The contributions of the humoral immune response to melanoma are now widely recognized, with reports of positive prognostic value ascribed to tumor-infiltrating B cells (TIL-B) and increasing evidence of B cells as key predictors of patient response to treatment. There are disparate views as to the pro- and anti-tumor roles of B cells. B cells appear to play an integral role in forming tumor-associated tertiary lymphoid structures (TLSs) which can further modulate T cell activation. Expressed antibodies may distinctly influence tumor regulation in the tumor microenvironment, with some isotypes associated with strong anti-tumor immune response and others with progressive disease. Recently, B cells have been evaluated in the context of cancer immunotherapy. Checkpoint inhibitors (CPIs), targeting T cell effector functions, have revolutionized the management of melanoma for many patients; however, there remains a need to accurately predict treatment responders. Increasing evidence suggests that B cells may not be simple bystanders to CPI immunotherapy. Mature and differentiated B cell phenotypes are key positive correlates of CPI response. Recent evidence also points to an enrichment in activatory B cell phenotypes, and the contribution of B cells to TLS formation may facilitate induction of T cell phenotypes required for response to CPI. Contrastingly, specific B cell subsets often correlate with immune-related adverse events (irAEs) in CPI. With increased appreciation of the multifaceted role of B cell immunity, novel therapeutic strategies and biomarkers can be explored and translated into the clinic to optimize CPI immunotherapy in melanoma. During early stages, primary melanoma lesions are removable through surgical intervention that is largely curative. In advanced disease however, melanoma can spread to regional lymph nodes and metastasize to distant sites. Historically, treatment options were limited in advanced disease to palliative cytotoxic chemotherapy, leading to poor 5-year survival rates. Over the last decade, immunotherapy, driven by checkpoint inhibitor antibodies, has transformed patient prognosis.via activation of different immune-inhibitory pathways, including via immune checkpoint molecules and their downstream signals. Physiologically, checkpoint pathways play a role in immune homeostasis, providing negative feedback stimuli to prevent autoimmune reactivity. The best-studied checkpoint pathways are those of negative regulatory molecules cytotoxic T lymphocyte associated protein-4 (CTLA-4) and of the Programmed Death Receptor 1 (PD-1) and its ligands PD-L1 and PD-L2.The rationale behind the use of checkpoint inhibitor therapy lies in the highly immunogenic nature of melanoma , 2. MelaCheckpoint inhibitors (CPIs) were designed to promote immune-mediated elimination of tumor cells through modulation of T cell responses. Anti-CLTA-4 (Ipilimumab approved in 2011)  or anti-Despite the undeniable clinical success of CPI therapy several challenges remain. There is currently a lack of biomarkers, limiting our ability to predict who will respond to treatment. Another important consideration is recognizing who will develop toxicities that are unfortunately common particularly with anti-CTLA-4 treatment and in the context of combination anti-CTLA-4 and anti-PD-1 therapy as compared with monotherapy. These toxicities, known as immune related adverse events (irAEs) vary in severity, can affect any organ system but most commonly target the skin, the gastrointestinal and the endocrine systems . AlthougThe role of T cells in CPI therapy has been extensively reviewed in literature. Contrastingly, B cell immunity has been less studied. B cells have a wide range of roles, including critical functions as professional antigen presenting cells (APCs), and they are capable of secreting cytokines and antibodies which enable them to conduct antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC). Considering their comprehensive functions and with B cells making up one of the two arms of the adaptive immune system, it is not surprising to envisage a role within melanoma immunity and response to CPI. However, the exact role B cells play in tumor immunity is unclear with the existence of different cell subsets with juxtaposing functions, such as activatory and regulatory B cells (Bregs), and the presence of B cells in tertiary lymphoid structures (TLSs). The potential pro- and anti-tumor roles of B cells in melanoma are summarized in via an APC, but also on the presence of a costimulatory second signal, typically through binding of CD28 on the T cell to CD80/86 on the APC. CTLA-4 is a competitive CD28 homolog that has a higher affinity to CD80 (B7-1) and to a lesser degree CD86 (B7-2) than CD28 therefore inhibiting T cell co-stimulation . Like CTLA-4, PD-1 is thought to be a negative regulator of T cell function, regulating peripheral tolerance and T cell responses. PD-1 is expressed more broadly than CTLA-4 and can be found on T cells, B cells, natural killer (NK) cells, and a variety of peripheral tissues . Expressvia SHP2 and converge to inhibit downstream P13K signaling  can be thought of as a distinct complex “organ” which contains an array of different cells, such as immune cells, tumor-associated vasculature and lymphatics, fibroblasts and pericytes . The immLymphocyte activation typically occurs in secondary lymphoid structures which include lymph nodes, the spleen and mucosal-associated lymphoid tissue . During + stromal B cells per mm2 tumor  or may reflect the confounding presence of immunosuppressive regulatory B cells.In accordance with expectations arising from these anti-tumor properties, the percentage of tumor-infiltrating and peritumoral B cells have been found to positively correlate with more favorable patient survival in the vast majority of melanoma cohort studies , 43, 44.Several studies have demonstrated the enriched presence of various differentiated B cell phenotypes such as memory, plasmablasts (PBs) and plasma cells within melanoma tumors. Elevated percentages of PBs (short-lived cells and part of the rapid antigen response) have been reported in the circulation of patients with melanoma compared to blood from healthy individuals. PBs are short-lived cells and part of a rapid antigen response. Although PBs have a potentially positive role in melanoma, the functional plasticity of these cells may result in transient switching to a “regulatory” pro-tumor phenotype owing to expression of TGFβ and IL-10 by this cell subset .+ CD19++ regulatory B cell subset has been identified in mice which strongly resembles MZ cells and protects against colitis, through interactions with Tregs (via their polarized cytokine expression.IL-10-producing “regulatory” B (B10) cells  have beeth Tregs . These ovia TGFβ secretion (+ and IL-10+), and that tumor-derived antigens in general could induce B cell IL-10-expression  into wild-type mice significantly exacerbated B16F10 melanoma growth, illustrating that IL-10-producing B cells can directly contribute to melanoma tumor-progression . A separTumor-infiltrating regulatory B cells therefore represent a potential mechanism of tumor-mediated immune escape that in certain conditions may outweigh antigen-presentation and anti-tumor antibody-mediated effector mechanisms to skew the overall impact of B cell infiltrates towards a neutral or even a negative effect, and mandates studies into the induction of regulatory B cells in melanoma patients and correlation with overall survival.Consistent with the contributions of regulatory B cells, the microenvironments of melanoma solid tumors are characteristically considered as harboring Th2-biased cytokine expression profiles , typicalin vitro that these Th2-biased microenvironments favor alternatively activated humoral immunity which confers a shift in B cell antibody expression towards IgG4  gene was found to correlate with lack of response to anti-CTLA-4 but this was not shown in the context of anti-PD-1 therapy (versus neoadjuvant Nivolumab plus Ipilimumab (n = 12)] ]  also hign = 12)]  and foun outcome . Since mlo B cell sub-populations : particularly a significant decrease in circulating B cells in patients who underwent combination CPI as opposed to those treated with a monotherapy anti-CLTA-4 or anti-PD-1 . No signulations . Of note-4 agent . Detailein vitro (There is some evidence that plasmablasts (PB) may be directly modulated by CPI. Circulating plasmablast levels were significantly enhanced in patients undergoing treatment with anti-CTLA-4 immunotherapy . When imin vitro . In thisin vitro .versus biopsies from non-responders  have the potential to engender anti-tumor effects, mediating ADCC, ADCP, CDC or to support antigen presentation by mediating the uptake of tumor antigens by APCs such as macrophages and dendritic cells . Furtherarcinoma . IgG claarcinoma  and othearcinoma –87.γRIIbs) in the TME and downstream immunoreceptor tyrosine-based inhibitory motif (ITIM) domain signalling  compared to other isotypes . Furthermore B cells expressing IgG3 isotype antibodies usually undergo less antibody SHM, and therefore IgG3 antibodies may have lower affinity for the target antigen while still able to occupy FcRs . IgG2 hamelanoma , 67, 71.β supporting IgA class switch. Melanoma-associated IgA has been found to correlate with poor clonality, suggesting that, in the tumor, class switching to IgA is a consequence of inflammation and not of an antigen-driven response, and that tumor-associated IgA could be non-tumor specific , Nivolumab plus Ipilimumab (10%), or Ipilimumab (4%). A positive correlation with progression-free survival (PFS) was found for high titers of total IgG, IgG1, IgG2, and IgG3, while a positive correlation with overall survival (OS) was found to be significant only for the IgG2 subclass . This rei.e., different patients raising antibodies against shared or similar epitopes. Moreover, when antibody lineages were analyzed and compared among patients, antibodies with high sequence similarity were found, suggesting these antibodies may have arisen from convergent selection and that the patients may be producing antibodies against shared epitopes. Of note, IgG2 was the most frequent isotype of these antibodies. Furthermore IgG2 was higher in responders compared to non-responders , the responder group had higher titers of antibodies specific for the tumor-associated antigens MDA  and the Cancer-Testis antigen NY-ESO-1 at baseline, compared to non-responders. Serum analyses showed that NY-ESO-1, TRP1/TYRP1, and TRP2/TYRP2 specific antibodies consisted of several IgG subclasses while the IgG antibodies for MelanA were mostly IgG1 and the antibodies for gp100 were of the IgG2 isotype . InteresIn patients with metastatic melanoma who were treated with anti-CTLA-4 combined with anti-angiogenic VEGF-A-targeted treatment, there was an increase of antibody titers recognizing the immunoregulatory protein Galectin-1 . Anti-Gal-1 antibody titers in turn correlated with better disease outcome. Higher frequencies of complete or partial responses and improved overall survival were seen in these individuals. Gal-1 has been reported to be up-regulated in many tumor types including melanoma and is usually associated with poorer survival, contributing to tumor growth, angiogenesis, metastasis, and immune evasion .Taken together, these results suggest that checkpoint blockade-associated restoration of T cell activity may also contribute to increasing the effectiveness of the humoral response and highlights the importance of the expressed antibodies as part of the anti-tumor activity. Furthermore, these studies suggest that melanoma-specific antibodies in pre-treatment sera may be promising indicators of CPI immunotherapy response and require further study.de novo events or whether they represent an unmasking of underlying immune mediated disease remains unclear , liver (hepatitis), lung (pneumonitis), and endocrine systems, including hypophysitis and insulin-dependent diabetes. The effects of anti-CTLA4 associated irAEs are generally more severe than those from anti PD-1 inhibition. Whether irAEs represent  unclear .lo B cell subsets in patients given combination checkpoint inhibition (Modulation of B cell phenotype has been correlated with irAE in patients undergoing CPI therapy. A recent study (described above) demonstrated an overall decline in B cell numbers but increased plasmablasts and CD21hibition Table 1 hibition . These Cvia multiple mechanisms including as antibody secreting cells, cytokine producers, APCs, and immunoregulators. Given the important role of the checkpoint pathways in regulating immune homeostasis, it is not surprising that activation of the CTLA-4 pathway has been extensively studied in the treatment of autoimmunity. Specifically Abatecept, a fusion protein of CTLA-4 and IgG1 Fc portion (CTLA-4-Ig), is approved for abrogating immune overactivity in rheumatoid arthritis ; The Guy’s and St Thomas’s Foundation Trust Charity Melanoma Special Fund (573); CRUK/NIHR in England/DoH for Scotland, Wales, and Northern Ireland Experimental Cancer Medicine Centre (C10355/A15587); Breast Cancer Now ; the Medical Research Council (MR/L023091/1); Cancer Research UK . The research was supported by the National Institute for Health Research (NIHR) Biomedical Research Centre (BRC) based at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London (IS-BRC-1215-20006). The authors are solely responsible for study design, data collection, analysis, decision to publish, and preparation of the manuscript. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, or the Department of Health.SNK and JFS are founders and shareholders of Epsilogen Ltd. HJB is now employed through a fund provided by Epsilogen Ltd.The remaining 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.


