A novel inhalable nanobody targeting IL-4Rα for the treatment of asthma.

BACKGROUND: Inhalable biologics represent a promising approach to improve the efficacy and safety of asthma treatment. Although several mAbs targeting IL-4 receptor α chain (IL-4Rα) have been approved or are undergoing clinical trials, the development of inhalable mAbs targeting IL-4Rα presents significant challenges. OBJECTIVE: Capitalizing on the distinctive advantages of nanobodies (Nbs) in maintaining efficacy during storage and administration, we sought to develop a novel inhalable IL-4Rα Nb for effectively treating asthma. METHODS: Three IL-4Rα immunized Nb libraries were used to generate specific and functional IL-4Rα Nbs. LQ036, a bivalent Nb comprising 2 HuNb103 units, was constructed with a high affinity and specificity for human IL-4Rα. The efficacy, pharmacokinetics, and safety of inhaled LQ036 were evaluated in B-hIL4/hIL4RA humanized mice. RESULTS: LQ036 inhibited secreted embryonic alkaline phosphatase reporter activity, inhibited TF-1 cell proliferation, and suppressed phosphorylated signal transducer and activator of transduction 6 in T cells from patients with asthma. Crystal structure analysis revealed a binding region similar to dupilumab but with higher affinity, leading to better efficacy in blocking the signaling pathway. HuNb103 competed with IL-4 and IL-13 for IL-4Rα binding. Additionally, LQ036 significantly inhibited ovalbumin-specific IgE levels in serum, CCL17 levels in bronchoalveolar lavage fluid, bronchial mucous cell hyperplasia, and airway goblet cell hyperplasia in B-hIL4/hIL4RA humanized mice. Inhaled LQ036 exhibited favorable pharmacokinetics, safety, and tissue distribution, with higher concentrations observed in the lungs and bronchi. CONCLUSIONS: These findings from preclinical studies establish the safety and efficacy of inhaled LQ036, underscoring its potential as a pioneering inhalable biologic therapy for asthma.

Preparation of a Dual-Functional Sulfated Galactofucan Polysaccharide/Poly(vinyl alcohol) Hydrogel to Promote Macrophage Recruitment and Angiogenic Potential in Diabetic Wound Healing.

A diabetic foot ulcer is a common high-risk complication in diabetic patients, but there is still no universal dressing for clinical treatment. In this study, a novel dual-functional sulfated galactofucan polysaccharide/poly(vinyl alcohol) hydrogel (DPH20) is developed during freeze-thaw cycles. Experimental results indicated that DPH20 had a high specific surface area, a dense porous structure, and a good swelling property, which could effectively adsorb the exudates and keep the wound moist. Furthermore, DPH20 exhibited remarkably recruited macrophage capability and accelerated the inflammation stage by improving the expression of the mRNA of CCL2, CCR2, and CCL22 in macrophages. DPH20 could promote cell migration and growth factor release to accelerate tube formation under hyperglycemic conditions in cell models of L929s and HUEVCs, respectively. Significantly, DPH20 accelerates the reconstruction of the full-thickness skin wound by accelerating the recruitment of macrophages, promoting angiogenesis, and releasing the growth factor in the diabetic mouse model. Collectively, DPH20 is a promising multifunctional dressing to reshape the damaged tissue environment and accelerate wound healing. This study provides an efficient strategy to repair and regenerate diabetic skin ulcers.

Antibiotic-associated vanishing bile duct syndrome: a real-world retrospective and pharmacovigilance database analysis.

