Background & Aims T cells play a critical role in viral infection. We examined whether T-cell effector and regulatory responses can define clinical stages of chronic hepatitis B (CHB). Methods We ...enrolled 200 adults with CHB who participated in the National Institutes of Health−supported Hepatitis B Research Network from 2011 through 2013 and 20 uninfected individuals (controls). Peripheral blood lymphocytes from these subjects were analyzed for T-cell responses (proliferation and production of interferon gamma and interleukin 10) to overlapping hepatitis B virus (HBV) peptides (preS, S, preC, core, and reverse transcriptase), influenza matrix peptides, and lipopolysaccharide. T-cell expression of regulatory markers FOXP3, programmed death-1, and cytotoxic T lymphocyte-associated antigen-4 was examined by flow cytometry. Immune measures were compared with clinical parameters, including physician-defined immune-active, immune-tolerant, or inactive CHB phenotypes, in a blinded fashion. Results Compared with controls, patients with CHB had weak T-cell proliferative, interferon gamma, and interleukin 10 responses to HBV, with increased frequency of circulating FOXP3+ CD127− regulatory T cells and CD4+ T-cell expression of programmed death-1 and cytotoxic T lymphocyte-associated antigen-4. T-cell measures did not clearly distinguish between clinical CHB phenotypes, although the HBV core-specific T-cell response was weaker in hepatitis B e antigen (HBeAg)+ than HBeAg− patients (percent responders: 3% vs 23%; P = .00008). Although in vitro blockade of programmed death-1 or cytotoxic T lymphocyte-associated antigen-4 increased T-cell responses to HBV, the effect was weaker in HBeAg+ than HBeAg− patients. Furthermore, T-cell responses to influenza and lipopolysaccharide were weaker in CHB patients than controls. Conclusions HBV persists with virus-specific and global T-cell dysfunction mediated by multiple regulatory mechanisms, including circulating HBeAg, but without distinct T-cell−based immune signatures for clinical phenotypes. These findings suggest additional T-cell−independent or regulatory mechanisms of CHB pathogenesis that warrant further investigation.
The etiology of deep dyspareunia in endometriosis is unclear. Our objective was to determine whether nerve bundle density in the cul-de-sac/uterosacrals (zone II) is associated with deep dyspareunia ...in women with endometriosis. We conducted a blinded retrospective immunohistochemistry study (n = 58) at a tertiary referral center (2011-2013). Patients were stringently phenotyped into a study group and 2 control groups. The study group (tender endometriosis, n = 29) consisted of patients with deep dyspareunia, a tender zone II on examination, and an endometriosis lesion in zone II excised at surgery. Control group 1 (nontender endometriosis, n = 17) consisted of patients without deep dyspareunia, a nontender zone II on examination, and an endometriosis lesion in zone II excised at surgery. Control group 2 (tender nonendometriosis, n = 12) consisted of patients with deep dyspareunia, a tender zone II on examination, and a nonendometriosis lesion (eg, normal histology) in zone II excised at surgery. Protein gene product 9.5 (PGP9.5) immunohistochemistry was performed to identify nerve bundles (nerve fibers surrounded by perineurium) in the excised zone II lesion. PGP9.5 nerve bundle density (bundles/high powered field HPF) was then scored by a pathologist blinded to the group. We found a significant difference in PGP9.5 nerve bundle density between the 3 groups (analysis of variance, F2,55 = 6.39, P = .003). Mean PGP9.5 nerve bundle density was significantly higher in the study group (1.16 ± 0.56 bundles/HPF ±standard deviation) compared to control group 1 (0.65 ± 0.36, Tukey test, P = .005) and control group 2 (0.72 ± 0.56, Tukey test, P = .044). This study provides evidence that neurogenesis in the cul-de-sac/uterosacrals may be an etiological factor for deep dyspareunia in endometriosis.
In small cell lung cancer (SCLC), acquired resistance to DNA-damaging therapy is challenging to study because rebiopsy is rarely performed. We used patient-derived xenograft models, established ...before therapy and after progression, to dissect acquired resistance to olaparib plus temozolomide (OT), a promising experimental therapy for relapsed SCLC. These pairs of serial models reveal alterations in both cell cycle kinetics and DNA replication and demonstrate both inter- and intratumoral heterogeneity in mechanisms of resistance. In one model pair, up-regulation of translesion DNA synthesis (TLS) enabled tolerance of OT-induced damage during DNA replication. TLS inhibitors restored sensitivity to OT both in vitro and in vivo, and similar synergistic effects were seen in additional SCLC cell lines. This represents the first described mechanism of acquired resistance to DNA damage in a patient with SCLC and highlights the potential of the serial model approach to investigate and overcome resistance to therapy in SCLC.
