Adoptive cell therapy (ACT), based on treatment with autologous tumor infiltrating lymphocyte (TIL)-derived or genetically modified chimeric antigen receptor (CAR) T cells, has become a potentially curative therapy for subgroups of patients with melanoma and hematological malignancies. To further improve response rates, and to broaden the applicability of ACT to more types of solid malignancies, it is necessary to explore and define strategies that can be used as adjuvant treatments to ACT. Stimulation of endogenous dendritic cells (DCs) alongside ACT can be used to promote epitope spreading and thereby decrease the risk of tumor escape due to target antigen downregulation, which is a common cause of disease relapse in initially responsive ACT treated patients. Addition of checkpoint blockade to ACT and DC stimulation might further enhance response rates by counteracting an eventual inactivation of infused and endogenously primed tumor-reactive T cells. This review will outline and discuss therapeutic strategies that can be utilized to engage endogenous DCs alongside ACT and checkpoint blockade, to strengthen the anti-tumor immune response. CheckpoBesides PD-1 and CTLA-4, other co-inhibitory receptors that influence anti-tumor immune responses have been discovered. In particular Lag-3 and T cell immunoreceptor with Ig and ITIM domains (TIGIT) are of interest in this respect and blockade of these receptors are being explored in clinical trials.Lag-3 is upregulated by activated CD4+ and CD8+ T cells, it structurally resembles the CD4 co-receptor and binds MHC class II molecules with high affinity . Lag-3 iTIGIT is expressed by activated T cells, NK cells memory T cells and a subset of Tregs. TIGIT binds to CD155 and CD112 that are expressed by antigen-presenting cells (APCs), T cells and other non-hematopoietic cells, including tumor cells. Engagement of TIGIT to CD155 on DCs has been shown to inhibit IL-12p40 production and instead induce IL-10 production, thereby rendering DCs tolerogenic rather than inflammatory . In thisThe use of PD-1 and CTLA-4 blockers has been shown to increase the durable response rates and overall survival of responding patients when administered as monotherapies. However, the clinical benefits of the checkpoint blockers as monotherapies are limited by relatively low objective response rates, e.g., 10–16% for ipilimimab and 30–40% for nivolumab and pembrolizumab in metastatic melanoma patients –40. The Clinical studies evaluating the efficacy of Lag-3 and TIGIT inhibition are still in early phases and despite encouraging results, the majority of patients still fail to respond to PD-1 or CTLA-4 blockade as mono- or combination therapy. This lack of sustained therapeutic responses can partly be attributed to the actions of other immune suppressive mechanisms in the tumor, e.g., secretion of amino acid depleting enzymes , TGF-β or IL-10 . For thiIn this review, we will discuss strategies, that have been described pre-clinically and clinically, to improve the efficacy of ACT and immune checkpoint blockade by engaging endogenous DCs to support the effector functions of infused and endogenous tumor-reactive T cells. Our primary focus will be on CD8+ T cells and the subsets of DCs that that are essential for stimulating a tumor-reactive T cell response, i.e. cross-presenting conventional type 1 dendritic cells (cDC1s) and type I interferon (IFN) producing plasmacytoid DCs (pDCs).In order to become activated and gain an effector phenotype, naïve T cells must to be introduced to their cognate antigen presented by activated APCs in the context of MHC molecules. DCs are the most effective type of APCs and they are indispensable for initiating an anti-tumor response . The outDue to the pivotal role of the DC activation status on T cell priming, tumor infiltration of immature or otherwise functionally deficient DCs presents an obstacle for efficient ACT. Non-activated tumor-associated DCs can contribute to the induction of a tolerable environment that both limits the effector functions of infused T cells and affects priming of endogenous T cells . It is tcDCs are the most effective types of APCs and they are dedicated to the continuous sampling of antigen. Precursors to cDCs are released from the bone marrow where after they enter lymphoid organs or other peripheral sites and develop into migratory or resident DC subsets . The migIn a cancer immunotherapy context, the important function of intra-tumoural CD103+ cDC1s has been well-described pre-clinically and via their function as the primary producers of the T cell recruiting chemokine CXCL10, they are known to be essential for tumor-homing of effector T cells . FurtherpDCs are unique in their ability to rapidly produce vast amounts of type I IFNs (IFN-α/β) in response to TLR stimulation . In contThe ability of pDCs to produce vast amounts of type I IFNs upon activation is important for induction of cancer immunity , 75. A pBecause engagement of DCs alongside ACT is pivotal for broadening the tumor-reactive response, it is relevant to explore treatment strategies that can be used to stimulate DC activation and/or antigen presentation. In this review, the focus will be on pre-conditioning with chemotherapeutics or radiotherapy, peptide or DC-based vaccination, stimulation with toll-like receptor (TLR) agonists or stimulator of interferon genes (STING) agonists, and CD40 ligation as adjuvant treatments to ACT and checkpoint inhibition.It is well-established that ACT following lymphodepletion can enhance anti-tumor reactivity in murine and human hosts –83, and in vitro. These results were in line with previous findings demonstrating that lymphodepletion with a single dose of CPX induced expansion of immature DCs that could be detected in the peripheral blood 8–16 days post treatment  antigen improved the persistence of CD19 CAR T cells modified to recognize EBV in relapsed pediatric acute lymphoblastic leukemia patients . In thisStimulating TAA-specific adoptively transferred T cells with post-infusion TAA vaccination has also shown promising results in pre-clinical and clinical trials. Recently, a pre-clinical study found that the persistence and activity of infused CAR-T cells could be enhanced by a tumor antigen vaccination . ResultsOther clinical trials have reported encouraging