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The major capsid protein of HPV, L1, assembles into pentamers that form a T = 7 icosahedral particle, but the location of the co-assembled minor capsid protein, L2, remains controversial. Several researchers have developed useful monoclonal antibodies targeting L2, but most react with linear epitopes toward the N-terminus. As a means to better define the virus capsid and better assess the localization and exposure of L2 epitopes in the context of assembled HPV, we have developed a panel of 30 monoclonal antibodies (mAbs) which target the N-terminus of L2 amino acids 11-200, previously defined as a broadly protective immunogen. Select mAbs were processed with enzymes and anti-L2 Fabs were generated. These new mAb/Fab probes will be beneficial in future studies to unravel the placement of L2 and to help better define the role of L2 in the HPV lifecycle and the nature of the broadly protective epitopes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Human papillomavirus (HPV) is well recognized as a causative agent for anogenital and oropharyngeal cancers, however, the biology of HPV infection at different mucosal locations, specifically the oral cavity, is not well understood. Importantly, it has yet to be determined if oral tissues are permissive for HPV infection and replication. We investigated for the first time the titers, infectivity, and maturation of HPV16 in oral epithelial versus genital epithelial tissue. We show that infectious HPV16 virions can be produced in oral tissue. This demonstrates, for the first time, that infectious virus could be spread via the oral cavity. HPV16 derived from oral tissue utilize a tissue-spanning redox gradient that facilitates the maturation of virions over time. Maturation is manifested by virion stability and increased susceptibility to neutralization with anti-HPV16 L1 antibodies. However, susceptibility to neutralization by anti-HPV16 L2 specific antibodies decreases during the maturation of HPV16 virions in oral tissue., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

We previously identified a set of 50 genes that were differentially transcribed in the hippocampal CA1 region of aged, learning-impaired rats compared to aged, superior learning animals during a Morris water maze paradigm. In the current study, we expressed three of these genes (Pctk1, Tcf12 and Ccnd1), which had shown increased transcription in aged, learning impaired rats, in the hippocampus of young rats using viral gene transfer and tested for learning and memory deficits at age 7-14months. Pctk1 injected animals displayed a modest deficit in acquiring latency in both the Morris water maze and the reverse Morris maze. In the radial arm water maze paradigm, Pctk1, Tcf12 and Ccnd1 expressing animals all showed significant deficits in spatial working memory compared to controls. Rats injected with Ccnd1 and Tcf12, but not Pctk1, also showed a significant deficit in spatial reference memory in the radial arm water maze. Electrophysiological experiments revealed no difference in LTP in Ccnd1 and Pctk1 animals. However, LTD induced by low frequency stimulation was observed in control and Ccnd1 animals, but not in Pctk1 treated animals. In addition, neither Ccnd1 nor Pctk1 expression produced any detectable neuropathology. In contrast Tcf12 expressing animals displayed significant neurodegeneration in both CA1 and dentate gyrus. Several Tcf12 animals also developed tumors that appeared to be glioblastomas, suggesting that aberrant Tcf12 expression in the hippocampus is tumorigenic. Thus, behavioral experiments suggested that overexpression of Pctk1 and Ccnd1 produce a deficit in learning and memory, but electrophysiological experiments do not point to a simple mechanism. In contrast, the learning and memory deficits in Tcf12 animals are likely due to neuropathology associated with Tcf12 gene expression., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We studied a panel of mutant viruses containing wild-type and chimeric capsid HPV16 and HPV18 proteins. The mutant capsid protein expression, genome amplification, and episomal maintenance were comparable with the wild-type virus. However, the chimeric viruses varied in their titers from wild-type. We show that the intertypical mutant chimeric capsid viruses, that L2 affects the structure of L1 and that L1 affects the structure of L2 in the virion. These effects were measured using a panel of conformation-dependent neutralizing L1 MAbs and an L2 capsid surface peptide derived neutralizing antibody. These data suggest that variation of one capsid gene not only affects its own structure and antigenicity, but also affects the structure and antigenicity of the other capsid protein. Implications of our data suggest that for the continued effectiveness of a vaccine, variation in both capsid proteins need to be considered and not just the protein the vaccine is directed against., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Using the HPV18 genome as the backbone, we exchanged the HPV18 L2 or L1 genes with those of HPV16. The intertypical exchange of HPV18 L1 with the HPV16 L1 produced genomes that efficiently replicated and produced infectious virus. Genomes containing an intertypical exchange of HPV18 L2 for the HPV16 L2 failed to produce infectious virus in multiple independently derived cell lines. Using chimeric constructs of individual capsid proteins, we identified a type-specific domain at the N-terminus of the HPV18L1 capsid protein, which interferes with its ability to cooperate with the HPV16 L2 protein to form infectious viral particles. Deletion of this domain allows for the cooperation of the HPV18 L1 protein and HPV16 L2 protein and production of infectious progeny. In addition, cooperation of this N-terminal HPV18 L1 deletion mutant protein with the wild-type HPV18 L2 protein efficiently replicates infectious virus but changes occur in the viral structure., (Copyright 2010 Elsevier Inc. All rights reserved.)

