Biomedical subjects

Simon C Watkins

Publications and source records attributed to Simon C Watkins.

At least 91 records · Page 5Linked to original sources

PubMed2003

Transforming growth factor beta blocks Tec kinase phosphorylation, Ca2+ influx, and NFATc translocation causing inhibition of T cell differentiation.

Transforming growth factor (TGF)-beta inhibits T cell proliferation and differentiation. TGF-beta has been shown to inhibit the expression of transcription factors such as GATA-3 and T-bet that play important roles in T cell differentiation. Here we show that TGF-beta inhibits T cell differentiation at a more proximal step. An early event during T cell activation is increased intracellular calcium levels. Calcium influx in activated T cells and the subsequent activation of transcription factors such as NFATc, events essential for T cell differentiation, are modulated by the Tec kinases that are downstream of the T cell receptor and CD28. We show that in stimulated CD4+ T cells, TGF-beta inhibits phosphorylation and activation of the Tec kinase Itk, increase in intracellular Ca2+ levels, NFATc translocation, and activation of the mitogen-activated protein kinase ERK that together regulate T cell differentiation. Our studies suggest that by inhibiting Itk, and consequently Ca2+ influx, TGF-beta limits T cell differentiation along both the Th1 and Th2 lineages.

Animals↗

PubMed2003

Aberrant development of motor axons and neuromuscular synapses in MyoD-null mice.

Myogenic regulatory factors (MRFs), muscle-specific transcription factors, are implicated in the activity-dependent regulation of nicotinic acetylcholine receptor (AChR) subunit genes. Here we show, with immunohistochemistry, Western blotting, and electron microscopy that MyoD, a member of the MRF family, also plays a role in fetal synapse formation. In the diaphragm of 14.5 d gestation (E14.5) wild-type and MyoD-/- mice, AChR clusters (the formation of which is under a muscle intrinsic program) are confined to a centrally located endplate zone. This distribution persists in wild-type adult muscles. However, beginning at E15.5 and extending to the adult, innervated AChR clusters are distributed all over the diaphragm of MyoD-/- mice, extending as far as the insertion of the diaphragm into the ribs. In wild-type muscle, motor axons terminate on clusters adjacent to the main intramuscular nerve; in MyoD-/- muscle, axonal bundles form extensive secondary branches that terminate on the widely distributed clusters. The number of AChR clusters on adult MyoD-/- and wild-type diaphram muscles is similar. Junctional fold density is reduced at MyoD-/- endplates, and the transition from the fetal (alpha, beta, gamma, delta) to adult-type (alpha, beta, delta, epsilon) AChRs is markedly delayed. However, MyoD-/- mice assemble a complex postsynaptic apparatus that includes muscle-specific kinase (MuSK), rapsyn, erbB, and utrophin.

Age Factors↗

PubMed2003

Necrotic cell death in response to oxidant stress involves the activation of the apoptogenic caspase-8/bid pathway.

Human epithelial (A549) cells exposed to hyperoxia die by cellular necrosis. In the current study, we demonstrated the involvement of apoptogenic factors in epithelial cell necrosis in response to hyperoxia, including the formation of the Fas-related death-inducing signaling complex and initiation of mitochondria-dependent apoptotic pathways. We showed increased activation of both Bid and Bax in A549 cells subjected to hyperoxia. Bax activation involved a Bid-assisted conformational change. We discovered that the response to hyperoxia in vivo predominantly involved the activation of the Bid/caspase-8 pathway without apparent increases in Bax expression. Disruption of the Bid pathway by gene deletion protected against cell death in vivo and in vitro. Likewise, inhibition of caspase-8 by Flip also protected against cell death. Taken together, we have demonstrated the involvement of apoptogenic factors in epithelial cell responses to hyperoxia, despite a final outcome of cellular necrosis. We have, for the first time, identified a predominant role for the caspase-8/Bid pathway in signaling associated with hyperoxic lung injury and cell death in vivo and in vitro.

Adenocarcinoma↗

PubMed2003

Caspase-8 expression and proteolysis in human brain after severe head injury.

