[Genetic analysis of three cases of acephalic spermatozoa syndrome caused by SUN5 mutation and the outcome of assisted reproductive technology].

To explore the genetic causes of 3 male infertility patients with acephalospermia and the outcome of assisted reproductive technology. Clinical diagnosis, sperm morphology examination, sperm transmission electron microscopy examination were performed on 3 patients, and the whole exome sequencing technology was used for screening, Sanger sequencing verification, mutation pathogenicity analysis, and protein sequence homology comparison. Assisted reproductive technology was implemented to assist pregnancy treatment. The 3 patients were all sporadic infertile men, aged 25, 42 and 26 years, and there was no obvious abnormality in the general physical examination. Male external genitalia developed normally, bilateral testicles were normal in volume, and bilateral epididymis and spermatic vein were palpated without nodules, cysts, and tenderness. Repeated semen analysis showed that a large number of immature sperm could be seen, and they had the ability to move. The SUN5 gene of the 3 male infertile patients was a case of homozygous missense mutation c.7C>T (p.Arg3Trp), a case of compound heterozygous missense mutation c.1067G>A (p.Arg356His) and nonsense mutation c.216G>A (p.Trp72*) and a case of homozygous missense mutation c.1043A>T (p.Asn348Ile), of which c.7C>T (p.Arg3Trp) and c.1067G>A (p.Arg356His) were new variants that had not been reported. SIFT, Mutation Taster and PolyPhen-2 software function prediction results were all harmful, the nonsense mutation c.216G>A (p.Trp72*) led to the premature termination of peptide chain synthesis which might have a greater impact on protein function. The homology regions in the protein sequence homology alignment were all highly conserved.The 3 male patients and their spouses obtained 4 biological offspring through intracytoplasmic sperm injection, all of which were boys, and one of them was a twin.Three male infertile patients might be caused by SUN5 gene mutations. Such patients could obtain their biological offspring through assisted reproductive technology. It was still necessary to pay attention to the genetic risk of ASS, it was recommended that both men and women conduct genetic counseling and screening at the same time. In clinical diagnosis, whole exome sequencing technology could be used to perform auxiliary examinations to determine the treatment plan and assisted reproductive methods as soon as possible to reduce the burden on the family and society. The newly discovered mutation sites of SUN5 gene provided clues and directions for elucidating the pathogenic mechanism, and at the same time expanded the pathogenic mutation spectrum of ASS.

Exosomes secreted by mice adipose-derived stem cells after low-level laser irradiation treatment reduce apoptosis of osteocyte induced by hypoxia.

OBJECTIVE: Kienböck's disease is a commonly seen posttraumatic avascular necrosis characterized by avascular necrosis of the lunate bone of the wrist which involves the dominant hand. In our study, we aimed to present midterm outcomes of 12 cases treated with radial metaphyseal core decompression. PATIENTS AND METHODS: In our clinic, 12 patients who applied to our outpatient clinic with intractable pain despite at least six weeks of conservative treatment were previously diagnosed and evaluated as Kienböck's disease between the years 2006 and 2014. Patients at early stage received radial metaphyseal core decompression. RESULTS: The patients were evaluated as postoperative grip strength, flexion-extension gap, ulnar-radial deviation gap, VAS, Quick DASH and MAYO wrist scoring and patient satisfaction. CONCLUSIONS: We determined that interventions performed for Kienböck's disease cannot halt radiological progression. We are of the opinion that radial metaphyseal core decompression, aiming at increasing blood perfusion, improve early diagnosis and treatment of Kienböck's disease, increasing the patient satisfaction.

Beneficial effects of low-level laser irradiation on senile osteoporosis in rats.