Cryoelectron microscopy images of HPV16 pseudovirions (PsV) depict that each pentamer of L1 can be occluded with a monomer of L2. Further research suggests that an N-terminal external loop of L2 exists, which is the target of neutralizing and cross-neutralizing antibodies. Here we show that N-terminal L2 cysteine residues, Cys22 and Cys28, have overlapping and independent structural roles, which affect both early- and late-stage assembly events. Substitution of either cysteine residue enhances infectivity markedly in comparison to wild-type HPV16. However, only Cys22Ser 20-day virions become nearly as stable as wild type. In addition, Cys22Ser, and Cys22,28Ser 20-day virions have lost their susceptibility to neutralization by anti-L2 antibodies, whereas Cys28Ser 20-day virions remain partially susceptible. These results suggest that Cys28 is necessary for late-stage stabilization of capsids, while Cys22 is necessary for proper display of L2 neutralizing epitopes.

The life cycle of papillomaviruses is tightly linked to differentiation of host keratinocytes, but the mechanisms and cues by which life cycle events are tied to differentiation remain obscure. We have begun a systematic study of the differentiation-dependent life cycle of HPV31. A variety of signaling pathways have been implicated in controlling keratinocyte differentiation, especially the protein kinase C (PKC) pathway. We have used pharmacological inhibitors to determine that genome amplification and late transcription depend on specific PKC isoforms, and that transcription and replication are independently controlled. We found that tyrosine kinases are necessary for viral amplification but not viral transcription. These studies indicate that the PKC pathway is an important regulator of differentiation-dependent HPV31 replication and transcription.

The study of the human papillomavirus (HPV) life cycle was hampered for more than 50 years by the lack of a conventional cell culture system for propagating HPV. Considerable progress has been made in the production of several HPV types using either organotypic rafts or human epithelial xenografts in immunocompromised mice. In this study, we demonstrated episomal maintenance of HPV-11 DNA in N-Tert cells. HPV-11 episomal DNA containing cell populations grown in raft culture showed induction of the productive viral life cycle. HPV-11 DNA amplification and viral capsid antigen synthesis were detected in differentiated layers of epithelia. The viruses generated were able to infect keratinocytes in vitro, which indicate that viruses generated were infectious. The demonstration of the productive HPV-11 life cycle in raft culture from cloned HPV-11 DNA will facilitate genetic analyses of viral gene functions that was not possible using the human xenograft athymic mouse model.

The role of the human papillomavirus oncoprotein E7 in carcinogenesis has been extensively studied. While the role of HPV E7 in the viral life cycle has also been studied, certain disparities exist, indicating that genotype differences may influence the role that E7 plays in the viral life cycle. In this study, we investigated the role of HPV18 E7 in the viral life cycle in order to gain a further understanding of this issue. To determine the role that HPV18 E7 plays in the viral life cycle, a translation termination substitution mutant of E7 in the context of the full HPV18 genome was created. We introduced linearized HPV18 E7-deficient genomic DNA into primary keratinocytes, where it recircularized and was maintained episomally at a range of five to several hundred copies of HPV genomic DNA. The mutant genomes failed to amplify following epithelial stratification and differentiation in organotypic culture. Moreover, virion morphogenesis did not occur. We found that the expression of HPV16 or HPV18 E7 in trans was able to rescue the amplification defect but not the defect in virion morphogenesis. These studies indicate that HPV18 E7 plays a critical role in the productive stage of the viral life cycle. In addition, these studies add further proof to the hypothesis that genotype differences exist for the role of E7 during the viral life cycle.