Programmed cell death involves a complex and interrelated cascade of cysteine proteases termed caspases that are synthesized as inactive zymogens, which are proteolytically processed to active enzymes. Caspase-8 is an initiator caspase that becomes activated when Fas death receptor-Fas ligand (FasL) coupling on the cell surface leads to coalescence of a "death complex" perpetuating the programmed cell death cascade. In this study, brain tissue samples removed from adult patients during the surgical management of severe intracranial hypertension after traumatic brain injury (TBI; n=17) were compared with postmortem control brain tissue samples (n=6). Caspase-8 mRNA was measured by semiquantitative reverse transcription and polymerase chain reaction, and caspase-8 protein was examined by Western blot and immunocytochemistry. Fas and FasL were also examined using Western blot. Caspase-8 mRNA and protein were increased in TBI patients vs. controls, and caspase-8 protein was predominately expressed in neurons. Proteolysis of caspase-8 to 20-kDa fragments was seen only in TBI patients. Fas was also increased after TBI vs. control and was associated with relative levels of caspase-8, supporting formation of a death complex. These data identify additional steps in the programmed cell death cascade involving Fas death receptors and caspase-8 after TBI in humans.

Age Factors↗

PubMed2003

Inducible expression of keratinocyte growth factor (KGF) in mice inhibits lung epithelial cell death induced by hyperoxia.

Oxidant-induced injury to the lung is associated with extensive damage to the lung epithelium. Instillation of keratinocyte growth factor (KGF) in the lungs of animals protects animals from oxidant-induced injury but the mechanism of protection is not well understood. An inherent problem in studying KGF function in vivo has been that constitutive overexpression of KGF in the lung causes embryonic lethality with extensive pulmonary malformation. Here we report the development of a stringently regulated, tetracycline-inducible, lung-specific transgenic system that allows regulated expression of KGF in the lung without causing developmental abnormalities from leaky KGF expression. By using this system, we show that exposure of KGF-expressing mice to hyperoxia protects the lung epithelium but not the endothelium from cell death in accordance with the selective expression of KGF receptor on epithelial and not on endothelial cells. Investigations of KGF-induced cell survival pathways revealed KGF-induced activation of the multifunctional pro-survival Akt signaling axis both in vitro and in vivo. Inhibition of KGF-induced Akt activation by a dominant-negative mutant of Akt blocked the KGF-mediated protection of epithelial cells exposed to hyperoxia. KGF-induced Akt activation may play an important role in inhibiting lung alveolar cell death thereby preserving the lung architecture and function during oxidative stress.

Animals↗

PubMed2003

Intra-mitochondrial poly(ADP-ribosylation) contributes to NAD+ depletion and cell death induced by oxidative stress.

Poly(ADP-ribosylation), primarily via poly(ADP-ribose) polymerase-1 (PARP-1), is a pluripotent cellular process important for maintenance of genomic integrity and RNA transcription in cells. However, during conditions of oxidative stress and energy depletion, poly(ADP-ribosylation) paradoxically contributes to mitochondrial failure and cell death. Although it has been presumed that poly(ADP-ribosylation) within the nucleus mediates this pathologic process, PARP-1 and other poly(ADP-ribosyltransferases) are also localized within mitochondria. To this end, the presence of PARP-1 and poly(ADP-ribosylation) were verified within mitochondrial fractions from primary cortical neurons and fibroblasts. Inhibition of poly(ADP-ribosylation) within the mitochondrial compartment preserved transmembrane potential (DeltaPsi(m)), NAD(+) content, and cellular respiration, prevented release of apoptosis-inducing factor, and reduced neuronal cell death triggered by oxidative stress. Treatment with liposomal NAD(+) also preserved DeltaPsi(m) and cellular respiration during oxidative stress. Furthermore, inhibition of poly(ADP-ribosylation) prevented intranuclear localization of apoptosis-inducing factor and protected neurons from excitotoxic injury; and PARP-1 null fibroblasts were protected from oxidative stress-induced cell death. Collectively these data suggest that poly(ADP-ribosylation) compartmentalized to the mitochondria can be converted from a homeostatic process to a mechanism of cell death when oxidative stress is accompanied by energy depletion. These data implicate intra-mitochondrial poly(ADP-ribosylation) as an important therapeutic target for central nervous system and other diseases associated with oxidative stress and energy failure.

Animals↗

PubMed2003

A role for class A scavenger receptor in dendritic cell nibbling from live cells.

