Limited diversity of gamma delta T-cell antigen receptor junctional region sequences in individuals with sarcoidosis compared to broad diversity in normal subjects.
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Amino Acid Sequence↗
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Uniform processing and analysis of IGVF massively parallel reporter assay data with MPRAsnakeflow.
As researchers and clinicians seek to identify human genomic alterations relevant to traits and disorders, identifying and aggregating evidence providing mechanistic support for associations between alterations and phenotypes remains challenging. In particular, the study of noncoding genomic variation remains a major challenge because of the lack of accurate functional annotation for activity in a given context and across alleles. Experimental evidence is critical for prioritizing and interpreting functional effects of genetic alterations. Massively parallel reporter assays (MPRAs) have emerged as a powerful high-throughput approach, enabling quantification of regulatory element activity and allelic effects, as well as systematic dissection of gene regulatory logic and variant effects across different contexts. However, the diversity of MPRA designs, lack of standardized formats, and many potential processing parameters hamper data integration, reproducibility, and meta-analyses across studies. To address these challenges, the Impact of Genomic Variation on Function (IGVF) Consortium established an MPRA focus group to develop community standards, including harmonized file formats, and robust analysis pipelines for a wide range of library types and experimental designs. Here, we present these formats and comprehensive computational tools, MPRAlib and MPRAsnakeflow, for uniform processing from raw sequencing reads to counts, processing, and visualization. Using diverse MPRA data sets, we investigated technical variability sources including barcode sequence bias, outlier barcodes, and delivery method (episomal vs. lentiviral). Our results establish best practices for MPRA data generation and analysis, facilitating robust, reproducible research and large-scale integration. The presented tools and standards are publicly available, providing a foundation for future collaborative efforts in regulatory genomics.
Humans↗
Quantitation of human immunodeficiency virus provirus and circulating virus: relationship with immunologic parameters.
Virologic and seroimmunologic parameters were determined in 56 persons infected with human immunodeficiency virus (HIV). The provirus level varied from 10 to 100,000/10(6) CD4+ lymphocytes, and genomic HIV RNA was detectable in 39 of 56 patients at a relative concentration varying from 10 to greater than 250 copies/mL of serum. Provirus expressed as copies per 10(6) CD4+ lymphocytes and as circulating virus per milliliter of serum increased with disease progression and decrease of CD4+ cell concentration. The mean provirus concentration expressed per milliliter of blood varied little among categories of patients with various levels of CD4+ cells, but there was a progressive increase of circulating HIV genomic RNA. These virologic data suggest that during the course of HIV infection, an increasing proportion of the remaining CD4+ lymphocytes harbor the HIV genome and produce infectious virus. Finally, there was a marked correlation between increased provirus and genomic RNA concentration and three seroimmunologic markers: decrease in CD4+ cell count, p24 antigenemia, and disappearance of antibodies to HIV core antigen.
Base Sequence↗
Phenotypic heterogeneity in osteogenesis imperfecta: the mildly affected mother of a proband with a lethal variant has the same mutation substituting cysteine for alpha 1-glycine 904 in a type I procollagen gene (COL1A1).
A proband with a lethal variant of osteogenesis imperfecta (OI) has been shown to have, in one allele in a gene for type I procollagen (COL1A1), a single base mutation that converted the codon for alpha 1-glycine 904 to a codon for cysteine. The mutation caused the synthesis of type I procollagen that was posttranslationally overmodified, secreted at a decreased rate, and had a decreased thermal stability. The results here demonstrate that the proband's mother had the same single base mutation as the proband. The mother had no fractures and no signs of OI except for short stature, slightly blue sclerae, and mild frontal bossing. As a child, however, she had the triangular facies frequently seen in many patients with OI. On repeated subculturing, the proband's fibroblasts grew more slowly than the mother's, but they continued to synthesize large amounts of the mutated procollagen in passages 7-14. In contrast, the mother's fibroblasts synthesized decreasing amounts of the mutated procollagen after passage 11. Also, the relative amount of the mutated allele in the mother's fibroblasts decreased with passage number. In addition, the ratio of the mutated allele to the normal allele in leukocyte DNA from the mother was half the value in fibroblast DNA from the proband. The simplest interpretation of the data is that the mother was mildly affected because she was a mosaic for the mutation that produced a lethal phenotype in one of her three children.
Alleles↗
Serum immunoassay of a small cell lung cancer associated ganglioside: development of a sensitive scintillation proximity assay.
