[Effect of hemoperfusion on protein energy wasting and long-term prognosis in patients on maintenance hemodialysis].

Objective: To explore the effect of hemoperfusion (HP) combined with hemodialysis (HD) (HD+HP) on protein energy wasting (PEW) and long-term prognosis in patients on maintenance HD (MHD). Methods: A prospective multicenter cohort study was conducted. Adult MHD patients who completed PEW assessment and underwent regular dialysis between July 2015 and July 2021 at 23 hemodialysis centers in Guizhou Province were selected. Demographic characteristics, physical indicators, laboratory indicators, 3-day diet diary and HP treatment data of the subjects were collected. The patients were divided into different groups according to the presence or absence of HP, the frequency of HP treatment and the type of cartridge, and then relevant indicators were compared. Multivariate logistic regression model and Cox proportional regression model were used to analyze the influence of HP treatment on PEW risk in MHD patients. Meanwhile, Kaplan-Meier method was used to plot the survival curve. Results: A total of 4 623 MHD patients (2 789 males and 1 834 females) aged (53.7±15.9) years were included in the study, with a median dialysis age of 64.3 (44.3, 92.3) months. There were 3 429 (74.2%) MHD patients treated with HD+HP, and 1 194 patients (25.8%) were not treated with HP. According to the 2008 diagnostic criteria of the International Society for Renal Nutrition and Metabolism (ISRNM), the incidence of PEW was 26.0% (1 204/4 623). Multivariate logistic regression analysis showed that female (OR=2.48, 95%CI: 1.55-3.95, P<0.001), diabetes (OR=1.75, 95%CI: 1.08-2.83, P=0.024) and high-sensitivity C-reactive protein (hs-CRP) (OR=1.02, 95%CI: 1.01-1.03, P=0.003) were risk factors for PEW, while treatment with HD+HP (OR=0.51, 95%CI: 0.31-0.87, P=0.012) and elevated triglyceride levels (OR=0.62, 95%CI: 0.48-0.80, P<0.001) were protective factors. Cox hazard ratio regression showed that among different HP treatment frequencies and cartridge types, 2 times/month (HR=0.40, 95%CI: 0.17-0.95, P=0.037), 3 times/month (HR=0.44, 95%CI: 0.23-0.85, P=0.014), 4 times/month (HR=0.54, 95%CI: 0.34-0.85, P=0.008), HA130 (HR=0.57, 95%CI: 0.36-0.89, P=0.014) and HA230 (HR=0.30, 95%CI: 0.15-0.63, P=0.001) had protective effects on the occurrence of PEW in MHD patients. The all-cause mortality rate was 11.3% (521/4 623) at 33 (24, 48) months of follow-up. Kaplan-Meier analysis showed that patients undergoing 4 times/month HP treatment (χ2=36.78, P<0.001) and using HA230 (χ2=9.46, P=0.002) had the highest survival rate. Conclusion: Treatment with HD+HP is a protective factor for PEW in patients with MHD, and 4 times/month HP treatment or HA230 significantly reduces the risk of PEW and all-cause mortality in patients with MHD.

Haploinsufficiency of cathepsin D leads to lysosomal dysfunction and promotes cell-to-cell transmission of α-synuclein aggregates.

Lysosomal dysfunction has been implicated both pathologically and genetically in neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease (PD). Lysosomal gene deficiencies cause lysosomal storage disorders, many of which involve neurodegeneration. Heterozygous mutations of some of these genes, such as GBA1, are associated with PD. CTSD is the gene encoding Cathepsin D (CTSD), a lysosomal protein hydrolase, and homozygous CTSD deficiency results in neuronal ceroid-lipofuscinosis, which is characterized by the early onset, progressive neurodegeneration. CTSD deficiency was also associated with deposition of α-synuclein aggregates, the hallmark of PD. However, whether partial deficiency of CTSD has a role in the late onset progressive neurodegenerative disorders, including PD, remains unknown. Here, we generated cell lines harboring heterozygous nonsense mutations in CTSD with genomic editing using the zinc finger nucleases. Heterozygous mutation in CTSD resulted in partial loss of CTSD activity, leading to reduced lysosomal activity. The CTSD mutation also resulted in increased accumulation of intracellular α-synuclein aggregates and the secretion of the aggregates. When α-synuclein was introduced in the media, internalized α-synuclein aggregates accumulated at higher levels in CTSD+/- cells than in the wild-type cells. Consistent with these results, transcellular transmission of α-synuclein aggregates was increased in CTSD+/- cells. The increased transmission of α-synuclein aggregates sustained during the successive passages of CTSD+/- cells. These results suggest that partial loss of CTSD activity is sufficient to cause a reduction in lysosomal function, which in turn leads to α-synuclein aggregation and propagation of the aggregates.

