Specific inhibition of protein kinase Cbeta expression by antisense RNA affects the activation of Jurkat T lymphoma cells.

Antisense RNA technology was employed to specifically inhibit the expression of the protein kinase Cbeta (PKCbeta) isoform in Jurkat cells, to explore its influence on the expression of surface antigens (CD69) and the cytokines interleukin-8 (IL-8), tumour necrosis factor (TNF)-alpha and beta, and to characterise its controversial involvement in the expression of IL-2 and its receptor (IL-2R). Transfection of cells with an antisense PKCbeta construct (as-PKCbeta-pREP3) significantly increased IL-2R/CD25 expression in phorbol 12-myristate 13-acetate (PMA)-stimulated as-PKCbeta-pREP3 transfectants, in contrast to Jurkat cells transfected with a control as-PKCalpha-pREP3 plasmid. IL-2 production, in contrast, was strongly inhibited in both transfectant populations stimulated by PMA plus the calcium ionophore ionomycin. Three clones (asb1/asb2/asb3), selected from as-PKCbeta-pREP3 transfectants, showed decreased PKCbeta protein levels (40 percent, 50 percent and 60 percent, respectively, as determined by western blotting) and mRNA levels. The specific inhibition was confirmed in immunoblots for other PKC (alpha, delta, epsilon, gamma, theta, and lambda lambda/tau) isoforms and in immunoprecipitates from representative (c2/asb2) clones. Stimulation of PKCbeta-depleted clones significantly increased CD25 expression but decreased IL-2 production (similarly to as-PKCbeta-pREP3 transfectants) and IL-2 message levels. CD69 expression and IL-8 secretion were significantly decreased, but TNFbeta message levels were highly increased in asb2/asb3 clones, without affecting TNFalpha secretion. Analysis of the mitogen-activated protein kinase (MAP Kinase) signalling pathway showed unaltered extracellular signal regulated kinase 1/2 (ERK1/2) and p38 phosphorylation but increased activation of c-Jun N-terminal kinase (JNK1) and its substrate, the transcription factor ATF-2 (activated transcription factor-2), which are involved in IL-2 gene expression. Our results revealed new PKCbeta functions, affecting CD69 expression and IL-8 production, and support the requirement for PKCbeta in IL-2 secretion/transcription and IL-2R regulation.

Engineering of major house dust mite allergens Der p 1 and Der p 2 for allergen-specific immunotherapy.

BACKGROUND: Specifically designed recombinant allergens with reduced IgE reactivity are promising candidates for a more defined, effective, and safer specific immunotherapy (SIT). OBJECTIVE: We sought to obtain hypoallergenic hybrid molecules which could potentially be applied to house dust mite (HDM) allergy treatment. METHODS: Two hybrid molecules (QM1 and QM2) derived from the two major Dermatophagoides pteronyssinus allergens, Der p 1 and Der p 2, were engineered by PCR, produced in Escherichia coli, and purified. The overall IgE-binding capacity of the hybrids was compared with their single components by Western blot, specific IgE, skin prick test (SPT), and IgE-inhibition assays. T cell proliferation assay were performed to confirm their retention of T cell reactivity. Immune responses to the hybrid molecules were studied in BALB/c mice. RESULTS: The IgE reactivity of both hybrid proteins was strongly reduced as evaluated by in vitro methods. Furthermore, in vivo SPTs performed on 106 HDM-allergic patients showed that the hybrid proteins had a significantly lower potency to induce cutaneous reactions than the individual components. Hybrid molecules induced higher T cell proliferation responses than those produced by an equimolecular mixture of Der p 1 and Der p 2. Immunization of mice with the hybrid proteins induced Der p 1- and Der p 2-specific IgG, which inhibited the binding of allergic patients' IgE to these natural allergens. CONCLUSION: QM1 and QM2 hybrids exhibited less IgE-binding activity but preserved immunogenicity and fulfilled the basic requirements for hypoallergenic molecules suitable for a future SIT of HDM allergy.

The oxidation handicap hypothesis and the carotenoid allocation trade-off.

