Studies on expression of aldehyde dehydrogenase in normal and cancerous tissues of thyroids.

Recently published articles have reported the controversial data regarding expression of aldehyde dehydrogenase isozyme 1A1 (ALDH1A1), a potential candidate marker for normal and cancer stem cells (CSCs), in thyroid tissues. These data prompted us to re-evaluate expression of ALDH1A1 in normal and cancerous thyroid tissues by 2 different means. The first method was immunohistochemistry with 2 different anti-ALDH1A1 antibodies from distinct companies. Following validating the integrity of these 2 antibodies by Western blotting with ALDH-expressing and nonexpressing cancer cell lines and immunohistochemistry with breast and colon tissues, we report here significant and comparable expression of ALDH1A1 in both normal and cancerous thyroid tissues with both antibodies. Next, relative expression levels of ALDH isozymes were evaluated by reverse transcription-polymerase chain reaction (RT-PCR), revealing that ALDH1A1 was the most highly expressed isozyme followed by ALDH9A1 and relative expression patterns of isozymes were very similar in normal and cancerous tissues. All these data demonstrate that thyroid cells of normal and cancer origins do express ALDH1A1 and to a lesser extent 9A1. Further study will be necessary to study functional significance of ALDH1A1 in the function and behaviors of thyroid normal and cancer stem cells.

Orthotopic neobladder reconstruction in elderly bladder cancer patients.

BACKGROUND: We compared the clinical results of orthotopic neobladder reconstruction in elderly patients and those in younger patients retrospectively in order to verify whether age is a critical factor in selecting a method of urinary diversion. METHODS: Following radical cystectomy for bladder cancer, 12 patients aged 75 or older and 17 patients under 75 who underwent orthotopic neobladder reconstruction between January 1992 and May 1999 were investigated in this study. The authors TS and BS were among the surgeons who performed operations for all cases. Of the 12 elderly patients, orthotopic neobladders were constructed according to Hautmann's method in nine cases, Studer's method in one case and Reddy's method in two cases. Of the 17 younger patients, these methods were employed in 12, one and four cases, respectively. Operative procedure, early and late complications, prognosis, continence and voiding pattern were investigated in these patients. RESULTS: The follow-up periods for elderly and younger groups ranged from 21.3 to 82.7 months and from 8.8 to 94.2 months, respectively. No difference in operation time, amount of bleeding or postoperative length of hospitalization was observed between elderly and younger patients. The rates of early complications in elderly and younger patients were 41.7% and 35.3%, respectively. Late complication rates were 33.3% and 47.1%, respectively. The difference in these complication rates was not statistically significant. One of the elderly and two of the younger patients had local recurrence and metastasis postoperatively. Those three patients had died of their bladder cancer. No statistically significant difference between groups was recognized in either cause-specific survival or overall survival, nor was there such a difference in relation to micturition/continence. CONCLUSION: Based on these results, we believe that because age is not a critical factor in the selection of urinary diversion method, neobladder reconstruction following cystectomy for bladder cancer is indicated in elderly patients. As stoma management is difficult for the patients, we consider orthotopic neobladder reconstruction to be the method of choice if the patients' general physical condition allows.

Prevalence of positive anticardiolipin antibody in benign infantile convulsion.

We report six anticardiolipin antibody (aCL)-positive cases among 18 children with epilepsy showing various seizure types in our initial study. These six cases revealed normal coagulation tests. As three of these six cases involved benign infantile convulsion (BIC), we further investigated the high frequency of positive aCL-Immunoglobulin (Ig) G in BIC in our subsequent study of nine cases that included three cases from the previous study and an additional six BIC cases followed and/or diagnosed by co-author (T.K.). As a result, eight of nine BIC cases were positive for aCL-IgG and the values of aCL-IgG decreased over long-term observation in three of these cases. The frequency of positivity for aCL-IgG in BIC was obviously higher than that of controls. Based on these results, we suggest that some immunological responses may be responsible for the pathogenesis of BIC.

Enhanced expression of complement C5a receptor mRNA in human diseased kidney assessed by in situ hybridization.

