Improving management of needle distress during the journey to dialysis through psychological education and training-the INJECT study feasibility pilot protocol.

BACKGROUND: Needle-related distress is a common yet poorly recognised and managed problem among haemodialysis (HD) patients. The aim of this pilot study is to test the feasibility and acceptability of the INJECT Intervention-an innovative psychology-based intervention to empower patients to self-manage needle distress with the support of dialysis nurses. METHODS: This investigator-initiated, single-arm, non-randomised feasibility study will take place in a large dialysis service in Adelaide, Australia. Participants will include patients aged ≥ 18 years, commencing or already receiving maintenance HD, recruited through dialysis physicians and nursing staff as individuals believed to be at risk of needle distress. They will be screened for inclusion using the Dialysis Fear of Injection Questionnaire (DFIQ) and enrolled into the study if the score is ≥ 2. The multi-pronged intervention encompasses (i) psychologist review, (ii) patient self-management program and (iii) nursing education program. The primary aim is to evaluate feasibility and acceptability of the intervention from patient and dialysis nurse perspectives, including recruitment, retention, engagement with the intervention and completion. Secondary exploratory outcomes will assess suitability of various tools for measuring needle distress, evaluate acceptability of the nursing education program and measure cannulation-related trauma and vascular access outcomes. CONCLUSION: The results will inform the protocol for larger trials addressing needle distress in HD patients. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (ANZCTR): ACTRN12621000229875, approved 4 April 2021, https://www.anzctr.org.au/ .

Signal recovery from stimulation artifacts in intracranial recordings with dictionary learning.

OBJECTIVE: Electrical stimulation of the human brain is commonly used for eliciting and inhibiting neural activity for clinical diagnostics, modifying abnormal neural circuit function for therapeutics, and interrogating cortical connectivity. However, recording electrical signals with concurrent stimulation results in dominant electrical artifacts that mask the neural signals of interest. Here we develop a method to reproducibly and robustly recover neural activity during concurrent stimulation. We concentrate on signal recovery across an array of electrodes without channel-wise fine-tuning of the algorithm. Our goal includes signal recovery with trains of stimulation pulses, since repeated, high-frequency pulses are often required to induce desired effects in both therapeutic and research domains. We have made all of our code and data publicly available. APPROACH: We developed an algorithm that automatically detects templates of artifacts across many channels of recording, creating a dictionary of learned templates using unsupervised clustering. The artifact template that best matches each individual artifact pulse is subtracted to recover the underlying activity. To assess the success of our method, we focus on whether it extracts physiologically interpretable signals from real recordings. MAIN RESULTS: We demonstrate our signal recovery approach on invasive electrophysiologic recordings from human subjects during stimulation. We show the recovery of meaningful neural signatures in both electrocorticographic (ECoG) arrays and deep brain stimulation (DBS) recordings. In addition, we compared cortical responses induced by the stimulation of primary somatosensory (S1) by natural peripheral touch, as well as motor cortex activity with and without concurrent S1 stimulation. SIGNIFICANCE: Our work will enable future advances in neural engineering with simultaneous stimulation and recording.

HIV-1 Nef blocks transport of MHC class I molecules to the cell surface via a PI 3-kinase-dependent pathway.

HIV causes a chronic infection by evading immune eradication. A key element of HIV immune escape is the HIV-1 Nef protein. Nef causes a reduction in the level of cell surface major histocompatibility complex class I (MHC-I) protein expression, thus protecting HIV-infected cells from anti-HIV cytotoxic T lymphocyte (CTL) recognition and killing. Nef also reduces cell surface levels of the HIV receptor, CD4, by accelerating endocytosis. We show here that endocytosis is not required for Nef-mediated downmodulation of MHC-I molecules. The main effect of Nef is to block transport of MHC-I molecules to the cell surface, leading to accumulation in intracellular organelles. Furthermore, the effect of Nef on MHC-I molecules (but not on CD4) requires phosphoinositide 3-kinase (PI 3-kinase) activity. We propose that Nef diverts MHC-1 proteins into a PI 3-kinase-dependent transport pathway that prevents expression on the cell surface.

HIV's evasion of the cellular immune response.

