Lymphatic filariasis (LF) is an incapacitating disease caused by three filarial nematodes belonging to the family Onchocercidae, namely Brugia timori, Wuchereria bancrofti and Brugia timori. An estimated 90% of lymphatic filariasis cases globally are caused by Wuchereria bancrofti. To evaluate the success of the Global Program to eliminate Lymphatic Filariasis it is essential to monitor the frequency of larval infection in the mosquito vector. Molecular methods to detect Wuchereria bancrofti DNA in mosquitoes have been in existence since 1996. However these methods have not been widely adopted due to the high cost associated with them and the inability of these assays to distinguish between immature and infectious stages in the mosquito vector. The overall aim of this project was to modify, as previously described in literature, the Laney real time PCR assay to permit it to be used in an end point Reverse Transcriptase (RT)-PCR ELISA format. The endpoint PCR-ELISA uses inexpensive conventional thermocyclers and inexpensive reagents and probes. To accomplish this overall goal the specific objectives were to produce a positive control RNAs for Wb-cut-1.2 L3 specific RT-PCR and Wb-TPH RT-PCR that detects any stage of the parasite, and to adapt the detection of both transcripts to a PCR-ELISA format. Positive RNA controls were prepared and purified using template cDNA made available through FR3, subsequent development and optimization of the RT PCR ELISA was achieved through the adaptation of the Onchocerca volvulus O150 PCR ELISA protocol. We found a 16-fold difference in the limit of detection between the ELISA assay and conventional end point RT-PCR when we did a 2-fold dilution series of PCR products for both Wb-Cut-1.2 and Wb-TPH. This indicates that our assay was 16 times more sensitive than the use of regular agarose gel electrophoresis to analyze PCR products. The limit of detection with ELISA and gel analysis were comparable when a 10-fold dilution series of the positive control RNA template was done. The RT-PCR ELISA takes a day to complete and up to three 96 well plates a day can be processed compared to the limited number of samples that can be analyzed by gel electrophoresis a day. It is anticipated that our assay will be used in the molecular xenomonitoring of Wuchereria bancrofti providing earlier time-point assessments of LF infection in endemic areas. In areas that were once endemic, our diagnostic tool will play a pivotal role in monitoring LF resurgence.

archives@tulane.edu / 1 / Kimberly Michelle Koporc

Mantle cell lymphoma (MCL) represents a heterogenous subtype of non-Hodgkin lymphoma (NHL), which can present in three distinct clinicopathologic variants: indolent type MCL, classic type MCL and blastoid type MCL. Despite the different variations, MCL, in general, is almost always associated with advanced-stage disease at diagnosis, with a strong predilection for significant extranodal involvement, usually to the bone marrow, CNS, peripheral blood and the gastrointestinal tract. However, the literature review reveals ocular involvement is a more rarely described extranodal site of involvement by MCL. Among the reported cases, the orbit was most commonly involved, followed by the eyelid and the lacrimal gland. We report a 63-year-old male who presented with a nine-month history of progressive symptoms of periorbital swelling and eyelid apraxia, causing bilateral visual disturbances. The patient was initially treated for presumed blepharospasm by his ophthalmologist with botulinum toxin injections; however, his periorbital edema continued to worsen, and he developed a discrete nodule in his right lower eyelid. Biopsy of the right eyelid nodule revealed classic type MCL with immunohistochemical testing positive for CD20, CD5, cyclin D1, SOX11 and Ki67 proliferative index of 40%. Fluorescence in situ hybridization (FISH) analysis detected (11;14) translocation. Mantle Cell Lymphoma International Prognostic Index Combined Biologic Index (MIPIb) score was calculated to be 6.5 points based on his age, ECOG performance status of 0-1, normal serum LDH, normal white blood cell count and elevated Ki67 proliferative index, stratifying patient into the high-risk group, with an estimated median overall survival of 37 months. Due to the bulky MCL involvement in the palpebral conjunctiva affecting his vision and eyelid function, he was immediately treated with radiation therapy to the bilateral orbits. PET-CT revealed adenopathy above and below the diaphragm. Bone marrow biopsy revealed focal involvement (5-10%) by MCL. Brain MRI revealed MCL infiltration in the bilateral orbits and lacrimal glands. Upper and lower endoscopy revealed multiple polyps positive for MCL. Given the advanced stage of the disease and his high-risk stratification, he was started on intensive induction chemotherapy with rituximab, dexamethasone, cytarabine, and carboplatin and received prophylactic intrathecal methotrexate. Systemic imaging after completion of four cycles of treatment revealed near resolution of the majority of the lymphadenopathy and all of the lymph nodes no longer demonstrated any significant metabolic activity. He completed two additional cycles of systemic chemotherapy and is currently being evaluated for autologous hematopoietic stem cell transplantation in complete remission-1 given his excellent response to treatment, his young age, high-risk disease at diagnosis, and good performance status. Despite the diffuse and extensive systemic disease, interestingly, our patient did not exhibit any constitutional or metastasis-associated symptoms and only presented with isolated periorbital swelling. Our case emphasizes the rare extranodal site of involvement by MCL and encourages all medical providers to remain cognizant of the varying ways in which MCL can present clinically.

