Acute motor and sensory axonal neuropathy (AMSAN) is a subtype of Guillain-Barré syndrome (GBS) differentiated by nerve conduction studies (NCS) and characterized by symmetric ascending paralysis often involving respiratory muscles. While opsoclonus, which is involuntary chaotic rapid eye movements, is not a common manifestation of GBS. Moreover, little published data are available on the relation between liver enzymes elevation and GBS.A 42-year-old man presented to Al Mouwassat University Hospital with weakness in all limbs and dyspnea. Examination showed an elevated respiratory rate, hyporeflexia, and decreased strength of upper and lower limbs. Analysis of cerebrospinal fluid revealed an albuminocyto-dissociation suggesting the diagnosis of GBS and subsequent plasmapheresis. NCS confirmed a diagnosis of AMSAN. Elevation in liver enzymes was noticed prompting further exploration with no positive findings. Despite treatment efforts, the patient developed severe dyspnea, deterioration in cognitive abilities, and opsoclonus with a normal brain MRI. Unfortunately, he developed respiratory failure which lead to his death.In this case, we highlight the occurrence of opsoclonus which is a rarely-encountered manifestation of GBS, in addition to an unexplained elevated liver enzyme, the thing that could contribute to larger research to further comprehend the pathophysiology of GBS.The online version contains supplementary material available at 10.1186/s12883-022-02599-0. Guillain-Barré syndrome (GBS) is a neurological disorder characterized by often ascending paralysis clinically and albumino-cytological dissociation in the cerebrospinal fluid (CSF) analysis. The Main etiology is that a trigger induces the generation of autoantibodies targeting peripheral nerve myelin or axonal membranes, leading to specific electrophysiological features and significantly reduced conduction velocities . GBS subThe exact pathogenesis of this phenomenon remains unclear, but it is proposed to be a result of either increased neuronal excitability in the paramedian pontine reticular formation (PPRF) which controls saccades or decreased omnipause neurons (OPNs) inhibition to the PPRF which results in ocular instability or oscillations .Liver function disturbances in GBS patients are usually attributed to viral hepatitis with Cytomegalovirus (CMV), Epstein-Barr virus (EBV), hepatitis A (HAV), B (HBV), C (HCV), or E (HEV) virus. However, some GBS patients have disturbed liver functions with no concurrent infection . UnderstA 42-year-old man presented to Al Mouwassat University Hospital with lower limb weakness that developed over the last 5 days followed by upper limbs weakness accompanied by paresthesia and numbness in upper and lower limbs. The patient was dyspneic at admission with New York Heart Association (NYHA) grade III (doing less than ordinary activity) . No cognLaboratory tests revealed an elevation in alanine transaminase (ALT) and aspartate transaminase (AST) on admission but normal other lab tests Table .Table 1b3, elevated total protein of (92 mg/dl), and a normal glucose level.Analysis of cerebrospinal fluid revealed an albuminocyto-dissociation with WBCs of 4 /mmSummarizing clinical symptoms and CSF analysis suggested the diagnosis of GBS and implicated plasmapheresis as a modality of treatment. However, elevated liver enzymes indicated further exploration. Echography of the liver and biliary tract revealed a 14 cm liver span and grade 1 fatty liver changes with no dilation of biliary tracts in and out of the liver, the thing that cannot by itself explain serum findings. Serology tests for viral hepatitis with HBV and HCV were negative. Toxicology screening and porphyria investigation were also negative.NCS demonstrated no sensory nor motor response after stimulating lower limbs nerves and F wave was absent. There was also a significant decrease in motor and sensory responses amplitude and absence F wave in upper limbs. These findings proved severe motor and sensory axonal injury confirming a diagnosis of AMSAN.Despite following therapeutic plasmapheresis for five sessions, the patient developed severe dyspnea , an SBCT of 10, and a decrease in motor strength in upper and lower limbs (MRC sum-score of 18) followed by CN 7 bilateral palsy. Concomitantly, the neurological exam revealed a deterioration in cognitive abilities with visual hallucinations, disorientation to people, and opsoclonus (showed in an additional movie file [see Additional file To our knowledge, clinical variants of GBS in the Syrian population have not been described in the literature which highlights the importance of reporting clinical extraordinary presentations. Our patient’s findings fulfilled the diagnostic criteria of GBS; the patient had a severe ascending weakness with hyporeflexia and bilateral facial nerve palsy and opsoclonus with NCS revealing the absence of motor and sensory response in addition to CSF test results. He didn’t have ataxia or ophthalmoplegia nor hyperreflexia or encephalopathy on admission the thing that shifted the diagnoses away from miller fisher syndrome and Bickerstaff encephalitis respectively. However, Bickerstaff encephalitis couldn’t be fully excluded despite the normal MRI imaging and due to lack of performance of GQ1b antibody screening. According to the few published data regarding opsoclonus in GBS patients, the pathophysiology of this clinical variant remains unexplained. However, multiple studies are suggesting an association between opsoclonus and immunological diseases with elevated antibodies titers which could account for the association between opsoclonus and GBS , 4. It cFrom another point, a published study suggested that liver function disturbances are present in up to 56% of GBS patients on admission . AnotherOur patient was negative for hepatitis B and C viruses and porphyria, with a clear toxicology screen and a negative history of alcoholism. Although HAV, HEV, CMV, EBV, and autoimmune hepatitis work up were planned, we did not get to complete the exploration for liver functions disturbances. Isolated ALT and AST elevation without hypoalbuminemia or prolonged prothrombin time suggested no liver failure. Administration of antibiotics to treat pneumonia, and clonazepam to treat the opsoclonus and the continuous deterioration of the patient neurological status decreased the cost-effectiveness of pursuing liver study leaving the etiology unclear and possibly attributed to either untested viral infection or due to the GBS autoimmune process itself.In conclusion, our case presents a severe form of GBS case with unique laboratories and clinical manifestation that might suggest new theories to be tested in more profound and bigger studies.We encourage similar reporting of cases to further understand the mechanisms and relations between these entities to improve diagnostic approaches and prognostic evaluation to such patients and avoid unfortunate results.Additional file 1. Additional file 2. Additional file 3. Additional file 4.


Endovascular thrombectomy (EVT) is part of first-line intervention for acute ischemicstroke management. Recent technological advances have demonstrated that large-borecatheters are an attractive approach for EVT. A multitude of approaches such as A DirectAspiration first Pass Technique (ADAPT) or in conjunction with stent retrieval (Solumbratechnique) have been developed with increasingly large-bore catheters, demonstratingsafety and efficacy. Furthermore, these techniques have demonstrated promise for theintervention of cerebral venous thrombosis as well as posterior circulation ischemicevents. Recently, advances in neurointerventional catheters have focused on improvedmaneuverability to navigate the neurovasculature, as well as larger inner diameters forimproved procedural versatility, including aspiration. We describe a case reporthighlighting our early institutional experience with the recently developed large-borecatheter, the BENCHMARK™ BMX™ 96. The case report entails near complete occlusion of theinternal carotid artery from acute thrombus and the utility of the BMX™ 96 catheter fortreatment of such extensive clot burden. The applicability of large-bore aspirationcatheters, with an emphasis on recent advances, for mechanical thrombectomy in arterial aswell as venous systems is discussed. To our knowledge, this is the first reported case ofuse of the BENCHMARK™ BMX™ 96 access system for EVT in acute ischemic stroke. Suchnew-generation large-bore catheters are a promising advance in neurointervention, and ourearly institution experience highlights the ease of use and versatility forneurointerventional procedures such as EVT. Continued recent technological advances have positioned increasingly large-borecatheters as an attractive approach for EVT in ischemic stroke. These catheters aretypically used either in the setting of A Direct Aspiration first Pass Technique (ADAPT) or in conjunction with stent retrieval (Solumbra technique). Stand-alone large-bore catheter aspiration has also shown efficacy for cerebralvenous thrombosis as well as posterior circulation EVT. Recent advances in neurointerventional catheters have been focused on improved systemmaneuverability and conformability to optimize navigation of the neurovasculature.Furthermore, larger inner diameters (ID) improve procedural versatility, such as aspiration,which has translated into safe and efficacious ADAPT approaches for acute ischemicstroke.8 Recent clinical trials (COMPASS and ASTER) have positioned ADAPT as an attractive first-line approach for EVT. We describe acase report highlighting our early institutional experience with the recently developedlarge-bore aspiration catheter, the BENCHMARK™ BMX™ 96 .Endovascular thrombectomy (EVT) is a mainstay of acute ischemic stroke management and isconsidered part of first-line intervention as supported by recent guidelines,A 66-year-old female presented to an outside hospital three hours after acute