Early dynamics of leptin plasma level in surgical critically ill patients. a prospective comparative study.

BACKGROUND: Leptin (LPT), a hormone secreted by adipocytes,plays a role in inflammation and infection. Our study aimed to characterize the early dynamics of LPT in comparison with CRP and IL-6 during systemic inflammatory response syndrome(SIRS) and sepsis in surgical patients. METHODS: Sixty patients were assigned into 3 equal groups:SIRS (SI) group with major abdominal elective surgery;sepsis (SE) group with community-acquired complicated intra-abdominal infection and controls (C). LPT, CRP and IL-6 were measured initially in all groups and repeated in groups SI and SE within 5 days after surgery (9 samples - 4 day 1, 2 day 2, 1 next 3 days). RESULTS: LPT increased at 12-24 hours in SI group, but stayed within normal range in SE group. CRP and IL-6 had higher values in SE group versus SI group with an early peak for IL-6 and a late peak for CRP. CONCLUSIONS: LPT has a different early dynamics during SIRS and sepsis. LPT measurement in association with CRP or IL-6 may be useful in the differential diagnosis and prognosis of surgical critical illness at different time courses.

POTENTIAL NEW ROLES OF LEPTIN IN HEALTH AND DISEASE.

Leptin is an adipose-tissue specific cytokine described 20 years ago and classically thought to be involved in regulation of food intake and energy homeostasis. More recently, leptin was proven to exert other multiple roles in carbohydrate and lipid metabolism, reproductive system, and inflammatory and immune reactions. Emerging data propose leptin to be a modulator of bone mass. In some particular circumstances leptin synthesis follow specific patterns in inflammation and sepsis, not being proportional with fat mass. Therefore, it may be used as diagnostic or prognostic marker for critical illness.

ATYPICAL PRIMARY HYPERPARATHYROIDISM DUE TO HYPOVITAMINOSIS D.

Vitamin D deficiency is nowadays very common in the general population and also in patients with primary hyperparathyroidism. Hypovitaminosis D may modify the clinical features and the severity of primary hyperparathyroidism. We present the case of a 75-year-old woman with a 10 year history of nephrolithiasis and severe osteoporosis, with multiple fragility fractures. Her bone and kidney status required a more thorough metabolic assessment. Despite minimal changes in serum calcium and phosphate levels, parathyroid hormone (PTH) level was markedly elevated. Ultrasound and specific Sesta-MIBI scintigraphy diagnosed and localized a left parathyroid adenoma. Vitamin D assessment showed levels in the range of hypovitaminosis. Vitamin D deficiency may mask hypercalcemia despite high serum PTH levels, and does not seem to diminish but on the contrary increases the risk of kidney lithiasis, as well as the deleterious effects of hyperparathyroidism on bone.

A renin-like activity in pineal gland and hypophysis.

The presence and distribution of renin-like activity in hypophysis, pineal gland and some neural tissues were studies in both normal and salt-loaded rats. The Boucher micromethod was used to detect enzymatic activity. In normal rats both the pituitary and pineal glands contained significantly higher values of renin-like activity than did the other tissues examined. In salt-loaded animals there was a significant decrease of the renin-like activity of the glandular tissue while in the other brain areas the activity increased. The results are discussed in terms of the possible physiological role of the central renin-angiotensin system in the regulation of fluid and electrolyte balance.

Combination of a 1,25-dihydroxyvitamin D3 analog and a bisphosphonate prevents experimental autoimmune encephalomyelitis and preserves bone.

The vitamin D analog TX527 (19-nor-14,20-bis epi-23-yne-1,25(OH)(2)D(3)), decreased disease severity (P < 0.001) and postponed disease onset (P < 0.0001) in SJL mice in which experimental autoimmune encephalomyelitis was induced. Levels of IFN-gamma and IL-2 mRNA were decreased in spinal cord and spleen in the analog-treated mice, suggesting a Th(1)-targeted effect. Adding the bisphosphonate pamidronate did not affect analog-protective efficacy, but completely prevented TX527-caused acceleration of bone turnover and increased total bone mineral content as well as femoral mineral and calcium content (P < 0.01). Less calcemic analogs of 1,25-dihydroxyvitamin D(3), in combination with bone sparing products such as bisphosphonates allow immune modulation in vivo without affecting bone.

Synergism between sirolimus and 1,25-dihydroxyvitamin D3 in vitro and in vivo.

