TACR1 gene polymorphism and sex differences in postoperative nausea and vomiting.

We hypothesised that the genetic effect of single nucleotide polymorphisms in the TACR1 gene, which encodes NK1 receptors, could influence the sex difference in postoperative nausea and vomiting. Thirty-two selected single nucleotide polymorphisms were genotyped by the Sanger sequencing method in 200 patients who underwent lower abdominal surgery. The incidence and severity of postoperative nausea and vomiting were evaluated after surgery. The rs3755468-SNP showed significant association with the incidence and severity of postoperative nausea and vomiting (p = 0.016). The TT haplotype defined by two single nucleotide polymorphisms, including the rs3755468-SNP, was associated with reduced incidence and severity of postoperative nausea and vomiting in female patients (p = 0.03). The rs3755468-SNP is located within the predicted oestrogen response element and a DNase I hypersensitive site. The single nucleotide polymorphisms in the TACR1 gene are associated with sex differences in postoperative nausea and vomiting and may help to elucidate the mechanisms underlying these differences.

Improved non-invasive total haemoglobin measurements after in-vivo adjustment.

We hypothesised that an in-vivo adjustment method and/or a newer sensor would increase the accuracy of non-invasive and continuous haemoglobin monitoring (SpHb) measurements. Two sensors, the R1-25 and R2-25a (the newer version), were used with laboratory total haemoglobin concentration (tHb) values simultaneously recorded. In-vivo adjusted SpHb (AdHb) was calculated by a simple formula: AdHb = SpHb - (1(st) SpHb - 1(st) tHb). The correlation coefficients between SpHb (or AdHb) and tHb were compared: SpHb in both sensors correlated strongly with tHb (p < 0.0001). In-vivo adjustment improved the correlation coefficient between SpHb and tHb from 0.86 to 0.95 for the R1-25 and from 0.83 to 0.93 for the R2-25a. There was no difference between the R1-25 and R2-25a sensors. The in vivo adjustment method improved the accuracy of SpHb measurements in both sensors.

PSK and OK-432-induced immunomodulation of inducible nitric oxide (NO) synthase gene expression in mouse peritoneal polymorphonuclear leukocytes and NO-mediated cytotoxicity.

We investigated whether PSK (a polysaccharide from the mycelia of Coriolus versicolor) or OK-432 (a streptococcal preparation) can up-regulate inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) production in mouse peritoneal polymorphonuclear leukocytes (PMNs). Six hrs after intraperitoneal injection of mice with PSK (2,500 microg/mouse) or OK-432 (100 microg/mouse), mouse peritoneal PMNs were restimulated with PSK (500 microg/ml) or OK-432 (10 microg/ml) plus 100 U/ml of mouse interferon-gamma (IFN-gamma) in vitro. Northern blot analysis showed strong synergism between both PSK and OK-432 and IFN-gamma for the induction of iNOS gene expression. NO production by PMNs was increased up to 20 microM (2 microM/10(6) PMNs/24 hrs) as measured by the Griess reagent method when PMNs were restimulated with PSK or OK-432 plus IFN-gamma for 24 hrs, although tumor cell killing was not detected. NO concentrations of more than 80 microM were required for P815 tumor cell killing. These results suggest that PMNs produce NO after stimulation with PSK or OK-432 in combination with IFN-gamma and may regulate the immune system in vivo, although the NO production induced by these agents is insufficient for tumor cell killing in vitro.

[Cesarean section using a temporary IVC filter in a pregnant woman with several histories of deep vein thrombosis].

A 31-year-old pregnant woman with a history of several episodes of deep vein thrombosis was scheduled for Caesarian section. The patient had an increased risk of intraoperative pulmonary thromboembolism due to thrombi isolated from deep veins. This is because a thrombus can be formed easily in deep veins by the influence of hypercoagulability in the third trimester and by the disturbance of venous return due to the pressure of the enlarged pregnant uterus. A temporary IVC filter (Filtre Thery, France) was inserted preoperatively. The filter basket was set in the IVC cephalad from the left renal vein and caudad from the hepatic vein. The operation was completed without any trouble. Rehabilitation was safely initiated just after the surgery by insertion of the filter and then the filter was removed on the third postoperative day. We conclude that the preoperative insertion of a temporary IVC filter is a useful technique to prevent intraoperative pulmonary thromboembolism in a patient with a history of deep vein thrombosis.

