There are several endogenous molecules that can trigger migraine attacks when administered to humans. Notably, calcitonin gene-related peptide (CGRP) has been identified as a key player in a signalling cascade involved in migraine attacks, acting through the second messenger cyclic adenosine monophosphate (cAMP) in various cells, including intracranial vascular smooth muscle cells. However, it remains unclear whether intracellular cAMP signalling requires CGRP receptor activation during a migraine attack in humans. To address this question, we conducted a randomized, double-blind, placebo-controlled, parallel trial using a human provocation model involving the administration of CGRP and cilostazol in individuals with migraine pretreated with erenumab or placebo. Our study revealed that migraine attacks can be provoked in patients by cAMP-mediated mechanisms using cilostazol, even when the CGRP receptor is blocked by erenumab. Furthermore, the dilation of cranial arteries induced by cilostazol was not influenced by the CGRP receptor blockade. These findings provide clinical evidence that cAMP-evoked migraine attacks do not require CGRP receptor activation. This discovery opens up new possibilities for the development of mechanism-based drugs for the treatment of migraine. et al. provide clinical evidence that cAMP-evoked migraine attacks do not require CGRP receptor activation. These findings open up new avenues for mechanism-based drug development for migraine.Using a human migraine provocation model, Do https://doi.org/10.1093/brain/awad390) for a scientific commentary on this article.See Levy ( By administering signalling molecules to patients and observing if it triggers an attack, we can gain insights into the underlying mechanisms.3 This is also known as a human provocation model.6 These type of studies, along with others, have provided evidence that calcitonin gene-related peptide (CGRP), a neuropeptide found in abundance in the trigeminovascular system, plays a crucial role.5 When CGRP binds to its receptor, it activates a downstream signalling cascade involving cyclic adenosine monophosphate (cAMP).5 In a series of clinical trials, we have shown that administering CGRP or cilostazol, a phosphodiesterase (PDE)-3 inhibitor that prevents cAMP metabolization, can provoke migraine attacks in patients.7-13 This highlights the importance of CGRP and cAMP signalling in migraine pathogenesis. Assuming a single-cell system, it is plausible for a downstream mechanism, such as PDE3 inhibition by cilostazol, to influence an upstream mechanism like CGRP receptor activation through positive or negative feedback loops. Such interactions, where downstream components affect upstream components, are common in human biology and illustrate the complexity of intracellular signalling regulation. However, it is important to recognize that migraine pathogenesis is more intricate than a single-pathway single-cell system. The trigeminovascular system comprises multiple cells from different tissues, adding complexity to the signalling pathways and interactions involved.5 Clinical trials have demonstrated that administration of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) can also provoke migraine attacks in humans.14 Similar to CGRP, both PACAP and VIP are able to increase intracellular cAMP levels.16 Moreover, erenumab, a fully human immunoglobulin (Ig) G2 monoclonal antibody (mAb) that targets and blocks the CGRP receptor, was introduced as a migraine preventative drug in 2018.18 Other drugs targeting the CGRP signalling pathway have followed.2 However, not all patients benefit from these treatments. These observations suggest that migraine attacks may not solely depend on the CGRP receptor but could involve a unified downstream cAMP-mediated mechanism. However, this hypothesis has not been directly tested in humans.5A key feature of migraine is that various environmental and endogenous trigger factors can initiate an attack.Thus, it is timely to ascertain whether migraine attack generation requires CGRP receptor activation. This can be established through the use of human provocation models. We hypothesized that treatment with erenumab would mitigate the physiological effects of administration of CGRP, but not those of cilostazol. To test this hypothesis, we conducted a randomized, double-blind, placebo-controlled, parallel trial.The protocol was approved by the Regional Health Research Ethics Committee of the Capital Region of Denmark (identifier: H-19073983), the Danish Data Protection Agency (P-2020-652) and the Danish Medicines Agency (identifier: 2020033418). We obtained a signed consent form at the time of screening before any protocol-related procedures or assessments. All study-related procedures complied with the Declaration of Helsinki, with later revisions. The study is registered in ClinicalTrials.gov (identifier: NCT04452929).4 were eligible for participation if they had a history of migraine for ≥12 months prior to screening with ≥4 migraine days per month in the 3 months prior to screening. Key exclusion criteria were history of any other primary headache disorder except for tension-type headache, any secondary headache disorder, daily consumption of any medication other than oral contraception and prior treatment with monoclonal antibodies or participation in clinical trials with monoclonal antibodies during the preceding 12 months. Study eligibility was ensured by review of medical records and evaluation by headache specialists at the Danish Headache Center. A full overview of inclusion and exclusion criteria is available on ClinicalTrials.gov (identifier: NCT04452929).Adults aged 18–65 years old with a diagnosis of migraine according to the International Classification of Headache Disorders, Third Edition (ICHD-3),We enrolled participants in a randomized, double-blind, placebo-controlled, parallel trial at a single centre in Denmark . ParticiPatients arrived between 08:00 a.m. and 12:00 p.m. on the experimental study days. Patients were in a supine-position during study-related procedures and assessments. Patients were not allowed to consume any medication within 24 h or four times the plasma half-life of the drug (whichever longest) on the experimental study days except for oral contraception. Furthermore, to remain eligible, participants could not have had a migraine attack during the preceding 48 h nor a headache during the preceding 24 h prior to administration of the experimental trigger.A headache specialist conducted a semi-structured interview at the screening visit. The interview included information on medical history, headache characteristics and frequency, family history of migraine and prior pharmacological treatment; these data were confirmed by review of medical records.On the experimental study days, investigators recorded data on headache characteristics, vital signs and adverse events every 10 min for 90 min after administration of the experimental trigger. After the in-hospital phase, patients were discharged with a 12-h headache diary for hourly recordings of headache features, accompanying symptoms, intake of any rescue medication and adverse events.20Patients rested in a quiet room for at least 30 min in a supine position before any measurements. During the in-hospital phase, we conducted electrocardiography and measured the blood pressure and heart rate. Furthermore, we used a high-resolution ultrasonography unit  as previously described to measure the diameter of the frontal branch of the superficial temporal artery and the radial artery.7-13 and that treatment prior to a provocation challenge lowers the expected induction rate by ∼20% .21 At 80% power and a 5% level of significance, we estimated that 72 patients would be needed in a two-arm parallel trial.Sample size calculations were based on the difference between two unpaired groups reporting migraine attacks. After administration of CGRP, we assumed that 51% of participants would report migraine attack in the placebo-treatment arm and 20% in the active-treatment arm. After administration of cilostazol, we assumed that 66% of participants would report migraine attack in the placebo-treatment arm and 20% in the active-treatment arm. These assumptions were based on previous migraine provocation studies, in which ∼66% of participants with migraine developed attacks after administration of CGRP and ∼86% after cilostazol,22 The secondary end points were difference in area under the curve (AUC) values for headache intensity scores, superficial temporal artery diameter, radial artery diameter, heart rate and mean blood pressure. Pearson’s chi-squared test was used to analyse the incidence of migraine attacks and headache as binary categorical data. We made calculations of AUC values in accordance with the trapezium rule.23 We used the Mann–Whitney test to analyse differences in headache AUC scores after administration of CGRP or cilostazol between the active-treatment arm versus placebo-treatment arm. Analyses of haemodynamic AUC values were analysed using unpaired, two-way t-tests. All statistical analyses were performed using R (version 4.1.0).24 A 5% level of significance was accepted for all comparisons. We present data as mean values with standard deviation (SD), except the headache score which is presented as the median. Time to onset of migraine attack, time to intake of rescue medication, time to peak headache intensity and duration of migraine-associated symptoms are presented as median values. Baseline was defined as the time of the start of infusion (T0).The primary end point was the incidence of migraine attacks in a 12-h observational period after administration of experimental triggers.A total of 80 patients provided informed consent from July 2020 to July 2021; 75 patients completed both experimental study days and were included in the final analysis . One patP = 0.024) ] followed by temporal [14 (38%)], occipital [11 (30%)] and parietal [7 (19%)] regions. In the placebo-treatment group, the most common localization was frontal region [30 (81%)] followed by temporal [25 (66%)], occipital [15 (41%)] and parietal [5 (13%)] regions. We did not find a difference in the area under the curve (AUC) for headache intensity between the