Plasma levels of soluble membrane attack complex are elevated despite viral suppression in HIV patients with poor immune reconstitution.

The complement system is now a therapeutic target for the management of serious and life-threatening conditions such as paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, glomerulonephritis and other diseases caused by complement deficiencies or genetic variants. As complement therapeutics expand into more clinical conditions, monitoring complement activation is increasingly important, as is the baseline levels of complement activation fragments in blood or other body fluid levels. Although baseline complement levels have been reported in the literature, the majority of these data were generated using non-standard assays and with variable sample handling, potentially skewing results. In this study, we examined the plasma and serum levels of the soluble membrane attack complex of complement (sMAC). sMAC is formed in the fluid phase when complement is activated through the terminal pathway. It binds the regulatory proteins vitronectin and/or clusterin and cannot insert into cell membranes, and can serve as a soluble diagnostic marker in infectious disease settings, as previously shown for intraventricular shunt infections. Here we show that in healthy adults, serum sMAC levels were significantly higher than those in plasma, that plasma sMAC levels were similar between in African Americans and Caucasians and that plasma sMAC levels increase with age. Plasma sMAC levels were significantly higher in virally suppressed people living with HIV (PLWH) compared to non-HIV infected healthy donors. More specifically, PLWH with CD4+ T cell counts below 200 had even greater sMAC levels, suggesting diagnostic value in monitoring sMAC levels in this group.

Biosynthetic homeostasis and resilience of the complement system in health and infectious disease.

BACKGROUND: The complement system is a central component of the innate immune system. Constitutive biosynthesis of complement proteins is essential for homeostasis. Dysregulation as a consequence of genetic or environmental cues can lead to inflammatory syndromes or increased susceptibility to infection. However, very little is known about steady state levels in children or its kinetics during infection. METHODS: With a newly developed multiplex mass spectrometry-based method we analyzed the levels of 32 complement proteins in healthy individuals and in a group of pediatric patients infected with bacterial or viral pathogens. FINDINGS: In plasma from young infants we found reduced levels of C4BP, ficolin-3, factor B, classical pathway components C1QA, C1QB, C1QC, C1R, and terminal pathway components C5, C8, C9, as compared to healthy adults; whereas the majority of complement regulating (inhibitory) proteins reach adult levels at very young age. Both viral and bacterial infections in children generally lead to a slight overall increase in complement levels, with some exceptions. The kinetics of complement levels during invasive bacterial infections only showed minor changes, except for a significant increase and decrease of CRP and clusterin, respectively. INTERPRETATION: The combination of lower levels of activating and higher levels of regulating complement proteins, would potentially raise the threshold of activation, which might lead to suppressed complement activation in the first phase of life. There is hardly any measurable complement consumption during bacterial or viral infection. Altogether, expression of the complement proteins appears surprisingly stable, which suggests that the system is continuously replenished. FUND: European Union's Horizon 2020, project PERFORM, grant agreement No. 668303.

Clusterin facilitates apoptotic cell clearance and prevents apoptotic cell-induced autoimmune responses.

Clusterin (Clu), an extracellular chaperone, exhibits characteristics of soluble innate immunity receptors, as assessed by its ability to bind some bacteria strains. In this study, we report that Clu also binds specifically to late apoptotic cells but not to live, early apoptotic, or necrotic cells. Histones, which accumulate on blebs during the apoptotic process, represent privileged Clu-binding motifs at the surface of late apoptotic cells. As a consequence, Clu potentiates, both in vitro and in vivo, the phagocytosis of late apoptotic cells by macrophages. Moreover, the increased phagocytosis of late apoptotic cells induced by Clu favors the presentation and cross-presentation of apoptotic cell-associated antigens. Finally, we observed that, in a model of apoptotic cell-induced autoimmunity, and relative to control mice, Clu(-/-) mice develop symptoms of autoimmunity, including the generation of anti-dsDNA antibodies, deposition of immunoglobulins and complement components within kidneys, and splenomegaly. These results identify Clu as a new molecule partner involved in apoptotic cell efferocytosis and suggest a protective role for Clu in inflammation and autoimmune diseases.

Clusterin Modulates Allergic Airway Inflammation by Attenuating CCL20-Mediated Dendritic Cell Recruitment.

