Insights into the differential proteome landscape of a newly isolated Paramecium multimicronucleatum in response to cadmium stress.

Employing microbial systems for the bioremediation of contaminated waters represent a potential option, however, limited understanding of the underlying mechanisms hampers the implication of microbial-mediated bioremediation. The omics tools offer a promising approach to explore the molecular basis of the bioremediation process. Here, a mass spectrometry-based quantitative proteome profiling approach was conducted to explore the differential protein levels in cadmium-treated Paramecium multimicronucleatum. The Proteome Discoverer software was used to identify and quantify differentially abundant proteins. The proteome profiling generated 7,416 peptide spectral matches, yielding 2824 total peptides, corresponding to 989 proteins. The analysis revealed that 29 proteins exhibited significant (p ≤ 0.05) differential levels, including a higher abundance of 6 proteins and reduced levels of 23 proteins in Cd2+ treated samples. These differentially abundant proteins were associated with stress response, energy metabolism, protein degradation, cell growth, and hormone processing. Briefly, a comprehensive proteome profile in response to cadmium stress of a newly isolated Paramecium has been established that will be useful in future studies identifying critical proteins involved in the bioremediation of metals in ciliates. SIGNIFICANCE: Ciliates are considered a good biological indicator of chemical pollution and relatively sensitive to heavy metal contamination. A prominent ciliate, Paramecium is a promising candidate for the bioremediation of polluted water. The proteins related to metal resistance in Paramecium species are still largely unknown and need further exploration. In order to identify and reveal the proteins related to metal resistance in Paramecia, we have reported differential protein abundance in Paramecium multimicronucleatum in response to cadmium stress. The proteins found in our study play essential roles during stress response, hormone processing, protein degradation, energy metabolism, and cell growth. It seems likely that Paramecia are not a simple sponge for metals but they could also transform them into less toxic derivatives or by detoxification by protein binding. This data will be helpful in future studies to identify critical proteins along with their detailed mechanisms involved in the bioremediation and detoxification of metal ions in Paramecium species.

Proteomic changes in the hippocampus of large mammals after total-body low dose radiation.

There is a growing interest in low dose radiation (LDR) to counteract neurodegeneration. However, LDR effects on normal brain have not been completely explored yet. Recent analyses showed that LDR exposure to normal brain tissue causes expression level changes of different proteins including neurodegeneration-associated proteins. We assessed the proteomic changes occurring in radiated vs. sham normal swine brains. Due to its involvement in various neurodegenerative processes, including those associated with cognitive changes after high dose radiation exposure, we focused on the hippocampus first. We observed significant proteomic changes in the hippocampus of radiated vs. sham swine after LDR (1.79Gy). Mass spectrometry results showed 190 up-regulated and 120 down-regulated proteins after LDR. Western blotting analyses confirmed increased levels of TPM1, TPM4, PCP4 and NPY (all proteins decreased in various neurodegenerative processes, with NPY and PCP4 known to be neuroprotective) in radiated vs. sham swine. These data support the use of LDR as a potential beneficial tool to interfere with neurodegenerative processes and perhaps other brain-related disorders, including behavioral disorders.

Spinal lymphoma in a goat.

A 3-year-old Pygmy Wether was presented for chronic hindlimb paralysis. A neurological exam revealed nonambulatory paraplegia with absent deep pain nociception, lack of hindlimb withdrawal reflexes, and paraspinal pain on palpation with T3 to L3 neurolocalization. MRI of the lumbar spine revealed an extensive, dorsal to dorsolateral, severely compressive, heterogeneously contrast-enhancing extradural lesion of the lumbar spine with intervertebral foraminal extension into the surrounding paraspinal musculature. Vertebral bone marrow involvement was also noted in the L5 and L6 vertebrae. A diagnosis of lymphoma was obtained after cytological sampling. This is the first case report describing specific MRI findings (signal characteristics, enhancement pattern, and perilesional changes) in a goat with paraspinal lymphoma.

