Purified CD34+ Lin- Thy+ stem cells do not contain clonal myeloma cells

High-dose therapy with autologous marrow or peripheral blood stem cell (PBSC) rescue has been extensively applied in the treatment of multiple myeloma (MM) patients during the past 10 years resulting in improved event-free and overall survival when compared with standard chemotherapy. However, relapses are common and cure is unlikely in the majority of patients. Because both bone marrow and PBSCs are contaminated with myeloma cells it is conceivable that relapse after autotransplantation originates at least in part from autografted tumor cells. In this study, mobilized PBSCs were examined for the presence of myeloma cells based on immunophenotyping and sensitive polymerase chain reaction (PCR)-based techniques. In addition, CD34+ Lin- Thy+ stem cells were purified from mobilized PBSC harvests of 10 MM patients by sequentially using counterflow elutriation centrifugation, treatment with phenylalanine methylester, and flow sorting, using 5-parameter gating (propidium iodide, forward scatter, side scatter, CD34+ v Lin- and CD34+ v Thy+). Virtually all mobilized unsorted PBSC preparations contained myeloma cells in sufficient quantities (range, < 0.01 to > 10%) potentially causing a disease relapse. Stem cell purification led to an overall enrichment by about 50-fold in all 10 patients; approximately 90% of the final cell population expressed CD34+ Lin- Thy+ with no evidence of myeloma cell contamination based on flow cytometric analysis of CD38bright cells (< 0.1%). Quantitative PCR amplification of patient-specific complementarity determining region III (CDRIII) DNA sequences showed depletion of clonal B cells by 2.7 to 7.3 logs, with the highest log reduction noted in the samples initially containing the most tumor cells. Our results show that purification of CD34+ Lin- Thy+ cells depletes myeloma cells to undetectable levels from up to 10% present in unsorted PBSCs, thus offering a tool to investigate whether MM relapse after autotransplantation can be reduced markedly.     

In silico dynamics of COVID-19 phenotypes for optimizing clinical management

Understanding the underlying mechanisms of COVID-19 progression and the impact of various pharmaceutical interventions is crucial for the clinical management of the disease. We developed a comprehensive mathematical framework based on the known mechanisms of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, incorporating the renin−angiotensin system and ACE2, which the virus exploits for cellular entry, key elements of the innate and adaptive immune responses, the role of inflammatory cytokines, and the coagulation cascade for thrombus formation. The model predicts the evolution of viral load, immune cells, cytokines, thrombosis, and oxygen saturation based on patient baseline condition and the presence of comorbidities. Model predictions were validated with clinical data from healthy people and COVID-19 patients, and the results were used to gain insight into identified risk factors of disease progression including older age; comorbidities such as obesity, diabetes, and hypertension; and dysregulated immune response. We then simulated treatment with various drug classes to identify optimal therapeutic protocols. We found that the outcome of any treatment depends on the sustained response rate of activated CD8⁺ T cells and sufficient control of the innate immune response. Furthermore, the best treatment—or combination of treatments—depends on the preinfection health status of the patient. Our mathematical framework provides important insight into SARS-CoV-2 pathogenesis and could be used as the basis for personalized, optimal management of COVID-19.

