Association between gut microbiota, bone metabolism, and fracture risk in postmenopausal Japanese women.

INTRODUCTION: We investigated the relationship between gut microbiota composition and osteoporosis/fracture risk in Japanese postmenopausal women using 16S rRNA gene sequencing, FRAX, bone mineral density, biochemical bone parameters, and a self-administered questionnaire. Variation in abundance of specific microbiota was found to be significantly associated with fracture risk and vitamin K levels. Gut microbiota data with respect to bone metabolism and fracture risk is limited. Vitamin K is produced by certain intestinal bacteria and has been reported to play a role in maintaining bone quality. PURPOSE: We investigated relationships among gut microbiota composition, bone metabolism, and fracture risk in postmenopausal Japanese women. METHODS: Bone mineral density (BMD) was evaluated in 38 postmenopausal women (mean age 62.9 years) using forearm dual-energy X-ray absorptiometry. We collected and analyzed serum bone turnover markers (vitamin K fraction and tartrate-resistant acid phosphatase 5b; TRACP-5b), gut microbiota profiling (16S rRNA gene sequencing), and self-administered questionnaire data, including fracture history and vitamin K intake. Vitamin K2, BMD, and TRACP-5b data were divided into high- and low-level groups using cutoff values of 0.06 ng/mL, 87.05%, and 420 mU/dL, respectively; the proportions of bacteria were analyzed. Fracture incidence and relative risk were investigated for each bacterium. RESULTS: The genus Bacteroides was predominant in the high vitamin K2 group (29.73% vs 21.58%, P = 0.022). Fracture incidence was significantly higher in the low Bacteroides group, with a 5.6-times higher risk ratio of fracture history. The family Rikenellaceae was more abundant in the low BMD group and more abundant in the high TRACP-5b group (2.15% vs 0.82%, P = 0.004; 2.38% vs 1.12%, P = 0.013, respectively). CONCLUSION: Bacteroides and Rikenellaceae may be involved in bone metabolism and fracture risk. Further investigations of the underlying microbiota-related pathways in bone metabolism may reveal treatment strategies, and facilitate the prevention of osteoporosis.

Detection of Colletotrichum acutatum Sensu Lato on Strawberry by Loop-Mediated Isothermal Amplification.

Colletotrichum acutatum, one of the most economically damaging pathogens of strawberry, is the primary causal agent of anthracnose fruit rot (AFR). A key challenge in managing AFR is detecting the pathogen on asymptomatic plants. To meet this need, a loop-mediated isothermal amplification (LAMP) assay was developed that incorporated two sets of primers: LITSG1, targeted on the intergenic transcribed spacer (ITS) region of ribosomal DNA, and Ltub2, on the ß-tubulin 2 gene. In pure culture assays, Ltub2 was specific for detection of C. acutatum, whereas LITSG1 detected C. acutatum and two additional anthracnose pathogens, C. gloeosporioides and C. fragariae. LITSG1 had 10-fold lower detection threshold (20 pg of mycelial DNA) than Ltub2 (200 pg mycelial DNA) in detection of C. acutatum from pure culture. For detection on asymptomatic leaves, two protocols for dislodging C. acutatum for DNA extraction were compared: i) the sonicate-agitate (SA) method and ii) the freeze-incubate-sonicate-agitate (FISA) method, which initially freezes tissues, followed by 2 days of incubation at 26°C in darkness, and then, sonication and agitation. Both methods were used for greenhouse-grown plant leaves that had been spray inoculated with serial dilutions ranging from 1.5 × 106 to 1.5 conidia ml-1. The FISA method produced more repeatable results than the SA method. For the FISA method, detection limits (expressed as initial inoculum concentrations) using LITSG1 and Ltub2 were 1.5 × 101 and 1.5 × 102 conidia ml-1, respectively. For composite samples comprised of inoculated (1.5 × 106 conidia ml-1) and noninoculated leaves of greenhouse-grown strawberry, the two sets of LAMP primers were compared using the SA method. Primer set LITSG1 consistently detected the pathogen from a single inoculated leaf in bulk samples of 50 or fewer pathogen-free leaves, whereas Ltub2 consistently detected one inoculated leaf in 20 or fewer pathogen-free leaves. Using primer set LITSG1, FISA was more sensitive than SA for detecting C. acutatum on leaves of field-grown plants from Florida. In an Iowa field trial using the FISA method, both primer sets detected C. acutatum in samples of asymptomatic leaves 6 days before fruit symptoms appeared. The results indicate that the LAMP assay has potential to provide a simplified method for detection of C. acutatum on asymptomatic strawberry plants.

