Influence of breast-feeding on the production of cytokines.

PROBLEM: Recently, we reported increases in the production of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and IL-4 during the postpartum period. The present study was undertaken to investigate whether these increases might be explained by increased prolactin while breast-feeding. METHOD: Whole blood from 41 women who were breast-feeding, 13 women not breast-feeding, and 31 healthy non-pregnant women was stimulated with phorbol 12-myristate 13-acetate and ionomycin, and the levels of cytokines in the supernatant were measured by enzyme-linked immunosorbent assay. Their serum levels of prolactin were measured by enzyme immunoassay. RESULTS: Increases in IFN-gamma, IL-2, IL-4, and IL-10 production were observed in women who were breast-feeding but not in women who were not breast-feeding. Serum levels of prolactin correlated with the levels of IFN-gamma in culture supernatant. CONCLUSIONS: These results suggest that breast-feeding induces production of cytokines and that IFN-gamma production is enhanced by physiological concentrations of prolactin.

Enzyme-linked immunosorbent assay for anti-tropomyosin antibodies and its clinical application to various heart diseases.

Tropomyosin is one of the key proteins for muscle contraction. We developed an enzyme-linked immunosorbent assay for antibodies to porcine muscle tropomyosin and measured serum anti-tropomyosin antibodies in patients with heart diseases and in normal controls. The mean values of absorbance in the ELISA assay of patients with ischemic heart disease (n=36, P<0.001), dilated cardiomyopathy (n=28, P<0.005), valvular heart disease (n=27, P<0.05), and collagen disease (n=38, P<0.05) were significantly higher than those of normal controls (n=53), but the value in patients with hypertrophic cardiomyopathy (n=19) was not significantly different from that of normal controls. When the cut-off value was fixed at the mean+2 SD of absorbance in normal controls, positive reactions were found in 19.4%, 7.1%, 18.5% and 15.8% of patients with ischemic heart disease, dilated cardiomyopathy, valvular heart disease, and collagen disease, respectively. An inhibition study revealed that anti-tropomyosin antibodies were different from anti-myosin antibodies, but there was a partial cross-reactivity between the two. Thus, there was a weak correlation of the titers of the two types of antibody within the group of heart diseases. These data indicate that measurement of anti-tropomyosin antibodies by ELISA is helpful for detecting autoimmune abnormalities in various heart diseases.

[Serum TSH measurement].

Serum TSH (thyrotropin) measurement is the first-line test to evaluate the thyroid function. Recent sensitive serum TSH assays are based on immunometric assays (IMAs) that use two or more antibodies. Electrochemiluminescent immunoassay expanded both the functional sensitivity potential and the range of serum TSH measurement. It is usually combined with serum free T4 measurement to evaluate the thyroid function, but can be used alone when the pituitary function is normal. Serum TSH levels do not reflect the thyroid function, i) when thyroid function is changing widely, ii) in low T4-T3 states, iii) in central hyperthyroidism or hypothyroidism, and iv) when antibodies such as heterophile antibodies, rheumatoid factors, and rarely anti-TSH antibodies are present.

CD30 expression and interleukin-4 and interferon-gamma production of intrathyroidal lymphocytes in Graves' disease.

