RETINAL MANIFESTATIONS OF WALKER-WARBURG SYNDROME IN TWO SIBLINGS WITH RXYLT1 MUTATIONS.

PURPOSE: We report two siblings with genetically confirmed Walker-Warburg syndrome (WWS), studied with multimodal imaging, who presented with different retinal manifestations. METHODS: This is a retrospective report of two WWS cases with ultra-widefield fundus photography, fluorescein angiography, and ultrasound. Molecular diagnosis was achieved using panel testing and targeted variant testing. RESULTS: Two siblings, one male and one female, born 17 months apart with a diagnosis of WWS underwent retinal examination with imaging. The 3-month-old female infant exhibited microphthalmia, persistent hyaloidal arteries, and retrolental membranes with total tractional retinal detachments on ultrasound in both eyes. The 22-day-old male newborn exhibited persistent hyaloidal arteries and extensive peripheral avascular retina on angiography in both eyes. Both were found to be positive for the same two pathogenic variants in the RXYLT1/TMEM5 gene, which accounts for approximately 9% of cases of genetically confirmed WWS. CONCLUSION: Siblings with genetically confirmed WWS can have variable presentations despite identical genotype. This highlights the phenotypic disease spectrum of WWS, which may be similar to that seen in familial exudative vitreoretinopathy.

Initial Experience With Biosimilar Bevacizumab-bvzr For Intravitreal Use in Children: A Case Series and Literature Review.

BACKGROUND AND OBJECTIVE: We report our initial experience with intravitreal bevacizumab-bvzr, a bevacizumab biosimilar approved by the US Food and Drug Administration in 2019 and recently introduced by our institution for off-label ophthalmologic use in children. PATIENTS AND METHODS: This was an Institutional Review Board-approved single-institution retrospective case series of pediatric patients 21 years or younger who received at least one intravitreal injection of biosimilar bevacizumab-bvzr. RESULTS: Twelve eyes of 9 patients were identified as having received intravitreal bevacizumab-bvzr, with a total of 13 injections performed. Indications for injection included retinopathy of prematurity (7/13), choroidal neovascularization (3/13), retinal vein occlusion (2/13), and Coats disease (1/13). Following injection of bevacizumab-bvzr, all patients experienced a positive clinical response. No occurrences of postinjection inflammation, intraocular pressure anomalies, or endophthalmitis were observed with a median follow-up of 18 weeks. CONCLUSION: In the absence of controlled studies, this case series supports the use of intravitreal bevacizumab-bvzr as an anti-vascular endothelial growth factor therapy option, including in the pediatric population and resource-poor settings. [Ophthalmic Surg Lasers Imaging Retina 2023;54:84-88.].

Cryptococcus neoformans releases proteins during intracellular residence that affect the outcome of the fungal-macrophage interaction.

Cryptococcus neoformans is a facultative intracellular pathogen that can replicate and disseminate in mammalian macrophages. In this study, we analyzed fungal proteins identified in murine macrophage-like cells after infection with C. neoformans. To accomplish this, we developed a protocol to identify proteins released from cryptococcal cells inside macrophage-like cells; we identified 127 proteins of fungal origin in infected macrophage-like cells. Among the proteins identified was urease, a known virulence factor, and others such as transaldolase and phospholipase D, which have catalytic activities that could contribute to virulence. This method provides a straightforward methodology to study host-pathogen interactions. We chose to study further Yeast Oligomycin Resistance (Yor1), a relatively uncharacterized protein belonging to the large family of ATP binding cassette transporter (ABC transporters). These transporters belong to a large and ancient protein family found in all extant phyla. While ABC transporters have an enormous diversity of functions across varied species, in pathogenic fungi they are better studied as drug efflux pumps. Analysis of C. neoformans yor1Δ strains revealed defects in nonlytic exocytosis, capsule size, and dimensions of extracellular vesicles, when compared to wild-type strains. We detected no difference in growth rates and cell body size. Our results indicate that C. neoformans releases a large suite of proteins during macrophage infection, some of which can modulate fungal virulence and are likely to affect the fungal-macrophage interaction.

