Search

Melanomas that have histopathologic features that overlap with those of Spitz nevus are referred to as spitzoid melanomas. However, the diagnostic concept is used inconsistently and genomic analyses suggest it is a heterogeneous category. Spitz tumors, the spectrum of melanocytic neoplasms extending from Spitz nevi to their malignant counterpart Spitz melanoma, are defined in the 2018 WHO classification of skin tumors by the presence of specific genetic alterations, such as kinase fusions or HRAS mutations. It is unclear what fraction of "spitzoid melanomas" defined solely by their histopathologic features belong to the category of Spitz melanoma or to other melanoma subtypes. We assembled a cohort of 25 spitzoid melanomas diagnosed at a single institution over an 8-year period and performed high-coverage DNA sequencing of 480 cancer related genes. Transcriptome wide RNA sequencing was performed for select cases. Only nine cases (36%) had genetic alterations characteristic of Spitz melanoma, including HRAS mutation or fusion involving BRAF, ALK, NTRK1, or MAP3K8. The remaining cases were divided into those with an MAPK activating mutation and those without an MAPK activating mutation. Both Spitz melanoma and spitzoid melanomas in which an MAPK-activating mutation could not be identified tended to occur in younger patients on skin with little solar elastosis, infrequently harbored TERT promoter mutations, and had a lower burden of pathogenic mutations than spitzoid melanomas with non-Spitz MAPK-activating mutations. The MAPK-activating mutations identified affected non-V600 residues of BRAF as well as NRAS, MAP2K1/2, NF1, and KIT, while BRAF V600 mutations, the most common mutations in melanomas of the WHO low-CSD category, were entirely absent. While the "spitzoid melanomas" comprising our cohort were enriched for bona fide Spitz melanomas, the majority of melanomas fell outside of the genetically defined category of Spitz melanomas, indicating that histomorphology is an unreliable predictor of Spitz lineage.

Spectral characteristics of fluorescent proteins (FPs) are well studied, and through protein engineering, several FP variants constituting entire visible spectrum have been created. One of the most common mechanisms attributed to spectral shifts in FP is excited state proton transfer (ESPT), hydroxyl moiety protonation and deprotonation, along with chromophore cis-trans isomerism. The most widely studied FPs are those derived from avGFP ( Aequorea victoria GFP) and Dsred (Discosoma coral). Apart from the above mechanism, certain interacting residues are said to play a vital role in altering the proton transfer pathway leading to numerous spectral variants. Similarly, the hydrogen-bonded networks solely cannot dictate the energy landscape of FPs. Non-bonded interactions also can create secondary harmonic shifts by dipole-dipole inductions. Side chain contacts tend to alter the topological and torsional geometry, thereby disturbing the chromophore's planarity. Side chain torsional variations have almost been unaccounted for their distortions in FPs. We hypothesize the torsional landscape and altered residual interactions as prominent factors for the spectral shifts. Through our 200 ns molecular dynamics investigation, we prospect that van der Waals packing in Dsred is more compact than that of avGFP, thus creating a low solvent occupiable environment and reduced solvent interactions having higher red spectral shift. The torsional changes of wild avGFP, S65T avGFP and Dsred have been studied to comprehend the inter-residual contact distance and the geometrical descriptors. Communicated by Ramaswamy H. Sarma.

Alveolar rhabdomyosarcoma (RMS) is associated with an underlying pathogenic translocation involving either PAX3 or PAX7 and FOXO1. The presence or absence of this fusion defines the biology and clinical behavior of this subtype of RMS and its identification in tumors is relevant to prognostication and treatment planning. To further explore the unique characteristics of fusion-driven RMS, we leveraged a published gene expression data set to perform an unbiased comparison of 33 fusion-positive and 25 fusion-negative RMS cases. Our analyses revealed 1790 expressed loci with more than two-fold differential expression at a threshold of P < .05. Genes with increased expression in fusion-positive relative to fusion-negative RMS were significantly enriched for those involved in "nervous system development," "neuron differentiation," and "neurogenesis," highlighting a neurodevelopmental gene expression signature driven by the alveolar RMS-associated fusion protein. We show that neurodevelopmental genes are enriched near PAX3-FOXO1 fusion protein binding sites, suggesting a genome-wide fusion protein-mediated activation of cis regulatory elements. Among the genes with differential expression in fusion-positive versus fusion-negative RMS, we identified expression of the transcriptional regulator of motor neuron and oligodendrocyte development, OLIG2, as a marker of the fusion protein-dependent neurodevelopmental signature. Immunohistochemical analysis of a cohort of 73 RMS specimens revealed OLIG2 expression in 96.4% of fusion-positive RMS (N = 27/28), but only in 6.7% of fusion-negative RMS (N = 3/45; P < .001). The proportion of OLIG2-expressing cells in fusion-negative cases did not exceed 5%, while 92.9% of fusion-positive cases showed expression in at least 5% of cells. Our findings identify OLIG2 expression as a unique manifestation of a neurodevelopmental gene expression signature driven by the oncogenic fusion protein characteristic of alveolar RMS, which may aid in the diagnostic and prognostic distinction of fusion-positive cases., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Acrylic acid (AA) is a widely used commodity chemical derived from non-renewable fossil fuel sources. Alternative microbial-based production methodologies are being developed with the aim of providing "green" acrylic acid. These initiatives will benefit from component sensing tools that facilitate rapid and easy detection of in vivo AA production., Results: We developed a novel transcriptional sensor facilitating in vivo detection of acrylic acid (AA). RNAseq analysis of Escherichia coli exposed to sub-lethal doses of acrylic acid identified a selectively responsive promoter (P yhcN ) that was cloned upstream of the eGFP gene. In the presence of AA, eGFP expression in E. coli cells harbouring the sensing construct was readily observable by fluorescence read-out. Low concentrations of AA (500 μM) could be detected whilst the closely related lactic and 3-hydroxy propionic acids failed to activate the sensor. We further used the developed AA-biosensor for in vivo FACS-based screening and identification of amidase mutants with improved catalytic properties for deamination of acrylamide to acrylic acid., Conclusions: The transcriptional AA sensor developed in this study will benefit strain, enzyme and pathway engineering initiatives targeting the efficient formation of bio-acrylic acid.

The cultural and ethnic landscape of North America is becoming increasingly diverse, with many refugees fleeing torture and persecution and seeking safety in the United States and Canada. In working with this population, clinicians must implement culturally appropriate methods of assessing and treating individuals from diverse backgrounds. Culture can exert a powerful and often misunderstood influence on psychological assessment, and the critical challenge is to account for both subjective experience of the client and the objective symptoms or behaviors present. The present review explores the literature on cross-cultural issues in the assessment of survivors of torture. I summarize best practices and review the theoretical and statistical bases for establishing the equivalence of constructs across cultures. Discussion centers around the utility of a cross-culturally valid measure of distress, and it is hoped that this review will encourage collaboration between clinicians and psychometricians to develop assessments for use with this vulnerable population.

