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PubMed1976

Study of human T and B lymphocytes with heterologous antisera. II. Partial characterization of a human T lymphocyte antigen.

Using a horse anti-human thymocyte serum made specific for T lymphocytes by absorption (anti-HTLA serum), a material reacting with it has been extracted from detergent-lysed peripheral blood lymphocytes. Acid elution and gel-filtration analysis allowed recovery of two peaks containing HTLA activity: one of 30,000-40,000 Daltons, the other being excluded by Sephadex G-200 and likely to be more than 300,000 Daltons. HTLA appeared to be released by lymphocytes incubated at 37 degrees, but not at 4 degrees, and significantly detectable after 10-24-h incubation. Finally co-capping experiments with anti-beta2m antibodies and absence of inhibition of cytolysis by anti-HTLA serum of celld beta2m.

Antigen-Antibody Reactions↗
PubMed1976

Distribution of erythrocyte receptors at the surface membrane of human T-lymphocytes.

During incubation at 4 degrees C, the binding sites for neuraminidase-treated human erythrocytes (nHRBC) and sheep red blood cells (SRBC) were diffusely distributed on the surface membrane of most rosette-forming normal human blood lymphocytes. Increasing the temperature to 37 degrees C resulted in a rapid rosette dissociation which was accompanied by a relative increase in cap formation and a corresponding decrease of ring-like rosettes. The conversion into caps was inhibited by reincubation of the cells at 4 degrees C. The results indicate a movement of the nHRBC and SRBC receptors in the plane of the human T lymphocyte plasma membrane.

Binding Sites↗
PubMed1999

Characterization of tyrosinase from the cap flesh of portabella mushrooms.

Tyrosinase, purified from the cap flesh tissue of portabella mushrooms, was characterized with regard to its physical and biochemical properties. A native molecular size of 41 kDa for the enzyme was obtained by size exclusion chromatography, whereas SDS-PAGE indicated that the enzyme contained a single subunit with a size of approximately 48 kDa under reduced and nonreduced conditions. The purified enzyme showed a single immunological cross-reacting protein after Western blotting when probed with antibodies against Agaricus bisporus tyrosinase. Isoelectric focusing demonstrated that the enzyme preparation, apparently homogeneous by electrophoresis, still contained three isoforms of pI 5.1, 5.2, and 5.3. The purified enzyme was able to oxidize a variety of mono-, di-, and triphenolic compounds. An apparent K(m) of 5 mM was obtained using catechol as the substrate, and an apparent K(m) of 9 mM was found using L-Dopa as a substrate. Ascorbic acid, kojic acid, tropolone, mercaptobenzothiazole, and salicylhydroxamic acid inhibited the enzyme severely at 100 microM.

Agaricus↗
PubMed1991

Sea urchin egg villin: identification of villin in a non-epithelial cell from an invertebrate species.

Fertilization of sea urchin eggs results in the rapid polymerization of actin filaments and subsequent formation of a brush border-like cortical cytoskeleton. A 110 x 10(3) Mr (110K) actin binding protein has been purified from extracts of unfertilized Strongylocentrotus purpuratus eggs. Analysis of polymerization kinetics using fluorescence and viscometry assays demonstrated that 110K accelerated the nucleation phase of actin assembly only in the presence of elevated Ca2+. The Ca(2+)-mediated effects were correlated with a decrease in sedimentable polymer and a decrease in average filament length. Addition of Ca2+ to solutions of 110K and F-actin, polymerized in the presence of EGTA, resulted in a precipitous drop in viscosity and the decreased viscosity was fully reversible upon chelation of Ca2+. The Ca2+ threshold for 110K activation was in the 10(-6) to 10(-7) M range. Nucleated assembly experiments using Limulus sperm acrosomal processes demonstrated that egg 110K capped the barbed ends of actin filaments. In the absence of Ca2+, 110K organized actin filaments into bundles at pH values less than 7.4. Anti-egg 110K antibody crossreacted with chicken intestinal epithelial cell villin and anti-porcine villin headpiece monoclonal antibody crossreacted with 110K. Further, 110K possesses an approximately 10 x 10(3) Mr terminal polypeptide segment that is immunologically related to villin headpiece. These studies demonstrate that sea urchin egg 110K is functionally, immunologically and structurally related to villin, an actin binding protein expressed in specific epithelial tissues in vertebrates. Consequently, this finding provides insight into the potential mechanisms that might determine the genesis of the cortical brush border cytoarchitecture in sea urchin eggs and further sheds light on the evolution of the villin protein family.

