Revisiting the Role of NAG across the Continuum of Kidney Disease.

Acute and chronic kidney diseases are an evolving continuum for which reliable biomarkers of early disease are lacking. The potential use of glycosidases, enzymes involved in carbohydrate metabolism, in kidney disease detection has been under investigation since the 1960s. N-acetyl-beta-D-glucosaminidase (NAG) is a glycosidase commonly found in proximal tubule epithelial cells (PTECs). Due to its large molecular weight, plasma-soluble NAG cannot pass the glomerular filtration barrier; thus, increased urinary concentration of NAG (uNAG) may suggest injury to the proximal tubule. As the PTECs are the workhorses of the kidney that perform much of the filtration and reabsorption, they are a common starting point in acute and chronic kidney disease. NAG has previously been researched, and it is widely used as a valuable biomarker in both acute and chronic kidney disease, as well as in patients suffering from diabetes mellitus, heart failure, and other chronic diseases leading to kidney failure. Here, we present an overview of the research pertaining to uNAG's biomarker potential across the spectrum of kidney disease, with an additional emphasis on environmental nephrotoxic substance exposure. In spite of a large body of evidence strongly suggesting connections between uNAG levels and multiple kidney pathologies, focused clinical validation tests and knowledge on underlining molecular mechanisms are largely lacking.

Benign fasciculation syndrome and migraine aura without headache as possible adverse events after BNT162b2 mRNA vaccination: a web-based survey.

AIM: To determine the characteristics of patients who experienced muscle fasciculations and migraine auras without headache after BNT162b2 immunization. METHODS: In January 2022, we published a case report that described a 48-year-old female patient who experienced muscle twitching and migraine auras without headache following BNT162b2 immunization. A self-administered online survey was sent to people who had written to us and complained of similar symptoms described in the case report (N=20). RESULTS: The survey was completed by 11 participants, of whom 10 reported muscle twitching following BNT162b2 immunization lasting a median of 14 (4-36.5) days. Five of these participants (50%) reported migraine auras without headache. Participants further reported on self-identified triggers that altered the intensity of their symptoms, such as anxiety or caffeine. Fifty percent of participants who got an acute SARS-CoV-2 infection (3/6) experienced increased muscle symptom intensity during the acute phase of the disease. CONCLUSION: To the best of our knowledge, our survey is the first to summarize patients' experiences of these phenomena occurring after BNT162b2 immunization. It is important to note that no causal relationship between vaccination and these phenomena can be inferred.

Ganglioside Composition Distinguishes Anaplastic Ganglioglioma Tumor Tissue from Peritumoral Brain Tissue: Complementary Mass Spectrometry and Thin-Layer Chromatography Evidence.

Gangliosides serve as antitumor therapy targets and aberrations in their composition strongly correlate with tumor growth and invasiveness. Anaplastic ganglioglioma is a rare, poorly characterized, malignant neuronal-glial tumor type. We present the first comparative characterization of ganglioside composition in anaplastic ganglioglioma vs. peritumoral and healthy brain tissues by combining mass spectrometry and thin-layer chromatography. Anaplastic ganglioglioma ganglioside composition was highly distinguishable from both peritumoral and healthy tissue despite having five to six times lower total content. Ten out of twelve MS-identified ganglioside classes, defined by unique glycan residues, were represented by a large number and considerable abundance of individual species with different fatty acid residues (C16-C24) in ceramide portions. The major structurally identified class was tumor-associated GD3 (>50%) with 11 species; GD3 (d18:1/24:0) being the most abundant. The dominant sphingoid base residue in ganglioside ceramides was sphingosine (d18:1), followed by eicosasphingosine (d20:1). The peritumoral tissue ganglioside composition was estimated as normal. Specific ganglioside composition and large variability of ganglioside ceramide structures determined in anaplastic ganglioglioma demonstrate realistic ganglioside expression patterns and correspond to the profile of high-grade malignancy brain tumors.

Plasma levels of soluble TGF ß receptor type III: no apparent promise as a marker in acute pancreatitis.

