The causes of hypercalcaemia in the neonate and infant are varied, and often distinct from those in older children and adults. Hypercalcaemia presents clinically with a range of symptoms including failure to thrive, poor feeding, constipation, polyuria, irritability, lethargy, seizures and hypotonia. When hypercalcaemia is suspected, an accurate diagnosis will require an evaluation of potential causes (e.g. family history) and assessment for physical features , as well as biochemical measurements, including total and ionised serum calcium, serum phosphate, creatinine and albumin, intact parathyroid hormone (PTH), vitamin D metabolites and urinary calcium, phosphate and creatinine. The causes of neonatal hypercalcaemia can be classified into high or low PTH disorders. Disorders associated with high serum PTH include neonatal severe hyperparathyroidism, familial hypocalciuric hypercalcaemia and Jansen’s metaphyseal chondrodysplasia. Conditions associated with low serum PTH include idiopathic infantile hypercalcaemia, Williams-Beuren syndrome and inborn errors of metabolism, including hypophosphatasia. Maternal hypocalcaemia and dietary factors and several rare endocrine disorders can also influence neonatal serum calcium levels. This review will focus on the common causes of hypercalcaemia in neonates and young infants, considering maternal, dietary, and genetic causes of calcium dysregulation. The clinical presentation and treatment of patients with these disorders will be discussed. Hypercalcaemia in the neonate and infant, although uncommon, can have serious long-term consequences, including nephrocalcinosis that may cause permanent kidney damage, osteoporosis and neurodevelopmental impairments, and has varied etiologies Table . Hyperca2D3 synthesis, which stimulates intestinal calcium absorption. The net effect is a normalisation of serum calcium levels [2D3, which can bind to the vitamin D receptor. Inactivation requires 24-hydroxylase (CYP24A1) [2D3. At the kidney proximal tubule, FGF binding to the FGF receptor (FGFR)–klotho complex stimulates signalling pathways that decrease PTH transcription and reduce plasma membrane expression of sodium–phosphate transporters resulting in reduced phosphate uptake and increased urinary excretion. FGF23 also reduces synthesis of 1,25(OH)2D by inhibiting CYP27B1 and stimulating CYP24A1 [2D3 and FGF23 are low in foetal circulation [Serum calcium levels are maintained by interplay between the parathyroid glands, kidney, gut and bone Fig. . When sem levels –9. Elevam levels –9. VitamCYP24A1) –9. FGF23culation .Fig. 1CThe causes of hypercalcaemia in the neonate are often distinct from those in older children and adults. This review will focus primarily on genetic causes of hypercalcaemia in neonates and young infants. Neonatal hypercalcaemia can be due to maternal or neonatal factors and can be divided into high or low PTH disorders Table .Neonatal severe hyperparathyroidism (NSHPT) (OMIM #239200) is a rare condition in which affected infants present with marked hypercalcaemia, skeletal demineralisation and failure to thrive, and can be fatal if untreated . In 1993Individuals with NSHPT usually present within the first 2 weeks post-partum with serum calcium levels of > 4.5 mmol/L . Serum cParathyroidectomy is the preferred treatment for NSHPT, with bisphosphonates used to reduce hypercalcaemia prior to surgery . SurgeryFHH , OMIM #145980) is an autosomal dominant disorder characterised by lifelong elevated serum calcium levels, high (approximately one-third) or inappropriately normal PTH concentrations and low kidney calcium excretion , 18, 19.FHH is rarely detected in infants, and the majority of patients with FHH are asymptomatic (~ 71%). However, some individuals have hypercalcaemic symptoms including constipation, fatigue, headaches, muscle cramps, nausea and vomiting, while other patients present with associated features including nephrocalcinosis/nephrolithiasis, osteoporosis and/or fractures and pancreatitis . TreatmeAP2S1 and GNA11 mutations [In the last decade, the genetic heterogeneity of FHH has emerged, with mutations in the Gα11 protein , by whicutations , 28.Foetal serum calcium levels are maintained at a higher concentration than maternal calcium concentrations to allow skeletal formation and mineralisation to take place . Materna2D3. This is rare since the introduction of breast milk fortifiers, which contain 30–40 mg kg−1 day−1 of phosphate [2+:PO4 ratios, along with careful monitoring of electrolytes including phosphorus have been recommended to prevent hypercalcaemia and hypophosphatemia [Hypercalcaemia can occur in the neonate or young infant following ingestion of excess calcium in enriched formulas. Low dietary phosphate can also cause hypercalcaemia, due to suppression of FGF23 or elevations in 1,25(OH)hosphate . In partphatemia , 34. Hypphatemia . Formulaphatemia .Excessive vitamin D intake, either by the infant or the breast-feeding mother can lead to increased intestinal calcium and phosphate absorption, enhanced bone resorption, and consequently hypercalcaemia and hyperphosphatemia . RehydraHypercalcaemia has been reported in some infants receiving enteral feeding formulas containing excess vitamin A. The metabolite of vitamin A, retinoic acid, can bind to receptors on osteoclasts and osteoblasts, resulting in increased bone resorption and decreased bone formation .Jansen’s metaphyseal chondrodysplasia  is an autosomal dominant disease characterised by short-limbed short stature, deformed, under-mineralised bones, chronic hypercalcaemia and hyperphosphaturia. Most patients have low or undetectable serum PTH levels and elevated serum markers of bone turnover .de novo, although some familial cases have been described [JMC is very rare, with fewer than 30 reported cases to date . It is cescribed . All repescribed , 41. Strescribed . Such a escribed . This isJMC initially presents as a generalised skeletal dysplasia, but as patients age it becomes apparent that the metaphyses are preferentially affected, with progressive expansion and irregular ossification, resulting in short and bowed legs and short hands with clubbed fingers , 40. Thi3, elevated serum alkaline phosphatase, and low or undetectable levels of PTH and PTH-related peptide (PTHrP) [The metaphyseal irregularities and skeletal hypo-mineralisation can be mistaken for hypophosphatemic rickets; however, the biochemical findings are inconsistent with this diagnosis . Neonate (PTHrP) . Serum c (PTHrP) ; however (PTHrP) .Attempts to perform genotype-phenotype correlations have been made, though there are inconsistencies in their findings. Early studies indicated that patients with His223 mutations may have more severe hypercalcaemia. However, it is now recognised that there is considerable variability even within families , 44. TheThere are currently no effective treatments for JMC, although the use of bisphosphonates in older children to reduce hypercalcaemia has been reported , 46. RecBetween the 1930s and 1950s, there was an increase in reported cases of hypercalcaemia in children by clinicians in the UK. Initial observations included failure to thrive, hypercalcaemia and distinct facial features (described as “elfin”). While the hypercalcaemia was transient in some, the facial abnormalities, developmental delay and mental retardation persisted . In 19532D3, although some have inappropriately normal levels [IIH classically presents in the first year of life with typical symptoms of hypercalcaemia, including failure to thrive, weight loss, dehydration, polyuria, vomiting and lethargy . Hypercal levels .CYP24A1 gene in several individuals with IIH [CYP24A1 encodes the cytochrome P450 enzyme 24-hydroxylase, which inactivates 1,25(OH)2D3 , indicating that IIH may go undetected if not challenged by vitamin D supplementation, resulting in increased lifetime risk of nephrocalcinosis and nephrolithiasis [Although early studies had established an association with vitamin D supplementation, an explanation for this link was only provided in 2011 when researchers identified recessive mutations in the with IIH . CYP24A1us state . Subsequs of PTH , 51. Funactivity . Some CY failure , 53, witithiasis .SLC34A1, which encodes the kidney proximal tubule sodium-phosphate co-transporter NaPi-IIa, were identified in patients with IIH [CYP27B1 expression and 1a-hydroxylase activity; while inhibiting CYP24A1 expression and 24-hydroxylase activity, which have the combined effect of increasing 1,25-(OH)2D3 and hypercalcaemia [SLC34A1 have also been described in a consanguineous family with an affected individual presenting with renal Fanconi’s syndrome, hypophosphatemic rickets, hypercalciuria and elevated 1,25(OH)2D3 levels [2+D3 levels [In 2016, mutations in a second gene, with IIH . Again, with IIH . This suwith IIH . Functiowith IIH . Subsequalcaemia . Mutatio3 levels , and in 3 levels . The cli3 levels . Thus, aWilliams-Beuren syndrome, also known as Williams syndrome (OMIM #194050), is a rare genetic multisystem disorder that affects the cardiovascular, connective tissue and central nervous systems. It affects ~1 in 7500–10,000 live births , 58. AltThere are discrepancies in the reported frequency of hypercalcaemia in association with Williams syndrome, ranging from as low as 5% to up to 50% , 61. TheELN) is seen in over 90% of cases and has been demonstrated to be responsible for the vascular and connective tissue defects in Williams syndrome [LIMK1 has been reported to contribute to the visuo-spatial cognition problems associated with Williams syndrome [CYP27B1 gene [Williams-Beuren syndrome is a microdeletion disorder caused by the loss of 26–28 genes on 7q11.23 , 62. Mossyndrome . Deletiosyndrome . DeletioCYP24A1 expression [CYP24A1 mutations showed corticosteroids fail to lower serum calcium levels, normalise PTH levels and decrease urinary calcium excretion in some individuals [Several treatment options have been used for hypercalcaemia in IIH and Williams syndrome. Vitamin D supplementation must be stopped immediately in all cases and rehydration performed. Rehydration therapy combined with furosemide is often successful in reducing serum calcium in patients with Williams syndrome; however, some children are unresponsive and require further treatment . Bisphospression . Howeverividuals . Use of ividuals .Descriptions of the successful use of calcitonin, which inhibits osteoclastic bone resorption and enhances kidney calcium excretion, have also been described to treat hypercalcaemia, in combination with thiazide diuretics to reduce hypercalciuria . HoweverALPL gene [ALPL providing confirmation [Hypophosphatasia (OMIM #171760) is caused by inactivating mutations in the tissue non-specific isoenzyme of alkaline phosphatase (TNSALP) encoded by the LPL gene . TNSALP LPL gene , 70. It irmation .ALPL mutations, with autosomal recessive inheritance more common in perinatal and infantile cases [Hypophosphatasia can present in various forms in neonates and infants and has a prevalence of 1:300,000 in Europe and 1:182,000 in Japan . The mosle cases .Pseudomonas aeruginosa and urinary tract infections [Blue diaper syndrome or Drummond’s syndrome (OMIM #211000) is so called as affected infants have inadequate intestinal tryptophan absorption, leading to excessive metabolism by intestinal bacteria and accumulation of indican, which gives urine a distinctive blue colour . The synfections , 75. ThePCSK1) was described [PCSK1 encodes prohormone convertase 1/3, a serine endoprotease that is required for the processing of precursors of many hormones including insulin and glucagon-like peptide-1. Mutations in PCSK1 have previously been associated with obesity and gastrointestinal disorders including malabsorptive diarrhoea [PCSK1 mutations are important in other cases of blue diaper syndrome with hypercalcaemia.Blue diaper syndrome is extremely rare and therefore estimating incidence is difficult. The inheritance pattern is most likely autosomal recessive. Defects in several proteins that transport amino acids including tryptophan have been proposed, although no mutations in these genes have been identified. Recently, a male infant with blue diaper syndrome with a homozygous frameshift mutation in pro-protein convertase subtilisin/kexin type 1 , which is predominantly expressed in the intestinal brush-border membrane [+-K+ pump, resulting in net transport of sodium, sugar and water across the epithelium. Consequently, patients with inactivating mutations in SGLT-1 present with severe life-threatening watery diarrhoea dehydration and abdominal distension in the first weeks of life.Congenital glucose-galactose malabsorption is an autosomal recessive disorder caused by inactivating