[Analysis of factors influencing lung function in patients with pneumoconiosis].

Objective: To analyze the influencing factors of lung function in pneumoconiosis patients, and to provide reference for clinical treatment. Methods: From July 2020 to December 2020, a questionnaire survey was conducted on pneumoconiosis patients in the jurisdiction by using the "Guangdong Province Occupational Disease Prevention and Control Institute" questionnaire, and the relevant items of patients were examined. The rate of counting data is expressed, and the measurement data is expressed by mean and standard deviation. Chi-square test was used for comparison between groups, trend chi-square test was used for trend analysis of ordered classified data. Multivariate analysis was carried out with binary logistic regression model. Results: A total of 1409 pneumoconiosis patients were enrolled. The abnormal rate of lung function in pneumoconiosis patients was 68.77%. The results of trend Chi-square test showed that the abnormal rate of lung function increased with the age of exposure to dust in different age groups (Chi Sqnare Trend=64.12、8.49、24.20, P<0.05) . In univariate analysis, there were statistical significance in different dust exposure age, working age, pneumoconiosis stage, complications and occupational pneumoconiosis diseases (P<0.05) . Multiple logistic regression results showed that age of exposure to dust, years of service, stage of pneumoconiosis and complications were the main influencing factors of lung function in pneumoconiosis patients (P<0.05) . Compared with patients aged 0-30 years, patients aged 50-70 years and older had a higher rate of abnormal lung function (OR=2.16, 95%CI: 1.12~4.16; OR=4.82, 95%CI: 2.05~11.35, all P<0.05) ; Compared with patients with 0~20 years of service, patients with 20~30 years of service and more than 30 years of service had a higher rate of abnormal lung function (OR=1.58, 95%CI: 1.10~2.25; OR=1.63, 95%CI: 1.28~2.40, P<0.05) ; Compared with stage Ⅰ patients, Stage Ⅱ and Stage Ⅲ patients had a higher rate of abnormal lung function (OR=1.62, 95%CI: 1.20~2.17; OR=2.23, 95%CI: 1.40~3.55, all P<0.05) ; Compared with patients without comorbidities, patients with comorbidities had a higher rate of abnormal lung function (OR=1.68, 95%CI: 1.20~2.38, P<0.05) . Conclusion: The factors such as age of exposure to dust, working age, stage of pneumoconiosis and complications may be the influencing factors of lung function in pneumoconiosis patients.

[Micro-CT analysis of tooth development of C57BL/6 mice strain].

Objective: To investigate the normal process of tooth development of C57BL/6 mouse strain by using micro-CT for better understanding about the tooth development of the human being and other species. Methods: A total of 54 C57BL/6 mice were used at postnatal day 1 (P1), P3, P7, P10, P14, P21, P28, P42 and P56 (n=6 for each age group). After euthanasia, the skulls and alveolar bones (with molars) were isolated and scanned by micro-CT scanner. After three dimensional reconstruction, the developmental status of the crown and root(s) for each tooth type was examined in different views. Results: The tooth development of mice from birth to mature (P56) could be divided into three stages. The first stage was from P1 to P14, in which the crowns of all the first, second and third molars had formed, while the roots had not fully developed yet. The second stage was from ablactation (P21) to P28, in which all the roots of the molars had reached their normal length, and the apical foramens had closed. Due to the mastication and occlusal abrasion, the incisors exhibited sharp cutting edges at the buccal enamel layer, and the corresponding molars formed a pit-to-fossa articulated relationship. The third stage was from P42 to P56, in which the root canal differentiation occurred, and 1-2 canal configuration was formed in several flat roots. The development of molar roots had completed and the apexes were enlarged due to the deposition of cementum around. Conclusions: In the process of mouse tooth development, the mineralization of the cusps, followed by crown formation and roots elongation, was precisely regulated in a spatial-temporal pattern. The incisors and the molars exhibited different modes of development.

[Immune checkpoint inhibitors induced pituitary immune-related adverse events: diagnosis and management].

