Blood clotting and traumatic injury with shock mediates complement-dependent neutrophil priming for extracellular ROS, ROS-dependent organ injury and coagulopathy.

Polymorphonuclear (PMN) leucocytes participate in acute inflammatory pathologies such as acute respiratory distress syndrome (ARDS) following traumatic injury and shock, which also activates the coagulation system systemically. Trauma can prime the PMN nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex for an enhanced respiratory burst, but the relative role of various priming agents in this process remains incompletely understood. We therefore set out to identify mediators of PMN priming during coagulation and trauma-shock and determine whether PMN reactive oxygen species (ROS) generated in this manner could influence organ injury and coagulation. Initial experiments demonstrated that PMN are primed for predominantly extracellular ROS production by products of coagulation, which was abrogated by CD88/C5a receptor(C5aR) inhibition. The importance of this was highlighted further by demonstrating that known PMN priming agents result in fractionally different amounts of extracellular versus intracellular ROS release depending on the agent used. Plasma from trauma patients in haemodynamic shock (n = 10) also primed PMN for extracellular ROS in a C5a-dependent manner, which correlated with both complement alternative pathway activation and thrombin generation. Furthermore, PMN primed by preincubation with products of blood coagulation directly caused loss of endothelial barrier function in vitro that was abrogated by C5aR blockade or NADPH oxidase inhibition. Finally, we show in a murine model of trauma-shock that p47phox knock-out (KO) mice with PMN incapable of generating ROS were protected from inflammatory end-organ injury and activated protein C-mediated coagulopathy. In summary, we demonstrate that trauma-shock and coagulation primes PMN for predominantly extracellular ROS production in a C5a-dependent manner that contributes to endothelial barrier loss and organ injury, and potentially enhances traumatic coagulopathy.

Cloning of the gene encoding rat JAK2, a protein tyrosine kinase.

A complete cDNA clone encoding the rat JAK2 protein tyrosine kinase was isolated from an Nb2-SP (rat pre-T lymphoma cell line) cDNA library. The nucleotide (nt) and deduced amino acid (aa) sequences for this clone were determined and an open reading frame of 3399 bp, encoding a protein of a deduced mass of 130 kDa, was found. The coding regions of the rat and murine Jak2 clones share 93.4% nt identity and 97.1% aa identity. Northern analysis demonstrated that the 5-kb mRNA is highly abundant in brain and spleen, less abundant in skeletal muscle and testis, and detectable in kidney, heart, lung and liver. Translation of the rat Jak2 mRNA in rabbit reticulocytes results in a protein which is specifically immunoprecipitated by antibodies (Ab) recognizing JAK2, but not by Ab recognizing JAK1.

Ultrastructural studies of implantation sites from mice deficient in uterine natural killer cells.

Implantation sites from three strains of immunodeficient mice [tgepsilon26, IL-2Rbeta nullxp56(lck)null and IL-2Rgamma null, now known as common cytokine chain gamma (gamma(c)) null], which lack uterine natural killer (uNK) cells, are histologically abnormal. The related anomalies (found from day 10 of gestation) include the absence of aggregation of lymphocytes in the mesometrial triangle, acellularity of the mesometrial decidua, decidual arteries with relatively thick walls and reduced lumen diameters, unusual prominence of the endothelium in the major decidual vessels, and an overall reduction in placental size. In this study we have characterized implantation sites in a new mouse strain (gammac(-)/RAG2(-)) that is deficient in all lymphoid lineages. We have compared implantation sites in tgepsilon26 to gammac(-)/RAG2(-)at the ultrastructural level in order to determine the earliest-time point at which implantation sites differed from those in immunocompetent mice, and the cell types affected. Implantation sites from both the uNK cell-deficient mice resemble those from random-bred, immunocompetent mice on days 6 and 7 of gestation. On day 8 of gestation, decidual cells on the mesometrial sides of implantation sites in both tgepsilon26 and gammac(-)/RAG2(-)revealed pleotrophic morphology and degeneration. In some vessels, endothelial cells were distorted or displaced from their supporting cells. Progressive changes, suggestive of loss of function of both the mesometrial decidua and endothelial cells, were seen to day 14 of gestation, the latest time-point analysed. In contrast to tgepsilon26 mice, homozygously-mated gammac(-)/RAG2(-)had normal litter sizes, with birthweights and weaning weights similar to congenic C57Bl/6J controls, and no significant perinatal loss. In both strains, the newly-documented endothelial cell lesions predict detrimental alterations to vasomotor function of the uterine vasculature. These studies add strength to the hypothesis that uNK cells may have specialized physiological, rather than classically immune, functions in the pregnant mammalian uterus.

