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Rapid and resource-efficient sample processing, high throughput, and high robustness are critical for effective scientific and clinical application of advanced antigen-specific immunoassays. Traditionally, such immunoassays, especially antigen-specific T-cell analysis by flow cytometry or enzyme-linked immunosorbent spot assays, often rely on the isolation of peripheral blood mononuclear cells. This process is time-consuming, subject to many pre-analytic confounders, and requires large blood volumes. Whole blood-based assays provide a facile alternative with increased pre-analytic robustness and lower blood volume requirements. Furthermore, whole blood-based assays allow for the preservation of inter-cellular interactions that are not captured by assays using isolated cell subsets. Recently, a refined whole blood immunoassay with dual anti-CD28 and anti-CD49d co-stimulation for comprehensive analysis of both antigen-specific T-cell functions and complex intercellular interactions in response to various fungal and viral antigens has been proposed. This protocol provides guidance for the preparation of stimulation tubes, blood stimulation, and downstream sample processing for flow cytometry, cytokine secretion assays, and transcriptional analyses. This includes a validated and functionally equivalent, previously unpublished, low-volume protocol (250 µL) to make flow cytometric and cytokine-based T-cell monitoring more accessible for studies in pediatric patients or preclinical studies in small animals (e.g., mice). Altogether, these protocols provide a versatile toolbox for complex antigen-specific immune analysis in both clinical and translational research settings.

Introduction: Pre-existent pools of coronavirus-specific or cross-reactive T cells were shown to shape the development of cellular and humoral immune responses after primary mRNA vaccination against SARS-CoV-2. However, the cellular determinants of responses to booster vaccination remain incompletely understood. Therefore, we phenotypically and functionally characterized spike antigen-specific T helper (Th) cells in healthy, immunocompetent individuals and correlated the results with cellular and humoral immune responses to BNT162b2 booster vaccination over a six-month period., Methods: Blood of 30 healthy healthcare workers was collected before, 1, 3, and 6 months after their 3rd BNT162b2 vaccination. Whole blood was stimulated with spike peptides and analyzed using flow cytometry, a 13-plex cytokine assay, and nCounter-based transcriptomics., Results: Spike-specific IgG levels at 1 month after booster vaccination correlated with pre-existing CD154+CD69+IFN-γ+CD4+ effector memory cells as well as spike-induced IL-2 and IL-17A secretion. Early post-booster (1-month) spike IgG levels (r=0.49), spike-induced IL‑2 (r=0.58), and spike-induced IFN‑γ release (r=0.43) correlated moderately with their respective long-term (6-month) responses. Sustained robust IgG responses were significantly associated with S-specific (CD69+±CD154+±IFN-γ+) Th-cell frequencies before booster vaccination (p=0.038), especially double/triple-positive type-1 Th cells. Furthermore, spike IgG levels, spike-induced IL‑2 release, and spike-induced IFN‑γ release after 6 months were significantly associated with increased IL‑2 & IL‑4, IP‑10 & MCP1, and IFN‑γ & IP‑10 levels at 1 month post-booster, respectively. On the transcriptional level, induction of pathways associated with both T-cell proliferation and antigen presentation was indicative of sustained spike-induced cytokine release and spike-specific IgG production 6 months post-booster. Using support vector machine models, pre-booster spike-specific T-cell frequencies and early post-booster cytokine responses predicted sustained (6-month) responses with F1 scores of 0.80-1.00., Discussion: In summary, spike-specific Th cells and T-cellular cytokine signatures present before BNT162b2 booster vaccination shape sustained adaptive cellular and humoral responses post-booster. Functional T-cell assays might facilitate early identification of potential non-responders., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Page, Dennehy, Mueller, Girl, Loell, Buijze, Classen, Messmann, Roemmele, Hoffmann, Wurster and Fuchs.)

Purpose: Comprehensive geriatric assessment (CGA) is the cornerstone of high-quality care for older adults. There is no current gold standard to guide what should be included as the baseline measure for CGAs. We examined what metrics are being captured in CGA baseline assessments completed by community based integrated care teams in Ireland., Methods: CGA's care pathways in Ireland are usually initiated with a written document that establish patients baseline in various assessment areas. These documents were the focus of this study. We completed a cross-sectional study of the components captured in CGA baseline assessments completed in a community setting. We contacted operational leads in each of the community health organisations in Ireland and requested a copy of their current initial baseline screening document for CGA., Results: We reviewed 16 individual CGA baseline documents for analysis in this study. Common assessment areas in all documents included frailty (with the Rockwood Clinical frailty scale used in 94%, n = 15), cognition (4AT-56% of CGAs, MMSE-25%, MOCA-25%, AMTS-19%, AD8-19%, Addenbrookes-13%, 6CIT-13%, mini cog-6%), mobility (100%, n = 16), falls (100%, n = 16), continence (100% n = 16), nutrition (100% n = 16). Mood (94%, n = 15), pain (44%, n = 7), bone health (63%, n = 10), sleep (62%, n = 10) and skin integrity (56%, n = 9). Formal functional assessment was completed in 94% (n = 15) of CGAs with the Barthel index being the tool most used 81% (n = 13). Half of the CGAs included a section describing carer strain (50%, n = 8). The majority of CGAs included a patient centred question which was some variation of 'what matters most to me' (75% n = 11). 87.5% of assessments included a care plan summary (n = 14)., Conclusions: This report highlights that the core tenets of CGA are being assessed across different community based initial CGA screening instruments. There was significant variability in the discussion of challenging topics such as carer strain and social well-being. Our results should prompt a discussion about whether a minimum dataset should be developed for inclusion in nationwide initial baseline CGA document, aiming to improve standardisation of assessments, which will impact areas highlighted for intervention and ultimately guide population health policy., (© 2024. The Author(s).)