Despite the prevalence of HPV16 in invasive cervical cancers, an in vitro system capable of producing infectious HPV16 is lacking. The organotypic (raft) culture system has allowed for the study of the entire differentiation-dependent life cycle of human papillomaviruses (HPVs). However, the use of this system with the prototype HPV16-containing cell line, W12, has failed to yield infectious virus. Our laboratory has introduced clinically derived HPV16(114/B) genomic DNA into primary keratinocytes, where it subsequently recircularized and maintained episomally at 50-100 copies per cell. Virion morphogenesis occurred after epithelial stratification and differentiation in raft culture. HPV16 virions were isolated that were able to infect keratinocytes in vitro. Infection was neutralized by monoclonal antibodies raised against HPV16 but not by monoclonal antibodies known to neutralize other HPV types.

Studies on the presence of human papillomavirus (HPV) DNA in cervical samples show that 10% or more of all clinical lesions contain at least two different HPV types. We have investigated if multiple HPV types can exist in the same cell and interact with one another or if they merely exist in the same tissue. Combinations of genital HPV genomes were electroporated into primary keratinocytes. Southern analyses of the electroporated cultures indicate that while a subset of high-risk HPV types can be stably maintained and replicate episomally in the same cell, interactions between types do occur, often to the detriment of one or both viruses in question. These studies provide insight into the interactions that may occur between HPV types in naturally occurring lesions.

We have investigated the interactions and consequences of superinfecting and coreplication of human papillomavirus (HPV) and herpes simplex virus (HSV) in human epithelial organotypic (raft) culture tissues. In HPV-positive tissues, HSV infection and replication induced significant cytopathic effects (CPE), but the tissues were able to recover and maintain a certain degree of tissue integrity and architecture. HPV31b not only maintained the episomal state of its genomic DNA but also maintained its genomic copy number even during times of extensive HSV-induced CPE. E2 transcripts encoded by HPV31b were undetectable even though HPV31b replication was maintained in HSV- infected raft tissues. Expression of HPV31b oncogenes (E6 and E7) was also repressed but to a lesser degree than was E2 expression. The extent of CPE induced by HSV is dependent on the magnitude of HPV replication and gene expression at the time of HSV infection. During active HSV infection, HPV maintains its genomic copy number even though genes required for its replication were repressed. These studies provide new insight into the complex interaction between two common human sexually transmitted viruses in an in vitro system, modeling their natural host tissue in vivo.

Multiple epidemiologic studies show that adeno-associated virus (AAV) is negatively associated with cervical cancer (CX CA), a cancer which is positively associated with human papillomavirus (HPV) infection. Mechanisms for this correlation may be by Rep78's (AAV's major regulatory protein) ability to bind the HPV-16 p97 promoter DNA and inhibit transcription, to bind and interfere with the functions of the E7 oncoprotein of HPV-16, and to bind a variety of HPV-important cellular transcription factors such as Sp1 and TBP. c-Jun is another important cellular factor intimately linked to the HPV life cycle, as well as keratinocyte differentiation and skin development. Skin is the natural host tissue for both HPV and AAV. In this article it is demonstrated that Rep78 directly interacts with c-Jun, both in vitro and in vivo, as analyzed by Western blot, yeast two-hybrid cDNA, and electrophoretic mobility shift-supershift assay (EMSA supershift). Addition of anti-Rep78 antibodies inhibited the EMSA supershift. Investigating the biological implications of this interaction, Rep78 inhibited the c-Jun-dependent c-jun promoter in transient and stable chloramphenicol acetyl-transferase (CAT) assays. Rep78 also inhibited c-Jun-augmented c-jun promoter as well as the HPV-16 p97 promoter activity (also c-Jun regulated) in in vitro transcription assays in T47D nuclear extracts. Finally, the Rep78-c-Jun interaction mapped to the amino-half of Rep78. The ability of Rep78 to interact with c-Jun and down-regulate AP-1-dependent transcription suggests one more mechanism by which AAV may modulate the HPV life cycle and the carcinogenesis process.

The organotypic (raft) culture system has allowed the study of the entire differentiation-dependent life cycle of human papillomaviruses (HPVs), including virion morphogenesis. We introduced linearized HPV45 genomic DNA into primary keratinocytes, where it recircularized and maintained episomally at a range of 10-50 copies of HPV genomic DNA. Following epithelial stratification and differentiation in organotypic culture, virion morphogenesis occurred. HPV45 virions were purified from raft cultures and were able to infect keratinocytes in vitro. By testing a panel of HPV VLP antisera, we were able to demonstrate that the infection was neutralized not only with human HPV45 VLP-specific antiserum, but also with human HPV18 VLP-specific antiserum, demonstrating serological cross-reactivity between HPV18 and HPV45.

