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To identify radiographic and MR features of hip osteoarthritis (OA) related to reduced hip extension during walking. Sixty six subjects, were stratified into those with (n = 36, KL = 2, 3) and without (n = 30, KL = 0, 1) radiographic hip OA. Cartilage and labrum lesions were graded semi-quantitatively on hip MRI. Alpha angle and lateral center edge (LCE) angle were measured. Sagittal kinematics and kinetics were calculated during walking at speed of 1.35 m/s using 3-D motion capture. All subjects completed Hip disability and Osteoarthritis Outcome Score (HOOS), timed up and go, and 6 min walk tests. Variables were compared between the two groups using one-way ANOVA (adjusting for age). Correlations of radiographic and MR parameters with peak hip extension were calculated. The OA group was older, had greater pain, and limitation of function. They also had lower peak hip extension and higher peak hip flexion; and worse acetabular and femoral cartilage lesions. Peak hip extension and flexion correlated with KL grade, cartilage lesions in the inferior and posterior femur. Reduced hip extension and greater hip flexion during walking are present in high functioning (HOOS > 85%) individuals with mild-moderate hip OA, and are associated with cartilage lesions.

ObjectiveTo evaluate the relationship of hip radiographic osteoarthritis (ROA) and MRI findings of cartilage lesions, labral tears, bone marrow edema-like lesions (BMELs) and subchondral cysts with self-reported and physical function.DesignEighty five subjects were classified as controls (n = 55, Kellgren-Lawrence (KL) 0, 1) or having mild-moderate ROA (n = 30, KL 2, 3). T2 weighted MRI images at 3-T were graded for presence of cartilage lesions, labral tears, BMELs and subchondral cysts. Posterior wall sign, cross-over sign, center-edge angle and alpha angle were also recorded. Function was assessed using Hip dysfunction and Osteoarthritis Outcome Score (HOOS), Timed-Up and Go (TUG) test and Y-Balance Test (YBT). Analysis compared function between subjects with and without ROA and those with and without femoral or acetabular cartilage lesions, adjusted for age. Non-parametric correlations were used to assess the relationship between radiographic scores, MRI scores and function.ResultsSubjects with acetabular cartilage lesions had worse HOOS (Difference = 5-10%, P = 0.036-0.004), but not TUG or YBT, scores. Acetabular cartilage lesions, BMELs and subchondral cysts were associated with worse HOOS scores (ρ = 0.23-0.37, P = 0.041-0.001). Differences in function between subjects with and without ROA or femoral cartilage lesions were not significant. Other radiologic findings were not associated with function.ConclusionsAcetabular cartilage defects, but not femoral cartilage defects or ROA, were associated with greater self-reported pain and disability. BMELs and subchondral cysts were related to greater hip related self-reported pain and disability. None of the radiographic or MRI features was related to physical function.

Abstract Viruses exploit various cellular processes for their own benefit, including counteracting anti-viral responses and regulating viral replication and propagation. In the past 20 years, protein sumoylation has emerged as an important post-translational modification that is manipulated by viruses to modulate anti-viral responses, viral replication, and viral pathogenesis. The process of sumoylation is a multi-step cascade where a small ubiquitin-like modifier (SUMO) is covalently attached to a conserved ΨKxD/E motif within a target protein, altering the function of the modified protein. Here we review how viruses manipulate the cellular machinery at each step of the sumoylation process to favor viral survival and pathogenesis.

Stromal cells promote extensive fibrosis in pancreatic ductal adenocarcinoma (PDAC), which is associated with poor prognosis and therapeutic resistance. We report here for the first time that loss of the RNA-binding protein human antigen R (HuR, ELAVL1 ) in PDAC cells leads to reprogramming of the tumor microenvironment. In multiple in vivo models, CRISPR deletion of ELAVL1 in PDAC cells resulted in a decrease of collagen deposition, accompanied by a decrease of stromal markers (i.e. podoplanin, α-smooth muscle actin, desmin). RNA-sequencing data showed that HuR plays a role in cell-cell communication. Accordingly, cytokine arrays identified that HuR regulates the secretion of signaling molecules involved in stromal activation and extracellular matrix organization [i.e. platelet-derived growth factor AA (PDGFAA) and pentraxin 3]. Ribonucleoprotein immunoprecipitation analysis and transcription inhibition studies validated PDGFA mRNA as a novel HuR target. These data suggest that tumor-intrinsic HuR supports extrinsic activation of the stroma to produce collagen and desmoplasia through regulating signaling molecules (e.g. PDGFAA). HuR-deficient PDAC in vivo tumors with an altered tumor microenvironment are more sensitive to the standard of care gemcitabine, as compared to HuR-proficient tumors. Taken together, we identified a novel role of tumor-intrinsic HuR in its ability to modify the surrounding tumor microenvironment and regulate PDGFAA., (© The Author(s) 2023. Published by Oxford University Press on behalf of NAR Cancer.)

Due to the limited heating efficiency of available magnetic nanoparticles, it is difficult to achieve therapeutic temperatures above 44 °C in relatively inaccessible tumors during magnetic hyperthermia following systemic administration of nanoparticles at clinical dosage (≤10 mg kg -1 ). To address this, a method for the preparation of magnetic nanoparticles with ultrahigh heating capacity in the presence of an alternating magnetic field (AMF) is presented. The low nitrogen flow rate of 10 mL min -1 during the thermal decomposition reaction results in cobalt-doped nanoparticles with a magnetite (Fe 3 O 4 ) core and a maghemite (γ-Fe 2 O 3 ) shell that exhibit the highest intrinsic loss power reported to date of 47.5 nH m 2 kg -1 . The heating efficiency of these nanoparticles correlates positively with increasing shell thickness, which can be controlled by the flow rate of nitrogen. Intravenous injection of nanoparticles at a low dose of 4 mg kg -1 elevates intratumoral temperatures to 50 °C in mice-bearing subcutaneous and metastatic cancer grafts during exposure to AMF. This approach can also be applied to the synthesis of other metal-doped nanoparticles with core-shell structures. Consequently, this method can potentially be used for the development of novel nanoparticles with high heating performance, further advancing systemic magnetic hyperthermia for cancer treatment., (© 2022 Wiley-VCH GmbH.)

