Monobody adapter for functional antibody display on nanoparticles for adaptable targeted delivery applications.

Vascular endothelial cells (ECs) play a central role in the pathophysiology of many diseases. The use of targeted nanoparticles (NPs) to deliver therapeutics to ECs could dramatically improve efficacy by providing elevated and sustained intracellular drug levels. However, achieving sufficient levels of NP targeting in human settings remains elusive. Here, we overcome this barrier by engineering a monobody adapter that presents antibodies on the NP surface in a manner that fully preserves their antigen-binding function. This system improves targeting efficacy in cultured ECs under flow by >1000-fold over conventional antibody immobilization using amine coupling and enables robust delivery of NPs to the ECs of human kidneys undergoing ex vivo perfusion, a clinical setting used for organ transplant. Our monobody adapter also enables a simple plug-and-play capacity that facilitates the evaluation of a diverse array of targeted NPs. This technology has the potential to simplify and possibly accelerate both the development and clinical translation of EC-targeted nanomedicines.

High susceptibility of p53(+/-) knockout mice in N-butyl-N-(4-hydroxybutyl)nitrosamine urinary bladder carcinogenesis and lack of frequent mutation in residual allele.

The loss of p53 functions is considered to compromise the growth-suppression machinery of the cell and facilitate neoplastic change. In humans, genetic alteration in the p53 gene is one of the most frequently observed molecular changes in tumors, including urinary bladder carcinomas. We have investigated the susceptibility of heterozygote p53 knockout mice to N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in terms of urinary bladder tumor induction. Both p53(+/-) knockout mice and C57BL/6 original parent strain were administered 0, 0.002, 0.004, 0.0075 and 0.025% BBN in the drinking water for 20 weeks. As compared with the C57BL/6 strain, greater lesion yields were observed in knockout mice after 20 weeks of treatment. Transitional cell carcinomas were found in 9 (75%) and 12 (100%) of each 12 mice of the 0.0075 and 0.025% BBN treatment groups, respectively, whereas only 1 (11%) and 6 (67%) of each 9 of the C57BL/6 mice demonstrated tumors. Preneoplastic lesions (dysplasia) were also observed more frequently in the lower dose groups in the knockout mice than C57BL/6 mice. PCR single-strand conformation polymorphism analysis followed by DNA direct sequencing of the p53 gene (exons 5-8) extracted from bladder tumors demonstrated mutations in 3 of 11 (27.3%; exon 7) and 8 of 29 (27.6%; exons 5-8) tumors in C57BL/6 and knockout mice, respectively. There was no significant difference in the mutation rates at the residual p53 gene between the two cases. All mutations observed in knockout mice were restricted to the normal allele, and none were present in the gene-targeted null allele. In a separate experiment, 5-bromo-2'-deoxyuridine labeling indices after treatment with BBN for 2 or 4 weeks were significantly higher in knockout mice than wild-type mice. Measurement of the urinary concentration of N-butyl-N-(3-carboxypropyl)nitrosamine, a proximate carcinogenic metabolite, revealed no significant differences between knockout and original parent strain after administration of 0.0075% BBN in the drinking water for 4 weeks. In conclusion, knockout mice are distinctly more sensitive to urinary bladder carcinogenesis induced by BBN than their original parent strain, as evidenced by elevated DNA synthesis during carcinogen administration and an increased tumor yield. The high susceptibility of p53 knockout mice appeared to be related to the high level of cell proliferation rather than that of N-butyl-N-(3-carboxypropyl)nitrosamine in the urine or that of mutations at the p53 gene.

Solution conformation and amyloid-like fibril formation of a polar peptide derived from a beta-hairpin in the OspA single-layer beta-sheet.

