Anastrepha fraterculus (Wiedemann) is a complex of cryptic species, the so-called “Anastrepha fraterculus complex”, for which eight morphotypes are currently recognized. A previous analysis of ITS1 in samples of the Anastrepha fraterculus complex, while revealing high distinctiveness among samples from different localities of South America, Central America, and Mexico, no direct association was made between sequence type and morphotype. In the present report, a correlated analysis of morphometry and ITS1 data involved individuals belonging to the same population samples. Although showing a low level of intra-populational nucleotide variability, the ITS1 analysis indicated numerous inter-population sequence type variants. Morphotypes identified by morphometric analysis based on female wing shape were highly concordant with ITS1 genetic data. The correlation of genetic divergence and morphological differences among the tested samples gives strong evidence of a robust dataset, thereby indicating the existence of various taxonomic species within the A. fraterculus complex. However, the data revealed genetic and morphological variations in some regions, suggesting that further analysis is still required for some geographic regions.The South American fruit fly  Anastrepha Schiner is endemic to the Neotropics and has been found to occur from the southern United States to northern Argentina and the Caribbean Islands [fraterculus group”, some are considered economically damaging, such as Anastrepha fraterculus (Wiedemann). In the 1940s, variations in wing pattern along its wide geographic distribution, similar to that of the genus, lead to the assumption that cryptic species might be involved [Anastrepha fraterculus populations have been analyzed for other biological parameters and significant data on the existence of different species have been accumulated over time.The genus  Islands . The gen Islands ,3,4. Amoinvolved ,6,7. SinAnastrepha fraterculus complex came from a karyotypic analyses of specimens collected in Brazil and Mexico, revealing marked differences in sex chromosomes [A. fraterculus s.l., thus giving support to the hypothesis that Anastrepha fraterculus actually includes a complex of cryptic species [The first non-morphological evidence indicating that different species could be involved in the omosomes ,9. Furthomosomes  and an iomosomes  also ideomosomes . Later, omosomes ,14. Phylomosomes , CO1 mitomosomes , and theomosomes , pointed species . However species ,9 and is species ,15 data Anastrepha fraterculus (AF) complex in Brazil: Anastrepha sp.1 aff. fraterculus, A. sp.2. aff. fraterculus, and A. sp.3 aff. fraterculus  [This difficulty was overcome after an integrated analysis of linear morphometry, genetics, and tests of reproductive isolation allowed the description of three biological entities within the A. sp.3) ,24,25,26A. fraterculus along its geographic distribution, a linear morphometric approach applied to samples from Mexico, Colombia, Brazil, and Argentina provided evidence of distinct morphotypes occurring in the different regions [A. sp.4), “Ecuadorian” (highlands of Peru and Ecuador), and three Brazilian morphotypes—“Brazilian-1” (including samples from Argentina), “Brazilian-2”, and “Brazilian-3”, corresponding to A. sp.1, A. sp.2 and A. sp.3, which have already been characterized [Apart from the description of the three species from Brazil and the evidences of genetic differences in samples of  regions . The ana regions ,29, resucterized ,20. Howecterized .The recognition of distinct morphotypes in the Neotropical Region stimulated the search for correlated molecular markers. Preliminary ITS1 sequence data suggested that differences do occur in samples of the AF complex throughout the whole region . FurtherA. fraterculus were only partially associated with morphotypes [Apart from the recent significant increase in knowledge on cryptic species characterization within the AF complex, the lack of comprehensive diagnostic features has made accurate recognition of distinct taxa difficult. For example, in a microsatellite analysis, the samples of photypes , and in photypes . Howeverphotypes ,20,34. HSpecimens of the AF complex from 23 samples were obtained from 20 localities spread throughout the Neotropics: Mexico, Guatemala, Colombia, Ecuador, Peru, Argentina, and Brazil . NineteeA. fraterculus (s.l.) (GenBank #AY775552) were used, i.e., 18SF-TAACTCGCATTGATTAAGTCCC and 5.8SR-GATATGCGTTCAAATGTCGATG, which bind to the 3′ end of 18S and the 5′ end of 5.8S genes, respectively. PCR reactions consisted of one cycle (2 min at 94 °C), 31 cycles  and a 5 min extension at 72 °C. Amplified products were electrophoresed in 0.8% agarose gel and visualized after ethidium bromide staining. Oneshot Escherichia coli cells were employed to generate five clones from each fragment by applying Topo TA Cloning . Plasmid DNA was extracted using the Perfectprep Plasmid Mini Kit . The BigDye reaction kit in an ABI Prism Automatic Sequencer  was employed for sequencing at the Institute of Chemistry, University of São Paulo, São Paulo, Brazil.DNA for the ITS1 analysis was extracted from 10 individuals of each sample collected in Brazil and from 5 individuals from each sample for the other localities. DNA was extracted from the isolated thorax of each individual in accordance with the Jowett protocol . For PCRA. fraterculus (l.s.) (GenBank #AY775552), the regions corresponding to 18S and 5.8S flanking genes were trimmed off. Amplification and sequencing of A or T homopolymeric regions are prone to the production of artifacts that are interpretable as either intra-individual polymorphisms or PCR slippage. Analysis of the ITS1 electropherograms of five clones from each sampled specimen with the Electropherogram Quality Analysis web tool [Amplified fragments of around 900 bp contained complete ITS1 and partial sequences of the flanking genes. After comparison with the ITS1 sequences of web tool  indicateSome of the samples analyzed herein  were colAnastrepha taxonomy  it is the most appropriate to precisely recognize homologous landmarks [Landmark-based geometric morphometry was applied to quantify and distinguish wing morphological features in 239 female specimens from 19 population samples of the AF complex . Morpholandmarks ,29.For permanent preparations, the right-wings of 10–12 specimens per sample were removed from their bases, washed with distilled water, and later dehydrated in a gradual alcohol series , with 20 min for each step. Then, they were placed in xylene for 2–3 min and mounted in a dorsal view between the slide and coverslip with Canada Balsam. Images of each wing were digitalized at an identical magnification with a high-resolution 5050 Olympus camera fitted to a Zeiss Stemi 508 stereomicroscope.1; (4) apex of vein R2+3; (5) apex of vein R4+5; (6) apex of vein M; (7) apex of vein CuA1 on the posterior margin; (8) apex of vein CuA2 on the posterior margin; (9) basal bifurcation of R2+3 and R4+5; (10) junction of R4+5 and cross-vein r-m; (11) basal angle of cell bm; (12) junction of M and cross-vein dm-bm; (13) junction of M and cross-vein r-m; (14) junction of M and cross-vein dm-cu; (15) junction of CuA1 and Cu2; (16) junction of CuA1 and cross-vein bm-cu; (17) junction of CuA1 and dm-cu; and (18) junction of A and Cu2 (=apex of cell bcu)  .A generalized Procrustes analysis using the MorphoJ program was conducted by overlapping landmark configurations that remove variations caused by differences in translation, orientation, and size into a common coordinate system . A CanonThe 32 ITS1 sequences obtained varied from 530 to 583 nucleotide (nt) positions, were rich in AT (ca. 70–84%),and showed complex structures. As described in detail for other samples of the AF complex [The representative ITS1 sequences obtained were aligned with previously distinguished representative sequence types—types TI, TIa, TII, TIIIABC, TIV —from sit9 (bases 291–298), C4 (bases 302–305), CACA (bases 493–496), and GGAAA (bases 602–606)), as well as several SNPs, and as such, it was named TIe. Genetic distances between these sequence types were low, the highest being those of ArHmol (mean of 0.014) and ArTuc (mean of 0.142) relative to the other samples , an ATT extension (bases 368–376), and SNPs at bases 353, 403, and 417. A variant of this sequence, TIa, besides the TI markers, has a unique ATT extension (bases 580–582) inside Highly Variable Region II in samples from Peru  and Bolivia (BoSCr). TIa was also found in the ArHmol sample from Argentina. Other samples of Brazilian morphotypes had the basic TI markers but showed differences characterizing other TI variants. The sequences from the Brazilian-1 morphotype had characteristic CCC (bases 129–131) and CCCC (bases 177–180) expansions, as well as a few other SNPs; sequences of Brazilian-2 morphotype had an additional G at positions 125 and 232, an additional A at position 668, and other nucleotide substitutions. The sequences found in the three samples of Brazilian-3 morphotype had a unique TAT extension at positions 359–361 and other SNPs. Hence, the sequences were considered new variants of TI associated specifically with the three Brazilian morphotypes and were named TIb (Brazilian-1), TIc (Brazilian-2), and TId (Brazilian-3). The sequence from Tucumán  was considered a variant of TI but was distinct from the other variants (TIa to TId) due to its unique extensions, (T samples .6 to (T)7 at position 249, and (ATT)n (n = 4 and 5) repeats (bases 565–579), as well as SNPs at bases 137, 190, 410, and 669. Sequence type TII was found in samples from Mexico, Central America, the lowlands of Venezuela, and San Andrés Island (Colombia). However, samples from the three localities from Mexico showed relevant differences in SNPs and unique insertions, namely MxJic, AAC (bases 143–145), GGGGG (bases 227–230), TTATAAA (bases 497–503); MxQro, TTT (bases 110–112), GAG (bases 478–480), AAA (bases 501–503), TG (bases 540–541); and MxTap, A6 (bases 560–565), and GGGGA (bases 353–356). The average intragroup genetic distance between sequences was 0.039 (These sequences are characterized by a putative reduction of TATAT to TTT (bases 110–114), a unique expansion of TGGGTGG (bases 211–217), another expansion of (T)as 0.039 . AlthougAlthough mutually very similar, TIII A was characterized by an expansion in the HVRI CAATATATA (bases 380–388) by insertions of an A (bases 391 and 569) and an AT (positions 419–420) and possible A expansions at base 605. This sequence type was found in the lowlands of Peru and Ecuador (<900 m) but showed little divergence between the sequences PeCaj, PePiu, and