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The selective Bruton tyrosine kinase (BTK) inhibitor poseltinib has been shown to inhibit the BCR signal transduction pathway and cytokine production in B cells (Park et al. Arthritis Res. Ther. 18, 91, https://doi.org/10.1186/s13075-016-0988-z , 2016). This study describes the translation of nonclinical research studies to a phase I clinical trial in healthy volunteers in which pharmacokinetics (PKs) and pharmacodynamics (PDs) were evaluated for dose determination. The BTK protein kinase inhibitory effects of poseltinib in human peripheral blood mononuclear cells (PBMCs) and in rats with collagen-induced arthritis (CIA) were evaluated. High-dimensional phosphorylation analysis was conducted on human immune cells such as B cells, CD8 + memory cells, CD4 + memory cells, NK cells, neutrophils, and monocytes, to map the impact of poseltinib on BTK/PLC and AKT signaling pathways. PK and PD profiles were evaluated in a first-in-human study in healthy donors, and a PK/PD model was established based on BTK occupancy. Poseltinib bound to the BTK protein and modulated BTK phosphorylation in human PBMCs. High-dimensional phosphorylation analysis of 94 nodes showed that poseltinib had the highest impact on anti-IgM + CD40L stimulated B cells, however, lower impacts on anti-CD3/CD-28 stimulated T cells, IL-2 stimulated CD4 + T cells and NK cells, M-CSF stimulated monocytes, or LPS-induced granulocytes. In anti-IgM + CD40L stimulated B cells, poseltinib inhibited the phosphorylation of BTK, AKT, and PLCγ2. Moreover, poseltinib dose dependently improved arthritis disease severity in CIA rat model. In a clinical phase I trial for healthy volunteers, poseltinib exhibited dose-dependent and persistent BTK occupancy in PBMCs of all poseltinib-administrated patients in the study. More than 80% of BTK occupancy at 40 mg dosing was maintained for up to 48 h after the first dose. A first-in-human healthy volunteer study of poseltinib established target engagement with circulating BTK protein. Desirable PK and PD properties were observed, and a modeling approach was used for rational dose selection for subsequent trials. Poseltinib was confirmed as a potential BTK inhibitor for the treatment of autoimmune diseases.Trial registration: This article includes the results of a clinical intervention on human participants [NCT01765478]., (© 2021. The Author(s).)

Pre-clinical murine models are critical for translating drug candidates from the bench to the bedside. There is interest in better understanding how anti-human CD3 therapy works based on recent longitudinal studies of short-term administration. Although several models have been created in this pursuit, each have their own advantages and disadvantages in Type-1 diabetes. In this study, we report a murine genetic knock-in model which expresses both a murine and a humanized-CD3ε-exon, rendering it sensitive to manipulation with anti-human CD3. These huCD3εHET mice are viable and display no gross abnormalities. Specifically, thymocyte development and T cell peripheral homeostasis is unaffected. We tested immune functionality of these mice by immunizing them with T cell-dependent antigens and no differences in antibody titers compared to wild type mice were recorded. Finally, we performed a graft-vs-host disease model that is driven by effector T cell responses and observed a wasting disease upon transfer of huCD3εHET T cells. Our results show a viable humanized CD3 murine model that develops normally, is functionally engaged by anti-human CD3 and can instruct on pre-clinical tests of anti-human CD3 antibodies., Competing Interests: The authors have read the journal’s policy and the authors of this manuscript have the following competing interests: the following authors were employed by Eli Lilly and Company at the time of conducting the research and may own stock: JC, YTK, NH, ALG, APM, and RJB. The following authors were employed by Macrogenics at the time of conducting the research and may own stock: PAM and EB. APM is an employee of Boehringer Ingelheim Pharmaceuticals Inc. However, this affiliation was not held at the time the study was conducted. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare.

