Quantitative Enzyme Immunoassay Research Articles

The effect of COX-2 inhibitor, nimesulide, on angiogenetic factors in primary endometrial carcinoma cell culture

Angiogenesis, the development of new blood vessels from preexisting capillaries, is essential for the development, growth and advancement of solid tumours. Angiogenesis is enhanced by prostaglandins (PGs) that are synthesised by the catalysis of cyclooxygenases (COX-1 and COX-2) from arachidonic acid. COX-2 is upregulated in a variety of malignancies and favours the growth of malignant cells by stimulating proliferation and angiogenesis. The aim of this study is to investigate the angiogenetic process by determining the levels of vascular endothelial growth factor (VEGF), monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-8 in endometrial cancer cells and to study the effect of nimesulide, a selective COX-2 inhibitor, on these mediators using cell culture. Endometrial tissue specimens were obtained from subjects with endometrial cancer and intramural leiomyoma. Cells were incubated with either 10 or 50 microM nimesulide for 24 h. VEGF, MCP-1 and IL-8 concentrations were determined by sandwich quantitative enzyme immunoassay (ELISA). VEGF concentration was significantly higher in cancer cells than normal endometrial cells. VEGF was decreased with 10 microM nimesulide in cancer cells whereas it remained unaltered in normal cells. Both MCP-1 and IL-8 concentrations were lower in cancer cells than normal cells. MCP-1 levels were decreased with both doses of nimesulide in normal cells, whereas IL-8 levels were significantly affected only by 50 microM of nimesulide. These results suggest that COX-2 inhibitors may be effective in the treatment of endometrial cancer via suppression of angiogenesis.

Serum Levels of Vascular Endothelial Growth Factor (VEGF) as a Prognostic Factor in Renal Cell Carcinoma

Purpose: Vascular endothelial growth factor (VEGF) is recognized as a potent constituent of the vascularization and growth of solid tumors. We assessed the serum levels of VEGF and we evaluated its correlation to the clinicopathologic findings and clinical outcome of patients with renal cell carcinoma (RCC). Materials and Methods: Serum samples were collected before surgery from patients with RCC. The levels of serum VEGF were assessed by using quantitative enzyme immunoassay. Comparison of the serum VEGF level with the tumor stage, grade, cell type and clinical outcome was performed, and the survival rate between the RCC patients above and below the mean VEGF level was evaluated. Results: The mean follow-up was 5.1 years. The serum VEGF level was significantly higher in the patients with RCC (mean: 497pg/ml) than that in the controls (mean: 211pg/ml) (p<0.001). The serum VEGF level was correlated with the tumor size, the pathologic T stage and the clinical stage and histologic nuclear grade, but not with the histologic subtype. Patients with a lower VEGF level (<497pg/ml) showed longer survival (p=0.0207 on univariate analysis, p=0.04 on multivariate analysis). Conclusions: Our study indicated that the serum VEGF level was significantly correlated with the clinical or pathologic stage, the nuclear grade and adverse survival. These results suggest that the serum VEGF level could be a comparable prognostic factor to the tumor stage and grade. (Korean J Urol 2007;48:1219-1223) 󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏

Circulating levels of the breast cancer growth factor GP88 in the serum of breast cancer (BC) patients

20050 Background: The 88 kDa autocrine growth factor PC-Cell Derived Growth Factor (GP88) plays a critical role in breast tumorigenesis. GP88 expression was low in estrogen receptor positive cells, whereas in ER negative cells, it was constitutively overexpressed. Increased GP88 expression was associated with anti-estrogen therapy resistance in ER+ cells and Herceptin resistance in Her-2 overexpressing breast tumors. Antisense inhibition of GP88 expression in human breast adenocarcinoma lead to inhibition of tumor growth in vivo. Immunohistochemical studies have shown that GP88 was expressed in 80% of invasive ductal carcinomas in correlation with expression of poor prognosis markers whereas normal tissues and benign breast lesions were negative. Since GP88 is secreted by breast cancer cells, we examined whether GP88 was found in the circulation at an elevated level in the sera of breast cancer patients when compared to healthy individuals. Methods: A blood sampling study was conducted to determine the serum level of GP88 in healthy volunteers (HV) and breast cancer patients (BC pts). Ten ml of blood was drawn every three months to obtain serum. GP88 serum concentration was determined in triplicate by quantitative enzyme immunoassay using human GP88 as standard. 126 BC pts were accrued. . In addition, sera from 53 healthy volunteers were obtained to establish a GP88 baseline in HV. BC pts characteristics: race: Caucasian- 61, African American-60, Asian-5; median age: 52.5 (range 26–84), stage I-32, II-34, III-18, IV-42. Results: Circulating GP88 was measurable in the serum. Median level of GP88 was 32.8 ng/ml (range 15.3–42.8) in HV and 43.8 ng/ml (range 15.4–158.4) in BC pts, (p-value = 0.0007). Conclusions: GP88 is measurable in the sera of HV and BC pts. Comparison between the two groups indicates that GP88 level is significantly higher in the sera of BC pts. These studies are important since it identifies GP88 as a measurable biomarker that is also a therapeutic target of malignant transformation or malignant progression of breast carcinoma (BC). Future studies will examine the correlation of GP88 level with BC prognostic factors. Correlation between the serum level of GP88 and therapeutic response to systemic therapy in breast cancer patients will also be assessed. [Table: see text]