Background: Deep vein thrombosis (DVT) and post-thrombotic syndrome (PTS) remain highly prevalent despite modern medical therapy. Contact activation is a promising target for safe antithrombotic anticoagulation. The anti-factor XI (FXI) monoclonal antibody 14E11 reduces circulating levels of FXI without compromising hemostasis. The human recombinant analog, AB023, is in clinical development. The role of FXI in mediation of inflammation during DVT resolution is unknown., Objectives: Investigate the effects of pharmacological targeting of FXI with 14E11 in an experimental model of venous thrombosis., Methods: Adult wild-type CD1 mice were treated with subcutaneous anti-FXI antibody (14E11, 5 mg/kg) versus saline prior to undergoing surgical constriction of the inferior vena cava (IVC). Mice were evaluated at various time points to assess thrombus weight and volume, as well as histology analysis, ferumoxytol enhanced magnetic resonance imaging (Fe-MRI), and whole blood flow cytometry., Results: 14E11-treated mice had reduced thrombus weights and volumes after IVC constriction on day 7 compared to saline-treated mice. 14E11 treatment reduced circulating monocytes by flow cytometry and macrophage content within thrombi as evaluated by histologic staining and Fe-MRI. Collagen deposition was increased at day 3 while CD31 and smooth muscle cell actin expression was increased at day 7 in the thrombi of 14E11-treated mice compared to saline-treated mice., Conclusion: Pharmacologic targeting of FXI enhances the early stages of experimental venous thrombus resolution in wild-type CD1 mice, and may be of interest for future clinical evaluation of the antibody in DVT and PTS., (© 2022 International Society on Thrombosis and Haemostasis. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Purpose: Seiffert spirals were recently explored as an efficient way to traverse 3D k-space compared to traditional 3D techniques. Several studies have shown the ability of 3D MR fingerprinting (MRF) techniques to acquire T 1 and T 2 relaxation maps in a short period of time. However, these sequences do not sample across a large region of 3D k-space every TR, especially in the way that Seiffert trajectories can., Methods: A 3D MRF sequence was designed using 8 Seiffert spirals rotated in 3D k-space, with flip angle modulation for T 1 and T 2 sensitivity. The sequence was compared to an MRF sequence using a 2D spiral rotated in 3D k-space using the tiny golden angle acquisition with similar resolution/readout duration. Both sequences were evaluated using simulations, phantom validation, and in vivo imaging., Results: In all experiments, the Seiffert spiral MRF sequence performed similar to if not better than the multi-axis 2D spiral MRF sequence. Strong intraclass correlation coefficients (> 0.9) were found between conventional and MRF sequences in phantoms, whereas the in vivo results showed slightly less aliasing artifact with the Seiffert trajectory., Conclusion: In this study, Seiffert spirals were used within the MRF framework to acquire high-resolution T 1 and T 2 relaxation time maps in less than 2.5 min. The reduced aliasing artifacts seen with the Seiffert sequence suggests that sampling over 3D k-space evenly each TR can improve quantification or shorten scan times., (© 2022 International Society for Magnetic Resonance in Medicine.)

Endometriosis is a devastating disease in which endometrial-like tissue forms lesions outside the uterus. It causes infertility and severe pelvic pain in ≈176 million women worldwide, and there is currently no cure for this disease. Magnetic hyperthermia could potentially eliminate widespread endometriotic lesions but has not previously been considered for treatment because conventional magnetic nanoparticles have relatively low heating efficiency and can only provide ablation temperatures (>46 °C) following direct intralesional injection. This study is the first to describe nanoparticles that enable systemically delivered magnetic hyperthermia for endometriosis treatment. When subjected to an alternating magnetic field (AMF), these hexagonal iron-oxide nanoparticles exhibit extraordinary heating efficiency that is 6.4× greater than their spherical counterparts. Modifying nanoparticles with a peptide targeted to vascular endothelial growth factor receptor 2 (VEGFR-2) enhances their endometriosis specificity. Studies in mice bearing transplants of macaque endometriotic tissue reveal that, following intravenous injection at a low dose (3 mg per kg), these nanoparticles efficiently accumulate in endometriotic lesions, selectively elevate intralesional temperature above 50 °C upon exposure to external AMF, and completely eradicate them with a single treatment. These nanoparticles also demonstrate promising potential as magnetic resonance imaging (MRI) contrast agents for precise detection of endometriotic tissue before AMF application., (© 2022 Wiley-VCH GmbH.)

Objectives: To assess emergency physician prescribing for simple extremity fractures-specifically, distal radius fractures-and describe the opportunity for reducing opioid prescribing., Methods: An electronic survey was distributed to 1238 emergency physicians employed by a nationwide practice serving 220 sites in 20 states. The survey presented two plain film views of a simple Colles fracture and asked: "For the last patient you discharged . . . with the above injury, which pain medications did you prescribe or recommend?" Responses were collected using a clickable checklist of common opioid and nonopioid pain medications. Respondents also specified the number of days covered by any prescription. We assessed associations between physician characteristics and opioid prescribing using the χ 2 test, the Wilcoxon rank-sum test, and multivariable regression models., Results: Responses were received from 447 (36%) physicians working in 18 states; 93% were trained in emergency medicine, 33% worked at academic sites, 68% had site volumes between 25,000 and 75,000, and the median experience was 10 (interquartile range 5-19) years. Overall, 92% (95% confidence interval 89%-95%) had prescribed an opioid for a median of 3 (interquartile range 3-4) days. The most commonly prescribed opioids were hydrocodone/acetaminophen (55%) and oxycodone/acetaminophen (20%). Physicians at academic sites prescribed opioids less frequently than those at nonacademic sites (88% vs 94%), but in multivariable regression there were no significant associations between physician characteristics and opioid prescribing., Conclusions: Emergency physicians commonly prescribe opioids for simple distal radius fractures. This represents a potential opportunity to reduce opioid prescribing.

