Granzyme B: a marker of risk for influenza in institutionalized older adults.

Risk for influenza increases with age while cellular immune responses decline. This was a prospective study to determine the relationship between cytokine and granzyme B levels in peripheral blood mononuclear cells stimulated with live influenza virus, and subsequent influenza illness. Granzyme B levels were lower in the group who later developed symptomatic laboratory-confirmed influenza (n=10) compared to the group who did not (n=90) (ANOVA, P=0.024). In contrast, none of the cytokine levels were related to the development of influenza. Thus, granzyme B is a potential marker of influenza risk in older adults.

Assessment of markers of the cell-mediated immune response after influenza virus infection in frail older adults.

The purpose of this study was to determine whether measures of the cell-mediated immune response to influenza virus could be used as markers of influenza virus infection. We studied 23 subjects who developed upper respiratory, lower respiratory, or systemic symptoms during a small outbreak of influenza in a nursing home population. Influenza virus culture from nasopharyngeal swabs yielded influenza virus isolates from 7 of the 23 subjects. Only three of the subjects had a fourfold rise in antibody titer to the influenza virus antigen positivity after the infection. Granzyme B and cytokine levels were measured in peripheral blood mononuclear cells (PBMC) obtained from all subjects and stimulated with live influenza virus. Elevated granzyme B levels in virus-stimulated PBMC in combination with lower respiratory tract or systemic symptoms in study subjects was a significant predictor of culture-confirmed influenza virus infection compared to those from whom influenza virus could not be identified. Cytokine levels did not distinguish between the two groups in a similar type of analysis. Granzyme B in combination with the clinical profile of symptoms may be a useful retrospective marker for influenza virus infection.

Responses to influenza vaccination in different T-cell subsets: a comparison of healthy young and older adults.

T-lymphocyte responses to influenza vaccination were measured in healthy young and older adult volunteers. All participants were vaccinated with the 1995-96 trivalent influenza vaccine. Cytokine and granzyme B levels were measured in peripheral blood mononuclear cells (PBMC) cultures after virus stimulation, prior to and 4 and 12 weeks after vaccination. The major findings in the older adult group were the different types of helper T-cell (Th) responses to each of the vaccine strains of virus and a very poor cytotoxic T lymphocyte (as measured by granzyme B) response to vaccination. IL-10, which is produced in a Th-type 2 response, was higher in PBMC stimulated with A/Texas/36/91 (H1N1) compared with A/Johannesburg/33/94 (H3N2); this difference was more marked in the PBMC from older compared with younger adults. In contrast, IL-2, which is produced in a Th-type 1 response, was measured in the same cultures and was significantly higher in A/Johannesburg/33/94-stimulated PBMC. IFN- gamma levels were highest in the PBMC stimulated with B/Harbin/7/94. The greatest age-related difference was the level of granzyme B in all virus-stimulated PBMC from the young compared with the older adult group. The strain of influenza virus contained in the vaccine, as well as the age of the subject, appear to be very important determinants of the T-cell response to vaccination.

Immune response to influenza vaccination in institutionalized elderly: effect on different T-cell subsets.

Humoral and cellular immunological responses to influenza vaccination were measured in volunteers in a long-term care facility. All participants were vaccinated with the commercially available 1994-95 trivalent influenza vaccine and blood samples were collected before and 6 and 12 weeks after vaccination. Cytokine and granzyme B in peripheral blood mononuclear cell (PBMC) cultures after virus stimulation, and serum antibody titres were measured for each of these time points. In general, the measures of the immunological response to vaccination were low and variably significant. The major finding was the difference with respect to post-vaccination measures for the two strains of influenza A contained in the vaccine. Geometric mean antibody titres were significantly higher for A/Texas/36/91 at all time points in the study when compared to A/Shangdong/09/93. There was a corresponding rise for interleukin-10 (IL-10) to the A/Texas/36/91 strain while no increase in IL-10 was observed in A/Shangdong/09/93-stimulated cultures after vaccination. In contrast, granzyme B rose after vaccination only in cultures stimulated with A/Shangdong/09/93. Interferon-gamma levels were also significantly higher in these PBMC cultures. There was a poor interleukin-2 (IL-2) response to both strains of influenza A. These data suggest that different strains or subtypes of influenza A may preferentially enhance T-helper type 1 versus type 2 responses through vaccination in institutionalized seniors.