Breaking tolerance is a key event leading to autoimmunity, but the exact mechanisms responsible for this remain uncertain. Here we show that the alarmin IL-33 is able to drive the generation of autoantibodies through induction of the B cell survival factor BAFF. A temporary, short-term increase in IL-33 results in a primary (IgM) response to self-antigens. This transient DNA-specific autoantibody response was dependent on the induction of BAFF. Notably, radiation resistant cells and not myeloid cells, such as neutrophils or dendritic cells were the major source of BAFF and were critical in driving the autoantibody response. Chronic exposure to IL-33 elicited dramatic increases in BAFF levels and resulted in elevated numbers of B and T follicular helper cells as well as germinal center formation. We also observed class-switching from an IgM to an IgG DNA-specific autoantibody response. Collectively, the results provide novel insights into a potential mechanism for breaking immune-tolerance via IL-33-mediated induction of BAFF.

The aim of this study was to evaluate the incidence, clinical features, and risk factors of sustaining inferior rectus (IR) palsy in a group of pediatric patients with orbital floor blowout fractures. We performed a retrospective case review of sequential cases of pediatric orbital floor blowout fractures (<18 years old) from 2000 to 2013 in a tertiary ophthalmic center in Singapore. A total of 48 patients were included in our study, of whom 5 had IR palsy (10.4%). Patients with IR palsy had a higher mean age (16.4 ± 1.5 years) compared with patients without IR palsy (12.4 ±3.3 years), had significantly ( p  < 0.05) worse preoperative motility, and had significantly greater proportion developing postoperative hypertropia (100%) compared with patients without IR palsy (4.7%). Our series of pediatric blowout fractures demonstrated IR palsy prevalence and clinical features for IR palsy which may be distinct to the pediatric group.

Polydimethylsiloxane (PDMS) has been extensively exploited to study stem cell physiology in the field of mechanobiology and microfluidic chips due to their transparency, low cost and ease of fabrication. However, its intrinsic high hydrophobicity renders a surface incompatible for prolonged cell adhesion and proliferation. Plasma-treated or protein-coated PDMS shows some improvement but these strategies are often short-lived with either cell aggregates formation or cell sheet dissociation. Recently, chemical functionalization of PDMS surfaces has proved to be able to stabilize long-term culture but the chemicals and procedures involved are not user- and eco-friendly. Herein, we aim to tailor greener and biocompatible PDMS surfaces by developing a one-step bio-inspired polydopamine coating strategy to stabilize long-term bone marrow stromal cell culture on PDMS substrates. Characterization of the polydopamine-coated PDMS surfaces has revealed changes in surface wettability and presence of hydroxyl and secondary amines as compared to uncoated surfaces. These changes in PDMS surface profile contribute to the stability in BMSCs adhesion, proliferation and multipotency. This simple methodology can significantly enhance the biocompatibility of PDMS-based microfluidic devices for long-term cell analysis or mechanobiological studies.

Purpose: The aim of this study was to report the characteristics and ophthalmic manifestations associated with dengue fever (DF) in the 2005-2006 DF epidemic in Singapore., Methods: A retrospective case series., Results: Eleven patients (7 males, 4 females) who were 14 to 40 years old (mean age, 27.3 years) with visual complaints occurring 1 to 2 weeks after the onset of DF underwent a complete ophthalmologic examination. The presenting visual acuity (VA) ranged from 6/6 to counting fingers. Five patients had bilateral eye involvement. Ophthalmic findings included retinal hemorrhages (15 eyes), cotton wool spots (15 eyes), retinal pigment epithelium alterations (5 eyes), optic disc swelling (3 eyes), foveolitis (3 eyes), and hyperemia (2 eyes). Impaired color vision was noted in 12 eyes. Visual field defects were noted in 13 eyes; 4 of these eyes were asymptomatic. Fundus fluorescein angiography was done in 9 patients; the findings include choroidal hyperfluorescence (9 eyes), blocked fluorescence (8 eyes), and capillary nonperfusion (1 eye). In 2 eyes, it was normal. Electrophysiology confirmed optic neuropathy in 3 eyes and maculopathy in 1 eye. The follow-up ranged from 12 days to 12 months. The VA at the last follow-up ranged from no light perception to 6/6. Visual field abnormalities were noted in 5 eyes despite a normal VA., Conclusions: Dengue fever is associated with a wide spectrum of ophthalmic manifestations. Rarely, ophthalmic complications in DF can result in permanent visual impairment. Further work can be done to elucidate the relationship between systemic and ocular manifestations of DF.