BACKGROUND: Anaphylatoxin C5a mediates inflammatory responses through interaction with a specific C5a receptor (C5aR), the expression of which is thought to be restricted to peripheral blood leukocytes. Although the presence of C5aR on cultured mesangial cells and tubular epithelial cells has recently been documented, the tissue distribution of C5aR in diseased kidney has not yet been determined. METHODS: Immunohistochemistry and nonradioactive in situ hybridization for C5aR were performed in 34 tissue samples of kidneys from patients with various renal diseases, including 4 with minimal change nephrotic syndrome (MCNS), 5 with membranous nephropathy (MN), and 25 with mesangial proliferative glomerulonephritis (mesGN; 15 patients with IgA nephropathy, 5 with non-IgA mesGN, and 5 with lupus nephritis). Normal portions of surgically resected kidney served as the control. RESULTS: In normal kidneys, C5aR protein was detected in tubular epithelial cells, while C5aR mRNA was detected in a few glomerular cells, tubular epithelial cells, and vascular endothelial and smooth muscle cells. In MCNS, the distribution of C5aR protein and mRNA was similar to that in normal kidneys. In MN and mesGN, C5aR protein and mRNA were detected in mesangial cells, glomerular epithelial and endothelial cells, Bowman's capsule cells, tubular cells, infiltrating cells, and vascular endothelial and smooth muscle cells. The glomerular expression of C5aR mRNA and protein correlated positively with the degree of mesangial hypercellularity and mesangial matrix expansion in mesGN. In the tubulointerstitium, interstitial expression of C5aR mRNA correlated positively with the degree of tubular atrophy and interstitial broadening in mesGN. Furthermore, the interstitial expression of C5aR mRNA correlated positively with the level of serum creatinine. CONCLUSIONS: Our results indicate that renal cells produce C5aR and that activation of C5a/C5aR pathway on renal cells may be involved in tissue injury in mesGN.

Differentiation stages of eosinophils characterized by hyaluronic acid binding via CD44 and responsiveness to stimuli.

To characterize interleukin (IL)-5-induced eosinophils, we examined the expression of CD44, very late antigen (VLA)-4, and the IL-5 receptor alpha chain, as well as the levels of eosinophil peroxidase and the generation of superoxide. Eosinophils were prepared from IL-5-transgenic mice, then characterized using electron microscopy to determine their responses to stimuli. Whereas CD44 densities remained almost constant, the level of VLA-4 increased in parallel with eosinophil maturation. Although a subset of IL-5-induced eosinophils with high side scatter recovered from bone marrow and rare ones found in blood recognized hyaluronic acid (HA), most did not have this property. Bone marrow eosinophils with high side scatter and lower density contained eosinophil peroxidase, not only in granules, but also in membranous structures for 30% of this population. This population developed HA-binding ability in response to IL-3, IL-4, IL-5, granulocyte-macrophage colony-stimulating factor, macrophage inflammatory protein (MIP)-2, monocyte chemotactic protein (MCP)-1, eotaxin, nerve growth factor (NGF), and opsonized zymosan (OZ). Peripheral blood eosinophils acquired HA-binding ability in response to the same stimuli, but their responses were less than those of bone marrow eosinophils with high levels of side scatter. However, splenic eosinophils did not respond to these stimuli. Although peripheral blood eosinophils did not proliferate when stimulated by IL-5, these were the only cells that released eosinophil peroxidase in response to IL-4, MIP-2, MCP-1, eotaxin, NGF, and OZ. With the exception of a subset of bone marrow eosinophils, the ability to acquire HA binding, but not the ability to generate superoxide, correlated with eosinophil peroxidase activity and major basic protein accumulation in the granules of maturing cells.

Ny-ESO-1 expression and immunogenicity associated with transitional cell carcinoma: correlation with tumor grade.

NY-ESO-1 mRNA expression in transitional cell carcinoma was investigated by reverse transcription-PCR and immunohistochemistry. NY-ESO-1 mRNA was detected in 20 of 62 (32%) tumor specimens. There was a correlation between NY-ESO-1 expression and tumor grade: 0 of 4 (0%) grade 1 (G1), 6 of 26 (23%) grade 2 (G2), and 14 of 32 (44%) grade 3 (G3) tumors were NY-ESO-1 mRNA positive. Immunohistochemical analysis using NY-ESO-1-specific monoclonal antibody ES121 showed that 2 of 14 NY-ESO-1 mRNA-expressing G3 tumors were positive for NY-ESO-1. No NY-ESO-1 staining was observed in the panel of 30 G1 or G2 tumor specimens, including 6 NY-ESO-1 mRNA-positive cases. Sera from an expanded panel of 124 patients with transitional cell carcinoma were tested for the presence of NY-ESO-1 antibody. Seropositivity was observed in 9 of 72 (12.5%) patients with G3 tumors, whereas none of 52 patients with G1 or G2 tumors produced antibody against NY-ESO-1. In the 9 positive patients with NY-ESO-1 antibody, 4 had muscular invasive tumors, and 5 had carcinoma in situ.

