Finally, we discuss our murine PET imaging experience and the challenges encountered.Acute coronary syndrome (ACS) describes a range of conditions associated with the rupture of high-risk or vulnerable plaque. Vulnerable atherosclerotic plaque is associated with many changes in its microenvironment which could potentially cause rapid plaque progression. Present-day PET imaging presents a plethora of radiopharmaceuticals designed to image different characteristics throughout plaque progression. Improved knowledge of atherosclerotic disease pathways has facilitated a growing number of pathophysiological targets for more innovative radiotracer design aimed at identifying at-risk vulnerable plaque and earlier intervention opportunity. This paper reviews the efficacy of PET imaging radiotracers  The goal of non-invasive imaging of atherosclerotic plaques is to enable better prediction of cardiovascular (CV) events by improving assessment of asymptomatic, at-risk plaque. Currently, the two main screening techniques in place are invasive coronary angiography and coronary computed tomography (CT) angiography (CCTA). The gold standard for identifying coronary artery stenosis is invasive coronary angiography, which is an invasive procedure with attending risks . CCTA utPET imaging has high sensitivity for the detection of sparse targets in the nanomolar range with low tracer doses . AlthougSpecific radiotracers in PET imaging have been extensively utilised in providing insight into patients with acute coronary syndrome (ACS). ACS represents an end-stage of atherosclerotic plaque development and rupture. Present-day PET imaging presents the option of choosing different radiopharmaceuticals to image different plaque characteristics throughout this process. The focus of this review is to discuss the utility of PET imaging in providing insight into assessing vulnerable plaque, its use in animal models, and its future direction.Atherosclerosis is initiated by the deposition and accumulation of lipids and fibrous elements in the arterial wall . Plaque FDG is a glucose analogue and the most-validated radiotracer for imaging high metabolically active inflammatory cells  and tissues  in animal models and humans . The resThe interpretation of the uptake of glucose by inflammatory cells and non-specific uptake of cells in the arterial wall could prove challenging. The different subtypes of inflammatory macrophages have divergent roles in plaque development and progression. M1 macrophages are pro-inflammatory and more glycolytically active than M2 anti-inflammatory cells . AnotherExperimental studies of FDG-PET in atherosclerosis have shown that distribution of FDG within atherosclerotic plaques occurs predominantly in macrophages, and FDG uptake correlates with plaque inflammation in clinical imaging . HoweverIn addition, uptake of FDG in the heart, an organ of high metabolic activity, can present challenges in assessing inflammation ,20. This18F-NaF is another radiotracer of interest. As the myocardium does not take up 18F-NaF, uptake can be easily detected in coronary plaque without confounding uptake from the myocardium (as observed with FDG). This practical consideration simplifies the application of 18F-NaF-PET imaging in patients [patients .18F-NaF-PET imaging targets this calcification process, and the uptake of NaF can assess plaque stability through measuring calcification. Arterial calcification is an independent predictor of an adverse CV event. It is now widely accepted that calcification associates with plaque progression and vulnerability. Microcalcifications provide further stimulus for inflammatory response and thus perpetuate the inflammatory cycle, leading to plaque instability [A characteristic feature of plaque development involves microcalcification (0.5–15 μm), a process dependent on inflammation resulting in the development of larger sheet-like deposits (> 3 mm) ,23. A nutability . Within tability . In latetability .18F-NaF-PET correlates with CV risk factors, and 18F-NaF uptake appears to be a good measure of the body’s atherosclerotic burden, potentially suited for assessment of anti-atherosclerosis therapy [18F-NaF may serve as a potential biomarker for vulnerability and CV risk [18F-NaF uptake in patients with high CV risk factors and thoracic fat volume [A recent review stated that  therapy . Studies CV risk ,33,34. Tt volume .18F-NaF-PET imaging was able to successfully identify vulnerable coronary lesions in 93% of patients with myocardial infarction [18F-NaF uptake in the patients correlated with intravascular ultrasound findings of microcalcification, a necrotic core, and positive remodelling.A prospective study of 80 patients utilising farction . Increas18F-NaF uptake compared to those without those characteristics [18F-NaF uptake correlated positively with coronary calcium score per patient, (2) patients with a history of myocardial infarction or unstable angina have a higher coronary artery 18F-NaF uptake, (3) increased 18F-NaF uptake in coronary atherosclerosis is independently correlated with partially calcified plaque components, and (4) coronary plaques with high-risk characteristics present with higher 18F-NaF uptake on PET than those without [Myung et al. demonstrated that coronary plaques with high-risk characteristics on intravascular imaging  had higher eristics . Moreove without .18F-NaF-PET in predicting coronary events. High 18F-NaF uptake is predictive of a coronary event within the next 2 years, correlating with advanced coronary calcification and presenting as a high-risk plaque on CCTA. The results support implementation of CCTA and 18F-NaF-PET for non-invasive identification of high-risk CAD [18F-NaF-PET could help predict myocardial infarction and provide any additional prognostic information to current methods of risk stratification [18F-NaF uptake was found to be better for predicting fatal or nonfatal myocardial infarction compared to coronary calcium scoring, the modified Duke CAD index, and the Reduction of Atherothrombosis for Continued Health (REACH) and Secondary Manifestations of Arterial Disease (SMART) risk scores.Several studies have investigated the utility of risk CAD . More refication . Measuri18F-NaF-PET imaging to quantify coronary artery plaque burden, the challenge of quantification due to low target to background ratios, partial volume effects and motion must also be considered [18F-NaF-PET imaging as an innovative approach to monitoring the disease progression and vulnerable plaques in atherosclerosis.When utilising nsidered . However68Ga-DOTATATE has become an attractive option in PET imaging due to its high specificity for the G-coupled receptor somatostatin receptor subtype-2 (SSTR2), which is up-regulated in activated macrophages [rophages ,41.ApoE-/- mouse model) at the tissue level [68Ga-DOTATATE to 18F-FDG evaluated in 42 patients with atherosclerosis was found to offer superior coronary imaging. 68Ga-DOTATATE demonstrated excellent macrophage specificity and better discriminative power to identify high-risk versus low-risk coronary lesions compared to 18F-FDG [68Ga-dotatate binding to SSTR2 within areas of CD68+ macrophage-rich carotid plaque regions with a strong correlation of carotid SSTR mRNA and in vivo 68Ga-DOTATATE activity [68Ga-DOTATATE as a valid investigation tool for unstable plaque identification.The low physiological expression of SSTR2 by the myocardium suggests that this tracer may be advantageous for imaging disease in the coronary arteries . Severalue level ,44. In h 18F-FDG . Furtheractivity . The stu68Ga-DOTATATE uptake in carotid plaque of patients with recent carotid events found no difference between recently symptomatic carotid plaques vs. contralateral plaques [+ macrophages in vitro, SSTR2 expression was not detected in excised plaques.In contrast, a prospective study evaluating  plaques . Despite68Ga-DOTATATE in preclinical and early human studies however, the radiotracer still warrants further characterisation to verify its role in vulnerable plaque risk stratification [The majority of studies have demonstrated potential use for fication ,47.68Ga-DOTATOC shares a similar SST binding profile to 68Ga-DOTATATE, but its binding affinity to SSTR is 10-fold lower [68Ga-DOTATOC-PET for assessing vulnerable plaque in the thoracic aorta was investigated, and the study found uptake correlated with CV risk factors [68Ga-DOTATOC significantly correlates with the Framingham risk score, a measure of CV risk, corresponding with earlier studies suggesting a role as a predictor of CV events and as a biomarker for vulnerable plaque assessment [ld lower ,49. The  factors . Furthersessment ,51,52,5368Ga-DOTATOC uptake with CV risk factors suggests its use as a potential predictor for CV events and a biomarker for the assessment of vulnerable plaque. Further studies exploring the clinical efficacy and relevance of 68Ga-DOTATOC are warranted to validate the value of 68Ga-DOTATOC PET/CT in atherosclerosis and its correlation with CV risk and events and PET indexes [The significant correlation of  indexes .64Cu-DOTATATE, like the other DOTATATE and DOTATOC variants, targets the SSTR expressed on activated macrophages which accumulate in active inflammatory lesions. A study in humans found that vascular uptake of 64Cu-DOTATATE was higher than 68Ga-DOTATOC, suggesting a potential role of 64Cu-DOTATATE in the assessment of atherosclerosis [clerosis .64Cu-DOTATATE correlated with gene expression of CD163, a surrogate marker of alternatively activated macrophages within atherosclerotic plaques [64Cu-DOTATATE uptake [In a study of 10 patients who underwent carotid endarterectomy, uptake of  plaques . This fi plaques . InteresE uptake . This obE uptake .64Cu-DOTATATE uptake [Markers of plaque vulnerability , and IL-18), previously found to be associated with FDG uptake, did not correlate with E uptake . This clThe C-X-C motif chemokine receptor 4 (CXCR4) is expressed on the surface of various cell types involved in atherosclerosis, including macrophages and T-cells . Its rol125I-pentixafor accumulated in inflamed plaques, which was verified histologically by the detection of macrophages and CXCR4 in plaques of the abdominal aorta and carotid artery [A study of human carotid plaques found CXCR4 expression was elevated in both stable and unstable atherosclerotic plaques, with the highest receptor expression found in macrophage-derived foam cells and macrophages . Similard artery . Togethe68Ga-pentixafor and 18F-FDG for the detection of arterial wall inflammation and calcification in lesions [68Ga-pentixafor identified a greater number of atherosclerotic lesions with higher uptake compared to 18F-FDG. 68Ga-pentixafor detected sites that were