VEGF-C and COX-2 expression in papillary thyroid cancer

In papillary thyroid cancer (PTC), age appears to be the most important single prognostic factor. Another characteristic feature is the lack of association between survival and lymph node metastases. Earlier, we found that expression of cyclooxygenase-2 (COX-2) is higher in older PTC patients, in agreement with the finding that older patients have a worse prognosis. Recent findings suggest that COX-2 can up-regulate vascular endothelial growth factor-C (VEGF-C) expression. Here, we investigated whether expression of VEGF-C differs between young and older PTC patients and whether expression of VEGF-C and COX-2 are correlated. Our retrospective study comprised 106 PTC patients selected by age: those under 35 or over 55 at diagnosis. Paraffin-embedded tissue samples were analysed by immunohistochemistry for VEGF-C protein expression. Furthermore, we investigated by quantitative RT-PCR and enzyme immunoassay the relationship between VEGF-C and COX-2 expression in papillary thyroid cancer cells (NPA cells). VEGF-C expression was significantly increased with age. In the tumours from older lymph node-positive (N1) patients, VEGF-C expression was significantly higher than in the tumours from young N1 patients. Moreover, all patients who died of cancer or who developed distant metastases were old, and most tumours from these patients (4 of 5) expressed VEGF-C and had had nodal metastases at the time of primary operation. Immunohistochemically, expression of COX-2 and VEGF-C correlated strongly. In cell culture, this correlation was not so clear, because the COX-2 selective inhibitor, NS-398, did not reduce VEGF-C expression. However, as both COX-2 and VEGF-C were induced by the tumour promoter phorbol 12-myristate 13-acetate (PMA), the same factors may control them both.

Switching from HPLC/UV to MEIA for whole blood sirolimus quantitation: comparison of methods

Sirolimus is a immunosuppressive agent for renal transplant recipients. Monitoring of whole blood sirolimus concentration is necessary in order to improve clinical outcomes. An increasing number of clinical laboratories (4-14% during 2005) are using microparticle enzyme immunoassay (MEIA) for sirolimus quantitation but previous reports indicated a high variability, with a mean difference of 17% for MEIA method vs. high-performance liquid chromatography/ultraviolet (HPLC/UV). This study was aimed at comparing the reliability of MEIA with the HPLC/UV method. Blood samples from transplant patients were processed using both HPLC/UV and MEIA assays. Comparison and Bland-Altman plots, as well as regression analysis and paired t-test were used to compare results of the assays. Concentrations were stratified into three groups and used to investigate whether any observed difference between methods could be influenced by sirolimus concentration. Regression analysis yielded a coefficient of correlation R of 0.9756, the line of best fit being y=0.9832x+0.1976. The statistical analysis showed no difference between the two sets of experimental data. The average percentage difference between the two methods was found to be -0.2+/-19.2%. On the basis of our present results, the tested MEIA assay is able to quantify sirolimus concentration with a clinically acceptable imprecision, similar to that of HPLC/UV method.

