Comparison of species, virulence genes and clones of Aeromonas isolates from clinical specimens and well water in Okinawa Prefecture, Japan.

AIMS: To reveal the sources of Aeromonas infection in Okinawa Prefecture of Japan, the species, virulence genes and clones of strains isolated from clinical specimens and well water were compared. METHODS AND RESULTS: The properties of both isolates were investigated by sequencing of rpoD, detection of 10 virulence genes using PCR and genotyping with pulsed-field gel electrophoresis. In all, 68 clinical and 146 well water strains of Aeromonas were isolated and the main species were A. caviae, A. dhakensis, A. hydrophila and A. veronii biovar sobria. Aeromonas dhakensis possessed various virulence genes; however, A. caviae possessed only fla. The same or similar clones were distributed in certain areas of Okinawa and one clone had survived several months in the biliary system of two patients, respectively. CONCLUSION: Although the same Aeromonas clone was not isolated from clinical and well water samples, our study revealed the detected patterns of virulence genes in both isolates, the distribution of identical/similar clones in the Okinawan environment and long-time survival in patient's organs. SIGNIFICANCE AND IMPACT OF THE STUDY: We investigated the association between Aeromonas patients and well water exposure. This study provides the properties of species, virulence genes and clones of Aeromonas isolated from samples of these origins.

Does preoperative enhanced CT predict requirement of intestinal resection in the patients with incarcerated myopectineal hernias containing small bowel?

PURPOSE: Bowel wall enhancement on CT imaging is considered one of the useful features for the prediction of the presence of irreversible ischemic change in patients with small bowel obstruction. However, the applicability of CT imaging in patients with incarcerated hernias has not been investigated in detail. The aim of this retrospective study was to evaluate the feasibility of preoperative CT findings for the prediction of the presence of irreversible ischemic change in patients with incarcerated hernias containing small bowel. METHODS: Included in this study were 76 patients who underwent surgery for preoperatively diagnosed incarcerated hernias containing small bowel (27 inguinal hernias, 37 femoral hernias and 12 obturator hernias) at our hospital between January 2011 and June 2020. The preoperative clinicoradiological features were compared between the groups, and predictors for intestinal resection were evaluated. RESULTS: Nineteen patients required intestinal resection (Resection group), and the other 57 patients did not require intestinal resection (Nonresection group). Multivariate analyses revealed that age ≥ 80 years (p = 0.018, odds ratio = 6.604) and the absence of bowel wall enhancement (p = 0.032, odds ratio = 51.200) were independent predictors for intestinal resection. In resected specimens, all patients with an absence of bowel wall enhancement on preoperative enhanced CT had ischemic changes extending beyond the muscularis propria. CONCLUSIONS: Preoperative enhancement CT yields useful information for the prediction of the presence of irreversible ischemic change in patients with incarcerated hernias containing small bowel.

Microendocytosis in eosinophilic leukocytes.

Rat peritoneal eosinophils were examined after intraperitoneal infusion either of a mixture of phosphate-buffered saline (PBS) and colloidal gold or of fetal calf serum. These cells characteristically contained vesiculotubular structures, cuplike structures, and small granules during centrifugation. The cup-shaped structures and elaborate labyrinths of vacuole-like spaces increased markedly after injection of the PBS-colloidal gold mixture, presumably as features of heightened microendocytic activity. The vesiculotubular structures increased greatly after infusion of fetal calf serum. A few cyrstalloid granules exhibited fine-structural changes after the PBS-colloidal gold injection, and more numerous crystalloid granules appeared altered after fetal calf serum. Infrequent small granules contained a lucent, crystal-like silhouette after the fetal calf serum injection. Eosinophils evidenced microendocytic uptake of gold spherules into coated vesicles, the cup-shaped structures, and the small granules, but not into the vesiculotubular structures or crystalloid granules after intraperitoneal infusion of the PBS-gold mixture. Strong unmasked acid phosphatase activity in small granules contrasted with the general lack of activity in normal-appearing crystalloid granules and moderate activity in apparently altered crystalloid granules, presumably reflecting active and latent forms of enzyme in the different granules.

Theophylline accelerates human granulocyte apoptosis not via phosphodiesterase inhibition.

