Delayed-Onset Anaphylaxis Caused by IgE Response to Influenza Vaccination

Influenza vaccine-associated anaphylaxis is a very rare allergic reaction to vaccines, but the most concerning and life-threatening adverse reaction. Although the safety of influenza vaccines has been well documented, occasional cases of anaphylaxis in vaccinated patients have been reported. In this study, we analyzed the immunoglobulin E (IgE) response to whole influenza vaccines in a pediatric case of delayed-onset anaphylaxis after influenza vaccination. The patient showed elevated specific IgE levels against whole influenza vaccines, especially with split virion from egg-based manufacturing process. Specific IgE levels to influenza vaccines showed decreased over. We evaluated a causal relationship between influenza vaccine and anaphylaxis event by enzyme-linked immunosorbent assay. Delayed-onset anaphylaxis after influenza vaccination can occur in children without predisposing allergic diseases. In addition, the results suggested that formulation and production system of influenza vaccines could affect the probability of severe allergic reaction to vaccines.

Development of a HA1-specific enzyme-linked immunosorbent assay against pandemic influenza virus A H1N1

PURPOSE: Enzyme-linked immunosorbent assay (ELISA) has been used in the diverse field to evaluate influenza virus infection; for the surveillance, diagnosis, efficacy evaluation, and development of the vaccine. The aim of this study was to establish an ELISA for detecting HA strain-specific antibodies using recombinant pandemic A H1N1 (pH1N1) HA1 (rHA1) protein. MATERIALS AND METHODS: rHA1 was produced in baculovirus system. The clinical performance of the developed ELISA was validated using human serum samples, by comparison with standard methods for detecting a neutralizing antibody; hemagglutination inhibition (HI) assay and microneutralization test (MNT). The ability of the ELISA system to evaluate the efficacy test of an influenza vaccine was explored by measuring antibody levels in the serum of vaccinated mice. RESULTS: Our ELISA could detect anti-rHA1 antibody in influenza-infected patients and vaccinated subjects. Compared to HI assay and MNT as reference methods, our method showed good performance in detection of anti-rHA1 antibody. Detection of the anti-rHA1 antibody in vaccinated mice and its correlation with titers in HI assay was also proved in a mice model. CONCLUSION: An ELISA system using rHA1 of pH1N1 influenza virus was developed, and showed good clinical performance in diagnosis of influenza virus infection and evaluation of the vaccination efficacy in both human and animal models.

Troglitazone Enhances the Apoptotic Response of DLD-1 Colon Cancer Cells to Photodynamic Therapy

PURPOSE: The aim of this study was to investigate whether the peroxisomal proliferator-activated receptor gamma (PPARγ) ligand troglitazone in combination with photodynamic therapy (PDT) enhances the apoptotic response of DLD-1 colon cancer cells. MATERIALS AND METHODS: The effects of troglitazone, PDT, and troglitazone in combination with PDT on cell viability and apoptosis were assessed in DLD-1 cells. Cell viability and proliferation were evaluated using the tetrazolium-based MTT assay, and apoptosis was evaluated via cell staining with propidium iodide (PI) and annexin V-FITC. The levels of pro-caspase-3 were measured via Western blot analyses. RESULTS: Treatment of troglitazone and PDT induced the growth retardation and cell death of DLD-1 cells in a dose-dependent manner, respectively. The combination treatment significantly suppressed cell growth and increased the apoptotic response of DLD-1 and resulted in apoptosis rather than necrosis, as shown by PI/annexin V staining and degradation of procaspase-3. CONCLUSION: Conclusion: These results document the anti-proliferative and apoptotic activities of PDT in combination with the PPARγ ligand troglitazone and provide a strong rationale for testing the therapeutic potential of combination treatment in colon cancer.