Recruitment and activation of dendritic cells (DCs) in the lungs are critical for Th2 responses in asthma, and CCL20 secreted from bronchial epithelial cells (BECs) is known to influence the recruitment of DCs. Because asthma is a disease that is closely associated with oxidative stress, we hypothesized that clusterin, an oxidative stress regulatory molecule, may have a role in the development of allergic airway inflammation. The aim of this study was to examine whether clusterin regulates CCL20 production from the BECs and the subsequent DC recruitment in the lungs. To verify the idea, clusterin knockout (Clu(-/-)), clusterin heterogeneous (Clu(+/-)), and wild-type mice were exposed intranasally to house dust mite (HDM) extract to induce allergic airway inflammation. We found that the total number of immune cells in bronchoalveolar lavage fluid and the lung was increased in Clu(-/-) and Clu(+/-) mice. Of these immune cells, inflammatory DCs (CD11b(+)CD11c(+)) and Ly6C(high) monocyte populations in the lung were significantly increased, which was accompanied by increased levels of various chemokines, including CCL20 in bronchoalveolar lavage fluid, and increased oxidative stress markers in the lung. Moreover, HDM-stimulated human BECs with either up- or downregulated clusterin expression showed that CCL20 secretion was negatively associated with clusterin expression. Interestingly, clusterin also reduced the level of intracellular reactive oxygen species, which is related to induction of CCL20 expression after HDM stimulation. Thus, the antioxidant property of clusterin is suggested to regulate the expression of CCL20 in BECs and the subsequent recruitment of inflammatory DCs in the airway.

The CLU-files: disentanglement of a mystery.

The multifaceted protein clusterin (CLU) has been challenging researchers for more than 35 years. The characterization of CLU as a molecular chaperone was one of the major breakthroughs in CLU research. Today, secretory clusterin (sCLU), also known as apolipoprotein J (apoJ), is considered one of the most important extracellular chaperones ever found. It is involved in a broad range of physiological and pathophysiological functions, where it exerts a cytoprotective role. Descriptions of various forms of intracellular CLU have led to further and even contradictory functions. To untangle the current state of knowledge of CLU, this review will combine old views in the field, with new discoveries to highlight the nature and function of this fascinating protein(s). In this review, we further describe the expression and subcellular location of various CLU forms. Moreover, we discuss recent insights into the structure of CLU and assess how structural properties as well as the redox environment determine the chaperone activity of CLU. Eventually, the review connects the biochemistry and molecular cell biology of CLU with medical aspects, to formulate a hypothesis of a CLU function in health and disease.

Absenteísmo-doença no serviço público municipal de Goiânia / Sickness absence in a municipal public service of Goiânia, Brazil

INTRODUÇÃO: O absenteísmo-doença, enquanto falta ao trabalho justificada por licença médica, é um importante indicador das condições de saúde dos trabalhadores. Em geral, características sociodemográficas e ocupacionais situam-se entre os principais fatores associados ao absenteísmo-doença. A administração pública é responsável por 21,8% dos empregos formais no Brasil. Esta população permite o estudo de uma grande variedade de categorias profissionais. OBJETIVO: Analisar o perfil e os indicadores de absenteísmo-doença entre servidores municipais de Goiânia, no Estado de Goiás, Brasil. Métodos: Estudo transversal das licenças certificadas para tratamento de saúde superiores a três dias, de todos os servidores, desde janeiro de 2005 a dezembro de 2010. Foram calculadas as prevalências, utilizando como critérios o número de indivíduos, os episódios e os dias de afastamento. RESULTADOS: Foram concedidas 40.578 licenças certificadas para tratamento de saúde a 13.408 servidores numa população média anual de 17.270 pessoas, o que resultou em 944.722 dias de absenteísmo. A prevalência acumulada de licença no período foi de 143,7%, com média anual de 39,2% e duração de 23 dias por episódio. A prevalência acumulada de absenteísmo-doença foi maior entre mulheres (52,0%) com idade superior a 40 anos (55,9%), com companheiro (49,9%), de baixa escolaridade (54,4%), profissionais de educação (54,7%), > 10 anos de serviço (61,9%) e múltiplos vínculos profissionais (53,7%). Os grupos de diagnósticos (CID-10) com as maiores prevalências acumuladas de licenças foram os do capítulo de transtornos mentais (26,5%), doenças osteomusculares (25,1%) e lesões (23,6%). CONCLUSÕES: Os indicadores de absenteísmo-doença expressam a magnitude desse fenômeno no serviço público e podem auxiliar no planejamento das ações de saúde do trabalhador, priorizando os grupos ocupacionais mais vulneráveis. .

