Type-specific Monoclonal Antibodies Research Articles

Upregulation of osteopontin expression in renal cortex of streptozotocin-induced diabetic rats is mediated by bradykinin.

The model of streptozotocin (STZ)-induced diabetes in Wistar rats was used to study the expression of osteopontin during development of diabetic nephropathy. Diabetes was confirmed by serum glucose levels exceeding 16 mmol/l during the experimental period of 12 weeks. During this period of time, diabetic nephropathy developed, as characterized by a reduced glomerular filtration rate (2.7 +/- 0.3 ml/min in controls vs. 1.7 +/- 0.1 ml/min in diabetic rats) and proteinuria (8.3 +/- 1.7 mg/24 h in controls vs. 22.0 +/- 4 mg/24 h in diabetic rats). Northern blot analysis revealed a time-dependent upregulation of renal cortical osteopontin expression reaching 138 +/- 6% of control levels after 2 weeks and 290 +/- 30% (mean +/- SE, n = 6-9) after 12 weeks. By immunostaining, the increased osteopontin expression could be located to the tubular epithelium of the renal cortex. Chronic treatment of animals with ramipril (3 mg/kg) during the 12-week experimental period led to a further increase in osteopontin mRNA expression in diabetic animals, amounting to 570 +/- 73% (mean +/- SE, n = 6) of controls. Increased levels of osteopontin were not associated with accumulation of monocyte/macrophages that were identified by the cell type specific monoclonal antibody ED-1. The increased osteopontin expression in ramipril-pretreated rats was abolished by application of the bradykinin B2-receptor antagonist, icatibant (0.5 mg/kg). In addition, increased osteopontin expression in diabetic rats, which did not receive any treatment after STZ injection, could as well be reduced by icatibant given for the final 2 weeks of the experimental period. These data suggest that a strong bradykinin B2-receptor-mediated upregulation of osteopontin occurs during the pathogenesis of experimental diabetic nephropathy in rats.

Identification of an immunodominant sequential epitope in glycoprotein G of herpes simplex virus type 2 that is useful for serotype-specific diagnosis.

A series of 67 oligopeptides that spanned the open reading frame of herpes simplex virus type 2 (HSV-2) glycoprotein G (gG2) were synthesized and tested for reactivity with 173 serum specimens collected from 117 individuals. The oligopeptides were made as multiple antigenic peptides consisting of four copies of a unique sequence attached to a branched lysine core and separated from the core by four glycine residues. The sera included HSV antibody-negative samples as well as sera from individuals from whom HSV had been isolated. Isolated viruses were typed by indirect fluorescence using a panel of type-specific monoclonal antibodies. One peptide, corresponding to residues 561 to 578 of gG2, did not react with any sera lacking HSV-specific antibodies of with sera from HSV-1-infected individuals, but did react with sera from HSV-2-infected individuals. For sera taken seven or more days after initialclinical lesions, the detection rate of the peptide was 92% (47/51), comparable with the 98% (50/51) of truncated glycoprotein D, a sensitive type-common reagent. We conclude that this peptide, of structure (PEEFEGAGDGEPPEDDDSG4)K3A, is an immunodominant type-specific epitope for human antibodies and should be useful for type-specific serodiagnosis of HSV-2. Surprisingly, the epitope lies within one of the most conserved regions of gG1 and gG2. The test can distinguish an initial HSV-2 infection in the presence of a preexisting HSV-1 infection.

Evaluation of the protective efficacy of a recombinant dengue envelope B domain fusion protein against dengue 2 virus infection in mice.

