The ultrastructure of the nasal polyps in patients with and without cystic fibrosis.

Nasal polyps are commonly associated with cystic fibrosis (CF) and also with idiopathic allergies, asthma, and aspirin intolerance. The pathogenesis of nasal polyp formation is controversial. The present study investigates the ultrastructure of thirteen nasal polyps surgically removed from seven CF patients and six non-CF (NCF) patients with allergic diseases, asthma, and aspirin intolerance. All nasal polyps showed focal edema, hyperplasia, atrophy, or squamous metaplasia of the epithelium. The lamina propria was moderately populated with small blood vessels and mucous glands and showed focal accumulation of inflammatory cells. The CF nasal polyps, however, revealed several specific characteristics: 1) minimal damage to the surface epithelium, 2) presence of a mucus blanket lining the apical epithelium, 3) occasional intracytoplasmic lumens, 4) continuous and fenestrated type capillaries, 5) numerous degranulated mast cells, 6) many plasma cells, often with atypical morphology and intracisternal Russell bodies, and 7) a smaller number of eosinophils as compared to the NCF nasal polyps. The results indicate significant differences between CF and NCF nasal polyps and support the multifactorial pathways theory of nasal polyp formation.

Detection of myocardial viability in ischemic-reperfused rat hearts by Tc-99m sestamibi kinetics.

BACKGROUND: The purpose of this study was to evaluate technetium 99m sestamibi (MIBI) kinetics in assessing myocardial viability in hearts subjected to different ischemia-reperfusion treatments, resulting in graded severity of injury. METHODS AND RESULTS: Sixteen isolated Krebs-Henseleit-perfused rat hearts were divided into 3 groups: control (flow, 12 mL/min; n = 5), ischemic-reperfused with glucose (IR+G, n = 6), and ischemic-reperfused without glucose (IR-G, n = 5). MIBI (11.1 mBq [300 microCi]) was infused for 60 minutes (uptake), followed by a 60-minute clearance. MIBI uptake (percent injected dose per gram) was significantly decreased in the IR+G (2.07 +/- 0.31) and IR-G groups (2.03 +/- 0.23; P = not significant with IR+G) compared with the control group (3.06 +/- 0.25, P <.05). Fractional washout of MIBI was more rapid in the IR-G group (72.7% +/- 3.9%, P <.05) than in the control (21.9% +/- 1.9%) and IR+G groups (20.3% +/- 1.7%). End retention (percent injected dose per gram) of MIBI in the IR-G (0.60 +/- 0.12) and IR+G groups (1.60 +/- 0.18) was significantly less than in the control group (2.30 +/- 0.11, P <.05), respectively. The retention in the IR-G group was less than in the IR+G group (P <.05). Creatine kinase assay, triphenyltetrazolium chloride staining, and transmission electron microscopy analysis demonstrated more serious myocardial damage in the IR-G group than in the IR+G group. End MIBI activity was highly correlated with myocardial viability determined by triphenyltetrazolium chloride staining (r = 0.94; P <.05) and creatine kinase assay (r = -0.86; P <.05). CONCLUSIONS: Clearance of Tc-99m sestamibi is sensitive to metabolic states and may be used for assessment of ongoing myocardial damage.

HL-91-technetium-99m: a new marker of viability in ischemic myocardium.

