Comparison of Human Papillomavirus RNA In Situ Hybridization and p16 Immunostaining in Diagnostically Challenging High-Grade Squamous Intraepithelial Lesions in the Background of Atrophy.

CONTEXT.­: Human papillomavirus (HPV) in the postmenopausal age group is complex, with infected patients in this age group at increased risk of progressing to invasive disease and showing decreased clearance of the virus. Additionally, atrophic changes of the cervix can make histologic distinction of high-grade squamous intraepithelial lesions (HSILs) difficult. OBJECTIVE.­: To determine morphologic and ancillary testing characteristics of atrophy and HSIL in postmenopausal patients. DESIGN.­: Files of patients at least 65 years of age were examined, with 81 patients (109 cases [53 benign, 56 HSIL]) included in the study. Results of morphology, immunostaining (p16 and Ki-67), and HPV RNA in situ hybridization (ISH) were noted on all cases with available material. RESULTS.­: Atrophy was present in 96 of 109 cases (88%) overall. Coarse nuclear chromatin was noted in none of the benign cases, in 19 of 30 HSIL biopsies (63%), and in 24 of 26 HSIL excisions (92%). All benign cases were negative for p16 and ISH. In the HSIL cases, 45 of 53 (89%) were positive for p16, and of cases with sufficient tissue for ISH, 44 of 45 (98%) were positive. Of the ISH/p16 discordant cases (n = 7), most were p16 negative/ISH positive (6 of 7; 86%), whereas 1 of 7 (14%) was p16 positive and ISH negative. A majority of HSIL cases showed near-full-thickness elevation of Ki-67 (45 of 54; 83%), whereas mitotic figures were less elevated. CONCLUSIONS.­: In postmenopausal patients with HSIL, mitotic activity is not reliably elevated, but Ki-67 is consistently high. ISH is a more direct method of HPV detection and should be considered in cases where morphology and immunolabeling show discordance.

Placental SARS-CoV-2 distribution correlates with level of tissue oxygenation in COVID-19-associated necrotizing histiocytic intervillositis/perivillous fibrin deposition.

INTRODUCTION: Recent evidence supports the - rare - occurrence of vertical transplacental SARS-CoV-2 transmission. We previously determined that placental expression of angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 receptor, and associated viral cell entry regulators is upregulated by hypoxia. In the present study, we utilized a clinically relevant model of SARS-CoV-2-associated chronic histiocytic intervillositis/massive perivillous fibrin deposition (CHIV/MPFVD) to test the hypothesis that placental hypoxia may facilitate placental SARS-CoV-2 infection. METHODS: We performed a comparative immunohistochemical and/or RNAscope in-situ hybridization analysis of carbonic anhydrase IX (CAIX, hypoxia marker), ACE2 and SARS-CoV-2 expression in free-floating versus fibrin-encased chorionic villi in a 20-weeks' gestation placenta with SARS-CoV-2-associated CHIV/MPVFD. RESULTS: The levels of CAIX and ACE2 immunoreactivity were significantly higher in trophoblastic cells of fibrin-encased villi than in those of free-floating villi, consistent with hypoxia-induced ACE2 upregulation. SARS-CoV-2 showed a similar preferential localization to trophoblastic cells of fibrin-encased villi. DISCUSSION: The localization of SARS-CoV-2 to hypoxic, fibrin-encased villi in this placenta with CHIV/MPVFD suggests placental infection and, therefore, transplacental SARS-CoV-2 transmission may be promoted by hypoxic conditions, mediated by ACE2 and similar hypoxia-sensitive viral cell entry mechanisms. Understanding of a causative link between placental hypoxia and SARS-CoV-2 transmittability may potentially lead to the development of alternative strategies for prevention of intrauterine COVID-19 transmission.

Increased placental expression of angiotensin-converting enzyme 2, the receptor of SARS-CoV-2, associated with hypoxia in twin anemia-polycythemia sequence (TAPS).