ZO-1 and -2 Are Required for TRPV1-Modulated Paracellular Permeability.

The tight junction-based paracellular pathway plays an important role in saliva secretion. Zonula occludens (ZO) proteins are submembranous proteins of tight junction complex; however, their function in salivary epithelium is poorly understood. Here, we found that activation of transient receptor potential vanilloid subtype 1 (TRPV1) by capsaicin increased rat saliva secretion both in vivo and ex vivo. Meanwhile, TRPV1 activation enlarged the width of tight junctions between neighboring acinar cells, increased the paracellular flux of 4-kDa fluorescein isothiocyanate (FITC)-dextran in submandibular gland (SMG) tissues, and decreased transepithelial electric resistance (TER) in SMG-C6 cells. ZO-1, -2, and -3 were distributed principally to the apical lateral region of acinar cells in SMG tissues and continuously encircled the peripheries of SMG-C6 cells in the untreated condition. TRPV1 activation obviously diminished ZO-1 and -2 staining, but not ZO-3 or ß-catenin, at the cell-cell contacts ex vivo and in vitro. Moreover, in untreated SMG-C6 cells, ZO-1 and -2 single or double knockdown by small interfering RNA (siRNA) increased the paracellular flux of 4-kDa FITC-dextran. In capsaicin-treated cells, ZO-1 and -2 single or double knockdown abolished, whereas their re-expression restored, the capsaicin-induced increase in paracellular permeability. Furthermore, TRPV1 activation increased RhoA activity, and inhibition of either RhoA or Rho kinase (ROCK) abolished the capsaicin-induced TER decrease as well as ZO-1 and -2 redistribution. These results indicate that ZO-1 and -2 play crucial roles in both basal salivary epithelial barrier function and TRPV1-modulated paracellular transport. RhoA-ROCK signaling pathway is responsible for TRPV1-modulated paracellular permeability as well as ZO-1 and -2 redistribution.

Pre- and Post-synaptic Effects of Botulinum Toxin A on Submandibular Glands.

Intraglandular injection of botulinum toxin type A (BoNT/A) is an effective treatment for sialorrhea. Despite numerous experimental and clinical studies on inhibition of saliva section by BoNT/A, the proteolysis of synaptosomal-associated protein 25 (SNAP-25) following BoNT/A treatment has not yet been confirmed in the salivary gland after injection of BoNT/A. More important, it is not known whether BoNT/A exerts a direct effect in acinar cells. Here, we show that injection of BoNT/A into the rat submandibular gland (SMG) decreased salivary flow in a dose-dependent manner; the inhibitory effect lasted at least 4 wk, and salivary flow recovered to normal levels by 12 wk. During the inhibitory period, SMG neurons and synapses expressed lower levels of full-length SNAP-25, and cleavage of SNAP-25 was observed, as indicated by detection of reduced molecular weight SNAP-25 using Western blotting. In addition, the water channel aquaporin 5 (AQP5) was downregulated and abnormally distributed in rat SMG after injection of BoNT/A. The direct effects of BoNT/A on AQP5 expression and distribution were assessed in vitro to exclude the influence of BoNT/A-induced inhibitory neurotransmission. In stable GFP-AQP5-transfected SMG-C6 cells, treatment with BoNT/A reduced the cell surface protein level of AQP5 in a dose- and time-dependent manner without affecting total AQP5 protein expression. Cell surface biotinylation and immunofluorescence demonstrated translocation of AQP5 from the membrane to the cytoplasm, which was confirmed by decreased levels of AQP5 protein in the membrane fraction and increased levels in the cytoplasmic fraction, suggestive of AQP5 redistribution. Taken together, these results indicated that BoNT/A reversibly decreased saliva secretion in rat SMGs through not only the presynaptic SNAP-25 cleavage but also the postsynaptic AQP5 redistribution. These data provide the first evidence for a direct effect of BoNT/A on the salivary gland.

