The impact of hepatitis B virus (HBV) infection on clinical outcomes of patients with diffuse large B-cell lymphoma.

We performed a retrospective study to analyse the characteristics and clinical outcomes of diffuse large B-cell lymphoma (DLBCL) patients with hepatitis B virus (HBV) infection and compare with those without HBV infection. The occurrence of hepatitis after withdrawal of prophylactic antiviral treatment on completion of chemotherapy was also assessed. The HBsAg-positive patients were given prophylactic antiviral treatment until 6 months after finishing chemotherapy. A total of 81 patients were recruited with 16 in the HBsAg-positive group and 65 in the HBsAg-negative group. The clinical characteristics were similar in both groups of patients. There was no significant difference in complete remission rate between the two groups (63% in HBsAg-positive group vs. 54% in HBsAg-negative group, P = 0.59). There was also no statistically significant difference in overall survival between the two groups (P = 0.23). Four of the 16 HBsAg-positive patients (25%) had hepatitis after cessation of chemotherapy and prophylactic lamivudine. The mean time of onset of hepatitis was 3 months after stopping lamivudine. In conclusion, HBV infection did not appear to affect the prognosis of DLBCL patients given antiviral prophylaxis. It is reasonable to consider prophylactic antiviral therapy to extend to at least one year on completion of chemotherapy.

Use of hydrophilic polymers with microcrystalline cellulose to improve extrusion-spheronization.

Microcrystalline cellulose 19 parts was combined with sodium carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxypropyl cellulose or polyvinylpyrrolidone one part, either by spray-drying or physical mixing. This combined excipient (20%) mixed with lactose (80%) and water was added to aid formation of pellets by the process of extrusion-spheronization. Spray-dry combined excipient produced pellets with higher yield, better sphericity and improved tolerance to minor variation in the level of water added, compared with the physical mix excipient. Physicochemical testing based on scanning electron microscopy with energy dispersive analysis, differential scanning calorimetry and X-ray diffraction analysis, indicated that the spray-drying with the hydrophilic polymer caused disintegration of the microcrystalline cellulose component into smaller crystallites, favouring its more uniform dispersion throughout the lactose during subsequent processing. The hydroxypropyl cellulose or polyvinylpyrrolidone containing excipients were the most satisfactory of the hydrophilic polymers examined, because they had the least adhesive strength favouring maximum yield of highly spherical pellets.

Comparison of two commercial brands of microcrystalline cellulose for extrusion-spheronization.

Two commercial brands of microcrystalline cellulose, the widely used Avicel PH-101 and the newly available Pharmacel 101, were compared for aqueous extrusion-spheronization of a model mix with lactose. Based on the results of multi-level experiments employing pellet size analysis by sieving and sphericity determination by image analysis, Avicel was shown to be less adversely affected by variation in added water or speed of spheronization. Physicochemical testing of powder samples from both brands was carried out using laser particle sized analysis, density determinations, differential scanning calorimetry and X-ray diffraction studies. The results indicated that the improved ease of processing with Avicel may be related to its smaller particle size with less aggregates, improved flow, lower depolymerization temperature range and absence of traces of cellulose II in its cellulose I content.

Effect of common classes of excipients on extrusion-spheronization.

Different classes of excipients with potential for use in the design of novel pelletized formulations manufactured by extrusion-spheronization were examined using factorial experiments. Among the various silicates examined in a model mix with microcrystalline cellulose and lactose wetted with water, kaolin, talc and Veegum F provided improved plasticity for the formation of spherical pellets. Weak bases such sodium bicarbonate and weak acids such as fumaric acid also aided spheronization. Whereas waxy materials such as hydrogenated castor oil and Precirol ATO5, and wetting agent such as sodium lauryl sulphate improved sphericity, these excipients reduced pellet yield by favouring agglomeration. Other materials promoting unwanted formation of over-size pellets were bentonite, citric acid and tartaric acid. The inclusion of Bentone 27, various hydroxide and carbonate bases, and fumaric acid favoured fines production. Collectively the results showed that within classes of excipients, it was not possible to predict the effect of different materials on pellet yield and sphericity. However, Carr's index and Hausner ratio calculated from density determinations correlated well with sphericity measured by image analysis.

