To support the clinical development of polatuzumab vedotin, we characterized the distribution, catabolism/metabolism, and elimination properties of polatuzumab vedotin and its unconjugated MMAE payload in Sprague Dawley rats. Several radiolabeled probes were developed to track the fate of different components of the ADC, with 125I and 111In used to label the antibody component and 3H to label the MMAE payload of the ADC. Following a single intravenous administration of the radiolabeled probes into normal or bile-duct cannulated rats, blood, various tissues, and excreta samples were collected over 7–14 days post-dose and analyzed for radioactivity and to characterize the metabolites/catabolites. The plasma radioactivity of polatuzumab vedotin showed a biphasic elimination profile similar to that of unconjugated polatuzumab but different from unconjugated radiolabeled MMAE, which had a fast clearance. The vast majority of the radiolabeled MMAE in plasma remained associated with antibodies, with a minor fraction as free MMAE and MMAE-containing catabolites. Similar to unconjugated mAb, polatuzumab vedotin showed a nonspecific distribution to multiple highly perfused organs, including the lungs, heart, liver, spleen, and kidneys, where the ADC underwent catabolism to release MMAE and other MMAE-containing catabolites. Both polatuzumab vedotin and unconjugated MMAE were mainly eliminated through the biliary fecal route (>90%) and a small fraction (<10%) was eliminated through renal excretion in the form of catabolites/metabolites, among which, MMAE was identified as the major species, along with several other minor species. These studies provided significant insight into ADC’s absorption, distribution, metabolism, and elimination (ADME) properties, which supports the clinical development of POLIVY.Polatuzumab vedotin (or POLIVY The development of antibody–drug conjugates (ADCs) has accelerated in recent years, resulting in many advancements to this class of therapeutic molecules . PolatuzThe pharmacokinetics (PK) of polatuzumab vedotin in rodents and cynomolgus monkeys were described by Li et al. , who shoUnlike the therapeutic antibodies, where they are often degraded into amino acids, small peptides, or small carbohydrates that are readily eliminated by renal excretion or return to the nutrient pool with minimal biological effects or safety concerns, ADCs contain a potent cytotoxic agent and are structurally more complex. Therefore, in addition to the characterization of the antibody and the cytotoxic payload, the understanding of linker stability is also critical, as a premature release of the payload can cause systemic toxicity ,8,9.For oncology indications, ADCs are likely to be used in combination with other chemotherapy agents that may interact with various cytochrome P450 (CYP) enzymes and drug transporters. Therefore, identifying the key catabolites of the ADC is valuable for assessing potential DDIs, determining the key drivers for efficacy and toxicity, and informing on which key analytes should be measured in a clinical setting.There are various approaches that are used to characterize the ADME properties of ADCs ; some grIn this study, we systemically characterized the distribution, catabolism, and elimination (DME) properties (no absorption with intravenous dosing) of polatuzumab vedotin and MMAE in rats using multiple radiolabeled probes to track the distribution, metabolism/catabolism, and elimination of polatuzumab vedotin, as well as the unconjugated MMAE payload. Rats were chosen as the model species as they do not cross-bind with polatuzumab vedotin, thus offering a general DME profile that is similar to patients who would have been dosed clinically in the linear range. These data provided insights into the DME properties of polatuzumab vedotin to support the clinical assessment of the potential DDIs.The polatuzumab and polatuzumab vedotin used for the in vivo studies presented here were generated at Genentech Inc. . The unconjugated polatuzumab is a humanized monoclonal IgG1 antibody that binds to human CD79b and does not cross-react with rodent CD79b. Polatuzumab vedotin was made as described previously .v/v (mobile phase B) run at a flow rate of 0.8 mL/min over a 12 min linear gradient with UV monitoring at 280 nm.The conjugation conditions were chosen to achieve an average drug-to-antibody ratio (DAR) of approximately 3.4–3.5. The ADC protein concentrations were calculated using absorbance at 280 nm (a 320 nm reference) and the molar extinction coefficient of the antibody. The average DARs were calculated from the integrated areas of the DAR species, which were resolved using hydrophobic interaction chromatography (HIC) on an analytical column . The HIC method used 1.5 M ammonium sulfate in 25 mM potassium phosphate pH 7.0 (mobile phase A) and 25 mM potassium phosphate pH 7.0 containing 25% isopropanol 3H]-MMAE was obtained from Seagen Inc. with a specific activity of 33.8 μCi/μg. [3H]-MMAE with the MC-vc-PAB linker with a specific activity of 19.9 μCi/μg  was conjugated to mAb, as described in the above 3H]-MMAE ADC had a specific activity of 33.3 μCi/mg and a DAR of 3.4  or [111In] with 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA)  as described previously [125I] or [111In]-DOTA was radiolabeled with intact polatuzumab vedotin (antibody with linker and payload).The resulting  radiolabeling, sodium-125iodine was added to an iodination tube  and, subsequently, conjugated to the polatuzumab vedotin. Then, to remove the excess unconjugated [125I], the solution was passed through a NAPTM-5, sephadex G-25 DNA grade, column . Similarly, DOTA was first conjugated to polatuzumab vedotin and purified with a NAPTM-5 column. The resulting DOTA-polatuzumab was then added to [111In] and chelated with ethylenediaminetetraacetic acid (EDTA) and purified with a NAPTM-5 column. The radiolabeled polatuzmab vedotin was characterized for purity, binding, specific activity, etc., before dosing the animals.For -MMAE mixed with MMAE at 200 μg/kg (≈80 μCi/kg radioactivity level). Blood samples (≈0.3 mL) were collected from the jugular cannula at multiple time points post IV administration. To obtain plasma, blood was collected into lithium heparin tubes and gently inverted several times to properly mix the anticoagulant (lithium heparin). Tissue samples from different organs  were also collected from animals euthanized via exsanguination under anesthesia at the designated time points. Of the 15 rats, 6 rats with terminal time points at 1 and 7 days post-dose  were housed in metabolism cages for the collection of urine and feces.Twenty-one female (15 with jugular vein cannulation and 6 with bile duct cannulation (BDC)) 6–8-week-old Sprague Dawley rats, each weighing about 200 g, were obtained from Charles River Laboratories. Fifteen rats with jugular vein cannulation were given an intravenous (IV) bolus dose of [3H]-MMAE mixed with MMAE at 200 μg/kg (16 μCi/rat radioactivity level). Bile was collected through the bile duct cannula from animals for 7 days at designated time intervals for the purpose of metabolite profiling and identification.The rats were housed in metabolism cages for one or two nights for acclimatization before dosing and were also kept in the metabolism cages post-dose. Urine and feces were collected from the rats at pre-dose, every 4 h from 0 to 8 h post-dose, once between 8 and 24 h, and then every 10 or 14 h until day 7 for the day 7 terminal time point animals. After each collection of urine and feces, the cages were rinsed with 10 mL of 50/50 methanol/water to remove any trace contaminants from previous collections. Another six rats with both a jugular vein cannula and a bile duct cannula were also given an IV bolus dose of -MMAE, the bile samples were analyzed across time points that covered the majority of the radioactivity  excreted. Due to the low radioactivity recovered in the urine, the urine samples were analyzed for up to 8 h. Both the bile and urine samples were investigated using Accela UPLC coupled with a Linear Trap Quadrupole (LTQ)-Orbitrap Velos system  and an online β-RAM 5C radiodetector  for radioprofiling.To characterize the metabolic profile of [3H]-MMAE, only the intact [3H]-MMAE was quantified.Chromatographic separation was performed on a Luna C18 column  with mobile phases A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) at a constant flow rate of 1 mL/min. The gradient was as follows: initial holding at 5% B for 2 min, increased to 15% B at 4 min, 42% B at 44 min, 75% B at 49 min, 95% B at 50 min, holding at 95% B until 55 min, decreasing to 5% B at 55.1 min, and then column re-equilibration until 60 min. The flow was split 3:1 post-column for radiomeasurements and mass spectrometry, respectively. As the majority of the metabolite was the intact [125I]- and [111In]-conjugated to the antibody of polatuzumab vedotin or polatuzumab vedotin conjugated with [3H]-MMAE. In the 125I and 111In study, sixty femoral-vein-cannulated female Sprague Dawley rats aged 6–8 weeks (≈200 g) were obtained from Charles River Laboratories and were divided evenly into four groups (n = 15 each group). In the first group, a mixture of [125I]- and [111In]- polatuzumab vedotin was given as a tracer  IV bolus dose.The distribution of polatuzumab vedotin was investigated in the rats using the [125I]- and [111In]- unconjugated polatuzumab antibodies instead of polatuzumab vedotin with the tracer and tracer plus 10 mg/kg unlabeled material, respectively. Blood and various tissues were collected at multiple intervals up to 14 days post-dose. To obtain the plasma, the blood was deposed into lithium heparin tubes and gently inverted several times to properly mix the anticoagulant. The total radioactivity of all samples was measured using a Perkin Elmer Wizard2 analyzer .The second group of rats received an IV bolus dose of the radiolabeled tracer polatuzumab vedotin, along with a 10 mg/kg dose of unlabeled polatuzumab vedotin. For the third and fourth groups, the rats were dosed with [3H]-MMAE. Twenty-one female 6–8-week-old Sprague Dawley rats  weighing about 200 g each were obtained from Charles River Laboratories. Fifteen normal rats were given an IV bolus dose of polatuzumab vedotin conjugated with [3H]-MMAE at 10 mg/kg (30 μCi/rat radioactivity level). Blood samples (≈0.3 mL) were collected from the tail vein of the animals at the terminal time points post IV administration.The tissue distribution of the polatuzumab vedotin was also assessed using polatuzumab conjugated with -MMAE at 10 mg/kg (30 μCi/rat radioactivity level). Bile was collected through the bile duct cannula from animals for 14 days at designated time intervals.Urine and feces were collected from the rats at pre-dose, every 4 h from 0 to 8 h post-dose, once between 8 and 24 h, and then every 10 to 14 h until day 7 or day 14, depending on the animal’s terminal time point. After each collection of urine and feces, the cages were rinsed with 10 mL of 50/50 methanol/water to remove any trace contaminants from previous collections. Another six rats with both a jugular vein cannula and a bile duct cannula were also given an IV bolus dose of polatuzumab vedotin conjugated with [TM 2900TR liquid scintillation analyzer. The processed samples underwent protein precipitation with a 4:1 (v/v) acetonitrile-to-sample ratio to obtain the soluble radioactivity (presumed as deconjugated [3H]-MMAE or its metabolite(s)) and the precipitable radioactivity (presumed as [3H]-MMAE still conjugated to polatuzumab) measured using a Tri CarbTM 2900TR liquid scintillation analyzer.The total radioactivity levels of the processed blood (plasma and cell pellet), tissues, urine, feces, and bile samples were measured using a Tri Carb3H]-MMAE, bile and urine samples from the study with [3H]-MMAE polatuzumab vedotin were pooled across time points that covered >90% of the radioactivity