DNA-mediated immunization with glycoprotein D of equine herpesvirus 1 (EHV-1) in a murine model of EHV-1 respiratory infection

DNA-mediated immunization was assessed in a murine model of equine herpesvirus 1 (EHV-1) respiratory infection. A single intramuscular injection with plasmid DNA encoding EHV-1 glycoprotein D (EHV-1 gD), including its predicted C-terminal membrane anchor sequence, induced a specific antibody response detectable by 2 weeks and maintained through 23 weeks post injection. A second injection at 4 weeks markedly enhanced the antibody response and all EHV-1 gD-injected mice developed neutralizing antibodies. A lymphocyte proliferative response to whole EHV-1 was observed and a predominance of IgG2a antibodies after DNA injection was consistent with the generation of a type 1 helper T-cell (Th1) response. Following intranasal challenge with EHV-1, mice immunized with EHV-1 gD DNA were able to clear virus significantly more rapidly from lung tissue and showed reduced lung pathology, in comparison to control mice.     

Tissue harmonic imaging: why does it work?

The recent utilization of harmonic frequencies in the imaging of both tissue and contrast agents has dramatically improved echocardiographic image quality. In contrast harmonics, the harmonic frequency energy is generated on reflection from the microbubble contrast agent. In tissue harmonics, the harmonic frequency energy is generated gradually as the ultrasonic wave propagates through the tissue. Critical to the utility of tissue-generated harmonic frequencies is their origin beyond the chest wall and their nonlinear relation to the fundamental frequency energy strength. These two characteristics of tissue-generated harmonics ensure that the echoes most likely to produce artifact are least likely to produce harmonic waves. Armed with an understanding of how these images are produced and with data emerging as to their clinical utility, we anticipate that harmonic imaging will become the standard for assessing regional and global left ventricular function in technically difficult studies.     

Standardization of the CFU-GM assay using hematopoietic growth factors

BACKGROUND: The recently discovered LRP protein has been shown to be involved in drug resistance and possibly in detoxification processes. MATERIALS AND METHODS: To study the relation between LRP expression and exposure to cigarette smoke, LRP immunoreactivity was evaluated in 39 paraffin embedded normal lung tissues derived from patients operated on for pneumothorax, and related to amount of pack years smoked. We also studied the LRP protein expression in 36 non-small-cell lung cancer (NSCLC) samples and related the expression to patient characteristics and survival. Furthermore 17 lung tumor samples (10 NSCLC and 7 SCLC) derived from patients treated with chemotherapy were analysed in order to investigate the relation between LRP or MRP expression and the patient's response to chemotherapy. RESULTS: In the normal lung tissues, LRP intensity levels were not correlated to the amount of pack years smoked, although a trend was seen for higher LRP intensity levels in patients who smoked more than 10 pack years. LRP expression was significantly higher in NSCLC samples than in SCLC samples, and all SCLC samples displayed very low LRP expression. Within NSCLC, squamous cell and adenocarcinomas had higher LRP expression than large cell undifferentiated and mixed tumors. In NSCLC patients LRP expression was not a prognostic factor for survival. At initial analysis LRP expression levels did not predict for the response to chemotherapy. Only 3 out of 17 patients expressed MRP, and all SCLC samples were MRP negative. CONCLUSIONS: Striking different expression levels were seen between NSCLC and SCLC for both LRP and MRP. In a preliminary analysis LRP expression was not predictive for response to chemotherapy in lung cancer patients. In pneumothorax patients LRP levels were not correlated with the amount of pack years smoked.     

Hand manifestations of oxalosis

Oxalosis is an unusual metabolic disease that results either from an inherited hepatic enzyme deficiency or as the result of poor oxalate clearance during chronic hemodialysis. We present two cases of oxalosis and describe the hand manifestations of this condition and their treatment. One patient had painful, progressive gangrene, whereas in the other the disease took an indolent course with small palmar crystalline deposits.     

A population-based incidence study of chronic fatigue

BACKGROUND: Although isoflurane may cause subendocardial hypoperfusion in the presence of coronary stenosis because of its coronary arteriolar dilatory effects, it is not known how the subendocardial microcirculation is affected. The authors examined the effects of isoflurane on poststenotic subendocardial microvessels with coronary stenosis. METHODS: The authors observed subendocardial microvessels in in situ beating swine hearts with or without critical stenosis of the left anterior descending coronary artery (LAD) with a needle-type videomicroscope during isoflurane- (ISO-H), adenosine- (ADE-H), and nitroglycerin- (NTG-H) induced hypotension (mean arterial pressure, 55 mmHg). Regional myocardial function, oxygen balance, and lactate metabolism in the region perfused by the LAD also were determined. RESULTS: In swine with stenosis, there were no differences in heart rate, cardiac output, and LAD blood flow among the three types of hypotension. Regional lactate production and anterior interventricular venous pO2 were similar during ISO-H and NTG-H but higher during ADE-H. With videomicroscopy, about half as many subendocardial microvessels could be visualized during ADE-H as with ISO-H and NTG-H. The average decrease in the systolic diameter of subendocardial microvessels of greater than 100 microm was 9 +/- 6% during ISO-H and 12 +/- 5% during NTG-H, but no consistent phasic diameter changes were observed during ADE-H. In swine without stenosis, a systolic diameter decrease was observed during all three types of hypotension. CONCLUSIONS: These findings suggest that hypotension induced by isoflurane or nitroglycerin preserves phasic diameter changes in subendocardial microvessels in the presence of critical coronary stenosis, whereas that induced by adenosine does not.     