results related to the objective clinical response of cancer patients treated with the ACT and post-infusion TAA vaccination. In line with these findings, a clinical phase II trial reported an improved 5 year recurrence-free and prolonged overall survival of patients with invasive hepatocellular carcinoma who received a post-operative DC based vaccine combined with ACT . This trRecent research has provided evidence of the existence of so-called “anti-regulatory T cells” that recognize immunosuppressive molecules , 111. StThe ability to combine ACT with vaccination, either with viral antigens or TAAs, represents an interesting approach to enhance the efficacy of ACT. The persistence of infused T cells have been linked to therapeutic efficacy , 119 andA well-established way to activate DCs is by stimulation with TLR agonists. TLRs are a type of pattern recognition receptors (PRRs) that comprise a group of endosomal and plasma-membrane associated proteins expressed on DCs and other innate immune cells, such as macrophages . TLRs arTLR stimulation can be used in combination with tumor antigen delivery to ensure activation of antigen presenting DCs. TLR stimulation can however also be used as an adjuvant treatment to other cancer therapies that induce tumor antigen release, such as RT. The combination of TLR stimulation and RT has been explored in pre-clinical studies with encouraging results. A study reported that intravenous administration of the TLR7 agonist resiquimod (R848) in combination with RT lead to clearance of established tumors in murine lymphoma models . The treTo the best of our knowledge, no clinical trials evaluating the effect of enhancing ACT efficacy by TLR stimulation have so far been completed. Pre-clinical studies have however described the use of TLR stimulation to augment ACT with encouraging results. A recent study reported that the administration of the TLR4 agonist Lipopolysaccharid (LPS) could augment the tumor-reactivity of adoptively transferred pmel-1 CD8+ T cells in mice with established B16.F10 tumors . AdminisAlthough TLR agonists are effective vaccine adjuvants, the overall beneficial effect of TLR stimulation as cancer immunotherapy is debated. The concept is complicated by the fact that TLRs are not only expressed by immune cells but also by cancer cells  and TLR −/− mice and that STING pathway activation and IFN-β production correlated with DNA detection in tumor infiltrating DCs  receptor family and is expressed by a range of different cell types including DCs, B cells, platelets and non-hematopoietic cells such as endothelial cells, fibroblasts and some types of cancer cells . LigatioThe ability of CD40 stimulation to enhance the efficacy of ACT has been evaluated in different pre-clinical models. Recently, a study showed that lymphodepleting WBI in combination with CD40 stimulation enhanced the accumulation of infused T cells in a murine pancreatic tumor model . Here itThe use of CD40 agonists has also been tested as cancer immunotherapy in clinical settings. A paper recently summarized the long-term outcomes of a phase I study of agonistic CD40 antibody and CTLA-4 treatment of metastatic melanoma patients . Here itin vitro expansion step of the process involves activating and stimulating T cell proliferation to reach a large number of cells. This activation and stimulation of T cells induce a stepwise differentiation process, which ultimately leads to generation of T cells with a terminally differentiated and short-lived effector phenotype that is suboptimal for ACT (The post-infusion performance of adoptively transferred T cells is critically dependent on several factors, in particular the composition of the TME that can favor or suppress the tumor-reactivity of the T cells. Studies have also linked the post-infusion performance to the differentiation status of the adoptively transferred T cells, with less differentiated T cells, such as naïve or stem cell memory T cells, having greater capacity for expansion and persistence after transfer. The T cells used for ACT typically have a more differentiated effector T cell phenotype at the time of infusion, because the  for ACT . Because for ACT , 12, eff for ACT , 153, sm for ACT , 155 or  for ACT , 157 to  for ACT , 159. ThIn addition to the examples given above, another emerging strategy is to modify the T cell product prior to infusion, thereby “arming” the T cells with the ability to engage endogenous DCs. Recent pre-clinical studies have investigated ACT with genetically modified T cells expressing membrane-anchored IL-12 , which iin vivo expansion, persistence and polyfunctionality of infused T cells. Engagement of activated DCs alongside ACT has also been associated with improved tumor control and prolonged survival in pre-clinical models. The addition of checkpoint blockade alongside ACT and DC stimulation can be utilized to counteract an eventual inactivation of tumor-reactive T cells and might provide an additional synergistic effect. The combination of ACT, treatments that activates and/or induces antigen presentation of DCs and checkpoint blockade therefore represents an interesting therapeutic strategy that potentially can enhance the efficacy and broaden the applicability of ACT in the future.The concept of ACT has been manifested as a promising therapeutic option for a subgroup of patients with melanoma and hematological malignancies. To further improve the therapeutic efficacy of ACT, and to broaden the application to other types of solid cancers, it is necessary to expand our knowledge on factors that can enhance the post-infusion persistence and functionality of transferred T cells. Engagement of DCs, as an adjuvant therapy to ACT, can stimulate a broader tumor-reactive response by priming endogenous T cells and facilitate post-infusion priming of adoptively transferred T cells. Accordingly, the combination of ACT and DC-activating treatments such as TLR or STING agonists, as well as CD40 stimulation and vaccination with viral or tumor antigens, has been found to have implications for the MH and DJ did the literature search and wrote the review. TA and MA revised and approved the final version of the review. All authors contributed to the article and approved the submitted version.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.