erenumab group and the placebo group following intravenous infusion of CGRP . The incidence of migraine-associated symptoms in the active-treatment group compared to the placebo-treatment group was, for nausea, 9 (24%) and 13 (34%), photophobia, 16 (43%) and 23 (61%), and phonophobia, 6 (16%) and 14 (37%), respectively. The median time to onset in the active-treatment group compared to the placebo-treatment group was, for nausea, 120 min  and 30 min , photophobia, 55 min  and 30 min , and phonophobia, 210 min  and 40 min , respectively. The median time of duration in the active-treatment group compared to the placebo-treatment group was, for nausea, 120 min  and 80 min , photophobia, 175 min  and 190 min , and phonophobia, 120 min  and 235 min , respectively.We demonstrated that erenumab can alleviate migraine attack induction with CGRP with 10 of 37 [27% ] participants developing migraine attacks compared to 20 out of 38 [53% ] participants who received placebo (= 0.024) . The incP = 0.533) ], followed by temporal [20 (53%)], occipital [14 (38%)] and parietal [14 (38%)] regions. In the placebo-treatment group, the most common localization was frontal region [30 (79%)], followed by temporal [29 (76%)], occipital [14 (37%)] and parietal [8 (21%)] regions. There was no difference in AUC for headache intensity .We found no difference in cilostazol-induced migraine attacks between participants who received erenumab versus placebo. Cilostazol-induced migraine attacks in 28 of 37 [76% ] participants in the erenumab group compared to 31 out of 38 [82% ] participants in the placebo group (= 0.533) . In the The incidence of migraine-associated symptoms in the active-treatment group compared to the placebo-treatment group was, for nausea, 17 (46%) and 22 (58%), photophobia, 24 (65%) and 23 (61%), and phonophobia, 17 (46%) and 19 (50%), respectively. The median time to onset in the active-treatment group compared to the placebo-treatment group was, for nausea, 240 min  and 300 min , photophobia, 310 min  and 240 min , and phonophobia, 180 min  and 240 min , respectively; the median time of duration in the active-treatment group compared to the placebo-treatment group was, for nausea, 210 min  and 150 min , photophobia, 300 min  and 420 min , and phonophobia, 315 min  and 240 min , respectively.0–90min, P < 0.00001) and the radial artery  compared to placebo  (P = 0.238) or radial artery (P = 0.984) in individuals randomized to erenumab compared to placebo   . Followi placebo . Further= 0.659) .The proportion of patients reporting warm sensations, palpitations and flushing in the erenumab group was lower than in the placebo group .11-13 Following administration of cilostazol, the proportion of patients reporting warm sensations and palpitations in the erenumab group was similar to the placebo group  patients who were pretreated with erenumab still experienced migraine attacks when cilostazol was administered; (ii) patients who were susceptible to migraine attacks following CGRP administration also had migraine attacks induced by cilostazol; and (iii) most participants who did not experience migraine attacks after receiving CGRP did have migraine attacks when cilostazol was administered.25 As a result, cilostazol leads to accumulation of intracellular cAMP. It is important to note that cilostazol does not directly increase cAMP generation but inhibits its degradation, thus relying on upstream regulators such as the CGRP receptor or other factors. Notably, the observation that cilostazol induces migraine attacks even in the presence of a CGRP receptor blockade suggests the existence of a CGRP-independent pathway for migraine attacks in humans. A preclinical study demonstrated that PACAP-induced hypersensitivity in a mouse model of migraine-like pain is independent of CGRP.26 However, it is important to interpret these findings cautiously as CGRP receptor antagonism was still able to inhibit pain induced by cilostazol in the same mouse model.27 This suggests that the translatability of the model may have limitations. Nonetheless, the PACAP signalling pathway is of interest as both the ligand and its receptors are expressed within the trigeminovascular system.5 In humans, PACAP binds to three different receptors , and experimental data have shown that activation of these receptors can increase intracellular levels of cAMP.5 Furthermore, a recent clinical trial demonstrated that Lu AG09222, an anti-PACAP mAb, was effective in inhibiting PACAP38-induced cephalic vasodilation and reducing concomitant headache in healthy volunteers.28Cilostazol, a PDE-3-inhibitor, blocks both isoforms of PDE3, which is responsible for metabolizing cAMP.29 Its inhibition of PDE3 leads to dilation cranial arteries in both healthy volunteers and individuals with migraine.31 Of note, cilostazol is used to relieve symptoms of intermittent claudication. Partly because of its vasodilatory effect.32 Our findings support a vascular site of action, as we observed a minor increase in arterial diameter during the 90 min in-hospital monitoring phase. However, further observation over a longer period is necessary to confirm these results, as previous studies with longer observational periods have shown a more pronounced increase in arterial diameter over time.31 These results align with the observation that stimulation of downstream targets of cilostazol, such as ATP-sensitive potassium (KATP) channels and large-conductance calcium-activated potassium (BKCa) channels, can induce migraine attacks in parallel with vasodilation in individuals with migraine.33-35 Interestingly, cilostazol can bind directly to the inner surface of BKCa channels, stimulating their activity.36 Sumatriptan (an anti-migraine drug) inhibits cAMP/PKA-signalling37-40 and constricts the superficial temporal artery and middle meningeal artery in parallel with pain relief during cilostazol-induced migraine attacks. This suggests a perivascular or peripheral site of action.30 Given that cilostazol is a lipophilic compound,42 we cannot exclude additional peripheral or central neuronal sites of action. Although both isoforms of PDE3 are co-localized with CGRP in the trigeminal ganglion,43 PDE3 activity does not regulate CGRP release.27 However, we cannot exclude a direct effect of cilostazol on the trigeminal ganglion as the PDE3A isoform is present in non-neuronal cells there.44 Furthermore, activation of the cAMP-PKA second messenger pathway can sensitize dural mechanonociceptors in rats, potentially independent of a vascular involvement. However, further data are needed to elucidate the potential effects of cilostazol within the nervous system, considering its limited ability to cross the blood–brain barrier in several species,45 which is consistent with the rarity of adverse events related to the CNS for cilostazol.32Cilostazol primarily inhibits the isoform PDE3A, which is predominantly found in vascular smooth muscle cells.46 This finding is clinically interesting as one-third of migraine patients do not report a substantial reduction in migraine frequency after treatment with an anti-CGRP receptor mAbs.48 It is worth mentioning that CGRP receptor blockade did partially mitigate CGRP-induced vasodilation, but there was still a residual vascular response. This suggests that CGRP signalling, although to a lesser extent, may also occur through receptors other than the canonical CGRP receptor. This residual effect could provide a partial explanation for why breakthrough migraine attacks can still occur despite treatment with anti-CGRP receptor mAbs, albeit at a lower frequency. It is possible that in patients treated with a CGRP receptor antagonist, residual CGRP receptors may still exert a minor physiological effect.49-53 Supposedly, circulating CGRP could bind to the AMY1 receptor, leading to mild vasodilation,49-53 as observed in our study. Interestingly, other peptides in the calcitonin family that share receptor affinity have also been implicated in migraine pathogenesis.49-51 Further data are needed to elucidate the role of the AMY1 receptor and its interaction with CGRP signalling in migraine pathophysiology.We found that CGRP receptor blockade did not completely prevent CGRP-induced migraine attacks. One-quarter of patients in the erenumab group still experienced a migraine attack following administration of CGRP, which exceeds the expected rate of a nocebo response [8.1% ].54 Differences in Tmax between participants are possible after oral administration of cilostazol. Nonetheless, the induction rate and median time to onset of cilostazol-induced migraine attacks is comparable to previous reports.54 As mentioned earlier, we noticed a slight increase in the diameter of the superficial temporary artery in both study arms after administering cilostazol. However, a longer observation period would have given us more information about potential vascular effects, as previous studies have shown a progressive increase in arterial diameter over time.31 We cannot exclude differences in bioavailability of erenumab between individuals. Nevertheless, mean Tmax is 5.5 days in healthy volunteers and consistent with an early onset of effect within the first week of treatment in patients.55-57 Experimental study days took place between Day 7 and Day 21 after randomization, enabling onset and maintenance of efficacy.Although we applied rigorous criteria during recruitment and standardized experimental procedures, we could not rule out various environmental factors  after discharge from the laboratory and the subsequent outpatient 12-h observation phase. However, alternative 12-h inpatient monitoring would have limited the feasibility of the study. In addition, the migraine attack induction rates for CGRP and cilostazol in the placebo arm were comparable to previously reported studies with these substances.The present data provide further evidence for the crucial role of second messenger signalling in a migraine attack. We demonstrate that migraine attack generation does not require CGRP receptor activation. These findings highlight the relevance of targeting downstream signalling pathways or other molecules involved in cAMP signalling, such as PACAP, as potential therapeutic targets for the treatment of migraine.


