Risk assessment on the carcinogenic potential of hybridoma cell DNA: implications for residual contaminating cellular DNA in biological products.

The aim of this study was to evaluate the possible carcinogenic potential of residual DNA derived from immortalized and possibly tumorigenic cell lines due to activated oncogenic sequences (oncogenes). These cell lines have been used for the production of biologicals, i.e. monoclonal antibodies, lymphokines and vaccines. The authors used hybridoma DNA as a first model. For this reason experiments in two species were performed, namely in 3-4 week-old female Balb/c mice and newborn Riv:TOX rats. Doses of 250 micrograms DNA, derived from Balb/c hybridoma cells, were injected subcutaneously (s.c.) in 200 mice. These mice also received a s.c. injection of the solvent only (TE buffer) at another site of the back skin (negative control for local tumour development). An additional group of 50 mice was treated intraperitoneally (i.p.) with the solvent only to serve as a negative control group for possible systemic tumorigenic effects. Doses of 5 micrograms plasmid pPy1 DNA, containing the entire Polyoma virus genome, served as positive control and were injected s.c. and i.p. in 20 and 50 mice, respectively. Doses of 50 micrograms hybridoma DNA or 5 micrograms pPy1 DNA were injected s.c. in rats too, using nine animals per group. During the experiment, animals were observed weekly, especially for the occurrence of subcutaneous tumours at the injection sites. The mouse study was terminated after more than 2 years, the rat study after 1 year. Gross necropsy was performed on all animals and histopathological examination of grossly suspected neoplastic lesions was performed. In the mouse experiment, tumour development at the s.c. injection site of the DNA was observed in one out of 20 animals in the pPy1-treated positive control group (neurofibrosarcoma) and one out of 200 animals in the hybridoma DNA-treated group (haemangioma-like lesion). Tumour development at or near the s.c. injection site of the solvent only was observed in two out of 200 animals. In the rat study none out of nine hybridoma DNA-treated rats developed tumours at the injection site, while three out of nine rats of the positive control group, injected with the pPy1 DNA, showed local tumour development (benign and malignant soft tissue tumours.) It is concluded that, at the high dose and numbers of animals tested, parenteral administration of hybridoma DNA does not induce local tumour development. Furthermore, no indications were found for systemic carcinogenic potential of the hybridoma DNA used. Based on a worst case approach of our data, the oncogenic risk of 100 pg residual DNA was estimated to be 2 x 10(-9), a value intermediate of the estimations of the WHO (1987) and the Dutch Health Council (1988) 5 x 10(-11) and 2 x 10(-7), respectively. Therefore, it is unlikely that the risk of 100 pg of DNA derived from other immortalized cell lines will exceed the level of generally accepted cancer risk of 10(-6).

Potency of veterinary rabies vaccines in The Netherlands: a case for continued vigilance.

Commercial rabies vaccines, used by veterinarians in the Netherlands, were collected for testing in the mouse potency test. Of the six vaccines tested, two were clearly below the minimal requirements for potency of 1.0 IU. Of these six vaccines the rabies virus glycoprotein (GP) and nucleoprotein (NP) contents were determined in an antigen competition ELISA. The GP content proved to correlate well with the potency found in the mouse potency test (r = 0.95, p < 0.01), whereas no such correlation was found for the NP content (r approximately 0, p > 0.05). After the manufacturers were told about the results, one of the two vaccines that did not comply with the requirements was withdrawn from the market. Measurement of the GP content of a second lot of the remaining vaccines indicated that sufficiently high levels of GP were present in all five. Additional in vivo testing in mice for efficacy against intracerebral challenge with the Dutch bat rabies virus EBL1-12 resulted in acceptable levels of protection with four of these five vaccines of the second lot. The data presented illustrate the need for continued potency evaluation of veterinary rabies vaccines in the Netherlands.

Poliomyelitis in The Netherlands: a review of population immunity and exposure between the epidemics in 1978 and 1992.

