Recombinant Full-length Plasmodium falciparum Circumsporozoite Protein-Based Vaccine Adjuvanted With Glucopyranosyl Lipid A-Liposome Quillaja saponaria 21: Results of Phase 1 Testing With Malaria Challenge.

BACKGROUND: Malaria is preventable yet causes >600 000 deaths annually. RTS,S, the first marketed malaria vaccine, has modest efficacy, but improvements are needed for eradication. METHODS: We conducted an open-label, dose escalation phase 1 study of a full-length recombinant circumsporozoite protein vaccine (rCSP) administered with adjuvant glucopyranosyl lipid A-liposome Quillaja saponaria 21 formulation (GLA-LSQ) on days 1, 29, and 85 or 1 and 490 to healthy, malaria-naive adults. The primary end points were safety and reactogenicity. The secondary end points were antibody responses and Plasmodium falciparum parasitemia after homologous controlled human malaria infection. RESULTS: Participants were enrolled into 4 groups receiving rCSP/GLA-LSQ: 10 µg × 3 (n = 20), 30 µg × 3 (n = 10), 60 µg × 3 (n = 10), or 60 µg × 2 (n = 9); 10 participants received 30 µg rCSP alone × 3, and there were 6 infectivity controls. Participants experienced no serious adverse events. Rates of solicited and unsolicited adverse events were similar among groups. All 26 participants who underwent controlled human malaria infection 28 days after final vaccinations developed malaria. Increasing vaccine doses induced higher immunoglobulin G titers but did not achieve previously established RTS,S benchmarks. CONCLUSIONS: rCSP/GLA-LSQ had favorable safety results. However, tested regimens did not induce protective immunity. Further investigation could assess whether adjuvant or schedule adjustments improve efficacy. CLINICAL TRIALS REGISTRATION: NCT03589794.

An inducible model for medial calcification based on matrix Gla protein deficiency.

Calcific deposits in the arterial media have been associated with a number of metabolic and genetic disorders including diabetes, chronic kidney disease and generalized arterial calcification of infancy. The loss of Matrix Gla protein (MGP) leads to medial elastic lamina calcification (elastocalcinosis) in both humans and animal models. While MGP-deficient (Mgp-/-) mice have been used as a reliable model to study medial elastocalcinosis, these mice are difficult to maintain because of their fragility. Also, these mice are unsuitable for long-term calcification studies in relation to age and sex as most often they die prematurely. In order to circumvent these problems we generated Mgp-/-;ApoE-FGF23 mice, which in addition to the ablation of Mgp alleles, carries a transgene expressing the phosphaturic hormone FGF23. Increased FGF23 levels in the circulation and ensuing hypophosphatemia in these mice lead to a complete prevention of medial calcification until late adulthood. Interestingly, upon feeding a high phosphorus diet for 10 days, we were able to induce medial calcification in 3-week-old Mgp-/-;ApoE-FGF23 mice. Our mineral analyses showed that the calcific deposits in these mice were Our mineral analyses showed that the Ca/P % in the calcific deposits in these mice were comparable to that of 5-week-old Mgp-/- mice although the level of crystallinity differed. The aorta explants from Mgp-/-;ApoE-FGF23 mice resulted in elastocalcinosis in the presence of 2mM phosphate in the culture medium which was completely prevented by pyrophosphate analogue alendronate. Mgp-/-;ApoE-FGF23 mice will be suitable for future in vivo or ex vivo studies examining the effects of age, sex and mineralization inhibitors on medial elastocalcinosis.

The c.-265G>A GLA gene promoter variant causes Fabry disease: The hidden culprit identified.

Here, we report the identification and functional characterization of a novel GLA variant, not detectable by routine molecular tests, in a family with FD suspicion.

Gla-domain mediated targeting of externalized phosphatidylserine for intracellular delivery.