PURPOSE: Vanishing bile duct syndrome (VBDS) is a rare, but potentially fatal adverse reaction triggered by certain medications. Few real-world studies have shown association between antibiotics and VBDS. We sought to quantify the risk and evaluate the clinical features of VBDS associated with antibiotics. METHODS: Data from 2004 to 2022 on VBDS events induced by antibiotics were retrieved from the FDA Adverse Event Reporting System (FAERS) database and disproportionality analyses were conducted. Furthermore, case reports from 2000 to 31 December 2022 on antibiotics-induced VBDS were retrieved for retrospective analysis. RESULTS: We collected 132 VBDS reports from the FAERS database. Fluoroquinolones had the greatest proportion and highest positive signal values of VBDS. The RORs (95% CIs) for antibiotics were fluoroquinolones 23.68 (18.12-30.95), macrolides 19.37 (13.58-27.62), carbapenems 17.39 (7.77-38.96), beta-lactam 13.28 (9.69-18.20), trimethoprim/sulfamethoxazole 9.05 (5.57-14.7), and tetracycline 4.02 (1.50-10.77). Twenty-three cases from 22 studies showed evidence of VBDS, beta-lactam (52.2%) was the most frequently reported agent. The median age was 45 years, the typical initial symptoms included rash (30.4%), fatigue/asthenia (26.1%), dark urine (21.7%) and Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN) (21.7%). The median time to onset of VBDS was 2 weeks. All cases had abnormal liver function test, and the median level of total bilirubin was 23.6 mg/dl (range 3.2-80 mg/dl). Cessation of culprit drugs and treatment with ursodeoxycholic acid (83.3%) were not associated with improved outcomes (57.1%). CONCLUSION: This study identified thirteen antibacterial agents with significant reporting associations with VBDS. Fluoroquinolones may be a neglected agent of inducing VBDS.

Spectrum of voriconazole-associated periostitis in clinical characteristics, diagnosis and management.

PURPOSE: Previous knowledge about the relationship between voriconazole exposure and periostitis was mainly based on limited case reports and few retrospective studies. The purpose of this study was to assess the clinical characteristics, diagnosis and management of voriconazole-associated periostitis. METHODS: Case reports and case series from 1998 to November 30, 2021 on periostitis induced by voriconazole were collected for retrospective analysis. RESULTS: Forty four patients (18 male and 26 female) from 34 studies were included in total. The median age was 58 years (29-74). The majority of patients had undergone organ transplantation (50.0%) or suffered from hematologic malignancy (31.81%). The median onset time of symptoms was 6 months after the start of voriconazole. The most common initial symptom was diffuse skeletal pain (68.28%) which can be severe and even disabling (66.7%). Ribs (37.21%), femurs (32.56%), scapulae (25.58%), humerus (23.26%), and clavicle (23.26%) were the common involved locations. Most cases were accompanied by different degrees of elevated serum alkaline phosphatase and fluoride level, while some presented with elevated bone-specific alkaline phosphatase. The main radiological features included periosteal reaction and multifocal high radiotracer uptake on bone scintigraphy. The formation of new bone was characterized with bilateral, irregular, nodular, as well as high density. The resolution of symptoms was observed with discontinuation of voriconazole in all patients, of whom 18 patients (52.94%) were relieved within a week. Itraconazole, posaconazole or isavuconazole were safe alternatives to voriconazole in voriconazole-induced periostitis. CONCLUSION: Voriconazole-induced periostitis is an infrequent complication characterized by bone inflammation involving one or multiple skeletal areas. Bony pain, elevated serum alkaline phosphatase as well as fluoride level are suspicious signs during voriconazole treatment.

Analysis of clinical characteristics of Kounis syndrome induced by contrast media.

BACKGROUND: Understanding the relationship between contrast agents and Kounis syndrome (KS) is mainly based on case reports. The purpose of this research is to explore the clinical characteristics of contrast media induced KS. METHODS: We searched for contrast-induced KS case reports through Chinese and English databases from 1991 to October 31, 2021. RESULTS: A total of 26 patients (19 men and 7 women,) were included, with a median age of 60 years (range 30-83). The contrast agents that cause KS mainly included gadolinium-based contrast agent (7 cases), iodine-containing contrast media (12 cases). KS mainly occurred within 30 min after administration and mainly manifests as chest pain and allergic reactions. Electrocardiogram (ECG) mainly showed ST elevation. Echocardiography mainly revealed normal. Coronary angiography showed normal, coronary vasospasm, stent thrombosis, occlusion and stenosis. After treatment with steroids, antihistamines and anti-ischemic therapy, 24 patients recovered completely and 2 patients died. CONCLUSIONS: KS is a rare adverse reaction of contrast media. Radiologists should recognize this rare but serious disease to ensure rapid diagnosis and proper management.

