CCL20-CCR6 axis directs sperm-oocyte interaction and its dysregulation correlates/associates with male infertility‡.

The interaction of sperm with the oocyte is pivotal during the process of mammalian fertilization. The limited numbers of sperm that reach the fallopian tube as well as anatomic restrictions indicate that human sperm-oocyte encounter is not a matter of chance but a directed process. Chemotaxis is the proposed mechanism for re-orientating sperm toward the source of a chemoattractant and hence to the oocyte. Chemokines represent a superfamily of small (8-11 kDa), cytokine-like proteins that have been shown to mediate chemotaxis and tissue-specific homing of leukocytes through binding to specific chemokine receptors such as CCRs. Here we show that CCR6 is abundantly expressed on human sperms and in human testes. Furthermore, radioligand-binding experiments showed that CCL20 bound human sperm in a specific manner. Conversely, granulosa cells of the oocyte-surrounding cumulus complex as well as human oocytes represent an abundant source of the CCR6-specific ligand CCL20. In human ovaries, CCL20 shows a cycle-dependent expression pattern with peak expression in the preovulatory phase and CCL20 protein induces chemotactic responses of human sperm. Neutralization of CCL20 in ovarian follicular fluid significantly impairs sperm migratory responses. Conversely, analyses in infertile men with inflammatory conditions of the reproductive organs demonstrate a significant increase of CCL20/CCR6 expression in testis and ejaculate. Taken together, findings of the present study suggest that CCR6-CCL20 interaction may represent an important factor in directing sperm-oocyte interaction.

Acute pulmonary embolism caused by highly aggregated intravenous immunoglobulin.

BACKGROUND AND OBJECTIVES: Six patients died and one patient survived following infusion of a specific lot of intravenous immunoglobulin (IVIG) within half an hour in May 2008. This study elucidated the underlying pathogenesis. MATERIALS AND METHODS: A variety of protein fractionation and identification approaches were employed to determine the abnormal components in IVIG products obtained from the hospital where the patients were treated. Animal studies using mice and monkeys were conducted to elucidate the pathophysiological mechanisms. In animal experiments, the effect and distribution of immunoglobulin was investigated using HE staining and immunohistochemistry (IHC) separately, while platelets and fibrinogen depletion were utilized to determine a possible link between thromboembolism formation in animals and the lethal effect of the IVIG. The size and distribution of the protein aggregates were determined with Coulter Counter Multisizer-3 after the dilution of the IVIG with plasma, and the lethal effect of the protein aggregates was simulated with artificial microparticles. RESULTS: The IVIG retrieved from the hospital was found to have striking similarities to the heat-treated IVIG in terms of protein aggregation profiles and lethal effects. Post-mortem examination indicated that immunoglobulin aggregates were mainly found in the lung of the animals, while depletion of platelets and fibrinogen from the IVIG preparations failed to prevent the death of the animals. Similar amount of artificial microparticles caused animal death in similar fashion. CONCLUSIONS: Our findings indicate that the retrieved IVIG exerted its lethal effects by blocking the pulmonary circulation without markedly altering the coagulation cascade or immunological events.

Development of the hybrid Sleeping Beauty: baculovirus vector for sustained gene expression and cancer therapy.

