In Vivo Comparison of Backscatter Techniques for Ultrasonic Bone Assessment at the Femoral Neck.

Ultrasonic techniques are being developed to detect changes in cancellous bone caused by osteoporosis. The goal of this study was to test the relative in vivo performance of eight backscatter parameters developed over the last several years for ultrasonic bone assessment: apparent integrated backscatter (AIB), frequency slope of apparent backscatter (FSAB), frequency intercept of apparent backscatter (FIAB), normalized mean of the backscatter difference (nMBD), normalized slope of the backscatter difference (nSBD), normalized intercept of the backscatter difference (nIBD), normalized backscatter amplitude ratio (nBAR) and backscatter amplitude decay constant (BADC). Backscatter measurements were performed on the left and right femoral necks of 80 adult volunteers (age = 25 ± 11 y) using an imaging system equipped with a convex array transducer. For comparison, additional ultrasonic measurements were performed at the left and right heel using a commercially available heel-bone ultrasonometer that measured the stiffness index. Six of the eight backscatter parameters (all but nSBD and nIBD) exhibited similar and highly significant (p < 0.000001) left-right correlations (0.51 ≤ R ≤ 0.68), indicating sensitivity to naturally occurring variations in bone tissue. Left-right correlations for the stiffness index measured at the heel (R = 0.75) were not significantly better than those produced by AIB, FSAB and FIAB. The short-term precisions of AIB, nMBD, nBAR and BADC (7.8%-11.7%) were comparable to that of the stiffness index measured with the heel-bone ultrasonometer (7.5%).

Optimization Rules for SARS-CoV-2 Mpro Antivirals: Ensemble Docking and Exploration of the Coronavirus Protease Active Site.

Coronaviruses are viral infections that have a significant ability to impact human health. Coronaviruses have produced two pandemics and one epidemic in the last two decades. The current pandemic has created a worldwide catastrophe threatening the lives of over 15 million as of July 2020. Current research efforts have been focused on producing a vaccine or repurposing current drug compounds to develop a therapeutic. There is, however, a need to study the active site preferences of relevant targets, such as the SARS-CoV-2 main protease (SARS-CoV-2 Mpro), to determine ways to optimize these drug compounds. The ensemble docking and characterization work described in this article demonstrates the multifaceted features of the SARS-CoV-2 Mpro active site, molecular guidelines to improving binding affinity, and ultimately the optimization of drug candidates. A total of 220 compounds were docked into both the 5R7Z and 6LU7 SARS-CoV-2 Mpro crystal structures. Several key preferences for strong binding to the four subsites (S1, S1', S2, and S4) were identified, such as accessing hydrogen binding hotspots, hydrophobic patches, and utilization of primarily aliphatic instead of aromatic substituents. After optimization efforts using the design guidelines developed from the molecular docking studies, the average docking score of the parent compounds was improved by 6.59 -log10(Kd) in binding affinity which represents an increase of greater than six orders of magnitude. Using the optimization guidelines, the SARS-CoV-2 Mpro inhibitor cinanserin was optimized resulting in an increase in binding affinity of 4.59 -log10(Kd) and increased protease inhibitor bioactivity. The results of molecular dynamic (MD) simulation of cinanserin-optimized compounds CM02, CM06, and CM07 revealed that CM02 and CM06 fit well into the active site of SARS-CoV-2 Mpro [Protein Data Bank (PDB) accession number 6LU7] and formed strong and stable interactions with the key residues, Ser-144, His-163, and Glu-166. The enhanced binding affinity produced demonstrates the utility of the design guidelines described. The work described herein will assist scientists in developing potent COVID-19 antivirals.

Effects of systemic immunosuppression on islet engraftment and function into a subcutaneous biocompatible device.

