Short-term inhalation of sargramostim with concomitant high-dose steroids does not hasten recovery in moderate COVID-19 pneumonia: a double-blind, randomised, placebo-controlled trial.

BACKGROUND: Granulocyte-macrophage colony stimulating factor (GM-CSF) inhalation may alleviate pulmonary inflammation caused by viral pneumonia. To investigate this, we evaluated its efficacy on COVID-19 pneumonia. METHODS: This double-blind, randomised, placebo-controlled study (ClinicalTrials.gov: NCT04642950) evaluated patients in the first half of 2021 at seven Japanese hospitals. Hospitalised patients with COVID-19 pneumonia with moderate hypoxaemia inhaled sargramostim or placebo for 5 days. The primary endpoint was days to achieve a ≥ 2-category improvement from baseline on a modified 7-category ordinal scale. Secondary endpoints included degree of oxygenation, defined by amount of oxygen supply, and serum CCL17 level. RESULTS: Seventy-five patients were randomly assigned in a 2:1 ratio to receive sargramostim or placebo, of which 47 and 23 were analysed, respectively. No difference was observed between groups regarding the primary endpoint (8.0 and 7.0 days for sargramostim and placebo, respectively) or in the secondary endpoints, except for CCL17. A post hoc sub-analysis indicated that endpoint assessments were influenced by concomitant corticosteroid therapy. When the cumulative corticosteroid dose was ≤500 mg during Days 1-5, recovery and oxygenation were faster in the sargramostim group than for placebo. Bolus dose corticosteroids were associated with temporarily impaired oxygenation and delayed clinical recovery. The increase in serum CCL17, a candidate prognostic factor, reflected improvement with sargramostim inhalation. The number of adverse events was similar between groups. Two serious adverse events were observed in the sargramostim group without causal relation. CONCLUSIONS: Inhaled sargramostim was likely to be effective for COVID-19 pneumonia unless the concomitant corticosteroid dose was high.

Absolute quantification of the α, α', and ß subunits of ß-conglycinin from soybeans by liquid chromatography/tandem mass spectrometry using stable isotope-labelled peptides.

ß-Conglycinin, a major protein in soybeans, shows improvement effect of lipid metabolism. Moreover, this protein influences the processing properties of soybeans. ß-Conglycinin is a hetero-trimer constituted by α, α', and ß subunits. In this work, a method for the selective quantification of these subunits was developed by means of protein absolute quantification (AQUA) technology using liquid chromatography/tandem mass spectrometry with the stable isotope-labelled internal standard peptides LQSGDALR[13C6,15N4], NILEASYDTK[13C6,15N2], and NPIYSNNFGK[13C6,15N2]. This method exhibited linear relationships (r2 > 0.99) in the concentration range of 1.2-300 fmol/µL for LQSGDALR[13C6,15N4] and NILEASYDTK[13C6,15N2], and of 4.7-300 fmol/µL for NPIYSNNFGK[13C6,15N2]. As a result, the content of these subunits in ß-conglycinin-rich and both α and α' subunit-deficient soybean cultivars was successfully determined. This quantitative assay is promising for the evaluation of the food functionality and processing properties of soybeans.

Quiescent adult stem cells in murine teeth are regulated by Shh signaling.

The mechanisms regulating the maintenance of quiescent adult stem cells in teeth remain to be fully elucidated. Our aim is to clarify the relationship between BrdU label-retaining cells (LRCs) and sonic hedgehog (Shh) signaling in murine teeth. After prenatal BrdU labeling, mouse pups were analyzed during postnatal day 1 (P1) to week 5 (P5W). Paraffin sections were processed for immunohistochemistry for BrdU, Sox2, Gli1, Shh, Patched1 (Ptch1) and Ki67 and for in situ hybridization for Shh and Ptch1. Dense LRCs, Gli1-(+) cells and Ptch1-(+) cells were co-localized in the outer enamel epithelium of the apical bud and apical dental papilla of incisors. In developing molars, dense LRCs were numerous at P1 but then decreased in number over the course of odontogenesis and were maintained in the center of pulp tissue. Gli1-(+) cells were maintained in the pulp horn during the examined stages, while they increased in number and were maintained in the center of pulp tissue during P2-5W. Ptch1-(+) cells were localized in the pulp horn at P1 and increased in number in the center of the pulp after P3W. Shh mRNA was first expressed in the enamel epithelium and then shifted to odontoblasts and other pulp cells. Shh protein was distributed in the epithelial and mesenchymal tissues of incisors and molars. These findings suggest that quiescent dental stem cells are regulated by Shh signaling, and that Shh signaling plays a crucial role in the differentiation and integrity of odontoblasts during epithelial-mesenchymal interactions and dentinogenesis.