Phosphatidylserine (PS) is a negatively charged phospholipid normally localized to the inner leaflet of the plasma membrane of cells but is externalized onto the cell surface during apoptosis as well as in malignant and infected cells. Consequently, PS may comprise an important molecular target in diagnostics, imaging, and targeted delivery of therapeutic agents. While an array of PS-binding molecules exist, their utility has been limited by their inability to internalize diagnostic or therapeutic payloads. We describe the generation, isolation, characterization, and utility of a PS-binding motif comprised of a carboxylated glutamic acid (GLA) residue domain that both recognizes and binds cell surface-exposed PS, and then unlike other PS-binding molecules is internalized into these cells. Internalization is independent of the traditional endosomal-lysosomal pathway, directly entering the cytosol of the target cell rapidly. We demonstrate that this PS recognition extends to stem cells and that GLA-domain-conjugated probes can be detected upon intravenous administration in animal models of infectious disease and cancer. GLA domain binding and internalization offer new opportunities for specifically targeting cells with surface-exposed PS for imaging and delivery of therapeutics.

Effectiveness and safety of insulin glargine 300 U/mL in insulin-naïve individuals according to diabetes duration: Results from the REALI European pooled data analysis.

AIM: To evaluate the effectiveness and safety of insulin glargine 300 U/mL (Gla-300) initiation according to diabetes duration (DD). MATERIALS AND METHODS: We analysed patient-level data from 2381 insulin-naïve individuals with type 2 diabetes (T2D), of whom 2349 (98.7%) were treated with Gla-300 for 24 weeks. Of the 2381 participants, 1048 (44.0%) had a DD of less than 8 years and 1333 (56.0%) had a DD of 8 years or longer. We further analysed the subgroups of participants having a DD of less than 4 years (N = 450), 4-8 years (N = 598), 8-12 years (N = 627) and 12 years or longer (N = 706). RESULTS: Mean ± standard deviation age was 60.2 ± 9.0 years in participants with a DD less than 8 years and 64.2 ± 8.8 years in those with a DD of 8 years or longer. At 24 weeks of Gla-300 therapy, HbA1c improved with a least-squares (LS) mean change from baseline of -1.88% (95% confidence interval [CI], -1.95 to -1.80) and -1.71% (95% CI, -1.77 to -1.65), respectively, resulting in a LS mean difference between groups of 0.17% (95% CI, 0.07 to 0.26; P = .0005). In the subgroup analysis, LS mean HbA1c reduction from baseline to week 24 was highest in participants with a DD of less than 4 years and lowest in participants with a DD of 12 years or longer. Overall, incidences of symptomatic and severe hypoglycaemia were low, irrespective of DD, without body weight changes. CONCLUSIONS: Gla-300 was effective and safe in insulin-naïve individuals with T2D, regardless of DD. Improvement in HbA1c was greater when Gla-300 was initiated in participants with a DD of less than 4 years, although the difference between the groups was modest.

Deciphering Breast Origin in Malignant Effusions: The Diagnostic Utility of an MGP, GATA-3, and TRPS-1 Immunocytochemical Panel.

INTRODUCTION: Defining the origin of metastatic cancer is crucial for establishing an optimal treatment strategy, especially when obtaining sufficient tissue from secondary malignancies is limited. While cytological examination is often used in this diagnostic setting, morphologic analysis alone often fails to differentiate metastases derived from the breast from other primaries. The hormone receptor, human epidermal growth factor receptor-2, gross cystic disease fluid protein 15, and mammaglobin immunohistochemistry are often used to diagnose metastatic breast cancer. However, their effectiveness decreases in estrogen receptor (ER)-negative breast cancers, including the triple-negative breast cancer (TNBC) subtype. METHODS: We conducted a comprehensive evaluation of GATA-binding protein 3 (GATA-3), trichorhinophalangeal syndrome type 1 (TRPS-1), and Matrix Gla Protein (MGP) immunochemistry across 140 effusion cytology specimens with metastatic adenocarcinoma derived from various primaries, including the breast, colon, pancreaticobiliary, lung, tubo-ovarian, and stomach. RESULTS: The expression rates of these immunomarkers were significantly higher in metastatic cancers originating from the breast than other primaries. In TNBC, TRPS-1 (80.00%) and MGP (65.00%) exhibited higher positivity rates compared to GATA-3 (40.00%). Additionally, our data suggest that an immunohistochemical panel comprising MGP, GATA-3, and TRPS-1 significantly enhances the detection of metastatic breast cancer in effusion cytology specimens, including TNBC in particular. When considering dual-marker positivity, the diagnostic accuracy was found to be 89.29% across all breast cancer subtypes and 92.93% for TNBC. CONCLUSIONS: MGP appears to be a robust marker for identifying metastatic breast cancer in malignant effusions, especially TNBC. MGP notably enhances diagnostic accuracy when incorporated together with GATA-3 and TRPS-1 in an immunohistochemical panel.

