The role of laminin-1 in the modulation of radiation damage in endothelial cells and differentiation.

Microvascular dysfunction due to endothelial damage is often associated with the ionizing radiation used during cancer therapy. This radiation-induced capillary injury is a major factor in the inhibition of new vessel growth (angiogenesis) and in disease states such as radiation-induced pneumonitis and nephropathy. Many studies have examined the effects of radiation on endothelial cell function; however, little is known regarding the role the basement membrane plays in radiation-induced endothelial cell damage and angiogenesis. Therefore, we examined the effects of gamma radiation on aortic explants, and in vitro on three endothelial cell types (of artery, vein and capillary origin) irradiated with or without the basement membrane glycoprotein laminin-1. As expected, irradiation inhibited angiogenic sprouting of the aortic explants, endothelial cell proliferation, attachment, migration and differentiation in vitro in a dose-dependent manner. However, the effect of radiation on several of these processes in angiogenesis was reduced when the cells were irradiated on laminin-1. To further evaluate the effects of radiation on endothelial cells, we examined the expression of the vascular endothelial cell growth factor (VEGF) kinase domain region receptor in endothelial cells irradiated in the presence and absence of laminin-1. In endothelial cells irradiated on laminin-1, KDR expression increased 2.5-fold over control levels. Therefore, although radiation has a dose-dependent inhibitory effect on processes associated with angiogenesis in vitro, the presence of the basement membrane glycoprotein laminin-1 during irradiation decreases these effects.

Cytokeratin positive T cell malignant lymphoma.

A high grade T cell malignant lymphoma is described in which weak staining of tumour cells for leucocyte common antigen and T cell markers coexisted with strong positive cytoplasmic staining with the anticytokeratin marker CAM 5.2. This is the first report of non-CD30 positive T cell lymphoma showing cytokeratin positivity. On ultrastructural examination there was no evidence of epithelial differentiation or of accumulation of cytokeratin-type intermediate filaments. The case adds to the range of malignant lymphomas which can, on rare occasions, show cytokeratin positivity. Pathologists should be aware of this potential diagnostic pitfall if inappropriate investigations and therapeutic regimens are to be avoided.

Marrow stromal cells as a source of progenitor cells for nonhematopoietic tissues in transgenic mice with a phenotype of osteogenesis imperfecta.

Marrow stromal cells from wild-type mice were infused into transgenic mice that had a phenotype of fragile bones resembling osteogenesis imperfecta because they expressed a human minigene for type I collagen. In mice that were irradiated with potentially lethal levels (700 cGy) or sublethal levels (350 cGy), DNA from the donor marrow stromal cells was detected consistently in marrow, bone, cartilage, and lung either 1 or 2.5 mo after the infusions. The DNA also was detected but less frequently in the spleen, brain, and skin. There was a small but statistically significant increase in both collagen content and mineral content of bone 1 mo after the infusion. Similar results were obtained with infusion of relatively large amounts of wild-type whole marrow cells into the transgenic mice. In experiments in which male marrow stromal cells were infused into a female osteogenesis imperfecta-transgenic mouse, fluorescense in situ hybridization assays for the Y chromosome indicated that, after 2.5 mo, donor male cells accounted for 4-19% of the fibroblasts or fibroblast-like cells obtained in primary cultures of the lung, calvaria, cartilage, long bone, tail, and skin. In a parallel experiment in which whole marrow cells from a male mouse were infused into a female immunodeficient rag-2 mouse, donor male cells accounted for 4-6% of the fibroblasts or fibroblast-like cells in primary cultures. The results support previous suggestions that marrow stromal cells or related cells in marrow serve as a source for continual renewal of cells in a number of nonhematopoietic tissues.

A regional audit of perinatal and infant autopsies in Northern Ireland.

