Hyone-Myong Eun

Therapeutic Effects of Recombinant Feline Interferon‐co on Feline Leukemia Virus (FeLV)‐Infected and FeLV/Feline Immunodeficiency Virus (FIV)‐Coinfected Symptomatic Cats

The clinical efficacy of a recombinant feline interferon, rFeIFN-omega, was evaluated for the treatment of cats presented with clinical signs associated with feline leukemia virus (FeLV) infection and FeLV/feline immunodeficiency virus (FIV) coinfection in the field. In this multicentric, double-blind, placebo-controlled trial, 81 cats meeting the inclusion criteria were randomly placed into 2 groups and treated subcutaneously with rFelFN-omega (1 million [M]U/kg per day) or placebo once daily for 5 consecutive days in 3 series (day 0, 14, 60). The cats were monitored for up to 1 year for clinical signs and mortality. During the initial 4-month period, interferon (IFN)-treated cats (n = 39) had significantly reduced clinical scores compared with placebo (n = 42), with all cats having received concomitant supportive therapies. Compared with the control, the IFN-treated group showed significantly lower rates of mortality: 39% versus 59% (1.7-fold higher risk of death for controls) at the 9-month time point and 47% versus 59% (1.4-fold higher risk of death for controls) at the 12-month time point. The IFN treatment was associated with minor but consistent improvement in abnormal hematologic parameters (red blood cell count, packed cell volume, and white blood cell count), apparently underlying the positive effects of IFN on clinical parameters. These data demonstrate that rFeIFN-omega initially has statistically significant therapeutic effects on clinical signs and later on survival of cats with clinical signs associated with FeLV infection and FeLV/FIV coinfection.

Treatment of canine parvoviral enteritis with interferon-omega in a placebo-controlled field trial

The clinical efficacy of a recombinant feline interferon (IFN) (type omega) was evaluated under field conditions for the treatment of dogs with parvoviral enteritis. In this multicentric, double-blind, placebo-controlled trial, 94 dogs from one to 28 months old were randomly assigned to two groups which were treated intravenously either with IFN (2·5 million units/kg) or placebo once a day for three consecutive days, and monitored for clinical signs and mortality for 10 days. Each dog received individual supportive treatment. The data from 92 interpretable cases (43 IFN-treated and 49 placebo) showed that the clinical signs of the IFN-treated animals improved significantly in comparison with the control animals, and that there were only three deaths in the IFN group compared with 14 deaths in the placebo group (P=0·0096) corresponding to a 4·4-fold reduction. Alternative analyses of the data taking into account the prior vaccination status of the dogs against canine parvovirus suggested that the IFN therapy resulted in a 6·4-fold reduction in mortality (P=0·044) in the unvaccinated cohort, a significant reduction when compared with the vaccinated cohort.

Treatment of canine parvoviral enteritis with interferon-omega in a placebo-controlled challenge trial

Canine parvoviral enteritis continues to cause significant morbidity and mortality in dogs worldwide, and efficacious antiviral therapies are lacking. The present trial was aimed at evaluating the therapeutic efficacy of a recombinant feline interferon (type omega) preparation in the treatment of parvoviral enteritis in dogs. A double-blind, placebo-controlled challenge trial was performed in beagle pups (8-9 weeks); clinical signs, body weight, hematologic parameters, and mortality were monitored for a period of 14 days after challenge. Fourteen animals were inoculated with virulent canine parvovirus; 10 animals that developed clinical signs thereby meeting the inclusion criteria were admitted to the treatment phase in two randomly selected groups (placebo and IFN) of equal size. The IFN group received daily intravenous injections of rFeIFN-omega (2.5 MU/kg) for three consecutive days. The placebo group received daily injections of saline without IFN. Both groups of animals received individual supportive treatment consisting of adjusted diet and electrolyte solution. All five dogs in the placebo group developed fulminating enteritis with typical clinical signs and died within 10 days post-inoculation (or 6 days post-treatment). In the IFN-treated group, one animal died on day 2 after the treatment was started, whereas the other four dogs survived the challenge and gradually recovered. Our data confirm that the rFeIFN-omega can exert a significant therapeutic effect on dogs with parvoviral enteritis by improving clinical signs and reducing mortality.

