Characterization of a Long-Acting Site-Specific PEGylated Murine GM-CSF Analog and Analysis of Its Hematopoietic Properties in Normal and Cyclophosphamide-Treated Neutropenic Rats.

Previously we reported that site-specific modification of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) A3C analog with polyethylene glycol (PEG) dramatically improved the pharmacokinetic properties of the protein in rats. However, we could not evaluate the hematological properties of the PEG-A3C protein in rats because human GM-CSF is inactive in rodents. To study the biological effects of PEGylated GM-CSF analogs in rodents we created a homologous site-specific PEGylated murine (mu) GM-CSF (T3C) protein. muGM-CSF and the T3C protein were expressed in Escherichia coli and purified by column chromatography. The purified T3C protein was covalently modified with a linear 20 kDa- or a branched 40 kDa-maleimide-PEG, and the monoPEGylated proteins purified by column chromatography. muGM-CSF, T3C and the two PEG-T3C proteins had comparable in vitro biological activities, as measured by stimulation of proliferation of the murine FDC-P1 cell line. The PEG-T3C proteins had 10- to 25-fold longer circulating half-lives than muGM-CSF and stimulated greater and longer lasting increases in neutrophils and white blood cells than muGM-CSF following a single intravenous or subcutaneous administration to rats. Treatment of rats made neutropenic with cyclophosphamide with the PEG-T3C proteins shortened the time for recovery of neutrophils to normal levels from 9 or 10 days to 5 or 6 days, whereas muGM-CSF showed no benefit versus vehicle solution. Acceleration of neutrophil recovery in cyclophosphamide-treated rats required a minimum of three PEG-T3C treatments over five days. The PEG-T3C proteins should prove useful for evaluating the potential therapeutic benefits of GM-CSF and long-acting GM-CSF proteins in rodent disease models.

Multidirectional flexible force sensors based on confined, self-adjusting carbon nanotube arrays.

We demonstrate a highly sensitive force sensor based on self-adjusting carbon nanotube (CNT) arrays. Aligned CNT arrays are directly synthesized on silicon microstructures by a space-confined growth technique which enables a facile self-adjusting contact. To afford flexibility and softness, the patterned microstructures with the integrated CNTs are embedded in polydimethylsiloxane structures. The sensing mechanism is based on variations in the contact resistance between the facing CNT arrays under the applied force. By finite element analysis, proper dimensions and positions for each component are determined. Further, high sensitivities up to 15.05%/mN of the proposed sensors were confirmed experimentally. Multidirectional sensing capability could also be achieved by designing multiple sets of sensing elements in a single sensor. The sensors show long-term operational stability, owing to the unique properties of the constituent CNTs, such as outstanding mechanical durability and elasticity.

Preoperative Computed Tomography-Determined Sarcopenia and Postoperative Outcome After Surgery for Non-Small Cell Lung Cancer.

BACKGROUND: Sarcopenia, reduced skeletal muscle mass, is associated with frailty, injuries, and mortality. The purpose of this study was to evaluate the impact of computed tomography-determined sarcopenia on surgical complications and outcomes after resection of non-small cell lung cancer. METHODS: For a total 272 non-small cell lung cancer patients that underwent surgery between 2011 and 2016, cross-sectional area of muscle at the third lumbar vertebra (L3) was retrospectively measured using preoperative chest computed tomography images. Sarcopenia was defined as an L3 muscle index of <55 cm2/m2 for men and of <39 cm2/m2 for women. Clinical characteristics, postoperative complications, disease-free survival, and overall survival of patients with or without sarcopenia were compared. RESULTS: A total of 60.3% ( n = 164) were male, and mean patient age was 62.9 ± 9.6 years. The prevalence of sarcopenia was 22.4% for all study subjects, 32.9% for men, and 6.5% for women. No significant difference was observed between patients with or without sarcopenia in terms of intensive care unit or hospital stay ( p = 0.502 and p = 0.378, respectively), and the presence of sarcopenia was not associated with postoperative complications. Furthermore, no significant difference was observed between the 3-year disease-free survival rate (74.3% vs 66.7%, p = 0.639) or 3-year overall survival rate (83.9% vs 87.7%, p = 0.563) of patients with or without sarcopenia. CONCLUSION: Sarcopenia as determined by preoperative computed tomography does not appear to have a negative impact on surgical outcome or overall survival for resected non-small cell lung cancer patients.

