Towards uterus tissue engineering: a comparative study of sheep uterus decellularisation.

Uterus tissue engineering may dismantle limitations in current uterus transplantation protocols. A uterine biomaterial populated with patient-derived cells could potentially serve as a graft to circumvent complicated surgery of live donors, immunosuppressive medication and rejection episodes. Repeated uterine bioengineering studies on rodents have shown promising results using decellularised scaffolds to restore fertility in a partially impaired uterus and now mandate experiments on larger and more human-like animal models. The aim of the presented studies was therefore to establish adequate protocols for scaffold generation and prepare for future in vivo sheep uterus bioengineering experiments. Three decellularisation protocols were developed using vascular perfusion through the uterine artery of whole sheep uteri obtained from slaughterhouse material. Decellularisation solutions used were based on 0.5% sodium dodecyl sulphate (Protocol 1) or 2% sodium deoxycholate (Protocol 2) or with a sequential perfusion of 2% sodium deoxycholate and 1% Triton X-100 (Protocol 3). The scaffolds were examined by histology, extracellular matrix quantification, evaluation of mechanical properties and the ability to support foetal sheep stem cells after recellularisation. We showed that a sheep uterus can successfully be decellularised while maintaining a high integrity of the extracellular components. Uteri perfused with sodium deoxycholate (Protocol 2) were the most favourable treatment in our study based on quantifications. However, all scaffolds supported stem cells for 2 weeks in vitro and showed no cytotoxicity signs. Cells continued to express markers for proliferation and maintained their undifferentiated phenotype. Hence, this study reports three valuable decellularisation protocols for future in vivo sheep uterus bioengineering experiments.

Validation of NMKL method No. 136--Listeria monocytogenes, detection and enumeration in foods and feed.

A collaborative study was organised to define the performance characteristics of the revised NMKL Method No.136 "Listeria monocytogenes. Detection and enumeration in foods". Chromogenic L. monocytogenes specific plating medium, Agar Listeria according to Ottaviani and Agosti (ALOA) was introduced in the revised method in order to improve the sensitivity and specificity of the method, and to shorten the analysis time. Efficacy of ALOA One Day from AES (ready-to-use agar in bottles), Listeria Chromogenic Agar (Agosti and Ottaviani Listeria agar) from Lab M (LCA) (dehydrated powder), Chromogenic Listeria Agar Plates from Oxoid (OCLA) (ready-to-use plates) and L. monocytogenes blood agar medium LMBA from Lab M (dehydrated powder) were tested. Three types of food matrices (vacuum-packed hot-smoked salmon, soft cheese and cooked ham) and one feed matrix (wheat grain) inoculated with two levels of L. monocytogenes with or without L. innocua were used in the study. A total of 24 samples were analysed both in the detection and enumeration part of the study by 18 and 17 Nordic laboratories, respectively. The sensitivities of ALOA, LCA, OCLA and LMBA in the detection of L. monocytogenes in food samples after one-step enrichment (Half-Fraser) were 94.4-96.4% and after two-step enrichment (Half-Fraser followed by Fraser) 97.7-100%. For wheat grain the respective figures were 84.7-88.9% and 90.3-93.1%, respectively. The precision characteristics were generally good for the enumeration of L. monocytogenes in the food samples with high levels of inoculation. Several poor values obtained from the food samples with low levels of inoculation probably reflect high uncertainty of measurement when less than 10 cfu/g was counted. Poor values obtained from the wheat grain samples by any of the media evaluated were due to poor precision for feed samples. According to the study, the revised NMKL Method No.136, 4th ed. showed excellent results in the detection and satisfactory results in the enumeration of L. monocytogenes in foods. The results for the detection of L.monocytogenes in wheat grain were good, but the method cannot be recommended for the enumeration of L. monocytogenes in feed-stuffs. Any one of the media evaluated can interchangeably be used as an obligatory isolation medium for the detection and enumeration of L. monocytogenes in foods, and for the detection in feed-stuffs. The L. monocytogenes specific plating media that were evaluated shorten the time of analysis and significantly reduce the work load. The detection of positive samples mostly after Half-Fraser enrichment, reduces the analysis time further, and makes it possible to skip the secondary enrichment. However, secondary enrichment cannot be totally left out, because samples with low levels of L. monocytogenes, with high levels of competing flora, and with injured L. monocytogenes, do need secondary enrichment.

