HLA-B*57 and IFNL4-related polymorphisms are associated with protection against HIV-1 disease progression in controllers.

Background: HIV-1-controllers maintain HIV-1 viremia at low levels (normally <2000 HIV-RNA copies/mL) without antiretroviral treatment. However, some HIV-1-controllers have evidence of immunologic progression with marked CD4+T-cell decline. We investigated host genetic factors associated with protection against CD4+T-cell loss in HIV-1-controllers. Methods: We analysed the association of interferon lambda 4 (IFNL4)-related polymorphisms and HLA-B haplotypes within Long Term Non-Progressor HIV-1-controllers ((LTNP-C), defined by maintaining CD4+T-cells counts >500 cells/mm3 for more than 7 years after HIV-1 diagnosis) versus non-LTNP-C, who developed CD4+T-cells counts <500 cells/mm3 Both a Spanish study cohort (n=140) and an international validation cohort (n=914) were examined. Additionally, in a subgroup of individuals HIV-1-specific T-cell responses and soluble cytokines were analysed RESULTS: HLA-B*57 was independently associated with the LTNP-C phenotype (OR=3.056 (1.029-9.069) p=0.044 and OR=1.924 (1.252-2.957) p=0.003) while IFNL4 genotypes represented independent factors for becoming non-LTNP-C (TT/TT, ss469415590, OR=0.401 (0.171-0.942) p=0.036 or A/A, rs12980275, OR=0.637 (0.434-0.934) p=0.021) in the Spanish and validation cohort, respectively, after adjusting for sex, age at HIV-1 diagnosis, IFNL4-related polymorphisms and different HLA-B haplotypes. LTNP-C showed lower plasma IP-10 (p=0.019) and higher IFN-γ (p=0.02) levels than the HIV-1-controllers with diminished CD4+T-cell numbers. Moreover, LTNP-C exhibited higher quantities of IL2+CD57- and IFN-γ+CD57- HIV-1-specific CD8+T-cells (p=0.002 and 0.041, respectively) than non-LTNP-C. Conclusions: We have defined genetic markers able to segregate stable HIV-1-controllers from those who experience CD4+T-cell decline. These findings allow for identification of HIV-1-controllers at risk for immunologic progression, and provide avenues for personalized therapeutic interventions and precision medicine for optimizing clinical care of these individuals.

Validation and comparison of instruments to identify frail patientes in primary care settings: Study protocol.

BACKGROUND: In the last few years several indices and tools, aimed at identifying frail subjects in various care settings have been developed. However, to date none of them has been incorporated into usual practice in the primary care setting. The purposes of this study are: 1) to evaluate the predictive capacity of the Tilburg Frailty Indicator (TFI), the Gérontopôle Frailty Screening Tool (GFST) and the KoS model together with two biomarker levels (SOX2 and p16INK4a) for adverse events related to frailty; 2) to determine differences in the use of healthcare services according to frailty. METHODS/DESIGN: Prospective multicentre cohort study with a 2-year follow-up. The study will be performed in primary care centres of Gipuzkoa and Costa del Sol, both located in Spain. Autonomous, non-institutionalized individuals aged 70 and over that agree to participate in this study will constitute the study population. A total of 900 individuals will be randomly selected from the healthcare administrative data bases of the participating health services. Data will be collected at baseline and at 1 and 2 years. The main independent variables assessed at baseline will be TFI outcomes, GFST and the KoS model, together with the expression of SOX2 and p16INK4a levels. During follow-up, loss of autonomy, the occurrence of death and consumption of healthcare resources will be assessed. DISCUSSION: The main focus of this work is the identification and evaluation of several instruments constructed under different rationales to identify frail subjects in primary care settings. The resulting outcomes have potential for direct application to the primary care practice. Early identification of the onset of functional impairment of elderly is an essential, still unresolved aspect in the prevention of dependence in the scope of primary care.

Hepatitis C virus replication in Caucasian HIV controllers.

