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The DNA of tumor tissue K1 obtained at autopsy from a case of hepatocellular carcinoma (HCC) in a 9-year-old boy contained integrated hepatitis B virus (HBV) DNA at a single site in the chromosome (case 2, Chang et al.: Hepatology 13:316-320, 1991). To characterize further the integrated viral DNA sequences, a genomic library of the K1 DNA was constructed in the lambda L47.1 vector. One phage clone, designated KTM-1, containing integrated HBV DNA and cellular flanking sequences was obtained from this library. The restriction map and DNA sequence of this clone showed that the integrated HBV DNA was partially deleted and rearranged. The most conserved viral DNA sequences were surface and X genes and arranged in the opposite orientation. The viral core gene was not present. Using chloramphenicol acetyltransferase (CAT) assay, the C-terminal truncated X open reading frame was demonstrated to retain its trans-activating ability. The result suggested that the functional integrated X gene may play a role in hepatocarcinogenesis. The study also showed that the right cellular flanking sequences were human alphoid repetitive sequences. © 1994 Wiley-Liss, Inc.

In order to assess seroprevalence of human T-lymphotropic virus type I (HTLV-I) infection among intravenous drug abusers in Taiwan, serum samples were collected from 858 male study subjects. Antibodies against HTLV-I (anti-HTLV-l) in sera were tested by enzyme-linked immu-nosorbent assay and confirmed by Western blotting. The overall prevalence of anti-HTLV-l (2.3%) in drug abusers was significantly higher than that in the general population in Taiwan with a relative risk of 4.9, but it was only slightly higher than that in prostitutes (1.9%). There was a statistically significant increase in prevalence with age. Drug abusers engaged in prostitution had a significantly higher prevalence (18.2%) than those who were not (2.1%). No significant association with anti-HTLV-l positivity was observed with marital status and educational level. Tattooed abusers had an increased prevalence (2.7%) compared with the untattooed (1.4%). Drug abusers tattooed before 1980 had a significantly higher prevalence (3.5%) than those tattooed after 1980 (0.8%). Anti-HTLV-l prevalence was higher for those who had been blood transfused (4.5%) than untransfused abusers (2.0%). © 1994 Wiley-Liss, Inc.

A hepatocellular carcinoma cell line, HCC36, was established from an adult HBV carrier in Taiwan. From Southern blot analysis, there were at least four sites of integration of HBV DNA, and no viral replicative intermediates were detected. A genomic library was constructed from HCC36 DNA, and two phage clones, designated lambda 36A and lambda 36B, were shown to contain HBV DNA and flanking cellular sequences. In lambda 36A, HBV DNA sequences were quite conserved, and 7.4% base variation was detected. The viral sequences in lambda 36A and lambda 36B differed in only four bases, in addition to the microdeletion and -insertion observed in lambda 36B. The flanking cellular sequences identified in lambda 36A were human Alu sequences and in lambda 36B satellite sequences.

We describe a Taiwanese woman with chronic progressive myelopathy, in whom Western blot analysis of the serum and cerebrospinal fluid (CSF) displayed positive reactions to human T-lymphotropic virus type I (HTLV-I) proteins, p19, p24, p28, p36, gp46 and p53. HTLV-I proviral genomes were detected in the peripheral blood mononuclear cells (PBMC) and CSF cells by nested polymerase chain reaction and Southern blot hybridization. HTLV-I was successfully isolated from PBMC stimulated with interleukin-2 (IL-2). The established cell line, named THAM-1, was an IL-2-independent T-cell line with CD2+, CD3+, CD4+, CD25+ and HLA-DR+. Retrovirus particles with type C morphology were observed in the THAM-1 cells by electron microscopy, and HTLV-I-related antigens were also demonstrated by immunocytochemical staining and Western blot assay. Southern blot analysis revealed that HTLV-I proviral genomes were integrated into the THAM-1 cellular DNA. In Northern blot analysis, two extra-species of RNA were detected in addition to three typical viral transcripts. For the first time, an HTLV-I-producing T cell line was established from a patient with HTLV-I-associated myelopathy in Taiwan, an HTLV-I non-endemic area.

