Residual carcinoma cells after chemoradiotherapy for esophageal squamous cell carcinoma patients: striving toward appropriate judgment of biopsy.

In esophageal squamous cell carcinoma (ESCC) patients who are treated with chemoradiotherapy (CRT), identification of the presence or absence of residual or recurrent carcinoma is usually pivotal in their clinical management. In addition, the extent of carcinoma invasion into the esophageal wall could determine the clinical outcome of these patients following CRT. Therefore, in this study, we evaluated the response to CRT both macroscopically and histologically in a consecutive series of 42 ESCC patients receiving neoadjuvant chemoradiotherapy following curative esophageal resection at Tohoku University Hospital between August 2011 and December 2012. The histological grading of tumor regression was as follows: grade 3, markedly effective (no viable residual tumor cells); grade 2, moderately effective (residual tumor cells in less than one-third of the tumor); grade 1, slightly effective (1b, residual tumor cells in one-third to two-thirds of the tumor; 1a, residual tumor cells in more than two-thirds of the tumor); and grade 0, ineffective. In this study, we selected grade 2 and 1b cases because they might show a complete response with definitive CRT. We evaluated the presence of any residual in situ lesions and tumor depth in detail. The grading of tumor regression in primary sites was as follows: grade 3 (7 cases), grade 2 (16 cases), grade 1b (13 cases), and grade 1a (6 cases). The concordance rate between macroscopic and histopathological evaluation on the depth of the tumor was 40% (17/42). Among 29 cases (grade 2 and grade 1b), intraepithelial lesions were not detected in 17 cases, and tumor nests were not detected in the lamina propria mucosae in 9 cases. The results of this study highlight the difficulties of detecting residual carcinoma cells using conventional endoscopic biopsy in patients who have received CRT. Therefore, when residual cancer is clinically suspected in patients who have received CRT, the biopsy specimen should be obtained from the deep layer of the esophagus whenever possible. Additionally, close follow-up is required using positron emission tomography/computed tomography, endoscopy, and other radiological evaluations.

Dehydroxymethylepoxyquinomicin (DHMEQ) can suppress tumour necrosis factor-α production in lipopolysaccharide-injected mice, resulting in rescuing mice from death in vivo.

Dehydroxymethylepoxyquinomicin (DHMEQ), a new nuclear factor (NF)-κB inhibitor, has several beneficial effects, including the suppression of tumour growth and anti-inflammatory effects. DHMEQ can also suppress the production of tumour necrosis factor (TNF)-α induced by lipopolysaccharide (LPS) in vitro. In the present study, we examine the effects of DHMEQ on TNF-α production in vivo and on the survival of mice injected with LPS. When DHMEQ was injected into mice 2 h before LPS injection, the survival of the LPS-injected mice was prolonged. When DHMEQ was injected twice (2 h before LPS injection and the day after LPS injection), all the mice were rescued. The injection of DHMEQ 1 h after LPS injection and the day after LPS injection also resulted in the rescue of all mice. The serum levels of TNF-α in the mice that received both LPS and DHMEQ were suppressed compared to the mice that received only LPS. These results suggest that DHMEQ can be utilized for the prevention and treatment of endotoxin shock.

A quantitative assay of oxidative metabolism by neutrophils in whole blood using flow cytometry.

A large number of purified and washed PMNLs are required to monitor generation of active oxygen derivatives in most in vitro studies and this can preclude investigations in small children. The present method has enabled us to measure the oxidative burst (generation of hydrogen peroxide) of PMNLs in a small amount of whole blood using 2',7'-dichlorofluorescein diacetate, phorbol myristate acetate and flow cytometry. Optimal conditions for this determination were evaluated and the reaction was found to be independent of the absolute numbers of PMNLs and other types of cell in whole blood. The present method will be of value in investigations of the leukocyte metabolism of patients not only with chronic granulomatous disease (CGD) but also with various infectious diseases.

FcgammaRIII b and FcgammaRIIa polymorphism may affect the production of specific NA1 autoantibody and clinical course of autoimmune neutropenia of infancy.