Background: Lifestyle interventions targeting households may be an effective means of promoting healthier cognitive function in later life, with extended benefit to other household members. In this systematic review and meta-analysis, we sought to assess the effect of targeting lifestyle behaviours of households on cognitive outcomes METHODS: An electronic search strategy was designed to identify randomised controlled trials (RCTs) where households were randomised to receive a lifestyle intervention for the prevention of cognitive decline, from database inception until April 2020. Our initial search identified no eligible studies, so we revised our search strategy to include trials enroling dyads. We reported the cognitive outcomes, functional outcomes, caregiver outcomes and long-term care (LTC) admissions for eligible studies., Findings: We identified no RCTs which randomised households to receive a lifestyle intervention for preventing cognitive decline. We identified five RCTs (n = 1721, with mean follow-up of 9.6 months) which randomised dyads, which evaluated diet (two trials) and physical activity (three trials)., Conclusion: Trials evaluating dietary and exercise interventions in dyads were identified. No trial demonstrated a significant association of interventions with change in cognitive testing, functional outcomes or long-term care admissions, although trials were small with short-term follow-up. Future studies should consider targeting lifestyle behaviours of households for prevention of dementia., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Objectives: An assessment of the comparative incidence of fatal or disabling stroke may influence choice of intervention for patients with severe aortic stenosis. We explored whether transcatheter aortic valve implantation (TAVI) is associated with a lower incidence of fatal or disabling stroke, compared to surgical aortic valve replacement (SAVR)., Materials & Methods: We classified stroke into two categories; fatal or disabling, or non-disabling, and completed meta-analyses for each. We explored randomised controlled trials to assess the effect publication year, predicted operative risk, and route of TAVI access., Results: There was no difference between treatment groups per 100 person years of follow up for disabling or non-disabling stroke outcomes. In a stratified analysis by year of publication, there was a lower rate of fatal or disabling stroke with TAVI in trials published after 2015, compared to those published in 2015 or before (p-interaction = 0.01 at 30 days). Higher proportions of transfemoral route access (>90%), more common in recent trials, were associated with a lower rate of fatal or disabling stroke (p-interaction = 0.03 at 30 days). Lower average surgical risk scores were associated with lower rates of fatal or disabling stroke (p = 0.02 at 30 days)., Conclusion: We found that treatment of aortic stenosis with TAVI compared with SAVR was not associated with an overall reduced risk in fatal or disabling stroke. Subgroup analyses suggested a lower risk of fatal or disabling stroke with TAVI in situations which reflect contemporary practice., Competing Interests: Declaration of Competing Interest None., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Schizophrenia is a severe neuropsychiatric disease that lacks completely effective and safe therapies. As a polygenic disorder, genetic studies have only started to shed light on its complex etiology. To date, the positive symptoms of schizophrenia are well-managed by antipsychotic drugs, which primarily target the dopamine D2 receptor (D2R). However, these antipsychotics are often accompanied by severe side effects, including motoric symptoms. At D2R, antipsychotic drugs antagonize both G-protein dependent (Gα i/o ) signaling and G-protein independent (β-arrestin) signaling. However, the relevant contributions of the distinct D2R signaling pathways to antipsychotic efficacy and on-target side effects (motoric) are still incompletely understood. Recent evidence from mouse genetic and pharmacological studies point to β-arrestin signaling as the major driver of antipsychotic efficacy and suggest that a β-arrestin biased D2R antagonist could achieve an additional level of selectivity at D2R, increasing the therapeutic index of next generation antipsychotics. Here, we characterize BRD5814, a highly brain penetrant β-arrestin biased D2R antagonist. BRD5814 demonstrated good target engagement via PET imaging, achieving efficacy in an amphetamine-induced hyperlocomotion mouse model with strongly reduced motoric side effects in a rotarod performance test. This proof of concept study opens the possibility for the development of a new generation of pathway selective antipsychotics at D2R with reduced side effect profiles for the treatment of schizophrenia.