Preliminary analysis indicated that a 19-amino-acid peptide sequence (435 to 453 of GtfC) within a highly conserved region of the glucosyltransferases of the cariogenic streptococci might be functionally important (J.-S. Chia, S.-W. Lin, T.-Y. Hsu, J.-Y. Chen, H.-W. Kwan, and C.-S. Yang, Infect. Immun. 61:1563-1566, 1993). To obtain antipeptide monoclonal antibodies (MAbs), the 19-amino-acid peptide was conjugated to bovine serum albumin and used as an antigen in BALB/c mice. Six immunoglobulin G-secreting hybridoma clones, CJSm18-S1 to -S6, specifically reacted with this peptide and with purified GtfC and GtfD but not with bovine serum albumin in an enzyme-linked immunosorbent assay. The concentrated hybridoma supernatant of all six MAbs inhibited GtfC enzymatic activity but failed to inhibit GtfD, although GtfD contains the same peptide sequence. Further analysis of a purified immunoglobulin G2b MAb from one of the clones, CJSm18-S3, confirmed that this MAb specifically inhibited GtfC enzymatic activity for insoluble-glucan synthesis in a dose-dependent manner. CJSm18-S3, even at high concentrations, had no effect on GtfD, which synthesizes water-soluble glucan exclusively. Furthermore, the in vitro sucrose-dependent adherence of Streptococcus mutans was also inhibited by CJSm18-S3 in a dose-dependent manner. Our results indicate that the peptide containing the N-terminal conserved region of glucosyltransferases is functionally important for both enzymatic activity and bacterial adherence.

In order to know the phylogenetic relationship and the route of transmission of a variant of coxsackievirus A24 (CA24v), an agent that caused four sequential outbreaks of acute hemorrhagic conjunctivitis from 1985 to 1989 in Taiwan, the nucleotide sequence variations in the virus-encoded proteinase 3C region (549 nucleotides) were studied with 19 isolates. The prototype strain (EH24/70), four isolates from Japan, and two isolates from Hong Kong were used for comparison. The nucleotide sequences of the Taiwan strains from the 1985-1986 and 1988-1989 epidemics were closely related within each epidemic, while they were more distantly related between strains from two epidemics. Phylogenetic analysis by the unweighted pairwise grouping method of the arithmetic average revealed that the 19 Taiwan isolates had diverged into two groups, 1985-1986 and 1988-1989 groups. The time at which these two groups diverged was estimated to be around May 1982, more than 3 years prior to the first appearance of the CA24v epidemic in Taiwan. On each occasion, the viruses caused a 2-year epidemic and then disappeared. The Taiwan isolates from 1985 to 1986 were closely related to the Japan isolates from 1985 to 1986 and the Taiwan isolates from 1988 to 1989 were phylogenetically close to the 1989 Japan isolates, indicating that Taiwan and Japan had two common-source outbreaks. However, none of the 1988 Taiwan isolates were phylogenetically close to the 1988 Japan or Hong Kong isolates. The evidence revealed that Taiwan has had two repeated but discontinuous introductions of CA24v since its first appearance in Taiwan in 1985. None of the other CA24v strains have been detected so far.

Two cDNA clones covering the N- and C-terminal portions of the EBV BXLFI open reading frame were selected from a cDNA library derived from P3HR1 cells. The two clones were ligated, the N-terminal untranslated region truncated, and the product inserted into an E. coli expression vector, pET3CP°. The fusion protein was expressed under control of the T7 phage phi 10 gene promoter and shown to possess thymidine kinase activity. The protein was then used as an antigen to detect antibody reactivities in serum samples of nasopharyngeal carcinoma patients and healthy blood donors. Using a 1 :400 dilution of serum samples in Western blot analyses, it was possible to differentiate the reactivities of serum IgA of NPC patients and healthy donors. The prevalence of positive reactivity to EBV TK in NPC was around 84%. The test was compared to others used for early diagnosis of NPC and was able to detect some patients who were negative in those tests. © 1992 Wiley-Liss, Inc.

A clone of the Epstein-Barr virus (EBV) thymidine kinase (TK) gene was derived from a cDNA library of P3HR1 cells. The gene product was expressed as a fusion protein in a procaryotic system by using T7 RNA polymerase. The recombinant TK showed a molecular mass of 67 kDa and was biologically active. Antiserum raised in mice immunized with partially purified TK recognized an antigen present in EBV-superinfected Raji cells using an indirect immunofluorescence assay.

