The CD4-centered universe of human T cell subsets.

Humans are continuously exposed to a high number of diverse pathogens that induce different types of immune responses. Primary pathogen-specific immune responses generate multiple subsets of memory T cells, which provide protection against secondary infections. In recent years, several novel T cell subsets have been identified and have significantly broadened our knowledge about T cell differentiation and the regulation of immune responses. At the same time the rapidly growing number of incompletely characterized T cell subsets has also generated some controversies. We therefore review here the current knowledge on features and functions of human α/ß T cell subsets, focusing on CD4(+) T cells classified according to cytokine production and tissue localization. The principal helper and regulatory T cell subsets can be identified by a limited number of relevant surface markers, which are an integral part of the T cell differentiation programs because they are directly induced by the relevant lineage-defining transcription factors. In vivo occurring human T cell subsets can thus be purified directly ex vivo from relevant tissues for molecular and functional studies, and represent not only an ideal model to study T cell differentiation, but they also offer important clinical opportunities.

[Laparoscopic-assisted versus open surgery for colorectal cancer: postoperative morbidity in a single center randomized trial]. / Laparoscopia vs approccio aperto in chirurgia colorettale: morbidità postoperatoria in un trial randomizzato monocentrico.

AIM: The primary objective of the study was to compare the effect of laparoscopic-assisted (LPS) versus open surgery (LPT) for colorectal cancer on postoperative morbidity. The secondary objectives were to evaluate immune response and intestinal wall oxygen perfusion. METHODS: A total of 610 patients with colorectal cancer were randomly assigned to receive colon resection by either LPS (n=306) or LPT (n=304). Four surgical staff members not involved in the study recorded postoperative complications up to 30 days after the operation. Immune response was evaluated by measuring lymphocytic proliferation after challenge with Candida albicans and phytohemoagglutinin before, at 3 and 15 days after the operation. Intestinal wall oxygen perfusion was continuously monitored using a probe. RESULTS: The conversion rate was 4.6% in the LPS group. Morbidity was 18.6% in the LPS group and 34.5% in the LPT group (P<0.0005). Infections developed in 9.1% of LPS-treated patients and in 20.2% of LPT-treated patients (P<0.0005). The mean length of stay was 9.7+/-2.6 days in the LPS group and 12.2+/-4.2 days in the LPT group (P<0.0001). In both groups lymphocytic proliferation was low at 3 days postoperative but returned to normal range at 15 days only in the LPS group. Interoperative intestinal oxygen perfusion values were higher in the LPS group. CONCLUSIONS: Laparoscopic colorectal surgery reduced both postoperative morbidity and length of stay. Lymphocytic proliferation and intestinal wall oxygen perfusion were higher in patients who underwent laparoscopic-assisted surgery.

Cytoadherence of Plasmodium falciparum-infected erythrocytes is mediated by a redox-dependent conformational fraction of CD36.

The adherence of Plasmodium falciparum-infected RBC (IRBC) to postcapillary venular endothelium is an important determinant of the pathogenesis of severe malaria complications. Cytoadherence of IRBC to endothelial cells involves specific receptor/ligand interactions. The glycoprotein CD36 expressed on endothelial cells is the major receptor involved in this interaction. Treatment of CD36-expressing cells with reducing agents, such as DTT and N-acetylcysteine, was followed by CD36 conformational change monitorable by the appearance of the Mo91 mAb epitope. Only a fraction of the surface expressed CD36 molecules became Mo91 positive, suggesting the presence of two subpopulations of molecules with different sensitivities to reduction. The Mo91 epitope has been localized on a peptide (residues 260-279) of the C-terminal, cysteine-rich region of CD36. Treatment with reducing agents inhibited the CD36-dependent cytoadherence of IRBC to CD36-expressing cells and dissolved pre-existent CD36-mediated IRBC/CD36-expressing cell aggregates. CD36 reduction did not impair the functionality of CD36, since the reactivity of other anti-CD36 mAbs as well as the binding of oxidized low density lipoprotein, a CD36 ligand, were maintained. The modifications induced by reduction were reversible. After 14 h CD36 was reoxidized, the cells did not express the Mo91 epitope, and cytoadherence to IRBC was restored. The results indicate that IRBCs bind only to a redox-modulated fraction of CD36 molecules expressed on the cell surface. The present data indicate the therapeutic potential of reducing agents, such as the nontoxic drug N-acetylcysteine, to prevent or treat malaria complications due to IRBC cytoadhesion.

