Polymorphisms of the tumor necrosis factor alpha locus among autoimmune disease susceptible and resistant inbred rat strains.

Inbred rat strains manifest remarkable differences in susceptibility/severity to autoimmune disease. MHC alleles strongly influence the pathogenesis of autoimmune disease in rats, but the precise mechanism(s) remain inadequately defined. The TNFalpha gene is located in the class III region of the MHC. Polymorphisms, influencing either the structure or expression of the TNF protein, might contribute to differences in autoimmune disease susceptibility/severity. We therefore sequenced the Tnf locus using genomic DNA from ACI, BB(DR), BN, DA, F344, and LEW rats that vary in susceptibility/severity to autoimmune diseases. We found 42 polymorphisms among these six strains. Although none of these polymorphisms are predicted to change the amino acid sequence of the TNF protein, several reside in potential non-coding regulatory regions and may influence expression levels. These polymorphisms may serve as good candidates for analysis of TNF expression to elucidate the mechanism(s) by which the MHC regulates susceptibility and/or severity of autoimmune diseases.

Genetic dissection of collagen-induced arthritis in Chromosome 10 quantitative trait locus speed congenic rats: evidence for more than one regulatory locus and sex influences.

Rat Chromosome 10 (RNO10) harbors Cia5, a non-MHC quantitative trait locus (QTL) that regulates the severity of type II collagen-induced arthritis (CIA) in DAxF344 and DAxBN F2 rats. CIA is an animal model with many features that resemble rheumatoid arthritis. To facilitate analysis of Cia5 independently of the other CIA regulatory loci on other chromosomes, DA recombinant QTL speed congenic rats, DA.F344(Cia5), were generated. These QTL congenic rats have a large chromosomal segment containing Cia5 (interval size < or =80.1 cM) from CIA-resistant F344 rats introgressed into their genome. Phenotypic analyses of these rats for susceptibility and severity of CIA confirmed that Cia5 is an important disease-modifying locus. CIA severity was significantly lower in the Cia5 congenic rats than in DA controls. We also generated DA Cia5 speed sub-congenic rats, DA.F344(Cia5a), which had a smaller segment of the F344 genome, Cia5a, comprising only the distal q-telomeric end (interval size < or = 22.5 cM) of Cia5, introgressed into their genome. DA.F344(Cia5a) sub-congenic rats also exhibited reduced CIA disease severity compared with the parental DA rats. The regulatory effects in both congenic strains were sex influenced. The disease-ameliorating effect of the larger fragment, Cia5, was greater in males than in females, but the effect of the smaller fragment, Cia5a, was greater in females. We also present an improved genetic linkage map covering the Cia5/Cia5a region, which we have integrated with two rat radiation hybrid maps. Comparative homology analysis of this genomic region with mouse and human chromosomes was also undertaken. Regulatory loci for multiple autoimmune/inflammatory diseases in rats (RNO10), mice (MMU11), and humans (HSA17 and HSA5q23-q31) map to chromosomal segments homologous to Cia5 and Cia5a.

Hemodynamic changes induced by liposomes and liposome-encapsulated hemoglobin in pigs: a model for pseudoallergic cardiopulmonary reactions to liposomes. Role of complement and inhibition by soluble CR1 and anti-C5a antibody.

