Improved Bioprocess with CHO-hTSH Cells on Higher Microcarrier Concentration Provides Higher Overall Biomass and Productivity for rhTSH

Since the recombinant thyroid-stimulating hormone (rhTSH) is secreted by stably transfected Chinese hamster ovary (CHO-hTSH) cells, a bioprocess consisting of immobilizing the cells on a substrate allowing their multiplication is very suitable for rhTSH recovering from supernatants at relative high degree of purity. In addition, such a system has also the advantage of easily allowing delicate manipulations of culture medium replacement. In the present study, we show the development of a laboratory scale bioprocess protocol of CHO-hTSH cell cultures on cytodex microcarriers (MCs) in a 1 L bioreactor, for the preparation of rhTSH batches in view of structure/function studies. CHO-hTSH cells were cultivated on a fetal bovine serum supplemented medium during cell growth phase. For rhTSH synthesis phase, 75% of supernatant was replaced by animal protein-free medium every 24 h. Cell cultures were monitored for agitation (rpm), temperature (A degrees C), dissolved oxygen (% DO), pH, cell concentration, MCs coverage, glucose consumption, lactate production, and rhTSH expression. The results indicate that the amount of MCs in the culture and the cell concentration at the beginning of rhTSH synthesis phase were crucial parameters for improving the final rhTSH production. By cultivating the CHO-hTSH cells with an initial cell seeding of four cells/MC on 4 g/L of MCs with a repeated fed batch mode of operation at 40 rpm, 37 A degrees C, 20% DO, and pH 7.2 and starting the rhTSH synthesis phase with 3 x 10(6) cells/mL, we were able to supply the cultures with enough glucose, to maintain low levels of lactate, and to provide high percent (similar to 80%) of fully covered MCs for a long period (5 days) and attain a high cell concentration (similar to 9 x 10(5) cells/mL). The novelty of the present study is represented by the establishment of cell culture conditions allowing us to produce similar to 1.6 mg/L of rhTSH in an already suitable degree of purity. Batches of produced rhTSH were purified and showed biological activity.

Correlation Between Autofluorescence Intensity and Tumor Area in Mice Bearing Renal Cell Carcinoma

Protoporphyrin IX (PpIX) is a porphyrin derivative that is accumulated in cancerous tissue in consequence of the tumor-specific metabolic alterations. The aim of this study was to evaluate the accumulation of PpIX in mice bearing renal cell carcinoma by spectroscopy analysis. A total of 24 male Balb/c mice, 6 weeks old, were divided into six groups: Normal (without inoculation of tumor cells) and 4, 8, 13, 16, and 20 days after inoculation of tumor cells. The orthotopic tumor model of renal cancer was used. Murine renal cell carcinoma (Renca cells) were inoculated into the subcapsular space of the kidney. Normal and tumor-bearing kidneys in different progression stages were removed and analyzed by ex-vivo spectroscopy and by microscopy, for tumor histometric analysis. Emission spectra were obtained by exciting the samples at 405 nm. Significant differences between normal and tumor-bearing kidneys in autofluorescence shape occurred in the 600-700 nm spectral region. A good correlation was found between emission band intensity at 635 nm and the tumor area.

Primary Antiphospholipid Syndrome and Thyroid Involvement

Background: Data on thyroid involvement in primary antiphospholipid syndrome are scarce and inconclusive. Objectives: The aim of this study was to evaluate the frequency of thyroid dysfunction and antibodies in patients with primary antiphospholipid syndrome ( PAPS) and the association of these alterations with clinical and immunologic features. Methods: The study group included 50 PAPS patients (44 females) with a mean age of 39.7 +/- 11.5 years and mean disease duration of 77.3 +/- 63.5 months. Clinical data related to thyroid dysfunction and PAPS were obtained by chart review, patient interview, and clinical examination. Serum levels of TSH, free T4, antithyroglobulin antibody (TgAb), antithyroperoxidase antibody (TPOAb), thyroid receptor antibody (TRAb), and antiphospholipid autoantibodies were analyzed by standard techniques. Results: We found no hyperthyroidism among patients and found 22% (11 patients) with hypothyroidism in this sample. There were no differences between the latter patients and the euthyroid group about demographic findings, disease duration, thrombotic or obstetric events, and frequency of antiphospholipid antibodies as well as prevalence of thyroid auto antibodies. The prevalence of thyroid autoantibodies found was 6 patients (12%) with TgAb, 5 with TPOAb (10%), and 2 patients (4%) with both TgAb and TPOAb, comprising 18% of positivity of at least one of the auto antibodies. Conclusion: Hypothyroidism is present among 22% of PAPS patients and thyroid autoantibodies in 18% of them. These findings suggest a common pathophysiologic mechanism between antiphospholipid syndrome and autoimmune thyroid diseases.

