Intravenous Challenge Research Articles

A novel murine wire injury based infectious endocarditis model aggravates aortic valve stenosis

Abstract Background With mortality rates up to 40% under optimal medical treatment, infectious endocarditis (IE) remains one of the deadliest infectious diseases worldwide. In the early stages of IE, diagnosis and treatment initiation are often difficult and research is limited due to unreliable models. Most murine IE models rely on permanent prosthetic wire placement and i.v. bacterial injection, thus making it inadequate to study IE pathophysiology with the aim to reflect the course of disease in humans. Purpose To develop a reproducible murine IE model, based on aortic valve injury preceding intravenous S. aureus bacterial challenge. Methods Using a wire-injury model we induced endothelial lesions. 72h after wire injury (WI), we performed intravenous bacterial challenge using S. aureus. Echocardiography was performed to confirm bacterial vegetations and assess valvular function. Cross sections of valvular leaflets were prepared for scanning electron microscopy (SEM). Macrophage, neutrophil and S. aureus-specific immunofluorescence staining was performed and visualized by immunofluorescence microscopy. Bacterial cultivation was performed from peripheral blood and valve tissue. Systemic immune response was analyzed using flow cytometry. Results WI induced endothelial damage in all mice verified using scanning electron microscopy imaging. The combination of wire injury and injection of 10^5 and 10^6 colonies-forming-units (CFU) S. aureus reliably caused IE validated via in vivo echocardiography as well as S.aureus immunofluorescence staining and SEM imaging. Mice undergoing bacterial challenge responded with significant neutrophilia in the blood. Using 10^6 CFU S. aureus was associated with an increased mortality, liver and kidney abscess formation due to systemic bacterial spreading compared to 10^5 CFU. Peak velocity across the aortic valve and aortic valve leaflet thickness as markers for aortic valve stenosis were increased in IE compared to WI only mice. Aortic regurgitation was more prevalent after bacterial challenge mediating increased left ventricular volumes, meanwhile ejection fraction was not altered. Immunofluorescence staining revealed a pro-inflammatory milieu including increased macrophage and neutrophil infiltration in IE compared to WI only mice. Conclusion In vivo echocardiography and ex vivo histological staining revealed reliable IE induction using our new model with S. aureus concentrations of 10^5 CFU. The additional bacterial challenge causes a more severe aortic valve stenosis compared to WI only mice, potentially due to an observed increase in valvular inflammation.Fig 1 Scanning electron microscopy after

Intranasal vaccination with engineered BCG expressing CCL2 induces a stronger immune barrier against Mycobacterium tuberculosis than BCG

Intradermal Mycobacterium bovis Bacillus Calmette-Guérin (BCG) vaccination is currently the only licensed strategy for preventing tuberculosis (TB). It provides limited protection against pulmonary TB. To enhance the efficacy of BCG, we developed a recombinant BCG expressing exogenous monocyte chemoattractant CC chemokine ligand 2 (CCL2), termed rBCG-CCL2. Co-culturing macrophages with rBCG-CCL2 enhances their abilities in migration, phagocytosis, and effector molecules expression. In the mouse model, intranasal vaccination with rBCG-CCL2 induced greater immune cells infiltration and a more extensive innate immune responses in lung compared to vaccination with parental BCG, as determined by multiparameter flow cytometry, transcriptomic analysis, and pathological assessments. Moreover, rBCG-CCL2 induced a high frequency of activated macrophages and antigen-specific Th1 and Th17 T cells in lungs. The enhanced immune microenvironment responded more effectively to intravenous challenge with Mycobacterium tuberculosis (Mtb) H37Ra, leading to significant reductions in H37Ra burden and pathological damage to the lungs and spleen. Intranasal rBCG-CCL2 vaccinated mice rapidly initiated pro-inflammatory Th1 cytokine release and reduced pathological damage to the lungs and spleen during the early stage of H37Ra challenge. The finding that co-expression of CCL2 synergistically enhances the immune barrier induced by BCG provides a model for defining immune correlates and mechanisms of vaccine-elicited protection against TB.

Downregulating miR-432-5p exacerbates adriamycin-induced cardiotoxicity via activating the RTN3 signaling pathway.