The oxidation handicap hypothesis proposes that testosterone mediates the trade-off between the expression of secondary sexual traits and the fight against free radicals. Coloured traits controlled by testosterone can be produced by carotenoid pigments (yellow-orange-red traits), but carotenoids also help to quench free radicals. Recently, it has been shown that testosterone increases the amount of circulating carotenoids in birds. Here, a testosterone-mediated trade-off in the carotenoid allocation between colour expression and the fight against oxidative stress is proposed. Male red-legged partridges were treated with testosterone, anti-androgens or manipulated as controls. Testosterone-treated males maintained the highest circulating carotenoid levels, but showed the palest red traits and no evidence of oxidative damage. Increased levels of a key intracellular antioxidant (i.e. glutathione) indicated that an oxidative challenge was in fact induced but controlled. The trade-off was apparently solved by reducing redness, allowing increased carotenoid availability, which could have contributed to buffer oxidative stress.

Inhibition of in vivo tumor growth by a monoclonal IgM antibody recognizing tumor cell surface carbohydrates.

We have shown previously that IgM from Ehrlich tumor (ET)-immunized mice, recognizing ET cell surface carbohydrates, protects control mice to a subsequent tumor challenge. The factors involved in such IgM-mediated protection were unknown, since it was independent of complement activation. Here, we have extended these in vivo studies by means of monoclonal IgM antibodies. Two of them (A10 and E1), strongly recognizing ET cells and with specificity to ET cell surface carbohydrates, were selected. The results show that A10 (but not E1 or unrelated IgM antibodies) is able to protect nonimmunized mice against ET growth. Protection by A10 was also seen by reducing 800-fold the initial dose; however, E1 was unsuccessful whatever the dose used. A10-mediated protection was observed in C3-defective mice (cobra venom factor treated) or in C5-deficient DBA/2, but not in silica-treated animals. Endotoxin removal did not affect the protection afforded by A10 while specific IgM depletion prevented any protective effect. In addition, the relationship between natural antibodies of IgM isotype recognizing ET cell surface carbohydrates and mouse strain resistance to this tumor is established. Similarly, this natural resistance seems to be complement independent but macrophage mediated. Therefore, these results indicate that some IgM molecules recognizing cell surface carbohydrates may participate in in vivo tumor suppression by a macrophage-dependent mechanism.

Inhibition of bone marrow-derived natural suppressor activity by glucocorticoids and its reversal by IFN-gamma or IL-2.

Natural suppressor (NS) activity is mediated by cells (NS cells) of bone marrow origin with ability to suppress nonspecifically proliferative responses of lymphocytes. Here we show that pharmacologic concentrations (10(-6)-10(-8) M) of glucocorticoids (GC) greatly inhibit NS activity, as detected by coculturing bone marrow and spleen cells stimulated with B cell (LPS) or T cell (concanavalin A) mitogens. Progesterone antagonizes GC-mediated inhibition of NS activity, suggesting that GC were acting through a receptor-dependent mechanism. A prior treatment of NS cells with GC (10(-5) M) has no effect on the NS activity mediated by these cells. GC are required in culture during the first 24 hr of the suppressor assay. Addition of low amounts of IFN-gamma to GC-treated cultures fully reverses NS cell-mediated suppression. IL-2 produces a reversion as well, while addition of IL-3 or IL-4 does not prevent the GC effect. Neutralizing anti-IFN-gamma antibodies, but not anti-IL-2 or anti-TGF-beta, greatly inhibit NS activity in absence of GC. Taken together, these results indicate that GC inhibit NS activity by impairing endogenous cytokine production required to obtain successful NS cell activation, and not by acting directly on NS cells (i.e., inhibiting the secretion of putative NS factors). Among the cytokines involved in NS cell activation, IFN-gamma appears to be critical, since its addition readily overrides the GC effect and its neutralization results in strong inhibition of NS activity.

Interleukin-12-dependent activation of human lymphocyte subsets.

