Tsunataro Kishida

Differential expression of granulysin and perforin by NK cells in cancer patients and correlation of impaired granulysin expression with progression of cancer.

Abstract. Granulysin has been identified as an effector molecule co-localized with perforin in the cytotoxic granules of cytotoxic T lymphocytes and natural killer (NK) cells, and has been reported to kill intracellular pathogens in infected cells in the presence of perforin and to induce a cytotoxic effect against tumor cells. The aim of the present study was to elucidate whether intracellular expression of granulysin and perforin by NK cells might be associated with progression of cancer. Flow cytometric analysis demonstrated high levels of perforin and granulysin expression by CD3– CD16+ cells in healthy controls. In contrast, cancer patients exhibited significantly decreased levels of granulysin expression (P<0.005), despite having equally high levels of perforin expression in comparison with healthy controls. The tumor-free patients expressed granulysin at levels similar to healthy controls, while the progressive tumor-bearing patients expressed remarkably lower levels of granulysin compared to healthy controls (P<0.0001). Similarly, patients with an advanced performance status had significantly fewer granulysin-positive NK cells than healthy controls. Meanwhile, a considerable number of the tumor-bearing patients showed a decrease in the number of circulating NK cells, and a correlation between impaired granulysin expression and reduced circulating NK cells was observed. These findings suggest that the tumor-bearing patients with impaired granulysin expression were in an immunosuppressive state. In conclusion, impaired expression of granulysin by NK cells correlates with progression of cancer, and determination of granulysin expression might prove informative for assessing the immunological condition of cancer patients.

Liver targeting of interferon through pullulan conjugation.

AbstractPurpose. The purpose of this study was to actively target interferon (IFN) to the liver through its chemical conjugation with pullulan, a water-soluble polysaccharide with a high affinity for the liver. Methods. Chemical conjugation of IFN with pullulan was achieved by a cyanuric chloride method. Following intravenous injection of the conjugates to mice, their body distribution and the activity of an IFN-induced enzyme, 2′,5′-oligoadenylate (2-5A) synthetase in the liver and other organs, were evaluated. Results. The cyanuric chloride method enabled us to prepare an IFN-pullulan conjugate that retained approximately 7–↑9 % of the biological activity of IFN. Pullulan conjugation enhanced the liver accumulation of IFN and the retention period with the results being reproducible. When injected intravenously to mice, the IFN-pullulan conjugate enhanced the activity of 2-5A synthetase in the liver. The activity could be induced at IFN doses much lower than those of free IFN injection. In addition, the liver 2-5A synthetase induced by conjugate injection was retained for 3 days, whereas it was lost within the first day for the free IFN-injected mice. Conclusions. IFN-pullulan conjugation was promising for IFN targeting to the liver with efficient exertion of its antiviral activity therein.

The effect of intraventricular interferon on subacute sclerosing panencephalitis

Two patients with subacute sclerosing panencephalitis (SSPE) were treated with intraventricular alpha interferon (IFN-alpha) via an Ommaya reservoir for 20-57 months. The clinical course of the disease was followed for 20-67 months. Clinical improvement was observed after daily intraventricular administration of IFN in one case. There were no serious complications or side effects during interferon therapy except for the fever. Intraventricular administration of IFN appears superior to intrathecal administration for long-term treatment in several respects and is considered to be a potential therapeutic modality for SSPE.

The use of BRM-activated killer cells in adoptive immunotherapy : A pilot study with nine advanced cancer patients