Approximately one half of patients develop ascites within 10 years of diagnosis of compensated cirrhosis. It is a poor prognostic indicator, with only 50% surviving beyond two years. Mortality worsens significantly to 20% to 50% at one year if the ascites becomes refractory to medical therapy. Pakistan has one of the highest prevalence of viral hepatitis in the world and patients with ascites secondary to liver cirrhosis make a major percentage of both inpatient and outpatient burden. Studies indicate that over 80% of patients admitted with ascites have liver cirrhosis as the cause.This expert opinion suggests proper assessment of patients with ascites in the presence of underlying cirrhosis. This expert opinion includes appropriate diagnosis and management of uncomplicated ascites, refractory ascites and complicated ascites  ascites, hepatorenal syndrome (HRS) and hyponatremia. The purpose behind this expert opinion is to help consultants, postgraduate trainees, medical officers and primary care physicians optimally manage their patients with cirrhosis and ascites in a resource constrained setting as is often encountered in a developing country like Pakistan. Approximately one half of patients develop ascites within 10 years of diagnosis of compensated cirrhosis.9The initial evaluation of ascites includes detailed history, physical examination, abdominal ultrasound, liver function tests including serum albumin and INR, renal function tests, serum electrolytes and spot urinary electrolytes. Ascitic fluid should be analyzed to calculate serum albumin to ascitic fluid albumin gradient (SAAG).Uncomplicated ascites is defined as ascites that is not infected and is not associated with refractoriness to conventional medical treatment, development of hyponatremia, spontaneous bacterial peritonitis, or hepatorenal syndrome.10Complicated ascites is defined as ascites with complications of one or more of the following: Spontaneous bacterial peritonitis (SBP) hepatorenal syndrome (HRS) and hyponatremia.10Ascites that cannot be mobilized with maximum dose of diuretics (furosemide 160 mg/day and spironolactone 400 mg/day) or the early recurrence (within 2 weeks) of which  cannot be satisfactorily prevented by medical therapy.12Ascites may be classified into three grades depending upon the quantity of free fluid in the peritoneal cavity. Choice of treatment depends upon this clinical quantification of fluid:14It is essential to carry out biochemical, microscopic and microbiologic analysis of ascitic fluid. Biochemistry includes total protein, albumin, LDH and glucose content of ascitic fluid. The total leucocyte count and differential leucocyte count (TLC and DLC) must be checked. Ascitic fluid ought to be sent in blood culture bottles for culture/sensitivity. Reagent strips for leucocyte esterase have been successfully used for bedside diagnosis of SBP in some studies.15It is important to rule out spontaneous bacterial peritonitis (SBP) if ascites in new in onset or worsening of ascites is noted. SBP should also be ruled out in those who get hospitalized with any complication of cirrhosis or for any other medical or surgical reason.All patients with ascites must undergo a complete history, examination and relevant investigations along with ascitic fluid analysis to calculate SAAG and to rule out spontaneous bacterial peritonitisAscitic fluid analysis is essential in cases of cirrhosis with new onset ascites, worsening ascites, or those admitted with other complications of cirrhosis.Treatment of moderate ascites is aimed at counteracting the increased renal reabsorption of sodium by restricting salt intake and increasing sodium excretion with diuretics.A diet with sodium content of less than 2g or 88mmol per day  must be stressed upon. This is roughly equal to one heaped teaspoon or two flat teaspoons of table salt. Diuretic dose can be increased stepwise every week, while maintaining a ratio of 40mg of furosemide for every 100mg of spironolactone. A maximum daily weight loss of 0.5kg/day is reasonable in the absence of pedal edema. There is no restriction to weight loss if massive pedal edema accompanies ascites.18Diuretics may require dose adjustment in summer months due to increased fluid losses. Diligent follow up of patients, every 1-2 weeks to begin with, is required as the dose of diuretics is stepped up for weight loss documentation, orthostatic hypotension, electrolyte imbalance, pre-renal azotemia, and signs of portosystemic encephalopathy as a result of diuretic overdose.19All patients with ascites should not be advised to restrict their fluid intake. Restriction of fluid intake should only be advised in the small minority who have documented serum sodium of below 125 mmol/L.19Spot urinary sodium to potassium ratio is a convenient alternative to 24-hour urinary sodium while assessing outpatient response to diuretic therapy. When this ratio is more than one, it suggests adequate diuresis.21Patients should be advised not to take more than 5 grams of table salt or 88 mmol/2g of sodium daily. (I-A)Diuretics should be given in a once daily dose (I-A)Diuretics should be introduced in a step-up dose at a ratio of 100:40 of spironolactone: furosemide(I-A)Weight, blood pressure, serum electrolytes, creatinine must be checked at every OPD visit along with examination for orthostatic hypotension and signs of portosystemic encephalopathy. (I-A)The frequency of outpatient visit should be once every one to four weeks depending upon the response to treatment(I-A)Spot urinary sodium to potassium ratio can be checked to assess response to diuresis. (II-A)Patients should be advised to stop diuretics if urinary sodium is < 30 mmol/day. (II-A)Diuretics should also be stopped if serum sodium is < 120 mmol/L (1-B)Regular use of intravenous furosemide should be best avoided. (I-B)Fluid restriction to 1000 ml/day is effective in increasing serum sodium concentration in only a minority of patients with hypervolemic hyponatremia, but may be effective in preventing a further reduction in serum sodium levels (1-A).Studies have shown that beta-blockers may increase mortality in patients with refractory ascites by causing hemodynamic instability.Angiotensin converting enzyme inhibitors and angiotensin receptor blockers are best avoided in patients with cirrhosis and ascites.It is best not to prescribe aminoglycosides, NSAIDs and other nephrotoxic drugs in patients with cirrhosis and ascites and should be used when other antibiotic or analgesics options are not available and under careful monitoring of renal function (II-A).27ParacentesisDiureticsLiver TransplantParacentesis is a procedure in which a needle or catheter is introduced into the peritoneal cavity to obtain ascitic fluid. It is either done to establish etiology of new-onset ascites or to rule out spontaneous bacterial peritonitis. It is also carried out for therapeutic purpose.30The left lower quadrant is the best site for a paracentesis. The anterior abdominal wall is thinner 3 cm above and 3 cm medial to the left anterior superior iliac spine. Most of the fluid gravitates at this site in comparison to the midline. This location also best avoids a distended cecum due to lactulose that is commonly used in patients with cirrhosis and the inferior epigastric arteries running in the rectus sheath. A 1.0 or 1.5 inch 20 or 22-gauge needle can be used for paracentesis. Patients with obesity may require a 3.5 inch needle. In those patients where fluid is difficult to localize, ultrasonography can be used for guidance. Paracentesis is best performed by a doctor who is trained for the procedure.33LVP ought to be followed by intravenous human albumin infusion to decrease the chances of