Melatonin ameliorates calcium homeostasis in myocardial and ischemia-reperfusion injury in chronically hypoxic rats.

Chronic hypoxia (CH) leads to the deterioration of myocardial functions with impaired calcium handling in the sarcoplasmic reticulum (SR), which may be mediated by oxidative stress. We hypothesized that administration of antioxidant melatonin would protect against cardiac and ischemia-reperfusion (I/R) injury by ameliorating SR calcium handling. Adult Sprague-Dawley rats that had received a daily injection of melatonin or vehicle were exposed to 10% oxygen for 4 wk. The heart of each rat was then dissected and perfused using a Langendorff apparatus. The ratio of heart-to-body weight, ventricular hypertrophy and hematocrit were increased in the hypoxic rats compared with the normoxic controls. Malondialdehyde levels were also increased in the heart of hypoxic rats and were lowered by the treatment of melatonin. The hearts were subjected to left coronary artery ischemia (30 min) followed by 120-min reperfusion. Lactate dehydrogenase leakage before ischemia, during I/R and infarct size of the isolated perfused hearts were significantly elevated in the vehicle-treated hypoxic rats but not in the melatonin-treated rats. Spectroflurometric studies showed that resting calcium levels and I/R-induced calcium overload in the cardiomyocytes were more significantly altered in the hypoxic rats than the normoxic controls. Also, the hypoxic group had decreased levels of the SR calcium content and reduced amplitude and decay time of electrically induced calcium transients, indicating impaired contractility and SR calcium re-uptake. Moreover, there were reductions in protein expression of calcium handling proteins, markedly shown at the level of SR-Ca(2+) ATPase (SERCA) in the heart of hypoxic rats. Melatonin treatment significantly mitigated the calcium handling in the hypoxic rats by preserving SERCA expression. The results suggest that melatonin is cardioprotective against CH-induced myocardial injury by improving calcium handling in the SR of cardiomyocytes via an antioxidant mechanism.

Melatonin ameliorates hippocampal nitric oxide production and large conductance calcium-activated potassium channel activity in chronic intermittent hypoxia.

Melatonin protects against hippocampal injury induced by intermittent hypoxia (IH). IH-induced oxidative stress is associated with decreases in constitutive production of nitric oxide (NO) and in the activity of large conductance calcium-activated potassium (BK) channels in hippocampal neurons. We tested the hypothesis that administration of melatonin alleviates the NO deficit and impaired BK channel activity in the hippocampus of IH rats. Sprague-Dawley rats were injected with melatonin (10 mg/kg, i.p.) or vehicle before daily IH exposure for 8 hr for 7 days. The NO and intracellular calcium ([Ca2+]i) levels in the CA1 region of hippocampal slices were measured by electrochemical microsenor and spectrofluorometry, respectively. The activity of BK channels was recorded by patch-clamping electrophysiology in dissociated CA1 neurons. Malondialdehyde levels were increased in the hippocampus of hypoxic rats and were lowered by the melatonin treatment. Levels of NO under resting and hypoxic conditions, and the protein expression of neuronal NO synthase (nNOS) were significantly reduced in the CA1 neurons of hypoxic animals compared with the normoxic controls. These deficits were mitigated in the melatonin-treated hypoxic rats with an improved [Ca2+]i response to acute hypoxia. The open probability of BK channels was decreased in the hypoxic rats and was partially restored in the melatonin-treated animals, without alterations in the expression of channel subunits and unitary conductance. Acute treatment of melatonin had no significant effects on the BK channel activity or on the [Ca2+]i response to hypoxia. Collectively, these results suggest that melatonin ameliorates the constitutive NO production and BK channel activity via an antioxidant mechanism against an IH-induced down-regulation of nNOS expression in hippocampal neurons.

Molecular cloning and characterization of full-length cDNAs encoding a novel high-molecular-weight Dermatophagoides pteronyssinus mite allergen, Der p 11.

