Toll-like receptor signalling induces the expression of serum amyloid A in epidermal keratinocytes and dermal fibroblasts.

BACKGROUND: Toll-like receptors (TLRs) play critical roles in innate immune response by sensing pathogen- or damage-associated molecular patterns. Epidermal keratinocytes and dermal fibroblasts also produce proinflammatory cytokines and chemokines under stimulation with TLR ligands. Serum amyloid A (SAA) is an essential factor in the pathogenesis of secondary amyloidosis, and also has immunomodulatory functions. SAA are produced mainly by hepatocytes but also by a variety of cells, including immune cells, endothelial cells, synoviocytes, and epidermal keratinocytes. However, SAA expression in human dermal fibroblasts has not been shown to date. AIM: To investigate the effect of TLR ligands on SAA expression in epidermal keratinocytes and dermal fibroblasts. METHODS: We investigated whether TLR ligands induce the expression of SAA in normal human epidermal keratinocytes (NHEKs) and normal human dermal fibroblasts (NHDFs) by real-time quantitative PCR and ELISA. The effect of SAA on its own expression in NHDFs was also studied. RESULTS: SAA expression was induced via nuclear factor-κB by TLR1/2, 3, 5 and 2/6 ligands in NHEKs. In NHDFs, TLR1/2 and TLR2/6 ligands increased SAA expression. SAA further induced its own expression via TLR1/2 and NF-κB in NHDFs, as previously reported for NHEKs. CONCLUSIONS: Our results provide new evidence that the skin's innate immune response contributes to the production of SAA, which might lead to an increased risk of systemic complications such as secondary amyloidosis of recessive dystrophic epidermolysis bullosa.

Interferon-Gamma Enhances TLR3 Expression and Anti-Viral Activity in Keratinocytes.

Toll-like receptors (TLRs) recognize specific microbial products in the innate immune response. TLR3, a double-stranded RNA sensor, is thought to have an important role in viral infections, but the regulation of TLR3 expression and its function in keratinocytes are not fully understood. Here we show the Th1 cytokine IFN-γ increased the TLR3 expression via STAT1 in cultured normal human epidermal keratinocytes (NHEKs). Co-stimulation with IFN-γ and the TLR3 ligand poly (I:C) synergistically increased the expression of IFN-ß, IL-6, IL-8, and human ß-defensin-2 in NHEKs compared with poly (I:C) or IFN-γ alone. These synergistic inductions were significantly inhibited by an endosomal acidification inhibitor, chloroquine, and by TLR3 siRNA. Co-stimulation with IFN-γ and poly (I:C) also significantly enhanced the anti-viral activity against herpes simplex virus type-1 in NHEKs compared with poly (I:C) or IFN-γ alone. In addition to the in vitro findings, an immunohistochemical analysis revealed IFN-γ-positive cells surrounding herpetic vesicles. These findings indicate that IFN-γ might contribute to the innate immune response to cutaneous viral infections by enhancing TLR3 expression and function in keratinocytes.

Cathelicidin antimicrobial peptide LL-37 augments interferon-ß expression and antiviral activity induced by double-stranded RNA in keratinocytes.

BACKGROUND: Cathelicidin antimicrobial peptide LL-37 has the capacity to kill a wide range of microbes and to modify host immunity. Recently, our group observed that the activation of keratinocytes by LL-37 and DNA greatly increases interferon (IFN)-ß through Toll-like receptor (TLR)9. However, the effect of LL-37 on the induction of IFN-ß through TLR3, a sensor of double-stranded (ds) RNA, in keratinocytes is not well known. OBJECTIVES: To investigate whether LL-37 could affect TLR3 signalling and antiviral activity in normal human epidermal keratinocytes (NHEKs). METHODS: We investigated the production of IFN-ß in NHEKs stimulated with a TLR3 ligand, poly (I:C), in the presence of LL-37. To examine the effect of LL-37 and poly (I:C) on antiviral activity, a virus plaque assay using herpes simplex (HS) virus type-1 was carried out. The uptake of poly (I:C) conjugated with fluorescein isothiocyanate (FITC) into the keratinocytes was observed in the presence of LL-37. Immunostaining for TLR3 and LL-37 was performed using skin samples from HS. RESULTS: LL-37 and poly (I:C) synergistically induced the expression of IFN-ß in NHEKs. Furthermore, co-stimulation with LL-37 and poly (I:C) significantly decreased the viral plaque numbers compared with poly (I:C) or LL-37 alone. LL-37 enhanced the uptake of FITC-conjugated poly (I:C) into cells. Immunohistochemical analysis demonstrated that the expression of TLR3 and LL-37 is upregulated in HS lesions. CONCLUSIONS: Our findings suggest that LL-37 augments the antiviral activity induced by dsRNA in keratinocytes, which may contribute to the innate immune response to cutaneous viral infections such as HS.