Efeitos adversos da poliquimioterapia para hanseníase

Quando há efeitos adversos da poliquimioterapia para a hanseníase utilizam-se drogas alternativas preconizadas pela OMS. Objetivos: Identificar o número de portadores de hanseníase e avaliar as condições clínicas dermatológicas dos que fizeram uso de tratamento alternativo (a retirada da drogas Rifampicina RMP ou Dapsona DDS).

Multivariate characterization of elements accumulated in King Bolete Boletus edulis mushroom at lowland and high mountain regions.

Based on ICP-MS, ICP-OES, HG-AAS, CV-AAS and elementary instrumental analysis of King Bolete collected from four sites of different soil bedrock geochemistry considered could be as mushroom abundant in certain elements. King's Bolete fruiting bodies are very rich in K (> 20 mg/g dry weight), rich in Ca, Mg, Na, Rb and Zn (> 100 microg/g dw), and relatively also rich in Ag, Cd, Cs, Cu, Fe, Mn and Se (> 10 microg/g dw). The caps of King Bolete when compared to stipes around two-to three-fold more abundant are in Ag, Cd, Cs, Cu, Hg, K, Mg, Mo, N, Rb, Se and Zn. King Bolete collected at the lowland and mountain sites showed Ag, Ba, Co, Cr, Hg, K, Mg, Mn, Mo and Na in caps in comparable concentrations, and specimens from the mountain areas accumulated more Cd and Sb. Elements such as Al, Pb and Rb occurred at relatively elevated concentration in King Bolete picked up at the metal ores-rich region of the Sudety Mountains. Because of high bioconcentration potential King Bolete at the background sites accumulate in fruiting bodies great concentrations of problematic elements such as Cd, Pb and Hg, i.e. up to nearly 20, 3 and 5 microg/g dw, on the average, respectively. The interdependence among determined mineral elements examined were using the principal components analysis (PCA) method. The PCA explained 56% of the total variance. The metals tend to cluster together (Ba, Cd, Cs, Cr, Ga, Rb, Se, Sr and V; K and Mg; Cu and Mo). The results provided useful environmental and nutritional background level information on 26 minerals as the composition of King Bolete from the sites of different bedrock soil geochemistry.

Detection of Ralstonia solanacearum by loop-mediated isothermal amplification.

Ralstonia solanacearum is a pathogenic bacterium that causes wilt in over 200 plant species. Here we report a rapid and sensitive detection of R. solanacearum using an isothermal method for copying DNA known as loop-mediated amplification (LAMP). A set of four primers was designed to replicate the gene coding for the flagellar subunit, fliC, and conditions for detection were optimized to complete in 60 min at 65 degrees C. Magnesium pyrophosphate resulting from the amplification reaction could be detected optically as an increase in the solution turbidity, and the DNA products spread in a reproducible ladder-like banding pattern after electrophoresis in an agarose gel. Replication of the fliC gene was detected only from R. solanacearum. The detection limit of this LAMP assay was between 10(4) to 10(6) colony forming units/ml, and the technique may be useful for developing rapid and sensitive detection methods for the R. solanacearum pathogen in soil and water.

Some mineral constituents of Parasol Mushroom (Macrolepiota procera).