BACKGROUND: Sickness absence, as work absenteeism justified by medical certificate, is an important health status indicator of the employees and, overall, sociodemographic and occupational characteristics are among the main factors associated with sickness absence. Public administration accounts for 21.8% of the formal job positions in Brazil. This population allows the study of a wide range of professional categories. OBJECTIVE: To assess the profile and indicators of sickness absence among public workers from the municipality of Goiania, in the State of Goiás, Brazil. METHODS: A cross-sectional study on certified sick leaves, lasting longer than three days, of all civil servants from January 2005 to December 2010. Prevalence rates were calculated using as main criteria the number of individuals, episodes and sick days. RESULTS: 40,578 certified sick leaves were granted for health treatment among 13,408 public workers, in an annual average population of 17,270 people, which resulted in 944,722 days of absenteeism. The cumulative prevalence of sick leave for the period was of 143.7%, with annual average of 39.2% and duration of 23 days per episode. The cumulative prevalence of sickness absence was higher among women (52.0%), older than 40 years old (55.9%), with a partner (49.9%), low schooling (54.4%), education professionals (54.7%), > 10 years of service (61.9%), and with multiple work contracts (53.7%). Diagnoses groups (ICD-10) with higher cumulative prevalence of sick leaves were those with mental disorders (26.5%), musculoskeletal diseases (25.1%), and injuries (23.6%). CONCLUSIONS: Indicators of sickness absence express the magnitude of this phenomenon in the public sector and can assist in planning health actions for the worker, prioritizing the most vulnerable occupational groups. .

Quantification of clusterin in paired cerebrospinal fluid and plasma samples.

BACKGROUND: Clusterin (ApoJ) is an amyloid-associated protein and plays an important role in Alzheimer's disease (AD) pathology. Recent genome-wide association studies have indicated that certain genetic variants increase the risk of developing AD. To determine if the expression of clusterin is different in AD patients, both systemically and locally in the brain, differs between (subgroups of) AD patients and non-AD cases, an assay available that detects clusterin in both plasma and cerebrospinal fluid (CSF) with equal sensitivity would be helpful. METHODS: We compared four different commercially available antibodies in their ability to detect recombinant clusterin and immune-purified human clusterin. Specificity was tested on western blot and in ELISA systems, and selection was based on the ability to detect clusterin in CSF and plasma. A sandwich ELISA was developed and validated with monoclonal antibody G7 as capture, and rabbit polyclonal (Alexis) antibodies for detection. RESULTS: Our ELISA measured clusterin concentrations in plasma and CSF with dynamic ranges of 2-70 mg/L and 0.5-40 mg/L, respectively. The assays showed 99.8% recovery in CSF and 97% recovery in plasma. Intra-assay coefficient of variation was 1.4% and inter-assay 8.8%. The assay shows no cross-reactivity with related apolipoproteins. Clusterin quantification is dependent on the type of storage for plasma samples. A single freeze/thaw cycle caused fluctuations of clusterin concentrations in plasma, while clusterin in CSF is stable for up to five cycles. CONCLUSIONS: We have successfully developed a clusterin ELISA that reliably measures CSF and plasma clusterin concentrations. In a pilot study, all samples gave results that were well within the dynamic range of the assay, with low variations. Freshly stored plasma samples are crucial for accurate clusterin quantification.

[The expression and purification of TRPM2 protein in E.coli and preparation of its polyclonal antibody].

