João Roberto Maciel Martins

Noninvasive serum markers in the diagnosis of structural liver damage in chronic hepatitis C virus infection.

Abstract: Aim: Several noninvasive markers are being used to assess the structural liver damage in patients with chronic hepatitis C (CHC). We evaluated the capacity of serum hyaluronic acid (HA), aspartate aminotransferase (AST)/ALT ratio, the AST to platelet ratio index (APRI) and γ‐glutamyltransferase (GGT) levels to predict the intensity of hepatic fibrosis in patients with CHC.

Practical determination of hyaluronan by a new noncompetitive fluorescence-based assay on serum of normal and cirrhotic patients.

A practical fluorescence-based assay method for determination of hyaluronan (HA) was developed. Plates were coated with hyaluronan-binding proteins (HABP) obtained from bovine cartilage and successively incubated with samples containing standard solutions of hyaluronan or serum from normal and cyrrhotic patients, biotin-conjugated HABP, and europium-labeled streptavidin. After release of europium from streptavidin with enhancement solution the final fluorescence is measured in a fluorometer. The method is specific for HA even in the presence of substantial amounts of other glycosaminoglycans (chondroitin, dermatan sulfate, and heparan sulfate, and heparin) or proteins. It is possible to quantify HA between 0.2 and 500.0 microg/L. Analyses of HA concentration in 545 normal subjects and 40 cirrhotic patients gave average values of 14.5 and 542.0 microg/L, respectively. It was also shown that older subjects (> or =51 years old) have more HA (28.0 microg/L) than younger subjects (12.0 to 14.0 microg/L). This new sandwich technique has shown high precision and sensitivity similar to those of a recently described fluorescence-based assay method, being able to measure HA in amounts as small as 0.2 microg/L. In addition, this noncompetitive assay avoids preincubation, consumes less time (<5 h) than the previous competitive fluorescence-based assay (>72 h), and avoids the use of radioactive materials.

Comparison of practical methods for urinary glycosaminoglycans and serum hyaluronan with clinical activity scores in patients with Graves' ophthalmopathy

objective  Immunosuppressive treatment of Graves’ opthalmopathy (GO) should be restricted to patients with active eye disease, but assessing disease activity is difficult. Several methods to evaluate GO activity have been introduced, but none of them is satisfactory. Glycosaminoglycans (GAGs) are complex polysaccharides that participate on the pathogenesis of GO and attempts to correlate its local increase to urinary GAGs (uGAGs) or serum hyaluronan (sHA) have been made, but the available techniques are labourious, time‐consuming and difficult for routine use. The aim of the present study is to develop practical and simple methods for uGAGs and sHA and compare them to the activity and severity of thyroid‐associated ophthalmopathy.

Concentration and distribution of hyaluronic acid in mouse uterus throughout the estrous cycle

OBJECTIVE To quantify and study the immunoexpression of hyaluronic acid (HA) in the uterine horns of the mouse throughout the estrous cycle phases. DESIGN Experimental study using an ELISA-like fluorometric assay to quantify HA and an avidin-biotin immunoperoxidase method using biotinylated hyaluronan-binding protein for histochemical studies. SETTING University-based laboratory. ANIMAL(S) Forty regularly cycling adult female mice were divided into four groups according to the diagnosed phase of the cycle: proestrus, estrus, metaestrus, and diestrus. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Histologic samples of the uterine horns. Immunohistochemical reaction was evaluated by detection of HA and CD44 distribution within the uterine horn. Tissue HA content was determined through an ELISA-like fluorometric assay with the same hyaluronan-binding protein and with europium-labeled streptavidin. RESULT(S) The immunohistochemical HA and CD44 reactions were most intense during diestrus, mainly below the luminal epithelium. HA was strongly stained in the connective tissue near the myometrium layer during metaestrus. The biochemical data showed that the highest concentration of HA in uterine horns occurred during diestrus (4053.0 +/- 651.4 ng/g dry tissue) compared with other phases. CONCLUSION(S) Our data show that the expression of HA in mouse uterine horns is highest during the diestrous phase. The fluctuations of HA in the mouse uterus during the estrous phase may be related to the varying estrogen and P levels during the cycle and may be important as far as embryo implantation is concerned.

Urinary glycosaminoglycans excretion and the effect of dimethyl sulfoxide in an experimental model of non-bacterial cystitis

PURPOSE We reproduced a non-bacterial experimental model to assess bladder inflammation and urinary glycosaminoglycans (GAG) excretion and examined the effect of dimethyl sulfoxide (DMSO). MATERIALS AND METHODS Female rats were instilled with either protamine sulfate (PS groups) or sterile saline (control groups). At different days after the procedure, 24 h urine and bladder samples were obtained. Urinary levels of hyaluronic acid (HA) and sulfated glycosaminoglycans (S-GAG) were determined. Also to evaluate the effect of DMSO animals were instilled with either 50% DMSO or saline 6 hours after PS instillation. To evaluate the effect of DMSO in healthy bladders, rats were instilled with 50% DMSO and controls with saline. RESULTS In the PS groups, bladder inflammation was observed, with polymorphonuclear cells during the first days and lymphomononuclear in the last days. HA and S-GAG had 2 peaks of urinary excretion, at the 1st and 7th day after PS injection. DMSO significantly reduced bladder inflammation. In contrast, in healthy bladders, DMSO produced mild inflammation and an increase in urinary HA levels after 1 and 7 days and an increase of S-GAG level in 7 days. Animals instilled with PS and treated with DMSO had significantly reduced levels of urinary HA only at the 1st day. Urinary S-GAG/Cr levels were similar in all groups. CONCLUSIONS Increased urinary levels of GAG were associated with bladder inflammation in a PS-induced cystitis model. DMSO significantly reduced the inflammatory process after urothelial injury. Conversely, this drug provoked mild inflammation in normal mucosa. DMSO treatment was shown to influence urinary HA excretion.

