Development of Squamous Cell Carcinoma After Pulmonary Aspergillosis in the Native Lung of a Lung Transplant Recipient: A Case Report.

Lung transplant recipients have a significant incidence of posttransplant lung nodules. Such nodules can occur from various etiologies, both in the lung allograft or in the native lung. They most commonly originate from infections, such as Pseudomonas or Aspergillus species, or from posttransplant lymphoproliferative disorder. Lung cancer is challenging to diagnose in a native lung, especially with an underlying fibrotic disease. We present a case of a 75-year-old woman who presented with classic clinical features of pulmonary aspergillosis in the native right lung with idiopathic pulmonary fibrosis 5 years after left-sided single-lung transplant. She required a right lower lobectomy and antifungal treatment with isavuconazonium sulfate and inhaled amphotericin. A persistent right upper lobe lung nodule was noted during surveillance imaging and was initially presumed to be recurrent Aspergillus infection; however, growth of the nodule and change in its characteristics prompted additional examination. A navigational bronchoscopic biopsy was positive for squamous cell carcinoma. Her options for stage IIIA squamous cell carcinoma were limited to chemotherapy with paclitaxel and carboplatin plus radiation. Although initial surveillance scans showed adequate tumor response, metastatic squamous cell carcinoma was found in the liver 6 months later. She was eventually transitioned to palliative care. This case highlights the importance of a high index of suspicion for examination of nodules in the native lung of lung transplant recipients, even in cases of a known diagnosis, owing to the high morbidity and mortality associated with primary lung cancer in this population.

Thoracoabdominal pressure gradient and gastroesophageal reflux: insights from lung transplant candidates.

Advanced lung disease is associated with gastroesophageal reflux disease (GERD). The thoracoabdominal pressure gradient (TAPG) facilitates gastroesophageal reflux, but the effects of TAPG on gastroesophageal reflux in patients with pulmonary disease have not been well defined. Patients diagnosed with end-stage lung disease are expected to have the most extreme derangement in respiratory mechanics. The aim of this study is to explore the relationship between TAPG and reflux in lung transplant (LTx) candidates. We reviewed LTx recipients who underwent pretransplant esophageal high-resolution manometry and a 24-hour pH study. Patients were excluded if they were undergoing redo LTx, had manometric hiatal hernia, or had previously undergone foregut surgery. TAPG was defined as the intra-abdominal pressure minus the intrathoracic pressure during inspiration. Adjusted TAPG was calculated by the TAPG minus the resting lower esophageal sphincter (LES) pressure (LESP). Twenty-two patients with normal esophageal function tests (i.e., normal esophageal motility with neither manometric hiatal hernia nor pathological reflux on 24-hour pH monitoring) were selected as the pulmonary disease-free control group. In total, 204 patients underwent LTx between January 2015 and December 2016. Of these, 77 patients met inclusion criteria. We compared patients with obstructive lung disease (OLD, n = 33; 42.9%) and those with restrictive lung disease (RLD, n = 42; 54.5%). 2/77 patients (2.6%) had pulmonary arterial hypertension. GERD was more common in the RLD group than in the OLD group (24.2% vs. 47.6%, P = 0.038). TAPG was similar between the OLD group and the controls (14.2 vs. 15.3 mmHg, P = 0.850); however, patients in the RLD group had significantly higher TAPG than the controls (24.4 vs. 15.3 mmHg, P = 0.002). Although TAPG was not correlated with GERD, the adjusted TAPG correlated with reflux in all 77 patients with end-stage lung disease (DeMeester score, rs = 0.256, P = 0.024; total reflux time, rs = 0.259, P = 0.023; total number of reflux episodes, rs = 0.268, P = 0.018). Additionally, pathological reflux was seen in 59.1% of lung transplant candidates with adjusted TAPG greater than 0 mmHg (i.e., TAPG exceeding LESP); GERD was seen in 30.9% of patients who had an adjusted TAPG ≤ 0 mmHg. In summary, TAPG varies based on the underlying cause of lung disease. Higher adjusted TAPG increases pathological reflux, even if patients have normal antireflux anatomy and physiology (i.e., no hiatal hernia and manometrically normal LES function). Adjusted TAPG may provide further insights into the pathophysiology of GERD.

Swallow-induced esophageal shortening in patients without hiatal hernia is associated with gastroesophageal reflux.