PeLmo. The average distance between TIIIA sequences was 0.002 ; consequn (n = 2 and 3) at positions 539–541. Every sequence from the highlands of Colombia (>1500 m) was type IV, and the average distance between samples was 0.006 as 0.006 . The samTo achieve an improved comparison between the sequences obtained with those previously described , the simAdditionally, the genetic relationships among the sequences were inferred by a maximum likelihood phylogenetic analysis by employing the entire set of ITS1 sequences. The ML tree showed that the sequences were also grouped in four clades, each of which congregated the same set of sequences as those included in the clusters of the UPGMA analysis . The cla = 15.327; p < 0.0001). The first two accounted for 75.7% of the group differentiation (CV1 = 55.7% and CV2 = 20%), whereas the remaining CV3, CV4, and CV5 contributed 11%, 9.8%, and 3.7%, respectively.A morphometric analysis of the female wing shapes of the 19 population samples showed the presence of at least six morphotypes within the AF complex. An overall examination indicated that all canonical variates (CV) were statistically significant , whereas for Brazilian-1/Brazilian-2 (SMD = 45.9) and Brazilian-2/Brazilian-3 (SMD = 49.2), the values were up to six times lower. In other geographical regions in the Americas, a close similarity was also noted for the wing-shape between Peruvian/Mexican (SMD = 9.18), as compared with the low similarity between Mexican/Andean morphotypes (SMD = 55). Meanwhile, minor similarities in wing shape were found between the Brazilian-3/Andean (SMD = 70.4) and Brazilian-3/Peruvian morphotypes (SMD = 74.2) .Partial-warp scores captured the shape variation to visualize the major trends associated with hypothetical changes found in the samples. These shifts in wing shape are depicted by an outline drawing and plot of the two first canonical variates by morphotype .2 = 50.93, p < 0.0001), showing that the Mexican and Peruvian groups possess smaller wing sizes than all the other morphotypes   . Furtherarisons) .A comparison between genetic and morphological data was conducted on the 19 population samples analyzed by the two methods .Clustering analyses revealed an aggregation into two widely discrete sets of samples, the first one consisting of four Argentinian populations , along with two Brazilian ones —all of them recognized as the Brazilian-1 morphotype   Andean, represented by a Colombian highland sample (CoIba) showing the largest divergence in wing shape, even among the nearest geographical samples, e.g., Peru and Ecuador; (2) Brazilian-2, depicted separately by only one Brazilian population (B2Isa); (3) Mexican, including all samples from Mexico and Guatemala ; and (4) Peruvian, composed of the Peruvian and Ecuadorian Pacific lowland samples . The clustering of ITS1 sequences from these samples yielded a highly similar dendrogram that separates the Brazilian-2 (Br2Isa) morphotype sequence from two other clades: one grouping the Mexican morphotype sequences and another one consisting of the three Peruvian morphotype samples, along with the Colombian (CoIba) sample from the Andean morphotype, which was not differentiated, as shown in the morphotype dendrogram.From a morphological point of view, the AF complex comprises at least eight morphotypes. The genetic divergence correlates with morphological differences for six of these morphotypes, as described in the present analysis, strongly confirming the existence of various taxonomic species within the AF complex. A previous analysis of ITS1 in samples of the AF complex revealed high distinctiveness throughout the Neotropical Region ,32,38. HThis molecular marker has been used in the genetic analysis of very closely related taxa for which a recent evolutionary history is assumed . The mulDifferences in the fragment size were not detected in a previous ITS1 analysis of Argentinian samples . If onlyAnastrepha samples has often been observed. Some hypotheses have been proposed to explain this phenomenon although the underlying cause(s) remain unknown [According to the data, the overall nucleotide variability of ITS1 sequences is generally low, varying from 0.006 (Brazilian-3) to 0.039 (Mexican). This is surprising, since non-coding regions have a tendency to accumulate mutations faster than coding sequences ,54. Rega unknown ,56,57,58 unknown ,20. NeveAnastrepha sp.1 aff. fraterculus (Brazilian-1 morphotype) [A. sp.2 and A. sp.3 coexist  [Sequence type TI and the variant TIa, detected in an extensive area throughout south/south-eastern Brazil, Argentina, Bolivia, the highlands of Peru (Cusco region), and in Paraguay, were putatively identifiable as photype) . Howeverphotype) . Moreovephotype)  collecte Br3Uba) ,20,34. D Br3Uba)  and the  Br3Uba) , the preIn Morphotype distinctiveness was in line with genetic differences revealed by the ITS-1 analysis, which, in turn, complied with the mutual reproductive incompatibility encountered in experimental crosses carried out with almost all of the morphotypes ,30. VariMorphological variation among samples from Mexico has already been described ,28,29. TThe existence of various biological entities within this species complex may be related to the wide continental distribution encompassing the diverse ecological conditions and geographic areas where they occur. Supposedly, the geographic distance could prevail in the maintenance of divergence. Nevertheless, reproductive isolation laboratory tests between distant geographic samples ,65,66,67A. sp. 1, A. sp. 2 and A. sp. 3) [Anastrepha fraterculus from Brazil [A. sp.1 and A.sp.2 [Since the Brazilian morphotypes, as a whole, have been well characterized through integrated analyses of several biological parameters, including reproductive isolation assays, they have been described as different species in previous studies (. sp. 3) ,20,67. Am Brazil . This ism Brazil ,20, and d A.sp.2  aligns wd A.sp.2 , their aA. sp.1 according to the authors) and flies from Argentina were found to be fertile [A. sp.3 is found in sympatry with A. sp.1 [A. fraterculus from south-eastern Brazil, which led to the proposition that homoploid hybrid speciation would be a prevalent process [No areas of sympatry have been described for the AF complex, except for the Brazilian morphotypes, and the knowledge of their host fruits is scarce. Crosses between specimens from south Brazil ( fertile , but it  A. sp.1 ,70,71. T process .Rhagoletis. It was postulated that initial allopatry would furnish the genetic architecture conducive to a sympatric host shift for Rhagoletis pomonella divergence [A. sp.1 and A. sp.2, in addition to an environmental factor related to altitude, that are involved in the distribution pattern currently observed [Considering the simplest model of ecological speciation, the occupation of different ecological niches or different geographic environments could result in reproductive isolation . It is rvergence . One mayvergence ,20,67. Tobserved . The detThe present report describes the results of an integrated genetic and morphologic comprehensive analysis of individuals belonging to the same population samples of the AF complex morphotypes. The morphometrical analysis clearly identified six morphotypes in the samples out of the eight described for the AF complex: Brazilian-1, Brazilian-2, Brazilian-3, Peruvian, Andean, and Mexican. The analysis of ITS-1 detected specific sequences for each morphotype, thus showing a strong congruence between genetic and morphological data. Moreover, the morphological and genetic variations revealed in the Argentinian and Mexican samples indicate that further research should be done to better understand its true meaning. Overall, the data clearly confirm that morphotypes are distinct biological species bearing genetic differences, and potential tools for recognizing the taxa of the AF complex are described, which may be useful for academic and applied purposes.


Pathologically, microangiopathic hemolytic anemia and thrombocytopenia lead to microvascular thrombosis occlusion and ischemia in the kidney and brain.3It seems an inhibitory autoantibody against a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) during INF-β therapy or some drugs  mediates ADAMTS13-acquired deficiency which leads to microvascular thrombus and platelet aggregation.We report a new TMA in a patient with multiple sclerosis (MS) who received treatment for 10 years with subcutaneous (SC) INF-β 1a. This emphasized that this risk will not decrease after a long period of time, hence clinical vigilance is necessary.She was a 38-year-old woman with right-handed MS. The patient had not been used any other drug except INF. She started developing epistaxis and gingival hemorrhage on June 29, 2017. Her blood pressure was 190/110 mmHg, and she was afebrile. Laboratory tests are summarized in 3, hemoglobin (Hgb) reached to 10.5 g/dl, and platelets increased to 65000/mm3, blood pressure has controlled to normal levels, and kidney has achieved normal function. After 1 month, red blood cells (RBCs) became elevated to 3810/mmLimited cases of TMA have been reported in patients with MS on treatment with INF-β. 2Broughton, et al. reported a late-onset TMA presented with hypertension, renal dysfunction, thrombocytopenia, and lactate dehydrogenase (LDH) elevation similar to our case. In their case, TMA was confirmed in kidney biopsy.4Olea, et al. reported an early-onset TMA presented with hypertension, thrombocytopenia, subnephrotic proteinuria, renal dysfunction, and elevated LDH. Kidney biopsy showed glomerular microangiopathy.5One other late-onset TMA presented with hypertension and renal dysfunction which was confirmed by kidney biopsy. Orvain, et al. postulated the possible role of anti-ADAMTS13 IgG antibody induced by INF-β.6In Vosoughi and Marriott study, the second late-onset TMA case presented with neurological manifestation, malignant hypertension, thrombocytopenia, pulmonary edema, and generalized tonic clonic seizure, thrombocytopenia and schizocytes in the blood smear. TMA diagnosis was confirmed clinically.Our case presented in a similar manner to other late-onset TMA cases with thrombocytopenia, hypertension, and renal dysfunction. Schizocytes in blood smear and therapeutic response to the classic treatment of TMA confirmed the diagnosis. TMA is a rare but actually life-threatening side effect of INF-β which could present late, even after 10 years of treatment. It is our opinion healthcare providers, who monitor and follow patients with MS, are supposed to consider the early presentation of TMA, especially any elevated unexplained hypertension.