Glucuronomannan GM2 from Saccharina japonica Enhanced Mitochondrial Function and Autophagy in a Parkinson's Model.

Parkinson's disease (PD), one of the most common neurodegenerative disorders, is caused by dopamine depletion in the striatum and dopaminergic neuron degeneration in the substantia nigra. In our previous study, we hydrolyzed the fucoidan from Saccharina japonica, obtaining three glucuronomannan oligosaccharides (GMn; GM1, GM2, and GM3) and found that GMn ameliorated behavioral deficits in Parkinsonism mice and downregulated the apoptotic signaling pathway, especially with GM2 showing a more effective role in neuroprotection. However, the neuroprotective mechanism is unclear. Therefore, in this study, we aimed to assess the neuroprotective effects of GM2 in vivo and in vitro. We applied GM2 in 1-methyl-4-phenylpyridinium (MPP+)-treated PC12 cells, and the results showed that GM2 markedly improved the cell viability and mitochondrial membrane potential, inhibited MPP+-induced apoptosis, and enhanced autophagy. Furthermore, GM2 contributed to reducing the loss of dopaminergic neurons in 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mice through enhancing autophagy. These data indicate that a possible protection of mitochondria and upregulation of autophagy might underlie the observed neuroprotective effects, suggesting that GM2 has potential as a promising multifunctional lead disease-modifying therapy for PD. These findings might pave the way for additional treatment strategies utilizing carbohydrate drugs in PD.

Preparation and Neuroprotective Activity of Glucuronomannan Oligosaccharides in an MPTP-Induced Parkinson's Model.

Parkinson's disease (PD), characterized by dopaminergic neuron degeneration in the substantia nigra and dopamine depletion in the striatum, affects up to 1% of the global population over 50 years of age. Our previous study found that a heteropolysaccharide from Saccharina japonica exhibits neuroprotective effects through antioxidative stress. In view of its high molecular weight and complex structure, we degraded the polysaccharide and subsequently obtained four oligosaccharides. In this study, we aimed to further detect the neuroprotective mechanism of the oligosaccharides. We applied MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) to induce PD, and glucuronomannan oligosaccharides (GMn) was subsequently administered. Results showed that GMn ameliorated behavioral deficits in Parkinsonism mice. Furthermore, we observed that glucuronomannan oligosaccharides contributed to down-regulating the apoptotic signaling pathway through enhancing the expression of tyrosine hydroxylase (TH) in dopaminergic neurons. These results suggest that glucuronomannan oligosaccharides protect dopaminergic neurons from apoptosis in PD mice.

Enzyme Kinetics for Complex System Enables Accurate Determination of Specificity Constants of Numerous Substrates in a Mixture by Proteomics Platform.

Many important experiments in proteomics including protein digestion, enzyme substrate screening, enzymatic labeling, etc., involve the enzymatic reactions in a complex system where numerous substrates coexists with an enzyme. However, the enzyme kinetics in such a system remains unexplored and poorly understood. Herein, we derived and validated the kinetics equations for the enzymatic reactions in complex system. We developed an iteration approach to depict the enzymatic reactions in complex system. It was validated by 630 time-course points from 24 enzymatic reaction experiments and was demonstrated to be a powerful tool to simulate the reactions in the complex system. By applying this approach, we found that the ratio of substrate depletion is independent of other coexisted substrates under specific condition. This observation was then validated by experiments. Based on this striking observation, a simplified model was developed to determine the catalytic efficiencies of numerous competing substrates presented in the complex enzyme reaction system. When coupled with high-throughput quantitative proteomics technique, this simplified model enabled the accurate determination of catalytic efficiencies for 2369 peptide substrates of a protease by using only one enzymatic reaction experiment. Thus, this study provided, in the first time, a validated model for the large scale determination of specificity constants which could enable the enzyme substrate screening approach turned from a qualitative method of identifying substrates to a quantitative method of identifying and prioritizing substrates. Data are available via ProteomeXchange with identifier PXD004665.

Belowground rhizomes in paleosols: The hidden half of an Early Devonian vascular plant.

