Hu8F4-CAR T cells with mutated Fc spacer segment improve target specificity and mediate anti-leukemia activity in vivo.

BACKGROUND AIMS: Hu8F4 is a T-cell receptor-like antibody with high affinity for the leukemia-associated antigen PR1/HLA-A2 epitope. Adapted into a chimeric antigen receptor (CAR) format, Hu8F4-CAR is composed of the Hu8F4 single-chain variable fragment, the human IgG1 CH2CH3 extracellular spacer domain, a human CD28 costimulatory domain and the human CD3ζ signaling domain. We have demonstrated high efficacy of Hu8F4-CAR-T cells against PR1/HLA-A2-expressing cell lines and leukemic blasts from patients with acute myeloid leukemia in vitro. Previous studies have shown that modification of the Fc domains of IgG4 CH2CH3 spacer regions can eliminate activation-induced cell death and off-target killing mediated by mouse Fc gamma receptor-expressing cells. METHODS: We generated Hu8F4-CAR(PQ) with mutated Fc receptor binding sites on the CH2 domain of Hu8F4-CAR to prevent unwanted interactions with Fc gamma receptor-expressing cells in vivo. RESULTS: The primary human T cells transduced with Hu8F4-CAR(PQ) can specifically lyse HLA-A2+ PR1-expressing leukemia cell lines in vitro. Furthermore, both adult donor-derived and cord blood-derived Hu8F4-CAR(PQ)-T cells are active and can eliminate U937 leukemia cells in NSG mice. CONCLUSIONS: Herein, we demonstrate that modification of the IgG1-based spacer can eliminate Fc receptor binding-induced adverse effects and Hu8F4-CAR(PQ)-T cells can kill leukemia in vivo.

Novel myeloperoxidase-derived HLA-A2-restricted peptides as therapeutic targets against myeloid leukemia.

BACKGROUND AIMS: Human myeloperoxidase has been shown to be overexpressed in many types of leukemia, such as chronic myeloid leukemia, acute myeloid leukemia and myelodysplastic syndrome. The authors identified two myeloperoxidase-derived HLA-A2-restricted peptides, MY4 and MY8, as novel leukemia-associated antigens. METHODS: Ex vivo-elicited MY4- and MY8-specific cytotoxic T lymphocytes were generated, and tested for leukemia cell lysis in vitro and in NOD/SCID AML xenograft model. RESULTS: These MY4- and MY8-specific cytotoxic T lymphocytes killed leukemic blasts while sparing healthy donor bone marrow cells. In addition, co-injection of MY4- and MY8-specific cytotoxic T lymphocytes into nonobese diabetic/severe combined immunodeficiency mice with acute myeloid leukemia drastically reduced tumor burden in vivo. The authors also found that MY4- and MY8-specific T cells could be detected in the peripheral blood mononuclear cells of allogeneic stem cell transplant recipients. CONCLUSIONS: These antigen-specific T cells were significantly increased in blood samples from patients compared with healthy donors, suggesting that both MY4 and MY8 are immunogenic and that MY4- and MY8-specific cytotoxic T lymphocytes may play a role in reducing leukemia in vivo. Thus, the discovery of MY4 and MY8 as novel leukemia-associated antigens paves the way for targeting these antigens in immunotherapy against myeloid leukemia.

Membrane-Associated Proteinase 3 on Granulocytes and Acute Myeloid Leukemia Inhibits T Cell Proliferation.