The present study aims to fill a gap in the study of resilience to trauma by examining resilience in a culturally diverse population. Approximately 70% of adults across the globe experience at least one traumatic event in their lifetime, yet resilience is a common response trajectory. This pilot study explored reactions to trauma and psychological resilience in an international sample of trauma-exposed participants. Participants were recruited online using the Amazon Mechanical Turk software and after completing an informed consent, were determined eligible to participate if they endorsed experiencing at least one traumatic event. Eligible participants then completed The Stressful Life Events Questionnaire, Brief Resiliency Scale, Ego Resiliency Scale, Posttraumatic Stress Disorder Checklist-Civilian Version, Brief Religious Coping Scale, and Multigroup Ethnic Identity Measure. The final sample included 200 trauma exposed adults from nineteen different countries worldwide, with a majority hailing from the United States, India, Sri Lanka and the Philippines. Results revealed that Asian participants scored significantly higher on resilience scales and endorsed higher levels of spiritually focused coping than other subgroups. Multivariate analyses revealed that these differences in resilience remained significant even after controlling for sense of ethnic identity and spiritual coping, suggesting that there may be culturally specific predictors of resilience within the Asian subgroup. Understanding variations in resilience will aid in developing culturally tailored interventions and pursuing a strengths-based approach to recovery from trauma. Limitations and implications are discussed.

Introduction: Mycosis fungoides (MF) with large cell transformation (LCT) is an advanced stage of cutaneous lymphoma with a poor prognosis. Identification of LCT is critical and especially challenging when the number of large abnormal lymphocytes is near but below 25%. We propose that Ki-67 and p53 may be useful in making this diagnosis., Methods: We identified 17 patients with advanced stage (T3 or T4) MF without LCT and 38 patients with a biopsy-confirmed new diagnosis of MF with LCT treated at our institution's cutaneous lymphoma clinic from 2012 to 2016. Seventeen patients underwent 22 biopsies with advanced stage MF (control), and 38 patients with 46 biopsies of MF with LCT were included in this study., Results: The MF cohort had an average CD30 expression of 4%, while the MF-LCT cohort had an average CD30 expression of 22% (P < 0.05). The MF cohort had an average Ki-67 staining of 13%, while the MF-LCT group had an average Ki-67 staining of 57% (P < 0.05). Forty-seven percent of the MF-LCT group was positive for p53; on the other hand, none of the MF control group showed increased p53 expression (P < 0.05)., Discussion: While CD30 shows some value in delineating large cell transformation, Ki-67 and p53 appear to be useful immunohistochemical markers in the diagnosis of LCT., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Optimum dose, route and duration of use of prophylactic magnesium sulphate in women with severe pre-eclampsia is still controversial. We compared the efficacy and safety of 'low-dose Dhaka' regime with 'Loading dose only' regime for seizure prophylaxis in severe preeclampsia using a randomised controlled trial in 402 women. The incidence of eclampsia in the 'low-dose Dhaka' regime group was 1.49% and that in the 'Loading dose only regime' was 2.98% (p = .321). In the low-dose Dhaka regime, injection site abscess and respiratory depression occurred in one woman each. Neonatal outcomes such as Apgar score at 5 minutes (5.0% vs. 8.05% p = .251) and perinatal mortality (20.4% vs. 21.9%, p = .724) were similar in both groups. Loading dose only regime may be considered an effective alternative regime for the prevention of eclampsia in women with severe preeclampsia. Impact statement What is already known on this subject: Efficacy of therapeutic short regime magnesium sulphate in eclampsia has already been reported. Data regarding prophylactic short regime in women with preeclampsia is sparse. What the results of this study add: We have shown that short regime of magnesium sulphate using only the loading dose in the prevention of seizure in preeclampsia is an effective alternative to the low-dose Dhaka regime. What the implications are of these findings for clinical practice and/or further research: The short regime is less resource-intensive. Further larger studies are needed to confirm the efficacy of this short regime and to establish its cost-effectiveness.

Mycosis fungoides is the most common and therefore quintessential cutaneous lymphoma and is typically characterized by an epidermotropic infiltrate of atypical monoclonal CD4+ lymphocytes. Classical histopathologic findings include epidermotropism, lymphocytes with convoluted nuclear contours and surrounding perinuclear "halos," and papillary dermal fibrosis. Atypical lymphocytes may occasionally form Pautrier's microabscesses with tagging of lymphocytes along the basal keratinocytes. Unfortunately, a variety of benign inflammatory infiltrates, as well as other cutaneous lymphomas, may demonstrate some similar histopathologic findings. Herein, we review the wide array of epidermotropic T-cell lymphomas and discuss distinguishing features between these entities. We also offer an algorithmic approach utilizing histopathologic, immunophenotypic, and molecular techniques that can be used for analyzing an epidermotropic T-cell infiltrate in order to render a specific diagnosis., (©2018 Frontline Medical Communications.)

The United States accepts more refugees than any other industrialized nation. As refugee populations grow, mental health professionals must implement culturally and ethnically appropriate strategies to assess and treat individuals from diverse backgrounds. Culture can exert a powerful and often misunderstood influence on psychological assessment, and few structured measures have been demonstrated to have adequate cross-cultural validity for use with diverse and vulnerable populations such as survivors of torture. This study examined the factor structure and equivalency of underlying construct(s) of psychological distress as measured by the Brief Symptom Inventory (BSI) in three samples who had survived torture and other severe trauma from Tibet, West Africa and the Punjab region of India. Confirmatory factor analyses provided support for configural invariance of a two-factor model across the three samples, suggesting that the two latent factors of Complex Dysphoria and Somatic Distress were present in each subgroup. The data provide additional support for the strict invariance model in the West African-Tibetan dyad suggesting that scores are comparable across those two groups. Implications for research and treatment are discussed.

