Expression of connective tissue growth factor (CCN2) in desmoplastic small round cell tumour.

BACKGROUND: Desmoplastic small round cell tumour (DSRCT) is a rare and often fatal abdominal tumour that is distinguished by well defined islands of cells, surrounded by prominent desmoplastic stroma. As in certain other tumours, the function of the Wilms's tumour protein (WT1) in repressing gene transcription is lost in DSRCT. AIMS: To assess the expression and localisation of connective tissue growth factor (CCN2) in DSRCT because this protein is transcriptionally repressed by WT1 and is associated with the production of abundant extracellular matrix. METHODS: CCN2 was assessed by in situ hybridisation and immunohistochemistry. RESULTS: CCN2 mRNA and protein were colocalised to the tumour cells themselves, in addition to stromal fibroblasts and vascular endothelial cells. CONCLUSIONS: These data show that CCN2 is produced in high amounts by several cell types in DSRCT, and highlight a potential role for this factor in the autocrine and paracrine regulation of tumour cell growth, matrigenesis, and angiogenesis.

Heparin-binding EGF-like growth factor decreases inducible nitric oxide synthase and nitric oxide production after intestinal ischemia/reperfusion injury.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been shown to protect intestine from ischemia/reperfusion (I/R) injury in vivo and to down-regulate inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production in intestinal epithelial cells in vitro. The present study was undertaken to investigate whether HB-EGF could modulate the iNOS/NO axis after total midgut I/R injury in rats. I/R injury induced a significant increase in iNOS gene expression (quantified by real-time RT-PCR) and protein production (detected by western blots), as well as elevation of serum NO levels (measured by chemiluminescence assay). Nitrotyrosine (NT) and iNOS production colocalized immunohistochemically, with positive staining found mainly in villous and crypt epithelial cells, as well as ganglion cells. Intraluminal administration of HB-EGF 45 min after the start of a 90-min ischemic interval significantly decreased I/R-induced iNOS gene expression and protein production, as well as serum NO levels. Immunohistochemically, HB-EGF administration led to elimination of iNOS and NT staining in crypt epithelial cells and ganglion cells, with only weak staining that remained in villous epithelial cells. Thus, HB-EGF protects the intestine from I/R injury, at least partially, through down-regulation of the iNOS/NO/NT pathway, a mechanism that is central to I/R injury in multiple organ systems.

Heparin-binding epidermal growth factor-like growth factor protects rat intestine from ischemia/reperfusion injury.

BACKGROUND: We have shown previously that heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is cytoprotective for intestinal epithelial cells exposed to hypoxia in vitro. We now examine the effects of HB-EGF on the recovery of small intestine from ischemic injury in vivo. METHODS: Segmental intestinal ischemia of 60-min duration was produced in adult rats by occlusion of a first-order branch of the superior mesenteric artery. Recombinant HB-EGF (100 microg) was injected intraluminally into the proximal small bowel after 45 min of ischemia in experimental animals, and buffered saline was injected in control animals. Animals were sacrificed after 48 h, and the affected bowel was resected, processed, and examined microscopically, with histologic grading of the ischemic injury. Additional animals were allowed to recover for up to 1 month to evaluate mortality differences. RESULTS: Intraluminal administration of HB-EGF resulted in significantly decreased extent and severity of ischemia/reperfusion injury, with significantly decreased grade of injury in the HB-EGF-treated compared with nontreated animals (average injury grade 0.66 compared with 2.44, respectively). Moreover, the mortality rate was significantly lower in the HB-EGF-treated animals compared with nontreated animals (0% vs 25%, respectively). HB-EGF-treated animals had increased weight gain in the postischemia recovery period. CONCLUSIONS: We conclude that HB-EGF, given intraluminally, reduces both the amount and the severity of ischemia/reperfusion injury in the small bowel, reduces the mortality associated with intestinal ischemia, and may enhance intestinal recovery. The in vitro and in vivo cytoprotective effects of this growth factor suggest that it may, in the future, be clinically useful in treating patients with intestinal ischemia.

