Search

Background/objective The criterion standard for anti-topoisomerase I antibody (anti-topo I antibody) testing in systemic sclerosis (SSc) uses immunodiffusion (ID) techniques, but enzyme-linked immunosorbent assay (ELISA) and multiple-bead technology are often used in current settings to save time and cost. Our aim was to assess the performance of the multiple-bead assay, ELISA, and ID testing methods. Methods We conducted a retrospective study of patients at the University of Michigan whose extractable nuclear antigen 10 autoantibody panel tested positive for the anti-topo I antibody by multiple-bead technology during a 1-year period. All samples positive by multiple-bead assay were sent to the RDL Laboratories and reflexed for ELISA, and all anti-topo I antibodies positive by ELISA were further tested by ID. Clinical data were reviewed by a rheumatologist and assessed for presence of SSc. Data were analyzed via frequency tables. Results Approximately 9500 extractable nuclear antigen 10 panels were ordered by physicians at the University of Michigan. Of these, 129 patients were positive for the anti-topo I antibody by multiple-bead assay, 51 were positive by multiple-bead assay and ELISA, and 21 were positive by multiple-bead assay, ELISA, and ID. We found that 26.4% of patients positive by multiple-bead assay, 47.1% positive by multiple-bead assay and ELISA, and 95.2% positive by multiple-bead assay, ELISA, and ID had SSc. Conclusions Multiple-bead assays have a high rate of false-positive results for the anti-topo I antibody in patients without clinical evidence of SSc. A stepwise approach of confirmation of positive multiple-bead assay results using both ELISA and ID improves the predictive value of antibody testing for the diagnosis of SSc.

In 2008, the University of Michigan Health System (UMHS) created a Laboratory Test Utilization Program that included the establishment of a Laboratory Formulary Committee under the imprimatur of the Faculty Group Practice, the Office of Clinical Affairs, the Department of Pathology, and UMHS hospital administration. A critical component of the program is UM-CareLink, an order entry system for inpatients and inpatient-like venues. UM-CareLink allows very basic decision support comment prompts. Through the application of peer-reviewed medical evidence, input by medical content experts, excellent cooperation by medical staff, and close oversight by Pathology of the Sendout Laboratory, this program has led to a robust process of test utilization oversight, excellent communication with clinical services, and significant UMHS activity-adjusted reductions in laboratory expense.

On the basis of anecdotal instances in which atypical laboratory findings suggested the possibility of unsuspected cryoglobulinemia, we applied predetermined criteria to determine how often such findings predict the presence of clinically significant cryoglobulinemia. The laboratory criteria are smeared M-spike (paraprotein) in agarose gel serum protein electrophoresis, precipitated protein at the serum application point of agarose electrophoresis gel, greater than 50% quantitative discrepancy between the densitometrically estimated M-spike and the relevant corresponding serum immunoglobulin isotype concentration from the same specimen, and smeared protein observed on an agarose electrophoresis immunofixation gel. Cases that fulfilled any of these criteria were prospectively collected for 2 years. Brouet types and clinical findings were determined in cases where cryoglobulins were subsequently identified and clinical data available. Among 83 patients in whom any of the above laboratory findings were identified, 52 had subsequent cryoglobulin evaluations. Fourteen of these 52 patients had cryoglobulinemia. Findings indicative of clinically significant cryoglobulinemia were present in 8 of the 10 patients in whom follow-up clinical data were available.

Emily C Somers,1–3 Seetha U Monrad,1 Jeffrey S Warren,4 Maritsa Solano,5 Lourdes Schnaas,6 Mauricio Hernandez-Avila,5 Martha Maria Tellez-Rojo,5 Howard Hu7 1Divison of Rheumatology, Department of Internal Medicine, 2Department of Environmental Health Sciences, 3Department of Obstetrics & Gynecology, 4Division of Clinical Pathology, Department of Pathology, University of Michigan, Ann Arbor, MI, USA; 5Center for Nutrition and Health Research, National Institute ofPublic Health, Cuernavaca, Morelos,6Department of Developmental Neurobiology, National Institute of Perinatology, Mexico City, Mexico; 7Occupational and Environmental Health, Dalla Lana School of Public Health, University ofToronto, Toronto, ON, Canada Objective: To characterize antinuclear antibody (ANA) prevalence according to distinct assay methodologies in a pediatric cohort from Mexico City, and to further examine associations with age and sex.Methods: Serum ANA were measured by indirect immunofluorescence assay (IFA) and multiplex immunoassay in 114 children aged 9–17 years. IFA was considered positive at a cutoff titer of ≥1:80. Agreement between assay methods was assessed by kappa statistic. Sensitivity, specificity, and 95% confidence intervals (CIs) of the multiplex were computed with IFA as the reference standard.Results: Of the 114 children (mean age 14.7 [standard deviation 2.1] years; 54 [47%] female), 18 of 114 (15.8%) were ANA positive by IFA, and 11 of 114 (9.6%) by 11-antigen multiplex assay. ANA prevalence was higher in females compared with males by both of the methods (ratios 1.6–1.9 to 1). Agreement between tests was classified as slight by kappa (κ=0.177 [95% CI −0.051, 0.406]). The multiplex immunoassay had sensitivity of 22.2% (95% CI 6.4, 47.6) and specificity of 92.7% (95% CI 85.6, 97.0), and failed to capture 3 of 4 (75%) of the high-titer (≥1:1280) IFA-positives.Conclusion: Up to 15% of children in this general population cohort were ANA positive, with a higher rate of positivity among females according to both assay methods. Substantial discordance in ANA results was found between IFA and multiplex methods, even for high-titer IFA positives. These findings underscore the need to sufficiently account for assay characteristics when interpreting ANA test results, and support IFA as the more appropriate assay for studies of subclinical autoimmunity. Keywords: Autoreactivity, biomarker, immune dysfunction, preclinical, subclinical autoimmunity, autoantibodies, pediatric, epidemiology

The operation of an effective utilization management program requires a governance structure that aligns both institutional and institution-wide goals and activities. It has been estimated that as much as 30 % of laboratory testing is likely to be wasteful. The prospect of broadly applied personalized or precision medicine holds great promise but with the attendant risk that, if misapplied, there is potential for extraordinary waste. As a vehicle for exploring the relationship between governance structure and effectiveness, this chapter outlines the four-phase evolution of laboratory utilization management in a large academic medical center (University of Michigan Health System). The consideration of failed early efforts and much more successful recent efforts illustrates the fundamental importance of alignment of governance in effective management of pathology and laboratory utilization.