excreted in that route and were investigated using a nanoACQUITY LC system  coupled with an LTQ-Orbitrap Velos system  and an online β-RAM 5C radiodetector  for radioprofiling.To characterize the catabolic profile of polatuzumab vedotin conjugated with  MMAE was first assessed following a single IV administration of 0.2 mg/kg [3H]-MMAE to rats. A rapid reduction of the MMAE radioactivity level was observed in the systemic circulation . The radioactivity level peaked at 10 min in most tissues; however, the exposure of unconjugated MMAE in tissues could only be maintained for a short period as the radioactivity was barely detectable by 144 h B.To understand the elimination mechanism of unconjugated MMAE, a mass balance study was performed in rats. At 144 h post single IV administration, 98.3% (±6.00%) of the injected dose was excreted in the feces, while 8.61% (±3.16%) of the injected dose was recovered in the urine . A complIn a separate study using bile-duct-cannulated rats to identify the metabolite, the collected bile samples showed similar radioactivity levels after 144 h, where 103% (±8.07%) of the injected dose was recovered. In the bile samples, six metabolites were identified , which i125I and 111In and the MMAE component with 3H in Sprague Dawley rats, which is a non-binding species. First, the systemic exposure of [125I] and [111In]-polatuzumab vedotin (tracking mAb component) was compared to the [125I] and [111In]-unconjugated polatuzumab antibody. The [125I]-polatuzumab vedotin showed a similar biphasic elimination profile to that of the radiolabeled unconjugated polatuzumab antibody following a single IV dose at both dose levels of a tracer only (≈3 μg/kg) and a tracer plus 10 mg/kg of unlabeled materials   A. -MMAE-polatuzumab vedotin also showed a biphasic elimination profile similar to the one with radioprobes tracking the antibody component , [3H]-MMAE-polatuzumab vedotin mainly existed in the plasma fraction of the blood instead of the RBC pellet . While both 125I and 111In probes can be used to assess the tissue distribution, only 125I can be released back to the extracellular space after the intracellular degradation of the antibody, while 111In residualized inside cells as the DOTA cannot cross the cell membrane.The tissue distribution of polatuzumab vedotin was also investigated following a single IV injection of radiolabeled polatuzumab vedotin with 125I]- and [111In]-polatuzumab vedotin was administered to rats, multiple tissues were recipients of the distribution as rats are not a binding species, with relatively higher radioactivity distribution observed in well-perfused organs, including the lungs, heart, and kidneys  of the total injected radioactivity was recovered in the feces samples, while only 5.21% (±1.03%) was found in the urine , followed by C4, C5, and C3, respectively.In the separated BDC rat study, the collected bile samples accounted for about 67% of the injected radioactivity dose, which corroborated with the feces data in the normal rat study. The lower recovery of radioactivity in BDC rats was likely due to the deteriorating health of the BDC rats after surgery. In these collected bile and urine samples, a total of eight catabolites (C1 to C8) of the polatuzumab vedotin were observed through liquid chromatography and mass spectrometry profiling with an online radiodetector, including MMAE (C7). The structures of six catabolites were identified and are summarized in ADCs are a therapeutic class that utilizes a highly potent cytotoxic payload that has a narrow therapeutic window if these cytotoxic payloads are administered alone. Upon the conjugation of cytotoxic payloads to a monoclonal antibody via a linker, the ADC improves in specificity, allowing for specific delivery to a tumor target and reducing the systemic exposure and normal tissue uptake, thus, increasing the therapeutic window. As such, it is important to gain ADME knowledge of the ADC and its cytotoxic payload.3H into the payload, while the antibody component, namely, polatuzumab, was radiolabeled with 125I or 111In.This study used a radiolabeling technique in a systematic approach with a preclinical species, namely, rats, to answer key questions related to the distribution, catabolism/metabolism, and elimination of polatuzumab vedotin and its cytotoxic payload, namely, MMAE, as a case study to aid in clinical development. Since an ADC contains multiple components, we took both large molecule and small molecule features into consideration for ADME studies by radiolabeling each component, as previously described ,13, wher3H]-MMAE exhibited fast systemic clearance and showed a wide distribution into multiple organs, as expected. Highly perfused organs, such as the liver, kidneys, and lungs, exhibited a higher distribution, while poorly perfused tissues, such as the muscles and skin, showed a lower amount of radioactivity. Interestingly, the brain showed minimal exposure, likely because it was protected by the blood–brain barrier. Rapid tissue distribution was followed by fast elimination with no persistency observed in any tissue examined in the study. Upon further analysis of the radioactivity in the plasma and cell pellet fraction, we found that the radioactivity level in the blood was six- to eight-fold higher than in the processed plasma, indicating that MMAE had high red blood cell partitioning, which is consistent with the results from the in vitro assessment (manuscript under preparation).Polatuzumab vedotin utilized a protease cleavable MC-vc-PAB linker joining a polatuzumab antibody and an MMAE cytotoxic payload. Upon internalization into the cell, we expected that MMAE would be released from the ADC via the cleavable linker via cathepsin B or through proteolytic degradation . TherefoWhile MMAE’s high blood partitioning can act as a depot for MMAE to remain systemically, the level of MMAE in the blood was much lower compared to the tissues we examined, and thus, its contribution to the tissues was minimal. The major elimination route of the IV administered unconjugated MMAE was through biliary–fecal secretion, where 98.3% (±6.00%) of the injected dose was excreted in the feces, whereas only 8.61% (±3.16%) of the injected dose was recovered in the urine over 7 days. Six metabolites were also observed and identified in the bile with unchanged MMAE as the primary species was eliminated, which accounted for 62.9% of the 74.2% total dosed radioactivity recovered in the bile up to 6 h post-dose.125I or 111In radiolabeling of the antibody. Following an IV administration of [125I]- or [111In]-polatuzumab vedotin or an unconjugated polatuzumab antibody, the plasma radioactivity–time profile followed a biphasic elimination with a short distribution phase, followed by a long elimination phase that was similar to the PK studies described by Li et al. [125I]- or [111In]-polatuzumab antibodies, both profiles showed a similar pattern.The distribution, catabolism, and elimination of polatuzumab vedotin was investigated in the next set of studies. The method we employed to track the antibody component of the ADC was through i et al. . When thHowever, the radioactivity level of the unconjugated polatuzumab antibody was higher than that of polatuzumab vedotin, which demonstrated that the systemic clearance of ADCs tended to be faster than that of the unconjugated mAbs, which was attributed to the “impact of conjugation” . Similar3H]-MMAE was conjugated to polatuzumab mAb to form the [3H]-MMAE-polatuzumab vedotin. After an IV administration, [3H]-MMAE-polatuzumab vedotin followed a biphasic elimination pattern similar to what was seen for [125I]- or [111In]-polatuzumab vedotin (tracking mAb component) but very different from the profile of unconjugated [3H]-MMAE, where [3H]-MMAE was cleared systemically within hours. This data suggested that the conjugation of [3H]-MMAE to the antibody could retain the exposure of MMAE, allowing for better tissue targeting, such as tumors, for the uptake.To track the fate of the cytotoxic payload, -MMAE-polatuzumab vedotin demonstrated a similar pattern to that of [125I]- or [111In]-polatuzumab vedotin and -unconjugated mAb, further confirming that the stability of ADC, as well as the antibody component, drove the ADC tissue disposition.Further analysis of the plasma using organic solvent extraction revealed that over 99% of the 3H]-MMAE or other 3H-containing catabolites were released in the tissues. The soluble radioactivity detected in the tissue dosed with [3H]-MMAE-polatuzumab vedotin decreased after the initial peak and throughout the course of the study without persistency over time, except in the dorsal root ganglion . The prolonged exposure of MMAE in the dorsal root ganglion might have contributed to peripheral neuropathy, which was reported as one of the toxic side effects of the MMAE–ADC treatment -MMAE-polatuzumab vedotin was mainly through biliary fecal secretion, with 103 ± 11.8% of the injected dose recovered in the feces and 5.21 ± 1.03% in the urine. Organic solvent precipitation of the feces samples resulted in over 80% of the radioactivity residing in the soluble fraction, suggesting that a catabolite of [3H]-MMAE-polatuzumab vedotin, such as deconjugated [3H]-MMAE or its downstream metabolite, were eliminated in a similar fashion to the unconjugated [3H]-MMAE when directly dosed. Further evidence of this claim was shown when we characterized the catabolites found in urine and bile .The elimination of [3H]-MMAE-polatuzumab vedotin, of which seven were identified. C7, which was identified as unconjugated MMAE, was the most abundant catabolite detected, while five other catabolites (C2 through C6) were also identified as metabolites from the unconjugated [3H]-MMAE study. C8, which was identified as cys-vc-MMAE, was the only catabolite not found in the unconjugated [3H]-MMAE study. Since unconjugated MMAE was the main catabolite eliminated over a long period, the role of the CYP enzyme in the metabolism would be minimal and, thus, would reduce the risk of major DDI adverse effects.There were eight catabolites detected in the urine and bile samples dosed with [As the liver played a critical role in the elimination of MMAE in rats, patients with hepatic impairment may experience an accumulation of MMAE in the body, leading to safety concerns. Limited mass balance data was reported by Han et al., who administered brentuximab vedotin to patients and found that MMAE was mostly eliminated as intact and through the feces , which cIn summary, we systematically characterized the distribution, catabolism, and elimination of polatuzumab vedotin and its unconjugated MMAE payload in rats using multiple radiometric approaches. Our studies demonstrated that polatuzumab vedotin had a stable linker in circulation and confirmed that the antibody component of the ADC dictated the overall ADC disposition in rats. Upon the distribution/internalization into the tissues, ADC underwent catabolism to release MMAE and several other catabolites over time, which were mainly eliminated via the biliary–fecal route, with a minor fraction through the urine in rats.These results imply that the liver plays a more important role than the renal clearance in the elimination of polatuzumab vedotin catabolites; therefore, additional hepatic impairment studies in patients might provide clinicians with further insight. Though unconjugated MMAE was identified as the major catabolite, the overall DDI risk was low given that the systemic and liver unconjugated MMAE levels were low, in conjunction with other assessments (in vitro DDI study not shown in this manuscript). In conclusion, these studies not only provide information to support the clinical development of polatuzumab vedotin but also provide a comprehensive and systematic approach to characterizing the ADC DME properties, which may help the development of other ADC therapeutics.