Background: Lignin is a potential biorefinery feedstock for the production of value-added chemicals including vanillin. A huge amount of lignin is produced as a by-product of the paper industry, while cellulosic components of plant biomass are utilized for the production of paper pulp. In spite of vast potential, lignin remains the least exploited component of plant biomass due to its extremely complex and heterogenous structure. Several enzymes have been reported to have lignin-degrading properties and could be potentially used in lignin biorefining if their catalytic properties could be improved by enzyme engineering. The much needed improvement of lignin-degrading enzymes by high-throughput selection techniques such as directed evolution is currently limited, as robust methods for detecting the conversion of lignin to desired small molecules are not available., Results: We identified a vanillin-inducible promoter by RNAseq analysis of Escherichia coli cells treated with a sublethal dose of vanillin and developed a genetically programmed vanillin-sensing cell by placing the 'very green fluorescent protein' gene under the control of this promoter. Fluorescence of the biosensing cell is enhanced significantly when grown in the presence of vanillin and is readily visualized by fluorescence microscopy. The use of fluorescence-activated cell sorting analysis further enhances the sensitivity, enabling dose-dependent detection of as low as 200 µM vanillin. The biosensor is highly specific to vanillin and no major response is elicited by the presence of lignin, lignin model compound, DMSO, vanillin analogues or non-specific toxic chemicals., Conclusions: We developed an engineered E. coli cell that can detect vanillin at a concentration as low as 200 µM. The vanillin-sensing cell did not show cross-reactivity towards lignin or major lignin degradation products including vanillin analogues. This engineered E. coli cell could potentially be used as a host cell for screening lignin-degrading enzymes that can convert lignin to vanillin.

Background: Hypertension in the postpartum period is a common phenomenon and is often a cause for concern. Following delivery, fluid that has been sequestered in the extravascular space is mobilized, producing a large auto-infusion of fluid from the extravascular to the intravascular compartment. As a result of this fluid mobilization process, there is an increase in central venous pressure and pulmonary capillary wedge pressure, which might favor the development of pulmonary edema. Thus, diuretics logically might be a better choice as antihypertensive medication in such a scenario., Methods: A total of 108 antenatal women diagnosed with having severe preeclampsia, with two high blood pressure recordings of ≥150/100 mm of Hg in the postpartum period within the first 24 hours of delivery, were enrolled in the study. These patients were randomly divided into two groups (Group A: furosemide 20 mg OD + nifedipine & Group B: nifedipine alone). Main outcome measures studied were reduction in systolic, diastolic, and mean arterial blood pressures, requirement of additional antihypertensive drugs to control blood pressure, duration of hospital stay, and antihypertensive requirement at discharge., Results: Both groups were comparable for distribution of age and parity and presence of imminent symptoms and eclampsia. There was no significant difference in mean systolic, diastolic, and mean arterial pressures (MAPs) between both the groups at admission. Mean gestational age at delivery was 36 weeks in both the groups. Requirement of additional antihypertensive was significantly higher in women in group B (26.0% vs. 8.0%, p = 0.017). Duration of hospital and postpartum stay and the use of antihypertensive at discharge were similar in both the groups Conclusion: In conclusion, the use of a short course of furosemide along with nifedipine significantly reduces the need of additional antihypertensive in severe preeclamptic women with postpartum hypertension when compared to women who received nifedipine alone.

Background: Multicomponent volar wrist lacerations of "spaghetti wrist" injuries are devastating injuries of the upper extremity. These patients require long-term commitment to rehabilitation. Patients presenting to our county hospital represent a unique and complex patient population in terms of psychosocial considerations. We aimed to identify obstacles to care and optimal recovery in this patient population., Methods: A patient database was queried for ICD-9 codes related to major upper extremity nerve injuries, which were treated by plastic surgery faculty at San Francisco General Hospital from 2008 to 2014. A retrospective chart review was performed to identify patients with spaghetti wrist injuries. Charts were reviewed for patient demographics including age, occupation, handedness, psychiatric illness, isolated versus polytrauma, and employment status. Injuries were categorized for mechanism of injury, structures involved, and timing and method of surgical treatment. Outcomes were assessed for motor recovery, sensory recovery, and tendon function., Results: We identified 18 patients with multicomponent volar wrist lacerations. Average patient age was 31 years. The most common mechanism of injury was accidental/work-related (n = 9, 50%), followed by self-inflicted (n = 4, 22%). Thirty-nine percent (n = 7) of patients had a psychiatric diagnosis, most commonly depression (n = 4, 22%). Eighty-nine percent (n = 16) of patients had an isolated injury to the upper extremity, and 39% (n = 7) had an injury to the dominant hand. Fifty percent (n = 9) of patients were lost to follow-up, with 28% (n = 5) having no known care plan. Motor, sensory, and tendon function outcomes for those with adequate follow-up were comparable to previously published studies., Discussion: Multicomponent volar wrist lacerations can be devastating, and although we are able to provide patients with appropriate timely surgical care, these patients require long-term care far beyond the operating room for optimal outcomes. Psychiatric illness, socioeconomic limitations, poor patient compliance, and irregular follow-up are obstacles to care. These issues highlight the need for better social support systems and mental health care to provide access to the services necessary to optimize recovery.

Gluteal abscess commonly follows intramuscular injections with contaminated needles. Carcinoma cecum is known to present with pericolic abscess due to microperforations and may rupture intraperitoneally. Gluteal abscess secondary to perforated carcinoma cecum with pericolic abscess is extremely uncommon. A 50-year-old woman who was receiving intramuscular iron injections for anemia presented with a 10×10-cm abscess in the right gluteal region and a vague mass in the right iliac fossa. After investigations, a diagnosis of perforated carcinoma cecum with pericolic abscess tracking into the right gluteal region was made, and incision and drainage were done. Fine-needle aspiration cytology from the cecal growth revealed adenocarcinoma. Unfortunately, the patient was not willing to undergo definitive treatment. This case is being reported for its rarity and as an uncommon etiology for a common condition.

Background: In mutilating hand injuries, tissue engineered tendon grafts may provide a reconstructive solution. We have previously described a method to decellularize cadaveric human flexor tendons while preserving mechanical properties and biocompatibility. The purpose of this study is to evaluate the immunogenicity and strength of these grafts when implanted into an immunocompetent rat model., Methods: Cadaveric human flexor tendons were divided into two groups. Group 1 was untreated, and Group 2 was decellularized by treatment with sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), and peracetic acid (PAA). Both groups were then analyzed for the presence of major histocompatibility complexes by immunohistochemistry (IHC). Pair-matched tendons from each group were then placed into the dorsal subcutaneous tissue and anchored to the spinal ligaments of Wistar rats for 2 or 4 weeks, and harvested. The infiltration of B-cells and macrophages was determined using IHC. The explants where then subjected to mechanical testing to determine the ultimate tensile stress (UTS) and elastic modulus (EM). Statistical analysis was performed using a paired Student's t-test., Results: The decellularization protocol successfully removed cells and MHC-1 complexes. At 2 weeks after implantation, there was increased infiltration of B-cells in Group 1 (untreated) compared with Group 2 (acellular), both in the capsule and tendon substance. There was improved ultimate tensile stress (UTS, 42.7 ± 8.3 vs. 22.8 ± 7.8 MPa, p<0.05) and EM (830.2 ± 206.7 vs. 421.2 ± 171.3 MPa, p<0.05) in tendons that were decellularized. At 4 weeks, there was continued B-cell infiltration in Group 1 (untreated) compared with Group 2 (acellular). There was no appreciable difference in macrophage infiltration at both time points. At 4 weeks Group 2 (acellular) demonstrated persistently greater UTS (40.5 ± 9.1 vs. 14.6 ± 4.2 MPa, p<0.05) and EM (454.05 ± 101.5 vs. 204.6 ± 91.3 MPa, p<0.05) compared with Group 1 (untreated)., Conclusions: Human flexor tendons that were decellularized with SDS, EDTA, and PAA resulted in removal of cellular antigens and a decreased immune response when placed into Wistar rats. These grafts showed better mechanical properties at 2 and 4 weeks when compared with control tendons. Decellularization is an important step toward the use of tissue engineered flexor tendons in upper extremity reconstruction.