Antiangiogenesis is an appealing anticancer approach but requires continued presence of the antiangiogenic agents, which can be remedied by gene therapy. Baculovirus is an emerging gene delivery vector but only mediates transient expression (<7 days); thus, this study primarily aimed to develop a hybrid baculovirus for sustained antiangiogenic gene expression and cancer therapy. We first constructed plasmids featuring adeno-associated virus inverted terminal repeats (AAV ITRs), oriP/Epstein-Barr virus-expressed nuclear antigen 1 (EBNA1) or Sleeping Beauty (SB) transposon and compared their efficacies in terms of persistent expression. In human embryonic kidney (HEK293) cells, AAV ITR failed to prolong the expression while oriP/EBNA1 moderately extended the expression to 35 days. In contrast, the SB system led to stable expression beyond 77 days even without antibiotic selection. Given this finding, we constructed a hybrid SB baculovirus expressing the SB transposase and harboring the transgene cassette flanked by inverted repeat/direct-repeat (IR/DR) elements recognizable by SB. The hybrid SB baculovirus efficiently transduced mammalian cells and mediated an expression duration longer than that by conventional baculoviruses, thanks to the transgene persistence and integration. The SB baculovirus (Bac-SB-T2hEA/w) expressing the antiangiogenic fusion protein comprising endostatin and angiostatin (hEA) also enabled prolonged hEA expression. With sustained hEA expression, Bac-SB-T2hEA/w repressed the angiogenesis in vivo, hindered the growth of two different tumors (prostate tumor allografts and human ovarian tumor xenografts) in mice and extended the life span of animals. These data altogether implicated the potential of the hybrid SB-baculovirus vector for prolonged hEA expression and for the treatment of multiple types of angiogenesis-dependent tumors.

Tetraspanins CD9 and CD81 are molecular partners of trimeric FcɛRI on human antigen-presenting cells.

BACKGROUND: Most functions of tetraspanins are not related to cell-surface receptor ligand binding, but are mediated by direct interactions with their partner proteins. Functions of trimeric FcɛRI, expressed by antigen-presenting cells (APCs), range from amplification of allergic inflammatory reactions to their active suppression. Cell-type-specific protein-protein interactions might play a role in the regulation of these bidirectional tasks. Therefore, we intended to study the interactions of trimeric FcɛRI with tetraspanins. METHODS: The expression levels of tetraspanins CD9, CD37, CD53, CD63, CD81, CD82, and CD151 on skin dendritic cells of atopic dermatitis (AD) patients or healthy individuals were detected by flow cytometry. Tetraspanin expression on FcɛRI(pos) and FcɛRI(neg) monocyte subpopulations was evaluated. Flow cytometry, confocal microscopy, immunoprecipitation, and immunoblotting experiments were performed to observe the relationship between tetraspanins CD9 and CD81 and FcɛRI. Furthermore, plate stimulation experiments were performed, and cytokines in the supernatants were detected. RESULTS: We found that human FcɛRI(pos) APCs expressed high amounts of tetraspanins and that the tetraspanins CD9 and CD81 were associated with FcɛRI. Concomitant activation of FcɛRI and CD9 on human monocytes increased FcɛRI-mediated cytokine release. CONCLUSION: Taken together, we show for the first time that CD9 and CD81 act as molecular partners of trimeric FcɛRI on human APC, which might be of importance in allergic diseases such as AD.

Association of single nucleotide polymorphisms in the diamine oxidase gene with diamine oxidase serum activities.

BACKGROUND: Histamine intolerance (HIT) is associated with an excess of histamine because of an impaired function of the histamine-degrading enzyme diamine oxidase (DAO). The genetic background of HIT is unknown yet. METHODS: Case-control association study of all haplotype tagging and four previously reported DAO SNPs and one HNMT Single nucleotide polymorphism with symptoms of HIT and DAO serum activity in 484 German individuals including 285 patients with clinical symptoms of HIT and 199 controls. RESULTS: Diamine oxidase serum activity was significantly associated with seven SNPs within the DAO gene. The minor allele at rs2052129, rs2268999, rs10156191 and rs1049742 increased the risk for a reduced DAO activity whereas showing a moderate protective effect at rs2071514, rs1049748 and rs2071517 in the genotypic (P = 2.1 × 10(-8) , 7.6 × 10(-10) , 8.3 × 10(-10) , 0.009, 0.005, 0.00001, 0.006, respectively) and allelic genetic model (P = 2.5 × 10(-11) , 5.4 × 10(-13) , 8.9 × 10(-13) , 0.00002, 0.006, 0.0003, 0.005, respectively). Reporter gene assays at rs2052129 revealed a lower promoter activity (P = 0.016) of the minor allele. DAO mRNA expression in peripheral blood mononuclear cells of homozygous carriers of the minor allele at rs2052129, rs2268999, rs10156191 was lower (P = 0.002) than homozygous carriers of the major allele. Diamine oxidase variants were not associated with the HIT phenotype per se, only with DAO activity alone and the subgroup of HIT patients displaying a reduced DAO activity. CONCLUSIONS: DAO gene variants strongly influence DAO expression and activity but alone are not sufficient to fully effectuate the potentially associated disease state of HIT, suggesting an interplay of genetic and environmental factors.