Immune checkpoint inhibitors (ICIs) have been used in treating a wide variety of cancers, but they challenge clinicians with a series of special immune related adverse events (irAEs) resulting from activated immune system. Since June 2018, when the first programmed cell death 1 (PD-1) inhibitor, nivolumab, was approved by the National Medical Products Administration (NMPA), abundant experience has been accumulated in coping with irAEs from PD-1 and PD-1 ligand 1 (PD-L1) blockade therapies. In October 2021, the first CTLA-4 inhibitor, ipilimumab, which has a different spectrum of irAEs was also approved by NMPA. The discrepancy in clinical features of pituitary irAEs is obvious between these two types of ICIs. Pituitary irAEs include hypophysitis and hypopituitarism. In this review of latest literature, we have summarized the incidence, possible mechanisms, time of onset, clinical presentations, hormone test, pituitary imaging, treatment strategies and recovery patterns of pituitary irAEs. By referring to domestic and foreign clinical guidelines, we have proposed practical suggestions for screening, diagnosing and treating pituitary irAEs.

[Pituitary immune-related adverse events induced by programmed cell death protein 1 inhibitors in advanced lung cancer patients: A report of 3 cases].

Pituitary immune-related adverse events induced by programmed cell death protein 1 inhibitors in advanced lung cancer patients: A report of 3 cases SUMMARY Programmed cell death protein 1 (PD-1) and its ligand 1 (PD-L1) have been widely used in lung cancer treatment, but their immune-related adverse events (irAEs) require intensive attention. Pituitary irAEs, including hypophysitis and hypopituitarism, are commonly induced by cytotoxic T lymphocyte antigen 4 inhibitors, but rarely by PD-1/PD-L1 inhibitors. Isolated adrenocorticotropic hormone(ACTH) deficiency (IAD) is a special subtype of pituitary irAEs, without any other pituitary hormone dysfunction, and with no enlargement of pituitary gland, either. Here, we described three patients with advanced lung cancer who developed IAD and other irAEs, after PD-1 inhibitor treatment. Case 1 was a 68-year-old male diagnosed with metastatic lung adenocarcinoma with high expression of PD-L1. He was treated with pembrolizumab monotherapy, and developed immune-related hepatitis, which was cured by high-dose methylprednisolone [0.5-1.0 mg/(kg·d)]. Eleven months later, the patient was diagnosed with primary gastric adenocarcinoma, and was treated with apatinib, in addition to pembrolizumab. After 17 doses of pembrolizumab, he developed severe nausea and asthenia, when methylprednisolone had been stopped for 10 months. His blood tests showed severe hyponatremia (121 mmol/L, reference 137-147 mmol/L, the same below), low levels of 8:00 a.m. cortisol (< 1 µg/dL, reference 5-25 µg/dL, the same below) and ACTH (2.2 ng/L, reference 7.2-63.3 ng/L, the same below), and normal thyroid function, sex hormone and prolactin. Meanwhile, both his lung cancer and gastric cancer remained under good control. Case 2 was a 66-year-old male with metastatic lung adenocarcinoma, who was treated with a new PD-1 inhibitor, HX008, combined with chemotherapy (clinical trial number: CTR20202387). After 5 months of treatment (7 doses in total), his cancer exhibited partial response, but his nausea and vomiting suddenly exacerbated, with mild dyspnea and weakness in his lower limbs. His blood tests showed mild hyponatremia (135 mmol/L), low levels of 8:00 a.m. cortisol (4.3 µg/dL) and ACTH (1.5 ng/L), and normal thyroid function. His thoracic computed tomography revealed moderate immune-related pneumonitis simultaneously. Case 3 was a 63-year-old male with locally advanced squamous cell carcinoma. He was treated with first-line sintilimab combined with chemotherapy, which resulted in partial response, with mild immune-related rash. His cancer progressed after 5 cycles of treatment, and sintilimab was discontinued. Six months later, he developed asymptomatic hypoadrenocorticism, with low level of cortisol (1.5 µg/dL) at 8:00 a.m. and unresponsive ACTH (8.0 ng/L). After being rechallenged with another PD-1 inhibitor, teslelizumab, combined with chemotherapy, he had pulmonary infection, persistent low-grade fever, moderate asthenia, and severe hyponatremia (116 mmol/L). Meanwhile, his blood levels of 8:00 a.m. cortisol and ACTH were 3.1 µg/dL and 7.2 ng/L, respectively, with normal thyroid function, sex hormone and prolactin. All of the three patients had no headache or visual disturbance. Their pituitary magnetic resonance image showed no pituitary enlargement or stalk thickening, and no dynamic changes. They were all on hormone replacement therapy (HRT) with prednisone (2.5-5.0 mg/d), and resumed the PD-1 inhibitor treatment when symptoms relieved. In particular, Case 2 started with high-dose prednisone [1 mg/(kg·d)] because of simultaneous immune-related pneumonitis, and then tapered it to the HRT dose. His cortisol and ACTH levels returned to and stayed normal. However, the other two patients' hypopituitarism did not recover. In summary, these cases demonstrated that the pituitary irAEs induced by PD-1 inhibitors could present as IAD, with a large time span of onset, non-specific clinical presentation, and different recovery patterns. Clinicians should monitor patients' pituitary hormone regularly, during and at least 6 months after PD-1 inhibitor treatment, especially in patients with good oncological response to the treatment.

