Defining priorities in the transition from paediatric to adult healthcare for rare bone disease patients: a dialogic approach.

The Italian patient association for Multiple Osteochondromas, Ollier Disease, and Maffucci Syndrome, Associazione Conto Alla Rovescia-ACAR Aps, conducted a mixed-methods study at its 2023 annual conference. The study included the Open Dialogue Approach and a feedback survey to identify the main priorities in the transitioning process from paediatric to adult healthcare for patients with Multiple Osteochondromas, Ollier Disease, and Maffucci Syndrome. The common needs identified by patients, families, caregivers, and healthcare professionals were coordination and continuity of care, patient empowerment and communication, social and practical support, and transition planning and support. This experience fostered a sense of collaboration and cooperation among stakeholders, helping to build trust and create a shared vision for improving the quality of care for these patients. Furthermore, it could be considered a starting point for other patient associations interested in using different approaches to identify the needs of their members and actively involve all stakeholders.

Inhibition of heat-induced denaturation of albumin by nonsteroidal antiinflammatory drugs (NSAIDs): pharmacological implications.

The activity of nonsteroidal antiinflammatory drugs (NSAIDs) in rheumatoid arthritis is not only due to the inhibition of the production of prostaglandins, which can even have beneficial immunosuppressive effects in chronic inflammatory processes. Since we speculated that these drugs could also act by protecting endogenous proteins against denaturation, we evaluated their effect on heat-induced denaturation human serum albumin (HSA) in comparison with several fatty acids which are known to be potent stabilizers of this protein. By the Mizushimas assay and a recently developed HPLC assay, we observed that NSAIDs were slightly less active [EC50 to approximately 10(-5)-10(-4) M] than FA and that the HPLC method was less sensitive but more selective than the turbidimetric assay, i.e. it was capable of distinguishing true antiaggregant agents like FA and NSAIDs from substances capable of inhibiting the precipitation of denatured protein aggregates. In conclusion, this survey could be useful for the development of more effective agents in protein condensation diseases like rheumatic disorders, cataract and Alzheimers disease.

Inhibition of calcium oxalate precipitation by bile salts.

BACKGROUND: Both urinary and biliary stones can contain calcium. Bile salts (BA), which are known to bind Ca2+, are commonly used to dissolve the latter but not the former. METHODS: The effect of physiologic BA on calcium oxalate (CaOx) precipitation was evaluated by a recently developed method. RESULTS: The Ca2+ binding properties of BA were confirmed by small but significant decreases in pH observed following addition of CaCl2 to bile acids solutions. More importantly, BA inhibited CaOx precipitation with effective concentrations of approximately 10-3 mol/L. The clinical relevance of the latter observation is presently unknown but it is of note that in the same in vitro assay, the activity of BA appeared comparable to that of citric acid, the most common drug for urolithiasis. Although BA do not reach mmol/L levels in urine, they are known to change the physicochemical properties of this fluid, possibly slowing down the crystal growth process. However, the hypothetical therapeutic use of BA in former stone patients would present at least two major problems: (i) hepatotoxicity and (ii) lithogenic activity, due to hyperoxaluria subsequent to increased intestinal absorption of oxalate. CONCLUSION: The ability of BA in effectively binding calcium ions and in inhibiting the precipitation of CaOx appears of interest from both a physiopathologic and a pharmacologic point of view, even if it does not currently seem exploitable for prophylactic or therapeutic purposes.

Analysis of lonidamine in rat serum and testis by high performance liquid chromatography.

A HPLC method for the determination of lonidamine in serum and testis, suitable for pharmacological studies in the rat and other mammals, has been developed. Briefly, 0.5 mL of serum or about 0.2 g of testicular tissue were extracted with ethyl acetate and evaporated to dryness under nitrogen. The residue was redissolved in methanol and an aliquot was injected onto a C18 column eluted with a mobile phase consisting of acetonitrile:water (51:49, v/v), containing 0.1% trifluoroacetic acid. The eluate was monitored at 230 nm with a sensitivity of 0.05 AUFS. By this method, the pharmacokinetics and the serum and testicular levels of the drug up to 120 h after the administration of one single dose (100 mg/kg body weight) of lonidamine to Sprague-Dawley rats have been studied. Results were highly variable, as previously reported, but a very good linear correlation was found between the serum and the testicular levels, suggesting that, in the rat, and possibly in the human, testicular levels could be estimated based on the serum concentrations.

Effect of lonidamine on alpha2-macroglobulin, hemopexin and alpha1-antitrypsin in the rat testis and epididymis.