The prognostic and therapeutic values of fibronectin have been reported in patients with renal cell carcinoma (RCC). However, the underlying mechanisms of malignancy in RCC are not completely understood. We found that silencing of fibronectin expression attenuated human RCC 786-O and Caki-1 cell growth and migration. Silencing of potential fibronectin receptor integrin α5 and integrin β1 decreased 786-O cell ability in movement and chemotactic migration. Biochemical examination revealed a reduction of cyclin D1 and vimentin expression, transforming growth factor-β1 (TGF-β1) production, as well as Src and Smad phosphorylation in fibronectin-silenced 786-O and Caki-1 cells. Pharmacological inhibition of Src decreased 786-O cell growth and migration accompanied by a reduction of cyclin D1, fibronectin, vimentin, and TGF-β1 expression, as well as Src and Smad phosphorylation. In 786-O cells, higher activities in cell growth and migration than in Caki-1 cells were noted, along with elevated fibronectin and TGF-β1 expression. The additions of exogenous fibronectin and TGF-β1 promoted Caki-1 cell growth and migration, and increased cyclin D1, fibronectin, vimentin, and TGF-β1 expression, as well as Src and Smad phosphorylation. These findings highlight the role of fibronectin in RCC cell growth and migration involving Src and TGF-β1 signaling. Renal cell carcinoma (RCC) is a lethal malignancy that originates in the kidney and is characterized by highly vascularized and metastatic features. Among the histological subtypes of this cancer, clear cell RCC is the most common type. Despite advances in early detection of small renal masses and the effectiveness of radical nephrectomy, approximately one third of patients with RCC have already developed metastases at diagnosis, eventually leading to metastatic lesions during the progression of this disease. Once metastasis has occurred, the five-year survival rate of patients with advanced RCC is unsatisfactory ,2. CliniExtracellular matrix forms a basic scaffold for the normal physiology of cells. The interaction between cell and extracellular matrix also plays a central role in the initiation and progression of malignancies. Among the extracellular matrix proteins identified, fibronectin is able to activate a series of signal transduction pathways by interacting with various receptors, thereby regulating cellular processes, including adhesion, migration, invasion, proliferation, differentiation, and morphogenesis. Accumulating evidence has indicated its carcinogenic and malignant role in several malignancies ,6,7,8,9.Fibronectin is a secreted extracellular matrix component and a member of the mesenchymal protein family, and, therefore, its biological activity is believed to be mainly governed via a process known as epithelial–mesenchymal transition (EMT). However, the engagement of fibronectin with integrin receptors might also actively initiate and transduce distinct postreceptor intracellular signaling, thereby contributing to dynamic regulation of cell adhesion, migration, invasion, and proliferation. Evidence indicates that focal adhesion kinase (FAK), Src, extracellular signal-regulated kinase (ERK), Akt, and transforming growth factor-β1 (TGF-β1) are concurrent or concomitant effectors of postreceptor actions of fibronectin ,19,20,21The importance of fibronectin in RCC cell biology was first investigated by RNA interfering (RNAi) strategy. Fibronectin silencing by siRNA caused a reduction of fibronectin protein expression in human RCC 786-O cells A and ledTo further investigate the effects of fibronectin on RCC cell growth and migration, 786-O cells were seeded onto fibronectin-coated cultured plates and Transwell inserts. The presence of exogenous fibronectin promoted cell growth A and cheThe aforementioned findings imply that fibronectin plays a substantial role in RCC cell growth and migration. Since FAK, Src, ERK, and Akt have been implicated in transducing signals of fibronectin to induce distinct cellular activities ,19,20,21To further demonstrate the cell growth and migration inhibitory effects of fibronectin silencing on RCC cells, human RCC Caki-1 cells were also investigated. As with 786-O cells, fibronectin silencing caused a reduction in Src phosphorylation, cyclin D1, vimentin, Smad phosphorylation A, cell gTo investigate whether a correlation existed between fibronectin level and cell growth/migration potential, a biochemical comparison was conducted in 786-O and Caki-1 cells. In subconfluent cells, 786-O cells expressed higher amounts of cytoplasmic and extracellular fibronectin along with integrin β1 compared with Caki-1 cells. However, the level of integrin α5 in 786-O cells was lower than in Caki-1 cells A. In parAs with 786-O cells, exogenous fibronectin promoted cell growth A, chemotIn addition to normoxic conditions, cell migration was evaluated under a hypoxic condition. Compared with normoxic condition, hypoxia for 8 h was able to promote 786-O cell migration towards 10% FBS A. WesterThe interaction between cancer cells and extracellular compartments plays a pivotal role in tumor progression. In the noncellular microenvironment, extracellular matrix proteins such as fibronectin, laminin, osteopontin, collagen, glycosaminoglycans, and proteoglycan are of considerable importance. Fibronectin is upregulated in RCC and is therefore of prognostic value. Moreover, it has been shown to be a promising biomarker for therapeutic responses ,15,16,17The effects of extracellular matrix proteins on cellular activities are multifactorial. Transmembrane protein receptors play a crucial role in transducing extracellular signals after ligand and receptor engagement. It has been demonstrated that tissue transglutaminase, osteopontin, integrin α5, integrin αv, integrin β1, and integrin β3 bind to and are activated by fibronectin, and have been implicated in tumor cell proliferation, adhesion, migration, invasion, stemness, and resistance in a number of cancers, including RCC. The roles and importance of dimeric integrin receptor subunits vary and depend on cell types and microenvironments ,19,21,22Fibronectin has been demonstrated to act as an extracellular cue, which facilitates the coordination of the functional and structural dynamics of cells through several intermediate effectors, including FAK, Src, ERK, and Akt, leading to the induction of transcriptional processes and cytoskeletal reorganization ,19,20,21The findings presented herein showed that knockdown of fibronectin expression in RCC cells caused a subsequent reduction in cell growth- and migration-associated molecules, including cyclin D1, vimentin, and TGF-β1, together with decreased Src and Smad phosphorylation. The addition of exogenous fibronectin reversed the abovementioned effects in terms of cell growth, migration, cyclin D1 expression, vimentin expression, TGF-β1 production, Src phosphorylation, and Smad phosphorylation. In its role as a master signaling molecule, Src is capable of converging diverse signals for the purpose of initiating the transcriptional program. The expression of cyclin D1, TGF-β1, vimentin, and fibronectin is under the control of Src ,23. The RUNX2), β-catenin, and Akt phosphorylation in 786-O cells compared with Caki-1 cells (data not shown). Although the findings of the current study suggest a role of integrin α5/integrin β1 and the existence of interaction among fibronectin, Src, and TGF-β1 signaling, and that these components play roles in the development of RCC, the complicated phenomena remind us that the roles of other uncharacterized molecules should not be ignored.786-O and Caki-1 are two common cell lines of human RCC with distinct genetic backgrounds . We founHypoxia represents an alternative factor in regulating the progression of RCC . The actThe regulation of cell proliferation and migration is multifactorial. Studies have highlighted the crucial role of fibronectin in RCC cell proliferation and motility, and have demonstrated its prognostic value ,15,16,172 at 37 °C [2, 5% CO2, and 94% N2 for 8 h [786-O ATCC CRL1932) and Caki-1 (ATCC HTB-46) cells were purchased from American Type Culture Collection  and propagated in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) by placing cells in an incubator with 5% COat 37 °C ,34. To e32 and Ca4 per well. During experiments, the CellTiter 96® AQueous One Solution Cell Proliferation Assay  was used to assess cell growth according to the manufacturer’s instructions.For the measurement of cell growth, cells were seeded onto 96-well plates at a density of 1 × 10Cells were seeded onto six-well plates at a density of 250 per well and maintained in DMEM containing 10% FBS for seven days. Afterwards, cells were fixed and cell colony was visualized by staining with crystal violet .5/mL and grown until confluence was reached. The confluent cell monolayer was then scratched with a tip of a 200-μL micropipette to create a wound and maintained in DMEM containing 0.5% FBS. Photomicrographs were taken immediately after the scraping and 16 h later. The areas of gaps without cell coverage were measured and the percentage of wound closure was calculated [Cells were seeded onto six-well plates at a density of 4 × 10lculated .4) in 200 μL DMEM containing 2% FBS was added to the top of the Transwell inserts without or with fibronectin coating (50 μg/mL), and 600 μL DMEM containing 10% FBS was put into the bottom well for 24 h. Furthermore, the upper wells and lower wells were replaced with DMEM containing 0.1% bovine serum albumin (BSA) and DMEM containing 0.1% BSA and fibronectin (50 μg/mL), respectively, in some studies and antibody neutralization assay. For the measurement of cell membrane receptors involved in fibronectin-mediated migration, 786-O cells were first incubated with isotype control IgG , integrin β1 IgG , or integrin α5 IgG  for 30 min before seeding to the Transwell inserts for migration assay. The migrating cells in the lower surfaces of the Transwell inserts were stained with Giemsa. For statistical comparison, six random fields per insert (10×) were selected for measurement [Cell migration was measured using a 24-well Transwell permeable chamber with 8 μm pore size . Briefly, cell suspension (2 × 10The siRNA against human fibronectin (sc-29315), integrin α5 (sc-29372), integrin β1 sc-35674), and control siRNA (sc-37007) were purchased from Santa Cruz Biotechnology . The delivery of siRNA into the cells was conducted using INTERFERinTM siRNA transfection reagent  in accordance with the manufacturer’s instructions [674, and The levels of TGF-β1 in supernatants were measured using an ELISA kit according to the manufacturer’s instructions .Cells were lysed in ice-cold Laemmli sodium dodecyl sulfate (SDS) sample buffer. Equal amounts of proteins were separated by 10% or 12% SDS-PAGE and transferred onto PVDF membranes which were incubated with specific antibodies followed by horseradish peroxidase-labeled IgG. All antibodies were reacted with fibronectin, integrin α5, integrin β1, FAK, phospho-FAK (Tyr-397), ERK, phospho-ERK (Thr-202/Tyr-204), Akt, phospho-Akt (Ser-473), Src, phospho-Src (Tyr-416), cyclin D1, vimentin, Smad , phospho-Smad , or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) . The signals in membranes were visualized using enhanced chemiluminescence (ECL) Western blotting reagents and the intensity of signals was determined by a computer image analysis system .t-test. A level of p < 0.05 was considered statistically significant.All data are expressed as mean values ± standard deviation. Statistical significance between groups was carried out using one-way analysis of variance followed by Student’s