In 228 patients with ventricular pacing burden >40%, LBBAP at last follow-up resulted in decreased left atrial diameter (LAD)  while RVP produced decreased left ventricular ejection fraction  when compared to baseline. After adjusting for age, the presence of atrial fibrillation, and other clinical factors, LBBAP was still associated with a decrease in LAD . We conclude that LBBAP might result in more preserved echocardiographic outcomes than RVP.The long-term lead stability and echocardiographic outcomes of left bundle branch area pacing (LBBAP) are not fully understood. This study aimed to observe the mid-long-term clinical impact of LBBAP compared to right ventricular pacing (RVP). Consecutive bradycardia patients undergoing LBBAP or RVP were enrolled. Pacing and electrophysiological characteristics, echocardiographic measurements, and procedural complications were prospectively recorded at baseline and follow-up. LBBAP was successful in 376 of 406 patients (92.6%), while 313 patients received RVP. During a mean follow-up of 13.6 ± 7.8 months, LBBAP presented with similar pacing parameters and complications to RVP, except a significantly narrower paced QRS duration (115.7 ± 12.3 ms vs. 148.0 ± 18.0 ms,  Left bundle branch area pacing (LBBAP), first reported by Huang et al. , has emeConsecutive patients receiving LBBAP or RVP procedures for symptomatic bradycardia were prospectively enrolled at Fuwai Hospital since 2019. All patients were indicated for pacemaker implantation per the American College of Cardiology, American Heart Association, and Heart Rhythm Society guidelines . PatientLBBAP was performed by using SelectSecure pacing lead  and a fixed-curve sheath  as previously published ,11. The RVP was performed with the active fixation lead positioned at the RV septum. Fluoroscopic radiographs from 45° left anterior oblique were used to confirm the lead position. The procedure and fluoroscopy duration for RVP lead were recorded from the advancement of the sheath to the end of successful implant of active-fixation pacing lead.Patients were followed up with at 3 months, 6 months, 12 months, and 24 months after implant. Pacing parameters  were recorded and compared between LBBAP and RVP. Echocardiography was performed at baseline, 6 months, 12 months, and 24 months after the procedure by using Vivid E9 systems  to evaluate left ventricular end-diastolic diameter (LVEDD) and left atrial diameter (LAD). Left ventricular ejection fraction (LVEF) was measured using biplane Simpson’s method in two-dimensional transthoracic echocardiography. Device-related complications were continuously tracked, including lead dislodgement, lead perforation, pacing system infection, and other procedure-related complications. t-test or the Mann–Whitney U-test. Categorical variables are reported as numbers and percentages and compared using chi-square or Fisher’s exact test. Paired t-tests were used to compare data at baseline and follow-up. Multiple linear regression analysis was used to investigate the impact of LBBAP and other clinical factors on LAD. A p-value of <0.05 indicates statistical significance. R version 3.5.1  was used to perform all analyses.Continuous variables are presented as mean ± SD or median with interquartile range and compared using Student’s p > 0.05). Compared with RVP, LBBAP was attempted more often in patients with RBBB or LBBB and patients with atrioventricular block (AVB) .A total of 406 patients underwent LBBAP procedures, and 313 patients received RVP. p > 0.05). The median procedural duration for 3830 lead implantation was longer than that in RVP , as was the fluoroscopy time .LBBAP was successfully achieved in 92.6% (376/406) of patients. The remaining 30 patients who failed LBBAP finally underwent LVSP with the lead located in the deep ventricular septum. LBB potential was present in 68.1% of patients with an average P-V interval of 27.7 ± 4.7 ms. The mean Sti-LVAT at the high output was 73.9 ± 13.4 ms, similar to that at low output. During the procedure, ring capture at 2 V/0.4 ms was achieved in 97.3% of patients, with a mean threshold of 1.04 ± 0.65 V/0.4 ms. LBBAP produced a significantly narrower pQRSd than RVP did (114 ± 10.7 ms vs. 148 ± 18.0 ms). As shown in p > 0.05) and remained stable during follow-up (p > 0.05) (p < 0.001) and remained stable during follow-up. LBBAP produced a significantly narrower pQRSd than RVP , and the difference in pQRSd persisted during follow-up (ollow-up A. There  > 0.05) B,C. Howeollow-up D.n = 169) resulted in a significantly decreased LAD  while no effect of RVP (n = 59) on LAD was observed (39.6 ± 6.5 mm vs. 40.8 ± 3.9 mm p > 0.05). The comparison of LAD did not differ significantly at implant between LBBAP and RVP groups. However, the mean LAD at last follow-up in patients with RVP was significantly larger than that in the LBBAP group  (p < 0.001), while LBBAP resulted in a stable LVEF  in patients with VP ≥ 40% (p > 0.05), nor was the tricuspid regurgitation .Echocardiographic parameters did not present a significant difference between LBBAP and RVP during the mean follow-up of 13.6 ± 7.8 months in the total study population A–C. Subg< 0.001) D. In addVP ≥ 40% E. Moderap = 0.036) when compared with RVP. In addition, baseline LAD and LVEF were also correlated with the negative change of ΔLAD (both p < 0.05). Atrial fibrillation was an independent risk factor for the enlarged ΔLAD .Multiple linear regression analysis for the impact of LBBAP on the change of LAD (ΔLAD) are shown in Lead dislodgement requiring lead revision occurred in one patient with LBBAP and two patients with RVP soon after the implantation procedure. One patient suffered from lead perforation after LBBAP procedure and had no symptoms except loss of pacing capture. All lead revision was successfully performed with no further symptoms or signs. RBB injury occurred in 30 of 376 patients (8.0%) and persisted in eight patients (2.1%) before discharge. During the follow-up, another two patients with RVP suffered lead dislodgement one month after the procedure and received successful ventricular lead repositioning. One patient with LVSP suffered an intermittently increased pacing threshold up to 4.0 V/0.4 ms at three months post-procedure due to severe septal fibrosis. Other device-related complications in both groups are summarized in This single-center prospective study demonstrated the mid-long-term lead stability and echocardiographic effect of LBBAP compared with RVP. The main findings are as follows: (1) LBBAP demonstrated favorable lead performance and pacing parameters similar to RVP during mid-long-term follow-up; (2) LBBAP resulted in significantly narrower QRSd, reduced LAD, and preserved LVEF in patients with VP ≥ 40% compared with RVP; (3) lead-related complications of LBBAP were low and similar to that of RVP. Our results provide evidence for mid-long-term lead stability and safety of LBBAP, and the potential effect of LBBAP on cardiac reverse remodeling compared with RVP.Data on LBBAP are limited due to the small sample size and short-term follow-up in most previous studies. Long-term lead stability is a significant concern for LBBAP, which might be affected by continuous myocardial contraction due to the deeply screwed LBBAP lead into the interventricular septum to capture the LBB area. Recently, Huang et al. reported the largest cohort of patients with LBBAP to date , and theMoreover, our study provided comparisons of procedure duration and fluoroscopy time between LBBAP and RVP. The mean procedure duration and fluoroscopy time of LBBAP were slightly longer but close to RVP in our study. Therefore, our results, together with previous studies, manifested the reliable pacing parameters of LBBAP similar to RVP in most patients requiring ventricular pacing.In our study, when patients were further stratified by VP%, a significantly decreased LVEF was found in patients receiving RVP with a high ventricular pacing burden. The deleterious effect of RVP on cardiac function and new-onset atrial fibrillation (AF) is widely known. Compared with RV apex pacing, HBP could result in a more physiological LV electromechanical activation and, consequently, better LA function  and is an = 376). Moreover, transient or persistent RBB injury may occur during the LBBAP procedure. Huang et al. reported that transient RBB injury occurred in 20.4% of patients while persistent RBB injury occurred in 8.9% of patients [Lead-related complications have been reported to be low , includiSeveral limitations need to be mentioned. Firstly, the non-randomized design is the main limitation of this single-center observational study, which necessitates cautious interpretation of our results. Individual option of pacing strategy was based on patients’ choice and physicians’ experience on device implantation. Moreover, although statistically significant differences were observed between the two groups regarding echocardiographic parameters, their magnitude and clinical significance seem limited. Outcomes with more than two years’ follow-up may provide more worthwhile data. Finally, our analysis included only bradycardia patients requiring ventricular pacing, while patients with heart failure were excluded from our study. LBBAP might play a more significant role in patients with heart failure and who require ventricular pacing. Large-scale, multicenter randomized controlled trials would provide robust evidence for the clinical application of LBBAP. If a leadless pacemaker with a suitable helix could be screwed into the ventricular septal and could capture the left bundle branch, the physiological pacing might be achieved without affecting the tricuspid function and pocket-related complications.LBBAP could produce stable pacing parameters and few lead-related complications comparable with RVP during mid-long-term follow-up. Compared with RVP, LBBAP may have a beneficial effect on LA function by reducing LAD in patients with a high ventricular pacing burden.