Theophylline, in addition to its bronchodilator effect, is reported to have an antiinflammatory action that may account for its clinical effectiveness in the reduction of inflammatory cells in the airway. In bronchial asthma, such inflammatory cytokines as GM-CSF and IL-5 are upregulated and have been proposed to cause granulocyte infiltration (neutrophils and eosinophils) in the airway by inhibition of granulocyte apoptosis. We examined the abilities of theophylline to counteract the prolongation of human granulocyte survival caused by cytokines. Theophylline was shown to shorten granulocyte survival in a dose-dependent manner. Upon incubation with a therapeutical concentration of theophylline (0.1 mM; 18 microg/ml), percentages of GM-CSF (10 ng/ml)-induced delayed apoptosis increased from 18+/-2% to 38+/-3% (p < 0.02) in neutrophils and from 21+/-2% to 35+/-2% (p < 0.02; 24-h incubation) in eosinophils. The percentage of IL-5 (5 ng/ml)-induced delayed eosinophil apoptosis also increased from 22+/-4% to 33+/-2% (P < 0. 05). In contrast, cyclic AMP (cAMP)-increasing agents (3-isobutylmethylxanthine, dibutyryl cAMP, and rolipram) inhibited granulocyte apoptosis in the control and anti-Fas antibody-treated cells. In eosinophils, the expression of bcl-2 protein decreased after incubation with theophylline. These findings suggest that theophylline accelerates granulocyte apoptosis, which may play an essential role in inflammation, and controls granulocyte longevity regardless of the elevation of intracellular cAMP levels.

Mitochondrial trifunctional protein deficiency. Catalytic heterogeneity of the mutant enzyme in two patients.

We examined the enzyme protein and biosynthesis of human trifunctional protein harboring enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase activity in cultured skin fibroblasts from two patients with long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency. The following results were obtained. (a) In cells from patient 1, immunoblot analysis and pulse-chase experiments indicated that the content of trifunctional protein was < 10% of that in control cells, due to a very rapid degradation of protein newly synthesized in the mitochondria. The diminution of trifunctional protein was associated with a decreased activity of enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase, when measured using medium-chain to long-chain substrates. (b) In cells from patient 2, the rate of degradation of newly synthesized trifunctional protein was faster than that in control cells, giving rise to a trifunctional protein amounting to 60% of the control levels. The 3-hydroxy-acyl-CoA dehydrogenase activity with medium-chain to long-chain substrates was decreased drastically, with minor changes in activities of the two other enzymes. These data suggest a subtle abnormality of trifunctional protein in cells from patient 2. Taken together, the results obtained show that in both patients, long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency is caused by an abnormality in the trifunctional protein, even though there is a heterogeneity in both patients.

Absence of IgD-CD27(+) memory B cell population in X-linked hyper-IgM syndrome.

The present study analyzed peripheral blood B cell populations separated by IgD and CD27 expression in six males with X-linked hyper-IgM syndrome (XHIM). Costimulation of mononuclear cells from most of the patients induced no to low levels of class switching from IgM to IgG and IgA with Staphylococcus aureus Cowan strain (SAC) plus IL-2 or anti-CD40 mAb (anti-CD40) plus IL-10. Measurable levels of IgE were secreted in some of the patients after stimulation with anti-CD40 plus IL-4. Costimulation with SAC plus IL-2 plus anti-CD40 plus IL-10 yielded secretion of significant levels of IgG in addition to IgM, but not IgA. The most striking finding was that peripheral blood B cells from all of the six patients were composed of only IgD+ CD27(-) and IgD+ CD27(+) B cells; IgD- CD27(+) memory B cells were greatly decreased. IgD+ CD27(+) B cells from an XHIM patient produced IgM predominantly. Our data indicate that the low response of IgG production in XHIM patients is due to reduced numbers of IgD- CD27(+) memory B cells. However, the IgG production can be induced by stimulation of immunoglobulin receptors and CD40 in cooperation with such cytokines as IL-2 and IL-10 in vitro.

24-Oxo metabolites of vitamin D3 analogues: disassociation of their prominent antileukemic effects from their lack of calcium modulation.