AIM: To assess the potential of the soluble transforming growth factor ß receptor type III (sTGFßrIII), a key regulator in TGFß signaling, as a biomarker for diagnosis and stratification of patients with acute pancreatitis (AP). METHODS: In this small prospective pilot study, patients' (N=22) plasma samples were obtained at three time points: the first and fourth day of hospitalization and the day of hospital discharge. Healthy controls' plasma (N=25) was obtained at a single time point. Concentration of sTGFßrIII in plasma was determined by ELISA. Data were analyzed by fitting linear or linear mixed models. RESULTS: Plasma sTGFßrIII levels at presentation (day 1) were similar in AP patients and healthy participants, irrespectively of the disease severity. sTGFßrIII levels in patients were constant during hospital stay. CONCLUSION: These observations do not support further evaluation of plasma sTGFßrIII levels in this setting, but do not exclude a potential biological role of TGFß and membrane-bound TGFßrIII in AP pathophysiology.

Identification of bone morphogenetic protein 4 in the saliva after the placement of fixed orthodontic appliance.

BACKGROUND: This study was conducted in order to explore the effects of orthodontic tooth movement (OTM) on the changes of salivary proteome. This prospective observational pilot study recruited 12 healthy teenage boys with malocclusion treated with a fixed orthodontic appliance and 6 appropriate control participants. Saliva samples were collected a day before and at 0, 2, 7, and 30 days after initialization of treatment, corresponding to the initial, lag, and post-lag phases of OTM. Pooled samples were analyzed by liquid chromatography-mass spectrometry, ELISA, and Western blotting. To date, there is no published data on the presence of BMP molecules or their antagonists in the saliva or in the gingival cervical fluid related to orthodontic conditions. RESULTS: A total of 198 identified saliva proteins were classified based on their functional characteristics. Proteins involved in bone remodeling were observed exclusively 30 days post appliance placement, including bone morphogenetic protein 4 (BMP4), a BMP antagonist BMP-binding endothelial regulator, insulin-like growth factor-binding protein 3, cytoskeleton-associated protein 4, and fibroblast growth factor 5. Based on the analysis of protein interactions, BMP4 was found to have a central position in this OTM-related protein network. CONCLUSIONS: The placement of a fixed orthodontic appliance induced occurrence of proteins involved in bone remodeling in the saliva at a time corresponding to the post-lag period of OTM. Limitations of this study include a relatively small sample size, limited time of monitoring patients, and the lack of interindividual variability assessment.

Plasma levels and tissue expression of soluble TGFßrIII receptor in women with early-stage breast cancer and in healthy women: a prospective observational study.

BACKGROUND: Mammary carcinogenesis is partly regulated by the transforming growth factor beta (TGFß) signaling pathway. Its function in cancer progression and metastasis is highly dependent on disease stage, and it is likely modulated by the ratio of membrane-bound vs. soluble TGFßrIII (sTGFßrIII). In this prospective observational study, we assessed tissue expression and plasma levels of sTGFßrIII in healthy women, women with benign breast lesions and in early-stage breast cancer patients. METHODS: In a preliminary study, plasma sTGFßrIII levels were determined in 13 healthy women (age 19-40 years) at different phases of the ovarian cycle, and in 15 patients (age 35-75 years) at different times of the day. The main study assessed plasma concentrations of sTGFßrIII in: (i) 158 healthy women in whom breast lesions were excluded; (ii) 65 women with benign breast lesions; (iii) 147 women with newly diagnosed breast cancer classified as American Joint Committee on Cancer (AJCC) stages 0 to IIB. Completers provided blood samples before surgery and at 10-30 and 160-180 days after surgery. Plasma sTGFßrIII concentrations were determined using an indirect ELISA kit. Part of the removed tissues underwent immunohistochemical (IHC) staining and analysis of tissue TGFßrIII expression. RESULTS: There appeared no relevant variations in plasma sTGFßrIII levels at different times of the day or different ovarian cycle phases. Before surgery, breast cancer patients had somewhat higher sTGFßrIII than healthy women, or those with benign breast lesions (by 14.5 and 26 ng/mL, respectively), with a tendency of larger differences at higher age. This correlated with lower expression of TGFßrIII in breast cancer vs. healthy tissue samples. At 160-180 days after surgery, plasma sTGFßrIII levels in breast cancer patients declined by 23-26 ng/mL. CONCLUSIONS: Plasma sTGFßrIII levels do not seem to relevantly vary during the day or the ovarian cycle. The coinciding higher plasma levels in newly diagnosed cancer patients than in healthy subjects and lower TGFßrIII expression in the malignant than in healthy breast tissue suggest ectodomain shedding as a source of circulating sTGFßrIII. Decline in plasma levels after tumor removal supports such a view.