OBJECTIVE: To investigate the rate and quality of perinatal/infant autopsies and their contribution to the final diagnosis. METHODS: The anonymised reports of autopsies performed on 174 of the 367 cases reported to the Northern Ireland Regional Confidential Enquiry into Stillbirths and Deaths in Infancy (CESDI) coordinator in 1993 were reviewed. They were scored using a modification of the CESDI Pathology Audit Form 93 and, based on the score obtained, ascrined to one of three groups: good, adequate or inadequate. Based on the information obtained, they were also assessed as providing diagnostic, confirmatory, additional or no diagnostic data. The pre-autopsy clinical extended Wigglesworth classification was compared with that based on autopsy findings. RESULTS: The autopsy rate was 47.4% and included 18 late fetal losses, 70 stillbirths, 57 neonatal deaths and 29 post neonatal deaths. The Regional Paediatric Pathology Centre performed 34.5% of the autopsies. Of the total number of autopsies, 46.6% failed to reach an adequate standard. Only 4.9% of the inadequate autopsies were performed in the Regional Centre. The Wigglesworth classification was altered in 20.7% of cases following autopsy. The autopsy findings of 49 cases were diagnostic, 75 confirmatory, 23 yielded additional information and 27 were useful in only a negative sense. CONCLUSION: The autopsy rate for this region is well below the recommended level of 75%. With the exception of the Regional Centre, the quality of the perinatal/infant autopsy did not reach the standard suggested in the CESDI Pathology Audit form 93. Despite this the autopsy yielded valuable diagnostic, confirmatory or additional findings in 84.5% and resulted in alteration to the pre-autopsy Wigglesworth classification in 20.7%.

Mammalian cells adapted to growth at pH 6.7 have elevated HSP27 levels and are resistant to cisplatin.

HSP27 levels are elevated in two Chinese hamster cell lines and in a human melanoma cell line adapted to growth at pH 6.7. The level of HSP72 is elevated in the melanoma cell line but not in the hamster cell lines adapted to growth at pH 6.7. HSC73 levels are not elevated in any of the adapted cell lines. Low pH adapted cells from all cell lines are resistant to cisplatin. It is proposed that elevated HSP27 levels in low pH-adapted cells may play a role in resistance to hyperthermia and resistance to cisplatin.

Extralobar sequestration and type II congenital cystic adenomatoid malformation in an infant with congenital diaphragmatic hernia.

This case report describes an infra-diaphragmatic sequestrated type II congenital cystic adenomatoid malformation (CCAM) containing striated muscle fibers in the stroma occurring in association with an extra-lobar sequestration (ELS) and a congenital diaphragmatic hernia in a female neonate. The classification and pathogenesis of CCAM and ELS are reviewed and the relationships between these lesions discussed.

Infantile haemangiopericytoma of the hand.

Infantile haemangiopericytomas (IHP) are rare subcutaneous tumours arising from pericytes. Clinically they are difficult to diagnose and pathologically they appear to be locally invasive, but they have a better prognosis than adult haemangiopericytomas. We report a case of IHP affecting the hand of a 7-week-old child that required urgent treatment.

Paraganglioma: an unusual tumour of the parathyroid gland.

A case of paraganglioma arising within a parathyroid gland is reported. The lesion was an incidental finding in a block dissection of neck performed for squamous carcinoma of the pharynx. A well-circumscribed lesion, exhibiting the characteristic pathological features of a paraganglioma, was embedded within the right inferior parathyroid gland. Due to its location, the chief histological differential diagnosis was an unusual variant of parathyroid adenoma. Immunohistochemistry and electron microscopy assisted in reaching a diagnosis. This, as far as we are aware, is the first reported case of a paraganglioma of the parathyroid gland.

The development and magnitude of thermotolerance during chronic hyperthermia in murine granulocyte-macrophage progenitors: II.

