Journal of Clinical Investigation最新文献

Ceramides are essential skin lipids for maintaining the mammalian skin permeability barrier, which protects against external stimuli. The precursor of epidermal ceramides, glucosylceramides (GlcCer), is synthesized within granular keratinocytes while its precise cellular transport mechanisms remain poorly characterized. Here, we identified three pathogenic variants in the GLTP gene, which encodes glycolipid transfer protein, in five unrelated families with nonsyndromic epidermal differentiation disorder presenting with generalized skin scaling. The biallelic GLTP variants resulted in loss of competent GLTP expression. CRISPR/Cas9-generated Gltp knockout mice exhibited lethal barrier defects, partially recapitulating the clinical features of our patients. We demonstrated that GLTP facilitated GlcCer transport in differentiated keratinocytes, with its deficiency causing impaired GlcCer trafficking and consequent aberrant retention in lysosomes, thereby disrupted lysosome function. The lysosomal dysfunction impaired autophagy flux, resulting in delayed keratinocyte terminal differentiation, which is expected to compromise the skin barrier integrity and ultimate abnormal scaling. Pharmaceutical inhibition of GlcCer synthesis effectively rescued both autophagy and keratinocyte differentiation defects. Our findings establish GLTP as a novel underlying gene for nonsyndromic epidermal differentiation disorders and unravel its essential role in maintaining skin homeostasis during terminal differentiation by mediating epidermal GlcCer transport.

Vision begins in the outer segment compartment of photoreceptor cells, which is constantly renewed through the addition of membrane material at its base and ingestion of mature membranes at its tip by the retinal pigment epithelium (RPE). The close apposition of outer segments to the RPE is believed to be critical for maintaining this renewal process. Yet, in several retinal diseases, expansion of the subretinal space separating photoreceptors from the RPE does not immediately impact photoreceptor functionality. Here, we analyzed outer segment function and renewal in the Adam9 knockout mouse characterized by a major expansion of the subretinal space. Surprisingly, photoreceptor-RPE separation affected neither the sensitivity of photoreceptor light-responses nor the normal rate of outer segment renewal in this mouse prior to the onset of photoreceptor degeneration. The latter is achieved through the formation of elongated RPE "pseudopods" extending across the enlarged subretinal space to ingest outer segment tips. This work suggests that pseudopod formation may underlie the persistence of photoreceptor function in human diseases accompanied by photoreceptor-RPE separation, such as vitelliform macular dystrophy or age-related macular degeneration associated with subretinal drusenoid deposits.

Background: Infection by Trypanosoma cruzi, the agent of Chagas disease, is endemic to the Americas and can irreparably damage the cardiac and gastrointestinal systems during decades of parasite persistence. Diagnosis of chronic infection requires confirmation by multiple serological assays due to the imperfect performance of existing tests. Current serology tests were developed using small specimen sets predominantly from South America, and lower performance has been observed in patients who acquired infection in Central America and Mexico.

Methods: To improve Chagas disease serology, we evaluated antibody responses against the entire T. cruzi proteome with phage display immunoprecipitation sequencing and further evaluated high prevalence antigens by immunoassay. We utilized specimen sets representing Mexico, Central America and South America and varying cardiac disease presentations, from 185 cases and 143 controls.

Results: We identified over 1,300 antigenic T. cruzi peptides. A trans-sialidase antigen demonstrated high seroprevalence across all regions and has not previously been described as a diagnostic target. Orthogonal validation of this peptide demonstrated increased antibody reactivity for infections originating from Central America.

Conclusion: This study provides proteome-wide identification of seroreactive T. cruzi peptides across a range of endemic populations not previously represented in antigen discovery and identifies a trans-sialidase peptide antigen (TS23) with potential for translation into diagnostic serological assays.

Trial registration: Not Applicable.