Troglitazone Enhances the Apoptotic Response of DLD-1 Colon Cancer Cells to Photodynamic Therapy

PURPOSE: The aim of this study was to investigate whether the peroxisomal proliferator-activated receptor gamma (PPARγ) ligand troglitazone in combination with photodynamic therapy (PDT) enhances the apoptotic response of DLD-1 colon cancer cells. MATERIALS AND METHODS: The effects of troglitazone, PDT, and troglitazone in combination with PDT on cell viability and apoptosis were assessed in DLD-1 cells. Cell viability and proliferation were evaluated using the tetrazolium-based MTT assay, and apoptosis was evaluated via cell staining with propidium iodide (PI) and annexin V-FITC. The levels of pro-caspase-3 were measured via Western blot analyses. RESULTS: Treatment of troglitazone and PDT induced the growth retardation and cell death of DLD-1 cells in a dose-dependent manner, respectively. The combination treatment significantly suppressed cell growth and increased the apoptotic response of DLD-1 and resulted in apoptosis rather than necrosis, as shown by PI/annexin V staining and degradation of procaspase-3. CONCLUSION: Conclusion: These results document the anti-proliferative and apoptotic activities of PDT in combination with the PPARγ ligand troglitazone and provide a strong rationale for testing the therapeutic potential of combination treatment in colon cancer.

Serum Dickkopf-1 as a Biomarker for the Diagnosis of Hepatocellular Carcinoma

PURPOSE: Dickkopf-1 (DKK-1) is a Wnt/beta-catenin signaling pathway inhibitor. We investigated whether DKK-1 is related to progression in hepatocellular carcinoma (HCC) cells and HCC patients. MATERIALS AND METHODS: In vitro reverse-transcription polymerase chain reaction (RT-PCR), wound healing assays, invasion assays, and ELISAs of patient serum samples were employed. The diagnostic accuracy of the serum DKK-1 ELISA was assessed using receiver operating characteristic (ROC) curves and area under ROC (AUC) analyses. RESULTS: RT-PCR showed high DKK-1 expression in Hep3B and low in 293 cells. Similarly, the secreted DKK-1 concentration in the culture media was high in Hep3B and low in 293 cells. Wound healing and invasion assays using 293, Huh7, and Hep3B cells showed that DKK-1 overexpression promoted cell migration and invasion, whereas DKK-1 knock-down inhibited them. When serum DKK-1 levels were assessed in 370 participants (217 with HCC and 153 without), it was significantly higher in HCC patients than in control groups (median 1.48 ng/mL vs. 0.90 ng/mL, p0.05). When three biomarkers were combined (DKK-1 plus AFP plus DCP), they showed significantly higher AUC (AUC=0.952) than single marker, DKK-1 plus AFP, or DKK-1 plus DCP (all p<0.001). CONCLUSION: DKK-1 might be a key regulator in HCC progression and a potential therapeutic target in HCC. Serum DKK-1 could complement the diagnostic accuracy of AFP and DCP.

Expression of Epstein-Barr Virus Gene and Clonality of Infiltrated T Lymphocytes in Epstein-Barr Virus-associated Gastric Carcinoma

BACKGROUND: Epstein-Barr virus associated gastric lymphoepithelioma-like carcinoma (LELC) is characterized by the intensive infiltration of lymphoid cells, the presence of EBV, and the better prognosis over typical adenocarcinoma. Thus, it was assumable that viral latent proteins may be responsible for the recruitment of a certain T cell repertoire to EBV-associated gastric carcinoma. METHODS: To examine above possibility, EBV gene expression in gastric carcinoma tissues and usage of TCR among the tumor infiltrating lymphocytes were analyzed. RESULTS: EBV specific DNA and EBERs RNA were detected in 4 out of 30 patients. RT-PCR analysis revealed that all 4 of EBV-positive tumor tissues expressed EBNA1 mRNA and BARTs and LMP2a was detected only one sample out of 4. However, the EBNA2 and LMP-1 transcripts were not detected in these tissues. CD8+ T cells were the predominant population of infiltrating lymphocytes in the EBV-positive gastric carcinoma. According to spectra type analysis of infiltrating T cells, 10 predominant bands were detected by TCR Vbeta CDR3 specific RT-PCR from 4 EBV-positive tumor tissues. Sequence analysis of these bands revealed oligoclonal expansion of T cells. CONCLUSION: These findings suggest that clonally expanded T cells in vivo might be a population of cytotoxic T cells reactive to EBV-associated gastric carcinoma.