Transplantation into genetically alymphoid mice as an approach to dissect the roles of uterine natural killer cells during pregnancy--a review.

Mice genetically deficient in the natural killer (NK) cell lineage lack uterine (uNK cells) and demonstrate morphometrically-quantifiable histopathology within their implantation sites. Two particular mouse strains, tg(epsilon),26 and RAG-2 null x gamma(c) null, have been used successfully as transplant recipients to address questions relating to the biology of uNK cells. uNK cells did not differentiate within decidualized uterine graft segments from normal mice, which were anastomosed orthotopically into immunodeficient hosts. uNK cells did appear in similar grafts placed into immunocompetent hosts, indicating that uNK cells or their progenitors must home to the uterus. This was confirmed by splenocyte transplantation into pregnant uNK cell deficient recipients. Only splenocytes from pregnant donors, not those from non-pregnant donors, homed to the uterus. Homing in this in vivo assay was independent of the CC-chemokine receptors, CCR-2 and CCR-5. Longer-term bone marrow cell reconstitution of neonatal or virgin adult uNK cell-deficient mice has identified a functional role for uNK cells in modification of the decidual arterioles which is mediated by IFN-gamma. By utilizing mutant and gene-ablated mice as donors for tissue or haematopoietic cell transplants to uNK cell deficient mice, it should be possible to fully characterize the in vivo regulation and functions of these pregnancy-specific uterine lymphocytes.

Can murine uterine natural killer cells give insights into the pathogenesis of preeclampsia?

These studies aimed to advance understanding of the functions of pregnancy-associated uterine lymphocytes of the natural killer (NK) cell lineage. The approach was morphometric analysis of implantation sites from timed pregnancies in genetically modified mice deficient in NK cells or in signaling associated with their major product, the cytokine interferon-gamma. In four different strains of pregnant, NK cell--deficient mice, the major decidual arterioles failed to undergo modifications to their smooth-muscle coats and displayed endothelial cell damage. Decidua lacked normal cell density. This pathology was observed by the end of the first trimester, before placental differentiation. By midgestation in these strains, placentas were smaller than in control strains. In normal mice, many uterine NK cells are perivascular in location and appear to be activated because they are the major sources of interferon-gamma and of the interferon-gamma--regulated enzyme inducible nitric oxide synthase. During pregnancy in mice genetically ablated for interferon-gamma, the interferon-gamma receptor chain-alpha or the transcription factor interferon regulatory factor-1, uterine NK cells differentiate but appear to be abnormal both morphologically and functionally. In these three strains, failure of pregnancy-induced vascular modifications and overt necrosis of decidua occur. Thus, in mice, lymphocytes of the NK cell lineage make specialized contributions to pregnancy-associated modification of the uterine vasculature and to maintenance of decidua. These contributions are achieved through interferon-gamma--mediated gene regulation and appear to enhance subsequent placental growth. Human CD56 bright decidual lymphocytes may have analogous functions. If so, changes in numbers or levels of activity of human uterine NK cells or mutations in genes regulated by uterine interferon-gamma could contribute to initiation of preeclampsia.

Oxygen uptake and heart rate relationship in persons with spinal cord injury.