COVID-19 has created unprecedented challenges for the health care system, and, until an effective vaccine is developed and made widely available, treatment options are limited. A challenge to the development of optimal treatment strategies is the extreme heterogeneity of presentation. Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) results in a syndrome that ranges in severity from asymptomatic to multiorgan failure and death. In addition to local complications in the lung, the virus can cause systemic inflammation and disseminated microthrombosis, which can cause stroke, myocardial infarction, or pulmonary emboli (1–4). Risk factors for poor COVID-19 outcome include advanced age, obesity, diabetes, and hypertension (5–13).Computational analyses can provide insights into the transmission, control, progression, and underlying mechanisms of infectious diseases. Indeed, epidemiological and statistical modeling has been used for COVID-19, providing powerful insights into comorbidities, transmission dynamics, and control of the disease (14–17). However, to date, these analyses have been population dynamics models of SARS-CoV-2 infection and transmission or correlative analyses of COVID-19 comorbidities and treatment response. Simple viral dynamics models have been also developed and used to predict the SARS-CoV-2 response to antiviral drugs (18, 19). These models, however, do not explicitly consider the biological or physiological mechanisms underlying disease progression or the time course of response to various therapeutic interventions, and only a few more-sophisticated models have been developed toward this direction (20, 21).Several therapies targeting various aspects of COVID-19 pathogenesis have been proposed and have either completed—or are currently being tested in—clinical trials (22). Despite strong biologic rationale, these treatments have generally produced conflicting results in the clinic. For example, trials of antiviral therapies (e.g., remdesivir) have been mixed: The original trial from China failed (23), a subsequent trial in the United States led to approval of remdesivir in the United States and other countries (24), and the recent results of the World Health Organization Solidarity trial again show no benefit (25). Other antiviral drugs alone or in combination are also showing promise (26).Other potential treatments include antiinflammatory drugs and antithrombotic agents. Because of the systemic inflammation seen in many patients, antiinflammatory drugs have been tested, including anti-IL6/IL6R therapy (e.g., tocilizumab, siltuximab) and anti-JAK1/2 drugs (e.g., barcitinib). It is not clear whether these drugs will be effective as stand-alone treatments, particularly after the recent failure of tocilizumab in a phase III trial (1, 27–29). In addition, given that a common complication of COVID-19 is the development of coagulopathies with microvascular thrombi potentially leading to the dysfunction of multiple organ systems (2, 3), antithrombotic drugs (e.g., low molecular weight heparin) are being tested. Recognizing the interactions of COVID-19 with the immune system (30), the corticosteroid dexamethasone has been tested, showing some promising results. Given the large range of patient comorbidities, disease severities, and variety of complications such as thrombosis, it is likely that patients will have heterogeneous responses to any given therapy, and such heterogeneity will continue to be a challenge for clinical trials of unselected COVID-19 patients (31).Here, we developed a systems biology-based mathematical model to address this urgent need. Our model incorporates the known mechanisms of SARS-CoV-2 pathogenesis and the potential mechanisms of action of various therapeutic interventions that have been tested in COVID-19 patients. In previous work, we have exploited angiotensin receptor blockers (ARBs) and angiotensin converting enzyme inhibitors (ACEis) for the improvement of cancer therapies and developed mathematical models of the renin−angiotensin system in the context of cancer desmoplasia (32–35). Using a similar approach, we developed a detailed model that includes lung infection by the SARS-CoV-2 virus and a pharmacokinetic/pharmacodynamic (PK/PD) model of infection and thrombosis to simulate events that take place throughout the body during COVID-19 progression (Fig. 1 and SI Appendix, Fig. S1). The model is first validated against clinical data of healthy people and COVID-19 patients and then used to simulate disease progression in patients with specific comorbidities. Subsequently, we present model predictions for various therapies currently employed for treatment of COVID-19 alone or in combination, and we identify protocols for optimal clinical management for each of the clinically observed COVID-19 phenotypes.Open in a separate windowFig. 1.Schematic of the detailed lung model. The model incorporates the virus infection of epithelial and endothelial cells, the RAS, T cells activation and immune checkpoints, the known IL6 pathways, neutrophils, and macrophages, as well as the formation of NETs, and the coagulation cascade. The lung model is coupled with a PK/PD model for the virus and thrombi dissemination through the body.     

The relationship between smartphone use and subjective musculoskeletal symptoms and university students

[Purpose] The purpose of this study was to investigate the use of smartphones by university students in selected areas, their musculoskeletal symptoms, and the associated hazard ratio. [Subjects and Methods] This involved the completion of a self-administered questionnaire by dental hygiene students in Seoul, Gyeonggido, and Gyeongsangbukdo. The 292 completed copies of the questionnaire were then analyzed. [Results] The most painful body regions after the use of smartphones were found to be the shoulders and neck. In the musculoskeletal system, back pain was found to have a positive correlation with the size of the smartphone’s liquid crystal display (LCD) screen, and pain in legs and feet were found to have a negative correlation with the length of time that the smartphone was used. As a result, it was revealed that the use of a smartphone was correlated with musculoskeletal symptoms. [Conclusion] Therefore, in today’s environment, where the use of smartphones is on the rise, it is necessary to improve the ways that they are used and to develop a preventive program to alleviate the symptoms of musculoskeletal damage.Key words: Smartphone, Musculoskeletal symptoms, Prevent     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Causes, consequences, and remedies for growth-induced solid stress in murine and human tumors