Background: Tissue-engineered flexor tendon grafts may allow reconstruction of severe tendon losses. One critical factor is the optimization of cell proliferation and reseeding. Use of growth factors--basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF)-1, and platelet-derived growth factor (PDGF)-BB--may improve culture conditions for human fibroblasts, tenocytes, and adipose-derived stem cells and increase repopulation of a tendon scaffold., Methods: All cell types were plated at a density of 10,000 cells per well and cultured in F12 media supplemented with varying concentrations of bFGF, IGF-1, and PDGF-BB. After 72 hours, cell proliferation was determined using the CellTiter assay. Human flexor tendon segments were acellularized and reseeded in a cell suspension of 5 × 10(5) cells/ml. After 5 days, tendon repopulation was determined using the MTS assay and histology. Statistical significance was determined with analysis of variance and a t test., Results: For all cell types, there was enhanced proliferation with growth factors. Among single growth factors, PDGF-BB at 50 ng/ml was the most efficient stimulator of proliferation. With multiple growth factors, the optimal concentration was determined to be 5 ng/ml bFGF, 50 ng/ml IGF-1, and 50 ng/ml PDGF-BB (increase when compared with control: fibroblasts, 2.92-fold; tenocytes, 2.3-fold; and adipose-derived stem cells, 2.4-fold; p < 0.05). Tendons reseeded with this optimal combination of growth factors showed improved reseeding compared with the control group (fibroblasts, 2.01-fold; tenocytes, 1.78-fold; and adipose-derived stem cells, 1.76-fold; p < 0.05)., Conclusions: bFGF, IGF-1, and PDGF-BB can be used to improve cellular proliferation and repopulation of an acellularized scaffold. The use of growth factors may be an important step in the tissue engineering of human flexor tendons.

Human tendon tissue engineering attempts to address the shortage of autologous tendon material arising from mutilating injuries and diseases of the hand and forearm. It is important to maximize the tissue-engineered construct's (TEC's) biomechanical properties to ensure that the construct is in its strongest possible state before reimplantation. In this study, we sought to determine the bioreactor treatment parameters that affect these properties. Using small- and large-chamber three-dimensional-construct bioreactors (SCB and LCB, respectively), we applied cyclic axial load to TECs comprising reseeded human flexor and extensor tendons of the hand. First, small-sample pilot studies using the LCB were performed on matched-paired full-length flexor tendons to establish proof of concept. Next, large-sample studies using the SCB were performed on matched-paired extensor tendon segments to determine how reseeding, load duty cycle, load magnitude, conditioning duration, and testing delay affected ultimate tensile stress (UTS) and elastic modulus (EM). We found that compared with reseeded matched-paired controls under dynamic-loading at 1.25 N per TEC for 5 days, (1) acellular TECs had lower UTS (p=0.04) and EM (p<0.01), (2) unloaded TECs had lower UTS (p=0.01) and EM (p=0.02), (3) static-loaded TECs had lower UTS (p=0.01) and EM (p<0.01), (4) TECs conditioned for 3 days had lower UTS (p=0.03) and EM (p=0.04), and (5) TECs conditioned for 8 days had higher UTS (p=0.04) and EM (p=0.01). However, TECs conditioned at higher loads (2.5 N per TEC) and lower loads (0.625 N per TEC) possessed similar UTS (p=0.83 and p=0.89, respectively) and EM (p=0.48 and p=0.89, respectively) as controls stimulated with 1.25 N per TEC. After cycle completion, there is attrition of UTS (p=0.03) and EM (p=0.04) over a 2-day period. Our study showed that the material properties of human allograft TECs can be enhanced by reseeding and dynamic-conditioning. While conditioning duration has a significant effect on material properties, the load magnitude does not. The issue of attrition in biomechanical properties with time following cycle completion must be addressed before bioreactor preconditioning can be successfully introduced as a step in the processing of these constructs for clinical application., (© Mary Ann Liebert, Inc.)

Objective: New training paradigms in vascular surgery allow for early specialization out of medical school. Surgical simulation has emerged as an educational tool for trainees to practice procedures in a controlled environment allowing interested medical students to perform procedures without compromising patient safety. The purpose of this study is to assess the ability of a simulation-based curriculum to improve the technical performance and interest level of medical students in vascular surgery., Design: Prospective observational cohort study of medical student performance., Setting: Academic medical center., Participants: Forty-one medical students (23 first year, 15 second year, 3 other) enrolled in a vascular surgery elective course. Students completed a survey of their interests and performed a renal stent procedure on an endovascular simulator (pretest). The curriculum consisted of didactic teaching and weekly mentored simulator sessions and concluded with a final renal stent procedure on the simulator (posttest). Objective procedural measures were determined during the pre- and posttest by the simulator, and subjective performance was graded by expert observers utilizing a structured global assessment scale. After the course, the students were surveyed as to their opinions about vascular surgery as a career option. Finally, 1 year after the course, all students were again surveyed to determine continued interest in vascular surgery., Results: The objective and subjective criteria measured on the simulator and structured global assessment scale significantly improved from pre- to posttest in terms of performer technical skill, patient safety measures, and structured global assessments. Before beginning the course, 8.5% of the students expressed high interest in vascular surgery, and after completing the course 70% were seriously considering vascular surgery as a career option (p = 0.0001). More than 95% of the students responded that endovascular simulation increased their knowledge and interest in vascular surgery. In the 1-year follow-up survey (n = 23 medical students), 35% had already entered their clinical years. Seventy percent of the students were still considering vascular surgery, while several other career options were still popular including the surgical subspecialties (70%), interventional cardiology (57%), and interventional radiology (48%). Most respondents indicated the major reasons for continued interest in vascular surgery were the ability to practice endovascular procedures on the simulator (100%) and mentorship from vascular surgery faculty (78%)., Conclusions: The use of high fidelity endovascular simulation within an introductory vascular surgery course improves medical student performance with respect to technical skill, patient safety parameters, and global performance assessment. Mentored exposure to endovascular procedures on the simulator positively impacts long term medical student attitudes towards vascular surgery. Simulator-based courses may have the potential to be an important component in the assessment and recruitment of medical students for future surgical training programs.

