Neutrophils injure cultured skeletal myotubes.

The purpose of the study was to test the hypothesis that neutrophils can injure cultured skeletal myotubes. Human myotubes were grown and then cultured with human blood neutrophils. Myotube injury was quantitatively and qualitatively determined using a cytotoxicity (51Cr) assay and electron microscopy, respectively. For the 51Cr assay, neutrophils, under non-in vitro-stimulated and N-formylmethionyl-leucyl-phenylalanine (FMLP)-stimulated conditions, were cultured with myotubes at effector-to-target cell (E:T) ratios of 10, 30, and 50 for 6 h. Statistical analyses revealed that myotube injury was proportional to the E:T ratio and was greater in FMLP-stimulated conditions relative to non-in vitro-stimulated conditions. Transmission electron microscopy, using lanthanum as an extracellular tracer, revealed in cocultures a diffuse appearance of lanthanum in the cytoplasm of myotubes and a localized appearance within cytoplasmic vacuoles of myotubes. These observations and their absence in control cultures (myotubes only) suggest that neutrophils caused membrane rupture and increased myotube endocytosis, respectively. Myotube membrane blebs were prevalent in scanning and transmission electron micrographs of cultures consisting of neutrophils and myotubes (E:T ratio of 5) and were absent in control cultures. These data support the hypothesis that neutrophils can injure skeletal myotubes in vitro and may indicate that neutrophils exacerbate muscle injury and/or delay muscle regeneration in vivo.

Actin cytoskeletal function is spared, but apoptosis is increased, in WAS patient hematopoietic cells.

Mutations in the Wiskott-Aldrich syndrome protein (WASP) have been hypothesized to cause defective actin cytoskeletal function. This resultant dysfunction of the actin cytoskeleton has been implicated in the pathogenesis of Wiskott-Aldrich syndrome (WAS). In contrast, it was found that stimulated actin polymerization is kinetically normal in the hematopoietic lineages affected in WAS. It was also found that the actin cytoskeleton in WAS platelets is capable of producing the hallmark cytoarchitectural features associated with activation. Further analysis revealed accelerated cell death in WAS lymphocytes as evidenced by increased caspase-3 activity. This increased activity resulted in accelerated apoptosis of these cells. CD95 expression was also increased in these cells, suggesting an up-regulation in the FAS pathway in WAS lymphocytes. Additionally, inhibition of actin polymerization in lymphocytes using cytochalasin B did not accelerate apoptosis in these cells. This suggests that the accelerated apoptosis observed in WAS lymphocytes was not secondary to an underlying defect in actin polymerization caused by mutation of the WAS gene. These data indicate that WASP does not play a universal role in signaling actin polymerization, but does play a role in delaying cell death. Therefore, the principal consequence of mutations in the WAS gene is to accelerate lymphocyte apoptosis, potentially through up-regulation of the FAS-mediated cell death pathway. This accelerated apoptosis may ultimately give rise to the clinical manifestations observed in WAS. (Blood. 2000;95:1283-1292)

Chemoprevention of vinyl carbamate-induced lung tumors in strain A mice.

The ability of potential chemopreventive agents to prevent vinyl carbamate-induced lung tumors was determined in 2 different experiments. Female strain A mice administered intraperitoneally either a single injection of 60 mg/kg vinyl carbamate that induced 24.0 +/- 1.72 tumors/mouse at 24 weeks or 2 injections of 16 mg/kg vinyl carbamate each (32 mg/kg total dose) that induced 43.2 +/- 3.2 tumors/mouse at 20 weeks. Lung carcinomas were found as early as 16 weeks. Dexamethasone and piroxicam provided in the diet were found to significantly inhibit lung tumors induced by 60 mg/kg vinyl carbamate at 24 weeks whereas myo-inositol also provided in the diet, did not significantly inhibit tumor formation. In animals given 6 16-mg/kg doses of vinyl carbamate, tumor multiplicity was reduced roughly 25% by alpha-difluoromethylornithine and green tea and reduced 50% by dexamethasone and piroxicam. Combinations of these agents were also tested using a total dose of 32 mg/kg of vinyl carbamate. Although alpha-difluoromethylornithine and green tea did not result in a significant inhibition of lung tumor formation if used alone, the combination of alpha-difluoromethylornithine and green tea resulted in a significant reduction of tumor multiplicity. The combinations of alpha-difluoromethylornithine or green tea with either dexamethasone or piroxicam or the combination of dexamethasone and piroxicam did not decrease tumor multiplicity greater than achieved by dexamethasone and piroxicam alone. In summary, selected chemopreventive agents previously shown to inhibit lung tumors by other chemical carcinogens also inhibited vinyl carbamate-induced lung tumors.