Relationship among risk factors of atherosclerosis, leukocyte count, and soluble intercellular adhesion molecule-1.

We investigated the effects of risk factors of atherosclerosis on soluble intercellular adhesion molecule-1 (sICAM-1) concentration and leukocyte count by using multivariate statistical analysis. The subjects were 90 people who were hospitalized for a complete check-up examination. Eight risk factors were selected as candidates for predictor variables: age, body mass index (BMI), uric acid, glycohemoglobin A1c, number of cigarettes smoked daily, total cholesterol, triglyceride, and high density lipoprotein (HDL)-cholesterol. The predictor variables were selected using a stepwise method, [criterion variable - predictor variable (standardized regression coefficient)]; sICAM1 - age (0.1859), number of cigarettes (0.2558), triglyceride (0.2447); leukocytes - number of cigarettes (0.2827), triglyceride (0.2526), HDL-cholesterol (-0.2800); stab leukocytes - number of cigarettes (0.2460); segmented leukocytes - glycohemoglobin A1c (0.1495), number of cigarettes (0.2716), HDL-cholesterol (-0.3254); lymphocytes - BMI (0.2639), number of cigarettes (0.1495), triglyceride (0.3520); monocytes - glycohemoglobin A1c (0.2617). These findings indicated that the risk factors of atherosclerosis may influence sICAM1 concentration and leukocyte count.

Detection and monitoring of simple renal cysts with computed tomography.

We retrospectively investigated the incidence of simple renal cysts in 1,288 healthy subjects, and monitored the growth and formation of such cysts in 39 subjects using abdominal computed tomography (CT). Simple renal cysts were detected in 138 (10.7%) of the 1,288 subjects. The incidence of simple renal cysts increased significantly with the subject's age (p < 0.001). We found that cyst size increased significantly with age and over time on serial CT studies (p < 0.001). Exoparenchymal cysts, comprising greater than 50% of extraparenchymal lesions, were significantly larger than endoparenchymal cysts (p < 0.001), and showed a greater tendency to increase in size over time.

Sensitivity and specificity of lung cancer screening in Osaka, Japan.

Sensitivity and specificity were evaluated for lung cancer screening conducted at 8 municipalities in Osaka Prefecture during 1981-1985. As a screening policy, all attendants were examined by miniature chest X-ray, and the high-risk group, defined as those who smoked cigarettes or had bloody sputum, were also examined by 3-day pooled sputum cytology. A total of 33,599 screening tests for 19,028 people who were 40 years old or more at the time of screening were conducted, resulting in 33,490 miniature chest X-ray examinations for 18,992 people and 11,420 sputum cytologies for 7,070 people. As a result, 43 lung cancer cases were detected. All test-negatives were followed by means of record linkage with the files of the Osaka Cancer Registry up to the end of 1986. There were 24 cases who were diagnosed as having lung cancer without having given a positive screening result in 1981-1986. Assuming the preclinical detectable phase of lung cancer to be one year uniformly, the sensitivity and specificity for the lung cancer screening were estimated to be 71.6% and 95.3%, respectively. The feasibility of increasing the sensitivity is discussed.

Analysis of oxygen equilibria of the giant hemoglobin from the earthworm Eisenia foetida using the Adair model.

