Culture conditions affect photoreactivating enzyme levels in human fibroblasts

Photoreactivation of pyrimidine dimers in mammalian cells occurs under our experimental conditions but has not been observed under conditions used by others. We have tested three possible differences in experimental procedures including dimer separation and analysis methods, illumination conditions and cell culture techniques. We show that out methods of dimer separation and analysis indeed measure cis-syn pyrimidine dimers and give results in quantitative agreement with the methods of others. We find that while light pre-illumination of fibroblasts from the xeroderma pigmentosum line XP12BE or of normal cells does not affect the cellular capacity for dimer photoreactivation. However, we show that cell culture conditions can affect photoreactivating enzyme levels and thus, cellular dimer photoreactivation capacity. Cells grown in Eagle's minimal essential medium (supplemented with 15% fetal bovine serum) contain very low levels of photoreactivating enzyme and cannot photoreactivate dimers in their DNA; however, companion cultures maintained in Dulbecco's modified Eagle's minimal medium do contain photoreactivating enzyme and can photoreactivate cellular dimers.     

Respiratory changes associated with rapid eye movements in normo- and hypercapnia during sleep

Rapid eye movements during rapid-eye-movement (REM) sleep are associated with rapid, shallow breathing. We wanted to know whether this effect persisted during increased respiratory drive by CO2. In eight healthy subjects, we recorded electroencephalographic, electrooculographic, and electromyographic signals, ventilation, and end-tidal PCO2 during the night. Inspiratory PCO2 was changed to increase end-tidal PCO2 by 3 and 6 Torr. During normocapnia, rapid eye movements were associated with a decrease in total breath time by -0.71 +/- 0.19 (SE) s (P < 0.05) because of shortened expiratory time (-0.52 +/- 0.08 s, P < 0.001) and with a reduced tidal volume (-89 +/- 27 ml, P < 0.05) because of decreased rib cage contribution (-75 +/- 18 ml, P < 0.05). Abdominal (-11 +/- 16 ml, P = 0.52) and minute ventilation (-0.09 +/- 0.21 ml/min, P = 0.66) did not change. In hypercapnia, however, rapid eye movements were associated with a further shortening of total breath time. Abdominal breathing was also inhibited (-79 +/- 23 ml, P < 0.05), leading to a stronger inhibition of tidal volume and minute ventilation (-1.84 +/- 0.54 l/min, P < 0.05). We conclude that REM-associated respiratory changes are even more pronounced during hypercapnia because of additional inhibition of abdominal breathing. This may contribute to the reduction of the hypercapnic ventilatory response during REM sleep.     

Trimidox-mediated morphological changes during erythroid differentiation is associated with the stimulation of hemoglobin and F-cell production in human K562 cells

Previous studies have demonstrated that stimulation of the ventral hippocampal (VH) formation (including the ventral CA1 and subicular areas) elicits increased locomotor activity in rats. The locomotor-activating effects of VH stimulation have been hypothesized to be mediated via hippocampal output to cortical and subcortical dopamine (DA) systems. This study examined whether increased locomotor activity produced by VH stimulation was blocked by pretreatment with a DA receptor antagonist, and whether DA metabolism in subdivisions of the nucleus accumbens, caudate-putamen, and prefrontal cortex was elevated by VH stimulation. Stimulation of the VH (defined as the ventral CA1 and its borders, ventral subiculum, and entorhinal cortex) with the cholinergic agonist carbachol was found to elevate locomotor activity, while pretreatment with the D2 receptor antagonist haloperidol blocked this effect. Stimulation of the VH did not alter DA metabolism (i.e., ratio of the DA metabolites DOPAC or HVA/DA) in any of the brain regions studied. These results indicate that the increased locomotor activity elicited by VH stimulation is not associated with dramatic increases in DA metabolism, but that it does require tonic activation of D2 receptors.     

Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis

Interleukin 1beta-converting enzyme-like proteases (caspases) are crucial components of cell death pathways. Among the caspases identified, caspase-3 stands out because it is commonly activated by numerous death signals and cleaves a variety of important cellular proteins. Studies in caspase-3 knock-out mice have shown that this protease is essential for brain development. To investigate the requirement for caspase-3 in apoptosis, we took advantage of the MCF-7 breast carcinoma cell line, which we show here has lost caspase-3 owing to a 47-base pair deletion within exon 3 of the CASP-3 gene. This deletion results in the skipping of exon 3 during pre-mRNA splicing, thereby abrogating translation of the CASP-3 mRNA. Although MCF-7 cells were still sensitive to tumor necrosis factor (TNF)- or staurosporine-induced apoptosis, no DNA fragmentation was observed. In addition, MCF-7 cells undergoing cell death did not display some of the distinct morphological features typical of apoptotic cells such as shrinkage and blebbing. Introduction of the CASP-3 gene into MCF-7 cells resulted in DNA fragmentation and cellular blebbing following TNF treatment. These results indicate that although caspase-3 is not essential for TNF- or staurosporine-induced apoptosis, it is required for DNA fragmentation and some of the typical morphological changes of cells undergoing apoptosis.     

Prevalence of morphological changes in the surfaces of the temporomandibular joint disc associated with internal derangement

PURPOSE: The purpose of this study was to determine the prevalence of morphological changes in the superior and inferior surfaces in the temporomandibular joint (TMJ) disc and relate them to disc displacement. MATERIALS AND METHODS: Thirty TMJs obtained from fresh cadavers were studied. The TMJs were dissected, and the superior and inferior surfaces of the disc were inspected and classified as intact, irregular, or perforated. These findings were corrolated to the position of the disc. RESULTS: There was a greater prevalence of morphologic changes in the inferior (57%) than in the superior surface (17%) of the disc (P < .001). This was found for joints both with normal disc position and those with disc displacement. There was no relationship between surface irregularities of the inferior surface and the position of the disc. Perforation was seen more frequently in joints with disc displacement than in those with normal disc position. CONCLUSION: Morphologic changes in the inferior surface of the TMJ disc are more prevalent than those in the superior surface, but this is not related to disc position. However, this needs to be considered when doing arthroscopy of only the superior joint compartment.     

Leukoregulin induction of protein expression in human orbital fibroblasts: evidence for anatomical site-restricted cytokine-target cell interactions

The molecular basis for the profound inflammatory response and the accumulation of hyaluronan in orbital connective tissues seen in thyroid-associated ophthalmopathy is unknown. Moreover, the link between the orbital manifestations of Graves' disease and those in the pretibial skin, localized dermopathy, has yet to be established. We have reported recently that leukoregulin, an activated T lymphocyte-derived cytokine, dramatically induces hyaluronan synthesis and prostaglandin-endoperoxide H synthase 2 in human orbital fibroblasts in culture. In the current studies, utilizing giant two-dimensional gel electrophoresis, we find that orbital fibroblasts express constitutively a protein profile that distinguishes them from skin fibroblasts derived from the abdominal wall and from the pretibium. We further demonstrate that leukoregulin, when present in culture medium for 16 hr, up-regulates a set of orbital fibroblast proteins not present in untreated cultures or in fibroblasts from the abdominal wall. However, some of the same protein inductions are present in the pretibial fibroblasts. These leukoregulin-induced changes in protein expression are completely blocked by dexamethasone (10 nM). Our findings are the first to identify proteins that appear to be expressed and differentially regulated in an anatomical site-restricted manner in orbital and pretibial fibroblasts and seem to establish a molecular link between fibroblasts from the orbit and those in pretibial skin.     

Parenteral versus enteral nutrition: morphological changes in human adult intestinal mucosa