A recombinant protein containing part of the dengue (DEN) 2 envelope protein was evaluated as a subunit immunogen for vaccination against DEN virus infection. A gene fragment encoding amino acids 298-400 (B domain) of the DEN-2 virus envelope was expressed as a fusion protein with the maltose binding protein (MBP) of Escherichia coli. This recombinant, DEN-2(B)/MBP, was purified and analyzed for its antigenicity, immunogenicity, and ability to protect mice against lethal challenge. The recombinant antigen reacted with a DEN-2 type-specific neutralizing monoclonal antibody (3H5), DEN-2 hyperimmune mouse ascitic fluid, and DEN-2 immune human sera. When administered to mice, DEN-2(B)/MBP elicited a DEN-2 virus neutralizing antibody response that conferred partial protection against challenge infection with a lethal dose of DEN-2 virus administered by intracranial inoculation. In addition, no replication of DEN-2 virus was detectable in the brains of the immunized mice as compared with control mice that were killed six days after challenge. Sera from immunized mice revealed no cross-neutralizing antibody to any of the other DEN serotypes in the plaque-reduction neutralization test. These findings warrant further studies with the DEN-2(B)/MBP antigen as a potential human vaccine candidate. An effective vaccine could prevent thousands of cases of illness and many deaths each year resulting from DEN virus infections.

Severe Gastroenteritis in Cincinnati: Role of Rotavirus ♦ 546

A rotavirus vaccine is likely to be licensed in the near future but the cost-effectiveness of the vaccine remains an issue. Recent advances in the outpatient management of diarrhea and the use of short (<24 hour) stay care may have altered the cost of medical care for rotavirus. To examine the impact of rotavirus infection prior to the licensure of a rotavirus vaccine, we conducted active rotavirus surveillance from March 16-September 17, 1997 on all hospitalized children <5 years of age with signs and symptoms of acute gastroenteritis (AGE). Stool samples were tested by ELISA for rotavirus antigen. Positive specimens were then serotyped using type-specific monoclonal antibodies directed against G-serotypes 1, 2, 3 and 4. Of the 148 children admitted with AGE, 39.2% were admitted to the short-stay unit and 15% were<6 months of age. Overall, 84.5% of AGE patients shed rotavirus. Short-stay patients were more likely to have rotavirus compared to inpatients (94.8% vs. 77.8%; p=.005). Children ≥.6 months of age were more likely to have rotavirus than those <6 months (88.9% vs. 59.1%;p<.001). Of the 125 specimens available for typing, 92% were typable. Of those infections with a known serotype, G1 was the predominant serotype accounting for 72.2% of infections, but unlike previous evaluations in Cincinnati, all 4 serotypes were identified [G4 (12.2%), G3 (10.4%), G2 (5.2%)]. There was no significant difference of the mean age by serotype. Rotavirus still accounts for a large proportion of cases of severe AGE, many of which are admitted to short-stay units which would be missed in hospital discharge surveys. Each of the four major serotypes were found during one season, indicating a need for a vaccine which provides protection against multiple serotypes.

Localization of inositol 1,4,5-trisphosphate receptors in the rat kidney

Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) serve as intracellular calcium release channels involved in signal transduction of various hormones in the kidney. Molecular cloning studies have shown that there are three types of IP3R, designated type 1, type 2, and type 3. To characterize their localizations in the rat kidney, we employed immunohistochemical studies using type-specific monoclonal antibodies that were raised against the 15 C-terminal amino acids of each type of IP3R. Type 1 was detected in glomerular mesangial cells and vascular smooth muscle cells. Type 2 was expressed exclusively in intercalated cells of collecting ducts from the cortex to the inner medulla. Type 3 was expressed in vascular smooth muscle cells, glomerular mesangial cells, and some cells of cortical collecting ducts, probably principal cells. As to the subcellular distribution, type 1 and type 2 showed a homogenous distribution in the cytoplasm, whereas type 3 was present mainly in the basolateral portion of the cytoplasm. These results indicate that IP3R isoforms were expressed in a cell-specific manner. The heterogeneous subcellular localizations among the IP3R types suggests compartmentalization of distinct IP3-sensitive Ca2+ pools.