We present a generally applicable unsupervised approach to this question based on semantic changepoints within a given collection of research papers. We illustrate the approach by a range of examples based on a nascent corpus of literature on COVID-19 as well as subsets of papers from PubMed on the World Health Organization list of neglected tropical diseases. The software is freely available at:  These range from finding the answer to a highly specific question to writing a general review of a topic. One of the motivations for a literature review might be to select a topic for research, where one may choose to perform research in a well-established area, pick an emerging area or aspire to be a pioneer in uncharted territory. Another possible motivation might be for a funding agency to keep track of emerging areas of research that might merit funding in the near future. Yet another motivation might be to keep track of new insights or technologies that address an acute health need such as a pandemic or diseases that are hard to treat effectively.1, clustering2, trend analysis4, citation network analysis5, and machine learning approaches for predicting high impact papers. We present a set of strategies from changepoint analysis and text embedding to address two questions. Which are the papers in a research area that are substantially different from previous work? Which papers are part of a related cluster that is substantially different from previous work? We use infectious diseases from two different time scales to illustrate the approach - COVID-196 over a temporal resolution of weeks, and leprosy7, considered a neglected tropical disease by the World Health Organization. We begin with a description of the methods used, followed by results and discussion, and end with an acknowledgment of the limitations and future work to address them.What if it were possible to identify papers that strayed from the mainstream? While many of these might end up as blind alleys, a subset of these might turn out to be harbingers of innovative, influential, or impactful directions in research. A few of the potential approaches to identify outliers, first-to-report, or first-in-field papers are topic modeling2.The overall summary of the methodology is shown in 2.18 The title, upload date, and abstract were retrieved for each paper published in 2020, yielding a total of 7151 analyzed papers.COVID papers were downloaded from the COVID-19 SARS-CoV-2 Preprints available from bioRxiv in JSON format on 7/31/2020.9.Leprosy papers were retrieved from the National Center for Biotechnology Information E-Utilities API. A list of UIDs for papers where the term “leprosy” appears in the title were retrieved using the ESearch method, and the title, abstract, and publication date for each UID were retrieved with the EFetch method. We only analyzed papers for which the title, abstract, and publication date were available. We also only considered papers published between 1980 and 2019 due to the low number of annual papers (less than 50 per year) published before 1980, yielding a total of 5068 analyzed papers. Plots were generated using the matplotlib package2.210 CountVectorizer tool to convert each title to a Bag-of-Words representation. We calculated the probability of each word in a year using We used the scikit-learnThe yearly entropy of word proportions in titles  was calculated by Shannon entropy, which is a popular measure of information content or variability of a distribution.2.313 The advantage of the bcp method is that it also provides a probability of there being a changepoint at a given time point. We performed changepoint detection for each word in the Bag-of-Words model of paper titles to analyze the word frequency per title vs time . Abstract changepoints were calculated in the same manner, except using abstracts as input to the Bag-of-Words model instead of titles.Changepoint detection aims to identify the point at which the probability distribution of a sequential variable changes. Changepoint analysis was conducting using the bayesian-changepoint-detection (bcp) Python package.2.414 Word clouds were created using the Wordcloud Python package,15 with weights for each word corresponding to the difference in frequency per title between Group B and Group A. Positive differences (increases in word frequency) are colored black, and negative differences (decreases in word frequency) are colored red.Differential word clouds are visual depictions of changes in research paper titles between two years, denoted Year A and Year B. Two groups are selected from the paper titles: Group A contains the titles of all papers published in or before Year A, and Group B contains the titles of all papers published in Year B. The Bag-of-Words model is applied to each group to determine the frequency per title of each word, and stop-words appearing in the NLTK stop-words set are removed.2.516. Abstracts were embedded in the same manner, except using the abstract as the input instead of the title. The embeddings were visualized using Principal Component Analysis as implemented by Scikit-Learn.Titles were first pre-processed by converting all words to lowercase and removing punctuation and stop-words found in the NLTK punctuation and stop-word sets. The processed titles were then converted into 700-dimensional vectors using the BioSentVec model2.6We use the following approaches to detect potentially novel papers or subtopics in one temporal window (or subcollection) with respect to another. For instance, one may compare papers in 2020 with all preceding years (novel compared to entire research legacy). Alternatively, one may compare papers published in 2020 with those published in 2019 (a change in direction of research compared to recent past). We employ 4 different strategies: T1, T2, Y1 and Y2 see .2.6.1We first analyzed the distribution of the pair-wise distance between all titles in embedded space for the COVID corpus and examined pairs sorted by distance to determine suitable thresholds for relatedness S-rel and, conversely, S-unrel for being semantically unrelated with high probability. There is a grey area in between the two thresholds where pairs of titles at the same distance from each other are sometimes related, and sometimes not. We confirmed the consistency of the thresholds by repeating the calibration check with the leprosy dataset. To determine if a title T is novel relative to a comparator collection C , we first determine the minimum Euclidean distance E-min between T and all titles in C. All titles T with E-min values higher than S-rel are potentially novel  but also be part of a ‘trendy’ neighborhood. In other words, a title T is considered to be part of a trend if it lies in a location that corresponds to low-density in the old neighborhood (blue dots) and high-density in the new neighborhood (green squares). This is quantified by requiring that a novel title T that is part of a trend be closer to 2.6.3Strategy Y1 tracks the location of the mean vector of papers in each time window. Long hops in embedded space may imply a substantial difference in the direction of research  may be gauged by estimating what proportion of papers in that window are at least distance S-rel from all papers in the past. When examining successive time windows, large upward oscillations of this proportion may signal the presence of a new trend.3.17We use two different approaches to detect changes over time in the focus of research papers on a particular topic. The first approach consists of using changepoint analysis to detect changes in the frequency of words within titles or abstracts. The second approach consists of embedding titles in vector space and using the distance between titles as an approximation of the corresponding semantic difference. To illustrate these approaches, we have chosen to focus on a pair of contrasting infectious diseases, COVID-19 and leprosy. COVID-19 has a short history as a pandemic affecting millions of people, while leprosy is one of the oldest human diseases. While much progress has been made, and effective treatment is available when diagnosed early, around 200,000 new cases continue to be reported each year.17The use of term frequencies as the basis of finding temporal changes in the focus of research papers has the following limitations:While considering terms independently might work well for categories such as drug names, it has limited ability to exploit the meaning of the entire title.Focusing only on the higher frequency terms may miss the true harbingers of change; the long tail of low frequency terms makes it harder to find the significant ones.20. More recently, it has been shown that models that directly embed a sentence are more accurate than taking the average of the constituent word vectors. Titles of papers typically encode more meaning than a set of words but often fail to form a well-formed sentence. In order to confirm if title embeddings retain inter-title similarity in the same manner as inter-sentence similarity21, we used BioSentVec16 to embed the titles from the bioRxiv COVID dataset and sorted pairs of titles by increasing Euclidean distance between the corresponding vectors. Representative pairs of related titles are shown in Ideally, we would like to project the key focus of research papers into a shared semantic space, without having to count frequencies first. This would make it possible to highlight newly populated regions of the space as containing papers representing new directions. Several models have been published for projecting words into vector spacesBased on the embedded space of titles, we used the following ways to identify titles that might be novel compared to the past:T1. Find titles that are furthest away from any title in the past .T2. Find titles whose location corresponds to a low density area of the set of past titles but high density area in the current time period.Strategy T1 is aimed at the identification of one of the earliest papers in a possibly new area. Each row in Leprosy control through multidrug therapy (MDT).Experience with WHO-recommended multidrug therapy (MDT) for multibacillary (MB) leprosy patients in the leprosy control program of the All Africa Leprosy and Rehabilitation Training Center in Ethiopia: appraisal of the recommended duration of MDT for MB patients.Bacillaemia in leprosy and effect of multidrug therapy.A search of PubMed confirms this trend in that