The colonization of terrestrial environments by rooted vascular plants had far-reaching impacts on the Earth system. However, the belowground structures of early vascular plants are rarely documented, and thus the plant-soil interactions in early terrestrial ecosystems are poorly understood. Here we report the earliest rooted paleosols (fossil soils) in Asia from Early Devonian deposits of Yunnan, China. Plant traces are extensive within the soil and occur as complex network-like structures, which are interpreted as representing long-lived, belowground rhizomes of the basal lycopsid Drepanophycus The rhizomes produced large clones and helped the plant survive frequent sediment burial in well-drained soils within a seasonal wet-dry climate zone. Rhizome networks contributed to the accumulation and pedogenesis of floodplain sediments and increased the soil stabilizing effects of early plants. Predating the appearance of trees with deep roots in the Middle Devonian, plant rhizomes have long functioned in the belowground soil ecosystem. This study presents strong, direct evidence for plant-soil interactions at an early stage of vascular plant radiation. Soil stabilization by complex rhizome systems was apparently widespread, and contributed to landscape modification at an earlier time than had been appreciated.

Inhibitory effects of oxymatrine on hepatic stellate cells activation through TGF-ß/miR-195/Smad signaling pathway.

BACKGROUND: Oxymatrine (OM), a quinolizidine alkaloid extracted from a herb Sophorae Flavescentis Radix, has been used to treat liver fibrotic diseases. However, the mechanism of its anti-fibrosis effects is still unclear. TGF-ß/Smad signaling and miR-195 have been proved to paly an important role in hepatic stellate cells (HSCs) activation and liver fibrosis. In this study, we investigated whether OM could inhibit HSCs activation through TGF-ß1/miR-195/Smads signaling or not. METHODS: First, the effects of OM on HSC-T6 in different concentrations and time points were tested by MTT assay. We choose three appropriate concentrations of OM as treatment concentrations in following experiment. By Quantitative Real-time PCR and Western Blot, then we investigated the effect of OM on miR-195, Smad7 and α-SMA's expressions to prove the correlation between OM and the TGF-ß1/miR-195/Smads signaling. Last, miR-195 mimic and INF-γ were used to investigate the relation between miR-195 and OM in HSC activation. RESULTS: Our results showed that the proliferation of HSC was significantly inhibited when OM concentration was higher than 200 µg/mL after 24 h, 100 µg/mL after 48 h and 10 µg/mL after 72 h. The IC50 of OM after 24, 48 and 72 h were 539, 454, 387 µg/mL respectively. OM could down-regulate miR-195 and α-SMA (P < 0.01), while up-regulate Smad7 (P < 0.05). In HSC-T6 cells transfected with miR-195 mimic and pretreated with OM, miR-195 and α-SMA were up-regulated (P < 0.05), and Smad7 was down-regulated (P < 0.05) . CONCLUSIONS: Given these results, OM could inhibit TGF-ß1 induced activation of HSC-T6 proliferation in a dose-dependent and time-dependent manner to some extent. We proved that OM inhibited HSC activation through down-regulating the expression of miR-195 and up-regulating Smad7.

Antihypertensive Effects of Two Novel Angiotensin I-Converting Enzyme (ACE) Inhibitory Peptides from Gracilariopsis lemaneiformis (Rhodophyta) in Spontaneously Hypertensive Rats (SHRs).

A variety of biologically active products have been isolated from Gracilariopsis lemaneiformis. In the present study, two novel angiotensin-converting enzyme (ACE) inhibitory peptides, FQIN [M(O)] CILR, and TGAPCR, were screened and identified from G. lemaneiformis protein hydrolysates by LC-MS/MS. The IC50 values of FQIN [M(O)] CILR and TGAPCR were 9.64 ± 0.36 µM and 23.94 ± 0.82 µM, respectively. In the stability study, both peptides showed stabilities of pH, temperature, simulated gastrointestinal digestion, and ACE hydrolysis. The Lineweaver⁻Burk plot showed that the two peptides were noncompetitive inhibitors of ACE. Molecular docking simulated the intermolecular interactions of two peptides and ACE, and the two peptides formed hydrogen bonds with the active pockets of ACE. However, FQIN [M(O)] CILR was more closely linked to the active pockets of ACE, thereby exerting better ACE inhibition. Spontaneously hypertensive rats (SHRs) were studied with an oral dose of 10 mg/kg body weight. Both peptides reduced systolic blood pressure (SBP) and diastolic blood pressure (DBP) in SHRs, of which FQIN [M(O)] CILR was able to reduce the systolic blood pressure by 34 mmHg (SBP) (p < 0.05). Therefore, FQIN [M(O)] CILR was an excellent ACE inhibitory peptide.

