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PubMed1989

Molecular characterization and protein analysis of the cap region, which is essential for encapsulation in Bacillus anthracis.

By using genetic complementation tests with various in vitro-constructed mutants with mutations in the cap region (which is essential for encapsulation in Bacillus anthracis), we identified three cistrons, capB, capC, and capA, in this order of arrangement. Minicell analysis revealed that these cistrons produce proteins of 44, 16, and 46 kilodaltons, respectively. The complete nucleotide sequence of 3,244 base pairs covering the whole cap region was determined and revealed the existence of the three open reading frames of capB (397 amino acid residues; molecular weight, 44,872), capC (149 amino acid residues; molecular weight, 16,522), and capA (411 amino acid residues; molecular weight, 46,420) arranged in the order predicted by complementation tests. These three cistrons were all transcribed in the same direction from promoters unique to each cistron. Judging from the predicted amino acid sequence of the three proteins and from their localization and their sensitivity to various physicochemical treatments, they appeared to be membrane-associated enzymes mediating the polymerization of D-glutamic acid via the membrane. Capsular peptides immunologically identical to that of B. anthracis were found in B. subtilis, B. megaterium, and B. licheniformis, but no sequence homologous to the cap region was found in any of these bacilli other than B. anthracis. Using strains of B. anthracis with or without insertional inactivation of the cap region, we found that the capsule of B. anthracis conferred strong resistance to phagocytosis upon the bacterial host.

Amino Acid Sequence↗
PubMed1986

Synthesis of epidermal growth factor (EGF) receptor in vitro using SP6 RNA polymerase-transcribed template mRNA.

The epidermal growth factor (EGF) receptor plays a key role in the control cellular proliferation, and its homology to the avian erythroblastosis virus erb B oncogene implicates its involvement in cellular transformation. The establishment of a correlation between the various structural domains of the EGF receptor and their functional counterparts would greatly advance our understanding of these processes. To this end, we have constructed an expression vector containing the SP6 viral promoter and an adjacent cDNA fragment encoding the full-length EGF receptor. Upon addition of SP6 RNA polymerase, this DNA is capable of generating large amounts of EGF receptor mRNA; this RNA can then be translated in vitro into immunoprecipitable EGF receptor protein. The translational efficiency of this EGF receptor RNA was found to be relatively low: approx. 100-fold lower than globin RNA synthesized using SP6 RNA polymerase. Use of these tools should now permit the synthesis and analysis of mutated EGF receptor protein in an effort to clarify the role of this receptor in growth control.

Cell-Free System↗
PubMed2006

5'-Triphosphate RNA is the ligand for RIG-I.

The structural basis for the distinction of viral RNA from abundant self RNA in the cytoplasm of virally infected cells is largely unknown. We demonstrated that the 5'-triphosphate end of RNA generated by viral polymerases is responsible for retinoic acid-inducible protein I (RIG-I)-mediated detection of RNA molecules. Detection of 5'-triphosphate RNA is abrogated by capping of the 5'-triphosphate end or by nucleoside modification of RNA, both occurring during posttranscriptional RNA processing in eukaryotes. Genomic RNA prepared from a negative-strand RNA virus and RNA prepared from virus-infected cells (but not from noninfected cells) triggered a potent interferon-alpha response in a phosphatase-sensitive manner. 5'-triphosphate RNA directly binds to RIG-I. Thus, uncapped 5'-triphosphate RNA (now termed 3pRNA) present in viruses known to be recognized by RIG-I, but absent in viruses known to be detected by MDA-5 such as the picornaviruses, serves as the molecular signature for the detection of viral infection by RIG-I.

Animals↗
PubMed1983

Mathematical modeling in immunology.

The first step in the sequence of events leading to antibody synthesis and secretion is antigen encounter by B cell receptors. The reaction leads to major membrane perturbations, including the capping and endocytosis of receptors. These events might play an important role in various aspects of the B cell response, but they are not well understood, and in any event it seems unlikely that a satisfactory explanation of response regulation to most antigens can be advanced in terms of these phenomena alone, without explicit consideration of other cell populations. Suppressor and helper T cell populations qualitatively and quantitatively regulate the response to almost all antigens. Knowledge of the antigen dose dependence of the response of these populations will undoubtedly be important for a complete understanding of major immune response phenomenology such as suppression and affinity regulation. T cell dose response patterns are currently unavailable, nor is much known of the molecular mechanisms mediating interactions among antigen responsive T cells. In order to explain why this information is important, and to connect disparate data on immunoregulation in a simple way, I introduced dose response patterns based on analogy with basophils, assumed cell interactions mediated by autoantiidiotypic antibodies, and briefly pursued the consequences. In the final section, the genetic experiments that are providing information on the molecular constraints on cell interactions were mentioned, and some of the outstanding theoretical questions were briefly discussed.

