Heparin resistance in COVID-19 patients in the intensive care unit.

Patients with COVID-19 have a coagulopathy and high thrombotic risk. In a cohort of 69 intensive care unit (ICU) patients we investigated for evidence of heparin resistance in those that have received therapeutic anticoagulation. 15 of the patients have received therapeutic anticoagulation with either unfractionated heparin (UFH) or low molecular weight heparin (LMWH), of which full information was available on 14 patients. Heparin resistance to UFH was documented in 8/10 (80%) patients and sub-optimal peak anti-Xa following therapeutic LMWH in 5/5 (100%) patients where this was measured (some patients received both anticoagulants sequentially). Spiking plasma from 12 COVID-19 ICU patient samples demonstrated decreased in-vitro recovery of anti-Xa compared to normal pooled plasma. In conclusion, we have found evidence of heparin resistance in critically unwell COVID-19 patients. Further studies investigating this are required to determine the optimal thromboprophylaxis in COVID-19 and management of thrombotic episodes.

Relationship of femoral neck areal bone mineral density to volumetric bone mineral density, bone size, and femoral strength in men and women.

UNLABELLED: Using combined dual-energy X-ray absorptiometry (DXA) and quantitative computed tomography, we demonstrate that men matched with women for femoral neck (FN) areal bone mineral density (aBMD) have lower volumetric BMD (vBMD), higher bone cross-sectional area, and relatively similar values for finite element (FE)-derived bone strength. INTRODUCTION: aBMD by DXA is widely used to identify patients at risk for osteoporotic fractures. aBMD is influenced by bone size (i.e., matched for vBMD, larger bones have higher aBMD), and increasing evidence indicates that absolute aBMD predicts a similar risk of fracture in men and women. Thus, we sought to define the relationships between FN aBMD (assessed by DXA) and vBMD, bone size, and FE-derived femoral strength obtained from quantitative computed tomography scans in men versus women. METHODS: We studied men and women aged 40 to 90 years and not on osteoporosis medications. RESULTS: In 114 men and 114 women matched for FN aBMD, FN total cross-sectional area was 38% higher (P < 0.0001) and vBMD was 16% lower (P < 0.0001) in the men. FE models constructed in a subset of 28 women and 28 men matched for FN aBMD showed relatively similar values for bone strength and the load-to-strength ratio in the two groups. CONCLUSIONS: In this cohort of young and old men and women from Rochester, MN, USA who are matched by FN aBMD, because of the offsetting effects of bone size and vBMD, femoral strength and the load-to-strength ratio tended to be relatively similar across the sexes.

Augmented environments for minimally invasive therapy: implementation barriers from technology to practice.

Augmented environments for medical applications have been explored and developed in an effort to enhance the clinician's view of anatomy and facilitate the performance of minimally invasive procedures. These environments must faithfully represent the real surgical field and require seamless integration of pre- and intra-operative imaging, surgical instrument tracking and display technology into a common framework centered around the patient. However, few image guidance environments have been successfully translated into clinical use. Several challenges that contribute to the slow progress of integrating such environments into clinical practice are discussed here in terms of both technical and clinical limitations.

A novel population of myeloid cells responding to coxsackievirus infection assists in the dissemination of virus within the neonatal CNS.

