Journal of Behavioral Medicine, VoL 7, No. 11 1984

Stress and the Transformation of Lymphocytes by Epstein-Barr Virus

J a n i c e K. K i e c o l l - G l a s e r , ~ 5 Carl E. S p e i c h e r , 2

J a n e E. H o l l i d a y , 3 and R o n a l d Glaser 3,4

Accepted forpublication: April 22, 1983

Although various stressors appear to influence herpesvirus infections, the underlying mechanisms have not been studied. A prospective design was used to examine the effects o f examination stress and loneliness on the transformation o f B lymphocytes in mixed cultures o f T and B lymphocytes by Epstein-Barr virus (EBV). Three blood samples were drawn from 42 EBV-seropositive medical students, with the baseline sample taken 1 month before their final examinations, the stress sample drawn on the first day of final examinations, and the third sample taken the first week after their return from summer vacation. A median split on the UCLA Loneliness Scale divided the subjects into high- and low-scoring loneliness groups. There were significant effects for change over trials, with the lowest trans-

formation levels (i.e., more virus required to transform cells)found in the stress sample. There was also a significant main effect for loneliness, in which high loneliness was associated with lower transformation levels. Possible immunological pathways for the observed changes are discussed.

KEY WORDS: Epstein-Barr virus; loneliness; stress; immune response.

~Department of Psychiatry, The Ohio State University College of Medicine, Columbus, Ohio 43210.

2Department of Pathology, The Ohio State University College of Medicine, Columbus, Ohio 43210.

3Department of Medical Microbiology and Immunology, The Ohio State University College of Medicine, Columbus, Ohio 43210.

4Comprehensive Cancer Center, The Ohio State University College of Medicine, Columbus, Ohio 43210.

5To whom correspondence should be addressed at Department of Psychiatry, The Ohio State University College of Medicine, 473 West 12th Street, Columbus, Ohio 43210.

1

0160-7715/84/0300-0001 $03.50/0 �9 1984 Plenum Publishing Corporation

2 Kiecolt-Glaser, Speicher, Holliday, and Glaser

INTRODUCTION

There is abundant anecdotal support for the impact of various stress- ors on the duration and intensity of human herpesvirus infections. How- ever, the immunological data which are necessary to demonstrate a linkage between psychosocial stressors and the herpesviruses have been lacking. This study was an attempt to demonstrate the joint impact of academic stress and loneliness on an immunological variable which is important in controlling the expression of one of the herpesviruses, while concurrently providing data on one potential immunological pathway through which stress might potentiate herpesvirus infections.

The Epstein-Barr virus (EBV) is a human oncogenic herpesvirus which has been shown to be the etiologic agent for infectious mononucleosis (IM) (Henle and Henle, 1982). Two previous studies have supported the associa- tion between academic stress and EBV infection. A prospective study of cadets entering the West Point Military Academy provided data on psy- chosocial risk factors which were predictive of the development of IM. Class members were screened on entry, and those who lacked antibody to EBV, i.e., had no previous infection, were followed for 4 years. Increased risk for infection as defined by seroconversion (subsequent presence of antibody) was associated with high levels of motivation, poor academic performance, and having fathers who were "overachievers" (Kasl et al., 1979).

In a recent study we found that elevated antibody titers to EBV anti- gens among students previously exposed to EBV, and in whom the virus was latent, appeared to be affected by academic pressures (Glaser et al., 1984). We obtained blood samples from medical students before and during ex- aminations and on their return from summer vacation. The EBV virus capsid antigen (VCA) IgG antibody titers were abnormally high in sera obtained from the first two samples and decreased significantly in the third. Subjects who were in the high-loneliness group had significantly higher VCA antibody titers than low-loneliness subjects on both samples. The ele- vated antibody titers presumably reflected changes in the control of latency of the EBV, although the pathways through which stressors may influence EBV latency have not been explored.

There are also data from human studies which suggest an association between reactivation of latent Herpes simplex virus (HSV) and level of distress. Although mood changes were unrelated to the recurrence of herpes labialis (RHL) in nursing students, general unhappiness was predictive of RHL frequency on a 1-year follow-up (Luborsky et al., 1976). Similarly, the level of distress appeared to affect the control of latent HSV and re- current genital lesions (Goldmeier and Johnson, 1982); more distressed subjects had significantly higher recurrence rates.