Human herpesvirus-6 (HHV-6) is a newly identified human pathogen. Currently clinicians rely mainly on blood lymphocyte culture and serological tests to diagnose HHV-6 infection. The polymerase chain reaction (PCR) was carried out on the plasma or sera of patients to determine the value of PCR in the diagnosis of HHV-6 infection. A total of 30 patients entered the study; 10 were experiencing acute HHV-6 infections and 20 were healthy and served as controls. HHV-6 DNA was detected by PCR in the serum or plasma of the 10 cases with acute HHV-6 infections. All 20 controls had no HHV-6 DNA in their sera. The time for serum to become PCR-positive coincided with the appearance of IgG HHV-6 antibody. The relatively late presence of HHV-6 DNA in serum might result from late lysis of infected cells by immune responses. It is concluded that detection of HHV-6 DNA by PCR in the serum is a valuable tool for the diagnosis of acute and/or active viral infection. © 1992 Wiley-Liss, Inc.

9614 Background: Gefitinib is a potential first-line treatment option for patients with advanced non-small cell lung cancer (NSCLC), especially for patients with activating mutations in the EGFR gene. However, little is known about patient-reported health-related quality of life (HRQOL) in this patient population. The aims of this study were to explore the prognostic values of baseline HRQOL for time-to-treatment failure (TTF), as well as the predictors of repeatedly measured posttreatment HRQOL, in advanced NSCLC patients receiving first-line gefitinib. Methods: A total of 106 chemonaive patients with advanced NSCLC were enrolled in a phase II trial. Gefitinib was given at a dose of 250 mg/d. HRQOL was assessed monthly with the EuroQoL instrument (EQ-5D) and the Lung Cancer Symptom Scale (LCSS) questionnaire. Baseline HRQOL and clinical/molecular predictors of TTF were jointly examined by multiple Cox's proportional hazards model. The associations between the clinical/molecular factors and repeatedly measured posttreatment HRQOL were analyzed by fitting marginal linear regression model using the generalized estimating equations (GEE) method. Results: In this prospective study, HRQOL data were obtained from 94 patients. Baseline EQ-5D index (estimated hazard ratio = 0.286, 95% C.I.: 0.135–0.603, p = 0.001) and the presence of L858R EGFR mutation in adenocarcinoma (estimated hazard ratio = 0.520, 95% C.I.: 0.307–0.880, p = 0.015) were retained as independent prognostic factors in the final multiple Cox's proportional hazards model for TTF. According to preliminary GEE analysis of repeatedly measured posttreatment HRQOL, the patients with wild-type EGFR consistently had worse HRQOL in EQ-5D index (p < 0.0001), EQ-5D VAS score (p = 0.0002), and LCSS global score (p < 0.0001), respectively. Conclusions: In advanced NSCLC patients receiving first-line gefitinib, better baseline EQ-5D index and L858R EGFR mutation in adenocarcinoma predict longer TTF. In addition, patients with wild-type EGFR had worse posttreatment HRQOL. No significant financial relationships to disclose.

An EBV-specific alkaline deoxyribonuclease (DNase) activity was first demonstrated in a few lymphoblastoid cell lines which containing both endonucleolytic and exonucleolytic activities (1,2). This enzyme acitivity was also detected in Raji cells superinfected with the virus (3). A retrospective study and a prospective study demonstrated that the anti-EBV DNase antibody in NPC patients araised before the appearance of clinical symptoms (4,5). In previous studies, we have identified a cDNA clone, BG9, from IUdR treated P3HR1 cells which encodes EBV specific DNase (6). When this 1.8 kb clone was used as a probe, 4 EBV DNase related transcripts have been revealed in P3HR1 cells by Northern analysis. In this study, the exact initiation and termination sites of the P3HR1 EBV DNase related transcripts were anaylsed.

