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Despite excellent vaccines, resurgent outbreaks of hepatitis A have caused thousands of hospitalizations and hundreds of deaths within the United States in recent years. There is no effective antiviral therapy for hepatitis A, and many aspects of the hepatitis A virus (HAV) replication cycle remain to be elucidated. Replication requires the zinc finger protein ZCCHC14 and noncanonical TENT4 poly(A) polymerases with which it associates, but the underlying mechanism is unknown. Here, we show that ZCCHC14 and TENT4A/B are required for viral RNA synthesis following translation of the viral genome in infected cells. Cross-linking immunoprecipitation sequencing (CLIP-seq) experiments revealed that ZCCHC14 binds a small stem-loop in the HAV 5′ untranslated RNA possessing a Smaug recognition-like pentaloop to which it recruits TENT4. TENT4 polymerases lengthen and stabilize the 3′ poly(A) tails of some cellular and viral mRNAs, but the chemical inhibition of TENT4A/B with the dihydroquinolizinone RG7834 had no impact on the length of the HAV 3′ poly(A) tail, stability of HAV RNA, or cap-independent translation of the viral genome. By contrast, RG7834 inhibited the incorporation of 5-ethynyl uridine into nascent HAV RNA, indicating that TENT4A/B function in viral RNA synthesis. Consistent with potent in vitro antiviral activity against HAV (IC 50 6.11 nM), orally administered RG7834 completely blocked HAV infection in Ifnar1 −/− mice, and sharply reduced serum alanine aminotransferase activities, hepatocyte apoptosis, and intrahepatic inflammatory cell infiltrates in mice with acute hepatitis A. These results reveal requirements for ZCCHC14-TENT4A/B in hepatovirus RNA synthesis, and suggest that TENT4A/B inhibitors may be useful for preventing or treating hepatitis A in humans.

Two infants, one girl, 5 months old, and one boy, 6 months old, presented with rhythmic and sustained motor activities of a stereotyped nature accompanied by moaning and grunting, facial flushing and altered awareness. The episodes occurred frequently and were initially believed to be epileptic. Normal electroencephalograms during the fits, lack of response to antiepileptic medication given to one child and careful reviewing of videotape recordings, enabled us eventually to diagnose the 'seizure-like' episodes as masturbatory activity.

Cloned natural suppressor (NS) cell lines derived from the spleens of TLI-treated adult BALB/c or neonatal BALB/c mice were assayed for their ability to inhibit acute GVHD in vivo. Two assay systems were used to measure GVHD: spleen enlargement of F1 hybrid neonates, and mortality of sublethally irradiated homozygous weanling mice after the i.p. injection of fresh allogeneic spleen cells. Both lines of NS cells significantly reduced GVHD, but the control HT-2 cell line (T cell line of BALB/c origin) did not affect GVHD. The NS cells reduced GVHD regardless of the strain combination of the donor and recipient. Thus, suppression occurred without restriction by the major histocompatibility complex, and without antigenic specificity.

During a short period of time after birth or after radiotherapy, the spleens of neonatal and adult TLI-treated mice contain suppressor cells of the mixed leukocyte reaction (MLR) and of graft-vs-host disease. The present report shows that the MLR suppressive activity of spleen cells from TLI-treated adult BALB/c mice can be maintained in long-term tissue culture by using conditioned medium. The suppressor cells can be cloned by limiting dilution, and reproducibly inhibit the [3H]TdR incorporation in the MLR at responder-to-suppressor cell ratios of 50:1. There is no antigen specificity or H-2 haplo-type restriction of the MLR suppression. The suppressor cells do not inhibit [3H]TdR per se, because no inhibition was observed in co-culture experiments with the EL4 tumor line or the IL 2-dependent HT-2 cell line. By using immunofluorescent staining techniques, the surface phenotype of the suppressor cells was found to be similar to that reported previously for cloned NK cells (Thy-1+, Lyt-1-, Lyt-2-, Ig-, Ia-, MAC-1-, asialo-GM1+). However, the suppressor lines showed no natural killer activity when YAC-1 target cells were used. Thus, the suppressor lines have been termed "natural suppressor" cells to indicate surface marker similarities to NK cells, both in vivo and in vitro, but different effector functions.