Recently, we reported that IL-12 increased expression and function of CD26/DPPIV, this may be a new cellular pathway of the Th1-like immune responses. Here, we looked for a specific subset which would respond to CD26 upregulation by IL-12. Contrary to previously described results, under our culture conditions (1 microg/ml of PHA), IL-12 enhanced preferentially the CD8 cell proliferation. By using dual fluorescence analysis, IL-12-dependent CD26 expression was found in both CD4 and CD8 (previously CD26+ or CD26-) activated T cells and, moreover, the CD45RO percentage was unaffected. However, the density of CD45RO Ag (which was reported to coexpress with CD26) was impaired. These effects can be implicated in the biological functions of IL-12 and provide some clinical possibilities.

Interleukin-12 enhances CD26 expression and dipeptidyl peptidase IV function on human activated lymphocytes.

Research of a cellular pathway activated by IL-12 which may result in new therapeutical approaches for IL-12, led us to find an intriguing relationship between IL-12 and CD26/DPPIV ectopeptidase on activated T cells. Both the percentage and median fluorescence intensity (MFI) of CD26+ cells in the PHA-stimulated PBMC or lymphoblasts increased when IL-12 (optimum dose, 2 ng/ml) was present. Maximum CD26 expression was observed on day-2 cultures of lymphoblasts, the presence of IL-12 receptor probably being necessary for this upregulation. In addition, CD26 upregulation correlated with enhanced DPPIV function. Enzyme affinity and secretion of the soluble form of DPPIV were not affected by IL-12. Kinetic behaviours of Ag expression and enzymatic activity support a different CD26 regulation pathway by IL-12. These data suggest that the correlation found in vivo between the CD26 expression and Th1-like immune responses is due to this IL-12-dependent upregulation.

Lymphocyte populations in tuberculous pleural effusions.

Different systemic and local responses to mycobacterial antigens suggest an active compartmentalization of responsive lymphocytes to tubercular antigens. This fact, observed in pleuritic processes, raises doubts about the accuracy of information obtained in the study of cells taken solely from peripheral blood. For this reason we decided to study the concept of compartmentalization in 140 patients suffering from pleural effusions. Patients were classified into six groups according to the aetiology of the effusion: group I, tuberculous, n = 23; group II, paraneoplastic, n = 41; group III, metapneumonic empyematous, n = 5; group IV, transudate, n = 38; group V, miscellaneous exudate, n = 19; group VI, unknown aetiology, n = 14. In each group we studied the lymphocyte population by using flow cytometry with doubly fluorescent monoclonal antibodies: B [expressing human lymphocyte antigen (HLA)-DR on the surface], T (CD3+), CD4+ and CD8+, and the subpopulation of activated T lymphocytes (together expressing CD3 and HLA-DR on the surface) (CD3+DR+). The study of these subpopulations in peripheral blood did not yield valuable results, but the CD3+DR+ population in pleural fluid demonstrated a diagnostic efficiency of 84% [positive predictive value (PPV) 51%, negative predictive value (NPV) 96%] at a cut-off value of 80.4 cells/mm3. The CD3+DR+ pleural fluid/peripheral blood ratio demonstrated an efficiency of 83% (PPV 50%, NPV 96%), and showed a statistically significant difference (P < 0.02) with regard to all the diagnostic groups, with the exception of the paraneoplastic effusions. The lymphocytic subpopulations study confirms the concept of compartmentalization in tuberculous pleuritis, as shown by the greater number of activated T lymphocytes present in pleural fluid in comparison with peripheral blood in tuberculous pleuritis, a 98% efficiency of adenosine deaminase (ADA) determination in pleural fluid versus a 50% value in peripheral blood, predominance of helper cells (CD4+) in pleural fluid and suppressor cells (CD8+) in peripheral blood, a greater CD4+/CD8+ ratio in pleural fluid than in peripheral blood, and a significant correlation of ADA-CD3+DR+ in pleural fluid, which does not occur in peripheral blood.

[Endoscopic fenestration as a treatment for asymmetrical hydrocephalus due to obstruction of the foramen of Monro]. / Fenestración endoscópica como tratamiento de la hidrocefalia asimétrica por obstrucción del foramen de Monro.