A dual role for CRTH2 in acute lung injury.

Acute respiratory distress syndrome (ARDS) is a life-threatening clinical condition defined by rapid onset respiratory failure following acute lung injury (ALI). Its increased incidence due to COVID-19 and high mortality rate (∻40%) make the study of ARDS pathogenesis a crucial research priority. CRTH2 is a G protein-coupled receptor with established roles in type 2 immunity and well-characterized inhibitors. Prior studies have shown it also promotes neutrophilic inflammation, indicating that CRTH2 inhibition may be a potential therapeutic strategy for ARDS. To test this hypothesis, we first examined the expression pattern of CRTH2 on murine neutrophils. We found it is expressed on neutrophils, but only after extravasation into the lung. Next, we showed that extravasated lung neutrophils generate inflammatory responses upon stimulation with the CRTH2-specific agonist DK-PGD2, as demonstrated by reactive oxygen species (ROS) production. This response was abrogated in CRTH2 KO neutrophils. Inhibition of CRTH2 with fevipiprant suppressed baseline ROS production, indicating an autocrine PGD2-CRTH2 signaling loop. We then evaluated the role of CRTH2 in vivo using a murine model of LPS-induced ALI. Despite the pro-inflammatory effects of CRTH2 on neutrophils in vitro, we observed worsening of lung injury in CRTH2-deficient mice in terms of neutrophilic inflammation, vascular leak, and survival. Bulk RNAseq of lung tissue indicated an impairment in type 2 immune signaling; qPCR and ELISA confirmed downregulation of the key type 2 effector cytokine, IL-4. Thus, CRTH2 appears to play a dual role in ALI, directly promoting neutrophil effector responses, but indirectly suppressing lung injury and neutrophilic inflammation through type 2 immunity. These findings reveal a novel protective function for CRTH2 during lung injury and argue against the use of CRTH2 inhibitors in ARDS.

Hypertension: A Cause of Bilateral Proliferative Retinopathy.

Purpose: To describe the case of a 67-year-old female with proliferative retinopathy secondary to uncontrolled hypertension. Methods: Retrospective case report including multimodal imaging. Results: A 67-year-old female presented with mild vitreous hemorrhage, retinal hemorrhage, hard exudate of the left eye and hard exudate, copper wiring of vessels, and retinal hemorrhages in the right eye. Optical coherence tomography depicted macular edema of both eyes. Fluorescein angiography revealed large areas of peripheral retinal ischemia and neovascularization with multiple areas of vascular leakage in both eyes. Conclusions: Proliferative hypertensive retinopathy has been rarely reported in the literature. Our patient exhibited findings consistent with proliferative retinopathy secondary to hypertensive retinopathy.

Association of the Pattern of Retinal Capillary Non-Perfusion and Vascular Leakage with Retinal Neovascularization in Proliferative Diabetic Retinopathy.

PURPOSE: To explore the correlation between retinal capillary non-perfusion and the distribution of retinal neovascularization and vascular leakage (VL) in patients with proliferative diabetic retinopathy (PDR). METHODS: Ultra-widefield angiograms of 96 eyes of 69 patients with PDR were reviewed for the proportion of non-perfused area to total gradable area, and for the presence of neovascularization and VL. RESULTS: Retinal neovascularization was distributed as such: neovascularization elsewhere (NVE), 57.3%; neovascularization of the disc (NVD), 11.5%; both neovascularization of the disc and elsewhere (NVED), 31.3%. The proportion of non-perfused retina, so-called ischemic index, was greater in eyes with NVED compared to eyes with NVE only, but not when compared to NVD only. Overall, 83% of eyes had VL. The presence and the extent of VL correlated with the proportion of the ischemic index. While VL and ischemic index were more severe in the mid-periphery and far-periphery, the majority of NVE was located in the posterior pole. CONCLUSIONS: The presence of both NVD and NVE is associated with a greater ischemic index than NVE alone. Although both VL and ischemic index is significantly higher in peripheral zones, the majority of neovascularization occurs at the posterior pole.