Surgical Techniques of Allogeneic Liver Transplantation in a Nonhuman Primate Model.

Herein, we report our experience of performing allogeneic orthotopic liver transplantation (LT) in nonhuman primates. We designed an allogeneic ABO-compatible orthotopic LT model in monkeys in a manner similar to that used in humans. We applied almost the same surgical procedures used for human conventional deceased donor LT. A total of 6 monkeys underwent allogeneic LT. One cynomolgus monkey aged 45 months (3.4 kg) and 5 rhesus macaque monkeys aged 50.2 ± 14.8 months (5.40 ± 0.33 kg) were used as recipients. In the donor surgery, the liver was perfused in situ through the aorta using cold histidine-tryptophan-ketoglutarate solution. The portal vein (diameter, 5-10 mm), supra- and infra-hepatic inferior vena cava (IVC) (diameter, 12-15 mm), and common bile duct (diameter, 1.5-3.0 mm) were dissected out. The hepatic artery was kept in continuity with the celiac trunk and abdominal aorta up to the iliac bifurcation (diameter, 5-6 mm). The mean graft weight was 102.0 g (94.8-111.0 g). Recipient surgery was conducted in parallel. After recipient hepatectomy, the graft was implanted. The suprahepatic IVC and portal vein were anastomosed to those of the graft. After reperfusion, the infrahepatic IVC was anastomosed. The aorta conduit of the graft was anastomosed to the infrarenal aorta of the recipient in a retrocolic end-to-side manner. Biliary reconstruction was performed in a duct-to-duct anastomosis with cholecystectomy. Mean operative time was 107.0 minutes for donor and 198.2 minutes for recipient. There was one operative death due to unknown cause. In conclusion, for allogeneic orthotopic LT in nonhuman primate model, we can apply almost the same procedure used for human conventional deceased donor LT in a similar manner.

Metastatic carcinoma of the oral region: An analysis of 21 cases.

BACKGROUND: Metastatic carcinoma to the jaws and oral region are very rare, representing less than 1% of all oral tumors. Unfortunately, oral metastasis is usually manifestation of an advanced stage of primary cancer, and indicates widespread disease and poor prognosis. MATERIAL AND METHODS: In this retrospective study, a total of 2039 patients with history of oral malignant tumor between 1980 and 2012 at Seoul National University Dental Hospital were evaluated. We analyzed the dental and medical records, and histopathological database of 2039 patients to assess the prevalence of oral metastasis of carcinoma in terms of sex and age, as well as, the most common origin of primary cancer, and prevalent site and histopathological type of metastatic carcinoma. RESULTS: Among 2039 patients, 21 (1.03%) were finally diagnosed with metastatic carcinoma of the jaws and oral region. Among the 21 patients, only 11 had a working diagnosis as oral metastasis upon clinical evaluation before performing a biopsy. The mean age at the time of diagnosis with a metastatic carcinoma was 56.86, and there was a male preponderance. Metastatic carcinoma was more frequent in the jaws than in the soft tissue, especially in the mandible compared to the maxilla. The most frequent primary site was the lungs, followed by the liver and breasts. The predominant histopathological types were hepatocellular carcinoma and adenocarcinoma. Patient outcomes indicated a poor prognosis with the time from the appearance of the metastasis to death was only 12 months. CONCLUSIONS: According to these cases, oral metastases of carcinoma were exceedingly rare in Koreans. It can allow the clinicians take into account the possible presence of metastases and lead to early diagnosis.

A randomized phase III trial of stereotactic radiosurgery (SRS) versus observation for patients with asymptomatic cerebral oligo-metastases in non-small-cell lung cancer.