Despite a strong cytotoxic T-lymphocyte (CTL) response directed against viral antigens, untreated individuals infected with the human immunodeficiency virus (HIV-1) develop AIDS. We have found that primary T cells infected with HIV-1 downregulate surface MHC class I antigens and are resistant to lysis by HLA-A2-restricted CTL clones. In contrast, cells infected with an HIV-1 in which the nef gene is disrupted are sensitive to CTLs in an MHC and peptide-specific manner. In primary T cells HLA-A2 antigens are downmodulated more dramatically than total MHC class I antigens, suggesting that nef selectively downmodulates certain MHC class I antigens. In support of this, studies on cells expressing individual MHC class I alleles have revealed that nef does not downmodulate HLA-C and HLA-E antigens. This selective downmodulation allows infected cells to maintain resistance to certain natural killer cells that lyse infected cells expressing low levels of MHC class I antigens. Downmodulation of MHC class I HLA-A2 antigens occurs not only in primary T cells, but also in B and astrocytoma cell lines. No effect of other HIV-1 accessory proteins such as vpu and vpr was observed. Thus Nef is a protein that may promote escape of HIV-1 from immune surveillance.

HIV-1 Nef protein protects infected primary cells against killing by cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTLs) lyse virally infected cells that display viral peptide epitopes in association with major histocompatibility complex (MHC) class I molecules on the cell surface. However, despite a strong CTL response directed against viral epitopes, untreated people infected with the human immunodeficiency virus (HIV-1) develop AIDS. To resolve this enigma, we have examined the ability of CTLs to recognize and kill infected primary T lymphocytes. We found that CTLs inefficiently lysed primary cells infected with HIV-1 if the viral nef gene product was expressed. Resistance of infected cells to CTL killing correlated with nef-mediated downregulation of MHC class I and could be overcome by adding an excess of the relevant HIV-1 epitope as soluble peptide. Thus, Nef protected infected cells by reducing the epitope density on their surface. This effect of nef may allow evasion of CTL lysis by HIV-1-infected cells.

The role of the 70 kDa subunit of human DNA polymerase alpha in DNA replication.

DNA polymerase alpha is the only enzyme in eukaryotic cells capable of starting DNA chains de novo and is required for the initiation of SV40 DNA replication in vitro. We have cloned the 70 kDa subunit of human DNA polymerase alpha (hereafter referred to as the B subunit) and expressed it as a fusion protein in bacteria. The purified fusion protein forms a stable complex with SV40 T antigen, both in solution and when T antigen is bound to the SV40 origin of DNA replication. Analysis of mutant forms of the B subunit indicates that the N-terminal 240 amino acids are sufficient to mediate complex formation. The B subunit fusion protein promotes formation of a complex containing T antigen and the catalytic subunit (subunit A) of DNA polymerase alpha, suggesting that it serves to tether the two proteins. These physical interactions are functionally significant, since the ability of T antigen to stimulate the activity of the catalytic subunit of DNA polymerase alpha is highly dependent upon the B subunit. We suggest that the interactions mediated by the B subunit play an important role in SV40 DNA replication by promoting DNA chain initiation at the origin and/or facilitating the subsequent priming and synthesis of DNA chains on the lagging strand template. The protein may play similar roles in cellular DNA replication.

Effects of T antigen and replication protein A on the initiation of DNA synthesis by DNA polymerase alpha-primase.

Studies of simian virus 40 (SV40) DNA replication in a reconstituted cell-free system have established that T antigen and two cellular replication proteins, replication protein A (RP-A) and DNA polymerase alpha-primase complex, are necessary and sufficient for initiation of DNA synthesis on duplex templates containing the SV40 origin of DNA replication. To better understand the mechanism of initiation of DNA synthesis, we analyzed the functional interactions of T antigen, RP-A, and DNA polymerase alpha-primase on model single-stranded DNA templates. Purified DNA polymerase alpha-primase was capable of initiating DNA synthesis de novo on unprimed single-stranded DNA templates. This reaction involved the synthesis of a short oligoribonucleotide primer which was then extended into a DNA chain. We observed that the synthesis of ribonucleotide primers by DNA polymerase alpha-primase is dramatically stimulated by SV40 T antigen. The presence of T antigen also increased the average length of the DNA product synthesized on primed and unprimed single-stranded DNA templates. These stimulatory effects of T antigen required direct contact with DNA polymerase alpha-primase complex and were most marked at low template and polymerase concentrations. We also observed that the single-stranded DNA binding protein, RP-A, strongly inhibits the primase activity of DNA polymerase alpha-primase, probably by blocking access of the enzyme to the template. T antigen partially reversed the inhibition caused by RP-A. Our data support a model in which DNA priming is mediated by a complex between T antigen and DNA polymerase alpha-primase with the template, while RP-A acts to suppress nonspecific priming events.

Reconstitution of simian virus 40 DNA replication with purified proteins.