An overview of serological and virological studies on poliomyelitis in the Netherlands between two epidemics in 1978 and 1992 is given. Three unvaccinated patients acquired poliomyelitis abroad. In the Netherlands vaccination coverage with quadruple DPT-IPV vaccine is very high. The strong immunogenicity of inactivated poliovirus vaccine was confirmed in a cohort of children, reflected in age-stratified antibody profiles of the population. Adults born in the pre vaccination era appeared in general protected, but 10-25% of persons born between 1930 and 1945 lacked neutralizing antibodies. Revaccination induced a booster type of antibody response in 75-90% of such persons, indicating immunological memory and protection. Virological studies on adopted children from other countries, patients with indications for viral examination, and river waters showed that the Netherlands was regularly exposed to polio virus (PV), without signs of indigenous transmission. Persons found to carry PV or their close contacts had travelled to a PV endemic country. Most of 557 isolates were vaccine-derived, only 8% were wild type viruses. Despite their presence, up to 1992 the well-known susceptibles for PV in the Netherlands were shielded by the herd immunity of the Dutch population.

Simultaneous administration of oral rhesus-human reassortant tetravalent (RRV-TV) rotavirus vaccine and oral poliovirus vaccine (OPV) in Thai infants.

Rhesus-human reassortant tetravalent (RRV-TV) oral rotavirus vaccine was given at the same time as oral poliovirus vaccine (OPV) or inactivated parenteral poliovirus vaccine (IPV) to Thai infants at 2, 4 and 6 months of age. Sera for rotavirus antibody studies were taken prior to and one month after each vaccination. After the first dose of vaccine at 2 months of age, 37% of the infants receiving rotavirus vaccine with IPV but only 10% of those receiving it with OPV showed a seroconversion by rotavirus IgA ELISA antibody test (p < 0.001). Likewise, neutralizing antibody seroconversion rates in initially seronegative subjects to rhesus rotavirus type 3 (RRV-3) after the first dose of RRV-TV vaccine were higher if the vaccine was given with IPV (74%) than if given with OPV (39%) (p = 0.0069). After the second and third doses of vaccine, the rotavirus IgA ELISA and RRV-3-neutralizing antibody response rates were not different between groups. Development of neutralizing antibodies to human rotavirus serotypes 1, 2 and 4 in the first seven months of life in vaccinees receiving rotavirus vaccine with OPV tended to occur at a lower rate than in those receiving rotavirus vaccine with IPV but the antibody levels were not significantly different at 7 months of age. Poliovirus type 2 and type 3 antibody responses were not different in infants receiving the rotavirus vaccine with OPV as compared with infants receiving only OPV. The mean poliovirus type 1 antibody level was slightly but not significantly lower at 5 and 7 months of age in infants that received both rotavirus vaccine and OPV.(ABSTRACT TRUNCATED AT 250 WORDS)

Poliomyelitis outbreak in an unvaccinated community in The Netherlands, 1992-93.

An outbreak of poliomyelitis occurred in the Netherlands between September, 1992, and February, 1993, after 14 years without endemic cases. The outbreak was due to poliovirus type 3 and involved 71 patients, of whom 2 died and 59 had paralysis. The patients were aged between 10 days and 61 years (median 18 years). None of the patients had been vaccinated, and all but 1 belonged to a socially and geographically clustered group of people who refuse vaccination for religious reasons. Control measures were taken within 5 days of notification of the first patient and included a wide offer of vaccination with the trivalent oral poliovirus vaccine to the population at risk. Sequence analysis of the viral genome showed closest similarity (96.7%) with a strain isolated in India in 1992, indicating that the virus probably originates from the Indian subcontinent. The difference, however, is still too large to assume direct import. Extensive outbreak investigation at schools, in the environment, at virus diagnostic laboratories, and in the general population showed no evidence of widespread circulation of the epidemic virus outside the groups at risk and area where these groups live. As in the previous outbreak in 1978, the general population, including the majority of unvaccinated people who live dispersed in the population, seemed to be well-protected against poliomyelitis.

