Proteome Characterization of BALF Extracellular Vesicles in Idiopathic Pulmonary Fibrosis: Unveiling Undercover Molecular Pathways.

In the longtime challenge of identifying specific, easily detectable and reliable biomarkers of IPF, BALF proteomics is providing interesting new insights into its pathogenesis. To the best of our knowledge, the present study is the first shotgun proteomic investigation of EVs isolated from BALF of IPF patients. Our main aim was to characterize the proteome of the vesicular component of BALF and to explore its individual impact on the pathogenesis of IPF. To this purpose, ultracentrifugation was chosen as the EVs isolation technique, and their purification was assessed by TEM, 2DE and LC-MS/MS. Our 2DE data and scatter plots showed considerable differences between the proteome of EVs and that of whole BALF and of its fluid component. Analysis of protein content and protein functions evidenced that EV proteins are predominantly involved in cytoskeleton remodeling, adenosine signaling, adrenergic signaling, C-peptide signaling and lipid metabolism. Our findings may suggest a wider system involvement in the disease pathogenesis and support the importance of pre-fractioning of complex samples, such as BALF, in order to let low-abundant proteins-mediated pathways emerge.

Emerging role of embryo secretome in the paracrine communication at the implantation site: a proof of concept.

OBJECTIVE: To assess the role of embryo secretome in modifying the molecular profile of glycodelin A (GdA) in endometrial organoids (ORG) mimicking the implantation window. To verify whether the use of embryo-conditioned culture medium at the time of the embryo transfer may increase in vitro fertilization outcome. DESIGN: Molecular study with human endometrial ORG and embryo-conditioned culture medium. Retrospective study using prospectively recorded data. SETTING: University hospital. PATIENT(S): For isolation and culture of endometrial glandular ORG, endometrial biopsy specimens from five white women of proven fertility undergoing laparoscopy for tubal sterilization. A total of 75 women undergoing intracytoplasmic sperm injection for tubal and/or male infertility factor. INTERVENTIONS(S): In vitro fertilization. MAIN OUTCOME MEASURE(S): Pinopodes presence in human endometrial ORG. Glycodelin A expression profile by means of two-dimensional electrophoresis. In vitro fertilization outcome. RESULT(S): This in vitro study demonstrated that the treatment of endometrial ORG with the secretome of medium conditioned by the growing embryo increased the GdA relative abundance and induced a different glycoform pattern. Biochemical and clinical pregnancy rate significantly increased when the spent medium was loaded during the transfer (17.5% vs. 36.6% and 16.5% vs. 35.1%, respectively). CONCLUSION(S): This study demonstrated that the secretome of implanting embryos is able to induce the expression as well as to determine the relative abundance and the glycosilation profile of endometrial GdA, a protein having a key role in the embryo-endometrial cross talk. Moreover, a significant increase in pregnancy rate was observed when the embryo transfer was performed by using the culture medium conditioned by the growing embryo.

Organoids of Human Endometrium: A Powerful In Vitro Model for the Endometrium-Embryo Cross-Talk at the Implantation Site.

Embryo implantation has been defined as the "black box" of human reproduction. Most of the knowledge on mechanisms underlining this process derives from animal models, but they cannot always be translated to humans. Therefore, the development of an in vitro/ex vivo model recapitulating as closely and precisely as possible the fundamental functional features of the human endometrial tissue is very much desirable. Here, we have validated endometrial organoids as a suitable 3D-model to studying epithelial endometrial interface for embryo implantation. Transmission and scanning electron microscopy analyses showed that organoids preserve the glandular organization and cell ultrastructural characteristics. They also retain the responsiveness to hormonal treatment specific to the corresponding phase of the menstrual cycle, mimicking the in vivo glandular-like aspect and functions. Noteworthy, organoids mirroring the early secretive phase show the development of pinopodes, large cytoplasmic apical protrusions of the epithelial cells, traditionally considered as reliable key features of the implantation window. Moreover, organoids express glycodelin A (GdA), a cycle-dependent marker of the endometrial receptivity, with its quantitative and qualitative features accounting well for the profile detected in the endometrium in vivo. Accordingly, organoids deriving from the eutopic endometrium of women with endometriosis show a GdA glycosylation pattern significantly different from healthy organoids, confirming our prior data on endometrial tissues. The present results strongly support the idea that organoids may closely recapitulate the molecular and functional characteristics of their cells/tissue of origin.

