A strategy for efficient cross-presentation of CTL-epitope peptides leading to enhanced induction of in vivo tumor immunity.

The activation of antitumor cytotoxic T-lymphocytes (CTLs) depends on how efficiently the relevant tumor antigen peptides are delivered into the major histocompatibility complex (MHC) class I presentation pathway in antigen presenting cells (APCs). An elegant approach to promote the peptide-MHC class I association has been described for enhanced peptide transportation into the endoplasmic reticulum (ER) by adding an ER insertion signal sequence (Eriss). Nevertheless, this approach does not appear potent enough to induce in vivo tumor protective immunity. Herein, we present a novel peptide-vaccine strategy based on the combined utilization of Eriss and fusogenic liposomes (FLs) capable of directly introducing encapsulated CTL-epitope peptides into the MHC class I pathway of APCs. APCs pulsed with free peptides, FL-encapsulated peptides, or FL-encapsulated Eriss-conjugated peptides exhibited comparable levels of antigen-presenting activity at early phases after pulsing. Interestingly, whereas in the first two methods the APC ability began to decline 40 to 60 h after pulsing, FL-encapsulated Eriss(+) peptides allowed APCs to retain peptide-presentation activity for at least 140 h. This advantage of FL-encapsulated Eriss(+) peptides correlated with the induction of more potent antitumor immunity compared with soluble Eriss(+) or Eriss(-) peptides or FL-encapsulated Eriss(-) peptides when they were administered in vivo. Thus, Eriss-conjugated CTL-epitope peptides encapsulated in FLs provide a highly efficient tumor-vaccine to enhance the induction of in vivo tumor immunity.     

Deficiency in systemic interleukin-1 receptor antagonist production as an operative risk factor in malnourished elderly patients with colorectal carcinoma

OBJECTIVE: Aging and tumor-related malnutrition are associated with increased inflammatory cytokine levels. However, it is unclear whether this influences the outcomes of surgery. We explored the relationships between cytokines and surgical complications among patients undergoing surgery for colorectal cancer. DESIGN: Laboratory experiment. SETTING: Surgery department of school of medicine. PATIENTS: One hundred patients with colorectal cancer. INTERVENTIONS: The perioperative circulating levels of interleukin (IL)-1beta, IL-6, and IL-1 receptor antagonist (Ra) were determined, and the numbers of circulating lymphocytes and neutrophils were counted. MEASUREMENTS AND MAIN RESULTS: Trends toward increasing postoperative infection were observed among patients who were older and had lower body mass index. Preoperative IL-1Ra and intraoperative blood loss, however, remained the only two independent predictors of postoperative infection. Clinically, patients with low preoperative IL-1Ra most frequently were the elderly with low body mass index. Postoperatively, elderly patients with low body mass index showed an exaggerated IL-6 response, followed by an exaggerated postoperative inflammatory response and increased postoperative loss of body weight. In contrast, normal immunoreactivity was preserved in well-nourished elderly patients. CONCLUSIONS: In colorectal cancer patients undergoing surgery, low preoperative IL-1Ra is associated with postoperative infection. In our patient population, lower IL-1Ra level is commonly observed in the elderly with low body mass index. These findings suggest that postoperative infection, frequently seen in the nutritionally deficient elderly, may be the result of defective immunoinflammatory adaptation system.     

A novel polymorphism of human CYP2A6 gene CYP2A6*17 has an amino acid substitution (V365M) that decreases enzymatic activity in vitro and in vivo

Cytochrome P450 (CYP) 2A6 is a major CYP responsible for the metabolism of nicotine and coumarin in humans. We identified a novel allele, designated CYP2A6*17 , which contains A51G (exon 1), C209T (intron 1), G1779A (exon 3), C4489T (intron 6), G5065A (V365M, exon 7), G5163A (intron 7), C5717T (exon 8), and A5825G (intron 8). We developed a genotyping method by polymerase chain reaction-restriction fragment length polymorphism for the CYP2A6*17 allele, targeting the G5065A mutation. The allele frequency in black subjects (n = 96) was 9.4% (95% confidence interval [CI], 5.3%-13.5%). The allele was not found in white subjects (95% CI, 0%-0.9%; n = 163), Japanese subjects (95% CI, 0%-1.6%; n = 92), and Korean subjects (95% CI, 0%-0.7%; n = 209). To examine the effects of the amino acid change in the CYP2A6*17 allele on the enzymatic activity, we expressed a wild-type or variant (V365M) CYP2A6 together with NADPH-CYP reductase in Escherichia coli . For coumarin 7-hydroxylation, the apparent Michaelis-Menten constant value of variant CYP2A6 (1.06 +/- 0.11 micromol/L) was significantly (P < .005) higher than that of wild type (0.60 +/- 0.05 micromol/L). The maximum velocity values of the wild-type and variant CYP2A6 were 0.61 +/- 0.06 and 0.64 +/- 0.07 pmol . min -1 . pmol -1 CYP, respectively. For nicotine C -oxidation, the apparent Michaelis-Menten constant values of the wild-type or variant CYP2A6 were 31.6 +/- 2.9 micromol/L and 31.3 +/- 3.1 micromol/L, respectively. The maximum velocity value of variant CYP2A6 (0.72 +/- 0.21 pmol . min -1 . pmol -1 CYP) was significantly (P < .05) lower than that of the wild type (1.80 +/- 0.42 pmol . min -1 . pmol -1 CYP). Thus the intrinsic clearance values for coumarin 7-hydroxylation and nicotine C -oxidation by the variant were both significantly (P < .05) decreased to 40% to 60% compared with the wild type. Furthermore, cotinine/nicotine ratios after 1 piece of nicotine gum was chewed, used as an index of in vivo nicotine metabolism, were significantly (P < .05) decreased in heterozygotes of the CYP2A6*17 allele (5.4 +/- 2.7, n = 12) compared with homozygotes of the wild type (11.5 +/- 10.5, n = 37). A subject with CYP2A6*17 / CYP2A6*17 revealed the lowest cotinine/nicotine ratio (1.8). We found a novel allele in black subjects that affects the nicotine metabolism in vitro and in vivo.     