Efficacies of osaterone and delmadinone in the treatment of benign prostatic hyperplasia in dogs

A multicentre randomised clinical trial was performed to compare the therapeutic potential of osaterone acetate with that of delmadinone acetate in the treatment of benign prostatic hyperplasia in dogs. The osaterone was administered orally at 0·25 mg/kg bodyweight once a day for seven days to 73 dogs. The delmadinone was administered by a single intramuscular or subcutaneous injection at 3 mg/kg bodyweight to 69 dogs. During the 180-day trial, the dogs were monitored five times for their clinical signs and prostate volume. The two drugs were similarly effective in reducing the clinical signs and inducing complete clinical remission, and both induced a similar level of minor, mostly transitory adverse effects. Osaterone reduced the volume of the prostate glands of the dogs significantly more quickly than delmadinone.

Protective Efficacy of the Calicivirus Valency of the Leucofeligen Vaccine against a Virulent Heterologous Challenge in Kittens.

Feline calicivirus (FCV) is a common feline pathogen with a potential for antigenic diversity. This study aimed to evaluate and characterize the protective efficacy of the FCV-F9 valency of a tetravalent vaccine, Leucofeligen, against challenge with an unrelated strain. Ten 9-week-old kittens were vaccinated while 10 remained as unvaccinated controls. The vaccinated cats received Leucofeligen twice subcutaneously with a 3-week interval. Four weeks after the second vaccination, all cats were challenged with virulent heterologous FCV and followed up for 21 days, monitoring their general condition, clinical signs, and immunological responses. During the vaccination phase, rectal temperatures and body weights were indistinguishable between the two groups. Only vaccinated cats showed FCV-specific seroconversion (both total and neutralizing antibodies). In the first week after challenge, the vaccinated cats had an 82.6% reduction in median clinical score compared to controls. Leucofeligen was thus shown to provide a significant clinical protection to kittens challenged with heterologous virulent FCV. This protection was similar whether the cats had neutralizing antibody or not, indicating a key role for cellular immunity in the overall protection. This also suggests that previously reported seroneutralisation studies may underestimate the level of cross-protection against field strains obtained with this modified live FCV-F9 vaccine.

4 – Restriction Endonucleases and Modification Methylases

Restriction endonucleases (R•ENases) are a part of the restriction (R) and modification (M) systems that bacteria operate for their protection against invading bacteriophages and foreign genetic elements. Restriction endonucleases are a group of DNases that recognize specific nucleotide sequences and cut dsDNA in a site-specific or nonspecific manner. The R–M systems are now amenable to a wealth of genetic analysis. Through this R–M system, bacteria destroy undesirable foreign DNAs that invade the cells through infection, conjugation, and transfection. Modification methylase (M•MTase) is responsible for modifying and protecting endogenous DNAs from similar digestions by R•ENase. The R and M activities may reside in a single multisubunit enzyme or in physically separate enzymes. This chapter focuses on type II R•ENases, which are commonly described as “restriction enzymes,” and their cognate M•MTases because of their paramount importance in molecular biology and recombinant DNA technology.

1 – Enzymes and Nucleic Acids: General Principles

Enzymes are bioreactors that run all biochemical reactions of a living cell in a steady, controlled manner with superlative efficiency and specificity. Physiologically, an enzyme is an essential, life-sustaining biocatalyst. Many chemical and biochemical reactions in vitro require enzymes to proceed at reasonable rates under mild conditions of temperature and pH. As a catalyst, enzyme can only promote a reaction in a thermodynamically favorable direction—it does not change the direction of a reaction. Therefore, enzymes are indispensable tools in recombinant DNA technology and in many other areas related to biochemical conversions. This chapter elucidates “classical” enzymes, as they are the present-day workhorses in recombinant DNA technology. The chapter begins with the explanation of some molecular biological and genetic aspects of protein biosynthesis. The chapter also reviews the fundamental concepts of enzymology—the physicochemical bases of protein structure and catalytic function. It also provides a background for relating the fundamental knowledge of protein biosynthesis, structure, and function to recombinant DNA technology.