A 59-year-old female patient with double mitral lesion was hospitalized with fever, coughand worsening dyspnea with shock.At 58 years old, the patient reported onset of dyspnea in medium exertions for fivemonths, associated with dry cough at night with dyspnea, which was relieved withorthostatism. Cardiac murmur was detected and the patient was referred to InCor, a heartspecialist hospital, for treatment (17/Sept/2010).There was no reference to rheumatic outbreaks in the past, and the patient had arterialhypertension and hypothyroidism.2, cardiacfrequency was 88 bpm, arterial blood pressure 140 x 90 mmHg; pulmonary auscultationresulted normal; cardiac auscultation revealed hypophonic 1st heart sound,hypophonic pulmonary component of 2nd heart sound and mitral holosystolicmurmur ++++/6+; abdomen exam resulted normal; there was no edema in the lower limbs andpulse palpation was normal.Physical exam when the patient was first examined (17/Sep/2010) showed the patientweighed 73 Kg, was 1.55 m tall, body mass index was 30.6 kg/mThe ECG (14/Sep/2010) showed sinus tachycardia, with 127 bpm frequency, PR interval 200ms, dQRS 92 ms, SÂQRS + 150º reverse, QTc 459 ms, overload of the left atrium andindirect signs of overload of the right atrium ,low-voltage front plane and overload of the right ventricle .3,hemoglobin 14.6 g/dL, hematocrit 45%, creatinine 1.08 mg/dL (FG = 55L/min/1.73m2), potassium 4.4 mEq/L and sodium 142 mEq/L.Laboratorial exams (14/Sep/2010) showed red blood cells 5.0 million/mmThe echocardiogram (25/Aug/2010) revealed aortic diameter 25 mm, left atrium 52 mm, rightventricle 44 mm, left ventricle 34/21 mm, ejection fraction 70%, septum thickness andposterior wall 11 mm; there was no alteration in segment contraction of the leftventricle; right ventricle’s systolic function was normal; mitral valve presentedthickened cusps with commissural fusion and reduced opening, compatible with a severelycompromised rheumatic condition, and there was significant valve insufficiency. Themaximum diastolic gradient between the left atrium and the ventricle was estimated at 30mm Hg, and the medium, at 18 mm Hg; the aortic valve showed discrete signs offibrocalcification without functional alterations; the tricuspid valve had severeinsufficiency. Pulmonary artery systolic pressure was estimated at 140 mmHg.Losartan 100 mg, furosemide 40 mg, digoxin 0,25 mg and acetylsalicylic acid 100 mg dailywere prescribed.Surgical treatment of the mitral valve was indicated.In December 2010 the patient sought emergency medical attention due to tachycardia anddyspnea.The ECG (16/Dec/2010) revealed nodal reentrant tachycardia, with 178 bpm frequency . The patAt the outpatient ward (5/Apr/2011) the patient was asymptomatic, with controlled bloodpressure (120/80 mmHg) and heart rate of 84 bpm, and the physical exam resulted normal,except for preexisting alterations in the cardiac auscultation. The patient used 200 mgof amiodarone, 40 mg of furosemide, 100 mg of losartan and 60 mg of diltiazem.The patient continued waiting to be operated and on 16/Sep/2011 she sought emergencymedical care, with dyspnea in small exertions and productive cough with purulent sputum,and no fever was reported.The physical exam showed a sleepy patient, with cold extremities and a heart rate of 98bpm, blood pressure 93 x 58 mmHg. Pulmonary auscultation revealed crackling rales in thelower third of both hemithorax; cardiac auscultation revealed rhythmic heart sounds,mitral regurgitation systolic murmur +++/6+ and diastolic arrhythmia ++/6+; the abdomenhad no abnormalities and there was edema ++/4+ in the lower limbs.The ECG (16/Sep/2011) showed sinus rhythm with 97 bpm frequency, PR 168 ms, dQRS 89 ms,SÂQRS + 150º reverse, QTc 513 ms, biatrial overload, giant P wave positive at V1and right ventricular overload .The echocardiogram (17/Sep/2011) revealed hypokinesia of the right and left ventricles,the latter with 55% ejection fraction, from moderate to strong mitral insufficiency,maximal mitral transvalve gradient at 22 mm Hg and medium, at 13 mmHg. Pulmonary arterypressure was estimated at 81 mmHg; however, the patient had systemic arterialhypotension, 53 mmHg medium pressure.Antibiotics were prescribed (ceftriaxone and clarithromycin), which later were changedfor the piperacillin/tazobactam and vancomycin association, in addition to vasoactiveamines, oxygen mask, and then orotracheal intubation for respiratory support.3,hemoglobin 12 g/dL, hematocrit 39%, VCM 93 fL, RDW-CV 17.9%, leukocytes13840/mm3 , platelets161000/mm3, urea 63m/dL, creatinine 1.44 m/dL (FG = 40 mL/min/1.73m2), magnesium 1.3 mEq/L, sodium 137 mEq/L, potassium 3.9 mEq/L,prothrombin time (INR) 1.7 and APTT rel 1.26.Laboratorial exams (17/Sep/2011) showed: red blood cells 4.2 million/mmThorax radiography (18/Sep/2011) revealed pulmonary congestion, opacification at theright base and increased cardiac area (presence of double contour and bulging unfoldingof the mid aortic arch) .The coronary angiography (20/Sep/2011) did not reveal coronary obstructions; and therewas severe calcification of the mitral valve.3 , platelets 142000/mm3, urea 74 mg/dL, creatinine 1.97 mg/dL (FG =28 mL/min/1.73 m2), AST 34 U/L, ALT 34 U/L, calcium 4 mEq/L, magnesium 1.3mEq/L, arterial lactate 155 m dL.Laboratorial exams (20/Sep/2011) revealed hemoglobin 11 g/dL, hematocrit 36%, VCM 92 fL,RDW-CV 17.8%, leukocytes 13440/mm3 , platelets 148000/mm3, urea 104 mg/dL, creatinine 2.88 mg/dl (FG =18 mL/min/1.73 m2), sodium 145 mEq/L, potassium 4.5 mEq/L, AST 1863 U/L, ALT426 U/L, gammaGT 87 U/L, alkaline phosphatase 152 U/L, magnesium 1.9 mE L, totalbilirubin 4.05 mg/dL, direct bilirubin 3.5 mg/dL, arterial lactate 173 mg/dL; arterialgasometry: pH 7, 16, pCO2 32.7 mm Hg, pO2 160 mm Hg, O2Saturation 99%, bicarbonate 11mEq/L, base excess (-) 16.2 mEq/L; TP (INR) 3.2; TTPA reltimes 3.13.Exams of 21/Sep/2011 showed hemoglobin 10.5 g/dL, hematocrit 37%, VCM 102 fL, RDW-CV16.8%, leukocytes 17840 mm/mmThorax radiography (21/Sep/2011) showed hypotransparency in the right pulmonary base andan increase in the cardiac area  .During hospitalization the patient developed hemodynamic instability, consumptioncoagulopathy, and presented cardiac arrest in pulseless electrical activity, with noresponse to resuscitation maneuvers, and she passed away .This is about a 59-year-old woman with double mitral lesions with pulmonaryhypertension who, while awaiting surgery, had arterial hypotension, respiratoryfailure with hypotransparency in the right lung field. It evolved withoutimprovement with vasoactive drugs, orotracheal intubation and antibiotic therapy,and she passed away in electrical activity without pulse.1-4The etiology of this patient's valve disease should be attributed to rheumatic fever,although there is no description of an acute outbreak of that disease in thepatient’s medical history. That is not unusual in a rheumatic disease scenario onceusing echocardiogram raises its frequency from 5 to 10 times when compared to aclinical diagnosis.5 which may go ahead with predominanceof humoral response mediated by Th2 lymphocytes, causing more symptoms and leadingmore easily to the use of secondary prophylaxis. Others show a predominance ofcellular response, mediated by Th1 lymphocytes with milder forms of clinicalmanifestations, but those where cardiac involvement predominates. Thus, patients whowould benefit the most from the use of that prophylaxis fail to do it and aresubject to relapses that aggravate valve lesions.6That difference in frequency between clinical diagnosis and echocardiography may bedue to the autoimmune response triggered by molecular mimicry,7-9Those subtypes of CD4 + (Th1 and Th2) lymphocytes produce different cytokines, thoseof Th1-type produce interleukin-2 and interferon-gamma cytokines, and those of Th2subtype secrete Interleukins 4, 5 and 10.10The guidelines of the World Health Organization and the US National Institute ofHealth (NIH) have defined the diagnosis of rheumatic heart disease by the presenceof cardiac murmur consistent with mitral or aortic insufficiency, andechocardiographic evidence of rheumatic valve damage, or history of acute rheumaticfever without echocardiogram done in the acute outbreak.11The predominant clinical onset of the heart rheumatic disease is dyspnea, whichoccurs between the third and fourth decade in life, mostly in women.12In this case, clinical manifestations happened later, but like in the rheumaticdisease, they presented alterations in the mitral valve, the valve most frequentlyaffected in the disease. Mitral regurgitation usually occurs earlier than stenosis,attributed to persistent or recurrent valvulitis.Despite its earlier onset, mitral regurgitation generally has a longer asymptomaticperiod due to increased atrial compliance due to its progressive increase,maintenance of cardiac output by dilatation of the left ventricle, and decreasedregurgitation fraction in exertions to decrease peripheral resistance in theexertion.Still on this case, the initial clinical scenario of de-compensation due to thepresence of tachycardia, once a shorter diastole is more detrimental to mitralstenosis, suggests its predominance. Additionally, the first echocardiogram (2010)was more compatible with the predominance of mitral stenosis with major hemodynamicrepercussion, once the dimensions of the left ventricle were normal and there was anincrease in the left atrium and in the pulmonary systolic pressure, as well asdilation of the right ventricle.2/m2 or< 1.5 cm2, there was fusion of the cusps, in addition to the pressuregradient between the left atrium and the upper ventricle at 10 mmHg, and pulmonaryarterial hypertension above 50 mmHg.13,14Echocardiographic criteria for severe stenosis were present, although the valve areawas not calculated, which would be less than 1.0 cmThe indication for percutaneous and/or surgical treatment is based on the presence ofsymptoms or atrial fibrillation, or systemic embolism, in patients usinganticoagulant and moderate or severe mitral stenosis, and in asymptomatic patientswith pulmonary systolic pressure ≥ 80 mmHg. Percutaneous valvuloplasty stillrequires the presence of favorable morphology - mobile thin cusps, free fromcalcification. In the second echocardiogram, one year after the accompaniment had started, thepredominance of mitral insufficiency was not discarded because there was malfunctionof the left ventricle.vena contracta -width of the jet next to the valve ≥ 0.7 cm; effective regurgitation orifice≥ 0.4 cm2; regurgitation volume ≥ 60 mL; regurgitationfraction ≥ 50%, ventricular filling pattern – ratio of the mitral valve’svelocity-time integral and the aortic valve above 1.3; wave velocity E ≥ 150cm/s; pulmonary artery systolic pressure ≥ 50 mm Hg; indexed atrial volume 60mL/m2; and left ventricle systolic diameter > 4.5 cm.3,15The right time of an indication for surgery in patients with severe mitralincompetence has always been controversial due to the symptoms’ late onset. Surgicaltreatment