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Objective: Low molecular mass hyaluronan causes inflammatory processes and can act as a pro-inflammatory cytokine in skin and other sites of activity in psoriatic arthritis (PsA). This study investigated whether the molecular mass distribution of hyaluronan (HA) in skin and the quantity of circulating HA are related to the clinical inflammatory picture in PsA with active disease and to the effect of treatment with anti-tumour necrosis factor-α (anti-TNF-α) adalimumab. Methods: Twenty patients with TNF-α-naïve active polyarticular PsA were included in this prospective clinical trial of treatment with 40 mg s.c. adalimumab according to standard procedure. Clinical activity, patients’ assessments, and skin biopsies were captured at inclusion and at the 12 week follow-up. Ten healthy individuals were recruited for comparison of HA analyses. Histochemistry of skin inflammation, serum HA, and molecular mass of HA were determined. Results: Overall improvements in clinical parameters were observed. Eight of 18 patients reached minimum disease activity after 12 weeks and disease activity was significantly reduced (p

To further elucidate the pathogenesis of systemic sclerosis (SSc) an experimental avian model was used. The University of California at Davis line 200 (UCD-200) chickens spontaneously develop a SSc-like disease that has most features of human SSc with vascular effects, inflammation, autoimmunity, and fibrosis. The first signs of disease in UCD-200 chickens are swelling and ischemic lesions of the comb and the presence of a tissue containing high amounts of glycosaminoglycan hyaluronan (HA). The aim of this study was to evaluate inflammatory and fibrotic processes of the disease with regard to the molecular weight of HA.Comb biopsies from UCD-200 and healthy White Leghorn (WL) chickens, as controls, at different ages were studied with the histochemical localization of HA, hyaluronidase-1 (Hyal-1), cluster of differentiation 3, immunoglobulin Y, and collagen I and III. The molecular weight distribution of HA was estimated with gas-phase electrophoretic analysis.At 2 days of age, HA was visualized in UCD-200 chickens at the dermal part of the comb with no simultaneous staining of Hyal-1. In adult UCD-200 chickens, the comb skin was almost totally devoid of HA compared to WL chickens of the same age. An increase of low molecular weight (LMW) HA was detected in comb tissue from UCD-200 at the age of 1 day, 1 week, 2 weeks, and 4 weeks, in contrast to adult animals.An early inflammatory process involving LMW HA was confirmed as a possible profibrotic process. This indicates that HA might be an important participant in the early inflammatory events of SSc in UCD-200 chickens and that the disappearance of HA in skin predisposes to fibrosis.

Scar tissues were collected from patients with keloids, hypertrophic scars and mature scars. Normal skin was obtained from healthy individuals. Clinical attributes were used to select which tissue to obtain but the distribution of the specific hyaluronan (HA) staining was then used for the definite classification of the various scar types. Light microscopic and ultrastructural analyses were performed with an HA-binding probe, antibodies for collagen I and III and staining for mast cells. Ultrastructural studies of keloids revealed an altered collagen structure in the dermal layers, with an abundance of collagen fibres of similar diameter in both the reticular dermis (RD) and the papillary dermis (PD) compared to normal skin. Furthermore, the keloids displayed epidermal changes, which involved the basement membrane (BM), with fewer hemidesmosomes and an altered shape of desmosomes in the entire enlarged spinous layer. The frequency of mast cells found in keloids was lower than in other scar tissues and normal skin. These alterations in epidermis could influence the hydrodynamic and cell regulatory properties of the wounded skin with impaired function and insufficient regulative capacity to hinder the ever-growing collagen tissue that is characteristic for keloids.

Introduction. The hypertrophic cardiomyopathy (HCM) disease process is not only limited to cardiomyocyte abnormalities but also engages the extracellular matrix. Hyaluronan (HA) and its receptor CD44 are involved in cellular growth and tissue proliferation but have so far been less studied in myocardial hypertrophy. In HCM, collagens are abundant but their histological distribution and relation to hyaluronan have not been described.Material and Methods. Myocardial specimens from 5 patients with symptomatic left ventricular tract obstruction undergoing myectomy due to HCM were processed for histochemistry and immunohistochemistry.Results. HA staining was more intense in HCM patients. The histological distribution of HA was the same in patients and controls, that is, interstitial staining including the space between cardiomyocytes, in fibrous septa, and in the adventitia of intramyocardial blood vessels. CD44 was not detected in the myocardium of patients or controls. Collagen I showed the same general localisation as HA but detailed distribution differed.Conclusions. This is the first study that describes the distribution of hyaluronan in human HCM. HA staining is more intense in HCM patients but without coexpression of its receptor CD44, at least not in the chronic phase of HCM. HA and collagen I have the same localisation.