The percent (%) peak oxygen uptake (VO2) and % peak heart rate (HR) relationships were determined in 13 persons with high (T1-T6) and 14 persons with low lesion (T7-T12) spinal cord injured paraplegia (SCI PARA) and 15 nonimpaired subjects during graded arm crank (AC) tests to exhaustion. Subjects were instructed to maintain a target cadence of 60 rpm on a modified electronically braked leg cycle ergometer. After 3 min of unloaded cranking, power output (PO) was increased by 8-16 W.min-1. VO2 and HR were determined via open-circuit spirometry and 12-lead ECG, respectively. Absolute HR and VO2 values for each PO were converted to % peak HR and % peak VO2 values. Linear regression slopes describing individual % peak HR and % peak VO2 relationships were calculated and compared between groups with one-way ANOVA. No significant differences (P > 0.05) were noted between the mean (+/- SD) regression slopes for persons with high lesion SCI PARA (1.48 +/- 0.21), persons with low lesion SCI PARA (1.48 +/- 0.26), and nonimpaired subjects (1.53 +/- 0.29). Regression equations derived using all data points within each group were as follows: High lesion SCI PARA: y = 1.3x-37.0, R = 0.85 Low lesion SCI PARA: y = 1.23x-30.9, R = 0.88 Nonimpaired subjects: y = 1.41x-46.2, R = 0.95 (y = % peak VO2, x = % peak HR). These equations are similar to those previously reported for nonimpaired men and women and cardiac patients during AC and leg cycle ergometry.(ABSTRACT TRUNCATED AT 250 WORDS)

A study on the engraftment and trafficking of bovine peripheral blood leukocytes in severe combined immunodeficient mice.

Bovine lymphoid cells were engrafted into untreated and into sublethally irradiated scid/scid mice, following intraperitoneal (i.p.) injection of bovine peripheral blood leukocytes (PBL). To distinguish bovine from murine cells, bovine PBL were stained prior to i.p. injection with a fluorescent cell linker compound, PKH26-GL and, after cell recovery, labelled with a monoclonal antibody (mAb) reactive with the bovine pan leukocyte cell surface marker, CD45, and analyzed by flow cytometry. This dual labelling system was used to monitor tissue localization and migration of bovine cells within PBL-SCID-bo from 30 min to 64 days after bovine PBL injection. Inoculated bovine PBL were rapidly cleared from the peritoneal cavity of PBL-SCID-bo over the first 24 h after injection, then bovine cell numbers within the peritoneal cavity increased gradually over the next 35 days. No bovine cells were observed in the peritoneal wash from either non-irradiated or irradiated mice after Day 56. Bovine cells were detected in the spleens of both non-irradiated and irradiated mice 12-14 days after inoculation, but were undetectable in non-irradiated and irradiated mice after Day 56, and in irradiated mice after Day 35. This decrease coincided with a significant reduction in both the numbers of bovine cells in the peritoneal cavity, and in the levels of bovine Ig detected in the sera. Bovine IgG and bovine IgM were detected in both normal and irradiated PBL-SCID-bo by 4 days after PBL injection, and remained detectable to 8 weeks after inoculation. T-cells appeared to be the predominant bovine cell population in the spleens. In a small proportion of both normal and irradiated PBL-SCID-bo, spleens three to 20 times the size of a normal SCID mouse spleen were observed. Relatively high levels of bovine cells were detected in the thymuses of some irradiated PBL-SCID-bo. Sporadic, low levels of bovine cells were observed in the mesenteric lymph nodes, peripheral blood, and kidneys of normal and irradiated mice, and in the lungs and livers of non-irradiated but not irradiated PBL-SCID-bo. Sublethal gamma irradiation of SCID recipients appeared to enhance engraftment of bovine cells into the murine spleen and thymus.

Offspring of xenogeneically-reconstituted scid/scid mice are capable of a primary xenogeneic immune response to DNP-KLH.