Clinical pharmacist interventions in managing Key Monitoring Drugs in China.

OBJECTIVE: Drug-related problems (DRPs) are common in hospitalized patients receiving Key Monitoring Drugs. Clinical pharmacy services have the potential to minimize drug-related harm and improve patient care. The aim of this study is to standardize the clinical application of Key Monitoring Drugs and reduce drug-related problems (DRPs) and associated costs, using clinical pharmacist interventions. PATIENTS AND METHODS: Clinical pharmacists formulate management measures for Key Monitoring Drugs using evidence-based medicine and analyze the DRPs of Key Monitoring Drugs in China at the Shandong Provincial Third Hospital over a period of five years, from 2015 to 2019. RESULTS: In 2019, the total cost of the use of Key Monitoring Drugs decreased by 10.12 million CNY, in comparison with the cost in 2015. The proportion of revenue generated from Key Monitoring Drugs also decreased by 11.49% compared with 2015. In addition, the cost per capita of Key Monitoring Drugs has gradually decreased; this resulted in a saving of 580.07 CNY per capita in 2019 compared with 2015. Over this time, the DRPs associated with Key Monitoring Drugs decreased by 45.50%. Through administrative intervention, prescription review, information management, and pharmaco-economic evaluation, a scientific management system for Key Monitoring Drugs has been established over this time, which standardizes the use of Key Monitoring Drugs and reduces their associated costs. CONCLUSIONS: Clinical pharmacists' interventions can assist in the early detection of drug-related problems associated with Key Monitoring Drugs and prevent any resulting harm to patients.

[Clinical analysis of 121 cases of urothelial carcinoma accompanied with multiple primary carcinoma].

Objective: To investigate the incidence and clinical characteristics of urothelial carcinoma (UC) accompanied with multiple primary carcinoma (MPC). Methods: The clinical data of 121 UC patients with MPC in Peking University Third Hospital from January 2010 to May 2018 were retrospectly analyzed. Results: UC patients with MPC accounted for 9.74% (121/1 242) of all the UC patients. The ratio of male to female patients was 2.10∶1 in the total MPC patients, but it was 1∶1 in the upper urinary tract MPC subgroup. The MPC patients were more common in elderly people, whose medium age was 68 (32-93) years old. Of all the location (131 person-time) of other tumors besides UC, the digestive system tumors occurred most frequently, accounting for 41.98% (55/131), followed by the urinary and male reproductive system tumors (20.61%, 27/131) and the female reproductive system (12.21%, 16/131). The proportion of the digestive system tumors (47.37%, 9/19) was the highest in the upper urinary tract MPC, with a total number of the other primary cancer of 19 person-time. However, the proportion of the urinary and male reproductive system tumors (37.14%, 13/35) was higher in the synchronous MPC group, with a total number of the other primary cancer of 35 person-time. Some patients had a history of radiotherapy and/or chemotherapy before UC was diagnosed. We also observed 2 cases of genetically confirmed Lynch syndrome. The median overall survival (mOS) of UC patients with MPC was 132 months, and the mOS of patients with UC as the first malignancy (including synchronous MPC and UC as the first malignancy in metachronous MPC) was 120 months. The mOS of the synchronous MPC group was 84 months, which was significantly shorter than 178 months of metachronous MPC group (χ(2) =14.029, P<0.001). Conclusions: The incidence of UC accompanied with MPC is not low, and the most common sites of MPC are the digestive system and reproductive system. Therefore, screening for MPC in UC patients, especially those with personal or family history of tumors, as well as elderly patients, may help early diagnosis and treatment of MPC patients and improve their prognoses.