Oxygen equilibrium curves of the giant hemoglobin from the earthworm Eisenia foetida were determined at various concentrations of cations. Using the Adair model of 12 oxygenation steps, we succeeded in fitting the data better than the simple concerted model (MWC model). Analysis of the Adair constants (K1 to K12) indicated that the increase in oxygen affinity occurs in the last six steps (K7 to K12) of the oxygen binding and that it is enhanced by increase in Ca2+ concentration. The Hill coefficient (nmax) at pH 7.5 attained a maximum value of 9.76 at 20 mM CaCl2. In the presence of physiological levels of Ca2+ (5 mM), the Bohr effect was similar to that seen in vertebrates. The data were consistent with the release of two Bohr protons being accompanied by the oxygen-linked binding of one Ca2+. Mg2+ and Na+ exerted a similar effect on the hemoglobin, though to a lesser extent. The stoichiometry of Ca2+ binding of the hemoglobin revealed the presence of two classes of binding sites, of which the affinities are high (Ka = 8.8 x 10(3) +/- 103 M-1) and low. The number of high affinity sites per heme was found to be 0.3, comparable to the number of oxygen-linked Ca2+ binding sites.

Dissociation of Limulus polyphemus (horseshoe crab) hemocyanin. I. Solution X-ray scattering study in equilibrium.

A solution X-ray scattering study has been performed on Limulus polyphemus (horseshoe crab) hemocyanin and its dissociated fragments at various pH values in the presence and absence of Ca2+. The scattering patterns of native hemocyanin (48-mer), the half molecule (24-mer), quarter molecule (12-mer) and monomer fraction were measured. The radii of gyration for the four molecular species were calculated from the Guinier plots to be 110.7, 91.3, 77.3, and 36.5 A, respectively. Models which yield good fits to the experimental data are presented. The models were constructed using eight, four and two spheres with a radius of 58 A, assuming the sphere to be the submultiple composed of six subunits. The radii of gyration were calculated on the basis of the model and the values found to be 106, 94 and 73 A, respectively, in good agreement with the experimental results.

[Roentgenographic manifestations of peripheral bronchogenic carcinomas].

Peripheral bronchogenic carcinomas in early clinical stage are discovered by plain chest roentgenogram, and good prognosis will be expected by surgical resection. 20 bronchogenic carcinomas (18 adenocarcinomas, one squamous cell carcinoma, and one large cell carcinoma) in 19 patients were examined. 9 cases presented noduler lesion on the plain chest roentgenogram, and the other presented 7 focal patchy shadow and 4 band-like shadow. Thin-section CT could demonstrated indented pleura (95%), notch (95%), convergence of peripheral vessels (70%), irregular border (75%). In adenocarcinomas, thin-section CT demonstrated airbronchogram or airbronchiologram (61%), multiple small cavitation (61%), and heterogeneous density (56%). CT-pathologic correlation proved thin-section CT could demonstrate tumor's internal texture and fine detail of tumor-lung interface. Small peripheral lung cancer in early stage could be discovered by plain chest roentgenogram, and thin-section CT was useful for the diagnosis of small peripheral bronchogenic carcinomas, especially adenocarcinomas.

[Airbronchogram sign in small peripheral bronchogenic carcinomas].

18 bronchogenic carcinomas (16 adenocarcinomas, one squamous cell carcinoma, and one large cell carcinoma) in 17 patients and the control group (benign nodules) were examined with high resolution and thin section CT. Thin-section CT images showed airbronchogram or airbronchiologram in 63%, multiple small cavitations in 63%, and heterogeneous densities 50% of the adenocarcinomas with excellent spatial and contrast resolution. Airbronchogram or airbronchiologram was not seen in other types of bronchogenic carcinomas and benign nodules. Therefore, demonstration of airbronchogram or airbronchiologram in nodular lesions can make the CT diagnosis of small peripheral bronchogenic carcinomas, especially adenocarcinomas.

[Computed tomography of gastric cancer].

