Impact of Prehospital Pain Management on Emergency Department Management of Injured Children.

OBJECTIVE: Provision of analgesia for injured children is challenging for Emergency Medical Services (EMS) clinicians. Little is known about the effect of prehospital analgesia on emergency department (ED) care. We aimed to determine the impact of prehospital pain interventions on initial ED pain scale scores, timing and dosing of ED analgesia for injured patients transported by EMS. METHODS: This is a planned, secondary analysis of a prospective multicenter cohort of children with actual or suspected injuries transported to one of 11 PECARN-affiliated EDs from July 2019-April 2020. Using Wilcoxon rank sum for continuous variables and chi-square testing for categorical variables, we compared the change in EMS-to-ED pain scores and timing and dosing of ED-administered opioid analgesia in those who did and those who did not receive prehospital pain interventions. RESULTS: We enrolled 474 children with complete prehospital and ED pain management data. Prehospital interventions were performed on 262/474 (55%) of injured children and a total of 88 patients (19%) received prehospital opioids. Children who received prehospital opioids with or without adjunctive non-pharmacologic pain management experienced a greater reduction in pain severity and were more likely to receive ED opioids in higher doses earlier and throughout their ED care. Non-pharmacologic pain interventions alone did not impact ED care. CONCLUSIONS: We demonstrate that prehospital opioid analgesia is associated with both a significant reduction in pain severity at ED arrival and the administration of higher doses of opioid analgesia earlier and throughout ED care.

Soluble receptor for advanced glycation end products in multiple sclerosis: a potential marker of disease severity.

OBJECTIVES: To compare serum levels of the receptor for advanced glycation end products (sRAGE) between multiple sclerosis (MS) patients and healthy control subjects, and to investigate whether serum sRAGE levels correlate with MS disease severity as indicated by the Kurtzke Expanded Disability Status Scale (EDSS). METHOD: 37 patients with clinical diagnosis of MS and 22 healthy control subjects were investigated in a cross-sectional study using enzyme-linked immunosorbent assays (ELISA). RESULTS: Serum levels of sRAGE were found to be significantly lower in MS patients compared to levels in healthy controls (p = 0.005). A trend toward lower levels of serum sRAGE was observed in female MS patients compared to their male counterparts (p = 0.05). A relationship between sRAGE and EDSS, and sRAGE and rate of clinical relapse was observed (p = 0.012). CONCLUSION: The significant reduction of sRAGE in MS patients relative to healthy controls supports the potential role for RAGE axis in MS clinical pathology. Lower levels of sRAGE may be associated with enhanced inflammatory responses. Based on these observations, further investigations into the role of sRAGE in MS clinical pathology is warranted.

Thiophilic interaction chromatography of prostate-specific antigen.

Prostate-specific antigen (PSA) protein and complexes of PSA with alpha1-antichymotrypsin (PSA-ACT) or alpha2-macroglobulin (PSA-A2M) prepared in vitro, have strong affinity for different thiophilic gels (T-gel). Free PSA, and these PSA complexes can be isolated due to their affinity for T-gels. The average recovery of PSA from several of the T-gels, based upon ELISA measurements, was 84 to 94%. The data suggest that T-gel affinity can be explored for the purification of free and complexed PSA from various biologic fluids.

Mild hyperthermia modulates biological activities of interferons.

A significant enhancement of antiviral activity of human IFN-alpha, -beta and -gamma and murine IFN-gamma is observed when cells are treated with mild hyperthermia (39 degrees C) during antiviral assays. Treatment of primary human fibroblast cells with mild hyperthermia for 4 and 24 hours prior to interferon antiviral assays (pre-assay hyperthermia) further enhances interferon antiviral activity. An enhancement of interferon induced enzyme, 2,5-oligoadenylate synthetase, is also observed in cells treated with interferon and mild hyperthermia. This increase in enzyme activity is, in part, responsible for the observed increase in interferon antiviral activity with hyperthermia. Besides antiviral activity, mild hyperthermia also increases interferon antiproliferative activity on different tumour cells beyond its effect at normal physiological temperatures. On the other hand, mild hyperthermia decreases human interferon production in both human and murine cells when challenged with a viral or non-viral inducer. Also, mild hyperthermia suppresses interferon-mediated enhancement of natural killer (NK) cell activity in human and murine cells. The findings demonstrate that, although mild hyperthermia has suppressive effects upon interferon production and NK cell activity, it significantly increases both antiviral and antiproliferative activities of all three human interferons. These observations have direct bearing upon clinical utilization of exogenously administered interferons to late stage cancer patients who for the most part have a weaker immune system. In these patients, the antiviral and antiproliferative efficacies of administered interferon can be enhanced by combining interferon and hyperthermia.

