Human iPSC-derived cerebral organoids model features of Leigh syndrome and reveal abnormal corticogenesis.

Leigh syndrome (LS) is a rare, inherited neurometabolic disorder that presents with bilateral brain lesions caused by defects in the mitochondrial respiratory chain and associated nuclear-encoded proteins. We generated human induced pluripotent stem cells (iPSCs) from three LS patient-derived fibroblast lines. Using whole-exome and mitochondrial sequencing, we identified unreported mutations in pyruvate dehydrogenase (GM0372, PDH; GM13411, MT-ATP6/PDH) and dihydrolipoyl dehydrogenase (GM01503, DLD). These LS patient-derived iPSC lines were viable and capable of differentiating into progenitor populations, but we identified several abnormalities in three-dimensional differentiation models of brain development. LS patient-derived cerebral organoids showed defects in neural epithelial bud generation, size and cortical architecture at 100 days. The double mutant MT-ATP6/PDH line produced organoid neural precursor cells with abnormal mitochondrial morphology, characterized by fragmentation and disorganization, and showed an increased generation of astrocytes. These studies aim to provide a comprehensive phenotypic characterization of available patient-derived cell lines that can be used to study Leigh syndrome.

Impact of alternative histopathology protocols in the dermatopathology laboratory: A prospective quality improvement study.

BACKGROUND: Histopathology protocols for processing dermatopathology specimens vary among laboratories. OBJECTIVE: To determine an optimal histopathology protocol to minimize cost and turnaround time (TAT) for biopsy specimens in a dermatopathology laboratory. METHODS: A prospective, 4-month study compared the mean cost and TAT of producing one versus two initial H&E slides, and zero versus three unstained slides that could be used for frequently used special or immunohistochemical (IHC) stains. RESULTS: For all cases, cost was lower for one versus two initial H&E slides, with an insignificant increase in TAT. Producing three vs zero unstained slides incurred higher cost, with no reduction in TAT. In a subset of cases in which frequently used special or IHC stains were performed, cost and TAT were optimized by producing one initial H&E and three unstained slides. CONCLUSION: A protocol of one initial H&E slide and zero unstained slides optimizes cost and TAT in our dermatopathology laboratory. Pigmented lesions and inflammatory dermatoses may benefit from the addition of unstained slides. Further study is needed to quantify this benefit and evaluate for other cases for which an alternative protocol is advantageous.

A retrospective analysis of diagnostic testing in a large North American cohort of patients with epidermolysis bullosa.

BACKGROUND: Accurate diagnosis of epidermolysis bullosa (EB) has significant implications for prognosis, management, and genetic counseling. OBJECTIVE: To describe diagnostic testing patterns and assess diagnostic concordance of transmission electron microscopy (TEM), immunofluorescence mapping (IFM), and genetic analysis for EB. METHODS: A retrospective cohort included patients enrolled in the Epidermolysis Bullosa Clinical Characterization and Outcomes Database from January 1, 2004, to July 8, 2019. Tests concluding the same EB type (EB simplex, junctional EB, dominant dystrophic EB, and recessive dystrophic EB) were considered concordant; those concluding different EB types were considered discordant; and those with nonspecific/nondefinitive results were equivocal. RESULTS: A total of 970 diagnostic tests were conducted from 1984 to 2018 in 771 patients. Genetic analyses were performed chronologically later than IFM or TEM (P < .001). The likelihood of undergoing genetic analysis was greater for junctional EB and recessive dystrophic EB, and the same for dominant dystrophic EB as compared with EB simplex. TEM results in 163 patients were equivocal (55%), concordant (42%), and discordant (3%). IFM results in 185 patients were equivocal (54%), concordant (42%), and discordant (4%). LIMITATIONS: Retrospective design. CONCLUSIONS: Diagnostic testing has shifted in favor of genetic analysis. TEM and IFM frequently offer equivocal findings when compared to the specificity afforded by genetic analysis.

Auditory dysfunction in type 2 Stickler Syndrome.

