Close relationship between T helper (Th)17 and Th2 response in murine allergic contact dermatitis.

BACKGROUND: Repeated exposure to allergens induces chronic allergic lesions in the skin and a shift in the cutaneous cytokine milieu to T helper (Th)2. AIM: To assess the relationships between Th17 and Th2 response during allergic contact dermatitis (ACD) in mice. METHODS: ACD was induced in C57BL/6 mice by single or repeated epicutaneous challenge of 2,4,6-trinitro-1-chlorobenzene. Relationships between Th17 and Th2 response were analyzed by immunohistochemical observations and activity of cytokines on days 8 (first challenge), 18 (11th challenge), 28 (21st challenge) and 38 (31st challenge). RESULTS: On day 8, tissue levels of interleukin (IL)-17 and IL-22 were high, whereas tissue levels of IL-4 and serum IgE concentration were low. Following acute contact dermatitis, mice developed chronic eczematous lesions on day 18, and gradually improved on days 28 and 38. Tissue IL-4 and serum IgE levels corresponded to the development and improvement of chronic eczematous lesions. Numbers of Th17 cells and tissue levels of IL-17 and IL-22 rapidly decreased as IL-4 and IgE levels increased on day 18. As levels of IL-4 and IgE decreased, the number of Th17 cells and tissue levels of IL-17 and IL-22 increased again on days 28 and 38. On day 18, tissue levels of Th17 response-inducing cytokines (IL-6, IL-23 and transforming growth factor-ß) were high, and IL-23-expressing cells appeared in abundance, when Th2 response was extremely high. IL-17 injection decreased tissue IL-4 and serum IgE levels. CONCLUSIONS: Th17 correlates closely with Th2 in murine chronic ACD induced by repeated epicutaneous challenge.

Elevated receptor for activated C kinase 1 expression is involved in intracellular Ca2+ influx and potentially associated with compromised regulatory T cell function in patients with asthma.

BACKGROUND: Regulatory T cells (T(regs)) are activated during anergy in response to T cell receptor (TCR) activation and functional immune suppression. Anergy of paediatric T(regs) is partially dependent on intracellular calcium mobility; following TCR activation, T(regs) do not exhibit increased intracellular Ca(2+) concentration ([Ca(2+) ](i)). OBJECTIVE: We determined whether [Ca(2+) ](i) in adult T(regs) defined their anergy, if intracellular Ca(2+) movement was linked to regulatory functions, whether [Ca(2+)](i) was indicative of asthma pathology, and the potential molecular mechanism responsible for Ca(2+) movement in T(regs). METHODS: T(regs) were purified by the magnetic bead method, and their regulatory functions were assessed by monitoring carboxyfluorescein succinimidyl ester-labelled responder T cell proliferation. The Ca(2+) response of Fura-2-labelled cells was measured using a video image analysis system. To analyse the functions of T(regs) at the molecular level, we generated Jurkat Tet-On(®) clones with doxycycline (Dox)-induced forkhead box P3 (FOXP3) protein expression. RESULTS: CD4(+) CD25(+) CD127(-/low) T(regs) from participants without asthma did not elicit Ca(2+) influx in response to TCR activation, exhibited little proliferation and suppressed proliferation of CD4(+) CD25(-) T cells. In contrast, under similar conditions, T(regs) from patients with asthma exhibited increased [Ca(2+)](i) and robust proliferation with partial loss of regulatory functions. FOXP3 protein levels in Tet-On(®) clones were high after both 2- and 5-day Dox treatment; however, 5-day cells were comparable with T(regs) from patients with asthma, whereas 2-day cells were similar to T(regs) from participants without asthma. Increasing [Ca(2+)](i) induced a high level of receptor for activated C kinase 1 (RACK1) expression in 5-day cells. CONCLUSIONS AND CLINICAL RELEVANCE: We confirmed that T(regs) in patients with asthma are functionally impaired and that the abnormal regulatory functions of these cells can be analysed by [Ca(2+)](i) following TCR engagement. Furthermore, the impaired functioning of T(regs) evident in patients with asthma may be due to a high level of RACK1.

TWEAK/Fn14 pathway promotes a T helper 2-type chronic colitis with fibrosis in mice.