INTRODUCTION: Recent reports suggest SARS-CoV-2, the virus causing COVID-19, may be transmittable from pregnant mother to placenta and fetus, albeit rarely. The efficacy of vertical transmission of SARS-CoV-2 critically depends on the availability of its receptor, ACE2, in the placenta. In the present study, we tested the hypothesis that placental ACE2 expression is oxygenation-dependent by studying the expression of ACE2 and associated cell entry regulators in the monochorionic twin anemia-polycythemia (TAPS) placenta, a model of discordant placental oxygenation. METHODS: We performed a retrospective comparative immunohistochemical, immunofluorescence and Western blot analysis of ACE2, TMPRSS2 and Cathepsin B expression in anemic and polycythemic territories of TAPS placentas (N = 14). RESULTS: ACE2 protein levels were significantly higher in the anemic twin territories than in the corresponding polycythemic territories, associated with upregulation of the key ACE2-related cell entry regulators, TMPRSS2 and Cathepsin B, immunolocalized to villous trophoblastic and stromal cells. Cellular colocalization of ACE2 and TMPRSS2, suggestive of functionality of this cell entry axis, was demonstrated by double immunofluorescence studies. DISCUSSION: Placental hypoxia is associated with upregulation of ACE2 expression, concomitant with increased expression of its key cell entry proteases. ACE2-regulated placental functions, both infection- and non-infection related, may be highly oxygenation-dependent.

Stromal cell-derived factor-1 (SDF-1) expression in very preterm human lungs: potential relevance for stem cell therapy for bronchopulmonary dysplasia.

Background: The axis formed by CXC chemokine receptor 4 (CXCR4), expressed on mesenchymal stromal cells (MSCs), and stromal cell-derived factor-1 (SDF-1), expressed in recipient organs, is a critical mediator of MSC migration in non-pulmonary injury models. The role and regulation of SDF-1 expression in preterm lungs, of potential relevance for MSC-based cell therapy for bronchopulmonary dysplasia (BPD), is unknown. The aim of this study was to determine the spatiotemporal pattern of CXCR4/SDF-1 expression in lungs of extremely preterm infants at risk for BPD.Methods: Postmortem lung samples were collected from ventilated extremely preterm infants who died between 23 and 29 wks ("short-term ventilated") or between 36 and 39 wks ("long-term ventilated") corrected postmenstrual age. Results were compared with age-matched infants who had lived <12 h or stillborn infants ("early" and "late" controls). CXCR4 and SDF-1 expression was studied by immunohistochemistry, immunofluorescence/confocal microscopy, and qRT-PCR analysis.Results: Compared with age-matched controls without antenatal infection, lungs of early control infants with evidence of intrauterine infection/inflammation showed significant upregulation of SDF-1 expression, localized to the respiratory epithelium, and of CXCR4 expression, localized to stromal cells. Similarly, pulmonary SDF-1 mRNA levels were significantly higher in long-term ventilated ex-premature infants with established BPD than in age-matched controls. The pulmonary vasculature was devoid of SDF-1 expression at all time points. Endogenous CXCR4-positive stromal cells were preferentially localized along the basal aspect of SDF-1-positive bronchial and respiratory epithelial cells, suggestive of functionality of the CXCR4/SDF-1 axis.Conclusions: Incipient and established neonatal lung injury is associated with upregulation of SDF-1 expression, restricted to the respiratory epithelium. Knowledge of the clinical associations, time-course and localization of pulmonary SDF-1 expression may guide decisions about the optimal timing and delivery route of MSC-based cell therapy for BPD.

Discordant placental oxygenation and autophagy in twin anemia-polycythemia sequence (TAPS).

