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Background: Moderate to severe atopic dermatitis (AD) is difficult to treat. There are rare reports on the treatment of moderate to severe AD by lowering serum IgE with a combination of methotrexate (MTX) and azathioprine (AZA) and hence improving symptoms. Objective: This study aimed to improve AD symptoms by lowering serum IgE with a combination therapy of MTX and AZA. Design: Retrospective cohort study of patients treated during 2013 and 2019. Setting: Patients recruited from a tertiary care university hospital in Taiwan. Participants: Sixty-five (case group) and 36 (control group) patients with moderate to severe AD, aged between 10 and 77 years. Measurements: Patients in the case group received a combination of MTX (15 mg weekly) and AZA (150 mg daily) for 1 to 5 years. Patients in the control group received any therapies other than this combination. Serum total IgE levels were checked periodically in both groups. Results: Serum total IgE was markedly reduced in the case group. For example, the cumulative success rates of a 50% IgE reduction in the case group in year 1 and year 5 were 69.2% and 98.5%, respectively, compared to 10.0% and 18.2% in the control group (hazard ratio 14.8; p < 0.001). At the end of year 4, the cumulative success rate of IgE reduction to below the normal range was 41.1% in the case group and 3.4% in the control group (hazard ratio 6.71; p = 0.033). Regarding adverse events, the rates of abnormal white blood cell counts, hemoglobin, platelet counts, and alanine aminotransferase were not increased in the case group compared to the control group. Conclusion: Combination therapy with MTX and AZA was effective in treating patients with intractable AD by reducing their serum total IgE levels.

Isoamylamine (IA) is an aliphatic monoamine molecule present in cheese, eggs, and wine. It belongs to the family of polyamines and also can be synthesized endogenously. It has been known that regulation of polyamines in cells is related to cell cycle and tumor formation. Malignant melanoma is difficult to treat and easily resistant to chemotherapy/radiotherapy through autophagy. In this study, we aim to clarify whether IA has a growth control effect on melanoma tumor cells and the regulatory mechanism. We treated B16-F1 melanoma cells with IA at concentrations of 0, 200, 400, and 600 ppm for 24 h. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was checked for cell viability and results showed that IA has an inhibitory effect on B16-F1 melanoma cells. The signaling molecules, which included Raf/MEK/ERK, were activated, while MSK1 and protein kinase B (AKT) were suppressed. Autophagy was also confirmed to be induced by IA. The acridine orange stain-positive cells were increased and BECN-1/LC3 upregulated. The data also showed that the autophagy regulatory molecule, 5'-adenosine monophosphate-activated protein kinase (AMPK), was induced after IA treatment, so we used dorsomorphin to inhibit AMPK and found that it could suppress autophagy. In conclusion, IA has an effect of inducing autophagy in B16-F1 cells and it is regulated through AMPK.

When a host suffers endotoxemic shock or septic shock, it results in many symptoms including disseminated intravascular coagulation (DIC). Septic shock (SS) causes coagulation time to decrease and then gradually increase, finally becoming prolonged and giving rise to DIC. Isoamylamine (IA) is one of the main components of grape products and can improve the survival rate of endotoxin lipopolysaccharide (LPS)-induced endotoxemic shock. The aim of this study was to elucidate if IA ameliorates coagulopathy in the early phase of LPS-induced damage. We studied the effects of IA on the coagulation system of extrinsic (prothrombin time [PT]) and intrinsic (activated partial thromboplastin time [aPTT]) pathways. PT and aPTT were tested in plasma drawn from mice following intraperitoneal (IP) injection of 1 mL of 1,000 ppm IA after LPS administration. Shortened PT was ameliorated by 1,000 ppm IA 1 h after LPS administration, but there was no effect on aPTT. In conclusion, IA 1,000 ppm partially intervenes in the early phase of LPS-induced damage, shortening plasma PT 1 h after LPS treatment in mice. Furthermore, we found 1,000 ppm IA also could attenuate MMP-9 expression through p-ERK/p-p38 signaling in mice hepatocyte extracts. This study focused on the effects of IA on blood coagulation function and inflammatory proteins. In the current situation of absence of effective treatment for SS, IA can increase survival rate and may offer another choice of patient avoiding causing death during endotoxemic shock.

Summary Objectives To document the patterns of first-order fertility rates associated with extreme maternal ages in Taiwan; determine if there is a gap in adverse birth outcomes (specifically low birth weight and prematurity) between adolescent mothers and older mothers; and determine which factors are important in explaining the differences in adverse birth outcomes between adolescent mothers and older mothers. Study design Government statistics and survey data [Taiwan Birth Cohort Survey (TBCS)]. Methods Descriptive statistics and logistic regression. Results Between 1989 and 2009, the first-order fertility rate for mothers aged 15–19 years decreased, whereas that for mothers aged 35–49 years increased. Analysis of TBCS data revealed that, in comparison with older mothers, adolescent mothers are not necessarily at higher risk for adverse birth outcomes. Birth complications, pregnancy-related risks, adequate number of prenatal care visits and weight gain are more important than socio-economic status and prevalence of smoking in determining birth outcomes among first-time mothers of extreme reproductive ages. Adolescent mothers are less likely to have birth complications and pregnancy-related risks, but are more likely to have an inadequate number of prenatal care visits and to gain an inadequate amount of weight compared with older mothers. Conclusions The risks associated with adolescent mothers are easier to manage than the risks associated with older mothers. Their birth outcomes can be improved through good policy. Taiwan's health policy should target specific risks to reduce the number of adverse birth outcomes among adolescent mothers rather than try to prevent all childbearing during adolescence.

Liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) were applied to characterize drug metabolites. Although these two methods have overcome the identification and structural characterization of metabolites analysis, they remain time-consuming processes. In this study, a novel multiple-stage tandem mass spectrometric method (MS(n) ) was evaluated for identification and characterization of new minor metabolism profiling of penicillin G, one of the β-lactam antibiotics, in human serum. Seven minor metabolites including five phase I metabolites and two phase II metabolites of penicillin G were identified by using data-dependent LC/MS(n) screening in one chromatographic run. The accuracy masses of seven identified metabolites of penicillin G were also confirmed by mass spectral calibration software (MassWorks™). The proposed data-dependent LC/MS(n) method is a powerful tool to provide large amounts of the necessary structural information to characterize minor metabolite in metabolism profiling.