Cytokines and fever.

Fever is induced in response to the entrance of pathogenic microorganisms into the body and is thought to be mediated by cytokines. Because these pathogens most commonly invade the body through its natural barriers and because body temperature is regulated centrally, these mediators are presumed to be produced peripherally and transported by the bloodstream to the brain, to act. It is generally considered that their febrigenic messages are further modulated there by prostaglandin E2 (PGE2). However, the detailed mechanism by which these cytokines signal the brain and activate the febrile response is not yet clear. Indeed, the specific role of each cytokine has been difficult to establish due to complex interactions among them. Furthermore, recent evidence suggests that different pyrogens may induce different cytokines; for example, i.v. LPS (a model of systemic bacterial infection) induces large increases in IL-6, but only small rises in IL-1 and TNF alpha plasma levels. Moreover, their appearance lags the fever onset. We recently found that subdiaphragmatic vagotomy, decomplementation, and blockade of Kupffer cells suppress the febrile response of guinea pigs to i.v. LPS, and that i.v. LPS rapidly stimulates the release of norepinephrine (NE) and, hence, of PGE2 in their preoptic-anterior hypothalamus (POA, the brain region containing the thermoregulatory controller). Based on these and other data in the literature, we hypothesize that LPS fever may be initiated as follows: i.v., LPS-->complement-->Kupffer cells-->cytokines?-->vagal afferents -->n. tractus solitarius?-->A1/A2 cell groups?-->ventral noradrenergic bundle? -->POA-->NE-->PGE2-->fever.

Prostaglandins and the release of the adrenergic transmitter.

Prostaglandins (PG) are synthesized from arachidonic acid, which is deesterified from tissue lipids in response to various stimuli including adrenergic transmitter, consequent to activation of one or more lipase(s). The profile of arachidonic acid metabolites generated in response to sympathetic nerve stimulation or administration of norepinephrine (NE) may vary in different tissues. For example, in the kidney and spleen, PGE2, is the major and PGI2 and PGF2 alpha the minor products; whereas in the heart and blood vessels, PGI2 is the principal product of arachidonic acid generated in response to sympathetic nerve stimulation. PGE2 and PGI2 inhibit release of NE and/or the postjunctional actions of this neurotransmitter in several tissues. These observations and the findings that inhibitors of cyclooxygenase enhance NE release and the response of effector organs to nerve stimulation suggest that PGs act as physiological modulators of adrenergic transmission. The mechanism by which PGs modulate release of the adrenergic transmitter has not yet been established. NE appears to be released from sympathetic fibers during depolarization by influx of Na+, which is associated with entry of Ca++ through omega-conotoxin-sensitive Ca++ channels that are distinct from those in the vascular smooth muscle, which are sensitive to nifedipine. Ouabain in low external K+ activates the former, whereas external Na+ depletion activates the latter type of Ca++ channels in the nerve fiber and promotes release of NE. PGs (PGE2) may inhibit release of NE from nerve fibers by interfering with the availability of Ca++ through these Ca++ channels or promoting efflux of Ca++ from the nerve terminal.

Blockade of lipopolysaccharide-induced fever by subdiaphragmatic vagotomy in guinea pigs.

It is generally believed that fever is mediated by certain cytokines produced by immune cells activated by exogenous pyrogens, e.g., lipopolysaccharides (LPS), released into the circulation and transported to the brain There, the cytokines are thought to stimulate prostaglandin (PG) E2 production within the organum vasculosum laminae terminalis region. PGE2 then may act as a febrigenic mediator locally or in the surrounding preoptic area (POA). However, whereas the increases in preoptic PGE2 and body (core) temperature (Tc) following the intravenous (i.v.) administration of LPS correlate temporally, cytokine levels in blood lag both these increases. From recent data in the literature, we have conjectured that a possible, alternative communication pathway between the i.v. LPS-activated immune system and brain PGE2 may be provided by the vagi. To test this possibility, we measured the levels of PGE2 in the extracellular fluid of the POA (collected by microdialysis) of conscious, subdiaphragmatically vagotomized or sham-operated guinea pigs following LPS administration (2 micrograms/kg; i.v.); controls received pyrogen-free saline (PFS). The effluents from the microdialysis probes were collected over 30-min periods throughout the experiments and the samples analyzed by radioimmunoassay; Tc was monitored continuously using thermocouples inserted 5 cm into the colon. LPS induced a biphasic fall in Tc and failed to increase preoptic PGE2 levels in the vagotomized guinea pigs (n = 10), whereas in their sham-operated controls (n = 10) it induced increases in both preopitc PGE2 and Tc within 15 min after its injection; PFS (n = 13) had no effect on either variable. We postulate that peripheral immune cell-derived signals may be transmitted via the vagi to the medulla. From other data, we suggest further that they may be conveyed from here via the ventral noradrenergic bundle to the POA region, where the released norepinephrine induces the local synthesis of PGE2 and, hence, fever onset.