Borderline personality disorder (BPD) is a common and serious mental disorder. Its prevalence is estimated to be around 20% among psychiatry inpatients and 11% in Psychiatric Outpatients. Patients with BPD present a wide range of psychopathological symptoms such as affective instability, impulsivity, interpersonal problems, psychotic-like symptoms and self-destructive behaviour. BPD also occurs as comorbid illness with number of other Psychiatric diseases. There is no psychotropic medication that has been approved by FDA or recommended by NICE guidelines nevertheless polypharmacy is routinely prescribed in patients with BPDTo domenstrate the possibility of using Asenapine in treating Border line personality disorderCase :A case seen in our practice of a patient with Borderline Personality presenting with symptoms of affective instability,impulsivity,quasipsychotic symptoms that have not responded to trial of many different antipsychotics.She was started on Asenapine and experienced significant improvement in symptoms and daily functioning.However her medication was changed due to Asenapine being non formulary and this caused relapse in her mental state.She reported erratic sleep,poor appetite,anxiety and aggression.Asenapine was restarted and she improved.2C , 5-HT2A , 5-HT7 , 5-HT2B , and 5-HT6. Asenapine’s favourable weight and metabolic profile are of clinical interest. Asenapine was generally safe and well tolerated in paediatric patients . One open label study that looked at efficacy of Asenapine in BPD showed improvement with Asenapine in not only affective but also improve impulsive and cognitive symptoms:120-3). The results of both the CGI-BPD and the BSL-23 scales, which reflect the view of clinician and patients, respectively, show a significant improvement in the BPD general symptomatology :120-3). In our case patient reported worsening of symptoms after Asenapine was discontinued.She experienced suicidal ideation, impulsivity, aggression, erratic sleep wake cycle and poor appetite.On restarting Asenapine there was significant improvement in her symptoms and marked subjective improvement in activities of daily living.Asenapine belongs to the chemical class of dibenzo-oxepino pyrroles and acts antagonistically at a number of receptors, and this combination of receptor-binding affinities differs from other available antipsychotics. Asenapine has high affinity for several 5-hydroxytryptamine (5-HT)-receptor subtypes, including 5- HTAsenapine has therapeutic efficacy as well as good tolerability and safety profile.It can be used in patients with BPD especially when other antipsychotics have caused undesirable side effects like weight gain.None Declared


Graft versus host disease (GVHD) can occur at any period post allogeneic hematopoietic stem cell transplantation as a common clinical complication contributing to significant morbidity and mortality. Acute GVHD develops in approximately 30-50% of patients receiving transplants from matched related donors. High doses of steroids are used as first-line treatment, but are unsuccessful in around 40% of patients, resulting in the diagnosis of steroid-refractory acute GVHD. Consensus has yet to develop for the management of steroid-refractory acute GVHD, and prognosis at six months has been estimated at around 50%. Thus, it is critical to find effective treatments that increase survival of steroid-refractory acute GVHD. This article describes the currently known characteristics, pathophysiology, and treatments for GVHD, with a special focus on recent advances in cell therapies. In particular, a novel cell therapy using decidua stromal cells (DSCs) was recently shown to have promising results for acute GVHD, with improved effectiveness over previous treatments including mesenchymal stromal cells. At the Karolinska Institute, severe acute GVHD patients treated with placenta-derived DSCs supplemented with either 5% albumin or 10% AB plasma displayed a one-year survival rate of 76% and 47% respectively. Furthermore, patients with steroid-refractory acute GVHD, displayed survival rates of 73% with albumin and 31% with AB plasma-supplemented DSCs, compared to the 20% survival rate in the mesenchymal stromal cell control group. Adverse events and deaths were found to be attributed only to complications of hematopoietic stem cell transplant and GVHD, not to the study intervention. ASC Therapeutics, Inc, in collaboration with the Karolinska Institute, will soon initiate a phase 2 multicenter, open-label study to further assess the efficacy and safety of intravenous DSC treatment in sixty patients with Grade II-IV steroid-refractory acute GVHD. This novel cell therapy represents a promising treatment to combat the poor prognosis that steroid-refractory acute GVHD patients currently face. NumbersGraft-versus-host disease (GVHD) is the most common life-threatening complication after allo-HCT , 7. Amon2Acute GVHD develops in 30-50% of patients after allo-HCT from a matched related donor , 11–14. cGVHD is the leading cause of late morbidity in recipients of allo-HCT and is associated with a higher risk of non-relapse mortality , 18. The2.1GVHD occurs when donor T cells recognize the host tissues as foreign due to histocompatibility differences between the donor and host. Even with matching major histocompatibility complex antigens, many patients develop GVHD because of minor histocompatibility antigen differences lying outside of the human leukocyte antigen (HLA) loci , 25, 26.The pathophysiology of aGVHD includes an initial stage of tissue damage from the conditioning regimen leading to activation of host antigen-presenting cells (APC) by danger-associated molecular patterns (DAMPs), such as ATP  and pathhttps://www.fda.gov/drugs/resources-information-approved-drugs/fda-approves-ruxolitinib-acute-graft-versus-host-disease) and by the European Commission (https://www.ema.europa.eu/en/medicines/human/EPAR/jakavi) for the treatment of Steroid refractory (SR) aGVHD.As with aGVHD, damage to host tissue and inflammatory cytokine release occurs due to pretransplant conditioning in the first phase of cGVHD. The second stage in the pathogenesis of cGVHD is suggested to include thymic injury and T and B cell dysregulation, followed by a third stage of aberrant tissue repair, often with fibrosis , 35, 36.2.2The standard backbone of GVHD prevention in patients undergoing allo-HCT includes the combination of a calcineurin inhibitor (cyclosporine or tacrolimus), which reduces the expansion of effector T cells by blocking interleukin (IL)-2, with a short course of methotrexate, which interferes with alloreactive T cell division –41. In DIn cases of aGVHD occurrence, the recommended first-line treatment is systemic high-dose steroid therapy. Of note, it is important to control GVHD as profound immunosuppression can lead to relapse of the original disease and increase the risk for opportunistic infections . CurrentThere is no consensus regarding the optimal approach for the management of SR-aGVHD; as such, there is no accepted standard of care treatment , 50, 51.As mentioned previously, GVHD prophylaxis commonly involves a calcineurin inhibitor combined with methotrexate. Meta-analysis of methotrexate for SR-aGVHD discovered an overall response rate of approximately 70% with a 59.2% complete response . MethotrAlemtuzumab and human chorionic gonadotropin are cost-effective treatment options particularly in low and middle income countries. In fact, the Campath Distribution Program provides it free for patients if found to be the necessary treatment . AlemtuzAs aGVHD patients typically present with increased TNF-α levels, another effective anti-inflammatory treatment is the use of anti-TNF-α antibodies. However, the lack of efficacy in a portion of patients and the potential for life-threatening infections have reduced its use in treating SR-aGVHD . InflixiThe use of anti-IL-2 receptor antibodies have varied in effectiveness between the four currently commercially available drugs . The IL-In addition, ruxolitinib, a selective Janus kinase 1/2 inhibitor, was approved by the FDA and the European Commission for the treatment of SR-aGVHD in adult and pediatric patients (≥12 years old). Based on results from the REACH-1 study, the median duration of response from ruxolitinib treatment was 0.5 months. The median time from the day‐28 response to either death or the need for a new therapy for aGVHD was 5.7 months –76. In aTreatment of cGVHD typically requires the prolonged (median 2 to 3.5 years) use of immunosuppressive agents. First-line treatment includes high-dose corticosteroids, typically 0.5 to 1 mg/kg per day. An approximate 50% response rate is seen with steroids; second-line therapy is required in more than half of patients within two years . The hetIbrutinib, a tyrosine kinase inhibitor targeting Bruton’s tyrosine kinase, was the first FDA-approved treatment for cGVHD (approved in August 2017), demonstrating an overall response rate (ORR) of 67% in patients with SR-cGVHD , 81.The Recent research has placed significant importance on the microbiota in various diseases, including for GVHD. At least six studies and five case reports of fecal transplantation have been published as of 2022 . Overall3Several cell types can be utilized to suppress alloreactive immune cells. Mesenchymal stromal cells (MSCs) and, to a lesser degree, regulatory T cells (Treg) have been efficacious in human immune reactions . Tregs a3.1The non-hematopoietic human MSC has the unique ability to differentiate into various mesodermal , ectodermal (neurocytes), and endodermal (hepatocytes) lineages . MSCs caMSCs have emerged as the most studied cell type for clinical and experimental cell therapy for various indications, including GVHD –98. The Multiple factors and mechanisms are involved in MSC-mediated immune modulation and regenerative function. They include paracrine activity, which involves the secretion of proteins/peptides and hormones, as well as mitochondrial transport via tunneling nanotubes or microvesicles and transport of exosomes or microvesicles . ExosomeThe first case report of MSCs used for the treatment of GVHD was reported from the Karolinska Institute in Sweden , followeThere are currently no FDA-approved MSC therapies on the market in the US. However, several MSC products have been granted regulatory approval in other countries  for the treatment of various conditions, including aGVHD , 119.