The intraflagellar transport protein IFT20 controls lysosome biogenesis by regulating the post-Golgi transport of acid hydrolases.

The assembly and function of the primary cilium depends on multimolecular intraflagellar transport (IFT) complexes that shuttle their cargo along the axonemal microtubules through their interaction with molecular motors. The IFT system has been moreover recently implicated in a reciprocal interplay between autophagy and ciliogenesis. We have previously reported that IFT20 and other components of the IFT complexes participate in the assembly of the immune synapse in the non-ciliated T cell, suggesting that other cellular processes regulated by the IFT system in ciliated cells, including autophagy, may be shared by cells lacking a cilium. Starting from the observation of a defect in autophagic clearance and an accumulation of lipid droplets in IFT20-deficient T cells, we show that IFT20 is required for lysosome biogenesis and function by controlling the lysosomal targeting of acid hydrolases. This function involves its ability to regulate the retrograde traffic of the cation-independent mannose-6-phosphate receptor (CI-MPR) to the trans-Golgi network, which is achieved by coupling recycling CI-MPRs to the microtubule motor dynein. Consistent with the lysosomal defect, an upregulation of the TFEB-dependent expression of the lysosomal gene network can be observed in IFT20-deficient cells, which is associated with defective tonic T-cell antigen receptor signaling and mTOR activity. We additionally show that the lysosome-related function of IFT20 extends to non-ciliated cells other than T cells, as well as to ciliated cells. Our findings provide the first evidence that a component of the IFT system that controls ciliogenesis is implicated in the biogenesis of lysosomes.

Clues to Non-Invasive Implantation Window Monitoring: Isolation and Characterisation of Endometrial Exosomes.

Despite the significant advances in the last decades, low implantation rate per transferred embryo still remains a major concern in assisted reproductive techniques, highlighting a need to better characterize endometrial receptivity also by mean of specific biomarkers. Based on physiology and on the intimate contact with endometrium as the tissue of interest, in this study we developed and validated an optimized protocol that uses extracellular vesicles (EVs) recovered from uterine flushings and from a cervical brush, the latter never used until now as an EVs source, as surrogates for endometrial biopsies. This method combines the safety of sampling with the ability to study the expression profile across the uterine cycle. We have compared the yield and composition of EVs recovered from different biofluids samples and fractions thereof, opting for chemical precipitation as the EV isolation procedure, assuring the highest yield without introducing any bias in specific EV recovery. Moreover, collected EVs, in particular exosome-like vesicles, express putative endometrial markers, such as glycodelin A and receptors for estrogen and progesterone, thus confirming their endometrial origin. We also identified uterine flushing EVs, in particular those recovered from its mucous fraction, as the richest source of endometrial transcripts, likely correlated to cellular (epithelial) origin of these vesicles. Finally, our pilot quantitative assessment of three endometrial gene profiles, in samples collected at different time points along the luteal phase, revealed the fluctuations apparently recapitulating gene expression variability prior reported during the menstrual cycle. Unlike tissue biopsy that is subjected to inter- and intra-sample differences, our data suggest that EVs from liquid biopsies (from uterine flushings and a cervical brush) obtained through less-invasive procedures, can be substrate to detect and track the tissue representative expression profiles, better depicting the total endometrium complexity.

Near-infrared quantum dots labelled with a tumor selective tetrabranched peptide for in vivo imaging.

