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PubMed2002

Comparison of molecular and immunological typing of isolates of Rice yellow mottle virus.

Isolates of Rice yellow mottle virus (RYMV) were typed at the molecular level through the sequences of the open reading frame (ORF) 4 (coding for the coat protein) and ORF1 (coding for the movement protein), and serologically by means of polyclonal and monoclonal antibodies. The overall patterns of diversity shown by molecular and serological analyses were similar: East-African isolates differed from West-African ones, and the West-African isolates from forest differed from the savannah ones. Each major strain had a different serological profile. However, molecular typing was more discriminating than immunological typing since several sequence variants belonged to the same serotype. In rare instances, there were explainable discrepancies between molecular and serological typing. Two amino acids at positions 115 (alanine vs threonine) and 191 (valine vs threonine) consistently discriminated between the major serotypes. These positions were located in antigenic sites as revealed by Spot-scan method and were recognised by discriminating monoclonal antibodies. One shared epitope, lying within a conserved region, may be responsible for the cross-reactivity between RYMV isolates. A rationale for the correlation between molecular and immunological typing of RYMV and other sobemoviruses is proposed.

Amino Acid Sequence↗
PubMed1993

Tracking of markers and onset of disease among HIV-1 seroconverters.

Repeated measurements on persons infected with HIV-1 indicate that infection has a dynamic impact on several markers of immune suppression and activation. The objectives of this report are: (a) to provide a statistical model for the correlation structure of serial measurements of immunological markers, and (b) to identify features of marker profiles associated with the timing of AIDS diagnoses. We analyse data obtained from 328 seroconverters participating in the Multicenter AIDS Cohort Study on whom the date of HIV-1 seroconversion is known within +/- 4.5 months. Immunological markers considered here are CD4 cell counts, serum beta 2-microglobulin and serum neopterin. The statistical model for HIV-related changes in markers consists of (1) a piecewise linear regression model for the trajectories of markers over time and (2) a two-parameter autocorrelation function that generalizes Markovian and simple random effects autocorrelation structures. Application of this model for marker measurements revealed a high degree of tracking, as the estimated autocorrelation function exhibited sub-exponential decay over time. Though current marker levels are most informative on future values, there is substantial information (memory) in previous measurements. A feature suggested by the analysis of groups formed according to the length of the AIDS-free period, is the sequential divergence of the CD4 trajectories where steeper declines occurred with a two-year lag prior to AIDS onset. For AIDS cases diagnosed 3-5 and 5-7 years after seroconversion, the rates of decline compared with those free of AIDS for at least 4 years were steeper by 95 and 46 per cent respectively at two years prior to AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1999

A noninvasive method for quantifying and distinguishing inflammatory skin reactions.

BACKGROUND: The ribonuclease protection assay (RPA) represents a technology that allows detection of small amounts of intact RNA. Recent progress in understanding cytokine networks in the skin suggests that measurements of cytokine mRNA levels could provide a method to distinguish various reactions such as irritant contact dermatitis and allergic contact dermatitis that can occur in the skin. OBJECTIVE: We attempted to differentiate and quantitate irritant and immunologic skin reactions by measuring mRNA levels. METHODS: We have used the technique of tape stripping human skin to remove superficial cell layers and have extracted RNA from these skin samples. This RNA was used for RPA analysis. RESULTS: By means of RPA analysis, we have demonstrated distinct cytokine profiles that appear to discriminate, for example, irritant from immunologic skin reactions. CONCLUSION: We have shown that multiple cytokine mRNA levels can be defined in these RNA samples obtained from the skin. This approach assesses not only the cytokine gene profiles, but at the same time may quantify the severity of common irritant versus allergic skin reactions.

Cytokines↗
PubMed2004

A global approach to tumor immunology.