Proteinase 3 (P3), a serine protease expressed by myeloid cells, localized within azurophil granules, and also expressed on the cellular membrane of polymorphonuclear neutrophils (PMN), is the target of autoimmunity in granulomatosis with polyangiitis. PR1, an HLA-A2 restricted nonameric peptide derived from P3, has been targeted effectively in myeloid leukemia. We previously showed (Molldrem et al. 2003. JClinInvest 111: 639-647) that overexpression of P3 in chronic myeloid leukemia induces apoptosis of high-affinity PR1-specific T cells, leading to deletional tolerance and leukemia outgrowth. In this study, we investigated the effect of membrane P3 (mP3)-expressing PMN and acute myeloid leukemia (AML) blasts on the proliferation of CD4 and CD8 T cells in vitro. We demonstrate that mP3-expressing PMN significantly inhibits autologous healthy donor T cell proliferation but does not affect cytokine production in activated T cells and that this effect requires cell proximity and was abrogated by P3 blockade. This inhibition required P3 enzyme activity. However, suppression was not reversed by either the addition of catalase or the inhibition of arginase I. In addition to P3 blockade, anti-low density lipoprotein receptor-related protein 1 (LRP1) Ab also restored T cells' capacity to proliferate. Last, we show dose-dependent inhibition of T cell proliferation by mP3-expressing AML blasts. Together, our findings demonstrate a novel mechanism whereby PMN- and AML-associated mP3 inhibits T cell proliferation via direct LRP1 and mP3 interaction, and we identify P3 as a novel target to modulate immunity in myeloid leukemia and autoimmune disease.

Sexually Transmitted Disease Partner Services Costs, Other Resources, and Strategies Across Jurisdictions to Address Unique Epidemic Characteristics and Increased Incidence.

BACKGROUND: Sexually transmitted disease (STD) partner services (PS) are a core component of STD programs. Data on costs are needed to support PS programming. METHODS: In Washington State STD PS programs, disease intervention specialists (DIS) conduct telephone-based interviews and occasional field visits, offer expedited partner therapy to heterosexuals with gonorrhea or chlamydia, and promote human immunodeficiency virus (HIV) testing, preexposure prophylaxis, and HIV care. We conducted activity-based microcosting of PS, including: observational and self-reported time studies and interviews. We analyzed cost, surveillance, and service delivery data to determine costs per program outcomes. RESULTS: In King, Pierce, and Spokane counties, respectively, DIS allocated 6.5, 6.4, and 28.8 hours per syphilis case and 1.5, 1.6, and 2.9 hours per gonorrhea/chlamydia case, on average. In 2016, each full-time DIS investigated 270, 268, and 61 syphilis and 1177, 1105, and 769 gonorrhea/chlamydia cases. Greater than 80% of syphilis cases in King and Pierce were among men who have sex with men versus 38% in Spokane. Disease intervention specialists spent 12% to 39% of their time actively interviewing cases and notifying partners (clients), and the remaining time locating clients, coordinating and verifying care, and managing case reports. Time spent on expedited partner therapy, HIV testing, and referrals to HIV treatment or preexposure prophylaxis, was minimal (<5 minutes per interview) at locations with resources outside PS staff. Program cost-per-interview ranged from US $527 to US $2210 for syphilis, US $219 to US $484 for gonorrhea, and US $164 to US $547 for chlamydia. DISCUSSION: The STD PS resource needs depended on epidemic characteristics and program models. Integrating HIV prevention objectives minimally impacted PS-specific program costs. Results can inform program planning, future budget impact, and cost-effectiveness analyses.

Neuropilin-1 mediates neutrophil elastase uptake and cross-presentation in breast cancer cells.

Neutrophil elastase (NE) can be rapidly taken up by tumor cells that lack endogenous NE expression, including breast cancer, which results in cross-presentation of PR1, an NE-derived HLA-A2-restricted peptide that is an immunotherapy target in hematological and solid tumor malignancies. The mechanism of NE uptake, however, remains unknown. Using the mass spectrometry-based approach, we identify neuropilin-1 (NRP1) as a NE receptor that mediates uptake and PR1 cross-presentation in breast cancer cells. We demonstrated that soluble NE is a specific, high-affinity ligand for NRP1 with a calculated Kd of 38.7 nm Furthermore, we showed that NRP1 binds to the RRXR motif in NE. Notably, NRP1 knockdown with interfering RNA or CRISPR-cas9 system and blocking using anti-NRP1 antibody decreased NE uptake and, subsequently, susceptibility to lysis by PR1-specific cytotoxic T cells. Expression of NRP1 in NRP1-deficient cells was sufficient to induce NE uptake. Altogether, because NRP1 is broadly expressed in tumors, our findings suggest a role for this receptor in immunotherapy strategies that target cross-presented antigens.