CT scans of 52 patients with advanced gastric cancer were reviewed, and the layer structure of the gastric wall was analyzed. Administration of 600 ml water, orally, expanded the gastric wall, while patients were in the prone position. Intravenous infusion of contrast media was routinely administered. We found that the normal layer structure of the gastric wall could be shown despite the cancer in the lesion. Borrmann 3 and 4 types. We guess that CT has a possibility in the assessment of the spread in the wall of gastric cancer.

Visualization of jugular vein on a thallium-201 scan for thyroid cancer.

A 52 year-old woman with recurrent thyroid cancer showed an accumulation of Tl-201 chloride in the left side of the neck. This proved to be reflux and retention of Tl-201 chloride in the left internal jugular vein and was verified with a Tc-99m HSA flow study. This phenomenon could be mistaken for metastases of thyroid cancer.

Oxygen exchange reaction during ATP hydrolysis by glycerinated muscle fibers, myofibrils, and synthetic actomyosin filaments.

The oxygen exchange during ATP hydrolysis by glycerinated muscle fibers, myofibrils, and synthetic actomyosin filaments was studied from the distribution of the [18O]Pi species produced by the hydrolysis of [gamma-18O]ATP. The products were mixtures of two species, one with a low extent of oxygen exchange and the other with a high extent. The low and high extents of oxygen exchange in these two Pi species were the same as those of the acto-S-1 ATPase reaction through the routes with and without the dissociation of actomyosin, respectively (Yasui, M., Ohe, M., Kajita, A., Arata, T., & Inoue, A. [1988] J. Biochem. 104, 550-559). During isometric contraction of glycerinated muscle fibers at 20 degrees C, the fraction of ATP hydrolysis with low extent of oxygen exchange was 0.83 and 0.70, respectively, in 0 and 120 mM KCl. In myofibrils, the fraction of ATP hydrolysis with a low extent of oxygen exchange was 0.72-0.88 in 0-120 mM KCl at 20 degrees C. Therefore, in glycerinated muscle fibers and myofibrils ATP seems to be mainly hydrolyzed through a route without the dissociation of actomyosin, especially at low ionic strength and at room temperature when the tension development is high. ATP hydrolysis through this route may be coupled with muscle contraction.

Cardiomyopathy characterized by abnormal accumulation of desmin-type intermediate filaments in cardiac muscle fibers. A case report and review of the literature.

A 42-year-old Japanese male, who had been suffering from congestive heart failure and electrocardiographic abnormalities (A-V block, intraventricular conduction disturbance, ventricular tachycardia), died after a clinical course of 2 years and 1 month. Macroscopic investigation revealed dilation of the left ventricle and thickening of the right ventricular wall. The unique finding in this case was a circumferential fibrous scar in the median circular layer and outer oblique layer of the left ventricular wall. Biopsy and autopsy materials revealed diffuse loss of myofibrils in the central zone of cardiac muscle fibers, and replacement with aniline blue-positive homogeneous material (17-35% of the area of one muscle fiber). Electron microscopy revealed abnormal accumulation of fine filamentous material (7.5-25 nm in diameter), which was immunohistochemically proved to be desmin-type intermediate filament. Moreover, sarcoplasmic reticulum-like material was detected in the degenerated area. At autopsy, degeneration was detected all over the heart. The ventricular muscle fibers were more severely affected than the atrial muscle fibers. The conduction system was also affected, in some parts more severely than the surrounding ordinary muscle fibers. The pathogenesis of this disorder remains to be clarified.

[Thin-section computed tomography of intrapulmonary metastases after surgical resection of lung cancer].

To evaluate the morphology of small peripheral intrapulmonary metastases of lung cancers, we studied thin-section computed tomography (CT) images of 12 lesions in 5 cases (1 squamous cell carcinoma, and 4 papillary adenocarcinomas). All lesions were resected, and histopathological diagnosis of them was performed in comparison with primary lesions to differentiate multiple primary lung cancers from intrapulmonary metastases. Thin-section CT images showed mildly lobulated nodules in connection with supplying pulmonary vessels, however, indented pleura and vascular convergence were less frequently seen in intrapulmonary metastases in contrast with primary lung cancers. Thin-section CT is helpful for distinguishing multiple primary lung cancers from intrapulmonary metastases in patients with a history of surgical resection for lung cancers.