strokesymptom onset with right hemiplegia and aphasia, with a National Institutes of Health StrokeScale (NIHSS) of 12. A non-contrast computed tomography (NCCT) scan of the head confirmedpreserved gray–white differentiation, with no signs of early infarction or hemorrhage(Alberta Stroke Program Early CT Score (ASPECTS) 10). A CT angiogram (CTA) of the arch tothe vertex revealed absent contrast filling from the mid-portion of the left common carotidartery (CCA) to the supraclinoid segment of the left internal carotid artery (ICA). The leftmiddle cerebral artery (MCA) circulation was preserved secondary to collateral flow via theanterior communicating artery. Intravenous thrombolytic therapy was contraindicated, as thepatient was taking warfarin for atrial fibrillation .Given the patient’s excellent ASPECTS score, good collaterals, and moderate stroke symptoms(a NIHSS score of 12), the patient was transported to our comprehensive stroke center foremergency EVT consideration. After arrival, approximately four hours later, a repeat NCCTscan of the head and CTA of the arch to the vertex showed no interval change, with no signsof early infarction (ASPECTS 10) and persistent left ICA occlusion . AdditioGeneral anesthesia was induced, and the patient was prepped in standard fashion. Afterright common femoral artery access, a co-axial system of a 90 cm BENCHMARK™ BMX™ 96 accesscatheter and 125 cm 6F Bern select catheter  was easily maneuvered into themid left CCA over a Terumo Advantage guidewire . Angiographic runs demonstrated near occlusion of the carotid bifurcation with astring sign and large amount of thrombus throughout the left ICA . The lef When paired with a suitable vacuum pump, even small increases in catheter IDtranslate into exponentially greater force applied to the thrombus for removal. ConsideringPoiseuille’s law, the rate of flow through a pipe for steady flow relates to the pressuredifference across the two ends of the pipe, the radius of the tube, as well as the fluidviscosity and the length of the tube. In particular, the radius has a direct relationship tothe 4th power to the flow  demonstrate efficacyand shorter procedure times in conjunction with large-bore aspiration catheters.13 Additionally, the aspiration cathetersize positively correlates with the rate of first-pass effect when used in conjunction withBCG and stent retrieval. However, the IDs of some of the larger BCG systems such as the MERCI 9F  remain smaller than the BMX™ 96 system, raising the issueof compatibility with large-bore catheters. With increasing advances in large-boreaspiration catheters, the need for larger-bore BCGs may be needed.This case report highlights the use of a BMX™ 96 catheter in the setting of extensivearterial clot burden. The BMX™ 96 has hydrophilic coating along the distal 11 cm, as well asprogressive laser-cut patterns along the entire length, contributing to the distal ease ofmaneuverability at the level of the aortic arch and cervical ICA while balancing proximalsupport. This can reduce the possible need to perform an exchange maneuver and thereforetranslates into reduced procedure times. The BMX™ 96 has an 8 Fr outer diameter  and a 0.096 inch ID. To our knowledge, this ID is the largest currently available foraspiration catheters used in EVT treatment and has favorable dimensions compared to otherwidely used aspiration catheters such as Neuron Max . The larger ID in the setting of ADAPT conceptually provides capacity toaspirate a greater thrombus burden from larger vessels such as the ICA with minimalrepositioning and potentially minimal passes, as recently supported in a meta-analysis.the flow . When co In particular, given the association of cerebral sinus thrombosis with infections,such as coronavirus disease 2019, continued refinement of approaches for effective treatmentis urgently required. One of the additional benefits of a large-bore aspiration system, suchas the BMX™ 96, in the setting of ADAPT is the reduced cost compared to more traditionalcatheters that are typically used in conjunction with stents and/or BCGs. Althoughpromising, further analysis is warranted to analyze the efficacy and complication risk oflarger-bore neurointerventional catheters as they gain more widespread use.Large-bore aspiration catheters are also a promising approach for the treatment of cerebralvenous thrombosis, which otherwise has high morbidity and mortality rates, with a proclivityfor younger patients. Small case series demonstrate the utility and safety of large-boreaspiration catheters in cerebral venous thrombosis. The use of an ADAPT approach with a large-bore catheter may minimize the risk ofintervention in these patients. Future studies, ideally using the most recent large-borecatheters, will likely aid to elucidate further safe and efficacious management options forpatients with isolated acute carotid occlusions.Finally, management approaches for symptomatic acute carotid artery occlusions withpreserved circle of Willis flow, as demonstrated in our case report, are not wellestablished. The natural history of such occlusions may be more favorable due to collateralflow via the circle of Willis, thus arguing against placing the patient at potential risk ofprocedure-related complications. However, these patients remain at increased risk ofinfarct, particularly in watershed regions. A recent retrospective analysis, using the MRCLEAN registry, suggests generally improved clinical outcomes for patients who underwent EVTfor isolated intracranial ICA occlusions. However, 20% of these patients did demonstratedistal clot migration with subsequent poor clinical outcome.The BENCHMARK™ BMX™ 96 access system is a promising advance in neurointervention, and toour knowledge, this is the first reported case of use in ischemic stroke. Our earlyinstitution experience highlights the ease of use and versatility for neurointerventionalprocedures such as EVT.


Low back pain (LBP) has been among the leading causes of disability for the past 30 years. This highlights the need for improvement in LBP management. Many clinical trials focus on developing treatments against degenerative disc disease (DDD). The multifactorial etiology of DDD and associated risk factors lead to a heterogeneous patient population. It comes as no surprise that the outcomes of clinical trials on intradiscal mesenchymal stem cell (MSC) injections for patients with DDD are inconsistent. Intradiscal MSC injections have demonstrated substantial pain relief and significant disability-related improvements, yet they have failed to regenerate the intervertebral disc (IVD). Increasing evidence suggests that the positive outcomes in clinical trials might be attributed to the immunomodulatory potential of MSCs rather than to their regenerative properties. Therefore, patient stratification for inflammatory DDD phenotypes may (i) better serve the mechanisms of action of MSCs and (ii) increase the treatment effect. Modic type 1 changes—pathologic inflammatory, fibrotic changes in the vertebral bone marrow—are frequently observed adjacent to degenerated IVDs in chronic LBP patients and represent a clinically distinct subpopulation of patients with DDD. This review discusses whether degenerated IVDs of patients with Modic type 1 changes should be treated with an intradiscal MSC injection. A fundamental challenge for improving the lives of chronic low back pain (CLBP) patients is the lack of effective targeted treatments. The development of novel targeted therapies for CLBP patients is hampered in part by the heterogeneity of the CLBP population. Pain may arise from several anatomical structures, including the intervertebral disc (IVD), the endplate, the vertebral body, the facet joints, the spinal ligaments, and the muscles. Central pain sensitization and psychosocial factors can further complicate the diagnosis.Degenerative disc disease (DDD) is one of the most common findings in CLBP patients. DDD is an inflammatory–catabolic process triggered by a long list of genetic, mechanical, and environmental factors that ultimately leads to the resorption of the IVD. Anti-inflammatory and regenerative approaches have been attempted to treat degenerated discs. In the past 15 years, many cell therapy approaches for DDD have been developed, several of which have reached phase I and II clinical trials, and a few phase III trials [Patient stratification is critical for showing a clinically meaningful treatment effect. However, the high prevalence of disc degeneration (DD) in the heterogeneous CLBP population  and the Modic changes (MC) are vertebral bone marrow lesions that are almost exclusively present at levels with DD. MC are frequently observed in CLBP patients. A systematic review investigated the prevalence of MC and reported a median prevalence of 43% in CLBP patients and 6% in a non-clinical population . AccumulVertebral bone marrow lesions adjacent to degenerated discs were first described by Assheuer et al. in 1987  and lateThe IVD and the vertebral endplate seem to play an important role in the pathomechanism of MC . MC onlyDespite an increasing understanding of the molecular and cellular changes in MC bone marrow and discs, the etiology of MC remains largely unknown. Autoinflammation against disc material and occult disc infection are both supported by clinical and experimental studies ,35,36,37In summary, clinical and experimental data suggest that disc inflammation can affect the adjacent bone marrow via an enhanced cross-talk through damaged endplates. Therefore, suppression of discal inflammation might represent a promising treatment strategy to protect the bone marrow from the vicious cross-talk with the inflammatory disc.ClinicalTrials.gov (accessed on 22 February 2022) database. Keywords used in the selection of clinical trials were: “modic”, “discopathy”, and “endplate changes”. All pharmaceutical interventional studies were considered and their relevance to the subject of this review was