BACKGROUND: Dermatophagoides pteronyssinus (Dp) and D. farinae (Df) mites are the most important source of indoor aeroallergens. Most Dp mite allergens identified to date have relatively low molecular weights (MWs). Identification of high-MW mite allergens is a crucial step in characterizing the complete spectrum of mite allergens and to provide appropriate tools for diagnostic and therapeutic application. METHODS: The full-length Der p 11 cDNA clone was isolated using cDNA library immunoscreening, the 5'-3' rapid amplification of cDNA ends (RACE) system and polymerase chain reactions (PCR). The whole cDNA insert and its PCR-derived DNA fragments (p1 to p4) were generated and expressed in the Escherichia coli expression system. The allergenicity of the recombinant protein and its peptide fragments was examined by IgE immunodot assays. The IgE-binding reactivity of rDer p 11 was analyzed in the serum of 50 asthmatic children with positive reactivity to Dp mite extract. Its recombinant peptide fragments were also examined by immunodot assays in 30 mite-allergic children. RESULTS: Der p 11 cDNA consists of a 2625-bp open reading frame encoding a 103-kDa protein with 875 amino acids. It exhibits significant homology with the paramyosin of other invertebrates. The protein sequence alignment of this newly identified Dp mite allergen (denominated as Der p 11) revealed over 89% identity with Der f 11 and Blo t 1. Among 50 Dp-sensitive asthmatic children, rDer p 11 showed positive IgE-binding reactivity to 39 patients (78%). Using immunodot assays, multiple human IgE-binding activities were demonstrated in all four fragments of Der p 11. Using immunoblot assays, the dominant IgG-binding epitope for monoclonal antibody (mAb642) was located in fragment p3 only. In immunoblot assays, cross-inhibition between rDer p 11 and rDer f 11 was up to 73-80% at concentrations of 100 microg/ml. CONCLUSIONS: This study confirms that the newly identified recombinant Der p 11 is a novel and important high-MW Dp mite allergen for asthmatic children. Our data also indicates that human IgE-binding major epitopes are scattered over the entire molecule of Der p 11.

Protein sequence analysis of a novel 103-kDa Dermatophagoides pteronyssinus mite allergen and prevalence of serum immunoglobulin E reactivity to rDer p 11 in allergic adult patients.

BACKGROUND: House dust mites are regarded as important indoor allergens. While the most studies mite allergens are low molecular weight (mw), a high mw Dermatophagoides farinae mite paramyosin (Der f 11) has recently been cloned. We have also cloned a novel high mw Dermatophagoides pteronyssinus (Dp) mite allergen, Der p 11. OBJECTIVE: The aim of this study was to isolate and express a cDNA gene coding for a Der p 11 allergen, to compare the sequence of Der p 11 with other antigens and to evaluate the presence of IgE reactivity to the recombinant protein (rDer p 11) in the sera of allergic adult patients. METHODS: The full-length Der p 11 gene was isolated by cDNA library screening, 5'-3' rapid amplification of cDNA ends and PCR. The cDNA gene was expressed as a glutathione-S-transferase fusion protein in Escherichia coli. The allergenicity of rDer p 11 was tested by human IgE immunodot or immunoblot assay in a large panel of 100 allergic patients with bronchial asthma, allergic rhinitis or eczema. RESULTS: Der p 11 is a 2965 bp cDNA gene with a 2625 bp open reading frame coding for a 875 amino acid protein. The deduced amino acid sequence of the Der p 11 showed significant homology with various invertebrate paramyosins. The prevalence of serum IgE reactivity to rDer p 11 on immunodot assay ranged from 41.7% to 66.7% in different allergic patient groups, whereas it was rare in non-atopic patients with urticaria (18.8%) and in normal individuals (8%). A high frequency (five out of eight) of MAST(Dp)- allergic serum samples had specific IgE-binding activity to rDer p 11 or its fragments on immunoblot assay, even though their IgE-binding activity to Dp extract was either weak or negative. CONCLUSION: The 103-kDa Der p 11 appears to be major Dp mite allergen with a high frequency of IgE reactivity in sera of patients allergic to mites.

