Increased stability and specificity through combined hybridization of peptide nucleic acid (PNA) and locked nucleic acid (LNA) to supercoiled plasmids for PNA-anchored "Bioplex" formation.

Low cellular uptake and poor nuclear transfer hamper the use of non-viral vectors in gene therapy. Addition of functional entities to plasmids using the Bioplex technology has the potential to improve the efficiency of transfer considerably. We have investigated the possibility of stabilizing sequence-specific binding of peptide nucleic acid (PNA) anchored functional peptides to plasmid DNA by hybridizing PNA and locked nucleic acid (LNA) oligomers as "openers" to partially overlapping sites on the opposite DNA strand. The PNA "opener" stabilized the binding of "linear" PNA anchors to mixed-base supercoiled DNA in saline. For higher stability under physiological conditions, bisPNA anchors were used. To reduce nonspecific interactions when hybridizing highly cationic constructs and to accommodate the need for increased amounts of bisPNA when the molecules are uncharged, or negatively charged, we used both PNA and LNA oligomers as "openers" to increase binding kinetics. To our knowledge, this is the first time that LNA has been used together with PNA to facilitate strand invasion. This procedure allows hybridization at reduced PNA-to-plasmid ratios, allowing greater than 80% hybridization even at ratios as low as 2:1. Using significantly lower amounts of PNA-peptides combined with shorter incubation times reduces unspecific binding and facilitates purification.

Protease-induced release of functional peptides from bioplexes.

Linking peptide functions directly to nucleic acids can be used to improve transfection. We have previously demonstrated this by sequence-specific hybridization of a bifunctional peptide nucleic acid (PNA) consisting of a nucleic acid binding moiety conjugated to a peptide. The resulting biological complex of PNA/DNA is called a Bioplex. The bifunctional PNA is continuously synthesized with one or more functional entities. For certain applications, it might be preferable to eliminate a functional entity after it has served its purpose. We have addressed this issue by adding a specific protease cleavage site to the construct. In this first approach, cathepsin L was used to cleave a linker sequence including a cathepsin L site: afrsaaq, thereby releasing the tri-peptide Arg-Gly-Asp (RGD) from the PNA anchor. In vitro and in vivo experiments showed an efficient cleavage of the peptide. Moreover, bifunctional PNA constructs were shown to retain activity of the second entity following removal of the first function. Since cathepsin L is ubiquitously expressed in eukaryotic cells and becomes active as the endosomal pH drops, inclusion of cathepsin sites makes it possible to remove functional entities in late endosomes/early lysosomes.

Adding functional entities to plasmids.

Non-viral gene therapy constitutes an alternative to the more common use of viral-mediated gene transfer. Most gene transfer methods using naked DNA are based upon non-sequence-specific interactions between the nucleic acid and cationic lipids (lipoplex) or polymers (polyplex). We have developed a technology in which functional entities hybridize in a sequence-specific manner to the nucleic acid (bioplex). This technology is still in its infancy, but has the potential to become a useful tool, since it allows the construction of highly defined complexes containing a variety of functional entities. In its present form the bioplex technology is based upon the use of peptide/nucleic acids (PNA) as anchors. Single, or multiple, functional entities are directly coupled to the anchors. By designing plasmids, or oligonucleotides, with the corresponding anchor target sequence, complexes with desired composition can easily be generated. The long-term aim is to combine functional entities in order to achieve optimal, synergistic interactions allowing enhanced gene transfer in vivo.

T-cell-derived malignant lymphoma in the boxer breed.

The boxer breed of dog is at high risk for a variety of neoplasms including lymphoma. In this observational study, tissue sections from boxer dogs with lymphoma were immunostained for T and B lymphocyte distinction, and the results compared with similar studies carried out on lymphoma tissues from temporally selected cohorts of golden retriever and rottweiler dogs. The frequency of T-cell lymphomas was significantly (P < 0.001 for all comparisons) higher in the boxers than in the rottweilers or golden retrievers. We are unaware of other reports linking immunotype of canine lymphoma with breed; whether other brachycephalic breeds of dogs have a similar preponderance of T-cell lymphoma awaits further study.

Evaluation of single-agent mitoxantrone as chemotherapy for relapsing canine lymphoma.

Many chemotherapeutic regimens will induce remission in dogs with lymphoma, but almost all dogs suffer relapse. Mitoxantrone was selected for evaluation as single-agent chemotherapy for relapsing canine lymphoma based on its use in humans undergoing salvage chemotherapy for non-Hodgkin's lymphoma and its tumoricidal effect against canine lymphoma. Dogs entered into study had multicentric lymphoma, and all had been treated solely with a standard combination chemotherapy protocol. At 1st relapse, all dogs were again staged and underwent lymph node biopsy. Mitoxantrone was administered IV at 6 mg/m2 every 21 days. Dogs were evaluated for lymphadenopathy before each dose of mitoxantrone. Fifteen dogs were entered into study. The average age (+/- SEM) of the dogs studied was 7.7 +/- 0.91 years, and most dogs were large (mean +/- SEM weight, 24.44 +/- 2.15 kg). Twelve dogs (80%) had B-cell lymphoma, and 3 had T-cell lymphoma. Dogs were staged IV (n = 12) or V (n = 3). The median duration of chemotherapy before entry into the study was 98 days. Overall median duration of response after mitoxantrone chemotherapy was 21 days. Complete responses were attained in 7 of 15 dogs (47%) with a median response duration of 84 days. Nine of 15 (60%) dogs attained a complete remission with additional chemotherapy after failing mitoxantrone chemotherapy. Mild toxicities were observed after mitoxantrone administration. No adverse reactions were observed during mitoxantrone infusions. The results of this study demonstrate that mitoxantrone, as a single agent, has limited value for dogs with lymphoma at 1st relapse after conventional multidrug chemotherapy.

