National survey of patient symptoms and therapies among 707 women with a lipedema phenotype in the United States.

BACKGROUND: National survey data exploring the patient experience with lipedema are lacking. METHODS: We conducted national surveys from 2016 to 2022 of women with lipedema as well as female controls. Surveys collected information on symptomatology, pain, and therapies. We performed logistic regression comparing symptoms among those with lipedema versus controls adjusting for age and BMI. RESULTS: A total of 707 women with lipedema and 216 controls completed the surveys. Those with lipedema had a mean age of 48.6 years and mean BMI of 40.9 kg/m2. Lipedema symptom onset occurred frequently at puberty (48.0%) or pregnancy (41.2%). Compared to controls, women with lipedema were more likely to report leg swelling in heat (odds ratio [OR], 66.82; 95% CI, 33.04-135.12; p < 0.0001), easy bruising (OR, 26.23; 95% CI, 15.58-44.17; p < 0.0001), altered gait (OR, 15.54; 95% CI, 7.58-31.96; p < 0.0001), flu-like symptoms (OR, 12.99; 95% CI, 4.27-39.49; p < 0.0001), joint hypermobility (OR, 12.88; 95% CI, 6.68-24.81; p < 0.0001), cool skin (OR, 12.21; 95% CI, 5.20-28.69; p < 0.0001), varicose veins (OR, 11.29; 95% CI, 6.71-18.99; p < 0.0001), and fatigue (OR, 9.59; 95% CI, 6.10-15.09; p < 0.0001). Additionally, 70.3% had upper arm involvement, 21.2% reported foot swelling, and 16.6% reported foot pain. Most (52.2%) reported no symptom improvement with diet or exercise. Common therapies used included compression therapy (45.0%), gastric bypass (15.7%), and lower-extremity liposuction (14.0%). CONCLUSION: In a large, national, symptom survey, women with lipedema reported excess pain, swelling, and fat in the legs along with numerous symptoms beyond those classically described. Symptom responses to common therapies remain understudied.

Characterizing thoracic morphology variation to develop representative 3D models for applications in chest trauma.

BACKGROUND: Rib fracture(s) occurs in 85% of blunt chest trauma cases. Increasing evidence supports that surgical intervention, particularly for multiple fractures, may improve outcomes. Thoracic morphology diversity across ages and sexes is important to consider in the design and use of surgical intervention devices in chest trauma. However, research on non-average thoracic morphology is lacking. METHODS: The rib cage was segmented from patient computed tomography (CT) scans to create 3D point clouds. These point clouds were uniformly oriented and chest height, width, and depth were measured. Size categorization was determined by grouping each dimension into small, medium, and large tertiles. From small and large size combinations, subgroups were extracted to develop thoracic 3D models of the rib cage and surrounding soft tissue. RESULTS: The study population included 141 subjects (48% male) ranging from age 10-80 with ∼20 subjects/age decade. Mean chest volume increased with age by 26% from the age groups 10-20 to 60-70, with 11% of this increase occurring between the youngest groups of 10-20 and 20-30. Across all ages, chest dimensions were ∼10% smaller in females and chest volume was highly variable (SD: ±3936.5 cm3). Representative thoracic models of four males (ages 16, 24, 44, 48) and three females (ages 19, 50, 53) were developed to characterize morphology associated with combinations of small and large chest dimensions. CONCLUSIONS: The seven models developed cover a broad range of non-average thoracic morphologies and can serve as a basis for informing device design, surgical planning, and injury risk assessments.

Semiautomated segmentation of lower extremity MRI reveals distinctive subcutaneous adipose tissue in lipedema: a pilot study.

Purpose: Lipedema is a painful subcutaneous adipose tissue (SAT) disease involving disproportionate SAT accumulation in the lower extremities that is frequently misdiagnosed as obesity. We developed a semiautomatic segmentation pipeline to quantify the unique lower-extremity SAT quantity in lipedema from multislice chemical-shift-encoded (CSE) magnetic resonance imaging (MRI). Approach: Patients with lipedema (n=15) and controls (n=13) matched for age and body mass index (BMI) underwent CSE-MRI acquired from the thighs to ankles. Images were segmented to partition SAT and skeletal muscle with a semiautomated algorithm incorporating classical image processing techniques (thresholding, active contours, Boolean operations, and morphological operations). The Dice similarity coefficient (DSC) was computed for SAT and muscle automated versus ground truth segmentations in the calf and thigh. SAT and muscle volumes and the SAT-to-muscle volume ratio were calculated across slices for decades containing 10% of total slices per participant. The effect size was calculated, and Mann-Whitney U test applied to compare metrics in each decade between groups (significance: two-sided P<0.05). Results: Mean DSC for SAT segmentations was 0.96 in the calf and 0.98 in the thigh, and for muscle was 0.97 in the calf and 0.97 in the thigh. In all decades, mean SAT volume was significantly elevated in participants with versus without lipedema (P<0.01), whereas muscle volume did not differ. Mean SAT-to-muscle volume ratio was significantly elevated (P<0.001) in all decades, where the greatest effect size for distinguishing lipedema was in the seventh decade approximately midthigh (r=0.76). Conclusions: The semiautomated segmentation of lower-extremity SAT and muscle from CSE-MRI could enable fast multislice analysis of SAT deposition throughout the legs relevant to distinguishing patients with lipedema from females with similar BMI but without SAT disease.