Serum specific IgE reactivity to recombinant Der f 11 in asthmatic children.

This study was designed to examine the prevalence of positive serum IgE reactivity to the recombinant group 11 Dermatophagoides farinae allergen (rDer f 11) in asthmatic children in Taiwan. Using immunoblot analysis in a preliminary study of 18 asthmatic children, 13 (72.2%) reacted positively to rDer f 11 and 16 (88.9%) showed positive reactivity to D. farinae extracts. The allergenicity of rDer f 11 was further evaluated with in vivo skin tests and in vitro IgE immunodot assays in 24 mite skin-test-positive asthmatic children. Whereas 17 (70.8%) had positive skin tests to rDer f 11, 18 (75.0%) had positive serum IgE reactivity to rDer f 11. A good coincidence (87.5%) between the immunodot assay and the skin test was confirmed in these asthmatic children. Moreover, the prevalence of serum IgE reactivity to rDer f 11 was further investigated in a large panel of 49 mite skin-test-positive asthmatic children. Again, 38 (77.6%) had positive serum IgE reactivity to rDer f 11 in immunodot assays. Taken together the positive IgE reactivity to rDer f 11 in immunodot analysis ranged from 75 to 77.6% in two groups of 73 mite skin-test-positive asthmatic children. High incidence of serum IgE antibodies specific for rDer f 11 in the present study suggests that Der f 11 is a novel major allergen of house dust mites.

Differential regulation of placental and germ cell alkaline phosphatases by glucocorticoid and sodium butyrate in human gastric carcinoma cell line TMK-1.

The expression and regulation of alkaline phosphatase (AP) was studied in the human gastric cancer cell line TMK-1. Biochemical analysis, reverse transcription-polymerase chain reaction, and Northern blot analysis demonstrated that the cells express placental, germ cell, and intestinal AP isozymes constitutively. Dexamethasone (Dex), a synthetic glucocorticoid, was shown to specifically induce the placental AP activity to about 10-fold and sodium butyrate (NaBu) induced germ cell AP activity to about 4-fold, respectively. In contrast, these two agents showed little effect on the level of intestinal isozymes. Dex and NaBu also differentially induced the mRNA levels of the placental and germ cell APs. Northern blot analysis of the placental AP transcript in the presence of the transcription inhibitor, 5, 6-dichloro-1-beta-D-ribofuranosyl benzimidazole, revealed that the half-life of placental AP mRNA is about 27 h for both the Dex-treated and untreated cells. Nuclear run-on transcription analysis indicated an apparent increase in the rate of placental AP gene transcription in Dex-treated cells. These results indicated that the effect of Dex occurred primarily by activation of the placental AP gene transcription in the cells. In order to study the direct Dex and NaBu effect on AP gene expression, the proximal promoter regions of AP genes were fused to luciferase reporter vectors. Despite the high similarity in nucleotide sequences of these two genes, transient transfection analysis demonstrated that Dex and NaBu exerted a specific stimulation only through the respective placental and germ cell AP gene promoter. Taken together, this study indicates that the expression of PAP and GCAP isozymes have specific regulatory mechanisms that can be differentially controlled by signals including glucocorticoid and NaBu.

Apoptosis induced by the sodium butyrate in human gastric cancer TMK-1 cells.