CD36 is a ditopic glycoprotein with the N-terminal domain implicated in intracellular transport.

The CD36 receptor sequence predicts two hydrophobic domains located at the N- and C-termini of the protein, but there are conflicting reports as to whether the N-terminal uncleaved leader sequence functions as a transmembrane domain. To investigate the topology of CD36, we generated a panel of mutants lacking either one or both hydrophobic regions and analyzed their folding and transport in COS-7 cells. The N- and the C-terminal hydrophobic regions were both sufficient to anchor CD36 in the membrane, and a FLAG epitope inserted at the N-terminus was located intracellularly. These results indicate that CD36 adopts a ditopic configuration. Accordingly, neither N- nor C-terminal truncation mutants were secreted. Analysis with conformation-specific monoclonal antibodies showed that the N-terminal transmembrane domain truncated molecule was slowly transported through the exocytic pathway and largely accumulated intracellularly. Thus, dual membrane insertion dictates the correct topogenesis and seems to be necessary for efficient folding and intracellular transport.

CD36 folding revealed by conformational epitope expression is essential for cytoadherence of Plasmodium falciparum-infected red blood cells.

CD36 is a membrane glycoprotein and a putative scavenger receptor expressed by several cell types. In capillary endothelial cells, it mediates the adherence of erythrocytes infected with Plasmodium falciparum. The CD36 sequence contains two hydrophobic domains located at the amino-and carboxyl-termini of the protein, but the topology of this protein and the functional significance of these domains are still not clearly defined. We generated soluble CD36-IgG chimeric molecules by fusion of the extracellular domains of CD36 with human immunoglobulin domains. The construct containing the N-terminal hydrophobic domain of CD36 was completely retained intracellularly as membrane-associated molecule, suggesting that the N-terminal hydrophobic domain of the CD36 is a real transmembrane domain and that CD36 has hairpin topology. A small amount of the CD36-IgG chimeric construct lacking both transmembrane domains escaped retention, was correctly processed, and accumulated in the extracellular medium as a soluble molecule. This CD36-IgG construct failed to bind Plasmodium falciparum-infected erythrocytes. Using monoclonal antibodies specific for either conformational or structural epitopes, we demonstrate that failure of this CD36-IgG construct to bind infected erythrocytes was due to incorrect folding of the soluble chimeric molecule.

Methionine 156 in the immunodominant domain of CD36 contributes to define the epitope recognized by the NL07 MoAb.

CD36 is a membrane glycoprotein expressed by several cell types, and play a role as a receptor for different physiological and pathological ligands. An immunodominant domain of CD36 has been described in the amino acidic region 155-183, where many ligands and monoclonal antibodies (MoAbs) react. MoAbs against CD36 have proved useful in structural as well as functional studies. One of these antibodies, MoAb NL07, recognizes a conformational epitope that is acquired in the late steps of the CD36 maturation. The NL07 epitope appears to be functionally relevant and blocks CD36-mediated binding to red blood cells infected with the malaria parasite Plasmodium falciparum (IRBC). In this work a mutant COS-7 clone expressing NL07-negative CD36 molecules on the cell surface was investigated. In the mutant, the methionine in position 156 of the wild type CD36 sequence was replaced by a valine. It was determined that methionine 156 was essential for NL07 reactivity, mapping the NL07 epitope to the vicinity of the functionally important immunodominant domain (aa 155-183) of CD36. Although methionine 156 is located in this region, the CD36V156 mutated molecule was apparently functional and able to bind IRBC and oxidized LDL.