Monocyte-derived dendritic cells (DC) possess the unique capacity to capture Ag from live cells through intimate cell contact, a process referred to as nibbling. We sought to define the receptor(s) mediating DC nibbling. Uptake of fluorescently labeled plasma membrane from live cells by DC was inhibited by protease treatment and by a panel of polyanionic ligands, implicating scavenger receptors (SR) in this process. Differential expression of SR on DC and macrophages correlated with the capacity to acquire membrane from live cells. Internalized membrane colocalized with SR ligand and entered the endosomal pathway. DC very efficiently acquired and internalized gp100 tumor Ag expressed at the surface of viable adenocarcinoma cells via recombinant adenoviral infection. Cross-presentation of gp100 by DC to MHC class I-restricted T cells was inhibited by polyanionic SR ligand and an Ab to type A SR (SR-A), whereas Ab to the class B SR CD36, which mediates uptake of apoptotic cells, induced no inhibition. DC capture of fluorescently labeled membrane from live cells was partially blocked by SR-A-specific Ab, suggesting that other SR may also be contributing to nibbling. DC maturation resulted in a switch in expression from type II SR-A (SR-AII) to the SR-AI splice variant. Finally, SR-A was identified on interdigitating DC isolated from monkey lymph nodes. These findings define a novel role for SR-A, and suggest that Ag uptake from live cells by DC may be important in the generation of immunity and in the maintenance of peripheral tolerance in vivo.

Animals↗

PubMed2003

Vascularization and tissue infiltration of a biodegradable polyurethane matrix.

Urethanes are frequently used in biomedical applications because of their excellent biocompatibility. However, their use has been limited to bioresistant polyurethanes. The aim of this study was to develop a nontoxic biodegradable polyurethane and to test its potential for tissue compatibility. A matrix was synthesized with pentane diisocyanate (PDI) as a hard segment and sucrose as a hydroxyl group donor to obtain a microtextured spongy urethane matrix. The matrix was biodegradable in an aqueous solution at 37 degrees C in vitro as well as in vivo. The polymer was mechanically stable at body temperatures and exhibited a glass transition temperature (Tg) of 67 degrees C. The porosity of the polymer network was between 10 and 2000 microm, with the majority of pores between 100 and 300 microm in diameter. This porosity was found to be adequate to support the adherence and proliferation of bone-marrow stromal cells (BMSC) and chondrocytes in vitro. The degradation products of the polymer were nontoxic to cells in vitro. Subdermal implants of the PDI-sucrose matrix did not exhibit toxicity in vivo and did not induce an acute inflammatory response in the host. However, some foreign-body giant cells did accumulate around the polymer and in its pores, suggesting its degradation is facilitated by hydrolysis as well as by giant cells. More important, subdermal implants of the polymer allowed marked infiltration of vascular and connective tissue, suggesting the free flow of fluids and nutrients in the implants. Because of the flexibility of the mechanical strength that can be obtained in urethanes and because of the ease with which a porous microtexture can be achieved, this matrix may be useful in many tissue-engineering applications.

Animals↗

PubMed2003

Identification of c-kit-positive cells in the mouse ureter: the interstitial cells of Cajal of the urinary tract.

The existence of a pacemaker system in the urinary tract capable of orchestrating the movement of filtrated urine from the ureteral pelvis to the distal ureter and lower urinary tract seems intuitive. The coordinated activity necessary for such movement or "peristalsis" would likely require an intricate network of cells with pacemaker-like activity, as is the case with the interstitial cells of Cajal (ICC) of the gut. We investigated whether these putative pacemaker cells of the urinary tract are antigenically similar to ICC of the gut by using immunofluorescence staining for c-kit, a cell-surface marker specific for ICC. Ureteral, urinary bladder, and urethral tissues were harvested from female mice of the WBB6F1 strain, and fixed sections were prepared and stained for c-kit. Cell networks composed of stellate-appearing, c-kit-positive, ICC-like cells were found in the lamina propria and at the interface of the inner longitudinal and outer circular muscle layers of the ureteral pelvis but not in the urinary bladder or urethra. Thus, like in the gut, c-kit-positive, ICC-like cells are present in the urinary tract but appear to be restricted to the proximal ureter of this murine species.

Animals↗

PubMed2003

Dendritic cells transduced to express interleukin-4 prevent diabetes in nonobese diabetic mice with advanced insulitis.