BACKGROUND: Intravenous administration of some liposomal drugs can trigger immediate hypersensitivity reactions that include symptoms of cardiopulmonary distress. The mechanism underlying the cardiovascular changes has not been clarified. METHODS AND RESULTS: Anesthetized pigs (n=18) were injected intravenously with 5-mg boluses of large multilamellar liposomes, and the ensuing hemodynamic, hematologic, and laboratory changes were recorded. The significant (P<0.01) alterations included 79+/-9% (mean+/-SEM) rise in pulmonary arterial pressure, 30+/-7% decline in cardiac output, 11+/-2% increase in heart rate, 236+/-54% increase in pulmonary vascular resistance, 71+/-27% increase in systemic vascular resistance, and up to a 100-fold increase in plasma thromboxane B2. These changes peaked between 1 and 5 minutes after injection, subsided within 10 to 20 minutes, were lipid dose-dependent (ED50=4. 5+/-1.4 mg), and were quantitatively reproducible in the same animal several times over 7 hours. The liposome-induced rises of pulmonary arterial pressure showed close quantitative and temporal correlation with elevations of plasma thromboxane B2 and were inhibited by an anti-C5a monoclonal antibody (GS1), by sCR1, or by indomethacin. Liposomes caused C5a production in pig serum in vitro through classic pathway activation and bound IgG and IgM natural antibodies. Zymosan- and hemoglobin-containing liposomes and empty liposomes caused essentially identical pulmonary changes. CONCLUSIONS: The intense, nontachyphylactic, highly reproducible, complement-mediated pulmonary hypertensive effect of minute amounts of intravenous liposomes in pigs represents a unique, unexplored phenomenon in circulation physiology. The model provides highly sensitive detection and study of cardiopulmonary side effects of liposomal drugs and many other pharmaceutical products due to "complement activation-related pseudoallergy" (CARPA).

The effects of biological and social risk factors on special education placement: birth weight and maternal education as an example.

The effects of birth weight (BW) and maternal education (ME) on special education placement at age 10 were studied. Epidemiologic methods quantified risk to the individual and to the population using an electronically linked, county-wide database of birth and school records. A dose-response relationship was found between BW and ME. High ME may serve as a buffer for children with a biological risk for developmental delays. A clinically important finding was that children born with very low BW to mothers with low ME were at a high level of individual risk for receiving special education services. However, such children accounted for a small number of the overall cases. The largest percentage of children receiving special education services had the single risk factor of low ME. From a public policy standpoint, children born to mothers with low levels of education are an important group to target for early intervention.

Receptor mechanisms of bradykinin-mediated activation of prenodal lymphatic smooth muscle.

We have previously shown that several endogenous vasoactive agents constrict prenodal lymph vessels in the canine forelimb. In this study, we assessed the receptor mechanisms by which bradykinin activates lymphatic smooth muscle. Intralymphatic (i.l.) infusion of bradykinin at concentrations of 3.82 x 10(-6), 3.82 x 10(-5) and 3.82 x 10(-4) molar significantly increased lymphatic perfusion pressure. To determine the potential role of lymphatic alpha-receptors in this response, we infused bradykinin at a concentration of 3.82 x 10(-4) molar i.l. before and during intra-arterial (i.a.) phentolamine administration. Prior to phentolamine, bradykinin resulted in a doubling of the lymphatic perfusion pressure. Phentolamine alone had no effect on the resting lymphatic pressure, but significantly reduced forelimb arterial pressures. When the infusion of bradykinin was repeated during phentolamine administration, there was no significant change in the lymphatic perfusion pressure. To determine the subclass of alpha-adrenergic receptors involved in this response, we infused bradykinin and the alpha1-receptor agonist phenylephrine i.l. before and during administration of i.a. prazosin, a specific alpha1-receptor antagonist, i.a. Prior to prazosin, both phenylephrine and bradykinin significantly increased lymphatic perfusion pressure. During prazosin administration, neither phenylephrine nor bradykinin significantly altered the lymphatic perfusion pressure. These data indicate that bradykinin-mediated increases in prenodal lymphatic smooth muscle tone are mediated by lymphatic alpha1-adrenergic receptors.

Receptor mechanisms of serotonin-induced prenodal lymphatic constriction in the canine forelimb.