mutations in the membrane . Glucosemembrane . The low2D3 [2D3 have been hypothesised to contribute to nephrocalcinosis and hypercalcaemia [The majority of mutations in SGLT-1 are nonsense, frameshift or splice site, resulting in truncated proteins that are not expressed . Missens2D3 . Upregulalcaemia . RestricLCT). A lactose-free diet usually leads to rapid recovery.Another rare autosomal recessive disorder, congenital lactase deficiency, has a similar clinical presentation to glucose-galactose malabsorption . NeonateSubcutaneous fat necrosis of the newborn is another rare cause of hypercalcaemia that normally arises within a few weeks of birth , 82. TheSLC12A1) and the potassium channel ROMK (encoded by KCNJ1) [2 (PGE2) in the plasma and urine. The basis of hypercalcaemia in these patients is uncertain, although elevated PGE2 may contribute as studies have shown it can stimulate bone resorption, increase PTH secretion and upregulate hydroxylation of 25-hydroxyvitamin D [Hypercalcaemia has been reported in neonates with the kidney tubular disorder Bartter’s syndrome. The affected infants had mutations in the sodium-potassium-chloride co-transporter NKCC2 (encoded by y KCNJ1) , 86. PatCDKN1C) gene have been identified in some cases, and inheritance may involve maternal imprinting [Hypercalciuria and/or hypercalcaemia can occur in children with the intrauterine growth retardation, metaphyseal dysplasia, adrenal hypoplasia congenita and genital anomalies IMAGe syndrome (OMIM #614732). Heterozygous mutations in the cyclin-dependent kinase inhibitor 1C 2D3 and hypercalciuria, contributing to hypercalcaemia [Hypercalcaemia may occur in association with a number of endocrine disorders including hyperthyroidism, hypothyroidism and adrenal insufficiency. Neonatal hyperthyroidism is usually associated with maternal Graves’ disease but can occur in rare endocrine disorders including autosomal dominant non-autoimmune hyperthyroidism due to activating mutations in the TSH receptor gene, McCune-Albright syndrome due to activating mutations in the alcaemia . In patialcaemia . Rare caalcaemia .Serum calcium levels are higher in neonates than older children and adults. Therefore age-appropriate normal ranges should be used.Transient hypercalcaemia may occur shortly after birth, caused by maternal hypocalcaemia.Accurate identification of the cause of hypercalcaemia may require combined assessment of the family history; biochemical parameters ; evaluation of physical features (to identify dysmorphology); and genetic analyses.As next-generation sequencing continues to improve, it is likely that further hypercalcaemia-associated genes will be identified. This may yield new treatment avenues and continue to improve neonatal care.Both caused by inactivating mutations in the CaSR gene only.Gα11 and AP2σ genes.NSHPT is caused by inactivating mutations in the CaSR only; FHH is caused by inactivating mutations in the CaSR, Gα11 and AP2σ genes; FHH is caused by inactivating mutations in the CaSR gene only.NSHPT is caused by inactivating mutations in the CaSR, Gα11 and AP2σ genes.Both are caused by inactivating mutations in the CaSR, Neonatal severe hyperparathyroidism (NSHPT) and familial hypocalciuric hypercalcaemia (FHH) are:Short-limbed short stature, deformed, under-mineralised bones, chronic hypercalcaemia and hypophosphaturia with elevated serum PTH levels and elevated serum markers of bone turnover.Short-limbed short stature, deformed, under-mineralised bones, chronic hypercalcaemia and hypophosphaturia with normal serum PTH levels and elevated serum markers of bone turnover.Short-limbed short stature, deformed, under-mineralised bones, transient hypercalcaemia and hyperphosphaturia with normal serum PTH levels and elevated serum markers of bone turnover.Short-limbed short stature, deformed, under-mineralised bones, chronic hypercalcaemia and hyperphosphaturia with normal serum PTH levels and elevated serum markers of bone turnover.Jansen’s metaphyseal chondrodysplasia is characterised by:CYP27B1 gene, encoding 1α-hydroxylase, which converts 25-hydroxyvitamin D3 to active 1,25(OH)2D3.The CYP24A1 gene, encoding 24-hydroxylase, which inactivates 1,25(OH)2D3.The CYP2R1 gene, encoding 25-hydroxylase, which converts vitamin D to 25-hydroxyvitamin D3.The CYP24A1 and CYP27B1 genes.The CYP2R1, CYP24A1 and CYP27B1 genes.The Idiopathic infantile hypercalcaemia (IIH) is caused by mutations in:>50%, is often symptomatic, may resolve within the first few years, but may recur during puberty.<50%, is often symptomatic, may resolve within the first few years, but may recur during puberty.<50%, is often asymptomatic, and always resolves within the first few years, with no recurrence.>50%, is often asymptomatic, and always resolves within the first few years, with no recurrence.In Williams syndrome the reported frequency of hypercalcaemia is:ALPL gene:Jansen’s metaphyseal chondrodysplasiaWilliam’s syndromeHypophosphatasiaBlue diaper syndromeSubcutaneous fat necrosis of the newbornWhich of these causes of hypercalcaemia is characterised by poor feeding, rachitic deformities that can result in respiratory complications, persistent rickets, bony craniosynostosis and is associated with mutations in the


In contrast, the mouse homolog of AHD11-AS1 (Abhd11os) overexpression had exhibited neuroprotective effects against mutant huntingtin-induced toxicity. Considering the emerging research reports, the authors attempted in this first review on ABHD11-AS1 to summarize and highlight its oncogenic potential and clinical significance in different human cancers. Lastly, we underlined the necessity for future mechanistic studies to unravel the role of ABHD11-AS1 in tumor development, prognosis, progression, and targeted therapeutic approaches.The aberrant expression of lncRNAs has been linked to the development and progression of different cancers. One such lncRNA is ABHD11 antisense RNA 1 (ABHD11-AS1), which has recently gained attention for its significant role in human malignancies. ABHD11-AS1 is highly expressed in gastric, lung, breast, colorectal, thyroid, pancreas, ovary, endometrium, cervix, and bladder cancers. Several reports highlighted the clinical significance of ABHD11-AS1 in prognosis, diagnosis, prediction of cancer progression stage, and treatment response. Significantly, the levels of ABHD11-AS1 in gastric juice had been exhibited as a clinical biomarker for the assessment of gastric cancer, while its serum levels have prognostic potential in thyroid cancers. The ABHD11-AS1 has been reported to exert oncogenic effects by sponging different microRNAs (miRNAs), altering signaling pathways such as PI3K/Akt, epigenetic mechanisms, and N6-methyladenosine (m Despite enormous progress in cancer research, most cancers are diagnosed at advanced stages resulting in high mortality due to a lack of methods for early diagnosis, effective treatment, and disease relapse. The currently available cancer biomarkers for diagnosis and treatment in clinical use are proteins. Since 2% of the genome only codes for proteins, the research has been focused more on the non-coding genome. Over the past decade, elucidation of the functional importance of non-coding DNA in the human genome has gained the attention of researchers. As a result, several non-coding RNAs (ncRNAs) and long non-coding RNAs (lncRNAs) have been identified that play a critical regulatory role in various cellular processes and biological functions [Cancer is a dangerous disease characterized by the uncontrollable proliferation of abnormal cells with high morbidity and mortality . As per ABHD11 antisense RNA 1 , which has gained attention over the years for its critical role in cancer progression, diagnosis, and treatment.LncRNAs are defined as non-protein coding RNA transcripts with over 200 nucleotides in length . LncRNAsABHD11-AS1 is an RNA gene known as long intergenic non-protein coding RNA 35 (LINC00035) and Williams–Beuren syndrome chromosome region 26 (WBSCR26), located at 7q11.23. Firstly, ABHD11-AS1 was reported to upregulate and correlate with clinicopathological features in gastric cancer progression . After tAmong numerous lncRNAs, a handful were characterized as they were very poorly expressed, poorly conserved, and more tissue specific. Existing literature indicates that some lncRNAs function in ‘trans’, far from their site of transcription, while others act in ‘cis’, modulating the expression of nearby genes. Some lncRNAs play functional roles in cellular homeostasis, growth, differentiation, and act as oncogenes or tumor suppressors . Though Gastric cancer (GC) is the most common gastrointestinal malignant tumor, with a high mortality and recurrence rate, typically originating from the gastric mucosal epithelium . GC is tThe expression levels of ABHD11-AS1 lncRNA in the plasma of GC patients of different stages were not high enough to differentiate from that of healthy control subjects . Very reThyroid cancer is the most typical endocrine-related malignant tumor. The incidence and mortality rate of thyroid cancer patients has continuously increased for several decades, with approximately 300,000 new cases and 40,000 deaths every year worldwide . ThyroidOvarian cancer (OC) represents the most lethal gynecological cancer of the female reproductive organs. Epithelial ovarian cancer (EOC) is prevalent, and accounts for more than 90% of OC cases worldwide . EOC is Colorectal cancer (CRC) is the most frequently diagnosed digestive system malignant tumor, and the leading cause of cancer deaths worldwide . CRC is Pancreatic cancer (PC) is the seventh most common malignant tumor worldwide, with high mortality due to lack of specific symptoms, high invasiveness, and metastasis. PC can develop in either exocrine cells or neuroendocrine cells (islet cells) of the pancreas. The exocrine cancers are most common in PC, and diagnosed at an advanced stage, whereas pancreatic neuroendocrine tumors (NETs) or islet cell tumors are less familiar with a better prognosis. Despite the current research and therapeutic development progress, the median survival time is only 3–6 months, with a five-year survival rate remaining under 5% ,57. The Moreover, the knockdown of ABHD11-AS1 in three PC cell lines  resulted in decreased cell proliferation, colony formation, and metastasis (migration and invasion) with increased apoptosis ,26. TargLACTB2, SPANXA1, SPANXA2, and SPANXC) hub genes [Breast cancer (BC) is a common cancer among women, accounting for 30% of all female cancer cases reported worldwide. BC is the second leading cause of cancer-related deaths . The lumub genes . Recentlub genes . ABHD11-ub genes . AlthougLung cancer incidence and mortality have increased over the past decade globally. Non-small cell lung cancer (NSCLC) is the most common histological type, accounting for 85% of reported lung cancer cases and deaths. Despite the tremendous progress in NSCLC diagnosis and treatment regimen over recent years, the five-year survival rate of NSCLC patients remains around 15% . Therefop < 0.001), tumor invasion depth (p = 0.001), and TNM stage. The authors have implicated different bladder cancer cell lines , which had higher ABHD11-AS1 expression than SV-HUC-1, a normal control cell line established from healthy ureter tissue. The siRNA-mediated knockdown of ABHD11-AS1 resulted in decreased cell proliferation (in MTT and EdU assays) and cell migration (scratch assay), along with enhanced apoptosis (Caspase-3 activity and flow cytometry) in bladder cancer cell lines. The overexpression of ABHD11-AS1 in bladder cancer cell lines exhibited contrary effects to its silencing. Considering these in vitro results, authors have concluded that ABHD11-AS1 lncRNA serves as an oncogene, and might be a potential therapeutic target in bladder cancer [Bladder cancer is the ninth most commonly diagnosed cancer globally, with an estimate of 500,000 cases annually. Since 2017, several studies have reported aberrant expression of lncRNAs that play a functional role in the prognosis, migration, and invasion of bladder cancer ,68,69. Rr cancer . FurtherEndometrial cancer (EC) is also called uterine cancer, in which malignant cells form in the endometrium tissue lining of the uterus. The incidence of EC is increasing every year, and leads to the second highest rates of death from gynecological cancers in women worldwide. EC is diagnosed at an average age of 60, and primarily affects post-menopausal women . In receCervical cancer (CC) is the primary gynecological malignant tumor that develops in a woman’s cervix. As per WHO statistics of 2018, 570,000 were diagnosed, and 300,000 women have died from CC globally. Mostly, the infection with high-risk human papillomaviruses (HR-HPV) has been linked