Assessment of antibody levels to SARS-CoV-2 in patients with idiopathic inflammatory myopathies receiving treatment with intravenous immunoglobulin.

Antibodies to Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2) have been reported in pooled healthy donor plasma and intravenous immunoglobulin products (IVIG). It is not known whether administration of IVIG increases circulating anti-SARS-CoV-2 antibodies (COVID ab) in IVIG recipients. COVID ab against the receptor binding domain of the spike protein were analyzed using a chemiluminescent microparticle immunoassay in patients with idiopathic inflammatory myopathies (IIM) both receiving and not receiving IVIG (IVIG and non-IVIG group, respectively). No significant differences in COVID ab levels were noted between IVIG and non-IVIG groups (417 [67-1342] AU/mL in IVIG vs 5086 [43-40,442] AU/mL in non-IVIG, p = 0.11). In linear regression models including all post-vaccination patient samples, higher number of vaccine doses was strongly associated with higher COVID ab levels (2.85 [1.21, 4.48] log AU/mL, regression coefficient [Formula: see text] [95% CI], p = 0.001), while use of RTX was associated with lower ab levels (2.73 [- 4.53, - 0.93] log AU/mL, [Formula: see text][95%CI], p = 0.004). In the IVIG group, higher total monthly doses of IVIG were associated with slightly higher COVID ab levels (0.02 [0.002-0.05] log AU/mL, p = 0.04). While patients on IVIG did not have higher COVID ab levels compared to the non-IVIG group, higher monthly doses of IVIG were associated with higher circulating levels of COVID ab in patients receiving IVIG, particularly in patients concomitantly receiving RTX. Our findings suggest that IIM patients, especially those at increased risk of COVID infection and worse COVID outcomes due to RTX therapy may have protective benefits when on concurrent IVIG treatment.

Identification of miR-34-3p as a candidate follicular phase serum marker for endometriosis: a pilot study.

OBJECTIVE: To identify early follicular phase microribonucleic acids (miRNAs) that are altered in serum of women with endometriosis. DESIGN: Case-control study. SETTING: Large university-affiliated in vitro fertilization center. PATIENT(S): Women with (n = 21) and without (n = 24) endometriosis. INTERVENTION(S): Serum samples were obtained from laparoscopy-confirmed patients with endometriosis. MAIN OUTCOME MEASURE(S): The differential expression of serum miRNAs relative to controls was measured using the NanoString nCounter technology and validated by quantitative real-time polymerase chain reaction in an independent cohort of 27 patients with endometriosis and controls (n = 24). Microribonucleic acid target signaling pathways and genes were analyzed bioinformatically. A chemically modified stable miR-34-3p oligonucleotide was used to examine the effect on proliferation of VK2E6/E7 endometrial cells in vitro. RESULT(S): Eighteen miRNAs were significantly up-regulated, and 1 miRNA (hsa-miR-34c-3p) was significantly down-regulated in the follicular phase of patients with endometriosis. The analysis of target signaling pathways using TargetScan predicted regulation of the mitogen-activated protein kinase, phosphoinositide 3-kinase/protein kinase B, Hippo, adenosine monophosphate-activated protein kinase, transforming growth factor beta, and endometrial cancer pathways, which have been implicated in the pathogenesis of endometriosis, by these miRNAs. The analysis of sequence complementarity identified prostaglandin E2 receptor 4, interleukin 6 signal transducer, and polo-like kinase 4 genes as possible direct targets of hsa-miR-34-3p. DSDI-1, a chemically modified stable miR-34-3p oligonucleotide, reduced cell proliferation in VK2E6/E7 endometrial cells in vitro. CONCLUSION(S): The follicular phase miRNA levels are altered in serum of women with endometriosis and may be useful as reproducible detection biomarkers for early diagnosis of endometriosis. hsa-miR-34-3p is significantly down-regulated in endometriosis, targets endometriosis genes, and reduces endometrial cell proliferation in vitro. These results support hsa-miR-34-3p as a potential therapeutic target in endometriosis.

Humoral responses to SARS-CoV-2 mRNA vaccines: Role of past infection.