checked. Multiple clinical trials investigating the efficacy of antibiotics or intradiscal steroid injections for the treatment of CLBP patients with MC were not listed in the ClinicalTrials.gov (accessed on 22 February 2022) database. Eligible studies were selected and are summarized in A literature review was carried out in January 2022 across the Various drugs have been tested in clinical trials to treat CLBP patients with and without MC. The AIM study investigated the use of amoxicillin to treat occult disc infections in a double-blind, randomized, phase III, placebo-controlled, multicenter clinical trial (NCT02323412) ,48. The Bisphosphonates might consolidate inflamed vertebral bodies, thereby improving the tolerance for mechanical load. A phase II, randomized, placebo-controlled, double-blinded clinical trial investigated the efficacy of zoledronic acid (ZA) for CLBP patients with MC (NCT01330238) ,43. BesiGlucocorticoids are potent anti-inflammatory and immunosuppressive drugs, frequently used to treat various inflammatory, allergic, and autoimmune disorders. Clinical trials investigated the efficacy of intradiscal steroid injection in a heterogeneous study population of patients suffering from discogenic LBP. These trials reported no significant clinical benefit in the use of intradiscal steroids ,53. MoreThe BackToBasic study is currently recruiting CLBP patients with MC1 to compare the effect of Infliximab—an intravenously administered TNF-α inhibitor—to a placebo (NCT03704363) . This phAntibiotics to treat occult disc infections, anti-inflammatory drugs to suppress discal inflammation, and bisphosphonates to consolidate vertebral bodies all represent valid causal treatment approaches. Current clinical trials seem to have recognized the pivotal role of inflammation in MC1 and therefore are looking into anti-inflammatory treatment approaches.A recent review highlighted the central role of inflammation in DDD independent of the presence of MC . The revMesenchymal stem cells (MSCs) have been used in numerous clinical trials for DDD independent of concomitant MC during the past decade. Clinical trials for DDD using MSCs have recently been reviewed . MSCs arThe underlying concept of regenerative treatment approaches in DDD assumes that MSC injection reconstitutes the healthy disc anatomy and thereby restores the normal functioning of the motion segment . The sucThe multifactorial etiology of DDD could manifest in various sources of discogenic pain. The key processes of DDD that are related to discogenic pain have been reviewed elsewhere . In summIn order to assess whether MSCs should be considered for ‘MC discs’, we next review the regenerative and immunomodulatory mode of action of MSCs and their contribution to pain relief.Structural damage of the IVD and cartilage endplate (CEP) might contribute to functional impairment of the vertebral motion segment, which in turn could result in painful discal inflammation ,76. TargThe cross-talk between MSCs and IVDCs was shown to downregulate the gene expression of pro-inflammatory cytokines in IVDCs and to significantly increase their insoluble collagen synthesis and proliferation rate in vitro . FurtherUnfortunately, in vitro studies are incapable of mimicking the complex IVD microenvironment. The hostile IVD microenvironment likely impairs the regenerative potential of MSCs . To enabThe following section summarizes the characteristics of the IVD microenvironment, which were discussed in a comprehensive review by Vadalà et al. . We revi2) and low glucose (1 mg/mL) were found to have positive effects on MSC-mediated IVD regeneration. MSCs cultured under hypoxia not only grew significantly faster than MSCs cultured under normoxia, but also had increased expression of genes associated with ECM assembly and improved differentiation potential [The avascular nature of IVDs creates a hypoxic microenvironment with limited nutrient availability. Hypoxia (2–5% Ootential ,102,103.otential ,104. Simotential ,107,108.otential . The nutotential . Endplatotential ,97,110. otential . No dataNon-degenerated IVDs have a pH between 7.1 and 7.4 but the pH can drop to 6.8 in mild DD and can reach values of 6.2 in severe DD ,112. TheThe IVD has a hyperosmolar environment. In non-degenerated IVDs, osmolarity ranges between 430 and 500 mOsm/L  but steaIn summary, it is challenging to restore a healthy IVD microenvironment by addressing single components of the complex network of cytokines, growth factors, catabolic enzymes, and neurotrophic factors found in degenerated IVDs . An adapThe exceptional potential of MSCs to modulate a broad range of immune cells makes them an interesting candidate for the treatment of inflammatory disorders. The impact of MSCs on the functional properties of various cells from the innate and adaptive immune system has been thoroughly reviewed elsewhere ,120. In The secretion of inflammatory factors by IVD cells and infiltrating immune cells not only shifts the balance between anabolic and catabolic processes towards ECM degradation but also promotes the secretion of neurotrophic factors by IVD cells, ultimately leading to nerve ingrowth and discogenic pain ,125,126.An ex vivo experimental study—using a bovine model of IVD degeneration—investigated the effect of a degenerative IVD microenvironment on the regenerative and immunomodulatory potential of human MSCs in co-culture with the bovine IVD . No notaThe clinical benefit of intradiscal MSC injections in clinical trials was inconsistent and had occasionally been reported to be restricted to a group of responders ,132. TheCutibacterium acnes [The three different types of MC may represent different stages of the same pathology . Howeverum acnes ,141. No um acnes .To address the complex pathogenesis of MC1, a broad treatment approach targeting multiple aspects of the disease might be favorable. Multimodal MSCs seem ideal to address the vicious IVD/bone marrow cross-talk through immunomodulation of the inflammatory DD environment, regeneration of the degenerated IVD, and repair of the CEP through chondrogenic differentiation or stimulation of host repair responses . HoweverDiscal inflammation seems to play an important role in MC1 pathology, as the increased diffusion of inflammatory molecules from ‘MC1 discs’ ,143 thro®—a composite of allogenic MSCs and hyaluronic acid—has already been approved by the Korean Ministry of Food and Drug Safety in 2012 for the treatment of cartilage degeneration including degenerative OA. A 7-year follow-up study demonstrated the persistence of the regenerated cartilage [The CEP is damaged in MC1, allowing a pro-inflammatory ‘MC1 disc’/bone marrow cross-talk. Repair of the leaky CEP might restore the IVD/bone marrow barrier, thereby disrupting the pro-inflammatory ‘MC1 disc’/bone marrow cross-talk. MSCs can promote cartilage regeneration by differentiating into chondrocytes to replace damaged cells or by promoting the proliferation and ECM deposition of resident chondrocytes through the secretion of cytokines, growth factors, and extracellular vesicles . There aartilage .The trabecular bone is thinned in MC1, possibly due to osteoclast activity. Suppression of osteoclast activity might consolidate inflamed vertebral bodies, thereby improving the tolerance for mechanical load. MSCs support osteoclastogenesis under physiological conditions through the secretion of RANKL and M-CSF . On the In summary, the regenerative and immunomodulatory properties of MSCs might counteract the painful molecular changes observed in DDD patients with MC1 . FirstlyWe next discuss whether patients with MC1 should be treated with MSCs.MSCs represent a valid treatment option for patients with MC1 for several reasons. Firstly, patients with MC1 represent a homogeneous subpopulation of DDD patients with a clear inflammatory phenotype. Treatment approaches in the homogeneous patient populations can result in larger and more consistent treatment effects, as seen in clinical trials on intradiscal and epidural steroid injections in CLBP patients with MC ,41,54,55A major concern regarding the use of MSCs for IVD repair is the harsh IVD microenvironment . AlthougThe positive outcomes in clinical trials on intradiscal MSC injection in DDD patients pointed to a regeneration-independent mode of action. Patients experienced substantial pain- and disability-related improvement despite the absence of discal regeneration ,76. HoweAs infectious MC1 may be treated with antibiotics ,37 and sThe efficacy of intradiscal MSC injections against MC1 has yet to be investigated in future clinical trials. The main goal of future trials on MC1 patients—as a homogeneous population with a clear inflammatory phenotype—should be to alleviate pain and slow down disease progression. These studies should focus on immunomodulation of the ‘MC1 discs’ and the repair of CEP damage to disrupt the inflammatory IVD/bone marrow cross-talk. MSCs might represent a safe and multimodal treatment approach with promising immunomodulatory and regenerative properties. Regeneration of structural damage to the IVD and CEP could prevent discal inflammation and possibly cure MC1 by disrupting the inflammatory IVD/bone marrow cross-talk. Advanced stages of DD might be an exclusion criterion for MSC injections, as the loss of structural integrity of the IVD and poor nutrient supply could impair the therapeutic efficacy of MSCs in this hostile IVD environment. No data on pH, osmolarity, and oxygen concentration in ‘MC1 discs’ are published, which might hamper the development of discal treatments. Assessing the condition of the CEP could be particularly important and should be considered when selecting patients for an MSC-based therapy. Therefore, novel tools to identify non-infectious MC1 and to assess CEP defects are required. Patient stratification, standardization of MSC preparation techniques, and selection of immunomodulation-related endpoints might pave the way for efficacious MC1 treatments.