Scanning electron microscopy of adult head lice (Pediculus humanus capitis) with focus on clinical implications.

BACKGROUND: The incidence of head lice infestations in North America is escalating with an estimated 12 million cases of head lice per year despite the existence of insecticidal therapies. OBJECTIVE: To evaluate certain characteristics of the human head louse, including their chitinous structure, nymphal developmental stages, legs with claw adaptations, antennae with sensory perceptions, and spiracles by which oxygen exchange occurs, for assessment of possible means to control the spread and growth of this insect. METHODS: Scanning electron-microscopic examination of head lice was performed. RESULTS: Newer treatments of head lice must acknowledge defense mechanisms that are based on anatomic structure and physiologic characteristics, details of louse transmission, and the insects' ability to 'resurrect' after sham death. CONCLUSIONS: An understanding of lice entomology is essential in the pursuit of novel means to control the lice epidemic.

Scanning electron microscopy of human head louse (Anoplura: Pediculidae) egg and its clinical ramifications.

Pediculosis affects more elementary school students than all other communicable childhood diseases combined, excluding the common cold. The current study using scanning electron microscopy visualized operculums through which developing embryos received oxygen, as well as fracture lines within the adherent sheath by which the egg is attached to the hair. Using microscopic techniques, including freeze-fracturing, the mode of attachment of the sheath to the hair follicle was observed, in addition to the existence of several inner membranes enclosing the embryo within the egg structure. The chemical nature of the sheath is also analyzed using electron dispersive x-ray analysis.

Strain differences in neointimal hyperplasia in the rat.

We performed an initial screen of 11 rat strains by use of a standard balloon injury to the left iliac artery to observe whether genetically determined differences existed in the development of neointimal hyperplasia. Neointimal hyperplasia was assayed 8 weeks after the vascular injury on coded microscopic sections. Statistically significant differences in the percentages of the vascular wall cross-sectional areas composed of intima (percentage intima) secondary to neointimal hyperplasia were noted among the different rat strains (P<0.02), with the Brown-Norway (BN), Dark Agouti, and Milan normotensive strain rats having the highest and the spontaneously hypertensive rats (SHR) having the lowest percentages of intima. In a separate experiment, F1 hybrids of SHRxBN strains and parental BN and SHR underwent the vascular injury, and the parental strains again showed a statistically significant difference from one another in the mean percentage of intima (P<0. 0001). The F1 hybrids showed an average percentage of intima intermediate between those of the parental strains. The average lumen size of the injured BN vessels were significantly smaller than that of the noninjured control vessels (P=0.044), but this significance disappeared when the circular areas of these vessels were calculated without taking neointimal growth into consideration (P=0.649). These results provide the groundwork for a genetic linkage analysis to identify the genes that influence the development of neointimal hyperplasia after vascular injury.

Infection by Rhodococcus equi in a patient with AIDS: histological appearance mimicking Whipple's disease and Mycobacterium avium-intracellulare infection.

Rhodococcus equi pneumonia with systemic dissemination is being reported increasingly in immunocompromised patients. This is the first case report of disseminated R equi infection with biopsy documented involvement of the large intestine. The patient was a 46 year old male with AIDS who was diagnosed with cavitating pneumonia involving the left lower lobe. R equi was isolated in culture from the blood and lung biopsies. Subsequently, the patient developed anaemia, diarrhoea, and occult blood in the stool. Colonoscopy revealed several colonic polyps. Histological examination of the colon biopsies showed extensive submucosal histiocytic infiltration with numerous Gram positive coccobacilli and PAS positive material in the histiocytes. Electron microscopy showed variably shaped intrahistiocytic organisms which were morphologically consistent with R equi in the specimen. Disseminated R equi infection may involve the lower gastrointestinal tract and produce inflammatory polyps with foamy macrophages which histologically resemble those seen in Whipple's disease and Mycobacterium avium-intracellulare infection.

Adrenocortical oncocytoma: two case reports and review of literature.

Oncocytomas of the adrenal gland are rare; only 9 cases are reported in the world literature. We report 2 new cases in which benign adrenal masses were detected during evaluation for microhematuria or flank pain. Subsequent to extirpation of the mass, pathologic examination established the diagnosis of adrenocortical oncocytoma.

