Alberto Beretta

Distinctive Features of the α1-Domain a Helix of HLA-C Heavy Chains Free of β2-Microglobulin

Only a few monoclonal antibodies are available with a restricted specificity to HLA-C products. In the present report, we demonstrate that antibody L31, previously shown to react with beta 2m-less (free) class I MHC heavy chains, binds to an epitope (residues 66-68 of the alpha 1 domain alpha helix) present on all the HLA-C alleles corresponding to the accepted (CW1 through CW8) serologic specificities, and on a few HLA-B heavy chains sharing with HLA-C an aromatic residue at position 67. Extensive IEF blot testing of HLA homozygous, EBV-transformed B-lymphoid cells indicates that HLA-C molecules are present at significantly lower levels than HLA-B polypeptides not only at cell surface, as previously demonstrated, but also in total cellular extracts. Testing of metabolically labeled HLA-CW1, -CW5, and -CW6 transfectants and HLA homozygous lymphoid cells, particularly HLA-CW1-expressing cells, demonstrates that the L31 epitope is present on a subpopulation of naturally occurring HLA-C molecules distinct from that identified by antibody W6/32 to beta 2m-associated heavy chains. Pulse-chase experiments demonstrate that this epitope is transiently made available to antibody binding at early biosynthetic stages, but becomes hidden upon assembly with beta 2m. Thus, free HLA-C and other Y/F67+ heavy chains are characterized by distinctive antibody binding features in a region (residues 66-68) included in a previously identified HLA-C restricted motif, which has been suggested to be the primary cause of distinctive features of the antigen-binding groove, low affinity for endogenous peptide antigens and beta 2m, and preferential uptake of exogenous peptides, possibly of viral origin. We also show that HLA-CW1 heavy chains, both free and beta 2m associated, acquire sialilation. Free HLA-CW1 heavy chains are expressed at the cell surface even when unsialilated, albeit at low levels.

Anti-Cell Antibodies in Exposed Seronegative Individuals with HIV Type 1-Neutralizing Activity

Despite repeated exposures to HIV-1, some individuals remain seronegative. This study reports that sera from a fraction of exposed seronegative (ESN) subjects showed HIV-neutralizing activity; 5 of 17 ESN sera and none of 17 controls neutralized two different HIV-1 primary isolates (range of neutralizing titers: 1/20 to 1/60). The neutralizing activity was associated with the IgG fraction of 4 of 4 neutralizing ESN sera. Moreover, in 11 of 17 and 9 of 17 ESN sera (but none of the control sera) we found antibodies against HLA class I and CD4, respectively. One of the ESN sera (EU22) neutralized efficiently the primary virus derived from the seropositive partner and showed a good broadly cross-reactive neutralization. Immunoadsorption of two IgG fractions from EU19 and EU22 on peripheral blood mononuclear cells (PBMC) removed virus-neutralizing antibodies. The correlations between the ESN status and neutralizing activity (p<0.05), anti-HLA antibodies (p<0.0002), and anti-CD4 antibodies (p<0.001) were statistically significant. However, there was no statistically significant correlation between neutralizing activity and either anti-HLA or anti-CD4 antibodies. It can therefore be said that exposure to HIV-1 without seroconversion is, in some individuals, associated with HIV-neutralizing antibodies (not directed against viral antigens) and/or with anti-cell autoantibodies, which are possibly specific for cellular antigens involved in the infection/entry process.

Selected Pool of Peptides from ESAT-6 and CFP-10 Proteins for Detection of Mycobacterium tuberculosis Infection

ABSTRACT We have validated a new test for detecting Mycobacterium tuberculosis infection. A pool of synthetic peptides derived from ESAT-6 and CFP-10 proteins was used to detect the number of specific gamma interferon-producing T cells by means of an enzyme-linked immunospot assay. Sixty-eight individuals positive for M. tuberculosis infection, either human immunodeficiency virus-seropositive or -seronegative, were studied. The test results were highly specific (87.5%) and sensitive (93.1%), more so than a classical lymphoproliferative assay (specificity and sensitivity of 77.27%), opening new possibilities for diagnosis and screening of tuberculosis. Moreover, the test allowed us to distinguish individuals infected with M. tuberculosis from those vaccinated with BCG.