The seco-steroid hormone, 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] inhibits proliferation and induces differentiation of malignant cells including those of the hematopoietic system. The 24-oxo metabolite of 1,25(OH)(2)D(3) also has prominent antiproliferative activities against various cancer cells. We chemically synthesized five novel 24-oxo vitamin D(3) analogues and evaluated their abilities both to inhibit clonal growth and induce differentiation of myeloid leukemia cells and to cause hypercalcemia. The 1alpha,25-dihydroxy-16-ene-D(3) [1,25(OH)(2)-16-ene-D(3)] and 1alpha,25-dihydroxy-16-ene-19-nor-D(3) [1,25(OH)(2)-16-ene-19-nor-D(3)] and their 24-oxo metabolites showed greater potency than 1,25(OH)(2)D(3) in their abilities to inhibit clonal proliferation of HL-60, NB4, and U937 leukemic cell lines as measured by methylcellulose soft-gel assay. Their inhibition of clonal growth was irreversible as analyzed by pulse exposure studies. The synthetic analogues also had greater potency than 1,25(OH)(2)D(3) to induce differentiation of HL-60 and NB4 cells as measured by generation of superoxide, nonspecific esterase production, and induction of CD11b and CD14 cell surface antigens and to increase the proportion of these cells in the G(0)-G(1) phase of the cell cycle. For most assays, the 24-oxo metabolite was slightly more potent than the unmodified analogue, and 50% activity was usually found in the nanomolar range. These analogues and their 24-oxo metabolites also inhibited fresh leukemic cell clonal proliferation. Expression of p27(KIP1), a cyclin-dependent kinase inhibitor that plays an important role in blocking the cell cycle, was found by Western blot analysis to be induced by the analogues and their 24-oxo metabolites in both HL-60 and U937 cells, suggesting a possible mechanism by which these analogues inhibit leukemic growth. Notably, the calcemic activity tested by injections of 1alpha,25-dihydroxy-16-ene-24-oxo-19-nor-D(3) in mice was at least 12-fold less than 1alpha,25(OH)(2)-16-ene-19-nor-D(3). Taken together, chemically synthesized 24-oxo metabolites of 1alpha,25(OH)(2)-16-ene-D(3) and 1alpha,25(OH)(2)-16-ene-19-nor-D(3) irreversibly inhibited proliferation and induced differentiation of acute myeloid leukemia cells with minimal toxicity; these compounds may have a role in the maintenance phase of therapy for acute myeloid leukemia.

Cytostatic effect of TNFalpha on cancer cells is independent of p21WAF1.

Tumor necrosis factor alpha (TNFalpha) is a cytotoxic/cytostatic compound for a variety of human cancer cells. The p21WAF1 protein is a cyclin-dependent kinase inhibitor (CDKI) that binds to cyclin/cyclin-dependent kinase (CDK) complexes and inhibits their kinase activities, thereby leading to cell cycle arrest. We found that the cytostatic effect of TNFalpha on the cervical cancer cell line, ME180, was concomitant with an arrest of these cells in the G0/G1 phase of the cell-cycle. This corresponded with an increase in both p21WAF1 mRNA and protein levels which likely occurred via a p53-independent pathway since ME180 is infected with the human papilloma virus. To elucidate the role of p21WAF1 in the TNFalpha-mediated growth and cell cycle arrest, we stably transformed ME180 cells with an antisense p21WAF1 expression vector. Two clones with reduced levels of p21WAF1 both in their basal state as well as after their exposure to TNFalpha were selected. The growth of these cells was still inhibited by TNFalpha and they arrested in G0/G1 similar to wildtype or empty vector transfected cells. These results indicate that although p21WAF1 expression increases dramatically with TNFalpha treatment, it may not play a critical role in the cytostatic effect of TNFalpha on ME180 cervical cancer cells.

Cloning and characterization of a novel gene encoding keratin-like protein from nematode Nippostrongylus brasiliensis.

Nippostrongylus brasiliensis (Nb) is one of the most important parasites in studying Th2 immune response of the host, but little is known about its antigenic structures of the excretory-secretory or structural proteins of the parasite. Here we report cloning and characterization of a novel antigenic gene from cDNA library of Nb adult worm by immunoscreening. The positive clone, KLP-Nb, had an open reading frame of 612 bp that encodes a 203-amino-acid protein and was homologous to 'similar to keratins in a glycine-rich region' of Caenorhabditis elegans. Its expression was confirmed by Northern blotting and IgG enzyme-linked immunosorbent assay. This protein seems to be one of the components of cuticle that covers the nematode body.

