Multicenter study of QuantiFERON®-TB Gold Plus in patients with active tuberculosis.

SETTING: QuantiFERON®-TB Gold Plus (QFT-Plus), recently approved for use in the United States, is a new-generation QuantiFERON assay that differs from its predecessors in that it uses an additional antigen tube containing peptides to elicit both CD8+ and CD4+ T-lymphocyte responses. OBJECTIVE: To assess the sensitivity of QFT-Plus compared with QuantiFERON®-TB Gold In-Tube (QFT-GIT) in participants with active TB. DESIGN: Adult patients with active TB at three US and two Japanese sites were eligible for this study if they had culture-confirmed TB and were either untreated or had received 14 days of anti-tuberculosis treatment. RESULTS: We enrolled 164 participants, nine of whom had indeterminate results. Excluding indeterminate values, there were 150 QFT-GIT-positive results among 159 tests and 146 QFT-Plus-positive results among 157 tests, with sensitivities of respectively 94.3% (95%CI 89.5-97.4) and 93.02% (95%CI 87.8-96.5%). The estimated sensitivities for the two tests were not significantly different (P = 0.16). Overall test agreement was 98.7%, with a κ statistic of 0.89 (95%CI 0.75-1.00). CONCLUSION: In this multisite study, we found that QFT-Plus had similar sensitivity to QFT-GIT in adult patients with active TB.

Mannose-binding lectin (MBL2) polymorphisms and inflammation in hypertensive patients.

We investigated the possible role of Mannose binding lectin 2 (MBL2) functional polymorphisms in the prevalence of hypertension and hypertensive end-organ damage in 300 hypertensive patients and 313 normotensive individuals from Southern Brazil. Hypertensive subjects with MBL2 AO/OO genotypes presented lower C-reactive protein levels than AA individuals and consequently lower inflammatory status.

A torque removal study on the primary stability of orthodontic titanium screw mini-implants in the cortical bone of dog femurs.

The aim of this study was to biomechanically evaluate the primary stability of pure titanium orthodontic mini-implants, inserted into pre-drilled cavities of differing diameters. Mini-implants (1.2 mm diameter) were placed into 1.0 mm and 1.2 mm diameter cavities prepared in the mid-region of the bilateral hind leg femurs of anesthetized beagles. Removal torque strengths were measured immediately, 1, 3, 6, 9 and 12 weeks post-insertion of the implant. For mini-implants placed into 1-mm cavities, removal torque values decrease over the first 6 weeks (p<0.01), after which values remained static. Average values obtained immediately, 1, 3 and 6 weeks post-insertion were 10.98, 8.83, 7.20 and 5.12 Ncm, respectively . Immediately post-insertion, removal torque values of mini-implants placed in a 1.2-mm cavity, were 11-fold lower than those placed in 1.0-mm cavities, which then demonstrated a significant increase in strength from 3 weeks (1.35 Ncm) to 6 weeks (5.17 Ncm) post-insertion (p<0.01). Measurements 6, 9 and 12 weeks post-insertion were similar to those in the 1.0-mm cavity. Initial stability of titanium mini-implants is considered necessary for immediate and early use in orthodontics, and an implant without this initial stability should be replaced or isolated until it develops the appropriate stability supported by osseointegration.

CD30+ large cell transformation of mycosis fungoides after psoralen plus ultraviolet A photochemotherapy.

Psoralen plus ultraviolet A (PUVA) photochemotherapy is widely used for the therapy of mycosis fungoides (MF). Clinical progression of MF is often associated with an increase in the size of tumour cells known as transformation. We report two patients with CD30+ large cell transformation that appeared after low-dose PUVA therapy for MF. Clinical data, histopathology, immunohistopathology and T-cell receptor gene rearrangement were studied. Nodules consisted of atypical large cells that expressed CD30. Monoclonal rearrangement of T-cell receptors was observed in one case. Low-dose PUVA therapy may be associated with CD30+ large cell transformation in patients with MF.

Idiopathic acquired generalized anhidrosis due to occlusion of proximal coiled ducts.

