High-throughput screening: advances in assay technologies

The expression and up-regulation of cell adhesion molecules on a human colonic epithelial cell line HT-29, and the peripheral blood T lymphocyte proliferation responses to bacterial superantigens presented by this cell line were investigated, compared with peripheral blood monocytes. In HT-29 cells, there was constitutive expression of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-3 (LFA-3) at a low level, but no constitutive expression of HLA-DR, LFA-1, B7-1 and B7-2 molecules. After stimulation with the supernatants of staphylococcal enterotoxin B (SEB)-stimulated peripheral blood mononuclear cells for 48 h, there was significant up-regulation of HLA-DR and ICAM-1 molecules (both > 90% positive). However, this stimulation had no effect on the expression of LFA-1, B7-1, B7-2 and LFA-3 molecules. In the presence of all tested superantigens SEB, toxic shock syndrome toxin-1, and streptococcal pyogenic exotoxin A, stimulated HT-29 cells caused significant T cell proliferation. When monocytes were used as antigen-presenting cells (APC), the MoAbs against HLA-DR, B7-2 and LFA-3 showed a significant inhibition of SEB-induced T cell proliferation. Anti-ICAM-1 MoAb had no effect on this response. On the other hand, when stimulated HT-29 cells were used as APC, the MoAbs against HLA-DR and ICAM-1 significantly inhibited SEB-induced T cell proliferation. In contrast to monocytes, anti-B7-2 and anti-LFA-3 had no effect on this response. SEB could not induce HT-29 cells to produce IL-8 directly; however, SEB significantly induced the stimulated HT-29 cells to produce IL-8 in the presence of T cells. Thus these data demonstrate that the products of superantigen-stimulated T cell activation can increase the expression of HLA-DR and ICAM-1 molecules on HT-29 cells significantly. Stimulated HT-29 cells can serve as APC to bacterial superantigens. This response is an HLA-DR- and ICAM-1-dependent, but B7-2- and LFA-3-independent process, which was different from professional APC monocytes.     

Detection of infrequent and multiple K-ras mutations in human tumors and tumor-adjacent tissues

A sensitive method was developed and applied to examine the distribution of K-ras gene mutations in histologically differing areas of lung tissues obtained from lung cancer patients. This method, which combines polymerase chain reaction (PCR), mutation allele enrichment (MAE), and denaturing gradient gel electrophoresis (DGGE), allows detection of one K-ras mutant allele present in 10(4) to 10(5) wild-type alleles. It was applied to analyze mutations in codon 12 of the K-ras gene in 43 tissue sites microdissected from paraffin-embedded sections obtained from 8 archival cases of lung cancer, all previously shown to have codon 12 K-ras mutations by direct sequencing. In four cases, mutations were detected only in the tumor, while in the other four cases, the same mutations were also found in tissues adjacent to tumors, using the MAE + DGGE method. No mutations were detected among normal-appearing cells in areas distant from the tumors in any of the cases studied. These findings demonstrate that K-ras mutations can be detected at low frequencies in normal-appearing cells from tissues adjacent to the tumor in some lung cancer cases. In addition, this approach also allowed detection of multiple mutations in colorectal tissues obtained from colorectal cancer patients. Thus, the MAE + DGGE method may be applicable to study of K-ras mutations in premalignant or morphologically suspicious lesions in bronchial mucosa or other types of human cancer.     

Osteoma of the base of the tongue

A 14-year-old girl developed a firm mass at the base of the tongue. Computed tomography indicated marked density suggesting either a foreign body or bony tissue. A thyroid scan confirmed the presence of a normally sized and positioned gland. The mass was removed in toto and found to be an osteoma. This is the first report of a case in which the diagnosis of this rare developmental lesion of the tongue was achieved preoperatively based on clinical and radiologic information. This experience should lead to greater awareness of this entity in the future. Recognition of this entity in the pediatric age group is especially useful in avoiding misdiagnosis of other, potentially more aggressive types of tongue mass lesions. Our case demonstrates that it is possible to detect this entity using computed tomography. The dense calcification is truly characteristic of the tumor and may be relied upon to exclude alternative soft tissue mass lesions. While other forms of osseous and cartilagenous neoplasms, such as extraskeletal osteosarcoma and chondrosarcoma, have been reported arising in the tongue, their malignant nature should otherwise be readily apparent. Osteoma of the tongue is the favored diagnosis when mature bone tissue is imaged at the base of the tongue.     