This article reports background concentrations of Ag, Ba, Cd, Co, Cr, Cs, Cu, Ga, Hg, Mn, Mo, Pb, Rb, Sb, Sr, Se, Tl, V and Zn in caps and stalks of M. procera collected from four spatially distant sites across Poland. The elements were determined using inductively coupled plasma-mass spectrometry (ICP-MS), hydride generation atomic absorption spectrometry (HG-AAS) or a cold vapor atomic absorption spectrometry (CV-AAS). Copper, zinc, rubidium, selenium, chromium and cobalt were the most abundant amongst elements determined in this mushroom. Some elements (Cu, Zn, Rb, Se, Pb, Hg, Cd, Mo) occurred at greater concentrations in the caps than stalks of M. procera and some (Ag, Ba, Sr, V, Tl) dominated in the stalks, while for some other this proportion was similar or varied (Mn, Cr, Co, Ga, Sb, Cs) depending on the sampling site. For elements such as copper, zinc, rubidium as well as selenium some spatial similarity in distribution and/or concentration values both in caps and stalks was noted. Cadmium and lead content in caps of M. procera was usually below the European Union tolerance limit value of 2.0 and 3.0 microg/g dw set for cultivated mushrooms, respectively. These two toxic metals have been found in elevated concentration in M. procera from unpolluted stands outside of Poland as reported by some authors, which implies the possibility of relatively high background levels in this species.

Selected elements in fly agaric Amanita muscaria.

Concentrations of Ag, Al, Ba, Ca, Cd, Co, Cu, Cr, Cs, Fe, Ga, Hg, K, Mg, Mn, Mo, Na, Pb, Rb, Se, Sb, Sr, V, Tl and Zn have been determined in the whole fruiting bodies, as well as separately in caps and stalks, of fly agaric collected from three geographically distant sites in northern part of Poland. The elements were determined using ICP-MS, ICP-OES, HG-AAS and CV-AAS, respectively. For elements such as Al, Ba, Cr, Fe, Ga, Mo, Mn, Pb, Sb, Sr, Tl, and V concentrations were similar in the caps and stalks, respectively, and for K, Zn, Ag, Ca, Cd, Cu, Hg, Mg, Rb and Se were greater in the caps, while for Co, Cs and Na in the stalks. For Ag, Al, Ba, Ca, Cd, Co, Cr, Cs, Fe, Ga, Hg, Mn, Mo, Pb, Rb, Sb, Sr, Tl and V concentration in the caps showed spatial variations (P<0.05), while for Cu, K, Mg, Na, Se and Zn was independent of the site. The elements such as K with median or mean in the caps between 37,000 and 43,000 microg/g.dm and Mg with 920 and 1,100 microg/g dm were most abundant. Next, within median values range from approximately 100 to 500 microg/g dm were such as Ca, Fe and Al, and in descending order they followed by Rb (100-400 microg/g dm); V, Na, Zn (50-200 microg/g dm); Cu, Mn (10-50 microg/g dm); Cd (10-20 microg/g dm); Se (5 microg/g dm); Ba (<1-3); Cr, Ag, Pb, Sr (<1-2 microg/g dm); Cs, Co, Hg (<1-1 microg/g dm); Ga (<0.5), Sb, Mo and Tl (<0.1 microg/g dm).

Selected elements of Poison Pax Paxillus involutus.

Concentrations of Ag, Al, Ba, Ca, Cd, Co, Cu, Cr, Cs, Fe, Ga, Hg, K, Mg, Mn, Mo, Na, Ni, Pb, Rb, Se, Sb, Sr, V, Tl and Zn have been determined in the whole fruiting bodies as well as separately in caps and stalks of Poison Pax collected from three geographically distant sites across Poland. The elements were determined using ICP-MS, ICP-OES, HG-AAS and CV-AAS, respectively. Based on arithmetic mean and median values for Poison Pax specimens from the Lezno site the elements such as Ag, Co, Cr, Cs, Mn, Mo, K, Pb, Rb, Sb, Se, V and Tl occur at similar concentration both in the caps and stalks, while for Cd, Cu, Hg, Mg and Zn around two-fold greater concentrations were noted in caps than stalks (cap/stalk concentration quotient > 1). Cs, Cd, Ni and Rb occurred at much greater concentration in specimens collected from the Klodzka Hollow in the Sudety Mountains when compared to the lowland site (Mann-Whitney U-test), and slightly greater values were noted also for Cr, Mo and Rb, while for Ca, Co, Mg and Mn were smaller The results provide useful environmental and biological baseline level of information for metallic elements of Poison Pax.