The aim of this study was to explore the effect of sirolimus (Sir) and tacrolimus (Tac) on islets implanted into a subcutaneous (SC), prevascularized device in syngeneic rats. Animals received a 40-day treatment with Tac and Sir (alone or in combination) starting either on day 0 or 40 days after islet transplantation. Controls received no treatment. A 40-day washout period was performed after immunosuppression (IS). Glycemia and intravenous glucose tolerance tests (IVGTT) were assessed at follow-up. In the control group, 75% of recipients achieved stable normoglycemia after islet transplantation, while none reversed diabetes with any IS regimen started on day 0. Graft dysfunction was irreversible after IS withdrawal. Glucose clearance (IVGTT) was significantly impaired among Tac-treated compared with control groups (P < .05 with IS; P < .01 after washout). Among animals with established grafts, islet dysfunction which occurred under IS treatment persisted after washout in animals treated with Tac and Sir plus Tac. When compared with controls, glucose clearance was significantly impaired in the Tac and Tac plus Sir groups before and after IS (P < .01, Tac; P < 0.01, Tac plus Sir). Sir and Tac showed profound deleterious effects on islet cell engraftment and function, which may hinder the success of implantation into biohybrid devices. Nondiabetogenic IS protocols must be developed for clinical application of islet transplantation into biohybrid devices.

Ischemic preconditioning improves islet recovery after pancreas cold preservation.

Increasing evidence supports the beneficial effects of ischemic preconditioning (IPC) of organs on subsequent ischemia. The aim of this study was to assess the effects of IPC of the pancreas on islet cell recovery after cold preservation using a rat model. The pancreas was deprived of perfusion (celiac artery and superior mesenteric artery occlusion) for 10 minutes followed by 10 minutes of reperfusion. Islet isolation was performed after 18 hours of cold ischemia. Glands undergoing IPC yielded significantly greater numbers of islets than controls. Following overnight culture, a significantly greater proportion of islets was recovered from IPC-treated pancreata. Microarray genomic analysis of pancreatic tissue revealed a significant differential expression of approximately 600 unique mRNA strands within IPC pancreata compared to only <100 unique mRNA strands within non-IPC pancreata (>2-fold change; P < .05). Proteomic analysis revealed significant differential expression of at least 5 proteins >1.5-fold change; P < .05) within the IPC vs control group. Our data indicated that IPC of the pancreas prior to cold preservation was associated with improved islet cell recovery after cold ischemia. IPC of the pancreas may represent a viable therapeutic intervention to increase islet transplantation success from a single donor and to maximize organ utilization.

Prolonged survival with FK778 (malononitrilamide) monotherapy after small bowel transplantation: a large animal study.

Malononitrilamide 715 (FK778) is a new class of immunosuppressant, derived from the active metabolite of leflunomide A77 1726. We investigated the efficacy of two different immunosuppressive induction protocols with tacrolimus plus FK778 followed by FK778 monotherapy. In a swine model of small bowel transplantation, we observed three groups, divided by different therapy regimens: group 1 (n = 5): no immunosuppressant (control group); group 2 (n = 10): oral tacrolimus (from postoperative day [POD] 0 to 30) and FK778 (from POD 0 to 60); group 3 (n = 8): oral tacrolimus, as group 2, and FK778 (from POD 7 to POD 60). Median survival was 11, 60, and 21 days in groups 1, 2, and 3, respectively. In group 1 all animals died of acute rejection; in group 2 the causes of death were technical complication (n = 1) and sepsis (n = 1); in group 3, one animal died from obstruction, two from pneumonia, one from peritonitis, one from sepsis. Group 2 accounted for 0.5 infection episode/animal versus 0.62 in group 3 (P < .05). Acute rejection was absent or mild in 66% and 75% of group 3 and 2 biopsies, respectively (P < .05). The D-xylose absorption curves from groups 2 and 3 were similar to those of the nontransplanted healthy animals. In conclusion, FK778 monotherapy after a consistent induction period with tacrolimus combined immunosuppression is able to extend survival and preserve optimal absorptive capacity of the small bowel allograft in our pig model. The association of tacrolimus and FK778 from day 1, compared to the delayed administration of FK778 from day 7, results in a significant reduction of infections and postoperative complications.

Elemental enteral nutrition preserves the mucosal barrier and improves the trophism of the villi after small bowel transplantation in piglets.