A complex nine base pair deletion in RET exon 11 common in sporadic medullary thyroid carcinoma

Genetic alteration of the RET proto-oncogene is associated with multiple endocrine neoplasia type 2A and 2B (MEN 2A and MEN 2B), familial medullary thyroid carcinoma (FMTC) and Hirschprung's disease. Oncogenically activated RET has also been demonstrated in sporadic medullary thyroid tumors, which in some cases show somatic missense mutations. We have recently described a complex 9 bp deletion in RET exon 11 in a single case of sporadic MTC. In order to determine the prevalence of this mutation among sporadic MTC tumors, we have now analysed 15 cases and five normal controls by PCR-based nonradioactive single-strand conformational polymorphism analysis (PCR-SSCP) and fragment size analysis of exon 11. DNA was extracted from microdissected tumor tissue or normal cells and subjected to nested PCR prior to analysis. A markedly divergent SSCP pattern and a PCR fragment 9 bp shorter than normal were demonstrated in 14 of the 15 MTC tumors. Sequencing revealed the deletion of nine bases encompassing a key cysteine at codon 634, often altered in MEN 2A. Four lymphocyte controls and normal thyroid tissue from one patient failed to show the deletion. Several factors in the DNA sequence environment immediately surrounding the deletions, including an extended inverted repeat, several direct repeats and a so-called symmetric element suggest that the deletional events may be non-random.     

Carcass maturity and dicationic salts affect preblended, low-fat, low-sodium restructured beef

Preblending A- and C-maturity muscles with MgCl(2) and/or CaCl(2) was investigated in low-fat, low-sodium restructured beef. Products were formulated to contain: 1) 80% chunks, preblended 12h with 0.05% MgCl(2), 0.05% CaCl(2), or a combination of each (0.1%) and 0.4% sodium tripolyphosphate (STPP) and 2) 20% mince preblended 12h with 0.05% of each dicationic salt or the combination of dicationic salts (0.1%), 0.4% STPP, and 1.0% NaCl. This formulation achieved a raw product NaCl content of 0.2%. Additionally, a control was formulated with chunks and mince that contained no dicationic salt. CaCl(2) decreased raw and cooked pH and cook yield, and increased cohesiveness; whereas, MgCl(2) increased cook yield and myosin solubility. Total protein solubility was not affected by muscle maturity or dicationic treatment. Myosin solubility of the combination treatment was greater for C-maturity muscle (57 months) compared to A-maturity muscle (20 months) formulations. Control, C-maturity muscle treatments contained more insoluble and total collagen (p < 0.05), and these treatments were more cohesive (p < 0.05) than control, A-maturity treatments. The combination of CaCl(2) and MgCl(2) increased hardness of A-maturity products, but it decreased hardness of C-maturity products. In addition to increasing hardness of A-maturity products, the combination treatment lowered (p < 0.05) cook yield for these products.     

Surface enhanced Raman spectroscopy for characterization of structural characteristics of carbon chains in alpha1-acid glycoprotein and pseudoglycoproteins]

Surface-enhanced Raman scattering (SERS) spectroscopy was used to study the structure of carbohydrate chains in glycosylated forms of alpha 1-acid glycoprotein (AGP) and in pseudoglycoproteins obtained by transferring the carbohydrate chains of AGP to a polyacrylamide carrier. It was found that AGP-D glycoform and pseudoglycoproteins containing three or more glycans per molecule, which possess high immunomodulating activity, have a specific spatial organization of carbohydrate chains. This organization is maintained by the interaction of neighboring glycans with each other and does not depend on the nature of the carrier (whether it is polypeptide or polyacrylamide).     