T 1ρ relaxation imaging is a quantitative imaging technique that has been used to assess cartilage integrity, liver fibrosis, tumors, cardiac infarction, and Alzheimer's disease. T 1 , T 2 , and T 1ρ relaxation time constants have each demonstrated different degrees of sensitivity to several markers of fibrosis and inflammation, allowing for a potential multi-parametric approach to tissue quantification. Traditional magnetic resonance fingerprinting (MRF) has been shown to provide quick, quantitative mapping of T 1 and T 2 relaxation time constants. In this study, T 1ρ relaxation is added to the MRF framework using spin lock preparations. An MRF sequence involving an RF-spoiled sequence with T R , flip angle, T 1ρ , and T 2 preparation variation is described. The sequence is then calibrated against conventional T 1 , T 2 , and T 1ρ relaxation mapping techniques in agar phantoms and the abdomens of four healthy volunteers. Strong intraclass correlation coefficients (ICC > 0.9) were found between conventional and MRF sequences in phantoms and also in healthy volunteers (ICC > 0.8). The highest ICC correlation values were seen in T 1 , followed by T 1ρ and then T 2 . In this study, T 1ρ relaxation has been incorporated into the MRF framework by using spin lock preparations, while still fitting for T 1 and T 2 relaxation time constants. The acquisition of these parameters within a single breath hold in the abdomen alleviates the issues of movement between breath holds in conventional techniques., (© 2020 John Wiley & Sons, Ltd.)

The use of quantitative imaging biomarkers in the imaging of various disease states, including cancer and neurodegenerative disease, has increased in recent years. T 1 , T 2 , and T 2 * relaxation time constants have been shown to be affected by tissue structure or contrast infusion. Acquiring these biomarkers simultaneously in a multi-parametric acquisition could provide more robust detection of tissue changes in various disease states including neurodegeneration and cancer. Traditional magnetic resonance fingerprinting (MRF) has been shown to provide quick, quantitative mapping of T 1 and T 2 relaxation time constants. In this study, T 2 * relaxation is added to the MRF framework using variable echo times (TE). To demonstrate the feasibility of the method and compare incremental and golden angle spiral rotations, simulated phantom data was fit using the proposed method. Additionally, T 1 /T 2 /T 2 */δf MRF as well as conventional T 1 , T 2 , and T 2 * acquisitions were acquired in agar phantoms and the brains of three healthy volunteers. Golden angle spiral rotation was found to reduce inaccuracy resulting from off resonance effects. Strong correlations were found between conventional and MRF values in the T 1 , T 2 , and T 2 * relaxation time constants of the agar phantoms and healthy volunteers. In this study, T 2 * relaxation has been incorporated into the MRF framework by using variable echo times, while still fitting for T 1 and T 2 relaxation time constants. In addition to fitting these relaxation time constants, a novel method for fitting and correcting off resonance effects has been developed., (© 2018 John Wiley & Sons, Ltd.)

Mutations in >30 genes are known to result in impairment of the adaptive immune system, causing a group of disorders collectively known as SCID. SCID disorders are split into groups based on their presence and/or functionality of B, T, and NK cells. Piglets from a line of Yorkshire pigs at Iowa State University were shown to be affected by T(-)B(-)NK(+) SCID, representing, to our knowledge, the first example of naturally occurring SCID in pigs. In this study, we present evidence for two spontaneous mutations as the molecular basis for this SCID phenotype. Flow cytometry analysis of thymocytes showed an increased frequency of immature T cells in SCID pigs. Fibroblasts from these pigs were more sensitive to ionizing radiation than non-SCID piglets, eliminating the RAG1 and RAG2 genes. Genetic and molecular analyses showed that two mutations were present in the Artemis gene, which in the homozygous or compound heterozygous state cause the immunodeficient phenotype. Rescue of SCID fibroblast radiosensitivity by human Artemis protein demonstrated that the identified Artemis mutations are the direct cause of this cellular phenotype. The work presented in the present study reveals two mutations in the Artemis gene that cause T(-)B(-)NK(+) SCID in pigs. The SCID pig can be an important biomedical model, but these mutations would be undesirable in commercial pig populations. The identified mutations and associated genetic tests can be used to address both of these issues., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Background: To develop a semi-quantitative MR-based hip osteoarthritis (OA) evaluation system (Scoring hip osteoarthritis with MRI, SHOMRI), and to test its reproducibility and face validity., Methods: The study involved 98 subjects with informed consent. Three-Tesla MR imaging of hip was performed in three planes with intermediate-weighted fat saturated FSE sequences. Two radiologists assessed cartilage loss, bone marrow edema pattern, subchondral cyst in 10 subregions, and assessed labrum in 4 subregions. In addition, presence or absence of ligamentum teres integrity, paralabral cysts, intra-articular body, and effusion in the hip joint were analyzed using the SHOMRI system. The reproducibility was assessed with intra-class correlation coefficient (ICC), Cohen's Kappa values and percent agreement. SHOMRI scores were correlated with radiographic Kellgren-Lawrence (KL) and OARSI atlas gradings, and clinical parameters, the hip osteoarthritis outcome score (HOOS) and hip range of motion (ROM), using Spearman's rank correlation and ordinal logistic regression., Results: ICC values were in the excellent range, 0.91 to 0.97. Cohen's Kappa values and percent agreement ranged from 0.55 to 0.79 and 66 to 99%, respectively. SHOMRI demonstrated significant correlations with KL and OARSI gradings as well as with clinical parameters, HOOS and ROM (P < 0.05). Among the SHOMRI features, subchondral cyst and bone marrow edema pattern showed the highest correlation with HOOS and ROM., Conclusion: SHOMRI demonstrated moderate to excellent reproducibility and significant correlation with radiographic gradings and clinical parameters., (© 2014 Wiley Periodicals, Inc.)