Our previous studies demonstrated that adoptive transfer of dendritic cells (DC) prevents diabetes in young nonobese diabetic (NOD) mice by inducing regulatory T(H)2 cells. In this report, as a means of treating NOD mice with more advanced insulitis, we infected DC with adenoviral vectors expressing interleukin (IL)-4 (Ad.IL-4), eGFP (Ad.eGFP), or empty vector (Ad psi 5). DC infected with any of the Ad vectors expressed higher levels of CD40, CD80, and CD86 molecules than uninfected DC and Ad.IL-4 DC produced IL-4 after lipopolysaccharide (LPS) and interferon (IFN)-gamma stimulation. Ad-infected DC efficiently stimulated allogeneic T cells, and cultures of T cells with Ad.IL-4 DC produced lower levels of IFN-gamma and marginally higher levels of IL-4. In vivo studies demonstrated that the Ad.eGFP DC trafficked to the pancreatic lymph nodes within 24 hr of intravenous administration, and could be visualized in the T cell areas of the spleen. The intrapancreatic IFN-gamma:IL-4 or IFN-gamma:IL-10 cytokine ratios were lower in 10-week-old mice treated with Ad.IL-4 DC, and these mice were significantly protected from disease. These results demonstrate, for the first time, that genetically modified DC can prevent diabetes in the context of advanced insulitis.

Adenoviridae↗

PubMed2003

Selective toxicity of engineered lentivirus lytic peptides in a CF airway cell model.

Lentivirus lytic peptides (LLPs) are derived from HIV-1 and have antibacterial properties. LLP derivatives (eLLPs) were engineered for greater potency against Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA). Minimum bactericidal concentration (MBC) was determined in low and physiologic salt concentrations. MBC was decreased against SA and equivalent against PA in physiologic salt when compared to the parent compound LLP1. In a novel cystic fibrosis (CF) airway cell model, one derivative, WLSA5, reduced the number of adherent PA and only moderately affected CF cell viability. Overall, eLLPs are selectively toxic to bacteria and may be useful against CF airway infections.

Anti-Bacterial Agents↗

PubMed2003

Carbon monoxide induces cytoprotection in rat orthotopic lung transplantation via anti-inflammatory and anti-apoptotic effects.

Successful lung transplantation has been limited by the high incidence of acute graft rejection. There is mounting evidence that the stress response gene heme oxygenase-1 (HO-1) and/or its catalytic by-product carbon monoxide (CO) confers cytoprotection against tissue and cellular injury. This led us to hypothesize that CO may protect against lung transplant rejection via its anti-inflammatory and antiapoptotic effects. Orthotopic left lung transplantation was performed in Lewis rat recipients from Brown-Norway rat donors. HO-1 mRNA and protein expression were markedly induced in transplanted rat lungs compared to sham-operated control lungs. Transplanted lungs developed severe intraalveolar hemorrhage, marked infiltration of inflammatory cells, and intravascular coagulation. However, in the presence of CO exposure (500 ppm), the gross anatomy and histology of transplanted lungs showed marked preservation. Furthermore, transplanted lungs displayed increased apoptotic cell death compared with the transplanted lungs of CO-exposed recipients, as assessed by TUNEL and caspase-3 immunostaining. CO exposure inhibited the induction of IL-6 mRNA and protein expression in lung and serum, respectively. Gene array analysis revealed that CO also down-regulated other proinflammatory genes, including MIP-1alpha and MIF, and growth factors such as platelet-derived growth factor, which were up-regulated by transplantation. These data suggest that the anti-inflammatory and antiapoptotic properties of CO confer potent cytoprotection in a rat model of lung transplantation.

Animals↗

PubMed2003

Identification of a synovial fibroblast-specific protein transduction domain for delivery of apoptotic agents to hyperplastic synovium.

Synovial hyperplasia, resulting in erosion of cartilage and bone, represents one of the major pathologies associated with rheumatoid arthritis. To develop an approach for efficient delivery of proteins or agents to synovium to induce targeted apoptosis of hyperplastic synovial tissue, we have screened an M13 peptide phage display library for synovial-specific transduction peptides. We identified a novel synovial-targeted transduction peptide, HAP-1, which is able to facilitate specific internalization of protein complexes into human and rabbit synovial cells in culture and rabbit synovial lining in vivo. HAP-1 and a non-tissue-specific cationic protein transduction domain, PTD-5, were fused to an antimicrobial peptide, (KLAK)(2), to generate two proapoptotic peptides termed DP2 and DP1, respectively. Administration of these peptides was able to induce apoptosis of rabbit and human synovial cells in culture, with DP2 inducing synovial cell-specific apoptosis. Intra-articular injection of DP1 and DP2 into arthritic rabbit joints with synovial hyperplasia induced extensive apoptosis of the hyperplastic synovium, while reducing the leukocytic infiltration and synovitis. These results suggest that proapoptotic peptides and, in particular, DP2 can be clinically useful for treatment of synovial hyperplasia, as well as inflammation. Moreover, the results demonstrate the feasibility of identifying tissue-specific transduction peptides capable of mediating efficient transduction in vivo.