We studied 18 children with autoimmune neutropenia (AIN) to evaluate whether there was a possible relationship between the specificity of granulocyte autoantibodies (anti-NA1,2) and the phenotype of the NA system. Direct granulocyte immunofluorescence test (D-GIFT) was positive in all patients, and indirect granulocyte immunofluorescence test (I-GIFT) was positive in 17 of these 18 patients, respectively. Fourteen of 18 patients showed preferential binding to neutrophils from NA(1+2-) phenotyped donors. Immunoblotting with anti-FcgammaRIIImAb showed that IgG prepared from 7 of 12 patients precipitated both FcgammaRIIIb from NA1 and NA2 neutrophil lysate, whereas the other 5 precipitated only NA1. Patients' IgG did not react with purified FcgammaRIIa. FcgammaRIIIb genotype were NA(1+2-) in 15 of 18 patients and NA(1+2+) in the other 3. FcgammaRIIa type of all patients were (H+R-). These distributions were significantly different from those of healthy Japanese blood donors (n = 608). The genotype of FcgammaRIIIb and FcgammaRIIa may affect the production of neutrophil specific auto-antibodies in AIN of infancy and influence its clinical course.

Serum eosinophil derived neurotoxin may reflect more strongly disease severity in childhood atopic dermatitis than eosinophil cationic protein.

Serum levels of eosinophil cationic protein (ECP) have been shown to be a good parameter of the disease severity of patients with atopic dermatitis (AD). However, the relationship between the disease severity and the eosinophil derived neurotoxin (EDN) has not been established in AD patients. The purpose of this study is to examine serum ECP and EDN levels in relation to the disease severity in AD children. Serum ECP and EDN levels were assessed in relation to the skin scores in 34 AD children (18 boys and 16 girls; age 0.6 to 7years: mean+/-S.D. 2.2+/-1.9) and six non-atopic control children (three boys and three girls; age 1 to 3years: mean+/-S.D. 1.7+/-0.9). Serum ECP and EDN levels of the patients with AD were significantly increased compared with the non-atopic controls. Serum EDN levels of the patients were also related to the disease severity. The skin scores were more significantly correlated with serum EDN levels than ECP levels. We concluded that serum EDN may reflect more strongly disease severity as eosinophilic activation in AD children than serum ECP.

Failure of the usual anti-Ig antibody method for quantitative measurement of low affinity antibodies.

The usual anti-Ig antibody method, consisting of the precipitation of soluble antigen-antibody complexes by heterologous anti-Ig antibody, was applied for quantitative estimation of guinea pig IgG2 anti-ovalbumin and anti-2,4-dinitrophenyl (DNP) antibodies by measuring the maximum amounts of antibody-bound antigens. However, the amounts of antibodies estimated were less than those obtained by other methods: the precipitin reaction, the precipitation of antigen-antibody complexes with 50% saturated ammonium sulfate, and equilibrium dialysis. In particular, the anti-Ig antibody method greatly underestimated the amount of anti-DNP antibody with low affinity for epsilon-DNP-L-lysine. Thus, it was concluded that partial dissociation of the antigen-antibody complexes occurring upon precipitation with anti-Ig antibody made the anti-Ig antibody method unsuitable for quantitative determination of antibodies.

Proteolytic cleavage of an activated subcomponent of the first component of rabbit complement, C1s.

When rabbit C1 purified by affinity chromatography on IgG-Sepharose 6B was chromatographed on DEAE-cellulose in the presence of ethylenediaminetetraacetate, C1s was isolated as two forms, C1s(I) and C1s(II), having different molecular weights. On the other hand, incubation of the C1 with soybean trypsin inhibitor before the chromatography resulted in the isolation of C1s(I) alone, indicating that, during the purification, C1s(II) was derived from C1s(I) by proteolytic cleavage of C1s(I) by a contaminating protease, probably plasmin [EC 3.4.21.7]. In fact, C1s(I) was completely converted to C1s(II) or a C1s(II)-like fragment by highly purified plasmin. Analysis of the polypeptide chain structures revealed that C1s(I), which consisted of H and L chains with molecular weights of 70,000 and 36,000, respectively, was converted to C1s(II) by cleavage of the H chain, since C1s(II) consisted of two chains each with a molecular weight of 37,000. This conversion proceeded without any alteration in C1 esterase activity, but was accompanied by loss of the ability to form C1r-C1s complex.

Transdiaphragmatic duodenal duplication in a premature infant.

A case of transdiaphragmatic duodenal duplication in a premature infant is presented. Vertebral abnormalities, which have invariably accompanied this disorder in reported cases so far, were characteristically absent. Therefore, the initial tentative diagnosis was right-sided diaphragmatic hernia. Barium examination was helpful in preoperative diagnosis.