Unilateral dilatation of the lateral ventricle is a rare condition. The most common causes are tumors of the lateral ventricles or in the area of the third ventricle, acute or chronic inflammatory gliosis, cysticercosis or congenital atresia of the foramen of Monro. We report a case of asymmetrical dilatation of the lateral ventricle in an adult patient presenting with a raised intracranial pressure syndrome caused by narrowing of the foramen of Monro which was occluded by a thin membrane. The patient underwent successful endoscopic fenestration of the Foramen of Monro.

[Wegener's granulomatosis: localization in the subglottic space]. / Granulomatosis de Wegener: presentación localizada en el espacio subglótico.

In the paper is presented a localized form of Wegener's granulomatosis. This entity being considered as incipient stage of the disease. Its better knowledge and consequent early diagnosis has contributed to increase the clinic pictures. We report one of these cases sitting on the subglottic space, in a 40-years-old woman, beginning with a serious dyspnea. Several clinic aspects of the disease are emphasized as well the immunologic procedures helping to the diagnosis and to day's classifications. Finally, the better prognosis of localized forms, because its excellent response to immunosuppresants, is stressed.

Possible interaction between a rodenticide treatment and a pathogen in common vole (Microtus arvalis) during a population peak.

A common vole (Microtus arvalis) population peak in Northern Spain in 2007 was treated with large scale application of chlorophacinone, an anticoagulant rodenticide of the indandione family. Voles found dead and trapped alive were collected in treated and untreated areas. Residues of chlorophacinone were analyzed in liver of voles by HPLC-UV. Also, the presence of the pathogen Francisella tularensis was analyzed by PCR in samples of vole spleen. Chlorophacinone (82-3800 ng/g; wet weight liver) was only detected in voles found dead in treated areas (55.5%). The prevalence of F. tularensis in voles found dead in treated areas was also particularly high (66.7%). Moreover, chlorophacinone levels were lower in voles that were PCR-positive for F. tularensis (geometric mean [95% CI], 418 [143-1219] ng/g) than in those that were PCR-negative (1084 [581-2121] ng/g). Interactions between pathogens and rodenticides might be considered to reduce the doses used in baits or to avoid the use of the more toxic 2nd generation anticoagulant rodenticides.

How effective is pre-release nematode control in farm-reared red-legged partridges Alectoris rufa?

Game bird farming is associated with high parasite levels that reduce farm productivity, reduce survival after releasing, and may pose a health risk for natural populations. The efficacy of albendazole (orally, 20 mg kg(-1) was evaluated in farmed red-legged partridges naturally infected with the nematodes Aonchotheca caudinflata and Heterakis gallinarum. In treated birds body condition improved, nematode egg deposition was reduced and the proportion of gravid A. caudinflata females was reduced, but not the overall worm burdens. Albendazole was found to be 36.8% and 17.1% effective against A. caudinflata and H. gallinarum, respectively. These results indicate that the anthelmintic treatment used normally in Spanish partridge farms is not effective enough to avoid the introduction of parasites into the field after release.

Avoiding bias in parasite excretion estimates: the effect of sampling time and type of faeces.

The study of host-parasite relationships usually requires reliable estimates of parasite intensity, which is often estimated from parasite propagule concentration in faeces. However, parasite excretion in faeces may be subject to variation due to endogenous or exogenous factors that must be identified to obtain reliable results. We analysed the effect of the hour of sample collection on propagule counts of 2 intestinal parasites infecting the red-legged partridge: the capillarid nematode Aonchoteca caudinflata and coccidia of the genus Eimeria (Protozoa). Also, we test whether there are differences in propagule counts between caecal and intestinal faeces. Individual faecal samples from infected birds were collected daily at 4 different hours during several days. The hour of the day exerted a very strong effect on propagule counts, excretion of both types of parasites showing a clear and constant increase from dawn to dusk. Also, capillarid eggs were more abundant in intestinal than in caecal faeces, whereas the inverse pattern was found for coccidian oocysts. Standardization of the hour of sample collection or statistical control of this variable is recommendable to prevent bias. Similarly, in bird species with long caeca, consistent collection of one type of faeces may avoid significant errors in parasite burden estimates.