Urinary Glycosaminoglycans as Biomarker for Urothelial Injury: Is It Possible to Discriminate Damage From Recovery?

OBJECTIVES The glycosaminoglycan (GAG) layer is referred to as a bladder protective factor. We reproduced an experimental model of urothelial damage to assess GAG metabolism in the process of injury and recovery of the urothelium. METHODS Wistar female rats were bladder catheterized and instilled with either protamine sulfate (PS groups) or sterile saline (control groups). At different days after the procedure, 24-hour urine samples were obtained. The urinary levels of hyaluronic acid (HA) and sulfated glycosaminoglycan were determined in all groups and in nonmanipulated rats (day 0). Additionally, sulfated-GAG synthesis was assessed by the incorporation of [(35)S]-inorganic sulfate. The bladders were analyzed by histochemical staining for HA and immunofluorescence for heparin sulfate and syndecan-4. RESULTS Urinary HA and sulfated-GAG were elevated after PS injection (P <0.05). A greater concentration of [(35)S]-labeled GAG in the PS group animals on the fifth day and, especially, on the seventh day represented increased GAG synthesis at these periods (P <0.05). Bladder sections from the PS group animals on day 1 showed a greater amount of HA in the urothelium. PS instillation damaged the urothelium layer of heparin sulfate and syndecan-4 seen in the control animals. On day 5, patchy areas of a restored layer were seen, and, on day 7, this layer had completely regenerated. CONCLUSIONS Urinary GAG cannot differentiate urothelial damage from recovery. Elevated levels of urinary GAG can result from either desquamation of the surface cell GAG layer or increased GAG synthesis to regenerate the damaged urothelium.

Optimizing nucleic acid extraction from thyroid fine-needle aspiration cells in stained slides, formalin-fixed/paraffin-embedded tissues, and long-term stored blood samples

OBJECTIVE Adequate isolation of nucleic acids from peripheral blood, fine-needle aspiration cells in stained slides, and fresh and formalin-fixed/paraffin-embedded tissues is crucial to ensure the success of molecular endocrinology techniques, especially when samples are stored for long periods, or when no other samples can be collected from patients who are lost to follow-up. Here, we evaluate several procedures to improve current methodologies for DNA (salting-out) and RNA isolation. MATERIALS AND METHODS We used proteinase K treatment, heat shock, and other adaptations to increase the amount and quality of the material retrieved from the samples. RESULTS We successfully isolated DNA and RNA from the samples described above, and this material was suitable for PCR, methylation profiling, real-time PCR and DNA sequencing. CONCLUSION The techniques herein applied to isolate nucleic acids allowed further reliable molecular analyses.

Concentration and distribution of hyaluronic acid in human vocal folds

Objective/Hypothesis: To evaluate the concentration and distribution of hyaluronic acid (HA) in human adult vocal folds.

Patients with head and neck tumors excrete a chondroitin sulfate with a low degree of sulfation: a new tool for diagnosis and follow-up of cancer therapy.

The chondroitin sulfate excreted in the urine of 10 patients with cancer of the head and neck and 27 healthy subjects was analyzed. The disaccharide products formed from chondroitin sulfate excreted by these 10 patients by action of chondroitinase ABC show a significant (P < 0.0001) relative increase of nonsulfated disaccharide (35.6% ± 5.7%) when compared with the nonsulfated disaccharide (10.0% ± 0.9%) present in the chondroitin sulfate of 27 healthy subjects. In 6 patients the structure of the excreted compound was analyzed up to 4 months after surgery. After removal of the cancer, the percent amounts of the nonsulfated disaccharide tend to approach the values found for the chondroitin sulfate of healthy subjects. A significant (P < 0.0001) change in the ratio of urinary chondroitin sulfate and heparan sulfate and a decrease in the electrophoretic migration of chondroitin sulfate were also observed. All of the patients with head and neck cancer analyzed so far have shown this structural anomaly of urinary chondroitin sulfate. This assay may be useful in the diagnosis and follow-up of cancer therapy. (Otolaryngol Head Neck Surg 2000;122:115–8.)

Hyaluronic acid concentration in postmenopausal facial skin after topical estradiol and genistein treatment: a double-blind, randomized clinical trial of efficacy.

ObjectiveThe aim of this work was to compare the effects of estradiol and genistein treatment on hyaluronic acid (HA) concentration in postmenopausal facial skin. MethodsIn this study, 30 postmenopausal women were evaluated in a prospective, randomized, double-blind trial. The volunteers were postmenopausal women treated in the Gynecology Department of the Federal University of São Paulo. The participants were divided into two groups: group E, treated with 0.01% 17&bgr;-estradiol gel (n = 15), and group G, treated with 4% genistein gel (isoflavones, n = 15). The treatment lasted for 24 consecutive weeks. Preauricular skin biopsies were performed for each participant at baseline (E1 and G1) and after treatment (E2 and G2) to evaluate HA concentration in tissue. The materials were processed using immunohistochemical and biochemical methods. ResultsAfter 24 weeks of treatment, HA concentration increased in both groups, but the effect was greater for estradiol treatment than for genistein treatment. ConclusionsOur data suggest that both treatments may enhance HA concentration in postmenopausal skin but that estrogen produces results that are greater than those produced by isoflavones.