Human peripheral blood leukocyte (PBL) reconstitution of severe combined immunodeficient (SCID) mice has provided a small animal model system (hu-PBL-SCID) useful for the study of the human immune system and disease pathogenesis. Transfer of xenogeneic PBL from donors other than humans has also been successful; however, the controversy remains regarding the capability of xenogeneically engrafted lymphocytes to mount a primary immune response. Human cells have been identified in offspring from hu-PBL-SCID but were not evaluated for a primary immune response. In the present study, offspring of bovine PBL-reconstituted SCID mice (F1-PBL-SCID-bo) were assessed for specific immune function. Sera from all of the F1-PBL-SCID-bo contained relatively low levels of bovine IgG 5 weeks after birth but bovine Ig became undetectable by 14 or 18 weeks. Eight F1-PBL-SCID-bo (23 or 27 weeks of age) were immunized with a single dose of 100 mu g dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH). Individual cells secreting bovine antibody were enumerated using the ELISA-plaque assay. One week after immunization, bovine cells secreting bovine immunoglobulin (IgG) specific for DNP-KLH were identified in the spleens from three of the F1-PBL-SCID-bo at a frequency of one antibody-secreting cell per 9 x 10(3) to 1 x 10(6) spleen cells. Thus, xenogeneic lymphocytes, passed from the mother to her offspring, retain the capacity for a primary immune response to DNP-KLH.

Xenogeneic (bovine) peripheral blood leukocytes engrafted into severe combined immunodeficient mice retain primary immune function.

The immune responsiveness of xenogeneic PBL engrafted into SCID mice was investigated using the bovine PBL-reconstituted SCID mouse model system (PBL-SCID-bo). Bovine PBL-reconstitution and B-cell activity were monitored by bovine serum Ig production. Bovine T-cell function was demonstrated by an antigen-specific immune response to bovine transplantation antigens provided by bovine skin allografts. Bovine allograft rejection was clearly evident in > 65% PBL-SCID-bo that received a bovine PBL inoculum either 30 days after bovine skin grafting, or 7-52 days before bovine skin grafting. Bovine allograft rejection was confirmed via histological examination and was characterized primarily by a band of infiltrating bovine lymphocytes at the periphery of the graft and tissue necrosis. A secondary immune response could be elicited if bovine cells in the PBL inoculum were presensitized to Ag from the bovine skin allograft donor. This study is the first to show that bovine cells engrafted in SCID mice after i.p. injection of bovine PBL retain some aspects of immune competency. These results confirm the value of the xenogeneic PBL-reconstituted SCID mouse model in the study of primary immunity.

Xenogeneic PBL-scid mice: their potential and current limitations.

The hu-PBL-scid has been constructed by intraperitoneal inoculation of lymphocytes from human peripheral blood into immunodeficient scid mice. Such scid mouse-human chimeras have proven useful as in vivo animal models for studies on human lymphocyte development and differentiation from pluripotent stem cells. Further, the hu-PBL-scid mouse provides a readily accessible model for the examination of immune cell function and involvement in autoimmune and infectious disease processes. In response to the growing need for model systems to examine the immune system and disease pathogenesis in agriculturally important animals, PBL engraftment of scid mice has expanded to include the bovine and equine species. This review discusses the properties and potential uses of the xenogeneic PBL-reconstituted scid mouse.

Individualism and the social in early American social psychology.

In this paper an attempt is made to specify the original conception of the social dimensions of cognition, emotion and behavior-and of a distinctively social psychology-that was held by early American social psychologists, but abandoned by later generations of social psychologists committed to Floyd Allport's individualistic experimental program. Two influential forms of "individualism" in the work of Floyd Allport are distinguished and detailed.

Expression of bovine immunodeficiency-like virus envelope glycoproteins by a recombinant baculovirus in insect cells.