Introduction: In recent years there has been a spurt of peer-reviewed publications on the ophthalmic complications of dengue fever. The authors aim to review the ocular manifestations, utility of relevant diagnostic tests, management, prognosis, and sequelae of dengue-related ocular complications., Methods: A comprehensive literature search was conducted on Medline, PubMed, and Google Scholar databases. Only articles published or translated into the English language were considered, and key data from the full article of each paper was extracted and evaluated., Results: The authors' search yielded 29 articles. A total of 686 patients with a mean age of 33.4 years (range 14-73 years) were included. Blurring of vision was the most common presenting symptom followed by scotoma. Ocular findings were mostly seen in the posterior pole of the fundus, manifesting as retinal hemorrhages, macular edema, foveolitis, vasculitis, and optic neuropathy. Most patients with dengue-related ophthalmic complications recover spontaneously without any treatment. Patients with severe visual loss or bilateral involvement were treated with systemic steroids and occasionally immunoglobulins. Prognosis of dengue-related ophthalmic complications is favorable; almost all patients had normal or showed improvement in visual acuity and complete resolution of dengue ophthalmic complications was noted in almost all cases., Conclusion: The pathological process of dengue ophthalmic complications is complex and clinical manifestations varied. Ophthalmic complications are usually seen in young adults who often present at the nadir of thrombocytopenia. Despite good recovery of vision and resolution of clinical signs in most patients, ophthalmologists and physicians should be aware and vigilant as isolated reports of cases of dengue ophthalmic complications with poor visual acuity refractory to treatment have been reported.

Background: This study was performed to report the prevalence of lower urinary tract symptoms (LUTSs), bother and related quality of life (QOL) and health-seeking behaviour in men 40 years old or older in Singapore., Methods: The study was a community-based, cross-sectional study targeting males 40 years and above in the township of Bedok North, Singapore. An interviewer-administered questionnaire inclusive of the International Prostate Symptom Score (IPSS) was used to score symptom severity objectively., Results: There were a total of 644 respondents. In all, 16.5% of patients with LUTS had moderate-to-severe LUTS (IPSS ≥8). The prevalence of LUTS was found to be significantly associated with age, diabetes mellitus and hypertension. The impact on QOL because of LUTS was proportional to the severity of LUTS. Nocturia was associated with the most bother. Out of the respondents with moderate-to-severe LUTS, 71.7% of them did not seek help for their symptoms., Conclusion: The investigators thus conclude that LUTS is common in our population and has significant impact on QOL. However, <30% of patients with moderate-to-severe LUTS sought medical attention for symptoms. We believe that steps should be taken to increase public awareness and variability of treatment options to address this problem facing the growing number of elderly men in the population.

Tregs expressing the transcription factor Foxp3 suppress self-reactive T cells, prevent autoimmunity, and help contain immune responses to foreign antigens, thereby limiting the potential for inadvertent tissue damage. Mutations in the FOXP3 gene result in Treg deficiency in mice and humans, which leads to the development of a multisystem autoimmune inflammatory disease. The contribution of dysregulated innate immune responses to the pathogenesis of Foxp3 deficiency disease is unknown. In this study, we examined the role of microbial signals in the pathogenesis of Foxp3 deficiency disease by studying Foxp3 mutant mice that had concurrent deficiencies in TLR signaling pathways. Global deficiency of the common TLR adaptor MyD88 offered partial protection from Foxp3 deficiency disease. Specifically, it protected from disease at the environmental interfaces of the skin, lungs, and gut. In contrast, systemic disease, in the form of unrestrained lymphoproliferation, continued unabated. The effect of MyD88 deficiency at environmental interfaces involved the disruption of chemokine gradients that recruit effector T cells and DCs, resulting in their entrapment in secondary lymphoid tissues. These results suggests that Tregs have a key role in maintaining tolerance at host-microbial interfaces by restraining tonic MyD88-dependent proinflammatory signals. Moreover, microbial factors may play a substantial role in the pathogenesis of human autoimmune disease resulting from Treg deficiency.