Hypersensitive mAChRs are involved in the epiphora of transplanted glands.

Autologous transplantation of the submandibular gland is an effective treatment for severe dry eye syndrome. However, more than 40% of patients experience epiphora 3 to 6 months after transplantation. The underlying mechanism of epiphora remains to be elucidated. To investigate the potential roles of muscarinic acetylcholine receptors (mAChRs) in the induction of epiphora in transplanted glands, we assessed and found elevated mRNA and protein expression of M1- and M3-mAChR in transplanted glands from epiphora patients. The content of inositol 1, 4, 5-trisphosphate was also elevated. Moreover, carbachol (5 and 10 µM) induced greater increase of [Ca(2+)]i in isolated epiphora submandibular cells than in controls. Although aquaporin-5 (AQP5) content and distribution in the apical and lateral plasma of epiphora glands did not change, AQP5 content was reduced in lipid microdomains (lipid rafts and caveolae) but increased in non-lipid microdomains compared with controls. Carbachol (10 µM) increased the ratio of non-lipid microdomain to total AQP5 in the cultured control submandibular gland tissue. Taken together, these results indicated that hypersensitive mAChRs might be involved in the epiphora of transplanted submandibular glands by modulating AQP5 trafficking.

Decreased submandibular adiponectin is involved in the progression of autoimmune sialoadenitis in non-obese diabetic mice.

OBJECTIVE: To investigate a possible role of adiponectin in the pathogenesis of autoimmune sialoadenitis in non-obese diabetic (NOD) mouse model of Sjögren's syndrome. MATERIALS AND METHODS: Expression of adiponectin and its receptors (AdipoR1/2) was detected by PCR, immunoblotting, or immunofluorescence. The level of adiponectin was quantified by ELISA. Adiponectin-related signaling molecules and pro-inflammatory cytokines were examined by PCR or immunoblotting. Apoptosis was evaluated by TUNEL staining, flow cytometry, and caspase 3 activation. RESULTS: Adiponectin and AdipoR1/2 mRNA and protein were expressed in submandibular glands. Adiponectin immunostaining was widely diffused in the cytoplasm of acinar and ductal cells. AdipoR1 was mainly distributed in acinar cytoplasm, while AdipoR2 was predominantly located at acinar cell membrane. Submandibular adiponectin levels were reduced during the progression of autoimmune sialoadenitis in 7-, 14-, and 21-week-old NOD mice, while AdipoR1/2 levels were unchanged. The levels of phosphorylated adenosine monophosphate-activated protein kinase, extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase were decreased, while interferon (IFN)-γ and glandular apoptosis were temporally increased at all time points. Moreover, exogenous adiponectin supplement inhibited, whereas neutralizing endogenous adiponectin by its antibody promoted IFN-γ-induced apoptosis and caspase 3 activation in cultured submandibular acinar cells. CONCLUSIONS: Adiponectin plays a protective role on submandibular cells. Decreased adiponectin might promote glandular destruction in autoimmune sialoadenitis.

Cytotoxic sesquiterpene lactones from Eupatorium lindleyanum.

Three new germacrane sesquiterpenes, eupalinolides C-E (1-3), along with three known germacrane sesquiterpenes, eupalinolide A (4), eupalinolide B (5), and 3beta-acetoxy-8beta-(4'-hydroxytigloyloxy)-14-hydroxycostunolide (6), were isolated from Eupatorium lindleyanum. They were tested for cytotoxicity against A-549, BGC-823, SMMC-7721, and HL-60 tumour cell lines. The results showed that these compounds demonstrated potent cytotoxicity. The structures of the compounds were elucidated by means of (1)H and (13)C NMR spectroscopic analysis, including 2D NMR experiments.

