Photon-counting 1.0 GHz-phase-modulation fluorometer.

We have constructed an improved version of a photon-counting phase-modulation fluorometer (PC-PMF) with a maximum modulation frequency of 1.0 GHz, where a phase domain measurement is conducted with a time-correlated single-photon-counting electronics. While the basic concept of the PC-PMF has been reported previously by one of the authors, little attention has been paid to its significance, other than its weak fluorescence measurement capability. Recently, we have recognized the importance of the PC-PMF and its potential for fluorescence lifetime measurements. One important aspect of the PC-PMF is that it enables us to perform high-speed measurements that exceed the frequency bandwidths of the photomultiplier tubes that are commonly used as fluorescence detectors. We describe the advantages of the PC-PMF and demonstrate its usefulness based on fundamental performance tests. In our new version of the PC-PMF, we have used a laser diode (LD) as an excitation light source rather than the light-emitting diode that was used in the primary version. We have also designed a simple and stable LD driver to modulate the device. Additionally, we have obtained a sinusoidal histogram waveform that has multiple cycles within a time span to be measured, which is indispensable for precise phase measurements. With focus on the fluorescence intensity and the resolution time, we have compared the performance of the PC-PMF with that of a conventional PMF using the analogue light detection method.

Selective ß2-adrenergic Antagonist Butoxamine Reduces Orthodontic Tooth Movement.

Recently, involvement of the sympathetic nervous system in bone metabolism has attracted attention. ß2-Adrenergic receptor (ß2-AR) is presented on osteoblastic and osteoclastic cells. We previously demonstrated that ß-AR blockers at low dose improve osteoporosis with hyperactivity of the sympathetic nervous system via ß2-AR blocking, while they may have a somewhat inhibitory effect on osteoblastic activity at high doses. In this study, the effects of butoxamine (BUT), a specific ß2-AR antagonist, on tooth movement were examined in spontaneously hypertensive rats (SHR) showing osteoporosis with hyperactivity of the sympathetic nervous system. We administered BUT (1 mg/kg) orally, and closed-coil springs were inserted into the upper-left first molar. After sacrifice, we calculated the amount of tooth movement and analyzed the trabecular microarchitecture and histomorphometry. The distance in the SHR control was greater than that in the Wistar-Kyoto rat group, but no significant difference was found in the SHR treated with BUT compared with the Wistar-Kyoto rat control. Analysis of bone volume per tissue volume, trabecular number, and osteoclast surface per bone surface in the alveolar bone showed clear bone loss by an increase of bone resorption in SHR. In addition, BUT treatment resulted in a recovery of alveolar bone loss. Furthermore, TH-immunoreactive nerves in the periodontal ligament were increased by tooth movement, and BUT administration decreased TH-immunoreactive nerves. These results suggest that BUT prevents alveolar bone loss and orthodontic tooth movement via ß2-AR blocking.

In situ visualization of plasma cells producing antibodies reactive to Porphyromonas gingivalis in periodontitis: the application of the enzyme-labeled antigen method.

Porphyromonas gingivalis is a keystone periodontal pathogen. Histologocally, the gingival tissue in periodontitis shows dense infiltration of plasma cells. However, antigens recognized by antibodies secreted from the immunocytes remain unknown. The enzyme-labeled antigen method was applied to detecting plasma cells producing P. gingivalis-specific antibodies in biopsied gingival tissue of periodontitis. N-terminally biotinylated P. gingivalis antigens, Ag53 and four gingipain domains (Arg-pro, Arg-hgp, Lys-pro and Lys-hgp) were prepared by the cell-free protein synthesis system using wheatgerm extract. With these five labeled proteins as probes, 20 lesions of periodontitis were evaluated. With the AlphaScreen method, antibodies against any one of the five P. gingivalis antigens were detected in 11 (55%) serum samples and 17 (85%) tissue extracts. Using the enzyme-labeled antigen method on paraformaldehyde-fixed frozen sections of gingival tissue, plasma cells were labeled with any one of the five antigens in 17 (94%) of 18 specimens, in which evaluable plasma cells were detected. The positivity rates in periodontitis were significantly higher than those found previously in radicular cysts (20% in sera and 33% in tissue extracts with the AlphaScreen method, and 25% with the enzyme-labeled antigen method). Our findings directly indicate that antibodies reactive to P. gingivalis are locally produced in the gingival lesions, and that inflammatory reactions against P. gingivalis are involved in periodontitis.