Tay-Sachs disease (TSD) is a progressive neurodegenerative disorder due to an autosomal recessively inherited deficiency of beta-hexosaminidase A (Hex A). Deficiency of Hex A in TSD is caused by a defect of the alpha-subunit resulting from mutations of the HEXA gene. To date, there is no effective treatment for TSD. Animal models of genetic diseases, similar to those known to exist in humans, are valuable and essential research tools for the study of potentially effective therapies. However, there is no ideal animal model of TSD available for use in therapeutic trials. In the present study, we report an animal model (American flamingo; Phoenicopterus ruber) of TSD with Hex A deficiency occurring spontaneously in nature, with accumulation of G(M2)-ganglioside, deficiency of Hex A enzymatic activity, and a homozygous P469L mutation in exon 12 of the hexa gene. In addition, we have isolated the full-length cDNA sequence of the flamingo, which consists of 1581 nucleotides encoding a protein of 527 amino acids. Its coding sequence indicates approximately 71% identity at the nucleotide level and about 72.5% identity at the amino acid level with the encoding region of the human HEXA gene. This animal model, with many of the same features as TSD in humans, could represent a valuable resource for investigating therapy of TSD.

Poly(orthoester) (POE) microspheres have been previously shown to possess certain advantages for the in vivo delivery of DNA vaccines. In particular, timing of DNA release from POE microspheres in response to acidic phagosomal pH was shown to be an important factor in determining immunogenicity, which was hypothesized to be linked to the natural progression of antigen-presenting cell uptake, transfection, maturation, and antigen presentation. Here we report in vitro characterization of the enhanced efficacy of POE microspheres by blending poly(ethylenimine) (PEI), a well-characterized cationic transfection agent, into the POE matrix. Blending of a tiny amount of PEI (approximately 0.04 wt%) with POE caused large alterations in POE microsphere properties. PEI provided greater control over the rate of pH-triggered DNA release by doubling the total release time of plasmid DNA and enhanced gene transfection efficiency of the microspheres up to 50-fold without any significant cytotoxicity. Confocal microscopy results of labeled PEI and DNA plasmids revealed that PEI caused a surface-localizing distribution of DNA and PEI within the POE microsphere as well as focal co-localization of PEI with DNA. We provide evidence that upon degradation, the microspheres of POE-PEI blends released electrostatic complexes of DNA and PEI, which are responsible for the enhanced gene transfection. Furthermore, blending PEI into the POE microsphere induced 50-60% greater phenotypic maturation and activation of bone marrow-derived dendritic cells in vitro, judged by the up-regulation of co-stimulatory markers on the cell surface. Physically blending PEI with POE is a simple approach for modulating the properties of biodegradable microspheres in terms of gene transfection efficiency and DNA release kinetics. Combined with the ability to induce maturation of antigen-presenting cells, POE-PEI blended microspheres may be excellent carriers for DNA vaccines.

Valve repair in children is technically demanding but more desirable than valve replacement. From April 2004 to September 2005, 1 boy and 8 girls with rheumatic heart disease, aged 2-13 years (median, 9 years), underwent valve repair for isolated mitral regurgitation in 5, combined mitral and aortic regurgitation in 2, mitral stenosis in 1, and mitral regurgitation associated with atrial septal defect in 1. Chordal shortening in 7, annular plication in 6, commissurotomy in 1, reconstruction of commissural leaflets in 7 were performed for mitral valve disease. Plication and reattachment of the aortic cusps was carried out in 2 patients. Annuloplasty rings were not used. All patients survived the operation, 8 had trivial or mild residual mitral regurgitation, and 1 had trivial aortic regurgitation. Mean left atrial pressure decreased from 14 to 7 mm Hg postoperatively. During follow-up of 3-18 months, all children were asymptomatic and enjoyed normal activity. None required reoperation. In addition to chordal shortening and annular plication, reconstruction of the commissural leaflets is considered the most important aspect of valve repair. It can be achieved without annuloplasty rings, giving good early and midterm results.

A systemic-pulmonary artery shunt in neonates with decreased pulmonary blood flow is technically demanding. We describe our surgical technique, postoperative management, and results in 19 neonates who underwent a modified Blalock-Taussig shunt between April 2003 and March 2006. Prostaglandin infusion was required in 8 patients who were critically cyanosed, and 5 were on inotropic support preoperatively. A 3.5 or 4.0-mm polytetrafluoroethylene graft was anastomosed with 8/0 polypropylene suture. Postoperatively, systemic pressure was kept slightly higher than normal, and heparin was started early. One patient required revision of the shunt, and one was reexplored for bleeding. There were 2 hospital deaths (mortality, 11%) in patients with preoperative hemodynamic instability. The mean follow-up period was 12 months, with no late postoperative shunt blockage or death. Meticulous surgical technique and judicious use of heparin and inotropic agents improved the outcome and reduced the incidence of shunt blockage and reexploration for bleeding.

Objective: To assess changes in metabolic parameters and body composition among 422 antiretroviral-naive patients randomized to 3 antiretroviral therapy (ART) strategies: protease inhibitor (PI; n = 141)-, nonnucleoside reverse transcriptase inhibitor (NNRTI; n = 141)-, or PI + NNRTI (n = 140)-based strategies with a median follow-up of 5 years., Methods: At baseline and 1-month (metabolic parameters only) and 4-month follow-up intervals, fat-free mass (FFM) and total body fat were calculated, anthropometric measurements were performed, and fasting metabolic parameters were obtained. Rates of change and mean change were compared., Results: The PI + NNRTI strategy resulted in greater increases in triglycerides and low-density lipoprotein cholesterol compared with the PI and the NNRTI strategies (P < 0.005), with no differences between the PI and NNRTI strategies. High-density lipoprotein cholesterol increased significantly more in the NNRTI strategy than in the PI strategy (P < 0.005). Insulin and insulin resistance increased similarly with all 3 strategies. Changes in total and regional body composition (loss of subcutaneous tissue area and gains in FFM, nonsubcutaneous tissue area, and visceral tissue area) were observed but did not differ by strategy., Conclusions: Long-term follow-up of participants initiating 3 ART strategies demonstrated similar changes in total and regional fat, with no differences by ART strategy. The differential effects on lipid metabolism by strategy and the overall increases in insulin and insulin resistance with all 3 strategies necessitate close monitoring of patients on ART.