We here report an enzyme linked immunosorbent assay (ELISA) and a scintillation proximity assay (SPA) for detection of the ganglioside FucGM1 in sera from small cell lung cancer (SCLC) patients. The SPA was more sensitive and reproducible than the ELISA. In this assay, monoclonal antibodies specific for FucGM1 were bound to SPA particles and incubated with labelled FucGM1 and 100 microliters test-serum overnight, and counted in a beta-counter. The sensitivity was 0.2 ng. Seven out of twenty sera from SCLC patients were positive, whereas none of twenty sera from healthy individuals were positive for FucGM1. The SPA was more sensitive than the previously reported HPTLC as well as a direct ELISA.
Carbohydrate Sequence↗
Protective studies in sheep immunized with cuticular collagen proteins and peptides of Haemonchus contortus.
Twenty-eight nonsibling sheep aged approximately 12 months and raised in a helminth-free environment were used in two protection studies. Immunizations were conducted by two intramuscular injections 30 days apart with a synthetic 18AA cuticle collagen peptide and native cuticle collagens derived from the third- and fourth-stage larvae of Haemonchus contortus. Ten days following the last immunization, the sheep were each given 500 infective H. contortus larvae per day for five consecutive days by intraruminal injection. Both collagen materials induced antibodies reactive with cuticle collagens; however, neither induced reproducible protection to H. contortus infections in vaccinated/infected sheep. In the most extensive test, there were no statistical differences in mean faecal worm egg count for 56 days post worm challenge, in mean numbers of H. contortus and female fecundity ratios at necropsy of immunized and unimmunized sheep. Failure to reproducibly immunize sheep with cuticle collagens may be due to the inability of antibodies or host immune cells to reach the collagen epitopes in the nematode cuticle without prior surface coat removal as postulated in human nematode studies.
Adjuvants, Immunologic↗
A hierarchical, count-based model highlights challenges in scATAC-seq data analysis and points to opportunities to extract finer-resolution information.
BACKGROUND: Data from Single-cell Assay for Transposase Accessible Chromatin with Sequencing (scATAC-seq) is highly sparse. While current computational methods feature a range of transformation procedures to extract meaningful information, major challenges remain. RESULTS: Here, we discuss the major scATAC-seq data analysis challenges such as sequencing depth normalization and region-specific biases. We present a hierarchical count model that is motivated by the data generating process of scATAC-seq data. Our simulations show that current scATAC-seq data, while clearly containing physical single-cell resolution, are too sparse to infer true informational-level single-cell, single-region of chromatin accessibility states. CONCLUSIONS: While the broad utility of scATAC-seq at a cell type level is undeniable, describing it as fully resolving chromatin accessibility at single-cell resolution, particularly at individual locus level, may overstate the level of detail currently achievable. We conclude that chromatin accessibility profiling at true single-cell, single-region resolution is challenging with current data sensitivity, but that it may be achieved with promising developments in optimizing the efficiency of scATAC-seq assays.
Single-Cell Analysis↗
Scintillation proximity enzyme assay. A rapid and novel assay technique applied to HIV proteinase.
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Amino Acid Sequence↗
Regulatory aspects of clonally expanded B-1 (CD5+ B) cells.
B-1 (CD5+ B) cells appear early in ontogeny, produce mainly unmutated polyreactive antibodies, and are capable of self-renewal. B-1 cells clonally expand with age and are the malignant cell in chronic lymphocytic leukemia. In this report immunological analysis of B-1 malignancies in NZB mice, a murine model of chronic lymphocytic leukemia, is related to current information on B-1 cells. B-1 clones from NZB mice produce high levels of interleukin-10, detected at the RNA level by semi-quantitative polymerase chain reaction. In addition, the B-1 malignant clones in NZB mice and their hybrids, are negative for B220/6B2 expression, the B-specific antigenic form of CD45 which is a membrane-associated phosphatase involved in lymphocyte activation. Both the autocrine production by B-1 cells of interleukin-10 and altered CD45 expression may be responsible for the clonal expansion of these cells, as well as the accompanying T cell expansion. We report the establishment of an in vitro cytotoxic CD8+ T cell line derived from an NZB with a B-1 malignancy. The effect of B-1 cell-derived interleukin-10 on subsets of T lymphocytes may account for the immunoregulatory properties of B-1 cells. In addition, the NZB malignancies were also characterized for immunoglobulin variable region sequence and antigen specificity. The B-1 malignancies produced immunoglobulin derived from unmutated germline sequences with no N base substitutions. It appears that both the immunoglobulin and interleukin-10 produced by the B-1 malignant cell in NZB mice may have immunoregulatory properties. A study of B-1 malignancies may shed light on the immunoregulatory properties of non-clonally expanded normal B-1 cells.
Animals↗
Homologous tachyphylaxis to bradykinin and its interference with allergic pleurisy in actively sensitized rats.
