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To investigate the involvement of T-cell response against hepatitis C virus (HCV) antigens in viral clearance after interferon therapy, we measured interleukin-2 (IL-2) production by peripheral mononuclear cells in response to HCV core in patients with chronic hepatitis C. In a cohort of 43 patients, we investigated the frequency of circulating core-specific T-helper (Th) cell precursors by the limiting-dilution assay, and in a second cohort of 60 patients, we analyzed the response to specific core epitopes using 52 synthetic 15-mer overlapping peptides. We observed that the frequency of core-specific Th cell precursors was significantly higher in patients with sustained biochemical and virological response (SR) after interferon (IFN) therapy (median, 1/55,736) than in untreated patients (1/274,023) or that in patients who remained viremic after completion of the treatment-nonresponders (NR) plus transient responders (TR) (1/1,909,972). Patients who failed to respond to IFN (NR) and those who relapsed after IFN discontinuation (TR) had a similarly low number of precursors. The number of core peptides recognized by SR, TR, NR, UT, and healthy controls was 8.2 +/- 1.5, 6.5 +/- 1.2, 2.0 +/- 0.5, 2.7 +/- 0.9, and 0.3 +/- 0.2, respectively. In SR, the intensity of the proliferative response to core peptides as estimated by the summation of stimulation indexes (sigmaSI) was significantly higher than in NR and than in UT, but not different from that of TR. Our results indicate that both expansion of HCV-specific Th cell precursors and Th cell recognition of multiple core epitopes seem to be important in the elimination of HCV after IFN therapy.
Loss of myocardial adenosine triphosphate (ATP) during ischemia can result in decreased cardiac function. Postischemic ATP levels remain low, and the reason for this is poorly understood. Previous attempts to enhance ATP recovery after ischemia have been only partially successful. To determine the long-term dynamics of ATP recovery and evaluate the effect of providing ATP precursors, we devised a method of obtaining sequential ventricular biopsies in dogs after 20 minutes of normothermic global ischemia on cardiopulmonary bypass. Our kinetic data show adenine (A) is metabolically favored over adenosine to regenerate ATP levels when adequate ribose (R) is present to produce phosphoribosylpyrophosphate. Therefore A (20 mM) plus R (80 mM) or saline (NS) was infused (1.0 ml X min-1) into the right atrium of dogs for 48 hours after ischemia. During A infusion myocardial tissue A was 0.19 +/- 0.07 nmol X mg-1, arterial A was 18.3 +/- 1.3 microM, coronary sinus A was 11.0 +/- 1.6 microM, and extraction of A by the myocardium was 38% +/- 10%. We found that while the decrease in ATP levels during ischemia was at least 50% in both groups, the postischemic ATP recovery rate in A/R dogs was more than eightfold greater than de novo synthesis (2.8 +/- 0.59 versus 0.34 +/- 0.06 nmoles X mg-1 X day). ATP levels in NS dogs were only 54% +/- 8% of preischemic values by 48 hours and required 9.9 +/- 1.4 days for full recovery. Recovery in A/R dogs required 1.2 +/- 0.2 days. Our results reveal that ATP recovery after a significant ischemic insult is slow, precursor availability is an important limiting factor in ATP recovery, and recovery time can be greatly shortened with precursor infusion even when started after the ischemic insult.
Within the limits of this study, it was found that 5 ppm of cobalt was adequate to give good levels of vitamin B(12). The vitamin B(12) precursor 5,6-dimethylbenzimidazole was found to be adequate at 10 ppm in the absence of aeration. In the presence of aeration, a zero level of precursor was found to be most desirable. The analysis of variance showed aeration to be highly significant, and the aeration and precursor interaction to be significant. No other significant effects were observed.