18118 Background: Skin rash is the most common toxicity of EGFR-targeted therapy. Skin rash of EGFR inhibitor is associated with longer survival or tumor response. However, the clinical and genetic factors associated with this skin rash are not well understood. Methods: Fifty-two non-small-cell lung cancer patients enrolled in a prospective clinical trial of first-line gefitinib treatment were genotyped for EGFR intron 1 CA repeat polymorphism (CAn) and single nucleotide polymorphisms at promoters G-216T, C-191A, and R521K. Grade 2 to 3 skin rash within 4 weeks of treatment (early G2/3 rash) was correlated with the genotype and clinicopathological features of the patients by multivariate logistic regression. Results: Seventeen patients (32.7%) developed early G2/3 rash. In multivariate logistic regression analysis, only the CAn genotype was associated with early G2/3 rash and the effect was modified by patient age. Early G2/3 rash developed in 21% of patients with homozygous long allele (19 to 22 repeats, L) genotype (4/19), 31% of heterozygous short allele (15 to 18 repeats, S) / L genotype (8/26), and 71% of S/S genotype (5/7), respectively. The median ages of patients with early G2/3 rash and patients without early G2/3 rash were 57 years (range: 39–77) and 69 years (range: 43–86), respectively. The estimated logarithm of odds ratio (ln OR) for early G2/3 rash, as compared to S/S genotype, for S/L genotype was -0.038 multiplied by patient age (P = 0.011); and the ln OR for L/L genotype was -0.050 multiplied by patient age (P = 0.004). Fifty patients were evaluable for response. In logistic regression analysis, early G2/3 rash correlated with tumor response (P = 0.027). However, the CAn genotype was not significantly correlated with tumor response (P = 0.43). Conclusions: Homozygous short allele of EGFR CAn is more likely to develop skin rashes on gefitinib treatment. Genotyping of EGFR CAn appears to be a useful predictive marker for development of skin rashes on gefitinib use. [Table: see text]

7167 Background: Gefitinib is an effective treatment for chemotherapy-treated nonsmall cell lung cancer (NSCLC) in East Asian patients. There were only limited prospective studies on the use of gefitinib in chemonaive advanced or metastatic NSCLC patients. Methods: Eligibility included histological or cytological proven chemonaive stage IIIB/IV NSCLC, measurable disease, ECOG performance status of 0–2, good organ functions and candidate for platinum-based chemotherapy. Patients were treated with 250mg gefitinib per day. Tumor assessments were performed every two months by RECIST criteria. Responding patients were treated until progression. Patients with stable disease more than 2 months may continue treatment or shift to chemotherapy. The primary objective of this study was overall response rate of patients. Secondary objectives included 1-year progression-free survival, overall survival, and ancillary biomarker studies. Simon’s two-stage design was used to aim at a response rate more than 20%. Forty-four patients were planned in stage one and the study will be terminated if 5 or less patients responded to the treatment. A total of 106 patients are planned. Results: The planned interim analysis on the first 44 patients is reported here. There were 13 male and 31 female, median age was 63 (range 39–86), ECOG performance status of 0/1/2 were 0/40/4. 33 patients were nonsmokers. 39 patients had adenocarcinoma. The side effects of the treatment were mild and well accepted by patients. There were 24/9/10/1 patients with PR/SD/PD/NE. Overall response rate was 54.5% and disease control rate was 75%. The response rates for female / adenocarcinoma / nonsmoker were 58%/59%/61% respectively. The response rates for male / non-adenocarcinoma / smoker patients were 46%/20%/36%. The median time to progression for all patients was 6.3 months. Conclusions: Gefitinib is a very effective treatment for East Asian chemonaive advanced or metastatic NSCLC patients. Although the patients’ number was small, the response rates for patients with one unfavorable predictive factor were comparable to the response rates of patients receiving combination chemotherapy. The response rates and duration for 106 patients will be presented in the meeting. [Table: see text]

The open circular genome of human hepatitis B virus (HBV) is known to contain a partially double-stranded DNA with a single-stranded gap region of variable length. This circular structure of the genome is maintained by base-pairing of the 5' ends of the two DNA stands, the long or L(-) strand and the short or S(+) strand. By cloning, mapping, and sequencing studies, we have localized three recombinational junctions of the integrated HBV in two hepatoma samples, HT14 and FOCUS. Breakpoints of recombination derived from these results and those of others appear to be clustered and coincidental with the identified 5' or the deduced 3' end of the long-strand DNA, respectively. Statistical analysis of these results supports the hypothesis that integration preferentially occurs in an extremely narrow region on the HBV genome. This site-specific recombinational mechanism appears to be conserved among different HBV subtypes. No extensive sequence homology was found between each pair of the recombining parental molecules; however, at the site of crossover, 2- to 3-base-pair junctional homology was consistently observed. Examination of the patterns of the integrated HBV DNAs allowed us to categorize these various patterns into four different groups according to their end specificity and strand polarity. The molecular form of relaxed circle is proposed to be one major substrate for HBV integration. The effect of free strand in the integration of HBV is emphasized in this model. Unlike any other known DNA animal viruses, the site specificity of HBV integration appears to be similar to that of the retroviruses.