to CC cases. In addition to HR-HPV infection, an individual’s genetic and epigenetic alterations significantly contribute to the development of CC ,75. The This section discusses various regulatory mechanisms through which ABHD11-AS1 contributes to the development and progression of different types of cancers. The modes of action of ABHD11-AS1 lncRNA are summarized in miRNAs are a class of small non-coding RNAs (sncRNAs) of 19–24 nucleotides in length, which can post-transcriptionally repress gene expression via binding to the 3’-untranslated region (3’-UTR) and translational inhibition of multiple target mRNAs and/or other RNAs . LncRNAsSOX4 (SRY-Box Transcription Factor 4) was a potential downstream target of miR-133a and ABHD11-AS1/miR-133a/SOX4 axis established as crucial for the prognosis of CRC [Lei et al. reported that ABHD11-AS1 interacts with miR-133a, which acts as a tumor suppressor in CRC by targeting LIM, SH3 protein 1, and inhibiting the mitogen-activated protein kinase (MAPK) pathway ,80. Amons of CRC . Accordis of CRC . Moreoves of CRC . Lately,s of CRC .STAT3 and SLC1A5, respectively [LPAR3) and epidermal growth factor receptor (EGFR) were direct targets of miR-133a-3p in thyroid cancer [PDPK1, CCNE1 (cyclin E1), and MARK2 in GC, PC, and CC, respectively. The current literature indicates that ABHD11-AS1 acts as ceRNA by sponging miRNAs to exert oncogenic effects in human cancers, and could be a potential therapeutic target.ABHD11-AS1 was found to sponge miRNAs miR-1301-3p and miR-199a-5p in PTC, and modulated the expression of their downstream targets ectively . In a duectively . The miRectively . Lysophod cancer . Howeverd cancer , miR-123d cancer , and miRd cancer  to exhibLncRNAs are known to regulate several oncogenic signaling pathways that control proliferation, growth, metastasis, invasion, and apoptosis by modulating critical signaling components and pathway activation ,83. The STAT3 axis [PDPK1 axis, and contribute to GC progression and metastasis [PI3K/Akt signaling is a vital pathway that regulates various cellular processes, including proliferation, differentiation, survival, and migration. This pathway is most frequently altered signaling in human cancers development and progression . It has AT3 axis , thus retastasis . Very retastasis . Thus, iEPS15L1) [The trans-membrane EGFR proteins are a family of receptor tyrosine kinases activated upon binding with peptide growth factors of the EGF-family of proteins. Due to genomic alterations, high expression and constitutive activation of EGFR often correlate positively with uncontrolled cell proliferation, tumor progression, and metastasis in human cancers, including OC, leading to a poor prognosis ,87. The EPS15L1) . A positRhoC is one of the Rho family of small Guanosine Triphosphatases (GTPases), which are key players regulating the cytoskeletal organization and affect cell division, migration, and polarity. Accumulated evidence indicates that adverse RhoC signaling affects angiogenesis and EMT processes, and is linked with cancer metastasis in different malignant tumors . In 2017LncRNAs are widely expressed in human cells, and regulate various biological processes such as proliferation, apoptosis, differentiation, and the cell cycle. A high-content RNAi screen identified several lncRNAs involved in genomic stability and cell division . The mamEpigenetics denotes the heritable changes in gene expression without modifications in DNA sequence. The epigenetic mechanisms include DNA methylation, histone modifications, chromatin remodeling, and non-coding RNAs. For several years, epigenetic dysregulation has contributed significantly to the development and progression of different human diseases, notably cancer. The epigenetic regulators are exploited as effective therapeutic targets for the treatment of cancer, and many molecules are in clinical development . The potKLF4 (Kruppel-Like Factor 4) transcription factor through the recruitment of EZH2, and thus H3K27me3 at its promoter to promote the Warburg effect [TIMP2 expression via EZH2-mediated H3K27me3 at its promoter [KLF4 and TIMP2 with a significant reduction in the occupancy of EZH2 and H3K27me3 at their promoters [Among different epigenetic alterations, histone modification, such as methylation, has been known to be critical for various biological processes in all eukaryotes. Histone methylation on a few residues, such as H3K4, H3K36, and H3K79, induces gene transcription, while methylation on H3K9, H3K27, and H4K20 inhibits gene expression ,95. The g effect . Similarpromoter ,32. ABHDromoters ,31. The 6A) modification is the most common form of dynamic and reversible RNA methylation in mammals. The m6A methylation accounting for 0.1–0.4% of adenosines in total RNA is modified [6A sites are enriched in 3′-UTR and near stop codons [6A methylation levels result in disease conditions, and significantly affect tumorigenesis and tumor progression. The dynamic equilibration in m6A levels were achieved by coordinated interaction of different enzymes, such as m6A methyltransferases (writers), m6A demethylases (erasers), and proteins that recognize m6A RNA modification (readers). The methyltransferase-like 3 (METTL3) is an important component of the classical complex of writers [6A levels by methylated RNA immunoprecipitation sequencing (MeRIP-Seq), and discovered that ABHD11-AS1 lncRNA transcript gained m6A modification in NSCLC cells (H1299), in comparison to normal cells [6A methylation on ABHD11-AS1 sequences. Moreover, overexpression of METTL3 significantly increased the m6A modification levels, and enhanced the stability of the ABHD11-AS1 lncRNA transcript. Therefore, it was concluded that METTL3-mediated m6A modification as a potential mechanism through which the ABHD11-AS1 is stably overexpressed, and contributes to the progression of NSCLC [Post-transcriptional modification of RNA gained the attention of researchers due to its emerging role both in physiological and pathological processes. N6-methyladenosine (mmodified . The higp codons . The abe writers . Very real cells . The bioof NSCLC .6A modification on ABHD11-AS1 enhances the stability of its transcript, and thus contributes to the development of lung cancer. In a few studies, ABHD11-AS1 was established as circulatory lncRNA, and its levels in body fluids serve as an essential biomarker for the early diagnosis of PC and PTC. The ABHD11-AS1 levels in gastric juice could serve as a biomarker for the early diagnosis of gastric cancer. Altogether, ABHD11-AS1 has excellent potential to serve as a molecular marker for early diagnosis, treatment, and improving human cancers’ overall prognosis. Considering the available literature, the authors warrant further mechanistic studies employing advanced technologies such as CRISPR/Cas9, RIP, ChIP, ChIRP, and transcriptomics to delineate the functions and molecular mechanisms of ABHD11-AS1 lncRNA in human malignancies. The development of gene-targeted therapies could exploit ABHD11-AS1 as a potential therapeutic target for cancer treatment.LncRNAs are aberrantly expressed in human malignancies and exploited as biomarkers for tumor diagnosis, metastasis, invasion, and treatment. One such lncRNA with oncogenic potential is ABHD11-AS1, which has gained a lot of attention in recent years due to its significant role in human cancers. This review summarizes the studies that reported the molecular mechanisms through which ABHD11-AS1 exerts its tumor-promoting effects in malignant tumors, and serves as a biomarker for the early diagnosis and treatment of cancers . ABHD11-


Hidradenitis suppurativa (HS) is a chronic, recurrent, inflammatory skin disease deriving from the hair follicles. The formation of inflammatory nodules, abscesses, fistulas, and sinus tracts is characterized by a large inflow of key pro-inflammatory mediators, such as IFN-γ, TNF-α, IL-1, IL-17, and IL-12/23. Adalimumab is currently the only Food and Drug Administration (FDA)- and European Medicines Agency (EMA)-approved biologic therapy for moderate to severe HS in adults and adolescents. However, the long-term effectiveness of this TNF-α inhibitor in HS patients has shown to be highly variable. This review aims to review the evidence for emerging therapies that target the main pro-inflammatory cytokines in HS pathogenesis. A review of the literature was conducted, using the PubMed and Google Scholar repositories, as well as Clinicaltrials.gov. Presently, the most promising biologics in phase III trials are anti-IL-17 antibodies, secukinumab, and bimekizumab. Furthermore, an anti-IL-1 biologic, bermekimab, is currently in phase II trials, and shows encouraging results. Overall, the clinical efficacies of all new targeted therapies published up to this point are limited. More studies need to be performed to clarify the precise molecular pathology, and assess the efficacy of biological therapies for HS. Hidradenitis suppurativa (HS) is a chronic, recurrent, inflammatory skin disease deriving from the hair follicles, affecting intertriginous skin, and commonly associated with various systemic comorbidities . Due to The clinical course is very variable, and ranges from mild clinical presentation, characterized by the formation of papules, pustules, and nodules, to severe cases of deep abscesses, draining sinus tracts, and keloid scars . The affHS has commonly been staged according to the Hurley staging system , a usefuConventional medical therapy, involving topical treatment and oral antibiotics, and lifestyle modifications, including weight loss and cigarette smoking cessation, are suitable for treating mild to moderate HS . With thThis review aims to provide current insights into the underlying molecular pathophysiology of HS, and the evidence for new and emerging therapies that specifically target cytokines involved in HS pathogenesis.The pathogenesis of HS is multifactorial, with genetic, environmental, and immunologic factors included in both the onset and maintenance of the condition .Up to 42% of patients with HS report a family history of the condition . The genPSEN1/PSEN2, PSENEN, NCSTN, and APH1A/APH1B, respectively . Fo. Fo38]. The second event is rupture of the dilated follicle , and disThe complement dysregulation is observed in HS, but it is uncertain whether elevated C5a and decreased C3b in serum of HS patients reflect a primary event in the HS pathogenesis, or result from advanced-stage disease. The primary function of C5 and C3 includes activation of the immune system via anaphylatoxins, opsonization, and bacterial lysis, all early events in HS. Elevated C5a in HS suggests complement hyperactivation, whereas decreased C3 results from using C3b for bacterial opsonization . ClinicaIn the Hurley stage II/III, nodules, abscesses, and fistulas are further formed, characterized by a massive infiltration of inflammatory cells and pro-inflammatory cytokines, including IFN-γ, TNF-α, IL-1, IL-17, and IL-12/23 . ActivatAfter follicular rupture, dispersed follicular content and the cytokine-driven feedback propagate and sustain chronic inflammation, the third event in the HS pathogenesis  38]. In. In38]. Despite many attempts to clarify the HS pathogenesis, it remains still poorly understood. Literature data describes the lack of antigen-specificity of T-cells involved in underlying pathophysiological mechanisms of HS, suggesting that HS is rather a cytokine-driven inflammatory process . RecentlTherapy for HS includes topical, systemic, surgical, and combined treatment. General measures and additional medical treatments are recommended regardless of the disease severity .The initial management of patients in Hurley stage I or mild HS usually includes a clindamycin 1% solution applied b.i.d.  during 3 months in the skin areas subject to recurrent flares ,54. PatiResorcinol is a topical peeling agent with keratolytic, antipruritic, and anti-inflammatory properties that can be administered for HS (Hurley I or II), where available . PatientAn intralesional corticosteroid injection involves the injection of a corticosteroid, such as triamcinolone acetonide, at a concentration of 5 to 10 mg/mL, directly into an inflammatory nodule . A noticA phase II open-label study (NCT04414514) assessing the efficacy of topical Janus Kinase (JAK) inhibitor (ruxolitinib 1.5% cream) in HS patients awaits recruitment of participants . Patients who do not respond to topical clindamycin are candidates for oral therapy. Tetracyclines are recommended as a first-line treatment for Hurley stage I or II . A commoClindamycin and rifampicin combination therapy is administered for patients who do not improve with oral tetracyclines treatment, or as first-line treatment for patients in Hurley stage II ,54. A tyShort-term