BACKGROUND: (Macro)autophagy is an important process of self-degradation of macromolecules and organelles that ensures cellular homeostasis and energy preservation during stressful conditions. Dysregulated placental autophagy has been implicated in a wide range of pregnancy complications. Recent studies identified hypoxia as a key regulator of trophoblast autophagy in vitro; however, its effects on placental autophagy in vivo remain incompletely understood. In this study, we evaluated the monochorionic twin anemia-polycythemia sequence (TAPS) placenta as model of discordant placental oxygenation to determine the effects of hypoxia on placental autophagy in utero. METHODS: We performed a retrospective comparative analysis of tissue oxygenation and autophagy in anemic and polycythemic territories of TAPS placentas (N = 12). Archival tissues were subjected to immunohistochemical, immunofluorescence and Western blot analyses of carbonic anhydrase (CA) IX (hypoxia marker) and key autophagy/lysosomal markers. RESULTS: CAIX protein levels were significantly higher in anemic twin territories than in corresponding polycythemic territories, consistent with relative tissue hypoxia. Anemic placental shares further displayed significantly higher levels of LC3I/II (autophagosome markers) and LAMP1/2 (lysosome markers), associated with upregulated expression of lysosome/autophagosome activity-associated markers, transcription factor EB and cathepsin D. The accumulation of autophagosomes and lysosomes in anemic shares was accompanied by elevated p62 protein expression, suggestive of inhibition of the downstream autophagy pathway. CONCLUSIONS: TAPS placentas display striking intertwin discordance in tissue oxygenation and autophagic activity and may provide a suitable model for study of the interrelationship between hypoxia, autophagy, and pregnancy outcome in a monochorionic twin setting.

Pathology of Neonatal Non- albicans Candidiasis: Autopsy Study and Literature Review.

INTRODUCTION/OBJECTIVES: Non- albicans Candida species such as Candida parapsilosis and Candida glabrata have emerged as prevalent pathogens in premature infants. The aim of this study was to systematically delineate the histopathologic findings in neonatal non- albicans candidiasis. METHODS: We performed a retrospective clinicopathologic analysis of extremely premature (23-28 weeks' gestation) infants diagnosed with invasive candidiasis. Archival autopsy tissues were subjected to periodic acid-Schiff, methenamine-silver and anti- Candida (immuno)histochemical stains, as well as dual anti- Candida and anti-cytokeratin or anti-CD31 immunofluorescence assays. In addition, we studied the prevalence of intestinal Candida colonization in a consecutive autopsy series of extremely premature infants. RESULTS: Based on positive postmortem blood and/or lung cultures, invasive candidiasis (3 non- albicans and 11 Candida albicans) was diagnosed in 14 of the 187 extremely premature infants examined between 1995 and 2017. In contrast to the well-known inflammatory and tissue-destructive phenotype of congenital C. albicans infection, invasive non- albicans candidiasis/candidemia caused by C. parapsilosis and C. glabrata was inconspicuous by routine hematoxylin-eosin-based histopathologic analysis despite a heavy fungal presence detected in intestines, lungs, and blood by targeted (immuno)histochemical assays. Intestinal colonization by Candida species was identified in 16 of the 26 (61%) extremely premature neonates who had lived for at least 1 week, as assessed by anti- Candida immunostaining. CONCLUSION: Invasive neonatal non- albicans candidiasis/candidemia appears to have no distinct histopathologic signature. Based on the notoriously low sensitivity of fungal blood cultures and the observed high frequency of Candida intestinal colonization (>50%), it is likely that non- albicans candidiasis/candidemia may be underdiagnosed in (deceased) preterm infants. Routine inclusion of targeted (immuno)histochemical fungal detection strategies in the perinatal autopsy may lead to deeper insight into the prevalence and clinical relevance of neonatal non- albicans candidiasis.

Redness discordance in monochorionic twin placentas: Correlation with clinical and placental findings.

