Association between transforming growth factor-ß1 expression and the clinical features of triple negative breast cancer.

The aim of the present study was to investigate the association between the expression levels of transforming growth factor-ß1 (TGF-ß1) and the clinical pathological characteristics and prognosis of triple negative breast cancer (TNBC) through study of TNBC patient tissue samples. The biological effects of TGF-ß1 on TNBC cells and the potential signal transduction pathway are additoinally investigated. Immunohistochemistry was utilized to investigate expression changes of the positive rate of TGF-ß1 in the TNBC, compared with the non-TNBC group, to explain the association between TGF-ß1 and clinical pathological characteristics and prognosis. MDA-MB-231 cells were treated with TGF-ß1 and subsequently the invasion and migration abilities, and the expression of proteins in certain signaling pathways were assessed before and after the treatment. Positive expression of TGF-ß1 was observed in 52.5% of TNBC tissue samples, which was higher than that observed in non-TNBC group (27.5%). High levels of TGF-ß1 expression were not significantly associated age, menopausal status, family history of cancer or tumor size; however, tumor histological grade and axillary lymph node metastasis were significantly associated (P<0.05). In addition, when the TGF-ß1 expression levels are higher, the 5-year disease-free survival rate is lower. TGF-ß1 expression promoted the invasion and migration of MDA-MB-231 cells, and the expression of Smad2 protein and P38 protein was increased, indicating that Smad2 protein and the P38 signaling pathway may serve an important role in TNBC.

Cutaneous larva migrans induced by swallowing live pond loaches.

Freshwater fish, specifically loaches, are potential hosts of Gnathostoma species. Consumption of uncooked freshwater loaches may result in cutaneous larva migrans. We report the case of a 70-year-old Taiwanese man who presented with a serpiginous skin rash on his abdomen. He had eaten live loaches intermittently (sometimes daily) for 5 months before his presentation. On histopathological examination, a transected parasite body and a headbulb were found in the mid-dermis. The parasite was confirmed as belonging to the genus Gnathostoma, most likely Gnathostoma doloresi. The patient recovered uneventfully without treatment, and had no symptoms at his 12-month follow-up visit.

Blood-brain barrier impairment with enhanced SP, NK-1R, GFAP and claudin-5 expressions in experimental cerebral toxocariasis.

Infection by Toxocara canis in humans may cause cerebral toxocariasis (CT). Appreciable numbers of T. canis larvae cross the blood-brain barrier (BBB) to invade the brain thus causing CT. In the present studies, we evaluated the BBB permeability and BBB injury as assessed by the cerebral Evans blue (EB) concentration as well as by pathological changes and glial fibrillary acidic protein (GFAP) expression in T. canis -infected mice monitored from 3 days (dpi) to 8 weeks post-infection (wpi). The vasodilation neuropeptides, the expressions of substance P (SP) and its preferred binding neurokinin-1 receptor (NK-1R) as well as claudin-5 of tight-junction proteins associated with BBB impairment were also assessed by Western blotting and reverse-transcriptase polymerase chain reaction. Results revealed that BBB permeability increased as evidenced by a significantly elevated EB concentration in brains of infected mice. BBB injury appeared due to enhanced GFAP protein and mRNA expressions from 4 to 8 wpi. Leukocytes might have been unrelated to BBB impairment because there was no inflammatory cell infiltration despite T. canis larvae having invaded the brain; whereas markedly elevated SP protein and NK-1R mRNA expressions concomitant with enhanced claudin-5 expression seemed to be associated with persistent BBB impairment in this experimental CT model.

Sero-epidemiology of Toxocara canis infection among aboriginal schoolchildren in the mountainous areas of north-eastern Taiwan.