Bleeding in the anterior pituitary lobe leading to tissue necrosis occurs in the acute stage of severe clinical forms of hemorrhagic fever with renal syndrome (HFRS), while atrophy of the anterior pituitary lobe with diminution of the gland function occurs after the recovery stage. The relationship between Hantaan virus infection and empty Sella syndrome (ESS) has rarely been reported.This patient was a 54-year-old previously healthy Chinese male. He presented with fever, headache, and backache with dizziness and oliguria. Physical examination was notable for hypotension and the signs of conjunctival suffusion. His platelets decreased, and his urine protein was positive. Hantaan virus IgM and virus RNA were positive.He was diagnosed as having HFRS. In his diuretic phase, his 24-hour urine volume was maintained at 10,000 mL, and his blood pressure was higher for a week. Then, he was diagnosed as having ESS after a series of examinations.Hormone replacement therapy was given to this patient after the diagnosis “ESS” was made.The patient's symptoms improved, and he was discharged from the hospital soon after hormone replacement therapy.Pituitary function examination and brain magnetic resonance imaging (MRI) need to be considered to scan for ESS and panhypopituitarism in the patients with HFRS accompanied by diabetes insipidus. The most common symptom of ESS is headache. Generally, the headache is deep, dull, and centrally situated. Sometimes headache is very severe, along with giddiness and vomiting before imaging examination.The term “empty sella” was first used in 1951 for the neuroradiological or pathological-anatomical exposure of an apparently empty sella turcica. Empty sella syndrome (ESS) is pathophysiologically characterized by either anatomic abnormalities in the diaphragma sellae (primary ESS), damage to the pituitary by irradiation/surgery, or autoimmunity leading to the availability of “empty” space in the sella (secondary ESS). Empty sella is the neuroradiological or pathological finding of an apparently empty sella turcica containing no pituitary tissue. The prevalence of primary empty sella, that is, empty sella without any discernible cause, is not precisely known; estimates range from 2% to 20%. Technical advances in neuroradiology have made empty sella an increasingly incidental finding. Improvements in neuroradiological imaging techniques have resulted in an increase in the incidental finding of “empty sella.” According to current data from India, an empty sella turcica without any detectable cause is an incidental finding in approximately 2% of all cerebral MRI scans. In asymptomatic adult patients, the question is whether and to what extent diagnostic hormone testing should be undertaken in an incidental finding of an empty sella, particularly as this does not necessarily have pathological significance.In primary ESS, the sella tends to be symmetrically ballooned without evidence of bony lesions. The suprasellar subarachnoid space herniates through an incomplete sellar diaphragm. Cisternography, high resolution computed tomography (CT) scanning, or magnetic resonance imaging (MRI) is used for diagnosis. The signs and symptoms depend upon the size, secretion, and pressure over the pituitary gland. Before the era of CT scan, many cases of empty sella were wrongly diagnosed.The causes of most of the primary ESS are still not clear. Hemorrhagic fever with renal syndrome (HFRS) is an acute infective multisystemic disease followed by fever, hemorrhages, and acute renal insufficiency. Bleeding in the anterior pituitary lobe leading to tissue necrosis occurs in the acute stage of severe clinical forms of HFRS, while atrophy of the anterior pituitary lobe with diminution of the gland function occurs after the recovery stage. The relationship between Hantaan virus infection and ESS has rarely been reported, especially in the acute stage.Herein, we present an adult case with ESS induced by HFRS. He was cured after supportive treatment and hormone replacement therapy.2This patient was a 54-year-old, previously healthy Chinese male with no significant past medical history and surgical history. He lived in a rural area with a high incidence of HFRS. He presented with fever, headache, and backache for 5 days in winter, the epidemic season of HFRS. He suddenly experienced fever up to 40°C. He sometimes felt ocular pain without visual disturbance. He felt dizzy, but he did not have his blood pressure examined. He exhibited oliguria during the last 3 days, but his urine output was not clear. Vomiting and abdominal pain were experienced.Physical examination was notable for a blood pressure of 88/53 mm Hg, weight = 62 kg, height = 5′7″, and body mass index = 21.5. His body temperature recovered to the normal level of 37°C. Signs of conjunctival suffusion  were found.Laboratory test results were as follows. Routine blood examination showed WBC of 55,000 cells/mL, percentage of neutrophils of 61.9%, and platelets of 93,000 cells/mL. The urine examination showed strong positive protein. Liver enzymes were increased . Renal function was abnormal . The reverse transcriptase-polymerase chain reaction (RT-PCR) Hantaan virus test result was positive. The Hantaan virus antibody IgM result was positive, while the Hantaan virus antibody IgG was negative. However, the result of antibody IgG to Hantaan virus was positive a week later and the titer of antibody IgG increased 4 times compared with that of the first test. The C-reactive protein (CRP) level was 105 mg/L (0–3.5), and the serum procalcitonin (PCT) was 1.3 μg/L (0.05–0.5).The urine output during the first 24-hour admission in our department was less than 200 mL. He was diagnosed as overlapping hypotensive phase and oliguric phase of HFRS. After supportive measures treatment, the patient progressed into the diuretic phase within a week. The regular blood cell test result was normal. His urine volume was significantly higher than normal, the 24-hour urine volume was maintained at 10,000 mL, and his blood pressure was obviously higher than his usual level for a week. He felt fatigued and lost his appetite. Then, the examination of pituitary function was performed. All of the results were much lower than the reference range . The brain MRI result showed that signals of cerebrospinal fluid could be observed in sella . His urine amount and the results of pituitary function tests recovered to the normal range, and the only discomfort – fatigue – disappeared 1 month later.3,6 During 1950 to 2014, the death rate of HFRS was reported as 2.89%, according to the statistical data from the National Health and Family Planning Commission of China.HFRS is a zoonotic disease caused by pathogenic hantaviruses. China is the most seriously affected country, with more than 10,000 cases reported annually, and accounts for over 90% of all cases worldwide. Serum PCT levels are much higher in bacterial, fungal, and parasitic infections than in viral infections or noninfected patients. In epidemic areas, HFRS patients with typical symptoms and routine laboratory findings could soon be diagnosed by experienced professional doctors. However, some cases need a differentiated diagnosis from severe bacterial infection, such as septicemia. The inflammatory biomarkers increased obviously in this case. Elevated levels of CRP and PCT are present in more than 90% of patients with HFRS. The elevated level of PCT in HFRS may be related to immune activation caused by hantavirus. Elevated CRP levels were observed in patients with hantavirus infection. Whether the elevated CRP and PCT are associated with the severity of HFRS remains a controversial issue.–12 It is well accepted that the main clinical hallmarks of HFRS are renal failure and hemorrhagic manifestations and that the disease is characterized by laboratory findings indicating renal function impairment, thrombocytopenia, and elevated ALT levels.CRP, secreted by the liver in response to bacterial infections, is a parameter used to diagnose infection. PCT, a precursor of calcitonin, is a diagnostic marker of the systemic inflammatory response, with a high sensitivity and specificity for infection. PCT and CRP have been used as new approaches to identify different types of infection. In a comparative analysis of 897 cases of HFRS, pituitary hemorrhage was revealed at autopsy in 36.8% of the patients, and pituitary necrosis in 5.2%. However, reports about pituitary involvement among survival cases with HFRS are not very common . For the most part, pituitary hemorrhage and necrosis were found in cases of death with HFRS via autopsy studies. On the contrary, treating hormonal dysregulation has a positive effect on morbidity and quality of life. In the diuretic phase of HFRS, patients need to be monitored carefully. The pituitary function and brain MRI examinations need to be considered to scan ESS and panhypopituitarism in the patients with HFRS accompanied by diabetes insipidus. Appropriate hormone replacement therapy could help the patients achieve relief quickly.It was recommended that overdiagnosis of empty sella should be avoided, as this might upset patients unnecessarily, and it also incurs substantial expense to the healthcare system.The authors thank the radiological technicians and neurosurgeons for their efforts in the clinical diagnosis and management of this patient.Conceptualization: Yang Wang.Data curation: Yuxiang Li, Yang Wang.Funding acquisition: Yang Wang.Investigation: Peng Zhang.Resources: Yuxiang Li.Software: Peng Zhang.Validation: Yang Wang.Visualization: Yang Wang.Writing – original draft: Haiying Chen, Peng Zhang.Writing – review & editing: Yang Wang.