Theophylline induces neutrophil apoptosis through adenosine A2A receptor antagonism.

This study was designed to determine whether theophylline would augment granulocyte apoptosis via a mechanism of adenosine A2A receptor antagonism. A selective adenosine A2 receptor agonist (CGS-21680, 1 microM) exhibited the most efficient potency for decreasing neutrophil apoptosis for 16 h from 63+/-5 to 19+/-4% (P < 0.001); it exerted poor and adverse effects on eosinophil survival. A selective protein kinase A inhibitor KT-5720 (10 microM) reversed the capacity of dibutyryl cAMP but not CGS-21680 to induce an inhibitory effect on neutrophil apoptosis, suggesting that occupancy of adenosine A2 receptors inhibit neutrophil apoptosis by a cAMP-independent mechanism. Theophylline derivatives show the following pattern of potency for inducing neutrophil apoptosis competing with CGS-21680: 8-phenyltheophylline = 8-p-sulfophenyltheophylline > theophylline >> enprofylline. This pattern is consistent with the affinity established for A2A receptors. Theophylline demonstrated an additive effect to that of anti-Fas antibody (CH11, 1 microg/mL) in inducing neutrophil apoptosis, but not to that of adenosine deaminase or KF-17837 (a selective A2 receptor antagonist; 1 microM), suggesting conflicting effects on the receptor antagonism. These findings suggest that theophylline has an immunomodulatory action on neutrophil apoptosis via a mechanism of A2A antagonism.

Effects of theophylline on human eosinophil functions: comparative study with neutrophil functions.

The understanding of theophylline as a bronchodilator has been reconsidered in recent years. We undertook to determine its immunomodulatory actions in granulocytes and elucidate their mechanism. Preincubation of neutrophils with theophylline (10(-5) to 5 x 10(-3) M) had a biphasic effect on O2(-) production stimulated with N-formyl-methionyl-leucyl-phenylalanine or C5a. Theophylline potentiates O2(-) production via adenosine A(2A) receptor antagonism induced by receptor-linked agonists from neutrophils, but not from eosinophils. The addition of theophylline caused a significant decline in neutrophil chemotaxis at lower concentrations than those for eosinophil motility. Theophylline reduces neutrophil chemotaxis via adenosine A1 receptor antagonism. At high concentrations, with an intracellular cAMP accumulation as a result of phosphodiesterase (PDE) inhibition, theophylline also exerts an inhibitory effect on the O2(-) production and chemotaxis of both types of cells. The difference in theophylline's effect on neutrophils and eosinophils appears to depend on the existence of specific adenosine receptors. Theophylline thus modulates granulocyte functions in association with specific adenosine receptor antagonism and cAMP-PDE inhibition.

Thrombopoietin enhances neutrophil production by bone marrow hematopoietic progenitors with the aid of stem cell factor in congenital neutropenia.

We examined the effects of granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), and thrombopoietin (TPO), alone or in combination, on the generation of neutrophils by bone marrow (BM) cells from three patients with severe congenital neutropenia (SCN) through the use of a serum-deprived liquid culture system. Synergistic effects of G-CSF and SCF on the neutrophil production by BM CD34+CD38+c-kit+ cells were observed in SCN patients as well as in normal controls. The addition of TPO to the culture containing G-CSF and SCF further augmented the growth of neutrophils in the two groups. Single-cell culture experiments revealed that the three-factor combination caused increases in both the number and size of neutrophil colonies compared with G-CSF + SCF in normal BM cells, whereas only a significant increment in the colony size was observed in SCN patients. Even in the presence of SCF or SCF + TPO, the concentrations of G-CSF necessary for the substantial production of neutrophils by CD34+CD38+c-kit+ cells were higher in two patients compared with the levels obtained by normal control cells. In addition, TPO did not accelerate the maturation of neutrophilic cells supported by G-CSF + SCF. When BM CD34+CD38-c-kit+ cells were targeted, the addition of TPO to the culture containing G-CSF and SCF was required for significant neutrophil colony growth in the two groups. These results suggest that TPO enhances the G-CSF-dependent neutrophil production with the aid of SCF in this disorder.

Possible role of stem cell factor as a serum factor: monoclonal anti-c-kit antibody abrogates interleukin-6-dependent colony growth in serum-containing culture.