A sero-epidemiological study of Toxocara canis infection was conducted among Atayal schoolchildren (aged 7-12 years) residing in the mountainous areas of north-eastern Taiwan. The 73 children investigated were each checked for anti-Toxocara IgG, in ELISA based on the larval excretory-secretory antigens of T. canis larvae. A short, self-administered questionnaire was then used to collect relevant information from each subject, including data on the keeping of dogs, playing in soil, eating raw vegetables, and whether the subjects normally washed their hands before eating. Once the seropositive children had been identified, odds ratios (OR), with their corresponding 95% confidence intervals (CI) and P-values, were calculated for each potential risk factor. When diluted 1:64, sera from 42 (57.5%) of the children gave a positive result in the ELISA, indicating that these 42 children were seropositive for T. canis infection. Seropositivity did not appear to be associated with the age or gender of the subject, the eating of raw vegetables, or the regular failure to wash hands prior to a meal. Compared with the other subjects, however, those who admitted living in a household where dogs were kept (OR = 3.79; CI = 1.23-11.69; P = 0.02) or playing in soil (OR = 3.00; CI = 1.10-8.16; P = 0.03) appeared at increased risk of seropositivity.

Enhanced inducible nitric oxide synthase expression and nitrotyrosine accumulation in experimental granulomatous hepatitis caused by Toxocara canis in mice.

The involvement of inducible nitric oxide synthase (iNOS) and nitrotyrosine (NT) in pathogenesis of toxocaral granulomatous hepatitis (TGH) in a murine host was quantitatively determined by biochemical, parasitological, pathological, and immunohistochemical assessments in a 42-week investigation. Mice were sacrificed for serum collection and histological processing as well as acid-pepsin digestion of the liver in a larval recovery study. Significantly increased levels of total serum NO were found in the trial, indirectly suggesting iNOS activation in the liver. iNOS reactivity was predominantly observed in infiltrating leucocytes in lesions and normal and apocrine-like cholangiocytes; in contrast, hepatocytes and multinucleated giant cells showed negative cytoplasmic staining in TGH. Strong iNOS-like reactivity was also detected on the body wall of larvae. The locations of NT reactivity were nearly identical to those of iNOS expression; infiltrating leucocytes or cholangiocytes stained for iNOS were also stained for NT in TGH. Enhanced iNOS expression, but not invading larvae (r = 0.256, P = 0.211), seemed to play a certain role in pathological damage in TGH due to a significant correlation between iNOS expression and serum alanine aminotransferase (ALT) levels (r =0.593, P = 0.021) in the trial. Our present results indicate a potential therapeutic strategy for treatment of GH caused by other nematodes through manipulation of iNOS expression.

The origin of the triploid in Paragonimus westermani on the basis of variable regions in the mitochondrial DNA.

Triploid, parthenogenetic forms of the lungfluke, Paragonimus westermani, occur in Japan, Korea and China. The origin(s) of triploidy has been debated over the years. Sequences of two regions in the mitochondrial DNA, i.e. partial lrRNA (16S), and a portion of the non-coding region, were obtained from natural populations of P. westermani. All triploid individuals (Japan, Korea, China) and a single tetraploid individual (China) had identical sequences in the 16S region studied. Some sequence variation was observed among diploids, with those from Taiwan being distinct from the remainder. Both neighbour joining and parsimony trees using the 16S region placed diploid individuals from southwestern Japan close to the triploids and the tetraploid. The fragment amplified from the mitochondrial non-coding region showed dimorphism. One form (type A) consisted of 239 bp comprising two identical tracts of 70 bp separated by a tract of 93 bp. The second form (Type B) consisted of only a single 70 bp tract. All diploid individuals from Taiwan, China and Korea possessed type A, while those from Japan were polymorphic; individuals from Oita and Hyogo had type B, those from Chiba had type A, but both types were found in Mie. On the other hand, all of the triploid individuals and two tetraploid individuals possessed type B. Both the form present in the non-coding region and the 16S sequence suggest an affinity between a south-eastern group of diploid populations in Japan and the triploid form. A possible mechanism responsible for the origin of the triploid is discussed.

Seroepidemiologic survey of Dirofilaria immitis infection among domestic dogs in Taipei City and mountain aboriginal districts in Taiwan (1998-1999).