Hypothalamic prostaglandin E2 during lipopolysaccharide-induced fever in guinea pigs.

Prostaglandin E2 (PGE2) is postulated to be a central mediator of fever. It is generally believed that it is produced in the preoptic area of the anterior hypothalamus (POA) because, among other evidence, its level increases both in the third ventricle and in the POA in response to pyrogens. However, lately, the question has arisen whether PGE2 might, in fact, be formed outside of the brain substance and then penetrate it, in particular through the organum vasculosum laminae terminalis. If produced outside the brain substance, the peripheral blockade of its synthesis should prevent lipopolysaccharides (LPS)-induced fever, whereas the intracarotid infusion of PGE2 should produce an increase in core temperature (T(C)) as well as in preoptic PGE2. To verify this hypothesis, continuous measurements of T(C) and preoptic PGE2 levels were made in conscious guinea pigs administered the PGE2 synthase inhibitor, indomethacin (10 or 50 mg/kg, im) 30 min before S. enteritidis LPS (2 mu g/kg, iv) or before PGE2 microdialyzed into the POA (1 mu g/mu l at 2 mu g/min for 2.5 h) and during PGE2 infused into a carotid artery (1 mu g and 10 mu g/mu l at 2 mu g/min for 1 h). LPS induced a biphasic 1.4 degrees C fever that was consistently associated with an increase in the level of PGE2 in the POA. Indomethacin at 10 mg/kg attenuated the course of the LPS-induced fever and prevented the associated increase in preoptic PGE2 for 90 min after fever onset; thereafter, PGE2 was significantly reduced by comparison with controls. Indomethacin at 50 mg/kg completely abolished both the fever and the increased levels of PGE2 in the POA; the fever induced by PGE2 microdialyzed into the POA was not affected by indomethacin pretreatment The intracarotid infusion of PGE2 produced T(C) falls and no increase in preoptic PGE2 levels. The indomethacin-induced blockade of fever and inhibition of the associated increase in preoptic PGE2 levels further substantiates the presumptive link between PGE2 in the POA and fever caused by LPS. The failure of exogenous PGE2 infusion to induce increases in T(C) and preoptic PGE2 levels excludes the possibility that PGE2 formed outside of the brain penetrates the POA and induces fever. Thus, in guinea pigs, the PGE2 associated with LPS-induced fever may be synthesized in the POA.

Effect of protein kinase C inhibitors on the actions of phorbol esters on vascular tone and adrenergic transmission in the isolated rat kidney.