Which U.S. States' Medicaid Programs Provide Coverage for Gender-Affirming Hormone Therapy and Gender-Affirming Genital Surgery for Transgender Patients?: A State-by-State Review, and a Study Detailing the Patient Experience to Confirm Coverage of Services.

BACKGROUND: To date, a comprehensive state-by-state assessment of transgender transition-related health care coverage for gender-affirming hormone therapy (GAHT) and genital gender-affirming surgery (GAS) has not been reported. AIMS: The aims of this study were 1) to verify which U.S. states' Medicaid systems do/do not cover GAHT and GAS; 2) to assess the ease/difficulty for patients to determine whether GAHT and GAS are Medicaid-covered benefits; and 3) to understand possible state-related predictors of Medicaid coverage for gender-affirming care. METHODS: We reviewed the official Medicaid Handbook and website for all 51 states (+D.C.) and 5 territories to confirm whether GAHT and GAS are covered benefits. When indeterminate, we called the Medicaid office in each state, and for many, Medicaid managed care organizations (MCOs), and individual in-state providers, to confirm coverage. We recorded our experiences, number of, and duration of phone calls to confirm coverage. OUTCOMES: The main outcome was a definitive answer from the state/territory's Medicaid program or MCOs regarding whether GAHT and GAS are/are not covered benefits. Secondary outcome measures included responses we received and the total number/duration of phone calls necessary to confirm coverage. RESULTS: Only 12 of 51 states and 0 of 5 territories featured their policy regarding coverage for GAHT in their Medicaid Handbook/webpages. We confirmed that 34 of 51 state Medicaid programs do cover GAHT, whereas 9 of 51 states' and 2 of 5 territories' do not. We could not confirm coverage of GAHT in 8 of 51 states and 3 of 5 territories. Only 26 of 51 states and 0 of 5 territories featured their policy regarding coverage for GAS in their Medicaid Handbook/webpages. We confirmed that 25 of 51 state Medicaid programs do cover GAS, whereas 22 of 51 states' and 3 of 5 territories' do not. We could not confirm coverage of GAS in 4 of 51 states and 2 of 5 territories. Up to 12 calls, lasting up to 125 minutes, were required to confirm coverage for GAHT/GAS. CLINICAL IMPLICATIONS: Our findings indicate that important health care access barriers/disparities exist today and warrant improvement. STRENGTHS & LIMITATIONS: To our knowledge, this is the most comprehensive assessment of transgender transition-related health care coverage. Limitations include possible bias, as it could be that we were more persistent than actual patients would be to determine service coverage, and a lack of specificity regarding which specific hormone formulations or procedures are/are not covered. CONCLUSION: Our findings show that only 34 of 51 (67%) states' Medicaid programs include GAHT and 25 of 51 (49%) include GAS as covered benefits. Our experience suggests that the process to confirm coverage can be especially time-consuming and frustrating for patients. Zaliznyak M, Jung EE, Bresee C, et al. Which U.S. States' Medicaid Programs Provide Coverage for Gender-Affirming Hormone Therapy and Genital Gender-Affirming Surgery for Transgender Patients?: A State-by-State Review, and a Study Detailing the Patient Experience to Confirm Coverage of Service. J Sex Med 2021;18:410-422.

Writing in the Margins of Sexual Function Questionnaires: A Qualitative Analysis of Data From Women With Pelvic Floor Disorders.