PPCD. To prevent PPCD, 8g of 20% human albumin is transfused for each liter of ascitic fluid removed when a LVP is performed.Paracentesis of over 5 litres has been shown to be safe in critically ill patients with tense ascites. It improves respiratory function in such patients including those who are mechanically ventilated with minimal circulatory compromise.42Paracentesis is safe in patients with hepatorenal syndrome and tense ascites. In fact, increased intra-abdominal pressure may add to renal compromise. Paracentesis can be performed in such cases with albumin infusion and results in improvement in renal function.44Albumin is known to reduce PPCD but its use after an LVP has not shown any difference in clinical outcomes including encephalopathy, hyponatremia, readmission, renal impairment and death. However in cirrhotic patients with any infection, albumin use may significantly reduce sepsis related mortality and renal impairment.46Thrombocytopenia and coagulopathy are not contraindications to paracentesis. Platelets or fresh frozen plasma need not be used to correct derangements. (I-A)Large volume paracentesis followed by diuretics is appropriate treatment in patients with diuretic sensitive tense ascites (I-A)Up to 5 litres of fluid can be removed without colloid support (I-A).Large volume paracentesis should be carried out under cover of 8 g of albumin for each litre of ascitic fluid tapped. (I-A).Patients must have their discharge weight documented and should be called for first follow up preferably after a week. (I-A)Patients must not be subjected to regular paracentesis if adequate weight reduction is achieved with diuretics and salt intake restriction after initial paracentesis. (I-A)Fresh frozen plasma (FFP) should not be used as an alternative form of colloid instead of albumin for paracentesis (I-A)Paracentesis  can be safely instituted in patients with tense ascites and spontaneous bacterial peritonitis (I-A)Paracentesis may be safely done in critically ill patients including those on mechanically assisted ventilation (I-B)Paracentesis with albumin infusion is safe in patients with hepatorenal syndrome and tense ascites (I-B)An indwelling peritoneal catheter can be used to perform paracentesis for up to 3 days (II-C)Ascites that cannot be mobilized or the early recurrence of which  cannot be satisfactorily prevented by medical therapy.47Ascites that cannot be mobilized or the early recurrence of which cannot be prevented because of a lack of response to sodium restriction and permissible dose of diureticsAscites that cannot be mobilized or the early recurrence of which cannot be prevented because of the development of diuretic induced complications that preclude the use of an effective diuretic dosageTreatment duration: Patients must be on maximum permissible dose of diuretics i.e. upto spironolactone 400 mg/day and furosemide 160 mg/day for at least 1 week. The patient must adhere to less than 88 mmol/day of sodium in the diet.Lack of response: Mean weight loss of less than 0.8 kg over 4 days and urinary sodium output less than the sodium intake.10Early ascites recurrence: Reappearance of grade 2 or 3 ascites within 4 weeks of initial mobilization.14Diuretic-induced complications:Diuretic-induced hepatic encephalopathy is the development of encephalopathy in the absence of any other precipitating factorDiuretic-induced renal impairment is an increase of serum creatinine by >100% to a value >2 mg/dl (177 mmol/L) in patients with ascites responding to treatmentDiuretic-induced hyponatremia is defined as a decrease of serum sodium by >10 mmol/L from baseline to a serum sodium of <125 mmol/LDiuretic-induced hypo- or hyperkalemia is defined as a change in serum potassium to <3 mmol/L or >6 mmol/L despite appropriate measuresDilutional hyponatremiaHepatorenal syndromeSpontaneous bacterial peritonitisHepatic hydrothoraxSpontaneous bacterial empyemaUmbilical herniaLarge volume paracentesisTIPSPeritoneovenous shuntLiver transplantationVaptansAutomated Low Flow Ascites (ALFA) PumpSerial therapeutic paracenteses are helpful in control of ascites.7Patients requiring paracenteses of approximately 10 L or more, less than two weeks interval, are likely not following a salt restricted diet.53Serial paracenteses can safely be carried out on outpatient basis for patients with proven refractory ascites every two weeks (I-A)Diuretics should be stopped if urinary sodium is less than 30 mmol/day (II-A)A salt restricted diet of less than 2g of sodium equivalent to 88 mmol per day must be adhered to even in patients with refractory ascites (I-A)Recommendations for colloid replacement and safety of paracentesis remain the same as mentioned for management of tense ascites. (II-A)Beta blockers ought to be discontinued in patients with refractory ascites. (II-A)TIPS is a side-to-side portocaval shunt placed between the portal vein that has high pressure owing to portal hypertension, and hepatic vein with low pressure, to decompress the portal system. It is used in patients with refractory ascites and for patients with variceal hemorrhage that is poorly controlled by endoscopic methods.56Earlier studies done with non-polytetrafluoroethylene (PTFE) covered stents showed conflicting data as regards patient survival post TIPS. TIPS did show a better control of ascites and better prevention of hepatorenal syndrome. However, encephalopathy was higher in patients undergoing TIPS compared to those undergoing serial paracentesis.11PTFE covered stents work better than uncovered stents in terms of failure rates.57To undergo TIPS, the patient should not have cardiopulmonary disease and must have normal cardiac ejection fraction ~60%). Patients due to undergo TIPS must have a MELD score of 18 or below.%. Patien61TIPS is a feasible option in patients not suitable for or as a bridge to transplantation in centres where expertise is available (I-A).It is recommended that patients be evaluated for MELD score and cardiac ejection fraction before undergoing TIPS. (I-B).Elective TIPS cannot be encouraged in patients with severe liver failure, concomitant infection, worsening renal failure or cardiopulmonary disease (II-B)Only PTFE coated stents are preferred over uncovered stents due to lower shunt failure rates (I-A)Regular outpatient paracentesis is a feasible option in patients not suitable for TIPS or liver transplant (I-A)Diuretics must not be withheld following TIPS as TIPS may in fact convert diuretic refractory ascites to diuretic sensitive ascites (I-A)A peritoneo-venous shunt is a shunt that drains peritoneal fluid from the peritoneum into either the internal jugular vein or the superior vena cava.51The LeVeen shunt was surgically placed and is not commercially available anymore.51The Denver shunt is a modified from of LeVeen shunt used for management of hydrocephalus. It is placed in a subcutaneous tunnel between the peritoneal cavity and the internal jugular vein by interventional radiologists under local anesthesia.51Potential complications of the peritoneovenous shunt are shunt occlusion, infection, disseminated intravascular coagulation (DIC) manifesting as post-shunt coagulopathy, deep vein thrombosis, catheter breakage, and leaks.51Peritoneovenous shunt placement is recommended for patients not suitable for regular outpatient paracentesis, TIPS or liver transplantation, in centres where expertise for performing such procedures is available. (I-A)Peritoneovenous shunt placement is not required in diuretic refractory patients who can come for regular outpatient paracentesis. (II-B)Liver transplantation is a procedure in which the patient’s diseased liver is replaced by a whole healthy liver (Deceased donor) or part of a healthy