NEDD4L intramolecular interactions regulate its auto and substrate NaV1.5 ubiquitination.

NEDD4L is a HECT-type E3 ligase that catalyzes the addition of ubiquitin to intracellular substrates such as the cardiac voltage-gated sodium channel, NaV1.5. The intramolecular interactions of NEDD4L regulate its enzymatic activity which is essential for proteostasis. For NaV1.5, this process is critical as alterations in Na+ current is involved in cardiac diseases including arrhythmias and heart failure. In this study, we perform extensive biochemical and functional analyses that implicate the C2 domain and the first WW-linker (1,2-linker) in the autoregulatory mechanism of NEDD4L. Through in vitro and electrophysiological experiments, the NEDD4L 1,2-linker was determined to be important in substrate ubiquitination of NaV1.5. We establish the preferred sites of ubiquitination of NEDD4L to be in the second WW-linker (2,3-linker). Interestingly, NEDD4L ubiquitinates the cytoplasmic linker between the first and second transmembrane domains of the channel (DI-DII) of NaV1.5. Moreover, we design a genetically encoded modulator of Nav1.5 that achieves Na+ current reduction using the NEDD4L HECT domain as cargo of a NaV1.5-binding nanobody. These investigations elucidate the mechanisms regulating the NEDD4 family and furnish a new molecular framework for understanding NaV1.5 ubiquitination.

Tympanic membrane perforations cannot be reliably detected using computed tomography based on 15 cadaver dogs.

The integrity of the tympanic membrane is an important factor when deciding treatment and therapeutic recommendations for dogs with ear disease; however, otoscopic examination may be difficult to perform due to features of external ear canal disease or patient compliance. CT is useful for the evaluation of middle ear disease, including cases in which middle ear disease is detected incidentally. The tympanic membrane is detectable using CT, but anecdotally, apparent focal defects or discontinuities of the tympanic membrane are often seen in patients with and without ear disease. The purpose of this prospective, observer agreement study was to determine if perforations of the tympanic membrane are reliably detectable on CT. Fifteen cadaver dogs underwent CT and video otoscopy to verify the integrity of each tympanic membrane. Cadavers were randomly assigned to have the tympanic membranes left intact or to undergo a myringotomy on either the left, the right, or both sides. CT was performed immediately following the myringotomies. Four blinded evaluators evaluated the pre- and post-myringotomy scans for a total of 30 scans (60 tympanic membranes). Average accuracy was low (44%), and interobserver agreement for all four evaluators was fair. Although the tympanic membrane is visible on CT, perforations of the tympanic membrane are unlikely to be accurately detected or excluded. The appearance of an intact tympanic membrane or defect in the membrane on CT should not be used as criteria to guide clinical treatment recommendations based on this cadaver model.

Comparison of atlantoaxial and lumbosacral cerebrospinal fluid centesis techniques in South American camelids.

BACKGROUND: Iatrogenic blood contamination during cerebrospinal fluid (CSF) centesis is common, which can limit the diagnostic usefulness of the sample. A novel ultrasound-guided CSF collection technique is described in horses, by which CSF is obtained from the atlantoaxial (AA) space. HYPOTHESIS/OBJECTIVES: To compare ultrasound-guided AA centesis with lumbosacral (LS) centesis in South American camelids (SAC). The hypotheses were that AA centesis would yield samples with less blood contamination although being technically more challenging than LS centesis. ANIMALS: Eight clinically healthy adult SAC from a university-owned teaching herd. METHODS: Single-blinded, randomized, 4-way, 4-period crossover study in which 2 veterinarians each performed both centesis techniques on each animal once. Cytological sample analysis was performed, and the technical difficulty of sample acquisition was assessed. RESULTS: The CSF was collected successfully and without complications by either technique during all collection attempts. Aspects of technical difficulty and concentrations of CSF analytes did not vary significantly between techniques. Median total nucleated cell and red blood cell counts were 1/µL and 0.5/µL and 167.5/µL and 155/µL for AA and LS techniques, respectively. The median total protein concentration was 32.9 mg/dL and 38 mg/dL for AA and LS centeses. A median of 1 attempt was necessary for both centesis techniques and the median number of needle repositioning events was 1 for AA and 0 for LS. CONCLUSION AND CLINICAL IMPORTANCE: Depending on clinical circumstances, ultrasound-guided AA centesis appears to be an acceptable alternative to other techniques for collection of CSF from SAC.