Enterovirus infection in newborn infants is a significant cause of aseptic meningitis and encephalitis. Using a neonatal mouse model, we previously determined that coxsackievirus B3 (CVB3) preferentially targets proliferating neural stem cells located in the subventricular zone within 24 h after infection. At later time points, immature neuroblasts, and eventually mature neurons, were infected as determined by expression of high levels of viral protein. Here, we show that blood-derived Mac3(+) mononuclear cells were rapidly recruited to the CNS within 12 h after intracranial infection with CVB3. These cells displayed a myeloid-like morphology, were of a peripheral origin based on green fluorescent protein (GFP)-tagged adoptive cell transplant examination, and were highly susceptible to CVB3 infection during their migration into the CNS. Serial immunofluorescence images suggested that the myeloid cells enter the CNS via the choroid plexus, and that they may be infected during their extravasation and passage through the choroid plexus epithelium; these infected myeloid cells ultimately penetrate into the parenchyma of the brain. Before their migration through the ependymal cell layer, a subset of these infected myeloid cells expressed detectable levels of nestin, a marker for neural stem and progenitor cells. As these nestin(+) myeloid cells infected with CVB3 migrated through the ependymal cell layer, they revealed distinct morphological characteristics typical of type B neural stem cells. The recruitment of these novel myeloid cells may be specifically set in motion by the induction of a unique chemokine profile in the CNS induced very early after CVB3 infection, which includes upregulation of CCL12. We propose that intracranial CVB3 infection may lead to the recruitment of nestin(+) myeloid cells into the CNS which might represent an intrinsic host CNS repair response. In turn, the proliferative and metabolic status of recruited myeloid cells may render them attractive targets for CVB3 infection. Moreover, the migratory ability of these myeloid cells may point to a productive method of virus dissemination within the CNS.

T cell growth factor receptors. Quantitation, specificity, and biological relevance.

To examine directly the hypothesis that T cell growth factor (TCGF) interacts with target cells in a fashion similar to polypeptide hormones, the binding of radiolabeled TCGF to various cell populations was investigated. The results indicate that TCGF interacts with activated T cells via a receptor through which it initiates the T cell proliferative response. Internally radiolabeled TCGF, prepared from a human T leukemia cell line and purified by gel filtration and isoelectric focusing, retained biological activity and was uniform with respect to size and charge. Binding of radiolabeled TCGF to TCGF-dependent cytolytic T cells occurred rapidly (within 15 rain at 37 degrees C) and was both saturable and largely reversible. In addition, at 37 degrees C, a receptor- and lysosome-dependent degradation of TCGF occurred. Radiolabeled TCGF binding was specific for activated, TCGF-responsive T cells. Whereas unstimulated lymphocytes of human or murine origin and lipopolysaccharide-activated B cell blasts expressed few if any detectable binding sites, lectin- or alloantigen-activated cells had easily detectable binding sites. Moreover, compared with lectin- or alloantigen-activated T cells, long-term TCGF-dependent cytolytic and helper T cell lines and TCGF-dependent neo-plastic T cell lines bound TCGF with a similar affinity (dissociation constant of 5-25 pM) and expressed a similar number of receptor sites per cell (5,000-15,000). In contrast, a number of TCGF-independent cell lines of T cell, B cell, or myeloid origin did not bind detectable quantities of radiolabeled TCGF. Binding of radiolabeled TCGF to TCGF-responsive cells was specific, in that among several growth factors and polypeptide hormones tested, only TCGF competed for binding. Finally, the relative magnitude of T cell proliferation induced by a given concentration of TCGF closely paralleled the fraction of occupied receptor sites. As the extent of T cell clonal expansion depends on TCGF and on the TCGF receptor, the dissection of the molecular events surrounding the interaction of TCGF and its receptor that these studies permit, should provide new insight into the hormonelike regulation of the immune response by this lymphokine.

Internalization of interleukin 2 is mediated by the beta chain of the high-affinity interleukin 2 receptor.

High-affinity IL-2-R correspond to a membrane receptor complex composed of two different IL-2-binding proteins, the Tac antigen (alpha chain) and a 70-75 kD beta chain. Using cell lines that express either the alpha or the beta protein, we demonstrate that IL-2 internalization occurs when ligand is bound to the isolated beta chain, but not when it is bound to the isolated alpha chain. The kinetics of IL-2 internalization mediated by the intermediate-affinity beta chain were nearly identical to those of the high-affinity alpha/beta heterodimer (t1/2 of 10-15 min), and each type of receptor targeted the bound IL-2 for intracellular degradation in lysosomes. The beta chain thus appeared to provide the essential element necessary for ligand internalization by both types of IL-2-R.

Direct demonstration of the identity of T cell growth factor binding protein and the Tac antigen.

