According to the encouraging results they could be proposed for the treatment of angle-open glaucoma, cancer regression and osteoporosis, in which both NO and CA activities are involved.Due to the recognized biological role of nitric oxide (NO) donating derivatives and of selective inhibitors of specific human carbonic anhydrase isoforms , promising compounds having an aromatic/heterocyclic primary sulfonamide and functionalized with NO-releasing moieties have been designed. These bifunctional agents have been tested  A large number of complex and multifaceted diseases strictly require multipotent molecules concurrently able to modulate two or more mechanisms of action according to the “one molecule, multiple targets” theory . Cancer,In this review, we explored the state of the art of some molecules effective towards two pivotal targets: nitric oxide (NO) network and specific human carbonic anhydrase (hCA) isoforms .The importance of NO is due to the fact that it plays a regulatory role in a wide array of biological effects, through or not the synthesis of cyclic guanosine monophosphate (cGMP) as a mediator . The NO-S-nitrosothiols and (benzo)furoxans [According to the reported properties, NO has a promising therapeutic potential and, currently, there is great interest in the design of NO-donating derivatives  for the furoxans . Moreovefuroxans ,7.On the other side, human carbonic anhydrase isoforms are responsible for specific physiological functions in humans and their selective inhibitors could have innovative clinical applications as diuretic, antiglaucoma, antiobesity or antitumor drugs. Sulfonamide and sulfamate derivatives represent the main and most explored chemotypes of classical CA inhibitors ,10,11,12Most of the literature data reported the effect of lowering intraocular pressure (IOP) in patients with open-angle glaucoma , but emein situ, likely for aqueous humor homeostasis in normal and pathological conditions [Open-angle glaucoma is a multifaceted disease associated with elevated IOP, damage of the optic nerve and loss of vision. IOP is based on the balance of aqueous humor production by the ciliary body and its drainage. The important role of NO in all compartments of the eye in maintaining basal resting tone of circulation has been recognized through cGMP-dependent pathways. Hypertensive glaucoma patients have a reduced NO/cGMP content in the aqueous humor. Despite these drugs are an underexplored class in the field of ocular therapeutics, glyceryl trinitrate decreases IOP and enhances cGMP levels nditions ,19.1–16) and atypical (compounds 17–28) hCA inhibitors linked to NO-donors can be an interesting approach in cases of glaucoma, as reported in Conversely, selective activation of hCA II, expressed in the ciliary bodies and elicited by β-adrenergic stimulation, improves aqueous formation and transport as well as its release into the posterior chamber, thus increasing IOP. For this reason, selective hCA II inhibitors are another class of drugs that has been used to reduce IOP ,21. Then®) or brinzolamide (Azopt®) are clinically used to regulate aqueous humor production and drainage and IOP in hypertensive glaucoma. On the contrary, hCA inhibitors such as acetazolamide (Diamox®), methazolamide, ethoxzolamide or dichlorophenamide have been used as systemic antiglaucoma drugs for more than 50 years, but suffered from side effects due to inhibition of the enzyme present in tissues other than the eye [1–5 reported in 5 was included to eventually measure the effect of increasing the theoretical deliverable amount of NO to eye tissues, whereas dorzolamide was the reference standard. Compounds 1–5 inhibited hCA II with Ki in the low nanomolar range. The alkyl nitrate linkers were more selective for hCA II than the benzyl nitrate spacer. Interestingly, compounds 1 and 3 were selective and equipotent to dorzolamide for hCA II. Conversely, hCA I inhibitory activity of these derivatives was lower (dorzolamide and isosorbide mononitrate were inactive). Compound 2 was also active against hCA IV in the low nanomolar range as dorzolamide. Crystallographic analysis of 5 in the CA active site showed some hydrogen bonds between secondary nitrate and carboxamide hydrogens of Asn62, between ester oxygen and carboxamide hydrogens and between primary nitrate oxygen with the imidazole moiety of His64.Accordingly, topical selective hCA II inhibitors such as dorzolamide  to five different sulfonamide scaffolds benzenesulfonamide and 4-hydroxybenzenesulfonamide) [in vivo. Compounds 8, 9, 11, 12 and 15 showed effective inhibition in the nanomolar range against the slow cytosolic isoform hCA I (an off-target enzyme for antiglaucoma agents), whereas the remaining compounds were less effective inhibitors. Other new sulfonamides acted as very good inhibitors of hCA II isozyme, with Kis in the range of 10–33 nM, in the same range as those of the systemically used antiglaucoma sulfonamides acetazolamide and dichlorophenamide or the topically acting dorzolamide. hCA IV was also effectively inhibited by some of them with respect to the clinically used drugs.Another series of derivatives onamide) . The hyd17–28 lack the sulfamoyl zinc-binding group and represent a class of mono- or polycyclic organic nitrate esters. Unfortunately, despite slight hCA II selectivity, their IC50 values ranged between 7.13 mM and 124 mM against hCA I and between 65.1 μM and 0.79 mM against hCA II  in animals treated for one month with these selective hCA IX inhibitors [Conversely, the selective sulfonamide-based hCA IX inhibitors were shown to exert antiproliferative effects in tumor cell lines cultured in hypoxic conditions. This hibitors .For the above mentioned reasons, some primary sulfonamide compounds have been preliminary investigated both as NO-donors and selective inhibitors of transmembrane hCA IX and XII isoforms that are almost overexpressed in hypoxic tumors as part of the network that cancer cells use to make the microenvironment favourable for solid tumor growth .6–16 in the low nanomolar range  were of medium inhibitory efficiency. Collectively, derivatives 6–16 were also effective as hCA XII inhibitors (Kis in the range of 19–147 nM) [6–16 generally lack a strong inhibitory selectivity towards hCA IX and XII (a common issue with sulfonamide-type inhibitors) with respect to the other off-target isoforms. Moreover, for these compounds, no in vivo studies have been performed so far demonstrating this biological activity under hypoxic conditions.From the reported data it is possible to extrapolate that the tumor-associated hCA IX and XII isozymes were efficaciously inhibited by compounds ar range . Against–147 nM) , contribin vivo data are strongly required for these derivatives to support this dual approach.While the effects of NO on osteoblasts are less characterized claiming the direct stimulation of osteoblast , regulation of bone formation and prevention of bone loss associated with the use of glucocorticoids , the rol1, 3 and 6 were also characterized for NO release by the analysis of their cGMP-mediated vasorelaxant properties on methoxamine-precontracted rabbit aortic rings and their EC50 were better than those of isosorbide mononitrate and dorzolamide used as comparators at clinically relevant doses  and higher water solubility for the topical administration, compared to other proposed sulfonamides . The NO-donor sulfonamide 6 showed a very strong IOP lowering effect at 60 min post-administration, such as for the reference drug dorzolamide in this animal model of glaucoma. Its biological effect could be attributed both to selective and potent CA inhibition (as regards the dorzolamide IOP reduction) and the NO-mediated action (as regards the non-CA inhibitor NO-donor isosorbide mononitrate).Both compounds in vitro and in vivo for the treatment of specific pathologies  showing that their effects could have a therapeutic role. As reported, additional biological data especially about cancer and osteoporosis treatment are required. Furthermore, it has been demonstrated that their strong efficacy as hCA inhibitors could arise not only from the sulfonamide moiety, coordinating to the Zn(II) ion from the enzymatic active site, but also from the same release of NO, which concurrently may bind itself to the enzyme or nitrosylate it, and may contribute to CA inhibition.Multifactorial diseases must be treated with a combination of drugs acting at different biological levels. In order to reduce side-effects, low compliance and pharmacokinetic issues, the possibility of incorporating two putative mechanisms of action  in only one molecule is the goal of the recent medicinal chemistry efforts pursuing the Multi-Target-Directed Ligands approach (MTDL). In this short review, we have analyzed some bifunctional agents characterized by primary sulfonamide groups and nitrate esters to provide selective hCA inhibition and NO release, respectively. These interesting derivatives have been studied