Numerous endogenous vasoactive agents have been shown to cause lymphatic smooth muscle contraction. In this study, we assessed the ability of serotonin (5-HT) to alter lymphatic smooth muscle activity and elucidated the receptor mechanisms of 5-HT's actions. Both intralymphatic and intra-arterial administration of 5-HT significantly increased lymphatic smooth muscle activity in lymphatics perfused at constant flow, as indicated by an increase in lymphatic perfusion pressure. The 5-HT-induced increase in lymphatic perfusion pressure is attenuated but not blocked by the intra-arterial infusion of phentolamine, suggesting the involvement of alpha-adrenoreceptors and 5-HT receptors. Intralymphatic infusion of the 5-HT2-receptor-agonist alpha-methylserotonin significantly increased lymphatic perfusion pressure, either alone or when administered into an alpha-receptor blocked preparation, whereas the 5-HT1-receptor-agonist carboxyami-dotryptamine maleate did not effect the prenodal lymphatics. These data indicate that the lymphatic smooth muscle contraction produced by 5-HT is mediated both by lymphatic alpha-adrenoreceptors and 5-HT2 receptors.

Pilot-scale demonstration of a two-stage methanotrophic bioreactor for biodegradation of trichloroethylene in groundwater.

A two-stage methanotrophic bioreactor system was developed for remediation of water contaminated with TCE and other chlorinated, volatile, aliphatic hydrocarbons. The first stage of the reactor was a suspended-growth culture vessel using a bubbleless methane-transfer device. The second stage was a plug-flow bioreactor supplied with contaminated groundwater and cell suspension from the culture vessel. The test objectives were to determine the applicability of microbial culture conditions reported in the literature for continuous, pilot-scale TCE treatment; the technical feasibility of plug-flow bioreactor design for treatment of TCE; and the projected economic competitiveness of the technology considering the cost of methane for growth of methanotrophs. The methanotrophic organism used in the study was Methylosinus trichosporium OB3b. Information on system operation was obtained in bench tests prior to conducting the pilot tests. In bench- and pilot-scale tests, variability in the degree of TCE degradation and difficulty in maintaining the microbial culture activity led to short periods of satisfactory biotreatment. Further development of the microbial culture system will be required for long-term operation. During transient periods of high TCE degradation activity, the bioreactor concept proved feasible by exhibiting both a high degree of TCE biodegradation (typically about 90% at influent TCE concentrations of 0.5-4 ppm) and a close approximation to first-order reactor kinetics throughout the length of the reactor. Actual methane usage in the pilot-scale reactor resulted in projected methane costs of $0.33 per 1000 gallons of water treated. This cost theoretically would be reduced by system modifications. The theoretical minimum methane cost was approximately $0.05 per 1000 gallons.

A comparison of microbial community characteristics among petroleum-contaminated and uncontaminated subsurface soil samples.

Measurements of microbial community size, including total cell counts and specific degrader enumerations, were conducted on subsurface soil samples from both petroleum-contaminated and pristine aquifers. Samples were collected from both uncontaminated and contaminated areas of the petroleum-contaminated sites. In pristine and uncontaminated samples, total cell counts (acridine orange direct counts) were related to depth. The deeper samples contained smaller total microbial populations. However, indices of microbial activity varied considerably from sample to sample and probably reflect soil and site heterogeneity. Exposure to petroleum contamination apparently altered the microbial community structure. In samples exposed to low levels of contaminants as vapors and/or dissolved phases (ppb concentrations), and not free product, the toluene-specific degrader populations were larger at greater depths, and the numbers of amino acid-specific degraders were highly correlated to the numbers of decane-specific degraders, indicating that petroleum-adapted microbial communities were present in the contaminated samples. In highly contaminated samples, total microbial population densities decreased with increasing depth; however, microbial activity tended to increase with depth. These results indicate that petroleum contaminants exert toxic effects on the active microbial community at high exposures and enrich specific degraders at ppb levels of dissolved contaminants.

Catecholamine-mediated lymphatic constriction: involvement of both alpha 1- and alpha 2-adrenoreceptors.

