The Mixed Epidermal Cell-Lymphocyte Reaction. II. Epidermal Langerhans Cells Are Responsible for the Enhanced Allogeneic Lymphocyte-Stimulating Capacity of Normal Human Epidermal Cell Suspensions

Earlier studies carried out in our laboratory which demonstrated that disaggregated human epidermal cells isolated from normal flexor forearm skin produced a greater degree of primary allogeneic lymphocyte blastogenic response than did autologous peripheral blood mononuclear cells have since been confirmed by others. We have now completed a series of additional studies designed to determine the basis for this difference. Blocking studies with anti-HLA-DR antibodies revealed that the allogeneic response triggered by epidermal cells was completely dependent on the presence of unbound HLA-DR molecules eliminating the possibility that nonspecific mitogenic effects produced by the epidermal cell suspension might be responsible for the difference. In addition we were unable to demonstrate that epidermal keratinocytes were supplying a nonspecific helper or growth-promoting effect to the interaction between stimulating HLA-DR bearing Langerhans cells and responding T lymphocytes. Since it has recently been suggested that the entire alloantigen-presenting capacity of unfractionated peripheral blood mononuclear cells can be attributed to a small population of HLA-DR antigen-bearing dendritic cells, we have considered the possibility that the greater degree of allogeneic lymphocyte response produced by epidermal cells could be due to the presence of a greater percentage of HLA-DR positive dendritic cells present in epidermal cell suspensions (i.e., Langerhans cells). Peripheral blood dendritic cell-enriched fractions and epidermal cell suspensions that contained similar percentages of dendritic cells produced equivalent amounts of allogeneic lymphocyte stimulation, whereas peripheral blood dendritic cell-enriched fractions that contained greater percentages of dendritic cells than were present in epidermal cell suspensions produced greater amounts of allogeneic stimulation. We therefore conclude that the enhanced mixed lymphocyte reaction stimulating capacity of human epidermal cell suspension could be explained by the fact that epidermal cell suspensions contain a greater percentage of HLA-DR bearing alloantigen-presenting dendritic cells than do unfractionated peripheral blood mononuclear cell suspensions

Amyopathic Dermatomyositis: A Review

Jim Gilliam's research interests throughout his career were forced upon better defining the relationships that exist between the cutaneous and systemic manifestations of the rheumatic diseases. Although the majority of his time was spent studying such relationships in lupus erythematosus patients, he was also intensely interested in dermatomyositis (DM) in this regard as well. He was particularly intrigued with the dissociation of the cutaneous and muscular manifestations of this disorder that occasionally occurs. The term ''dermatomyositis sine myositis'' has been used in the past to describe patients who present with only the cutaneous manifestations of DM; however, very little published data is available from systematic examinations of such patients. For several reasons, we have preferred the term ''amyopathic dermatomyositis" to describe that rare patient who for long periods of time suffers from the classical skin lesions of DM as the only clinically significant manifestation of their disease. In this presentation, we review our own personal experience with a group of six such patients and compare and contrast it to that of other workers who have dealt with this subject over the past two decades

A Macrophage Phenotype for a Constitutive, Class II Antigen-Expressing, Human Dermal Perivascular Dendritic Cell

A previously uncharacterized population of class II antigen-bearing dendritic cells that are intimately associated with the dermal microvasculature was identified in normal human skin using a double-label, indirect immunofluorescence technique. The only other major HLA-DR positive dermal cell type noted in these studies, the dermal microvascular endothelial cell (DMVEC), appeared to express lesser amounts of HLA-DR region gene product than did this dermal perivascular dendritic cell (DPDC). These DPDC were particularly common around small vessels in the superficial vascular plexus of the papillary dermis and were distinct from the mast cell, another cell type normally seen in a similar location. Phenotypic and ultrastructural studies have determined that the DPDC is more closely related to the monocyte/macro-phage lineage than the dendritic cell lineage. The perivascular location and phenotype of this cell distinguishes it from other previously described constitutive dermal cell types such as the classic “histiocyte,” veiled cell, and dendrocyte. The relatively rich expression of all three major HLA-D region gene products by this dermal perivascular dendritic macro-phage would suggest that it could play a significant role in the immunobiology of the dermal microvascular unit