INTRODUCTION/OBJECTIVES: Recent studies suggest redness (color) discordance of the placental basal plate may be a marker for twin anemia-polycythemia sequence (TAPS), a recently described complication of diamniotic-monochorionic twinning characterized by marked intertwin hemoglobin (Hb) discordance in the absence of oligohydramnios-polyhydramnios. In this study, we determined the clinicoplacental and choriovascular correlates of basal plate color discordance in monochorionic twin placentas, and assessed its value as postnatal indicator of TAPS. METHODS: We performed a clinicoplacental analysis of 100 consecutive non-TTTS diamniotic-monochorionic twin placentas with available photographic documentation of the basal plate. Basal plate redness was quantified by computer-assisted analysis of digital images and expressed as intertwin color difference ratio (CDR). RESULTS: The CDR ranged between 1.00 and 3.58 (median CDR: 1.14; 90th %ile: 1.98). Compared to twins with low CDR (N = 90), twins with high CDR (≥2.0; N = 10) had significantly higher hemoglobin difference (11.25 g/dL versus 2.55 g/dL) and significantly fewer and smaller artery-to-artery (AA) and artery-to-vein (AV) anastomoses. Apgar scores and birth weights were equivalent in both groups. Among the 10 twin sets with high CDR, six (60%) qualified as TAPS, as defined by intertwin Hb difference >8 g/dL and absent or very small AA and AV anastomoses. Conversely, 6 of 8 (75%) twin sets with TAPS had a CDR ≥ 2.0. CONCLUSION: Intertwin CDR correlates with intertwin hemoglobin difference and chorionic angioarchitecture. A CDR value ≥ 2.0 (the 90%ile value for CDR derived from the present cohort) has high specificity (96%), but relatively low positive predictive value (60%) as indicator of TAPS, as currently defined.

Clinicoplacental correlates of amniocyte vacuolization in association with gastroschisis.

INTRODUCTION/OBJECTIVES: Gastroschisis has been associated with a characteristic type of amniocyte vacuolization. In this study, we determined the frequency and clinicoplacental correlates of this apparently unique alteration of the amniotic epithelium. METHODS: We performed a retrospective clinicopathologic analysis of 74 consecutive cases of isolated gastroschisis. Placental membrane sections were reviewed for presence and extent of amniocyte vacuolization, and immunostained for adipophilin, a lipid droplet-associated protein. Controls included placentas from pregnancies complicated by omphalocele, meconium exposure or chorioamnionitis. RESULTS: A distinct type of diffuse, fine and homogeneous amniocyte vacuolization was present in 15/74 (20%), absent in 41/74 (55%), and equivocal in 18/74 (24%) gastroschisis cases. Similar amniocyte vacuolization was seen in only 1/30 meconium-stained controls, and in none of the other non-gastroschisis controls. Adipophilin immunostaining enhanced the visualization of the cytoplasmic vacuoles and confirmed their lipid nature. Compared to gastroschisis cases without such vacuolization, cases with typical, extensive amniocyte vacuolization had a tendency to lower birth weight percentile (26% versus 40%; P < 0.08), a significantly lower fetal:placental weight ratio (4.72 versus 5.51; P < 0.01), and a significantly higher frequency of associated meconium exposure (14/15 versus 15/41, P < 0.001) and/or chorioamnionitis (8/15 versus 6/41; P < 0.01). The length of hospital stay was equivalent for infants with or without amniocyte vacuolization. CONCLUSION: Diffuse, fine and homogeneous lipid droplet accumulation in amniocytes is highly characteristic of gastroschisis, but only seen in about 20% of cases. The functional implications of excessive lipid accumulation, and the exact mechanisms underlying the strong association between amniocyte lipid accumulation and chorioamnionitis/meconium exposure in a subset of gastroschisis cases remain undetermined.

Effects of human umbilical cord blood mononuclear cells on respiratory system mechanics in a murine model of neonatal lung injury.

BACKGROUND: Mononuclear cells (MNCs) have well-documented beneficial effects in a wide range of adult pulmonary diseases. The effects of human umbilical cord blood-derived MNCs on neonatal lung injury, highly relevant for potential autologous application in preterm newborns at risk for bronchopulmonary dysplasia (BPD), remain incompletely established. The aim of this study was to determine the long-term morphologic and functional effects of systemically delivered MNCs in a murine model of neonatal lung injury. MATERIALS AND METHODS: MNCs from cryopreserved cord blood (1 × 106 cells per pup) were given intravenously to newborn mice exposed to 90% O2 from birth; controls received cord blood total nucleated cells (TNCs) or granular cells, or equal volume vehicle buffer (sham controls). In order to avoid immune rejection, we used SCID mice as recipients. Lung mechanics (flexiVent™), engraftment, growth, and alveolarization were evaluated eight weeks postinfusion. RESULTS: Systemic MNC administration to hyperoxia-exposed newborn mice resulted in significant attenuation of methacholine-induced airway hyperreactivity, leading to reduction of central airway resistance to normoxic levels. These bronchial effects were associated with mild improvement of alveolarization, lung compliance, and elastance. TNCs had no effects on alveolar remodeling and were associated with worsened methacholine-induced bronchial hyperreactivity. Granular cell administration resulted in a marked morphologic and functional emphysematous phenotype, associated with high mortality. Pulmonary donor cell engraftment was sporadic in all groups. CONCLUSIONS: These results suggest that cord blood MNCs may have a cell type-specific role in therapy of pulmonary conditions characterized by increased airway resistance, such as BPD and asthma. Future studies need to determine the active MNC subtype(s), their mechanisms of action, and optimal purification methods to minimize granular cell contamination.