PR1-specific cytotoxic T lymphocytes are relatively frequent in umbilical cord blood and can be effectively expanded to target myeloid leukemia.

BACKGROUND AIMS: PR1 is an HLA-A2 restricted leukemia-associated antigen derived from neutrophil elastase and proteinase 3, both of which are normally stored in the azurophil granules of myeloid cells but overexpressed in myeloid leukemic cells. PR1-specific cytotoxic lymphocytes (PR1-CTLs) have activity against primary myeloid leukemia in vitro and in vivo and thus could have great potential in the setting of adoptive cellular therapy (ACT). Adult peripheral blood-derived PR1-CTLs are infrequent but preferentially lyse myeloid leukemia cells. We sought to examine PR1-CTLs in umbilical cord blood (UCB) because UCB units provide a rapidly available cell source and a lower risk of graft-versus-host disease, even in the setting of mismatched human leukocyte antigen (HLA) loci. METHODS: We first determined the frequency of PR1-CTLs in HLA-A2(+) UCB units and then successfully expanded them ex vivo using repeated stimulation with PR1 peptide-pulsed antigen-presenting cells (APCs). After expansion, we assessed the PR1-CTL phenotype (naive, effector, memory) and function against PR1-expressing target cells. RESULTS: PR1-CTLs are detected at an average frequency of 0.14% within the CD8(+) population of fresh UCB units, which is 45 times higher than in healthy adult peripheral blood. UCB PR1-CTLs are phenotypically naive, consistent with the UCB CD8(+) population as a whole. In addition, the cells can be expanded by stimulation with PR1 peptide-pulsed APCs. Expansion results in an increased frequency of PR1-CTLs, up to 4.56%, with an average 20-fold increase in total number. After expansion, UCB PR1-CTLs express markers consistent with effector memory T cells. Expanded UCB PR1-CTLs are functional in vitro as they are able to produce cytokines and lyse PR1-expressing leukemia cell lines. CONCLUSIONS: This study is the first report to show that T cells specific for a leukemia-associated antigen are found at a significantly higher frequency in UCB than adult blood. Our results also demonstrate specific cytotoxicity of expanded UCB-derived PR1-CTLs against PR1-expressing targets. Together, our data suggest that UCB PR1-CTLs could be useful to prevent or treat leukemia relapse in myeloid leukemia patients.

A novel TCR-like CAR with specificity for PR1/HLA-A2 effectively targets myeloid leukemia in vitro when expressed in human adult peripheral blood and cord blood T cells.

BACKGROUND AIMS: The PR1 peptide, derived from the leukemia-associated antigens proteinase 3 and neutrophil elastase, is overexpressed on HLA-A2 in acute myeloid leukemia (AML). We developed a T-cell receptor (TCR)-like monoclonal antibody (8F4) that binds the PR1/HLA-A2 complex on the surface of AML cells, efficiently killing them in vitro and eliminating them in preclinical models. Humanized 8F4 (h8F4) with high affinity for the PR1/HLA-A2 epitope was used to construct an h8F4- chimeric antigen receptor (CAR) that was transduced into T cells to mediate anti-leukemia activity. METHODS: Human T cells were transduced to express the PR1/HLA-A2-specific CAR (h8F4-CAR-T cells) containing the scFv of h8F4 fused to the intracellular signaling endo-domain of CD3 zeta chain through the transmembrane and intracellular costimulatory domain of CD28. RESULTS: Adult human normal peripheral blood (PB) T cells were efficiently transduced with the h8F4-CAR construct and predominantly displayed an effector memory phenotype with a minor population (12%) of central memory cells in vitro. Umbilical cord blood (UCB) T cells could also be efficiently transduced with the h8F4-CAR. The PB and UCB-derived h8F4-CAR-T cells specifically recognized the PR1/HLA-A2 complex and were capable of killing leukemia cell lines and primary AML blasts in an HLA-A2-dependent manner. CONCLUSIONS: Human adult PB and UCB-derived T cells expressing a CAR derived from the TCR-like 8F4 antibody rapidly and efficiently kill AML in vitro. Our data could lead to a new treatment paradigm for AML in which targeting leukemia stem cells could transfer long-term immunity to protect against relapse.

