Gene expression analysis of Ca2+ channels and pumps expressed in T cells by RT-qPCR identified overexpression of the plasma membrane CRAC channel subunit ORAI1 and PMCA in older T cells. To test whether overexpression of the plasma membrane Ca2+ channel is sufficient to explain the kinetic information, we adapted a previously published computational model by Maurya and Subramaniam to include additional details on the store-operated calcium entry (SOCE) process to recapitulate Ca2+ dynamics after T cell receptor stimulation. Simulations demonstrated that upregulation of ORAI1 and PMCA channels is not sufficient to explain the observed alterations in Ca2+ signaling. Instead, modeling analysis identified kinetic parameters associated with the IP3R and STIM1 channels as potential causes for alterations in Ca2+ dynamics associated with the long term ex vivo culturing protocol. Due to these proteins having known cysteine residues susceptible to oxidation, we subsequently investigated and observed transcriptional remodeling of metabolic enzymes, a shift to more oxidized redox couples, and post-translational thiol oxidation of STIM1. The model-directed findings from this study highlight changes in the cellular redox environment that may ultimately lead to altered T cell calcium dynamics during immunosenescence or organismal aging.T cells reach a state of replicative senescence characterized by a decreased ability to proliferate and respond to foreign antigens. Calcium release associated with TCR engagement is widely used as a surrogate measure of T cell response. Using an ex vivo culture model that partially replicates features of organismal aging, we observe that while the amplitude of Ca T cells sustain elevated cytoplasmic Ca2+ levels for gene transcription, by balancing store-operated Ca2+ entry (SOCE) through the plasma membrane and Ca2+ buffering by the mitochondria. Calcium dynamics encode information from the antigenic peptide:TCR interaction for instructing T cells to activate cytokine production, such as IFN-γ i traces using the kinetic module of FlowJo . Consistent instrument voltage settings were used throughout data collection. All Ca2+ traces were first registered to ensure stimulation occurred at the same time. Time courses were smoothed using Savitzky-Golay filtering and normalized to the baseline (0–180 seconds) fluorescence. Peak time, peak amplitude and integral under the curve were calculated using custom Matlab scripts ). Decay parameters were obtained by fitting the decay portion of the dynamics to a sum of exponentials:Primary CD8+ T cells was extracted using the RNeasy Mini isolation kit  with RNase-free DNase set  according to the manufacturer's protocol. The integrity and concentration of intact total RNA was verified with a NanoDrop 1000 Spectrophotometer (Thermo Scientific). Real-time PCR was performed with a StepOnePlus RT-qPCR system instrument  using predesigned gene-specific primer and probe sets (SA Biosciences) for Actin, ORAI1, SERCA2b, SERCA3, PMCA, IP3R2 and IP3R3 or the Human Oxidative Stress and Antioxidant Defense PCR Array containing 84 predesigned gene-specific primer and probe sets following the manufacturer’s protocols. Briefly, 1 μg of total RNA was reverse transcribed and amplified using the RT-PCR kit (Qiagen) following the manufacturer’s instructions. Each 20 μL reaction mixture aliquot contained 1 μL of primer mixture (SA Biosciences), 2 μL of universal PCR Master Mix (Qiagen) and 1–4 μL of cDNA or water as a negative control. Initial denaturation of DNA was carried out at 95°C for 10 min. Forty amplification cycles were performed, each cycle consisting of denaturation  and annealing and extension . Relative expression levels were calculated using the ΔCT method (2-ΔCT). For individual calcium gene probe sets, each sample was amplified in triplicate and results were normalized using the housekeeping gene actin [Total RNA from CD8ne actin . For the6 cells were lysed in 100 μl 2% NP-40-based lysis buffer for standard SDS-PAGE analysis or were lysed in a guanidine HCl lysis buffer with 9.3 mg/mL iodoacteic acid (IAA) as previously described for redox western blotting [For western blotting, 8 × 10blotting . Bands wGlutathione (GSH) and glutathione disulfide (GSSG) were measured by HPLC as S-carboxmethyl N-dansyl derivatives using γ-glutamylglutamate as an internal standard .-1 mol-1), T = 298.15 K, z the number of transferred electrons (2), and F is the Faraday constant (96.485 C mol-1). The standard redox potentials of GSH and Trx at pH 7 used for the calculations were Cellular redox potential with respect to glutathione and thioredoxin was calculated using the Nernst equation: + T cells were isolated from whole blood and cultured as described. After 5 and 21 days in culture, primary CD8+ T cells were lysed at room temperature in degassed RIPA lysis buffer containing iodoacetamide . After lysing for 20 min in the dark, the lysates were sonicated on ice for 10 min. Cellular debris was removed by centrifugation at 10000 x g for 20 min at 4°C. The lysates were passed through a Zeba spin desalting column (Pierce) to remove excess iodoacetamide. Samples were then frozen so that all further assay steps could be performed on young and old cells simultaneously. The lysates were pre-cleared with PureProteome Protein A magnetic beads (Millipore) for 30 min at room temperature. STIM1 was immunoprecipitated overnight at 4°C using 1:40 dilution of anti-STIM1  and subsequently pulled down for 30 min at room temperatures. Beads were washed three times with lysis buffer without iodoacetamide. To reduce oxidized thiols and elute STIM1, lysis buffer containing 1 mM DTT and 2% SDS were added to the IP beads. The samples were heated for 15 min at 37°C and then for 10 min at 90°C. The supernatant was separated from the beads and nascent thiols were biotinylated using 3 mM of PEO-biotin-iodoacetamide (BIAM) (Cyanogen) in the dark at room temperature for 1 h. The reaction was quenched with the addition of 4X reducing Laemmlis sample buffer. For assay validation and controls, Jurkat cells were used instead of primary cells. Oxidized and reduced assay controls were prepared by either lysing in buffer without iodoacetamide or omitting DTT from the elution buffer, respectively. STIM1 oxidation was analyzed by standard Western blotting techniques. STIM1 was detected with . To detect oxidized cysteines, blots were stripped, blocked overnight at 4°C with Odyssey blocking buffer, and probed with 1:10000 IRDye® 800CW streptavidin (LI-COR Biotechnology) in Rockland blocking buffer for 1 h at room temperature. Blots were imaged with an Odyssey Scanner (LI-COR Biotechnology) and were quanitified in ImageStudio with the background calculated on a lane-by-lane basis according to standard procedures.Primary human CD82 in the plasma membrane to generate diacylglycerol (DAG) and 1, 4, 5-inositol triphosphate (IP3). Binding of IP3 to the IP3 receptor (IP3R) triggers the release of Ca2+ stored in the ER (JIP3). The resulting drop in ER Ca2+ levels activates the ER Ca2+ sensor STIM1, which translocates to the ER-PM (plasma membrane) junctions to activate a more sustained influx in the cytosol through the calcium release activated Ca2+ channels (CRAC) on the PM (Jcrac) [2+ ATP-ase (PMCA) pumps Ca2+ out of the cytosol and maintains a steep gradient of Ca2+ concentration from 50 nM inside the cell to 1.5 mM in the extracellular space (Jpmca). Because of this steep gradient, we assume there is a very small Ca2+ leak into the cytosol from the extracellular space (JPMleak). The Sarco/ER Ca2+ ATP-ase (SERCA) pumps cytosolic Ca2+ back in the ER stores to maintain an ER luminal concentration of 350 μM (Jserca). Similarly, we consider a small leak of Ca2+ ions from the ER to the cytosol (JERleak). Mitochondria are essential for the activation and maintenance of the store-operated calcium entry (SOCE) by buffering Ca2+ ions and preventing the negative feedback of Ca2+ on the CRAC channels [2+ ions in the mitochondria is mediated through the Ca2+ uniporter (Jmitin) and extrusion through the Na+/Ca2+ exchanger (Jmitout). The fundamental equations of Ca2+ kinetics in the various cellular compartments are described as follows:The binding of a peptide/MHC complex to the TCR triggers the recruitment of tyrosine kinases Lck, ITK and Zap70 to the TCR/CD3 complex, ultimately resulting in the phosphorylation and activation of PLC-γ The model represents these receptor-initiated events as a one-step input to phospho-PLC-γ levels. Activated PLC-γ cleaves PIP (Jcrac) ,29. The channels . Uptake cyt, Camit and CaER denote the concentration of free Ca2+ in the cytosol, mitochondria and ER respectively. βi, βer, βmit are the ratio of free to total Ca2+, assuming fast buffering with calcium-binding proteins in the cytosol, ER and mitochondria respectively [2+ is constant in the three cellular compartments and do not model explicitly the dynamics of free calcium-binding proteins. ρer, ρmit are the ratios of the ER and mitochondria volume to that of the cytosol.Caectively ,32. In t2+ signaling after TCR binding requires formation of IP3 through PLC-γ phosphorylation. We modeled PLC-γ activation as a simplified one step mass action kinetics (R) unbinding from the TCR , is a function of cytoplasmic Ca2+ and Q, the effective affinity of Ca2+ to the inhibitory site.A is a variable controlling the relative time scales between the differential equations, Kinh, the Ca2+ affinity to the Ca2+ inhibitory site and KIP3inh the affinity of IP3 to the IP3 binding site when the Ca2+ inhibitory site is occupied.The fraction of inactivated IP2+ ions from the ER to the cytoplasm. JERleak can be described as:Because of the gradient of concentration between the ER and the cytosol, there is a constant leakage of CaVserca is the maximum flux across the SERCA pump and Kserca is the concentration of Cacyt at which the reaction rate is half of Vserca. Although T cells express both SERCA 2b and SERCA 3 isoforms, which have different affinities for Ca2+ and maximal pumping rate, we simplified the model by lumping these two isoforms into one average SERCA pump with a unique