The main goals for a successful small bowel transplantation (SBTx) are the control of acute rejection and maintenance of the mucosal barrier, which plays a key role in preventing bacterial translocation and preserving absorptive capacity. According to recent evidence that sustaining enteral nutrition (EN) as rehabilitative therapy improves the integrity of the mucosal barrier after SBTx, we studied the trophic effect of a new elemental enteral solution whose proteinic supply is represented by oligomeric-aminoacidic chains. In a swine SBTx model we studied three groups, divided by the different postoperative feeding: group 1 (n = 5): standard swine chow, group 2 (n = 5): polymeric enteral solution, group 3 (n = 5): elemental enteral solution (Peptamen, Nestlè Corp). All animals were immunosuppressed with a tacrolimus/FK778 combined oral therapy. The nutritional indices evaluated were: body weight, episodes of diarrhea, D-xylose absorption test, and histopatological and villi morphometric analysis. Three pigs died before the end of the study, two in group 1 (pneumonia and sepsis), one in group 2 (pneumonia). Mean days of diarrhea were 15, 10, and 3 in groups 1, 2, and 3, respectively (P < .05). The final/starting weight ratio was 1.08 for group 3 and 0.92 for group 2 (P < .05); the D-xylose curves showed a statistically significant difference for group 3 versus the groups 2 and 1 (P < .05), as well as for the villi height (P < .01) and width (P < .05). In conclusion, elemental enteral solution, with its basic protein supply, does not require a very complex enzymatic system to be metabolized. Thus, it may contribute to a faster recovery of the mucosal barrier and to limit the hypercatabolic state.

FK778 does not impair intestinal allograft absorption in a preclinical model of total small bowel transplantation.

Malononitrilamide 715 (FK778), a new low-molecular weight immunosuppressant, inhibits both T-cell and B-cell function by acting on the pathway for de novo pyrimidine biosynthesis. Pyrimidines are important for intestinal trophism; their inhibition may predispose to metabolic and functional impairments, such as diarrhea and malabsorption. In this study we assessed the absorptive capacity of intestinal allografts in a large-animal model of small bowel transplantation (SBTx) in pigs chronically treated with FK778. Ten outbred pigs underwent total orthotopic SBTx. Immunosuppression consisted of oral tacrolimus (trough levels 5-15 ng/mL) and oral FK778 (4 mg/kg per day) administered for 60 days. The D-xylose absorption test was performed at day 60 to evaluate carbohydrate absorption. Results were compared to normal controls. Eight of the 10 animals were alive and in good condition at day 60. All of their allografts were free of rejection. The animals had a mean maximal weight loss of 6.4% during the study period; the final weight was comparable to the initial weight (P > .05). Diarrhea was present in all animals (mean 16% of postoperative days). The D-xylose curves showed that absorption in the transplanted animals at day 60 was similar to that in the untreated controls (P > .05). The absence of differences was confirmed by the statistical analysis. In conclusion, our preclinical study in pigs showed that chronic treatment with FK778 in combination with tacrolimus did not impair carbohydrate absorption by the allograft after SBTx.

FK778 and tacrolimus combination therapy to control acute rejection after pig intestinal transplantation.

Malononitrilamide 715 (FK778) is a new class of low molecular weight immunosuppressant. Experimental studies in heart, liver, and kidney transplantation have shown a strong synergism when FK778 is used in combination with tacrolimus and when its administration is delayed by 7 days after the transplant. Following this indication, in a swine model of orthotopic small bowel transplantation (SBT), we assessed the efficacy of combined low dose tacrolimus and FK778 administered from day 0 or day 7. The entire small bowel was replaced in 16 piglets: group 1 (n = 5), no immunosuppression; group 2 (n = 6) oral tacrolimus to maintain whole blood trough levels between 5 and 15 ng/mL plus FK778 4 mg/kg per day; group 3 (n = 6) oral tacrolimus as in group 2 plus FK778 4 mg/kg per day administered after a 7-day delay posttransplant. The median survival was 8 days in group 1, 60 days in group 2, and 13 days in group 3. The differences between group 2 and 1 and between group 2 and 3 are statistically significant. Three episodes of major bacterial infection were detected in both group 2 and 3 (0.5 episode/animal). The infectious-related mortality was 0% in group 2 and 50% in group 3 (P < .05). Acute cellular rejection was absent or mild in all group 2 and 3 stomal biopsies. In conclusion, combining tacrolimus and FK778 allowed prolonged survival after SBT in swine when FK778 was started at the time of SBT. The delayed administration of FK778 resulted in a high incidence of lethal infectious complications.