Cocaine differentially modulates chemokine production by mononuclear cells from normal donors and human immunodeficiency virus type 1-infected patients.

Earlier studies have supported a significant role for cocaine in the susceptibility to and the progression of human immunodeficiency virus type 1 (HIV-1) infection. Recently, several unique HIV-1 entry coreceptors (e.g., CCR5 and CCR3) and a trio of HIV-1-specific suppressor chemokines, namely, RANTES (regulated-upon-activation T expressed and secreted), macrophage inflammatory protein 1alpha (MIP-1alpha) and MIP-1beta, were identified. Although cocaine has been linked to the immunopathogenesis of HIV-1 infection, the corresponding cellular and molecular mechanism(s) have not been well defined. We hypothesize that cocaine mediates these pathologic effects through the downregulation of HIV-1-suppressing chemokines and/or upregulating HIV-1 entry coreceptors in HIV-1-infected subjects, resulting in disease progression to AIDS. Our results show that cocaine selectively downregulates endogenous MIP-1beta secretion by normal peripheral blood mononuclear cells (PBMC), while cocaine did not affect the MIP-1beta production by PBMC from AIDS patients. Cocaine also selectively suppresses lipopolysaccharide-induced MIP-1beta production by PBMC from HIV-infected patients. Further, cocaine significantly downregulates endogenous MIP-1beta gene expression, while it upregulates HIV-1 entry coreceptor CCR5 by normal PBMC. These studies suggests a role for cocaine as a cofactor in the pathogenesis of HIV infection and support the premise that cocaine increases susceptibility to and progression of HIV-1 infection by inhibiting the synthesis of HIV-1 protective chemokines and/or upregulating the HIV-1 entry coreceptor, CCR5.

Mechanism(S) of interferon inhibitory activity in blood from patients with AIDS and patients with lupus erythematosus with vasculitis.

We have earlier reported that patients suffering from acquired immuno-deficiency syndrome (AIDS), systemic lupus erythematosus (SLE) with vasculitis, Wegner granulomatosis and certain types of late stage cancer have interferon inhibitory activity in their serum. The purpose of this study was to identify the factor(s) involved in this interferon inhibitory activity. Twenty patients with advanced AIDS, twenty patients with SLE and vasculitis and twenty normal healthy controls between ages 25-40 years were studied. In contrast to normal, healthy controls, significant interferon inhibitory activity was found in AIDS and SLE patients. This appears to be largely due to: (a) increased soluble circulating interferon alpha/beta receptors, (b) increased prostaglandin E2 levels which inhibits interferon and (c) a interferon inhibitory protein. Further understaging of the nature of interferon inhibitory activity in the patient's sera and development of anti-interferon inhibitory agents would greatly enhance interferons potential as a treatment modality.

Immunoregulatory effects of morphine on human lymphocytes.

It is now well established that parenteral drug abuse is a significant risk factor for contracting human immunodeficiency virus type 1 (HIV-1) infection and subsequently developing AIDS. Earlier studies have shown that morphine can modulate various immune responses and therefore support the premise that morphine is a cofactor in susceptibility to and progression of HIV infection. Dysregulation of interferon (IFN) production, nonspecific apoptosis of T cells, and the immune response to soluble HIV gene products have been associated with potential mechanisms of pathogenesis in HIV disease. The present study was undertaken to examine the immunomodulatory role of morphine on HIV protein-induced lymphocyte proliferative responses, Sendai and Newcastle disease virus-induced alpha IFN (IFN-alpha) and IFN-beta production by lymphocytes and fibroblast cells, respectively, and induction of apoptosis of normal lymphocytes in vitro. Our results demonstrate that HIV protein-induced human lymphocyte proliferative responses were significantly inhibited by morphine in a dose-dependent manner. Furthermore, morphine significantly inhibited both IFN-alpha and IFN-beta production by normal lymphocytes and fibroblasts but induced apoptosis of normal lymphocytes. Inhibition of IFN-alpha production by morphine could be reversed by the opiate receptor antagonist naloxone. This suggests that the immunomodulatory effects of morphine are mediated through the opioid receptor. These studies support a role of morphine as a cofactor in the pathogenesis of HIV infection and describe some of the possible pathologic mechanisms which underlie the immunoregulatory effects of morphine.

Mapping of epitopes on porcine zona pellucida-3 alpha by monoclonal antibodies inhibiting oocyte-sperm interaction.