Gestational and pre-gestational diabetes are frequent problems encountered in obstetrical practice and their complications may influence both the mother  and the foetus . Given the important implications for mothers and their offspring, screening and appropriate management of diabetes during pregnancy are essential. This is a review of articles published between 2015 and 2018 on Medline via Ovid that focus on advances in the management of diabetes in pregnancy. Recent data have concentrated predominantly on optimising glycaemic control, which is key for minimising the burden of maternal and foetal complications. Lifestyle changes, notably physical exercise and diet adjustments, appear to have beneficial effects. However, data are inconclusive with respect to which diet and form of exercise provide optimal benefits. Oral glycaemic agents—in particular, metformin—are gaining acceptance as more data indicating their long-term safety for the foetus and newborn emerge. Recent reviews present inconclusive data on the efficacy and safety of insulin analogues. New technologies such as continuous insulin pumps for type 1 diabetes and telemedicine-guided management of diabetes are significantly appreciated by patients and represent promising clinical tools. There are few new data addressing the areas of antenatal foetal surveillance, the timing and need for induction of delivery, and the indications for planned caesarean section birth. Gestational diabetes mellitus (GDM) is defined as any degree of glucose intolerance that is first diagnosed during pregnancy, whereas pre-gestational diabetes is defined as diabetes mellitus (DM) (type 1 or 2) present before conception3. The incidence of diabetes has been increasing worldwide, and the prevalence of hyperglycaemia, as defined by the 2013 World Health Organization (WHO) diagnostic criteria, is estimated to be as high as 16.6% during pregnancy; GDM represents 84% of these cases4.Hyperglycaemia during pregnancy is a common condition associated with maternal and foetal adverse outcomes such as pre-eclampsia, macrosomia, shoulder dystocia, increased risk of stillbirth, and neonatal hypoglycaemiaGiven the important maternal and foetal complications, identifying and optimally treating diabetes during pregnancy are of paramount importance. The goal of this review is to highlight new evidence in the antepartum management of hyperglycaemia during pregnancy. A search and review of articles published between 2015 and 2018 on Medline via Ovid were conducted and salient points were summarised. The article will discuss glycaemic surveillance and control using non-pharmacological and pharmacological methods as well as advances in the obstetrical management in the antepartum period.3. However, because this approach is perceived to result in an increase in GDM prevalence, many organisations have persisted with a two-step approach. In 2016, considering recommendations of the Canadian Diabetes Association (now known as Diabetes Canada), the Society of Obstetricians and Gynaecologists of Canada (SOGC) endorsed a two-step screening approach with an initial 50-g glucose challenge test (GCT) for all pregnant women2. Although the American College of Obstetricians and Gynecologists (ACOG) recommends screening all women, the choice of approach and cut-off values are not standardised, but a two-step approach is favoured5. The Royal Australian and New Zealand College of Obstetricians and Gynaecologists (RANZCOG) suggests screening all women but is against using a two-step approach and instead advises direct use of a 75-g OGTT6. In contrast, the Royal College of Obstetricians and Gynaecologists/National Institute for Health and Care Excellence (RCOG/NICE) advises screening only women with risk factors for GDM using a single step 75-g OGTT7.Although the need to screen for GDM is universally accepted, the approach through which this should be achieved remains contentious. The International Association of Diabetes and Pregnancy Study Groups (IADPSG) recommendation of a single-step 75-g oral glucose challenge test (OGTT) screening strategy has been adopted by the WHO7. One recent study has evaluated the use of a two-step approach using the 2013 WHO adopted criteria and this has not supported the continuing use of a 50-g GCT8. However, the benefits of using the WHO criteria, which have increased the prevalence of GDM some four-fold over the rate previously diagnosed with the two-step approach, need a more robust prospective evaluation. First-trimester screening for pre-existing hyperglycaemia presents an even greater dilemma. The glycaemia threshold used to identify women who will benefit from early intervention is not known. The concept of early GDM, as opposed to abnormal results being interpreted as indicative of pre-existing DM, is increasingly recognised, but there is a paucity of data to define this9.The most recent of many Cochrane reviews addressing the best approach determines that there are still insufficient data to conclude which approach is best and that only large-volume well-conducted randomised control trials (RCTs) will resolve this10. Although control of glycaemia during pregnancy has been shown to reduce adverse maternal and neonatal outcomes, no absolute threshold at which adverse risks occur has been identified. The most recent societal guidelines in glucose monitoring and glycaemia targets are reported in14.Maternal hyperglycaemia is associated with adverse maternal and foetal outcomes and there is a well-established association between increasing glycaemia and the occurrence of adverse outcomes15, there are currently no standardised criteria regarding the precise steps that should be taken to optimise glucose control. A recent meta-analysis identified RCTs which used various levels of strictness for intensifying glucose control, ranging from a single recorded value exceeding target values to more than 50% of recordings being above target values, and concluded that there is not enough evidence to recommend one approach over the other given the wide variations seen across the included studies16. A retrospective study by Scifreset al. suggests that hyperglycaemia in gestational diabetes might require a tighter control in the obese population, in whom the effects of hyperglycaemia on pregnancy seem amplified17. The use of continuous glucose monitors in pregnancy has been gaining popularity, although the data are sparse; an underpowered RCT showed inconclusive benefits with this monitoring method21.Although therapy adjustment based on postprandial blood glucose levels is associated with improved outcomeset al. found no difference in obstetrical outcomes with restricting weight gain beyond the 2009 Institute of Medicine criteria for patients with gestational diabetes22. Lifestyle modification alone is sufficient in about 70 to 85% of women with diagnosed GDM to achieve glycaemic targets11. Although most guidelines recommend a 1- to 2-week trial of lifestyle modification, pharmacotherapy should not be delayed, as euglycaemia is important in reducing adverse outcomes25. To date, there is inconclusive evidence as to when to initiate pharmacotherapy in cases of failure of the first-line approach; however, the conclusions of a meta-analysis suggest that pharmacotherapy should be considered in women with GDM when one or two glucose values exceed target levels at 1 or 2 hours postprandial during a 1- or 2-week trial period24.Lifestyle changes represent the first-line approach to therapy in gestational diabetes and include dietary modification and physical activity with the aim of limiting gestational weight gain and improving glycaemic control. Although there is still controversy regarding optimal gestational weight gain, a retrospective study by Wong25. A Cochrane review evaluated the impact of lifestyle modifications on weight gain and showed less gestational weight gain, decreased risks of macrosomia and caesarean delivery but no impact on incidence of pre-eclampsia or preterm birth26. There is evidence showing a beneficial effect of a low glycaemic index diet, but more studies are required to define precisely what a low glycaemic index diet should entail28. Although diet is the cornerstone of treatment, good data are lacking; previous limited-power RCTs show a benefit with low-carbohydrate, high-vegetable and whole-grain diets30. Small studies suggest that carbohydrate restriction may be associated with unintended adverse effects32. Other dietary approaches with probiotics and vitamin supplements have gained popularity, but the evidence is insufficient to recommend their generalised use34. One RCT with 140 patients with GDM showed that co-supplementation with vitamin D and fatty acids was associated with lower glycaemia; however, maternal and foetal outcomes were not evaluated35. Larger RCTs comparing different dietary approaches are still required before guidelines on the use of supplements can be developed.Most international obstetrical associations advocate for an immediate referral to a certified dietician and increased physical activity