Validation of Gene Expression Changes of Osteopontin and MMP-1 in Primary and Metastatic Colorectal Carcinomas

BACKGROUND: Metastasis is one of the most important characteristics of cancer in terms of its impact on patient survival. Unfortunately, identification of altered genes during tumor metastasis is limited. METHODS: Using high-throughput microarrays containing 19K spotted human oligonucleotides, gene expression of primary and matched metastatic colon cancer were compared in previous study. Although DNA microarray analysis did not demonstrate complete classification of primary and metastatic carcinoma, 80 differentially expressed genes were identified. Among these, expression of osteopontin, matrix metalloproteinase-1 (MMP-1) and serpin A1 was assessed using immunohistochemistry in a validation set containing 43 pairs from tissue microarrays. RESULTS: The expression of osteopontin was significantly higher in metastatic carcinoma than in primary carcinoma, as indicated by mRNA expression. The expression of MMP-1 was significantly lower in metastatic carcinoma. Expression of serpin A1 was not correlated with the microarray results. CONCLUSIONS: Osteopontin and MMP-1 expression successfully classified primary and metastatic colorectal carcinomas and further studies on their clinical application is encouraged.

Detection of micrometastasis in fixed paraffin-embedded Sentinel Lymph Nodes of Breast cancer using RT-PCR / 한국유방암학회지

PURPOSE: Sentinel lymph node (SLN) biopsy is considered a highly accurate and very economic method of assessing the axillary nodal status in breast cancer patients. Recently immunohistochemical (IHC) staining and reverse transcriptase polymerase chain reaction (RT-PCR) are commonly used to evaluate micrometastasis in the sentinel lymph node. However, most of the RT-PCR studies have been performed using fresh tissue. This study was conducted to assess micrometastasis in clinically node-negative breast cancer by using RT-PCR technique on the paraffin embedded sentinel lymph nodes. METHODS: Sixty patients who undergone SLN biopsy followed by axillary lymph node dissection due to breast carcinoma were evaluated from February 2000 to January 2001 at the Breast Cancer Center, Department of Surgery, Yongdong Severance Hospital. Serial sections were made from all sentinel lymph nodes for the H&E staining and for the IHC staining with monoclonal anti-cytokeratin antibody. RNA was extracted from the paraffin embedded sentinel lymph nodes and RT-PCR was performed using cytokeratin 19 mRNA, MUC-1 mRNA, and MAGE-A3 mRNA. RESULTS: In 32 out of 60 cases, beta-actin mRNA was detected after RT-PCR, and the 28 cases which had no product after RT-PCR for beta-actin were excluded from this study. Twenty five cases showed as being metastasis positive and 7 cases showed as being metastasis negative by serial section (SS) H&E staining. Three out of 25 negative cases tested for by SS H&E staining were found to be positive by IHC. Ten, six and, eight cases out of the 25 negative cases tested for by SS H&E were found to be positive by RT-PCR for cytokeratin 19, MUC-1, and MAGE-A3, respectively. Among the 22 cases that were found to be negative by both SS H&E staining and IHC staining, 9, 4, and 6 cases were converted to positive by RT-PCR for cytokeratin 19, MUC-1, and MAGE-A3, respectively. Using the combination of two or three markers for performing RT-PCR was more sensitive than any single marker to detect micrometastasis (p < 0.05). CONCLUSION: Even though we failed to extract RNA in 46% of the paraffin embedded tissues, it may be possible to detect micrometastasis by using RT-PCR with the paraffin embedded tissue. RT-PCR is far more sensitive than IHC for detecting microme tastasis, and when we combine multiple markers, the detection rate is higher than for any one marker.

