Discovery of 342 putative new genes from the analysis of 5'-end-sequenced full-length-enriched cDNA human transcripts.

In this work we describe the process that, starting with the production of human full-length-enriched cDNA libraries using the CAP-Trapper method, led us to the discovery of 342 putative new human genes. Twenty-three thousand full-length-enriched clones, obtained from various cell lines and tissues in different developmental stages, were 5'-end sequenced, allowing the identification of a pool of 5300 unique cDNAs. By comparing these sequences to various human and vertebrate nucleotide databases we found that about 40% of our clones extended previously annotated 5' ends, 662 clones were likely to represent splice variants of known genes, and finally 342 clones remained unknown, with no or poor functional annotation. cDNA-microarray gene expression analysis showed that 260 of 342 unknown clones are expressed in at least one cell line and/or tissue. Further analysis of their sequences and the corresponding genomic locations allowed us to conclude that most of them represent potential novel genes, with only a small fraction having protein-coding potential.

CD69 and regulation of the immune function.

CD69, also known as activation inducer molecule, very early activation antigen, MLR-3 and Leu-23, is a member of the natural killer (NK) cell gene complex family of signal transducing receptors. CD69 is as a type II transmembrane glycoprotein with a C-type lectin binding domain in the extracellular portion of the molecule. CD69 expression is induced in vitro on cells of most hematopoietic lineages, including T and B lymphocytes, NK cells, murine macrophages, neutrophils and eosinophils, while it is constitutively expressed on human monocytes, platelets and epidermal Langerhans cells. Although a specific ligand for CD69 has not been identified, its wide cellular distribution and the induction of intracellular signals upon CD69 crosslinking suggest a role for the receptor in the biology of hematopoietic cells. Moreover, certain results indicate that CD69 may be involved in the pathogenesis of such diseases as rheumatoid arthritis, chronic inflammatory liver diseases, mild asthma, and acquired immunodeficiency syndrome.

Expression and function of the early activation antigen CD69 in murine macrophages.

CD69, a member of the natural killer cell gene complex family of signal transducing receptors, represents one of the earliest activation antigens in human and murine lymphocytes. In contrast, human monocytes may express CD69 in a constitutive fashion. We have evaluated the expression and function of CD69 in murine bone marrow-derived macrophages. CD69 expression as determined by flow cytometry was not constitutive but was induced by stimulation with interferon-gamma (IFN-gamma) plus bacterial lipopolysaccharide (LPS) or tumor necrosis factor a (TNF-alpha). Stimulation with LPS alone was equally effective. Infection with the protozoan parasite Leishmania did not induce CD69 expression nor influence CD69 up-regulation by IFN-gamma plus LPS. Induction of CD69 expression was significantly inhibited in the presence of prostaglandin E2 or dibutyryl-cAMP. Stimulation of macrophages with anti-CD69 monoclonal antibody in the presence of IFN-gamma induced both nitric oxide production and TNF-alpha release. Moreover, anti-CD69 stimulation of Leishmania-infected macrophages resulted in elimination of the intracellular parasite. These results suggest that CD69 is an activation antigen for murine macrophages and may serve as a signaling receptor for an as yet uncharacterized ligand.

[Leishmania-macrophage interactions: role of cytokines and molecules co-involved in killing]. / Interazioni Leishmania-macrofago: ruolo delle citochine e delle molecole coinvolte nel killing.

In this review we have summarized the main data concerning Leishmania-macrophage interactions, with particular emphasis on receptors involved in adhesion, activating or deactivating cytokines and toxic molecules responsible for parasite killing. At present it is also known that a different T helper (Th)1- or Th2-cell response may be critical for the outcome of Leishmania infection in human and in murine models. Therefore, we have mentioned the recent studies on cytokines, such as IL-2, which are able to cause the switch from a Th2, disease-promoting immune response, to a Th1, protective response. In fact, in the light of these findings, these molecules may be used in the future for immunotherapeutical or immunoprophylactic purposes.

Biochemical host response to interferon-beta.