Animals↗
PubMed2003

Serum human glandular kallikrein (hK2) and insulin-like growth factor 1 (IGF-1) improve the discrimination between prostate cancer and benign prostatic hyperplasia in combination with total and %free PSA.

BACKGROUND: There is growing evidence describing an association of hK2 and IGFs with cancer. The aim of this study is to investigate the differences in serum levels of hK2 and IGFs in a large group of patients with benign prostatic hyperplasia (BPH) or prostatic carcinoma (CaP) and to examine the value of these variables, as well as their various combinations with PSA, for discriminating between these two clinical entities. METHODS: Human glandular kallikrein 2 (hK2), insulin-like growth factor-1 (IGF-1), free and total PSA concentrations were measured with non-competitive immunological procedures. Receiver operating characteristic (ROC) analysis as well as univariate and multivariate logistic regression analysis were performed to investigate the potential utility of the various markers and their combinations for discriminating between BPH and CaP. RESULTS: hK2 and IGF-1 concentrations were increased in CaP patients, in comparison to BPH patients. hK2/free PSA and free/total PSA ratios (area under the curve, AUC = 0.70) were stronger predictors of prostate cancer than the IGF-1/total PSA ratio (AUC = 0.56) in the group of patients with total PSA <4 microg/L. The hK2/free PSA ratio (AUC = 0.74) was found to have significant discriminatory value in patients with total PSA within the "gray zone" (4-10 microg/L). Multivariate logistic regression models confirmed the observed relationships and identified IGF-1/free PSA and hK2/free PSA as independent predictors of CaP. CONCLUSIONS: hK2/free PSA and IGF-1/free PSA ratios may be useful adjuncts in improving patient selection for prostate biopsy.

Aged↗
PubMed1986

Normal human serum restores the expression of Fc gamma receptors in immune complex-blocked human mononuclear cells.

A progressive inhibition of both antibody-dependent cellular cytotoxicity (ADCC) and Fc gamma R expression was observed when peripheral blood mononuclear cells (PBMC) were incubated with soluble immune complexes (IC) at 37 degrees. Treatment of these IC-blocked PBMC with normal human serum (NHS) reversed both effects and increased the release of attached IC from the cell surface when compared to heat-inactivated serum (HI-NHS) or culture medium (TCM) treatments. Immunofluorescence studies demonstrated a redistribution of IC, predominantly in the form of caps in IC-blocked cells treated with HI-NHS (56%) or TCM (51%). However, when these IC-blocked cells were treated with NHS, only 11% of them showed caps redistribution. We propose that NHS regulation of Fc gamma R expression may be a physiological way of modulating the different immunological events triggered by IC.

Animals↗
PubMed1999

Chemokine RANTES in atopic dermatitis.

Chemokines play a key role in inflammatory diseases. The aim of this study was to estimate chemokine RANTES in the sera of patients with atopic dermatitis (AD) and to analyze the correlation between RANTES serum level and the immunological and clinical parameters of the disease. Serum levels of RANTES (ELISA; R&D Systems), total IgE and specific IgE (FEIA; Pharmacia CAP System) were estimated in 24 patients with AD, 28 patients with pollinosis (PL) and 22 healthy nonatopic subjects (HC). The division of the AD group into a pure AD (pAD) subgroup, without a coexisting respiratory allergy, and a subgroup of patients with AD and a respiratory allergy (AD+AO) was done according to Wütrich. Levels of RANTES were higher in the AD group than in the HC group and the PL group. RANTES levels did not differ among subgroups with various clinical scores and between the pAD and AD+AO subgroups. There were no correlations between levels of RANTES and total IgE. Significant positive correlations between serum levels of RANTES and Dermatophagoides farinae and cat dander-specific IgE were found in the AD group. We conclude that the serum level of chemokine RANTES differs patients with AD from patients with PL. The increase of RANTES concentration in the serum of patients with AD depends neither on a clinical picture nor an IgE system.

Adolescent↗
PubMed2000

Analysis of free prostate-specific antigen (PSA) after chemical release from the complex with alpha(1)-antichymotrypsin (PSA-ACT).