®, Osiris Therapeutics Inc.), a human BM-derived MSC product, was well tolerated in clinical studies with no identified infusion-related toxicities or other safety concerns , a manufactured MSC product equivalent to remestemcel-L, was approved in Japan to treat patients of all ages with acute GVHD. Within the first three years after approval, Temcell was evaluated in 381 patients. The ORR was 61% for the 151 evaluable patients who received it as second-line therapy following first-line steroid therapy for aGVHD  and the lymphocyte profiles of 16 aGVHD patients given MSC treatment, no obvious markers for MSC therapy response were revealed . In a coIn a small cohort of SR-aGVHD patients, the increase of serum PGE2 after MSC treatment was significantly greater in the responders compared to the non-responders. Hence, it was suggested that PGE2 monitoring could estimate the immunological activity of MSC therapy in GVHD patients . In addi3.2Placenta-derived decidua stromal cells (DSCs) are a type of stromal cells of maternal origin isolated from the fetal membranes of term placenta. The placenta includes the amnion, chorionic plate, villous and smooth chorion, decidua basalis, and umbilical cord. The decidua is a maternal uterine tissue that plays an important role in maternal-fetal immune tolerance , 154. DSNon-clinical studies have shown that placenta-derived DSCs differ from BM-MSCs in several aspects. They are smaller in size, show less differentiation capacity for chondrogenic and osteogenic fates, have a stronger inhibitory effect on allogeneic T cell proliferation, and in vitro data shows that they promote coagulation more effectively than BM-MSCs –162. FurIn vitro evaluation of DSCs shows a great immunosuppressive capacity for the proliferation of alloreactive T cells, production of proinflammatory cytokines, and induction of anti-inflammatory IL-10 secretion . In addiErkers et al. (2013) reports that DSCs need to be near alloreactive lymphocytes to mediate a suppressive effect via paracrine mechanisms or increase the frequency and/or expression level of the Treg population. Thus, DSCs may not use paracrine factors solely for systemic immunosuppression but more specifically to target T cells directly in affected tissues . DSCs inDSCs may influence stromal cell-mediated immune modulation and changes in activated T cell phenotypes through IL-2 production and IL-2R signaling. Sirolimus and cyclosporine A target the IL-2 signaling pathway, diminishing the antiproliferative effect of DSCs in vitro . The chaComprehensive non-clinical short- and long-term toxicity studies of systemic DSC infusions were conducted in two animal models (rat and mouse) and in series of in vitro assays employing human blood . No throA major obstacle in stromal cell therapy is the trapping of IV infused MSCs into the lung microvasculature, impairing their homing to target tissue due to the first-passage effect , which is a function of the cell size and deformability. DSCs have smaller diameters, being approximately half the volume of MSCs, which could encourage microvascular passage and reduce microvascular organ and pulmonary embolism risks , 165. In3.2.1DSCs have been clinically investigated for the treatment of severe aGVHD at the Karolinska Institute in Sweden , 166–169The distribution of DSCs after IV infusion has also been investigated in severe cGVHD. The pilot study by Erkers et al. (2015) used DSCs labeled with 111Indium (111In), and the in vivo distribution was tracked for 48 hours in two patients. The labeled DSCs were initially located in the lungs, followed by dissemination to the liver and spleen . MSCs ha3.2.1.1In the investigator-initiated study , a total of 38 subjects with severe aGVHD Grade 2-4 were enrolled and treated with placenta-derived DSCs, as a second-line therapy. Of the subjects, 21 (ages 1.6-72.4 years) received DSCs supplemented with 5% albumin , and 17 (ages 0.9-65.6 years) received DSCs supplemented with 10% AB plasma . The number of patients who were steroid-refractory in the DSC/Albumin and DSC/AB Plasma groups was 11 and 13, respectively and were treated between 2011-2015. In both study groups, the majority of subjects were males (52.9% - 76.2%) and most subjects had aGVHD Grade 3 . Control groups, including only patients with acute steroid-refractory GVHD, included 15 subjects receiving BM-MSC treatment (ages 34-65 years) and 32 historical controls receiving all available treatment as a second-line therapy except cells (ages 3.7-67.7 years). All control patients were from the Karolinska Institute Database, treated between the years 2000–2010, and were not matched to the treatment group.The results reported in this review paper were taken from the manuscript Ringden et al. . The FisIn a subgroup of subjects with SR-aGVHD , the one-year survival rate aGVHD was significantly higher in the DSC/Albumin group (73%), versus the DSC/AB Plasma group , the MSC group , and the historical control group , based on the Fisher exact test. A complete or partial response was observed in all SR-aGVHD subjects (100.0%) in the DSC/Albumin group at four weeks after the first DSC dose , which was significantly higher than in the DSC/AB Plasma group and the MSC group with approximately only 25% seeing a response in the historical control group. The differences in response  vs. no response between the DSC/Albumin group and all other study groups were statistically significant at all timepoints evaluated.The most common adverse events in the DSC groups were relapse , fungal infection , and pneumonia . Lower rate of bacterial infection and multiple organ failure were reported in DSC groups versus control groups (7.9% and 34.4% respectively). Death events were reported for all study groups and occurred at lower rates in the DSC groups (44.7%) versus the control groups (96.9%). The majority of subjects in the DSC group died from either acute GVHD (23.7%), relapse (5.3%) or bacterial infection (5.3%); GVHD was the main cause of death in the control groups. Overall, based on the limited safety data available for analysis, no safety signals detected were attributable to the study intervention (DSC). The causes of adverse events and deaths were associated with common complications seen among subjects undergoing HCT and with severe aGVHD.3.3ASC Therapeutics, Inc. is developing ASC930, an allogeneic off-the-shelf investigational product consisting of live human placenta-derived DSCs in collaboration with the Karolinska team who originally developed DSCs as described. ASC930 is being developed for the treatment of patients with SR-aGVHD Grade II-IV following allo-HCT. ASC930 will be given to the hospitalized patients in an infused IV on a weekly basis. Each dose consists of 1 ± 0.2 x 10^6 cells/kg body weight and will be repeated for four weeks based on the patient’s condition.The clinical development program of ASC930 was initiated with a re-analysis of safety and efficacy data from open-label studies conducted in academic settings  in subjects with aGVHD , 178. AnDue to the limited knowledge of how DSCs act in the body and how they activate the immune system, biomarkers are vital to determine the mechanisms of DSC treatments. Biomarkers for disease, immune, and cellular response to DSCs could help identify the optimal patients, treatment, and monitoring strategies for safety and efficacy of cell therapy for aGVHD. DSCs can be measured in the body, but also the donor-derived cell-free DNA can be quantified to understand the cellular actions or predict organ rejection. Inflammation also needs to be monitored because it can be a precursor to organ injury. Flow cytometry is the typical method for measuring the immune response by identifying and quantifying types of immune cells. Endothelial dysfunction biomarkers in the blood, such as ST2 and REG3α, can predict long-term outcomes and have been incorporated into the MAGIC algorithm probability to determine mortality post aGVHD treatment. High levels of these two biomarkers in the blood are found in aGVHD patients and predict mortality.4Steroid refractory acute GVHD is a life-threatening complication that limits the success of HCT. HCT is widely used in curing many hematological malignancies and other fatal disorders of the immuno-hematological system. The only FDA-approved drug for SR-aGVHD, ruxolitinib, cures just a portion of patients with SR-aGVHD. MSCs are rare cells in all body tissues but may be cultured and expanded to a large number of cells. Among several other features, MSCs are immunosuppressive and have been used to treat aGVHD, among other immunological disorders. MSCs are an attractive candidate for therapy because there are almost no reported side effects. DSCs have a greater immunosuppressive capacity than other sources of MSCs, and preliminary in vitro and clinical data suggest they more effectively treat alloreactivity and aGVHD compared to other sources of MSCs . Further clinical phase 1/2 trials and prospective randomized trials compared to the best available therapies, including ruxolitinib, are needed to establish DSCs as a therapy for SR-aGVHD.5Finding safe and efficacious treatments for acute Graft versus Host Disease (aGVHD) after stem cell transplantation in patients with hematological malignancies is crucial because of its high mortality rate and the limited success of current treatment options. Over approximately two decades, autologous and allogeneic cell therapies have been used in clinical trials to treat aGVHD with encouraging responses. The introduction of decidua stromal cells (DSCs) has shown a 50% higher survival rate compared to those observed with mesenchymal stem cells (MSCs) in both pediatric and adult populations experiencing steroid-refractory aGVHD (SR-aGVHD). Interestingly, the vehicle used for DSCs was found to be relevant, since albumin (ASC930) rather than AB plasma significantly increased the survival rates. The FDA has cleared ASC930 for a phase 2 trial for Grade II-IV SR-aGVHD, to confirm the manufacturability, safety and efficacy data already demonstrated in academic trials of DSCs.The future of allogeneic cell therapies in SR-aGVHD will be a crucial milestone, not only for this indication, also for the demonstration that placenta-derived DSCs can exert a potent immunoregulatory role in patients with life-threatening conditions and potentially with autoimmune diseases. With the promise of these therapies also comes the expansion of therapeutic indications, determination of relevant endpoints, both clinical and surrogate biomarkers, as well as inclusion/exclusion criteria to target the right patient population in bespoke clinical trials. Among the most difficult challenges for allogeneic cell therapies is ensuring reproducibility and large-scale manufacturability. Scaling up these therapies is time-intensive and costly with significant obstacles halting or slowing down their path to clinical use. This potential bottleneck in the availability of clinical materials requires the collaborative efforts of academia and industry to avoid supply chain disruptions and ensure accelerated progress in the cell therapy space. Future automation solutions may alleviate some of these issues, including reducing human error, enhancing reproducibility, and achieving standardization. Automation holds the potential to assist with the critical quality control necessary for cell therapy. Interest from investors and manufacturers is increasing with successful clinical trials and hopefully, will assist successful therapies in reaching the production and manufacturing phases.The advantages of using DSCs over MSCs make them an attractive alternative to inflammatory-related diseases. Although still under investigation, some efficacy and safety limitations seen with MSC-based therapy have been found to be circumvented when using DSCs instead. More research is also required to identify the long-term effects of DSC-based therapies beyond one-to-five-year survival data for various clinical indications. We also need to determine the potential of repeated treatment cycles, compared with a potentially safer ‘one-and-done’ single infusion, given the pharmacokinetic and pharmacodynamic profile of the ‘hit-and-run’ nature of DSCs.The progress already achieved with DSC-based therapies in pre- and clinical development programs for aGVHD so far is paving the way toward the approval and mainstream use of these therapies in the clinic. DSC-based treatments will reach full-scale production and manufacturing phases over the next decade. The present indications for most cell therapies include diseases with few to no current treatments, high mortality rates, and substantial threats to the quality of life. It will also be interesting to see the use for cell therapy treatments broaden in the near future, which is bound to occur with the safety and efficacy data reported by current clinical trials.The authors confirm substantial contributions and revisions of this manuscript and approve it for publication.