A standardized model of partial thickness scald burns in mice.

BACKGROUND: Large mammal partial thickness wound models were developed primarily for their anatomical similarity to human wounds, yet lack the economy, ease of handling, and statistical power afforded with rodent models. Previous small mammal models of partial thickness burn injury have failed to demonstrate complete reepithelialization in less than 3 weeks. We present a murine partial thickness scald model with a reepithelialization rate comparable to that of porcine wound models. METHODS: Thirty-eight adult male mice were secured in a burn template allowing exposure of a 2 x 3-cm area of the shaved dorsum to 60 degreesC water for 45 s, followed by 4 degreesC water for 45 s. Four wounds were harvested daily on Postburn Days 1-7, 10, and 14 for histologic evaluation. RESULTS: Histologic evidence of partial thickness dermal injury with sparing of dermal appendage epithelial cells was seen in all wounds. Of 134 wound sections evaluated 26 contained some areas of full thickness dermal injury, with only 8 of these 26 sections showing full thickness injury in 50% or more of the cross-sectional area of the wounds. Complete wound reepithelialization was seen between Postburn Days 10 and 14. The viable dermal thickness in all burn cross sections was at least 40-80 micrometers, and up to 1400 micrometers in reepithelialized wounds. CONCLUSIONS: This murine model of partial thickness scald injury provides a standardized thermal wound with consistent depth of injury, low mortality, and a reepithelialization rate between 10 and 14 days. A simple protocol allows easy production of 30-50 wounds daily with one technician.

Segmental intestinal ischemia: an improved method of producing small bowel injury.

Previous animal models of intestinal ischemia-reperfusion have been successful in causing considerable mucosal damage, cellular destruction and sepsis. However, this often results in the death of the animal, making it impossible to examine the effects of modulators of the ischemic event. The sequence of morphologic and physiologic changes in the bowel from such injuries continues to be an area of intense examination. We have studied these changes by producing segmental intestinal ischemia in vivo in a rat model. By occluding a first-order branch of the superior mesenteric artery (SMA) and by selectively ligating terminal collateral branches, reproducible segmental intestinal ischemia was achieved. Bowel damage ranged from alterations in the villus structure to frank hemorrhagic necrosis of the intestinal wall. This model allows the study of hypoperfusion injury to the small intestine without total SMA occlusion, thus reducing the overall mortality.

Cystic degeneration of heterotopic pancreas.

The case of a large cyst arising from heterotopically-situated pancreatic tissue in an 11-month-old girl is reported. This is the first published report of childhood pancreatic cyst that developed in heterotopic pancreatic tissue. There is strong evidence to suggest that the cyst became symptomatic as a result of secondary infection, an additional unreported phenomenon. This case serves to underscore the fact that pancreatic cysts should be considered in the differential diagnosis of intestinal duplication cysts of childhood.

Acceleration of partial-thickness burn wound healing with topical application of heparin-binding EGF-like growth factor (HB-EGF).

Heparin-binding EGF-like growth factor has been identified in human burn-wound fluid and in the epithelial cells of excised human partial-thickness burns. In the present study, the effect of heparin-binding EGF-like growth factor on burn-wound healing was evaluated by incorporating purified, recombinant heparin-binding EGF-like growth factor into slow-release cholesterol-lecithin pellets that were applied topically to partial-thickness burns in mice. Both experimental (heparin-binding EGF-like growth factor-treated) and control (untreated) mice were sacrificed on days 3, 5, and 10 after burn. Total burn-wound area, histology, keratinocyte proliferation, and in situ hybridization analysis for transforming growth factor-alpha were determined for each wound. The mean wound area of the experimental group on day 5 after burn was 1.07 cm2, compared with 2.20 cm2 for controls (p=0.04). Cellular proliferation (as measured by immunohistochemical detection of 5-Bromo-2-deoxyuridine) on day 5 after burn in marginal keratinocytes and follicular epithelial cells was greater in the experimental group than in the control group. In situ hybridization showed up-regulation of transforming growth factor-alpha mRNA levels in experimental animals by day 5 after burn. Topical application of heparin-binding EGF-like growth factor significantly accelerates the reepithelialization of murine partial-thickness burns, increases keratinocyte proliferative activity, and enhances production of endogenous transforming growth factor-alpha mRNA.