Replication of plasmid DNA molecules containing the simian virus 40 (SV40) origin of DNA replication has been reconstituted with seven highly purified cellular proteins plus the SV40 large tumor (T) antigen. Initiation of DNA synthesis is absolutely dependent upon T antigen, replication protein A, and the DNA polymerase alpha-primase complex and is stimulated by the catalytic subunit of protein phosphatase 2A. Efficient elongation of nascent chains additionally requires proliferating cell nuclear antigen, replication factor C, DNA topoisomerase I, and DNA polymerase delta. Electron microscopic studies indicate that DNA replication begins at the viral origin and proceeds via intermediates containing two forks that move in opposite directions. These findings indicate that the reconstituted replication reaction has many of the characteristics expected of authentic viral DNA replication.

Human monocyte Arg-Serpin cDNA. Sequence, chromosomal assignment, and homology to plasminogen activator-inhibitor.

An LPS-stimulated, human monocyte cDNA library was screened for stimulation-specific clones. One clone (pcD-1214) contained a 1.9-kb pair insert that hybridized to a 2,000-nucleotide mRNA expressed by peripheral blood monocytes, the histiocytic lymphoma cell line U937, and umbilical cord endothelial cells. The 415-amino-acid precursor polypeptide predicted from the cDNA (46,596 molecular weight) has a putative 22-residue signal peptide and approximately 35% homology with members of the serine protease inhibitor (Serpin) superfamily. On the basis of amino acid homology and alignment of COOH-terminal residues within the Serpin-reactive center, the clone pcD-1214 was identified as coding for an Arg-Serpin. Southern blot analysis of human-mouse somatic cell hybrid DNA locates the Arg-Serpin gene on human chromosome 18. A perfect match between amino acid residues 347-376 in this Arg-Serpin and the published sequence of a 30-residue, tryptic peptide from the COOH-terminus of a monocyte plasminogen activator-inhibitor (PAI-2), strongly suggests that the Arg-Serpin encoded by pcD-1214 is PAI-2.

Transcriptional regulation of the human prointerleukin 1 beta gene.

Interleukin 1 (IL 1) is a protein produced by monocytes in response to certain antigens which produces a wide variety of cellular responses in various tissues. We have studied the regulation of the human proIL-1 beta gene in THP-1 human monocytic leukemia cells. Lipopolysaccharide (LPS) induction of this gene results in an immediate and transient increase of message that rapidly falls to a low, but constant, level within 6 hr. This decrease results from a specific repression of transcription by 2 hr after stimulation. Cycloheximide inhibition of new protein synthesis causes a superinduction of IL 1 message, but does not alter the initial kinetics of message production. This presumably delays the synthesis of a labile transcriptional repressor protein and implies that the proIL-1 beta gene is under the control of both a transcriptional activator and a newly synthesized transcriptional repressor. The transient increase in mRNA production and the sustained low-level synthesis beyond the initial transient response suggest that the IL 1 protein itself may act intracellularly in a manner analogous to that described for several proto-oncogenes and cellular competence factors.

Genomic sequence for human prointerleukin 1 beta: possible evolution from a reverse transcribed prointerleukin 1 alpha gene.

We have isolated the human prointerleukin 1 (proIL-1) beta gene from leukocyte and fetal liver libraries. The nucleotide sequence and its gene organization reveals that the proIL-1 beta gene is composed of seven exons with a primary transcription product length of 7,008 nucleotides. The exon sequence agrees well with that of the human proIL-1 beta cDNA. Features of interest within the transcriptional unit include positioned TATA, CAT, and poly-adenylation signals for gene regulation, as well as the signatures of gene duplication via retrotransposition in the form of flanking direct repeats and a genomic poly A tail. The genomic organization of the proIL-1 beta gene with respect to the number and position of exon boundaries is strikingly similar to that of the recently reported human proIL-1 alpha gene. Therefore, we hypothesize that the proIL-1 beta may have arisen by a reverse transcriptase mediated duplication of the related alpha gene.

Interleukin-1 gene (IL1) assigned to long arm of human chromosome 2.

A complementary DNA (cDNA) probe for the predominant (pI 7) form of human monocyte-derived interleukin-1 (IL1) and a collection of 30 human-mouse somatic cell hybrids were used to assign the IL1 gene to human chromosome 2 by Southern blot analysis of hybrid cell DNA digested with the restriction endonuclease BglII. In situ hybridization to human metaphase chromosomes localized the IL1 gene to the long arm of chromosome 2 at position 2q13-2q21 between two fragile sites.