An enzyme-linked immunosorbent assay for the detection of mouse polyomavirus-specific antibodies in laboratory mice.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection and quantification of IgM and IgG serum antibodies to mouse polyomavirus (MPV). To evaluate the potential of this ELISA for the screening of laboratory rodents, serum samples from specific pathogen free (SPF) BALB/c RIVM mice, collected after experimental intraperitoneal infection with MPV, were tested by this assay. The results were compared with those obtained from the same sera in an immunofluorescence assay (IFA) and a haemagglutination inhibition assay (HIA). The ELISA proved to be the most sensitive of the 3 assays, allowing the detection of seropositive animals within 7 days post-infection and giving antibody titres that were about 4 to 8 times higher than those found in the IFA and HIA respectively. More than 5000 serum samples from non-infected specific pathogen free laboratory mice and 90 from 10 SPF N:NIH/RIVM mice experimentally infected with K-papovavirus, were negative in this assay, thus confirming the specificity of the ELISA.

Comparison of enhanced potency inactivated poliovirus vaccine (EIPV) versus standard oral poliovirus vaccine (OPV) in Thai infants.

Enhanced potency inactivated poliovirus vaccine (EIPV), combined with diphtheria-tetanus-pertussis (DTP) vaccine, was compared with oral poliovirus vaccine (OPV) regarding immunogenicity in Thai infants, vaccinated at 2, 4 and 6 months of age. EIPV induced significantly higher seroconversion rates than OPV to all 3 poliovirus types after the second and third immunization. After 3 doses of each vaccine, at 7 months of age, all infants receiving EIPV proved seropositive for poliovirus type 1, type 2 and type 3 neutralizing antibodies, whereas of those receiving OPV, 9% remained seronegative (titre < 1:4) for type 1 (p = 0.0042) and 11% for type 3 (p = 0.0013). All participating children were given an additional dose of OPV at the age of 9 months and tested again at 12 months of age. At that point, virtually all infants had poliovirus neutralizing antibodies, but the geometric mean titres to each poliovirus type were significantly higher in the vaccinees who had received EIPV. It is concluded that the greater immunogenicity of EIPV vis-à-vis 3 doses of OPV may be biologically significant for protection against poliovirus types 1 and 3 in countries where cases of poliomyelitis occur in young children. These findings warrant considering EIPV, alone or in combination with OPV, for an immunization programme in Thailand and similar countries in the future.

Effects of iodized oil on trivalent oral polio vaccine in vitro.

Iodine deficiency can be effectively reversed by administration of iodized oil (IO). Since 1989, the WHO Expanded Programme on Immunization recommended delivery of oral IO along with vaccines, in appropriate situations. However, administration of IO at the same time as trivalent oral polio vaccine (TOPV) has not been recommended because of absence of studies addressing potential harmful effects of iodized oil on immuno-responses to TOPV. In this study an in-vitro cell culture system was used to measure possible adverse effects of IO on TOPV. The system itself was shown not to be influenced by either IO or a standard non-iodized oil (NIO). However, marked effects of IO on virus infectivity was measured when a 50% mixture of IO and TOPV was incubated for 48 h at 37 degrees C, as compared to incubation without IO. Clear differences were found in effects of IO on vaccines of different sources, suggesting differences in composition of these vaccines, e.g. in stabilizers. Principally Sabin 3 appeared susceptible to IO under the given extreme conditions. Pre-dilution of the IO-TOPV mixture prior to incubation clearly reduced the effects of IO on virus infectivity Inoculation of the IO/TOPV-mixture directly onto susceptible cells, a model approaching the in vivo situation, resulted in no detrimental effects on virus infectivity. Further studies are planned looking at interactions of iodized oil and TOPV in children at immunization contacts.

[Poliomyelitis in The Netherlands, 1979-1991: immunity and exposure]. / Poliomyelitis in Nederland, 1979-1991: immuniteit en blootstelling.

An overview is presented of serological and virological studies on poliovirus immunization and circulation in the Netherlands, performed between 1979 and 1991. In this period, only three patients with poliomyelitis were notified. All had acquired the infection abroad. The vaccinations in the national immunization programme, using inactivated poliovirus vaccine, build a strong immunity. This can also be seen in age-stratified serological profiles of the Dutch population. In these surveys, persons from the time at which vaccination was offered have neutralizing antibodies. Older persons, especially those born between 1930 and 1945, sometimes lack antibodies. However, 85-90% of them show a rapid booster response upon vaccination, demonstrating immunological memory. Hence, they will be protected against poliomyelitis upon contact with wild poliovirus. Virological data show a regular import of poliovirus, especially in adoptive children tested on entry into the Netherlands, coming from developing countries. Nearly all other virus isolates in Dutchmen were related to import from such countries. None of the imported patients or other persons in whom poliovirus was detected spread the virus over the country. It demonstrates that as a rule the herd immunity of the well-vaccinated Dutch population is good. Exceptions occur, however, as demonstrated by the epidemics in 1978 and 1992. Large socio-geographic clusters of susceptible people who refuse vaccinations are not sufficiently protected.

