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Introduction: Risk factors for seriously ill coronavirus disease 19 (COVID-19) patients have been reported in several studies. However, to date, few studies have reported simple risk assessment tools for distinguishing patients becoming severely ill after initial diagnosis. Hence, this study aimed to develop a simple clinical risk nomogram predicting oxygenation risk in patients with COVID-19 at the first triage., Methods: This retrospective study involved a chart review of the medical records of 84 patients diagnosed with COVID-19 between February 2020 and March 2021 at ten medical facilities. The patients were divided into requiring no oxygen therapy (non-severe group) and requiring oxygen therapy (severe group). Patient characteristics were compared between the two groups. We utilized univariate logistic regression analysis to confirm determinants of high risks of requiring oxygen therapy in patients with moderate COVID-19., Results: Thirty-five patients ware in severe group and forty-nine patients were in non-severe group. In comparison with patients in the non-severe group, patients in the severe group were significantly older with higher body mass index (BMI), and had a history of hypertension and diabetes. Serum blood urea nitrogen (BUN), lactic acid dehydrogenase (LDH), and C-reactive protein (CRP) levels were significantly higher in the severe group. Multivariate analysis showed that older age, higher BMI, and higher BUN levels were significantly associated with oxygen requirements., Conclusions: This study demonstrated that age, BMI, and BUN were independent risk factors in the moderate-to-severe COVID-19 group. Elderly patients with higher BMI and BUN require close monitoring and early treatment initiation., (Copyright © 2021 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Introduction: There is an increasing incidence of Pneumocystis pneumonia (PcP) among individuals without human immunodeficiency virus (HIV) infection (non-HIV PcP). However, prognostic factors for patients with non-HIV PcP have not been identified. Moreover, A-DROP (for classifying the severity of community-acquired pneumonia) or the blood urea nitrogen-to-serum albumin ratio (BUN/Alb), which is reported to be a predictor of mortality of community-acquired pneumonia, has not been established as an efficient prognostic factor in patients with non-HIV PcP. In this study, we analyzed the prognostic factors for non-HIV PcP and evaluated the prognostic ability of A-DROP and the BUN/Alb ratio., Methods: This retrospective study involved a chart review of the medical records of 102 patients diagnosed with non-HIV PcP between January 2003 and May 2019 at five medical facilities., Results: Overall, 102 patients were involved in this study. The 30-day mortality rate for non-HIV PcP was 20.5% in this study population. Compared with survivors, non-survivors had significantly lower serum albumin levels and significantly higher age, corticosteroid dosage at the PcP onset, alveolar-arterial oxygen gradient, A-DROP score, lactate dehydrogenase levels, blood urea nitrogen levels, and BUN/Alb ratio. Multivariate analysis showed that a high BUN/Alb ratio at treatment initiation was significantly associated with 30-day mortality risk. The receiver operating characteristic curves showed that A-DROP score had the highest prognostic ability in estimating 30-day mortality., Conclusions: In patients with non-HIV PcP, a high BUN/Alb ratio is an independent prognostic predictor of mortality risk, and A-DROP is useful for classifying the severity., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

The effect of systemic corticosteroids on clinical outcomes in patients with coronavirus disease 2019 (COVID-19) remains controversial. While the use of corticosteroids raises concerns regarding delayed viral clearance, secondary infections, and long-term complications that can lead to increased mortality, corticosteroids have the potential to reduce mortality if used appropriately. Herein, we report good outcomes in two patients with COVID-19 who received systemic corticosteroids as adjunctive therapy. An 83-year-old man with hypertension and smoking history and a 62-year-old man with a drinking habit were transferred to our hospital with a diagnosis of COVID-19. The patients developed general malaise and loss of appetite with persistent high fever. Despite the prescription of antiviral drugs, their hypoxemia progressed rapidly. However, after the introduction of systemic corticosteroids, their symptoms improved as the fever decreased, and their hypoxemia gradually improved. These results suggest that some patients with COVID-19 may benefit from the appropriate use of systemic corticosteroids as adjunctive therapy., Competing Interests: Declaration of competing interest None., (Copyright © 2020. Published by Elsevier Ltd.)

Here, an indomethacin (IMC)/saccharin (SAC)/polyvinylpyrrolidone (PVP) ternary solid dispersion (SD) prepared by spray-drying was characterized to clarify its dissolution mechanism. Solid-state NMR spectroscopy revealed that IMC and SAC in the ternary SD interacted via hydrogen bonding in a similar manner as that in the IMC/SAC co-crystal. Initial IMC dissolution from the ternary SD was slower than that from the binary SD, although IMC supersaturation was maintained for a relatively longer time in the ternary SD. Solid- and solution-state NMR measurements for dispersed particles in the dissolution test revealed that the particle for IMC/PVP binary SD was composed of amorphous IMC dispersed in PVP matrix and α-form IMC. In contrast, the particles for the ternary SPD was composed of amorphous IMC dispersed in PVP matrix and IMC/SAC co-crystals. Scanning electron microscopy indicated that in the binary SD, amorphous IMC microfiber-gel was generated on the surface of particles after the dissolution test, preventing amorphous IMC from contacting with water. In contrast, in the ternary SD, IMC/SAC co-crystals were generated on the surface of particles, and intermediate spaces were formed on the surface, which allowed water intrusion into the particles and continuous dissolution of IMC., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Background: Peripheral pulmonary lesions are rarely observed directly before transbronchial biopsy. This study aimed to characterize the differences between malignant and benign peripheral pulmonary lesions according to the findings of direct observation using probe-based optical fiberscopy., Methods: Thirty patients who underwent probe-based optical fiberscopy in combination with bronchoscopy using endobronchial ultrasonography with a guide sheath for the evaluation of peripheral pulmonary lesions were prospectively included in this study. The patients were divided into the malignant and benign groups according to their final diagnosis. The findings of probe-based optical fiberscopy in the two groups were compared., Results: The numbers of patients who were diagnosed using histological or bacteriological analyses via bronchoscopic sampling in the malignant and benign groups were 20/23 (87.0%) and 2/7 (28.6%), respectively. On probe-based optical fiberscopy, angiogenesis and vascular engorgement were observed only in the malignant group. The disappearance of subepithelial microvessel transparency and presence of bronchiolar stenosis were observed more frequently in the malignant group (78.3% and 60.9%) than in the benign group (28.6% and 28.6%), whereas increased mucus secretion was observed more frequently in the benign group (71.4%) than in the malignant group (8.7%)., Conclusions: These results suggest that the findings of direct observation using probe-based optical fiberscopy are useful for differentiating malignant from benign peripheral pulmonary lesions., Trial Registry: UMIN-CTR; UMIN000018796; URL: https://www.umin.ac.jp/ctr/index.htm., (Copyright © 2019 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.)