Psoriatic arthritis is a chronic systemic disease in which patients develop persistent inflammation of the skin and joints, leading to disability and joint damage. The extracellular component hyaluronan (HA) plays an important role in regulatory processes such as inflammation, wound healing and tumour progression. At any site of inflammation HA can be depolymerized to low-molecular weight fragments, which, in turn, induce an array of inflammatory mediators that can lead to chronic inflammation. This study describes the serum concentration and dermal distribution of HA, its receptor CD44 and the metalloproteinases 3 and 9 in skin biopsies from patients with different types of psoriatic arthritis. Fifty-one patients with psoriatic arthritis were included in the study and classified as oligo- or poly-arthritic PsA with and without treatment. Biopsies were obtained from both involved and non-involved skin and compared with biopsies from healthy individuals. Serum HA was analysed for estimation of the total turnover of HA. The main findings were an overall redistribution of HA in both involved and non-involved psoriatic skin and an epidermal imbalance between HA and CD44. The structurally and functionally important basement membrane zone was found to be disintegrated and devoid of HA irrespective of the type of articular involvement, treatment or skin affection.

Objectives To perform a multicentre study to evaluate the performance of the colorimetric redox indicator (CRI) assay and to establish the MICs and critical concentrations of rifampicin, isoniazid, ofloxacin, kanamycin and capreomycin. Methods The study was carried out in two phases. Phase I determined the MIC of each drug. Phase II established critical concentrations for the five drugs tested by the CRI assay compared with the conventional proportion method. Results Phase I: a strain was considered resistant by the CRI assay if the MIC was >/=0.5 mg/L for rifampicin, >/=0.25 mg/L for isoniazid, >/=4.0 mg/L for ofloxacin and >/=5.0 mg/L for kanamycin and capreomycin. Sensitivity was 99.1% for isoniazid and 100% for the other drugs and specificity was 97.9% for capreomycin and 100% for the other drugs. Phase II: the critical concentration was 0.5 mg/L for rifampicin, 0.25 mg/L for isoniazid, 2.0 mg/L for ofloxacin and 2.5 mg/L for kanamycin and capreomycin giving an overall accuracy of 98.4%, 96.6%, 96.7%, 98.3% and 90%, respectively. Conclusions Results demonstrate that the CRI assay is an accurate method for the rapid detection of XDR Mycobacterium tuberculosis. The CRI assay is faster than the conventional drug susceptibility testing method using solid medium, has the same turnaround time as the BACTEC MGIT 960 system, but is less expensive, and could be an adequate method for low-income countries.

The role of hyaluronan in cardiac growth has become evident, previously shown by increased myocardial levels of hyaluronan in a rat model of cardiac hypertrophy.To further investigate the role of hyaluronan and regulation of its synthesis in cardiac hypertrophy, quantitative measurements of myocardial hyaluronan concentration was correlated to gene transcription in hypertrophic cardiac tissue. Factor analysis was used to study this correlation over time. A subset of differentially expressed genes was identified with a transcriptional regulation correlating to the increased synthesis of hyaluronan, suggesting a common regulatory pathway.Four transcription factors, Myc, Fos, Junb and Egr1, were also up-regulated. Furthermore, the Ace gene was up-regulated, representing increase of angiotensin II, an inducer of these transcription factors and fetal genes in cardiac hypertrophy. This demonstrates a coordinated synthesis of hyaluronan and pro-hypertrophic gene expression, regulated by immediate early genes, with angiotensin II as a possible mediator.

We report overprinting stable isotope evidence of fluid–rock interaction below two detachment faults along which mantle rocks were exhumed to the seafloor, between the respective landward and seawa ...

Tuberculosis (TB) is an ancient disease, but not a disease of the past. The increasing prevalence of drug-resistant strains of Mycobacterium tuberculosis, the causative agent of TB, demands new measures to combat the situation. Rapid and accurate detection of the pathogen, and its drug susceptibility pattern, is essential for timely initiation of treatment, and ultimately, control of the disease. Molecular-based methods offer a great chance to improve detection of drug-resistant TB; however, their development and usage should be accompanied with a profound understanding of drug resistance mechanisms and circulating M. tuberculosis strains in specific settings, as otherwise, the usefulness of such tests may be limited. This review gives an overview of the history of TB treatment and drug resistance, drug resistance mechanisms for the most commonly used drugs and molecular methods designed to detect drug-resistant strains.

Tuberculosis (TB) remains a global health problem. The increasing prevalence of drug-resistant Mycobacterium tuberculosis, the causative agent of TB, demands new measures to combat the situation. Rapid and accurate diagnosis of the pathogen and its drug susceptibility pattern is essential for timely initiation of optimal treatment, and, ultimately, control of the disease. We have developed a molecular method for detection of first- and second-line drug resistance in M. tuberculosis by Pyrosequencing(®). The method consists of seven Pyrosequencing assays for the detection of mutations in the genes or promoter regions, which are most commonly responsible for resistance to the drugs rifampicin, isoniazid, ethambutol, amikacin, kanamycin, capreomycin, and fluoroquinolones. The method was validated on clinical isolates and it was shown that the sensitivity and specificity of the method were comparable to those of Sanger sequencing. In the protocol in this chapter we describe the steps necessary for setting up and performing Pyrosequencing for M. tuberculosis. The first part of the protocol describes the assay development and the second part of the protocol describes utilization of the method.