Early outcome of different steroid-free regimens in small bowel transplantation: a large-animal study.

The intestine is a highly immunogenic organ that requires heavy immunosuppression (IS); therefore corticosteroid withdrawal after clinical small bowel transplantation (SBT) has not been standardized. In this study, we compared different immunosuppressive regimens (none with steroid or induction treatment) in a SBT pig model. Large White unrelated piglets were transplanted and divided into four groups as follow: group 1 (n = 3): no IS; group 2 (n = 10): IS with tacrolimus only; group 3 (n = 10): IS with tacrolimus and mycophenolate mofetil; group 4 (n = 5): IS with tacrolimus and rapamycin. Follow-up time was 30 days. All IS drugs were given orally; tacrolimus whole blood levels ranged between 5 and 15 ng/mL in all groups except for group 2 whose tacrolimus whole blood levels ranged between 15 and 25 ng/mL. Group 1 pigs died of graft acute rejection (ACR) after a median of 12 days. Overall survival in groups 2, 3, and 4 at day 30 was 40%, 80%, and 60%, respectively. Biochemical parameters, including glycemia and cholesterol, were into the normal range with no significant differences between groups. At the end of the study, one animal in group 2 and another one in group 4 showed histological signs of moderate to severe ACR. The incidence of infection was higher in group 2 (2.1 episodes/pig) compared to group 3 (1.25) and group 4 (1.6). This large-animal study demonstrates that tacrolimus-based IS without corticosteroids allows, in the early postoperative period (30 days) after SBT, good survival rates without an increased risk in the incidence of rejection.

Incidence of graft rejection in small bowel transplanted pigs after immunosuppression withdrawal.

As intestinal grafts require heavy immunosuppression, there are no reports of immunosuppression withdrawal after clinical small bowel transplantation. In this large-animal study, we investigated the occurrence of graft rejection in intestinal-transplanted pigs after withdrawal. Large-White unrelated piglets were transplanted and divided in three groups: group 1 (n = 5), intestinal transplantation (ITx) with no immunosuppression; group 2 (n = 7), Itx and 60 days of treatment with tacrolimus and mycophenolate mofetil; group 3 (n = 5), Itx and donor bone marrow infusion (DBMi) and 60 days of treatment with tacrolimus and mycophenolate mofetil. Follow-up time after withdrawal was 120 days. Group 1 pigs died of graft acute cellular rejection (ACR) after a median of 11 days. In group 2, two pigs died of ACR-related infection and another two of ACR within 90 days. The remaining three animals (43%) were sacrificed at day 180, and their grafts showed no signs of ACR. In group 3, two pigs died of ACR-related infection and one of graft versus host disease within 80 days; at day 180 the two surviving animals showed signs of chronic rejection in the allograft. This study demonstrates that total withdrawal after ITx is followed by sudden and lethal ACR (or ACR-related infection) in more than 50% of the recipients. When a tolerance-inducing strategy as DBMi is applied, lethal graft versus host disease may also occur. In group 3, the intestinal allograft, to which the recipients were partially tolerant, developed chronic rejection that was probably associated with a decline with time of donor-leukocytes chimerism, as recently demonstrated in rats.

Platelet activation by diacylglycerol or ionomycin is inhibited by nitroprusside.