overlooked on FDG-PET, suggesting that the pentixafor uptake originates from cell types beyond inflammation [68Ga-pentixafor-positive uptake sites might represent very early-stage lesions without markedly elevated inflammation [A recent study evaluated the performance of  lesions . The retammation . CXCR4-eammation .68Ga-pentixafor was associated with an increased incidence of CV risk factors [68Ga-pentixafor and FDG uptake with the degree of calcification [In a study of oncology patients, increased uptake of  factors . In line factors , the autfication . Non-calfication .68Ga-pentixafor uptake was observed in 1411 sites in 51 patients and was significantly associated with calcified plaque burden and CV risk factors, including age, arterial hypertension, hypercholesterolemia, history of smoking, and prior cardiovascular events [r events . Increasr events .68Ga-pentixafor uptake represent the sum of all CXCR4-expressing cells localised within or near a particular lesion? [68Ga-pentixafor to FDG resulted in only a weak correlation between tracers. Further studies are highly warranted to elucidate the underlying biological mechanisms and sources of CXCR4 to improve understanding of the clinical utility of this radiotracer.While insightful into CV risk and identifying early lesion development, no definitive conclusions can be drawn about its exact cellular source. Does  lesion? ,59,61,64Animal models have facilitated the understanding of underlying mechanisms contributing to atherosclerotic plaque stability and monitoring disease progression. Moreover, using mice, or other animals for that matter, can be used for proof-of-concept studies, or to assess radiotracer behaviours in vivo. Furthermore, ex vivo validation of readings can be confirmed via gamma-counting, autoradiography, and immunohistochemistry for improved quantification . Here, wHypoxia has been reported in plaques from humans and animal models of atherosclerosis. In atherosclerosis and vascular disease of larger arteries, hypoxia occurs within layers of the arterial wall . HypoxiaIn concert with inflammation, hypoxia also triggers metabolic glucose changes to maintain ATP production in cells. Under these conditions, HIF-1α triggers glycolytic gene activation in endothelial cells, giving rise to enhanced cell proliferation and inflammation ,70. HIF-18F-fluoromisonadazole uptake has been demonstrated. Uptake is increased and correlates with advanced disease progression and aligns in regions rich with macrophage population and neovascularization [In a rabbit model of atherosclerosis, rization   play a key role throughout all stages of atherosclerosis and are involved in vascular inflammation, smooth muscle cell migration, endothelial dysfunction, extracellular matrix degradation, vascular calcification, and plaque activation and destabilization . They arPrevious studies examining the role of MMP associate increased expression with morphological changes in diseased arteries of experimental models of atherosclerosis . Increas123I- or 125I-labelled CGS 27023A, a broad spectrum MMP inhibitor [99mTC-labelled broad-spectrum MMP-inhibiting macrocyclic compound, have been shown to bind to atherosclerotic plaque and show early promise in translatable MMP imaging [In models of atherosclerosis, nhibitor , and RP- imaging ,83  uptake in atherosclerotic plaques of ApoE-/- mice. The study also observed 111In-tilmanocept accumulation in macrophage rich organs [The mannose receptor is over-expressed in activated macrophages. Authors utilising the novel h organs  , demonstrated ex vivo binding capability to peritoneal murine macrophages and in the aorta of atherosclerotic rabbit models [68Ga-NOTA-MSA PET/CT was higher in atherosclerotic animals compared to control and were not different from 18F-FDG-PET/CT imaging.Another novel mannose receptor-targeting radiotracer, t models  , 18F-NaF (n = 3), or 68Ga-DOTATATE (n = 3) radiotracer  and the University of Western Australia (F71731).ApoE-/- murine model. In mice given 18F-FDG, we observed 18F-FDG uptake in the ascending and arch of the aorta, where the plaque burden was expected to be high , we assessed the efficacy of these three imaging agents in plaque identification in the  be high . In our 18F-FDG, which exhibited intense homogenous myocardial uptake, we observed no background abnormal myocardial binding in mice given 68Ga-DOTATATE. Only one mouse showed low-grade uptake in the ascending aorta in agreement with the 18F-FDG PET CT , resulting in lower-than-expected whole body tracer concentration further jeopardising the image quality. The physical characteristics of 68Ga also likely contribute to the poor image quality due to the longer positron range and higher positron energy [68Ga less ideal for imaging, especially in mice as they can limit special resolution [Unlike G PET CT . We founn energy . This masolution .18F-NaF-imaged mice produced excellent skeletal images; however, they did not identify any plaque calcification (image not shown). This was likely due to the mice being too young, or the imaging being performed too early in the disease course to observe changes from atherosclerosis. 18F-NaF by far produced the cleanest quality images out of all three radiopharmaceuticals.18F-FDG visualisation and quantification in coronary plaques. Furthermore, image clarity is of concern due to cardiac and respiratory motion. In efforts to mitigate these issues, some investigators perform end-diastolic imaging using only part of the PET scan at the cost of increased image noise. Additionally, PET images require co-registration with CT or MRI for localisation, which are both hindered by cardiac and respiratory motion [Unfortunately, while PET is widely flexible and applicable to many disease diagnoses, PET imaging has a number of limitations. With a limited spatial resolution of 3–5 mm, making reproducible measurements in the right coronary artery and mid to distal vessels becomes problematic. The inability to optimally suppress glucose uptake by the myocardium can interfere with y motion .18F-FDG or 18F-NaF. This is in part due to the ease of tracer synthesis, their availability, and their history in clinical applications.Another challenge of PET imaging is the availability of radiotracers. Although many radiotracers have been proven to be useful for identifying atherosclerotic plaque in preclinical models of disease, the majority of the clinical studies imaging coronary plaque have been performed using either The expanding number of tracers available that can target receptors expressed by macrophages, luminal endothelial cells, hypoxia, and angiogenesis have been used to detect active atherosclerotic plaque, but only in small clinical cohorts. The successful translation of these tracers to clinical practice will depend on availability, affordability and ease of synthesis.In addition, confirmation of plaque vulnerability needs to be correlated with increased plaque tracer uptake after the atherothrombotic event has occurred. To demonstrate the association, it will be necessary to show that increased tracer uptake by the coronary artery plaque is associated with a future cardiac event.Finally, to really enable feasible clinical molecular imaging, major advances in image acquisition and processing are required to address limitations imposed by cardiac and respiratory motion when assessing plaque.The scope of imaging inflammation needs to increase to incorporate more inflammatory pathways identified by basic science. The understanding of particular inflammatory cytokines and their role in atherogenesis provides additional opportunity for radiotracer development.Altogether, enhanced inflammation has been identified as a marker of the risk of post-infarct ventricular dysfunction and heart failure . OveractThe study of C-reactive protein (CRP), interleukin (IL)-1β, and NLR family pyrin domain-containing 3 (NLRP3) inflammasome has revealed critical roles for each in post-infarction systemic inflammation and progression of atherosclerosis ,97,98,99Therapeutically, administration of IL-1β-inhibiting agents  result in a significantly lower rate of recurrent cardiovascular events and prevent future hospitalisation for heart failure . MoreoveBuilding on these strong correlations, utilising baseline systemic NLRP3 concentration is a promising prognostic utility and, through correlation with GRACE and TIMI risk scores, could prove an efficient event predictor for MACE . DevelopDespite progress in understanding the complex underlying biology of atherosclerosis, it remains a global health problem. Improved knowledge of the disease mechanisms has translated to an increase in novel radiotracer development and leaps forward in plaque characterisation imaging. However, to prospectively recognise vulnerable plaque and prevent the occurrence of adverse events, an imaging strategy that targets the molecular changes in vulnerable plaque is needed. While biomarkers assess systemic inflammation, a plaque marker of susceptible rupture would be invaluable in the process of targeted local intervention and primary prevention of adverse events. Moreover, including inflammatory pathways in therapeutic targets could bring PET imaging diagnosis and intervention closer together to effectively target cardiovascular outcomes and improve patient outcome.


Age and disease prevalence are the two biggest risk factors for COVID-19 symptom severity and death. We therefore hypothesized that increased biological age, beyond chronological age, may be driving disease-related trends in COVID-19 severity. Using the UK Biobank England data, we tested whether a biological age estimate (PhenoAge) measured more than a decade prior to the COVID-19 pandemic was predictive of two COVID-19 severity outcomes . Logistic regression models were used with adjustment for age at the pandemic, sex, ethnicity, baseline assessment centers, and pre-existing diseases/conditions. 613 participants tested positive at inpatient settings between March 16 and April 27, 2020, 154 of whom succumbed to COVID-19. PhenoAge was associated with increased risks of inpatient test positivity and COVID-19 related mortality  adjusting for demographics including age at the pandemic. Further adjustment for pre-existing disease s/conditions at baseline  and at the early pandemic  decreased the association. PhenoAge measured in 2006-2010 was associated with COVID-19 severity outcomes more than 10 years later. These associations were partly accounted for by prevalent chronic diseases proximate to COVID-19 infection. Overall, our results suggest that aging biomarkers, like PhenoAge may capture long-term vulnerability to diseases like COVID-19, even before the accumulation of age-related comorbid conditions.