THE ROLE OF NEW TECHNOLOGIES IN DEFINING SALIVARY PROTEIN COMPOSITION FOLLOWING PLACEMENT OF FIXED ORTHODONTIC APPLIANCES - BREAKTHROUGH IN THE DEVELOPMENT OF NOVEL DIAGNOSTIC AND THERAPEUTIC PROCEDURES.

Human saliva is rich in proteins of variable functions (e.g., enzymes, immunoglobulins, cytokines) and origin (blood plasma, salivary glands, or oral microflora). Circadian dynamics, volume and composition (electrolytes, pH, protein, etc.) of secreted saliva vary with local and systemic physiological and pathophysiological conditions. Therefore, the composition of saliva, protein in particular, has been intensively investigated to identify the potential markers and/or mechanisms of systemic and local diseases. Proteomic techniques used for the analysis of biological fluids have enabled great advances in salivary protein stabilization (as the main precondition for their analysis) and detection of those found in saliva in very low concentrations, including small proteins and peptides. This review brings the main characteristics of current proteomic techniques such as liquid chromatography-mass spectrometry, two-dimensional electrophoresis-mass spectrometry, and surface-enhanced laser desorption ionization/time of flight/mass spectrometry. These techniques enable simultaneous identification of hundreds and thousands of protein molecules, as well as identifying those of a potential biological value in particular states. This literature review is focused on the state-of-the-art and possibilities offered by proteomic techniques in analyzing the effects of orthodontic appliances on salivary protein composition and searching for potential markers of therapeutic success/failure or for the molecules by which therapeutic effects are achieved.

Tumor tissue hnRNP M and HSP 90α as potential predictors of disease-specific mortality in patients with early-stage cutaneous head and neck melanoma: A proteomics-based study.

BACKGROUND: Breslow tumor thickness and mitotic rate are standardly used for risk stratification of patients with malignant melanoma. However, their prognostic value is relatively limited and a need for improved prognostication has been advocated. We aimed to screen the tumor tissue proteome in a search for potentially useful prognostic factors in early-stage cutaneous head and neck melanoma. METHODOLOGY AND FINDINGS: Proteomic profiles of archival formalin-fixed tissue samples of 31 patients (age 23-90 years) with early-stage head and neck cutaneous malignant melanoma (American Joint Committee on Cancer, AJCC, stage I/II) were determined and expression intensities were compared to those of melanocytic nevi, yielding ratios used in data analysis. Medical charts were retrospectively reviewed to determine time elapsed since diagnosis to disease-specific death or censoring. In a multivariate recursive partitioning analysis (as per AJCC guidelines), higher expression levels of heterogeneous nuclear ribonucleoprotein M (hnRNP M) [n = 18, HR = 1.94 vs. the entire cohort; HR = 5.95 (95%CI 2.43-14.5) for "high" vs. "low" (n = 13)] and of heat shock protein 90 alpha (HSP 90α) [n = 17, HR = 2.09 vs. the entire cohort; HR = 4.59 (95%CI 1.87-11.2) for "high" vs. "low" (n = 14)] were each independently strongly associated with higher mortality (accounting for clinical and standard pathohistological features). Higher Breslow thickness and mitotic rate were associated with higher mortality only when proteomic data were disregarded. CONCLUSIONS AND SIGNIFICANCE: Data suggest that tumor tissue expression of hnRNP M and/or of HSP 90α deserve further investigation and clinical validation as potential novel risk stratification aids in patients with stage I-II cutaneous head and neck malignant melanoma.

Elevated plasma RANTES in fibrodysplasia ossificans progressiva - A novel therapeutic target?