Therapeutic Monitoring of Tacrolimus Concentrations in Blood of Renal and Liver Transplant Recipients: Comparison of Microparticle Enzyme Immunoassay and Enzyme Multiplied Immunoassay Methods

Monitoring of tacrolimus blood concentrations is of utmost importance in the management of organ transplant recipients due to narrow therapeutic index of the drug and its considerable interpatient variability in pharmacokinetics. Thus therapeutic monitoring of tacrolimus plays a crucial role not only in the evaluation of the drug efficacy but also in the control of possible side effects. We compared immunoassay-based methods, quantitative enzyme multiplied immunoassay (EMIT) with quantitative microparticle enzyme immunoassay (MEIA), using blood samples from renal and liver transplant recipients ( n = 40) treated with tacrolimus. Blood samples were obtained for diagnostic routine measurements. The tacrolimus concentrations measured by EMIT for all the transplant patient samples were higher (2.8 to 28.5 ng/mL) than results obtained in MEIA (3.0 to 25.0 ng/mL). The mean difference expressed in percentage was 13.94% and correlation coefficient EMIT versus MEIA was 0.97. The tacrolimus concentrations measured by EMIT for renal graft recipients ( n = 8) were higher (6.0 to 13.3 ng/mL) than those measured by MEIA (6.1 to 12.2 ng/mL), mean difference expressed in percentage was 14.1% and correlation coefficient was 0.85. The tacrolimus concentrations for liver transplant recipients ( n = 32) measured by EMIT (2.8 to 28.5 ng/mL) were higher than results obtained in MEIA (3.0 to 25.0 ng/mL), the mean difference expressed in percentage was 13.89%, and the correlation coefficient was 0.98. The results obtained in the study show an insignificant difference in specificity of both methods used to determine the concentration of an active drug. Thus both methods, EMIT and MEIA, seem to have similar diagnostic value.

PC-cell derived growth factor (PCDGF/GP88): A novel circulating biomarker in breast cancer (BC) patients

551 Background: The identification of a measurable biomarker that is also a therapeutic target of malignant transformation or malignant progression of breast carcinoma (BC) is needed. PCDGF/GP88, an 88 kDa glycoprotein autocrine growth factor is expressed in human BC tumors in a positive correlation with their tumorigenicity. In estrogen receptor positive (ER+) cells, PCDGF/GP88 expression is low and is stimulated by estradiol whereas in ER negative cells, it is constitutively overexpressed. In ER+ cells, PCDGF/GP88 mediates estrogen mitogenic activity and its overexpression renders cells estrogen-independent and tamoxifen-resistant. Inhibition of PCDGF/GP88 expression by antisense transfection leads to inhibition of breast tumor incidence and growth in nude mice. PCDGF/GP88 is expressed in 80% of human invasive ductal carcinomas and correlates with expression of markers of poor prognosis whereas normal tissues and benign breast lesions are negative. Methods: A blood sampling study was conducted to determine the serum level of PCDGF/GP88 in healthy volunteers (HV) and BC pts. Approximately 10 ml of blood was drawn every three months and processed to obtain serum. PCDGF/GP88 serum concentration was determined in triplicate by quantitative enzyme immunoassay using human PCDGF/GP88 as standard. One hundred twenty three subjects were accrued: 16 HV and 107 BC pts. BC Patient characteristics: race, Caucasian- 51, African American-51, Asian-5; median age, 52.5 (range 29–84); stage I - 24, II - 29, III - 13, IV - 41. Results: Circulating PCDGF/GP88 was measurable in the serum. Mean level of PCDGF was 33.6 ng/ml (range 23.8–42.8) in HV and 47.4 ng/ml (range 19.9–158.4) in BC pts, (p- value= 0.004). Conclusions: PCDGF/GP88 is measurable in the sera of HV and BC pts. Comparison between the two groups of subjects indicates that PCDGF/GP88 level is significantly higher in the sera of BC pts. Future studies will examine the correlation of PCDGF/GP88 level with BC prognostic factors such as stage, tumor size, lymph node involvement, tumor grade and presence of ER and HER-2neu. We will also attempt to correlate the serum level of PCDGF/GP88 with median survival of BC pts. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration A&G A&G

Relationship of Helicobacter Pylori Infection with Different Digestive System Malignant Tumors

Studies have shown a close association between Helicobacter pylori infection and gastric cancer while its relationship with other digestive system tumors has not been clarified. This study evaluated whether H. pylori infection affected risk for different digestive system malignancies. Sera were tested for lgG antibodies against H. pylori using quantitative enzyme immunoassay in 222 subjects: 82 patients with digestive system malignancies including gastroduodenal, pancreatic, hepatic, and colorectal carcinomas, 82 health controls, and 58 patients with other malignancies (cancer controls). H. pylori infection was noted in 58.8% of health controls, 73.2% of patient with digestive cancer and 82.8% of cancer controls. H. pylori infection imposed 2.8 folds increased risk for colorectal carcinoma. H. pylori infection was associated with increased but non-significant risk for developing genitourinary, bone, and breast cancers. Thus large-scale epidemiological studies to confirm the association and the possible causative role of H. pylori infection in the etiopathogenesis of various digestive and non-digestive system cancers are needed to help to develop preventive measures and appropriate interventions.