Adriamycin (ADR) is a widely used chemotherapy drug in clinical practice and it causes toxicity in the myocardium affecting its clinical use. miR-432-5p is a miRNA primarily expressed in myocardial cells and has a protective effect in the myocardium. We aim to explore the protective effect of miR-432-5p on ADR-caused impaired mitochondrial ATP metabolism and endoplasmic reticulum stress (ERs). The primary cardiomyocytes were obtained from neonatal mice and the ADR was added to cells, meanwhile, a mice model was constructed through intravenous ADR challenge, and expression levels of miR-432-5p were examined. Subsequently, the miR-432-5p was introduced in vitro and in vivo to explore its effect on the activity of mitochondrial ATP synthesis, autophagy, and ER stress. The bioinformatics analysis was performed to explore the target of miR-432-5p. ADR decreased the expression of miR-432-5p in cardiomyocytes. It also decreases mitochondrial ATP production and activates the ER stress pathway by increasing the expression of LC3B, Beclin 1, cleaved caspase 3, and induces cardiac toxicity. miR-432-5p exogenous supplementation can reduce the cardiotoxicity caused by ADR, and its protective effect on cardiomyocytes depends on the down-regulation of the RTN3 signaling pathway in ER. ADR can induce the low expression of miR-432-5p, and activate the RTN3 pathway in ER, increase the expression of LC3B, Beclin 1, cleaved caspase 3, CHOP, and RTN3, and induce cardiac toxicity.

Modernizing Endocarditis Treatment Through a Multidisciplinary Team Approach

Fostering a multidisciplinary team approach in cardiovascular care to manage valve disease and heart failure is not a new concept; however, it is underused in the setting of infective endocarditis (IE). IE is one of the deadliest medical conditions largely due to a delay in diagnosis and treatment as a result of the time used to individually coordinate care between multiple specialties. Research shows a multidisciplinary endocarditis team decreases mortality rates by decreasing the time between admission and initiating treatment. Patients with IE secondary to intravenous substance abuse pose complex challenges to developing an effective, comprehensive, multidisciplinary endocarditis team.

Long-Term Protective Immunity against Ehrlichia chaffeensis Infection Induced by a Genetically Modified Live Vaccine.

Human monocytic ehrlichiosis, an emerging tick-borne disease, is caused by Ehrlichia chaffeensis. Infections with the pathogen are also common in the canine host. Our previous studies demonstrated that functional disruption within the E. chaffeensis phage head-to-tail connector protein gene results in bacterial attenuation, creating a modified live attenuated vaccine (MLAV). The MLAV confers protective immunity against intravenous and tick transmission challenges one month following vaccination. In this study, we evaluated the duration of MLAV protection. Dogs vaccinated with the MLAV were challenged with wild-type E. chaffeensis via intravenous infection at 4-, 8-, and 12-months post-vaccination. Immunized dogs rapidly cleared the wild-type pathogen infection and tested positive for bacteremia less frequently than unvaccinated controls. While immune responses varied among dogs, vaccinees consistently mounted IgG and CD4+ T-cell responses specific to E. chaffeensis throughout the assessment period. Our findings demonstrate that MLAV-mediated immune protection persists for at least one year against wild-type bacterial infection, marking a major advancement in combating this serious tick-borne disease. The data presented here serve as the foundation for further studies, elucidating the molecular mechanisms underlying virulence and vaccine development and aiding in preventing the diseases caused by E. chaffeensis and other tick-borne rickettsial pathogens.

Non-invasive ventral cervical magnetoneurography as a proxy of in vivo lipopolysaccharide-induced inflammation

Maintenance of autonomic homeostasis is continuously calibrated by sensory fibers of the vagus nerve and sympathetic chain that convey compound action potentials (CAPs) to the central nervous system. Lipopolysaccharide (LPS) intravenous challenge reliably elicits a robust inflammatory response that can resemble systemic inflammation and acute endotoxemia. Here, we administered LPS intravenously in nine healthy subjects while recording ventral cervical magnetoneurography (vcMNG)-derived CAPs at the rostral Right Nodose Ganglion (RNG) and the caudal Right Carotid Artery (RCA) with optically pumped magnetometers (OPM). We observed vcMNG RNG and RCA neural firing rates that tracked changes in TNF-α levels in the systemic circulation. Further, endotype subgroups based on high and low IL-6 responders segregate RNG CAP frequency (at 30-120 min) and based on high and low IL-10 response discriminate RCA CAP frequency (at 0-30 min). These vcMNG tools may enhance understanding and management of the neuroimmune axis that can guide personalized treatment based on an individual’s distinct endophenotype.