Background Since its creation in 1992 by gene inactivation via gene targeting, the apolipoprotein E “knockout” mouse has become the most widely used rodent model for the study of atherosclerosis. Commercially available apolipoprotein E(−/−) mice are bred on a C57BL/6J background. The goal of the present study was to investigate the development of atherosclerosis in apolipoprotein E-deficient mice generated on a Balb/c background. Methods We compared serum cholesterol concentrations and the development of atherosclerotic lesions in heterozygous Balb/c [apolipoprotein E(+/−)] mice fed regular rodent chow, Balb/c apolipoprotein E-deficient mice fed regular chow, and Balb/c apolipoprotein E-deficient mice fed a high-fat diet for up to 30 weeks. Expression of the chemokine JE (murine homologue of MCP-1), as well as the adhesion molecules E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, in the aortas of knockout mice fed a high-fat diet was measured by enzyme-linked immunosorbent assay. Results Balb/c apolipoprotein E-deficient mice develop atherosclerotic lesions in a reproducible temporal and morphological pattern. Total serum cholesterol concentrations in Balb/c apolipoprotein E-deficient mice fed regular chow or a high-fat diet, respectively, closely parallel those reported for C57BL/6J apolipoprotein E-deficient mice. The expression of all three adhesion molecules in the aorta follows a similar temporal pattern, peaking in the first 15 weeks, whereas JE concentrations peak around 23 weeks. Conclusion The availability of Balb/c apolipoprotein E-deficient mice will facilitate the study of atherosclerosis in a mouse strain that can concomitantly develop other pathological states that are not readily inducible in mice with the C57BL/6J background.

Quantification of intact serum immunoglobulins has proven useful in the evaluation of patients with suspected immunodeficiency disorders, lymphocyte and plasma cell neoplastic diseases, allergic conditions, and some chronic inflammatory and autoimmune disorders. Since the advent of quantitative immunoglobulin assays nearly 50 years ago (1), increasingly robust analytical methods have been developed. The armamentarium of intact immunoglobulin assays was first substantively expanded by the development of immunoglobulin light-chain measurements in which the light chains are bound to heavy chains (intact immunoglobulins). The last decade has seen the development of both free (unbound) immunoglobulin light-chain assays and heavy/light-chain (HLC) or junctional epitope assays, the latter of which allows for individual measurements of IgGκ, IgGλ, IgAκ, IgAλ, IgMκ, and IgMλ (2, 3). Despite these advances in immunoglobulin and light-chain quantification methods, there remain technical complexities that can influence accuracy and, in turn, proper clinical application. Equally important, even with robust assays, is the need for thorough understanding of the clinical indications for, and limitations to, immunoglobulin and related measurements.

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of endothelial nitric oxide synthase and a proposed cardiovascular risk factor, is elevated in chronic kidney disease (CKD). Pharmacological strategies that lower plasma concentration of ADMA may be expected to increase nitric oxide (NO.) bioavailability and potentially limit atherosclerosis. We hypothesized that the antioxidant alpha-tocopherol (vitamin E) reduces ADMA levels in CKD.An open-label pilot interventional study using 800 IU of vitamin E was undertaken in eight stable out-patients with non-diabetic CKD (creatinine clearance30 ml/min/1.73 m(2)) and six healthy controls, with the objective of measuring plasma ADMA levels at baseline and after 8 weeks of treatment. Plasma ADMA, symmetric dimethylarginine (SDMA) and alpha-tocopherol concentrations were determined at study entry and exit using high-performance liquid chromatography, while plasma total F2-isoprostanes, an index of oxidative stress, were measured using a commercially available enzyme-linked immunosorbent assay kit.ADMA and SDMA concentrations were significantly higher in the plasma of patients compared with that of controls (P/= 0.001). After treatment with vitamin E, ADMA decreased by 23% in six of eight patients (P0.001). The remaining two patients showed either an increase or no change (overall, P = 0.16). There was no significant change in plasma F2-isoprostanes with vitamin E treatment for 8 weeks.Antioxidant therapy with vitamin E has the potential to lower ADMA levels in CKD patients, implying increased NO. availability. This strategy merits further exploration in the setting of CKD prior to renal replacement.

Monocyte chemoattractant protein-1 (MCP-1) is a pivotal mediator of angiocentric granuloma formation in glucan-induced pulmonary granulomatous vasculitis. Based on the rationale that mononuclear phagocytes retrieved from granulomas are rich sources of nitric oxide (NO) and that the recruitment of mononuclear phagocytes into lesions abates as granuloma formation slows, we tested the hypothesis that MCP-1 gene expression is regulated by a NO-sensitive mechanism. Preexposure of endothelial cell (EC) monolayers to NO donor compounds markedly reduced cytokine-induced MCP-1 expression and cytosolic-to-nuclear translocation of nuclear factor-kappa B (NF-kappaB), reversed fluctuations in endothelial reduced glutathione (GSH) pools but did not affect cGMP concentrations. The lungs of mice bearing targeted disruptions of the inducible nitric oxide synthase (iNOS) gene exhibited significantly higher concentrations of MCP-1 following glucan infusion than did those of wild-type mice. Cumulatively, these data suggest that NO suppresses MCP-1 expression by blunting the redox changes associated with cytokine-induced EC activation.

Background Immune dysregulation associated with mercury has been suggested, although data in the general population are lacking. Chronic exposure to low levels of methylmercury (organic) and inorganic mercury is common, such as through fish consumption and dental amalgams. Objective We examined associations between mercury biomarkers and antinuclear antibody (ANA) positivity and titer strength. Methods Among females 16–49 years of age (n = 1,352) from the National Health and Nutrition Examination Survey (NHANES) 1999–2004, we examined cross-sectional associations between mercury and ANAs (indirect immunofluorescence; cutoff ≥ 1:80). Three biomarkers of mercury exposure were used: hair (available 1999–2000) and total blood (1999–2004) predominantly represented methylmercury, and urine (1999–2002) represented inorganic mercury. Survey statistics were used. Multivariable modeling adjusted for several covariates, including age and omega-3 fatty acids. Results Sixteen percent of females were ANA positive; 96% of ANA positives had a nuclear speckled staining pattern. Geometric mean (geometric SD) mercury concentrations were 0.22 (0.03) ppm in hair, 0.92 (0.05) μg/L blood, and 0.62 (0.04) μg/L urine. Hair and blood, but not urinary, mercury were associated with ANA positivity (sample sizes 452, 1,352, and 804, respectively), after adjusting for confounders: for hair, odds ratio (OR) = 4.10 (95% CI: 1.66, 10.13); for blood, OR = 2.32 (95% CI: 1.07, 5.03) comparing highest versus lowest quantiles. Magnitudes of association were strongest for high-titer (≥ 1:1,280) ANA: hair, OR = 11.41 (95% CI: 1.60, 81.23); blood, OR = 5.93 (95% CI: 1.57, 22.47). Conclusions Methylmercury, at low levels generally considered safe, was associated with subclinical autoimmunity among reproductive-age females. Autoantibodies may predate clinical disease by years; thus, methylmercury exposure may be relevant to future autoimmune disease risk. Citation Somers EC, Ganser MA, Warren JS, Basu N, Wang L, Zick SM, Park SK. 2015. Mercury exposure and antinuclear antibodies among females of reproductive age in the United States: NHANES. Environ Health Perspect 123:792–798; http://dx.doi.org/10.1289/ehp.1408751

Hyperhomocysteinemia is an independent risk factor for atherosclerosis and atherothrombosis. While in vitro studies have revealed a number of homocysteine-mediated alterations in the thromboregulatory properties of endothelial cells, comparatively little is known about homocysteine-modulated smooth muscle cell function. We observed that exposure of human aortic smooth muscle cells to pathophysiologically relevant concentrations of homocysteine results in concentration-dependent increases in cytokine-induced MCP-1 and IL-8 secretion. RNase protection assays revealed that both MCP-1 and IL-8 mRNA concentrations are increased in homocysteine-treated smooth muscle cells when compared to cells activated with cytokines alone. Homocysteine treatment also increased cytosolic-to-nuclear translocation of the p65 and p50 subunits of the Rel/NF-κB family of transcription factors but had no effect on AP-1 activation. Cumulatively, these data suggest that homocysteine may increase monocyte recruitment into developing atherosclerotic lesions by upregulating MCP-1 and IL-8 expression in vascular smooth muscle cells.

