Mycobacterium avium Subsp. paratuberculosis Induces Specific IgE Production in Japanese People with Allergies.

Background. The prevalence of allergies is steadily increasing worldwide; however, the pathogenesis is still unclear. We hypothesized that Mycobacterium avium subsp. paratuberculosis (MAP) may contribute to allergy development. This organism can be present in dairy foods, it can elicit an immunomodulatory switch from a Th1 to a Th2 response, and it has been speculated that it is linked to several human autoimmune diseases. To determine the contribution, sera from 99 individuals with various atopic disorders and 45 healthy nonallergic controls were assessed for total IgE levels and successively for MAP-specific IgE by ELISA. Results. The mean total serum IgE level in allergic patients was 256 ± 235 IU/mL, and in the healthy controls it was 62 ± 44 IU/mL (AUC = 0.88; p < 0.0001). Among the patient groups, 50 of the 99 subjects had increased IgE total level ≥ 150 IU/mL, while 49 subjects had IgE ≤ 150 IU/mL (mean level: 407 ± 256 IU/mL versus 106 ± 16 IU/mL; p < 0.0001). Additionally, 6 out of 50 subjects (12%) with IgE ≥ 150 IU/mL and none (0%) with IgE ≤ 150 IU/mL were positive for specific MAP IgE (AUC = 0.63; p = 0.03). Conclusion. The present study revealed that MAP has the ability to induce specific IgE and might contribute to the induction of allergic inflammation in genetically predisposed individuals.

Molecular identification and characterisation of catalase and catalase-like protein genes in urease-positive thermophilic Campylobacter (UPTC).

BACKGROUND: Thermophilic Campylobacter are important bacterial pathogens of foodborne diseases worldwide. These organisms' physiology requires a microaerophilic atmosphere. To date, little is known about the protective catalase mechanism in urease-positive thermophilic campylobacters (UPTC); hence, it was the aim of this study to identify and characterise catalase and catalase-like protein genes in these organisms. MATERIALS AND METHODS: Catalase (katA) and catalase (Kat)-like protein genes from the Japanese UPTC CF89-12 strain were molecularly analysed and compared with C. lari RM2100 and other C. lari and thermophilic Campylobacter reference isolates. RESULTS: A possible open reading frame of 1,422 base pairs, predicted to encode a peptide of 474 amino acid residues, with calculated molecular weight of 52.7 kilo Daltons for katA, was identified within UPTC CF89-12. A probable ribosome binding site, two putative promoters and a putative ρ-independent transcription terminator were also identified within katA. A similar katA cluster also existed in the C. lari RM2100 strain, except that this strain carries no DcuB genes. However, the Kat-like protein gene or any other homologue(s) were never identified in the C. lari RM2100 strain, or in C. jejuni and C. upsaliensis. CONCLUSIONS: This study demonstrates the presence of catalase/catalase-like protein genes in UPTC organisms. These findings are significant in that they suggest that UPTC organisms have the protective genetic capability of helping protect the organisms from toxic oxygen stress, which may help them to survive in physiologically harsh environments, both within human and animal hosts, as well as in the natural environment.

Transformation and characterization of an arsenic gene operon from urease-positive thermophilic Campylobacter (UPTC) in Escherichia coli.

An arsenate susceptibility test was performed with transformed and cultured Escherichia coli DH5α cells, which carried recombinant DNA of full-length arsenic (ars) operon, namely a putative membrane permease, ArsP; a transcriptional repressor, ArsR; an arsenate reductase, ArsC; and an arsenical-resistance membrane transporter, Acr3, from the Japanese urease-positive thermophilic Campylobacter lari (UPTC) CF89-12. The E. coli DH5α transformant showed reduced susceptibility to arsenate (~1536 µg/mL), compared to the control. Thus, these ars four-genes from the UPTC CF89-12 strain cells could confer a reduced susceptibility to arsenate in the transformed and E. coli DH5α cells. E. coli transformants with truncated ars operons, acr3 (acr3) and arsC-acr3 (∆arsC-acr3), of the ars operon, showed an MIC value of 384 µg/mL (~384 µg/mL), similar to the E. coli cells which carried the pGEM-T vector (control). Reverse transcription PCR confirmed in vivo transcription of recombinant full-length ars operon and deletion variants (∆acr3 and ∆arsC-acr3) in the transformed E. coli cells.