Few studies have examined preventative behaviour practices with respect to COVID-19 among people living with HIV (human immunodeficiency virus). Using a cross-sectional survey from a Canadian Institutes of Health Research Canadian HIV Trials Network study (CTN 328) of people living with HIV on vaccine immunogenicity, we examined the relationships between participant characteristics and behavioural practices intended to prevent COVID-19 infection. Participants living in four Canadian urban centers were enrolled between April 2021–January 2022, at which time they responded to a questionnaire on preventative behaviour practices. Questionnaire and clinical data were combined to explore relationships between preventive behaviours and (1) known COVID-19 infection pre-enrolment, (2) multimorbidity, (3) developing symptomatic COVID-19 infection, and (4) developing symptomatic COVID-19 infection during the Omicron wave. Among 375 participants, 49 had COVID-19 infection pre-enrolment and 88 post-enrolment. The proportion of participants reporting always engaging in preventative behaviours included 87% masking, 79% physical distancing, 70% limiting social gatherings, 65% limiting contact with at-risk individuals, 33% self-isolating due to symptoms, and 26% self-quarantining after possible exposure. Participants with known COVID-19 infection pre-enrolment were more likely to self-quarantine after possible exposure although asymptomatic . Participants with multiple comorbidities more likely endorsed physical distancing , although this was not significant in logistic regression analysis adjusted for age, sex, race, number of household members, number of bedrooms/bathrooms in the household per person, influenza immunization, and working in close physical proximity to others. Overall, participants reported frequent practice of preventative behaviours.The online version contains supplementary material available at 10.1186/s12981-023-00571-7. Globally, many people have faced challenges during the COVID-19 pandemic including loss of income and employment, worsened mental health, and decreased access to medical care . The panDoes previous known COVID-19 infection influence preventative behaviours among PLWH?Is participant multimorbidity (presence of multiple comorbidities) associated with preventative behaviour practices among PLWH?Are preventative behaviour practices, living in a crowded space, and working in close proximity to others associated with COVID-19 transmission among PLWH?Are preventative behaviour practices and/or uptake of COVID-19 vaccination associated with developing symptomatic COVID-19 infection during the highly contagious Omicron wave among PLWH?Since the first global COVID-19 immunization campaign was launched, attitudes and uptake of the COVID-19 vaccine in people living with HIV have been much more extensively researched than behavioural practices . In this study, we sought to understand the relationships between preventative behaviours and COVID-19 infection in a multi-centre, cross-sectional study of people living with HIV in Canada. We addressed this topic through four questions:Our study population comprised people living with HIV living in Montréal, Ottawa, Toronto and Vancouver, Canada aged at least 16 years who had received no more than two COVID-19 immunizations at the time of enrolment. The immunization requirement was part of the inclusion criteria for a separate study on COVID-19 vaccine immunogenicity . ParticiFrom the total study population, responses from appropriate subsets of participants were analyzed to address each of the four aforementioned questions, as described in Table 3, suppressed HIV viral load), and low number of comorbidities (one or fewer) was 52%. One-third of participants (28.1%) had two or more comorbidities. Most participants (69.1%) had achieved more than a secondary school education (secondary school education comprises schooling up to age 17–18 in Canada). A total of 49 participants had documented COVID-19 infection before study enrolment based on either self-report  or laboratory testing . Eighty-eight participants contracted COVID-19 infection during the study period, up until April 2022, based on either of self-report  and laboratory testing . Of note, the vaccine immunogenicity study is still ongoing so the total number of COVID-19 infections during the total study period is currently unknown. Overall, preventative behaviours were frequently practiced in the cohort, with 87% masking in public, 79% distancing, 70% avoiding large gatherings, and 65% limiting contact with vulnerable persons.Among the 375 participants enrolled in the study, the mean age was 52.0 years (standard deviation 13.3 years) with a range of 19.7–83.5 years. Detailed characteristics of participants are presented in Table To address this question, we excluded individuals with positive serum COVID-19 antibody testing (presumed prior infection) but no knowledge of prior infection (n = 24). A detailed explanation of the participant subsets used in each of the four questions is found in Fig. Participants in the multimorbidity group were more likely to be older , live in a household with fewer members (p = 0.020), have more bedrooms and bathrooms in the household per person , usually get an influenza immunization (p = 0.045), and less likely to be performing paid or unpaid work in close physical proximity to others (p = 0.035). They were more likely to be vaccinated with four doses against COVID-19 by September 2022 . In response to the preventative behaviours survey, participants in the multimorbidity group were more likely to be practicing physical distancing . This difference, however, was no longer statistically significant after adjusting for participant characteristics  , and no other significant differences in preventative behaviours between groups were noted.The participants in the COVID-19 infection group were more likely to have fewer bedrooms per person . There were no identified differences in the proportion of participants performing paid or unpaid work in close physical proximity to others between those with and without COVID-19 infection . There were no identified differences in preventative behavior practices between those with and without baseline COVID-19 infection.In Canada, the Omicron wave began in late November 2021 . ParticiUsing data from our cohort of people living with HIV, we examined four questions regarding COVID-19 preventative behaviours. Another study done in the Canada in general population assessed determinants of adherence to major coronavirus preventive behaviours, including demographics, attitudes and concerns and showed that adherence to COVID-19 prevention behaviours was worse among men, younger adults, and workers, and deteriorated over time . We did Preventative behaviours including masking, physical distancing, and limiting gatherings have had high uptake globally in people living with HIV. In a South African cohort of people living with HIV, 80% changed one or more activities based on public health recommendations . One UniLimited data exist on the effects of prior COVID-19 infection on preventative behaviours or on the influence of working in close physical proximity to others on COVID-19 behaviours in people living with HIV. Greater vaccine uptake among those with multimorbidity and/or older age has been reported in a South African cohort of people living with HIV . In contWe observed no difference in vaccination status between participants sustaining Omicron infection and those not infected. We note that studies in the general population have shown a less protective effect of original vaccine formulations against the Omicron variants although behavioural differences during the Omicron wave may have also played a role in our cohort .Our study has several limitations: the entire cohort was participating in COVID-19 vaccination programs to some degree and had easy access to provincial and federal public health programs for testing and education. This may limit the generalization of our results to settings where public health infrastructure is not available to disseminate information and vaccines. It also encapsulates behaviours for only a portion of people living with HIV who consented to vaccination. Data was collected only at the beginning of Omicron wave resulting in small number of participants being infected by COVID-19 Omicron variant and therefore may not be fully generalizable to Omicron and subsequent waves.In summary, our Canadian cohort of people living with HIV reported high rates of preventative behaviour practices. We found differences in preventative behaviours among those with prior COVID-19 infection and in those with multimorbidity suggesting these are key motivating factors in facilitating preventative behaviours.Additional file 1. HIV-COV CITF CDE baseline questionnaire.


This review examines differences and similarities between upper tract urothelial carcinoma (UTUC) and bladder urothelial carcinoma (BUC) with respect to their epidemiological, clinical, pathological, and biological features and discusses the resulting therapeutic consequences. Systemic treatments for invasive and metastatic diseases are considered, and an overview of the expected developments in this field is provided.Upper tract urothelial carcinoma (UTUC) is a rare disease included, along with the much more frequent urothelial bladder cancer (BUC), in the family of urothelial carcinomas (UCs). However, while UTUCs and BUCs share several features, their epidemiological, clinical, pathological, and biological differences must be considered to establish an optimal therapeutic strategy. This review examines the clinical differences between UTUC and BUC, as well as the main results obtained by molecular screening of the two diseases. The findings of clinical trials, performed in peri-operative and metastatic settings and assessing systemic treatments in UC, are summarised. A comparison of the data obtained for UTUC and BUC suggests improved therapeutic approaches, both in regards to routine practice and future drug development. Upper tract urothelial carcinoma (UTUC) is a rare cancer which is part of a much more frequent group of tumours known as urothelial carcinomas (UCs). Among the latter, bladder urothelial carcinoma (BUC) accounts for 90–95% of the cases . While tThe incidence of UTUC is approximately 2 per every 100,000 inhabitants/year , and thaSmoking is a major risk factor for UC. Studies of UTUC have estimated an increase in the relative risk from 2.5 to 7% ,17,18, aAristolochic acid (AA) is the active element of the Aristolochiaceae family of herbaceous plants. Its accidental ingestion and its use in traditional pharmacopoeia are associated with the higher incidence of UTUC in the Balkans and on the Asian continent  . DespiteMLH1, MSH2, MSH6, PMS2), predisposes patients to several cancers transmitted by autosomal dominant inheritance. In terms of its localisation, UTUC is the third most common (~5%) tumour on the spectrum of Lynch syndrome tumours, after colorectal and endometrial localisations  [The standard of care for the peri-operative treatment of BUC is cisplatin-based neoadjuvant chemotherapy . The sam< 0.001] . Among t< 0.001] . The ben2) and gemcitabine (GC) (1000 mg/m2 on days 1 and 8 of each cycle) or carboplatin (AUC 4.5 or AUC5) and gemcitabine (GP). The results showed an improved DFS . At 3 years, 71% (95% CI: 61–78) and 46% (95% CI: 36–56) of patients receiving chemotherapy and surveillance, respectively, were event-free. This benefit was consistent across the subgroups, even for the 28% of patients who received GP [p = 0.11).Beyond the question of benefit, a majority of UTUC patients are not eligible for neoadjuvant chemotherapy because of the unreliability of preoperative staging and histopathology, as well as the difficulty in proving the invasive nature of the tumour based on the biopsy. For BUC, conclusive evidence for the benefits of adjuvant chemotherapy is lacking, since all of the relevant trials showed significant methodological bias . For UTUeived GP . This fieived GP . An updaGiven the anti-tumour activity of immune checkpoint inhibitors (ICIs) in metastatic BUC, their efficacy has been assessed in the adjuvant setting. The phase 3 Checkmate 274 trial randomised patients with muscle-invasive UC who had undergone radical surgery to receive nivolumab or placebo every 2 weeks for one year . The priThe question of peri-operative treatment for UTUC is being addressed in several ongoing clinical trials. As discussed above, the main issue regarding neoadjuvant regimens in UTUC is the need for biopsy-based proof of muscle invasion. Since most high-grade UTUCs