Investigating the Relationship between the Substrates' Consumption and Their Abundances in a Complex Enzymatic System.

The enzymatic process involving the incubation of a library of substrates with an enzyme is the key step for a few important experiments for bioanalytical chemistry including proteomics analysis, enzymatic labeling, substrate screening, etc. The relationship between the substrates' consumption and their abundances in a complex enzymatic system with a huge number of coexisting substrates of different abundances was not well-known. In this study, we have demonstrated theoretically and experimentally that the priority of substrate consumption depended on their specificity constants but not abundances. We derived the expression between the fractions of the substrates consumed (pi) and their specificity constants. Using the enzymatic system of five synthetic peptide substrates of trypsin, we validated through 24 experiments that the ln(1 - pi) values of competing substrates have linear correlation with their specificity constants, and thus, the priority of substrate depletion has no relation with their abundances. Using a state of the art quantitative proteomics approach, we found that the ln(1 - pi) values of 144 competing substrates between any two of four experiments have a linear relationship and the prioritization of substrates can be achieved by sorting their consumption rates in the experiment. This study will improve our understanding of the enzymatic kinetics in the complex system and will benefit the design of enzymatic analytical approaches.

Sensitive, Robust, and Cost-Effective Approach for Tyrosine Phosphoproteome Analysis.

Albeit much less abundant than Ser/Thr phosphorylation (pSer/pThr), Tyr phosphorylation (pTyr) is considered as a hallmark in cellular signal transduction. However, its analysis at the proteome level remains challenging. The conventional immunopurification (IP) approach using antibodies specific to pTyr sites is known to have low sensitivity, poor reproducibility and high cost. Our recent study indicated that SH2 domain-derived pTyr-superbinder is a good replacement of pTyr antibody for the specific enrichment of pTyr peptides for phosphoproteomics analysis. In this study, we presented an efficient SH2 superbinder based workflow for the sensitive analysis of tyrosine phosphoproteome. This new method can identify 41% more pTyr peptides than the previous method. Its excellent performance was demonstrated by the analysis of a variety of different samples. For the highly tyrosine phosphorylated sample, for example, pervanadate-treated Jurkat T cells, it identified over 1800 high confident pTyr sites from only 2 mg of proteins. For the unstimulated Jurkat cells, where the pTyr events rarely occurred, it identified 343 high confident pTyr sites from 5 mg of proteins, which was 31% more than that obtained by the antibody-based method. For the heterogeneous sample of tissue, it identified 197 high confident pTyr sites from 5 mg protein digest of mouse skeletal muscle. In general, it is a sensitive, robust and cost-effective approach and would have wide applications in the study of the regulatory role of tyrosine phosphorylation in diverse physiological and pathological processes.

Biphasic Affinity Chromatographic Approach for Deep Tyrosine Phosphoproteome Analysis.

Tyrosine phosphorylation (pTyr) is important for normal physiology and implicated in many human diseases, particularly cancer. Identification of pTyr sites is critical to dissecting signaling pathways and understanding disease pathologies. However, compared with serine/threonine phosphorylation (pSer/pThr), the analysis of pTyr at the proteome level is more challenging due to its low abundance. Here, we developed a biphasic affinity chromatographic approach where Src SH2 superbinder was coupled with NeutrAvidin affinity chromatography, for tyrosine phosphoproteome analysis. With the use of competitive elution agent biotin-pYEEI, this strategy can distinguish high-affinity phosphotyrosyl peptides from low-affinity ones, while the excess competitive agent is readily removed by using NeutrAvidin agarose resin in an integrated tip system. The excellent performance of this system was demonstrated by analyzing tyrosine phosphoproteome of Jurkat cells from which 3,480 unique pTyr sites were identified. The biphasic affinity chromatography method for deep Tyr phosphoproteome analysis is rapid, sensitive, robust, and cost-effective. It is widely applicable to the global analysis of the tyrosine phosphoproteome associated with tyrosine kinase signal transduction.

