Induction of inner ear hair cell-like cells from Math1-transfected mouse ES cells.

Math1, a basic helix-loop-helix transcription factor homolog of the Drosophila atonal gene, is considered to be a key factor for induction of sensory hair cells (HCs) during development of the organ of Corti or cochlea. Although embryonic stem (ES) cells are able to produce HC-like cells, the role of Math1 in induction of those cells has not been thoroughly elucidated. In the present study, we introduced Math1 into ES cells in order to achieve efficient generation of HC-like cells. ES cells carrying Tet-inducible Math1, Math1-ES cells, were generated using a Tet-On gene expression system. Embryoid bodies (EBs) formed in the absence of doxycycline (Dox) for 4 days were allowed to grow for an additional 14 days in the dishes in the presence of 400 µg/ml of Dox. At the end of those 14-day cultures, approximately 10% of the cells in EB outgrowths expressed the HC-related markers myosin6, myosin7a, calretinin, α9AchR, and Brn3c (also known as Pou4f3) and showed formation of stereocilia-like structures, whereas few cells in EB outgrowths grown without Dox showed those markers. Reporter assays of Math1-ES cells using a Brn3c-promoter plasmid demonstrated positive regulation of Brn3c by Math1. Furthermore, such HC-related marker-positive cells derived from Math1-ES cells were found to be incorporated in the developing inner ear after transplantation into chick embryos. Math1-ES cells are considered to be an efficient source of ES-derived HC-like cells, and Math1 may be an important factor for induction of HC-like cells from differentiating ES cells.

In vitro differentiation of mouse embryonic stem cells into inner ear hair cell-like cells using stromal cell conditioned medium.

Hearing loss is mainly caused by loss of sensory hair cells (HCs) in the organ of Corti or cochlea. Although embryonic stem (ES) cells are a promising source for cell therapy, little is known about the efficient generation of HC-like cells from ES cells. In the present study, we developed a single-medium culture method for growing embryoid bodies (EBs), in which conditioned medium (CM) from cultures of ST2 stromal cells (ST2-CM) was used for 14-day cultures of 4-day EBs. At the end of the 14-day cultures, up to 20% of the cells in EB outgrowths expressed HC-related markers, including Math1 (also known as Atoh1), myosin6, myosin7a, calretinin, α9AchR and Brn3c (also known as Pou4f3), and also showed formation of stereocilia-like structures. Further, we found that these cells were incorporated into the developing inner ear after transplantation into chick embryos. The present inner ear HC induction method using ST2-CM (HIST2 method) is quite simple and highly efficient to obtain ES-derived HC-like cells with a relatively short cultivation time.

Febrile period and maximum body temperature in patients with measles over 15 years old.

The medical records of Japanese patients with measles, age 15 to 39 years old, were analyzed for febrile period in 200 patients and maximum body temperature in 211 patients. The febrile periods by age group were as follows: 7.7 +/- 1.4 days, 7.8 +/- 1.7 days, 8.1 +/- 1.7 days, 8.1 +/- 2.4 days and 7.5 +/- 1.4 days in 15-19, 20-24, 25-29, 30-34 and 35-39 year-olds, respectively. No significant differences in febrile periods were identified among the different age groups. A maximum body temperature over 40 degrees C was seen in 61, 58, 59, 50 and 33% of the 15-19, 20-24, 25-29, 30-34 and 35-39 year-olds, respectively. The results of our study indicate age is not a determining factor for febrile period, but maximum body temperature may be lower in older than in younger patients with measles.

Galbeta1-6Gal, antigenic epitope which accounts for serological cross-reaction in diagnosis of Echinococcus multilocularis infection.

Parasitic helminths express various antigenic carbohydrates, which often account for serological cross-reactions. In serodiagnosis, it is essential to inspect cross-reactivity between the target parasite and other parasites in order to assess diagnostic performance. Our previous study showed that the Galbeta1-6Gal sequence was a common epitope between Echinococcus multilocularis (Em) and E. granulosus (Eg). Furthermore, compounds with this sequence from Fasciola hepatica (Fh) reportedly were recognized by sera with Eg infection. Our aim is to investigate whether this sequence is one of the widely common epitopes in many kinds of parasites. For various parasites, sera with Fh infection cross-reacted at the highest frequency (71.4%) against Em antigen. In patients with other parasitic infections, sera showed cross-reactions against Fh antigen bound to Em antigen with a high frequency (23.7%). Binding inhibition tests with commercial Galbeta1-6Gal disaccharide showed that Galbeta1-6Gal was the common epitope between not only Em, Eg and Fh, but also between various other parasites. Furthermore, the presence of the Galbeta1-6Gal epitope in Em antigen was confirmed by immunoblot testing with the specific antibody for this sequence. This study showed that the Galbeta1-6Gal sequence is one of the antigenic epitopes that accounts for serological cross-reactivity between Em and various other parasites.

