Assessment of Occult Pulmonary Involvement in Ulcerative Colitis.

INTRODUCTION: Nearly 50% of patients with inflammatory bowel disease (IBD) experience at least one extraintestinal manifestation. Bronchopulmonary involvement is rare in IBD. Pulmonary function test (PFT) abnormality in cases of ulcerative colitis (UC) has been reported to be 17-55%. Occult pulmonary disease may be diagnosed using variables of the PFT. Hence, we aim to evaluate the frequency and type of pulmonary dysfunction in patients with UC in remission. METHODS: Eighty-three patients of UC in remission and 48 controls underwent the PFT including forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC), Tiffeneau value (FEV1/FVC), and midexpiratory flow (MEF 25-75%) rate with a spirometer. The patients were divided based on the age of onset of UC into A1 (<16 years), A2 (16-40 years), and A3 (>40 years) and based on the extent of disease into E1 (proctitis), E2 (left-sided colitis), and E3 (extensive colitis). RESULTS: Patients with UC had significantly abnormal PFT compared with controls (51 [61.5%] vss. 8 [16.67%]; p = 0.000). Patients with UC commonly had a restrictive pattern (33 [64.47%]) of PFT followed by small airway disease (11 [21.56%]) and obstructive pattern (7 [13.72%]). Pulmonary involvement in cases of UC was more in E3 followed by E2 and E1. Pulmonary involvement was more in the late age of onset of disease. BMI was positively and significantly correlated with FEV1 and FVC. Hemoglobin had a positive and significant correlation with FEV1 while a negative correlation with FEV1/FVC and MEF 25-75%. All predictors except for age were found to contribute in higher risk (OR > 1) for PFT abnormality. CONCLUSION: Patients with UC have chronic pulmonary inflammation leading to different patterns of lung involvement in the form of restrictive, obstructive airway, and small airway disease. Patients with UC commonly have a restrictive pattern of pulmonary involvement. Impairment of the PFT is related to the disease extent and the age of onset of disease. Assessment of the PFT using a spirometer is a noninvasive, simple, cost-effective, and reliable method for early detection of occult pulmonary involvement in patients of UC.

Yield of p53 expression in esophageal squamous cell cancer and its relationship with survival.

BACKGROUND/AIMS: Esophageal squamous cell carcinoma (ESCC) is the most aggressive type of cancer. Mutation of tumor suppressor gene p53 is observed in many gastrointestinal malignancies including ESCC. The immunohistochemical protein expression of mutant p53 has been proposed as a potential tool to evaluate the biological behavior of ESCC. Predictive value of p53 for survival is debatable, hence this study was formulated to know the survival of patients with p53 expression in ESCC. PATIENTS AND METHODS: We prospectively included 91 consecutive patients of ESCC from August 2014 to August 2016. Biopsy specimens were treated immunohistochemically and expression of p53 gene was analyzed by Immunoreactive Score (IRS). These findings were then compared with clinicopathological parameters such as age, gender, histological grades, and TNM stages. All patients received treatment and were kept under regular follow-up. RESULTS: M: F ratio was 2.03:1. p53 expression analyzed by IRS showed low expression (score ≤6) in 35 patients (38.46%) and high expression (>6) in 56 patients (61.54%). Level of p53 expression increased significantly with increasing histological grades of ESCC and TNM stage (P ≤ 0.001). Multivariate analysis shows p53 expression as independent predictor of survival. After 1 year of follow up, survival in the p53 high-expression group was 67.86% [standard error (SE) = 0.0473, confidence interval (CI) = 0.75-0.97) and in low p53 expression group was 91.43% (SE = 0.06, CI = 0.53-0.78) with statistically significant difference P = 0.0001 when analyzed with Kaplan-Meier method. CONCLUSION: Expression of p53 correlates with the survival and is a simple, effective and reproducible modality to determine the prognosis and survival in ESCC.

Synchronous Dual Primary Malignancies of Liver and Pancreas with Colonic Metastasis: An Unique Presentation.