In animal experiments total parenteral nutrition induces an atrophy of the small intestinal mucosa. In humans morphological data are few and controversial. Therefore, the aim of this study was to investigate the effect of parenteral nutrition on the intestinal mucosa of human adults. For this purpose samples of the proximal jejunum of a) patients with chronic pancreatitis receiving total parenteral nutrition as presurgical treatment, b) enterally nourished patients without (controls) and c) with chronic pancreatitis were compared using light and scanning electron microscopy. Statistical differences were assessed applying computer-assisted morphometry. The results demonstrated that the thickness of the jejunal mucosa decreased already in enterally nourished patients with chronic pancreatitis. However, after total parenteral nutrition the decrease (atrophy) was enhanced due to a strong reduction in villus height albeit the crypt length increased. In addition, scanning electron microscopy revealed distinctive changes in mucosal surface pattern, whereby finger-like villi were replaced by leaf-like villi and by long, winding bifurcating ridges. Cell shedding was absent. In conclusion, total parenteral nutrition in humans induces 1) an atrophy and 2) a remodelling of the intestinal mucosa (epithelium and lamina propria) with a decrease in the absorbing surface. These alterations involve both cell proliferation and cell shedding. The response of the mucosa to parenteral nutrition is immediate and the effect of the treatment in bringing about morphological alterations is more efficacious at the beginning than in the successive period. The basic disorder (chronic pancreatitis) of the patients nourished parenterally contributes to mucosal atrophy, but not to remodelling.     

A rapid and sensitive radioimmunoassay for the detection of human cytomegalovirus binding and infection of human fibroblasts

A rapid and sensitive radioimmunoassay for the quantitation of HCMV binding and infection of human fibroblasts (HFF) was developed. The protocol involves the use of a monoclonal antibody (27-156) reactive with HCMV gB (alpha-gB), followed by an 125I-labeled second antibody to mouse IgG. Antibody to gB bound specifically to HFF inoculated with HCMV when compared to sham inoculated cells or cells inoculated with HSV (strain KOS). Antibody to gB also bound to HFF infected with HCMV 48 h prior to assay. The binding of antibody to HFF inoculated with HCMV was found to be dependent on antibody concentration and to demonstrate saturable kinetics. Moreover, antibody binding was directly dependent on the concentration of the virus inoculum, using either conventional viral preparations or gradient purified HCMV. The binding of antibody to HFF inoculated with HCMV at 4 degrees C was found to be dependent on antibody concentration and to demonstrate saturable kinetics. Displacement of HCMV binding to HFF with the proteoglycan heparin sulfate could be detected, thus allowing for competitive binding studies. This binding assay allows for the relative quantitation of HCMV binding to cells and will be useful for examining the early events of cell-viral interactions.     

Plasma levels of arginine vasopressin elevated in patients with major depression

Mentally healthy subjects show increased plasma concentrations of the neuropeptides, arginine vasopressin (AVP) and oxytocin (OT), under conditions of stress, but data are lacking about plasma concentrations of AVP and OT in patients with major depression. We thus assessed plasma concentrations of AVP and OT in patients with major depression (n = 52) and healthy controls (n = 37). Mean plasma AVP concentrations were higher in the group of depressed patients than in controls. A subgroup of 16 patients showed very high levels of plasma AVP, but no other feature differentiating this subgroup from the other patients was found. In-patients showed higher plasma AVP levels than out-patients, and melancholic patients had higher plasma AVP levels than did nonmelancholic patients. Plasma AVP levels were slightly related to psychomotor retardation and significantly inversely to neuroticism. Patients' plasma OT concentrations had a wider range than in controls. AVP and AVP-mediated functions may be a factor in the clinical picture of depression, possibly by influencing the activity of the hypothalamic-pituitary-adrenal axis.     

Ocular vascular endothelial growth factor levels in diabetic rats are elevated before observable retinal proliferative changes

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor. VEGF levels in ocular tissue of 6-, 12-, 18- and 28-week-old Goto-Kakizaki (GK) rats, a well-known model of non-insulin-dependent diabetes, were evaluated by highly sensitive ELISA. VEGF concentrations in the GK rat as well as in non-diabetic Wistar rat significantly decreased from the age of 6 weeks to 18 weeks. However, although VEGF concentrations in the Wistar rat continued to fall significantly from 18 to 28 weeks of age, the levels were maintained between 18 and 28 weeks of age in GK rats. Levels were significantly different between the GK and Wistar rats at 28 weeks of age. Results of immunohistochemical studies of the eyes of Wistar and GK rats at 28 weeks of age suggest diffuse distribution of this cytokine in cells of neural origin. Weak to moderate VEGF immunoreactivity was exhibited mainly in the ganglion cell layer, inner plexiform layer and inner/outer nuclear layers in rats with and without diabetes. However, in the retinal optic nerve fiber layer, retinal pigment epithelium and choroid, strong VEGF immunoreactivity was noted only in the GK rat. In conclusion, increased VEGF production in certain ocular tissue, similar to that in humans, is observed quite early, at least before the appearance of observable retinal changes in the diabetic GK rat. This also suggests that the GK rat can be used as a model of initial or latent phase diabetic retinopathy.     