Background: Pulmonary function tests (PFTs) comprise the traditional method for detecting central airway obstruction (CAO) and evaluating therapeutic effects, but are effort-dependent. By contrast, the forced oscillation technique (FOT) is performed during tidal breathing in an effort-independent mode and is universally used to assess respiratory function in patients with chronic obstructive pulmonary disease (COPD) and asthma. We used the FOT to measure airway resistance and reactance in patients with CAO before and after interventional bronchoscopy and compared the results to data obtained using PFTs., Methods: Twelve patients with CAO were recruited from December 2013 to July 2016. The FOT, PFTs, chest computed tomography (CT), COPD Assessment Test (CAT), and the modified Medical Research Council (mMRC) dyspnea scale were employed before and after interventional bronchoscopy. The minimum airway cross-sectional area (MACSA) was calculated using a CT image calculator., Results: Of the 12 patients, 6 had tracheal obstruction and 6 had bronchial obstruction. All FOT measurements, except ΔX5, were significantly improved after interventional bronchoscopy in all cases. The significance of the improvement was greater with the FOT than PFTs. The MACSA, CAT, and mMRC dyspnea scale scores also significantly improved in all cases. Furthermore, only alteration of resistance at 20 Hz (R20) significantly correlated with the alteration of the MACSA after intervention. No significant correlations were found for PFTs., Conclusions: The FOT is suitable and convenient for assessing therapeutic results in patients with tracheobronchial CAO. The alteration of R20 is useful for estimating the airway dilation of CAO after interventional bronchoscopy., (Copyright © 2018 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.)