Background This study aimed to examine the associations between aboriginality, age, demographic and socioeconomic factors of the mother and the risk of low birth weight (LBW) in mountain townships of Taiwan. Methods We analyzed the LBW proportion of single live babies born to 2032 first-time mothers between 2004 and 2005. Data were analyzed using the chi-square test, analysis of variance, the Scheffe test and logistic regression. Results About 14.8% of Aboriginal mothers and 18.7% of Aboriginal teen mothers gave birth to infants of LBW. Aboriginal mothers were found to be at higher risk of delivering LBW infants; however, after controlling for marital status and education, the influence of aboriginality and age was no longer significant. Conclusions Marital status and education are more important determinants of LBW than aboriginality and age in mountain townships of Taiwan.

Background Arginase I blood levels elevate in cancerous patients and correlate with cancer stages and poor prognosis. Since arginase is capable of enhancing cell growth, it is unclear whether its ominous effect on cancer progression is through the inhibition of immunity or through direct enhancement of cancer cell growth. We tried to clarify this question. Methods NS-1 mouse myeloma cells were inoculated intraperitoneally (i.p.) into mice. Purified mouse arginase I was injected daily either intravenously (i.v.) or i.p. for 6 d. A tumor-only control group received i.p. tumor cells without arginase. The survival rates of all mice were recorded. Results Survival rates were significantly lower in the i.v. group than in the i.p. group ( P =0.017) or in the tumor-only control group ( P =0.034). As spleen is readily exposed to i.v. arginase, its natural killer cells were studied and were found to have been significantly suppressed by arginase in vitro ( P Conclusion Our results indicate that the direct inhibition of the immune system by i.v. arginase is more significant in shortening the survival of tumor-bearing mice than localized (i.p.) arginase promotion of tumor cell growth. Thus, an elevation of arginase in a patient's blood is very harmful to the host immune system, e.g. splenic natural killer cells.

Background Penicillium species are prevalent airborne fungi. However, the prevalence of allergic sensitization to Penicillium antigens and the true impact of these ubiquitous fungi on atopic respiratory disorders remain to be determined. Objectives The purpose of this study was to analyze the prevalence of immunoglobulin (Ig)E and IgG antibodies against Penicillium chrysogenum (Pen ch 13), the alkaline serine protease major allergen of P. chrysogenum , in asthmatic patients of different age groups. Methods Pen ch 13 was purified from a culture medium of P. chrysogenum . The reactivity of IgE and IgG antibodies to Pen ch 13 in the serum samples of 212 asthmatic patients was analyzed by immunoblotting methods. Results Sixty-nine (33%) of the 212 sera analyzed showed IgE and/or IgG immunoblot reactivity to Pen ch 13. Significant differences in the prevalence of IgE and/or IgG antibody reactivity to Pen ch 13 were found among eight different age groups of 212 asthmatic patients. The frequency of IgE-binding reactivity to Pen ch 13 increased significantly with the age of the patients. It was 7% for the group less than 10 years old and 42% for the group older than 70 years old. In addition, a significant difference between the prevalence of IgE (7%) and IgG (33%) antibodies against Pen ch 13 in the group aged 10 or less was also found. Conclusions Our study demonstrates that IgE and IgG antibodies specific for Pen ch 13 were detected in approximately one-third of the 212 asthmatic patients analyzed. Our results suggest that allergic sensitization to Pen ch 13, and possibly to other airborne Penicillium species, is more common in older asthmatic patients.

Background:Penicillium chrysogenum (Penicillium notatum) is a prevalent airborne Penicillium species. A 34-kD major IgE-reacting component from P. chrysogenum has been identified as an alkaline serine protease (Pen ch 13, also known as Pen n 13 before) by immunoblot and N-terminal amino acid sequence analysis. Methods: In the present study, Pen ch 13 was further characterized in terms of cDNA cloning, protein purification, enzymatic activity, histamine release and IgE cross-reactivity with alkaline serine protease allergens from two other prevalent fungal species – P. citrinum (Pen c 13) and Aspergillus flavus (Asp fl 13). Results: A 1,478-bp cDNA (Pen ch 13) that encodes a 398-amino-acid alkaline serine protease from P. chrysogenum was isolated. This fungal protease has pre- and pro-enzyme sequences. The previously determined N-terminal amino acid sequence of the P. chrysogenum 34-kD major allergen is identical to that of residues 116–125 of the cDNA. Starting from Ala116, the deduced amino acid sequence (283 residues) of the mature alkaline serine protease has a calculated molecular mass of 28.105 kD with two cysteines and two putative N-glycosylation sites. It has 83 and 49% sequence identity with the alkaline serine proteases from P. citrinum and A. fumigatus, respectively. The recombinant Pen ch 13 was recovered from inclusion bodies and isolated under denaturing condition. This recombinant protein reacted with IgE antibodies in serum from an asthmatic patient and with monoclonal antibodies (PCM8, PCM10, PCM39) that reacted with the 34-kD component from P. chrysogenum. The N-terminal amino acid sequence of the purified native Pen ch 13 is identical to that determined previously for the 34-kD major allergen in crude P. chrysogenum extracts. The purified native Pen ch 13 has proteolytic activity with casein as the substrate at pH 8.0. This enzymatic activity was inhibited by phenylmethylsulfonyl fluoride or diethylpyrocarbonate. Pen ch 13 was also able to degrade gelatin and collagen but not elastin. Basophils from 5 asthmatic patients released histamine (12–73%) when exposed to the purified Pen ch 13. In ELISA (enzyme-linked immunosorbent assay) experiments, IgE for Pen ch 13 was able to compete with purified Pen ch 13, Pen c 13 or Asp fl 13 in a dose-related manner. Conclusions: These results demonstrated that the 34-kD major allergen of P. chrysogenum is an alkaline serine protease. These results also indicated that atopic patients primarily sensitized by either of these prevalent fungal species may develop allergic symptoms by exposure to other environmental fungi due to cross-reacting IgE antibodies against this protease.