Immunodeviation towards a Th17 immune response associated with testicular damage in azoospermic men.

Infection and inflammation of the male reproductive tract are thought to be a primary aetiological factor of male infertility. Furthermore, several studies suggest that T lymphocytes are critically involved as regulator in the pathogenesis of male infertility under these conditions and are thought to induce autoimmune orchitis. In this context of autoimmunity the recently described T helper (Th) 17 subset has been suggested to play an essential role so that the aim of this study was to investigate the expression and characteristics of Th17 cells as well as the presence of Th17 inducing antigen presenting cells (APCs) in azoospermic testis with chronic inflammation (ATCI) compared with normal spermatogenesis. By stereological analysis, we detected base line expression of Th17 cells in Con. However, increased expression intensity and number of Th17 cells and their cytokines [interleukin (IL)-17A, IL-21, IL-22] and a decreased level of Foxp3(+) and interferon-γ(+) cells could be demonstrated in ATCI. Moreover, along with these data, increased numbers of Th17-inducing IL-23 producing CD11c(+) and CD68(+) APCs could be detected in ATCI. From these data, a picture emerges that Th17 cells orchestrated by IL-23 producing APCs are critically involved in chronic inflammation in ATCI.

Dysbaric osteonecrosis by X-ray and CT scan in Chinese divers.

OBJECTIVE: To compare the results of X-ray and CT scan for dysbaric osteonecrosis in Chinese divers. METHODS: Both shoulders, hips and knees of 66 asymptomatic divers with diving duration of more than one year were examined by X-ray and CT scan. RESULTS: The most frequent locations of dysbaric osteonecrosis were the upper femurs, followed by the upper humerus, lower femurs and upper tibias, and the most frequent radiographic lesions were calcification spots and cystic changes. Of the lesions detected, 38% (27/71) and 42% (95/229) werejuxta-articular of the femoral and humeral heads by X-ray and CT respectively. The detection rates of dysbaric necrosis (juxta- and/or other lesions) of X-ray and CT scan were 42.4% (95% confidence interval: 30.5%-54.3%) and 81.8% (95% CI: 72.4%-91.2%) respectively (p<0.05). If CT scan was used as the gold standard, the sensitivity of X-ray was 100% and the specificity was 31.6%. CONCLUSION: CT scan showed a higher detection rate of dysbaric necrosis than X-ray. We recommend that CT scan be used for early diagnosis of dysbaric osteonecrosis.

Matrix-specific FAK and MAPK reorganization during Caco-2 cell motility.

We have previously reported that Caco-2 cell motility redistributes FAK, paxillin, and activates p38. However, the subcellular organization of these intracellular signals during cell migration is unclear. We, therefore, investigated the organization of actin, FAK, paxillin, and activated ERK and activated p38 during Caco-2 motility across collagen I, fibronectin, laminin, and tissue culture treated glass. Differential density seeding generated homogeneous static and migrating populations. Expression of actin, FAK, paxillin, phospho-ERK, and phospho-p38 were examined by immunofluorescent staining in static and motile cells. Actin was concentrated toward the peri-nuclear central area of cells migrating on matrix proteins studied. Actin immunoreactivity was decreased in the leading edge of lamellipodia. FAK immunoreactivity was weaker in migrating cells than in static cells on the same matrix. FAK was expressed along cell-cell contacts of both cell populations, but absent in migrating lamellipodia of matrix-cultured cells. Paxillin staining was diffuse in static cells but organized toward migrating lamellipodia in a radial manner. Like FAK, phosphorylated ERK was expressed in the central region of migrating cells but was dramatically decreased at areas of cell-cell contact and free lamellipodia. Fibronectin exerted the greatest effect on ERK activation in all matrix proteins studied. In contrast, phosphorylated p38 staining was stronger in migrating cells on matrix than in static cells on the same matrix. Phosphorylated p38 was expressed in the nuclear of migrating cells and disappeared in the cell-cell contact side and free lamellipodia. Interestingly, the reorganization of these proteins was distinctly different on tissue culture treated glass without a physiologic matrix substrate. For instance, FAK staining increased rather than decreased in motile cells on plastic, and lamellipodial FAK staining could be discerned. Matrix may influence Caco-2 biology during migration not only by triggering intracellular phosphorylation events but also by reorganizing the cytoskeleton and the subcellular localization of these intracellular signals.