A point prevalence study of the use of psychotropic medication in an acute general hospital.

BACKGROUND: There are high rates of cognitive problems and organic and functional psychiatric disorders in patients admitted to general hospital wards, which may necessitate the use of psychotropic medications. There is evidence of over-prescription of medications such as antipsychotics and antidepressants in community settings such as residential care. However, the prevalence of psychotropic use in general hospitals is unknown. METHODS: A point prevalence study of the use of psychotropic medications in an acute general hospital was conducted by auditing medication charts for type, dose, indication, and other clinical processes associated with their use. RESULTS: The files of 197 patients were audited, 139 (70%) were aged 65 years and over. Eighty-nine (45%) of patients were prescribed a psychotropic, with 35 (17.7%) antidepressants; 21 (11%) antipsychotics; 19 (10%) benzodiazepines; six (3%) mood stabilizer; five (2%) cholinesterase inhibitors; one (0.5%) anti-parkinsonian medication, and one (1%) patient on zolpidem and another patient melatonin (1%). Most prescriptions (72%) were in people 65 years and over and 27 (20%) of indications were found to be off label prescribing. There were deficits in the documentation of indications for the use of psychotropic medications and follow up arrangements. CONCLUSIONS: Rates of benzodiazepines use were less than that reported incomparable settings. Concerns regarding off label prescribing, under-dosing, and lack of adequate documentation of indications and follow-up instructions were raised. Given the potential adverse effects of psychotropic medication, improved governance, and education regarding their use is required.

CD13-positive bone marrow-derived myeloid cells promote angiogenesis, tumor growth, and metastasis.

Angiogenesis is fundamental to tumorigenesis and an attractive target for therapeutic intervention against cancer. We have recently demonstrated that CD13 (aminopeptidase N) expressed by nonmalignant host cells of unspecified types regulate tumor blood vessel development. Here, we compare CD13 wild-type and null bone marrow-transplanted tumor-bearing mice to show that host CD13(+) bone marrow-derived cells promote cancer progression via their effect on angiogenesis. Furthermore, we have identified CD11b(+)CD13(+) myeloid cells as the immune subpopulation directly regulating tumor blood vessel development. Finally, we show that these cells are specifically localized within the tumor microenvironment and produce proangiogenic soluble factors. Thus, CD11b(+)CD13(+) myeloid cells constitute a population of bone marrow-derived cells that promote tumor progression and metastasis and are potential candidates for the development of targeted antiangiogenic drugs.

Broad cross-presentation of the hematopoietically derived PR1 antigen on solid tumors leads to susceptibility to PR1-targeted immunotherapy.

PR1 is a HLA-A2-restricted peptide that has been targeted successfully in myeloid leukemia with immunotherapy. PR1 is derived from the neutrophil granule proteases proteinase 3 (P3) and neutrophil elastase (NE), which are both found in the tumor microenvironment. We recently showed that P3 and NE are taken up and cross-presented by normal and leukemia-derived APCs, and that NE is taken up by breast cancer cells. We now extend our findings to show that P3 and NE are taken up and cross-presented by human solid tumors. We further show that PR1 cross-presentation renders human breast cancer and melanoma cells susceptible to killing by PR1-specific CTLs (PR1-CTL) and the anti-PR1/HLA-A2 Ab 8F4. We also show PR1-CTL in peripheral blood from patients with breast cancer and melanoma. Together, our data identify cross-presentation as a novel mechanism through which cells that lack endogenous expression of an Ag become susceptible to therapies that target cross-presented Ags and suggest PR1 as a broadly expressed tumor Ag.