Severe combined immunodeficiency (SCID) is the result of a set of inherited genetic defects which render components of the immune response nonfunctional. In Arabian horses, Jack Russell terriers, and mice, the disorder is a consequence of the absence of T and B lymphocytes, while natural killer (NK) cell and other leukocyte populations remain intact. Preliminary analysis of a naturally acquired form of inherited SCID in a line of pigs showed several defects in the architecture and composition of secondary lymphoid organs. In this study, a quantitative assessment of lymphocyte populations in affected and normal littermates showed depleted T or B lymphocyte populations in affected pigs; however, NK cells and neutrophils were present in numbers comparable to unaffected littermates. The results indicate that the immune defect in pigs shares the same features as other SCID-affected species., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Weaned pigs from a line bred for increased feed efficiency were enrolled in a study of the role of host genes in the response to infection with Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). Four of the pigs were euthanatized early in the study due to weight loss with illness and poor body condition; 2 pigs before PRRSV infection and the other 2 pigs approximately 2 weeks after virus inoculation. The 2 inoculated pigs failed to produce PRRSV-specific antibodies. Gross findings included pneumonia, absence of a detectable thymus, and small secondary lymphoid tissues. Histologically, lymph nodes, spleen, tonsils, and Peyer's patches were sparsely cellular with decreased to absent T and B lymphocytes.

Animal models for cancer therapy are invaluable for preclinical testing of potential cancer treatments; however, therapies tested in such models often fail to translate into clinical settings. Therefore, a better preclinical model for cancer treatment testing is needed. Here we demonstrate that an immunodeficient line of pigs can host and support the growth of xenografted human tumors and has the potential to be an effective animal model for cancer therapy. Wild-type and immunodeficient pigs were injected subcutaneously in the left ear with human melanoma cells (A375SM cells) and in the right ear with human pancreatic carcinoma cells (PANC-1). All immunodeficient pigs developed tumors that were verified by histology and immunohistochemistry. Nonaffected littermates did not develop tumors. Immunodeficient pigs, which do not reject xenografted human tumors, have the potential to become an extremely useful animal model for cancer therapy because of their similarity in size, anatomy, and physiology to humans.

Purpose: Breathing motion can create large errors when performing magnetic resonance (MR) thermometry of the breast. Breath holds can be used to minimize these errors, but not eliminate them. Between breath holds, the referenceless method can be used to further reduce errors by relying on regions of nonheated fatty tissue surrounding the heated region. When the surrounding tissue is heated (i.e., for a hyperthermia treatment), errors can result due to phase changes of the small amounts of water in the tissue. Therefore, an extension of the referenceless method is proposed which fits for the field in fatty tissue independent of temperature change and extrapolates it to the water-rich regions., Methods: Nonheating experiments were performed with male volunteers performing breath holds on top of a phantom mimicking a breast with a tumor. Heating experiments were also conducted with the same phantom while mechanically simulated breath holds were performed. A nonheating experiment was also performed with a healthy female breast. For each experiment, a nonlinear fitting algorithm was used to fit for temperature change and B0 field inside of the fatty tissue. The field changes were then extrapolated into water-rich (tumor) portions of the image using a least-squares fit to a fifth-order equation, to correct for field changes due to breath hold changes. Similar results were calculated using the image phase, to mimic the use of the referenceless method., Results: Phantom results showed large reduction of mean error and standard deviation. In the non-heating experiments, the traditional referenceless method and our extended method both corrected by similar amounts. However, in the heating experiments, the average deviation of the temperature calculated with the extended method from a fiber optic probe temperature was approximately 50% less than the deviation with the referenceless method. The in vivo breast results demonstrated reduced standard deviation and mean., Conclusions: In this paper, we have developed an extension of the referenceless method to correct for breathing errors using multiecho fitting methods to fit for the B0 field in the fatty tissue and using measured field changes as references to extrapolate field corrections into a water-only (tumor) region. This technique has been validated in a number of situations, and in all cases, the correction method has been shown to greatly reduce temperature error in water-rich regions. The method has also been shown to be an improvement over similar methods that use image phase changes instead of field changes, particularly when temperature changes are induced.

Cryptosporidiosis in calves is an ongoing problem, primarily because of the high prevalence and high morbidity associated with the infection. This article summarizes current knowledge of the host/parasite interactions associated with cryptosporidiosis. The infection process in intestinal mucosa, the pathophysiology of the disease process, and the immune responses initiated in the calf to control the infection are discussed. Methods for diagnosing C. parvum infection, treatments that have been tried, and management controls are also examined., (Copyright 2010 Elsevier Inc. All rights reserved.)

Purpose: To establish accuracy of real time noninvasive temperature measurements using magnetic resonance thermal imaging in patients treated for high grade extremity soft tissue sarcomas., Methods: Protocol patients with advanced extremity sarcomas were treated with external beam radiation therapy and hyperthermia. Invasive temperature measures were compared to noninvasive magnetic resonance thermal imaging (MRTI) at 1.5 T performed during hyperthermia. Volumetric temperature rise images were obtained using the proton resonance frequency shift (PRFS) technique during heating in a 140 MHz miniannular phased array applicator. MRTI temperature changes were compared to invasive measurements of temperature with a multisensor fiber optic probe inside a #15 g catheter in the tumor. Since the PRFS technique is sensitive to drifts in the primary imaging magnetic field, temperature change distributions were corrected automatically during treatment using temperature-stable reference materials to characterize field changes in 3D. The authors analyzed MRT images and compared, in evaluable treatments, MR-derived temperatures to invasive temperatures measured in extremity sarcomas. Small regions of interest (ROIs) were specified near each invasive sensor identified on MR images. Temperature changes in the interstitial sensors were compared to the corresponding ROI PRFS-based temperature changes over the entire treatment and over the steady-state period. Nonevaluable treatments (motion/imaging artifacts, noncorrectable drifts) were not included in the analysis., Results: The mean difference between MRTI and interstitial probe measurements was 0.91 degrees C for the entire heating time and 0.85 degrees C for the time at steady state. These values were obtained from both tumor and normal tissue ROIs. When the analysis is done on just the tumor ROIs, the mean difference for the whole power on time was 0.74 degrees C and during the period of steady state was 0.62 degrees C., Conclusions: The data show that for evaluable treatments, excellent correlation (deltaT < 1 degrees C) of MRTI-ROI and invasive measurements can be achieved, but that motion and other artifacts are still serious challenges that must be overcome in future work.