maximum velocity and Ca2+ affinity.where 2+ intake in the mitochondria through the uniporter is modeled with a 4th order Hill function [Vmitin is the maximum rate of Ca2+ uptake in the mitochondria and Kmitin is the concentration of Cacyt at which the reaction rate is half of Vmitin.Cafunction ,39:Jmit2+ efflux from the mitochondria through the Na+/Ca2+ exchanger and permeability transition pores (PTP) is given by the lumped expression [Vmitout is the maximum rate of Ca2+ efflux and Kmitout is the concentration of Cacyt at which the reaction rate is half of Vmitout.Capression ,32:JmitJcrac include second order Hill dynamics with respect to either IP3 levels [2+ levels [2+ diffusible factor (CIF) [et al. [et al. [et al. designed SOCE as a feedback controller that rejects disturbances and tracks Ca2+ levels in the cytosol and in the ER [2+ in the ER. Therefore, Jcrac can be expressed as:Vcrac is the maximum Ca2+ influx through the CRAC channels, Ksoc is the concentration of Caext at which the half maximal observed reaction rate is achieved and Kstim is the dissociation constant of ER Ca2+ to STIM1.The details of store operated calcium entry (SOCE) have only been uncovered recently. Previous mathematical descriptions of 3 levels  or cytop+ levels , as wellor (CIF) . More re [et al.  and Chen [et al.  have attn the ER . We simp2+ influx through the plasma membrane is also permitted through a plasma membrane leak and is given by:KPMleak is the rate of leakage through the plasma membrane.Ca2+ efflux from the cytosol to the extracellular space is mainly due to the PMCA pumps and is described as:Vpmca is the maximal PMCA efflux rate and Kpmca is the concentration of Caext at which the reaction rate is half of Vpmca.CaThe series of differential equations were solved using Matlab R2014b . The ODE solver for stiff system ode23s was used. Initial conditions were chosen according to published experimental data before parameter optimization or computed at steady state .+ T Cell Model:tsim is the maximal simulation time, N the number of state variables used for optimization and C the number of experimental conditions being optimized. The Old CD8+ T Cell Model was fit with a similar, slightly altered error function to account for differences in peak amplitude :Parameter estimation was performed by estimating the difference between the experimental data and the corresponding model prediction (sum of squared error) using a genetic algorithm followed by a combination of Matlab Global Optimization Toolbox functions for constrained nonlinear programing (fmincon) and pattern search algorithm (patternsearch). Since the model parameters were estimated to fit different experimental conditions, the objective function consisted in the sum of errors across experimental conditions for fitting the Jurkat and Young CD82+ dynamics were experimentally measured following TCR ligation in three different conditions: no inhibitor, 50 μM EGTA or 100 μM TMB-8.The parameter bounds were selected based on previously published experimental or modeling parameter data . For fitp is the specific parameter used to perform the sensitivity analysis.Sensitivity analysis was performed by perturbing each parameter value (one at a time) by 1 to 20% and comparing the new peak time, amplitude and decay constant to the feature values without perturbation:2+ signaling in lymphocytes was first developed for Ca2+ signaling in Jurkats and then adapted for primary CD8+ T cells . The initial parameter set was populated from the best parameter fit +/- 20% of the Jurkat Ca2+ model. The model was fit to conditions without inhibitors and was validated by predicting Ca2+ dynamics in the presence of chemical inhibitors. Similar to the Jurkat Model fitting, 15 runs were completed of this optimization algorithm and the results are compiled using the shadedErrorBar function from MATLAB® Central.Our computational model of TCR-induced Ca+ T cells [2+ and dynamic responses to TCR activation by flow cytometry. In previous work with this culturing procedure, we established that immunosenescence, as defined by an inability to divide, is achieved within 12 population doublings in approximately 24 days in culture [+ T cells, we observed elevated resting levels of cytoplasmic Ca2+ in young cells after only a few days in culture (days 5–6) and old cells after a prolonged time in culture (days 20–24) when the cells had plateaued in division rate . Older T cells reached their peak amplitude faster and had a faster decay time constant , mitochondria as well as the extracellular space as shown in We then developed a computational model for calcium signaling after T cell receptor ligation to investigate the differences in the calcium signaling network between the young and old primary CD82+ signaling in T cell activation, we optimized the model to fit Ca2+ signaling dynamics in activated Jurkat cells for a total of 17 different parameter sets , the optimized parameter values are quite different for those two cell types, especially the maximum velocities  for young (days 4–8 in culture) and old cells (days 20–24 in culture). Out of these six targets, PMCA and ORAI1 showed significant upregulation (p < 0.05) with age . These two parameters alone were not sufficient to fit simultaneously the time-to-peak, decay time constant, and amplitude /nYoung)*100%, where nYoung is the parameter value in the Young CD8+ T cell Model and nOld is the parameter value in the Old CD8+ T Cell Model.To ensure these parameters were the drivers of the observed old T cell phenotype, we used the parameter set from the Young CD8e course , with adex vivo aging protocol that may induce oxidative post-transcriptional changes in SERCA, IP3, and STIM1 and correspondingly alter kinetic parameters. In Vcrac and Kstim, individually to determine the effect on the calcium traces. We found that both of these parameters alone altered the peak time and decay of the calcium signaling when varied +/- 20% of the optimized Young CD8+ T cell model, which supports our model predictions that STIM1 may be involved with age related changes in T cells.Because we typically associated changes in Vmax with altered protein abundance and changes in binding rate constants with intrinsic changes to proteins , this motivated our investigation of redox metabolic reprogramming during the + T cells of healthy adult donors. Out of these 84 genes, only 58 were expressed in T cells (ex vivo expansion. Among downregulated genes in older T cells (p<0.1) were Glutaredoxin 2 (Glrx2), and Thioredoxin reductase 1 (Txnrd1). To validate the PCR array results and compare mRNA levels to protein expression, we measured the relative levels of the H2O2-producing enzyme Duox1 by Western blot analysis, which confirmed increased levels of Duox1  and thioredoxin 1 (Trx-1) redox couples, as measures of cellular oxidation. The glutathione thiol/disulfide redox couple (GSH/GSSG) is the predominant mechanism for maintaining the intracellular microenvironment in a highly reduced state that is essential for antioxidant/detoxification capacity, redox enzyme regulation, transcription of antioxidant response elements (ARE) and adequate immune response. Total levels of glutathione did not change significantly with time in culture (p = 0.13); however, the ratio of oxidized to reduced glutathione did increase, as indicated by changes in the GSH/GSSG redox potential . The thi2+ signaling dynamics in older T cells, our findings that T cells shift towards a more oxidizing intracellular environment with days in culture, and literature reports of STIM1 as a redox sensor [ex vivo expansion protocol. We developed an assay to detect changes in cysteine redox regulation of STIM1 by measuring reversibly oxidized cysteines including sulfenic acids, S-glutathionylation, and disulfides. By protecting reduced cysteines via alkylation during cell lysis and then reducing reversibly oxidized cysteines after STIM1 immunoprecipitation, we quantified cysteine oxidation of STIM1 via biotinylation of these nascent thiols with PEO-biotin-iodoacetamide (BIAM). Jurkat cell lysates were used as assay controls as shown in Based on our model’s predictions of STIM1-mediated PMCA inhibition as a key factor in describing Cax sensor , we hypo+ T cells from three different donors were subjected to the accelerated cell divisions ex vivo and changes in redox regulation of STIM1 were quantified using this assay. The streptavidin signal was normalized to the STIM1 signal across each sample. As shown in ex vivo.Primary CD82+ release with immunosenescence [2+ dynamics in T cells have been associated with several age-related diseases, such as neurodegenerative, autoimmune and inflammatory disorders [2+ signaling are a marker of T cell function in response to stimuli and are known to affect downstream cellular response, we asked whether Ca2+ signaling dynamics change after a cell population has undergone multiple population doublings. In our culture model, we did not observe a clear trend in baseline Ca2+ levels, or a reduction in peak amplitude or sustained levels after stimulation Click here for additional data file.S2 FigPlots represent 17 different optimized parameter sets that were obtained from comparing the model prediction to experimental data.(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S7 FigKstim was varied +/- 20% the fit value of 178.(PDF)Click here for additional data file.S8 FigVcrac was varied +/- 20% the fit value of 2.37.(PDF)Click here for additional data file.S9 Figa) Representative Western Blot. b) Quantification of the Western Blots. Protein levels are normalized to the young cells protein expression level. * p<0.05 (paired 2-tail t-test).(PDF)Click here for additional data file.S10 Fig(PDF)Click here for additional data file.S1 TableRed genes represent targets that are not expressed in CD8+ T cells.(PDF)Click here for additional data file.S2 Table-ΔCT).A fold change below 1 corresponds to a downregulation (2(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 TableThis parameter set was used for all sensitivity analysis performed on the Young CD8+ T Cell Model.(PDF)Click here for additional data file.