Intractable Q fever treated with recombinant gamma interferon.

A 3-year-old boy with Q fever received several kinds of antibiotics including minocycline, but spiking fever and positive PCR of Coxiella burnetii continued for several months. He became asymptomatic and his abnormal laboratory data normalized after the administration of gamma interferon three times a week.

Identification of proacrosin binding protein sp32 precursor as a human cancer/testis antigen.

Serological expression cloning of antigens eliciting a humoral immune response to a syngeneic mouse sarcoma identified pem (mouse placenta and embryonic expression gene) as a new member of the cancer/testis family. To identify the human homologue of pem, mouse pem sequences and pem-related expressed sequence tags from human testis were used as PCR primers for amplification using human testis cDNA. However, rather than pem, another gene, designated OY-TES-1, was isolated and found to be the human homologue of proacrosin binding protein sp32 precursor originally identified in mouse, guinea pig, and pig. OY-TES-1 maps to chromosome 12p12-p13 and contains 10 exons. Southern blot analysis suggests the presence of two OY-TES-1-related genes in the human genome. In normal tissues, OY-TES-1 mRNA was expressed only in testis, whereas in malignant tissues, a variable proportion of a wide array of cancers, including bladder, breast, lung, liver, and colon cancers, expressed OY-TES-1. Serological survey of 362 cancer patients with a range of different cancers showed antibody to OY-TES-1 in 25 patients. No OY-TES-1 sera reactivity was found in 20 normal individuals. These findings indicate that OY-TES-1 is an additional member of the cancer/testis family of antigens and that OY-TES-1 is immunogenic in humans.

A boy with normal growth in spite of growth hormone deficiency after resection of a suprasellar teratoma.

We reported a boy with panhypopituitarism after removal of a suprasellar teratoma and pituitary stalk transection at the age of 3 months. His growth was accelerated after 5 years of age without growth hormone (GH) therapy, although he had poor height growth until age 4 under treatment with hydrocortisone, levothyroxine sodium, and desamino-D-arginine vasopressin (DDAVP). Hyperphagia and obesity developed after surgery. Endocrinological examination revealed no GH response to glucagon, low serum levels of insulin-like growth factor (IGF)-1 and IGF binding protein-3 (IGFBP-3). Serum prolactin was normal, but serum insulin was high. Some patients who received an operation for craniopharyngioma were reported to achieve normal growth without GH secretion, but the mechanism is still unknown. High serum levels of prolactin or insulin can be associated with normal IGF in GH deficient patients. This patient had obesity and high serum insulin, which may be related to growth without GH secretion.

Evaluation of the pharmacological activity of extracts from amomi semen on the gastrointestinal tracts.

We have investigated the effects of methanolic and alcoholic extracts from Amomi Semen on gastric secretion, as well as gastrointestinal propulsion or the prokinetic activities. The methanolic extract from Amomi Semen dose dependently decreased the volume output, acid output, and pepsin output in rat's gastric juice with increasing pH value, while the alcoholic extract had no influence on basal gastric acid secretion. Furthermore, the alcoholic extract improved the L-dopa to induce a delay of gastrointestinal transit in mice, while the methanolic extract did not improve it. However, both extracts had no influence on gastrointestinal transit in intact mice. These results suggest that Amomi Semen has an inhibitory effect on gastric acid secretion and that it has effects as the gastrointestinal prokinetics rather than propulsion. The present study pharmacologically elucidates a belief that Amomi Semen has been used in Chinese medicine for the treatment of gastrointestinal dyspepsia, which includes hyperchlorhydria, stomachache, abdominal distention, anorexia, gastric atony, etc.

A simple approach for the analysis of intracellular movement of oxidant-producing intracellular compartments in living human neutrophils.