Allogeneic hematopoietic stem cell transplantation  is used for the treatment of hematologic cancers and disorders. However, graft-versus-host disease (GVHD) in which the donor immune cells attack the genetically-disparate recipient is a significant cause of morbidity. Acute GVHD is an inflammatory condition and the gastrointestinal system is a major organ affected but is also tied to beneficial graft-versus-tumor (GVT) effects. There is increasing interest on the role of the microbiome on immune function as well as on cancer progression and immunotherapy outcomes. However, there are still significant unanswered questions on the role the microbiome plays in GVHD progression or how to exploit the microbiome in GVHD prevention or treatment. In this review, concepts of HSCT with the focus on GVHD pathogenesis as well as issues in preclinical models used to study GVHD will be discussed with an emphasis on the impact of the microbiome. Factors affecting the microbiome and GVHD outcome such as obesity are also examined. The bridging of preclinical models and clinical outcomes in relation to the role of the microbiome will also be discussed along with possibilities for therapeutic exploitation. Before receiving the donor cells, recipients are treated with cytoreductive or immunosuppressive conditioning regimens including either total body irradiation, chemotherapy, or a combination of both to eradicate the malignant or abnormal cells, create a niche for the donor hematopoietic stem cells (HSCs) as well as facilitate engraftment and reconstitution.2 There are two principal types of HSCT: allogeneic HSCT , in which there is genetic disparity between the donor HSCs and the recipient, and autologous HSCT (auto-HSCT) in which the HSC’s are from the recipient. The primary advantage of allo-HSCT is the potent antitumor response due to a graft-versus-tumor (GVT) effect generated by donor T cells to the tumor. However, this beneficial GVT is also tied to one of the primary disadvantages of an allo-HSCT revolving around the development of recipient tissue damage in the gastrointestinal (GI) tract, skin, and liver due to the attack by the allo-reactive donor T cells, called graft-versus-host disease (GVHD). GVHD represents a major cause of morbidity following allo-HSCT. The extent of genetic disparity, the types and extent of cytoreductive conditioning used on the recipient as well as the presence of co-morbidities all impact the occurrence and severity of GVHD.3 GVHD represents a major limitation in allo-HSCT and necessitates the need for immunosuppression which also impacts the beneficial GVT effects resulting in higher relapse.Hematopoietic stem cell transplantation (HSCT) is primarily used for the treatment of hematological malignancies such as leukemia and lymphoma, but also for hematopoietic disorders5 Outside of direct tissue attack, delayed hematopoiesis and immune reconstitution post-transplant which also result during GVHD are additional life-threatening complications of allo-HSCT due to susceptibility to opportunistic infections. Immune cell reconstitution is critical but is also dependent on the age of the recipient but NK cells (30–100 d) followed by T cells , and B cells (1–2 y) all impact susceptibility to opportunistic infections as well as relapse.6 Increased susceptibility to bacterial infections such as Pseudomonas, Enterobacter, Klebsiella, parasites, fungi, and reactivation of latent viruses such as Cytomegalovirus, Herpes Simplex, and Epstein Barr virus all rise.7 To counteract these conditions, the uses of hematopoietic growth factors  to promote myeloid recovery, administration of prophylactic antibiotics, and anti-viral therapies (for CMV reactivation) are routinely applied.9 Patient demographics are being increasingly appreciated as factors such as obesity, diet, age and other co-morbidities clearly affect the allo-HSCT outcome but are still poorly understood as well as the impact of these different prophylactic regimens on the various aspects of HSCT and recovery.The cytoreductive conditioning regimens play a crucial role in HSCT outcome but also result in significant toxicities, particularly in older patients. The myeloablative regimen consists of intensive radiation therapy alone or in combination with chemotherapy but can result in significant GI, hepatic and pulmonary toxicities also fueling the GVHD processes. Therefore, non-myeloablative immunosuppressive conditioning using agents such as fludarabine as well as lower doses of cytoreductive agents are now used in order to reduce toxicities and facilitate engraftment of donor HSCs, especially for children or older patients and this has greatly increased application of allo-HSCT to these patient cohorts.3 GVHD is the immunological phenomena mediated by the donor-derived T cells in the graft targeting the major histocompatibility complex (MHC) and minor-MHC antigen differences  and immunosuppressed recipient resulting in host multi-organ organ attack including the GI system, lung, liver, skin resulting in tissue damage, organ failure, and mortality. GVHD is the cause of 15–30% of deaths post-allogeneic HSCT.10 GVHD prevention can be successful by simply removing donor T cells from the HSC graft but unfortunately, it also abrogates beneficial GVT and the dominant procedures use T cell-replete grafts. An effective means to treat ongoing GVHD outside of blanket immunosuppression is still lacking especially with severe GVHD due to the nature of a “cytokine storm” that occurs and these immunosuppressive regimens also impact GVT and therefore relapse. Therefore, understanding the mechanisms of GVHD induction as well as evaluation of multiple approaches to ameliorate this disease is in need.Although allo-HSCT is a potential treatment for hematological malignancies, GVHD still remains one of the most difficult obstacles for the success of this approach.12 The pathologic processes of aGVHD and cGVHD are clearly distinct. aGVHD is primarily an inflammatory disease and is due to many factors such as tissue toxicities arising after conditioning regimen which culminates with donor T cell-mediated target tissue damage due to MHC disparities and massive pro-inflammatory cytokine release originally called a “cytokine storm” and which is extremely difficulty to treat.13 The primary aGVHD target organs are the GI tract or gut, liver, lung, and skin with severe acute gut GVHD representing a major cause of early morbidity. It is the pro-inflammatory cytokine storm consisting of IL-1, TNF, IL-6, and other cytokines and recruitment of innate cells such as macrophages which amplify the process that makes aGVHD extremely difficult to control or treat. In contrast, cGVHD typically results later after HSCT and is associated with tissue fibrosis resembling an autoimmune-like syndrome attacking skin and mucosal tissues.3 As opposed to aGVHD, cGVHD is associated with a dominant role for donor B cells in its progression. The last phase of cGVHD involves in tissue fibrosis regulated mainly by macrophages with transforming growth factor β and platelet-derived growth factor α. These pathological changes can severely impact quality of life and cGVHD is notoriously difficult to treat as it can become resistant to steroids or immunosuppressive regimens resulting in extremely limited options for treatment. It is important to note that these processes are not exclusionary and a patient can present with both types necessitating determination by biopsy.There are two principal types of GVHD with distinct features in pathobiology and clinical phenotypes: acute and chronic GVHD. Clinically, acute GVHD (aGVHD) usually appears at the first 100 d after transplant, while chronic GVHD (cGVHD) was classically defined as occurring later although this is now shown not to be absolute and both processes can also occur at the same time.14 Radiation and chemotherapeutics such a cyclophosphamide cause extensive DNA damage and apoptosis in the GI tract, along with reduced proliferation of intestinal stem cells. This leads to an increase in intestinal permeability, which results in bacterial translocation from the microbiome in the gut, culminating in systemic infection and reduced survival post-HSCT.13 Endotoxin from bacterial translocation further active myeloid cells via toll receptor engagement and fuel the allo-reactive processes of the donor T cells and exacerbate the gut damage causing the cascade to progress. The gut tissue damage by the conditioning regimens, also promote recruitment of allo-reactive donor T cells to the intestinal tract.15 Acute GVHD with the destruction of the intestinal mucosa leads to a failure of fluid absorption, particularly in the ileum, and voluminous diarrhea in patients.14 One of the earliest symptoms of GI GVHD is the tissue damage happened in duodenum, leading to the early bleeding, appetite loss, nausea, vomiting, and weight loss.16–18 The continuous development and use of anti-fungi, anti-viral, and GVHD prophylaxis has helped in reducing the incidence of severe GI bleeding after HSCT.19 The pathology of aGVHD in the gut is distinctive with neutropenic enterocolitis diagnosed by the thickening of the ileum and colon wall using abdominal computed tomographic (CT) scan.20 CT scan with intravenous and oral contrast confirms thickened bowel segments in patients with acute GVHD.21 Similar to CT scan, magnetic resonance enterography (MRE) also could be used as an alternative method for assessing GI acute GVHD with its capability to detect long-segment bowel wall thickening, submucosal edema, and mucosal hyperemia.22 Treatment options primarily consist of blanket immunosuppression although more targeted approaches including cytokine blockade or blocking T cell homing to the gut via blockade of specific integrins are being evaluated.Cytoreductive conditioning regimens are the predominant cause of mucositis early post-HSCT, followed by the development of acute GVHD.1,24 Although GVHD and GVT effect share several biology processes such as antigen-presenting cell activation and donor T cell activation possibly to similar if not identical antigens, it has been difficult even in preclinical models to delineate the two processes which nonetheless remains the primary goal in studies. Lympho-depletion has been shown to enhance the efficacy of adoptively transferred tumor-specific CD8 + T cells by using antibody to remove the γC cytokine-responsive endogenous cells.25 Interestingly, in cancer immunotherapy, depletion of the patient’s lymphocytes has been increasingly studied to improve efficacy of adoptive immune cell transfer. Lympho-depleting conditioning regimen prior to adoptive cell transfer in patients with metastatic melanoma significantly improved the efficacy of therapy with in vitro expanded tumor-infiltrating lymphocytes.26 These cytoreductive conditioning regimens are also routinely applied with chimeric antigen receptor (CAR)-T cell therapy and similar toxicities are being observed including impact on cytokine storm or “cytokine release syndrome.”28 Outside of merely creating a niche for the transferred T cells, with T cell immunotherapy outside of HSCT, it has been postulated that bacterial translocation due to conditioning regimen potentially contributed to improve efficacy by activating dendritic cells augmenting the function of adoptively transferred CD8 T cells.29 It has been demonstrated that an intact microbiome is essential to maintain anti-tumor effects in preclinical models. Use of gnotobiotic or germ-free mice as well mice treated with antibiotics all have reduced responses to immunotherapies indicating an important role of the microbiome in immune regulation and function.31 Additionally, it was observed that certain types of bacteria potentially contribute to modulate the clinical outcomes with immunotherapies. Bifidobacterium spp. is associated with delayed tumor progression and increased responses to programmed death-ligand 1 (PD-L1) checkpoint blockade.32 Using another preclinical model, Bacteroides spp. is found to be required for optimal anti-tumor responses of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) checkpoint blockade.33 These results indicate that the microbiome can play pivotal roles in cancer immunotherapy efficacy regarding T cell immunotherapies as well as potential GVT effects post-allo-HSCT.Allo-HSCT is used as a cancer immunotherapy because it generates the GVT effect in which donor-derived cells attack the host hematologic malignant cells.H2 phenotype.35 Gnotobiotic mice are animals with defined microorganisms and are also used in studies to study the impact of particular bacterial species. However, the complexity of handling protocols, quality control of bacterial colonization, and extremely high cost are the main