The effect of protein kinase C (PKC) inhibitors 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (H-7), polymyxin B (PMB), D-sphingosine (SPH), sangivamycin (SNG) and staurosporin (ST) on the action of PKC activators phorbol 12,13-dibutyrate (PDBu) and 12-o-tetradecanoylphorbol-13-acetate (TPA), on adrenergic neuroeffector events was investigated to determine the contribution of PKC in adrenergic transmission in the rat kidney. Infusion of TPA (5 x 10(-6) mM) or PDBu (6 x 10(-6) mM) produced renal vasoconstriction and enhanced the overflow of tritium elicited by periarterial renal nerve stimulation (RNS) (2 Hz) in the isolated rat kidney perfused with Tyrode's solution and prelabeled with [3H]norepinephrine. H-7 (2.7 x 10(-3) mM) and ST (2 x 10(-5) mM) did not alter RNS-induced overflow of tritium but attenuated the vasoconstrictor response to RNS and exogenous NE. PMB (1 x 10(-8) mM) and SPH (3.3 x 10(-4) mM) but not SNG (3.3 x 10(-3) mM) attenuated the RNS-induced overflow of tritium but increased the basal renal vascular tone and enhanced the vasoconstrictor response to RNS and exogenous NE. H-7, PMB, SPH, SNG or ST failed to alter the effects of PDBu to increase basal vascular tone and the overflow of tritium and the increase in renal vasoconstriction to RNS. PMB at 1 x 10(-9) mM but not at 1 x 10(-8) mM and SPH (3.3 x 10(-4) mM) but not H-7, SNG or ST inhibited the effect of TPA to increase the overflow of tritium. The effect of TPA on the vasoconstrictor response to RNS or to increase basal vascular tone was not altered by PKC inhibitors. These data suggest that in the rat kidney, PKC is either resistant to the actions of H-7, PMB, SPH, SNG and ST, or PDBu and TPA produce renal vasoconstriction and facilitate adrenergic transmission by a mechanism unrelated to PKC activation.

Influence of protein kinase C activators on vascular tone and adrenergic neuroeffector events in the isolated rat kidney.

To investigate if protein kinase C (PKC) activation is involved in mediating or modulating adrenergic transmission, we have investigated the effect of phorbol esters, PKC activators, on the release of adrenergic transmitter elicited by periarterial renal nerve stimulation (RNS) in the isolated rat kidney perfused with Tyrode's solution and prelabeled with [3H]norepinephrine. Infusion of 12-o-tetra-decanoyl-phorbol 13-acetate (TPA) at 5 x 10(-7) mM produced renal vasoconstriction and a rise in basal perfusion pressure without any consistent effect on the rise in tritium efflux or the perfusion pressure elicited by RNS. Higher concentrations of TPA (5 x 10(-6) to 5 x 10(-5) mM) increased both the basal as well as RNS-induced efflux of tritium; the basal perfusion pressure was increased so dramatically that the effect of RNS to raise perfusion pressure was reduced compared to that observed in the vehicle group. Infusion of phorbol-12,13-dibutyrate at 6 x 10(-6) mM reduced the basal but increased the RNS-induced efflux of tritium; the basal as well as the rise in perfusion pressure caused by RNS was increased. Phorbol-13-monoacetate that does not activate PKC failed to alter the basal or the increase in tritium efflux and perfusion pressure elicited by RNS. The rise in perfusion pressure produced by TPA or phorbol-12,13-dibutyrate was reduced by nifedipine (1.4 x 10(-6) mM) but it was abolished by omission of Ca++ from the perfusion medium. These data suggest that PKC activation with phorbol esters produces renal vasoconstriction by promoting influx of extracellular Ca++.(ABSTRACT TRUNCATED AT 250 WORDS)

Pyrogen sensing and signaling: old views and new concepts.

Fever is thought to be caused by endogenous pyrogenic cytokines, which are elaborated and released into the circulation by systemic mononuclear phagocytes that are activated by exogenous inflammatory agents and transported to the preoptic-anterior hypothalamic area (POA) of the brain, where they act. Prostaglandin (PG) E2 is thought to be an essential, proximal mediator in the POA, and induced by these cytokines. It seems unlikely, however, that these factors could directly account for early production of PGE2 following the intravenous administration of bacterial endotoxic lipopolysaccharides (LPS), because PGE2 is generated before the cytokines that induce it are detectable in the blood and the before cyclooxygenase-2, the synthase that they stimulate, is expressed. Hence other, more quickly evoked mediators are presumed to be involved in initiating the febrile response; moreover, their message may be conveyed to the brain by a neural rather than a humoral pathway. This article reviews current conceptions of pyrogen signalling from the periphery to the brain and presents new, developing hypotheses about the mechanism by which LPS initiates fever.

Interaction between norepinephrine and prostaglandin E2 in the preoptic area of guinea pigs.