[The heart and diabetes. An anatomo-clinical study]. / Coração e diabetes. Estudo anátomo-clínico.

We performed a retrospective study in 72 autopsies of diabetic patients (DMP) selected out of 2,239 adult autopsies, comprehending the period between 1966 to 1982. In order to analyse the possible Diabetic Cardiomyopathy, the DMP were divided into 8 groups according to the presence or the absence of Myocardial Fibrosis (MF) and Congestive Heart Failure (CHF). The Diabetes Mellitus (DM) incidence according to the race, sex, age and the presence of Kimmestiel-Wilson (KW) were in agreement with the literature data. The majority of the deaths occurred after the sixth-decade and we did not find any DMP with Malignant Hypertension. Hypertension and Coronary Artery Disease (CAD) increased the frequency of anatomical cardiac alterations, as follows: 1. MF was more associated with CAD, 2. Hypertension was more frequent in DMP with KW in the nodular form; 3. Hypertension increased the frequency of left ventricular hypertrophy; 4. Myocardial Infarction occurred in the absence of occlusive vascular phenomena. The Myocardial Fibrosis (MF) observed in DMP without ACD and without hypertension may be final anatomic demonstration of a gradual metabolic-functional process, and not the basic mechanism of the CHF in the possible Diabetic Cardiomyopathy.

Putative association of a TLR9 promoter polymorphism with atopic eczema.

BACKGROUND: Toll-like receptors (TLR) play a pivotal role in the induction of first-line defense mechanisms of the innate immune system and trigger adaptive immune responses to microbial pathogens. Genetic variations in innate immunity genes have been reported to be associated with a range of inflammatory disorders. Deficiencies on the level of immunity receptors such as pathogen-recognition receptors are suspected to affect the maturation of our immune system and to avail thereby the high prevalence of atopic diseases and susceptibility of atopic patients to microbial infections. AIMS OF THE STUDY: We evaluated TLR9 as susceptibility gene for atopic eczema (AE). METHODS: Analyses of four tag single-nucleotide polymorphisms in two panels of families containing a total of 483 parent-affected offspring trios as well as a cohort of 274 unrelated adult AE cases and 252 hypernormal population-based controls have been performed. RESULTS: In both family cohorts, polymorphism C-1237T, which is located within the promoter region of the TLR9 gene, was significantly associated with AE, in particular the intrinsic subtype of AE. No associations were seen in the case-control cohort. Luciferase reporter gene assays revealed significantly higher promoter activity of the TT allelic variant at this single nucleotide polymorphism site. CONCLUSION: These observations suggest that the TLR9 promoter polymorphism C-1237T might affect AE susceptibility in particular in patients with the intrinsic variant of AE.

Protein tyrosine phosphatase interacting protein 51 (PTPIP51) is a novel mitochondria protein with an N-terminal mitochondrial targeting sequence and induces apoptosis.