Stress and EBV 3

Certain unique characteristics of the herpesviruses suggest that they might be particularly sensitive as markers of stress-related changes in cellu- lar immunocompetency. When an otherwise healthy person develops a viral infection such as influenza or mumps, the cellular immune response is mobilized and the virus is eventually completely eliminated. However, the herpesviruses are able to escape eradication in the host because of their ability to escape detection while they reside in latently infected cells after the initial period of active infection. Indeed, HSV can be reactivated to induce lesions even in the presence of high circulating antibody levels. The cellular immune response is responsible for both the limitation of the primary infec- tion and the control of the latent virus. It is therefore not surprising that stress-related changes in the potency of the cellular immune response would alter its control over herpesvirus infections (Glaser and Gottleib-Stematsky, 1982).

If peripheral blood lymphocytes from an adult seropositive (i.e., pre- viously infected and still latently infected) donor are infected with a strain of transforming EBV and allowed to incubate in vitro, lymphoblastoid cell lines (LCL) can be established which are capable of indefinite growth; EBV DNA becomes stably and latently associated with the host cell (Pope, 1979). However, it is more difficult to demonstrate this phenomenon when cells from EBV seropositive individuals are used because specific killer T cells are activated in vitro to lyse the EBV-infected B lymphocytes (Rickinson et al., 1981).

In this study we measured the ability of EBV to transform B lym- phocytes in mixed cultures of T and B lymphocytes obtained from the peri- pheral blood of EBV-seropositive medical students. Three samples were obtained, which varied according to presumed stressfulness: (1) 1 month before final examinations, (2) on the first day of finals, and (3) on return from summer vacation. The ability of EBV to transform B lymphocytes from these individuals was measured and compared among the three blood samples. Using this prospective design, it was expected that the most extreme changes would occur on the second sample, which was expected to represent the most acute distress point. These expected changes in trans- formation would provide evidence linking academic stress and the trans- formability of B lymphocytes. The data were also studied in relationship to loneliness, since higher EBV antibody titers have been found in high- loneliness subjects (Glaser et al., 1984). Significant differences in transformation levels were expected between high- and low-loneliness subjects, given past research which has demonstrated differences in cellular immunocompetency as a function of loneliness (Kiecolt-Glaser et al., 1983a, b). Self-report distress data were also collected from subjects in order to provide an independent measure against which to validate the presumed stressfulness of the three sample points.

Kiecolt-Glaser, Speicher, Holliday, and Glaser

METHOD

Subjects and Timing o f Samples. The first-year medical student class of The Ohio State University College of Medicine was asked for volunteers for research on stress and the immune response. Of 175 students in the class, 76 responded to the initial announcement, 75 returned for the second sample, and 70 returned for the third sample. The average age for the sample of 23 women and 47 men was 23 years.

The initial blood draw occurred 1 month before the final examination period at the end of April and was designated baseline, or Bleed 1. The second sample was obtained on the first day of final examinations at the end of May and represented an acute stress sample, or Bleed 2. The third sample was drawn 3 months later, during the students' first week in school following their summer vacation, in early September. It was presumed to represent the least stressed sample and was designated Bleed 3.

Self-Report Measures. The Brief Symptom Inventory (BSI), a short form of the Symptom Checklist-90 (SCL-90; Derogatis, 1977), provided data on nine primary symptom dimensions and three global distress in- dices. Each item was rated on a five-point scale according to the amount of discomfort the problem had caused in the last week. It was administered at the same time the blood samples were obtained, in order to provide an in- dependent measure of distress.

The UCLA Loneliness Scale (Russell et al., 1980), administered at the time of Bleed 1, provided a measure of the subjective adequacy of inter- personal contacts. Scores were divided at the median, 34.5, to form high (mean = 41.62)- and low (mean = 28.6)-scoring loneliness groups.

We were also interested in transient changes in loneliness, so the brief State Loneliness Scale was included for all three samples (Shaver et al., 1981). On this questionnaire respondents rated their agreement with state- ments based on their feelings during the "last few days."

Cells. The Burkitt LCL B95-8 was grown in RPMI 1640 medium supplemented with 8~ fetal bovine serum (FBS), 25 tzg/ml Gentamicin, and 0.025~ sodium bicarbonate in 16-oz glass prescription bottles. The cells were maintained at 35~

Preparation o f EBV Stocks. The B95-8 cells were grown in 3-liter spinner flasks in preparation for virus harvest. Cells were treated with 13-O- tetradecanoyl-phorbol-13-acetate (TPA), 40 ng/ml, for 7 days without feeding in order to increase the amount of virus synthesis. The super- natants were then harvested and virus concentrates were prepared by pro- cedures previously described (Henry et al., 1978). The B95-8 virus stocks were titrated using human cord blood B lymphocytes with an end point of 30 days.