Experiments were performed to elucidate the role of cyclic guanosine monophosphate (cGMP) on platelet activation induced by protein kinase C (PKC) activators and calcium ionophore. Human platelets were pretreated with acetylsalicylic acid and with hirudin and apyrase. Aggregation and ATP secretion in response to the PKC activators 4 beta-phorbol 12-myristate 13-acetate (PMA) and 1-oleoyl 2-acetylglycerol (OAG) were inhibited by the nitrovasodilator sodium nitroprusside (SNP), an activator of guanylate cyclase, and by 8-bromo-cyclic GMP (8-Br-cGMP). The experiments were performed in the presence of M&B 22948, an inhibitor of cGMP phosphodiesterase. SNP and 8-Br-cGMP also inhibited platelet aggregation and secretion evoked by the ionophore ionomycin. In fura-2 loaded platelets SNP did not affect basal cytosolic Ca2+ level nor the rise induced by low concentrations of ionomycin, both in the presence and absence of extracellular Ca2+. The phosphorylation of the 47 and 20 kDa protein induced by ionomycin or PMA were not significantly decreased by SNP or 8-Br-cGMP. The present results suggest that cGMP is able to inhibit both the PKC and the Ca(2+)-dependent pathways leading to platelet activation by interfering, similarly to cAMP, with processes following protein phosphorylation, close to the effector systems.

Salicylate fails to prevent the inhibitory effect of 5,8,11,14-eicosatetraynoic acid on human platelet cyclo-oxygenase and lipoxygenase activities.

Sodium salicylate is inactive both on cyclo-oxygenase and lipoxygenase prepared from human platelets. It prevents the inhibition of cyclo-oxygenase induced by aspirin, but does not counteract the inhibitory effect of 5,8,11,14-eicosatetraynoic acid on both enzymes. It also fails to interfere with the inhibitory activity of nordihydroguaiaretic acid on lipoxygenase. These data indicate that, unlike eicosatetraynoic acid, non-steroidal anti-inflammatory drugs interact with a site on cyclo-oxygenase distinct from the catalytic site, although related to it. Such a supplementary binding site is lacking on lipoxygenase.

Cyclic GMP and nitroprusside inhibit the activation of human platelets by fluoroaluminate.

Sodium nitroprusside, an activator of the soluble guanylate cyclase, inhibits the intracellular Ca2+ mobilization, ATP secretion and aggregation of human platelets evoked by fluoroaluminate. Similar results are obtained with 8-bromo-cyclic GMP (8-Br-cGMP). Both nitroprusside and 8-Br-cGMP inhibit the protein kinase C-dependent phosphorylation of the 47 and 20 kDa proteins induced by fluoroaluminate, but not by the protein kinase C activators phorbol ester and diacylglycerol. Since fluoroaluminate interacts directly with a G protein, the present results suggest that the cGMP interferes with platelet activation at the level of G protein-phospholipase C.

Platelet responses promoted by the activation of protein kinase C or the increase of cytosolic Ca2+ are potentiated by adrenaline. Effects of cAMP and staurosporine.

We studied the action of the alpha 2 adrenergic agonist adrenaline on the platelet responses evoked by the activation of protein kinase C or by the ionophore induced increase of cytosolic Ca2+. Both the phorbol ester and ionomycin-induced aggregation are strongly potentiated by adrenaline which per se does not behave as an activating agonist. The potentiation by adrenaline is observed both when added before and after the aggregating agent; in the latter case the effect increases on increasing the delay of adrenaline addition. Adrenaline also reverses the inhibition by cAMP of the PMA (or ionomycin) induced aggregation. It also has a strong potentiating effect (over 100%) on the phorbol ester induced ATP secretion and a weaker effect on the secretion induced by ionomycin. The effect on secretion is visible only when adrenaline is added prior to the stimulus. The inhibition by cAMP of the PMA or ionomycin induced secretion is also counteracted by adrenaline. In no case adrenaline modifies the pattern of platelet phosphoproteins. Ionomycin induces some platelet aggregation also in the presence of the protein kinase inhibitor staurosporine; also this phosphoprotein independent aggregation is strongly stimulated by adrenaline.

Efficacy of malononitrilamide FK778 in a preclinical model of small bowel transplantation.