Zona pellucida glycoprotein 3 alpha (ZP3 alpha) has been designated as the primary sperm receptor ligand in porcine gamete interaction. In this study, epitopes were mapped on porcine ZP3 alpha (pZP3 alpha) by using monoclonal antibodies (mAbs) possessing in vitro contraceptive efficacy. Using Western blots, we tested recombinant pZP3 alpha fragments expressed as fusion proteins in Escherichia coli and corresponding to pZP3 alpha precursor protein amino acid residues 18-142 (F1), 140-243 (F2), 239-363 (F3), and 359-462 (F4), for reactivities with mAbs. MAb-403 reacted with F3, and mAbs-412 and -421 with F2. MAb-420 showed weak reactivity with F1. Synthesis of overlapping 12-mer peptides further resolved the epitope for mAb-420 to amino acid residues 133-144, mAb-421 to 157-168, mAb-412 to 205-216, and mAb-403 to 301-312. MAbs-412 and -420 inhibited the binding of boar sperm to zona-encased porcine oocytes. These results, the first to define peptide epitopes of pZP3 alpha, should assist in the design of a synthetic peptide-based immunocontraceptive vaccine.

Differential effects of human immunodeficiency virus type 1 envelope protein gp120 on interferon production by mononuclear cells from adults and neonates.

While considerable progress in examining the course of human immunodeficiency virus (HIV) infection in adults has been made, a better understanding of the natural history of perinatal HIV infection remains to be obtained. Dysregulation of the production and functions of various cytokines, especially the interferons (IFNs), during HIV infections has been reported. Using an in vitro model system, we examined the effects of the HIV type 1 envelope protein, gp120 (10, 50, and 100 ng/ml), on gamma IFN (IFN-gamma) and IFN-alpha production by lymphocytes from neonates and adults and also examined the potential regulatory effects of gp120 on phorbol 12-myristate acetate (PMA)- and Sendai virus-induced IFN-gamma and IFN-alpha production by lymphocytes. PMA at a concentration of 50 ng/ml plus 50 ng of calcium ionophore A23187 per ml was used to induce IFN-gamma, while 150 hemagglutinating units of Sendai virus was used to induce IFN-alpha production. The antiviral activity of both IFN-alpha and IFN-gamma in leukocyte culture supernatants was assayed on BG-9 cells by a dye uptake technique using vesicular stomatitis virus as a challenge virus. Placental cord blood leukocyte (CBL) samples from healthy, term infants and adult peripheral blood leukocytes (APBL) produced no IFN in response to gp120. However, CBL produced significantly decreased levels of IFN-gamma compared with APBL in response to PMA plus ionophore. gp120 significantly suppressed both Sendai virus-induced IFN-alpha and PMA-induced IFN-gamma production by both CBL and APBL in a dose-dependent manner. However, gp120-induced suppression of IFN-alpha and IFN-gamma was significantly greater with CBL than with APBL. Treatment of CBL and APBL with gp120 did not induce any phenotypic alteration of the CD45 RO+ subset. Increased suppression of IFN-alpha and IFN-gamma production by gp120 in neonates may partially explain their apparent increased susceptibility to the clinical progression of HIV infections compared with that of adults.

Segregation of normal and pathological human red blood cells, lymphocytes and fibroblasts by immobilized metal-ion affinity partitioning.

Immobilized metal-ion affinity partitioning (IMAP) is shown to be useful as a preliminary screening test and for the separation of different cell populations based upon recognition of the differences in the proteins on cell surfaces. The feasibility of using IMAP to segregate a spectrum of normal human cells (red blood cells, lymphocytes and fibroblasts) from their counterpart pathological cells has been demonstrated. A clear segregation between normal and sickle-cell anemia red blood cells (RBC), or malaria (Plasmodium vivax) infected RBCs was obtained. Further, the partition differences were found to depend on the nature and the concentrations of metal used. Cells from breast cancer and those from the lung adenocarcinoma showed differences in their partition pattern as compared to normal fibroblasts when PEG-IDA-M(II) was added to the phase system. Maximum differences between the three cell populations were observed in the presence of 10% PEG-IDA-Ni(II). Normal lymphocytes and Burkitt's lymphoma cells (Raji and Namalwa cell lines) were shown to partition differently in the presence of PEG-IDA-M(II) in the phase system. Normal lymphocytes could be distinguished from the Burkitt's lymphoma cell lines in all three phases (top, interface and bottom), in the presence of 10% PEG-IDA-Ni(II) in the system. These differences in the partition behavior could mainly be attributed to the density, surface exposure and micro-environment of histidine residues of cell membrane-associated proteins. These data, along with those obtained for normal and pathological human cells show that IMAP could be a simple and versatile tool for the segregation and study of cells.