To study the hyaluronan occurrence in human vocal folds, with special regards to gender and smoking and to discuss the implications of findings.This is a descriptive/morphologic study.Sixteen cadaveric vocal folds from eight individuals between 58 and 90 years old (six women and two men) were removed and studied morphologically. Three of the individuals had been cigarette smokers. A direct method for hyaluronan histochemistry using a hyaluronan-binding protein probe (HABP) was used to visualize the polysaccharide. Five examiners performed an analysis of the intensities of hyaluronan staining, independently.We observed intense hyaluronan staining of the vocal folds of which those from women stained considerably stronger than those from men. Stratified squamous epithelium stained for hyaluronan in all sections, whereas respiratory epithelium only stained weakly or not at all. The highest accumulation of hyaluronan occurred subepithelially in the lamina propria, corresponding to Reinke's space. It was observed that vocal folds from smokers were more intensively stained than those from nonsmokers.Hyaluronan is found in all layers of the human vocal fold. Contradictory to earlier studies, hyaluronan was visualized in squamous epithelium, where it may function as an impact protector. The occurrence of hyaluronan in smokers may have implications in the development of vocal fold inflammation and tumor initiation as hyaluronan is an important molecule in these processes.

Tuberculosis (TB) remains a global health problem. The increasing prevalence of drug-resistant Mycobacterium tuberculosis, the causative agent of TB, demands new measures to combat the situation. Rapid and accurate diagnosis of the pathogen and its drug susceptibility pattern is essential for timely initiation of optimal treatment, and, ultimately, control of the disease. We have developed a molecular method for detection of first- and second-line drug resistance in M. tuberculosis by Pyrosequencing(®). The method consists of seven Pyrosequencing assays for the detection of mutations in the genes or promoter regions, which are most commonly responsible for resistance to the drugs rifampicin, isoniazid, ethambutol, amikacin, kanamycin, capreomycin, and fluoroquinolones. The method was validated on clinical isolates and it was shown that the sensitivity and specificity of the method were comparable to those of Sanger sequencing. In the protocol in this chapter we describe the steps necessary for setting up and performing Pyrosequencing for M. tuberculosis. The first part of the protocol describes the assay development and the second part of the protocol describes utilization of the method.

The extracellular matrix is a dynamic space and a prerequisite for the function of cardiomyocytes. We have previously reported on the distribution of the glycosaminoglycan hyaluronan (HYA) and its cellular receptor CD44 in the vascular system. In newborn rats, HYA and its receptor were colocalized, but in the adult animals, no such colocalization was observed. Furthermore, the distribution of both HYA and CD44 differed between newborn and adult animals. In this study, the distribution of HYA and its receptor CD44 is explored in the heart. Hearts from newborn and adult rats were stained for visualization of HYA and CD44 using histochemistry and immunohistochemistry. HYA stained the interstitium of the myocardium heterogeneously. Strong staining was seen in the heart valves of both newborn and adult animals. CD44 staining was sparse in hearts from both newborn and adult animals. There are no major changes in the distribution of HYA in the myocardium during the postnatal development in contrast to the blood vessels. Thus, the structure of the interstitium does not change after birth when the heart starts to grow mainly through cardiomyocyte hypertrophy rather than hyperplasia. The abundance of HYA in the heart valves is probably related to their unique physiological properties to withstand repetitive mechanical stress. Anat Rec Part A 288A:587–592, 2006. © 2006 Wiley-Liss, Inc.

Acute myeloid leukemia (AML) is a clonal disorder characterized by abnormal proliferation of myeloid cell precursors. Research has mainly focused on the cellular events, but the bone marrow matrix has attracted minor interest. In this study bone marrow biopsies were obtained from 35 newly diagnosed AML patients. The bone marrows were analyzed regarding the occurrence and distribution of hyaluronan (HYA) and reticulin fibers (type III collagen). The bone marrow sections were analyzed histochemically and compared with bone marrows from 30 healthy controls. The HYA staining was significantly stronger in the AML patients compared with the controls. Only one patient demonstrated abnormal reticulin staining score, but in the group of patients with antecedent myelodysplastic syndrome (MDS), the reticulin staining score was significantly higher compared with the patients with de novo AML. There was a significant correlation between the HYA staining and reticulin staining scores in the AML patients as was seen in the control group.

Specimens of basal cell carcinomas collected from 28 patients were classified into three groups: superficial, nodular, and infiltrative, according to their microarchitecture. The specimens were then subjected to histological characterization by means of a biotinylated hyaluronan-binding probe (HABP). By using Ki-67 and PCNA the proliferative activity of the BCC tumours was evaluated with immunohistological techniques. In superficial BCC the tumour islands displayed moderate hyaluronan (HA) staining. Feeble proliferation, denoted by modest mitotic activity and weak Ki-67 and PCNA immunoreactivity, occurred within the tumour islands. The surrounding connective tissue resembled normal skin, and no differentiated tumour stroma was observed. In nodular BCC, the HA staining of the tumour strands was weak to moderate, denoting increased proliferative activity. The differentiated surrounding tumour stroma stained strongly for HA. Tumour islands of infiltrative BCC stained weakly to moderately to HA and evidenced intense proliferation. The intensely HA-stained tumour stroma ended abruptly and the adjacent areas were almost devoid of HA. This study showed that the proliferative activity of BCC cells is associated with increased expression of HA in the tumour stroma. Modification of tumour-associated connective tissue indicates a close relationship between the tumour cells and the adjacent matrix. In particular, in infiltrative BCC, such alterations include degeneration and possible modification and remodelling of the surrounding extracellular matrix. These processes involving areas of probable importance for tumour progression, should be considered when deciding the extent of intended surgical resection.