Excessive Iodine Intake and Ultrasonographic Thyroid Abnormalities in Schoolchildren

High nutritional levels of iodine may induce a higher prevalence of autoimmune thyroiditis,hypothyroidism, goiter, as well as hyperthyroidism, mostly in the elderly. This study assessed thyroid volume and ultrasonographic abnormalities as well as urinary iodine excretion (UIE) in 964 schoolchildren living in an iodine-sufficient area in southern Brazil. Thyroid volume correlated with age and body surface area in boys and girls. In 76.8% of the children, UIE was above 300 mu g/l, with higher levels among boys compared to girls (484.2 mu g/l vs 435.3 mu g/l, p <0.001). Thyroid abnormalities detected by ultrasonography included hemiagenesis (0.5%), nodules (0.2%), cysts (0.7%), and hypoechogenicity (11.7%). Goiter was present in 1.9% of the children. Hypoechogenicity, a relevant marker of autoimmune thyroiditis, was the most common abnormality found in our study, and this may be linked to excessive iodine intake.

Phase I trial of DNA-hsp65 immunotherapy for advanced squamous cell carcinoma of the head and neck

Considering that mycobacterial heat-shock protein 65 (hsp65) gene transfer can elicit a profound antitumoral effect, this study aimed to establish the safety, maximum-tolerated dose (MTD) and preliminary efficacy of DNA-hsp65 immunotherapy in patients with advanced head and neck squamous cell carcinoma (HNSCC). For this purpose, 21 patients with unresectable and recurrent HNSCC were studied. Each patient received three ultrasound-guided injections at 21-day intervals of: 150, 600 or 400 mu g of DNA-hsp65. Toxicity was graded according to CTCAE directions. Tumor volume was measured before and after treatment using computed tomography scan. The evaluation included tumor mass variation, delayed-type hypersensitivity response and spontaneous peripheral blood mononuclear cell proliferation before and after treatment. The MTD was 400 mg per dose. DNA-hsp65 immunotherapy was well tolerated with moderate pain, edema and infections as the most frequent adverse effects. None of the patients showed clinical or laboratory alterations compatible with autoimmune reactions. Partial response was observed in 4 out of 14 patients who completed treatment, 2 of which are still alive more than 3 years after the completion of the trial. Therefore, DNA-hsp65 immunotherapy is a feasible and safe approach at the dose of 400 mg per injection in patients with HNSCC refractory to standard treatment. Further studies in a larger number of patients are needed to confirm the efficacy of this novel strategy.

Immunohistochemical analysis of p53, APE1, hMSH2 and ERCC1 proteins in actinic cheilitis and lip squamous cell carcinoma

Aims: This study has compared the tissue expression of the p53 tumour suppressor protein and DNA repair proteins APE1, hMSH2 and ERCC1 in normal, dysplastic and malignant lip epithelium. Methods and results: Morphological analysis and immunohistochemistry were performed on archived specimens of normal lip mucosa (n = 15), actinic cheilitis (AC) (n = 30), and lip squamous cell carcinoma (LSCC) (n = 27). AC samples were classified morphologically according to the severity of epithelial dysplasia and risk of malignant transformation. LSCC samples were morphologically staged according to WHO and invasive front grading (IFG) criteria. Differences between groups and morphological stages were determined by bivariate statistical analysis. Progressive increases in the percentage of epithelial cells expressing p53 and APE1 were associated with increases in morphological malignancy from normal lip mucosa to LSCC. There was also a significant reduction in epithelial cells expressing hMSH2 and ERCC1 proteins in the AC and LSCC groups. A higher percentage of malignant cells expressing APE1 was found in samples with an aggressive morphological IFG grade. Conclusions: Our data showed that epithelial cells from premalignant to malignant lip disease exhibited changes in the expression of p53, APE1, hMSH2 and ERCC1 proteins; these molecular change might contribute to lip carcinogenesis.