Testosterone increases bioavailability of carotenoids: insights into the honesty of sexual signaling.

Androgens and carotenoids play a fundamental role in the expression of secondary sex traits in animals that communicate information on individual quality. In birds, androgens regulate song, aggression, and a variety of sexual ornaments and displays, whereas carotenoids are responsible for the red, yellow, and orange colors of the integument. Parallel, but independent, research lines suggest that the evolutionary stability of each signaling system stems from tradeoffs with immune function: androgens can be immunosuppressive, and carotenoids diverted to coloration prevent their use as immunostimulants. Despite strong similarities in the patterns of sex, age and seasonal variation, social function, and proximate control, there has been little success at integrating potential links between the two signaling systems. These parallel patterns led us to hypothesize that testosterone increases the bioavailability of circulating carotenoids. To test this hypothesis, we manipulated testosterone levels of red-legged partridges Alectoris rufa while monitoring carotenoids, color, and immune function. Testosterone treatment increased the concentration of carotenoids in plasma and liver by >20%. Plasma carotenoids were in turn responsible for individual differences in coloration and immune response. Our results provide experimental evidence for a link between testosterone levels and immunoenhancing carotenoids that (i) reconciles conflicting evidence for the immunosuppressive nature of androgens, (ii) provides physiological grounds for a connection between two of the main signaling systems in animals, (iii) explains how these signaling systems can be evolutionary stable and honest, and (iv) may explain the high prevalence of sexual dimorphism in carotenoid-based coloration in animals.

[Dendritic cells in chronic adult periodontal disease]. / Las células dendriticas en la enfermedad periodontal crónica del adulto.

At the gingiva there are two cellular populations with mediation capacity in the immunoregulation, whose mechanisms seen to be implicated in the pathogenesis of the periodontal disease. One of these populations is formed by the macrophages and the other one by the Langerhans cells belonging to the epithelia. In this paper wr are demonstrating that both populations keep an inverse proportional relation in the two sides of gingival epithelium. In the oral side, the Langerhans cells are predomiating, while in the periodontal pocket they are in small number, in comparison with the macrophages, which are highly abundant and forming a subepithelial band.

Interleukin-2 killer cells: in vitro evaluation of combination with prothymosin alpha.

Interleukin-2 (IL-2) is a potent inducer of lymphokine-activated killer (LAK) activity against both NK-sensitive and NK-resistant tumor cell lines. IL-2 therapy has been associated with clinical responses, but these responses have occurred only with high, toxic doses of the cytokine. Since prothymosin alpha (ProT alpha) is able to enhance the spontaneous NK activity of cells from normal donors, we studied the effect of ProT alpha on LAK activity by IL-2. The lysis of K562 and Daudi cells by effector cells cultured with IL-2 was increased time dependently when cultures also contained ProT alpha. PBLC was separated by discontinuous density centrifugation in the LGL-enriched fractions. Within the CD16+ population, all effector cell precursors were CD2+, but the effector population after IL-2 or IL-2 + ProT alpha activation also contained CD16+CD2- cells; the CD2 molecule is thus indispensable for induction of LAK activity by IL-2 or IL-2 + ProT alpha but not for the action of activated LAK cells (or for the enhancing effect of ProT alpha). Within the CD3- CD16+ LGL population, 5 micrograms/ml ProT alpha plus 50 U/ml IL-2 was able to induce p70 IL-2R expression to a similar extent to 100 U/ml of IL-2. The use of ProT alpha to enhance the induction of LAK activity by IL-2 may be able to improve immunotherapy of cancer.

Increased serum IgE in alcoholics: relationship with Th1/Th2 cytokine production by stimulated blood mononuclear cells.