Identification and evaluation of antioxidants in Japanese parsley.

Two cultivars of Japanese parsley were harvested in different seasons; their antioxidant capacities were evaluated by oxygen radical absorbance capacity (ORAC) methods, and the contents of hydrophilic and lipophilic antioxidants were compared. Japanese parsley possessed potent antioxidant capacities both in hydrophilic and lipophilic extracts when evaluated by ORAC methods. LC/MS/MS analyses revealed that chlorogenic acid and four kinds of quercetin glycosides were major antioxidants in the hydrophilic extract. Lutein was the main contributor to the antioxidant capacity of the lipophilic extract. Antioxidant capacities of the hydrophilic extracts of both cultivars tended to be higher in winter because of the increase in the contents of chlorogenic acid and quercetin glycosides. An obvious trend in the lipophilic antioxidant capacities or lutein contents was not observed irrespective of the cultivar.

Improvement and Interlaboratory Validation of the Lipophilic Oxygen Radical Absorbance Capacity: Determination of Antioxidant Capacities of Lipophilic Antioxidant Solutions and Food Extracts.

A lipophilic oxygen radical absorbance capacity (L-ORAC) assay is an evaluation of the antioxidant capacity of solutions of lipophilic compounds. The concentrations of fluorescein, radical generator, and Trolox standard solutions were optimized to improve the precision of the assay. An interlaboratory study using two antioxidant solutions and three food extracts as test samples conducted in accordance with harmonized protocol demonstrated satisfactory L-ORAC measurements; the intermediate precision relative standard deviations (RSD(int)) ranged from 7.0 to 16.7%, the reproducibility relative standard deviations (RSD(R)) ranged from 14.8 to 19.4%, and the HorRat values ranged from 1.35 to 1.78.

Extraction efficiency of hydrophilic and lipophilic antioxidants from lyophilized foods using pressurized liquid extraction and manual extraction.

The efficient extraction of antioxidants from food samples is necessary in order to accurately measure their antioxidant capacities. α-Tocopherol and gallic acid were spiked into samples of 5 lyophilized and pulverized vegetables and fruits (onion, cabbage, Satsuma mandarin orange, pumpkin, and spinach). The lipophilic and hydrophilic antioxidants in the samples were sequentially extracted with a mixed solvent of n-hexane and dichloromethane, and then with acetic acid-acidified aqueous methanol. Duplicate samples were extracted: one set was extracted using an automated pressurized liquid extraction apparatus, and the other set was extracted manually. Spiked α-tocopherol and gallic acid were recovered almost quantitatively in the extracted lipophilic and hydrophilic fractions, respectively, especially when pressurized liquid extraction was used. The expected increase in lipophilic oxygen radical absorbance capacity (L-ORAC) due to spiking with α-tocopherol, and the expected increase in 2,2-diphenyl-1-picrylhydrazyl radical scavenging activities and total polyphenol content due to spiking with gallic acid, were all recovered in high yield. Relatively low recoveries, as reflected in the hydrophilic ORAC (H-ORAC) value, were obtained following spiking with gallic acid, suggesting an interaction between gallic acid and endogenous antioxidants. The H-ORAC values of gallic acid-spiked samples were almost the same as those of postadded (spiked) samples. These results clearly indicate that lipophilic and hydrophilic antioxidants are effectively extracted from lyophilized food, especially when pressurized liquid extraction is used.

Protein expression, gene amplification, and mutational analysis of EGFR in triple-negative breast cancer.

