Human T cell growth factor (TCGF) produced by repeated stimulation of non-adherent human lymphocytes.

T cell growth factor (TCGF) has become a valuable means of maintaining T lymphocytes in long-term culture and of studying T cell function. Numerous problems have been met in the production of TCGF of consistently good quality and in the maintenance of human T cell lines over long periods. We have investigated optimal conditions for TCGF production, and simplified assay systems for TCGF activity. The best TCGF production was obtained by short-term treatment with high concentrations of phytohemagglutinin (PHA). The TCGF producing lymphocytes could be re-used for TCGF production up to 1 month after the first treatment course. Human cultured T cell lines, fresh lymphocytes, short-term PHA stimulated lymphocytes and cultured marmoset T lymphocyte lines were all used for assay of TCGF. We recommend PHA stimulation of human lymphocytes for this assay on a routine basis, comparing results with a standard TCGF batch and calculating a growth index. Adherent cells impair TCGF production. Optimal TCGF production was seen when lymphocyte preparations without adherent cells from different donors were used.

Acyclovir efficiently inhibits oropharyngeal excretion of Epstein-Barr virus in patients with acute infectious mononucleosis.

Shedding of Epstein-Barr virus (EBV) into saliva was studied in 31 patients with verified acute infectious mononucleosis. The patients had been randomized for intravenous treatment with acyclovir (ACV) at 10 mg/kg body weight at 8 h intervals for 7 days, or placebo, in a double-blind trial. EBV in centrifuged throat washings was detected by transformation of umbilical cord lymphocytes and by immunofluorescence staining for EBV-associated nuclear antigen in fixed cell smears. Saliva samples were obtained before and during treatment, and after 4 weeks and 6 months, respectively. ACV effectively but transiently interrupted EBV production (P less than 0.001), but virus shedding resumed at the initial level within 3 weeks of cessation of the treatment. Initially, 93.5% of the patients had detectable EBV in the saliva compared with 83% in the 4th week and 58% after 6 months.

C-terminal domain of the Epstein-Barr virus LMP2A membrane protein contains a clustering signal.

The latency-regulated transmembrane protein LMP2A interferes with signaling from the B-cell antigen receptor by recruiting the tyrosine kinases Lyn and Syk and by targeting them for degradation by binding the cellular E3 ubiquitin ligase AIP4. It has been hypothesized that this constitutive activity of LMP2A requires clustering in the membrane, but molecular evidence for this has been lacking. In the present study we show that LMP2A coclusters with chimeric rat CD2 transmembrane molecules carrying the 27-amino-acid (aa) intracellular C terminus of LMP2A and that this C-terminal domain fused to the glutathione-S-transferase protein associates with LMP2A in cell lysates. This molecular association requires neither the cysteine-rich region between aa 471 and 480 nor the terminal three aa 495 to 497. We also show that the juxtamembrane cysteine repeats in the LMP2A C terminus are the major targets for palmitoylation but that this acylation is not required for targeting of LMP2A to detergent-insoluble glycolipid-enriched membrane microdomains.

Effect of acyclovir on infectious mononucleosis: a double-blind, placebo-controlled study.

Thirty-one patients with clinical and laboratory diagnoses of infectious mononucleosis who had had symptoms for seven or fewer days were randomized for intravenous treatment with acyclovir (10 mg/kg) or placebo at 8-hr intervals for seven days in a double-blind trial. Clinical signs and symptoms were registered, and excretion of virus in the saliva as well as antibody responses in sera and saliva were assessed before, during, and at regular intervals in the six months after treatment. Acyclovir significantly (P less than .001), but reversibly, inhibited oropharyngeal shedding of Epstein-Barr virus. The humoral and cellular immune responses, however, did not differ between the two groups; nor did the development of viral latency. There were no significant (P greater than .05) differences in individual clinical symptoms or in laboratory parameters between the two groups; however, when data concerning duration of fever, weight loss, tonsillar swelling, pharyngitis, and self-assessment by the patient were combined, a significant (P less than or equal to .01) effect of treatment with acyclovir was evident.

Direct identification by PCR of EBV types and variants in clinical samples.