therapy with different antibiotics  can be administered to prevent impairment, or reduce pain . AmoxiciAcitretin has been used to treat HS in the early stages of HS (Hurley I or mild II), and the chronic stages of HS with recurrent nodules, abscesses, sinus tracts, and scarring . AcitretDapsone is a sulphone drug with antibacterial and anti-inflammatory effects. This drug should be administered if a patient with mild to moderate disease (Hurley stage I or II) fails to respond to the standard first- or second-line treatment . EfficacZinc salts have anti-inflammatory and antiandrogenic properties, and may be administered as maintenance therapy in Hurley I and II stages . At initCiclosporin A is a calcineurin inhibitor and a potent immunosuppressant. It should be administered to HS patients who fail to respond to standard first-, second-, and third-line therapies . PreviouLong-term therapy with systemic corticosteroids can result in rebound flare on withdrawal. On the other hand, short-term systemic therapy (0.5–0.7 mg/kg daily) with rapid tapering can be beneficial in the reduction of lesions in acute flares .Surgical therapy is recommended in more severe cases of HS, with abscesses, fistulas, and sinus tracts formation, or in a longstanding HS refractory to other treatments . The typ2) laser, long-pulsed neodymium-doped yttrium aluminum garnet (Nd:YAG) laser, and photodynamic therapy (PDT) ..78].Infliximab is a chimeric mouse/human IgG1 monoclonal that binds to soluble and transmembrane TNF-α with high affinity. It is FDA-approved for use in IBD, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, and plaque psoriasis .In a phase II double-blind, placebo-controlled trial, 38 patients with moderate to severe HS were randomized to receive 5 mg/kg of intravenous infliximab on weeks 0, 2, 4, 6, 14, and 22, or a placebo. By week 8, 57% of patients had a 50% or greater decrease in HSSI compared to 5% of patients in the placebo group. The infliximab use also improved the pain VAS and DLQI scores .A long-term study evaluated the efficacy of a single course of infliximab  on 10 patients, and followed up for at least 1 year. Three patients had no recurrence of lesions in a 2-year follow-up period .A study conducted to determine the optimal dosing of infliximab included 52 patients with HS. Patients tolerated infliximab well, and achieved significant improvement in abscess and nodule count, most of them (67%) at a 10 mg/kg schedule intravenously every 6 or 8 weeks . A prospGemella morbillorum bacteremia, and a metastatic cutaneous squamous cell carcinoma during infliximab treatment ..88]..Etanercept is a fully human recombinant molecule consisting of two soluble TNF receptor subunits (p75) fused to the Fc portion of human IgG1. It is FDA-approved for use in rheumatoid arthritis, juvenile idiopathic arthritis, ankylosing spondylitis, plaque psoriasis, and psoriatic arthritis In a phase II placebo-controlled study, 20 patients with moderate to severe HS were randomized to receive etanercept 50 mg or placebo subcutaneously twice a week for 12 weeks. Afterwards, 50 mg of etanercept subcutaneously twice a week for 12 more weeks was administered to all participants. The results showed that etanercept was not efficient enough for HS treatment  90]..90].Golimumab is a fully human anti-TNF-α IgG1 monoclonal antibody that binds to soluble and membrane-bound TNF-α with high affinity. It is FDA-approved for use in rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and ulcerative colitis . Our literature search found only two case reports regarding the use of golimumab for HS. One described the use of golimumab, 50 mg subcutaneously every 4 weeks, in a patient with severe HS and psoriatic arthritis who had failed adalimumab and anakinra previously. After administering golimumab, the patient’s HS worsened despite the improvement in her psoriatic arthritis . The secCertolizumab pegol (CZP) is a pegylated humanized monoclonal anti-TNF-α agent, which lacks the fragment crystallizable (Fc) region, thus preventing active placental transfer. It is FDA-approved for use in Crohn’s disease, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and plaque psoriasis .Several case reports described the use of certolizumab pegol (400 mg subcutaneously every other week) in HS patients refractory to other biological drugs with good disease control ,96,97.A retrospective study on the use of TNF-α inhibitors described no efficacy of certolizumab pegol (200 mg subcutaneously every 2 weeks) treatment in two HS patients .One of the published case reports described the treatment of a pregnant HS patient with certolizumab pegol. From 19 weeks of gestation, certolizumab pegol 400 mg was administered subcutaneously every other week. At the 8-week follow-up, the patient achieved HiSCR. However, at the next visit, the patient presented with worsening pain and one new abscess. Therefore, the dose was increased to 400 mg subcutaneously weekly at 32 weeks of gestation, and symptoms significantly improved. The medication was well-tolerated, and adequate pain control was achieved  95]..95].Secukinumab is a fully human IgG1κ monoclonal antibody that binds to IL-17A with high affinity. It is FDA-approved for moderate to severe plaque psoriasis, psoriatic arthritis, and ankylosing spondylitis .In a 24-week-long, open-label pilot trial of nine patients with moderate to severe HS, secukinumab 300 mg was administered subcutaneously weekly for 5 weeks, and then every 4 weeks for 24 weeks. Even though HiSCR was achieved in 78% of patients by week 24, the authors concluded that the psoriasis dosing regimen might not be sufficient for treating HS, as inflammatory levels are higher in HS .n = 14) of all patients achieved HiSCR by week 24 ..113].The extremely rapid clinical response of 20 HS patients treated with brodalumab and a significant reduction in dermal tunnel drainage ,112 are Bimekizumab is a humanized IgG1κ monoclonal antibody that neutralizes both IL-17A and IL-17F. Studies have shown that bimekizumab is effective in patients with psoriasis .A phase II clinical trial randomized 90 patients with moderate to severe HS to receive bimekizumab , a placebo, or adalimumab. At week 12, 46% of patients treated with bimekizumab achieved a 75% reduction in total abscesses and nodules count (HiSCR 75), and 32% of them achieved HiSCR 90, in comparison to 10% of patients treated with placebo who achieved HiSCR 75, and none of whom achieved HiSCR 90. Thirty-five percent of patients treated with adalimumab achieved HiSCR 75, and 15% of them achieved HiSCR 90 .Currently, three phase III clinical studies to evaluate the efficacy and safety of bimekizumab in HS treatment are underway   116,117117,118.Ixekizumab is a humanized IgG4 monoclonal antibody that neutralizes soluble IL-17A and IL-17 A/F. It is FDA-approved for use in moderate to severe plaque psoriasis, active psoriatic arthritis, active ankylosing spondylitis, and active non-radiographic axial spondyloarthritis with objective signs of inflammation .Two case reports of HS patients with concomitant psoriasis treated with ixekizumab were published. The results suggested ixekizumab as a safe and effective treatment for both psoriasis and HS ,121.Another case report of a female HS patient with concomitant herpes simplex virus (HSV) infection reported ixekizumab administration. After HSV resolution, the patient was initiated on subcutaneous ixekizumab 160 mg at week 0, and 80 mg at weeks 2, 4, 6, 8, 10, and 12, followed by 80 mg every 4 weeks. Thirteen months after initiating ixekizumab, the patient demonstrated significant improvement in her condition, suggesting that ixekizumab may represent another promising therapeutic modality  122]..122].CJM112 is a human IgG1κ monoclonal antibody that neutralizes soluble IL-17 and IL-17A/F .A phase II randomized controlled study was conducted in patients with moderate to severe HS to evaluate the efficacy and safety of multiple doses of CJM112 (NCT02421172). HS-PGA response rate was 32.3% in patients treated with CJM112 at week 16, compared to 12.5% in patients treated with placebo  124]..124].Ustekinumab is a fully human IgG1κ monoclonal antibody that neutralizes the p40 subunit of IL-12 and IL-23. It is FDA-approved for use in plaque psoriasis, psoriatic arthritis, and Crohn’s disease . A phase II open-label study included 17 patients with moderate to severe HS treated with 45 or 90 mg ustekinumab subcutaneously at weeks 0, 4, 16, and 28. In 82% of patients, moderate to marked improvement of the mSS was reported at week 40, whereas 47% of patients achieved HiSCR. A low concentration of leukotriene A4-hydrolase (LTA4H) was observed in good responders to the biologic, which may be predictive of ustekinumab effectiveness .A multicentric retrospective review reported treatment of 14 HS patients with an ustekinumab dosage regimen for Crohn’s disease. Patients received an intravenous infusion of ustekinumab adjusted by weight , and then a subcutaneous maintenance dose of 90 mg every 8 weeks. The study involved the patients with recalcitrant HS, resistant to at least one prior biologic drug. Fifty percent of the patients achieved HiSCR. A significant pain reduction and quality of life improvement were observed in 71.42% of patients at week 16 . The equSeveral case reports and case series studies described the variable efficacy of ustekinumab in treating HS patients. In one case series study, the improvement in the HS-PGA score was noticed in 70% of patients, and the improvement in the Numerical Pain Rating Scale (NPRS) was recorded in 80% of patients . AnotherBased on the published results so far, it cannot be concluded that the ustekinumab dosage regimen for Crohn’s disease results in better clinical outcomes than the psoriasis dosage regimen. However, this requires further investigation in randomized controlled trials.Guselkumab is a human IgG1κ monoclonal antibody that neutralizes the p19 subunit of IL-23. It is FDA-approved for use in adults with moderate to severe plaque psoriasis .Several case reports and case series reported achieving HiSCR after administering 100 mg guselkumab subcutaneously at weeks 0 and 4 (induction phase), followed by injections every 8 weeks in HS patients ,135,136.In a case series that included four patients, subcutaneous injections of 100 mg guselkumab were administered in higher frequency at week 0, and then every 4 weeks. Two patients (50%) only slightly improved after the guselkumab treatment, one patient showed no response to therapy, and one patient had increased HS severity after the treatment .In a phase II placebo-controlled, double-blind study (NCT03628924), patients with moderate to severe HS were randomized to receive two different dosages of guselkumab or a placebo. The results showed that HiSCR was achieved at week 16 in 50.8% of the participants treated with 200 mg of guselkumab subcutaneously at weeks 0, 4, 8, and 12. In comparison, 38.7% of the participants who received a placebo, and 45% of participants who received 1200 mg of guselkumab intravenously at weeks 0, 4, and 8, followed by 200 mg of guselkumab subcutaneously at week 12, achieved HiSCR at week 16  138]..138].Risankizumab is a fully human IgG1κ monoclonal antibody that neutralizes the IL-23 . It is FWe found four case reports describing the successful use of risankizumab  in HS patients ,142,143.In a phase II placebo-controlled study, patients with moderate to severe HS were randomized to receive one of two dose levels of risankizumab or a placebo. No results have been posted yet (NCT03926169)  144]..144].Tildrakizumab is a humanized IgG1κ monoclonal antibody that neutralizes the p19 subunit of IL-23. It has been FDA-approved for use in moderate to severe plaque psoriasis .As found by our literature search, there are only two case series studies describing the use of tildrakizumab 100 mg subcutaneously at weeks 0 and 4, and then 200 mg every 4 weeks, for the treatment of moderate to severe HS. All patients achieved HiSCR at week 8, and half of them from every study reported improvement in DLQI scores  146,147147.Anakinra is a fully human recombinant IL-1 receptor (IL-1R) monoclonal antibody that inhibits IL-1α and IL-1β from interacting with IL-1R. It is FDA-approved for use in rheumatoid arthritis, neonatal-onset multisystem inflammatory disease, and deficiency of IL-1 receptor antagonist (DIRA) ,149.In a 24-week-long, placebo-controlled trial, 20 HS patients were randomized to receive 100 mg of anakinra subcutaneously g.d. or a placebo for 12 weeks. At week 12, 78% of HS patients treated with anakinra achieved HiSCR, compared to 30% in the placebo group. However, the difference in the HiSCR rates after 12 weeks was not significant .A case