Current Mechanistic Understandings of Lymphedema and Lipedema: Tales of Fluid, Fat, and Fibrosis.

Lymphedema and lipedema are complex diseases. While the external presentation of swollen legs in lower-extremity lymphedema and lipedema appear similar, current mechanistic understandings of these diseases indicate unique aspects of their underlying pathophysiology. They share certain clinical features, such as fluid (edema), fat (adipose expansion), and fibrosis (extracellular matrix remodeling). Yet, these diverge on their time course and known molecular regulators of pathophysiology and genetics. This divergence likely indicates a unique route leading to interstitial fluid accumulation and subsequent inflammation in lymphedema versus lipedema. Identifying disease mechanisms that are causal and which are merely indicative of the condition is far more explored in lymphedema than in lipedema. In primary lymphedema, discoveries of genetic mutations link molecular markers to mechanisms of lymphatic disease. Much work remains in this area towards better risk assessment of secondary lymphedema and the hopeful discovery of validated genetic diagnostics for lipedema. The purpose of this review is to expose the distinct and shared (i) clinical criteria and symptomatology, (ii) molecular regulators and pathophysiology, and (iii) genetic markers of lymphedema and lipedema to help inform future research in this field.

Iota-carrageenan and xylitol inhibit SARS-CoV-2 in Vero cell culture.

Last year observed a global pandemic caused by SARS-CoV-2 (severe acute respiratory syndrome-coronavirus 2) infection affecting millions of individuals worldwide. There is an urgent unmet need to provide an easily producible and affordable medicine to prevent transmission and provide early treatment for this disease. Since the nasal cavity and the rhinopharynx are the sites of initial replication of SARS-CoV-2, a nasal spray may be an effective option to target SARS-CoV-2 infection. In this study, we tested the antiviral action of three candidate nasal spray formulations against SARS-CoV-2 in vitro. We determined that iota-carrageenan in concentrations as low as 6 µg/mL inhibits SARS-CoV-2 in vitro. The concentrations of iota-carrageenan with activity against SARS-CoV-2 in vitro may be easily achieved through the application of nasal sprays as commonly used in several countries. Recently a double-blind, placebo-controlled study showed that iota-carrageenan in isotonic sodium chloride reduces ca. five times the risk of infection by SARS-CoV-2 in health care personnel. Further, xylitol at a concentration of 50 mg/mL (ca. 329 mM) was found to exert some antiviral action, though this preliminary finding needs further confirmation.

NiTi-Nb micro-trusses fabricated via extrusion-based 3D-printing of powders and transient-liquid-phase sintering.

We present a novel additive manufacturing method for NiTi-Nb micro-trusses combining (i) extrusion-based 3D-printing of liquid inks containing NiTi and Nb powders, solvents, and a polymer binder into micro-trusses with 0/90° ABAB layers of parallel, ∼600 µm struts spaced 1 mm apart and (ii) subsequent heat-treatment to remove the binder and solvents, and then bond the NiTi powders using liquid phase sintering via the formation of a transient NiTi-Nb eutectic phase. We investigate the effects of Nb concentration (0, 1.5, 3.1, 6.7 at.% Nb) on the porosity, microstructure, and phase transformations of the printed NiTi-Nb micro-trusses. Micro-trusses with the highest Nb content exhibit long channels (from 3D-printing) and struts with smaller interconnected porosity (from partial sintering), resulting in overall porosities of ∼75% and low compressive stiffnesses of 1-1.6 GPa, similar to those of trabecular bone and in agreement with analytical and finite element modeling predictions. Diffusion of Nb into the NiTi particles from the bond regions results in a Ni-rich composition as the Nb replaces Ti atoms, leading to decreased martensite/austenite transformation temperatures. Adult human mesenchymal stem cells seeded on these micro-trusses showed excellent viability, proliferation, and extracellular matrix deposition over 14 days in culture. STATEMENT OF SIGNIFICANCE: Near-equiatomic NiTi micro-trusses are attractive for biomedical applications such as stents, actuators, and bone implants because of their combination of biocompatibility, low compressive stiffness, high surface area, and shape-memory or superelasticity. Extrusion-based 3D-printing of NiTi powder-based inks into micro-trusses is feasible, but the subsequent sintering of the powders into dense struts is unachievable due to low diffusivity, large particle size, and low packing density of the NiTi powders. We present a solution, whereby Nb powders are added to the NiTi inks, thus forming during sintering a eutectic NiTi-Nb liquid phase which bonds the solid NiTi powders and improves densification of the struts. This study investigates the microstructure, porosity, phase transformation behavior, compressive stiffness, and cytocompatibility of these printed NiTi-Nb micro-trusses.