BACKGROUND: It is unclear whether treating brain metastasis before starting systemic chemotherapy can improve survival compared with upfront chemotherapy in non-small-cell lung cancer (NSCLC) with asymptomatic cerebral oligo-metastases. PATIENTS AND METHODS: We undertook a randomized, controlled trial of 105 patients with one to four brain metastases, admitted to Samsung Medical Center between 2008 and 2013. Patients were randomly assigned to receive stereotactic radiosurgery (SRS) (49 patients) followed by chemotherapy or upfront chemotherapy (49 patients). The primary end point was overall survival (OS) and secondary end points included central nervous system (CNS) progression-free survival, progression to symptomatic brain metastasis and brain functional outcome. RESULTS: The median age was 58 years (range, 29-85) with ECOG 0-1 performance status, and 40% of patients were never smokers. Most patients had adenocarcinoma, and about half of patients had only one brain metastasis, while the rest had multiple cerebral metastases. The median OS time was 14.6 months [95% confidence interval (CI), 9.2-20.0] in the SRS group and 15.3 months (95% CI, 7.2-23.4) for the upfront chemotherapy group (P = 0.418). There was no significant difference in time to CNS disease progression [median, 9.4 months (SRS) versus 6.6 months (upfront chemotherapy), P = 0.248]. Symptomatic progression of brain metastases was observed more frequently in the upfront chemotherapy group (26.5%) than the SRS group (18.4%) but without statistical significance. CONCLUSIONS: Although this study included smaller sample size than initially anticipated due to early termination, SRS followed by chemotherapy did not improve OS in oligo-brain metastases NSCLC patients compared with upfront chemotherapy. Further study with large number of patients should be needed to confirm the use of upfront chemotherapy alone in this subgroup of patients. CLINICAL TRIALS NUMBER: NCT01301560.

Postoperative hypoalbuminemia as a risk factor for surgical site infection after oral cancer surgery.

OBJECTIVES: Postoperative surgical site infection (SSI) is a frequent postoperative complication in patients with oral cancer and significantly affects patient recovery and medical expenses. The aim of this study was to examine the predictors of SSI in patients undergoing major surgery for oral or oropharyngeal squamous cell carcinoma (OSCC) and to determine the relationship between perioperative albumin and the development of SSI. SUBJECTS AND METHODS: In 337 consecutive patients who underwent clean-contaminated surgery for OSCC, serum albumin, glucose, and hemoglobin levels were perioperatively measured. Differences between the groups were examined using Fisher's exact test, Mann-Whitney U-test, and multiple logistic regression analysis. RESULTS: Surgical site infection was detected in 88 (26.1%) patients with median time to development of 10 (2-25) days. Multiple logistic regression analysis showed that only postoperative serum albumin < 2.5 g dl(-1) was an independent variable predictive of SSI (P = 0.003). The duration of hospital stay was negatively correlated with postoperative albumin (R(2) = -0.302, P < 0.001). CONCLUSION: Early postoperative hypoalbuminemia <2.5 g dl(-1) is an independent risk factor for the development of SSI in patients undergoing oral cancer surgery. Clinicians should be aware of the implications of postoperative hypoalbuminemia and consider more intensive postoperative care in these patients.

Site-specific PEGylation enhances the pharmacokinetic properties and antitumor activity of interferon beta-1b.

Interferon beta (IFN-ß) is widely used to ameliorate disease progression in patients with Multiple Sclerosis. IFN-ß has a short half-life in humans, necessitating frequent administration for optimum effectiveness. Covalent modification of IFN-ß with polyethylene glycol (PEG) improves the pharmacokinetic properties of the protein, but can adversely affect the protein's in vitro bioactivity. Random modification of lysine residues in IFN-ß with amine-reactive PEGs decreased the in vitro bioactivity of the protein 50-fold, presumably due to modification of lysine residues near critical receptor binding sites. PEGylated IFN-ß proteins that retained high in vitro bioactivity could be obtained by selective modification of the N-terminus of the protein with PEG. Here we use site-specific PEGylation technology (targeted attachment of a cysteine-reactive-PEG to an engineered cysteine residue in IFN-ß) to identify several additional amino acid positions where PEG can be attached to IFN-ß without appreciable loss of in vitro bioactivity. Unexpectedly, we found that most of the PEG-IFN-ß analogs showed 11- to 78-fold improved in vitro bioactivities relative to their unPEGylated parent proteins and to IFN-ß-1b. In vivo studies showed that a lead PEG-IFN-ß protein had improved pharmacokinetic properties compared to IFN-ß and was significantly more effective than IFN-ß at inhibiting growth of a human tumor xenograft in athymic mice.

Porcine islet adaptation to metabolic need of monkeys in pig-to-monkey intraportal islet xenotransplantation.