Two mRNA vaccines (BNT162b2 and mRNA-1273) against severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) are globally authorized as a two-dose regimen. Understanding the magnitude and duration of protective immune responses is vital to curbing the pandemic. We enrolled 461 high-risk health services workers at the University of California, Los Angeles (UCLA) and first responders in the Los Angeles County Fire Department (LACoFD) to assess the humoral responses in previously infected (PI) and infection naïve (NPI) individuals to mRNA-based vaccines (BNT162b2/Pfizer- BioNTech or mRNA-1273/Moderna). A chemiluminescent microparticle immunoassay was used to detect antibodies against SARS-CoV-2 Spike in vaccinees prior to (n = 21) and following each vaccine dose (n = 246 following dose 1 and n = 315 following dose 2), and at days 31-60 (n = 110) and 61-90 (n = 190) following completion of the 2-dose series. Both vaccines induced robust antibody responses in all immunocompetent individuals. Previously infected individuals achieved higher median peak titers (p = 0.002) and had a slower rate of decay (p = 0.047) than infection-naïve individuals. mRNA-1273 vaccinated infection-naïve individuals demonstrated modestly higher titers following each dose (p = 0.005 and p = 0.029, respectively) and slower rates of antibody decay (p = 0.003) than those who received BNT162b2. A subset of previously infected individuals (25%) required both doses in order to reach peak antibody titers. The biologic significance of the differences between previously infected individuals and between the mRNA-1273 and BNT162b2 vaccines remains uncertain, but may have important implications for booster strategies.

Clonally Focused Public and Private T Cells in Resected Brain Tissue From Surgeries to Treat Children With Intractable Seizures.

Using a targeted transcriptomics approach, we have analyzed resected brain tissue from a cohort of 53 pediatric epilepsy surgery cases, and have found that there is a spectrum of involvement of both the innate and adaptive immune systems as evidenced by the differential expression of immune-specific genes in the affected brain tissue. The specimens with the highest expression of immune-specific genes were from two Rasmussen encephalitis cases, which is known to be a neuro-immunological disease, but also from tuberous sclerosis complex (TSC), focal cortical dysplasia, and hemimegalencephaly surgery cases. We obtained T cell receptor (TCR) Vß chain sequence data from brain tissue and blood from patients with the highest levels of T cell transcripts. The clonality indices and the frequency of the top 50 Vß clonotypes indicated that T cells in the brain were clonally restricted. The top 50 Vß clonotypes comprised both public and private (patient specific) clonotypes, and the TCR Vß chain third complementarity region (CDR3) of the most abundant public Vß clonotype in each brain sample was strikingly similar to a CDR3 that recognizes an immunodominant epitope in either human cytomegalovirus or Epstein Barr virus, or influenza virus A. We found that the frequency of 14 of the top 50 brain Vß clonotypes from a TSC surgery case had significantly increased in brain tissue removed to control recurrent seizures 11 months after the first surgery. Conversely, we found that the frequency in the blood of 18 of the top 50 brain clonotypes from a second TSC patient, who was seizure free, had significantly decreased 5 months after surgery indicating that T cell clones found in the brain had contracted in the periphery after removal of the brain area associated with seizure activity and inflammation. However, the frequency of a public and a private clonotype significantly increased in the brain after seizures recurred and the patient underwent a second surgery. Combined single cell gene expression and TCR sequencing of brain-infiltrating leukocytes from the second surgery showed that the two clones were CD8 effector T cells, indicating that they are likely to be pathologically relevant.

MiR130b from Schlafen4+ MDSCs stimulates epithelial proliferation and correlates with preneoplastic changes prior to gastric cancer.

The myeloid differentiation factor Schlafen4 (Slfn4) marks a subset of myeloid-derived suppressor cells (MDSCs) in the stomach during Helicobacter-induced spasmolytic polypeptide-expressing metaplasia (SPEM). OBJECTIVE: To identify the gene products expressed by Slfn4+-MDSCs and to determine how they promote SPEM. DESIGN: We performed transcriptome analyses for both coding genes (mRNA by RNA-Seq) and non-coding genes (microRNAs using NanoString nCounter) using flow-sorted SLFN4+ and SLFN4- cells from Helicobacter-infected mice exhibiting metaplasia at 6 months postinfection. Thioglycollate-elicited myeloid cells from the peritoneum were cultured and treated with IFNα to induce the T cell suppressor phenotype, expression of MIR130b and SLFN4. MIR130b expression in human gastric tissue including gastric cancer and patient sera was determined by qPCR and in situ hybridisation. Knockdown of MiR130b in vivo in Helicobacter-infected mice was performed using Invivofectamine. Organoids from primary gastric cancers were used to generate xenografts. ChIP assay and Western blots were performed to demonstrate NFκb p65 activation by MIR130b. RESULTS: MicroRNA analysis identified an increase in MiR130b in gastric SLFN4+ cells. Moreover, MIR130b colocalised with SLFN12L, a human homologue of SLFN4, in gastric cancers. MiR130b was required for the T-cell suppressor phenotype exhibited by the SLFN4+ cells and promoted Helicobacter-induced metaplasia. Treating gastric organoids with the MIR130b mimic induced epithelial cell proliferation and promoted xenograft tumour growth. CONCLUSION: Taken together, MiR130b plays an essential role in MDSC function and supports metaplastic transformation.