Effect of human hydrosalpinx fluid on the development of mouse embryos and role of the concentration of growth factors in culture medium with and without hydrosalpinx fluid

The aim of this study was to investigate the effect of human hydrosalpinx fluid (HF) on the development and blastulation of mouse embryos and the role of the concentration of growth factors in culture medium with and without HF. In total, 2100 mouse embryos were cultured. Female mice were induced to superovulate and then mated with males. Two-cell-stage embryos were recovered from the oviduct and cultured in Ham's F-10 medium with bovine serum albumin and HF. Epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) were analyzed by quantitative enzyme immunoassay. Mean blastulation index of 1.11, 0.97 and 0.98 was found at HF concentration of 5%, 20% and 30%, respectively (p = 0.8). The mean value of EGF in the control culture medium without HF was 11.2 pg/ml, which was statistically significantly different from that in culture medium containing HF (p < 0.001). The mean value of IGF-I in the control group without HF was 1.30 pg/ml and was not statistically significantly different from that in culture medium containing HF. Development of the two-cell-stage embryos was not affected at low (< 30%) HF concentrations. In conclusion, the present study demonstrates that even apparently normal blastulation is affected by any concentration of HF because of low embryonic EGF.

Stability of Frozen Serum Levels of Insulin-like Growth Factor-I, Insulin-like Growth Factor-II, Insulin-like Growth Factor Binding Protein-3, Transforming Growth Factorβ, Soluble Fas, and Superoxide Dismutase Activity for the JACC Study

BACKGROUND: Subjects of the Japan Collaborate Cohort Study (JACC Study) gave peripheral blood samples collected between 1988 and 1990. We conducted to investigate whether levels of serum components measured after 9 years of frozen storage are stable or not.METHODS: To assess the degradation of frozen serum components in the JACC Study, we compared levels of various components (IGF-I, IGF-II, IGFBP-3, TGF-β1, sFas, and total SOD activity) between fresh and stored sera collected from other inhabitants. Serum levels of constituents were measured by immunoradiometric assay (IGF-I, IGF-II and IGFBP-3), quantitative enzyme immunoassay (TGF-β1), enzyme-linked immuno-adsorbent assay (sFas), and an improved nitrite method (SOD activity).RESULTS: The coefficients of variation for intra- and inter-assay precisions of the measurements were less than 9%. Levels of IGF-I, IGF-II, IGFBP-3, TGF-β1 and sFas in sera after storage for 9 years at -80°C were similar to those of fresh sera newly collected from inhabitants. The distributions of serum IGF-I, IGF-II, IGFBP-3, TGF-β1, sFas and SOD activity for specimens collected from different individuals tended to be similar to those of serum levels for frozen specimens collected from different individuals and stored for 9 years.CONCLUSIONS: There was no statistically significant difference in distribution of measured values of IGF-I, IGF-II, IGFBP-3, TGF-β1, and sFas between newly collected sera and frozen specimens stored for 9 years. Thus, measurements of these serum constituents of specimens stored for the JACC Study can be reliably used in nested case-control study.

Possible Relation of Crohn's Disease and Hereditary Thrombophilia: Detection of ASCA Seropositivity. (Preliminary Results).