In this article, we proposed a risk assessment strategy for CYP3A time-dependent inhibition (TDI) during drug discovery based on a thorough retrospective study of 13 reference drugs, some of which are known to have in vitro TDI potential but have unknown clinical relevance. First, the traditional parameter k inact /K I , recommended by regulatory authorities for necessity decision making in clinical drug-drug interaction (DDI) studies, was investigated as a predictive index for clinical TDI liability. The cutoff value of 1.1 for k inact /K I , established by the Food and Drug Administration, tended to produce false-positive prediction results for clinical DDI occurrence. The value of 1.25 recommended in the European Medicines Evaluation Agency draft guideline yielded better predictions with only 1 false negative for diltiazem. Second, to enable earlier risk assessment, remaining activity, defined as the residual CYP3A activity in vitro obtained in the screening conditions, was investigated as an alternative index. As a result, the ratios of unbound C max or area under the curve to remaining activity precisely predicted clinical DDI occurrence. In conclusion, we demonstrated the predictive power of k inact /K I and remaining activity values for clinical DDIs. These findings provide insights that enable TDI risk assessment, even during drug discovery., (Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Background: The purpose of this study was to investigate the relationship between psychological competitive ability and the incidence of noncontact ACL injuries among high school female athletes., Methods: A three-year prospective cohort study was conducted using 300 15-year-old high school female athletes with no previous injuries or symptoms in their lower limbs (106 handball players and 194 basketball players). At baseline, their psychological competitive abilities were assessed using a self-administered questionnaire-the Diagnostic Inventory of Psychological Competitive Ability (DIPCA.3). After the baseline examination was performed at high school entry, all players were prospectively followed for 36months to document any subsequent incidence of ACL injury, according to their coaches. An unpaired t-test with Welch's correction was performed to compare the differences in the psychological competitive abilities between the injured and uninjured players., Results: Of the 300 players, 25 (8.3%) experienced a noncontact ACL injury during the three-year observation period. The injured players had significantly higher total DIPCA.3 scores for psychological competitive ability than the uninjured players (169.9±18.8 vs. 159.2±21.6, P=.036). Additionally, the injured players had significantly higher scores than the uninjured players in the following categories: aggressiveness, volition for self-realization, volition for winning, judgment, and cooperation. However, no significant differences were observed in patience, self-control, ability to relax, concentration, confidence, decision, and predictive ability., Conclusions: High psychological competitive ability was associated with the incidence of noncontact ACL injuries in high school female athletes., Level of Evidence: Level II (prospective cohort study)., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: This study aimed to present a new technique for anatomical single bundle anterior cruciate ligament (ACL) reconstruction. We developed an original rounded rectangular dilator set to create rounded rectangular femoral tunnels. This technique can increase the femoral tunnel size without roof impingement, and has the potential to reduce the graft failure rate. We investigated the tunnel position and the incidence of intraoperative complications., Method: The presented technique is anatomical single bundle ACL reconstruction using a semitendinosus graft (with or without the gracilis tendon). The tunnel was drilled via an additional medial portal. Rounded rectangular tunnels were created using a special dilator. Tibial tunnels were created using conventional rounded tunnels. Fixation was achieved using a suspensory device on the femoral side and a plate and screw on the tibial side., Patients: Fifty patients underwent this surgery, and intraoperative complications were investigated. The femoral tunnel positions were documented postoperatively from computed tomography scans using the quadrant method. The tibial tunnel positions (anterior-to-posterior, medial-to-lateral) were documented using intraoperative X-ray scans., Results: Only one patient had a partial posterior tunnel wall blowout. The femoral tunnel length varied between 30 and 40mm (mean, 34.9±3.3mm). All femoral and tibial tunnels were located within the area of the anatomical ACL insertions., Conclusion: We did not experience any serious intraoperative complications during anatomical single bundle ACL reconstruction using a rounded rectangle dilator, and the resulting locations of the femoral and tibial tunnels were within the anatomical ACL footprint., Level of Evidence: Level IV., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The present study was conducted to evaluate the suitability of a repeated-dose liver micronucleus (LMN) assay in young adult rats as a collaborative study by the Mammalian mutagenicity study (MMS) group. All procedures were performed in accordance with the standard protocols of the MMS Group. Six-week-old male Crl:CD(SD) rats (5 animals/group) received oral doses of the hepatocarcinogen N-nitrosomorpholine (NMOR) at 0 (control), 5, 10, and 30mg/kg/day (10mL/kg) for 14 days. Control animals received vehicle (water). Hepatocytes were collected from the liver 24h after the last dose, and the number of micronucleated hepatocytes (MNHEPs) was determined by microscopy. The number of micronucleated immature erythrocytes (MNIMEs) in the femoral bone marrow was also determined. The liver was examined using histopathologic methods after formalin fixation. The results showed statistically significant and dose-dependent increases in the number of MNHEPs in the liver at doses of 10mg/kg and greater when compared with the vehicle control. However, no significant increase was noted in the number of MNIMEs in the bone marrow at doses of up to 30mg/kg. Histopathology of the liver revealed hypertrophy and single cell necrosis of hepatocytes at doses of 5mg/kg and above. These results showed that the induction of micronuclei by NMOR was detected by the repeated-dose LMN assay, but not by the repeated-dose bone marrow micronucleus assay., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: The purpose of this study was to investigate the efficacy of additional oblique magnetic resonance imaging (MRI) for the diagnosis of anterior cruciate ligament (ACL) tear and evaluation of ACL remnant tissue., Methods: We retrospectively reviewed the records of 54 knees. Three independent readers evaluated the MR images by the use of three methods: orthogonal sagittal images only (method A); orthogonal sagittal and additional oblique sagittal images (method B); and orthogonal sagittal and oblique coronal images (method C). The sensitivity, specificity, and accuracy for the diagnosis of an ACL tear and the detection of the condition of the ACL remnant tissue by the use of each method were calculated in comparison with arthroscopic findings as the reference standard., Results: The arthroscopic records revealed 27 knees with intact ACLs and 27 with torn ACLs. Among the 27 knees with torn ACLs, 9 did not have continuous remnant tissue and 18 had certain remnant tissue attached to the femur or the posterior cruciate ligament. The specificities and accuracies of methods B and C for diagnosing an ACL tear were higher than those for method A. The sensitivity, specificity, and accuracy of method C for the detection of ACL remnant tissue were higher than those for method A and B., Conclusions: Additional use of oblique MRI improved the accuracy of diagnosis of ACL tear and showed a reasonable level of efficacy in detecting ACL remnant tissue., Level of Evidence: Level IV (case series)., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Colonoscopic examination is the common pathway for positive screening tests detecting colorectal lesions. We evaluated a specific, quantitative high-throughput automatic immunochemical fecal occult blood test (Auto iFOBT) method for colorectal cancer (CRC) screening and to determine its concordance with physician assessments informed by complete colonoscopy, the gold-standard technique for evaluation of the colonic mucosa., Methods: 1200 CRC symptomatic patients were recruited for a retrospective investigation. Colorectal neoplasia were localized by colonoscopy and cancer outcomes were enumerated according to severity. In addition, stool samples were collected and analyzed by Auto iFOBT to derive sensitivity, specificity, and positive predictive value. Qualitative colonoscopy and Auto iFOBT results were correlated, as were cancer severities and quantitative hemoglobin concentrations., Results: Ninety-one patients were found positive for CRC; 50 mucosal, 20 submucosal, and 21 advanced. At standard cutoff, sensitivity was 60%, 90%, and 95%, respectively. Specificity and positive predictive value for all neoplasia and cancers were 89.6% and 86.4%, and 60.9% and 33.7%, respectively. Cancer severities could be approximated roughly according to hemoglobin concentrations., Conclusions: Specific qualitative 2-day Auto iFOBT is an accurate tool for the detection of colorectal cancer and therefore provides the basis for a large-scale screening program., (Copyright 2010 Elsevier B.V. All rights reserved.)

Introduction: The authors developed the 3D-cephalogram via combination of 3D-CT and cephalometric analysis., Methods: The 3D-cephalogram can directly express cephalometric data on the surface of skeletal structures in a manner similar to a conventional cephalogram., Results: This analysis revealed complex facial asymmetries in particular, on either side of the mandible. Images displayed no geometric error due to magnification nor any overlap effect inherent in the conventional cephalogram., Conclusions: The 3D-cephalogram is helpful in instructing physicians, and patients themselves regarding pathological anatomy and treatment procedures.