First findings on the seroepidemiology of human paragonimosis at the anti-tuberculosis centre of Divo, Repubuc of Ivory Coast (West Africa).

An epidemiological study was carried out in 2004-2005 at the anti-tuberculosis centre of Divo (Ivory Coast) to collect sera from patients who consulted for tuberculosis suspicion and to estimate the seroprevalence of human paragonimosis in the context of a systematic screening. No Paragonimus egg was found in the stools and/or sputa of the 167 persons investigated. In contrast, 41 sera were ascertained with antibodies against Paragonimus africanus using ELISA testing. As the optical density (OD) values related to seropositive findings were found under 0.6 (the minimal OD to detect an active paragonimosis), the above antibody titres might originate from patients in chronic or in convalescent stages, or might result of cross reactions with trematodes. Concomitantly, dissection of local crabs (Callinectes marginatus) demonstrated the presence of Paragonimus metacercariae in six out of 34 examined. The parasite burdens in crabs ranged from two to 35 cysts with a mean diameter of 302 microm. In Ivory Coast, the locality of Divo must be considered an at-risk zone in reason of the presence of anti-Paragonimus antibodies in several human sera and the presence of infected crabs at the local market.

Plasma D-dimer levels in patients with typhoid fever.

The plasma levels of D-dimer can be used as a marker of fibrin formation and degradation. Plasma D-dimer levels in the febrile phase of 6 patients with typhoid fever and in the afebrile convalescent phase of 4 of them were measured. D-dimer levels were high in the febrile phase of all 6 patients and within normal range in the afebrile convalescent phase of all 4 patients. Our results indicate that thrombus formation and fibrinolysis may occur in the febrile phase of patients with typhoid fever.

Evaluation of the antigenic similarities of adult-worm extracts from three Gnathostoma species, using sera from Mexican and Japanese patients with Gnathostoma infections.

The antigenic similarities of adult-worm extracts of Gnathostoma spinigerum, G. hispidum and G. doloresi, all of which are important food-borne parasites causing larva migrans in humans, were evaluated. The 40 sera used came from gnathostomiasis cases in Mexico, where G. binucleatum is endemic, or in Japan, where G. doloresi predominates. When used as the fixed antigens in microplate-ELISA, the adult-worm extracts from the three different species of Gnathostoma were found to have equal binding capacity to the Gnathostoma-specific IgG antibodies in the sera of the Mexican and Japanese patients. The correlation coefficients for the optical densities seen in the ELISA, between any two of the three Gnathostoma extracts, were all >0.900. The dose-response curves produced when four sera were tested, in the microplate-ELISA, against the three different Gnathostoma extracts were nearly identical, indicating that the antigens in each of the extracts had similar avidity. Furthermore, the results of competitive-inhibition ELISA indicated that the antigenic specificities of the three extracts were almost identical. An antigen of 40 kDa, which SDS-PAGE and immunoblot analysis revealed to be present in all three extracts, was recognized by the sera from the gnathostomiasis cases. When the sera were investigated by dot-blot ELISA, they also gave similar results whichever extract was used as the antigen source. It appears that, in the serodiagnosis of gnathostomiasis by microplate- or dot-ELISA, each of the three adult-worm extracts would be equally useful, regardless of the causative species of Gnathostoma.

Elevated levels of thymus and activation-regulated chemokine (TARC) in pleural effusion samples from patients infested with Paragonimus westermani.

To investigate the pathogenic mechanisms of eosinophilic pleural effusion in patients with paragonimiasis, we measured the levels of various chemokines including thymus and activation-regulated chemokine (TARC), eotaxin, RANTES and IL-8 in pleural effusion samples. Samples were obtained from 11 patients with Paragonimus westermani infection, six patients with pleural transudate, eight with tuberculous pleurisy and five with empyema. High percentages of eosinophils were detected in pleural fluid (range 9-100%, median 81%) of patients with paragonimiasis. TARC concentrations in pleural effusions of paragonimiasis were markedly higher than those of other groups. Eotaxin levels were also higher in pleural effusions of paragonimiasis patients, although significant difference was noted only against transudate samples. There was a significant correlation between TARC concentrations and percentages of eosinophils, and between TARC and eotaxin concentrations in pleural effusion. There were also significant correlations between TARC concentration and the titre of anti-P. westermani IgG and between eotaxin concentration and the titre of anti-P. westermani IgG. Our findings suggest that TARC contributes to the pathogenesis of eosinophilic pleural effusion in paragonimiasis.