at the time of diagnosis of GDM36. In addition, the data were insufficient to evaluate what form of exercise was most beneficial. Therefore, future studies will be required to validate and assess the efficacy and safety of standardised exercise regimens, especially since data on the safety of exercise in the first trimester are scarce37. Nonetheless, in the absence of contraindications, physical activity, in combination with dietary changes, can be encouraged as an integrated part of the non-pharmacological approach.A Cochrane review has evaluated the role of exercise in pregnancy on glycaemic control. Exercise was associated with lower fasting and postprandial blood glucose values but remained inconclusive with respect to long-term maternal or foetal effects25, certain oral glycaemic agents are gaining attention. For instance, the NICE in the UK recommended metformin as a first-line treatment in its 2015 guidelines, except in cases where the fasting plasma glucose level exceeds 7.0 mmol/L at diagnosis38. Meanwhile, Diabetes Canada (formerly the Canadian Diabetes Association) describes metformin as a promising glycaemic agent given its side effect profile and efficacy25, and the medication is gaining ground in Australian obstetrical practice39.Although most national obstetrical associations continue to recommend insulin as first-line pharmacotherapy for diabetes in pregnancy given its inability to cross the placenta42. Data suggest that between 14 and 50% of cases treated with metformin will require additional insulin to reach the target blood glucose levels, making it difficult for any meta-analysis to evaluate the effect of metformin alone given the frequent use of additional insulin42. To date, very few studies have evaluated the impact of metformin use during pregnancy on long-term maternal and foetal health43. One RCT (n = 97) found that children exposed to metformin in the prenatal period were heavier and taller at 18 months of age and had similar body compositions and no differences in social or linguistic development compared with controls44. Another RCT (n = 146) found no differences in neurodevelopmental outcomes at 2 years of age between toddlers within utero exposure to metformin versus those exposed only to insulin45. This RCT also showed no difference in offspring body fat percentage at 2 years, although several skinfold measures were larger in metformin-exposed offspring46. A further follow-up found similar total and abdominal body fat percentages at 7 to 9 years of age and no differences in metabolic measures between the offspring of mothers who received either metformin or insulin in pregnancy. However, in one subgroup population, children of mothers who received metformin were larger on several measures at 9 years of age than those who received insulin47. These data, though somewhat reassuring, highlight the need for further investigation in this area.Several meta-analyses have studied the efficiency of metformin, showing its superiority to insulin in terms of reducing the risk of foetal hypoglycaemia, large-for-gestational-age foetuses, pregnancy-associated hypertension, and maternal gestational weight gain49. In the meta-analysis by Farraret al., glyburide was estimated to be most effective in reducing caesarean section rate but less effective than metformin or insulin for other adverse outcomes related to GDM41. When compared with insulin, glyburide appears to have worse neonatal outcomes, including more hypoglycaemia, macrosomia, birth injuries, and respiratory distress syndrome, and no improvement in glycaemic control50. Given these conclusions, glyburide should not be considered as a first-line treatment but rather should be held in reserve in cases where neither insulin nor metformin is tolerated or in cases where metformin is insufficient to control the glycaemia25.In meta-analyses comparing oral pharmacotherapy in the treatment of GDM, glyburide is associated with higher birth weights and rates of macrosomia when compared with other agents, making its use less favourable43.Overall, oral glycaemic agents, particularly metformin, appear to be efficient in treating diabetes during pregnancy, but they do cross the placenta and the long-term effects on the foetus are not yet well defined. This information needs to be conveyed to the parents if an oral glycaemic agent is chosen52. Although insulin analogues are gaining clinical ground53, the data to support their use are sparse. Specifically, the above Cochrane reviews have limited results on the benefits and safety of newer analogues, including glargine, lispro, and detemir52. Another meta-analysis concluded that there is a lack of information on the efficacy and safety of rapid-acting analogues lispro and aspart. A literature review found no association of lispro, aspart, or detemir with increased congenital anomalies compared with human insulin54.When glycaemic control does not meet pregnancy goals with lifestyle changes, insulin is added as an adjuvant therapy. Recent Cochrane reviews have found no evidence to recommend one specific insulin type or regimen over any other in pregnancy55. For type 1 DM, the closed-loop insulin delivery approach has been shown to provide better glycaemic control over sensory-augmented pump therapy in an initial study of 16 patients; however, data on the efficacy, safety and feasibility of closed-loop therapies during pregnancy are lacking57. Although this new regimen appears to be well perceived by mothers with type 1 DM58, additional larger RCTs are required to evaluate the effects of this approach on maternal and foetal outcomes.Limited review data illustrate that continuous insulin infusion pumps, though increasingly popular, offer no maternal or foetal advantages or disadvantages over the traditional multiple daily injection approach25. Examples of such approaches are web uploads of capillary blood glucose measurements on cell-phone apps59, apps which include lifestyle and dietary counseling60, or even clinical decision support systems which suggest insulin adjustments based on glycaemic values61. Telemedicine allows for prompt management of care across distances with fewer face-to-face medical visits62 and has been associated with high patient satisfaction65. In 2016, Minget al. published a meta-analysis of seven RCTs that involved telemedicine in gestational diabetes [62]. The authors showed similar maternal and neonatal outcomes such as glycaemic control, caesarean rates, macrosomia, and neonatal intensive care admissions, concluding that the evidence at the time was insufficient to show that telemedicine in gestational diabetes results in improved clinical outcomes. This was believed to be due to underpowered studies and the heterogenicity of e-platforms66. A randomised study by Mackillopet al. included 208 patients with gestational diabetes followed via traditional glycaemic control or via an app and found that the app group had more satisfaction with care, better glycaemic control, lower incidence of preterm delivery, fewer caesarean section births, and similar costs64.The use of information technology and web platforms for pregnant women with diabetes is rapidly increasing worldwideTherefore, the use of e-platforms in gestational diabetes management shows promising results with respect to patient satisfaction and no detrimental effect on pregnancy outcomes. Whether such healthcare tools are cost-effective or can help improve care in urban or remote areas remains to be determined by adequately powered RCTs.2 and therefore represent a population that requires more antenatal surveillance. The perfect surveillance strategy is not known and as such there are slight variations amongst societal guidelines, as illustrated in25. No recent developments have been reported in the literature.Gestational and pre-gestational diabetes are associated with an increased risk of stillbirth14. In 2017, the GINEXMAL trial randomly assigned 425 patients with low-risk gestational diabetes to induction of labour at 38 + 0 to 39 + 0 weeks versus expectant management until 41 weeks. Of note, they excluded patients with an estimated foetal weight above 4000g or with an unfavourable cervix. There was no difference in the rates of caesarean section  or foetal or maternal morbidities other than increased rates of hyperbilirubinemia in the newborns in the induction group67. A separate retrospective cohort study found that routine induction of labour at 38 or 39 weeks in women with gestational diabetes was associated with a lower incidence of caesarean section and a higher incidence of neonatal intensive care unit admission when induction was prior to 39 weeks68. A Cochrane review published in 2018 included only the GINEXMAL trial and as such concluded that there is insufficient evidence regarding benefits of induction in gestational diabetes69. In terms of mode of delivery, caesarean section is recommended above 4500g by the American College of Obstetricians and Gynecologists12, whereas for the International Federation of