To assess influence of host response to interferon-beta (IFN-beta), on biochemical parameters, beta 2-microglobulin (beta 2-M) and neopterin were evaluated in 15 and 12 patients respectively before and 24 h after 1-46 X 10(6) IU intravenously (i.v.) IFN-beta given every other day. In 4 additional patients, both molecules were determined before and after 24, 48, 72, and 96 h of weekly IFN-beta injections. Serum beta2-M levels significantly increased 24 h after IFN-beta administration in the overall group of 15 patients treated with the alternate day schedule (p = 0.003) as well as in the group of patients treated with the weekly schedule (p = 0.00003). Maximum induction of beta 2-M was observed 24 h after a single weekly IFN-beta injection, but the levels of this protein 72 h after still remained significantly higher than baseline values (p = 0.001). This demonstrates the progressive accumulation of beta 2-M in the circulation produced by the continuous IFN administration. Nevertheless, in patients treated with both IFN treatment schedules, a clear correlation between the increments of beta 2-M and the IFN-beta doses was observed (p = 0.00002 and p = 0.0016 for the alternate day and the weekly schedule respectively). Furthermore the under curve area (AUC) of 48 h beta 2-M levels after IFN administration significantly rose (p less than 0.05) with increasing IFN doses in 4/6 patients. In spite of the accumulation of beta 2-M in the circulation, the overall serum values of this protein 24 h after each successive IFN-injection, in the 15 patients receiving the alternate-day treatment, were significantly higher than before the immediate preceding dose both in patients with initially normal and those with initially high base levels (p = 0.00055 and p = 0.011, respectively). As with beta 2-M, neopterin levels significantly rose during IFN treatment (p less than 0.05) in the group of patients as a whole. After single weekly IFN-beta injections, maximum induction of neopterin was observed 24 h after administration, then the levels of this molecule slowly declined towards the baseline levels, but 96 h after, its levels were still significantly elevated (p less than 0.00001). Neopterin induction was not related to IFN-beta doses, but the levels of this molecule both before and after IFN administration were correlated with an increase in the number of IFN injections (p = 0.0006 and p = 0.0009, respectively).(ABSTRACT TRUNCATED AT 400 WORDS)

A randomized trial to evaluate the immunorestorative properties of thymostimulin in patients with Hodgkin's disease in complete remission.

A total of 19 Hodgkin's disease (HD) patients (12 male, 7 female) aged 26-67 years, who had been in complete unmaintained remission for 6 months or more when the study was initiated, were randomly given 50 mg thymostimulin (TS) i.m. daily (G1) or every other day (G2) for 35 days. A third group (G3) was not treated. Then TS, at the same dose was administered twice a week for the following 22 weeks in patients both initially receiving loading or intermittent TS treatment. When compared with age- and sex-matched controls, as a group, the patients' circulating OKT3+, OKT4+, OKT11+ and E-AETR+ cells were depressed (P less than 0.001 for both proportions and absolute numbers), whereas their OKT8+ cell population was not. Following 5 weeks of daily TS administration, the proportions and numbers of all T cell fractions significantly increased in G1 patients (P less than 0.03 for all the comparisons tested), while following intermittent TS treatment (G2) only the proportions of OKT3+ and OKT11+ cells (P less than 0.03), but not of other T cell fractions, significantly increased. In addition, no significant changes in the absolute numbers of T cell fractions were observed in this group of patients. Furthermore, no spontaneous variations in the T cell pool size occurred in untreated patients. TS maintenance therapy did not produce any further improvement in the size of overall T cells and T cell subsets but sustained percentage and absolute numbers of these cells during administration and the absolute number of T cells even after discontinuation of therapy. The TS-induced improvement in the T cell pool was not associated with any change in the size of circulating non-T lymphocytes and monocytes. In vitro phytohemagglutinin-induced interleukin-2 (IL-2) and gamma-interferon (IFN-gamma) synthesis was assessed in 11 patients (3 G1, 4 G2, and 4 G3). Although it was not statistically significant, a rise in IL-2 and IFN-gamma production was observed in TS-treated patients, but not in untreated controls. TS failed to exert any effect on the serum circulating levels of neopterin, type I and II IFN, beta-2 microglobulin (B2-M) and immunoglobulins (Ig). TS can thus improve defective T cell frequences and numbers and may modulate IL-2 and IFN-gamma production.

Immunologic profile in patients with Hodgkin's disease in complete remission.

Mononuclear cell subsets in peripheral blood, in vitro production of interleukin-2 (IL-2) and gamma interferon (IFN gamma), spontaneous cell-mediated cytotoxicity (SCMC) and circulating levels of Type I IFN, neopterin, beta-2 microglobulin (B2-M), immunoglobulins and complement fractions were studied in 33 patients with Hodgkin's disease (HD) in complete remission. The mean percentages, but not the absolute numbers, of T-lymphocytes expressing pan-T markers (OKT11, OKT3, ER, E-AET R) were significantly decreased compared with control values. Furthermore, patients showed a selective loss of OKT4+ cells, as well as increased percentages and numbers of Leu7+ and OKIa+ lymphocytes, and of OKM1+, LeuM2+, and LeuM3+ cells. OKT4+ cell depletion was a characteristic of patients with shorter time since beginning of remission as well as of those with nodular sclerosis (NS), mixed cellularity Hodgkin's disease (MC-HD), and systemic symptoms at diagnosis. Multifactorial statistical analysis carried out to investigate the effect of disease characteristics and the time since remission began on peripheral mononuclear blood cell (PMBC) subsets showed that histologic condition was the single best predictor of T-cell pool or OKT4+ cell subset size. Time since remission duration and other disease-related factors determined differences in the percentages, but not in the absolute numbers, of T-cell fractions. In addition, neither the disease features nor the time since remission duration determined significant differences in the absolute number of non-T-mononuclear cells in the various patient groups. Patients displayed decreased in vitro synthesis of IL-2 and IFN gamma. The values of SCMC, Type I IFN, neopterin, B2-M, immunoglobulins, and complement fractions did not differ greatly from those of controls.