Introduction: Hereditary thrombophilia (Factor V Leiden mutation -FV G1691A, FVL, FII G20210A -prothrombin mutation), has been reported to be correlated to Crohn's disease (CD) by some studies. The determination of anti- Saccharomyces Cerevisiae IgA and IgG Antibodies (ASCA) have been described as important serological marker for the differential diagnosis of CD. The aim of the study was to evaluate ASCA seropositivity in a high-risk population (i.e. thrombophilic patients, pts) for CD.Material-Methods: From April to July 2004, 37 patients (16 men, 21 women, mean age 52,5± 9.6 years old) with hereditary thrombophilia and 28 healthy control subjects (16 men, 12 women, mean age 51,3 ± 8.5 years) were included in the study. Fasting blood samples were drawn from patients and control subjects were examined for serum p-ANCA, ASCA IgA and IgG titers. Pts and control subjects were matched for age, sex and smoking and had to fill a questionnaire concerning history of rectal bleeding, diarrhea lasting longer than 4 weeks, inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS).Colonoscopy or flexible sigmoidoscopy was proposed to all patients and controls when reported an alarm symptom. ASCA IgA and IgG titers were measured with quantitative enzyme immunoassay method (Medizym ASCA IgG, Medizym ASCA IgA, and MEDIPAN, GERMANY).Results: In the patient group 8 pts reported IBS symptoms, 2 had history of rectal bleeding while none IBD or chronic diarrhea. In the control group 6 patients reported IBS symptoms while none IBD, rectal bleeding or chronic diarrhea (p>0,05). Ten and 15 pts were found to be positive for ASCA IgG and IgA respectively. In the control group, 6 and 10 subjects were found to be positive for ASCA IgG and IgA respectively (p<0,05 and p<0,1 respectively).Conclusions: Thrombophilic pts, a high-risk population for CD, present an important rate of ASCA IgG and IgA seropositivity. Although these are preliminary results, thrombophilic pts should be screened for ASCA and some of them with colonoscopy. ASCA seropositivity in the control group was found to be greater than in the literature but this may be explained by the small sample.

Evaluation of recombinant antigen-based assays for diagnosis of bullous autoimmune diseases.

The diagnosis of autoimmune bullous diseases is based on clinical observation and on the presence of autoantibodies directed to molecules involved in the adhesion systems of the skin. Immunofluorescence assays are the currently accepted method for detection of autoantibodies; such assays depend greatly on the skill of operators and are difficult to standardize. Recombinant desmoglein-1 (Dsg1), Dsg3, and BP180 peptides, the main autoantigens in pemphigus or bullous pemphigoid, have been used to develop new quantitative enzyme immunoassays (EIA) for the detection of specific antibodies. The present study was undertaken to evaluate the sensitivity and specificity of these immunoassays and to determine the correlation between the results and the clinical aspects of diseases. Serum samples from patients with pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, or mucous membrane pemphigoid, from healthy individuals, and from patients with unrelated autoimmune conditions were tested. Anti-desmoglein reactivity was detected in all the patients with pemphigus and in none of the controls. Patients with the more benign form of cutaneous disease had anti-Dsg1 antibodies, while patients with deeper cutaneous lesions or with mucosal involvement had anti-Dsg3 reactivity also, or exclusively. The BP180-based assay was positive for 66.6% of patients with bullous pemphigoid and for none of the patients with mucous membrane pemphigoid, and no reactivity was detected in the control sera. In conclusion, the anti-Dsg1 and anti-Dsg3 assays are useful in the diagnosis of pemphigus and provide information on the clinical phenotype of the disease. However, the sensitivity of EIA for detection of autoantibodies in bullous pemphigoid should be improved by the use of additional antigens or epitopes.

Use of quantitative and objective enzyme immunoassays to investigate the possible association between Chlamydia pneumoniae and Mycoplasma pneumoniae antibodies and asthma

Sera from 150 consecutive patients with established asthma and 150 matched controls were examined for Chlamydia pneumoniae IgG and IgA with commercially available enzyme immunoassays (EIAs) detecting immune response solely to surface proteins of elementary bodies. The assays were also modified to measure combined immune response to surface proteins and family-specific lipopolysaccharide antigen. Mycoplasma pneumoniae IgG and IgA were measured with new commercial EIAs utilising P1-enriched protein fraction as an antigen. No statistically significant differences between the patient groups in terms of prevalence or levels of antibodies to either organism were found with these methods.