We reported that serum levels of interleukin-5 (IL-5) and soluble CD30, mainly secreted from T helper 2 (Th2) cells, were increased in Graves' disease. To clarify the immune balance of Th1/Th2 within the Graves' thyroid gland, we have compared the expression of CD30, a preferential marker for T cells producing type 2 cytokines, and the production of interferon-gamma (IFN-gamma) and IL-4 between intrathyroidal lymphocytes (ITL) and peripheral blood lymphocytes (PBL). In PBL, none of these parameters were different between patients and normal subjects. The proportion of CD30+ cells in ITL was markedly higher (5.1%+/-2.8%, p < 0.0001) than that in patients' PBL (0.4%+/-0.3%). Likewise, both the proportions of IFN-gamma+ (14.8%+/-5.5%) and IL-4+ cells (2.4%+/-0.5%) in ITL were higher than those in PBL (9.6%+/-2.5%; p < 0.01, 1.5%+/-0.4%; p < 0.0001, respectively). The proportion of type 0 (both IFN-gamma and IL-4 positive, 1.0%+/-0.4% p < 0.001), type 1 (IFN-gamma positive, 14.0%+/-5.6%, p < 0.01) or type 2 cells (IL-4 positive, 1.4%+/-0.5%, p < 0.05) in ITL was significantly higher as compared with those in PBL (0.4%+/-0.1%, 9.0%+/-2.4%, 1.1%+/-0.3%, respectively). The ratios of ITL/PBL in CD30+ (23.3+/-30.6) and type 0 cells (2.5+/-1.2) were higher than the ratios in other subsets. The proportion of CD30+ cells correlated with the proportion of type 0 cells (r = 0.686, p < 0.01), but not with type 1 or type 2 cells. These findings suggest that there is no obvious deviation of Th2/Th1 profile in the Graves' thyroid gland, although intrathyroidal CD30+ T cells and Th0 cells may play some role in the development of autoimmune abnormalities in Graves' disease.

Increased serum concentration of interleukin-12 in patients with silent thyroiditis and Graves' disease.

We previously reported that interleukin-5 (IL-5), secreted from Th2 cells, was increased in patients with Graves' disease, but not in patients with silent thyroiditis. In this study, we investigated serum levels of interleukin-12 (IL-12) in order to examine the role of Th1-type immune response in the pathogenesis of autoimmune thyroid diseases. Serum levels of IL-12 were determined by a highly sensitive sandwich enzyme-linked immunosorbent assay in 68 patients with Hashimoto's thyroiditis (26 of whom had silent thyroiditis), 74 patients with Graves' disease, 8 patients with subacute thyroiditis, and 27 normal controls. Serum levels of IL-12 in thyrotoxic patients with silent thyroiditis (385.2 +/- 164.5 pg/mL, mean +/- SD), and in thyrotoxic patients with Graves' disease (343.6 +/- 163.8 pg/mL) were significantly increased compared with serum levels in normal subjects (163.9 +/- 66.8 pg/mL, p < 0.0001, p < 0.0001, respectively) or in thyrotoxic patients with subacute thyroiditis (241.9 +/- 46.5 pg/mL, p < 0.01, < 0.05, respectively). The ratio of IL-12 to IL-5 in thyrotoxic patients with silent thyroiditis (64.2 +/- 39.7) was significantly higher than that in normal controls (33.7 +/- 13.3, p < 0.01) or in thyrotoxic patients with Graves' disease (40.6 +/- 36.0, p < 0.05). These data suggest that Th1-type immune response is predominant in silent thyroiditis, and that not only Th2-type immune response but also Th1-type immune response is important in the pathogenesis of Graves' disease.

Intraluminal duodenal diverticulum in a child: incidental onset possibly associated with the ingestion of a foreign body.

UNLABELLED: Funnel-type intraluminal duodenal diverticulum (windsock web) is a rare congenital malformation. A 4-year-old boy with vomiting and abdominal pain for several weeks was referred to the hospital. A plain abdominal X-ray on admission disclosed a double bubble sign. Abdominal echography and CT disclosed a foreign body lodged in the alimentary tract. After the foreign body was removed with a fibrescope, endoscopy showed a stenotic descending portion where the foreign body was located. An upper gastro-intestinal contrast study demonstrated a post-bulbar duodenal stenosis with a barium-filled pear-shaped sac in the descending portion of the duodenum. Surgical exploration was done under the diagnosis of windsock web of the duodenum. A simple excision of the web at its base was carried out. A hole 7 mm in diameter was found at the edge of the web. The microscopic appearance of the resected specimen was characterized by the duodenal mucosa with an extensive chronic inflammation lining both sides of the diverticulum and the lack of muscular layer of mucosa. CONCLUSION: If an ingested material is not excreted in the stool, possible clogging in the intestinal tract should always be considered and a further intensive examination is warranted.

The in vitro effect of macrolides on the interaction of human polymorphonuclear leukocytes with Pseudomonas aeruginosa in biofilm.