Sustainable veterinary medicine for the new era.

Sustainability aims to harmonise life on Earth without compromising the essential natural resources that should be the birthright of future generations. 'Sustainable medicine' (SM) is just one component of the wide range of possible sustainable approaches to peaceful co-existence. Sustainable medicine envisions an uncomplicated system of maintaining the health of people and animals, both now and for many years to come. This type of medicine is based on ancient wisdom, knowledge and healing arts, combined with the advantages and technical achievements of modern science and other areas of medicine; it is an integrated approach to preventive, safe and affordable healing. The term sustainable medicine also implies that the main therapeutic materials used in the course of practising this type of medicine can be replaced or replenished with minimal environmental damage after harvesting. The aim of sustainable medicine is to maintain the balance of nature, allowing an estimated 7 to 100 million species of life forms to co-exist and reproduce, and to sustain the long-term future of this planet. The world is in the midst of an environmental crisis: anthropogenic environmental damage in the last century was greater than in any previous century. One of the major concerns is the misuse of medicines, and the resulting immune depletion in people and animals. Many traditional medical systems have taught that appropriate adaptation by, and of, an effective defence system is the key to health and survival. This is only possible if priority is given to a preventive rather than a curative approach to health care; the very same approach that is advocated by proponents of SM: an approach based on proper diagnosis and the use of personalised, tailor-made medicine. The authors propose SM (the combination of the advantages of modern, traditional and complementary medical systems) as the best approach to providing better health care services for people and animals. The article presents a brief history of traditional medicines and outlines strategies for developing SM. The authors highlight some important factors in the development of SM in animal health care and attempt to encourage veterinarians to adopt a sustainable approach to treating animals.

Aquapuncture therapy of repeat breeding in dairy cattle.

Repeat breeders cause great economic loss in dairy herds. This study determined the efficacy of simple aquapuncture therapy (acupuncture combined with injection) to treat repeat breeders in two dairy herds (one in Hsinchu County and the other in Tainan County). Both herds had used gonadotropin-releasing hormone (GnRH) beforehand to treat the symptom but the success rate was poor--about 30%. Therefore, cows that failed to respond to GnRH were given the aquapuncture treatment. A total of 18 animals (two heifers and 16 cows) that had failed to conceive to 3-9 services were treated by aquapuncture, in which 10 ml and 5 ml of 50% glucose solution were injected at Baihui (Hundred Meetings, the depression in the center of the lumbosacral space) and Shenpeng (Kidney Shelf, the depression in the space between the lumbar vertebral transverse processes 5-6) acupoints, respectively with a 21G, 1.5-inch hypodermic needle. Both acupoints belong to the traditional acupuncture points of bovine and equine systems. Most animals showed heat within 14 days after aquapuncture and were inseminated artificially. Serum progesterone concentrations and rectal palpation were used for pregnancy diagnosis. The pregnancy rate after the treatment was 14/18 (77.7%) and 12/18 (66.6%) based on progesterone concentration and rectal palpation respectively, suggesting acupuncture as a simple and effective method to treat repeat breeders in dairy herds. However, the final result obtained from actual delivery of fetus in the experimental animals was only 8/18 (44.4%).

Biological activity in the repopulating rat spermatocyte after the withdrawal of gossypol treatment. VI. Alteration in nuclear factors for interaction with histone gene promoter.

