In vitro-selected drug-resistant varicella-zoster virus mutants in the thymidine kinase and DNA polymerase genes yield novel phenotype-genotype associations and highlight differences between antiherpesvirus drugs.

Varicella zoster virus (VZV) is usually associated with mild to moderate illness in immunocompetent patients. However, older age and immune deficiency are the most important risk factors linked with virus reactivation and severe complications. Treatment of VZV infections is based on nucleoside analogues, such as acyclovir (ACV) and its valyl prodrug valacyclovir, penciclovir (PCV) as its prodrug famciclovir, and bromovinyldeoxyuridine (BVDU; brivudin) in some areas. The use of the pyrophosphate analogue foscarnet (PFA) is restricted to ACV-resistant (ACV(r)) VZV infections. Since antiviral drug resistance is an emerging problem, we attempt to describe the contributions of specific mutations in the viral thymidine kinase (TK) gene identified following selection with ACV, BVDU and its derivative BVaraU (sorivudine), and the bicyclic pyrimidine nucleoside analogues (BCNAs), a new class of potent and specific anti-VZV agents. The string of 6 Cs at nucleotides 493 to 498 of the VZV TK gene appeared to function as a hot spot for nucleotide insertions or deletions. Novel amino acid substitutions (G24R and T86A) in VZV TK were also linked to drug resistance. Six mutations were identified in the "palm domain" of VZV DNA polymerase in viruses selected for resistance to PFA, PCV, and the 2-phophonylmethoxyethyl (PME) purine derivatives. The investigation of the contributions of specific mutations in VZV TK or DNA polymerase to antiviral drug resistance and their impacts on the structures of the viral proteins indicated specific patterns of cross-resistance and highlighted important differences, not only between distinct classes of antivirals, but also between ACV and PCV.

Dual infection with polyomavirus BK and acyclovir-resistant herpes simplex virus successfully treated with cidofovir in a bone marrow transplant recipient.

A hematopoietic stem cell transplant recipient developed a mucosal herpes simplex virus-1 (HSV-1) infection while under acyclovir (ACV) treatment (HSV was later shown to be resistant to ACV). Concomitantly, the patient presented a hemorrhagic cystitis (HC) due to polyomavirus BK, for which intravenous cidofovir (CDV) was prescribed. The patient benefited from the broad-spectrum anti-DNA virus activity of CDV, and not only the HC resolved without signs of nephrotoxicity but also the HSV-1 lesions disappeared. This is the first report describing the effect of CDV on 2 simultaneous and unrelated DNA viral infections in an immunosuppressed transplant recipient. In addition, we describe here that this HSV-1 isolate possesses a unique phenotype and genotype.

Gene cloning of a new plasma CC chemokine, activating and attracting myeloid cells in synergy with other chemoattractants.

Chemokines are important mediators of cell migration during inflammation and normal leukocyte trafficking. Inflammatory chemokines are induced in multiple cell types at sites of infection. Here, we describe a novel bovine CC chemokine, designated regakine-1, that is constitutively present at high concentrations in plasma. Cloning of its gene revealed an expected two intron/three exon organization, with a rather long first intron. In addition to a 21-residue signal peptide, the coding sequence corresponded to a 71-residue secreted protein. However, the natural regakine-1 protein missed the COOH-terminal lysine residue. Regakine-1 has only weak sequence similarity (<50% identical residues) with other animal or human chemokines. Northern blot analysis demonstrated regakine-1 RNA expression in spleen and lung. At physiological concentrations (30-100 ng/mL), natural 7.5 kDa regakine-1 stimulated gelatinase B release from neutrophils and chemoattracted immature myeloid HL-60 cells, as well as mature granulocytes. Regakine-1 was more potent on human myeloid cells than the human plasma CC chemokine hemofiltrate CC chemokine-1 (HCC-1). Moreover, regakine-1 synergized with the bacterial peptide N-formylmethionylleucylphenylalanine (fMLP), yielding a 10-fold increase in neutrophil chemotactic response above their additive effect. Regakine-1 did not compete with interleukin-8 (IL-8) for binding to neutrophils, nor did it affect fMLP-induced calcium signaling, suggesting that regakine-1 recognizes a different receptor. In view of its high constitutive plasma concentration, regakine-1 is believed to recruit myeloid cells into the circulation, whereas its synergy with other neutrophil chemoattractants suggests that it also enhances the inflammatory response to infection.