BACKGROUND: Technetium 99m-HL91 is a new hypoxia imaging agent that demonstrates increased uptake in ischemic, viable myocardium. This study was performed to determine whether HL91 is taken up by nonviable myocardium. METHODS: Twenty-three Krebs-Henseleit buffer-perfused, isolated rat hearts were studied. Tc-99m-HL91 300 microCi was infused over 10 minutes, followed by a 60-minute clearance. Myocardial activity was monitored by use of an NaI crystal. Four groups were studied: control (flow = 12 mL/min, n = 7), low flow (flow = 1 mL/min, n = 6), no flow/reflow (60 minutes no flow/60 minutes reflow before Tc-99m-HL91 infusion, flow = 12 mL/min, n = 5), and cyanide-treated (before Tc-99m-HL91 infusion, flow = 12 mL/min, n = 5). Injury was assessed by creatine kinase, transmission electron microscopy, and triphenyltetrazolium chloride. RESULTS: Control (no injury) and cyanide-treated (severe injury) hearts demonstrated low uptake (6.3+/-0.5 mean+/-SEM and 5.7+/-1.2 microCi, respectively) and low 60-minute retention (13.8%+/-2.2% and 13.7%+/-3.9%, respectively). Low-flow hearts (minimal injury) demonstrated markedly increased uptake (43.5+/-2.8 microCi, P < .01) and increased 60-minute retention (33.2%+/-2.9%, P < .01) compared with control. No-flow/reflow hearts (moderate injury) demonstrated intermediate uptake (8.7+/-0.5 microCi, P < .05 to control), although retention was not significantly different (18.9%+/-3.5%, P = ns). Severely and rapidly injured myocardium demonstrated Tc-99m-HL91 peak uptake and retention indistinguishable from normal. Moderately injured myocardium demonstrated uptake intermediate between severely injured and low-flow-induced ischemic, viable myocardium. CONCLUSION: Thus Tc-99m-HL91 is not taken up or retained in nonviable and irreversibly injured myocardium.

Kinetics of technetium-99m-teboroxime in reperfused nonviable myocardium.

UNLABELLED: This study evaluates 99mTc-teboroxime uptake and clearance kinetics in reperfused infarcted myocardium. METHODS: In 47 isolated buffer perfused rat hearts, 17 had normal flow (Control), 13 had 30 min of no flow followed by reflow (Noflow30) and 11 had 60 min of no flow followed by reflow (Noflow60). A 1-hr uptake phase was begun by normally perfusing all 41 hearts with 99mTc-teboroxime-doped buffer. After uptake, a 1-hr clearance phase was begun by switching to a 99mTc-teboroxime-free buffer. Technetium-99m activity was monitored with a Nal probe. Triton X-100, a membrane detergent, was given after tracer loading to six additional hearts. RESULTS: Control and Noflow30 hearts showed near linear and rapid uptake, while Noflow60 hearts showed curvilinear and significantly less uptake than predicted. All three of these groups showed biexponential clearance. Early t1/2 was not significantly different for the three groups (Control = 6.3 +/- 1.9 sem min, Noflow30 = 5.4 +/- 1.3 min, Noflow60 = 8.9 +/- 2.8 min). Late t1/2 was significantly shorter for Noflow30 (52.3 +/- 5.3 min) and the Noflow60 (50.9 +/- 4.3 min), compared to the Control hearts (74.1 +/- 6.6 min, p < 0.05). One-hour fractional clearances were significantly greater for the Noflow30 and Noflow60 hearts (0.65 +/- 0.01 and 0.65 +/- 0.01, respectively) compared to the Controls (0.55 +/- 0.01, p < 0.05). In hearts given Triton X-100, there was a markedly increased fractional clearance of 0.96 +/- 0.01 (p < 0.01 compared to Controls). Electron microscopy showed evidence of mild injury in the Noflow30 hearts, more extensive damage in the Noflow60 hearts and severe irreversible injury in Triton X-100 hearts. CONCLUSION: Myocardial 99mTc-teboroxime uptake and clearance kinetics are significantly altered in mildly and moderately injured reperfused myocardium. Technetium-99m-teboroxime clearance is markedly accelerated in the setting of overt damage to cell and organelle membranes induced by Triton X-100.

Preproinsulin I and II mRNAs and insulin electron microscopic immunoreaction are present within the rat fetal nervous system.