BACKGROUND: The impact of pelvic floor disorders (PFDs) on female sexual function is not well understood, partly due to difficulties in measurement and evaluation. AIM: We sought to assess how women with PFDs respond to sexual function questionnaires through an analysis of survey marginalia, or the comments written in the margins of fixed-choice surveys. METHODS: 94 women with PFDs completed validated written sexual function questionnaires (Global Study of Sexual Attitudes and Behaviors survey, Female Sexual Function Index, and the Pelvic Organ Prolapse/Urinary Incontinence Sexual Questionnaire, International Urogynecological Association-Revised). Marginalia, or the additions, eliminations, and changes subjects made (by hand) to survey items, were collected. Data were coded and analyzed qualitatively using grounded theory methodology. OUTCOMES: Themes and emergent concepts related to the content of survey marginalia were the primary outcomes of this study. RESULTS: We observed 177 instances of marginalia across all questionnaires. Qualitative analysis revealed 7 preliminary themes and 2 emergent concepts. Preliminary themes included partner-related topics, loss, problems during intercourse, emotional problems, other medical problems, and survey answer choices failing to capture the spectrum of patient experiences. Emergent concepts revealed highly diverse sexual function in this population and a wide range of factors that influence sexual function. CLINICAL IMPLICATIONS: Conducting qualitative studies alongside sexual function questionnaires can allow for a more meaningful assessment of the sexual function of women with various underlying conditions, such as PFDs. STRENGTHS & LIMITATIONS: This is the first study of its kind to analyze survey marginalia from sexual function questionnaires among women with PFDs. The limitations of this study include the inherently spontaneous nature of marginalia data. In addition, the ways in which study participants responded to sexual function questionnaires in our study may not be reflective of all potential subjects. CONCLUSION: Analysis of survey marginalia from sexual function questionnaires amongst women with PFDs revealed new information regarding patients' histories, concerns, and thoughts. Over half of the women in this study felt the need to expand, explain, or eliminate responses from the questionnaires. Many subjects were no longer sexually active, which accounted for a large majority of participants leaving questions blank or responding with "N/A." Standard sexual evaluation tools may fail to capture the complexity, spectrum, and depth and breadth of patient experiences. Parameshwar PS, Borok J, Jung E, et al. Writing in the Margins of Sexual Function Questionnaires: A Qualitative Analysis of Data From Women With Pelvic Floor Disorders. J Sex Med 2020;17:1705-1714.

ZEB1 Mediates Fibrosis in Corneal Endothelial Mesenchymal Transition Through SP1 and SP3.

Purpose: ZEB1 is induced during endothelial-mesenchymal transition (EnMT) in the cornea. Induction of SP1 and SP3 by ZEB1 along with identification of putative SP1 and SP3 binding sites in promoters of EnMT-associated gene lead us to investigate their roles in retrocorneal membrane formation in the corneal endothelium. Methods: Expressions of SP1, SP3, and EnMT associated genes were analyzed by immunoblotting and semiquantitative reverse transcription polymerase chain reaction. Accell SMARTpool siRNAs targeting ZEB1, SP1, and SP3 were used for gene knockdown. SP1 and SP3 binding to promoters of EnMT associated genes was investigated by chromatin immunoprecipitation assay. Corneal endothelium in mice was surgically injured in vivo under direct visualization. Results: Transient Fibroblast Growth Factor 2 stimulation increased the expression of both SP1 and SP3 in the human corneal endothelium ex vivo. ZEB1 siRNA knockdown inhibited FGF2-induced SP1 mRNA and protein but not the expression of SP3. FGF2-induced expression of EnMT-related genes, such as fibronectin, vimentin, and type I collagen, was reduced by both SP1 and SP3 siRNA knockdown, with inhibition of SP1 having a greater inhibitory effect than SP3. Additionally, although SP1 and SP3 proteins were found to bind together, SP1 and SP3 could bind to the same promoter binding sites of EnMT-related genes in the absence of the other. Moreover, siRNA knockdown of Zeb1 inhibited injury-dependent RCM formation in mouse corneal endothelium in vivo. Conclusions: Zeb1, through SP1 and SP3, plays a central role in mesenchymal transition induced fibrosis in the corneal endothelium and suggests that Zeb1 could be targeted to inhibit anterior segment fibrosis.