Oryctolagus cuniculus) are a popular companion animal, food animal, and animal model of human disease. Abnormal red cell shapes (poikilocytes) have been observed in rabbits, but their significance is unknown. The objective of this study was to investigate the prevalence and type of poikilocytosis in pet rabbits and its association with physiologic factors, clinical disease, and laboratory abnormalities. We retrospectively analyzed blood smears from 482 rabbits presented to the University of California-Davis Veterinary Medical Teaching Hospital from 1990 to 2010. Number and type of poikilocytes per 2000 red blood cells (RBCs) were counted and expressed as a percentage. Acanthocytes (>3% of RBCs) were found in 150/482 (31%) rabbits and echinocytes (>3% of RBCs) were found in 127/482 (27%) of rabbits, both healthy and diseased. Thirty-three of 482 (7%) rabbits had >30% acanthocytes and echinocytes combined. Mild to moderate (>0.5% of RBCs) fragmented red cells  were found in 25/403 (6%) diseased and 0/79 (0%) healthy rabbits (P = 0.0240). Fragmentation and acanthocytosis were more severe in rabbits with inflammatory disease and malignant neoplasia compared with healthy rabbits (P<0.01). The % fragmented cells correlated with % polychromasia, RDW, and heterophil, monocyte, globulins, and fibrinogen concentrations (P<0.05). Echinocytosis was significantly associated with renal failure, azotemia, and acid-base/electrolyte abnormalities (P<0.05). Serum cholesterol concentration correlated significantly with % acanthocytes (P<0.0001), % echinocytes (P = 0.0069), and % fragmented cells (P = 0.0109), but correlations were weak (Spearman ρ <0.02). These findings provide important insights into underlying pathophysiologic mechanisms that appear to affect the prevalence and type of naturally-occurring poikilocytosis in rabbits. Our findings support the need to carefully document poikilocytes in research investigations and in clinical diagnosis and to determine their diagnostic and prognostic value.Rabbits ( Poikilocytosis is the presence of abnormally shaped erythrocytes in peripheral blood. Identification of poikilocytes is an important part of blood smear evaluation because shape changes often are associated with specific diseases, providing clues to underlying pathogenesis and facilitating diagnosis and treatment. Poikilocytes can result from biochemical changes, toxins, or physical damage to erythrocytes; regardless of cause, they can shorten erythrocyte survival and contribute to anemia Rabbits are popular companion animals, are raised for meat, and are used extensively as animal models of human disease, including atherosclerosis, disorders of lipid metabolism, diabetes, and cardiovascular disease The objectives of this study were to retrospectively characterize the prevalence, type, and severity of poikilocytes in a large cohort of rabbits and to investigate associations between poikilocytes and physiologic factors , clinical disease, and CBC and biochemical findings. We hypothesized that poikilocytes in rabbits would be associated with specific diseases or laboratory abnormalities.Electronic medical records from the University of California-Davis VMTH were searched retrospectively for rabbit visits between 1990 and 2010 at which a complete blood count (CBC) was done. Cases were excluded if a blood smear was not available or if a clinical diagnosis was not provided. Repeat visits by the same rabbit were excluded. Patient number, date, signalment , clinical and pathologic diagnoses, and CBC and biochemistry results were recorded. Age was reported in years and categorized as adult (≥1 year old) or juvenile (<1 year). Clinical diagnoses were those reported in the visit summary by the clinician, and were based on the results of physical examination, laboratory tests, imaging, and occasionally, histopathology. Pathology results (biopsy and necropsy) were recorded if they were obtained within 6 months of the CBC and/or were relevant to the primary clinical disease. Healthy rabbits were those presented for routine physical examination or elective spay or neuter.c from 1997 to 2005, and Coulter Dacos analyzer  from 1990 to 1996. When instruments were upgraded, results were calibrated to retain consistency in results between analyzers.From January, 1990 to September, 2001 hematology results were obtained using a Baker Systems 9110 Plus hematology analyzer . From September, 2001 to December, 2010 hematology results were obtained using an ADVIA 120 hematology system  with the rabbit setting in MultiSpecies System Software. Differential counts were obtained manually by counting 200 leukocytes in Wright-Giemsa-stained blood smears. Total plasma protein concentration was determined by refractometry and fibrinogen concentration was determined using the heat precipitation method. Hemolysis (pink plasma color) was qualitatively evaluated as mild, moderate, or marked. Biochemical results were obtained on a Roche Hitachi 917 analyzer  from 2006 to 2010, Hitachi 717Original blood samples had been collected and processed according to our laboratory’s standard operating procedure. Whole blood was placed into tubes containing EDTA and smears were prepared, air-dried, and stained by certified medical technologists within ∼1 hour of collection. Poikilocytes were reported semiquantiatively  by the technologists. Smears were coverslipped prior to storage in the laboratory slide archive.On each original stained smear, 2000 RBCs were counted and characterized at 1000X magnification by a senior clinical pathology resident (AGB) blinded to information about the rabbit. Poikilocytes were defined based on standard morphology and counting was limited to representative monolayer fields in which about half the erythrocytes were touching but did not overlap th slide (according to laboratory accession number) and all samples where the qualitative poikilocyte results in the original hematology report were widely discrepant from the quantitative results.A board-certified clinical pathologist (MMC) independently determined % poikilocytes in a subset of the smears, also in a blinded manner; results from the two observers were averaged for analysis. The subset included a random sample consisting of every 10t test or ANOVA. Chi square analysis was used to compare nominal data. A multivariate model using Spearman’s rank test was used to evaluate correlations among % poikilocytes and CBC and biochemistry variables. Principal component analysis was done using % poikilocytes and selected CBC and biochemistry variables. A P-value of.05 was used to indicate statistical significance.Data were compiled in an Excel  spreadsheet and examined for aberrant entries. Statistical analyses were done using JMP 10.0.0 . Poikilocyte percentages were tested for normality by examination of histograms and Shapiro-Wilk tests. Differences in % poikilocytes between groups were evaluated using Wilcoxon/Kruskal-Wallis rank sums tests. Rabbits with none, rare, and mild (few) poikilocytes were combined and compared with those having moderate to marked poikilocytosis. Differences in age and hematologic and biochemical data were analyzed using Student’s Sylvalagus bachmani); and 14 were excluded because a clinical diagnosis was not reported. In total, 482 rabbits were included in the study.Nine-hundred-seventy-five rabbit visits with at least partial CBC results were identified during the 20-year period. Of these, 406 samples were excluded as repeat CBCs; 48 were excluded because smears were missing; 25 were excluded because they were native brush rabbits (Rabbits ranged from 3 months to 12 years of age (median 4 years), with 428 adults and 54 juveniles. Rabbits included 205 females  and 277 males . Rabbit breeds included Netherland Dwarf (n = 64), Lop (60), Mini-Lop (30), Mini-Rex (28), Holland Lop (20), New Zealand White (16), Dutch (13), Rex (10), Angora (8), French Lop (8), Flemish Giant (6), English Spot (5), English Lop (4), Dwarf (4), Dwarf Hotot (3), Lionhead (2), Chinchilla Rabbit (2), 1 each Dwarf Lionhead, Finnish Giant, Florida White, Havana, Hottot, Jersey Wooly, Lop-eared Angora, Norwegian Dwarf, and crossbred (21); breed was not specified for 170 rabbits.Of the 482 rabbits, 79 were healthy and 403 were diseased. The mean (± SD) age of healthy rabbits (1.6±1.9 years) was significantly lower than that of diseased rabbits (4.6±2.8 years) . No difference was found in the proportion of females and males or in breed distribution between healthy and diseased rabbits.A total of 155/482 (32%) smears were quantified by two observers, with good agreement in % poikilocytes (average difference 0.05%±5% over a range of 0 to 70%). In the remaining smears, quantitative findings were similar to poikilocytes noted (or not) in the original laboratory report.A majority of rabbits 251/482 (52%) had none, rare, or mild <3%) poikilocytosis; 90/482 (19%) rabbits had moderate and 141 (29%) had marked poikilocytosis. Acanthocytes and echinocytes were the most frequently observed poikilocytes, with no significant difference between healthy and diseased rabbits –3. One-h% poikiloSchistocytes, microcytes, keratocytes, and spherocytes (subsequently combined as “fragmented” red cells) were observed in low numbers, with 457/482 (95%) rabbits having none to rare fragmented cells and 25/482 (5%) having mild to moderate fragmentation –3. Only No significant difference in % poikilocytes was observed between samples with none (n = 374), slight (n = 101), or moderate (n = 7) sample hemolysis. Further, neither moderate to marked acanthocytosis or echinocytosis nor mild to moderate fragmentation were associated with the presence of sample hemolysis. None of the 7 samples with moderate hemolysis had increased fragmentation and only 3/98 samples with mild to moderate fragmentation had slight hemolysis.Associations between poikilocytes and physiologic factors were evaluated in clinically healthy rabbits (n = 79). Healthy rabbits included 47 adults and 32 juveniles ranging from 3 months to 8 years of age. No significant difference in % poikilocytes or in the proportion of samples with moderate to marked poikilocytosis was found between adult and juvenile rabbits; no correlation was found between age and % poikilocytes.Healthy rabbits included 32 females  and 47 males . Female rabbits had a slightly higher % echinocytes  than male rabbits  . A higher proportion of females than males had moderate