Human colostrum contains an activity that inhibits the production of IL-2.

The effect of human colostrum on T cell immune function was investigated. Colostrum inhibited the proliferation of human T cells activated by allogeneic, concanavalin A (Con A) or phytohaemagglutinin (PHA) stimulation. Colostrum also inhibited the production of IL-2 by Con A-activated human peripheral blood T cells and by Con A-activated Jurkat cells, a human T lymphoma line. Similarly, human colostrum inhibited the production of IL-2 by EL4 cells, a murine thymoma line, when stimulated with phorbol myristate acetate. The inhibitory activity was not cytotoxic and could not be neutralized by antibody to transforming human growth factor beta.

Randomization tests: statistics for experimenters.

Randomization (permutation) tests free the experimenter from the constraints of random sampling, a known error distribution and equal variances, to give a direct answer to the question "how likely is such a large (or small) result if the applied treatments had no effect?" The "result" may be the difference in mean responses, a correlation coefficient or any other value of interest. A randomization test is not a different statistical test but a different, and always valid, method of determining statistical significance. The familiar t-test and F-test can be carried out by data permutation without any parametric assumptions being fulfilled. A particular advantage of this method is that unbalanced designs and missing values are easily accommodated. Even with only a small number of subjects the number of permutations will be large and a computer is necessary if the randomization test is to be of practical value. To make this method of determining statistical significance generally available an interactive microcomputer program, forming a comprehensive package for the design and analysis of experiments, has been prepared.

Development of precytotoxic T cells in cyclosporine-suppressed mixed lymphocyte reactions.

Cyclosporine (CsA) blocked the generation of cytolytic activity in a primary MLR of mouse spleen cells. As expected from the known mechanism of action of this drug, it also blocked the accumulation of IL-2 during the MLR. Addition of human rIL-2 did not overcome the inhibition of CTL generation, even when it was added daily to keep its level similar to that produced in a normal MLR. Daily addition was necessary, because the CsA-inhibited MLR consumed IL-2, either by utilization or degradation. The outcome of a 5-day MLR in the presence of CsA (CsA-MLR) depended on whether or not IL-2 was continuously present. In the presence of IL-2, there was no generation of CTL activity, probably because such cultures contained IL-2-dependent suppressive elements described previously. However, when day 5 CsA-MLR cells generated in the absence of IL-2 were washed and recultured with human rIL-2, there was a burst of CTL activity, with a more than 50-fold increase in alloantigen-specific cytotoxicity within 24 to 48 h. This increase is not explainable simply by the proliferation of existing effector CTL. The noncytotoxic cells produced in an MLR in the presence of CsA, and which can be rapidly activated to cytotoxic effector cells by IL-2, are termed "precursor-effector CTL" (peCTL). They could be detected by day 3 of a primary CsA-MLR culture. Their conversion to effector CTL by IL-2 was not inhibited by CsA. Exposure of peCTL to IL-4 also generated CTL activity, to a somewhat lesser degree than IL-2, but the IL-4-induced activation was inhibited by CsA, suggesting that it depended on the induction of another CsA-sensitive lymphokine. The intracellular levels of mRNA encoding the CTL-specific serine esterases CCP1 and CCP2 (granzymes B and C, respectively) increased rapidly during the IL-2-driven conversion of peCTL to effector CTL. This study demonstrates that in the presence of CsA precursors for CTL can accumulate, and that these can be rapidly converted to cytotoxic effector cells by IL-2.

IL-4 potentiates the IL-2-dependent proliferation of mouse cytotoxic T cells.

In addition to the regulation of B lymphocyte growth and differentiation, the cytokine IL-4 (BSF-1) exerts effects on T lymphocytes and other bone marrow-derived lineages. We show here that recombinant mouse IL-4 synergizes with low levels of IL-2 to increase the yield of cytotoxic activity in a primary MLR, and the proliferation of both cloned IL-2-dependent CTL lines and cells obtained from a primary MLR. IL-4 did not induce the proliferation of any of several cloned CTL cell lines on its own. It also did not replace IL-2 in stimulating the growth or reactivation of quiescent, antigen-dependent CTL clones. However, IL-4 was synergistic with IL-2 after reactivation of the quiescent cells with antigen plus IL-2. Enhancement by IL-4 of the IL-2-driven proliferation of an antigen-independent line was blocked by the addition of anti-IL-4 monoclonal antibody. Although incubation of the CTL clones with IL-4 or with IL-2 plus IL-4 induced a transient increase in the expression of the mRNA encoding the 55 kDa IL-2 receptor, no change in the number or affinity of IL-2 receptors because of IL-4 was detected. This suggests that IL-4 does not potentiate the IL-2 response by altering IL-2 receptor levels. Instead, we propose that the synergistic effect of IL-4 is mediated by a different signalling mechanism from that used by IL-2.