Evaluating phenotype and genotype of drug-resistant strains in herpesviruses.

The isolation of drug-resistant strains of herpesviruses, including Herpes Simplex Virus type I (HSV-1) and type 2 (HSV-2), Varicella-Zoster Virus (VZV), and cytomegalovirus (CMV), has been reported with increasing frequency in immunocompromised patients and is a matter of major concern. Determination of antiviral drug susceptibilities is a prerequisite for the management of drug-resistant herpesvirus infections. Phenotyping studies should be correlated with genotyping, i.e., characterization of the mutations in the target genes. The isolation of drug-resistant virus in the laboratory and the determination of their phenotype and genotype may be useful to clarify the mechanisms of selective drug action. We describe here the procedures used for in vitro selection of drug-resistant herpesvirus mutants and the determination of their patterns of drug-susceptibility. The subcloning of the HSV-1 DNA polymerase gene is described as an example of the methodology followed to determine the mutation(s) in the drug-target viral gene that are associated with the resistant phenotype. To avoid the introduction of mutations by PCR amplification, all subcloning experiments were executed directly on viral DNA. Viral DNA was prepared from each plaque-purified viral strain and a 3.4 kb BamHI fragment containing 87% of the HSV-1 DNA polymerase gene coding region was purified and further digested with SacI; the two resulting fragments were subcloned into pU18 and propagated in Escherichia coli. Plasmid DNA was isolated and the inserts were sequenced using dideoxynucleotide chain termination method with T7 DNA polymerase and Taq DNA polymerase in an automated laser fluorescent DNA sequencer. pUC/M13 reverse, universal primers and oligonucleodite primers based on the wild-type virus sequence were used. The nucleotide sequences of the DNA polymerase genes of the different mutants was then compared with the nucleotide sequence of the wild-type HSV-1 KOS strain.

PECAM1, MPO and PRKAR1A at chromosome 17q21-q24 and susceptibility for multiple sclerosis in Sweden and Sardinia.

Using genome screen, DNA sequence and mapping data, we scanned the human chromosomal region 17q21-q24 for polymorphic markers in single copy genes. Three such genes were identified: the gene for myeloperoxidase (MPO) at 17q21.3-q23.2, containing a CA-microsatellite in the eighth intron and a functional single base substitution (G to A) in the promoter region, the platelet endothelial cell adhesion molecule-1 gene (PECAM1) at 17q23, which has a CA-repeat sequence in the sixth intron, and the gene for the regulatory subunit RIalpha of cAMP-dependent protein kinase (PRKAR1A) at 17q23-q24, in which a GA-microsatellite was detected in the 5'-flanking region. Association of these polymorphisms with multiple sclerosis (MS) was studied in a Swedish case-control population of 199 MS patients and 145 control subjects, and in 203 simplex families from Sardinia. None of these polymorphic genes was found to be a genetic marker for disease susceptibility. These results are in contrast with previous studies on the involvement of MPO in MS and suggest that the elevated expression of PECAM-1 in MS, as earlier documented, is related to transactivation by other gene products.

Polymorphism analysis suggests that the gelatinase B gene is not a susceptibility factor for multiple sclerosis.

The human gelatinase B (MMP-9) gene promoter region contains a CA microsatellite repeat and a single nucleotide polymorphism which are known to influence transcriptional activity. These two polymorphisms were used to investigate the existence of an association between multiple sclerosis (MS) susceptibility and the MMP-9 gene. In a case-control analysis of 345 Swedish individuals and in a study of 125 Sardinian simplex families no genetic associations between the gelatinase B gene polymorphisms and MS susceptibility were found. These data reinforce the suggestion of epistasis in the regulation of the metalloproteinase-inhibitor balance in MS.

Resistance of herpes simplex virus type 1 against different phosphonylmethoxyalkyl derivatives of purines and pyrimidines due to specific mutations in the viral DNA polymerase gene.