A 23-residue peptide termed BH(9-10) was designed based on a beta-hairpin segment of the single-layer beta-sheet region of Borrelia OspA protein. The peptide contains a large number of charged amino acid residues, and it does not follow the amphipathic pattern that is commonly found in natural beta-sheets. In aqueous solution, the peptide was highly soluble and flexible, with a propensity to form a non-native beta-turn. Trifluoroethanol (TFE) stabilized a native-like beta-turn in BH(9-10). TFE also decreased the level of solubility of the peptide, resulting in peptide precipitation. The precipitation process accompanied a conformational conversion to a beta-sheet structure, as judged with circular dichroism spectroscopy. The precipitate was found to be fibrils similar to those associated with human amyloid diseases. The fibrillization kinetics depended on peptide and TFE concentrations, and had a nucleation step followed by an assembly step. The fibrillization was reversible, and the dissociation reaction involved two phases. TFE appears to induce the fibrils by stabilizing a beta-sheet conformation of the peptide that optimally satisfies hydrogen bonding and electrostatic complementarity. This TFE-induced fibrillization is quite unusual, because most amyloidogenic peptides form fibrils in aqueous solution and TFE disrupts these fibrils. Nevertheless, the BH(9-10) fibrils have similar structure to other fibrils, supporting the emerging idea that polypeptides possess an intrinsic ability to form amyloid-like fibrils. The high level of solubility of BH(9-10), the ability to precisely control fibril formation and dissociation, and the high-resolution structure of the same sequence in the beta-hairpin conformation in the OspA protein provide a tractable experimental system for studying the fibril formation mechanism.

The fibronectin type III domain as a scaffold for novel binding proteins.

The fibronectin type III domain (FN3) is a small autonomous folding unit which occurs in many animal proteins involving in ligand binding. The beta-sandwich structure of FN3 closely resembles that of immunoglobulin domains. We have prepared a phage display library of FN3 in which residues in two surface loops were randomized. We have selected mutant FN3s which bind to a test ligand, ubiquitin, with significant affinities, while the wild-type FN3 shows no measurable affinity. A dominant clone was expressed as a soluble protein and its properties were investigated in detail. Heteronuclear NMR characterization revealed that the selected mutant protein retains the global fold of FN3. It also has a modest conformational stability despite mutations at 12 out of 94 residues. These results clearly show the potential of FN3 as a scaffold for engineering novel binding proteins.

Formation of the single-layer beta-sheet of Borrelia burgdorferi OspA in the absence of the C-terminal capping globular domain.

Borrelia outer surface protein A (OspA) contains a unique single-layer beta-sheet that connects N and C-terminal globular domains. This single-layer beta-sheet segment (beta-strands 8-10) is highly stable in solution, although it is exposed to the solvent on both faces of the sheet and thus it does not contain a hydrophobic core. Here, we tested whether interactions with the C-terminal domain are essential for the formation of the single-layer beta-sheet. We characterized the solution structure, dynamics and stability of an OspA fragment corresponding to beta-strands 1-12 (termed OspA[27-163]), which lacks a majority of the C-terminal globular domain. Analyses of NMR chemical shifts and backbone nuclear Overhauser effect (NOE) connectivities showed that OspA[27-163] is folded except the 12th and final beta-strand. (1)H-(15)N heteronuclear NOE measurements and amide H-(2)H exchange revealed that the single-layer beta-sheet in this fragment is more flexible than the corresponding region in full-length OspA. Thermal-denaturation experiments using differential scanning calorimetry and NMR spectroscopy revealed that the N-terminal globular domain in the fragment has a conformational stability similar to that of the same region in the full-length protein, and that the single-layer beta-sheet region also has a modest thermal stability. These results demonstrate that the unique single-layer beta-sheet retains its conformation in the absence of its interactions with the C-terminal domain. This fragment is significantly smaller than the full-length OspA, and thus it is expected to facilitate studies of the folding mechanism of this unusual beta-sheet structure.

The roles of turn formation and cross-strand interactions in fibrillization of peptides derived from the OspA single-layer beta-sheet.