[KRAS mutations analysis in mucinous epithelial lesions of the endometrium].

Objective: To investigate the frequency of KRAS mutation in mucinous epithelial lesions of the endometrium, and analyze the correlation between KRAS mutation and the clinicopathologic features. Methods: The cohort included forty-three cases of mucinous epithelial lesions of the endometrium selected from July 2015 to October 2017 from Beijing Obstetrics and Gynecology Hospital, and 22 control cases. Genomic DNA was extracted from formalin-fixed paraffin-embedded tissue sections. Polymerase chain reaction amplification for KRAS exons 2 and 3 was performed, followed by sequencing using capillary electrophoresis. The Fisher exact test was used to compare the prevalence of KRAS mutation among the different groups. Results: The patients'age ranged from 33 to 77 years [mean (55.12±9.34) years, median 55 years]. None of the eight cases of endometrial hyperplasia with mucinous differentiation without atypia showed KRAS mutation. The frequency of KRAS mutations was 1/10 in endometrial atypical hyperplasia, 1/12 in endometrioid carcinoma, 4/11 in endometrial atypical hyperplasia with mucinous differentiation (EAHMD), 6/15 in endometrioid carcinoma with mucinous differentiation (ECMD) and 8/9 in mucinous carcinoma (MC), respectively. The differences were statistically significant between MC versus EC (P<0.01) and MC versus ECMD (P<0.05). Conclusion: The high frequency of KRAS mutation in EAHMD, ECMD and MC indicates that KRAS mutational activation is implicated in the pathogenesis of endometrial mucinous carcinoma.

[Effects of glycyrrhizic acid on ERK1/2 and p38 MAPK signaling pathway in a murine model of asthma].

Objective: To explore the effects of glycyrrhizic acid (GA) on Extracellular regulated protein kinases (ERK1/2) and p38 mitogen-activated protein kinases (p38 MAPK) signaling pathways in a murine model of asthma. Methods: Sixty female BALB/c mice were randomly divided into 6 groups (n=10 each): a control group, an asthmatic group, two treatment groups with low and high doses of GA, U0126 group and SB203580 group. Within 24 hours after the last OVA challenge, histological studies of lung were conducted with the hematoxylin and eosin staining (HE) and alcian blue-periodic acid-Schiff (AB-PAS), the relative protein expression of ERK1/2 and p38 MAPK were detected by immunohistochemistry and Western blotting in vivo. CD4(+) T cells were purified from spleens of OVA-sensitized and challenged mice by using the Mouse CD4 Cell Positive Isolation Kit and incubated with anti-CD3 mAb (1 µg/ml) in the presence of various concentrations of GA (10 and 100 µg/ml), U0126 (10 µmol/L) or SB203580(10 µmol/L). After 72 h of incubation, the relative protein expression of ERK1/2 and p38 MAPK of CD4(+) T cells were detected by Western blotting in vitro. Results: The asthmatic mice induced infiltration of inflammatory cells around airways and blood vessels, airway goblet cell hyperplasia and mucus production. Administration of GA at a dose of 100 mg/kg, U0126 or SB203580 significantly reduced the infiltration of inflammatory cells in the peribronchial areas and goblet cell hyperplasia compared with the asthmatic mice. The protein expressions of p-ERK1/2 were lower in GA at a dose of 100 mg/kg (0.090±0.022) and U0126 group (0.072±0.017) than those in asthmatic group (0.143±0.022) (all P<0.05). The protein expressions of p-p38 MAPK were lower in GA at a dose of 100 mg/kg (0.072±0.019) and SB203580 group (0.061±0.015) than those in asthmatic group (0.121±0.022) (all P<0.05) by immunohistochemistry. Compared with asthmatic group (0.783±0.133, 0.649±0.095), the protein expressions of p-ERK1/2 and p-p38 MAPK in GA at a high dose group (0.385±0.186, 0.275±0.089) and in U0126 group (0.117±0.051) or in SB203580 group (0.108±0.043) were decreased by Western blotting (all P<0.05). The expressions of p-ERK1/2 in CD4(+) T cells after 72 h incubation were lower in 100 µg/ml concentrations of GA (0.579±0.184) and group U0126 (0.249±0.082) and the expressions of p-p38 MAPK were much lower in 100 µg/ml concentrations of GA (0.445±0.081) and group SB203580 (0.249±0.082) compared with those in group CD3 (1.028±0.147, 0.902±0.107) (all P<0.05). Conclusion: ERK1/2 and p38 MAPK signaling pathways are activated in asthmatic mice and GA may negative regulate this activation.