Polymerase chain reaction facilitates the cloning, CDR-grafting, and rapid expression of a murine monoclonal antibody directed against the CD18 component of leukocyte integrins.

Two novel approaches of recombinant PCR technology were employed to graft the complementarity determining regions from a murine monoclonal antibody (mAb) onto human antibody frameworks. One approach relied on the availability of cloned human variable region templates, whereas the other strategy was dependent only on human variable region protein sequence data. The transient expression of recombinant humanized antibody was driven by the adenovirus major late promoter and was detected 48 hrs post-transfection into non-lymphoid mammalian cells. The application of these new approaches enables the expression of a recombinant humanized antibody just 6 weeks after initiating the cDNA cloning of the murine mAb.

Generation of helper-free amphotropic retroviruses that transduce a dominant-acting, methotrexate-resistant dihydrofolate reductase gene.

We constructed several retroviruses which transduced a mutant dihydrofolate reductase gene that was resistant to methotrexate inhibition and functioned as a dominant selectable marker. The titer of dihydrofolate reductase-transducing virus produced by virus-producing cells could be increased to very high levels by selection of the cells in increasing concentrations of methotrexate. Helper virus-free dihydrofolate reductase-transducing virus was also generated by using a broad-host-range amphotropic retroviral packaging system. Cell lines producing helper-free dihydrofolate reductase-transducing virus with a titer of 4 X 10(6) per ml were generated. These retroviral vectors should have general utility for high-efficiency transduction of genes in cultured cells and in animals.

Interaction of K papovavirus with hamster cells: transformation of glial cells in vitro but failure of the virus to produce central nervous system tumors in vivo.

Primary cultures of lungs, kidneys, and glial cells derived from midgestation Syrian hamsters were inoculated with 10(5) hemagglutinating units of murine K-papovavirus and were serially subcultivated to allow appearance of lines of persistently infected or transformed cells. K virus did not replicate in renal cell cultures and produced only transient productive infection of lung cells. Evidence of K virus-induced cell transformation was not detected in either of these cultures. Inoculation of glial cultures with K virus, however, resulted initially in a protracted infection in which 80--100 percent of cells expressed K virus V antigen for 18 subcultivations and in which cloning experiments suggested that all cells in the culture contained the viral genome. After 18 subcultivations numbers of positive cells rapidly diminished, and cells appeared which exhibited altered morphology and density dependence. These altered cells (KVHG3 cells) grew well in serum-free media, could be cloned in soft agar, and were negative for infectious virus or K virus V antigen. Although KVHG3 cells did not exhibit staining when reacted with antisera to K virus T antigen, Southern blot analysis of these cultures demonstrated the presence of K virus DNA integrated into the host chromosomal DNA and indicated that some rearrangement of the viral genome had occurred. Attempts to produce tumors in hamsters with these cells were unsuccessful, as were attempts to induce tumors in newborn hamsters by intracranial inoculation of K virus. The present study demonstrates that K virus is capable of causing productive infection and cell transformation in primary cultures of fetal hamster glial cells but that other hamster cell types are relatively resistant to the virus and that both K virus and K virus-transformed hamster cells are poorly oncogenic for hamsters in vivo.

Persistent infection and transformation of mouse glial cultures by K virus, a murine papovavirus.

Foetal mouse glial cultures were inoculated with murine K papovavirus and subjected to serial subcultivation. Two cell lines were developed. The first of these, KVBCG2A, remained positive for viral infectivity and K virus capsid (V) antigen for over 30 subcultivations. Productive infection was not abolished by serial subcultivation in the presence of antiviral antibody. The second cell line, KVBCG1B, became negative for infectious virus and K virus V antigen, could be cloned from single cells and produced tumours in mice. Sera from tumour-bearing animals produced nuclear fluorescence of KVBCG1B cells and K virus-infected mouse embryo cells but did not react with uninfected mouse embryo cells or with cells infected by polyoma virus. DNA hybridization studies confirmed the presence of K virus DNA in KVBCG1B cells and suggested integration of the viral genome into host chromosomal DNA. K virus produces both persistent infection and cell transformation in glial cultures derived from its natural host.