We previously demonstrated that a beta-hairpin peptide, termed BH(9-10), derived from a single-layer beta-sheet of Borrelia OspA protein, formed a native-like beta-turn in trifluoroethanol (TFE) solution, and it assembled into amyloid-like fibrils at higher TFE concentrations. This peptide is highly charged, and fibrillization of such a hydrophilic peptide is quite unusual. In this study, we designed a circularly permutated peptide of BH(9-10), termed BH(10-9). When folded into their respective beta-hairpin structures found in OspA, these peptides would have identical cross-strand interactions but different turns connecting the strands. NMR study revealed that BH(10-9) had little propensity to form a turn structure both in aqueous and TFE solutions. At higher TFE concentration, BH(10-9) precipitated with a concomitant alpha-to-beta conformational conversion, in a similar manner to the BH(9-10) fibrillization. However, the BH(10-9) precipitates were nonfibrillar aggregation. The precipitation kinetics of BH(10-9) was exponential, consistent with a first-order molecular assembly reaction, while the fibrillization of BH(9-10) showed sigmoidal kinetics, indicative of a two-step reaction consisting of nucleation and molecular assembly. The correlation between native-like turn formation and fibrillization of our peptide system strongly suggests that BH(9-10) adopts a native-like beta-hairpin conformation in the fibrils. Remarkably, seeding with the preformed BH(10-9) precipitates changed the two-step BH(9-10) fibrillization to a one-step molecular assembly reaction, and disrupted the BH(9-10) fibril structure, indicating interactions between the BH(10-9) aggregates and the BH(9-10) peptide. Our results suggest that, in these peptides, cross-strand interactions are the driving force for molecular assembly, and turn formation limits modes of peptide assembly.

Decreased VLDL apoprotein CII/apoprotein CIII ratio may be seen in both normotriglyceridemic and hypertriglyceridemic patients on chronic hemodialysis treatment.

Pathogenetic factors that may be related to uremic hypertriglyceridemia were studied in 27 patients who had been undergoing chronic hemodialysis treatment for over two years. They were divided into two groups consisting of 14 hypertriglyceridemic (HTG) patients with fasting serum triglycerides (TG) of 170 mg/dL or higher, aged 45 +/- 11 yr (mean +/- SD) and 13 normotriglyceridemics (NTG) with serum TG less than 170 mg/dL aged 42 +/- 9 yr. Serum lipid, lipoprotein [low density lipoprotein (LDL) and very low density lipoprotein (VLDL)] and apoprotein (Apo) levels, as well as ultracentrifugally obtained VLDL apo subfractions and serum carnitine were compared between the two groups, which enabled us to rule out various factors inherent to uremic state and present in both groups. The HTG group of patients, who showed (by definition) significantly elevated TG (300 +/- 167 mg/dL v 123 +/- 30 mg/dL in NTG) and VLDL levels, concomitantly showed significantly increased serum total cholesterol (P less than .001) and LDL (P less than .001), and significantly decreased apo AI/apo B, or an index of risk of atherogenesis (P less than .05). Serum apo CII (7.3 +/- 3.3 mg/dL v 3.6 +/- 1.0 mg/dL in NTG), apo E (4.8 +/- 2.8 mg/dL v 2.9 +/- 1.3 mg/dL) and VLDL/serum apo CII (38 +/- 18 v 22 +/- 12), ie, the amount of VLDL covered by a unit of apo CII, were elevated in the HTG compared with the NTG group of patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Harmful effects of inotropic agents on myocardial protection.

Using an isolated working rat heart model, the pretreatment effects of positive inotropic agents on ischemia-reperfusion injury were investigated. The experiment consisted of (1) working control perfusion; (2) working perfusion with isoproterenol (I), milrinone (M), a combination of these drugs (I + M) and dibutyl-cyclic adenosine monophosphate (DB) followed by ischemic arrest for 33 minutes at 37 degrees C or 150 minutes at 20 degrees C and Langendorff reperfusion; and (3) working perfusion. Under conditions of normothermic ischemia, percent recoveries of postischemic cardiac output (mean +/- standard error of the mean) in the I, M, I + M, and DB groups were 37.8% +/- 12.7%, 61.3% +/- 3.1%, 0%, and 53.1% +/- 5.2%, respectively. Under conditions of hypothermic ischemia, the percent recoveries in I + M and DB groups were 10.9% +/- 7.9% and 29.8% +/- 9.5%; they were all significantly lower than that in the control group. The addition of diltiazem or ryanodine at several concentrations and lowering of the Ca2+ concentration in the St. Thomas' cardioplegic solution did not prevent I + M-induced injury. Our data suggest that pretreatment by I + M aggravated ischemia-reperfusion injury, and adjustments in Ca2+ concentration were not sufficient to prevent that injury.

Excision of cavernous angioma with preservation of coexisting venous angioma. Case report.

The case of a patient presenting with dysphasic seizures due to a cavernous angioma coexisting with a venous malformation is reported. The cavernous angioma was resected with preservation of the venous malformation, as confirmed by postoperative studies. The patient was seizure-free following surgery.