Stress and EBV 5

EBVAntibody Assays. The indirect immunofluorescence (IF) test was used to determine which individuals were antibody positive for EBV. The procedure used has been described previously (Glaser et al., 1973).

EBV Transformation Studies. Heparinized whole blood was obtained from each individual. The lymphocytes were separated from the red blood cells using Hypaque-Ficoll gradient centrifugation. The lymphocytes (a mixture of T and B cells) were seeded into 24-well Linbro tissue culture plates at a concentration of 1 x 106 total cells per well. Human cord blood lymphocytes originally used to titrate the transforming titers of the EBV stock used were infected as controls to confirm the transforming activity of the virus. Log dilutions, from 10 .2 to 10 6, of a stock of EBV with trans- formation activity of 106 transforming units (TU/ml), were prepared in RPMI 1640 medium. One milliliter of each virus dilution was placed in each well. Infected cells were incubated at 37~ for 30 days and were fed twice a week by the removal of approximately 1 ml of medium and replacement with 1 ml of fresh medium. The competence of the cellular immune response was measured indirectly by determining the amount of EBV necessary to induce transformation of B lymphocytes. Transformation titers were determined by visibly observing for cells dividing to form colonies (typical of cells transformed by EBV) in the well with the highest dilution of EBV. One well per sample was set aside as a control, to account for spontaneous transformation. One milliliter of medium was placed in this Well as substitution for 1 ml of virus dilution. Ability to transform the B lymphocytes in the mixed cell cultures is defined here as those EBV-infected B lymphocytes actively growing 30 days postinfection. If no transformation was observed at any dilution, the transformation level was designated 10 All samples were blind coded. The transformation control using human cord blood lymphocytes confirmed a 106 TU/ml transforming titer with our EBV stock. The same virus stock, the same lot of 24-well plates, and the same lot of media and FBS were used for all transformation assays through- out the study. Therefore, while it was not possible to perform transforma- tion experiments using cells from the three blood samples on the same day, all other conditions were essentially identical and controlled.

Data Analysis and Transformation of Data. Both the immunological and the self-report data were analyzed using a repeated-measures analysis of variance design (ANOVA), with one within-subjects variable (change over trials) and one between-subjects variable (loneliness). This produced a 3 x 2 ANOVA design which permitted the evaluation of the expected changes over time in transformation levels, the effect of loneliness, and any interac- tions between the two variables.

The transformation data were analyzed following a base 10 loga- rithmic conversion. This logarithmic trasnformation reflected the loga- rithmic dilutions of virus used in this study.

6 Kiecolt-Glaser, Speicher, Holliday, and Glaser

Fig. 1. (a) Photomicrograph of EBV-infected blood mononuclear cells showing B tymphocytes transformed by EBV 30 days postinfection. Note the large clump of lymphoblastoid cells. (b) Photomicrograph of control (mock-infected) blood mononuclear ceils 30 days postinfection.

Stress and EBV 7

RESULTS

Transformation of Lymphocytes by EBV. Of the 70 subjects who par- ticipated in all three samples, 12 were EBV seronegative, i.e., they did not have VCA IgG antibody, and were therefore excluded from the analyses. Lymphocytes from an additional 8 subjects spontaneously transformed in one of the three blood samples and therefore were excluded, resulting in incomplete data for that particular subject; in no case did we observe spontaneous transformation of control cell cultures from two consecutive blood samples. Data from an additional 8 subjects were excluded because of fungal contamination of ceil cultures of one or more samples, leaving a total of 42 subjects for data analysis. The remaining subjects were equally distributed between the high- and the low-loneliness groups.

Transformation of B lymphocytes was observed in Bleeds 1-3 (Figs. 1 a and 1 b). There were significant effects for both change over trials and loneliness, as shown in Fig. 2. More virus was required to t ransform cells

- 4

W !- t- z - 3 o

@ z

n,,

0 - 2 --1

:Z ]~ LOW t

LONELINESS /

' / IS

,(

I 2 3 BLOOD SAMPLES

Fig. 2. Mean (~ SE) transformation levels of peripheral blood B lymphocytes in- fected with EBV obtained from medical students at three different times.