A stable bovine papillomavirus hybrid plasmid that expresses a dominant selective trait.

We describe a bovine papillomavirus hybrid plasmid containing the neomycin resistance gene from Tn5 inserted into a mammalian cell transcriptional unit. This plasmid is maintained as a stable extrachromosomal element (20 to 100 copies per diploid genome) in mouse cells selected either for the transformed phenotype or for resistance to the aminoglycoside G418. Cells selected for G418 resistance initially display a flat, nontransformed phenotype before exhibiting the gross morphological characteristics of transformation. The delay in the appearance of the transformed phenotype indicated that some intracellular event or series of events subsequent to the establishment of transcriptionally active bovine papillomavirus 1 hybrid plasmid is required for the manifestation of the transformed phenotype.

Bovine papilloma virus deoxyribonucleic acid: a novel eucaryotic cloning vector.

A novel eucaryotic vector derived from the transforming region of bovine papilloma virus was established and demonstrated to be highly effective for introducing foreign genes into animal cells. The foreign deoxyribonucleic acid (DNA) is replicated and actively transcribed as an episome, and the transcripts are translated into an authentic gene product. We have constructed a DNA hybrid molecule, BPV69T-rI1, containing the transforming region of bovine papilloma virus DNA and the rat preproinsulin gene I (rI1), and used it to transform susceptible mouse cells. DNA hybridization analysis has demonstrated the presence of multiple unintegrated copies of hybrid DNA molecules, with the bovine papilloma virus 1 DNA segment and the rI1 gene covalently linked in selected transformed cell lines. S1 nuclease analysis revealed the presence of a correctly spliced coding segment of the preproinsulin transcript similar or identical in its electrophoretic mobility to that of messenger ribonucleic acid produced in rat insulinoma cells. Significant levels of a protein immunoreactive with anti-insulin serum were detected by radioimmunoassay in the culture medium of transformed cells. Immunoprecipitation analysis in conjunction with competitive binding to bovine proinsulin established the identity of the protein as that of rat proinsulin.

Mouse cells transformed by bovine papillomavirus contain only extrachromosomal viral DNA sequences.

The viral DNA sequences in mouse C127 cells transformed by bovine papillomavirus type 1 (BPV-1) virions, by full-length linear BPV-1 DNA, or by a defined transforming subgenomic DNA segment of BPV-1 were examined by reassociation kinetics and blot hybridization. In all cases, the transformed cells contained multiple copies of BPV-1 DNA, present exclusively as supercoiled or nicked circular extrachromosomal molecules or as a slowly migrating complex of circular viral DNA molecules. In the transformed cell lines established from cells transfected with full-length linear BPV-1 DNA, there was recircularization of the input DNA which in some cases resulted in the loss of the restriction site used in the linearization of the DNA. In the transformed cell lines established with the defined subgenomic segment there was circularization of the DNA accompanied by the acquisition of new sequences or duplication and rearrangement of the BPV-1 sequences. In contrast to other well-studied virus transformation systems, no integration of the BPV-1 genome into the host chromosome could be detected under conditions sensitive enough to detect 0.1-0.2 viral genome equivalent. It was concluded that maintenance of transformation may be mediated by nonintegrated viral DNA.

Cloned human polyomavirus JC DNA can transform human amnion cells.

The genome of the human polyomavirus JC (Mad-1 strain) was molecularly cloned in Escherichia coli by using the plasmid vector pBR322. Recombinant DNA molecules were constructed with the entire JC genome inserted either at its unique EcoRI site at 0.0 map units or at its unique BamHI site at 0.51 map units. Viral DNA from each of these recombinant plasmids was capable of transforming human amnion cells, and cell lines established from transformed foci were positive for JC tumor antigen as assayed by indirect immunofluorescence.

Cloning of human papilloma virus genomic DNAs and analysis of homologous polynucleotide sequences.