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), a TNF superfamily member, induces damage of the epithelial cells (ECs) and production of inflammatory mediaters through its receptor Fn14 in a model of acute colitis. In our current study of chronic colitis induced by repeated rectal injection of a hapten, we found that inflammation, fibrosis, and T helper 2 (Th2)-type immunity were significantly reduced in Fn14 gene knockout (KO) mice when compared with wild-type (WT) control mice. Expression of thymic stromal lymphopoietin (TSLP) was lower in Fn14 KO colon ECs than in WT ECs. TWEAK potentiates the induction of TSLP by interleukin-13 (IL-13) in colon explants from WT but not in Fn14 KO tissue. TSLP receptor KO mice exhibit milder chronic colitis, similar to that in Fn14 KO mice. TWEAK and IL-13 synergistically promote fibroblast proliferation. Thus we propose an IL-13-TWEAK/Fn14-TSLP axis as a key mechanism underlying chronic colitis with fibrosis.

Molecular phylogenetic analysis of Barley yellow mosaic virus.

Complete nucleotide sequences of three strains (I, III, and IV) of Barley yellow mosaic virus (BaYMV) isolated in Japan were determined. The length of the genome was the same among the three strains; RNA1 was 7,642 nt and RNA2 was 3,585 nt. The molecular phylogenetic analysis showed that strain I was most closely related to the Chinese isolate, and these two strains formed one cluster with European isolates. Strains II, III, and IV, and the Korean isolate formed another cluster. Amino acid sequences of each viral gene product were compared among strains. The sequences of the VPg protein showed less identity among almost strains (less than 92%) than the sequences of other proteins (more than 93%). VPg is thought to be involved in interactions with host factors, especially initiation factor 4E (eIF4E) or eIF(iso)4E, and infection. Therefore, the relationship between amino acid substitutions and infection of host plants is analyzed.

Optimal antidiarrhea treatment for antitumor agent irinotecan hydrochloride (CPT-11)-induced delayed diarrhea.

PURPOSE: An antitumor camptothecin derivative CPT-11 has proven a broad spectrum of solid tumor malignancy, but its severe diarrhea has often limited its more widespread use. We have demonstrated from a rat model that intestinal beta-glucuronidase may play a key role in the development of CPT-11-induced delayed diarrhea by the deconjugation of the luminal SN-38 glucuronide, and the elimination of the intestinal microflora by antibiotics or dosing of TJ-14, a Kampo medicine that contains beta-glucuronidase inhibitor baicalin, exerted a protective effect. In the present study, we assessed the efficacy of several potential treatments in our rat model to clarify which is the most promising treatment for CPT-11-induced delayed diarrhea. METHODS AND RESULTS: Oral dosing (twice daily from days -1 to 4) of streptomycin 20 mg/kg and penicillin 10 mg/kg (Str/Pen), neomycin 20 mg/kg and bacitracin 10 mg/kg (Neo/Bac), both of which inhibited almost completely the fecal beta-glucuronidase activity, or TJ-14 1,000 mg/kg improved the decrease in body weight and the delayed diarrhea symptoms induced by CPT-11 (60 mg/kg i.v. from days 1 to 4) to a similar extent. The efficacy was less but significant in activated charcoal (1,000 mg/kg p.o. twice daily from days -1 to 4). In a separate experiment using rats bearing breast cancer (Walker 256-TC), TJ-14, Neo/Bac, and charcoal at the same dose regimen improved CPT-11-induced intestinal toxicity without reducing CPT-11's antitumor activity. In contrast, oral dosing (twice a day) of cyclosporin A (50 mg/kg), a P-glycoprotein and cMOAT/MRP2 inhibitor or valproic acid (200 mg/kg), a UDP-glucuronosyltranferase inhibitor, exacerbated the intestinal toxicity without modifying CPT-11's antitumor activity. CONCLUSIONS: The result clearly demonstrated the ability of Neo/Bac, Str/Pen, and TJ-14, less but significant ability of activated charcoal, to ameliorate CPT-11-induced delayed-onset diarrhea, suggesting the treatments decreasing the exposure of the intestines to the luminal SN-38 are valuable for improvement of CPT-11-induced intestinal toxicity. In contrast, the treatments affecting the biliary excretion of CPT-11 and its metabolites might have undesirable results.

Cytological study of early cervical adenocarcinoma: special reference to the depth of invasion.