liver of a willing donor (Living donor).The presence of refractory complicated ascites is one of the indications for liver transplantation in patients with decompensated cirrhosis.38Since survival after refractory ascites develops is measured in months, such patients should be advised liver transplantation (I-A)Arginine vasopressin (AVP) has a crucial role in regulation of water and sodium in the body. It acts via three receptor subtypes V1a, V1b, and V2 distributed throughout the body. Vaptans are nonpeptide vasopressin receptor antagonists (VRA). Tolvaptan and Conivaptan are examples.62Due to the ability to cause loss of excess water, the Vaptans have a role in correction of hyponatremia and fluid overload in patients with heart failure and cirrhosis. Some studies recommend the agent Tolvaptan .65There are concerns regarding the safety of such agents as regards side effects such as worsening of portal hypertension leading to variceal hemorrhage, hypernatremia, dehydration, deranged renal function, and central pontine demyelination.67Vaptans are newer agents but have potential side effects. The agent Tolvaptan may be used in cirrhotic patients with ascites and hyponatremia but patients need to be carefully monitored for side effects (III-C)There is no local evidence on use of vaptans in refractory asciteshttp://www.alfapump.com/alfapump).A surgically implanted pump that automatically transfers peritoneal fluid to the bladder has been undergoing trials for some time , infection, ascitic fluid leakage through wounds, bladder perforation and renal impairment.73Insertion of ALFA pump should only be performed in specialist centres where expertise to place the pump and to deal with its potential complications is available. Pump placement should currently be ideally performed in the setting of a controlled clinical trial (II-C)No local evidence is available on results of ALFA pumpTo compare 4 grams of albumin versus 8 grams of albumin per litre of fluid removed during large volume paracentesis (LVP) in prevention of post paracentesis circulatory dysfunction.To study the number of patients with cirrhosis and ascites who go on to develop refractory ascites.To compare Terlipressin with Albumin versus albumin alone during paracentesis to prevent post paracentesis circulatory dysfunction.To diagnose diuretic resistant ascites by the 80 mg IV furosemide challenge test.To ascertain benefit of combination of IV albumin and IV furosemide in hypo-albuminemic patients.To study the likelihood of spontaneous bacterial peritonitis in patients undergoing large volume paracentesis with use of indwelling catheter.To study the efficacy of regular albumin infusions as an adjunct to diuretics for improved survival.To study timing and benefits of discontinuation of diuretics in patients with clinically undetectable ascites.To study efficacy of eplenerone in patients with cirrhosis and refractory ascites.To study the effect of peritoneovenous shunts by interventional radiologists in the local scenario.>16 corresponds to higher incidence of SBP.90The incidence of SBP in cirrhotic patients in Pakistan averages 27-38 % of all patients admitted to hospitals, as noted in various studies.One and two-year survival rates following an episode of SBP are 30-40% and 20-30%, respectively.91Ascitic fluid should be sent for total and differential leukocyte counts, Gram stain and cultures with antibiotic sensitivity in all patients with cirrhosis who are admitted to hospital for any reason, regardless of the interval between current and last admission. This should be done even if recent admission’s ascitic fluid reports have ruled out SBP. This should be done prior to the administration of any antibiotics. Ascitic fluid samples must be transferred to culture bottles at the bedside and transferred for analysis to the laboratory within 30 minutes (I-A)Administration of antibiotics to cirrhotic patients immediately following admission should be discouraged and these should only be started once a sample has been obtained for leucocyte counts. (I-A)All patients with cirrhosis and ascites presenting with signs of infection such as fever, abdominal pain or encephalopathy should receive empirical antibiotic treatment for possible SBP after ascitic fluid sampling. (I-A)Cefotaxime intravenously at a dose of 2g 8 hourly remains the first line antibiotic for all cases of confirmed or suspected SBP.(I-A)Ceftriaxone I.V at a dose of 1g 12 hourly is an alternative to the above regime. It is cost effective and has been shown to effectively control SBP. (I-A)Switching to a different antibiotic may be done if the patient does not clinically improve and shows a non-significant reduction in leukocyte counts on a repeat ascitic fluid sample obtained 48 hours after the first sample. (I-B)Modification to antibiotic regime must preferably be done in light of culture and sensitivity reports. (I-B)All patients should receive 1.5 g albumin per kg body weight within 6 hours of diagnostic paracentesis and 1.0 g/kg on day 3. I.V albumin remains very expensive and lower doses of albumin need to be considered in light of new trials. (II-B)Proton pump inhibitors are massively over-prescribed  and they may contribute to the development of SBP in patients with cirrhosis and ascites. PPIs must be used only in patients with clear documented dyspepsia and/or reflux and their use must be short duration as and when symptoms develop. (I-B)Secondary bacterial peritonitis must be ruled out in patients with very high leucocyte counts. (I-B)In such cases, ascitic fluid must be analysed for protein, glucose, LDH, Gram Stain and CEA along with a contrast enhanced CT of the abdomen & pelvis area to rule-out/rule-in a focal source of infection. (I-B)Prophylaxis against SBP must be done in all cases of GI bleeding in patients with liver cirrhosis. (I-A)Prophylaxis against SBP in cases of GI bleeding in cirrhotic patients can be effectively done starting with 1g daily of ceftriaxone and switching to oral therapy after endoscopy. (I-B)Oral therapy in aforementioned scenario is continued to day 7. (I-B)Oral agents that can be used include ciprofloxacin 500mg BID, levofloxacin 500mg OD or norfloxacin 400mg twice daily. (I-A)Prophylaxis against SBP is also required for the following two groups:Patients who have had one episode of SBP: Such patients require prophylaxis till death or liver transplantation. (I-A)<1.5 g/dL along with renal dysfucntion  or advanced cirrhosis (Child score 9 and bilirubin 3). Such patients should preferably receive prophylaxis for one year. (I-A)Patients with ascitic fluid protein Prophylaxis against SBP can be done using once daily doses of ciprofloxacin. It is advisable to use different antibiotics for treatment and prophylaxis in the same patient (II-A)Intermittent dosing (such as once weekly) of antibiotics for prophylaxis is not recommended as it may result in emergence of resistant strains of bacteria. (II-C)To study 6-month mortality in patients with and without regular antibiotic prophylaxis against SBP.To study the efficacy of ofloxacin and Trimethoprim-Sulfamethoxazole in prophylaxis against SBP.To study the difference in yield of TLC/DLC in ascitic fluid collected in vial with EDTA as against a plain syringe in SBP.To study the decrease in TLC in ascitic fluid after first dose of antibiotic in patients diagnosed with SBP.Hepatorenal syndrome (HRS) is defined as the occurrence of renal failure in a patient with advanced liver disease, with portal hypertension and ascites, in the absence of an identifiable cause of renal failure.93Two studies carried out in tertiary care hospitals in Sindh, Pakistan, revealed a 15% incidence of hepatorenal syndrome in patients with