Vitamin K: a potential missing link in critical illness-a scoping review.

BACKGROUND: Vitamin K is essential for numerous physiological processes, including coagulation, bone metabolism, tissue calcification, and antioxidant activity. Deficiency, prevalent in critically ill ICU patients, impacts coagulation and increases the risk of bleeding and other complications. This review aims to elucidate the metabolism of vitamin K in the context of critical illness and identify a potential therapeutic approach. METHODS: In December 2023, a scoping review was conducted using the PRISMA Extension for Scoping Reviews. Literature was searched in PubMed, Embase, and Cochrane databases without restrictions. Inclusion criteria were studies on adult ICU patients discussing vitamin K deficiency and/or supplementation. RESULTS: A total of 1712 articles were screened, and 13 met the inclusion criteria. Vitamin K deficiency in ICU patients is linked to malnutrition, impaired absorption, antibiotic use, increased turnover, and genetic factors. Observational studies show higher PIVKA-II levels in ICU patients, indicating reduced vitamin K status. Risk factors include inadequate intake, disrupted absorption, and increased physiological demands. Supplementation studies suggest vitamin K can improve status but not normalize it completely. Vitamin K deficiency may correlate with prolonged ICU stays, mechanical ventilation, and increased mortality. Factors such as genetic polymorphisms and disrupted microbiomes also contribute to deficiency, underscoring the need for individualized nutritional strategies and further research on optimal supplementation dosages and administration routes. CONCLUSIONS: Addressing vitamin K deficiency in ICU patients is crucial for mitigating risks associated with critical illness, yet optimal management strategies require further investigation. IMPACT RESEARCH: To the best of our knowledge, this review is the first to address the prevalence and progression of vitamin K deficiency in critically ill patients. It guides clinicians in diagnosing and managing vitamin K deficiency in intensive care and suggests practical strategies for supplementing vitamin K in critically ill patients. This review provides a comprehensive overview of the existing literature, and serves as a valuable resource for clinicians, researchers, and policymakers in critical care medicine.

Fabry Disease with Genetic Variants of Unknown Significance and Concomitant Immunoglobulin A Nephropathy.

INTRODUCTION: The diagnosis of Fabry disease (FD) with genetic variants of unknown significance (VUSs) is relatively difficult. We explored patients with novel VUS variants and concomitant immunoglobulin A nephropathy (IgAN) to improve the understanding of VUS. METHODS: The study retrospectively investigated patients with genetically confirmed FD. Probands with VUS were selected from the database of FD patients who underwent genetic analysis. Demographic, clinicopathological, and laboratory data from probands and family members were collected and analyzed. RESULTS: Fourteen probands and their family members were included in the study. The probands were divided into group 1 (patients with VUS, n = 5) and group 2 (patients with pathologic/likely pathologic variants, n = 9). The group 1 included 2 missense mutations and 1 deletion mutation, while the group 2 included 6 missense mutations and 2 deletion mutations. There were no significant differences in gender, age, serum creatinine, eGFR, and proteinuria between the two groups. IgA deposition with myeloid bodies was found in all VUS patients. The cardiac involvement in group 2 was more severe than that in group 1. Seven families performed the pedigree analysis, and after the comprehensive evaluation, two GLA variants (c.479C>A, p.Ala160Asp; c.1032-1058 del, p.Ser345_Met353del) were upgraded from VUS to the likely pathogenic. CONCLUSION: The clinical manifestations of FD are heterogeneous. FD often coexists with nephrotic disorders, such as IgAN and MCD. Comprehensive evaluation, especially tissue-specific biopsy, is necessary for patients with GLA-VUSs. Two GLA variants (c.479C>A, p.Ala160Asp; c.1032-1058 del, p.Ser345_Met353del) were upgraded from VUS to the likely pathogenic after the comprehensive evaluation.