Radiolabeled molecules from detergent-solubilized human T cell blasts were fractionated on affinity supports coupled with T cell growth factor (TCGF) and anti-Tac antibody. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that a glycoprotein of approximately 58,000 mol wt was bound in both cases. Sequential binding to the two affinity supports demonstrated that the molecules recognized in each instance were identical. Thus, the Tac antigen contains the cellular binding site for TCGF. Note added in proof: Preliminary binding experiments using high concentrations of [3H]leucine, lysine TCGF with a low specific radioactivity indicate the existence of a sizeable pool of receptor sites with an affinity 2,000-10,000 times lower than that of the high affinity receptors measured in Fig. 1. Such sites may explain the numerical discrepancy between early TCGF binding experiments (5) and the binding of the anti-Tac antibody. The hypothesis that the anti-Tac antibody apparently reacts with both classes of receptor would explain its effect on the physiological response (high affinity binding) and the high level of Tac antigen on the cell surface.

Characterization of the interleukin 2 receptors (IL-2R) expressed on human natural killer cells activated in vivo by IL-2: association of the p64 IL-2R gamma chain with the IL-2R beta chain in functional intermediate-affinity IL-2R.

Interleukin 2 (IL-2) receptors expressed on the surface of activated T cells and natural killer (NK) cells exhibit a variety of affinity states depending on their subunit composition. Low-affinity binding is associated with a 55-kDa alpha chain, intermediate-affinity binding with a 70-75-kD beta chain, and high-affinity binding with a bimolecular complex of the alpha and beta subunits. In a previous study of the IL-2 receptors expressed on NK cells obtained from cancer patients after in vivo IL-2 therapy, we documented a discrepancy between the level of beta chain and the level of intermediate-affinity IL-2 binding sites expressed on the cell surface. Based on this result, we postulated that formation of intermediate-affinity receptor sites required a component in addition to the beta chain, and that this component was present at limiting levels in the patient NK cells. In the present study we have examined the structure of the intermediate-affinity receptor complex using monoclonal antibodies that recognize the beta chain, but that do not interfere with its ability to bind IL-2. Evidence is presented establishing the physical association of a novel protein of 64 kD with the beta chain in intermediate-affinity IL-2 binding sites. This molecule, termed IL-2R gamma chain, coprecipitated with beta chains prepared from cells that had been incubated with IL-2, but was undetectable in immunoprecipitates prepared in the absence of IL-2. Examination of gamma chain expression in post-IL-2 therapy NK cells, where only low levels of intermediate-affinity IL-2 binding were detectable, revealed that the gamma chain was associated with, on average, only 10-12% of the beta chains expressed on such cells. This contrasted with approximately equal levels of beta and gamma chain expression on YT cells, a cell line that has both high levels of cell surface beta chain expression and high levels of IL-2 binding. Thus, the ratio of gamma chain to beta chain present in the immunoprecipitates roughly correlated with the proportion of beta chain involved in intermediate-affinity receptor sites. This result suggests that the 64-kD gamma chain is the component responsible for regulating the affinity of IL-2 association with the beta subunit. By further defining the structural components necessary for IL-2 receptor formation, these studies provide additional insight into mechanisms whereby lymphocytes might regulate their responsiveness to IL-2.

Low and high affinity cellular receptors for interleukin 2. Implications for the level of Tac antigen.

Interleukin 2 promotes proliferation of T cells by virtue of its interaction with a high-affinity cell surface receptor. This receptor is a 55,000 mol wt glycoprotein that is also recognized by the murine monoclonal antibody, anti-Tac. Quantitative binding studies with radiolabeled IL-2 and anti-Tac, however, initially indicated far more antibody binding sites per cell than IL-2 binding sites. Extension of the IL-2 binding analysis to concentrations several thousand-fold higher than that necessary for the T cell proliferative response demonstrated the existence of a class (or classes) of low-affinity IL-2 binding sites. Inclusion of the low-affinity IL-2 binding greatly reduced the quantitative discrepancy in the ligand binding assays. That the low-affinity binding, as well as the high-affinity interaction, was associated with the Tac molecule was indicated by the finding that the antibody could substantially or totally block the entire spectrum of IL-2 binding and by the finding that IL-2 could in turn block all radiolabeled anti-Tac binding. The low-affinity sites were found on activated T cells, several human and murine T cell lines and two examples of Tac-positive B cells. The physiological role of the low-affinity IL-2 binding sites and the molecular changes in the Tac protein that give rise to the affinity differences remain open to investigation.

