Evaluation of thyroglobulin expression in murine reproductive organs during pregnancy

Citation Moravej A, Jeddi‐Tehrani M, Salek‐Moghaddam AR, Dokouhaki P, Ghods R, Rabbani H, Kazemi‐Sefat GE, Shahbazi M, Zarnani AH. Evaluation of thyroglobulin expression in murine reproductive organs during pregnancy. Am J Reprod Immunol 2010; 64: 97–103 Problem In pregnant women with antithyroglobulin antibody, prevalence of abortion is 2–4 fold higher compared to normal controls. Direct effect of such harmful autoantibodies on female reproductive organs may serve a role in pregnancy loss. Method of study Expression of thyroglobulin in decidua, placenta, and ovary of pregnant Balb/c mice ((Balb/c×Balb/c and Balb/c×C57BL/6) during early, middle, and late stages of pregnancy was evaluated. Expression of thyroglobulin was investigated in these tissues by semi‐quantitative RT‐PCR. In addition, polyclonal antithyroglobulin antibody was produced, and expression of thyroglobulin protein in aforesaid tissues was evaluated by immunohistochemistry and dot‐blot analysis. Results The results showed that thyroglobulin message is not expressed in placenta, decidua, or ovary in any stages of pregnancy. The same results were obtained at the protein level. Conclusion It is likely that antithyroglobulin antibodies have no direct detrimental effect on such organs in patients with thyroid autoimmunity suffering from recurrent abortion.     

Prevalence, genetics and clinical presentation of chronic granulomatous disease in Sweden

To estimate the prevalence of chronic granulomatous disease (CGD) in Sweden, an inquiry asking for known and possible CGD cases was mailed to paediatric, internal medicine and infectious disease departments all over Sweden. The detected patients were characterized as to genetics and the clinical presentation. Twenty–one patients (belonging to 16 different families) were found, corresponding to a prevalence of ∼ 1/450 000 individuals. The patients with X–linked disease, lacking a functional gp91^phox protein ( n = 12), comprised 57% and 43% of the patients had an autosomal recessive (AR) disease lacking p47^phox ( n = 7) or p67^phox ( n =1), respectively. All unrelated patients with X–linked disease displayed different gene abnormalities such as point mutations predicting nonsense ( n = 3), missense ( n = 1) or splice site mutations ( n = 2), but also a total deletion and a unique 40 base pair duplicature insertion. The patients with p47^phox–deficiency showed a GT deletion at a GTGT tandem repeat, and the p67^phox–deficient patient displayed a heterozygous in–frame deletion of AAG combined with a large deletion in the other allele. Three patients died during the study period, two from Pseudomonas cepacia infections. Patients with X–linked disease had more frequent infections (mean of 1.7 per year), than the patients with AR inheritance (0.5 infections per year). The most common infections were dermal abscesses ( n =111), followed by lymphadenitis ( n =82) and pneumonias ( n =73). Inflammatory bowel disease–like symptoms, mimicking Crohn's disease of the colon, was seen in three CGD patients.     

Urokinase receptor antibody can reduce tumor volume and detect the presence of occult tumor metastases in vivo