The pharmacologic effects of histamine and isoproterenol (autacoids) were studied on clones of murine T helper (TH) and natural suppressor (NS) cells. The data are consistent with receptors for the autacoids being nonrandomly distributed on phenotypically and functionally distinct clones. The effects of histamine on IL 2 secretion by TH cells could be either inhibitory or stimulatory, depending on the conditions of incubation with the autacoid. When TH cells were pretreated with histamine, their secretion of IL 2 was augmented; conversely, if histamine was added to the TH cells in the presence of antigen, IL 2 secretion was inhibited. The suppressor function of NS cells was enhanced by preincubation with histamine (10(-4) M) for 4 hr before the washed NS cells were added to responder and stimulator cells in mixed lymphocyte cultures. Two H1 receptor antagonists, mepyramine (10(-6) M) and pyrobutamine (10(-7) M), each competitively blocked the histamine activation of the NS cells, but an H2 receptor antagonist cimetidine (10(-5) M) did not alter the suppressor-enhancing function of histamine. Activation of NS cells did not occur if histamine was added with responder, stimulator, and co-cultured cells in MLR. The effects of each autacoid were additive in cytolytic T cells alone. The adenylate cyclase pools that can be stimulated by isoproterenol and histamine in cytolytic T cells may be independent of each other, but further work will be needed to prove this point.

The present study is an attempt to produce xenogeneic anti-idiotype antibodies (Id) against mouse anti-H2 antibodies. The purpose is to obtain such anti-Id antibodies with reactivity against both B and T cells in quantities which will allow thorough investigation of the biochemical nature of the T cell receptor for alloantigen. Data are presented from experiments performed with an antiserum 5936 which was obtained after immunization with C57B1/6 anti-CBA antibodies.

The present study describes the qualitative reactions of a xenogeneic anti-idiotype (Id) antiserum produced in a mouse-gamma-globulin-tolerant rabbit (5,936) against B6 anti-CBA IgG antibodies. The results showed that such an anti-Id antiserum reacts specifically against anti-H-2k antibodies and against H-2k alloantigen-activated T cells from the following pairs of congenic mice: B10 (H-2b) and B10.D2 (H-2d); and A.BY (H-2b) and A.SW (H-2s), but not against C3H.SW (H-2b) and C3H.OH (H-2o); and BALB/b (H-2b) and BALB/c (H-2d). CB 20 (BALB/c mice with the Ig-1b allotype) anti-CBA T blasts also express idiotypic determinants that react with rabbit 5,936 antiserum. Thus, positive reactions are obtained between rabbit 5,936 anti-Id antiserum and anti-H-2k IgG preparations and T blasts from mice carrying the Ig-1b or Ig-1e allotype, but not from mice carrying the Ig-1a allotype. These reactions are qualitatively independent of the H-2 genotype of the Id-producing mice. Such a finding strongly suggests that the Id-bearing receptor molecules on mouse T cells are coded for by genes that are associated with the Ig heavy-chain-linkage group and not to the mouse histocompatibility complex. Furthermore, the anti-Id antibodies studied react preferentially against anti-H-2k antibodies or T cells with specificity toward the IAk-region-associated serological specificities. Thus, genes associated with the Ig heavy-chain-linkage group seem to be structural genes for at least T-cell receptors with specificity for IA-region-coded membrane antigens.

The present experiments showed 1) that it was possible to produce mouse T cell lines against MHC determinants with a relatively high success rate by stimulation of purified T cells with allogeneic cells in the presence of irradiated syngeneic spleen cells; 2) that these lines could be led to react against selected H-2 specificities; 3) that only T cell lines established from Ig-1b allotype mice contained 5936-Id+ T cells (5936-Idiotypes are defined by an antiserum against B6 anti-CBA IgG produced in rabbit no 5936, which was tolerant to mouse gamma-globulin); and 4) that antigenic determinants coded by IAk genes induce the 5936-Idiotype(s). The latter data are in accordance with the 5936-idiotype characteristics of primary MLC T blasts. All T cell lines contained both specific MLC-responding cells and cytolytic cells. However, studies on the functional capacity of 5936-Id+ T cells from both primary MLC and the T cell lines showed that neither MLC-responding cells nor cytolytic cells directed against H-2Kk, IAk, or H-2Dk were 5936-Id+. Thus, 5936-Id+ T cells may be regulator cells induced by IAk antigens.