The active form of vitamin D, 1 alpha, 25-(OH)2D3, displays immunomodulatory effects in vitro and in vivo at pharmacological levels. We evaluated the dose-effect relationship of 1,25(OH)2D3 and sirolimus (rapamycin, RAP) in vitro, on the inhibition of PHA-stimulated PBMC proliferation, by using the median effect analysis. Pharmacological concentrations of 1,25(OH)2D3 (between 10(-9) and 3 x 10(-6) M) interacted synergistically with RAP (combination index value of 0.01 for 50% suppression of PBMC proliferation). In vivo, the effect of 1,25(OH)2D3 and RAP combinations on the evolution of experimental allergic encephalomyelitis in SJL mice was analyzed. 1,25(OH)2D3, given i.p., in monotherapy, at a dose of 2 micrograms/kg every two days, from day -3 until day +19 after disease induction, or RAP, injected daily at a dose of 0.3 mg/kg for the same period, decreased EAE incidence (paralysis in 70 and 55% of the animals, respectively, versus 98% in the placebo treated group, p < 0.001). The combination treatment using the two drugs in these subtherapeutical doses provided near-total clinical (8% paralysis, p < 0.001 compared to monotherapy with 1,25(OH)2D3 or RAP) and histological protection, comparable to that obtained with RAP in monotherapy at a threefold higher dose (1 mg/kg/d). When the two drugs were given using an alternate day administration schedule (RAP at 0.6 mg/kg and 1,25(OH)2D3 at 2 micrograms/kg, each given on alternate days from day -3 to 19), near total protection was again obtained (13% paralysis, p < 0.001 versus control). These in vitro and in vivo data support the existence of synergistic interactions between 1,25(OH)2D3 and RAP. Considering the narrow therapeutic windows of both RAP and vitamin D-related compounds in autoimmune disease models, combinations of these drugs could find clinical application in reducing their individual therapeutically efficient doses to non-toxic levels.

Analogs of 1,25-dihydroxyvitamin D3 as dose-reducing agents for classical immunosuppressants.

BACKGROUND: Most immunosuppressants have a narrow margin between efficacy and side effects. A major goal in the development of immunomodulatory strategies is the discovery of combinations of drugs exerting synergistic immunomodulatory effects. The active form of vitamin D, 1,25(OH)2D3, is an immunomodulator that interacts with T cells but mainly targets antigen-presenting cells. We have demonstrated synergism between 1,25(OH)2D3 and cyclosporine, rapamycin, and FK506. The aim of this study was to investigate whether this synergism could be observed with other immunosuppressants (mycophenolate mofetil, leflunomide, and the methylxanthine A802715) and whether analogs of 1,25(OH)2D3 share this synergistic capacity in vivo. METHODS: In vitro, the median effect analysis was applied to the inhibition of phytohemagglutinin A-induced lymphocyte proliferation. In vivo, synergism between analogs of 1,25(OH)2D3 and cyclosporine or mycophenolate mofetil was evaluated in experimental autoimmune encephalomyelitis. RESULTS: In vitro, all combinations with 1,25(OH)2D3 were synergistic. The strongest synergism was seen with the inhibitors of interleukin 2 secretion, cyclosporine and FK506 (indexes 0.16 and 0.27, respectively). The weakest synergism was observed in combinations using A802715, a second-signal inhibitor (index 0.52), or the nucleotide synthesis inhibitor mycophenolate mofetil (index 0.43). In vivo, analogs of 1,25(OH)2D3 share the in vitro-observed synergism with 1,25(OH)2D3. Moreover, the differences in synergism with different immunomodulators were also present in vivo, where the best synergism was again seen in combination with cyclosporine (up to 100% paralysis protection). CONCLUSIONS: These data confirm that 1,25(OH)2D3 and its analogs are potent dose-reducing drugs for other immunomodulators, making them potentially interesting for clinical use in autoimmunity and transplantation.

Prevention of murine experimental allergic encephalomyelitis: cooperative effects of cyclosporine and 1 alpha, 25-(OH)2D3.