the first hit for the query “leprosy AND MDT” is only in 1985.Based on the embedded space of titles, we used the following ways to identify years that might be novel compared to the past:Find the mean title vector for each year, and trace the path from year to year. The longer paths may represent a significant change between adjacent years.Estimate the proportion of titles in a given year that are located far away from any title in the preceding time period. A large change in the proportion of such titles in a given year may suggest a new and growing area of research.A low-dimensional projection of strategy Y1 is shown in While the title might be the most succinct ‘sentence’ representation of the topic of a paper, a rhetorical or terse title may fail to convey the essence of a paper. We therefore attempted to embed entire abstracts as an alternative version of strategy Y1. While this shows a possibly less noisy path from year to year, the variance  decreases so that the semantic ‘hops’ from year to year become smaller . Results4.We have presented and illustrated approaches to the detection of semantic changepoints within a set of research publications. Admittedly, a novel paper is not synonymous with a high impact paper. Nor is it synonymous with a novel conclusion. False negatives are also possible. For instance, analogous sentences differing in only one term  may have similar embeddings, especially within long titles. More specialized embedding schemes that are domain and problem specific may be necessary. Changepoint analysis is based on acute differences, and therefore less able to detect steady growth in a new area over a longer time period. As proof of concept, we have focused on a subset of the publications for each disease. For detection of truly novel papers, multiple databases will need to be considered together with sophisticated ontology and machine-based querying to maximize recall without sacrificing precision. The approach can be potentially enhanced by using named entity recognition approaches for more insightful analysis of the underlying reasons for an observed changepoint. A more detailed modeling of content based on incorporating topic modeling and/or the analysis of full text journal papers can help provide more granular changepoints and corresponding interpretations. Ultimately, the approach presented in this paper could potentially be incorporated into a real time system for the detection of novel information as it appears. Clinicians could potentially use it to find new vistas in their specific disciplines, especially in the context of hitherto incurable diseases. Researchers could find nascent topics worth expanding into.Supp-table


Rheumatoid arthritis (RA) is the most commonly occurring chronic inflammatory arthritis, the exact mechanism of which is not fully understood. Tumor Necrosis Factor (TNF)-targeting drugs has been shown to exert high effectiveness for RA, which indicates the key importance of this cytokine in this disease. Nevertheless, the response to TNF inhibitors varies, and approximately one third of RA patients are non-responders, which is explained by the influence of genetic factors. Knowledge in the field of pharmacogenomics of anti-TNF drugs is growing, but has not been applied in the clinical practice so far. Different genome-wide association studies identified a few single nucleotide polymorphisms associated with anti-TNF treatment response, which largely map genes involved in T cell function. Studies of the gene expression profile of RA patients have also indicated specific gene signatures that may be useful to develop novel prognostic tools. In this article, we discuss the significance of TNF in RA and present the current knowledge in pharmacogenomics related to anti-TNF treatment response. RA is a complex and heterogenous disease; what differentiates patients with RA are the number of affected joints, antibodies and serum cytokines levels, and the severity of joint destruction . TNF  by bone marrow stromal cells, mainly fibroblasts and activated synovial T cells. RANKL is crucial for propagating osteoclasts maturation. TNF can also stimulate osteoclast precursors directly through TNFR1 signalling. The soluble TNF is responsible for mobilizing osteoclasts from the bone marrow ,21. In aTNFAIP3 (A20), BCL2A1, CFLAR (FLIP), and FAS, while expression of some pro-apoptotic genes, including APAF1, CASP8, CASP10, FADD, TNFRSF1A and TNFRSF1B, is down-regulated. These transcriptional changes allow neutrophils to delay apoptosis and thereby enhance inflammation. Interestingly, these effects of TNF on human neutrophils seem to be concentration-dependent, since high concentrations may paradoxically promote apoptosis. During continuous exposure to TNF, desensitization of TNF-inducible expression in neutrophiles occurs, which is presumably a result of a down-modulation of both TNFR1 and TNFR2 receptors [In RA, TNF exposure on neutrophils up-regulates several genes involved in the NF-κB signaling pathway, particularly TNF itself. In addition, TNF induces expression of anti-apoptotic genes, e.g., eceptors .The relationship between TNF and T cell responses is complex, since T cells both respond to and produce TNF. TNF is of a key importance for proper T cells development. It is responsible for modulation of T cells development in thymus, as well as generation of efficient adaptive immune responses in lymph nodes. TNF is necessary for the activation of effector, memory, and regulatory T cells. Extensive production of TNF influence maturation of the antigen-presenting dendritic cells, and thus recalls efficient activation of naïve T cells. In addition, TNF was experimentally shown to promote leukocyte adhesion and diapedesis, which enable T cells invading the inflamed tissue .Expansion of Th17 cell subset is hypothesized to be an important mechanism of peripheral tolerance breakdown in RA. The increase in Th17 was reported in RA peripheral blood, synovial fluid and tissue, however Th17 lymphocytes are rarely found at inflammatory sites in comparison with other T cell subsets. This is presumably due to the rapid transition of Th17 lymphocytes to the non-classic Th1 phenotype, induced by TNF. Shifted Th17 cells become particularly aggressive and presumably are of particular importance in pathogenesis of inflammatory disease, including RA .Much knowledge about the influence of TNF on lymphocytes in RA is provided also by data from studies evaluating the effect of treatment with anti-TNF drugs. Interestingly, in long-term observation of anti-TNF-treated patients, the number of circulating Th1, Th2 and Th17 lymphocytes was reported to increase ,29. Non-Dulic et al. also showed that, as compared with non-responders, anti-TNF responders in long-term observation were characterized with lower proportions of total CD4+ T cells and higher proportion of late activation CD4+HLA-DR+ T cells. In the same study, T cells expressing the early activation marker CD69 were indicated as promising predictive marker of anti-TNF blocker response .TNF appears to inhibit the suppressive functions of Tregs, which are essential in preventing autoimmunity. Tregs prevent optimal costimulation by antigen presenting cells, and further response related to cytotoxic T cells . The mecThe TNF inhibitor golimumab was shown to largely affect peripheral memory T cell responses. In long-term observation, patients treated with golimumab displayed increased effector memory T cells and antigen-specific T cell cytokine production .As for B cells, conflicting results have been reported. TNF blocking with infliximab was found to increase the frequencies of circulating memory B-cells, whereas another study with etanercept showed the opposite effect .Formation of new capillaries from existing vessels, termed angiogenesis, is a key event in the formation and maintenance of pannus . ProinflTNF contributes to the increased risk of cardiovascular comorbidities in RA patients. Vascular injury is a result of the disruption of the endothelial barrier. TNF via TNFR1 signaling destabilizes the endothelial skeleton by actin rearrangement and microtubule destabilization . In addiAccording to a recent 2019 update of the European League Against Rheumatism (EULAR) recommendations for the management of rheumatoid arthritis, TNF inhibitors should be applied in patients with insufficient response to an initial course of conventional synthetic (cs) DMARDs  and the presence of poor prognostic factors .Each of the TNF inhibitors show substantial structural differences . TNF inhmax of about five days, and mean bioavailability of 64%. The elimination half-life (t1/2) is approximately 14 days [ADA is a fully human monoclonal IgG1 antibody which binds specifically to TNF, and inhibits its activity by blocking its binding to the p55 and p75 TNF receptors on the cell surface. ADA also influences a biological response induced or regulated by TNF, including changes in the concentration of intercellular adhesion molecules responsible for leukocyte migration . In RA patients, a rapid decrease in the levels of acute phase reactants , cytokines (IL-6) and matrix metalloproteinases  is observed. ADA is administered subcutaneously (s.c.) at a dose of 40 mg every two weeks. After administering s.c., the drug is absorbed and distributed slowly, with t 14 days ,43.max is two to six days and the mean absolute bioavailability is 51%. t1/2 is approximately 9 days [GOL is a fully human monoclonal IgG1 antibody that forms high affinity complexes with both sTNF and tmTNF, preventing TNF from binding to its receptors. Similarly to ADA, the binding of human TNF by golimumab neutralizes TNF-induced cell surface expression of the E-selectin adhesion molecule, the vascular intercellular adhesion molecule (VCAM-1), and the intercellular adhesion molecule (ICAM-1) of endothelial cells. In vitro, golimumab inhibits the secretion of IL-6, IL-8 and TNF-induced granulocyte colony stimulating factor (GM-CSF). In patients receiving golimumab, a decrease in C-reactive protein levels is observed, resulting in significant reductions in IL-6, ICAM-1 molecules, MMP-3) and vascular endothelial cell growth factor (VEGF). GOL is administered either s.c. at a dose of 50 mg every month, or intravenously (i.v.). After administering s.c., the mean ty 9 days ,44.1/2 ranges from 7.7 to 9.5 days [IFX is a chimeric monoclonal antibody (mAb) composed of a murine variable region and a human IgG1 