The measurement of lower back mobility is essential in the assessment of lower back pain including ankylosing spondylitis. Original Schober’s test (OST) and modified Schober’s test (MST) are popularly conducted in daily rheumatology and orthopedics clinical practices. To our knowledge, this report is the only anthropometric reference study in a normal oriental population. The OST declined with age from 5.0 cm in the youngest (20–30 years old) to 3.1 cm in the aged (70–80 years old) male subjects and from 3.6 cm to 2.4 cm in the female subjects. The male OST was significantly more than the female OST. There was a good correlation between OST and MST in each of the three age groups of both sexes.

Aspergillus species are common airborne fungi that have been identified as causative agents of extrinsic bronchial asthma. More than 10 allergens from A. fumigatus have been recently characterized by cDNA cloning. The objective of this study is to identify A. fumigatus allergens through immunoblot analysis using sera from asthmatic patients. IgE-binding components of A. fumigatus and IgE cross-reactivity among allergens of different prevalent airborne fungal species were analysed by immunoblot and immunoblot inhibition, respectively, using sera from asthmatic patients. The N-terminal amino acid sequences of major allergens identified were determined by Edman degradation. Among two batches (70 and 41 sera) of asthmatic sera tested, 19 (27%) and 14 (34%), respectively, have IgE immunoblot reactivity towards components of A. fumigatus. A 34-kDa protein that reacts with IgE antibodies in 15 (79%) and 11 (79%) of the 19 and 14 positive samples, respectively, may be considered a major allergen of A. fumigatus. The N-terminal amino acid sequences of the 34 kDa major allergen and the 30.5 and 30 kDa IgE-binding components of A. fumigatus showed sequence identity to that of the vacuolar serine proteinase from A. fumigatus. The results from immunoblot inhibition show IgE cross-reactivity among major allergens of A. fumigatus, P. notatum and P. oxalicum. Results obtained suggest that the 34 kDa major allergen of A. fumigatus may be a vacuolar serine proteinase. There is IgE cross-reactivity among serine proteinase allergens of A. fumigatus, P. notatum and P. oxalicum.

Background: Penicillium citrinum has been identified as the most prevalent airborne Penicillium species in the Taipei area. It is important to understand the allergenic composition of this ubiquitous fungal species. Objective: The complementary DNA (cDNA) clone of an allergen from P citrinum was isolated and expressed in Escherichia coli as a fusion protein. mAbs were prepared with the recombinant protein as antigen. The corresponding natural allergen in the fungal extracts was identified with the mAbs. Methods: A Uni-Zap XR P citrinum cDNA library was screened with sera from asthmatic patients. An IgE-binding cDNA clone was isolated and expressed as a glutathione-S-transferase fusion protein. The frequency of IgE binding to the expressed protein was analyzed by immunoblotting. Spleen cells from BALB/c mice immunized with the recombinant protein were fused with NS-1 cells for mAb generation. Results: A P citrinum cDNA library was screened with a mixture of serum samples from 4 asthmatic patients. An IgE-binding cDNA clone was obtained and designated as PCE2. PCE2 has a 694-bp insert that contains a 167 amino acids open reading frame. The deduced amino acid sequence of the encoded protein has 82.6% (138 amino acids) identity with an Aspergillus fumigatus peroxisomal membrane protein allergen (Asp f 3). PCE2 was expressed in E coli as a fusion protein and designated as Pen c 3. Sera from 13 (46%) of the 28 Penicillium -sensitized asthmatic patients demonstrated IgE binding to Pen c 3. In addition, 11 of the 13 Pen c 3–positive serum samples have IgE immunoblot reactivity to recombinant Asp f 3. The presence of IgE cross-reactivity between Pen c 3 and Asp f 3 was also detected by immunoblot inhibition. Four of the 6 mAbs generated against Pen c 3 cross-react with Asp f 3. The presence of the corresponding 18-k natural allergens in the crude extracts of P citrinum and A fumigatus were detected by immunoblot with use of the mAbs and sera from asthmatic patients. Conclusion: Results obtained suggest that the peroxisomal membrane protein (Pen c 3) is an important allergen of P citrinum. PCE2 is a full-length cDNA clone encoding this allergen. In addition, the mAbs generated may be useful in standardizing the diagnostic allergenic extracts. (J Allergy Clin Immunol 2000;105:827-33.)

Background: Aspergillus species are prevalent indoor airborne fungi and have been identified to be a causative agent of human allergic disorders. In the present study, we identified, purified and characterized the allergen(s) from Aspergillus flavus, a predominant airborne Aspergillus species in the Taipei area. Methods: The IgE–binding components of A. flavus were identified by SDS–PAGE immunoblotting with sera from asthmatic patients. The N–terminal amino acid sequences of the major allergens were determined by Edman degradation. The allergenic cross–reactivity among allergens from different fungi was analyzed by immunoblot inhibition using sera from asthmatic patients. The detected major allergen was purified from the culture medium. It was further characterized in terms of its N–terminal amino acid sequence, its IgE–binding activity and its enzymatic activity. Results: The results of the immunoblot analysis indicate that a 34–kD component that has high IgE–binding (63%) frequency is a major allergen of A. flavus. The N–terminus of this 34–kD major allergen (GLTTQKSAP) has high sequence identity with that of the 34–kD alkaline serine proteinase major allergen of A. oryzae. Results from immunoblot inhibition studies indicate that IgE cross–reactivity occurs among the 34–kD major allergens of A. flavus, A. fumigatus and Penicillium citrinum. The 34–kD major allergen of A. flavus was purified from the culture medium by ammonium sulfate precipitation and DEAE ion exchange chromatography. The N–terminal amino acid sequence of the purified allergen (Asp fl 13) is identical to that determined previously for the 34–kD major allergen in the crude extract of A. flavus. The IgE immunoblot reactivity to the 34–kD major allergen in the crude extract can be dose–dependently inhibited by the purified Asp fl 13. The degree of IgE binding to the 34–kD major allergen in the crude extract correlates with that of the purified Asp fl 13 in sera of 8 asthmatic patients. The purified Asp fl 13 has proteolytic activity with casein as substrate at pH 8.0. This enzymatic activity can be inhibited by either phenylmethylsulfonyl fluoride or diethylpyrocarbonate. Conclusion: Our results suggest that the 34–kD alkaline serine proteinase is a major allergen of A. flavus. There was IgE cross–reactivity among allergens of A. flavus, A. fumigatus and P. citrinum.