at biopsy are found to show muscle invasion, the issue of whether tumour grade, when used as a criterion for neoadjuvant treatment, could lead to survival improvements remains to be determined. The phase II NAUTICAL trial  04574960) randomises patients with high-grade UTUC to neoadjuvant or adjuvant chemotherapy. Another phase II/III trial (NCT04628767) also uses the criterion of high tumour grade to evaluate neoadjuvant chemotherapy, with or without durvalumab, in patients with localised UTUC. The ABACUS-2 phase 2 trial will assess the effect of neoadjuvant atezolizumab for patients with rare histological subtypes of bladder cancer or with UTUC who are at high risk of relapse (NCT04624399) .The abovementioned anti-Nectin-4 antibody-drug conjugate enfortumab vedotin, shown to be effective in metastatic BUC [FGFR alterations are a more frequent feature of UTUC, especially in the early stages of the disease, and constitute a therapeutic target. The phase III PROOF 302 trial (NCT04197986) [FGFR3 alterations and a high risk of recurrence who received neoadjuvant cisplatin or who are cisplatin-ineligible. Patients have been randomised to the placebo group or to receive anti FGFR infigratinib for up to one year in the adjuvant setting.As also noted above, 4197986)  includesHER2 overexpression is frequently found in UTUC (36% of score 2 or 3+ on the HercepTest) [Since cepTest) , a phaseThe main trials are summarised in For patients with advanced/metastatic disease, the standard method of care for those with BUC is a platinum-containing regimen, with a slight benefit for cisplatin over carboplatin. As the initial trials testing platinum did not include UTUC patients ,73, platIn the second-line setting, mono-chemotherapy with taxanes was historically proposed for BUC patients, albeit based on retrospective studies, with few patients and deceptive results. In 2009, Bellmunt et al. published a phase III randomised trial comparing vinflunine  with best supportive care in the second line setting for 370 BUC patients. While the study did not find an OS benefit in the intent-to-treat population, a statistically significant benefit was identified when the 13 patients exhibiting significant protocol deviations were excluded. In that case, the median OS was 6.9 vs. 4.3 months  and the overall response rate (ORR) was 8.6%. Whether the study included patients with UTUC is unclear, as no data for this population are available.n = 18) and BUC (n = 59), with a median OS of 5.0 and 8.2 months and an ORR of 22.2% and 23.7%, respectively [n = 44) or after (n = 61) immunotherapy showed an improved clinical benefit  and a trend toward OS improvement. This study included 23 (22%) patients with UTUC, but no conclusion could be drawn from this subgroup analyses.In 2015, a prospective, observational study investigated the safety and efficacy of vinflunine in patients pre-treated with platinum-based chemotherapy . Vinflunectively . These rectively . A retroIn 2017, the KEYNOTE-045 study showed that, compared to mono chemotherapy, pembrolizumab significantly improved OS for BUC patients with disease progression after platinum-based chemotherapy (without avelumab maintenance) . This trIn 2020, the Javelin-100 trial randomised 700 patients without disease progression after first-line chemotherapy (4–6 cycles of GC or GP) to receive either maintenance avelumab or surveillance . The stuIn the first-line setting, 374 cisplatin ineligible patients received pembrolizumab within the KEYNOTE-052 phase 2 trial. The ORR was 24% for the overall population, of which 19% of patients suffered from UTUC. The ORRs for UTUC and BUC were similar, at 22% and 28%, respectively. Based on these results and those from the KEYNOTE-361 trial, the US Food and Drug Administration (FDA), but not the EMA, approved pembrolizumab for patients with metastatic urothelial carcinoma (BUC or UTUC) who are not eligible for platinum-containing regimens.The phase 2 IMvigor210 trial enrolled 119 patients with advanced UC who were ineligible for cisplatin to receive atezolizumab as a first-line therapy. The results led to FDA, but not EMA, approval of this regimen for cisplatin-ineligible patients with PD-L1-expressing UC or any patients who are platin-ineligible in the first-line setting, regardless of the tumour’s anatomic site. While the study included à significant proportion of UTUC patients (28%), no subgroup analyses were published.In case of progression after chemotherapy and immunotherapy (maintenance or second-line), the anti-Nectin-4 ADC enfortumab vedotin is the standard of care for BUC patients. The phase 3 EV-301 trial randomised 608 patients with locally advanced or metastatic UC who had previously received platinum-containing chemotherapy, but who had experienced disease progression during or following PD-1/L1 inhibitor treatment to receive enfortumab vedotin or chemotherapy . In thisFGFR2 or FGFR3 alteration were shown to benefit from treatment with a pan-FGFR tyrosine kinase inhibitor. In a phase 2 study, 99 patients with UC (23 with UTUC) pretreated with chemotherapy received 8 mg of erdafitinib daily [FGFR alterations [FGFR alterations, that has progressed despite chemotherapy and immunotherapy, regardless of the primary site. Given the higher incidence of FGFR alterations in UTUC and the clinical activity observed in this population, erdafitinib can be considered as the treatment of choice for UTUC.Patients with metastatic UC harbouring an ib daily . The stuerations . ErdafitClinical trials dedicated to metastatic UTUC are very rare, but several molecules are currently being studied in trials that include both BUC and UTUC patients. These trials are summarised in In the phase 2 mono-arm TROPHY-U-01, 113 patients with metastatic UC and disease progression after prior platinum-based and anti PD(L)-1 therapies were allocated to receive sacituzumab govitecan, an anti-Trop2 antibody conjugated to SN-38 (an active metabolite of irinotecan) . While tThe results of the development of immunotherapy for UTUC and BC are currently indissociable, as there is no specific trial for UTUC. Ongoing trials with immunotherapy are evaluating several combinations of ICIs, or ICIs with other molecules, in the first-line setting as maintenance, or in the late stages of the disease . The molContrary to the subgroup analyses of the neoadjuvant trial Checkmate 274, the outcomes were better for UTUC than for BUC in the KEYNOTE-045 trial. These differences reflected the presence among the UTUC population of patients with MSI-high tumours, known to be very good responders to ICIs . To dateSome trials for UC in general are also of specific interest for UTUC because of its unique biology, as noted in previous sections. This issue is further examined below.FGFR alterations is particularly interesting for patients with UTUC, as FGFR alterations are more frequent in these tumours. New anti FGFR inhibitors, such as ICP-192 (gunagratinib) or TYRA-300, are currently being evaluated for UC in phase 2 trials (NCT04492293 and NCT05544552). Other anti-FGFR agents, such as AZD4547 in combination with tislelizumab (anti PD-1) and futibatinib in combination with pembrolizumab, are being tested in association with ICIs to enhance the anti-tumour effect in UC. Both are currently being evaluated in phase 2 trials (NCT05775874 and NCT04601857).The promising clinical activity of erdafitinib in UCs with If HER2 amplifications are of low frequency in UC and even lower in UTUC, then the development of new antibody-drug conjugates targeting low-HER2 tumours may offer new treatment opportunities for UC. A recent study reported that 64% of 130 UTUC tumours analysed by IHC were at least HER2 1+ . MRG002 The HRR pathway is frequently altered in both BUC and UTUC, suggesting the efficacy of poly(ADP-ribose) polymerase (PARP) inhibitors in these patients. In the mono-arm phase II TALASUR trial (NCT04678362), talazoparib was added to avelumab (regardless of HRR mutations) as a maintenance treatment in patients with metastatic UC without disease progression after chemotherapy consisting of a first-line platinum-regimen . To imprHRAS mutations, although rare, are twice as frequent in UTUC than in BUC. Tipifarnib is a quinolinone that inhibits the enzyme farnesyl protein transferase and prevents the activation of Ras oncogenes. A phase 2 mono-arm trial is currently assessing tipifarnib in UCs harbouring HRAS or STK11 mutations for patients pre-treated with platinum-based chemotherapy (NCT02535650). In preliminary results from 21 patients, the ORR was 24%, but there was no response for patients with tumours harbouring STK11 mutations [utations .TSC1 mutations are three times more frequent in UTUC than in BC. Sapanisertib is a dual mTORC1/2 inhibitor that was tested in a phase 2 mono-arm trial (NCT03047213) in patients with metastatic UC. However, due to the absence of an objective response and poor tolerance of the drug, the trial was suspended [uspended .While the common features of BUC and UTUC suggest shared therapeutic targets, the differences between these tumours should be taken into account in clinical practice and in trial design.In the neoadjuvant setting, it is tempting to extrapolate the benefit of a neo adjuvant cisplatin-based regimen demonstrated in BUC to UTUC, especially because many patients will become cisplatin ineligible after nephroureterectomy. However, several issues specific to UTUC merit consideration. First, unlike BUC, there is no level 1 evidence for the benefit of neoadjuvant chemotherapy in UTUC. In 2022, a systematic review of 24 studies using neoadjuvant therapy in UTUC were analysed. Neoadjuvant treatment seemed to be associated with improved survival and better pathological response compared to the results for surgery alone. However, this result applied to retrospective or single arm trials, and there was no clear advantage when this method was compared to surgery followed by adjuvant treatment . The lowThe second main issue for neoadjuvant treatment in UTUC is the need to clearly identify muscle invasion, since the biopsies are much narrower and more difficult to perform than in BUC. A correlation with tumour grade was reported, as muscle invasive tumours at nephroureterectomy were found in 60% of patients with biopsies showing high-grade tumours . Thus, sIn the adjuvant setting, the benefit of platin-based chemotherapy was well demonstrated in the POUT trial. The DFS benefit was significant for patients who received cisplatin or carboplatin, a crucial finding for clinical practice, since most patients exhibit renal impairment after nephroureterectomy. The Checkmate 274 trial showed that nivolumab improved DFS for the overall population in the adjuvant setting, but subgroup analyses showed no benefit for UTUC patients. Since most UTUCs are of the luminal-papillary molecular subtype, characterised by immune cell infiltration, they are probably less responsive to immunotherapy . FurtherIn the metastatic setting, the anatomic specificities of UTUC are a less informative determinant of the therapeutic strategy, and clinical trials have often mixed UTUC and BUC patients. However, the biological differences between the two entities, as discussed herein, can be useful in clinical practice. For instance, a higher proportion of UTUCs than BUCs are MSI-high tumours. The MSI-high status should then be assessed for UTUC, since it can predict immunotherapy efficacy, but also the screening of patients and their families for germline mutations should also be recommended. While several targetable gene alterations are over-represented in UTUC compared to BUC, they are nonetheless generally present in both diseases. Thus, the rarity of dedicated trials for metastatic UTUC is not an issue, if these patients can be included in trials gathering all UCs. Nevertheless, since the UTUC population is likely to exhibit distinct responses in clinical trials, the respective subgroup data and analyses should be systematically presented.Although the similarities between UTUC and BUC have allowed for the rapid development and use of effective therapies in this rare group of diseases, the more recent understanding of the nature of these “disparate twins” raises critical issues concerning UTUC treatment. The lack of substantial evidence for neoadjuvant chemotherapy in UTUC has to be taken into account in routine practice, and there is an unmet need for dedicated trials in this setting. Comprehensive data from UTUC subgroup patients in mixed clinical trials should also be systematically published. Therapeutic strategies using molecular targets specific to UTUC could also lead to more precise medicine and improved outcomes for these patients.