Boosting Immunogenicity of a Recombinant Mycobacterium smegmatis Strain via Zinc-Dependent Ribosomal Proteins.

Tuberculosis (TB) continues to be a major global health burden and kills over a million people annually. New immunization strategies are required for the development of an efficacious TB vaccine that can potentially induce sterilizing immunity. In this study, we first confirmed that a live vaccine strain of Mycobacterium smegmatis, previously designated as IKEPLUS, conferred a higher survival benefit than the Bacillus Calmette-Guerin (BCG) in a murine model of intravenous Mycobacterium tuberculosis (Mtb) infection. We have shown that there was a significant increase in the expression of the Rv0282 gene, which is encoded in the esx-3 locus, which played an important role in iron uptake when IKEPLUS was grown in both low zinc and iron-containing Sauton medium. We then confirmed using in vitro assays of biofilm formation that zinc plays a vital role in the growth and formation of M. smegmatis biofilms. IKEPLUS grown in low zinc media led to the better protection of mice after intravenous challenge with a very high dosage of Mtb. We also showed that various variants of IKEPLUS induced apoptotic cell-death of infected macrophages at a higher rate than wild-type M. smegmatis. We next attempted to determine if zinc containing ribosomal proteins such as rpmb2 could contribute to protective efficacy against Mtb infection. Since BCG has an established role in anti-mycobacterial efficacy, we boosted BCG vaccinated mice with rmpb2, but this did not lead to an increment in the protection mediated by BCG.

Consumer experience and outcomes of insertion, removal, and management practices of short and midline peripheral intravenous catheters in acute health care: a mixed methods systematic review protocol.

This review will synthesize and integrate current research on the practices relating to the insertion, removal, and management of short and midline peripheral intravenous catheters on consumer experiences and outcomes in acute health care. The aim is to provide consolidated evidence to support policy decision-makers and health care workers in overcoming peripheral intravenous catheter device management challenges. Implementing the evidence provided by this review could increase positive consumer outcomes and reduce the gap between research evidence and clinical practice. Peripheral intravenous catheterization is a process in which a device is inserted into a patient's peripheral vein to administer medication, blood products, and therapeutic fluids. Recent studies have demonstrated the positive and negative impacts of peripheral intravenous catheter site placement, pain relief for insertion, complications, consumer engagement, and routine versus clinically indicated removal of these devices on consumers and the health care system. Managing these impacts in relation to research-practice gaps and conflicting evidence remains challenging. Moreover, consumer experiences and outcomes concerning these challenges are unclear. This review will consider quantitative, qualitative, and mixed methods studies on consumer experiences and outcomes in acute health care. The quantitative component will consider outcomes of patients' pain, satisfaction, anxiety, distress, preferences, fear, and comfort. The qualitative component will consider consumer experiences, including perceptions, preferences, perspectives, attitudes, expectations, and satisfaction. This study will follow JBI's convergent segregated approach for mixed method systematic reviews. A search of 11 databases will be conducted for peer-reviewed published articles, theses, and dissertations. There will be no age, date, or language limitations. PROSPERO CRD42023410214.

Effects of systemic or uterine endotoxin challenge in Holstein cows at 5 or 40 days postpartum on clinical responses, uterine and systemic inflammation, and milk yield