Massachusetts faces an opioid and substance abuse crisis at the same time the U.S. and Massachusetts have some of highest rates of incarceration in the world. This issue brief examines the problem and economic costs and consequences of untreated substance abuse. It examines the benefits of expanding access to treatment in the community, at arrest and initial detention, within the courts, within jails and prisons, at re‐entry and under community supervision, with the intent to reduce substance abuse, incarceration and recidivism and thereby improve health and public safety. The report recommends (1) implementing a pre‐arrest program to divert low‐level drug offenders to treatment, (2) enhancing and expanding specialty courts throughout the state, (3) increasing access to medication‐assisted treatment (MAT), and (4) expanding a Medicaid enrollment program in DOC and HOC facilities to improve access to healthcare services immediately upon release. To facilitate change and judicious invest of resources, it also recommends instating governance structures to coordinate efforts between health and criminal justice organizations within the Executive, Legislative and Judiciary branches of government.

The E3 transcription unit of human adenoviruses (Ads) encodes immunomodulatory proteins. Interestingly, the size and composition of the E3 region differs considerably among Ad species, suggesting that distinct sets of immunomodulatory E3 proteins may influence their interaction with the human host and the disease pattern. However, to date, only common immune evasion functions of species C E3 proteins have been described. Here we report on the immunomodulatory activity of a species D-specific E3 protein, E3/49K. Unlike all other E3 proteins that act on infected cells, E3/49K seems to target uninfected cells. Initially synthesized as an 80- to 100-kDa type I transmembrane protein, E3/49K is subsequently cleaved, with the large ectodomain (sec49K) secreted. We found that purified sec49K exhibits specific binding to lymphoid cell lines and all primary leukocytes, but not to fibroblasts or epithelial cells. Consistent with this binding profile and the molecular mass, the sec49K receptor was identified as the cell surface protein tyrosine phosphatase CD45. Antibody-blocking studies suggested that sec49K binds to the membrane proximal domains present in all CD45 isoforms. Functional studies showed that sec49K can suppress the activation and cytotoxicity of natural killer cells as well as the activation, signaling, and cytokine production of T cells. Thus, we have discovered an adenovirus protein that is actively secreted and describe immunomodulatory activities of an E3 protein uniquely expressed by a single Ad species.

Angiotensin (ANG) II activating type 1 receptors (AT(1)Rs) enhances superoxide anion (O(2)*(-)) and arachidonate (AA) formation. AA is metabolized by cyclooxygenases (COXs) to PGH(2), which is metabolized by thromboxane (Tx)A(2) synthase to TxA(2) or oxidized to 8-isoprostane PGF(2alpha) (8-Iso) by O(2)*(-). PGH(2), TxA(2), and 8-Iso activate thromboxane-prostanoid receptors (TPRs). We investigated whether blood pressure in a rat model of early (3 wk) two-kidney, one-clip (2K,1C) Goldblatt hypertension is maintained by AT(1)Rs or AT(2)Rs, driving COX-1 or -2-dependent products that activate TPRs. Compared with sham-operated rats, 2K,1C Goldblatt rats had increased mean arterial pressure (MAP; 120 +/- 4 vs. 155 +/- 3 mmHg; P < 0.001), plasma renin activity (PRA; 22 +/- 7 vs. 48 +/- 5 ng x ml(-1) x h(-1); P < 0.01), plasma malondialdehyde (1.07 +/- 0.05 vs. 1.58 +/- 0.16 nmol/l; P < 0.01), and TxB(2) excretion (26 +/- 4 vs. 51 +/- 7 ng/24 h; P < 0.01). Acute graded intravenous doses of benazeprilat (angiotensin-converting enzyme inhibitor) reduced MAP at 20 min (-36 +/- 5 mmHg; P < 0.001) and excretion of TxA(2) metabolites. Indomethacin (nonselective COX antagonist) or SC-560 (COX-1 antagonist) reduced MAP at 20 min (-25 +/- 5 and -28 +/- 7 mmHg; P < 0.001), whereas valdecoxib (COX-2 antagonist) was ineffective (-9 +/- 5 mmHg; not significant). Losartan (AT(1)R antagonist) or SQ-29548 (TPR antagonist) reduced MAP at 150 min (-24 +/- 6 and -22 +/- 3 mmHg; P < 0.001), whereas PD-123319 (AT(2)R antagonist) was ineffective. Acute blockade of TPRs, COX-1, or COX-2 did not change PRA, but TxB(2) generation by the clipped kidney was reduced by blockade of COX-1 and increased by blockade of COX-2. 2K,1C hypertension in rats activates renin, O(2)*(-), and vasoconstrictor PGs. Hypertension is maintained by AT(1)Rs and by COX-1, but not COX-2, products that activate TPRs.