The release of norepinephrine (NE) and prostaglandin E2 (PGE2) in the preoptic-anterior hypothalamus (POA) by systemically administered pyrogens suggests that both substances may mediate the febrile response. To investigate their possible interaction, we measured directly the levels of PGE2 in the extracellular fluid of the POA of conscious guinea pigs microdialyzed intrapreoptically with exogenous NE over the entire course of their febrile response to endotoxin. Acidified and buffered NE (NEa, NEb), artificial cerebrospinal fluid (aCSFa, aCSFb), and vehicle (Veha, Vehb) were tested. All but aCSFb depressed the febrile response to endotoxin. The microdialysis of aCSFa, aCSFb, Veha, Vehb, and NEa did not change basal preoptic PGE2 levels. However, NEb, at a dose that by itself did not affect body temperature (Tb), caused a large elevation in preoptic PGE2. The intravenous injection of endotoxin increased the level of PGE2 in the POA. NEb potentiated this increase, whereas NEa, aCSFa, and Vehb reduced it; Veha reduced it for the first 60 min and enhanced it for the last 90 min of the experiment. Thus these data suggest that the low pH of the NE solute and/or its Veh may confound the observed effects of NE on the Tb and preoptic PGE2 induced by endotoxin. We surmise that this is due to a neurotoxic action of the antioxidants and the acidity of the solution on thermosensitive neurons in the POA. Hence, the results of experiments using exogenous, usually acidified, NE preparations that often also contain additives should be interpreted with caution.

Mechanism of resistance to alpha-adrenergic receptor antagonists of renal nerve stimulation-induced vasoconstriction at low frequencies.

To determine why renal vasoconstriction elicited by periarterial nerve stimulation (RNS) at lower frequencies (< 4 Hz) is resistant to alpha-adrenergic receptor blockade in the rat kidney, we reevaluated the effect of alpha-receptor antagonists on the vasoconstrictor response to norepinephrine (NE) and to RNS and on the release of adrenergic transmitter. The alpha-receptor antagonist prazosin (PZ) at 0.2 and 7 nM reduced the vasoconstrictor response to NE, and 2.4 microM PZ abolished it. PZ (0.2 or 7 nM) reduced RNS-induced vasoconstriction without altering the fractional tritium overflow. PZ (2.4 microM) enhanced fractional tritium overflow and reduced the vasoconstrictor response to RNS at 2-10 Hz, but not at 0.5 or 1 Hz. The effect of 0.2 nM PZ to reduce RNS-induced vasoconstriction was reversed by increasing the concentration to 2.4 microM. Corynanthine (COR; 2.6 microM), a preferential alpha-receptor blocker, or phenoxybenzamine (PBZ; 30 nM) abolished the vasoconstrictor response to NE but only partially reduced response to RNS and enhanced the fractional tritium overflow. Rauwolscine (RW; 2.5 nM), a preferential alpha 2-receptor antagonist, did not alter the vasoconstrictor response to NE but potentiated RNS-induced vasoconstriction and fractional tritium overflow. RW (7.7 microM) inhibited NE-induced vasoconstriction but potentiated the vasoconstrictor response to RNS and fractional tritium overflow. PZ (7 nM) abolished the potentiation by RW and reduced the vasoconstrictor response to RNS. These data suggest that a component of RNS-induced vasoconstriction in the rat kidney is attributable to co-release of a nonadrenergic transmitter with NE. The diminished effect of alpha-receptor antagonists at higher concentrations (e.g., PZ 2.4 microM) to reduce RNS-induced vasoconstriction is caused by their prejunctional action to enhance co-release of the nonadrenergic transmitter.

Attenuation by alpha,beta-methylenadenosine-5'-triphosphate of periarterial nerve stimulation-induced renal vasoconstriction is not due to desensitization of purinergic receptors.

We investigated in the isolated rat kidney the modulation of vasoconstrictor responses to ATP (0.05-0.5 mumol), renal nerve stimulation (RNS) (0.5-10.0 Hz), norepinephrine (NE) (0.15-0.9 nmol), angiotensin II (2 pmol) and arginine vasopressin (3 pmol) by alpha,beta-methylenadenosine-5'-triphosphate (alpha beta mATP) infused at 6 microM (Procedure I) or for short intervals (5 min) at a low concentration (60 nM) gradually increased to 6 microM to reduce the dramatic initial vasoconstriction (Procedure II). Infusion of alpha beta mATP (Procedure I) produced a marked, transient rise in perfusion pressure of 146 to 198 mm Hg that returned to basal level within 10 min and thereafter inhibited the vasoconstrictor response to ATP, RNS (0.5-6.0 Hz), NE, angiotensin II and arginine vasopressin. Infusion of alpha beta mATP by Procedure II produced a smaller maximal transient increase in perfusion pressure (< 100 mm Hg) and reduced the vasoconstrictor responses to RNS at 0.5 to 2.0 Hz and to the lower dose of NE (0.15 nmol) only. ATP infusion reduced the vasoconstrictor response to both RNS and NE. In animals pretreated with reserpine, the effect of RNS to produce vasoconstriction was inhibited. These data suggest that ATP does not contribute to the renal vasoconstrictor response elicited by RNS, and that attenuation of renal vasoconstrictor responses by alpha beta mATP is not due to desensitization of purinergic receptors.