BACKGROUND: Physiologic regulation of glucose metabolism is different between donor and recipient of xenogeneic pancreatic islet transplantation. We sought to assess whether the capacity of donor islets to adapt to recipient metabolic requirements should be considered in determining the success of pancreatic islet xenotransplantation. METHODS: Rhesus macaque hosts rendered diabetic by streptozotocin were transplanted with porcine islets into the liver. Porcine c-peptide and insulin levels as well as intravenous glucose tolerance test (IVGTT) were measured at intervals. RESULTS: At 2 months after islet transplantation, glucose responses on IVGTT showed a normoglycemic pattern. There was a 2.48 fold increase in C-peptide level during the initial 15 minutes of IVGTT in normal monkeys: from 3.122 ng/mL at baseline to 7.728 ng/mL at 15 minutes. Monkeys transplanted with porcine islets showed 2.38- and 2.45-folds the initial increases in C-peptide on IVGTT at 2 and 4 months after transplantation, respectively. Histopathologic evaluation identified the host endothelial cells having well lined the vessels of the porcine islets in the monkey liver. CONCLUSIONS: The glucose response on IVGTT of porcine islets engrafted in the monkey liver resembled the normal monkey pattern rather than that of pigs. The presence of monkey endothelial cells suggested that porcine islets were well adapted to the local environment of the recipient.

Acute necrotic stomatitis (noma) associated with methicillin-resistant Staphylococcus aureus infection in a newly acquired rhesus macaque (Macaca mulatta).

BACKGROUND: A newly acquired rhesus macaque was suffering from rapid destruction of the left cheek caused by necrotizing stomatitis. METHODS: To restore reconstructive surgery and intensive care with antibiotics, wound protection, wound healing agents, and debridement were applied. RESULTS: Staphylococcus aureus and Enterococcus faecalis were isolated from the culture of the lesion, and the antibiotic susceptibility test revealed methicillin-resistant Staphylococcus aureus infection. Vancomycin and ampicillin-sulbactam effectively treated the bacterial infections, and reconstructive surgery was performed once the infection was cleared. Topical application of recombinant human epidermal growth factor (rhEGF) was useful to treat exposed wound of the noma lesion. CONCLUSIONS: Simian noma associated with methicillin-resistant Staphylococcus aureus (MRSA) had not previously been reported in non-human primates. Although noma associated with MRSA is hard to cure because of its rapid and destructive progress, the aggressive therapy used in this study led to the successful resolution of an acute necrotic stomatitis lesion in a rhesus macaque.

Diagnostic dilemma of pseudoprogression in the treatment of newly diagnosed glioblastomas: the role of assessing relative cerebral blood flow volume and oxygen-6-methylguanine-DNA methyltransferase promoter methylation status.

BACKGROUND AND PURPOSE: Methylation of the MGMT gene promoter is associated with a favorable prognosis in adult patients with GBM treated with TMZ. We determined the incidence of pseudoprogression according to the MGMT methylation status and the potential value of DSC perfusion MR images for predicting pseudoprogression. MATERIALS AND METHODS: New or enlarged enhancing lesions after CCRT in adult patients with newly diagnosed GBMs were prospectively assessed by measuring their rCBV by using DSC perfusion MR images. Tumor tissue was assayed to determine MGMT promoter methylation status. All patients were regularly followed up at an interval of 2 months by MR images, including DSC perfusion MR images. RESULTS: Ninety eligible patients were enrolled in this study. After CCRT, new or enlarged enhanced lesions were found in 59 of 90 patients, which were subsequently classified as pseudoprogression (26 patients, 28.9%) and real progression (33 patients, 36.7%). Overall, there was a significant difference in the mean rCBV between pseudoprogression and real tumor progression (P = .003). The ROC curve revealed that an rCBV ratio >1.47 had an 81.5% sensitivity and a 77.8% specificity. The unmethylated MGMT promoter group had a significant difference of mean rCBV between pseudoprogression and real progression (P = .009), though the methylated MGMT promoter group had no significant difference (P = .258). CONCLUSIONS: The current study suggests that rCBV measured by DSC perfusion MR images has a differential impact on the predictability of pseudoprogression in patients with GBM.

A cluster of Teflon pledgets manifesting as an intrathoracic cavitary mass following lung resection.

Teflon pledgets are widely used for hemostasis and the reinforcement of friable tissue in surgery. However, rare but serious complications caused by the erosion of Teflon pledgets have been reported. We present an unusual case of an intrathoracic cavitary mass that was formed by the erosion of a cluster of Teflon pledgets into the lung parenchyma eight years after a lung resection.