This article reports the effects of gossypol at the genomic level in rat spermatogenic cells. After gossypol treatment for various times (8, 12, and 19 weeks), the spermatogonial cells were allowed to rest for 2 to 4 weeks. The function of histone H4 gene promoter (H4GP) in the repopulating pachytene spermatocytes (RPS) was investigated. The sequences of the oligonucleotides for the H4GP binding sites 1 and 2 were synthesized by an ABI-392 DNA synthesizer. RPS and the control pachytene spermatocytes (CPS) were obtained by centrifugal elutriation and subsequently they were used for the preparation of nuclear protein extracts (NPE). The NPE interaction with the DNA fragment of site 1 or 2 was studied by an electrophoresis mobility shift assay (EMSA). EMSA with NPE-CPS revealed ten major gel shift bands for site 1 and 2. The presence of extra unlabelled DNA fragments competed with 6 of the bands. After 2 to 4 weeks recovery from 8, 12, and 19 weeks of gossypol treatment, NPE-RPS failed to shift four bands (b through e) in site 1. These results suggested that gossypol treatment affected the transcription factors for interaction with site 1. On the contrary, no effect was demonstrated in NPE that interacted with site 2. Furthermore, gossypol treatment did not change the nucleotide sequence in the H4GP site 1 and 2.

Fatal infection of silvered leaf monkeys with a virus-like infectious agent (VLIA) derived from a patient with AIDS.

Four silvered leaf monkeys, inoculated with a virus-like infectious agent (VLIA) derived from transformed NIH/3T3 cells (sb51) transfected with Kaposi's sarcoma DNA of an AIDS patient, showed wasting syndromes and died in 7-9 months. Two monkeys had a transient lymphadenopathy in earlier stages. Two moribund animals showed lymphopenia. Although 3 of the VLIA inoculated monkeys had persistent low grade fever early in the infection, the animals became afebrile in the later stages. One VLIA inoculated animal had a prominent antibody response, which occurred 7 months after VLIA inoculation. The other 3 monkeys had a transient or poor antibody response in the later stages. These 3 animals revealed periodic VLIA antigenemia during the course of the experiment. A control monkey was killed 8 months after the last VLIA inoculated monkey succumbed and showed neither an antibody response nor evidence of antigenemia. VLIA-specific DNA could be directly detected in necropsy tissues of all 4 monkeys inoculated with VLIA using the polymerase chain reaction method. VLIA infection was identified in all 4 spleens, 2 of 4 livers, 1 of 2 kidneys, and all 3 brains tested from these 4 animals, but not in the tissues from the control monkey. The necropsy examination of the 4 VLIA inoculated animals revealed no opportunistic infections, acute inflammatory lesions, malignancy or cause of death other than VLIA infection. We believe that the VLIA caused a fatal systemic infection in these monkeys.

A novel virus-like infectious agent in patients with AIDS.

A novel virus-like infectious agent (VLIA), obtained by direct transfection of DNA from Kaposi's sarcoma of a patient with acquired immune deficiency syndrome (AIDS), was transmissible from culture to culture by cell-free filtrate. VLIA contained an outer limiting membrane and had a buoyant density of 1.17-1.20 g/ml in a sucrose gradient. The DNA genome of VLIA was estimated to be greater than 150 kilobase (kb) pairs and carried repetitive sequences. An 8.6 kb pair cloned probe (psb-8.6) and a 2.2 kb pair cloned probe (psb-2.2) of VLIA detected specific sequences in DNA of VLIA infected cells, but not in DNA of uninfected NIH/3T3 cells. By Southern blot hybridization analysis, VLIA was distinct from all known members of human herpes virus, from vaccinia virus, monkey herpes virus saimiri (HVS), and mouse cytomegalovirus (MCMV). Using synthetic primers with the VLIA specific DNA sequences and the polymerase chain reaction (PCR) method, we detected VLIA sequences in DNA isolated from 7 out of 10 patients with AIDS. VLIA infection was identified in spleen, liver, brain, lymph node, Kaposi's sarcoma tissues, or peripheral blood mononuclear cells from these patients, but not in 5 different organs and a tumor from 5 subjects without AIDS. Antiserum raised against VLIA in rabbit positively immunostained brain and lymph node tissues from these AIDS patients.

Development of a direct-binding enzyme-linked immunosorbent assay as an alternate assay for anti-hepatitis B core.