The regeneration of lens tissue from the iris of newts has become a classical model of developmental plasticity, although little is known about the corresponding plasticity of the mammalian iris. We here demonstrate and characterize multipotent cells within the iris pigment epithelium (IPE) of postnatal and adult rodents. Acutely-isolated IPE cells were morphologically homogeneous and highly pigmented, but some produced neurospheres which expressed markers characteristic of neural stem/progenitor cells. Stem/progenitor cell markers were also expressed in the IPE in vivo both neonatally and into adulthood. Inner and outer IPE layers differentially expressed Nestin (Nes) in a manner suggesting that they respectively shared origins with neural retina (NR) and pigmented epithelial (RPE) layers. Transgenic marking enabled the enrichment of Nes-expressing IPE cells ex vivo, revealing a pronounced capacity to form neurospheres and differentiate into photoreceptor cells. IPE cells that did not express Nes were less able to form neurospheres, but a subset initiated the expression of pan-neural markers in primary adherent culture. These data collectively suggest that discrete populations of highly-pigmented cells with heterogeneous developmental potencies exist postnatally within the IPE, and that some of them are able to differentiate into multiple neuronal cell types.

Neural stem cells/progenitors that give rise to neurons and glia have been identified in different regions of the brain, including the embryonic retina and ciliary epithelium of the adult eye. Here, we first demonstrate the characterization of neural stem/progenitors in postnatal iris pigment epithelial (IPE) cells. Pure isolated IPE cells could form spheres that contained cells expressing retinal progenitor markers in non-adherent culture. The spheres grew by cell proliferation, as indicated by bromodeoxyuridine incorporation. When attached to laminin, the spheres forming IPE derived cells were able to exhibit neural phenotypes, including retinal-specific neurons. When co-cultured with embryonic retinal cells, or grafted into embryonic retina in vivo, the IPE cells could also display the phenotypes of photoreceptor neurons and Muller glia. Our results suggest that the IPE derived cells have retinal stem/progenitor properties and neurogenic potential without gene transfer, thereby providing a novel potential source for both basic stem cell biology and therapeutic applications for retinal diseases.

Aim: The purpose of the study was to assess the efficacy of bone grafting from the mandibular outer cortex for reconstructing the orbital walls., Material and Methods: Bone grafting was performed in 75 patients. The site the transplants were harvested from were: A: mental region, B: area posterior to the mental foramen, C: ramus region. In order to obtain the appropriate curvature for the orbital floor, proper selection of the donor area is required. The bony defect size was confirmed pre-operatively from 3D-CT data. Bone, characteristically 2-3 mm thick, was harvested from each area and grafted into the blow-out fractures., Results: Out of the 75 patients 13 cases underwent reconstruction using mandibular outer cortex bone from area A, 8 from area B, and 54 from area C. The maximum size available for harvest from area C was 7 x 4 cm; material from this area could also be used for the repair of both medial and inferior orbital wall defects if necessary., Conclusion: Bone harvest from the mandible affords several advantages including (1) ease of harvest, (2) ease of trimming, (3) appropriate size and curvature, (4) absence of functional disability, (5) no secondary deformity, (6) no visible scars, (7) post-operative immobilization not necessary, (8) absence of post-operative difficulties with respect to breathing and walking and (9) major complications are rare.

Members of a series of diarylmethanols, diarylmethyl pivalates, and diarylmethyl acetates (analyte sets 1-26) were enantioresolved with the (S,S)-Whelk-O1 chiral stationary phase (CSP). An analogue of the (S,S)-Whelk-O1 selector was combined with enantioenriched samples of the various diarylmethyl pivalates and thereby used as a chiral solvating agent (CSA) for high field 1H NMR studies. The absolute configurations of a number of chiral diarylmethyl pivalates were assigned using this approach, and hydrolysis of the pivalates allowed assignment of the absolute configurations of the corresponding diarylmethanols. Chromatographic, 1H NMR, and X-ray evidence are given in support of a chiral recognition model for the enantioresolution of diarylmethyl esters on this CSP.

We investigated the ability of adult human RPE cells to differentiate into neurons. Two cell lines were evaluated. The cells were cultured in medium with 8% serum, transferred to a neural stem cell maintenance culture, and induced to differentiate with retinoic acid. The cells were immunocytochemically examined at each step. The cells showed epithelial-like morphology with 8% serum and all were immunoreactive for beta-III tubulin. After transfer to the stem cell maintenance culture, they changed morphologically and became immunoreactive for MAP5 and neurofilament200 after inducement with retinoic acid. The ratio of MAP5 positive cells was higher in the young adult RPE cell line. No GFAP or rod-opsin immunoreactive cells were observed. Adult human RPE cells even from old person are capable of differentiating into neurons, although the ratio of mature neurons was greater in the young than in the old cell line in this condition.

Background: We encountered two out of 100 cases in which implanted biodegradable plates and screws had fractured within 1 month postoperatively., Material: Failure of the material was confirmed through clinical symptoms, radiographs or CT findings. In addition, four specimens obtained from these two cases were examined with regard to their ultrastructure using scanning electron microscopy., Results: Several principal patterns of the fractured surface were found: (1) gradual cracking, i.e. 'circular stair' and, (2) tortuous threads, i.e. a wavy line. It is conceivable that the material may not have been hit by major sudden forces but a disproportion between the thread configuration and the drilled hole may have led to screw loosening and torsion. Subsequently, the threads were deformed in a 'wavy' manner, finally leading to cracking and fracture of plates and screws. Fractures of plates and screws due to these instabilities are thought to be distinguishable from material resorption., Conclusion: In the application of biodegradable materials, more than two screws per single bone segment should be used as a principle of plate-fixation technique in order to avoid a stability-compromising situation, particularly in the stress-bearing areas of the maxillofacial region. Moreover, three-dimensional fixation using more than two plates is recommended in the facial skeleton e.g. zygomatic tripod. Intermaxillary fixation should also be considered to reinforce initial stability in stress-bearing areas.