Whether HIV controllers, patients who spontaneously control HIV viraemia, are able to control hepatitis C virus (HCV) infection, in terms of spontaneous clearance or lower HCV replication, is not well understood. To assess to what extent Caucasian HIV controllers are able to control HCV replication and potential associated factors, plasma HIV-1 and HCV RNA levels, anti-HCV antibodies, HCV genotype and human leucocyte antigens (HLA) typing were determined in samples from 75 HIV controllers (33 viraemic controllers, <1000 HIV-1 RNA copies/mL, and 42 elite controllers, <40 HIV-1 RNA copies/mL) and compared with 261 HIV-infected noncontrollers. We did not find differences in the HCV spontaneous clearance rates between groups. However, we interestingly found a lower HCV viral load in HIV controllers, alongside a different distribution of HCV genotypes in relation to the comparison group. In addition, HLA-B57 was associated with a lower HCV viral load in the control group and HIV controllers, and conversely, HLA-B35 with higher HCV viral load in HIV controllers. The subrepresentation of HCV genotype 1 and the overrepresentation of HLA-B57 only partly explained the lower HCV viral load found in HIV controllers. In fact, HIV controller status was independently associated with lower HCV viral load, together with HCV genotype non-1, the presence of HLA-B57 and absence of HLA-B35. Caucasian HIV controllers are able to better control HCV replication, in terms of lower HCV viral load levels. These findings support the idea that some common host mechanisms are involved in the defence against these two persistent infections.

Major histocompatibility complex class I viral antigen processing in the secretory pathway defined by the trans-Golgi network protease furin.

Classical antigen presentation by major histocompatibility complex class I molecules involves cytosolic processing of endogenously synthesized antigens by proteasomes and translocation of processed peptides into the endoplasmic reticulum (ER) by transporters associated with antigen presentation (TAP). Alternative pathways for processing of endogenous antigens, generally involving the ER, have been suggested but not fully proved. We analyzed the potential for class I presentation of proteolytic maturation of secretory antigens in the exocytic pathway. We found that hepatitis B (HB) virus secretory core protein HBe can efficiently deliver COOH-terminally located antigenic peptides for endogenous class I loading in the absence of TAP. Antigen presentation to specific cytotoxic T lymphocytes correlates with protein maturation at the COOH terminus, since modification of maturation and transport of HBe through the secretory pathway alters antigen presentation. Both maturation and a necessary processing step occur in the Golgi or post-Golgi compartment. Antigen presentation is independent of proteasome activity, but inhibitors of the trans-Golgi network resident protease furin inhibit both HBe maturation and antigen presentation. These results define a new antigen processing pathway located in the secretory route, with a central role for proteolytic maturation mediated by the subtilisin protease family member furin as an efficient source for antigen presentation.

Cytomegalovirus prevents antigen presentation by blocking the transport of peptide-loaded major histocompatibility complex class I molecules into the medial-Golgi compartment.

Selective expression of murine cytomegalovirus (MCMV) immediate-early (IE) genes leads to the presentation by the major histocompatibility complex (MHC) class I molecule Ld of a peptide derived from MCMV IE protein pp89 (Reddehase, M.J., J. B. Rothbard, and U.H. Koszinowski. 1989. Nature (Lond.). 337:651). Characterization of endogenous antigenic peptides identified the pp89 peptide as the nonapeptide 168YPHFMPTNL176 (del Val, M., H.-J. Schlicht, T. Ruppert, M.J. Reddehase, and U.H. Koszinowski. 1991. Cell. 66:1145). Subsequent expression of MCMV early genes prevents presentation of pp89 (del Val, M., K. Münch, M.J. Reddehase, and U.H. Koszinowski. 1989. Cell. 58:305). We report on the mechanism by which MCMV early genes interfere with antigen presentation. Expression of the IE promoter-driven bacterial gene lacZ by recombinant MCMV subjected antigen presentation of beta-galactosidase to the same control and excluded antigen specificity. The Ld-dependent presence of naturally processed antigenic peptides also in nonpresenting cells located the inhibitory function subsequent to the step of antigen processing. The finding that during the E phase of MCMV gene expression the MHC class I heavy chain glycosylation remained in an Endo H-sensitive form suggested a block within the endoplasmic reticulum/cis-Golgi compartment. The failure to present antigenic peptides was explained by a general retention of nascent assembled trimolecular MHC class I complexes. Accordingly, at later stages of infection a significant decrease of surface MHC class I expression was seen, whereas other membrane glycoproteins remained unaffected. Thus, MCMV E genes endow this virus with an effective immune evasion potential. These results also indicate that the formation of the trimolecular complex of MHC class I heavy chain, beta 2-microglobulin, and the finally trimmed peptide is completed before entering the medial-Golgi compartment.