In human neutrophils, superoxide is generated primarily within specialized oxidant-producing intracellular compartments. The present study employs a simple methodological approach to evaluate the intracellular movement of these structures in living human neutrophils. Using a CCD camera system, we monitored fluorescence in cells loaded with the succinimidyl ester of dichlorodihydrofluorescein diacetate, which is nonfluorescent until oxidized by reactive oxygen species. Fluorescence-positive intracellular compartments became detectable after neutrophils were stimulated with phorbol myristate acetate for 1 min. Further stimulation increased the intracellular compartments in both number and size in a time-dependent manner. Upon stimulation with phorbol myristate acetate, no fluorescence was seen in intracellular compartments of neutrophils isolated from patients with X-linked chronic granulomatous disease lacking gp91-phox, a membrane component of NADPH oxidase. The method enables tracking of the movement of a single oxidant-producing intracellular compartment following cell stimulation and visualization of the intracellular structures formed by fusion of oxidant-producing intracellular compartments with endocytotic vesicles and phagosomes. Therefore, it is considered to be an informative tool for evaluation of the intracellular dynamics of oxidant-producing intracellular compartments in living human neutrophils and may have a diagnostic value.

An immunohistochemical study of developing glomeruli in human fetal kidneys.

BACKGROUND: In the glomerulonephritis, mesenchymal cells frequently repeat the expression of fetal immunohistochemical phenotypes. However, in human glomerulogenesis the phenotypic alteration of mesangial and other types of glomerular cells has not been clearly defined. Our aim was to clarify the characteristics of fetal mesangial cells and glomerular capillary endothelial cells, as well as their changes during glomerulogenesis using immunohistochemistry. METHODS: We examined the renal tissues of 34 autopsied fetuses and neonates, 5 children, and 5 adults using immunohistochemistry and immunoelectron microscopy, using antibodies for cytoskeletons, contraction-associated proteins, and endothelial cell markers. RESULTS: In the V and S stages, there were no cells showing mesangial and endothelial features within the vesicles and the S-shaped bodies. In the S stage, small blood vessels, consisting of endothelial cells (CD31+, CD34+) and primitive perivascular mesenchymal cells (alpha-smooth muscle actin+, low molecular caldesmon+, vimentin+), were branched from developing interlobular arteries and appeared to extend to the lower clefts of the S-shaped bodies. In the C stage, the perivascular mesenchymal cells aggregated at the root of the immature glomeruli. In the M stage, they migrated toward the periphery of immature glomeruli and gradually lost their fetal immunohistochemical features. Similarly, with further maturation, the fetal glomerular capillary endothelial cells gradually lost the immunostaining for CD34, while the strong staining intensity of CD31 remained unchanged, just as that in the adult glomerular capillary endothelial cells. CONCLUSIONS: In human glomerulogenesis, we demonstrate that fetal mesangial and capillary endothelial cells change their immunohistochemical phenotypes with maturation. They gradually lose fetal immunohistochemical phenotypes. Already before birth, the mesangial cells in almost all glomeruli at the late M stage acquire the adult phenotype.

Polymyositis associated with primary cytomegalovirus infection.

A case of polymyositis associated with primary cytomegalovirus infection in a 17-y-old girl is reported. The girl exhibited fever, sore throat, progressive myalgia and muscle weakness with elevated creatine kinase, atypical lymphocytosis and myopathic features in the electromyogram. Histopathologically, biopsied muscle met the criteria for polymyositis. Primary cytomegalovirus infection was proven by seroconversion of IgG as well as IgM antibodies. This is the first report of an association between cytomegalovirus infection and polymyositis.

A variant of myelokathexis with hypogammaglobulinemia: lymphocytes as well as neutrophils may reverse in response to infections.

A 7-year-old boy with prolonged and marked leukopenia diagnosed at 6 months of age is described. The polymorphonuclear cells presented no hypersegmented nuclei or concentrated nuclear chromatin, although vacuolated myeloid cells appeared in bone marrow smears. Neutrophils reversed in response to administration of G-CSF. His leukocyte counts were 400-1000/microL during afebrile periods and increased to 2000-3000/microL in response to infections. The increased leukocyte was usually neutrophils, but lymphocytes also increased at EB-virus infection. The serum IgG decreased gradually and was 364 mg/dL at 7 years of age. Antibody responses were normal and recurrent otitis media has been the patient's only problem. Granulocytopenia with hypogammaglobulinemia of this patient mimics myelokathexis with hypogammaglobulinemia, and lymphocytes also increased at viral infections.

Analysis of IgG subclasses in chronic active Epstein-Barr virus infection.