Lifetime comorbidity, lifetime history of psychosis and suicide attempts, and current symptoms of patients with deteriorated affective disorder

Expression of insulin-like growth factor-I (IGF-I) receptors and insulin-like growth factor-II/mannose-6-phosphate (IGF-II/Man6P) receptors in cultured bovine alveolar macrophages (BAM) was demonstrated by competitive binding studies and crosslinking experiments. Western blotting of protein extracts from cultured BAM using an anti bovine IGF-II/Man6P receptor antiserum (#66416) confirmed the presence of IGF-II/Man6P receptors on BAM. The effects of IGFs and Man6P on generation of inositol phosphates was measured by HPLC analysis of perchloric acid extracts from myo-[3H]inositol-labelled cultured BAM. IGF-I at nanomolar concentrations and Man6P (10[-8]-10[-3] M) stimulated the accumulation of both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 after 30 sec. IGF-II (up to 2.3 x 10[-8] M) had no significant effect on inositol phosphate accumulation under the same conditions. Both IGFs and Man6P induced a rise in [Ca2+]i in cultured BAM. In addition, using the fluorescent dye SNARF-1/AM we could demonstrate rapid but small IGF-II (10[-9] M) triggered acidification (0.07 pH units) of cultured BAM. Taken together, our results indicate not only the presence of both IGF-I and IGF-II/Man6P receptors on BAM, but also provide evidence of the linkage of the IGF-I receptor to the inositol phosphate system.     

Cell-permeable ceramides prevent the activation of phospholipase D by ADP-ribosylation factor and RhoA

The mechanism of inhibition of phospholipase D (PLD) by ceramides was determined using granulocytes differentiated from human promyelocytic leukemic (HL-60) cells. In a cell-free system, hydrolysis of phosphatidylcholine by membrane-bound PLD depended upon phosphatidylinositol 4,5-bisphosphate, guanosine 5'-3-O-(thio)triphosphate) (GTPgammaS), and cytosolic factors including ADP-ribosylating factor (ARF) and RhoA. C2-(N-acetyl-), C8- (N-octanoyl-), and long-chain ceramides, but not dihydro-C2-ceramide, inhibited PLD activity. Apyrase or okadaic acid did not modify the inhibition of PLD by ceramides, indicating that the effect in the cell-free system was unlikely to be dependent upon a ceramide-stimulated kinase or phosphoprotein phosphatases. C2- and C8-ceramides prevented the GTPgammaS-induced translocation of ARF1 and RhoA from the cytosol to the membrane fraction. In whole cells, C2-ceramide, but not dihydro-C2-ceramide, inhibited the stimulation of PLD by N-formylmethionylleucylphenylalanine and decreased the amounts of ARF1, RhoA, CDC42, Rab4, and protein kinase C-alpha and -beta1 that were associated with the membrane fraction, but did not alter the distribution of protein kinase C-epsilon and -zeta. It is concluded that one mechanism by which ceramides prevent the activation of PLD is inhibition of the translocation to membranes of G-proteins and protein kinase C isoforms that are required for PLD activity.     

Inhibition of nitric oxide production. Mechanisms of vascular albumin leakage

The mechanisms by which nitric oxide modulates microvascular albumin exchange were investigated by monitoring leukocyte-endothelial cell adhesion and fluorescein isothiocyanate-albumin leakage in rat mesenteric venules exposed to NG-nitro-L-arginine methyl ester (L-NAME). L-NAME elicited an initial rapid increase followed by a slower rate of albumin accumulation in the interstitial space. The initial phase of albumin leakage preceded the L-NAME-induced leukocyte adherence and emigration, whereas the magnitude of the albumin leakage observed in the later phase of L-NAME exposure was highly correlated with the number of adherent and emigrated leukocytes in the same segment of venule. Monoclonal antibodies (MAbs) directed against adhesion molecules CD11/CD18, ICAM-1, or P-selectin, but not a nonbinding MAb, attenuated the albumin leakage induced by L-NAME. WEB2086, a platelet activating factor antagonist, and 8-bromoguanosine 3',5'-cyclic monophosphate (8-br-cGMP) reduced the leukocyte adherence and emigration as well as the increased albumin leakage. Only 8-br-cGMP and the P-selectin MAb attenuated the platelet-leukocyte aggregation elicited by L-NAME. Phalloidin, which promotes endothelial junctional integrity, inhibited both the early and late phases of albumin leakage. Overall, these findings suggest that the increased albumin leakage observed in postcapillary venules after inhibition of nitric oxide production involves a mechanism that includes a role for cGMP, platelet activating factor, leukocyte-endothelial cell adhesion, and the endothelial cell cytoskeleton.