Brown Urology: A Historical Perspective.

The first Urology Residency Program in the United States was founded at the Johns Hopkins Hospital in the wake of the first structured surgery residency as established by Dr. William Halsted in the early 20th Century.1 Dr. Hugh Hampton Young was selected to lead the Genitourinary Division and the foundation for the first urology residency training program was established.2 The Brown University Medical School, initially opened in 1811, effectively closed circa 1827, and re-established in the 1970s, has a long tradition in training surgeons.3,4 The Rhode Island Hospital Urology Residency Training Program was organized in the early 1950s and will be explored in this article. Brown University affiliated with the residency program in the mid-1980s to establish the first and only academic urology residency program in Rhode Island. Today, this program provides state-of-the-art urologic care for thousands of patients in the state.

Vision loss, gaze palsy, and nystagmus in a patient with leukemia.

A 22-year old male with a history of B-cell acute lymphoblastic leukemia with recent bone marrow transplantation and on immunosuppressive therapy presented with painless, subacute vision loss of two weeks duration. He exhibited a horizontal gaze palsy, nystagmus, and mildly swollen and hyperemic optic discs with peripapillary flame hemorrhage on retinal exam. He had bilateral cecocentral scotomas on visual field exam, and MRI of his brain/orbits demonstrated hyperintensities in the hypothalamus, periaqueductal gray, and dorsal rostral medullary regions. After continued progression of symptoms despite discontinuation of the patient's tacrolimus, an empiric trial of IV thiamine treatment was started before the patient's lab vitamin levels were available, given strong clinical suspicion for a nutritional etiology. The patient's clinical presentation improved dramatically, and he achieved a final visual acuity of 20/20, full visual fields bilaterally, and resolution of nystagmus. A final diagnosis of Wernicke's encephalopathy was supported by his clinical course, imaging findings, and further confirmation with blood thiamine levels. This case presents unique ocular manifestations of Wernicke's encephalopathy and highlights the importance of early diagnosis in this potentially reversible condition.

Synchronous recurrence of concurrent colon adenocarcinoma and dedifferentiated liposarcoma.

A 62-year-old man presented with concurrent sigmoid colon adenocarcinoma and small bowel mesenteric dedifferentiated liposarcoma. Following surgical resection of the colon cancer, complete excision of the mesenteric sarcoma and adjuvant folinic acid, fluorouracil and oxaliplatin (FOLFOX) chemotherapy, the patient demonstrated no radiological evidence of disease for more than 2 years. The patient then developed synchronous recurrence of both cancers: the colon cancer metastasised to the liver and a pelvic lymph node, and the liposarcoma recurred in the original location. The patient underwent additional chemotherapy with complete response of the metastatic colon cancer and stable disease for the liposarcoma. The recurrent mesenteric tumour was subsequently resected. Although concurrent cancers have been reported, this unique case of synchronous recurrence raises interesting hypotheses regarding host-tumour interaction and immune surveillance.

Injury induces endothelial to mesenchymal transition in the mouse corneal endothelium in vivo via FGF2.