Human Tamm-Horsfall glycoprotein (T-H) is produced by renal cells of ascending limb of loop of Henle and is largely excreted in urine. N-linked glycans account for close to 30% of the weight of T-H. We studied the biosynthesis of recombinant T-H permanently expressed in HeLa cells. The conversion from the precursor (84 kDa) to the mature form (97 kDa) mainly depends on the processing of glycans from the high-mannose to polyantennary type. The conversion from precursor to mature form is very slow and the glycan structure of precursor appears to be that of a glycoprotein not yet processed by Golgi alpha 1,2 mannosidase. Since T-H has a very high number of disulfide bridges (more than 50 cysteine residues/mol) one may infer that the rate limiting step for the precursor export out of ER is the formation of a correct set of disulfide bonds. Mature T-H isolated from HeLa cells retained one N-linked chain with the high-mannose structure similarly to urinary T-H. This result indicates that the occurrence of one unprocessed high-mannose chain in mature T-H is host-cell independent and very likely related to the T-H primary structure.
The B-cell mitogens LPS and lipoprotein stimulate 20-35 percent of all B cells in the spleen of 6- to 8-wk old C3H/Tif mice, as determined by limiting dilution analysis of precursors. Each reactive cell grows to a clone of IgM-secreting PFC, enumerated in a hemolytic plaque assay detecting all IgM secreting cells, regardless of v-region specificity. We have used these mitogens to reveal the total repertoire of Ig specificities produced by these mitogen-reactive B cells. We have determined in plaque assays with six different target erythrocytes the number of spleen cells limiting to one the number of mitogen-reactive B cells detected as specific IgM-secreting clones in each of these plaque assays. By this method, the absolute frequencies of precursor B cells with defined v-gene specificities could be calculated, for at least, one third of all B cells. The frequencies of specific IgM-plaque-forming B-cell clones within the total pool of mitogen-reactive B cells was 1 in 10 for NIP(12),-SRC, 1 in 50 for TNP(12)- SRC, 1 in 100 for NIP(1)-SRC, 1 in 160 for TNP(3)- SRC, 1 in 500 for HRC, and 1 in 1,000 for SRC. These frequencies were the same in the LPS- and in the lipoprotein-reactive B-cell population for TNP(30)- SRC and SRC.
The nature of the in vitro human cytotoxic T-cell responder population to HSV type 1 (HSV-1) was studied. In 5-day HSV-1-stimulated cultures that contained MHC-restricted activity, two phenotypically distinct populations of cells were present that were capable of lysing HSV-1-infected B cell lines in a 5-h 51Cr-release assay. The first was CD4+, CD8-, CD16- cell typical of class II-restricted T cells, whereas the other population bore a CD4-, CD8-, CD16+ NK-cell phenotype. Elimination of the NK cell fraction from bulk cultures by using anti-CD16 plus C frequently resulted in cell populations that killed in an Ag-specific, HLA-DR-restricted fashion. In some cases the anti-CD16-pretreated cultures retained a killing population that was unrestricted to MHC products. In no instance were any cytotoxic T cells that were restricted to class I Ag in evidence. Limiting dilution analysis of precursor frequency indicated that about 1 in 4000 to 1 in 8000 cells from peripheral blood are specific for HSV-1 in seropositive individuals. Comparisons of HLA class I-matched and HLA class II-matched targets with the autologous target by using limiting dilution analysis yielded results entirely consistent with those obtained in the bulk culture assay system.
In vivo administration of interleukin-2 (IL-2)-secreting tumor cells results in complete protection against persistent infection by Theiler's murine encephalomyelitis virus (TMEV) in susceptible DBA/2 mice. The IL-2-mediated protection was found to depend on the inoculum size as well as the timing of IL-2 administration. IL-2-treated and TMEV-infected mice displayed a three- to fourfold relative increase in virus-specific cytotoxic T-lymphocyte (CTL) precursors. Thus, we postulate that the persistence of TMEV infection in susceptible mice reflects limited numbers of relevant CTL precursors and their time course of induction and activation.