Actins↗
PubMed1985

Insulin receptor capping and its correlation with calmodulin-dependent myosin light chain kinase.

Both fluorescence microscopy and fluorometric analysis techniques have been used to characterize insulin receptor capping in IM-9 human lymphoblastoid cells. Morphologically, insulin caps appear similar to lectin or antiimmunoglobulin-induced caps displaying a preferential accumulation of actin, myosin, and actin-binding protein directly underneath the cap structure. Using the fluorescent calcium indicator quin2 we have detected no change in the calcium activity following insulin stimulation. However, in the presence of a number of calmodulin inhibitors, such as W-5, W-7, W-12, and trifluoperazine (TFP), insulin capping is significantly inhibited, which implies that a calmodulin-regulated process is involved. Using double immunofluorescence microscopy, we have found that the calmodulin-dependent myosin light chain kinase (MLCK) is concentrated directly beneath insulin caps. Upon treatment with trifluoperazine (TFP), the redistribution of both MLCK and insulin receptors are inhibited concomitantly. Our data indicate that the calmodulin-dependent myosin light chain kinase may be directly responsible for the activation of actomyosin-mediated contractility during insulin receptor capping.

Actomyosin↗
PubMed1996

Sequences within a small yeast RNA required for inhibition of internal initiation of translation: interaction with La and other cellular proteins influences its inhibitory activity.

We recently reported purification, determination of the nucleotide sequence, and cloning of a 60-nucleotide RNA (I-RNA) from the yeast Saccharomyces cerevisiae which preferentially blocked cap-independent, internal ribosome entry site (IRES)-mediated translation programmed by the poliovirus (PV) 5' untranslated region (UTR). The I-RNA appeared to inhibit IRES-mediated translation by virtue of its ability to bind a 52-kDa polypeptide which interacts with the 5' UTR of viral RNA. We demonstrate here that the HeLa 52-kDa I-RNA-binding protein is immunologically identical to human La autoantigen. Moreover, I-RNA-mediated purified La protein. By using I-RNAs with defined deletions, we have identified sequences of I-RNA required for inhibition of internal initiation of translation. Two smaller fragments of I-RNA (16 and 25 nucleotides) inhibited PV UTR-mediated translation from both monocistronic and bicistronic RNAs. When transfected into HeLa cells, these derivatives of I-RNA inhibited translation of PV RNA. A comparison of protein binding by active and inactive I-RNA mutants demonstrates that in addition to the La protein, three other polypeptides with apparent molecular masses of 80, 70, and 37 kDa may influence the translation-inhibitory activity of I-RNA.

Autoantigens↗
PubMedDate not supplied

Regional accumulations of T cells, macrophages, and smooth muscle cells in the human atherosclerotic plaque.

The cellular composition of human atherosclerotic plaques was analyzed by immunologic techniques. Plaques were removed from the internal carotid artery during surgery, and a panel of monoclonal antibodies was used to identify cell types. Macrophages stained by Anti-Leu-M3 were found throughout the plaque, particularly in the lipid core region, where 60% of the cells reacted with this antibody. T cells expressing the T3 antigen were most abundant in the fibrous cap, where they constituted 20% of the cell population. T cells were also isolated from the plaque and detected by a rosetting test; many of these T cells were activated, as indicated by the expression of HLA-DR. Other types of leukocytes were uncommon in the plaque. An antibody to the intermediate filament protein, desmin, was used as a marker for smooth muscle cells since some, but not all, vascular smooth muscle cells contain this protein. The desmin-positive cells were uncommon in the nonatherosclerotic intima but were more numerous in the plaque. In conclusion, atherosclerotic plaques are heterogeneous with respect to cellular composition. The smooth muscle cell dominates in the fibrous cap, which also contains many T cells; the lipid core is dominated by macrophages. We suggest that interactions between smooth muscle cells and blood-borne cells are important in the pathogenesis of atherosclerosis.

Animals↗
PubMed1989

Natural killer cell activity and recycling capacity of natural killer cells in patients with carcinoma of the prostate.