Bone marrow trephine biopsies from 30 healthy volunteers, 10 men and 20 women aged 18-60 yr were obtained for identification and localisation of hyaluronan (HYA). Fixation, decalcification and embedding were performed by two different methods, with identical results in both. For comparison bone marrow trephine biopsies from three patients with different haematological diseases and known fibrosis were studied. All bone marrow specimens were also stained for reticulin grading. HYA was found in the bone marrow specimens from healthy individuals in a pattern that was concordant with the reticulin staining, the common way of visualising bone marrow fibrosis. In bone marrow from the patients with known fibrosis the HYA and reticulin staining were both more intense and abundant. Interestingly, HYA was also found intracellularly in eosinophilic cells in normal bone marrow. HYA is a polysaccharide unique both in structural and biological properties, and in excess it may predict bone marrow fibrosis.

Control of the global epidemic tuberculosis is severely hampered by the emergence of drug-resistant Mycobacterium tuberculosis strains. Molecular methods offer a more rapid means of characterizing resistant strains than phenotypic drug susceptibility testing. We have developed a molecular method for detection of rifampicin-resistant M. tuberculosis based on padlock probes and magnetic nanobeads. Padlock probes were designed to target the most common mutations associated with rifampicin resistance in M. tuberculosis, i.e. at codons 516, 526 and 531 in the gene rpoB. For detection of the wild type sequence at all three codons simultaneously, a padlock probe and two gap-fill oligonucleotides were used in a novel assay configuration, requiring three ligation events for circularization. The assay also includes a probe for identification of the M. tuberculosis complex. Circularized probes were amplified by rolling circle amplification. Amplification products were coupled to oligonucleotide-conjugated magnetic nanobeads and detected by measuring the frequency-dependent magnetic response of the beads using a portable AC susceptometer.

The histochemical distribution of hyaluronan was analysed in various urogenital organs of male and female (non-pregnant and pregnant) rats by use of a hyaluronan-binding protein and avidin biotin/peroxidase staining. Microwave-aided fixation was used to preserve the extracellular location of hyaluronan. The concentrations of hyaluronan in the different tissues were measured with a highly sensitive radio-assay. Hyaluronan accumulated predominantly in the connective tissue around smooth muscle fibres and in the subepithelial lamina propria. Abundant hyaluronan also occurred in perivascular and perineural connective tissue. In the female urogenital organs, hyaluronan content was high in the vagina and urinary bladder, and highest in the vagina during pregnancy. In the uterus, the surface epithelium of the endometrium stained intensely. In the ovary, the zona pellucida of the oocyte and the theca interna cell layer of the follicles and the follicular fluid of mature follicles exhibited prominent staining. The corpus luteum was devoid of hyaluronan, whereas enlarged corpora lutea of pregnancy exhibited weak, patchy staining. In male urogenital organs, staining for hyaluronan was absent from the testis and epididymis, whereas the erectile connective tissue of the penis stained intensely. The hyaluronan concentrations were high in penile tissue and urinary bladder, while testis, epididymis and the ductus deferens contained only little hyaluronan.

ckground : The health burden related to obesity is rising among children and adolescents along with the general population worldwide. For the individual as well as the society this trend is alarming. Several factors are driving the trend, and the solution seems to be multifaceted because long-lasting treatment alternatives are lacking. This study aims to explore adolescents’ and young adults’ motivation for attending group-based obesity treatment and social and environmental factors that can facilitate or hinder lifestyle change. Methods : In this study, we arranged three focus groups with 17 participants from different obesity treatment programs in the west and south of Norway. The content in these programs differed, but they all used Motivational Interviewing as a teaching method. We conducted a data-driven analysis using systematic text condensation. Self-determination theory has been used as an explanatory framework. Results : We identified four major themes: 1) motivation, 2) body experience and self-image, 3) relationships and sense of belonging, and 4) the road ahead . Many of the participants expressed external motivation to participate but experienced increasing inner motivation and enjoyment during the treatment. Several participants reported negative experiences related to being obese and appreciated group affiliation and sharing experiences with other participants. Conclusion : Motivation may shift during a lifestyle course. Facilitating factors include achieving and experiencing positive outcomes as well as gaining autonomy support from other course participants and friends. Obstacles to change were a widespread obesogenic environment as well as feelings of guilt, little trust in personal achievements and non-supporting friends. Key words: Weight management, motivation, self-determination theory, adolescents, lifestyle (Published: 9 November 2016) Citation: Int J Qualitative Stud Health Well-being 2016, 11: 32744 - http://dx.doi.org/10.3402/qhw.v11.32744

Objective. To investigate the production of type II collagen (CII) antibodies in the synovial fluid (SF) of rheumatoid arthritis (RA) patients, and to examine the HLA dependence of this local production. Methods. The ELISPOT method was used for enumerating anti-CII—reactive cells. Serologic tissue typing was performed. Results. Anti-CII—reactive cells were found in the SF of 16 of 31 patients, but not in any of the peripheral blood samples obtained in parallel. SF anti-CII antibody production showed no correlation with clinical parameters, but its frequency increased significantly with age. The IgG anti-CII response occurred exclusively in patients who were positive for HLA—DR4 and was significantly associated with DR4. Conclusion. Anti-CII production may be important in local immune complex formation. The indirect demonstration of a DR4-restricted T cell response to CII is an indication of a pathogenetic role of collagen autoimmunity in RA.

