Mari Tanigawa

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.

Enhancement of nitric oxide generation by low frequency electromagnetic field

Oxidative stress is implicated in the intracellular signal transduction pathways for nitric oxide synthase (NOS) induction. The electromagnetic field (EMF) is believed to increase the free radical lifespan [S. Roy, Y. Noda, V. Eckert, M.G. Traber, A. Mori, R. Liburdy, L. Packer, The phorbol 12-myristate 13-acetate (PMA)-induced oxidative burst in rat peritoneal neutrophils is increased by a 0.1 mT (60 Hz) magnetic field, FEBS Lett. 376 (1995) 164-6; F.S. Prato, M. Kavaliers, J.J. Carson, Behavioural evidence that magnetic field effects in the land snail, Cepaea nemoralis, might not depend on magnetite or induced electric currents, Bioelectromagnetics 17 (1996) 123-30; A.L. Hulbert, J. Metcalfe, R. Hesketh, Biological response to electromagnetic fields, FASEB 12 (1998) 395-420]. We tested the effects of EMF on endotoxin induced nitric oxide (NO) generation in vivo. Male BALB/C mice were injected with lipopolysaccharide (LPS) intraperitoneously (i.p.), followed by the exposure to EMF (0.1 mT, 60 Hz). Five hours and 30 min after the LPS administration, mice were administered with a NO spin trap, ferrous N-methyl-D-glucaminedithiocarbamate (MGD-Fe). Thirty minutes later, mice were sacrificed, and their livers were removed. The results were compared to three control groups: group A (LPS (-) EMF(-)); group B (LPS(-) EMF(+)); group C (LPS(+) EMF(-)). The ESR spectra of obtained livers were examined at room temperature. Three-line spectra of NO adducts were observed in the livers of all groups. In groups A and B very weak signals were observed, but in groups C and D strong spectra were observed. The signal intensity of the NO adducts in Group D was also significantly stronger than that in Group C. EMF itself did not induce NO generation, however, it enhanced LPS induced NO generation in vivo.

Carbohydrate Antigenic Determinant (CA 19–9) and Other Tumor Markers in Gastrointestinal Malignancies

The serum carbohydrate antigenic determinant (CA 19-9) was assayed in patients with various diseases, and it provides excellent sensitivity for adenocarcinoma of the pancreas (25/27, 93%), while only 4% (2/54) of the patients with benign diseases and none of the 40 healthy subjects showed elevated CA 19-9 concentrations over 37 units/ml as upper normal value. Increased serum carcinoembryonic antigen (CEA) levels over 2.5 ng/ml were observed in patients with pancreatic cancer (18/22, 82%), compared to 22% (12/54) of the patients with benign diseases and 10% (4/40) of the healthy subjects. 12 of the 19, 6 of the 19 and none of the 22 patients with pancreatic cancer exhibited high serum ferritin, beta 2-microglobulin, or alpha-fetoprotein levels, respectively. A significant difference in CA 19-9 was found between patients with pancreatic cancer and gastric cancer, other gastrointestinal (GI) malignancies, other non-GI malignancies, benign digestive diseases or normal populations.

Changes in Cerebral Blood Flow during Olfactory Stimulation in Patients with Multiple Chemical Sensitivity: A Multi-Channel Near-Infrared Spectroscopic Study

Multiple chemical sensitivity (MCS) is characterized by somatic distress upon exposure to odors. Patients with MCS process odors differently from controls. This odor-processing may be associated with activation in the prefrontal area connecting to the anterior cingulate cortex, which has been suggested as an area of odorant-related activation in MCS patients. In this study, activation was defined as a significant increase in regional cerebral blood flow (rCBF) because of odorant stimulation. Using the well-designed card-type olfactory test kit, changes in rCBF in the prefrontal cortex (PFC) were investigated after olfactory stimulation with several different odorants. Near-infrared spectroscopic (NIRS) imaging was performed in 12 MCS patients and 11 controls. The olfactory stimulation test was continuously repeated 10 times. The study also included subjective assessment of physical and psychological status and the perception of irritating and hedonic odors. Significant changes in rCBF were observed in the PFC of MCS patients on both the right and left sides, as distinct from the center of the PFC, compared with controls. MCS patients adequately distinguished the non-odorant in 10 odor repetitions during the early stage of the olfactory stimulation test, but not in the late stage. In comparison to controls, autonomic perception and negative affectivity were poorer in MCS patients. These results suggest that prefrontal information processing associated with odor-processing neuronal circuits and memory and cognition processes from past experience of chemical exposure play significant roles in the pathology of this disorder.

Treatment of malignant ascites and pleurisy by a streptococcal preparation OK-432 with fresh frozen plasma—a mechanism of polymorphonuclear leukocyte (PMN) accumulation

A single injection of a streptococcal preparation, OK-432, with fresh frozen plasma (FFP) (or fresh human serum) into the peritoneal or pleural cavity for the treatment of malignant ascites or pleurisy resulted in a complete reduction of ascitic fluid or pleural effusion in 5 out of 11 patients. FFP was used a further source of complement for the effective accumulation of antitumor polymorphonuclear leukocytes (PMNs) by complement-derived chemotactic factors in the cavity. C5a increased in the fluids 3-9 h after the injection and preceded a massive increase in PMNs. C1 inhibitor (C1INH) and C3b inactivator (C3bINA) decreased in several cases 6 h after the treatment. Chemotactic arachidonic acid metabolites, thromboxane B2(TXB2) as a characteristics of TXA2, and leukotriene B4(LTB4) also increased at the same time even in cases where C5a changed only minimally, and may play a role in accumulating antitumor PMNs in the cavity.

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.