Successful treatment of ruptured hepatocellular carcinoma with intraperitoneal injection of OK-432

We report a 51-year-old man with a ruptured hepatocellular carcinoma (HCC). He was admitted to the hospital with abdominal pain and distension. Imaging studies revealed massive ascites, liver cirrhosis, and a 3-cm tumor at the inferior edge of the medial segment of the liver, with adhesions to the greater omentum. Abdominal paracentesis showed bloody ascites, and the patient was diagnosed with a ruptured HCC. OK-432, an immunomodulatory agent prepared from an attenuated strain of Streptococcus pyogenes, was injected (10?KE) into the peritoneal cavity four times within 1 week; the massive ascites disappeared, and the serum alpha-fetoprotein (AFP) level decreased to within the normal limits. Afterwards, he underwent a curative operation for HCC. His postoperative course was uneventful and he was discharged from the hospital on the twenty-second postoperative day. He had shown no evidence of recurrence or metastases at the time he died of hepatic failure related to alcohol abuse 9 months after the operation.     

Effect of intravenous or intra-amniotic injection of dehydroepiandrosterone sulphate on oestrogen levels in urine and serum in late pregnancy with live anencephalic foetuses.

Dehydroepiandrosterone sulphate (DHAS) was injected intravenously or intra-amniotically into eight volunteers carrying live anencephalic foetuses (including one microcephalic foetus). Urinary and unconjugated serum oestrone, oestradiol and oestriol were measured before and after DHAS administration. In seven pregnant women with live anencephalic foetuses the urinary excretion of oestriol was very low, and the ratio of oestriol to oestrone + oestradiol was much less than that during normal pregnancy. Increases of urinary oestrone and oestradiol but no significant change in the ratio of oestriol to oestrone + oestradiol were observed 24 h after i.v. administration of DHAS to five patients. In three patients, between 1 and 12 h after i.v. administration of DHAS (100-200 mg), the concentrations of serum oestrone, oestradiol and oestriol increased to 13-5, 6-8 and 3-1 times the control values, respectively. After injection of DHAS (200 mg) intra-amniotically into two patients, the urinary excretion of all three oestrogens increased much more on day 2 than on day 1, and the ratio of urinary oestriol to oestrone + oestradiol rose greatly. On the other hand, the concentrations of unconjugated serum oestrogens in these patients increased progressively between 1 and 12 h or more after DHAS administration, and the maximal level of serum oestriol was 9-8 times the control value while those of oestrone and oestradiol were 4-6 times and 5-0 times the control values, respectively. These results suggest that in late human pregnancy DHAS in the circulation of the mother is converted to oestriol largely via the phenolic pathway (DHAS leads to oestrone leads to oestriol), whereas DHAS circulating within the foeto-placental compartment is converted to oestriol via both the phenolic and the neutral intermediates.     

Analysis of chronic heart failure registry in the Tohoku district: third year follow-up.

BACKGROUND: Because the real prognosis of Japanese patients with chronic heart failure (CHF) is still unknown, the aim of the present study was to clarify the prognosis and predictors for mortality of CHF patients using the Chronic Heart failure Analysis and Registry in Tohoku district (CHART). METHODS AND RESULTS: As of February 2003, 1,154 stable CHF patients with optimum standard therapy have been enrolled in the registry since February 2000 and of these, 175 died of some cause during the follow-up period (mean follow-up period, 1.9+/-0.9 years), giving 1- and 3-year all-cause mortality rates of 7.3% and 20.9%, respectively. Multivariate analysis showed that age at entry, diabetes, ventricular tachycardia, plasma concentration of brain natriuretic peptide (BNP), New York Heart Association (NYHA) functional class and rural residence were significantly associated with all-cause mortality (hazard ratio: 1.028, 1.940, 1.650, 1.001, 1.713, and 2.226). The 1-year all-cause mortality rates of CHF patients with a left ventricular ejection fraction <25%, left ventricular end-diastolic diameter > or =60 mm, BNP > or =500 pg/ml, NYHA > or = III, or with underlying coronary artery disease were 15.0%, 11.4%, 16.8%, 16.3%, and 10.8%, respectively. CONCLUSIONS: The prognosis of Japanese CHF patients with these predictors remains poor. A stratified approach is necessary to improve their survival and quality of life.     