PURPOSE: To present the extent and site of lesion of auditory dysfunction in a large cohort of individuals with type 2 Stickler Syndrome. Type 2 Stickler Syndrome results from a mutation in the gene coding for α-1 type XI pro-collagen, which has been identified in the human vitreous, cartilage and the cochlea of the mouse. The condition is characterised by classic ocular abnormalities, auditory dysfunction, osteoarthropathy and oro-facial dysplasia. METHODS: This is a population study which used a combination of audiometric, tympanometric, and self-report measures on a series of 65 individuals (mean age 29.2 years, range 3-70, female 63.1%) with genetically confirmed type 2 Stickler Syndrome. RESULTS: Hearing impairment was identified in at least one ear for 69% of individuals. Analysis against age-matched normative data showed that reduced hearing sensitivity was present across all test frequencies. Sensorineural hearing loss was most common (77% of ears), with conductive (3%), mixed (7%) and no hearing loss (13%), respectively. The proportion of hypermobile tympanic membranes (24%) was less than previously documented in type 1 Stickler Syndrome. When present, this appears to arise as a direct result of collagen abnormalities in the middle ear. Self-report measures of speech and spatial hearing in sound were comparable to a non-syndromic cohort with similar audiometric thresholds. CONCLUSIONS: Auditory impairment in type 2 Stickler Syndrome is predominantly associated with cochlear hearing loss of varying severities across affected individuals. The impact on hearing thresholds can be seen across the frequency range, suggesting a contribution of defective collagen throughout the cochlea. Self-report questionnaires showed that difficulties understanding speech, and spatial information in sound (such as that used for localisation), were worse than a young, normal-hearing population but comparable to a non-syndromic cohort with similar audiometric thresholds. Therefore, it is likely that hearing loss in type 2 Stickler Syndrome arises in the auditory periphery, without significant central processing deficits.

Comparison of ELISA with electro-chemiluminescence technology for the qualitative and quantitative assessment of serological responses to vaccination.

BACKGROUND: Profiling immune responses induced by either infection or vaccination can provide insight into identification of correlates of protection. Furthermore, profiling of serological responses can be used to identify biomarkers indicative of exposure to pathogens. Conducting such immune surveillance requires readout methods that are high-throughput, robust, and require small sample volumes. While the enzyme-linked immunosorbent assay (ELISA) is the classical readout method for assessing serological responses, the advent of multiplex assays has significantly increased the throughput and capacity for immunoprofiling. This report describes the development and assay performance (sensitivity, linearity of detection, requirement for multiple dilutions for each sample, intra- and inter-assay variability) of an electro-chemiluminescence (ECLIA)-based multiplex assay. METHODS: The current study describes the development of a multiplex ECLIA-based assay and characterizes the sensitivity, linear range, and inter- and intra-assay variability of the ECLIA platform and its agreement with the traditional ELISA. Special emphasis was placed on potential antigenic competition when testing closely related antigens in the multiplex format. RESULTS: Multiplexing of antigens in ECLIA provides significant practical benefits in terms of reducing sample volume requirements and experimental time. Beyond the practical advantages of multiplexing, the ECLIA provides superior assay performance when compared to the ELISA. Not only does ECLIA show good agreement with the ELISA assay, but the linear range of ECLIA is also sufficiently wide to permit single-dilution measurements of concentration without the need to do serial dilutions. The lack of antigenic competition allows the simultaneous testing of closely related antigens, such as plate antigens representing different alleles of the same protein, which can inform about cross-reactivities-or lack thereof-of serological responses. CONCLUSION: The advantages of the newly developed tool for assessing the antigen profiles of serological responses may ultimately lead to the identification of biomarkers associated with various disease stages and or protection against disease.

The G2 research R.I.P. fragmenting bullet-radiographic features of a recently encountered projectile.

Gunshot wounds (GSWs) are an important cause of disability and death in the USA. Although radiography is limited in its ability to detect bullet types, a projectile introduced during the last decade, the R.I.P. bullet by G2 Research, consists of a base slug connected to 6 to 8 sharp trocars designed to diverge within soft tissue following impact, resulting in what we believe to be a unique imaging appearance that can be confusing to those not familiar with this particular projectile. Furthermore, this bullet is 100% copper, which may allow for safe imaging with magnetic resonance imaging if correctly identified prior to scanning.

Soft-Tissue Tumors of the Hand-Imaging Features.

Imaging studies of the hands and fingers are common, and radiologists are generally comfortable with traumatic and degenerative conditions which arise frequently in daily practice. However, a variety of common and uncommon soft-tissue tumors also occur in the hand, the appropriate diagnosis of which can be a source of confusion for both clinicians and radiologists. These lesions often have overlapping imaging characteristics; however, a structured approach can help provide a focused differential diagnosis and impact further workup and management. We discuss several such tumors, categorizing them as cystic-appearing, noncystic masses along tendons and aponeuroses, adipocytic tumors, vascular lesions, and miscellaneous lesions with imaging features that can aid diagnosis.