BACKGROUND: Prostate-specific antigen (PSA), a marker for prostate cancer (CaP), forms a covalent complex with alpha(1)-antichymotrypsin (ACT) in human blood. Structural analysis of the PSA-ACT complex is difficult, and complexation may be a reason for biased immunological assays when compared with the analysis of free PSA. We developed a method to cleave the PSA-ACT complex chemically. The liberated PSA was thus available for analysis as free PSA (F-PSA). METHODS: PSA was released from the PSA-ACT complex by cleaving the interprotein ester bond with ethanolamine under alkaline conditions. The release was followed by reversed-phase HPLC and an immunoassay for F-PSA. Released PSA obtained from human blood was further immunopurified and analyzed by matrix-assisted laser desorption-induced time of flight (MALDI-TOF) mass spectrometry. RESULTS: In vitro-prepared PSA-ACT complex was completely cleaved by treatment with nucleophilic compounds such as ethanolamine at pH 9-10. The released PSA was stable under these conditions and could be measured by reversed-phase HPLC as well as the ENZYMUN immunoassay for F-PSA. When plasma from a CaP patient [containing 190 microg/L F-PSA and 1890 microg/L total PSA (T-PSA)] was treated under similar conditions, a concentration of approximately 1600 microg/L F-PSA was measured at the end of the incubation, indicating that the PSA-ACT complex was completely cleaved. Two benign prostatic hyperplasia and CaP sera panels (12 and 13 sera, respectively) containing 4-45 microg/L T-PSA were similarly treated. The concentrations of F-PSA measured after incubation were, on average, 85% of the T-PSA values of the untreated sera. Finally, the PSA released from the complex of the CaP plasma was isolated by immunosorption, analyzed by MALDI-TOF mass spectrometry, and compared to PSA obtained from semen. The intact PSA as well as the peptides observed after digestion with endoproteinase Lys C did not reveal any structural difference between the PSA from these two sources. CONCLUSIONS: PSA complexed to ACT in plasma of a CaP patient seems to be structurally very similar to the PSA reference material from semen. The release of PSA from the PSA-ACT complex allows F-PSA and T-PSA to be measured by the same immunological assay, thus eliminating any possible bias between two different assays.

Biomarkers, Tumor↗
PubMed1989

Ca2+-independent F-actin capping proteins. Cap 32/34, a capping protein from Dictyostelium discoideum, does not share sequence homologies with known actin-binding proteins.

Polyclonal and monoclonal antibodies against purified 32/34-kDa F-actin-capping protein from Dictyostelium discoideum were generated and used to isolate clones coding for both subunits from a lambda gt11 expression library. In addition genomic clones were isolated for the 34-kDa subunit. The sequences of the corresponding inserts were determined and the amino acid sequences of the proteins deduced. The amino acid sequences have been confirmed by sequencing tryptic peptides of both subunits. As judged from immunological data and sequence comparison, the subunits are completely different. Each of them is encoded by a single copy gene. The mRNAs are present throughout all stages of development. Severing proteins, which exert also capping activity, show no sequence similarity with the cap 32/34-kDa protein. However, a pronounced homology between the 32-kDa subunit and a hypothetical yeast protein was found indicating the presence of a protein homologous to the 32-kDa subunit of the D. discoideum capping protein in yeast.

Actin Depolymerizing Factors↗
PubMed2004

Observations on the proposed relationship between infection burden and early malignancy in developing countries (e.g., India).

Sastry and Parikh [Med. Hypotheses 60(4) (2003) 573] have recently sought an explanation for the fact that the occurrence of a particular cancer in populations in a developing country such as India takes place at a younger age (about one decade) than in populations in Western countries. They have hypothesized that a higher infectious burden in India gives rise to repeated cell divisions leading to early senescence of immune cells, and, thence their reduced ability for immune surveillance against cancer, resulting in earlier onset of cancer. The analysis presented here points out to some difficulties with this interpretation, both on empirical and theoretical grounds. The reduced surveillance ability, caused by higher infectious burden, of the immune cells postulated by Sastry and Parikh [loc. cit.] would also mean that populations in India should suffer higher incidence of cancer, as compared to people in Western countries; the empirical data show that, in fact, quite the opposite is true - in the present communication shows that for many common cancers, typical cities in India show the lowest incidence. Theoretically, it is postulated here that repeated heavy infections in India, in fact, challenge the immune system, particularly the adaptive immune system and create an immunological memory: this trains and strengthens the immune system against the future battles. Also it is shown that the shortening of the telomeric cap by repeated cell divisions caused by heavy infectious attacks, as argued by Sastry and Parikh [loc. cit.], is not the cause of earlier onset of cancers among Indians; in fact, when telomeric caps become shortened to a critical point, a danger signal is generated arresting the cell cycle - thus, it provides a fundamental mechanism for ordering the cell to cease proliferation. It is suggested that the root of occurrence of cancers at an earlier age in India perhaps lies in the accumulation of mutations at an earlier age among Indians who do develop cancers; the factors responsible for these accelerated mutations are not clear at the present time and need further investigation.