Axon and ganglion cell injury in rabbits after percutaneous trigeminal balloon compression.

New Zealand white rabbits were used to determine whether the changes in the Vth cranial nerve sensory root after compression were associated with the loss of a specific subclass of Vth cranial nerve ganglion cells, the disappearance of a distinct subset of primary afferent terminals in Vth cranial nerve nucleus caudalis, and/or injury to a specific axonal fiber type. There was no significant difference in the size of surviving ganglion cells after Vth cranial nerve compression, as measured 2 to 3 months after injury (P > 0.5, n = 4). Densitometric analysis of the nerves of rabbits that survived > 2 months after compression showed no significant difference in the immunoreactivity of substance P and calcitonin gene-reactive protein between compressed and control sides (P > 0.1, n = 4). Fink-Heimer staining of the Vth cranial nerve subnucleus caudalis revealed that transganglionic degeneration was most dense in the deeper layers, which are the sites of termination of large myelinated fibers. Ultrastructural evaluation of the type of myelinated axons injured by Vth cranial nerve compression in rabbits killed 7, 14, 37, and 270 days after injury was studied, and morphometric analysis was performed. The frequency distribution of axon diameters was significantly different for injured and control areas. The injured areas had higher ratios of small (< 3-microns diameter) to large-diameter axons compared to control distribution. These data indicate that balloon compression results in loss of fibers from the Vth cranial nerve sensory root and extensive transganglionic degeneration in the Vth cranial nerve brain stem complex. Cell size measurements and immunocytochemical data suggest that there is no specific loss of small ganglion cells or fine-caliber primary afferents. These experiments suggest that balloon compression relieves trigeminal pain by injuring the myelinated axons involved in the sensory trigger to the pain.

Antioxidant enzyme gene transcription in copper-deficient rat liver.

Antioxidant enzymes, Cu/Zn- and Mn-superoxide dismutase, catalase, and glutathione peroxidase, constitute an important defense mechanism against cytotoxicity of reactive oxygen species. Copper is essential for the activity of Cu/Zn-superoxide dismutase. Oxidative stress, therefore, is expected in organs of rats fed copper-deficient diet due to reduced Cu/Zn-superoxide dismutase activity. Our previous studies have shown that the expression of antioxidant enzymes was altered in copper-deficient rat liver. The present report was undertaken to study further the transcription of these enzymes in liver nuclei of rats made copper-deficient for 4 weeks. While copper deficiency decreased the copper in liver by about 80%, it did not alter the copper content in liver nuclei. In spite of a 100% elevation in nuclear iron concentration, liver nuclei from copper-deficient rats showed normal appearance. The transcriptional rates for Cu/Zn-superoxide dismutase, glutathione peroxidase, and glyceraldehyde-3-phosphate dehydrogenase were not altered by dietary copper deprivation. In contrast, transcriptional rates for Mn-superoxide dismutase and beta-actin were increased but that for catalase was reduced in the nuclei isolated from the copper-deficient rat liver. These results suggest that oxidative stress, resulting from copper deficiency, differentially modulates the gene transcription for the antioxidant enzymes in rat liver.

Ki-ras mutations in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-initiated and butylated hydroxytoluene-promoted lung tumors in A/J mice.

The promotional effects of butylated hydroxytoluene (BHT) on lung tumorigenesis induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was evaluated in a two-stage model of lung tumorigenesis in the A/J mouse. Mice were treated in two separate experiments with 1.54 mmol/kg (9.1 mg/mouse) NNK, which induced an average of 8.4 and 9.1 tumors/mouse in the experiments. Animals fed a diet that contained 1 g/kg BHT after administration of the carcinogen in these two experiments demonstrated an increase of the tumor multiplicity by 63% and 43%. Multiplicity of forestomach tumors was not effected by BHT in the diet. No differences in lung tumor morphology were seen as a result of the promoting effect of BHT. Mutations in the Ki-ras oncogene from lung tumors induced by NNK (19 tumors) or by NNK plus a diet containing BHT (34 tumors) were characterized by polymerase chain reaction, single-strand conformational polymorphism, and direct sequencing. All 19 NNK-induced tumors not promoted with BHT contained activated Ki-ras genes with GC-->AT transitions at the second base of codon 12. Only 11 of 34 NNK-induced and BHT-promoted tumors (32%) had this characteristic Ki-ras alteration. These data suggest that the NNK-initiated mouse lung tumorigenesis pathway, which involves the specific mutation of the Ki-ras oncogene, is altered to a predominantly non-ras mechanism when these tumors are promoted by BHT in the diet.