OBJECTIVE: Early cervical adenocarcinoma (ECA) with a tumour depth of <3 mm has a good prognosis. To clarify the cytological features of ECAs with depth <3 mm, these were compared with those of ECA with 3-5 mm and invasive adenocarcinoma (IA) invading the cervical wall with more than 5 mm in depth. METHODS: The cervical cytological features of ECAs with depth <3 mm (14 cases) were compared with those of ECA with 3-5 mm (four cases) and IA (13 cases). Cytologically, the presence or absence of tumour diathesis, number of atypical cells, crowded cell groups, groups with glandular structures, feathering, groups with palisading borders, rosettes, clusters, cell shape and size, nuclear shape and size, nucleolar shape and size, chromatin distribution, border and character of cytoplasm, and single cell pattern were evaluated. RESULTS: A tumour diathesis was seen in five of 14 ECA <3 mm in depth (36%), all four ECA with 3-5 mm (100%) and 11 of 13 IA with more than 5 mm (85%). Single cells, macronucleoli and coarsely granular chromatin pattern were less frequent in ECA of <3 mm than that in ECA with 3-5 mm and IA. The number of atypical cells and glandular structures in ECA was significantly less than that in IA. Cell crowding, feathering, palisading and rosettes were common in both ECA and IA. CONCLUSION: The characteristic cytological features of ECA with depth <3 mm, having a good prognosis, were clean background, fewer single cells and macronucleoli, and less frequent coarsely granular chromatin pattern compared with those in ECA with 3-5 mm and IA. The number of atypical cells and glandular structures in ECA was significantly less than that in IA. Familiarity with the cytological features of ECA and its mimics is essential.

Clinico-cytological study of uterine papillary serous carcinoma.

OBJECTIVE: The aim of this study was to determine whether or not we could distinguish uterine papillary serous carcinoma (UPSC) from other types of endometrial cancer by cytology. METHODS: We examined the cytological findings of the endometrium from five cases with UPSC and compared them with 10 cases with endometrioid adenocarcinoma, grade 1 (G1). A morphometric analysis was performed. Cytological samples from the cervix and ascites of the patients with UPSC were also reviewed. RESULTS: All five patients had FIGO stage III and IV tumours. Three patients died of the disease and two are still alive with disease. The tumour cells of UPSC tended to be shed in papillary clusters with a tumour diathesis. Psammoma bodies were seen only in UPSC. The frequency of irregular-shaped nuclei, membrane thickness and eccentric nuclei in UPSC was higher than in G1. The chromatin pattern was coarsely granular, and both anisonucleosis and bare nuclei were prominent in UPSC. Cytomorphometrically, the maximum diameter of the nuclei in UPSC was significantly greater than that in G1. The nucleoli were also more often seen in UPSC than in G1. The findings of the nuclei and nucleoli in the cervical and peritoneal fluid cytology closely resembled those in endometrial smears. The features of the cervical smears and peritoneal fluid cytology were different from those of endometrial cytology regarding clear background and small clusters of cells. CONCLUSION: As the endometrial cytology findings accurately suggested the histological diagnosis of UPSC, the diagnosis of UPSC was confirmed in this study by endometrial cytology. The cytological diagnosis of UPSC should be based on the findings of tumour diathesis, psammoma bodies and papillary clusters composed of tumour cells with enlarged nuclei and numerous nucleoli.

Development of endometrial cancer following radiation therapy for cervical carcinoma.

The clinical and histologic findings in five cases of endometrial cancer, which developed following radiation therapy for squamous cell carcinoma of the cervix, are described. The mean age at endometrial cancer diagnosis was 69 years and average latency period from initial therapy to development of endometrial carcinoma was 13.4 years. For endometrial cancer, one patient had Stage Ib, one patient had Stage IIIa, two patients had Stage IIIc, and one patient did not undergo laparotomy. The histological types were carcinosarcoma in two patients, endometrioid adenocarcinoma, grade 3 in one patient, and clear cell carcinoma in one patient. All patients died of disease within 33 months of diagnosis. Endometrial cancers that develop after radiation treatment have a preponderance of high-risk histological subtypes, and consequently have a poor prognosis. Long-term follow-up should be mandatory for patients surviving radiation therapy for cervical cancer in order to detect and effectively treat second malignancies.

Inactivation of Chlamydia trachomatis and Chlamydia (Chlamydophila) pneumoniae by ozone.