House dust mites have been reported as one of the most important allergens in Taiwan especially in asthmatic patients. This study was conducted to determine the allergenicity of Blomia tropicalis and sensitization of asthmatic patients in Taiwan. Serial dust samples were collected every month between July 1993 and June 1994 from 13 houses of mite-allergic patients. About 1 m2 surface area of a quilt was vacuumed. The floating method was used to collect mites, then identification and counting were performed. Results showed that Dermatophagoides pteronyssinus and B. tropicalis were the two most common species of mites found in allergenic patients’ houses in Taipei. D. pteronyssinus accounted for 52.1% of the total number of mites and was found in every house. B. tropicalis, although not present in every sample, accounted for 44.3% of the total number of mites. The skin test positive reaction to B. tropicalis, D. pteronyssinus and Dermatophagoides farinae were 73.3, 88.3 and 85.0% as determined from 60 allergic patients who attended our allergy clinics. The extract prepared from B. tropicalis was used to determine the allergenicity and contained at least 30 protein components when silver stained. The most frequently detected allergens were proteins with molecular weights of 14.3, 106.5, 94.0, 72.0, 91.9, 63.7, 100.3, 43.6, 27.3, 62.0, 34.7, 18.3, 41.1 and 21.9 kD. The frequency of IgE binding of patient sera to those proteins were 87.0, 65.2, 56.5, 43.4, 39.1, 39.1, 34.8, 30.4, 30.4, 17.4, 17.4, 17.4, 13.0 and 8.7%. The results from immunoblot inhibition showed that there was IgE cross-reactivity among the B. tropicalis and D. pteronyssinus. However, there were two major allergenic components of B. tropicalis not inhibited by D. pteronyssinus with molecular weights of about 14.3 and 27.3 kD. The use of B. tropicalis extract for diagnostic purposes to identify patients with specific sensitivity should be considered in Taiwan.

The therapeutic effect and mechanism of action of tetrandrine on asthma are not defined. Recently, it has been proposed that mononuclear cell (MNC) infiltration in the airway plays a role in the pathogenesis of asthma. In this study, we evaluated the effect of tetrandrine on the cell receptor expression and cytokine production of MNC from two groups (young atopic and old non-atopic) of stable asthmatic patients. MNC separated from peripheral blood of both asthmatic patients and normal individual were cultured in serum free RPMI-1640, with or without phytohemagglutinin (5 μg/ml) and tetrandrine (2 μg/ml). After culture, MNCs were harvested and stained with monoclonal antibodies for HLA-DR, CD23, CD25 and CD3. MNC supernatants were collected for the measurement of IL-2, IL-4 and interferon-gamma (IFN-γ). The results show that tetrandrine may inhibit (1) MNC proliferation, (2) the production of IL-2, IL-4 and IFN-γ, and (3) the expression of HLA-DR, CD23 and CD25 on CD3 positive T cells. They were inhibited to a similar extent in both groups of asthmatic patients. These results suggest that tetrandrine might have some therapeutic role in relation to the suppression of lymphocyte function in asthmatics.

We investigated the contributing effect of Dermatophagoides pteronyssinus-stimulated mononuclear cell (MNC) cultured supernatants on the differentiation and density alteration of eosinophils. MNC, obtained from either normal subjects or asthmatics, were cultured with or without D. pteronyssinus. The supernatants were tested for the activity of eosinophil differentiation and density alteration. The results showed a significant increase in eosinophil differentiation activity in D. pteronyssinus-stimulated MNC supernatants of asthmatics when compared with normal subjects. This activity can be blocked by anti-IL-5 antibodies. Eosinophil hypodensity change was also noted after treatment with D. pteronyssinus-stimulated MNC supernatants or IL-5, but not after D. pteronyssinus treatment. In conclusion, MNCs, activated by D. pteronyssinus, might contribute to the eosinophil differentiation and hypodensity change in asthmatics.

To determine whether nasal allergic symptoms can cause bronchial hyperresponsiveness to methacholine, 30 subjects with allergic rhinitis (22 with allergic rhinitis and 8 with allergic asthmatic rhinitis) were studied. All subjects were skin test positive to Dermatophagoides pteronyssinus (DP) and underwent nasal allergic provocation with DP. After provocation, there was a severe nasal allergic reaction in the challenged nostril with a significant increase in nasal resistance both immediately and long (7 h) after DP exposure. There were no significant changes in the forced expiratory volume in 1 s and the forced expiratory flow rate between 25 and 75% of the forced vital capacity and in both allergic rhinitis and allergic asthmatic rhinitis patients. There was also no change in bronchial hyperresponsiveness to methacholine. The eosinophil counts, leukotriene B4, leukotriene C4 and platelet-activating factor levels in nasal discharges also showed no differences in both groups of patients. Our studies suggest that nasal provocation with limited allergen is a safe diagnostic technique. However, the relationship between nasal resistance and airway hyperreactivity is not obvious in this study. The similar concentrations of nasal inflammatory mediators in both allergic rhinitis and allergic asthmatic rhinitis indicate that bronchial hyperreactivity is not solely due to nasal drainage of inflammatory mediators.

Acute massive pulmonary hemorrhage (AMPH) is a rare and highly fatal complication in systemic lupus erythematosus (SLE). We report here survival in a case of AMPH in a SLE patient with both rapidly progressive glomerulonephritis and lupus anticoagulant. The AMPH occurred while the nephritis was refractory to 2 courses of pulse methylprednisolone therapy. After combined therapy with plasmapheresis plus cyclophosphamide, circulating immune complex levels declined, AMPH recovered, and serum creatinine levels returned to normal. In conclusion, the combined therapy of plasmapheresis plus cyclophosphamide should be considered for treating AMPH especially in those SLE patients with rapidly progressive glomerulonephritis.