Monocyte chemoattractant protein-1 is a chemokine with potent monocyte activating and chemotactic effects. Monocyte chemoattractant protein-1 gene and protein expression is rapidly up-regulated in response to a variety of acute and chronic central nervous system disorders. The activation and recruitment of microglia and monocytes into areas of inflammation may play a critical role in the pathogenesis of acute brain injury. Monocyte chemoattractant protein-1 could be a pathophysiologically important mediator of the microglial and monocyte responses in the brain. Using a well-characterized model of acute excitotoxic brain injury in neonatal rats, experiments were designed to evaluate whether monocyte chemoattractant protein-1 plays a role in the progression of tissue damage. Direct co-administration of recombinant monocyte chemoattractant protein-1 with the excitotoxin N -methyl- d -aspartate exacerbated injury, both in the striatum and in the hippocampus, by 55% and 167%, respectively. Complementary experiments to determine the effect of functional inhibition of monocyte chemoattractant protein-1, using an anti-monocyte chemoattractant protein-1-neutralizing antibody, revealed that co-administration of the antibody with N -methyl- d -aspartate attenuated tissue injury in the striatum and hippocampus by 57% and 39%, respectively. Together, these data suggest that monocyte chemoattractant protein-1 is a mediator of acute excitotoxic brain injury in neonatal rats and that inflammatory mechanisms contribute significantly to the pathogenesis of acute neonatal brain injury. Whether chemokines are pathophysiologically relevant mediators of neuronal injury in human neonates remains to be determined.

Evenomation by arachnids of the genus Loxosceles frequently results in disfiguring necrotic skin lesions. The cellular and molecular mechanisms which contribute to lesion development are incompletely defined but appear to involve participation of several pro-inflammatory mediators. We have recently observed that Loxosceles deserta venom induces the production of chemokines in human umbilical vein endothelial cells (HUVECs) and human pulmonary epithelial cells. In the present study we observed that Loxosceles deserta venom induces the expression of vascular endothelial growth factor (VEGF) in human keratinocytes but little in smooth muscle cells and none in pulmonary epithelial cells. A potent endothelial cell-specific mitogen, VEGF induces angiogenesis and vascular permeability in vivo. RNase protection assay data indicate that VEGF mRNA concentrations in keratinocytes are significantly increased at 2 h following venom exposure. These data suggest that keratinocyte-derived VEGF may contribute to the vasodilation, edema and erythema which occur following Loxosceles evenomation.

In hypophysectomized rats, prolactin induces regression of the corpora lutea. Luteal regression is accompanied by infiltration of monocytes/macrophages, declines in luteal mass and plasma progestins, and increased staining for monocyte chemoattractant protein-1 (MCP-1). We investigated whether similar events are induced during the estrous cycle, after the proestrous prolactin surge. Rats were killed on proestrus or on estrus, and one ovary was frozen for immunohistochemical detection of MCP-1, monocytes/macrophages (ED1-positive), and differentiated macrophages (ED2-positive) and for in situ detection of apoptotic nuclei. Corpora lutea of the current (proestrus) or preceding (estrus) cycle were dissected from the ovaries of additional rats and frozen for the same analyses and for determination of total protein content. In sections of whole ovaries, intensity and distribution of MCP-1 staining were increased in corpora lutea of multiple ages on estrus as compared to proestrus, as were numbers of differentiated macrophages and apoptotic nuclei per high-power field. Sections of isolated corpora lutea showed these increases on estrus, and the number of monocytes/macrophages per high-power field was also significantly increased. Accompanying these inflammatory/immune events, the corpora lutea on estrus showed decreased weight and total protein per corpus luteum, as compared to corpora lutea on proestrus. These changes are consistent with a proposed role for prolactin in the initiation of luteal apoptosis and of a sequence of inflammatory/immune events that accompany regression of the rat corpus luteum during the normal estrous cycle.

Intra-articular injection of streptococcal cell wall Ag followed by i.v. challenge (“reactivation”) results in a destructive lymphocyte-dependent monoarticular arthritis. To further define the role of immune mechanisms in the model, Abs to Th1 and Th2-related cytokines were evaluated. Treatment of rats with antibodies to IL-4 reduced swelling, while treatment with anti-IL-10 or anti-IFN-γ either had no effect or slightly enhanced the inflammatory response. These results suggest that Th-2 immune mechanisms may be, at least in part, operative in the model. To more precisely define the role of IL-4, the effects of anti-IL-4 on monocyte chemoattractant protein-1 (MCP-1) expression were evaluated. Initial studies demonstrated that mRNA (as determined by in situ hybridization) and protein (as determined by immunofluorescence) for MCP-1 were detectable in inflamed synovial tissue in a time-dependent manner. Anti-IL-4 treatment significantly reduced the expression of mRNA for MCP-1 24 and 72 h after reactivation. In addition, anti-MCP-1 inhibited swelling and reduced influx of 111In-labeled T cells. These data suggest that the reactivation model of streptococcal cell wall Ag-induced arthritis is Th-2 dependent, and that an inter-relationship exists between IL-4 and the expression of MCP-1.

A variety of inflammatory diseases are accompanied by activation of the complement system. We examined the role of the membrane attack complex (MAC) in mediating neutrophil adhesion to endothelial cells. To assemble the MAC in endothelial cell monolayers, a C5b-like molecule was created through the treatment of purified C5 with the oxidizing agent chloramine-T, followed by addition of the remaining components (C6-C9) that constitute the MAC. Use of this method abrogated potentially confounding effects mediated by other complement components (e.g., C5a). MAC assembly resulted in a rapid (30 min), concentration-dependent increase in neutrophil adherence. A monoclonal antibody directed against P-selectin inhibited MAC-mediated neutrophil adhesion. A whole cell EIA confirmed P-selectin expression after formation of the MAC. Incubation of neutrophils with the platelet-activating factor receptor antagonist, CF 3988, also significantly decreased adhesion, indicating that PAF plays a role in MAC-mediated adhesion. These results suggest that the MAC can promote neutrophil adhesion through P-selectin and PAF-mediated mechanisms.