In a swine model of orthotopic small bowel transplantation, we assessed the efficacy of combined immunosuppressive therapy with low-dose tacrolimus plus FK778, compared with high-dose tacrolimus monotherapy. The small bowel was replaced in 23 piglets: group 1 (n = 5), no immunosuppression; group 2 (n = 12), oral tacrolimus to maintain whole blood trough levels between 15 and 25 ng/mL; and group 3 (n = 6), oral FK778 4 mg/kg/d, plus oral tacrolimus to maintain whole blood trough levels between 5 and 15 ng/mL. Follow-up time was limited to 60 days. Overall survival rates at 30 and 60 days were 0% and 0% in group 1, 30% and 0% in group 2, and 66% and 66% in group 3, respectively. The median survival time was 11 days in group 1, 28 days in group 2, and more than 60 days in group 3. The differences between groups 3 and 1 and between groups 3 and 2 were statistically significant. The numbers of major bacterial infections were 19 in group 2 (1.9 episodes per animal) and 3 in group 3 (0.75 episodes per animal). The infectious-related mortality rate was 70% in group 2 (7 cases) and 0% in group 3 (P < .05). Acute cellular rejection was absent or mild in 85% of group 2 stomal biopsy specimens and in 100% of group 3 biopsy specimens. In conclusion, combination therapy of low-dose tacrolimus is potentiated by FK778 to prevent acute cellular rejection and prolong small bowel transplant survival in pigs.

Chemical synthesis and structural characterization of the RGD-protein decorsin: a potent inhibitor of platelet aggregation.

Decorsin is a 39-residue RGD-protein crosslinked by three disulfide bridges isolated from the leech Macrobdella decora belonging to the family of GPIIb-IIIa antagonists and acting as a potent inhibitor of platelet aggregation. Here we report the solid-phase synthesis of decorsin using the Fmoc strategy. The crude polypeptide was purified by reverse-phase HPLC in its reduced form and allowed to refold in the presence of glutathione. The homogeneity of the synthetic oxidized decorsin was established by reverse-phase HPLC and capillary zone electrophoresis. The results of amino acid analysis after acid hydrolysis of the synthetic protein, NH2-terminal sequencing and mass determination (4,377 Da) by electrospray mass spectrometry were in full agreement with this theory. The correct pairing of the three disulfide bridges in synthetic decorsin was determined by a combined approach of both peptide mapping using proteolytic enzymes and analysis of the disulfide chirality by CD spectroscopy in the near-UV region. Synthetic decorsin inhibited human platelet aggregation with an IC50 of approximately 0.1 microM, a figure quite similar to that determined utilizing decorsin from natural source. In particular, the synthetic protein was 2,000-fold more potent than a model RGD-peptide (e.g., Arg-Gly-Asp-Ser) in inhibiting platelet aggregation. Thermal denaturation experiments of synthetic decorsin, monitored by CD spectroscopy, revealed its high thermal stability (Tm approximately 74 degrees C). The features of the oxidative refolding process of reduced decorsin, as well as the thermal stability of the oxidized species, were compared with those previously determined for the NH2-terminal core domain fragment 1-41 or 1-43 from hirudin. This fragment shows similarity in size, pairing of the three disulfides and three-dimensional structure with those of decorsin, even if very low sequence similarity. It is suggested that the less efficient oxidative folding and the enhanced thermal stability of decorsin in respect to those of hirudin core domain likely can be ascribed to the presence of the six Pro residues in the decorsin chain, whereas none is present in the hirudin domain. The results of this study indicate that decorsin can be obtained by solid-phase methodology in purity and quantities suitable for structural and functional studies and thus open the way to prepare by chemical methods novel decorsin derivatives containing unusual amino acids or even non-peptidic moieties.

Warfarin and heparin interaction with prostacyclin-like activity generated by arterial wall in the rat.

A study was performed to evaluate the hypothesis that heparin opposes uncontrolled PGI2 production by vessels due to thrombin generation. We treated animals with warfarin, an inhibitor of liver synthesis of prothrombin and other vit. K-dependent clotting factors. In fact, if thrombin is presumed to be the stimulus for PGI2 production, warfarin should suppress this effect. As warfarin treatment did not affect significantly PGI2-like activity, we conclude that a different hypothesis is necessary to explain the phenomenon. On the other hand, i.p. sodium heparin effectively induced a decrease in PGI2-LA synthesis, whereas s. c. calcium heparin did not significantly influence it.