Arginase has been detected in high levels in gastric cancer tissues. The effect of arginase on the activities of splenic natural killer (NK) cell, phytohemagglutinin activated killer (PAK) cell, and interleukin-2 activated killer (LAK) cell in patients with gastric cancer (N = 12) was evaluated in vitro. These activities in patients (N = 10) with trauma and benign lesions were used as control. The splenic NK and PAK cell activities in patients with gastric cancer were significantly lower than in the controls (P0.05), whereas LAK cell activity did not have significant difference. Arginase inhibited all splenic killer cell activities to a similar degree between patients with gastric cancer and the controls. The inhibition was dose-related. These data suggest that arginase may play a positive role in the spread of gastric cancer cells. However, LAK may be a potential approach of immunoadoptive therapy in the future.

Human liver arginase has many biological effects on lymphocytes, macrophages, liver cells, and tumor cells, in addition to its major role in the liver urea cycle. We have developed a sandwich enzyme-linked immunosorbent assay (ELISA) method to quantitate arginase concentrations in plasma that can be applied to various body fluids. The sensitivity was 2.5 ng/mL. The coefficients of variation were good both in intra- and inter-assay. Using this method to study the stability of an arginase preparation, we found that plasma arginase was stable for only 1 or 2 days even at temperatures as low as 4 degrees C. The mean plasma level was 41.0 +/- 3.3 ng/mL (mean +/- SE) in 143 normal subjects. There was no age difference in the general population and in the male group. However, in the female group, the plasma arginase level increased with age (p = 0.05). Its biological significance was unclear. As a whole, the ELISA method for the measurement of arginase concentration in the body fluid is convenient and reliable.

Methotrexate pneumonitis is one of the most unpredictable and potentially serious adverse effects associated with the use of low-dose pulse methotrexate in treating rheumatoid arthritis. However, its occurrence in treating bronchial asthma has never been reported. A patient with steroid-dependent bronchial asthma developed interstitial pneumonitis during methotrexate therapy. Dyspnea, fever and oral ulcer occurred successively during the initial 4 months of intermittent low-dose methotrexate pulse therapy. Despite severe hypoxemia and interstitial infiltration in both lung fields, the lung lesions disappeared after treatment with corticosteroid and discontinuation of methotrexate therapy. In conclusion, methotrexate pneumonitis might develop after treatment. Once pneumonitis is suspected, methotrexate should be withdrawn.

Liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) were applied to characterize drug metabolites. Although these two methods have overcome the identification and structural characterization of metabolites analysis, they remain time-consuming processes. In this study, a novel multiple-stage tandem mass spectrometric method (MS(n) ) was evaluated for identification and characterization of new minor metabolism profiling of penicillin G, one of the β-lactam antibiotics, in human serum. Seven minor metabolites including five phase I metabolites and two phase II metabolites of penicillin G were identified by using data-dependent LC/MS(n) screening in one chromatographic run. The accuracy masses of seven identified metabolites of penicillin G were also confirmed by mass spectral calibration software (MassWorks™). The proposed data-dependent LC/MS(n) method is a powerful tool to provide large amounts of the necessary structural information to characterize minor metabolite in metabolism profiling.

Arginase, a potent immune inhibitor, existed in much greater abundance in the cytoplasm of cancer cells than in normal cells. Serum arginase levels from 31 patients with colorectal adenocarcinoma were determined by using enzyme immunoassay (mean +/- standard error = 18.96 +/- 4.83 ng/ml) and showed to be significantly higher than levels from control subjects (n = 115, 3.09 +/- 0.22 ng/ml) (P less than 0.005). Surgical samples of 15 patients were individually homogenized and assayed by the same method and revealed that the arginase level in tissues with colorectal cancer was two times greater than the level found in normal mucosal tissues (1.74 +/- 0.31 micrograms/g tissue versus 0.77 +/- 0.09 micrograms/g tissue, P less than 0.005). However, the serum arginase levels in patients with colorectal cancer were independent of their carcinoembryonic antigen (CEA) levels (n = 27, arginase 11.81 +/- 1.88 ng/ml, CEA 17.31 +/- 4.24 ng/ml, r = 0.084, P = 0.666). The results suggested that serum arginase level can be a valuable criterion for preoperative evaluation and possibly postoperative follow-up study. It can also combine with CEA determination to intensify the clinical assessment for colorectal cancer.

Gastric cancer tissues have high levels of glucocorticoid receptors (GR) and arginase. To investigate the interrelation of glucocorticoid, GR and arginase, three human gastric cancer cell lines (AZ-521, NUGC-3, KATO-III) were treated with hydrocortisone in the presence or absence of a glucocorticoid antagonist RU38486. GR were found to be present in all three lines, and hydrocortisone significantly increased the production of total arginase in all 3 lines. The induction of arginase production by hydrocortisone was inhibited by RU38486. These findings suggest that the regulation of arginase production by hydrocortisone in gastric cancer cells is mediated through GR.

Components of the crude extract of Penicillium notatum recognized by human IgE antibodies (Abs) were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The allergenic components were identified with sera from 19 allergic patients and 20 blood donors. The allergen-Ab complexes were visualized by 125I-labeled monoclonal antihuman IgE and autoradiography. A total of 11 allergenic components, ranging in molecular weights (MWs) from 94,000 to 20,000 daltons, were identified. Heterogeneity in the IgE-binding patterns of the serum samples tested was also observed. However, the major allergen appears to be the component with an MW of about 68,000 daltons that was recognized by IgE Abs in 56% of the 39 sera analyzed. Furthermore, the component with an MW of about 64,000 daltons that was recognized by IgE Abs in 46% of the sera analyzed was also considered as an important allergen. Results obtained from this study will be useful in additional characterization of allergens of P. notatum and related fungal species.