The presence of growth-induced solid stresses in tumors has been suspected for some time, but these stresses were largely estimated using mathematical models. Solid stresses can deform the surrounding tissues and compress intratumoral lymphatic and blood vessels. Compression of lymphatic vessels elevates interstitial fluid pressure, whereas compression of blood vessels reduces blood flow. Reduced blood flow, in turn, leads to hypoxia, which promotes tumor progression, immunosuppression, inflammation, invasion, and metastasis and lowers the efficacy of chemo-, radio-, and immunotherapies. Thus, strategies designed to alleviate solid stress have the potential to improve cancer treatment. However, a lack of methods for measuring solid stress has hindered the development of solid stress-alleviating drugs. Here, we present a simple technique to estimate the growth-induced solid stress accumulated within animal and human tumors, and we show that this stress can be reduced by depleting cancer cells, fibroblasts, collagen, and/or hyaluronan, resulting in improved tumor perfusion. Furthermore, we show that therapeutic depletion of carcinoma-associated fibroblasts with an inhibitor of the sonic hedgehog pathway reduces solid stress, decompresses blood and lymphatic vessels, and increases perfusion. In addition to providing insights into the mechanopathology of tumors, our approach can serve as a rapid screen for stress-reducing and perfusion-enhancing drugs.     

Tolerance to apoptotic cells is regulated by indoleamine 2,3-dioxygenase

Tolerance to self-antigens present in apoptotic cells is critical to maintain immune-homeostasis and prevent systemic autoimmunity. However, mechanisms that sustain self-tolerance are poorly understood. Here we show that systemic administration of apoptotic cells to mice induced splenic expression of the tryptophan catabolizing enzyme indoleamine 2,3-dioxygenase (IDO). IDO expression was confined to the splenic marginal zone and was abrogated by depletion of CD169(+) cells. Pharmacologic inhibition of IDO skewed the immune response to apoptotic cells, resulting in increased proinflammatory cytokine production and increased effector T-cell responses toward apoptotic cell-associated antigens. Presymptomatic lupus-prone MRL(lpr/lpr) mice exhibited abnormal elevated IDO expression in the marginal zone and red pulp and inhibition of IDO markedly accelerated disease progression. Moreover, chronic exposure of IDO-deficient mice to apoptotic cells induced a lupus-like disease with serum autoreactivity to double-stranded DNA associated with renal pathology and increased mortality. Thus, IDO limits innate and adaptive immunity to apoptotic self-antigens and IDO-mediated regulation inhibits inflammatory pathology caused by systemic autoimmune disease.     

The diagnostic value of fine needle aspiration in parotid lumps

Introduction

Fine needle aspiration (FNA) is a safe and quick method of diagnosing superficial lumps, which aids preoperative planning. However, FNA of the parotid gland has not gained the widespread acceptance noted in other head and neck lumps. The aim of this study was to determine the ability of FNA of the parotid gland to differentiate benign and malignant disease, and to determine the impact on surgical outcome.

Methods

A retrospective analysis of 201 consecutive parotid operations with preoperative FNA in a large district hospital in the UK was performed. The diagnostic characteristics were calculated for benign and malignant disease, and the impact on surgical procedure was determined.

Results

In identifying benign disease, FNA has a sensitivity of 85% and a specificity of 76%. In detecting malignant disease, FNA has a sensitivity and specificity of 52% and 92% respectively. A false positive on FNA was associated with a higher incidence of neck dissection.

Conclusions

FNA is a useful diagnostic test. However, owing to low sensitivity, it is necessary to interpret it in the context of all other clinical information.     

Cell-autonomous control of interferon type I expression by indoleamine 2,3-dioxygenase in regulatory CD19+ dendritic cells

Following CD80/86 (B7) and TLR9 ligation, small subsets of splenic dendritic cells expressing CD19 (CD19(+) DC) acquire potent T cell regulatory functions due to induced expression of the intracellular enzyme indoleamine 2,3-dioxygenase (IDO), which catabolizes tryptophan. In CD19(+) DC, IFN type I (IFN-alpha) is the obligate inducer of IDO. We now report that IFN-alpha production needed to stimulate high-level expression of IDO following B7 ligation is itself dependent on basal levels of IDO activity. Genetic and pharmacologic ablation of IDO completely abrogated IFN-alpha production by CD19(+) DC after B7 ligation. In contrast, IDO ablation did not block IFN-alpha production by CD19(+) DC after TLR9 ligation. IDO-mediated control of IFN-alpha production depended on tryptophan depletion as adding excess tryptophan also blocked IFN-alpha expression after B7 ligation. Consistent with this, DC from mice deficient in general control of non-derepressible-2 (GCN2)-kinase, a component of the cellular stress response to amino acid withdrawal, did not produce IFN-alpha following B7 ligation, but produced IFN-alpha after TLR9 ligation. Thus, B7 and TLR9 ligands stimulate IFN-alpha expression in CD19(+) DC via distinct signaling pathways. In the case of B7 ligation, IDO activates cell-autonomous signals essential for IFN-alpha production, most likely by activating the GCN2-kinase-dependent stress response.     