[Role and mechanism of Th17/Treg in obstructive sleep apnea hypopnea syndrome].

Objective: To explore the role and the mechanism of Th17/Treg in obstructive sleep apnea hypopnea syndrome (OSAHS). Methods: A total of 100 patients who completed polysomnography (PSG) in the sleep lab of Affiliated Wujiang Hospital of Nantong University from Mar. 2015 to Apr. 2016 were enrolled and divided into four groups (primary snoring as the control group, mild OSAHS, moderate OSAHS and severe OSAHS) according apnea hypopnea index (AHI). The proportion of Th17, Treg (of CD4+ T cells) and the expression of interleukin (IL)-17A, IL-6 were detected and the relevant data were analyzed by the correlation analysis and the multiple stepwise regression analysis. Results: Compared with the control group, the OSAHS patients had higher Th17% [(1.36±0.46)%, (1.68±0.30)%, (2.23±0.03)% vs (1.02±0.22)%], Th17/Treg [(0.22±0.07), (0.28±0.10), (0.29±0.00) vs (0.13±0.03)], IL-17A [(2.53±0.89), (2.99±1.96), (7.77±1.63) vs (1.45±0.78) ng/L], IL-6 [(6.14±4.37), (9.41±4.66), (12.58±6.65) vs (5.44±3.13) ng/L] and lower Treg% [(7.57±0.16)%, (6.46±1.57)%, (6.10±1.19)% vs (8.02±1.45)%] (all P<0.05). A positive correlation could be seen between Th17%, Th17/Treg, IL-17A, IL-6 and AHI, oxygen desaturation index (ODI) respectively, there was a negative correlation between Th17%, Th17/Treg, IL-17A, IL-6 and the lowest oxygen saturation (SpO2) (all P<0.05). The proportion of Treg had a negative correlation with AHI or ODI and a positive correlation with the lowest SpO2 (all P<0.05). The lowest SpO2 was the most important factor which could influence Th17%, Treg% and the radio of Th17/Treg. Conclusions: There is an imbalance of Th17/Treg in OSAHS. Therefore, Th17 and the relevant inflammatory cytokines may be involved in the occurrence and development of OSAHS.

Distribution and Characteristics of Colletotrichum spp. Associated with Anthracnose of Strawberry in Hubei, China.

Anthracnose caused by Colletotrichum spp. is a serious disease of strawberry. The etiology of anthracnose of strawberry is complex, and several Colletotrichum spp. have been regarded as causal agents. In the present study, multilocus (actin, ß-tubulin, calmodulin, glyceraldehyde-3-phosphate dehydrogenase, and chitin synthase) phylogenetic analysis revealed that 100 isolates of Colletotrichum associated with anthracnose of strawberry in central China belong to five species. In total, 97 isolates were identified belonging to the Colletotrichum gloeosporioides species complex, with C. murrayae, C. gloeosporioides, C. fructicola, and C. aenigma accounting for 81, 8, 4, and 4% of the total isolates, respectively. Three isolates belonging to the C. acutatum complex were identified as C. nymphaeae. On inoculated strawberry plants, isolates of C. fructicola and C. murrayae species showed strong pathogenicity to both leaves and petioles of strawberry, with plant mortality 30 days after inoculation of 77.8 and 55.6%, respectively. C. gloeosporioides, C. aenigma, and C. nymphaeae showed strong pathogenicity to leaves but weak pathogenicity to petioles, with plant mortality 30 days after inoculation of 5.6, 16.7, and 11.1%, respectively. The five species were divided into four classes based on their maximum growth temperatures. Isolates of C. murrayae and C. gloeosporioides were more tolerant to high temperature (>34°C) than isolates of other species, followed by C. fructicola and C. aenigma. Isolates of C. nymphaeae, which are only distributed in areas of higher altitude (1,100 m), were highly sensitive to higher temperature. These results indicate that pathogenicity and adaptation to temperature are important factors in the distribution of Colletotrichum spp. on strawberry plants. This research may increase our understanding of how Colletotrichum spp. emerge and spread to geographical regions with different latitudes or elevations.