Cytokines are peptide hormones that regulate a wide variety of immune and inflammatory processes. Individual cytokines are produced by many different cell types and exhibit pronounced pleiotropism. Cytokines such as tumor necrosis factor alpha interleukins -5, -10, and -12 play important roles in immunity. Interleukins -2 and -4, and transforming growth factor gamma mediate lymphocyte activation and differentiation.

Monocyte chemoattractant protein 1 mediates glomerular macrophage infiltration in anti-GBM Ab GN. Monocyte chemoattractant protein 1 (MCP-1) is a C-C chemokine with potent monocyte chemotactic and activating properties that may contribute to glomerular macrophage infiltration in anti-GBM Ab GN. We have previously reported increased glomerular steady state expression of MCP-1 mRNA relative to GAPDH mRNA in the heterologous phase of experimental anti-GBM Ab GN. In this report, we expand upon these data by demonstrating that the increase in MCP-1 mRNA correlated with MCP-1 protein expression at 24 hours that was determined with an ELISA (2069 ± 147 pg/mg glom lysate). This increase in MCP-1 expression was associated with glomerular monocyte/ macrophage infiltration which peaked at 24 hours (8.2 ± 1.0 ED-1 cells/glom). The site of MCP-1 mRNA production was localized by combining immunohistochemistry with in situ hybridization. The majority of cells which expressed MCP-1 mRNA at three hours were intrinsic glomerular cells, while 55% of the cells that expressed MCP-1 mRNA at 15 hours were monocytes/macrophages. To determine if MCP-1 affected glomerular macrophage infiltration, rats with α -GBM Ab GN were administered a polyclonal neutralizing Ab to rat MCP-1. This resulted in a 38% decline in glomerular macrophage infiltration (3.3 ± 0.3 vs. 1.8 ± 0.2 ED-1 cells/glom, P = 0.0001) that was associated with a 45% reduction in urinary protein excretion (260 ± 53 vs. 162 ± 46 mg/d, P = 0.0001). These data demonstrate an important role for MCP-1 in the pathogenesis of glomerular macrophage infiltration in anti-GBM Ab GN.

This study investigates the expression and function of monocyte chemoattractant protein-1 (MCP-1) in rats with aminonucleoside nephrosis induced by a single intraperitoneal injection of puromycin aminonucleoside (PAN). On Day 7, PAN-treated rats had a sixfold increase in renal MCP-1 messenger (m)RNA levels and a twofold increase in interleukin-1 beta mRNA levels. During the course of PAN nephrosis, most of the de novo MCP-1 protein resembled protein droplets that were prominent in glomeruli between Days 3 and 14 and weaker but visible in tubules between Days 5 and 10. In addition, occasional tubules showed a cytoplasmic staining pattern for MCP-1. Two studies evaluated the effect of MCP-1 neutralization on renal monocyte recruitment. In the first study, PAN-treated rats were treated with affinity-purified MCP-1-neutralizing rabbit IgG on Days 0, 1, 3, and 5; kidneys were harvested on Day 7. There was no difference in the mean number of interstitial macrophages [119 +/- 28 vs 88 +/- 9 ED-1+ cells/1000 tubulointerstitial (TI) cells; 106 +/- 28 vs 119 +/- 33 Ia+ cells/1000 TI cells] or intraglomerular macrophages [2.0 +/- 0.9 vs 1.7 +/- 0.5 ED-1+ cells/glomerular cross section (gcs); 1.2 +/- 0.3 vs 1.1 +/- 0.4 Ia+ cells/gcs] compared with nephrotic rats treated with nonimmune rabbit IgG. In the second study, a group of PAN-treated rats was treated with MCP-1-neutralizing IgG administered continuously by an intraperitoneal miniosmotic pump for 7 days and was compared with a control group treated in an identical fashion with PAN and nonimmune IgG. On Day 7 there was no difference in the mean number of interstitial macrophages (55 +/- 45 vs 67 +/- 16 ED-1+ and 70 +/- 63 vs 61 +/- 13 Ia+ cells/1000 TI cells) and intraglomerular macrophages (1.0 +/- 0.4 vs 1.6 +/- 0.9 ED-1+ and 0.6 +/- 0.1 vs 1.1 +/- 0.7 Ia+ cells/gcs). The results of this study suggest that although MCP-1 gene and protein expression are increased in the kidneys of rats with aminonucleoside nephrosis, MCP-1 does not appear to play an essential role in early renal monocyte recruitment in this model.

Chemotactic cytokines coordinate the recruitment of leukocytes into the lung during pulmonary inflammation. In a previous study, we determined that rat pulmonary alveolar macrophages (PAMs) facilitate monocyte recruitment and activation in the lung during acute inflammatory lung injury, in part, through the inducible expression of monocyte chemoattractant protein-1 (MCP-1). MCP-1 is an 11 to 15 kD basic peptide that specifically mediates monocyte chemotaxis and activation. Inflammatory mediators that regulate the expression and secretion of MCP-1 by rat PAMs have not been identified. We determined that stimulation of resident rat PAMs with bacterial lipopolysaccharide (LPS), murine tumor necrosis factor-alpha, or human interleukin-1 beta resulted in the inducible expression of MCP-1 mRNA and the secretion of biologically active MCP-1. In contrast, phorbol myristate acetate, a nonphysiologic leukocyte activator, was significantly less effective in stimulating either enhanced MCP-1 mRNA expression or secretion of MCP-1. These results indicate that the expression of MCP-1 mRNA and the secretion of MCP-1 by rat PAMs are regulated by bacterial products (LPS) and inflammatory cytokines. Further, these results suggest PAMs are regulated by bacterial products (LPS) and inflammatory cytokines. Further, these results suggest that resident PAMs, through elaboration of MCP-1, may play a pivotal role in regulating recruitment and activation of monocytes in the lung during acute inflammatory lung injury.

Recent clinical trials have raised concern that therapy with recombinant human erythropoietin (EPO) may increase cardiovascular disease risk, event rate, and mortality. Endothelial cell (EC) apoptosis has been implicated in both atherogenesis as well as in the destabilization and rupture of atheromatous plaques. In the current study we observed that EPO and the EPO-mimetic peptide EMP-1 markedly suppressed lipopolysaccharide-induced apoptosis in EC monolayers. Therapeutic concentrations of EPO upregulated Bcl-2 expression while concurrently diminishing expression of Bax, resulting in a net decrease in the ratio of Bax to Bcl-2 protein concentrations. In vivo studies demonstrated that erythropoietin receptor (EPOR) is abundantly expressed in murine aorta and that EPO treatment for 10 weeks markedly decreased the ratio of Bax to Bcl-2 protein in the aortas of apolipoprotein E-deficient (APO E-KO) mice fed a high-fat diet. To our knowledge these data are the first to reveal a modulation of regulators of the apoptotic pathway in murine aorta by chronic EPO treatment. These observations imply that long-term administration of EPO may have the potential to affect plaque stability.