Amino Acid Sequence↗

PubMed2003

Alterations in inducible 72-kDa heat shock protein and the chaperone cofactor BAG-1 in human brain after head injury.

The stress response in injured brain is well characterized after experimental ischemic and traumatic brain injury (TBI); however, the induction and regulation of the stress response in humans after TBI remains largely undefined. Accordingly, we examined injured brain tissue from adult patients (n = 8) that underwent emergent surgical decompression after TBI, for alterations in the inducible 72-kDa heat shock protein (Hsp70), the constitutive 73-kDa heat shock protein (Hsc70), and isoforms of the chaperone cofactor BAG-1. Control samples (n = 6) were obtained postmortem from patients dying of causes unrelated to CNS trauma. Western blot analysis showed that Hsp70, but not Hsc70, was increased in patients after TBI versus controls. Both Hsp70 and Hsc70 coimmunoprecipitated with the cofactor BAG-1. The 33 and 46, but not the 50-kDa BAG-1 isoforms were increased in patients after TBI versus controls. The ratio of the 46/33-kDa isoforms was increased in TBI versus controls, suggesting negative modulation of Hsp70/Hsc70 protein refolding activity in injured brain. These data implicate induction of the stress response and its modulation by the chaperone cofactor and Bcl-2 family member BAG-1, after TBI in humans.

Adolescent↗

PubMed2003

Elevated hepatocyte levels of the Forkhead box A2 (HNF-3beta) transcription factor cause postnatal steatosis and mitochondrial damage.

The Forkhead box (Fox) transcription factor Foxa2 (HNF-3beta) and related family members Foxa1 (HNF-3alpha) and Foxa3 (HNF-3gamma) act in concert with other hepatocyte nuclear factors (HNF) to coordinately regulate liver-specific gene expression. To circumvent the hepatic functional redundancy of the Foxa proteins, we used the T-77 transgenic (TG) mouse line in which the -3-kb transthyretin (TTR) promoter functioned to increase hepatocyte expression of the Foxa2 cDNA. Adult TG mice exhibited reduced hepatic glycogen and progressive liver injury, but maintained normal serum levels of glucose, insulin, and glucagon. In this study, we further characterized the postnatal liver defect in TTR-FoxA2 TG mice. The postnatal TG mice displayed significant reduction in serum glucose levels and in hepatocyte glycogen storage without increased serum levels of ketone bodies and free fatty acid suggesting that they are not undergoing a starvation response. We show that TG liver developed a substantial transient steatosis, which reached a maximum at postnatal day 5 and is associated with increased expression of hepatic genes involved in fatty acid and triglyceride synthesis, lipid beta-oxidation, and amino acid biosynthesis. Furthermore, transmission electron microscopy analysis of postnatal TG liver revealed extensive mitochondrial membrane damage, which is likely due to reactive oxygen species generated from lipid beta-oxidation. In conclusion, our model proposes that in response to reduction in hepatocyte glycogen storage, the TTR-Foxa2 TG mice survive by maintaining sufficient serum levels of glucose through gluconeogenesis using deaminated amino acids with dicarboxylate products of peroxisomal lipid beta-oxidation shuttled through the tricarboxylic acid cycle.

Animals↗

PubMed2003

Cellular mechanisms of interleukin-12-mediated neuroblastoma regression.