Drug-resistant strains of herpes simplex virus type 1 (HSV-1) were selected under the pressure of (S)-3-hydroxy-2-phosphonylmethoxypropyl (HPMP) derivatives of cytosine (HPMPC, cidofovir) and adenine (HPMPA) and 2-phosphonylmethoxyethyl (PME) derivatives of adenine (PMEA, adefovir) and 2,6-diaminopurine (PMEDAP). HPMPC-resistant (HPMPC(r)) and HPMPA(r) strains were cross-resistant to one another, but they remained sensitive to foscarnet (PFA), acyclovir (ACV) and the PME derivatives, while the PMEA(r) and PMEDAP(r) strains showed cross-resistance to PFA and ACV. The PMEA(r), PMEDAP(r) and PFA(r) mutants all revealed a single nucleotide change resulting in a Ser-724 to Asn mutation within the conserved region II of the DNA polymerase. Two HPMPA(r) clones and one HPMPC(r) clone possessed single amino acid changes in the DNA polymerase (HPMPA(r) clone D1, Leu-1007 to Met; HPMPA(r) clone B5, Ile-1028 to Thr; HPMPC(r) clone C3, Val-573 to Met). The HPMPC(r) clone A4 contained two mutations, Ala-136 to Thr and Arg-700 to Met. The mutation at position 136, located outside the catalytic domain of the enzyme, was not detected in other HPMPC(r) clones, suggesting that this mutation may not be responsible for the resistant phenotype. Residue 573 is located within the 3'-->5' exonuclease editing domain close to the catalytically important residues Tyr-577 and Asp-581. Similarly, residue 700 is located in the palm subdomain of the catalytic domain, adjacent to the Asp residues 717, 886 and 888 that are vital for polymerase activity. The HPMPA(r) mutations at residues 1007 and 1028, beyond the last conserved region, still fall within the thumb subdomain of the catalytic domain. The different drug-resistant mutants varied in neurovirulent behaviour, the HPMPC(r) strains showing reduced neurovirulence compared with the wild-type.

High-resolution analysis of IL-6 minisatellite polymorphism in Sardinian multiple sclerosis: effect on course and onset of disease.

A minisatellite polymorphism located in the 3' flanking region of the interleukin-6 (IL-6) gene was analysed in 192 Sardinian simplex families with multiple sclerosis (MS). By applying a high-resolution sizing approach, 9 alleles were identified. None of these were associated with in globo susceptibility to MS as shown by transmission disequilibrium testing. Analysis of clinically different groups showed that the A5 allele was associated with a benign (P = 0.007) but not with a malignant (P = 0.45) course of disease. In particular, the frequency of the A5/A5 genotype was significantly higher in patients with benign MS (P = 0.002). In addition, carriage of any of the larger alleles (A6-->A9) was associated with accelerated onset of disease (P = 0.025). Our results suggest that allelic variations in the IL-6 gene may predispose to alterations in the course and initial onset of MS.

Lack of association between the interferon regulatory factor-1 (IRF1) locus at 5q31.1 and multiple sclerosis in Germany, northern Italy, Sardinia and Sweden.

Interferon regulatory factor-1 (IRF-1) is a transcriptional inducer of the interferon-beta (IFN-beta) gene and other interferon-stimulated genes. A GT repeat polymorphism in the 7th intron of the IRF-1 gene was used as a marker to test for association with multiple sclerosis (MS) in a case-control study including individuals from Germany, Northern Italy and Sweden. In none of these populations, did we find any significant allelic association with disease. This lack of association was confirmed by testing transmission disequilibrium of individual IRF1 alleles in a representative sample of Sardinian simplex MS families. No deviation of the expected 50% transmission rates was seen. Therefore, our work does not provide evidence in favor of IRF1 being a candidate for conferring genetic susceptibility to, or protection against, MS in Europe.

Monitoring drug resistance for herpesviruses.

Herpes simplex type 1 (HSV-1) and type 2 (HSV-2), varicella-zoster virus (VZV) and human cytomegalovirus (CMV) cause diseases that are usually self-limiting in the immunocompetent host. However, HSV-1, HSV-2, VZV, and CMV are major causes of morbidity and mortality in the immunocompromised patient. Prolonged antiviral treatment is often required for the clinical management of herpesvirus infections in the immunocompromised patient, and this favors the emergence of drug-resistant strains. The isolation of acyclovir-resistant (ACV(r)) HSV-1, HSV-2, and VZV strains as well as ganciclovir-resistant (GCV(r)) CMV strains has been reported with increasing frequency and is a major concern (1-3). Resistance to foscarnet (phosphonoformic acid [PFA]), the drug of choice when ACV or GCV fails, has also been described in the clinic (4,5). Furthermore, double resistance to both GCV and PFA (for CMV) and to both ACV and PFA (for HSV) has been observed in immuno-compromised patients after sequential and concomitant treatment with either or both drugs.