To estimate the seroprevalence of Dirofilaria immitis infection in domestic dogs in Taiwan, we utilized a commercial ELISA kit (Snap, IDEXX, USA) for detecting circulating antigens released by adult female worms. Serum specimens of 664 domestic dogs sampled from Taipei City in northern Taiwan and 14 mountain aboriginal districts in eastern Taiwan were screened for D. immitis antigens. Multivariate-adjusted odds ratios (ORs) with their 95% confidence intervals (CIs) were estimated by multiple logistic regression analysis. A total of 89 subjects were antigen-positive, giving a seroprevalence of 13.4%, of which the seroprevalence in Taipei City and mountain aboriginal districts were 13.8 and 12.1%, respectively. The mean overall seropositive rates were 6.3% in 1-3-year-old age group, 14.1% in 3-6-year-old age group and 23.7% in the > or =6-year-old age group. The older the age, the higher the seroprevalence (OR=4.6, 95% CI=2.4-9.0 for the > or =6-year-old age group versus 1-3-year-old age group, P<0.001) for all the dogs in the present study. Moreover, seroprevalence was not different between female and male dogs in either Taipei City or mountain aboriginal districts (P>0.05). Also, no significant difference in seroprevalence among dogs between the two geographical areas was found (P>0.05). In the logistic regression analysis, the seroprevalence of D. immitis remained significantly increased with age after multivariate adjustment in the present study. To our knowledge, this is the first report on the status of D. immitis infection in domestic dogs in Taipei City and mountain aboriginal districts in Taiwan to date.

Toxoplasma gondii infection: relationship between seroprevalence and risk factors among inhabitants in two offshore islands from Taiwan.

A seroepidemiological study of toxoplasmosis among inhabitants of Penghu Island and Kinmen Island offshore of Taiwan was performed using the latex agglutination test from July 1999 to June 2000. In order to determine risk factors for Toxoplasma gondii (T. gondii) infection, the effects of a history of eating raw/undercooked meats and raising pets were focused on using a self-administrated questionnaire. The seroprevalence (28.2%; 190/673) in Kinmen Island was significantly higher than that (2.71%; 8/293) in Penghu Island (P < 0.001). A significant difference in seroprevalence between both sexes was found in Kinmen Island (P < 0.05), but not in Penghu Island. The results of multiple logistic regression analysis showed that the older the age, the higher the OR in both Islands, yet a significant difference in seroprevalence between children and adults or the elderly was observed in Kinmen Island (P < 0.001). Moreover, those who had histories of raising cats or eating raw/undercooked meats seemed to have greater opportunities to become infected with T. gondii (OR = 2.9, 95% CI = 1.9-4.5, P < 0.001; OR = 1.5, 95% CI = 1.1-2.1, P < 0.05). In Penghu Island, a significant association between seroprevalence and a history of raising cats was also observed (OR = 4.6, 95% CI = 1.1-20.1, P < 0.05). Furthermore, workers, farmers, and fishermen seemed to be more susceptible to T. gondii infection than students in Kinmen Island.

Characterization by pulse-field electrophoresis of a new region of DNA amplification containing the M2 subunit of ribonucleotide reductase in hydroxyurea-resistant Leishmania.

An extrachromosomal circular DNA of of approximately 50-kb size was amplified in the hydroxyurea-resistant variant of Leishmania mexicana amazonensis. The amplicon carried the M2 gene of ribonuleotide reductase as part of the gene encoding resistance to hydroxyurea. The amplicon was unstable. It disappeared rapidly as shown in pulse-field gradient electrophoresis gels after reversion of the cells for 20-80 days. This loss of amplified DNA was accompanied by a rapid loss of resistance to hydroxyurea during the same period. The amplicon was not hybridized to specific probes from any of the four regions of DNA amplification previously reported for Leishmania. This region of amplification thus appears to be a new region of DNA amplification in Leishmania.

[Larval distribution in different organs of ICR strain mice infected with Toxocara canis].