Recent observations suggest a lack of humoral response after SARS-CoV-2 vaccination in multiple sclerosis (MS) patients treated with fingolimod or ocrelizumabTo assess serological response to SARS-CoV-2 vaccination in MS patients receiving these disease-modifying treatments (DMTs) in a real-life setting.Retrospective clinical data collection from MS patients followed at San Raffaele Hospital MS Centre . All patients treated with fingolimod or ocrelizumab who had received a complete anti-COVID-19 vaccination course, with no clinical history suggestive of previous SARS-CoV-2 infection and with an available post-vaccination serological assay obtained at least 14 days after vaccination completion were considered for the study.We collected data from 32 MS patients, 16 treated with fingolimod and 16 receiving ocrelizumab. Among the fingolimod group 10 patients (62.5%) had a positive serological response after vaccination and among ocrelizumab-treated patients a positive serological test was found in six cases (37.5%). No relation between serological response and clinical features  was identified.Our initial real-life experience suggests a variable antibody production in MS patients receiving these DMTs. At present, there are no sufficient data to do not recommend anti-SARS-CoV-2 vaccine in these patients. SARS-CoV-2 infection has been responsible of an unprecedented pandemic all over the world, with consequences on several social aspects. Several concerns have been raised in relation to an increased susceptibility to COVID-19 in patients affected by Multiple sclerosis (MS), considering the many potential interactions with the immune system, disease-modifying treatments (DMTs) and neurological complications described in association with SARS-CoV-2 infection , with a Inactivated vaccines are generally safe in MS, with the majority of data deriving from previous experiences with flu vaccines . DespitePost-marketing experiences in the field are gradually becoming available. Achiron and colleagues recently assessed humoral response to mRNA COVID-19 vaccines on an Israeli cohort of 93 MS patients-receiving second-line DMTs  and 32 untreated MS patients . After vWe report our initial clinical experience, with the aim to investigate COVID-19 vaccines response in MS patients receiving ocrelizumab or fingolimod. We retrospectively collected data from 32 MS patients followed at San Raffaele Hospital MS Centre , 16 treated with fingolimod and 16 with ocrelizumab. All patients received a complete anti-SARS-CoV-2 vaccination course, consisting of two separate doses  and no one had a clinical history suggestive of previous SARS-CoV-2 infection. Anti-Spike protein antibody test was obtained at least 2 weeks  after vaccination course completion. Serology tests have been performed in different laboratories as part of routine clinical assessment in order to evaluate humoral responses to vaccination (detailed techniques for each single patient are reported in Tables Our experience, in comparison to previously published data , suggestClearly, this study is not without limitations: the sample size is relatively small and data collection is retrospective, with serological exams performed with different techniques. In addition, we do not have pre-vaccinations serological tests available; northern Italy had a very high prevalence of SARS-CoV-2 infection over the last year, therefore we cannot exclude a previous asymptomatic infection possibly influencing the serological response.In this historical moment, it is of outmost importance that a very large proportion of the population, including people with MS, adheres to mass vaccination campaigns, in order to get through the current pandemic condition. With the rapid progression of SARS-CoV-2 vaccination programs all over the world, more extensive real-life data from different geographic regions are likely to become available in the near future. The assessment, in the context of prospective clinical trials, of humoral and in particular T cell response to SARS-CoV-2 vaccination will be also crucial to tailor the clinical management of MS patients.


COVID-19, a respiratory viral infection, has affected 388 million individuals worldwide as of the February 4, 2022. In this review, we have outlined the important liver manifestations of COVID-19 and discussed the possible underlying pathophysiological mechanisms and their diagnosis and management. Factors that may contribute to hepatic involvement in COVID-19 include direct viral cytopathic effects, exaggerated immune responses/systemic inflammatory response syndrome, hypoxia-induced changes, vascular changes due to coagulopathy, endothelitis, cardiac congestion from right heart failure, and drug-induced liver injury. The majority of COVID-19-associated liver symptoms are mild and self-limiting. Thus management is generally supportive. Liver function tests and abdominal imaging are the primary investigations done in relation to liver involvement in COVID-19 patients. However, imaging findings are nonspecific. Severe acute respiratory syndrome coronavirus 2 RNA has been found in liver biopsies. However, there is limited place for liver biopsy in the clinical context, as it does not influence management. Although, the management is supportive in the majority of patients without previous liver disease, special emphasis is needed in those with nonalcoholic fatty liver disease, cirrhosis, hepatocellular carcinoma, hepatitis B and C infections, and alcoholic liver disease, and in liver transplant recipients. The article title and abstract were read for the initial selection and then the full-text article was read. Reference lists of the full-text articles were scanned to identify any additional studies. All types of research articles, including original research articles, reviews, case series, short communications, and case reports were considered. Of the 103 articles identified, 59 were analyzed further .N = 77) of AIH patients.The current literature has several studies on liver-related outcomes in COVID-19. However, the definition of liver injury tends to vary among the different studies. Furthermore, specifying liver-related outcomes in COVID-19 patients is made difficult because of the studies describing different etiologies, different disease severities, small numbers of study participants from a single geographical location, and the lack of correlation of liver test results with preexisting liver conditions. Preexisting chronic liver disease (CLD) may predispose a person to adverse outcomes following COVID-19 because of immune dysregulation.,Factors that may contribute to liver involvement in COVID-19 include direct viral cytopathic effects, exaggerated immune responses/systemic inflammatory response syndrome (SIRS), hypoxia-induced changes, vascular changes due to coagulopathy,Angiotensin-converting enzyme 2 (ACE2) receptors provide a gateway for viral entry, and its tissue distribution determines the pattern of viral tropism. There is high expression of ACE2 on cholangiocytes  and low expression on hepatocytes, Kupffer cells (liver macrophages), and endothelial cells.The renin-angiotensin system (RAS) plays a major role in liver inflammation, tissue remodeling, and fibrosis. Angiotensin-converting enzyme 2 is a key negative regulator of the RAS and limits fibrosis through the degradation of Angiotensin II and the formation of Angiotensin (1–7). Upon binding of the SARS-CoV-2 virus, ACE2 is endocytosed and levels are reduced on the cell surface. Murine studies found reduced ACE2 levels to worsen liver fibrosis in chronic liver injury models.,9/L are independent risk factors for liver injury. Lymphopenia is noted in 63–70.3% of COVID-19 patients. Postmortem liver histology shows microvesicular steatosis and T cell accumulation, pointing to the presence of immune-mediated damage.An exaggerated inflammatory response in COVID-19 leads to lymphocyte activation, neutrophilia, and an increase in C-reactive protein (CRP) and inflammatory cytokines. Levels of serum interleukin (IL)-2, IL-6, IL-7, IL-10, tumor necrosis factor (TNF)-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-inducible-protein-10, monocyte chemotactic protein-1, and macrophage-inflammatory-protein-1 alpha are significantly higher in severe COVID-19.,Liver hypoxia (because of microvascular thrombosis and gas exchange defects secondary to lung injury) may cause hepatic damage. Ischemic injury to the gut with resulting intestinal endotoxemia, and activation of the sympathetic nervous and adrenocortical systems may further contribute to liver damage.–As most COVID-19 patients have fever, antipyretics containing acetaminophen are frequently used. Higher doses of this medication are known to cause liver damage. Many antiviral drugs are administered  and some of them may have adverse effects on the liver .31,Recent studies on gut microbiota have suggested an alteration in intestinal microbiota composition  contributes to different immune-mediated inflammatory diseases.Preliminary observations suggest that SARS‐CoV‐2 affects mitochondrial activity.,,–The COVID-19-associated liver injury is defined as liver damage occurring due to the virus or its treatment in those with or without preexisting liver damage.,,The commonest liver test abnormalities reported are mild to moderate elevations in ALT and AST levels (seen in 14–53% of cases).,Elevated bilirubin levels are observed in 20–40% of patients and 10% had very high levels.,,Up to 4% of patients with severe COVID-19 had reduced albumin levels.,,Elevations of serum GGT levels point to the presence of cholangiocyte injury,The described pathological changes in liver histology are mainly ascertained from postmortem studies. Most of the studies do not indicate whether the patients had preexisting liver disease or the severity of the liver derangement, precluding useful interpretation of the pathological findings. The liver histology changes noted in COVID-19 include moderate microvascular and macrovascular steatosis and mild lobular portal inflammation.,,–The majority of patients with COVID-19 have no or mild liver function abnormalities during the illness.Liver function tests and abdominal imaging are the primary investigations done in relation to liver involvement in COVID-19 patients. Liver biochemistry including the liver enzymes (ALT and AST), serum bilirubin, albumin, and PT should be monitored for diagnosing liver damage.Lei et al. found liver hypo-echogenicity (homogeneous or heterogeneous) and peri-cholecystic fat stranding to be common positive findings on abdominal computed tomography. However, such findings are only detected in a subset of patients with liver derangement and hence such investigations should be used very selectively.The American Association for the Study of Liver Diseases (AASLD) recommends the consideration of causes unrelated  to COVID-19 and other causes  for any liver test abnormalities.Liver functions should be monitored in COVID-19 patients at admission and during hospitalization.Those with preexisting liver disease, the elderly, or individuals who consume high amounts of alcohol or are obese should be monitored closely. The AASLD recommends the consideration of causes unrelated to COVID-19  and other causes  for any observed liver test abnormalities. Patients receiving immunosuppressive medications (liver transplant recipients or those with autoimmune hepatitis) should be managed as they were prior to the pandemic. Those with chronic HBV should continue their treatment and in HCV patients without decompensated cirrhosis treatment may be delayed. Hepatocellular carcinoma should be treated without delay.Some preexisting liver diseases are risk factors for poorer prognosis in COVID-19. Preexisting liver disease increases ACE2 expression on hepatocytes (observed in murine and human studies) and may thus increase the hepatic tropism of SARS-CoV-2.Patients with NAFLD have a higher risk of COVID-19 progression (6.6% versus 44.7%) and a higher likelihood of abnormal LFTs (70% versus 11.1%).These patients are more susceptible to SARS-CoV-2 infections, owing to their immunocompromised status. Increased disease severity and complications lead to higher mortality. A multicenter study of 50 cirrhosis patients with COVID-19 found a 30-day mortality rate of 34%.Patients with hepatocellular carcinoma (HCC) often need to visit a hospital for their treatment (chemotherapy and or immunotherapy) and thus need to be managed and monitored carefully. They have a higher risk of getting hospital-associated COVID-19, especially those who underwent surgery or received systemic treatment in the prior month. Post-epatectomy liver failure (PHLF) is a life-threatening situation following hepatectomy. Increased inflammation during COVID-19 may predispose the patient to PHLF. Secretion of IL-6 after hepatectomy and during PHLF, may further increase inflammation during COVID-19.Globally, there are over 250 million people living with HBV infection.,Thus, it is important to study the clinical characteristics of COVID-19 patients with preexisting HBV infection. These patients tend to have a more severe form of COVID-19.Both alcohol-associated liver disease (ALD) and alcohol use disorders (AUD) have been affected during the COVID-19 pandemic. Economic and social stresses resulting from the pandemic increased alcohol consumption in some individuals and delays in care have led to increased mortality from alcohol-associated hepatitis.A meta-analysis comparing 1522 COVID-19-infected liver transplant (LT) patients and around 240,000 COVID-19-infected non-LT patients showed similar mortality rates.––,,,,,Surgical services, especially routine surgeries for both benign and malignant conditions, have been affected worldwide during the COVID-19 pandemic.Liver transplantation has been affected due to COVID-19 infection worldwide.In Italy, though liver transplantation was carried out during COVID-19, a 25% reduction in procured organs was observed during the first 4 weeks of the outbreak.,,Drug–drug interaction is a problem associated with LT during the COVID-19 era. Patients who underwent LT are usually poly-medicated mainly with immunosuppressive drugs. Since a number of drugs used in COVID-19 have only been recently authorized, monitoring for potential drug interactions is important.Patients with CLD (predominantly cirrhosis), hepatobiliary malignancies, candidates for liver transplantation, and immunosuppressed individualsA limitation of this review is that the majority of studies are observational and have small numbers of subjects making it difficult to provide more definitive conclusions. It is possible that subtle liver findings were not documented (and thus underestimated) during the early part of the pandemic. Well-conducted studies from different regions of the world would help expand the evidence base and provide better answers to the many questions at hand. Ours is a broad overview of the main reported hepatic manifestations in COVID-19 and their management. A more comprehensive and detailed profile of specific aspects should emerge as more data are published from different countries.In conclusion, liver involvement is observed in COVID-19 patients and may influence disease prognosis and outcomes. The factors that may contribute to liver involvement in COVID-19 include direct viral cytopathic effects, exaggerated immune responses, hypoxia-induced changes, vascular changes due to coagulopathy, endothelitis, cardiac congestion from right heart failure, and drug-induced liver injury. Further clinical and laboratory studies should help ascertain more details on the potential mechanisms of SARS-CoV-2 infections and the liver. The COVID-19 vaccines should be administered as early as possible to patients with CLD and a third dose of the vaccine needs to be considered for LT recipients.