In vivo eradication of MLL/ENL leukemia cells by NK cells in the absence of adaptive immunity.

It remains unclear how the immune system affects leukemia development. To clarify the significance of the presence of immune systems in leukemia development, we transferred MLL/ENL leukemia cells into immune-competent or immune-deficient mice without any preconditioning including irradiation. The wild-type mice did not develop leukemia, whereas all the Rag2(-/-)γc(-/-) mice lacking both adaptive immune cells and natural killer (NK) cells developed leukemia, indicating that leukemia cells were immunologically rejected. Interestingly, leukemia cells were also rejected in 60% of the Rag2(-/-) mice that lacked adaptive immune cells but possessed NK cells, suggesting that NK cells play a substantial role in the rejection of leukemia. Moreover, engraftment of leukemia cells was enhanced by NK cell depletion in Rag2(-/-) recipients and inhibited by transfer of NK cells into Rag2(-/-)γc(-/-) recipients. Upregulation of NKG2D (NK group 2, member D) ligands in MLL/ENL leukemia cells caused elimination of leukemia cells by NK cells. Finally, we found that leukemia cells resistant to elimination by NK cells had been selected during leukemia development in Rag2(-/-) recipients. These results demonstrate that NK cells can eradicate MLL/ENL leukemia cells in vivo in the absence of adaptive immunity, thus suggesting that NK cells can play a potent role in immunosurveillance against leukemia.

CD138-negative clonogenic cells are plasma cells but not B cells in some multiple myeloma patients.

Clonogenic multiple myeloma (MM) cells reportedly lacked expression of plasma cell marker CD138. It was also shown that CD19(+) clonotypic B cells can serve as MM progenitor cells in some patients. However, it is unclear whether CD138-negative clonogenic MM plasma cells are identical to clonotypic CD19(+) B cells. We found that in vitro MM colony-forming cells were enriched in CD138(-)CD19(-)CD38(++) plasma cells, while CD19(+) B cells never formed MM colonies in 16 samples examined in this study. We next used the SCID-rab model, which enables engraftment of human MM in vivo. CD138(-)CD19(-)CD38(++) plasma cells engrafted in this model rapidly propagated MM in 3 out of 9 cases, while no engraftment of CD19(+) B cells was detected. In 4 out of 9 cases, CD138(+) plasma cells propagated MM, although more slowly than CD138(-) cells. Finally, we transplanted CD19(+) B cells from 13 MM patients into NOD/SCID IL2Rγc(-/-) mice, but MM did not develop. These results suggest that at least in some MM patients CD138-negative clonogenic cells are plasma cells rather than B cells, and that MM plasma cells including CD138(-) and CD138(+) cells have the potential to propagate MM clones in vivo in the absence of CD19(+) B cells.

Histopathological diagnosis of Japanese spotted fever using formalin-fixed, paraffin-embedded skin biopsy specimens: usefulness of immunohistochemistry and real-time PCR analysis.

Japanese spotted fever (JSF) is caused by Rickettsia japonica, and lethal cases are reported yearly in southwest Japan. We thus established the method of diagnosing JSF by immunohistochemistry (IHC) and real-time PCR (RT-PCR) using formalin-fixed, paraffin-embedded skin biopsy specimens. Two monoclonal antibodies were used for IHC, and the 17k genus common antigen gene served as the target of RT-PCR. We collected skin biopsy (n = 61) and autopsy (n = 1) specimens from 50 patients clinically suspected of JSF. Immunohistochemically, the rickettsial antigens were localized as coarse dots in the cytoplasm of endothelial cells and macrophages. Thirty-one seropositive cases plus one autopsy case (group A) and nine seronegative cases but with positive IHC and/or RT-PCR (group B) were judged as JSF. Nine cases were regarded as non-JSF disorders based on negative serology, IHC and RT-PCR (group C). Of 50 biopsies (eschar 34, eruptions 10, and scabs 6) from groups A and B, IHC and RT-PCR positivities were 94% (32/34) and 62% (21/34) for eschar, 80% (8/10) and 30% (3/10) for eruptions, and 33% (2/6) and 50% (3/6) for scabs. For IHC, eschar was most suitable, and scabs were insufficient. Unexpectedly, 18 biopsies happened to be fixed in 100% formalin, and this lowered the detection rate by RT-PCR, but IHC was tolerant. Sequence analysis using five skin biopsy specimens confirmed a 114 bp DNA stretch homologous to that reported for the target gene of R. japonica. In 26 (84%) of the 31 seropositive patients, the diagnosis was made by IHC and/or RT-PCR earlier than serology.