Insulin-like substance has been found within the nervous system. In the rat, preproinsulin II mRNA was shown within the brain and preproinsulin I mRNA within the retina. The present study demonstrates the presence of preproinsulin mRNAs within the 15, 17 and 19 day gestational age fetal rat brain, spinal cord and dorsal root ganglia (DRG), employing RNA template-specific polymerase chain reaction (RS-PCR), semi-nested PCR and RNase protection assay. Preproinsulin I mRNA was present in the 17 and 19 day gestational age brain, spinal cord and DRG, and only in the brain of the 15 day gestational age brain. Preproinsulin II mRNA was present in all the gestational ages studied in the brain, spinal cord and DRG. The RS-PCR and the semi-nested PCR demonstrated products that co-migrated with the pancreatic control. The semi-nested products were characterized as preproinsulin I and II by restriction enzyme digestion and sequence. RNase protection assay using specific cRNA for preproinsulin I and II showed a band that co-migrated with pancreatic preproinsulin I and II mRNAs, and confirmed the PCR results. In addition, insulin receptor mRNA was detected by RS-PCR. Ultrastructural studies showed insulin immunoreaction within the endoplasmic reticulum, Golgi apparatus, cytoplasm, axon, dendrites, and in relation to the synapses. Thus, we demonstrated the presence of preproinsulin I and II mRNA, insulin receptor mRNA and insulin immunoreaction within the rat fetal central and peripheral nervous system.

Clearance of technetium 99m N-NOET in normal, ischemic-reperfused, and membrane-disrupted myocardium.

BACKGROUND: Technetium 99m-labeled bis(N-ethoxy, N-ethyl dithiocarbamato) nitrido technetium(v) (99mTcN-NOET) is a new neutral cardiac perfusion imaging agent that has been shown to have very high uptake and retention in vitro. The purpose of this study was to determine the clearance kinetics of 99mTcN-NOET in control, ischemic-reperfused, and membrane-disrupted myocardium. METHODS AND RESULTS: After a 100 microCi (3.7 x 10(6) Bq) bolus of 99mTcN-NOET was injected, myocardial clearance was monitored for 1 hour by the use of a sodium iodide detector in 30 isolated, Krebs-Henseleit (KH) perfused rat hearts. Seven hearts were used as controls (group 1). In seven ischemic-reperfused hearts, tracer administration and uptake was followed by 30 minutes of no flow and 1 hour of reflow (group 2). In six additional ischemic-reperfused hearts, tracer administration was followed by deprivation of flow for 1 hour followed by 1 hour of reflow (group 3). Six hearts were perfused with a 0.5% Triton X-100 KH perfusate for 1 hour (group 4). Four hearts were perfused with KH for 10 minutes, followed by cyanide for 10 minutes (group 5). This cycle was repeated three times. Activities remaining in each heart at the end of each experiment were quantitated, and activity at peak uptake was calculated. The 99mTcN-NOET myocardial clearance was near linear in the control (0.6 +/- 0.4) and both ischemic-reperfused groups with virtually no fractional clearance (1.2% +/- 0.6% and 2.1% +/- 0.6%, respectively; p = NS). In the Triton X-100 membrane-disrupted hearts, clearance was substantial (94.2% +/- 4.0%; p < 0.0001 compared with the control and ischemic-reperfused groups). Cyanide treatment produced rapid clearance, which was arrested by a return to the standard KH perfusate. Peak uptake as a percentage of injected dose was 74.9% +/- 1.4% for all groups combined. CONCLUSION: Thus 99mTcN-NOET has extremely high myocardial retention after 1 hour in normal myocardium and is not significantly affected by ongoing myocardial ischemia or reperfusion injury in this model. Clearance is increased markedly in extreme conditions of membrane disruption. These data are consistent with the concept that 99mTc-NOET is localized predominantly in or on cell membranes. 99mTcN-NOET is a promising, new myocardial perfusion imaging agent that exhibits a stable myocardial distribution in the setting of acute developing injury.

Effects of no flow and reperfusion on technetium-99m-Q12 kinetics.