Functional evaluation of chondrocyte sheeting immunodelusive immunoisolated bioartificial pancreas.

In islet transplantation, encapsulation of immunoisolated islets may provide a way to protect the graft from immune attacks with no immunosuppression. To develop an immunodelusive immunoisolated bioartificial pancreas (BAP), chondrocyte sheets were prepared by cell sheet engineering. We made an immunoisolated BAP encapsulated with rodent-derived chondrocyte sheets and then evaluated its function. Sprague-Dawley rats were used as the source of auricular cartilage and chondrocytes were maintained and expanded by passages. Lewis rats were prepared for islet isolation. A 3-dimensional chondrocyte sheeting immunodelusive immunoisolated BAP (CSI-BAP) was created by multi-layering and unifying the chondrocyte sheets. Subsequently, islets were embedded between each multi-layer sheet. To evaluate the function of the CSI-BAP, a glucose challenge test was performed and secretion of insulin in the culture medium was measured by an enzyme-linked immunosorbent assay. When observed by phase-contrast microscopy, the CSI-BAP maintained close connections between chondrocyte sheets. Islets in the CSI-BAP maintained viability at day 10 and showed good insulin secretion, as revealed by a prompt reaction to increased concentrations of glucose at days 5 and 10. In long-term culture, the CSI-BAP maintained its ability to secrete insulin for 8 weeks. This BAP technology could be an important tool for successful islet transplantation without immunosuppressive drugs.

Proliferation of pancreatic endocrine cells using disaggregation-expansion-reaggregation technology in isolated rat islets.

Donor scarcity is a major obstacle for clinical islet transplantation. Hence, the effective use of the limited number of available islets is necessary for successful islet transplantation. We have developed a new technology that could produce pseudo-islets. Morphologic and functional evaluation was performed to test the feasibility of using these cells for transplantation. A 3-step procedure known as disaggregation-expansion-reaggregation (DER) was employed for pseudo-islet preparation. Islets isolated from 200 to 250-g male Lewis rats by collagenase digestion were separated into single cells by trypsinization. These pancreatic endocrine cells (PECs) were expanded by serial passages in culture before being aggregated at a high cell-density in a suspended state. After DER, cells were morphologically analyzed over time, and gene expression evaluated by reverse transcriptase polymerase chain reaction (RT-PCR). Through expansion by passage for 2 weeks in continuous cultures, approximately 1 million PECs were recovered after aggregation. By phase-contrast microscopy, they presented with spherical shapes and similar sizes compared with naïve islets (50-800 microm). RT-PCR results indicated expression of insulin, glucagon, and pancreatic and duodenal homeobox gene 1, which were observed in primary isolated islets as well. The insulin secretion capacity of pseudo-islets was confirmed by enzyme-linked immunosorbent assay. In conclusion, PECs treated with DER showed potential to serve as a cell source for pseudo-islet generation after in vitro cellular expansion. These cells were both morphologically and genetically similar to naïve islets. Our new technique could be a potential method to overcome the scarcity of donor islets in the near future.

Therapeutic protein-drug interactions and implications for drug development.

Many intrinsic and extrinsic factors can affect an individual patient's drug exposure and response. The US Food and Drug Administration (FDA) has published a number of guidances that recommend how and when to evaluate these factors during drug development. The most recent FDA draft guidance on drug interactions provides advice for in vitro and in vivo drug interaction studies, including suggestions for study design, dosing strategies and analysis, and interpretation of data for medical product labels. The draft guidance updated the FDA's recommendations on the evaluation of important cytochrome P450 (CYP) enzyme- and transporter-based drug interactions during drug development.

Difficult diagnosis of a double omental hernia that led to extensive small bowel infarction.

A 47-year-old female complained of abdominal pain in the epigastrium for about 2 h after a meal. At the initial abdominal radiograph, there were no findings of remarkable bowel loops. On the following day of hospitalization, the pain became worse; moreover, it could not be controlled by medicine. Also, a dilated bowel loop was detected on the imaging studies. When exploring the peritoneal cavity, we found a strangulated small bowel that protruded through the lesser omental sac within the defects of the gastrocolic or gastrohepatic ligaments. After performing manual reduction, the restoring viability of herniated small bowel failed; consequently, extensive small bowel resection was mandatory. Herein we reported a case of extensive small bowel hemorrhagic infarction due to a double omental hernia that was not diagnosed at the time of visiting the emergency department.