Background: In the craniofacial skeleton, osseous fixation techniques for stabilization include stainless steel wire, miniplates or bioresorbable plates; in some cases, stainless steel wires are still indicated. Most recently, several case reports have demonstrated that microplates or stainless steel wires migrate intracranially when used in the craniofacial skeleton in neonates., Patient: We present the case of a 7-year-old male who underwent treatment of brachycephaly at the age of 5 months. In this patient, internal migration of wires was observed., Conclusion: In our opinion, there is a difference between migration of wires and of plates in the growing cranium. Factors affecting the incidence of material migration in cranioplasty are believed to include (1) age, (2) site and (3) the material itself.

The incidence of solitary neurofibroma of the facial nerve originating in the parotid region is extremely low. We report a case of a solitary neurofibroma in a 30-year-old male, who initially presented with a parotid mass without facial paresis or paralysis. A chain of small nodules had been palpable in the right parotid region for the previous 2-3 years. MRI and CT scans revealed several small ovoid lesions extending from the frontal margin of the parotid gland to the retromandibular region. The lesions were surgically removed. The main trunk of the facial nerve was adherent to the dorsal side of the largest nodule; however, this mass was resected atraumatically. Histopathological examination indicated neurofibroma. The incidence, presentation, diagnosis and surgical treatment of intraparotid neurofibroma are discussed and compared with those of Schwannoma., (Copyright 2002 The British Association of Plastic Surgeons. Published by Elsevier Science Ltd. All rights reserved.)

Multiple myeloma (MM) cells cause devastating bone destruction by activating osteoclasts in the bone marrow milieu. However, the mechanism of enhanced bone resorption in patients with myeloma is poorly understood. In the present study, we investigated a role of C-C chemokines, macrophage inflammatory protein (MIP)-1alpha and MIP-1beta, in MM cell-induced osteolysis. These chemokines were produced and secreted by a majority of MM cell lines as well as primary MM cells from patients. Secretion of MIP-1alpha and MIP-1beta correlated well with the ability of myeloma cells to enhance osteoclastic bone resorption both in vitro and in vivo as well as in MM patients. In osteoclastogenic cultures of rabbit bone cells, cocultures with myeloma cells as well as addition of myeloma cell-conditioned media enhanced both formation of osteoclastlike cells and resorption pits to an extent comparable to the effect of recombinant MIP-1alpha and MIP-1beta. Importantly, these effects were mostly reversed by neutralizing antibodies against MIP-1alpha and MIP-1beta, or their cognate receptor, CCR5, suggesting critical roles of these chemokines. We also demonstrated that stromal cells express CCR5 and that recombinant MIP-1alpha and MIP-1beta induce expression of receptor activator of nuclear factor-kappaB (RANK) ligand by stromal cells, thereby stimulating osteoclast differentiation of preosteoclastic cells. These results suggest that MIP-1alpha and MIP-1beta may be major osteoclast-activating factors produced by MM cells.

Oculocardiac reflex has been recognized as the result of mechanical stimulation to the orbital tissue. The authors encountered a case of severe arrhythmia due to oculocardiac reflex in a patient with a zygomatic fracture. Previous health examinations suggested no abnormalities in the heart in his schooldays, and the initial diagnosis of his arrhythmia as complete A-V block due to injury (using ECG and cardiac ultrasonography). Because his arrhythmia did not improve spontaneously, he underwent cardiac pacing. After repair of the fracture, his arrhythmia completely disappeared. The pacemaker was removed on the first postoperative day. The pathogenesis of this rare case will be discussed.

To develop a new immunotherapy for multiple myeloma, we have generated a monoclonal antibody (MoAb) that detects a human plasma cell-specific antigen, HM1.24. Our previous study has shown that mouse anti-HM1.24 MoAb inhibits the proliferation of human myeloma cells implanted into severe combined immunodeficiency mice. In this report, we evaluated the antitumor activity of the humanized anti-HM1.24 MoAb (IgG1kappa), which was constructed by grafting the complementarity-determining regions. In contrast to the parent mouse MoAb, humanized anti-HM1.24 MoAb mediated antibody-dependent cellular cytotoxicity (ADCC) against both myeloma cell lines and myeloma cells from patients in the presence of human peripheral blood mononuclear cells (PBMCs). The PBMCs from untreated myeloma patients exhibited ADCC activity as efficiently as those of healthy donors. Although decreased ADCC activity of PBMCs was observed in patients who responded poorly to conventional chemotherapy, it could be significantly augmented by the stimulation with interleukin-2 (IL-2), IL-12, or IL-15. There was a strong correlation between the percentage of CD16(+) cells and ADCC activity in the PBMCs of myeloma patients. Moreover, peripheral blood stem cell collections from myeloma patients contained higher numbers of CD16(+) cells than PBMCs and exhibited ADCC activity that was enhanced by IL-2. These results indicate that humanized anti-HM1.24 MoAb has potential as a new therapeutic strategy in multiple myeloma and that treatment of effector cells with immunomodulating cytokines can restore the effect of humanized anti-HM1.24 MoAb in patients with diminished ADCC activity.