The hormone 1 alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3) has immune modulatory activities in vitro and in vivo, and can prevent or delay the onset of experimental or spontaneous autoimmune diseases. At therapeutical doses, however, hypercalcemic side effects are found. The present experiments examined the effects of combined treatment with subtherapeutic doses of cyclosporine A (CsA) and 1,25(OH)2D3 on the evolution of experimental autoimmune encephalomyelitis (EAE) in SJL mice. 1,25(OH)2D3 at 5 micrograms/kg body weight (given by i.p. injection every 2 days) prevented the appearance of paralysis in 70% of the treated mice. The treatment with 1,25(OH)2D3 at 2 micrograms/kg/2 days alone had less substantial protective effects (22% disease-free animals versus 5% in the control group). However, when this subtherapeutic dose was associated to treatment with a daily dose of CsA (2 or 5 mg/kg/day), which by itself was subtherapeutic (24 and 50% disease-free animals, respectively), the association of both drugs led to near-total protection (86% disease-free animals when combined with the highest dose of CsA). When an alternate day administration schedule (CsA at 10 mg/kg and 1,25(OH)2D3 at 2 micrograms/kg, each given on alternate days from day -3 to +19 after disease induction) was used, all treated mice were completely protected clinically and histologically. The two drugs also showed additive effects on serum osteocalcin and urinary calcium and desoxypyridinoline excretion, but not on serum calcium concentration. Our experiments demonstrate that 1,25(OH)2D3 might be a potential dose-reducing agent for CsA in immunosuppressive therapy.

Cyclic GMP and protein kinase G inhibit the quantal transmitter release induced by protein kinase C.

Protein kinase G inhibits the spontaneous release of acetylcholine quanta at the frog neuromuscular junction as shown by the effects of H-8, a G kinase blocking agent. Moreover, the permeant dibutyryl cGMP blocked the frequency increase obtained in the presence of protein kinase C activators (diacylglycerol and phorbol ester) while the cAMP activated protein kinase A did show only an additive effect.

Adenosine alters the vascular effects of a diacylglycerol analogue and polymyxin B.

Endothelium-denuded rat aorta rings were used to study the possible relationship between protein kinase C and the mechanism of adenosine-induced smooth muscle relaxation. Adenosine (5 x 10(-4) M) partially relaxed the aortic rings contracted by either a depolarising amount of KCl (4 x 10(-2) M) or activation of protein kinase C with 1-oleoyl-2-acetyl-sn-glycerol (10(-6) M). The same amount of adenosine blocked the further relaxation obtained in the presence of polymyxin B (5 x 10(-5) M), a protein kinase C blocking agent. These results suggest a possible interaction in vascular smooth muscle between adenosine and protein kinase C.

Effects of liposome-entrapped adenosine in the isolated rat aorta.

This study examined the effects of adenosine- and adenosine deaminase-loaded liposomes upon the contractile activity of the vascular smooth muscle, using the isolated, de-endothelised rat aorta ring as in vitro model. While control liposomes had no effect, intraliposomal adenosine (5 x 10(-3) M) induced contraction of the preparation. Intraliposomal adenosine deaminase induced partial relaxation of high K(+)-precontracted rings. The adenosine-induced contraction seems to involve Ca2+ influx through L-type channels as an essential component, but protein kinase C may also have a modulatory role.

Effects of liposome-entrapped D-myo-inositol 1,4,5-trisphosphate and D-myo-inositol 1,3,4,5-tetrakisphosphate in the isolated rat aorta.

This study examined the effects of D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)- and D-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4)-loaded liposomes upon the contractile activity of vascular smooth muscle, using the isolated (endothelium removed) rat aortic ring as in vitro model. While control liposomes had no effect, the administration of Ins(1,4,5)P3-containing liposomes contracted the smooth muscle preparation. Furthermore, a similar effect was seen with the administration of Ins(1,3,4,5)P4-filled liposomes but, in this case, the rings developed a significantly higher level of active tension. Pretreatment of the aortic preparation with heparin-loaded liposomes blocked the contractions induced by both Ins(1,4,5)P3- and Ins(1,3,4,5)P4-containing liposomes.

Multiple effects of tyrosine kinase inhibitors on vascular smooth muscle contraction.