Glycosylated components induced in African swine fever (ASF) virus-infected Vero cells.

African swine fever (ASF) virus production was inhibited more than 100 fold by 5 mM glucosamine, 2 mM 2-deoxyglucose and 3 microM tunicamycin. ASF virus induced in Vero cells the synthesis of 19 glycosylated components of molecular weights ranging from 9K to 220K, the major ones being those of 9K, 13K, 14K, 74K and 220K. At least five of the induced glycosylated components, of molecular weights 13K, 33K, 34K, 38K and 220K, were probably virus-coded glycoproteins, as suggested by a comparative analysis of the time course of synthesis and the antigenicity of these components in extracts from [35S]methionine or [14C]glucosamine-labeled infected cells. The non-protein glycosylated components present in extracellular ASF virus particles had a cellular origin.

Molecular analysis of herpesviral gene products recognized by protective cytolytic T lymphocytes.

The infection of the mouse with murine cytomegalovirus (MCMV) served as a model system to understand the biology of human CMV infection. The contribution of cytolytic T lymphocytes (CTL) to the recovery from infection was studied. Protection against lethal MCMV disease could be conferred on immunodepleted hosts by adoptive transfer of lymphocytes. The antiviral effect was mediated by specifically sensitized T lymphocytes of the CD8+ subset. These cells limited viral spread, prevented tissue destruction by viral cytopathic effects, and protected from lethal disease. Transferred cells have protective therapeutic function even when the virus has already colonized host tissues. CD8+ cells do not require the contribution of CD4+ cells for in vivo function. Selective expression of immediate-early (IE) phase genes in target cells allowed the detection of the immunodominant IE antigen recognized by CTL. The major IE gene ieI encodes a non-structural viral phosphoprotein, pp89, which resides in the nucleus of infected cells where it acts as transcriptional regulator. Expression of gene ieI is under temporal control, and membrane presentation of the protein domain detected by CTL is down-regulated by MCMV early-phase products. A recombinant vaccinia virus expressing gene ieI induced immunity that protected mice against a subsequent challenge with a lethal dose of MCMV. The protective effect was entirely mediated by CD8+ T lymphocytes. Thus, an experimental vaccine expressing a single nonstructural herpesvirus protein can induce a protective cellular immune response.

An erythroid species-specific antigen of swine detected by a monoclonal antibody.

A monoclonal antibody (1AC11) has been produced which recognized the glycophorin of swine red blood cells. 1AC11 was specific for swine membrane erythrocytes. No other swine cells (leukocytes, macrophages, kidney and testis cells) nor red blood cells from all the tested mammalian species (goat, human, sheep, cattle, horse, rabbit, cat and guinea pig) were recognized. There was no blood group activity detected. Immunocytochemical analysis of blood vessel in the swine pituitary tissue showed that besides membrane erythrocytes, cytoplasmic molecules were recognized in some cells. Immunoblot analysis of both membrane and aqueous phase of chloroform/methanol fractions from swine erythrocytes showed that the monoclonal antibody 1AC11 reacts with the major sialoglycoprotein of apparent molecular weight 45,000 daltons.

[Fibronectin as a diagnostic marker in several determined neurological diseases]. / Fibronectina como marcador diagnóstico de determinadas enfermedades neurológicas.

