Perforated colorectal neoplasms: correlation of clinical, contrast enema, and CT examinations

Results of clinical, contrast enema (CE), and computed tomographic (CT) examinations in 39 patients with perforated colorectal neoplasms were retrospectively reviewed. Twenty patients were toxemic at initial presentation, but in only four patients was the diagnosis of perforated colorectal neoplasm initially suspected clinically. CE study was performed in 22 patients and enabled the diagnosis of perforated neoplasm in 11 cases, neoplasm alone in eight, and neither neoplasm nor perforation in three. CT was performed in 38 patients and enabled the diagnosis of perforated neoplasm in 36; pericolic phlegmon but no mass lesion was evident in two. In 16 patients, CT also demonstrated metastatic disease. Because of its reliability in establishing the diagnosis and staging the extent of the inflammatory and neoplastic disease, CT is indicated in cases of suspected or proved perforated colorectal neoplasm and in cases in which CE study findings are indeterminate or suggestive of perforated neoplasm.     

Novel strategies for enhancing tissue integration in cartilage repair

Introduction Articular cartilage is unable to initiate a spontaneous repair response when injured due to its avascular and aneural properties. Within adult cartilage, chondrocytes are entrapped within an extensive extracellular matrix and are unable to migrate to sights of injury to regulate tissue repair. Injury to this tissue therefore inevitably leads to degeneration of the cartilage and the development of degenerative diseases such as osteoarthritis. The surgical technique of autologous chondrocyte transplantation (ACT) was developed for the treatment of full‐thickness cartilage defects ( Brittberg et al. 1994 ). Implantation of chondrocytes into the defect site repairs the injury site with a mixture of fibrocartilaginous and hyaline‐like tissue that poorly integrates with the existing cartilage and frequently degenerates with time. In this current study, we have developed an in vitro model to investigate methods for enhancing this integration and the development of a more biomechanically stable repair tissue. Materials and methods Bovine articular cartilage explants from the metacarpalphalangeal joint were experimentally injured using a stainless steel trephine and cultured for a period of 28 days. Autologous chondrocytes in an agarose suspension were injected into the interface region at the injury site. Media was collected and analysed for proteoglycan and collagen content using the DMMB and hydroxyproline assays, respectively. Matrix metalloproteinase (MMP) expression was also analysed using zymography and an adapted collagen fibril assay. Results Morphological analyses indicate attempts at repair and integration within both control and experimental treatment groups, although the presence of autologous chondrocytes appeared to amplify this repair response. Although not statistically significant, considerable differences in proteoglycan release between injured explants and the intact control group were seen. Collagen release into the media was only seen at day 28 within experimental cultures. An up‐regulation of MMP‐2 and MMP‐9 was seen within the experimental cultures compared to the controls. Preliminary data also suggest up‐regulation of collagenases in the experimental group when compared to controls. Discussion As seen with clinical ACT treatment, the presence of autologous chondrocytes appears to enhance repair and integration attempts; however, morphologically, this repair tissue appears to be fibrocartilaginous. Further analysis will establish whether the repair tissue is true hyaline cartilage and monitor the synthesis and turnover of macromolecules within the established culture system.     

A novel keratanase-generated keratan sulfate antibody and its application to structural analysis of skeletal and corneal keratan sulfate