BACKGROUND: Although elevated serum levels of immunoglobulins are frequently observed in patients with chronic active Epstein-Barr virus (EBV) infection, there have been no reports concerning levels of IgG subclasses. METHODS: Serum levels of IgG subclasses were measured by the enzyme-linked immunosorbent assay (ELISA) in 30 children with severe chronic active EBV infection. RESULTS: Serum levels of IgG1 were elevated in most patients, except for one who showed an abnormally low level of IgG1 and progressive hypogammaglobulinemia. Serum levels of IgG2, IgG3 and IgG4 in the patients were comparable to those in control children, while abnormally low levels of IgG2, IgG3 and IgG4 were observed in six, three and four cases, respectively. CONCLUSION: Although not always susceptible to bacterial infections, low levels of IgG2 were frequently observed in patients with chronic active EBV infection and elevated IgG1 is responsible for the increase of serum IgG in these patients.

Age-related changes in the posterior limb of the internal capsule revealed by magnetic resonance imaging.

In this study, we examined the age-related differences in the corticospinal tract (CST) of the posterior limb (PL) of the internal capsule (IC) by analyzing 53 magnetic resonance (MR) images from 45 subjects. Regions in the posterior part of the PL of the IC were observed as hypointense areas in T1-weighted images of axial sections at 4 years of age. These regions became clear, spotty hyperintense areas in T2-weighted images of subjects older than 9 years. These regions persisted in all cases beyond 9 years of age, level on both T1- and T2-weighted images. The region is consistent with the CST from MR images of coronal and sagittal sections in our study. These results suggest that differences among the region may occur, such as an increase in fiber size and an increase in the thickness of the myelin sheaths, depending on age after the completion of general myelination in the CST.

Mesangial myofibroblastic transformation in steroid-dependent minimal change nephrotic syndrome.

Patients with minimal change nephrotic syndrome (MCNS) occasionally show frequent relapses with proteinuria after cessation of steroid treatment, even though no significant pathological abnormalities are found in the glomeruli, compared with those in nonrelapsed and good-prognosis cases of MCNS. To resolve this contradiction, we immunohistochemically and ultrastructurally examined a biopsied renal tissue of a patient who showed glomerular features of MCNS and frequent clinical relapses. Immunohistochemistry demonstrated the overexpression of alpha-smooth muscle actin (ASMA) and vimentin in glomerular mesangial cells despite no mesangial cell proliferation, compared with nine nonrelapsed cases of MCNS. These facts may be an important clue to the investigation of the pathogenesis of steroid-dependent MCNS with frequent relapses. Furthermore, the immunohistochemical examination of ASMA and vimentin may be useful to detect mesangial myofibroblastic transformation that is not demonstrated in conventional light microscopy and immunofluorescence study.

Functional expression of transmembrane 4 superfamily molecules on human eosinophils.

BACKGROUND: Transmembrane 4 superfamily (TM4SF) molecules are exclusively found on hematopoietic cells. Several members of the TM4SF are reported to be associated with other cell surface molecules, including integrins, and might participate in signal transduction, but little is known about their role on eosinophils. In the present study, we determined the expression and function of TM4SF molecules on human eosinophils. METHODS: Surface expression of TM4SF molecules on purified peripheral blood eosinophils was examined using indirect immunofluorescence and flow cytometry. Purified eosinophils were incubated with anti-TM4SF monoclonal antibodies (mAbs) for up to 24 h. Eosinophil activation was evaluated by measuring eosinophil homotypic aggregation as well as changes in surface expression of CD11b or CD62L by flow cytometry. RESULTS: Freshly isolated eosinophils expressed CD9, CD37, CD53, CD63 and CD81. Incubation with anti-CD9 mAb but not with anti-CD37, CD53, CD63 or CD81 mAb induced significant eosinophil homotypic aggregation. Incubation with any of the anti-TM4SF mAb for 30 min failed to alter the expression of either CD11b or CD62L on eosinophils. In contrast, the expression of CD11b was significantly enhanced after 24 h of incubation with anti-CD53 mAb, while the expression of CD62L was significantly reduced with anti-CD81 mAb. CONCLUSIONS: Cross-linking of some surface TM4SF molecules induced significant eosinophil homotypic aggregation, upregulation of CD11b expression, or CD62L shedding, consistent with activation of eosinophils. Our data suggest that several TM4SF molecules are functionally expressed on human eosinophils, and therefore might participate in allergic inflammation.