Fibronectin/α5 Integrin Contribute to Hypertension-Associated Arterial Ageing and Calcification through Affecting BMP2/MGP Imbalance and Enhancing Vascular Smooth Muscle Cell Phenotypic Transformation.

INTRODUCTION: Hypertension can accelerate and aggravate the process of arterial ageing and calcification. However, the mechanism behind has yet to be well elucidated. METHODS: Here, we monitored the dynamic changes of fibronectin (FN)/α5 integrin, bone morphogenetic protein 2/matrix Gla protein (BMP2/MGP), and Runx2 in the aorta of spontaneously hypertensive rats (SHRs) and thoracic aortic vascular smooth muscle cells (VSMCs), also the phenotypic transformation of VSMCs during the process of arterial ageing and calcification. Further, study on arterial ageing and calcification through antagonist experiments at the molecular level was explored. RESULTS: We found extracellular FN and its α5 integrin receptor expressions were positively associated with arterial ageing and calcification in SHR during ageing, as well in VSMCs from SHR in vitro. Integrin receptor inhibitor of GRGDSP would delay this arterial ageing and calcification process. Moreover, the elevated FN and α5 integrin receptor expression evoked the disequilibrium of BMP2/MGP, where the expression of BMP2, a potent osteogenic inducer, increased while MGP, a calcification inhibitor, decreased. Furthermore, it was followed by the upregulation of Runx2 and the phenotypic transformation of VSMCs from the contractile phenotype into the osteoblast-like cells. Notably, BMP2 antagonist of rmNoggin was sufficient to ameliorate the ageing and calcification process of VSMCs and exogenous BMP2-adding accelerate and aggregate the process. CONCLUSION: Our study revealed that hypertension-associated arterial ageing and calcification might be a consequence that hypertension up-regulated FN and its high binding affinity integrin α5 receptor in the aortic wall, which in turn aggravated the imbalance of BMP2/MGP, promoted the transcription of Runx2, and induced the phenotypic transformation of VSMCs from the contractile phenotype into the osteoblast-like cells. Our study would provide insights into hypertension-associated arterial ageing and calcification and shed new light on the control of arterial calcification, especially for those with hypertension.

Extrahepatic Vitamin K-Dependent Gla-Proteins-Potential Cardiometabolic Biomarkers.

One mechanism to regulate pathological vascular calcification (VC) is its active inhibition. Loss or inactivation of endogenic inhibitors is a major inductor of VC. Such inhibitors are proteins rich in gamma-glutamyl residues (Gla-proteins), whose function strongly depends on vitamin K. The current narrative review is focused on discussing the role of extrahepatic vitamin K-dependent Gla-proteins (osteocalcin, OC; matrix Gla-protein, MGP; Gla-rich protein, GRP) in cardio-vascular pathology. Gla-proteins possess several functionally active forms whose role in the pathogenesis of VC is still unclear. It is assumed that low circulating non-phosphorylated MGP is an indicator of active calcification and could be a novel biomarker of prevalent VC. High circulating completely inactive MGP is proposed as a novel risk factor for cardio-vascular events, disease progression, mortality, and vitamin K deficiency. The ratio between uncarboxylated (ucOC) and carboxylated (cOC) OC is considered as an indicator of vitamin K status indirectly reflecting arterial calcium. Despite the evidence that OC is an important energy metabolic regulator, its role on global cardio-vascular risk remains unclear. GRP acts as a molecular mediator between inflammation and calcification and may emerge as a novel biomarker playing a key role in these processes. Gla-proteins benefit clinical practice as inhibitors of VC, modifiable by dietary factors.

Diagnosis of Fabry Disease Using Alpha-Galactosidase A Activity or LysoGb3 in Blood Fails to Identify Up to Two Thirds of Female Patients.