No description available.

The histologic distinction of follicular patterned lesions of thyroid, that is follicular adenoma, follicular carcinoma, and the follicular variant of papillary thyroid carcinoma can be extremely difficult. The differential diagnostic criteria regarding nuclear features of papillary thyroid carcinoma are subjective, resulting in high interobserver variability. Although papillary thyroid carcinoma metastasizes mainly via lymphatic vessels, whereas follicular carcinoma spreads mostly hematogenously, there are no data regarding utility of objective quantitative criteria such as lymphatic and general blood vessel density for the differential diagnosis of these lesions. In this study, 35 follicular patterned lesions of thyroid (14 follicular adenomas, 10 follicular carcinomas, and 11 of the follicular variant of papillary thyroid carcinomas) were evaluated immunohistochemically. An assessment of intra- and peritumoral lymphatic vessel density was performed using novel lymphatic endothelium-specific marker D2-40, and the intra- and peritumoral general vessel density was determined by the panendothelial marker CD31. There were no significant differences in the intra- and/or peritumoral general vessel densities, and peritumoral lymphatic vessel densities among follicular adenoma, follicular carcinoma and the follicular variant of papillary thyroid carcinoma. In contrast, the intratumoral lymphatic vessel density was significantly higher in the follicular variant of papillary thyroid carcinoma than in either follicular adenoma or follicular carcinoma (34.63, 15.04, and 0.11 respectively; P<0.0001). The results of the study show that intratumoral lymphatic vessel density may serve as a useful tool in the differential diagnosis of follicular patterned lesions of thyroid. © 2005 USCAP, Inc All rights reserved.