is indicated for the onset of symptoms, of dilatation (systolic diameter> 4.5 cm), or left ventricular dysfunction (ejection fraction < 60%). Theguidelines define severe mitral regurgitation based on several parameters: valvemorphology - ring dilation ≥ 3.5 cm; characteristics of the regurgitation jet- a rotating jet (multicolor) that reaches the posterior atrial wall, or lateral jetthat fills at least 40% of the atrial surface; the Thus, whichever was the predominance of valvular dysfunction, the patient already hadindication for surgical treatment of the mitral valve because there was alreadysevere hemodynamic repercussion and, as it usually happens in valvopathy with arheumatic origin, there was a double lesion.Other ways in which the disease may appear are atrial arrhythmias, the most commonbeing atrial fibrillation, embolic events, acute heart failure, or infectiveendocarditis.In this case there was de-compensation of heart failure due to nodal reentranttachycardia, arrhythmia is usually not associated with mitral valvopathy. In mitralstenosis there is usually atrial fibrillation due to dilation and atrial fibrosis,in addition to an inflammatory process in the acute phase (Aschoff nodules).16Atrial phenomena in mitral insufficiency are similar to those of stenosis asinterstitial fribrosis and inflammation; however, there is no hypertrophy, myolysisand necrosis of atrial myocytes.As to the patient's final condition, there are three possible causes: infectiousendocarditis, pulmonary infection or pulmonary thromboembolism.For the diagnosis of endocarditis there would be only fever and de-compensation ofheart failure, lacking worsened murmur and vegetation in the valves.17In other words, the involvement of the endocardium in systemic infection has not beenproven. Blood culture is not a diagnostic criterion. The clinical criteria of strongsuspicion are: new valve injury (insufficiency), embolic events of unknown origin,sepsis of unknown origin, hematuria and fever in a patient who has a prosthesis,previous valvopathy and dental or endoscopic manipulation of the colons, newconduction disorders , firstepisode of cardiac de-compensation, positive blood cultures, skin complications or ophthalmic complications (Roth), peripheral abscesses.18According to their frequency, the most common complications - heart failure >systemic embolization > stroke > intracardiac abscess.However, endocarditis cannot be ruled out because in rheumatic valvular disease itcan be difficult to diagnose the vegetation.Infection at any place can be responsible for the de-compensation of heart failure inpatients with severe valvopathy. In this case, pneumonia was suspected due to thepresence of suggestive image at the right base  and leukAs the last and most probable cause of the onset of the patient's final hemodynamicstatus there is pulmonary thromboembolism.The clinical situation is very non-specific and may be mixed up with acute coronarysyndrome or pneumonia. Favoring it there is the image at the base of the right lungand dysfunction of the right ventricle.19In the “International Cooperative Pulmonary Embolism Registry (ICOPER)” pulmonarythromboembolism is associated with the presence of heart failure (hazard ratio 2.4),right ventricle hypokinesia (2.0), systolic arterial hypotension < 90 mmHg (2.9),age > 70 years (1.6), cancer (2.3), chronic obstructive pulmonary disease (1.8).In the same sense, pulmonary thromboembolism with right ventricle hypokinesiadoubled the mortality within 3 months.The exam deemed "gold standard" in the diagnosis of pulmonary thromboembolism isangiotomography, but failing that, or due to the patients’ hemodynamic instability,the finding of dilation and right ventricle dysfunction on the echocardiogram can bediagnostic alternatives.20 (Dr.Desiderio Favarato)And as to hepatic alterations – elevation of transaminases and disorders incoagulation (elevation of TAP-INR- and relation of APTT times) – they are compatiblewith ischemic hepatitis with extensive liver necrosis due to low cardiac output inpatients with high ventricular diastolic pressures. Its onset takes on average oneweek after the episode of low hepatic flow leading to centrilobularnecrosis.Diagnostic hypotheses: Rheumatic mitral valvopathy (double lesion),pulmonary thromboembolism, cardiogenic shock, multiple organ failure. (Dr.Desiderio Favarato)The exam of the heart showed increased weight 470 g) as well as moderate tosignificant increase in volume of both atria. The mitral valve showed a lesioncharacterized by fusion of commissures and marked calcification of the cusps,compatible with rheumatic disease sequelae  and 7. T0 g as weIn the other organs there were signs compatible with shock, such as, for instance,recent centrilobular hepatic necrosis, acute renal tubular necrosis, and smallsub-endocardial infarcts on ventricular walls.Anatomopathological diagnoses: rheumatic heart disease with mitral valvesequelae ; chronic passive pulmonary congestion withsigns of passive pulmonary hypertension; hemothorax on the left without a definedcausal factor.Cause of death: Pulmonary thromboembolism (Prof. Dr. Vera DemarchiAiello)The involvement of the mitral valve in this case is typical of rheumatic diseasesequelae, and the heart showed signs of de-compensation, such as marked dilation ofthe atria. Signals of terminal shock were found in the various organs.21The involvement of only the mitral valve is common in rheumatic disease, and it canbe found in over 50% of the cases of chronic rheumatic diseases as doubledysfunction (stenosis and insufficiency) or just insufficiency.No lesions were found in arterial or venous thoracic vessels able to explainhemothorax.22 In addition to the lesions in thecardiac valves on the left side, myocardial diseases can also evolve chronicallywith secondary pulmonary hypertensive involvement. Individuals thus affected canhave a troubled evolution in the postoperative period of valve surgery, or that of acardiac transplantation, when this is a therapeutic option. A recent study by ourlaboratory revealed that venous lesions of pulmonary hypertension group 2 arefrequent and that the appearance of phosphodiesterase 5 in pulmonary vessels ofthose patients is greater than in the normal ones.23 (Prof. Dr. Vera Demarchi Aiello)The pulmonary situation included recent aspects, such as thromboembolism of hilarvessels and pulmonary infarcts, with other chronic ones, characterized by long-termchronic passive congestion. Passive congestion ends up by causing passive pulmonaryhypertension, which starts in the venous territory. The case under discussionpresented lesions in venules characteristic of this type of impairment. In the lastclassification of pulmonary hypertension, this group (of hypertension secondary tolesions of the left heart) is known as pulmonary hypertension group 2.


This is mediated through activation of the hypoxia-inducible factor (HIF) pathway. At physiological oxygen levels (normoxia), HIF-prolyl hydroxylases (PHDs) hydroxylate proline residues on HIF-α subunits leading to their destabilization by promoting ubiquitination by the von-Hippel Lindau (VHL) ubiquitin ligase and subsequent proteasomal degradation. HIF-α transactivation is also repressed in an O2-dependent way due to asparaginyl hydroxylation by the factor-inhibiting HIF (FIH). In hypoxia, the O2-dependent hydroxylation of HIF-α subunits by PHDs and FIH is reduced, resulting in HIF-α accumulation, dimerization with HIF-β and migration into the nucleus to induce an adaptive transcriptional response. Although HIFs are the canonical substrates for PHD- and FIH-mediated protein hydroxylation, increasing evidence indicates that these hydroxylases may also have alternative targets. In addition to PHD-conferred alterations in protein stability, there is now evidence that hydroxylation can affect protein activity and protein/protein interactions for alternative substrates. PHDs can be pharmacologically inhibited by a new class of drugs termed prolyl hydroxylase inhibitors which have recently been approved for the treatment of anemia associated with chronic kidney disease. The identification of alternative targets of HIF hydroxylases is important in order to fully elucidate the pharmacology of hydroxylase inhibitors (PHI). Despite significant technical advances, screening, detection and verification of alternative functional targets for PHDs and FIH remain challenging. In this review, we discuss recently proposed non-HIF targets for PHDs and FIH and provide an overview of the techniques used to identify these.All metazoans that utilize molecular oxygen (O Because of the absolute reliance on a continuous supply of O2, animals have evolved a mechanism to adapt to hypoxia at a cellular level α and -β h HIF-1β  pathwaysh HIF-1β . In addih HIF-1β . Each HIh HIF-1β ,5,10. In2), iron (Fe2+), α-ketoglutarate  and ascorbic acid (vitamin C) -carboxyl-labeled substrates, such as radioactively labeled 2-OG [2 capture assays are based on the knowledge that hydroxylation by 2-OG-dependent dioxygenases results in the decarboxylation of 2-OG and the release of CO2. Hydroxylation in the presence of 2-OG radiolabeled with 14C at the carbon position leads to the release of radioactive CO2, which can then be captured with filters that are pre-saturated with calcium hydroxide (Ca(OH)2) [2-sensing electrode or quantify unreacted α-ketoglutarate by post-reaction derivatization that forms a fluorescent product [One method to identify prolyl hydroxylation conferred by PHDs is to monitor their activity by measuring led 2-OG ,58,59. TCa(OH)2) ,59. Vari product ,57,60. A product . Alterna product ,63. One In silico screening of putative hydroxylation targets for the HIF-α “consensus sequence”, LXXLAP (with X referring to any amino acid), present in the oxygen-dependent degradation domain (ODD) of HIF-α subunits allows the identification of other putative substrates for PHD-mediated hydroxylation based on sequence similarities with known targets ,64. PoteMass spectrometry (MS) is one of the most frequently applied methods to detect protein hydroxylation in cellular extracts. In MS, the digested peptides are ionized and analyzed in terms of their specific masses and charges (mass/charge ratio) . In geneThe combination of MS and a pharmacological substrate-trapping strategy can facilitate the detection of, for example, transient FIH-mediated enzyme-substrate interactions . To do tAnother way to detect putative prolyl hydroxylation targets is the von Hippel-Lindau protein (pVHL)-capture-based approach . VHL, whFinally, the verification of hydroxylated peptides can be determined by a spectrum of different in vitro and in vivo immunodetection methods including advanced cloning techniques, immunoprecipitation, western blotting using for example specific anti-hydroxyproline (Pro402 and Pro564 ) and antAs outlined above, HIF-igenesis ,71. In togenesis ,73,74. Goduction . Here, wications ,45,46,762) and iron (Fe2+), thus, identifying PHDs as cellular oxygen sensors [In 2001, two research groups published papers reporting that, under normoxia, the HIF-1 complex ,44. Usin sensors . The Kaeresidues .In 2002, Hewitson and colleagues first-described that the newly identified FIH  is respoα . After cof HIF-α . Next, sof HIF-α . Further binding . Interestingly, there are significant differences between the oxygen-dependent regulation of each hydroxylation site in human HIF-1n levels . In addid Pro564 . Surprisn levels . This mi regions . 