It has been proposed that catecholamines may regulate lymphatic function by altering lymphatic resistance. In this study we perfused lymphatics in the paw of the anesthetized dog, administering catecholamines intralymphatically before and during intra-arterial administration of adrenoreceptor antagonists. Epinephrine and norepinephrine both increased lymphatic perfusion pressure. When the infusions were repeated during intra-arterial phentolamine, neither catecholamine significantly altered lymphatic perfusion pressure, thus implicating lymphatic adrenoreceptors. Intralymphatic infusion of these catecholamines during the intra-arterial infusion of prazosin significantly increased lymphatic perfusion pressure, but the increase was markedly less than that seen prior to prazosin. In these same animals, the lymphatic constriction produced by intralymphatic phenylephrine was completely blocked, indicating an effective blockade of the alpha 1-receptors. The intralymphatic infusion of phenylephrine or alpha-methylnorepinephrine both significantly increased lymphatic perfusion pressure, confirming the presence of both alpha 1- and alpha 2-receptors. These data demonstrate that the prenodal lymph vessels of the canine forelimb contain both alpha 1- and alpha 2-receptors and that epinephrine and norepinephrine interact with both classes of receptors to produce lymphatic constriction.

Neuropeptide modulation of lymphatic smooth muscle tone in the canine forelimb.

Neurokinin A and B are putative inflammatory mediators. We assessed their ability to alter prenodal lymphatic resistance. Intralymphatic neurokinin A (3.0 x 10(-6), 3.0 x 10(-5) and 3.0 x 10(-4) mol l(-1)) significantly constricted lymphatics at the two highest doses. Preliminary experiments suggested that neurokinin B might dilate lymphatics. To test this, lymphatic pressure was increased by norepinephrine (3.1 x 10(-6) mol l(-1)). Neurokinin B (2.7 x 10(-4) mol l(-1)) was then infused intralymphatically during norepinephrine infusion. Norepinephrine increased perfusion pressure from 5.6 +/- 0.6 mmHg to 12.1 +/- 1.4 mmHg. Subsequent infusion of neurokinin B significantly decreased lymphatic perfusion pressure from 11.9 +/- 1.3 mmHg to 9.9 +/- 1.1 mmHg. These data indicate that neurokinin A and B can alter lymphatic resistance and are consistent with the hypothesis that lymph vessel function may be subject to modulation by neurokinins.

Receptor mechanisms of PAF mediated lymphatic constriction in the canine forelimb.

Platelet activating factor (PAF) is a potent inflammatory lipid. In this study we assessed the ability of PAF to impact lymphatic vessel function by altering prenodal lymphatic resistance. Intralymphatic PAF (7.47 x 10(-6), 7.47 x 10(-5) and 7.47 x 10(-4) M) increased lymphatic perfusion pressure at the two highest infusion rates. PAF mediated lymphatic constriction was not altered by the intra-arterial infusion of phentolamine but was blocked by the intra-arterial infusion of the PAF receptor antagonist WEB 2170. These data indicate that in addition to PAF's effects on microvascular permeability, this agent may also impact the ability of the lymphatics to transport fluid through alterations in lymphatic smooth muscle tone. PAF mediated lymphatic constriction is not mediated by alpha-receptors but rather through PAF receptor mediated mechanism.

Endothelin-mediated constriction of prenodal lymphatic vessels in the canine forelimb.

Endothelin is a 21 amino acid peptide which is produced by the vascular endothelium and is believed to be the mediator of endothelium-dependent vasoconstriction. In the current study we assessed the ability of synthetic human endothelin-1 to affect prenodal lymphatic vessel contractility in the canine forelimb. Intralymphatic infusion of endothelin at 1.09 x 10(-9), 1.09 x 10(-8) and 1.09 x 10(-7) M significantly constricted lymphatic vessels as evidenced by dose-dependent increases in lymphatic perfusion pressure. The increase in lymphatic perfusion pressure seen during intralymphatic infusion of endothelin at 1.09 x 10(-8) M during the intra-arterial infusion of phentolamine was not significantly different from that seen prior to phentolamine, indicating that endothelin-mediated lymphatic constriction is not alpha-receptor mediated. Intra-arterial infusion of endothelin at three infusion rates significantly increased forelimb arterial, systemic and lymphatic perfusion pressures. The constriction seen when endothelin (1.09 x 10(-8) M) was infused intralymphatically in the intact lymphatic system was not significantly different from that observed when only the prenodal lymph vessel was perfused. This indicated that the lymph nodes and efferent lymph vessels do not contribute significantly to the lymphatic constriction produced by endothelin. These data are consistent with the hypothesis that endothelin may modulate lymphatic function under either normal or pathophysiological conditions.

