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.

Differential diagnosis and clinical course of autoimmune neutropenia in infancy: comparison with congenital neutropenia.

AIM: Autoimmune neutropenia in infancy (AIN) is caused by granulocyte-specific autoantibodies. Clinical presentation and diagnosis have not been well studied, resulting in cumbersome diagnostic investigations and unnecessary treatment such as granulocyte colony-stimulating factor (G-CSF) therapy. METHODS: Clinical, laboratory and immunological data of 18 infants with AIN were evaluated. Granulocyte-specific autoantibodies were detected by the direct granulocyte immunofluorescence test (D-GIFT), indirect granulocyte immunofluorescence test (I-GIFT) and immunoblotting. RESULTS: The average age of onset and resolution of neutropenia in AIN was 7.4 +/- 3.4 mo (mean +/- SD) and 20.4 +/- 4.9 mo, respectively. Sixteen of the 18 patients presented with mild infectious symptoms; the other 2 patients were detected by chance and presented with no infectious symptoms. D-GIFT was positive in all patients, and I-GIFT was positive in 17 of these 18 patients. Most patients showed preferential binding to neutrophils from NA(1 + 2-)-phenotyped donors by 1-GIFT and immunoblotting. An antibiotic (sulfamethoxazole-trimethoprim) was given to 15 patients for prophylaxis. G-CSF was given to only one infectious patient. CONCLUSION: A combination of diagnostic tests for the detection of granulocyte-specific autoantibodies was useful in diagnosing AIN, thus avoiding unnecessary investigations. Continuous treatment with G-CSF was not necessary for prophylaxis, even if neutrophil counts were extremely low.

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.

Increased serum nitrate levels in infants with atopic dermatitis.

BACKGROUND: The pathogenesis of atopic dermatitis (AD) is still unknown. A recent study has shown that inducible nitric oxide synthase (iNOS) is expressed in the atopic skin lesion, suggesting the involvement of nitric oxide in the skin inflammation of AD. The purpose of the study was to examine serum nitrate (NO3) levels in relation to the disease severity in children with AD. METHODS: Serum nitrate levels were assessed in relation to the skin scores in 88 patients with atopic dermatitis (AD) (aged 0.4-8 years: mean+/-SD, 2.2+/-1.9, 41 boys and 47 girls) and 12 nonatopic children (aged 0.8-4 years: mean+/-SD, 1.8+/-0.9, seven boys and five girls). RESULTS: Serum nitrate levels of patients with AD were significantly increased as compared to nonatopic controls and were also correlated with the disease severity. The skin scores were significantly correlated with serum nitrate levels as well as peripheral eosinophil counts. CONCLUSION: Our results indicate that nitric oxide may be involved in the pathogenesis of vasodilation and erythema in AD skin.

T-cell lines from 2 patients with adenosine deaminase (ADA) deficiency showed the restoration of ADA activity resulted from the reversion of an inherited mutation.

Inherited deficiency of adenosine deaminase (ADA) results in one of the autosomal recessive forms of severe combined immunodeficiency. This report discusses 2 patients with ADA deficiency from different families, in whom a possible reverse mutation had occurred. The novel mutations were identified in the ADA gene from the patients, and both their parents were revealed to be carriers. Unexpectedly, established patient T-cell lines, not B-cell lines, showed half-normal levels of ADA enzyme activity. Reevaluation of the mutations in these T-cell lines indicated that one of the inherited ADA gene mutations was reverted in both patients. At least one of the patients seemed to possess the revertant cells in vivo; however, the mutant cells might have overcome the revertant after receiving ADA enzyme replacement therapy. These findings may have significant implications regarding the prospects for stem cell gene therapy for ADA deficiency.

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.

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.

Plasma thrombomodulin levels in children with atopic dermatitis.

UNLABELLED: Plasma thrombomodulin (TM) levels were measured in 68 patients with atopic dermatitis (AD) and 35 controls. Plasma TM levels in patients with AD were significantly higher than those of controls (p < 0.01). A significant correlation was observed between plasma TM levels and skin scores of AD or peripheral eosinophil counts (p < 0.01). There was also a positive correlation between plasma TM and vascular cell adhesion molecule-1 levels (p < 0.05). CONCLUSION: These results suggest that plasma TM levels may reflect a severity of AD and/or endothelial cell activation induced by an allergic inflammation.

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.

Two cases of autoimmune neutropenia possibly induced by beta-lactam antibiotics in infants.

This report describes two infants who had neutropenia develop after treatment with beta-lactam antibiotics. The first patient, a 9-month-old girl, was administered flomoxef (FMOX); the second patient, a 14-month-old boy, was administered cefotiam (CTM). Both infants were found to have neutropenia 4 to 5 days after they were placed on respective antibiotics, and neutropenia had persisted despite antibiotics withdrawal for 1 to 3 months. Drug-induced lymphocyte stimulation test (DLST), granulocyte-bound antibodies, serum granulocyte antibodies, and immunoblotting analysis indicated that beta-lactam antibiotics possibly triggered production of granulocyte autoantibody with resultant autoimmune neutropenia.

