Quantitation of feline immunodeficiency proviruses in doubly infected cats using competitive PCR and a fluorescence-based RFLP

A nested polymerase chain reaction assay, which amplifies a region of the gag gene, was developed for the direct detection of feline immunodeficiency virus (FIV) DNA sequences in the blood of infected cats. This method detects as few as ten copies of a plasmid containing the whole genome of the FIV-Pet isolate on agarose gel. To distinguish two FIV isolates in double infected cats: we devised an RFLP analysis on PCR amplified products exploiting sequence differences in the gag gene of the two strains. To quantitate the two strains, a fluorescent inner-sense primer was used in the second amplification step. Amplicons were subsequently digested, heat-denatured and loaded on a polyacrylamide gel in an automated DNA sequencer. The proportion of the two isolates was determined using the laser-excited fluorescence of labelled strain specific fragments. These data were used to extrapolate the numbers of proviral genomes from the total viral load as estimated by using a competitive PCR assay, These sensitive and specific assays complement virological detection of FIV and enable superinfection studies to be evaluated; a prerequisite for the testing of live attenuated immunodeficiency virus vaccines

COMPETITIVE POLYMERASE CHAIN-REACTION FOR QUANTITATING FELINE IMMUNODEFICIENCY VIRUS LOAD IN INFECTED CAT TISSUES

To quantitate the FIV provirus copy numbers present in tissue of infected cats, we have applied a competitive polymerase chain reaction (cPCR) recently described for HIV. The method consists in coamplifying a fixed amount of the DNA to be examined with graded copy numbers of a DNA competitor incorporating a short deletion and bearing the same primer recognition sequences. These conditions ensures almost identical thermodynamic and amplification efficiency for both template species but permit a prompt recognition of the two amplification products by gel electrophoresis. Since the amounts of the two amplicons are dependent on relative initial template concentrations, the number of FIV genomes in the sample can be calculated by densitometric analysis of the electrophoretics bands. After validation, the method has been applied to study the provirus loads in the tissues of cats infected with Pisa-M2 isolate of FIV

PARTIAL NUCLEOTIDE SEQUENCING OF 6 SUBTYPE 2C HEPATITIS-C VIRUSES DETECTED IN ITALY

The great majority of 121 hepatitis C virus (HCV) isolates obtained from 117 Italian patients with community-acquired infection could readily be typed by genotype-specific PCR. Subtype Ib was dominant (74 isolates); subtypes 2b, 2a, and la followed, with 19, 14, and 8 isolates, respectively. The six isolates that remained untyped by this method were classified as subtype 2c on the basis of sequence analysis of PCR amplicons obtained from the core and NS5 genes. These findings indicate that HCV subtype 2c has a relatively high prevalence in Italy. Sequencing the core region from positions 160 to 259 is sufficient to distinguish subtype 2c from other known HCV genotypes

A neutralizing antibody-inducing peptide of the V3 domain of feline immunodeficiency virus envelope glycoprotein does not induce protective immunity.

Specific-pathogen-free cats, immunized with a 22-amino-acid synthetic peptide designated V3.3 and derived from the third variable region of the envelope glycoprotein of the Petaluma isolate of feline immunodeficiency virus (FIV), developed high antibody titers to the V3.3 peptide and to purified virus, as assayed by enzyme-linked immunoassays, as well as neutralizing antibodies, as assayed by the inhibition of syncytium formation in Crandell feline kidney cells. V3.3-immunized animals and control cats were challenged with FIV and then monitored for 12 months; V3.3 immunization failed to prevent FIV infection, as shown by virus isolation, anti-whole virus and anti-p24 immunoglobulin G antibody responses, and positive PCRs for gag and env gene fragments. Sequence analysis of the V3 region showed no evidence for the emergence of escape mutants that might have contributed to the lack of protection. The sera of the V3.3-hyperimmunized cats and two anti-V3.3 monoclonal antibodies neutralized FIV infectivity for Crandell feline kidney cells at high antibody dilutions but paradoxically failed to completely neutralize FIV infectivity at low dilutions. Moreover, following FIV challenge, V3.3-immunized animals developed a faster and higher antiviral antibody response than control cats. This was probably due to enhanced virus replication, as also suggested by quantitative PCR data

Activity and safety of epirubicin plus paclitaxel in advanced breast cancer.

We performed a dose-escalation study to evaluate the maximum tolerated dose (MTD) of paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) plus a fixed dose of epirubicin. Epirubicin was administered as a 90 mg/m2 bolus immediately followed by a 3-hour infusion of paclitaxel starting at 135 mg/m2 and escalating by 20mg/m2 for each triplet of patients as long as no dose-limiting toxicity had occurred; courses were repeated every 3 weeks. The MTD was defined as that at which any of the following toxicities occurred in at least two of six patients: absolute neutrophil count less than 500/microliter for more that 7 days or less than 100/microliter for more than 3 days; any episode of febrile neutropenia requiring intravenous antibiotics and hospitalization; grade 4 thrombocytopenia requiring platelet transfusion; failure to recover absolute neutrophil count to > or = 1,500/microliter and/or platelets to > or = 100,000/microliter by day 28; and any grade > or = 3 nonhematologic toxicity. Two MTDs were defined: the first without granulocyte colony-stimulating factor (MTD 1) and the second with granulocyte colony-stimulating factor given either to accelerate recovery of grade 4 neutropenia lasting more than 72 hours or immediately in case of febrile neutropenia (MTD 2); granulocyte colony-stimulating factor was never used prophylactically. To date, 22 patients have been entered into the study; the median patient age was 55 years (age range, 30 to 66 years). Nineteen (86\%) patients had received adjuvant chemotherapy that included anthracyclines in 12 cases (55\%). The viscera were the dominant sites of disease in 55\% of patients. Median baseline ventricular ejection fraction was 58\% (range, 53\% to 67\%). Short-lasting grade 4 neutropenia occurred in 61\% of courses; however, only four episodes of febrile neutropenia were recorded. Grade 4 thrombocytopenia was reported in 8\% and grade 3 anemia in 3\% of courses; four patients experienced peripheral neuropathy (three patients grade 1, one patient grade 2); complete alopecia was universal. The cardiac effects of the combination were surprisingly low: median ejection fraction at study entry was 58\%, and after a cumulative dose 1,080 mg/m2 it was 56\%. Three complete responses and 12 partial responses have been documented for an overall response rate of 83.3\% (95\% confidence interval, 58\% to 96\%). In conclusion, neutropenia is the most frequent toxicity of this novel combination. However, the MTD has not yet been reached. The combination of epirubicin plus paclitaxel is highly active, and no signs of cumulative myocardiopathy have been observed