Small cell osteosarcoma and mesenchymal chondrosarcoma are 2 primary bone tumors with a small round blue cell component, which can mimic the appearance of Ewing sarcoma. Distinguishing these tumors ...from each other on biopsy material is important clinically, as optimal therapy differs according to the tumor type. However, separating these entities on morphology alone can be challenging. FLI-1 has been described to be a useful marker for Ewing sarcoma, particularly when hematolymphoid markers are negative. In small cell osteosarcoma and mesenchymal chondrosarcoma, the FLI-1 staining pattern has not been adequately characterized. Using a monoclonal FLI-1 antibody, nuclear immunoreactivity in tumor cells was evaluated in 10 small cell osteosarcomas, 10 mesenchymal chondrosarcomas, and 8 Ewing sarcomas, together with a number of other small, round, blue cell tumors. None of the small cell osteosarcomas or mesenchymal chondrosarcomas exhibited FLI-1 staining in the tumor cells, in contrast to the positive nuclear FLI-1 staining in the stromal endothelial cells. In comparison, 6 of the 8 Ewing sarcomas showed moderate-to-strong nuclear FLI-1 staining of the tumor cells in addition to strong staining of the stromal endothelial cell nuclei. With the exception of lymphoblastic lymphomas, FLI-1 positivity was not seen in the other small round blue cell tumors examined. These findings show that, in contrast to Ewing sarcoma, small cell osteosarcoma and mesenchymal chondrosarcoma lack FLI-1 immunoreactivity. FLI-1 is therefore useful in the differential diagnosis of small round blue cell tumors of the bone.
The coordination of the therapeutically interesting AuCl(PEt3) to the de novo designed peptide, TRIL23C, under aqueous conditions, is reported here. TRIL23C represents an ideal model to investigate ...the binding of AuCl(PEt3) to small proteins in an effort to develop novel gold(I) phosphine peptide adducts capable of mimicking biological recognition and targeting. This is due to the small size of TRIL23C (30 amino acids), yet stable secondary and tertiary fold, symmetric nature and the availability of only one thiol binding site. AuCl(PEt3) was found to react readily with the Cys side chain in a 1:1 ratio as confirmed by UV-visible, 31P NMR and mass spectrometry. Circular dichroism confirmed that the coiled coil structure was retained on coordination of the {Au(PEt3)}+ unit. Redesign of the exterior of TRIL23C based on a biologically relevant recognition sequence found in GCN4, did not alter the coordination chemistry of AuCl(PEt3). To the best of our knowledge, this represents the first report on the coordination of gold(I) phosphine compounds to de novo designed peptides, and could lead to the generation of novel gold(I) phosphine peptide therapeutics in the future.
The coordination of gold triethylphosphine through the cysteine side chain of a de novo designed coiled coil peptide has been investigated under mild aqueous conditions. The secondary structure is retained in the gold–peptide adduct and could ultimately be exploited to develop novel gold–peptide therapeutics capable of biomolecular recognition. Display omitted
IntroductionThe independent and causal cardiovascular disease risk factor lipoprotein(a) (Lp(a)) is elevated in >1.5 billion individuals worldwide, but studies have prioritised European ...populations.MethodsHere, we examined how ancestrally diverse studies could clarify Lp(a)’s genetic architecture, inform efforts examining application of Lp(a) polygenic risk scores (PRS), enable causal inference and identify unexpected Lp(a) phenotypic effects using data from African (n=25 208), East Asian (n=2895), European (n=362 558), South Asian (n=8192) and Hispanic/Latino (n=8946) populations.ResultsFourteen genome-wide significant loci with numerous population specific signals of large effect were identified that enabled construction of Lp(a) PRS of moderate (R2=15% in East Asians) to high (R2=50% in Europeans) accuracy. For all populations, PRS showed promise as a ‘rule out’ for elevated Lp(a) because certainty of assignment to the low-risk threshold was high (88.0%–99.9%) across PRS thresholds (80th–99th percentile). Causal effects of increased Lp(a) with increased glycated haemoglobin were estimated for Europeans (p value =1.4×10−6), although inverse effects in Africans and East Asians suggested the potential for heterogeneous causal effects. Finally, Hispanic/Latinos were the only population in which known associations with coronary atherosclerosis and ischaemic heart disease were identified in external testing of Lp(a) PRS phenotypic effects.ConclusionsOur results emphasise the merits of prioritising ancestral diversity when addressing Lp(a) evidence gaps.