Anti-hepatitis B core (anti-HBc) screening is now used in some countries as a surrogate test to reduce the incidence of posttransfusion non-A, non-B hepatitis. The purpose of this study was to develop and standardize an alternate assay, for the detection of anti-HBc, based on a direct-binding enzyme-linked immunosorbent assay (db-ELISA). Microtiter plates were coated with recombinant DNA hepatitis B core antigen. Patient antibody was detected spectrophotometrically using a goat, anti-human immunoglobulin conjugated with horseradish peroxidase. The db-ELISA was compared to a standard competitive-binding ELISA (cb-ELISA). A competitive-binding radioimmunoassay (cb-RIA) was used as the reference assay for this study. The specificity of the cb-ELISA was 50% and the positive predictive value was 64% when compared to the cb-RIA. The specificity and positive predictive value of the db-ELISA were both 97% when compared to the cb-RIA. Based on titration studies, the sensitivity of the db-ELISA is at least equal to the cb-RIA. Based on the data generated in this study, the db-ELISA appears to be an acceptable screening test for anti-HBc.

Inapparent infection of hepatitis A virus.

To detect inapparent infection with hepatitis A virus, serial sera were collected from patients with hepatitis A and their contacts in two waterborne epidemics in China. Epidemic 1 occurred in a rural village near Hangzhou during August 1978-January 1979, and epidemic 2 took place in a rural primary school in Pinghu County in Zhejiang in April-May 1985. These sera were tested for antibodies against hepatitis A virus (anti-HAV), serum glutamic pyruvic transaminase (SGPT) activity, and icteric index. Feces also were collected in epidemic 1 to test for hepatitis A virus antigen. Both anti-HAV immunoglobulin M (IgM) and total anti-HAV were assayed in sera from "healthy persons" (symptomless persons without icterus and with normal SGPT level) who were in close contact with hepatitis A patients. In epidemic 1, among 18 "healthy persons", 12 were anti-HAV IgM positive, two were immune, and four susceptibles escaped infection. In epidemic 2, among 32 "healthy children", three were anti-HAV IgM positive, five had been infected by hepatitis A virus in the past, and 24 were not infected. These results demonstrate that inapparent infections occur along with overt and subclinical infections during epidemics of hepatitis A. The proportions of inapparent, subclinical, and overt infections were, respectively, 34.3%, 45.7%, and 20% in epidemic 1, and 25%, 50%, and 25% in epidemic 2. In addition, hepatitis A virus particles were demonstrated in the feces of all infected subjects who were examined and who included all levels of clinical response. These particles were identified with immuno-electron microscopy and enzyme-linked immunoassay.

Reproductive consequences of mega vitamin E supplements in female rats.

Female rats fed 0, 25, 2500 and 10,000 IU vitamin E/kg diet for 3 months were examined for reproductive performance. On 10,000 IU vitamin E/kg diet, the fertility of inseminated rats was significantly reduced as compared to rats given normal or nutritional levels of vitamin E.

Effect of high levels of dietary vitamin E on liver and plasma lipids and fat soluble vitamins in rats.

The effect of low, moderate and high dietary vitamin E (ranging from 0 to 25,000 IU/kg diet) on the levels of alpha-tocopherol, total lipids, cholesterol and vitamin A in liver and plasma of rats fed for 8 and 16 months was studied. A logarithmic relationship was observed between dietary levels of vitamin E and the concentrations of this vitamin in liver and plasma. The total alpha-tocopherol in whole liver of rats fed different levels of dietary vitamin E for 16 months was approximately double in comparison to rats fed for 8 months. Totoal lipids in liver were significantly increased by excess vitamin E supplementation in rats fed for 8 months, but not in rats fed for 16 months. There was no significant change observed in liver cholesterol level at 16 months. Plasma total lipids and cholesterol were lowered by vitamin E deficiency and also by dietary levels higher than 2,500 IU vitamin E/kg diet in rats fed for 16 months. Liver vitamin A storage was 4.5 times higher in rats supplemented with vitamin E than in rats without any supplement, but the effect of excess dietary vitamin E was no different from that of normal level (25 IU/kg diet). The findings of our long-term study are compared with the results of other short-term studies and the implications are discussed.