Our objective was to investigate whether the presence of a tumor increases hyaluronan (HA) levels in surrounding prostate tissues and whether this extratumoral HA influences tumor growth and outcome. From a series of 287 men diagnosed with prostate cancer at transurethral resection and followed up with watchful waiting, tissue microarrays were constructed, stained, and scored for HA. A high HA staining score in the tumor stroma or in nonmalignant prostate tissue stroma were both associated positively with higher Gleason score and larger tumor volume, and was associated with a poor outcome. HA staining score was not an independent marker for outcome (multivariate Cox, with Gleason score, tumor volume, stage, and HA variables). In an orthotopic rat prostate cancer model, hyaluronic acid synthase-1 mRNA levels and HA staining were increased in normal prostate tissue surrounding prostate cancer. Orthotopic prostate cancer growth was increased by intraprostatic injection of HA. In conclusion, cancer in the prostate apparently stimulates HA synthesis both in tumor stroma and in the surrounding normal tissue. This promoted tumor growth and was associated with an unfavorable outcome.

Myelofibrosis is commonly seen in patients with chronic myeloproliferative diseases and sometimes in myelodysplastic syndrome, acute leukaemia and lymphoproliferative diseases. The fibrotic process is evaluated by grading the amount of collagen deposited in the bone marrow interstitium. The established method to evaluate bone marrow fibrosis is staining for reticulin to visualise the collagen fibres. However, the extra cellular matrix does not only contain collagens but also other components, e.g. glycosaminoglycans of which hyaluronan is the most abundant. Hyaluronan is important for structural and cellular functions. Earlier studies have shown that there is a positive correlation between hyaluronan and reticulin staining in healthy volunteers and in patients with de novo acute myeloid leukaemia. In this study bone marrow biopsies from 43 patients with a malignant disease involving the bone marrow were compared with 18 patients with a malignant disease not involving the bone marrow. The intensity of hyaluronan grading was significantly higher in the patients with disease involving the bone marrow compared to the healthy controls but not compared to the patients without disease involving the bone marrow. The staining intensity of reticulin in the bone marrow was significantly higher in the patients with disease involving the bone marrow, compared to those without disease involving the bone marrow and to the controls. In all patients and the controls there was a correlation between hyaluronan and reticulin.

Proteins which specifically bind hyaluronan have been used to develop sensitive immunoassay-like analytical techniques. These assays have made it possible to analyse hyaluronan concentrations in nanogram amounts in tissues and body fluids. The normal adult human circulating level of hyaluronan varies between 10 and 100 micrograms/l. Increased levels of serum hyaluronan (greater than 1 mg/l) have been found in inflammatory rheumatic diseases such as rheumatoid arthritis, scleroderma and psoriatic arthritis. Even higher levels occur in serum from patients with cirrhotic liver diseases and in patients with malignant mesothelioma. Elevated serum hyaluronan levels are a sign either of increased production and/or outflow from the tissues or of decreased elimination of the polysaccharide in the liver endothelial cells or in other parts of the reticuloendothelial system or through the kidneys. The serum concentration is also influenced by increased age and by the degree of physical activity. Important information on pathological processes has been gained by analysing the hyaluronan concentration in other body fluids such as urine, perfusion fluid from the intestine and bronchoalveolar lavage fluid. In the future these analyses should also be useful in clinical work.

The importance of glycosaminoglycan hyaluronan (HA) and its receptor CD44 in cell proliferation is becoming increasingly evident. Expression of the genes coding for hyaluronan synthase 1 (HAS1), HAS2, HAS3, CD44, fibroblast growth factor-2 (FGF-2), and FGF receptor-1 (FGFR-1) and the histological evidence for increases of HA and CD44 were investigated in an experimental rat model of cardiac hypertrophy. The abdominal aorta was ligated to induce cardiac hypertrophy, and mRNAs prepared from heart tissue were analyzed after 1, 6, and 42 days. The total concentration of HA was quantified, and HA and CD44 were studied histochemically. The expression of HAS1, HAS2, CD44, and FGF-2 was considerably up-regulated at days 1 and 6 and returned to basal levels after 42 days. FGFR-1 was up-regulated at day 1 but at basal levels once more at days 6 and 42. The concentration of HA significantly increased in aorta-ligated rats. Histochemical analysis showed increased expression of CD44 in hypertrophied myocardium mainly in and around the coronary arteries. These results agree well with other studies of tissue growth (malignancies and wound healing). The increase of HA, its synthases, and receptor in parallel with FGF-2 and its receptor illustrates their complicated interplay in the development of cardiac hypertrophy. The up-regulation of both HAS1 and HAS2 indicates the importance of HA production in the hypertrophic process and the possibility that HA is needed for two different purposes for the heart to be able to adapt to the increased afterload caused by aortic ligature.