Penicillium chrysogenum (Penicillium notatum) is a prevalent airborne Penicillium species. A 34-kD major IgE-reacting component from P. chrysogenum has been identified as an alkaline serine protease (Pen ch 13, also known as Pen n 13 before) by immunoblot and N-terminal amino acid sequence analysis.In the present study, Pen ch 13 was further characterized in terms of cDNA cloning, protein purification, enzymatic activity, histamine release and IgE cross-reactivity with alkaline serine protease allergens from two other prevalent fungal species--P. citrinum (Pen c 13) and Aspergillus flavus (Asp fl 13).A 1,478-bp cDNA (Pen ch 13) that encodes a 398-amino-acid alkaline serine protease from P. chrysogenum was isolated. This fungal protease has pre- and pro-enzyme sequences. The previously determined N-terminal amino acid sequence of the P. chrysogenum 34-kD major allergen is identical to that of residues 116-125 of the cDNA. Starting from Ala116, the deduced amino acid sequence (283 residues) of the mature alkaline serine protease has a calculated molecular mass of 28.105 kD with two cysteines and two putative N-glycosylation sites. It has 83 and 49% sequence identity with the alkaline serine proteases from P. citrinum and A. fumigatus, respectively. The recombinant Pen ch 13 was recovered from inclusion bodies and isolated under denaturing condition. This recombinant protein reacted with IgE antibodies in serum from an asthmatic patient and with monoclonal antibodies (PCM8, PCM10, PCM39) that reacted with the 34-kD component from P. chrysogenum. The N-terminal amino acid sequence of the purified native Pen ch 13 is identical to that determined previously for the 34-kD major allergen in crude P. chrysogenum extracts. The purified native Pen ch 13 has proteolytic activity with casein as the substrate at pH 8.0. This enzymatic activity was inhibited by phenylmethylsulfonyl fluoride or diethylpyrocarbonate. Pen ch 13 was also able to degrade gelatin and collagen but not elastin. Basophils from 5 asthmatic patients released histamine (12-73%) when exposed to the purified Pen ch 13. In ELISA (enzyme-linked immunosorbent assay) experiments, IgE for Pen ch 13 was able to compete with purified Pen ch 13, Pen c 13 or Asp fl 13 in a dose-related manner.These results demonstrated that the 34-kD major allergen of P. chrysogenum is an alkaline serine protease. These results also indicated that atopic patients primarily sensitized by either of these prevalent fungal species may develop allergic symptoms by exposure to other environmental fungi due to cross-reacting IgE antibodies against this protease.

Penicillium species are prevalent indoor airborne fungi that have been identified as causative agents of human extrinsic bronchial asthma. In the preparation of standardized diagnostic reagents, it is imperative to define the allergens of these ubiquitous fungi. Results from our previous study on P. oxalicum suggest that the 34-kd major immunoglobulin E-reacting component of this prevalent Penicillium species is probably a vacuolar serine protease. The purpose of the present study was to define this major P. oxalicum allergen (Pen o 18) through cDNA cloning and immunologic characterization. The cDNA of Pen o 18 was isolated through a combination of reverse transcriptase-polymerase chain reaction and 5'- and 3'-rapid amplification cDNA ends reactions. The primers used in these reactions were constructed according to the internal amino acid sequences of Pen o 18 and the conserved amino acid sequences of fungal serine proteases. Our results showed that a 1897-bp cDNA with an open reading frame of 503 residues was isolated for the proenzyme of Pen o 18. The encoded protein has a 16-residue signal peptide and a 119-residue prosequence. On maturation, the protein has an N-terminal glutamate that is the 136th residue encoded by the cDNA. Apparently the precursor also undergoes C-terminal processing with the cleavage of about 47 amino acids. The cDNA for Pen c 18 (the vacuolar serine protease allergen from P. citrinum ) was also isolated for comparison. Contrary to a previous report, the C-terminal region of Pen c 18 is similar to that of Pen o 18. Recombinant proteins (rPen o 18 and rPen c 18) with the putative mature N-termini and a his-tag were obtained by expressing the corresponding cDNAs in Escherichia coli. Serum samples from 7 asthmatic patients with immunoglobulin E reactivity to the 34-kd component of P. oxalicum also react to his-tagged recombinant Pen o 18. The presence of immunoglobulin E cross-reactivity between rPen o 18 and rPen c 18 was detected by immunoblot inhibition. Two monoclonal antibodies (PCM39 and FUM20) against fungal serine proteases react with rPen o 18, rPen c 18, and the 35/34-kd components in the corresponding crude fungal extracts. These components also react with immunoglobulin E antibodies in serum samples from asthmatic patients. In conclusion, results obtained confirm that the 34-kd major allergen of P. oxalicum is a vacuolar serine protease. The cDNAs of Pen o 18 and Pen c 18 encode precursor molecules that appear to undergo both N-terminal and C-terminal processing. Constructs beginning with mature N-terminal can be expressed in E. coli to produce recombinant polypeptides that are reactive to monoclonal antibodies or immunoglobulin E antibodies in serum samples from asthmatic patients. Results obtained may provide useful information and materials for preparation of standardized diagnostic reagents in clinical mold allergy.

Background:Aspergillus species are common indoor airborne fungi and have been considered as causative agents of human allergic disorders. However, allergens of different Aspergillus species have not been effectively characterized. The object of this study was to identify and characterize IgE-binding components of Aspergillus oryzae. Methods. Allergens of A. oryzae were identified by immunoblot analysis using sera from asthmatic patients. The N-terminal amino acid sequences of allergens thus identified were determined by Edman degradation. The antigenic and the allergenic cross-reactivities between allergens of different fungi were analyzed by immunoblotting and immunoblot inhibition analysis, respectively, using a monoclonal antibody (MoAb) 55A against the 33-kD major allergen of Penicillium citrinum and a mixture of IgE-containing asthmatic serum samples. Results: Thirteen components of A. oryzae ranging in apparent molecular weight from 16 to 42 kD react with IgE antibodies. A 34-kD component that showed intense IgE-binding reactivity and was detectable in the highest frequency in our asthmatic serum samples tested was considered a major allergen of A. oryzae. The 34-kD component also reacted with MoAb 55A. Results from immunoblot inhibition studies also demonstrated the IgE cross-reactivity between the 34-kD major allergens of A. oryzae and P. citrinum. In addition, the sequence of the N-terminal 18 amino acid residues of the 34-kD major allergen of A. oryzae was found to be identical to that of the alkaline serine proteinase from the same Aspergillus species. Conclusion: The 34-kD major allergen of A. oryzae is an alkaline serine proteinase. There is IgE cross-reactivity between the major serine proteinase allergens of A. oryzae and P. citrinum.