Gynecology and Obstetrics the threshold is 4000g14.Given the concerns related to the increased risks of stillbirth, macrosomia, caesarean section and shoulder dystocia in pregnancies complicated by diabetes, there is ongoing discussion as to whether earlier induction would be beneficial in this patient population and, if so, at what gestational age. The timing of induction varies amongst obstetrical organisations and their specific recommendations are illustrated inImportant information regarding the optimal management of diabetes in pregnancy is still emerging. This review illustrates some encouraging advances, including the use of oral hypoglycaemic agents—in particular, metformin—and insulin analogues. Diabetic tools such as continuous glucose monitoring and closed-loop insulin delivery show promising outcomes in small populations of patients with type 1 DM, whereas e-health technologies, such as online platforms for glycaemic monitoring, show encouraging results as modern approaches to glucose management. There is no new strong evidence to advocate for any significant changes in the existing recommendations for antenatal surveillance and labour induction.DM, diabetes mellitus; GCT, glucose challenge test; GDM, gestational diabetes mellitus; NICE, National Institute for Health and Care Excellence; OGTT, oral glucose challenge test; RCT, randomised control trial; WHO, World Health Organization


Traditional epidemiological investigation of nosocomial transmission of influenza involves the identification of patients who have the same influenza virus type and who have overlapped in time and place. This method may misidentify transmission where it has not occurred or miss transmission when it has. We used influenza virus whole-genome sequencing (WGS) to investigate an outbreak of influenza A virus infection in a hematology/oncology ward and identified 2 separate introductions, one of which resulted in 5 additional infections and 79 bed-days lost. Results from WGS are becoming rapidly available and may supplement traditional infection control procedures in the investigation and management of nosocomial outbreaks. Influenza virus whole-genome sequencing (WGS) was used to investigate an outbreak of influenza A virus infection in a hematology/oncology ward; 2 separate introductions were identified. The role of WGS for nosocomial outbreak investigation is discussed. Nosocomial transmission of influenza A virus is of significant concern since infection in individuals who are immunocompromised, immunosuppressed, at extremes of age, or pregnant have an increased risk of severe illness, morbidity and death , 2. The Commonly, nosocomial transmission of influenza virus within the healthcare setting has been identified through traditional molecular diagnostic methods  for the detection of viral species, combined with data collected on patient and staff movement within the hospital. Patients who have overlapped in time and space and who have evidence of infection with the same viral type and, if available, subtype are assumed to have transmitted to each other. Direct Sanger sequencing methods have more recently been used to identify possible clusters of nosocomial influenza virus infection , but nexTwo patients with hematological/oncological malignancies  platform.Amplification of influenza A virus RNA was performed using the SuperScript III One-Step RT-PCR kit (Invitrogen) and a modified 8-segment PCR method . Fifty-mLibrary preparations were generated using the Nextera XT DNA sample preparation kit (Illumina) according to manufacturer’s instructions, using 1 ng of input DNA. Tagged PCR samples were purified with 30 µL of AMPure XP beads . Sample library normalization and MiSeq sample loading were carried out according to the Nextera XT protocols. Pooled normalized samples, including a Phi-X control at a final concentration of 0.1 pM, were loaded onto a Mi-Seq reagent kit V3 600 cycle (Illumina) and sequenced on a MiSeq (Illumina).Consensus genomes were generated from short reads, using ICONIC’s in-house de novo assembly pipeline, applying a read depth cutoff of ≥20 reads to the final sequences. Phylogenetic analysis was undertaken first by separately aligning each set of segments using MAFFT  and thenSeven samples from 7 hematology/oncology patients, 1 sample from a visiting relative of patient B, and 9 samples from other patients in the same hospital tested positive for influenza A virus, according to multiplex RT-PCR analysis. WGS was successful for 15 of 17 samples, with a median read depth across all samples of 800 . Attempts to sequence the sample from the relative of patient A were unsuccessful. The sample from patient D failed to generate sequence across all 8 segments of the genome with sufficient depth, resulting in a reliable sequence for NS1 alone (segment 8). Despite this limitation, when a reliable sequence from this patient’s sample was included in a maximum-likelihood phylogenetic tree analysis, samples from patients B, C, D, E, F, and G clustered tightly with high bootstrap support (>95%), with almost identical sequences . The samThere was no evidence of transmission between patient A and patient B, who both had influenza A virus detected by RT-PCR analysis on day 1. These 2 patients were likely infected via separate nosocomial transmissions. Incidentally, a further probable (unrecognized) nosocomial transmission was identified on a separate ward  who were initially considered to be index cases, based on findings of traditional epidemiological methods, were not linked and that one of these individuals was not involved in the outbreak. The use of traditional epidemiological infection control measures erroneously linked these 2 unrelated infections and missed spread of the infection from the north to the south side of the ward, leading to a delay in infection control action. Had WGS data been available in real time, the cross-ward transmission would have been identified on day 1 or 2, and the ward would have been closed. Identification of the separate source of influenza A virus introduction  would additionally have led to a separate investigation and instigation of infection control procedures specific to this introduction.The integration of WGS data with epidemiological analysis in nosocomial infections could identify common pathways of transmission on wards, such as communal areas, bays, shared patient equipment, healthcare staff, or visitors interacting with each other and other patients. Early identification of pathways of transmission could prevent further nosocomial cases of influenza. Interventions would include earlier initiation of ward closure; prophylaxis; emphasis on influenza vaccination of staff and, if they are unwell, treatment; enhanced equipment or room cleaning; and the limiting of visitor-visitor or visitor-patient contact. Prophylaxis has been shown to reduce the risk of symptomatic influenza virus infection in immunocompetent and immunocompromised adults by up to 80% . Staff vWe achieved full-length genome sequencing for all but 2 isolates in this case, allowing more-accurate linkage of infections, compared with the accuracy achieved via limited sequencing of the hemagglutinin or neuraminidase genes. One of these 2 failed attempts at sequencing may be attributable to sampling the relative of patient B close to the end of their illness, when the quantity and quality of viral RNA is less robust.Staphylococcus aureus, but it has been used less frequently to identify transmission of respiratory viruses, including influenza virus [NGS has been used to identify common sources of nosocomial outbreaks of norovirus and methicillin-resistant za virus . PossiblTwo large studies from an epidemiological surveillance unit in Germany and a retrospective analysis of nosocomial transmissions in care facilities in Canada have confirmed nosocomial influenza virus transmission where it was thought to have occurred, using WGS , 14. AltAt this hospital, during the 2016 influenza season, WGS allowed early identification of the predominant circulating influenza A virus subtype as H1N1, rather than H3N2, and led to a local policy change from the recommendation of oseltamivir as first-line treatment to zanamivir. Public Health England altered the national guideline in a similar way, 1 month later . Moving We have described an outbreak of influenza in a hematology/oncology ward in which real-time use of WGS may have mitigated propagation of the outbreak and certainly would have increased understanding of it. The use of WGS during the influenza season, either for all hospitalized patients or specifically for patients who develop influenza during an inpatient stay, may identify transmission where it was not suspected and allow rapid implementation of infection control procedures.The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at Supplementary Figure LegendClick here for additional data file.Supplementary Figure 1Click here for additional data file.Supplementary MethodsClick here for additional data file.