Antiallergic effect of apple polyphenols on the allergic model mouse.

We studied here the antiallergic effect of apple condensed tannins (ACT) administered orally to a type I allergy model mouse transplanted with an IgEL a2 hybridoma secreting anti-2,4,6-trinitrophenyl (TNP) immunoglobulin E (IgE). The oral administration of ACT significantly inhibited the ear swelling responses at 1 h after antigen-stimulation with picryl chloride. The response was dose dependent within 0.1 to 10 mg/mouse. The inhibition of the ear swelling response reached the maximal level (90% inhibition) when ACT was administered 2 h before the antigen challenge. These findings suggest that ACT has an antiallergic effect on type I allergic symptoms.

[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.

Dizygotic twin sisters with myelokathexis: mechanism of its neutropenia.

Dizygotic twin sisters were first found to have neutropenia at 1 year of age when evaluated for recurrent pulmonary infections. Since then they have remained neutropenic (0.05 approximately 0.5 x 10(9)/l). Despite of their neutropenia, myeloid hyperplasia was evident on a marrow smear examination, and a number of cells were hypersegmented with fine interlobular bridging with chromatin strands and cytoplasmic vacuolation. Electron microscopy showed apoptotic cells with condensed nuclei and apoptotic bodies in the cytoplasm. Although life span, hydrogen peroxide production, phagocytosis, spreading, and chemotaxis of peripheral neutrophils were normal, the survival of bone marrow neutrophils in both infants was markedly decreased when compared with that of normal bone marrow neutrophils. During the bone marrow culture apoptotic neutrophils were observed at an earlier stage in both patients than in normal controls, biochemically and morphologically. Morphology of bone marrow neutrophils in both patients resembled that of cultured control bone marrow neutrophils. Peripheral neutropenia and appearance of characteristic neutrophils in the bone marrow in myelokathexis are considered to be an expression of apoptosis of bone marrow neutrophils.

Heterogeneity in F-actin polymerization of cord blood polymorphonuclear leukocytes stimulated by N-formyl-methionyl-leucyl-phenylalanine.

BACKGROUND: To elucidate the mechanism responsible for defects of polymorphonuclear leukocyte (PMNL) chemotaxis of neonates, we determined actin polymerization of NBD (7-nitrobenz-2-oxa-diazol)-phallacidin-stained PMNL following stimulation with either N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA) in cord blood and adult controls. METHODS: We measured F-actin content in PMNL stained with NBD-phallacidin using flow cytometry. RESULTS: Relative F-actin polymerization, that is, a ratio of stimulated F-actin to basal F-actin, was significantly decreased in cord blood PMNL when compared with that of adult PMNL. Distribution of fMLP-stimulated F-actin showed a bimodal pattern, while adult PMNL disclosed a single pattern. Following stimulation with PMA, however, F-actin levels were equal in both cord and adult PMNL. A fluorescein isothiocyanate-conjugated fMLP receptor assay showed no significant difference in binding capacity of fMLP receptors between adult and cord PMNL. CONCLUSION: These results indicate that a deficiency of PMNL chemotaxis in neonates may be due, in part, to decreased relative F-actin polymerization, which may be caused by functional heterogeneity in cord blood PMNL.

Reduction of Staphylococcus aureus in atopic skin lesions with acid electrolytic water--a new therapeutic strategy for atopic dermatitis.

The subjects studied were 22 pediatric patients newly diagnosed with atopic dermatitis (AD); 11 were treated with acid electrolytic water (AEW), which has a strong bactericidal activity (AEW group), and the other 11 with tap water (placebo group). AEW or tap water, 1 ml/cm2 (body surface area), was sprayed on their skin lesions with a spray gun each twice a day for a week. There were no significant differences between the two groups in regard to sex, age, serum IgE, peripheral eosinophil counts, grading scores of AD, and duration of AD. The study was designed as a randomized, placebo-controlled, double-blind clinical trial. Colony counts of Staphylococcus aureus on skin lesions in the AEW group, both 3 min after spraying (P < 0.05) and after 1 week of skin treatment (P < 0.01), were significantly decreased as compared with colony counts before treatment, while there was no significant difference in the placebo group before and after treatment. Grading scores of AD also decreased in the AEW group (P < 0.01), but not in the placebo group. Both the subjects' guardians' evaluation and a referee physician's evaluation of treatment effect were significantly higher in the AEW group than in the placebo group (P < 0.01). AEW may be potentially effective in preventing a staphylococcal chronic inflammation in AD because of its strong bactericidal activity.