constant region, which has high affinity for both sTNF and tmTNF. In vivo, IFX rapidly forms stable complexes with human TNF, resulting in the loss of biological activity by TNF, and a decrease in the concentration of IL-6 and C-reactive protein. In RA patients, the standard dose of IFX is 3 mg/kg i.v. at weeks 0, 2, and 6 (induction therapy), followed by maintenance therapy every eight weeks. t9.5 days ,45.1/2 to values comparable to the t1/2 of a complete antibody. CZP has a high affinity for both sTNF and tmTNF. CZP does not contain the fragment crystallizable region (Fc) that is present in complete antibodies, therefore it does not bind complement, and does not cause antibody dependent cellular cytotoxicity. In addition, it does not induce apoptosis in vitro in human monocytes or peripheral blood lymphocytes, nor does it induce neutrophil degranulation. The recommended starting dose of CZP for adult patients is 400 mg at weeks 0, 2 and 4, followed by a maintenance regimen every four weeks. Bioavailability is approximately 80%, and t1/2 is approximately 14 days [Certolizumab pegol is a PEGylated Fab’ fragment of a humanized monoclonal antibody, obtained by expression in E. coli cells and conjugation with polyethylene glycol (PEG). PEG conjugation prolongs the t 14 days ,46.1/2, which is approximately 70 h. ETN works by competitive inhibition of TNF to prevent attaching to TNFR receptors, rendering TNF biologically inactive. The drug is administered s.c. at a standard dose of 50 mg every week. The estimated bioavailability is 76% [ETA is a human fusion protein composed of the extracellular domain of TNF receptor (TNFR2/p75) and the Fc region of IgG1. Soluble receptors in dimeric form, such as ETA, have a greater affinity for TNF than monomeric receptors and are therefore much more potent competitive inhibitors of TNF. The presence of an immunoglobulin Fc fragment as a binding element prolongs serum ty is 76% ,47.It is estimated that around 25% of anti-TNF-treated RA patients fail to achieve initial treatment targets (primary non-response) ,49. If anucleotide-binding protein-like (NUBPL) gene, which is required for the assembly of the respiratory chain NADH dehydrogenase (complex I), and rs1813443, located in the intronic region of contactin 5 (NCTN5), a member of the immunoglobulin superfamily. The other identified markers are rs4411591, rs7767069, rs1447722, and rs1568885, the biological roles of which remain unclear. Nevertheless, none of the identified eight SNP reached a genome-wide level of significance, thus their occurrence tends to be more prognostic than predictive [LINC02549 rs7767069 and either copy of the LARRC55 rs717117G allele. Carriers of the rs7767069 allele show an increase in the number of T cells expressing the memory markers CD45RO and CD45RA, as well as the increased serum levels of soluble scavenger CD5 and CD6 receptors.The genome wide association study (GWAS) approach is useful for identifying potentially causal single-nucleotide polymorphisms, which is predictive for an anti-TNF therapy outcome. An analysis of the large Dutch Rheumatoid Arthritis Monitoring (DREAM) registry indicated that eight genetic loci explain 3.8% of the variance in treatment response. Two single nucleotide polymorphisms (SNPs), rs12142623 and rs4651370, are located in close proximity to the PLA2G4A gene . The proLARRC55 rs717117G allele show decreased levels of IL-6 after stimulation of PBMCs, which suggest the possible link between reduced IL6-mediated anti-inflammatory response and inadequate response to iTNF in this group of patients [MED15 gene (rs113878252), which is presumably responsible for polymerase II transcription. The other highly significant SNP in this study was in the MAFB locus (rs6065221), encoding the basic leucine zipper transcription factor that plays an important role in the regulation of lineage-specific hematopoiesis [CD84 gene expression in peripheral blood mononuclear cells, were indicated as a potential predictors of response to etanercept [TNF gene, rs17301249 in the EYA4 gene, rs1532269 mapping to the PDZD2 gene, and rs2812378 in the CCL21 gene, were also associated with responsiveness to TNF inhibitors [Carriers of the patients . The genopoiesis . Two SNPanercept . In singhibitors ,57.TNF, IL-1β, IL-1RA, ICAM1, and MyD88), the inflammatory chemokines , as well as neutrophiles, macrophage and osteoclast-associated genes . The lympho-myeloid pathotype was associated with the highest expression of B cell- and plasmablast lineage genes . The fibroid pathotype is characterized by high expression of fibroblast growth factor genes , bone homeostasis–associated genes  and the Wnt and TGFβ pathways genes. The pauci-immune pathotype has the most diverse expression profile and all of the above-mentioned patterns may be found, but the additional expression of the SFRP3/FRZB gene, involved in Wnt signaling and the regulation of bone development, may be elevated. All groups show increased expression of IL-6, the IL-6 receptor, and associated STAT3 protein transcription factor pathway genes [Synovial biomarkers may be useful tools to predict anti-TNF clinical response . In RA, ay genes ,60.sICAM1 and high expression of CXCL13 corresponding to the lymphoid pathotype were shown to have a better clinical response to anti-TNF treatment as compared with myeloid pathotype-associated sICAM1-high/CXCL13-low patients. Interestingly, the opposite relationships have been found in IL-6R–treated patients [In general, the lymphoid pathotype is linked to more aggressive disease, RF and ACPA seropositivity, and a higher proportion of patients requiring biological therapy ,62. Furtpatients . In contpatients .Aterido et al. performed the analysis of transcriptomic association between selected gene coexpression modules in the RA synovium and the clinical response to TNF inhibitors, including ADA and IFX. Two of the 13 analyzed modules, which were significantly enriched for genes involved in the nucleotide metabolism and epigenetic marks from CD4+ regulatory T cells, have been found to be significantly associated with the response to anti-TNF drugs .IFI27, ISG15, IFI44L, OASL, USP18, RSAD2, and LY6E) in peripheral whole blood and the lympho-myeloid pathotype has been shown [In general, the peripheral whole-blood transcriptome in RA patients is less variable than the synovial one. However, in RNA-seq analysis a substantial association between type I IFN response genes expression , (2) TMEM176A and (3) TMEM176B , (4) IFI44  [PLSCR1  [Beyond the above, the link between gene expression patterns of peripheral blood mononuclear cells (PBMCs) and response to treatment have also been found. PBMCs include lymphocytes , monocytes and dendritic cells. Póliska et al. have identified five genes whose baseline expression is upregulated in PBMCs in TNF-responders: (1) sponses) , and (5)ophages) ,67.JAK1, IFI27 IFNAR1, IRF1, TNFA, TLR4, MYD88, CD86, and IL8 [In peripheral blood monocytes of RA patients, differential IFN pathway activation prior to anti-TNF treatment has been observed in subsets of responders and non-responders. In addition, TNF inhibitor response groups, which were defined by type I IFN, were shown to correlate with the expression of  and IL8 .Only recently, Tao et al. reported building the novel machine learning model based on the analysis of transcriptome signatures from PBMCs, monocytes, CD4+ T cells, and methylation signatures from PBMCs, which enable prediction of anti-TNF response in patients with RA. In the study, gene expression and DNA methylation profiling was performed in two cohorts of patients, who were going to begin either ADA or ETN therapy. In both cohorts of patients, genes differentially expressed between responders and non-responders included those involved in DNA and nucleotide binding, such as RFX2, IRF8, and TAF1, TAF11, FOXO4. Additionally, in the ETN cohort TRAF6, representing the TNF receptor signaling gene, was also differentially expressed. The interesting issue was that, in both cohorts, a distinct hypermethylation pattern between responders and non-responders was observed, and this may be of particular importance to understanding the importance of epigenetics in non-responsiveness to TNF inhibitors. The tool presented by the authors is undoubtedly interesting; however, it is worth noting that the indicated model reached an overall accuracy of 85.9% in the ADA cohort and 79% in the ETN cohort, which may represent insufficient levels of reliability for use in clinical practice . TherefoCMPK2, IFI44L, IFI6, IFIT1B, LY6E, OAS1, OAS2, OAS3, RSAD2, and USP18 predicted a good response. Of the 47 non-responder genes, 13 genes were related to neutrophil granule proteins (NGPs): AZU1, BPI, CEACAM8, CRISP3, CTSG, DEFA4, ELANE, LCN2, LTF, MMP8, MPO, RNASE2 and RNASE3. The combination of three genes: CMPK2, IFIR1B and RNASE3, showed the greatest predictive power [The specific gene expression profiles in peripheral blood neutrophiles can also predict a subsequent response to TNF blockers. In a study by Wright et al., the type I IFN signalling was identified as the most enhanced pathway in anti-TNF responders. The expression levels of the 10 IFN-regulated genes ve power . The expve power .Knowledge regarding anti-TNF pharmacogenetics has not been applied in clinical settings. This is mostly due to the large genetic variability of individuals with RA. The heterogeneity of RA implicates the need for discrimination of different RA subsets with various anti-TNF treatment responses. The other obstacle is that the above-mentioned studies examined only the effects of a single SNP, while identifying the common risk haplotypes may be necessary. Furthermore, the problem with identified specific transcriptional signatures is that their complexity makes them hard to interpret. Applying knowledge of TNF inhibitors’ pharmacogenomics into clinical practice also requires consideration of the importance of environmental factors , which are known to affect clinical outcomes. The growing availability of next generation sequencing (NGS) technology and whole genome/exome sequencing (WGS/WES) data, accompanied with statistical tools, can facilitate the identification of rare and low-penetrant causal variants in RA, which is of key importance for the design of genomic tests. Nevertheless, the introducing of specific genomic tests in anti-TNF-treated patients will require prior multicenter validation and standardization.