Steroid hormone receptors have been shown to bind to response elements in the upstream regulatory region (URR) of human papillomavirus (HPV) in a ligand-dependent manner to affect viral promoter activity. To better understand how the enhancer activity of the URR differs between high risk HPV types, we chose to compare the basal and glucocorticoid-dependent activities of the URRs of HPV18 and HPV31. We found that the URR of HPV18 is a stronger enhancer than the URR of HPV31 in six different cell lines of epithelial origin. Furthermore, the activity of the URR of HPV31 was not inducible by the synthetic glucocorticoid dexamethasone (dex) in any cell line tested, while the URR of HPV18 was dex-inducible in the majority of these lines. These studies indicate significant differences between the URRs of high risk HPV types.

Adeno-associated virus (AAV), a common genital virus, may have a "protective" role against human papillomavirus (HPV)-associated cervical cancer. Epidemiological studies indicate a negative correlation between AAV infection and the incidence of cervical cancer. In contrast, HPV is positively associated with cervical cancer. To investigate interactions between these two viruses we used the organotypic "raft" culture system. The raft culture system is capable of supporting the complete HPV life cycle. Raft tissues that were actively replicating HPV were superinfected with AAV type 2 (AAV-2). We observed a multiplicity of infection (m.o.i.)-dependent enhancement and inhibition of HPV DNA replication, concomitant with AAV-2 replication. The data suggest that at low m.o.i. of AAV-2 infection, HPV DNA replication was slightly increased compared to controls and AAV-2 replicated poorly. At high AAV-2 m.o.i., HPV DNA replication was reduced and AAV-2 replicated to high levels. AAV-2 replication was increased in the presence of HPV compared to primary human keratinocyte, squamous cell carcinoma, and HaCat raft cultures infected with AAV-2 alone. These data suggest that HPV may provide types of "enhancer/helper" functions for AAV-2 replication and progeny formation. Infection with AAV-2 had significant effects on epithelial morphology. During infection with low m.o.i. of AAV-2 the epithelium stratified to a greater extent than in controls. With high m.o.i. of AAV-2 infections, tissue cytopathic effects were observed, indicating an additional factor responsible for the effect of AAV-2 on HPV replication and infection. Our results demonstrate a complex interaction between AAV-2, HPV, and skin during dual infection., (Copyright 2001 Academic Press.)

Since its discovery in 1966, adeno-associated virus type 2 (AAV) has been described as a helper-dependent parvovirus. However, in this study we demonstrate that AAV undergoes its complete life cycle, devoid of helper viruses or genotoxic agents, in the organotypic epithelial raft tissue culture system, a model of normal skin. AAV progeny production directly correlated with epithelial differentiation, as nondifferentiating keratinocytes were defective for this activity. Large nuclear virus arrays of particles of approximately 26 nm (parvovirus size) were observed in the granular layers of the raft epithelium by electron microscopy. Additionally, dosage-dependent histologic changes, some of which might be interpreted as cytopathology, were induced in the AAV-infected epithelial tissues. These data suggest a new biological model for AAV; that is, AAV is an epithelial-tropic autonomous parvovirus that can alter normal squamous differentiation., (Copyright 2000 Academic Press.)

Human papillomaviruses (HPVs) are epitheliotropic viruses, and their life cycle is intimately linked to the stratification and differentiation state of the host epithelial tissues. Defining a role for the E5 gene product in the differentiation-dependent viral life cycle has been difficult due to the lack of a suitable culture system. We used the organotypic (raft) culture system to investigate the spatial and temporal expression pattern of the E5 protein during the differentiation-dependent life cycle of HPV-31b. We report the generation of antisera specific to the HPV-31b E5a protein. The HPV-31b E5a protein was detected throughout the viral life cycle in raft cultures as determined by immunostaining analyses, and the protein was localized predominantly to the basal and granular layers. Expression of epidermal growth factor receptor or platelet-derived growth factor receptors, two proteins with which E5 has been shown to interact in cell culture, did not specifically colocalize with E5a expression. However, HPV-31b E5a expression did colocalize with the epithelial differentiation-specific marker filaggrin. The kinetics of E5a protein expression during the complete viral life cycle was analyzed by immunoblotting, and the highest level was found to be coincidental with the onset of virion morphogenesis., (Copyright 1998 Academic Press.)