Idiopathic acquired generalized anhidrosis is a very rare disease of unknown pathogenesis. We report a 25-year-old man with acquired generalized anhidrosis due to occlusion of the coiled ducts. He did not have sweat secretion over the entire surface of the body, including the palms and soles. Sweat-inducing stimuli provoked tingling pain on the skin. Pilocarpine iontophoresis on the forearm did not induce sweat secretion. Neurological examination did not reveal any abnormality in the central or peripheral nervous system. Skin biopsy showed that the coiled ducts were occluded by an amorphous eosinophilic substance. This amorphous eosinophilic substance was positive with periodic acid-Schiff (PAS) staining and was resistant to digestion by diastase. Electron microscopy demonstrated that the coiled ducts were completely occluded by an amorphous substance. The substance occluding the coiled ducts contained fibrous structures. These findings suggested that the acquired generalized anhidrosis in this patient was caused by occlusion of the coiled ducts by a PAS-positive substance probably derived from dark cell granules.

Localized lymphomatoid papulosis.

A 50-year-old Japanese male visited our clinic in April 1999 with a 2-year history of self-healing, reddish papules on his right palm. On examination, there were grouped erythematous papules, 2-4 mm in size, which formed a relatively well-circumscribed erythematous plaque. A biopsy specimen showed a wedge-shaped, dense dermal infiltrate consisting of variously sized mononuclear lymphoid cells mixed with few large CD30-positive cells and inflammatory cells, suggesting the diagnosis of regional lymphomatoid papulosis (LyP). Analysis of the T cell receptor gene revealed a polyclonal pattern on lesional skin. Only 5 cases of LyP presenting in a regional distribution have been reported previously. Although the etiology of localized LyP remains unknown, considering that 2 of 5 reported patients developed widespread lesions regional LyP may be the initial presentation of typical LyP.

Rapid RNA cleavage using an antisense system with two terpyridine.Cu(II) complexes.

An effective approach to promote sequence-specific RNA cleavage by an antisense 2'-O-methyloligonucleotide with a terpyridine.Cu(II) complex attached at the 5'-end was developed. We have synthesized a Cu(II) complex 3'-conjugate, which when used in a tandem fashion, greatly enhanced the RNA cleavage efficiency.

Lysophosphatidic acid positively regulates the fluid flow-induced local Ca(2+) influx in bovine aortic endothelial cells.

Using real-time confocal microscopy, we have demonstrated that lysophosphatidic acid (LPA), a bioactive phospholipid existing in plasma, positively regulates fluid flow-induced [Ca(2+)](i) response in fluo 4-loaded, cultured, bovine aortic endothelial cells. The initial increase in [Ca(2+)](i) was localized to a circular area with a diameter of <4 microm and spread concentrically, resulting in a mean global increase in [Ca(2+)](i). The local increase often occurred in a stepwise manner or repetitively during constant flow. The percentage of cells that responded and the averaged level of increase in [Ca(2+)](i) were dependent on both the concentration of LPA (0.1 to 10 micromol/L) and the flow rate (25 to 250 mm/s). The response was inhibited by removing extracellular Ca(2+) or by the application of Gd(3+), an inhibitor of mechanosensitive (MS) channels, but not by thapsigargin, an inhibitor of the endoplasmic reticular Ca(2+)-ATPASE: It was also inhibited by 8-bromo-cGMP, and the inhibition was completely reversed by KT5823, an inhibitor of protein kinase G (PKG). These results suggest that the [Ca(2+)](i) response arises from Ca(2+) influx through Gd(3+)-sensitive MS channels, which are negatively regulated by the activation of PKG. The spatiotemporal properties of the [Ca(2+)](i) response were completely different from those of a Ca(2+) wave induced by ATP, a Ca(2+)-mobilizing agonist. Therefore, we called the phenomenon Ca(2+) spots. We conclude that LPA positively regulates fluid flow-induced local and oscillatory [Ca(2+)](i) increase, ie, the Ca(2+) spots, in endothelial cells via the activation of elementary Ca(2+) influx through PKG-regulating MS channels. This indicates an important role for LPA as an endogenous factor in fluid flow-induced endothelial function.