Sugar-sweetened beverages (SSBs) have little nutritional value and a robust body of evidence has linked the intake of SSBs to weight gain and risk of type 2 diabetes (T2D), cardiovascular disease (CVD), and some cancers. Metabolic Syndrome (MetSyn) is a clustering of risk factors that precedes the development of T2D and CVD; however, evidence linking SSBs to MetSyn is not clear. To make informed recommendations about SSBs, new evidence needs to be considered against existing literature. This review provides an update on the evidence linking SSBs and cardiometabolic outcomes including MetSyn. Findings from prospective cohort studies support a strong positive association between SSBs and weight gain and risk of T2D and coronary heart disease (CHD), independent of adiposity. Associations with MetSyn are less consistent, and there appears to be a sex difference with stroke with greater risk in women. Findings from short-term trials on metabolic risk factors provide mechanistic support for associations with T2D and CHD. Conclusive evidence from cohort studies and trials on risk factors support an etiologic role of SSB in relation to weight gain and risk of T2D and CHD. Continued efforts to reduce intake of SSB should be encouraged to improve the cardiometabolic health of individuals and populations.  Metabolic syndrome (MetSyn) is known as a clustering of interrelated risk factors for type 2 diabetes (T2D) and cardiovascular disease (CVD) that occur together more often than by chance alone. Although there is some confusion regarding the clinical definition of MetSyn and whether it is a unique syndrome or a mixture of unrelated phenotypes, the most widespread consensus for a diagnosis is the presence of at least three of five risk factors including hyperglycemia, raised blood pressure, elevated triglyceride levels, low high-density lipoprotein cholesterol levels, and central adiposity . Given tSugar-sweetened beverages (SSBs) are the largest source of added sugar in the diet. They include carbonated and non-carbonated soft drinks, fruit drinks, and sports drinks that contain added caloric sweeteners, and they are low in nutritional quality. To date, a large body of evidence supports a strong link between intake of SSBs and weight gain  and riskConsumption of SSBs has decreased modestly in the US since around 2002 ; howeverIn contrast to the US and other high-income countries where consumption of SSBs is either declining or plateauing, intake of SSBs is increasing in many LMICs as a consequence of widespread urbanization and beverage marketing. A report based on survey data from adults in 187 countries found that SSB consumption was higher in upper–middle-income countries and lower–middle-income countries compared to high-income or low-income countries . Of the Many observational studies have evaluated the relationship between consumption of SSBs and weight gain or obesity. The majority ,21,22,23The association between SSBs and obesity is strengthened by our previous analysis of gene–SSB interactions . Based oCompared to observational studies, most trials have evaluated short-term effects on weight change rather than long-term patterns. In our previous meta-analysis of five trials, we found that adding SSBs to the diet significantly increased body weight . AnotherAnother meta-analysis evaluating the effects of dietary sugars on body weight found that, in trials of adults with ad libitum diets, reducing intake of free sugar or SSB was associated with a decrease in body weight, while increasing intake was associated with a comparable weight increase . BecauseFew prospective studies have examined intake of SSBs in relation to the development of MetSyn, most likely due to challenges in outcome assessment. However, these along with studies of individual risk factors generally show adverse associations that are consistent with studies linking SSBs to weight gain and risk of T2D. Our previous meta-analysis of three cohort studies found a higher risk of about 20% (relative risk (RR), 1.20; 95% confidence interval (CI), 1.02–1.42) comparing highest to lowest categories of SSB intake . HoweverStudies examining individual risk factors rather than MetSyn tend to be more consistent. In the Coronary Artery Risk Development in Young Adults (CARDIA) study, higher SSB consumption was associated with a number of cardiometabolic outcomes: high waist circumference , high low-density lipoprotein (LDL) cholesterol , high triglycerides , and hypertension  . AlthougIn a systematic review including five prospective cohort studies examining SSB intake in relation to vascular risk factors, positive associations were observed for blood pressure, triglycerides, LDL cholesterol, and blood glucose, and an inverse association was observed for high-density lipoprotein (HDL) cholesterol . These fFindings from short-term trials and experimental studies also provide important evidence linking SSBs with cardiometabolic risk factors, and they provide mechanistic support for the epidemiologic evidence linking intake of SSBs to higher risk of T2D and coronary heart disease (CHD). Many of these studies explored the effects of sugars used to flavor SSBs such as high-fructose corn syrup (HFCS)  or sucrose (50% fructose and glucose) in liquid form. A meta-analysis of 39 RCTs found that higher compared to lower intakes of dietary sugars or SSB significantly raised triglyceride concentrations (mean difference (MD): 0.11 mmol/L; 95% CI: 0.07, 0.15), total cholesterol , LDL cholesterol , and HDL cholesterol  . The mosp = 0.005), triglycerides (p = 0.02), and insulin (p = 0.002) [In a two-week parallel-arm trial, Stanhope and colleagues showed that consuming beverages containing 10%, 17.5%, or 25% of energy requirements from HFCS produced a significant linear dose–response increase in postprandial triglycerides, fasting LDL cholesterol, and 24-h mean uric acid concentrations . In anot= 0.002)  In a 10-= 0.002) . A rando= 0.002) . Other t= 0.002) ,53.Although experimental evidence from RCTs is lacking due to high cost and other feasibility considerations, findings from prospective cohort studies have shown strong and consistent associations in well-powered studies. A meta-analysis of 17 prospective cohort studies evaluating SSB consumption and risk of T2D found that a one-serving-per-day increment in SSB was associated with an 18% higher risk of T2D (95% CI: 9% to 28%) among studies that did not adjust for adiposity  Figure . Among sEmerging evidence linking intake of SSBs to CVD is strengthened by consistent associations of SSBs with cardiometabolic risk factors, in addition to weight gain and risk of T2D. A meta-analysis of nine prospective cohort studies found that a one-serving-per-day increase in SSB was associated with a 13% higher risk of stroke  based on one study, and 22% higher risk of myocardial infarction (MI)  based on two studies . In the Building on the clinical evidence, a few studies have also shown a link between SSB intake and risk of all-cause or CVD mortality. We recently found that, among over 118,000 women and men from the NHS and HPFS, intake of SSBs was positively associated with risk of death from any cause in a dose-dependent manner . CompareSSBs contribute to weight gain through decreased satiety and an incomplete compensatory reduction in energy intake at subsequent meals following ingestion of liquid calories . A typicSSBs contribute to the development of T2D and cardiometabolic risk in part through their ability to induce weight gain, but also independently through metabolic effects of constituent sugars . ConsumpSeveral beverages have been suggested as alternatives to SSBs including water, 100% fruit juice, coffee, tea, and ASBs. Unlike SSBs, water does not contain liquid calories and, for most people with access to safe drinking water, it is the optimal calorie-free beverage. We found that replacement of one serving per day of SSBs with one serving of water was associated with less weight gain  and a loAlthough 100% fruit juice might be perceived as a healthy choice since juice contains some vitamins and nutrients, they also contain a relatively high number of calories from natural sugars. Previous cohort studies have found positive associations between consumption of fruit juice and weight gain  and T2D Numerous studies have shown that regular consumption of coffee (decaffeinated or regular) and tea can have favorable effects on T2D and CVD risk ,101, posASBs provide few to no calories but retain a sweet flavor, making them an attractive alternative to SSBs. Paradoxically, some cohort studies have reported positive associations between ASB consumption and weight gain and risk of T2D and CVD ,36. ThesConsumption of ASBs in place of SSBs could be a helpful strategy to reduce cardiometabolic risk among heavy SSB consumers with the ultimate goal of switching to water or other healthful beverages. Further studies are needed to evaluate potential metabolic consequences of consuming ASBs over the life course and better understand underlying biological mechanisms. Understanding potential health impacts of ASB consumption is especially important in the context of sugar reduction policies such as taxation and