Anagrelide is a second-line option for reduction of thrombocythemia in patients with chronic myeloproliferative disorders (CMPDs). A multicenter, open, phase II study of anagrelide treatment in 60 patients during 2 yr was performed by the Swedish Myeloproliferative Disorder Study Group. Adequate bone marrow biopsies were obtained from 53 of the CMPD patients [36 essential thrombocythemia (ET), 16 polycythemia vera (PV), 1 chronic idiopathic myelofibrosis (CIMF)] before treatment and compared with biopsies from 30 healthy volunteers and 34 patients with acute myeloid leukemia (AML). Higher reticulin and hyaluronan (HYA) scores were found before anagrelide therapy in the CMPD patients than in the normal controls (p0.001 and p0.001, respectively) and AML patients (p0.001 and p = 0.011, respectively). At the end of the study 30 CMPD patients were still on anagrelide treatment and in 19 of these patients, all diagnosed as ET (n = 16) or PV (n = 3), pretreatment bone marrow biopsies were compared with follow-up samples. After 2 yr of anagrelide therapy the reticulin and HYA scores were significantly higher than before treatment (p = 0.02 and p = 0.002, respectively). The cellularity was significantly higher (p = 0.014), although the number of megakaryocytes did not change significantly. The increase of reticulin and HYA in the bone marrow after 2 yr of treatment with anagrelide indicated progression of fibrosis. Although anagrelide is a valuable drug for reduction of platelet levels, it seems unable to stop progression of bone marrow fibrosis and hypercellularity in ET and PV.

The CD44 receptor is a transmembrane glycoprotein expressed on a variety of cells like endothelial, epithelial and smooth muscle cells. This molecule has many important functions, e.g. in cell-cell and cell-matrix interactions and signal transduction. The main ligand for CD44 is hyaluronan (HYA). HYA is a glycosaminoglycan with structural and cell biological properties. The localization of HYA in the vessel wall of arteries and veins in the healthy adult and newborn rat has been described earlier. In this study the occurrence of the CD44 receptor was investigated in the same vessels and compared to the localization of HYA. Both CD44 and its ligand showed an increased expression in the vessel wall of newborn rats compared to that of adult rats. Although HYA is abundant in the adventitia of adult rats, virtually no expression of CD44 was observed. Our results indicate that the CD44 receptor expression is increased during the stage of maturation of the vessel tree whereas the CD44 receptor is less needed by HYA in the healthy vessel wall.

The major site for elimination of HA from the bloodstream is via receptor mediated endocytosis by liver endothelial cells (LEC) (Fraser and Laurent 1989, Laurent and Fraser 1992). The HA receptor (HAR) on LEC has been characterized and isolated from rat LEC membranes (Forsberg and Gustafson 1991). A monospecific polyclonal antibody against the 90–100 kD receptor, which could inhibit binding of HA to LEC and LEC membranes, (termed anti-HARLEC) was raised (Forsberg and Gustafson 1991). In immunohistochemical studies the antibody stains mainly endothelium such as the sinusoids in the liver, spleen and lymph nodes, some capillaries in kidney and corneal endothelial cells (Gustafson et al. 1994, Forsberg et al. 1994). Tryptic peptides of the receptor were sequenced and found to be identical to intercellular adhesion molecule-1 (ICAM-1) (McCourt et al. 1994). ICAM-1 is normally expressed at low levels, but has been found on normal liver endothelium at the sinusoids and on the endothelium of lymph nodes, spleen ...

The matrix components of the vessel wall are of great importance for the function of the vessel system of which both endurance and elasticity are prerequisites. One component of the vessel wall is the glycosaminoglycan hyaluronan (HYA) with its unique physico-chemical properties, e.g. viscoelasticity and barrier function. The present study aimed to map and compare the normal localisation and distribution of hyaluronan in the arteries and veins of both adult and newborn rats, using a specific staining method utilizing a hyaluronan-binding protein. The hyaluronan stained clearly different in veins and arteries both in newborn and adult rats. In the veins, the tunica intima stained intensely whereas the corresponding area in the arteries only showed a weak and scattered staining pattern. In the adult rats, the matrix between the smooth muscle cells of the tunica media of the veins had a clearly positive staining pattern compared to the media in the arteries which showed only a few scattered areas of positive staining. In the newborn rats, the media of the arteries stained more intensely. In both newborn and adult rats, the adventitia stained intensely both in veins and arteries. Moreover, the HYA-staining pattern differed in veins in different regions of the body. Since HYA is known to be involved in many different biological processes, the results are of importance to understand physiological properties of the vessel tree and to explain the development of vascular diseases.

Glycosaminoglycans are important components of all extracellular matrices. One of the glycosaminoglycans is hyaluronan, which is ubiquitously distributed throughout the connective tissue. Hyaluronan is especially abundant in the skin, in which it is of both structural and functional importance. This study describes the localization and distribution of hyaluronan in the skin of healthy individuals and of 23 patients with insulin-dependent diabetes mellitus and various degrees of limited joint mobility. In normal skin, hyaluronan staining was seen in all layers but most prominently in the papillary dermis and the basement membrane zone. In the skin from diabetic patients with normal or only moderately restricted mobility of the hands (limited joint mobility grades 0 and 1), the distribution of hyaluronan was similar to that of normal skin. In the skin of patients with severe restriction in joint mobility (limited joint mobility grade 2) the staining pattern was significantly different with weak hyaluronan staining in the papillary dermis and the basement membrane zone almost devoid of hyaluronan. Moreover, an increased epidermal thickness in the latter patients was evident as well as a pronounced hyaluronan staining compared with normal epidermis.