Physiological and pharmacological role of lysophosphatidic acid as modulator in mechanotransduction.

The mechanotransduction mechanism is believed to play an important role in maintenance of cellular homeostasis in a wide variety of cell types. In particular, the mechanotransduction system in vascular endothelial cells may be an essential mechanism for local hemodynamic control. Elevations in intracellular free Ca2+ concentration ([Ca2]i) are an important signal in the initial step of mechanotransduction and mechanosensitive (MS) cation channels are thought to be a putative pathway; however, the molecular mechanisms remain unclear. We found that lysophosphatidic acid (LPA), a bioactive phospholipid, sensitizes the response of [Ca2+]i to mechanical stress in several cell types. Employing real-time confocal microscopy, local increases in [Ca2+]i in several regions within the cell during application of mechanical stress were clearly visualized in bovine lens epithelial and endothelial cells in the presence of LPA. The phenomenon was termed "Ca2+ spots". Pharmacological studies revealed that Ca2+ spots arise due to influx through MS channels. In this report, our data indicating the possible significance of LPA as an endogenous factor involved in regulation of mechanotransduction is reviewed. Furthermore, our findings suggest that the Ca2+ spot is a novel phenomenon occurring as an elementary Ca2+-influx event through MS channels directly coupled with the initial step in mechanotransduction.

Downregulation of CXCR-2 but not CXCR-1 expression by human keratinocytes by UVB.

Interleukin-8 (IL-8) belongs to the CXC chemokine family. IL-8 exerts its biological activities by binding to specific cell surface receptors, CXCR-1 and CXCR-2. Both receptors bind IL-8 with high affinity but they have different affinities for MGSA/Groalpha and NAP-2. It has been shown that the expression of epidermal CXCR-2 is increased in psoriasis, suggesting that activation of KC mediated by CXCR-2 contributes to the characteristic epidermal changes observed in psoriasis. In order to examine the mechanism(s) by which UVB therapy is effective for several dermatoses including psoriasis, we sought to examine if UVB would modulate the expression of CXCR-1 and CXCR-2 in human keratinocytes (KC). Constitutive expression of CXCR-1 and CXCR-2 mRNA was detected by RT-PCR in normal cultured human KC. After 100 or 300 J/m(2) irradiation, a decrease in CXCR-2 mRNA was detectable from 12 h after irradiation; this downregulation was observed until 48 h after irradiation. In contrast, the CXCR-1 mRNA level was unchanged. Immunohistochemical studies and flow cytometry analysis confirmed the suppressive effect of UVB on the expression of CXCR-2 protein in cultured human keratinocytes. Immunohistochemical studies on two minimal erythema doses (2MED)-exposed and 2MED-unexposed skin from healthy volunteers revealed that CXCR-2 staining occurred over the whole layer of the epidermis but at 24 h after 2MED irradiation, the positive staining of CXCR-2 was decreased. A faint CXCR-1 staining was observed in the lower part of the epidermis both in unexposed and exposed skins. Our results indicate that UVB-induced growth inhibition of KC in hyperproliferative skin disorders may, in part, be related to downregulation of CXCR-2.

Adsorption of glycosaminoglycans onto hydroxyapatite using chromatography.

Proteoglycans are known to play an important role in the mineralization process, acting either as promoters or inhibitors. In this study the binding affinity of a variety of constituent glycosaminoglycan to hydroxyapatite was studied. Glycosaminoglycans (10-1000 microg ml(-1) in 0.02 M sodium acetate (pH 6.8) were constantly circulated through a hydroxyapatite column for 1 h. The total amount of glycosaminoglycan bound was determined by dimethylmethylene blue assay. The relative affinities of the different glycosaminoglycans remaining bound to hydroxyapatite was investigated by examining their release in a 0-1 M sodium phosphate gradient. Differences were noted between the desorption profiles of dermatan sulfate with two elution peaks and chondroitin 4-sulfate and chondroitin 6-sulfate each with a single peak. Dermatan sulfate and chondroitin 6-sulfate had a higher affinity for hydroxyapatite than chondroitin 4-sulfate possibly due to the presence of differing di-sulfated disaccharide ratios in the glycosaminoglycan chains. These findings suggest the presence of a variety of binding forms of each glycosaminoglycan or the differing orientation of these forms to yield different complexes with hydroxyapatite. The Ca2+ co-ordinates of the glycosaminoglycans are known to vary and may in part explain these findings.

