Particle-bombardment-mediated DNA vaccination with rotavirus VP4 or VP7 induces high levels of serum rotavirus IgG but fails to protect mice against challenge

We recently reported that epidermal immunization using the PowderJet particle delivery device with plasmid vector pcDNA1/EDIM6 encoding rotavirus VP6 of murine strain EDIM induced high levels of serum rotavirus IgG but failed to protect mice against EDIM infection (Choi, A. H., Knowlton, D. R., McNeal, M. M., and Ward, R. L. (1997) Virology 232, 129-138.). This was extended to determine whether pcDNA1/EDIM4 or pcDNA1/EDIM7, which encode either rotavirus VP4 or VP7, the rotavirus neutralization proteins, could also induce rotavirus-specific antibody responses and if these responses resulted in protection. Titers of rotavirus serum IgG increased with the first dose in mice immunized with pcDNA1/EDIM7, but little or no serum rotavirus IgG was detected in mice immunized with pcDNA1/EDIM4. In vitro assays with these plasmids in rabbit reticulocyte lysates showed that VP4 was expressed but the amount was considerably lower than VP6 or VP7. To improve expression of VP4 and induction of rotavirus-specific humoral responses, the coding region of VP4 was cloned into the high-expression plasmid WRG7054 as a fusion protein containing the 22-amino-acid secretory signal peptide of tissue plasminogen activator (tPA) at its N terminus. In vitro expression of tPA::VP4 was significantly higher than unmodified VP4, and mice inoculated with WRG7054/EDIM4 generated high titers of rotavirus IgG. The coding sequence of VP7 without the first 162 nucleotides was also cloned into WRG7054, but no difference was observed between titers of serum rotavirus IgG in mice immunized with this plasmid (WRG7054/EDIM7Delta1-162) and pcDNA1/EDIM7. The rotavirus-specific IgG titers in all immune sera were predominantly IgG1 indicating induction of Th 2-type responses. None of the mice immunized with any of the VP4 or VP7 plasmids developed serum or fecal rotavirus IgA or neutralizing antibody to EDIM. When immunized mice were challenged with EDIM virus, there was no significant reduction in viral shedding relative to unimmunized controls. Therefore epidermal immunization with VP4 or VP7 alone elicited rotavirus IgG responses but did not protect against homologous rotavirus challenge.     

Solitary pulmonary papillomas in adults: a clinicopathologic and in situ hybridization study of 14 cases combined with 27 cases in the literature

Solitary endobronchial papillomas in adults are rare neoplasms. Only sporadic cases have been documented. The histologic classification of these tumors remains problematic, and little is known about their clinical behavior. The clinical and pathologic features of 13 endobronchial papillomas and a single endobronchiolar papilloma were reviewed. In situ hybridization for human papillomavirus (HPV) types 6/11, 16/18, and 31/33/51 was performed on seven cases. Twenty-seven additional well-documented cases were identified in a literature review. Human papillomavirus studies were performed in four of the previously reported cases. The 41 neoplasms combined from the Armed Forces Institute of Pathology and literature review were divided into three groups according to their histologic features. Thirty-one of 41 (76%) patients were men. The ages of the patients ranged from 26 to 74 years (median, 57 years). Three morphologically distinct histologic types were recognized; 27 squamous cell papillomas, 7 glandular papillomas, and 7 mixed squamous and glandular papillomas. Squamous papillomas: 23 of 27 (85%) patients were men, and the median age was 54 years. Six of eleven (55%) of these patients smoked. Twenty-six lesions were exophytic and a single lesion had an inverted pattern. Seven of 24 (29%) lesions featured cytologic atypia and 5 of 24 (14%) had viral cytopathic effect. Five of seven (71%) cases examined for HPV DNA were positive. Three of 18 (17%) recurred. Glandular papillomas: Four of seven (57%) patients were women. The mean age was 67 years. One of five (20%) patients smoked. Five lesions were central, and two were peripheral. Four lesions had columnar epithelium, and three had ciliated epithelium. One of six (17%) lesions recurred. Mixed papillomas: five of seven (71%) patients were men. The median age was 64 years. Three of five (60%) patients smoked. Three of seven (43%) lesions featured cytologic atypia. Four of five lesions were examined for HPV DNA and all were negative. No lesions recurred. This study demonstrates that solitary endobronchial papillomas can be separated into three distinct morphologic categories. Squamous cell and mixed papillomas are predominantly lesions of male smokers in their 6th decade. Although cytologic atypia is observed in many cases, the rarity of these tumors and difficulty in separating papillomas from endobronchial papillary squamous carcinomas make generalizations regarding the risk of progression to carcinoma tenuous at best. Human papillomavirus appears to play a pathogenetic role in some squamous cell papillomas, but not in mixed papillomas, yet its presence in the squamous lesions does not correlate with recurrence or malignancy. The first report of an inverted squamous cell papilloma indicates clinical features similar to the more common exophytic squamous cell papillomas. Glandular papillomas, the rarest of all endobronchial papillomas, are found in an older age group than squamous and mixed papillomas, and most-patients are nonsmokers. Based on these findings, all endobronchial papillomas should be completely excised.     