Spectrum of Voriconazole-Induced Periostitis With Review of the Differential Diagnosis.

OBJECTIVE: Voriconazole is an antifungal medication used primarily for the treatment of Candida and Aspergillus infections. A fairly newly described side effect of long-term voriconazole use is periostitis. The purpose of this article is to describe the main differential consideration-hypertrophic osteoarthropathy-and other differential diagnoses, including venous stasis, thyroid acropachy, and hypervitaminosis A. CONCLUSION: With knowledge of imaging appearance, clinical manifestations, and outcomes, radiologists can make an accurate diagnosis of voriconazole-induced periostitis, and clinical teams can initiate appropriate management.

Update on Imaging of Knee Arthroplasties: Normal Findings and Hardware Complications.

Total knee arthroplasty (TKA) is the most common joint replacement performed. This article reviews the normal appearance of TKA including the most common types of arthroplasties as well as complications. Common complications at the present time are infection, aseptic loosening, and instability. Rarer complications such as polyethylene wear, periprosthetic fracture, and soft tissue pathology are also discussed. Although the mainstay of imaging is radiographs, newer techniques in TKA imaging such as computed tomography and magnetic resonance imaging are also reviewed.

From tumor to trauma: etiologically deconstructing a unique differential diagnosis of musculoskeletal entities with high signal intensity on T1-weighted MRI.

OBJECTIVE: This article reviews the neoplastic and nonneoplastic abnormalities of the musculoskeletal system that contain high signal intensity on T1-weighted MRI. The physical properties accounting for the increased signal intensity as well as the key clinical and imaging characteristics of each entity are discussed. CONCLUSION: Recognition of high signal intensity within musculoskeletal lesions on T1-weighted MRI can limit the differential diagnosis and can also have important clinical implications.

Infarct-Associated Bone Sarcomas: Multimodality Imaging Findings.

OBJECTIVE: The objective of our study was to characterize infarct-associated bone sarcoma and its imaging features. MATERIALS AND METHODS: Our databases were searched for instances of sarcoma arising in association with osteonecrosis. Demographic and imaging data were recorded. The imaging studies of 258 patients with sarcomas were reviewed to determine whether underlying osteonecrosis was present. Radiographic and MRI studies of patients with bone infarction were reviewed to categorize the various appearances of infarction and to determine if sarcomas tended to arise in a particular pattern. A literature review was performed. RESULTS: Nine infarct-associated bone sarcomas were found in eight patients: seven malignant fibrous histiocytomas (MFHs) and two osteosarcomas. All occurred in the femur or tibia; multifocal infarction was documented in all patients except one. Sarcomas were commonly associated with a so-called "mature"-type pattern of osteonecrosis-that is, with well-defined calcified margins. Osteolysis of infarct-associated MFHs was often overlooked at initial presentation and was often detected only after pathologic fracture. CT and MRI revealed cortical penetration in all cases; infarct margin disruption was evident, but preservation of fat within the infarct was typical. Increased radiotracer activity with relative central photopenia was characteristic of large infarct-associated bone sarcomas on scintigraphy. All lesions, including those treated at our institution and those found in the literature, were metaphyseal or diaphyseal, and although epiphyseal extension of sarcoma from a metadiaphyseal infarct was common, no purely epiphyseal lesions were encountered. CONCLUSION: Radiologists must remain vigilant for this rare occurrence, especially in patients with new pain in an area of known bone infarction.

Imaging Diagnosis of Solitary Tumors of the Phalanges and Metacarpals of the Hand.

OBJECTIVE: Tumors and tumorlike lesions of the tubular bones of the hand, often incidentally discovered lesions, present a unique but challenging differential diagnosis. CONCLUSION: Imaging, including both radiography and cross-sectional imaging, can allow the radiologist to generate a clinically useful differential diagnosis based on the distinguishing features of these entities. Recognition of these lesions is important because clinical management and treatment by orthopedic oncologists vary depending on the diagnosis.

Osteoma of long bone: an expanding spectrum of imaging findings.