Immune guinea pig lymph node cells were fractionated on Ig anti-Ig or HSA anti-HSA affinity columns or on plastic surface in medium containing carbonyl iron. These techniques selectively removed B lymphocytes, K lymphocytes or adherent cells. The residual cells (Fc receptor-negative T lymphocytes) responded to soluble antigen in vitro in the same way or even better compared with nonfractionated cells. In addition, there was no indication that antigen-antibody complexes were superior to antigen in triggering lymph node cells or purified lymph code T lymphocytes into DNA synthesis. The results obtained suggested that memory T lymphocytes can be stimulated by antigen autonomously.

Lethally irradiated mice injected with syngeneic thymocytes and immunized with protein antigens develop specific helper T cells. If injected with semiallogeneic thymocytes, such mice generate H-2 antigen-specific cytotoxic T cells. Most spleen cells from these chimeric mice possess Fc receptors. The present results demonstrate that the development of Fc-receptor-bearing cells in thymocyte-injected irradiation chimeras seemingly is due to the physiological conditions in the mice rather than to the specific immunization. As a corollary, both helper T cells and cytotoxic T cells did not have Fc receptors, at least not in their effector state. Thus, Fc receptors on T cells would seem irrelevant to their immune function.

Cloned natural suppressor (NS) cells derived from spleens of total lymphoid irradiated BALB/c mice were incubated with the phorbol ester, PMA, and calcimycin for 4 h. After thorough washing, the induced NS cells were incubated in serum-free medium for 24 h and the supernatants were collected. The supernatants suppressed the MLR between normal adult responder and stimulator spleen cells. There was no Ag specificity or H-2 haplotype restriction of the MLR suppression. The supernatants did not inhibit [3H]thymidine incorporation per se, because they did not suppress mitogen stimulation of spleen cells. Protease digestion of the supernatants removed the suppressive activity, and dialysis studies indicated that the molecular size of the suppressive factor was larger than 50,000 Da and smaller than 100,000 Da. The suppressive activity was stable at 56 degrees C, pH 2, for 1 h. Thus, NS cell clones can be induced to secrete an immunosuppressive lymphokine, NS factor.

Naturally occurring suppressor cells of the in vitro mixed leukocyte culture reaction and of in vivo graft-vs.-host disease have been identified in the spleens of neonatal mice (1) and of adult mice recovering from total lymphoid irradiation (2), whole-body irradiation (3), and syngeneic marrow transplantation (4), or cyclophosphamide therapy (5). Using both positive and negative selection procedures, the suppressors were reported to be null lymphocytes that did not express mature macrophage surface markers, nor differentiate into mature macrophages in vitro, nor demonstrate natural killer (NK) activity (1). Subsequently, cloned lines of these natural suppressor (NS) cells were derived from either adult mice given total lymphoid irradiation (TLI) (2) or from neonates (6). The cloned NS cell lines expressed a surface phenotype (2, 6) similar to that reported previously for cloned NK cells (Thy-1(+), asialo-GM1(+), Ig(-), Lyt-1(-), Lyt-2(-), Ia(-), MAC-1(-)) (7-9). However, the NS cells did not show NK activity in the standard assay with YAC-1 target cells. The cloned NS lines suppressed the proliferation of responder cells and the generation of cytolytic cells in the mixed leukocyte reaction (MLR), and suppressed lethal graft-vs.-host disease in vivo (10, 11). In view of the unusual function and surface phenotype of the cells, the lineage of these cells remained unclear. To determine the lineage of the cloned NS cells, we searched for expression and rearrangement of the α and β chain genes of the T cell antigen receptor, as well as that of the γ chain gene. Studies of the phenotypically similar NK cell yielded conflicting results. Thus, cloned lines of murine NK cells were reported to have rearrangements of the β chain genes, and to express mRNA for all three chains (12). In contrast, freshly purified rat or human large granular lymphocytes (LGL) were shown to express only the 1.0 kb mRNA species of the β chain gene (13), indicative of D-J joining (14). Thus, some but not all cells with NK function express the T cell receptor and are members of the T cell lineage. The current report shows that the NS lines express full-length mRNA transcripts for the a and β chain of the T cell receptor, as well as the γ chain gene.