Potential genotoxicity from integration sites in CLAD dogs treated successfully with gammaretroviral vector-mediated gene therapy.

Integration site analysis was performed on six dogs with canine leukocyte adhesion deficiency (CLAD) that survived greater than 1 year after infusion of autologous CD34+ bone marrow cells transduced with a gammaretroviral vector expressing canine CD18. A total of 387 retroviral insertion sites (RIS) were identified in the peripheral blood leukocytes from the six dogs at 1 year postinfusion. A total of 129 RIS were identified in CD3+ T-lymphocytes and 102 RIS in neutrophils from two dogs at 3 years postinfusion. RIS occurred preferentially within 30 kb of transcription start sites, including 40 near oncogenes and 52 near genes active in hematopoietic stem cells. Integrations clustered around common insertion sites more frequently than random. Despite potential genotoxicity from RIS, to date there has been no progression to oligoclonal hematopoiesis and no evidence that vector integration sites influenced cell survival or proliferation. Continued follow-up in disease-specific animal models such as CLAD will be required to provide an accurate estimate of the genotoxicity using gammaretroviral vectors for hematopoietic stem cell gene therapy.

Conversion of the severe to the moderate disease phenotype with donor leukocyte microchimerism in canine leukocyte adhesion deficiency.

Leukocyte adhesion deficiency-1 (LAD-1), a genetic immunodeficiency disease characterized by life-threatening bacterial infections, results from the defective adherence and migration of leukocytes due to mutations in the leukocyte integrin CD18 molecule. Canine LAD (CLAD) represents the canine homologue of the severe phenotype of LAD-1 in children. In previous studies we demonstrated that non-myeloablative stem cell transplantation from matched littermates resulted in mixed donor-host chimerism and reversal of the disease phenotype in CLAD. In this study, we describe two CLAD dogs with less than 2% donor leukocyte chimerism following non-myeloablative transplant. Both dogs are alive more than 24 months after transplant with an attenuated CLAD phenotype resembling the moderate deficiency phenotype of LAD. The improvement in the CLAD phenotype with very low levels of donor CD18(+) leukocytes correlated with the preferential egress of the CD18(+) neutrophils into extravascular sites. The clinical response with very low levels of donor CD18(+) leukocytes in CLAD supports using this model for testing gene therapy strategies since the low levels of gene-corrected hematopoietic cells expected with hematopoietic gene therapy would likely have a therapeutic effect in CLAD.

Identification of phase I and phase II metabolites of Guanfu base A hydrochloride in human urine.

Guanfu base A is a novel arrhythmic drug candidate isolated from the tuber of a traditional Chinese herb. Phase I and Phase II metabolites of Guanfu base A (GFA) Hydrochloride were studied in human urine by means of liquid chromatography mass spectrometry (LC/MSD) and tandem mass spectrometry (MS/MS). For phase I metabolites, Guanfu base I (GFI) was separated by HPLC and identified by comparison with authentic reference for their retention times, molecular ion peaks, fragment ions, and UV spectra. GFA oxide was also indicated to exist in human urine. For phase II metabolites, after human urine was treated either with glucuronidase or sulfatase, GFA occured in the chromatograms. It was suggested that there were GFA glucuronide and GFA sulfate in human urine. Further more, positive molecular ions, m/z 606 and m/z 510, of the two conjugates were detected in human urine by LC/MSD. In addition, characteristic ion of m/z 606 was identified as the precursor ion of m/z 177 [Glucuronic acid+H]+ by using MS/MS. Characteristic ion of m/z 430 [GFA+H]+ was also identified as a product ion of m/z 606 [GFA glucuronide+H]+. It was concluded that there were GFI. GFA oxide, GFA glucuronide and GFA sulfate in human urine.