BACKGROUND: Although triple-negative breast cancer (TNBC) with epidermal growth factor receptor (EGFR) expression has been extensively studied, few studies have simultaneously examined EGFR expression and EGFR gene amplification. Here, we examined the correlations of EGFR expression with EGFR gene amplification, EGFR-activating mutations, and the expression of components of the Akt pathway. METHODS: Tumor tissues were obtained from 84 patients with TNBC. We analyzed the expression of EGFR, phosphorylated Akt (p-Akt), phosphorylated mammalian target of rapamycin (p-mTOR), and other relevant proteins using immunohistochemistry. We also analyzed EGFR gene and chromosome 7 copy numbers by dual-color in situ hybridization. DNA was extracted from formalin-fixed paraffin-embedded samples. Analysis of EGFR gene-activating mutations was performed using the smart amplification process version 2 assay. RESULTS: Most TNBCs expressing EGFR are non-specialized invasive ductal carcinomas, whereas others are likely to be rare specialized carcinomas, such as typical medullary carcinoma, apocrine carcinoma, metaplastic carcinoma, and adenoid cystic carcinoma. EGFR was expressed in samples from 28 of 84 (33.3%) patients, but the EGFR gene was not amplified in any of the 84 samples. There were significant correlations between EGFR expression and the number of polysomic cells and the presence of high polysomy of chromosome 7. However, EGFR expression was not correlated with p-Akt or p-mTOR expression, nor with the other clinicopathological factors recorded in this study. We found no evidence of EGFR gene-activating mutations. CONCLUSIONS: EGFR gene amplification and EGFR-activating mutations might not be the mechanisms leading to the constitutive activation of EGFR in TNBC. Further investigation is needed to clarify the other molecular mechanisms for oncogenic activation of EGFR in TNBC.

Genetic clonal mapping of in situ and invasive ductal carcinoma indicates the field cancerization phenomenon in the breast.

Nearly 80% of well-differentiated in situ duct carcinomas (g1 DCIS) have been shown to be multicentric (multilobar) lesions, while most in situ poorly differentiated duct carcinomas (g3 DCIS) were unifocal (unilobar) lesions. Here we present a clonality study of 15 cases of DCIS, all showing multiple foci. Twelve of these cases were associated with an invasive duct carcinoma. Fifteen cases of female breast cancer patients all showing multiple DCIS foci (5 g1 DCIS, 5 g2 DCIS, 5 g3 DCIS) were randomly selected and histologically studied using large histological sections. Care was taken to laser-microdissect DCIS foci that were most distantly located from one another in the same large section, and pertinent cells were genetically studied. Invasive duct carcinoma and ipsilateral lymph node metastases and/or contralateral lesions, whenever present, were additionally microdissected. DNA of neoplastic cells was purified, and the mtDNA D-loop region was sequenced. Genetic distance of different foci from the same case was visualized by phylogenetic analyses using the neighbor-joining method. Patients ranged in age from 36 to 87 years (mean 65.1). All 9 cases of widely spread DCIS were not clonal. Four of 6 cases that showed multiple adjacent foci were clonally related on mtDNA analysis. In the present series, 11/15 DCIS appeared as multiple synchronous primary breast tumors, genetically not related to one another. The present data enhance the view that breast can also show the field cancerization phenomenon, paralleling what has already been proposed in other organs.

The value of large sections in surgical pathology.

Large format sections (LS) first have been introduced in breast pathology more than a century ago. Since then, they constituted for longtime a research tool to better understand breast microanatomy and the relationship between radiological images and pathological features. Similarly LS have been used to study neoplastic, inflammatory, and degenerative diseases affecting various organs, as brain, lung, gastrointentinal tract, bone, urinary tract, prostate, and placenta. Currently LS are mostly applied to diagnostic routine to better stage tumours such as prostate and breast carcinomas or to correlate radiologic imaging to gross specimens. The purpose of the present paper is to review the historical background and the basis of the applications of LS in surgical pathology, with special emphasis on breast tumours.

Synovial sarcoma involving the median nerve: a case report.

Synovial sarcoma may arise from different and unusual sites. Here a case of biphasic synovial sarcoma arising or invading the radial nerve in a 59-year-old female classically showing chromosomal reciprocal translocation (X; 18) is reported. The differential diagnosis from similar tumors is discussed.