After thermocycling, Novaloc’s RFs decreased by 33% (p < 0.001) while the Locators’ RFs increased by 34% . In contrast to LP, the RFs of Novaloc abutments and LOs predominantly showed no clinically relevant dependence on implant angulation. Ageing processes tended to result in lower RFs at higher implant angulation. Thus, the Novaloc attachment system offers an alternative to Locator attachments. It is characterized by a comparatively continuous RF-curve over the entire wearing period. Future clinical studies have to be conducted to verify the in vitro demonstrated advantages of the Novaloc system.Removable implant-anchored dentures have become an established treatment concept especially for older, multimorbid patients. This study investigates the retention force (RF) of two different attachment systems. A total of 96 specimens (n = 8 for each condition) were fabricated and RF was measured under different conditions: fatigue , thermal undulation  and implant-angulation . The Novaloc system ((N), 0° and 15° abutments, yellow matrix (Y)) was compared to the Locator system ((L), pink (P) and orange (O)). Initial RFs (8.57 ± 0.99 N (NY), 19.39 ± 8.10 N (LP), 8.8 ± 5.28 N (LO)) were reduced by ageing simulation (26% (NY), 66% (LP), 89% (LO);  In geriatric dentistry, the removable implant-anchored denture represents an established treatment concept. The insertion of two interforaminally positioned endosseous implants has become an established method for secure anchorage of a full denture ,2,3,4, yDepending on the jawbone content, deviations of the implant axes ranging from 0.5° to 27° in the horizontal and between 0.1° and 12.9° in the sagittal direction were found in everyday clinical practice ,15. MoreWear and the consequent loss of retention is basically the most frequent prosthetic complication with all individual connecting elements. Since retention and wear behavior are crucial for patient satisfaction , many stThus, the objective of the present study was to compare the Novaloc and Locator attachment systems with respect to retention behavior before, during, and after simulated ageing at different implant angulations. Matrix inserts with approximately equal retention forces according to the manufacturer’s specifications were used. As a primary null hypothesis, it was assumed that for the selected matrices, the initially measured pull-off forces correspond to the manufacturer’s specifications, regardless of the implant angulation, and that the retention forces among the different abutment systems are approximately the same, according to the selection. The second null hypothesis assumed that no clinically relevant retention force changes occur due to ageing simulations on the attachment systems. As the third null hypothesis, it was assumed that with the Straumann Novaloc Retentive System, there is no difference between a 15° secondary part at an implant angulation of 15° (20°) and a 0° secondary part at 0° (5°) angulation.A detailed list of the system-specific components used can be found in Sample size calculation was based on the results of Stephens et al., Mínguez-Tomás et al., and Elsyad et al. ,27,28, wA specimen basically consisted of two parts. The lower part of the specimen simulated the patient’s jaw. It contained the implant analog  with the screw-retained abutments (N and L). Especially for this purpose, metal blocks of stainless steel (68 mm × 20 mm × 20 mm) were fabricated with exactly matching drill holes for all implant angulations to be tested b. The maAll retention force measurements were obtained on the universal testing machine. Part 1 of the specimen was attached to the lower traverse, and part 2 of the sample was fixed manually onto the patrix. A hook that could be screwed into the axial course of the specimen holder part 2, ; I facilAll specimens were subjected to an ageing simulation after the initial pull-off forces were determined. For this purpose, 10,000 insertions and removals of a denture were simulated in the chewing simulator. After every 100, 200, 500, 1000, 5000 and 10,000 cycles, the specimens were removed and retention force measurements were taken on the universal testing machine. The simulation was performed in a saliva bath mixture . Finally, all specimens (part 2) were artificially aged in the thermocycler with 5000 alternating cycles between a hot (55 °C) and cold (5 °C) bath of distilled water. The immersion time was 30 s, and the dripping time was 17 s.x-axis) were taken and, using the formula proposed by Petropoulus et al. [To verify a statement about the release period and possible differences in the separation of the respective attachment systems, three force-displacement diagrams of each initial specimen combination from the initial pull-off tests were evaluated, as shown in s et al. , the relApart from the material, the geometry of the abutments or matrices influences the retention force. Dimensional changes caused by wear can cause retention force changes. To verify these, all matrices were measured by light microscopy initially and after simulated ageing  in accordance with the illustrations .At all measurement time points, 20 pull-off values were created for each specimen; thus, the sample size per test series and measurement time point was n = 160. All data were listed in Excel, transferred to the SPSS program , and descriptively analyzed in terms of the following problems:In accordance with the matrix color coding, the initially measured values were averaged and compared with the manufacturer’s specifications. Any deviations were stated as percentages.t-test for paired samples was used to check for significances.On the basis of the matrix color coding, the initial mean values were compared with the mean values after insertion and removal simulation and after thermal cycling. The Kolmogorov–Smirnov and Shapiro–Wilk tests were used to check the results of the individual test series for normal distribution. Levene’s test was used to check for homogeneous variances. The t-test was applied to independent samples. For all comparisons, the effect sizes were calculated using values from the test statistics (t-tests) according to Dunlap et al. [Since, according to the manufacturer’s specifications, matrices of medium retention forces were used in this study, the various test series were also compared with each other at the same measurement time points. Here, the p et al. .Influence of artificial ageing according to matrix color coding and implant angulationp-values due to an extremely large sample size, the 20 pull-off values per sample and measurement time point were averaged for statistical comparisons. After testing for normal distribution and variance homogeneity , single-factor analysis of variance (ANOVA) with repeated measurements was used to check for significances. In the absence of homogeneity of variances, Welch ANOVA with Dunett T3 post-hoc test was used.In order to avoid distorted Under the given conditions, the t-test (ANOVA) for independent samples was used to compare the mean retention force values within an attachment system at different angulations. This comparison of the retention forces was performed per angulation initially, after 10,000 insertions and removals, and after thermocycling.p ≤ 0.05), the comparison was carried out by means of the Mann–Whitney U test.For the comparisons, the release periods per attachment system were summarized on average. Due to missing preconditions  of the differently coded matrices are shown in The pull-off value of the yellow Novaloc matrix is specified as medium in the product description and defined at 12.01 N. On average, the yellow Novalocs initially achieved a 28.64% lower value, independent of the angulation. In contrast, the initially measured retention value of 19.39 N of the pink Locator matrices was 45.35% higher than the specified 13.34 N. On average, only the orange Locator matrices initially reached the specified manufacturer value.p < 0.001) in the pink Locators and 89% (p < 0.001) in the orange matrices. For the Novaloc system, the retention force was reduced by an average of 26% (p < 0.001). For all three attachment systems, a strong effect is confirmed with d > 1 in the mean value comparisons.In comparison with the average initially measured retention force values , simulatp = 0.002) and orange (p = 0.148) Locators, the thermocycling process produced an average retention force increase of 34%. On the Novalocs, on the other hand, the values decreased by 33% (p < 0.001) compared to the initial values. The significances of these differences (p ≤ 0.05) were confirmed by a strong effect size (d > 0.9).Compared to the initial measurements for the pink (p = 0.824). After 10,000 cycles, there was no difference in retention force between the pink Locator system and the Novaloc system (p = 0.227). All remaining comparisons as well as the average retention forces after thermocycling showed differences with clinical relevance (p < 0.001).Mutual comparisons of the independent test series show that, on average, only the initial values of the Locator system with the orange matrix inserts did not differ significantly from the Novaloc system with yellow matrix inserts . Some of them showed slightly lower retention forces after the ageing process of 10,000 cycles in specimen combinations with higher implant angulations (p ≥ 0.05).On the straight Novaloc abutments, no significant differences were initially verified with various angulations (ulations . This cop ≥ 0.05). Only after 10,000 insertion and removal cycles, a slightly (~1.13 N) smaller average retention force (p = 0.031) was determined at an implant angulation of 20°.The orange Locator matrix inserts showed no significant differences initially and after thermocycling . The only exception was the comparison of NY-15°/IA-15° to NY-0°/IA-0° with 10,000 insertions and removals .Comparisons of the Novaloc combinations (NY-15°/IA-15° to NY-0°/IA-0° and NY-15°/IA-20° to NY-0°/IA-5°) with theoretically identical positioning of the Novaloc abutments relative to the matrix or denture pull-off direction showed no significant differences (p < 0.001). The longest release periods were found in the Locator system with the pink matrix inserts . Between these groups, the mean value of the Locator system with the orange matrices was 0.00368 min and that of the angled Novaloc abutment was 0.00397 min. Statistically, the differences between NY-0° and LO-0° (p = 0.658) and between NY-15° and LO-0° (p = 0.065) could not be confirmed.As shown in In general, wear is characterized by mechanically and partially chemically induced surface material loss . Wear-inThe null hypothesis that the pull-off forces, according to the matrix color coding, correspond to the manufacturers’ specifications had to be partially rejected. As shown in Both the average retention forces  and the retention force curves of the individual test series basically showed a continuous, statistically significant loss of retention force from 1000 insertion and removal cycles onwards. Consequently, the second null hypothesis also had to be rejected. However, in the opinion of the authors, a retention force loss of 26% is not clinically relevant, or