[Degree of protection against poliomyelitis of Dutch subjects born before 1945 and of allochthonous subjects in Utrecht, and the relationship to socioeconomic status]. / Beschermingsgraad tegen poliomyelitis van Nederlanders geboren vóór 1945 en van allochtonen in de stad Utrecht, en het verband met de sociaal-economische status.

In 1992 a seroepidemiological survey regarding the immune status for poliomyelitis was carried out amongst the population of the city of Utrecht: Dutch people born before 1945 and migrant workers and their families of all ages. Migrant workers and their families were well protected against poliomyelitis, using the WHO criteria (titre > or = I:8). The Dutch people born before 1945 were also well protected. Protection of Dutch people born before 1945 was better as their socioeconomic status was lower.

[Virological and serological aspects of the Dutch polio epidemic in 1992]. / Virologische en serologische aspecten van de Nederlandse polio-epidemie in 1992.

During the 1992 poliovirus type 3 outbreak in the Netherlands virological and serological investigations were conducted. No molecular epidemiological link was traced between poliovirus type 3 that caused the outbreak of poliomyelitis in the Netherlands and isolates from previous epidemics investigated. Serological neutralization assessments indicate that the inactivated poliomyelitis vaccine used in the Dutch national immunization schedule induces immunity to the causative agent.

[The value of cytological studies in nodular goiter: results in 495 patients]. / De waarde van cytologisch onderzoek bij nodulair struma: resultaten bij 495 patiënten.

The accuracy of clinical diagnosis and fine-needle aspiration biopsy (FNAB) was evaluated in a total of 495 patients of whom 183 were operated upon within 6 months after FNAB and 312 were not. Operated patients were divided into three subgroups with high, moderate or low suspicion of malignant neoplasms on clinical grounds. Histological examination revealed an overall malignant neoplasm rate of 23%. The rate, i.e. the positive predictive value, was 74% for the subgroup with high clinical suspicion, vs 14% and 10% respectively for the subgroups with moderate and low clinical suspicion. The sensitivity of a high clinical suspicion was 60% and the specificity 93%. The overall sensitivity of FNAB was 93% in the operated group and probably not less than 87% in all patients studied; specificity was 71% and 84% respectively for these groups. The overall positive predictive value of a positive cytology result (malignant or uncertain) was 48%. In the subgroups with moderate or low clinical suspicion which are more representative of a non-university setting, the average predictive value was 27%. In our opinion all patients in the group with high clinical suspicion need surgical treatment, regardless of the FNAB result; those with lower degrees of clinical suspicion and malignant or uncertain FNAB result should also undergo diagnostic surgical exploration.

Purification process monitoring in monoclonal antibody preparation: contamination with viruses, DNA and peptide growth factors.

Administration in vivo of monoclonal antibodies to humans is challenged by considerations regarding their safety. Contamination with viruses, potentially oncogenic nucleic acids and biologically active components like growth factors and hormones forms a serious point of concern in this respect. We have investigated the potential risk of viral contamination by measuring the reduction of 12 different viruses (after spiking) in the standard downstream purification process of ascitic fluid. Depending on the type of virus added and the purification step employed, the reduction of infectious virus particles varies considerably. The overall reduction ranges from about 10(3), observed for a member of the family of Papovaviridae, to more than 10(12) for members of the families of Herpesviridae and Orthomyxoviridae. Using hybridization analysis with a mouse (genomic) DNA probe, we show that the amount of residual DNA in ascitic fluids may also vary considerably, ranging from 75 ng/ml to 1 microgram/ml. In crude preparations produced in cell culture, much lower DNA concentrations are found (0.3 ng/ml). When standard downstream purification procedures are applied to ascitic fluid, a significant reduction of residual DNA levels is observed in the purified monoclonal antibody preparations and in intermediate fractions. The overall reduction factors vary from about 10(3) to 10(4), which is also confirmed by spiking experiments with either purified DNA or crude chromatin-like DNA. Using in-vitro cellular assays, we further show that peptide growth factors like PDGF and TGF beta are present in considerable amounts in ascitic fluids. The observed biological activities, however, are completely eliminated during the purification steps applied.