OBJECTIVE: To purify the prokaryotically expressed GST-TRPM2N fusion protein and prepare specific polyclonal antibody against human transient receptor potential melastatin 2(TRPM2) protein. METHODS: The DNA fragment encoding evolutionarily conserved N-terminus (1-334 amino acids) of TRPM2 was amplified by PCR. The amplicon was then subcloned into prokaryotic expression plasmid pGEX-4T-3 and the recombinant plasmid was transformed into BL21 (DE3) cells. The expression of GST-TRPM2N fusion protein with a molecular weight about 70 000 Da was induced with 1 mmol/L IPTG and purified by GST affinity chromatography. The purified protein was mixed with complete Freund's adjuvant and used to immunize the New Zealand white rabbits with classical 4-injection protocol to generate specific anti-TRPM2 polyclonal antibody. The specificity and titer of the anti-TRPM2 antibody was analyzed by Western blotting. RESULTS: The cDNA fragment encoding N-terminus of human TRPM2 was amplified by PCR and directionally subcloned into pGEX-4T3 plasmid. Under the induction of IPTG, we observed the expression of GST-TRPM2N fusion protein. Polyclonal antibody against human TRPM2 protein was successfully prepared in the rabbits immunized with the purified GST-TRPM2N fusion protein. And the preliminary analysis showed that the anti-TRPM2 antibody could specifically identify transiently expressed TRPM2-EE in HEK293 cells. CONCLUSION: The specific polyclonal antibody against human TRPM2 protein has been successfully prepared, which facilitates our future research on the function of TRPM2 channel.

Regulation of the expression of CLU isoforms in endometrial proliferative diseases.

Clusterin (CLU) is a nearly ubiquitous multifunctional protein synthesized in different functionally divergent isoforms, sCLU and nCLU, playing a crucial role by keeping a balance between cell proliferation and death. Studying in vivo CLU expression we found a higher mRNA expression both in neoplastic and hyperplastic tissues in comparison to normal endometria; in particular, by RT-qPCR we demonstrated an increase of the specific sCLU isoform in the neoplastic and hyperplastic endometrial diseases. On the contrary, no CLU increase was detected at the protein level. The CLU gene transcriptional activity was upregulated in the hyperplastic and neoplastic tissues, indicating the existence of a fine post-trans-criptional regulation of CLU expression possibly responsible for the protein decrease in the malignant disease. A specific CLU immunoreactivity was present in all the endometrial glandular cells in comparison to the other cellular compartments where CLU immunoreactivity was lower or absent. In conclusion, our results suggest the existence of a complex regulatory mechanism of CLU gene expression during the progression from normal to malignant cells, possibly contributing to endometrial carcinogenesis. Moreover, the specific alteration of the sCLU:nCLU ratio associated with the pathological stage, suggests a possible usage of CLU as molecular biomarker for the diagnosis/prognosis of endometrial proliferative diseases.

Clusterin in Alzheimer's disease.

Clusterin, also known as apolipoprotein J, is a ubiquitous multifunctional glycoprotein. Following its identification in 1983, clusterin was found to be clearly increased in Alzheimer's disease (AD). Later research demonstrated that clusterin could bind amyloid-beta (Abeta) peptides and prevent fibril formation, a hallmark of AD pathology. In addition to preventing excessive inflammation, intracellular clusterin was found to reduce apoptosis and oxidative stress. Although early studies were inconclusive, two recent large-scale genome-wide association studies (GWAS) independently identified variants within the clusterin gene as risk factors for developing AD. This review focuses on the characteristics of clusterin and possible mechanisms of its relationship to AD.

Complement receptor 1 (CR1) and Alzheimer's disease.

Alzheimer's disease (AD) is the most common neurodegenerative disease and it poses an ever-increasing burden to an aging population. Several loci responsible for the rare, autosomal dominant form of AD have been identified (APP, PS1 and PS2), and these have facilitated the development of the amyloid cascade hypothesis of AD aetiology. The late onset form of the disease (LOAD) is poorly defined genetically, and up until recently the only known risk factor was the ε4 allele of APOE. Recent genome-wide association studies (GWAS) have identified common genetic variants that increase risk of LOAD. Two of the genes highlighted in these studies, CLU and CR1, suggest a role for the complement system in the aetiology of AD. In this review we analyse the evidence for an involvement of complement in AD. In particular we focus on one gene, CR1, and its role in the complement cascade. CR1 is a receptor for the complement fragments C3b and C4b and is expressed on many different cell types, particularly in the circulatory system. We look at the evidence for genetic polymorphisms in the gene and the possible physiological effects of these well-documented changes. Finally, we discuss the possible impact of CR1 genetic polymorphisms in relation to the amyloid cascade hypothesis of AD and the way in which CR1 may lead to AD pathogenesis.