Alveolar macrophages from PRRSV-infected and naïve pigs were placed into culture and infected with PRRSV laboratory strain SD-23983. Permissiveness increased with time in culture, and macrophages from infected pigs could be superinfected. Addition of actinomycin D, an inhibitor of mRNA synthesis, blocked infection. Interferon-gamma reduced infection in cultures, while the addition of tumor necrosis factor-alpha or interleukin (IL)-10 did not affect permissiveness. IL-4 produced a marginal increase in the percentage of infected cells, but without a detectable increase in virus yield. These results suggest that the PRRSV-permissive population of cells in culture arises from a non-permissive precursor population and depends on new mRNA synthesis.

A nonradioactive multi-parameter flow cytometry assay was developed to identify antigen-specific lymphocytes in human subjects previously vaccinated against rabies virus and was subsequently compared to the standard tritiated thymidine method. A cell tracking dye, carboxyfluorescein succinimidyl ester, was used in combination with surface label for CD4 and CD8 cells in order to determine the response of lymphocytes to killed rabies virus in an antigen recall assay. The rabies virus-specific lymphocyte response was compared to the humoral immune response in each of ten vaccinated and five non-vaccinated subjects. Lymphocyte responses to rabies virus were observed in all ten vaccinated subjects; some noted as early as 3 days after stimulation while others were not until 7 days after stimulation. There was good agreement between the proliferation index of the CFSE assay and the simulation index of the [3H]thymidine assay (kappa statistic=0.73). An inverse relationship was detected between the level of rabies virus neutralizing antibody (RVNA) and the lymphocyte response to inactivated rabies virus in the vaccinated subjects. The association between cytokines production and level of humoral and cellular response was investigated in four representative subjects. Two vaccinated subjects with high proliferation indices and low RVNA titers produced Th1 type cytokines to rabies virus stimulation, whereas two vaccinated individuals with low proliferation indices and high RVNA titers responses did not produce these cytokines.

In this study bovine alveolar macrophage neurokinin-1 (NK-1) and the in vitro response to substance P (SP) exposure were investigated. Bovine alveolar macrophage membrane extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted using anti-NK-1 antiserum demonstrated the presence of an approximately 60kDa band. Phagocytosis of fluorescent bioparticles by SP-exposed macrophages was 39% greater than that of non-exposed macrophages (P=0.0089). Likewise, there was 28% greater TNF production by macrophages following SP exposure compared to non-exposed controls (P=0.116). These results suggest that bovine alveolar macrophages respond to SP at least in part by enhancing phagocytosis and TNF production.

We had previously demonstrated that a Type-1-like immune response involving interferon-gamma expression in lamina propria lymphocytes accompanied by IgG2 subclass fecal antibodies to Cryptosporidium parvum p23 emerged in gut mucosa of calves recovering from cryptosporidiosis. Because a recombinant p23 had been shown to protect calves from cryptosporidiosis when administered as a vaccine antigen to late gestation cattle, this study was undertaken to determine if the same vaccine antigen could induce a Type-1-like, in vitro response by T cells from calves that had recovered from C. parvum infection. We isolated peripheral blood mononuclear cells from calves that had been previously infected with C. parvum oocysts and incubated them in the presence or absence of the recombinant C. parvum p23 vaccine antigen. We used flow cytometry to simultaneously detect cells in cell cycle and identify the T cell subset containing cycling cells. We also used flow cytometry to identify interferon-gamma positive cells and 2-dimensional gel electrophoresis to profile proteins made by PBMC stimulated with the recombinant p23 vaccine antigen. The results demonstrated that CD4+ T lymphocytes proliferated and that interferon-gamma was synthesized by a subset of stimulated cells. Two-dimensional gel electrophoresis revealed the presence of several cytoplasmic proteins in a size range of approximately 25-80 kDa that were detected in p23-stimulated, but not in unstimulated, cytoplasmic samples. Together, the results show that the recombinant p23 vaccine antigen can stimulate a Type-1-like immune response by T cells from calves that have recovered from C. parvum infection.

Passive transfer of maternal antibodies via colostrum is important to protect newborn ruminants against microbial pathogens. In this study, 10 sets of calf serum, a sample of the colostrum fed to the calf, and serial fecal samples through the first 6 days after birth were collected from arbitrarily selected newborn Holstein heifers. A recombinant Cryptosporidium parvum p23, termed rC7, was used to determine whether anti-C. parvum antibodies can be detected in clinically normal neonates. The results demonstrated that serum, the associated colostrum, and fecal samples contained anti-rC7 antibodies. IgM and IgG1 anti-rC7 tended to be present in highest titers. The presence of specific antibodies to C. parvum was confirmed using Western blots of purified sporozoite membranes probed with serum and colostral whey. Collectively, the results indicated that neonatal calves had antibodies to C. parvum as early as 1 day after birth and suggested that the antibodies were passively transferred.

The effect of salivary gland extract of the stable fly, Stomoxys calcitrans (L), on bovine lymphocyte proliferation was determined, and antibody reactivity to salivary gland proteins was characterized in cattle exposed to stable flies. Salivary glands were dissected from male and female flies (4-8 d after eclosion), and protein extracts were made by freeze-thaw cycles. Salivary gland extract (SGE, 1 and 5 microg) significantly inhibited mitogen-driven proliferation of bovine lymphocytes, compared with 1 and 5 microg of identically prepared midgut extract (ANOVA, P < 0.05). Phytohemagglutinin A (PHA) stimulated lymphocyte responses were suppressed by 61.7 and 79.5% (mean values) with 1 and 5 microg of SCE, whereas concanvalin A (Con A) stimulated responses were suppressed by 62.9 and 77.1% (1 and 5 microg). In contrast, midgut extract (1 and 5 microg) minimally suppressed PHA (12.7% +/- 12.6 and 18.7% +/- 15.5) and Con A-driven responses (13.8% +/- 20.5 and 24.6% +/- 14.9), respectively. Viability studies using propidium iodide and flow cytometry demonstrated that SGE was not cytotoxic. Two-color immunofluorescence studies identified T and B lymphocytes as the nonviable cells in the cultures. Western blot analysis of serum collected from five dairy cows during periods of low and high fly exposure identified an immunodominant 27 kDa protein among the salivary gland proteins. These results indicate that exposure of cattle to stable fly saliva during blood feeding results in an antibody response to salivary proteins and that the saliva has a potential to modulate T lymphocyte function.