The in vitro effect of macrolides at concentrations below the minimum inhibitory concentration (sub-MIC) on the interaction between Pseudomonas aeruginosa biofilm and human polymorphonuclear leukocytes (PMNs) was investigated by using a chemiluminescence assay. The PMN response to either mucoid or nonmucoid P. aeruginosa biofilm was strongly reduced compared with the response to planktonic bacteria (p < 0.01, p < 0.001, respectively). When biofilms were treated with erythromycin, clarithromycin, roxithromycin and azithromycin prior to incubation with PMNs, the chemiluminescence response was markedly enhanced as compared to untreated controls, and a dose-dependent effect was noted over the range of sub-MIC concentrations studied. In general, macrolides appeared to be slightly more active against mucoid biofilm. Azithromycin was shown to be the most active macrolide against P. aeruginosa biofilms. However, the treatment with sub-MICs of rokitamycin did not have any effect. On the other hand, treatment of planktonic bacteria with macrolides before being exposed to the PMNs did not affect the chemiluminescence response as compared to untreated controls. These findings suggest that macrolides inhibiting the biofilm formation of P. aeruginosa may facilitate the phagocytosis of bacteria by PMNs.

Increased serum concentration of interleukin-5 in patients with Graves' disease and Hashimoto's thyroiditis.

We investigated serum levels of interleukin-5 (IL-5) in order to examine the role of T-helper 2 (Th2)-type immune response in the pathogenesis of autoimmune thyroid diseases. Serum levels of IL-5 were determined by a highly sensitive sandwich enzyme-linked immunosorbent assay in 42 patients with Graves' disease, 32 patients with Hashimoto's thyroiditis, 12 patients with silent thyroiditis, and 21 normal controls. Compared with serum levels in normal subjects (5.8 +/- 4.2 pg/mL), IL-5 was increased in patients with Graves' disease (16.4 +/- 16.7 pg/mL, p < .01), and in patients with Hashimoto's thyroiditis (10.0 +/- 7.6 pg/mL, p < .05), but not in patients with silent thyroiditis. There was no correlation between serum free thyroxine (FT4) and IL-5 levels. These data suggest an important role of the Th2-type immune response in the pathogenesis of Graves' disease and Hashimoto's thyroiditis.

Serum concentration of soluble Fas in patients with autoimmune thyroid diseases.

Fas is an apoptosis-signaling receptor molecule found on the surface of a number of cell types. Malfunction of the Fas system accelerates autoimmune diseases, whereas its exacerbation may cause tissue destruction. Soluble Fas (sFas) molecule lacks the transmembrane domain due to alternative splicing and blocks Fas-mediated apoptosis. This study investigated serum levels of sFas in autoimmune thyroid diseases. Serum levels of sFas were determined by enzyme-linked immunosorbent assay in 46 patients with Graves' disease, 32 patients with Hashimoto's thyroiditis, 14 patients with silent thyroiditis, and 24 normal controls. Compared with normal subjects (1.43+/-0.37 ng/mL), sFas was increased in thyrotoxic patients with Graves' disease (1.89+/-0.47 ng/mL, p < 0.001), and was decreased in patients with Graves' disease in remission (1.02+/-0.41 ng/mL, p < 0.001) and in euthyroid patients with Hashimoto's thyroiditis (0.97+/-0.25 ng/mL, p < 0.0001), but was normal in hypothyroid patients with Hashimoto's thyroiditis and in thyrotoxic patients with silent thyroiditis. Thus, changes in serum levels of sFas could not be explained by changes in serum thyroid hormones, although sFas concentration correlated with free thyroxine (r = 0.692, p < 0.0001). Also, the levels of sFas significantly correlated with the activities of TSH receptor antibody in Graves' disease (r = 0.671, p < 0.0001). Increased sFas in Graves' disease suggests increased expression of alternatively spliced Fas mRNA variant that produces sFas protein and decreased of cell surface expression of Fas, and may induce thyroid cell growth and production of TSH receptor antibody by protecting against apoptosis of thyroid cells and autoreactive B cells. Decreased sFas in Hashimoto's thyroiditis suggests decreased Fas mRNA variant and increased full-length Fas mRNA and membrane Fas, and may induce destruction of thyroid cells by promoting apoptosis of thyroid cells.