Hu8F4-CAR T cells with mutated Fc spacer segment improve target-specificity and mediate anti-leukemia activity in vivo.

Hu8F4 is a T cell receptor (TCR)-like antibody with high affinity for leukemia-associated antigen PR1/HLA-A2 epitope. Adapted into a chimeric antigen receptor (CAR) format, Hu8F4-CAR is comprised of the Hu8F4 scFv, the human IgG1 CH2CH3 extracellular spacer domain, a human CD28 costimulatory domain, and the human CD3ζ signaling domain. We have demonstrated high efficacy of Hu8F4-CAR-T cells against PR1/HLA-A2-expressing cell lines and leukemic blasts from AML patients in vitro. Previous studies have shown that modification of the Fc domains of IgG4 CH2CH3 spacer regions can eliminate activation-induced cell death and off-target killing mediated by mouse Fc gamma receptor (FcgR)-expressing cells. We generated Hu8F4-CAR(PQ) with mutated Fc receptor binding sites on the CH2 domain of Hu8F4-CAR to prevent unwanted interactions with FcgR-expressing cells in vivo. The primary human T cells transduced with Hu8F4-CAR(PQ) can specifically lyse HLA-A2+ PR1-expressing leukemia cell lines in vitro. Furthermore, both adult donor-derived and cord blood-derived Hu8F4-CAR(PQ)-T cells are active and can eliminate U937 leukemia cells in NSG mice. Herein, we demonstrate that modification of the IgG1-based spacer can eliminate Fc receptor-binding-induced adverse effects and Hu8F4-CAR(PQ)-T cells can kill leukemia in vivo.

The Global ALPL gene variant classification project: Dedicated to deciphering variants.

BACKGROUND: Hypophosphatasia (HPP) is an inherited multisystem disorder predominantly affecting the mineralization of bones and teeth. HPP is caused by pathogenic variants in ALPL, which encodes tissue non-specific alkaline phosphatase (TNSALP). Variants of uncertain significance (VUS) cause diagnostic delay and uncertainty amongst patients and health care providers. RESULTS: The ALPL gene variant database (https://alplmutationdatabase.jku.at/) is an open-access archive for interpretation of the clinical significance of variants reported in ALPL. The database contains coding and non-coding variants, including single nucleotide variants, insertions/deletions and structural variants affecting coding or non-coding sequences of ALPL. Each variant in the database is displayed with details explaining the corresponding pathogenicity, and all reported genotypes and phenotypes, including references. In 2021, the ALPL gene variant classification project was established to reclassify VUS and continuously assess and update genetic, phenotypic, and functional variant information in the database. For this purpose, the database provides a unique submission system for clinicians, geneticists, genetic counselors, and researchers to submit VUS within ALPL for classification. An international, multidisciplinary consortium of HPP experts has been established to reclassify the submitted VUS using a multi-step process adhering to the stringent ACMG/AMP variant classification guidelines. These steps include a clinical phenotype assessment, deep literature research including artificial intelligence technology, molecular genetic assessment, and in-vitro functional testing of variants in a co-transfection model to measure ALP residual activity. CONCLUSION: This classification project and the ALPL gene variant database will serve the global medical community, widen the genotypic and phenotypic HPP spectrum by reporting and characterizing new ALPL variants based on ACMG/AMP criteria and thus facilitate improved genetic counseling and medical decision-making for affected patients and families. The project may also serve as a gold standard framework for multidisciplinary collaboration for variant interpretation in other rare diseases.

An anti-PR1/HLA-A2 T-cell receptor-like antibody mediates complement-dependent cytotoxicity against acute myeloid leukemia progenitor cells.

