A novel somatic mutation in GNAQ in a capillary malformation provides insight into molecular pathogenesis.

Sturge-Weber syndrome (SWS) is a sporadic, congenital, neuro-cutaneous disorder characterized by a mosaic, capillary malformation. SWS and non-syndromic capillary malformations are both caused by a somatic activating mutation in GNAQ encoding the G protein subunit alpha-q protein. The missense mutation R183Q is the sole GNAQ mutation identified thus far in 90% of SWS-associated or isolated capillary malformations. In this study, we sequenced skin biopsies of capillary malformations from 9 patients. We identified the R183Q mutation in nearly all samples, but one sample exhibited a Q209R mutation. This new mutation occurs at the same residue as the constitutively-activating Q209L mutation, commonly seen in tumors. However, Q209R is a rare variant in this gene. To compare the effect of the Q209R mutation on downstream signaling, we performed reporter assays with a GNAQ-responsive reporter co-transfected with either GNAQ WT, R183Q, Q209L, Q209R, or C9X (representing a null allele). Q209L showed the highest reporter activation, with R183Q and Q209R showing significantly lower activation. To determine whether these mutations had similar or different downstream consequences we performed RNA-seq analysis in microvascular endothelial cells (HMEC-1) electroporated with the same GNAQ variants. The R183 and Q209 missense variants caused extensive dysregulation of a broad range of transcripts compared to the WT or null allele, confirming that these are all activating mutations. However, the missense variants exhibited very few differentially expressed genes (DEGs) when compared to each other. These data suggest that these activating GNAQ mutations differ in magnitude of activation but have similar downstream effects.

Transabdominal Intrapericardial Approach in Liver Transplantation for Unresectable Primary Hepatic Functioning Paraganglioma With Invasion Into Hepatic Veins and Suprahepatic Vena Cava: A Surgical and Anesthesia Management Challenge.

Primary hepatic functional paraganglioma is a rare form of extra-adrenal catecholamine-secreting tumor. Definitive treatment of functioning paraganglioma is challenging because of the critical location of the tumor frequently in close proximity to vital structures and risk of excessive catecholamine release during operative manipulation. We report the multidisciplinary management approach for a case of unresectable primary hepatic functional paraganglioma with invasion into the hepatic veins and suprahepatic vena cava. To our knowledge, this is the first report showing that orthotopic liver transplantation is curative for patients with unresectable primary hepatic paraganglioma. For locally advanced unresectable hepatic paraganglioma that involves the intrapericardial vena cava, a meticulous pre- and intraoperative medical management and transabdominal intrapericardial vascular control of the suprahepatic vena cava during orthotopic liver transplantation allows for complete extirpation of the tumor and achieves optimal outcome.

Previsual symptoms of Xylella fastidiosa infection revealed in spectral plant-trait alterations.

Plant pathogens cause significant losses to agricultural yields and increasingly threaten food security1, ecosystem integrity and societies in general2-5. Xylella fastidiosa is one of the most dangerous plant bacteria worldwide, causing several diseases with profound impacts on agriculture and the environment6. Primarily occurring in the Americas, its recent discovery in Asia and Europe demonstrates that X. fastidiosa's geographic range has broadened considerably, positioning it as a reemerging global threat that has caused socioeconomic and cultural damage7,8. X. fastidiosa can infect more than 350 plant species worldwide9, and early detection is critical for its eradication8. In this article, we show that changes in plant functional traits retrieved from airborne imaging spectroscopy and thermography can reveal X. fastidiosa infection in olive trees before symptoms are visible. We obtained accuracies of disease detection, confirmed by quantitative polymerase chain reaction, exceeding 80% when high-resolution fluorescence quantified by three-dimensional simulations and thermal stress indicators were coupled with photosynthetic traits sensitive to rapid pigment dynamics and degradation. Moreover, we found that the visually asymptomatic trees originally scored as affected by spectral plant-trait alterations, developed X. fastidiosa symptoms at almost double the rate of the asymptomatic trees classified as not affected by remote sensing. We demonstrate that spectral plant-trait alterations caused by X. fastidiosa infection are detectable previsually at the landscape scale, a critical requirement to help eradicate some of the most devastating plant diseases worldwide.

C560Rß3 caused platelet integrin αII b ß3 to bind fibrinogen continuously, but resulted in a severe bleeding syndrome and increased murine mortality.