Canavan disease (CD), an autosomal recessive neurodegenerative disorder, is caused by mutations in the aspartoacylase (ASPA) gene. In the present study, the ASPA gene was analyzed in 24 non-Jewish patients with CD from 23 unrelated families. Within this cohort, we found three large novel deletions of approximate 92, 56, and 12.13 kb in length, using both self-ligation of restriction endonuclease-digested DNA fragments with long-distance inverse PCR and multiplex dosage quantitative PCR analysis of genomic DNA. The 92 kb large deletion results in complete absence of the ASPA gene in one homozygous and one compound heterozygous patient, respectively. The 56 kb large deletion causes absence of the majority of the ASPA gene except for exon 1 alone in a compound heterozygous patient. The 12.13 kb deletion involves deletion of the ASPA gene from intron 3 to intron 5 including exons 4 and 5 (I3 to E4E5I5) in a compound heterozygous patient. Patients with the three large deletions clinically manifested severe symptoms at birth, including seizures. Our study showed that the combined use of long-distance inverse PCR and multiplex dosage quantitative PCR analysis of genomic DNA is a helpful and rapid technique to search for large deletions, particularly for detection of large deletions in compound heterozygous patients.

The tremor rat is a spontaneous epilepsy model with a seizure phenotype caused by a deletion in the aspartoacylase (ASPA) gene. The absence of ASPA expression in these animals results in undetectable levels of enzyme activity and the accumulation of the substrate N-acetyl-aspartate (NAA) in brain, leading to generalized myelin vacuolation and severe motor and cognitive impairment. In support of human gene therapy for CD, recombinant adeno-associated viral vector (AAV-2) expressing ASPA was stereotactically delivered to the tremor rat brain and effects on the mutant phenotype were measured. AAV-ASPA gene transfer resulted in elevated aspartoacylase bioactivity compared to untreated mutant animals and elicited a significant decrease in the pathologically elevated whole-brain NAA levels. Assessment of motor function via quantitative rotorod testing demonstrated that rats injected with AAV-ASPA significantly improved on tests of balance and coordinated locomotion compared to animals receiving control vectors. This study provides evidence that AAV-2-mediated aspartoacylase gene transfer to the brain improves biochemical and behavioral deficits in tremor rat mutants (tm/tm) and supports the rationale of human gene transfer for Canavan disease.

Depletion of body cell mass (BCM) in human immunodeficiency virus (HIV)-infected patients is strongly associated with disease progression and death. Although whole-body protein turnover is increased in HIV infection, it is not known whether protein intake is independently associated with BCM. The purpose of this study was to determine the associations, if any, between protein intake and several body composition variables in 467 weight-stable, HIV-infected men with CD4 <200 cells/mm(3) enrolled in a multicenter nutritional supplementation trial. Baseline BCM, total body fat and extracellular mass as measured by bioelectrical impedance analysis, dietary intake (24 h food recall) and muscle building activity assessed by structured interview were analyzed to determine association(s) between body composition variables and macronutrient intake. Multiple regression analysis showed that BCM was positively associated with body weight (P = 0.001), height (P < 0.001), protein intake (P < 0.001), muscle-building activity (P < 0.001) and African-American ethnicity (P < 0.05) and negatively associated with carbohydrate intake (P < 0.05), age (P < 0.001) and number of prior AIDS-related diagnoses (P < 0.001). We conclude that protein intake is associated with increased BCM, whereas carbohydrate intake is negatively associated with BCM in HIV-infected men, independently of muscle building activity.

Objective: To compare the efficacy of three nutritional regimens in the prevention of weight loss., Design: A three-arm randomized controlled trial with primary outcome measure percent change in weight over four months., Patients: A total of 536 patients with CD4 count <200 cells/mm3 and stable weight, defined as <5% weight loss as determined by a weight measurement 3 to 6 months before randomization were recruited at fourteen administrative units in the United States, each unit consisting of multiple primary care sites., Intervention: The three arms were 500 kcal daily of caloric supplement with peptides and medium-chain triglycerides plus a multivitamin and mineral supplement, 500 kcal of a caloric supplement with whole protein and long-chain triglycerides plus a multivitamin and mineral supplement, and a multivitamin and mineral supplement only., Results: There were no significant differences among the three regimens in the percent change in weight (p = .74) and body cell mass (p = .63). On average, 65% of the recommended 500 kcal/day of caloric supplements containing peptides with medium-chain triglycerides and 82% of the 500 kcal/day of the caloric supplement containing whole protein and long-chain triglycerides were consumed., Conclusions: Caloric supplements do not promote increases in average weight or body cell mass in weight-stable, HIV-infected adults beyond that offered by a multivitamin and mineral supplement.

Thermolabile forms of the lysosomal enzyme beta-hexosaminidase B (Hex B), likely to result from different genetic defects, have been described. Ten individuals in five generations of a family of Oriental Jewish ancestry were identified biochemically as carriers of a thermolabile Hex B form. The beta-chain thermolability was found to be associated with the presence of a G --> A transition at nucleotide 1627 of the HEX B gene causing the substitution of Ala543 with a threonine. Oriental Jew whose Hex B was heat labile. Since thermolabile Hex B has been shown to occur more frequently among Jews of Oriental origin, the Ala543 --> Thr mutation may be the common mutation associated with beta-chain thermolability in this ethnic group.

Mucolipidosis Type IV is a rare, autosomal recessive disorder characterized by corneal opacification, mental retardation, and delayed motor milestones. Whereas lysosomal storage material has been demonstrated in biopsied tissues and leukocytes, the complete autopsy pathology, including neuropathology, is unknown. The metabolic defect remains speculative. We report the general and neuropathologic findings of the only known autopsy. In the central nervous system, neuronal loss in the cerebral cortex, basal ganglia, deep cerebellar nuclei, and brainstem nuclei was marked by astrocytosis; the cytoplasm of residual neurons had brown granules. These granules were positive with periodic acid-Schiff, Concanavalia ensiformis, and Sudan black, but not with Luxol-fast blue. Ultrastructurally, neurons contained lysosomes laden with osmiophilic, amorphous and granular material, and few lamellated membrane structures. Hepatocytes, epithelia, endothelia, chondrocytes, and tissue macrophages also stained positively with Datura stramonium and Ricinus communis-I agglutinins, with renal glomeruli also staining with peanut agglutinin; most non-neural cells contained osmiophilic granules on toluidine blue-stained, plastic embedded sections, corresponding to lamellated membrane structures. These findings complement the previously reported ocular morphology and brain and liver biochemistry performed in the same patient, and suggest that the storage material in neurons differs from that in non-neural cells. Furthermore, the underlying defect is not likely to be a deficiency of a single enzyme (i.e. a lysosomal hydrolase).

Advanced abdominal pregnancy is a rare event with a high fetal and maternal morbidity and mortality. We present 3 cases of abdominal pregnancy seen at our hospital. The perinatal mortality was 100% with no maternal deaths. Literature has been reviewed and management of abdominal pregnancy is discussed. Awareness of this condition and its clinical features is important in preventing abdominal pregnancy related morbidity, mortality. Careful clinical examination coupled with investigatory aids like X-rays and ultrasound will clinch the diagnosis. Most of the patients require early laparotomy following diagnosis and the morbidity and mortality are high particularly for the fetus.