Selective inhibition by alcohol and cortisol of natural killer cell activity of lymphocytes from cord blood.

1. The immunosuppressive effects of drugs such as alcohol or hormones such as cortisol may be age-related. To test this hypothesis, the authors investigated the in vitro effects of ethanol (EtOH) and cortisol on Natural Killer (NK) cell activity of lymphocytes from normal cord blood in comparison with that of lymphocytes from normal adult peripheral blood. 2. K562, an erythroleukemia cell line, was used as a target in a 4 hr 51Cr release assay. 3. Ethanol at 0.3% (V/V) and cortisol at 0.05, 0.1 and 0.2 microgram/ml concentrations, added directly to a mixture of effector and target cells significantly suppressed the NK activity of cord blood lymphocytes in a dose dependent fashion, whereas similar concentrations of either EtOH or cortisol did not manifest significant immunoregulatory effects on NK cell activity of normal adult lymphocytes. 4. Pre-treatment of the target with either EtOH or cortisol for 4 hours did not affect cytotoxicity. Inhibition of cytotoxicity was also not due to direct toxicity of effector cells because lymphocytes treated with either EtOH or cortisol showed normal 51Cr release and their viability was comparable to that of untreated control cells. 5. This suggests a selective inhibitory effect of EtOH and cortisol on NK activity of neonatal lymphocytes that may be of clinical significance.

Pentoxifylline and meclofenamic acid treatment reduces clinical manifestations in a murine model of AIDS.

C57/BL/6 mice infected with LP-BM5 MuLV virus developed an AIDS-like disease (MAIDS) with splenomegaly, leukopenia, thrombocytopenia, anemia, decreased numbers of helper/inducer and suppressor/cytotoxic T-cells and decreased production of interferon alpha. We have shown previously that HIV-associated Kaposi's sarcoma tissue contains high levels of prostaglandin E2 (PgE2), and this inhibits interferon synthesis through a cAMP-dependent second-messenger process. In this study we treated groups of MAIDS-infected mice with combinations of pentoxifylline, an agent which increases cAMP and inhibits phosphodiesterases, and sodium meclofenamic acid, a PgE2 inhibitor. Treated mice showed: 1) significantly higher total leukocyte and platelet counts, 2) higher total L3T4+ (helper/inducer) and Lyt-2+ (suppressor-cytotoxic) T-cell population. Pathologic examination also showed significantly less hepatosplenomegaly and lymphadenopathy in animals treated with pentoxifylline and meclofenamic acid. Partly, PgE2-induced suppression of interferon alpha production may mediate expression of retrovirus infection in this murine model of AIDS.

Mapping of immunogenic domains on porcine zona pellucida 3 alpha and beta glycoproteins by murine monoclonal antibodies.

PROBLEM: Immunization with zona pellucida (ZP) leads to block in fertility with variable degree of ovarian dysfunctions. To design an immunocontraceptive vaccine based on synthetic peptides of zona pellucida, it is imperative to identify and define epitopes involved in sperm binding. METHOD: Epitopic domains recognized by monoclonal antibodies (MAbs) specific to either porcine ZP3 alpha or ZP3 beta glycoproteins were delineated in an enzyme-linked immunosorbent assay (ELISA) based on the ability of a MAb in solution to inhibit the binding of biotinylated ZP3 to another MAb coated on a microtitration plate. Immunoblot studies were carried out to determine the nature of reactive determinants. Porcine oocytes preincubated with MAbs were tested for sperm binding in vitro. RESULTS: Out of 23 MAbs generated, 10 had specificity for ZP3 alpha and 13 for ZP3 beta. By using these antibodies, eight epitopic domains on both ZP3 alpha and ZP3 beta were discernible. On ZP3 beta, epitopic domain DI partially overlaps with DII and DV with DVI, whereas on ZP3 alpha domains DI to DV were in close proximity with a partial overlap, suggesting the dominance of this region. All 10 MAbs against ZP3 alpha, and 10 out of 13 against ZP3 beta recognized deglycosylated forms of antigens. Seven antibodies having specificities for ZP3 alpha and ZP3 beta respectively recognized linear epitopes. MA-30, having specificity for ZP3 beta and MA-420 for ZP3 alpha and recognizing linear epitopes significantly inhibit the binding of boar sperm to porcine oocytes in vitro. CONCLUSIONS: Collectively, these studies indicate the value of utilizing MAbs for identifying and characterizing functionally significant ZP determinants. MAbs recognizing sequential epitopes will help in the elucidation of the amino acid sequence of the epitopes, which will subsequently help in design of synthetic immunocontraceptive vaccines.