PR1 (VLQELNVTV) is a human leukocyte antigen-A2 (HLA-A2)-restricted leukemia-associated peptide from proteinase 3 (P3) and neutrophil elastase (NE) that is recognized by PR1-specific cytotoxic T lymphocytes that contribute to cytogenetic remission of acute myeloid leukemia (AML). We report a novel T-cell receptor (TCR)-like immunoglobulin G2a (IgG2a) antibody (8F4) with high specific binding affinity (dissociation constant [K(D)] = 9.9nM) for a combined epitope of the PR1/HLA-A2 complex. Flow cytometry and confocal microscopy of 8F4-labeled cells showed significantly higher PR1/HLA-A2 expression on AML blasts compared with normal leukocytes (P = .046). 8F4 mediated complement-dependent cytolysis of AML blasts and Lin(-)CD34(+)CD38(-) leukemia stem cells (LSCs) but not normal leukocytes (P < .005). Although PR1 expression was similar on LSCs and hematopoietic stem cells, 8F4 inhibited AML progenitor cell growth, but not normal colony-forming units from healthy donors (P < .05). This study shows that 8F4, a novel TCR-like antibody, binds to a conformational epitope of the PR1/HLA-A2 complex on the cell surface and mediates specific lysis of AML, including LSCs. Therefore, this antibody warrants further study as a novel approach to targeting leukemia-initiating cells in patients with AML.

Characterization of immunologic properties of a second HLA-A2 epitope from a granule protease in CML patients and HLA-A2 transgenic mice.

The serine proteases, neutrophil elastase (HNE) and proteinase 3 (PR3), are aberrantly expressed in human myeloid leukemias. T-cell responses to these proteins have been correlated with remission in patients with chronic myeloid leukemia (CML). Human PR3/HNE-specific CD8(+) T cells predominantly recognize a nonameric HLA-A2-restricted T-cell epitope called PR1 which is conserved in both Ags. However, CML patients have CD8(+) T cells in peripheral blood recognizing an additional HLA-A2 epitope termed PR2. To assess immunologic properties of these Ags, novel recombinant vaccinia viruses (rVV) expressing PR3 and HNE were evaluated in HLA-A2 transgenic (Tg) mice (HHDII). Immunization of HHDII mice with rVV-PR3 elicited a robust PR3-specific CD8(+) T-cell response dominated by recognition of PR2, with minimal recognition of the PR1 epitope. This result was unexpected, because the PR2 peptide has been reported to bind poorly to HLA. To account for these findings, we proposed that HHDII mice negatively selected PR1-specific T cells because of the presence of this epitope within murine PR3 and HNE, leading to immunodominance of PR2-specific responses. PR2-specific splenocytes are cytotoxic to targets expressing naturally processed PR3, though PR1-specific splenocytes are not. We conclude that PR2 represents a functional T-cell epitope recognized in mice and human leukemia patients. These studies are registered at www.clinicaltrials.gov as NCT00716911.

Multi-faceted role of LRP1 in the immune system.

Graft versus host disease (GVHD) represents the major complication after allogeneic hematopoietic stem cell transplantation (Allo-SCT). GVHD-prone patients rely on GVHD prophylaxis (e.g. methotrexate) and generalized anti-GVHD medical regimen (glucocorticoids). New anti-GVHD therapy strategies are being constantly explored, however there is an urgent need to improve current treatment, since GVHD-related mortality reaches 22% within 5 years in patients with chronic GVHD. This review is an attempt to describe a very well-known receptor in lipoprotein studies - the low-density lipoprotein receptor related protein 1 (LRP1) - in a new light, as a potential therapeutic target for GVHD prevention and treatment. Our preliminary studies demonstrated that LRP1 deletion in donor murine T cells results in significantly lower GVHD-related mortality in recipient mice with MHC (major histocompatibility complex) -mismatched HSCT. Given the importance of T cells in the development of GVHD, there is a significant gap in scientific literature regarding LRP1's role in T cell biology. Furthermore, there is limited research interest and publications on this classical receptor molecule in other immune cell types. Herein, we endeavor to summarize existing knowledge about LRP1's role in various immune cells to demonstrate the possibility of this receptor to serve as a novel target for anti-GVHD treatment.