Twenty-two cases of bilateral internal iliac artery ligation done in the Department of Obstetrics and Gynecology, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry, India over a period of 10 years (1981-1990) were studied. Various indications and complications were analysed. Internal iliac artery ligation is a life-saving procedure in pelvic hemorrhage. Complications are infrequents.

An adult-onset lysosomal storage disorder was diagnosed in a 5-year-old Schipperke dog with progressive cerebellar and central vestibular signs. It was characterized by cerebellar atrophy with extensive loss of Purkinje and granular cells, and hydrocephalus. Enlarged and vacuolated neurons were observed in spinal cord and brain; pancreatic centrolobular and islet cells were also vacuolated. Ultrastructurally, enlarged secondary lysosomes laden with lamellated membrane structures were present in neurons and empty enlarged vacuoles were found in pancreatic centroacinar, ductal, and islet cells. On frozen sections neurons stained with Ricinus communis agglutinin-I and wheat germ agglutinin. On paraffin sections neurons stained with luxol fast blue, periodic acid-Schiff, Concanavalia ensiformis agglutinin, and were autofluorescent. These findings indicate an accumulation of glycolipids containing terminal beta-galactosyl and alpha-sialyl residues, and N-linked oligosaccharides. Tissue activity of lysosomal beta-galactosidase was 50% of normal and the activity of beta-hexosaminidase was elevated. Brain lipid-bound sialic acid was twice normal, with a small increase of GM1-ganglioside, but there was a significant elevation of GM2 (GD2) and GM3 (GD3). In addition, significant elevations of sialylated and non-sialylated oligosaccharides were noted. These clinical, biochemical and pathological findings are similar to those observed in human patients with adult-onset galactosialidosis.

Magnetic resonance imaging (MRI), pathologic examinations, and biochemical analyses were performed on 2 different canine mutants with GM1 gangliosidosis (i.e., English Springer Spaniel and Portuguese Water Dog) and on age- and sex-matched controls. Serial MRI studies were also performed on a child with infantile-onset GM1 gangliosidosis. The affected dogs had abnormalities on MRI, including a relative increase in gray matter and an abnormal signal intensity of cerebral and cerebellar white matter observed on T2-weighted MRI. White matter changes on MRI were similar to white matter abnormalities observed in a 15-month-old boy with GM1 gangliosidosis. The weight ratio of white to gray matter from the frontal lobe was markedly reduced. Microscopic examination revealed characteristic ballooned neurons which stained lightly with Luxol-fast blue. The central cerebral and cerebellar folia white matter exhibited pallor and gliosis, while the corpus callosum and fornix stained normally with Luxol-fast blue. Axons appeared intact on Bodian staining. Ultrastructural studies revealed fewer myelinated axons in affected puppies. Total gangliosides in gray matter were elevated. Thin-layer chromatography demonstrated GM1 ganglioside as the predominant ganglioside. The amount of cerebrosides and sulfatides was reduced in the gray and white matter when compared to controls but the ratio in gray and white matter remained unchanged. Immunostaining of neutral glycolipids disclosed increased amounts of stage-specific embryonic antigen-1 glycolipid in gray matter. These findings suggest that canine models for GM1 gangliosidosis are associated with abnormal myelin development which may be similar to the human disease.

The clinical, morphologic, histochemical, and biochemical features of GM1-gangliosidosis in two canine models, English Springer Spaniel (ESS) and Portuguese Water Dog (PWD), have been compared. The disease onset, its clinical course, and survival period of the affected dogs were similar in both models. Skeletal dysplasia was noted radiographically at 2 months of age, whereas at 4 1/2 months of age there was progressive neurologic impairment. However, dwarfism and coarse facial features were seen only in ESS. Both models had similar deficiency in activity of lysosomal beta-galactosidase, but possessed a normal protein activator for GM1-beta-galactosidase. Both models stored GM1-ganglioside, asialo-GM1, and oligosaccharides in brain. Furthermore, only the PWD stored glycoproteins containing polylactosaminoglycans in visceral organs, and neither model stored them in the brain. Morphologically, both models demonstrated similar storage material in multiple tissues and cell types. The ultrastructure of the storage material was cell-type specific and identical in both models. However, some differences in the lectin staining pattern were noted. Our clinical, biochemical, and histochemical findings indicate that PWD and ESS may represent two different mutations of the beta-galactosidase gene. Moreover, the authors conclude that it is difficult, and inappropriate, to apply the human classification of GM1-gangliosidosis (i.e. infantile, juvenile, and adult forms) to these canine models.

We describe a novel late-onset lysosomal lipid storage disease affecting a Tibetan terrier. The principal clinical manifestations include visual loss, progressive cerebellar ataxia and dementia. A necropsy of an affected 10-year-old dog demonstrated cerebellar atrophy. Histological analysis revealed extensive loss of retinal ganglion cells and cerebellar Purkinje cells, and mild to moderate loss of neurons in the cerebrum, basal ganglia and spinal cord. There were generalized neuronal hypertrophy and multifocal neuronal necrosis associated with the presence of enlarged macrophages. Neurons and perineuronal macrophages contained cytoplasmic granules that stained with PAS, luxol fast blue and several lectins. The granules were sudanophilic and autofluorescent. Electron microscopic analysis revealed lysosomes laden with lamellated membrane structures in neurons, pancreatic ductal and centroacinar cells and in cultured fibroblasts. These findings indicate lysosomal storage of both lipid and carbohydrate. Biochemical analysis of brain lipids and numerous lysosomal enzyme assays of leukocytes and cultured fibroblasts were unsuccessful in elucidating the underlying enzyme defect, although a generalized increase of brain gangliosides was noted.

The clinical, neurophysiological, morphological and biochemical manifestation of eyes from Persian kittens affected with alpha-mannosidosis were studied. Clinically the disease is characterized by progressive corneal and lenticular opacification. In addition there is asymmetry in shape and latency of signal conductions which were demonstrated by visual evoked potential studies. Morphological and histochemical studies revealed vacuolization of various ocular cell types which stained positively with Concanavalia ensiformis agglutinin (Con A) and wheat germ agglutinin (WGA). Biochemical studies illustrated low activity of acid alpha-mannosidase in cultured keratocytes and abnormal storage of partially degraded oligosaccharides in these cells, in vitreous humor and lens. This comprehensive study of ocular alpha-mannosidosis demonstrates enzyme deficiency which leads to abnormal storage of oligosaccharides in affected cells and is manifested by morphological alterations and functional impairment.