Lack of inhibitory effects of several fluoroquinolones on cytochrome P-450 3A activities at clinical dosage in dogs.

Inhibitory effects of several fluoroquinolones (FQs) on liver CYP3A activities were examined by in vitro and in vivo tests in dogs. Midazolam (MDZ) hydroxylation rate was used to determine the CYP3A activities in liver microsomes. Enrofloxacin (EFX), ofloxacin (OFX) orbifloxacin (OBFX) and ciprofloxacin (CFX) were tested. None of the FQs changed Vmax, Km or intrinsic clearance (Vmax/Km) of MDZ. For in vivo test, we examined the effects of oral administration of EFX and OFX on the pharmacokinetics of quinidine (QN), a CYP3A substrate. EFX or OFX (10 mg/kg) was administered once a day for 3 days. QN (2 mg/kg) was intravenously injected at 2 h after the final dose of FQs administration. The same dose of QN was intravenously injected 3 weeks before the start of FQs administration for control. Neither EFX nor OFX changed the pharmacokinetic parameters of QN. These in vitro and in vivo consisted results suggest that these FQs lack the inhibitory effects on CYP3A activities in dogs. Hence, given these results, the risk of drug-drug interaction is unlikely to occur between FQs and CYP3A substrates in clinical situation in dogs.

Effect of ofloxacin on theophylline pharmacokinetics at clinical dosage in dogs.

We examined the effects of ofloxacin (OFX) and norfloxacin (NFX) on theophylline (TP) pharmacokinetics in dogs. OFX, as a noncompetitive and mechanism-based inhibitor, and NFX, as a noncompetitive inhibitor, were orally administered (5 mg/kg) for a single dose or multiple doses (12 hourly for 3 days). TP (5 mg/kg, i.v) was injected at 2 h after the final dose of the fluoroquinolones (FQs). The same dose of TP was injected (i.v) 3 weeks before the start of FQs treatment for control. Multiple doses of OFX significantly reduced the total body clearance (Cl(B)) of TP from 0.117 to 0.085 L/h/kg, although a single dose did not change it. Neither a single dose nor multiple doses of NFX changed the TP pharmacokinetics. Plasma NFX concentrations increased after multiple doses. Those of OFX also increased but were still two orders of magnitude below the K(i) for noncompetitive inhibition of CYP1A in dogs. Time-dependent reduction in Cl(B) of TP suggests that mechanism-based inhibition of OFX was the major mode to decrease Cl(B) of TP. The mechanism-based inhibition may result in substantial inhibition of CYP1A activities in clinical conditions.

A comparative study of neurogenesis in the retinal ciliary marginal zone of homeothermic vertebrates.

The retina of many fish and amphibians grows throughout life, roughly matching the overall growth of the animal. The new retinal cells are continually added at the anterior margin of the retina, in a circumferential zone of cells, known as the ciliary marginal zone, or CMZ. Recently, Fischer and Reh [Dev. Biol. 220 (2000) 197] have found that new neurons are added to the retina of the chicken via proliferation and subsequent differentiation of neurons and glia at the retinal margin in a zone highly reminiscent of the CMZ of lower vertebrates. In addition, other groups have reported that putative retinal stem cells could be isolated from the ciliary margin of the adult mouse. In light of these findings, we have re-investigated the eyes of three additional species to determine whether other homeothermic vertebrates also possess CMZ cells and whether we could detect evidence for addition of neurons at the retinal margin in mature animals. We examined one additional avian species, the quail, one marsupial, the opposum, and one mammal, the mouse. We find that the CMZ cells have been gradually diminished during vertebrate evolution. The quail has a reduced CMZ as compared to the chicken, while the opposum has only a few cells likely related to the CMZ and we failed to find evidence of CMZ cells at the margin of the mouse retina.