to marked acanthocytosis  and echinocytosis  .Healthy rabbits included Netherland Dwarf (14), Mini-Rex (5), Dutch (4), Holland Lop (4), and Lop (4), New Zealand White (2), Mini-Lop (2), Lionhead (2), English Spot (2), 1 each of Angora, Dwarf, Dwarf Lionhead, English Lop, Flemish Giant, Hottot, and Rex; and cross-breed (5), breed was not reported for 28 rabbits. No significant breed difference was found in % poikilocytes or in the proportion of samples with moderate to marked poikilocytes, however samples sizes were small.Diseased rabbits were classified into 12 organ groups based on primary diagnosis . PatholoNo difference in the % or severity of acanthocytes and echinocytes was found on the basis of organ system, but a significantly lower % fragmented cells was found in healthy rabbits and in rabbits with ophthalmic disease compared with other organ systems . Moderate to marked acanthocytosis and mild to moderate fragmentation were observed significantly more often in rabbits with septic and inflammatory disorders compared with healthy rabbits . A signiSignificant differences were observed in laboratory values in rabbits with moderate to marked acanthocytosis or echinocytosis and with mild to moderate fragmentation . BecauseBased on Spearman rank correlation for all rabbits combined, % acanthocytes correlated positively with % polychromasia, red cell distribution width (RDW), and mean platelet volume (MPV) (P<0.02); % echinocytes correlated positively with RBC count, hematocrit (HCT), hemoglobin concentration (HGB), mean cell hemoglobin concentration (MCHC), MPV, anion gap, and sodium, potassium, chloride, and creatinine concentrations, and negatively with bicarbonate and glucose concentrations (P<0.01); % fragmented cells correlated positively with % polychromasia, MCHC, RDW, and heterophil, monocyte, and potassium concentrations, and negatively with bicarbonate and albumin concentrations (<0.02). Cholesterol correlated significantly with % acanthocytes (P<0.0001), % echinocytes (P = 0.0069), and % fragmented cells (P = 0.0109), but was not highly predictive (Spearman ρ <0.2).We used principal component analysis to further visualize correlation patterns . In compTo our knowledge, this study is the first to quantify red cell morphology in a large population of rabbits. Acanthocytes and echinocytes comprised >3% of RBCs in about one-third of healthy and diseased rabbits and were slightly more frequent in healthy female than male rabbits. Fragmented red cells  occurred in low numbers and were more frequently observed in diseased rabbits, especially those with abscesses, bronchopneumonia, cellulitits, gastrointestinal inflammation, and malignant neoplasia. In addition, moderate to marked echinocytosis was associated with renal failure and electrolyte abnormalities. These findings suggest common pathophysiologic mechanisms of poikilocyte formation in sepsis (fragmentation) and uremia (echinocytes). Our findings warrant further investigation into the pathogenesis of red cell shape change in rabbits and assessment of the diagnostic or prognostic value of poikilocytes.Acanthocytes and echinocytes were the most frequent poikilocytes observed in rabbits in this study; they often occurred together and formed a morphologic spectrum that sometimes made differentiation difficult Rabbits fed high cholesterol (atherogenic) diets routinely develop acanthocytes (“spur cells”) or echinocytes An important finding in this study was the association of fragmentation with inflammation (often septic) and with malignant neoplasia in rabbits. This could be the result of bacterial toxins or microangiopathy, with endothelial fibrin deposition and microthrombi causing physical damage to erythrocytes Sepsis and inflammation-mediated oxidative damage and cytokines also alter red cell membranes, reducing deformability and increasing phagocytosis by macrophages Interestingly, high cholesterol diets can induce inflammation and oxidative stress together with acanthocyte formation in rabbits We combined spherocytes, keratocytes, microcytes, and schistocytes as fragmented red cells because they often appeared together and in low numbers Strong associations between echinocytes, renal disease, and electrolyte abnormalities suggested uremia as one mechanism of moderate to marked echinocytosis in rabbits, as in other species A limitation of this retrospective study was the inability to verify underlying disease in all of the rabbits. In addition, we selected the primary disease process, but some rabbits had multiple problems. However, pathologic confirmation of disease was obtained in many of the rabbits, including diseases with potential relevance to poikilocyte formation. Further, many rabbits had diseases  that are readily diagnosed clinically or with imaging and laboratory tests. Despite this limitation, the large size of the database and consistent relationships between poikilocytes and clinicopathologic abnormalities helped support the primary findings of our study.In conclusion, acanthocytes and echinocytes comprised >3% of erythrocytes in about one-third of healthy and diseased rabbits; a small proportion of rabbits had >30% poikilocytes. Echinocytosis also was associated with renal disease and electrolyte abnormalities, consistent with the effect of uremia on red cells. Rabbits with abscesses, other inflammatory disorders, and malignant neoplasia had more red cell fragmentation, which was associated with mild regenerative anemia and hematologic and biochemical evidence of inflammation. Serum cholesterol concentration also correlated poikilocytosis, but was not strongly predictive. Future research is warranted to prospectively evaluate the pathophysiologic mechanisms of poikilocyte formation, the role of diet, and the diagnostic and prognostic value of poikilocytes in rabbits.


There are, however, no such models for retinal pigment epithelium (RPE). Our aim was to construct a Ca2+ signaling model for RPE based on our experimental data of mechanically induced Ca2+ wave in the in vitro model of RPE, the ARPE-19 monolayer.Computational models of calcium , P2Y2 receptors, IP3 receptors, ryanodine receptors, Ca2+ pumps, and gap junctions. The cells in our epithelial model are connected to each other to enable transport of signaling molecules. Parameterization was done by tuning the above model components so that the simulated Ca2+ waves reproduced our control experimental data and data where gap junctions were blocked.We combined six essential Ca2+ signaling in ARPE-19 cells, and the basic mechanism was found to be as follows: 1) Cells near the stimulus site are likely to conduct Ca2+ through plasma membrane SSCCs and gap junctions conduct the Ca2+ and IP3 between cells further away. 2) Most likely the stimulated cell secretes ligand to the extracellular space where the ligand diffusion mediates the Ca2+ signal so that the ligand concentration decreases with distance. 3) The phosphorylation of the IP3 receptor defines the cell’s sensitivity to the extracellular ligand attenuating the Ca2+ signal in the distance.Our model was able to explain Ca2Y2 receptors or accelerate P2Y2 receptor phosphorylation, which may partially be the reason for Ca2+ wave attenuation by suramin. Being the first RPE Ca2+ signaling model created based on experimental data on ARPE-19 cell line, the model offers a platform for further modeling of native RPE functions.The developed model was able to simulate an array of experimental data including drug effects. Furthermore, our simulations predict that suramin may interfere ligand binding on P The distance (x) defines the distance of the NB layer from the MS cell centre that was calculated using the idealized hexacon RPE cell architecture (Three parameters were assumed to vary according to the location of the cell with respect to the MS cell: stretch (θ) activating the stretch-sensitive Caion ([L]),16,29,30i are presented in the model as a combination of Ca2+ fluxes2+ channels (JSSCC), inositol 1,4,5-trisphosphate (IP3) receptor type 3 (RyR). JPump combines the Ca2+ pumping functions of sarco/endoplasmic reticulum ATPase (SERCA) and the plasma membrane Ca2+ ATPase (PMCA). Leak Ca2+ current (JLeak) describes the total leakage from the extracellular space and the endoplasmic reticulum (ER) to the cytoplasm. 2+ flux through gap junctions.Time dependent changes in intracellular Ca2+ channels (SSCCs) on the cell membrane are activated, when exposed to mechanical stimulation. Their closure is caused either by relaxation in the mechanical force or by their adaption to that mechanical force defined byT] denotes the total number of surface receptors, K1 is the dissociation constant for unphosphorylated receptors, and [L] is the extracellular ligand concentration. The total number of phosphorylated surface receptors 2 is the dissociation constant for phosphorylated receptors. The binding of the ligand to the G-protein coupled receptor P2Y2 results in a cascade of events leading to the activation of enzyme phospholipase C (PLC). This enzyme then hydrolyses the phosphatidylinositol 4,5-bisphosphate (PIP2) to IP3. The activation rate (ka) of the G-protein is proportional to two ratios: the ratio of the activities of the ligand unbound and bound receptor species (δ), and the ratio of the number of ligand bound receptors and the total number of receptors (pr). Denoting the deactivation of G-protein to occur at a deactivation rate of kd, the equations for the amount of Gα∙GTP labeled as [G] as well as for the ratio pr can be expressed asThe agonist-induced activation of the second messenger system, here P3 isdeg is the degradation rate of IP3 and 3 flux through gap junctions. The rate coefficient for PIP2 hydrolysis (rh) includes the effective signal gain parameter (α) and the dissociation constant for Ca2+ binding to PLC (K3) that can be expressed asEquation for the concentration of IP2 is required for IP3 production to be maintained over sustained periods of agonist stimulation. The equation for the number of PIP2 molecules [PIP2] isr represents the PIP2 replenishment rate and [(PIP2)T] the total number of PIP2 molecules.