cirrhosis.93Proposed mechanisms of HRS are splanchnic vasodilation which is accompanied by renal vasoconstriction.104There are two types of HRS. Hepatorenal syndrome type of acute kidney injury (HRS-AKI) formerly Type 1 HRS is a rapidly progressive renal failure in patients with advanced liver failure and is associated with poor prognosis. Hepatorenal syndrome type of chronic kidney disease (HRS type 2) is a slowly progressive renal failure in patients with cirrhosis and refractory ascites.106The diagnostic criteria for diagnosis of HRS-AKI have recently been revised by the International ascites club in 2015. The previous definition of an absolute reading of serum creatinine ≥1.5mg/dl for diagnosis of HRS was discarded based on knowledge that patients with cirrhosis have a decreased muscle mass and also have an impaired urea cycle. Moreover, milder derangements in renal function were being missed as a result of this cut-off serum creatinine level. The criteria for urine output was also removed since patients with cirrhosis tend to retain sodium and water as a result of renin-angiotensin-activation-system and tend to be oliguric despite having normal renal function.107The new definitions for AKI in cirrhosis are as follows108There is a retrospective study that diagnosed HRS-AKI on the basis of the current criteria and was able to pick HRS-AKI at stage 1 in 40% of in-hospital patients with cirrhosis and ascites. Those patients who had stage 1 AKI despite a serum creatinine less than 1.5mg/dl still had 3.5-fold 30 day mortality than those without AKIIn a study carried out at a tertiary care hospital in Islamabad, mortality was found to be higher in patients with advanced liver disease with renal failure as compared to those without renal failure (31% vs 4.5%). Acute kidney injury was the most common presentation and the most common etiologies of renal dysfucntion were infection and hypovolemia. Reversibility was higher with hypovolemia but greater number of deaths were seen in patients with HRS and sepsis.109The HRS-AKI is defined as International club of ascites (ICA) criteria AKI-ICA stage 2 or more while other causes of renal failure have been excluded. Thus other causes of AKI such as hypovolemia, shock, urinary tract obstruction, use of nephrotoxic medication and parenchymal diseases of kidney have been ruled out.109The box below gives the revised diagnostic criteria for HRS-AKIHRS-AKI fails to respond to volume expansion with IV albumin or withdrawal of diuretics105There are a few biomarkers that can be utilized for diagnosis of HRS-AKI and make it less of a diagnosis of exclusion. Urinary neutrophil gelatinase-associated lipocalin (NGAL) levels and urinary Interleukin-18 (IL-18) levels have shown a significant difference between each category of AKI.HRS type 2 is a slowly progressive form of renal failure where there is slowly progressive deterioration of renal function in patients who have decompensated cirrhosis and refractory ascites. Patients have oliguria and salt and water retention over the course of several months with a slow but steady decline in renal function. The rest of the criteria remain the same as for HRS-AKI.117Patients with cirrhosis and refractory ascites may have other causes of kidney injury and HRS type 2 is a diagnosis of exclusion. Other etiologies of CKD may complicate HRS type 2. It is therefore difficult to diagnose. The prognosis remains poor, albeit slightly better than HRS-AKI.118It is important to diagnose AKI in cirrhosis and to identify the possible etiology (I-A)There are two types of HRS. HRS-AKI and HRS type 2. (I-A)Before a diagnosis of HRS-AKI is reached, other etiologies that must be excluded are hypovolemia, shock, use of nephrotoxic medicines and agents and underlying renal parenchymal disease. (I-A)HRS-AKI is diagnosed by noticing even a slight rise in serum creatinine. Careful monitoring of renal function in patients with cirrhosis and ascites is crucial especially if they are hospitalized. (I-A)An effort must be made to identify the precipitating cause of HRS-AKI. To name a few, systemic bacterial infections, spontaneous bacterial peritonitis in particular, variceal bleed and use of non-selective beta blockers are common. (II-A)An early diagnosis of HRS-AKI is known to decrease mortality (I-II)Hallmark of HRS type 2 is a slow but constant decline in renal function and carries a poor prognosis. (I-A)Urinary biomarkers may be used to predict HRS-AKI and can be of help in differentiating HRS from other causes of AKI. (II-B)Once diagnosed, HRS-AKI should be treated promptly to prevent further deterioration of renal function. It is important to assess for other complications of cirrhosis and to monitor vital signs, urine output, serum electrolytes, renal and liver function tests. Such patients should ideally be managed in an intensive treatment unit and an effort should be made to measure the central venous pressure.Once HRS-AKI has been established, patients are best managed in the ICU setting with careful monitoring of vital signs and urine output (I-A)Diuretics must be stopped and intravenous fluid should be monitored to avoid fluid overload and dilutional hyponatremia. (I-A)Once HRS has been diagnosed, a trial of furosemide may be given to maintain urine output in patients who show signs of fluid overload. Spironolactone, however, is best avoided to prevent life threatening hyperkalemia (I-A)Since beta blockers have been identified as potential triggers in a recent study, it is better to stop them in patients who might have been taking them earlier as prophylaxis against variceal bleed. (II-B)All patients with HRS-AKI should undergo screening for sepsis. Thus blood, urine and ascitic fluid cultures must be sent at first presentation. Antibiotics should be continued even if no signs of bacterial infection are found. (I-A)Large volume paracentesis is best avoided in an acute setting but it can be safely carried out to make patients with tense ascites more comfortable. (I-A)Vasoconstrictor agents such as terlipressin, midodrine/octreotide and norepinephrine have been evaluated in combination with albumin for treatment of HRS-AKI. These agents bring about vasoconstriction in the splanchnic vasculature and increase the mean arterial pressure. Albumin is of benefit in patients with sepsis owing to its scavenging, anti-oxidant as well as endothelial stabilizing effect apart from its additional benefit of volume expansion.117Terlipressin is initiated at a dose of 1mg 4 to 6 hourly. Dose can go up to 2mg every 4-6 hours if a reduction of more than 25% is not seen in serum creatinine on day 3. Albumin is started at 1g per kg body weight going upto 100g on day 1 and then continued at 40g/day.A combination of albumin with octreotide at a dose of 200 microgram subcutaneously three times a day and oral at a titrated dose of upto 12.5mg three times a day has been of benefit in a few studies from the US and Europe.124Noradrenaline at a dose of 0.5-3mg/h is infused continuously dose is gradually increased to achieve a raise in arterial blood pressure and improvement in renal parameters in HRS-AKI.131These therapies can also be of short term benefit in patients with HRS-CKD but HRS tends to recur once vasoconstrictor therapy is withdrawn.133Very few studies are available on prevention of HRS. Pentoxifylline showed some promise at a dose of 400mg three times a day in alcoholic hepatitis.82For prevention of HRS-AKI and HRS-CKD, albumin should be administered at a dose of 8g for every litre removed when large volume paracentesis (>5L) is carried out.117TIPS has been of benefit in only a handful of patients with HRS-AKI. A strong statement cannot therefore be made on it being used as