Analysis of an IFN-gamma gene (IFNG) polymorphism in multiple sclerosis in Europe: effect of population structure on association with disease.

An intronic dinucleotide polymorphism in the IFN-gamma gene (IFNG) was used as a marker for testing association with multiple sclerosis (MS). Disease association was analyzed in case-control sets sampled from four geographically separate European populations (Germany, Northern Italy, Sardinia, and Sweden). Only in the Swedish was a weak disease association of the IFNG allele pattern found, mainly due to a higher frequency of IFNG allele I1 in MS patients. No evidence for association was found in the German or Northern Italian populations. These results contrast with the situation in Sardinia. We have recently reported transmission disequilibrium of IFNG allele I2 in Sardinian MS siblings not carrying the predisposing DRB1 *03 or *04 alleles (Ann. Neurol. 44, 841-842, 1998). Further analysis now shows that I2 is significantly more often transmitted to DRB1 *03-/*04- males, than to DRB1 *03-/*04- females. The odds ratio (OR) for IFNG-associated susceptibility to MS in the total Sardinian DRB1*03-/*04- group was 1.88 for I2 heterozygotes but amounted to 8.235 for I2 homozygotes, suggestive of a recessive mode of inheritance. Score test-based statistics pointed to an I2 allele dosage effect acting in susceptibility. Comparison of the IFNG allele frequencies in seven European populations (Northern Finnish, Southern Finnish, Swedish, Danish, German, Italian, and Sardinian) revealed a highly different distribution pattern. We introduced latitude as a score variable in order to test for trend in binomial proportions. This test statistic showed that for both most common alleles, I1 and I2 (compiled allele frequency about 85%), a significant opposite north-to-south trend is seen throughout Europe. This effect is primarily due to the extreme values found in the outlier populations of Finland and Sardinia. Our findings are discussed with respect to recent literature pertinent to the role of the IFNG chromosome region in autoimmune diseases.

Transcriptional control of the human MCP-2 gene promoter by IFN-gamma and IL-1beta in connective tissue cells.

Human monocyte chemotactic protein-2 (MCP-2) is a member of the CC chemokine family. It is produced by mononuclear leukocytes, diploid fibroblasts, and tumor cells after induction with IL-1beta or IFN-gamma. To understand the transcriptional regulation of the gene, we have analyzed the structure and function of the promoter region. The sequence of the 5'-flanking region was determined and the transcription start site was found to be located at 68 nucleotides upstream of the ATG translation start codon. 5'-Deletion mutants were generated and transfected into E6SM diploid fibroblasts and MG-63 osteosarcoma cells. Expression was measured by luciferase assay in transfected unstimulated cells and after stimulation with IL-1beta, IFN-gamma, or a combination. The region between nucleotides -143 and -73 (relative to the transcription initiation site), containing putative cis-elements for GATA-1, H-APF1, AP-1, and GAS, is important for basal transcription levels in both cell lines. Stimulation for 18 h with IL-1beta alone failed to affect expression of any of the constructs both in diploid fibroblasts and in osteosarcoma cells. In both cell lines IFN-gamma increased the activity of all mutants that possessed the region between -340 and -301. In MG-63 cells, stimulation with the combination of IL-1beta and IFN-gamma caused an additional increase in expression of the constructs from -340 onward. Finally, the presence of transcription factors in nuclear extracts of MG-63 cells and their specificity to bind to various oligonucleotide probes in this [-340; -301] region were evidenced by electromobility shift assays. These results show that IFN-gamma, produced by lymphocytes and NK cells, induces the transcription of the MCP-2 gene in fibroblasts and thereby can indirectly contribute to recruitment of various leukocyte cell types to inflammatory sites.

Microsatellite polymorphisms in the gene promoter of monocyte chemotactic protein-3 and analysis of the association between monocyte chemotactic protein-3 alleles and multiple sclerosis development.