Identification of a Novel Core Type in SalmonellaLipopolysaccharide: COMPLETE STRUCTURAL ANALYSIS OF THE CORE REGION OF THE LIPOPOLYSACCHARIDE FROM SALMONELLA ENTERICA sv. ARIZONAE O62

For the first time, the complete structure of a lipopolysaccharide (LPS) core region from Salmonella enterica has been identified that is different from the Ra core type generally thought to be present in all Salmonella LPS. The LPSs from two rough mutants and the smooth form of S. enterica sv. Arizonae IIIa O62, which all failed to react with an Ra core type-specific monoclonal antibody and were resistant to phage FO1, were analyzed after chemical modification using monosaccharide analysis, mass spectrometry, and NMR spectroscopy. In the novel core type, the terminal D-GlcNAc residue present in the Ra core type, is replaced by a D-Glc residue. The O-specific polysaccharide is alpha1-->4-linked to the second distal Glc residue of the core. Furthermore, phosphoryl substituents attached to O-4 of L-glycero-D-manno-heptose (Hep) I and II were identified as 2-aminoethyl diphosphate (on Hep I) and phosphate (Hep II). [structure: see text] Abbreviations in Structure I are as follows: Hepp, L-glycero-D-manno-heptopyranose; Kdo, 3-deoxy-D-manno-oct-2-ulopyranosonic acid; PPEA, 2-aminoethyl diphosphate; R, O-specific polysaccharide. The presence of this novel core type in LPS of S. enterica should be taken into account in the development of a general antibody-based diagnostic system for Salmonella.

Differentiation and quantification of Theileria sergenti piroplasm types using type-specific monoclonal antibodies.

Antigenic properties of two representative allelic products of the major piroplasm surface protein (MPSP) of Theileria sergenti were studied. Sera from cattle infected with either of Ikeda and Chitose types of the parasite reacted strongly with homologous but weakly with heterologous recombinant antigens in immunoblotting. Monoclonal antibodies (MoAbs) produced against the both allelic products of MPSP parasites reacted only to the immunizing antigen. These results suggested that crossreactivity between two allelic products is very low inspite of relatively high homology in their amino acid sequences. Double staining of parasitized erythrocyte smear using type-specific MoAbs by an indirect immunofluorescent assay revealed that the set of MoAbs was useful for quantitative and differential detection of each type of parasite in mixed population.

Fine mapping of bovine herpesvirus 1 (BHV-1) glycoprotein C neutralizing epitopes by type-specific monoclonal antibodies and synthetic peptides

Bovine herpesvirus glycoprotein C (gC) functions as a major virus attachment protein. Here, two BHV-1 gC-specific epitopes that are specified by complement-dependent neutralizing MAbs are mapped. The BHV-1 gC-specific peptides and MAbs were used to specifically localize continuous epitopes by direct binding to the MAbs and by blocking the Mab reactivity (competitive ELISA) to authentic viral antigen. The results of competitive ELISA indicated that the complement-dependent neutralizing epitopes recognized by MAbs F2 and 24 were located between BHV-1 gC amino acids (aa) 47–69 and (aa) 109–119, respectively.

Intracellular Targeting with Low pH-triggered Bispecific Antibodies

Bispecific antibodies were designed to deliver a reversibly bound ligand into target cells and then spontaneously release it upon passage into acidified vesicles. These reagents were assembled by coupling monoclonal antibodies that recognize acid-sensitive epitopes on diphtheria toxin to cell type-specific monoclonal antibodies. The dual binding capacity of the bispecific antibodies was confirmed by delivery of 125I-diphtheria toxin to target molecules present on intact cells. Bispecific antibodies directed against transferrin receptors on human cells were loaded with toxin and tested for cytotoxicity. The mutant diphtheria toxins CRM107 and CRM45 were used since their inability to bind cell receptors renders them ordinarily nontoxic. Their full cytotoxic potential, however, was restored via bispecific antibody-mediated delivery and release within low pH intracellular vesicles. Cytotoxicity was shown to be specific by blocking receptor sites and to be acidification-dependent by protection using NH4Cl to raise endosomal pH. Kinetics for inhibition of cellular protein synthesis was identical for native diphtheria toxin and the bispecific antibody. CRM107 combination. The rate of inhibition (t1/2 = 20 min) indicated that release of CRM107 from the antibody combining site was fast, and its toxic action was unimpeded by this delivery mechanism.

Detection and typing of herpes simplex viruses by using recombinant immunoglobulin fragments produced in bacteria.