Generation and characterization of monoclonal antibodies to porcine zona pellucida-3 alpha glycoprotein.

All 10 monoclonal antibodies generated reacted with deglycosylated ZP3 alpha both in ELISA and Western blots, thereby suggesting that these do not recognize carbohydrate determinants. Moreover, 7 antibodies (MAs -402, -403, -405, -412, -420, -421 and -423) recognized reduced and carboxyamidomethylated ZP3 alpha in Western blot suggesting that these antibodies recognized linear epitopes. The epitopes recognized by MAs -410, -413 and -425 were sequential or conformational, stabilized by disulphide bonds. Three antibodies namely, MA -405, -420 and -421 inhibited in vitro, zona lysis by trypsin.

The alpha-interferon system in patients undergoing renal transplantation and treated with cyclosporine and prednisone.

The interferon system was investigated in 65 normal control patients and ten patients with chronic renal disease, approximately a half year after renal transplantation and treatment with prednisone and cyclosporine. In previous studies, comparable doses of prednisone had no effect on the interferon system. It was assumed that the changes observed were primarily the result of cyclosporine therapy. In contrast with normal persons, low levels of interferon-alpha (IFN-alpha) were found in the circulation of patients. It was thought that this may be related to low grade infections in immunosuppressed persons. In patients, the IFN-alpha synthesizing capacity of leukocytes was significantly decreased. IFN-alpha inhibitor level was slightly increased in two patients. Inhibition of IFN-alpha (IFN-alpha) synthesizing capacity may be part of the immunosuppression mechanism of action of cyclosporine in patients undergoing transplant.

A discriminant function analysis of various interferon parameters among alcoholics and heavy smokers.

A three-group design of alcoholics, heavy smokers and controls was used to investigate the status of the interferon system, including natural killer cell activity. Group differences were indicated via discriminant function analysis which correctly classified 86% of subjects. Test-retest relations were investigated for 14 subjects following a 30-day alcoholic inpatient program. Whereas significant immune suppression was indicated at the time of detoxification, recovery was evident at the 30-day follow-up. Results are discussed in terms of the significance of alcohol abuse on immune system functioning with consequences for susceptibility to viruses, bacteria and medical illnesses.

Effect of alcohol on spleen cells and their functions in C57BL/6 mice.

Spleen cells from C57BL/6 mice maintained on alcohol containing liquid diet for two weeks were evaluated for different immune functions. On an average, 22% fewer spleen cells were recovered from alcohol-fed mice when compared to cells from control animals. In alcohol-fed mice, the relative frequency of B cells increased, whereas total T cells including CD4+ cells decreased significantly. Alcoholic mice, when challenged with poly(rI) poly(rC), produced significantly less interferon than control mice. In vitro production of interferon alpha and gamma by the spleen cells of alcoholic mice was reduced by 67-90%. No significant differences were seen in the level of natural killer cell activity in spleen cells of control and alcoholic mice. These results suggest that chronic alcohol intake can result in not only changes in the number of immune cells, but more importantly affect their biological functions such as their ability to produce interferons.

Effect of an interferon inhibitor on the antiproliferative signal of interferon-alpha.

We have described a nonantibody type inhibitor of interferons (IFN) in the blood of patients with AIDS, advanced neoplastic disorders, and lupus erythematosus in earlier reports (1,2). In the present study we show that the semipurified inhibitor blocks the antiproliferative signal of IFN-alpha in Daudi cells and the membrane potential shifting ability of IFN-alpha is modulated by the interferon inhibitor preparation (IFI).

Effects of meclofenamate on natural killer cell activity.

The effect of meclofenamate on natural killer cell activity was studied in a four hour 51Cr release assay. Meclofenamate, a nonsteroidal anti-inflammatory drug, had no effect on natural killer cell activity when added to the mixture of target (K562) and effector (peripheral blood mononuclear) cells either at the beginning of the reaction or when effector cells were pretreated with meclofenamate for 12-24 hours. However, a significant increase (p less than 0.0005) in the natural killer cell activity was seen when target cells were pretreated with meclofenamate for 12-24 hours prior to their mixing with effector cells. This increase in natural killer cell activity was observed consistently when target cells were taken from different donors.