Construction and expression of a recombinant urease gene cluster from Campylobacter sputorum biovar paraureolyticus.

Recombinant full-length urease gene cluster and seven 100% deletion recombinant variants of urease subunits genes, (ureG, ureH, ureA, ureB, ureC, ureE and ureF) were constructed in vitro from the Campylobacter sputorum biovar paraureolyticus LMG17591 strain and expressed in Escherichia coli JM109 cells. A urease-positive reaction (1.885 micromol/min/mg protein) in the log-phase cultured E. coli cells transformed with pGEM-T vector carrying the recombinant full-length urease genes cluster was detected. Among the seven 100% deletion recombinant variants, each of the ureG-, ureH(D)-, ureA-, ureB-, ureC-, ureE- and ureF-deletion variants showed no change in assay of the urease reaction, and similarly as in the E. coli cell lysate with pGEM-T vector only. Recombinant full-length urease gene cluster and 100% deletion recombinants of the ureE gene in the transformed and log-phase cultured E. coli cells from the C. sputorum showed positively accelerated urease activities when cultured in the medium containing NiCl2 (750 micromol/L), but no activity was accelerated in the C. sputorum cultured in NiCl2. In addition, thiourea (20 mmol/L) completely inhibited urease activities from all C. sputorum examined. The putative recombinant urease subunits A and C were immunologically identified by Western blot analysis with polyclonal anti-urease alpha (A) and beta (B), raised against Helicobacter pylori.

Reliability of nucleotide sequence information of full-length flagellin A gene (flaA) and flaA short variable region (SVR) for molecular discrimination of Campylobacter lari organisms.

We aimed to clarify if the thermophilic Campylobacter lari organisms including urease-negative (UN) C. lari and urease-positive thermophilic Campylobacter (UPTC) can be differentiated at the species and/or subspecies levels by employing the full-length flaA gene and flaA short variable region (SVR) nucleotide sequence information or not. Thermophilic Campylobacter isolates (n = 45) including UN C. lari (n = 17), UPTC (n = 18), and Campylobacter jejuni (n = 10) were well discriminated at the isolate level by the unweighted pair group method using arithmetic means analysis and neighbor joining procedures constructed based on the full-length flaA gene and flaA SVR nucleotide sequence information. Thus, these procedures may possibly be useful for epidemimological studies for C. lari and C. jejuni.

Molecular analysis and characterization of a urease gene operon from Campylobacter sputorum biovar paraureolyticus.

When recombinant plasmid DNA from a genomic DNA library and inverse PCR products of Campylobacter sputorum biovar paraureolyticus LMG17591 strain were analyzed, an approximate 6.5-kb pair region, encoding a urease gene operon, was identified. Within the operon, seven closely spaced and putative open reading frames for ureG, ureH(D), ureA, ureB, ureC, ureE, and ureF were detected in order. A possible overlap was detected between ureG and ureH(D), ureH(D) and ureA, and ureE and ureF. In addition, two putative promoter structures, probable ribosome-binding sites and a putative ρ-independent transcriptional terminator structure were identified. The urease gene operon transcription in the cells was confirmed by the reverse transcription-PCR analysis. A neighbor-joining tree constructed based on the nucleotide sequence information of urease genes showed that C. sputorum biovar paraureolyticus formed a cluster with Arcobacter butzleri, urease-positive thermophilic Campylobacter and some Helicobacter spp., separating those from the other urease-producing bacteria, suggesting a commonly shared ancestry among these organisms.

EMMPRIN (CD147) expression and differentiation of papillary thyroid carcinoma: implications for immunocytochemistry in FNA cytology.