Age Distribution↗
PubMed2002

Reactivity to potential cross-reactive foods in fruit-allergic patients: implications for prescribing food avoidance.

BACKGROUND: Prescribing therapeutic elimination diets in patients with fruit allergy should include recommendations on which other foods of the same family or group may be safely consumed. Evidence-based data on the management of fruit allergy are lacking; therefore, advice may vary from just avoiding the offending fruit, to overly restrictive diets of the entire botanical family. The aims of this investigation were to assess clinical reactivity to potential cross-reactive foods in fruit-allergic patients, and the implications for prescribing specific therapeutic elimination diets. METHODS: Sixty-five adults diagnosed with clinical allergy to one or more fruits were evaluated for IgE-mediated allergy to other related foods, which might share cross-reactive antigens. Those with actual allergy to some Rosaceae fruit (including peach, apple, apricot, plum, and almond) underwent skin prick testing (SPT), food-specific IgE assessment by the Pharmacia CAP-FEIA system, and oral challenges with the entire group. For those with allergy to other fruits (chestnut, melon, banana, kiwi, or avocado) immunologic and clinical reactivity to all five were evaluated. Since a number of people in the study also had proven clinically allergy to some nut or legume (peanut, sunflower seed, walnut, pistachio, or hazelnut), the reactivity to the entire set was investigated. RESULTS: Thirty-four of those tested (52%) were found to be clinically allergic to more than one fruit, so 125 allergic reactions occurred in the 65 patients. Peach, melon, kiwi, apple, and banana accounted for 72% of allergic reactions. Forty-two (65%) had sensitization to pollens, causing seasonal allergic rhinitis and/or asthma, and 18 (28%) were sensitized to latex. The 65 with IgE-mediated fruit allergy underwent 351 additional SPT and food-specific IgE determinations with potential cross-reactive foods considered in this study; 223 (64%) of these results were positive. The routine challenges with potential cross-reactive foods uncovered 18 further reactions in 14 (22%) out of 65 (to avocado in seven; apricot in three; plum in one; almond and peanut in one; banana and hazelnut in one; avocado, banana and kiwi in one). Only 8% (18/223) of positive results for SPT, or food-specific IgE levels of greater than 0.35 kU(A)/l to potential cross-reactive foods investigated in this study, were clinically relevant. CONCLUSION: Elimination diets that rely on total group avoidance, or only on the results of allergy testing, might have resulted in unnecessary restriction of 205 foods in the 65 people studied. The lack of routine oral challenges with related fruits--before prescribing dietary restriction--may have allowed 18 food reactions in one-fifth (14/65) of patients to be overlooked.

Adolescent↗
PubMed1981

Thoracic duct cannulation via a transjugular vascular endoscope (lymphoscope) in the dog.

We describe the feasibility of transjugular endovascular visualization and cannulation of the canine cervical thoracic duct using a flexible cholangioscope modified for lymphoscopy. The lymphoscope is composed of a prismatic side-view optic with a catheter guide channel attached by a metal cap to the terminal end. The technique has clinical applicability to the study of neoplastic diseases, examination and depletion of lymphocyte subpopulations in immunologic disorders, and perhaps the overcoming of increased resistance to lymph flow at the cervical lympho-venous junction in edematous conditions characterized by excessive lymph formation.

Animals↗
PubMed1995

Purification, partial characterization and immunolocalization of a proteophosphoglycan secreted by Leishmania mexicana amastigotes.

The intracellular amastigote form of the parasitic protozoon Leishmania mexicana expresses a high-molecular weight phosphoglycan, which is antigenically related to the surface glycolipid lipophosphoglycan and the secreted enzyme acid phosphatase of Leishmania promastigotes. This antigen was purified from a cell-free homogenate of infected mouse tissue and from amastigotes. Compositional and immunological analysis of the purified components indicate a proteophosphoglycan structure consisting of serine-rich polypeptide chains and mild acid-labile phosphooligosaccharides capped by mannooligosaccharides. Immunofluorescence and immunoelectron microscopy of parasitized mouse peritoneal macrophages and infected mouse tissue suggest that the proteophosphoglycan is secreted in large amounts by amastigotes via their flagellar pockets into the parasitophorous vacuoles of host cells. In some infected macrophages proteophosphoglycan is also located in vesicles apparently originating from the parasitophorous vacuole, which demonstrates redistribution of a secreted amastigote antigen in parasitized host cells.