Longitudinal esophageal body shortening with swallow-induced peristalsis has been reported in healthy individuals. Esophageal shortening is immediately followed by esophageal re-elongation, and the lower esophageal sphincter (LES) returns to the baseline position. High-resolution manometry (HRM) allows for objective assessment of extent of shortening and duration of shortening. In patients without hiatal hernia at rest, swallow-induced esophageal shortening can lead to transient hiatal hernia (tHH) which at times may persist after the completion of swallow. This manometric finding has not been investigated in the literature, but a question arises whether this swallow-induced transient herniation can effect on the likelihood of gastroesophageal reflux. This study aims to assess the relationship between gastroesophageal reflux and the subtypes of swallow-induced esophageal shortening, i.e. tHH and non-tHH, in patients without hiatal hernia at rest. After Institutional Review Board (IRB) approval, we queried a prospectively maintained database to identify patients who underwent HRM evaluation and 24-hour pH study between January to December 2015. Patients with type-I esophagogastric junction (EGJ) morphology (i.e. no hiatal hernia) according to the Chicago classification v3.0 were included. The patterns of the esophageal shortening with swallows were divided into two subtypes, i.e. tHH and non-tHH. tHH was defined as an EGJ double high-pressure zones (≥1 cm) at the second inspiration after the termination of swallow-induced esophageal body contraction. The number of episodes of tHH was counted per 10 swallows and tHH size was measured for each patient. In total, 41 patients with EGJ morphology Type-I met the inclusion criteria. The mean age was 47.2 years, 35 patients (85.4%) were women, and the mean body mass index was 33.9 kg/m2. The mean number of tHH episodes was 3 out of 10 swallows; mean maximal tHH size was 1.3 cm. Patients who had tHH in ≥3 out of 10 swallows (n = 16; 39.0%) were more likely to have abnormal DeMeester scores than patients with <3 swallows (56% vs. 28%; P = 0.070). Patients with maximal tHH ≥2 cm in at least 1 swallow (n = 17; 41.5%) were more likely to experience pathological reflux than patients with maximal tHH <2 cm (59% vs. 25%; P = 0.029). In conclusion, we showed that, in a subset of patients with Type-I EGJ morphology, swallowing induced transient EGJ double high-pressure zones (≥1 cm) after peristalsis. We have named this new manometric finding the swallow-induced tHH. A high prevalence of pathological reflux disease was observed in patients with maximal tHH ≥2 cm. The degree of swallow-induced tHH could be an early indicator of lower esophageal sphincter dysfunction in patients without manometric hiatal hernia.

Donor-Derived Exosomes With Lung Self-Antigens in Human Lung Allograft Rejection.

The immunological role of exosomes in allograft rejection remains unknown. We sought to determine whether exosomes are induced during lung allograft rejection and to define the antigenic compositions of HLA, lung-associated self-antigens (SAgs) and microRNAs (miRNAs). Exosomes were isolated from sera and bronchoalveolar lavage fluid from 30 lung transplant recipients (LTxRs) who were stable or who had acute rejection (AR) or bronchiolitis obliterans syndrome (BOS). Exosomes were defined by flow cytometry for CD63 and western blotting for annexin V SAgs, collagen V (Col-V) and Kα1 tubulin were examined by electron microscopy; miRNAs were profiled by a miRNA array. Donor HLA and SAgs were detected on exosomes from LTxRs with AR and BOS but not from stable LTxRs. Exosomes expressing Col-V were isolated from sera from LTxRs 3 mo before AR and 6 mo before BOS diagnosis, suggesting that exosomes with SAgs may be a noninvasive rejection biomarker. Exosomes isolated from LTxRs with AR or BOS also contained immunoregulatory miRNAs. We concluded that exosomes expressing donor HLA, SAgs and immunoregulatory miRNAs are present in the circulation and local site after human lung transplantation and play an important role in the immune pathogenesis of acute allograft rejection and BOS.

A CDK2 activity signature predicts outcome in CDK2-low cancers.