OBJECTIVE: Extracellular matrix metalloproteinase inducer (EMMPRIN) and its induced matrix metalloproteinases (MMPs) play a crucial role in tumour progression, invasion and metastasis. EMMPRIN expression has been demonstrated in several tumours, but its expression profile in thyroid cancer remains unclear. METHODS: We evaluated the expression profile of EMMPRIN at various stages of differentiation of thyroid carcinoma, including 20 cases of well-differentiated papillary carcinoma (WDPC), 15 cases of papillary carcinoma with a poorly differentiated carcinoma component (PC/PDC) and four cases with an undifferentiated carcinoma (UDC) component, using paraffin-embedded sections for immunohistochemical stains. Also, we used 32 fine needle aspiration cytology and imprint smears from the same cases for immunocytochemical stains. The staining results were evaluated with a scoring system. RESULTS: Immunohistochemical staining showed that EMMPRIN expression was absent or weak in almost all WDPC specimens, whereas it was moderate or strong in PDC and UDC components. In tumours that showed a gradual morphological transformation from WDPC to PDC components, the expression of EMMPRIN was progressively stronger from the areas of WDPC to those of PDC. WDPC, PC/PDC and UDC had expression scores of 4.9, 45.0 and 245.7, respectively. Results of immunocytochemical staining showed almost the same staining profile as those of immunohistochemical staining. The cytological atypia of EMMPRIN-positive cells was greater than that of negative cells. CONCLUSION: These results indicated that EMMPRIN expression correlates significantly with the degree of dedifferentiation of thyroid carcinoma. This study demonstrates the feasibility of expression of EMMPRIN using fine needle aspiration samples. Therefore, immunocytochemical analysis of EMMPRIN may be a novel aid to evaluate the differentiation of thyroid carcinoma.

Shock-wave mach-reflection slip-stream instability: a secondary small-scale turbulent mixing phenomenon.

Theoretical and experimental research, on the previously unresolved instability occurring along the slip stream of a shock-wave Mach reflection, is presented. Growth rates of the large-scale Kelvin-Helmholtz shear flow instability are used to model the evolution of the slip-stream instability in ideal gas, thus indicating secondary small-scale growth of the Kelvin-Helmholtz instability as the cause for the slip-stream thickening. The model is validated through experiments measuring the instability growth rates for a range of Mach numbers and reflection wedge angles. Good agreement is found for Reynolds numbers of Re 2 x 10(4). This work demonstrates, for the first time, the use of large-scale models of the Kelvin-Helmholtz instability in modeling secondary turbulent mixing in hydrodynamic flows, a methodology which could be further implemented in many important secondary mixing processes.

Enhanced vagal modulation and exercise induced ischaemia of the inferoposterior myocardium.

OBJECTIVE: To determine whether the Bezold-Jarisch reflex or enhancement of vagal nerves, which are preferentially distributed in the inferoposterior myocardium, results from exercise induced ischaemia in this region. METHODS: On the basis of exercise myocardial scintigraphy and coronary angiography, 145 patients were classified as follows: group I, 34 patients with inferoposterior ischaemia; group A, 32 with anterior ischaemia; and control, 79 without ischaemia. The relation between ischaemic areas and ECG leads with ST segment changes and vagal modulation assessed by heart rate variability (HRV) (high frequency (HF) component (0.15-0.40 Hz) and coefficient of HF component variance (CCVHF), which is the square root of HF divided by mean RR interval) were assessed. RESULTS: The rate of ST segment depression in any lead did not differ between group I and group A. HF and CCV(HF) were similar before exercise but higher in group I than in group A and the control group after exercise (mean (SEM) HF: 94 (17) ms2, 41 (7) ms2, and 45 (6) ms2, respectively, p = 0.021; CCV(HF): 1.18 (0.09)%, 0.81 (0.07)%, and 0.89 (0.05)%, p = 0.0053). Furthermore, the percentage change in CCV(HF) before and after exercise was higher in group I than in group A or controls (mean (SEM) 22 (10)%, -24 (4)%, and -21 (3)%, p < 0.0001). The optimal cut off for diagnosis of inferoposterior ischaemia was -5% with a sensitivity of 74%, specificity 75%, and accuracy 75%. CONCLUSIONS: Vagal modulation as assessed by HRV analysis was enhanced in association with exercise induced inferoposterior ischaemia. Exercise ECG testing combined with HRV analysis would increase accuracy in the diagnosis of ischaemic areas in selected patients with angina pectoris.

Bone growth oscillation: longitudinal metabolic process of bone growth in congenital adrenal hyperplasia and nonendocrine short stature.

To clarify the longitudinal metabolic process of bone growth in children, we observed the relationship between the level of serum osteocalcin (OC), a marker of bone metabolism, and growth velocity in 10 prepubertal patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency and 9 prepubertal patients with nonendocrine short stature (NESS), but no major hormonal abnormalities influencing bone metabolism. Observations were made every 6 months over a 7-year period. In patients with CAH who exhibited a wide variation in growth velocity during the course of the investigation, the levels of OC fluctuated over a wide range, suggesting metabolically variable bone growth. In contrast, in patients with NESS who exhibited a relatively stable growth velocity, the OC level remained within a narrow range, suggesting metabolically stable bone growth. The meaning of such divergent metabolic processes of bone growth observed in CAH and NESS and its relationship to actual bone structure or bone intensity should be further investigated.