8 �9 Kiecolt-Giaser, Speicher, Holliday, and Glaser

Table I. Means and Standard Deviations of BSI TScores Across Samples

Sample

BSI scale 1 2 3

Somatization* 50.34 (7.43) 53.51 (9.03) 48.51 (7,97) Obsessive-Compulsive** 59.06 (9.83) 63.23 (10.65) 53.87 (10.78) Interpersonal Sensitivity* 59.11 (11.43) 58.89 (12.18) 55.83 (10.97) Depression*** 58.66 (10.25) 60.15 (9.80) 55.04 (10.04) Anxiety** 57.85 (10.58) 67.47 (9.25) 56.30 (9.96) Hostility*** 52.89 (10.03) 58.94 (9.76) 53.13 (8.57) Phobic Anxiety 53.83 (9.18) 55.94 (9.29) 53.36 (8.60) Paranoia 53.28 (10.59) 54.64 (10.72) 52.77 (10.39)

Psychoticism* 58.68 (11.56) 60.60 (11.24) 54.96 (10.43) General Symptom Index*** 53.89 (7.94) 55 .51 (8:74) 51.13 (9.54) Positive Symptom Total** 58.06 (8.87) 61.17 (9.17) 54.40 (11.65) Positive Symptom Distress Index** 55.06 (6.76) 59.11 (6.85) 49.83 (10.49)

*P < 0.01. **P < 0.0001.

***P < 0.001.

from the second bleed, compared to the first and third bleeds [F(2,80) = 12.53, P < 0.0001]. There were significant differences between

the high- and the low-loneliness groups IF(I,40) = 4.16, P < .05], with the high-loneliness group having lower transformation levels, reflecting the requirement for more virus. An interaction between change over trials and loneliness approached significance [F(2,80) -- 2.36, P < 0.10].

Self-Report Measures. There were significant changes over trials for all BSI scales except phobic anxiety and paranoia, as shown in Table I. The pattern of scores on most of the scales fit the presumed stressfulness of the three sample points, with the highest scores on Bleed 2, followed by Bleed 1, with the lowest scores on Bleed 3. Although�9 high-loneliness group had higher scores on most of the BSI scales, the effects did not reach statistical significance.

As would be expected from scales tapping similar dimensions, high scorers on the UCLA Loneliness Scale had higher State Loneliness scores across the three bleeds [F(1,40) = 8.91, P < 0.005]. There was not a sig- nificant change in State Loneliness scores over trials [F(2,80) = 2.10]. The interaction between loneliness group membership and change over trials was not significant [F(2,80) -- 2.021.

DISCUSSION

In this study, we measured the effects of examination stress and loneliness on the ability of B lymphocytes infected with EBV (in mixed cultures with T lymphocytes) to transform into growing LCLs. Blood

Stress and EBV 9

samples were obtained from medical students on three separate occasions. The data obtained suggest that the transformation of lymphocytes into LCLs by EBV can be significantly affected by both stress and loneliness. These data are particularly noteworthy in that this young and healthy subject sample had ample prior exposure to such examination stress. These data provide preliminary evidence for one mechanism through which stress could have an impact on herpesvirus latency.

No occurrence of IM was reported at any time immediately preceding or during the course of the study. Thus, all the students who were EBV seropositive were antibody positive due to the presence of latent EBV, presumably associated with their B lymphocytes.

There are several possible interpretations of these data in regard to the immune response. There may have been physiological differences in the susceptibility of the B lymphocytes to transformation by EBV across the three different blood samples. Alternatively, the B lymphocytes may have had the same ability to be transformed by EBV, but the memory T lym- phocytes in these EBV-seropositive individuals may have varied in their ability to inhibit the outgrowth of the EBV-infected B lymphocytes. It is also possible that the transformation levels could be a function of the total numbers of memory T lymphocytes present at the time of sampling (Rickinson et al., 1981) and/or an interaction with other T lymphocyte subpopulations (e.g., helper and suppressor cells). While the second and third hypotheses are more probable alternatives, studies are underway to determine which of these alternatives are specifically associated with the stress-related changes which were observed in this study.