BACKGROUND AND OBJECTIVES: ß(3)-Deficient megakaryocytes were modified by human ß(3)-lentivirus transduction and transplantation to express sufficient levels of a C560Rß(3) amino acid substitution, for investigation of how an activated αII b ß(3) conformation affects platelets in vivo in mice. PATIENT/METHODS: As in our previous report of an R560ß(3) mutation in a patient with Glanzmann thrombasthenia, R560ß(3) murine platelets spontaneously bound antibody that only recognizes activated αII b ß3 bound to its ligand, fibrinogen. RESULTS: With this murine model, we showed that αII b -R560ß3 mutation-mediated continuous binding of fibrinogen occurred in the absence of P-selectin surface expression, indicating that the integrin was in an active conformation, although the platelets circulated in a quiescent manner. Remarkably, only 35% of R560ß(3) 'mutant' mice survived for 6 months after transplantation, whereas 87% of C560ß(3) 'wild-type' mice remained alive. Pathologic examination revealed that R560ß(3) mice had enlarged spleens with extramedullary hematopoiesis and increased hemosiderin, indicating hemorrhage. R560ß(3) megakaryocytes and platelets showed abnormal morphology and irregular granule distribution. Interestingly, R560ß(3) washed platelets could aggregate upon simultaneous addition of fibrinogen and physiologic agonists, but aggregation failed when platelets were exposed to fibrinogen before activation in vitro and in vivo. CONCLUSIONS: The results demonstrate that continuous occupancy of αIIb ß3 with fibrinogen disrupts platelet structure and function, leading to hemorrhagic death consistent with Glanzmann thrombasthenia rather than a thrombotic state.

Topo IIIalpha and BLM act within the Fanconi anemia pathway in response to DNA-crosslinking agents.

The Bloom protein (BLM) and Topoisomerase IIIalpha are found in association with proteins of the Fanconi anemia (FA) pathway, a disorder manifesting increased cellular sensitivity to DNA crosslinking agents. In order to determine if the association reflects a functional interaction for the maintenance of genome stability, we have analyzed the effects of siRNA-mediated depletion of the proteins in human cells. Depletion of Topoisomerase IIIalpha or BLM leads to increased radial formation, as is seen in FA. BLM and Topoisomerase IIIalpha are epistatic to the FA pathway for suppression of radial formation in response to DNA interstrand crosslinks since depletion of either of them in FA cells does not increase radial formation. Depletion of Topoisomerase IIIalpha or BLM also causes an increase in sister chromatid exchanges, as is seen in Bloom syndrome cells. Human Fanconi anemia cells, however, do not demonstrate increased sister chromatid exchanges, separating this response from radial formation. Primary cell lines from mice defective in both Blm and Fancd2 have the same interstrand crosslink-induced genome instability as cells from mice deficient in the Fancd2 protein alone. These observations demonstrate that the association of BLM and Topoisomerase IIIalpha with Fanconi proteins is a functional one, delineating a BLM-Topoisomerase IIIalpha-Fanconi pathway that is critical for suppression of chromosome radial formation.

Retinal neovascular markers in retinopathy of prematurity: aetiological implications.

AIM: (1) To determine if expression of the blood-tissue barrier associated glucose transporter GLUT1 is preserved by the neovasculature of retinopathy of prematurity (ROP), in contrast with the reported loss of GLUT1 expression in preretinal vessels of proliferative diabetic retinopathy. (2) To compare the vascular immunophenotype of ROP to juvenile haemangioma, another perinatal neovascular disorder that has recently been shown to express placental type vascular antigens, including GLUT1 and Lewis Y antigen. METHODS: A retrospective case report was carried out. Immunoreactivities for GLUT1 and Lewis Y antigen were assessed in a human eye with stage 3 ROP and compared with those in a control (paediatric) eye. The presence or absence of endothelial GLUT1 and Lewis Y immunoreactivity was determined in preretinal and intraretinal vessels. RESULTS: Immunoreactivity was positive for GLUT1 and negative for Lewis Y in the intraretinal and preretinal neovasculature of the ROP affected eye and in the normal retinal vessels of the control eye. CONCLUSIONS: Retention of immunoreactivity for GLUT1 distinguishes ROP from proliferative diabetic retinopathy. Furthermore, absence of Lewis Y antigen co-expression distinguishes ROP from juvenile haemangioma, a perinatal form of GLUT1 positive neovascularisation that has recently been linked to placental vasculature.

Congenital nonprogressive hemangioma: a distinct clinicopathologic entity unlike infantile hemangioma.