Imaging plays a crucial role in the accurate staging of prostate cancer. Prostate-specific membrane antigen (PSMA) is overexpressed in prostate cancer cells, and targeting the PSMA protein for diagnostic purposes has become of great clinical value. Another valuable feature of PSMA is its opportunity to serve as a target for delivering radionuclide therapy to cancer cells. PSMA-ligands can be labeled with various radionuclides, such as alpha and beta-emitters. This review offers an overview of the literature on recent developments in nuclear medicine regarding PSMA in prostate cancer diagnostics and targeted radionuclide therapy.177Lu), to deliver high radiation doses to tumor cells locally. Treatment with 177Lu-PSMA RLT has demonstrated a superior overall survival rate within randomized clinical trials as compared to routine clinical care in patients with metastatic castration-resistant prostate cancer (mCRPC). The current review provides an overview of the literature regarding recent developments in nuclear medicine related to PSMA-targeted PET imaging and Theranostics.Targeting the prostate-specific membrane antigen (PSMA) protein has become of great clinical value in prostate cancer (PCa) care. PSMA positron emission tomography/computed tomography (PET/CT) is increasingly used in initial staging and restaging at biochemical recurrence in patients with PCa, where it has shown superior detection rates compared to previous imaging modalities. Apart from targeting PSMA for diagnostic purposes, there is a growing interest in developing ligands to target the PSMA-protein for radioligand therapy (RLT). PSMA-based RLT is a novel treatment that couples a PSMA-antibody to  radionuclide, such as Lutetium-177 ( Prostate cancer (PCa) is the second-most common malignancy worldwide, and it is the fifth leading cause of cancer-related mortality among men . When deCurrently, imaging plays a pivotal role in assessing the disease extent, particularly through targeting the prostate-specific membrane antigen (PSMA) . PSMA is225Ac), Lead-212) or beta-emitting radionuclides ), with both having different characteristics in terms of physics and radiobiology . Al. Al10]. 177Lu-PSMA-617 . 225Ac-PSMA-617 . In total, 63% of patients had a PSA decline of more than 50%, and 87% had any PSA response. Remarkably, five patients (13%) showed a response for over two years. Unfortunately, four patients (10%) dropped out of this study because of (severe) side effects (xerostomia), and five patients (13%) terminated treatment due to lack of response following the first cycle [225Ac-PSMA-617 . A total of 82% of patients had any PSA response in this cohort, and 70% had a PSA decline of >50%. Grades I and II xerostomia were reported in 85% of the patients, not leading to treatment discontinuation [The most commonly used alpha-emitter for PSMA-ligand treatment is see also . A retrost cycle . Sathekginuation . 225Ac-PSMA-617 could benefit patients who did not respond to prior 177Lu-PSMA-RLT. Several studies included patients previously treated with 177Lu-PSMA-RLT. Yadav et al. prospectively enrolled 28 men with mCRPC to receive 225Ac-PSMA-617 treatment (median of three cycles). A total of 54% of these had prior exposure to 177Lu-PSMA therapy. After the first treatment cycle, 25% of the patients had a PSA decline of ≥50%, which increased to 39% at the end of follow-up. Any PSA decline was found in 78.6%. Patients’ refractory to 177Lu-PSMA less frequently showed a PSA decline of ≥50% than patients with no history of 177Lu-PSMA therapy (26.6% versus 53.8%). Half of the patients reported fatigue and 29% xerostomia (grade I/II) as AE [225Ac-PSMA-617 was offered every eight weeks (median of two cycles) to 26 patients with mCRPC who progressed after a median of four cycles of 177Lu-PSMA treatment. In 88% of the patients, any PSA decline was described, and 65% had a PSA decline of ≥50%. Grade I/II xerostomia was observed in all patients, leading to study discontinuation in six patients (23%). The reported hematological AE (grade III/IV) were thrombocytopenia (19%), leucopenia (27%), and anemia (35%) [225Ac-PSMA RLT is sparse due to the limited availability of 225Ac [I) as AE . In the ia (35%) . Althougof 225Ac .In recent years, PSMA PET has gained an increasingly important role in both initial diagnosis and at the biochemical recurrence of disease in patients with prostate cancer. In addition, PSMA PET/CT is being used more frequently during follow-up of the disease to assess treatment response. Aside from targeting the PSMA protein for diagnostic purposes, PSMA may also be a target for combined diagnostics and therapeutic purposes, the Theranostics approach. PSMA radioligand therapy has shown to be an effective and safe therapeutic option for patients with metastatic castration-resistant prostate cancer. Its oncological effect is currently being investigated in patients presenting with metastatic hormone-sensitive prostate cancer.


Whole-body positron emission tomography/computed tomography (PET/CT) using Subsequently, the patient underwent chemotherapy with two cycles of escalated BEACOPP, resulting in CT-based partial response. Therefore, two cycles of ABVD and local radiotherapy were added in curative intent.While the formerly affected lymph nodes showed a complete metabolic response at end-of-treatment PET/CT (Deauville score: 2), newly enlarged mediastinal and bilateral hilar lymph nodes with increased FDG-uptake as well as several hypermetabolic bone lesions were detected . A biopsy from a tracer-avid lesion in the anterior superior iliac spine was obtained. Histology revealed only non-caseating epitheloid cells and giant cells  adjacent to blood vessels , indicative of sarcoidosis. Since bronchoalveolar lavage and endobronchial ultrasound-guided transbronchial needle aspiration did not reveal malignant cells either, no further therapy was initiated. Due to the absence of clinical symptoms, a sarcoid-like reaction was favored over actual sarcoidosis as the possible diagnosis.After 3 months, follow-up PET/CT showed almost complete remission of the lymphadenopathy and bone lesions (Deauville score: 2) supporting the hypothesis of a tumor-related sarcoid-like reaction.Sarcoid-like reactions are observed in up to 13.8% of HL patients and should be taken into account as a differential diagnosis for hilar and/or mediastinal lymphadenopathy in patients with a history of malignancy [


Atherosclerotic ischemic coronary artery disease (CAD) is a significant community health challenge and the principal cause of morbidity and mortality in both developed and developing countries for all ethnic groups. The progressive chronic coronary atherosclerosis is the main underlying cause of CAD. Although enormous progress occurred in the last three decades in the management of cardiovascular diseases, the prevalence of CAD continues to increase worldwide, indicating the need for discovery of deeper molecular insights of CAD mechanisms, biomarkers, and innovative therapeutic targets. Recently, several research groups established that microRNAs essentially regulate various cardiovascular development and functions, and a deregulated cardiac enriched microRNA profile plays a vital role in the pathogenesis of CAD and its biological aging. Numerous studies established that over- or downregulation of a single miRNA gene by ago-miRNA or anti-miRNA is enough to modify the CAD disease process, significantly prevent age-dependent cardiac cell death, and markedly improve cardiac function. In the light of more recent experimental and clinical evidences, we briefly reviewed and discussed the involvement of miRNAs in CAD and their possible diagnostic/therapeutic values. Moreover, we also focused on the role of miRNAs in the initiation and progression of the atherosclerosis plaque as the strongest risk factor for CAD. Ischemic coronary artery disease (CAD) is the major public health problem in both developed and developing countries for both genders in all human races. Coronary heart disease (CHD) is responsible for one in seven deaths in the USA, killing over 366,800 people per year. Almost every forty seconds, a USA citizen will have a heart attack, and in the United Kingdom, 1 in 3 men and 1 in 4 women die from acute myocardial infarction (AMI) .Although the mortality rates from CAD in Asian countries are comparatively less than those in the western communities, recently, the number of CAD patients significantly increased due to an increased rate of dyslipidemia, overweight, type-2 diabetes, high blood pressure, and population aging. Around 80% of people who die of CHD are ≥65 years of age. Lifestyle and environmental agents such as unbalanced diets, high consumption of cholesterol-containing fast foods, preserved food with excess sodium chloride, hypovitaminosis D, lack of regular physical exercise, and air pollution are other risk factors –5. MoreoAccumulating evidence reveals that microinterfering/inhibitory ribonucleic acids  could be worth pursuing for new alternative diagnostic and/or treatment strategies for CAD. MicroRNAs are very small noncoding RNAs of about ~22 nucleotide size that regulate target gene expression through posttranscription mechanisms, mainly translational repression and/or mRNA destabilization. It has been accredited that microRNAs are playing essential roles in all human physiological functions including cell cycling, aging, and cellular apoptosis that affect stem cell homeostasis, angiogenesis, and immune function. In this concern, a single miRNA can target several mRNAs. Therefore, the dysregulated biogenesis and homeostasis of miRNAs in tissues have been linked to various diseases, particularly cardiovascular disorders in which they have been examined. The latter include myocardial infarction, remodeling after myocardial infarction, heart failure, dilated cardiomyopathy, cardiac hypertrophy, arrhythmias, atrial fibrillation, valvular heart disease, hypertension, atherosclerosis, and peripheral artery disease. Recently, several clinical research groups acknowledged that miRNAs are detectable in different body fluids including blood plasma and serum. They are extraordinarily protected from degradation by plasma ribonucleases (RNAases) as they are mostly carried out encapsulated within microvesicles –11. The Several excellent studies recognized that miRNAs are critically engaged in various cardiopathophysiological processes and that they have significant diagnostic, prognostic, and therapeutic applications for cardiovascular diseases , 26–30. We carefully consulted various publications regarding microRNAs and CAD up to January 2021 through searching of different important scientific databases such as microRNA target databases , Scopus, MEDLINE, PubMed Central, and Embase. CAD and microRNAs, IHD and circulating microRNAs, stable angina and microRNAs, unstable angina and microRNAs, acute