BACKGROUND: Near-infrared quantum dots (NIR QDs) are a new class of fluorescent labels with excellent bioimaging features, such as high fluorescence intensity, good fluorescence stability, sufficient electron density, and strong tissue-penetrating ability. For all such features, NIR QDs have great potential for early cancer diagnosis, in vivo tumor imaging and high resolution electron microscopy studies on cancer cells. RESULTS: In the present study we constructed NIR QDs functionalized with the NT4 cancer-selective tetrabranched peptides (NT4-QDs). We observed specific uptake of NT4-QDs in human cancer cells in in vitro experiments and a much higher selective accumulation and retention of targeted QDs at the tumor site, compared to not targeted QDs, in a colon cancer mouse model. CONCLUSIONS: NIR QDs labelled with the tetrabranched NT4 peptide have very promising performance for selective addressing of tumor cells in vitro and in vivo, proving rising features of NT4-QDs as theranostics.

Redox imbalance and morphological changes in skin fibroblasts in typical Rett syndrome.

Evidence of oxidative stress has been reported in the blood of patients with Rett syndrome (RTT), a neurodevelopmental disorder mainly caused by mutations in the gene encoding the Methyl-CpG-binding protein 2. Little is known regarding the redox status in RTT cellular systems and its relationship with the morphological phenotype. In RTT patients (n = 16) we investigated four different oxidative stress markers, F2-Isoprostanes (F2-IsoPs), F4-Neuroprostanes (F4-NeuroPs), nonprotein bound iron (NPBI), and (4-HNE PAs), and glutathione in one of the most accessible cells, that is, skin fibroblasts, and searched for possible changes in cellular/intracellular structure and qualitative modifications of synthesized collagen. Significantly increased F4-NeuroPs (12-folds), F2-IsoPs (7.5-folds) NPBI (2.3-folds), 4-HNE PAs (1.48-folds), and GSSG (1.44-folds) were detected, with significantly decreased GSH (-43.6%) and GSH/GSSG ratio (-3.05 folds). A marked dilation of the rough endoplasmic reticulum cisternae, associated with several cytoplasmic multilamellar bodies, was detectable in RTT fibroblasts. Colocalization of collagen I and collagen III, as well as the percentage of type I collagen as derived by semiquantitative immunofluorescence staining analyses, appears to be significantly reduced in RTT cells. Our findings indicate the presence of a redox imbalance and previously unrecognized morphological skin fibroblast abnormalities in RTT patients.

Amyloidosis, inflammation, and oxidative stress in the heart of an alkaptonuric patient.

BACKGROUND: Alkaptonuria, a rare autosomal recessive metabolic disorder caused by deficiency in homogentisate 1,2-dioxygenase activity, leads to accumulation of oxidised homogentisic acid in cartilage and collagenous structures present in all organs and tissues, especially joints and heart, causing a pigmentation called ochronosis. A secondary amyloidosis is associated with AKU. Here we report a study of an aortic valve from an AKU patient. RESULTS: Congo Red birefringence, Th-T fluorescence, and biochemical assays demonstrated the presence of SAA-amyloid deposits in AKU stenotic aortic valve. Light and electron microscopy assessed the colocalization of ochronotic pigment and SAA-amyloid, the presence of calcified areas in the valve. Immunofluorescence detected lipid peroxidation of the tissue and lymphocyte/macrophage infiltration causing inflammation. High SAA plasma levels and proinflammatory cytokines levels comparable to those from rheumatoid arthritis patients were found in AKU patient. CONCLUSIONS: SAA-amyloidosis was present in the aortic valve from an AKU patient and colocalized with ochronotic pigment as well as with tissue calcification, lipid oxidation, macrophages infiltration, cell death, and tissue degeneration. A local HGD expression in human cardiac tissue has also been ascertained suggesting a consequent local production of ochronotic pigment in AKU heart.

p66Shc regulates vesicle-mediated secretion in mast cells by affecting F-actin dynamics.