Effects of induced hypertension on transcranial Doppler ultrasound velocities in patients after subarachnoid hemorrhage

BACKGROUND AND PURPOSE: Transcranial doppler ultrasound (TCD) is used after subarachnoid hemorrhage to detect cerebral vasospasm and is often treated with induced hypertension. Cerebral autoregulation, however, may be disturbed in this population, raising the possibility that TCD velocities may be elevated by induced hypertension. To study this possibility, we performed continuous TCD monitoring of the middle cerebral artery during the induction and withdrawal of induced hypertension in patients after subarachnoid hemorrhage. METHODS: Twenty-eight patients were studied during the induction and withdrawal of hypertension using primarily phenylephrine. Continuous monitoring was performed on the middle cerebral artery with the highest flow velocity. Treatment was based on rising TCD velocities or clinical evidence for cerebral vasospasm. Mean arterial pressure and mean TCD velocities were recorded every minute. A change of > 15% from starting TCD values was considered significant. Cerebral autoregulation was calculated as a percentage of intact autoregulation. Patients were subsequently divided into groups of disturbed and intact autoregulation. RESULTS: In 10 of 19 patients (53%), TCD velocities changed by > 15% and paralleled changes in mean arterial pressure. This directly altered the TCD interpretation of the grade of vasospasm in 7 of 19 patients (36%). Three additional patients had smaller absolute changes in TCD velocities. No clinical difference could be identified between patients with disturbed and intact autoregulation. CONCLUSIONS: In patients with disturbed autoregulation after subarachnoid hemorrhage, induced hypertension can alter cerebral blood flow velocities. The level of autoregulation needs to be considered when interpreting TCD velocities in patients after subarachnoid hemorrhage.     

Cervical spondylotic myelopathy: surgical results and factors affecting prognosis

OBJECTIVE: A variety of factors may affect surgical outcome in patients with cervical spondylotic myelopathy. The aim of this study is to determine these factors on the basis of preoperative radiological and clinical data. METHODS: To assess the factors affecting postoperative outcome after surgery for cervical spondylotic myelopathy, the clinical and radiological data of 27 patients with cervical spondylotic myelopathy were reviewed. Functional and neurological statuses were assessed using the Japanese Orthopaedic Association (JOA) scale modified by Benzel. In all patients, the effect of age, symptom duration, cervical curvature, presence or absence of preoperative high signal intensity within the spinal cord as revealed by T2-weighted magnetic resonance imaging, and diameters of the spinal canal and vertebral body on pre- and postoperative neurological statuses were investigated. Plain radiographs were obtained for all patients, magnetic resonance images for 21 patients (77.8%), computed tomographic scans for 13 patients (48.1%), myelograms for 6 patients (22.2%), and computed tomographic myelograms for 4 patients (14.8%). There were five patients with a JOA score of 10, six patients with a JOA score of 11, six patients with a JOA score of 12, four patients with a JOA score of 13, four patients with a JOA score of 14, one patient with a JOA score of 15, and one patient with a JOA score of 16. All patients underwent cervical laminectomies. The mean follow-up period was 54.1 months. The final neurological examinations revealed improvement in the JOA scores of 85.1 % of the patients. RESULTS: Statistical analysis of all patients revealed mean JOA scores of 12.185 +/- 1.618 and 14.370 +/- 2.15 before surgery and at final examination, respectively. The difference between the preoperative JOA score and the final JOA score was determined to be statistically significant (P < 0.0001). Statistical analyses also showed better neurological improvement in patients younger than 60 years and in patients with normal preoperative cervical lordosis. Although patients without preoperative high signal intensity of the spinal cord showed a better improvement rate than did patients with preoperative high signal intensity, the determined difference was statistically insignificant. CONCLUSION: It can be concluded that age and abnormal cervical curvature predict less postoperative neurological improvement. The presence of preoperative high signal intensity within the spinal cord may also reflect less neurological improvement.     