Characteristics of cell-membrane and extracellular proteoglycans in monkey submandibular gland.

We investigated the characteristics of cell-membrane and extracellular proteoglycans in the monkey submandibular gland using enzymatic digestion and Western blot analysis. Extracellular proteoglycans and cell-membrane proteoglycans were extracted from the phosphate-buffered saline (PBS) soluble fraction and microsomal fraction of submandibular glands, respectively, and purified partially by ion exchange chromatography. Cellulose acetate membrane electrophoresis and immunoblotting with 3G10 monoclonal antibody or 6B6 monoclonal antibody showed that the extracellular proteoglycans contain several heparan sulfate proteoglycans (117, 67, 47 and 35 kDa core proteins) and a dermatan sulfate proteoglycan (45-50 kDa core protein), while the cell-membrane proteoglycans contain only a heparan sulfate proteoglycan with 69 kDa core protein. These results indicate that the several extracellular heparan sulfate proteoglycans may be released ectodomains of the cell-membrane proteoglycans from cell surface. It is suggested that the cell-membrane and extracellular proteoglycans may regulate the function of growth factors and the microenvironment in the normal submandibular gland.

Expression of the cell-surface heparan sulfate proteoglycan mRNA in monkey submandibular gland.

We investigated the mRNA expression of cell-surface heparan sulfate proteoglycans, syndecans and glypican, in the adult female monkey submandibular gland using the reverse transcription-polymerase chain reaction (RT-PCR) technique. Agarose gel electrophoresis of the PCR products of the cDNA generated from RNA was carried out to demonstrate the expression of mRNA in syndecan-1, syndecan-2, syndecan-4 and glypican in this study. In order to compare the mRNA expression level among the cell-surface heparan sulfate proteoglycans, we measured changes in the relative intensity of PCR products with increasing thermal cycle number. The expression levels were syndecan-4 > syndecan-1, syndecan-2 > glypican. Considering these results together with our previous report, we found that the cell-surface heparan sulfate proteoglycans, syndecan-1, syndecan-2, syndecan-4 and glypican, are synthesized in the monkey submandibular glands, and that their ectodomains are released into the extracellular matrix. It was speculated that control of the expression patterns of the cell-surface proteoglycans may regulate the cellular function and behavior in the submandibular gland.

[Evaluation on the clinical background on early death in patients with pulmonary tuberculosis during the past five years].

We evaluated the clinical background of early death (within 3 months after admission to our hospital) in patients with active pulmonary tuberculosis during the past five years (1992-1996). Among 65 active pulmonary tuberculosis patients who died during the past five years, 32 (49%) died directly of tuberculosis. Thirteen (41%) of those 32 patients died of acute respiratory failure and 9 patients (28%) died in emacitation state. Twenty two patients (69%) died within 3 months after admission to our hospital (the early death group) and 10 patients (31%) died after 3 months (the late death group). Thirteen patients (59%) in the early death group died of acute respiratory failure. On the other hand, none in the late death group died of acute respiratory failure but 4 patients died of chronic heart and/or respiratory failure and 4 patients died in emarciation state. Compared to the patients in the late death group, more patients in the early death group had long total delays (patient's and doctor's delays), had coexisiting diseases, had fallen into acute respiratory failure, and were under malnutrition. We evaluated the nutritional condition of patients using the Onodera's PNI (Prognostic Nutritional Index; 10 x serum almumin concentration + 0.005 x peripheral lymphocyte count) and the PNI value was lower among the patients in the early death group than among those in the late death group. To prevent death due to tuberculosis, we emphasize that it is important to start anti-tuberculosis therapy before patients fall into acute respiratory failure and/or malnutrition.