Natural killer (NK) cell activity and recycling capacity of NK cells in patients with carcinoma of the prostate (CaP) was determined by combining the 51Cr-release cytotoxicity assay and single cell cytotoxicity assay in agarose. The NK cell activity in patients with advanced CaP (stage D) was significantly lower than the NK cell activity of normal controls and that of patients with localized CaP (stage B and C). The number of active NK cells in the peripheral blood lymphocytes was similar in all groups tested including normal controls, patients with localized CaP, and patients with advanced CaP. Maximal killing potential (Vmax) and maximal recycling capacity (MRC) were significantly reduced in patients with advanced CaP as compared to patients with localized CaP or normal controls. Obtained results indicated that the depressed NK cell activity in advanced CaP was not due to a reduced number of active NK cells but due to the decreased recycling capacity of the individual NK cells.

Adult↗
PubMed2006

Long-term immunologic effects of broad-spectrum aeroallergen immunotherapy.

BACKGROUND: Limited data exist regarding extended, long-term immunologic effects of immunotherapy in polysensitized individuals. To study possible long-term effects, skin tests and specific IgE levels were obtained from subjects who had previously received broad-spectrum aeroallergen immunotherapy years before. METHODS: Eighty-two subjects (78% male, mean age 23 years) previously enrolled in a randomized, placebo-controlled trial of immunotherapy for treatment of childhood allergic asthma were reevaluated in adulthood (mean follow-up interval, 10.8 years) by puncture skin tests and CAP-RAST levels for major aeroallergens. All completed at least 18 months (median 27 months) of maintenance active treatment or placebo injections without subsequent immunotherapy. RESULTS: At adult follow-up, 36% of all skin tests to treatment allergens among subjects who received immunotherapy (n = 41) had significantly reduced intensity versus 26% of skin tests among placebo recipients (n = 41; p = 0.03). No significant differences were noted for individual treatment allergens. No significant differences were observed in the long-term changes of serum-specific IgE antibody levels for all treatment allergens between immunotherapy treatment and placebo groups (p = 0.43). The treatment and placebo groups had a similar acquisition of new skin test sensitivities from time of randomization in the original childhood trial to debriefing (15 vs. 20%; p = 0.28) and to adult follow-up (30 vs. 31%; p = 0.75). CONCLUSIONS: Immunotherapy suppresses skin test sensitivity 8-16 years after discontinuation of treatment, but long-term effects on specific IgE levels in serum are not observed. Broad-spectrum immunotherapy does not appear to affect the acquisition of new inhalant sensitivities.

Adolescent↗
PubMed1998

[Severe community-acquired pneumonia in immunocompetent children].

Severe community-acquired pneumonia (CAP) is still a serious disease with a high mortality rate, especially in developing countries. Children under 5 years are more prone to severe CAP. In this article, the authors review the definition and clinical criteria used in the initial evaluation and decision to hospitalize patients. The diagnosis approach requires only a limited number of laboratory tests before initiating the supportive measures and wide spectrum antimicrobial therapy. If the patient fails to respond favorably 48-72 hours after initiating therapy, more invasive investigations are indicated. After resolution, an immunological evaluation is warranted.

Child↗
PubMed1978

A syndrome associating partial albinism and immunodeficiency.

Two unrelated patients with partial albinism, frequent pyogenic infections and acute episodes of fever, neutropenia and thrombocytopenia are described. Their pigmentary dilution was characterized by large clumps of pigments in the hair shafts and an accumulation of melanosomes in melanocytes. Melanocytes had few short dendritic expansions, and keratinocytes were hypopigmented. No or few Langerhans' cells were detected in skin by electron microscopy and ATP-ase reactions. This pigmentary dilution, different from all other human albinisms, resembles the unique defect of the mutant dilute (d-d) mouse. Despite the presence of an adequate number of T and B lymphocytes, the patients were hypogammaglobulinemic, deficient in antibody production and incapable of manifesting delayed skin hypersensitivity or of rejecting skin grafts. Their leukocytes did not stimulate normal lymphocytes and could not generate cytotoxic cells during mixed leukocyte reaction. T lymphocytes of one patient were unable to exert a helper effect on the maturation of B lymphocytes into immunoglobulin-containing cells following in vitro stimulation with pokeweed mitogen. This suggests that the humoral deficiency might be secondary to a defect of helper T lymphocytes. Granulocytes did not show any morphologic abnormality, and their bactericidal activity was only moderately reduced. An increased number of polymorphonuclear leukocytes with polar distribution of Concanavaline A (Con A) receptors (capping) was found in one patient and her parents. The family histories suggest that this syndrome is transmitted as an autosomal recessive character.