Polymorphism of the polyalanine tract of thyroid transcription factor-2 gene in patients with thyroid dysgenesis.

OBJECTIVE: One of the thyroid-specific transcription factors, thyroid transcription factor-2 (TTF-2), performs a crucial role in the development of the thyroid gland. We performed genetic analysis of the TITF2 gene (encoding TTF-2) in patients with thyroid dysgenesis. METHODS: By direct sequencing of the PCR products of TITF2, we screened the genomic DNA from 46 patients with thyroid dysgenesis (five had agenesis, six had hypoplasia, 15 had ectopy, and 20 were undetermined). We also studied the transcriptional activities of TITF2 by co-expressing the luciferase gene directed by the human thyroglobulin gene promoter. RESULTS: Human TITF2 consists of a forkhead domain, a polyalanine tract, and unique C-terminal residues. In one of the patients with an ectopic sublingual thyroid, we found a polyalanine tract of 11 alanine residues on one chromosome instead of the 14 alanine residues found in normal controls. In one patient with hypoplasia, the polyalanine tract consisted of 12 heterozygous alanine residues. The reduced polyalanine tracts were not detected in 101 normal individuals. However, the expression study showed that the transcriptional activities of TITF2 with reduced polyalanine-tract lengths were equal to that of TITF2 with an unreduced polyalanine tract. CONCLUSION: These results suggest that the polymorphism of the polyalanine tract of TITF2 is not a frequent cause of developmental defects of the human thyroid gland.

Systemic effects of transdermal testosterone for the treatment of microphallus in children.

OBJECTIVES: To elucidate the metabolic effects of topical testosterone for the treatment of microphallus in children. METHODS: We administered 5% testosterone ointment to 50 prepubertal boys for the treatment of microphallus, allowing us to observe its metabolic effect on plasma concentrations of testosterone as a marker of transdermally absorbed testosterone, insulin-like growth factor (IGF)-I as a marker of growth hormone secretion status, and osteocalcin as a marker of bone metabolic turnover. RESULTS: Transdermal application of testosterone for 30 days at a dose that affects penile growth increased mean (+/-SD) plasma testosterone concentrations from 7.5+/-5.1 to 31.0+/-8.2 ng/dL (pre- vs. post-treatment, respectively; P<0.01). This was associated with a slight but statistically significant elevation of IGF-I concentrations (117.2+/-76.9 vs. 154.4+/-81.5 ng/mL; P<0.05). No significant change in osteocalcin levels was found. CONCLUSIONS: When using testosterone ointment as a treatment for microphallus, it should be borne in mind that this application has systemic effects.

Preliminary report: effect of adrenal androgen and estrogen on bone maturation and bone mineral density.

To clarify the independent physiological roles of adrenal androgen and estrogen on bone growth, we compared the lumbar spine bone mineral density (BMD) in prepubertal girls with virilizing congenital adrenal hyperplasia (CAH) (n = 17) and girls with central precocious puberty (CPP) (n = 18). When BMD was analyzed according to chronologic age, no significant differences were found between CPP and CAH patients. However, when adjusted to bone age, BMD was statistically higher in CAH than in CPP subjects. This finding suggests that adrenal androgen, as well as estrogen, plays an important role in increasing BMD. Adrenal androgen may act on bone not only as androgen, but as estrogen after having been metabolized into an aromatized bone-active compound in peripheral tissues, such as bone and fat. Therefore, adrenal androgen may have a more important role in increasing BMD than previously realized.

[Neonatal transient hypothyroidism].

Neonatal screening for congenital hypothyroidism can detect permanent and transient hypothyroidism. The latter condition is called neonatal transient hypothyroidism. This may result from various causes, but, especially, it should be suspected whenever there is a history of maternal autoimmune thyroid disease, including Hashimoto thyroiditis, Graves disease, hypothyroidism on replacement therapy, or recurrent congenital hypothyroidism of a transient nature in subsequent siblings. Transplacental TSH-receptor antibodies may affect fetal or neonatal thyroid function. Although thyroid dysfunction may be transitory, long-term follow-up is desirable.