The effects of three tyrosine kinase inhibitors: genistein, quercetin and psi-tectorigenin, were investigated on contractions evoked in de-endothelised rat aortic rings, either by phenylephrine or 70 mM K+. A dose-dependent inhibition of both contractions by all three compounds was observed, the phenylephrine-mediated contractions being more sensitive to genistein. No differences between genistein or quercetin effects in pre-treatment or post-treatment protocols were found. Ca2+ store refilling, expressed in terms of phenylephrine-induced tension in Ca(2+)-free medium, was dose-dependently blocked by quercetin and genistein. Sodium orthovanadate, an inhibitor of tyrosine phosphatase, contracted the rat aortic rings with an IC50 of 0.66 microM. Its presence during the refilling period after exposure to Ca(2+)-free medium completely prevented the subsequent response to phenylephrine. One can conclude that the use of the above-mentioned protein tyrosine kinase inhibitors in the rat aorta blocks a step involved in Ca2+ entry and Ca2+ store refilling. A definite conclusion regarding the vanadate effects is not possible due to the fact that this compound also affects Ca2+ ATP-ases.

Effects of alpha-trinositol administered extra- and intracellularly (using liposomes) on rat aorta rings.

The effects of alpha-trinositol, a D-myo-inositol [1,2,6]trisphosphate derivative, were studied on de-endothelised rat aorta rings. The substance was applied extracellularly as well as intracellularly (by using liposomes as drug carriers). Upon extracellular administration, the drug reduced the level of contraction induced by 40 mM K+ or by phenylephrine (10(-5) M). No effects were observed on relaxed preparations. Liposomes containing alpha-trinositol induced a dose-dependent contraction of the preparations under resting tension with a threshold of 10(-5) M in the aqueous phase. These contractions were heparin-insensitive but were significantly blocked by D-600 (10(-5) M) (an L-type Ca2+ channel blocker) or in Ca(2+)-free medium. Our data suggest that alpha-trinositol has a plasmalemmal mechanism of action which could involve Ca2+ influx from the extracellular space.

Vasorelaxant properties of brefeldin A in rat aorta.

The effects of brefeldin A, a putative specific agent that disassembles the Golgi apparatus were assessed on the contractility of de-endothelised rat aorta. Brefeldin A inhibited, either as pre- or as post-treatment, the contractions elicited by K+ (75 mM) or phenylephrine (10 microM), being significantly more potent upon the latter. The thapsigargin (1 microM)-induced rat aorta contraction was less sensitive to brefeldin A inhibition. Pre-treatment with brefeldin A (30-100 microM) did not affect phenylephrine-induced transient contractions in Ca2+-free medium, but strongly inhibited the phenylephrine-induced sustained contractions upon re-admission of Ca2+ to the medium. Brefeldin A was unable to prevent Ca2+ stores refilling. We concluded that brefeldin A inhibits Ca2+ entry but not the pathways activated after Ca2+ stores depletion or the pathways responsible for replenishment of these stores in rat aorta, presumably by disassembling the Golgi apparatus network.

Effects of liposome-entrapped platelet-activating factor in the isolated rat trachea.

The effects of platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine)-filled liposomes upon rat tracheal rings in vitro were examined. The capture of liposomes by the smooth muscle cells of the isolated tracheal rings as well as the release of their content into the cytoplasm was shown by using Evans blue (5 x 10(-4) M)-loaded liposomes. Administration of PAF (10(-3) M)-filled liposomes contracted the preparations, in contrast with extracellular administration of PAF and control liposomes, which had no effect. Administration during the plateau or pretreatment with liposomes containing BN 52021 (3-t-butylhexahydro-4,7b-trihydroxy-8-methyl-9H-1,7a-epoxymethano- 1H,6aH- cyclopenta[c]furo(2,3-b)furo[3',2':3,4]cyclopental [1,2-d]furan-5,9,12(4H)-trione) ((10(-3) M, a selective PAF receptor antagonist) or heparin (5 x 10(-5) M) blocked this contraction. BN 52021 and heparin, not entrapped in liposomes, had no such effect. Our data suggest an intervention of PAF in the mechanisms of contraction of tracheal smooth muscle, involving a direct or indirect intervention (intracellular receptors for PAF cannot be excluded). At the same time, the rat trachea contraction induced by PAF-loaded liposomes could be linked to the PtdIns(1,4,5)P3-dependent Ca2+ channels from the endoplasmic reticulum and/or to the interaction with G proteins, as shown by the blocking effects of heparin-containing liposomes.

Adenosine effects upon the spontaneous quantal transmitter release at the frog neuromuscular junction in the presence of protein kinase C-blocking and -activating agents.