The role of N-terminal phosphorylation of DGK-θ.

Diacylglycerol kinases (DGKs) are lipid kinases that mediate the phosphorylation of diacylglycerol (DAG) leading to the production of phosphatidic acid (PtdOH). To examine the role of phosphorylation on DGK-θ, we first identified the phosphorylated sites on endogenous DGK-θ from mouse brain and found four sites: S15, S17, which we refer to phosphomotif-1 sites, and S22 and S26 which we refer to as phosphomotif-2 sites. This study focused on the role of these phosphorylated sites on enzyme activity, membrane binding, thermal stability, and cellular half-life of DGK-θ. After generating a construct devoid of all non-catalytic phosphorylation sites (4A), we also generated other constructs to mimic phosphorylation of these residues by mutating them to glutamate (E). Our data demonstrate that an increase in membrane affinity requires the phosphorylation of all four endogenous sites as the phosphomimetic 4E but not other phosphomimietics. Furthermore, 4E also shows an increase in basal activity as well as an increase in the Syt1-induced activity compared to 4A. It is noteworthy that these phosphorylations had no effect on the thermal stability or cellular half-life of this enzyme. Interestingly, when only one phosphorylation domain (phosphomotif-1 or phosphomotif-2) contained phosphomimetics (S15E/S17E or S22E/S26E), the basal activity was also increased but membrane binding affinity was not increased. Furthermore, when only one residue in each domain mimicked an endogenous phosphorylated serine (S15E/S22E or S17E/S26E), the Syt1-induced activity as well as membrane binding affinity decreased relative to 4A. These results indicate that these endogenous phosphorylation sites contribute differentially to membrane binding and enzymatic activity.

Computed tomographic characteristics of confirmed and presumed noncutaneous pythiosis in 25 dogs.

Pythium insidiosum is an aquatic oomycete that causes granulomatous infection in dogs, most commonly cutaneous and gastrointestinal. Ultrasonographic characteristics of gastrointestinal pythiosis have been described; occasionally, CT is utilized in the clinical setting, and CT features of pythiosis have not been published. The purpose of this retrospective, multicenter, descriptive study is to describe CT characteristics of noncutaneous canine pythiosis. The following CT parameters were recorded: lesion anatomic location, number, shape, margination, size, attenuation pre- and postcontrast, enhancement pattern, lymph nodes affected, other lesions identified, and presence of peritoneal effusion or steatitis. Descriptive statistics demonstrating the frequency of lesion appearances were performed. Twenty-five dogs with noncutaneous pythiosis lesions that underwent CT were included; 19 had primarily gastrointestinal infections, four primarily arterial infections, one intrathoracic and intra-abdominal infection, and one primary pulmonary infection. In dogs with primary gastrointestinal infection, lesions were most common at the ileocolic junction and were most frequently focal, well-defined, moderate to marked circumferential wall thickening that was homogeneous and smoothly marginated precontrast, with moderate heterogeneous contrast enhancement. Most dogs had involvement of multiple gastrointestinal regions. Of four dogs with primary arterial involvement, three had large aneurysmal dilatations of the cranial mesenteric artery with severe mural thickening. All dogs had regional lymphadenopathy, which was variable but generally mild. Nine dogs had peritoneal effusion; six dogs had steatitis. CT features of pythiosis can overlap with neoplasia, but pythiosis should be considered as a differential, especially in young dogs. Findings supported using CT as an adjunct imaging test for increasing clinical suspicion of noncutaneous pythiosis.