Apoptosis is a genetically determined cell suicide program. Mitochondria play a central role in this process and various molecules have been shown to regulate apoptosis in this organelle. In the present study, we firstly identified that protein tyrosine phosphatase interacting protein 51 (PTPIP51) is a novel mitochondrial protein, which may induce apoptosis in HEK293T and HeLa cell lines. PTPIP51 transfection resulted in the externalization of phosphatidylserine (PS), activation of caspase-3, cleavage of PARP, and condensation of nuclear DNA. Further investigation revealed that PTPIP51 over-expression caused a decrease in mitochondrial membrane potential and release of cytochrome c, suggesting that it may be involved in a mitochondria/cytochrome c mediated apoptosis pathway. We also found that a putative TM domain near the N terminus of PTPIP51 is required for its targeting to mitochondria, as evidenced by the finding that deletion of the PTPIP51 TM domain prevented the protein's mitochondiral localization. Furthermore, this deletion significantly influenced the ability of PTPIP51 to induce apoptosis. Taken together, the results of the present study suggest that PTPIP51 is a mitochondrial protein with apoptosis-inducing function and that the N-terminal TM domain is required for both the correct targeting of the protein to mitochondria and its apoptotic functions.

Dynamics of mode competition in the gyrotron backward-wave oscillator.

The axial modes of the gyrotron backward-wave oscillator (gyro-BWO) each exhibit a distinctive asymmetry in axial field profile. As a result, and in sharp contrast to the behavior of the familiar resonator-based gyrotron oscillator, particle simulations of the gyro-BWO reveal a radically different pattern of mode competition in which a fast-growing and well-established mode is subsequently suppressed by a later-starting mode with a more favorable field profile. This is verified in a Ka-band experiment and the interaction dynamics are elucidated with a time-frequency analysis.

BMP2 gene therapy on the repair of bone defects of aged rats.

Age-related decline in the number of mesenchymal stem cells (MSCs) and their reduced capability to differentiate osteogenically, along with diminished availability of growth factors, may be major factors accounting for reduced bone formation in the aging mammalian body. In the first part of the study, we compared the number of MSCs in bone marrow (BM) and the content of bone morphogenetic protein 2 (BMP2) in cortical bone tissue in juvenile, adult, and aged (1, 9, and 24 months, respectively) male rats. To assay the influence of aging on osteogenic differentiation ability, MSCs from the three age groups were transduced with the BMP2 gene. Following gene transduction, the production of BMP2 in culture media, expression of osteogenic proteins (e.g., alkaline phosphatase, type Ialpha1 collagen, osteopontin, and bone sialoprotein), as well as ectopic bone formation in athymic mice were compared. Results showed that the number of MSCs in BM as well as the content of BMP2 in cortical bone tissue decreased with age, but no significant differences between the three age groups were found with regard to production of BMP2 or capability of BMP2 gene-modified MSCs to differentiate osteogenically. The second part of the study applied BMP2 gene-modified autologous MSCs/beta-tricalcium phosphate for repair of bone defects in aged rats with positive results. Our data indicate that the osteogenic potential of MSCs of aged rats can be restored following BMP2 gene transduction and that this technique may be a useful approach in the future planning of gene therapy for age-related osteoporotic fractures.

Repairing of goat tibial bone defects with BMP-2 gene-modified tissue-engineered bone.

Bone defects larger than a critical size are major challenges in orthopedic medicine. We combined tissue-engineered bone and gene therapy to provide osteoprogenitor cells, osteoinductive factors, and osteo-conductive carrier for ideal bone regeneration in critical-sized bone defects. Goat diaphyseal bone defects were repaired with tissue and genetically engineered bone implants, composed of biphasic calcined bone (BCB) and autologous bone marrow derived mesenchymal stem cells (BMSC) transduced with human bone morphogenetic protein-2 (hBMP-2). Twenty six goats with tibial bone defects were divided into groups receiving implants by using a combination of BCB and BMSCs with or without the hBMP-2 gene. In eight goats that were treated with BCB that contained hBMP-2 transduced BMSC, five had complete healing and three showed partial healing. Goats in other experimental groups had only slight or no healing. Furthermore, the area and biochemical strength of the callus in the bone defects were significantly better in animals treated with genetically engineered implants. We concluded that the combination of genetic and tissue engineering provides an innovative way for treating critical-sized bone defects.

Human caco-2 motility redistributes FAK and paxillin and activates p38 MAPK in a matrix-dependent manner.