[A case of acute exacerbation of idiopathic interstitial pneumonia after pneumonectomy].

A 52-year-old male was scheduled for right upper lobectomy due to lung cancer. The patient was diagnosed as having stable idiopathic interstitial pneumonia (IIP) based on preoperative lung biopsy, physical examination and laboratory data. He received prophylactic steroids to prevent an acute exacerbation of IIP. However, he fell into severe hypoxia 5 days after operation, and died of respiratory failure 43 days after operation. It must be born in mind that the patients with IIP may suffer exacerbation even if their lungs have been diagnosed as in stable stage. Prophylactic use of steroids may cause a deterioration in IIP due to delayed healing and infection.

Muscle fiber immaturity and inactivity reduce myonecrosis in Duchenne muscular dystrophy.

We report on the first case of X-linked recessive myotubular myopathy (MTM1) coinciding with Duchenne muscular dystrophy (DMD). The muscle biopsy specimens of the patient show only the characteristic findings of MTM1, without the findings of DMD. We theorize that this was caused by the muscle fiber immaturity and inactivity.

Efficacy of in vitro sensitized cells generated by in vivo priming with OK-432 for adoptive immunotherapy of the poorly immunogenic B16-Bl6 melanoma.

We investigated the efficacy of the streptococcal preparation OK-432 as an adjuvant for in vivo priming in induction of sensitized cells for adoptive immunotherapy of the poorly immunogenic B16-BL6 (BL6) melanoma. C57BL/6 (B6) mice were immunized subcutaneously (s.c.) with 3 x 10(6) viable BL6 tumor cells admixed with various doses of OK-432 ranging from 1 to 100 micrograms in the foot-pad. Draining popliteal lymph nodes (LNs) were harvested 7 days after immunization and LN cells were further sensitized with irradiated tumor cells in the presence of 60-300 IU/ml of IL-2 for 11 days. These in vitro sensitized (IVS) cells (2 x 10(6)) were transferred intravenously (i.v.) to B6 mice bearing 4-day pulmonary metastases established by i.v. injection of 2-4 x 10(5) viable BL6 cells. The mice were also received intraperitoneally (i.p.) 4 x 10(4) IU/day of IL-2 for 4 days after adoptive transfer. Transfer of IVS cells from mice immunized by s.c. injection of tumor cells admixed with 10 micrograms of OK-432 significantly reduced the numbers of BL6 pulmonary metastases compared with that of control IVS' cells without the administration of OK-432 (P = 0.003). These effective IVS cells also significantly prolonged the survival of treated animals (P = 0.003). Functional IVS cells required in vitro stimulation with tumor cells. However, addition of OK-432 in the vaccine resulted in no enhancement of in vitro cytotoxicity and no characteristic change of phenotype of IVS cells. These results suggest that in vivo priming of OK-432 facilitates the sensitization of tumor-reactive T-cells. The procedure of in vivo priming with OK-432 may be beneficial in the adoptive immunotherapy of melanoma.

Morphological study of cytotoxicity produced by PSK-induced polymorphonuclear leukocytes (PMNs) and Nocardia rubra cell wall skeleton.

The morphologic changes in PMNs induced by an i.p. injection of PSK, a polysaccharide from the mycelia of Coriolus versicolor, and tumor cells undergoing cell death, were evaluated by immunohistochemical staining and electron microscopy. Male C3H/He mice, 8-10 -weeks old, received an i.p. injection of 125 mg/kg of PSK. Their PMNs were obtained 6 h after the PSK injection by peritoneal lavage. N-CWS (Nocardia rubra cell wall skeleton) was added at the start of the chromium release assay using the MM46 mammary carcinoma cell line, which is syngeneic to C3H/He mice, as target cells. During the cytotoxic assay, the cells were fixed at various time points. The MM46 cells expressed ICAM-1 while the PMNs expressed both ICAM-1 and LFA-1 as determined by immunohistochemical staining and immunoelectron microscopy using anti-ICAM-1 and anti-LFA-1 antibodies. PMNs with ruffle-like microvilli adhered to the MM46 tumor cells 30 min after the addition of N-CWS. Immunoelectron microscopic findings suggested that the adhesion molecules were LFA-1 on the PMNs and ICAM-1 on the MM46 tumor cells, but cell fusion between the PMNs and tumor cells was not observed. The MM46 tumor cells gradually lost their microvilli, which showed cell damage, and died 6-7 h after the addition of the N-CWS. This time course of tumor cell death is compatible with the results of the cytotoxic assay. Pretreatment of PMNs by anti-LFA-1 antibody suppressed 1% lysis of MM46 tumor cells from 90% to 10% (p < 0.01). These data suggest that adhesion molecule on the surface of PMNs such as LFA-1 might play an important role on signal transduction of these PMNs cytotoxic function in this experimental system.