The COVID-19 pandemic caused by SARS-CoV-2 exposed a global problem, as highly effective vaccines are challenging to produce and distribute, particularly in regions with limited resources and ...funding. As an alternative, immunoglobulins produced in eggs of immunized hens (IgY) can be a simple and inexpensive source for a topical and temporary prophylaxis. Here, we developed a method to extract and purify IgY antibodies from egg yolks of hens immunized against viral pathogen-derived proteins using low-cost, readily available materials, for use in resource-limited settings.
Existing protocols for IgY purification and equipment were modified, including extraction from yolks and separation of water-soluble IgY using common household reagents and tools. A replacement for a commercial centrifuge was developed, using a home food processor equipped with a 3D printed adapter to enable IgY precipitation. IgY purification was verified using standard gel electrophoresis and Western blot analyses.
We developed a step-by-step protocol for IgY purification for two settings in low- and middle-income countries (LMIC): a local laboratory, where commercial centrifuges are available, or a more rural setting, where an alternative for expensive centrifuges can be used. Gel electrophoresis and Western blot analyses confirmed that the method produced highly enriched IgY preparation; each commercial egg produced ~ 90 mg of IgY. We also designed a kit for IgY production in these two settings and provided a cost estimate of the kit.
IgY purified from eggs of immunized local hens can offer a fast and affordable prophylaxis, provided that purification can be performed in a resource-limited setting. Here, we created a low-cost method that can be used anywhere where electricity is available using inexpensive, readily available materials in place of costly, specialized laboratory equipment and chemicals. This procedure can readily be used now to make an anti-SARS-CoV-2 prophylaxis in areas where vaccines are unavailable, and can be modified to combat future threats from viral epidemics and pandemics.
Poorly differentiated chordoma (PDC) is a recently recognized subtype of chordoma characterized by expression of the embryonic transcription factor, brachyury, and loss of INI1. PDC primarily affects ...children and is associated with a poor prognosis and limited treatment options. Here we describe the molecular and immune tumour microenvironment profiles of two paediatric PDCs produced using whole-genome, transcriptome and whole-genome bisulfite sequencing (WGBS) and multiplex immunohistochemistry. Our analyses revealed the presence of tumour-associated immune cells, including CD8+ T cells, and expression of the immune checkpoint protein, PD-L1, in both patient samples. Molecular profiling provided the rationale for immune checkpoint inhibitor (ICI) therapy, which resulted in a clinical and radiographic response. A dominant T cell receptor (TCR) clone specific for a brachyury peptide-MHC complex was identified from bulk RNA sequencing, suggesting that targeting of the brachyury tumour antigen by tumour-associated T cells may underlie this clinical response to ICI. Correlative analysis with rhabdoid tumours, another INI1-deficient paediatric malignancy, suggests that a subset of tumours may share common immune phenotypes, indicating the potential for a therapeutically targetable subgroup of challenging paediatric cancers.
Pulmonary arterial (PA) stiffness is associated with increased mortality in patients with pulmonary hypertension (PH); however, the role of PA stiffening in the pathogenesis of PH remains elusive. ...Here, we show that distal vascular matrix stiffening is an early mechanobiological regulator of experimental PH. We identify cyclooxygenase-2 (COX-2) suppression and corresponding reduction in prostaglandin production as pivotal regulators of stiffness-dependent vascular cell activation. Atomic force microscopy microindentation demonstrated early PA stiffening in experimental PH and human lung tissue. Pulmonary artery smooth muscle cells (PASMC) grown on substrates with the stiffness of remodeled PAs showed increased proliferation, decreased apoptosis, exaggerated contraction, enhanced matrix deposition, and reduced COX-2-derived prostanoid production compared with cells grown on substrates approximating normal PA stiffness. Treatment with a prostaglandin I
analog abrogated monocrotaline-induced PA stiffening and attenuated stiffness-dependent increases in proliferation, matrix deposition, and contraction in PASMC. Our results suggest a pivotal role for early PA stiffening in PH and demonstrate the therapeutic potential of interrupting mechanobiological feedback amplification of vascular remodeling in experimental PH.
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