Anderson-Fabry disease is a lysosomal storage disorder caused by mutations in the GLA gene, which encodes the enzyme α-galactosidase A. The GLA gene is located on the X-chromosome, causing an X-linked pathology: due to lyonization, female patients usually manifest a variable symptomatology, ranging from asymptomatic to severe phenotypes. The confirmation of the clinical diagnosis of Fabry disease, achieved by measuring α-galactosidase A activity, which is usually the first test used, shows differences between male and female patients. This assay is reliable in male patients with causative mutations in the GLA gene, in whom the enzymatic activity is lower than normal values; on the other hand, in female Fabry patients, the enzymatic activity is extremely variable between normal and pathological values. These fluctuations are also found in female patients' blood levels of globotriaosylsphingosine (LysoGb3) for the same reason. In this paper, we present a retrospective study conducted in our laboratories on 827 Fabry patients with causative mutations in the GLA gene. Our results show that 100% of male patients had α-galactosidase A activity below the reference value, while more than 70% of female patients had normal values. It can also be observed that almost half of the female patients with pathogenic mutations in the GLA gene showed normal values of LysoGb3 in blood. Furthermore, in women, blood LysoGb3 values can vary over time, as we show in a clinical case presented in this paper. Both these tests could lead to missed diagnoses of Fabry disease in female patients, so the analysis of the GLA gene represents the main diagnostic test for Fabry disease in women to date.

Differences in Bone Metabolism between Children with Prader-Willi Syndrome during Growth Hormone Treatment and Healthy Subjects: A Pilot Study.

Despite therapy with growth hormone (GH) in children with Prader-Willi syndrome (PWS), low bone mineral density and various orthopedic deformities have been observed often. Therefore, this study aimed to analyze bone markers, with an emphasis on vitamin K-dependent proteins (VKDPs), in normal-weight children with PWS undergoing GH therapy and a low-energy dietary intervention. Twenty-four children with PWS and 30 healthy children of the same age were included. Serum concentrations of bone alkaline phosphatase (BALP), osteocalcin (OC), carboxylated-OC (Gla-OC), undercarboxylated-OC (Glu-OC), periostin, osteopontin, osteoprotegerin (OPG), sclerostin, C-terminal telopeptide of type I collagen (CTX-I), and insulin-like growth factor-I (IGF-I) were determined using immunoenzymatic methods. OC levels and the OC/CTX-I ratios were lower in children with PWS than in healthy children (p = 0.011, p = 0.006, respectively). Glu-OC concentrations were lower (p = 0.002), but Gla-OC and periostin concentrations were higher in patients with PWS compared with the controls (p = 0.005, p < 0.001, respectively). The relationships between IGF-I and OC (p = 0.013), Gla-OC (p = 0.042), and the OC/CTX-I ratio (p = 0.017) were significant after adjusting for age in children with PWS. Bone turnover disorders in children with PWS may result from impaired bone formation due to the lower concentrations of OC and the OC/CTX-I ratio. The altered profile of OC forms with elevated periostin concentrations may indicate more intensive carboxylation processes of VKDPs in these patients. The detailed relationships between the GH/IGF-I axis and bone metabolism markers, particularly VKDPs, in children with PWS requires further research.

Vitamin K2 and D in Patients With Aortic Valve Calcification: A Randomized Double-Blinded Clinical Trial.