Albinism↗
PubMed1984

A monoclonal antibody detecting a new human T cell antigen, HuLy-m2.

HuLy-m2 is a new T cell antigen detected by a monoclonal antibody. The antibody (anti-HuLy-m2) reacts with 85% of human T cells, 20% B cells, and 50% null cells in peripheral blood. The antibody is cytotoxic, binds protein A, and is of the IgG2a subclass. HuLy-m2 antigen consists of a glycoprotein dimer Mr 37,000 subunit structure, in which sialic acid and carbohydrate play some role in antigenicity. Capping studies showed the HuLy-m2 antigen to be distinct from the previously described OKT-3, 4, 8, HuLy-m1 (OKT11), and HuLy-m3 antigens.

Animals↗
PubMed1992

Comparative evaluation of the pharmacia CAP system and the DPC AlaSTAT system for in vitro detection of allergen-specific IgE with the skin prick test.

Allergen-specific IgE concentrations for 6 inhalant allergens were compared in 54 subjects, using the Pharmacia CAP System and the DPC AlaSTAT System. Similar results were obtained in 91.6% of the tests. In 8% of the tests a positive score was obtained for the Pharmacia CAP System whereas a negative result was found by use of the DPC AlaSTAT System. A negative Pharmacia CAP System result combined with a positive DPC AlaSTAT System result occurred only in 0.4% of the tests. Sensitivity and specificity was established for the Pharmacia CAP System and the DPC AlaSTAT System by comparison with results of the skin prick test. Results obtained with the Pharmacia CAP System were found to be of slightly higher sensitivity than those obtained by DPC AlaSTAT System and of similar or slightly lower specificity.

Allergens↗
PubMed2002

Neopterin levels and dexamethasone suppression test in posttraumatic stress disorder.

Neopterin has recently gained growing importance as an immunological marker in psychiatric disorders. In the present study, we aimed to evaluate whether the dexamethasone suppression test (DST) and neopterin were associated with posttraumatic stress disorder (PTSD). Fourteen patients with PTSD and 14 controls were enrolled in the study. A clinical evaluation and measurements of cortisol and neopterin levels before and after DST were performed. Additionally, all patients were assessed by Clinician Administered PTSD Scale (CAPS). There was a significantly higher DST nonsuppression in the patient group than control group. There were positive correlations between the duration of illness and CAPS, basal cortisol or postdexamethasone cortisol levels in the patient group. The mean neopterin levels for both before and after DST were significantly lower in the patient group than control group. In conclusion, our results suggest that not only the patients with PTSD have considerable DST nonsuppression but also PTSD may be associated with neopterin.

Adolescent↗
PubMed1997

Dynamic distribution of chemoattractant receptors in living cells during chemotaxis and persistent stimulation.

While the localization of chemoattractant receptors on randomly oriented cells has been previously studied by immunohistochemistry, the instantaneous distribution of receptors on living cells undergoing directed migration has not been determined. To do this, we replaced cAR1, the primary cAMP receptor of Dictyostelium, with a cAR1-green fluorescence protein fusion construct. We found that this chimeric protein is functionally indistinguishable from wild-type cAR1. By time-lapse imaging of single cells, we observed that the receptors remained evenly distributed on the cell surface and all of its projections during chemotaxis involving turns and reversals of polarity directed by repositioning of a chemoattractant-filled micropipet. Thus, cell polarization cannot result from a gradient-induced asymmetric distribution of chemoattractant receptors. Some newly extended pseudopods at migration fronts showed a transient drop in fluorescence signals, suggesting that the flow of receptors into these zones may slightly lag behind the protrusion process. Challenge with a uniform increase in chemoattractant, sufficient to cause a dramatic decrease in the affinity of surface binding sites and cell desensitization, also did not significantly alter the distribution profile. Hence, the induced reduction in binding activity and cellular sensitivity cannot be due to receptor relocalization. The chimeric receptors were able to "cap" rapidly during treatment with Con A, suggesting that they are mobile in the plane of the cell membrane. This capping was not influenced by pretreatment with chemoattractant.

Animals↗
PubMed2000

[Recommendations of GESIDA (Grupo de Estudio de SIDA)/National Plan on AIDS with respect to the anti-retroviral treatment in adult patients infected with the human immunodeficiency virus in the year 2000 (II)].