Cells exhibit diverse sizes and shapes, tailored for functional needs of tissues. Lung alveoli are lined by large, extremely thin epithelial alveolar type-1 cells (AT1s). Their characteristic morphology is essential for lung function and must be restored after injury. The mechanisms underlying small, cuboidal alveolar type-2 cells (AT2s) differentiation into thin AT1s remain elusive. Here, we demonstrated that AT2s undergo a stepwise morphological transformation characterized by the development of a unique thick microtubule (MT) bundle organization, critical for AT1 morphology. Using AT2 cultures and in vivo genetic loss of function models, we found that MT bundling process occurs in a transitional cell state during AT2 differentiation and was regulated by the TP53/TAU signaling axis. Notably, TAU underwent a linear clustering process, forming beads-on-a-string-like pattern that preceded thick MT-bundle formation. Genetic gain or loss of function of TAU in mouse or human models, prevented the formation of thick MT-bundles, highlighting the critical role of precise TAU levels in generating ultra-thin AT1s. This defect was associated with increased tissue fibrosis following bleomycin-induced injury in vivo. GWAS analysis revealed risk variants in MAPT locus in lung diseases. Moreover, TP53 controlled TAU expression and its loss phenocopied TAU deficiency. This work revealed an unexpected role for TAU in organizing MT-bundles during AT2 differentiation.

Radiotherapy (RT) is a central treatment for prostate cancer (PCa), acting by inducing DNA double-strand breaks (DSBs). Tumor ability to repair these breaks limits RT efficacy, making DSB repair inhibitors potential radiosensitizers. Therefore, tumor-specific radiosensitization strategies are critically needed for PCa. Approximately 50% of PCa cases harbor the TMPRSS2-ERG gene fusion, leading to overexpression of the ERG transcription factor (ERG+). We demonstrate that ERG+ tumors shift DSB repair toward the PARP1-dependent end-joining (PARP1-EJ) pathway. Proteomic and western blot analyses revealed elevated PARP1, XRCC1, and LIG3 in ERG+ cells. PARP inhibition with olaparib increased residual γH2AX/53BP1 foci post-irradiation in ERG+ cells, indicating enhanced radiosensitization. In tissue-slice-cultures (TSCs) from 53 tumors of 40 high-risk PCa patients, olaparib selectively increased H2AX/53BP1 foci selectively in ERG+ samples. ERG+ patient-derived organoids also showed significantly delayed growth and survival when treated with olaparib plus RT compared to either treatment alone. Interestingly, ERG-negative cells within ERG+ TSCs were similarly radiosensitized by olaparib, likely through bystander effect, with residual 53BP1 foci levels comparable to ERG+ cells, confirmed by medium exchange experiments. These findings suggest that ERG expression promotes dependency on PARP1-EJ, rendering ERG+ PCa more susceptible to PARP inhibition. Combining PARP inhibitors with RT may offer a tumor-selective radiosensitization for ERG+ PCa patients.

Aging commonly causes decline of testosterone or estrogen, leading to overaccumulation of fatness in males or females, respectively. Although such phenomenon can be readily explained by estrogen's direct action on adipocytes in females, accumulative evidence does not support the direct action of testosterone in adipocyte lipid metabolism, suggesting that there is a missing intermediary link. Herein, we propose that glycoprotein hormone β5 (GPHB5) is the intermediary linkage between testosterone and the regulation of adiposity. In clinical samples, blood levels of GPHB5 were correlated negatively with men's ages, and positively with circulating testosterone. Testosterone directly stimulated the expression of GPHB5 in cultured cells, pharmacological blockade of androgen receptor (AR) functions abrogated such effect. Knockout of AR led to not only development of obesity but also reduction of GPHB5 expression. Genetic ablation of GPHB5 in the males, but not in the females, lowered the browning of white adipose tissue, diminished energy expenditure and caused severe obesity. Importantly, elevated blood testosterone didn't exert its catabolic actions in GPHB5 null mice, and yet, recombinant GPHB5 protein was able to stimulate energy expenditure and reduce adiposity. Taken together, these results provided the strong proof that GPHB5 is the "missing" intermediary hormone linking testosterone (and aging) and its well-known catabolic effect on adipose tissue.

Mycobacterium tuberculosis (Mtb) remains a global health crisis, ranking among the deadliest infectious diseases worldwide. In response to the World Health Organization's call for therapeutic vaccines to complement antibiotic regimens and reduce tuberculosis (TB) treatment duration, we developed an intranasal DNA vaccine fusing the Mtb stringent response gene relMtb with the gene encoding the dendritic cell-targeting chemokine Mip3a/CCL20. Administered alongside the first-line regimen, this vaccine accelerated stable cure in immunocompetent murine TB models, reducing lung inflammation and eliciting robust and sustained RelMtb-stimulated T-cell responses systemically and locally. The Mip3a/relMtb vaccine enhanced dendritic cell recruitment, activation, and spatial coordination with T cells, suggesting improved innate-adaptive immune synergy. Notably, it augmented the efficacy of a novel drug-resistant TB regimen as well. Critically, the vaccine induced analogous antigen-stimulated T-cell immunity in nonhuman primates, the gold standard for preclinical TB vaccine evaluation, with responses detected in blood and bronchoalveolar lavage mirroring those observed in the murine models. These findings underscore the potential of this strategy to advance therapeutic TB vaccine development targeting Mtb persisters while providing a framework to define correlates of vaccine-mediated protection.