Loss of heterozygosity of the APC gene in oral squamous cell carcinoma

The aim of this study was to investigate loss of heterozygosity (LOH) of the APC tumor suppressor gene loci, using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) in 40 cases of oral squamous cell carcinoma (OSCC). Observed informativity was 72.5% for APC exon 11 and 82.5% for APC exon 15. LOH at APC exon 11 was observed in 2 (6.9%) of 29 informative cases, and no LOH was observed for APC exon 15. Our results suggest that inactivation of the APC gene plays a minor role in the carcinogenesis of OSCC. (C) 2008 Elsevier GmbH. All rights reserved.

Relationship between the appearance of tongue carcinoma on intraoral ultrasonography and neck metastasis

To evaluate the usefulness of intraoral ultrasonography (IOUS) as a tool for predicting neck metastasis. Squamous cell carcinoma (SCC) of the tongue is aggressive and has a great propensity to metastasize to cervical lymph nodes. SCC of the oral cavity has a worse prognosis when associated with metastatic cervical nodes. Therefore, the metastatic potential of tongue carcinoma should be graded preoperatively to help determine the requirement for neck dissection. Nineteen patients (11 men, 8 women) between 36 and 79 years of age (mean age 60) with T1 to T4a TNM-stage tongue carcinomas were evaluated preoperatively with IOUS. Clinical and pathological TNM classifications were performed. The average tumor thicknesses measured using histological sections were significantly (p < 0.01) lower than those with IOUS (1.3 vs. 1.6 cm, respectively). A significant correlation was observed between the tumor thickness measured using ultrasonography and that measured using histological sections (pathology). Based on this greater accuracy, the cutoff point of tumor thickness based on IOUS evaluation for predicting neck metastasis was determined to be 1.8 cm. Some factors may influence neck metastasis. A knowledge of these would help to avoid unnecessary surgical intervention for N0 patients. The results of this study indicates that there is a significant correlation between neck metastasis and tumor thickness. Intraoral ultrasonography is useful tool for identifying tongue tumors and measuring their thickness, with the thickness measured by IOUS showing a very good correlation with histological measurements. Moreover, IOUS provides prognostic information prior to surgical treatment since tumor thickness can predict the chance of recognizing metastatic cervical nodes.

ERCC1 protein, mRNA expression and T19007C polymorphism as prognostic markers in head and neck squamous cell carcinoma patients treated with surgery and adjuvant cisplatin-based chemoradiation

Adjuvant cisplatin-based chemoradiation improves survival in HNSCC patients presenting with risk features. ERCC1 (excision repair cross-complementation group 1) is associated with resistance to chemo- and radiation therapy and may have a prognostic value in HNSCC patients. Here we studied ERCC1 expression and the polymorphism T19007C as prognostic markers in these patients. This is a retrospective and translational analysis, where ERCC1 protein expression was evaluated by immunohistochemistry, using an H-score, and mRNA expression was determined by RT-PCR. T 19007C genotypes were detected by PCR-RFLP carried out using DNA template extracted from normal lymph nodes. A high H-score was seen in 32 patients (54%), who presented better 5-year overall survival (5-y OS: 50% vs. 18%, HR 0.43, p=0.026). Fifteen out of 45 patients (33%), with high mRNA expression, presented better 5-year overall survival (OS) (86% vs. 30%, HR 0.26, p=0.052). No OS difference was detected among T 19007C genotypes. High H-score and mRNA expression remained significant as favorable prognostic factors in a multivariate analysis. Collectively, our results suggest that high ERCC1 expression seems to be associated with better OS rates in HNSCC patients submitted to adjuvant cisplatin-based chemoradiation.