Effect of cigarette smoke on the mutagenic activation of environmental carcinogens by rodent liver.

In order to assess the effect of cigarette smoke (CS) on metabolic enzymes, male hamsters and rats were exposed for two weeks to smoke produced in a Hamburg type II smoking machine. The livers were then used for Ames liquid incubation and western immunoblot assays. Mutagenic activities of seven heterocyclic amines (HCAs) in Salmonella typhimurium TA98 in the presence of rat or hamster liver S9 were elevated up to 3.7 times above controls (including sham smoke control). Enhancement of mutagenic activities of PhIP and aflatoxin B(1) was observed only in CS-exposed hamster, whereas no significant alteration of mutagenicity was observed with 2-aminofluorene, benzo[a]pyrene, and 3'-hydroxymethyl-N, N-dimethyl-4-aminoazobenzene in strain TA98 or with six N-nitrosodialkylamines in strain TA100. 7,8-Benzoflavone and/or furafylline considerably inhibited the mutagenic activation of IQ and Trp-P-1 in the presence of liver S9 from untreated hamsters and sham smoke- or CS-exposed hamsters and rats, indicating the predominant involvement of hamster cytochrome P450 (CYP) 1A enzymes in the metabolic activation of HCAs. In addition, the data suggest that CS-exposure may selectively induce hepatic CYP1A1/1A2 isoforms. Western immunoblot analyses of liver microsomes using anti-rat CYP antibodies revealed that CS-exposure increased the levels of hamster CYP1A2 (3.9-fold) and rat CYP1A2 (3.0-fold) and CYP1A1, without significant change in the levels of CYP2E1 and CYP2B and 3A isoforms in each species. The presently observed selective induction of HCA activation and CYP isozymes due to CS supports the idea that CS may contribute to enhancing effects on initiation by carcinogens which are metabolically activated by hepatic CYP1A1/1A2. In conjunction with results observed for smokers, the present findings indicate that the hamster is a good animal for studies with CS, and that cigarette smoking in combination with intake of heating protein-rich foods as a life style may markedly contribute to the human carcinogenesis by HCAs.

Primary intracranial solitary leptomeningeal glioma: a report of 3 cases.

Primary intracranial solitary leptomeningeal gliomas are exceedingly rare. We, therefore, performed a detailed clinical, radiological and pathological analysis to better characterize these tumors in 3 patients (33- and 72-year-old men and a 72-year-old woman). Two of the tumors were located in the frontal region and 1 in the temporal region. Magnetic resonance imaging revealed a well circumscribed large lesion (maximal diameter 4 - 6 cm) with peritumoral edema, mixed low- and isosignal intensity on T1-weighted images, hypersignal intensity on T2-weighted images and non-homogeneous contrast enhancement. External carotid angiography demonstrated a vascular supply to these tumors via branches of the middle meningeal artery. Gross total resection was achieved in all patients. The pathological diagnosis was glioblastoma in 2 patients and oligodendroglioma in 1. The MIB-1 nuclear labeling index ranged from 11.8% - 23.6% (mean 18.2%). Local tumor recurrence was documented in 2 patients after 8 and 11 months, respectively. The other patient with glioblastoma developed a metastasis to the femur 39 months after craniotomy. A definitive diagnosis can be made by careful radiological assessment and histopathological examination.

The importance of the real-time fluoroscopic intraoperative direct cholangiogram in the laparoscopic cholecystectomy using a new instrument.

BACKGROUND/AIMS: Laparoscopic cholecystectomy (LC) has become an accepted standard operative technique for gallstone treatment worldwide. On the other hand, complications, such as bile duct injuries, have been reported recently with the expansion of indication for LC. Intraoperative cholangiogram (IOC), to minimize the risk of bile duct injury, is now considered to be essential for safe LC. There are disadvantages to IOC such as increased operating time, the possibility of bile duct injury and the difficulties of manipulation. MATERIAL AND METHODS: We have developed a method for real-time fluoroscopic cholangiograms using a new instrument designed by our group for safe LC. First, a round-tip stylet is inserted through a sheath to coax it gently through the spiral valves of the cystic duct. Secondly, the stylet is removed and the cholangiogram catheter is inserted smoothly. Digital C-arm fluoroscopy provides "real-time" imaging of biliary tree. As a result, we became able to obtain a clear cholangiogram easily in a very short time. RESULTS: In the first 136 patients, direct cholangiograms were attempted in 106 cases and successfully completed in 102 cases (96.2%). CONCLUSION: With the development of real-time fluoroscopic intraoperative direct cholangiogram, we are able to cope with bile duct injuries and anomalies, and unsuspected bile duct stones.