Using a well-characterized rat model of immune complex-mediated acute inflammatory lung injury, we determined that there is a time-dependent elaboration of monocyte chemotactic activity in bronchoalveolar lavage fluid. Monocyte chemotactic activity is also significantly enhanced in culture supernatants from pulmonary alveolar macrophages (PAMs) from injured rat lungs. Northern hybridization analysis revealed markedly increased expression of rat monocyte chemoattractant protein 1 (MCP-1) mRNA in PAMs obtained from rats with immune complex-induced lung injury. The increased expression of MCP-1 mRNA and associated increase in monocyte chemotactic activity present in culture supernatants of PAMs from injured rat lungs suggest that PAMs may participate in the pathogenesis of acute inflammatory lung injury by the secretion of monocyte chemoattractants including MCP-1.

We have compared the role of tumor necrosis factor (TNF) in the pathogenesis of analogous acute immune complex-induced lung and dermal vascular injury models in rats. Intratracheal administration of IgG anti-bovine serum albumin (BSA), followed immediately by intravenous infusion of BSA, results in acute neutrophil-mediated alveolitis. Neutralization of intrapulmonary TNF activity with anti-TNF antibodies resulted in reduced pulmonary neutrophil recruitment and marked attenuation of lung injury. Intradermal injection of IgG anti-BSA, followed by intravenous BSA, results in acute neutrophil-mediated dermal vasculitis. Neither locally nor systemetically administered anti-TNF antibodies reduced dermal vascular injury as measured by local hemorrhage and vasopermeability changes. Based on morphometric analysis and measurements of myeloperoxidase in tissue extracts, anti-TNF antibodies had no effect on dermal neutrophil recruitment. Intradermal and intrapulmonary administration of physiologic concentrations of recombinant human TNF resulted in modest, dose-dependent increases in local vasopermeability accompanied by negligible neutrophil recruitment. Administration of higher concentrations of TNF resulted in increases in both local vasopermeability and neutrophil recruitment. These data suggest that while both rat pulmonary and dermal blood vessels can respond to TNF-triggered proinflammatory events, endogenous TNF plays a much greater role in acute alveolitis than in dermal vasculitis.

C ONSIDERABLE new insights have been obtained regarding mechanisms of leukocyte-mediated tissue injury. Since endothelial cell injury often occurs during the acute inflammatory response, much work has been done to define in vitro mechanisms involving the killing of endothelial cells by activated neutrophils. At the same time, the use of in vivo models of acute lung injury has allowed definition of events that can be linked to the development of acute vascular injury by products of activated phagocytic cells. We will briefly review recent studies that affect both topics.

It is becoming increasing clear that injury to cells or tissues related to the inflammatory reaction can be ascribed to a complex array of mediators generated and released from activated phagocytes such as neutrophils, monocytes and macrophages. Furthermore, there is now accumulating evidence for cell-cell interactions which involve phagocytic cells and result in amplified damage at the cellular or tissue level. We will provide insights into mechanisms of injury to tissues or cells at two levels: in vivo lungs or dermal inflammatory reactions initiated by the local deposition of immune complexes and in vitro killing of endothelial cells by activated neutrophils.

Intense research efforts have been directed toward characterizing mediators that control the inflammatory response and regulate the growth, differentiation, and function of cells involved in inflammation. Tumor necrosis factor, or cachectin, and members of a heterogeneous group of peptides called interleukins exhibit a wide spectrum of activities, some of which appear to influence the evolution of inflammatory processes. This review outlines the observations that have led to our current understanding of the biology of tumor necrosis factor and the interleukins. Particular attention is directed toward the evidence suggesting that these cytokines function as mediators of inflammatory responses.

Patients with advanced chronic renal disease (CRD) suffer from excessive morbidity and mortality due to complications of accelerated atherosclerosis. Approximately 90% of dialysis-dependent end stage renal disease patients suffer from anemia. Recombinant human erythropoietin (EPO) in combination with iron has become widely used to treat anemic CRD patients. While treatment with EPO results in improved quality of life it may also contribute to the development of atherosclerosis. Recent studies suggest that a reduction in nitric oxide (NO) availability may be linked to EPO-induced vascular dysfunction. Furthermore, CRD per se is thought to result in a state of NO deficiency. The present study suggests that EPO may exert proatherogenic activity by augmenting the cytokine-induced expression of monocyte-chemoattractant protein-1 (MCP-1) in human umbilical vein endothelial cells (HUVECs) and by stimulating the proliferation of HUVECs and human vascular smooth muscle cells (HVSMCs). Augmentation of MCP-1 expression appears to be linked to EPO-induced downregulation of endothelial NO synthase (ecNOS). NO released from a series of synthetic donor compounds suppressed the EPO-mediated augmentation of cytokine-induced MCP-1 expression. In vitro studies revealed that EPO reduces ecNOS expression at both the protein and mRNA levels and that EPO also mediates a reduction in ecNOS enzymatic activity. These observations suggest potential mechanisms through which EPO may contribute to the development of accelerated atherosclerosis, particularly in the setting of CRD where NO availability may already be compromised.

Monocyte chemoattractant protein-1 (MCP-1) is a pivotal mediator of angiocentric granuloma formation in glucan-induced pulmonary granulomatous vasculitis. Based on the rationale that mononuclear phagocytes retrieved from granulomas are rich sources of nitric oxide (NO) and that the recruitment of mononuclear phagocytes into lesions abates as granuloma formation slows, we tested the hypothesis that MCP-1 gene expression is regulated by a NO-sensitive mechanism. Preexposure of endothelial cell (EC) monolayers to NO donor compounds markedly reduced cytokine-induced MCP-1 expression and cytosolic-to-nuclear translocation of nuclear factor-kappa B (NF-kappaB), reversed fluctuations in endothelial reduced glutathione (GSH) pools but did not affect cGMP concentrations. The lungs of mice bearing targeted disruptions of the inducible nitric oxide synthase (iNOS) gene exhibited significantly higher concentrations of MCP-1 following glucan infusion than did those of wild-type mice. Cumulatively, these data suggest that NO suppresses MCP-1 expression by blunting the redox changes associated with cytokine-induced EC activation.

OBJECTIVES Envenomation by Loxosceles species (brown recluse) spiders results in large dermal inflammatory lesions. Venom-induced dermal inflammation occurs indirectly via soluble mediators of inflammation. This study aimed to explore whether the anatomic extent of dermonecrotic arachnidism is due to the cascade of soluble proinflammatory mediators elicited by venom deposited at the bite site, or due to diffusion of the venom per se. METHODS Three New Zealand white rabbits received intradermal L. reclusa venom (3-microg) injections in the flank. At the time of maximum dermal inflammation (24 hr), paired 4-mm dermal biopsies were obtained in 2-cm intervals extending 0 to 12 cm from the inoculation site. Normal dermal tissue was obtained from the opposite flank to serve as a negative control. One biopsy sample from each interval was homogenized and assayed for myeloperoxidase (MPO) activity and for the presence of venom via an enzyme immunoassay (EIA). The other paired dermal biopsy was sectioned, and examined for the presence of polymorphonuclear neutrophils (PMNs) by microscopy. Lesional areas were measured using digital images imported into imaging software. RESULTS Mean +/- SD lesional diameter 24 hours post inoculation measured 9.18 +/- 0.64 cm. Venom was detected in biopsies 0 to 10 cm from the injection site. As expected, the highest venom concentrations were measured at the inoculation site (4.28 +/- 3.9 ng/4 mm). In addition, PMNs and MPO were detected up to 8 and 10 cm from the inoculation site, respectively. Neither PMNs nor MPO was detected in tissue absent of venom (kappa = 0.88, p < 0.001). CONCLUSIONS Loxosceles venom diffuses from the envenomation site. The extent of dermal inflammation mirrors the extent of Loxosceles venom diffusion. This observation implies that the venom itself defines the extent and magnitude of tissue injury following Loxosceles envenomation.