The role of cyclin-dependent kinase 2 (CDK2) in cancer is controversial. A major hurdle is the availability of tools to easily assess its activity across many samples. Here, we introduce a transcriptional signature to specifically track CDK2 activity. It responds to genetic and chemical perturbations in the CDK-RB-E2F axis, correlates with mitotic rate in vitro and in vivo and reacts rapidly to changes in CDK2 activity during cell cycle progression. We find that CDK2 activity is specifically elevated in human testes, mirroring its critical function in mice, and report very distinct profiles across human cancers. Increased CDK2 activity decreases risk in colon cancer, but elevates poor outcome two- to fivefold in specific tumors, including low grade glioma, kidney, thyroid, adrenocortical and prostate cancer. These are typically 'CDK2-low' cancers, suggesting that above a certain threshold CDK2 promotes progression, but further increases do not influence outcome. Multivariate analysis revealed that the CDK2 signature is the most important predictive feature in these cancers versus dozens of other clinical parameters, such as tumor grade or mitotic index. Thus, transcriptome data provides a novel, straightforward method to monitor CDK2 activity, implicates key roles for the kinase in a subset of human tissues and tumors and enhances cancer risk prediction. The strategy used here for CDK2 could be applied to other kinases that influence transcription.

Indications for gastrointestinal endoscopy in childhood.

Endoscopic examination of the gastrointestinal tract (GIT) for diagnostics and therapy in children has evolved markedly over the last 20 or so years and is now usually undertaken by paediatric endoscopists. Updated diagnostic and management guidelines for common disorders including coeliac disease, gastro-oesophageal reflux disease, eosinophilic oesophagitis and inflammatory bowel disease highlight the central role of endoscopy. Therapeutic endoscopic approaches are also now widely available and further broaden the referral spectrum to include treatment of GIT bleeding, gastrostomy insertion, dilation of strictures and polypectomy. Lastly, the advent of newer technologies allows the examination of hitherto inaccessible areas of the GIT such as the mid-small bowel by wireless capsule video-endoscopy and enteroscopy. We summarise recent current practice and clinical guidelines, focussing on the key indications for referrals that are likely to require endoscopic assessment.

Peptides derived from the dependence receptor ALK are proapoptotic for ALK-positive tumors.

ALK is a receptor tyrosine kinase with an oncogenic role in various types of human malignancies. Despite constitutive activation of the kinase through gene alterations, such as chromosomal translocation, gene amplification or mutation, treatments with kinase inhibitors invariably lead to the development of resistance. Aiming to develop new tools for ALK targeting, we took advantage of our previous demonstration identifying ALK as a dependence receptor, implying that in the absence of ligand the kinase-inactive ALK triggers or enhances apoptosis. Here, we synthesized peptides mimicking the proapoptotic domain of ALK and investigated their biological effects on tumor cells. We found that an ALK-derived peptide of 36 amino acids (P36) was cytotoxic for ALK-positive anaplastic large-cell lymphoma and neuroblastoma cell lines. In contrast, ALK-negative tumor cells and normal peripheral blood mononuclear cells were insensitive to P36. The cytotoxic effect was due to caspase-dependent apoptosis and required N-myristoylation of the peptide. Two P36-derived shorter peptides as well as a cyclic peptide also induced apoptosis. Surface plasmon resonance and mass spectrometry analysis of P36-interacting proteins from two responsive cell lines, Cost lymphoma and SH-SY5Y neuroblastoma, uncovered partners that could involve p53-dependent signaling and pre-mRNA splicing. Furthermore, siRNA-mediated knockdown of p53 rescued these cells from P36-induced apoptosis. Finally, we observed that a treatment combining P36 with the ALK-specific inhibitor crizotinib resulted in additive cytotoxicity. Therefore, ALK-derived peptides could represent a novel targeted therapy for ALK-positive tumors.

E2f2 induces cone photoreceptor apoptosis independent of E2f1 and E2f3.

The 'activating' E2fs (E2f1-3) are transcription factors that potently induce quiescent cells to divide. Work on cultured fibroblasts suggested they were essential for division, but in vivo analysis in the developing retina and other tissues disproved this notion. The retina, therefore, is an ideal location to assess other in vivo adenovirus E2 promoter binding factor (E2f) functions. It is thought that E2f1 directly induces apoptosis, whereas other activating E2fs only induce death indirectly by upregulating E2f1 expression. Indeed, mouse retinoblastoma (Rb)-null retinal neuron death requires E2f1, but not E2f2 or E2f3. However, we report an entirely distinct mechanism in dying cone photoreceptors. These neurons survive Rb loss, but undergo apoptosis in the cancer-prone retina lacking both Rb and its relative p107. We show that while E2f1 killed Rb/p107 null rod, bipolar and ganglion neurons, E2f2 was required and sufficient for cone death, independent of E2f1 and E2f3. Moreover, whereas E2f1-dependent apoptosis was p53 and p73-independent, E2f2 caused p53-dependent cone death. Our in vivo analysis of cone photoreceptors provides unequivocal proof that E2f-induces apoptosis independent of E2f1, and reveals distinct E2f1- and E2f2-activated death pathways in response to a single tumorigenic insult.