Primary ex vivo culture of keratinocytes isolated from hypertrophic scars as a means of biochemical characterization of CD36.

The CD36 antigen is a molecule which is ectopically expressed on epidermal keratinocytes of hypertrophic scars and is a good candidate for a marker for a broad range of skin pathologies. Most marker studies have been performed using immunohistochemical techniques on fixed skin sections. Our aim was to investigate the biochemical features of the CD36 expressed in pathological keratinocytes and to find an in vitro model for the study of the regulation of its expression. Here we show how keratinocytes isolated from hypertrophic scars can be cultivated in vitro and employed as a model for the study of these cells. We demonstrated that the antigenic features of the CD36 expressed on keratinocytes of hypertrophic scars are identical to those described for the CD36 expressed by other cell types. The molecule was expressed on the surface of keratinocytes which were non-adherent in vitro. Adherent and proliferating keratinocytes, as well as normal keratinocytes, were CD36 negative both at the surface and intracellularly. The in vitro proliferating cells from hypertrophic scars, but not the normal keratinocytes, showed intracellular expression of CD36 after long-term culture and cell stratification, suggesting a regulated expression of CD36 in pathological keratinocyte differentiation.

Formation of one or more intrachain disulphide bonds is required for the intracellular processing and transport of CD36.

In monocytes/macrophages, CD36 is thought to have a role as a scavenger receptor, mediating the phagocytosis of apoptotic cells and the endocytic uptake of oxidized low-density lipoproteins and fatty acids. The proposed topology of CD36 predicts that, of ten cysteine residues, six lie in the extracellular domain, whereas four are equally distributed in the two short terminal tails flanking the N-terminal and C-terminal hydrophobic stretches. Here we investigate the formation of intrachain disulphide bonds, on the basis of the assumption that the cysteine residues present in the luminal domains are generally oxidized, whereas those in the cytosol are reduced. As revealed by gel mobility-shift assays, disulphide bonds are present in the extracellular domain of the CD36 molecule. The formation of these bonds is required for the transport of CD36 from endoplasmic reticulum to Golgi. Furthermore reactive thiol groups are present in the CD36 sequence, which upon lysis form an intrachain extra loop as an artifact. This disulphide bond is not formed in either (1) truncated CD36 lacking the two C-terminal cysteine residues or (2) Triton X-100-insoluble wild-type CD36 molecules, suggesting that, in this fraction, the C-terminal thiol groups are modified.

Synthesis, processing, and intracellular transport of CD36 during monocytic differentiation.

CD36 is an integral membrane glycoprotein expressed by several cell types, including endothelial cells of the microvasculature, erythrocytes, platelets, and monocytes. In the monocytic lineage, CD36 is expressed during the late stages of differentiation in the bone marrow, in circulating monocytes, and in some tissue resident macrophages, and it is thought to mediate the phagocytosis of apoptotic cells and the endocytic uptake of modified lipoproteins. Here we analyze the synthesis, processing, and intracellular transport of CD36 in U937 and THP-1, two human cell lines representing different stages of monocytic maturation. In both cell lines, phorbol 12-myristate 13-acetate induces the expression of CD36. A 74-kDa intracellular precursor is first synthesized that has the hallmarks of a resident protein of the endoplasmic reticulum. The precursor protein is later processed into a mature form of 90-105 kDa which is transported to the cell surface. The kinetics of processing differ significantly in U937 and THP-1. These differences are specific for the CD36, as two unrelated proteins (CD11b and CD45R) are processed and transported to the surface at similar rates in the two cell lines. A 33-kDa endoglycosidase H-sensitive glycoprotein specifically associates with the 74-kDa precursor. Coprecipitation of gp33 correlates with slow processing of CD36 precursor, suggesting that gp33 may play a role in regulating the intracellular transport of CD36, during monocyte maturation.