In situ hybridization detection of homeobox genes reveals distinct expression patterns in oral squamous cell carcinomas

Aims: To analyse the expression of three homeobox genes (HOXA7, PITX1 and PRRX1) in oral squanous cell carcinomas (OSCC) and the relationship of such expression to certain distinct histopathological features of OSCC and in comparison to adjacent non-neoplastic epithelium (NT). Methods and results: Digoxigenin-labelled riboprobes that are specific for each homeobox gene were generated and in situ hybridization was carried out on frozen sections. In NT samples, HOXA7 and PITX1 transcripts were found more frequently in all epithelial layers, while PRRX1 was expressed in the basal layer. With OSCC samples, expression of the three genes was associated with all histological features. However, the HOXA7 and PITX1 signals were more intense in sheets and nests and PRRX1 in small nests and isolated cells. Conclusion: HOXA7, PIXT1 and PRRX1 homeobox genes have different patterns of expression in OSCC depending on its histological features.

Expression Profile of p63 in 127 Patients with Laryngeal Squamous Cell Carcinoma

Objectives: To evaluate p63 expression in laryngeal squamous cell carcinoma and its prognostic significance. Methods: p63 expression was examined by immunohistochemistry and scored in 127 patients with laryngeal squamous cell carcinomas. Results: Sixty-two cases had scored 3, sixty had scored 2, four had scored 1 and one case did not show any expression (48.8, 47.2, 3.1 and 0.8%, respectively). Overall survival was 73.9% at 24 months and 59.5% at 60 months. The disease-free survival was 77.2 and 75.1%, and the disease-specific survival was 79 and 67% at 24 and 60 months, respectively. Uni- and multivariate analysis identified that decreased immunoexpression of protein p63 was a statistically significant factor for the risk of recurrence and death by cancer. Conclusions: p63 expression was highly prevalent in laryngeal squamous cell carcinomas, and its underexpression was correlated with a worse prognosis. Copyright (C) 2010 S. Karger AG, Basel

Thyroid Function After Unilateral Total Lobectomy

Objective: To evaluate the incidence of postoperative hypothyroidism among patients who underwent unilateral total lobectomy and identify related factors. Design: Retrospective medical record analysis. Setting: Oncological center and private clinic. Patients: From March 1996 to July 2005, 228 euthyroid patients underwent unilateral total lobectomy for benign diseases; 168 had all the information required for inclusion in this study. Main Outcome Measures: Serum levels of thyrotropin and antithyroidal antibodies were assessed, as well as ultrasonographic evaluation of the remaining thyroid lobe and review of all histological specimens, with emphasis on lymphocytic infiltration. Hypothyroidism was defined as thyrotropin level greater than 5.5 mU/L. Results: Most patients were female (88%), with a median (range) age of 45 (16-72) years. Hypothyroidism occurred in 61 cases (32.8%), during a median follow-up period of 29 months (range, 6-108 months). Statistically related factors included higher preoperative thyrotropin levels (2.1 mU/L among hypothyroid patients vs 1.2 mU/L in euthyroid patients; P<.001), smaller thyroid remnant volume (3.9 mL vs; 6.0 mL, respectively; P = .003); right vs left lobectomy (P = .006), and higher thyroperoxidase antibody serum levels (P = .009). Conclusions: Postoperative hypothyroidism appeared in 32.8% of the cases in this series, especially among patients with elevated preoperative thyrotropin and postoperative thyroperoxidase antibody levels, after right lobectomy and when a smaller thyroid remnant was left. After confirmation with larger prospective series, these results may support the indication for early postoperative hormone supplementation in these instances.

Mutations of the thyroglobulin gene and its relevance to thyroid disorders

Purpose of review To perform an update review on thyroglobulin gene mutations associated with congenital hypothyroidism, thyroid cancer, and autoimmunity. Recent findings Forty-two thyroglobulin mutations have been identified in dyshormonogenetic congenital hypothyroidism. Clinical and laboratory criteria defining defective thyroglobulin synthesis are mostly related to thyroglobulin mutations, generally caused by intracellular thyroglobulin transport defects to the colloid rather than defects in thyroid hormones synthesis. Some mutated thyroglobulin may escape the rigorous chaperone control and reach the colloid, allowing a wide phenotypic spectrum that includes euthyroidism in an adequate iodine environment. In some patients, continuous levothyroxine treatment does not reduce elevated serum thyroid-stimulating hormone (TSH) levels that may lead to goiter development. Prenatally, inactive mutant thyroglobulin will not be able to synthesize thyroid hormones and may increase pituitary thyrotroph threshold for thyroid hormone feedback. Congenital goiter is a risk factor for thyroid cancer and some thyroglobulin variants may confer susceptibility to thyroid autoimmunity. Summary Advances in the understanding of thyroglobulin genetic defects and its severity should allow researchers to perform adequate molecular diagnosis, genetic counseling, and intrauterine treatment to prevent subtle deficits in central nervous system development. This knowledge should improve the understanding of physiological functions of the thyroid and influence of nutritional iodine.