Monocyte chemotactic protein 3 (MCP-3) is a chemokine that attracts mononuclear cells, including monocytes and lymphocytes, the inflammatory cell types that predominate in multiple sclerosis lesions. We studied the possible association between the presence of a CA/GA microsatellite repeat polymorphism in the promoter/enhancer region of the MCP-3 gene and the occurrence of multiple sclerosis. DNA samples from 192 Swedish multiple sclerosis (MS) patients and 129 healthy controls were analysed by an automated fluorescent technique. In the whole sample population, five MCP-3 allele variants (MCP-3*A1 to MCP-3*A5) were detected with an allele frequency ranging between 0.3% and 46%. The individual MCP-3 allele frequencies did not differ significantly between MS patients and control individuals. The relative MS risk, attributable to HLA-DRB1*15 was 3.05 (chi2 = 22.25, p < 0.0001). The phenotype frequency (PF) of none of the MCP-3 alleles was significantly altered in the population of controls versus unselected MS patients. When MS patients and control subjects were stratified according to positivity for HLA-DRB1*15, the MCP-3*A4-associated risk for developing MS decreased to 0.36 (p = 0.011). In the stratified groups of patients who were negative for both HLA-DRB1*15 and HLA-DRB1*03, and hence possessed a lower risk to develop MS, the MCP-3*A2-associated risk for MS development decreased significantly (p = 0.018). We conclude that the MCP-3*A4 allele might protect against MS development on the background of the increased risk in HLA-DRB1*15+ individuals and the MCP-3*A2 allele seems protective in low-risk individuals, who are both negative for DRB1*03 and DRB1*15.

A competitive RT-PCR method for the quantitative analysis of cytokine mRNAs in mouse tissues.

The authors describe the design and validation of a competitive RT-PCR method for the efficient and reproducible quantitation of mRNA molecules of IFN-gamma, TNF-alpha, IL-4 and IL-10 in mouse spleen RNA extracts. Before being subjected to RT-PCR, the RNA extracts were supplemented with internal control RNAs (IC-RNAs), which were constructed by inserting DNA fragments in the cDNA of the respective cytokines. The efficiency of amplification of the target and the IC-RNA was shown to remain equal over a wide range of cycle numbers. Reproducibility was such that differences in mRNA contents that were greater than 17% could be detected between two RNA samples run in parallel. Normal mouse spleen tissue was found to contain 10(7)-10(8) molecules of TNF-alpha, IFN-gamma, IL-4 and IL-10 mRNA per micrograms total RNA extracted. Injection of animals with anti-CD3 antibody, a well-known cytokine inducer, resulted in a moderate increase in TNF-alpha and IL-10 mRNA levels (14- and 24-fold, respectively), and in a substantially greater increase in the levels of mRNA for IL-4 and IFN-gamma (199- and 851-fold, respectively). These results demonstrate an accurate and reliable quantitation of cytokine mRNA levels in animal tissues.

The human MCP-2 gene (SCYA8): cloning, sequence analysis, tissue expression, and assignment to the CC chemokine gene contig on chromosome 17q11.2.

Monocyte chemotactic proteins (MCPs) form a subfamily of chemokines that recruit leukocytes to sites of inflammation and that may contribute to tumor-associated leukocyte infiltration and to the antiviral state against HIV infection. With the use of degenerate primers that were based on CC chemokine consensus sequences, the known MIP-1 alpha/LD78 alpha, MCP-1, and MCP-3 genes and the previously unidentified eotaxin and MCP-2 genes were isolated from a YAC contig from human chromosome 17q11.2. The amplified genomic MCP-2 fragment was used to isolate an MCP-2 cosmid from which the gene sequence was determined. The MCP-2 gene shares with the MCP-1 and MCP-3 genes a conserved intron-exon structure and a coding nucleotide sequence homology of 77%. By Northern blot analysis the 1.0-kb MCP-2 mRNA was predominantly detectable in the small intestine, peripheral blood, heart, placenta, lung, skeletal muscle, ovary, colon, spinal cord, pancreas, and thymus. Transcripts of 1.5 and 2.4 kb were found in the testis, the small intestine, and the colon. The isolation of the MCP-2 gene from the chemokine contig localized it on YAC clones of chromosome 17q11.2, which also contain the-eotaxin, MCP-1, MCP-3, and NCC-1/MCP-4 genes. The combination of using degenerate primer PCR and YACs illustrates that novel genes can efficiently be isolated from gene cluster contigs with less redundancy and effort than the isolation of novel ESTs.