The effects of sodium butyrate on cell proliferation, gene expression, and apoptosis were investigated. Upon exposure to sodium butyrate the cells exhibited marked morphological changes, reduced cell proliferation and most cells died through apoptosis within 48 hours. In the presence of dexamethasone, however, the sodium butyrate-triggered apoptosis was markedly reduced. Studies using the glucocorticoid receptor antagonist RU486 indicated that the protective effect of dexamethasone was mediated through glucocorticoid receptor. Sodium butyrate markedly induced the c-jun proteins level, whereas the c-Myc protein was down-regulated rapidly. c-Jun protein may play an important role in the action of sodium butyrate since its induction preceded the onset of DNA fragmentation. In addition, preincubation of the cells with dexamethasone markedly delayed the induction of c-jun levels by sodium butyrate. Analysis of the expression of bel-2-related genes indicated that the Bcl-xS protein level was increased in the presence of sodium butyrate and the up-regulation of Bcl-xS by sodium butyrate was also blocked by dexamethasone. Taken together, these results indicate that c-myc, c-jun and Bcl-xS proteins may be involved in the mechanism of sodium butyrate-triggered apoptosis in these cells.

Significance of specific IgG subclass antibodies to house dust mites in asthmatic children.

BACKGROUND: Dermatophagoides pteronyssinus (Dp) and Dermatophagoides farinae (Df), the major components of house dust, are considered to be etiologic factors of extrinsic asthma. The relationships between immunoglobulin (Ig) G subclass antibodies specific for Dp (or Df) were compared in specific IgE-positive and -negative asthmatic children. METHODS: Serum levels of IgG and IgE subclass antibodies specific for Dp and Df were studied in 52 children (age, 3-13 years; mean age, 8.4 years) with asthma using enzyme-linked immunosorbent assays. The skin prick test was also used in diagnosis of the reactivity of allergic disease. RESULTS: The levels of serum-specific IgG1 and IgG2 to Dp and Df in mite-specific IgE-(or skin-test) positive asthmatic children were significantly higher than those in mite-specific IgE- (or skin test) negative children (p < 0.01). Significant correlations between the level of the specific IgE and IgG1 (r = 0.6067; p = 0.0001) or IgG2 (r = 0.5851; p = 0.0002) to Dp, and IgG1 (r = 0.3823; p = 0.0214) or IgG2 (r = 0.5057; p = 0.0017) to Df were found. The specific IgE level and skin test reactivity showed a high correlation of greater than 96% (50/52). CONCLUSIONS: The levels of mite-specific IgG subclasses were partially compatible to specific IgE levels and skin test reactivity. We conclude that house dust mite allergy is significantly associated with specific IgG1, IgG2 and IgE responses.

Protein sequence analysis and mapping of IgE and IgG epitopes of an allergenic 98-kDa Dermatophagoides farinae paramyosin, Der f 11.

BACKGROUND: A 98-kDa mite paramyosin (Der f 11) from Dermatophagoides farinae (Df) is highly allergenic, and its cDNA (Df642) has been cloned. This paper describes the sequence characteristics and the mapping of the immunodominant human IgE and IgG epitopes of Der f 11. METHODS: The protein sequence analysis was performed with a combination of FASTA, GCG, and CLUSTAL W computing packages. The whole cDNA insert and its PCR-derived DNA fragments were generated and expressed in E. coli. These overlapping recombinant peptides (F1 to F5) were used for B-cell epitope mapping with 18 mite-allergic sera by dot immunoassays. RESULTS: Df642 cDNA encodes a partial sequence that contains the 2nd to 26th 28-residue repeats and lacks the N-terminus and the C-terminus. The sequence identity of Der f 11 with other known paramyosins is 34-60%. The dominant IgE epitopes are located in peptides F1 and F4, whereas the dominant IgG epitopes are located in peptides F1 and F2. These peptides are more reactive than whole rDf642. CONCLUSIONS: Mite paramyosin is very similar to other known paramyosins. The human IgE and IgG epitopes are scattered throughout the entire molecule. Data also indicate the presence of unique IgE and IgG epitopes in Der f 11.

Expression and regulation of alkaline phosphatases in human breast cancer MCF-7 cells.