Asymmetric dimethylarginine (ADMA), which inhibits NO synthase, is inactivated by N(G),N(G)-dimethylarginine dimethylaminohydrolase (DDAH). We tested whether DDAH-1 or -2 regulates serum ADMA (S(ADMA)) and/or endothelium-derived relaxing factor (EDRF)/NO. Small inhibitory (si)RNAs targeting DDAH-1 or -2, or an siRNA control were given intravenously to rats. After 72 hours, EDRF/NO was assessed from acetylcholine-induced, NO synthase-dependent relaxation and 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate for NO activity in isolated mesenteric resistance vessels (MRVs). Expression of mRNA for DDAH-1 versus -2 was 2- and 7-fold higher in the kidney cortex and liver, respectively, whereas expression of DDAH-2 versus -1 was 5-fold higher in MRVs. The proteins and mRNAs for DDAH-1 or -2 were reduced selectively by 35% to 85% in the kidney cortex, liver, and MRVs 72 hours following the corresponding siRNA. S(ADMA) was increased only after siDDAH-1 (266+/-25 versus 342+/-39 [mean+/-SD] nmol x L(-1); P<0.005), whereas EDRF/NO responses and NO activity were not changed consistently by siDDAH-1 but were greatly reduced after siDDAH-2. Mean arterial pressure was not changed significantly by any siRNA. In conclusion, S(ADMA) is regulated by DDAH-1, which is expressed at sites of ADMA metabolism in the kidney cortex and liver, whereas EDRF/NO is regulated primarily by DDAH-2, which is expressed strongly in blood vessels. This implies specific functions of DDAH isoforms.

Beta-glucans are structural components of fungal cell walls which are involved in the immune recognition of fungal pathogens and possess beneficial immunomodulatory activities in isolated form. Here we have developed a soluble chimeric form of the major mammalian beta-glucan receptor, Dectin-1, and demonstrate its application for the detection and characterisation of soluble and insoluble beta-glucans, including fungal particles, using ELISA, flow cytometric and fluorescence-based microscopy assays.

Tempol is an amphipathic radical nitroxide (N) that acutely reduces blood pressure (BP) and heart rate (HR) in the spontaneously hypertensive rat (SHR). We investigated the hypothesis that the response to nitroxides is determined by SOD mimetic activity or lipophilicity. Groups (n = 6-10) of anesthetized SHRs received graded intravenous doses of Ns: tempol (T), 4-amino-tempo (AT), 4-oxo-tempo (OT), 4-trimethylammonium-2,2,6,6-tetramethylpiperidine-1-oxyl iodide (CAT-1), 3-carbamoyl-proxyl (3-CP), or 3-carboxy-proxyl (3-CTPY). Others received native or liposomal (L) Cu/Zn SOD. T and OT are uncharged, AT is positively charged and cell-permeable, and CAT-1 is positively charged and cell-impermeable. 3-CP and 3-CTPY have five-member pyrrolidine rings, whereas T, AT, OT, and CAT-1 have six-member piperidine rings. T and AT reduced mean arterial pressure (MAP) similarly (-48 +/- 2 mmHg and -55 +/- 8 mmHg) but more (P < 0.05) than OT and CAT-1. 3-CP and 3-CTPY were ineffective. The group mean change in MAP with piperidine Ns correlated with SOD activity (r = -0.94), whereas their ED(50) correlated with lipophilicity (r = 0.89). SOD and L-SOD did not lower BP acutely but reduced it after 90 min (-32 +/- 5 and -31 +/- 6 mmHg; P < 0.05 vs. vehicle). Pyrrolidine nitroxides are ineffective antihypertensive agents. The antihypertensive response to piperidine Ns is predicted by SOD mimetic action, and the sensitivity of response is by hydrophilicity. SOD exerts a delayed hypotensive action that is not enhanced by liposome encapsulation, suggesting it must diffuse to an extravascular site.

Some CD28-specific monoclonal antibodies (mAbs) have the surprising property to activate T-cells without the need for TCR ligation. This review summarizes the differences between these "superagonistic" and conventional CD28-specific mAbs with regard to binding specificity and signalling properties. Furthermore, the dramatic effects of in vivo application of CD28 superagonists with regard to the induction of regulatory T-cells and polyclonal T-cell expansion in lymphopenic settings are discussed under the aspect of potential therapeutic applications.