[Clinical evaluation of a new parenteral cephem, cefozopran, in children].

Cefozopran (CZOP, SCE-2787), a new parenteral cephem, was evaluated for its antibacterial activity and clinical efficacy. CZOP, 24.0-78.0 mg/kg/day, was given to 11 pediatric patients in 3 dose a day via 30-minute drip infusion. Clinically evaluated were nine patients including 4 with acute pneumonia, 2 with urinary tract infections, 2 with lymphadenitis and 1 with sepsis. Two patients were excluded because of possible non-bacterial infections. Clinical efficacies were excellent in 5, good in 3 and fair in 1. Bacteriological responses were confirmed for 5 strains in 5 patients. Four strains were eradicated, but one strain was not. MICs of CZOP were equal to those of ceftazidime. Side effects or abnormal laboratory test results were observed in 3 patients; diarrhea in 1, elevated GPT in 1 and thrombocytosis in 1, but none of them was significant.

[Intestinal flora of infants with cow milk hypersensitivity fed on casein-hydrolyzed formula supplemented raffinose].

We studied the intestinal flora of infants with cow milk hypersensitivity fed on casein-hydrolyzed formula (MA-1) and the influence of that supplemented with Raffinose (MA-1[R]). Infants with cow milk hypersensitivity were fed with MA-1 for 2 weeks, after which the formula was changed to MA-1[R]. Fourteen subjects were enrolled in this study and divided into two groups; three who fed with breast or conventional milk in addition to MA-1 or MA-1[R](BM group) and 11 mainly fed with MA-1 or MA-1[R] (TF group). Intestinal flora was investigated at two weeks after MA-1 feeding and at two weeks after MA-1[R] feeding, respectively. Bifidobacterium was detected as the most predominant bacteria in all examples in the BM group, and that count and the ratio in all bacteria remained high even after changing MA-1 to MA-1[R]. On the other hand, bacteria count and ratios of Bifidobacterium in all bacteria were conspicuously low in the TF group as compared with the BM group. And with the change from MA-1 to MA-1[R] in the TF group, the bacterial number and the occupation ratio of Bifidobacterium were increased, and Enterobacteriaceae bacterial count and the occupation ratio were decreased. The change of the intestinal flora with MA-1[R] feeding was mainly caused by the breeding action of Raffinose on bifidobacteria. Further studies are needed from a viewpoint of clinical effectiveness about the influence of normalization of the intestinal flora for the treatment of food hypersensitivity.

[Genetic analysis of two patients with adenosine deaminase (ADA)-deficiency: detection of three novel mutations and characterization of two paradoxical carriers].

Genetic basis of two patients (AT, MT) with ADA deficiency was studied. We identified three novel mutations (119 Q-->Stop, 235 R-->Q, one base deletion in Exon 4) from the patients. 119 Q-->Stop was detected in AT and her father. Deletion of one base in Exon 4 which would change the reading frame after codon 105 H, was detected in MT, her father and brother. There was no relation between the two families, however, 235 R-->Q was also detected in both the patients and their mothers. Extremely low ADA activity of PBMCs was revealed in healthy MT's mother and brother just as MT, although their dAXP levels of RBCs showed significantly lower than that of MT. We defined that they shared an additional mutation (310 M-->T) together with the mutation described above, respectively. EBV-transformed B-cell line (EBV-B) were established from the carriers. To our surprise, ADA activity of their lines was 1/10-1/5 of normal. The result of heat treatment studies using the EBV-B showed that the mutant ADA rapidly lose its enzyme activity without degradation of the protein. It suggests that 310 M-->T mutant ADA rapidly lost its enzyme activity due to conformational change of the catalytic site of ADA.

[Effect of dexamethasone on osteoblastic cells derived from mouse calvaria].