OBJECTIVE: To investigate the effect of low-level laser irradiation (LLLI) on bone mineral density (BMD), bone structures, bone biomechanical properties and bone metabolism in senile osteoporosis, and to explore a relatively more secure and effective way to prevent and treat osteoporosis. MATERIALS AND METHODS: Sprague-Dawley (SD) male rats at different age stages (4 months old, 12 months old and 20 months old) were selected and randomly divided into six groups. The rats in the treatment group were treated with LLLI for 12 weeks, and then the microstructure of bones was analyzed by micro-computed tomography (micro-CT) scanning. The biomechanical indexes of the femur were detected by the three-point bending test. Levels of the blood calcium (Ca)2+, blood phosphorus (P)3+, urine Ca, urine P and urine creatinine (CREA) were detected using an automatic biochemical analyzer. The contents of serum osteocalcin (OCN) and bone alkaline phosphatase (BAP) were measured by enzyme-linked immunosorbent assay (ELISA). The bone formation rate (BFR) was analyzed by double fluorescent labeling with calcein and tetracycline. Hematoxylin and eosin (HE) staining and toluidine blue staining were used to analyze the number of bone marrow osteoblasts and adipocytes. RESULTS: Micro-CT results showed that compared with those in the young group, the bone mineral density (BMD) in the old group was significantly decreased, and the trabecular microstructure was seriously damaged. LLLI could significantly enhance the BMD and improve the damage to the trabecular microstructure; the three-point bending test revealed that LLLI could significantly improve the biomechanical properties and enhance the mechanical strength of the femur in the old group; the biochemical analysis showed that LLLI could significantly reduce Ca and P losses and elevate the levels of serum BAP and OCN; the bone histomorphology analysis results indicated that LLLI could increase BFR and mineral apposition rate (MAR), increase the number of osteoblasts and decrease the number of adipocytes in the bone marrow in the old group. CONCLUSIONS: LLLI can effectively improve osteoporosis, increase BMD, improve bone structure and improve bone biomechanical properties in old rats; at the same time, it increases the levels of serum BAP and OCN and the number of osteoblasts in the bone marrow, suggesting that the osteogenesis function of osteoblasts is enhanced.

OS054. Differential strain sensitivities to ischemia/reperfusion in two mouse models of fetal growth restriction.

INTRODUCTION: Uterine ischemia/reperfusion (I/R) in rodents is a common model for the study of fetal growth restriction (FGR). It has been observed that different strains of mice vary in their response to uterine I/R. OBJECTIVES: Our objective was to characterize fetal and placental growth, and uterine and placental inflammation, in pregnant mice from 2 strains (C3H/HeOuJ and C57BL/6J), in response to different uterine I/R modalities. METHODS: Timed-pregnant mice (6-8 in each experimental group) were subjected to unilateral uterine I/R by either total flow restriction (TFR,right ovarian and uterine arterial clamping,5 or 30min) or partial flow restriction (PFR,right ovarian artery clamping,30min), or to sham-operation, on the 14th (for C57 mice) or 15th (for C3H mice) day of gestation. Four days later, fetal and placental weights and fetal loss were evaluated, and myeloperoxidase (MPO) activity was assayed in uterine and placental tissues. Data were analyzed by ANOVA with p<0.05 considered significant. RESULTS: In C3H/HeOuJ mice, TFR/30 min induced significantly (p<0.05) lower fetal and placental weights, and higher MPO activity in both uterus and placenta, compared to sham-operated controls. PFR/30min produced fetal but not placental growth restriction in the C3H mice. In contrast, C57BL/6J mice exhibited much greater sensitivity to uterine I/R: TFR/5 min was adequate to induce FGR in the C57 mice, which was equivalent in proportion to FGR caused by TFR/30min in C3H. C57 mice also exhibited significantly greater fetal loss and higher uterine, but not placental, MPO activity in response to I/R compared to sham-operated controls. CONCLUSION: Reliable FGR can be induced in different strains of mice by uterine I/R through adjustment of intensity, duration, and gestation day of the challenge. Mice of different strains have different tolerance to uterine I/R. These strain disparities would lend themselves to identifying the role of mouse strain-specific genetic determinants which form the bases for the observed differences in sensitivity to I/R-induced FGR.