Purpose: To determine whether the mouse corneal endothelium enters endothelial to mesenchymal transition (EndoMT) following surgical injury in vivo. Methods: The corneal endothelium in anesthetized mice was surgically injured in vivo under direct visualization. The secretion of interleukin-1 beta (IL-1ß) and fibroblast growth factor 2 (FGF2) into the aqueous humor was analyzed with western blotting. The expression of FGF2, Snai1, Zeb1, Col1a1, Col1a2, Fn1, Vim, Cdk2, Ccne1, and Cdh1 was analyzed with semiquantitative RT-PCR in the mouse corneal endothelium ex vivo and in vivo. Knockdown of FGF2 was done using siRNA. Col8a2 was used as a corneal endothelial marker, and Keratocan (Ktcn) was used as a stromal marker. ß-actin was used as a loading control. Results: Sequential expression of IL-1ß and FGF2 was detected in the aqueous humor after surgical injury. FGF2 treatment induced expression of endothelial to mesenchymal transition-related genes including Snai1, and Zeb1 in the mouse ex vivo corneal endothelium. This led to increased expression of Col1a1, Col1a2, Fn1, and Vim and suppression of Cdh1 in a time-dependent manner. Expression of FGF2, Snai1, Zeb1, Col1a1, Col1a2, Fn1, Vim, Cdk2, and Ccne1 was completely abolished by FGF2 siRNA knockdown in the mouse corneal endothelium ex vivo. Surgical injury induced FGF2 expression in the in vivo mouse corneal endothelium. The injury-dependent expression of FGF2, Snai1, Zeb1, Col1a1, Col1a2, Fn1, Vim, Cdk2, and Ccne1 and the suppression of Cdh1 were inhibited by siRNA knockdown of FGF in the mouse corneal endothelium in vivo. Moreover, siRNA knockdown of FGF2 inhibited the formation of the injury-dependent retrocorneal membrane in the in vivo mouse corneal endothelium. Conclusions: These findings suggest that after surgical injury, FGF2 induces the expression of EndoMT-related genes Snai1, Zeb1, Col1a1, Col1a2, Fn1, Vim, Cdk2, and Ccne1 in the mouse corneal endothelium in vivo, similar to the human corneal endothelium ex vivo.

Ureteral stenting practices following routine ureteroscopy: an international survey.

PURPOSE: Stent omission after routine ureteroscopy (rtURS) is accepted by current guidelines and may result in decreased patient morbidity and treatment costs. In a value-based healthcare model, the added morbidity and cost of routine stent placement may be scrutinized. Furthermore, data are limited on urologist cost knowledge and it is effect on ureteral stent placement. As such, we seek to describe ureteral stenting practices and urologist cost knowledge amongst US and non-US-based urologists. METHODS: The ureteroscopic practice patterns and cost awareness of members of the Endourological Society were surveyed using an international email listserv. Respondents were grouped by practice location (US vs non-US). Logistic regression was used to evaluate the associations of surgeon practice location with stenting practices. RESULTS: 233 completed responses were received with a response rate of 13.5%. Results revealed that 55% and 71% of respondents reported ureteral stent insertion after rtURS more than 75% of the time for ureteral and renal stones, respectively. Reporting stent insertion following more than 75% of rtURS was more common among US participants for both ureteral and renal stones. Overall, reported cost knowledge was high, but lower among US participants. On multivariable analysis, US respondents were more likely to place ureteral stents after rtURS for ureteral stones more than 75% of the time when compared to those abroad (OR 3.43 p < 0.01). CONCLUSION: Ureteral stenting after rtURS is over utilized in the US compared to other countries. While this phenomenon is multifactorial in nature, cost knowledge may be under recognized as a determinant of ureteral stent placement following rtURS.

The capsule of Cryptococcus neoformans.

The capsule of Cryptococcus neoformans is its dominant virulence factor and plays a key role in the biology of this fungus. In this essay, we focus on the capsule as a cellular structure and note the limitations inherent in the current methodologies available for its study. Given that no single method can provide the structure of the capsule, our notions of what is the cryptococcal capsule must be arrived at by synthesizing information gathered from very different methodological approaches including microscopy, polysaccharide chemistry and physical chemistry of macromolecules. The emerging picture is one of a carefully regulated dynamic structure that is constantly rearranged as a response to environmental stimulation and cellular replication. In the environment, the capsule protects the fungus against desiccation and phagocytic predators. In animal hosts the capsule functions in both offensive and defensive modes, such that it interferes with immune responses while providing the fungal cell with a defensive shield that is both antiphagocytic and capable of absorbing microbicidal oxidative bursts from phagocytic cells. Finally, we delineate a set of unsolved problems in the cryptococcal capsule field that could provide fertile ground for future investigations.