Clusterin immunoreactivity as a predictive factor for progression of non-muscle-invasive bladder carcinoma.

INTRODUCTION: There is a need for prognostic markers which can predict the subset of patients who will not respond sufficiently to conservative management in non-muscle-invasive bladder carcinoma. We analyzed the association of clusterin (CLU) with clinicopathological factors. MATERIALS AND METHODS: Immunohistochemical CLU expression was investigated in paraffin-embedded archival tissues of initial transurethral resection specimens of 46 patients with non-muscle-invasive bladder carcinoma. The result was expressed as the proportion of the number of CLU-containing tumor cells to the total number of tumor cells detected in each slide and 'percent CLU expression' was calculated for each patient. RESULTS: Of the 46 cases (35 male, 11 female), 18 were ≥ 65 years of age. CLU expression was significantly higher in male and elderly patients. Following the initial transurethral resection, 39 patients showed tumor recurrence, and progression was seen in 25 patients, of whom 17 progressed to muscle invasion during follow-up. Although there was no significant correlation between CLU expression and recurrence, significant correlation with overall progression and progression to muscle-invasive disease was observed in this cohort of patients (p = 0.001 and p = 0.014, respectively). Among the patients with progression to muscle invasion, 13 underwent radical cystectomy with pT2 tumor in 5 patients in the final pathology of surgical specimens and pT3 and higher in the remainder. CONCLUSIONS: CLU immunoreactivity showed correlation with age, gender and progression, mainly progression to muscle invasion. Thus, CLU can be used as a molecular marker to predict the potential of progression to muscle-invasive disease in a particular tumor which in turn may prove useful in the decision-making process for early cystectomy without losing time with conservative management.

Detecting protein aggregates on untreated human tissue samples by atomic force microscopy recognition imaging.

We apply topography and recognition (TREC) imaging to the analysis of whole, untreated human tissue for what we believe to be the first time. Pseudoexfoliation syndrome (PEX), a well-known cause of irreversible blindness worldwide, is characterized by abnormal protein aggregation on the anterior lens capsule of the eye. However, the development of effective therapies has been hampered by a lack of detailed knowledge of the protein constituents in these pathological deposits and their distribution. Using both TREC and immunofluorescence, one of the proteins implicated in the PEX pathology--the apolipoprotein clusterin--was detected, and differences in its distribution pattern on the surface of untreated human lens capsule tissue in both PEX and normal control samples were investigated. Our study shows the potential of TREC imaging for the analysis of whole, untreated human tissue samples.

Resistance of neonatal porcine Sertoli cells to human xenoantibody and complement-mediated lysis is associated with low expression of alpha-Gal and high production of clusterin and CD59.

BACKGROUND: Porcine Sertoli cells have an inherent resistance to human xenoreactive antibody and complement-mediated lysis, however, the mechanisms of protection are still unclear. METHODS: Neonatal porcine Sertoli cells (NPSCs) were isolated from testes of 10 to 15-day-old piglets. Immortalized porcine aortic endothelial cells (iPECs) were used as control cells. An in vitro humoral injury model was used to assess whether NPSCs could resist human xenoantibody and complement-mediated lysis. Expressions of alpha-Gal, clusterin, CD59, CD46, and CD55 were examined to investigate the possible mechanisms of the immunoprotection of NPSCs. RESULTS: Compared with iPECs, NPSCs significantly resisted human natural antibody and complement-mediated lysis when incubated with 20% normal human serum (NHS) in vitro (24.38 +/- 0.50 vs. 53.13 +/- 14.53%, P < 0.01). Mechanistically, NPSCs expressed lower level of alpha-Gal (Gmean: 41.78 +/- 2.39 vs. 95.17 +/- 2.39, P < 0.01), which was correlated with decreased binding of human xenoreactive IgG and IgM. Additionally, NPSCs expressed higher level of clusterin measured by both Western blot and fluorescence-activated cell sorter analysis and expressed more CD59 mRNA detected by reverse-transcription polymerase chain reaction. CONCLUSIONS: These data demonstrate that the resistance of NPSCs to human xenoantibody and complement-mediated lysis is associated with low expression of xenoantigen alpha-Gal and high production of the complement inhibitors clusterin and CD59.