series of six HS patients treated with 100 mg of anakinra subcutaneously g.d. showed improvement in the mSS, HS-PGA, and DLQI scores after 8 weeks for five patients who completed the treatment course. However, relapse of HS occurred eight weeks after the end of the treatment course .Due to the reported rapid rebound of HS after anakinra cessation ,151, lonOne case report described the administration of higher doses of anakinra (200 mg subcutaneously g.d.) to a female patient with refractory HS. This led to the disease remission, which was maintained 1 year after the end of anakinra treatment .Several case reports reported the failure of anakinra in treating severe HS  92,153,,153,154.Bermekimab, also named MABp1, is a fully human recombinant IgG1κ monoclonal antibody that neutralizes IL-1α . High coIn a prospective trial, 20 HS patients were randomized to receive a placebo or bermekimab (7.5 mg/kg intravenously every other week) for 12 weeks. By the end of week 12, 60% of patients treated with bermekimab achieved HiSCR, compared to 10% of patients treated with placebo (NCT02643654) . When reIn a recent phase II open-label study, HS patients who were naive to anti-TNF therapy (18), or had failed prior anti-TNF therapy (23), were treated with 400 mg of bermekimab subcutaneously weekly (13 doses). A high percentage of patients achieved HiSCR at week 12  .A new, phase II, randomized, placebo and active comparator-controlled dose range study to evaluate the safety and efficacy of bermekimab for the treatment of HS patients is currently recruiting participants (NCT04988308)  159]..159].Further phase II and phase III randomized controlled studies are necessary to confirm the efficacy and safety of this promising biologic in the HS treatment.Canakinumab is a fully human IgG1κ monoclonal antibody that targets IL-1β. It is FDA-approved for use in cryopyrin-associated periodic syndromes and systemic juvenile idiopathic arthritis .Our literature search found controversial results regarding canakinumab use  in HS patients. Several case reports described a positive response, demonstrated by an improvement in mSS and reduced pain score without any adverse effect ,162, wheApremilast is an orally administered selective PDE-4 inhibitor that regulates inflammatory mediators, such as TNF-α, IL-10, and IL-23. It is FDA-approved for use in patients with moderate to severe plaque psoriasis and active psoriatic arthritis .A trial in which 20 patients with moderate to severe HS were randomized to receive either 30 mg of apremilast b.i.d. (15 patients) or placebo (5 patients) reported that 53.3% of patients treated with apremilast achieved HiSCR at week 16 compared to 0% of patients treated with placebo .After the study completion, 100% of the responders chose to continue treatment. Fifty percent (4/8) of initial responders discontinued treatment within the first year, and the 2-year follow-up data were available for four of the initial responders. All patients who continued the treatment maintained HiSCR during the 2-year follow-up .In a case series study of nine HS patients who failed to respond to other treatments, five out of six patients who persisted with treatment showed a significant improvement in the mSS, the pain VAS, and DLQI scores .IFX-1 is an IgG4κ monoclonal antibody that binds to C5a with high affinity, and blocks its biological activity .An open-label trial, which included 12 HS patients, reported that 75% of patients treated with IFX-1 achieved HiSCR after 50 days. After 134 days, this increased to 83.3% .A phase II controlled trial (NCT03487276) randomized the patients with moderate to severe HS to receive four different doses of IFX-1 intravenously  or placebo. The highest HiSCR response rate (51.5%) was achieved after vilobelimab 800 mg intravenously every 4 weeks. The HiSCR response rate in patients treated with placebo was 47.1% .Avacopan is an orally administered small molecule C5a receptor antagonist. Several studies are underway for the treatment of HS, antineutrophil cytoplasmatic antibodies-associated vasculitis, and atypical hemolytic uremic syndrome with avacopan .A placebo-controlled trial (NCTO3852472) randomized HS patients into three groups . The results of this study are yet to be published .Signals from IL-2 receptor (IL-2R), IL-4 receptor (IL-4R), IL-5 receptor (IL-5R), IL-6 receptor (IL-6R), IL-13 receptor (IL-13R), and type I interferons activate JAKs, which subsequently activate signal transducers and activators of transcription proteins (STATS). After activation, STATs enter the nucleus to bind to transcriptional regulatory sites of target genes, and induce inflammation. Several studies evaluated the efficacy of JAK inhibitors in HS .INCB054707 is a JAK1 inhibitor assessed in two phase II trials  evaluated how well upadacitinib works to treat adult patients with moderate to severe HS. The results of this study are yet to be published .Tofacitinib is a JAK inhibitor that has been used with good clinical outcomes in two HS patients resistant to other biologics .IRAK4 functions downstream of multiple innate immune cell receptors, such as TLR and IL-1Rs .KT-474 is an orally administered small molecule that targets IRAK4, and is under development for the treatment of interleukin-1 receptor (IL-1R)/ TLR-driven inflammatory diseases . A phasePF-06650833 is an IRAK4 inhibitor  investigSignals from cytokines IL-23, IL-12, and type I interferons activate TYK2 . RopsaciBrepocitinib (PF-06700841) is a TYK2/JAK1 inhibitor that prevents IL-12 and IL-23 signaling . SeveralRituximab is a chimeric mouse/human monoclonal antibody that targets CD20 protein. It is FDA-approved for use in non-Hodgkin’s lymphoma, chronic lymphocytic leukemia, rheumatoid arthritis, granulomatosis with polyangiitis, microscopic polyangiitis, and pemphigus vulgaris .A recent case report described rituximab administration in low doses (200 mg intravenously in two courses) in a kidney transplant recipient with idiopathic carpotarsal osteolysis who suffered from chronic active antibody-mediated rejection (CAAMR). After the kidney transplantation, the patient was diagnosed with HS. Dramatic improvement of HS was reported after the rituximab administration, but the dose was insufficient for CAAMR .CFZ533  is a fully human monoclonal antibody that blocks the CD40 pathway . A phaseSpesolimab is a humanized monoclonal antibody that blocks the activation of the IL-36 receptor (IL-36R) , and is Imsidolimab is a humanized monoclonal antibody that blocks the activation of IL-36R . It is cLYS 006 is an orally administered LTA4 hydrolase inhibitor that is currently being investigated for the treatment of HS and inflammatory acne . A phaseLY 3041658 is a monoclonal antibody that neutralizes chemokines that bind to the CXCR1 or CXCR2 receptors . A phaseCSL 324 is a recombinant anti-G-CSF receptor monoclonal antibody that might be effective in treating diseases associated with increased numbers of neutrophils at sites of inflammation . A phaseCurrently, there are several available treatment options for HS. A combination of conservative therapy and surgical treatment ensured the best clinical outcome before the biologic era. Nowadays, biologic therapy is the most effective pharmacological treatment of moderate to severe HS .An anti-TNF-α biologic, adalimumab, was approved by the FDA and the EMA 7 years ago, and has significantly improved the clinical outcomes in HS patients since then . HoweverMost exposure–response relationship studies show a positive correlation between biologics concentration and optimal clinical outcomes in immune-mediated diseases . One of Combination therapy with a TNF-α inhibitor and an oral immunomodulator is often utilized for patients with rheumatoid arthritis or IBD to enhance the efficacy of the biologic drug, or reduce the risk of immunogenicity . MethotrSince HS has some similar features to psoriasis, the identification of potential drugs for HS has often been based on preexistent drugs for psoriasis rather than on HS pathogenesis . AlthougThe registration of a new biologic drug requires evidence for effectiveness and safety in randomized controlled trials . HoweverMost biologics and other investigated immunomodulatory therapies for HS have a generally good safety profile . HoweverPresently, the most promising biologics in phase III trials are anti-IL-17, secukinumab, and bimekizumab. The biologic currently in phase II trials that targets IL-1, bermekimab, shows encouraging results too. Treatment should be started as early as possible to prevent fibrosis and sinus tracts formation. An inverse correlation was established between adalimumab use and clinical response .HS patients commonly have other immune-mediated diseases, so determining the optimal therapy for both HS and comorbidities is often a real-life problem. A round-table session held in 2020 during the 9th Conference of the European Hidradenitis Suppurativa Foundation in Athens concluded that targeting TNF-α and IL-12/23 results in better clinical outcomes in psoriasis than in psoriatic arthritis, HS, and Crohn’s disease, whereas targeting IL-17 is significantly more effective in psoriasis than in psoriatic arthritis, and it is obviously not effective in Crohn’s disease . Furthervice versa, patients with psoriasis may develop HS during adalimumab treatment. Other authors reported similar experiences with adalimumab [The additional complications that may occur during the treatment with biologic drugs are paradoxical responses, i.e., when targeting an involved cytokine may exacerbate another disease originally mediated through the same cytokine. For example, according to our experience, paradoxical development of psoriasis may develop in patients treated with adalimumab due to their HS; and alimumab . In addialimumab . Severalalimumab . TherefoBiologics are often used before the surgery to minimize the area required for surgical resection in patients with severe HS that do not respond well enough to biologics alone . The resSuccessful use of the new therapeutic options depends on the proper stratification of patient groups. Clinically relevant biomarkers could be helpful. A recent systematic review critically evaluated biomarkers in HS, and showed that only one diagnostic (serum IL-2R), one monitoring , and two predictive biomarkers  achieved a high GRADE , but none of them had sufficient clinical validity to be recommended for routine use in the clinical setting .The abundance of different methods measuring clinical outcomes of new, targeted treatments for the HS has led to difficulties in comparing the efficacy of different drugs. HiSCR, the most commonly used validating score in randomized controlled trials, is more dynamic than Hurley staging, easy to perform contrary to mSS, and in good correlation with patient-reported outcomes. The main disadvantage of HiSCR is in high placebo rates in randomized controlled trials, probably due to its construction as a binary variable . An ideaFurther research on novel therapeutic agents using validated HS scoring systems, further longitudinal studies assessing efficacy and safety of new drugs, and proper stratification of patient groups, hopefully with the help of HS biomarkers, are required to achieve adequate clinical response, and to improve the quality of life of HS patients significantly.The literature search was conducted using the PubMed and Google Scholar repositories. Only relevant meta-analyses, observational studies, randomized controlled trials, and systematic reviews published in English and Croatian and between 1998–2022 were taken into consideration, utilizing the keywords: hidradenitis suppurativa, pathophysiology, treatment, biologics, TNF-α inhibitors, IL-17 inhibitors, IL-12/23 inhibitors, IL-23 inhibitors, IL-1 inhibitors, JAK inhibitors, small molecule inhibitors. An additional review of phase I, II, and III randomized clinical trials registered at Clinicaltrials.gov was conducted up to February 2022, and all of the studies evaluating the efficacy and safety of biologic drugs and small molecule therapeutics were included in the review. Two authors (A.M.Č. and Z.B.M.) collected sources of information, read the reference abstracts, and included a total of 196 articles and published results of registered randomized controlled trials primarily focusing on the molecular pathophysiology of hidradenitis suppurativa, conservative therapy of hidradenitis suppurativa, as well as current biologic and other immunomodulatory therapeutics used to target underlying pathogenesis of HS. After the literature search, the authors  conceptualized the report in the form of a narrative review. Authors with high expertise in this field (B.M. and Z.B.M.) reviewed and revised the manuscript.