Fibrodysplasia ossificans progressiva (FOP) is a rare hereditary disease caused by a mutation in the intracellular domain of the activin A receptor type I and is characterized by episodes (flare-ups) of progressive heterotopic endochondral ossification (HO) in the soft tissues. The mutation alone is not sufficient for the occurrence of HO since flare-ups are triggered by inflammation and activation of the innate immune system. A number of cellular and humoral mediators have been implicated in animal and in vitro models. Observations in humans support the inflammatory nature of the condition, but data on the involved mediators are variable. We hypothesize that for induction of flare-ups in patients with FOP increase in at least one of the pro-inflammatory cytokines is both essential and sufficient to trigger the entire process of the inflammatory cells influx resulting in the novel ectopic bone formation and we suggest that C-C motif ligand 5 (CCL5), a pro-inflammatory chemokine also known as Regulated on activation, normal T-cell expressed and secreted (RANTES), might be the key candidate. CCL5 is a chemoattractant for all cellular types implicated in HO and is produced by the cells of the tissue microenvironment at the sites of HO as well as by the pro-inflammatory cellular mediators. CCL5 induces ossification in cultured human pluripotent mesenchymal cells (hMSCs) and in the primary culture of monocytes from FOP patients (but not from their healthy relatives), stimulation with lipopolysaccharide induces CCL5 expression. Finally, in a pilot study we used a panel of 23 cytokines and chemokines to screen the plasma samples of three subjects: a female patient with FOP during a flare-up; a female patient with hyperostosis corticalis generalisata (van Buchem disease), another rare disease characterized by excessive bone formation at the sites where it regularly occurs that does not include inflammatory events; and a healthy woman without bone disorders. There appeared a rather clear-cut signal of a 2-fold higher level of CCL5 in the FOP patient vs. the healthy subject and the van Buchem patient. Evaluation of the hypothesis would require an international prospective study, with main motivation being the lack of a conclusive treatment as the major unmet need in FOP. A treatment targeting CCL5 receptor already exists and is used in HIV-infected patients.

Differential expression of heat shock proteins and activation of mitogen-activated protein kinases in A549 alveolar epithelial cells exposed to cigarette smoke extract.

NEW FINDINGS: What is the central question of this study? What is the effect of cigarette smoke on cell death, oxidative damage, expression of heat shock proteins (HSPs) and activation of mitogen-activated protein kinases (MAPKs) in A549 alveolar epithelial cells? What is the main finding and its importance? Cigarette smoke induces cytotoxicity and oxidative damage to A549 cells, increases expression of different HSPs and activates MAPK signalling pathways. This could be related to inflammatory response and apoptosis observed in lungs of patients with smoking-related diseases. ABSTRACT: Cigarette smoking is one of the main risk factors for development of chronic obstructive pulmonary disease (COPD). We previously reported that cigarette smoke (CS) induces damage to proteins and their ineffective degradation. Here, we hypothesize that CS could induce oxidative stress and cytotoxicity in lung epithelial cells through alterations of heat shock protein (HSP) expression and mitogen-activated protein kinase (MAPK) signalling pathways. We exposed A549 alveolar epithelial cells to various concentrations of cigarette smoke extract (CSE). Higher concentrations of CSE caused apoptosis of A549 cells after 4 h, while after 24 h cell viability was decreased, and lactate dehydrogenase in cell culture medium was increased as well as the number of necrotic cells. Concentrations of malondialdehyde (MDA) were elevated, while total thiol groups were decreased. Changes in the expression of HSPs (HSP70, HSP32 and HSP27) were time-dependent. After 6 h, CSE caused an increase in the expression of HSP70 and HSP32, while after 8 h all examined HSPs were up-regulated and remained increased up to 48 h. Treatment of A549 cells with CSE stimulated phosphorylation of extracellular signal-regulated kinase and p38 in a dose-dependent manner, while c-Jun N-terminal kinase activation was not detected. By using specific inhibitors, we demonstrated that MAPKs and HSPs interplay in CSE effects. In conclusion, our results show that MAPKs and HSPs are involved in the mechanism underlying CSE-induced cytotoxicity and oxidative damage to A549 alveolar epithelial cells. These processes could be related to inflammatory response and apoptosis observed in lungs of patients with smoking-related diseases, such as COPD.