Trypanosoma carassii calreticulin binds host complement component C1q and inhibits classical complement pathway-mediated lysis.

Trypanosoma carassii is an extracellular parasite of economically important fish species that has evolved several strategies to circumvent host immune responses. Proteomic analysis of the excreted/secreted (ES) and surface molecules of the parasite has revealed a number of proteins that may be involved in host-parasite interactions. Among the parasite molecules identified in the ES of T. carassii was calreticulin. We cloned and produced T. carassii calreticulin (rTcaCRT), and generated a rabbit polyclonal antibody to the recombinant protein. The incubation of parasites with rabbit anti-rTcaCRT affinity-purified IgG antibody indicated substantial CRT levels on the surface of trypanosomes, as well as internal structures of permeabilized organisms. Recombinant parasite calreticulin bound several molecules in host serum including the first complement component, C1q. The host C1q specifically interacted with parasite CRT since the C1q-dependent lysis of sensitized sheep erythrocytes was inhibited by rTcaCRT. Our findings suggest that CRT may be used by the parasite to inhibit hosts' classical complement pathway.

Transcriptome profiling of a TGF-beta-induced epithelial-to-mesenchymal transition reveals extracellular clusterin as a target for therapeutic antibodies.

Transforming growth factor (TGF)-beta plays a dual role in tumorigenesis, switching from acting as a growth inhibitory tumor suppressor early in the process, to a tumor promoter in late-stage disease. Since TGF-beta's prometastatic role may be linked to its ability to induce tumor cell epithelial-to-mesenchymal transition (EMT), we explored TGF-beta's EMT-promoting pathways by analysing the transcriptome changes occurring in BRI-JM01 mammary tumor epithelial cells undergoing a TGF-beta-induced EMT. We found the clusterin gene to be the most highly upregulated throughout most of the TGF-beta time course, and showed that this results in an increase of the secreted form of clusterin. By monitoring several hallmark features of EMT, we demonstrated that antibodies targeting secreted clusterin inhibit the TGF-beta-induced EMT of BRI-JM01 cells, as well as the invasive phenotype of several other breast and prostate tumor cell lines (4T1, NMuMG, MDA-MB231LM2 and PC3), without affecting the proliferation of these cells. These results indicate that secreted clusterin is a functionally important EMT mediator that lies downstream within TGF-beta's EMT-promoting transcriptional cascade, but not within its growth-inhibitory pathways. To further investigate the role played by secreted clusterin in tumor metastasis, we assessed the effect of several anti-clusterin monoclonal antibodies in vivo using a 4T1 syngeneic mouse breast cancer model and found that these antibodies significantly reduce lung metastasis. Taken together, our results reveal a role for secreted clusterin as an important extracellular promoter of EMT, and suggest that antibodies targeting clusterin may inhibit tumor metastasis without reducing the beneficial growth inhibitory effects of TGF-beta.

Chapter 8: Clusterin: A multifacet protein at the crossroad of inflammation and autoimmunity.

For years, clusterin has been recognized as a secreted protein and a large number of works demonstrated that this ubiquitously expressed protein has multiple activities. Among the described activities several were related to inflammation and immunity such as its regulatory activity on complement. Then it became clear that a nuclear form of the protein with proapoptotic property existed and more recently that a cytoplasmic form could regulate NF-kappaB pathway. Again, these activities have a strong repercussion in inflammation and immunity. On the other hand, data available on the exact role of CLU in these processes and autoimmunity were quite scarce until recently. Indeed, in the last few years, a differential CLU expression in subtype of T cells, the regulation of CLU expression by proinflammatory cytokines and molecules, the regulation of expression and function of CLU depending on its subcellular localization, the interaction of CLU with nuclear and intracellular proteins were all reported. Adding these new roles of CLU to the already reported functions of this protein allows a better understanding of its role and potential involvement in several inflammatory and immunological processes and, in particular, autoimmunity. In this sense, rheumatoid arthritis appears to be a very attractive disease to build a new paradigm of the role and function of CLU because it makes the link between proliferation, inflammation, and autoimmunity. We will try to see in this review how to bring altogether the old and new knowledge on CLU with inflammation and autoimmunity. Nevertheless, it is clear that CLU has not yet revealed all its secrets in inflammation and autoimmunity.