Increased serum concentration of soluble CD30 in patients with Graves' disease and Hashimoto's thyroiditis.

This study investigated serum levels of the soluble form of CD30 (sCD30), which is mainly secreted from T helper 2(Th2) cells, in autoimmune thyroid diseases. The possible relationship of sCD30 to autoantibody production was also evaluated. Serum levels of sCD30 were determined by an enzyme-linked immunosorbent assay in 71 patients with Graves' disease, 37 patients with Hashimoto's thyroiditis, and 21 normal donors. Compared with normal subjects (7.1 +/- 4.5 U/mL), sCD30 was increased in patients with Graves' disease (29.2 +/- 25.2 U/mL, P < 0.0001) and in patients with Hashimoto's thyroiditis (29.9 +/- 26.9 U/mL, P < 0.0001). In Graves' disease, sCD30 levels were higher in thyrotoxic patients (41.7 +/- 31.2 U/mL, P < 0.001) than in remission patients (15.8 +/- 11.0 U/mL), and a significant correlation was observed between sCD30 levels and serum activities of TSH receptor antibody (r = 0.444, P < 0.0001). In Hashimoto's thyroiditis, sCD30 levels were higher in patients with transient destructive thyrotoxicosis caused by the aggravation of the disease (48.8 +/- 34.4 U/mL, P < 0.05) than in euthyroid patients (24.2 +/- 19.4 U/mL). These data suggest that serum sCD30 is a valuable marker of disease activity and support an important role of the Th2-type immune response in the pathogenesis in Graves' disease and Hashimoto's thyroiditis.

Preparation and Characterization of New Self-Assembled Polyamide Film Containing Disulfide Bonds on Gold Electrode

New polyamides containing disulfide bonds in their main chains are prepared by condensation between 3,3'-dithiodipropionic acid and various alkyldiamines (NH2-(CH2)n-NH2, n = 4 and 12, polyamide I and II). The polyamides form a thin layer on a gold substrate. The surface of polyamide modified electrodes were investigated by AFM and XPS measurements. The surface structure depends on the polyamide structure, especially alkyl chain length of the polyamide. Furthermore, adsorption of ferrocene derivatives [1-ferrocenylmethanol (FME) or 2-amino-3-ferrocenylpropionic acid (AFP)] on the polyamide modified electrodes and their electrochemical responses were investigated. Interaction between the polyamide on the electrode and the ferrocene derivatives depends on the structure of the ferrocene derivatives and polyamide chain length.

Behçet's disease associated with myelodysplastic syndromes. A case report and a review of the literature.

BACKGROUND: Behçet's disease has rarely been reported in association with myelodysplastic syndromes (MDS). Increased production of reactive oxygen species (ROS) by neutrophils has a primary role in the pathogenesis of Behçet's disease. However, decreased production of ROS by neutrophils has frequently been reported in patients with MDS. The current study was undertaken to determine the role of ROS production in a patient with Behçet's disease and MDS. METHODS: A patient with MDS with trisomy 8 who developed Behçet's disease is described and a review of the literature of patients with Behçet's disease in MDS is presented. The production of ROS by neutrophils was investigated by luminol-enhanced chemiluminescence (CL) assay. RESULTS: Based on a review of the literature, 10 cases of Behçet's disease associated with MDS have been reported to date. Nine patients had undergone cytogenetic analysis of bone marrow cells, 7 of whom (78%) had trisomy 8. Neutrophils taken from the authors' patient during the active phase of Behçet's disease demonstrated an increased CL response. Moreover, serum from this patient increased the CL emission of neutrophils from healthy volunteers. CONCLUSIONS: These data suggest that trisomy 8 predisposes to Behçet's disease in patients with MDS. Furthermore, an increased ROS production by neutrophils may be associated with the diverse clinical findings in this disease. In this study, neutrophils were activated directly by serum factors.