Procoagulant Activity of Blood and Microvesicles Is Disturbed by Pneumococcal Pneumolysin, Which Interacts with Coagulation Factors.

The coagulation and contact systems are parts of the innate immune system as they prevent bleeding and dissemination of pathogens and also contribute to microbial killing by inflammatory reactions and the release of antimicrobial peptides. Here, we investigated the influence of Streptococcus pneumoniae on the coagulation and contact system. S. pneumoniae (pneumococci), but no other investigated streptococcal species, impairs coagulation of blood by autolysis and release of pneumolysin. Defective blood coagulation results from the lysis of tissue factor-producing mononuclear cells and their procoagulant microvesicles, which are the main trigger for blood coagulation during sepsis. In addition, pneumolysin binds coagulation and contact system factors, but this does not result in activation. Thus, pneumococci modulate activation of the coagulation system by releasing pneumolysin, which could potentiate lung injury during pneumonia.

Human late memory CD8+ T cells have a distinct cytokine signature characterized by CC chemokine production without IL-2 production.

Late memory T cell skewing is observed in the setting of immune recovery after cord blood transplantation, and may be associated with inferior control of viral reactivation and cancers. Therefore, we sought to understand how late memory cells differ functionally from earlier stage memory T cells, and whether surface phenotypes associated with differentiation stages were predictably associated with functional signatures. Higher order cytokine flow cytometry allows characterization of human T cells based on complex phenotypic markers and their differential capacity to simultaneously secrete effector proteins, including cytokines and chemokines. We used 8-color, 10-parameter cytokine flow cytometry to characterize the functional activation of human late memory CD8(+) T cells defined by CD45RA and CD27 expression (CD27(-)CD45RA(+)). We assessed the 15 possible functional signatures of cells defined by production of IL-2, IFN-gamma, TNF-alpha, and MIP-1beta alone or in combination, following activation with Ags stimulating bypassing surface proteins (PMA:ionomycin) or through the TCR (e.g., viral Ags). Late memory CD8(+) T cells produced abundant amounts of CC chemokines (MIP-1beta, MIP-1alpha, and RANTES) but not IL-2. IL-2/IFN-gamma coproduction, characteristic of protective immune responses to viral infections, was absent in late memory CD8(+) T cells. These data demonstrate that functional cytokine signatures are predictably associated with CD8(+) maturation stages, and that the polarization of late memory CD8(+) T cells toward CC chemokine production and away from IL-2 production suggests a unique functional role for this subset.

In training emergency physicians the carotid artery Doppler with passive leg raise, does previous sonographic experience influence scan time and competency?

BACKGROUND: Determination of fluid responsiveness (FR) associated with intravascular fluid resuscitation in hypotensive patients poses a challenge, with current best evidence methods fraught with poor retest reliability and difficulty in image acquisition (Osman, Crit Care Med 2007; 35: 64; Marik, Crit Care Med 2009; 37: 2642). Doppler carotid blood flow with passive leg raise (PLR) is a recent modality for determining FR (Marik, Chest 2013; 143: 364). PURPOSE: This study aimed to determine whether emergency physicians with limited ultrasound experience can reliably acquire this skill. METHOD: This prospective study recruited 60 emergency physicians with varying experience, who underwent a 3-step learning programme. Participants performed carotid velocity time integral (VTi) Doppler on healthy subjects, followed by repeat measurements in the PLR position. A 16-point checklist and time recorded were assessed for each sonographer, with each participant completing a post-study questionnaire to evaluate perceived competence and ease of skill acquisition. RESULTS: Of the 60 emergency physicians recruited, 37 (61.6%) were inexperienced and 23 (38.4%) were experienced. Against the 16-point assessment, 61% completed assessment without any errors. Fifty-six out of 60 (94.3%) completed the assessment to acceptable standard with errors recognised and corrected, and four participants (6.7%) made critical errors without correction (Figure 1). Average (±SEM) total scan time was 4:52 ± 0:19, with no significant difference found between inexperienced and experienced groups. CONCLUSIONS: This study demonstrated feasibility to train emergency physicians, demonstrating that average FR assessment was obtained within 5 min, with no difference between prior experience in scan quality/time taken. 94% completed the scan to acceptable standards, demonstrating ease of carotid Doppler flow with PLR to provide critical information in management of the hypotensive patient.