Injection of thymocytes into lethally irradiated mice generates in the spleen of the hosts a lymphoid cell population which consists mainly of T lymphocytes (1–5) This procedure has been used frequently in studies on the induction of T lymphocyte effector functions. Thus, the injection of parental thymocytes into 800 R irradiated F1 hybrid mice produces a cytotoxic T lymphocyte population specific for the H-2 antigens possessed by the other parent. The induction and expression of this particular T lymphocyte effector function have been shown not to depend on Fc receptor, C3′ receptor or membrane immunoglobulin (Ig) positive cells (2, 4). A majority of spleen cells from irradiated F1 hybrid mice injected with parental thymocytes acquire Fc receptors (3). Since, as stated above, cytotoxic T lymphocytes did not carry Fc receptors, we looked for Fc receptors (as measured by the EA-RFC assay; E = sheep erythrocytes, A = CBA×Balb/c anti-SRBC 7S antibodies, RFC = rosette forming cells (4)) on helper T lymphocytes induced by injecting syngeneic thymocytes into 800 R irradiated F1 hybrid mice together with the protein antigens: human serum albumin (HSA) or ovalbumin (OA), emulsified in complete Freund’s adjuvant (CFA). In addition we investigated the development of Fc receptor bearing cells in the spleens of 800 R irradiated F1 hybrid mice injected with 1) saline, 2) syngeneic thymocytes, 3) syngeneic thymocytes and protein antigen in CFA, or 4) parental thymocytes. This was done in order to determine whether the Fc receptor bearing cells were of host origin or derived from the thymocyte inoculum and whether the Fc receptor bearing cells developed as a consequence of immunization.

The survival of peritoneal exudate macrophages after 3 to 10 days in culture was examined by measuring the numbers of phagocytes per culture. This was determined by letting the cultured cells phagocytize Latex particles. The number of Latex particle-containing cells was taken as a measure of the survival of phagocytes. It was found that one tenth of the cells judged by light microscopy as macrophage-like survived the culture period. Thus, the calculated plating factor of 9.3 was used to estimate the actual number of macrophages in suspensions of spleen, lymph node or thymus cells by culturing these cells and subsequently counting Latex particle-containing cells. In addition, the acridine orange technique was used to determine actual numbers of macrophages in freshly prepared cell suspensions of lymphoid organs. Latex studies on spleen and thymus cells gave results correlating well with data obtained by the acridine orange technique. By contrast, many more acridine orange positive cells than phago-cytizing cells were found when lymph node cells were cultured.

The visual and somatosensory evoked potentials were delayed in two cases of the adult form of metachromatic leukodystrophy. Brainstem auditory evoked potentials were normal. The conduction velocity along peripheral nerves was 50% slowed in one case and near normal in the other. The findings are compatible with demyelination in the central and peripheral nervous systems. The diagnosis of metachromatic leukodystrophy should be considered in cases of early dementia, with or without psychosis or other neurologic deficits, in which evoked potentials are delayed and peripheral nerve conduction is slowed.

A total of 42 EEGs from five patients with subacute sclerosing panencephalitis were studied. Periodic complexes were noticed in 35 (83%) of these. The interval between the complexes shortened in all patients with progression of the illness. The gradual EEG changes may reflect the increasing number of infected cells as well as an on-going accumulation of immature virus structures. The records without complexes were either from the early onset (one record) or terminal stage (six records).

The present experiments were carried out in order to answer the question whether the precursor T cells of cytotoxic T lymphocytes (PCTL) are Fc receptor positive (FcR+), Fc receptor negative (FcR-), or both. The data show that cytotoxic T lymphocytes (CTL) can be generated in vitro from both FcR+ and FcR- PCTL. Furthermore, we investigated if the function of FcR+ and FcR- CTL differed in the allograft response in vitro. Qualitatively, FcR- PCTL differentiate into FcR- CTL only, whereas FcR+ PCTL differentiate into both FcR+ and FcR- CTL. However, quantitatively, CTL generated from FcR- PCTL display a higher T cell mediated cytotoxicity than CTL generated from FcR+ PCTL. Mixing experiments indicate that FcR+ T cells regulate the differentiation of FcR- PCTL into CTL. These conclusions hold true for PCTL in both normal and memory responder cell populations.

The present report deals with our attempts to characterize the potent stimulator cell(s) that induce Fc receptor (FcR)-negative T lymphocytes to differentiate into cytotoxic T lymphocytes in vitro. We found two categories of such cells: [1] resting B cells, FcR+ T cells, and macrophages and [2] T cells either activated in 800-R-irradiated mice against H-2 antigens or M-locus antigens or activated in vitro by concanavalin A. The distinction between potent and weak stimulator cells is discussed in relation to the surface antigenic markers and the immunological function of these cells.