Protocols to Assess Coagulation Following In Vitro Infection with Hemorrhagic Fever Viruses.

During the course of infection with a hemorrhagic fever virus (HFV), the checks and balances associated with normal coagulation are perturbed resulting in hemorrhage in severe cases and, in some patients, disseminated intravascular coagulopathy (DIC). While many HFVs have animal models that permit the analyses of systemic coagulopathy, animal infection models do not exist for all HFVs and moreover do not always recapitulate the pathology observed in human tissues. Furthermore, molecular analyses of how coagulation is affected are not always straightforward or practical when using ex-vivo animal-derived samples, thus reinforcing the importance of cell culture studies. This chapter highlights procedures utilizing human umbilical vein endothelial cells (HUVECs) as a model system to evaluate components of the intrinsic (prekallikrein (PK), factor XII (FXII), kininogen, and bradykinin (BK)) and extrinsic (Tissue Factor (TF)) systems. Specifically, protocols are included for the generation of a coculture blood vessel model, plating and infection of HUVEC monolayers and assays designed to measure activation of PK and FXII, cleavage of kininogen, and to measure the expression of TF mRNA and protein.

A fusion-inhibiting peptide against Rift Valley fever virus inhibits multiple, diverse viruses.

For enveloped viruses, fusion of the viral envelope with a cellular membrane is critical for a productive infection to occur. This fusion process is mediated by at least three classes of fusion proteins (Class I, II, and III) based on the protein sequence and structure. For Rift Valley fever virus (RVFV), the glycoprotein Gc (Class II fusion protein) mediates this fusion event following entry into the endocytic pathway, allowing the viral genome access to the cell cytoplasm. Here, we show that peptides analogous to the RVFV Gc stem region inhibited RVFV infectivity in cell culture by inhibiting the fusion process. Further, we show that infectivity can be inhibited for diverse, unrelated RNA viruses that have Class I (Ebola virus), Class II (Andes virus), or Class III (vesicular stomatitis virus) fusion proteins using this single peptide. Our findings are consistent with an inhibition mechanism similar to that proposed for stem peptide fusion inhibitors of dengue virus in which the RVFV inhibitory peptide first binds to both the virion and cell membranes, allowing it to traffic with the virus into the endocytic pathway. Upon acidification and rearrangement of Gc, the peptide is then able to specifically bind to Gc and prevent fusion of the viral and endocytic membranes, thus inhibiting viral infection. These results could provide novel insights into conserved features among the three classes of viral fusion proteins and offer direction for the future development of broadly active fusion inhibitors.

Endothelial cell permeability during hantavirus infection involves factor XII-dependent increased activation of the kallikrein-kinin system.

Hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) are diseases caused by hantavirus infections and are characterized by vascular leakage due to alterations of the endothelial barrier. Hantavirus-infected endothelial cells (EC) display no overt cytopathology; consequently, pathogenesis models have focused either on the influx of immune cells and release of cytokines or on increased degradation of the adherens junction protein, vascular endothelial (VE)-cadherin, due to hantavirus-mediated hypersensitization of EC to vascular endothelial growth factor (VEGF). To examine endothelial leakage in a relevant in vitro system, we co-cultured endothelial and vascular smooth muscle cells (vSMC) to generate capillary blood vessel-like structures. In contrast to results obtained in monolayers of cultured EC, we found that despite viral replication in both cell types as well as the presence of VEGF, infected in vitro vessels neither lost integrity nor displayed evidence of VE-cadherin degradation. Here, we present evidence for a novel mechanism of hantavirus-induced vascular leakage involving activation of the plasma kallikrein-kinin system (KKS). We show that incubation of factor XII (FXII), prekallikrein (PK), and high molecular weight kininogen (HK) plasma proteins with hantavirus-infected EC results in increased cleavage of HK, higher enzymatic activities of FXIIa/kallikrein (KAL) and increased liberation of bradykinin (BK). Measuring cell permeability in real-time using electric cell-substrate impedance sensing (ECIS), we identified dramatic increases in endothelial cell permeability after KKS activation and liberation of BK. Furthermore, the alterations in permeability could be prevented using inhibitors that directly block BK binding, the activity of FXIIa, or the activity of KAL. Lastly, FXII binding and autoactivation is increased on the surface of hantavirus-infected EC. These data are the first to demonstrate KKS activation during hantavirus infection and could have profound implications for treatment of hantavirus infections.