Comparing NGS and NanoString platforms in peripheral blood mononuclear cell transcriptome profiling for advanced heart failure biomarker development.

In preparation to create a clinical assay that predicts 1-year survival status of advanced heart failure (AdHF) patients before surgical/interventional therapies and to select the appropriate clinical assay platform for the future assay, we compared the properties of next generation sequencing (NGS) used in the gene discovery phase to the NanoString platform used in the clinical assay development phase. In 25 AdHF patients in a tertiary academic medical center from 2015 to 2016, PBMC samples were collected and aliquoted for NGS RNA whole transcriptome sequencing and compared to 770 genes represented on NanoString's PanCancer IO 360 Gene Expression research panel. Prior to statistical analysis, NanoString and NGS expression values were log transformed. We computed Pearson correlation coefficients for each sample, comparing gene expression values between NanoString and NGS across the set of matched genes and for each of the matched genes across the set of samples. Genes were grouped by average NGS expression, and the NanoString-NGS correlation for each group was computed. Out of 770 genes from the NanoString panel, 734 overlapped between both platforms and showed high intrasample correlation. Within an individual sample, there was an expression-level dependent correlation between both platforms. The low- vs. intermediate/high-expression groups showed NGS average correlation 0.21 vs. 0.58-0.68, respectively, and NanoString average correlation 0.07-0.34 vs. 0.59-0.70, respectively. NanoString demonstrated high reproducibility (R 2 > 0.99 for 100 ng input), sensitivity (probe counts between 100 and 500 detected and quantified), and robustness (similar gene signature scores across different RNA input concentrations, cartridges, and outcomes). Data from NGS and NanoString were highly correlated. These platforms play a meaningful, complementary role in the biomarker development process.

Overexpressing Long Noncoding RNAs Using Gene-activating CRISPR.

Long noncoding RNA (lncRNA) biology is a new and exciting field of research, with the number of publications from this field growing exponentially since 2007. These studies have confirmed that lncRNAs are altered in almost all diseases. However, studying the functional roles for lncRNAs in the context of disease remains difficult due to the lack of protein products, tissue-specific expression, low expression levels, complexities in splice forms, and lack of conservation among species. Given the species-specific expression, lncRNA studies are often restricted to human research contexts when studying disease processes. Since lncRNAs function at the molecular level, one way to dissect lncRNA biology is to either remove the lncRNA or overexpress the lncRNA and measure cellular effects. In this article, a written and visualized protocol to overexpress lncRNAs in vitro is presented. As a representative experiment, an lncRNA associated with inflammatory bowel disease, Interferon Gamma Antisense 1 (IFNG-AS1), is shown to be overexpressed in a Jurkat T-cell model. To accomplish this, the activating clustered regularly interspaced short palindromic repeats (CRISPR) technique is used to enable overexpression at the endogenous genomic loci. The activating CRISPR technique targets a set of transcription factors to the transcriptional start site of a gene, enabling a robust overexpression of multiple lncRNA splice forms. This procedure will be broken down into three steps, namely (i) guide RNA (gRNA) design and vector construction, (ii) virus generation and transduction, and (iii) colony screening for overexpression. For this representative experiment, a greater than 20-fold enhancement in IFNG-AS1 in Jurkat T cells was observed.

Factors in the Selection of Surface Disinfectants for Use in a Laboratory Animal Setting.