coronary syndrome (ACS) and microRNAs, acute myocardial infarction (AMI) and microRNAs, role of microRNAs/miR in atherosclerosis, and therapeutic advantages of microRNAs in CAD were used as keywords during searching the databases. In addition, manual searches were also conducted for related bibliographies from the retrieved articles and very recent review reports.Caenorhabditis elegans. They also discovered lin-4 gene, which was known to control the timing of C. elegans larval development by suppressing the lin-14 gene. They also discovered two small RNAs that regulate the timing of C. elegans development in 2000. Whereas the first-to-second larval stage transition requires lin-4 miRNA, the late larval stage-to-mature nematode transition requires let-7 miRNA. The lin-4 and let-7 miRNAs are homologous to each other, but they are complementary to 3′-untranslated sequences of specific protein-coding genes and negatively controlled the expression-coding genes. Similarly, let-7 miRNA and other small temporally orchestrated miRNAs have the potential to be involved in the regulation of the progressive developmental stages of all organisms including humans , 4645, 4Moreover, NGS is also able to detect unknown miRNAs which the other two generally used miRNA methods (RT-qPCR and microarray) are incapable of doing. Therefore, the NGS technique has been widely used for gene regulation research and profiling of miRNAs in diagnosis, prognosis, and drug development  45, 46], 4645, 4Atherosclerosis is a chronic inflammatory disease of the arterial wall caused by endothelial injury and subendothelial accumulation of lipid and extracellular matrix proteins and calcium, particularly at sites of disturbed blood flow that finally form atherosclerotic plaque . SeveralAccumulating research studies over the last 3 decades recognized that miRNAs play an essential role in regulating pathways controlling lipid homeostasis. Therefore, they are significantly involved in the molecular mechanism of initiation and progression of pathophysiological processes of coronary atherosclerotic plaques. MicroRNAs presented them as promising new diagnostic, therapeutic, and prognostic targets. Normal lipid homeostasis is essential to maintain cellular integrity. Deregulated LDL and HDL metabolism/levels both in the cell and in the circulation are directly linked to various atherosclerotic disease processes. Recent clinical and preclinical studies have identified that microRNAs control LDL and HDL abundance and function and significantly regulate plasma lipoprotein levels. Generation and excretion of various lipoproteins are usually maintained by the hepatocytes. Several liver-associated miRNAs  have been well established to functionally regulate lipoprotein metabolism. Therefore, the altered homeostasis of these miRNAs is critically involved in the production of dyslipidemia . MoreoveκB (NF-κB) activity in endothelial cells and markedly suppressed leukocyte recruitment and reduced atherosclerotic lesion formation in mice [κB-linked dependent proteins that include tissue factor, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. Therefore, the anti-inflammatory microRNA-181b is a potential target for the prevention of thrombus formation [A very recent study noticed that microRNA-30c-5p significantly protected human aortic endothelial cells from oxidized low-density lipoprotein-associated plaque macrophage caspase-1-mediated pyroptotic cell death via downregulation of the NLRP3 protein expression through the forkhead box O3 (FOXO3) pathway. This could be a new treatment approach for atherosclerotic diseases . Angiopo in mice . Moreoveormation . The invChanges in the levels of the endothelium-specific microRNA-126, microRNA-17, and microRNA-92a and the vascular smooth muscle-specific microRNA-145, along with the inflammation-linked microRNA-155, were inversely associated with atherosclerosis plaque formation, while being positively correlated with plaque necrotic and necrolipidic tissue content, and the expression levels were markedly reduced in CAD patients. Therefore, augmentation of these miRNAs by systemic delivery of mimic miRNAs was dramatically reduced by atherosclerosis plaque progression through modulating their target mRNAs , 60. A rApoE−/− mouse models, while the mimic expression of miR-124 remarkably reduced anti-inflammatory cytokines and proinflammatory cytokines and inhibited macrophage apoptosis through the suppression of p38, suggesting that overexpression of miR-124 may be considered a promising treatment for atherosclerosis and CAD patients [The expressions of miR-21, miR-92a, and miR-99a were significantly upregulated, whereas miR-let-7f, miR-1, and miR-22 were markedly downregulated in human advanced coronary atherosclerotic plaques. Inhibition or overexpression of these miRNAs significantly reduced coronary atherosclerotic plaque progression and rupture . Very repatients . The antβ-hydroxy, β-methylglutaryl-CoA (HMG-CoA) reductase, and cytochrome P450 3A and inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) to lower LDL uptake via suppressing LDL receptors [Recent reports showed that changes in the expression of microRNAs are closely linked with the therapeutic actions of statins and effectively reduced atherosclerosis plaque formation/rupture through direct or indirect regulation of eceptors . Statinseceptors . Though A prospective therapeutic benefit of miRNAs is that they target multiple genes involved in the same pathological pathway process. In contrast, conventional therapies mainly target a single protein, leaving the others unaffected and potentially able to maintain the pathological mechanism. Currently, tools for such a new therapeutic approach utilize microRNA mimics (miR mimics), overexpression, anti-miRNAs (anti-miRs), or inhibitors. In this concern, the anti-miR approach is more appropriate to favor gene suppression, whereas miR mimics are utilized for the upregulation of gene expression. Up to date, the only miRNA-based therapy that reached the clinical application is a Locked Nucleic Acid- (LNA-) modified anti-miR directed against the liver-specific miR-122. The latter is necessary for the replication of hepatitis C virus (HCV) where it binds to two adjacent sequences close to the 5′-end of the HCV genome to guide the RISC-like molecule containing Ago2 binding. Subcutaneous administration of anti-miR-122 at 3-7 mg/kg body weight in chronic hepatitis C genotype 1 virus-infected patients for 5 weeks in a phase II clinical trial showed evidently reduced HCV RNA expression—even in some patients, no HCV RNA was detected up to 14 weeks of follow-up following the treatment. Furthermore, primary safety and the adverse effect profile were excellent. However, the use of anti-miRs in the cardiovascular medicine is still in the preclinical stage , 66.A clinical research found that microRNA-34 levels in the plasma of CAD patients were significantly higher than those in non-CAD subjects, whereas the levels of silent information regulator protein-1  were lower in CAD patients compared with non-CAD subjects. After treatment with the lipid-lowering agent atorvastatin for eight months in CAD patients, the plasma level of miR-34a was significantly reduced while the SIRT1 level was dramatically increased. Therefore, through inhibiting miR-34a, atorvastatin was suggested to play an important protective role for endothelial function in CAD patients . MicroRNIt is well established that endothelial progenitor cells (EPCs) and vascular endothelial growth factors (VEGF) are essential components for angiogenesis, and circulatory VEGF concentrations are negatively associated with CAD development. Plasma levels of VEGF levels and angiogenic activities were markedly reduced while concentrations of miR-23 and miR-361-5p were significantly elevated in EPCs from CAD patients , 72. On Serum levels of each of microRNA-499a-3p and microRNA-135b-5p were 4.7- and 3.9-times, respectively, higher in CAD groups than in normal healthy volunteer subjects. It was also found that the high levels of these two miRNAs could jointly promote ECs and vascular smooth muscle cell (VSMC) proliferation and migration by repressing the myocyte enhancer factor 2C (MEF2C) gene expression. This presents microRNA-499a-3p and microRNA-135b-5p as potential therapeutic targets for atherosclerotic CAD patients .β1 (AP2B1), and downregulation of miR-502 also ameliorates myocardial insult [β (HADHB), ATP-citrate lyase (ACLY), protein kinase AMP-activated catalytic subunit α1 (PRKAA1), and acetyl-CoA carboxylase α (ACACA). They orchestrate normalization of dyslipidemia and lower CAD risk [γ-catenin. Moreover, miR-939 controls angiogenesis and is a new treatment option for CAD [Myogenic miR-502 suppresses the autophagy process and plays an atheropreventive role in CAD by directly targeting the ras oncogene-related GTPase Rab1b and adaptor-related protein complex 2 subunit l insult . Rayner CAD risk . The blo for CAD . Relativ for CAD .  Table Utilizing several targets, microRNA-126 significantly protects cardiomyocytes against injury. EC-derived apoptotic bodies are produced during atherosclerosis and convey paracrine alarm signals to recipient vascular cells. The latter are induced to produce CXCL12, a process tightly regulated with the EC-enriched microRNA-126. Moreover, systemic delivery of microRNA-126 reduced atherosclerosis plaque, recruited more hematopoietic stem and progenitor cells expressing stem cell antigen 1, and stabilized atherosclerotic plaque in various mouse models. These actions culminate into a significant prevention of atherosclerotic CAD . After bEstablishment of coronary collateral circulation enhances circulation in ischemic cardiac tissue and reduces cardiac mortality. In such a state, levels of circulatory microRNA-146 were noticed to be high in a positive correlation with the extent of coronary collaterals. In addition, miR-146 positively correlates levels of vascular endothelial growth factor A (VEGF-A) with a potential favorable therapeutic effect in CAD patients . In a twExtensive heart remodeling with fibroblasts happens during myocardium injury that significantly reduces heart function. There are a number of cardiomyocyte-specific microRNAs that regulate the biology of cardiac fibroblasts and its transformation. Adult human cardiac tissue has a very low capacity to regenerate after cardiac tissue injury. The Eulalio research group injected human-microRNA-590 and human-microRNA-199a into myocardial infarction mice. They reported marked enhancement of cardiomyocyte proliferation and an outstanding repair of injured cardiac tissue. This resulted in almost complete recovery of cardiac functional parameters in both neonatal and adult infarcted mice. These two miRs executed their cardiac regeneration therapeutic actions through their target genes, Homer1, homeodomain-only protein X (HOPX), and chloride intracellular channel 5 (Clic5) , 86.