Established and new mouse models reveal E2f1 and Cdk2 dependency of retinoblastoma, and expose effective strategies to block tumor initiation.

RB(+/-) individuals develop retinoblastoma and, subsequently, many other tumors. The Rb relatives p107 and p130 protect the tumor-resistant Rb(-/-) mouse retina. Determining the mechanism underlying this tumor suppressor function may expose novel strategies to block Rb pathway cancers. p107/p130 are best known as E2f inhibitors, but here we implicate E2f-independent Cdk2 inhibition as the critical p107 tumor suppressor function in vivo. Like p107 loss, deleting p27 or inactivating its Cdk inhibitor (CKI) function (p27(CK-)) cooperated with Rb loss to induce retinoblastoma. Genetically, p107 behaved like a CKI because inactivating Rb and one allele each of p27 and p107 was tumorigenic. Although Rb loss induced canonical E2f targets, unexpectedly p107 loss did not further induce these genes, but instead caused post-transcriptional Skp2 induction and Cdk2 activation. Strikingly, Cdk2 activity correlated with tumor penetrance across all the retinoblastoma models. Therefore, Rb restrains E2f, but p107 inhibits cross talk to Cdk. While removing either E2f2 or E2f3 genes had little effect, removing only one E2f1 allele blocked tumorigenesis. More importantly, exposing retinoblastoma-prone fetuses to small molecule inhibitors of E2f (HLM006474) or Cdk (R547) for merely 1 week dramatically inhibited subsequent tumorigenesis in adult mice. Protection was achieved without disrupting normal proliferation. Thus, exquisite sensitivity of the cell-of-origin to E2f and Cdk activity can be exploited to prevent Rb pathway-induced cancer in vivo without perturbing normal cell division. These data suggest that E2f inhibitors, never before tested in vivo, or CKIs, largely disappointing as therapeutics, may be effective preventive agents.

A rapid simple approach to quantify chromosome conformation capture.

Chromosome conformation capture (3C) is a powerful tool to study DNA looping. The procedure generates chimeric DNA templates after ligation of restriction enzyme fragments juxtaposed in vivo by looping. These unique ligation products (ULPs) are typically quantified by gel-based methods, which are practically inefficient. Taqman probes may be used, but are expensive. Cycle threshold (Ct) determined using SYBR Green, an inexpensive alternative, is hampered by non-specific products and/or background fluorescence, both due to high template/ULP ratio. SYBR Green melting curve analysis (MCA) is a well-known qualitative tool for assessing PCR specificity. Here we present for the first time MCA as a quantitative tool (qMCA) to compare template concentrations across different samples and apply it to 3C to assess looping among remote elements identified by STAT1 and IRF1 ChIP-chip at the interferon-gamma responsive CIITA and SOCS1 loci. This rapid, inexpensive approach provided highly reproducible identification and quantification of ULPs over a significant linear range. Therefore, qMCA is a robust method to assess chromatin looping in vivo, and overcomes several drawbacks associated with other approaches. Our data suggest that basal and induced looping is a involving remote enhancers is a common mechanism at IFNgamma-regulated targets.

Regional topical hypothermia of the beating heart: preservation of function and tissue.

BACKGROUND: Protection of the myocardium during beating heart operations is paramount. The goal of this study is to determine if regional topical hypothermia (RTH) preserves myocardial viability and function during periods of temporary coronary artery occlusion. METHODS: Sixteen pigs were divided into two groups (RTH and control). Each group received 40 minutes of midleft anterior descending coronary occlusion followed by 3 hours of reperfusion. The RTH group (n = 10) received RTH and the control group (n = 6) received no cooling. Myocardial and core temperatures were measured with thermistors. Sonomicrometers and micromonameters were used to determine load independent indices of myocardial function. These indices were measured at base line, during coronary occlusion, and at 3 hours of reperfusion. The myocardium at risk and the infarct area were determined with monastral blue dye and triphenyl tetrazolium chloride staining. RESULTS: The mean myocardial temperature in the risk zone during coronary occlusion was significantly less in the RTH group (29.4 degrees C +/- 5.6 degrees C versus 35.7 degrees C +/- 1.1 degrees C, p < 0.05). After 40 minutes of coronary occlusion, both the RTH group and control had a significant reduction in regional elastance (9.38 +/- 3.54 and 11.05 +/- 1.67 mm Hg/mm) compared with base line measurements (14.70 +/- 2.42 and 16.80 +/- 4.79 mm Hg/mm), p < 0.05. However, after 3 hours of reperfusion, the elastance returned to base line levels in the RTH group (15.83 +/- 3.06 mm Hg/mm) but remained significantly depressed in the control group (9.97 +/- 3.63 mm Hg/mm, p < 0.04). Myocardial necrosis as a percentage of the risk zone was significantly less in the hypothermia group (25% +/- 2% versus 62% +/- 5%, p < 0.001). CONCLUSIONS: Regional topical hypothermia during isolated temporary coronary occlusion provides regional myocardial protection expressed as a return of function and decreased necrosis. Regional topical hypothermia may be clinically applicable to myocardial preservation during beating heart operations.