Serum testosterone levels are not elevated in patients with ankylosing spondylitis

OBJECTIVE: Studies in patients with ankylosing spondylitis (AS) describe slightly elevated serum testosterone levels, but these studies were not properly controlled for possible confounders. METHODS: In a case-control study serum levels of sex steroids, luteinizing hormone, and sex hormone binding globulin (SHGB) were measured in patients with AS and in age and sex matched controls. The body mass index, smoking status, use of alcohol, and fat intake were recorded. RESULTS: Testosterone levels measured in serum extracts did not differ in 50 male patients with AS compared to controls (mean +/- SD 16 +/- 4 vs 15 +/- 5 nmol/l, respectively; p = 0.54). In unextracted serum, however, male patients showed elevated testosterone (p < 0.001) and dehydroepiandrosterone sulfate levels (p = 0.003), even after controlling for confounders (p < 0.001). One of 10 female patients had an elevated testosterone level in unextracted serum. The 17 male users and one of the 2 female users of phenylbutazone had the highest testosterone levels in unextracted serum, and all showed a significant decline after extraction. Serum levels of other sex steroids, luteinizing hormone, and SHGB did not differ significantly between patients and controls. CONCLUSION: Serum testosterone levels are not elevated in male patients with AS. Spuriously elevated testosterone levels in unextracted serum might be related to the use of phenylbutazone in our patient sample.     

Significance of elevated IgG anticardiolipin antibody levels in patients with Budd-Chiari syndrome

OBJECT: Congenital spinal hamartomas are defined as tumors of well-differentiated mature elements situated in an abnormal location. In this report, the authors document the clinical and pathological features of spinal hamartomas in 10 patients. METHODS: Ten patients presented with midline dorsal malformations at birth, initially diagnosed as teratomas or myelomeningoceles. The locations of the masses were variable: two were located in the thoracic region, four at the thoracolumbar junction, two in the lumbar region, one at the lumbosacral junction, and one in the sacral region. The results of the neurological examination were normal in nine patients. All but one mass had intact skin and seven had palpable bone components. Neuroimaging studies revealed widening of the spinal canal, heterotopic bone located dorsally in some patients, and varying degrees of involvement of the intraspinal contents. During surgery, six patients were found to have involvement of the spinal cord or cauda equina. The pathological characteristics of the masses included three or more of the following: bone, cartilage, synovial membrane, urinary tract tissue, cyst wall, yellow or brown fat, and nerves. The well-differentiated cellular elements, which formed mature structures, along with the absence of primitive cellular components and neoplastic characteristics are more consistent with a diagnosis of hamartoma than teratoma. CONCLUSIONS: In this series, the authors describe a lesion that is overt on physical examination, yet can have occult spinal canal involvement. Complete neurosurgical evaluation is essential to provide appropriate treatment and prognosis.     

Characteristics associated with rapid decline in forced expiratory volume

Though the mechanism of tissue damage induced by colonic inflammation in ulcerative colitis is unknown, it has been established that the inflammatory mediator and potent neutrophil (PMN) chemotaxin, leukotriene B4(LTB4), is present in elevated amounts in the inflamed mucosa. The unique role of 5-lipoxygenase in the production of leukotrienes has made it a target for inhibition. This study used a rat model of acute colonic inflammation induced by a single IP injection of Mitomycin-C to test the efficacy of a specific and potent 5-lipoxygenase inhibitor zileuton in the treatment of colonic inflammation. We hypothesized that after inducing colitis in rats with mitomycin-C, the administration of oral zileuton would inhibit leukotriene production, thus preventing PMN infiltration and subsequent tissue damage. Zileuton decreased colonic tissue damage as measured by Histological score. However, zileuton did not significantly decrease neutrophil infiltration measured by mucosal PMN or myeloperoxidase (MPO) levels. Although zileuton was successful in significantly decreasing the frequency of severe colitis in our model, the fact that the decrease in PMN count and MPO level was not statistically significant suggests that another mechanism may be involved in its anti-inflammatory effect.     