Inhibition of TNF-alpha-induced activation of NF-kappaB by hantavirus nucleocapsid proteins.

Hantaviruses cause two human diseases thought to be immune-mediated: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). We recently reported that the nucleocapsid (N) protein of HFRS-causing Hantaan virus (HTNV) inhibits tumor necrosis factor-alpha (TNF-alpha) activation of nuclear factor-kappaB (NF-kappaB). Here we measured the ability of other hantaviral N proteins to similarly interfere with the inflammatory process of TNF-alpha. We found that like HTNV N, the N proteins of HFRS-causing Seoul and Dobrava viruses inhibited TNF-alpha activation of NF-kappaB and translocation of the NF-kappaB p65 subunit, but did not interfere with degradation of inhibitor of NF-kappaB (IkappaB). In contrast, the HFRS-causing Puumala virus and the HPS-causing Andes and Sin Nombre viruses did not prevent TNF-alpha activation of NF-kappaB or nuclear translocation of p65. These studies provide evidence that hantaviruses differ in their abilities to interfere with host innate immune responses.

Hantaan virus nucleocapsid protein binds to importin alpha proteins and inhibits tumor necrosis factor alpha-induced activation of nuclear factor kappa B.

Hantaviruses such as Hantaan virus (HTNV) and Andes virus cause two human diseases, hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome, respectively. For both, disease pathogenesis is thought to be immunologically mediated and there have been numerous reports of patients with elevated levels of proinflammatory and inflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha), in their sera. Multiple viruses have developed evasion strategies to circumvent the host cell inflammatory process, with one of the most prevalent being the disruption of nuclear factor kappa B (NF-kappaB) activation. We hypothesized that hantaviruses might also moderate host inflammation by interfering with this pathway. We report here that the nucleocapsid (N) protein of HTNV was able to inhibit TNF-alpha-induced activation of NF-kappaB, as measured by a reporter assay, and the activation of endogenous p65, an NF-kappaB subunit. Surprisingly, there was no defect in the degradation of the inhibitor of NF-kappaB (IkappaB) protein, nor was there any alteration in the level of p65 expression in HTNV N-expressing cells. However, immunofluorescence antibody staining demonstrated that cells expressing HTNV N protein and a green fluorescent protein-p65 fusion had limited p65 nuclear translocation. Furthermore, we were able to detect an interaction between HTNV N protein and importin alpha, a nuclear import molecule responsible for shuttling NF-kappaB to the nucleus. Collectively, our data suggest that HTNV N protein can sequester NF-kappaB in the cytoplasm, thus inhibiting NF-kappaB activity. These findings, which were obtained using cells transfected with cDNA representing the HTNV N gene, were confirmed using HTNV-infected cells.

Ovarian tumor domain-containing viral proteases evade ubiquitin- and ISG15-dependent innate immune responses.

Ubiquitin (Ub) and interferon-stimulated gene product 15 (ISG15) reversibly conjugate to proteins and mediate important innate antiviral responses. The ovarian tumor (OTU) domain represents a superfamily of predicted proteases found in eukaryotic, bacterial, and viral proteins, some of which have Ub-deconjugating activity. We show that the OTU domain-containing proteases from nairoviruses and arteriviruses, two unrelated groups of RNA viruses, hydrolyze Ub and ISG15 from cellular target proteins. This broad activity contrasts with the target specificity of known mammalian OTU domain-containing proteins. Expression of a viral OTU domain-containing protein antagonizes the antiviral effects of ISG15 and enhances susceptibility to Sindbis virus infection in vivo. We also show that viral OTU domain-containing proteases inhibit NF-kappaB-dependent signaling. Thus, the deconjugating activity of viral OTU proteases represents a unique viral strategy to inhibit Ub- and ISG15-dependent antiviral pathways.

Replication of varicella-zoster virus in human skin organ culture.