Bone marrow trephine biopsies from 30 healthy volunteers, 10 men and 20 women aged 18-60 yr were obtained for identification and localisation of hyaluronan (HYA). Fixation, decalcification and embedding were performed by two different methods, with identical results in both. For comparison bone marrow trephine biopsies from three patients with different haematological diseases and known fibrosis were studied. All bone marrow specimens were also stained for reticulin grading. HYA was found in the bone marrow specimens from healthy individuals in a pattern that was concordant with the reticulin staining, the common way of visualising bone marrow fibrosis. In bone marrow from the patients with known fibrosis the HYA and reticulin staining were both more intense and abundant. Interestingly, HYA was also found intracellularly in eosinophilic cells in normal bone marrow. HYA is a polysaccharide unique both in structural and biological properties, and in excess it may predict bone marrow fibrosis.

Hyaluronan is a major component of synovial fluid. It is synthesized in the joint and partly degraded in joint capsule and partly carried by lymph to lymph nodes and the general circulation. During various joint disorders, especially inflammatory ones, the amount of hyaluronan in the joint and its turnover rate are increased leading to pathologically high serum levels of the polysaccharide. These levels are a complex function of synovitis mass and the physical activity of the patient. Intra-articular injection of hyaluronan has been used in the treatment of osteoarthritis. Although it results in a relief of pain, controlled clinical trials have not yet convincingly shown any beneficiary effects of the treatment.

Hyaluronan (HA) and aminoterminal propeptide of type III procollagen (PIIINP), two biochemical connective tissue markers, were determined in 76 patients with primary biliary cirrhosis (PBC). The HA and PIIINP concentrations were significantly increased compared with controls (P less than 0.001). Both HA and PIIINP levels correlated significantly with conventional liver-function tests. All patients with stage IV PBC showed increased concentrations of both these variables. However, HA was a better marker with regard to prediction of development of cirrhosis as well as prediction of symptoms. Furthermore, HA also showed a negative correlation with time of survival (P less than 0.05). The present data indicate that HA is a more sensitive marker of liver damage in PBC than PIIINP.

Catabolism of hyaluronan was studied by injecting hyaluronan labelled with [125I]-tyramine cellobiose ([125I]-TC) into knee joints of rabbits. After endocytosis [125I]-TC remains intracellularly allowing localization of the site of catabolism. At 6 hours after injection 63% could be recovered in and around the joint, while at 48 hours 32% remained locally. Chromatography showed that 12% of the injected tracer was degraded in joint tissues at 6 hours, increasing to 33% at 24 hours. There was no apparent degradation within the joint fluid. No tracer was found in the regional lymph glands, but 16% of the injected tracer was detected in the liver at 24 hours. This investigation demonstrates that hyaluronan in the joint can be degraded both locally and in the liver.

It has been suggested that a high level of hyaluronan (hyaluronic acid, HYA) in pleural fluid is an indicator of malignant mesothelioma. In 78 consecutive patients with pleural effusion of various causes the HYA concentration was measured in pleural fluid samples and in serum. Nine patients had malignant pleural mesothelioma, and in three of them the HYA level in pleural fluid was 100 mg/l or more. In 42 patients with effusions due to metastatic malignancy, the mean HYA in the pleural fluid was 75 mg/l, and in five the HYA level was above 100 mg/l. Cardiac insufficiency caused the effusion in 11 patients, of whom two had a level above 100 mg/l in pleural fluid. Four patients had a serologically confirmed viral infection and had HYA levels in pleural fluid of 8, 157, 335, and 554 mg/l, respectively. One patient had postinfectious effusion with an HYA level in pleural exudate of 748 mg/l, the highest in this investigation. Two patients had benign asbestos pleural effusions, and both had high pleural HYA levels (256 and 490 mg/l, respectively). The serum HYA values were much lower than in the pleural fluid, namely from 15 to 480 micrograms/l; the levels were independent of the levels in the pleural fluid. Thus, a high level of HYA in pleural fluid is not specific for mesothelioma but can occur in other malignant or benign diseases, and a low level does not exclude mesothelioma.

The histochemical distribution of hyaluronan (hyaluronic acid, HYA) was analysed in various types of muscles in the rat by use of a hyaluronan-binding protein (HABP) and the avidin-biotin/peroxidase complex staining procedure. Microwave-aided fixation was used to retain the extracellular location of the glycosaminoglycan. In skeletal muscles, HYA was detected in the connective tissue sheath surrounding the muscles (epimysium), in the septa subdividing the muscle fibre bundles (perimysium) and in the connective tissue surrounding each muscle fibre (endomysium). HYA was heterogeneously distributed in all striated muscles. In skeletal muscles with small fibre dimensions (e.g., the lateral rectus muscle of the eye and the middle ear muscles), HYA was predominantly accumulated around the individual muscle fibres. Perivascular and perineural connective tissue formations were distinctly HYA-positive. In cardiac muscles, HYA was randomly distributed around the branching and interconnecting muscle fibres. In comparison, smooth muscle tissue was devoid of HYA.