Our objective was to investigate the effects of intravenous (IV) or intrauterine (IU) lipopolysaccharide (LPS) challenge at 5 or 40 d postpartum (DPP) on clinical signs, systemic and uterine inflammation, dry matter intake (DMI), and milk yield (MY). Holstein cows at 5 DPP (n = 23) or at 40 DPP (n = 24) were blocked by parity and randomly assigned to 1 of 3 treatments: (1) IV-LPS (0.0625 μg/kg BW [5 DPP] or 0.1 μg/kg BW [40 DPP] over 1h), (2) IU-LPS (100 μg [5 DPP] or 300 μg [40 DPP] in 20 mL of saline), or (3) 20 mL of saline IU (IU-SAL; same for 5 and 40 DPP). The proportion of polymorphonuclear (PMN) cells was measured by endometrial cytology at d -1, 1, 4, and 7 relative to treatment. Blood haptoglobin (Hp), serum amyloid A (SAA), and LPS-binding protein (LBP), DMI, and MY were measured from d -1 through d 7. Data were analyzed separately for each DPP group in multivariable linear regression models accounting for repeated measures. Both DPP groups showed increases in rectal temperature and heart and respiratory rates, and decrease in rumination rate following IV-LPS, but not following IU-LPS. At 5 DPP, endometrial PMN proportion was similar in IU-LPS and IU-SAL. Serum Hp was unaffected by LPS challenge, SAA was greater in IV-LPS from 12 h to 24 h after challenge, and LBP was greater in IV-LPS from 8 h to 24 h. At 40 DPP, PMN was greater in IU-LPS (37 ± 4%) than in IU-SAL (15 ± 4%) 1 d after LPS challenge. Serum Hp was greater from 24 h to 72 h after challenge in IV-LPS than in the other groups, SAA was greater in IV-LPS from 6 h to 48 h, and LBP was greater in IV-LPS from 8 h to 24 h. At both 5 and 40 DPP, treatment did not affect DMI, but MY was lesser in IV-LPS cows at 12 h and 24 h than in IU-SAL or IU-LPS. The IV-LPS challenge resulted in more pronounced changes in clinical signs and acute phase protein (APP) concentrations than IU-LPS or IU-SAL at 40 DPP, but more subtle or inconsistent changes at 5 DPP. These may be due to the different doses of LPS used at 5 and 40 DPP or possibly due to the high variation in baseline clinical signs and APP observed in all groups at 5 DPP. The IU-LPS increased uterine PMN 1 d after challenge at 40 DPP but not at 5 DPP. At each time, IU-LPS did not produce changes in clinical signs or markers of systemic inflammation.

Prophylactic vaccination inducing anti-Env antibodies can result in protection against HTLV-1 challenge in macaques

HTLV-1 infection occurs by cell-to-cell transmission and can induce fatal adult T-cell leukemia (ATL). Vaccine development is critical for the control of HTLV-1 transmission. However, determining whether vaccine-induced anti-Env antibodies can prevent cell-to-cell HTLV-1 transmission is challenging. Here, we examined the protective efficacy of a vaccine inducing anti-Env antibodies against HTLV-1 challenge in cynomolgus macaques. Eight of ten vaccinated macaques produced anti-HTLV-1 neutralizing antibodies (NAbs) and were protected from an intravenous challenge with 108 HTLV-1-producing cells. In contrast, the two vaccinated macaques without NAb induction and ten unvaccinated controls showed HTLV-1 infection with detectable proviral load after challenge. Five of the eight protected macaques were administered with an anti-CD8 monoclonal antibody, but proviruses remained undetectable and no increase in anti-HTLV-1 antibodies was observed even after CD8+ cell depletion in three of them. Analysis of Env-specific T cell responses did not suggest involvement of vaccine-induced Env-specific T cell responses in the protection. These results indicate that anti-Env antibody induction by vaccination can result in functionally sterile HTLV-1 protection, implying the rationale for strategies aimed at anti-Env antibody induction in prophylactic HTLV-1 vaccine development.

Expert Opinions on the Management of Hemophilia A in India: The Role of Emicizumab.

Hemophilia A (HA) is a genetic disorder of hemostasis associated with a deficiency or reduced activity of clotting factor VIII (FVIII). This disorder remains unacceptably underdiagnosed in India. Early diagnosis and appropriate management of HA can substantially prevent morbidity and mortality. Currently, HA is managed with regular replacement therapy using standard or extended half-life FVIII concentrates or non-factor drug products. The challenges associated with FVIII concentrates include plateauing of drug effect, issues with its administration and adherence to treatment, breakthrough bleeds, and the development of inhibiting antibodies against administered clotting factors. Emicizumab is a bispecific antibody, launched in India in April 2019, for managing patients with HA. To investigate the role of emicizumab in Indian patients with HA, opinions were sought from 13 eminent hematologists and experts from India on the effectiveness of emicizumab in preventing all bleeds, spontaneous bleeds, perioperative bleeds, and intracranial hemorrhage; resolving target joints; and reducing the rate of hospitalizations and fatality associated with HA in children and adults, with or without inhibitors. The benefits of emicizumab over traditional FVIII concentrates include the subcutaneous route of delivery, less frequent dosing, and a lack of inhibitor development, in addition to providing sustained hemostasis without in-depth monitoring. It is a safe and effective management option for all HA patients, especially for patients with certain archetypes, such as those with inhibitors, those with high annualized bleed rates, those living far away from hemophilia care centers, pediatric patients and infants with intravenous access challenges, and those with a history of life-threatening bleeding events.