Rod and cone degeneration in the rd mouse is p53 independent.

PURPOSE: To determine whether p53 is required for the death of rod and cone photoreceptors in rd mice, a model of human retinitis pigmentosa, and/or for the natural degeneration of inner nuclear layer (INL) cells in the developing retina. METHODS: Rod photoreceptor and INL apoptosis was assessed by TUNEL staining of mouse sagittal sections from post natal day (P) 10, 13, 15, 17, and 20 day p53+/+ and p53-/- rd retinas. Cone photoreceptor survival was measured by counting the total number of peanut agglutinin (PNA) positive cells in eighty four 0.25 mm x 0.25 mm bins in each eye, distributed equally across the four quadrants of whole mount retinas from 3 month old p53+/+ and p53-/- rd retinas. RESULTS: Both the kinetics of rod and INL cell death as well as the survival of cones were essentially unaffected by the absence of p53. CONCLUSIONS: Despite established links with retinal apoptosis, p53 is not essential for rod or cone cell degeneration in the rd mouse or for the elimination of bipolar and Muller cells during late retinal development.

The effect of pleural adhesions on pediatric cystic fibrosis patients undergoing lung transplantation.

The degree of pleural scarring complicating cystic fibrosis (CF) lung disease is thought to impact on the outcome of adult lung transplantation. This has not been previously studied in the pediatric population. We studied all patients undergoing lung transplantation at Children's Hospital Los Angeles from 1993 through 2000. Operative times, grade of pleural scarring, blood product transfusion requirements, and perioperative mortality were compared for patients with cystic fibrosis (35) versus those without this diagnosis (11). Patients with CF were slightly older (14.7+/-3.8 vs 10.6+/-5.6 years; P = 0.01) but had similar weights (34.8+/-8.7 vs 34.4+/-12.3 kg). The degree of pleural scarring was greater in the CF group but was only severe in four patients. Scarring did not impact on operative times (237+/-46 vs 219+/-39 minutes; P = 0.22) or cardiopulmonary bypass times (127+/-40 vs 133+/-49 minutes). Total perioperative blood requirements for the two groups were similar (35.6+/-14.9 vs 42.8+/-76.7 cm3/kg; P = 0.82). Pleural scarring in the pediatric CF patients undergoing lung transplantation is only severe in a minority of patients. It does not increase duration of operation nor blood transfusion requirements. CT scanning is consequently unnecessary in the preoperative workup of CF patients being evaluated for transplantation. CF patients undergoing transplantation have perioperative outcomes similar to those of noncystic patients.

Laparoscopic ultrasonic epithelial ablation of the lower esophagus after nissen fundoplication in a porcine model: assessment of tissue damage and healing process.

A substantial population of patients with Barrett's esophagus has undergone antireflux surgery but still requires annual surveillance endoscopy. These patients would benefit from a definitive ablation of the Barrett's mucosa, which would remove the malignant potential of this disease. This study evaluates the efficacy of applying ultrasonic energy to remove the epithelium of the lower esophagus in a porcine model with prior Nissen fundoplication. Four Yakutan minipigs underwent laparoscopic Nissen fundoplication. After 2 weeks they underwent transgastric Cavitron ultrasonic surgical aspirator (CUSA; Valleylab, Boulder, CO) ablation of the lower esophageal epithelium. Healing of the mucosa was assessed by endoscopy at 2 weeks and pathological examination at 4 weeks after ablation. All pigs underwent successful laparoscopic Nissen fundoplication. Complete lower esophageal epithelial ablation was accomplished through the fundoplication in three animals. One pig developed a bezoar that prohibited ablation. At 2 weeks endoscopy showed patchy squamous epithelial regeneration, which was confirmed histologically. Esophageal specimens at 4 weeks showed complete regeneration of squamous epithelium with a partially healed small ulcer in one animal. No stricture formation was seen. We conclude that the CUSA technique can completely ablate Barrett's mucosa in the setting of a prior antireflux procedure. Healing with squamous mucosal regeneration is rapid and complete.