We characterized the antigenic cross-reactivity of two medically important North American Loxoxceles species: L. reclusa (native to southeastern US) and L. deserta (native to southwestern US). Dermonecrosis resulting from bites from these two North American spider species are indistinguishable clinically. Polyclonal IgG antivenins directed against L. reclusa and L. deserta were raised in rabbits and used to develop specific enzyme immunoassays (EIAs). Antigenic differences in the two venoms were evaluated as follows: (1) Comparison of the sensitivities and correlation coefficient (R(2)) of anti-L. reclusa (alpha LoxR) and anti-L. deserta antibodies (alpha LoxD) in the detection of varying concentrations of the two venoms; (2) separation and western blot comparison of venom components; (3) protein sequence analysis of L. desertavenom and comparison to the L. reclusa protein sequence analysis present in a US national database; and (4) in vivo evaluation of alpha LoxR and alpha LoxD antivenins in attenuating dermal lesions (rabbit model). Correlation coefficients for alpha LoxR (R(2)=0.99) and alpha LoxD (R(2)=0.99) polyclonal antibodies in the measurements of standard concentrations of venoms were virtually identical. Western blot analysis revealed multiple common bands between the two venoms. Amino acid data (amino acids 1-35, N-terminal) of the active venom components of the two venoms revealed only three non-identical amino acids. alpha LoxR and alpha LoxD antivenins were similarly effective in blocking the development of rabbit skin lesions (ANOVA p

Loxosceles spiders, of which the brown recluse is the best known, are indigenous to southcentral and southwestern regions of the United States. Loxosceles spider envenomation frequently results in painful, centrally necrotic, erythematous skin lesions that evolve over 24 to 48 hours and may take several weeks to completely heal. The diagnosis of loxoscelism is typically is based on the presence of the characteristic dermal lesion, because no definitive clinical diagnostic assay exists, and the spider is generally not available for identification. We used a rapid Loxosceles-specific enzyme immunoassay to detect spider venom in a dermal biopsy and hairs plucked from a suspicious skin lesion on the lower extremity of a 52-year-old man. This report indicates that in using a novel Loxosceles-specific immunoassay, venom can be detected in dermonecrotic skin and hair specimens for up to 4 days after envenomation.

Chemokines are a family of structurally related cytokines that activate and recruit leukocytes into areas of inflammation. The “CC” chemokine, monocyte chemoattractant protein (MCP)-1 may regulate the microglia/monocyte response to acute brain injury. Recent studies have documented increased expression of MCP-1 in diverse acute and chronic experimental brain injury models; in contrast, there is little information regarding expression of the MCP-1 receptor, CCR2, in the brain. In the neonatal rat brain, acute excitotoxic injury elicits a rapid and intense microglial response. To determine if MCP-1 could be a regulator of this response, we evaluated the impact of excitotoxic injury on MCP-1 and CCR2 expression in the neonatal rat brain. We used a reproducible model of focal excitotoxic brain injury elicited by intrahippocampal injection of NMDA (10 nmol) in 7-day-old rats, to examine injury-induced alterations in MCP-1 and CCR2 expression. RT-PCR assays demonstrated rapid stimulation of both MCP-1 and CCR2 mRNA expression. MCP-1 protein content, measured by ELISA in tissue extracts, increased >30-fold in lesioned tissue 8–12 h after lesioning. CCR2 protein was also detectable in tissue extracts. Double-immunofluorescent labeling enabled localization of CCR2 both to activated microglia/monocytes in the corpus callosum adjacent to the lesioned hippocampus and subsequently in microglia/monocytes infiltrating the pyramidal cell layer of the lesioned hippocampus. These results demonstrate that in the neonatal brain, acute excitotoxic injury stimulates expression of both MCP-1 and its receptor, CCR2, and suggests that MCP-1 regulates the microglial/monocyte response to acute brain injury.

Bites from the brown recluse spider and other arachnids from the genus Loxosceles frequently induce necrotic skin lesions that can be recalcitrant to treatment and disfiguring. The authors used a rabbit model of dermonecrotic arachnidism to address the therapeutic efficacy of intradermal (id) polyclonal anti-Loxosceles Fab fragments (alphaLoxd Fab) raised against Loxosceles deserta spider venom.Fab fragments were prepared by papain digestion and affinity chromatography from the IgG fraction of L. deserta antivenom raised in rabbits. Eighteen inbred New Zealand white rabbits were assigned to six groups of three. The rabbits received L. deserta venom (3 microg, id) injections into each flank. Cohorts of rabbits received single id injections (at one venom site/rabbit) of 30 microg alphaLoxd Fab at different times (T = 0, 1, 2, 4, 8, and 12 hours) after venom injection. In each rabbit the opposite flank was left untreated. As an additional control, one group of rabbits (T = 0) received nonspecific Fab (30 microg, id) in the opposite flank. Dermal lesions were quantified as a function of time through the use of a series of digital photographs and imaging software. In addition, myeloperoxidase (MPO) activity, a measure ofneutrophil accumulation, was determined in lesion biopsies. Lesion areas and MPO activities were analyzed by repeated-measures analysis of variance (ANOVA).Lesion areas and MPO activity were markedly reduced when alphaLoxd Fab was administered very early after venom injections. As the interval between venom inoculation and antivenom treatment increased, the therapeutic benefit of alphaLoxd Fab decreased. The final time tested that demonstrated therapeutic efficacy of alphaLoxd Fab was T = 4 hours. Lesion attenuation was no longer apparent when alphaLoxd Fab was given 8 hours post inoculation.Intradermal administration of alphaLoxd Fab attenuates Loxosceles-induced dermonecrotic lesion formation when given up to 4 hours after venom inoculation in this rabbit model.