Aged people are considered prone to gram-negative bacteremia and septic shock. This relationship was tested in murine endotoxin shock.Balb/c mice of various ages (1.4-13.4 months) were intraperitoneally injected with lipopolysaccharide (LPS), and rates of survival were observed. The production of TNF-alpha in vivo induced by LPS was measured.The survival rates were the smallest in the oldest and youngest groups. Production of TNF-alpha attained a maximum at 2 h after LPS injection and was smaller in the oldest group; it had a reciprocal relationship to survival rates in each group except the youngest group.Old and young mice had smaller rates of survival and greater production of TNF-alpha following endotoxin shock induced by LPS.

Hepatic arteritis is a rare complication in systemic lupus erythematosus (SLE). Information about its clinical manifestations is still very limited. Elevated serum r-glutamyl transpeptidase and alkaline phosphatase levels, but without elevated bilirubin and transaminase levels, were found in the present report to be the clinical presentation of hepatic arteritis. This clinical picture originally suggested a disease of the biliary tree. Hepatic arteritis must be included in the differential diagnosis of biliary tract disorders in SLE.

Since arginine can stimulate lymphocyte proliferation in the healthy human, its effect on lymphocyte proliferation in vitro was studied in 7 patients with far advanced gastric cancer. These patients with normal nourishment were ambulatory and could consume a regular diet. A daily dietary supplement of 30 g arginine for 7 days did not alter the total lymphocyte counts or the T/B cell ratio in the peripheral blood. Enhancement of lymphocyte proliferation in response to mitogen stimulation was not observed. Furthermore, an in vitro study on the effect of arginine on phytohemagglutinin-stimulated lymphocyte proliferation showed that lymphocytes from gastric cancer patients had poorer responses than those obtained from normal subjects, despite the supplement in the culture medium with normal serum, patient serum, or fetal bovine serum. Arginine ingestion did not impair liver function and had no detectable side effects except transient nausea in 1 patient. These results indicate that dietary arginine supplement appears safe but does not stimulate lymphocyte function in far advanced gastric cancer patients. The suppressed immune function in gastric cancer patients may be the result of their intrinsic lymphocyte defect.

A total of 21 multiply transfused patients with severe aplastic anemia (SAA) were treated with bone marrow transplantation between March 1985 and September 1990: 20 allogeneic and one syngeneic transplants. A positive response in mixed lymphocyte culture (MLC) was also noted in 7 allogeneic recipients. Pregraft conditioning included high-dose cyclophosphamide (CY) 200 mg/kg over 4 consecutive days, followed by 300 cGy total-body irradiation the day before BMT. Seventeen patients older than 14 years received additional donor buffy-coat cells infusion for 5 days posttransplant. A combination of methotrexate and cyclosporine was used for prophylaxis of graft-versus-host disease. Seventeen patients were alive with a functional graft, and Kaplan-Meier product limit estimates showed a 80.95% probability of survival at 67.7 months. There were 4 deaths: two died of primary graft failure, one from secondary rejection, and the other from chronic GVHD-related complications. Acute GVHD, grade I was noted in only one patient (5.6%). In contrast, chronic GVHD was observed in 10 out of 18 (55.6%) evaluable patients. Venoocclusive liver disease and interstitial pneumonitis were not diagnosed. Our findings indicate that the combination of CY/TBI/BC is well tolerated and results in a low incidence of graft failure/rejection in multiply transfused Chinese patients who received transplants for SAA. The MTX/CsA combination was confirmed as being remarkable in reducing the incidence and severity of acute GVHD. For patients with SAA under the age of 40, with an HLA-identical sibling, we highly recommend BMT as the treatment of choice.

The activities of thymidine kinase and uridine kinase (enzymes for pyrimidine salvage pathway) in phytohemagglutinin (PHA) - prestimulated lymphocytes were inhibited by arginase in a similar pattern to the inhibition on thymidine incorporation. Further study revealed that arginase did not directly affect the activities of these enzymes in the cell-free system. Thymidine kinase and uridine kinase activities of PHA-prestimulated lymphocytes were inhibited by arginase making their activities as low as that cultured in arginine-free RPMI-1640 medium. These results suggest that arginine-depletion in the culture medium is the primary mode of action of arginase on the inhibition of mitogen-stimulated lymphocyte proliferation.

Anticardiolipin antibodies purified from serum from patients with systemic lupus erythematosus (SLE) by cardiolipin micelles were studied for their effects on lymphocytes and neutrophils. At a concentration of 160 micrograms/ml they markedly suppressed the [3H]thymidine incorporation of mononuclear cells stimulated by phytohaemagglutinin (4.9 (SEM 1.9%) of the control) and pokeweed mitogen (26.7 (10.5%) of the control). In addition, anticardiolipin antibodies changed the cell cycle of phytohaemagglutinin stimulated lymphocytes such that the S and G2+M phases were significantly diminished (G0/G1 = 64.62%, S = 20.59%, G2+M = 14.78% in the presence of normal human IgG v G0/G1 = 86.07%, S = 10.32%, G2+M = 3.59% in the presence of anticardiolipin antibodies). The suppression of lymphocyte proliferation by anticardiolipin antibodies was shown not to be caused by an alteration of T cell subpopulations. However, the interleukin 2 receptors on the cell surface and the soluble interleukin 2 receptors in the supernatant of phytohaemagglutinin stimulated mononuclear cells were decreased in the presence of anticardiolipin antibodies. On the other hand, the phagocytic activity of neutrophils was 40% inhibited at a higher concentration of anticardiolipin antibodies (300 micrograms/ml) through suppression of C3b/C4b and Fc receptors on polymorphonuclear leucocytes. These results suggest that anticardiolipin antibodies exert inhibitory effects on both lymphocytes and phagocytes in addition to the coagulation cascade. These newly found activities of anticardiolipin antibodies were mediated by the non-specific membranotropic property of the antibodies.

Effects of the extracts from Ganoderm lucidum (GL-P) to influence immune status of the hemophiliacs with positive HIV antibody and reversed helper/suppressor T-lymphocyte ratio were studied. Since the extracts from G. lucidum have been reported to contain high levels of adenosine, the untoward antiplatelet effect of the extracts on hemophiliacs were highly concerned. Five patients of hemophilia A voluntarily received the extracts which has been analyzed to contain 150 mg of adenosine in 100 gm of the extracts. Patients were estimated to take 1.35 mg of the adenosine daily. Platelet aggregation tests before and after the trial of the extracts showed no significant change. Our crude extracts of the Ganoderma lucidum was considered not to have untoward antiplatelet effect in vivo despite the high contents of adenosine.