Molecular Characteristics of SS-B/La and SS-A/Ro Cellular Antigens

Anti-SS-B/La and anti-SS-A/Ro antibodies coexist in certain patients with connective tissue diseases such as systemic lupus erythematosus or Sjögren's syndrome. The respective antigenic structures with which these autoantibodies bind have not been fully characterized. The present study was conducted to better define these two different cellular antigens. WiL2 cell extracts were used to obtain partially purified SS-B/La and SS-A/Re antigens, Both were found to be present in most fractions obtained after sequential purification with ammonium sulfate salt precipitation, G-200 gel filtration. DE-52 ion exchange chromatography, and preparative slab gel electrophoresis. However, SS-B/La antigenic activity was also found to be present in some fractions that did not contain detectable SS-A/Re activity. These findings suggested the existence of two different forms of SS-B/La antigen: one containing the SS-B/La antigen only and the other containing both the SS-B/La and SS-A/Re antigens. The RNA and protein components of these two ribonuclear protein particles were further defined by immunoprecipitation experiments using 32P-labeled WiL2 cell extract. The SS-B/La antigen was found to be associated with several RNAs while the SS-A/Re antigen was associated with several other distinct RNAs. Both antibodies precipitated a common 43K molecular weight phosphoprotein. The antigenic peptides of these 2 antibodies were analyzed using an immunoblot system, The SS-B/La antigen was present on a 43K peptide which was unstable and could be degraded to several peptides of lower molecular weight (40K, 38K, 30K), while the SS-A/Ro antigen occurred on a peptide having a molecular weight of about 60K

Blanks, a nuclear siRNA/dsRNA-binding complex component, is required for Drosophila spermiogenesis

Small RNAs and a diverse array of protein partners control gene expression in eukaryotes through a variety of mechanisms. By combining siRNA affinity chromatography and mass spectrometry, we have identified the double-stranded RNA-binding domain protein Blanks to be an siRNA- and dsRNA-binding protein from Drosophila S2 cells. We find that Blanks is a nuclear factor that contributes to the efficiency of RNAi. Biochemical fractionation of a Blanks-containing complex shows that the Blanks complex is unlike previously described RNA-induced silencing complexes and associates with the DEAD-box helicase RM62, a protein previously implicated in RNA silencing. In flies, Blanks is highly expressed in testes tissues and is necessary for postmeiotic spermiogenesis, but loss of Blanks is not accompanied by detectable transposon derepression. Instead, genes related to innate immunity pathways are up-regulated in blanks mutant testes. These results reveal Blanks to be a unique component of a nuclear siRNA/dsRNA-binding complex that contributes to essential RNA silencing-related pathways in the male germ line

C-BERST: Defining subnuclear proteomic landscapes at genomic elements with dCas9-APEX2 [preprint]

Mapping proteomic composition at distinct genomic loci and subnuclear landmarks in living cells has been a long-standing challenge. Here we report that dCas9-APEX2 Biotinylation at genomic Elements by Restricted Spatial Tagging (C-BERST) allows the rapid, unbiased mapping of proteomes near defined genomic loci, as demonstrated for telomeres and centromeres. By combining the spatially restricted enzymatic tagging enabled by APEX2 with programmable DNA targeting by dCas9, C-BERST has successfully identified nearly 50% of known telomere-associated factors and many known centromere-associated factors. We also identified and validated SLX4IP and RPA3 as telomeric factors, confirming C-BERST\u27s utility as a discovery platform. C-BERST enables the rapid, high-throughput identification of proteins associated with specific sequences, facilitating annotation of these factors and their roles in nuclear and chromosome biology