BACKGROUND: Menaquinone-7 (MK-7), also known as vitamin K2, is a cofactor for the carboxylation of proteins involved in the inhibition of arterial calcification and has been suggested to reduce the progression rate of aortic valve calcification (AVC) in patients with aortic stenosis. METHODS: In a randomized, double-blind, multicenter trial, men from the community with an AVC score >300 arbitrary units (AU) on cardiac noncontrast computer tomography were randomized to daily treatment with tablet 720 µg MK-7 plus 25 µg vitamin D or matching placebo for 24 months. The primary outcome was the change in AVC score. Selected secondary outcomes included change in aortic valve area and peak aortic jet velocity on echocardiography, heart valve surgery, change in aortic and coronary artery calcification, and change in dp-ucMGP (dephosphorylated-undercarboxylated matrix Gla-protein). Safety outcomes included all-cause death and cardiovascular events. RESULTS: From February 1, 2018, to March 21, 2019, 365 men were randomized. Mean age was 71.0 (±4.4) years. The mean (95% CI) increase in AVC score was 275 AU (95% CI, 225-326 AU) and 292 AU (95% CI, 246-338 AU) in the intervention and placebo groups, respectively. The mean difference on AVC progression was 17 AU (95% CI, -86 to 53 AU; P=0.64). The mean change in aortic valve area was 0.02 cm2 (95% CI, -0.09 to 0.12 cm2; P=0.78) and in peak aortic jet velocity was 0.04 m/s (95% CI, -0.11 to 0.02 m/s; P=0.21). The progression in aortic and coronary artery calcification score was not significantly different between patients treated with MK-7 plus vitamin D and patients receiving placebo. There was no difference in the rate of heart valve surgery (1 versus 2 patients; P=0.99), all-cause death (1 versus 4 patients; P=0.37), or cardiovascular events (10 versus 10 patients; P=0.99). Compared with patients in the placebo arm, a significant reduction in dp-ucMGP was observed with MK-7 plus vitamin D (-212 pmol/L versus 45 pmol/L; P<0.001). CONCLUSIONS: In elderly men with an AVC score >300 AU, 2 years MK-7 plus vitamin D supplementation did not influence AVC progression. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT03243890.

Prevention of Arterial Elastocalcinosis: Differential Roles of the Conserved Glutamic Acid and Serine Residues of Matrix Gla Protein.

BACKGROUND: Inactivating mutations in matrix Gla protein (MGP) lead to Keutel syndrome, a rare disease hallmarked by ectopic calcification of cartilage and vascular tissues. Although MGP acts as a strong inhibitor of arterial elastic lamina calcification (elastocalcinosis), its mode of action is unknown. Two sets of conserved residues undergoing posttranslational modifications-4 glutamic acid residues, which are γ-carboxylated by gamma-glutamyl carboxylase; and 3 serine residues, which are phosphorylated by yet unknown kinase(s)-are thought to be essential for MGP's function. METHODS: We pursued a genetic approach to study the roles of MGP's conserved residues. First, a transgenic line (SM22a-GlamutMgp) expressing a mutant form of MGP, in which the conserved glutamic acid residues were mutated to alanine, was generated. The transgene was introduced to Mgp-/- mice to generate a compound mutant, which produced the mutated MGP only in the vascular tissues. We generated a second mouse model (MgpS3mut/S3mut) to mutate MGP's conserved serine residues to alanine. The initiation and progression of vascular calcification in these models were analyzed by alizarin red staining, histology, and micro-computed tomography imaging. RESULTS: On a regular diet, the arterial walls in the Mgp-/-; SM22α-GlamutMgp mice were not calcified. However, on a high phosphorus diet, these mice showed wide-spread arterial calcification. In contrast, MgpS3mut/S3mut mice on a regular diet recapitulated arterial calcification traits of Mgp-/- mice, although with lesser severity. CONCLUSIONS: For the first time, we show here that MGP's conserved serine residues are indispensable for its antimineralization function in the arterial tissues. Although the conserved glutamic acid residues are not essential for this function on a regular diet, they are needed to prevent phosphate-induced arterial elastocalcinosis.

All reported non-canonical splice site variants in GLA cause aberrant splicing.

BACKGROUND: Fabry disease is an X-linked lysosomal storage disorder caused by insufficient α-galactosidase A (GLA) activity resulting from variants in the GLA gene, which leads to glycosphingolipid accumulation and life-threatening, multi-organ complications. Approximately 50 variants have been reported that cause splicing abnormalities in GLA. Most were found within canonical splice sites, which are highly conserved GT and AG splice acceptor and donor dinucleotides, whereas one-third were located outside canonical splice sites, making it difficult to interpret their pathogenicity. In this study, we aimed to investigate the genetic pathogenicity of variants located in non-canonical splice sites within the GLA gene. METHODS: 13 variants, including four deep intronic variants, were selected from the Human Gene Variant Database Professional. We performed an in vitro splicing assay to identify splicing abnormalities in the variants. RESULTS: All candidate non-canonical splice site variants in GLA caused aberrant splicing. Additionally, all but one variant was protein-truncating. The four deep intronic variants generated abnormal transcripts, including a cryptic exon, as well as normal transcripts, with the proportion of each differing in a cell-specific manner. CONCLUSIONS: Validation of splicing effects using an in vitro splicing assay is useful for confirming pathogenicity and determining associations with clinical phenotypes.