limitations for extensive application of this model in current research and similar immune deficits.36–38 It is also important to keep in mind that, even under SPF housing conditions, mice obtained from different vendors display distinct microbiome profiles which has been demonstrated to significantly affect their responses to cancer immunotherapies.32 Importantly, these differences were found to be normalized when mice are co-housed or even bedding from one are transferred given that mice exhibit coprophagy (the eating of their feces) which normalizes the microbiome within a cage.32 Interestingly, feral mice and mice obtained from pet stores have extensive bacterial and pathogen exposure and present with notably more diverse microbiota than SPF mice. Furthermore, the immunological profile of such mice has been demonstrated to more appropriately model the complexity of human immune system demonstrating the importance of the microbiome on immune functions but also the complexities in attempting to model reflecting the human condition.39The inbred laboratory mouse model is the most common preclinical model used to study allo-HSCT. The development of inbred laboratory mouse husbandry and genetic modification tools facilitated the broad use of murine models of GVHD and GVT. Mice are inbred , fed with controlled diets, and housed in highly regulated specific pathogen free (SPF) environments which provide researchers the ability to generate more reproducible data. However, these factors also give rise to significant discrepancies between the clinical scenario which is not as controlled given that humans are outbred and exposed to multiple pathogens throughout life and often as they age, have the presence of co-morbidities including obesity. Another one of these variables that is increasingly appreciated concerns the microbiome of laboratory mice and its difference between humans. Outside of clear species differences and dietary habits, there are multiple studies providing evidences of how mouse gut microbiota shifts in various housing conditions and affects the immune system. In addition, compared to mice housed under SPF conditions, the use of germ-free mice often used in microbiome transfer studies, lack all microorganisms and have various immunological abnormalities with significant defects in immune development. These include deficits in development of lymphoid organs, altered mucosal immunity, impaired innate cell number and function, and adaptive responses being in a naïve state as well as demonstrated decreases of total CD4+ helper T cells while shifting to T40 and aging41 are associated with a heightened inflammatory state which predisposes individuals to after HSCT to cytokine-induced pathologies due to increased gut permeability and bacterial translocation43 and condition regimen complications targeting the GI tract .44 Obesity (BMI > 30) has reached pandemic proportions in the U.S. with greater than one-third of U.S. adults are obese according to Center for Disease Control and Prevention. Obesity is associated with meta-inflammation exacerbated by metabolic complications such as glucose intolerances, diabetes, hypertension etc. The inflammation in the obese environment is also fueled by adipocyte factors and the effects on the immune system have been called “inflammaging” due to suppressed adaptive immune responses.45 This is in part due to alteration of immune subsets47 as well as increased production of inflammatory cytokines such as IL-1β, TNF-α, IL-6 from macrophages, monocytes, and T cells.48 Obesity also impacts the microbiome with less diversity being reported.50 Obesity of the recipient has been shown to correlate with poor outcomes after allogeneic HSCT in both mouse preclinical models and human clinical outcome data50 in which high BMI was associated with a significantly greater risk of grade II–IV acute GVHD51  as well as the presence of co-morbidities such as cancer, all of these can impact outcomes.54 Apart from obesity, aging also plays a critical role in inducing microbiota changes. The decreased abundances of Bifidobacterium and Lactobacillus, together with an increased abundance of Enterobacteriaceae are the key microbiota shifts in elderly individuals.55 Possible reasons for microbiome shifts in aging could be explained based on diet adjustment, lessened exercise, reduced mobility, residence locations, less muscle mass (sarcopenia), etc.56 Although obesity and aging correlate with restricted microbiota diversity in mouse and human58 and the less-diverse microbiome profile has been proved to be associated with poor outcomes after HSCT,59–61 recent studies suggested that there are certain types of bacteria that could serve as beneficial factor for health and longevity.62Bifidobacteria supplementation reduced the accumulation of aging biomarkers (carbonyls and lipofuscin),63 while transfer of Christensenella minuta to germ-free mice significantly reduced adiposity gain.64 Thus, there is an interplay between the gut microbiome on body weight gain and immune outcomes but also in reverse where the diet can impact the microbiome.Outside of the not insignificant species differences, human populations exhibit considerable MHC diversity, wide ranges in age and different pathogen exposure as well as the existence of preexisting conditions  all of which can be influenced by the microbiome and have profound immunological consequences. Both obesitye GVHD51 . However66 These data indicate the necessity to modify our current murine HSCT models to be more reflective of human conditions  that significantly affect HSCT outcomes. Clinical data have suggested that obese patients are at higher risk of infection-associated complications and transplantation-related toxicity post-HSCT, especially in allogeneic HSCT.nditions . One exc74–76 In canine models, GI tract acute GVHD displayed a distinct punctate GI hemorrhage with blunted villous architecture, sloughing, mucosal destruction progressing from crypt abscess formation to denudation, and CD3 infiltration representing typical GI acute GVHD symptoms also representing a useful model but mechanistic studies are extremely difficult to perform.78 Thus, both small animal and large animal models offer advantages and disadvantages in HSCT studies  is also a DNA sequencing method but amplifies all genomic DNA in a sample  by gene fragmentation, tagging, PCR amplification, sample pooling in equal proportions, DNA sequencing, and bioinformatics analysis to classify results into taxonomic levels.81 Because of broader sequencing capabilities, shotgun NGS sequencing allows researchers to identify multiple bacteria, fungi, viruses and many other types of microorganisms in their samples.82 Diversity is one of the most important parameters in microbiome study. Microbiome diversity has been well-established as a “biomarker” for HSCT outcome. High microbiome diversity correlates with better survival rate, lower incidence and severity of GVHD.84 Alpha-diversity (definition of the species composition within samples) and beta-diversity  are key factors to study the gut microbial ecosystem.85 In alpha-diversity, Shannon diversity index was used to describe evenness and diversity by measuring both the number of species and the inequality between species abundances. In contrast, Shannon evenness index is independent of species richness and provides information about how evenly the microbes are distributed in a sample. Beta diversity represents the differences between microbial communities from different environments with the main focus on the differences in taxonomic abundances from different samples.86 Because of new molecular techniques, statistic and bio-informatics analysis methods are discovered every few years; microbiome characterization is expected to reach the next level soon with potential linkage with microbiome alteration factors such as antibiotics, strain dynamics, and homeostasis.Given the predominance of gut GVHD and the importance of the microbiome on immune functions, particularly in the gut, there has been considerable interest in defining the gut microbiome in both preclinical and clinical samples. The most commonly used method in microbiome studies uses sequencing of 16S ribosomal RNA (rRNA), the highly conserved gene in all bacteria. Sequencing of regions of hyper-variability in 16s rRNA gene helps us identify different bacterial taxa but this technique could result into inaccuracies at species level classification. The most common hyper-variable regions in 16S rRNA is V3-V4 region. A simple DNA sequencing procedure involves bacterial DNA extraction from samples, bacterial DNA amplification from variable regions of 16S rRNA using polymerase chain reaction (PCR), library preparation, DNA sequencing, bioinformatics, and biostatistics analyses.60 some pathogenic bacterial taxa have been shown to increase in the GVHD recipients such as Enterococcaceae, Akkermansia muciniphila, and Lactobacillales.87 In contrast, Blautia has been shown as the beneficial bacterial genus for HSCT88 as well as the correlation of increased GVHD with reduced abundances of some bacterial genera such as Faecalibacterium, Bacteroides, and Parabacteroides.89 Certain bacterial taxa also have been shown to contribute to GI tract recovery after radiation. Lachnospiraceae and Enterococcaceae are essential to maintain GI tract integrity and facilitate immune reconstitution long-term after radiation exposure.90 Therefore, multiple selective microbiota alteration approaches have been extensively studied to ameliorate GVHD and improve HSCT outcomes  or mice fed with high-fat diet,103 indicating the benefit of antibiotic treatment on controlling obesity-associated “meta-inflammation”. More importantly, antibiotic treatment prevented bacterial translocation from the intestine into the bloodstream after radiation, a critical step for the activation of innate immune cells (e.g. dendritic cells) and cytokine storm triggering, therefore reducing gut permeability and tissue damage due to radiation sensitivity.104 These evidences provide different perspectives of how one might consider choosing the “right type” of antibiotics to preserve the beneficial bacteria taxa without compromising treatment efficacy.The microbiome recovery after antibiotic treatment and HSCT is another parameter which can be variable and is impacted by factors intrinsic to the patient as well as diet and regimens applied all affect the gut and microbiome repopulation dynamics. It was reported that the recovery of the gut microbiota started around day 50, but even by day 100 post-HSCT, the composition and bacterial abundance were less diverse compared to the pre-transplant microbiota.84 Continuous prophylactic treatment using broad-spectrum antibiotic cocktail with ampicillin, vancomycin, and neomycin demonstrated a protective effect in gut permeability and GVHD outcomes in high-fat diet fed obese mice.50 Short-term treatment with a similar antibiotic combination after HSCT also showed a complete protection from diarrhea, weight loss, and death in mice.94 However, there are evidences about prophylactic antibiotics resulted into microbiome disruption and higher transplant-related mortality than those who had antibiotics administered on or after day 0 or untreated patients indicating that more still needs to be delineated on the impact of different antibiotics as well as timing of administration.102Besides the type of antibiotics, timing of treatment also affects outcomes post-HSCT. Short-term discontinuous administration of ampicillin followed by a recovery period before HSCT resulted in worse survival outcomes in mice underwent allogeneic MHC mismatched HSCT.105 GVHD in the digestive tract has been shown to correlate with malnutrition, protein losing enteropathy, magnesium derangements, and deficiencies of zinc, vitamin B12 and vitamin D.106 Prebiotics are indigestible carbohydrates or substances metabolized by beneficial intestinal bacteria that help inhibiting the growth of other pathogenic bacteria, boosting the immune responses, and reducing the risk of various diseases.107 Short-chain fatty acids (SCFAs) are the product from the fermentation process of bacteria that recently been linked with prebiotics metabolism and health benefit.108 One of the most important SCFAs is butyrate, which has been shown to mitigate GVHD through the signaling by G-protein-coupled receptor 43 (GPR43) on the intestinal epithelial cells.109 Propionate and tryptophan has been shown to markedly increase in mice that survived long-term radiation exposure and administration of these metabolites also protected mice from cytokine storm and GI tract permeability.90 Dietary fiber changed the gut microbiota profiles by increasing diversity (Bacteroidaceae and