Evidence of transmission of hepatitis B virus to spouses from sequence analysis of the viral genome

Despite advances in postnatal care, patients born with a congenital diaphragmatic hernia (CDH) suffer substantial morbidity and mortality. The present study was undertaken to determine the prognostic influence of prenatally-diagnosed liver herniation in the hemithorax in fetuses with CDH. The medical records of 48 patients evaluated for a prenatally-diagnosed left CDH were retrospectively reviewed. Patients were analysed according to the position of the liver by prenatal ultrasound; 32 fetuses had a major portion of the liver herniated into the left hemithorax ('liver up') and 16 had an intra-abdominal liver ('liver down'). Liver position was determined using colour-flow Doppler ultrasonography. There were two fetal deaths in the liver-up group and one in the liver-down group. The liver-up group more frequently required extracorporeal membrane oxygenation (ECMO) support (53 per cent) compared with the liver-down group (19 per cent). Postnatal survival was significantly less in the liver-up group (43 per cent) vs. the liver-down group (93 per cent). Fetuses with congenital diaphragmatic hernia and liver herniated into the hemithorax have a much worse prognosis than similarly afflicted fetuses without liver herniation. Prenatal ultrasonographic diagnosis of congenital diaphragmatic hernia allows for preparation for a critically ill newborn and aids in prenatal family counselling.     

A constipation scoring system to simplify evaluation and management of constipated patients

PURPOSE: Constipation is a common complaint; however, clinical presentation varies with each individual. The aim of this study was to assess a standard scoring system for evaluation of constipated patients. MATERIALS AND METHODS: All consecutive patients with idiopathic constipation who were referred for anorectal physiologic testing were assessed. A subjective constipation score was calculated based on a detailed questionnaire that included over 100 constipation-related symptoms. Based on the questionnaire, scores ranged from 0 to 30, with 0 indicating normal and 30 indicating severe constipation. The constipation score was then compared with the objective findings of the physiology tests, which include colonic transit time (CTT), anal manometry (AM), cinedefecography (CD), and electromyography (EMG). Colonic inertia was defined as diffuse marker delay on CTT without evidence of paradoxical contraction on AM, CD, or EMG. Pelvic outlet obstruction was defined as paradoxical puborectalis contraction, rectal prolapse or rectoanal intussusception, rectocele, or sigmoidocele. RESULTS: A total of 232 patients (185 females and 47 males) of a mean age of 64.9 (range, 14-92) years were evaluated. All patients had a score of more than 15; on evaluation of the significance of different symptoms in the constipation score with the Pearson's linear correlation test, 8 of 18 factors were identified as significant (P < 0.05). These factors included frequency of bowel movements, painful evacuation, incomplete evacuation, abdominal pain, length of time per attempt, assistance for evacuation, unsuccessful attempts for evacuation per 24 hours, and duration of constipation. All 232 patients had objective obstruction attributable to one or more of the following causes: paradoxical puborectalis contraction (81), significant rectocele or sigmoidocele (48), rectoanal intussusception (64), and rectal prolapse (9). CONCLUSION: The proposed constipation scoring system correlated well with objective physiologic findings in constipated patients to allow uniformity in assessment of the severity of constipation.     

Natural killer cell-mediated eradication of neuroblastoma metastases to bone marrow by targeted interleukin-2 therapy

Targeted interleukin-2 (IL-2) therapy with a genetically engineered antidisialoganglioside GD2 antibody-IL-2 fusion protein induced a cell-mediated antitumor response that effectively eradicated established bone marrow and liver metastases in a syngeneic model of neuroblastoma. The mechanism involved is exclusively natural killer (NK) cell-dependent, because NK-cell deficiency abrogated the antitumor effect. In contrast, the fusion protein remained completely effective in the T-cell-deficient mice or immunocompetent mice depleted of CD8+ T cells in vivo. A strong stimulation of NK-cell activity was also shown in vitro. Immunohistology of the leukocytic infiltrate of livers from treated mice revealed a strong staining for NK cells but not for CD8+ T cells. The therapeutic effect of the fusion protein was increased when combined with NK-cell-stimulating agents, such as poly I:C or recombinant mouse interferon-gamma. In conclusion, these data show that targeted delivery of cytokines to the tumor microenvironment offers a new strategy to elicit an effective cellular immune response mediated by NK cells against metastatic neuroblastoma. This therapeutic effect may have general clinical implications for the treatment of patients with minimal residual disease who suffer from T-cell suppression after high-dose chemotherapy but are not deficient in NK cells.