is relevant only to a limited extent. Moreover, in the literature, the reported relative retention force losses after artificial ageing range from 21% to 78.62% for Locator attachment systems ,36,37,38Besides the angulation of implant abutments, varying designs of matrices were presented in the last years to compensate implant divergence. Thus, future studies might evaluate the retentive behavior of angulated abutments of the Novaloc system in comparison to abutments with another construction principle, for example the OT Equator system . The Equator system in combination with the smart-box abutment system allows due to a tilting mechanism with a rotation fulcrum the passive insertion up to 50° implant angulation. Both in vitro and in vivo studies found this system to reveal acceptable results ,44.In assessments based on the mean values, thermocycling produced a significant and clinically relevant increase in retention force, specifically for the Locators, in both the initial and in the retention force values after artificial ageing. For the Novalocs, on the other hand, the initial values and the values after artificial ageing decreased . These vBy adding additives (fibers etc.), the mechanical properties, or possibly the retention force, can be improved or changed. Consequently, however, the sensitivity to hydrolysis would increase due to the formation of macroscopically or microscopically small gaps between the matrix and additive. Liquids can be absorbed into the resulting spaces by capillary action. However, the manufacturer did not provide a definitive statement regarding the composition; thus, there is no information on changes to any new batches. Clinical applications often show heavy wear of the Locators ,42, whicp ≤ 0.05), were not clinically relevant  difference after 10,000 insertions and removals was nearly 1 N, which is not clinically relevant. The retention force is ensured by the angle compensation of the Novaloc abutment, such that the same conditions exist with regard to undercuts and the pull-off direction.The third null hypothesis, that there is no difference between a 15° secondary part at an implant angulation of 15° (20°) and a 0° secondary part at 0° (5°) angulation on the Novaloc attachment system, could be confirmed. The statistically quantified , but is not reflected by a noticeably greater loss of retention force. The nonetheless higher wear verified in the marginal area of the yellow matrices may be due to the different shapes of the occlusal and lateral surfaces on the part of the manufacturer when compared to the straight Novaloc abutment. This is especially pertinent since microscopically, a tendency toward increased widening with greater implant angulation can be noticed  with currently introduced attachment systems that consist of matrices with a tilting mechanism .Within the limits of this study design, the following conclusions can be drawn:


Therapeutic lymphangiogenesis in an orthotopic lung transplant model has been shown to improve acute allograft rejection that is mediated at least in part through hyaluronan drainage. Lymphatic vessel endothelial hyaluronan receptor (LYVE-1) expressed on the surface of lymphatic endothelial cells plays important roles in hyaluronan uptake. The impact of current immunosuppressive therapies on lung lymphatic endothelial cells is largely unknown. We tested the hypothesis that FK506, the most commonly used immunosuppressant after lung transplantation, induces lung lymphatic endothelial cell dysfunction..Lung lymphatic endothelial cells were cultured in vitro and treated with FK506. Telomerase activity was measured using the TRAP assay. Protein expression of LYVE-1 and senescence markers p21 and β-galactosidase was assessed with western blotting. Matrigel tubulation assay were used to investigate the effects of FK506 on TNF-α-induced lymphangiogenesis. Dual luciferase reporter assay was used to confirm NFAT-dependent transcriptional regulation of LYVE-1. Flow cytometry was used to examine the effects of FK506 on LYVE-1 in precision-cut-lung-slices ex vivo and on hyaluronan uptake in vitro, FK506 downregulated telomerase reverse transcriptase expression, resulting in decreased telomerase activity and subsequent induction of p21 expression and cell senescence. Treatment with FK506 decreased LYVE-1 mRNA and protein levels and resulted in decreased LEC HA uptake. Similar result showing reduction of LYVE-1 expression when treated with FK506 was observed ex vivo. We identified a putative NFAT binding site on the LYVE-1 promoter and cloned this region of the promoter in a luciferase-based reporter construct. We showed that this NFAT binding site regulates LYVE-1 transcription, and mutation of this binding site blunted FK506-dependent downregulation of LYVE-1 promoter-dependent transcription. Finally, FK506-treated lymphatic endothelial cells show a blunted response to TNF-α-mediated lymphangiogenesis.In vitro. The implications of our findings on the long-term health of lung allografts merit more investigation.FK506 alters lymphatic endothelial cell molecular characteristics and causes lymphatic endothelial cell dysfunction in vitro and ex vivo. These effects of FK506 on lymphatic endothelial cell may impair the ability of the transplanted lung to drain hyaluronan macromolecules in vivo Lung transplant carries the worst outcome of any solid organ transplant with a 60% 5-year survival  is a mainstay of immune-suppression in lung   were cultured in microvascular cell culture media with supplements . All experiments were done with LEC between passages 3 to 5. FK506  and Cyclosporin A  were reconstituted with DMSO and used at indicated concentrations. At the 48 h time point, FK506 did not affect LYVE-1 protein levels  assay  per manufacturer’s instruction and as we have previously described -124/+ 124 wildtype (NFATc binding site – ttttcc) and P-124/+ 125 mutant (NFATc binding site – ttgtcc) constructs. Reverse transfection was carried out on a total of 20,000 HEK-293 T cells using X-tremeGENE HP DNA Transfection Reagent (Sigma). Cells were transfected with 5 ng of pRL_CMV vector  and 95 ng of P-124/+ 214 (wildtype or mutant) construct or pGL3_Basic vector (negative control). Cells were treated with 15 ng/mL of FK506 at 6 h post-transfection, and luciferase activity was tested 24 h after transfection using Dual-Glo Luciferase Assay System kit (Promega) as per manufacturer’s protocol. Biotek Synergy HT microplate reader  with Biotek Gen5.1.1 microplate data collection software was used for luciferase luminescence detection. Each transfection was carried out in triplicates in 4 independent experiments.Matrigel tubulation assays were conducted as we previously described 12Nrud/J mice  were obtained as previously described , hyalurodinase , and DNase I  for an hour at 37 °C. Cells were then washed twice with cold FACS buffer (PBS with 1% BSA), and incubated with purified rat anti-mouse CD16/CD32 Fc block solution  for 20 min at 4 °C, followed by incubation with anti-LYVE1 monoclonal antibody conjugated with PE-Cyanine7  for 30 min at 4 °C. Lastly, cells were washed twice with FACS buffer and subjected to flow cytometry analysis.t-tests were conducted. Telomerase activity in control and FK506 (15 ng/ml)-treated cells was compared using a paired, two-tailed Student’s t-test. A P value of less than 0.05 was considered significant.All experimental data are presented as the mean ± SEM. For analysis of western blot and q-PCR data, one-way repeated measure ANOVAs (RM ANOVA) (within-subjects factor: treatment) were run, followed by Tukey’s post-hoc test to evaluate treatment effect according to dose of FK506 (10 or 15 ng/ml). To compare immunofluorescent intensity values between positive control and FK506 (15 ng/ml)-treated cells, two-tailed Student’s FK506 inactivates calcineurin, which blocks the dephosphorylation of NFAT, a necessary step for its nuclear translocation and subsequent transcription activation. To confirm that FK506 blocks NFAT nuclear translocation in lung LEC, we treated LEC with FK506 and showed with cell fractionation that treatment resulted, as expected, in decreased NFAT nuclear translocation  is a key enzyme involved in telomere maintenance . Treatment with FK506 resulted in a reduction in luciferase activity, which was not observed in pGL3_Basic vector with no NFAT binding sites , and a significant reduction (p < 0.05) in the response to FK506 compared to the wildtype construct  compared to pGL3_Basic vector alone  decreased TERT expression and telomerase activity, which was associated with increase in LEC senescence; 2) decreased LYVE-1 expression and LEC HA-uptake and 3) inhibited TNF-α-induced lymphangiogenesis.Calcineurin inhibitors, including FK506, are currently the mainstay of immunosuppression in lung transplantation, with important effects on T-cell function and prevention of allograft rejection  for 48H. Molecular weight (kDa) for each protein is indicated on the right. Ratio of LYVE-1 to β-actin density was expressed as fold-change relative to control. Data represent mean ± SEM of three independent experiments, consisting of one technical replicate each. Supplementary Figure 2. FK506 prevents NFAT nuclear translocation. Lung lymphatic endothelial cells were treated as indicated. Samples of protein lysates (Entry: 27%), and nuclear (27%) fractions were separated by SDS-PAGE and transferred to nitrocellulose membranes, and reacted with antibodies against NFAT, PARP (nuclear marker) and GAPDH (cytoplasmic marker). FK506 resulted in a marked decrease in NFAT in nuclear fraction. Data represent mean ± SEM of 3 independent experiments (p < 0.05). Supplementary Figure 3. Effect of Cyclosporin A on TERT and LYVE-1 expression. Real-time PCR analysis of TERT (A) and LYVE-1 (B) mRNA in lung lymphatic endothelial cells treated with control or Cyclosporin A (10 μg/mL) for 48 h. Results were expressed as the fold change compared to control. Graphs represent the mean ± SE from three independent experiments. p < 0.05 (*) and p < 0.01 (**) by T-Test. Supplementary Figure 4. Effect of FK506 on other lymphatic markers. Real-time PCR analysis of podoplanin (PDPN) (A) and PROX1 (B) mRNA in control and FK506-treated (48 h) lung lymphatic endothelial cells. Results were expressed as fold change compared to control. Graphs represent the mean ± SE from three independent experiments. Supplementary Figure 5. Effects of LYVE-1 inhibition with function blocking antibodies on HA uptake in vitro and FK506 treatment on CD44 expression. LEC were plated in 6-well plates and treated with Isotype (control) and LYVE-1 monoclonal antibodies (10 μg/mL) for 72 h. Cells were then incubated in media containing 1000 μg/mL of FITC-HA for 5 h. Percentage of FITC-positive cells (A) were analyzed by flow cytometry. Real-time PCR analysis of CD44 (B) mRNA in control and FK506-treated (48 h) lung lymphatic endothelial cells. Results were expressed as the fold change compared to control. Graphs represent the mean ± SE from three independent experiments. Supplementary Figure 6. Western blot full images. Supplementary Table 1. Primary antibodies used in these studies. Supplementary Table 2. Real-time PCR primers.