HM1.24 antigen has been identified as a surface molecule preferentially expressed on terminally differentiated B cells, and its overexpression is observed in multiple myeloma cells. The HM1.24 antigen is, therefore, expected as a most potent target molecule for antibody-based immunotherapy for multiple myeloma. Here, we have identified the cDNA for human HM1.24 antigen and also analyzed its gene structure including the promoter region. The HM1.24 antigen is a type II membrane glycoprotein, which has been reported as a bone marrow stromal cell surface antigen BST2, and may exist as a homodimer on myeloma cell surface. Although a reason for the overexpression in myeloma cells is not understood, very interestingly, the promoter region of the HM1.24 gene has a tandem repeat of three cis elements for a transcription factor, STAT3, which mediates interleukin-6 (IL-6) response gene expression. Since IL-6 is a differentiation factor for B cells, and known as a paracrine/autocrine growth factor for multiple myeloma cells, the expression of HM1.24 antigen may be regulated by the activation of STAT3. Importantly, a humanized anti-HM1.24 antibody effectively lysed the CHO transformants which expressed HM1.24 antigen as high as human multiple myeloma cells, but not the cells with lower antigen expression. This evaluation shows that ADCC heavily depends on the expression level of target antigens and, therefore, the immunotherapy targeting the HM1.24 antigen should have a promising potential in clinical use., (Copyright 1999 Academic Press.)

Erythropoietin is known to be an essential hemopoietic growth factor for maturation of erythroid progenitor cells. Like other hemopoietic growth factors, erythropoietin acts as a survival factor that supports maturation of the erythroid progenitor through the suppression of apoptosis. It is unclear whether erythropoietin can also induce differentiation, or if another external regulator is needed to initiate this process. The present study using murine cell lines revealed that maturation of the erythroid lineage requires costimulation by activin A and erythropoietin. Erythropoietin alone dose not induce differentiation and cells stimulated by activin A alone undergo apoptotic death. Costimulation with erythropoietin and activin A, however, rescues the cells from apoptotic death and permits differentiation. Two-step cultivation showed that cells pretreated with activin A no longer need activin A and differentiate in the presence of erythropoietin alone. The action of activin A commits the cell to death or to differentiation, and the presence of erythropoietin enables differentiation through suppression of apoptosis.

Multiple myeloma remains an incurable malignancy because of marked resistance of tumor cells to conventional chemotherapeutic agents. Alternative strategies are needed to solve these problems. To develop a new strategy, we have generated a monoclonal antibody (MoAb), which detects a human plasma cell-specific antigen, HM1.24. In this report, we evaluated the in vivo antitumor effect of unconjugated anti-HM1.24 MoAb on human myeloma xenografts implanted into severe combined immunodeficiency (SCID) mice. Two models of disseminated or localized tumors were established in SCID mice by either intravenous or subcutaneous injection of human myeloma cell lines, ARH-77 and RPMI 8226. When mice were treated with a single intraperitoneal injection of anti-HM1.24 MoAb 1 day after tumor inoculation, the development of disseminated myeloma was completely inhibited. In mice bearing advanced tumors, multiple injections of anti-HM1.24 MoAb reduced the tumor size and significantly prolonged survival, including tumor cure, in a dose-dependent manner. The proliferation of cultured human myeloma cells was inhibited in vitro by anti-HM1.24 IgG-mediated complement-dependent cytotoxicity, but not by the antibody alone. Moreover, spleen cells from SCID mice mediated antibody-dependent cell cytotoxicity against RPMI 8226 cells. These results indicate that anti-HM1.24 MoAb can be used for immunotherapy of multiple myeloma and related plasma cell dyscrasias.

Human pancreatic ribonuclease 1 (hRNase 1) in the mature form has been produced in E. coli using T7 expression system. The recombinant hRNase 1 protein was solubilized from the inclusion bodies, refolded in glutathione redox system, and purified through chromatographic procedures by utilizing cation-exchange and reversed-phase columns. The ribonucleolytic activity of recombinant hRNase 1 was examined on yeast RNA and cytidylyl-3',5'-adenosine revealing the distinctive ribonucleolytic activity. The activity was perfectly inhibited by human placental RNase inhibitor. Truncation of 7 amino acid residues in the amino-terminal sequence resulted in much reduction in ribonucleolytic activity and in affinity to human placental RNase inhibitor with the disintegration of secondary structures of the protein observed by circular dichroism spectra. The present study has revealed the important contribution of the amino-terminal sequence of hRNase 1 to the characteristics of the protein.

V(D)J recombination and somatic hypermutations are developmentally regulated during B-cell differentiation; therefore, DNA analysis of the Ig gene delineates the cellular origin of B-cell neoplasms. We analyzed the third complementarity-determining region and adjacent regions of the Ig heavy-chain gene of tumor cells from 7 patients with Waldenström's macroglobulinemia (WM) and from 10 patients with B-cell chronic lymphocytic leukemia (CLL), 2 of whom progressed to high-grade non-Hodgkin's lymphoma (NHL), ie, Richter's syndrome (RS). There were no intraclonal variations resulting from VH replacements or ongoing somatic mutations in both WM and CLL. We found replacement mutations in the D and/or JH segments in all patients with WM and in 4 of the 10 patients with CLL, including the 2 RS patients. Replacement mutations were clustered in codon 102 of the JH segment. Preferential utilization of the JH4 gene was found in WM (5 of 7 [71.4%]) and in CLL (7 of 10 [70.0%]), and DXP family genes in CLL (5 of 10 [50.0%]). In conclusion, WM and CLL with RS are generated under the influence of antigenic stimulation and selection. However, the majority of CLL may arise from a distinct subpopulation that has the restricted repertoire of nonmutated Ig genes.