In a recent study (Leone et al., 2000) we reported that lonidamine (LND), an antispermatogenic drug, affected the concentration of selected testicular and epididymal proteins in the rat. Thus, the effect of LND on alpha2-macroglobulin (alpha2-M) and on other two acute phase proteins (APP), hemopexin (HPX) and alpha1-antitrypsin (alpha1-AT) was examined here. LND was administered orally at the dose of 100 mg/kg, the animals were killed after 24 and 48 hr and the samples were analyzed by immunoblotting. The drug did not induce any significant change of alpha2-M in the serum or testis and of HPX and alpha1-AT in the serum, testis or epididymis. Thus, the antispermatogenic action of LND was not accompanied by a significant change of these inflammatory markers, even if it did cause a decrease of alpha1-inhibitor-3, a negative APP, as previously reported (Leone et al., 2000).

In vitro evaluation of antioxidant activity by electrophoresis and high performance liquid chromatography.

Two methods for the analysis of antioxidants, based on polyacrylamide gel electrophoresis (PAGE) and gel permeation high performance liquid chromatography (HPLC) were developed. Both of them exploit the variations of the signal (band or peak) given by human serum albumin (0.2% w/v in 100 mM sodium phosphate pH 7) upon oxidation with hypochlorite (1% of a solution containing 4% active Cl), quantitatively determined by densitometric analysis or peak integration. Based on such changes, two formulas were defined which allowed the determination of the antioxidant activity of ascorbic acid (EC(50,PAGE)=4.8x10(-4) M, EC(50,HPLC)=3.6x10(-4) M), glutathione (EC(50,PAGE)=1.5x10(-4) M, EC(50,HPLC)=2.0x10(-4) M) and melatonin (EC(50,PAGE)=5.2x10(-4) M, EC(50,HPLC)=3.2x10(-4) M), chosen as reference compounds. A good correlation was found between the activities of these substances in the two assays, which are also in good agreement with literature data, indicating that the two methods are essentially equivalent. These assays could be useful for the screening of new antioxidant drugs for pathological conditions such as cataract, rheumatic diseases, atherosclerosis and Alzheimer's disease.

Changes of acute-phase proteins in streptozotocin-induced diabetic rats.

Quantitative and qualitative changes of serum proteins, apart from glycation, have not been sufficiently studied in streptozotocin-induced diabetic rats (D), the most common experimental model for diabetes. Thus, we decided to analyze the serum of diabetic rats by concanavalin A-blotting in comparison with rats with acute inflammation induced by fermented yeast (Y), in which characteristic alterations of serum proteins have been described. Two months after the streptozotocin treatment, the blood glucose levels were highly elevated (456+/-24 vs. 124+/-10 mg/dl, p<0.001, n=12), the body weight was significantly lower than normal (279+/-10 vs. 392+/-6 g, p<0.001, n=12), and serum proteins appeared to be highly glycated (p<0.001) when analyzed by the fructosamine assay, without any significant change in the total serum protein concentration. Analysis by concanavalin A-blotting, revealed a significant decrease of alpha1-inhibitor-3 (alpha1-I3, p<0.05) and an increase of the beta chain of haptoglobin (beta-Hp, p<0.05) in both D and Y rats (n=3) compared with control animals. However, acute inflammation caused a marked rise of two prominent acute phase proteins, alpha2-macroglobulin and hemopexin, which did not change appreciably in diabetic rats. Further work will be necessary to evaluate the physiopathological significance of these phenomena which could result from changes of both concentration and glycosylation of the aforementioned proteins.

Effects of lonidamine on testicular and epididymal proteins in the rat.

The mechanism responsible for the antispermatogenic activity of lonidamine (LND) [1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid], a drug with low systemic toxicity and lack of significant hormonal effects, is still unclear but may be related to alterations of Sertoli cell proteins. Here, we confirmed that a single oral dose of LND (100 mg/kg b.w.) to sexually mature Sprague-Dawley rats causes shrinkage and weight reduction of the testes after 48 h. These macroscopic changes correlated with histologic alterations revealed by light microscopy, consistent with partially reversible inhibition of spermatogenesis. When the testes and the epididymides of animals treated with or without LND were homogenized and analyzed by the Bradford assay, a significant increase of total protein content was observed after 24 and 48 h. When these homogenates were analyzed by concanavalin blotting, specific changes of the major rat macroglobulins, i.e. alpha(1)-inhibitor-3, alpha(2)-macroglobulin, and alpha(1)-macroglobulin, were noted. In particular, LND caused a decrease of testicular alpha(1)-inhibitor-3, but not an increase of testicular alpha(2)-macroglobulin, indicating a mild local inflammatory response to the drug.