labeling, which may lead to product reformulation and more ASBs in the food supply.In response to the strong evidence linking consumption of SSBs to weight gain and risk of T2D and CVD, national and international organizations are already calling for reductions in intake of these beverages to help curb obesity and improve cardiometabolic health . Both thBox 1Governments should impose financial incentives such as taxation of SSBs of at 1east a 10% price increase, and implement limits for use of Supplemental Nutrition Assistance Program (SNAP) benefits for SSBs or subsidizing SNAP purchases of healthier foods, to encourage healthier beverages choices.Regulations are needed to reduce exposure to marketing of unhealthy foods and beverages in the media and at sports events or other activities, particularly in relation to children.Front-of-package labelling or other nutrition labeling strategies should be implemented to help guide consumers to make healthy food and beverage choices. These changes should be accompanied by concurrent public health awareness campaigns.Policies should be adopted to reduce the availability of SSBs in the workplace, healthcare facilities, government institutions, and other public spaces, and ensure access to safe water and healthy alternatives. Policies that make healthful beverages the default choice should also be adopted.Educational campaigns about the health risks associated with overconsumption of SSBs should be aimed at healthcare professionals and clinical populations.National and international campaigns targeting obesity and chronic disease prevention should include the health risks associated with overconsumption of SSBs.National and international dietary recommendations should include specific guidelines for healthy beverage consumption.Intake of SSBs remains high in the US and is rising in many parts of the world. Based on findings from prospective cohort studies and short-term experimental trials of cardiometabolic risk factors, there is strong evidence for an etiological relationship between intake of SSBs and weight gain and risk of T2D and CHD. The evidence for a link with stroke is less clear and warrants further research, including the potential sex difference. Few studies have investigated intake of SSBs in relation to MetSyn, and this may be due to challenges in assessment and controversy about its clinical utility. However, findings on individual risk factors suggest a link. Since development of MetSyn often precedes onset of T2D and CHD, preventing or reversing MetSyn could be an effective way to curtail rising T2D and CHD rates.SSBs are thought to promote weight gain through incomplete compensation for liquid calories at subsequent meals. These beverages may increase T2D and CHD in part through weight gain and independently through metabolic effects of constituent sugars. A mechanistic area that warrants future research is exploring the health effects of sugar consumed in solid form compared to SSB, and further elucidating compensatory effects of liquid vs. solid sugars. With the strength of evidence sufficient to call for reductions in intake of SSB for optimal cardiometabolic health, important research gaps exist regarding suitable alternative beverages, including the long-term health effects of consuming ASBs. Continued evaluation of SSB policies that are already in place is needed, as are more and higher-quality RCTs to identify effective strategies to reduce intake of SSBs at the individual and population level. SSBs present a clear target for health policy; however, chronic disease prevention should focus on improving overall diet quality by consuming more healthful foods and limiting unhealthy ones. Given the high levels of intake across the globe, reducing consumption of SSBs is an important step in improving diet quality that could have a measurable impact on weight control and improving cardiometabolic health.


Objective. Corneal neovascularization is a sight-threatening condition affecting more than 1.4 million people per year. Left untreated, it can lead to tissue scarring, oedema, lipid deposition, and persistent inflammation that may significantly affect visual prognosis and quality of life. The aim was to review the recent evidence relating to the pathophysiology, investigations and management of corneal neovascularization.Methods. Literature review of prospective and retrospective studies, clinical trials and animal models relating to the pathophysiology, investigation and management of corneal neovascularization.Results. Corneal neovascularization is characterized by the invasion of new blood vessels into the cornea caused by an imbalance between angiogenic and antiangiogenic factors that preserve corneal transparency as a result of various ocular insults and hypoxic injuries. Risk factors that have been implicated in the pathogenesis of the disease include contact lens wear, ocular surface disease, trauma, previous surgery and herpes. The results highlighted the current and future management modalities of corneal neovascularization, which includes corneal transplantation, laser - phototherapy, injections and topical treatment. Conclusion. The future of corneal neovascularization is promising and this paper discusses the upcoming revolution in local gene therapy.Abbreviations. HSK = herpes stromal keratitis, VEGF = vascular endothelial growth factor, VEGFR-1 = VEGF Receptor-1, FGF = Fibroblast growth factor, PDGF = Platelet-derived growth factor, IL-6 = interleukin-6, IL-7 = interleukin-7, IL-8 = interleukin-8, IRS-1 = insulin receptor substrate-1  A healthy cornea is a transparent, avascular tissue located anterior to the iris and the pupil. Maintaining transparency and avascularity is essential to preserve optimal vision as well as protect the eye against infections and structural damage. 1]. Abnormal new vessels can invade the corneal stroma from pre-existing pericorneal structures as a result of a disruption in the balance of angiogenic and antiangiogenic factors that normally preserve corneal transparency and subsequently lead to corneal neovascularization .This occurs due to a wide variety of ocular insults, including infection, inflammation, ischemia, degeneration, trauma, and loss of the limbal stem cell barrier. Corneal pathologies that can lead to neovascularization include lipid keratopathy, corneal ulcers and scars, herpes eye disease, infectious keratitis, chemical burns, graft rejections and hypoxic insults from contact lens wear [4]. Moreover, 20% of corneal specimens taken from corneal transplant procedures have shown evidence of corneal neovascularization [3]. Corneal neovascularization is a sight-threatening condition and a growing public health concern. One study reported the estimated incidence rate of 1.4 million people per year, 12% of whom suffered subsequent loss of vision [5]. The pathophysiology, investigations and various treatment options currently being undertaken as well as future therapeutic potentials are discussed in this review. Currently, the treatment methodology depends on the state of maturation of the blood vessels at presentation. Established mature blood vessels do not require angiogenic growth factors, whereas immature blood vessels are dependent on them for proliferation, hence treatment is aimed at either removal of established vasculature or preventing neoangiogenesis . The cornea is avascular in healthy individuals; however, under specific pathological circumstances, new capillaries can grow within the cornea. There are three categories of neovascularization based on severity: superficial neovascularization, vascular pannus and deep stromal vascularisation. The mechanisms of corneal neovascularization are observed in significant detail in animal models. It has been hypothesized from these models that corneal neovascularization commences as a result of insult or injury. It is known that a number of diseases and conditions can lead to the development of corneal neovascularization. The most common causes highlighted have been the wearing of contact lenses, inflammation of the eyelid, trauma, previous surgery and herpes [6]. The problem arises as the conjunctival epithelium is rich in goblet cells and highly vascularized. Consequently, the resulting phenotype is optically inferior and leads to the deterioration of vision [7]. Furthermore, the process also leads to an irregular optical surface, weakened tensile strength, and incompetent barrier function.When the cornea is damaged, the epithelial defects are normally healed by the corneal and limbal epithelium. The corneal limbus is located at the corneoscleral junction. The limbal epithelium is rich in stem cells with the capacity to differentiate from normal corneal epithelium. However, defects can occur, leading to these cells undergoing apoptosis and repaired abnormally by the conjunctival epithelium [8]. Strieter et al. demonstrated the relationship to be dose dependent [9]. High doses of 400ng/ cornea did not give rise to neovascularization, whereas doses in the range of 2-40ng/ cornea resulted in neovascularization. Furthermore, the study interestingly found regression of vascularity after 14 days, which suggested that IL-8 