Low-temperature synthesis of single-walled carbon nanotubes by alcohol gas source growth in high vacuum.

Carbon nanotube (CNT) growth was carried out on SiO2/Si substrates with a Co catalyst using an alcohol gas source method in an ultra-high vacuum chamber. The resulting CNTs were characterized by scanning electron microscopy (SEM), Raman spectroscopy and transmission electron microscopy (TEM). Reducing the ethanol pressure decreased the optimum growth temperature for maximum yield, enabling single-walled carbon nanotube (SWNT) growth at 400 degrees C. By employing an Al2Ox buffer layer, SWNT yield increased several times, even at 400 degrees C. Under TEM observation, the Co particle size on the Al2Ox layers did not show a significant dependence on the growth temperature between 400 and 700 degrees C. Raman and TEM results confirmed activation of Co particles with larger diameter (>1 nm) by the Al2Ox buffer layer.

Autoerythrocyte sensitization syndrome with vertigo and hemilateral auditory impairment.

Autoerythrocyte sensitization syndrome (AES) is characterized by recurrent, painful purpura or ecchymosis. Testing for the reappearance of lesions after injection of the patient's own erythrocytes is usually useful for the diagnosis of AES, but the significance of this test is still controversial. As the lesions often appear in patients with psychiatric disorders, mental factors such as depression and stress are considered to be involved in the occurrence and exacerbation of AES. We report a 28-year-old woman who presented recurrent episodes of painful purpura with vertigo and hemilateral auditory impairment after difficulties at her workplace. After the diagnosis of AES, she was referred for psychiatric counselling, after which the symptoms disappeared. These findings suggest that treatment for psychological disorders is important in patients with AES.

Antineutrophil cytoplasmic antibody-associated vasculitis with oculomotor nerve palsy.

We report a patient with antineutrophil cytoplasmic antibody-associated vasculitis with oculomotor nerve palsy. The patient presented with a high fever, diplopia, blepharoptosis and impairment of ocular movement of the left eye except for lateral gaze. Multiple erythematous and livedoid lesions were observed on the forehead, both cheeks and both legs. Laboratory examination showed positive results for myeloperoxidase antineutrophil cytoplasmic antibodies. Skin biopsy revealed leucocytoclastic vasculitis of the small arteries in the lower dermis. The patient was successfully treated with systemic corticosteroids.

Conjunctival displacement to the corneal side for oblique-parallel insertion in 25-gauge vitrectomy.

PURPOSE: To assess the usefulness of the method of oblique-parallel trocar insertion with conjunctival displacement to the corneal side in 25-gauge (G) transconjunctival vitrectomy. METHODS: 25-G vitrectomy was performed in 77 consecutive eyes. Before making obliqueparallel trocar insertions, the conjunctiva was conventionally displaced superiorly in 35 eyes, but was displaced toward the corneal side in 42 eyes. After surgery, the distance between the scleral and conjunctival wounds was measured with calipers. The frequency of scleral wound exposure was assessed. RESULTS: After cannula removal at the end of surgery, inferior repositioning of the superiorly displaced conjunctiva was observed, while marked posterior repositioning of the corneal side caused displacement of the conjunctiva due to gravity. The superior displacement distances between the sclera and conjunctival wounds were 2.4+/-0.3 mm at the infusion port, 2.0+/-0.4 mm at the superior temporal port, and 1.9+/-0.4 mm at the superior nasal port, while the corresponding distances for corneal side displacement were 3.6+/-0.5, 3.5+/-0.5, and 2.5+/-0.5mm, and were all significantly (p<0.0001) greater with corneal side displacement. The frequency of scleral wound exposure due to conjunctival damage around the cannula (infusion port) was significantly (p=0.0164) lower for corneal side displacement (0/42; 16.7%) than superior displacement (5/35; 14.3%). There was no postoperative endophthalmitis in all 77 patients studied. CONCLUSIONS: In 25-G transconjunctival vitrectomy, using oblique-parallel trocar insertions with the conjunctiva displaced toward the corneal side results in marked posterior repositioning of the conjunctiva after cannula extraction. Corneal side conjunctival displacement is technically easy and completely covers the scleral wound. This method is expected to be effective in preventing endophthalmitis.

Vitreous prolapse through the scleral wound in 25-gauge transconjunctival vitrectomy.