The human hepatoma-derived cell line, HepG2, synthesized and secreted functional complement proteins C1r, C1s, C2, C3, C4, C5, factor B, C1 inhibitor, C3b inactivator, a small amount of C6, and trace amounts of C8; but failed to produce detectable C1q, C7, or C9. Immunochemically, C2, C3, C4, C5, and B were isolated from culture medium as proteins with molecular sizes and subunit structures identical to the corresponding components isolated from serum. C2 and factor B from cellular lysates had slightly lower molecular weights than the corresponding proteins in culture medium. C3, C4, and C5 were detected as single chain precursor molecules in cellular lysates. These results demonstrate that human C5, like C3 and C4, is synthesized as a single chain precursor that is converted by limited proteolysis to the native two-chain molecule. It also establishes the precursor-product relationship for human pro-C4 and native C4, pro-C5, and native C5.
Peptide presentation by autologous dendritic cells (DCs) is a new tool to activate tumor antigen-specific T cells in melanoma patients. However, it is not known whether autologous DCs, differentiated by two of the most efficient protocols (from CD34+ progenitors or from monocytes), are equally effective as professional antigen-presenting cells (APCs) when the patients have a low frequency of peptide-specific precursors. To this end, a limiting dilution assay was applied to evaluate the frequency of antigen-specific CTL precursors (CTLps) in peripheral blood of HLA-A*0201+ melanoma patients. Then, from two melanoma patients showing low frequency of CTLps to melanoma antigen-A/melanoma antigen recognized by T cell (Melan-A/Mart-1)(27-35) peptide, autologous DCs were differentiated from granulocyte colony-stimulating factor-mobilized CD34+ progenitors or from monocytes. CD34+- and monocyte-derived DCs were characterized by a similar proportion of CD1a+ cells expressing HLA class II antigens and CD54, CD80, and CD86 molecules. Both types of DC presented Melan-A/Mart-1(27-35) and tyrosinase(369-377) peptides to melanoma-specific CTL clones and were equally effective as peptide-pulsed APCs in the activation of influenza A matrix(58-66)-specific CTLs from high-frequency precursors (1294/10(6) and 1789/10(6) lymphocytes in the two patients). However, efficient activation of Melan-A/Mart-1(27-35)-specific CTLs from low-frequency precursors (158/10(6) and 77/10(6) lymphocytes) of the two patients was markedly dependent on the use of peptide-loaded CD34+-derived DCs. These results suggest that CD34+- and monocyte-derived DCs are not functionally equivalent APCs for the activation of low-frequency peptide-specific CTLps.
A method has been developed for detecting anti-beta-galactosidase antibodies after isoelectric focusing in thin layers of polyacrylamide gel. By "staining" with wild-type enzyme, all antibodies against beta-galactosidase are detected, while a subset of antibodies able to activate a mutant enzyme is detected by staining with that enzyme. Limiting dilutions of beta-galactosidase-primed or unprimed spleen cells of BALB/c mice were transferred together with antigen into sublethally irradiated syngeneic hosts. The limiting role of the precursor B cells has been judged by the analysis of the clonal distribution of galactosidase-specific antibodies in recipient sera. The frequency of anti-wild-type beta-galactosidase precursor cells was one in 0.42 x 10(6) in the primed and one in 0.93 x 10(6) in the unprimed spleen. The frequency of precursor cells for antibodies activating the mutant enzyme was one in 1.5 x 10(6) in the primed and one in 4.6 x 10(6) in the unprimed spleen. Therefore four and five times less anti-M (mutant) than anti-B (wild-type) precursor cells exist in the spleens of primed and unprimed BALB/c mice, respectively. Comparing 51 clones derived from one primed donor mouse, it was possible to demonstrate that at least 43 different mutant-beta-galactosidase-activating antibodies can be produced in one mouse. Comparing these 43 clones with 27 clones derived from another donor mouse, only one clone seemed to be common to both mice. From this the repertoire of the BALB/c strain has been estimated to consist of over 1000 different mutant enzyme-activating antibodies.