Mutant GATA3 Actively Promotes the Growth of Normal and Malignant Mammary Cells.

BACKGROUND/AIM: GATA3, a transcription factor expressed in luminal breast epithelial cells, is required for mammary gland development. Heterozygous GATA3 mutations occur in up to 15% of estrogen receptor (ER)-positive breast tumors and have been proposed to be null alleles resulting in haploinsufficiency; however, the mutation spectrum of GATA3 in breast cancer is in sharp contrast to that found in HDR syndrome, a true GATA3 haploinsufficiency disease. MATERIALS AND METHODS: Transgenic mice, 3D cultures and xenografts were used to examine the effect of mutant GATA3 expression on mammary cell proliferation. RESULTS: Mutant GATA3 accelerated tumor growth of ZR751 cell xenografts and promoted precocious lobuloalveolar development in transgenic mouse mammary glands. CONCLUSION: GATA3 mutations, recently observed in breast cancer, encode active transcription factors, which elicit proliferative phenotypes in normal mammary epithelium and promote the growth of ER-positive breast cancer cell lines.

Second Primary Malignancies in Patients with Well-differentiated/Dedifferentiated Liposarcoma.

BACKGROUND: Well-differentiated/dedifferentiated (WD/DD) liposarcoma is a rare malignancy of putative adipocyte origin. To our knowledge, there have only been isolated case reports describing second primary cancer in patients with this disease. We report on a combined case series of such patients and explore the frequency of this occurrence using a national cancer database. MATERIALS AND METHODS: Demographics and clinicopathological data were collected from patients with WD/DD liposarcoma who were found to have a concurrent or subsequent second primary cancer, at one of three sarcoma referral centers from 2014-2016. The Surveillance, Epidemiology and End Results (SEER) database was also queried to identify adult patients diagnosed with WD/DD liposarcoma between 1973-2012. Observed/expected (O/E) ratios of second primary malignancies among these cases were calculated by comparison to the age-adjusted cancer incidence in the general population using SEER*stat software. RESULTS: In total, 26 out of 312 consecutive patients (8.3%) with WD/DD liposarcoma at our centers had a second primary cancer identified within 2 years of liposarcoma diagnosis. In the SEER database, among 1,845 patients with WD/DD liposarcoma, 75 (4.1%) had a second cancer within 2 years after liposarcoma diagnosis (O/E ratio=1.81, 99% confidence interval(CI)=1.33-2.40). Patients less than 50 years old at the time of liposarcoma diagnosis had a higher O/E ratio for second primary malignancy compared to older patients. A total of 269 patients (14.6%) developed a second cancer (O/E=1.33, 99% CI=1.15-1.54). CONCLUSION: In some patients with WD/DD liposarcoma, there appears to be an increased risk of having a second primary cancer. Further validation and investigation is needed, as this finding may have implications (e.g. closer screening) for patients with this disease.

Cryptococcus neoformans urease affects the outcome of intracellular pathogenesis by modulating phagolysosomal pH.

Cryptococcus neoformans is a facultative intracellular pathogen and its interaction with macrophages is a key event determining the outcome of infection. Urease is a major virulence factor in C. neoformans but its role during macrophage interaction has not been characterized. Consequently, we analyzed the effect of urease on fungal-macrophage interaction using wild-type, urease-deficient and urease-complemented strains of C. neoformans. The frequency of non-lytic exocytosis events was reduced in the absence of urease. Urease-positive C. neoformans manifested reduced and delayed intracellular replication with fewer macrophages displaying phagolysosomal membrane permeabilization. The production of urease was associated with increased phagolysosomal pH, which in turn reduced growth of urease-positive C. neoformans inside macrophages. Interestingly, the ure1 mutant strain grew slower in fungal growth medium which was buffered to neutral pH (pH 7.4). Mice inoculated with macrophages carrying urease-deficient C. neoformans had lower fungal burden in the brain than mice infected with macrophages carrying wild-type strain. In contrast, the absence of urease did not affect survival of yeast when interacting with amoebae. Because of the inability of the urease deletion mutant to grow on urea as a sole nitrogen source, we hypothesize urease plays a nutritional role involved in nitrogen acquisition in the environment. Taken together, our data demonstrate that urease affects fitness within the mammalian phagosome, promoting non-lytic exocytosis while delaying intracellular replication and thus reducing phagolysosomal membrane damage, events that could facilitate cryptococcal dissemination when transported inside macrophages. This system provides an example where an enzyme involved in nutrient acquisition modulates virulence during mammalian infection.