Using the Unc93b1 3d mutation that selectively abolishes nucleic acid-binding Toll-like receptor (TLR) (TLR3, -7, -9) signaling, we show these endosomal TLRs are required for optimal production of IgG autoAbs, IgM rheumatoid factor, and other clinical parameters of disease in 2 lupus strains, B6-Fas(lpr) and BXSB. Strikingly, treatment with lipid A, an autoAb-inducing TLR4 agonist, could not overcome this requirement. The 3d mutation slightly reduced complete Freund's adjuvant (CFA)-mediated antigen presentation, but did not affect T-independent type 1 or alum-mediated T-dependent humoral responses or TLR-independent IFN production induced by cytoplasmic nucleic acids. These findings suggest that nucleic acid-sensing TLRs might act as an Achilles' heel in susceptible individuals by providing a critical pathway by which relative tolerance for nucleic acid-containing antigens is breached and systemic autoimmunity ensues. Importantly, this helps provide an explanation for the high frequency of anti-nucleic acid Abs in lupus-like systemic autoimmunity.

Background: Salivary eosinophil cationic protein (ECP) level has the potential to be an assessment tool for asthma. Its measurement is not well-established and needs standardization. We studied how passive drool (PD) and two commercial devices, Salivette(®) (cotton-based device) and Sorbette(®) (cellulose-cotton-based-device), may affect ECP levels during collection among healthy subjects., Methods: Study I (n = 10) involved direct sampling of healthy adult subjects with Salivette(®) and Sorbette(®). Study II (n = 33) involved 'indirect' sampling of previously collected PD by both devices., Results: In study I, ECP levels were detected in all PD samples but only in three with Sorbette(®) and none with Salivette(®) (collection order: PD, Sorbette(®) and Salivette(®)). We changed the order of collection (Salivette(®), Sorbette(®), PD) and the results were similar (ECP levels detected in all PD samples, three with Sorbette(®) and only one with Salivette(®)). In study II, ECP levels in saliva collected by PD was 12.8 μg/L (median) and using Sorbette(®) and Salivette(®) were < 2.0 μg/L and 3.4 μg/L respectively (p < 0.01). ECP levels in PD correlated with Sorbette(®) (r(s) = 0.79, p < 0.01) and Salivette(®) (r(s) = 0.62, p < 0.01)., Conclusion: Compared to PD, saliva collected using cotton or cellulose-based collection devices resulted in lower measurable ECP levels.

Incomplete Freund's adjuvant (IFA) serves as a carrier for water-in-oil emulsion (W/O) vaccines. The stability of such emulsions greatly affects vaccine safety and efficacy since continued presence of antigen depots at lymphoid organs releasing low-level antigens is known to stimulate a potent immune response and high-level systemic release of antigens can lead to tolerance. W/O emulsions for the purpose of clinical and laboratory peptide-based vaccinations have been prepared using the techniques of syringe extrusion, vortex or high-speed homogenization. There is no consensus in the field over which technique would be best to use and no immunological data are available that compare the three techniques. In this study, we compared the immune responses induced by a peptide-based vaccine prepared using vortex, syringe-extrusion and homogenization. The vaccination led to tumor rejection by mice vaccinated with the peptide-based vaccine prepared using all three techniques. The immunological data from the in vivo cytotoxicity assay showed a trend for lower responses and a higher variability and greater range in the immune responses induced by a vaccine that was emulsified by the vortex or homogenizer techniques as compared to the syringe-extrusion technique. There were statistically significant lower numbers of IFNgamma-secreting cells induced when the mice were vaccinated with a peptide-based vaccine emulsion prepared using the vortex compared to the syringe-extrusion technique. At a suboptimal vaccine dose, the mice vaccinated with a peptide-based vaccine emulsion prepared using the vortex technique had the largest tumors compared to the syringe-extrusion or the homogenizer technique. In the setting of a busy pharmacy that prepares peptide-based vaccine emulsions for clinical studies, the vortex technique can still be used but we urge investigators to take special care in their choice of mixing vessels for the vortex technique as that can influence the stability of the emulsion. However, in instances where the optimal dose is unknown, we caution investigators against using the vortex technique to prepare the peptide-based vaccine emulsions. Overall, we report that all three techniques can be used to prepare peptide-based vaccine emulsions under optimal dose conditions and we discuss important details regarding the proper preparation of the emulsions.