Introduction The objective of this study was to make monoclonal antibodies specific for keratanase‐generated neoepitopes in keratan sulfate (KS) and to use them along with existing KS monoclonal antibodies (e.g. 5D4, IB4) to investigate KS sulfation pattern motifs in connective tissue proteoglycans during development, ageing and disease. Methods Bovine nasal cartilage aggrecan (BNC A1D1) was trypsin digested, generating a range of GAG‐peptide fragments. The sample was then subjected to anion‐exchange and size exclusion chromatography to separate KS peptides from CS attachment domain fragments. Fractions were analysed by Western blotting for positive immunoreactivity for KS, then pooled and keratanase digested to generate ‘KS stub’ antigens. Immunization and fusions were carried out as previously described ( Caterson et al. 1983 ; Hughes et al. 1992 ). Screenings involved the use of a range of antigens; including keratanase vs. keratanase II‐digested bovine cartilage aggrecan and bovine corneal KS‐PGs. A new monoclonal antibody, BKS‐I, was identified that specifically recognized a keratanase‐generated neoepitope on both skeletal and corneal KS. This novel monoclonal antibody was used along with existing KS monoclonal antibodies 5D4 and 1B4 to investigate KS structure. Results and discussion Bovine trypsin‐generated aggrecan KS‐peptides were chondroitinase ABC treated and either keratanase or keratanase II treated. The digests were run on SDS‐PAGE and immunolocated with monoclonal antibody 5D4 (that recognizes linear disulfated N‐acetyl lactosamine disaccharide‐containing segments in KS) and the new ‘KS‐stub’ monoclonal antibody BKS‐I. Our results indicated that there was reduced monoclonal antibody 5D4 immunostaining after keratanase pretreatment. However, keratanase II digestion completely removed all 5D4 structural epitopes. In contrast, BKS‐I showed no immunostaining on the untreated KS‐peptides but strong staining on keratanase treated samples and no staining after keratanase II digestion. Similar patterns of immunoreactivity were observed with Western blot analysis of untreated, keratanase treated and keratanase II treated corneal KS‐PGs. Conclusion These data indicate that monoclonal antibody BKS‐I recognizes a nonreducing terminal neoepitope‐containing sulfated N‐acetylglucosamine adjacent to a nonsulfated lactosamine disaccharide. We also conclude that skeletal KS must have a structure with four possible variations opposed to the generic structures, proposed as being made of disulfated disaccharides at the nonreducing end, followed by a series of monosulfated disaccharides at the middle and nonsulfated disaccharides nearer the linkage region. 5D4 staining, observed after keratanase digestion, indicates that there must be a minimum structure of a pentasulfated hexasaccharide remaining on the KS chain ‘stubs’ near the linkage region of skeletal and corneal KS. The BKS‐I monoclonal antibody can be used to demonstrate differential substitution of KS GAG chains in the CS attachment region of cartilage aggrecan with ageing. It has also proven useful for immunohistochemical analyses identifying the sites of KS–PG association with collagen lamellae of cornea.     

Frequent ongoing T-cell receptor rearrangements in childhood B- precursor acute lymphoblastic leukemia: implications for monitoring minimal residual disease

Crosslineage T-cell receptor delta (TCR delta) rearrangements are widely used as tumor markers for the follow up of minimal residual disease in childhood B-precursor acute lymphoblastic leukemia (ALL) by polymerase chain reaction (PCR). The major drawback of this approach is the risk of false-negative results due to clonal evolution. We investigated the stability of V delta 2D delta 3 rearrangements in a group of 56 childhood B-precursor ALL patients by PCR and Southern blot analysis. At the PCR level, V delta 2D delta 3-to-J alpha rearranged subclones (one pathway for secondary TCR delta recombination) were demonstrated in 85.2% of V delta 2D delta 3-positive patients tested, which showed that small subclones are present in the large majority of patients despite apparently monoclonal TCR delta Southern blot patterns. Sequence analysis of V delta 2D delta 3J alpha rearrangements showed a biased J alpha gene usage, with HAPO5 and J alpha F in 26 of 32 and 6 of 32 clones, respectively. Comparison of V delta 2D delta 3 rearrangement status between diagnosis and first relapse showed differences in seven of eight patients studied. In contrast, from first relapse onward, no clonal changes were observed in six patients studied. To investigate the occurrence of crosslineage TCR delta rearrangements in normal B and T cells, fluorescence-activated cell sorter-sorted peripheral blood CD19+/CD3- and CD19-/CD3+ cell populations from three healthy donors were analyzed. V delta 2D delta 3 rearrangements were detected at low frequencies in both B and T cells, which suggests that V delta 2-to-D delta 3 joining also occurs during normal B-cell differentiation. A model for crosslineage TCR delta rearrangements in B-precursor ALL is deduced that explains the observed clonal changes between diagnosis and relapse and is compatible with multistep leukemogenesis of B-precursor ALL.     

Sequence comparison of human and yeast telomeres identifies structurally distinct subtelomeric domains

We have sequenced and compared DNA from the ends of three human chromosomes: 4p, 16p and 22q. In all cases the pro-terminal regions are subdivided by degenerate (TTAGGG)n repeats into distal and proximal sub- domains with entirely different patterns of homology to other chromosome ends. The distal regions contain numerous, short (<2 kb) segments of interrupted homology to many other human telomeric regions. The proximal regions show much longer (approximately 10-40 kb) uninterrupted homology to a few chromosome ends. A comparison of all yeast subtelomeric regions indicates that they too are subdivided by degenerate TTAGGG repeats into distal and proximal sub-domains with similarly different patterns of identity to other non-homologous chromosome ends. Sequence comparisons indicate that the distal and proximal sub-domains do not interact with each other and that they interact quite differently with the corresponding regions on other, non- homologous, chromosomes. These findings suggest that the degenerate TTAGGG repeats identify a previously unrecognized, evolutionarily conserved boundary between remarkably different subtelomeric domains.