angiogenesis underwent dynamic modulation as it was observed in normal wound healing, suggesting a dynamic relationship between inflammation and wound healing. Research suggests that IL-8 may also contribute to the manifestation of corneal neovascularization [10]. Another source of VEGF is infected cells stimulating the production of VEGF as a result of IL-6 expression [11]. A similar relationship has been observed in response to infected cells expressing IL-7, which also stimulates nearby cells to release VEGF [12]. The excessive release of VEGF leads to the development of fragile blood vessels in the cornea.As previously mentioned, HSK can lead to the development of corneal neovascularization. HSK is classed as an immune mediated disease and due to the eye being immune privileged, has been considered a target tissue for HSK. It is believed that VEGF has a significant role in the development of corneal neovascularization as a result of HSK. It has been suggested that the presence of HSK leads to inhibition of VEGF receptor (sVEGFR-1) synthesis at a higher rate compared to VEGF resulting in a ratio imbalance between sVEGFR-1 and VEGF and therefore, the release of VEGF is accelerated to consequently cause angiogenesis . Therefore, preparing and conditioning the vascularized cornea before transplantation is a hopeful potential therapeutic development.Meta-analysis on 24,000 corneal grafts revealed that rejection of transplanted corneas is higher in patients with neovascularization. The analysis estimates that “presence of corneal neovascularization before surgery is 30% more likely that the transplant will fail, and more than doubles the risk of graft rejection”, in other words, the greater the neovascularization the higher risk of rejection [Treatment of Corneal Neovascularization- Laser/ Phototherapy14]. Studies have shown its efficacy in regression of corneal neovascularization [15]. Photodynamic therapy involves a photosensitizing compound, light and oxygen. The compound is absorbed by the neovascular tissue and is activated through laser treatment, which causes free radicals to be released thus destroying the surrounding neovascular tissue and reversing corneal neovascularization [16]. It has been shown that photodynamic therapy is safe and has a high efficacy within humans; however, it is a very costly method of treatment as well as time consuming [16]. Argon laser therapy for corneal neovascularization is the use of an argon laser beam, which passes through a clear cornea, but, when there are many vessels present, the haemoglobin (within the blood) absorbs the argon energy allowing corneal vessels to coagulate, which causes reversal of the corneal neovascularization [17]. Therefore, the hope is that these therapies will be introduced as an adjunct and usage will increase.Both laser and phototherapy need further study to determine their efficacy when compared to other therapeutic strategies. Currently, safety concerns associated with laser therapy and the cost and time of phototherapy have been the negative issues coupled with this innovative treatment, resulting in the relatively low uptake in clinical practice. However, a recent study by Gerten et al. has shown that the combination therapy of bevacizumab with argon laser-therapy causes a marked decrease in corneal neovascularization, this being because the argon laser-induced coagulation closes the mature pathological blood vessels whilst the bevacizumab prevents new angiogenesis [Injections18]. 15 eyes randomized to receive 2.5mg/ 0.1ml subconjunctival injections and 15 eyes randomized to 0.9% saline. A standard therapy of preservative-free dexamethasone 0.1% drops four times a day was prescribed for all patients at baseline. As described previously, treatment can be administered in many ways, also including the administration of steroids and anti-VEGF agents through subconjunctival injections with similar efficacy to topical treatment. Petsogulu C et al. carried out a randomized control trial looking at the outcomes of subconjunctival bevacizumab in 30 eyes of 30 patients with corneal neovascularization . The safety concerns include the potential of replication-deficient viral vectors such as adenoviruses and retroviruses to become replication-competent and pathogenic again. Furthermore, retroviral vectors randomly integrate their genome into host cells, which can lead to insertional mutagenesis to occur [20]. Gene therapies that influence angiogenic factors like VEGF have been investigated, for example Lai and colleagues transduced corneal epithelial cells with an adenovirus vector containing the VEGFR-1 gene in a rodent model and found that it successfully inhibited corneal neovascularization [19]. Gene therapy can also occur through intrasomal or subconjunctival injections or via electroporation and gene gun [21]. However, the use of viral vectors has the highest efficiency in transduction of genes [22]. Furthermore, when the adenovirus vector containing VEGFR-1 was subconjunctivally injected in a rat model of corneal neovascularization there was inhibition of the corneal neovascularization [23]. Likewise, when an adeno-associated viral vector containing the gene for human angiostatin (protein-angiogenesis inhibitor) was subconjunctivally injected in a rat model, the rats showing a significant decrease in corneal neovascularization [24]. Although gene therapy has shown promise in effectiveness there are still technical and safety issues which have to be overcome first [25].Moreover, this method of treatment also allows the incorporation of gene therapy strategies. Gene therapy involves transferring therapeutic genes to the cornea through different vectors. There are safety concerns regarding viral vectors  but they are the most efficient in infecting the corneal epithelial cells with infection rates of 80-100%, allowing higher gene transfer rates compared to non-viral vectors [Topical Treatments25]. Topical steroids such as cortisone, dexamethasone and prednisolone have all been shown to have an antiangiogenic effect and hence inhibit corneal neovascularization [25-28]. However, there are studies suggesting that steroids do not inhibit the development of corneal vascularisation [29]. This was however demonstrated in response to corneal neovascularization post chemical injury, with recent research suggesting positive outcomes in other scenarios [30]. Klintworth has shown that steroid use is most effective in suppressing angiogenesis when applied directly after or before corneal injury and if applied any later it has no effect on the development of corneal vascularisation [29]. It is thought that steroids work by inhibiting cell chemotaxis and by inhibiting pro-inflammatory cytokines like interleukin-1 and -6 [31]. They also cause lymphocytes to be killed and inhibit vascular dilation, which all amounts to their antiangiogenic effect [31]. The use of steroids (such as cortisone) in conjunction with heparin and cyclodextrins causes a greater antiangiogenic effect, this leading to the development of ‘angiostatic steroids’, which are thought to modulate collagen metabolism that can completely disintegrate the basement membrane of the blood vessels . Heparin modulates the expression of anti-angiogenic and pro-angiogenic factors [25]. However, steroids have a considerable side effect profile with negative associations such as glaucoma and increased infection susceptibility due to their immune suppressive effect.Steroids and anti-VEGF agents are currently the mainstay initial treatment for corneal neovascularization [34]. The eye is a site which has ‘angiogenic privilege’ meaning it has a balance of pro-angiogenic and anti-angiogenic factors. Pro-angiogenic factors include VEGF, FGF and PDGF [25]. Selectively targeting these angiogenic growth factors is desirable over steroids due to their side effect profile and more selective action. Anti-VEGF drugs work by inhibiting VEGF which prevents new blood vessel formation through down regulation of endothelial cell proliferation. Bevacizumab is a humanized monoclonal antibody which binds to all VEGF isoforms [35].VEGF has been shown to be crucial in inflammatory corneal neovascularization through the rat experimental model [36]. Lin and colleagues have similarly shown that early treatment with bevacizumab inhibits corneal neovascularization but late treatment does not display these features [37]. This shows that anti-VEGF therapy is not as effective in individuals who have mature blood vessels as they do not rely on pro-angiogenic factors [37]. Anti-VEGF treatment is important during active vessel growth which is characterized by the presence of immature blood vessels relying on pro-angiogenic factors for proliferation [38]. This is in line with the findings by Lin that anti-VEGF treatment (bevacizumab) is effective when used in early treatment of patients with corneal neovascularization [37]. Anti-VEGF treatment can have undesirable effects, including suppression of wound healing, corneal nerve regeneration and can systemically cause hypertension and cardiovascular disease [25]. Krizova showed that the use of bevacizumab is effective and very safe in treating active corneal neovascularization