The extracellular vesicular compartment has emerged as a novel system of intercellular communication; however, the mechanisms involved in membrane vesicle biogenesis and secretion are as yet unclear. Among immune cells releasing membrane vesicles-mast cells that reside near tissues exposed to the environment-are master modulators of immune responses. Here, we have addressed the role of p66Shc, a novel regulator of mast cell activation and homeostasis, in the dynamic reorganization of the actin cytoskeleton that is associated with morphological changes during secretion. We show that p66Shc is recruited as a complex with the lipid phosphatase SHIP1 to the F-actin skeleton and impairs antigen-dependent cortical F-actin disassembly and membrane ruffling through the inhibition of Vav and paxillin phosphorylation. We also show that in addition to acting as a negative regulator of antigen-dependent mast cell degranulation, p66Shc limits the basal release of granule contents by inhibiting microvesicle budding from the plasma membrane and piecemeal degranulation. These findings identify p66Shc as a critical regulator of actin dynamics in mast cells, providing a basis for understanding the molecular mechanisms involved in vesicle-mediated secretion in these cells.

Alkaptonuria is a novel human secondary amyloidogenic disease.

Alkaptonuria (AKU) is an ultra-rare disease developed from the lack of homogentisic acid oxidase activity, causing homogentisic acid (HGA) accumulation that produces a HGA-melanin ochronotic pigment, of unknown composition. There is no therapy for AKU. Our aim was to verify if AKU implied a secondary amyloidosis. Congo Red, Thioflavin-T staining and TEM were performed to assess amyloid presence in AKU specimens (cartilage, synovia, periumbelical fat, salivary gland) and in HGA-treated human chondrocytes and cartilage. SAA and SAP deposition was examined using immunofluorescence and their levels were evaluated in the patients' plasma by ELISA. 2D electrophoresis was undertaken in AKU cells to evaluate the levels of proteins involved in amyloidogenesis. AKU osteoarticular tissues contained SAA-amyloid in 7/7 patients. Ochronotic pigment and amyloid co-localized in AKU osteoarticular tissues. SAA and SAP composition of the deposits assessed secondary type of amyloidosis. High levels of SAA and SAP were found in AKU patients' plasma. Systemic amyloidosis was assessed by Congo Red staining of patients' abdominal fat and salivary gland. AKU is the second pathology after Parkinson's disease where amyloid is associated with a form of melanin. Aberrant expression of proteins involved in amyloidogenesis has been found in AKU cells. Our findings on alkaptonuria as a novel type II AA amyloidosis open new important perspectives for its therapy, since methotrexate treatment proved to significantly reduce in vitro HGA-induced A-amyloid aggregates.

Morphological changes and oxidative damage in Rett Syndrome erythrocytes.

BACKGROUND: Hypoxemia and increased oxidative stress (OS) have been reported in Rett Syndrome (RTT), a genetical neurodevelopmental disorder. Although OS and hypoxemia can lead to red blood cells (RBCs) shape abnormalities, no information on RBCs morphology in RTT exists. Here, RBCs shape was evaluated in RTT patients and healthy subjects as a function of OS markers, blood oxygenation, pulmonary gas exchange, and cardio-respiratory parameters. METHODS: RBCs morphology was evaluated by Scanning Electron Microscopy. Intraerythrocyte and plasma non protein-bound iron (NPBI), esterified F(2)-Isoprostanes (F(2)-IsoPs), 4-HNE protein adducts (4-HNE PAs) were measured. Pulmonary oxygen gradients and PaO(2) were evaluated by gas analyzers and cardiopulmonary variables by pulse oximetry. In RTT patients these parameters were assessed before and after ω-3 polyunsaturated fatty acids (ω-3 PUFAs) administration. RESULTS: Altered RBCs shapes (leptocytes) and increased NPBI were present in RTT, together with increased erythrocyte membrane esterified F(2)-IsoPs and 4-HNE PAs. Abnormal erythrocyte shapes were related to OS markers levels, pulmonary gas exchange, PaO(2) and cardio-respiratory variables. After ω-3 PUFAs, a decrease of leptocytes was accompanied by a progressive increase in reversible forms of RBCs. This partial RBCs morphology rescue was related to decreased OS damage markers, improved pulmonary oxygen exchange, and cardiopulmonary physiology. CONCLUSIONS: These findings indicate that in RTT 1) RBCs shape is altered; 2) the OS-hypoxia diad is critical in generating altered RBCs shape and membrane damage; 3) ω-3 PUFAs are able to partially rescue RBCs morphology and the OS-derived damage. GENERAL SIGNIFICANCE: RBCs morphology is an important biosensor for OS imbalance and chronic hypoxemia.

