affinity, MOG
tetramer-positive T cells and promoted consistent experimental autoimmune encephalomyelitis induction, unlike mice challenged with NFM
Although NFM
is immunogenic and cross-reactive with MOG at the polyclonal level, it fails to expand a threshold level of encephalitogenic, high-affinity MOG-specific T cells.
Recognition of self-peptide-MHC (pMHC) complexes by CD4 T cells plays an important role in the pathogenesis of many autoimmune diseases. We analyzed formation of immunological synapses (IS) in ...self-reactive T cell clones from patients with multiple sclerosis and type 1 diabetes. All self-reactive T cells contained a large number of phosphorylated T cell receptor (TCR) microclusters, indicative of active TCR signaling. However, they showed little or no visible pMHC accumulation or transport of TCR-pMHC complexes into a central supramolecular activation cluster (cSMAC). In contrast, influenza-specific T cells accumulated large quantities of pMHC complexes in microclusters and a cSMAC, even when presented with 100-fold lower pMHC densities. The self-reactive T cells also maintained a high degree of motility, again in sharp contrast to virus-specific T cells. 2D affinity measurements of three of these self-reactive T cell clones demonstrated a normal off-rate but a slow on-rate of TCR binding to pMHC. These unusual IS features may facilitate escape from negative selection by self-reactive T cells encountering very small amounts of self-antigen in the thymus. However, these same features may enable acquisition of effector functions by self-reactive T cells encountering large amounts of self-antigen in the target organ of the autoimmune disease.
Malignant melanoma incidence has been increasing for over 30 years, and despite promising new therapies, metastatic disease remains difficult to treat. We describe preliminary results from a Phase I ...clinical trial (NCT01586403) of adoptive cell therapy in which three patients received autologous CD4
+
and CD8
+
T cells transduced with a lentivirus carrying a tyrosinase-specific TCR and a marker protein, truncated CD34 (CD34t). This unusual MHC Class I-restricted TCR produces functional responses in both CD4
+
and CD8
+
T cells. Parameters monitored on transduced T cells included activation (CD25, CD69), inhibitory (PD-1, TIM-3, CTLA-4), costimulatory (OX40), and memory (CCR7) markers. For the clinical trial, T cells were activated, transduced, selected for CD34t
+
cells, then re-activated, and expanded in IL-2 and IL-15. After lymphodepleting chemotherapy, patients were given transduced T cells and IL-2, and were followed for clinical and biological responses. Transduced T cells were detected in the circulation of three treated patients for the duration of observation (42, 523, and 255 days). Patient 1 tolerated the infusion well but died from progressive disease after 6 weeks. Patient 2 had a partial response by RECIST criteria then progressed. After progressing, Patient 2 was given high-dose IL-2 and subsequently achieved complete remission, coinciding with the development of vitiligo. Patient 3 had a mixed response that did not meet RECIST criteria for a clinical response and developed vitiligo. In two of these three patients, adoptive transfer of tyrosinase-reactive TCR-transduced T cells into metastatic melanoma patients had clinical and/or biological activity without serious adverse events.
Coxiella burnetii is a zoonotic pathogen that causes Q fever in humans and is transmitted primarily from infected goats, sheep, or cows. Q fever typically presents as an acute febrile illness; ...however, individuals with certain predisposing conditions, including cardiac valvulopathy, are at risk for chronic Q fever, a serious manifestation that may present as endocarditis. In response to a cluster of Q fever cases detected by public health surveillance, we evaluated C. burnetii infection in a community that operates a large-scale cow and goat dairy. A case was defined as an individual linked to the community with a C. burnetii phase II IgG titer ≥ 128. Of 135 participants, 47 (35%) cases were identified. Contact with or close proximity to cows, goats, and their excreta was associated with being a case (relative risk 2.7, 95% confidence interval 1.3-5.3). Cases were also identified among individuals without cow or goat contact and could be related to windborne spread or tracking of C. burnetii on fomites within the community. A history of injection drug use was reported by 26/130 (20%) participants; follow-up for the presence of valvulopathy and monitoring for development of chronic Q fever may be especially important among this population.
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