After recovery from a first intraplantar or intrathoracic stimulation with bradykinin, repeated daily provocation with this peptide resulted in a progressive loss of its ability to cause paw or pleural oedema, reaching 0-20% of the control within seven and four consecutive provocations, respectively. The phenomenon was shown to be time reversible, since the unresponsiveness ceased when stimulations were discontinued, and localized, since paw oedema evoked by the peptide was not modified after desensitization of either the contralateral paw or the pleural cavity. Furthermore desensitization to bradykinin did not influence the pleurisy elicited by either histamine (200 micrograms/cavity), 5-hydroxytryptamine (5-HT) (100 microgram/cavity) or platelet-activating factor (PAF) (1 microgram/cavity), suggesting that the desensitization was selective. In contrast, when actively sensitized animals were submitted to bradykinin-induced tachyphylaxis, pleural exudation and leukocyte influx induced by antigen were drastically reduced, strongly implying bradykinin in this process. We demonstrated that repeated daily stimulation with bradykinin cause selective, local and reversible auto-refractoriness, which may be useful as a tool in attempting to evaluate the role of this peptide in inflammation.
Amino Acid Sequence↗
A computer-aided procedure for measuring Lashley III maze performance.
As an animal swims through the Lashley III maze, an observer types into a Macintosh computer the path taken. The computer program, Observe Software, then breaks the string of choices into two-step sequences and counts the number of such sequences. These data are then sent to a spreadsheet, where the sequences are sorted into forward and backward responses. Forward choices are Correct Path, T Choice Errors and Cul Entry Errors. All backward choices are errors, by definition. They are classified as T Choices, Cul Entries, Cul Exits, and Return to Start. The animal's behavior is then described by the various error classes plus a measure called Learning Index. Examples of learning by rats and mice are presented.
Animals↗
Disseminated "Mycobacterium genavense" infection in patients with AIDS.
We describe 18 patients with advanced HIV infection, most of whom had a chronic illness characterised by fever, diarrhoea, and massive loss of weight. Biopsy and necropsy samples revealed abundant acid-fast microorganisms in intestines, liver, spleen, lymph nodes, and many other tissues, which did not grow on solid media, although limited growth was observed in liquid blood cultures. Using primers complementary to bacterial 16S rRNA we amplified DNA sequences from tissue and leucocyte extracts and from blood-culture bottles. The sequences obtained were unique and suggest that the microorganism is a new member of the genus Mycobacterium, for which we propose the name "Mycobacterium genavense". Disseminated infection with "M genavense" should be considered in the differential diagnosis of HIV-infected patients with extreme immunosuppression, wasting, and fever.
Acquired Immunodeficiency Syndrome↗
Isolation of a specific granulopoiesis inhibitor from calf spleen and identification as glutathione.
A small peptide was isolated from calf spleen, specifically inhibiting murine granulopoiesis in vitro. Purification involved ultrafiltration, anion-exchange chromatography with a FPLC system and reversed-phase chromatography on HPLC. Determination of the amino acid composition, following acid hydrolysis and phenyl isothiocyanate and dabsyl chloride derivatization, revealed the amino acids glutamic acid, cysteine and glycine. Although the N-terminal amino group was not blocked, peptide sequencing with common techniques was not possible. Comparison of the isolated peptide with the well-known tripeptide glutathione by HPLC and fast atom bombardment (FAB)/tandem mass spectrometry showed the identity of both substances. Moreover, glutathione was found to be a specific granulopoiesis inhibitor in vitro at 10-100 nM, a so far unknown property of this well-known peptide.
Amino Acid Sequence↗
Proportionality-based association metrics in count compositional data.
Compositional data comprise vectors that describe the constituent parts of a whole. Data arising from various -omics platforms such as 16S and RNA sequencing are compositional in nature. In this kind of data, correlations between features on raw counts have no meaningful interpretation. Metrics of proportionality were formulated to address this problem. However, an inherent bias arises when these metrics are calculated empirically on count-based measures due to variability in read depths. We quantify the bias introduced by empirically calculating proportionality-based association metrics in count data. Additionally, we propose a means of estimating these metrics within a logit-normal multinomial model in pursuit of more accurate estimates. The model-based estimates are shown to outperform empirical estimates in simulated data and are applied to a mouse embryonic stem cell single-cell sequencing dataset, as well as a pediatric-onset multiple sclerosis metagenomic dataset.
Animals↗
A possible correlation between the type of bcr-abl hybrid messenger RNA and platelet count in Philadelphia-positive chronic myelogenous leukemia.