Adoptive immunotherapy of poorly immunogenic tumors with in vitro sensitized cells generated by intratumoral administration of biological response modifiers.

We investigated the efficacy of intratumoral administration of biological response modifiers (BRM) in induction of in vitro sensitized (IVS) cells for adoptive immunotherapy of the poorly immunogenic MCA 102 sarcoma and B16-BL6(BL6) melanoma. We used the bacterial immunoadjuvant Nocardia rubra cell wall skeleton (N-CWS), and a streptococcal preparation, OK-432, for MCA 102 and BL6, respectively. After C57BL/6(B6) mice were inoculated subcutaneously (s.c.) with viable MCA 102 or BL6 tumor cells in the foot-pad, mice were injected intratumorally (i.t.) with N-CWS ranging from 10 to 400 micrograms or OK-432 ranging from 1 to 100 micrograms. Draining popliteal lymph nodes (LN) were harvested 7 days after i.t. administration of BRM, and LN cells were cultured with irradiated tumor cells in the presence of IL-2 for 11 days. These IVS cells (7.5 x 10(6) or 2 x 10(6)) were transferred intravenously (i.v.) to B6 mice with 4 day pulmonary metastases established by i.v. injection of viable MCA 102 cells (1 x 10(6)) or viable BL6 cells (3 x 10(5)). The mice were also received intraperitoneally 4 x 10(4) IU/day of IL-2 for 4 days after adoptive transfer. The transfer of IVS cells from mice immunized by i.t. injection of 100 micrograms of N-CWS 1 week after inoculation of tumor cells significantly reduced MCA 102 pulmonary metastases, compared with control IVS cells without administration of N-CWS. Moreover, the transfer of IVS cells from mice immunized by i.t. injection of 10 micrograms of OK-432 3 days after inoculation of tumor cells significantly reduced BL6 pulmonary metastases compared with control IVS cells without administration of OK-432. The administration of N-CWS resulted in no enhancement of in vitro cytotoxicity. Although the administration of 10 micrograms of OK-432 augmented in vitro cytotoxicity of IVS cells against BL6, cytotoxic activity was lower than that of IVS cells immunized with N-CWS. The major phenotype was CD8+ cells in IVS cells immunized with N-CWS or OK-432. These results suggest that i.t. administration of N-CWS and OK-432 facilitates the production of sensitized T-cells, and this administration route of BRM may be useful in the adoptive immunotherapy of human cancer.

[Suppressed increase of C3 receptors on polymorphonuclear leukocytes by stimulation with C5a in diabetes mellitus].

This study was undertaken to clarify the susceptibility to infection of patients with diabetes mellitus. The complement system is activated through the classical and/or the alternative pathways to produce many kinds of anaphylatoxins in the inflammatory process. One of the anaphylatoxins, C5a, possesses both the strong biological activity of a chemotactic factor and the increasing effect of CR1 and CR3 expression on polymorphonuclear leukocytes (PMNs). It is well known that the complement receptors, CR1 and CR3, on PMNs play important roles in the phagocytosis. We studied the changes of the expression of these receptors on PMNs in non-insulin dependent diabetes mellitus after the stimulation with recombinant human C5a. PMNs from 11 patients with non-insulin dependent diabetes mellitus and 11 normal controls were tested. There was no significant difference of CR1 and CR3 expression on PMNs after the addition of 10 ng/ml C5a between patients with diabetes mellitus and normal controls. However, by the stimulation with 50 ng/ml C5a, the increase of the CR3 expression on PMNs of patients with diabetes mellitus was significantly smaller than normal controls (p < 0.02). The increase of the expression of CR1 on PMNs was not significantly different the two groups. It is suggested that the small increase of the CR3 expression on PMNs by the stimulation with C5a is one of the factors of the susceptibility to infection in patients with diabetes mellitus.