Biological and clinical advances in the understanding of tumor immunology suggest that immune responsiveness of human tumors is a complex biological phenomenon that could be best studied by a real-time comparison of tumor/host interactions in the tumor microenvironment through a high-throughput discovery-driven approach. This conclusion is derived from our recognition that too many hypotheses or, in other words, no solid single hypothesis exist, based on experimental results, to further drive experimentation in human subjects. Functional genomic studies entertained during the last few years consolidated the belief that in humans the interactions between tumor and immune cells are too complex to be approached exclusively with a hypothesis driven method. We believe that immune cells suit cancer cells in a Yin and Yang balance by opposing and yet mutually depending on each other. Indeed, immune infiltration in tumors may play a dual role modulating in different circumstances cancer cell growth or destruction through a physiological modulation of inflammation. It is reasonable to question what induces inflammation at the tumor site. We hypothesize that inflammation is primarily driven by the phenotype of tumor cells that can modulate their microenvironment through cell-to-cell interactions or the secretion of soluble factors. Thus, in analogy the observation of immune cells within tumors parallels the presence of paramedics, police and firemen at the scene of an accident, which is reactive to and not causative of the occurrence. In this review we will explore this hypothesis by reporting and summarizing most of our recent work in the frame of available literature on the subject.

Adjuvants, Immunologic↗
PubMedDate not supplied

Second generation antidepressants: a comparative review.

The authors review four "second generation" antidepressants (maprotiline, amoxapine, trazodone, and nomifensine) in terms of action on biogenic amines and receptors, antidepressive efficacy, and adverse effects. Doxepin is used as a comparative agent and is similar to the prototypical tricyclic agents in all the above categories. Maprotiline is a selective noradrenergic agent, but shares a similar adverse effect profile with doxepin and may be associated with a high frequency of seizures in overdose. Amoxapine is a mixed action antidepressant with significant neuroleptic activity in vivo. Its adverse effect profile is highlighted by symptoms related to its neuroleptic activity, and seizures and acute renal failure in overdose. Trazodone is a selective serotonergic agent with low anticholinergic activity, and minimal morbidity/mortality in overdose. Reports of priapism, leading to impotence in some men, however, is of concern. Nomifensine is a potent noradrenergic and dopaminergic agent with low anticholinergic activity, and minimum cardiotoxicity and low morbidity/mortality in overdose. Its most important adverse effects include overstimulation and infrequent, usually reversible, immunologic hypersensitivity reactions. Trazodone and nomifensine have favorable profiles for use in the elderly. Trazodone may be more favorable in the anxious/agitated patient due to its sedative effects, whereas nomifensine may be more beneficial in the retarded, apathetic patient.

Aged↗
PubMed2002

Detection of single molecules: solution-phase single-molecule fluorescence correlation spectroscopy as an ultrasensitive, rapid and reliable system for immunological investigation.

More sensitive techniques in molecular and clinical immunology are essential for the development of reproducible profiles. We have developed a novel methodology named solution-phase single-molecule fluorescence correlation spectroscopy (SPSM-FCS) that fulfils these demands. It is based on the quantification of the probability density of single molecule events in solution is necessary. For example, the Brownian motion of the fluorophore rhodamine-green is detected. Counting about 100,000 photon counts per second and per molecule permits the identification of one single compound. In order to study the applicability of SPSM-FCS in immunology, we have detected and identified a larger nonfluorescent substance in a very complex mixture. The 'unknown' molecules studied were the autoantibodies in serum samples directed against the antigen alpha 3 chain of type IV collagen. In both systems, we were able to characterize the probability density of single fluorescent molecules by means of the averaged absolute molecule number without any calibration. The specific molecules exhibited a Poisson distribution in solution in terms of their 'critical' bulk concentration below about 1 nM. This proof of principle indicates how the SPSM-FCS methodology could be used in immunoassays.

Animals↗
PubMed1998

Biological and immunological properties of nerve growth factor from snake venoms.