Stable expression of cDNA encoding the human interleukin 2 receptor in eukaryotic cells.

Human interleukin 2 (IL-2) receptor cDNA derived from HUT 102B2 cells was stably expressed in murine L cells. These L cell transfectants (a) displayed surface receptors of the aberrant size of the IL-2 receptors on HUT 102B2 cells, (b) did not respond to exogenous IL-2 with augmented proliferation, and (c) expressed low affinity but not high affinity receptors for IL-2.

Increased expression of the interleukin 2 (IL-2) receptor beta chain (p70) on CD56+ natural killer cells after in vivo IL-2 therapy: p70 expression does not alone predict the level of intermediate affinity IL-2 binding.

The expression of the 70-kD beta subunit of the interleukin 2 receptor (IL-2R) has been examined on peripheral blood lymphocytes (PBL) obtained from patients receiving systemic infusions of IL-2. Using monoclonal antibodies directed against p70, flow cytometric analyses revealed a greater than threefold increase in expression of the IL-2R beta chain on CD56+ natural killer (NK) cells from post-IL-2 therapy PBL relative to pre-therapy cells. The level of p70 expression on the post-therapy cells was three- to fourfold greater (based on fluorescence intensity) than the level of p70 expression on YT cells, an NK-like cell line that expresses approximately 12,000 intermediate affinity IL-2 binding sites/cell. Despite the high level of p70 expression, in 125I-IL-2 binding assays only 790-1,290 intermediate affinity IL-2 binding sites/cell were detected on post-therapy cells from six patients. These data represent the first report of increased p70 expression after in vivo IL-2 administration and suggest a requirement for at least one additional subunit for the formation of functional intermediate affinity IL-2Rs. Furthermore, the presence on the surface of post-therapy NK cells of excess p70 that does not bind IL-2 with intermediate affinity implies that the formation of intermediate affinity IL-2Rs is not solely determined by the level of p70 expression, and that the response of NK cells to IL-2 might be regulated by altering the expression of p70 or some other IL-2R subunit.

Lymphokine-activated killer cell phenomenon. III. Evidence that IL-2 is sufficient for direct activation of peripheral blood lymphocytes into lymphokine-activated killer cells.

Purified interleukin 2 (IL-2) was found to be sufficient for direct activation of peripheral blood lymphocytes into lymphokine-activated killer (LAK) cells. The LAK activation factor was directly and consistently associated with IL-2 activity using classic protein purification techniques, adsorption to IL-2-dependent cell lines, and inhibition with anti-Tac antibody. As yet, no other cytokines have been found that perform the same role.

Expression of interleukin 2 receptors on activated human B cells.

Using anti-Tac, a monoclonal anti-interleukin 2 (IL-2) receptor antibody, we have explored the possibility that certain activated B cells display receptors for IL-2. Resting normal B cells and unselected B cell lines established from normal individuals were Tac antigen negative. In contrast, the cell surface Tac antigen expression was demonstrable on 6 of 10 B cell lines from patients with Burkitt's lymphoma, 5 of 6 B cell lines derived from patients with HTLV-I-associated adult T cell leukemia (including all four that had integrated HTLV-I into their genome), and on certain normal B cells activated with pokeweed mitogen. Furthermore, cloned Epstein-Barr virus-transformed B cell lines derived from Tac-positive normal B cells continued to express the Tac antigen in long-term cultures and manifested high affinity IL-2 receptors identified in binding studies with purified radiolabeled IL-2. The line 5B4 developed in the present study could be induced with purified JURKAT-derived or recombinant IL-2 to express a larger number of IL-2 receptors. Furthermore, the addition of IL-2 to the 5B4 B cell line augmented IgM synthesis, which could be blocked by the addition of anti-Tac. The size of the IL-2 receptors expressed on the cloned normal B cell lines was similar (53,000-57,000 daltons) to that of receptors on phytohemagglutinin-stimulated T cell lymphoblasts. Thus, certain malignant and activated normal B cells display the Tac antigen and manifest high affinity receptors for IL-2. These data suggest that IL-2 may play a role in the differentiation of activated B cells into immunoglobulin-synthesizing and -secreting cells.