OBJECTIVE: To determinate if fibronectin in CSF can be a useful parameter in the diagnosis of some neurologic illnesses. MATERIAL AND METHODS: We have studied 30 patients, subdivided in four groups, depending on the type of neurologic illness. We have chosen as control a 10-patient group, which came to the Emergency Service and were diagnosed as a suspicious of neurologic illness, but after this it was discarded. In the whole group we practiced a lumbar puncture, with cytology, systematic, biochemistry, cultures, immunoglobulins determination and fibronectin quantification by ELISA. RESULTS: We want to emphasize the increase in fibronectin levels in CSF in both the patients with bacterial meningitis and the multiple sclerosis groups, when it's compared with the control group. CONCLUSION: For this, the determination of fibronectin levels in CSF might be a useful parameter in the diagnosis of some neurologic illnesses.

Intracellular rate-limiting steps in MHC class I antigen processing.

Quantitative aspects of the endogenous pathway of Ag processing and presentation by MHC class I molecules to CD8+ CTL were analyzed over a wide range of Ag expression in recombinant vaccinia virus-infected cells expressing beta-galactosidase as model Ag. Only the amount of starting Ag was varied, leaving other factors unaltered. Below a certain level of Ag synthesis, increasing protein amounts led to a sharp rise in recognition by CTL. Higher levels of Ag expression led to a saturation point, which intracellularly limited the number of naturally processed peptides bound to MHC and thereby also CTL recognition. The rate-limiting step was located at the binding of the antigenic peptide to MHC inside the vaccinia virus-infected cell or before this event.

Selective involvement of proteasomes and cysteine proteases in MHC class I antigen presentation.

CTL recognize peptides derived from protein Ags bound to MHC-class I molecules. Proteasomes probably participate in the generation of these peptide epitopes. We investigated the role of proteasomes in the presentation of endogenously synthesized short viral proteins. To this end, we employed proteasome and cysteine protease inhibitors and two closely related recombinant vaccinia viruses that code for 17- and 19-amino acid-long products encompassing murine CMV 9pp89 epitope. Presentation of both minigene products required processing to shorter peptides and was independent of ubiquitination. Proteasomes were necessary for processing the 17-mer product, and cysteine proteases were not required. In contrast, the 19-mer product could be processed in parallel either by proteasomes or by cysteine proteases independently. These results highlight the diversity of alternative processing pathways even for short peptidic Ags, provide evidence for the involvement of cysteine proteases in MHC class I presentation, and show that cleavage by cysteine proteases is governed by sequences flanking the epitope.

Glycosylated components of African swine fever virus particles.

Extracellular African swine fever (ASF) virus particles were specifically agglutinated by several lectins, suggesting the presence of surface glycosylated component(s) containing at least glucose, mannose, or both; galactose, N-acetylgalactosamine, or both; N-acetylneuraminic acid and N-acetylglucosamine, but not fucose. When virions were purified from infected Vero cells labeled with [14C]glucosamine, [14C]galactose and analyzed by polyacrylamide gel electrophoresis, no major structural glycoproteins were detected. However, several species of glycolipids were found when virions were extracted with organic solvents and analyzed by thin layer chromatography. These, plus two minor glycosylated structural components, of apparent mol wt 230K and 95K, could account for the agglutination of ASF virions with concanavalin A.

Principles of cytomegalovirus antigen presentation in vitro and in vivo.

Cytomegaloviruses are members of the ubiquitous family of herpesviruses. They escape immunological clearance and persist throughout life in the infected host. Yet, the stability of the balance of this virus-host interaction is dependent upon the state of the cellular immune response, and usually requires the function of specific CD8 T lymphocytes. In a mouse model, the major antigen that triggers protective CD8 T lymphocytes has been characterized as a nonameric amino acid motif of a nonstructural virus protein. Analysis of the naturally presented peptide has led to the conclusion that the sequence of an antigenic epitope is not the only parameter that decides upon its efficient presentation. Furthermore, the virus has developed regulatory functions that can prevent antigen presentation in productively infected cells. Contradictions between in vivo and in vitro findings are resolved in a hypothesis postulating an essential contribution of cytokines to the in vivo control of productive cytomegalovirus infection.