Proteomic Changes in the Hippocampus after Repeated Explosive-Driven Blasts.

Repeated blast-traumatic brain injury (blast-TBI) has been hypothesized to cause persistent and unusual neurological and psychiatric symptoms in service members returning from war zones. Blast-wave primary effects have been supposed to induce damage and molecular alterations in the brain. However, the mechanisms through which the primary effect of an explosive-driven blast wave generate brain lesions and induce brain consequences are incompletely known. Prior findings from rat brains exposed to two consecutive explosive-driven blasts showed molecular changes (hyperphosphorylated-Tau, AQP4, S100ß, PDGF, and DNA-polymerase-ß) that varied in magnitude and direction across different brain regions. We aimed to compare, in an unbiased manner, the proteomic profile in the hippocampus of double blast vs sham rats using mass spectrometry (MS). Data showed differences in up- and down-regulation for protein abundances in the hippocampus of double blast vs sham rats. Tandem mass tag (TMT)-MS results showed 136 up-regulated and 94 down-regulated proteins between the two groups (10.25345/C52B8VP0X). These TMT-MS findings revealed changes never described before in blast studies, such as increases in MAGI3, a scaffolding protein at cell-cell junctions, which were confirmed by Western blotting analyses. Due to the absence of behavioral and obvious histopathological changes as described in our previous publications, these proteomic data further support the existence of an asymptomatic blast-induced molecular altered status (ABIMAS) associated with specific protein changes in the hippocampus of rats repeatedly expsosed to blast waves generated by explosive-driven detonations.

Plasma proteomic analysis on neuropathic pain in idiopathic peripheral neuropathy patients.

BACKGROUND AND AIMS: Why only half of the idiopathic peripheral neuropathy (IPN) patients develop neuropathic pain remains unknown. By conducting a proteomics analysis on IPN patients, we aimed to discover proteins and new pathways that are associated with neuropathic pain. METHODS: We conducted unbiased mass-spectrometry proteomics analysis on blood plasma from 31 IPN patients with severe neuropathic pain and 29 IPN patients with no pain, to investigate protein biomarkers and protein-protein interactions associated with neuropathic pain. Univariate modeling was done with linear mixed modeling (LMM) and corrected for multiple testing. Multivariate modeling was performed using elastic net analysis and validated with internal cross-validation and bootstrapping. RESULTS: In the univariate analysis, 73 proteins showed a p-value <.05 and 12 proteins showed a p-value <.01. None were significant after Benjamini-Hochberg adjustment for multiple testing. Elastic net analysis created a model containing 12 proteins with reasonable discriminatory power to differentiate between painful and painless IPN (false-negative rate 0.10, false-positive rate 0.18, and an area under the curve 0.75). Eight of these 12 proteins were clustered into one interaction network, significantly enriched for the complement and coagulation pathway (Benjamini-Hochberg adjusted p-value = .0057), with complement component 3 (C3) as the central node. Bootstrap validation identified insulin-like growth factor-binding protein 2 (IGFBP2), complement factor H-related protein 4 (CFHR4), and ferritin light chain (FTL), as the most discriminatory proteins of the original 12 identified. INTERPRETATION: This proteomics analysis suggests a role for the complement system in neuropathic pain in IPN.

The C-Terminal of NaV1.7 Is Ubiquitinated by NEDD4L.