Viral persistence and chronic immunopathology in the adult central nervous system following Coxsackievirus infection during the neonatal period.

Coxsackieviruses are significant human pathogens, and the neonatal central nervous system (CNS) is a major target for infection. Despite the extreme susceptibility of newborn infants to coxsackievirus infection and viral tropism for the CNS, few studies have been aimed at determining the long-term consequences of infection on the developing CNS. We previously described a neonatal mouse model of coxsackievirus B3 (CVB3) infection and determined that proliferating stem cells in the CNS were preferentially targeted. Here, we describe later stages of infection, the ensuing inflammatory response, and subsequent lesions which remain in the adult CNS of surviving animals. High levels of type I interferons and chemokines (in particular MCP-5, IP10, and RANTES) were upregulated following infection and remained at high levels up to day 10 postinfection (p.i). Chronic inflammation and lesions were observed in the hippocampus and cortex of surviving mice for up to 9 months p.i. CVB3 RNA was detected in the CNS up to 3 months p.i at high abundance ( approximately 10(6) genomes/mouse brain), and viral genomic material remained detectable in culture after two rounds of in vitro passage. These data suggest that CVB3 may persist in the CNS as a low-level, noncytolytic infection, causing ongoing inflammatory lesions. Thus, the effects of a relatively common infection during the neonatal period may be long lasting, and the prognosis for newborn infants recovering from acute infection should be reexplored.

Three-dimensional imaging of heart, lungs, and circulation.

A new imaging device, the dynamic spatial reconstructor (DSR), is described. It differs from commercially available computed tomography scanners in several ways. It images a volume rather than a slice; it images the volume in stop-action to minimize blurring due to motion; and it repeats the scan 60 times per second so that the functional movements of heart muscle and lung tissue and the distribution of roentgen contrast medium in blood can be quantitated in any portion of the body, especially in the heart, great vessels, and lungs. The system is under evaluation as a research tool for physiologic and, ultimately, clinical investigations.

Patient specific physical anatomy models.

The advent of small footprint stereo-lithographic printers and the ready availability of segmentation and surface modeling software provide a unique opportunity to create patient-specific physical models of anatomy, validation of image guided intervention applications against phantoms that exhibit naturally occurring anatomic variation. Because these models can incorporate all structures relevant to a procedure, this allows validation to occur under realistic conditions using the same or similar techniques as would be used in a clinical application. This in turn reduces the number of trials and time spent performing in-vivo validation experiments. In this paper, we describe our general approach for the creation of both non-tissue and tissue-mimicking patient-specific models as part of a general-purpose patient emulation system used to validate image guided intervention applications.

Analysis of prostaglandin E2 effect on T lymphocyte activation. Abrogation of prostaglandin E2 inhibitory effect by the tumor promotor 12.0 tetradecanoyl phorbol-13 acetate.

We have investigated the inhibitory potential of prostaglandin E2 (PGE2) with respect to intracellular messengers implicated in the signaling system of T-lymphocyte activation pathway. Using the fluorescent indicator Quin 2, it is demonstrated that PGE2 inhibits the increase in cytosolic-free calcium concentration [Ca2+]i. Reconstitution of calcium mobilization in the presence of PGE2 by the calcium ionophore A23187 results in a partial restoration of both interleukin 2 (IL2) production and cell proliferation and has no effect on the inhibition of transferrin receptor expression. In contrast, the treatment of cell cultures with the tumor promotor 12.0 tetra decanoyl phorbol-13-acetate (TPA) abrogates the suppressor activity of PGE2. When T lymphocyte stimulation is provided by the combination of A23187 and TPA, the PGE2 inhibitory effect does not occur. These data also indicate that the down regulation of transferrin receptor by PGE2 is proximal to protein kinase C activation and is not associated with decreased expression of the functional IL2 receptor.