Deletion of the central proline-rich repeat domain results in altered antigenicity and lack of surface expression of the Streptococcus mutans P1 adhesin molecule

Members of the family of surface adhesins of oral streptococci, including P1 of Streptococcus mutans, contain two highly conserved repeat domains, one rich in alanine (A region) and the other rich in proline (P region). To assess the contribution of the P region to the biological properties of P1, an internal deletion in spaP was engineered. In addition, the P region was subcloned and expressed as a fusion partner with the maltose binding protein of Escherichia coli and liberated by digestion with factor Xa. Results of Western blot experiments in which recombinant polypeptides were probed with a panel of 11 monoclonal antibodies indicated that the P region is a necessary component of conformational epitopes within the central portion of P1. Antibodies reactive with the P region were detected in a polyclonal rabbit antiserum generated against whole S. mutans cells but not in two rabbit antisera generated against purified P1 (Mr approximately 185,000), suggesting that this domain is immunogenic on the surface of intact bacteria but not as part of a soluble full-length molecule. Finally, transformation of a spaP-negative mutant with a shuttle vector containing an internally deleted spaP lacking P-region DNA resulted in a complete absence of surface-localized P1 and substantially less P1 in sonicated cells compared to the case for the mutant complemented with the full-length gene. These results suggest that the P region is an integral component contributing to the conformation of the central region of P1 and indicate that its presence is necessary for surface expression of the molecule on S. mutans.     

VCAM (IGSF) adhesion molecule expression in breast carcinomas detected by automated and quantitative immunocytochemical assays

Expression of vascular cell adhesion molecules (VCAM) in tumors is associated with endothelial cell activation and may facilitate adherence of carcinomatous cells to the vessel wall, promoting bloodborne metastases. Expression of VCAM was investigated in 202 breast carcinomas using automated (Ventana System) and quantitative (SAMBA image analyzer) immunoperoxidase staining of frozen sections. Positive VCAM immunoreactivity was observed in 83 tumors (41%) (mean immunostained surface, 12.4%; SD, 10.5). The mean area of immunostaining was correlated with clinical and pathologic prognostic indicators and with the immunohistochemical expression in tissue sections of various indicators of cell proliferation, metastatic potential, and drug resistance or sensitivity, evaluated according to the same method. There was no correlation of VCAM immunoreactivity with tumor size, type, or grade or with nodal status. Also, no significant correlation was observed between VCAM and MIB1/Ki67, p53, Bcl-2, E cadherin, CD44v, cathepsin D, CD31, P-gp, ER, PR, or pS2. However, VCAM immunoreactivity was significantly correlated with ELAM and VLA2 (P = .001) and VLAs (P = .008) expression. The results suggest that VCAM expression in breast carcinoma tissue sections is likely not a prognostic indicator. Its practical clinical relevance, if any, must be established by correlation with patients' outcomes and tumor sensitivity to drugs.     

Identification of marker chromosomes and translocations in man using a computerized diagnostic database and fluorescent in situ hybridization