In the connective tissues the two major structural components are collagens and glycosaminoglycans (GAGs). When assessing pathological processes in the extracellular matrix, e.g. fibrosis, attention is primarily directed to the collagens. Hyaluronan (HYA) is a GAG of great interest not only as a tentative marker of early fibrosis but also of importance for the disease process in various malignancies. Acute myeloid leukaemia (AML) is a clonal disorder characterised by abnormal proliferation of myeloid cell precursors. Research has mainly focused on the cellular events but the bone marrow matrix has attracted minor interest. In this study bone marrow biopsies were obtained from 35 newly diagnosed AML patients and 30 healthy individuals. The bone marrow sections were analysed histochemically for HYA and reticulin (type III collagen). The HYA staining was significantly stronger, p=0.003, in the AML patients compared to the healthy controls. Only one patient demonstrated abnormal reticulin staining score, but in the group of 8 patients with antecedent myelodysplastic syndrome (MDS), the reticulin staining score was significantly higher, p=0.003, compared to the patients with de novo AML. There was a significant correlation between the HYA and reticulin staining scores, p=0.005, in the AML patients as was seen in the control group, p=0.0005, indicating that the variables HYA and reticulin are not independent. No correlation was found between HYA grading and lactate dehydrogenase (LDH), number of blast cells, sex, age or antecedent blood disease before AML diagnosis. In the control group there was a significant correlation, p=0.042, between HYA and age. Our findings establish that there is a deposition of HYA in bone marrow in healthy individuals and that HYA is increased in AML patients. It is discussed whether HYA could be an early marker of impending fibrosis and also exert important effects on cellular events in the disease process.

SummaryMiddle-ear effusions of the mucoid type were collected from 14 children with secretory otitis media and analysed by a sensitive assay for hyaluronic acid. The concentration of the polysaccharide varied between three and 42 μg/g. in different patients but was in the same range in effusion material taken from the two ears of the same patient. As the hyaluronic acid concentrations were high compared to the serum levels, the results indicate that the effusion material contains a component derived from connective tissue.

The removal of hyaluronic acid (HA) from the blood-stream was studied in four normal human subjects after injection of high molecular weight preparations labelled with 3H in the acetyl position. The plasma half-life of the injected material ranged between 2.5 and 5.5 min. The daily turnover of HA in the circulation was estimated to be at least 150 mg. Its elimination was predominantly extrarenal, the upper molecular weight limit for renal excretion being 25 000. Evidence for rapid degradation was provided by the identification of 3H2O in urine. Calculations from the specific activity of urinary 3H2O indicated that approximately 55% of the acetyl content of the injected HA was completely oxidized within 3 h, and 85% within the first day. It is concluded that hyaluronic acid in the amounts currently used for therapeutic purposes should not accumulate significantly in the circulation.

SUMMARY Hyaluronate concentrations were measured in suction blister fluid from blisters raised on the abdominal skin of 12 healthy control subjects, on unaffected skin of 14 patients with psoriasis, and on lesional skin from 12 of these patients. The concentrations of hyaluronate in blister fluid from the controls and from the uninvolved skin of patients with psoriasis were within the same range (2.9–10.5 and 2.4–7.8 mg/1, respectively). Six of the patients had active, widespread, untreated psoriasis and in these cases the hyaluronate concentration in blister fluid from lesional skin was greatly increased (24–30 mg/1). Patients with stable or regressing psoriasis had no significant increase in hyaluronate levels in the blister fluid from lesional skin compared with the controls (range 4.6–12.4 mg/1). Patients with active psoriasis had significantly higher serum hyaluronate concentrations than the controls and those with inactive psoriasis.

• Patients (n = 10) at the acute phase of farmer's lung were investigated with chest roentgenography, lung function tests, and bronchoalveolar lavage (BAL) fluid analysis (n = 9). They had diffuse interstitial lung infiltrates and a reduction of the diffusion capacity. The dominating recovered cell types during BAL were lymphocytes; and in two patients, granulocytes. A prominent increase in mast cell numbers was seen in all patients. After avoidance of contact with moldy plant material for four to ten weeks (n = 7), lung function started to improve; and the BAL cell counts, to decrease. At clinical remission six to 14 months later (n = 7), the chest roentgenogram was normal and the diffusion capacity was slightly subnormal. The BAL numbers of mast cells and lymphocytes had further decreased but still remained increased compared with those in the healthy controls. Parallel to the normalization of the lung function and the recovery of BAL fluid cells, the increased BAL fluid concentrations of hyaluronic acid and procollagen III peptide started to decrease. These potential markers of fibroblast activation were significantly related to the mast cell number, but not to the lymphocyte number. The study has demonstrated that pulmonary mastocytosis is a prominent finding in farmer's lung and is related to the disease activity. The observed relationship between pulmonary mastocytosis and biochemical signs of lung fibroblast activation is further evidence to support the hypothesis of a mast cell interaction with lung connective tissue. ( Arch Intern Med 1988;148:1362-1365)