Early antiretroviral therapy in SIV-infected rhesus macaques reveals a multiphasic, saturable dynamic accumulation of the rebound competent viral reservoir.

The rebound competent viral reservoir (RCVR)-virus that persists during antiretroviral treatment (ART) and can reignite systemic infection when treatment is stopped-is the primary barrier to eradicating HIV. We used time to initiation of ART during primary infection of rhesus macaques (RMs) after intravenous challenge with barcoded SIVmac239 as a means to elucidate the dynamics of RCVR establishment in groups of RMs by creating a multi-log range of pre-ART viral loads and then assessed viral time-to-rebound and reactivation rates resulting from the discontinuation of ART after one year. RMs started on ART on days 3, 4, 5, 6, 7, 9 or 12 post-infection showed a nearly 10-fold difference in pre-ART viral measurements for successive ART-initiation timepoints. Only 1 of 8 RMs initiating ART on days 3 and 4 rebounded after ART interruption despite measurable pre-ART plasma viremia. Rebounding plasma from the 1 rebounding RM contained only a single barcode lineage detected at day 50 post-ART. All RMs starting ART on days 5 and 6 rebounded between 14- and 50-days post-ART with 1-2 rebounding variants each. RMs starting ART on days 7, 9, and 12 had similar time-to-measurable plasma rebound kinetics despite multiple log differences in pre-ART plasma viral load (pVL), with all RMs rebounding between 7- and 16-days post-ART with 3-28 rebounding lineages. Calculated reactivation rates per pre-ART pVL were highest for RMs starting ART on days 5, 6, and 7 after which the rate of accumulation of the RCVR markedly decreased for RMs treated on days 9 and 12, consistent with multiphasic establishment and near saturation of the RCVR within 2 weeks post infection. Taken together, these data highlight the heterogeneity of the RCVR between RMs, the stochastic establishment of the very early RCVR, and the saturability of the RCVR prior to peak viral infection.

Serial direct sodium removal in patients with heart failure and diuretic resistance.

Loop diuretics may exacerbate cardiorenal syndrome (CRS) in heart failure (HF). Direct sodium removal (DSR) using the peritoneal membrane, in conjunction with complete diuretic withdrawal, may improve CRS and diuretic resistance. Patients with HF requiring high-dose loop diuretics were enrolled in two prospective, single-arm studies: RED DESERT (n = 8 euvolaemic patients), and SAHARA (n = 10 hypervolaemic patients). Loop diuretics were withdrawn, and serial DSR was utilized to achieve and maintain euvolaemia. At baseline, participants required a median 240 mg (interquartile range [IQR] 200-400) oral furosemide equivalents/day, which was withdrawn in all participants during DSR (median time of DSR 4 weeks [IQR 4-6]). Diuretic response (queried by formal 40 mg intravenous furosemide challenge and 6 h urine sodium quantification) increased substantially from baseline (81 ± 37 mmol) to end of DSR (223 ± 71 mmol, p < 0.001). Median time to re-initiate diuretics was 87 days, and the median re-initiation dose was 8% (IQR 6-10%) of baseline. At 1 year, diuretic dose remained substantially below baseline (30 [IQR 7.5-40] mg furosemide equivalents/day). Multiple dimensions of kidney function such as filtration, uraemic toxin excretion, kidney injury, and electrolyte handling improved (p < 0.05 for all). HF-related biomarkers including N-terminal pro-B-type natriuretic peptide, carbohydrate antigen-125, soluble ST2, interleukin-6, and growth differentiation factor-15 (p < 0.003 for all) also improved. In patients with HF and diuretic resistance, serial DSR therapy with loop diuretic withdrawal was feasible and associated with substantial and persistent improvement in diuretic resistance and several cardiorenal parameters. If replicated in randomized controlled studies, DSR may represent a novel therapy for diuretic resistance and CRS. RED DESERT (NCT04116034), SAHARA (NCT04882358).

Comparison of the immunogenicity and protective efficacy of ACAM2000, MVA, and vectored subunit vaccines for Mpox in rhesus macaques.