Natural resistance of 129/SvJ mice to Strongyloides venezuelensis infection.

The susceptibility of 129/SvJ mice to infection with Strongyloides venezuelensis was compared with that of C57BL/6 mice. After a primary infection, daily egg output in faeces (EPG) from 129/SvJ mice was lower and terminated earlier than that from C57BL/6 mice. Adult worm recovery from the small intestine of 129/SvJ mice on day 7 was also lower than that of C57BL/6 mice. When the numbers of larvae recovered from the lungs were examined on days 2, 3 and 4 after a primary infection, they were comparable between the two strains. On the other hand, when an equal number of larvae recovered from the lungs of each strain on day 3 were implanted orally into homologous strain mice, the magnitude of EPG and the number of adult worms in the small intestine on day 5 after implantation were significantly lower in 129/SvJ than in C57BL/6 mice. The number of mucosal mast cells in the jejunum was not significantly different between 129/SvJ and C57BL/6 naive mice. Total chondroitin sulphate concentration in the gut washings obtained from naive mice was significantly higher in 129/SvJ (11.34 +/- 9.48) than in C57BL/6 mice (1.09 +/- 0.77, P < 0.05). These results indicate that the natural resistance of 129SvJ mice to S. venezuelensis infection is expressed at the intestine, probably due to higher concentration of chondroitin sulphate, which prevents establishment of S. venezuelensis.

Clinical features of paragonimiasis cases recently found in japan: parasite-specific immunoglobulin M and G antibody classes.

We retrospectively analyzed clinical features in 30 patients who were referred to our laboratory and given a diagnosis of Paragonimus westermani infection in 1999. Our results indicate that pleurisy with eosinophilia and dominant immunoglobulin (Ig) M antibody are characteristic features of the early stage of paragonimiasis, whereas IgG antibody is dominant in the late stage. Thus, in addition to tests for parasite-specific IgG antibody, tests for IgM-class antibody should always be considered for patients with pleurisy in whom paragonimiasis is suspected.

Clinical features and parasite-specific IgM/IgG antibodies of paragonimiasis patients recently found in Japan.

Clinical features of a total of 30 paragonimiasis westermani patients referred to and diagnosed in our laboratory in 1999 were analyzed retrospectively. Most patients were middle-aged (average: 48 years, range: 13-72 years) with the male/female ratio of 19/11. Over 70% of the patients had respiratory symptom and over 80% had peripheral blood eosinophilia and high serum IgE level. All but two cases had radiologic abnormalities on the chest X-ray. Only in 3 cases were Paragonimus eggs detected in the sputum smear. We classified the patients into two groups depending on the chest X-ray findings: patients having pleurisy alone and those having nodular/cavitating lesions in the lung parenchyma. We measured parasite specific IgM/IgG antibodies in all patients sera by microplate ELISA. The mean parasite-specific IgM/IgG antibody ratio was significantly higher in the parenchymatous lesion group than in the pleurisy group. While IgM antibody titer had a strong positive correlation with the degree of eosinophilia in peripheral blood, IgG antibody titer had an inverse correlation. Although the degree of eosinophilia in peripheral blood was higher in the pleurisy group than in the parenchymatous lesion group, total IgE level in serum was comparable between the two groups. The present results indicate that pleurisy with eosinophilia and dominant IgM antibody are the characteristic features of the early stage of paragonimiasis, whereas parenchymatous lesions in lungs with low grade eosinophilia and dominant IgG antibody are of the late stage. These results suggest that detection of IgM antibody should always be considered for the immunodiagnosis for paragonimiasis-suspected patients with pleurisy.

Amblyomma testudinarium tick bite: one case of engorged adult and a case of extraordinary number of larval tick infestation.

This paper reports two recent cases of tick bite due to Amblyomma testudinarium. The first case was an 86-year-old farmer infested with a fully engorged adult tick attached on his inguinal region. The second case was a 57-year-old male infested with an extraordinarily large number of larval ticks (> 100 larvae). The ticks were identified as A. testudinarium based on morphological characteristics. To our knowledge, the latter case is the eleventh case of larval tick bites among all tick species and the fourth case with larval A. testudinarium in Japan.