Onset of Graves' thyrotoxicosis after an attack of allergic rhinitis.

We previously reported that allergic rhinitis was an aggravating factor for Graves' disease and that thyrotoxicosis relapsed 2 months after an allergic attack. In this paper, we report a patient who showed onset of Graves' thyrotoxicosis after an attack of allergic rhinitis. The patient, a 30-year-old woman, was initially diagnosed with subclinical autoimmune thyroiditis. Interestingly, the patient showed weak activity of thyroid-stimulating antibody (TSAb), while TSH-binding inhibitory immunoglobulin (TBII) was negative and her thyroid function tests, including TSH, were completely normal. The patient developed severe allergic rhinitis in response to Japanese cedar pollen lasting from February until April in 1995 with an increase in serum antigen-specific immunoglobulin E and peripheral blood eosinophils. Two months later, she developed thyrotoxicosis in association with increase in TSAb and TBII. These findings suggest that allergic rhinitis not only aggravates Graves' disease but also induces the clinical onset of Graves' thyrotoxicosis.

The influence of azithromycin on the biofilm formation of Pseudomonas aeruginosa in vitro.

The influence of azithromycin on biofilm formation by Pseudomonas aeruginosa, a cause of refractory chronic respiratory tract infection, was investigated. Alginic acid produced by a mucoid strain of P. aeruginosa was quantified by high-performance liquid chromatography from colonies growing on an agar medium. Polysaccharides in the biofilm formed on silicon chips by a nonmucoid strain were determined by a tryptophan reaction. The effect of azithromycin was examined at concentrations below the minimum inhibitory concentration (sub-MIC) for each strain. Azithromycin significantly inhibited the production of alginic acid from the mucoid strain at > or = 1/256 MIC, and the production of exopolysaccharides from the nonmucoid strain at > or = 1/16 MIC. The inhibition of biofilm formation by azithromycin was also observed by scanning electron microscopy. These findings suggest that azithromycin inhibits biofilm formation by P. aeruginosa at concentrations well below the MIC.

A new technique for quantitative imaging of cerebrovascular reserve capacity using a double injection method with N-isopropyl-p-[123I]iodoamphetamine.

Estimation of local cerebrovascular reserve capacity is an important aspect of the management of patients with cerebrovascular occlusive disease. Regional normal values of cerebrovascular reserve capacity have not yet been established. Recently, Mori et al. (1994) introduced a method to quantitatively measure cerebral blood flow twice in 30 min after a double injection of N-isopropyl-p-[123I]iodoamphetamine using a background subtraction method. We utilized this double injection method in tandem with acetazolamide (20 mg/kg, intravenous injection) in 10 normal volunteers to produce a map of quantitative cerebrovascular reserve capacity using single photon emission computed tomography. The normal regional cerebrovascular reserve capacity was: frontal lobe, 51.0 +/- 19.8%; temporal lobe, 53.6 +/- 12.4%; parietal lobe, 69.3 +/- 31.8%; basal ganglia, 85.1 +/- 42.7%; cerebellum, 49.6 +/- 17.9% (mean +/- SD). One-way analysis of variance showed that the cerebrovascular reserve capacity did not differ significantly among structures. This new imaging technique and normal values of regional cerebrovascular reserve capacity may provide information about the pathophysiology and surgical indication in patient with occlusive cerebrovascular disease.

[Intra-individual and seasonal variations of thyroid function tests in healthy subjects].

We report intra-individual and seasonal variations of thyroid function tests in healthy subjects. Blood samples were obtained from thirteen healthy males and seven healthy females every two weeks over a period of one year, and totally 25 samplings of each were made. Serum thyrotropin (TSH), free thyroxine (FT4) and free triiodothyronine (FT3) were measured after the completion of the sampling. The 25 samples from each subject were always assayed with the same assay run. Variations of FT4 and FT3 in each subject were narrow and approximately one-third of normal reference ranges. The magnitude of individual variation of TSH values was proportional to the average of TSH in each individual. Serum TSH and FT3 values during winter were significantly higher than those during summer, but such change was not observed on serum FT4.