Human papillomavirus (HPV) genome replication is dependent on the expression of E1 and E2 proteins. The organotypic (raft) culture system was used to investigate changes in viral early gene expression and vegetative genome replication during the complete life cycle of HPV type 31b (HPV31b). We have previously shown the synthesis of HPV31b viral particles as early as 10 days of growth of CIN-612 9E raft tissues (Ozbun, M. A., and Meyers, C. (1997). J. Virol. 71, 5161-5172). In the present study, we investigated the structures and temporal expression levels of HPV31b E1 and E2 transcripts, as well as the replication of the viral genome during the viral life cycle. The amplification state of the HPV31b genome was maximal at 10 days of raft tissue growth. Furthermore, the expression levels of E1 and E2 RNAs correlated with vegetative viral DNA replication. Levels of E1- and E2-specific transcripts were dissimilar throughout the viral life cycle. E2 RNA levels remained relatively constant, whereas E1 RNA levels were upregulated during the maximal amplification of viral genomes and the biosynthesis of virions. These data indicate that E1 may be the major regulator of viral genome amplification in preparation for DNA packaging and virion morphogenesis., (Copyright 1998 Academic Press.)

Infection by adeno-associated virus (AAV) is associated with lower cervical cancer rates. We have been investigating the hypothesis that AAV interacts with and inhibits the role of human papillomaviruses (HPV) in cervical cancer. We have been studying the response of bovine papillomavirus type 1 (BPV) oncogenic transformation and DNA replication to AAV as a prototype system. The AAV Rep 78 gene product is responsible for this inhibition. Here, it is demonstrated that in two assay systems, focus formation of C127 cells and chloramphenicol acetyl-transferase (CAT, measuring P89 promoter expression) assays, the smaller the time interval between AAV introduction relative to BPV introduction, the higher the level of inhibition resulted. Preinfection with AAV was also effective in inhibiting BPV, but the effectiveness also decreased with increasing time intervals. These differences in inhibition demonstrate that the efficiency of AAV's inhibition of BPV changes dramatically with time (as much as 10(4) when delaying AAV infection) and possibly reflect temporal changes in viral gene expression by AAV, BPV, or both, which affect the AAV-papillomavirus interaction. It is further found that two different chimeric BPV/AAV genomes, equivalent to a specific simultaneous infection by these viruses at a specific ratio (which can't be duplicated by virus infection), were fully defective for oncogenic transformation and DNA replication. These chimeric BPV/AAV genomes were also able to trans-inhibit the wild-type BPV genome. Finally, and surprisingly, C127 cells with resident AAV Rep78 positive provirus were found to have increased sensitivity to oncogenic transformation by BPV. These data define the conditions under which an inhibitory affect of the AAV Rep78 gene on BPV phentotypes can be expected. However, under certain conditions AAV appears able to stimulate BPV oncogenic transformation. This final observation is not totally unexpected as Rep78 is a transcription factor known to both stimulate or repress AAV's own gene expression depending upon adenovirus coinfection.

Viral vector-mediated gene transfer in brain can provide a means for gene therapy and functional studies. However, robust and persistent transgene expression in specific populations of the adult brain has been difficult to achieve. In an attempt to produce localized and persistent transduction in rat brain, we compared recombinant adeno-associated virus (rAAV) vectors incorporating either the immediate early cytomegalovirus (CMV) promoter or the neuron-specific enolase (NSE) promoter. Transduction in hippocampus resulting from the NSE promoter-containing construct was more efficient and persistent than that resulting from the CMV promoter-containing construct. Most hippocampal cells transduced with the NSE promoter had multipolar neuron morphology. Neurons with glutamatergic morphology were transduced weakly. In order to produce a local supply of neurotrophic factor to cells that degenerate under certain disease and experimental conditions, the NSE promoter was utilized to drive expression of brain-derived neurotrophic factor (BDNF) in medial septum or substantia nigra. In this construct, the NSE promoter drives dicistronic expression of BDNF and an enhanced version of green fluorescent protein (GFP). We estimated 3000-15,000 GFP-positive cells per injection of rAAV into septum or substantia nigra, a transduction ratio of 5-20 infectious virus particles per transduced cell. This frequency may be sufficient for trophic factor gene therapy as well as for investigating specific protein function in "topical (i.e., localized) transgenic" animals produced by rAAV., (Copyright 1998 Academic Press.)