Both Epstein-Barr virus (EBV) type A and type B, and variants of type A, were identified simultaneously by polymerase chain reaction (PCR) amplification of a DNA region coding for a 13 amino acid repeat in the Epstein-Barr virus nuclear antigen (EBNA) 6. Whereas this region varies extensively in type A isolates, no variation was seen in type B isolates. When a repetitive region in the LMP1-coding region was amplified by PCR, it was possible to distinguish individual variants of type B isolates from each other. Forty-two saliva samples from HIV-1-carrying individuals were examined for the presence of type A and type B virus. Both types and multiple variants of each type were found with a much higher frequency than in the saliva samples from healthy individuals. Type A EBV alone was detected in mouthwash samples from 6 infectious mononucleosis (IM) patients. Both type A and B were detected in the peripheral blood B-lymphocytes (PBL) from 1 healthy individual. The same type A variant was demonstrated both in PBL and in the mouthwash sample from another healthy individual. In this study it was shown that a combination of the EBNA 6- and LMP 1-specific PCRs followed by Southern hybridisation can be used to identify both type A and type B virus, as well as to distinguish between multiple variants of the same strain, in saliva and B-cells from both healthy and immunosuppressed individuals.

Detection of multiple Epstein-Barr viral strains in allogeneic bone marrow transplant recipients.

We have previously shown in 3 allogeneic bone-marrow transplant (BMT) recipients that complete replacement of recipient marrow was associated with the elimination of the pretransplant Epstein-Barr virus (EBV) strain of the recipient. To study the kinetics of EBV elimination and reinfection in more detail, we have performed a longitudinal study of BMT recipients combining serology, virus isolation from mouthwashes and peripheral blood, and EBV strain characterization. Oropharyngeal EBV excretion was found to persist after the cytoreductive therapy prior to BMT, whereas EBV-carrying cells in the blood were detected only after 5 weeks following BMT. During the first month post-BMT, 2 different EBV strains could be isolated from sequential mouth-washes of 3 patients. The initial strains were found to persist up to 7, 21, and 29 days post-BMT, whereas the subsequent strains appeared at 21, 42, and 34 days post-BMT, respectively. Thus, the original EBV strain may persist only for a limited time after BMT, and the oropharyngeal epithelium may be reinfected by a new EBV strain from the blood within 3 weeks. With respect to the coexistence of multiple EBV strains, 2 patterns were evident. From the day 62 mouthwash of 1 patient, 1 Type A and 1 Type B strain were isolated. From the day 180 mouthwash of a second patient, a dominant Type A strain was recovered, together with 6 "variant" strains that differed from each other by only a single EBNA protein (EBNA 1). This pattern may be explained by viral recombinations during replication, which may form the basis for the vast polymorphism of EBV observed in unrelated individuals.

Acyclovir treatment in infectious mononucleosis: a clinical and virological study.

Fifty-six patients with a clinical and laboratory diagnosis of infectious mononucleosis who had not been ill for more than seven days, were randomised for peroral treatment with acyclovir (800 mg five times daily) or placebo for seven days in a double blind trial. Clinical, virological and immunological parameters were monitored in each patient for six months. During treatment, shedding of Epstein-Barr virus' as assessed in 36 patients, was significantly reduced (p less than 0.001). However, virus production in the oropharynx returned to pre-treatment levels one week after the cessation of therapy. Virus was detected in 35 patients at enrollment and in 28 of 36 patients at the six-month control. No effect on the clinical course of the disease was noticed. The virus-specific antibody response was also unaffected. A significant reduction in spontaneous outgrowth of in vivo Epstein-Barr virus-infected B-lymphocytes was found at 180 days after treatment in four acyclovir-treated patients compared to six controls (p less than 0.001). In another three patients with over-whelming clinical symptoms causing airway obstruction and/or disseminated intravascular coagulopathy, treatment with intravenous acyclovir (10 mg/kg three times daily) was combined with prednisolone (0.7 mg/kg daily) for ten days. Virus shedding ceased transiently during treatment, but returned to initial levels within one week. A dramatic clinical effect on the pharyngeal oedema and general health of the two patients with airway obstruction was noticed, but was much less evident in a patient with intravascular coagulopathy.