Intussusceptive-like angiogenesis in human fetal lung xenografts: Link with bronchopulmonary dysplasia-associated microvascular dysangiogenesis?

BACKGROUND: Human fetal lung xenografts display an unusual pattern of non-sprouting, plexus-forming angiogenesis that is reminiscent of the dysmorphic angioarchitecture described in bronchopulmonary dysplasia (BPD). The aim of this study was to determine the clinicopathological correlates, growth characteristics and molecular regulation of this aberrant form of graft angiogenesis. METHODS: Fetal lung xenografts, derived from 12 previable fetuses (15 to 22 weeks' gestation) and engrafted in the renal subcapsular space of SCID-beige mice, were analyzed 4 weeks posttransplantation for morphology, vascularization, proliferative activity and gene expression. RESULTS: Focal plexus-forming angiogenesis (PFA) was observed in 60/230 (26%) of xenografts. PFA was characterized by a complex network of tortuous nonsprouting vascular structures with low endothelial proliferative activity, suggestive of intussusceptive-type angiogenesis. There was no correlation between the occurrence of PFA and gestational age or time interval between delivery and engraftment. PFA was preferentially localized in the relatively hypoxic central subcapsular area. Microarray analysis suggested altered expression of 15 genes in graft regions with PFA, of which 7 are known angiogenic/lymphangiogenic regulators and 5 are known hypoxia-inducible genes. qRT-PCR analysis confirmed significant upregulation of SULF2, IGF2, and HMOX1 in graft regions with PFA. CONCLUSION: These observations in human fetal lungs ex vivo suggest that postcanalicular lungs can switch from sprouting angiogenesis to an aberrant intussusceptive-type of angiogenesis that is highly reminiscent of BPD-associated dysangiogenesis. While circumstantial evidence suggests hypoxia may be implicated, the exact triggering mechanisms, molecular regulation and clinical implications of this angiogenic switch in preterm lungs in vivo remain to be determined.

Intranasal versus intraperitoneal delivery of human umbilical cord tissue-derived cultured mesenchymal stromal cells in a murine model of neonatal lung injury.

Clinical trials investigating mesenchymal stromal cell (MSC) therapy for bronchopulmonary dysplasia have been initiated; however, the optimal delivery route and functional effects of MSC therapy in newborns remain incompletely established. We studied the morphologic and functional effects of intranasal versus i.p. MSC administration in a rodent model of neonatal lung injury. Cultured human cord tissue MSCs (0.1, 0.5, or 1 × 10(6) cell per pup) were given intranasally or i.p. to newborn severe combined immunodeficiency-beige mice exposed to 90% O2 from birth; sham controls received an equal volume of phosphate-buffered saline. Lung mechanics, engraftment, lung growth, and alveolarization were evaluated 8 weeks after transplantation. High-dose i.p. MSC administration to newborn mice exposed to 90% O2 resulted in the restoration of normal lung compliance, elastance, and pressure-volume loops (tissue recoil). Histologically, high-dose i.p. MSC administration was associated with alveolar septal widening, suggestive of interstitial matrix modification. Intranasal MSC or lower-dose i.p. administration had no significant effects on lung function or alveolar remodeling. Pulmonary engraftment was rare in all the groups. These findings suggest that high-dose systemic administration of human cultured MSCs can restore normal compliance in neonatally injured lungs, possibly by paracrine modulation of the interstitial matrix. Intranasal delivery had no obvious pulmonary effects.