In this study 80 ICR mice were infected per os with 260 embryonated Toxocara canis eggs each through a stomach tube. Ten mice were sacrificed at weekly intervals over an eight-week period. Internal organs including livers, lungs, hearts, spleens, kidneys, brains, and carcasses were pooled separately and T.canis larvae from each category treated with artificial digestive fluid were recovered and later collected by the Baermann technique. Larval distributions at a given week in different organs or in a given organ at different weeks were compared and analyzed. Results revealed that total larval recovery increased from week one (11.6%) to four(19.7%); thereafter it decreased gradually and dropped to 13.4% by week eight. Larval distribution in different organs varied with time. No larvae were recovered from the heart or spleen during the study period, except for a small number recovered from the spleen in the 4th and 5th weeks after infection. In general, the larvae were recovered mainly from the brain, carcass and liver. Larval recovery from the liver was high in the first two weeks (4.5% and 3.6%, respectively) and in week 4(5.6%). Recovery from the brain increased drastically in week 4(6.8%) and remained high. Recovery from the carcass increased with time. In the case of the lungs, a sharp peak was observed in week 3(4.8%), but recovery from the kidneys was low (0.7-1.6%). Our results indicated that the ICR strain mouse is also a suitable animal model for the study of toxocariasis in paratenic hosts.

[Seroepidemiology of Toxoplasma gondii infection among Atayal aborigines and local animals in Nan-ao district, Ilan county and Jen-ai district, Nan-tou county, Taiwan].

In this study, latex agglutination test (LAT) was used to detect sera anti-toxoplasma antibodies of Atayal aborigines and local animals in Nan-ao district, Ilan county and Jen-ai district, Nan-tou county as a measure of exposure to the Toxoplasma gondii. Out of 422 Atayal aborigines and 64 different animals were tested in Nan-ao district and 82 Atayal children in Jen-ai district were also screened, the positive rates for sera anti-toxoplasma antibodies were 21.8%, 17.2% and 15.9%, respectively. In Nan-ao district, neither were the positive rates significantly different in males (22.1%) and females (21.4%), nor in humans (21.8%) and dogs (19.6%) (P > 0.05). The seroprevalence in adults (28.3%) was significantly higher than that in children (18.7%) (P < 0.05), and the highest seropositive rate (38.1%) was in the age group 50-59 years and the lowest (7.7%) was in the age group 1-9 years. In general, the age pattern of prevalence is consistent with increasing duration of exposure to Toxoplasma gondii with age. For animals, the seropositive rate in dogs (19.6%) was also significantly higher than that in wild rats (7.7%) (P < 0.05). No significant difference in seropositive rate of Atayal children was observed between Nan-ao and Jen-ai districts (P > 0.05).

Worm recovery and precipitin antibody response in guinea pigs and rats infected with Clonorchis sinensis.

Guinea pigs (Hartley strain) and rats (Wistar strain) were each fed 200 and 100 Clonorchis sinensis metacercariae, respectively. Five animals from each species were sacrificed weekly between 1-8 weeks postinfection (WPI) and then at 12, 16, 20 and 30 WPI for collection of worms, bile and sera. The overall worm recovery rates for guinea pigs and rats were 18.7% and 12.4%, respectively. Only one of the five rats examined at 20 WPI still harbored one worm, while all were worm-free at 30 WPI. By a double diffusion test, no antibodies were detected against C. sinensis adult antigens in the bile juice. Serum antibodies were detected in at least 95% of the infected guinea pigs between 4-30 WPI and rats between 3-16 WPI. Precipitin antibodies seemed to be correlated with the presence of live worms in rats that had been infected for more than 12 weeks.

Seroprevalence of Toxoplasma gondii antibodies among Atayal aboriginal people and their hunting dogs in northeastern Taiwan.

Atayal aborigines, living at an altitude of 1,500-1,600 m in northeastern Taiwan, still hunt for wild animals with the help of hunting dogs. In this study, the latex agglutination test (LAT) was used to detect sera anti-toxoplasma antibodies in this community as a measure of their exposure to Toxoplasma gondii. The positive rates for sera anti-toxoplasma antibodies were 21.8% and 19.6%, respectively, in 422 Atayal and 51 hunting dogs tested. Neither of the positive rates were found to be significantly different between male (22.1%) and female Atayal (21.4%), or between humans (21.8%) and dogs (19.6%) (P > 0.05) when compared by the Chi-Squared test (chi 2-test). A significant difference was observed between the positive rates in adults (28.3%) and children (18.7%) (P < 0.05), and the age pattern of prevalence is consistent with an increasing duration of exposure to Toxoplasma gondii with age. The consumption of raw liver of wild animals or insufficiently cooked meat may be the major mode of transmission of toxoplasmosis in Atayal.