Fine morphological changes in the penicillate nerve endings of human hairy skin during prolonged itching

Morphological changes in cutaneous nerve endings were investigated electron microscopically in patients suffering from certain kinds of urticaria with associated itching and in normal skin in which wheals and local itching were induced either by application of nettle hairs or by intracutaneous injections of a timothy pollen extract. Skin samples were obtained with a high speed dermal punch without anesthesia from the wheal areas. It was found that some subepidermal free nerve endings derived from non-myelinated nerve fibers (penicillate endings) showed accumulations of extra-cytoplasmic glycogen which was localized in the distended spaces between the axolemma, the Schwann cell membrane and the nerve basement membrane. In some cases, the glycogen was found to be so abundant that it occupied most of the cross sectional area of the ending. No morphological changes were observed in the pappilary endings, in nerve endings of the hairs or in the autonomic terminals. The conducting segments of all cutaneous nerve fibers showed normal morphology. The unusual morphological changes that occur in some penicillate nerve endings during the wheal development indicate that these endings are involved in the skin reaction and therefore they may be the specific end organs that are associated with itch, at least in urticaria.     

Acidic urine pH is associated with elevated levels of free urinary benzidine and N-acetylbenzidine and urothelial cell DNA adducts in exposed workers

We evaluated the influence of urine pH on the proportion of urinary benzidine (BZ) and N-acetylbenzidine present in the free, unconjugated state and on exfoliated urothelial cell DNA adduct levels in 32 workers exposed to BZ in India. Postworkshift urine pH was inversely correlated with the proportions of BZ (r = -0.78; P < 0.0001) and N-acetylbenzidine (r = -0.67; P < 0.0001) present as free compounds. Furthermore, the average of each subject's pre- and postworkshift urine pH was negatively associated with the predominant urothelial DNA adduct (P = 0.0037, adjusted for urinary BZ and metabolites), which has been shown to cochromatograph with a N-(3'-phosphodeoxyguanosin-8-yl)-N'-acetylbenzidine adduct standard. Controlling for internal dose, individuals with urine pH < 6 had 10-fold higher DNA adduct levels compared to subjects with urine pH > or = 7. As reported previously, polymorphisms in NAT1, NAT2, and GSTM1 had no impact on DNA adduct levels. This is the first study to demonstrate that urine pH has a strong influence on the presence of free urinary aromatic amine compounds and on urothelial cell DNA adduct levels in exposed humans. Because there is evidence that acidic urine has a similar influence on aromatic amines derived from cigarette smoke, urine pH, which is influenced by diet, may be an important susceptibility factor for bladder cancer caused by tobacco in the general population.     

Testicular morphological changes in children with acute lymphoblastic leukemia following chemotherapy

Morphological changes in the testis induced by chemotherapy given according to the Tokyo Children's Cancer Study Group (TCCSG) regimens were studied in children with acute lymphoblastic leukemia (ALL). After informed consent, testicular biopsies were performed 14 times in 12 patients at the end of treatment. The testicular morphology in all cases had sustained a degree of damage. The tubular fertility index (TFI), calculated as the percentage of seminiferous tubules containing identifiable spermatogonia, was from 0 to 42.8% (mean 33.4%) below the normal value. Infiltration of leukemic cells was the most significant factor contributing to the decrease in TFI. There were no differences in the TFI among the TCCSG protocols. Formation of sperm was recognized in six cases, whose ages were 7, 8, 9, 10, 15 and 19 years. In two children, testicular biopsy was performed twice. In the second biopsy, TFI was elevated and sperm formation with the maturation of Leydig cells was observed. A number of other pathological changes were observed: modification of spermatogonia, Sertoli cells and inclusion bodies in spermatogonia, abnormal maturation of Leydig cells, evidence of interstitial fibrosis and thickening of the basement membrane. These results suggest that recent strong chemotherapy for the treatment of ALL might cause severe but not fatal damage to children's testicular tissue. As chemotherapy escalates, more investigation of testicular function will be necessary.