Random assignment of treatments in a 96-well (8 X 12) microtiter plate. A practical method.

A computer program, written in BASIC, has been developed to assign treatments strictly at random to the wells of a microtiter plate. A hard copy is printed of the treatment numbers with their assigned wells, followed by an 8 X 12 matrix of the treatment numbers. When a microtiter plate is placed over the matrix these numbers may be viewed directly through the wells. A program listing is reproduced. The effects of randomization on within-plate variation have been investigated and compared with two systematic methods of assignment.

Interaction of interleukin 2 with cells: quantitative analysis of effects.

The response of T lymphocytes to interleukin 2 (IL 2) is accurately described by a four-parameter logistic function. Both data generated by a theoretical model of IL 2-driven proliferation and experimental data conformed to this function for all doses of IL 2. Assays measuring either the rate of DNA synthesis or cellular metabolism were well described. The variance of response was not constant but increased in a predictable way. Weighting was therefore included in deriving a nonlinear curve-fitting program. The effects on response of cell density, time, and the T lymphocyte line used were examined. Assays gave reproducible estimates of potency when test preparations were compared with a standard preparation, but not otherwise. A model for IL 2 proliferation was derived on the basis of the two-state model of the cell cycle, with cells leaving a quiescent state randomly and then traversing the other stages of the cell cycle in a determinate way.

Is IL-2 regulated by a serum inhibitor?

Normal murine serum inhibits the proliferation of cloned cytotoxic T lymphocytes driven by pure interleukin 2 (IL-2), indicating that a component of normal murine serum is directly inhibitory to IL-2-dependent proliferation. However, the effect is not specific to such cells, since an IL-2-independent variant cell, and a number of lymphoid tumor cell lines are similarly inhibited. Addition of purified IL-2 does not overcome the inhibition, although its degree is reduced. Fractionation of murine serum showed that there are at least two inhibitory activities, which migrate with globular proteins of molecular weights greater than or equal to 10(6) and 4 X 10(4), respectively, on gel chromatography. Neither of the activities was specific for IL-2-dependent cells. Furthermore, murine IL-2 is stable in murine serum in vitro, although it disappears rapidly from the circulation after intravenous injection. It is therefore unlikely that serum inhibitor of IL-2 is an important immunoregulator in vivo.

Mechanism by which antibodies to non-AgB antigens mediate rejection of rat leukaemia cells.

The August and Hooded rat strains are compatible at the major histocompatibility locus (both are AgB5 or Rtlc). Antisera against the minor histocompatibility antigens of Hooded rats were raised by immunizing August rats with grafts of tumours or normal tissue. Such antisera, if transferred to normal unimmunized August rats, cause them to reject i.v. administered Hooded rat leukemia (HRL) cells within a few hours, and X-irradiated August rats, for whom a graft of HRL is lethal, can survive indefinitely if pretreated with the antiserum. The distribution of 125I-labelled HRL cells in the tissues of August rats was followed at times after their injection, and it was found that, in the presence of antiserum, i.v. administered leukaemic cells are rapidly destroyed in the liver and spleen. The active component of the antiserum is IgG antibody, and its action is independent of the lytic elements of complement. Antibody-mediated splenic and hepatic clearance of the leukaemia cells is unaffected by total-body X-irradiation but reduced by treating the rats with colloidal carbon. The data are consistent with the hypothesis that the rejection of HRL across the histocompatibility barrier studied is, in the presence of antibody, effected by immunophagocytosis.

DNA synthesis in isolated lymphocyte nuclei. Effects of megaloblastic anaemia due to folate or vitamin B-12 deficiency or antimetabolite drugs.