Bifidobacteriaceae), which enhances SCFAs production.110 Microbiome changes also are dependent on the components of diets. The use of the “Western Diet,” which involves high fat and high sugar as opposed to only high fat, reduces Bacteroidetes and Actinobacteria, but increases Firmicutes, Proteobacteria, and Deferribacteres,72 while a high-fat only diet increases Bacteroides, Enterobacteriaceae, Escherichia, Klebsiella, and Shigella.111 With regard to diets, the length of time on the diet impacts outcome as does the composition of the diet with high-fat only diets ranging in the percent of fat (from 25–60%) in the diet.Dietary intake has been shown to influence the component of the trillions of intestinal microorganisms.Bifidobacterium and Lactobacilli and the decrease of the genus Bacteroides112 which potentially beneficial for HSCT patients (https://doi.org/10.1101/2020.04.08.20058198) or cancer treatment with checkpoint blockade.32 Enteral supplementation of glutamine, fiber, and oligosaccharide in a retrospective study of allogeneic HSCT showed less mucositis grade 3–4, reduced weight loss and diarrhea, and improved survival percent at day 100 post-HSCT.113 In a recent article studying impact of pre-biotics and HSCT outcomes, resistant starch and prebiotics mixture  were used pre-transplant and throughout 4 weeks post-HSCT. Patients that consumed prebiotics had reduced mucosal tissue damage and ameliorated acute GVHD.114Prebiotics consist of starch, fructans (inulin and fructo-oligosaccharides), and galacto-oligosaccharides that contribute to the increase of genus Lactobacillus rhamnosus GG in drinking water before and after HSCT resulted in reduced bacterial translocation, improved survival, and reduced acute GVHD pathogenesis.115 Administration of 17 butyrate-producing Clostridia spp. strains by oral gavage before and after allogeneic HSCT with an MHC mismatched model demonstrated a significant increase of butyrate in the intestine and better survival rate.116 A cocktail of Clostridium bolteae, Ruminococcus gnavus, Ruminococcus torques, and Blautia producta delivered by oral gavage also showed significant survival in mice.117 However, the efficacy of probiotics for HSCT in human is still questionable. The use of probiotics-enriched yogurt in a case report of a patient with autologous HSCT for treating mantle-cell lymphoma resulted in unexpected Lactobacillus acidophilus sepsis.118 In line with that, another clinical study reported the bloodstream infections with Lactobacillus bacteremia in patients with autologous and allogeneic HSCT within the first 100 d post-HSCT which implied that the toxicities from immunosuppression by conditioning regimens and mucosal disruption could contribute to bacteremia from probiotics consumption.119 In contrast, a study in children and adolescents undergoing allogeneic HSCT suggested that administration of probiotics with Lactobacillus plantarum was safe and feasible with no bacteremia case recorded.120Probiotics are live microorganisms administered to improve health and have long been used as part of traditional diets. Supplementation of probiotics restores commensal flora in the intestine and has been shown to promote growth of beneficial species, thereby improving intestinal microbiome diversity and HSCT outcomes. In murine models, probiotics has been studied with some positive results. Oral administration of Clostridium difficile infections post-HSCT. Early clinical studies used FMT to treat patients with Clostridium difficile infections with high efficacy and minimal complications.121–127 FMT was also performed in steroid-resistant acute GVHD patients with no adverse effect and resulted in the restored microbiome diversity, an increase of peripheral regulatory T cells and better progression-free survival.128–130 Most recently, when FMT was used in patients with steroid-refractory or steroid-dependent intestinal GVHD, researchers observed a significant increase of microbiome alpha-diversity, increased abundances of butyrate-producing Clostridiales and Blautia.131 Taken together, FMT is a promising approach for intestinal GVHD patients and worth to be investigated further in a larger scale.Aside from probiotics, another approach to introduce beneficial bacteria for HSCT patients for microbiome preservation or restoration is fecal microbiota transplantation (FMT). This is the method that is potentially applicable if patients have Although microbiome characterization in HSCT studies has been investigated in the past decades, there are many questions remain unanswered regarding the “cause and effect” of specific bacterial taxa with the immune system, cell metabolism, and physiological consequences. Although obesity or high-fat diet consumption has been shown to affect microbiota profile and vice versa, little is known about whether microbiome changes in obesity, or high-fat diet exposure in either short-term or long-term could influence the HSCT outcomes. Most of the current concepts are descriptive and there is an urgent need on finding mechanism of how the different bacteria interact and how it affects immune parameters. The inbred laboratory mouse is still the cornerstone of preclinical HSCT modeling, but there is clearly a need to have it better reflect the human condition and involve human modifying factors such as obesity and prior infectious challenges, particularly in performing microbiome studies. This is also important with regard to finding a balance between having sufficient incorporation of the variables involved in human HSCT and also allowing for reproducibility of results. The gut pathology in the preclinical GVHD models does reflect the clinical scenario with common immune pathways and even similarities in microbiome content. Nonetheless, much more is needed to be known before targeted manipulation of the intestinal microbiome-immune system axis could be successfully applied as a therapeutic approach to limit HSCT complications such as GVHD and opportunistic infections, improve GVT effect and survival outcomes for patients. This not only applies to aGVHD affecting the gut but also in other organs and cGVHD given the linkage of the gut microbiome throughout every organ and immune parameter.


Background: The COVID-19 pandemic has contributed to a decline in mental health globally. Compared to the general population, university students have been identified as a group vulnerable to developing depression symptoms during the pandemic. Social isolation, a signature mental health consequence under physical-distancing regulations, is a known predictor of depression symptoms during the pandemic. Yet, more research is required to understand the mechanism that underpins the isolation–depression association and identify psychological factors that may attenuate the association. The current study aimed to understand the role of stress and resilience in the isolation–depression association among university students. Methods: Data were collected from 1718 university students between 28 and 31 May 2020. Partial least squares structural equation modelling (PLS-SEM) was used to examine the mediating role of perceived stress and the moderating role of resilience in the isolation–depression association. Results: We found that perceived stress partially mediated the association between social isolation and depression symptoms. Both the direct and indirect effects were moderated by participants’ resilience levels. Conclusions: Social isolation during the pandemic may contribute to depression symptoms both directly and through elevated stress levels. As an internal strength, resilience may buffer the adverse effects of isolation and stress on depression symptoms. Targeted interventions including mindfulness and physical exercise training may provide promising results in reducing depression symptoms among university students and should be considered by university administrators particularly during times of imposed physical-distancing measures. The COVID-19 pandemic has caused public health concerns globally. Movement restrictions, including stay-at-home orders and physical-distancing rules, have been taken in most countries to some degree to reduce the spread of COVID-19. Despite its advantages in flattening the curve, the restriction regulations may bring up unfavourable mental health outcomes, including symptoms of depression. Research showed that the prevalence of moderately severe and severe depression symptoms among the general population  was 3.7-fold higher and 7.5-fold higher during the lockdown period than pre-pandemic, respectively [University students may be at higher risk for experiencing depression symptoms during the pandemic. Compared to the general population, university students often face additional stressors, including changes in learning mode, uncertainty about academic trajectory and living arrangements, as well as a faltering job market after graduation. Previous research found that university students reported the highest prevalence of depression symptoms compared to medical staff and the general population; 38.6% vs. 21.2% vs. 15.8%, respectively ,5. GivenSocial isolation  has beeOne construct that may explain the association between social isolation and depression symptoms is perceived stress . SpecifThe indirect relationship between isolation, stress and depression may be due to differences in coping. Isolated individuals have been found to use less adaptive coping strategies  compared to their non-isolated counterparts. Additionally, the stress-buffer model suggests that individuals may be able to harness support from their social networks when facing stressful situations . These sAs previously noted, not all individuals experience the adverse mental health outcomes associated with social isolation. This inconsistency may suggest potential moderators that buffer the negative impact from isolation. One candidature moderator is resilience. Resilience is defined as the ability to bounce back from adversity and adapt to the changing environmental demands flexibly . ResilieTaken together, the current study examined the mechanism linking isolation, stress and depression symptoms; we further explored whether the association may be moderated by resilience. Through a structural equation model, we proposed that (1) the relationship between social isolation and depression symptoms may be mediated by perceived stress, and (2) resilience may moderate the negative impacts of social isolation and stress on depression symptoms.Data were collected between 28 and 31 May 2020. All students who were currently enrolled with Monash University received invitation links to the online Qualtrics survey. Inclusion criteria for the current study are: (1) currently enrolled with Monash University; (2) aged 18 and above. Participants who did not provide informed consent were excluded from the current study. Only participants who completed all measures of interest were included in the data analysis, resulting in a final sample of 1718 participants. No reimbursement was provided for participation. This study followed the Helsinki Declaration and was approved by the Human Research Ethics Committee at Monash University (Project Number 23969).Brief Resilience Scale : This isPatient-Reported Outcomes Measurement Information System (PROMIS) Depression Scale : This isPatient-Reported Outcomes Measurement Information System (PROMIS) Social Isolation Scale : This isPerceived Stress Scale 10 items : This isThis study aimed to examine the mediating role of perceived stress between social isolation and depressive symptoms among university students and whether resilience may moderate the effect of social isolation and perceived stress on depressive symptoms. The proposed model was examined via PLS-SEM. PLS-SEM is the second generation technique to analyse the causal relationship between latent variables . CompareAmong 1718 participants, 1227 were females (71.4%). The median age of participants was 21 . In total, 736 (42.8%) participants exhibited clinical levels of depression symptoms (T > 60). Descriptive characteristics and correlations of included variables were presented in 2 values). As a prediction oriented approach, R2 was commonly used as an indicator of model fit for PLS-SEM. Based on [2 values of 0.19, 0.33, and 0.67 refer to weak, moderate, and substantial variance explained by endogenous latent variables, respectively. For the current model, social isolation, stress and resilience accounted for 68.1% of the variance of depression symptoms. Meanwhile, social isolation accounted for 30.7% of the variance of perceived stress. The results of PLS-SEM analysis are presented in Based on , R2 valuTo assess the reliability and validity of the measurement model, we calculated the internal