Acute reactions in dogs treated with doxorubicin: increased frequency with the use of a generic formulation.

During a 4-month period, 34 dogs with tumors received a total of 60 doses of a single generic formulation of doxorubicin; 13 acute drug reactions were observed in these 34 dogs, and no acute reactions were observed after replacing the product with the proprietary brand. These reactions were characterized by one or more of the following signs: pruritus; head-shaking; urticaria; erythema of the pinnal, axillary, or inguinal regions; vocalization; vomiting; hyperemic or pale mucous membranes; high heart rate; and high respiratory rate. We propose that a component unique to generic doxorubicin was responsible for the unusually high number of acute drug reactions observed.

Osteogenic sarcoma and cisplatin chemotherapy in dogs: 16 cases (1986-1989).

Sixteen dogs, given adjuvant cisplatin chemotherapy after amputation for osteogenic sarcoma of the appendicular skeleton, had a median survival time of 413 days. Ten dogs (62%) were alive 1 year after amputation. Dogs were given cisplatin at a dosage of 50 mg/m2 of body surface every 4 weeks for a total of 6 cisplatin treatments, or until metastatic disease was detected. Cisplatin chemotherapy was well-tolerated by most dogs, with only 1 dog developing serious gastrointestinal toxicosis, requiring hospitalization. Results of this study support other investigators' findings that when a cisplatin chemotherapy-based protocol is administered, survival times after amputation can be prolonged for dogs with osteogenic sarcoma.

The electrocardiogram in epidemiologic investigations. A new classification system.

A new coding system for ECG abnormalities was developed, based on Frank's orthogonal ECG leads. In contrast to other systems, such as the Minnesota Code (MC), the new system was based on data collected prospectively in a cooperative study of 5031 records. The records were classified solely on the basis of non-ECG information. A record sample from normal women was also available. The large data base allowed stratification of ECG criteria according to sex and race. ECG criteria were determined at two levels of sensitivity and specificity. Specificity was 80-100% at the first level and 90-90% at the second. The new system has fewer criteria than other codes, which leads to reduction of coding errors and coding time. For common problems in differential diagnosis, optional criteria were included. A computer program for automated coding was also developed.

Analysis of discrepancies between VCG and ECG interpretation of anterior wall myocardial infarction.

In 247 cases in which an old anterior myocardial infarction (MI) was suspected and in which SVEC III-lead system vectorcardiograms (VCGs) and matching conventional 12-lead electrocardiograms (ECGs) were available, records were analyzed to investigate the diagnostic agreement or discrepancy between the two techniques. The frequency of agreement (155 cases) exceeded the frequency of disagreement (92 cases), but the frequency of disagreement even with lumped subgroups was high. Eighty percent of this disagreement was between positive VCG and negative ECG diagnostic consensus. The positive MI group by ECG diagnosis showed smaller R waves and larger S waves and larger S waves in leads V2 and V3 and smaller R/S ratios in leads V2 and V4 than the negative MI groupe by ECG, while small R waves or QS patterns and large S waves in just lead V1 were the only positive finding in the positive MI group by VCG. The important VCG characteristic for positive MI diagnosis was abnormal posterior deviation of the initial QRS vectors. The high incidence of discrepancies shown by positive VCG and negative ECG diagnoses resulted from the fact that, in spite of the presence of a fairly well developed R wave in precordial leads, the initial QRS vectors were displaced posteriorly. In 8 of 18 cases classified into negative VCG and positive ECG diagnostic group, poor R wave progression did not necessarily result in the posterior displacement of the initial QRS vectors. In contrast to the difficulties in applying ECG criteria for anterior MI (variability of QRS patterns in precordial leads), an orthogenal VCG (representing the phasic changes in the depolarization process) seems to clarify the equivocal situation in the ECG diagnosis of MI.

Hans Schaefer's asystole: two case reports.

Two cases of myocardial infarction are reported with transient (case 1) or permanent (Case 2) recovery of an R wave in anterior chest leads in the acute epsiode. This observation confirms H. Schaefer's hypothesis of asystole of the affected area, i.e. electrical inexcitability without necrosis. This phase precedes necrosis in any acute infarction; in the large majority of cases, myocardial infarction ultimately develops; therefore, demonstration in a clinical follow-up is quite rare.