Retinoblastoma: the disease, gene and protein provide critical leads to understand cancer.

Retinoblastoma has contributed much to the understanding of cancer. The protein product of the RB gene, pRB, is a multifaceted regulator of transcription which controls the cell cycle, differentiation and apoptosis in normal development of specific tissues. Elucidating the mechanisms in which pRB plays a critical role will enable novel therapies and strategies for prevention, not only for retinoblastoma, but for cancer in general.

Involvement of retinoblastoma family members and E2F/DP complexes in the death of neurons evoked by DNA damage.

Neuronal death evoked by DNA damage requires cyclin-dependent kinase 4 (Cdk4) and 6 activity and is accompanied by elevation of cyclin D1-associated kinase activity. Because Cdk4/6 phosphorylates retinoblastoma protein (pRb) family members that then modulate the transcriptional activity of E2F/DP1 complexes, we examined the involvement of these components in DNA damage-evoked neuronal death. Camptothecin induced rapid pRb and p107 phosphorylation at a Cdk4/6 phosphorylation site followed by selective loss of Rb and p107. The CDK inhibitor flavopiridol suppressed pRb and p107 phosphorylation and loss, implicating CDK activity in these events. Moreover, the loss of pRb and p107 appeared to be mediated by caspases because it was blocked by general caspase inhibitors. The role of phosphorylation and pRb and p107 loss in the death pathway was indicated by observations that virally mediated expression of pRb mutated at sites of phosphorylation, including the Cdk4/6 site, inhibited death. Finally, expression of dominant-negative versions of DP1, known to compromise E2F transcriptional activity, protects cortical neurons from death induced by camptothecin and sympathetic neurons from death evoked by UV treatment. Taken together, these results implicate the CDK-pRb/E2F/DP pathway as a required element in the neuronal death evoked by DNA damage.

pRB is required for interferon-gamma-induction of the MHC class II abeta gene.

pRB is required for IFN-gamma-induction of MHC class II in human tumor cell lines, providing a potential link between tumor suppressors and the immune system. However, other genes, such as cyclin D1, show pRB-dependency only in tumor cells, so by analogy, pRB may not be necessary for cII-regulation in normal cells. Here, we demonstrate that induction of the mouse MHC class II I-A heterodimer is normal in RB+/+ mouse embryonic fibroblasts (MEFs), but deficient in RB-/- MEFs. Inducibility is restored in RB-/- MEFs stably transfected with wild type RB cDNA or infected with an adenovirus expressing pRB. Thus, involvement of pRB in MHC class II expression is conserved in the mouse and is not an aberrant feature of tumorigenic, aneuploid, human tumor cells. Although cII genes are generally induced in a coordinate fashion, suggesting a common mechanism, we found that pRB was specifically required for induction of the Abeta, but not Aalpha or other MHC cII genes including Ebeta, Ii and H2-Malpha. Finally, IFN-gamma-induction of class II transactivator (CIITA), was pRB-independent, suggesting that pRB works downstream of this master-regulator of MHC class II expression.

Rapid, high level protein production using DNA-based Semliki Forest virus vectors.

Semliki Forest virus (SFV) vectors can be produced faster, and have a wider host range, than baculovirus vectors. However, the original SFV system requires in vitro manipulation of RNA. We have generated a system that is wholly DNA-based. Both the replicon vector, encoding SFV polymerase and the protein of interest, and the helper vector, encoding viral structural proteins, were modified so that expression was RNA polymerase II-dependent. Transfection of the modified replicon plasmid alone generated 20-30-fold more protein than obtained from a simple expression vector. Expression required the SFV replicase, which amplifies replicon RNA. The SFV-based vector generated 10-20-fold more protein than a plasmid based on Sindbis virus. Cotransfection of SFV replicon and helper vectors generated viral titers of around 10(6) infectious particles/ml. A single electroporation, plated on one 10-cm plate, generated enough virus (10(7) particles) to produce >500 microg of protein. Wild type, replication proficient virus was not detected in three tests utilizing almost 10(8) viral particles, a distinct advantage over a DNA Sindbis-based system in which over half the virus particles generated are fully infectious. The new SFV vectors significantly enhance the utility of this expression system.