Actions of nitric oxide on the release of prostacyclin from bovine endothelial cells in culture.

Endothelial cells release the potent vasodilator prostacyclin, as well as the highly labile endothelium-derived relaxing factor (EDRF) which mediates vascular relaxation induced by some vasodilators including acetylcholine and bradykinin. EDRF has recently been characterised as nitric oxide (NO). The effects of NO on prostacyclin release, measured as 6-keto-PGF1 alpha, from endothelial cells obtained from bovine thoracic aorta, have now been investigated. Incubation of endothelial cells in culture with bradykinin (10-100 nM) stimulated the release of 6-keto-PGF1 alpha. Pre-incubation (0.5-2 min) with NO (13-130 microM) caused a significant dose-dependent inhibition of 6-keto-PGF1 alpha release, reaching a maximum of 29 +/- 4% inhibition. Pre-incubation with superoxide dismutase (30 units ml-1) which prevents the breakdown of NO, significantly augmented the degree of inhibition, as did the selective inhibitor of cyclic GMP phosphodiesterase, M & B 22948 (5 microM), reaching 51 +/- 2% inhibition. The potentiation by M & B 22948 suggests that this inhibitory effect of high concentrations of NO is brought about by elevation of intracellular cyclic GMP levels following activation of guanylate cyclase. Whether endogenous NO is produced by endothelial cells under physiological conditions in sufficient quantities to modulate prostacyclin release remains to be established.

Platelet activation and modulation of the induction of nitric oxide synthase in the conscious rat.

Injection of lipopolysaccharide (LPS) (Salmonella W. Typhosa i.v. bolus) into conscious rats, induced a rapid drop of circulating platelets analogous to that induced by ADP. The animals showed a small fall in mean arterial blood pressure (MABP), an increase in heart rate and a significant increase in plasma nitrite and nitrate level. This result is consistent with the stimulation of an inducible NO synthase (i-NOS). The administration of the stable prostacyclin analogue, iloprost plus ADP or LPS, significantly protected against the decrease in free platelet number induced by ADP or LPS. The plasma nitrite and nitrate level stimulated by LPS was significantly reduced by iloprost and also by prostacyclin. These results are consistent with an inhibition of i-NOS by agents that increase the intracellular level of cAMP. The administration of the NO donor S-Nitroso-N-acyl-D-penicillamine (SNAP) plus ADP or LPS, significantly prevented thrombocytopenia induced by ADP and by LPS. SNAP did not decrease the plasma nitrite and nitrate level stimulated by LPS; furthermore it induced a significant increase of heart rate, without affecting MABP, suggesting a direct accelerating effect of NO on the sino-atrial node. The administration of S-nitroso-glutathione (GSNO), a stable nitrosothiol, plus ADP or LPS, significantly prevented thrombocytopenia induced by ADP but not by LPS. GSNO significantly reduced the plasma nitrite and nitrate level stimulated by LPS. These data demonstrate that the L-Arginine: NO pathway in vivo may be modulated by prostanoids and that compounds which increase cAMP, such as iloprost, are able to protect against LPS-induced early thrombocytopenia.

[Light chain deposition disease]. / La malattia da deposito di catene leggere.

We report the case of a rather rare form of K-light chain deposition disease (LCDD) in a 61-year-old man with hypertension and rapidly progressing nephropathy. Laboratory findings prompted suspicion of the diagnosis which was confirmed by light-microscopic and immunofluorescent studies of samples taken by percutaneous renal and liver biopsy. Hepatic and urinary K-light chains were present; no circulating light chains were detected. Bone marrow examination evidenced mild infiltration of lymphoid cells, all positive for K-light chain staining. Plasma cells were within normal ranges. LCDD appeared as nodular glomerulosclerosis with rare crescents and extensive tubular involvement with K-light chain deposits. There was no evidence of altered liver function, nor was amyloid found in the bone marrow, kidney or liver. After one year of continuous therapy with melphalan and prednisone, the patient's renal function has not worsened. We conclude with a review of the clinical and physiopathological features of the light chain subgroup of monoclonal immunoglobulin deposition diseases (MIDD).