Nonclinical safety evaluation of two vaccine candidates for herpes simplex virus type 2 to support combined administration in humans.

Herpes simplex virus type 2 (HSV-2) is the most common cause of genital disease worldwide. The development of an effective HSV-2 vaccine would significantly impact global health based on the psychological distress caused by genital herpes for some individuals, the risk transmitting the infection from mother to infant, and the elevated risk of acquiring HIV-1. Five nonclinical safety studies were conducted with the replication defective HSV529 vaccine, alone or adjuvanted with GLA-SE, and the G103 subunit vaccine containing GLA-SE. A biodistribution study was conducted in guinea pigs to evaluate distribution, persistence, and shedding of HSV529. A preliminary immunogenicity study was conducted in rabbits to demonstrate HSV529-specific humoral response and its enhancement by GLA-SE. Three repeated-dose toxicity studies, one in guinea pigs and two in rabbits, were conducted to assess systemic toxicity and local tolerance of HSV529, alone or adjuvanted with GLA-SE, or G103 containing GLA-SE. Data from these studies show that both vaccines are safe and well tolerated and support the ongoing HSV-2 clinical trial in which the two vaccine candidates will be given either sequentially or concomitantly to explore their potential synergistic and incremental effects.

Atractylodin targets GLA to regulate D-mannose metabolism to inhibit osteogenic differentiation of human valve interstitial cells and ameliorate aortic valve calcification.

Atractylodin (ATL) has been reported to exert anti-inflammatory effects. Osteogenic changes induced by inflammation in valve interstitial cells (VICs) play a key role in the development of calcified aortic valve disease (CAVD). This study aimed to investigate the anti-calcification effects of ATL on aortic valves. Human VICs (hVICs) were exposed to osteogenic induction medium (OM) containing ATL to investigate cell viability, osteogenic gene and protein expression, and anti-calcification effects. Gas chromatography-mass spectroscopy (GC-MS) metabolomics analysis was used to detect changes in the metabolites of hVICs stimulated with OM before and after ATL administration. The compound-reaction-enzyme-gene network was used to identify drug targets. Gene interference was used to verify the targets. ApoE-/- mice fed a high-fat (HF) diet were used to evaluate the inhibition of aortic valve calcification by ATL. Treatment with 20 µM ATL in OM prevented calcified nodule accumulation and decreases in the gene and protein expression levels of ALP, RUNX2, and IL-1ß. Differential metabolite analysis showed that D-mannose was highly associated with the anti-calcification effect of ATL. The addition of D-mannose prevented calcified nodule accumulation and inhibited succinate-mediated HIF-1α activation and IL-1ß production. The target of ATL was identified as GLA. Silencing of the GLA gene (si-GLA) reversed the anti-osteogenic differentiation of ATL. In vivo, ATL ameliorated aortic valve calcification by preventing decreases in GLA expression and the up-regulation of IL-1ß expression synchronously. In conclusion, ATL is a potential drug for the treatment of CAVD by targeting GLA to regulate D-mannose metabolism, thereby inhibiting succinate-mediated HIF-1α activation and IL-1ß production.

Deciphering the diagnostic dilemma: A comprehensive review of the Taiwanese cardiac variant in Fabry disease.