During mouse embryogenesis the production of "primitive" erythrocytes (EryP) precedes the production of "definitive" erythrocytes (EryD) in parallel with the transition of the hematopoietic site from the yolk sac to the fetal liver. On a macrophage colony-stimulating factor-deficient stromal cell line OP9, mouse embryonic stem cells were shown to give rise to EryP and EryD sequentially with a time course similar to that seen in murine ontogeny. Studies of the different growth factor requirements and limiting dilution analysis of precursor frequencies indicate that most EryP and EryD probably developed from different precursors by way of distinct differentiation pathways.
During vertebrate development many types of precursor cells divide a limited number of times before they stop dividing and terminally differentiate. It is unclear what causes the cells to stop dividing when they do. We have been studying this problem in the oligodendrocyte cell lineage, which is responsible for myelination in the vertebrate central nervous system. Here we show for the first time that in clonal cultures of oligodendrocyte precursor cells purified from embryonic day 18 (E18) rat optic nerves the first oligodendrocytes develop within 3-4 days, equivalent to the time they first differentiate in the nerve, and that this timely differentiation depends on the presence of thyroid hormone. These findings suggest that a cell-intrinsic, thyroid-hormone-regulated timer determines when the first oligodendrocytes develop. Whereas the first oligodendrocytes develop asynchronously within clones, the vast majority develop after the first week in culture and do so more synchronously within clones. We show that beta1 thyroid hormone receptors in the precursor cells increase in clonal cultures in the absence of thyroid hormone in parallel with the increasing sensitivity of the cells to the cell-cycle-arresting activity of thyroid hormone; moreover, the increase in beta1 receptors, like the timer itself, is accelerated at 33 degrees C compared to 37 degrees C, suggesting that the increase in receptors may be part of the intrinsic timer. Finally, we show that the precursor cells do not divide indefinitely when stimulated to divide extensively in the absence of thyroid hormone but, instead, eventually stop dividing and either die or differentiate.
Oxidative-decarboxylation rates of branched-chain amino acids in rat hemidiaphragm and of branched-chain 2-oxo acids in hemidiaphragm, soleus muscle and heart slices of 110-120 g rats were increased considerably by 3-4 days of starvation, when they were calculated from the specific radioactivity in the medium. When the supply from endogenous protein degradation to the oxidation-precursor pool was severely limited by transaminase inhibitors, oxidative-decarboxylation rates of branched-chain 2-oxo acids rose significantly. Since this apparent increase was relatively larger in preparations from fed rats than from 3-days-starved rats, the differences in oxidation rates with nutritional state became less or even not significant. With rat heart the smaller dilution of the oxidation precursor pool after starvation is in accordance with the reported decrease in protein breakdown. Since protein degradation increases with starvation in skeletal muscles, we suggest that the amino acid pool arising from protein degradation is more segregated from the oxidation precursor pool in muscles from starved than from fed rats. We conclude that starvation increases branched-chain amino acid and 2-oxo acid oxidation in skeletal and cardiac muscle considerably less than has been suggested by previous studies.
Controversy concerning the immunologic role of antigen-binding cells (ABC) has prompted us to attempt to quantitate the proportion of stimulable ABC, in immunized animals, which are precursors for cells producing antibody specific for the antigen bound. Using a lipopolysaccharide (LPS)-driven limiting dilution analysis system, the precursor frequency (PF) of cells secreting IgM and IgG and sheep erythrocyte (SRBC)-specific IgM and IgG was established for highly purified SRBC antigen-binding cell (SRBC-ABC) and unfractionated populations taken from CBA/J mouse spleens on days 5, 12 and 180 of the in vivo primary immune response to SRBC. At all these times, almost all SRBC-ABC spontaneously secreting immunoglobulin (Ig) secreted SRBC-specific Ig, and almost all precursors of Ig-secreting cells in the ABC populations were precursors of cells secreting specific anti-SRBC antibody. In SRBC-ABC populations, the PF for total and SRBC-specific Ig secretion was seen to decrease on days 5 and 12 after immunization and to increase to 3.5 to 7 times nonimmune levels 180 days after immunization. The absolute number of precursors, within the SRBC-ABC population, for the secretion of SRBC-specific Ig decreased on day 12 after immunization. In the unfractionated population, the PF for SRBC-specific Ig secretion temporarily increased after immunization, reaching peak levels 5 days (IgM) and 12 days (IgG) after immunization. These two changes may be related, representing the progress of stimulated cells out of the ABC pool as they lose receptors en route to full maturation. The small clone sizes on days 5 and 12 indicate that ABC divide less in response to LPS when already engaged in a response to antigen. In contrast, the PF for total IgM and IgG secretion in the unfractionated population was not greatly affected by immunization.