Ex vivo expanded human cord blood-derived hematopoietic progenitor cells induce lung growth and alveolarization in injured newborn lungs.

BACKGROUND: We investigated the capacity of expanded cord blood-derived CD34+ hematopoietic progenitor cells to undergo respiratory epithelial differentiation ex vivo, and to engraft and attenuate alveolar disruption in injured newborn murine lungs in vivo. METHODS: Respiratory epithelial differentiation was studied in CD34+ cells expanded in the presence of growth factors and cytokines ("basic" medium), in one group supplemented with dexamethasone ("DEX"). Expanded or freshly isolated CD34+ cells were inoculated intranasally in newborn mice with apoptosis-induced lung injury. Pulmonary engraftment, lung growth and alveolarization were studied at 8 weeks post-inoculation. RESULTS: SP-C mRNA expression was seen in 2/7 CD34+ cell isolates expanded in basic media and in 6/7 isolates expanded in DEX, associated with cytoplasmic SP-C immunoreactivity and ultrastructural features suggestive of type II cell-like differentiation. Administration of expanding CD34+ cells was associated with increased lung growth and, in animals treated with DEX-exposed cells, enhanced alveolar septation. Freshly isolated CD34+ cells had no effect of lung growth or remodeling. Lungs of animals treated with expanded CD34+ cells contained intraalveolar aggregates of replicating alu-FISH-positive mononuclear cells, whereas epithelial engraftment was extremely rare. CONCLUSION: Expanded cord blood CD34+ cells can induce lung growth and alveolarization in injured newborn lungs. These growth-promoting effects may be linked to paracrine or immunomodulatory effects of persistent cord blood-derived mononuclear cells, as expanded cells showed limited respiratory epithelial transdifferentiation.

Correlation between cord insertion type and chorionic villus vascularization of the co-twin in diamniotic-monochorionic twin pregnancies.

BACKGROUND: Recent evidence suggests that cord insertion type of one twin correlates with chorionic plate vascularization of the monochorionic co-twin. Specifically, for twins with paracentral cords, chorionic plate vascularization is significantly greater when the co-twin has a velamentous, rather than paracentral cord insertion. AIMS: To determine whether this correlation between cord insertion type and vascularization of the co-twin also extends to the deeper chorionic villus tree. STUDY DESIGN: Morphometric analysis of chorionic villus vascularization in CD31-immunostained sections of a retrospective cohort of gestational age-matched third trimester monochorionic placentas with discordant paracentral/velamentous (PC/V) or concordant paracentral/paracentral (PC/PC) cord insertions. OUTCOME MEASURES: Vascular numerical density (number of vascular profiles per unit villus stromal area) of intermediate villi (>80 µm diameter) and terminal villi (<80 µm). RESULTS: For twins with paracentral cord insertion, the vascular numerical density of intermediate villi was significantly higher for twins in a discordant PC/V relationship than for those in a concordant PC/PC relationship (P<0.05), thus replicating previous findings in superficial chorionic vessels. For terminal villi, in contrast, the vascular numerical density of twins with paracentral cords in a PC/V combination was significantly lower than of those in a PC/PC combination, and similar to that of their co-twins with velamentous cord insertion. CONCLUSIONS: Early placental angiogenesis in monochorionic twin gestations may be influenced by implantation and cord localization of the co-twin. The regulation of terminal villus angiogenesis appears to be dissociated from more proximal villus angiogenesis and independent of cord insertion of the co-twin.

Long-term outcome of human cord blood-derived hematopoietic progenitor cells in murine lungs.