PCR and immunoblot analyses of dystrophin in Becker muscular dystrophy.

The dystrophin gene was examined by PCR analysis in 30 Japanese patients with Becker muscular dystrophy (BMD). Fifteen PCR of these patients had exon deletion, generally, less than three exons. Muscle biopsies were also performed in 20 BMD patients (10 with sequence deletions, and 10 without detectable sequence deletions) in order to correlate PCR findings with immunoblot and immunostaining data. A patchy, heterogeneous membrane immunostaining pattern of reduced intensity was found, irrespective of the presence or absence of deletions. Immunoblotting studies demonstrated dystrophin of low molecular mass and quantity in BMD patients with deletion mutations, while a low quantity of dystrophin with an apparent wild type molecular mass was observed in nearly half the BMD patients without detectable deletions. However, these dystrophins were also found to have slightly abnormal molecular masses when the standard electrophoresis time was prolonged. This suggests that immunoblots and PCR data correlate well in patients with BMD. Additionally, it is suggested that immunoblot assays can detect abnormalities in dystrophin in the absence of detectable PCR deletions.

Recombinant human granulocyte colony-stimulating factor augments cytotoxicity of OK-432-induced polymorphonuclear leukocytes.

Polymorphonuclear leukocytes (PMNs) recovered from the peritoneal cavity of mice treated with the streptococcal preparation OK-432, exhibited strong cytotoxicity after the in vitro addition of Nocardia rubra cell wall skeleton (N-CWS). In this study, we investigated whether recombinant human granulocyte colony-stimulating factor (rhG-CSF) could augment the cytotoxicity of OK-432-induced PMNs after the addition of N-CWS in vitro. PMNs recovered from the peritoneal cavity of 8- to 10-week-old, male C3H/He mice induced by intraperitoneal (i.p.) injection of 50 KE/kg (1 KE = 0.1 mg) of OK-432 were used in a 51Cr release assay against MM46 mammary carcinoma cells. While addition of rhG-CSF in vitro did not augment the cytotoxicity of OK-432-induced PMNs, marked augmentation of the cytotoxicity of OK-432-induced PMNs was observed following a single subcutaneous (s.c.) or i.p. injection of 125 micrograms/kg of rhG-CSF. The effect of in vivo administered rhG-CSF was dependent on the timing of the injection with respect to OK-432 administration and differed from s.c. or i.p. injections. Interestingly, the cytotoxicity of OK-432-induced PMNs was rather weak following consecutive s.c. or i.p. administration of rhG-CSF for 7-14 days. H2O2 is likely involved in mediating the cytotoxicity of OK-432-induced PMNs since activity was significantly reduced by the in vitro addition of low concentration of catalase. Generation of H2O2 by the PMNs correlated with cytotoxicity. These results suggest that in vivo administration of rhG-CSF augments the cytotoxicity of OK-432-induced PMNs in a time dependent fashion and that H2O2 plays an important role in mediating their cytotoxicity.

Protein truncation test (PTT) for rapid detection of translation-terminating mutations.

Currently available techniques used to recognize point mutations in genetic disease are time consuming and are capable of screening only small pieces of DNA. Moreover, they detect all sequence differences including phenotypically silent changes. Consequently, they are not convenient to analyse mutations in large, multi-exonic genes, where a large fraction of pathological point mutations arises from early termination, as is the case for the one third non-deletion/duplication cases of Duchenne Muscular Dystrophy. We have developed a rapid and sensitive method, the Protein Truncation Test (PTT). PTT is based on a combination of RT-PCR, transcription and translation and selectively detects translation-terminating mutations. We demonstrate its effectiveness to detect point mutations in DMD-patients and carrier females. PTT should be widely applicable diagnostically in any disease where early terminations contribute substantially to the disease cause.

Selective IgM deficiency in a patient with Hashimoto's disease.

A 70-year-old man with Hashimoto's disease had selective IgM deficiency, while other immunoglobulin levels were normal. In vitro mixing experiments were carried out in which B cells and T cells from the patient and from a healthy control donor were co-cultured in the presence of pokeweed mitogen, in order to investigate the etiology of IgM hypoproduction. The results indicated that the patient had B-cell dysfunction, involving the impairment of B-cell differentiation. In addition, both IgG of the healthy control donor and thyroid hormone may play important roles in the pathogenesis of this case.