Juvenile onset systemic lupus erythematosus thyroid dysfunction: A subgroup with mild disease?

The aim of this study was to evaluate the frequency of thyroid dysfunction and thyroid antibodies in patients with juvenile onset Systemic Lupus Erythematosus (JOSLE) and its association with clinical and immunological features. Seventy-seven patients with JOSLE, 64 females, median age 19 years, were consecutively enrolled from March to December 2007. Clinical data related to thyroid dysfunction and lupus were obtained by chart review and patient interview. Serum levels of TSH, free T4, anti-thyroglobulin (TgAb), anti-thyroperoxidase (TPOAb), TRAb and lupus related autoantibodies were analyzed by standard techniques. Nine patients were diagnosed as hypothyroidism and 4 hyperthyroidism. 28% JOSLE patients had moderate/high titer of thyroid antibodies: 23% TgAb, 2.6% TPOAb and 3.9% TRAb. JOSLE patients with positive thyroid autoantibodies had higher frequency of anti-U1RNP antibodies than patients without these antibodies (40.9 vs. 14.5%, OR:0.25, CI:0.08-0.76, p = 0.017). Furthermore, renal/neurological/hematological involvement was less frequently observed in patients with hypothyroidism (55.6 vs. 87.5%, OR:0.18, CI:0.04-0.81, p = 0.035) and with thyroid antibodies (68.4 vs. 90.9%, OR:0.22, CI:0.06-0.82. p = 0.027) than in patients without these alterations. No association with PTPN22 polymorphism was found. In conclusion, JOSLE patients have high prevalence of subclinical hypothyroidism. The novel association of anti-thyroid antibodies with anti-U1RNP antibodies in JOSLE seems to identify a subgroup of patients with less life-threatening organ involvement. (C) 2009 Elsevier Ltd. All rights reserved.

Uptake by breast carcinoma of a lipidic nanoemulsion after intralesional injection into the patients: A new strategy for neoadjuvant chemotherapy

Objective. Previously we showed that after intravenous injection a lipidic nanoemulsion concentrates in breast carcinoma tissue and other solid tumors and may carry drugs directed against neoplastic tissues. Use of the nanoemulsion decreases toxicity of the chemotherapeutic agents without decreasing the anticancer action. Currently, the hypothesis was tested whether the nanoemulsion concentrates in breast carcinoma tissue after locoregional injection. Methods. Three different techniques of injection of the nanoemulsion were tested in patients scheduled for surgical treatment: G1 (n=4) into the mammary tissue 5 cm away from the tumor; G2 (n=4) into the peritumoral mammary tissue; G3 (n=6) into the tumoral tissue. The nanoemulsion labeled with radioactive cholesteryl oleate was injected 12 h before surgery; plasma decay of the label was determined from blood samples collected over 24 h and the tissue fragments excised during the surgery were analyzed for radioactivity uptake. Results. Among the three nanoemulsion injection techniques, G3 showed the greatest uptake (data expressed in c.p.m/g of tissue) by the tumor (44,769 +/- 54,749) and by the lymph node (2356 +/- 2966), as well as the greatest concentration in tumor compared to normal tissue (844 +/- 1673). In G1 and G2, uptakes were, respectively, tumor: 60 +/- 71 and 843 +/- 1526; lymph node: 263 +/- 375 and 102 +/- 74; normal tissue: 139 +/- 102 and 217 +/- 413. Conclusions. Therefore, with intralesional injection of the nanoemulsion, a great concentration effect can be achieved. This injection technique may be thus a promising approach for drug-targeting in neoadjuvant chemotherapy in breast cancer treatment. (C) 2008 Published by Elsevier Inc.