Allergens and antigens of Bermuda grass pollen fractionated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes were identified using twenty-one sera of Bermuda grass pollen-allergic patients. The IgE- and IgG-binding pollen components transferred to nitrocellulose were detected by reaction with enzyme-labelled anti-human IgE and anti-human IgG, respectively. There was heterogeneity in both IgE- and IgG-binding patterns of the allergic sera tested. Fourteen pollen components, ranging in molecular weight from 16,000 to 88,000 daltons, bound to IgE antibodies. Only two of the fourteen allergens identified reacted with IgE antibodies of more than 50% of the twenty-one allergic sera. The pollen component with a molecular weight of 32,000 daltons showed by far the highest frequency of IgE binding, being recognized by sixteen (76%) of the twenty-one sera examined. Fifteen (71%) of the twenty-one sera tested had IgE antibodies that reacted with more than one of the fourteen allergenic components identified. Pollen components recognized by IgE antibodies also reacted with IgG antibodies, and there were components only recognized by IgG antibodies. Results obtained from this study should be useful both clinically and in research.

The content of glucocorticoid receptor (GR) and arginase in human gastric cancer and the corresponding normal gastric mucosal tissues was determined. Among the 25 patients studied, the GR content in gastric cancer tissues was 33.2 +/- 10.2 fmol/mg protein versus 7.6 +/- 3.4 fmol/mg protein in gastric mucosal tissues. This difference is statistically significant (P less than 0.005). Of the 25 paired samples, 19 cancer tissues contained GR, whereas only seven of the normal mucosal tissues had GR. The level of arginase in gastric cancer tissues in 19 patients was assayed, it was 26.6 +/- 4.2 ng/mg protein which is also significantly higher than that in normal gastric mucosal tissues (13.5 +/- 1.8 ng/mg protein) (P less than 0.005). Since glucocorticoids and arginase are potent immune suppressive agents, the increased level of GR and arginase in gastric cancer tissue suggest that these glucocorticoid-related factors in gastric cancer tissue may play a partial role in regulating cellular immunity.

Liver aqueous extract (LEx) can powerfully inhibit phytohaemagglutinin (PHA)-induced lymphocyte proliferation. The extracellular mechanism of inhibition by LEx was studied. There are three possibilities. (1) The possibility of extracellular inactivation of PHA by LEx was excluded by incubating cells with PHA first, followed by washing, and then incubating cells with LEx. The result was that LEx was still able to inhibit cell proliferation completely. (2) The possibility of competition for cell surface PHA receptors by LEx was excluded by the above experiments plus the use of N-acetyl-D-galactosamine to remove surface-bound PHA. Following this treatment, LEx was still able to inhibit cell proliferation completely. (3) The possibility of arginase-induced arginine depletion resulting in lymphocyte suppression was supported by the following experiments. Cells were incubated in media in which arginine was depleted either by reacting with LEx, and the media were afterwards bound with anti-arginase antibody, or by amino acid constituted media without arginine. The degrees of proliferation inhibition were similar in both treatments. These results indicate the important role of arginine-depletion by LEx, and may account for the LEx-induced lymphocyte inhibition.

Peripheral polymorphonuclear leukocytes and mononuclear leukocytes from normal subjects and subjects with asthma were studied for their production of slow-reacting substance (SRS) after nonimmunologic stimulation with the calcium ionophore A23187. The amount of SRS generated was determined in both polymorphonuclear leukocyte fractions and mononuclear leukocyte fractions from four groups of subjects: 10 with extrinsic asthma, five with mixed-type asthma, 10 with intrinsic asthma, and 10 normal control subjects. In either cell type, the SRS produced was much more in cells from subjects with extrinsic asthma and less in cells from subjects with the mixed-type asthma, intrinsic asthma, and the normal subjects in order of decreasing quantity. These results indicate the amount of SRS generated by nonimmunologic stimulation with calcium ionophore is related to the degree of atopy and suggest the existence of an intrinsic cellular defect in cells from subjects with atopic asthma in addition to the effects of higher serum levels of IgE antibodies.

The incorporation of labeled precursors into DNA, RNA and protein in phytohaemagglutinin (PHA)-prestimulated human lymphocytes was maximally inhibited by liver extract (LEx) or arginase at 24 h. The activities of DNA polymerase α, β and γ were less inhibitable by these agents than was [ 3 H]thymidine incorporation. The inhibition of DNA, RNA and protein syntheses by either LEx or arginase is probably due to arginine depletion by arginase activity, since their syntheses were similarly inhibited when cultured in an arginine-free medium in the absence of arginase. These results indicate that arginase nonspecifically inhibits the activities of DNA polymerase. The inhibition is probably due to arginine depletion.

Various doses of glucocorticoids given in vivo caused a similar degree of maximal lymphopenia. The sensitivities of mitogen-induced proliferation to the suppressive effects of glucocorticoid added in vitro were studied in residual lymphocytes obtained after steroid injection. Methylprednisolone (MP) administered intravenously depleted circulatory lymphocytes and reduced markedly the proliferative responses of residual cells to mitogens (phytohemagglutinin and concanavalin A) 4 to 8 hr after the injection. The addition of MP in vitro to the residual cells further inhibited the cell proliferation. The degrees of proliferation inhibition induced by in vitro MP were compared in cells obtained at various intervals after MP injection. At each specific mitogen concentration, lymphocytes obtained at various intervals were inhibited to a similar degree by MP in the cultures. There was no evidence that cells obtained at the period of maximal lymphopenia, 4 to 8 hr after MP injection, were more resistant to the inhibition of glucocorticoid added in vitro . Hence, the residual lymphocytes were not “steroid-resistant” in the sense of proliferative responses to T-cell mitogens. These results indicate the mechanism of lymphocyte sequestration is unrelated to the suppressive effects of glucocorticoid on cell proliferation.