Different factors affecting human ANP amyloid aggregation and their implications in congestive heart failure.

AIMS: Atrial Natriuretic Peptide (ANP)-containing amyloid is frequently found in the elderly heart. No data exist regarding ANP aggregation process and its link to pathologies. Our aims were: i) to experimentally prove the presumptive association of Congestive Heart Failure (CHF) and Isolated Atrial Amyloidosis (IAA); ii) to characterize ANP aggregation, thereby elucidating IAA implication in the CHF pathogenesis. METHODS AND RESULTS: A significant prevalence (85%) of IAA was immunohistochemically proven ex vivo in biopsies from CHF patients. We investigated in vitro (using Congo Red, Thioflavin T, SDS-PAGE, transmission electron microscopy, infrared spectroscopy) ANP fibrillogenesis, starting from α-ANP as well as the ability of dimeric ß-ANP to promote amyloid formation. Different conditions were adopted, including those reproducing ß-ANP prevalence in CHF. Our results defined the uncommon rapidity of α-ANP self-assembly at acidic pH supporting the hypothesis that such aggregates constitute the onset of a fibrillization process subsequently proceeding at physiological pH. Interestingly, CHF-like conditions induced the production of the most stable and time-resistant ANP fibrils suggesting that CHF affected people may be prone to develop IAA. CONCLUSIONS: We established a link between IAA and CHF by ex vivo examination and assessed that ß-ANP is, in vitro, the seed of ANP fibrils. Our results indicate that ß-ANP plays a crucial role in ANP amyloid deposition under physiopathological CHF conditions. Overall, our findings indicate that early IAA-related ANP deposition may occur in CHF and suggest that these latter patients should be monitored for the development of cardiac amyloidosis.

The spermatogenesis and the sperm structure of Terebrantia (Thysanoptera, Insecta).

Spermatogenesis and the sperm structure of the terebrantian Aeolothrips intermedius Bagnall are described. Spermatogenesis consists of two mitotic divisions; the second is characterized by the loss of half of the spermatids, which have pyknotic nuclei. Early spermatids have two centrioles, but when spermiogenesis starts, a third centriole is produced. The three basal bodies give rise to three flagella; later these fuse into a single flagellum which contains three 9+0 axonemes. The basal bodies are surrounded by a large amount of centriole adjunct material. During spermiogenesis this material contributes to the shifting of the three axonemes towards the anterior sperm region parallel to the elongating nucleus, and it is transformed into a dense cylinder. In the mature spermatids the three axonemes amalgamate to create a bundle of 27 doublet microtubules. Near the end of spermiogenesis the dense cylinder of the centriole adjunct lies parallel to the nucleus and the axonemes. It ends where the mitochondrion appears at half-sperm length. We confirm that Terebrantia testes have a single sperm cyst; their sperm are characterized by a cylindrical nucleus, three axonemes fused into one, a small mitochondrion and a short cylindrical centriole adjunct which corresponds to the dense body described in a previous work. The acrosome is lacking. At the midpoint of the anterior half of the sperm the outline of the cross-section is bilobed, with the nucleus contained in a pocket evagination of the plasma membrane. These characters are discussed in light of a comparison between Tubulifera and Terebrantia.

Are dialysis devices usable as ozone gas exchangers?