Heart failure (HF) remains a public health concern as it is associated with high morbidity and death rates. In particular, heart failure with preserved ejection fraction (HFpEF) represents the dominant (>50%) form of HF and mostly occurring among postmenopausal women. Hence, the initiation and progression of the left ventricular diastolic dysfunctions (LVDD)  in postmenopausal women have been attributed to estrogen deficiency and the loss of its residue cardioprotective effects. In this review, from a pathophysiological and immunological standpoint, we discuss the probable multiple pathomechanisms resulting in HFpEF, which are facilitated by estrogen deficiency. The initial discussions recap estrogen and estrogen receptors (ERs) and β-adrenergic receptors (βARs) signaling under physiological/pathological states to facilitate cardiac function/dysfunction, respectively. By reconciling these prior discussions, attempts were made to explain how the loss of estrogen facilitates the disruptions both ERs and βARs-mediated signaling responsible for; the modulation of intra-cardiomyocyte calcium homeostasis, maintenance of cardiomyocyte cytoskeletal and extracellular matrix, the adaptive regulation of coronary microvascular endothelial functions and myocardial inflammatory responses. By scaffolding the disruption of these crucial intra- and extra-cardiomyocyte physiological functions, estrogen deficiency has been demonstrated to cause LVDD and increase the incidence of HFpEF in postmenopausal women. Finally, updates on the advancements in treatment interventions for the prevention of HFpEF were highlighted. Heart failure (HF) remains a public health concern as it is associated with high rates of morbidity and deaths that ultimately result from cardiovascular diseases . Left ve2+ in cardiomyocytes for proper cardiac contractions, are downstream of the βARs , cardiac cytoskeletal proteins, and coronary microvascular endothelial (CME) dysfunctionalities, plus maladaptive inflammatory responses in the myocardia. Regardless, estrogen (E2) and estrogen receptors (ERs) have been shown to adaptively modulate the expressions, responses, and activities of the aforementioned factors implicated in the initiation and progression of HFpEF  under physiological state. However, hyperactivity of the sympathetic nervous system (SNS) during stress upregulates circulating catecholamines, which overstimulates βARs, causing the receptors to become dysfunctional, leading to their desensitization and downregulation . Unfortuthe βARs . Hence, of HFpEF . Hence, of HFpEF .This review seeks to discuss the probable mechanisms aggravating the incidence of HFpEF in postmenopausal women from a pathophysiological and immunological standpoint. The initial discussion recaps E2 and ERs and βARs signaling under physiological/pathological states to facilitate cardiac function/dysfunction, respectively. Finally, by reconciling these prior discussions, we explain the possible pathomechanisms of how E2 deficiency scaffolds signaling cascades that hastens the incidence of HFpEF in postmenopausal women and provided updates on the advancements in treatment interventions.1AR, and β2AR) are both predominate seven transmembrane-spanning receptors expressed in cardiomyocytes, cardiac fibroblast, and vascular tissues, and mediates signaling crucial for their cellular functions , and sodium-calcium exchanger (NCX), as well as myofilament proteins  to facilitate inotropy, chronotropic, and relaxation  and βARs  in the pathogenesis of LVDD and, ultimately, HFpEF  and ultimately induce HFpEF  at Ser1177 when β2AR/Gai/PI3K/Akt-induced NO production is dysregulated, thereby, reduces the initiation or progression toward HFpEF  in postmenopausal women have revealed that the synergy of βARs hyperstimulation and E2 deficiency permits maladaptive signaling cascades which drives the adverse cardiac remodeling . Appareny, HFpEF . Meanwhiy, HFpEF . (3) Sigce HFpEF . (4) Pre in LVDD . (5) Susrd HFpEF . (6) Dowrd HFpEF . HoweverHerein, the discussions will be centered on explaining the probable pathomechanisms involved in the initiation and progression of HFpEF in postmenopausal women, mostly due to the synergy of E2 deficiency and βARs dysfunctionalities.2+ cycle that might explain the predisposition of postmenopausal women to HFpEF.Focusing on individual calcium handling proteins to assess the extent of their alterations and dysfunctionalities resulting from E2 deficiency reveals multiple vulnerabilities in the Ca2+ regulates both mitochondrial and contractile activities; hence, it plays a crucial role in sustaining the energy demands and supplies of the myocardium. Although primarily regulated by signaling via βARs, E2 and ERs have also been shown to influence the expression of the receptor and its downstream proteins  involved in maintaining the intracellular calcium homeostasis (2+ into cardiomyocytes upon phosphorylation and the influx of Ca2+ into the cytosol from the sarcoplasmic reticulum (SR) via RyR to facilitate contraction. The latter phase, which ensures relaxation, requires that the excess Ca2+ be taken back into the SR via SERCA and/or expelled out of the myocyte via NCX  of the Ca2+ cycle are responsible for contraction, any inefficiencies occurring here may indirectly impact negatively on the latter phase cascade (Ca2+ uptake and Ca2+ expulsion), as the amount of Ca2+ influx in the cytosol might exceed the Ca2+ hold capacity plus Ca2+ efflux capability. Consistent with these inferences, Miranda et al. suggested that SR Ca2+ release and SR Ca2+ uptake are impaired during HFpEF  and improved myocardial contraction to attenuate HFpEF. Altogether, it has been suggested and shown that E2 by signaling via non-genomic (GPR30) and/or genomic (ERα and ERβ) could directly or indirectly exert these cardioprotective effects to prevent HFpEF by preserving the ECM and cardiovascular function . Hence, 2AR has been demonstrated to induce NO production via Gai/PI3K/Akt  are dependent on functionalities of their intima layer made of endothelial cells, which in turn depend on NO bioavailability. The production of NO by eNOS is crucial for the regulation of several cardiovascular physiology such as; endothelial cell proliferation and migration, ECM degradation, angiogenesis, platelet function, vascular inflammation modulation, and vascular tone regulation . Again, PI3K/Akt . Nonethe2AR might induce LVDD, as Matsubara et al. demonstrated in their study  Als the ECM . (4) Las the ECM . Meanwhi the ECM . This su the ECM . However the ECM , might iIn summary, elucidating the underlying cause of the prevalence of HFpEF in postmenopausal women over the recent decades has revealed that the loss of E2 and its residual cardioprotective effects permits the initiation of maladaptive alterations intra- and extra-cardiomyocytes. These adverse alterations tend to affect the physiological functions of the cardiomyocytes as well as other constituents of the myocardia, such as; fibroblasts, inflammatory cells, coronary vessels, and epicardial adipose . The synTaken together, decades of research works focused on elucidating the underlying cause of increased HFpEF in postmenopausal women have found that E2 exerts cardioprotection via the adaptive modulation of multiple homeostatic functions that sustains proper cardiac health and functions. Therefore, its deficiency during menopause permits dysregulations of cascades and physiological functions crucial for facilitating cardiac inotropy and chronotropic, should there be any stressful event.Therapeutic interventions for the prevention and management of HFpEF in postmenopausal women has solely been the use of menopausal hormonal treatment until the findings of the Women’s Health Initiative randomized controlled trial and others, reporting the side effects and discouraged their usage significantly . ExploraAlso, due to the complexity of the disease mechanism of HFpEF, it is suggested that multiple pathomechanisms may be targeted with their appropriate drug combinations to improve treatment outcomes. Meanwhile, employing E2 replacement therapy might circumvent the use of multiple drugs as it has been shown to adaptively modulate these multiple pathways to exert cardioprotection and/or ameliorate adverse cardiac remodeling .2+ handling. Conversely, when the E2 replacement therapy was initiated early (1 month) after ovariectomy, E2 was potent at inhibiting the aforementioned dysfunctions, which hasten the incidence of HFpEF (Clinical data have shown that the severity of LVDD/HFpEF is increased mostly after 6 years of menopause . In lineof HFpEF . These sof HFpEF . TherefoAOA conceived the review idea. GKA and AOA fine-tuned the review idea and drafted and wrote the manuscript. GKA, AOA, RM, MLNN, JA-A, RR, and NA proofread and revised the manuscript with the supervision of HS. All authors accepted the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background In early March 2020, a SARS-CoV-2 outbreak in the ski resort Ischgl in Austria triggered the spread of SARS-CoV-2 throughout Austria and Northern Europe. In a previous study, we found that the seroprevalence in the adult population of Ischgl had reached 45% by the end of April, representing an exceptionally high level of local seropositivity in Europe. We performed a follow-up study in Ischgl, which is the first to show persistence of immunity and protection against SARS-CoV-2 and some of its variants at a community level.Methods Of the 1259 adults that participated in the baseline study, 801 have been included in the follow-up in November 2020. The study involved the analysis of binding and neutralizing antibodies and T cell responses. In addition, the incidence of SARS-CoV-2 and its variants in Ischgl was compared to the incidence in similar municipalities in Tyrol until April 2021.Findings For the 801 individuals that participated in both studies, the seroprevalence declined from 51.4%  47.9–54.9) to 45.4% (95% CI 42.0–49.0). Median antibody concentrations dropped considerably  but antibody avidity increased . Only one person had lost detectable antibodies and T cell responses. In parallel to this persistent immunity, we observed that Ischgl was relatively spared, compared to similar municipalities, from the prominent second COVID-19 wave that hit Austria in November 2020. In addition, we used sequencing data to show that the local immunity acquired from wild-type infections also helped to curb infections from variants of SARS-CoV-2 which spread in Austria since January 2021.Interpretation The relatively high level of seroprevalence (40–45%) in Ischgl persisted and might have been associated with the observed protection of Ischgl residents against virus infection during the second COVID-19 wave as well as against variant spread in 2021.Funding Funding was provided by the government of Tyrol and the FWF Austrian Science Fund. In patient cohorts, several studies have recently shown persistence of immunity against SARS-CoV-2 using laboratory surrogate parameters such as antibodies and T cell responses, for up to 8 months.However, this is, to our knowledge, the first study at the community-level, where we show that the persistence of the laboratory markers of immunity was concomitant with reduced virus transmission within a population, for the wild-type virus (D614G) as well as for the B.1.1.7 variant.Our findings suggest that seropositivity levels of around 40–45% can persist in a community for over 8 months, which in turn may have helped to reduce the spread of both wild-type and B.1.1.7 transmission at the local level.Alt-text: Unlabelled box1Early on in the COVID-19 pandemic, it had already become clear that individuals previously infected with SARS-CoV-2 virus are immune to re-infection. Consequently, well-documented re-infections have been extremely