Adoptive immunotherapy using MHC-nonrestricted-lymphocytes, peripheral blood γδT cells and NK cells was devised. Peripheral blood mononuclear cells (3 x 107) were selected by immobilization to anti-CD3 monoclonal antibody for 4 days and cultured for 2 weeks in the presence of IL-2. Thereafter they were reactivated by 500 U/ml of IFN-α and 1000 U/ml of IL-2 for 1 hour. Enhancement of NK and LAK activities was confirmed. Peripheral blood γδT cells proliferated in response to immobilized anti-CD3 antibody (3% to 30%). Approximately 6 x 109 BRM-activated killer (BAK) cells composed of CD56+ γδT cells and CD56+ NK cells, were dispensed to cancer patients via intravenous drip infusion. Nine patients were treated with BAK cells every 2 weeks or every month on an outpatient basis. During the course of adoptive immunotherapy, the crossed affinity immunoelectrophoresis (CAIE) pattern of serum immunosuppressive acidic protein (IAP) was analysed. Both the production and glycosylation pattern of IAP is changed in response to tumor enlargement and may therefore act as a marker of the disease progression. During the course of BAK therapy, the glycosylation IAP pattern of 6 patients changed from tumor (T) to normal (N). In addition, the performance status of all patients was maintained at 90–100% of the Karnofsky scale and any side effects including fever were not observed during treatments with BAK cells. Moreover, the overall quality of life (QOL) of the patients, scored at the Face scale was favorable. In addition, blood levels of activated γδT cells producing IFN-γ were assayed as an indication marker of BAK therapy. The normal range of IFN-γ producing γδT cells comprised 6.9 ± 0.9% of peripheral blood mononuclear cells (PBMC), according to a single cell FACScan analyses of PBMCs derived from normal individuals. IFN-γ producing γδT cells of Patients No. 8 and 9, who received extensive chemotherapy before initiation of BAK therapy, comprised only 0.2% and 2% of PBMC, respectively. These patients died 3 and 6 months after beginning BAK therapy. Peripheral blood γδT cells of Patients Nos. 1–7 proliferated in response to immobilized anti-CD3 antibody and the frequency of IFN-γ producing γδT cells in PBMC preparation of these patients were over 3% before initiation of BAK therapy. Since our data show a positive correlation between survival time and initial γδT cell counts, a low frequency of these cells may contraindicate BAK therapy.

Impaired IFN-α Production and the Risk of Cancer Development

Type I interferons (IFNs) play a pivotal role not only in antiviral immunity but also in the surveillance of cancer development. In order to quantify the critical function of type I IFNs in the suppression of human cancer development, IFN-alpha production in response to Sendai virus stimulation has been compared between healthy control subjects and hepatitis C virus (HCV)-infected patients, the latter being an ideal population for longterm monitoring of cancer development. Data for IFN-alpha production were obtained retrospectively over a 17-year period by examining medical records in a study population of 2315 individuals, of which 112 healthy controls and 20 HCV-infected patients were selected. Sixty percent of the HCV-infected patients had impaired or declining IFN-alpha production, in comparison to 17% in the healthy control group. Mean IFN-alpha levels were lower in patients who developed hepatocellular carcinoma than in the HCV-infected patients who remained cancer free. Our findings suggest that impairment of IFN-alpha production may be linked to an increased cancer risk and that dysfunction of the IFN system is associated with some types of cancer. Therefore, periodic assessment and quantification of IFN-alpha production can be a potential test for the early detection of cancer in humans.

A series of immune responses leading to the induction ofT cell IL-12/IL-18 responsiveness in patientswith relatively large tumor burdens

Abstract. The induction of interleukin-12 (IL-12) responsiveness in T cells depends on T cell receptor (TCR) triggering, and is regarded as a parameter of recently TCR-sensitized T cells. Here, we investigated whether IL-12 responsiveness could be detected in freshly prepared T cells from tumor-bearing patients, and if so whether such patients exhibited additional immunological parameters related to IL-12 responsiveness. CD4+ and CD8+ T cell populations from an appreciable proportion of tumor-bearing patients exhibited high levels of IL-12 responsiveness as evaluated by IL-12-stimulated interferon-gamma (IFN-γ) production. T cell populations with high IL-12 responsiveness were observed in the group of patients with moderate to large tumor mass or tumor metastases rather than in patients with small tumors. The frequency of such a T cell population was also lower in post-surgery tumor-free patients, showing the correlation between IL-12 responsiveness and the presence of a certain extent of tumor burden. More importantly, a higher incidence of IL-12 responsiveness was observed in tumor-bearing patients exhibiting detectable plasma IL-12 levels, and correlated with IL-18 responsiveness. T cell IL-12 and IL-18 responsiveness is induced by TCR triggering and subsequent IL-12 stimulation respectively. Furthermore, TCR-triggered T cells stimulate antigen-presenting cells (APC) to produce IL-12. Therefore, the present observations suggest that an immune response loop from TCR sensitization to the induction of IL-12/IL-18 responsiveness via IL-12 production operates in tumor-bearing patients, particularly in those with relatively large tumor burdens.