Method validation by interlaboratory studies of improved hydrophilic oxygen radical absorbance capacity methods for the determination of antioxidant capacities of antioxidant solutions and food extracts.

Hydrophilic oxygen radical absorbance capacity (H-ORAC) is a method for evaluating antioxidant capacities of solutions of hydrophilic compounds. In this study, we improved the original method for H-ORAC determination, and evaluated the precision of the two improved methods (methods A and B) by interlaboratory studies using 5 antioxidant solutions and 5 food extracts as test samples. An interlaboratory study of method A, in accordance with the harmonized protocol, demonstrated satisfactory performance (intermediate precision relative standard deviations (RSD(int)) ranging from 4.6 to 18.8%; the reproducibility relative standard deviations (RSD(R)) ranging from 7.0 to 21.1%, and the HorRat values ranging from 0.40 to 1.93). However, methodological problems remained, and a further improved method, method B, was thus developed. An interlaboratory study of method B by 5 participating laboratories showed better intermediate precision and reproducibility (RSD(int) and RSD(R) ranging from 1.8 to 9.4%, and from 4.4 to 13.8%, respectively), and all HorRat values for the test samples were less than 1.3, suggesting good performance for the H-ORAC measurement.

Differentiation capacity of BrdU label-retaining dental pulp cells during pulpal healing following allogenic transplantation in mice.

Our recent study has demonstrated the localization of putative dental pulp stem cells in the developing molar by chasing 5-bromo-2'-deoxyuridine (BrdU)-labeling. However, their differentiation capacity subsequent to the tooth transplantation remains to be elucidated. This study aims to clarify the differentiation capacity of BrdU label-retaining dental pulp cells and their relationship to cell proliferation and apoptosis during pulpal healing following allogenic transplantation in mice. Following extraction of the mouse molar in BrdU-labeled animals, the roots and pulp floor were resected and immediately allo-grafted into the sublingual region in non-labeled animals, and vice versa. In the labeled transplants, label-retaining cells (LRCs) were increased in number and committed in nestin-positive newly differentiated odontoblast-like cells, whereas they were not committed in osteoblast-like cells. In the labeled host, on the contrary, LRCs were committed in neither odontoblast- nor osteoblast-like cells, although they were transiently increased in number and finally disappeared in the pulp tissue of the transplants. Interestingly, numerous apoptotic cells appeared in the pulp tissue including LRCs during the experimental period. These results suggest that transplanted LRCs maintain their proliferative and differentiation capacity in spite of extensive apoptosis occurring in the transplant, whereas transiently increased host-derived LRCs finally disappear in the pulp chamber following apoptosis.

[Evaluation of the side effects of intravenous patient controlled analgesia after spine surgery].

BACKGROUND: We have adopted intrravenous patient controlled analgesia (IV-PCA) for spine surgery. We could not find reports about detailed examinations of the side effects of IV-PCA using morphine after spine surgery, so we investigated retrospectively side effects in cases using morphine IV-PCA. METHODS: Eighty-five patients underwent IV-PCA after spine surgery. The contents of PCA pump were morphine 20 mg (= 2 ml), droperidol 2 mg (= 0.8 ml), and saline 77 ml. We fixed continuous infusion at 2 ml x hr(-1), bolus infusion at 2 ml x hr(-1), and lockout time at 15 minutes. Respiration time, SpO2, blood pressure, pulse rate, nausea and vomiting, and VAS were monitored while IV-PCA was in use. When severe side effects were noticed, IV-PCA was discontinued by physician in charge. We judged discontinuation of IV-PCA as occurrence of severe side effects. RESULTS: IV-PCA was discontinued in seven patients. The causes of discontinuation were nausea and vomiting, hypotension, and bradycardia. Nausea and vomiting was the most common cause and found mostly in women. CONCLUSIONS: Because IV-PCA was discontinuated in 8.2% of patients, it was thought that its management depending on patients' personal state was necessary to utilize IV-PCA as a method of postoperative analgesia.

Triple-negative breast cancer: histological subtypes and immunohistochemical and clinicopathological features.