UNLABELLED: The purpose of this study were to determine if 99mTc-Q12 tracer kinetics could be affected by the insult of total no flow followed by reflow and the effects of viability on clearance. METHODS: In six control buffer perfused rat hearts, flow was maintained at 12 ml/min throughout uptake and clearance. In six hearts (IR30), 30 min of no flow was followed by 60 min of reflow. In six hearts (IR60), 60 min of no flow was followed by 60 min of reflow. One millicurie 99mTc-Q12 was injected in control hearts or during reflow in the IR30 and IR60 hearts and myocardial clearance was monitored for 1 hr using a Nal detector. RESULTS: Control hearts demonstrated biphasic 99mTc-Q12 myocardial clearance with an early rapid clearance phase ending 5-10 min after injection (73.5% +/- 1.5% retention) followed by a late slow clearance phase (90.5% +/- 0.2% retention). IR30 hearts demonstrated a near identical clearance curve (74.3% +/- 0.9% early retention, 90.9% +/- 0.6% late retention). IR30 heart electron micrographs demonstrated predominantly ischemia insulted but viable cells. IR60 hearts also demonstrated a biphasic myocardial clearance, with a late slow phase similar to controls (91.9% +/- 0.6% retention). The early rapid phase was significantly faster than controls (61.1% +/- 3.4%). IR60 heart electron micrographs demonstrated predominantly injured nonviable cells. Well counting confirmed decreased retention in the IR60 rats compared to controls and IR30 rats. CONCLUSION: Technetium-99m-Q12 myocardial clearance is normally biphasic, with an early rapid phase ending after 5-10 min and a late slow phase. Ischemicaly insulted but viable myocardium created by 30 min of no flow followed by reflow has no effect on either clearance phase. This tracer warrants further study to determine its potential utility in assessing myocardial viability.

An immunohistochemical and in situ hybridization study of insulin-like growth factor I within fetal neuron cell cultures.

Fetal neuron cell cultures (NCC) from 22 day gestation and 18 day gestation fetal rabbit brain were studied for the presence of insulin-like growth factor I (IGF I). The 22 day gestation NCC were incubated in an IGF I free/insulin free/serum free medium. The 18 day gestation NCC were incubated in: (1) IGF I free/insulin free/serum free medium, (2) IGF I containing medium (100 ng)/serum free medium, and (3) serum containing medium. The 22 day gestation NCC survived in the IGF I free/insulin free/serum free medium. Furthermore, IGF I was detected in the medium by RIA from day one to day ten of incubation. In contrast, the 18 day gestation NCC did not survive in the IGF I free/insulin free/serum medium, but survived in the serum medium. When the 18 day gestation NCC were incubated in the serum free medium containing 100 ng IGF I the cells survived for a period of 2-3 days. Immunoreactive IGF I was found within the 22 day gestation NCC incubated in the IGF I free/insulin free/serum free medium and 18 day gestation NCC in serum medium. Likewise, IGF I mRNA was found only within the 22 day gestation NCC. Internalization studies of IGF I have shown that the peptide was internalized from the medium by the two different gestational age NCC's studied. IGF I receptors were found in both 22 day gestation and 18 day gestation NCC. In conclusion IGF I may promote cell survival in early stages of brain development, and may be of exogenous origin. In contrast the 22 day gestation NCC are capable of producing and secreting IGF I, and indeed appear to respond to this growth factor in an autocrine fashion.

Immunohistochemical and in situ hybridization study of an insulin-like substance in fetal neuron cell cultures.