The signals involved in restitution during mucosal healing are poorly understood. We compared focal adhesion kinase (FAK) and paxillin protein and phosphorylation, extracellular signal-regulated kinase (ERK) 1, ERK2, and p38 activation, as well as FAK and paxillin organization in static and migrating human intestinal Caco-2 cells on matrix proteins and anionically derivatized polystyrene dishes (tissue culture plastic). We also studied effects of FAK, ERK, and p38 blockade in a monolayer-wounding model. Compared with static cells, cells migrating across matrix proteins matrix-dependently decreased membrane/cytoskeletal FAK and paxillin and cytosolic FAK. Tyrosine phosphorylated FAK and paxillin changed proportionately to FAK and paxillin protein. Conversely, cells migrating on plastic increased FAK and paxillin protein and phosphorylation. Migration matrix-dependently activated p38 and inactivated ERK1 and ERK2. Total p38, ERK1, and ERK2 did not change. Caco-2 motility was inhibited by transfection of FRNK (the COOH-terminal region of FAK) and PD-98059, a mitogen-activated protein kinase-ERK kinase inhibitor, but not by SB-203580, a p38 inhibitor, suggesting that FAK and ERK modulate Caco-2 migration. In contrast to adhesion-induced phosphorylation, matrix may regulate motile intestinal epithelial cells by altering amounts and distribution of focal adhesion plaque proteins available for phosphorylation as well as by p38 activation and ERK inactivation. Motility across plastic differs from migration across matrix.

Differential dietary effects on colonic and small bowel neoplasia in C57BL/6J Apc Min/+ mice.

The effects of fiber on colon cancer are controversial. Twenty 5-week old C57BL/6J Apc Min/+ mice were fed for 60 days with a commercial mouse diet (Teklad LM-485) and eight semidefined diets containing 5-10% various fibers and 20% soybean oil. Ten additional C57BL/6J congenic litter-mates were fed each diet to assay colonic SCFA. SCFA, stool bulk, and colonic tumor incidence differed only slightly among the semidefined diets despite variations in fiber content and source. However, food consumption, caloric intake, stool bulk, and SCFA were substantially increased by the Teklad diet compared with all other groups. The Teklad diet significantly increased the number of mice with colonic tumors, average number of tumors/mouse, total tumor burden, colonic atypical hyperplasia, and small bowel tumors. Mice fed high-fat, no-fiber diets had more small bowel tumors (29.8 +/- 3.1) than mice fed diets with fiber (8.2 +/- 2.1) or with low fat and no fiber (18.1 +/- 3.4) (P < 0.05 for each group). These studies suggest that fat predisposes to and fiber protects against small bowel tumors but not colon tumors in these mice. Thus, diets high in fiber or yielding high colonic luminal SCFA may not necessarily protect against colonic cancer. Furthermore, the effects of dietary fiber in Teklad appear overshadowed by some other biologically active factors in this animal model.

ERK regulates the hepatocyte growth factor-mediated interaction of Gab1 and the phosphatidylinositol 3-kinase.

Based on our previous observations that active ERK associates with and phosphorylates Gab1 in response to HGF, and the prediction that the ERK phosphorylation site is adjacent to one of the phosphatidylinositol 3-kinase (PI3K) SH2 binding motifs, we examined the possibility that ERK phosphorylation can regulate the Gab1/PI3K association. The HGF-mediated association of Gab1 with either full-length GST-p85 or its isolated N- or C-terminal SH2 domains was inhibited by approximately 50% in the setting of ERK inhibition, a result confirmed by co-immunoprecipitation of the native proteins. A 14-amino acid peptide encoding (472)YVPMTP(477) (one of the major p85 binding sites in Gab1 and the predicted ERK phosphorylation site) was synthesized with either phosphotyrosine alone (pY), or phosphotyrosine + phosphothreonine (pYT). In both pull-down assays and competition assays, pYT demonstrated a higher affinity for p85 than did pY alone. Finally, examination of the phosphorylation state of Akt after HGF stimulation revealed that ERK inhibition resulted in a decrease in Akt activation at both 5 and 10 min. These results suggest that activated ERK can phosphorylate Gab1 in response to HGF stimulation and thereby potentiate the Gab1/PI3K association and subsequent PI3K activation.