Detection of micrometastasis in fixed paraffin-embedded Sentinel Lymph Nodes of Breast cancer using RT-PCR / 한국유방암학회지

PURPOSE: Sentinel lymph node (SLN) biopsy is considered a highly accurate and very economic method of assessing the axillary nodal status in breast cancer patients. Recently immunohistochemical (IHC) staining and reverse transcriptase polymerase chain reaction (RT-PCR) are commonly used to evaluate micrometastasis in the sentinel lymph node. However, most of the RT-PCR studies have been performed using fresh tissue. This study was conducted to assess micrometastasis in clinically node-negative breast cancer by using RT-PCR technique on the paraffin embedded sentinel lymph nodes. METHODS: Sixty patients who undergone SLN biopsy followed by axillary lymph node dissection due to breast carcinoma were evaluated from February 2000 to January 2001 at the Breast Cancer Center, Department of Surgery, Yongdong Severance Hospital. Serial sections were made from all sentinel lymph nodes for the H&E staining and for the IHC staining with monoclonal anti-cytokeratin antibody. RNA was extracted from the paraffin embedded sentinel lymph nodes and RT-PCR was performed using cytokeratin 19 mRNA, MUC-1 mRNA, and MAGE-A3 mRNA. RESULTS: In 32 out of 60 cases, beta-actin mRNA was detected after RT-PCR, and the 28 cases which had no product after RT-PCR for beta-actin were excluded from this study. Twenty five cases showed as being metastasis positive and 7 cases showed as being metastasis negative by serial section (SS) H&E staining. Three out of 25 negative cases tested for by SS H&E staining were found to be positive by IHC. Ten, six and, eight cases out of the 25 negative cases tested for by SS H&E were found to be positive by RT-PCR for cytokeratin 19, MUC-1, and MAGE-A3, respectively. Among the 22 cases that were found to be negative by both SS H&E staining and IHC staining, 9, 4, and 6 cases were converted to positive by RT-PCR for cytokeratin 19, MUC-1, and MAGE-A3, respectively. Using the combination of two or three markers for performing RT-PCR was more sensitive than any single marker to detect micrometastasis (p < 0.05). CONCLUSION: Even though we failed to extract RNA in 46% of the paraffin embedded tissues, it may be possible to detect micrometastasis by using RT-PCR with the paraffin embedded tissue. RT-PCR is far more sensitive than IHC for detecting microme tastasis, and when we combine multiple markers, the detection rate is higher than for any one marker.

Expression Patterns of Cytokines and Chemokines Genes in Human Hepatoma Cells

Various cytokines and chemokines play a role in carcinogenesis. However, no study has previously been undertaken to investigate comprehensively the expressions of cytokines and chemokines in hepatoma cells. In this study, we determined which cytokines and chemokines are expressed in hepatoma cells. Recently, it was reported that the expressions of several chemokines could be increased by Fas stimulus in many normal and cancer cells. Therefore, we also investigated whether chemokines expression is regulated by Fas ligation. To address this issue, we performed RNase protection assays upon 13 cytokines and 8 chemokines genes in 10 human hepatoma cell lines, comprising 8 hepatitis B virus (HBV)-associated hepatoma cell lines. Transforming growth factor-beta2 (TGF-beta2) was found to be expressed in 8 HBV-associated hepatoma cell lines, and to be potently expressed in 5 cell lines; however, the mRNA expressions of interleukin-10 (IL-10), IL-12, interferon-gamma(IFN-gamma) and tumor necrosis factor-alpha(TNF-alpha) were not detected in any cell lines examined. Among the chemokines investigated in this study, IL-8 was expressed by 8 HBV- associated hepatoma cell lines, and monocyte chemoattractant protein-1 (MCP-1) by 7 HBV-associated hepatoma cell lines. However, the mRNA expressions of macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-beta, interferon-inducible protein-10 (IP-10), RANTES, lymphotactin and I-309 were either very weak or undetectable. Fas ligation did not increase chemokines expression in hepatoma cells. Conclusively, TGF-beta2, IL-8 and MCP-1 were overexpressed in HBV-associated hepatoma cells, and the expressions of chemokines were not increased by Fas ligation in human hepatoma cells.