Potentiation of in vivo Antitumor Effects of Recombinant Interleukin‐1α by Gelatin Conjugation

Chemical conjugation of a recombinant human interleukin‐1α (IL‐1) with gelatin was conducted using a water‐soluble carbodiimide in an attempt to augment the indirect effect of IL‐1 on in vivo tumor cell growth in mice. Chromatographic studies of the IL‐1‐gelatin conjugate demonstrated that the apparent molecular weight of IL‐1 was increased by the gelatin conjugation and about 60% of IL‐1 activity was retained in the prepared conjugate. Intraperitoneal (i.p.) injection of the conjugate significantly suppressed the intraperitoneal growth of a subline of Meth A fibrosarcoma cells (RR1 cells), compared with the effect of free IL‐1 at the same dose, although the cells per se were resistant not only to free IL‐1 but also to gelatin‐conjugated IL‐1. Simple mixing of gelatin with free IL‐1 did not augment the in vivo antitumor effect as compared with that of free IL‐1. Gelatin conjugation improved the in vivo stability of IL‐1. Prolonged retention of IL‐1 activity in the peritoneal cavity as well as the circulation of mice was observed after i.p. injection of the IL‐1‐gelatin conjugate in comparison with free IL‐1 injection, irrespective of the presence of tumor cells. Gelatin conjugation was effective in augmenting the in vivo antitumor effects of IL‐1 to activate host cells, e.g. macrophages (Mø). The i.p. injection of the conjugate enhanced Mø infiltration into the peritoneal cavity of tumor‐bearing mice and peritoneal Mø were strongly activated to inhibit the in vitro growth of RR1 cells. Thus, gelatin conjugation was effective in augmenting the indirect effect of IL‐1 via host cells, leading to a high suppressive effect on in vivo growth of tumor cells.

Quantitative and qualitative characteristics of colony-forming unit-erythroid colonies in myelodysplastic syndrome patients

The microcytofluorometrical method was applied to determine the relative hemoglobin (Hb) content in the bone marrow colony-forming unit-erythroid (CFU-E) colonies from 6 patients with myelodysplastic syndromes (MDS) and 10 healthy subjects. This method relies on a photochemical reaction, by which intracellular Hb is converted into fluorescent porphyrin using a 0.2 M mercaptoethylamine solution (an SH donor) and violet light (lambda = 405 nm). The relative Hb content was determined as a function of the intensity of emitted porphyrin fluorescence. The number of colonies identified by porphyrin fluorescence was smaller in MDS patients than in normal subjects. The relative Hb content was also lower in MDS patients than in normal subjects. In addition, the coefficient of variation of the relative Hb content in the CFU-E colonies was larger in MDS patients than in normal subjects. These findings suggest that colonies with low relative Hb content undergo impaired erythropoiesis and that the CFU-E colonies undergoing the impaired erythropoiesis are mixed with CFU-E colonies showing normal erythropoiesis in the bone marrow of MDS patients.

Susceptibility to Infection Due to Diminished Interferon-α-Producing Capacity in Patients with Refractory Anemia with Excess of Blasts or Refractory Anemia with Excess of Blasts in Transformation

Peripheral whole blood cells from normal subjects and from 8 patients with refractory anemia with excess of blasts (RAEB) and one patient with RAEB in transformation (RAEB-t) were studied to ascertain their interferon (IFN)-α-producing capacity. This capacity was determined by time-resolved immuno-fluoroassay 20 hrs after the blood cells had been incubated with 500 HA/ml of Sendai virus. The mean IFN-α-producing capacity in the peripheral whole blood cells of 35 normal males was 8718 ± 4683 IU/ml, while that of 49 normal females was 7549 ± 4731 IU/ml; not a significant difference. All of the RAEB and RAEB-t patients examined showed a significantly low (<2500 IU/ml) IFN-α-producing capacity in the peripheral whole blood cells. In addition, the levels of IFN-α-producing capacity in 1 ml cell suspension containing 1 x 10(6) peripheral mononuclear blood cells were markedly lower in all 5 RAEB patients and the RAEB-t patient examined than those in the normal subjects, when 5 HA of Sendai virus was used. The activities of natural killers were lower in the RAEB/RAEB-t patients than those in the normal subjects. These findings suggest that IFN-α-producing capacity is diminished in the peripheral whole blood cells of RAEB and RAEB-t patients, and great care is advised regarding infection by virus and bacteria in treatment of these patients.