Production and characterization of recombinant active mouse gelatinase B from eukaryotic cells and in vivo effects after intravenous administration.

Gelatinase B is a matrix metalloproteinase involved in tissue remodelling. When mouse cells are triggered in vitro with interleukin-1, bacterial endotoxin, virus-mimicking double-stranded RNA or cytokine inducers, they produce gelatinase B. To test the effects of gelatinase B in vivo, the enzyme was expressed in Chinese hamster ovary (CHO) cells. Hybrid genomic DNA-cDNA constructs under the control of two constitutive viral promoters were generated by PCR-mediated exon amplification. In vitro transcription and translation of the mRNA in reticulocyte lysate yielded the correct 79-kDa protein, and expression in CHO cells resulted in an intact glycosylated 110-kDa gelatinase B which was enzymically active. However, the production yields of recombinant enzyme from 50 tested clones were low and cell-culture supernatants contained significant amounts of copurifiable endogenous CHO gelatinase B. Therefore, the enzyme was expressed in the yeast Pichia pastoris. Recombinant proenzyme was secreted and recovered from the yeast culture medium at 10 mg/l. Amino-terminal sequence analysis indicated that affinity purification of the recombinant protein on gelatin-Sepharose yielded the expected N-glycosylated proenzyme form (110 kDa) in addition to an amino-terminally truncated unglycosylated variant (69 kDa). Both forms had gelatinolytic activity on zymography. The recombinant mouse gelatinase B was used to determine its pharmacokinetics and its haematological effects in vivo. After intravenous injection in rabbits, gelatinase B disappeared from the circulation within 6 h. In addition to a transient leukopenia, we observed a rapid increase in leukocytosis, which indicates that gelatinase B might be a factor involved in the desorption of adherent leukocytes from the vascular bed and in the release of leukocytes from the bone marrow. Gelatinase B secretion and activation might well be one of the crucial molecular mechanisms explaining leukocytosis which is associated with infections and almost all types of inflammation.

Structural and functional analysis of the promoter region of the human MCP-3 gene: transactivation of expression by novel recognition sequences adjacent to the transcription initiation site.

Human monocyte chemotactic factor-3 (MCP-3) belongs to the C-C chemokines, which are cytokines involved in cell recruitment in inflammation and cancer. Northern blotting and reverse transcriptase polymerase chain reaction (RT-PCR) analyses showed that the MCP-3 gene is expressed in many human tissues and tumor cell lines and that the expression level is increased by various stimuli. Measles virus and phorbol 12-myristate 13-acetate (PMA) induced MCP-3 mRNA after 6 hr of stimulation. Interferon-beta (IFN-beta) induced MCP-3 mRNA after 16 hr, a time point when the PMA-induced mRNA had the tendency to level off. No significant increase in MCP-3 mRNA levels was observed in MG-63 cells after stimulation with interleukin-1beta (IL-1beta). To elucidate the regulation of MCP-3 gene expression, we determined the sequence of 5 kb of the MCP-3 promoter. This sequence contained a microsatellite that was shown to be polymorphic in various cell lines. Next 5'-deletion mutants of the promoter were generated and transfected into MG-63 cells, demonstrating the presence of several positive and negative transcriptional regulatory elements. One of the positive elements was located at -37, only 21 bp upstream from the TATAA box. This element was similar to an AP-1 element and also to a homeodomain protein Pbx1 binding site. A deletion mutant from -110 to +52 possessed the highest promoter activity, and the longer deletion mutants had relatively low activities. The region between -190 and -172 contained an Ets-like element and inhibited promoter activity. Stimulation with PMA dramatically increased promoter activity through activation of a positive element present between -172 and -100. The same 5'-deletion mutants were transfected into HeLa and Jurkat cells. None of the deletion mutants had any significant activity in Jurkat cells. In HeLa cells, low levels of MCP-3 mRNA were detected by RT-PCR, but the profile of the promoter activities of the deletion mutants was different from that seen in MG-63 cells.