The Philadelphia (Ph1) chromosome, in which the hybrid bcr-abl gene is formed, is thought to be the initial event in chronic myelogenous leukemia (CML). The position of the breakpoint within the breakpoint cluster region (bcr) on Ph1 chromosome and the splicing pattern determine the species of the fused bcr-abl messenger RNA (mRNA). We tried to detect the two types of fused mRNAs in 57 chronic-phase cases of Ph1-positive CML using the polymerase chain reaction procedure (RT-PCR). The bcr exon 2/abl exon 2 fused mRNA (b2-a2) was detected in 17 patients, the bcr exon 3/abl exon 2 fused mRNA (b3-a2) was detected in 34 patients, and both types of mRNA were detected in six patients. The platelet counts of patients who expressed b3-a2 mRNA or both types were significantly higher than those of patients who expressed only b2-a2 (841.5 v 373.5 x 10(9)/L; P less than .015), although there was no significant difference in the white blood cell counts or hemoglobin. This finding suggests a possibility that the type of bcr-abl mRNA may affect the thrombopoietic activity in CML.
Base Sequence↗
Immunization-induced decrease of the CD4+:CD8+ ratio in cats experimentally infected with feline immunodeficiency virus.
In a previous experiment a group of 15 specified pathogen free (SPF) cats were experimentally infected with a Swiss isolate of feline immunodeficiency virus (FIV). A group of 15 SPF cats served as FIV negative controls. Nine cats of each group were vaccinated with a recombinant feline leukemia virus (FeLV) vaccine, six cats in each group with a placebo vaccine. All vaccinated cats developed high antibody titers to FeLV and were protected against subsequent FeLV challenge infection. In both control groups five of six cats became persistently infected with FeLV. Unexpectedly, the primary immune response to the vaccine antigen was significantly higher in the FIV positive group than in the FIV negative. The secondary response was stronger in the FIV negative cats. The goal of the present investigation was to further study the immune response in these 30 cats. They were immunized twice with the synthetic peptide L-tyrosine-L-glutamic acid-poly(DL-alanine)-poly(L-lysine) (TGAL) 21 days apart. Blood samples were collected on four occasions during the immunization process. They were tested for antibodies to TGAL, complete blood cell counts and CD4+, CD8+ and pan-T-lymphocyte counts. The following observations were made: (1) in contrast to the FeLV vaccine experiment, the primary immune response to TGAL was not significantly stronger in the FIV positive cats when tested by enzyme-linked immunosorbent assay (2). The absolute size of the CD4+ lymphocyte population was distinctly smaller in the FIV positive than in the FIV negative cats. The lowest CD4+ values were found in the dually FIV/FeLV infected cats. (3) A population of CD8+ lymphocytes was identified that was characterized by a distinctly weaker fluorescence. The size of this population increased in FIV positive and decreased in FIV negative cats during the TGAL immunization experiment. (4) The CD4+:CD8+ ratio increased in FIV negative cats during TGAL immunization from 1.9 to 2.3. In contrast, in FIV positive animals the CD4+:CD8+ ratio decreased significantly from 1.9 to 1.3 during the same period. From these and earlier data it was concluded that in short-term FIV infection the immune response to T-cell dependent antigens may be increased over that of the controls. Immune suppression develops gradually with duration of the infection. The significant drop of the CD4+:CD8+ ratio over a 5 week immunization period suggests that antigenic stimulation may accelerate the development of immune suppression in FIV positive cats. If this is a general feature, FIV infection may provide a particularly interesting model for studying the pathogenesis of AIDS.
Amino Acid Sequence↗
Partial amino acid sequence of erythrocyte carbonic anhydrase from tiger shark.
1. A partial primary structure (197 residues) of carbonic anhydrase from tiger shark (Galeocerdo cuvieri) erythrocytes has been determined. 2. The amino acid sequence of the enzyme is identical to those of human cytoplasmic carbonic anhydrases (CA I-III) by as much as 52-60%. 3. It is shown that tiger shark CA most closely resembles the CA II isoenzyme of amniotes. 4. Isoelectric focusing and inhibition studies on carbonic anhydrase from dogfish (Squalus acanthias) blood and muscle indicate the presence of the same isoenzyme in shark blood and muscle.
Alkylation↗
Targeted disruption of the mouse transforming growth factor-beta 1 gene results in multifocal inflammatory disease.
Transforming growth factor-beta 1 (TGF-beta 1) is a multifunctional growth factor that has profound regulatory effects on many developmental and physiological processes. Disruption of the TGF-beta 1 gene by homologous recombination in murine embryonic stem cells enables mice to be generated that carry the disrupted allele. Animals homozygous for the mutated TGF-beta 1 allele show no gross developmental abnormalities, but about 20 days after birth they succumb to a wasting syndrome accompanied by a multifocal, mixed inflammatory cell response and tissue necrosis, leading to organ failure and death. TGF-beta 1-deficient mice may be valuable models for human immune and inflammatory disorders, including autoimmune diseases, transplant rejection and graft versus host reactions.
Animals↗