Human sewage contamination of waterways is a major issue in the United States and throughout the world. Models were developed for estimation of two human-associated fecal-indicator and three general fecal-indicator bacteria (HIB and FIB) using in situ optical field-sensor data for estimating concentrations and loads of HIB and FIB and the extent of sewage contamination in the Menomonee River in Milwaukee, Wisconsin. Three commercially available optical sensor platforms were installed into an unfiltered custom-designed flow-through system along with a refrigerated automatic sampler at the Menomonee River sampling location. Ten-minute optical sensor measurements were made from November 2017 to December 2018 along with the collection of 153 flow-weighted discrete water samples (samples) for HIB, FIB, dissolved organic carbon (DOC), and optical properties of water. Of those 153 samples, 119 samples were from event-runoff periods, and 34 were collected during low-flow periods. Of the 119 event-runoff samples, 43 samples were from event-runoff combined sewer overflow (CSO) influenced periods (event-CSO periods). Models included optical sensor measurements as explanatory variables with a seasonal variable as an interaction term. In some cases, separate models for event-CSO periods and non CSO-periods generally improved model performance, as compared to using all the data combined for estimates of FIB and HIB. Therefore, the CSO and non-CSO models were used in final estimations for CSO and non-CSO time periods, respectively. Estimated continuous concentrations for all bacteria markers varied over six orders of magnitude during the study period. The greatest concentrations, loads, and proportion of sewage contamination occurred during event-runoff and event-CSO periods. Comparison to water quality standards and microbial risk assessment benchmarks indicated that estimated bacteria levels exceeded recreational water quality criteria between 34 and 96% of the entire monitoring period, highlighting the benefits of high-frequency monitoring compared to traditional grab sample collection. The application of optical sensors for estimation of HIB and FIB markers provided a thorough assessment of bacterial presence and human health risk in the Menomonee River. Human sewage contamination of recreational waterways is a potential health concern, especially in urban waterways throughout the United States and world. Sources of sewage contamination include sanitary and combined sewer overflows, as well as leaking sewage conveyance systems caused by aging infrastructure and sewer misconnections, which can lead to contamination in the stormwater system and receiving waterways ,2. A totE. coli, enterococci, and fecal coliforms [Bacteroides and human Lachnospiraceae [Bacteroides), uncertainties involved in these relations, reliability of sensors in long-term deployments, and transferability of optical sensor-to-sewage relationships among watersheds. Continuous fixed-point deployment of such optical fluorescent sensors, along with water collection and analysis of HIB when coupled with flow measurements will help to expand the information regarding the utility of optical sensors and fill gaps in knowledge for techniques to estimate sewage concentrations and loads to receiving water bodies.Traditionally, fecal indicator bacteria (FIB) such as oliforms ,10 and aoliforms ,12 have piraceae ,14, pharpiraceae , and humpiraceae ,17. Althpiraceae –20. A rapiraceae –26. Realpiraceae . Municippiraceae ,26,28–30The overall goal of this study was to use optical sensor technology to characterize the dynamics of non-effluent human sewage contamination continuously on a fine time scale in the Menomonee River. Specific objectives are as follows: 1) implement an in-situ sensor package comprised of commercially available off the shelf optical fluorometer sensors to measure specific optical properties of water, 2) collect water samples for HIB and FIB concurrently with measurements of optical properties of water during all seasons and throughout different hydrologic conditions 3) use resulting optical data to develop surrogate regressions for estimation of magnitude, and variability of HIB and FIB on a fine time scale and determine frequency of exceedance of primary-contact recreation standards, 4) estimate the proportion of sewage contamination within the stream as determined by estimation of HIB; and 5) estimate continuous loadings of sewage contamination in the stream.th St. at Milwaukee, Wisconsin, U.S. Geological Survey (USGS) station identification number, 04087142 (hereafter referred to as MRM). The sampling location was along the south bank of the Menomonee River 1.15 miles (1.85 kilometers) upstream from the confluence with the Milwaukee River. The watershed draining to MRM was chosen because it contained no upstream water reclamation facility (WRF) effluent locations, allowing the evaluation of contamination from illicit discharges and imperfect sanitary conveyance infrastructure. Additionally, the sampling location was within the combined sewer service area and periodically received discharge from Combined Sewer Overflows (CSO) (The study was conducted from November 2017 to December 2018. Samples were collected at the Menomonee River at 16ws (CSO) .Escherichia coli  and culture measurements for fecal coliforms (FC). Analysis of HIB included qPCR measurements of human Bacteroides (HB) and human Lachnospiraceae (L3). Optical properties of water included fluorescence and absorbance spectral analysis.Water samples were collected monthly between December 2017 and December 2018 during periods of low flow and during periods of increased flow from rainfall and snowmelt. Samples were analyzed for three FIBs, two HIBs, dissolved organic carbon (DOC), and optical properties of water. Analysis of FIB included culture and quantitative polymerase chain reaction (qPCR) measurements of enterococci (EN) and Multiple discrete samples were collected during each sampling event to represent variability throughout the runoff hydrograph or during a 24-h low-flow period. Discrete samples were collected using a refrigerated automatic sampler and Teflon-lined polyethylene sample tubing . An alternating 24-bottle configuration was used to allow for sequential sample collection of 300 ml samples for optical properties and DOC (round glass bottle) and bacteria (autoclaved wedge-shaped polypropylene bottle) for a potential 12 samples in each bottle type. Four to six samples were collected during individual 24-h low-flow sampling periods, and six to eleven samples were collected during individual runoff events, varying by flow response and duration of event.Human sewage influent from one WRF was sampled monthly throughout the year to equally represent sewage influent in each season . A totalImmediately upon arrival to the laboratory, a volume of 200 mL from each surface water sample was filtered through a 0.22 μm pore mixed cellulose ester filter  with the exception of three samples that had a sample volume of 100 mL filtered. Filters were then folded and placed in 2 mL screw-cap vials and immediately stored at -80°C until DNA extraction. Additional details for computing the limit of quantification based on the volume of water filtered are presented in Bacteroides  [Lachnospiraceae  [Escherichia coli (EC) [For qPCR analyses, DNA was extracted from frozen filters using MPBIO FastDNA® SPIN Kit for Soil  within six months of filtering. Prior to extraction, salmon testes DNA was added to extraction buffer according to USEPA Method 1611 . In prev, HF183) , human LLachno3) , enterocLachno3) , and Escoli (EC)  assays a2 = 0.998; the L3 assay standard curve slope = -3.45, y = 38.38, efficiency 94.99%, and r2 = 0.996; the EN assay standard curve slope = -3.43, y = 39.77, efficiency 95.82%, and r2 = 1.00; the EC assay standard curve slope = -3.48, y = 39.27, efficiency 93.76%, and r2 = 0.999. Additional details for DNA extraction, qPCR assays, and culture-based analysis are provided in Assays for HB, L3, EN, and EC were carried out by Applied Biosystems StepOne Plus Real-Time PCR System Thermal Cycling Block  with Taqman hydrolysis probe chemistry, and assay conditions followed manufacturer’s instructions. Inter-run controls consisted of environmental samples that were previously run on a minimum of three separate occasions. Concentrations were determined from standard curves. The HB assay standard curve slope = -3.36, y = 37.11, efficiency 98.62%, rStream water from the 300 mL glass bottle was used for dissolved organic carbon (DOC) analysis and simultaneous analysis of fluorescence and absorbance . DOC and optical analysis were completed at the U.S. Geological Survey Upper Midwest Water Science Center in Madison, Wisconsin. Environmental samples for DOC and optical analyses were filtered  into two pre-combusted 40 ml amber glass vials. Samples were stored in the dark at 4°C until sample analysis. All samples for optical analysis were analyzed within 5 days of collection, and DOC samples were analyzed within 14 days of collection .CSH analyzer, coupled with the Shimadzu ASI-V auto sampler. The non-purgeable organic carbon (NPOC) analysis method was employed, in which samples were sparged with 2 M HCl to remove all inorganic carbon prior to combustion. Based on a 1 mg C L-1 DOC standard  measured from 2017 to 2018, a method detection limit (MDL) of 0.108 mg C L-1 was computed.DOC samples were analyzed using a Shimadzu TOC-VFluorescence excitation-emission matrix (EEM) and absorbance scan measurements were performed using a Jobin Yvon Aqualog benchtop Spectrofluorometer . EEMs were generated using excitation (ex) wavelengths of 240–800 nm at intervals of 3 nm and emission (em) wavelengths of 247–827 nm at intervals of 2.33 nm with an integration time of 1 s and a CCD gain set at medium. For absorbance at each wavelength and select fluorescence signals, the minimum reporting limit (MRL) was calculated as the mean absorbance, or fluorescence signal, for the field blank samples (ultra-pure 18.2 megohm water) plus three standard deviations. The MRLs for the select absorbance and fluorescence signals for the present study are available online [Two quality assurance field blank samples were collected; DOC and optical property results were below the method detection levels. Additional information on quality assurance sample collection can be found in Three commercially available optical sensor platforms were included in a custom flow-through system at MRM that was designed to deliver unfiltered water for real-time measurement of turbidity, water temperature, and six unique measurements of optical properties . A WET LThe potential for L3 and HB assays to cross-react with samples from other