Thirty-seven bacterial clones producing human recombinant monoclonal antibody Fab fragments (rFabs) reactive to herpes simplex virus (HSV) antigens were selected from a human combinatorial antibody library constructed in a phage-display vector by a panning procedure against an HSV lysate. Thirty-four of the HSV-specific rFabs were able to specifically recognize HSV-infected cells in indirect immunofluorescence (IF) assays; of these, 25 recognized cells infected by either HSV type 1 (HSV-1) or HSV-2, while 9 recognized only HSV-1-infected cells. One HSV type-common rFab (rFab H37) and one HSV-1-specific rFab (rFab H85) were further evaluated as reagents for viral detection and typing by IF staining in 134 HSV-positive (72 HSV-1 and 62 HSV-2) viral cultures from clinical specimens. The results obtained with these two rFabs were fully consistent with those obtained with a commercial preparation of fluorescein-labeled anti-HSV type-specific murine monoclonal antibodies. The detection sensitivity with the type-common rFab in indirect IF assays was higher overall than that provided by the type-specific murine monoclonal antibodies. Preparations of rFabs suitable for IF staining can be easily and inexpensively obtained in a clinical microbiology laboratory from Escherichia coli cultures. Similar HSV-specific rFabs, therefore, could be advantageous for in vitro diagnostic purposes.

Antigenic and Plaque Variations of Serotype II Feline Infectious Peritonitis Coronaviruses.

Three feline coronavirus (FCoV) isolates KUK-H, M91-266, and M91-267 were examined to elucidate their biological and antigenic properties as well as disease potential in cats. Immune stainings of virus-infected cells by using FCoV type-specific monoclonal antibodies indicated that their antigenic specificity was serotype II. However, antigenic variations among these serotype II FCoVs were detected by neutralization assay with hyperimmune antisera against FCoVs and canine coronaviruses, and with experimentally infected cat sera; there were two subtypes in serotype II FCoVs. The isolates efficiently grew in fcwf-4 cell culture showing lytic CPE enough to form distinct plaques: when measured 48 hr after infection, plaque sizes of both M91-266 and M91-267 were approximately 1 mm in diameter, and a mixture of small (less than 1 mm in diameter) and large (approximately 3 mm in diameter) plaques were produced in the case of KUK-H. Strains KUK-H, M91-266 and M91-267 produced feline infectious peritonitis (FIP) in 50%, 67% and 89% of experimentally inoculated kittens, respectively. Furthermore, 80% of the kittens inoculated with the small plaque former of KUK-H developed FIP accompanied by more prominent clinical signs as well as pathological changes when compared with 28.6% of kittens inoculated with the large plaque former. These results suggest that serotype II FIPVs producing smaller size of plaques are more virulent than those producing larger size of plaques.

Demonstration of antigenic and genotypic variation in Orientia tsutsugamushi which were isolated in Japan, and their classification into type and subtype.

A total of 40 strains of Orientia tsutsugamushi (34 isolates from patients and trombiculid mites in Japan, and 6 prototype strains of antigenic variants) were examined for classification based on the reactivities with type-specific monoclonal antibodies in indirect immunofluorescence tests, and on the restriction fragment length polymorphism of a polymerase chain reaction (PCR)-amplified 56-kilodalton type-specific antigenic protein gene. By these methods, several antigenic and genotypic variants were found among the strains, and these variants were classified into types and further into subtypes. These results suggest that there are many variants in O. tsutsugamushi, and the methods used here seem to be useful for the systematic classification of the numerous variants. A strain which may be a new type distinguishable from those identified previously was also found in this study. Furthermore, variety in the degree of pathogenicity in mice related to type and/or subtype classification were observed.

Ultrastructural aspects of the replication of dengue virus type 2 isolated in Brazil.