Human monocyte chemotactic protein-2: cDNA cloning and regulated expression of mRNA in mesenchymal cells.

Stimulated MG-63 osteosarcoma cells have been used as a source to purify and identify the monocyte chemokines MCP-1, MCP-2 and MCP-3. In comparison with MCP-1, the production yields of MCP-2 and MCP-3 in these cells are rather low and variable. Although the protein sequence of human MCP-2 is identified, its DNA sequence remains elusive. A degenerate primer set was used to isolate an MCP-2 gene fragment from the chemokine YAC contig on human chromosome 17. Based on the gene sequence of MCP-2, a unique primer set was synthesized and used to screen cDNA libraries for the presence of MCP-2 transcripts by PCR. The complete MCP-2 cDNA was cloned from a human bone marrow cDNA library and sequenced. The cDNA-derived protein sequence was identical to that of purified natural MCP-2, except for Gln46 which replaced Lys46. There seem thus to exist two MCP-2 allelic variants because at position 46 the codons of two residues (Lys46 and Gln46) were detected in individual genomes. As shown by Northern hybridization, the MCP-2 steady-state mRNA levels in normal diploid fibroblasts were increased by IL-1 beta, IFN-gamma and the double-stranded RNA poly rI:rC. RT-PCR analysis showed induction of MCP-2 mRNA in MG-63 cells by IFN-gamma and IL-1 beta. The regulated production of MCP-2 by tumor cells and normal mesenchymal cells is indicative of a role in neoplasia and inflammatory host responses.

Effect of VH and VL consensus sequence-specific primers on the binding and neutralizing potential of a single-chain FV directed towards HuIFN-gamma.

We have previously reported on the cloning and bacterial expression of a biologically active scFv antibody fragment (scFv-D9D10) derived from the mouse anti-human interferon-gamma (HuIFN-gamma) antibody, D9D10. Since the variable (V) regions were isolated by means of VH and VL consensus sequence-specific PCR primers and cloned in an expression vector relying on primer-incorporated restriction sites, some amino acids (aa) at the N- and C-terminal ends of the cloned V domains were expected to differ from the corresponding ones in the natural D9D10 antibody. Therefore, the naturally occurring sequences of both V domains were isolated by means of traditional cDNA synthesis procedures. In comparison with scFv-D9D10, the "natural" V sequences differed in three aa in VH and three in VL. The V domains of scFv-D9D10 were adapted to their natural sequence by means of PCR-directed mutagenesis to yield scFv-D9D10N. Comparison of the binding and neutralizing potentials of both antibody fragments did not reveal differences in either of both activities. In addition, their affinities for HuIFN-gamma were found to be equal. These results show that murine VH and VL consensus-specific primers can yield antibody fragments having functional properties equivalent to those of the natural scFv. Information on the impact of the use of V-specific primers on kinetics of interaction between the recombinant antibody and the corresponding antigen is important for the development of most engineered antibodies or their fragments.

Localization and reactivity of an immunodominant domain in the NS3 region of hepatitis C virus.

Analysis of the amino acid sequences of the nonstructural region 3 (NS3) of the hepatitis C virus type 1 revealed four points with a high average hydrophilicity (Ah). Two of these potential antigenic sites were expressed in E. coli as short fragments. The first fragment of 91 residues (NS3f3: residues 1359-1449) harbors the hexapeptide K-K-K-C-D-E with an Ah of 2.33; the second fragment is 73 residues long (NS3f4: residues 1460-1532) and encompasses the heptapeptide R-S-N-R-R-G-R with an Ah of 1.79. Both fragments were expressed with truncated hepatitis B core (tHBc) as a carrier protein. The fusion proteins were purified from the bacterial lysates by affinity chromatography on immobilized monoclonal antibodies against HBc, and evaluated as antigens in an enzyme immunoassay for the detection of HCV antibodies. In a specificity control panel, reactivity with NS3f3 was only found in proven HCV carriers, while reactivity with NS3f4 was weak in HCV carriers but accounted for some of the nonspecific serological reactions. In a group of 48 genotyped HCV-infected volunteer blood donors, antibodies against NS3f3 were detected in 90% (27/30) of HCV-type 1 infections and in all HCV-type 4 infections (5/5).(ABSTRACT TRUNCATED AT 250 WORDS)