Cynomolgus macaque model of TB, we revealed that transcriptional signatures of different molecular TB endotypes did not depend on TB progression post-infection. Moreover, we provide evidence that patients with molecular endotypes characterized by high levels of IFN responses (IFN-rich), suffered from more severe lung pathology than those with lower levels of IFN responses (IFN-low). Harnessing machine learning (ML) models, we derived gene signatures classifying IFN-rich and IFN-low TB endotypes and revealed that the IFN-low signature allowed slightly more reliable overall classification of TB patients from non-TB patients than the IFN-rich one. Using the paradigm of molecular endotypes and the ML-based predictions allows more precisely tailored treatment regimens, predicting treatment-outcome with higher accuracy and therefore bridging the gap between conventional treatment and precision medicine.Group-aggregated responses to tuberculosis (TB) have been well characterized on a molecular level. However, human beings differ and individual responses to infection vary. We have combined a novel approach to individual gene set analysis (GSA) with the clustering of transcriptomic profiles of TB patients from seven datasets in order to identify individual molecular endotypes of transcriptomic responses to TB. We found that TB patients differ with respect to the intensity of their hallmark interferon (IFN) responses, but they also show variability in their complement system, metabolic responses and multiple other pathways. This variability cannot be sufficiently explained with covariates such as gender or age, and the molecular endotypes are found across studies and populations. Using datasets from a  Mycobacterium tuberculosis (Mtb) fall sick with active TB (Tuberculosis (TB) remains a major threat to human health with 10 million new cases and 1.4 million deaths in 2019  samples with Mtb-specific antigens. The false negative rate of IGRA among Mtb infected individuals is in the order of 15%  expression of different genes may be highly correlated and thus, selecting one or another gene does not influence the performance of the model; (2) different molecular mechanisms dominate the response to TB in different cohorts; (3) various cohorts contain varying numbers of individuals with a certain type of dominant response which influences outcome of comparison of ‘all TB patients’ to ‘all healthy subjects’. For example, the study of Maertzdorf et al. identified JAK-STAT signaling and TLR signaling pathways next to IFN response as dominant in TB  which determines the release of IFN γ r of 15% . Thus, bnt in TB . In contnt in TB . Studiesnt in TB , 15. It Cynomolgus macaques  on the integrated transcriptome data with various gene set collections, including pre-defined blood transcriptional modules (BTMs) , 17 to rmacaques  and obsemacaques  and MSigmacaques  based GSmacaques , 21.The overview of the workflow of the study and the used statistical methods can be found in the via the R-package GEOquery (All utilized datasets are publicly available in Gene Expression Omnibus (GEO) data repository . Study-nGEOquery .BiomaRt R package (Included studies met the following criteria: (i) they contained WB data from untreated TB patients and healthy controls (including LTBI) each; (ii) they contained at least eight samples from TB patients and healthy controls each; (iii) they were performed using platforms which measured expression of at least 16,000 overlapping genes; (iv) they were performed using platforms with annotations available in  package , 25. Sevhttps://github.com/terkaterka/immune-response-to-TB). Datasets were analyzed with R package limma for differential expression analysis  and samples of untreated TB patients were included. MDS was created out of the training sets from each study using only common genes. Nonparametric standardization based on median and interquartile range (IQR) values (Equation 1) was used to standardize the expression values measured in each study, in order to minimize batch effects and heterogeneity between the experiments.Where:.,jIQR – IQR for expression measurement of gene i across all samples.All utilized data underwent initial quality controls which comprised outliers and artifact-detection and quality-assurance. We ascertained that case and control cohorts clustered according to TB and IFN status and not by their study of origin. Umap-algorithm  derived To perform GSA for individual patients, row-wise z-transformation of gene expression values was applied. For each gene, mean expression and standard deviation of its expression were calculated for healthy individuals from every cohort. Subsequently, the mean gene expression of healthy individuals was subtracted from the expression measurements of every individual present in the MDS and the result was divided by standard deviation of gene expression for healthy individuals. The z-score was calculated based on all samples from healthy individuals. Thus, for each patient and gene, the expression z-score is the number of standard deviations below or above the average for healthy individuals. The larger the absolute value of the z-score, the higher the deviation of the expression of that gene from the average in the healthy population.tmod .Two previously published sets of BTMs were utilized , 17. A tGSA was performed on the list of genes from every individual included in MDS sorted by increasing z-score using the three created module sets. Individuals presenting no significant enrichment in any of the IFN I modules were defined as IFN-low and are represented graphically as ‘IFN I-’. Individuals presenting enrichment in at least one IFN I module were defined as IFN-rich and are further represented graphically as ‘IFN I+’. Similarly, the ‘IFN II-’ and ‘IFN I and II-’ individuals presented no enrichment in the IFN II or IFN I and II module set, respectively. Those presenting respective enrichments were defined as ‘IFN II+ or ‘IFN I+ and II+’. Ultimately, the overlaps between study participants classified as IFN I+, IFN II+, and IFN I+ and II+ were analyzed and their classification was compared to IFN+ and IFN- participant groups based on the original BTMs , 17.To investigate the influence of discrete features  on the IFN status the chi-square test was performed and Cramér’s V effect size was calculated. Moreover, the odds ratio (OR) was calculated with 95% CI and the test for OR was conducted (H0: OR=1). For continuous variables (age), the Mann-Whitney test was performed and rank biserial correlation was calculated as effect size.stats, pca3d and tmod  curves using R package pROC (Random Forest (RF) models were generated using R package omForest , 37 to cage pROC .We ranked the transcripts found in either model according to the amount of statistical importance in the RF model to identify a cut-off for the number of transcripts required to effectively discriminate TB patients from non-TB samples. TB IFN+ and TB IFN- signatures were defined consisting of top (i) 5, (ii) 7, (iii) 10, (iv) 20, (v) 50 or (vi) 200 ranked transcripts, and new models which were trained only on the transcripts that surpassed the cut-off threshold were created. The new models were tested using 10-fold cross validation within the training MDS and their performance was evaluated using ROC plot. The optimal signature size was chosen when the increase of the number of selected transcripts ranked by highest statistical importance did not cause further significant improvement of signature’s area under curve (AUC) in classification of TB patients and non-TB disease controls.For the identification of TB IFN+ and TB IFN- signatures two new class balanced RF models retaining the proportion of one TB to three non-TB cases were trained using the subsets of the complete training MDS subsets containing (i) all TB IFN+ and non-TB (Signature Model 1), (ii) all TB IFN- and non-TB (Signature Model 2). A signature consisting of the 20 top ranking transcripts from the Signature Model 1 was defined as IFN+ signature. A signature of 50 top ranking transcripts from the Signature Model 2 was defined as IFN- signature. Obtained TB IFN+ and TB IFN- signatures were tested on the test MDS and their performance was evaluated by a ROC curve analysis.The obtained TB IFN+ and TB IFN- signatures were tested on the external dataset from Cai et al.  and BlanCynomolgus macaques  for successful integration .To further benchmark the implemented division into IFN+ and IFN- groups we tested the differences between the TB patients categorized into the two groups using PCA. The results indicated that although the clusters of IFN+ and IFN- TB patient groups overlapped, the two centers of the clusters were geometrically shifted in regards to each other as best shown by PC2 and PC7 , or ISGs. To avoid using the genes on which the division into IFN+ and IFN- patient groups was based, we identified genes described in IFN signaling pathways, but not included in the original BTMs , 17, andInterestingly, despite IFN type I and type II signaling pathways being the most studied in the immune response to TB, we found that also IFN λ receptor gene (IFNLR1), but not IFN λ gene (IFNL) itself, which both belong to type III IFN signaling pathway was significantly overexpressed in IFN+ compared to IFN- TB patients or healthy .Based on X-Ray images of their lungs, Berry et al.  assignedGene signatures are frequently used to differentiate between TB patients and healthy individuals , 9, 46. versus healthy individuals.The IFN- signature showed a slightly better overall performance in the identification of TB patients  as surrogate marker of severity of pulmonary pathology . This suggests that the “IFN-rich” endotype is distinguished not only by its pronounced IFN response but also by pronounced NF-kappa B signaling and complement system signaling, while the genes belonging to T cell receptor signaling pathway and Th17 cell