Basophil Recruitment to Skin Lesions of Patients with Systemic Lupus Erythematosus Mediated by CCR1 and CCR2.

BACKGROUND/AIMS: Basophils have been reported to infiltrate skin lesions in various skin diseases, but not in systemic lupus erythematosus (SLE). This study investigated basophil infiltration in SLE and its mechanism. METHODS: Twenty newly diagnosed SLE patients and twenty healthy controls were enrolled. Nine SLE patients underwent skin biopsies. Flow cytometric analysis the phenotype of peripheral basophils and their migration rate toward RANTES and MCP-1 were analyzed with the transwell culture system, also the expression of these two chemokines in skin tissue were analyzed with immunohistochemistry. RESULTS: Increased activation and decreased numbers of peripheral basophils were observed in SLE patients compared with controls. Basophil migration into skin lesions of SLE patients were observed, but not in normal skin tissue. This migration was related to the upregulation of chemokine receptors CCR1 and CCR2 on basophils. In vitro studies showed that migration rate toward RANTES and MCP-1 increased significantly in basophils from SLE patients compared with those from controls. Consistently, high levels of RANTES and MCP-1 expression were observed in skin lesions from SLE patients but not in normal skin tissue. CONCLUSION: Basophil recruitment to skin lesions of SLE patients mediated by CCR1 and CCR2, which may contribute to tissue damage in SLE.

The proteomic analysis improved by cleavage kinetics-based fractionation of tryptic peptides.

Selective enrichment of specific peptides is an effective way to identify low abundance proteins. Fractionation of peptides prior to mass spectrometry is another widely used approach to reduce sample complexity in order to improve proteome coverage.In this study, we designed a multi-stage digestion strategy to generate peptides with different trypsin cleavage kinetics. It was found that each of the collected peptide fractions yielded many new protein identifications compared to the control group due to the reduced complexity. The overlapping peptides identified between adjacent fractions were very low, indicating that each fraction had different sets of peptides. The multi-stage digestion strategy separates tryptic peptides with different cleavage kinetics while RPLC separates peptides with different hydrophobicity. These two separation strategies were highly orthogonal, and showed an effective multidimensional separation to improve proteome coverage.

Latisemenia longshania, gen. et sp. nov., a new Late Devonian seed plant from China.

The earliest known ovules in the Late Devonian (Famennian) are borne terminally on fertile branches and are typically enclosed in a cupule. Among these ovules are some that have terete integumentary lobes with little or no fusion. Here, we report a new taxon, Latisemenia longshania, from the Famennian of South China, which bears cupulate ovules that are terminal as well as opposite on the fertile axis. Each ovule has four broad integumentary lobes, which are extensively fused to each other and also to the nucellus. The cupule is uniovulate, and the five flattened cupule segments of each terminal ovule are elongate cuneate and shorter than the ovule. Associated but not attached pinnules are laminate and Sphenopteris-like, with an entire or lobate margin. Latisemenia is the earliest known plant with ovules borne on the side of the fertile axis and may foreshadow the diverse ovule arrangements found among younger seed plant lineages that emerge in the Carboniferous. Following the telome theory, Latisemenia demonstrates derived features in both ovules and cupules, and the shape and fusion of integumentary lobes suggest effective pollination and protection to the nucellus. Along with other recent discoveries from China, Latisemenia extends the palaeogeographic range of the earliest seed plants.

Geographical distribution, a risk factor for the incidence of lupus nephritis in China.