In the present study, the effect of dexamethasone on MC3T3-E1 cells, a strain of osteoblasts derived from mouse cranial bone, was determined. The following results were obtained. 1) Dexamethasone showed dose-dependent suppression of the growth of MC3T3-E1 cells at concentration of 1 microgram/ml or more. 2) The alkaline phosphatase activity was increased 12, 24, and 48 hours after treatment with dexamethasone at 1, 10 or 30 micrograms/ml. The activity was highest at 48 hours, the level being 311% of the control value at a dose of 10 micrograms/ml. When dexamethasone at a dose of 60 micrograms/ml or more was used, the activity was increased at 12 hours, but was lower than the control at 48 hours. 3) Synthesis of collagenous protein was facilitated after 24-hour treatment with dexamethasone at 1, 10 or 30 micrograms/ml. In particular, the level of synthesis was highest, 232% of the control value, at 10 micrograms/ml. Such synthesis, however, was suppressed at a dose of 60 micrograms/ml or more. 4) Synthesis of collagenous protein was facilitated by 48-hour treatment with dexamethasone at a dose of 1 or 10 micrograms/ml and suppressed at a dose of 30 micrograms/ml or more. 5) Microscopic observation of stained preparations revealed that dexamethasone caused vacuolar degeneration, deep staining of the nucleus, and pyknosis at 60, 150, and 200 micrograms/ml, respectively.

Isolation of functional polymorphonuclear leukocytes from rhesus monkeys using discontinuous Ficoll-Hypaque density gradients.

A simple, rapid method for separating polymorphonuclear leukocytes (PMNs) from adult rhesus monkey blood, based on the use of a discontinuous gradient of Ficoll-Hypaque (densities, 1.100, 1.077) has been developed. Using this method, 71.1% of the PMNS were recovered in a layer with a purity of 91% PMNs with very few contaminating erythrocytes. The added advantage of the method described here is that a higher chemotactic activity was retained.

Cartilage-hair hypoplasia associated with IgG2 deficiency.

We report on a 1 year old boy with cartilage-hair hypoplasia (CHH). He suffered from recurrent upper respiratory infections and short-limbed dwarfism. As with most patients with CHH, he had impaired cellular immunity as determined by lymphocyte reactivity. In addition, he had a selective IgG2 deficiency. This combination of immunodeficiencies has not previously been reported for patients with CHH. His recurrent upper respiratory infections were likely to be associated with cellular immunodeficiency and IgG2 deficiency.

Propofol depressed neutrophil hydrogen peroxide production more than midazolam, whereas adhesion molecule expression was minimally affected by both anesthetics in rats with abdominal sepsis.

The treatment of sepsis may require mechanical ventilation of the lungs and sedation. Because neutrophils are the most important effector cells for protecting against sepsis, and propofol and midazolam are the most widely used anesthetics for sedation, we studied the effects of these two anesthetics on the neutrophil function during sepsis. Sepsis was induced in rats by cecal ligation and puncture. At either 4 h or 24 h after cecal ligation and puncture, blood and peritoneal neutrophils were obtained, incubated with the test anesthetics, and the hydrogen peroxide (H(2)O(2)) production and CD11b/c expression were determined by flow cytometry. In both early (at 4 h) and late (at 24 h) sepsis, propofol and midazolam depressed H(2)O(2) production by blood and peritoneal neutrophils at clinical concentrations. Propofol caused more depression than midazolam (P < 0.005). In both early and late sepsis, the effect of the anesthetics on the up-regulation of the stimulation-induced CD11b/c expression on blood neutrophils was minimal at clinical concentrations. If these results ultimately become clinically relevant, midazolam may be preferable to propofol for sedation during sepsis.

Role of reactive oxygen species in neutrophil apoptosis following ingestion of heat-killed Staphylococcus aureus.

Neutrophils, short-lived leucocytes that die by apoptosis, play an important role in the first stage of defense against bacterial infections. It has been reported that phagocytosis of intact bacteria or Candida albicans can accelerate neutrophil apoptosis. However, the mechanism of phagocytosis-mediated neutrophil apoptosis is not well characterized. In this study, we evaluated whether ingestion of heat-killed Staphylococcus aureus (S. aureus) enhances neutrophil apoptosis and whether this type of apoptosis is mediated by oxidative stress by using antioxidants and polymorphonuclear leucocytes (PMNs) from patients with chronic granulomatous disease (CGD). Co-culture of PMNs with varying doses of S. aureus resulted in accelerated PMN death in a dose- and time-dependent manner. Increased PMN apoptosis was observed by both Annexin V and PI staining. Similar results were observed in PMNs of CGD patients. Dimethyl sulphoxide (DMSO, an OH* scavenger) did not significantly inhibit either S. aureus-ingested PMN apoptosis or spontaneous PMN apoptosis. On the other hand glutathione (GSH, an H2O2 scavenger) significantly inhibited both types of apoptosis. Our findings suggest that oxygen-independent pathways may mainly operate in the process of phagocytosis-induced apoptosis.