A micro-51chromium-release assay was used to examine the possible relationship between antibodies cytotoxic to lymphoid cells and EBV infections of the donors. No correlation was found between sera capable of killing lymphoid cells pre-labelled with51 chromium and the presence or absence of antibodies to the viral capsid antigen (anti-VCA). Sera obtained from patients with infectious mononucleosis, Burkitt's lymphoma, nasopharyngeal carcinoma and Hodgkin's disease known to have elevated titers to anti-VCA showed cytotoxicity similar to control values. There appeared to be both heat-stable and heat-labile complement-dependent cytotoxic antibodies.

Serum samples obtained from 13 individuals who were found to have a previous history of or to be suffering from nasopharyngeal carcinoma (NPC) were examined for the presence of antibody to Epstein-Barr virus (EBV) DNase activity. Significant to high levels of antibody to EBV DNase activity were detected in most serum samples obtained from four patients prior to the diagnosis of NPC. The samples from the other nine patients showed variable levels of antibody. The majority of the samples collected at the remission stage of the disease, especially those from long-term survivors, contained little or no antibody to the DNase activity. Data presented here suggest that antibody to EBV DNase activity may be a useful marker for the early diagnosis as well as the prognosis of NPC.

We have used particle induced X-ray emission (PIXE) to analyze the elemental compositions and uptakes of a lanthanide, yttrium in this report, in tumors and normal tissues of nude mice. A small amount of yttrium nitrate was injected into nude mice with tumors. Samples of normal and malignant tissues taken from these mice were bombarded by the 2 MeV proton beam from a 3 MeV Van de Graaff accelerator with a Ge detector system to determine the relative elemental compositions of tissues and the relative concentrations of yttrium taken up by these tissues. We found that the uptakes of yttrium by tumors were at least five times more than those by normal tissues. Substantial differences were often observed between the trace element weight (or concentration) pattern of the cancerous and normal tissues. The present result is compared with human tissues.

A retrospective study of nasopharyngeal carcinoma (NPC) revealed that smoking, working under poor ventilation, use of nasal balms or oil for nasal and throat troubles, use of herbal drugs, and anti-EBV antibody titer were found statistically associated. The dural interactions of these factors to the risk of NPC were presented. Except in work conditions with poor ventilation and when herbal drugs are used, all the combinations were synergistic. The synergistic actions were especially remarkable with smoking and other factors. The possible etiological mechanisms of NPC are discussed.

The consistent association of elevated antibody titers to the Epstein-Barr virus associated antigens in patients with nasopharyngeal carcinoma has led to correlations of antibody titers with tumor presence and extent. Recently, serum levels of specific glycoproteins and immunoglobulins have been correlated with tumor presence and extent in patients with head and neck squamous carcinomas, and elevation of the immunoglobulin IgA has been described in patients with nasopharyngeal carcinoma. These correlations prompted a study of 69 untreated patients with nasopharyngeal carcinoma and 53 healthy normal subjects in Taiwan to determine the relations among specific proteins (haptoglobin, α1-acid glycoprotein, α1-antitrypsin, α2-HS-glycoprotein, prealbumin, and albumin), immunoglobulin levels (IgA, IgG, IgM), anti-EBV associated antibody titers, and clinical tumor stage. Elevated antibody titers to EBV-associated antigens (EBV-associated nuclear antigen, viral capsid antigen, and early antigen) were related to tumor presence but not significantly related to clinical tumor stage. Compared with the levels in normal subjects, serum levels of acute-phase proteins (haptoglobin, α1-acid glycoprotein, α1-antitrypsin) were significantly increased (p < 0.001) in patients with nasopharyngeal carcinoma, and levels of α2HS-glycoprotein were significantly decreased (p < 0.001). Uniquely, serum levels of acute-phase proteins, particularly haptoglobin, were directly related to clinical tumor stage (p < 0.05 − 0.001). Furthermore, serum haptoglobin levels correlated directly with anti-EBNA antibody titers (p < 0.03) and serum levels of IgA (p < 0.01). Serum levels of α2HS-glycoprotein correlated inversely with titers of IgA antibodies to early antigen (p < 0.03). The findings demonstrate that measurement of specific serum glycoprotein and immunoglobulin levels may be useful adjuncts to anti-EBV antibody titers in the serodiagnosis and monitoring of response to treatment of nasopharyngeal carcinoma and aid in the derivation of improved staging systems for these tumors.