Neuronal nitric oxide synthase (NOS) regulates the expression of inducible NOS in rat small intestine via modulation of nuclear factor kappa B.

We previously reported that neuronal nitric oxide synthase (nNOS) is the predominant NOS in the intestine. Inducible NOS (iNOS), an enzyme involved in the inflammatory response, is regulated by cytokines via the transcriptional factor NF-kappaB. We examined a new mechanism of intestinal iNOS regulation with respect to the role of nNOS and its effect on NF-kappaB. Young Sprague-Dawley rats were treated for 4 days with 1) saline, 2) 7-nitroindazole (7-NI, specific nNOS inhibitor), 3) 7-NI + pyrrolidine dithiocarbamate (PDTC, NF-kappaB inhibitor), or 4) PDTC. Intestinal iNOS mRNA, NF-kappaB activity, and the tissue content of the regulatory IkappaBalpha were examined. We found that 7-NI-treated animals had higher intestinal NF-kappaB (p50-p65) activity, lower IkappaBalpha content, and increased intestinal iNOS mRNA, iNOS protein, and iNOS activity compared with controls. All of these changes were abolished when PDTC was given together with 7-NI. PDTC alone had no effect. 7-NI induces a delayed increase in intestinal myeloperoxidase activity (after elevation in NF-kappaB and iNOS), which could be abrogated by PDTC. We conclude that in normal rat small intestine, nNOS suppresses the gene expression of iNOS through NF-kappaB down-regulation and that nNOS suppression leads to IkappaBalpha degradation, NF-kappaB activation, and iNOS expression.

Platelet-activating factor increases mucosal permeability in rat intestine via tyrosine phosphorylation of E-cadherin.

1. Platelet-activating factor (PAF), an inflammatory mediator, plays an important role in mediating intestinal injury. However, it remains unclear whether PAF has a function in the intestine. The production of PAF by normal intestine and by unstimulated intestinal epithelial cell lines suggests that PAF may have a regulatory function in the normal bowel. 2. In this study we investigated the role of PAF in modulating intestinal mucosal permeability in rats. Lumen-to-blood transit of FD-4 (dextran 4400), (an index of intestinal permeability), was assessed in sham-operated rats and rats injected with PAF (1.25 microg kg(-1), i.v., a dose insufficient to induce intestinal injury). 3. PAF-induced villus cytoskeletal changes were examined by staining the intestine for F-actin. The effect of PAF on tyrosine phosphorylation of the junctional protein E-cadherin was examined by immunoprecipitation. Some rats were pretreated with AG1288 (a tyrosine kinase inhibitor) before PAF injection, and mucosal permeability change was assessed. 4. To investigate the role of endogenous PAF upon mucosal permeability, we studied the effect of PAF antagonists on (intraluminal) glucose-induced increase in mucosal permeability. 5. We found that low dose PAF: (a) alters the cytoskeletal structure of intestinal epithelium, (b) causes the influx of FD4 from intestinal lumen to systemic circulation, (c) induces tyrosine phosphorylation of E-cadherin and cadherin-associated proteins. Glucose-induced mucosal permeability increase is abolished by using two structurally different PAF antagonists. 6. These results suggest that endogenous PAF modulates macromolecular movement across the intestinal mucosal barrier, probably via tyrosine phosphorylation of E-cadherin and cytoskeletal alteration of enterocytes.

Mechanism for the isotype dependence of antibody-mediated toxicity in Cryptococcus neoformans-infected mice.