Synchronous appearance of the primary gastrointestinal malignancies is rare. Coexistence of primary pancreatic and hepatocellular carcinoma as synchronous malignancy is even rarer. We report a case of such combination in a 50 year old female who presented with bleeding per rectum and while evaluating we found simultaneous appearance of primary malignancies of pancreas, liver and solitary colonic metastasis from the pancreas in the background of chronic calcific pancreatitis. To the best of our knowledge this combination of synchronous pancreatic and hepatocellular carcinoma and colonic metastasis from the pancreas is unique.

Targeted inactivation of integrin-linked kinase in hair follicle stem cells reveals an important modulatory role in skin repair after injury.

Integrin-linked kinase (ILK) is key for normal epidermal morphogenesis, but little is known about its role in hair follicle stem cells and epidermal regeneration. Hair follicle stem cells are important contributors to newly formed epidermis following injury. We inactivated the Ilk gene in the keratin 15--expressing stem cell population of the mouse hair follicle bulge. Loss of ILK expression in these cells resulted in impaired cutaneous wound healing, with substantially decreased wound closure rates. ILK-deficient stem cells produced very few descendants that moved toward the epidermal surface and into the advancing epithelium that covers the wound. Furthermore, those few mutant cells that homed in the regenerated epidermis exhibited a reduced residence time. Paradoxically, ILK-deficient bulge stem cells responded to anagen growth signals and contributed to newly regenerated hair follicles during this phase of hair follicle growth. Thus ILK plays an important modulatory role in the normal contribution of hair follicle stem cell progeny to the regenerating epidermis following injury.

Bilateral thalamic lesions.

OBJECTIVE: Bilateral thalamic lesions are rare and relatively obscure neoplasms. We present our experience with nine cases of bilateral thalamic lesions and attempt to analyse them in the background of available literature. MATERIALS AND METHODS: Retrospective analyses of the case records of 9 cases of bilateral thalamic lesions treated in our department since January 2002, which have a minimum of 1 year follow-up. RESULTS: The study group included four males and five females with a mean age of 14.6 years (5 years to 29 years). Seven of these patients had radiological evidence of bilateral thalamic lesions at presentation and 2 patients had involvement of the opposite thalamus at a later stage of the disease. All patients except one presented with raised intracranial pressure symptoms. Focal motor deficits (4/9), behavioral and memory disturbances (3/9) were the other major presenting symptoms. Biopsy confirmation was possible in six patients and histopathology was suggestive of low grade fibrillary astrocytoma in all six patients. Seven patients required CSF diversion procedure for associated hydrocephalus. Eight of our nine patients underwent radiotherapy. On last follow-up, 3 patients were clinically stable with images suggestive of arrested disease, four patients had evidence of progressive disease both clinically and radiologically and there were two recorded cases of mortality. CONCLUSION: Primary bilateral thalamic lesions have characteristic neuroradiological properties and are distinct from unilateral thalamic tumours with bilateral progression. Almost all of these lesions on histology prove to be gliomas but decompressive surgery is seldom feasible. Surgical intervention is limited to biopsy and CSF diversion for hydrocephalus. Bilateral thalamic lesions remain unresponsive to adjuvant therapy and generally carry a poor prognosis.

Meningiomas of the lateral ventricle - a report of 15 cases.

Lateral ventricular meningiomas are rare tumours that pose considerable surgical challenge. This study attempts to analyse some of the important clinical features of these tumours and review technical considerations in surgery for lateral ventricular meningiomas. A retrospective analysis of the case records of patients with lateral ventricular meningiomas operated in our institute since 1998 with a minimum of one year follow up was done. The variables analysed included age, sex, clinical presentation, imaging characteristics, histopathology and operative details. Outcome was analysed using the Glasgow outcome score (GOS). Fifteen patients with a mean age of 40.6 years formed the study group. A female preponderance was observed (M:F 5:10). Raised intracranial pressure was the predominant symptom at presentation (10/15; 66%) followed by visual field deficits (6/15; 40%) and contralateral motor deficits (5/15; 33.3%). One patient presented with evidence of intratumoural bleed. The tumour was on the right side in 7 patients and on the left side in 8 patients. The lesion was located in the trigone of the lateral ventricle in 13 patients and in the body of the ventricle in two. The tumours were excised through a parietooccipital approach in 11 (73.33%) patients and through a middle temporal gyrus approach in 4 (26.66%).The tumour recurred in 2 patients, both tumours being histologically fibroblastic variants. Fresh operative complications included motor deficits in 3, contralateral homonymous hemianopia in 2, dysphasia in 1, refractory seizures in 2 and loculated hydrocephalus in one. We had no operative mortality. At last follow-up for 10 patients were in GOS 5, two were in GOS 4 and three in GOS 3. Lateral ventricular meningiomas are difficult tumours to operate. Total surgical excision through a superior parietal lobule or middle temporal gyrus approach is possible in most cases with minimal morbidity.