Osteoma of long bone is an extremely rare, benign bone-forming surface lesion with the largest published case series consisting of only 14 patients. The most important and often most difficult lesion to differentiate from osteoma of long bone radiographically is parosteal osteosarcoma, which is a rare, low-grade surface osteosarcoma with the potential for dedifferentiation. Reports of imaging studies of osteoma of long bone depict a well-defined ossified mass arising from the surface of the diaphysis or metadiaphysis of a long bone. A characteristic feature is the homogeneity of the mass, with uniform density near or equal to that of cortical bone from the base of the lesion to its periphery. The 45-year-old female in this case presented with left hip fullness and was subsequently found to have a proximal femoral osteoma, which was unique in that it contained large fatty marrow spaces that corresponded to bands of relatively low density on plain radiography and computed tomography, giving it a heterogeneous appearance atypical of osteoma of long bone. Furthermore, the osteoma reported here was associated with a small but separate nodular focus of ossification in the adjacent soft tissue. These findings led to a presumptive diagnosis of parosteal osteosarcoma with a local soft tissue metastasis or satellite nodule resulting in radical resection of the tumor. Definitive diagnosis of osteoma was made on histology of both the parent lesion and ossified nodule as no neoplastic spindle cell proliferation was present to establish a diagnosis of low-grade osteosarcoma. This represents, to the best of our knowledge, the first such presentation of osteoma of long bone.

Narrative Capacity.

What develops in adulthood? More specifically, what develops in adult analysis, not just in terms of thwarted childhood capacities, not just through accrued experience, but even more fundamentally in terms of abilities or structures not possible until the present moment? In this paper, I posit narrative capacity-the capacity to organize conflictual aspects of self and other in a temporary causal-motivational sequence-as a core feature of what develops in the clinical encounter between the analyst and adult patient. It develops, as I demonstrate, through play with narrative fragments, contrasts, and integrations in the analytic field. I present a clinical process note to show how these elements texture and problematize one another. A successful analysis leads not to any one life story but to the more basic ability to weave and unweave our stories.

Cubonavicular Coalition: Magnetic Resonance Imaging Findings and Associated Pathology of a Rarely Reported Condition in 27 Patients.

RATIONALE AND OBJECTIVES: (1) Describe multimodality imaging of cubonavicular coalition (CNC) with magnetic resonance imaging (MRI) focus, (2) evaluate CNC associated foot and ankle pathology, (3) examine clinical presentation/symptoms associated with CNC, (4) record CNC treatment. MATERIALS AND METHODS: Retrospective Institutional Review Board (IRB) approved study. Picture Archiving and Communication System (PACS) databases searched for CNC. Final study population: 34 cases in 27 patients. Each CNC was reviewed for: coalition type (osseous versus non-osseous- cartilaginous versus fibrous), tendon and ligament pathology, bone marrow edema at CNC and adjacent joints, presence and severity of degenerative changes at CNC and adjacent joints, fractures, additional coalitions, laterality, and pes planus. MRI planes and radiographic views on which coalitions were best identified were recorded. Each CNC EMR was reviewed for: symptoms, trauma, management, patient demographics. Inter-reader reliability was performed for type of non-osseous coalition. RESULTS: Final cohort included 34 cases in 27 patients (average age: 34.7, range: 10-76; 71% female). No CNC was completely osseous. On MRI, 89.5% of coalitions were non-osseous and 5.3% were partially osseous. 76.5% of patients had referable symptoms including pain, limited motion, inability to bear weight. 23.5% of patients were surgically managed/pathologically proven. On MRI, 36.8% of patients had tendon pathology, 52.6% had ligamentous pathology, 100% had bone marrow edema-like signal abnormality about the CNC, and 88.2% had CNC degenerative changes. There was bone marrow edema-like signal abnormality at bones adjacent to the CNC in 52.6% and adjacent joint degenerative disease present in 50%. CNC was best identified on oblique radiographs and axial MRI. Inter-reader reliability for non-osseous coalition type was poor, Cronbach's alpha 0.554. CONCLUSION: CNC is subtle and findings of osteoarthritis or bone marrow edema-like about the cubonavicular articulation should raise suspicion for underlying coalition.

Musculoskeletal manifestations of neurofibromatosis type 1.

OBJECTIVE: We will describe and illustrate various musculoskeletal manifestations of neurofibromatosis type 1 (NF1) encountered on imaging studies. CONCLUSION: Because NF1 is one of the most common genetic disorders, radiologists should be familiar with its imaging manifestations.

Contaminated heparin associated with adverse clinical events and activation of the contact system.