Structural basis for inhibition of protein tyrosine phosphatases by Keggin compounds phosphomolybdate and phosphotungstate

Protein-tyrosine phosphatases (PTPs) constitute a family of receptor-like, and cytoplasmic enzymes, which catalyze the dephosphorylation of phosphotyrosine residues in a variety of receptors and signaling molecules. Together with protein tyrosine kinases (PTKs), PTPs are critically involved in regulating many cellular signaling processes. In this study, diverse compounds were screened for PTP inhibition and selectively screened for inhibitors with the end product inhibition properties. Among phosphate analogues and their derivatives for PTP inhibition, Keggin compounds phosphomolybdate (PM) and phosphotungstate (PT) strongly inhibited both PTP-1B and SHP-1, with K(i) values of 0.06-1.2 micromM in the presence of EDTA. Unlike the vanadium compounds, inhibition potencies of PM and PT were not significantly affected by EDTA. PM and PT were potent, competitive inhibitors for PTPs, but relatively poor inhibitors of Ser/Thr phosphatase. Interestingly, PM and PT did not inhibit alkaline phosphatase at all. The crystal structure of PTP-1B in complex with PM, at 2.0 A resolution, reveals that MoO(3), derived from PM by hydrolysis, binds at the active site. The molybdenium atom of the inhibitor is coordinated with six ligands: three oxo-ligands, two apical water molecules and a S atom of the catalytic cysteine residue. In support of the crystallographic finding, we observed that molybdenium oxides (MoO(3), MoO(2), and MoO(2)Cl(2)) inhibited PTP-1B with IC(50) in the range 5-15 micromM.

The Effects of Swimming Training on Lymphocyte Proliferation and ROS Production in Spleen Lymphocytes of BALB/c Mice

BACKGROUND: Aerobic training can be defined as any physical exercise that increases the heart rate and enhances the body's intake of oxygen long enough to benefit the condition of body. Running, cycling, and swimming are examples of aerobic activities. This type of exercise optimises immune functions. Recently several experimental findings suggested that the regular swimming training increase immune response, but there have been very few reports which compare warm water exercise with cold water exercise in spleen lymphocytes. METHODS: This study was designed to examine the effects of regular swimming training on Index, the number of lymphocytes, proliferative activity and production of reactive oxygen species (ROS) by splenocytes in BALB/c mice. Thirty six mice (6 week old) were performed 10 weeks of regular swimming training and they were divided into 6 groups according to the regular swimming training (CRG: control resting group, CEG: control exercise group, WRG: warm water trained resting group, WEG: warm water trained exercise group, CORG: cold water trained resting group, COEG: cold water exercise group). Analytical items were weight change, spleen index, the number of lymphocytes, proliferative activity and production of ROS. All data were expressed as mean and standard deviation by using SPSS package program (ver. 10.0). RESULTS: The swimming training significantly decreased body weight, and increased spleen index, the number of lymphocytes and proliferative activity in the presence or absence of Con A and LPS added conditions. For the WRG and CORG, the quantity of ROS from splenocytes was higher than CRG, whereas, ROS by spleen lymphocytes was lower following 90 min acute exercise stress. CONCLUSION: These results suggested that the swimming training not only increases the number of lymphocytes but also increases proliferative activity by splenocytes in vitro.

Apoptosis of Chondro cytes and Its Regulation in Pathogenesis of Osteoarthritis / 대한류마티스학회지

No abstract available.

Polymerase Chain Reaction in the Diagnosis of Infectious Endophthalmitis

PURPOSE: We evaluated the efficacy of the PCR which is known as more sensitive method than culture in the diagnosis of causal microorganisms of the infective endophthalmitis. METHODS: We used 0.3 ml of aqueous humor and 0.5 ml of vitreous sample in 3 cases of postoperative and 1 case of endogenous endophthalmitis for detecting causal microorganisms. To detect the bacteria we used universal, Gram positive and negative primers, and to detect the fungus we used fungal primer. RESULTS: Three cases of endophthalmitis, there was no bacteria in the bacterial culture for 10 days but PCR results identified causal microorganisms in all cases. CONCLUSIONS: PCR is effective in the fast and accurate diagnosis of infective endophthalmitis and especially in the culture-negative endophthalmitis.