The serine protease urinary plasminogen activator or urokinase (uPA), produced in abundance by many malignancies, plays a key role in tumor cell invasion and metastasis. uPA is localized within the malignant cell milieu via its cell surface receptor [uPA receptor (uPAR)], which is expressed by tumor and tumor-associated cells. In the present study, we have used a syngeneic model of rat breast cancer to directly evaluate the role of uPAR as a diagnostic and therapeutic target in metastatic breast cancer. A polyclonal antibody against the ligand-binding NH(2)-terminal domain of rat uPAR (ruPAR) was developed. This antibody recognizes ruPAR by both immunofluorescence and Western blot analysis. Recombinant ruPAR and ruPAR IgG displaced the binding of (125)I-labeled ruPAR IgG to rat prostate cancer cells (Dunning R3227 Mat Ly Lu) and breast cancer cells (Mat B-III) overexpressing ruPAR (Mat B-III-uPAR). ruPAR IgG also blocked the invasive capacity of these tumor cells in a dose-dependent manner. Mat B-III-uPAR cells were inoculated s.c. into the mammary fat pad of syngeneic female Fischer rats. On day 10 after tumor cell inoculation, animals were injected with (125)I-labeled preimmune or ruPAR IgG and then sacrificed at timed intervals. Maximum (125)I uptake was observed in primary tumors and in tissues commonly affected by tumor metastases (liver, spleen, lungs, and lymph nodes) at 12 h. Injection of (125)I-labeled preimmune or ruPAR IgG into normal non-tumor-bearing animals resulted in minimal basal levels of uPAR expression and established the specificity of the ruPAR IgG. Similar results were obtained by Northern blot and PCR analysis of mRNA isolated from tissues of normal and tumor-bearing animals. To evaluate the effectiveness of this antibody in tumor progression, ruPAR IgG (50-100 microg/day) was injected s.c. for 7 days (day 1-7) at the site of tumor cell inoculation (mammary fat pad), and animals were sacrificed at various time points for evaluation of tumor growth and metastases. Animals receiving ruPAR IgG showed a marked decrease in tumor growth and metastases as compared with control tumor-bearing animals receiving the same dose of preimmune rabbit IgG. Histological analysis of experimental primary tumors showed marked tumor necrosis that was due to increased tumor cell apoptosis as determined by terminal deoxynucleotidyl transferase-mediated nick end labeling assay. Together, these studies demonstrate the ability of anti-uPAR antibody to decrease tumor volume and detect the presence of microscopic occult tumor metastases in malignancies where uPA/uPAR play a key role in tumor progression.     

Impact of alterations affecting the p53 pathway in bladder cancer on clinical outcome, assessed by conventional and array-based methods.

This study was designed to define the potential clinical relevance of identifying alterations affecting p53 pathway in bladder cancer and to test a new, low-cost, high-throughput, and array-based TP53 sequencing technology. Tumor samples from 140 evaluable patients with bladder cancer were analyzed with two methods to detect TP53 gene mutations, including single-stranded conformational polymorphism followed by direct sequencing and an oligonucleotide array-based sequencing method. Immunohistochemistry was used to assess patterns of expression of p53, p21/WAF1, and mdm2. Median follow-up time was 27.6 months. Results from the above analyses were correlated with clinicopathological parameters and outcome. Combining the mutation-detection assays, 79 cases (56.4%) were found to harbor TP53 gene mutations. Direct sequencing identified 66 point mutations and five frameshift mutations. The p53 oligonucleotide array detected 65 point mutations and four splice site mutations in different exons but missed all five frameshift mutations. p53 nuclear overexpression was observed in 71 cases (50.7%), lack of p21 nuclear expression was found in 81 cases (57.9%), and mdm2 nuclear overexpression was seen in 64 cases (45.7%). In multivariate analysis, 17 patients (12.1%) had an altered p53 pathway, defined by the detection of mutant TP53 and/or p53 nuclear overexpression, loss of p21 nuclear expression, and mdm2 nuclear overexpression, and exhibited the worst clinical outcome in the observation period (P = 0.015), and it appears to be a significant prognostic factor associated with patient survival.     

Phase I trial of intravesical gemcitabine in bacillus Calmette-Guérin-refractory transitional-cell carcinoma of the bladder.

PURPOSE: The aim of this phase I study was to determine the safety and toxicity profile of gemcitabine administered as an intravesical agent in patients with transitional-cell carcinoma (TCC) of the bladder. PATIENTS AND METHODS: Patients with superficial bladder cancer refractory to intravesical bacillus Calmette-Guérin (BCG) therapy and refusing a cystectomy were considered eligible for the trial. Gemcitabine was given in the bladder for 1 hour twice weekly in 100 mL sodium chloride for a total of six treatments. After a 1-week break, a second course of six treatments over 3 weeks was given, followed by response assessment. Four dose levels were explored: 500 mg, 1,000 mg, 1,500 mg, and 2,000 mg. RESULTS: Eighteen patients completed therapy: three at 500 mg, six at 1,000 mg, three at 1,500 mg, and six at 2,000 mg. No grade 3 or 4 toxicity was observed at 500 mg. At 1,000 mg, three patients developed hematuria and one had a skin reaction resembling grade 3 hand-foot syndrome. Three patients at 1,500 mg had no grade 3 or 4 toxicity. Of six patients at 2,000 mg, one had grade 3 thrombocytopenia and neutropenia without infection. Seven patients had a complete response (negative cytology and posttreatment biopsy), and four patients had a mixed response (negative bladder biopsy but positive cytology). CONCLUSION: Gemcitabine has substantial activity as an intravesical agent in BCG-refractory TCC and warrants further investigation. Therapy given twice weekly was associated with minimal bladder irritation and tolerable myelosuppression. The recommended phase II dose for twice-weekly therapy is 2,000 mg.     