Some of the properties of a partially purified particle bound and soluble beta-glucosidase (EC 3.2.1.21) from pig kidney were compared. The soluble beta-glucosidase (1) hydrolyzed 4-methylumbelliferyl-beta-D-glucoside (4-MU-beta-D-glucoside) 17 alpha-estradiol 3beta-glucoside. 17 alpha-estradiol 17beta-glucoside, and salicin, but not glucosylceramide, (2) possessed a broad pH optimum (5.5-7.0), (3) had an isoelectric point of 4.9, and (4) was inhibited by Triton X-100. Several compounds were found to be competitive inhibitors of its hydrolytic activity, gluconolactam and estrone beta-glucoside being the most effective. In contrast, a particulate beta-glucodidase purified from the same tissue (1) had an acidic pH optimum (5.0), (2) was stimulated by sodium taurocholate and 'Gaucher's factor' for the hydrolysis of both 4-MU-beta-glucosidase and glucosylceramide, and (3) was capable of catalyzing a transglucosylation reaction employing 4-MU-beta-D-glucoside or glucosylceramide as the glucosyl donor, and [14C]ceramide as acceptor.

Histopathologic, ultrastructural and Golgi impregnation studies disclosed lesions characteristic of a neuronal lysosomal storage disease in related sheep with onset of neurologic signs at 4-6 months. Biochemical and enzymatic evaluation disclosed storage of GM1 ganglioside, asialo-GM1, and neutral long chain oligosaccharides in brain, urinary excretion of neutral long chain oligosaccharides, and deficiencies of lysosomal beta-galactosidase and alpha-neuraminidase. Retrospective and limited prospective genetic studies suggested autosomal recessive inheritance. A gene-dosage effect on beta-galactosidase levels was documented in fibroblasts from putative heterozygous sheep. Fibroblasts from affected sheep did not have increased beta-galactosidase activity after incubation with the protease inhibitor, leupeptin. In some aspects this disease is similar to GM1 gangliosidosis, but is unique in that a genetic defect in lysosomal beta-galactosidase may cause the deficiency of lysosomal alpha-neuraminidase.

The morphology and ultrastructure of circulating white blood cells from six Persian and from five Russian Blue/Siamese cats deficient in lysosomal activity of alpha-mannosidase and arylsulfatase B, respectively, were studied and compared to cells from corresponding normal and carrier cats. In cats with mannosidosis, light microscopic examination revealed vacuoles in lymphocytes and monocytes, whereas electron microscopic studies demonstrated additional vacuoles in neutrophils, eosinophils, and basophils. In cats with mucopolysaccharidosis VI (MPS VI), vacuoles containing metachromatic granules were observed in lymphocytes, neutrophils, eosinophils, and monocytes. Ultrastructural studies of these cells identified the accumulation of fibrillar material, which often was associated with lamellated membrane structures.

Human leukocytes contain at least two isozymes of 4-methylumbelliferyl-beta-glucosidase acting optimally at pH 4.0 and 4.8; in Gaucher disease, only the former is deficient. Brief exposure of the leukocyte homogenate to pH 4.0 at room temperature results in irreversible inactivation of the pH 4.8 activity, while the activity at pH 4.0 remains unaffected. The more acidic isozyme is stimulated four- to fivefold by 0.2% sodium taurodeoxycholate (TDC) with a shift in the pH optimum to 5.0. The less acidic isozyme is completely suppressed in the presence of this detergent. Both leukocyte isozymes appear to be membrane-bound since gel filtration of Sephadex G-200 produces only one peak of activity located at the void volume, unlike in liver and kidney where a second peak also can be demonstrated. Heat inactivation analysis indicated that in controls, assayed in the absence of detergent, pH 4.0 activity is more thermostable than pH 4.8 activity. However, in Gaucher disease, the residual beta-glucosidase at pH 4.0 is just as thermolabile as the unaffected pH 4.8 activity. Heat inactivation of the enzyme in the presence of TDC resulted in rapid loss of activity, suggesting a direct effect of the bile salt on the configuration of the enzyme decreasing its thermal stability. In the absence of detergent, acid beta-glucosidase shows two K(m)'s, one at 3.2 mM and another at 0.9 mM. In the presence of detergent, only the higher K(m) at 3.3 mM is obtained. In patients with Gaucher disease and in obligate carriers, the K(m) remains essentially unaffected while the V(max) shows the expected deficiency.A reliable and reproducible selective assay technique has been developed for the diagnosis of Gaucher disease homozygotes and obligate heterozygotes and for the carrier screening of individuals at risk for this inherited disorder. The efficacy of this technique has been demonstrated by studying the activity in 42 controls, 26 patients, 32 obligate heterozygotes, and 23 healthy relatives of patients with Gaucher disease.

A simple assay technique for the determination of sulfatidase activity in leukocytes has been developed for the reliable diagnosis of metachromatic leukodystrophy (MLD). Sulfatide is tritiated in sphingosine and fatty acid by reduction with [3H]sodium borohydride in alkali in the presence of palladium chloride. This labeled natural substrate for aryl sulfatase A (AsA) is hydrolyzed by normal human leukocytes in 25 mM-acetate buffer, pH 5.0, in the presence of 0.3% sodium taurodeoxycholate. The enzyme activity is greatly improved after dialysis, exhibiting better linearity with protein concentration. It is stimulated maximally by 5 mM-MnCl2 with an apparent Km of 0.17 mM for the substrate. Patients with MLD exhibited virtually no detectable sulfatidase activity although they had residual AsA activity that was measured with the synthetic substrate, p-nitrocatechol sulfate (NCS). Potential heterozygotes could be identified by the sulfatidase assay in instances where the NCS assay for AsA was inconclusive. Several individuals with levels of AsA activity characteristic of MLD, including a few healthy carriers and certain patients with unknown neurological diseases, were shown not to have MLD by the presence of measurable levels of sulfatidase in their leukocytes.

In Gaucher disease the genetic lack of acid beta-glucosidase activity causes glucocerebroside to accumulate in the lysosomes of macrophage-derived cells, producing large characteristic Gaucher cells. The formation of Gaucher cells seems to be central to the pathobiology of this lysosomal storage disease. To develop a model simulating this process, cultured murine peritoneal macrophages were treated with conduritol B epoxide, a specific irreversible inhibitor of acid beta-glucosidase, for 6, 15, and 24 days. The conduritol B epoxide-treated macrophages accumulated glucocerebroside as a function of time, progressing to a fivefold elevation over control values after 24 days of treatment. Electron microscopy of the cells treated for 24 days reveals characteristics of Gaucher cells, including striations consisting of oriented fibrils. With conventional staining techniques, these fibrils have an appearance considered highly characteristic of Gaucher disease. Thus, macrophages treated with conduritol B epoxide are a useful model for studying the metabolic consequences and morphologic features associated with glucocerebroside accumulation in Gaucher cells.