To reveal heterogeneous properties of triple-negative (TN) breast cancers (estrogen receptor negative, progesterone receptor negative and HER2 negative) and to clarify whether the developmental pathways to TN breast cancer are single or multiple, we conducted clinicopathological and immunohistochemical studies on TN breast cancers, with special reference to comparison of the invasive component (iIC) and the ductal component (dcIC) of invasive TN breast cancer and pure TN ductal carcinoma in situ (TNDCIS). Tumor tissues were obtained from 97 patients with TN invasive carcinoma and 10 patients with TNDCIS. Two histological subclassifications, "atypical" medullary carcinoma (type A, n=16) and carcinoma with a central acellular zone (type B, n=11), were distinguished from conventional ductal carcinoma. Other invasive ductal carcinomas were classified as type C (n=64) and special types were classified as type D (n=5). The follow-up period for the 96 patients ranged from 5 to 147.8 months (mean, 47.6 months). Out of 97 cases, dcIC was present in 29 (30%) cases and type A and B had significantly few ductal components, 0% and 18%, respectively. There were only six (6%) cases with non-TN cells in dcIC and TN cells in iIC and five of them were type C. In 13 (13%) cases, epidermal growth factor receptor (EGFR) expression existed only in iIC. Therefore, most of the TN carcinoma develops originally and rapidly invades at the early stage, especially in types A and B. The relapse rate of type B was the highest (36.4%) and the overall survival of patients with type B was the shortest (P=0.02), which indicates that the prognosis of type B is significantly worse than the other types.

Epitope analysis of peanut allergen Ara h1 with human monoclonal IgM antibody clone #86.

A human-mouse hybridoma clone #86 secreting IgM-class human monoclonal antibody to peanut allergen protein Ara h1 was newly established. To detect an antibody-binding sequence (epitope) on Ara h1, the monoclonal antibody #86 was reacted with multi-pin apparatus with a series of peptides synthesized from the amino acid sequence of Ara h1. The antibody #86 was found to bind to a peptide with amino acid sequence of 481EEEEDEDEEEEGSNREVRRY500. Further analysis with shorter pin-peptides with ten amino acid-long showed that the peptides reacted with the antibody #86 contained a sequence of 485DEDEEEE491. This might be an essential linear sequence of this epitope. When the 485DED487 part of the peptide was replaced by alanine, decreased binding of antibody #86 was observed.

Mapping of BrdU label-retaining dental pulp cells in growing teeth and their regenerative capacity after injuries.

Recent studies have demonstrated that human dental pulp contains adult stem cells. A pulse of the thymidine analog BrdU given to young animals at the optimal time could clarify where slow-cycling long-term label-retaining cells (LRCs), putative adult stem cells, reside in the pulp tissue. This study focuses on the mapping of LRCs in growing teeth and their regenerative capacity after tooth injuries. Two to seven peritoneal injections of BrdU into pregnant Wistar rats revealed slow-cycling long-term dense LRCs in the mature tissues of born animals. Numerous dense LRCs were postnatally decreased in number and reached a plateau at 4 weeks after birth when they mainly resided in the center of the dental pulp, associating with blood vessels. Mature dental pulp cells were stained with Hoechst 33342 and sorted into (<0.76%) side population cells using FACS, which included dense LRCs. Some dense LRCs co-expressed mesenchymal stem cell markers such as STRO-1 or CD146. Tooth injuries caused degeneration of the odontoblast layer, and newly differentiated odontoblast-like cells contained LRCs. Thus, dense LRCs in mature pulp tissues were supposed to be dental pulp stem cells possessing regenerative capacity for forming newly differentiated odontoblast-like cells. The present study proposes the new hypothesis that both granular and dense LRCs are equipped in the dental pulp and that the dense LRCs with proliferative capacity play crucial roles in the pulpal healing process following exogenous stimuli in cooperation with the granular LRCs.

Axillary lymph node dissection can be avoided in women with breast cancer with intraoperative, false-negative sentinel lymph node biopsies.