To understand the prevalence and incidence of hepatitis A virus (HAV) infection in Chinese children in Taiwan, we determined the age-specific prevalence of hepatitis A antibodies (anti-HAV) in 823 children, ranging from birth (cord blood) to 19 years of age. The frequency of anti-HAV rose with increasing age with three different slopes, probably reflecting different age-specific incidences: lowest under 4 years of age, intermediate between 4 and 10 years, and highest above 10 years. We obtained follow-up specimens on 618 of the preschool children under 6 years of age. 11 (1.9%) of the 573 without antibodies had seroconversions after an average of 1.9 years of follow-up. The annual incidence was 1.0% and increased with advancing age of the children after infancy. None of the 11 seroconversions had clinical hepatitis. Among the 28 children 1-24 months of age who had antibodies, only 1 was positive on follow-up, reflecting loss of passively acquired maternal antibody in most children.

A salt-dependent DNA polymerase activity was demonstrated in the culture of an EBV-producing, lymphoblastoid cell line (NPC-204 cells) treated with 5-iodo-2'-deoxyuridine (IUdR). There was a high frequency of levels of antibody to this enzyme in sera of patients with nasopharyngeal carcinoma (NPC). In contrast, sera from healthy subjects had little or no neutralizing activity. The high antibody level appeared as early as stage 1 of the disease in many NPC patients. The levels of the antibody increased with the progression of the disease and declined in treated patients. The results strongly suggest that tests measuring serum antibody against EBV DNA polymerase activity can be used for early diagnosis and prognosis of NPC.

SummaryAdenovirus stocks prepared by serial passage of the virus in rhesus monkey kidney cells were found to be contaminated with the simian papovavirus SV40. The infectivity titer and number of particles of SV40 in each of 4 virus stocks examined were higher than those of adenovirus. The virus stocks had an average adenovirus titer of 103.6 TCD50/ml and an adenovirus particle count of about 107.6 particles/ml. The same stocks had an average SV40 titer of 106.0 TCD50/ml and an SV40 particle count of about 109.0 particles/ml. Antigenicity of the adenovirus and SV40 components was tested by inoculation of rabbits and guinea pigs, using the untreated stock, and stocks heated in water and in MgCl2. When the virus stock was heated at 50°C for 30 minutes in water, the adenovirus component lost all of its infectivity and a large part of its antigenicity, while the SV40 component retained both infectivity and antigenicity. When the virus stock was heated in M MgCl2 at 50°C for 30 minutes, the adenovirus component...

Summary Three DNA viruses, vaccinia, herpes simplex, and adenoviruses, have been studied for thermal inactivation in the presence of monovalent and divalent cations. Na+ protected vaccinia and adenoviruses from inactivation at 50°C, but Mg++ and Ca++ markedly increased the rate of inactivation of all three agents. This confirms our earlier observation with another DNA virus (vacuolating SV40) (12) and is in contrast to the remarkable stabilizing effect exerted by divalent cations on the small RNA viruses (9, 10).

The following parameters of the Taiwan monkey,Macaca cyclopis, are presented and compared with other species ofMacaca: the menstrual cycle, sexual skin and vaginal desquamation changes during the menstrual cycle, time of ovulation, gestation period, breeding season, body weight of the newborn, age and body weight at menarche, body weight at first conception, spermatozoa count in the ejaculate, and the body weight at sexual maturity of the male. The many similarities in reproductive biology ofMacaca cyclopis, the rhesus monkey (Macaca mulatta), and the cynomolgus monkey (Macaca irus) include sexual skin and vaginal desquamation changes during the menstrual cycle, time of ovulation, gestation period, and placental sign. Body weight ofM. cyclopis is lower thanM. mulatta at birth, at menarche and at the first conception in females and at sexual maturation in males, is heavier thanM. irus at birth and is not different fromM. radiata at birth. No difference in menstrual cycle ofM. cyclopis was observed in animals housed in air-conditioned rooms compared to those housed in nonair-conditioned rooms. Summer amenorrhea was not observed inM. cyclopis but a high incidence of low vaginal desquamation was noted to occur in summer months. The mode of length of the menstrual cycle ofM. cyclopis is shorter thanM. mulatta and other species ofMacaca. The breeding season ofM. cyclopis in the wild extends from the end of September to January. In laboratory conditions their fertile period extends throughout the year.