The bovine immunodeficiency-like virus (BIV) env open reading frame (ORF) contains both sequences encoding env and sequences for exon 1 of the putative rev gene. Recombinant baculoviruses incorporating BIV env ORF sequences were constructed to characterize the expression, processing, and immunogenicity of products of the BIV env ORF in insect cells and to develop reagents to study native BIV Env glycoproteins. A recombinant baculovirus containing the entire env ORF synthesized a nonglycosylated, 20-kDa, BIV-specific protein, apparently unrelated to native BIV Env proteins. In contrast, a recombinant baculovirus containing a truncated env ORF in which the coding sequences for rev exon 1 were deleted synthesized three size classes of glycosylated proteins in insect cells related to the BIV Env precursor (gp145), surface (gp100), and transmembrane (gp45) glycoproteins observed in BIV-infected mammalian cells. Oligomers of recombinant BIV Env proteins also formed in these baculovirus-infected insect cells. Immunofluorescence staining of intact insect cells infected by the baculovirus expressing BIV Env with BIV-specific serum demonstrated that the recombinant Env glycoproteins were expressed on the cell surface. Antisera raised to recombinant Env glycoproteins immunoprecipitated native gp145, gp100, and gp45 in BIV-infected bovine cells similar to sera from animals naturally or experimentally infected with BIV.

Analysis of the transcription pattern and mapping of the putative rev and env splice junctions of bovine immunodeficiency-like virus.

The bovine immunodeficiency-like virus (BIV) genome contains the obligatory structural genes of all retroviruses and, in addition, the complex central region of lentiviruses; this novel region may code for at least five nonstructural/regulatory genes in BIV (K.J.Garvey, M.S. Oberste, J.E. Elser, M.J. Braun, and M.A. Gonda, Virology 175:391-409, 1990). As a prelude to determining the function of these novel open reading frames, the transcriptional pattern of BIV was studied by Northern analysis of RNA from BIV-infected cells. Five size classes of BIV-specific RNAs of 8.5, 4.1, 3.8, 1.7, and 1.4 kb were detected. The 8.5-kb RNA contains sequences from all regions of the genome; it is the virion RNA and probably serves as the gag-pol transcript as well. By using gene-specific probes, subgenomic viral RNAs of 3.8, 1.7, and 1.4 kb were tentatively identified as the env, tat, and rev spliced messages, respectively. The 4.1-kb RNA could not be unambiguously identified but may encode vif. The hybridization patterns of the putative tat and rev mRNAs suggest that they are the products of multiple splicing events. Discrete transcripts for the BIV W and Y central region open reading frames were not defined. The characterization of partial cDNA clones has permitted the mapping of the env and putative rev splice junctions.

Vibrio vulnificus biogroup 2: new biogroup pathogenic for eels.

Clinical and nonclinical isolates of the lactose-positive Vibrio vulnificus were compared with Vibrio strains isolated from lesions on eels (Anguilla japonica) cultured commercially in Japan. Strains were compared phenotypically and antigenically, for pathogenicity to mice and eels, and for genetic relatedness. The strains isolated from diseased eels differed phenotypically from the original species description of V. vulnificus in that they were negative for indole production, ornithine decarboxylase activity, growth at 42 degrees C, and acid production from mannitol and sorbitol. No relationship between the surface antigens of V. vulnificus strains from environmental and clinical sources and the strains from diseased eels was observed. Typical V. vulnificus strains and the eel isolates were pathogenic to mice; however, only those strains originally isolated from diseased eels were found to be pathogenic to eels. Results of DNA-DNA competition experiments revealed that there was greater than 90% relative reassociation between clinical and nonclinical V. vulnificus and strains from diseased eels. Based on the results of the DNA-DNA competition experiments, we conclude that the strains isolated from diseased eels were V. vulnificus; however, the differences in phenotypic characteristics and eel pathogenicity indicated that these strains represent a different biogroup. Therefore, we propose that strains phenotypically similar to the type strain of the species (ATCC 27562) be classified as V. vulnificus biogroup 1 and the strains phenotypically similar to those isolated from diseased eels be classified as V. vulnificus biogroup 2 represented by the reference strain ATCC 33148.

Influence of posture on arm exercise tolerance and physiologic responses in persons with spinal cord injured paraplegia.