Our purpose here is to describe and compare ultrastructural aspects of DEN-2 virus replication in the C6/36 mosquito cell line of the two otbreaks in the states of Ceara and Bahia. Sera from four patients from Fortaleza (Ceara) and two from Eunapolis (Bahia) were isolated and typed previously as DEN-2 at the Laboratory of Flaviviruses, Department of Virology, Instituto Oswaldo Cruz (Nogueira et al. 1995 Rev Inst Med Trop Sao Paulo submitted) by indirect immmunofluorescence test with type specific monoclonal antibodies. Cells were inoculated with 50 μl of the original human sera containing virus and maintained at 28°C in Leibovitz-15 medium. Showing 30-50% of cytopathic effect, the cell cultures were fixed in glutaraldehyde and processed as described previously (Barth et al. 1994 loc. cit.). Ultrathin sections were observed with a Zeiss EM-900 electron microscope. At least 50 cells were analyzed for virus replication in each sample. The Brazilian DEN-2 virus replication in mosquito cell cultures of all examined sera present some peculiar morphological aspects, when compared with the reference New Guinea C strain. Typical featured virus particles, that occur in great number inside the rough endoplasmatic reticulum (RER) and derived vesicles after about three days of inoculation, are observed at high magnification in ultrathin sections and present always a smooth surface (Fig. 1); small granules on the virus particle surfaces can be observed by the negative staining technique at higher magnifications, corresponding to the virus membrane and envelope proteins (Fig. 2 arrow). Beside these classical structured DEN-2 virus particles, virus-like particles occur in the RER and derived vesicles, also with the isolates of patient sera from State of Rio de Janeiro, but not in the cells infected with the reference New Guinea C strain. These particles, characterized by a fuzzy coated surface, are less numerous but persistent just to the 5th passage level in cells (Barth et al. 1994 loc. cit.). Cell controls were always free from these particles, as well as the same cell line infected by DEN-1 viruses. No antibodies, that may be engulfed by the cells during endocytosis, were found inside the infected cells; this means that the last described particles are not virus particles covered by antibodies. Human sera from Fortaleza and Eunapolis cities, show the same morphological aspects of the virus particles as described for DEN-2 from Rio de Janeiro (Fig. 3). Classical structured virus particles occur beside the fuzzy coated ones in the same RER vesicles. Smooth membrane structures, RESEARCH NOTE

Antigenic differences between HSV-1 and HSV-2 glycoproteins and their importance for type-specific serology.

The ability to discriminate between herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) infections by serological means is of increasing importance for clinical virological diagnostics. HSV type-specific serology has a number of applications, e.g. to guide duration and dosage of antiviral therapy, to allow for stringent epidemiological analyses, to evaluate efficacy of HSV vaccine candidates, and to help the clinician during counseling of couples where one has genital herpes. The genomes of the two HSV types appear to be sufficiently intratypically stable and intertypically discrepant to permit such a discrimination. Despite recent advances in methodology, the ideal HSV type-specific serological assays still remain to be developed. The viral antigens utilized for such an assay should evoke strong antibody responses, but only against unique determinants, in order to be able to combine high sensitivity and specificity. Of all the HSV envelope proteins, the most promising candidate antigens, i.e. HSV-1 glycoprotein G (gG-1) and its HSV-2 counterpart gG-2, contain relatively long type-unique stretches of amino acids. However, whether the type-specific determinants preferentially localize to the unique or homologous stretches of gG is still unknown. Paradoxically, type-specific monoclonal antibodies to the moderately type-specific antigen, HSV-1 glycoprotein C, have hitherto been mapped to homologous rather than to type-unique stretches. A definition of human type-specific epitopes on gG-1 and gG-2, as well as a broader search for new candidate HSV antigens, might be needed to fully discriminate dual infections from high titer single HSV-1 or HSV-2 infections.