α, and ephrin A3 (EFNA3) genes. These effects nominate miR-210 as a promising novel therapeutic target of AMI [MicroRNA-210 is highly expressed in normoxic HL-1 cardiomyocytes of live mice compared with the hypoxic cells. MicroRNA-210 upregulates several angiogenic factors, inhibits caspase activity and apoptosis, significantly improves left ventricular ejection fraction rate, and improves the survival rate in a mouse AMI model. For that, it regulates protein tyrosine phosphatase 1B (PTP1B), hypoxia-inducible factor- (HIF-) 1t of AMI , 88.Senescence is a major nonmodifiable risk factor for different types of cardiovascular pathology and contributes significantly bad prognosis in patients with AMI. The aging heart has a high expression rate of miR-34a, and its inhibition significantly reduces the age-associated cardiomyocyte cell death and fibrosis following AMI. This improves the recovery of myocardial function through the regulation of contractile function of the aging cardiac muscle and that of the post-AMI patients. Protein phosphatase 1 regulatory subunit 10 (PPP1R10) is the target gene for miR-34a . VasculoWhen AMI is induced in neonatal and adult mouse hearts by ligation of coronary artery, the expression of miR-34a was increased 7 days later only in adult but not in neonatal hearts. miR-34a regulates cell cycle and cell death through the modulation of its target genes Bcl2, cyclin D1, and Sirt1. Delivery of an anti-miR-34a to heart tissues diminished such post-AMI miR-34a upregulation and significantly improved post-AMI remodeling . Circulaγ-lyase (CSE) expression and hydrogen sulfide (H2S) production and aggravated hypoxic cardiomyocyte injury. Oppositely, silencing miR-30 prevents the hypoxic cardiomyocyte injury by increasing CSE and H2S levels. Systemic administration of antisense LNA-miR-30 inhibitor increased H2S and CSE levels simultaneously with a noticeable decrease in cardiac infarct size and cell death and a markedly enhanced cardiac function in response to AMI [Hullinger et al. demonstrated that systemic administration of the inhibitory microRNA-15 in AMI murine and swine models significantly decreased the cardiomyocyte infarct region, prevented heart remodeling, and improved heart function . Decrease to AMI .β1 also happened. Increases in miR-101a/b expression reduce collagen production and proliferation of rat neonatal cardiac fibroblasts. Cotransfection with antisense inhibitors of miR-101a/b abrogated these effects. Four weeks after adenovirus-mediated overexpression of miR-101a in rats with chronic MI, there was noticeable improvement in cardiac function [In the aging heart with MI, interstitial cardiac fibrosis is the major cause for deterioration of heart function. After coronary artery ligation in rats, the expression of miR-101a/b in the peri-infarct area is decreased. Overexpression of miR-101a decreased the expression of c-Fos, at protein and mRNA levels. A consequent reduction in transforming growth factor- (TGF-) function .+ progenitor cells in patients with STEMI. In particular, miR-378 through controlling CD34+ progenitor cells in vivo presents a new endogenous repair mechanism for AMI [The increases in cardiac endothelial-specific microRNA-24 following acute heart attack is correlated with enhanced apoptosis of cardiac ECs and reduced endothelial new capillary formation. MicroRNA-24 works through modulating the activities of p21-activated kinase 4 (PAK4) and EC transcription factor GATA2. Blocking miR-24 reduces infarct size that showed reduced EC apoptosis and enhanced vascularity with a consequent preservation of cardiac performance and improved survival after AMI in mice . Cardiac for AMI . In the  for AMI .H9c2 cardiomyocytes exposed to hypoxia show high miR-122 expression, and knockdown of microRNA-122 enhances the viability of these cells and reverses the apoptotic effect of hypoxia through modulating the PTEN/PI3K/AKT and cell autophagy pathways . Chun etLi et al. reported a higher expression of microRNA-93 in CAD and AMI patients, a mouse AMI model, and cultured cardiac tissue exposed to hypoxia for 2 hrs. Its role was evidently protective since upregulation of microRNA-93 decreases remodeling with increased vascularity and polarization of the macrophage that caused significant improvement in cardiac activity. On the contrary, knockdown of microRNA-93 significantly aggravated cardiac remodeling. This caused increased left ventricular systolic and diastolic diameter and a reduced ejection fraction rate .α, interleukin- (IL-) 1, and IL-17 signaling. A recent report demonstrated that miRNA-146 knockdown significantly reduced cardiac apoptosis and improved cardiac function in an AMI rat model [κB inflammatory response, apoptosis, and fibrosis. To inhibit TLR4/NF-κB, miR-146a targets mRNA of early growth response factor 1 (EGR1) and prevents its posttranscriptional expression (EGR1) [β1 pathway [miRNA-146 regulates tumor necrosis factor- (TNF-) at model . When hyn (EGR1) . Researcn (EGR1) . It has  pathway .Our recent study demonstrated that miR-375 levels were noticeably downregulated in AMI patients and HR-exposed H9c2 cells, whereas overexpression of miR-375 markedly protected cardiac cell death by inhibiting the expression of caspase-3 via regulating the NLK target, suggesting a potential therapeutic target for AMI patient . The rolThe exciting world of miRNA-based research and its potential clinical applications is developing very rapidly. To date, most of the studies dissected the pathogenetic, diagnostic, and prognostic values of miRNAs in cardiovascular diseases. Multicenter larger-scale animal experiments as well as human clinical trials are necessary to establish the therapeutic value of microRNAs as cardiovascular drug targets and/or drugs.Despite the many reasons to be enthusiastic for miRNAs as a new class of molecular targets or drugs for the treatment of CHD, there are several obstacles that need to be overcome before the clinical application of miRNAs. Although various microRNAs are critically involved in multisystem diseases including CHD, the molecular mechanism of some microRNAs/their mimics and anti-miRs are still largely unknown. Off-target effects for several miRNAs are known. Therefore, further studies on sensitivity, specificity, and potential complications of such effects for cardiac-specific microRNAs must be scrutinized before application.Since most studies systemically administered these cardiac-specific anti- and mimic miRNAs, they may have secondary effects on other tissues. Therefore, tissue-targeting studies are urgently needed to lower by-standard effects and induce efficiency. Additionally, larger scale multicenter animal studies are needed to confirm the efficacy, half-life, and reversibility of anti-/mimic microRNAs. The detection of miRNAs by the use of qRT-PCR or droplet digital PCR (ddPCR) techniques is time consuming, expensive, and even gives some false-positive reports. Therefore, it is necessary to develop faster and cost-effective modified PCR machines.However, currently, most of the advanced clinical laboratories and highly sensitive and specific, more accurate, comparatively cheaper and less time consuming, enormous potential, and breakthrough NGS techniques have been used for the detection of miRNAs as clinical biomarkers for diagnosis, prognosis, and potential pharmaceutical targets for the management of CAD patients , 112. Ho


Chimeric Antigen Receptor (CAR) T-cells represent a breakthrough in personalized cancer therapy. In this strategy, synthetic receptors comprised of antigen recognition, signaling, and costimulatory domains are used to reprogram T-cells to target tumor cells for destruction. Despite the success of this approach in refractory B-cell malignancies, optimal potency of CAR T-cell therapy for many other cancers, particularly solid tumors, has not been achieved. Factors such as T-cell exhaustion, lack of CAR T-cell persistence, cytokine-related toxicities, and bottlenecks in the manufacturing of autologous products have hampered the safety, effectiveness, and availability of this approach. With the ease and accessibility of CRISPR-Cas9-based gene editing, it is possible to address many of these limitations. Accordingly, current research efforts focus on precision engineering of CAR T-cells with conventional CRISPR-Cas9 systems or novel editors that can install desired genetic changes with or without introduction of a double-stranded break (DSB) into the genome. These tools and strategies can be directly applied to targeting negative regulators of T-cell function, directing therapeutic transgenes to specific genomic loci, and generating reproducibly safe and potent allogeneic universal CAR T-cell products for on-demand cancer immunotherapy. This review evaluates several of the ongoing and future directions of combining next-generation CRISPR-Cas9 gene editing with synthetic biology to optimize CAR T-cell therapy for future clinical trials toward the establishment of a new cancer treatment paradigm. The utility of CRISPR-Cas9 technology has led to a surge in applying genome editing approaches to combat a variety of genetic disorders and cancers. In these cases, inherited genetic diseases with known gene mutations can be corrected , 2. ThisCAR T-cell therapy has led to sustained remissions in populations of otherwise refractory patients and, more specifically, has demonstrated complete response rates of > 80–97% in certain B-cell malignancies such as acute lymphoblastic leukemia (ALL) , 6. BecaDespite these developments, the causes of unsuccessful CAR T-cell therapy are multifactorial and may not be addressed with synthetic biological improvements alone. Major limitations to successful CAR T-cell therapy occur both during the manufacturing process in vitro and during CAR T-cell proliferation in vivo. In many B-cell malignancies, complete responses are associated with robust CAR T-cell proliferation, with a clear advantage of long-term CAR T-cell persistence , 17–21. Additional hurdles are specific to solid tumors where CAR T-cells must traffic to tumor sites and surmount stromal barriers to infiltrate the tumor bed and elicit tumor-specific cytotoxicity. Even if trafficking and infiltration are successful, T-cells can become dysfunctional due to a toxic tumor microenvironment (TME) characterized by metabolic perturbations, the presence of inhibitory soluble factors and cytokines, as well as elevated frequencies of suppressive immune cells or tumor cells that secrete these mediators and overexpress ligands for negative immune checkpoint receptors. Malignant cells also undergo antigen loss or downregulation to escape and evade CAR T-cell recognition at the tumor site. Potential CAR T-cell therapy patients may also experience advanced disease progression during the prolonged process of gaining enough