differentiation are strongly down-regulated in this endotype . These databases have a broader  scope than the BTMs. Next, we performed GSA using those gene set collections and tested the independence from IFN gene set enrichment in individual proportions. For several of them, the enrichment in individual patients strongly correlated (positively or negatively) with the IFN status. This was the case among others for “Hallmark p53 pathway” module (correlation coefficient of the eigengenes r=0.98), “NF-kappa B signaling pathway” (r=0.96), “Hallmark complement” (r=0.96), “T cell receptor signaling pathway” (r=-0.94), “Th17 cell differentiation” (r=-0.94). Likewise, the proportion of patients presenting enrichment in those modules significantly overlapped with the enrichment in the IFN gene set  and occurred far less commonly among TB patients from different cohorts than the enrichment in the IFN modules. To unravel these, for each given gene set, we tested the correlation of enrichments across all TB patients between the given gene set and the IFN response. This yielded 16 gene sets  which showed enrichment  in at least 10% of TB patients in the MDS and with no enrichment in the remaining TB patients and, at the same time, no significant correlation with the IFN response  the IFN I signaling pathway is thought to be mostly detrimental and (ii) the IFN II pathway is considered to play a major role in protection . Yet, ouUnsupervised analysis identified that samples collected from IFN- and IFN+ TB patient groups cluster together but are shifted with regard to each other. GSE on the weights of genes revealed contribution of T cells, which are potent producers of IFN γ and IFN α cytokines . PCA of Our analysis demonstrates that differences in the enrichment of IFN related modules is not a consequence of varying abundances of IFN α, IFN β or IFN γ in WB since the IFN+ and IFN- patient groups presented similar expression of IFN α, IFN β and IFN γ genes. Rather, significant differences in the expression of IFN α and IFN γ receptors and ISGs such as CXCL10 between the IFN+ and IFN- TB patient groups were critical. We conclude that regulation of gene expression in IFN+ and IFN- TB patient groups was not caused by differential expression of IFN α, β or γ, but by differential expression of IFNR genes and ISGs.The abundance of the transcript BATF2 contributed to the differences in enrichment observed between IFN+ and IFN- TB patient groups. The BATF2 levels were significantly higher in IFN+ than IFN- TB patient groups. This leucine zipper transcription factor has been shown to exacerbate lung pathology in an experimental mouse model  and has We defined diagnostic signatures of IFN+ and IFN- TB patient groups using ML methods. The selected IFN+ signature comprised 20 transcripts, while the optimal IFN- signature consisted of 50 transcripts. 7 transcripts were present in both IFN+ and IFN- TB signatures: GBP5, AIM2, GBP2, POLB, WARS1, LHFPL2, DUSP3. Several of these genes are related to IFN-signaling which emphasizes the important role of IFN in TB even in patients in whom IFN signaling is enriched marginally.versus sarcoidosis patients, which was only satisfactory for the IFN+ TB signature. TB and sarcoidosis have been shown to present a remarkably high overlap between biomarkers that discriminate versus healthy controls  the transcriptomes of eight out of 52 LTBI patients clustered with profiles of TB patients and four out of 21 TB patients presented transcriptional profiles resembling those of LTBI. Similar observations were reported by Blankley et al.  who deteTo this end, we next explored the possibility of further novel, molecular endotypes of TB, which are not directly linked to IFN responses. We found that TB patients differ in the activity of genes associated with calcium signaling, insulin signaling and amino acid metabolism. These findings introduce an exciting new avenue of exploring pathways linked to TB. We are aware that our results are based on lack of an observed correlation with the IFN response – and thus might indicate lack of evidence for association with IFN rather than evidence of lack. However, given the large number of patients on which we based our study, we feel reasonably confident that if such effect exists, its magnitude must be small. Clearly, as these findings are based on an exploratory analysis, a further validation targeted directly at our hypotheses will be essential. The concept of endotypes to describe subgroups of patients on the basis of distinct transcriptomic, epigenetic or metabolic features has been applied to several diseases, most recently also to TB . A combihttps://github.com/terkaterka/immune-response-to-TB).Although our study provides deeper insight into individual variability among TB patients at the level of gene expression, there are limitations: first, human cohorts are highly variable due to numerous factors including genetic variability, conditions of life and circumstances of infections . Additional confounders include varying study designs and conduct, as well as technical variation. To partly account for these confounders, we validated our results in several ways including cross-validation and leaving 20% of the acquired studies unprocessed for independent testing, acquisition of independent validation datasets and testing the gene signatures of IFN+ and IFN- TB on a different disease - sepsis. Application of our data collection, normalization and analytical methods in numerous external datasets revealed that the proposed analytical framework is robust and can be used in other multi-cohort studies. Our dataset collection selected out of published TB datasets and newly defined sets of IFN I, IFN II as well as IFN I and II inducible genes can be accessed on the website:  data repository.Publicly available datasets were analyzed in this study. This data can be found here: All of the used, publicly available datasets are referenced in the manuscript. The datasets are found in the Gene Expression Omnibus .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.


We present an open and isolated palmar dislocation of the head of the fifth metacarpal bone without fracture. The diagnosis, which was initially made based on the X-rays, was confirmed during the operation. The patient was satisfactorily treated with open reduction, Kirschner wires fixation and casting followed with hand physiotherapy. The volar plate and collateral ligaments prevent MCP joints  of dislocation due to protection against hyperextension, radial and ulnar deviation. The most common dislocation of MCP joint is a dorsal dislocation  and the Therefore, the purpose of this report is to present a patient with an isolated palmar dislocation of the head of the fifth metacarpal bone without a fracture, satisfactorily treated with open reduction, Kirschner wires fixation, casting, and followed with hand physiotherapy and hand occupational therapy.A 62-year-old right-handed male presented to the Emergency Department (ED) with palmar protrusion of the head of the fifth metacarpal bone through the skin following a fall with injury to the right hand, On examination, patient had besides an open wound with palmar protrusion the fifth metacarpal bone through the skin, pain around the wound. However, capillary refill and sensation were normal, which suggested an intact neurovascular bundle of the fifth finger. Further, normal flexion of distal interphalangeal joint and the proximal interphalangeal joint was observed. Wrist and other movements were normal. The patient had no previous history of trauma to the right hand.Standard radiographs were obtained which revealed isolated palmar dislocation of the head of the fifth metacarpal bone without any other associated injury or fracture, An attempt to perform a closed reduction under local anesthesia was unsuccessful. The patient was admitted to undergo an open reduction under regional anesthesia the same day.Extending the wound, we observed a dislocation of flexor digitorum superficialis tendon (FDS) and flexor digitorum profundus tendon (FDP) to radio dorsal from the head of the fifth metacarpal bone. After replacing tendons to palmar, dislocation dissolved. Neither reconstruction of the volar plate nor exploration of the intermetacarpal ligaments were performed. After achieving hemostasis, the wound was closed with Ethilon 5-0. Internal stabilization with Kirschner- wires through MCP joint in 90 degrees of flexion was performed, see An isolated open palmar dislocation of the head of the fifth metacarpal bone without any fracture is rare. Reposition of this type of dislocations can be practically impossible due to volar plate, collateral ligament, tendinous juncture connecting the fourth and fifth extensor digitorum communis tendons, sesamoid bone, or as observed in our case luxation of FDS and FDP to dorsoradial to the head of the metacarpal bone . ConsideThe volar plate wasn’t reconstructed during the surgery, because it required a total exposure of the joint capsule. During the operation, some stability of the joint was noticed due to the surrounding structures, and given the obtained results we can assume that during the healing process affected joint developed more stability due to the scar tissue and the formation of adhesions.To the best of our knowledge, this is the first published case of open isolated palmar dislocation of the head of the fifth metacarpal bone without fracture.In our opinion, a closed reduction of rare palmar MCP joint dislocations have little chance of success due to volar plate, collateral ligament, tendinous juncture connecting the fourth and fifth extensor digitorum communis tendons, sesamoid bone, or as observed in our case luxation of FDS and FDP to dorsoradial to the head of the metacarpal bone. The open reposition and fixation showed, in our case, good results after sessions of hand physiotherapy and hand occupational therapy.