OBJECTIVE: To update the recommendations for antiretroviral therapy (ART) in adult HIV-infected persons according to the new scientific advances and the existence of new antiretroviral drugs in the last two years. METHODS: The ART recommendations have been condensed by a panel of experts from the Spanish AIDS Study Group (Grupo de Estudio de Sida-GESIDA) of the Spanish Infectious Diseases and Clinical Microbiology Society (SEIMC) and from the Clinical Advisory Panel (CAP) of the Secretariat of the Spanish National Plan on AIDS (SPNS) of the Ministry of Health. Three levels of evidence have been established depending if the data came from randomized and controlled studies, from cohort or case-control studies or from descriptive studies and expert opinions, for that purpose we have reviewed the advanced in HIV pathophysiology and results of efficacy (clinical, virologic and immunologic) and security (toxicity) from clinical trials involving ART lasting at least 12 months, from cohort studies and pharmacokinetic and security data of antoiretrovírico drugs, presented in international conferences or published in biomedical journals in the last two years. In each situation we have established either to recommend or to consider or not recommend ART. RESULTS: Nowadays, ART consistent of at least three drugs constitutes the election therapy for chronic HIV infection, since it delays clinical progression, increases significantly the survival and diminishes hospital admissions and associated costs. The decision to start ART must be based upon three elements: presence or absence of symptoms, plasma vírica load and CD4+ cells counts. Thus, in asymptomatic cases with a high CD4+ cells count (> 500/microliter) and low vírica load (< 10,000 copies/ml by branched DNA bDNA or < 20,000 copies/ml by reverse-transcription polymerase chain reaction [RT-PCR] or nucleic acid sequence based amplification [NASBA]) we recommend to delay ART. In symptomatic patients we recommend to start it, and in asymptomatic patients, we could recommend or consider ART initiation depending on the risk of progression, established by the vírica load and the CD4+ cells count. In any case, if therapy is started, the objective must be to reach an indetectable vírica load (< 50 copies/ml). The adherence to ART plays a key role for its initial moment and for the duration of the antiviral response. ART can achieve a restoration of cellular immunity inb the advanced patients. There are few therapeutic options in failing patients due to cross-resistance. Resistance studies can be useful in this setting. The toxicity (lypodistrophy) is a new and limiting factor of ART which requires to look for new therapeutic options. ART criteria for acute infection, pregnancy, post-exposure prophylaxis and when to use resistance testing are discussed. CONCLUSIONS: In this moment, there is a more conservative attitude towards starting ART than in previous recommendations in which a virus eradication was considered. On the other hand, the high number of disposable drugs, the more sensitive monitorization methods (plasma vírica load) and the possibility of performing resistance studies make therapeutic strategies more dynamic and individualized for each patient and situation. In any case, it is mandatory to ensure a perfect adherence to ART from the patients.

Acquired Immunodeficiency Syndrome↗
PubMed1986

The development of a contraceptive vaccine by purification of antigens from gametes.

There are several ways to make immunological contraceptive vaccines. We are trying to purify gamete antigens from human seminal plasma and porcine zona pellucida (ZP) by using several monoclonal antibodies (Mabs) for this purpose. A human seminal antigen relevant to sperm immobilization with corresponding antibody was purified by immunoaffinity chromatography-bound Mab 1C4. This antigen was common to human milk protein but is different from lactoferrin. It was termed "ferrisplan" and was secreted mainly from the epithelial cells of seminal vesicle and adhered to the midpiece and postnuclear cap region of spermatozoa. Another Mab 2C6 reacted only to the SP component but not to other organ extract including human milk protein. The corresponding antigen to Mab 2C6 is aimed to purify by using high performance liquid chromatography and immunoaffinity chromatography-bound Mab 2C6. Several Mabs to porcine ZP antigens common to human ZP did not block human in vitro fertilization unless the second antibody (anti-mouse gamma-globulin) was used. It is speculated that porcine ZP will not contain the antigenic component that relates to sperm receptor of human ZP. Therefore, an antigenic molecule located close to sperm receptor of human ZP must be purified from the porcine ZP by using immunoaffinity chromatography-bound Mab and other biochemical methods, expecting production of a corresponding antibody that blocks the fertilization in humans.

Animals↗