Molecular basis of antigenic variation between the glycoproteins C of respiratory bovine herpesvirus 1 (BHV-1) and neurovirulent BHV-5

Herpesvirus glycoprotein C (gC) functions as a major virus attachment protein. The gC sequence of the neurovirulent bovine herpesvirus type 5 (BHV-5) virus was determined and compared with the gC sequence of the nonneurovirulent BHV-1. Alignment of the predicted amino acid sequences of BHV-1 and BHV-5 gC ORFs showed that the amino-terminal third of the protein differed between the two viruses. Whole or subgenomic fragments of gC coding regions from both viruses were expressed as trpE—gC fusion proteins in Escherichia coli to map linear epitopes defined by type-specific murine monoclonal antibodies (MAbs). Based on the reactivity of BHV-1-specific MAbs with the recombinant proteins, two epitopes were mapped between BHV-1 gC residues 22 and 172. Unidirectional deletion of these residues at the carboxy end mapped one within residues 22–69 and the other within residues 103–122. Two BHV-5-specific MAbs identified an epitope coding region within BHV-5 gC residues 31–78. Bovine antisera against BHV-1 and BHV-5 showed specificity to BHV-1 gC residues 22–69 and to BHV-5 gC residues 31–78, respectively, in a type-specific manner.

Enzyme-linked immunosorbent assay-based inhibition test for neutralizing antibodies to polioviruses as an alternative to the neutralization test in tissue culture

A poliovirus-binding inhibition test (PoBI test) was established for the quantitative determination of antibodies to polioviruses and was evaluated in comparison with the conventional neutralization test (NT). The first step of the PoBI test is an incubation of serial dilutions of test samples with inactivated poliovirus followed by the detection of free viral epitopes by a double antibody sandwich enzyme-linked immunosorbent assay with type-specific capture polyclonal antisera and type-specific neutralizing monoclonal indicator antibodies. A comparison of the PoBI test with the conventional NT for antibodies to all three types in 100 human serum samples showed excellent correlations (r > 0.95) over a wide range of antibody concentrations. The PoBI test, not necessitating live virus and tissue culture facilities, could be a simple alternative to the NT, and the principle of the assay is potentially applicable to other microbial systems.

Comparative study of five methods for intratypic differentiation of polioviruses

A coded panel of 90 poliovirus isolates, 30 of each of the three known serotypes, was used to evaluate five methods for the intratypic differentiation of polioviruses: (i) an enzyme-linked immunosorbent assay with polyclonal cross-absorbed antisera (PAb-E), (ii) a neutralization assay with type-specific monoclonal antibodies (MAb-N), (iii) a restriction fragment length polymorphism (RFLP) assay, (iv) a Sabin vaccine strain-specific PCR assay, and (v) a Sabin vaccine strain-specific cRNA probe hybridization (ProHyb) assay. Sequence analysis was used for the definitive characterization of the strains. The panel was distributed to five laboratories; each laboratory analyzed the strains by at least two methods. Each method was used by three or four laboratories. The total performance scores (percentage correct results per number of tests) of the five methods were 96.7% for PAb-E, 93.9% for MAb-N, 91.9% for RFLP assay, 93.3% for Sabin vaccine strain-specific PCR, and 97.4% for Sabin vaccine strain-specific ProHyb. Consistent results were obtained by each laboratory for 88 of 90 isolates (97.8%) examined by PAb-E, 81 of 90 isolates (90.0%) examined by MAb-N, 78 of 90 isolates (86.7%) examined by RFLP assay, 81 of 90 isolates (90.0%) examined by PCR, and 89 of 90 isolates (98.9%) examined by ProHyb assay. Six strains were classified differently by different methods. It is recommended that at least two methods be used for the intratypic differentiation of poliovirus isolates, and each method should be based on a different principle (i.e., antigenic properties and nucleotide sequence composition). If two assays yield discrepant results, further characterization, preferably by partial sequence determination, will be required for correct identification.

Virological Study on Dengue Epidemic in Vientiane Municipality, Lao PDR, 1994.

In May 1994, an epidemic of dengue fever (DF) / dengue hemorrhagic fever (DHF) was reported in Ban Horn commune in Vientiane municipality. This was after a six-year period of non-epidemic DF/ DHF in the municipality. In early June, we collected 81 serum specimens from children with fever of unknown origin (FUO) in the commune. Of 81 specimens, 48 were taken from children within 5 days after the onset of FUO and subjected to dengue (DEN) virus isolation. Four strains of DEN-1 and 12 strains of DEN-2 viruses were isolated from 15 serum specimens (31.3%). A case of dual infection with DEN-1 and DEN-2 viruses was detected by enzyme antibody method using type-specific monoclonal antibodies and reverse transcription polymerase chain reaction (RT-PCR). The percentage incidences of the positive virus isolation from the serum specimens taken on the second and the third day of fever were 41.8% and 21.1%, respectively, and no virus was isolated from the specimens other than those from these two days. All the specimens were examined by the IgM-capture ELISA. Thirty-two specimens (39.5%) were positive for DEN IgM antibody.