BACKGROUND: Geographical variation in lupus nephritis epidemiology may indicate important environmental factors contributions to the etiology of lupus nephritis. This paper first describes the epidemiology of biopsy-proven lupus nephritis in China by performing a systematic literature review and the possible social-environmental influential factors. METHODS: The keywords "lupus nephritis", "renal biopsy" and "systemic lupus erythematous" were searched in the three largest Chinese electronic databases and Medline/PubMed. The data of the patients with biopsy-proven lupus nephritis were extracted. The possible environmental influential factors including the population density, ethnic group populations, the ratio of females to males, the average sunshine per year, annual average temperature and annual relative humidity, in different regions of China were analyzed. RESULTS: Forty-one study centers with 34574 renal disease patients, and 3699 lupus nephritis patients met the inclusion criteria. Lupus nephritis accounts for 2.37% to 25% of all renal disease and 27.2% to 80.65% of renal disease associated with secondary glomerular diseases. The male-to-female ratio is approximately 1:5 in lupus nephritis patients. The included period is predominantly from 1995 to 2010. The proportion ratio of biopsy-proven lupus nephritis in all renal disease or in secondary glomerular disease significantly increased with decreasing latitude from the north to the south part of China. The population is predominantly Han Chinese. CONCLUSIONS: Geographical distribution appears to be a risk factor for the incidence of biopsy-proven LN in China.

Effects of red blood cell lysing solutions on the detection of peripheral basophils of healthy normals and SLE patients by flow cytometry.

To evaluate the effect of four widely used red blood cell lysing solutions on counting and measurement of activation marker of peripheral basophils in normals and systemic lupus erythematosus (SLE) patients by flow cytometry. Our results showed that the light scatter properties including FS and SS value of leukocytes in whole blood were preserved when whole blood samples were lysed in RBC Lysis Buffer and FACS Lysing Solution, while were affected when lysed in distilled water or ACK. By counting basophils, RBC Lysis Buffer and FACS Lysing Solution were almost the same level, while were significantly lower when lysed in distilled water or ACK. The expressions of CD203c on peripheral basophils of SLE patients were significantly higher than those of normals. Comparing the data of CD203c expression obtained demonstrated that there were no significant differences among them, while FACS Lysing Solution treatment leads to a slightly lower staining intensity of CD203c. We provide a solid description that the widely used red blood cell lysing reagents may influence the light scatter properties of leukocytes, the accuracy of quantity of absolute number of the existence of basophil subsets and the quantity of staining intensity of cell-activated marker CD203c fluorescence when measured by flow cytometry.

Transforming growth factor-ß1 regulates the telomerase reverse transcriptase in rat hepatic stellate cells.

OBJECTIVE: To determine the effect of transforming growth factor-ß1 (TGF-ß1) on the expression of telomerase in hepatic stellate cells (HSCs) in rats and the role of TGF-ß1 in the development of liver fibrosis. METHODS: Primary HSCs were isolated from normal rats by density gradient separation and divided into 2 groups for culturing. The morphology of HSCs was identified by the inverted fluorescence microscope. The purity of HSCs was identified by immunohistological expression and fluorescence analysis. One group of HSCs was treated with different concentrations (0, 0.1, 1, and 10 ng/mL) of TGF-ß1 for 24 h, while the other group was treated with 1 ng/mL TGF-ß1 and cultured for 3, 6, and 9 days. The mRNA expression of telomerase reverse transcriptase (TERT) was assessed and compared by polymerase chain reaction. RESULTS: Cell morphology showed that TGF-ß1 triggered the differentiation of HSCs from a quiescent phenotype into highly activated myofibroblasts. TERT mRNA expression in the primary HSCs showed slight increase with the culture time, though with no statistical difference between the results at various time points (P>0.05). TGF-ß1 at 0.1 ng/mL did not significantly affect the TERT mRNA level compared with the 0 ng/mL group, while 1 ng/mL and 10 ng/mL TGF-ß1 significantly decreased the level of TERT mRNA (P<0.05). TGF-ß1 at 1 ng/mL had only weak effect on TERT mRNA expression after the 3 day treatment compared with the 0 ng/mL group (P>0.05). TGF-ß1 at 1 ng/mL significantly inhibited TERT mRNA expression 6 days after the treatment (P<0.05). TGF-ß1 inhibited the expression of TERT mRNA level in the HSCs in both dose- and time-dependent manner. CONCLUSION: TGF-ß1 may contribute to the transdifferentiation of HSCs by reducing TERT levels to develop hepatic fibrosis.