Coxsackievirus targets proliferating neuronal progenitor cells in the neonatal CNS.

Type B coxsackieviruses (CVB) frequently infect the CNS and, together with other enteroviruses, are the most common cause of viral meningitis in humans. Newborn infants are particularly vulnerable, and CVB also can infect the fetus, leading to mortality, or to neurodevelopmental defects in surviving infants. Using a mouse model of neonatal CVB infection, we previously demonstrated that coxsackievirus B3 (CVB3) could infect neuronal progenitor cells in the subventricular zone (SVZ). Here we extend these findings, and we show that CVB3 targets actively proliferating (bromodeoxyuridine+, Ki67+) cells in the SVZ, including type B and type A stem cells. However, infected cells exiting the SVZ have lost their proliferative capacity, in contrast to their uninfected companions. Despite being proliferation deficient, the infected neuronal precursors could migrate along the rostral migratory stream and radial glia, to reach their final destinations in the olfactory bulb or cerebral cortex. Furthermore, infection did not prevent cell differentiation, as determined by cellular morphology and the expression of maturation markers. These data lead us to propose a model of CVB infection of the developing CNS, which may explain the neurodevelopmental defects that result from fetal infection.

An integrated system for real-time image guided cardiac catheter ablation.

Minimally invasive cardiac catheter ablation procedures require effective visualization of the relevant heart anatomy and electrophysiology (EP). In a typical ablation procedure, the visualization tools available to the cardiologist include bi-plane fluoroscopy, real-time ultrasound, and a coarse 3D model which gives a rough representation of cardiac anatomy and electrical activity. Recently, there has been increased interest in incorporating detailed, patient specific anatomical data into the cardiac ablation procedure. We are currently developing a prototype system which both integrates a patient specific, preoperative data model into the procedure as well as fuses the various visualization modalities (i.e. fluoroscopy, ultrasound, EP) into a single display. In this paper, we focus on two aspects of the prototype system. First, we describe the framework for integrating the various system components, including an efficient communication protocol. Second, using a simple two-chamber phantom of the heart, we demonstrate the ability to integrate preoperative data into the ablation procedure. This involves the registration and visualization of tracked catheter points within the cardiac chambers of the preoperative model.

Has control of hypercholesterolemia and hypertension in type 1 diabetes improved over time?

OBJECTIVE: To determine the extent to which patients' awareness, treatment, and control of hypertension and hypercholesterolemia have changed over time and to examine factors associated with awareness and treatment in a type 1 diabetes population. RESEARCH DESIGN AND METHODS: Data from six examinations conducted over 10 years from the Pittsburgh Epidemiology of Diabetes Complications Study, a prospective study of subjects with childhood-onset (<17 years of age) type 1 diabetes diagnosed between 1950 and 1980 and followed since 1986, were analyzed. Hypertension and hypercholesterolemia were defined according to the concurrent Joint National Committee and National Cholesterol Education Program Adult Treatment Panel criteria, respectively. RESULTS: Results demonstrated that awareness of both conditions has improved; however, control is not optimal (e.g., only 32.1 and 28% of those with hypertension in 1986-1988 and 1996-1998 were controlled, while for hypercholesterolemia, the rates were 0 and 5.5%, respectively). Stratified by age-group (18-29, 30-39, and >40 years), the youngest subjects with hypercholesterolemia were least likely to be treated and controlled to goal levels. Older age and physician contact were correlates of awareness and treatment of hypertension at baseline, while presence of renal or coronary complications was also associated with awareness and treatment of both hypertension and hypercholesterolemia at the 10-year follow-up. CONCLUSIONS: There is a considerable treatment gap, particularly for hypercholesterolemia. Improved treatment of both hypertension and hypercholesterolemia are clearly needed, particularly hypercholesterolemia in younger age-groups who have not yet experienced long-term complications.