C B Pettinelli

Optimal conditions were established for the adoptive transfer of experimental allergic encephalomyelitis (EAE) in SJL/J mice. Lymph node cells from SJL/J mice primed in vivo with myelin basic protein (BP) were incubated in vitro with BP. These cells proliferated specifically to BP and when transferred at the optimal conditions into syngeneic mice induced EAE in 100% of the recipients. The in vitro proliferative response to BP was dependent on the presence of Lyt 1+ 2- T lymphocytes. Furthermore, when the activated LNC were treated before transfer with anti-Thy 1 or anti-Lyt 1 antibody and C, neither clinical nor histologic signs were observed in the recipients, whereas treatment with anti-Lyt 2 antibody and C had no effect. These results indicated that Lyt 1+ 2- T cells are responsible for the transfer of EAE.

OBJECTIVES: (1) To document rates and patterns of adherence from enrollment until week 24 of an AIDS clinical trial; (2) to examine the association of adherence to clinical end-points including plasma HIV-1 RNA level and CD4 cell count; and (3) to identify predictors of adherence from clinical, behavioural, psychosocial and demographic factors. DESIGN: Sub-study of a multicentre, randomised, open-label, comparison-controlled trial; 21 collaborating units of the Adult AIDS Clinical Trials Group. Observational, prospective analysis. METHODS: Ninety-three subjects with baseline plasma HIV-1 RNA levels >500 copies/ml, who completed clinical assessment, plasma HIV-1 RNA titres and CD4 cell counts at study entry, weeks 2, 4 and every 4 weeks thereafter until week 24. All patients were antiretroviral-experienced but were naive to non-nucleoside reverse transcriptase inhibitors and protease inhibitors. Self-reported adherence to antiretroviral therapies prescribed as part of the trial was assessed every 4 weeks from trial, week 4 until week 24. RESULTS: Average adherence was high, with 63% of subjects reporting >95% adherence across the trial. However, there was a significant decline in adherence over time on trial. After controlling for potential confounding variables, patients who were less than 95% adherent to medications were 3.5-times more likely to have treatment failure (HIV-1 RNA >50 copies/ml) than subjects with adherence rates of 95-100%. The strongest predictor of adherence was adverse clinical events (for example, dermatological, gastrointestinal symptoms): patients with adverse events were 12.8-times less likely to have 95-100% adherence. Other clinical, demographic, psychosocial and behavioural factors were also significant predictors of adherence. CONCLUSIONS: Adherence influences virological outcome even in AIDS clinical trials where overall adherence rates are high and should therefore be monitored in future trials. Intervention may be warranted to enhance adherence for subjects who have early toxicities, express concern about taking medications as directed, and for women and minorities.

GPBP was shown to be encephalitogenic in SJL mice by direct challenge and in experiments in which an adoptive transfer system was employed. The three fragments obtained by treating GPBP with pepsin were assessed in the same manner. The encephalitogenic activity resided in the C terminal half of the molecule (residues 89-169). LNC also proliferated to the same fragment in vitro. Fragments 1-37, and, to a lesser extent, 44-48 stimulated sensitized LNC to proliferate but did not induce disease.