Studies on cryopreservation of Toxoplasma gondii and its antigenicity in mice.

Two strains (RH and GC, the latter of which is a Taiwan isolate of porcine origin) of Toxoplasma gondii were kept at -20 degrees C, -60 degrees C, and in liquid nitrogen (-196 degrees C) to follow the time course change in viability and virulence of the parasites by direct count and animal inoculation methods. Changes in antibody titers in some of the mice inoculated with the thawed organisms were assayed by the indirect immunofluorescent antibody test. Viability and virulence of T. gondii were best preserved by storage in liquid nitrogen. Tachyzoites kept in liquid nitrogen for eight years still can lead to the death of the injected mice in 2-3 weeks. Virulence of the tachyzoites could be maintained for eight weeks at most at -20 degrees C and -60 degrees C. Dimethylsulfoxide (DMSO) seemed to be a better cryoprotectant for T. gondii than glycerol, but the DMSO-preserved organisms resulted in fewer tachyzoite-containing peritoneal exudates in inoculated mice than the glycerol-preserved organisms. The local isolate (GC strain) tachyzoites tolerated cryopreservation less well than the RH strain parasites. Low antibody titers (at most 1:64) were produced in mice that survived more than 16 days after inoculation with thawed tachyzoites.

[Application of staphylococcal protein A for immunodiagnosis of early toxoplasma infection].

RH strain Toxoplasma gondii was used to inoculate mice and rats. Sera collected at intervals were assayed by the indirect fluorescent antibody test (IFAT) and the latex agglutination test (LAT) to monitor the time-course change in titers of anti-toxoplasma IgM and IgG antibodies. In addition, the above sera and some anti-toxoplasma seropositive human sera (with IFAT antibody titers greater than or equal to 1:16) were adsorbed with staphylococcal protein A (SpA) and assayed for changes in IgM and IgG antibody titers so as to evaluate the usefulness of SpA adsorption in detecting anti-toxoplasma IgM which shows up early in toxoplasma infections. Samples assayed included 262 sera from mice, 65 sera from 5 rats and 85 human sera. The results revealed that parasite specific IgM-IFAT antibodies were detectable in 40% of the sera from mice 3 days after infection. After SpA adsorption, however, the IgM-IFAT antibody could be detected in a few specimens as early as 2 days post infection. IgG-IFAT and LAT antibodies first appeared in the sera on the 5th day of infection. In mice inoculated with freeze-killed tachyzoites (immunized mice), IgG-IFAT and LAT antibodies remained high from 16-35 days after inoculation, whereas IgM-IFAT antibodies were undetectable. Even after SpA treatment, only about half of the 16-day samples showed IgM antibody titers and the other specimens still remained negative. In general, IgM antibody titers increased 2-6 fold after SpA adsorption while IgG antibodies were almost completely removed after the treatment with a residual IgG-IFAT titer of less than or equal to 1:4.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibody to Trichomonas vaginalis in human cervicovaginal secretions.

Human cervicovaginal secretions were obtained from patients at the Gynecology and Obstetrics Clinics at National Taiwan University Hospital and Cathay General Hospital, Republic of China. Among the 500 patients examined, 33 (6.6%) were infected with Trichomonas vaginalis as determined by the culture method. Secretions from 24 of the infected patients and 30 noninfected women were assayed for anti-T. vaginalis immunoglobulins by the indirect immunofluorescent antibody technique. A few serum samples from both infected and noninfected persons were also included in this study. Immunoglobulin G (IgG) antibody against T. vaginalis was detected in 17 (70.8%) secretions from the infected women. Among the 17 positive secretions, anti-parasite IgA was found in two specimens, IgE was found in three, and IgM was found in one. Of the 30 secretions, 7 (23.3%) from noninfected women also contained anti-parasite IgG. Low levels of natural anti-trichomonad IgG and IgM were detected in the sera of normal persons. Infection with T. vaginalis caused an increase in the serum IgG antibody titer. Cross-reaction between T. vaginalis and Pentatrichomonas hominis was also observed.