animal species  does exist . TherefoRaw in-situ field sensor time-series data were foul- and drift-corrected following approved U.S. Geological Survey techniques and methods ,44. BrieExamination of the relation between sensor-measured values and laboratory-measured values indicated that temperature and turbidity effects on FDOM results likely influenced final values. Data were therefore adjusted using temperature and turbidity based on established methods: This included a linear relationship with the difference in field-measured temperature and the temperature at which samples were analyzed in the laboratory ,46 and atau statistic [Statistical significance was determined with a pair-wise Wilcoxon rank sum test with corrections for multiple comparisons for FIB and HIB concentrations . Statisttatistic –52. In aRegression models were developed to explore the relationship between the optical signals , turbidity, mean one-hour flow from the MRM sample location, seasonal interaction variables (explanatory variables), and three FIB, two HIB, and one sum of HIBs (sHM) bacteria marker measurements (response variables). Sensors S2-F and S3-CF were correlated (r = 0.896). To avoid overfitting the model, only one of these signals was used in model development: S2-F was chosen over S3-CF because S2-F had a more complete data record than S3-CF, and the data record for S3-CF required more extensive post-monitoring adjustments to account for biofouling that occurred in the absence of a wiper system. Sensor S2-F had a wiper system that minimized biofouling.The model selection process included several steps to minimize prediction error. Given the prevalence of censored data in the response variable results (bacteria), survival regression with an assumed Gaussian distribution for the response variables was used for development of models. The response data were log-base 10 transformed. Explanatory variables for the optical sensors, S1-CF, S1-A, and S2-F, also included seasonal variables (sine and cosine of (Julian day/365.25)2π) as interaction terms to account for the potential of background dissolved organic matter changes through the year, consistent with a previous effort that has demonstrated this approach to be effective . ResultsThe non-CSO model was used for estimating concentrations except during six CSO discharge periods . The proportion of sewage estimates are unit-less and represent an estimate of sewage within the stream. The proportion of sewage for each bacteria marker was computed by dividing the estimated concentration for each bacteria marker by the mean concentration of the bacteria marker in WRF sewage influent. A mean annual proportion of sewage was computed for sHM by taking the sum of daily sHM loads and dividing by the sum of daily MRM streamflow to get the average annual sHM estimated stream concentration, which was then divided by the average WRF influent concentration. Using the proportion of sewage and MRM streamflow, an estimate of the annual volume of sewage delivered to the MRM sampling location was computed.Bacteria concentration estimates resulting from this study can be used to evaluate the potential for human health risk. The Menomonee River in Milwaukee, Wisconsin flows into the Milwaukee River estuary and can have a negative impact on these downstream recreational waters. First, recreational water quality criteria have been developed by the U.S. Environmental Protection Agency (U.S. EPA) for fecal indicator bacteria . Second,E. coli and enterococci, have been established by the U.S. EPA for primary contact designated uses [E. coli in fresh waters.Recreational water quality criteria for the FIB, ted uses . For theted uses . Data frIn addition, human-associated indicators of fecal contamination (HB) have been used to monitor human sewage impacted waters ,38,58,59Turbidity, water temperature, and fluorescence sensors were deployed from November 2017 to December 2018, and 153 discrete water samples were collected from December 2017 to December 2018, to evaluate real-time sensor measurements and to quantify FIB, HIB, and laboratory-measured optical properties of water. Of those 153 water samples, 119 samples were collected during event-runoff periods and 34 were collected during low-flow periods. Further, of the 119 event-runoff samples, 43 samples were collected during CSO-influenced event-runoff periods (referred to as event-CSO periods) . In addiCorrected time-series for field sensors S1-A, S1-CF, and S2-F, along with mean one-hour streamflow and turbidity were used as explanatory variables in the regression modeling to estimate FIB and HIB . Final cMedian concentrations of L3 and HB in stream samples were 1,100 and 1,800 CN/100 mL with detection rates of 79% and 89%, respectively . Median Concentrations of EN (CN/100 mL) in WRF influent samples were on average 1.5 orders of magnitude greater than the other HIB and FIB markers with all bacteria markers occurring in 100% of WRF influent samples  to 0.028 (2.8%) sewage content in the stream depending on the stream conditions. The flow-weighted annual mean proportion of sewage for CSO periods were an order of magnitude greater, 0.0011 (0.11%), then during non-CSO periods 0.00034 (0.034%) for the sHM.The proportion of sewage in the Menomonee River was estimated using the mean WRF influent concentration for the study period and the continuous estimated concentrations for HIB markers and sHM  [Previous monitoring and modeling with human-associated bacteria in the Great Lakes region, including the Menomonee River watershed, indicated that the incidence of human-specific pathogenic viruses increased with human-associated bacteria concentration  . The ava3 during the study period using sHM loadings. For individual HIB, there was not much difference in sewage contamination volume estimates using HB (4.9 million m3) as compared to L3 (5.5 million m3). Despite the consistency in results between HIB markers, including more than one measure of sewage influence is warranted given that each technique has associated uncertainties: these human-associated markers do have potential to cross react with fecal material from other animals even though they are thought to be primarily human-derived [HIB loads have previously been computed during defined monitoring periods on the Milwaukee River using discrete samples collected during baseflow and runoff events ,37. The -derived ,66–68.The use of surrogate models is not uncommon for predicting FIB in a more timely manner than the 18–24 hours that it takes for laboratory analysis. This has most commonly been done for prediction of FIB using models developed with explanatory variables chosen from web-available data , manually measured physical and water quality characteristics, or deployed water quality sensors  with telemetry at beaches –71 and i−7 (0.000026%) and 0.028 (2.8%) estimated from Dec 2017 to Dec 2018 in the current study. Applying this approach to sewage content estimation in multiple areas of a watershed could help facilitate identification of areas and time periods with the greatest sewage contamination, enabling remediation projects to focus efforts. For example, consistent with previous research [Previous research for this monitoring location used two human-associated bacteria, three human-specific viruses, and the sum of the human-specific viruses (Sum of HSV) to compute the mean sewage contamination from all samples collected (low-flow and runoff-event periods) from April 2009 to March 2011 . The estresearch ,36,37, lThe current study established that the relation between optical properties and sewage was strengthened by including seasonality, flow conditions, and the nature of sewage contamination (CSO vs non-CSO periods) in the modeling structure . PreviouThe monitoring and modeling system described in the current research indicates that HIB, FIB, and sewage contamination can be estimated with these methods, but there are limitations of the monitoring system and the models derived from resulting data that should be understood. For working models used for real-time predictions, initial discrete HIB and FIB measurements are necessary for model development and continuing discrete HIB and FIB measurements are necessary for model validation or refinement after the initial model development. In addition, it can be a challenge to represent the full range of HIB and FIB concentrations with discrete samples, and without representing the full range of variability, model extrapolation can have high levels of uncertainty. Therefore, it is judicious to limit the extent of extrapolation beyond the model calibration range. It has been demonstrated that models can be developed for a diverse variety of watersheds , but to In addition, it is important to understand that the variables used to estimate bacteria in this study are surrogates that are not direct measurements of bacteria presence. Models provided here are a statistical simplification of the relation between bacteria concentrations and the optical properties of water, turbidity, and season. These surrogate relationships may change over time based on changes in the watershed, and even without any changes in the watershed, the fit of each model has uncertainty associated with it. Sources of uncertainty may include climate and weather-related variability , hydrologic variability , seasonal variability, anthropogenic activities , non-human fecal matter interferences , and more ,86–89. VE. coli, enterococci, and HB, and regardless of the benchmark bacteria and concentration level chosen, the concentration exceedances were more prevalent during colder-weather months as opposed to warmer months with the exception being during runoff events, active CSO periods, and later in the summer. The monitoring and modeling system described in the current research indicates that HIB, FIB, and sewage contamination can be estimated with these methods, but there are limitations of the monitoring system and the models derived from resulting data that can be watershed specific. As a result, applying this technique elsewhere will require validation in each watershed to support development of these models. However, as shown in the current study, the application of optical sensors for estimation of HIB and FIB markers did provide a thorough assessment of bacterial presence, and human health risk in the Menomonee River.Surrogate models developed in the current study provided a means to estimate bacteria concentrations over short temporal scales, incorporating variability driven by seasonal, diurnal, hydrologic, and other environmental changes. Recreational water quality criteria for primary contact designated uses were assessed for S1 Appendix(DOCX)Click here for additional data file.