NaV1.7, the neuronal voltage-gated sodium channel isoform, plays an important role in the human body's ability to feel pain. Mutations within NaV1.7 have been linked to pain-related syndromes, such as insensitivity to pain. To date, the regulation and internalization mechanisms of the NaV1.7 channel are not well known at a biochemical level. In this study, we perform biochemical and biophysical analyses that establish that the HECT-type E3 ligase, NEDD4L, ubiquitinates the cytoplasmic C-terminal (CT) region of NaV1.7. Through in vitro ubiquitination and mass spectrometry experiments, we identify, for the first time, the lysine residues of NaV1.7 within the CT region that get ubiquitinated. Furthermore, binding studies with an NEDD4L E3 ligase modulator (ubiquitin variant) highlight the dynamic partnership between NEDD4L and NaV1.7. These investigations provide a framework for understanding how NEDD4L-dependent regulation of the channel can influence the NaV1.7 function.

Native lamin A/C proteomes and novel partners from heart and skeletal muscle in a mouse chronic inflammation model of human frailty.

Clinical frailty affects ∼10% of people over age 65 and is studied in a chronically inflamed (Interleukin-10 knockout; "IL10-KO") mouse model. Frailty phenotypes overlap the spectrum of diseases ("laminopathies") caused by mutations in LMNA. LMNA encodes nuclear intermediate filament proteins lamin A and lamin C ("lamin A/C"), important for tissue-specific signaling, metabolism and chromatin regulation. We hypothesized that wildtype lamin A/C associations with tissue-specific partners are perturbed by chronic inflammation, potentially contributing to dysfunction in frailty. To test this idea we immunoprecipitated native lamin A/C and associated proteins from skeletal muscle, hearts and brains of old (21-22 months) IL10-KO versus control C57Bl/6 female mice, and labeled with Tandem Mass Tags for identification and quantitation by mass spectrometry. We identified 502 candidate lamin-binding proteins from skeletal muscle, and 340 from heart, including 62 proteins identified in both tissues. Candidates included frailty phenotype-relevant proteins Perm1 and Fam210a, and nuclear membrane protein Tmem38a, required for muscle-specific genome organization. These and most other candidates were unaffected by IL10-KO, but still important as potential lamin A/C-binding proteins in native heart or muscle. A subset of candidates (21 in skeletal muscle, 30 in heart) showed significantly different lamin A/C-association in an IL10-KO tissue (p < 0.05), including AldoA and Gins3 affected in heart, and Lmcd1 and Fabp4 affected in skeletal muscle. To screen for binding, eleven candidates plus prelamin A and emerin controls were arrayed as synthetic 20-mer peptides (7-residue stagger) and incubated with recombinant purified lamin A "tail" residues 385-646 under relatively stringent conditions. We detected strong lamin A binding to peptides solvent exposed in Lmcd1, AldoA, Perm1, and Tmem38a, and plausible binding to Csrp3 (muscle LIM protein). These results validated both proteomes as sources for native lamin A/C-binding proteins in heart and muscle, identified four candidate genes for Emery-Dreifuss muscular dystrophy (CSRP3, LMCD1, ALDOA, and PERM1), support a lamin A-interactive molecular role for Tmem38A, and supported the hypothesis that lamin A/C interactions with at least two partners (AldoA in heart, transcription factor Lmcd1 in muscle) are altered in the IL10-KO model of frailty.

Ultrasonographic diagnosis of a lymphomatous lymph node presumptively infiltrating the ureter causing hydronephrosis in a 3-year-old dog.

A 3-year-old male neutered mixed breed dog was presented for chronic vomiting and diarrhea. Abdominal ultrasound revealed a large amorphous, heterogeneous mass within the left mid to caudal abdomen most consistent with medial iliac lymph node. It appeared to invade the left ureter and extend distally causing ureteral obstruction and hydronephrosis. Concurrent additional ultrasound findings were consistent with metastatic or multicentric neoplasia. Fine needle aspirates of the lymph node and spleen both confirmed large cell lymphoma. These findings present evidence of lymphoma invading directly from an organ into the ureter which has not previously been reported in dogs.

α-lipoic acid ameliorates consequences of copper overload by up-regulating selenoproteins and decreasing redox misbalance.