BACKGROUND: Many species of bats have two sets of submandibular glands, principal and accessory. The accessory gland may resemble the principal one but more often shows wide morphological divergence. The free-tailed bat, Tadarida thersites, is very unusual in that it has two sets of parotid glands rather than binary submandibular glands. We studied the ultrastructure of the principal parotid gland to establish a baseline for comparison with the accessory parotid. METHODS: Two specimens of adult free-tailed bats, one male and one female, were live-trapped in western Kenya. Parotid glands were fixed for electron microscopy using a protocol expressly designed for field fixation and then embedded by conventional means. RESULTS: Histologically, the principal parotid is a typical serous gland. The secretory granules of the endpiece cells have an unusual substructure in that they contain variable numbers of lucent halos and one or several spherules. Intercalated duct cells contain a significant number of dense, serous-like granules. Striated ducts have the usual basal configuration of mitochondria and folded plasma membranes, but the supranuclear cytoplasm contains many small, dense granules, so that these ducts resemble the granular convoluted tubules found in the submandibular glands of many families of rodents. The apices of the duct cells have a peculiar contour--the luminal surfaces obliquely invaginate into the apical cytoplasm, so that in thin section the luminal membranes appear to be underlaid by a layer of vacuoles. CONCLUSION: Although the principal parotid gland of the free-tailed bat shows some distinctive, species-specific ultrastructural features, it basically is similar to the parotid gland in two other molossid bats, Tadarida brasiliensis and Molossus molossus. The distinctive features in the principal parotid gland of T. thersites might relate to its feeding on hard-bodied insects and perhaps to the production of lysozyme.     

Hyperhydration: tolerance and cardiovascular effects during uncompensable exercise-heat stress

This study examined the efficacy of glycerol and water hyperhydration (1 h before exercise) on tolerance and cardiovascular strain during uncompensable exercise-heat stress. The approach was to determine whether 1-h preexercise hyperhydration (29.1 ml H2O/kg lean body mass with or without 1.2 g/kg lean body mass of glycerol) provided a physiological advantage over euhydration. Eight heat-acclimated men completed three trials (control euhydration before exercise, and glycerol and water hyperhydrations) consisting of treadmill exercise-heat stress (ratio of evaporative heat loss required to maximal capacity of climate = 416). During exercise ( approximately 55% maximal O2 uptake), there was no difference between glycerol and water hyperhydration methods for increasing (P < 0.05) total body water. Glycerol hyperhydration endurance time (33. 8 +/- 3.0 min) was longer (P < 0.05) than for control (29.5 +/- 3.5 min), but was not different (P > 0.05) from that of water hyperhydration (31.3 +/- 3.1 min). Hyperhydration did not alter (P > 0.05) core temperature, whole body sweating rate, cardiac output, blood pressure, total peripheral resistance, or core temperature tolerance. Exhaustion from heat strain occurred at similar core and skin temperatures and heart rates in each trial. Symptoms at exhaustion included syncope and ataxia, fatigue, dyspnea, and muscle cramps (n = 11, 10, 2, and 1 cases, respectively). We conclude that 1-h preexercise glycerol hyperhydration provides no meaningful physiological advantage over water hyperhydration and that hyperhydration per se only provides the advantage (over euhydration) of delaying hypohydration during uncompensble exercise-heat stress.     

Characterization of a baculovirus-encoded ATP-dependent DNA ligase

Sequence analysis of the Lymantria dispar multicapsid nucleopolyhedrovirus (LdMNPV) genome identified an open reading frame (ORF) encoding a 548-amino-acid (62-kDa) protein that showed 35% amino acid sequence identity with vaccinia virus ATP-dependent DNA ligase. Ligase homologs have not been reported from other baculoviruses. The ligase ORF was cloned and expressed as an N-terminal histidine-tagged fusion protein. Incubation of the purified protein with [alpha-32P]ATP resulted in formation of a covalent enzyme-adenylate intermediate which ran as a 62-kDa labeled band on a sodium dodecyl sulfate-polyacrylamide gel. Loss of the radiolabeled band occurred upon incubation of the intermediate with pyrophosphate, poly(dA) . poly(dT)12-18, or poly(rA) . poly(dT)12-18, characteristics of a DNA ligase II or III. The protein was able to ligate a double-stranded synthetic DNA substrate containing a single nick and inefficiently ligated a 1-nucleotide (nt) gap but did not ligate a 2-nt gap. It was able to ligate short, complementary overhangs but not blunt-ended double-stranded DNA. In a transient DNA replication assay employing six plasmids containing the LdMNPV homologs of the essential baculovirus replication genes, a plasmid containing the DNA ligase gene was neither essential nor stimulatory. All of these results are consistent with the activity of type III DNA ligases, which have been implicated in DNA repair and recombination.