Glucocorticoids down-regulate the extracellular matrix proteins fibronectin, fibulin-1 and fibulin-2 in bone marrow stroma.

Glucocorticoids regulate hematopoietic cell interactions with the bone marrow microenvironment, but the molecules involved in the regulation are still largely unknown. We have studied the effect of glucocorticoids on mRNA expression and protein synthesis of the major extracellular matrix adhesion protein fibronectin and three other extracellular proteins, fibulin-1, fibulin-2 and nidogen-1, in mouse bone marrow cultures and in a hematopoiesis supporting the stromal MC3T3-G2/PA6 cell line. Glucocorticoids suppressed mRNA expression and protein synthesis of fibronectin, fibulin-1 and fibulin-2, but not nidogen-1, in adherent cells of bone marrow cultures, as shown by Northern blot analysis and immunoprecipitation. mRNA levels of all four proteins were down-regulated by dexamethasone in MC3T3-G2/PA6 cells, indicating a direct glucocorticoid effect on cells synthesizing extracellular matrix proteins. Dexamethasone down-regulated fibronectin mRNA rapidly, within 2 h of treatment, in the stromal cells. This effect did not require mRNA or protein synthesis, as shown by Northern blot analysis after treatment by actinomycin D and cycloheximide. Interferon-alpha, which also has been reported to modulate haematopoietic cell-matrix interactions, did not affect mRNA expression of the proteins in MC3T3-G2/PA6 cells. Our results indicate that glucocorticoids down-regulate expression of several mesenchymal-type extracellular matrix molecules in bone marrow, but with a variable effect on different proteins. Thus one mechanism by which glucocorticoids regulate haematopoiesis may be by altering the relative proportions of extracellular matrix proteins.

Association of extracellular matrix proteins fibulin-1 and fibulin-2 with fibronectin in bone marrow stroma.

Extracellular matrix (ECM) molecules, together with growth factors and stromal cells, regulate haematopoietic cell development in bone marrow (BM). We report here expression of ECM proteins fibulin-1 and fibulin-2 in mouse BM. In other tissues, fibulin-1 and fibulin-2 associate with fibronectin and other ECM proteins. Fibulin-2 has also been found to adhere to cells via beta3 integrins. We studied the association of fibulins with fibronectin in BM stroma. By confocal microscopy, fibulin-1 and fibulin-2 immunostainings were co-localized with fibronectin in the adherent layer of long-term BM cultures. In cell adhesion assays using recombinant proteins, mouse fibulin-2 adhered to human erythroid-megakaryocytic leukaemia cell line HEL. This adhesion was mediated by beta3 integrins. However, HEL cells did not adhere to human fibulin-2. We therefore studied a possible species-specific cell-adhesive activity of mouse fibulin-2 by using mouse megakaryocytes, obtained by culture of BM cells in the presence of thrombopoietin. These megakaryocytes did not adhere to mouse fibulin-2. Our findings suggested that the functional role of fibulin-1 and fibulin-2 in BM stroma is related to binding to the major cell adhesion protein fibronectin, whereas adhesion of mouse fibulin-2 to human cells containing the integrin beta3 chain is not related to an apparent physiological function of the protein.

Exploration of the immunoreactivity of the Traditional Chinese medicine Shenrouyangzhentang to vasoactive intestinal polypeptide.

AIM: To study the immunoreactivity of the Chinese medicine Shenrouyangzhentang to vasoactive intestinal polypeptide (VIP) and its therapeutic mechanism. METHODS: The immunoreactivity of the Chinese medicine Shenrouyangzhentang to VIP was detected in the plasma of 20 normal people and 20 patients with Piyinxu (Spleen Yin deficiency) using the radioimmunoassay (RIA) method. RESULTS: The maximum binding rate B0/T was 53.29%, the non-specific binding rate N0/T was 1.170%, and the VIP standard curve was Y = 0.81983 + 0.44319X - 0.28927X(2), R(2) = 0.990. The VIP content in Shenrouyangzhentang was 106.6 ng/L ± 20 ng/L), while it was 90.16 ng/L ± 15 ng/L in normal human plasma and 63.25 ng/L ± 11 ng/L in the plasma of Pixinxu patients. The difference between normal plasma and Pixinxu patient plasma was statistically significant (P < 0.05). CONCLUSION: The Chinese medicine Shenrouyangzhentang demonstrated VIP immunoreactivity similar to that of normal plasma. The (vasoactive intestinal polypeptide) VIP content in Pixinxu patient plasma was lower than that in healthy subjects (P < 0.05).