PURPOSE: To examine the effectiveness of excising peripheral vitreous until the cannula tip is exposed to prevent vitreous prolapse through the scleral wound in 25-gauge transconjunctival vitrectomy. METHODS: Twenty-five-gauge vitrectomy was performed in 60 consecutive eyes. Peripheral vitrectomy was conducted until the cannula tip was exposed in 30 eyes and with conservation of the vitreous around the cannula in 30 eyes. Vitreous prolapse through the scleral wound was examined using a suction stick. RESULTS: Vitreous prolapse through the scleral wound was transparent, fine and short, and detectable only with the suction stick. The incidence of vitreous prolapse through the scleral wound was 0% (0 of 30 eyes) when peripheral vitreous was excised until the cannula tip was exposed, and 20% (6 of 30 eyes) when the vitreous around the cannula was conserved, with a significant difference between two groups (p=0.0237). In two of six eyes with vitreous prolapse, the scleral wound was open, but there was no leakage of intraocular fluid and normal ocular pressure was maintained. CONCLUSIONS: If peripheral vitrectomy is performed without excising the vitreous surrounding the cannula, there is a 20% risk of the vitreous prolapsing through the scleral wound. Vitreous prolapse through the scleral wound is difficult to detect because it is transparent, fine and short, and there is no intraocular fluid leakage. Therefore, detecting vitreous prolapse with a suction stick and appropriate intervention are important for preventing endophthalmitis.

Severe hypoglycaemia in a person with insulin autoimmune syndrome accompanied by insulin receptor anomaly type B.

AIMS: A rare case of the insulin autoimmune syndrome (IAS) accompanied by insulin receptor anomaly is reported. METHODS: Antibodies to insulin and insulin receptor were determined in the patient with severe hypoglycaemia before and after the treatment with prednisolone. RESULTS: Titers of antibody to insulin and insulin receptors were 73.0% and 41.5%, respectively. Drug-induced lymphocyte stimulation tests were all negative for the suspicious drugs. Her HLA-DR was DRB1*0403/04051. Following steroid therapy, the formation of antibodies was suppressed and alleviated her symptoms. Scatchard analysis yielded findings specific to polyclonal antibodies. CONCLUSIONS: The changes in autoantibodies resulted in alleviation of the hypoglycemic symptoms as a result of steroid therapy.

25-Gauge peripheral iridectomy during vitrectomy.

PURPOSE: To report peripheral iridectomy using a 25-gauge vitreous cutter in a 42-year-old man with pupillary block due to adhesion of the internal iris surface to the continuous circular capsulorhexis. METHODS: A corneal opening was made at 10 o'clock during vitrectomy. A 25-gauge vitreous cutter was inserted into the anterior chamber with the port facing downward, and peripheral iridectomy at the 12 o'clock position was performed. The vitreous cutter was set at a cutting speed of 2500 cpm and the aspiration pressure at 600 mmHg. RESULTS: A 25-gauge vitreous cutter with a fine shaft could easily be inserted into the peripheral anterior chamber, and there was no contact with the corneal endothelium even when the anterior chamber became shallow in association with iridectomy. In this patient, pupillary block resolved with peripheral iridectomy, and ocular pressure was also controlled. CONCLUSIONS: 25-gauge peripheral iridectomy is a simple technique that permits iridectomy of appropriate size at any desirable location.

Effect of low-intensity back exercise on quality of life and back extensor strength in patients with osteoporosis: a randomized controlled trial.

UNLABELLED: Randomized controlled study in 80 postmenopausal women with osteoporosis was conducted to investigate the effect of a home-based, simple, low-intensity exercise. Low-intensity back-strengthening exercise was effective in improving the quality of life and back extensor strength. INTRODUCTION AND HYPOTHESIS: Back-strengthening exercise is effective in increasing back extensor strength and decreasing risk of vertebral fractures. We hypothesized that a home-based, simple, low-intensity exercise could enhance back extensor strength and improve the quality of life and/or spinal range of motion in postmenopausal women in a short-term follow-up. METHODS: Eighty postmenopausal women with osteoporosis were randomly assigned to a control group (n = 38) or an exercise group (n = 42). Subjects were instructed to lift their upper trunk from a prone position antigravity and maintain the neutral position. Isometric back extensor strength, spinal range of motion, and scores for quality of life were evaluated at baseline and 4 months. RESULTS: Back extensor strength significantly increased both in the exercise group (26%) and in the control group (11%). Scores for quality of life increased in the exercise group (7%), whereas it remained unchanged in the control group (0%). There was a significant difference in quality of life score between the groups (p = 0.012). CONCLUSIONS: Low-intensity back-strengthening exercise was effective in improving the quality of life and back extensor strength in patients with osteoporosis.