Functional improvement of pig islet with exocrine encapsulation.

Porcine-specific obstacles in islet isolation frequently result from the low purity or contamination with exocrine tissues. We implemented a new technique involving as capsulation of islets with excess exocrine tissue as a beneficial material to address those difficulties. Pig islets were hand-picked as high purity (HI) or low purity (LO) islets containing significant amounts of exocrine tissue. We performed static (ST) or shaking (SK) cultures of HI and LO islets. Islet function was examined after 24 hours by a glucose challenge test. Insulin secretion into the culture media was continuously measured using ELISA during a 6-day culture period. Islet function after 24 hours exhibited better maintenance under SK than ST culture as assessed by a stimulation index. The ideal islet morphology was seen in LO islets at 3 days of SK culture with typical islet shapes of a smooth surface and a spherical configuration. In contrast, typical islet morphology was not observed in HI islets under SK culture; maintenance of typical spherical appearances was difficult. Insulin secretion from LO islets under SK culture was higher than under other conditions during the 6-day period. Under SK culture conditions, exocrine-encapsulated LO islets showed enhanced islet function by condensing loose islet aggregates into firm spheroids with native exocrine tissues as a natural scaffold.

Trastuzumab treatment improves brain metastasis outcomes through control and durable prolongation of systemic extracranial disease in HER2-overexpressing breast cancer patients.

In patients with human epidermal growth factor receptor-2 (HER2)-overexpressing breast cancer, treatment with trastuzumab has been shown to markedly improve the outcome. We investigated the role of trastuzumab on brain metastasis (BM) in HER2-positive breast cancer patients. From 1999 to 2006, 251 patients were treated with palliative chemotherapy for HER2-positive metastatic breast cancer at Samsung Medical Center. The medical records of these patients were analysed to study the effects of trastuzumab on BM prevalence and outcomes. Patients were grouped according to trastuzumab therapy: pre-T (no trastuzumab therapy) vs post-T (trastuzumab therapy). The development of BM between the two treatment groups was significantly different (37.8% for post-T vs 25.0% for pre-T, P=0.028). Patients who had received trastuzumab had longer times to BM compared with patients who were not treated with trastuzumab (median 15 months for post-T group vs 10 months for pre-T group, P=0.035). Time to death (TTD) from BM was significantly longer in the post-T group than in the pre-T group (median 14.9 vs 4.0 months, P=0.0005). Extracranial disease control at the time of BM, 12 months or more of progression-free survival of extracranial disease and treatment with lapatinib were independent prognostic factors for TTD from BM.

Enhanced circulating half-life and antitumor activity of a site-specific pegylated interferon-alpha protein therapeutic.

Recombinant interferon alpha-2 (IFN-alpha2) has proven useful for treating a variety of human cancers and viral diseases. IFN-alpha2 has a short circulating half-life in vivo, which necessitates daily or thrice weekly administration to patients. It is possible to extend the circulating half-life of IFN-alpha2 by random modification of lysine residues in the protein with polyethylene glycol (PEG); however, such preparations have heterogeneous structures and low specific activities, and may not provide optimal therapeutic benefits to patients. A long-acting, site-specific, monoPEGylated IFN-alpha2 protein has now been created by targeted attachment of a 20 kDa or a 40 kDa maleimide-PEG to a cysteine analogue of IFN-alpha2, M111C. In vitro bioactivities of the purified 20 kDa and 40 kDa PEG-M111C proteins were within 2- to 3-fold of those of wild type IFN-alpha2 and 7- to 10-fold better than that of a 40 kDa PEG IFN-alpha2 protein created using nontargeted, amine-PEGylation methodology. The 20 kDa and 40 kDa PEG-M111C proteins demonstrated 26- to 38-fold longer half-lives, respectively, than IFN-alpha2 following subcutaneous administration to rats. The 20 kDa PEG M111C protein inhibited growth of human NIH:OVCAR-3 cells transplanted into nude mice by >90%, as measured by tumor size, tumor weight, and number of animals with detectable tumors at necropsy, and was significantly more effective than a comparable dose of IFN-alpha2. These data extend our previous findings that bioactivity of IFN-alpha2 can be largely preserved by targeted attachment of PEG moieties to nonessential sites in the protein and provide evidence that site-specific PEGylated IFN-alpha2 proteins possess enhanced tumoricidal properties in vivo.