BACKGROUND: Increased serum immunoglobulin (Ig) E values are frequently found in alcoholics. Cytokines produced by T-helper-2 (Th2) lymphocytes are required for IgE synthesis. Chronic alcoholism is associated with altered cytokine balance. This study analyzed the relationship between Th1 and Th2 cytokine production by stimulated peripheral blood mononuclear cells (PBMCs) and serum IgE levels, both in atopic and nonatopic alcoholics. METHODS: Twenty-five patients admitted to the hospital with alcohol withdrawal syndrome were included in the study. Five were classified as atopic and 20 as nonatopic by means of skin-prick tests. Interleukin-4 (IL-4), IL-10, IL-12, IL-13, and interferon gamma were measured in the supernatants of 48-hr cultures of PBMCs stimulated with phytohemagglutinin. Total serum IgE was measured by chemiluminescent enzyme immunoassay. Results were compared with those of 15 healthy controls (seven atopics and eight nonatopics). RESULTS: Total serum IgE concentrations were higher in alcoholics than in controls, in both atopic and nonatopic subjects. The ratio of IL-4 to interferon gamma production by phytohemagglutinin-stimulated PBMCs (as an approach to Th2/Th1 balance) was significantly lower in alcoholics than in healthy controls, both in the atopic and in the nonatopic group. No difference was observed regarding IL-10, IL-12, and IL-13 production between alcoholics and controls. No correlation was demonstrated between cytokine production and total serum IgE levels in any group. CONCLUSIONS: Increased total serum IgE is observed in alcoholics together with a paradoxically low ratio of Th2 to Th1 cytokine production by phytohemagglutinin-stimulated PBMCs. These findings are independent of the atopic status of patients.

Development of splenic natural suppressor (NS) cells in Ehrlich tumor-bearing mice.

Spleen cells from C57BL/6J mice bearing Ehrlich carcinoma growing as a solid tumor show progressive unresponsiveness to concanavalin A (Con A) and lipopolysaccharide (LPS) mitogens. This is accompanied by striking spleen enlargement with marked hematopoietic activity. Lymphoproliferative assays of normal spleen cells in co-culture with tumor-bearing spleen cells (TBSC) show that: (a) TBSC contain non-specific suppressor cells able to abrogate both Con A and LPS responses, or mixed lymphocyte reaction, of normal spleen cells and (b) suppression by TBSC is MHC-unrestricted, non-prostaglandin-mediated and greatly enhanced by Con A supernatants. Suppressor cells associated with TBSC are large, low-density cells without markers of mature B or T lymphocytes or of the mononuclear phagocyte system. Most appear to be asialo-GM1-negative, as suppression was only partially inhibited by treatment with anti-asialo-GM1 and complement. Since NK activity is lacking in TBSC, our data strongly suggest that these "null" suppressor cells are related to the natural suppressor (NS) cells found described in normal bone-marrow and neonatal spleens, or induced in adult spleens by total lymphoid irradiation, graft-vs.-host disease, or cyclophosphamide treatment.

Overexpression of prothymosin alpha accelerates proliferation and retards differentiation in HL-60 cells.

Prothymosin alpha (ProTalpha) is an acidic nuclear protein the expression of which is related to the proliferation and differentiation processes in mammalian cells. In the present study we have stably transfected HL-60 cells, a biological system that allows the study of both proliferation and differentiation, with recombinant vectors encoding sense and antisense ProTalpha mRNA. In the HL-60 cell clones overexpressing ProTalpha we observed an acceleration in the growth rate, whereas expression of the antisense orientation showed the opposite effect. Moreover, cell-cycle analysis demonstrated that the G1-phase was shortened in the cells expressing the sense construct. Before studying how ProTalpha affects differentiation, we showed that the down-regulation of ProTalpha gene during differentiation occurs in all mammalian cell lines (HL-60, K562, U937, MEL C88, N2A and PC12) analysed. The biological effect evoked by the induction of the ProTalpha sense vector was the retardation of cell differentiation, although expression of the antisense construct showed no effect on differentiation. In conclusion, our findings provide evidence that ProTalpha is directly implicated in cellular proliferation and that the maintenance of high levels of ProTalpha inside HL-60 cells is incompatible with their ability to differentiate.