The effect of retinoic acid and dexamethasone on alkaline phosphatase (AP) expression was investigated in human breast cancer MCF-7 cells. Cellular AP activity was induced significantly by retinoic acid or dexamethasone in a time-dependent and dose-dependent fashion. A marked synergistic induction of AP activity was observed when the cells were incubated with both agents simultaneously. Two AP isozymes, tissue-nonspecific (TNAP) and intestinal (IAP), were shown to be expressed in MCF-7 cells as confirmed by the differential rate of thermal inactivation of these isozymes and RT-PCR. Based on the two-isozyme thermal-inactivation model, the specific activities for TNAP and IAP in each sample were analyzed. TNAP activity was induced only by retinoic acid and IAP activity was induced only by dexamethasone. Whereas dexamethasone conferred no significant effect on TNAP activity, retinoic acid was shown to inhibit IAP activity by approximately 50%. Interestingly, TNAP was found to be the only isozyme activity superinduced when the cells were costimulated with retinoic acid and dexamethasone. Northern blot and RT-PCR analysis were then used to demonstrate that the steady-state TNAP mRNA level was also superinduced, which indicates that the superinduction is regulated at the transcriptional or post-transcriptional levels. In the presence of the glucocorticoid receptor antagonist RU486, the dexamethasone-mediated induction of IAP activity was blocked completely as expected. However, the ability of RU486 to antagonize the action of glucocorticoid was greatly compromised in dexamethasone-mediated superinduction of TNAP activity. Furthermore, in the presence of retinoic acid, RU486 behaved as an agonist, and conferred superinduction of TNAP gene expression in the same way as dexamethasone. Taken together, these observations suggest that the induction of IAP activity by dexamethasone and the superinduction of TNAP by dexamethasone were mediated through distinct regulatory pathways. In addition, retinoic acid plays an essential role in the superinduction of TNAP gene expression by enabling dexamethasone to exert its agonist activity, which otherwise has no effect.

Isolation and characterization of a novel 98-kd Dermatophagoides farinae mite allergen.

BACKGROUND: Exposure to allergens from house dust mites is a significant cause of immediate hypersensitivity. Thus far, the active mite allergens defined are low molecular weight (MW) proteins or glycoproteins. However, other important mite allergens remain to be investigated. In this study a high MW mite antigen with a high IgE-binding activity was characterized. METHODS: An anti-Dermatophagoides farinae (Df) monoclonal antibody, mAb642, which recognized a 98-kd allergenic mite protein, was used for affinity chromatography. The purified Df642 was characterized biochemically and immunologically. RESULTS: Competitive ELISA demonstrated that mAb642 was inhibited by the interaction between serum IgE from allergic patients and Df642 antigen in a dose-dependent fashion. The IgE reactivity to both 98-kd and 92-kd components was removed or diminished by preincubation of asthmatic sera with Df642-coated CNBr-activated cellulose-4B gel. Two-dimensional immunoblot analysis revealed that there are at least 4 isoforms of Df642 that represent a minor component in the crude mite extract. The allergenicity of Df642 was assayed by IgE immunoassay with a large panel of 67 sera from asthmatic patients with positive skin reactions, and Df 642 showed positive IgE reactivity with more than 80% of the sera tested. Thus it should be classified as an important allergen. In addition, amino acid sequence analysis revealed that Df642 shares more than 50% homology with paramyosin from invertebrates. CONCLUSION: We have identified and characterized a 98-kd house dust mite allergen that showed greater than 80% IgE reactivity with sera from patients allergic to mites. This is the first high MW allergen characterized to date, and it shares high sequence homology with paramyosins in invertebrates.

Elevated cytokeratin-19 expression associated with apoptotic resistance and malignant progression of human cervical carcinoma.