Introduction: Tempol is a permeant nitroxide superoxide dismutase (SOD) mimetic that lowers mean arterial pressure (MAP) in spontaneously hypertensive rats (SHRs). We investigated the hypothesis that the antihypertensive response entails a negative salt balance, blunting of plasma renin activity (PRA), endothelin-1 (ET-1), or catecholamines or correction of oxidative stress as indexed by 8-isoprostane prostaglandin F(2alpha) (PGF(2alpha)) (8-Iso)., Methods: Groups (N= 6 to 8) of SHRs were infused for 2 weeks with vehicle or tempol (200 nmol/kg/min) or given tempol (2 mmol/L) in drinking water., Results: Tempol infusion reduced the MAP of anesthetized SHRs (150 +/- 5 vs. 126 +/- 6 mm Hg) (P < 0.005). Oral tempol did not change the heart rate but reduced the MAP of conscious SHRs (-23 +/- 6 mm Hg) (P < 0.01) but not Wistar-Kyoto (WKY) rats. Tempol infusion increased the PRA (2.2 +/- 0.2 vs. 5.0 +/- 0.9 ng/mL/hour) (P < 0.005), did not change excretion of nitric oxide (NO) [NO(2)+ NO(3) (NOx)], ET-1, or catecholamines but reduced excretion of 8-Iso (13.2 +/- 1.4 vs. 9.6 +/- 0.9 ng/24 hours; P < 0.01). Cumulative Na(+) balance and gain in body weight were unaltered by tempol infusion. Tempol prevented a rise in MAP with high salt intake., Conclusion: Tempol corrects hypertension without a compensatory sympathoadrenal activation or salt retention. The response is independent of nitric oxide, endothelin, or catecholamines and occurs despite increased PRA. It is accompanied by a reduction in oxidative stress and is maintained during increased salt intake.

We used cyclooxygenase-1 (COX-1)-deficient mice to test the hypothesis that COX-1 regulates blood pressure (BP) and renal hemodynamics. The awake time (AT) mean arterial pressures (MAPs) measured by telemetry were not different between COX-1(+/+) and COX-1(-/-) (131+/-2 versus 126+/-3 mm Hg; NS). However, COX-1(-/-) had higher sleep time (ST) MAP (93+/-1 versus 97+/-2 mm Hg; P<0.05) and sleep-to-awake BP ratio (+8.6%; P<0.05). Under anesthesia with moderate sodium loading, COX-1(-/-) had higher MAP (109+/-5 versus 124+/-4 mm Hg; P<0.05), renal vascular resistance (23.5+/-1.6 versus 30.7+/-1.7 mm Hg . mL(-1) . min(-1) . g(-1); P<0.05) and filtration fraction (33.7+/-2.1 versus 40.2+/-2.0%; P<0.05). COX-1(-/-) had a 89% reduction (P<0.0001) in the excretion of TxB2, a 76% reduction (P<0.01) in PGE2, a 40% reduction (P<0.0002) in 6-ketoPGF1alpha (6keto), a 27% reduction (P<0.02) in 11-betaPGF2alpha (11beta), a 35% reduction (P<0.01) in nitrate plus nitrite (NOx), and a 52% increase in metanephrine (P<0.02). The excretion of normetanephrine, a marker for sympathetic nervous activity, was reduced during ST in COX-1(+/+) (6.9+/-0.9 versus 3.2+/-0.6 g . g(-1) creatinine . 10(-3); P<0.01). This was blunted in COX-1(-/-) (5.1+/-0.9 versus 4.9+/-0.7 g . g(-1) creatinine . 10(-3); NS). Urine collection during ST showed lower excretion of 6keto, 11beta, NOx, aldosterone, sodium, and potassium than during AT in both COX-1(+/+) and COX-1(-/-), and there were positive correlations among these parameters (6keto versus NOx; P<0.005; 11beta versus NOx; P<0.005; and NOx versus sodium; P<0.005). In conclusion, COX-1 mediates a suppressed sympathetic nervous activity and enhanced NO, which may contribute to renal vasodilatation and a reduced MAP while asleep or under anesthesia. COX-1 contributes to the normal nocturnal BP dipping phenomenon.

We investigated the hypothesis that thromboxane A2 (TxA2)-prostaglandin H2 receptors (TP-Rs) mediate the hemodynamic responses and increase in reactive oxygen species (ROS) to ANG II (400 ng x kg(-1) x min(-1) sc for 14 days) using TP-R knockout (TP -/-) and wild-type (+/+) mice. TP -/- had normal basal mean arterial blood pressure (MAP) and glomerular filtration rate but reduced renal blood flow and increased filtration fraction (FF) and renal vascular resistance (RVR) and markers of ROS (thiobarbituric acid-reactive substances and 8-isoprostane PGF2alpha) and nitric oxide (NOx). Infusion of ANG II into TP +/+ increased ROS and thromboxane B2 (TxB2) and increased RVR and FF. ANG II infusion into TP -/- mice reduced ANG I and increased aldosterone but caused a blunted increase in MAP (TP -/- : +6 +/- 2 vs. TP +/+: +15 +/- 3 mmHg) and failed to increase FF, ROS, or TxB2 but increased NOx and paradoxically decreased RVR (-2.1 +/- 1.7 vs. +2.6 +/- 0.8 mmHg x ml(-1) x min(-1) x g(-1)). Blockade of AT1 receptor of TP -/- mice infused with ANG II reduced MAP (-8 mmHg) and aldosterone but did not change the RVR or ROS. In conclusion, during an ANG II slow pressor response, AT1 receptors activate TP-Rs that generate ROS and prostaglandins but inhibit NO. TP-Rs mediate all of the increase in RVR and FF, part of the increase in MAP, but are not implicated in the suppression of ANG I or increase in aldosterone. TP -/- mice have a basal increase in RVR and FF associated with ROS.