In vitro augmentation of cytotoxicity by N-CWS in peritoneal polymorphonuclear leukocytes (PMNs) induced by PSK, OK-432 or N-CWS

Peritoneal polymorponuclear leukocytes (PMNs) were collected from the peritoneal cavity of C3H/He mice 6 hrs after intraperitoneal (i.p.) injection of 2.5 mg/head of PSK, 1 KE (100 µg)/head of OK-432 or 200 µg/head ofNocardia rubra cell wall skeleton (N-CWS). Withoutin vitro stimulation, these PMNs did not show cytotoxicity to syngeneic MM46 mammary carcinoma cells in51Cr release assay. Cytotoxicity of these PMNs was augmented by the addition of 25 µg/ml of N-CWS but not of PSK or OK-432 to cultures for the assay at the beginning of the culture. H2O2 production of PSK-induced PMNs was increased by thein vitro addition of 25 µg/ml of N-CWS but not of PSK. These results suggest that PSK as well as OK-432 and N-CWS can induce PMNs capable of responding further to N-CWS as the second stimulant.

Assessment of cerebral blood flow in patients with multiple chemical sensitivity using near-infrared spectroscopy—recovery after olfactory stimulation: a case–control study

ObjectivesMultiple chemical sensitivity (MCS) is a chronic acquired disorder characterized by non-specific symptoms in multiple organ systems associated with exposure to odorous chemicals. We previously observed significant activations in the prefrontal cortex (PFC) during olfactory stimulation using several different odorants in patients with MCS by near-infrared spectroscopy (NIRS) imaging. We also observed that the patients with MCS did not adequately distinguish non-odorant in the late stage of the repeated olfactory stimulation test. The sensory recovery of the olfactory system in the patients with MCS may process odors differently from healthy subjects after olfactory stimulation.MethodsWe examined the recovery process of regional cerebral blood flow (rCBF) after olfactory stimulation in patients with MCS. NIRS imaging was performed in 6 patients with MCS and in 6 controls. The olfactory stimulation test was continuously repeated 10 times. The study also included a subjective assessment of the physical and psychological status and of the perception of irritating and hedonic odors.ResultsAfter olfactory stimulation, significant activations were observed in the PFC of patients with MCS on both the right and left sides compared with controls. The activations were specifically strong in the orbitofrontal cortex (OFC). Compared with controls, autonomic perception and feelings identification were poorer in patients with MCS. OFC is associated with stimuli response and the representation of preferences.ConclusionsThese results suggest that a past strong exposure to hazardous chemicals activates the PFC during olfactory stimuli in patients with MCS, and a strong activation in the OFC remains after the stimuli.

Association of Odor Thresholds and Responses in Cerebral Blood Flow of the Prefrontal Area during Olfactory Stimulation in Patients with Multiple Chemical Sensitivity

Multiple chemical sensitivity (MCS) is a disorder characterized by nonspecific and recurrent symptoms from various organ systems associated with exposure to low levels of chemicals. Patients with MCS process odors differently than controls do. Previously, we suggested that this odor processing was associated with increased regional cerebral blood flow (rCBF) in the prefrontal area during olfactory stimulation using near-infrared spectroscopic (NIRS) imaging. The aim of this study was to investigate the association of odor thresholds and changes in rCBF during olfactory stimulation at odor threshold levels in patients with MCS. We investigated changes in the prefrontal area using NIRS imaging and a T&T olfactometer during olfactory stimulation with two different odorants (sweet and fecal) at three concentrations (zero, odor recognition threshold, and normal perceived odor level) in 10 patients with MCS and six controls. The T&T olfactometer threshold test and subjective assessment of irritating and hedonic odors were also performed. The results indicated that the scores for both unpleasant and pungent odors were significantly higher for those for sweet odors at the normal perceived level in patients with MCS than in controls. The brain responses at the recognition threshold (fecal odor) and normal perceived levels (sweet and fecal odors) were stronger in patients with MCS than in controls. However, significant differences in the odor detection and recognition thresholds and odor intensity score between the two groups were not observed. These brain responses may involve cognitive and memory processing systems during past exposure to chemicals. Further research regarding the cognitive features of sensory perception and memory due to past exposure to chemicals and their associations with MCS symptoms is needed.

IFN production ability and healthy ageing: mixed model analysis of a 24 year longitudinal study in Japan

Objective To track changes in interferon (IFN) production in healthy individuals to shed light on the effect these changes have on the course of healthy ageing. Design Study is based on data that were collected over 24 years from a cohort of individuals whose IFN-α production was quantified as a part of their annual routine health check-up. Setting All individuals in this study underwent regular health check-ups at Louis Pasteur Center for Medical Research. Participants 295 healthy individuals (159 males and 136 females) without a history of cancer, autoimmune diseases and hepatitis C virus (HCV) whose IFN-α production was quantified more than five times within 24 years were selected. Finally, 29 males and 4 females whose IFN-α production was quantified more than 25 times were selected and their data were analysed using a mixed model. Main outcome measures HVJ stimulated IFN-α production was quantified. Healthy individuals periodical log transformed IFN-α values (y) were plotted versus age (x) and fitted to linear (y=mx+n) and quadratic formula (y=ax2+bx+c) expressions to reveal changes in the IFN-α production in these healthy individuals. Results The linear expression showed that log (IFN-α) had a slight tendency to decline (3% over 10 years). However, the quadratic formula analysis showed the quadratic expression to be more positive than negative (a concave U-shaped pattern) which means that individuals’ once declining IFN production recovered as they aged. Conclusions Although we observed a marginal decline in IFN-α production, we also observed that IFN production recovered even in individuals in their mid50s to early 60s. These results combined with our previous cross-sectional studies of patients with various diseases suggest that in healthy individuals, the impairment of IFN production is triggered more by the onset of disease (notwithstanding the cause) rather than by ageing.