Integrin-linked kinase interactions with ELMO2 modulate cell polarity.

Cell polarization is a key prerequisite for directed migration during development, tissue regeneration, and metastasis. Integrin-linked kinase (ILK) is a scaffold protein essential for cell polarization, but very little is known about the precise mechanisms whereby ILK modulates polarization in normal epithelia. Elucidating these mechanisms is essential to understand tissue morphogenesis, transformation, and repair. Here we identify a novel ILK protein complex that includes Engulfment and Cell Motility 2 (ELMO2). We also demonstrate the presence of RhoG in ILK-ELMO2 complexes, and the localization of this multiprotein species specifically to the leading lamellipodia of polarized cells. Significantly, the ability of RhoG to bind ELMO is crucial for ILK induction of cell polarization, and the joint expression of ILK and ELMO2 synergistically promotes the induction of front-rear polarity and haptotactic migration. This places RhoG-ELMO2-ILK complexes in a key position for the development of cell polarity and forward movement. Although ILK is a component of many diverse multiprotein species that may contribute to cell polarization, expression of dominant-negative ELMO2 mutants is sufficient to abolish the ability of ILK to promote cell polarization. Thus, its interaction with ELMO2 and RhoG is essential for the ability of ILK to induce front-rear cell polarity.

Childhood and adolescent meningiomas: a report of 38 cases and review of literature.

BACKGROUND: The aim is to study the clinical, radiological and pathological features of childhood and adolescent meningiomas and analyse outcome prognosticators. METHOD: A retrospective analysis of the case records of patients less than 20 years of age operated for a meningioma in our institute since 1982 was performed. The variables analysed included age, sex, presentation, associated neurofibromatosis (NF), imaging characteristics, extent of resection and histopathology. RESULTS: The study group included 20 males and 18 females with a mean age of 15.53 years. Eleven children (28.9%) had evidence of NF of whom three had NF2 with bilateral vestibular schwannomas. The common presenting symptoms were seizures (76.3%), raised intracranial tension (71%), and focal neurological deficits (39.4%). The location of the operated tumours were as follows: ten skull base (24.4%), ten falx/parasagittal (24.4%), eight spinal (19.5%), five convexity (12.2%), three posterior fossa (7.3%), three intraventricular (7.3%) and two optic nerve sheath (4.9%). Two children (4.9%) had cystic meningiomas. Grade I excision was achieved only in twenty tumours (48.8%). On histopathology, thirty (73.2%) were grade I, nine (21.9%) were grade II and two (4.9%) were grade III meningiomas. Seven tumours recurred of which six were located at the skull base. During the mean follow up period of 4.74 years, the majority, 32 (84.2%) had a good outcome and five (13.2%) had a poor outcome. One child (2.6%) expired due to post-operative sepsis. CONCLUSION: Childhood meningiomas are uncommon but not rare lesions with a marginal male predominance. Absence of large series with long follow up precludes any definite conclusions on the clinical course and outcome. Uniform observations made in different series including ours, include a higher incidence of the skull base location and tumours with atypical histopathology. Favourable prognostic factors include younger age (< than 10 years), superficial location, total excision and absence of neurofibromatosis. Location and extent of excision appear to be more important than histopathology grade in predicting outcome.

Modulation of integrin-linked kinase nucleo-cytoplasmic shuttling by ILKAP and CRM1.