Background: Plasma heparan sulfate, a glycosaminoglycan released during endothelial glycocalyx degradation, predicts sepsis mortality. Chondroitin sulfate is a circulating glycosaminoglycan not specific to glycocalyx degradation; its relevance to sepsis is unknown.

Methods: We studied the associations of plasma chondroitin sulfate with (a) mortality in patients with sepsis-associated hypotension and (b) the relative effectiveness of a randomly-assigned liberal versus restrictive intravenous fluid resuscitation strategy. We selected 574 patients enrolled in the Crystalloid Liberal or Vasopressors Early Resuscitation in Sepsis trial using an outcome-enriched sampling strategy. We used liquid chromatography-mass spectrometry to quantify plasma chondroitin sulfate. In comparison, we measured hyaluronic acid as a glycocalyx degradation marker and IL-6 as an inflammatory biomarker. We conducted Cox proportional hazards regression analyses to examine associations of baseline biomarker concentrations with mortality and resuscitation strategy effectiveness. We used inverse probability of selection weights and generalized raking to account for the non-representative sampling design.

Results: Plasma chondroitin sulfate, hyaluronic acid, and IL-6 were associated with mortality within 90 days. As baseline chondroitin sulfate increased, subsequent randomization to a restrictive strategy was increasingly beneficial (p = 0.022): treatment effect hazard ratio (restrictive versus liberal) for mortality was estimated as 1.49 (95% CI 0.98-2.27), 1.30 (1.00-1.69), 1.09 (0.82-1.44), 0.88 (0.66-1.16), and 0.71 (0.52-0.97) for 10th, 25th, 50th, 75th and 90th percentiles of baseline chondroitin sulfate.

Conclusions: Plasma chondroitin sulfate predicts sepsis mortality and may modify the response to a subsequent liberal vs. restrictive intravenous fluid resuscitation strategy.

Trial:

Clinicaltrials: gov NCT03434028.

Chordoma are rare malignant osseous neoplasms with a striking rate of recurrence. Primary chordomas typically originate from embryonic notochord remnants, whereas recurrent chordomas usually stem from tumor cells infiltrating bone or cartilage post-surgery. Clinically, the recurrent chordomas exhibit stiffer extracellular microenvironment (ECM) than primary tumors. Intriguingly, this study identified cytoskeleton rearrangement, stress fiber reorganization, enhanced stemness, and Notch signaling activation in recurrent chordoma tissues or cell lines surviving stiff substrates, indicating the critical roles of mechanical remodeling and tumor stemness in stiffness-resistance. We propose a novel recurrence model where tumor cells experience mechanoadaptive organization to resist stiff microenvironment-induced cell death. O-GlcNAcylation of Notch1 intracellular domain (NICD1) is central to this process. Mechanistically, the stiff ECM-driven ligand-independent phosphorylation of EPHA2 sequentially activates LYN kinase, and subsequently triggers OGT activity by phosphorylating Y989 and Y418, the newly revealed critical residues for OGT glycosyltransferase activity; this induces NICD1 O-GlcNAcylation at T2063, T2090, and S2162, specifically promoting transcription of mechanical and stemness-related genes. MIR31 deletion upregulates LYN, enhancing stiffness perception and tipping the balance toward O-GlcNAc addition to NICD1, finally resulting in mechanoadaptation- and tumor stemness-driven recurrence. Consequently, MIR31 deletion is a potential biomarker for recurrence and patient stratification in Notch- or OGT-targeted therapies.

The fingertip is one of the only known complex structures in mammals that can fully regenerate following amputation. This phenomenon can be studied in mice using the amputation of the digit tip, the regenerative success of which has been shown to be reliant on effective bone clearance prior to new bone formation. In this issue of the JCI, Vishlaghi et al. investigated whether local lymphatic vessels are involved in this process. Interestingly, they found that inhibiting lymphangiogenesis resulted in accelerated clearance of damaged tissue and bone, thereby improving subsequent digit regeneration. This study is the first to our knowledge to report lymphatic involvement in digit regeneration and raises questions regarding the underlying mechanisms at play.