Serological and virological assessment of oral and inactivated poliovirus vaccines in a rural population in Kenya.

A study was carried out in a rural community in Kenya to compare the humoral and intestinal immunity provided by three doses of oral poliovirus vaccine (OPV) and two or three doses of enhanced-potency inactivated poliovirus vaccine (IPV). The immunization series was started at 8-12 weeks of age and the interval between doses was 2 months. In children with low levels of maternal antibodies (i.e., those most at risk), the first dose of either vaccine stimulated antibody response. Children with high levels of maternal antibodies responded to the first dose of OPV, but not to that of IPV. Subsequent doses led to increases in the mean antibody titres with both vaccines. After three doses of OPV, the proportion of children with antibody titres of greater than or equal to 1:8 was 92% for type 1 virus, 98% for type 2, and 90% for type 3. After two doses of IPV the proportion of children with antibody titres of greater than or equal to 1:8 was 94%, 88%, and 97% for type 1, type 2, and type 3, respectively; after three doses of IPV, 100% of children had antibodies greater than or equal to 1:8 for types 1 and 3, and 98% for type 2. Intestinal immunity was tested with a challenge dose of type 1 OPV, but the dose used was too small to detect a significant difference between the vaccines.

The value of fine-needle aspiration biopsy in patients with nodular thyroid disease divided into groups of suspicion of malignant neoplasms on clinical grounds.

The accuracy of clinical diagnosis and fine-needle aspiration biopsy (FNAB) was evaluated in 169 patients surgically treated for nodular thyroid disease. Patients were divided into three groups with high, moderate, or low suspicion of malignant neoplasms on clinical grounds without previous knowledge of cytologic or histologic results. Histologic examination revealed an overall malignant neoplasm rate of 23%; the rate was 71%, 14%, and 11% for the groups with high, moderate, and low suspicion, respectively. The FNAB diagnostic interpretations of malignant and uncertain were considered positive. Overall sensitivity, specificity, and accuracy for FNAB were 92%, 71%, and 75%, respectively. Sensitivity in the high-, moderate-, and low-suspicion groups was 95%, 89%, and 88%, respectively; specificity was 88%, 72%, and 67%, respectively; and accuracy was 93%, 75%, and 69%, respectively. In our opinion, all patients in the group with high clinical suspicion need surgical treatment whatever the FNAB result; those with lower degrees of clinical suspicion and malignant or uncertain FNAB result [corrected] should also undergo surgery.

Tumor markers for ovarian cancer. A comparative immunohistochemical and immunocytochemical study of two commercial monoclonal antibodies (OV632 and OC125).

A comparative immunohistologic and immunocytologic study was performed to assess the immunoreactivity of the monoclonal antibodies OC125 and OV632, both directed at antigens present on epithelial ovarian tumors. OC125 reacted with 53 of 59 ovarian carcinomas, 20 of 20 uterine carcinomas, and 25 of 111 nongynecologic tumors (including 20 of 38 breast carcinomas). OV632 was demonstrated in 47 of 59 ovarian carcinomas, 11 of 20 uterine carcinomas, and only 7 of 111 nongynecologic tumors. With OV632 no reactivity was found in carcinomas of the breast or the gastrointestinal tract. Cytologic preparations of malignant effusions of patients with ovarian cancer showed reactivity with OC125 in 32 of 35 cases, and OV632 with positivity in 34 of 35 cases. Mesothelial cells in reactive effusions were OC125 positive in 16 of 20 cases but never showed positivity with OV632. The authors conclude that for histopathology a combination of OC125 and OV632 offers high sensitivity (0.86) and specificity (0.89) for ovarian cancer. For cytology, OV632 is the most specific tumor marker available.