A monoclonal antibody (MoAb) that defines a novel terminal B-cell-restricted antigen, termed HM1.24, was developed against a human plasma cell line. The MoAb, designated anti-HM1.24, reacted with five different human myeloma cell lines, as well as with monoclonal neoplastic plasma cells obtained from the bone marrow or peripheral blood of patients with multiple myeloma or Waldenström's macroglobulinemia. The HM1.24 antigen was also expressed by mature Ig-secreting B cells (plasma cells and lymphoplasmacytoid cells) but not by other cells contained in the peripheral blood, bone marrow, liver, spleen, kidney, or heart of normal individuals or patients with non-plasma-cell-related malignancies. The anti-HM1.24 MoAb bound to human myeloma RPMI 8226 cells with an affinity constant of 9.2 x 10(8) M-1, indicating approximately 84,000 sites/cell. By immunoprecipitation assay under reducing conditions, this MoAb identified a membrane glycoprotein that had a molecular weight of 29 to 33 kD. Our studies indicate that the HM1.24-related protein represents a specific marker of late-stage B-cell maturation and potentially serves as a target antigen for the immunotherapy of multiple myeloma and related plasma cell dyscrasias.

The effect of erythroid differentiation factor (EDF) on the maintenance of human hematopoietic progenitors in a microenvironment was examined by co-culture of adherent- and E rosette-depleted mononuclear cells from the bone marrow (BM) or peripheral blood (PB) with allogenic stromal cells. EDF had no effect on colony formation of erythroid burst-forming units (BFU-E) from the BM cultured without a stromal layer. The number of BFU-E cultured with the stromal layer was decreased less in the presence of EDF than in its absence. This activity of EDF was also observed when the mononuclear cells were separated from the stromal layer by a filter membrane. These data suggest that EDF facilitates maintenance of the number of BFU-E through a humoral factor(s) secreted by the stromal layer. The number of BM erythroid colony-forming units (CFU-E) was decreased on addition of EDF, which promotes differentiation of CFU-E. The number of PB CFU-E was increased irrespective of the presence or absence of EDF over 2 weeks, suggesting that BFU-E, which are more abundant in PB than in BM, differentiate to supply CFU-E. However, the addition of EDF resulted in less increase of PB CFU-E, indicating that it inhibited the proliferation of CFU-E progenitors to suppress colony formation. On the other hand, CFU-GM was consistently decreased by addition of EDF to this culture system. These data indicate that EDF acts as a commitment factor and/or a promoter of erythroid progenitors in a hematopoietic microenvironment.

We examined the effects of activin A on the proliferation and differentiation of immature hematopoietic progenitors prepared from peripheral blood (PB) using methylcellulose and liquid-suspension culture. In a kinetic analysis, colony formation by PB granulocyte-macrophage colony-forming unit (CFU-GM) was delayed in a dose-dependent manner by the addition of activin A only when stimulated with interleukin-3 (IL-3), but not when stimulated with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), or stem cell factor (SCF) plus G-CSF. DNA-synthesizing CFU-GM was increased by IL-3, but this effect was abolished by activin A. In contrast, PB erythroid burst-forming unit (BFU-E) was accelerated by the addition of activin A only when exposed to IL-3 plus erythropoietin (Epo), but not when exposed to Epo or Epo plus SCF. DNA-synthesizing BFU-E was increased by IL-3 and activin A, alone and additively in combination. In a mixed culture of myeloid and erythroid progenitors, activin A increased the numbers of BFU-E and CFU-Mix colonies at concentrations of 1 and 10 ng/mL and decreased the number of CFU-GM colonies in a dose-dependent manner. However, in a liquid-suspension culture of erythroid progenitors, activin A decreased total cell count and the percentage of hemoglobin-containing cells only when cells were exposed to IL-3 plus Epo. These results indicate that activin A suppresses the proliferation of IL-3-responsive CFU-GM progenitors and stimulates the proliferation and differentiation of IL-3-responsive BFU-E progenitors, and suggest that activin A acts as a commitment factor of immature hematopoietic progenitors for erythroid differentiation.

The pathophysiologic significance of proteasomes in hematologic malignancies was examined by comparison of the proteasome levels in normal subjects and patients with benign liver diseases. The serum proteasome level measured by enzyme-linked immunosorbent assay was found to be positively correlated with the tumor burden of the patients with hematologic malignancies such as acute leukemia, chronic myelogenous leukemia, non-Hodgkin's lymphoma, and myeloma. Immunohistochemical staining showed that proteasomes were strongly expressed in these tumor cells, especially in the nuclei. These data suggest that the elevated levels of serum proteasomes in these patients are derived from tumor cells, reflect the tumor burden, and so provide prognostic information. However, in patients with benign liver diseases, serum proteasome levels correlated with serum alanine aminotransferase activities, suggesting that in hematologic malignancies associated with liver injury some of the serum proteasomes may originate from hepatocytes. The marked production of proteasomes by malignant blood cells may be involved in transformation and proliferation of these cells.