rare to date, one year after the pandemic emerged . HoweverIn late April 2020, we studied the seroprevalence for SARS-CoV-2 in Ischgl, a popular ski resort in the Tyrolean Alps . Ischgl In this paper, we report the results of a follow-up study (Ischgl 2) performed in the first week of November 2020, among adults, 6.5 months after the first study and up to 8 months after the first infection wave in Ischgl. First, we found that T-cell and antibody responses could still be detected in most individuals that had recovered from infection, and that Ischgl was relatively spared from the second SARS-CoV-2 wave that hit Austria in November 2020, compared to similar municipalities. Second, we showed that community protection also worked against some of the variants of SARS-CoV-2 and that this protection persisted until April 2021.22.1st and 27th, 2020 and follow-up (Ischgl 2) with EC number: 1330/2020, which took place between November 2nd and 8th, 2020. All inhabitants of Ischgl at the time point of the respective study were invited. After a patient briefing written informed consent was obtained from all participants. This cross-sectional epidemiological survey targeted all residents of Ischgl/Tyrol older than 18. At baseline, n = 1259 adults (82.4%) of all eligible adults (n = 1527) in Ischgl enrolled in the study . The funders had no role in study design, data collection, data analysis, interpretation, writing the manuscript or the decision to submit it for publication. The authors have not been paid by a pharmaceutical company or other agency to write this article. The corresponding authors had access to all the data of the study and had final responsibility for the decision to submit for publication.33.1In the first week of November 2020, 6.5 months after our first seroprevalence study in the ski-resort Ischgl (baseline study = Ischgl 1), we again invited all adult Ischgl residents to participate in a study on the level of local immunity to SARS-CoV-2 (follow-up study = Ischgl 2). In April 2020, an estimated 1527 adults lived in Ischgl, of these 1259 participated in Ischgl 1 (82.4%). In November, an estimated 1304 adults were living in Ischgl, this decrease reflects the absence of seasonal workers. Of these, 904 adults participated in November (69.3% participation rate). Of the 1259 study participants at baseline, 801 individuals participated again in November and were included in the analysis in the follow-up study presented here . Age distribution was similar in the adult population in both studies, while there was a slight bias towards female participants in Ischgl 2 (54.9%) relative to Ischgl 1 (51.9%) (Supplementary Table 1).3.2The testing strategy for antibodies to SARS-CoV-2 is depicted in n = 1259) [n = n = 801) of 51.4% (95% CI 47.9–54.9). Here, we determined the seroprevalence for all adult participants at Ischgl 2 (n = 891) with 44.7% (95% CI 41.4 - 48.0). For individuals that participated in both studies (n = 801), the level of seropositivity declined between both studies to 45.4% (95% CI 42.0–49.0) in Ischgl 2 (Supplementary Table 3). From the 90 individuals that only participated in Ischgl 2 and not in Ischgl 1, 37.8% (95% CI 27.8–48.6) were seropositive in Ischgl 2 (Supplementary Table 3). The individuals that only participated in Ischgl 2 were not otherwise included in the following analysis.For all individuals with serostatus d and a, neutralizing antibodies were determined. The following individuals were considered seropositive: p (anti-S IgG and anti-N IgG positive), d (discordant) plus neutralizing antibody positive; a (anti-S IgA only) plus neutralizing antibody positive. We have previously described a seroprevalence of 45.0% (95% CI 42.2 – 47.8) for all adult participants at Ischgl 1 ( = 1259) . For then = 801, Z =22.0, p< 0.001, Wilcoxon signed-rank test), 88.9% in the anti-N-IgG-Abbott  and 58.6% in the anti-N-IgG-Roche assay  (`Median antibody levels to SARS-CoV-2 significantly declined in each assay - by 50% in the anti-S IgG assay (nk test) e. In sevnk test) . The majnk test) . Interesnk test) . Of the nk test) .Table 1SAlso, the neutralizing antibody titers declined in most individuals that were neutralizing antibodies positive in Ischgl 1, and 90 of 430  individuals completely lost neutralization capacity in our assay f,g. InteWe then studied the avidity of the antibodies in individuals that were anti-S IgG positive in Ischgl 1 and 2 i. While 3.3n = 28, all NTpos ), became discordant ), or IgA only  or became negative  in Ischgl 2. Individuals that were antibody negative in both studies  served as a control group. As positive controls, we used pepCEF and PHA, and T cell response to these stimulations were comparable in all investigated groups .In Ischgl 1, T cell response had not been analysed. To study if the decline in humoral immunity was compensated at least partially by T cell responses in Ischgl 2 participants, we analyzed the response to peptide pools derived from S, M and N protein of SARS-CoV-2 by ELISPOT a,b and iAlthough approximately one third of individuals in the seronegative group showed T cell responses to at least one peptide pool , Fig. 2bp = 0.007, χ² test, with Fisher's exact test). When comparing baseline negative participants with baseline positives in the different subsets in the follow-up, the number of reactive peptide pools in the p-p group but not p-d/NTpos and p-d/NTneg groups was significantly higher than in the n-n group . Thus, T cell response in term of the number of reactive peptide pools correlated with declining serostatus with p-p > p-d/NTpos > p-d/NTneg (neg), 5 still had a detectable T cell response, thus those individuals could potentially still have some T cell-mediated immune protection. Of the four individuals in the p-n group that had lost all antibodies in the commercial assays, 3 still retained neutralizing antibodies, two of which had detectable T cell responses and only one individual had lost all binding and neutralizing antibodies and also showed no T cell responses .th/ 6th, 2020 were very similar between Ischgl and the control municipalities (28 vs. 33%). This suggests that the willingness of Ischgl citizens to test did not differ compared to other municipalities.We then investigated whether the high-level immunity in Ischgl, which was still detected in November, had limited virus transmission during the second wave of SARS-CoV-2, which hit Austria in autumn 2020. We compared daily numbers of confirmed SARS-CoV-2 PCR-positive cases of Ischgl with 13 control municipalities selected from Tyrol. Notice that information on the number of undertaken PCR-tests was not available at the municipality level. However, participation rates in a free and voluntary mass antigen test conducted in the province of Tyrol on the weekend of December 53.5In the end of 2020, first reports about the spread of two SARS-CoV-2 Variants-of-Concern (B.1.1.7 and B.1.351) in Europe surfaced (rd and April 6th (end of sample period). Sequencing these cases, we found that 222 were wild-type , 110 the B.1.1.7 variant  and 99 the B.1.351 variant . Using this data, we first analysed whether the overall number of cases (B.1.351 + B.1.1.7 + wild-type) continued to be significantly larger in the control municipalities compared to Ischgl from January to April 2021. Specifically, we applied a two-sample t-test  to test for a significant difference in the 7-day moving average of daily new confirmed COVID-19 cases per 100,000 inhabitants between Ischgl and the control municipalities. We found the mean of the 7-day moving average of daily new cases to be 17.4 (95% CI 15.9–18.8) for the control municipalities, and 7.5 (95% CI 5.6–9.4) for Ischgl. The difference amounts to 9.9 and is statistically significant . Applying the same two-sample t-test on B.1.1.7 cases only, we again found that Ischgl had a significantly lower number of cases compared to the control municipalities (p < 0.07). The mean of the 7-day moving average of daily new B.1.1.7 cases is 4.1 (95% CI 3.1–5.2) for the control municipalities, and 2.8 (95% CI 1.3– 4.3) for Ischgl. Regarding the wild-type, we observed a statistically significant difference of 4.4 . While we observed 99 B.1351 cases in our control municipalities between January and April 2021, we did not observe a single case of this variant in Ischgl. B.1351 was mostly found in the eastern districts of Tirol rather than in the west, where Ischgl is located.Overall, we observed 495 SARS-CoV-2 PCR-positive cases, with 10 of those in Ischgl and 485 in our control municipalities between December 23These results suggest that Ischgl had relatively less spread of both the wild-type and the B.1.1.7 variant compared to the control municipalities. However, the two-sample t-test cannot tell us whether the wild-type and B.1.1.7 spread similarly within Ischgl. Recent research based on neutralization assays suggest that antibodies acquired from wild-type infections work similarly well against B.1.1.7 . However4Our findings suggest that the immunity to SARS-CoV-2 on the community level can persist for at least eight months. We found that both antibodies and T cell responses persisted and that this might translate into a reduced local virus transmission.Our study extends the previous research in three important ways: First, we confirm that immunity as defined by T cell and/or neutralizing antibody response lasts for up to several months and that T cell and antibody responses can be discordant ,26. The Second, since our study was performed in a population within a defined narrow geographical area, we were able to analyze the epidemiological transmission of SARS-CoV-2 within this population along with immunity patterns in the laboratory. We found that the incidence of SARS-CoV-2 in Ischgl was lower than in comparable municipalities during the second wave that hit Austria in November 2020. The reduced incidence in Ischgl relative to comparable municipalities (as well as to the rest of Austria) indicates that the level of immunity to SARS-CoV-2 helped to curb new infections in Ischgl.Finally, our findings suggest that seropositivity levels of around 40-45% can persist in a community for over 8 months, which in turn may have helped to reduce the spread of both wild-type and B.1.1.7 transmission at the local level.There are some limitations of our study. In the follow-up study relative more of the baseline study seropositives returned compared to the baseline study negatives. This might have introduced a bias in the seroprevalence calculation of the follow-up study. However, seroprevalence of the study participants that were new in the follow-up study was in a similar range. Additionally, T cells were only analyzed in the follow-up study; hence development of T cell responses is difficult to judge. Lastly, we cannot distinguish between the effects of the high seroprevalence and of non-pharmacological interventions on virus transmission in Ischgl as at the time of our follow-up study lockdown light measures were in place. However, the lockdown light measures were identical in Ischgl and the rest of Austria.5WB, ZB, HW, RW, DvL, JP, JK designed experiments; ZB, LR, AR, LP, DP, MB performed experiments; WB, ZB, KB, HW, IT, BF, HU, LK, RW, DvL, JP, JK analyzed data; WB, KB, SS, LF designed questionnaire; AW the local general practitioner, supervised the study in Ischgl; WB, ZB, KB, HW, DvL, JP, JK had access to all data and reviewed them; HW, DvL, JP, JK wrote the paper; all authors read and approved the manuscript.6Upon publication of our study, the data and codes will be available from the corresponding authors upon receipt of a suitable request.IT has contracts with Kymab and Sierra Oncology and received consulting fees from Kymab. For the other authors, no conflicts of interests exist. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.