The 2022-2023 mpox outbreak triggered vaccination efforts using smallpox vaccines that were approved for mpox, including modified vaccinia Ankara (MVA; JYNNEOS), which is a safer alternative to live replicating vaccinia virus (ACAM2000). Here, we compare the immunogenicity and protective efficacy of JYNNEOS by the subcutaneous or intradermal routes, ACAM2000 by the percutaneous route, and subunit Ad35 vector-based L1R/B5R or L1R/B5R/A27L/A33R vaccines by the intramuscular route in rhesus macaques. All vaccines provided robust protection against high-dose intravenous mpox virus challenge with the current outbreak strain, with ACAM2000 providing near complete protection and JYNNEOS and Ad35 vaccines providing robust but incomplete protection. Protection correlated with neutralizing antibody responses as well as L1R/M1R- and B5R/B6R-specific binding antibody responses, although additional immune responses likely also contributed to protection. This study demonstrates the protective efficacy of multiple vaccine platforms against mpox virus challenge, including both current clinical vaccines and vectored subunit vaccines.

Intravenous lipopolysaccharide challenge in early- versus mid-lactation dairy cattle. II: The production and metabolic responses

Most immunometabolic research uses mid-lactation (ML) cows. Cows in early lactation (EL) are in a presumed state of immune suppression/dysregulation and less is known about how they respond to a pathogen. Study objectives were to compare the production and metabolic responses to i.v. LPS and to differentiate between the direct effects of immune activation and the indirect effects of illness-induced hypophagia in EL and ML cows. Cows in EL (n = 11; 20 ± 2 DIM) and ML (n = 12; 131 ± 31 DIM) were enrolled in a 2 × 2 factorial design containing 2 experimental periods (P). During P1 (3 d), cows were fed ad libitum and baseline data were collected. At the initiation of P2 (3 d), cows were randomly assigned to 1 of 2 treatments by lactation stage (LS): (1) EL (EL-LPS; n = 6) or ML (ML-LPS; n = 6) cows administered i.v. a single bolus of 0.09 µg LPS/kg of BW; Escherichia coli O55:B5 or (2) pair-fed (PF) EL (EL-PF; n = 5) or ML (ML-PF; n = 6) cows administered i.v. saline. Administering LPS decreased DMI and this was more severe in EL-LPS than ML-LPS cows (34% and 11% relative to baseline, respectively). By design, P2 DMI patterns were similar in the PF groups compared with their LPS counterparts. Milk yield decreased following LPS (42% on d 1 relative to P1) and despite an exacerbated decrease in EL-LPS cows on d 1 (25% relative to ML-LPS), remained similar between LS from d 2 to 3. The EL-LPS cows had increased milk fat content, but no difference in protein and lactose percentages compared with ML-LPS cows. Further, cumulative ECM yield was increased (21%) in EL-LPS compared with ML-LPS cows. During P2, EL-LPS cows had a more intense increase in MUN and BUN than ML-LPS and EL-PF cows. Administering LPS did not cause hypoglycemia in either EL-LPS or ML-LPS cows, but glucose was increased (33%) in EL-LPS compared with EL-PF. Hyperinsulinemia occurred after LPS, and insulin was further increased in ML-LPS than EL-LPS cows (2.2-fold at 12 h peak). During P2, circulating glucagon increased only in EL-LPS cows (64% relative to all other groups). Both EL groups had increased NEFA at 3 and 6 h after LPS from baseline (56%), but NEFA in EL-LPS cows gradually returned to baseline thereafter and were reduced relative to EL-PF until 36 h (50% from 12 to 24 h). Alterations in BHB did not differ between ML groups, but EL-LPS had reduced BHB compared with EL-PF from 24 to 72 h (51%). Results indicate that there are distinct LS differences in the anorexic and metabolic responses to immune activation. Collectively, EL cows are more sensitive to the catabolic effects of LPS than ML cows, but these exacerbated metabolic responses appear coordinated to fuel an augmented immune system while simultaneously supporting milk synthesis.