DNA synthesis has been studied in nuclei isolated from phytohaemagglutinin-stimulated lymphocytes from normal subjects and patients with megaloblastic anaemia. Lymphocytes were incubated for 72 h, nuclei isolated and incorporation of tritiated deoxythymidine triphosphate ([3H]TTP) into DNA measured, usually over a 10 min incubation period. Preincubation of normal phytohaemagglutinin-stimulated lymphocytes with methotrexate (1 - 10(-5) M, 48--72 h), 5-fluorouracil (1 - 10(-6) M, 70--72 h), and 1-beta-D-arabinofuranosyl cytosine (cytosine arabinoside) (4 - 10(-5) M, 71--72 h) caused a mean rise in [3H]TTP incorporation of 1.7 (P less than 0.01), 1.7 (P less than 0.05) and 2.4 (P less than 0.0025) fold, respectively. Hydroxyurea (3 - 10(-4) M, 48--72 h) in two experiments caused a mean increase of 1.6 fold. Untreated vitamin B-12- and folate-deficient cells showed a 2.0-fold (P less than 0.05) increase above the incorporation when the deficiencies were corrected by addition of vitamin B-12 and folic acid between 0 and 72 h in vitro. The mean percentages of the incorporation due to ATP-independent synthesis in nuclei from normal untreated cells, 5-fluorouracil-treated, cytosine arabinoside treated and vitamin B-12- or folate-deficient cells were 56 +/- 7% S.E., 41 +/- 7%, 84 +/- 3% and 28 +/- 6%, respectively. 5-Fluorouracil caused a two-fold increase in the cytoplasmic fraction of DNA polymerase when added to phytohaemagglutinin-stimulated lymphocytes between 48 and 72 h of culture but had no significant effect when added between 70 and 72 h.

Thymidine kinase in megaloblastic anaemia.

Thymidine kinase has been measured in phytohaemagglutinin (PHA)-stimulated lymphocytes from 13 normal subjects and eight patients with megaloblastic anaemia. The levels in normal subjects ranged from 0.20 to 2.10 units/mg protein (mean 0.903 units/mg protein) and in megaloblastic anaemia from 2.99 to 9.97 units/mg protein). All the patients showed raised levels of the enzyme which were partly but not completely reduced to normal by addition of folic acid in vitro. Vitamin B12 in vitro had a lowering effect in the five vitamin-B12-deficient patients and two patients with combined deficiencies but not in one 'pure' folate-deficient patient. Thymidine kinase activity was highest in the cells of the least anaemic patients, suggesting that the degree of anaemia in megaloblastic anaemia may be determined in part by the ability of the cells to utilize thymidine by the 'salvage' pathway when the de novo pathway of thymidylate synthesis is failing. The rise in thymidine kinase activity in megaloblastic anaemia is presumably due to induction of the enzyme. Addition of methotrexate or 5-fluorouracil, drugs known to inhibit de novo thymidylate synthesis, caused an increase in thymidine kinase activity in normal PHA-stimulated lymphocytes after 24 h (but not after 1 h) which could be completely blocked by addition of puromycin. Thymidine mono- and di-phosphate kinases were also measured in normal PHA-stimulated lymphocytes. The activities were substantially higher than that of thymidine kinase and their activities were unaffected by methotrexate addition.

Effect of iron deficiency and desferrioxamine on DNA synthesis in human cells.

Desferrioxamine (10(-3) M) caused a fall in the deoxyadenosine triphosphate level after 4 h incubation in normal phytohaemagglutinin-stimulated lymphocytes. There was a rise in the concentrations of the other three deoxyribonucleoside triphosphates (deoxythymidine-,deoxycytidine-and deoxyguanosine-triphosphate). The changes are similar to those caused by hydroxyurea, a known inhibitor of ribonucleotide reductase. Desferrioxamine (10(-3 M) was found to inhibit human lymphocyte ribonucleotide reductase to a mean of 11% of control activity after 45 min incubation. Both drugs, desferrioxamine and hydroxyurea, inhibited incorporation of [3H]thymidine DNA into lymphocytes in the presence or absence of deoxyuridine, and inhibited production of lymphocytic thymidine kinase, having opposite effects to methotrexate on both [3H]thymidine incorporation and thymidine kinase activity. Phytohaemagglutinin-stimulated lymphocytes from patients with chronic iron deficiency showed lower levels of all our deoxyribonucleoside triphosphates than normal lymphocytes. It is suggested that this may be due to reduced ribnucleotide reductase activity of the iron-deficient cells.