This paper gives experimental evidence involving protein kinase C (PKC) in the inhibitory effects of adenosine (ADO) upon the spontaneous transmitter release at the frog neuromuscular junction. In the presence of two PKC inhibitors--polymyxin B (5 x 10(-6) mol/l) and H-7 (10(-5) mol/l), both adenosine (5 x 10(-5) mol/l) and its stable analogue 1-PIA (5 x 10(-8) mol/l), significantly increased the rate of the spontaneous release of acetylcholine quanta. Even when PKC was activated with OAG (5 x 10(-6) mol/l) or TPA (162 x 10(-9) mol/l) and quantal release was increased greatly, ADO still inhibited release. ADO deaminase increased the PKC-induced activation of the transmitter release significantly.

Trifluoperazine-sensitive activation of the spontaneous transmitter release at the frog motor endplates by low doses of procaine.

Low concentrations of procaine (10(-6)-5 X 10(-5) mol/l) induced a significant increase of the spontaneous quantal transmitter release in the neuromuscular junctions of the frog cutaneous pectoris nerve-muscle preparation. The frequency of miniature endplate potentials (mepps) was increased although their size slightly decreased probably on the account of a partial block of Na+-channels at the postsynaptic membrane. The activatory effect of pre-caine was not altered under experimental conditions known to change the Ca2+ fluxes across the nerve terminal membrane such as using a Ca2+-free Ringer, or a Ca2+-channel blocker (D600), a high K+ Ringer or, finally, a low Na+ Ringer. In the presence of caffeine no change of procaine-induced activation appeared. Trifluoperazine (TFP), in a concentration known to specifically block calmodulin, completely blocked the procaine-induced increase of mepp frequency. These data suggest that procaine presumably by way of a calmodulin-dependent mechanism is related to the free cytosolic Ca2+ equilibrium. It is possible that procaine increases the free cytosolic Ca2+ concentration by blocking an active calmodulin-dependent Ca2+ extrusion mechanism.

Quantal parameters of transmission at the frog neuromuscular junction.

Estimates of quantal release parameters at frog neuromuscular junctions showed that alterations in [Ca2+]o affected m (number of quanta), p (probability of quantal release) and n (stores of quanta available for release). The effect of [Ca2+]o depended upon the initial value for p. When p was low, raising [Ca2+]o increased m and p, but not n. However, when p was large, raising [Ca2+]o had no further effect on p but increased m and n. During prolonged repetitive nerve stimulation to cause a decrease in m, n was decreased and p was increased. This finding was attributed to a failure of transmitter mobilization to maintain the stores of transmitter available for release.

Characteristics and possible mechanisms of low-Na+ induced contractions in rat aorta.

The influence of reducing external Na+ concentration ([Na+]ex) upon vascular smooth muscle contractility was investigated using the rat isolated aorta. NaCl from the physiological saline solution (PSS) was replaced with either choline-Cl, sucrose, or LiCl to give the following [Na+]ex (mM): 115, 85, 55, and 25 (115NaPSS to 25NaPSS). Small reductions in [Na+]ex (115NaPSS) induced a biphasic contraction, comparable in amplitude with the control one induced by phenylephrine 10(-6) M. Elimination of the endogenous catecholamine participation using either phentolamine 10(-5) M or guanethidine 3.10(-6) M similarly reduces these contractions to 25% (sucrose replacement). A similar relaxing effect was obtained with D600 10(-5) M, an antagonist of the voltage operated Ca2+ channels (25-30% residual tension for all the substitutes). Large reductions in [Na+]ex (25NaPSS) induced contractions comparable in amplitude and shape, but less sensitive to phentolamine and guanethidine (residual tension 65-75%, sucrose replacement) and insensitive to D600 (all the substitutes). The Na+/K+ ATPase inhibitor ouabain (10(-4) M) elicited slowly developing contractions, the amplitude being 115% of the phenylephrine 10(-6) M control. Phenylephrine further contracted the 115NaPSS precontracted preparations, but was significantly less effective in 25NaPSS, although the precontraction levels were similar for the same substitute used. The amplitude of the superimposed phenylephrine contractions exhibited [Na+]ex dependence. Phenylephrine 10(-6) M failed to further contract the ouabain 10(-4) M precontracted rings. We conclude that relatively small reductions in [Na+]ex are able to induce contractions of rat aorta primarily through release of endogenous catecholamines, probably through neural Na+/Ca2+ exchange. Larger reductions in [Na+]ex appear to cause contraction through muscular Na+/Ca2+ exchange.