A 3 year old Hispanic male presented with fevers, skin rash, left neck swelling and refusal to walk of several days duration. Physical exam revealed a febrile, fussy toddler with a tender, cystic lesion in the left submandibular region. Both ankles had tender cystic lesions on the lateral malleolar regions. Labs: WBC 33,000 with neutrophilia, bandemia, thrombocytosis, and increased ESR and CRP. MRI of the neck and ankles revealed cystic lymphatic malformations with no communication with the joints.ENT specialist was consulted and neck cystic lesion was aspirated to rule out a septic focus. Bone scan of the lower extremities ruled out infectious or malignant etiology. He was started on multiple antibiotics with a presumed diagnosis of sepsis. An ECHO on the 4th hospital day showed a pericardial effusion which required a pericardial window. He also developed bilateral pleural effusions which resolved with supportive treatment. Aspirates from the cystic lesions, pericardial fluid, blood and urine cultures were sterile. Even in the second week of hospital stay, he continued to spike high fevers (Tmax 107) with high white counts and left shift inspite of treatment with antibiotics. A diagnosis of systemic onset juvenile idiopathic arthritis (SOJIA) was made with input from rheumatologist. Antibiotics were discontinued and steroids were started with good response. Cystic lesions were treated with percutaneous sclerotherapy with doxycycline. He was discharged home on oral steroids, NSAID’s and weekly methotrexate. Etanercept was added to decrease dependence on oral steroids. He remains in good health 2 years since initial presentation. Discussion: A febrile toddler who refuses to walk is a common clinical presentation in pediatrics. Differential diagnosis of such a patient includes osteomyelitis, septic arthritis, acute rheumatic fever, leukemia and non-accidental trauma. The presence of systemic extra-articular symptoms as in our patient must alert the pediatrician to systemic onset juvenile rheumatoid arthritis (SOJIA). It accounts for 10-20% of all juvenile idiopathic arthritis (JIA) patients with an incidence of 0.4-0.8 per 100,000. SOJIA differs from other conditions in its multisystem involvement. Clinical features like pleurisy, pericarditis, spiking fevers, hepatosplenomegaly and lymphadenopathy overshadow the joint symptoms. The joint involvement may be completely absent or may be a late clinical feature. These patients have leukocytosis, thrombocytosis and high inflammatory markers simulating a septic focus. In our patient, the accurate diagnosis was complicated by the confounding presence of multiple cystic lymphatic lesions. Treatment of SOJIA is challenging. Oral steroids, NSAID’s, methotrexate, etanercept and the newer anakinra have been used with varying success. Our case underlines the importance of considering a diagnosis of systemic onset JIA in a febrile toddler even in the absence of overt joint involvement.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive, rare malignancy. Exact incidence is unknown due to lack of diagnostic criteria. Typically, it involves skin and bone marrow and less likely, lymph nodes and visceral organs. We present a 76 year old male who started having a lesion on the left side of his back that was progressively enlarging. He initially started on antibiotic and topical medications for more than a month which did not help. Punch biopsy of the lesion was consistent with blastic plasmacytoid dendritic cell neoplasm, positive for CD2, CD5, CD7, CD43, weak CD58,Tdt, bcl-6. Patient denies fever, chills, night sweats, weight loss, change in appetite. Physical exam revealed a purplish lesion raised in the left upper back with multiple satellite-like purple lesions throughout the back. Laboratory showed white cell count 3.2 K/uL, hemoglobin 13 g/dL, platelet 135 K/uL. Bone marrow biopsy shows immature blastic neoplasm involving 15% of the bone marrow. Cytogenetics showed normal karyotype. Flow cytometry shows an immature lymphoid population with expression of CD4, CD56, and CD 123, negative for FLT3, IDH1, IDH2, NPM1 mutations. Positron emission tomography (PET) scan showed skin thickening with minimal FDG uptake in left posterior skin soft tissue of the chest near the shoulder with no other abnormal focal uptake and splenomegaly. BPDCN is a rare aggressive malignancy that is more common in older populations. The origin is from type 2 dendritic cells. Typical presentations are skin lesions, cytopenia, lymphadenopathy, and splenomegaly. Some of the cytological features of BPDCN include cloudy sky (blue cytoplasm with clearer areas), pseudopods, and microvacuoles. Confirmation of diagnosis is with immunophenotyping. Workup after diagnosis include complete blood count, liver and renal function, hepatitis panel, peripheral blood smear, bone marrow evaluation, systemic imaging, cerebrospinal fluid cytology. Treatment of BPDCN is challenging in this era. Most patients respond to chemotherapy, although they relapse. Tegraxofusp is suggested for remission induction therapy following allogeneic hematopoietic cell transplantation. Median overall survival is about one year. Only patients who underwent hematopoietic stem cell transplant had prolonged survival. Myelemia, old age and altered general state have worse prognosis.

No description available.

No description available.