Complement reduction impairs the febrile response of guinea pigs to endotoxin.

Although it is generally believed that circulating exogenous pyrogens [e.g., lipopolysaccharides (LPS)] induce fever via the mediation of endogenous pyrogens (EP) such as cytokines, the first of these, tumor necrosis factor-alpha, is usually not detectable in blood until at least 30 min after intravenous administration of LPS, whereas the febrile rise begins within 15 min after its administration. Moreover, although abundant evidence indicates that circulating LPS is cleared primarily by liver macrophages [Kupffer cells (KC)], these do not secrete EP in immediate response. This would imply that other factors, presumably evoked earlier than EP, may mediate the onset of the febrile response to intravenous LPS. It is well known that blood-borne LPS very rapidly activates the intravascular complement (C) system, some components of which in turn stimulate the quick release into blood of various substances that have roles in the acute inflammatory reaction. KC contain receptors for C components and are in close contact with afferent vagal terminals in the liver; the involvement of hepatic vagal afferents in LPS-induced fever has recently been shown. In this study, we tested the hypothesis that the initiation of fever by intravenous LPS involves, sequentially, the C system and KC. To test this postulated mechanism, we measured directly the levels of prostaglandin E2 (PGE2) in the interstitial fluid of the preoptic anterior hypothalamus (POA), the presumptive site of the fever-producing controller, of conscious guinea pigs over their entire febrile course, before and after C depletion by cobra venom factor (CVF) and before and after elimination of KC by gadolinium chloride (GdCl3). CVF and GdCl3 pretreatment each individually attenuated the first of the biphasic core temperature (Tc) rises after intravenous LPS, inverted the second into a Tc fall, and greatly reduced the usual fever-associated increase in POA PGE2. We conclude, therefore, that C activation may indeed be pivotal in the induction of fever by intravenous LPS and that substance(s) generated presumably by KC in almost immediate reaction to the presence of LPS and/or C may transmit pyrogenic signals via hepatic vagal afferents to the POA, where they rapidly induce the production of PGE2 and, hence, fever.

Relation between complement and the febrile response of guinea pigs to systemic endotoxin.

We reported recently that the complement (C) system may play a role in the febrile response of guinea pigs to intravenous lipopolysaccharide (LPS) administration because C depletion abolished the LPS-induced rise in core temperature (T(c)). The present study was designed to investigate further the relation between C reduction [induced by cobra venom factor (CVF); 20, 50, 100, and 200 U/animal iv] and the fever of adult, conscious guinea pigs produced by LPS injected intravenously (2 microg/kg) or intraperitoneally (8, 16, 32 microg/kg) 18 h after CVF; control animals received pyrogen-free saline. Serum C levels were measured as total hemolytic C activity before and 18 h after CVF injection and expressed as CH(100) units. In other experiments, serum C levels were determined at various intervals after the intravenous and intraperitoneal injections at different doses of LPS alone. LPS produced fevers generally of similar heights but of different onset latencies and durations, depending on the dose and route of administration. CVF caused dose-related reductions in serum C, from approximately 1,136 U to below detection. These reductions proportionately attenuated the fevers induced by intraperitoneal LPS, but not by intravenous LPS. Intravenous and intraperitoneal LPS per se caused reductions in serum C of 25 and 40%, respectively, indicating activation of the C cascade. These decreases were transient, however, occurring early during the febrile rise approximately 30 min after LPS injection. These data thus support the notion that the C system may be critically involved in the febrile response of guinea pigs to systemic, particularly intraperitoneal, LPS.