We have used the organotypic culture system as a model to study the initial infectious process and spread of herpes simplex virus type 1 (HSV-1) in fully stratified and differentiated human epithelial tissue. The growth kinetics of HSV-1 were determined in organotypic tissues of human epidermal or ectocervical origin. Concurrently, we followed the spread of HSV-1 by immunostaining thin sections of infected organotypic tissue. After HSV-1 was applied to the top cornified epithelial layer, virus penetrated to the basal layer of replicating epithelium and grew to high titers. The virus was limited in its spread in that not all cells within the tissue had demonstrable infection. A ribonucleotide reductase mutant, ICP6 delta, could infect and replicate in basal layers of the organotypic tissues. However, we found that spread was limited in, and to, the basal cell layer. Peak ICP6 delta titers were 100-fold less than in cultures infected with wild-type HSV-1. Studies of HSV mutants should allow us to further define the role of specific viral genes which are associated with infection and spread in a tissue culture system that mimics the initial portal of entry for certain HSV infections.

The purpose of this study was to determine if oxygen delivery to rabbit cardiac allografts arrested and stored in University of Wisconsin solution (UWS) at 4 degrees C would affect preservation. Nineteen isolated rabbit hearts were rapidly excised and perfused at 80 mm Hg on an isovolumic modified Langendorff apparatus. A micromanometer was placed within a balloon and inserted into the left ventricle through the mitral valve orifice. Digitized pressure waveforms were collected at 11 known balloon volumes from 0.8 to 1.2 ml. Baseline data were obtained for all hearts while perfused with Krebs-Henseleit solution equilibrated with 95% O2:5% CO2 at 37 degrees C. All hearts were arrested with 30 ml of UWS (290 mOsm). The control group (N = 10) was stored in UWS at 4 degrees C for 8 hr, and the experimental group (N = 9) was perfused with oxygenated UWS (O2 content = 5.6 ml O2) at 4 degrees C for 8 hr at a pressure of 60 mm Hg (5-10 ml/min). Both groups were then reperfused with Krebs-Henseleit buffer at 80 mm Hg for 15 min at for postpreservation data acquisition. Left ventricular developed pressures over a physiologic range (pressure-volume area) and maximum positive and negative dP/dt were calculated. Recovery of left ventricular parameters as a percentage of the baseline values was determined. Mean pressure-volume area recovery in the nonperfused group was 40 +/- 7.9% versus the perfused group (71 +/- 7.0%, P = 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Perioperative right ventricular (RV) dysfunction remains a significant problem following single lung transplantation (SLT), especially in patients with pulmonary hypertension. Total RV power (Wt), a determinant of RV function, is the sum of the mean component (Wm) which contributes to actual blood flow and the oscillatory component (Wo) which is the energy expended on arterial pulsation. Calculation of Wo is possible only through harmonic analysis of pulmonary arterial (PA) pressure and flow waveforms, and as much as 33% of RV power is attributed to it. The purpose of this study was to precisely quantify changes in RV power output using Fourier analysis of PA pressure and flow waveforms after SLT. Fourteen dogs (donors) were instrumented with a PA ultrasonic flow probe, PA and left atrial (LA) micromanometers, and LA epicardial pacing leads. Control (Pre-Tx) pressure-flow data were acquired during transient occlusion of the right PA at a heart rate of 140. The PA was cannulated, the lungs were flushed with 1 liter of modified Euro-Collins solution at 4 degrees C, and the left lung was harvested and transplanted to 14 recipient dogs in a standard manner. After 1 hr of reperfusion, PA (Post-Tx) pressure-flow data were acquired as above. All recipient animals survived SLT with a mean ischemic time of 183 +/- 3 min. Following SLT, both the mean, Wm, (69 +/- 9 to 161 +/- 23 mW) and oscillatory, Wo, (23 +/- 3 to 46 +/- 10 mW) components of RV power output increased significantly after SLT (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Single lung transplantation (SLT) is emerging as definitive therapy for end-stage pulmonary disease of varying etiology, yet a complete description of the hemodynamic properties of the transplanted lung has not been reported. In this study, Fourier analysis was used to calculate the pulmonary arterial (PA) impedance spectrum before and immediately after SLT to define precisely the pulmonary pressure-flow relationship. Median sternotomies were performed in 18 dogs (donors): an ultrasonic flow probe was placed around the PA and micromanometers were placed in the PA and left atrium (LA). Control PA pressure and flow (PAQ) and LA pressure were measured during transient occlusion of the right PA. The lungs were harvested using cold modified Euro-Collins solution for preservation. After thoracotomy and pneumonectomy, left SLT was performed in 18 recipient dogs with a mean ischemic time of 179 +/- 6 min. After reperfusion for 1 hr, PA pressure and flow data were again collected. Characteristic impedance (Z0), a measure of resistance to pulsatile flow, was compared to input resistance (Rin), a measure of resistance to mean flow, and pulmonary vascular resistance (PVR), the conventional index. Rin is defined as the zeroth harmonic of the impedance spectrum and Z0 as the mean of impedance moduli from 2-12 Hz. All recipients survived transplantation. Both PVR and Rin increased significantly after transplantation (11 +/- 1 vs 19 +/- 3 Wood U, P less than 0.05, and 1352 +/- 121 vs 1964 +/- 244 dyne.sec.cm-5, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Papilloma induced by the cottontail rabbit papillomavirus (CRPV) progress at a high frequency to cancers. This property makes the CRPV system unique to study the role of papillomaviruses in cancer development. In contrast to bovine papillomavirus type 1, no convenient in vitro transformation system for CRPV has been available. Here, we describe two in vitro systems that we have developed. Transformation of NIH 3T3 cells is greatly facilitated by deletions in the CRPV late region. Specifically, a 300-bp segment located in the 5' half of open reading frame L2 inhibits transformation of NIH 3T3 cells by CRPV DNA. In a second system involving the natural host cell of CRPV, rabbit skin epithelial cells, transformation is a two-stage phenomenon leading first to cells with an increased growth potential which subsequently become morphologically transformed. Transformation of rabbit skin epithelial cells does not require deletions in the L2 ORF. However, late region sequences located in the 3' half of ORF L1 are needed, as they are for papilloma induction in rabbits.