Monitoring indexes for early renal injury in the workers exposed to mercury

To study the diagnostic method for early renal injury in the workers exposed to mercury (Hg). The contents of urinary Hg were determined by chemical method. Urinary microalbumin (mALB), beta(2)-microglodulin (beta(2)-MG) and retinol binding protein (RBP) levels were measured with total quantitative enzyme immunoassay. The activities of urinary N-acetyl-beta-D-glucosaminidase (NAG) and gamma-glutamyl transferase (gamma-GT) were determined by rate methods. Urinary creatinine (Cr) was measured by using picric acid method. The levels of urinary BRP, beta(2)-MG, NAG and gamma-GT in exposed workers [(439.7 +/- 201.4), (141.4 +/- 56.3) micro g/g Cr and (12.3 +/- 5.7), (60.3 +/- 18.5) U/g Cr respectively] were significantly higher than those in controls (P < 0.05, P < 0.01). The levels were increased gradually with the increasing contents of urinary Hg. The positive detection rate for single or two combined indexes was rather lower whereas that for 4 combined indexes was as high as 85.5%. A positive correlation was noted between the contents of urinary Hg and urinary BRP, beta(2)-MG, NAG and gamma-GT (r: 0.466, 0.379, 0.323, 0.311, P < 0.05). Urinary RBP was correlated to urinary beta(2)-MG, NAG and gamma-GT (r: 0.362, 0.354, 0.332, P < 0.05). Combined detection of urinary RBP, beta(2)-MG, NAG and gamma-GT is a sensitive method for the diagnosis of early renal injury in the workers exposed to Hg.

Development of a rapid enzyme immunoassay for the detection of retinol-binding protein

Retinol-binding protein (RBP) was chosen as a surrogate marker for retinol because of the close correspondence between retinol and RBP. To meet the need for rapid, cost-effective determination of vitamin A status in populations, a quantitative enzyme immunoassay (EIA) for detection of RBP was developed. The resulting RBP EIA, a competitive assay, uses RBP adsorbed to microtest strip wells to compete with RBP in serum. The assay takes approximately 40 min. With a reference panel of sera, test accuracy was found to be within 4% of expected values through the calibrated range of 0.48-1.92 micro mol RBP/L (10-40 micro g RBP/mL). Intraassay and interassay variability averaged 6.7% and 8.9%, respectively. Specificity testing showed no interference from other serum proteins, prealbumin, rheumatoid factor, bilirubin, estrogen, or C-reactive protein. The RBP EIA provided linear results between 0.43 and 1.80 micro mol RBP/L (9 and 38 micro g RBP/mL). Preliminary laboratory evaluations indicated that the RBP EIA correlates well with radial immunodiffusion for RBP and with HPLC for retinol, the current reference standard. A field evaluation in a population at risk for vitamin A deficiency (VAD) resulted in close correlation between RBP EIA measures and retinol measures by HPLC (R(2) = 0.82). The RBP EIA is as reliable in estimating VAD as is HPLC retinol. After successful validations, the test should enable public health authorities to rapidly monitor VAD and track vitamin A status in populations.

Presence of glomerular basement membrane (GBM) antibodies in HIV- patients with Pneumocystis carinii pneumonia.

Following the unexpected finding of antibodies to GBM in a patient with Pneumocystis carinii pneumonia in the absence of kidney abnormalities, the presence of anti-GBM antibodies was analysed in 14 patients with pulmonary P. carinii infection who did not have clinical evidence of autoimmune glomerulonephritis. Patients were divided into three groups: HIV- with P. carinii pneumonia (n = 4), HIV+ with P. carinii pneumonia (n = 5) and HIV- carriers of P. carinii without pneumonia (n = 5). As control groups, HIV- patients with community-acquired non-P. carinii pneumonia (n = 6) and healthy individuals (n = 16) were included. Anti-GBM antibodies, studied with a quantitative enzyme immunoassay (EIA) for anti-alpha3 chain of collagen IV antibodies, were detected in three out of the four HIV-patients with P. carinii pneumonia, but not in any individuals of the other categories. These results suggest that P. carinii alveolar injury or the host response to the organism could affect the basal membrane Goodpasture antigen or a similar antigen, and induces anti-GBM antibody production in HIV- patients, and support the hypothesis that, at least in some cases, Goodpasture's syndrome could be triggered by an alveolar lesion induced by a P. carinii pneumonia.

8-isoprostane increases expression of interleukin-8 in human macrophages through activation of mitogen-activated protein kinases.