BACKGROUND: There is an urgent need to determine whether oversulfated chondroitin sulfate (OSCS), a compound contaminating heparin supplies worldwide, is the cause of the severe anaphylactoid reactions that have occurred after intravenous heparin administration in the United States and Germany. METHODS: Heparin procured from the Food and Drug Administration, consisting of suspect lots of heparin associated with the clinical events as well as control lots of heparin, were screened in a blinded fashion both for the presence of OSCS and for any biologic activity that could potentially link the contaminant to the observed clinical adverse events. In vitro assays for the activation of the contact system and the complement cascade were performed. In addition, the ability of OSCS to recapitulate key clinical manifestations in vivo was tested in swine. RESULTS: The OSCS found in contaminated lots of unfractionated heparin, as well as a synthetically generated OSCS reference standard, directly activated the kinin-kallikrein pathway in human plasma, which can lead to the generation of bradykinin, a potent vasoactive mediator. In addition, OSCS induced generation of C3a and C5a, potent anaphylatoxins derived from complement proteins. Activation of these two pathways was unexpectedly linked and dependent on fluid-phase activation of factor XII. Screening of plasma samples from various species indicated that swine and humans are sensitive to the effects of OSCS in a similar manner. OSCS-containing heparin and synthetically derived OSCS induced hypotension associated with kallikrein activation when administered by intravenous infusion in swine. CONCLUSIONS: Our results provide a scientific rationale for a potential biologic link between the presence of OSCS in suspect lots of heparin and the observed clinical adverse events. An assay to assess the amidolytic activity of kallikrein can supplement analytic tests to protect the heparin supply chain by screening for OSCS and other highly sulfated polysaccharide contaminants of heparin that can activate the contact system.

Bronchoalveolar lavage fluid review in acute promyelocytic leukemia differentiation syndrome.

The patient was a 62-year-old Caucasian man with blood smear and flow cytometry concerning for acute promyelocytic leukemia with FISH ultimately confirming PML-RARA translocation. He had a 30-year history of employment at a nuclear power plant. He presented with diffuse intravascular coagulation, hyperleukocytosis, and quickly developed acute respiratory distress syndrome. On day four of ATRA + Hydrea, a bronchoalveolar lavage was performed and was non-bloody. On microscopic fluid review, abnormal immature cells with bilobed nuclear contours were identified, similar in morphology to those seen on the diagnostic blood smear review, amidst background alveolar-type macrophages. Subsequent flow cytometric analysis showed these cells to be abnormal promyelocytes; however, they differed from the blood flow cytometry study performed prior to initiation of ATRA by showing maturational immunophenotypic changes. While the leukemic promyelocytes on bronchoalveolar lavage were morphologically immature, these immunophenotypic changes somewhat recapitulated those seen in normal granulocyte maturation and were thus suggestive of so-called differentiation syndrome. Unfortunately, the patient passed away during induction chemotherapy due to complications from diffuse intravascular coagulation and differentiation syndrome. Important pathobiological information can be gathered from fluid review and concomitant flow cytometric analysis.

Delayed fractional dosing with RTS,S/AS01 improves humoral immunity to malaria via a balance of polyfunctional NANP6- and Pf16-specific antibodies.

BACKGROUND: Malaria remains a key cause of mortality in low-income countries. RTS,S/AS01 is currently the most advanced malaria vaccine, demonstrating ∼50% efficacy in controlled human malaria infection (CHMI) studies in malaria-naive adults and ∼30%-40% efficacy in field trials in African infants and children. However, a higher vaccine efficacy is desirable. METHODS: Modification of the vaccine regimen in a CHMI trial in malaria-naive individuals resulted in significant increase in protection. While three equal monthly RTS,S/AS01 doses (RRR) were used originally, the administration of a delayed third dose with 20% of the original antigen dose (RRr) resulted in ∼87% protection, linked to enhanced antibody affinity maturation. Here, we sought to identify a novel molecular basis for this higher protective efficacy using Systems Serology. FINDINGS: We demonstrate that the delayed fractional dose maintains monocyte phagocytosis and NK activation mediated by NANP6-specific antibodies, key correlates of protection for the RRR regimen. However, it is also marked by a higher breadth of C-term Fc effector functions, including enhanced phagocytosis. The RRr regimen breaches immunodominance of the humoral immune response, inducing a balanced response across the C-terminal (Pf16) and NANP region of CSP, both of which were linked to protection. CONCLUSIONS: Collectively, these data point to an unexpectedly concordant evolution in Fab avidity and expanded C-term Fc effector functions, providing novel insights into the basis for higher protection conferred by the delayed fractional dose in malaria-naive individuals. FUNDING: This research was supported by PATH's Malaria Vaccine Initiative and the MGH Research Scholars program.