Because surface disinfectants are an important means of pathogen control within laboratory animal facilities, these products must have an appropriate spectrum of antimicrobial activity. However, many other factors must also be considered, including effects on human health, environmental safety, and animal behavior. Aqueous solutions of sodium hypochlorite often are considered to be the 'gold standard' for surface disinfection, but these products can be corrosive, caustic, and aversive in odor. This study was designed to identify disinfectants that are as effective as hypochlorite solutions but more acceptable for use in a laboratory animal setting. An antiviral disinfectant-efficacy assay was developed by using viral vectors that expressed green fluorescence protein as surrogates for wild-type viruses of concern in laboratory animals. Efficacy testing revealed that most of the products were highly effective when used against viral vectors in suspension. However, when the disinfectants were challenged by buffering virus in protein or drying virus on nonporous surfaces, the hypochlorite and peroxymonosulfate products performed the best. Review of safety data sheets for the agents indicated that a peroxide-based product was considerably safer than the other products tested and that the pH of most products was not conducive to disposal down a drain. Behavioral testing of Swiss Webster, C57Bl/6, and BALB/c mice showed that the hypochlorite- and peroxide-based products were clearly aversive, given that the mice consistently avoided these products. All of these factors must be considered when choosing the appropriate disinfectant.

Short conserved sequences of HIV-1 are highly immunogenic and shift immunodominance.

UNLABELLED: Cellular immunity is pivotal in HIV-1 pathogenesis but is hampered by viral sequence diversity. An approach to minimize this diversity is to focus immunity on conserved proteome sequences; therefore, we selected four relatively conserved regions (Gag amino acids 148 to 214 and 250 to 335, Env amino acids 521 to 606, and Nef amino acids 106 to 148), each created in three mosaics, to provide better coverage of M-group HIV-1 sequences. A conserved-region vaccine (CRV) delivering genes for these four regions as equal mixtures of three mosaics each (each region at a separate injection site) was compared to a whole-protein vaccine (WPV) delivering equimolar amounts of genes for whole Gag, Env, and Nef as clade B consensus sequences (separate injection sites). Three rhesus macaques were vaccinated via three DNA primes and a recombinant adenovirus type 5 boost (weeks 0, 4, 8, and 24, respectively). Although CRV inserts were about one-fifth that of WPV, the CRV generated comparable-magnitude blood CD4+ and CD8+ T lymphocyte responses against Gag, Env, and Nef. WPV responses preferentially targeted proteome areas outside the selected conserved regions in direct proportion to sequence lengths, indicating similar immunogenicities for the conserved regions and the outside regions. The CRV yielded a conserved-region targeting density that was approximately 5-fold higher than that of the WPV. A similar pattern was seen for bronchoalveolar lymphocytes, but with quadruple the magnitudes seen in blood. Overall, these findings demonstrate that the selected conserved regions are highly immunogenic and that anatomically isolated vaccinations with these regions focus immunodominance compared to the case for full-length protein vaccination. IMPORTANCE: HIV-1 sequence diversity is a major barrier limiting the capability of cellular immunity to contain infection and the ability of vaccines to match circulating viral sequences. To date, vaccines tested in humans have delivered whole proteins or genes for whole proteins, and it is unclear whether including only conserved sequences would yield sufficient cellular immunogenicity. We tested a vaccine delivering genes for four small conserved HIV-1 regions compared to a control vaccine with genes for whole Gag, Env, and Nef. Although the conserved regions ranged from 43 to 86 amino acids and comprised less than one-fifth of the whole Gag/Env/Nef sequence, the vaccines elicited equivalent total magnitudes of both CD4+ and CD8+ T lymphocyte responses. These data demonstrate the immunogenicity of these small conserved regions and the potential for a vaccine to steer immunodominance toward conserved epitopes.

Reduction of HLA class I expression by ribonucleic acid interference mitigates allogenicity of human primary and Immortalized cells.

Unmatched human leukocyte antigens (HLA) expressed by allogeneic donor cells are the major target for immunological rejection. In order to reduce the immunogenicity of allograft cells, we have developed lentiviral vectors for delivery of short hairpin ribonucleic acid (shRNA) against Class I HLA. This approach was evaluated in both an established human embryonic kidney cell line and primary human CD34+ hematopoietic stem/progenitor cells. Target cells transduced with lentiviral vectors expressing either HLA-A*0201 allele-specific or HLA-A, -B, -C consensus sequence-specific shRNA showed effective knockdown of cell surface HLA expression. Mixed lymphocyte-target cell reactions showed significantly reduced interferon-gamma production from alloreactive cytotoxic T lymphocytes and significantly reduced levels of target cell apoptosis after shRNA-mediated knockdown of HLA expression and target cell survival correlated with vector transduction efficiency. Furthermore, increasing resistance to complement-dependent cytotoxicity mediated by anti-HLA antibodies was observed to correlate with increasing levels of shRNA vector transduction in primary human CD34+ cells. Notably, non-HLA restricted killing by lymphokine-activated killer cells was not incurred after HLA knockdown. These data demonstrate the potential for genetic engineering strategies targeting incompatible HLA alleles to reduce both cellular and humoral responses and enable graft survival after transplantation of allogeneic cells and tissues.