P < 10−3). Compared to the 43 patients alive at the end-of follow-up, patients who died were older , with 82% of them older than 72 years vs only 23% of live patients (P < 10−3). Age  and age over 72 years (OR) = 14.85; 95%CI = 2.7–80; P = .002) were independently associated with mortality. TCZ in addition to SOC for severe COVID-19 patients did not reduce mortality, subsequent need for invasive mechanical ventilation nor did it shorten the time of oxygen support, despite better control of the inflammatory response. More powerful and randomized controlled trials are warranted to determine if TCZ is effective in the management of COVID-19.To assess tocilizumab (TCZ) efficacy associated to standard of care (SOC) compared to SOC alone in severe coronavirus associated disease 2019 (COVID-19) patients. In a matched case-control study from 3 French Hospital COVID-19 Departments, 27 patients with severe COVID-19 treated with TCZ and SOC were matched for baseline epidemiological and clinical features and compared to 27 severe COVID-19 patients treated with SOC alone. Baseline characteristics of the study population were comparable between groups. Eleven patients (20%) died. TCZ was not associated with clinical improvement as compared to SOC regarding oxygen-free status (44% vs 63%) and death (18.5% vs 22%), despite a higher decrease of the C-reactive protein at Day 7 (10.7 vs 52 mg/L;  There are currently no effective therapies to prevent severe forms of COVID-19 and no treatment has yet proven effective in reducing mortality in these patients.The emergent outbreak of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a global pandemic.–6 as suggested by the correlation between plasma IL-6 levels and COVID-19 severity.,8 Before the demonstration of efficacy of corticosteroids in COVID-19 by the Recovery and Solidarity trials,–11 there was an urgent need for novel therapies to treat COVID-19 associated/induced CRS.Elevated plasmatic levels of inflammatory cytokines such as interleukin-6 (IL-6) and others, in COVID-19, also known as cytokine release syndrome (CRS) plays a key role in the pathology of severe COVID-19 Considering the key role of IL-6 blocking by TCZ in the treatment of CAR T-cells CRS and the role of IL-6 in COVID-19 CRS, TCZ was rapidly considered as a potential benefit treatment for COVID-19 CRS. Preliminary published investigations (retrospective uncontrolled and case-controlled) reported unconclusive results.–16 In July 2020, an online published metanalysis of 7 controlled studies showed no difference on death or ICU admission between TCZ treated groups and SOC groups, whereas 2 recent papers, published in August 2020, reported a reduced risk of invasive mechanical ventilation (IMV) and of death in TCZ treated patients when compared to those receiving SOC.–20Tocilizumab (TCZ) is an anti-human IL-6 receptor (IL-6R) monoclonal antibody that inhibits IL-6 signaling by binding soluble IL-6R and membrane IL-6R, blocking by this way its pro-inflammatory proprieties. TCZ is approved for rheumatoid arthritis, juvenile inflammatory arthritis and refractory giant cell arteritis, with a good tolerance except for the risk of bacterial infection and sigmoiditis. TCZ is also considered to be effective for the treatment of chimeric antigen receptor T cell therapy (CAR T-cells) related CRS.We herein report the results of a retrospective matched case-control study looking at the efficacy of a TCZ treatment plus SOC compared to SOC alone in COVID-19 patients, on mortality, necessity of ICU admission and need for IMV. We also discuss the results of the available studies on the use of TCZ in severe COVID-19.22.1We performed a multicenter retrospective analysis of all SARS-CoV-2 infected patients treated with TCZ (n = 27) in addition to SOC (TCZ group) from 3 French hospitals  between March and April 2020. These patients were matched for baseline epidemiological  and clinical features  to 27 patients who received only SOC (non-TCZ group). Of note, most of the 27 patients included in the control group were admitted in March 2020, before we started to introduce TCZ for severe COVID-19.All 54 included patients had clinical symptoms of respiratory infection and a positive nasopharyngeal swab polymerase chain reaction test for SARS-CoV-2 (Cobas SARS-CoV-2 – ROCHE). A chest Computed Tomography – scan was performed in 48 patients and was consistent with SARS-CoV-2 infection with variable degrees of lung involvement . Epidemiological, clinical, laboratory and therapeutic data were recorded from patients’ medical records. These collected data are anonymized, stored and made available if necessary.2.2At the time the study was designed (between March and April 2020), the SOC for the treatment of COVID-19 patients in France consisted in oxygen therapy (adapted to oxygen saturation objectives), antibiotics (mostly ceftriaxone or cefotaxime plus azithromycin or rovamycin), antivirals  according to physician's decision and intensive life support  care when needed. All patients received anticoagulant treatment: standard prophylactic low molecular weight heparin for 22 patients, prophylactic body mass index-adapted dosing for eight patients and therapeutic dosing for 14 patients (one patient received apixaban). As their benefit in COVID-19 management was debatable, only 14 patients (26%) received steroids treatment.2 ≥ 94%, 7 to 15 days after symptoms onset. Confirmed bacterial or fungal co-infection before TCZ were exclusion criteria. Twenty-four patients received 1 dose of intravenous TCZ (8 mg/kg) and 3 patients received 1 subcutaneous injection (162 mg). Concomitant corticosteroid therapy was allowed. The study was classified as a category 3 (MR004) according to the French Jardé law and it was approved by the Ethics Committee of each of the 3 institutions.TCZ was compassionately administered to 27 patients with severe COVID-19, following local guidelines and ethical procedures, after multidisciplinary concertation. Severe COVID-19 was diagnosed in patients requiring at least 6 L/min of oxygen flow to reach a SpO2.3Patients were evaluated at Day (D) 1 , D7, D14 and at the end of follow-up. Patients were followed from admission to the date of the last news or death. The primary endpoint was mortality. The secondary endpoints were: mortality at day D7 and D14, subsequent ICU admission (for patients not in ICU at baseline) and need of IMV, percentage of oxygen-free patients at D7, D14 and at the end of follow-up as well as biological parameters , ferritin) at D7 and D14.2.4P value <0.20 were included in a stepwise multivariate logistic regression analysis in order to assess independent associations. The level of significance  was set at .050 for all comparisons and analyses. Statistical analyses were performed using MedCalc software version 10.0.1.0 . The propensity score was performed using inverse probability of treatment weighting and double robust methods, further forcing several parameters already identified as death risk factors  in a large cohort of SARS-CoV2 infected patients .Continuous variables were expressed as mean ± standard deviation (SD) or median with 95% confidence interval (CI). Categorical variables were expressed as numbers and percentages. Groups were compared using Mann-Whitney, Kruskal-Wallis or Friedman tests for continuous variables and Fisher's exact test for categorical variables. On the basis of the results of univariate analyses, variables with 3P = .150). However, the rate of patients with invasive mechanical ventilation at admission was comparable . TCZ patients also received steroids more frequently . The use of hydroxychloroquine (48% vs 33%), lopinavir/ritonavir (11% vs 26%) and the anticoagulation dosing were not statistically different between the TCZ and non-TCZ groups.Baseline characteristics of the study population are presented in Table 3.13 vs 1450/mm3; D14: 1760/mm3 vs 1260/mm3, respectively)  and a slight improvement of the lymphocyte count . There was no significant difference between groups, at D7 and D14, regarding the proportion of oxygen-free patients  and death . At the end of follow-up, both groups remained statistically similar regarding oxygen-free status (44% vs 63%) and death (18.5% vs 22%).3.2P < 10−3); 82% of the deceased were older than 72 years, compared to only 23% of live patients (P < 10−3). The deceased patients were also more likely to have underlying chronic heart disease  and lower lymphocyte count  at baseline. We did not find any significant difference regarding IMV (45.5% vs 46.5%), the use of hydroxychloroquine (45.5% vs 39.5%), lopinavir/ritonavir (18% vs 19%), corticosteroids (36% vs 23%), TCZ (36% vs 23%) or anticoagulation dosing between these 2 groups.During the follow-up period , 11 patients (20%) died within a 4 days-median interval (95%CI = 2.8–20.1). When compared to the 43 live patients at the end of follow-up, in a univariate analysis (Table P = .008) and age above 72 years  were the only independent variables associated with death , with chronic respiratory disease and ongoing tobacco consumption. The other 4 patients were 73, 77, 83 and 89-year-old. The 6 control-patients who died were aged between 71 and 84.Using a stepwise logistic regression analysis, we found that age (odds ratio (OR) = 1.15, 95%CI, 1.04–1.3,  but recently published Recovery and Solidarity trials brought enough proof of the efficacy of steroids (dexamethasone) in reducing COVID-19 mortality. Because the TCZ treated group more frequently received corticosteroids (44% vs 7%), we hypothesized that the potential efficacy of TCZ might have been tempered by a negative impact of corticosteroids on death rate. Thus, we generated a propensity score model using the balance diagnostic of inverse probability weighted treatment (IPWT) model . This IPWT logistic model did not ascertain a favorable effect of TCZ treatment on death occurrence , even after using a double robust method .When the study was designed, the benefit of corticosteroids for COVID-19 management was debatable,4,21 These data suggested that the use of cytokine inhibitors such as