A simplified flow cytometry method of CD4 and CD8 cell counting based on thermoresistant reagents: implications for large scale monitoring of HIV-infected patients in resource-limited settings.

To validate a simplified flow cytometry assay for CD4 and CD8 T cell counting based on monoclonal antibodies which are made resistant to high temperatures (simplified thermoresistant assay (STRA)).

Expression of β2m-Free HLA Class I Heavy Chains in Neuroblastoma Cell Lines

Flow cytometry with the specific monoclonal antibody (MoAb) L31 was used to analyse the expression of HLA class I heavy chains not bound with β2‐microglobulin (β2m) by neuroblastoma (NB) cell lines IMR‐32 and LA‐N‐1. The cells, which express barely detectable amounts of β2m‐free (L31‐positive molecules) and β2m‐complexed HLA class I antigens (W6.32‐ and BBM. I‐reactive molecules), expressed MHC class I molecules not bound to light chains upon differentiation with either retinoic acid or serum starvation. The expression was not accompanied by an increase of surface heterodimers. Conversely, recombinant interferon‐γ (rIFN‐γ) treatment led IMR‐32 and LA‐N‐1 cells to almost exclusively express β2m‐complexed HLA class I heavy chains. Surface β2m‐free MHC class I molecules displayed a molecular mass of ~45 kDa and did not bind exogenously added β2m. No changes in the synthesis of either HLA class I and β2m mRNAs or of L31 proteins were observed in differentiated NB cells, thus suggesting that the surface exposure of unusual HLA class I antigens is regulated post‐translationally. These findings indicate that, in addition to activated lymphocytes, the surface expression of β2m‐free class I heavy chains is a feature of other cell types, such as NB cells.

HLA-C and HIV-1: friends or foes?

The major histocompatibility complex class I protein HLA-C plays a crucial role as a molecule capable of sending inhibitory signals to both natural killer (NK) cells and cytotoxic T lymphocytes (CTL) via binding to killer cell Ig-like receptors (KIR). Recently HLA-C has been recognized as a key molecule in the immune control of HIV-1. Expression of HLA-C is modulated by a microRNA binding site. HLA-C alleles that bear substitutions in the microRNA binding site are more expressed at the cell surface and associated with the control of HIV-1 viral load, suggesting a role of HLA-C in the presentation of antigenic peptides to CTLs. This review highlights the role of HLA-C in association with HIV-1 viral load, but also addresses the contradiction of the association between high cell surface expression of an inhibitory molecule and strong cell-mediated immunity. To explore additional mechanisms of control of HIV-1 replication by HLA-C, we address specific features of the molecule, like its tendency to be expressed as open conformer upon cell activation, which endows it with a unique capacity to associate with other cell surface molecules as well as with HIV-1 proteins.

Comment on "Influence of HLA-C expression level on HIV control".

Apps et al. (Reports, 5 April 2013, p. 87) found that high human leukocyte antigen C (HLA-C) expression favors HIV-1 control. However, as noted here, HLA-C was assessed with a monoclonal antibody (DT9) that cross-reacts with HLA-E. In the context of the available evidence, this is consistent with the idea that the two leukocyte antigens collaborate to keep the HIV-1 virus at bay.

Selective granulocyte/monocyte apheresis in the treatment of HIV-infected patients: short-term and long-term effects on immunological and virological parameters.

CD4 T cells constitute the major cellular target of HIV infection and, in the natural evolution of the disease, are gradually lost, leading to severe immunodeficiency. Highly active antiretroviral therapy (HAART) is generally effective in reducing HIV replication to undetectable levels and allows the recovery of CD4 T cells in the majority of patients treated. However, some sanctuaries of HIV replication persist even after years of effective HAART and are responsible for the rebound of viral replication when treatment is interrupted. Monocytes/macrophages are also infectable by HIV and are less susceptible to its cytopathic effects compared to CD4 T cells. Replication- competent HIV DNA is detectable in peripheral monocytes of patients under HAART. These cells may therefore contribute to the maintenance of HIV replication during treatment. In addition, both monocytes and granulocytes are abnormally activated during HIV infection and this results in overproduction of some pro-inflammatory cytokines, among them TNF-. For these reasons we tested whether the renewal of the pool of circulating monocytes and granulocytes by selective apheresis (G1 apheresis) could influence some key immunological and virological parameters in HAART-treated patients. The results showed that treatment with G1 apheresis without discontinuation of HAART results in an accelerated immune reconstitution with sustained increases in CD4 T cell counts in those patients who respond virologically to HAART. G1 apheresis is also followed by a strong reduction of TNF- and a reduction of cells bearing integrated HIV DNA. Taken together, the results indicate that G1 apheresis could be used to improve the immunological and virological response to HAART.