quality T-cells for manufacturing. The above issues cannot be addressed with a single approach. However, many of these challenges may be overcome by using site-specific genetic editing in combination with next-generation cellular engineering approaches. With the genesis of easily multiplexable precision genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) (CRISPR-Cas9) technology , there iThe emergence of CRISPR-Cas9 technology with its simplicity, flexibility, and effectiveness has considerably improved the process and time frame of gene editing. Briefly, the Cas9 DNA endonuclease enzyme can be directed to virtually any site in the genome to create a double-stranded DNA break. The cleavage region for Cas9 is selected based on a 20-base pair single guide RNA sequence (sgRNA) that directs Cas9 to the target DNA cut site, which possesses sequence complementarity to the sgRNA. This site is additionally specified by a protospacer adjacent motif (PAM) sequence within the target DNA downstream of the cleavage site, consisting of any variation of 5’-NGG. Together with the core sgRNA, there are additional structures and RNA features like loops and hairpins needed for active and stable complex formation with Cas9 as detailed in Fig. The conventional Cas9 protein consists of six domains, REC I, REC II, Bridge Helix, PAM-Interacting, HNH and RuvC , 35. RecDSB induction permits gene disruption and/or transgene insertion into target loci. However, there are risks associated with such breakage when it occurs at off-target sites, potentially introducing unintended genome alterations. Thus, precise control of when and where DSBs occur is beneficial for reducing the frequencies of unintended indels and translocations  and prevSeveral Cas9 enzyme variants have also been developed to overcome the disadvantages of DNA DSB cleavage. For example, Nickase Cas9 (nCas9) variants contain mutations in either the RuvC or HNH domain which render Cas9 capable of cleaving only the targeted or non-targeted DNA strand, respectively  Fig. 2bb2b. The In the process of base editing Fig. b, introdA major limitation of base editing is that it cannot be applied to introduce transversion mutations  as well as small deletions or insertions. To address this constraint, an alternative technology known as ‘prime editing’ was developed to facilitate all possible base-to-base conversions and targeted insertions or deletions without inducing DSBs . In thisdCas9 can be used to promote or attenuate transcriptional activity based on recruitment of transcriptional activators or repressors to specific sites in the processes of CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi), respectively –70 Fig.c. CRISPRNumerous factors often collectively prevent durable remissions following CAR T-cell therapy, including autologous CAR T-cell manufacturing issues, limited CAR T-cell expansion and/or persistence as well as various T-cell-intrinsic and -extrinsic resistance mechanisms Fig. a. RecentSeveral approaches have been taken to improve the survival and function of CAR T-cells, including optimization of costimulatory endodomains , preventGenetic strategies to modulate cytokine signaling during CAR T-cell activation and expansion have the potential to bolster antitumor activity, enhance T-cell persistence and/or reduced toxicity. CRISPR-Cas9 systems that incorporate transgene knock-in approaches have been exploited for this purpose. More specifically, CRISPR/Cas9-based gene editing combined with viral or non-viral DNA delivery permits simultaneous bi-allelic or sequential gene targeting to engineer T-cells with expression cassettes in a site-specific manner , 91 Figb. Using TRAC) locus, resulting in uniform CAR expression, reduced tonic signaling, decreased exhaustion and increased antitumor efficacy [TRAC also gives the added benefit of producing a potential universal product.Many current CAR T-cell manufacturing protocols involve ex vivo autologous T-cell expansions followed by transduction with a viral vector containing the chimeric receptor sequence . While lefficacy  Fig. 4bbTRAC locClinical development of novel CAR T-cell therapies is often hampered by the low yield and poor functionality of mature, autologous peripheral blood T-cells from many elderly and heavily-pretreated patients. A proposed solution is the procurement of healthy donor leukocytes to produce ‘universal’ T-cells with optimized in vivo persistence and antitumor potency. However, generating off-the-shelf CAR T-cell products is challenging because multiple genome edits within finite numbers of differentiated T-cells are needed to prevent alloreactivity and immunogenicity as well as potentiate robust tumor-specific activity. The problem has been further exacerbated by a gap in the development of renewable sources of precision-engineered T-cells, largely resulting from bottlenecks in manufacturing and facile multiplexed genetic ablation strategies.The primary challenge to overcome in the setting of allogenic products is the induction of graft-versus-host-disease (GvHD), where the endogenous donor T-cell receptor recognizes ‘non-self’ surface human leukocyte antigen (HLA) molecules in the patient, eliciting an immune reaction. Healthy donor allogeneic CAR T-cells can be derived from patients’ previous HLA-matched hematopoietic stem cell transplant (HSCT) donor or from gene-edited cells which have been modified to allow them to be given to non-HLA matched subjects. There is increasing enthusiasm for the use of so-called ‘third party CAR T-cells’ . An off-the-shelf product would allow one to start with T-cells from healthy donors to create large quantities of universal tumor-specific T-cells that could be used in any patient without the need for HLA matching. This strategy would reduce costs, speed drug administration, and make the T-cell products accessible to lymphopenic and critically ill cancer patients that often do not have sufficient numbers of healthy T-cells for treatment. As proof-of-concept for this approach, CAR T-cells derived from healthy unrelated donors have conferred anti-leukemic efficacy in children and adults with relapsed ALL .Ideally, universal T-cell products should have 3 essential characteristics: 1) resistance to recognition/rejection by host natural killer (NK) cells and T-cells, 2) optimized for potency, persistence and safety, and 3) available as on-demand therapeutics. Without the ability to incorporate such features, large-scale and reproducible low-cost production of high quality, safely engineered CAR T-cells cannot be achieved and the promise of cell therapies will remain elusive to vast populations of cancer patients around the world..The T-cell receptor α constant (TRAC) locus has been extensively studied as an ideal target for both gene knockout and CAR knock-in , 91. PlaFig. b, c.PDCD1, PD-1) and those encoding death receptors such as CD95/Fas to prevent TME-mediated inhibition or apoptosis [TRAC, PDCD1 and B2M triple knockout CAR T-cells have demonstrated robust anti-tumor function in in vitro and in vivo models incorporating CD19 and prostate stem cell antigen (PSCA)-directed CARs [TRAC, TRBC and PDCD1 knockout) T-cells with a transgenic TCR specific for tumor-associated antigen NY-ESO-1 in patients with myeloma and sarcoma [PDCD1 was also demonstrated to be feasible in the setting of clinical product scalability, and this product proved to be safe in a phase I trial for advanced non-small-cell lung cancer patients [Successful multiplex CRISPR-Cas9 editing of CAR T-cells has been done with targeting inhibitory genes . As these therapies require several genetic edits within mature T-cells, inefficiencies and combinatorial stochasticity in production result in a final product that typically contains many different populations of cells with variable editing combination efficiencies that need to be evaluated and optimized to increase efficacy [The challenges of producing a universal CAR T-cell product from finite numbers of mature, fully differentiated healthy donor T-cells are compounded by the anticipated engineering demands, particularly for solid tumors in which multiple CRISPR-Cas9-based gene edits are needed to be combined with CAR-based recognition to achieve safety and efficacy. For example, in the setting of allogeneic T-cell therapies involving checkpoint blockade, expression of a transgene encoding a synthetic antigen receptor is required, in addition to knockout of several inhibitory molecules  as well as T-cell receptor (TCR) alpha and beta constant regions  amenable to genetic modification and differentiation into mature tumor-targeted T-cells. iPSCs are obtained from adult somatic cells by inducing expression of a combination of transcription factors  that transform cells into a pluripotent state , 110  genes as well as genes encoding inhibitory molecules such as PD-1 to create unique pools of allogenic T-cell products with superior antitumor functionality, as has been previously seen in edited primary T-cells directed against glioblastoma and acute lymphoblastic leukemia in vivo models , 115. IdThe clinical efficacy of CAR T-cell technology has been proven in the setting of human cancer. However, there are major limitations to accessing this technology. Currently, it is a bespoke product made for individual patients, and therefore, the time to manufacture can prevent access as can the cost. Further, the T-cells used as starting material from patients are likely to have developed cancer associated T-cell dysfunction, which may not be reversible. Advances in CRISPR-Cas9-based genome editing will help address the several current unmet needs in CAR T-cell therapy. Such advances may involve DSB-free genome editing methods like base and prime editing to allow precise and controllable genetic modification in a robust manner. In addition, CRISPR-Cas9-induced multiplex knockout of inhibitory molecules potentiates enhanced CAR T-cell expansion and persistence that may allow for circumvention of T-cell-intrinsic as well as -extrinsic resistance mechanisms operative in both hematopoietic and non-hematopoietic malignancies. Targeted knock-in approaches also have the potential to fine-tune transgene insertion during CAR T-cell engineering to generate effective and potent cell products with temporally regulated effector functions. Finally, procurement of normal donor leukocytes or iPSCs to produce a ‘universal’ CAR T-cell product that can be CRISPR-Cas9 engineered to overcome certain histocompatibility barriers and with enhanced persistence/antitumor function will significantly improve the manufacturing of cellular immunotherapies and therapeutic durability. Development of next-generation therapies with CAR T-cells, involving innovative genetic editing with CRISPR-Cas9 technology, will ultimately improve affordability and reduce off-target toxicities while enhancing antitumor effectiveness.