α-lipoic acid (LA) is an essential cofactor for mitochondrial dehydrogenases and is required for cell growth, metabolic fuel production, and antioxidant defense. In vitro, LA binds copper (Cu) with high affinity and as an endogenous membrane permeable metabolite could be advantageous in mitigating the consequences of Cu overload in human diseases. We tested this hypothesis in 3T3-L1 preadipocytes with inactivated Cu transporter Atp7a; these cells accumulate Cu and show morphologic changes and mitochondria impairment. Treatment with LA corrected the morphology of Atp7a-/- cells similar to the Cu chelator bathocuproinedisulfonate (BCS) and improved mitochondria function; however, the mechanisms of LA and BCS action were different. Unlike BCS, LA did not decrease intracellular Cu but instead increased selenium levels that were low in Atp7a-/- cells. Proteome analysis confirmed distinct cell responses to these compounds and identified upregulation of selenoproteins as the major effect of LA on preadipocytes. Upregulation of selenoproteins was associated with an improved GSH:GSSG ratio in cellular compartments, which was lowered by elevated Cu, and reversal of protein oxidation. Thus, LA diminishes toxic effects of elevated Cu by improving cellular redox environment. We also show that selenium levels are decreased in tissues of a Wilson disease animal model, especially in the liver, making LA an attractive candidate for supplemental treatment of this disease.

Implementation and impact of a point of care electroencephalography platform in a community hospital: a cohort study.

Objective: To determine the clinical and financial feasibility of implementing a poc-EEG system in a community hospital. Design: Data from a prospective cohort displaying abnormal mentation concerning for NCSE or rhythmic movements due to potential underlying seizure necessitating EEG was collected and compared to a control group containing patient data from 2020. Setting: A teaching community hospital with limited EEG support. Patients: The study group consisted of patients requiring emergent EEG during hours when conventional EEG was unavailable. Control group is made up of patients who were emergently transferred for EEG during the historical period. Interventions: Application and interpretation of Ceribell®, a poc-EEG system. Measurement and main results: 88 patients were eligible with indications for poc-EEG including hyperkinetic movements post-cardiac arrest (19%), abnormal mentation after possible seizure (46%), and unresponsive patients with concern for NCSE (35%). 21% had seizure burden on poc-EEG and 4.5% had seizure activity on follow-up EEG. A mean of 1.1 patients per month required transfer to a tertiary care center for continuous EEG. For the control period, a total of 22 patients or a mean of 2 patients per month were transferred for emergent EEG. Annually, we observed a decrease in the number of transferred patients in the post-implementation period by 10.8 (95% CI: -2.17-23.64, p = 0.1). Financial analysis of the control found the hospital system incurred a loss of $3,463.11 per patient transferred for an annual loss of $83,114.64. In the study group, this would compute to an annual loss of $45,713.05 for an overall decrease in amount lost of $37,401.59. We compared amount lost per patient between historical controls and study patients. Implementation of poc-EEG resulted in an overall decrease in annual amount lost of $37,401.59 by avoidance of transfer fees. We calculated the amount gained per patient in the study group to be $13,936.44. To cover the cost of the poc-EEG system, 8.59 patients would need to avoid transfer annually. Conclusion: A poc-EEG system can be safely implemented in a community hospital leading to an absolute decrease in transfers to tertiary hospital. This decrease in patient transfers can cover the cost of implementing the poc-EEG system. The additional benefits from transfer avoidance include clinical benefits such as rapid appropriate treatment of seizures and avoidance of unnecessary treatment as well as negating transfer risk and keeping the patient at their local hospital.