Primary pulmonary hypertension in infancy: report of two autopsy cases.

Two cases of unexplained plexogenic pulmonary arteriopathy in infancy were studied by patho-anatomical approach. The patients were boys, age 7 months and 2 years 2 months. Pulmonary arteries revealed Grade 3 and Grade 4 hypertensive changes, respectively, after Heath and Edwards' criteria. The configuration of the media of the pulmonary trunk suggested that though these cases were very young, pulmonary arterial pressure had been elevated since some time after birth.

Oxygen exchange during the acto-subfragment-1 ATPase reaction: evidence for the two-route mechanism of the actomyosin ATPase reaction.

The oxygen exchange occurring during the acto-S-1 ATPase reaction was analyzed based on the distribution of 18O-labeled species of P1 using [gamma-18O]ATP as a substrate. Evidence was found for the two-route mechanism in which ATP is hydrolyzed via the dissociation of acto-S-1 into F-actin and the S-1-phosphate-ADP complex, S-1PADP, and their recombination, and also hydrolyzed without the dissociation of acto-S-1 (Inoue, A., Shigekawa, M., & Tonomura, Y. (1973) J. Biochem. 74, 923-934; Inoue, A., Ikebe, M., & Tonomura, Y. (1980) J. Biochem. 88, 1663-1677). When ATP was mainly hydrolyzed without the dissociation of acto-S-1, the extent of oxygen exchange was low. When ATP was hydrolyzed by both routes, the distribution of product P1 with 3, 2, 1, and 0 18O atoms showed a mixture resulting from low and high oxygen exchange. The rate of ATPase without the dissociation of acto-S-1 can be estimated from the rate of the overall reaction (v), the rate of recombination of S-1PADP with F-actin (vr), and the extent of dissociation of acto-S-1 (a). The distribution of the P1 species measured was almost equal to that calculated from the ratio of ATP hydrolysis via the two pathways as avr and v-avr, respectively. This result indicates that the rates of the dissociation of acto-S-1PADP into S-1PADP and F-actin and their recombination are much lower than the rate of decomposition of the acto-S-1PADP complex into acto-S-1 + ADP + Pi.

Adrenocortical carcinoma in infancy. Report of a case with immunohistochemical and ultrastructural studies.

A case of adrenocortical carcinoma associated with congenital heart defect in a 6-month-old Japanese girl is reported. A fist-sized tumor was incidentally noted in the right hypochondrium upon admission for cardiac surgery. No clinical endocrinopathy was evident in this case. The resected tumor was encapsulated with smooth surface and no invasion to adjacent tissues or organs was observed. Histologically, the tumor was composed of small cells with granular or clear cytoplasm, and occasional giant cells with single or multiple nuclei. By electron microscopy, the tumor cells showed various nuclear contours with distinct nucleoli and had a moderate amount of cytoplasm containing abundant rough endoplasmic reticulum and mitochondria with variable-sized electron-dense granules. Intercellular desmosome-like junctions were observed in some tumor cells. Immunohistochemically, the tumor cells contained granules positive for estriol, progesterone and cortisol. These morphological findings including electron microscopic features suggested that the tumor cells had a malignant character.

An autopsy case of diffuse myelomatosis associated with systemic kappa light chain deposition disease (LCDD). A patho-anatomical, immunohistochemical and immunobiochemical study.

A case of systemic light chain deposition disease in a 48-year-old man is presented. Clinically, the patient showed the signs of multiple organ (liver, heart and kidney) failure, but multiple myeloma was not diagnosed. Autopsy revealed generalized deposition of hyaline, a Congo red-negative substance, especially in the arterial walls of various organs. In the bone marrow, myelomatous proliferations of plasma cells positive for kappa light chain were recognized. The deposited substance was ultrastructurally different from amyloid fibrils, and was identified as kappa light chain by immunohistochemistry. A liver tissue extract was immunobiochemically examined and the deposited substance was confirmed to consist of kappa light chain, its molecular weight being approximately 14,000 to 17,000 Da.