The monoclonal rat anti-c-kit antibody (ACK2), which abrogates colony growth supported by stem cell factor (SCF), significantly inhibited the interleukin-6 (IL-6)-dependent growth of hematopoietic progenitors derived from spleen cells of normal and 5-fluorouracil (5-FU)-treated mice and from bone marrow cells of normal mice in serum-containing culture. The numbers and types of colonies supported by IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF), however, were not influenced by the addition of ACK2 to the cultures of the bone marrow cells from normal mice. In replating experiments with pooled blast cells, ACK2 caused a partial, but significant, inhibition of GM colony growth supported by a combination of IL-6 and fetal bovine serum (FBS), which suggests that FBS is one source of the SCF activity. Conversely, the addition of SCF or FBS with IL-6 to a serum-free culture had significant synergistic effects on the total number of colonies derived from post-5-FU spleen cells and from pooled blast cells. The dose response study showed that the ability of 30% FBS to interact with IL-6 on the colony growth by post-5-FU spleen cells was equivalent to that of approximately 5 ng/mL SCF. These findings suggest that c-kit plays an important role in the growth of hematopoietic progenitors responding to IL-6, and that SCF in the serum affects the development of hematopoietic progenitors in serum-containing cultures.

The twitcher mouse: immunocytochemical study of Ia expression in macrophages.

The cells expressing immune response associated antigen (Ia) were investigated in the nervous system of the twitcher mouse (an authentic murine model of globoid cell leukodystrophy in humans). With immunocytochemistry using a monoclonal antibody against Mac-1 antigen, many Mac-1 immunoreactive cells (Mac-1 positive cells) were detected in the central as well as the peripheral nervous systems (CNS and PNS). In the CNS, Mac-1 positive cells in the gray matter showed cellular morphology of ramified microglia with delicate cellular processes, while in the white matter Mac-1 positive cells were more plump in shape. Ia expressing cells (Ia positive cells) were also largely confined to the white matter. About 10% of the Mac-1 positive cells were Ia positive. The Ia and Mac-1 positive cells were slender and spindle shaped, and morphologically similar to Ia positive cells in the peripheral nerves while the cells expressing Mac-1 only were more plump in shape. With immunoelectron microscopy, however, both slender Ia positive and plump Ia negative and Mac-1 positive cells revealed electron lucent cytoplasmic vacuoles containing characteristic tubular inclusions of globoid cell leukodystrophy. The results suggest that Ia positive cells are a subset of macrophages in the CNS. Whether Ia expression was induced to "endogenous" microglia or whether Ia expressing cells were exogenous cells infiltrated in the CNS in response to pathological lesions is yet to be determined.

Myasthenia gravis and alopecia areata.

Of 202 patients with myasthenia gravis (MG), 6 (3%) developed alopecia areata. All six patients had a thymoma verified by pathology; the frequency of alopecia areata rose to 17% of 35 MG patients with a thymoma. In one patient who had no recognizable tumor in the mediastinum, an ectopic thymoma was present in the anterior neck.

Anti-IL-12 antibody prevents the development and progression of multiple sclerosis-like relapsing--remitting demyelinating disease in NOD mice induced with myelin oligodendrocyte glycoprotein peptide.

Treatment with monoclonal anti-IL-12 antibody injected on day 0, 7 and 10 after immunization with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 in NOD mice resulted in significant suppression of the development and the severity of the chronic relapsing-remitting experimental autoimmune encephalomyelitis (EAE) both clinically and histologically. The spleen cells from anti-IL-12 antibody treated mice displayed markedly inhibited MOG35-55 specific proliferation and IFN-gamma production. MOG35-55 specific antibody production was enhanced by anti-IL-12 antibody treatment. These results suggest that IL-12 is critically involved in the pathogenesis of MOG-induced EAE and that antibody to IL-12 could be an effective therapeutic agent in the clinical treatment of autoimmune demyelinating diseases such as multiple sclerosis (MS).

Delayed in vitro immunoglobulin production by cord lymphocytes.