consistency/reliability  and composite reliability; see The HTMT values for measurement constructs were below 0.85 see , indicatp < 0.01). Further, resilience moderated the association between social isolation and depression symptoms  as well as the association between stress and depression symptoms . Detailed direct and indirect effects of predictors on the outcomes variable are presented in To examine the mediation and moderation hypothesis, we used a bias-corrected bootstrap procedure to determine the statistical significance of coefficients . Based oTo visually illustrate results from two moderation analyses, simple slope tests were conducted at three levels  of resilience . Restriction regulations during the pandemic may increase social isolation, leading to worsened mental health outcomes including depression symptoms. To provide empirical evidence that supports the development of mental health policies and interventions at universities, the current study examined the mediating role of stress and the moderating role of resilience in the isolation–depression association among university students. Based on our results, stress partially mediated the relationship between social isolation and depression symptoms. Further, we found resilience buffered the negative impact of social isolation and stress on depression symptoms. Consistent with previous studies, we found a positive association between social isolation and depression symptoms ,41. A hiMore importantly, we found that perceived stress mediates the association between social isolation and depression symptoms. Specifically, social isolation may lead to increased stress levels, which further contributes to depression symptoms. The mediating effect may be explained by differences in coping. It has been found that lonely individuals tend to use rumination as a coping strategy for negative situations . RuminatOur results demonstrate that resilience may buffer the negative impact of social isolation and stress on depression symptoms. Compared to students with low resilient levels, students high in resilience reported fewer depression symptoms during the pandemic despite the occurrence of social isolation and stress. As an ability to adapt to stressful situations, higher resilience level enables individuals to consciously appraise adverse conditions, reframe negative thoughts in a more useful manner, and utilize active strategies of coping with stressors  when faBy highlighting the moderating role of resilience, our findings provide important implications for university administrators. Specifically, resilience is deemed as a trainable target for interventions ,54,55. MDespite its input to existing literature, several limitations should be considered when interpreting the current findings. Firstly, there is a high proportion of female participants  in the current study, which may impact participants’ self-reported distress symptoms . The oveSocial isolation  during the pandemic may contribute to university students’ depression symptoms both directly and through elevated stress levels. Students may need more internal/external resources to mitigate the negative consequences caused by the COVID-19 crisis and related measures. Resilience, a modifiable internal strength that may buffer the adverse effects of social isolation and stress on depression symptoms, should be targeted when developing prevention and early interventions. This may be especially important under physical-distancing measures when individuals have limited access to coping resources from external social networks. When dedicating resources during the pandemic, it will be important for university administrators to consider investing in building resilience among students. Online resources promoting awareness and mental health literacy, or online training for mindfulness and physical exercise that can be practised at home may be promising as preventions/early interventions to reduce depression symptoms among university students. Such interventions have advantages, including remote accessibility, cost-effectiveness and may serve as valuable add-ons to face-to-face counselling services that are considerably disrupted by restriction measures.


Cell therapies using immune cells or non-parenchymal cells of the liver have emerged as potential treatments to facilitate immunosuppression withdrawal and to induce operational tolerance in liver transplant (LT) recipients. Recent pre-clinical and clinical trials of cellular therapies including regulatory T cells, regulatory dendritic cells, and mesenchymal cells have shown promising results. Here we briefly summarize current concepts of cellular therapy for induction of operational tolerance in LT recipients. Transplantation of organs, including the liver, across the HLA barrier induces strong alloimmune responses in recipients. Both cellular and humoral alloresponses contribute to rejection. Current immunosuppressive (IS) therapies including calcineurin inhibitors (cyclosporin and tacrolimus) and corticosteroids target recipient T cell activation, expansion, and cytotoxicity effectively, and reduce acute rejections to less than 15% of transplants but their long-term use is associated with increased risk of infections and malignancies . While oMore than half a century ago, Billingham and Medawar described the phenomenon of acquired immunologic tolerance to transplant antigens by successfully grafting the skin of a calf onto its fraternal twin . InductiLiver transplantation is presently the only proven treatment for end-stage liver disease. Liver transplant was first performed clinically by Starzl in 1963 . In 1969These and similar observations opened the gateway to IS weaning in LT which could address the long-term toxicities and cost of therapy. A meta-analysis summarized the accumulated clinical experience with IS-weaning protocols in LT from 1993–2006 and the available data indicated that elective IS withdrawal is possible in 19.4% of recipients . HoweverOver the past three decades, researchers have been studying ways to induce tolerance to solid organ transplants using hematopoietic stem cells. In theory, donor bone marrow infusion should produce donor pluripotent hematopoietic stem cells, inducing donor-specific hyporeactivity . Rao et Immune cell therapies have been shown to induce stable tolerance in clinical liver transplantation and several transplant centers are conducting tolerance-inducing clinical trials in LT recipients . They arIt is well recognized that the liver is a uniquely immunologically privileged organ . LTs req10 lymphocytes [Thirdly, there is a large population of migratory immune cells in liver allografts compared to other solid organs that can further explain its privileged status. In fact, the liver can be described as a lymphoid organ . Immune phocytes . These cphocytes . NPCs ofphocytes ,29.Finally, the liver is constantly exposed to gut-derived pathogens and antigen metabolic byproducts which is evident from high levels of lipopolysaccharides (LPS) in the portal system. DCs, KCs, and LSECs expressing toll-like receptors (TLR) recognize pathogen-associated molecular patterns (PAMPs) as part of their innate defense from foreign pathogens. In response to TLR stimulation due to constant exposure to low levels of LPS, these cells release anti-inflammatory cytokines such as IL-10. This phenomenon is known as LPS tolerance and TLR signaling plays a critical role in maintaining the balance between immune activation and tolerance . This coLiver MSCs have been isolated from biopsies of livers prepared for transplant and have a higher expression of PD-L1 and PTGS1 which are known inhibitors of T cell proliferation compared to bone marrow and adipose-derived MSCs . They arMultiple studies to date demonstrate that liver DCs possess superior tolerogenic properties compared to their counterparts isolated from other tissues. For example, freshly isolated liver DCs induce higher numbers of Tregs than their blood-derived DCs . In addiHSCs are pericytes in the space of Disse, between the sinusoids and basal surface of the hepatocytes. They play a role in maintaining the extracellular matrix, sinusoidal blood flow, storage of vitamin A, and function as APCs . On stimSinusoids are lined by LSECs. They are 5–7 µm in diameter and provide close contact with lymphocytes for effective immune interaction with LSECs. LSECs express minimal levels of CD80, CD86, CD40, and MHC class II hence are poor stimulators of T cells . T cellsKCs are resident macrophages in the liver described by Karl von Kupffer. They mature from circulating bone marrow-derived monocytes and account for 80–90% of tissue macrophages present in the body and directly interact with passenger leukocytes . After lNK cells, defined by the presence of CD56 and absence of CD3, are extensively present in the healthy liver . HepaticTregs were first reported by Sakaguchi et al. in thymectomized mice . They coTregs control transplant rejection by first migrating to the graft to induce graft tolerance and then drain into lymph nodes and help maintain tolerance . Two typThe first clinical trial of T-cell-based therapy to induce OT was conducted by Todo et al., using an ex vivo-generated regulatory T-cell-enriched cell product in 10 adult patients post-LT . The culKing’s group  proved tTregs demonstrate promise for targeted allograft tolerance and an alternative to classical IS. The first successful pilot study by Todo led to multiple OT induction trials currently using Treg cell therapy in LT . Tregs dDCs are stellate-shaped APCs expressing CD11c and CD1a. They arise from the bone marrow and seed into peripheral tissue. Mature DCs produce pro-inflammatory cytokines, while liver-derived DCs have a lower maturity status and produce high levels of regulatory factors including IL-10, IL-27, retinoic acid, and prostaglandin E2 . DCs witIn vitro generation of DCregs has been achieved by utilizing cytokines , pharmaceutical agents , and IS  . The firOf note, ex-vivo expansion of DCregs yielded high numbers of viable cells, constituting a possible advantage over other types and sources of regulatory cells  which require several weeks to expand. Currently, donor-derived DCregs are restricted to live-donor organ transplantation and alternative therapies such as autologous DCregs loaded with donor antigen are promising for deceased donors . GeneticMesenchymal stem cells (MSCs) are nonhematopoietic multipotent progenitor cells that play a role in tissue remodeling and reside in various tissues. The International Society for Cell Therapy committee defines MSCs as (1) adhering to plastic and being fibroblast-like after culture in vitro; (2) being positive for CD105, CD73, and CD90 but negative for CD45, CD34, CD14, CD19, or HLA-DR by flow cytometry; (3) differentiating in vitro into osteoblasts, adipocytes, and chondroblasts . MSCs inMSCs are not immunogenic as they do not express HLA hence they evade recipient T cells and can be a potent cellular therapy tool regardless of the immunogenic differences between MSCs and recipients . In the Induced pluripotent stem cells (iPSCs) are the result of reprogramming somatic cells to generate pluripotent progenitor cells and have great potential as a major cell source for producing various types of cells to induce tolerance in the field of transplantation . iPSCs hThe induction of operational tolerance after transplant has been a critical goal ever since the first solid organ transplants were completed nearly 70 years ago. After the discovery of the liver as an immunologically privileged organ, our understanding of the immunomodulatory roles of the different cells in the liver continues to grow and we turn to cell therapy as a solution outside the purview of classic IS. We are closer than ever before to achieving the goal of operational tolerance in liver transplantation. Treg infusion after LT is currently undergoing multiple clinical trials. While we await these results, larger and longer-term trials are the next step in bringing this practice into widespread clinical use and hopefully will enhance the understanding of long-term Treg viability and the applications of chimeric antigen receptor technology. DCregs demonstrate a promising ability to blunt the memory immune response, as well as an important role in preventing hyperacute rejection; however, until clinical trial results are available in 2023, no definitive recommendation can be made. In addition, the role of recipient DCregs for deceased donor transplants needs further investigation. MSC administration has so far not demonstrated a consistent path for IS withdrawal, but more research into the timing and dosing of this powerful immune regulator could provide a promising cellular therapy for tolerance induction.