2 and VHL capture assays, and in silico methods in combination with substrate trapping strategies, which can serve as a model for the screening, detection and verification of other alternative hydroxylation targets. In conclusion, the detection and verification of PHD- and FIH-mediated HIF-Since the discovery of HIF, hundreds of genes have been reported to be sensitive to hypoxia. While HIF is a master-regulator of hypoxia-dependent gene expression, the transcriptional repertoire in response to low oxygen is not restricted to HIF, with multiple other transcription factors affected by hypoxic conditions and/or oxygen levels ,84. Of p2 release assay was not provided in this study [When investigating the molecular mechanisms underpinning the hypoxia-dependent regulation of NF-κB, it was observed that the pan-hydroxylase inhibitor, DMOG, promoted IkBactivity . Using P81 IKKβ) . This mois study .Subsequent studies have supported alterations in hydroxylase-dependent signaling with changes in NF-κB activity ,89,90,91lleagues . They eming IKKβ . While IWhile it was proposed that IKKtability . Alternapothesis . This st of IKKβ .The hypothesis that P191 of IKKcontrols . As parton assay . The reaon assay ,89. ThisThus, in summary, IKKThe tumor suppressor gene, p53, is important in the regulation of a multitude of cellular responses including apoptosis, senescence, DNA repair, and cell cycle arrest ,93,94,95However, it seems that this is only part of the story, as PHDs influence p53, localization, stability and activity on multiple levels. Within this context, it has been shown that PHD1 regulates p53 binding to p38α kinase in a hydroxylation-dependent manner which can cause resistance to chemotherapy in a xenograft colorectal cancer model . Loss ofMore recently, co-immunoprecipitation studies confirmed that PHD1 interacts with p53. In addition, tandem mass spectrometry (MS/MS) revealed that PHD1 most probably hydroxylates p53 at proline 142 . SubsequThere is mounting evidence that both PHD1 and PHD3 can affect p53 protein stability and most probably function through the hydroxylation of proline 359 (PHD3) and 142 (PHD1). However, some controversy exists as to whether the subsequent effects of PHD1 on cellular chemotherapy resistance  or the i2 capture assays) combined with recombinant PHD1 was used to detect novel PHD1 hydroxylation substrates. Using mass spectrometry, the authors could subsequently show that PHD1 hydroxylates FOXO3a at two proline residues (Pro426 and 437), which results in proteasomal degradation [The transcription factor forkhead box O (FOXO) is of relevance to diverse cancer regulatory pathways as it suppresses cell proliferation and survival by activating the expression of cancer-specific target genes ,102,103.radation . The regradation . Furtherradation ,109,110.Notably, FOXO transcription factors repress Cyclin D1 expression, which plays an important role during cell proliferation ,111. ThiIn conclusion, there is evidence to support FOXO3a as an alternative target for PHD1-mediated hydroxylation. Strategies to induce FOXO3a signaling by targeting PHD1 expression or PHI might have therapeutic potential, especially in chronic kidney diseases and in cancers such as breast cancer, in which the expression of FOXO3a or loss of Cyclin D1 inhibits cell proliferation ,111. Mitogen-activated protein kinase (MAPK) 6 is involved in angiogenic pathways, as MAPK6 controls the expression of VEGFR2, induces endothelial cell migration, proliferation and vascular tube formation . In addiUsing a new pharmacological trapping strategy by treating cells with DMOG, combined with quantitative interaction proteomics, Rodriguez and colleagues provided evidence that both RIPK4 and MAPK6 are alternative targets for FIH- and PHD3-mediated hydroxylation, respectively . ApplyinSeveral studies have demonstrated the crucial impact of hypoxia on the cell cycle by, for example, inducing G1/S1 cell cycle arrest both by HIF-dependent and HIF-independent mechanisms ,121,122.A recent study demonstrated that PHD1 hydroxylates centrosomal protein (Cep) 192, which is a critical centrosome component, on the proline residue 1717 . The autRecently, it has been shown that the transcription factor, zinc fingers and homeoboxes 2 (ZHX2), functions as a tumor suppressor during the development of hepatocellular cancer and lymphoma ,126. By Ovarian tumor domain containing ubiquitin aldehyde binding protein 1 (OTUB1) is a deubiquitinase involved in cell metabolism ,127 and 2 capture assay) was decreased compared to wildtype cells [Focusing on the metabolic aspects of OTUB1 signaling, Scholz and colleagues demonstrated, using MS and immunoprecipitation, that OTUB1 is hydroxylated by FIH on asparagine 22 (Asn22) which could be prevented by pharmacological PHD/FIH inhibition with DMOG and hypoxia . DMOG-mepe cells . Notablype cells . Thus, rpe cells . This st2 capture assay also revealed that p105 is a target for FIH-mediated hydroxylation. Of note, the CO2 capture assay detected several other putative FIH targets for hydroxylation including tankyrase-1 or gankyrin [As discussed above, NF-κB signaling is oxygen-dependent ,133. Botgankyrin . Sequencyrin [β) . MS/MS cyrin [β) . Subsequyrin [β) . Inhibityrin [β) . HoweverGiven the fact that FIH likely hydroxylates other ARD-containing proteins , that arReceptor-interacting serine/threonine kinase (RIPK) 4 is known to interact with two isozymes of the protein kinase C family (PKCβ and PKCδ) ,137. MorRodriguez and colleagues demonstrated that RIPK4 is hydroxylated by FIH using a pharmacological trapping strategy with DMOG in combination with quantitative interaction proteomics . FIH-medNon-HIF hydroxylation targets of PHDs and FIH are of great interest in terms of understanding the in vivo pharmacology and further clinical applications of PHIs, as they potentially increase the treatment spectrum of PHIs and could predict putative “off-target” effects of PHIs. By applying in silico genomic screening analysis (for example for the consensus sequence LXXLAP), protein association methods (for example immunoprecipitation) and pharmacological substrate-trapping with PHI followed by mass spectrometry analysis, increasing numbers of candidate targets for PHDs and FIH have been discovered, see 2 capture assay, is that the qualitative and quantitative amounts of enzymes and co-substrates are pre-determined by the experimental design and most likely do not always represent in vivo stoichiometry. The identification of alternative targets will, therefore, be limited by the experimental conditions under which the assay is performed. This explains, in part, conflicting results between the CO2 capture assays that are performed under different conditions , detection  and verification  assays should be applied along with functional assays as a gold standard to identify novel functional non-HIF targets of PHDs and FIH, see One disadvantage of screening techniques that detect enzymatic activity, such as the COl lysate ). Yet unThe enzymatic reaction of PHD- and FIH-mediated HIF hydroxylation, see Most studies to date have shown that the hydroxylation of proteins is directly associated with modified protein stability by either changing the interaction with protein deubiquitinases  or ubiqIn summary, protein hydroxylation by PHDs and FIH is neither unique nor ubiquitous but does affect a number of pathways, both HIF and non-HIF related. There is now mounting evidence that PHDs and FIH mediate protein hydroxylation of alternative targets other than HIF, see


We are reporting a case of a 63-year-old Chinese female who presented to therheumatology clinic with positive antinuclear antibody and unintentional weightloss along with lymphadenopathy. Further workup revealed eosinophilia, elevatedanti–double stranded DNA, serum protein, and serum IgG4 (immunoglobulin G4). Thepatient was diagnosed with systemic lupus erythematosus. Due to the raised IgG4level along with eosinophilia and diffuse lymphadenopathy, IgG4-related systemicdisease was suspected. It was confirmed with IgG4 staining on lymph node biopsy.Our case is presenting the fact that systemic lupus erythematosus andIgG4-related disease can be present in the same patient with multipleoverlapping features making accurate diagnosis challenging. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with a variableclinical presentation and can affect any part of the body. IgG4 (immunoglobulinG4)-related disease (IgG4-RD) is a systemic fibroinflammatory disease with proteanmanifestations involving virtually any organ in the body. Hallmarks of IgG4-relateddisease are lymphoplasmacytic tissue infiltration, fibrosis (often in storiformpattern), obliterative phlebitis, and elevated serum IgG4 concentration. Treatmentmainly involves use of steroids and immunosuppressive agents. We are reporting acase of a 63-year-old female presenting with joint pains, fatigue, unintentionalweight loss along with lymphadenopathy with an unusual overlap of SLE andIgG4-RD.A 63-year-old Chinese female presented to rheumatology clinic with positiveantinuclear antibody 1:80 homogeneous pattern, severe fatigue, hair loss, jointpains for approximately last 3 years, unintentional weight loss of 24 pounds  in last 8 months, and lymphadenopathy. Her joint pains arelocalized to bilateral hands, elbows, shoulders, hips, and knees. Of all the jointsher hands hurt her the most. Joint pains are associated with intermittent swellingand early morning stiffness lasting at least for 30 minutes. Her symptoms were worseduring winter and cold weather. She is unable to do her activities of daily livinglike holding a coffee mug, eating with a spoon, opening bottles, and so on.She was evaluated for underlying malignancy in the setting of generalizedlymphadenopathy and significant unintentional weight loss. Her past medical historywas significant for hypertension, thyroid nodule status post ultrasound-guided fineneedle aspiration cytology consistent with benign follicular colloidal nodule andbilateral carpal tunnel syndrome on electromyography status post nerve release.3 2000 international units. Family historywas significant for cancer in paternal grandmother and hypertension andhypercholesteremia in mother. She is an ex-smoker with 30 pack-year smoking historyand quit smoking 2 years ago.Her medications include benazepril-hydrochlorothiazide 20 mg–12.5 mg, diclofenacpotassium 50 mg PRN, vitamin DPhysical examination was positive for thin, cachectic female with palpable posteriorcervical, supraclavicular, and bilateral axillary lymphadenopathy. Tenderness wasfelt in multiple proximal inter phalangeal joints of bilateral hands with mildsynovitis. Diffuse thinning of hair on scalp was noted.Further review of records showed intermittent eosinophilia on the complete bloodcount. Subsequent work-up showed elevated anti–double stranded DNA 1:40 byimmunofluorescence assay , elevated IgG4 level of 452 mg/dL , elevated serum protein 9.7 g/dL, serum globulin 7.1 g/dL, serumprotein electrophoresis showing polyclonal increase in the gamma region with noM-spike, and erythrocyte sedimentation rate of 67 mm/h.Other testing was negative for smith antibody, ribonuclear