Varying degrees of Prlr recombination in the hypothalamus with these Cre lines left open the possibility that central actions of prolactin could contribute to the pregnancy-induced changes in glucose homeostasis. Targeted deletion of Prlr specifically from the forebrain, including areas of expression induced by Pdx-Cre and Rip-cre, had no effect on pregnancy-induced adaptations in glucose homeostasis. These data emphasize the pancreas as the direct target of prolactin/placental lactogen action in driving adaptive changes in glucose homeostasis during pregnancy.Adaptive changes in glucose homeostasis during pregnancy require proliferation of insulin-secreting beta-cells in the pancreas, together with increased sensitivity for glucose-stimulated insulin secretion. Increased concentrations of maternal prolactin/placental lactogen contribute to these changes, but the site of action remains uncertain. Use of Cre-lox technology has generated pancreas-specific prolactin receptor (Prlr) knockouts that demonstrate the development of a gestational diabetic like state. However, many Cre-lines for the pancreas also express Cre in the hypothalamus and prolactin could act centrally to modulate glucose homeostasis. The aim of the current study was to examine the relative contribution of prolactin action in the pancreas and brain to these pregnancy-induced adaptations in glucose regulation. Deletion of prolactin receptor (Prlr) from the pancreas using Pdx-cre or Rip-cre led to impaired glucose tolerance and increased non-fasting blood glucose levels during pregnancy. Prlr During pregnancy, glucose regulation undergoes major adaptations to ensure a consistent supply of glucose to the fetus . GlucoseIn vitro studies demonstrate that prolactin increases insulin secretion and beta-cell proliferation  have “ectopic” Cre expression in the brain and there has been no assessment in either study examining the potential contribution of deletion of Prlr in the brain.The expansion of the beta-cells occurs in anticipation of increased insulin demand, and as such is a hormone-induced adaptation to prepare for future changes in physiological requirements. The anterior pituitary hormone prolactin and its pregnancy-specific homologue placental lactogen, which also acts through the prolactin receptor (Prlr), play key roles in mediating the adaptations of the beta-cells during pregnancy –6. Thereferation , and in feration . Furtherferation , due to feration . More referation , 12. WhiThere is extensive evidence that, in addition to the beta-cells, the Rip-Cre transgene drives recombination of floxed genes in the brain, particularly in parts of the hypothalamus involved in regulation of glucose homeostasis –15. IndeThe aim of this study is to address this deficit, investigating the contribution of prolactin/placental lactogen action in the pancreas and the hypothalamus in the pregnancy-induced adaptations of glucose homeostasis. The hypothalamus plays a major role in metabolic homeostasis, including regulation of insulin secretion and whole-body glucose metabolism –20. We hPrlrlox/lox mice . To generate mice with Prlr deleted from the pancreas and beta-cells, female Prlrlox/lox mice were crossed with male mice expressing Cre-recombinase (Cre) in Rip containing cells 25Mgn/J, obtained from The Jackson Laboratory #003573) or pdx containing cells 6Tuv/J, obtained from The Jackson Laboratory #014647) respectively. As both of these Cre lines also express Cre in the forebrain to varying degrees, we also generated mice with Prlr deleted specifically from the forebrain by crossing female Prlrlox/lox mice with mice in which Cre expression was driven by the neuron-specific calcium/calmodulin dependent protein kinase II-alpha . This study only used mice in their first pregnancy.Experiments used female mice aged 8-12 weeks old, housed in a temperature- and lighting- controlled environment  and allowed access to food and water Mice were anesthetized with sodium pentobarbital and perfused transcardially with 4% paraformaldehyde. Brains and pancreata were removed and post-fixed for one hour in 4% paraformaldehyde, then cryoprotected in 30% sucrose at 4°C overnight. For brains, coronal sections (30µm) were cut through the hypothalamus at the level of the arcuate nucleus and sections were then processed for free-floating chromogen immunohistochemistry for GFP using an anti-GFP antibody  as previously described . For thein situ hybridization assessment of Prlr mRNA, mice were perfused with 2% paraformaldehyde and pancreata were collected and cryoprotected in 30% sucrose at 4°C overnight. Sections (16 µm) were collected through the pancreas and underwent in situ hybridization using an S35-labelled RNA probe targeted to the mRNA for the long form of the Prlr as described previously . Glucose tolerance tests (GTT) and glucose stimulated insulin secretion (GSIS) experiments were performed in mice after an overnight fast. For GTTs, basal blood glucose levels were measured at -30 minutes and then immediately before mice received an i.p. injection of glucose  and then at various time points after injection. For GSIS, mice were injected with either saline or glucose  and were decapitated 10 minutes later. Trunk blood was collected, spun down and plasma was stored at -20°C until insulin concentration was determined using an ultra-sensitive mouse insulin ELISA . For non-fasting blood glucose levels, mice were rapidly decapitated and blood glucose was measured immediately in trunk blood, which was then collected, and processed as described above for determination of insulin concentration.Pancreata were collected for immunohistochemistry as described above and stored at -80°C until further processing. Pancreatic tissue was embedded in paraffin and cut in 4 µm thick sections. Pancreatic sections used for quantitative analysis were systematically selected after a random start and three sections (200 µm apart) per pancreas were included in analysis. To identify beta-cells, pancreatic sections underwent chromogen immunohistochemistry for insulin using an anti-insulin antibody . Gills Hematoxylin was used as a counter stain. To determine beta-cell fraction, each 4 µm section (n=3 per mouse) was imaged using an Olympus Montaging Microscope, and 10-30 non-overlapping fields of view were photographed per section. These images were adjacent to one another and covered the entire pancreatic area on the slide. The images were put together into a montage using Autopano Pro software and then analyzed using ImageJ software to determine the area of total pancreatic tissue and the area stained for insulin. Beta-cell fraction was determined by dividing total area stained for insulin by total area of pancreatic tissue in the section.Prlrlox/lox/Rip-Cre mice and controls (both Cre-negative/flox-positive (Prlrlox/lox) and Cre-positive/flox-negative (Rip-Cre) mice) underwent a GTT on day 15 or day 16 of pregnancy. Non-fasting blood glucose levels were determined in another cohort of pregnant Prlrlox/lox/Rip-Cre mice and controls. GFP immunohistochemistry was carried out on pancreas and brain sections from a third cohort of pregnant Prlrlox/lox/Rip-Cre mice and Prlrlox/lox mice. Body weight (day 1 to day 18 pregnancy) and food intake (day 9 to day 18 pregnancy) was monitored in another cohort of Prlrlox/lox/Rip-Cre mice and controls (Cre-negative/flox-positive only).Pregnant female Prlrlox/lox), Prlrlox/lox/Rip-Cre, Prlrlox/lox/Pdx-Cre and Prlrlox/lox/CamK-Cre mice underwent immunohistochemistry to detect GFP positive cells as described above. Immunohistochemistry was examined and photographed using an Olympus AX70 research light microscope . GFP-positive cells in the arcuate nucleus [-1.34 to -2.30 from bregma (The extent of Rip-Cre-mediated recombination in the brain was such that we decided to compare with both a forebrain-specific Cre (CamK-Cre) and a second pancreas-specific Cre (Pdx-Cre). Sections collected from the hypothalamus of pregnant and non-pregnant female control (m bregma ] were coPrlrlox/lox), Prlrlox/lox/Pdx-Cre and Prlrlox/lox/CamK-Cre mice underwent a GTT both prior to mating and on day 15/16 of pregnancy. A second cohort of female control (Prlrlox/lox), Prlrlox/lox/Pdx-Cre and Prlrlox/lox/CamK-Cre mice were perfused either as virgin controls or on day 16 of pregnancy and pancreata were collected for immunohistochemistry for insulin. GSIS, non-fasting blood glucose levels and insulin concentrations were assessed in further cohorts of non-pregnant and pregnant Prlrlox/lox and Prlrlox/lox/Pdx-Cre mice. Non-fasting plasma insulin concentration were measured in trunk blood samples from a second cohort of day 16/17 pregnant Prlrlox/lox/CamK-Cre and control (Prlrlox/lox) mice. The pancreata of pregnant Prlrlox/lox and Prlrlox/lox/Pdx-Cre mice were collected and processed for immunohistochemistry for either GFP or pSTAT5 to characterize the deletion of Prlr in the pancreas of this transgenic mouse. Pancreas tissue was collected from another group of pregnant Prlrlox/lox and Prlrlox/lox/Pdx-Cre mice for in situ hybridization for Prlr mRNA.Control . GFP-positive cell counts, GTT area under the curve, fasting and non-fasting glucose concentrations were analyzed by Student’s t-test or one-way ANOVA if there were more than two groups followed by Prlrlox/lox/Rip-cre (Prlr gene. No positive GFP staining was observed in the pancreas from control (Prlrlox/lox) mice (Using a Prlr reporter line (Prlr-iCre X ROSA26-CAGS-τGFP), GFP staining indicative of Prlr expression was localized to islets within the pancreas (Prlrlox/lox/Rip-Cre mice had elevated non-fasting blood glucose concentrations compared to either pregnant Rip-cre or Prlrlox/lox mice (one-way ANOVA P=0.0036)   had significantly lower body weight than Cre-negative Prlrlox/lox mice (controls) at the same stage of pregnancy  (Prlrlox/lox/Rip-Cre and control (Prlrlox/lox) mice increased food intake during the second half of pregnancy  (Prlrlox/lox/Rip-Cre had lower food consumption compared to controls (Effect of genotype P=0.0029) (Prlrlox/lox/Rip-Cre mice (Prlrlox/lox/Rip-Cre and control Prlrlox/lox mice (Effect of genotype P=0.5516)  , while fasted blood glucose levels were similar =6.341) =5.332) (Prlrlox/lox/CamK-Cre had significantly lower fasting blood glucose than control (Prlrlox/lox) mice =15.27)   nor Prlrlox/lox/CamK-Cre (Sidak’s multiple comparison test Prlrlox/lox/CamK-Cre non-pregnant vs pregnant P=0.438), suggesting that this effect was driven by prolactin action directly on the beta cells (Prlrlox/lox/CamK-Cre mice had similar beta-cell fraction as pregnant control mice (Sidak’s multiple comparison test P=0.5727), confirming that this action of prolactin was not mediated by Prlr in the forebrain =7.373, Sidak’s multiple comparison test control non-pregnant vs pregnant P=0.0022) Figure 6Increasing concentrations of prolactin/placental lactogen play a key role in the adaptations of the beta-cell to pregnancy , 2. IdenPrlrlox/lox/Rip-Cre mice, our data replicated earlier findings that showed impaired glucose regulation during pregnancy in mice with deletion of Prlr from beta-cells  mice. Non-pregnant Prlrlox/lox/CamK-Cre mice are hyperprolactinemic since the removal of Prlr from tuberoinfundibular dopamine neurons in the arcuate nucleus leads to impaired negative feedback of prolactin secretion  and Maurice Wilkins Centre for Molecular Biodiscovery provided grant funding to cover salaries and working expenses for this project.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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