Three crystalliferous (Cry+) strains of Bacillus thuringiensis subsp. israelensis (serotype 14) that produce parasporal protein crystals toxic to dipteran larvae and several acrystalliferous (Cry-) mutants, either induced or spontaneously derived from a single Cry+ parent, were examined for the presence of covalently closed circular (CCC) DNA in attempts to correlate toxin production with the presence of a specific plasmid. The plasmid profiles of both Cry+ and Cry- variants were analyzed by both a cleared lysate- and a modified Eckhardt lysate-electrophoresis technique. All of the Cry- mutants derived from the Cry+ parental strain had lost a 4.0- to 4.4-megadalton (Mdal) plasmid. Bioassay data confirmed loss of toxin production by the Cry- variants. All three Cry+ strains, including the parent of the Cry- strains, contained CCC plasmids DNAs of the following approximate molecular weights: 4.0 to 4.4, 5.2 to 6.0, and 11.4 to 13.0 Mdal. One Cry+ strain contained an additional CCC plasmid of 6.7 to 7.2 Mdal. The plasmid patterns for several Cry- derivatives differed in other respects from the pattern for their parent strain. The various Cry+ and Cry- strains could be distinguished either by phenotypical differences in antibiotic sensitivity, crystal production, and toxicity, or by differences in their plasmid profiles.

Genetic studies to elucidate the role of papillomaviruses in the development and progression of tumors have been severely hampered because the viruses cannot be grown in tissue culture and therefore mutants are not available. We have employed recombinant DNA for papilloma induction to identify essential sequences involved in papillomavirus pathogenesis. Here, we demonstrated that deleting most of the open reading frame (ORF) L2 did not affect the potential of viral cottontail rabbit papillomavirus (CRPV) DNA to induce papillomas. The extrachromosomally maintained DNA in the papillomas was not rearranged and the major transcripts of 1.3 and 2.0 kb encoded E7 and E6, respectively. A recombinant DNA containing a larger deletion lacking the 3' terminal half of ORF L2 and all of ORF L1 (pdlBc/l) did not induce papillomas. The results indicate that sequences in the late region not required for transformation of NIH 3T3 cells by bovine papillomavirus type-1 essential in CRPV for induction of papillomas.