A study aimed to compare the efficiency of the ozone transfer of four hydrophilic dialysis filters, and one hydrophobic gas-exchange device (GED) has been performed. Obviously, the former should be specifically used only for dialysis. Unfortunately, some clinicians incautiously use them as GEDs. It has been shown that: (i) dialysis filters present a wide range of gas-exchange yield (from 0 up to 70%), often related to variability according to the treatment time; (ii) by scanning microscopy, it has been noticed that hollow fibers are somewhat altered by ozone; and (iii) because their constitutive materials may not be ozone-resistant, they may release toxic compounds harmful for the patients. On the contrary, the appropriate GED is ozone-transfer efficient, is ozone-resistant, and is suitable for blood autotransfusion and ozonation.

Carboplatin-induced alteration of the thiol homeostasis in the isolated perfused rat kidney.

Carboplatin elicits minor side effects with respect to its first generation analogue cisplatin. Nevertheless, a dose-dependent nephrotoxicity of the drug has been reported to occur both in patients and in rats and a possible pathogenic role have been attributed to oxidative stress. We have studied the effect of carboplatin administration on the thiol/disulfide balance, on other biomarkers of oxidative stress and on antioxidant enzymes in the isolated perfused rat kidney. A 5-500microM carboplatin dose range did not alter renal function but significantly decreased levels of cysteine, glutathione and exposed protein sulfhydryl groups. Only a minimal increment in disulfides was observed, whereas malonyldialdehyde and protein carbonyls did not increase significantly. Among the antioxidant enzymes studied, only thioltransferase was inhibited by the treatment. Our data suggest that a minimal oxidative stress is present under our experimental conditions, thus indicating that platinum-based drugs do not produce significant amount of reactive oxygen species.

New findings on sperm ultrastructure in thrips (Thysanoptera, Insecta).

Sperm ultrastructure of several species in each of the two suborders of Thysanoptera Tubulifera and Terebrantia shows a distinctive and unusual architecture. Members of the whole order share a bizarre axoneme consisting of 27 microtubular elements derived from the amalgamation of 3 (9+0) axonemes present in each spermatid at the beginning of spermiogenesis. The reciprocal shifting of these axonemes along the length of the sperm, together with their possible shortening and overlapping for short distances, could explain why in some species it is never possible to observe the complete set of 27 microtubular elements in any one cross section. Tubuliferan sperm have a small elliptical (in cross section) acrosome extending the length of the sperm. In Bolothrips insularis and Compsothrips albosignatus this structure is larger and is associated with an external, flattened vesicle throughout its length. Terebrantian sperm lack an acrosome, but display for half their length a dense body running parallel to the nucleus. The sperm, in members of this suborder, are also characterized by possession of a small mitochondrion and by the unusual bilobed outline of cross sections through the anterior sperm region, with the nucleus located in one of the two lobes. Structures serving to anchor sperm to the inner surface of the cyst cell have been observed at their anterior tips in the testes of tubuliferans. In B. insularis, an anterior appendage is formed in immature sperm and is maintained in the mature spermatozoon parallel to its long axis in the most anterior region. Such an anchoring structure has not been observed in sperm of the terebrantian species examined, probably because the testis of terebrantians contains only a single cyst of developing gametes.

Nanostructured HA crystals up-regulate FGF-2 expression and activity in microvascular endothelium promoting angiogenesis.