Stability and expression levels of HLA-C on the cell membrane modulate HIV-1 infectivity

ABSTRACT HLA-C expression is associated with a differential ability to control HIV-1 infection. Higher HLA-C levels may lead to better control of HIV-1 infection through both a higher efficiency of antigen presentation to cytotoxic T lymphocytes and the triggering of activating killer immunoglobulin-like receptors on NK cells, whereas lower levels may provide poor HIV-1 control and rapid progression to AIDS. We characterized the relative amounts of HLA-C heterotrimers (heavy chain/β2 microglobulin [β2m]/peptide) and HLA-C free heavy chains on peripheral blood mononuclear cells (PBMCs) from healthy blood donors harboring both alleles with stable or unstable binding to β2m/peptide. We analyzed the stability of HLA-C heterotrimers of different allotypes and the infectivity of HIV-1 virions produced by PBMCs with various allotypes. We observed significant differences in HLA-C heterotrimer stability and in expression levels. We found that R5 HIV-1 virions produced by PBMCs harboring unstable HLA-C alleles were more infectious than those produced by PBMCs carrying the stable variants. We propose that HIV-1 infectivity might depend both on the amounts of HLA-C molecules and on their stability as trimeric complex. According to this model, individuals with low-expression HLA-C alleles and unstable binding to β2m/peptide might have worse control of HIV-1 infection and an intrinsically higher capacity to support viral replication. IMPORTANCE Following HIV-1 infection, some people advance rapidly to AIDS while others have slow disease progression. HLA-C, a molecule involved in immunity, is a key determinant of HIV-1 control. Here we reveal how HLA-C variants contribute to the modulation of viral infectivity. HLA-C is present on the cell surface in two different conformations. The immunologically active conformation is part of a complex that includes β2 microglobulin/peptide; the other conformation is not bound to β2 microglobulin/peptide and can associate with HIV-1, increasing its infectivity. Individuals with HLA-C variants with a predominance of immunologically active conformations would display stronger immunity to HIV-1, reduced viral infectivity and effective control of HIV-1 infection, while subjects with HLA-C variants that easily dissociate from β2 microglobulin/peptide would have a reduced immunological response to HIV-1 and produce more infectious virions. This study provides new information that could be useful in the design of novel vaccine strategies and therapeutic approaches to HIV-1.

Efficacy of a Standardized Extract of Prunus mume in Liver Protection and Redox Homeostasis: A Randomized, Double-Blind, Placebo-Controlled Study

The antioxidant, anti‐inflammatory and hepatoprotective effects of Prunus mume (PM) have previously been demonstrated. This double‐blind, placebo‐controlled study was designed to evaluate the influence of two doses of a food supplement, made of 150 mg of a standardized PM extract on liver transaminases, lipid profile, glycemia, neopterin and reduced and oxidized thiols in plasma and erythrocytes, during a 3‐month treatment period, in healthy subjects with transaminases levels between 20 and 40 UI/L. Forty‐five subjects (56.0 ± 11.6 years) were enrolled. The results showed a beneficial and statistically significant effect versus placebo of PM extract on liver function, with a decrease versus baseline in alanine aminotransferase (47%), aspartate aminotransferase (7%), gamma‐glutamyl transpeptidase (15%) and glycemia (11%). The lipid profile modification was also positive with an increase versus baseline in HDL cholesterol (13%), and a decrease in LDL/HDL ratio (12%) and triglycerides (8%). The antioxidant action of PM translated into a decrease in oxidized glutathione, reduced/oxidized cysteine‐glycine, oxidized cysteine (intracellular pro‐oxidant) and neopterin (inflammation biomarker), was associated with an increase in reduced glutathione. These results are in favor of the use of a standardized extract of P. mume for the support of liver health and prevention of common metabolic and inflammation‐based diseases. Copyright