Influence of the sequence variations of the HLA-DR promoters derived from human melanoma cell lines on nuclear protein binding and promoter activity

In previous studies we reported that the expression of HLA-DR on melanoma cell lines was differentially modulated by IFN- gamma and that the transcription rate was responsible for this differential modulation. We have also reported the nucleotide sequence variations in the promoter region of HLA-DR genes, and proposed that differences in the promoter activity by the sequence variations of the HLA-DR promoters might contribute to such a differential transcriptional regulation at the promoter level. In this study, in order to assess whether the sequence variations of the HLA-DR promoters affect the factor binding and exert influence on the promoter activity, nuclear factor binding to our previous six HLA-DRA and fourteen HLA-DRB promoter clones was evaluated with the nuclear protein extracted from a B-lymphoblastoid cell line (BLCL), BH, together with the chloramphenicol acetyltransferase (CAT) reporter assay. In the HLA-DRA promoters, clone 35 containing one bp nucleotide sequence variation at the octamer binding site (OCT) (GATTTGC to GATCTGC) showed relatively weak factor binding. In the HLA-DRB promoters, clusters I, III, and IV of our previous HLA-DRB promoter homologues, containing one bp nucleotide sequence variation (GATTCG) in their Y boxes exhibited weak factor binding and CAT activity compared to other clusters (GATTGG) that showed strong factor binding and CAT activity. This data suggests chat the binding patterns of transcription factors influenced by the nucleotide sequence variations of the HLA-DR promoter could affect the promoter activity and the DNA sequence elements in the HLA-DR promoter could mediate transcriptional regulation.

Restoration of P-glycoprotein function is involved in the increase of natural killer activity with exogenous interleukin-15 in human immunodeficiency virus-infected individuals

A depressed level of natural killer (NK) activity is one of the various immunologic abnormalities in human immunodeficiency virus (HIV) infection. Interleukin-15 (IL-15), an immunotherapeutic candidate in HIV infection, increases NK activity and induces the excretion of CC-chemokines from divergent immune cells, but the mechanisms of NK activity enhancement by IL-15 stimulation is not clearly established in HIV infection. This study examined whether CC-chemokines, which are known to increase NK activity, are secreted adequately in HIV-infected individuals, and also investigated whether P-glycoprotein is involved in NK activity enhancement after IL-15 administration. NK activity increased with IL-15 stimulation in NK cells of HIV-infected individuals, as it does in normal NK cells. IL-15 stimulates NK cells to secrete CC-chemokines, such as, macrophage inflammatory protein-1alpha (MIP-1alpha), macrophage chemotactic protein-1alpha (MCP-1alpha) and regulated upon activation, normal T cells expressed and secreted (RANTES) in both HIV-infected individuals and controls with no significant difference. P-glycoprotein expression and function is decreased in HIV-infected individuals and restored only in NK cells of HIV-infected individuals after IL-15 stimulation. P-glycoprotein may play a role in the mechanism of increased NK cell activity in HIV-infected individuals after IL-15 stimulation.