Elevated concentrations of trace elements in Caspian seals (Phoca caspica) found stranded during the mass mortality events in 2000.

Concentrations of V, Mn, Fe, Cr, Co, Cu, Zn, As, Se, Mo, Ag, Cd, Tl, Hg, Pb, and organic mercury (Org-Hg) were determined in liver, kidney, and muscle of healthy Caspian seals ( Phoca caspica) collected in 1998. These concentrations were compared with those of seals infected with canine distemper virus (CDV) found stranded along the coastal areas in 2000. Concentrations of toxic elements (As, Ag, Cd, Tl, Hg, Pb, and Org-Hg) in Caspian seals stranded in 2000 were comparable or lower than those of samples collected in 1998 and in other pinnipeds. Thus it may be inferred that these elements were not the causative agents in the deaths of the seals. In contrast, concentrations of Zn and Fe were much higher in diseased Caspian seals than those in other pinnipeds. Zinc concentrations in all tissues of Caspian seals also increased during 1993-2000. Furthermore, negative correlations were found between blubber thickness and hepatic and renal Zn concentrations. These results imply the disturbance in homeostatic control and nutritional status of essential elements in Caspian seals stranded in 2000.

Characterization of new epidemic strains of influenza B virus by using neutralizing monoclonal antibodies.

During the 1998-1999 influenza season, two distinct influenza B virus Yamagata group strains were isolated from the patients of a private clinic. Each responded differently to monoclonal antibodies (Mabs) 5H4 and 8B3 on staining, and hemagglutination inhibition and neutralizing tests. When the analysis of nucleotide sequences was undertaken, the identity of deduced amino acid sequences of the HA1 region was 94%, which suggested that they derived from different strains. They were termed 5H4-responding strains and 5H4-nonresponding strains, respectively. The analysis of laboratory-induced antigenic variants suggested that the amino acid at position 149 is important to the reactivity to 5H4. This residue was "Arg" in 5H4-responding strains and "Lys" in nonresponding strains. During the 1998-1999 season, a total of 100 influenza B virus strains were isolated and 5H4-responding strains were the major type (94 strains). In the 1999-2000 influenza season, only two influenza B virus strains were isolated. Neither responded to 5H4. However, analysis of the deduced amino acid sequences of the HA1 region suggested that one of the two strains was derived from the 5H4-responding strains of the previous season. The amino acid residue at position 149 was "Lys" in place of "Arg." These observations suggested that 5H4-nonresponding strains will increase in coming seasons.

Arsenic accumulation in the liver tissue of marine mammals.

Arsenic concentrations were determined in livers of 226 individuals representing 16 different marine mammal species to elucidate its accumulation with age, sex, and feeding habits. Arsenic concentrations varied widely among species and individuals, and ranged from < 0.10 to 7.68 micrograms g-1 dry weight. Marine mammals feeding on cephalopods and crustaceans contained higher arsenic concentrations than those feeding on fishes. No significant gender difference in arsenic concentration was found for almost all the species. Also, no apparent trend with age (or body length) in arsenic accumulation was found for most of the species. It was noted that two seal species, Baikal seal and Caspian seal, from landlocked water environments, contained lower arsenic concentrations than the marine species. To our knowledge, this is the first comprehensive study of arsenic accumulation in a wide range of marine mammal species.

Evaluation of the method for nifedipine administration for a rapid onset of clinical effect: a clinical study in normal volunteers.