We have previously reported that murine granulocyte-macrophage progenitors (CFU-GM) are capable of developing thermotolerance during chronic hyperthermia at temperatures of 40 to 42 degrees C. However, a differential profile of intrinsic thermal response and, in particular, the capability of developing thermotolerance during chronic heating was identified between CFU-GM and macrophage colony-forming units (CFU-M) stimulated respectively, by lung conditioned medium (LCM) and L929 cell conditioned medium (CCM). Nucleated marrow cells treated in vitro were cultured in McCoy's 5A medium plus 15% fetal bovine serum (FBS) in semisolid agar with 10% of CCM. Two different treatment protocols were used in this study to determine the kinetics of thermotolerance in CFU-M: (1) nucleated marrow from mouse tibia and femur were chronically heated in vitro at temperatures of 40, 41 and 42 degrees C (up to 480 min) or (2) nucleated marrow cells were heated over a period of 90 min stepwise from 37 to 42 degrees C, at a heating rate of 0.056 degrees C/min, before exposure to 42 degrees C. The amount of thermotolerance developed was analysed at various times after chronic incubation at 40-42 degrees C by a challenge with 15 min at 44 degrees C. In contrast to CFU-GM, the surviving fraction of CFU-M heated with 15 min at 44 degrees C did not increase during chronic hyperthermia at 40 degrees C for up to 480 min indicating failure to develop thermotolerance. However, CFU-M were able to develop thermotolerance during prolonged incubation at 41 and 42 degrees C, although to a much less extent than observed in CFU-GM. In other words, there was much less development of thermotolerance in murine CFU-M compared to that in CFU-GM. Furthermore, a slow temperature transit from 37 to 42 degrees C over 90 min before exposure to 42 degrees C induced CFU-M to develop thermotolerance. The thermotolerance ratio (TTR, the ratio of the surviving fraction at maximum tolerance versus normotolerance) increased from a maximum of 3.5 after 180 min at 42 degrees C (no warm-up) to a maximum of 4.1 after 60 min at 42 degrees C when the cells received a slow warm-up to 42 degrees C. This implies that in the murine bone marrow granulocyte/macrophage lineage, CFU-M does not normally develop thermotolerance during hyperthermia and that the colony forming unit-granulocyte (CFU-G) and CFU-GM play a more critical role than CFU-M in the initiation and promotion of thermotolerance during chronic hyperthermia. However, in a situation that simulates the slow heat-up used clinically in wholebody hyperthermia, e.g., the 90 min slow warm-up from 37 to 42 degrees C, stimulated CFU-M to develop greater thermotolerance more rapidly than during rapid heating.

The development and magnitude of thermotolerance during chronic hyperthermia in murine bone marrow granulocyte-macrophage progenitors: I.

Murine bone marrow granulocyte-macrophage progenitors (CFU-GM) are capable of developing thermotolerance during exposure to temperatures < 42.5 degrees C. Bone marrow from the tibia and femora was heated to 40-42 degrees C (i.e. chronic hyperthermia), and challenged immediately with 15 min at 44 degrees C at regular intervals during treatment (step-up heating). CFU-GM were heated and cultured in McCoy's 5A medium + 15% FBS (fetal bovine serum) and lung-conditioned medium (source of colony stimulating factor) in semisolid agar. The kinetics of thermotolerance development and decay, and the magnitude of the thermotolerance during chronic heating with temperatures of 40-41.5 degrees C were similar. Survival increased rapidly to a maxima by approximately 120 min of hyperthermia (temperatures of 40-41.5 degrees C) and thereafter decreased with a slope similar to the controls. Normalization for cell killing by chronic hyperthermia that occurred during "step-up' heating permitted analysis of thermotolerance in the surviving cells. The surviving fraction after 15 min at 44 degrees C, during incubation at 40, 41 and 41.5 degrees C increased from 0.13 to maxima of 0.56 +/- 0.04, 0.71 +/- 0.03 and 0.82 +/- 0.03 respectively, by 150 min and did not decrease for up to 480 min during chronic hyperthermia. The surviving fraction after 15 min at 44 degrees C during incubation at 42 degrees C increased more slowly than during incubations at 40-41.5 degrees C. The survival of thermotolerant cells after exposure to 15 min at 44 degrees C during 42 degrees C chronic hyperthermia was maximal at 0.87 +/- 0.08 by 120 min and then decreased after approximately 150 min of exposure to 42 degrees C. The thermotolerance ratios (TTR's) were 4.0, 5.4, 6.7 and 6.9 for temperatures of 40, 41, 41.5 and 42 degrees C respectively. The results suggest that chronic hyperthermia temperatures (i.e. 40-42 degrees C) induce rapid thermotolerance development in CFU-GM during the thermal exposure and protect this normal marrow progenitor during whole body hyperthermia or ex vivo purging of leukaemic cells.

Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice.