The objective of this study was to determine whether changes in the ileal intraepithelial lymphocyte (TEL) phenotype and function occurred prior to development of diarrhea in Cryptosporidium parvum-infected calves. Calves were orally inoculated with 10(8) oocysts and maintained in enteric pathogen-free conditions until their use in experiments. Age-matched uninfected calves were used for comparisons. Ileal IELs were isolated and phenotyped to determine whether changes in lymphocyte population dynamics had occurred by 3 days postinoculation (PI). Ex vivo reverse transcriptase-polymerase chain reaction of messenger ribonucleic acid (mRNA) from IELs from infected calves was compared with controls to determine whether changes in cytokine expression had occurred by 3 days PI. No significant changes in lymphocyte population dynamics were documented, however, IELs isolated from 4 out of 8 infected calves, but not from 8 out of 8 control calves, expressed mRNA for interleukin-10 (IL-10). IL-10 expression by IELs was associated with the expression of a significantly larger (P < 0.001) proportion (0.75) of monoclonal antibody-defined C. parvum epitopes within infected ileal epithelium, as compared with a much smaller proportion (0.30) of epitopes with IL-10 lymphocytes. The results suggest that a temporal association exists between the expression of IL-10 by ileal IELs and the expression of C. parvum antigens in infected calf epithelium prior to development of cryptosporidiosis.

This study was undertaken to characterize the mucosal response to Cryptosporidium parvum in infected calves that had recovered from diarrhea. Flow cytometric surface phenotypes of lamina propria lymphocyte (LPL) suspensions from infected calves and age-matched controls revealed the presence of a significantly larger proportion of CD25+ LPL in infected calves than in controls. Freshly isolated LPL from infected calves expressed more iNOS and interferon (IFN)-gamma than did controls. Infected calves excreted IgG1 and IgG2 isotype antibodies to C. parvum p23 by the end of the experiment. Moreover, immunohistochemistry of ileal sections revealed the presence of IgG1+ and IgG2+ B lymphocytes in the villi and IgG1+ but not IgG2+ B lymphocytes in continuous Peyer's patch nodules. These data are consistent with the emergence of a type-1-like mucosal immune response in terminal ileal mucosa as calves recover from cryptosporidiosis.

Fecal samples obtained at intervals from six calves with acute cryptosporidiosis contained antibodies of multiple isotypes to p23. IgM-, IgA-, and IgG(1)-isotype anti-p23 appeared before IgG(2)-isotype antibodies. All anti-p23 antibodies had declined by 2 months after infection. One calf that failed to shed oocysts following initial exposure developed IgG(1)-isotype anti-p23 antibodies. One calf that died following exposure to Cryptosporidium parvum oocysts lacked detectable anti-p23 antibodies. Re-inoculation with C. parvum resulted in a brief, marked recall response to p23.

Cryptosporidium parvum is an important zoonotic protozoan pathogen that causes acute infection and self-limiting gastrointestinal disease in neonatal calves. There are currently no consistently effective antimicrobials available to control cryptosporidiosis. Therefore, immunotherapeutic and vaccination protocols offer the greatest potential for long-term control of the disease. In order to devise effective control measures, it is important to better define mucosal immunity to C. parvum in young calves. This review summarizes the information that has accumulated over the last decade which helps to define the intestinal mucosal immune system in neonatal calves, and the events that occur in the intestinal mucosa after infection by C. parvum.

We have developed an assay to detect mucosally delivered antibody to Cryptosporidium parvum sporozoite antigens. We absorbed a recombinant 23-kD sporozoite protein to polystyrene microspheres, and used flow cytometry to detect, titer, and determine the isotype of antibody to p23 that was shed in the feces of experimentally infected calves. Noninoculated calves have low levels of mucosal antibody to p23, with IgG1 as the predominant isotype. Antibody titers rise in inoculated calves as the animals recover from cryptosporidiosis. A calf that was naturally protected from cryptosporidiosis had mucosal IgM and IgG1 isotype anti-p23 antibodies prior to challenge with C. parvum oocysts. Ten days after challenge, the calf had high titers of IgM, IgA, IgG1, and IgG2 anti-p23 antibodies. Together, the data show that this method can be used to assess mucosally delivered antibody to C. parvum.

We had previously shown that ileal intraepithelial lymphocytes isolated from calves with cryptosporidiosis include significantly increased numbers of CD8+ T lymphocytes and activated CD4+ cells. These increases could result from redistribution of resident mucosal lymphocytes or from homing of peripheral T cells to ileal mucosa. To determine whether resident mucosal lymphocytes can redistribute to Cryptosporidium parvum-infected epithelium, oocysts were inoculated in vitro onto ileum explants taken from 1-2-wk-old noninfected calves. After 24 hr of incubation, the explants were collected and frozen in liquid nitrogen. Immunohistochemical analysis of T-lymphocyte subpopulations was performed on sections, and labeled lymphocytes adjacent to villous epithelial cells were counted. Compared with uninoculated explants, there was a statistically significant increase in the number of CD8+ T lymphocytes per 100 epithelial cells in oocyst-inoculated tissue. In addition, there were increased numbers of CD4+ T cells and activated (CD25+) lymphocytes adjacent to C. parvum-infected epithelium. These results show that resident mucosal T lymphocytes can accumulate at the epithelium during C. parvum infection.

Ileal intraepithelial lymphocyte (IEL) suspensions from suckling calves (1-3 weeks old) and weaned calves (3-6 months old) were phenotyped to determine whether there were differences in the lymphocyte populations consistent with postnatal maturation of the mucosal immune system. Flow cytometric comparisons of IEL from the two age groups revealed the presence of significantly larger proportions of CD4+ T lymphocytes and CD8+ T cells in the weaned animals. In contrast, there was a significantly larger proportion of B-B2+ IEL in the suckling calves. Freshly isolated IEL from both groups of calves expressed mRNA for TNF-alpha and IFN-gamma, but not IL-4 or IL-10. The B-B2+ IEL population was more closely examined by flow cytometry. These cells co-expressed IgM and CD21. However, they did not express IgA, IgG1, nor any of several additional leukocyte differentiation molecules. Immunohistochemical data confirmed the presence of IgM+ lymphocytes, and the paucity of IgA+ and IgG1+ lymphocytes in suckling calf ileum. However, substantial numbers of IgA+ and IgG1+ cells were observed in weaned calf ileum. Together, the data are consistent with ongoing postnatal maturation of the gut mucosal immune system.