Novel thyrotropin (TSH)-TSH antibody complex in a healthy woman and her neonates.

A novel high-molecular-weight (MW) form of immunoreactive TSH [35,000 Da on Sephacryl S-200HR gel chromatography (S-200 chromatography)] was documented in a 32-year-old healthy woman who delivered two neonates with transient hyperthyrotropinemia. Her TSH levels ranged from 21.2 to 53.9 mU/L on different days or from 11.0 to 48.1 mU/L by the different immunoradiometric assay methods. The IgG fractions showed specific 125I-labeled hTSH binding and inhibited in vitro cAMP increase induced by hTSH but not that induced by bTSH. On protein G Superose HR affinity chromatography (protein G chromatography) equilibrated with 10 mmol/L sodium/potassium phosphate buffer (PB) followed by elution with 0.1mol/L glycine buffer, 95-99% of her TSH immunoreactivity eluted in the latter (bound) fraction while almost all was in the former (unbound) fraction in the control serum containing authentic hTSH. However, after dialysis of this bound fraction overnight with PB adding 0.5 mol/L NaCl (PB/NaCl), which exhibited greater ionic strength than PB, almost all TSH immunoreactivity changed from the bound fraction into the unbound fraction on the protein G chromatography equilibrated with PB/NaCl. These data indicate that the novel immunoreactive TSH was due to hTSH and hTSH-specific antibody complex, and dissociation of the complex may be incomplete on direct S-200. The immunoreactive TSH showed high MW form (35,000 Da). The dissociation may be almost complete during dialysis with greater ionic strength; the native TSH then appeared to be of formal size.

Pathogenic role of asialo human chorionic gonadotropin in gestational thyrotoxicosis.

We reported that gestational thyrotoxicosis is induced by thyroid-stimulating activity (TSA) of circulating hCG. However, the serum immunological hCG concentration did not correlate to TSA. To elucidate this, we examined the relation of carbohydrate moieties of hCG to bioactivity in 79 early pregnant women, divided into 4 groups: no emesis, mild emesis, hyperemesis, and gestational thyrotoxicosis with hyperemesis. Serum free T4 (FT4) and free T3 (FT3) levels were significantly higher and TSH was lower in the hyperemesis (FT4, 23.42 +/- 5.02 pmol/L; FT3, 6.26 +/- 1.80 pmol/L; TSH, 0.30 +/- 0.44 mU/L) and in gestational thyrotoxicosis (FT4, 48.65 +/- 14.80 pmol/L; FT3, 14.71 +/- 3.47 pmol/L; TSH, < 0.04 mU/L) groups than in the no emesis group (FT4, 16.99 +/- 2.48 pmol/L; FT3, 5.51 +/- 0.75 pmol/L; TSH, 1.37 +/- 1.23 mU/L; P < 0.0005). TSA was also significantly higher in the hyperemesis (566 +/- 187%) and gestational thyrotoxicosis (832 +/- 168%) groups than in the no emesis group (321 +/- 135%). We found no significant difference among serum hCG concentrations measured by immunoassay in the four groups. To characterize the carbohydrate chains, serum hCG was fractionated by Concanavalin-A and ricin lectin affinity chromatography. The fraction firmly bound to Con-canavalin-A, which contains hCG with high mannose and hybrid-type carbohydrate chains, was significantly higher in the hyperemesis group (91.07 +/- 2.06%; n = 15) than in the no emesis group (89.61 +/- 2.38%; n = 24; P < 0.04). The fraction firmly bound to ricin column, which contains hCG with asialo-carbohydrate chains, was significantly increased in the gestational thyrotoxicosis group (3.44 +/- 1.70%; n = 5) compared with that in the no emesis group (1.77 +/- 0.49%; n = 24; P < 0.03). Serum FT4 positively correlated to the hCG fraction firmly bound to ricin column (r = 0.61; P < 0.001). We conclude that thyrotoxicosis with hyperemesis may be caused by circulating asialo-hCG with higher thyrotropic bioactivity.