Conservation of Intracellular Pathogenic Strategy among Distantly Related Cryptococcal Species.

The genus Cryptococcus includes several species pathogenic for humans. Until recently, the two major pathogenic species were recognized to be Cryptococcus neoformans and Cryptococcus gattii We compared the interaction of murine macrophages with three C. gattii species complex strains (WM179, R265, and WM161, representing molecular types VGI, VGIIa, and VGIII, respectively) and one C. neoformans species complex strain (H99, molecular type VNI) to ascertain similarities and differences in the yeast intracellular pathogenic strategy. The parameters analyzed included nonlytic exocytosis frequency, phagolysosomal pH, intracellular capsular growth, phagolysosomal membrane permeabilization, and macrophage transcriptional response, assessed using time-lapse microscopy, fluorescence microscopy, flow cytometry, and gene expression microarray analysis. The most striking result was that the intracellular pathogenic strategies of C. neoformans and C. gattii species complex strains were qualitatively similar, despite the species having separated an estimated 100 million years ago. Macrophages exhibited a leaky phagolysosomal membrane phenotype and nonlytic exocytosis when infected with either C. gattii or C. neoformans Conservation of the intracellular strategy among species that separated long ago suggests that it is ancient and possibly maintained by similar selection pressures through eons.

Novel Antifungal Compounds Discovered in Medicines for Malaria Venture's Malaria Box.

Similarities in fungal and animal cells make antifungal discovery efforts more difficult than those for other classes of antimicrobial drugs. Currently, there are only three major classes of antifungal drugs used for the treatment of systemic fungal diseases: polyenes, azoles, and echinocandins. Even in situations where the offending fungal organism is susceptible to the available drugs, treatment courses can be lengthy and unsatisfactory, since eradication of infection is often very difficult, especially in individuals with impaired immunity. Consequently, there is a need for new and more effective antifungal drugs. We have identified compounds with significant antifungal activity in the Malaria Box (Medicines for Malaria Ventures, Geneva, Switzerland) that have higher efficacy than some of the currently used antifungal drugs. Our best candidate, MMV665943 (IUPAC name 4-[6-[[2-(4-aminophenyl)-3H-benzimidazol-5-yl]methyl]-1H-benzimidazol-2-yl]aniline), here referred to as DM262, showed 16- to 32-fold-higher activity than fluconazole against Cryptococcus neoformans. There was also significant antifungal activity in other fungal species with known antifungal resistance, such as Lomentospora prolificans and Cryptococcus gattii. Antifungal activity was also observed against a common fungus, Candida albicans. These results are important because they offer a potentially new class of antifungal drugs and the repurposing of currently available therapeutics. IMPORTANCE Much like the recent increase in drug-resistant bacteria, there is a rise in antifungal-resistant strains of pathogenic fungi. There is a need for novel and more potent antifungal therapeutics. Consequently, we investigated a mixed library of drug-like and probe-like compounds with activity in Plasmodium spp. for activity against two common fungal pathogens, Cryptococcus neoformans and Candida albicans, along with two less common pathogenic species, Lomentospora prolificans and Cryptococcus gattii. We uncover a previously uncharacterized drug with higher broad-spectrum antifungal activity than some current treatments. Our findings may eventually lead to a compound added to the arsenal of antifungal therapeutics.