anti-IL-6  may reduce the risk of death in severe COVID-19 independently of ICU admission or mechanical ventilation.,22 Our controlled study, comparing 2 well-matched groups with respect to comorbidities and risk factors for severe disease, shows no effect of TCZ as an add-on therapy to SOC for severe COVID-19 patients in reducing mortality or subsequent need for IMV, despite a better control of the inflammatory response.The deleterious role of the cytokine storm accompanied by uncontrolled macrophage and monocyte cells activation and high levels of serum IL-6, interleukin 1 (IL-1) and Tumor Necrosis Factor (TNF) α was recognized early in the SARS-CoV2 epidemic. and some suggested that low doses of TCZ might prevent disease progression in patients with moderate COVID-19 and high inflammation. Some patients in our study may have thus received TCZ too late. Indeed, the interval between symptom onset and aggravation was 1 day longer in the TCZ group than in the non-TCZ group . Two different patterns of immune dysfunction were suggested in SARS-CoV2-related ARDS; the first, driven by interleukine 1β (IL1β), suggestive of macrophage activation syndrome (hyperferritinemia and elevated H score for reactive hemophagocytic syndrome in up to 25% of patients); the second, driven by IL-6, consisting of immune dysregulation associating hypercytokinemia, immuneparalysis (as indicated by decreased Human Leukocyte Antigen – DR molecules on CD14 monocytes), and global lymphopenia. We hypothesize that the efficacy of anti-IL-1β (such as anakinra) or anti-IL6 (such as TCZ) molecules may be influenced by these patterns which may be present at different extents at the time the drug is initiated.Nevertheless, we cannot rule out that TCZ could be of use in some patients, as illustrated by Figure –16,26 but none were prospective or randomized. A metanalysis of 7 controlled studies showed no difference on death (relative risk (RR) = 0.61; 95%CI = 0.31–1.22) or ICU admission  between TCZ treated groups and SOC groups. Another study, not included in the metanalysis, also failed to show a beneficial effect of TCZ on death or need for IMV; age equal or above 75 years was the only predictive factor of death in the TCZ patients.We focused our discussion on published manuscripts and excluded submitted pre-published papers. Several studies dealing with the use of TCZ have been published with different methodological designs (case-controlled or uncontrolled),P = .020) in TCZ treated patients, a 45% decreased likelihood of death  despite higher superinfection occurrence  in patients under mechanical ventilation and decreased hospital-related mortality  in TCZ treated ICU patients  when compared to SOC. Another study found a non-significant tendency towards reduced mortality among ICU TCZ treated patients  when compared with SOC patients. Finally, all awaited prospective randomized studies came just to be published, reporting the absence of TCZ efficacy in reducing COVID-19 mortality,–33 thus confirming our first results.Very recent papers were published which showed a reduced risk of IMV and of death (adjusted hazard ratio (HR) = 0.61; 95%CI = 0.40–0.92; –36 Age above 72 years was the only independent factor associated with death, yielding a 15-fold superior risk. Indeed, when looking at the electronic health records of 17 million adult American patients, a 2-fold increase of the death risk was found in COVID-19 patients in the 60 to 70 year-old group, when compared with the 50 to 59 year-old group; this risk dramatically increased in patients older than over 80 years . Noteworthy, male gender, grade II obesity and chronic respiratory disease such as chronic obstructive pulmonary disease were also associated with an increased risk of death in COVID-19. These 3 factors were the main features of the 54-year-old patient treated with TCZ who died in our study.In our study, TCZ was not associated with an improvement of the survival rate. The analysis of factors associated with death clearly indicated that older age was the main risk factor for death in all these reports, as we did. All other parameters including concomitant treatment with antibiotics, antiviral and presumably anti-inflammatory treatments were mostly non associated with death.Despite 2 well-matched groups with respect to comorbidities and risk factors for severe disease, our study has several limitations including the retrospective, multicenter design as well as the limited number of included patients, which requires us to analyze and consider our results with caution. A center bias might be discussed as the 3 centers may have had different SOC. However, the baseline clinical parameters were comparable . We can however note that patients included in one of the 3 participating hospitals were more severe than those of the 2 other centers. A time frame bias may be discussed. Indeed, 85% (23/27) of the control patients were recruited during March 2020 as compared to only 52% (14/27) of TCZ treated patients, whereas the SOC evolved between March and April 2020 with the better understanding of SARS-CoV2 infection. Also, in the propensity score analysis used to evaluate the impact of TCZ on death occurrence taking into account the higher proportion of corticosteroids treated patients in the TCZ group, TCZ failed to significantly influence death rate, but this should be considered with caution given the limited number of events (only 11 deaths).In summary, despite an attractive and consistent rationale, supported by evidence of the association of increased serum IL-6 with severe SARS-CoV2 infection, our study failed to demonstrate a benefit of TCZ treatment for severe COVID-19 patients. Considering the limitations of our study and the discrepancies of new published results from well-matched or randomized controlled studies, the interest of TCZ treatment on death and ICU admission reduction is still debatable and deserves to be more clearly demonstrated.Lariboisière COVID-19 group: ALBERTINI Mathieu, AMADOR BORRERO Blanca, BOUAJILA Sara, BRITANY Kimbimbi, BURLACU Ruxandra, CACOUB Léa, CHAMPION Karine, CHAUVIN Anthony, DELCEY Véronique, DILLINGER Jean-Guillaume, FERON Florine, FRAZIER Aline, Thomas FUNCK-BRETANO, GALLAND Joris, GAUTHIER Diane-Cecile, GAUTIER Jean-François, HENRY Patrick, HUSCENOT Tessa, Sarah IZABEL Mathilde, JAULERRY, JOUABLI Moenes, JULLA Jean-Baptiste, KEVORKIAN Jean-Philippe, LALOI MICHELIN Marie, LEROY Pierre, LOPES Amanda, MANGIN Olivier, MEGARBANE Bruno, MICHON Maxime, MOULY Stephane, MUNIER Anne-Lise, NAHMANI Yoram, NICOL Martin, NICOLAS Eroan, POULAT Audrey, REVUE Eric, RICHETTE Pascal, RIVELINE Jean-Pierre, RUBENSTEIN Emma, SELLIER Pierre-Olivier, SENE Damien, THOREAU Benjamin, VODOVAR Dominique, ZANIN Adrien, AVENEAU Clément, BASTARD Paul, BEAUVAIS Diane, BOGHEZ Loredana, BORDERIOU Alix, CONWAY Paul, COSMA Lavinia, DAVY Vincent, DESJARDIN Clément, DEVATINE Sandra, DUCROZ GERARDIN Christel, DUPE Charlotte, GOBERT Chloé, GROS Clotilde, KADIRI Soumaya, KHAN Enmat, ONGNESSEK Sandrine, RHMARI Fatima, SACCO Isabelle, SAPTEFRAT Natalia, SCHAUPP Pauline, SERRE Justine, SICA Gabriel, SIDERIS Georgios, SMATI Sonia, TOURNIER Marine, TRECA Pauline, TRUONG Tony, TUFFIER Mathilde, ARCELLI Mattéo, BOUE Yvonnick, COPIE Alban, DEYE Nicolas, EKHERIAN Jean-Michel, ERRABIH, Zaccaria, GONDE Antoine, GRANT Caroline, GUERIN Emmanuelle, MAGALHAES Adèle, MALISSIN Isabelle, MEGARBANE Bruno, MEURISSE Edouard, MRAD Aymen, NAIM Giulia, NGUYEN Philippe, NITENBERG Kiyoko, PEPIN-LEHALLEUR Adrien, PERAULT Arthur, PERRIN Lucile, RENAUD Maxime, SUTTERLIN Laetitia, VOICU Sebastian.Diaconesses Croix Saint-Simon Hospital COVID-19 Group: Thierry LAZARD, Benjamin SUBRAN, Karine MALEY, Pascal CHAZERAIN, Castille DECATHELINEAU, Grégoire SALTIEL, Arnaud VANJAK, Théo DHOTE, Yunyun MIAO, Louise GILLARD, Christiane STRAUSS, Nathalie LE GUYADER, Elisabeth KLEIN, Beate HEYM, Isabelle ETIENNEY, Jean-Michel DEVYS, Simon MARMOR, Roselyne DI MARCO, Isabelle VERBEKE, Aurélie DERVAUX, Françoise JULIEN, Nora CHOPFENBERG, Jeanine LAVIOLLE, Virginie CASTEL, Laurence MARSAL, Charles HOUDEVILLE, Vanessa POLIN, Axel EGAL, Elsa LAMBRESCAK, Thomas AUBERT, Antoine MOUTON, Florence AIM, Marie PROTAIS, Laure BERNARD, Andrea MECCARIELLO.Rothschild Hospital Foundation COVID-19 group: Thomas SENÉ (Internist) and Pierre TROUILLER (ICU) would like to thank all the medical, paramedical and administrative staff involved in the management of the COVID-19 pandemic at the Rothschild Foundation Hospital.All co-authors would like to thank all the medical, paramedical and administrative staffs involved in the management of the COVID-19 pandemic at Lariboisière Fernand Widal, Diaconesses Croix Saint-Simon and Rothschild Foundation Hospitals.Conceptualization: Ruxandra Burlacu, Jonathan London, Thomas Sene, Wladimir MAUHIN, Damien Sene.Data curation: Ruxandra Burlacu, Jonathan London, Audrey Fleury, Thomas Sene, Vanina MEYSSONNIER, Valérie ZELLER, Joris GALLAND, Tessa Huscenot, Emma Rubenstein, Pierre Trouiller, Roland AMATHIEU, Johannes KUTTER, David BLONDEEL, Gabriel LEJOUR, Stephane Mouly, Olivier Lidove, Lariboisière Rothschild Diaconesses COVID19 Groups, Wladimir MAUHIN.Formal analysis: Abdourahmane Diallo, Wladimir MAUHIN, Damien Sene.Investigation: Joris GALLAND, Tessa Huscenot, Emma Rubenstein, Damien Sene.Methodology: Ruxandra Burlacu, Abdourahmane Diallo, Valérie ZELLER, Emma Rubenstein, Wladimir MAUHIN, Damien Sene.Supervision: Gabriel LEJOUR.Validation: Jonathan London, Audrey Fleury, Olivier Lidove, Wladimir MAUHIN.Visualization: Audrey Fleury, Roland AMATHIEU.Writing – original draft: Ruxandra Burlacu, Jonathan London, Olivier Lidove, Lariboisière Rothschild Diaconesses COVID19 Groups, Damien Sene.Writing – review & editing: Ruxandra Burlacu, Jonathan London, Audrey Fleury, Thomas Sene, Abdourahmane Diallo, Vanina MEYSSONNIER, Valérie ZELLER, Joris GALLAND, Tessa Huscenot, Emma Rubenstein, Pierre Trouiller, Roland AMATHIEU, Johannes KUTTER, David BLONDEEL, Gabriel LEJOUR, Stephane Mouly, Olivier Lidove, Lariboisière Rothschild Diaconesses COVID19 Groups, Wladimir MAUHIN, Damien Sene.