Intravenous lipopolysaccharide challenge in early- versus mid-lactation dairy cattle. I: The immune and inflammatory responses

Cows in early lactation (EL) are purportedly immune suppressed, which renders them more susceptible to disease. Thus, the study objective was to compare key biomarkers of immune activation from i.v. LPS between EL and mid-lactation (ML) cows. Multiparous EL (20 ± 2 DIM; n = 11) and ML (131 ± 31 DIM; n = 12) cows were enrolled in a 2 × 2 factorial design and assigned to 1 of 2 treatments by lactation stage (LS): (1) EL (EL-LPS; n = 6) or ML (ML-LPS; n = 6) cows administered a single LPS bolus from Escherichia coli O55:B5 (0.09 µg/kg of BW), or (2) pair-fed (PF) EL (EL-PF; n = 5) or ML (ML-PF; n = 6) cows administered i.v. saline. After LPS administration, cows were intensely evaluated for 3 d to analyze their response and recovery to LPS. Rectal temperature increased in LPS relative to PF cows (1.1°C in the first 9 h), and the response was more severe in EL-LPS relative to ML-LPS cows (2.3 vs. 1.3°C increase at 4 h post-LPS; respectively). Respiration rate increased only in EL-LPS cows (47% relative to ML-LPS in the first hour post-LPS). Circulating tumor necrosis factor-α, IL-6, monocyte chemoattractant protein-1, macrophage inflammatory protein (MIP)-1α, MIP-1β, and IFN-γ-inducible protein-10 increased within the first 6 h after LPS and these changes were exacerbated in EL-LPS relative to ML-LPS cows (6.3-fold, 4.8-fold, 57%, 93%, 10%, and 61%, respectively). All cows administered LPS had decreased circulating iCa relative to PF cows (34% at the 6 h nadir), but the hypocalcemia was more severe in EL-LPS than ML-LPS cows (14% at 6 h nadir). In response to LPS, neutrophils decreased regardless of LS, then increased into neutrophilia by 24 h in all LPS relative to PF cows (2-fold); however, the neutrophilic phase was augmented in EL- compared with ML-LPS cows (63% from 24 to 72 h). Lymphocytes and monocytes rapidly decreased then gradually returned to baseline in LPS cows regardless of LS; however, monocytes were increased (57%) at 72 h in EL-LPS relative to ML-LPS cows. Platelets were reduced (46%) in LPS relative to PF cows throughout the 3-d following LPS, and from 24 to 48 h, platelets were further decreased (41%) in EL-LPS compared with ML-LPS. During the 3-d following LPS, serum amyloid A (SAA), LPS-binding protein (LBP), and haptoglobin (Hp) increased in LPS compared with PF groups (9-fold, 72%, and 153-fold, respectively), and the LBP and Hp responses were more exaggerated in EL-LPS than ML-LPS cows (85 and 79%, respectively) whereas the SAA response did not differ by LS. Thus, our data indicates that EL immune function does not appear "suppressed," and in fact many aspects of the immune response are seemingly functionally robust.

Establishing RTS,S/AS01 as a benchmark for comparison to next-generation malaria vaccines in a mouse model

New strategies are needed to reduce the incidence of malaria, and promising approaches include vaccines targeting the circumsporozoite protein (CSP). To improve upon the malaria vaccine, RTS,S/AS01, it is essential to standardize preclinical assays to measure the potency of next-generation vaccines against this benchmark. We focus on RTS,S/AS01-induced antibody responses and functional activity in conjunction with robust statistical analyses. Transgenic Plasmodium berghei sporozoites containing full-length P. falciparum CSP (tgPb-PfCSP) allow two assessments of efficacy: quantitative reduction in liver infection following intravenous challenge, and sterile protection from mosquito bite challenge. Two or three doses of RTS,S/AS01 were given intramuscularly at 3-week intervals, with challenge 2-weeks after the last vaccination. Minimal inter- and intra-assay variability indicates the reproducibility of the methods. Importantly, the range of this model is suitable for screening more potent vaccines. Levels of induced anti-CSP antibody 2A10 equivalency were also associated with activity: 105 μg/mL (95% CI: 68.8, 141) reduced liver infection by 50%, whereas 285 μg/mL (95% CI: 166, 404) is required for 50% sterile protection from mosquito bite challenge. Additionally, the liver burden model was able to differentiate between protected and non-protected human plasma samples from a controlled human malaria infection study, supporting these models’ relevance and predictive capability. Comparison in animal models of CSP-based vaccine candidates to RTS,S/AS01 is now possible under well controlled conditions. Assessment of the quality of induced antibodies, likely a determinant of durability of protection in humans, should be possible using these methods.