[Replenishment of PIPolecules.3 receptor type 3 (IP3R3) function is represented by the Eqs 3R3 is shown in 3-induced release of Ca2+ from the ER through IP3R3  for the receptor to conduct. The steady-state proportion of open receptors (O) isϕ function controls the sensitivity of IP3R3 to [IP3], and it can be expressed as-1, k2, k3, and k5 being constants. Coefficient k1 describes a rate for IP3R3 transition from shut state (S) to open state (O)1 is the maximum rate of S to O transition, and β1 is the [Ca2+]i at which the rate is half of its maximum. Coefficient k4 expresses the rate for IP3R3 from the first inactivated state (I1) to the second inactivated state (I2). It can be expressed as1 to I2 transition is agonist specific and involves a phosphorylation of IP3R3 by kinase activity. This is defined by parameter α4 that denotes the maximum rate of I1 to I2 transition, while β4 denotes the value of [IP3] at which the rate is half maximal.iKb)3),and wherPump combines the pumping functions of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) and plasma membrane Ca2+ ATPase (PMCA)Pump indicates the maximum flux rate of the pumps and KPump states the [Ca2+]i for half-maximal pumping rate.J2+ flux via GJs  and the CaParameter A describes the area of the cell membranes connecting the neighbouring NB layers in the monolayer. The value for A is received by multiplying the area of one hexagon side, that is the length of the hexagon side (l = 7μm) times the height of the cell (h = 12μm), by the number of hexagon edges between the two NB layers asides see . In othePolarization of the ARPE-19 monolayer was demonstrated by immunolabeling the tight junctions in the monolayer. Confocal microscopy image  shows th2+ wave width at half maximum and end Ca2+ concentration in NB1-NB9 layers. In NB10 layer, however, time to peak was longer in the simulation results than in the data. In the control data set  (3R3 phosphorylation rate regulated by the kinase activity (α4) increased following a shallow exponential, almost linear function, from NB1 to NB10. The corresponding values of α4 with the distance were lower in the control data set  . The strdata set  than in data set  indicatiB layers .2+ dynamics is: 1) Cells near the stimulus site conduct Ca2+ through plasma membrane SSCCs, and gap junctions conduct the Ca2+ and IP3 between cells further away from stimulated cell. 2) The MS cell secretes one or several types of ligand to the extracellular space where the ligand diffusion mediates the Ca2+ signal so that the ligand concentration decreases with distance. 3) The phosphorylation of the IP3 receptor defines the cell’s sensitivity to the extracellular ligand attenuating the Ca2+ signal in the distance.The resulting model of mechanical stimulus induced Ca2+ wave features described in Materials and Methods was studied for a set of parameters that were fitted in this study for NB1, NB5 and NB10 layers  resulted in faster Ca2+ waves in NB1  on Ca2+ wave features were complex: with decreasing α4 Ca2+ wave peak amplitude increased , but changes in their values affected significantly the investigated Ca2+ wave features.The sensitivity of the four Ca0 layers . From ths in NB1 . Increass in NB1  and incr maximum . The effncreased , time toncreased , the Ca2NB layer , and theNB layer . The Ca2 maximum  and end  maximum , and the2+ wave features were compared between the experimental GA-treated and GA-suramin-treated data sets as well in NB1, NB5 and NB10 layers  by changing their values by ±25%. Similarly, G-protein cascade parameters were studied as well to consider the possible effect of suramin to disrupt the coupling between the receptor in the cell membrane and the G-protein. Our aim was to investigate the degree to which the observed differences in the Ca2+ wave features between GA-treated and GA-suramin-treated data sets could be accounted for by the changes in these parameters. The sensitivity analysis revealed that the modifications in P2Y2 unphosphorylated receptor dissociation constant (K1) and P2Y2 receptor phosphorylation rate (kp) indeed induced changes that were similar to the experimental observations  (d)   and G-pr(ka) (d)  had dive1 and kp. The values of these parameters ranged as follows: K1 values decreased from 8.83μM in NB1 to 5.15μM in NB10 and kp values decreased from 0.19s-1 in NB1 to 0.05s-1 in NB10. Overall, the values of K1 and kp were higher in the GA-suramin-treated data set than in the GA-treated data set. The simulated curves seem to fit well to the experimental data, except in NB1 layer at the end of the Ca2+ wave. In GA-suramin-treated data set, R2 values were higher than 0.8 in NB1-NB7 and lower than 0.8 in NB8-NB10  cells are on the monolayer; 2) they are connected to each other by GJs permeating Ca2+ and IP3, and 3) the cells are most probably experiencing different stretching and chemical conditions depending on their distance from the mechanical stimulation site. To the best of our knowledge, this is the first time as Ca2+ signaling model has been implemented for the ARPE-19 monolayer. The model uses a set of location specific parameters including stretch, extracellular ligand concentration, and IP3R3 phosphorylation rate as well as the Ca2+ and IP3 fluxes through GJs.The ARPE-19 cell line is an important biological model of human RPE despite its certain limitations . This pa2+ signaling as extracellular signal mediators has been mathematically modeledi induced by connective tissue growth factor (CTGF)[2+]ii. The effdels e.g.,21,29, p2+ signaling molecules between the NB layers based on the Ca2+ and IP3 concentration gradients, and permeated Ca2+ and IP3 selectively. As expected, NB layers near the MS cell were more sensitive to IP3 input than the distant NB layers, and this was seen especially in the end Ca2+ concentration at 90 seconds’ time point.GJs connect the adjacent cells together and allow the diffusion of signaling molecules between them. The diffusion through GJs has previously been modeled, for example, in airway epithelium. In our 2Y2 receptors to enhance their desensitization. This may indicate that suramin targets on P2Y2 receptors as an unspecific P2 receptor antagonist attenuating the Ca2+ wave. This intriguing model hypothesis driven from the model results needs to be confirmed experimentally. It is worth noting, however, that suramin has also been considered to disrupt the coupling between the receptor in the cell membrane and the G-protein by blocking the association of the G-protein α and βγ subunits[2+ wave width at half maximum. Despite the observed diversity in their effects between the NB layers, it is possible that suramin targets the G-protein cascade as well, by acting as an attenuator of the Ca2+ wave.In the GA-suramin-treated data set, the experimental data was reproduced in our model by increasing the unposphorylated receptor dissociation constant, which likely reflects disrupted ligand binding, and by increasing the phosphorylation rate of the P subunits. In our 2+ signaling based on experimental work on the ARPE-19 cell line. This cell line is used extensively as a model of RPE, although it differs from it to some extent. The limitations of ARPE-19 compared to native human RPE arise, for example, from cell organization and metabolism[2+ stores, melanosomes, and needs to be taken into account when expanding our model to describe native RPE. In addition, we confirmed the polarity of the ARPE-19 monolayer with confocal microscopy. Trans-epithelial resistance (TER) that is a general measure of epithelial integrity was not measured due to technical challenges to perform the measurements on glass cover slips with our present equipment [2+ activity. Thus it provides a good basis to address the native RPE in the future, even though it, being based on an in vitro model of RPE, needs to be considered only as a model. To improve the model further, experimental data and model implementations on certain additional Ca2+ related mechanisms, such as P2X receptors[2+ channels[+/Ca2+ exchangers[2+ concentrations, and therefore our model also features only relative Ca2+ activity.Our work presents a computational model of epithelial Caetabolism. Importaquipment . Neverthreceptors, voltage channels and Na+/xchangers would bexchangers did not 2+ signaling would allow one to simulate cellular Ca2+ responses under several physiological, pathological, and experimental conditions. Our present model represents significant progress towards this goal since it is able to reproduce the experimental data from an RPE type epithelium, ARPE-19 cell line, in different conditions, simulate several epithelial Ca2+ signaling mechanisms, and predict drug responses in the epithelia. Our future work will include further development of the model especially focusing on the role of the voltage sensitive Ca2+ channels in the RPE.A full mathematical understanding of RPE and epithelial Ca


During labour, hormones like oxytocin and prostaglandins potentiate uterine contractions by increasing their duration, strength and frequency. The most informative approach to studying the mechanisms underlying hormonal modulation of uterine contractility is to record [Ca2+]i responses to hormones in intact myometrial samples that have not been subjected to enzymatic treatment for cell isolation or cell culture conditions. However, the spatio-temporal resolution of such recording is limited due to the motion artifacts occurring in contracting tissue. Here we describe the application of our newly developed motion correction algorithm to investigate the [Ca2+]i dynamics in control and oxytocin stimulated slices of human myometrium on a cellular level. We present evidence that oxytocin induces asynchronous [Ca2+]i oscillations in individual myocytes within intact myometrium which are similar to those observed in cultured cells. The oscillations occur between synchronous action potential-driven [Ca2+]i transients but appear to be unrelated to contractions. Furthermore, the oxytocin-triggered [Ca2+]i oscillations wane within 30–50 min of hormone application, while the action potential induced [Ca2+]i transients remain augmented. We conclude that oxytocin-induced [Ca2+]i oscillations are not relevant to the acute regulation of myometrial contractility but may play a role in longer-term regulatory processes, for example, by triggering gene expression.Elucidation of cellular mechanisms regulating myometrial contractility is crucial for improvement in management of many obstetric abnormalities, such as premature delivery, uterine dystocia and post-partum haemorrhage. Myometrial contractions are triggered by periodic synchronous rises in intracellular calcium concentration ([Ca The rate of cross-bridge cycling