A case of small cell carcinoma of the stomach.

A case of small cell carcinoma of the stomach is reported. A 53-year-old male was referred to our hospital for elective surgery for gastric cancer. Pre-operative examinations revealed no metastases. Gastrectomy was performed curatively, and there were no gross findings of metastases. Histologically, the tumor was composed of intermediate-sized cells with hyper-chromatic nuclei and scanty cytoplasm. These cells were argyrophilic and positive for chromogranin A. A small portion of the tumor consisted of conventional adenocarcinoma (signet ring cell carcinoma and tubular adenocarcinoma). No lymph node metastasis was observed microscopically. However, 7 months after the operation, splenic and hepatic metastases were detected, and the patient died very soon thereafter. Small cell carcinoma of the stomach is a very rare disease. In literature, only 15 cases have been cured surgically. Among them, only one case had been diagnosed as small cell carcinoma before the operation, which suggests the difficulty of pre-operative diagnosis. The prognosis of this disease is very poor compared with the common type of gastric carcinoma. Considering the poor prognosis of this particular disease, adjuvant chemotherapy might be mandatory in all cases even if surgically curative resection is performed.

[The roles of anesthetics and daily used drugs in cardiovascular changes during normovolemic hemodilution (NVHD)].

The changes of cardiovascular parameters and serum cathecholamine levels associated with normovolemic hemodilution (NVHD) were studied under three different conditions: Group 1; the patients for cardiac surgery who were taking cardiac drugs, and were anesthetized with fentanyl 30 micrograms.kg-1, Group 2; the patients with no-cardiac disease and taking no drugs, who were anesthetized with fentanyl 30 micrograms.kg-1 and Group 3; the patients with no-cardiac disease and taking no drugs, who were anesthetized with 0.75% halothane. Cardiac function was compared among three groups. After NVHD, blood pressure and heart rate of group 3 were significantly higher than those of group 1 and 2. Moreover, serum epinephrine and norepinephrine were elevated significantly after NVHD in only group 3. From this study, we conclude that, (1) daily used drugs do not predict hypotension during NVHD, and (2) high dose fentanyl anesthesia is associated with hypotension during NVHD.

[Clinical experience of a subcutaneously implantable drug delivery catheter (PORT-A-CATH)].

A drug delivery catheter system with subcutaneously implantable port, PORT-A-CATH, was applied for intra-arterial, intravenous and intraperitoneal chemotherapy and hyperalimentation in treating 99 cancer patients. The average implantation period was 135.1 days and this system was applied for more than one year in 5 cases. Any troubles due to port or catheter materials were not observed during the study. Catheter occlusion occurred in 11 cases infection in 7 (catheter-related infection 4, pocket infection 3), and skin necrosis in 4. This system was proved useful to reduce the risk of infection and enabled easy and safe long-term repeated administration, compared to the catheters with external end. Intra-arterial chemotherapy became possible to the outpatients with the use of this system, which seemed to contribute for the improvement of quality of life of the patients.

[Clinical evaluation of cisplatin in children with malignant solid tumors. Pediatric Cisplatin Study Group].

A cooperative multicenter clinical study on cisplatin in children with malignant solid tumors was conducted in seventeen institutions. Of 63 children entered into the study, 18 patients were treated with cisplatin alone, 33 with a VCAP regimen (VCR, CPA, ADM and CDDP) and 12 with other combination regimens. The numbers of evaluable patients were 14, 27 and 7, respectively. Response rates for neuroblastoma were 37.5% (3/8) with cisplatin alone and 79.2% (19/24) for the VCAP regimen. Major adverse effects were gastrointestinal symptoms, bone marrow suppression and renal impairment. Hearing difficulty, electrolyte imbalance and transient elevation of transaminase were also observed. However, these adverse effects were within a tolerable range of severity. The results of this study demonstrate that cisplatin is a useful drug in the treatment of neuroblastoma.