In mineralized tissue the process of angiogenesis is required for normal osteogenesis during bone repair and in reconstructive and substitutive surgery, for proper biomaterial/tissue integration. Nanotechnologies have been proposed to improve the compatibility of biomaterials for use in orthopaedic and reconstructive surgery (e.g., nanocrystals). The aim of this study was to determine the effect of nanostructured hydroxyapatite (HA) on angiogenesis. Microvascular endothelial cell survival, proliferation and migration, crucial events in the angiogenic process, were evaluated together with cytoskeleton and biochemical signalling markers. Induction of migration, metalloproteinase (MMP-2) and focal adhesion Kinase (FAK) activity documented the ability of HA nanocrystals to stimulate capillary endothelium toward an angiogenic phenotype. HA concentrations, ranging from 2 to 10 microg/ml, promoted endothelium survival and proliferation, preserved alphavbeta3-integrin localization, stimulated beta-actin reorganization and Akt phosphorylation (98% vs control). Immunoassays for key signalling pathways in angiogenesis (i.e., endothelial nitric oxide synthase (eNOS) and fibroblast growth factor-2 (FGF-2)) demonstrated that HA increased their expression. Moreover, quantitative RT-PCR and Western blotting analysis confirmed that HA nanocrystals exposure up-regulated FGF-2 mRNA by 6 fold and increased 18 kDa protein isoform by 40%. HA enhanced cell responsiveness to vascular endothelial growth factor (VEGF) in terms of NOS activity (1.5 fold over control), increasing the ability of microvascular endothelium to differentiate into capillary-like structures when grown in 3D fibrin gel. In conclusion our data document the proangiogenic properties of HA nanocrystals. This material stimulates endothelial cell functions and biochemical pathways to an extent similar to VEGF, and primes them to VEGF stimulation, leading to differentiation in pseudocapillary formations in 3D matrices.

Ultrastructural analysis of the aberrant axoneme morphogenesis in thrips (Thysanoptera, Insecta).

Thrips spermiogenesis is characterized by unusual features in the differentiating spermatid cells. Three centrioles from which three individual short flagella are initially assembled, make the early spermatid a tri-flagellated cell. Successively, during spermatid maturation, the three basal bodies maintain a position close to the most anterior end of the elongating nucleus, so that the three axonemes are progressively incorporated in the spermatid cytoplasm, where they run in parallel to the main nuclear axis. Finally, the three axonemes amalgamate to form a microtubular bundle. The process starts with the formation of rifts at three specific points in each axonemal circumference, corresponding to sites 1,3,7 and leads to the formation of 9 microtubular rows of different length, i.e. 3 "dyads", 3 "triads" and 3 "tetrads". In the spermatozoon, the nucleus, the mitochondrion and the bundle of microtubules are arranged in a helicoidal pattern. The elongation of the spermatozoon is allowed by the deep anchorage of the spermatid to the cyst cell through a dense mass of material which, at the end of spermiogenesis, becomes a long anterior cylindrical structure. This bizarre "axoneme" does not show any trace of progressive movement but it is able to beat. According to the presence of dynein arms, sliding can take place only within each row and not between the rows. The possible molecular basis underlying the peculiar instability of thrips axonemes is discussed in light of the present knowledge on the organization of the axoneme in mutant organisms carrying alterations of the tubulin molecule.

Substance P antagonist blocks leakage and reduces activation of cytokine-stimulated rat brain endothelium.

We recently demonstrated that substance P mediates increased permeability of brain endothelium exposed to HIV-1 gp120. To test whether substance P is involved in immune processes at the blood-brain barrier (BBB), we stimulated rat brain endothelial cultures prepared from cerebral microvessels with Interferon-gamma (IFN-gamma) and Tumor necrosis factor-alpha (TNF-alpha), two proinflammatory cytokines that alter the BBB and measured permeability to albumin and expression of adhesion molecule ICAM-1 and MHC class II antigen in the presence and absence of spantide, a powerful substance P antagonist. In a dose-dependent manner, spantide completely neutralized increased permeability induced by TNF-alpha and IFN-gamma and expression of MHC class II molecule induced by IFN-gamma and prevented associated cell morphological changes as revealed by scanning electron microscope. Spantide also reduced expression of ICAM-1 induced by TNF-alpha and IFN-gamma by 35% and 30%, respectively. Substance P mRNA was found in unstimulated brain endothelial cells and was upregulated after stimulation with TNF-alpha and IFN-gamma. These in vitro findings demonstrate that substance P plays a major pathogenetic role in damaging and activating the BBB vascular component in the presence of proinflammatory cytokines.