In the study reported here, we used RT-PCR with primers specific for interleukin-1 (IL-1), IL-6, IL-10, tumor necrosis factor-alpha (TNF-alpha), and nitric oxide synthase (iNOS) to assess the cytokine mRNA expression associated with bovine blood monocytes during their differentiation to macrophages cultured on plastic (1 week). In addition, we used RT-PCR to assess the contribution of gammadelta T cells as a source of interferon-gamma (IFN-gamma), the induction signal for iNOS. Further, we evaluated cytocentrifuge preparations from the cultures for the production of IL-10 using specific antibody. We previously demonstrated that iNOS can be induced in cultured bovine monocytes in response to IFN-gamma and TNF-alpha but lose this capability in a short period of time. However, we demonstrate here that iNOS induction from monocytes cultured with IFN-gamma secreting gammadelta T cells is prolonged, suggesting that this source of IFN-gamma primes the monocytes before exogenous stimulation. Based on mRNA expression, placement of monocytes in culture resulted in activation, followed by quiescence. By 6 days in culture, the iNOS message was reduced below the basal level. In addition, the TNF-alpha message was substantially reduced, and IL-1 and IL-6 messages were reduced below detectable levels. This correlated with an increase in IL-10 message. Downregulation of these same cytokine messages as well as IFN-gamma message occurred within a 20-h period when IL-10 was added exogenously to cultures of total leukocytes. At the same time, there was an increase in the number of IL-10-positive cells and an increase in the intensity of anti-IL-10 staining within adherent cells. These results provide evidence for IL-10 regulation of some bovine mononuclear phagocyte effector functions.

We describe the characterization of two subsets of bovine gamma delta T cells having distinct cell surface phenotype and tissue distribution. One population expresses the previously described 215,000 MW WC1 antigen and is negative for the cell-surface differentiation antigens CD2, CD4, and CD8. The second population expresses CD2 and CD8 but not WC1 and appears to have a T-cell receptor (TCR) rearrangement distinct from that of the WC1+ population. The WC1- population is found in large numbers in spleen and intestine. In addition, this subset is not recognized by a number of monoclonal antibodies (mAbs) specific for TCR families that are well represented in the WC1+ population. The results indicate that the gamma delta T-cell population in cattle is considerably larger than previously described and that this population can be subdivided into two distinct subsets based on cell-surface phenotype and tissue distribution.

The objective of this study was to identify disease-related changes in lymphocyte populations within ileal mucosae of calves with cryptosporidiosis. Groups of five neonatal calves were orally infected at 3 days of age with 10(8) oocysts and maintained in enteric-pathogen-free conditions until clinical disease was established or until the animals had recovered from disease. Age-matched uninfected calves were used for comparison. Ileal mucosal lymphocytes were collected, quantitated, and phenotyped to determine whether changes in lymphocyte composition occurred in infected animals. We observed significantly larger numbers of intraepithelial CD8+ T lymphocytes in ileal mucosae from acutely infected calves compared with those from control animals. In addition, a proportion of intraepithelial CD4+ T cells from acutely infected calves coexpressed CD25, whereas there was an absence of coexpressed CD25 on CD4+ T cells from control calves. Ex vivo reverse transcriptase PCR of RNA from intraepithelial lymphocytes from control calves showed a cytokine expression pattern consisting of tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma), while intraepithelial lymphocytes from calves with cryptosporidiosis expressed IFN-gamma but not TNF-alpha. Together, the results indicate that changes occur in the ileal intraepithelial lymphocyte population coincidently with Cryptosporidium parvum-induced enteric disease.

Microbicidal activity of reactive oxygen intermediates and reactive nitrogen intermediates has been described from both murine and human cytokine activated macrophages. An L-arginine-dependent pathway of nitric oxide generation has recently been described from bovine bone marrow-derived and monocyte-derived macrophages in response to a phagocytic stimulus. We have investigated the induction and release of both reactive oxygen intermediates and reactive nitrogen intermediates from bovine neutrophils, and blood and spleen mononuclear phagocytes in response to either a phagocytic or cytokine stimulus. Mononuclear phagocytes were poor producers of hydrogen peroxide (a measure of reactive oxygen intermediate production) under conditions that readily caused release by neutrophils. In contrast, nitrite, as a measure of nitric oxide production, could not be induced from neutrophils under any stimulation conditions, while mononuclear phagocytes responded to both a phagocytic stimulus and cytokines with the induction of nitric oxide synthase message and production of nitric oxide. There appeared to be two populations of monocytes that differed both in their adherent characteristics and their level of cytokine-induced nitric oxide production. Both populations stained with a single monoclonal antibody. However, the population that had not adhered to plastic within 3 h responded to cytokine stimulation, producing up to 3 times more nitric oxide on a per cell basis than the readily adherent population. Cytokine induction required the presence of interferon-gamma and either tumor necrosis factor-alpha or lipopolysaccharide. L-arginine dependence was demonstrated by inhibition with an L-arginine analog and restoration with addition of excess L-arginine.