is controlled by the relative equilibrium of activity between myosin light-chain kinase that phosphorylates the regulatory myosin light chains and myosin light-chain phosphatase that dephosphorylates them  and approval from the Local Ethics Committee at University Hospital Coventry and Warwickshire (REC-05/Q2802/107) from term-pregnant women (≥37 weeks gestation) undergoing elective caesarean section before the onset of labour.3. The strip was then ligatured with braided 2-0 Mersilk suture  at both ends before being stretched and fixed to the base of a stainless steel tissue holder using cyanoacrylate glue. From this, 200 μm-thick slices were cut using an oscillating vibroslicer  in oxygenated ice-cold Krebs solution. Slicing was performed with razor blades at an oscillating speed of 86 Hz with lateral amplitude of 1 mm and an advance speed of 0.10–0.20 mm s−1. First cuts and the glued base of the strip were discarded. Each slice was then separated by cutting the extremity of the slice using fine dissecting scissors and transferred into Krebs solution. After 1 h incubation at room temperature for equilibration and recovery and 30 min loading with Fluo-4/AM (see below) the slices were used in experiments within 4–6 h.Myometrial specimens used in this study were obtained from 12 different patients. Samples were collected into sterile 25 ml plastic tubes filled with cold Krebs solution, placed on ice and delivered to the laboratory within 20–30 min. On arrival, each biopsy specimen was trimmed under a stereo microscope into a strip of approximately 1.0×0.5×0.3 cm2.22+]i recording, slices were incubated for 30 min at 37 °C in Krebs solution containing 13 μM Fluo-4/AM . Non-ionic detergent Pluronic F127  was included to aid the dye loading. This protocol produced homogeneous staining of cells throughout the slice and gave better tissue viability compared to loading at room temperature, which required up to 4 h incubation to achieve the same intensity of Fluo-4 signal. The loaded slice was placed in a glass-bottomed Petri dish and weighted down with a 250 mg slice grid . Since no stretch was applied to slices used in these experiments, the resting tension was estimated to be zero mN. The dish was then secured in a spring-loaded holder in a temperature-controlled environmental chamber on the stage of an inverted microscope (Axiovert 200 M) equipped with an LSM 510 META confocal scanner  and superfused with pre-warmed (35 °C) Krebs solution at a flow rate of 2 ml min−1 for 30–40 min until stable spontaneous contractions developed. Slices that failed to develop spontaneous activity within this timeframe were excluded.For i corresponding to F/F0=1 and upward deflections corresponding to [Ca2+]i increase.Examination of recorded time series in ImageJ revealed that substantial motion artifacts were present. To remove these artifacts, the image sequences were imported into MATLAB  and processed using our motion-correction algorithm. A detailed description of the motion correction procedure has been given elsewhere . BrieflyF/F0=1 and downward deflections follow the time course of contractions.To monitor the time course of contraction, a larger ROI was placed over the moving edge of the slice in the corresponding uncorrected image sequence. The mean intensity obtained from this ROI is proportional to the displacement of the slice as it moves in and out of the ROI during contraction–relaxation cycle. In contrast to other traces, the displacement trace was normalised to the highest values of intensity between contractions, when the slice was fully relaxed. The relaxed state therefore corresponds to 32+]i transients occurring in all cells at the same time. As illustrated in the bottom left panel in 2+]i traces making it difficult to obtain reliable single cell data. In most cases, application of the image processing algorithm eliminated these artifacts almost completely, thus allowing single-cell data to be extracted from the image sequences.2+]i dynamics in spontaneously contracting slices of human myometrium   while variation in mean amplitude was not significant.As shown in e (FWHM) . Interes2+]i transients in all cells tested. Oxytocin also elicited low-amplitude [Ca2+]i oscillations in many but not all cells (2+]i but they had a higher amplitude and were present in a larger number of cells.Application of 10 nM oxytocin greatly increased the amplitude and duration of the global [Call cells A. Simila2+]i transients continued to increase in their amplitude and duration from cycle to cycle in the presence of oxytocin, the individual-cell [Ca2+]i oscillations disappeared after 2–3 cycles. After 1 h treatment with oxytocin, the amplitude and duration of global [Ca2+]i transients was increased by 127% and 683% respectively. 2+]i oscillations were not accompanied by any measurable contractions. That is, only augmented phasic contractions were observed in the presence of oxytocin. The rate of relaxation of phasic contractions was substantially lower after prolonged incubation with oxytocin compared to that at the beginning of oxytocin application. In summary, the sequence of events induced by oxytocin was as follows. Immediately after the application of oxytocin, a global [Ca2+]i transient of dramatically increased duration and slightly potentiated amplitude was initiated. That was followed by induction of [Ca2+]i oscillations in a subset of cells and by further progressive increase in the amplitude of global [Ca2+]i transients and phasic contractions. After 2–3 contraction–relaxation cycles, the [Ca2+]i oscillations in individual cells have subsided (see While global i oscillations have been demonstrated in primary cultures of human uterine myocytes (2+]i oscillations in intact cells residing within the myometrium have not been demonstrated due to methodological limitations associated with [Ca2+]i imaging in contractile multicellular preparations. In the present study, we managed to overcome these limitations by combining confocal [Ca2+]i imaging and advanced image processing to investigate the dynamics [Ca2+]i on a cellular level in control and oxytocin stimulated slices of human myometrium. For the first time, we present evidence that oxytocin induces asynchronous [Ca2+]i oscillations in individual myocytes within intact myometrium which are similar to those observed in cultured cells. Surprisingly, these oscillations seem to be unrelated to the oxytocin-induced augmentation of myometrial contractions and the tissue-level [Ca2+]i transients as they occur only in a proportion of cells and quickly disappear, while augmentation of contractility in the presence of oxytocin persists for much longer. Our findings are compatible with the idea that myometrial contractions are regulated by the membrane oscillator (2+ sensitivity of contractile machinery. Based on the data obtained in this study, we conclude that oxytocin-induced [Ca2+]i oscillations are not relevant to the acute regulation of myometrial contractility but may play a role in longer-term regulatory processes, for example, by triggering gene expression. In the present study, we did not investigate the effects of stretch on [Ca2+]i and contractile responses to oxytocin. Undoubtedly, mechanical stretch is an important variable, especially at the end of pregnancy and during labour. More research on intact myometrium is needed to elucidate the effects of mechanical stretch on myometrial contractility and gene expression.Elucidation of cellular mechanisms regulating myometrial contractility is crucial for improvement in management of many obstetric abnormalities, such as premature delivery, uterine dystocia and post-partum haemorrhage. Oxytocin is widely used by obstetricians for labour induction and for prevention of post-partum haemorrhage . The phymyocytes . Howevercillator , likely,The authors declare that they have no conflict of interest in regard to the above manuscript.


Here, we utilise ratiometric imaging of Ca2+ indicator dyes and genetically encoded Ca2+ indicator proteins to identify and characterise a single, rapid, transient wave of Ca2+ in the Drosophila egg at activation. Using genetic tools, physical manipulation and pharmacological treatments we demonstrate that the propagation of the Ca2+ wave requires an intact actin cytoskeleton and an increase in intracellular Ca2+ can be uncoupled from egg swelling, but not from progression of the cell cycle. We further show that mechanical pressure alone is not sufficient to initiate a Ca2+ wave. We also find that processing bodies, sites of mRNA decay and translational regulation, become dispersed following the Ca2+ transient. Based on this data we propose the following model for egg activation in Drosophila: exposure to lateral oviduct fluid initiates an increase in intracellular Ca2+ at the egg posterior via osmotic swelling, possibly through mechano-sensitive Ca2+ channels; a single Ca2+ wave then propagates in an actin dependent manner; this Ca2+ wave co-ordinates key developmental events including resumption of the cell cycle and initiation of translation of mRNAs such as bicoid.Activation is an essential process that accompanies fertilisation in all animals and heralds major cellular changes, most notably, resumption of the cell cycle. While activation involves wave-like oscillations in intracellular Ca For allFor live imaging, oocytes were prepared as described previously . OvariesAs described previously , embryosd for Ca2+ = 190 nM. For artificial elevation of cytoplasmic Ca2+ levels, 20–100 pl of 10 mM CaCl2 was injected.Preparation for microinjection was carried out as described previously . Stage 1ex vivo, a standard hypotonic activation buffer, first developed by Mahowald and colleagues  for control experiments, 10 mM CaCl2 and activation buffer with 10 µg/ml final concentration cytochalasin-D (Sigma).To activate the oocytes lleagues  and alsolleagues , was use2+ measurements were performed using two fluorescent dyes- Calcium Green-1 Dextran (Ca2+ sensitive) and Texas Red Dextran (Ca2+ insensitive volume marker). Ratio image calculation was performed in Fiji (Image J) and displayed in pseudocolour. All other images were acquired with an inverted Leica SP5 Confocal microscope under a 20× 0.7NA immersion objective. Experiments requiring mechanical pressure utilised a mounted microinjection apparatus (Olympus). Myr-GCaMP5 was driven by the Gal4/UAS system. GCaMP5 Kd for Ca2+ = 660 nM . Ca= 660 nM . Fiji . Prior to addition of activation buffer, both Calcium Green-1 Dextran (10 kDa) and Texas Red Dextran (10 kDa) were micro-injected in equal concentrations and allowed to evenly diffuse through the cytoplasm (45 minutes) to provide a baseline measure of intracellular Ca2+ concentration. Upon activation, we observed a consistent, rapidly propagating increase in intracellular Ca2+, followed by a slower “recovery phase”, decrease .Although mutations in the Cativation , to datedecrease , n = 8. chambers , n = 8. 