We studied the ability of fetal neuron cell cultures from different rabbit fetal brain gestational ages to produce and secrete an insulin-like substance (ILS). Neurons from 22-day gestation were incubated in serum-containing medium or insulin-free/serum-free medium, and 18-day gestation fetal rabbit neurons were also incubated in serum-free/insulin containing medium and serum-containing medium. The 22-day cultures survived in the serum-containing medium and the insulin-free/serum-free medium. The 18-day cultures died when incubated in the insulin-free/serum-free or serum-free/insulin-containing medium, but survived when incubated in serum-containing medium. Using immunohistochemical and in situ hybridization techniques, ILS and insulin-like mRNA were demonstrated within the 22-day cultures incubated in all media compositions, but not within the 18-day cultures incubated in the serum-containing medium. Ultrastructural studies of the 22-day cultures demonstrated an ILS in the endoplasmic reticulum, Golgi and cytoplasm. Northern blots showed the presence of an insulin-like mRNA within the 22-day gestation neuron cell cultures. Insulin receptor was present in the 22-day cultures, but was absent in the 18-day cultures. In addition, we characterized the ILS from the 22-day cultures incubated in the insulin-free/serum-free medium employing high-performance liquid chromatography (HPLC), radioimmunoassay and Western blots. Analysis by HPLC and Western blots demonstrated the presence of an ILS in the extract. We have shown that while 22-day fetal neuron cultures produce and secrete an insulin-like substance indistinguishable from authentic insulin, neuron cell cultures from early brain development do not express this capability.

High-resolution electron microscopy of interlysosomal junctions.

Five different malignant tumors and two nasal polyps were examined by electron microscopy for diagnosis purposes. In all cases occasional large cells containing lysosomes interconnected by characteristic junctions were observed. The interlysosomal junctions appeared organized in zig-zag and ladder like configurations. The ultrastructural and morphometric analyses of these junctions postulated that the two configurations represented the same structure viewed in two different sectioning planes. High-resolution electron microscopic evidence suggests that the interlysosomal junction is organized as a 35-40 A thick pleated ribbon extending between a 200-A wide interlysosomal space, in close contact with the adjoining lysosomal membranes, without apparent integration into the membrane.

A quantitative comparison between in vivo-and in vitro-derived Friend leukemia virus.

The biological activities of RNA viruses derived from Friend leukemia cells in culture (TCV) were compared with those of viruses derived from the plasma (PV) of mice infected with Friend leukemia virus (FLV). The comparison was quantitatively based on the actual number of viruses used in each experiment as determined by counting under the electron microscope. Electron microscopy also provided a qualitative assessment of the structural integrity of the concentrated virus particles used in various bioassays. The data shows that the leukemogenic and spleen-focus-forming (SFF) activities of TCV, although demonstrable, are respectively 10(5) and 10(4) lower than those of PV. Moreover, TCV has 10(4) less helper activity (S+L- test) than PV. The level of reverse transcriptase activity is ten times lower in TCV than in PV which indicates that there is little correlation between polymerase activity and the other biological activities measured. The decreased biological activity of the in vitro grown virus is thought to be intrinsic to this type of virus although all extrinsic factors have not been ruled out.

Tumorigenicity, immunogenicity, and virus production in mouse melanoma cells treated with 5-bromodeoxyuridine.

Bromodeoxyuridine (BrdU), whether administered in a 30-hr pulse of 30 mug/ml or continuously in low concentrations (1-3 mug/ml), significantly increased production of particles with the morphology of murine leukemia virus in a mouse melanoma (B16) cell line. Particles were very rare in control cells, detectable only by electron microscopy. By contrast, in many experiments with BrdU-treated cells the numbers of virus particles counted by electron microscopy increased over 100-fold, and other tests for murine leukemia virus (plaque assay and tests for group-specific antigens 1 and 3 and for Gross cell-surface antigens) became positive. All BrdU-treated cells, regardless of drug concentration or length of treatment, in addition to showing loss of both pigment and of piled-up morphology, were suppressed in tumorigenicity compared with the control cells. These effects were all reversible. A significant percentage of mice injected with BrdU-treated cells were protected against subsequent tumor formation when challenged with malignant control cells. The degree of protection conferred on the mice correlated well with the number of virus particles counted in the injected cells. There was also good correlation between the amount of cell-associated virus and the degree of suppression of malignancy for cells treated continuously with 1 mug of BrdU per ml, but not as good for cells treated for short periods with higher concentrations of BrdU.