whether applied topically or given as a subconjunctival injection [39]. However, they also show that bevacizumab does not have the same effect on mature corneal neovascularization and this treatment does not cure the disorder.Another study has shown that bevacizumab does have an immediate inhibitory effect on corneal neovascularization and inflammation, but the effects are very short-lived [New Topical Therapeutic Advancement40]. Aganirsen is an antisense oligonucleotide, which inhibits the expression of IRS-1 mRNA, mRNA of interleukin-1beta and the mRNA of VEGF [40]. Cursiefen et al. conducted a phase 3 trial and found that topical administration of Aganirsen eye drops massively inhibits corneal neovascularization in patients with keratitis and that the need for future transplantation is not needed. They have also shown that Aganirsen is very safe and well tolerated in individuals [41].It has been shown that the activation of the IRS-1 proteins is vital in angiogenesis and it is overexpressed in corneal neovascularization sites [42]. Jovanovic and Nikolic have shown that the use of topical doxycycline on human corneal neovascularization is effective in reducing the neovascularization and effective in the healing process without any side effects [43]. Combination therapy of anti-VEGF agents, steroids and doxycycline have been investigated and have been shown to have higher efficiency in inhibiting corneal neovascularization compared to solitary use [32]. The theory behind combination therapy is that this method will target various mechanisms involved in maintaining corneal neovascularization and hence will be much more efficient in inhibiting the disease and its reoccurrence.Another novel advance in the treatment of corneal vascularisation is the use of matrix metalloproteinase inhibitors such as use of tetracyclines. Doxycycline is a tetracycline analogue, which has an antibiotic effect but is also a matrix metalloproteinase inhibitor. Matrix metalloproteinase are enzymes that degrade collagen, basement membranes and the extracellular matrix. Doxycycline also inhibits angiogenesis in a non-metalloproteinase-dependent mechanism, and has an anti-inflammatory effect [New Injectable Therapeutic Advancements44]. This technique has been modified through using an electrolysis needle that is much more flexible and precise [45]. Trikha et al. carried out a 5 year retrospective study on individuals who underwent fine needle diathermy and found that this treatment is safe and very effective in regressing corneal neovascularization [46]. This suggests that this method of treatment can be used in conjunction with anti-VEGF drugs to allow an angioregressive treatment of corneal neovascularization . Pillai et al. first described the technique of fine needle diathermy, which involves using a needle to cauterize individual vessels; this method is effective in occluding mature vessels that are not dependent on angiogenic growth factors [49]. Furthermore, when the adenovirus vector was subconjunctivally injected in a rat model, there was inhibition of the corneal neovascularization [50]. Likewise, when an adeno-associated viral vector containing the gene for human angiostatin (protein-angiogenesis inhibitor) was subconjunctivally injected in a rat model, the rats showed a significant decrease in corneal neovascularization [51]. Although gene therapy has shown promise in effectiveness, there are still current technical concerns.Gene therapy targeting VEGF has had successful results, for example Lai et al. have shown that an adenovirus vector carrying VEGFR genes caused regression of the corneal neovascularization [The ever-expanding knowledge of the mechanisms involved in corneal neovascularization are allowing different treatment options to be developed. Anti-VEGF drugs have been the centre of discussion as have matrix-metalloproteinase inhibitors. These methods of treatments for corneal neovascularization currently still depend on the blood vessel maturity stage. Therefore, local gene therapy may be a promising universal treatment of corneal neovascularization, with the hope that safety concerns can be allayed by continuing and impending research.Conflict of interestsThe authors declare no conflict of interests.AcknowledgementsAll authors have equal contribution. There are no financial or proprietary interests.


T helper 17 (Th17) cells represent a distinct population of immune cells, important in the defense of the organism against extracellular infectious agents. Because of their cytokine profile and ability to recruit other immune cell types, they are highly pro-inflammatory and are involved in the induction of several autoimmune disorders. Recent studies show that Th17 cells and their signature cytokine IL-17 have also a role in a wide variety of neurological diseases. This review article will briefly summarize the evidence linking Th17 cells to brain diseases associated with cognitive impairment, including multiple sclerosis (MS), ischemic brain injury and Alzheimer’s disease (AD). We will also investigate the mechanisms by which these cells enter the brain and induce brain damage, including direct effects of IL-17 on brain cells and indirect effects mediated through disruption of the blood-brain barrier (BBB), neurovascular dysfunction and gut-brain axis. Finally, therapeutic prospects targeting Th17 cells and IL-17 will be discussed. It is well documented that T helper 17 (Th17) cells are important in the defense of the organism against opportunistic pathogens of fungal or bacterial origin, making them a fundamental part of the adaptive immune system. Th17 cells have also a role in barriers protection, especially in the bowel, where they regulate the host response to microbiota and maintain the intestinal homeostasis  and other costimulatory molecules , homodimers or heterodimers  from fibroblasts, endothelial cells and epithelial cells . These mIL-17A and other IL-17 family cytokines are not only produced by Th17 cells but also by other immune and non-immune cells. Indeed, under different conditions involving the activation of the immune system, other selective cell subtypes, such as macrophages, dendritic cells, natural killer cells and γδ T cells produce IL-17 , have revealed the role of Th17 cells in MS  is the most common cause of cognitive impairment in the elderly. AD classical pathological hallmarks are intracellular neurofibrillary tangles and extracellular amyloid-β (Aβ) plaques. It is however well established that cerebrovascular alterations coexist in determining the development of the disease . The effect could be mediated by promoting the transition of lymphocyte function associated antigen 1 (LFA-1) integrin from the low- to the high-affinity binding state, thus enhancing neutrophil adhesion to the cerebral endothelium , however, Th17 cells migration acts with a caveolin-independent TJ remodeling occurring in early phase of EAE , compared to peripheral endothelial cells  and underlying actin cytoskeleton , which is essential for the brain’s functional and structural integrity. All these components of NVU interact in response to physiological stimuli and assure adequate brain perfusion through different mechanisms  Thr495 inhibitory phosphorylation , a population of innate immune cells , and to their influence on mediated T-cells immunity.The gut microbiota influences the activities of astrocytes and microglia, which in turn act on various neuropsychological processes such as neuronal development, neurotransmission, BBB integrity and CNS immune system activation  and serum/glucocorticoid-regulated kinase 1 , whose production is influenced by the activity of Th17 cells. Therefore, targeting Th17 cells instead of individual effector cytokines would be a greater benefit. Some efforts have been made regarding targeting RORγt, Moreover, as already mentioned, the gut-brain axis has shown great relevance in CNS functions and in cognitive impairment. Avoiding excessive salt intake may be a strategy to prevent the damage deriving from brain microvasculature impairment, which leads to altered brain function and cognitive impairment (Faraco et al., In conclusion, emerging evidence point to a role of Th17-IL-17 in a wide variety of neurological diseases associated with cognitive impairment, ranging from neurovascular to neurodegenerative diseases. However, several arguments need to be discussed concerning mainly the mechanism of their pathogenic effect and their potential value as therapeutic targets. Considering the expanding role of adaptive and innate immunity in a growing number of cerebral and systemic pathologies, additional research in this field is required and may lead to new insights into the pathogenesis and treatment of these highly prevalent and devastating neurological disorders.CI contributed substantially to the conception and design of this review and provided final approval of the version to publish. VC took the lead in writing the manuscript. CI, JA and FO provided critical feedback and helped shape the research, analysis and manuscript. All authors revised the manuscript critically for important intellectual content.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.