2+ transient during activation by standard hypotonic activation buffer, we used the genetically encoded Ca2+ indicator Ca2+-sensitive GFP (GCaMP) . Using UAS-myrGCaMP5, we could clearly see that the Ca2+ transient propagated as a wave   under th (GCaMP) , targetis a wave . Initiats a wave , t = 70′myrGCaMP5 Ca2+ reporter did not interfere with the expected morphological changes in the oocyte associated with activation, including swelling, rounding at the poles and increased rigidity , which were similar to control experiments where the reporter was not expressed  did not trigger a Ca2+ transient or activation. However, addition of 10 mM CaCl2, which is five times more concentrated than the CaCl2 in activation buffer, caused a rapid increase in intracellular Ca2+ from all regions of the cortex, accelerated swelling and lysis within minutes (2+ change (data not shown). Finally, we showed that hypo-osmotic shock by addition of dH2O promotes accelerated swelling as well as an increase in Ca2+ in the egg . These results demonstrate a role for the cytoskeleton in coupling swelling and propagation of the Ca2+ wave.Previous work has shown that the actin cytoskeleton is re-organised at activation . We testDrosophila heralds a cascade of events, including translational initiation of several mRNA's: bcd, nanos, hunchback, caudal, Toll, torso, smaug, and string . We observe a similar dispersed pattern of Me31B in the early embryo when compared to the large foci in stage 14 oocyte  a stage 14 oocyte enters the lateral oviduct; (2) fluid in the oviduct initiates a change in intracellular Ca2+ at the posterior pole; (3) osmotic swelling and likely IP3 mediated release of intracellular Ca2+; (4) Ca2+ release by IP3 is in part controlled by a functional actin cytoskeleton; (5) a wave of Ca2+ propagates through the cell by Ca2+-induced Ca2+ release; (6) Ca2+ is reabsorbed into internal stores; (7) resumption of the cell cycle and translation of maternal mRNAs.In this study we visualised a rapid wave of intracellular Ca2+ release, in the form of a wave or oscillation, at egg activation is initiated by the sperm -bisphosphate (PIP2), inositol 1,4,5-trisphosphate (IP3) cascade as in other systems .While we cannot completely rule out a role for physical pressure or additional contribution from a specific factor for activation, our data supports a model where an increase in Ca2+ release  elongation at activation in a sra dependent manner (bcd mRNA is released from anchoring in large aggregates at anterior pole and is no longer observed in P bodies (2+ mediates the observed changes in mRNA and P bodies. While Ca2+ signalling typically requires transaction proteins, such as calmodulin, another possibility is that Ca2+ mediates change through direct binding to its target. In this model, which is not restricted to Drosophila or egg activation, calcium bound to mRNA or P body components would thus alter their conformation, charge or association. Further examination of mRNA untranslated regions and protein dissociation in different calcium concentrations are required to test this hypothesis. Drosophila, with its advantages for live cell imaging and the availability of genetic tools will be an ideal system to approach these questions and determine whether similar mechanisms operate in other systems.Now that we know that a calcium signalling event accompanies activation in tivation . The antt manner . VisualiP bodies . The los


We demonstrate that NFAT1 has a private line of communication with Orai1, activating in response to Ca2+ microdomains near the open channels. By contrast, NFAT4 stimulation requires both local Ca2+ entry and a nuclear Ca2+ rise. We mapped differences in nuclear location to amino acids within the SP-3 motif of the NFAT regulatory domain. The different Ca2+ dependencies enable agonists to recruit different isoform combinations as stimulus strength increases. Our study uncovers a mechanism whereby co-existing cytoplasmic transcription factor isoforms are differentially activated by distinct sub-cellular Ca2+ signals.Protein isoforms are widely expressed in biological systems. How isoforms that co-exist within the same sub-cellular domain are differentially activated remains unclear. Here, we compare the regulatory mechanism of two closely related transcription factor isoforms, NFAT1 and NFAT4, that migrate from the cytoplasm to the nucleus following the increase in intracellular Ca  •2+ activates NFAT1 and NFAT4 transcription factorsCytoplasmic Ca•2+ microdomains near plasma membrane Ca2+ channelsNFAT1 is stimulated by Ca•2+ microdomains and a nuclear Ca2+ riseNFAT4 requires both Ca•2+ dependencies impart distinct NFAT nuclear residency timesDifferent Ca 2+ signatures. NFAT1 has a private line of communication with plasma membrane Ca2+ channels, whereas NFAT4 additionally requires a nuclear Ca2+ rise.Protein isoforms are often co-expressed in cells, occupy the same spatial domain, and are activated by the same intracellular signal. How selective recruitment of one isoform occurs is unclear. Here, we have compared the activation of NFAT1 and NFAT4, two closely related vertebrate transcription factors. We report that the different isoforms are stimulated by distinct sub-cellular Ca Caenorhabditis elegans, and vertebrates all express protein isoforms reinforces the view that this tight evolutionary conservation underlies an important, isoform-specific biological function. Critical questions are therefore how some isoforms can be activated, but not others, when they are co-expressed and how different isoforms evoke distinct effects. A partial answer has been provided by the finding that isoforms of protein kinases are corralled to different sub-cellular locations  are activated by cytoplasmic Caus genes . In restpression .2+ entry through store-operated Ca2+ release-activated Ca2+ (CRAC) channels  increases InsP3 levels and generates cytoplasmic Ca2+ oscillations that are accompanied by CRAC channel opening  that had been engineered to express only in the nucleus by virtue of insertion of a nuclear localization sequence (NLS) (referred to as PV-NLS). PV-NLS-GFP was retained exclusively in the nucleus  with NFAT4-GFP and stimulated cells with thapsigargin. NFAT4-GFP accumulation within the nucleus was now virtually undetectable  selectively deplete stores and thus open CRAC channels, higher concentrations (μM) raise cytoplasmic Ca2+ mainly through direct Ca2+ entry by the ionophore. In intact RBL-1 cells, for example, the sustained Ca2+ response to 2 μM ionomycin is weakly affected by La3+, a CRAC channel blocker . However, BTP2 inhibited store-operated Ca2+ entry by 82% ± 9% when applied after Ca2+ influx had been evoked by thapsigargin. Although stimulation with thapsigargin in cells expressing PV-NLS caused little NFAT4 translocation, subsequent exposure to 2 μM ionomycin in these same cells rescued NFAT4-GFP accumulation within the nucleus  so that mutant PV-NLS was unable to bind Ca2+ , casein kinase 1, and glycogen synthase kinase 3. DYRKs directly phosphorylate the serine-proline repeat (SP-3) motif, which primes for further phosphorylation of the SP-2 and serine-rich region 1 (SRR-1) by the other kinases . No Ca2+rylation . To mimirylation A. The ti nucleus , we hypo nucleus B, co-locear Ca2+ B. In cel reduced B. PV-NLStectable E, top. A blocker . Consist nucleus J. Applicg PV-NLS A and S3Bind Ca2+ . Express (referred to as cytosolic PV-NES) resulted in a predominantly cytoplasmic distribution . Stimulation with either 50 nM . Compared with wild-type NFAT1-GFP, S276V-NFAT1-GFP moved out of the nucleus slightly faster  or when the Ca2+ rise is sustained. A simple model that simulates the nuclear dynamics of NFAT1 and NFAT4 is shown in 4) of NFAT4 relative to NFAT1 leads to marked differences in the nuclear levels of each transcription factor. Previous work in T cells showed that different transcription factors responded to different periodicities of the Ca2+ signal  and were cultured at 37°C with 5% COescribed .Cells were transfected using lipofectamine, as described . cDNA foNFAT1-GFP, NFAT4-GFP, and NFAT4-cherry levels in the cytosol and nucleus were measured using the IMAGO charge-coupled device camera-based system from TILL Photonics, with a ×100 oil immersion objective . Regions of interest of identical size were drawn in the cytosol and nucleus of each cell, and fluorescence was computed. Nuclear localization was confirmed by co-staining with the nuclear dye DAPI . Unless 2+ measurements were carried out at room temperature using the IMAGO charge-coupled device camera-based system from TILL Photonics , with NaOH. Ca2+-free solution had the following composition: 145 mM NaCl, 2.8 mM KCl, 2 mM MgCl2, 10 mM D-glucose, 10 mM HEPES, and 0.1 mM EGTA (pH 7.4), with NaOH. Ca2+ signals are plotted as R, which denotes the 356/380 nm ratio.Cytoplasmic Cahotonics . Cells, 2+ were performed with fluo-4. Cells were plated onto glass coverslips and either were not transfected or co-transfected with untagged PV-NLS and mcherry (marker for transfection) or PV-NES-DsRed. Cells were loaded with 2 μM fluo4-AM in a standard external solution for 40 min at room temperature. The changes in fluo-4 fluorescence (488 nm) in the cytosolic and nuclear compartments were monitored with a Zeiss LSM 510 Meta confocal microscope.Simultaneous measurements of thapsigargin-induced changes in cytoplasmic and nuclear CaCells were loaded with EGTA-AM (20 μM) or solvent (0.1% DMSO) for 45 min in the dark, as described .Total cell lysates (40 μg) were analyzed by SDS-PAGE on either a 10% D or 12% ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001).Data are presented as the mean ± SEM. Statistical significance was determined by using the Student’s t test, except in P.K. and A.B.P. designed the experiments. P.K. carried out the experiments, and P.K. and A.B.P. analyzed the data. A.B.P. wrote the manuscript.