In vivo stable transduction of humanized liver tissue in chimeric mice via high-capacity adenovirus-lentivirus hybrid vector.

We developed hybrid vectors employing high-capacity adenovirus as a first-stage carrier encoding all the components required for in situ production of a second-stage lentivirus, thereby achieving stable transgene expression in secondary target cells. Such vectors have never previously been tested in normal tissues, because of the scarcity of suitable in vivo systems permissive for second-stage lentivirus assembly. Here we employed a novel murine model in which endogenous liver tissue is extensively reconstituted with engrafted human hepatocytes, and successfully achieved stable transduction by the second-stage lentivirus produced in situ from first-stage adenovirus. This represents the first demonstration of the functionality of adenoviral-lentiviral hybrid vectors in a normal parenchymal organ in vivo.

Lentiviral vectors with CMV or MHCII promoters administered in vivo: immune reactivity versus persistence of expression.

Lentiviral vectors (LVs) are potential tools for genetic vaccination. To improve the safety of LV vaccines, we evaluated the selectivity, bio-distribution, persistence of expression, and immune potency of vesicular stomatitis virus G (VSV-G)-pseudotyped vectors transcriptionally targeted to antigen presenting cells (APCs) through a major histocompatibility complex class II (MHCII) promoter. Control vectors contained the ubiquitous cytomegalovirus (CMV) promoter. Bio-distribution studies after intravenous injections of LVs expressing green fluorescent protein (GFP) or luciferase were conducted by a combination of flow cytometry, immunofluorescence, real-time quantitative polymerase chain reaction (RT-Q-PCR) and whole-body bioluminescence analyses. GFP-expressing vectors showed selective expression in MHCII(+) cells of spleen and LV-CMV-GFP administration produced noticeable spleen inflammation, whereas LV-MHCII-GFP did not. Long-term optical imaging analyses of C57BL/6 mice injected with LV-CMV-LUC showed diminishing luciferase expression in the liver and spleen over time. Vaccination/boost with LV-CMV expressing the melanoma antigen tyrosinase-related protein 2 (TRP2) yielded dose-dependent antigen-specific CD8(+) T-cell reactivity and high protection against B16 melanoma challenge. Unexpectedly, administration of LVs containing the MHCII promoter resulted in persistence of luciferase expression and viral integration in MHCII(+) splenocytes and virtually no CD8(+) T-cell responses against TRP2. These studies reveal that APC transduction by LVs could lead to immune reactivity (LV-CMV) or persistence of transgene expression (LV-MHCII), providing a relevant paradigm for vaccination and gene replacement approaches.

Lentiviral vector-mediated autonomous differentiation of mouse bone marrow cells into immunologically potent dendritic cell vaccines.

Approaches facilitating generation of dendritic cell (DC) vaccines for clinical trials and enhancing their viability, bio-distribution, and capacity to stimulate antigen-specific immune responses are critical for immunotherapy. We programmed mouse bone marrow (BM) cells with lentiviral vectors (LV-GI4) so that they produced granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) in an autonomous manner. DC/LV-GI4 cells underwent autonomous trans-differentiation to yield typical phenotypic characteristics of DCs. DC/LV-GI4 cells that self-differentiated either ex vivo or in vivo showed persistent and robust viability and stimulated high influx of DCs into draining lymph nodes (LNs). The immunostimulatory efficacy of DC/LV-GI4 cells was evaluated using MART1 and TRP2 as co-expressed melanoma antigens. Mice vaccinated with DC/LV-GI4 cells that self-differentiated in vitro or in vivo produced potent antigen-specific responses against melanoma, which correlated with protective and long-term therapeutic anti-tumor effects. Thus, DC precursors can be genetically engineered after a single ex vivo manipulation, resulting in DC vaccines with improved activity.