Intranasally delivered human cord blood-derived CD34+ hematopoietic progenitor cells have the capacity to engraft and undergo transdifferentiation to surfactant-containing alveolar epithelial type II cell-like cells in lungs of newborn mice. The aim of this study was to determine the long-term fate of such transplanted cells as well as their effects on alveolar development in neonatally injured lungs. Double transgenic CCSP+/FasL+ mice with inducible lung-specific FasL expression, targeted to induce respiratory epithelial apoptosis in the perinatal period, served as model of neonatal lung injury. Non-injured single transgenic CCSP+/FasL- littermates served as controls. Freshly isolated umbilical cord blood CD34+ cells (0.5 to 1.0×10(6)) were administered at postnatal day 5 by intranasal inoculation; sham controls received equal volume PBS. Engraftment, alveolar epithelial differentiation, lung growth, and alveolarization were evaluated one year after transplantation. Engrafted cord blood-derived cells, detected by human-specific FISH (fluorescent in situ hybridization) analysis, and cord blood-derived alveolar type II-like cells, detected by double immunofluorescence analysis, while sparse, were seen in all conditions and more frequent in double than single transgenic recipients. The total lung volume and volume of air-exchanging parenchyma, assessed by stereological volumetry, were significantly greater in CD34-treated double transgenic animals than in PBS-treated double transgenic controls. Alveolarization, assessed by histomorphometry, was equivalent in these groups. These results suggest that transdifferentiated alveolar epithelial cells, derived from cord blood CD34+ cells, can persist up to one year after intrapulmonary delivery. Cord blood-CD34+ cell administration appears to have growth-promoting effects in injured newborn lungs, without affecting alveolar development in this model.

The human fetal lung xenograft: validation as model of microvascular remodeling in the postglandular lung.

BACKGROUND: Coordinated remodeling of epithelium and vasculature is essential for normal postglandular lung development. The value of the human-to-rodent lung xenograft as model of fetal microvascular development remains poorly defined. AIM: The aim of this study was to determine the fate of the endogenous (human-derived) microvasculature in fetal lung xenografts. METHODS: Lung tissues were obtained from spontaneous pregnancy losses (14-22 weeks' gestation) and implanted in the renal subcapsular or dorsal subcutaneous space of SCID-beige mice (T, B, and NK-cell-deficient) and/or nude rats (T-cell-deficient). Informed parental consent was obtained. Lung morphogenesis, microvascular angiogenesis, and epithelial differentiation were assessed at 2 and 4 weeks post-transplantation by light microscopy, immunohistochemical, and gene expression studies. Archival age-matched postmortem lungs served as control. RESULTS: The vascular morphology, density, and proliferation of renal subcapsular grafts in SCID-beige mice were similar to age-matched control lungs, with preservation of the physiologic association between epithelium and vasculature. The microvasculature of subcutaneous grafts in SCID-beige mice was underdeveloped and dysmorphic, associated with significantly lower VEGF, endoglin, and angiopoietin-2 mRNA expression than renal grafts. Grafts at both sites displayed mild airspace dysplasia. Renal subcapsular grafts in nude rats showed frequent infiltration by host lymphocytes and obliterating bronchiolitis-like changes, associated with markedly decreased endogenous angiogenesis. CONCLUSION: This study demonstrates the critical importance of host and site selection to ensure optimal xenograft development. When transplanted to severely immune suppressed, NK-cell-deficient hosts and engrafted in the renal subcapsular site, the human-to-rodent fetal lung xenograft provides a valid model of postglandular microvascular lung remodeling.

Efficacy of a non-hypercalcemic vitamin-D2 derived anti-cancer agent (MT19c) and inhibition of fatty acid synthesis in an ovarian cancer xenograft model.