Increasing sexually transmitted infection rates in young men having sex with men in the Netherlands, 2006–2012

Background

Men having sex with men (MSM) remain the largest high-risk group involved in on-going transmission of sexually transmitted infections (STI), including HIV, in the Netherlands. As risk behaviour may change with age, it is important to explore potential heterogeneity in risks by age. To improve our understanding of this epidemic, we analysed the prevalence of and risk factors for selected STI in MSM attending STI clinics in the Netherlands by age group.

Methods

Analysis of data from the national STI surveillance system for the period 2006–2012. Selected STI were chlamydia, gonorrhoea, infectious syphilis and/or a new HIV infection. Logistic regression was used to identify factors associated with these selected STI and with overall STI positivity. Analyses were done separately for MSM aged younger than 25 years and MSM aged 25 years and older.

Results

In young MSM a significant increase in positivity rate was seen over time (p?<?0.01), mainly driven by increasing gonorrhoea diagnoses, while in MSM aged 25 and older a significant decrease was observed (p?<?0.01). In multivariate analyses for young MSM, those who were involved in commercial sex were at higher risk (OR: 1.5, 95% CI: 1.2-1.9). For MSM aged 25 years and older this was not the case. Having a previous negative HIV test was protective among older MSM compared to those not tested for HIV before (OR: 0.8, 95% CI: 0.8-0.8), but not among younger MSM.

Conclusions

MSM visiting STI clinics remain a high-risk group for STI infections and transmission, but are not a homogenous group. While in MSM aged older than 25 years, STI positivity rate is decreasing, positivity rate in young MSM increased over time. Therefore specific attention needs to be paid towards targeted counselling and reaching particular MSM sub-groups, taken into account different behavioural profiles.

     

Differential mobilization of myeloma cells and normal hematopoietic stem cells in multiple myeloma after treatment with cyclophosphamide and granulocyte-macrophage colony-stimulating factor

Peripheral blood stem cells (PBSCs) mobilized with high-dose chemotherapy and hematopoietic growth factors are now widely used to support myeloablative therapy of multiple myeloma and effect complete remissions in up to 50% of patients with apparent extension of event- free and overall survival. Because tumor cells are present not only in bone marrow, but also in virtually all PBSC harvests, it is conceivable that autografted myeloma cells contribute to relapse after autotransplants. In this study, the kinetics of mobilization of normal hematopoietic stem cells were compared with those of myeloma cells present in PBSC harvests of 12 patients after high-dose cyclophosphamide and granulocyte-macrophage colony-stimulating factor administration. CD34+ and CD34+Lin-Thy+ stem cell contents were measured by multiparameter flow cytometry, and myeloma cells were quantitated by immunostaining for the relevant Ig light chain and by a quantitative polymerase chain reaction for the myeloma-specific CDRIII sequence. Results indicated marked heterogeneity in the percentages of mobilized stem cells among different patients (0.1% to 22.2% for CD34+ cells and 0.1% to 7.5% for CD34+Lin-Thy+ cells, respectively). The highest proportions of hematopoietic progenitor cells were observed early during apheresis, with 9 of 12 patients mobilizing adequate amounts of CD34+ cells for 2 autotransplants (> 4 x 10(6)/kg) within the first 2 days, whereas peak levels (percent and absolute numbers) of myeloma cells were present on days 5 and 6 (0.5% to 22.0%). During the last days of collection, mobilized tumor cells exhibited more frequently high labeling index values (1% to 10%; median, 4.4%) and an immature phenotype (CD19+). The differential mobilization observed between normal hematopoietic stem cells and myeloma cells can be exploited to reduce tumor cell contamination in PBSC harvests.