The clinical use of bone morphogenetic proteins revisited: a novel biocompatible carrier device OSTEOGROW for bone healing.

PURPOSE: The purpose of this study was to revise the clinical use of commercial BMP2 (Infuse) and BMP7 (Osigraft) based bone devices and explore the mechanism of action and efficacy of low BMP6 doses in a novel whole blood biocompatible device OSTEOGROW. METHODS: Complications from the clinical use of BMP2 and BMP7 have been systemically reviewed in light of their role in bone remodeling. BMP6 function has been assessed in Bmp6-/- mice by µCT and skeletal histology, and has also been examined in mesenchymal stem cells (MSC), hematopoietic stem cells (HSC) and osteoclasts. Safety and efficacy of OSTEOGROW have been assessed in rats and rabbits. RESULTS: Clinical use issues of BMP2 and BMP7 have been ascribed to the limited understanding of their role in bone remodeling at the time of device development for clinical trials. BMP2 and BMP7 in bone devices significantly promote bone resorption leading to osteolysis at the endosteal surfaces, while in parallel stimulating exuberant bone formation in surrounding tissues. Unbound BMP2 and BMP7 in bone devices precipitate on the bovine collagen and cause inflammation and swelling. OSTEOGROW required small amounts of BMP6, applied in a biocompatible blood coagulum carrier, for stimulating differentiation of MSCs and accelerated healing of critical size bone defects in animals, without bone resorption and inflammation. BMP6 decreased the number of osteoclasts derived from HSC, while BMP2 and BMP7 increased their number. CONCLUSIONS: Current issues and challenges with commercial bone devices may be resolved by using novel BMP6 biocompatible device OSTEOGROW, which will be clinically tested in metaphyseal bone fractures, compartments where BMP2 and BMP7 have not been effective.

Galectin-3 expression in response to LPS, immunomodulatory drugs and exogenously added galectin-3 in monocyte-like THP-1 cells.

Galectin-3, a structurally unique beta-galactoside-binding lectin, through the specific protein-protein and protein-carbohydrate interactions participates in numerous biological processes, such as cell proliferation and apoptosis, adhesion and activation. Its expression and secretion by until now an unknown mechanism are modulated by diverse molecules and are dependent on different physiological and pathophysiological conditions. By autocrine and paracrine actions, galectin-3 modulates many immune reactions and affects various immune cells, particularly those of monocyte-macrophage lineage. This is why galectin-3 has recently become an attractive therapeutic target. However, molecular mechanisms of its actions as well as regulatory mechanism of its expression and activation are still largely unknown. In this study, we show that lipopolysaccharide (LPS) provokes upregulation of galectin-3 expression on both gene and protein level in monocyte-like THP-1 cells, which can be inhibited by dexamethasone, but not with non-steroidal anti-inflammatory drugs aspirin and indomethacin. Resting and LPS-challenged monocyte-like THP-1 cells do not have detectable amount of surface-bound galectin-3, but are able to bind exogenously added galectin-3 with the same capacity. Although galectin-3 is generally considered to be a pro-inflammatory molecule, here we show that the exogenously added galectin-3 does not affect interleukin (IL)-1ß, IL-6, IL-8, IL-10, IL-12p70 and TNF-α production in resting and LPS-activated monocyte-like THP-1 cells nor influences its own gene expression level in those cells.

Galectin-3 endocytosis by carbohydrate independent and dependent pathways in different macrophage like cell types.