Mutagenesis of the K-ras protooncogene in mouse lung tumors induced by N-ethyl-N-nitrosourea or N-nitrosodiethylamine.

The role of ras gene activation in the development of lung tumors induced by N-ethyl-N-nitrosourea (ENU) and N-nitrosodiethylamine (DEN) was evaluated in the A/J mouse, a strain susceptible to chemically induced lung tumors. DNAs isolated from both ENU- and DEN-induced lung tumors were screened for activating mutations in the K-ras gene by utilizing the polymerase chain reaction (PCR) and direct sequence analysis. Mutations in the K-ras gene were detected in 11 of 11 ENU-induced tumors and 23 of 28 DEN-induced tumors. In ENU-induced tumors, there were three GC----AT transitions in the second base of codon 12, and seven AT----GC transitions and one AT----TA transversion in the second base of codon 61. A similar spectrum of K-ras mutations was observed in DEN-induced lung tumors: five GC----AT transitions and two GC----TA transversions in the second base of codon 12, and sixteen AT----GC transitions at the second base of codon 61. Ninety-one percent (31/34) of the observed mutations are consistent with the formation of the promutagenic O4-ethylthymine and O6-ethylguanine adducts in DNA. Therefore, lung tumors from the A/J mouse induced by DEN and ENU could be initiated by the interaction of reactive metabolites with specific sites in the K-ras gene. This is the first clear example of activation of the K-ras gene by ethylating agents in a rodent lung tumor system.

Keratin expression in chemically induced mouse lung adenomas.

Chemically induced mouse lung tumors exhibit distinctive growth patterns, characterized by an alveolar or solid appearance, a papillary appearance, or a combination of the two. Lung tumors induced in strain A/J mice by either benzo(a)pyrene (BP) or by N-nitrosoethylurea (ENU) were examined for expression of low- and high-molecular-weight cytokeratins. Simple cytokeratins (low molecular weight) were found in all epithelial cells of the normal mouse lung and in all tumor types, whereas higher-molecular-weight cytokeratins were found only in normal bronchiolar cells and in papillary tumor cells. These data lend support to the hypothesis that chemically induced papillary lung tumors in strain A/J mice are derived from bronchiolar Clara cells.

A human bronchial epithelial cell strain with unusual in vitro growth potential which undergoes neoplastic transformation after SV40 T antigen gene transfection.

Bronchial epithelial cells were cultured from an individual with no evidence of malignant disease. These cells, designated HB56B, had a greatly extended in vitro life-span, being able to undergo 50 passages and 200 population doublings in contrast to the usual 3 to 4 passages and 20 to 30 population doublings characteristic of normal human bronchial epithelial cells. HB56B cells had karyotypic evidence of an amplified region on the short arm of chromosome II. Unlike normal bronchial epithelial cells, which undergo terminal squamous differentiation in vitro in response to fetal bovine serum, HB56B cells were only minimally affected by serum. These cells were readily established as an immortalized cell line, HB56B/5T, following transfection with a plasmid containing SV40 early region DNA. HB56B cells were non-tumorigenic in athymic nude mice, but HB56B/5T cells within a few passages of transfection with the SV40 plasmid formed tumors of which 28/37 regressed. HB56B cells may offer an experimental system for the study of proliferation, differentiation, and senescence control in human bronchial epithelial cells.

Carnitine deficiency associated with ornithine transcarbamylase deficiency.

An infant with X-linked recessive ornithine transcarbamylase deficiency is described who also had severe deficiency of plasma and liver carnitine during normoammonemic periods. Treatment with L-carnitine (100 mg/kg/day) for 12 months decreased the frequency of hospitalizations for hyperammonemia, although it did not alter his neurologic status. This report demonstrates that persistent carnitine deficiency may be present in patients with ornithine transcarbamylase deficiency even when plasma ammonia is normal. Carnitine evaluation and supplementation may be important in the treatment of patients with this metabolic disorder.

Glyceraldehyde-3-phosphate dehydrogenase and other enzymatic activity in normal mouse lung and in lung tumors.