Homogeneous preparation of nerve growth factor (NGF) was isolated in purity by two steps HPLC fractionation from venoms of snakes belonging to the major families: Crotalidae, Elapidae, and Viperidae. Biological activity of NGF was tested on PC12 cells for neurite outgrowth and molecular weights were determined by PAGE. Antisera raised against NGFs in Balb/C mice. Immunological cross reactivity for antisera was assayed by ELISA and immunoprecipitin tests. HPLC profiles for the venoms from the species belonging to the same family were identical. The biological and immunological properties of NGFs from different species of snake belonging to the same family were also found to be identical. However, NGFs of venoms from different families of snakes showed differences in properties. Neurite outgrowth on PC12 cells due to NGF from the family Elapidae, especially the cobra species, was greater than NGF from the venoms of Crotalidae and Viperidae, with the exception of N. n. nivea which showed poor activity and C. polystictus of Crotalidae family having very good activity.

Animals↗
PubMed2001

Application of differential display to immunological research.

The majority of immunological processes are mediated by cell-to-cell contact or receptor-ligand interactions that transmit intracellular signals and affect the regulation of transcription in the nucleus. As a consequence, precursor cells develop into their respective lineages and cells differentiate further during an immune response. In order to study changes in normal cells or even cells that have been isolated from diseased tissue, a number of approaches have been developed. One such method, differential display (DDRT-PCR), is a versatile technique for the analysis of gene expression that is based on RT-PCR and denaturing polyacrylamide gel electrophoresis. This technique is applicable to multiple samples of clonal or purified cell populations as well as to complex tissues and can be used to provide mRNA fingerprints. However, the main purpose of DDRT-PCR is to isolate differentially regulated genes in biological systems. The method is carried out without prior hypothesis as to which genes should be examined and so increases the possibility of identifying completely novel and unexpected changes in transcription. A major drawback has been the isolation of false positive clones and the need to confirm the results of analysis by another method. This makes DDRT-PCR labour intensive. A number of strategies have been recommended to reduce these problems, including reverse-northern analysis as a confirmatory step for screening putative differentials. In order to reduce the number of gel fingerprints that would be required to cover all the mRNAs in a cell, several focused approaches have been suggested. These include targeted differential display for the isolation of multigene families that have conserved protein domains or gene signatures and subtractive differential display whereby one population is subtracted from the other prior to screening. The purpose of this review is to provide some guidance to the immunologist who might wish to apply DDRT-PCR in their research. A number of examples where DDRT-PCR has been used successfully in immunological research are included.

Animals↗
PubMed1983

Foreign body granulomatosis. Clinical and immunologic findings.

We studied the clinical and immunologic features of 10 patients with foreign body granulomatosis associated with injection of pentazocine. Five had typical micronodules on chest radiographs, 9 had abnormal 67Ga lung images, and 9 had elevated serum angiotensin-converting enzyme levels. Pulmonary function tests showed significant decreases in diffusing capacity in 8 subjects, but were otherwise normal. Bronchoalveolar lavage fluid had significantly (p less than 0.02) increased total cell numbers (13.8 +/- 7.5 x 10(7) and significantly lower percentages of lymphocytes (2 +/- 1) and neutrophils (2 +/- 1) (p less than 0.02) when compared with that from control subjects. Peripheral blood lymphocytes were normal when expressed as percentages of total leukocytes or as percent T-lymphocytes. Monoclonal antibody determinations of T-lymphocyte subsets (helper and suppressor) were similar to those in control subjects (p = NS). Lymphocyte transformation responses to a variety of antigens and skin test reactivity were normal. Foreign body granulomatosis contrasts with other interstitial lung diseases in its bronchoalveolar lavage cellular profile and in the absence of altered peripheral immunologic indexes.

Adult↗
PubMed2006

Extrinsic allergic alveolitis: comparative study of the bronchoalveolar lavage profiles and radiological presentation.