Ab-based therapies have undergone a renaissance in recent years, but infusion-related reactions are a significant clinical problem. Administration of certain mAbs to Swiss Webster mice infected with Cryptococcus neoformans can result in acute lethal toxicity (ALT) characterized by cardiovascular collapse. The ability of a mAb to produce ALT is isotype dependent and occurs with IgG1 but not IgG3. To investigate this phenomenon, we measured spleen and liver cytokine responses and platelet-activating factor (PAF) content in mice given C. neoformans glucuronoxylomannan (GXM) followed by specific Ab of IgG1 or IgG3 isotype. We found no evidence to suggest that the differences in IgG1 and IgG3 toxicity were due to differences in chemokine or cytokine response. In contrast, liver and spleen tissue PAF content was significantly greater in mice IgG1. Furthermore, our results show differences in the response to IgG1- and IgG3-GXM complexes regarding: 1) macrophage-inflammatory protein-1alpha and monocyte chemoattractant protein-1 regulation, 2) splenic and hepatic PAF content, and 3) hepatic PAF content in infected mice. IgG1-associated ALT appears to be the result of greater production of PAF in response to IgG1-GXM complex formation. The results are consistent with the view that IgG1 and IgG3 interact with different Fc receptors. Our findings strongly suggest that the mechanism for Ab-mediated ALT is different from the cytokine release syndrome described after administration of other therapeutic mAbs.

Type I nitric oxide synthase (NOS) is the predominant NOS in rat small intestine. Regulation by platelet-activating factor.

Constitutive nitric oxide synthase (cNOS) may play an important protective role in the intestine, since our previous study has shown that the degree of bowel injury induced by platelet-activating factor (PAF), a potent inflammatory mediator, is inversely related to the cNOS content of the intestine. This study aims to examine the composition of the cNOS system in rat small intestine, and its regulation by PAF. We found that an approximately 120 kDa NOS I (neuronal NOS) is the predominant NOS in rat intestine, as evidenced by the following: (a) immunoblotting with specific antibodies detected a NOS I of approximately 120 kDa, but little NOS III; (b) the Ca(2+)-dependent, constitutive NOS (cNOS) activity of the rat intestine was removed by immunoprecipitation with the anti-NOS I, but not anti-NOS II or anti-NOS III antibodies; (c) RT-PCR revealed constitutive expression of NOS I in the intestinal tissue, but only a minute amount of NOS III. Immunofluorescent staining with anti-NOS I located NOS in the Auerbach plexus and nerve fibers in the muscle layer. We also found that this 120 kDa NOS I is rapidly (within 1 h) down-regulated in response to PAF administration. The protein level, enzyme activity as well as mRNA of nNOS were all decreased in the intestine.

Roles of nitric oxide synthases in platelet-activating factor-induced intestinal necrosis in rats.

OBJECTIVE: To examine the role of constitutive and inducible nitric oxide synthases (cNOS and iNOS) in platelet-activating factor (PAF)-induced shock and intestinal injury. DESIGN: Prospective, randomized, controlled experimental study. SETTING: Hospital research laboratory. SUBJECTS: Young adult male Sprague-Dawley rats were anesthetized and studied. INTERVENTIONS: Rats were injected with PAF, either alone or after the following pretreatments: a) selective iNOS inhibitors aminoguanidine or S-methylisothiourea; b) 3-morpholinosydnonimine, a NO donor; c) S-methylisothiourea + 3-morpholinosydnonimine; and d) antineutrophil antibody (to deplete neutrophils). MEASUREMENTS AND MAIN RESULTS: Blood pressure, hematocrit, white blood cell counts, intestinal injury, and intestinal cNOS and iNOS activities were assessed. We found that: a) cNOS is the predominant NOS in the intestine and its activity is inversely correlated to the level of tissue injury; b) there is a time-dependent increase in cNOS activity in sham-operated animals, which was abolished by PAF; c) Western blotting and immunohistochemistry showed iNOS present in the normal intestine, localizing mainly in crypt cells; d) iNOS inhibitors attenuated PAF-induced injury in animals with high cNOS activity, but had no protective effect in animals with low cNOS activity; e) 3-morpholinosydnonimine, alone or together with S-methylisothiourea, alleviated PAF-induced injury; and f) neutrophil depletion blocked the suppressive effect of PAF on cNOS and prevented injury. CONCLUSIONS: We conclude that cNOS and iNOS play different roles in PAF-induced intestinal injury. Caution should be exerted concerning potential therapeutic uses of iNOS inhibitors.