Cells from transgenic mice expressing a human mini-gene for collagen I were used as markers to follow the fate of mesenchymal precursor cells from marrow that were partially enriched by adherence to plastic, expanded in culture, and then injected into irradiated mice. Sensitive PCR assays for the marker collagen I gene indicated that few of the donor cells were present in the recipient mice after 1 week, but 1-5 months later, the donor cells accounted for 1.5-12% of the cells in bone, cartilage, and lung in addition to marrow and spleen. A PCR in situ assay on lung indicated that the donor cells diffusely populated the parenchyma, and reverse transcription-PCR assays indicated that the marker collagen I gene was expressed in a tissue-specific manner. The results, therefore, demonstrated that mesenchymal precursor cells from marrow that are expanded in culture can serve as long-lasting precursors for mesenchymal cells in bone, cartilage, and lung. They suggest that cells may be particularly attractive targets for gene therapy ex vivo.

Thermal response and hyperthermic radiosensitization of scid mouse bone marrow CFU-C.

PURPOSE: Scid mice are severely immunodeficient as a result of a defective recombinase system. Mice with the scid mutation have been shown to have an increased sensitivity to ionizing radiation, presumably as a result of an inability to repair DNA damage. Little is known of the impact of this mutation on the thermal response and on hyperthermic radiosensitization. This investigation established the thermal response (42-44 degrees C), patterns of thermotolerance development, and the impact of hyperthermia (60 min at 40 degrees C or 42 degrees C) on the radiation response of bone marrow colony forming unit-culture cells (CFU-C) in scid mice. METHODS AND MATERIALS: Anesthetized scid mice (pentobarbital, 90 mg/kg) were killed by cervical dislocation and the nucleated marrow obtained from both tibia and femora by passing 2 ml of cold McCoy's 5A medium supplemented with 15% fetal bovine serum through each bone. Single cell suspensions of nucleated marrow were heated in 12 x 75 mm sterile tissue culture tubes at a concentration of approximately 5 x 10(6) cells/ml. Radiation, when used, was delivered immediately prior to hyperthermia by a 137Cs irradiator (dose rate of 1.20 Gy/min). Colony forming unit-culture were cultured in semisolid agar in the presence of colony stimulating factor (conditioned medium from L929 cells) for 7 days. RESULTS: The slope of the radiation dose-response curve for CFU-C in scid mice was biphasic, the Dos (+/- SE) were 0.29 +/- 0.03 Gy and 1.09 +/- 0.20 Gy, respectively. The Dos of the radiation dose-response curve for wild type marrow from CB-17 and Balb/c mice were 1.28 +/- 0.05 Gy and 1.47 +/- 0.15 Gy, respectively. The Dos of the hyperthermia dose-response curves for scid mice were 75 +/- 5, 10 +/- 1.4, and 4 +/- 0.2 min, respectively, for temperatures of 42 degrees, 43 degrees, and 44 degrees C. Thermotolerance development at 37 degrees C increased to a maximum at approximately 240 min after acute hyperthermia (15 min at 44 degrees C) and thereafter, decreased to control levels within 15 h. Thermotolerance did not develop in scid CFU-C during chronic hyperthermia at temperatures < 42.5 degrees C. Hyperthermia (60 min at 40 degrees or 42 degrees C) immediately after ionizing radiation did not significantly alter the terminal slope of the radiation dose-response curve of scid CFU-C (Do = 1.28 +/- 0.08 Gy). By contrast, hyperthermia following radiation of wild type CFU-C resulted in a decrease in the Do from 1.47 +/- 0.05 Gy (Balb/c, rad only) to 1.31 +/- 0.08 or 1.06 +/- 0.18 Gy for 60 min at 40 degrees or 42 degrees C, respectively. CONCLUSION: These results show that the thermal response and the pattern of thermotolerance development of scid CFU-C were similar to that of wild type Balb/c CFU-C, but that hyperthermia given immediately after ionizing radiation did not alter the radiation response of scid CFU-C. The scid mutation does not increase hyperthermic sensitivity or change the pattern of thermotolerance development of scid mouse CFU-C, implying that the scid mutation is not involved with thermal response, but does render the already radiation-sensitive scid cells incapable of thermal radiosensitization.

Varicella zoster gastritis in a bone marrow transplant recipient.