NmeCas9 is an intrinsically high-fidelity genome editing platform [preprint]

Background: The development of CRISPR genome editing has transformed biomedical research. Most applications reported thus far rely upon the Cas9 protein from Streptococcus pyogenes SF370 (SpyCas9). With many RNA guides, wild-type SpyCas9 can induce significant levels of unintended mutations at near-cognate sites, necessitating substantial efforts toward the development of strategies to minimize off-target activity. Although the genome-editing potential of thousands of other Cas9 orthologs remains largely untapped, it is not known how many will require similarly extensive engineering to achieve single-site accuracy within large (e.g. mammalian) genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large (~4.2 kb) open reading frame, limiting its utility in applications that require size-restricted delivery strategies such as adeno-associated virus vectors. In contrast, some genome-editing-validated Cas9 orthologs (e.g. from Staphylococcus aureus, Campylobacter jejuni, Geobacillus stearothermophilus and Neisseria meningitidis) are considerably smaller and therefore better suited for viral delivery. Results: Here we show that wild-type NmeCas9, when programmed with guide sequences of natural length (24 nucleotides), exhibits a nearly complete absence of unintended editing in human cells, even when targeting sites that are prone to off-target activity with wildtype SpyCas9. We also validate at least six variant protospacer adjacent motifs (PAMs), in addition to the preferred consensus PAM (5′-N4GATT-3′), for NmeCas9 genome editing in human cells. Conclusions: Our results show that NmeCas9 is a naturally high-fidelity genome editing enzyme and suggest that additional Cas9 orthologs may prove to exhibit similarly high accuracy, even without extensive engineering

NmeCas9 is an intrinsically high-fidelity genome-editing platform

BACKGROUND: The development of CRISPR genome editing has transformed biomedical research. Most applications reported thus far rely upon the Cas9 protein from Streptococcus pyogenes SF370 (SpyCas9). With many RNA guides, wildtype SpyCas9 can induce significant levels of unintended mutations at near-cognate sites, necessitating substantial efforts toward the development of strategies to minimize off-target activity. Although the genome-editing potential of thousands of other Cas9 orthologs remains largely untapped, it is not known how many will require similarly extensive engineering to achieve single-site accuracy within large genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large open reading frame, limiting its utility in applications that require size-restricted delivery strategies such as adeno-associated virus vectors. In contrast, some genome-editing-validated Cas9 orthologs are considerably smaller and therefore better suited for viral delivery. RESULTS: Here we show that wildtype NmeCas9, when programmed with guide sequences of the natural length of 24 nucleotides, exhibits a nearly complete absence of unintended editing in human cells, even when targeting sites that are prone to off-target activity with wildtype SpyCas9. We also validate at least six variant protospacer adjacent motifs (PAMs), in addition to the preferred consensus PAM (5\u27-N4GATT-3\u27), for NmeCas9 genome editing in human cells. CONCLUSIONS: Our results show that NmeCas9 is a naturally high-fidelity genome-editing enzyme and suggest that additional Cas9 orthologs may prove to exhibit similarly high accuracy, even without extensive engineering

Developing User Personas to Aid in the Design of a User-Centered Natural Product-Drug Interaction Information Resource for Researchers

Pharmacokinetic interactions between natural products and conventional drugs can adversely impact patient outcomes. These complex interactions present unique challenges that require clear communication to researchers. We are creating a public information portal to facilitate researchers’ access to credible evidence about these interactions. As part of a user-centered design process, three types of intended researchers were surveyed: drug-drug interaction scientists, clinical pharmacists, and drug compendium editors. Of the 23 invited researchers, 17 completed the survey. The researchers suggested a number of specific requirements for a natural product-drug interaction information resource, including specific information about a given interaction, the potential to cause adverse effects, and the clinical importance. Results were used to develop user personas that provided the development team with a concise and memorable way to represent information needs of the three main researcher types and a common basis for communicating the design’s rationale