Simultaneous determination of hydrocortisone, dexamethasone, indomethacin, phenylbutazone and oxyphenbutazone in equine serum by high-performance liquid chromatography.

Ethyl acetate extracts of equine serum, containing 0-5 microg/ml of hydrocortisone (HYD), dexamethasone (DEX), oxyphenbutazone (OPB), indomethacin (IND), phenylbutazone (PB) and probenecid as internal standard, were evaporated with nitrogen, resuspended in methanol and analyzed by HPLC, using a C-18 column equilibrated with 51:49 acetonitrile-water, 0.1% trifluoroacetic acid, at 1 ml/min. The eluate was monitored at 254 nm. The selectivity (inter-assay C.V.<4%), sensitivity (limits of quantitation of 0.25 microg/ml for HYD, DEX and IND, 0.5 microg/ml for PB and 1 microg/ml for OPB, despite the occurrence of significant degradation of OPB and PB during the analysis) and precision (intra-assay and inter-assay C.V.'s of about 3-6 and 9-15%, respectively) of the method appeared appropriate for anti-doping control of racehorses.

Inhibition of heat-induced aggregation of beta- and gamma-crystallin by alpha-crystallin evaluated by gel permeation HPLC.

The capability of alpha-crystallin (alpha-C), a known molecular chaperon, of protecting beta-C and gamma-C against heat-induced aggregation was studied by gel permeation high performance liquid chromatography. The activity was calculated using a formula based on the changes in the areas under the chromatographic peaks of these proteins, which appeared well separated. When heat-induced aggregation was studied in the range 22-90 degrees C, beta-C appeared more stable than gamma-C. The activity of alpha-C in stabilizing gamma-C but not beta-C was already relevant at 60 degrees C, but the maximum activity was higher (about 35%) for beta-C than for gamma-C. This method could be useful for studying the effect of drugs with potential anti-cataract activity on heat-induced aggregation of individual lens proteins.

Inhibition of Candida rugosa lipase by berberine and structurally related alkaloids, evaluated by high-performance liquid chromatography.

It is known that certain microorganisms produce extracellular lipase to better colonize the skin and mucosal surfaces. Since different extracts from medicinal plants have anti-lipase activity (Shimura et al., Biosci. Biotechnol. Biochem., 56: 1478-1479, 1992), we examined the effects of selected natural substances on Candida rugosa lipase. In the presence of the compounds under examination, the enzyme was incubated with beta-naphthyl laurate, and beta-naphthol, produced by the enzymatic reaction, was extracted with ethyl acetate and analyzed by reversed phase HPLC, using a C-18 column. Thus, the inhibitory activity was calculated by a proper formula based on the variations of the area under the chromatographic peak of beta-naphthol. The method was validated by analyzing substances with known anti-lipase activity such as saturated fatty acids (C10-16) and tetracycline. Berberine and a number of structurally related alkaloids such as chelidonine, chelerythrine, and sanguinarine appeared active. This property of berberine and sanguinarine is of interest because they are used in pathological conditions in which microbial lipases could play a pathogenic role.

Stabilization of rat serum proteins following oral administration of fish oil.

The mechanism of action of fish oil (FO), currently used in different chronic inflammatory conditions such as rheumatoid arthritis (RA), is not completely understood, although it is thought that it could alter the metabolism of endogenous autacoids. In addition, we hypothesized that the known capability of fatty acids (FA) of stabilizing serum albumin and perhaps other proteins, may be of pharmacological relevance considering that it is shared by other anti-rheumatic agents (e.g. nonsteroidal antiinflammatory drugs). Thus, we studied the effect of oral administration of FO and corn oil (CO), a vegetable oil with a different composition, on the stability of rat serum proteins, evaluated by a classical in vitro method based on heat-induced protein denaturation. FO, and, to a lower extent, CO inhibited heat-induced denaturation of rat serum (RS): based on the inhibitory activity (EC50) of the major fatty acids against heat-induced denaturation of RS in vitro, it was possible to speculate that in vivo effects of palmitic acid (C16:0) and eicosapentaenoic acid (EPA, C20:5, n-3) may be more relevant than that of linolenic acid (C18:2). To better investigate this phenomenon, we extracted albumin from the serum of animals treated or not with FO with a one-step affinity chromatography technique, obtaining high purity rat serum albumin preparations (RSA-CTRL and RSA-FO), as judged by SDS-PAGE with Coomassie blue staining. When these RSA preparations were heated at 70 degrees C for 30 min, it was noted that RSA-FO was much more stable than RSA-CTRL, presumably due to higher number of long chain fatty acids (FA) such as palmitic acid or EPA. In conclusion, we provided evidences that oral administration of FO in the rat stabilizes serum albumin, due to an increase in the number of protein bound long chain fatty acids (e.g. palmitic acid and EPA). We speculate that the stabilization of serum albumin and perhaps other proteins could prevent changes of antigenicity due to protein denaturation and glycosylation, which may trigger pathological autoimmune responses, suggesting that this action may be involved in the mode of action of FO in RA and other chronic inflammatory diseases.