Nifedipine is frequently used for patients who require an immediate reduction of blood pressure elevated temporarily by various administration techniques including sublingual route without administrating intravenous infusion of vasodilator. A cross-over clinical study was conducted to investigate the optimal administration method of nifedipine for rapid management of hypertension. Four method of administering 10 mg nifedipine (the capsule was bitten and swallowed, sublingually with a hole in it or the contents administered orally or intranasally with a syringe) were evaluated with regarded efficacy, safety, and usefulness in 6 normal volunteers. Systolic and diastolic blood pressures were correlated with the nifedipine serum concentration in each method. Nifedipine pharmacokinetic parameters differed among the 4 administration methods. Nifedipine was absorbed rapidly by not only intestinal mucosa but also the nasal or oral mucosa. The pharmacological effect of intranasal or sublingual administration was superior. However, mint oil which is present in nifedipine capsules stimulates nasal mucosa when administered intranasally. For clinical usage, nifedipine capsules in which a hole is made with a needle, administered sublingually, can be effectively and safely used for rapid management of systemic hypertension.

Enhanced inhibition of lymphocyte activation by Mycobacterium avium complex in human T lymphotrophic virus type I carriers.

BACKGROUND: We have previously reported that disseminated pulmonary Mycobacterium avium complex (MAC) infection is more common in human T lymphotrophic virus type I (HTLV-I) carriers than in non-carriers. However, the reason for this remains unclear. It has been shown that glycopeptidelipid (GPL), one of the lipid components of the cell envelope of MAC, is able to reduce the lymphocyte blastogenic response to mitogens. The purpose of this study was to clarify whether or not the inhibitory effect of GPL differs between HTLV-I carriers and non-carriers. METHODS: Peripheral blood lymphocytes were obtained from 29 patients who had recovered from pulmonary MAC infection (10 of whom also had HTLV-I infection) and the lymphocyte counts and T cell subpopulations of the peripheral blood lymphocytes in HTLV-I carriers and non-carriers were compared. The inhibitory effect of GPL on the lymphocyte blastogenic response to phytohaemagglutinin (PHA) was tested in these 29 cases and in 15 healthy controls who had never suffered from MAC (seven of whom also had HTLV-I infection). All HTLV-I positive cases were carriers. RESULTS: There was no significant difference in the numbers or subset proportions of T cells between HTLV-I carriers and non-carriers. Lymphocyte activation by PHA was significantly inhibited by GPL in MAC positive and negative HTLV-I carriers compared with MAC negative non-carriers and MAC negative healthy controls (p<0.001). CONCLUSIONS: We suggest that MAC infection leads to strong inhibition of lymphocyte activation in HTLV-I carriers. This may account, in part, for the severity of pulmonary MAC infection in HTLV-I carriers.

Existence of escape mutant in HTLV-I tax during the development of adult T-cell leukemia.

Although Tax protein is the main target of cytotoxic T lymphocyte (CTL) on human T-cell lymphotropic virus type I (HTLV-I)-infected cells, and Tax peptide 11 through 19 binding to HLA-A*02 has been shown to elicit a strong CTL response, there are patients with adult T-cell leukemia (ATL) bearing HLA-A*02. To explore whether there is genetic variation in HTLV-I tax that can escape CTL recognition during the development of ATL, the HTLV-I tax gene was sequenced in 55 patients with ATL, 61 patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and 62 healthy carriers, and it was correlated with the presence of HLA-A*02. First, a premature stop codon in the 5' half of the tax gene that looses transactivation activity on the viral enhancer was observed in 3 patients with acute and 1 patient with chronic ATL. This stop codon was revealed to emerge after the viral transmission to the patient from sequence analysis in family members with ATL. Second, amino acid change in Tax peptide 11-19 was observed in 3 patients with ATL. CTL assays demonstrated that this altered Tax 11-19 peptide, observed in ATL patients with HLA-A*02, was not recognized by Tax 11-19-specific CTL. Two patients with ATL had large deletions in tax by sequencing, and 5 patients with ATL had deletions in HTLV-I by Southern blotting. These findings suggest that at some stage of ATL development, HTLV-I-infected cells that can escape the host immune system are selected and have a chance to accumulate genetic alterations for further malignant transformation, leading to acute ATL.