Background and Objectives: Administration of analgesic medication before surgery, rather than at the completion of the procedure, may reduce postoperative pain. Similarly, administration of multiple analgesics, with different mechanisms of action, may provide improved postoperative pain control and functional recovery. The purpose of our study was to compare pain scores and intravenous opioid consumption after outpatient anterior cruciate ligament (ACL) reconstruction in patients who received a multimodal drug combination (intravenous [IV] ketorolac, intra-articular morphine/ropivacaine/epinephrine, and femoral nerve block with ropivacaine) either before surgery or immediately at the completion of the surgical procedure., Methods: Forty patients presenting for same-day arthroscopic ACL repair using a semitendinosis tendon graft were included in this study. The patients were randomized to receive the following drugs either 15 minutes before skin incision or immediately after skin closure: (1) Ketorolac 30 mg IV. (2) Intra-articular injection of 20 mL ropivacaine 0.25% + morphine 2 mg and epinephrine 1:200,000. (3) Femoral nerve block with 20 mL ropivacaine 0.25%. Verbal pain scores were obtained in the postanesthesia care unit (PACU) and on postoperative days 1, 3, and 7. IV patient controlled analgesia (PCA) morphine consumption in the PACU was also recorded., Results: Verbal pain rating scores were lower in group I (preemptive) for 2.0 hours after arrival in the PACU. There was no difference between groups in pain scores on postoperative days 1, 3, and 7. Mean IV PCA morphine consumption in the PACU was lower in group I (6.4 mg) versus group II (12.3 mg), P <.05., Conclusion: Preemptive, multimodal administration of our 3-component analgesic drug combination resulted in lower pain scores during the initial stay in the PACU unit and lower consumption of IV PCA morphine in the PACU. However, pain scores were similar in both groups on postoperative days 1, 3, and 7; thus, there was no measurable long-term advantage associated with preemptive multimodal drug administration.

Studies of CD5-deficient mice indicate that the transmembrane glycoprotein CD5 negatively regulates antigen receptor-mediated signals in thymocytes, lymph node T cells and B1a cells. CD5 contains four tyrosine residues in its cytoplasmic domain and is phosphorylated on tyrosine residues following antigen receptor ligation. Recently it has been proposed that CD5 function is dependent on the recruitment of the tyrosine phosphatase SHP-1 to tyrosine-phosphorylated CD5 and subsequent dephosphorylation of signaling molecules. In this study we investigated the requirements for, and sites of, CD5 tyrosine phosphorylation. Using a T cell line deficient in the tyrosine kinase p56(lck) and the same cell line reconstituted with this kinase, we show that p56(lck) expression is required for efficient CD5 tyrosine phosphorylation. Using tyrosine-phosphorylated peptides corresponding to CD5 cytoplasmic sequences we also show that the Src homology 2 (SH2) domain of p56(lck) binds prominently to pY429SQP, with 30-fold less affinity to pY463DLQ and not to pY441PAL. A number of murine CD5 Y --> F and deletion mutants were expressed in Jurkat T cells. The Y441F mutant was tyrosine phosphorylated at levels comparable to wild-type, but the Y429F and Y463F mutants were phosphorylated at lower levels. Two deletion mutants, which contain only one tyrosine residue (Y378) located at the interface of the transmembrane and cytoplasmic domains, were not tyrosine phosphorylated, suggesting that Y378 is not readily available for phosphorylation. Taken together these results suggest that both Y429 and Y463 can recruit p56(lck), and that these residues are the only prominent sites for CD5 tyrosine phosphorylation.

Thy-1, a glycosylphosphatidylinositol (GPI)-anchored glycoprotein expressed at high levels on thymocytes, has been implicated in positive and negative signal transduction. We show that Thy-1 associates with a protein of 85--90 kDa, which is prominently phosphorylated in vitro as well as in vivo following the stimulation of thymocytes with pervanadate. pp85--90 is not identical to known proteins that are phosphorylated following T cell activation. The SH2 domains of fyn, csk, phosphatidylinositol 3'-kinase, rasGAP, vav and lck bind to pp85--90 with varying affinities. The SH2 domains of ZAP70, SHP-1 and PLC gamma 1 and the SH3 domains of lck, vav and HS1 did not bind to pp85--90. The molecular weight, iso-electric point, efficient phosphorylation by fyn and lck and preferential binding to the SH2 domain of fyn compared to that of lck indicate that Thy-1-associated pp85-90 may be identical to a recently cloned, fyn-associated transmembrane adaptor protein, PAG-85., (Copyright 2001 Academic Press.)