We further showed that the presence of cholesterol in membrane will alter the distribution of Vpr in the complex membrane environments, which may explain the change of the Vpr induced membrane permeability in the presence of cholesterol.Being a major metabolite for maintaining cellular homeostasis, as well as an important structural component in lipid membrane, cholesterol also plays critical roles in the life cycles of some viruses, including human immunodeficiency virus-1 (HIV-1). The involvement of cholesterol in HIV-1 infectivity, assembly and budding has made it an important research target. Viral protein R (Vpr) is an accessory protein of HIV-1, which is involved in many major events in the life cycle of HIV-1. In addition to its multi-functional roles in the HIV-1 life cycle, it is shown to interact with lipid membrane and form a cation-selective channel. In this work, we examined the effect of cholesterol on the interaction of Vpr and lipid membrane. Using calcein release assay, we found that the membrane permeability induced by the membrane binding of Vpr was significantly reduced in the presence of cholesterol in membrane. In addition, using solid-state NMR (ssNMR) spectroscopy, Vpr was shown to experience multiple chemical environments in lipid membrane, as indicated by the broad line shape of carbonyl  Cholesterol is generally viewed as a structural component in cell membranes, which plays an important role in membrane stability and fluidity. It is also an essential precursor for the biosynthesis of several steroid hormones and bile acids . RecentlE. coli expression system and further demonstrated that Vpr has a stronger binding affinity to DOPG lipids compared to DOPC lipids, suggesting a lipid-composition dependent of Vpr-membrane interaction  Cysteine selective labeled Vpr embedded in liposome, we showed the existence of complex Vpr-membrane environments and the distribution of Vpr among these environments was altered with the presence of cholesterol in membrane. We believe that more insights into cholesterol modulating Vpr-membrane interaction can be revealed by other methods, such as computational simulations. In summary, we show that cholesterol, without interacting with Vpr directly, can affect the Vpr-membrane interaction.HIV-1 viral protein R (Vpr) is a functional accessory protein, which is involved in many aspects of the viral life cycle, including the nuclear import of the pre-integration complex ,15,16, ceraction . During eraction ,10. In aeraction . Once theraction . Moreoveeraction , which ceraction ,34. Thuseraction  and CPPseraction . The ana15N, 99%] ammonium chloride,  D-glucose, and  L-Cystine were purchased from Cambridge Isotope Laboratories .  dodecylphosphocholine (DPC) was purchased from Frankenstein Bio Reagents, . Chloroform was purchased from Avantor, Radnor, PA, USA. 2,2,2-Trifluoroethanol (TFE) was purchased from Alfa Aesar, Hersham, UK. 2,2,2-Trifluoroacetic acids (TFA) was purchased from ACROS Organics, Morris Plains, NJ, USA. Deuterium oxide (D2O), Triton X-100 (TX-100), calcein, cholesterol, 3-(trimethylsilyl)-1-propanesulfonic acid sodium salt (DSS) were purchased from Sigma Aldrich, St. Louis, MO, USA. ISOGRO was purchased from Silantes, Munich, DE or Sigma Aldrich, St. Louis, MO, USA.1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were purchased from Avanti Polar Lipids .  labeled Vpr protein, 1 g of  ammonium chloride, 2 g of  D-glucose and 1 L of D2O (98%) per liter culture were used to replace the natural abundance chemicals in the growth medium. This culture was also supplemented with the 1 g of  ISOGRO per liter after induction with 0.1 mM isopropyl β-D-thiogalactoside (IPTG). To prepare  Cysteine selective labeled Vpr protein, a mixture of amino acids and nucleobases was used to replace the ammonium chloride in the defined growth medium. The mixture of amino acids and nucleobases contains 125 mg of  L-Cystine, 200 mg of each of the rest of nineteen non-labeled amino acids, and the five nucleobases, including 125 mg of adenine, 125 mg of guanine, 125 mg of thymine, 125 mg of cytosine, and 50 mg thymine per liter.We followed the established protocol  to produ600 reached the range of 0.7–0.9, and the culture was continued to grow at 18 °C for 24 h to allow for the expression of the soluble His-tagged GB1-fused Vpr protein. Pure Vpr protein precipitates can be obtained following the established protocol  labeled His-tagged GB1-fused Vpr with tobacco etch virus (TEV) protease to remove His-tagged GB1 tag. The  labeled Vpr protein precipitates dissolved in a denaturing buffer, consisting of 50 mM sodium phosphate at pH 6.5, 50 mM sodium chloride (NaCl), 0.5 mM tris(2-carboxyethyl)phosphine (TCEP), 1 mM ethylenediaminetetraacetic acid (EDTA), and 6 M guanidine hydrochloride (GdnHCl), to the concentration of 1 mg/mL. The protein solution was then added in a dropwise manner at a flow rate of 50 µL/min to a refolding buffer, consisting of 50 mM sodium phosphate at pH 6.5, 50 mM NaCl, 0.5 mM TCEP, 1 mM EDTA, and 9 mM of the tail deuterated  DPC. The protein solution was further dialyzed against a buffer containing 25 mM sodium phosphate at pH 6.5, 25 mM NaCl, 1 mM dithiothreitol (DTT), and 1 mM EDTA at 4 °C using a 6–8 kDa molecular weight cut-off (MWCO) dialysis membrane . The protein solution was concentrated and further purified by size exclusion chromatography using a Superdex increase s200 (10/300) GL column  equilibrated with a buffer containing 25 mM sodium phosphate at pH 6.5, 25 mM NaCl, 1 mM TCEP, 1 mM EDTA, and 1.8 mM  DPC. The NMR samples were further concentrated with 5 kDa molecular weight cut-off (MWCO) centricon . The samples for the 3D triple resonance experiments and for the cholesterol titration experiment were of the concentrations of 552 µM and 280 µM, respectively. D2O, containing DSS at the concentration of 20 mg/mL, was included in each NMR sample to 6% (wt %).The Vpr protein precipitates were obtained by treating [U-13C] cysteine selective labeled Vpr protein was used in the proteoliposomes production, and a DOPG liposome without Vpr protein incorporated was also prepared as a reference ssNMR sample. The size distribution of Vpr proteoliposomes were characterized by dynamic light scattering to ensure the size homogeneity , as indicated in To produce Vpr incorporated in DOPG liposomes, DOPG lipid was dissolved in chloroform (50 mg/mL) and dried on the side of a round bottom flask using a gentle stream of nitrogen gas. To produce Vpr incorporated in DOPG containing 5%, 10%, and 30% of cholesterol liposomes, the lipid mixes were first solubilized in chloroform with the molar ratio of DOPG lipid to cholesterol as 95 to 5, 90 to 10 and 70 to 30, respectively. Vpr protein precipitates were dissolved in a TFE solution containing 0.1% TFA to the concentration of 1 mg/mL, and then added to the dried lipid film containing flask. The lipid and protein mixture were prepared with the molar ratio of lipid to Vpr as 50 to 1. A gentle stream of nitrogen gas was used to remove the organic solvent, followed by placing the flask under vacuum overnight. The protein-lipid film formed on the glass wall was re-suspended in a buffer containing 5 mM sodium phosphate at pH 7.5, 5 mM potassium sulfate, and 1 mM TCEP. The Vpr proteoliposomes were further homogenized using an extruder with polycarbonate membrane with a defined pore size of 0.4 µm in diameter. For secondary structure analyses using circular dichroism spectroscopy, the Vpr proteoliposomes were purified by size exclusion chromatography using an ENrich 70 (10/300) column  in a buffer containing 5 mM sodium phosphate at pH 7.5, 5 mM potassium sulfate, and 1 mM TCEP. To prepare the samples for the ssNMR experiment,  TROSY-HSQC and 3D TROSY-HNCA, 3D TROSY-HNCOCA, 3D TROSY-HNCACB spectra of  Vpr solubilized in  DPC detergent micelles were recorded at 37 °C using Bruker AVANCE III 850 MHz NMR spectrometer  equipped with a TCI (1H/13C/15N) 5mm CryoProbe with Z gradient. The assignment was performed with computer aided resonance assignment (CARA) program  TROSY-HSQC spectrum was recorded to examine if cholesterol binds specifically to Vpr. To further increase the amount of cholesterol in the sample, additional cholesterol was first dissolved in ethanol to the concentration of 6 mg/mL due to the low solubility of cholesterol in DPC buffer. Then, 10 μL of the cholesterol in ethanol was added to the Vpr sample reaching the final ratio of cholesterol to Vpr as 2.4 to 1. An additional 2D  TROSY-HSQC spectrum was recorded for the comparison with cholesterol-free spectrum.For sequence-specific resonance assignment, a set of standard NMR spectra, including 2D  labeled Vpr protein solubilized in  DPC detergent micelles for solution NMR characterizations. The 2D  TROSY-HSQC spectrum of Vpr in  DPC micelles in 1H,15N] TROSY-HSQC spectrum of Vpr stayed unchanged for several weeks, suggesting the sample was of very good stability. The strip plot of TROSY-HNCA and TROSY-HNCACB spectra with the residues assigned from W54 to A59 is shown in As indicated in calcein release assay, membrane permeability was induced by the binding of Vpr to membrane. The fact that cholesterol plays a role in modulating membrane permeability indicates cholesterol alter the interaction between Vpr and membrane. Here, we attempted to employ solution NMR spectroscopy to examine if cholesterol binds to Vpr specifically. We prepared [U-13C] cysteine selective labeled Vpr embedded in liposomes with and without cholesterol were prepared and characterized using 13C{31P} REDOR NMR spectroscopy. This strategy has been used to study the insertion of HIV fusion peptides in membranes  cysteine selective labeled Vpr since no significant contribution of the carbonyl carbons of lipids was observed as shown in 13C signal observed around 170 ppm reflect the chemical environments of the cysteine residue, Cys-76, of Vpr since it is the only one cysteine residue in Vpr sequence (13C signal from a single-site amino acid residue would be less than 5 ppm [13C chemical shift, the observed broad peak ranging from 165 to 178 ppm indicated the multiple chemical environments which the Vpr Cys-76 residue experiences. Lipid membranes have been shown to play active roles in modulating the functions of membrane associated proteins or membrane proteins [0 spectra, as shown in To further examine Vpr in lipid environment, the samples of [1-embranes ,45,46. Heference . It is isequence . We obsean 5 ppm . Since dproteins ,50,51,52proteins . As suggBased on the results of calcein release assay, the cholesterol concentration in membrane affects the Vpr induced membrane permeability, suggesting a role of cholesterol in the Vpr-membrane interaction. No obvious evidence of specific cholesterol binding to Vpr was observed in DPC detergent micelles based on the result of the NMR titration experiments. Nonetheless, ssNMR spectroscopy was further used to show that the presence of cholesterol in membrane would affect the distribution of Vpr in the various membrane environments, which may partially account for cholesterol playing a role in the Vpr-membrane interaction. We believe that lipid membrane is as an active component regulating membrane protein functions, rather than just a passive component to support membrane protein. Cholesterol, being an active player in the membrane, can affect the function of membrane protein directly or indirectly.