Molecular diagnosis has undergone rapid and significant advancements in recent years. But because molecular diagnosis can be conducted independently of phenotype, it can engender ambiguity and potential misinterpretations in disease diagnosis. Fabry disease, an X-linked lysosomal storage disorder, arises from a deficiency in α-galactosidase A. In 2002, Ishii and colleagues uncovered a variant (IVS4+919G > A) deep within intron 4 of the GLA gene that could lead to aberrant splicing of the GLA mRNA. This variant is present in 1:875 males in Taiwan, and many patients with hypertrophic cardiomyopathy and the IVS4+919G > A variant are currently treated by enzyme replacement therapy, an expensive treatment. Unfortunately, till now only one article published in 2013 described the outcome of treatment. This review summarized the conflicting evidence about the clinical relevance of the IVS4+919G > A variant, and suggest a multifactorial model, rather than a monogenic model, for the involvement of the IVS4+919G > A variant in hypertrophic cardiomyopathy. The diagnostic dilemma for this Taiwanese cardiac variant in Fabry disease clearly emphasizes the need for precise interpretation and application of molecular diagnostic results.

Enzyme Replacement Therapy for FABRY Disease: Possible Strategies to Improve Its Efficacy.

Enzyme replacement therapy is the only therapeutic option for Fabry patients with completely absent AGAL activity. However, the treatment has side effects, is costly, and requires conspicuous amounts of recombinant human protein (rh-AGAL). Thus, its optimization would benefit patients and welfare/health services (i.e., society at large). In this brief report, we describe preliminary results paving the way for two possible approaches: i. the combination of enzyme replacement therapy with pharmacological chaperones; and ii. the identification of AGAL interactors as possible therapeutic targets on which to act. We first showed that galactose, a low-affinity pharmacological chaperone, can prolong AGAL half-life in patient-derived cells treated with rh-AGAL. Then, we analyzed the interactomes of intracellular AGAL on patient-derived AGAL-defective fibroblasts treated with the two rh-AGALs approved for therapeutic purposes and compared the obtained interactomes to the one associated with endogenously produced AGAL (data available as PXD039168 on ProteomeXchange). Common interactors were aggregated and screened for sensitivity to known drugs. Such an interactor-drug list represents a starting point to deeply screen approved drugs and identify those that can affect (positively or negatively) enzyme replacement therapy.

Endothelial Cell Dysfunction and Hypoxia as Potential Mediators of Pain in Fabry Disease: A Human-Murine Translational Approach.

Fabry disease (FD) is caused by α-galactosidase A (AGAL) enzyme deficiency, leading to globotriaosylceramide accumulation (Gb3) in several cell types. Pain is one of the pathophysiologically incompletely understood symptoms in FD patients. Previous data suggest an involvement of hypoxia and mitochondriopathy in FD pain development at dorsal root ganglion (DRG) level. Using immunofluorescence and quantitative real-time polymerase chain reaction (qRT PCR), we investigated patient-derived endothelial cells (EC) and DRG tissue of the GLA knockout (KO) mouse model of FD. We address the question of whether hypoxia and mitochondriopathy contribute to FD pain pathophysiology. In EC of FD patients (P1 with pain and, P2 without pain), we found dysregulated protein expression of hypoxia-inducible factors (HIF) 1a and HIF2 compared to the control EC (p < 0.01). The protein expression of the HIF downstream target vascular endothelial growth factor A (VEGFA, p < 0.01) was reduced and tube formation was hampered in the P1 EC compared to the healthy EC (p < 0.05). Tube formation ability was rescued by applying transforming growth factor beta (TGFß) inhibitor SB-431542. Additionally, we found dysregulated mitochondrial fusion/fission characteristics in the P1 and P2 EC (p < 0.01) and depolarized mitochondrial membrane potential in P2 compared to control EC (p < 0.05). Complementary to human data, we found upregulated hypoxia-associated genes in the DRG of old GLA KO mice compared to WT DRG (p < 0.01). At protein level, nuclear HIF1a was higher in the DRG neurons of old GLA KO mice compared to WT mice (p < 0.01). Further, the HIF1a downstream target CA9 was upregulated in the DRG of old GLA KO mice compared to WT DRG (p < 0.01). Similar to human EC, we found a reduction in the vascular characteristics in GLA KO DRG compared to WT (p < 0.05). We demonstrate increased hypoxia, impaired vascular properties, and mitochondrial dysfunction in human FD EC and complementarily at the GLA KO mouse DRG level. Our data support the hypothesis that hypoxia and mitochondriopathy in FD EC and GLA KO DRG may contribute to FD pain development.