Pokeweed mitogen-induced immunoglobulin (Ig) production by cord lymphocytes was studied in vitro by Ig-secreting plaque-forming cell (Ig-PFC) assay. Although adult mononuclear cells generated all of IgM-, IgG-, and IgA-PFC, cord mononuclear cells generated only IgM-PFC when cultured for seven days. The number of cord IgM-PFC was 102 +/- 26/10(4) mononuclear cells, being about one fourth of that of adult IgM-PFC. When cultured for 14 days, cord mononuclear cells formed increased numbers of IgM-PFC in contrast to adult cells and yielded IgG-PFC as well, indicating delayed Ig production. Cord T cells were much less effective at helping adult B cells to differentiate into Ig-PFC as compared with adult T cells. Substitution of adult T cells for cord T cell markedly improved the response of cord B cells. The present study demonstrates Ig secretion by cord lymphocytes in response to pokeweed mitogen stimulation. The results further indicate that the delayed Ig production by cord lymphocytes is largely due to functional immaturity of the T cells.

Natural killer cell immunodeficiency in siblings: defective killing in the absence of natural killer cytotoxic factor activity in natural killer and lymphokine-activated killer cytotoxicities.

A immunodeficiency of natural killer cells as effectors for natural killer and lymphokine-activated killer cytotoxicities was first demonstrated in siblings. Two of three male siblings persistently lacked natural killer activity against K562 target cells as assayed by a 51Cr-release assay: percent lysis values were less than 1.0% as compared to the normal lymphocyte values of 43.5% +/- 6.2% (mean +/- SD). Their lymphocytes did not develop natural killer cell activity by changing effector to target ratios, prolonging the incubation time, or stimulating them with interferon-alpha or interleukin 2. Numbers of lymphocytes bearing Leu-7, CD16, or NKH-1 were normal but those of Leu-7-, CD16+ cells were decreased as estimated by flow cytometry. Single cell-in-agarose assays showed normal numbers of natural killer cells capable of binding to a target cell but incapable of killing it. They had depressed levels of lymphokine-activated killer activity, which was totally eliminated by the treatment with OKT3 and complement. This result indicates that the patients' natural killer cells are also defective in the capacity to work as effectors for lymphokine-activated killer activity. The patients' natural killer cells did not produce natural killer cytotoxic factor activity. Antibody-dependent cellular cytotoxicity and cytotoxic T lymphocyte cytotoxicity were normal. These results demonstrate a selective natural killer cell deficiency as effectors for natural killer and lymphokine-activated killer cytotoxicities with a familial tendency, in which there is defective killing with the absence of natural killer cytotoxic factor activity.

Decrease in number of CD16(Leu 11)+CD45RA(2H4)+ cells and defective production of natural killer cytotoxic factor in childhood acute lymphoblastic leukemia.

CD16 or CD45RA is known to be a functional molecule, which provides activation signals in natural killer (NK) cells or T cells, respectively. To dissect the decreased NK activity in childhood acute lymphoblastic leukemia (ALL), the expression of CD16 (Leu 11) or CD45RA (2H4) and the production of natural killer cytotoxic factor (NKCF) were investigated. CD16+ cells or CD45RA+ cells in peripheral blood lymphocytes were not decreased as compared with controls, however, CD16+CD45RA+ cells in ALL (4%) were lower (p less than 0.05) than controls (17%). The production of NKCF in ALL patients (9.2%) was lower (p less than 0.05) than controls (16.5). These data suggest that the decreased NK activity in ALL patients can be attributable at least in part to the functional impairment of NK cells to produce NKCF.

Decreased cytotoxic potential of fresh and recombinant interleukin 2-cultured large granular lymphocytes in childhood acute lymphoblastic leukemia.

Cytotoxic potential (cytotoxic efficiency and binding affinity) of large granular lymphocytes (LGL) in childhood acute lymphoblastic leukemia (ALL) patients was calculated by Michaelis-Menten's kinetic equation. Cytotoxic efficiency (Vmax) of fresh or 3-day recombinant interleukin 2 (IL-2)-cultured LGL, not of similarly cultured T cells, in on-therapy ALL patients was decreased (31% in fresh LGL, P less than 0.05 and 28% in cultured LGL, P less than 0.01) as compared with that in controls, although the binding affinity (Km) of these cells were not decreased. The cytotoxic efficiency and the binding affinity of IL-2-cultured LGL in off-therapy ALL patients was not decreased. These data indicate that the cytotoxic potential of LGL was selectively decreased in on-therapy ALL patients due to the impairment in the post-binding cytotoxic process, not due to the decrease in the binding affinity.