BACKGROUND: Infantile hemangiomas are common tumors, distinctive for their perinatal presentation, rapid growth during the first year of life, and subsequent involution-and for their expression of a unique immunophenotype shared by placental microvessels. Occasional "hemangiomas" differ from the classic form in presenting fully formed at birth, then following a static or rapidly involuting course. These congenitally fully developed lesions have generally been assumed to be clinical variants of more typical, postnatally developing hemangiomas. This assumption has not been tested by rigorous histologic and immunophenotypic comparisons. OBJECTIVE: To compare the histologic and immunohistochemical features of congenital nonprogressive hemangiomas with those of typical, postnatally proliferating, hemangiomas. DESIGN: All cellular vascular tumors resected from infants younger than 4 months at Arkansas Children's Hospital, Little Rock, over the past 20 years (43 lesions from 36 patients) were first characterized histologically and immunohistochemically, then clinically by chart review. SETTING: A university-affiliated pediatric hospital. MAIN OUTCOME MEASURES: Histologic appearance, immunoreactivity for the infantile hemangioma-associated antigens GLUT1 and LeY, and clinical behavior. RESULTS: Congenital nonprogressive hemangiomas differed from postnatally proliferating infantile hemangiomas in histologic appearance and immunohistochemical profile. Distinguishing pathologic features of these tumors were lobules of capillaries set within densely fibrotic stroma containing hemosiderin deposits; focal lobular thrombosis and sclerosis; frequent association with multiple thin-walled vessels; absence of "intermingling" of the neovasculature with normal tissue elements; and lack of immunoreactivity for GLUT1 and LeY. CONCLUSION: Congenital nonprogressive hemangiomas are histologically and immunophenotypically distinct from classically presenting hemangiomas of infancy, unlikely to be related to the latter in pathogenesis.

A unique microvascular phenotype shared by juvenile hemangiomas and human placenta.

BACKGROUND: Juvenile hemangiomas are common, benign tumors, distinctive for their perinatal presentation, rapid growth during the first year of life, and subsequent involution. We recently reported that endothelia of hemangiomas highly express GLUT1, a glucose transporter normally restricted to endothelia with blood-tissue barrier function, as in brain and placenta. OBJECTIVE: To investigate possible further similarities between hemangioma and placental vessels. DESIGN: In a retrospective study of a variety of vascular tumors and anomalies, we assessed lesional immunoreactivities for the placenta-associated vascular antigens FcgammaRII, Lewis Y antigen (LeY), merosin, and GLUT1. SETTING: A university-affiliated pediatric hospital. MAIN OUTCOME MEASURE: Immunoreactivities scored for each antigen were summarized according to lesional type, compared with those of normal skin, brain, and placenta, and correlated with patient age, sex, and lesional location. RESULTS: All of 66 hemangiomas (patients aged 22 days to 7 years) showed intense immunoreactivity for FcgammaRII, merosin, LeY, and GLUT1. No immunoreactivities for these markers were seen in any of 26 vascular malformations, 4 granulation tissue specimens, 13 pyogenic granulomas, or in the tumor vasculature of 6 malignant tumors of nonvascular origin. Microvascular immunoreactivity for all 4 markers was observed in placental chorionic villi, but was absent in microvessels of normal skin and subcutis. Brain microvessels expressed only GLUT1 and merosin. CONCLUSIONS: A distinct constellation of tissue-specific markers is uniquely coexpressed by hemangiomas and placental microvessels. These findings imply a unique relationship between hemangioma and the placenta and suggest new hypotheses concerning the origin of these tumors.

Role of the Bloom's syndrome helicase in maintenance of genome stability.

The RecQ family of DNA helicases has members in all organisms analysed. In humans, defects in three family members are associated with disease conditions: BLM is defective in Bloom's syndrome, WRN in Werner's syndrome and RTS in Rothmund-Thomson syndrome. In each case, cells from affected individuals show inherent genomic instability. The focus of our work is the Bloom's syndrome gene and its product, BLM. Here, we review the latest information concerning the roles of BLM in the maintenance of genome integrity.

Histopathological diagnosis of infantile hemangiomas and vascular malformations.

Current histologic and immunophenotypic criteria useful in the pathological diagnosis of benign vascular tumors and anomalies of childhood, with emphasis on infantile hemangiomas and vascular and lymphatic malformations, have been described and correlated with clinical presentation. The authors have emphasized recent advances in research that portend an emerging revolution in our understanding of these perplexing lesions. These latter findings, particularly in the case of hemangiomas, suggest possible new preventive strategies and therapeutic avenues involving targeted use of angiogenic modulators.

The Bloom's syndrome gene product interacts with topoisomerase III.