Cette thèse de doctorat concerne le développement et la caractérisation d’une plateforme de microaiguilles (MNs) pour la livraison de microtatouages fluorescents fonctionnels pour la surveillance de santé à domicile. Ceci est présenté dans le contexte d’un intérêt croissant pour les systèmes de surveillance de la santé à distance et de précision, mis en évidence par la littérature scientifique et par la popularité des produits commerciales comme les Fitbits et les moniteurs de glucose en continu (CGMs). Si ces dispositifs offrent un accès sans précédent à des informations sur la santé en temps réel, ils mettent aussi en évidence le fossé qui existe entre les paramètres de santé faciles à surveiller et ceux qui ont une utilité clinique. Les Fitbits, bien qu’entièrement non invasifs, sont limités en termes de surveillance. Les CGMs peuvent fournir des mesures précises de la glycémie en temps réel, mais ils nécessitent le port permanent d’un lecteur implanté. Un système idéal combinerait la nature non-invasive d’un Fitbit avec la précision et l’utilité d’un CGM. Au coeur de ce problème est la peau; la majorité des informations intéressantes sur la santé ne sont accessibles qu’en franchissant cette barrière, ce qui nécessite généralement une prise de sang, qui peut être douloureuse, limitée à un environnement clinique et nécessitant un personnel qualifié. Les MNs — aiguilles miniatures capables de traverser sans douleur la couche externe de la peau — offrent la possibilité de contourner ce problème. En particulier, les MNs en polymères solubles sont simples à fabriquer, faciles à utiliser pour les patients et ne génèrent pas de déchets dangereux, ce qui les rend idéaux pour l’utilisation à domicile. En utilisant ces MNs pour livrer des capteurs fluorescents — formant un microtatouage fonctionnel dans la peau — il est possible de surveiller beaucoup d’informations de santé de manière non invasive. Avec un lecteur de fluorescence portable, cette technologie pourrait améliorer de manière significative l’accessibilité des informations de santé à domicile. Afin de mettre cette technologie en contexte, cette thèse commence par un article de perspective décrivant les développements récents et les tendances dans le domaine des MN polymères pour les applications diagnostiques (Chapitre 1). Il est suivi d’une série d’articles décrivant le développement et les essais itératifs de la technologie du microtatouage. Le premier iv article porte sur l’administration d’un colorant fluorescent inerte pour le suivi du drainage lymphatique dans le contexte de la surveillance du lymphoedème (Chapitre 2). Il est suivi d’un article décrivant un microtatouage fonctionnel pour surveiller les espèces réactives de l’oxygène (ROS) dans le contexte de l’inflammation dermique (Chapitre 3), suivi par l’utilisation de ce microtatouage pour caractériser et étudier une réponse inflammatoire médiée par les neutrophiles dans la peau (Chapitre 4). Le chapitre suivant s’éloigne des applications du microtatouage pour discuter une nouvelle méthodologie de synthèse organique qui servira à faciliter le développement et la synthèse de nouveaux capteurs fluorescents à utiliser dans les microtatouages (Chapitre 5). Enfin, la dernière partie de la thèse décrit la progression vers le développement d’un microtatouage pour le marquage continu et réversible de la peau, utilisant le pH dermique comme modèle (Chapitre 6). / This PhD thesis concerns the development and characterization of a microneedle (MN)-based platform for the delivery of functional fluorescent microtattoos for at-home or point-of-care diagnostics and monitoring. This topic is positioned within the context of increasing interest in remote and precision health monitoring systems, as evidenced by both a growing body of scientific literature and the rise in popularity of consumer health monitoring products such as Fitbits and continuous glucose monitors (CGMs). While such devices offer unprecedented access to real-time health information, they also serve to highlight a gap that exists between health parameters that are easy to monitor, and those that are of clinical utility. Fitbits, though entirely non-invasive, are extremely limited in terms of what they can monitor. CGMs can provide accurate real-time blood glucose measurements, but they require continuously wearing an implanted sensor. An ideal system would combine the non-invasive nature of a Fitbit with the precision and utility of a CGM. At the heart of this issue is the skin; the majority of interesting health information can only be accessed by breaching this barrier, and this generally requires blood sampling, which can be painful, is limited to a clinical setting, and requires trained personnel to conduct. MNs — miniature needles able to painlessly cross the skin’s outer layer — offer the opportunity to circumvent this issue. To this end, there has been growing interest in using MNs for diagnostic applications. In particular, dissolving polymeric MNs are simple to manufacture, easy for patients to use, and generate no hazardous waste, rendering them ideal for at-home or point-of-care use. By using these MNs to deliver specifically designed fluorescent sensors — forming a functional microtattoo within the skin — it is possible to monitor a wide range of health information in a non-invasive manner. Paired with a portable or wearable fluorescence reader, this technology could meaningfully improve the accessibility of health information from home. To provide context for this technology, this thesis begins with a perspective article outlining recent developments and trends in the field of polymeric MNs for diagnostic applications (Chapter 1). This is followed by a series of articles outlining the iterative development and testing of microtattoo technology. The first involves the delivery of an inert fluorescent dye for tracking lymphatic drainage in the context of lymphedema monitoring (Chapter 2). This is followed by an article outlining a functional microtattoo for monitoring reactive oxygen species (ROS) in the vi context of dermal inflammation (Chapter 3), followed by the use of this microtattoo to characterize and study a neutrophil-mediated inflammatory response in the skin (Chapter 4). The next chapter digresses from the applications of microtattoo technology to discuss a novel organic synthesis methodology that will serve to facilitate the development and synthesis of novel fluorescent sensors for use in microtattoos (Chapter 5). Finally, the last part of the thesis outlines the progression towards the development of a microtattoo for the continuous and reversible monitoring of physiological data, using dermal pH as a model analyte (Chapter 6).