an option in HRS-AKI.It, however, has been of benefit in patients with HRS-CKD in view of improvement in renal function as well as in control of ascites.Randomized–controlled trials have not shown a survival benefit of renal replacement therapy  or extracorporeal liver support (ELS) for HRS-AKI and HRS-CKD. Continuous venovenous hemodialysis use may, however, be of some benefit in patients who are hemodynamically unstable.Owing to the poor prognosis of HRS-AKI and HRS-CKD, these patients must be referred for liver transplantation as soon as possible.146Pharmacologic therapy of terlipressin combined with albumin should be administered in patients with HRS-AKI (I-A)Dose of terlipressin can be administered in an incremental manner upto a maximum of 2mg/4 h if there is less than 25% reduction of serum creatinine on day 3 (I-A)For those patients who do not respond, treatment should be discontinued within 14 days (I-A)Patients on terlipressin must be carefully watched for cardiac dysrrhythmias and for signs of ischemia of extremities and splanchnic vasculature. (I-A)Alternatives to terlipressin and albumin are combination of albumin with octreotide and midodrine. Unfortunately, midodrine is not available in Pakistan (II-B)A combination of norepinephrine and albumin is also an alternative but very little international and local data is available to support these therapies (II-B)There is insufficient data to prove that TIPS can be used in the setting of HRS-AKI not responding to pharmacologic therapy( II-B)Renal replacement therapy may be useful only as a bridge to liver transplantation. (II-B)HRS-AKI patients must be considered for liver transplantation (I-A)Terlipressin and albumin is of temporary benefit only in patients with HRS type 2 since HRS tends to recur once therapy is withdrawn (I-A)Patients with HRS-CKD should be evaluated for TIPS which might improve renal function and control ascites.(I-A)Patients must undergo cardiac evaluation, renal and liver function for MELD before undergoing TIPS (I-A)Liver transplantation improves survival in patients with HRS-CKD (I-A)Patients with HRS type 2 who require renal support for >12 weeks must be considered for combined kidney and liver transplantation. (I-A) Prevention of HRSPentoxifylline has proven to be of some patients with HRS in the setting of alcoholic hepatitis (II-A)Early diagnosis and management of SBP with antibiotics and albumin is known to prevent HRS (I-A)Large volume paracentesis is best done under cover of albumin (I-A)To study the incidence of HRS-AKI and HRS-CKD in light of the new International Club Ascites definitionsTo study the efficacy of lower dose of albumin in combination with terlipressin for management of HRS-AKITo study the response of combination of albumin and norepinephrine in the setting of HRS-AKITo ascertain the utility of urinary biomarkers as predictors of HRS-AKI and its diagnosisTo study efficacy of TIPS as treatment for HRS-AKI not responding to pharmacologic therapyTo study efficacy and adverse event profile for TIPS for management of HRS type 2BA: Revised manuscript and gave final approval, AS: Wrote a section of manuscriptAA: Wrote a section of manuscript, BFZ: Revised and did final editing of manuscript, literature search & updated citations and references, ZA: Wrote a section of manuscriptLK: Wrote a section of manuscript, JIF: Wrote a section of manuscript, MS: Wrote a section of manuscript, AAN: Wrote a section of manuscript, AAC: Wrote a section of manuscript, ZYH: Wrote a section of manuscript, MS: Wrote a section of manuscript.


This cohort study evaluates the association between the coronavirus disease 2019 (COVID-19) stay-at-home orders and suicide deaths in Massachusetts. While increases in psychiatric stressors during the coronavirus disease 2019 (COVID-19) pandemic have been reported, it is presently unknown whether suicide rates similarly changed during stay-at-home periods.STROBE) reporting guideline was followed.In this cohort study, we assembled suicide death data for persons aged 10 years and older from the Massachusetts Department of Health Registry of Vital Records and Statistics from January 2015 through May 2020. This study was not subject to institutional review board approval or the requirement for informed consent because it used public data. The Strengthening the Reporting of Observational Studies in Epidemiology  and SAS version 9.4 (SAS Institute).We used autoregressive integrated moving average (ARIMA) and seasonal ARIMA models to analyze suicide deaths in Massachusetts, with yearly population as the covariate. We used the Akaike information criterion (AIC) to select the best model. We plotted suicide deaths during each month of 2020 for which adequate data exist (January to May). To be conservative, we plotted an alternative scenario in which the number of deaths pending investigation by the state medical examiner exceeding the corresponding monthly averages from 2015 to 2019 were ascribed to suicide. Incident rates and rate ratios for the stay-at-home period  and the corresponding period in 2019 were determined. Statistical significance was set at During the pandemic period, the incident rate for suicide deaths in Massachusetts was 0.67  per 100 000 person-months vs 0.80  per 100 000 person-months during the corresponding period in 2019 . Because data for 2019 and 2020 are preliminary, a sensitivity analysis including all deaths still pending final cause adjudication as of November 14, 2020, was performed. The addition of the 47 deaths pending cause determination occurring from March through May 2020 and the 32 cases still pending determination from the corresponding period in 2019 did not change these findings. The conservative assumption that all pending investigations for March to May were suicides yielded an incident rate of 0.89  deaths per 100 000 person-months for 2020 and 0.95  deaths per 100 000 person-months for 2019 .The number of suicide deaths during the stay-at-home period did not deviate from projected expectations using either preliminary data or an alternate scenario in which deaths pending investigation that exceeded the average remaining number of deaths that occurred during the corresponding period in 2015 to 2019 were ascribed to suicide . Deceden3 Early in the outbreak, social distancing–created stressors may have been offset by a sense of shared purpose in flattening the curve. There were efforts at bolstering connections through video platforms; anticipation regarding governmental support, including unemployment benefits and stimulus aid; and mental health awareness campaigns about the risks of isolation, loneliness, and despair that could accompany the public health imperative of physical distancing. One limitation to this study is its reliance on cause-of-death adjudication. However, unlike other causes of death, every suicide is investigated by a medical examiner, rendering resulting death certificates more reliable than many other causes. As the pandemic persists, uncertainty about its scope and economic impact may increase.4 Suicide risk often increases with rising unemployment and related strains, access to firearms, substance use, and interpersonal violence.5 Individuals with serious mental illness whose illness worsens may also contribute to related morbidity and mortality. However, our data suggest that an increase in suicide deaths in Massachusetts was not associated with the stay-at-home advisory. Moving forward, effective prevention efforts will require comprehensive attention to the full spectrum of mental health services.The stable rates of suicide deaths during the stay-at-home advisory in Massachusetts paralleled findings following ecological disasters.