Loxosceles spider evenomation in man frequently results in disfiguring necrotic skin lesions. Recent studies suggest that several proinflammatory mediators participate in lesion development. We have observed that Loxosceles deserta venom induces production of the chemokines interleukin-8, growth-related oncogene alpha, and monocyte chemoattractant protein-I by human umbilical vein endothelial cells. Members of the Rel/Nuclear factor (NF)-kappaB family of transcription factors are important regulators of many genes involved in immune and inflammatory responses. We hypothesized that Loxosceles-venom-induced chemokine expression in human umbilical vein endothelial cells is mediated by NF-kappaB.Human umbilical vein endothelial cell monolayers were exposed to activating concentrations of Loxosceles deserta venom. Nuclear extracts of these monolayers were analyzed by electrophoretic mobility shift assay. A direct cause and effect linkage between NF-kappaB activation and chemokine expression by Loxosceles venom was established through examination of the effect of SN50 on interleukin-8 and monocyte chemoattractant protein-1 production using a whole-cell enzyme immunoassay. SN50 is a cell-permeable peptide that specifically blocks cytosolic to nuclear translocation of NF-kappaB. Furthermore, the venom-induced synthesis of chemokine mRNAs was investigated by RNase protection assays.Loxosceles deserta venom induces the activation of NF-kappaB in human umbilical vein endothelial cells. Antibodies to p50 and p65, but not to p52, c-Rel, or Rel B, induce supershifts of the DNA-protein complexes formed by oligonucleotide probes and nuclear extracts from venom-activated human umbilical vein endothelial cells. SN50 peptide inhibits NF-kappaB translocation and interleukin-8 and monocyte chemoattractant protein-1 production in activated human umbilical vein endothelial cells.Loxosceles deserta venom induces synthesis of interleukin8 and monocyte chemoattractant protein-1 mRNAs in human umbilical vein endothelial cells. The expression of chemokines occurs via an NF-kappaB-dependent pathway.

Monocyte chemoattractant protein-1 (MCP-1) regulates monocyte accumulation in several macrophage-dependent experimental disease models. In the neonatal brain, activated microglia accumulate rapidly after hypoxic-ischemic injury. These cells produce potentially neurotoxic factors that may contribute to the progression of injury. To determine whether MCP-1 could be one of the molecular signals that influences the microglial response to hypoxic-ischemic injury in the neonatal brain, we examined the impact of acute hypoxic-ischemic injury on MCP-1 mRNA and protein expression. Seven-day-old rats underwent right carotid artery ligation, followed by 3 hours of 8% oxygen exposure, to elicit ipsilateral forebrain hypoxic-ischemic injury. To detect MCP-1 mRNA in situ hybridization assays were performed using 35S-labeled antisense riboprobes generated from rat MCP-1 cDNA. Animals were evaluated 0, 1, 2, 4, 8, 16, 24, 48, and 120 hours after hypoxic exposure (N > or = 3/group). Immunocytochemistry (with a polyclonal rabbit antirat MCP-1 antibody) was used to determine the anatomic and temporal distribution of MCP-1, in samples obtained 10 minutes to 5 days after hypoxic exposure (N > or = 3/group). Monocyte chemoattractant protein-1 mRNA was first detected in periventricular regions of the lesioned hemisphere 1 hour after hypoxia-ischemia; periependymal and intraparenchymal MCP-1 mRNA expression were detected at 4 hours; hybridization signal peaked at 8 to 24 hours; and no MCP-1 mRNA was detected at 48 and 120 hours. In lesioned forebrain, MCP-1 protein expression were consistently detected at 2.5 to 48 hours after hypoxia-ischemia. Many immunoreactive cells appeared to be neurons. These results suggest that in the developing brain, MCP-1 could represent a functionally important molecular signal for the microglial response to hypoxic-ischemic injury.

Objective To determine the relative effect of an ultrasonic scalpel on reproductive tissue compared with CO 2 laser and electrosurgery. Design Prospective, randomized animal study. Setting University laboratory setting. Animals Sixteen New Zealand White rabbits. Intervention(s) A steel scalpel, an ultrasonic scalpel, a CO 2 laser, or electrosurgery were used to perform an ovarian wedge resection and to remove the distal uterine horn. A 3-cm longitudinal incision also was made in the uterine horn. Main Outcome Measure(s) The number of 1-second bursts of needle-tip electrosurgery required for hemostasis, the depth and degree of coagulation necrosis, degree of fibrin deposition, and postoperative adhesion formation. Result(s) The amount of electrosurgery needed to achieve hemostasis was less for any of the four power techniques than for the steel scalpel, with the exception of the ultrasonic scalpel at level 5 when used on the ovary. The depth (range: 0.30 to 0.38 mm) and the degree of coagulation necrosis was not different for any of the power techniques. The fibrin score was greatest for the ultrasonic scalpel at level 5 in both the ovarian tissue and the uterine tissue. There was no difference in adhesion scores for the power techniques and the steel scalpel. Conclusion(s) The ultrasonic scalpel at level 3 is not different from either CO 2 laser or electrosurgery in terms of hemostatic properties, coagulation necrosis, or adhesion formation in the rabbit model. Fertil Steril ® 1997;67:382-6

Objectives Missing laboratory data is a common issue, but the optimal method of imputation of missing values has not been determined. The aims of our study were to compare the accuracy of four imputation methods for missing completely at random laboratory data and to compare the effect of the imputed values on the accuracy of two clinical predictive models. Design Retrospective cohort analysis of two large data sets. Setting A tertiary level care institution in Ann Arbor, Michigan. Participants The Cirrhosis cohort had 446 patients and the Inflammatory Bowel Disease cohort had 395 patients. Methods Non-missing laboratory data were randomly removed with varying frequencies from two large data sets, and we then compared the ability of four methods—missForest, mean imputation, nearest neighbour imputation and multivariate imputation by chained equations (MICE)—to impute the simulated missing data. We characterised the accuracy of the imputation and the effect of the imputation on predictive ability in two large data sets. Results MissForest had the least imputation error for both continuous and categorical variables at each frequency of missingness, and it had the smallest prediction difference when models used imputed laboratory values. In both data sets, MICE had the second least imputation error and prediction difference, followed by the nearest neighbour and mean imputation. Conclusions MissForest is a highly accurate method of imputation for missing laboratory data and outperforms other common imputation techniques in terms of imputation error and maintenance of predictive ability with imputed values in two clinical predicative models.

Glucan-induced pulmonary granulomatous vasculitis in the rat mimics several human lung diseases (e.g., Wegener's granulomatosis, intravenous talcosis). We sought to clarify the role of intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of glucan-induced granulomatous vasculitis. Immunohistochemical analysis of lung sections from rats with florid vasculitis (48 hours) revealed marked alveolar septal and lesional expression of ICAM-1. An ex vivo binding analysis with isotope-labeled antibodies and lung sections taken at various times up to 48 hours after glucan infusion revealed a progressive increase in whole-lung ICAM-1 expression. In vivo measurements of vascular wall-associated ICAM-1 expression revealed an earlier rise that began less than 6 hours after glucan infusion, peaked at 24 to 48 hours, and then declined to near baseline during the ensuing 24 to 96 hours. To assess whether ICAM-1 expression both within blood vessel walls and within lesions per se is important in granuloma development, we carried out in vivo neutralization experiments with several different routes of administration of antibody to ICAM-1. Monoclonal antibody to rat ICAM-1 was either infused intravenously at time 0 (when glucan was infused), infused intravenously at time 0 and after 24 hours, instilled only intratracheally 24 hours after glucan infusion, or given both intravenously (time = 0 and 24 hours) and intratracheally (time = 24 hours). Infusions of monoclonal antibody to rat ICAM-1 resulted in dose-dependent reductions in mean granuloma number and cross-sectional area. Intrapulmonary instillation of antibody to rat ICAM-1 (via tracheostomy 24 hours after glucan infusion) resulted in a modest reduction in mean granuloma number and cross-sectional area. When antibody to ICAM-1 was both infused and instilled via the trachea, we found an additive reduction in mean granuloma size and number. There was a 12-fold increase in adhesion of ED-1-positive peripheral blood mononuclear cells (monocytes) to granuloma-bearing frozen lung sections prepared 48 hours after glucan infusion. Moreover, 73% of the additional adherent monocytes were bound specifically to granulomas per se. The increase in ex vivo monocyte binding to lung sections prepared at 48 hours was reduced 62% when sections were incubated with monoclonal antibody to ICAM-1. Taken together, these data indicate that ICAM-1 expression in evolving glucan-induced granulomatous vasculitis occurs first within blood vessel walls and then within lesional cells per se. The in vivo blocking studies suggest that ICAM-1 expression in both anatomic sites is important in granuloma development.