Generation of MHC class I peptide antigens by protein processing in the secretory route by furin.

Cytosolic degradation of endogenously synthesized proteins by the proteasome and translocation of processed peptides to the endoplasmic reticulum by the transporters associated with antigen presentation constitutes the classical route for antigen presentation by MHC class I proteins. We have previously defined an alternative pathway in the secretory route involving proteolytic maturation of precursor proproteins for chimeric hepatitis B virus secretory core protein HBe containing a class I epitope at its carboxy-terminus. We extend those results by demonstrating that intracellular delivery of the trans-Golgi network protease furin increases both proteolytic maturation and antigen presentation of the chimeric HBe proteins. An additional class I epitope from the HIV envelope gp160 protein was inserted into this COOH-terminal region of two different chimeric HBe proteins. This epitope was also presented to CTL in a transporter-independent manner involving furin, and protein maturation and antigen presentation were also enhanced by furin over-expression. Presentation of this second epitope was restricted by a different class I allele, thus suggesting that antigen presentation by this new pathway may apply to any antigenic epitope and class I molecule. These results define the furin proteolytic maturation pathway of HBe in the secretory route as a general antigen processing route for MHC class I presentation.

Efficient processing of an antigenic sequence for presentation by MHC class I molecules depends on its neighboring residues in the protein.

Processing of endogenously synthesized proteins generates short peptides that are presented by MHC class I molecules to CD8 T lymphocytes. Here it is documented that not only the sequence of the presented peptide but also the residues by which it is flanked in the protein determine the efficiency of processing and presentation. This became evident when a viral sequence of proven antigenicity was inserted at different positions into an unrelated carrier protein. Not different peptides, but different amounts of the antigenic insert itself were retrieved by isolation of naturally processed peptides from cells expressing the different chimeric proteins. Low yield of antigenic peptide from an unfavorable integration site could be overcome by flanking the insert with oligo-alanine to space it from disruptive neighboring sequences. Notably, the degree of protection against lethal virus disease related directly to the amount of naturally processed antigenic peptide.

Dietary myo-inositol effect on sugar cataractogenesis.

The lens myo-inositol (MI) content is known to be depleted during initial cataractogenesis in both streptozocin (STZ)-induced diabetic and 50% galactose-maintained rats. The objective of this study is to establish whether dietary MI supplementation protects lens transparency, MI content and individual fiber cell ultrastructure is both model systems of sugar cataract. In the diabetic study, after induction with STZ, rats were immediately placed on normal chow supplemented with 2% MI for 14 weeks while additional age-matched control and diabetic rats remained untreated. Within 14 weeks, untreated diabetic rat lenses were totally opaque with undetectable MI content; MI was undetectable by 1 month. These opaque lenses were devoid of fiber cells and exhibited only acellular, amorphous cortical regions between 0 and 500 microns from the capsule. In contrast, 14-week, MI-treated diabetic rat lenses exhibited only cortical vacuolation indicative of initial cataractogenesis; MI content was 0.41 +/- 0.26 mumol/g wet weight of lens. Scanning electron micrographs indicated a granulated, acellular region subadjacent to the capsule and confirmed the presence of cortical fiber cells, approximately 100 microns from the capsule. In 50% galactose-maintained rats, daily administration of MI for 1 month was unable to prevent total opacification or reverse initial cataractogenesis indicating that in rapidly progressing galactose cataracts, MI was unable to protect lens transparency, MI content and cortical fiber ultrastructure. The combined results suggest that MI may exert a protective effect on the slowly developing diabetic cataract. Of the 2 models, the time course and polyol content in STZ diabetic lenses more closely correlate to the human diabetic lens which has a low activity of aldose reductase; therefore, it is possible that MI may exert a protective effect in human diabetic cataract.