Perfused prenodal lymphatics are constricted by prostaglandins.

Prostaglandins may contribute to the control of lymph flow by affecting lymphatic vessel contractility. We measured the pressure in perfused prenodal lymphatic vessel in the paw of the anesthetized dog as affected by administration of prostaglandins E1, E2, F2 alpha or arachidonic acid. The forelimb was perfused at constant flow with blood obtained from a femoral artery. Systemic arterial, central venous, and forelimb vascular pressures were measured. When added to the lymphatic perfusate, all of the prostaglandins and arachidonic acid caused constriction of lymphatic vessels. Perfusion of prenodal lymphatics separated from downstream nodes and vessels showed that this constriction occurred primarily in prenodal vessels. However, only prostaglandin F2 alpha caused lymphatic constriction when infused into the blood to the forelimb. Because prostaglandins are a common component of the lymph leaving an area of tissue damage, these results are compatible with the possibility that prostaglandins, by directly affecting lymphatics, help modulate lymph flow following local injury.

Constriction of perfused lymphatics by acetylcholine, bradykinin and histamine.

We have previously reported that perfused lymphatic vessels in the canine forelimb constrict in response to increased sympathetic nerve activity or local infusions of endogenous vasoconstrictor substances. In the present study we have assessed the effects of three endogenous vasodilators; acetylcholine, bradykinin and histamine on lymphatic vessel contractility. Each one of these agents, when infused intralymphatically, produced lymphatic constriction as evidenced by significant increases in lymphatic perfusion pressure. The threshold concentrations which produced lymphatic constriction were between 10(-6) and 10(-5) molar for acetylcholine and bradykinin and between 10(-5) and 10(-4) molar for histamine. Surgical exclusion of the lymph nodes and efferent lymph vessels from the perfused tissue did not significantly affect the observed response, indicating that the response occurs predominately in the prenodal segments of the lymphatic system. Infusion of acetylcholine and bradykinin into the arterial supply to the forelimb did not significantly alter lymphatic perfusion pressure, unlike the response seen when catecholamines are infused intra-arterially. Histamine displayed an unusual property in that it constricts lymph vessels upon initial administration but was thereafter completely ineffective. Constriction of lymphatic vessels by substances which are potent vasodilators clearly indicates that significant functional differences exist in endothelial cell/smooth muscle relationships between blood vessels and lymph vessels.

CGRP-mediated changes in segmental resistances in the canine forelimb.

Calcitonin gene-related peptide (CGRP) is a 37 amino acid peptide which is found in high concentrations in the perivascular nerves innervating the resistance vessels of the peripheral circulation. In the current study we have infused CGRP at three infusion rates (.01, .1 and 1.0 micrograms/min into the brachial artery for thirty minutes at each infusion rate) in the isolated, innervated canine forelimb perfused at natural flow. We measured large artery and vein pressures, small artery and vein pressures and blood flows in both the skin and skeletal muscle circulations for the calculation of total and segmental (large artery, small vessel and large vein) vascular resistances. Infusion of the lowest dosage of CGRP produced slight vasodilation in some animals but did not significantly alter the mean resistances of all the animals as a group. The middle dosage resulted in a 55% decrease in total forelimb resistance and a small but significant decrease in systemic arterial pressure. The highest dosage of CGRP resulted in a 65% decrease in total forelimb resistance and a 34% decrease in systemic arterial pressure. The decreases in forelimb resistances were equally distributed between skin and muscle and were manifested in both large artery and small vessel resistances. The potent vasodilatory effects of CGRP and its concentration in perivascular nerves innervating the resistance vessels of the peripheral circulation suggests a potential role for CGRP in control of circulatory function under normal and/or pathophysiological conditions.