Sera collected from patients with nasopharyngeal carcinoma (NPC), in Taiwan, were titrated for antibodies to herpes-type virus (HTV) in a Burkitt's lymphoma cell line (P3HR-1) by the indirect immunofluorescence antibody technique. The results were compared with those of neighborhood controls matched by age and sex, with patient families and with neighborhood control families. Antibody liters were higher in the NPC cases than in any of the 3 control groups. Dissociations in the frequency distributions of antibody titers in the NPC and healthy control groups were found to be maximum when the limiting value was set at 1:640. There were no significant differences in the frequency distribution among the 3 control groups. Of 117 NPC patients, 40% had anti-HTV titers equal to or greater than 1:640, while such values occurred in only 5, 5, and 6%, respectively, of the neighborhood controls, patient families, and control families. Though not statistically significant, there was difference in the percentages of “sero-positive” cases for the NPC patients by sex—42% for males and 35% for females. There was no sex difference, or it was a little higher in females, in the 3 control groups. The geometric mean titers were 1:242, 1:65, 1:69, and 1:61, respectively, for the NPC patients, neighborhood controls, patient families, and neighborhood control families. There was a difference in the geometric means by sex in the patients, i.e., 1:253 and 1:210, respectively, for males and females, while no differences by sex were noted in the 3 control groups. Ridit analysis and chi square test showed definitely different results between the NPC patients and the 3 control groups. The relative risks indicated that persons with antibody titer of 1:2560 had more than 200 times the NPC risk of those with antibody titer of less than 1:40. There were no indications of household aggregation of high anti-HTV titer (≥ 1:640) in the patients and control families. The significance and implications of these findings are discussed.

Summary MgCl2-inactivated adenovirus vaccine (type 7) was prepared by heating at 50°C for 30 min, six times longer than necessary to inactivate the infective moiety of the virus. The vaccine proved to be as antigenic in rabbits as the live virus, producing the same neutralizing and complement-fixing antibody titers. Infectivity could not be restored by removal of the MgCl2 through dialysis.

Antibodies to Epstein-Barr virus capsid antigen (anti-VCA) and early antigen (anti-EA) were measured in 263 patients with nasopharyngeal carcinoma (NPC), 624 age- and sex-matched neighborhood controls, 570 family members of NPC patients and 830 family members of neighborhood controls in Taiwan. The distribution of antibody titers was significantly different between NPC patients and the other three groups. More than 55% and 45% of NPC patients had titers of greater than or equal to 1:640 and greater than or equal to 1:80 for anti-VCA and anti-EA, respectively, while less than 6.7% and 2.5% of the other three groups had such high titers. The geometric means of anti-VCA and anti-EA titers were 1:352 and 1:45, respectively, in NPC patients compared to less than 1:77 and 1:12, respectively, in the comparison groups. Anti-VCA and anti-EA titers were significantly correlated. The association of EBV with NPC is discussed.

We have previously shown that high levels of antibody to Epstein-Barr virus (EBV)-specific DNase may be a useful marker for the early diagnosis of nasopharyngeal carcinoma (NPC). Sera from 3,368 males in an area with a high risk for the development of NPC were examined for the presence of antibody to EBV-specific DNase activity. Significant levels of the antibody were found in 430 of these sera. As a result, 32 individuals have attended the out-patient department of otolaryngology for clinical examination. Among them, one individual was found to have a stage II nasopharyngeal carcinoma. This result supports the value of determination of antibody to EBV-specific DNase as a marker for the early detection of NPC.

DNA extracted from two series of nude mouse passaged nasopharyngeal carcinoma tissues was digested with restriction erdonuclease Bam HI, then electrophoresed on agarose gel and analysed for the EBV DNA fragments by Southern hybridization with twenty-seven radioactive Bam HI DNA fragments from cloned EBV DNA of B95–8 cell line. Constant hybridization patterns were revealed in these experiments and the results suggest a stable existence of EBV DNA in these tissues even after a long time of passage. When EBV DNA Bam HI F, H, I, W and Y fragments were used as probes, extra hybridization bands were found in the autoradiographs. The significance of these divergencies is discussed.