Arginine methylation of RNA-binding proteins is impaired in Huntington's disease.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion in the HD gene, coding for huntingtin protein (HTT). Mechanisms of HD cellular pathogenesis remain undefined and likely involve disruptions in many cellular processes and functions presumably mediated by abnormal protein interactions of mutant HTT. We previously found HTT interaction with several protein arginine methyl-transferase (PRMT) enzymes. Protein arginine methylation mediated by PRMT enzymes is an important post-translational modification with an emerging role in neurodegeneration. We found that normal (but not mutant) HTT can facilitate the activity of PRMTs in vitro and the formation of arginine methylation complexes. These interactions appear to be disrupted in HD neurons. This suggests an additional functional role for HTT/PRMT interactions, not limited to substrate/enzyme relationship, which may result in global changes in arginine protein methylation in HD. Our quantitative analysis of striatal precursor neuron proteome indicated that arginine protein methylation is significantly altered in HD. We identified a cluster highly enriched in RNA-binding proteins with reduced arginine methylation, which is essential to their function in RNA processing and splicing. We found that several of these proteins interact with HTT, and their RNA-binding and localization are affected in HD cells likely due to a compromised arginine methylation and/or abnormal interactions with mutant HTT. These studies reveal a potential new mechanism for disruption of RNA processing in HD, involving a direct interaction of HTT with methyl-transferase enzymes and modulation of their activity and highlighting methylation of arginine as potential new therapeutic target for HD.

Differential Detection of O-GlcNAcylated proteins in the heart using antibodies.

Thousands of mammalian intracellular proteins are dynamically modified by O-linked ß-N-acetylglucosamine (O-GlcNAc). Global changes in O-GlcNAcylation have been associated with the development of cardiomyopathy, heart failure, hypertension, and neurodegenerative disease. Levels of O-GlcNAc in cells and tissues can be detected using numerous approaches; however, immunoblotting using GlcNAc-specific antibodies and lectins is commonplace. The goal of this study was to optimize the detection of O-GlcNAc in heart lysates by immunoblotting. Using a combination of tissue fractionation, immunoblotting, and galactosyltransferase labeling, as well as hearts from wild-type and O-GlcNAc transferase transgenic mice, we demonstrate that contractile proteins in the heart are differentially detected by two commercially available antibodies (CTD110.6 and RL2). As CTD110.6 displays poor reactivity toward contractile proteins, and as these proteins represent a major fraction of the heart proteome, a better assessment of cardiac O-GlcNAcylation is obtained in total tissue lysates with RL2. The data presented highlight tissue lysis approaches that should aid the assessment of the cardiac O-GlcNAcylation by immunoblotting.

The Interaction of Borrelia burgdorferi with Human Dendritic Cells: Functional Implications.

Dendritic cells bridge the innate and adaptive immune responses by serving as sensors of infection and as the primary APCs responsible for the initiation of the T cell response against invading pathogens. The naive T cell activation requires the following three key signals to be delivered from dendritic cells: engagement of the TCR by peptide Ags bound to MHC molecules (signal 1), engagement of costimulatory molecules on both cell types (signal 2), and expression of polarizing cytokines (signal 3). Initial interactions between Borrelia burgdorferi, the causative agent of Lyme disease, and dendritic cells remain largely unexplored. To address this gap in knowledge, we cultured live B. burgdorferi with monocyte-derived dendritic cells (mo-DCs) from healthy donors to examine the bacterial immunopeptidome associated with HLA-DR. In parallel, we examined changes in the expression of key costimulatory and regulatory molecules as well as profiled the cytokines released by dendritic cells when exposed to live spirochetes. RNA-sequencing studies on B. burgdorferi-pulsed dendritic cells show a unique gene expression signature associated with B. burgdorferi stimulation that differs from stimulation with lipoteichoic acid, a TLR2 agonist. These studies revealed that exposure of mo-DCs to live B. burgdorferi drives the expression of both pro- and anti-inflammatory cytokines as well as immunoregulatory molecules (e.g., PD-L1, IDO1, Tim3). Collectively, these studies indicate that the interaction of live B. burgdorferi with mo-DCs promotes a unique mature DC phenotype that likely impacts the nature of the adaptive T cell response generated in human Lyme disease.