Anti–neutrophil cytoplasmic antibody (ANCA) determinations appear to be useful in the diagnostic evaluation and therapeutic monitoring of patients with necrotizing vasculitis. The purpose of this study was to determine whether semiquantitative ANCA measurements are useful in distinguishing between increases in primary disease activity and complications of cytotoxic or immunosuppressive therapy. The authors reviewed clinical data from 27 consecutive ANCA-positive patients who had at least three ANCA determinations performed at the University of Michigan Medical Center. Eleven patients had Wegener’s granulomatosis (WG), 3 had “limited” WG; 9 had “ANCA-positive” vasculitis; and 4 had “pauciimmune” rapidly progressive glomerulonephritis. During a period of 3 months to 3.8 years, 159 ANCA determinations were performed, primarily for the purposes of diagnosis and monitoring disease activity. Eleven episodes were identified in which a single ANCA assay was used to distinguish between increased primary disease activity and a suspected therapeutic complication. The suspected therapeutic complications included pneumonia (8 of 11), bacterial sinusitis (1 of 11), cyclophosphamide-induced pneumonitis (1 of 11), and cyclophosphamide-induced vestibular toxicity (1 of 11). In 10 of 11 instances in which an ANCA assay was used to distinguish between an exacerbation in vasculitic disease activity and a therapeutic complication, interpretation of the ANCA titer was pivotal in arriving at a therapeutically appropriate decision either to increase or discontinue cytotoxic therapy. This study suggests that ANCA determinations can be used reliably to distinguish between increased primary disease activity and complications of therapy that mimic an increase in disease activity.

Using a well-characterized model of bleomycin-induced pulmonary fibrosis in the rat, we determined that there was a time-dependent elaboration of monocyte chemotactic activity in bronchoalveolar lavage fluid. Northern hybridization analysis revealed markedly increased expression of rat monocyte chemoattractant protein-1 (MCP-1) mRNA in alveolar macrophages (AMs) from rats following induction of pulmonary fibrosis. Monocyte chemotactic activity was also significantly increased in conditioned media from AMs retrieved from injured rat lungs. These data suggest that one important role of AMs in the pathogenesis of chronic inflammatory lung injury and pulmonary fibrosis is the regulation of monocyte recruitment and activation within the lung secondary to secretion of monocyte chemoattractants including MCP-1.

We report here the production of systemic levels of tumor necrosis factor (TNF) and interleukin 6 (IL-6) and associated changes in serum macroglobulin to albumin ratios in a nonlethal rat model of partial hepatic ischemia/reperfusion (I/R). Plasma IL-6 was detectable and elevated at 1 hr of reperfusion as compared to sham-operated controls (I/R rats = 12,100 +/- 3860 pg/ml; sham rats = 5260 +/- 842 pg/ml; IL-6 values = means +/- SEM) and reached maximal levels at 6 hr of reperfusion (I/R rats = 47,400 +/- 25,700 pg/ml; sham rats = 3370 +/- 394 pg/ml), in contrast to maximal TNF levels at 30 min of reperfusion (I/R rats = 72 +/- 15 pg/ml; sham rats = 2 +/- 2 pg/ml; TNF values = means +/- SEM). Pretreatment with neutralizing TNF antisera prior to ischemia resulted in a reduction of IL-6 at 1 hr of reperfusion (anti-TNF = 3870 +/- 2550 pg/ml; control serum = 7650 +/- 1670 pg/ml), but was without effect on IL-6 levels at subsequent time points over the 24 hr of reperfusion. Electrophoretic determination of macroglobulin (alpha 1 + alpha 2) and albumin concentrations in sham-operated and ischemia/reperfusion animals demonstrated an elevation in the macroglobulin/albumin ratio in both groups over time, suggestive of an acute phase response, and the ratio was unchanged by immunoneutralization of TNF prior to ischemia/reperfusion. We conclude that this model of hepatic ischemia/reperfusion results in temporally distinct systemic elevations in IL-6 and TNF; however, the induction of IL-6 and the associated changes in serum macroglobulin concentration are independent of TNF.

Acute lung injury is a common feature of the adult respiratory distress syndrome in which there is cellular injury involving both pulmonary microvascular endothelial cells and alveolar epithelial cells. In an attempt to define in greater detail the pathophysiological events that underlie the development of acute lung injury, we have employed the rat model of acute lung injury following intraalveolar deposition of immunoglobulin G (IgG) immune complexes. In this model, injury involves both microvascular endothelial cells as well as alveolar epithelial cells in a manner that is complement dependent, neutrophil dependent, and related to the formation of toxic oxygen species, inasmuch as interventions involving catalase, superoxide dismutase, and scavengers of HO· are all protective [1–3]. It is now becoming evident that products of both neutrophils and lung macrophages are involved in the processes leading to injury.

We have analyzed leukocyte to lung adhesive interactions and neutrophil-mediated lung injury using a rat lung organ culture system. Rat lung slices were maintained in tissue culture on gelatin sponges floating at the gas-liquid interface. Maintenance of three-dimensional alveolar structure, critical to the viability of lung tissue, was achieved by instilling 0.5% agarose (in 37 degrees C RPMI 1640) into the lungs during tissue explanation. Quantitative neutrophil to lung adhesive interactions were examined using an adaptation of the Woodruff-Stamper frozen section binding assay. Pretreatment of organ cultures with recombinant human tumor necrosis factor (rhTNF) resulted in a protein synthesis-dependent three- to fourfold increase in adhesiveness for neutrophils. Time course and mononuclear leukocyte binding experiments revealed that TNF-induced rat lung adhesiveness peaks at 4 h and is largely neutrophil-specific. Agonist-induced activation of neutrophils in the presence of [3H]leucine-labeled organ cultures resulted in lung injury as assessed by radioisotope release. These observations are consistent with endothelial cell culture data that indicate that TNF-induced endothelium exhibits a protein synthesis-dependent increase in adhesiveness for neutrophils. These data validate rat lung organ cultures as a model system that can be used to assess mechanisms of neutrophil adhesion and leukocyte-mediated tissue injury.