Fine mapping of bovine herpesvirus-1 (BHV-1) glycoprotein D (gD) neutralizing epitopes by type-specific monoclonal antibodies and sequence comparison with BHV-5 gD

Overlapping fragments of the bovine herpesvirus-1 (BHV-1) glycoprotein (gD) ORF were expressed as trpE-gD fusionproteins in Escherichia coli to map linear neutralizing epitopes defined by BHV-1-specific MAbs. The MAbs 3402 and R54 reacted with the expressed fragments on Western blots that located the epitopes between the amino acids 52–126 and 165–216, respectively, of gD. Bovine covalescent sera with high neutralizing antibody titers against BHV-1 reacted with these bacterially expressed proteins containing both of the epitopes. Alignment of these sequences from BHV-1 with the corresponding region of the BHV-5 gD ORF sequences (reported here) identified several amino acid mismatches. Since the MAbs 3402 and R54 neutralize the BHV-1 and not BHV-5, it was presumed that these were important amino acids in defining the epitope. To further localize the neutralizing epitopes, synthetic peptides corresponding to these regions in the BHV-1 gD ORF were tested for their capacity to block monoclonal antibody neutralization of BHV-1 infectivity. The peptides encompassing amino acids 92—106 (3402 epitope) and amino acids 202–213 (R54 epitope) of the BHV-1 gD competed with BHV-1 for the binding by MAbs 3402 and R54, respectively, in a dose-dependent manner. Antisera produced in rabbits to these peptides conjugated to a carrier reacted strongly with a 30-kDa protein by Western blotting and had neutralizing antibody titers against BHV-1.

Synergistic Differentiation-Promoting Activity of Interferon and Tumor Necrosis Factor- : Role of Receptor Regulation on Human Neuroblasts

Interferon gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF) synergize in inducing human neuroblastoma cells to differentiate terminally in vitro into mature nonproliferating neurons. The mechanisms by which this synergistic activity takes place are still obscure. To understand the basis of IFN-gamma-TNF synergism, we investigated the constitutive equipment of receptors to IFN-gamma and TNF in two human neuroblastoma cell lines (i.e., LAN-5 and GI-LI-N) and their quantitative and functional variations following treatment with IFN-gamma or TNF. IFN-gamma receptors and TNF receptors were assessed and functionally characterized by radioreceptor-binding assay before and after treatment of the cells with IFN-gamma or TNF. The TNF receptor subtypes were identified by the reverse transcriptase-polymerase chain reaction, chemical cross-linking of receptors to iodinated TNF, and inhibition of TNF binding by type-specific anti-TNF receptor monoclonal antibodies. The effects of cytokines on cell differentiation were assessed by thymidine incorporation inhibition and morphologic maturation. No quantitative or functional modification of IFN-gamma receptors was observed in TNF-treated cells. However, after treatment with IFN-gamma, TNF receptor numbers were enhanced to a different extent in both cell lines. The two neuroblastoma cell lines expressed, both constitutively and after IFN-gamma induction, only one species of TNF receptor, i.e., the p80 form in LAN-5 and the p60 form in GI-LI-N. Sequential treatment with IFN-gamma followed by TNF, but not in the opposite order, could reproduce the early effects of differentiation in neuroblastoma cells, supporting a role for TNF receptor up-regulation as a basis for the cooperation between the two cytokines. The results strongly suggest that receptor regulation can be at least one mechanism by which IFN-gamma and TNF exert their synergistic effects. Moreover, it appears that the two TNF receptor types are redundant in signaling neuroblastoma cell differentiation. Our findings can provide a guideline for a rational design of experimental differentiation-based therapeutic protocols in patients with neuroblastoma.