Cytokeratin-19 is an intermediate filament protein associated with the integrity of cell structure, and its elevated expression has been reported to correlate with the disease progression of oesophagus and lung cancers. In this study, we examined the level of cytokeratin-19 in five cervical cancer cell lines by immunobinding and Western blotting analyses. Compared with two control cell lines, FS-4 (foreskin cell line) and G9T (glioma cell line), all five cervical carcinoma cell lines (Caski, CC7T, ME180, HeLa and SIHA) showed higher cytokeratin-19 expression. By double-staining flow cytometry, expression of cytokeratin-19 in cervical cancer cells was suggested to be in a cell cycle-independent manner. Furthermore, we could specifically localize the SIHA cell-derived tumours in nude mice by injecting with cytokeratin-19-recognized radiolabelled MAb Cx-99 antibody, suggesting the possibility of using cytokeratin-19 as a marker of cervical carcinoma. A clinical investigation was therefore performed on 19 patients (11 patients with cervical carcinoma and eight patients with benign neoplasia). In the 11 patients having cervical carcinoma, all eight patients with advanced stages and one out of three patients with early stage diseases showed higher cytokeratin-19 protein contents than the other 10 patients with benign neoplasia. This suggested that elevation of cytokeratin-19 level was associated with cervical cancer staging. In addition, we have studied the biological significance of elevated cytokeratin-19 level in malignant cervical cancer. The apoptotic rate of cervical carcinoma cells in response to cisplatin was increased if their cellular cytokeratin-19 level was reduced by specific antibody MAb Cx-99. These results indicated that elevation of cytokeratin-19 expression could associate with the apoptotic resistance and malignant progression of cervical carcinoma.

Dexamethasone suppresses apoptosis in a human gastric cancer cell line through modulation of bcl-x gene expression.

Treatment of human gastric cancer TMK-1 cells with transcription and translation inhibitors rapidly triggered cell apoptosis. Along with cell apoptosis, the Bcl-xS level was markedly upregulated suggesting a crucial role of this protein in promoting the apoptotic process. In the presence of dexamethasone, however, cell apoptosis was greatly attenuated as demonstrated by DNA histogram shift and DNA fragmentation. Studies using the glucocorticoid receptor antagonist RU486 indicated that attenuation of apoptosis was mediated through glucocorticoid receptors. Dexamethasone not only suppressed the apoptosis-associated upregulation of Bcl-xS but also enhanced the basal level of Bcl-xL in the cells. In addition, bcl-x mRNA stability was significantly extended in the presence of dexamethasone. These results indicate that dexamethasone exerted a protective effect and delayed apoptosis of TMK-1 cells by modulating bcl-x gene expression.

Effects of transcription and translation inhibitors on a human gastric carcinoma cell line. Potential role of Bcl-X(S) in apoptosis triggered by these inhibitors.

The effects of the macromolecular synthesis inhibitors 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB), actinomycin D, and cycloheximide on the human gastric cancer TMK-1 cell line were studied. These agents inhibited DNA, RNA, or protein synthesis efficiently and induced cell death rapidly in a wide range of concentrations. After 8 hr of exposure to these agents, the cells exhibited morphological features of apoptosis, including cell shrinkage, nuclear condensation, DNA fragmentation, and formation of apoptotic bodies. Western blot analysis revealed that these inhibitors altered the protein levels of apoptosis-related gene products such as c-Myc, Bcl-X(S), and the mutant p53 (mp53) in TMK-1 cells markedly. The c-myc mRNA and protein levels were decreased initially and were then induced markedly to a new level after 4 hr of exposure to DRB, a RNA polymerase II inhibitor. The Bcl-X(S) levels were increased rapidly after treatment with all of these agents, whereas the levels of Bcl-X(L) and Bax remained largely unchanged. Northern blot analysis indicated that the c-myc overexpression is concomitant to DRB-induced DNA fragmentation and that the increased mp53 protein level was mainly a posttranscriptional event. Our observations suggest that the up-regulation of Bcl-X(S) may serve as an important mechanism for the apoptosis triggered by these inhibitors. This study also provides evidence for the notion that interference with the cellular survival pathway may lead to apoptosis.