Activin A/EDF, initially found as a differentiation inducer of murine Friend erythroleukemia, also has a stimulatory effect on erythropoiesis in vitro and in vivo. Here we proved activin A/EDF activity in human serum. The activin A/EDF level in 18 normal human serum samples was measured by a specific bioassay and was found to be 8.3 +/- 4.6 ng/ml, indicating that there exists sufficient activity to affect erythropoiesis in normal serum. In contrast, activin A/EDF activity was reduced in the chronic renal failure patients and 23/26 serum samples examined showed levels below 1.2 ng/ml. Further analysis using HPLC revealed that chronic renal failure serum actually contained as much activin A/EDF as normal serum, and that the difference between normal and patient serum existed in the content of a specific inhibitor of activin A/EDF. This observation suggests the possibility that the inhibitor is participating in the regulation of activin A/EDF activity in vivo in chronic renal failure patients and also the possibility of activin A/EDF could be utilized in the therapy of the anemia of such patients.

The effects of erythroid differentiation factor (EDF) on the levels of zeta-globin and several proto-oncogene mRNAs and transferrin receptors (Tf-R) of K-562 cells were examined. EDF decreased Tf-R expression and increased the level of zeta-globin mRNA. The mRNA level of c-fos began to rise within 3 hours and continued to increase up to 72 hours, but the levels of c-myb and c-abl decreased to 23 and 19%, respectively, of their initial levels after 48 hours. In contrast, the mRNA levels of c-myc and c-fms decreased transiently, but recovered within 48 hours. The modulation of several proto-oncogene mRNA levels was observed much earlier than the differentiation induction and the growth inhibition of K-562 cells by EDF.

Embryonal carcinoma(EC) cells, the undifferentiated stem cells of teratocarcinomas, have many properties in common with pluripotent embryonic cells, and thus provide an excellent system for studying the early events involved in embryonic development and stem cell differentiation. We have isolated three novel mutants with temperature-sensitive(ts) cell growth that were able to differentiate at a non-permissive temperature for cell growth. These mutations affect the progression of the cell cycle, leading to the transient accumulation of cells in a specific phase, the S phase, of the cell cycle, which is likely to be the primary cause of stem cell differentiation of EC cells at non-permissive temperature. Isolation of these mutants strongly supports the notion that there is a close association between the inhibition of DNA synthesis and EC cell differentiation.

The in vivo effect of human EDF on erythroid precursors (CFU-E and BFU-E) was investigated in normal and bled mice. In anemic (bled) mice, EDF treatment led to significant dose-dependent rises in the CFU-E and BFU-E levels of bone marrow. An elevation in the level of CFU-E was also seen in the spleen. In normal mice, a significant elevation in the level of bone marrow BFU-E was observed. Thus, EDF has an effect on erythropoiesis in anemic and normal mice in vivo.

Nucleoside analogues inhibiting DNA synthesis can induce cell differentiation in teratocarcinoma cells. We have examined how their abilities to induce F9 cell differentiation were specifically counterbalanced by their corresponding normal nucleosides. We have also compared the differentiation inducing ability of the wild type F9 cells with that of its thymidine kinase-less mutant using plasminogen activator, as a differentiation marker, which is expressed at a very early stage of endodermal cell differentiation and can be assayed quantitatively. The results obtained were clearly explainable by the conventionally accepted action mechanisms of the nucleoside analogues, thus strongly suggesting that their abilities to induce cell differentiation were direct consequences of the inhibition of DNA synthesis; thus this confirms the notion that a close association exists between the inhibition of DNA synthesis and the induction of teratocarcinoma stem cell differentiation.

Peripheral blood stem cells (PBSC) were collected for autotransplantation by a total of 46 continuous-flow leukaphereses in 17 children with various types of cancer in whom the stem-cell pool had been expanded by chemotherapy. As the cells collected by leukapheresis were contaminated with many visible cell clumps, platelets, and erythrocytes, they were separated from the platelet-rich plasma (PRP) by slow-speed centrifugation and fractionated on a discontinuous gradient of Percoll. All the hematopoietic progenitors (CFU-GM, CFU-GEMM) in the starting samples were recovered at the interface of 40% and 60% Percoll solutions largely free of other cellular components and with a substantial reduction in volume. The separation and freezing procedures could be completed within three hours after obtaining cells by leukapheresis. After their fractionation and storage, these PBSC were shown to be able to reconstitute normal hematopoiesis in ten children with poor prognosis leukemia or neuroblastoma for whom no HLA-compatible marrow donors were available and who had been subjected to marrow-ablative therapy. This separation procedure is simple, efficient, and readily available and can be used for children as a routine procedure for PBSC autotransplantation.

A sensitive enzyme immunoassay (EIA) for the detection of anti-thyroglobulin (Tg) antibodies was developed using Fab'-horseradish peroxidase (HRP) conjugate. Anti-Tg antibody was assayed by incubation with a thyroglobulin-coated polystyrene ball and then with affinity-purified anti-IgG Fab'-HRP conjugate. The HRP activity was assayed fluorimetrically. The sensitivity was 625 amol/tube and anti-Tg antibody levels between 0.5 and 200 ng/ml could be determined. The recoveries of anti-Tg antibody added to human sera at three different concentrations were 94.2-101.0%. Both within- and between-assay coefficients of variation were below 10%. Significant correlation was observed between values by the EIA and TGHA method (Kendall's rank correlation coefficient = 0.712, P less than 0.001). The present EIA for anti-Tg antibody is sensitive enough to determine anti-Tg antibody synthesized in vitro by the lymphocytes from patients with autoimmune thyroid disease and normal subjects. The amounts of anti-Tg antibody synthesized by peripheral lymphocytes from patients with Hashimoto's disease were significantly greater than those from patients with Graves' disease, although serum levels of anti-Tg antibody were usually elevated in both groups of patients. The results obtained suggest that anti-Tg antibody is synthesized in a different manner in patients with Hashimoto's disease and in patients with Graves' disease.