INTRODUCTION: Extrinsic allergic alveolitis (EAA) is an immunologically mediated interstitial lung disease. The abnormalities in the bronchoalveolar lavage (BAL) fluid cell counts are almost always seen in patients with EAA according to the stage of the disease. The aim of this retrospective study was to find out how the BAL lymphocyte count, percentage of lymphocytes expressing HLA-DR, CD4/CD8 T cell ratio in BAL fluid, and the concentration of immunoglobulin G in serum correspond to the inflammatory activity of the disease. METHODS: The study included 14 patients with EAA. BAL fluid samples were obtained and processed for cytological and cytometric analysis. Immunoglobulin G serum concentrations were measured. High resolution computed tomography (HRCT) scoring system modified by Gay was used for establishing the alveolar and interstitial score in each patient. CONCLUSIONS: It was found that subjects with normal value of CD4/CD8 ratio in BAL fluid had higher interstitial HRCT score. Clinical presentation, continuous exposure to the causative antigens, and BAL lymphocyte count positively correlated with the alveolar HRCT score. It is proposed that the increased BAL lymphocyte count could be the predictor of the inflammatory activity of the disease, especially in people with lasting exposure to the offending antigen.

Acute Disease↗
PubMed1990

[The immunological cellular phenotype and the prognosis in neuroblastoma in children].

Ganglioside profile was evaluated in 19 samples of tumor tissue obtained from 13 surgical patients with various morphological patterns of neuroblastoma. In six of those cases, two samples from each proving most different in terms of cell maturity were selected for examination. The relative content of GD2 gangliosides was 27.0-37.6% in sympathoblastoma and as low as 5.1-14.8% in ganglioneuroblastoma. Ganglioneuroblastomas showed fairly high levels of GM1 and GD1a gangliosides which were almost completely absent in sympathoblastomas. Ganglioside profile variations seen within each tumor type were incomparable with differences in profile established between morphological patterns of neuroblastoma studied.

Biomarkers, Tumor↗
PubMed1986

A diazepam binding inhibitor (DBI)-like neuropeptide is detected in human brain.

Diazepam binding inhibitor (DBI), a 11,000 MW neuropeptide, which coexists with GABA and elicits proconflict responses in the rat, has been purified and partially sequenced from rat brain. We now report purification and characterization of a DBI-like neuropeptide from human brain. Its molecular weight and pharmacological profile is identical to that of rat DBI but differs in the amino acid composition and immunologically. The tryptic fragments of human DBI differ from rat DBI in the HPLC elution profile and in the amino acid sequence. Using high affinity specific human DBI antibodies, the distribution of DBI-like immunoreactivity in bioptic samples of human brain appeared to be similar to that of DBI found in rat brain. DBI-like immunoreactivity was also found in spinal fluid of human volunteers. The cerebrospinal fluid content of this peptide might be used as a probe to study whether spinal fluid DBI content changes in neuropsychiatric disorders.

Amino Acid Sequence↗
PubMed2005

Identification of high and low responders to lipopolysaccharide in normal subjects: an unbiased approach to identify modulators of innate immunity.

LPS stimulates a vigorous inflammatory response from circulating leukocytes that varies greatly from individual to individual. The goal of this study was to use an unbiased approach to identify differences in gene expression that may account for the high degree of interindividual variability in inflammatory responses to LPS in the normal human population. We measured LPS-induced cytokine production ex vivo in whole blood from 102 healthy human subjects and identified individuals who consistently showed either very high or very low responses to LPS (denoted lps(high) and lps(low), respectively). Comparison of gene expression profiles between the lps(high) and lps(low) individuals revealed 80 genes that were differentially expressed in the presence of LPS and 21 genes that were differentially expressed in the absence of LPS (p < 0.005, ANOVA). Expression of a subset of these genes was confirmed using real-time RT-PCR. Functional relevance for one gene confirmed to be expressed at a higher level in lps(high), adipophilin, was inferred when reduction in adipophilin mRNA by small interfering RNA in the human monocyte-like cell line THP-1 resulted in a modest but significant reduction in LPS-induced MCP-1 mRNA expression. These data illustrate a novel approach to the identification of factors that determine interindividual variability in innate immune inflammatory responses and identify adipophilin as a novel potential regulator of LPS-induced MCP-1 production in human monocytes.