Multicenter study of universal prophylaxis versus pre-emptive therapy for patients at intermediate risk (R+) for CMV following heart transplantation.

INTRODUCTION: Heart transplant (HT) recipients with prior exposure to cytomegalovirus (CMV R+) are considered intermediate risk for CMV-related complications. Consensus guidelines allow for either universal prophylaxis (UP) or preemptive therapy (PET) (serial CMV testing) approaches to CMV prevention in such patients. Whether an optimal approach to mitigate CMV related risks exists in this setting remains uncertain. We therefore assessed the utility of PET as compared to UP in CMV R+ HT recipients. METHODS: Retrospective analysis of all CMV R+ HT recipients from 6 U.S. centers between 2010 and 2018 was performed. The primary outcome was the development of CMV DNAemia or end-organ disease resulting in the initiation/escalation of anti-CMV therapy. The secondary outcome was CMV-related hospitalization. Additional outcomes included incidence of acute cellular rejection (ACR) ≥ grade 2R, death, cardiac allograft vasculopathy (CAV), and leukopenia. RESULTS: Of 563 CMV R+ HT recipients, 344 (61.1%) received UP. PET was associated with increased risk for the primary (adjusted HR 3.95, 95% CI: 2.65-5.88, p < .001) and secondary (adjusted HR 3.19, 95% CI: 1.47-6.94, p = .004) outcomes, and with increased ACR ≥ grade 2R (PET 59.4% vs. UP 34.4%, p < .001). Incidence of detectable CAV was similar at 1 year (PET 8.2% vs. UP 9.5%, p = .698). UP was associated with increased incidence of leukopenia within 6 months post-HT (PET 34.7% vs. UP 43.6%, p = .036). CONCLUSION: The use of a PET CMV prophylaxis strategy in intermediate risk HT recipients associated with increased risk of CMV infection and CMV-related hospitalization, and may associate with worse post-HT graft outcomes.

Three year post heart transplant outcomes of desensitized durable mechanical circulatory support patients.

BACKGROUND: The risks and benefits of desensitization therapy (DST) in highly sensitized mechanical circulatory support (MCS) patients are not well known. We investigated 3 year post-transplant outcomes of desensitized durable MCS patients. METHODS: Among 689 consecutively enrolled heart transplantation recipients between 2010 and 2016, we categorized them into Group A (desensitized MCS patients, n = 21), Group B (desensitized non-MCS patients, n = 28) and Group C (all nondesensitized patients, n = 640). Post-transplant outcomes included the incidence of primary graft dysfunction, 3-year survival, freedom from cardiac allograft vasculopathy, nonfatal major adverse cardiac events, any treated rejection, acute cellular rejection, antibody mediated rejection (AMR) and infectious complications. RESULTS: The types of DST in Groups A and B were similar and included combinations of rituximab/intravenous immunoglobulin and plasmapheresis/bortezomib. Group A, compared with Group B, showed significantly higher pre-DST panel reactive antibody (PRA) (92.2 ± 9.8 vs. 83.3 ± 15.6, P = 0.007) and higher PRA reduction after DST (-22.2 ± 26.9 vs. -6.3 ± 7.5, P = 0.015). Groups A and C showed comparable primary graft dysfunction, 3-year survival, freedom from cardiac allograft vasculopathy, nonfatal major adverse cardiac events, any treated rejection, acute cellular rejection, and AMR. Although statistically not significant, Group A showed numerically higher 3-year freedom from AMR than Group B. Infectious complications were similar in both Groups A and B. CONCLUSIONS: DST for MCS patients showed significant PRA reduction, resulting in an expansion of the donor pool. The post-transplant outcome of desensitized MCS patients showed comparable clinical outcomes to non-desensitized control patients in the same study period, revealing the safety and efficacy of DST.