Dystrophin expression in a Duchenne muscular dystrophy patient with a frame shift deletion.

The exon 45 deletion is a common dystrophin gene deletion. Although this is an out-of-frame deletion, which should not allow for protein synthesis, it has been observed in mildly affected patients. We describe a patient with an exon 45 deletion who produced protein, but still had a severe Duchenne muscular dystrophy phenotype. RT-PCR analysis and cDNA sequencing from the muscle biopsy sample revealed that the exon 45 deletion induced exon skipping of exon 44, which resulted in an in-frame deletion and the production of dystrophin. A conformational change in dystrophin induced by the deletion is proposed as being responsible for the severe phenotype in the patient. We feel that the variable clinical phenotype observed in patients with the exon 45 deletion is not due to exon splicing but may be the result of other environmental or genetic factors, or both.

Immunohistochemical localization of heparin-binding epidermal growth factor-like growth factor in normal skin and skin cancers.

Heparin-binding epidermal growth factor (EGF)-like growth factor is a 22-kDa glycoprotein that was originally identified as a secreted product of cultured human macrophages. Although the growth factor mRNA has been identified in various cells and tissues, the tissue distribution of the protein itself has rarely been demonstrated. In this study, the EGF-like growth factor was detected immunohistochemically in a variety of human skin samples by indirect immunofluorescence using a polyclonal rabbit antiserum raised against residues 26-41 of mature heparin-binding EGF. The keratinocytes of a variety of epithelium-derived structures demonstrated reproducible, specific staining for the EGF. In normal tissues, this staining was prominent in the basal cells of the epidermis and in the epithelial cells lining epidermal appendages such as hair follicles, sebaceous sweat glands and eccrine sweat glands. In addition, specific staining was detected in skin cancers derived from the basal epithelial cell layer, including basal and squamous cell carcinomas of the skin, with no staining detected in melanoma specimens. Immunoreactive heparin-binding EGF was characteristically associated with the surface of cells. With minor exceptions, the immunoreactive sites are identical to the known EGF receptor distribution in the skin, and suggest that keratinocyte-derived heparin-binding EGF may act in concert with other EGF family members in processes such as skin morphogenesis and wound repair, as well as in the development of skin cancers.

Production of heparin-binding epidermal growth factor-like growth factor (HB-EGF) at sites of thermal injury in pediatric patients.

Fluids that accumulate at wound sites may be an important reservoir of growth factors that promote the normal wound healing response. The presence of heparin-binding growth factors was studied in burn wound fluid (BWF) from 45 pediatric patients who had sustained partial thickness burns. One of the growth factors present was similar to platelet-derived growth factor (PDGF) based on its heparin affinity, inhibition of bioactivity by a PDGF antiserum, and detection in a PDGF-AB enzyme-linked immunosorbent assay. A second growth factor was identified as heparin-binding epidermal growth factor-like growth factor (HB-EGF) based on its heparin affinity, competition with 125I-labeled epidermal growth factor (EGF) for EGF receptor binding, and recognition in biological assays and Western blots by two HB-EGF antisera. Amino acid sequence analysis of one form of this second growth factor verified its identity as an N-terminally truncated form of HB-EGF. Immunohistochemical analysis of partial thickness burns demonstrated the presence of HB-EGF in the advancing epithelial margin, islands of regenerating epithelium within the burn wound, and in the duct and proximal tubules of eccrine sweat glands. HB-EGF in the surface epithelium of burn wounds was uniformally distributed, whereas it was restricted to the basal epithelium in nonburned skin. These data support a role for PDGF and HB-EGF in burn wound healing and suggest that the response to injury includes deposition of HB-EGF and PDGF into blister fluid and a redistribution of HB-EGF in the surface epithelium near the wound site.