BACKGROUND: Galectin-3 (the Mac-2 antigen) is abundantly expressed in both macrophage like cells and certain non-macrophage cells. We have studied endocytosis of galectin-3 as one important step relevant for its function, and compared it between variants of a macrophage like cell line, and non-macrophage cells. METHODS: Endocytosis of galectin-3 was observed by fluorescence microscopy and measured by flow cytometry. The endocytosis mechanism was analysed using galectin-3 mutants, galectin-3 inhibitors and endocytic pathways inhibitors in the human leukaemia THP-1 cell line differentiated into naïve (M0), classical (M1) or alternatively activated (M2) macrophage like cells, and the non-macrophage cell lines HFL-1 fibroblasts and SKBR3 breast carcinoma. RESULTS: Galectin-3 endocytosis in non-macrophage cells and M2 cells was blocked by lactose and a potent galectin-3 inhibitor TD139, and also by the R186S mutation in the galectin-3 carbohydrate recognition domain (CRD). In M1 cells galectin-3 endocytosis could be inhibited only by chlorpromazine and by interference with the non-CRD N-terminal part of galectin-3. In all the cell types galectin-3 entered early endosomes within 5-10 min, to be subsequently targeted mainly to non-degradative vesicles, where it remained even after 24 h. CONCLUSIONS: Galectin-3 endocytosis in M1 cells is receptor mediated and carbohydrate independent, while in M2 cells it is CRD mediated, although the non-CRD galectin-3 domain is also involved. General significance The demonstration that galectin-3 endocytosis in M1 macrophages is carbohydrate independent and different from M2 macrophages and non-macrophage cells, suggests novel, immunologically significant interactions between phagocytic cells, galectin-3 and its ligands.

Galectin-1 and galectin-3 expression profiles in classically and alternatively activated human macrophages.

BACKGROUND: Galectins have been identified as modulators of many monocyte/macrophage functions. In the response to a wide range of environmental cues macrophages may exhibit different biochemical and biological characteristics, but two main subtypes, classically (M1) and alternatively (M2) activated macrophages have been recognized. To contribute to elucidation of role and regulation of galectin-1 and galectin-3 in differently activated macrophages we explored their expression profiles in these cells. METHODS: Human monocytes obtained from blood donors were differentiated into classically (M1) and alternatively (M2a/M2c) activated macrophages. Gene and protein expression levels of intra- and extracellular galectins were investigated by qRT-PCR, Western-blot, flow cytometry, and ELISA while cytokine and surface receptor expression profiling was performed by flow cytometry. RESULTS: Differentiation/polarization of human monocytes into classically (M1) and alternatively (M2a/M2c) activated macrophages was followed by profound changes of galectin-3 expression and its proteolytic cleavage. Expression and secretion of Gal-3 was tightly regulated and significantly differed among classically (M1) and alternatively (M2a/M2c) activated macrophages, while the differences of galectin-1 expression profiles were not as pronounced. Human monocytes exhibited high amount of free galectin-3 receptors, while on both types of activated macrophages were fully saturated. CONCLUSIONS: Galectin-3 is more distinctive descriptor of macrophages differentiation/activation than galectin-1. Its specific expression and secretion pattern in M1 vs. M2a/M2c macrophages contributes to better understanding of its role and regulation in these cells. GENERAL SIGNIFICANCE: Recognition of distinct galectin-1 and galectin-3 expression profiles in differently activated macrophages provides a new insight on biological characteristics of these cells and sheds a new light of galectin-3 as a modulator of individual macrophage subset. This article is part of a Special Issue entitled Glycoproteomics.

Hyperserotonemia in autism: the potential role of 5HT-related gene variants.

Increased platelet serotonin level (PSL) has been consistently found in a portion of autistic patients. Suggested mechanisms for hyperserotonemia in autism have been increased synthesis of serotonin (5HT) by tryptophan hydroxylase (TPH), increased uptake into platelets through 5HT transporter (5HTt), diminished release from platelets through 5HT2A receptor (5HT2Ar) and decreased metabolism by monoamine oxydase (MAOA). The allelic influence of genes, encoding the mentioned 5HT elements, on PSL was investigated in 63 autistic subjects. Our study shows that 5HTt-LPR and -1438AG 5HT(2Ar) genotypes did not significantly affect PSL. However, significantly higher PSLs were observed in subjects with "cc" genotype of a218c TPH and subjects with "4" genotype of uVNTR MAOA. In addition, when TPH-cc and MAOA-4 were combined as "high 5HT" genotypes, a correlative increase in PSL was observed with the increase in the number of "high 5HT" genotypes. These results suggest a possible synergistic effect of genes regulating 5HT synthesis/degradation in dysregulation of the peripheral 5HT homeostasis of autistic patients.