This study compared metabolic and cardiopulmonary responses to incremental supine and upright sitting arm crank ergometry (ACE) in nine men with spinal cord injured paraplegia ranging from T1-T5. Both tests consisted of continuous graded ACE from rest to volitional fatigue on a modified electronically braked cycle ergometer with the work rate increased by 8.2 W.min-1. No significant differences (P > 0.05) existed for peak ACE power output (W), oxygen uptake, pulmonary ventilation, respiratory exchange ratio, and O2 pulse between the two tests. Heart rate and O2 pulse responses at six submaximal work rates representing 0-58% peak W were also not significantly different between postures. These data indicate that ACE tolerance in persons with high-lesion paraplegia was not enhanced when ACE was performed in the supine posture.

Characterization of bovine immunodeficiency virus rev cDNAs and identification and subcellular localization of the Rev protein.

One of the six putative accessory genes of bovine immunodeficiency virus (BIV) is similar to those identified as rev in the human immunodeficiency virus and visna virus genomes. To further analyze the BIV rev gene locus, protein, and function, rev cDNAs were cloned and characterized. BIV rev mRNA is derived from the full-length transcript by multiple splicing events and consists of three exons, including the untranslated leader sequence and two coding exons. BIV rev cDNA was expressed in bacteria and in a mammalian in vitro translation expression system. A 23-kDa Rev protein (p23rev) was immunologically detected in lysates from both systems by using an antiserum made to a synthetic Rev peptide. Recombinant p23rev made in bacteria was purified and used to make a polyvalent antiserum. Antisera to Rev peptide and recombinant p23rev immunoprecipitated p23rev from BIV-infected mammalian cells but not from virions. A mammalian expression vector using the BIV rev cDNA was constructed; p23rev was immunoprecipitated with anti-Rev serum from 32P-labeled lysates of monkey cells transfected with this plasmid, demonstrating that BIV Rev is phosphorylated. Immunofluorescence and immunoelectron microscopy with anti-BIV Rev antisera localized Rev in the nucleus and, particularly, in the nucleoli of BIV-infected cells. In functional studies, the expression of BIV Rev was shown to positively regulate the appearance both of Gag protein, which is translated from the unspliced primary viral transcript, and of singly spliced env mRNA but not that of the multiply spliced tat mRNA. These results demonstrate that BIV Rev activity correlates with the known function of lentivirus Rev proteins.

A therapeutic human IgG4 monoclonal antibody that depletes target cells in humans.

It is traditionally held that human IgG4 MoAbs should not deplete target cells in vivo, as this isotype is inactive in a number of in vitro assays that measure effector function. We have previously challenged this dogma, and the current study was designed to investigate the in vivo biological effects in humans of a MoAb of human IgG4 isotype. Nine patients with refractory rheumatoid arthritis (RA) fulfilling ARA criteria, and one with ankylosing spondylitis (AS) received a human IgG4 Campath-1 MoAb (with specificity against the pan-lymphocyte antigen CD52) as part of a two-stage therapeutic protocol. In stage 1, patients received a single dose of this MoAb. Stage 2, starting 48 h later, comprised a 5-day course of a human IgG1 Campath-1 MoAb with identical V-region (CAMPATH-1H), as previously used in the management of RA patients. The intervening 48 h provided a window of opportunity to monitor the biological effects of the IgG4 MoAb for comparison with the IgG1. The two MoAbs were also compared for in vitro biological activity. IgG4 depleted peripheral blood lymphocytes (PBL), albeit less efficiently than IgG1. It produced a first-dose reaction of similar intensity, although associated circulating tumour necrosis factor-alpha (TNF-alpha) levels were lower. TNF-alpha release from whole blood in vitro was also greater with the IgG1 MoAb. The study design did not permit conclusions to be drawn regarding therapeutic efficacy of the IgG4 MoAb. In summary, a human IgG4 Campath-1 MoAb depletes target cells in vivo. Importantly, this study demonstrates for the first time in humans that in vitro assays may not predict the in vivo effector function of therapeutic MoAbs.