AIMS: To clarify the inhibitory effects of ozone on Chlamydia trachomatis and C. pneumoniae. METHODS AND RESULTS: Cell culture was performed using HeLa229 cells for C. trachomatis, and Human Line cells for C. pneumoniae. C. trachomatis strain D/UW-3/Cx and C. pneumoniae strain AR-39 were used. Ozone water was generated by an ozone water dispenser and diluted to desired concentration just before each experiment. Preinoculation minimum cidal concentration (MCC) and postinoculation MCC methods were employed. In preinoculation MCC, chlamydial strains were treated with serially diluted ozone water followed by inoculation to cells. In postinoculation method, chlamydial strains were inoculated to cells and incubated for 24 h. Then infected cells were treated with ozone water, followed by additional incubation for 48 h. Complete inactivation was obtained in preinoculation MCC method at 0.5 ppm of ozone water for 30 s, or 4 ppm for 5 s. CONCLUSION: Ozone at a concentration of 4 ppm was enough for immediate inactivation of both C. trachomatis and C. pneumoniae. SIGNIFICANCE AND IMPACT OF THE STUDY: Ozone water at 4 ppm should be applicable for prevention of C. trachomatis urogenital infections.

[Tsutsugamushi disease (scrub typhus) in Japan: epidemiological aspects].

Epidemiological aspects of tsutsugamushi disease (scrub typhus) in Japan in 1998 were analyzed using questionnaires. Four hundred and sixteen scrub typhus cases were reported in 24 prefectures in 1998. The annual number of the patients in 1998 was similar to those in the preceding three years. There was no sex difference. The patients at the age of 51 or greater accounted for 72% of the total cases. Patients engaged in farming and forestry accounted for 32% and 14% cases, respectively. Fifty-six, 21 and 19% were reported in Kyusyu, Kanto and Tohoku-Hokuriku districts, respectively, 96% of the total cases being reported in these 3 districts. Most cases were reported from April through June with some from October through December in Tohoku-Hokuriku districts, while most cases were reported from October through December in other districts, including Kyusyu and Kanto districts. Thus, there was a difference in epidemic seasons among the districts. Serotypes of scrub typhus rickettsia were analyzed by serum antibody titers in the Kyusyu district. The novel Kawasaki and Kuroki types were major strains; however, no geographical difference was seen within the Kyusyu district. Interestingly, 24 cases were diagnosed only by the new serotypes not by the classical serotypes (Kato, Karp and Gilliam) in serological tests. This result suggests that further investigations are required to determine the prevalent serotypes in each district and to improve the serological tests. This was the first comprehensive report of epidemiology of scrub typus in entire Japan. Information obtained in the present study provides deep insight into prediction, diagnosis, treatment and prevention of scrub typhus in Japan.

[Tsutsugamushi disease (scrub typhus) in Japan: clinical features].

Clinical features of tsutsugamushi disease (scrub typhus) were analyzed, based on 416 cases reported in Japan in 1998. Three major clinical symptoms: eschar, fever and rash were found in 87%, 98% and 92% of the cases, respectively. Elevated levels of CRP, GOT, GPT and LDH were observed in 96%, 85%, 78% and 91%, respectively. These clinical and laboratory findings were observed in the majority of the cases and considered important for diagnosis. Disseminated intravascular coagulation developed in 21 cases, indicating that scrub typhus can be life threatening. Lymphadenopathy was observed in 51% of the cases. Enlarged lymph nodes were limited to the local sites in 75% of these lymphadenopathy cases and most of these sites were adjacent to eschars. Most eschars were scabbed and located in the abdomen and the lower half of the body, especially the feet. This suggests that these parts are frequently exposed to tsutsugamushi mites. Furthermore, the skin is soft in these parts and covered by cloth. These factors may make it possible for mites to keep biting without being noticed for several hours, long enough for rickettsial transmission. Interestingly, eschar and rash were absent in 14% and 8% of the cases, respectively. This result suggests that the cases without the unique symptoms may have been misdiagnosed as common cold or other febrile illnesses. One hundred and fifty-four suspected cases were not scrub typhus cases by the serological tests. The three major clinical symptoms were present in approximately a half of these negative cases, eschar being observed in approximately 70%. This may suggest the presence of new type of scrub typhus can not be diagnosed by the present laboratory tests. Clinical features of scrub typhus in Japan were well revealed, and information obtained in the present study is useful for improving clinical diagnosis. It should, however, be stressed that there were cases that could not be correctly diagnosed only by the clinical symptoms, suggesting that it is important to improve the serological tests.