Perfluoroalkyl substances exposure and immunity, allergic response, infection, and asthma in children: review of epidemiologic studies.

BACKGROUND: Increased exposure to perfluoroalkyl substances (PFAS) potentially affects infant and childhood health through immunosuppression. Given rapidly evolving research on PFAS, it is important to comprehensively examine the impact of PFAS exposure among the pediatric population as new research becomes available due to potential fragility of the developing immune system. OBJECTIVES: This review assessed the effects of PFAS fetal, infant and childhood exposures upon the development of immune function during early life stages. METHODS: Researchers completed a literature review, searching PubMed for human studies published since 2010 for PFAS and health outcomes among infants and children. Included articles incorporated key search terms in the title or abstract; non-research reports and non-English papers were excluded. The search identified 518 studies for possible inclusion. Following hands-on review, 34 were determined relevant. Subsequent analyses found 8 additional relevant articles, totaling 42 studies. RESULTS: Major immune-related sequelae from PFAS exposures on infant and child health outcomes documented in recent literature include:• Strong indication of immunosuppression, with diminished childhood antibody response to vaccination, particularly with PFOA, PFOS and PFHxS exposures.• Some indication of increased risks of childhood infectious diseases/infections, particularly from PFOS exposures.• Limited indication of an effect of PFAS exposure on allergic reactions/allergen specific IgE antibodies.• Limited indication of an effect of PFAS exposure on atopic dermatitis (AD).• Limited indication of an effect of PFAS exposure on asthma and lung function. CONCLUSION: This review summarizes recent findings of PFAS effects on infant and childhood immune health. Evidence of immunosuppression, diminished vaccine efficacy, and increased risk of infections, allergies, asthma and AD were described following in utero, infant, and early childhood PFAS exposures. Further investigation is warranted to characterize PFAS exposure pathways and potential modes of action in relation to PFAS effects on the developing immune system. Incontrovertible proof of PFAS immunotoxic effects could optimally be obtained by a large prospective study cohort of mothers and children from infancy through school-age. Regular assessments of circulating antibodies and response to infant and childhood vaccines during growth years could prove invaluable.

What Motivates Smokers to Switch to ENDS? A Qualitative Study of Perceptions and Use.

Switching completely from cigarettes to electronic nicotine delivery systems (ENDS) may reduce health risks for addicted smokers. This paper provides information about perceptions and other factors that may influence smokers' ENDS use and substitution for cigarettes. We conducted 12 online focus groups (N = 61) among smokers who had never tried using ENDS (Never Users, N = 11), currently used both cigarettes and ENDS (Dual Users, N = 21), used but discontinued ENDS (Rejectors, N = 14), and switched completely to ENDS use (Switchers, N = 15). Thematic analysis was used to interpret the transcripts. Participants described initial interest in trying ENDS in hopes of quitting smoking and because of convenience (i.e., due to rules, regulations, or social norms). Risk perceptions and higher prices relative to cigarettes were reported as disadvantages of ENDS that discouraged initiation. Dual Users and Rejectors reported product problems (e.g., products breaking) and dissatisfaction (i.e., inability to satisfy cravings for cigarettes) as factors that lowered their substitutability for cigarettes or led to discontinuing ENDS use. Switchers indicated that satisfaction, lack of product problems, and perceived safety facilitated successfully switching from cigarette smoking to exclusive ENDS use. However, Switchers reported trying many products before they found ones that satisfied their needs. We recommend that policymakers consider the potential impact of tobacco control policies on smokers' motivation and ability to switch completely from cigarettes to ENDS.