8-isoprostane is a marker of oxidative stress in vivo and increased plasma and urine levels are found in patients with vascular disease and in atherosclerotic plaques. Inflammatory chemokines such as interleukin (IL)-8 seem to play an important pathogenic role in atherogenesis. We therefore investigated the effects of 8-isoprostane on the expression of inflammatory chemokines with consciousness on IL-8 (mRNA and protein) in human macrophages. In addition, we studied the involvement of mitogen-activated protein kinases (ERK 1/2 and p38 MAPK) and nuclear factor-kappaB (NF-kappaB) in this process. 8-isoprostane (10 microM) induced IL-8 expression (mRNA and protein), measured by real-time quantitative RT-PCR and enzyme immunoassay, respectively, in both THP-1 macrophages and human monocyte-derived macrophages. Moreover, 8-isoprostane increased mRNA expression of macrophage inflammatory protein-1alpha as determined by RNase protection assay. In this process, 8-isoprostane induced the activation of two major MAP-kinases; ERK 1/2 and p38 MAPK. Furthermore, the ERK 1/2 inhibitor, PD98059, and the p38 MAPK inhibitor, SB203580, markedly reduced 8-isoprostane-induced IL-8 expression (mRNA and protein), while inhibition of NF-kappaB activation and translocation had no significant effect on IL-8 expression. We show that 8-isoprostane increases IL-8 expression in human macrophages involving both ERK 1/2 and p38 MAPK, but not NF-kappaB signaling pathway. These findings further support a link between oxidative stress/lipid peroxidation and inflammation in human macrophages and suggest a role for 8-isoprostane in this process. This 8-isoprostane-induced chemokine expression might be involved in the pathogenesis of atherosclerosis as well as other inflammatory disorders.

Serum concentrations of tumour necrosis factor-alpha (TNF-alpha) and soluble TNF-alpha receptor p55 in patients with hypothyroidism and hyperthyroidism before and after normalization of thyroid function.

Tumour necrosis factor-alpha (TNF-alpha) is a cytokine with numerous immunological and metabolic activities. Receptors for TNF-alpha have been demonstrated in thyroid follicular cells and TNF-alpha and its receptors have been implicated in the cytotoxic mechanisms that characterize the thyroid destruction in autoimmune thyroid disease. In patients with Graves' disease, serum levels of TNF-alpha have been reported to be elevated and administration of TNF-alpha to humans has been shown to induce hormonal alterations resembling those seen in the nonthyroidal illness syndrome. To evaluate serum concentrations of TNF-alpha and the soluble receptor for TNF-alpha (sTNFR-I) in a group of patients with thyroid dysfunction before and after normalization of thyroid function with appropriate therapy. We studied 20 patients with hypothyroidism (18 women and 2 men, mean age +/- SD, 48.8 +/- 16.1 years) and 20 patients with hyperthyroidism (14 women and 6 men, age 44.6 +/- 15.9 years). Patients were assessed at the time of diagnosis and again after normalization of thyroid function tests with appropriate therapy. A group of 20 healthy subjects (15 women and 5 men, age 44.9 +/- 15.1 years) were also studied as a control group. All subjects were ambulatory and were studied as outpatients during visits to the endocrinology clinic. Serum concentrations of free T4 (FT4), total T3, TSH, TNF-alpha and sTNFR-I were measured in all subjects. TNF-alpha and sTNFR-I were measured using a quantitative enzyme immunoassay. In patients with hypothyroidism serum concentrations of TNF-alpha (3.17 +/- 1.18 pg/ml) and sTNFR-I (1273 +/- 364 pg/ml) were significantly higher than those found in controls (2.42 +/- 0.76 pg/ml, P < 0.05, and 971 +/- 235 pg/ml, P < 0.01, respectively). Normalization of thyroid function with l-thyroxine therapy did not significantly modify TNF-alpha or sTNFR-I levels. There were no differences in pre- and post-therapy values of TNF-alpha and sTNFR-I in patients with autoimmune (n = 14) or nonautoimmune (n = 6) hypothyroidism. Before therapy, patients with hyperthyroidism showed elevated serum concentrations of TNF-alpha (3.36 +/- 1.21 pg/ml; P < 0.01) and sTNFR-I (2274 +/- 579 pg/ml; P < 0.001) in relation to the control group. Treatment of hyperthyroidism was accompanied by a normalization of TNF-alpha levels (2.46 +/- 0.89 pg/ml; P < 0.001) and by a significant decrease in sTNFR-I concentrations (1369 +/- 475 pg/ml; P < 0.001). Post-therapy levels of TNF-alpha and sTNFR-I showed a significant correlation with loss of weight (r = 0.674, P < 0.01, and r = 0.629, P < 0.01, respectively) in hypothyroid patients. No correlation between these parameters was found in the group of patients with hyperthyroidism. In summary, these results confirm the relevance of activation of the TNF-alpha system in patients with thyroid dysfunction, as high plasma concentrations of TNF-alpha and sTNFR-I have been demonstrated in patients with hypothyroidism or hyperthyroidism. Treatment of hyperthyroidism is accompanied by a significant reduction in the previously elevated concentrations of both TNF-alpha and sTNFR-I. However, these changes are not seen when normalizing thyroid function in patients with hypothyroidism.