Increased activated human T cell lymphotropic virus type I (HTLV-I) Tax11-19-specific memory and effector CD8+ cells in patients with HTLV-I-associated myelopathy/tropical spastic paraparesis: correlation with HTLV-I provirus load.

To discern the T cell subtype associated with T cell differentiation, the expression of CD45RA and CD27 was measured from total CD8(high) cells and from human T cell lymphotropic virus type I (HTLV-I) Tax11-19 peptide-specific CD8(+) cells in peripheral blood lymphocytes of patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Phenotypically defined memory and/or effector cells (CD45RA(-)CD27(+), CD45RA(+)CD27(-), and CD45RA(-)CD27(-)) were increased in HAM/TSP CD8(+) cells, compared with those of HTLV-I-seronegative healthy control subjects. The percentage of human leukocyte antigen (HLA)-DR-positive cells was also increased in CD8(+) cells of HAM/TSP, compared with those in HLA-DR(+)CD8(+) cells of healthy control subjects. HTLV-I provirus load correlated with the frequency of Tax11-19-specific CD8(+) cells. The high frequency of memory and/or effector type HTLV-I Tax11-19-specific CD8(+) cells suggests that continuous restimulation driven by HTLV-I antigens in vivo may be associated with the pathogenesis of HAM/TSP.

Increased HTLV type 1 tax specific CD8+ cells in HTLV type 1-asociated myelopathy/tropical spastic paraparesis: correlation with HTLV type 1 proviral load.

Less than 1% of individuals infected with the human T lymphotropic virus type 1 (HTLV-1) develop an inflammatory neurological disorder, termed HTLV-1-associated myelopathy/tropical spastic (HAM/TSP), while the vast majority of those infected remain asymptomatic HTLV-1 carriers (ACs). The fundamental viroimmunological differences between these groups are not well understood. To address this issue, we have investigated HTLV-1-specific T cell responses and measured the proviral load in these groups. Frequencies of HTLV-1-specific CD8(+) cells were demonstrated to be significantly higher in HAM/TSP patients than in ACs by using intracellular cytokine staining and soluble divalent HLA-A2/Ig fusion protein loaded with HTLV-1 Tax 11-19 peptide. It is consistent with the observed increase in HTLV-1-specific cytotoxic T lymphocytes in HAM/TSP patients. These CD8(+) cells produced interferon (IFN)-gamma in recognition of HTLV-1 antigens bound to HLAs on the infected CD4(+) cells. Using phenotypic markers indicative for T cell differentiation, memory and/or effector HTLV-1 Tax-specific CD8(+) cells were found to be increased in HLA-A2 HAM/TSP patients. HTLV-1 proviral load was elevated in HAM/TSP patients when compared to ACs. In addition, the proviral load in HAM/TSP patients correlated with the frequency of HTLV-1-specific IFN-gamma(+)CD8(+) cells or Tax-HLA-A2/Ig(+)CD8(+) cells, especially with the effector cells. In contrast, the proviral load inversely correlated with memory cells. These results suggest that HTLV-1 antigens may continuously stimulate HTLV-1-specific CD8(+) cells and differentiate them from memory cells into effector cells in vivo. These differentiated HTLV-1-specific CD8(+) cells may play a role in the pathogenesis of HAM/TSP.

Myocilin expression in the astrocytes of the optic nerve head.

We investigated the expression of myocilin in the optic nerve head of porcine eyes by Western blotting and immunohistochemical staining. Myocilin was localized in the nucleus, centrosome, glial filament, mitochondria, and some parts of the cell membranes of the astrocytes. Myocilin was also detected at the edge-feet portion of the processes of astrocytes adjacent to the inner limiting membrane and blood vessel wall. The astrocytes are the major cell population in the optic nerve head, contributing to the architecture of the nerve axon and blood vessels. Therefore, myocilin gene mutation and change of myocilin protein are likely to affect the architecture of the optic nerve head and induce various forms of glaucomatous optic nerve damage.