Micromethods for the analysis of tear proteins in pharmacological studies.

Although it is well established that ocular mucins and other proteins are essential for tear film stability, whether certain drugs, like non steroidal antiinflammatory drugs (NSAIDs), could cause ocular dryness by inhibiting their secretion is not known. To perform these and other studies of pharmacological interest, we evaluated several micromethods for the analysis of tear samples. The major proteins of the tear fluid collected in capillaries, i.e. IgA, lactoferrin, tear specific prealbumin and lysozyme, were analyzed by SDS-PAGE and gel permeation HPLC, using 2.5-5 microliters of sample. Gastric mucin (PGM), examined as a standard, was analyzed by solid phase assays based on previously described histochemical staining methods: dot blot assays were performed using small disks of polyvinylidene difluoride or nylon membranes, prepared by an ordinary paper punch, which were coated with PGM and stained by Alcian blue or the periodic acid Schiff's reagent. The densitometric analysis was carried out using an ordinary flat scanner controlled by a personal computer equipped with an inexpensive software. The sensitivity of these simple assays was low (100-500 micrograms) but considered sufficient for certain studies. A more sensitive assay (5-20 micrograms) was developed by immobilizing PGM in small agarose gels (100 microliters), prepared in the wells of 96-well microplates, which could by stained by stains-all and analyzed by an automatic plate reader at 595 nm.

Development of an enzyme-linked immunosorbent assay, using a monoclonal antibody against alpha2-macroglobulin, for the diagnosis of systemic lupus erythematosus.

OBJECTIVES: To develop an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (mab) directed against abnormally glycosylated serum alpha2-macroglobulin (alpha2-M) from patients with systemic lupus erythematosus (SLE). DESIGN AND METHODS: Serum alpha2-M purified by HPLC from patients with SLE was injected in a Balb/c, CB6 F1 female mouse and hybrid cell lines were screened using alpha2-M Glu-C fragments derived from SLE and normal donors (NHS). A mab was selected and used to develop an ELISA by which sera from NHS (n = 14), SLE (n = 34), rheumatoid arthritis (n = 15), Sjögren's syndrome (n = 11), mixed connective tissue diseases (n = 12), and liver diseases (n = 11) were analyzed. RESULTS: The affinity of the mab for alpha2-M from SLE, but not from the other diseases, was higher compared to NHS, as demonstrated by immunoblotting and ELISA. CONCLUSIONS: The ELISA was capable of recognizing changes of glycosylation of alpha2-M in SLE and may be useful for its differential diagnosis.

Differential changes in alpha2-macroglobulin and hemopexin in brain and liver in response to acute inflammation.

Changes in serum and cerebrospinal fluid (CSF) proteins following generalized acute inflammation induced by fermented yeast in the rat was examined by concanavalin A-blotting, immunoblotting, and radioimmunoassay. Using alpha2-macroglobulin (alpha2-M) and hemopexin (HPX) as marker proteins, the concentration alpha2-M was found to increase in serum and CSF by 150- and 5-fold, respectively, whereas the concentration of HPX increased by about 4-fold in both fluids following yeast-induced inflammation. The lesser increase in alpha2-M in the CSF versus the systemic circulation is not likely to be the result of changes in the permeability of the blood--brain barrier, since no change in the total protein content of CSF was detected in inflamed rats when compared to control animals. These results, however, illustrate the regulation of the same protein, such as alpha2-M, in two separate organs within the same animal can be drastically different. These results also suggest a possible protective role of alpha2-M in the brain during acute inflammation. Moreover, these observations are consistent with the previous observation that there is a differential response in the level of alpha2-M between the testis and the systemic circulation during inflammation.

Changes in concanavalin A-reactive proteins in neurological disorders.