Purpose: To illustrate the multiple causes of hypoxemia to be considered following cardiopulmonary bypass and how therapy given to improve oxygen delivery may have contributed to a decrease in arterial oxygen saturation to life-threatening levels., Clinical Features: A 61 yr old man with severe mitral regurgitation and chronic obstructive lung disease underwent surgery for mitral valve repair. A pulmonary artery catheter with the capacity to measure cardiac output and mixed venous oxygen saturation (SvO2) continuously was used. Two unsuccessful attempts were made to repair the valve which was finally replaced, requiring cardiopulmonary bypass of 317 min. Dobutamine 5 micrograms.kg-1.min-1 and sodium nitroprusside 1 microgram.kg-1.min-1 were used to increase cardiac output. Soon after, the SvO2 decreased progressively from 55 to 39%. The patient became cyanotic with a PaO2 of 39 mmHg. Sodium nitroprusside was stopped and amrinone 100 mg bolus followed by 10 micrograms.kg-1.min-1 was given in addition to adding PEEP to the ventilation. With these measures PaO2 could be maintained of safe levels but PEEP and high inspired oxygen concentrations were needed postoperatively until the trachea could be extubated on the third postoperative day., Conclusion: The profound hypoxemia in this case was likely due to a combination of intra- and extrapulmonary shunt, both augmented by sodium nitroprusside. The desaturation of mixed venous blood amplified the effect of these shunts in decreasing arterial oxygen saturation. The interaction of these factors are analyzed in this report.

Background and Objectives: To determine the analgesic efficacy of preoperative tumescent infiltration with lidocaine for reduction mammoplasty., Methods: Women with mammary hypertrophy were randomly allocated to one of two study groups in a double-blind clinical trial. Patients in group 1 received preincision infiltration with 5 mL/kg of 0.35% lidocaine with 1:1,000,000 epinephrine into each breast after induction of general anesthesia. Group 2 patients received similar injections of 5 mL/kg of saline with 1:1,000,000 epinephrine. Intravenous patient-controlled analgesia (PCA) morphine (1.0 mg bolus with 5-minute lockout) was available for 9.5 hours in the postoperative period. Visual analog pain scores were recorded during the postoperative period, and hourly morphine consumption data were retrieved from the PCA apparatus. Fitness for discharge was evaluated by the postanesthesia care unit nurse using standardized discharge criteria., Results: Visual analog pain scores were higher in group 2 patients until 3.5 hours after surgery. Patients in the saline group had higher intravenous morphine consumption during all 1-hour postoperative intervals, although the differences between groups were statistically significant only until 4.5 hours after the operation. Total intravenous morphine consumption during the first 9.5 hours after surgery in group 1 was 16.9+/-11.9 mg versus 31.1+/-18.0 mg in group 2 (P < .05). Postoperative nausea and vomiting occurred with equal frequency (87%) in both study groups, and there was no difference between groups in time to achieve fitness for discharge, i.e., a postanesthesia discharge score of > or = 9., Conclusion: Preoperative tumescent infiltration with lidocaine results in reduced pain and lower postoperative opioid requirements in the initial hours after reduction mammoplasty.

Purpose: To determine, after Caesarean delivery, uterine contractility and blood pressure following intravenous (i.v.) and intramyometrial (imy) injection of oxytocin., Methods: In a double-blind clinical trial 40 parturients scheduled for elective Caesarean section with spinal anaesthesia were randomized into two equal groups. One litre Ringer's lactate was administered i.v. before intrathecal injection of 1.7 ml bupivacaine 0.75% and 0.3 mg morphine. All patients received simultaneous i.v. and imy injections after removal of the placenta. Patients in Group 1 received 5 IU (10 IU.ml-1) oxytocin i.v. and 2 ml saline imy: Group 2 patients received 0.5 ml saline i.v. and 20 IU oxytocin into the myometrium. Baseline systolic blood pressure (SBP) and heart rate were measured before delivery and at one minute intervals for 15 min after injection of study solutions. Uterine contractility was assessed at 1, 2, 4, 6, 8, 10 and 15 min after oxytocin injection. Haemoglobin concentration before surgery and on first post-operative day was also recorded., Results: Mean decrease in systolic blood pressure (SBP) one minute after oxytocin was 8.4 mmHg in Group vs 14.6 mmHg in Group 2 (P < 0.001). Systolic blood pressure returned to baseline two minutes after oxytocin in Group 1 and after three minutes in Group 2. Uterine contractility and change in haemoglobin concentration were similar in both groups., Conclusion: Intramyometrial administration of 20 IU oxytocin after Caesarean delivery is associated with more severe hypotension than is i.v. injection of 5 IU oxytocin. Route of oxytocin injection did not affect uterine tone.