Expression of c-erbB2 and HLA-A2 in Breast Cancer Patients / 한국유방암학회지

PURPOSE: Oncogene c-erbB2 produces a transmembrane protein similar in structure to the tyrosine kinase family. Overexpression of c-erbB2 is known to lower the survival rate of breast cancer patients. c-erbB2 protein is an important antigen for tumor specific cytotoxic T lymphocytes induction that is dependent on its presentation as stably complexed with HLA-A2. In 1997, Nistico P reported low frequency of c-erbB2 proto-oncogene overexpression in HLA A2 positive breast cancer patients. And then in this study, correlation of HLA-A2 and the c-erbB2 expression was investigated in breast cancer patients. MATERIALS AND METHODS: HLA-A DNA typing by locus-specific generic PCR and by hybridization with sequence-specific oligonucleotide probes (SSOP) was performed on peripheral blood lymphocytes from 52 breast cancer patients (a PCR-SSOP typing method, involving a PCR amplification in conjunction with digoxigenin labelled sequence-specific oligonucleotide probes). To determine c-erbB2 expression, immunohistochemistry from paraffin-embedded tissues in a series of 47 patients with available tissue blocks was performed by use of rabbit anti-human c-erbB2 oncoprotein (DAKO, Glostrup, Denmark). And then we statistically analyzed the relation between the expressions of HLA-A2 and c-erbB2 in breast cancer patients. RESULTS: 29 out of 52 patients (55.8%) were HLA-A2 positive. 23.4% (11out of 47 patients) of breast cancer patients overexpressed c-erbB2. The patients with c-erbB2 overexpression showed lower estrogen receptor positivity compared to those without c-erbB2 overexpression (10.5%, vs 33.3%). HLA-A2 positive patients showed 18.5% (5/27) of overexpression and HLA-A2 negative patients showed 30.0% (6/20) of c-erbB2 overexpression (p=0.283). CONCLUSIONS: We observed no correlation between HLA-A2 and prognostic factors in breast cancer such as tumor size, axillary nodal status. However, our results showed a tendency without statistical significance between HLA-A2 and high frequency of c-erbB2 overexpression. More accumulation of patients will be needed for better conclusions.

The effect of cytokines and endotoxin on the nitric oxide production and its relation to mitochondrial aconitase activity in cultured rat lung microvascular endothelial cells / 대한내과학회지

OBJECTIVE: Both constitutive and inducible forms of nitric oxide synthase exist in endothelial cells. Disorders that produce acute lung injury frequently release endotoxin and cytoknes, such as interferon(IFNgamma) and tumor necrosis factor (TNFalpha). Endotoxin and these cytokines likely act as important mediators of cell injury. Because nitric oxide (NO) avidly reacts with iron, it may affect the activity of key enzymes, such as mitochondrial aconitase, which contain an iron-sulfur structure as a prosthetic group. METHOD: We studied the effect of IFNgamma, TNFalpha and E. coli lipopolysaccharide(LPS) on NO production and mitochondrial aconitase activity in cultured rat lung microvascular endothelial cells(RLMVC). RESULT: Exposing RLMVC for 24 hours to IFNgamma(500 U/mL), TNFalpha(300 U/mL) and LPS(5 microgram/mL) significantly increases nitrite production to 20+/-1 micrometer compared to 0.07 micrometer in control cells(P<0.05, n=4). Cytokine treatment also reduced mitochondrial aconitase activity from 196+/-8 to 102+/-34 nmole/min/mg of cell protein(P<0.05, n=4). Treatment with the inhibitor of nitric oxide synthase N-monomethyl-L-arginine(NMMA) (0.5 mM) not only significantly blunted the cytokine-mediated increase in nitrite formation (3+/-0.5 micrometer vs 20+/-1 micrometer with cytokines, P<0.05, n=4), but also prevented the cytokine-mediated drop in aconitase activity (161+/- 24 vs. 196+/-8 nmole/min/mg of cell protein, NS). CONCLUSION: Exposing RLMVC to IFNgamma, TNFalpha and E. coli LPS substantially decreases mitochondrial aconitase activity. Nitric oxide appears to mediate this effect. Our results suggest that the excessive production of NO by endothelial cells, in response to cytokines and endotoxin, may inhibit the function of the endothelial cell itself.