Serum samples from 154 patients with nasopharyngeal carcinoma (NPC), 374 with other cancers, 1,000 normal controls from Government Employees' Clinic Center (GECC), and 3,642 individuals of various ethnic-dialect groups living in high-risk areas for NPC were collected and the concentration of antibodies to Epstein-Barr virus (EBV)-specific DNase activity was determined. Taking a serum sample where 1 ml will neutralize two or more units of the DNase activity as positive, 2-4 units as low level, 4-6 units as medium level, and more than 6 units as a high level of antibody, 90.3% of the NPC patients contained significant amounts of antibodies to EBV-specific DNase activity and most of those had high levels of the antibody. In contrast, only 11% of sera from patients with cancers other than NPC contained antibodies to EBV-specific DNase activity, and high levels were very rare (2.1%). The difference in positive rates between these two groups is highly significant according to the chi 2 test (P less than 0.001). The positive rate of this antibody in the control group (GECC) was 5.3% with 0.0%, 0.8%, and 4.5% having high, medium, and low levels of antibodies, respectively. Again, the difference in positive rates between the GECC group and the NPC group is statistically significant (P less than 0.001). Taken separately, the positive rates of anti-EBV DNase activity in the three high-risk groups were 11.7%, 13.0%, and 13.1%. No significant difference in age distribution for the levels of this antibody was observed in the control GECC group or the three high-risk groups. However, the positive rates of the three high-risk groups are more than twice those of the GECC group (11.7% approximately 13.1% vs 5.3%). This ratio coincides with the ratio of the probability of developing NPC in high-risk groups compared to that of the GECC group (also more than two times). The significance of this coincidence is discussed.

Sera from 126 patients with nasopharyngeal carcinoma (NPC), 48 other cancers (OC) and 54 normal controls (NC) were collected at the National Taiwan University Hospital and tested for immunoglobulin levels as well as for IgG and IgA antibodies against Epstein-Barr virus (EBV) capsid antigen (VCA), early antigen (EA) and nuclear antigen (EBNA) by the immunofluorescence antibody technique. The mean concentrations of IgA and IgG in untreated NPC patients were higher than those of OC and NC groups, but IgM level was lower than that of OC group. The incidence and geometric mean titers (GMT) of anti-EA and anti-VCA in serum IgG and IgA, and anti-EBNA were substantially higher in NPC patients than in OC and NC groups. The GMT of anti-VCA in IgG and IgA, and anti-EA in IgG increased with advancing clinical stages of NPC but no such an increment was observed in anti-EBNA. After successful treatment the GMT of anti-VCA in IgG and IgA, anti-EA in IgG, and anti-EBNA became lower than those of pre-treatment and these antibody titers except anti-EBNA titers increased again in recurrent cases, indicating that tests for EBV-associated antibody titers are useful for monitoring the clinical status of NPC. In untreated and treated NPC patients, anti-VCA in IgG and IgA were significantly correlated (correlation coefficient r = 0.65 and 0.72 respectively, i.e. p < 0.005 in both groups). A correlation was also observed in anti-EA in IgG and IgA in untreated NPC patients (r = 0.2, p < 0.05). Among 11 throat washings of untreated NPC patients tested, anti-EA and anti-VCA in both IgG and IgA were detected in 1, 5, 1 and 2 cases, respectively.

A complementary DNA (cDNA) library of mRNAs obtained from P3HR1 cells treated with IUDR was constructed in λgt10 and screened with radiolabeled B95–8 EBV DNA Bam HI fragment B and G probes. One clone, designated BG9, containing a 1.7 kb cDNA was further studied. Results of complete DNA sequencing revealed that BG9 emcompassed the B95–8 EBV DNA sequences from 120,747 to 122,412 base pairs and corresponded to the BGLF5 open reading frame of EBV DNase gene. A detailed comparison of the sequences of BG9 with those of published B95–8 EBV DNA indicated that there were 14 different base pairs which may result in 7 amino acid residue changes.. The products of in vitro transcription and translation of a subclone, pGEM3–BG9, appeared to contain EBV DNase activity and a 52 kd protein was immunoprecipitated with a NPC patient’s serum containing high level of antibody to this enzyme activity.

The prevalence of Taiwan monkeys with antibodies to Epstein-Barr virus, as detected by the indirect immunofluorescence technique, was lower than that of healthy persons in Taiwan.