Using a wearable patch to develop a digital monitoring biomarker of inflammation in response to LPS challenge.

Remote inflammation monitoring with digital health technologies (DHTs) would provide valuable information for both clinical research and care. Controlled perturbations of the immune system may reveal physiological signatures which could be used to develop a digital biomarker of inflammatory state. In this study, molecular and physiological profiling was performed following an invivo lipopolysaccharide (LPS) challenge to develop a digital biomarker of inflammation. Ten healthy volunteers received an intravenous LPS challenge and were monitored for 24 h using the VitalConnect VitalPatch (VitalPatch). VitalPatch measurements included heart rate (HR), heart rate variability (HRV), respiratory rate (RR), and skin temperature (TEMP). Conventional episodic inpatient vital signs and serum proteins were measured pre- and post-LPS challenge. The VitalPatch provided vital signs that were comparable to conventional methods for assessing HR, RR, and TEMP. A pronounced increase was observed in HR, RR, and TEMP as well as a decrease in HRV 1-4 h post-LPS challenge. The ordering of participants by magnitude of inflammatory cytokine response 2 h post-LPS challenge was consistent with ordering of participants by change from baseline in vital signs when measured by VitalPatch (r = 0.73) but not when measured by conventional methods (r = -0.04). A machine learning model trained on VitalPatch data predicted change from baseline in inflammatory protein response (R2 = 0.67). DHTs, such as VitalPatch, can improve upon existing episodic measurements of vital signs by enabling continuous sensing and have the potential for future use as tools to remotely monitor inflammation.

Candida albicans extracellular vesicles trigger type I IFN signalling via cGAS and STING.

The host type I interferon (IFN) pathway is a major signature of inflammation induced by the human fungal pathogen, Candida albicans. However, the molecular mechanism for activating this pathway in the host defence against C. albicans remains unknown. Here we reveal that mice lacking cyclic GMP-AMP synthase (cGAS)-stimulator of IFN genes (STING) pathway components had improved survival following an intravenous challenge by C. albicans. Biofilm-associated C. albicans DNA packaged in extracellular vesicles triggers the cGAS-STING pathway as determined by induction of interferon-stimulated genes, IFNβ production, and phosphorylation of IFN regulatory factor 3 and TANK-binding kinase 1. Extracellular vesicle-induced activation of type I IFNs was independent of the Dectin-1/Card9 pathway and did not require toll-like receptor 9. Single nucleotide polymorphisms in cGAS and STING potently altered inflammatory cytokine production in human monocytes challenged by C. albicans. These studies provide insights into the early innate immune response induced by a clinically significant fungal pathogen.

Lymphocyte transformation tests predict delayed-type allergy to piperacillin/tazobactam in patients with cystic fibrosis

BackgroundAntibiotic treatment is crucial for patients with chronic bacterial infections. Suspected drug allergies often lead to inconsistent therapies and challenging clinical management for patients and caregivers. The objective of this study was to evaluate the value of lymphocyte transformation tests in comparison to skin tests for the prediction of delayed-type allergic reactions. MethodsThis prospective, observational study tested the diagnostic value of skin prick tests, intradermal tests (reading: 15 min and 72 h) and lymphocyte transformations tests for the prediction of allergic reactions in CF patients with physician reported allergy to piperacillin/tazobactam, meropenem and ceftazidime. The tests were performed directly before a 14d intravenous drug challenge. ResultsWe performed 33 drug challenges in 29 subjects. 21 drug challenges were negative (63 %); 12 lead to a reaction (37 %), of those 2 were immediate and 10 were delayed-type. 100 % of the skin prick tests were negative. 97 % (33/34) of the intradermal tests with early reading and 100 % of the intradermal tests with late reading yielded negative results. 5/11 patients who experienced a delayed-type reaction during the drug challenge had a positive lymphocyte transformations test. All 17 patients who did not react had a negative lymphocyte transformations test. For piperacillin/tazobactam, 4/5 patients who experienced a delayed-type reaction during the drug challenge had positive lymphocyte transformations tests. Hence, for piperacillin/tazobactam, the sensitivity of the lymphocyte transformation test for prediction of reactions was 80.0 % and the specificity 100 %. ConclusionWe demonstrate that the lymphocyte transformation test predicts delayed-type allergy to piperacillin/tazobactam in contrast to skin tests.