Paradoxical regional myocardial uptake between 201Thallium and 123I-BMIPP SPECT in patients with cardiomyopathy

 The clinical significance of the paradoxical mismatched phenomenon between ²⁰¹Tl and ¹²³I-BMIPP is still unknown. We report two cases that revealed paradoxical regional myocardial uptake between two tracers in patients with cardiomyopathy. There may be abnormal myocardium in these patients where active transportation of ²⁰¹Tl is disturbed and passive transportation of ¹²³I-BMIPP is not disordered. Received: July 31, 2002 / Accepted: September 21, 2002 Correspondence to K. Ueshima     

Classifying genotype F of hepatitis B virus into F1 and F2 subtypes

AIM: To explore the propriety of providing hepatitis B virus (HBV) genotypes F and H with two distinct genotypes. METHODS: Eleven HBV isolates of genotype F (HBV/F) were recovered from patients living in San Francisco, Japan, Panama, and Venezuela, and their full-length sequences were determined. Phylogenetic analysis was carried out among them along with HBV isolates previously reported. RESULTS: Seven of them clustered with reported HBV/F isolates in the phylogenetic tree constructed on the entire genomic sequence. The remaining four flocked on another branch along with three HBV isolates formerly reported as genotype H. These seven HBV isolates, including the four in this study and the three reported, had a sequence divergence of 7.3-9.5% from the other HBV/F isolates, and differed by > 13.7% from HBV isolates of the other six genotypes (A-E and G). Based on a marked genomic divergence, falling just short of > 8% separating the seven genotypes, these seven HBV/F isolates were classified into F2 subtype and the former seven into F1 subtype provisionally. In a pairwise comparison of the S-gene sequences among the 7 HBV/F2 isolates and against 47 HBV/F1 isolates as well as 136 representing the other six genotypes (A-E and G), two clusters separated by distinct genetic distances emerged. CONCLUSION: Based on these analyses, classifying HBV/F isolates into two subtypes (F1 and F2) would be more appropriate than providing them with two distinct genotypes (F and H).     

PBC-AIH overlap syndrome with concomitant ITP and Hashimoto’s disease with positivity for anti-centromere antibody

We report a case of primary biliary cirrhosis (PBC)-autoimmune hepatitis (AIH) overlap syndrome with concurrent idiopathic thrombocytopenic purpura (ITP) and Hashimotos disease with positivity for anticentromere antibody. The patient was a 64-year-old woman with symptoms of jaundice and general fatigue. About 30 years earlier, she had been diagnosed as having ITP and had undergone splenectomy. As part of her present history, she had exhibited liver dysfunction in 1995, during the follow-up of Hashimotos disease, and a liver biopsy led to the diagnosis of PBC. In March 2000, she was admitted to hospital because of general fatigue and jaundice. Blood tests revealed: total protein (TP), 6.6g/dl; -globulin (glb), 35.9%; total bilirubin (T-bil), 9.41mg/dl; direct bilirubin (D-bil), 7.52mg/dl; aspartate aminotransferase (AST), 957U/l; alanine aminotransferase (ALT), 651U/l; alkaline phosphatase (ALP), 595U/l; -guanosine triphosphate (GTP), 129U/l; IgG, 2620mg/dl; IgM, 223mg/dl; hepatitis B surface antigen (HBsAg), negative; anti-hepatitis C virus (HCV), negative; antinuclear antibody, positive; antimitchondrial antibody (AMA), negative (by the immunofluorescence [IF] method); and anti-pyruvate dehydrogenase complex (PDC)-E2 antibody, positive (by Western blotting). Anticentromere antibody (ACA), which is an alternative diagnostic marker for PBC, was detected in this patient. Prednisolone was administered after admission and liver function test results improved markedly. The liver biopsy in 1995 had revealed infiltration of lymphocytes and plasma cells in the portal areas with fibrous expansion and periportal necrosis. Destructive cholangitis was observed, as well as scattered epitheloid cell granulomas in some portal areas. Liver biopsy after the steroid treatment revealed alleviated necrotic inflammatory responses of hepatocytes, while the destructive cholangitis persisted. This is a very rare case of PBC-AIH overlap syndrome accompanied by ITP and Hashimotos disease which provides a possible insight into the mechanisms and interplay of autoimmune diseases.