A novel method for detecting HIV-1 by non-radioactive in situ hybridization: application of a peptide nucleic acid probe and catalysed signal amplification.

A novel in situ hybridization (ISH) method for detecting human immunodeficiency virus-1 (HIV-1) was developed by applying a peptide nucleic acid (PNA) probe and a catalysed signal amplification (CSA) method. The PNA probe used in the present study possessed 15 base sequences of the HIV-1 protease gene, and the 5' end of the probe was labelled with the fluorescein isothiocyanate (FITC) molecule. The hybridized probe was detected by sequential reactions of the following antibodies and reagents: horseradish peroxidase (HRP)-conjugated anti-FITC antibody, biotinylated tyramide (first amplification), HRP-labelled streptavidin, biotinylated tyramide (second amplification), and streptavidin-conjugated Alexa 488. The signal of Alexa 488 was finally detected by fluorescence microscopy. HIV-1-related dotted signals were clearly obtained in HIV-1 persistently infected cell lines, MOLT4-III(B) and ACH-2, and CD4-positive T lymphocytes from AIDS patients. For light microscopy, HRP-labelled streptavidin was reacted instead of streptavidin-conjugated Alexa 488 at the final treatment, followed by diaminobenzidine as chromogen. This method can detect HIV-1 in either blood smear samples or paraffin-embedded autopsy tissue and is useful as a sensitive non-radioactive method for in situ hybridization.

A comparative study of the fluoroquinolone antibacterial agents on the action potential duration in guinea pig ventricular myocardia.

We examined the effects of ten fluoroquinolone antibacterial agents, levofloxacin, sitafloxacin, trovafloxacin, ciprofloxacin, gemifloxacin, tosufloxacin, gatifloxacin, grepafloxacin, moxifloxacin and sparfloxacin, on action potentials recorded from guinea pig ventricular myocardia. Sparfloxacin prolonged action potential duration (APD) by about 8% at 10 microM and 41% at 100 microM. Gatifloxacin, grepafloxacin and moxifloxacin also prolonged APD at 100 microM by about 13%, 24% and 25%, respectively. In contrast, levofloxacin, sitafloxacin, trovafloxacin, ciprofloxacin, gemifloxacin and tosufloxacin had little or no APD-prolonging effect at concentrations as high as 100 microM. These findings suggest that there are differences in potency to prolong QT interval among the fluoroquinolones.

Diastereoselective radical hydrogenation of alpha-(1-Hydroxyalkyl)vinyl sulfoxides and sulfones controlled by intramolecular hydrogen bonding

The reaction of (S)-alpha-(1-hydroxyalkyl)vinyl sulfoxides (S)-5 with alkyl radicals and tributyltin hydride gave the addition-hydrogenation products with high diastereoselectivity, whereas the reaction with (R)-alpha-(1-hydroxyalkyl)vinyl sulfoxides (R)-5 resulted in complete recovery of the starting sulfoxides. Stereoselective intramolecular hydrogen bonding between the hydroxy group and the diastereotopic sulfonyl oxygen led to high diastereoselectivity in the radical reaction of alpha-(1-hydroxyethyl)vinyl sulfone 12. An important role of intramolecular hydrogen bonding on the diastereoselectivity as well as the reactivity toward alkyl radicals is discussed.

In vitro analysis of the change in resistance of Chlamydia trachomatis under exposure to sub-MIC levofloxacin for a therapeutic term.

BACKGROUND: While fluoroquinolone-resistant Chlamydia trachomatis strains have not been clinically isolated, they were isolated in an in vitro study recently. METHODS: To determine whether C. trachomatis strains develop resistance under sub-MIC antibacterial exposure in a clinical therapeutic term, C. trachomatis strains were exposed to sub-MIC levofloxacin (LVFX) for about 2 weeks. The MIC of LVFX was measured and DNA fingerprinting was performed every 72 h by PCR using random primers. RESULTS: There was almost no change in the MIC under exposure to 0.125 microg/ml LVFX. However, some mutational changes in DNA fingerprints developed. CONCLUSIONS: In clinical therapeutic terms, resistant strains of C. trachomatis will probably not develop, even if sub-MIC LVFX is employed.