Live attenuated vaccines have been observed to have particularly beneficial effects for child survival when given in the presence of maternally transferred immunity (priming). We aimed to test this finding and furthermore explore the role of paternal priming.In an exploratory, retrospective cohort study in 2017, parental Bacillus Calmette-Guérin (BCG) scars were assessed for infants from the Bandim Health Project (BHP) who had participated in a 2008–2013 trial of neonatal BCG vaccination. Parental scar effects on mortality were estimated from birth to 42 days, the age of the scheduled diphtheria-tetanus-pertussis (DTP) vaccination, in Cox proportional hazard models adjusted with Inverse Probability of Treatment Weighting.For 66% (510/772) of main trial infants that were still registered in the BHP area, at least one parent was located. BCG scar prevalence was 77% (353/461) among mothers and 63% (137/219) among fathers. In the first six weeks of life, maternal scars were associated with a mortality reduction of 60%  and paternal scars with 49% (-68% to 84%). The maternal scar association was most beneficial among infants that had received BCG vaccination at birth (73% (-1% to 93%)). Although priming was less evident for paternal scars, having two parents with scars reduced mortality by 89% (13% to 99%) compared with either one or none of the parents having a scar.Parental BCG scars were associated with strongly increased early-life survival. These findings underline the importance of future studies into the subject of inherited non-specific immunity and parental priming.Danish National Research Foundation; European Research Council; Novo Nordisk Foundation; University of Southern Denmark. We searched PubMed without restrictions to English language publication using the search string:  AND  AND (non-specific OR nonspecific OR heterologous). BCG and other live-attenuated vaccines are associated with reduced overall mortality and morbidity among children. Maternal BCG vaccination/scarification increases this beneficial effect among their BCG-vaccinated offspring, possibly due to a ‘trained immunity’ phenotype with increased pro-inflammatory cytokine production. No studies on paternal BCG vaccination were found, nor studies on non-BCG-vaccinated infants.Both maternal and paternal BCG scars are associated with improved early-life survival of their offspring. For maternal scar, associations were especially beneficial among those children who received BCG at birth. This was less apparent for paternal scar, but having a second parent with a BCG scar greatly improved associations with survival.Our findings highlight the need for studies on inherited non-specific immunity, including paternal effects. The studies on parental BCG vaccination indicate that its non-specific effects are not limited to the vaccinated individual, but could extent to their offspring. This makes universal BCG vaccination policies, as well as efforts to increase BCG scarring by revaccination of scar-negative individuals possibly low-cost interventions to improve health for current and future generations.Alt-text: Unlabelled box1Bacillus Calmette-Guérin (BCG) and other live attenuated vaccines have been associated with beneficial non-specific effects (NSEs); they increase child survival by protecting against unrelated infections Interestingly, it has been shown that beneficial NSEs of live attenuated vaccines can be enhanced if the infant is primed by maternal immunity Subsequently, maternally primed BCG effects have been described in both low- and high-income settings. In Danish neonates, BCG vaccination reduced hospital admission due to infections by 35% (6% to 55%) when their mother had received BCG, whereas there was no effect in neonates born to BCG-unvaccinated mothers (p for interaction=0.01) To further explore the role of parental BCG priming and examine the potential interaction with infant BCG vaccination we took advantage of the most recent of three BCG trials,22.1The Bandim Health Project (BHP) maintains a Health and Demographic Surveillance System (HDSS) in six urban districts in Bissau, Guinea-Bissau. Births are registered by BHP staff at the country's main hospital and during regular trimonthly home-visits. Routine vaccinations are provided at three HDSS health centres in accordance with the WHO-recommended immunization schedule.2.2The original RCT design has been described in detail previously 2.3N = 1258) were sought for in the HDSS system in February 2017. Families of infants who had not moved according to the latest census information (N = 722) were visited, including parents of infants who had died during the original trial. We did not perform a sample size calculation as it was impossible to estimate the proportion of parents that could be reached and would be willing to participate. We conducted up to three home visits to find both parents. Parents who were present, received an explanation about the present study being an extension of the original BCG trial. After informed oral consent, BCG scars were assessed and measured with a ruler by one of two field assistants. Field assistants were unaware of the hypothesis regarding parental BCG scars. We have previously shown that a BCG scar is an indicator for a correctly applied BCG vaccineStreptococcus pneumoniae, but lower responses of monokine induced by IFN-γ to Haemophilus influenzae and macrophage inflammatory protein 1α to Pam3CSK4 and peptidoglycan The immune mechanisms underlying NSEs of BCG are not fully understood and most likely feature both heterologous innate and T-cell responses of which the former are termed ‘trained immunity’ and are most widely described for the NSEs of BCG Trained immunity after vaccination is probably mediated by epigenetic modifications, such as DNA methylation, miRNAs or histone modifications, Our study corroborate several initial studies, indicating that the combination of maternal BCG priming and infant BCG vaccination reduces offspring mortality and hospital admissions in low- and high-income settings, respectively ,8. ThereIn addition to the benefits of maternal priming, this study is the first to describe possible beneficial effects of paternal BCG scars. The paternal BCG scar estimate on early-life survival was similar to that of maternal scars. Although the interaction with infant BCG vaccination was less evident, having a father with a BCG scar amplified the beneficial association of a maternal scar on mortality and vice versa. In BCG unvaccinated infants, parental scar effects were underpowered. However, both paternal and maternal scars were associated with large reductions in early-life mortality. Since infants often receive delayed BCG vaccinations,In conclusion, both maternal and paternal BCG vaccine scars were associated with decreased mortality in the first six weeks of life, the effect being enhanced when both parents had a scar. While both parental scars were associated with reduced mortality among BCG unvaccinated infants, interaction with infant BCG vaccination was more evident for maternal BCG scars. The possibility of inherited health benefits of BCG underlines the importance of a general BCG vaccination policy and the need for future studies into the subject of inherited non-specific immunity.CSB and PA conceived and designed both the original BCG trial and the parental BCG scar study. SBS, KJJ and FSB supervised the field data collection of the original trial. MLTB, PB and IS supervised the additional field data collection conducted for the parental BCG scar study. IM was responsible for the data collection and data entry within the original trial and the parental BCG scar study. MLTB and PB verified the underlying data and MLTB performed all statistical analyses. FSB and IS assisted in conceptualizing the manuscript and MLTB wrote the first draft of the manuscript. All authors contributed to the final version of the manuscript.MLTB and FSB received support from the University of Southern Denmark in the form of a scholarship. CSB holds an ERC starting grant and PA holds a research professorship from Novo Nordisk. PB, SBS, KJJ, IM, and IS declare no conflict of interest.