Purification of heparin-binding epidermal growth factor-like growth factor from pig uterine luminal flushings, and its production by endometrial tissues.

Pig uterine luminal flushings contain at least four heparin-binding growth factors (HBGF) that stimulate fibroblast [3H]thymidine incorporation. One of these factors, which appeared to be a relatively minor HBGF, was eluted from heparin affinity columns by 1.0 M NaCl and was found to compete with 125I-epidermal growth factor (EGF) for binding to an endometrial carcinoma cell line. This EGF receptor (EGF-R)-binding property was abolished by an antiserum to heparin-binding EGF-like growth factor (HB-EGF) that specifically blocks binding of HB-EGF to the EGF-R. Reverse-phase HPLC resulted in the purification of two EGF-R-binding activities correlated with 13,500 and 17,000 M(r) proteins that reacted with an antiserum raised against residues 9-26 of human HB-EGF. Uterine extracts also contained an EGF-R-binding factor that was eluted from heparin by 1.0 M NaCl and was antagonized by HB-EGF antiserum. Endometrial mRNA subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR through the use of HB-EGF-specific primers yielded fragments of the predicted size. Cloning of the nested PCR product revealed a 380-bp porcine HB-EGF cDNA sequence that was 78-85% homologous to primate or rodent HB-EGF. HB-EGF was immunohistochemically localized primarily to the luminal epithelium in both pregnant and nonpregnant animals.

Reduced receptor binding by a human interferon-gamma fragment lacking 11 carboxyl-terminal amino acids.

Treatment of recombinant human interferon-gamma (IFN-gamma) with either 1) the arginine-specific proteases clostripain or submaxillaris protease or 2) the broadly specific enzyme pronase produced a stable fragment with m.w. of 15,500. Structural analysis showed that the cleavage occurred between residues 129 and 130 and thus produced a fragment lacking only 11 carboxyl-terminal amino acids. The fragmented and untreated molecules showed identical amino-terminal amino acid sequences and were equally reactive with either polyclonal or monoclonal anti-IFN-gamma. IFN-gamma lacking carboxyl-terminal amino acids displayed a 1000- to 2000-fold reduction in its capacity to bind to cellular IFN-gamma receptors at 4 degrees C. Functionally the fragment showed a 50-fold reduction in its ability to induce antiviral activity in fibroblasts and a 10-fold reduction in its ability to induce Fc receptors on the human histiocytic lymphoma cell line U937. These results thus suggest that the carboxyl terminus of human IFN-gamma contributes significantly to the formation of the receptor-binding site of the molecule.

Vascular sarcomas (probably angiosarcomas) transplanted from suspensions of liver cells from diethylnitrosamine-treated rats.

F344 male rats were given 90 ppm diethylnitrosamine in their drinking water ad libitum in two cycles. Livers containing neoplastic nodules, hepatomas, and no sarcomas in the sections sampled were digested in parallel with 0.05% collagenase, 0.1% Pronase, or 0.25% trypsin. Cells were transplanted into 9- to 19-day-old F344 rats. Despite the absence of sarcomas in the sections examined microscopically from each such liver before digestion and the presence of multiple hepatomas in all sections examined, vascular sarcomas, probably angiosarcomas, were observed in a large proportion of animals injected with the suspensions of cells; hepatomas were not observed in these animals. Morphology by light microscopy, immunohistochemical demonstration of factor VIII, histochemical demonstration of alkaline phosphatase, and the presence of Weibel-Palade bodies strongly suggest that these tumors are angiosarcomas. Similar tumors developed from cells obtained in parallel with the aid of Pronase, collagenase, or trypsin. Cell suspensions obtained with Pronase yielded tumors with the shortest latent period between the injection of cells and the death of one-half of the transplant recipients. The procedure that we used provides a consistent method for the production of transplantable sarcomas. The absence of sarcomas in the single sections taken from donor livers and multiple sections of similar livers not used for transplantation suggests that transplantability of these sarcoma cells is acquired very early in this neoplasm.