Further investigation of the modifying effect of various chemopreventive agents on apoptosis and cell proliferation in human colon cancer cells.

Recent preclinical assays using animal models have shown that naturally-occurring and synthetic chemicals such as auraptene (AUR), nobiletin (NOB), hesperidin (HE), diosmin (DIO), indole-3-carbinol (I3C), 1'-acetoxychavicol acetate (ACA), 2,5-di-O-acetyl-D-1,4-glucaro-6,3-dilactone (ACE), D-glucuronic acid gamma-lactone (GL), chlorogenic acid (CGA), protocatechuic acid (PA), and sinigrin (SIN) are possible preventive agents against the development of cancer. However, the mode of action of such preventive agents remains to be elucidated. The current study, therefore, was conducted to analyze whether these agents induce apoptosis and/or inhibit DNA synthesis in human colorectal cancer cell lines. We performed an 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay to evaluate the modifying effects of the chemicals on cell viability as the first screening. Then, induction of apoptosis was detected by means of a DNA fragmentation assay, a quantitative enzyme immunoassay, and morphological analysis using 4-diamidino-2-phenylindole staining. In addition, the modulating effects of the compounds on DNA synthesis of the cells with fixed doses of the compounds were analyzed by scoring the 5-bromo-2'-deoxyuridine labeling index. AUR, NOB, I3C, ACA, and ACE had apoptosis-inducing effects in a concentration- and time-dependent manner, some of which were followed by a reduction in replicating DNA synthesis. CGA, PA, SIN, GL, DIO, and HE had little modulating effect on cell viability, apoptosis, and DNA synthesis in this cell system. Our results suggest that AUR, I3C, ACA, NOB, and ACE might exert tumor-preventive action through apoptosis- and/or cell proliferation-dependent mechanisms and, on the other hand, CGA, PA, SIN, HE, DIO, and GL might be apoptosis- and cell proliferation-independent. These assays provided an initial tool for further mechanical studies of tumor-preventive agents and future applications to mechanism-based chemopreventive studies.

IDENTIFICATION OF ANTIGENIC AND ALLERGENIC NATURAL RUBBER LATEX PROTEINS BY IMMUNOBLOTTING1*

ABSTRACT Quantitation of proteins in finished natural rubber latex (NRL) products is essential in predicting their allergenic potential. The ASTM standard Modified Lowry method for measuring total protein content has been used for several years. Most recently, ASTM published a standard for more sensitive and more specific enzyme immunoassay for quantitation of antigenic NRL proteins. It is an ELISA inhibition assay, using rabbit anti NRL sera. Since the measurement of proteins in this method depends on recognition capacity of rabbit antibodies, the selection of an appropriate protein source for rabbit immunization is crucial for the accuracy of such test. In this study, we evaluated the composition of NRL proteins from ammoniated (AL) and nonammoniated (NAL) raw latex and from finished NRL products, and compared the effectiveness of sera from rabbits immunized with NRL proteins, to react with those extracts. Immune rabbit sera were analyzed by immunoblotting against extracts of several samples of AL, NAL, and glove proteins. In the NAL extracts, we identified 26–28 protein bands by SDS-PAGE. AL samples had between 6 and 9 bands with a great variation in the band positions among the samples. The Western blot analysis showed that anti-AL rabbit serum reacted with 4–9 protein bands in various AL extracts. The highest intensity of reaction was observed with the extract used to immunize the rabbits. Similar reaction was observed with anti-NAL serum. However, when the antisera were blotted against NAL extracts, anti-NAL serum reacted more strongly and with a larger number of proteins than anti-AL serum. In summary, anti-NAL serum recognized an equal number of proteins in AL extract as anti-AL serum. However, anti-AL serum recognized fewer protein molecules in NAL extract than anti-NAL serum. Our findings suggest that NAL extract contains more individual proteins than other extracts, and sera from rabbits immunized with this antigen have a greater capacity to react with a wide spectrum of NRL proteins. This finding may be helpful in selecting the representative reference antigen and antiserum for further efforts in NRL protein quantitation. *The submitted manuscript has been authored by a contractor of the U.S. Government. Accordingly, the U.S. Government retains a non-exclusive, royalty-free license to publish or reproduce the published form of this contribution, or allow others to do so, for Government purposes.