The role of xanthine oxidase in platelet activating factor induced intestinal injury in the rat.

BACKGROUND: Xanthine oxidase (XO) is an important source of reactive oxygen species in the small intestine. AIMS: To examine the interaction of platelet activating factor (PAF), XO, and neutrophils in mediating intestinal injury in rats. METHODS: Two doses of PAF were used to induce either reversible hypotension, or irreversible shock with intestinal necrosis. The activities of XO, and its precursor xanthine dehydrogenase (XD), in both the whole intestinal tissue and epithelial cells, were measured. XO was localised by histochemical staining. RESULTS: PAF dose dependently induced an increase in XO activity, predominantly in the ileal epithelium, without altering the total activity of XD+XO. Most of the XD to XO conversion was via proteolysis. PAF induced XO activation and intestinal injury were prevented by prior neutrophil depletion. PAF induced XO activation is probably not due to reperfusion, as XO activation preceded the recovery of mesenteric flow. Allopurinol pretreatment substantially inhibited intestinal neutrophil sequestration induced by high dose (but not low dose) PAF. CONCLUSIONS: PAF rapidly activates intestinal XO through proteolytic XD-XO conversion, predominantly in the ileal epithelium. This effect is mediated by neutrophils. XO activation promotes PAF induced polymorphonuclear leucocyte sequestration in the intestine.

Interaction of platelet-activating factor, spleen and atrial natriuretic peptide in plasma volume regulation during endotoxaemia in rats.

1. We studied endotoxin (lipopolysaccharide, LPS)-induced platelet-activating factor (PAF) production in various visceral organs, and the effect of PAF antagonists or splenectomy on LPS-induced changes. 2. PAF production in response to LPS was highest in the spleen, followed by ileum, heart, lung and kidneys. None was found in the liver. The splenic response was rapid, reaching 10 times the basal level at 30 min. The increased PAF content in each organ was unrelated to the enzyme activity of either macrophages or neutrophils. 3. LPS-induced hypotension and haemoconcentration were largely prevented by PAF antagonists and splenectomy. 4. Plasma volume fell, and plasma atrial natriuretic peptide (ANP) rose, after LPS administration. Splenectomy or pretreatment with PAF antagonists almost completely prevented these LPS-induced changes at 30 min, but only partially reversed them at 90 min. 5. These results suggest that during endotoxaemia: (a) the spleen is the site of the highest endogenous PAF production; (b) the initial release of ANP is dependent on the production of endogenous PAF, and a PAF-ANP interaction mediates the early plasma volume reduction; (c) plasma volume reduction as well as ANP release depend on the spleen; (d) PAF mediated the hypotensive response and its action in the spleen; and (e) sequestered neutrophils are probably not the main source of PAF in the spleen.

Intestinal NF-kappaB is activated, mainly as p50 homodimers, by platelet-activating factor.

NF-kappaB, a transcription factor, upregulates gene transcription of many inflammatory mediators. Here, we examined the activity of NF-kappaB in the rat small intestine, and how it may be affected by platelet-activating factor (PAF), an important mediator for intestinal injury and inflammation. Ileal nuclear extracts from sham-operated and PAF (1.5 microg/kg)-injected rats were prepared for the assessment of NF-kappaB DNA-binding activity, and the identification of NF-kappaB subunits. The experiment was also performed on neutrophil-depleted rats to examine whether the PAF effect is neutrophil-dependent. Cellular NF-kappaB was localized by immunohistochemistry. We found that: (a) NF-kappaB is constitutively active in rat small intestine; (b) PAF at a dose below that causing shock and bowel necrosis enhances DNA-binding activity of NF-kappaB within 30 min after injection; activated NF-kappaB contains predominantly p50 subunits; (c) immunohistochemistry showed that PAF induced translocation of p50 into the nucleus of cells of the lamina propria, as well as of the epithelium; and (d) the effect of PAF is abrogated by neutrophil depletion, suggesting a role of neutrophils in NF-kappaB activation. Our study suggests that NF-kappaB is weakly active constitutively in the intestine, and inflammatory stimuli such as PAF activate NF-kappaB and enhance its DNA-binding activity in the intestine, which contains predominantly p50 subunits.