Francisella tularensis is a highly virulent bacterium that is capable of causing severe disease (tularemia) in a wide range of species. This organism is characterized into two distinct subspecies: tularensis (type A) and holarctica (type B) which vary in several crucial ways, with some type A strains having been found to be considerably more virulent in humans and laboratory animals. Cottontail rabbits have been widely implicated as a reservoir species for this subspecies; however, experimental inoculation in our laboratory revealed type A organisms to be highly virulent, resulting in 100% mortality following challenge with 50–100 organisms. Inoculation of cottontail rabbits with the same number of organisms from type B strains of bacteria was found to be rarely lethal and to result in a robust humoral immune response. The objective of this study was to characterize the protection afforded by a prior challenge with type B strains against a later inoculation with a type A strain in North American cottontail rabbits (Sylvilagus spp). Previous infection with a type B strain of organism was found to lengthen survival time and in some cases prevent death following inoculation with a type A2 strain of F. tularensis. In contrast, inoculation of a type A1b strain was uniformly lethal in cottontail rabbits irrespective of a prior type B inoculation. These findings provide important insight about the role cottontail rabbits may play in environmental maintenance and transmission of this organism. Francisella tularensis (F. tularensis) is an intracellular, zoonotic bacterium, infection which induces tularemia [50 for some type A strains of F. tularensis has been found to be as low as one colony-forming unit (cfu) in mice), is classified as a Tier 1 Select Agent by the Centers for Disease Control and Prevention (CDC) [F. tularensis is classified into two subspecies: tularensis and holarctica which are referred to as type A and B respectively, and are responsible for the vast majority of human tularemia cases [F. tularensis cause mortality in 7% of human cases [ularemia . This oron (CDC) , 3. F. tia cases ,5. Despiia cases . Type A ia cases –9. Basedia cases . Furtheria cases , 11. Typia cases . These ria cases , 14. Typan cases , 11.Sylvilagus spp).Tularemia has been recognized for over a century and has long been associated with cottontail rabbits primarily due to rabbit die-offs or contact with a rabbit preceding human cases of tularemia , 16. OurAll aspects of this work, including experimental manipulations and sampling were conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee at Colorado State University . Rabbits were trapped on public lands using Havahart traps baited with grain with approval from the Colorado Department of Natural Resources, Parks and Wildlife; cottontail rabbits are not endangered or protected. At the end of the study period, rabbits were euthanized by intravenous overdose of pentobarbital.F. tularensis used in this study were provided by the CDC and were subsequently maintained in our laboratory; passage number is unknown. For simplicity, the strains are abbreviated to include the U.S. state in which they were originally isolated and their clade distinction  were wild-trapped along the front range of Colorado. Rabbits were transported to an ABSL-3 facility at Colorado State University and acclimatized for 2–3 weeks prior to infection. During that time they were treated for ectoparasites, a IPTT-300 temperature transponder  was implanted subcutaneously under lidocaine anesthesia, and an LPS-based ELISA (see below) performed on pre-inoculation serum to provide some assurance that the rabbits were naïve. The rabbits were individually housed in standard size, stainless steel rabbit cages and provided free access to alfalfa hay, commercial rabbit pellets, and water.F. tularensis on day 0 and one of four strains on day 28  at both time points in order to quantify the organ burden. Duplicate samples (100μL) from each of the three dilutions were plated on CHAB agar plates, incubated at 37°C with 5% CO2 for 24–48 hours, and colony counts recorded. For each rabbit with a positive culture, DNA was extracted from a bacterial colony and PCR was used to confirm its identity as F. tularensis using a protocol described by Long et al. [Organ burden and identity of the infecting organism were evaluated by collecting 100 mg samples of liver and spleen in a vial with 0.9mL of MMH broth containing 15% glycerol and 2 stainless steel BBs; these samples were immediately homogenized in a mixer mill and frozen at -80°C. We did not evaluate organ burdens from lung or kidney because in previous studies we found that those tissues did not provide additional information over what obtained from liver and spleen . Serial g et al. . For rabg et al.  was usedF. tularensis LPS obtained from BEI Resources , rinsed and blocked with 5% non-fat dry milk. Serum samples were diluted 1:1,000 in incubation buffer and loaded in duplicate wells. Following a 1-hour incubation, the plate was emptied and rinsed. Goat anti-rabbit horseradish-peroxidase conjugate  was used as the secondary antibody and incubated for 1-hour. The plate was again emptied and rinsed prior to the application of substrate  and proceeded for 15–20 minutes before the reaction was stopped via the addition of 1N hydrocholoric acid. Pooled serum from laboratory rabbits was used as a negative control.Rabbits were manually restrained and bled from the jugular vein prior to infection and on 14, 28, and 42 dpi-1, or upon euthanasia due to a moribund condition. In rare instances when rabbits died prior to the observation of a moribund condition (hunched/recumbent position or hypo-responsive) blood was not collected. Humoral antibody response was evaluated using an ELISA developed in our laboratory based on the World Health Organization Guidelines on Tularaemia  and descF. tularensis. Median survival time (in days) and 95% confidence intervals were calculated using a Kaplan Meier survival function and univariate non-parametric analyses were conducted using the log rank test to compare the survival function among cottontail rabbits infected with a combination of Francisella strains. Results were considered statistically significant with p-values <0.05.Descriptive statistics and survival analysis were performed using STATA software . Time to death was measured in days from the time of inoculation with different strains of Baseline body temperature was found to be between 38.3 and 39.4°C for all rabbits. Initial inoculation with either type B strain (KY-B or OR-B) resulted in elevated body temperatures starting 2–3 dpi-1 and peaking between 40.6 and 41.7°C. Overt clinical disease was not observed with the exception of one rabbit (#19) that became moribund following the initial inoculation with OR-B, and was euthanized at 8 dpi-1.Body temperature following MA-A1b inoculation at day 0 dpi-2 appeared to have been influenced by which type B strain the rabbit had originally received. Rabbits inoculated with OR-B at the first time point were found to develop a fever 3 to 5 dpi-2, whereas rabbits that received KY-B at the first time point developed a fever 5 to 7 dpi-2. Fever associated with inoculation with WY-A2 was found to be the same for both groups and was observed 4 to 6 dpi-2. The peak body temperature for rabbits inoculated with MA-A1b and WY-A2, irrespective of which type B strain had been administered previously, was found to be between 40.6 and 41.7°C (data not shown).The median time to euthanasia  and range following inoculation with a type A strain is summarized in F. tularensis was cultured in the urine from each rabbit, type A and type B, respectively. Rabbits challenged with type B at both time points were found to have cleared the organism from the liver and spleen upon euthanasia at the end of the study period, with an exception for one rabbit (#13) that was found to have 700 organisms/gram in the spleen.All rabbits inoculated with a type B strain followed by challenge with a type A strain at day 28 were found to have type A organism in the spleen upon necropsy . Urine wThe primary gross lesions observed were microabscesses on the liver or spleen and splenomegaly . RabbitsWith a few exceptions, rabbits developed a robust antibody response characterized by a moderate rise in antibodies by 14 dpi-1 which peaked at 28 dpi-1 and remained stable until euthanasia at 42 dpi-1 or earlier . SerologF. tularensis. Infection of laboratory mice with both type A and B strains results in uniform mortality [F. tularensis has important implications for further understanding the role of cottontail rabbits in the maintenance and transmission of F. tularensis. Clearly, humoral immunity per se likely does not mediate protective immunity to an intracellular pathogen like F. tularensis, but was used in this study as an index of immune response to infection.This study provided an initial characterization in cottontail rabbits of the effect of a prior type B infection on the outcome of subsequent inoculation with a virulent type A strain of ortality  and it wF. tularensis is, in some cases, sufficient to lengthen the survival time following infection with a virulent type A strain. Inoculation with MA-A1b was uniformly lethal prior to 14 dpi regardless of whether or not the rabbit was previously inoculated with a type B strain; however, rabbits that were previously exposed were found to survive for a longer duration as compared to those that received PBS at the first time point. Inoculation with WY-A2 alone was found previously [Our study establishes that a robust immune response, as assessed by antibody production, initiated by challenge with a type B strain of eviously  to causeeviously .F. tularensis. This observed difference indicates that a robust antibody response is associated with partially protecting rabbits from inoculation with a virulent strain of F. tularensis (WY-A2), although is insufficient to afford protection against highly virulent strains (MA-A1b).Interestingly, the two rabbits (#26 and #27) that were euthanized from the OR/WY group prior to 14 dpi-2) were found to have antibody levels similar to the rabbits that survived which, as has been previously observed, provides an additional indication that antibody production is not the major influence on host outcome following exposure to F. tularensis. [The pattern and magnitude of the antibody response proved to be fairly uniform irrespective of the inoculating strain and length of survival following inoculation. Sequential exposure to a type B strain at days 0 and 28 was found to result in a humoral immune response of a similar magnitude to those that received only one inoculation with a type B strain. Additionally, fever was observed between 3 and 7 dpi in the majority of rabbits regardless of the inoculated strain. This febrile response was similar to that described following aerosol exposure of New Zealand White rabbits with the Schu S4 strain of arensis. .Surprisingly, all rabbits inoculated with type A strains were found to have spleens that were culture positive for this organism (either MA-A1b or WY-A2) at the time of necropsy, even in instances where the rabbit survived until 14 dpi-2. Because both type A strains have been found to be 100% fatal when administered solely, the finding of culture positive tissues in apparently healthy animals is suggestive of an important role for the adaptive immune response.F. tularensis in its natural host and thus, we believe our findings to be representative of a natural setting. Secondly, the likelihood in a natural setting of a sequential infection in cottontail rabbits of a type B strain of F. tularensis followed by a type A strain is unknown. These strains do overlap geographically and thus, it is certainly a possibility that a single animal could be infected with several strains of the organism. Next, captivity for cottontail rabbits is indisputably a stressful environment. We attempted to alleviate the stress of the laboratory setting by handling the rabbits gently and infrequently. Finally, chocolate agar plates were used only for the WY-A2 strain due to growth requirements which may have confounded the culture results.This study was subject to some limitations. First, the cottontail rabbits utilized in this study were wild-caught and thus, were undoubtedly harboring various organisms that could influence the immune responses observed in our study. However, our intent was to study F. tularensis does not provide full protection against challenge with a virulent type A strain, it does lengthen the survival period for rabbits inoculated with either KY-B or OR-B followed by WY-A2 and in some cases, rabbits survive infection altogether. These findings are important and help to shape our understanding of the role that cottontail rabbits may play in the maintenance and transmission of F. tularensis amongst humans and other animals.Our results strongly suggest that although a previous exposure to a type B strain of