Studies of knockout mice indicate that the glycoprotein CD5, which is expressed on Tcells, most thymocytes and a subset of B cells, down-regulates TCR- and B cell receptor (BCR)-mediated signaling. CD5 is associated with the TCR and BCR, and is phosphorylated on cytoplasmic tyrosine residues following antigen receptor ligation. Cross-linking of CD5 or pervanadate stimulation of thymocytes induces the association of a 120-kDa tyrosine-phosphorylated protein with CD5. The proto-oncoprotein c-cbl associates with CD5 in pervanadate-stimulated thymocytes, and reprecipitation analysis demonstrates that the major proportion of CD5-associated pp120 is c-cbl. The GTPase-activating protein for ras (ras GAP), which is not tyrosine phosphorylated following CD5 cross-linking, associates with CD5 in pervanadate-stimulated thymocytes. Using tyrosine-phosphorylated peptides we show that ras GAP interacts in an SH2-mediated manner with the phosphorylated Y429SQP sequence of CD5. Both c-cbl and ras GAP have been proposed to suppress receptor-mediated signaling, and may contribute to CD5-mediated suppression of TCR or BCR signaling.

Purpose: To describe the anaesthetic management and report the incidence of PDPH in three parturients who had experienced accidental dural puncture during labour and the subsequent deliberate intrathecal insertion of an epidural catheter., Clinical Features: Inadvertent dural puncture with a 16-gauge Tuohy needle occurred during the first stage of labour at 3-4 cm cervical dilatation in all three women. The 20-gauge epidural catheter was immediately inserted into the subarachnoid space after accidental dural penetration. Intermittent intrathecal injections of lidocaine or bupivacaine with fentanyl were administered to provide analgesia during labour and delivery. Two of the women had spontaneous vaginal deliveries, whereas Caesarean section was performed in one case due to acute fetal distress during the second stage of labour. The intrathecal catheter was left in-situ for 13-19 hr after delivery and the women were questioned daily for symptoms of PDPH. None of the three women developed PDPH after dural puncture and intrathecal catheterisation with the epidural catheter., Conclusion: Immediate intrathecal insertion of the epidural catheter after accidental dural puncture during labour proved to be an effective prophylactic technique to prevent PDPH in these three parturients.

CD5 is a glycoprotein expressed on thymocytes, T cells, and a subset of B cells. Antibody-mediated cross-linking studies or studies on CD5 knockout mice implicate CD5 as a co-stimulatory or negative regulatory molecule. CD5 is rapidly phosphorylated on tyrosine (Y) residues following Tcell activation. Y429 and Y441 occur in an imperfect immunoreceptor tyrosine-based activation motif (ITAM)-like sequence. We investigated whether phosphatidylinositol (PI) 3-kinase, which binds to tyrosine-phosphorylated ITAM, interacts with CD5 following T cell activation. PI 3-kinase activity and the regulatory p85 subunit of PI 3-kinase associated with CD5 in pervanadate-stimulated, but not in unstimulated thymocytes. Cellular p85 as well as the recombinant Src homology 2 (SH2) domains of p85 bound a tyrosine-phosphorylated peptide encompassing Y463 with approximately threefold greater affinity than a doubly tyrosine-phosphorylated Y429-Y441 peptide. Binding of the C-SH2 domain to the Y463 phosphopeptide, together with preferential binding of the N-SH2 domain to the Y429-Y441 phosphopeptide, suggests a bivalent interaction. A 120-kDa phosphoprotein (pp120) associated with CD5 and specifically with the Y429-Y441 phosphopeptide in stimulated thymocytes. We conclude that stimulation of thymocytes with pervanadate induces the recruitment of PI 3-kinase and pp120 to CD5.

Cathepsin L was purified from the liver of a higher primate, the baboon (Papio ursinus), largely in a single-chain form and in the form of proteolytically active complexes with an endogenous cystatin. This mimics the situation found in both human and sheep livers. Both forms of cathepsin L were active at physiological pH. Physicochemical characterization and N-terminal amino sequencing of baboon cathepsin L showed a close relationship with the human enzyme. Cystatins with characteristics similar to those found for stefins A and B could also be purified from baboon livers. Proteolytically active, SDS-stable complexes could be shown to form in vitro with the molecules characterized as stefin B, but not with stefin A type cystatins. The non-inhibitory complexes could be shown to require less cysteine for activation than free cathepsin L and this, together with the above result, might indicate that a sulfhydryl interchange mechanism is responsible for the formation of covalent, non-inhibitory complexes.

In addition to their usual responsibilities, many nurses share valuable time, knowledge, experience, and compassion working as community volunteers. Thousands of Americans depend on services that could not be provided without the many hours of work contributed by volunteers. This article describes one day in the experience of a nurse who is a regular volunteer at a day center for the homeless. Patients' names have been changed to protect their privacy.