The complete DNA genomes of four distinct human papilloma viruses (human papilloma virus subtype 1a [HPV-1a], HPV-1b, HPV-2a, and HPV-4) were molecularly cloned in Escherichia coli, using the certified plasmid vector pBR322. The restriction endonuclease patterns of the cloned HPV-1a and HPV-1b DNAs were similar to those already published for uncloned DNAs. Physical maps were constructed for HPV-2a DNA and HPV-4 DNA, since these viral DNAs had not been previously mapped. By using the cloned DNAs, the genomes of HPV-1a, HPV-2a, and HPV-4 were analyzed for nucleotide sequence homology. Under standard hybridization conditions (Tm = --28 degrees C), no homology was detectable among the genomes of these papilloma viruses, in agreement with previous reports. However, under less stringent conditions (i.e., Tm = --50 degrees C), stable DNA hybrids could be detected between these viral DNAs, indicating homologous segments in the genomes with approximately 30% base mismatch. By using specific DNA fragments immobilized on nitrocellulose filters, these regions of homology were mapped. Hybridization experiments between radiolabeled bovine papilloma virus type 1 (BPV-1) DNA and the unlabeled HPV-1a, HPV-2a, or HPV-4 DNA restriction fragments under low-stringency conditions indicated that the regions of homology among the HPV DNAs are also conserved in the BPV-1 genome with approximately the same degree of base mismatch.

Conserved polynucleotide sequences among the genomes of papillomaviruses.

The DNAs of different members of the Papillomavirus genus of papovaviruses were analyzed for nucleotide sequence homology. Under standard hybridization conditions (Tm - 28 degrees C), no homology was detectable among the genomes of human papillomavirus type 1 (HPV-1), bovine papillomavirus type 2 (BPV-2), or cottontail rabbit (Shope) papillomavirus (CRPV). However, under less stringent conditions (i.e., Tm - 43 degrees C), stable hybrids were formed between radiolabeled DNAs of CRPV, BPV-1, or BPV-2 and the HindIII-HpaI A, B, and C fragments of HPV-1. Under these same conditions, radiolabeled CRPV and HPV-1 DNAs formed stable hybrids with HincII B and C fragments of BPV-2 DNA. These results indicate that there are regions of homology with as much as 70% base match among all these papillomavirus genomes. Furthermore, unlabeled HPV-1 DNA competitively inhibited the specific hybridization of radiolabeled CRPV DNA to bpv-2 DNA fragments, indicating that the homologous DNA segments are common among these remotely related papillomavirus genomes. These conserved sequences are specific for the Papillomavirus genus of papovaviruses as evidenced by the lack of hybridization between HPV-1 DNA and either simian virus 40 or human papovavirus BK DNA under identical conditions. These results indicate a close evolutionary relationship among the papillomaviruses and further establish the papillomaviruses and polyoma viruses as distinct genera.

A rapid method for detecting and mapping homology between heterologous DNAs. Evaluation of polyomavirus genomes.

A new approach for evaluating homologous sequences among related DNAs is presented. Conventional filter hybridization techniques are employed at 35 degrees C in a range of formamide concentrations in order to perform annealings at effective temperatures as low as Tm -50 degrees C which permits the detection of regions of homology with as much as 33% base mismatch. Under such nonstringent conditions, high levels of specific annealing can be obtained at plateau levels. In combination with the Southern "blotting" technique (1975), this approach can be used to perform biochemical heteroduplex melting experiments. The homology among the genomes of the murine polyoma virus (Py), the simian virus 40 (SV40), and the human papovavirus BK was evaluated using this new methodology.

Characterization of the genome of the murine papovavirus K.

The DNA genome of the murine papovavirus K virus (KV) was characterized and compared with the genome of polyoma virus. A physical map of the KV genome was constructed by analysis of the size of DNA fragments generated by sequential cleavage with combinations of restriction endonucleases. By using one of the three EcoRI sites in the KV genome as the 0 map position, the KV physical map was then oriented to the polyoma virus genome. Of 42 restriction sites mapped within the KV genome, 7 were localized within 0.01 map unit of their respective sites in the polyoma virus genome; an eighth site mapped within 0.02 map unit. KV replication was examined and found to be bidirectional, initiating at approximately 0.70 map unit. This corresponds well to the origin of replication within the polyoma virus genome and further supports the orientation of the KV physical map.