Characteristics of saliva secreted by patients with TCM-Piyinxu.

AIM: To investigate various characteristics of saliva secreted by patients with TCM-Piyinxu (Spleen-yin deficiency). METHODS: Twenty-five individuals with Piyinxu (15 males and 10 females; age range 26-70 years, mean age = 45 years) diagnosed based on criteria used in traditional Chinese medicine, were compared with 20 individuals with Shenyinxu (Kidney-yin deficiency) (11 males, 9 females; age range 35-75 years, mean age = 50) and 30 normal individuals (17 males, 13 females; age range 35-65 years, mean age = 49 years). After acid stimulation, the saliva flow in each group was measured, and the levels of amylase and protein in saliva were determined using an automatic biochemical analyzer. The resultant data were analyzed using the Kruskal-Wallis test and one-way factorial ANOVA test. RESULTS: The flow rates of saliva and amylase in Piyinxu patients (0.27 ± 0.016 mL/min and 2134.13 ± 343.51 IU/min, respectively) were lower than those in normal subjects (0.46 ± 0.027 mL/min and 3501.63 ± 1099.63 IU/min, respectively, P < 0.01), but higher than those in the Shenyinxu group (0.13 ± 0.051 mL/min and 951.62 ± 383.17 IU/min, respectively, P < 0.01). The three groups showed no significant difference in their level of total salivary protein (Piyinxu group, 3.07 ± 0.60 g/L; Shenyinxu group, 3.01 ± 0.90 g/L, and control group, 2.94 ± 1.13 g/L, P = 0.869), amount of amylase per saliva volume, or their ratio of amylase to protein in secreted saliva (P = 0.173 and P = 0.436, respectively). CONCLUSION: Piyinxu patients showed altered rates of saliva and amylase secretion when compared with those parameters in patients with Shenyinxu and normal subjects.

Genomic restriction endonuclease analysis and mapping of murine guanylate cyclase-A/atrial natriuretic factor receptor gene.

The membrane-bound form of guanylate cyclase represents a biologically active atrial natriuretic factor receptor (GC/ANF-R). We have constructed genomic map of murine GC-A/ANF-R gene using 17 different restriction endonucleases. The restriction mapping results indicated that murine GC-A/ANF-R gene is approximately 20 kb single copy with multiple smaller exons and bigger introns. The Kpn I and Sfu I restriction digests produced 27 kb and 35 kb fragments, respectively, which hybridized with 5'- and 3'-flanking cDNA probes. Both of these fragments should cover the entire murine GC-A/ANF-R genomic sequences. The southern blot hybridization of genomic DNA from human, rat and mouse, using murine 5'-flanking cDNA probe indicated the presence of higher variant sequences in the 5'-flanking region of GC-A/ANF-R gene among different species. The noncoding 5'-flanking probe (350 bp) hybridized only to mouse genomic DNA but not to the human or rat DNA. These sequence variations located in the noncoding 5'-flanking region of GC-A/ANF-R gene may explain the divergent evolutionary development among different species. This is the first demonstration of the restriction endonuclease digestion and genomic mapping of murine GC-A/ANF-R gene which should be valuable to the understanding of its regulation and function.

A new alpha-thalassemia-2 deletion resulting in microcytosis and hypochromia and in vitro chain imbalance in the heterozygote.

We have detected a novel alpha-thalassemia-2 with a large (18+ kb) deletion involving the alpha 1- and theta 1-globin genes and the 3' hypervariable region sequence. Unexpectedly, the heterozygote had a mild anemia with a marked microcytosis and hypochromia, and an in vitro alpha/beta chain synthesis ratio of 0.62-0.66. It is suggested that the deletion includes a sequence that is involved in the in cis regulation of the alpha 2-globin gene.