Adolescent↗
PubMed2005

Isolation and preliminary characterization of a novel Helicobacter species from swine.

OBJECTIVE: To determine whether a Helicobacter sp similar to Helicobacter pylori in the stomachs of humans could be isolated from the stomachs of pigs. ANIMALS: 4 young conventionally reared and 21 gnotobiotic pigs. PROCEDURE: Gastric mucosal homogenates (10% wt/vol) from 4 young conventionally reared pigs were cultured on Skirrow medium under microaerophilic conditions to assess the presence of Helicobacter spp. Colonies with morphologic features compatible with Helicobacter organisms were selected, tested for urease activity, and subpassaged on Skirrow medium. Isolates were examined via SDS-PAGE electrophoresis and reciprocal western blot analyses involving convalescent sera from monoinfected gnotobiotic pigs. RESULTS: Urease- and catalase-positive, gram-negative, microaerophilic, small, curved rod bacteria were isolated from the gastric mucosa of young healthy pigs. The first isolate (2662) was structurally and immunologically closely related to H pylori isolated from humans. The second isolate (1268) displayed an SDS-PAGE profile dissimilar to that of H pylori and isolate 2662, yet it shared limited immunologic cross-reactivity with these microbes. CONCLUSIONS AND CLINICAL RELEVANCE: Findings of this study indicate that development of gastric mucosal ulcers and ulceration of the nonglandular pars esophagea in pigs may be associated with gastric colonization by swine-origin Helicobacter spp, which are similar to H pylori isolated from humans.

Animals↗
PubMed2006

Expression profile and clonality of T-cell receptor beta variable genes in normal human endometrium.

PROBLEM: In spite of their key immunological role, alphabeta+ T cells residing in endometrium have not been extensively explored. We analyzed here expression profile of TCRBV genes in normal human endometrium compared with peripheral blood. METHODS: Samples were taken from normal reproductive women. RT-PCR using BV-gene specific primers was performed on blood and endometrial samples. After blotting, hybridization with radiolabelled probe and autoradiography, relative expression of each TCRBV family was determined. Clonal expansions of the over-expressed genes were assessed by CDR3 length polymorphism. RESULTS: Only one gene (TCRBV7) was expressed significantly and two other genes marginally more in the endometrium compared with blood. All three TCRBV genes examined showed a rather restricted pattern in the endometrium in contrast to polyclonal patterns in the blood. CONCLUSION: Our results stress the similarities between T cells residing in different mucosal tissues and provide a basis for future investigations about endometrial T cells and their antigen specificities in gynecological problems.

Adult↗
PubMed2006

CD4+ T cells and the Th1/Th2 imbalance are implicated in the pathogenesis of Graves' ophthalmopathy.