The experimental results demonstrate that the introduced protecting group could help augment the selectivity of such on-surface reaction, which can be applied to the precise fabrication of functional surface nanostructures.The on-surface C–H bond activation and coupling reaction is a powerful approach to constructing fine-tuned surface nanostructures. It is quite challenging to control its regioselectivity due to the inertness of the C–H bond involved. With scanning tunneling microscopy/spectroscopy and theoretical calculations, the C–H activation and sequential intramolecular dehydrocyclization of  On-surface synthesis emerged in the last decade as an efficient approach to achieving novel chemical reactions/products and fabricating elaborate surface molecular nanostructures ,3,4,5,6.β-H of the pyrrole ring or the para-H of the meso-phenyl group in these molecules can be activated at elevated temperatures and interlinked with adjacent groups, molecules and nano-entities on the surface. This leads to the fact that intramolecular and intermolecular couplings take place along different reaction paths, resulting in the regioselectivity of the C–H BAC reaction of porphyrins on the surface. Several reports have described the selective control of intra and inter-molecular products of porphyrins by thermal   on Au(111). In Co(TAP), the para-positions of its four meso-phenyl groups were substituted with methoxy groups. As the bond strength is comparable between the C–O and C–H bonds, such methoxy groups are likely to survive high temperature treatment that are necessarily required to activate the C–H bonds. Therefore, the intermolecular coupling at the para-sites of meso-phenyl groups could be suppressed. Our STM measurements clearly identified the intramolecular DHC products of Co(TAP) on Au(111) at elevated temperatures. Combined scanning tunneling spectroscopy (STS) and theoretical calculations further supported the reaction products formed. Experimentally observed and resolved reaction intermediates indicated that methoxy substituents could stay intact at a temperature whereat the DHC reaction started. The peculiar observation of the intermolecular coupling products demonstrated the profound selectivity of the reaction based on statistical analyses.In this study, low temperature scanning tunneling microscopy (LT-STM) was employed to investigate on-surface reactions of meso-phenyl groups. (3) Claw-shaped protrusions around four dim spots stem from the methoxy groups    on Au molecules, intramolecular DHC products and intermolecular products were further analyzed, aiming at a quantitative understanding of the reaction of the Co(TAP) molecule on Au(111). Before the statistics, the rule for counting each type of molecules is defined as follows. For the DHC reaction, a Co(TAP) molecule either partially or fully turned into the product was counted as one molecule in the statistical process. For intermolecular coupling reaction, whether or not the molecules in an intermolecularly connected chain already underwent DHC reaction, they were counted as intermolecular coupling products only. This ensured that sum of molecular proportions for Co(TAP), intramolecular DHC and intermolecular coupling exactly equaled 100%. For each sample annealed at a different temperature, the STM images collected at different regions of the substrate surface were used for the statistical analyses. In total, 220–250 molecules were counted for each sample. As displayed in meso-phenyl and pyrrole groups can be activated. Additionally, the methoxy groups are about to dissociate as well. However, because the intermolecular Co(TAP) coupling requires the dissociation of the methoxy groups in advance, the intramolecular DHC reaction becomes more feasible. Given that the annealing time is relatively short (2 min), it is proposed that the steric hindrance effect of methoxy group kinetically promotes the selectivity of the intramolecular DHC reaction against the intermolecular counterpart. Once all methoxy groups dissociate, further thermal annealing of the as-prepared sample at 750 K results in the increase of the intermolecular coupling products  molecule on Au(111) is as follows: At an elevated temperature of about 523 K, the C–H bonds in the products .−10 Torr. An atomically flat Au(111) single crystal (MaTeck GmbH) surface was prepared by cycled argon ion beam bombardment at 2 kV and subsequent high temperature annealing at 750–800 K. The cleanness of the surface was confirmed by the STM imaging; only traces (<3‰) of contaminates (adsorbed H or C atoms) were observed on the Au(111) surface. If present, the contaminating H or C atoms normally appeared as dark depressions in the STM images. Co(TAP)  was deposited onto the surface via thermal evaporation from a home-made tantalum boat. The deposition rate of the molecule was monitored with a quartz crystal microbalance (Inficon SQM-160) and kept at 1–2 ML/min during the deposition. Thermal annealing of the sample was conducted by radiation heating at the back of the single crystal. The sample temperature was measured with an infrared thermometer which was calibrated by a thermocouple in advance. The used STM tips were mechanically sheared Pt/Ir alloy wires. All STM and STS measurements were carried out at liquid helium temperature (~4.4 K). All STM images were raw images directly exported from the Rev9 software by RHK Technology. The STS signals were recorded at constant tip height using a lock-in amplifier (SRS SR830) with a bias modulation of Vrms = 10 mV at 1720 Hz. The STS data were processed with the Origin software.All experiments were performed on a LT-STM (Unisoku USM1300) system with a base pressure lower than 3 × 10The DFT calculations were performed with the Gaussian 09 software  , using tIn summary, we employed LT-STM and DFT calculations to explore the DHC and intermolecular coupling reactions of Co(TAP) on Au(111). Both STS measurements and DFT calculations strongly supported the proposed chemical structures of the DHC products. At 523 K, the DHC reaction carried on with the dissociation of the methoxy groups. Rapid annealing at higher temperatures led to selective formations of intramolecular DHC products over intermolecular coupling products, implying that the substitution of a protective group such as methoxy could kinetically mediate the selectivity of intra and inter-molecular reactions.