Expression and localization of HGF mRNA in monkey submandibular gland.

We investigated the characteristics and mRNA expression of hepatocyte growth factor (HGF) in the adult female monkey submandibular gland using Western blot analysis and the reverse-transcriptase PCR (RT-PCR) technique, and observed HGF mRNA localization using the in situ RT-PCR technique. HGF was extracted with PBS containing protease inhibitors, and purified partially by heparin affinity chromatography. With Western blot analysis, the HGF fraction showed an immunopositive protein band of 69-kDa corresponding to the alpha-subunit of HGF. The gene expression for HGF was revealed in the monkey submandibular gland using RT-PCR, and the PCR products showed high homology to cDNA for human HGF. Furthermore, the mRNA signal for HGF was localized in the striated and excretory ductal cells. These results unequivocally confirmed both the synthesis and existence of HGF in monkey submandibular glands.

Chondroitin sulfate isomers in synovial fluid of healthy and diseased human temporomandibular joints.

Synovial fluid was collected from the superior articular cavity of the temporomandibular joint in patients with unilateral internal derangement and joint pain whose contralateral joint was healthy. Glycosaminoglycans were liberated by digestion with pronase E, and precipitated with cetylpyridinium chloride and ethanol. Unsaturated disaccharide isomers of chondroitin sulfate, obtained following chondroitinase ACII digestion, were analyzed by high-performance liquid chromatography. Analytic data indicated that deltaDi-0S and deltaDi-6S were often found in chondroitin sulfate from the fluid of the diseased joints. The amounts of deltaDi-0S and deltaDi-6S differed significantly between synovial fluid samples from the diseased and healthy joints. Comparison of the relative proportions of the unsaturated disaccharides in the synovial fluid with previously reported values for several tissues, indicated that the chondroitin sulfate originated from articular cartilage, with possibly some contributions from soft connective tissues and serum present in the synovial fluid. These results suggest that chondroitin sulfate in the synovial fluid provides a useful indicator of the degree of internal derangement of the temporomandibular joint.

A pharmacokinetic comparison of two oral liquid glucocorticoid formulations.

To compare pharmacokinetics of liquid prednisolone and prednisone solutions and to assess relative bioavailability, six healthy adult men were administered 15 mg of each formulation. Blood samples were obtained and assayed for plasma prednisolone concentrations by high-performance liquid chromatography. Peak concentration was significantly higher with liquid prednisolone (mean +/- SD 430.3 +/- 62.5 vs 333.0 +/- 27.8 ng/ml, p = 0.013), with similar times to peak concentration. Prednisolone liquid gave higher concentrations at every time point (statistically significant for all except 0.25 hrs after the dose), resulting in a significantly greater total area under the curve (2029.8 +/- 246.9 vs 1633.3 +/- 221.1 ng/ml.hour, respectively, p = 0.002). Clearance was slower for prednisolone (128.3 +/- 15.1 vs 149.1 +/- 17.6 ml/min/1.73 m2, p = 0.01), and the relative bioavailability of the prednisolone liquid using prednisone liquid as the reference standard was 116 +/- 14%. Thus, prednisolone liquid has similar pharmacokinetic characteristics as prednisone liquid, with improved bioavailability.

The safety of inhaled corticosteroid therapy in children.

Although discussed for many years, systemic adverse effects from inhaled corticosteroid therapy remains a complicated and controversial topic. It is by now well-known that inhaled corticosteroids may affect growth, bone, and adrenal function, yet the clinical relevance and the risks for such adverse effects are poorly defined. Earlier intervention, use of higher doses, differing potency claims, and new inhalers also affect the potential for systemic effects following inhaled administration. Until further research better characterizes these risks and identifies appropriate monitoring, caution in the use of this highly effective therapy is advised.