Molecular analysis of HLA-DR gene expression induced by IFN-gamma in malignant melanoma cell lines

Human leukocyte antigen (HLA) class II molecules are polymorphic cell surface glycoproteins that are crucial for the cellular interaction in immune response. The expression of class II molecules is regulated in a tissue-specific and cytokine-inducible manner, and is mainly restricted to the antigen presenting cells. However, some tumor cells also express class II molecules, and in some class-II-negative tumor cells, class II expression is inducible by interferon (IFN)-gamma. However, their expression varies, even though the tumor cells originate from the same histological origin; some tumor cells show strong expression, others show weak or no expression. To determine whether this differential expression of class II molecules on tumor cells is transcriptionally regulated, FACS analysis and Northern hybridization were performed using a panel of melanoma cell lines, IGR3, Malme-3M, SK-Mel-24, and SK-Mel-28 to analyze the cell surface expression and mRNA transcription rate of HLA-DR before and after treatment with IFN-gamma. FACS analysis showed that before IFN-gamma treatment, IGR3 and Malme-3M cells barely expressed HLA-DR. On the contrary, almost all of the SK-Mel-24 cells (> 90%) and a relatively high rate (> 50%) of SK-Mel-28 cells expressed HLA-DR. After IFN-gamma treatment, HLA-DR expression was induced in Malme-3M cells and SK-Mel-28 cells which displayed elevated levels of HLA-DR expression in a time-dependent manner. However, IGR3 cells never responded to IFN-gamma. Northern analysis showed that treatment with IFN-gamma led to the steady-state mRNA augmentation of the HLA-DR gene in Malme-3M and SK-Mel-28, whereas in IGR3, IFN-gamma did not augment the transcriptional rate of the HLA-DR gene. To further clarify this differential modulation, sequencing analysis of PCR product of the HLA-DR proximal promoter region was done, since the transcription rate of the class II gene is controlled by the well-conserved proximal promoter region. Six independent clones from PCR products of the HLA-DRA proximal promoter region and 16 clones from PCR products of the HLA-DRB proximal promoter region were isolated from the above cell lines and sequenced. Comparison of the nucleotide sequences of all 6 clones of DRA promoter showed that the sequences are extremely similar in both regulatory sequences and their intervening sequences. Sixteen clones of HLA-DRB promoter showed sequence variations such as substitution and insertion/deletion, and these 16 clones could be further grouped into 6 homologues with sequence homology. These data established that the melanoma cell lines studied here showed a differential susceptibility to IFN-gamma on the modulation of HLA-DR molecules, that this modulation is transcriptionally regulated, and that the difference in promoter activity by sequence variation might contribute to such a differential transcriptional regulation at the promoter level.

Isolation of a Partial Human cDNA Encoding a Factor Binding to the Perfect Palindrome of Enhancer A of HLA Class I Promoter, Homologous to NF-kB2

Genes encoding sequence-specific DNA binding proteins have been isolated by screening cDNA libraries constructed in rgt11 expression vector with recognition site DNAs. We isolated a rgt11 recombinant human cDNA clone, designated to C2, using a DNA probe consisted of heptamer of the perfect palindrome (PP; GGGGATTCCCC) of enhancer A (Enh A) of HLA dass I promoter. Sequencing analysis showed that this clone contained a partial cDNA homologous to NF-kB2. Lysogenic E. coli containing the C2 was generated and crude cell extract was prepared. Immunoblot using anti-B-galactosidase antibody showed that this lysogenic E. coli expressed B-galactosidase fusion protein. Electrophoretic mobility shift assay (EMSA) and DNase I footprinting assay were done using crude cell extract and their patterns were compared with nuclear protein extracted from an EBV transformed B lymphoblastoid cell line (BLCL). EMSA showed that crude cell extract prepared from E. coli lysogen speci5cally bound to the PP of Enh A region of HLA class I gene. DNase I footprinting assay showed that the binding sequence of this recombinant B-galactosidase fusion protein was identical to that of nuclear protein extracted from a BLCL. Our data indicate that a Agt11 recombinant cDNA clone was isolated from a human cDNA library using the PP of Enh A of the HLA class I promoter and this clone encoded a B-galactosidase fusion protein capable of binding to the PP and belongs to a NF-xB subunit.