Animals↗
PubMed1979

H-2L: demonstration of four new allelic products and independence of H-2D and H-2L molecules.

The H-2L molecules were detected for the first time in the Dd region products using antisera against the public specificity H-2.28. This specificity was analyzed because its presence in K as well as in D region products and its apparent allelism with another public specificity, H-2.1, indicated that these two specificities may have a special position may have a special position in the H-2 system. This was corroborated by subsequent identification of H-2L molecules in Dq and Dk products using anti-H-2.28 AND ANTI-H-2.1 sera, respectively, while none of the other previously known public or private specificities was detected on H-2L molecules. We tested the products of four D region alleles which had not been analyzed previously. In each of them we identified two distinct types of molecules: H-2D, which reacts with sera agains the D region private specificity, and H-2L, which does not react with these sera, but which is detectable either by anti-H-2.28 sera (H-2Lb, H-2Lf, H-2LS) or by anti-H-2.1 sera (H-2Ldx). This increases the number of identified H-2L alleles to seven (five H-2.8+, two H-2.1+). No association between H-2D and H-2D and H-2L molecules on the cell surface was detected in capping experiments.

Alleles↗
PubMed2004

T-cell activation is accompanied by an ubiquitination process occurring at the immunological synapse.

The immunological synapse (IS) is a specialized signaling area formed at the contact site between T-cells and antigen-presenting cells (APC), where sustained engagement and signaling of TCR and accessory molecules occur. A key feature of T-cell antigen recognition is that the process of TCR/peptide-MHC interaction is self-limited by the internalization and degradation of triggered TCR and recruited signaling components. The mechanism of signaling component degradation involves their ubiquitination and targeting for degradation. Yet, the relationship between the ubiquitination process and TCR signaling as well as the cellular localization of TCR-induced ubiquitination are still elusive. In the present work, we visualize for the first time ubiquitination at the TCR signaling area. We show an enrichment of ubiquitin staining in TCR/CD3 caps in T-lymphocytes stimulated by anti-CD3 antibodies. Remarkably, we also show the recruitment of the ubiquitin ligase Cbl-b and a significant ubiquitination at the immunological synapse in antigen-stimulated T-cells. Our results identify the immunological synapse as the cellular area where TCR-induced protein ubiquitination occurs. They imply that the synapse is a specialized site where the activation process is not only triggered, but also controlled via ubiquitination of signaling actors.

Adaptor Proteins, Signal Transducing↗
PubMed2000

A case of Haemophilus parainfluenzae pneumonia.

A 41 year old woman presented with community acquired pneumonia (CAP) which failed to resolve following treatment with amoxycillin and cefaclor prior to referral. Quantitative culture of sputum revealed a pure growth of Haemophilus parainfluenzae and, following antibiotic susceptibility testing of the isolate, ciprofloxacin was prescribed resulting in resolution of the infection. Immunological investigations showed that the patient had a high titre of H parainfluenzae specific IgM. The combination of a pure growth of H parainfluenzae, a response to appropriate antimicrobial therapy, and the presence of a specific antibody response indicated that this organism had a pathogenic role in the patient's pneumonia and should be considered in the differential diagnosis of CAP.

Adult↗
PubMed1986

The histopathology of experimental trachoma: ultrastructural changes in the conjunctival epithelium.

Experimental acute conjunctivitis was produced in cynomolgus monkeys by ocular inoculation with serovar B of Chlamydia trachomatis. The cellular responses to chlamydial conjunctivitis infection were examined by light, transmission, and scanning electron microscopy. A self-limited, acute conjunctivitis resulted from a single primary inoculation. A moderate lymphocytic infiltrate was present in the conjunctiva. After repeated inoculation, a chronic conjunctivitis (trachoma) developed. Prominent lymphoid follicles with distinct germinal centers were present in these tissues. Scanning electron microscopy revealed patchy areas of cellular alteration and loss of microvilli. Intracellular injury, documented by transmission electron microscopy, included disruption of the plasmalemmal membranes and rupture of the cytoplasmic organelles. The inflammatory infiltrate consisted of plasma cells, polymorphonuclear cells, lymphocytes, eosinophils, and degranulating mast cells. The immune response following single and repeated chlamydial infections was characterized by immunoperoxidase staining with monoclonal antibodies to pan-leukocytes, macrophages, and B cells. The center of the follicle was comprised of B cells, with T cells in the cap region. Large macrophages were also found in the germinal center. Further study is required to determine which cellular mechanisms are involved in the histopathologic and immunologic alterations induced in the conjunctiva after chlamydial infection.

Animals↗