Changes of glycosylation of cerebrospinal fluid proteins such as alpha2-macroglobulin, and prostaglandin D synthase were studied by lectin blotting, using concanavalinA, in multiple sclerosis (n = 42) and neuropathies (n = 20) in comparison to neurological controls (n = 22). The concanavalinA-reactivity of alpha2-macroglobulin, which was increased in the neuropathies but not in multiple sclerosis compared to controls, correlated with the total concanavalinA-reactivity in controls and neuropathies but not in multiple sclerosis, indicating that the protein could be abnormally glycosylated in the latter disease. Although the concentration and the concanavalinA-reactivity of prostaglandin D synthase were not significantly different in the three groups, the two parameters correlated only in neuropathies but not in controls or multiple sclerosis, probably due to the high heterogeneity of the protein. These changes deserve to be studied in further detail in view of their potential clinical applications.

Inhibition of protein denaturation by fatty acids, bile salts and other natural substances: a new hypothesis for the mechanism of action of fish oil in rheumatic diseases.

Natural hydrophobic substances like bile salts (cholate, deoxycholate, chenodeoxycholate, lithocholate and their conjugates with glycine and taurine), fatty acids (caprylic, capric, lauric, myristic, palmitic, stearic, oleic, linoleic, arachidonic, eicosapentaenoic and docosahexaenoic acid) were much more active (EC50 approximately 10(-4)-10(-5) M) than selected amino acids (EC50 > 10(-2) M) and inorganic salts (EC50 approximately 10(-1) M) in inhibiting heat-induced denaturation of human serum albumin in vitro. Fish oil, rich in n-3-polyunsaturated acids such as eicosapentaenoic acid and docosahexaenoic acid, administered p.o. (1 ml/kg) in the rat, protected ex vivo (after 2 hr) serum against heat-induced denaturation more than bendazac, a known antidenaturant drug. Thus, we speculated that the antidenaturant activity of fish oil may be partly (in addition to the known effect on endogenous eicosanoid composition) responsible for its beneficial effects in rheumatoid arthritis and other rheumatic conditions. In this connection, it is of note that the in vitro antidenaturant activity of fish oil fatty acids was higher than that of known antidenaturant drugs such as bendazac and bindarit and nonsteroidal anti-inflammatory drugs like phenylbutazone and indomethacin which could exert beneficial effects in chronic inflammatory conditions by stabilizing endogenous proteins.

Interaction of hyaluronic acid with mucin, evaluated by gel permeation chromatography.

Hyaluronic acid (HA) is known to increase the ocular bioavailability of ophthalmic drugs not only for its viscous properties but also for its specific affinity for ocular mucins. This phenomenon, called bio- or mucoadhesion, can be evaluated in vitro by mechanical tests which, however, require considerable amounts of mucin (M) that are difficult to obtain from ocular surfaces. Thus, we developed an alternative method, based on gel permeation liquid chromatography, to examine the interaction of HA with microgram quantities of mucin. HA (from human umbilical cord or rooster comb) were fractionated using a Sepharose CL-4B column, before and after incubation with porcine gastric mucin (PGM), and the fractions were analyzed by a specific assay based on the histological dye Stains-all. PGM interacted with high molecular weight (M.W). HA, causing the displacement of low M.W., non-covalently bound, HA fragments, which were eluted under a distinct chromatographic peak. By quantitating the relative area of this peak, an evaluation of the mucoadhesion of HA could be obtained. This method could be useful to study the interaction between HA and microgram quantities of ocular M (mucin), obtained from individual patients or normal subjects.

Serum amine oxidase can specifically recognize and oxidize aminohexyl (AH) chains on AH-Sepharose support: single-step affinity immobilization.

Preparative affinity chromatography of bovine serum amine oxidase (SAO) on aminohexyl (AH)-Sepharose was often associated with an unexpected irreversible SAO retention on the support. This particular enzyme immobilization, occurring without coupling reagents, was supposed to be due to a SAO ability to: (i) recognize alkylamine groups of the support as macro-molecularized substrate; (ii) catalyse their oxidation to the corresponding aldehydes, with release of NH3 and H2O2; and (iii) be immobilized on the activated support by a coupling between the nascent aldehyde groups and SAO free amine groups. This affinity immobilization procedure, with the self-activation of the support, being mild, allows by simple incubation for 24 h, the enzyme immobilization with the retention of 80% from original specific activity of free SAO. Immobilized SAO on AH-Sepharose microcolumns, viewed as a continuous flow-system reactor, was able to catalyse benzylamine oxidation for several weeks.