[A case of secondary myelodysplastic syndrome following chemotherapy for lung cancer].

A case of secondary myelodysplastic syndrome (MDS) following chemotherapy for lung cancer is reported. A 78-year-old man, with a smoking history of 20 cigarettes/day for 55 years, was incidentally, diagnosed as having stage IV squamous cell carcinoma of the lung in 1987 during admission for transurethral resection of bladder cancer. He received combination chemotherapy of mitomycin C, vincristin, and cisplatin for his lung cancer between July and September 1988. His clinical course remained almost stable until October 1989, when his blood count showed severe anemia and thrombocytopenia. He was diagnosed as having secondary MDS induced by cytotoxic agents used for the treatment of lung cancer, based on the dysplastic findings of precursor cells in the bone marrow and the chromosome abnormality of 51XY, +8, +9, +21, 3p-, 5q-, +2mar. He died of infection with the progression of MDS in March 1990.

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

Peritoneal polymorphonuclear 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 micrograms)/head of OK-432 or 200 micrograms/head of Nocardia rubra cell wall skeleton (N-CWS). Without in vitro stimulation, these PMNs did not show cytotoxicity to syngeneic MM46 mammary carcinoma cells in 51Cr release assay. Cytotoxicity of these PMNs was augmented by the addition of 25 micrograms/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 the in vitro addition of 25 micrograms/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.

Effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on cytotoxicity of PSK-induced peritoneal polymorphonuclear leukocytes (PMNs).

We investigated whether recombinant human granulocyte colony-stimulating factor (rhG-CSF) enhanced the cytotoxicity of PSK-induced polymorphonuclear leukocytes (PMNs) in the peritoneal cavity. Male C3H/He mice, 8- to 10-week-old, received single subcutaneous (s.c.) or intraperitoneal (i.p.) injection of 2.5 micrograms/animal of rhG-CSF at different time points before or after an i.p. administration of PSK. In other experiments, mice were s.c. or i.p. treated with the same dosage of rhG-CSF every day for 7 or 14 consecutive days and i.p. injected with 2.5 mg/animal of PSK on the last day. Peritoneal PMNs were harvested 6 hrs after the administration of PSK and purified to more than 95% by Ficoll-Paque for in vitro cytotoxic assay. In vitro cytotoxic assays with 51Cr labeled MM46 mammary carcinoma cells were added with 5-20 micrograms/ml of Nocardia rubra cell wall skeleton (N-CWS) at the beginning of the assay to augment the cytotoxic activity of PMNs. In vitro addition of rhG-CSF to the assay did not enhance the cytotoxicity of PSK-induced PMNs. However, the cytotoxicity was significantly increased when rhG-CSF was s.c. administered 12 hrs before a PSK injection or 2 or 5 hrs after that. On the other hand, the cytotoxicity was rather weak when mice s.c. or i.p. received consecutive injections of rhG-CSF. This cytotoxicity may be mediated by H2O2, since H2O2 production of PMNs during the cytotoxic assay appears to correlate with the levels of cytotoxicity under suppressed H2O2 generation by catalase or enhanced generation by rhG-CSF. These results suggest that rhG-CSF augments the cytotoxicity of PSK-induced PMNs when administered in vivo timely.

[Role of oxygen species and lipid peroxidation for anti-tumor effect of intra-arterial injection with degradable starch microspheres].

The role of superoxide and lipid peroxidation for anti-tumor effect of intra-arterial injection with degradable starch microspheres (DSM) was investigated in rabbits. The anti-tumor effect of intra-arterial injection with DSM was studied in rabbits with VX2 carcinoma of the hind leg. The tumor growth in rabbit treated with DSM 5 times was completely suppressed, and thiobarbituric acid (TBA)-reactive substances in the tumor tissue treated with DSM were significantly increased. But the anti-tumor effect of DSM and the increase in TBA reactive substances in the tumor tissue treated with DSM were significantly inhibited by treatment with superoxide dismutase combined with catalase. These results suggest that the anti-tumor effect of intra-arterial injection with DSM may be due to ischemia-reperfusion injury and that active oxygen species and lipid peroxidation may play an important role in the anti-tumor effect of intra-arterial injection with DSM.