Rapamycin enriches for CD4(+) CD25(+) CD27(+) Foxp3(+) regulatory T cells in ex vivo-expanded CD25-enriched products from healthy donors and patients with multiple sclerosis.

BACKGROUND: CD4(+) CD25(bright+) regulatory T cells (Treg) can be expanded to clinical doses using CD3/CD28 Ab-coated beads plus IL-2. However, this method requires high purity of the starting population to prevent overgrowth by non-regulatory T cells. Rapamycin, an agent that inhibits T-cell proliferation but selectively spares Treg, may be a means to expand Treg from less pure CD25-enriched cells. METHODS: CD25-enriched cells were prepared by a single-step immunomagnetic-selection using anti-CD25 microbeads. The cells were activated with a single addition of anti-CD3/CD28 beads and expanded in ex vivo 15-5% HS and autologous CD4(+) CD25(-) feeder cells,+/-rapamycin (0.01-20 ng/mL). IL-2 was added on day 3. Cells were rested for 2 days in ex vivo 15-5% HS and tested for phenotype, intracellular Foxp3 protein and suppressor activity. RESULTS: In the absence of rapamycin, CD25-enriched fractions expanded >17 000-fold by 21 days. Although suppressor activity was detected to day 14, it declined significantly by 21 days as non-regulatory cells expanded. The addition of rapamycin inhibited expansion of non-regulatory T cells at doses > or =1 ng/mL while increasing suppressor activity and the percentage of CD4(+) CD25(+) CD27(+) Foxp3(+) cells. Rapamycin did not enrich for Foxp3(+) cells in expanded cultures of CD4(+) CD25(-) cells. Treg were also readily expanded in cultures of CD25-enriched cells obtained from patients with multiple sclerosis in the presence of rapamycin. DISCUSSION: The addition of 1-20 ng/mL rapamycin to CD25-enriched cultures increased the purity of cells with the phenotype and function of Treg. This approach may alleviate the need for rigorous enrichment of Treg prior to activation and expansion for potential clinical use.

Suppression of EBV release from irradiated B lymphoblastoid cell-lines: superior activity of ganciclovir compared with acyclovir.

BACKGROUND: B lymphoblastoid cell-lines (BLCL), generated by exposure of PBMC to a laboratory strain of EBV, are commonly utilized in the preparation of T cells used for immunotherapy. Although most B cells are latently infected, BLCL contain a subset of cells that harbor infectious virus, which could be released into the infusion product during preparation. To reduce this known risk, laboratories have pretreated BLCL for > or = 14 days with 100 microM acyclovir (ACV), an inhibitor of viral DNA polymerase, prior to use. We tested the effectiveness of ACV in preventing the release of infectious virus from irradiated fresh and previously frozen BLCL, and compared its effects with those of ganciclovir (GCV). METHODS: BLCL were grown for 14 days in medium containing various doses of ACV or GCV, washed, irradiated, and tested for the presence of infectious virus in co-culture assays with cord blood mononuclear cells(CBMC) (21 CBMC to BLCL). B-cell transformation was assessed at 3-4 weeks of culture. RESULTS: Both fresh and previously frozen BLCL released infectious virus, which transformed nearly all (92%) of CBMC co-cultures (n = 52). Transformation was not prevented by treatment with 100 microM ACV (88%, n = 52). Increasing the ACV dose to 200 microM (or 50 microg/mL) still allowed transformation in 4/9 (44%) cultures, while this and higher doses severely reduced the proliferation rate of the BLCL during ACV exposure. Infectious virus release was detectable within 1 day of ACV removal and BLCL irradiation. In contrast, GCV was able to prevent infectious virus release in 12/12 co-cultures at a concentration (15 microM) that only modestly reduced BLCL growth. DISCUSSION: These results indicate that GCV is more effective at preventing release of infectious EBV from irradiated BLCL than ACV at concentrations that do not severely inhibit B-cell growth.

[Bacterial endocarditis. Clinical picture, treatment and course in 37 patients (author's transl)]. / Bakterielle Endokarditis. Klinik, Therapie und Verlaufsform bei 37 Patienten

Between March 1971 and April 1976 37 patients were seen with manifest bacterial endocarditis. The main signs were high temperature and cardiac murmurs whereas other "classical" signs such as splenomegaly, anaemia, leucocytosis, and positive anti-streptolysin titres were much less frequent. In 35 cases bacteriological proof was possible. As causative organism a total of 30 gram-positive organisms (of which 15 were Streptococcus viridans and 8 were Staphylococcus species) and 10 gram-negative bacteria (4 of which were Pseudomonas aeruginosa) could be demonstrated. Treatment was mainly with beta-lactam and/or aminoglycoside antibiotics. Use of the combination of penicillin and streptomycin or gentamicin was based on the results of in-vitro bactericidal activity. The main complications were emboli, penicillin allergies, pulmonary involvement and cardiac complications. 13 patients died; the main cause was cardiac failure which was irreversible even despite operative valve replacement during the acute infection in two cases.