Effects of tamoxifen and retinoic acid on cell growth and c-myc gene expression in human breast and cervical cancer cells.

The effects of estradiol, tamoxifen, and retinoic acid on the proliferation of breast and cervical cancer cells were investigated. Estrogen stimulated only MCF-7 cell growth, whereas tamoxifen and retinoic acid inhibited the proliferation of all cells studied. Northern blot analysis indicated that estradiol up-regulates c-myc mRNA level in all cell lines studied regardless of the estrogen receptor status in the cells. On the contrary, tamoxifen inhibits c-myc gene expression in all cell lines studied except in MCF-7 cells where the c-myc transcript was not affected. The inhibitory effect of tamoxifen on c-myc gene expression and cell proliferation in estrogen receptor-negative cells suggest an estrogen receptor-independent mechanism. The results also suggest that different mechanisms are involved in the regulation of cell growth and c-myc gene expression in different cancer cells by estrogen and tamoxifen.

Growth suppression of human colorectal carcinoma in nude mice by monoclonal antibody C27-abrin A chain conjugate.

PURPOSE: The aim of this study was to assess an immunotoxin, monoclonal antibody C27-abrin A chain conjugate (MAAAC), that might be effective in the treatment of colorectal carcinoma. METHODS: The immunotoxin was prepared by a specific monoclonal antibody against carcinoembryonic antigen (CEA), monoclonal antibody C27, linked to N-succinimidyl-3-(2-pyridyldithio)propionate and then coupled covalently to the toxic abrin-A chain to synthesize MAAC. The therapeutic role of this immunotoxin in suppressing the in vitro and in vivo growth of CEA-secreting human colorectal cancer cells (LS174T) was assayed by methods of protein biosynthesis inhibition, cell colony proliferation, and treatment of tumor cells before and after inoculation in nude mice. RESULTS: We found that MAAC effectively suppressed the growth of LS174T in culture medium and completely eradicated cells in inoculated nude mice. In contrast, irrelevant immunotoxin antiferritin-abrin A chain conjugate and isotype-matched monoclonal immunoglobin (MOPC21IgG1)-abrin A chain conjugate did not cause such effects. The in vitro toxicity was highly specific because the conjugate (MAAC) inhibited de novo protein biosynthesis, impeded growth, and caused death of cells possessing surface CEA determinants. The 50 percent inhibition dose values of the conjugate for colonogenic survival and for protein biosynthesis in LS174T cells were 0.09 microgram/ml and 0.06 microgram/ml, respectively. Colon survival was inhibited 96.3 percent after prolonged MAAC treatment. MAAC showed selective cytotoxicity; the inhibitory effect of MAAC to the CEA-secreting LS174T cells over the CEA-nonsecreting human embryonic kidney cells was 16-fold. CONCLUSION: These results indicate that MAAC may be of benefit in therapy during or soon after resection of colorectal carcinoma or in patients who have micrometastasis.

Regulation of the expression of alkaline phosphatase in a human breast-cancer cell line.

Treatment of the cultured human breast-cancer cells BC-M1 with dexamethasone induced a placental-type alkaline phosphatase (ALP). Both the ALP activity and the mRNA level in the cells were increased. The induction of ALP activity by dexamethasone was time- and dose-dependent. The accumulation of ALP mRNA was inhibited by both actinomycin D and cycloheximide, indicating that its induction is a complex event and may involve other regulatory proteins. Retinoic acid showed opposing effects with dexamethasone on the expression of alkaline phosphatase. Retinoic acid (RA) and phorbol 12-myristate 13-acetate also substantially reduced the dexamethasone-induced expression of ALP. Studies on thermostability and sensitivity to various amino acid inhibitors indicated that the BC-M1 ALP is most similar to the placental form. Northern hybridization analysis also revealed that ALP mRNA transcripts in BC-M1 and term placenta are similar in size and are distinct from that of the placental-like mRNA transcript in choriocarcinoma cells. Analysis of the degradation of BC-M1 ALP mRNA showed a similar half-life of 27 h in the untreated and in dexamethasone- or RA-treated cells. These findings demonstrated that the induction of ALP in BC-M1 cells by dexamethasone is mainly due to the increase in the transcription of the ALP gene.