Clusterin facilitates exchange of glycosyl phosphatidylinositol-linked SPAM1 between reproductive luminal fluids and mouse and human sperm membranes.

Glycosyl phosphatidylinositol (GPI)-linked proteins, which are involved in post-testicular maturation of sperm and have a role in fertilization, are acquired on the sperm surface from both vesicular and membrane-free soluble fractions of epididymal luminal fluid (LF) and uterine LF. Herein, we investigate the mechanism of uptake of these proteins from the soluble fraction of LFs using sperm adhesion molecule 1 (SPAM1) as a model. Ultracentrifugation and native Western blot analysis of the soluble fraction revealed that SPAM1 is present in low-molecular-weight (monomeric) and high-molecular-weight (oligomeric) complexes. The latter are incapable of transferring SPAM1 and may serve to produce monomers. Monomers are stabilized by hydrophobic interactions with clusterin (CLU), a lipid carrier that is abundantly expressed in LFs. We show that CLU is involved in the transfer of SPAM1 monomers, whose delivery was decreased by anti-CLU antibody under normal and apolipoprotein-enhanced conditions. Coimmunoprecipitation revealed an intimate association of CLU with SPAM1. Both plasma and recombinant CLU had a dose-related effect on transfer efficiency: high concentrations reduced and low concentrations enhanced delivery of SPAM1 to human and mouse sperm membranes, reflecting physiological states in the epididymal tract. We propose a lipid exchange model (akin to the lipid-poor model for cholesterol efflux) for the delivery of GPI-linked proteins to sperm membranes via CLU. Our investigation defines specific conditions for membrane-free GPI-linked protein transfer in vitro and could lead to technology for improving fertility or treating sperm pathology by the addition of relevant GPI-linked proteins critical for successful fertilization in humans and domestic animals.

Transcriptional and posttranslational regulation of clusterin by the two main cellular proteolytic pathways.

Clusterin/apolipoprotein J (CLU) is a secreted glycoprotein associated with many severe physiological disturbances that represent states of increased oxidative stress, such as aging, cancer, atherosclerosis, diabetes, and renal and neurodegenerative diseases. The aim of our work was to examine the effect of proteasome and lysosome inhibition on CLU expression and to determine whether those proteolytic pathways are implicated in CLU gene regulation and protein degradation. To this end we used two different model systems, namely the U-2 OS osteosarcoma cell line and the WI38 primary human embryonic lung fibroblasts. We report that proteasome inhibition promotes both heat-shock factor 1 (HSF-1)-dependent CLU gene expression induction and protein accumulation due to reduced degradation. In contrast, lysosome inhibition results in elevated levels of CLU protein but does not affect the CLU mRNA levels. We also provide direct evidence that both the intracellular precursor, psCLU, and the mature secreted, sCLU, isoforms constitute proteolytic substrates of the proteasome and the lysosome. Overall our findings indicate that CLU overexpression after proteasome inhibition relates to both positive gene transcriptional regulation by HSF-1 and posttranslational protein accumulation due to reduced proteasomal and lysosomal degradation.

TGF-beta indirectly favors the development of human Th17 cells by inhibiting Th1 cells.

Human Th17 clones and circulating Th17 cells showed lower susceptibility to the anti-proliferative effect of TGF-beta than Th1 and Th2 clones or circulating Th1-oriented T cells, respectively. Accordingly, human Th17 cells exhibited lower expression of clusterin, and higher Bcl-2 expression and reduced apoptosis in the presence of TGF-beta, in comparison with Th1 cells. Umbilical cord blood naïve CD161(+)CD4(+) T cells, which contain the precursors of human Th17 cells, differentiated into IL-17A-producing cells only in response to IL-1beta plus IL-23, even in serum-free cultures. TGF-beta had no effect on constitutive RORgamma t expression by umbilical cord blood CD161(+) T cells but it increased the relative proportions of CD161(+) T cells differentiating into Th17 cells in response to IL-1beta plus IL-23, whereas under the same conditions it inhibited both T-bet expression and Th1 development. These data suggest that TGF-beta is not critical for the differentiation of human Th17 cells, but indirectly favors their expansion because Th17 cells are poorly susceptible to its suppressive effects.