The emergence and spread of multiresistant methicillin-resistant Staphylococcus aureus (MRSA) strains, especially those resistant to fusidic acid and rifampicin, in Malaysian hospitals is of concern. In this study DNA fingerprinting by PFGE was performed on fusidic acid- and rifampicin-resistant isolates from Malaysian hospitals to determine the genetic relatedness of these isolates and their relationship with the endemic MRSA strains. In all, 32 of 640 MRSA isolates from 9 Malaysian hospitals were resistant to fusidic acid and rifampicin. Seven PFGE types (A, ZC, ZI, ZJ, ZK, ZL and ZM) were observed. The commonest type was type ZC, seen in 72% of isolates followed by type A, seen in 13%. Each of the other types (ZI, ZJ, ZK, ZL and ZM) was observed in a single isolate. Each type, even the commonest, was found in only one hospital. This suggests that the resistant strains had arisen from individual MRSA strains in each hospital and not as a result of the transmission of a common clone.

The incidence of food-borne salmonellosis due to Salmonella enterica serotype Weltevreden is reported to be on the increase in Malaysia. The pulsed-field gel electrophoresis (PFGE) subtyping method was used to assess the extent of genetic diversity and clonality of Salmonella serotype Weltevreden strains from humans and the environment. PFGE of XbaI-digested chromosomal DNA from 95 strains of Salmonella serotype Weltevreden gave 39 distinct profiles with a wide range of Dice coefficients (0.27 to 1.00), indicating that PFGE is very discriminative and that multiple clones of Salmonella serotype Weltevreden exist among clinical and environmental isolates. Strains of one dominant pulsotype (pulsotype X1/X2) appeared to be endemic in this region, as they were consistently recovered from humans with salmonellosis between 1996 and 2001 and from raw vegetables. In addition, the sharing of similar PFGE profiles among isolates from humans, vegetables, and beef provides indirect evidence of the possible transmission of salmonellosis from contaminated raw vegetables and meat to humans. Furthermore, the recurrence of PFGE profile X21 among isolates found in samples of vegetables from one wet market indicated the persistence of this clone. The environment in the wet markets may represent a major source of cross-contamination of vegetables with Salmonella serotype Weltevreden. Antibiotic sensitivity tests showed that the clinical isolates of Salmonella serotype Weltevreden remained drug sensitive but that the vegetable isolates were resistant to at least two antibiotics. To the best of our knowledge, this is the first study to compare clinical and environmental isolates of Salmonella serotype Weltevreden in Malaysia.

Aim of Study: To determine and compare the pulsed-field gel electrophoresis (PFGE) patterns of endemic MRSA strains in 2 major Malaysian hospitals and to compare the PFGE patterns with antibiotypes of the strains studied., Methods: Fifty-six MRSA strains selected randomly between September 1997 and July 1998 from Hospital Queen Elizabeth (HQE) and Hospital Umum Sarawak (HUS) were tested for antimicrobial resistance and DNA fingerprinting was carried out by pulsed-field gel electrophoresis (PFGE) technique., Results: Seven PFGE types were recognised (A, B, C, D, E and F). All 7 PFGE types were observed in HQE while only 2 PFGE types (B, C) were noted in HUS strains. There is a predominance of a single PFGE pattern (type B) in both hospitals, as seen in 46% of HQE strains and 89% of HUS strains. Subtype B2 was the commonest subtype in HQE while subtype B1 predominated in HUS. Strains resistant to fusidic acid and rifampicin exhibited PFGE type F that is unique to HQE. All strains were resistant to penicillin, erythromycin, cotrimoxazole, tetracycline and gentamicin. Strains with the same antibiotic susceptibility pattern can be different PFGE types., Conclusion: Molecular typing of the MRSA by PFGE is a useful tool in the study of endemic strains present in an institution. Strains in HQE were found to be more heterogeneous than HUS strains. Common PFGE types can also be seen in both hospitals suggesting that some of the strains was genetically related and has propagated within and between the 2 hospitals. Our findings also indicate that the relationship between antibiotic susceptibility and PFGE patterns was not close and antibiograms should not be relied upon for typing strains in epidemiological studies. By knowing the DNA fingerprints of the isolates endemic in each hospital, the spread of MRSA with a particular PFGE type can be monitored within and between hospitals.