Murine spleen cells from normal donors were cultured in vitro with trinitrobenzene sulfonate (TNBS)-conjugated soluble proteins, i.e., bovine gamma globulin (TNP-BGG) or bovine serum albumin (TNP-BSA). Addition of 100 mug of any of these TNP-proteins to the spleen cell cultures led to the generation of cytotoxic T-cell effectors which were H-2-restricted and TNP- specific. The lytic potential of such effectors was comparable to that generated by sensitization with TNBS-modified syngeneic cells, and was restricted to haplotypes shared at the K or K plus I-A, or the D regions of the H-2 complex. Greater effecter cell activity was generated by addition of TNP-BGG against TNBS-modified targets which shared K plus I-A than against modified targets which shared the D region with the responding cells, which suggests that the same immune response genes are involved when the response is generated by the addition of TNP-conjugated soluble proteins or of TNBS- modified cells. H-2-restricted, TNP-specific effecter cells were generated by culturing mouse spleen cells with syngeneic cells which had been preincubated with TNP- BGG or TNP-BSA for 1.5 h. The addition of unconjugated soluble proteins to the cultures did not result in cytotoxic effectors detectable on H-2-matched targets, whether the targets were prepared by modification with TNBS, or by incubation with either the unconjugated or TNP-conjugated proteins. Depletion of phagocytic cells in the tumor preparation by Sephadex G-10 column fractionation before incubation with TNP-BSA had no effect on their lysis by the relevant effector cells. Immunofluorescent staining of tumor target cells with anti-TNP antibodies indicated that TNP could be detected on the tumor cells within 10 rain of incubation with TNP-BSA. The cytotoxic response generated by addition of the TNP-proteins to spleen cell cultures was found to be T-cell dependent at the effector phase, as shown by the sensitivity of the lytic phase to absorbed RAMB and complement. Furthermore, the response did not appear to be attributable to antibody-dependent cellular cytotoxicity. Three mechanisms were considered which could account for the generation of H-2-restricted, TNP-specific, cytotoxic T-cell effectors by the addition of soluble TNP-proteins. These include covalent linkage of activated TNP groups from the soluble proteins to cell surface components, macrophage processing of the soluble conjugates and presentation to the responding lymphocytes in association with H-2-coded self structures, or hydrophobic interaction of the TNP-proteins to cell surfaces. Results obtained from sodium dodecyl sulfate gel patterns indicating that cell-bound TNP was still linked to BSA, and the observation that phagocytic-depleted cells could interact with the soluble TNP-proteins and function as H-2-restricted targets, appear not to favor the first two proposed mechanisms.

Spleen cells from B10.BR and C57BL/10 (B10) mice were compared for their ability to generate primary in vitro cytotoxic responses to syngeneic cells modified with different concentrations (from 10 to 0.031 mM) of trinitrobenzene sulfonate (TNBS) (TNP-self). Although both strains generated effector cells to TNP-self in the range of 10-0.25 mM TNBS modification, effector activity of B10 cells was weaker than that of B10.BR cells. B10 spleen cells did not respond to syngeneic stimulating cells modified at 0.1 mM or lower, whereas B10.BR cells generated effector activity even when stimulated by TNP-self modified with as low as 0.031 mM TNBS. Fluorescence analysis of the modified cells using the FACS II indicated that equivalent quantities of TNP were conjugated to the surfaces of B10.BR and B10 spleen cells for any given concentration of TNBS modification. Similar strain-dependent differences were observed when the TNP was diluted out in the cultures by reducing the number of stimulating cells modified with 10 mM TNBS. These response patterns were verified by stimulating cultures of B10.BR and B10 spleen cells either with TNP conjugated to bovine serum albumin or bovine gamma globulin (B10.BR but not B10 cells responded to TNP-conjugated proteins) or with TNBS-modified glass-adherent spleen cells. The strain-dependent differences could also be detected at the effector phase, because optimally stimulated B10.BR, but not B10 effector cells, could lyse 0.1 mM TNBS-modified syngeneic target cells. The genetic parameters associated with the response and nonresponse patterns of B10.BR and B10 mice were further investigated by comparing the cytotoxic responses to low doses of TNP-self of spleen cells from the following strains: (a) C3H/HeJ (H-2k) and C3H.SW (H-2b); (b) BALB.K (H-2k) and BALb.b (h-2b); and (c) B10.A (H-2a) and B10.D2 (H-2d). The H-2k and H-2a, but not the H-2b and H-2d, strains generated cytotoxic responses to TNP-self when the syngeneic stimulators were modified with 0.1 mM TNBS. Further studies using (B10 X B10.BR)F1 responding cells and parental or F1-modified stimulating cells, indicated that the F1 cells generated cytotoxic activity to low doses of TNP in association with H-2k but not in association with H-2b self products. The results of this study indicate that H-2-linked genetic factors, expressed in the target as well as in the responding and/or stimulating cell populations, control the ability of inbred mouse strains to generate cytotoxic effector cells to low doses of TNP-self. Such dose-dependent genetic effects may be important in the regulation of immune responses activated in vivo by chronic exposure to infectious agents.