Blood mononuclear cells (lymphocytes and monocytes) were isolated from infected calves during in vivo control of acute anaplasmosis and cultured with Anaplasma marginale organisms. Supernatants from the cultures reduced the proportion of erythrocytes containing viable A. marginale in vitro, indicating that an antibody-independent mechanism of rickettsemia control might occur during acute anaplasmosis.

gamma delta T cells in ruminants can be subdivided in two or more subpopulations on the basis of the expression of surface antigen WC1, which can exist in different isoforms. In this study, 18 monoclonal antibodies (mAbs) submitted to the Third International Workshop that were predicted to react with gamma delta TcR molecules were analysed and expression of their antigens was investigated on the different gamma delta T cell subpopulations. A set of control mAbs positive for TcR1 (86D), BoCD3 (MM1A), WC1 (B7A1, BAQ4A, CACTB32A, and BAQ89A) was included for comparative studies. Previous investigations demonstrated eight of the mAbs immunoprecipitated peptides with apparent M(r)s of 37 and/or 47 kDa, indicating they recognized determinants on the T cell receptor, TcR1. Two color flow cytometric analyses in the present study demonstrated the mAbs formed three groups; group 1, a set of mAbs that recognize TcR1 determinants expressed on all gamma delta T cells and groups 2 and 3, sets of mAbs that recognize TcR1 determinants on some gamma delta T cells: TcR1-N6 and TcR1-N7 respectively. mAbs from the latter groups define families of TcR1 molecules that express either one or both of the determinants. These antigenically distinct forms of TcR1 are expressed in equal proportion on the two gamma delta T cell populations that express one of the mutually exclusive isoforms of WC1, WC1-N3 and WC1-N4. The data indicate usage of the mAb-defined families of the gamma delta TcR is primarily restricted to the WC1+ subpopulation of gamma delta T cells. However, a small subpopulation of CD2+, WC1- gamma delta T cells expresses a form of TcR1 positive for the determinant TcR1-N6.

Products released from activated macrophages have been demonstrated to have microbicidal activity against a variety of microorganisms. Reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) have been shown to affect the induction of degenerate (crisis) forms of Plasmodium spp. Polyamines are degraded into acrolein which has also been shown to be toxic to Plasmodium spp. We have investigated the possibility that these products act similarly with Babesia bovis. Crisis forms of B. bovis developed in erythrocyte cultures after the introduction of supernatants containing ROI, RNI, and acrolein. Xanthine degradation by xanthine oxidase leads to the formation of superoxide anion, hydrogen peroxide, and hydroxyl radicals. The degradation in the presence of B. bovis was toxic to the parasite. The toxicity was partially reversed by the addition of the ROI scavenger catalase. However, H2O2 added directly had little effect, suggesting a role for the other ROI products. Spermine degradation by polyamine oxidase and direct addition of acrolein was toxic in a dose-dependent manner. Finally, spontaneous generation of nitric oxide from sodium nitroprusside or S-nitroso-N-acetyl-penicillamine was also toxic in a dose-dependent manner. These data lead us to suggest a role for activated macrophages in the primary immune response against B. bovis.

Ileal intraepithelial and lamina propria lymphocytes from newborn, 1.5-week-old, and 3-week-old calves were compared to determine to what extent the mucosa becomes populated after birth. Single and dual fluorescence flow cytometry were used with monoclonal antibodies to bovine (Bo) CD molecules to identify lymphocyte subpopulations. Few ileal mucosal lymphocytes were present in calves at birth. However, by 1.5 weeks of age, the villi were populated with large numbers of lymphocytes, and by 3 weeks of age, the numbers had increased further. These included a prominent subpopulation of gamma delta T cells. Several subsets of gamma delta T cells populated ileal mucosa after birth. The predominant subset coexpressed BoCD2, and a smaller subset coexpressed BoCD8. WC1+ gamma delta T cells comprised the smallest subset. All gamma delta T cell subsets coexpressed ACT2, a molecule expressed on activated WC1+ and WC1- gamma delta T cells from cattle.

A panel of monoclonal antibodies to bovine leukocyte differentiation molecules was used to evaluate peripheral blood and splenic lymphocytes from cattle of various ages. The major population of peripheral blood lymphocytes from neonatal calves was gamma delta T-cell receptor (TCR1) positive, as determined by TCR1-N12 expression. TCR1-N12+ lymphocytes were decreased in number in older calves, and were lowest in adult cattle. The major subpopulation of TCR1-N12+ cells from peripheral blood coexpressed WC1, but not BoCD2. A small subpopulation of peripheral blood TCR1-N12+ cells from cattle of all ages coexpressed BoCD2, but not WC1. The TCR1-N12+ BoCD2+ lymphocytes made up the largest TCR1-N12+ lymphocyte subpopulation in spleens of both calves and adults. The TCR1-N12+WC1+ splenic lymphocytes were present as a small population. The data indicate that two subpopulations of TCR1+ lymphocytes are present in cattle of all ages. These two subpopulations are differentially distributed between blood and spleen, with TCR1-N12+WC1+ lymphocytes predominating in blood, and TCR1-N12+BoCD2+ cells predominating in spleen.

A monoclonal antibody (mAb), MM1A, that identifies a molecule expressed on a large percentage of bovine lymphocytes (60-80%) was examined to determine its specificity. Two and three colour immunofluorescence analysis using flow cytometry revealed the molecule is highly expressed on all CD4+ and CD8+ lymphocytes and lymphocytes that express WC1 and the gamma/delta TCR. In contrast to mAbs reactive with BoCD5, MM1A did not react with B lymphocytes. Biochemical analysis revealed that the mAb immunoprecipitates a molecular complex comprising a set of peptides with M(r) approximately 12, 16, 22, 32, 36, and 44 kDa under reducing conditions and an additional 96 kDa peptide complex under non-reducing conditions. The data indicate that MM1A recognizes the bovine orthologue of CD3 (BoCD3).

Fluorescence flow cytometry was employed to assess the potential of a vital dye, hydroethidine, for use in the detection and monitoring of the viability of hemoparasites in infected erythrocytes, using Babesia bovis as a model parasite. The studies demonstrated that hydroethidine is taken up by B. bovis and metabolically converted to the DNA binding fluorochrome, ethidium. Following uptake of the dye, erythrocytes containing viable parasites were readily distinguished and quantitated. Timed studies with the parasiticidal drug, Ganaseg, showed that it is possible to use the fluorochrome assay to monitor the effects of the drug on the rate of replication and viability of B. bovis in culture. The assay provides a rapid method for evaluation of the in vitro effect of drugs on hemoparasites and for analysis of the effect of various components of the immune response, such as lymphokines, monocyte products, antibodies, and effector cells (T, NK, LAK, ADCC) on the growth and viability of intraerythrocytic parasites.