BACKGROUND: It is currently unclear which patients with breast cancer with sentinel lymph node (SLN) metastases do not need axillary lymph node dissection (ALND). PATIENTS AND METHODS: A cohort of 1,132 women who had unilateral invasive breast cancer with clinically negative nodes or nodes suspicious for metastasis, were intraoperatively diagnosed as having negative SLNs, and did not undergo an immediate ALND. Our intraoperative histological investigation uses H&E staining of a frozen section from a maximum cut surface of each SLN. Of these 1,132 women, 132 (11.7%) were postoperatively diagnosed as having positive SLNs, which classifies them as having an intraoperative, false-negative SLN biopsy (SLNB). Patient and tumor characteristics, treatment methods, and the prognoses of these patients were investigated and compared with the remaining 1,000 patients who were negative for SLNB. RESULTS: Of the 132 patients with intraoperative, false-negative SLNB, none underwent a further ALND. With a median follow-up period of 58.1 months, none of these patients exhibited recurrence in the axillary nodes. Their recurrence-free survival rates were not statistically different from those of patients with negative SLNB. CONCLUSIONS: ALND can be avoided in most patients with breast cancer with intraoperative, false-negative SLNB.

True recurrences and new primary tumors have different clinical features in invasive breast cancer patients with ipsilateral breast tumor relapse after breast-conserving treatment.

Ipsilateral breast tumor relapse (IBTR) after breast-conserving treatment (BCT) may represent two distinct types of lesion, including a true recurrence (TR) or a new primary tumor (NPT). The aim of this study was to ascertain the difference between TRs and NPTs and to show the clinical significance of classifying IBTR into these two types of recurrence. Patients (n = 2,075) with unilateral invasive breast cancer who underwent BCT between 1987 and 2005 at Saitama Cancer Center were analyzed. IBTR was classified into TR and NPT, which was based on all clinical and pathological features of both a primary tumor and IBTR that can be evaluated. IBTR-free survival and the risk factors were analyzed in order to compare the findings for TR and NPT. In addition, the salvage surgical methods for IBTR and overall survival after IBTR were analyzed. Sixty patients with IBTR were classified into 52 with TR and eight with NPT. IBTR-free survival was significantly shorter in the patients with TR than those with NPT. Young age, tumor size, a positive surgical margin, and omission of radiation therapy (RT) were significant risk factors for TR. Omission of RT was the only significant risk factor for NPT. In 27 patients who underwent a repeat lumpectomy for TR, four had a second IBTR. The overall survival after IBTR was worse in patients with TR than NPT. TR and NPT show quite different clinical features. Classifying IBTR into TR or NPT can therefore help to select the most appropriate treatment for IBTR.

Ipsilateral breast tumor relapse after breast conserving surgery in women with breast cancer.

Ipsilateral breast tumor relapse (IBTR) is a potentially a significant problem after breast conserving surgery (BCS). With a median follow-up period of 64.7 months, IBTR occurred as a first relapse in 67 (3.0%) of a total of 2243 patients and distant recurrence occurred in 167 (7.4%). A positive surgical margin and the omission of radiotherapy (RT) were independently associated with IBTR. The five-year cumulative IBTR rates were 5.1% in patients with positive margins and 2.0% in the patients with negative margins. The five-year cumulative IBTR rates were 1.8% in patients with RT and 8.1% in patients without RT. IBTR was independently associated with distant-recurrence-free survival rates as well as age, nodal metastasis, lymphovascular invasion and progesterone receptor status. The five-year distant-recurrence-free survival rates were 81.9% in patients with IBTR and 93.2% in patients without IBTR. In order to prevent IBTR, a negative margin and the administration of RT are therefore considered to be important in patients who undergo BCS.

Positive sentinel lymph node biopsy predicts the number of metastatic axillary nodes of breast cancer.

It remains to be clarified whether a positive sentinel lymph node biopsy (SLNB) can predict the number of metastatic axillary nodes. This study examined a consecutive series of women with unilateral invasive breast cancer who underwent axillary lymph node dissection after an intra-operative positive SLNB. The numbers of positive and negative sentinel lymph nodes (SLNs) were analyzed for a likelihood of pN1a, pN2a, and pN3a diseases as per the UICC TNM classification. Of the 368 study patients, 165 (45%) had one positive SLN and one or more negative SLNs. This result represented the most common combination of positive and negative SLNs. It was also the most predictive indicator (93%) of pN1a disease and the least predictive indicator (7% or 0%) of pN2a or pN3a disease, respectively. The numbers of positive and negative SLNs can predict the number of metastatic axillary nodes in breast cancer patients.