BACKGROUND/PURPOSE: Interleukin-12 (IL-12) is a proinflammatory cytokine with potent antitumor effects. Previous studies from the authors laboratory showed regression of established neuroblastoma in mice vaccinated with IL-12 transduced dendritic cells (DC). Although regression was associated with intense T cell infiltration, the precise role of T cells is unknown. The purpose of this work is to study the cellular mechanisms in IL-12-mediated tumor regression. METHODS: Three groups of mice (n = 12) received subcutaneous inoculation with 1 x 10(6) murine neuroblastoma cells (TBJ). Anti-CD4 (T helper), anti-CD8 (T cytotoxic), or antiasialo-GM1 (natural killer) antibodies were injected intravenously at 3-day intervals to deplete various immune effector cell populations. Mice in each depletion group and the control (nondepleted) group were injected intratumorally on day 7 with 1 x 10(6) DC IL-12-transduced DC. Tumors were harvested for morphometry and immunohistochemistry at 21 days. RESULTS: CD4 depletion had no effect on tumor growth in either control or IL-12-vaccinated animals. In contrast, CD8-depleted animals treated with IL-12-transduced DC underwent initial regression followed by progressive tumor growth (P <.01). These tumors were smaller in size at the same time-point. However, NK cell depletion (antiasialo GM1) completely abrogated the antitumor effects of IL-12-transduced DC, leading to progressive tumor growth from the outset. There was no difference between the control and treated animals in this group. CONCLUSIONS: Contrary to our hypothesis that IL-12 DC primarily function to stimulate a T cell-mediated response, these data suggest that NK cells are essential for the initial antitumor response of animals treated with IL-12-transduced DC. CD8+ T cells appear to be necessary effector cells for complete rejection of tumor and possibly memory. NK cells are responsible for the early immune response. Furthermore, CD4+ (T helper) cells did not play any role in IL-12-induced regression. These results imply that for DC to generate an effective antitumor response against neuroblastoma both acquired and innate effector cells are required.

Animals↗

PubMed2003

Characterization of the expression of inducible nitric oxide synthase in rat and human liver during hemorrhagic shock.

It has been previously shown that the inducible nitric oxide (NO) synthase (iNOS; NOS-2) is elevated after hemorrhage, and that iNOS-derived NO participates in the upregulation of inflammation as well as lung and liver injury postresuscitation from shock. The purpose of this study was to elucidate the time course of iNOS mRNA expression, as well as the cellular and subcellular localization of iNOS protein in the liver posthemorrhage in rats subjected to varying durations of hemorrhagic shock (HS; mean arterial blood pressure [MAP] = 40 mmHg) with or without resuscitation. Expression of iNOS mRNA in rat liver by real-time reverse transcriptase (RT)-PCR demonstrated iNOS upregulation in shocked animals as compared with their sham counterparts as early as 60 min after the initiation of hemorrhage. By 1 h of HS, iNOS protein was detectable in rat liver by immunofluorescence, and this expression increased with time. Immunofluorescence localized iNOS primarily to the hepatocytes, and in particular to hepatocytes in the centrilobular regions. This analysis, confirmed by immunoelectron microscopy, revealed that iNOS colocalizes with catalase, a peroxisomal marker. Furthermore, we determined that iNOS mRNA is detectable by RT-PCR in liver biopsies from human subjects with HS (MAP < 90 mmHg) associated with trauma (n = 18). In contrast, none of the seven nontrauma surgical patients studied had detectable iNOS mRNA in their livers. Collectively, these results suggest that hepatic iNOS expression, associated with peroxisomal localization, is an early molecular response to HS in experimental animals and possibly in human patients with trauma with HS.

Animals↗

PubMed2003

Some morphological, growth, and genomic properties of human cells chronically infected with porcine endogenous retrovirus (PERV).

A major concern in using porcine organs for transplantation is the potential of transmission of porcine endogenous retrovirus (PERV). To investigate the long-term effects of PERV infection on human cells, human embryonic kidney cell line HEK-293 infected with PERV PK-15 was maintained for up to 72 passages and samples were harvested at intervals for use in morphological, growth, and genomic analyses. Morphology, DNA content/cell, and doubling time of uninfected and infected cells were similar. Restriction fragment length polymorphism (RFLP) analysis of PCR-amplified nearly full-length PERV genome showed no alterations in band pattern. RFLP analysis of the long terminal repeats (LTR) showed some changes in band pattern, but not in length. Southern blot analysis of genomic DNA of infected cells indicated random integration of PERV without structural alterations in proviral genome. Semi-quantitative PCR demonstrated a gradual increase of proviral load in the infected cells. Sequence analysis of the LTR region of PERV from infected cells indicated a relatively low rate (6.0 x 10(-4)/bp or about 2 x 10(-6)/bp/generation) of mutation. There were also indications of recombination of PERV strains A and B. Finally, PERV infection had no effect on transcription of human endogenous retrovirus-K (HERV-K) genes. Together, no significant effect attributable to PERV infection was evident on chronically PERV-infected HEK-293 cells.

Animals↗