protein antibody,anti-SSA, anti-SSB, rheumatoid factor, anti-cyclic citrullinated peptide,anti-phospholipid antibody panel, anti-neutrophilic cytoplasmic antibody withmyeloperoxidase, and proteinase 3 antibody. C3 and C4 levels within normal limits.Urinalysis and renal function were normal. Infection workup was negative forhepatitis, HIV, tuberculosis (quantiferon gold), syphilis (rapid plasma reagin), andcoccidiomycosis. Peripheral blood flow cytometry showed no flow cytometric evidenceof monoclonality, acute leukemia, or lymphoproliferative disorder.A computed tomography scan of the neck, chest, abdomen, and pelvis with and withoutcontrast showed prominent cervical , axillarAge-specific cancer screening was negative for malignancy including pap smear,colonoscopy, and bilateral screening mammogram.Ultrasound-guided fine needle aspiration cytology of bilateral axillary lymph nodeswas negative for malignancy with abundant plasma cells and positive IgG4 staining and 3.She was diagnosed with overlapping syndrome of IgG4 related disease and SLE. She wasstarted on 10 mg prednisone and 200 mg plaquenil daily, which significantly improvedher symptoms. Her prednisone was tapered slowly and completely discontinued in 5months. She is currently on maintenance plaquenil 200 mg daily back to her usualstate. She gained weight, and her other symptoms of fatigue, hair loss, joint pains,and lymphadenopathy completely resolved. Her repeat anti–double stranded DNA, IgG4,and eosinophil levels were within normal limits .1SLE is a chronic autoimmune disease with a variable clinical presentation and canaffect any part of the body. The most common presentation is a mixture of symptomsaffecting the skin, musculoskeletal system, hematologic, and fatigue along withpositive serologies. The diagnosis of SLE should be based on a combination ofclinical and immunologic findings as described in Our patient satisfied 4 criteria with at least 1 clinical and 1 immunologic criteriaas mentioned in the case presentation and was labelled as SLE.3 Serum IgG4 levels are elevatedin approximately 60% of patients. Up to 40% of patients with IgG4-RD have aperipheral eosinophilia and elevated IgE levels.3 Our patient had elevation ofIgG4 level and peripheral blood eosinophilia. Her IgE level was not checked.IgG4-RD is a systemic fibroinflammatory disease with protean manifestations involvingvirtually any organ in the body. Hallmark of IgG4-RDs are lymphoplasmacytic tissueinfiltration, fibrosis (often in storiform pattern), obliterative phlebitis, andelevated serum IgG4 concentration. Most commonly seen in middle-aged and olderpeople; mean age from 59 to 68 years and more common in men (70% to 80%).IgG4 is an antibody that has a very unique structure and function. It is the leastabundant antibody and comprises less than 5% of antibody in human class. Generally,IgG4 level in human body remains always stable in normal condition.4Autoimmunity is one of the commonest triggers for IgG4-RD with key role of T helper-2cell (Th2) cell involvement in the pathophysiology. Th2 cells overexpressinterleukins 4, 5, 10, and 13 as well as transforming growth factor-β, whichresulted in increased eosinophils as well elevated IgG4 and IgE concentrations. Italso contributes to activation of fibroblasts leading to fibrosis.IgG4-related systemic disease can involve pancreas, biliary tree, salivary glands,periorbital tissues, kidneys, lungs, lymph nodes, meninges, aorta, prostate, breast,thyroid gland, pericardium, and skin.5 proposed a set of criteria for the diagnosis of systemic IgG4-RD designed tobe used independently of the predominant organ: (1) serum IgG4 concentration >135mg/dL and (2) >40% of IgG+ plasma cells being IgG4+ and >10 cells/high-poweredfield of biopsy sample. Validation of these criteria needs to be tested further byconducting international multidisciplinary collaboration with an expert panel. Ourpatient had elevated serum IgG4 level along with abundant IgG4-positive plasma cellson the biopsy.The diagnostic approach of IgG4-related systemic disease should be based on acombination of clinical evaluation, laboratory parameters, imaging, and biopsyresults. Initially proposed criteria were centered on specific organs rather thansystemic IgG4-RD. In 2011, Umehara et al6 first described a form of IgG4-RD as sclerosing pancreatitis in 1961. It waslater termed as autoimmune pancreatitis. Since then, IgG4-RD has been described innearly every organ system. Enlargement of organ is the key finding for clinicalsuspicion of IgG4-RD. Organomegaly is detected either through physical examinationor in incidental finding on diagnostic imaging.7 Symptoms related to specificorgans can be also the primary features like abdominal pain, respiratory symptoms,diarrhea, and pruritis.7Lymphadenopathy is seen in 25% patients approximately.4 In laboratory tests, mild tomoderate eosinophilia is present in one third patients.4 Computed tomography and magnetic resonance imaging findings are veryimportant to support the diagnosis of IgG4-RD. Due to heterogeneous characteristicsof IgG4-RD signs and symptoms, diagnosis is very challenging.Sarles et al8The main therapeutic option in IgG4-RD is glucocorticoids. Based on diseasepresentation, immunosuppressive drugs are also being used. In 90% reported cases,azathioprine was used as an immunosuppressive agent. Anecdotal data and multiplecase reports mention the use of other immunosuppressive agents such ascyclophosphamide methotrexate, mycophenolate mofetil, and tacrolimus. Biologicagents (Rituximab) and surgery are also available options to treat depending on thesymptoms and clinical findings. Because of rarity of the disease, no randomizedcontrolled trails have been conducted and most of the treatment options are derivedfrom individual experience, case reports, and case series.We report an unusual case of overlap of SLE without any kidney involvement andIgG4-RD. Review of the literature shows only one case describing an overlap ofIgG4-RD and lupus nephritis. Our case highlights that these 2 diseases can overlapin some patients, which was never reported in the past.


Objective: Since decreased brain-derived neurotrophic factor (BDNF) and increased dipeptidyl peptidase-4 (DPP4) activity have both been implicated in the pathogenesis of mild cognitive impairment (MCI), the aim of our study was to evaluate the association of MCI with plasma DPP4 activity to BDNF ratio (DBR) in an elderly population with normal glucose tolerance.Methods: We cross-sectionally measured C-reactive protein, interleukin-6, nitrotyrosine, 8-iso-PGF2a, DPP4 activity BDNF and calculated the DBR in a total of 1,066 elderly participants in China. MCI was determined by the Montreal Cognitive Assessment and finally confirmed by neurologists.Results: An inverse correlation was found between DPP4 activity and BDNF  and this inverse correlation was partly mediated by nitrotyrosine and 8-iso-PGF2a. Across rising quartiles of DBR, nitrotyrosine, 8-iso-PGF2a, C-reactive protein and interleukin-6 progressively increased, whereas the Montreal Cognitive Assessment score progressively decreased. Subjects in the lowest quartile of BDNF and highest quartiles of DBR and DPP4 activity, had higher MCI risk compared with subjects in the highest quartile of the BDNF and lowest quartiles of DBR and DPP4 activity, respectively . The odds ratio for MCI became more pronounced with decreased BDNF and increased DPP4.Conclusion: In conclusion, a negative correlation was found between DPP4 activity and BDNF, and this negative correlation was partly mediated by oxidative stress, not inflammation. The DBR was positively associated with MCI and thus may be used as a novel risk biomarker for MCI in an elderly population with normal glucose tolerance. Mild cognitive impairment (MCI) is a transitional step between normal cognition and dementia, in which patients have objective disturbances and cognitive complaints on cognitive examinations, but in which their ability to carry out daily life activities are not affected . Once MCDipeptidyl peptidase-4 (DPP4) is a serine exopeptidase belonging to the S9b DPP family that exists as a membrane-anchored cell surface protein or circulates as a soluble form in the peripheral circulation , 2018b. Therefore, the aim of this study was to determine (1) whether plasma DPP4 activity is inversely related to, (2) what factors might mediate this inverse relationship, (3) whether DBR could be considered as a novel biomarker for MCI. Previous studies have found significant correlations between hyperglycemia and DPP4 activity, BDNF and cognitive impairment , to exclThe study participants included 1066 elderly non-diabetic subjects aged 60 years or older, who visited the Medical Examination Department of the Affiliated Hospital of Guilin Medical University, between 2013 and 2016. Subjects were excluded if they met any of the following criteria: (1) presence of diseases comprising of hyperglycemia, head trauma, hypertensive crisis, malignancy, hypothyroidism, non-alcoholic fatty liver disease, chronic or acute inflammatory diseases, dementia, heart, liver, respiratory, and kidney dysfunction, (2) history of psychological disturbances, visual/auditory disorders and central nervous system disease that might result in dementia, (3) those who had taken medication affecting circulating levels of DPP4 activity or cognitive function within one year before enrollment, (4) drug or alcohol abuse. Our study was approved by the Drugs and Medical Apparatus Ethics Committee at the Affiliated Hospital of Guilin Medical University, and all participants provided signed written informed consent .Demographic and clinical data were recorded in a standardized interview by trained staff. Venous blood specimen was collected from fasting participants to measure circulating levels of CRP, IL-6, 8-iso-PGF2a, nitrotyrosine, BDNF, and DPP4 activity. The plasma levels of BDNF were determined using an ELISA kit , BDNF concentrations were obtained against a standard curve and expressed as ng/ml. DPP4 activity, 8-iso-PGF2a, nitrotyrosine, CRP, and IL-6 were assayed as previously described , 2015a. t-test, chi-square and ANCOVA to compare clinical and biochemical parameters. ANCOVA was conducted to compare continuous variables across DBR quartiles after adjusting for age, gender, and BMI. The relationships between parameters were investigated with partial correlation analysis and multivariate logistic regression analysis.Statistical analyses were performed with SPSS version 16.0. Data were presented as proportions, frequencies, median (interquartile range) or mean ± standard deviation. We used the Y = cX + e1 (2) M = aX + e2 (3) Y = c’X + bM + e3, X represents DPP4 activity, Y represents BDNF, M represents the mediator, a represents the regression coefficient for the relationship between DPP4 activity and mediator, b represents the regression coefficient for the relationship between mediator and BDNF, c represents the regression coefficient for the relationship between DPP4 and BDNF, and c’ represents the direct effect of DPP4 on BDNF after controlling for the indirect effect. A Sobel Test was conducted to further test mediation. An indirect ratio was calculated to estimate the strength of mediation: ([a∗b]/c)  [a∗b]/c) .P < 0.05), with a lower BDNF and MoCA score and higher 