Platelet-activating factor (PAF) up-regulates plasma and tissue PAF-acetylhydrolase activity in the rat: effect of cycloheximide.

Platelet-activating factor (PAF) is a proinflammatory phospholipid mediator implicated in necrotizing enterocolitis. Regulation of PAF-acetylhydrolase (AH), the enzyme degrading PAF, is poorly understood. In this study we found that administration of a dose of PAF (1.5 microg/kg, i.v.), which does not cause gross intestinal injury, increased plasma and intestinal PAF-AH in the rat. Cycloheximide (CHX, 5 mg/kg, i.v.) reduced the activity of plasma (but not intestinal tissue) AH in control, as well as in PAF-injected rats, and aggravated systemic inflammation and tissue injury in the latter. The intestinal necrosis induced by PAF and CHX was ameliorated by posttreatment with WEB2170 (a PAF antagonist), indicating a role of endogenous PAF in mediating injury. Both WEB2170 and anti-TNF antibody reduced PAF-induced AH activity in intestinal tissue, but not in the plasma. Allopurinol largely prevented the injury induced by PAF and CHX, but had no effect on the up-regulation of AH. We conclude: 1) de novo protein synthesis is required to maintain physiologic AH level in the plasma; 2) PAF up-regulates plasma and intestinal AH activity; 3) CHX enhances the injurious effect of PAF; 4) endogenous PAF and TNF also play a role in the up-regulation of intestinal AH; the former probably mediating the intestinal injury by PAF; and 5) reactive oxygen species may mediate the injurious effect of PAF plus CHX, but do not contribute to the regulation of AH by PAF.

Role of leukocyte beta 2-integrin in PAF-induced shock and intestinal injury.

Leukocyte adhesion and diapedesis, critical steps in the inflammatory process, depend on the expression of integrin CD11b/CD18. In this study, we examined the preventive effect of monoclonal antibodies (MAb) against CD11b (1B6), CD11a, or CD18 (CL26) on platelet-activating factor (PAF)-induced bowel injury. Young male Sprague-Dawley rats were anesthetized and injected with either of two doses of PAF (2.5 or 3 micrograms/kg iv) to induce transient hypotension and irreversible shock. Some rats wee also injected intravenously with 1B6 (anti-CD11b), anti-CD11a, CL26 (anti-CD18), or combined anti-CD11a and 1B6, 30 min before PAF. Animals receiving a low dose of PAF developed mild hypotension, hemoconcentration, increased intestinal myeloperoxidase, and bowel injury after 1 h. These effects were completely prevented by pretreatment with 1B6. A high dose of PAF induced irreversible shock and gross intestinal necrosis. Both CL26 and 1B6 were partially effective in attenuating PAF-induced bowel injury. Addition of anti-CD11a to 1B6 in the treatment further ameliorated the systemic adverse effects of PAF and intestinal injury. However, focal minor injury still developed. Anti-CD11a alone, fucoidin, or anti-P-selectin was ineffective. Rats depleted of neutrophils were also largely protected from the adverse effects of PAF at high doses, although minor intestinal injury often persisted. We conclude that leukocyte beta 2-integrins play an important role in PAF-induced hypotension, leukopenia, hemoconcentration, and intestinal necrosis, and that CD11b/CD18 is the main adhesion molecule involved in the pathogenesis of injury. However, CD11/CD18- and neutrophil-independent pathways exist for mediating PAF-induced bowel injury, although their role is probably a minor one.