Lead nitrate induced apoptosis in alveolar macrophages from rat lung

In this work we have attempted to characterize the programmed cell death (apoptosis) in alveolar macrophages exposed to various concentrations of lead nitrate. It was found that after 3 h of exposure a significant increase in superoxide anion production was observed, i.e. the number of trypan blue - exculding cells, was unchanged (< or = 95%) with any dose of lead employed. Agarose gel electrophoresis and diphenylamin reaction analysis revealed the occurrence of internucleosomal DNA fragmentation evaluated using cytological analysis by fluorescence dyes, suggesting that lead nitrate at low concentrations and short periods of exposure leads macrophages into apoptosis. However, time course studies showed that beyond 3 h, toxicity occurs, which could be attenuated by phosphodiesterase inhibitors, such as caffeine, suggesting a possible mechanism involving cAMP.     

Anti-human leukocyte antigen antibodies are associated with restenosis after percutaneous coronary intervention for cardiac allograft vasculopathy

BACKGROUND: Percutaneous coronary intervention (PCI) to palliate cardiac allograft vasculopathy (CAV) has been associated with high restenosis rates, possibly related to increased inflammation associated with this disease. Whether markers of immunologic rejection are associated with restenosis in this population is unknown. The goal of the study was to determine the predictors of restenosis after PCI for CAV. METHODS: Records were reviewed retrospectively from a single, high-volume cardiac transplant center. Clinical, angiographic, and immunologic data were collected on all patients postorthotopic heart transplantation (OHT) that had subsequent PCI. Restenosis was defined as greater than 50% stenosis at the previous intervention site. RESULTS: PCI was successfully performed on 62 de novo lesions in 40 patients an average of 6.8+/-3.9 years after OHT. Angiographic follow-up data was available for 79%, with an average follow-up of 1.54+/-1.22 years. The 1-year restenosis rate was 49% (64% for balloon percutaneous transluminal coronary angioplasty and 33% for coronary stenting [P=0.09 for difference]). The frequency of immunoglobulin (Ig)G antibody to major histocompatibility complex (MHC) class I antigen was highly associated with risk of restenosis (hazard ratio [HR] 11.33, P=0.01). Greater stenosis severity and smaller target vessel diameter were also predictors of restenosis as in the nontransplant population. CONCLUSIONS: The findings suggest that in patients postPCI for CAV, humoral allo-immunity may contribute to restenosis and that IgG antibodies to MHC class I antigen may help predict the risk of restenosis after PCI in this population.     

Evidence for C-Reactive Protein's Role in (CRP) Vascular Disease: Atherothrombosis,Immuno-Regulation and CRP

For clinicians plasma C-reactive protein (CRP) levels are the only widely available tests that provide a tangible link between inflammation and atherosclerosis. New AHA/CDC joint guidelines from 2002-03 now include the measurement of CRP as a class IIa recommendation for stratifying patients with known cardiovascular disease (CVD) at a moderate (10-20%) 10-year event risk and a class IIb recommendation for patients without known CVD [1]. While the association of CRP and atherosclerosis is by now accepted, the molecular biology behind the association is evolving rapidly into a fascinating story. While some of the story remains obscure, this review aims to bridge the clinical and basic science and identify what is known about the role of this ancient molecule in atherosclerosis. The review covers CRP's interaction with atherosclerosis' major ingredients and cell types including the endothelium, monocytes and neutrophils, lipoproteins and the complement system. Taken together, the clinical and basic science leave the tantalizing impression that CRP has a fundamental role in atherogenesis, and hint at a more complex immunomodulatory effect which transforms the acute inflammatory response to vascular injury into the chronic inflammation seen in atherosclerosis.