Graves' ophthalmopathy (GO) is considered to be an organ-specific autoimmune disease. However, the pathogenesis of GO is incompletely understood at the present time. To clarify the immunological differences between newly diagnosed GO and Graves' disease (GD) without ophthalmopathy or healthy controls (HC), we examined T-cell profile and the Th1/Th2 profile cell balance in GO (n=20), GD (n=20) and HC (n=20) using flow cytometry. We also assessed the influence of methimazole on the immunocyte profiles in patients with GO and GD and analyzed the relationship of the immunologic changes with CAS, FT3, FT4, TRAb, TMA and TGA among the three investigated groups. We report in this study that: 1) The percentage of CD4+ T cells and the ratio of CD4+/CD8+ cells were higher, but the population of CD8+ T cells was lower in both GO and GD than those of HC (P<0.05); 2) The percentage of CD8-/IFNgamma+ T cells (Th1) and the ratio of CD8-/IFNgamma+ to CD8-/IL-4+ T cells (Th1/Th2) in GO were considerably higher as compared to those in GD and HC (P<0.05). On the contrary, the population of Th1 cells, as well as the ratio of Th1/Th2 cells, was lower in GD than that of GO and HC (P<0.05); 3) There were no significant differences in T-cell profile and the Th1/Th2 cell balance in either GO or GD patients before and after methimazole treatment; 4) There was a positive correlation of Th1 cell percentage and the Th1/Th2 cell ratio with the clinical activity score (CAS) in GO (P<0.05), whereas CAS in GO had no correlation with the T-cell profile, the percentage of Th2 cells, and TRAb (P>0.05); 5) T-cell subset and the ratio of Th1/Th2 cells did not correlate significantly with FT3, FT4, TRAb, TMA, or TGA in GO and GD (P>0.05). Finally, 6) there were no statistical differences in TRAb, TMA, and TGA between early GO and GD without ophthalmopathy (P>0.05). Collectively, these results indicate that the balance of Th1/Th2 in GO shifts to Th1 dominance and that the cellular immune responses mediated by the Th1-type CD4+ cells might play a dominant role in the pathogenesis of GO, and thus suggest that the Th1 cell percentage and the ratio of Th1/Th2 cell subsets may be potentially utilized as clinical parameters for disease activity, for monitoring the effectiveness of immunosuppressive treatment, or for developing immunospecific forms of therapy for Graves' ophthalmopathy.

Adolescent↗
PubMed2004

Gene expression profile of B cells from Xid mice and Btk knockout mice.

Bruton's tyrosine kinase (Btk) is important for B lymphocyte development. To identify genes that are differentially expressed in primary B cells lacking functional Btk, splenocytes from X-linked immunodeficiency (Xid), Btk knockout (Btk KO) and immunocompetent CBA mice were used in microarrays containing more than 12000 genes and expressed-sequence tags. We found 4515 common transcripts expressed in duplicate experiments in the three strains. Out of these, 38 were differentially expressed genes (21 were up-regulated >2-fold and 17 were down-regulated <-2-fold) between CBA and Btk defective (Xid or Btk KO) mice. Ten out of these genes were selected and quantitative real-time PCR was conducted for validation and further investigation. Real-time experiments correlated nicely with the microarray data. No definitive phenotypic difference has previously been reported between Xid and Btk KO mice. We found 7 genes whose expression differed (>2-fold) between the two strains. Moreover, when the 38 genes that differed between immunocompetent CBA mice and Btk defective mice were ranked according to fold-increase, the levels in Btk KO mice were significantly more altered. This suggests that the defect in Btk KO mice is more severe and demonstrates that the mutant Btk protein in Xid mice does not generally function as dominant-negative form.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1977

Fractionation of primary amyloid fibrils. Characterization and chemical interaction of the subunits.

Amyloid fibrils of kappa origin from a patient with primary amyloidosis are dissociated in various denaturants and fractionated into their subunit components on Sepharose 6B. Solubilization of the fibrils in 4 M guanidine-HCl followed by reduction and alkylation produced 22 000 and 17 000 dalton fractions. Without prior reduction and alkylation, these fractions exist as a high molecular weight protein which can be separated on Sepharose 6B. A high molecular weight protein can be directly dissociated from the amyloid fibril with 1% sodium dodecyl sulfate or 1 M NaCl. Reduction and alkylation of this material produces the two lower molecular weight fractions, i.e., 22 000 and 17 000. These have in the first 20 residues identical N-terminal amino acid sequences; they share immunologic identity and have similar tryptic peptide map profiles. Amino acid analysis of the 22 000 dalton fraction is identical with the intact immunoglobulin light chain isolated from the patient's serum. These data suggest that the insoluble amyloid fibril is the result of aggregation by disulfide linkages between the 22 000 and 17 000 dalton fractions.

Amino Acid Sequence↗