BACKGROUND: Numerous vitamin-D analogs exhibited poor response rates, high systemic toxicities and hypercalcemia in human trials to treat cancer. We identified the first non-hypercalcemic anti-cancer vitamin D analog MT19c by altering the A-ring of ergocalciferol. This study describes the therapeutic efficacy and mechanism of action of MT19c in both in vitro and in vivo models. METHODOLOGY/PRINCIPAL FINDING: Antitumor efficacy of MT19c was evaluated in ovarian cancer cell (SKOV-3) xenografts in nude mice and a syngenic rat ovarian cancer model. Serum calcium levels of MT19c or calcitriol treated animals were measured. In-silico molecular docking simulation and a cell based VDR reporter assay revealed MT19c-VDR interaction. Genomewide mRNA analysis of MT19c treated tumors identified drug targets which were verified by immunoblotting and microscopy. Quantification of cellular malonyl CoA was carried out by HPLC-MS. A binding study with PPAR-Y receptor was performed. MT19c reduced ovarian cancer growth in xenograft and syngeneic animal models without causing hypercalcemia or acute toxicity. MT19c is a weak vitamin-D receptor (VDR) antagonist that disrupted the interaction between VDR and coactivator SRC2-3. Genome-wide mRNA analysis and western blot and microscopy of MT19c treated xenograft tumors showed inhibition of fatty acid synthase (FASN) activity. MT19c reduced cellular levels of malonyl CoA in SKOV-3 cells and inhibited EGFR/phosphoinositol-3kinase (PI-3K) activity independently of PPAR-gamma protein. SIGNIFICANCE: Antitumor effects of non-hypercalcemic agent MT19c provide a new approach to the design of vitamin-D based anticancer molecules and a rationale for developing MT19c as a therapeutic agent for malignant ovarian tumors by targeting oncogenic de novo lipogenesis.

Resilience of the human fetal lung following stillbirth: potential relevance for pulmonary regenerative medicine.

Recent advances in pulmonary regenerative medicine have increased the demand for alveolar epithelial progenitor cells. Fetal lung tissues from spontaneous pregnancy losses may represent a neglected, yet ethically and societally acceptable source of alveolar epithelial cells. The aim of this study was to determine the regenerative capacity of fetal lungs obtained from second trimester stillbirths. Lung tissues were harvested from 11 stillborn fetuses (13 to 22 weeks' gestation) at postdelivery intervals ranging from 10 to 41 hours and grafted to the renal subcapsular space of immune-suppressed rats to provide optimal growth conditions. Histology, epithelial and alveolar type II cell proliferation, and surfactant protein-C mRNA expression were studied in preimplantation lung tissues and in xenografts at posttransplantation week 2. All xenografts displayed advanced architectural maturation compared with their respective preimplantation tissues, regardless of gestational age and postdelivery interval. The proliferative activity of the grafts was significantly higher than that of the preimplantation tissues (mean Ki-67 labeling index 26.7%±7.7% versus 14.7%±10.5%; P<.01). The proliferative activity of grafts obtained after a long (>36 hours) postdelivery interval was significantly higher than that of the corresponding preimplantation tissue, and equivalent to that of grafts obtained after a short postdelivery interval (<14 hours). The regenerative capacity of fetal lung tissue was greater at younger (13 to 17 weeks) than at older (19 to 22 weeks) gestational ages. The presence of inflammation/chorioamnionitis did not appear to affect graft regeneration. All grafts studied displayed robust surfactant protein-C mRNA expression. In conclusion, fetal lung tissues from second trimester stillbirths can regain their inherent high regenerative potential following short-term culture, even if harvested more than 36 hours after delivery.

Cardiac surgery in a patient with paroxysmal nocturnal hemoglobinuria.

A 44-year-old woman, who had been diagnosed with paroxysmal nocturnal hemoglobinuria, presented with chest pain. Angiography revealed critical distal left main stenosis and proximal stenoses of the left anterior descending and circumflex arteries. Prophylactic antibiotics, infusion of blood products and mannitol intraoperatively, and antithrombotic therapy postoperatively ensured a good outcome of coronary artery bypass graft surgery under cardiopulmonary bypass.

Emergency surgical embolectomy for pulmonary emboli after failed thrombolysis.

A 34-year-old man presented in cardiogenic shock secondary to massive pulmonary embolism. Surgical embolectomy was performed after failed thrombolysis. Intraoperatively, a right atrial lesion and pulmonary emboli were removed. Histopathology revealed myxoma complicated by pulmonary emboli. The patient made a good recovery. This case suggests that surgical embolectomy should be considered as first-line treatment for all patients with acute massive pulmonary embolism, and not reserved for those with severe hemodynamic compromise or failed conservative management.