Restless legs syndrome in hemodialysis patients: health-related quality of life and laboratory data analysis.

AIMS: To compare clinical data, sleep quality and health-related quality of life (HRQOL) with and without RLS in HD patients. MATERIALS AND METHODS: The international RLS study group diagnosis questionnaire was completed by 228 HD patients. The Pittsburg Sleep Quality Index (PSQI) for the evaluation of sleep quality and the Kidney Disease Quality of Life (KDQOL-SF) for the analysis of HRQOL were also used. RESULTS: 53 (23%) patients were diagnosed as RLS. Age and age at the initiation of HD were significantly younger in the RLS group. Serum calcium concentration (Ca) was significantly higher in the RLS group. Sleep quality evaluated by PSQI was significantly lower in the RLS group. In SF-36 domains of KDQOL-SF, bodily pain, general health perceptions, vitality, role functioning emotional, mental health and mental component score were significantly lower in the RLS group. In kidney targeted scales of KDQOL-SF, symptoms/problems, burden of kidney disease, cognitive function, quality of social interaction, sleep and patient satisfaction were significantly lower in the RLS group. CONCLUSION: High Ca was possibly connected to the pathophysiology of RLS which impaired sleep quality as well as HRQOL including mental health and many kidney disease related scales.

Efficacy of sildenafil for Japanese patients with audio-visual sexual stimulation (AVSS) test by the RigiScan Plus.

Sildenafil is most effective in men with mild-to-moderate ED, but not severe ED in Japan. In order to evaluate the efficacy of sildenafil, we conducted the present study using the AVSS test by the RigiScan Plus. The subjects were 56 patients (age: 34-82 years, mean: 60.5 years) with ED. The IIEF5 questionnaire and the AVSS test were conducted before and after administration of sildenafil. The penile rigidity could not be measured in 19 patients. Of these 19, sildenafil was effective in 7 and not effective in 12. The 7 cases in whom sildenafil was effective were all false-negatives. The sensitivity of sildenafil was 84%, and its specificity was 100%. This study suggests that the AVSS test by RigiScan Plus can objectively evaluate the efficacy of sildenafil, and shows potential for predicting that efficacy.

Do motorcyclists have erectile dysfunction? A preliminary study.

The aim of the present study was to evaluate the relationship between motorcycling and erectile dysfunction (ED). We investigated the relationship between motorcycling and erectile function using the 5-items version of the International Index of Erectile Function (IIEF5) in 234 motorcyclists (response rate 75%) and 752 healthy controls (response rate 66%). In all, 161 (69%) of 234 motorcyclists were diagnosed as ED based on IIEF5. The prevalence of ED in the motorcycle group increased by age as: 58, 63, 76 and 93%, for motorcyclists in 20-29, 30-39, 40-49 and 50-59 years, respectively. There was a significant difference in the prevalence of ED between the motorcycle group and the control group in all age groups. On stepwise logistic regression analysis, motorcycling was the strongest risk factor for ED. Although the severity of ED in motorcyclists was not so severe, motorcycling may be one of risk factors for ED.

Can we develop biomarkers that predict response of cancer patients to immunotherapy?

PRIMARY OBJECTIVE: The primary objective is to delineate the potential utility of cancer biomarkers that correlate and predict response to immunotherapy in cancer patients who are refractory to conventional therapeutics. Unlike significant development of biomarkers that predict response to chemotherapy, very few biomarkers have been developed to predict the response to immunotherapy. MAIN OUTCOMES AND RESULTS: This article describes briefly the importance of characterizing and validating biomarkers for immunotherapy. A few examples have been provided, such as the transcription factor NF-kappaB, the transcription repressor Yin-Yang 1 (YY1), the pro-apoptotic gene product (Smac/DIABLO) and the circulating Fas and Fas ligand. These biomarkers have been determined to be of prognostic significance in different cancers. CONCLUSIONS: Immunotherapy is considered as an alternative therapy in the treatment of cancer patients who are refractory to chemotherapy/radiation/hormonal therapies. Cross-resistance to apoptosis develops between cancer cells that are resistant to conventional therapeutics and immunotherapy. Therefore, it is important to develop biomarkers that will determine patient response to immunotherapy.