The EBV data may have implications for certain kinds of cancer and neurologic diseases. The EBV is a prime human tumor virus candidate because of its strong association with African Burkitt lymphoma and naso- pharyngeal carcinoma (Tuckwiller and Glaser, 1983). Linkages are being in- vestigated between EBV and certain lymphoproliferative diseases, e.g., renal transplant " l ymphomas , " the X-linked lymphoproliferative syndrome, etc. (Hanto et al., 1981; Henle and Henle, 1981; Purtillo et al., 1981; Saemundsen et al., 1981). The EBV is also associated with certain neuro- logic diseases, e.g., Guiltain-Barre syndrome (Gottleib-Stematsky and Glaser, 1982). The significant changes in transformation levels could have greater EBV-associated risks in an immunologically compromised popula- tion, as well as greater risks associated with other herpesvirus infections (Glaser and Gottleib-Stematsky, 1982).

One variable which may have significantly affected the results is the chronicity of the stressor. At the time of the first bleed the students de- scribed themselves as chronically distressed, due to unrelenting academic pressures. Monjan (1981), summarizing literature on the temporal relation- ships between stress and immunocompetency, suggested that acute stressors

10 Kiecolt-Glaser, Speicher, Hoiliday, and Glaser

are more likely to produce decreased immunological function, while chronic stressors may actually lead to an enhancement of the immune response.

In this study we found that high-loneliness subjects did not describe themselves as significantly more distressed than the low-loneliness group. These data are in contrast to previous loneliness research (Pepalu and Perlman, 1982). It may be that there are dimensions of loneliness apart from those associated with distress which have an impact on immunocom- petency.

We have previously found an association between loneliness and poorer immunological competence. Both medical students and psychiatric inpatients who were above their group's median in loneliness had sig- nificantly lower levels of natural killer cell (NK) activity (Kiecolt-Glaser et al., 1983a, b). Lonelier psychiatric inpatients also had a significantly poorer response to phytohemagglutinin, a mitogen which stimulates T-lymphocyte proliferation in vitro.

This significant association between loneliness and immunocom- petency in two different populations is striking. The results of this study support the importance of loneliness as an important mediator of health and illness which deserves further study.

ACKNOWLEDGMENTS

This study was supported in part by funds from the Samuel J. Roessler Foundation, the Academic Enrichment Fund, Division of Clinical Psy- chology, Department of Psychiatry, federal funds administered through the Work-Study Program of The Ohio State University, and The Ohio State University Comprehensive Cancer Center Grant CA-16058-09 from the National Cancer Institute, National Institutes of Health.

We thank Mr. Jay Stoerker, Ms. Lynn Wilcox, Ms. Trudy Hupp, Ms. Denise Ricker, Mr. William Briner, Ms, Lisa Ims, Mr. Jeff Crain, Ms. Jan Curtis, Mr. Martin Dunn, Ms. Sue Romick, Ms. Allison Post, and Mr. Dan Green for laboratory assistance. We also wish to thank Ms. Nancy Nicholls, Ms. Vera Garofalo, Ms. Connie Gillam, Ms. Patti Warren, Ms. Kathy Reger, Mr. Jae Lee, Ms. Carolyn Pittenger, and Ms. Nellie Cherevas for drawing blood.

We are grateful to our medical student subjects who entered the lecture-discussion program in 1981 at The Ohio State University. Without their enthusiastic support this project would not have been possible.

Stress and EBV 11

R E F E R E N C E S

Derogatis, L. R. (1977). SCL-90 R-(Revised) Version Manual-L Clinical Psychometrics Research, Baltimore.

Glaser, R., and Gottleib-Stematsky, T. (eds.) (1982). Human Herpesvirus Infections: ClinicalAspects, Marcel Dekker, New York.

Glaser, R., Kiecolt-Glaser, J. K., Holliday, J., and Speicher, C. (1984). The relationship of stress and loneliness to Herpesvirus antibody titers (submitted for publication).

Glaser, R., Nonoyama, M., Decker, B., and Rapp, F. (1973). Synthesis of Epstein-Barr virus antigens and DNA in activated Burkitt somatic cell hybrids. Virology 55: 62-69.

Goldmeier, D., and Johsnon, Ao (1982). Does psychiatric illness affect the recurrence rate of genital herpes. Br. J. Vener. Dis. 58: 40-43.

Gottleib-Stematsky, T., and Glaser, R. (1982). Association of Epstein-Barr virus with neurologic diseases. In Glaser, R., and Gottleib-Stematsky, T. (eds.), Human Herpes- virus Infections: ClinicalAspects, Marcel Dekker, New York.

Hanto, D. W., Friaaera, G., Purtilo, D. T., Sakamoto, K., Sullivan, J. L., Saemundsen, A. K., Klein, G., Simmons, R. L., and Najarian, J. S. (1981). Clinical spectrum of lymphoproliferative disorders in renal transplant recipients and evidence for the role of Epstein-Barr virus. CancerRes. 41:4253-4261.