8-iso-PGF2a, nitrotyrosine, CRP, IL-6 and DPP4 activity . Compared with controls, subjects with MCI had a lower BDNF and MoCA score and higher 8-iso-PGF2a, nitrotyrosine, DPP4 activity and a DBR (P < 0.01) (2.36 ± 1.11 ng/ml in men vs. 2.65 ± 1.07 ng/ml in women) (nd a DBR . Comparen women) .Partial correlation analyses revealed that (1) DPP4 was inversely related to BDNF in all participants, (2) 8-iso-PGF2a, nitrotyrosine, CRP, and IL-6 were all positively correlated with DBR and DPP4 activity and negatively with BDNF, (3) BDNFs were positively associated, and DPP4 and DBRs negatively, with the MoCA score .P < 0.01), in the second model, BDNF was inversely correlated with DPP4 activity (P < 0.01), in the third model, DPP4 activity, nitrotyrosine or 8-iso-PGF2a were all inversely correlated with BDNF (P < 0.01). A Sobel test revealed that 8-iso-PGF2a and nitrotyrosine both had an indirect effect, representing 20.4 and 26.0% of the total effect on the correlation between BDNF and DPP4 activity, respectively. When the participants were further divided into two subgroups according to gender, the results of the mediating role of oxidative stress parameters in the inverse correlation between DPP4 activity and BDNF in male and female participants showed similar results   . The odd < 0.01) .The novel findings of this study were that (1) circulating levels of BDNF were inversely correlated with DPP4 activity, (2) the inverse correlation between BDNF and DPP4 was partially mediated by oxidative stress, not inflammation (3) higher levels of DBR and DPP4 activity and lower levels of BDNF were positively related to the increased risk of MCI.Previous researches suggested that decreased BDNF and increased DPP4 activities were found in the periphery circulation of patients with MCI , suggestD-aspartate channel  since this study was a cross-sectional research, inferences about causalities are limited, (2) some unknown factors other than oxidative stress might also exist to mediate the negative correlation between BDNF and DPP4, (3) the presence of some unmeasured confounders may also have had some effects on the relationship between MCI and DBR limiting generalizations of this study, however, after adding a wide range of potential confounders in the statistical analyses, the strong relationship between DBR and MCI are unlikely to be an artifact, (4) we failed to discuss how other diseases may influence DBR. Previous studies have reported higher levels of DPP4 activity in patients with diabetes, obesity and depression , in addiIn conclusion, our current data supported the negative relationship between BDNF and DPP4 activity in MCI, from a clinical perspective, we extrapolated that this negative relationship was partly mediated by oxidative stress, not inflammation. In particular, DBR might be more suitable to serve as a risk biomarker for MCI than either BDNF or DPP4 activity alone, in an elderly non-diabetic population. However, because of the nature of this study, the feasibility of identifying DBR as a novel biomarker or even a therapeutic target for MCI still needs to be confirmed by future studies.TZ, BG, LX, and XC conceptualized and designed the study. TZ, BG, BC, XC, LQ, XH, LX, HP, YC, and LQ performed the data acquisition and analysis, or interpreted the data. TZ, LX, LT, and YG drafted the manuscript. TZ, LT, and YG critically revised the manuscript for important intellectual content. LQ performed the statistical analysis. TZ obtained funding and supervised the study. All authors approved the final manuscript to be published and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


There is a morphological overlap among renal epithelial tumors, particularly chromophobe renal cell carcinoma (CHRCC), clear cell renal cell carcinoma (CCRCC), renal oncocytoma (RO), and papillary renal cell carcinoma (PRCC). Discriminating between these tumors is important but sometimes challenging. This study is aimed at evaluating the clinical usefulness of the combined immunochemistry for the “three 7” markers  to distinguish chromophobe renal cell carcinoma from these mimics.  Immunochemical staining for CK7, CD117, and Claudin-7 was performed in 68 CHRCCs, 199 CCRCCs, 32 ROs, and 30 PRCCs. Fluorescence in situ hybridization (FISH) was performed in some cases to exclude CCRCC and PRCC. The sensitivity (SE) and specificity (SP) for CHRCC as well as the immunoreactivity of each marker and their combinations were statistically evaluated.  High positive rates for CK7 (94%), CD117 (87%), Claudin-7 (94%), and their combinations  were observed in CHRCC compared to those in CCRCC, RO, and PRCC, with increasingly higher SP when combinations of the “three 7” markers were applied .  CK7, CD117, and Claudin-7 are frequently expressed in CHRCC with high specificity. We recommend the routine use of these 3 markers as a routine panel when making a differential diagnosis of CHRCC and excluding other mimics. Chromophobe renal cell carcinoma (CHRCC) is the third most common renal cell carcinoma RCC, 5%) and is inferior to clear cell renal cell carcinoma  and papillary renal cell carcinoma PRCC, 15%) [5% [1]. C% and is β/S100A1, etc. [The diagnosis of renal cell carcinoma is sometimes challenging and troubling for pathologists because of the frequent histologic overlapping among each carcinoma type. The distinction of CHRCC from clear cell renal cell carcinoma (CCRCC), renal oncocytoma (RO), papillary renal cell carcinoma (PRCC), and renal cell carcinoma with XP11.2 translocation/TEF3 fusion (XP11.2 tRCC) may cause a diagnostic dilemma. Numerous immunochemical markers have been reported, including CK7, CD117 (KIT), parvalbumin, DOG1 cyclin D1, vimentin, EMA, S1001A, kidney-specific cadherin (Ksp-cad), Claudin-7, and Claudin-8 –9. HowevA1, etc. , 11–15. WHO Classification of Tumours of the Urinary System and Male Genital Organs [This study was approved by the Institutional Review Board of the Department of Pathology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine. Cases diagnosed as CHRCC, CCRCC, RO, and PRCC with complete clinicopathologic data were selected from May 1, 2010, to May 1, 2019. All hematoxylin and eosin- (HE-) stained slides were independently reviewed by 2 experienced pathologists (X.Y. and C.F.W.). A diagnostic consensus on each case was achieved according to the 2016 l Organs .Each surgical specimen was specifically resectioned, and the markers CD7, CD117, and Claudin-7 were stained. Four-micrometer thick sections were obtained from 10% formalin-fixed and paraffin-embedded tissue blocks, followed by immunohistochemical staining using the following commercially available antibodies: anti-CK7 , anti-CD117 , and anti-Claudin-7 . Antibody binding was detected using a universal immunoperoxidase polymer method . A Dako automated immunohistochemistry system  was used according to the manufacturer's protocol. The IHC results were independently interpreted by 2 experienced pathologists (J.Z. and C.F.W.). More than 10% of tumor cells showing membranous or both membranous and cytoplasmic staining for CK7, CD117, and Claudin-7 were considered positive: focal, 10%-50%; diffuse, more than 50%.For a subset of difficult cases showing overlapping morphological and immunohistochemical features, FISH was additionally applied . The procedure has been previously described , 19. TheThe sensitivity (SE) and specificity (SP) of CHRCC compared to those of the other 3 types of renal cell tumors  were calculated using standard formulas. The differences in the immunoreactivity of each marker and their combinations were evaluated using the chi-squared test between CHRCC and CCRCC, PRCC (type 1 and type 2), or RO.n = 24), grade 2 , grade 3 , and grade 4 . Grading was not performed in ROs (n = 3), grade 2 , grade 3 , and grade 4 . To confirm the separation of a subset of renal tumors, the gene status of 3p (n = 10) or the combinations of chromosomes 7, 17, and Y for PRCC (n = 14) were detected using FISH. All CCRCCs showed a loss of 3p , except for the expression of CK7 or Claudin-7 between CHRCC and type 1 PRCC and CD117 between CHRCC and RO.The SE and SP of the “three 7” markers in CHRCC and comparisons of CHRCC with CCRCC, PRCC, and RO are summarized in There are overlapping morphological features among CHRCC, PRCC, CCRCC, and RO, often leading to diagnostic challenges when encountering difficult cases. Immunohistochemical staining, compared to electron microscopy or Hale colloidal iron staining, is an easier way to facilitate the discrimination of these carcinomas . Althougp = 0.5497). Some negative cases can show marked focal positivity for CK7 in either CCRCC or RO [Notably, 60-100% of CHRCCs are positive for CK7, with a typically diffuse (90-100% of tumor cells) and strong staining pattern , 22. ConCC or RO . The scaCC or RO . AccordiCC or RO . The SP CC or RO . AnalogoCC or RO –30. AlthCC or RO , 28–31, The recently described HOCTs have been observed in distinct clinical settings, including renal oncocytosis, Hogg–Dubé syndrome, or sporadic RCC . HOCTs aAlthough there were papers applying CK7, CD117, or Claudin-7 with other markers to make differential diagnoses, the combination of these 3 markers has never been reported.In diagnostic practice, differential diagnoses of CHRCC, PRCC, CCRCC, and RO are routinely performed before the given diagnosis of any of these 4 tumors is made. In this scenario, the specificity of immunostaining markers is a priority but the sensitivity is secondary. The diagnostic SP for CHRCC of the combination of any two of the markers CK7, CD117, and Claudin-7 was significantly enhanced compared to that of a single immunostaining marker. The application of these “three 7” markers together reached 100% SP in our study. In the past 10 years of practice, we have routinely used these 3 markers when differentiating CHRCC from other renal cell carcinomas. When histologic overlapping exists between CHRCC and other renal cell carcinomas, more than 2 positive markers are more likely to support the diagnosis of CHRCC . If only one or none of the 3 markers was positive, then the diagnosis of CHRCC is suggestive but uncertain, and therefore, further investigations should be carried out. We usually perform special staining colloidal iron and FISH tests  to exclude the other mimics. However, morphology is a priority, when there is a paradox between the typical histological features of a given carcinoma and the immunohistochemical staining. Cases that do not benefit from “three 7” immunostaining (only 1 or no marker positive) without superimposed classical morphology and distinct molecular markers present truly difficult situations, and most of these cases are designated as unclassified renal cell carcinomas. However, additional markers need to be investigated in the future.In conclusion, our study and experience demonstrated that CK7, CD117, and Claudin-7 are frequently expressed in CHRCC with high specificity. Knowing the expression features and patterns facilitates the interpretation of positive staining in the renal cell carcinomas. Application of the “three 7” markers, if necessary, with some special staining and/or molecular tests can resolve a majority of the diagnostic issues for CHRCC. We recommend that these 3 markers are used as a routine panel in the differential diagnosis of CHRCCs from other mimics.