One way to improve the delivery of oncology palliative care in low and middle-income countries (LMICs) is to leverage mobile technology to support healthcare providers in implementing pain management guidelines (PMG). However, PMG are often developed in higher-resourced settings and may not be appropriate for the resource and cultural context of LMICs.This research represents a collaboration between the University of Virginia and the Nepalese Association of Palliative Care (NAPCare) to design a mobile health application (‘app’) to scale-up implementation of existing locally developed PMG.We conducted a cross-sectional survey of clinicians within Nepal to inform design of the app. Questions focused on knowledge, beliefs, and confidence in managing cancer pain; barriers to cancer pain management; awareness and use of the NAPCare PMG; barriers to smart phone use and desired features of a mobile app.n = 48) had training in palliative care/cancer pain management and the majority  reported high confidence levels (scores of 8 or higher/10) in managing cancer pain. Highest ranked barriers to cancer pain management included those at the country/cultural level, such as nursing and medical school curricula lacking adequate content about palliative care and pain management, and patients who live in rural areas experiencing difficulty accessing healthcare services . Most nurses and physicians use an Android Smart Phone , had heard of the NAPCare PMG , and reported frequent use of apps to provide clinical care . Key barriers to smart phone use differed by discipline, with nurses reporting greater concerns related to cost of data access  and being prohibited from using a mobile phone at work .Surveys were completed by 97 palliative care and/or oncology healthcare providers from four diverse cancer care institutions in Nepal. 49.5% (Smart phone apps can help implement PMG and support healthcare providers in managing cancer pain in Nepal and similar settings. However, such tools must be designed to be culturally and contextually congruent and address perceived barriers to pain management and app use. High quality palliative care, which emphasizes pain management, can greatly reduce global suffering from non-communicable diseases, such as cancer –6. This Professional oncology organizations have created pain management guidelines (PMG) to help health care providers effectively manage pain –29. UnfoSince its establishment in 2009, the Nepalese Association of Palliative Care   Institutional Review Board and the Nepal Health Research Council and adhered to all relevant guidelines and regulations. It is important to note that methodological decisions for this study were guided, in part, by the intent of the funding mechanism to strengthen research capacity within Nepal, and to facilitate learning and mentoring opportunities between the University of Virginia (UVA) and Nepal interdisciplinary teams.Our 36-item survey was collaboratively developed by the Nepal and UVA research teams, in consultation with the UVA Center for Survey Research. The survey took approximately 7 months to design (September 2018 – April 2019) and was a highly iterative and collaborative process between the Nepal and UVA research teams, working both remotely and during in-person fieldwork visits. Survey items were informed by the study aims and the broader literature related to known barriers to cancer pain management and challenges related to opioid availability in low and middle-income countries across various levels of the Social Ecological Model   many terms did not have precise Nepali equivalents; 2) Nepali healthcare providers are trained in English and a high degree of English fluency was expected from respondents; and 3) the surveys would be administered face-to-face with a Nepali research team member available to assist with any translation needs. Particular attention was paid to be sure items were clear and contextually and culturally congruent/relevant. Questions were primarily quantitative , with a few optional write-in responses if a participant wanted to expand upon a response and organized in four sections: 1) participant demographics; 2) knowledge, beliefs and perceived barriers related to cancer pain management; 3) awareness and use of the NAPCare PMG; and 4) smart phone use, barriers, and desired features of a mobile app.We recruited a quota sample of healthcare providers over age 18 who provide direct care to patients with cancer. The target number of respondents for each site was determined by the size of the institution  and took into account what Nepal research team members felt were realistic recruitment goals. Potential respondents were identified by Nepal research team members, screened for eligibility, and offered a chance to participate in the survey. All participants provided verbal informed consent, which was approved by respective ethical committees , prior to any data collection. Participants reviewed an approved study information document and had the opportunity to discuss any questions with a trained research team member before proceeding with the survey. The sample was stratified across a public general hospital; a public cancer hospital; a private cancer hospital; and a hospice within Kathmandu Valley and Chitwan District, Nepal. The 4 study sites were selected because they see a high volume of cancer patients and represent diverse practice contexts.Pencil-and-paper surveys were administered to consented participants between May – August 2019 by trained Nepali research team members. We used paper surveys due to factors related to cost, convenience and flexibility in the field. Participants received a small cash incentive for completing the survey . Completed survey data were entered into Qualtrics for data storage and management. We elected to use Qualtrics for initial data entry due to ease of user interface and to familiarize the Nepal team with the Qualtrics platform.Qualtrics survey data were exported into SPSS v.26.1 . Data analysis was conducted both at UVA and by members of the Nepal team, with mentorship and support from researchers at UVA.During a fieldwork trip to Nepal in August 2019, the UVA and Nepal team conducted a thorough data audit, which included: 1) visual inspection of all paper surveys and cross-checking study identification numbers with the master participant log from each site; and 2) validating data entered into SPSS with paper survey responses with a random sample of at least 15% of surveys from each site. If data entry errors were discovered, we performed additional random validity checks to ensure accurate data entry of that site. Also, if two answers were marked on an item that only allowed for one response – and it was difficult/impossible to know which response the participant truly intended – then we used a coin toss to determine the response. We also reviewed all open text items and ‘other’ write-in options across the entire data set to ensure responses were correctly captured in SPSS; we made slight corrections for clarity and/or completeness.Descriptive statistics were calculated for all quantitative items. Additionally, inferential comparative analyses were run to explore how variables of interest assessed by the survey (such as confidence in pain management tasks) may vary by institution  and healthcare provider role . Specifically, Pearson chi-squared tests and independent sample t-tests were conducted to check for statistically significant relationships (α = 0.05). Our survey measures aimed to understand perceptions and experiences of respondents across individual survey items rather than present composite scales or indices. However, for the grouped personal, institutional, and country/cultural barriers to pain management, we did calculate Cronbach’s Alpha and all had values above 0.8,  indicating strong internal reliability.Most open text responses were brief, one phrase responses  and were therefore simply counted, quantified, and presented with corresponding quantitative items. The last survey item, ‘is there anything else you would like to share about your experience with cancer pain in Nepal?’ yielded more detailed text responses which were exported into Microsoft Word, organized into groups based on similarity of key phrases, and then reviewed for patterns using a basic qualitative descriptive approach . For exan = 92), as they are expected to be the primary mobile app users. Findings are presented below by survey section.With the exception of Tables n = 64; 66%), female , identified palliative care as their current practice area , and reported spending over 80% of their time caring for patients with cancer .A total of 97 healthcare providers  completed the survey across all four study sites. Overall, the majority of respondents were nurses  compared to those who did not . Overall, a higher percentage of nurses completed training  compared to physicians  and nurses rated its helpfulness  as higher than physicians . This difference was statistically significant in the overall sample and within the public general hospital.Almost equal numbers completed formal training in palliative care or cancer pain management (n = 92) the average mean confidence score for managing cancer pain, in general, was 7.8. Assessing the patient’s need for morphine, calculating breakthrough morphine doses, and adjusting/titrating morphine doses also all had high mean confidence scores .Participants were asked to rate their confidence  in specific cancer pain management activities. For physicians and nurses  and administering (for nurses) was also assessed per common opioid Fig. . The mosn = 92), followed by institutional . The lowest category of barriers involved personal factors .Potential barriers to cancer pain management were assessed in the categories of personal, institutional, and country/cultural factors . By overall category, the highest rated barriers were country/cultural  and by nurses  and physicians  was ‘nursing and medical school curriculums in Nepal do not contain enough content about palliative care and pain management.’ Other top ranked system-level barriers included ‘patients in rural areas have trouble accessing healthcare services’  and ‘stigma and fear of cancer cause delayed diagnosis/treatment’ . The lowest ranked country/cultural barrier overall  and by nurses  and physicians  was ‘patient believes pain is deserved for wrongs committed in past lives.’ Statistically significant differences were found between nurses and physicians overall, and within the public general hospital, related to the barriers ‘patient or patient’s family worries the patient will get addicted to pain medication’ and ‘patient believes they should bear pain without complaint,’ with nurses rating these barriers with greater impact.The highest rated country/cultural barrier, overall  and by nurses  was ‘no palliative care outpatient department.’ For physicians, the highest rated institutional barrier was ‘no separate beds or wards for palliative care’ . The lowest ranked institutional barrier, overall  and by nurses  and physicians  was ‘nurses and doctors do not communicate well;’ this barrier was significantly different within the private cancer hospital, with all physician respondents ranking this factor as having no impact .The highest rated institutional barrier, overall . This barrier was statistically significant between nurses  and physicians  at the public general hospital. The lowest rated personal barriers included “I worry I will get in trouble for giving patients pain medication,”  and “I worry patients will get addicted to pain medication,” . Ratings for “I am not sure of the best action to take to manage cancer pain” varied significantly between nurses and physicians at both the public cancer hospital and the private cancer hospital. However, the physicians at the public cancer hospital rated it significantly higher than nurses at their institution , whereas physicians at the private cancer hospital rated the barrier significantly lower than nurses .The highest rated personal barrier was ‘I have to care for too many patients,’ , there was a statistically significant difference between nurses  and physicians . 92% of respondents also agreed or strongly agreed (8 or higher) that morphine is a beneficial and helpful medication . 82% of respondents disagreed or strongly disagreed (2 or lower) that morphine is only appropriate for dying patients . 45% of respondents also disagreed or strongly disagreed (2 or lower) that ‘regular morphine use will cause physical dependence,’ . 67% of respondents disagreed or strongly disagreed (2 or lower) that ‘regular morphine use will cause addiction.’ Lastly, 66% of respondents disagreed or strongly disagreed (2 or lower) that ‘morphine commonly causes respiratory depression’  and this difference was statistically significant between nurses and physicians at the public cancer hospital  and the private cancer hospital .On a scale of 0, strongly disagree to 10, strongly agree, 95% of respondents reported an agreement rating of 8 or higher with the statement ‘it is my role/my job to manage cancer pain.’ While this item had a high overall rating  endorsed the statement ‘cancer pain can be difficult but usually can be controlled.’ No respondents endorsed the statement “cancer pain is unavoidable and cannot be controlled.’The majority of respondents  is included in Table The entire sample (n = 96), followed by morphine 10 mg prolonged release , morphine 30 mg prolonged release and morphine injectable , and then morphine 10 mg immediate release . No respondent selected the option ‘none of these morphine formulations are regularly available at my institution’ or ‘unsure.’Specifically, the most frequently reported regularly available morphine formulation overall was morphine syrup, which was available at every institution  reported no morphine shortages within the past 6 months at their institution; a small number of respondents indicated a morphine shortage had occurred at their institution in the past 6 months  or they were unsure . Overall, respondents indicated that if morphine shortages had occurred, they generally lasted a few days  or they were unsure of duration . Shortages within the past 6 months of codeine and tramadol were also infrequently reported , and if they did occur, respondents were more unsure of duration . Overall and between institutions there was the most variance regarding fentanyl, with reported shortages and duration of fentanyl shortages statistically different among the study sites  and more respondents overall reporting fentanyl shortages  or being unsure if fentanyl shortages occurred . Of note, the survey did not specify formulation of fentanyl, e.g., transdermal patch versus parenteral.The majority of respondents , paracetamol , and steroids . The most commonly reported non-pharmacological treatments for cancer pain were heat/cold packs ; massage ; and meditation/yoga .The most commonly reported non-opioid pharmacological treatments for cancer pain were non-steroidal anti-inflammatory drugs  had heard of the NAPCare PMG; of those, 97% (n = 85) had read at least some, or all, of the guidelines. There were statistically significant differences between nurses and physicians related to ‘helpfulness of the guidelines in your clinical practice’  and regarding frequency of use of the NAPCare guidelines in clinical practice, with nurses more likely to use the guidelines multiple times a day  compared to physicians . 37% (n = 32) of respondents indicated that they use other PMG guidelines to help manage cancer pain, usually the World Health Organization (WHO) guidelines (n = 23).96% of nurses and physicians (n = 90) reported having access to a smart phone; of-those, all respondents (n = 89) reported having daily access to a smart phone, and most  use an Android phone. 46% of physicians (n = 13) reported “I encounter no barriers in using a smart phone” compared to 3% (n = 2) of nurses. The most frequent barriers to smart phone use reported by physicians were cost of data access  and concern about using smart phones in the presence of patients . Nurses reported more barriers to smart phone use in general, the top three being cost of data access ; not allowed to use mobile phone at work ; and concern about using smart phones in the presence of patients .All but two participants , followed by ‘sharing information with/learning from other healthcare providers’ and ‘understanding cancer pain physiology’ . Physicians most frequently selected ‘prescribing/giving opioid medications’ , followed by ‘prescribing/giving non-opioid medications’ , and ‘sharing information with/learning from other healthcare providers’ . Both nurses and physicians selected the option ‘prescribing/giving non-pharmacological treatments’ as the least important way a mobile app could help them manage cancer pain . Overall, nurses and physicians most frequently selected the option ‘cancer pain management guidelines will be followed more consistently’  and ‘healthcare providers will feel more confident prescribing/giving pain therapies’  as the top indictors to evaluate app effectiveness; ‘morphine will be more available to patients in need’ received the fewest responses as a way to evaluate the app’s effectiveness .When asked how a mobile app could help healthcare providers better manage cancer pain, nurses most frequently selected ‘educating patients and family members’ , and higher for physicians  than nurses . Average mean frequency of mobile app use for personal reasons, overall, was higher than app use for clinical care , and was higher for physicians  compared to nurses .Average mean frequency  of reported mobile app use for clinical care, overall, was 6.38  answered the final survey item . Respondents shared key challenges related to managing cancer pain in Nepal, and also offered suggestions for improvement. Responses focused on four overarching themes, including: access to care; cancer pain as common and challenging; need to improve training and awareness; and concerns related to opioids. Table In total, 90 respondents  and female, consistent with global gender demographics of nurses . StatistOur overall findings related to general opioid confidence, knowledge and beliefs are encouraging. Across all sites, providers rated average helpfulness of palliative care/cancer pain management training as high  and this rating was statistically significantly higher, overall, for nurses compared to physicians. This finding underscores the importance of ensuring interdisciplinary engagement in the creation and implementation of palliative care training content , 48–51.Variations in confidence levels related to specific cancer pain management tasks were seen across institutions and by roles . Reasons for variation across institutions and roles are likely related to multiple factors, including previous palliative care training , autonomy and scope of practice expectations within the specific institution , anAcross all study sites both physicians and nurses endorsed that cancer pain is difficult but can usually be controlled and strongly agreed that it is their role to manage cancer pain. Additionally, providers perceived morphine to be a beneficial and helpful medication, and not only for patients who are actively dying. Both nurses and physicians at the public general hospital most accurately agreed that regular morphine use will cause physical dependence, however other respondents largely disagreed, which could have worrying patient care implications, especially for opioid withdrawal. Most respondents disagreed that morphine use in cancer patients commonly causes respiratory depression; this knowledge could prevent concern over this side effect leading to a failure to prescribe morphine, but conversely care is also needed as respiratory depression is possible if safe prescribing practices are not followed. Collectively, these findings suggest information within pain management apps related to opioid pharmacology  is likely always important to include to support busy healthcare professionals and prevent patient harm – even for users with prior palliative care training.Lack of opioid availability is a serious concern in the delivery of palliative care and can be especially difficult in LMICs . HoweverOur results contribute to a more complete understanding of barriers to cancer pain management within LMICs, particularly between private and public sector hospitals which represent very different care contexts. Public/government sector hospitals generally provide subsidized care to a large volume of patients of lower socioeconomic status and must cope with significant resource constraints; private sector hospitals generally care for more financially secure patients who can pay out of pocket for treatment and operate with greater resources. This resource disparity is likely represented in our finding that respondents, regardless of role, at the private cancer hospital rated both institutional and personal barriers to cancer pain management very low compared to other hospitals and the overall ratings. These institutional differences are important to understand as they influence contextual barriers to cancer pain management, and thus, potential adherence or non-adherence to PMG.Key institutional barriers across all sites, and for both nurses and physicians, related to the high volume of patients they care for and lack of dedicated palliative care beds or outpatient department. Mobile app design can account for these barriers to some extent by ensuring that user interfaces are as quick and efficient as possible to accommodate brief patient care interactions and include clear, concise recommendations. It is encouraging that concerns regarding addiction and repercussions for prescribing/administering opioids were not highly rated barriers. It is especially encouraging that these barriers were the lowest rated among nurses, as they are often the ones to decide whether to administer an as-needed (PRN) pain medication. We also find this encouraging as a contrast to prior research that indicates fears related to addiction and/or regulatory surveillance can limit opioid prescribing/administration, even for patients with legitimate, severe pain, in both high and low income countries , 53. HowConsistent with prior research  our findIt is encouraging that such a large proportion of respondents had heard of the NAPCare PMG, reported using them multiple times a day, and strongly agreed that the guidelines were helpful in their day-to-day clinical practice. We are optimistic that such a degree of pre-existing awareness and receptivity to the NAPCare PMG bodes well for use of a mobile app. It is also validating that the most frequently used ‘other’ cancer pain management guideline by participants were the WHO guidelines, since they serve as the foundation for the NAPCare PMG.Our survey confirms the ubiquity of smart phones in LMICs  and the Overall, and especially for nurses, the most desired feature to better manage cancer pain was the ability of the app to educate patients and family members. While this feature is not within scope for the first iteration of our app, it is clearly a critical future component to include. Regarding evaluation of the app, key user-defined metrics include frequency of app use; confidence levels in prescribing/administering pain therapies; and adherence to the NAPCare PMG, including documenting reasons when PMG cannot be followed  to help inform future tailored interventions.The primary limitation of this study is the convenience (versus probability-based) sample of palliative care sensitized healthcare providers and the respondent quotas we established prior to survey administration. Our sample size also limited our ability to conduct more detailed multivariate analyses to more robustly explore significant differences among groups; this would be an important area of future research. However, our sampling strategy was in keeping with the pilot nature of this grant and the overall intention to mentor, and not overburden, new investigators within Nepal. In hindsight we underestimated the number of participants willing to complete our survey, and could have likely surveyed a larger, more diverse sample of clinicians, particularly at the public general and cancer hospitals. As with any survey, there is the risk of response and recall bias; we attempted to mitigate this by taking care to write our questions in neutral, clear language and by providing both remote and in-person training and support to Nepal team members who administered the survey.In this paper we describe a survey designed to identify gaps and opportunities in cancer pain management in Nepal with a focus on informing design of a tailored digital health intervention to support clinicians providing care to patients with cancer. However, it is important to note that any digital health intervention can have unintended consequences , such asOur survey of diverse cancer care institutions within Nepal emphasizes that healthcare providers view cancer pain as an important symptom management concern, use smart phones and apps frequently, and are receptive to a mobile app to provide PMG support. Mobile apps must be informed by a clear understanding of contextual barriers to both cancer pain management and smart phone usage that are influenced by institutional resource and disciplinary differences.