The histogenesis of chemically induced mouse lung adenomas is currently being debated. Tumors induced by a variety of chemicals and in a number of different strains exhibit growth patterns having a solid/alveolar appearance, a papillary appearance, or a mixture of both. Ultrastructural observations suggest that solid tumors are derived from the alveolar type II pneumocyte and that papillary tumors arise from the bronchiolar Clara cell. However, recent immunocytochemical investigations have concluded that most mouse lung tumors are derived solely from the alveolar type II cell. Enzyme histochemical methods have previously been utilized to identify Clara cells in pulmonary cell isolates and also to characterize mouse lung tumors. This report demonstrates a difference in glyceraldehyde-3-phosphate dehydrogenase (G3PD) activity in type II pneumocytes and Clara cells. Solid tumors and type II cells appear to have a similar G3PD activity, and this activity is different from that observed in papillary tumors and bronchiolar cells. These findings support morphological evidence that suggests mouse lung tumors are phenotypically different and may arise from at least two different cells of origin.

Strain A/J mouse lung adenoma growth patterns vary when induced by different carcinogens.

The histogenesis of mouse lung adenomas is currently being investigated in several laboratories. Based upon studies of a limited number of carcinogens in different mouse strains, some investigators suggest that all lung adenomas in mice are derived from alveolar type II cells, whereas others suggest a Clara cell origin for a majority of the tumors. This report differs from previous investigations in that 12 different carcinogens were evaluated for the types of lung tumor growth patterns they induced in a single mouse strain (strain A mice). The carcinogens aflatoxin B1 (AFB1), benzo(a)pyrene (BP), 1,2-dimethylhydrazine (DMH), 3-methylcholanthrene (MCA), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and N-nitrosomethylurea (MNU) induced tumors with a predominantly solid/alveolar growth pattern, whereas N-nitrosodiethylamine (NDEA) induced predominantly papillary tumors. Most of the other carcinogens induced a higher proportion of lung tumors with the solid/alveolar growth pattern than with the papillary growth pattern; however, ratios between the 2 growth patterns varied. If, as suggested by others, solid tumors are derived from alveolar type II cells and papillary tumors from Clara cells, then carcinogens may differ with respect to their ability to transform one cell type or the other.

Comparative properties of untreated and N-nitrosobenzylmethyl-amine-transformed rat esophageal epithelial cell lines.

A culture system utilizing rat esophageal epithelial cells has been developed. Four normal and eight N-nitrosobenzylmethylamine-treated lines were compared with respect to chromosome number, anchorage-independent growth in agarose, and tumorigenic potential in syngeneic rats. All cell lines were aneuploid with nine in the near-tetraploid range and three in the near-diploid range. No relation between tumorigenic potential and chromosome number or structure was apparent. Similarly, anchorage-independent growth in agarose did not correlate with tumorigenic potential. Three of the 12 immortalized lines (two carcinogen-treated and 1 untreated) induced well-differentiated squamous cell carcinomas in syngeneic rats. These tumors had weak metastatic potentials suggesting that tumorigenic potential and metastatic ability are separately controlled. These cell lines will be useful for the investigation of factors involved in the conversion of immortalized rat esophageal epithelial cell lines to lines of high metastatic potential.

Metabolic studies of carnitine in a child with propionic acidemia.

Carnitine metabolism was studied in a 7-y-old boy with propionic acidemia due to an almost total deficiency of propionyl-CoA carboxylase. The initial diagnosis was made at 3 wk of age followed by numerous episodes of metabolic acidosis despite a low-content branch-chain amino acid diet containing supplemental biotin. Although clinically stable and in a nonacidotic state, the plasma concentration of total carnitine was normal (38.9 microM; normal = 46 +/- 10, mean +/- SD, n = 30) whereas free carnitine was decreased (5.7 microM; normal = 37 +/- 8) and short-chain acylcarnitines were increased (28.6 microM; normal = 5.7 +/- 3.5). Skeletal muscle and liver specimens obtained at open biopsy had low total and free carnitine contents and increased ratio of short-chain acylcarnitines to free carnitine. Short-chain acylcarnitine content was low in liver but increased in skeletal muscle. The liver contained fatty vacuoles, enlarged mitochondria with paracrystalline inclusions, and numerous peroxisomes whereas the skeletal muscle also had lipid vacuoles and an increase in number and size of mitochondria. A carnitine challenge test (100 mg L-carnitine/kg body wt via a gastrostomy tube) resulted in a peak plasma carnitine concentration at 120 min. With maintenance therapy of 100 mg L-carnitine/kg/day the plasma free carnitine remained relatively low, the plasma glycine concentration decreased, and urinary acylcarnitine excretion increased. This study demonstrates that the alterations in carnitine and its derivatives observed in plasma and urine reflect the same type of altered distribution in tissue and provides further data on the effects of L-carnitine therapy.