Pioneering work on insulin and on lipotropin, published in 1967, led to the formulation of the hypothesis that biologically active peptides such as peptide hormones are derived from larger precursor molecules by enzymatic cleavage at basic amino acid pairs. Since then, an ever-increasing number of hormones and active peptides including neuropeptides have been found to be contained within the sequence of a larger precursor protein and are released by limited proteolytic cleavage. The hypothesis concerning the posttranslational maturation of precursor molecules into smaller active entities is now firmly established in many tissues (e.g., pituitary gland, pancreas) and for all kinds of organisms (e.g. mammals, fish), though the nature of the enzyme(s) responsible for this maturation process still remains unknown. This article presents current knowledge of the intracellular processing of precursor molecules, especially the conversion of prohormone to hormone. The discussion deals principally with the evidences concerning the localization of the maturation process mainly in the secretory granules, the role of basic amino acid pairs at the cleavage site, and the possible involvement of serine and (or) thiol endopeptidases in that process.
We have determined the frequency of splenic interleukin 2-producing T-cell precursors that respond to H-2-identical, Mls-disparate stimulating cells. Gene products of the Mlsa and Mlsd alleles are highly stimulatory in primary mixed lymphocyte reactions and activate substantial numbers of T-cell precursors in normal spleens. Although the product of the Mlsc allele has been described as weakly stimulatory in primary mixed lymphocyte reactions, we detected abundant T-cell precursors responsive to Mlsc under our limiting dilution conditions; H-2k Mlsa T-cell precursors responding to Mlsc were as frequent (2.605 per 1000) as H-2k Mlsb or Mlsc T-cells responsive to Mlsd (1.975 per 1000). In contrast, using the frequency of self-responding cells as a threshold, we were unable to detect precursors for Mlsa, Mlsb, Mlsc, or Mlsd in H-2b mice, for Mlsb in H-2k mice, or for Mlsc in H-2k Mlsb or Mlsd mice. Thus, we conclude that in select strain combinations and under our experimental conditions Mlsc stimulators can activate T-cell precursors that are as abundant as precursors reactive to strong stimulators Mlsa and Mlsd.
There has been a controversy for many years over the functional status of cells that leave the thymus (thymus migrants) to populate the peripheral lymphoid organs. Are they immunoincompetent like cortical thymocytes and so probably derived from them, or are they functionally mature like medullary thymocytes? Until recently the techniques used to assess putative thymus migrants have been indirect, but it is now possible to measure the function of recent thymus migrants directly. We used intrathymic injection of a solution of fluorescein isothiocyanate to label thymocytes, and used electronic cell sorting to purify the fluorescent cells that accumulate in the periphery over the following 3 to 4 hr. The migrants have been enriched from an original frequency of about 1:1000 in lymph nodes and spleen, to greater than 98% purity. These cells have been compared with normal peripheral T cells for proliferative and cytotoxic precursor activity in a high cloning efficiency, lectin-induced, limit dilution culture system and in an allospecific limit dilution system. The frequency of precursors of proliferative lymphocytes and cytotoxic lymphocytes and the size of the clones produced is the same for recent migrants and peripheral T cells. Thus by the criteria of proliferation and cytotoxic responses to mitogens and generation of allospecific CTL, thymus migrants, a few hours after their emigration from the thymus, are fully immunocompetent; we therefore see no evidence of a post-thymic precursor-type cell that requires major maturation steps after leaving the thymus.