Bloom's syndrome is a rare genetic disorder associated with loss of genomic integrity and a large increase in the incidence of many types of cancer at an early age. The Bloom's syndrome gene product, BLM, belongs to the RecQ family of DNA helicases, which also includes the human Werner's and Rothmund-Thomson syndrome gene products and the Sgs1 protein of Saccharomyces cerevisiae. This family shows strong evolutionary conservation of protein structure and function. Previous studies have shown that Sgs1p interacts both physically and genetically with topoisomerase III. Here, we have investigated whether this interaction has been conserved in human cells. We show that BLM and hTOPO IIIalpha, one of two human topoisomerase III homologues, co-localize in the nucleus of human cells and can be co-immunoprecipitated from human cell extracts. Moreover, the purified BLM and hTOPO IIIalpha proteins are able to bind specifically to each other in vitro, indicating that the interaction is direct. We have mapped two independent domains on BLM that are important for mediating the interaction with hTOPO IIIalpha. Furthermore, through characterizing a genetic interaction between BLM and TOP3 in S. cerevisiae, we have identified a functional role for the hTOPO IIIalpha interaction domains in BLM.

GLUT1: a newly discovered immunohistochemical marker for juvenile hemangiomas.

Juvenile hemangiomas are common, benign vascular tumors of infancy. These lesions enlarge rapidly through cellular hyperplasia during the first year of life and then involute over several years. Distinctive histopathologic features of hemangiomas diminish during this evolution, and differentiation from vascular malformations becomes increasingly difficult. This distinction has important therapeutic implications, as juvenile hemangiomas differ from malformations in natural history and in potential for recurrence. We report here that high endothelial immunoreactivity for the erythrocyte-type glucose transporter protein GLUT1 is a specific feature of juvenile hemangiomas during all phases of these lesions. In a retrospective study, we found intense endothelial GLUT1 immunoreactivity, involving more than 50% of lesional microvessels, in 97% (139 of 143) of juvenile hemangiomas from patients aged 1 month to 11 years. No endothelial GLUT1 immunoreactivity was found in any of 66 vascular malformations (17 arteriovenous, 33 venous, 11 lymphatic, and 5 port-wine) from patients aged 5 days to 75 years, or in any of 20 pyogenic granulomas or 7 granulation tissue specimens. Abundant Ki-67 positivity in these latter lesions established that GLUT1 expression does not simply reflect mitotically active endothelium. Focal GLUT1 immunoreactivity was found in 3 of 12 angiosarcomas, but not in any of 5 hemangioendotheliomas (epithelioid or infantile kaposiform). These findings establish GLUT1 immunoreactivity as a highly selective and diagnostically useful marker for juvenile hemangiomas. Because high levels of endothelial GLUT1 expression in normal tissue are restricted to microvessels with blood-tissue barrier function, these findings also have implications for the molecular and developmental pathogenic mechanisms of juvenile hemangiomas.

GLUT1 immunoreaction patterns reliably distinguish hemangioblastoma from metastatic renal cell carcinoma.

BACKGROUND: Hemangioblastoma and metastatic renal cell carcinoma (RCC) may show striking histologic similarities, and the distinction between these two tumors can be difficult. Both occur in middle age, and both occur with increased incidence in von Hippel-Lindau disease (vHL). GLUT1 is an erythrocyte-type glucose transporter protein that is highly expressed by endothelia in brain--but not most peripheral--microvasculature, and by tumor cells in many epithelial malignancies. GLUT1 is expressed by endothelial cells in juvenile hemangiomas, and endothelial GLUT1 expression has been reported for 2 hemangioblastomas arising in a single patient with vHL. METHODS: We performed immunoreactions for GLUT1 on archival hemangioblastomas from 12 patients (one with vHL), and on RCCs metastatic to brain of 9 patients. RESULTS: Hemangioblastomas showed intense endothelial GLUT1 reactivity in 11/12 tumors resections; the only GLUT1-negative tumor was one for which only previously frozen material was available for immunoreaction, and this tissue showed poor GLUT1 immunoreactivity of internal erythrocyte controls. Hemangioblastoma stromal cell reactivity was found in only 1 case, and was weak and focal. RCCs, in contrast, showed no intralesional endothelial GLUT1 reactivity, but did show intense tumor cell membrane reactivity in 9/9 cases. CONCLUSION: that GLUT1 immunoreactivity patterns reliably distinguish hemangioblastoma from RCC.

Immunogenic and antigenic dominance of a nonneutralizing epitope over a highly conserved neutralizing epitope in the gp41 envelope glycoprotein of human immunodeficiency virus type 1: its deletion leads to a strong neutralizing response.