A case is reported of a patient who had previously undergone autologous bone marrow transplantation for recurrent Hodgkin's disease. The patient developed a generalised vesicular skin eruption. The clinical diagnosis was of disseminated shingles. Herpes viral particles were identified within the vesicular fluid by electron microscopy and using a specific monoclonal antibody to varicella zoster virus (VZV), positive immunofluorescence was detected in scrapings from the base of a vesicle. Gastroscopy and biopsy were performed because of severe abdominal pain and vomiting. The histological features were of non-specific active inflammation. Despite the histological absence of viral inclusions electron microscopy of the gastric biopsy revealed the presence of intranuclear herpes viral particles with a diameter of 90-100 nm. VZV specific DNA was detected by the polymerase chain reaction in the gastric biopsy extract. The patient was treated with acyclovir and made a full recovery.

Surfactant replacement therapy in preterm neonates: a comparison of postmortem pulmonary histology in treated and untreated infants.

New histological lesions have been reported in the lungs of preterm neonates treated with surfactant for respiratory distress syndrome (RDS). Globular deposits of hyaline maternal in parenchymal air spaces, absence of hyaline membranes, and increased interstitial cellularity and edema without associated fibrosis have been described. Fifteen histological findings were assessed in the lung pathology of 76 infants with RDS from three study groups. Group I (24 infants) died in the presurfactant era (before 1982), group II (26 infants) died despite having surfactant treatment, and group III (26 infants) were either untreated controls or did not receive surfactant for other reasons. The three groups were comparable in respect of sex and survival time. All infants were 34 weeks of gestation or less. Infants with a significant congenital abnormality or pulmonary hypoplasia were excluded. The 76 cases were assessed independently and "blindly" by two pathologists. The histological findings assessed were alveolar collapse; epithelial necrosis, proliferation, and metaplasia; hyaline membranes; dilated lymphatics; pulmonary interstitial emphysema; interstitial edema, inflammation, and fibrosis; arteriolar muscular hyperplasia; interstitial and intra alveolar hemorrhage; massive pulmonary hemorrhage; and pneumonia. No significant differences were found in any of the histological findings between the three groups. The hyaline membranes seen in the surfactant-treated infants were identical to those in the untreated lungs and were of the characteristic linear type. Interstitial fibrosis, inflammation, and edema were present in all three groups. It has also been suggested that surfactant therapy protects preterm infants from interstitial hemorrhage but predisposes them to intra-alveolar hemorrhage. No significant difference in the incidence of intra-alveolar and interstitial hemorrhage in the three groups was identified.

Influence of biological response modifiers: measurement of tumor blood flow and temperature.

Human recombinant interferon-alpha-2b (IFN) is known to inhibit growth of both normal and tumor cells and to stimulate immune effector cell function. We have previously shown that IFN and other biological response modifiers augment accumulation of radiolabeled antibodies in tumors. This investigation demonstrates that 30 min post i.m. administration of IFN significantly (p < 0.01) enhances tumor perfusion in tumor-bearing mice and persists for a much longer time than in normal tissue, which, in part, may contribute to the enhanced tumor uptake of radiolabeled monoclonal antibodies within 1.5 h of their administration. Histological examinations of tumors obtained from animals receiving IFN 72 h previously did not show changes in inflammatory cells. Our investigation shows that laser Doppler flowmetry and color Doppler imaging can provide an excellent means of measuring tumor perfusion changes in small living animals where radioactive tracers cannot be used.

Fatal inhalation injury following an industrial accident involving acetic anhydride.

A 22-year-old man sustained 35 per cent burns to his skin and an inhalation injury in an industrial accident involving acetic anhydride. Although the skin burns healed following irrigation and conservative treatment, the inhalation injury proved fatal.

Congenital heart disease and urinary tract abnormalities in two siblings with DOOR syndrome.

The acronym DOOR was first used by Cantwell in 1975 to describe a syndrome comprising sensorineural deafness, osteodystrophy, onychodystrophy, and mental retardation. To date, 16 cases of the syndrome have been documented in the literature. We present two sisters who died in early infancy with the clinical features of DOOR syndrome, both of whom in addition had cardiac defects and urinary tract abnormalities. Both infants had the classical clinical features of sensorineural deafness, seizures, hypoplastic nails, finger-like thumbs, and the characteristic facies of the syndrome. Autopsy in each case revealed the additional findings of a membranous ventricular septal defect and a septum secundum atrial septal defect. The first child had left-sided hydronephrosis and hydroureter, and the second sibling had bilateral hydronephrosis, hydroureter, and dilatation of the bladder. Congenital heart disease and renal abnormalities have not to our knowledge been previously described in association with the DOOR syndrome.