Production and characterisation of a monoclonal antibody (Cx-99) against cervical carcinoma.

An IgG1 monoclonal antibody (MAb Cx-99) has been established which recognises a surface antigen on epithelial cells, but not on fibroblastic or hematopoietic cells. Immunohistochemical studies showed that this antigen was present in all 37 squamous cell carcinomas (SCC) including 33 cervical SCC, and 30 of the 32 adenocarcinomas examined; most of the 33 cervical SCC were stained extensively. It was also detected in the culture medium of cervical cancer cell lines. In the normal cervix, this antigen was restricted to the undifferentiated basal cells. This observation suggests that the widespread expression of the antigen was triggered by oncogenesis. The MAb Cx-99 recognised an epitope on an asialyted glycoprotein which has an apparent molecular weight of 37 kilodaltons (kD) (and 2 minor proteins at 18 and 27 kD) and an isoelectric point (pI) of 5.3. It may have potential for studies on differentiation and oncogenesis and for diagnostic applications.

Changes in the levels of house dust mite specific IgG4 during immunotherapy in asthmatic children.

Serum levels of IgG subclass and house dust mite (Dermatophagoides pteronyssinus, Dpt) specific IgG4 were evaluated during immunotherapy in asthmatic children. Asthmatic children undergoing long-term immunotherapy (more than 2 years) posed a mean value of total serum IgG4 or Dpt-specific IgG4 antibodies significantly higher than that of patients prior to receiving immunotherapy, asthmatic (placebo) controls, or patients undergoing short-term immunotherapy (less than 1 year) (P less than 0.05). The mean levels of serum Dpt-specific IgG4 in all asthmatic groups were also significantly higher than in the non-allergic controls (P less than 0.01). Moreover, the mean level of Dpt-specific IgG4 tended to increase during immunotherapy. A significant correlation between total serum IgG4 and Dpt-specific IgG4 antibodies was noted (r = 0.6243; P less than 0.001). Serial follow-up reveals that Dpt-specific IgG4 levels usually rose significantly with clinical improvement in asthmatic children during immunotherapy. These results suggest that the anti-mite-specific IgG4 antibody may serve as an indicator for clinical outcome of mite allergy during immunotherapy.

The prevalence of allergic disease and IgE antibodies to house dust mite in schoolchildren in Taiwan.

The prevalence of positive specific IgE antibodies to house dust mites (Dermatophagoides pteronyssinus; D. farinae) was determined by enzyme-linked immunosorbent assay (ELISA) in 5097 (61%) volunteers of 8345 schoolchildren aged between 7 and 14 yr from two government schools. All of them filled out a questionnaire concerning allergic symptoms. Among them, 412 (8.1%) children showed a positive reaction to at least one of the two mite allergens, the range varying between 5.6 and 11.2% according to the child's age. Boys had higher prevalence of positive mite specific IgE than girls (9.8% vs. 6.4%, P less than 0.01), with the overall male to female ratio 1.5:1. The prevalence of bronchial asthma in boys and girls was 5.3% and 3.3% respectively. The positive mite specific IgE antibody in children with asthma and allergic rhinitis was 52% (103 of 198) and 28.7% (193 of 673) respectively. The mean levels of mite specific IgE were not significantly related to the age of onset and severity of asthmatic symptoms (P greater than 0.1), but were significantly different among subjects with current and past asthma (P less than 0.001). It is suggested that the mite-specific IgE may play a role in the pathogenesis of bronchial asthma in children.