The Kennedy peptide, (731)PRGPDRPEGIEEEGGERDRDRS(752), from the cytoplasmic domain of the gp41 transmembrane envelope glycoprotein of HIV-1 contains a conformationally dependent neutralizing epitope (ERDRD) and a linear nonneutralizing epitope (IEEE). No recognized murine T cell epitope is present. The peptide usually stimulates virus-specific antibody, but this is not always neutralizing. Here we show that IEEE (or possibly IEEE plus adjacent sequence) is immunogenically and antigenically dominant over the ERDRD neutralizing epitope. Thus rabbits immunized in a variety of routes, doses, and adjuvants with a chimeric cowpea mosaic virus (CPMV) expressing the Kennedy peptide on its surface (CPMV-HIV/1) synthesized IEEE-specific serum antibody but no ERDRD-specific or HIV-1-neutralizing antibody. To test if this resulted from immunodominance or from a hole in the antibody repertoire, we immunized rabbits with chimera CPMV-HIV/29, which expresses the GERDRDR part of the Kennedy sequence. This chimera readily stimulated ERDRD-specific, neutralizing antibody. In mice the situation was less extreme, but individual animals with low neutralizing titers had a high ratio of IEEE-specific:ERDRD-specific antibody. Data are consistent with immunodominance of IEEE over ERDRD in the Kennedy peptide. IEEE-specific antibody was also antigenically dominant and prevented ERDRD-specific antibody from binding to its epitope and from neutralizing HIV-1. It may be that HIV-1 has evolved a nonneutralizing immunodominant epitope that allows it to possess a neutralizing epitope without suffering the consequences, and this idea is supported by the covariance of both epitope sequences. To our knowledge this is the first example of a defined sequence that controls the activity of an adjacent epitope.

Evidence of residual disease in ossicles of patients undergoing cholesteatoma removal.

For the past several years there has been much debate regarding the advisability of reusing the incus for ossicular reconstruction in cases involving cholesteatoma. There appears to be some evidence that microscopic foci of cholesteatoma in the incus could lead to reimplantation of the cholesteatoma should the incus be used in the reconstruction phase. In an effort to elucidate the incidence of microscopic residual cholesteatoma, the incudes of patients with cholesteatoma were examined both grossly in the operating room and microscopically in the laboratory for erosion and residual cholesteatoma. Our examination showed that a number of specimens apparently free of cholesteatoma after macroscopic examination had microscopic evidence of cholesteatoma. Likewise, microscopic examination of an incus that appeared to be free of residual cholesteatoma revealed epithelial cells deeply invading the bone. Macroscopic examination consistently underestimated the amount of erosion that was clearly evident upon histologic examination. In light of these findings, gross examination of the incus after removal of cholesteatoma is not reliably predictive of invasive microscopic disease. Reusing the ossicles in this situation creates the potential of reimplanting the disease.

Pseudomonas aeruginosa outer-membrane protein F epitopes are highly immunogenic in mice when expressed on a plant virus.

A synthetic peptide (peptide 10) representing a surface-exposed, linear B cell epitope from outer-membrane (OM) protein F of Pseudomonas aeruginosa was shown previously to afford protection in mice from P. aeruginosa infection. This peptide was expressed in tandem with the protein F peptide 18 on each of the two coat proteins of cowpea mosaic virus (CPMV). The chimaeric virus particles (CVPs) expressing the peptides on the S (small) coat protein (CPMV-PAE4) and L (large) coat protein (CPMV-PAE5) were used to immunize mice. Following subcutaneous immunization in Freund's and QuilA adjuvants, CPMV-PAE4 induced antibodies predominantly against peptide 18, whereas CPMV-PAE5 produced antibodies exclusively against peptide 10, indicating that the site of peptide expression on CPMV influences its immune recognition. The anti-peptide antibodies elicited by CPMV-PAE5 were predominantly of the IgG2a isotype, indicating a highly polarized TH1-type response. The peptide-specific IgG2a strongly recognized the whole F protein, but more importantly, recognized protein F in all seven Fisher-Devlin immunotypes of P. aeruginosa. Furthermore, the peptide-specific IgG2a in CVP/QS-21 adjuvant-immunized mice was shown to bind complement and to augment phagocytosis of P. aeruginosa by human neutrophils in vitro. The ability of CPMV-PAE5 to induce P. aeruginosa-specific opsonic IgG2a gives it potential for further development as a protective vaccine against P. aeruginosa.