The inflammatory actions of platelet activating factor are blocked by levorotatory terbutaline.

Platelet activating factor (PAF), a potent vasoactive lipid, may play an important role in the inflammatory process. In this study, we infused PAF intra-arterially to characterize its edematogenic potency in the canine forelimb. We have also assessed the ability of the beta 2-receptor agonist l-terbutaline to block PAF-mediated edema formation. The infusion of PAF at .25 micrograms/min, .5 micrograms/min and 1 micrograms/min increased forelimb arterial pressures and, at the two higher dosages, significantly decreased systemic arterial pressure. PAF infusions increased transvascular fluid and macromolecular flux as indicated by significant increases in skin lymph flow, protein concentration and protein transport. The intra-arterial infusion of l-terbutaline at 1 micrograms/min significantly decreased forelimb arterial pressures but did not affect small vein pressure, systemic pressure or forelimb lymph parameters. The subsequent infusion of PAF at .5 micrograms/min, during the continued infusion of l-terbutaline, failed to significantly affect forelimb lymph parameters. These data indicate that PAF is significantly more potent as an edematogenic agent in the forelimb than histamine or bradykinin. Furthermore, the blockade of PAF-mediated edema formation by l-terbutaline suggests that beta 2-receptor agonists may be capable of antagonizing the inflammatory actions of a wide variety of putative inflammatory mediators.

Neurokinin A and B: potent vasodilators in the canine forelimb.

Neurokinin A and neurokinin B may play a role in control of the peripheral circulation in either physiological or pathophysiological conditions. We have infused these peptides intra-arterially at three infusion rates each to assess their actions on vascular pressures, blood flows and total and segmental resistances in skin and skeletal muscle in the canine forelimb. Neurokinin A infusions (.01, .1, and 1 micrograms/min) decreased total forelimb resistance; transiently, 26% and 57%, respectively. The decrease in resistance was equally distributed between the skin and skeletal muscle circulations and was manifest in both large artery and small vessel resistances. Systemic and forelimb arterial pressures were decreased in a dose-dependent manner. Neurokinin B infusions (.5, 1 and 5 micrograms/min) decreased total forelimb resistance 29%, 31%, and 52%, respectively. The decrease in resistance was equally distributed between the skin and skeletal muscle circulations and was the result of decreases in both large artery and small vessel resistances. Systemic and forelimb arterial pressures were decreased in a dose-dependent manner. The potent effect of neurokinins on vascular resistance and their concentration in perivascular nerves innervating the resistance vessels of the circulation suggests a potential role for these neuropeptides in circulatory control.

Renal vascular response to amino acids: effect of pancreatectomy.

We ascertained the importance of glucagon in modulating the renal hemodynamic response to amino acid (AA) infusion in anesthetized dogs. In controls (n = 6), AAs (L-serine, alanine, and proline; 0.051 mmol.kg-1.min-1 iv) elevated renal blood flow (RBF) and glomerular filtration rate (GFR) by 35 and 34%, respectively, while elevating arterial plasma glucagon-like immunoreactivity (AGLI) by 96 pmol/l. In control pancreatectomized (PX) dogs (n = 6), all parameters remained at control values over 2 h. In PX dogs, AAs (n = 6) failed to reproduce the renal hemodynamic and AGLI responses elicited by AAs in controls. In PX dogs infused with AAs, replacement of AGLI (n = 6) to an incremental plasma level of 111 pmol/l, a level no different than that produced by AAs in controls, elevated RBF and GFR by 25 and 26.5%, respectively. These hemodynamic responses were 71 and 78%, respectively, of the total responses elicited by AAs in controls. In PX dogs infused with glucagon alone (0.86 pmol.kg-1.min-1; n = 6), an incremental change in AGLI of 112 pmol/l was accompanied by only small increases in RBF and GFR (9%). These data suggest the importance of glucagon in modulating the renal hyperemia and hyperfiltration ascribed to AA infusion in anesthetized dogs.