Varicella-zoster virus (VZV) infection is restricted to humans, which hinders studies of its pathogenesis in rodent models of disease. To facilitate the study of VZV skin tropism, we developed an ex vivo system using human fetal skin organ culture (SOC). VZV replication was analyzed by plaque assay, transmission electron microscopy, and histology. The yield of infectious VZV from SOC increased approximately 100-fold over 6 days, virions were abundant, and lesions developed that contained VZV antigens and resembled varicella and zoster lesions. The SOC system for VZV replication has applications for testing virus mutants and antiviral drugs.

Human cytomegalovirus genes in the 15-kilobase region are required for viral replication in implanted human tissues in SCID mice.

Since animal models for studying human cytomegalovirus (HCMV) replication in vivo and pathogenesis are not available, severe combined immunodeficiency mice into which human tissues were implanted (SCID-hu mice) provide an alternative and valuable model for such studies. The HCMV clinical isolates, including those of the Toledo strain, replicate to high titers in human tissue implanted into SCID mice; however, the attenuated AD169 strain has completely lost this ability. The major difference between Toledo and AD169 is a 15-kb segment, encoding 19 open reading frames, which is present in all virulent strains but deleted from attenuated strains. This fact suggests that crucial genes required for HCMV replication in vivo are localized to this region. In this study, the importance of this 15-kb segment for HCMV replication in vivo was determined. First, Toledo(BAC) virus (produced from a Toledo bacterial artificial chromosome) and AD169 virus were tested for growth in SCID-hu mice. Toledo(BAC), like Toledo, grew to high titers in implanted human thymus and liver tissues, while AD169 did not. This outcome showed that the Toledo genome propagated in bacteria (Toledo(BAC)) retained its virulence. The 15-kb segment was then deleted from Toledo(BAC), and the resulting virus, Toledo(Delta15kb), was tested for growth in both human foreskin fibroblast (HFF) cells and SCID-hu mice. Toledo(Delta15kb) had a minor growth defect in HFF but completely failed to replicate in human thymus and liver implants. This failure to grow was rescued when the 15-kb region was inserted back into the Toledo(Delta15kb) genome. These results directly demonstrated that the genes located in the 15-kb segment are crucial for HCMV replication in vivo.

Roscovitine, a cyclin-dependent kinase inhibitor, prevents replication of varicella-zoster virus.

Understanding the interactions between varicella-zoster virus (VZV) and host cells can be addressed by using small molecule inhibitors of cellular enzymes. Roscovitine (Rosco) is a purine derivative that inhibits cyclin-dependent kinase 1 (cdk1), cdk2, cdk5, cdk7, and cdk9, which are key regulators of the cell cycle and transcription. Herpesviruses are known to interact with cell cycle proteins; thus, the antiviral effects of Rosco on VZV growth were evaluated. In a plaque reduction assay, 25 micro M Rosco prevented VZV replication, and the antiviral effect was reversible for at least up to 24 h posttreatment. Rosco also reduced expression of the major transactivator, IE62, over 48 h. Confocal microscopy studies indicated that Rosco caused the immediate-early proteins ORF4 and IE62 to abnormally localize in infected cells and prevented cell-cell spread of VZV over 48 h. Rosco was found to inhibit VZV DNA synthesis as measured by real-time PCR, and this technique was used to estimate the 50% effective concentration (EC(50)) of 14 micro M. This value was close to the EC(50) estimate of 12 micro M determined from plaque reduction assays. At 25 micro M, Rosco was not cytotoxic over 48 h in a neutral red uptake assay, and proliferation was slowed as the cells accumulated in a G(2)-like state. These results demonstrate the importance of cdk's in VZV replication and suggest that cdk inhibitors could serve as useful VZV antivirals.

Viral and cellular kinases are potential antiviral targets and have a central role in varicella zoster virus pathogenesis.

Herpesviruses utilize viral and cellular kinases for replication, and these mediate essential functions that are important for viral pathogenesis. Elucidating the roles of kinases in herpesvirus infections may highlight virus-host interactions that are possible targets for kinase inhibitors with antiviral activity. Varicella zoster virus (VZV) encodes two kinases that phosphorylate viral proteins involved in regulation, assembly, and virulence. VZV infection also induces the activity of host cell cyclin-dependent kinases (cdk4 and cdk2) in nondividing cells, causing a disregulation of the cell cycle. Roscovitine and Purvalanol, kinase inhibitors that target cdks, prevent VZV replication at concentrations with few cytotoxic effects. Cdk inhibitors therefore have potential as antivirals that may extend to a broad range of viruses and have the added advantage that resistance does not arise easily.