Henle, W., and Henle, G. (1981). Epstein-Barr virus-specific serology in immunological com- promised individuals. Cancer Res. 41 : 4222-4225.

Henle, W., and Henle, G. (1982). Epstein-Barr virus and infectious mononucleosis. In Glaser, R., and Gottleib-Stematsky, T. (eds.), Human Herpesvirus Infections: Clinical Aspects, Marcel Dekker, New York.

Henry, B. E., Glaser, R., Hewetson, J., and O'Callaghan, D. J. (1978). Expression of altered ribonucleotide reductase activity associated with the replication of the Epstein- Barr virus. Virology 89: 262-271.

Kasl, S. V., Evans, A. S., and Niederman, J. C. (1979). Psychosocial risk factors in the development of infectious mononucleosis. Psychosom. Med. 41: 445-466.

Kiecolt-Glaser, J. K., Garner, W., Speicher, C., Penn, G. M., Holliday, J., and Glaser, R. (1984a). Psychosocial modifiers of immunocompetence in medical students. Psy- chosom. Med. 46: 7-14.

Kiecolt-Glaser, J. K., Ricker, D., Messick, G., Speicher, C., Holliday, J., Garner, W., and Glaser, R. (1984b). Urinary cortisol levels, cellular immunocompetency, and loneliness in psychiatric inpatients. Psychosom. Med. 46:15-24.

Luborsky, L., Mintz, J., Brightman, V. J., and Katcher, A. H. (1976). Herpes simplex and moods: A longitudinal study. J. Psychosom. Res. 20: 543-548.

Monjan, A. A. (1981). Stress and immunologic competence: Studies in animals. In Ader, R. (ed.), Psychoneuroimmunology, Academic Press, New York.

Peplau, L. A., and Perlman, D. (eds.) (1982). Loneliness': A Sourcebook of Current Theory, Research and Therapy, John Wiley and Sons, New York.

Pope, J. H. (1979). Transformation by the virus in vitro. In Epstein, M. A., and Achong, B. G. (eds.), The Epstein-Barr Virus, Springer-Verlag, New York.

Purtilo, D. T., Sakarnoto, K., Saemundsen, A. K., Sullivan, J. L., SYnnerholm, A. C., Anvret, M., Pritchard, J., Sloper, C., Sieff, C., Pincott, J., Pachman, L., Fich, K., Cruzi, F., Cornet, J. A., Collins, R., Barnes, N., Knight, J., Sandstedt, B., and Klein, G. (1981). Documentation of Epstein-Barr virus infection in immunodeficient patients with life-threatening lymphoproliferative diseases by clinical, virological and immunopathological studies. Cancer Res. 41:4226-4236.

Rickinson, A. B., Moss, D. J., Wallace, L. E., Rowe, M., Misko, I. S., Epstein, M. A., and Pope, J. H. (1981). Long-term T-cell-mediated immunity to Epstein-Barr virus. Cancer Res. 41 : 4216-4221.

12 KiecolI-Glaser, Speicher, Holliday, and Glaser

Russell, D., Peplau, L. A., and Cutrona, C. E. (1980). The revised UCLA Loneliness Scale: Concurrent and discriminant validity evidence. J. Pers. Soc. Psychol. 39: 472-480.

Saemundsen, A. K., Purtillo, D. T., Sakaniti, K., Sullivan, J. L., Synnerholm, A. C., Hanto, D., Simmons, R., Anvret, M., Collins, R., and Klein, G. (1981). Documentation of Epstein-Barr virus infection in immunodeficient patients with life-threatening lym- phoproliferative diseases by Epstein-Barr virus complementary RNA/DNA and viral RNA/DNA hybridization. Cancer Res. 41 : 4237-4242.

Shaver, P., Furman, W., Buhrmester, D., and Williams, T. (1981). State and trait loneliness during the transition into college. Paper presented at the annual meeting of the American Psychological Association, Los Angeles, Calif., August 1981.

Thoits, P. A. (1982). Conceptual, methodological and theoretical problems in studying social supports as a buffer against life stress. J. Health Soc. Behav. 23: 145-159.

Tuckwi!ler, L. S., and Glaser, R. (1983). Epstein-Barr virus and nasopharyngeal carcinoma. In Reznick-Schuller (ed.), Comparative Respiratory Tract Carcinogenesis, CRC Press, Cleveland, Ohio.