Integrin-linked kinase (ILK) plays key roles in a variety of cell functions, including cell proliferation, adhesion and migration. Within the cell, ILK localizes to multiple sites, including the cytoplasm, focal adhesion complexes that mediate cell adhesion to extracellular substrates, as well as cell-cell junctions in epidermal keratinocytes. Central to understanding ILK function is the elucidation of the mechanisms that regulate its subcellular localization. We now demonstrate that ILK is imported into the nucleus through sequences in its N-terminus, via active transport mechanisms that involve nuclear pore complexes. In addition, nuclear ILK can be rapidly exported into the cytoplasm through a CRM1-dependent pathway, and its export is enhanced by the type 2C protein phosphatase ILKAP. Nuclear localization of ILK in epidermal keratinocytes is associated with increased DNA synthesis, which is sensitive to inhibition by ILKAP. Our studies demonstrate the importance for keratinocyte proliferation of ILK regulation through changes in its subcellular localization, and establish ILKAP and CRM1 as pivotal modulators of ILK subcellular distribution and activity in these cells.

Impaired hair follicle morphogenesis and polarized keratinocyte movement upon conditional inactivation of integrin-linked kinase in the epidermis.

Integrin-linked kinase (ILK) is key for cell survival, migration, and adhesion, but little is known about its role in epidermal development and homeostasis in vivo. We generated mice with conditional inactivation of the Ilk gene in squamous epithelia. These mice die perinatally and exhibit skin blistering and severe defects in hair follicle morphogenesis, including greatly reduced follicle numbers, failure to progress beyond very early developmental stages, and pronounced defects in follicular keratinocyte proliferation. ILK-deficient epidermis shows abnormalities in adhesion to the basement membrane and in differentiation. ILK-deficient cultured keratinocytes fail to attach and spread efficiently and exhibit multiple abnormalities in actin cytoskeletal organization. Ilk gene inactivation in cultured keratinocytes causes impaired ability to form stable lamellipodia, to directionally migrate, and to polarize. These defects are accompanied by abnormal distribution of active Cdc42 to cell protrusions, as well as reduced activation of Rac1 upon induction of cell migration in scraped keratinocyte monolayers. Significantly, alterations in cell spreading and forward movement in single cells can be rescued by expression of constitutively active Rac1 or RhoG. Our studies underscore a central and distinct role for ILK in hair follicle development and in polarized cell movements, two key aspects of epithelial morphogenesis and function.

Differential expression of regulator of G-protein signaling R12 subfamily members during mouse development.

Regulators of G-protein Signaling (RGS proteins) are a multigene family of GTPase-accelerating proteins for the Galpha subunit of heterotrimeric G-proteins. The mammalian R12 RGS protein subfamily is composed of RGS12 and RGS14, two proteins characterized by their multidomain architecture of hallmark RGS domain, tandem Ras-binding domains (RBDs), and a second Galpha interacting domain, the GoLoco motif. The Rgs12 gene generates multiple splice variants, the largest of which encodes N-terminal PDZ and PTB domains in addition to the core RGS/RBD/GoLoco motifs. The Rgs14 gene encodes a protein similar to the non-PDZ/PTB domain RGS12 splice variants. The spatiotemporal expression patterns of RGS12 and RGS14 proteins were examined by immunohistochemistry in a developmental series of postimplantation mouse embryo. We report that RGS12 splice variants exhibit differential spatiotemporal patterns of expression during postimplantation embryogenesis, suggesting nonoverlapping roles. In contrast, RGS14 is found ubiquitously throughout the postimplantation period. We conclude that R12 subfamily RGS proteins likely play significant and different roles in specific tissues and periods of mouse embryogenesis.

A novel role for integrin-linked kinase in epithelial sheet morphogenesis.

Integrin-linked kinase (ILK) is a multidomain protein involved in cell motility and cell-extracellular matrix interactions. ILK is found in integrin-containing focal adhesions in undifferentiated primary epidermal keratinocytes. Induction of keratinocyte differentiation by treatment with Ca(2+) triggers formation of cell-cell junctions, loss of focal adhesions, and ILK distribution to cell borders. We now show that Ca(2+) treatment of keratinocytes induces rapid (6 h) localization of tight junction (TJ) proteins. The kinetics of ILK movement toward the cell periphery mimics that of AJ components, suggesting that ILK plays a role in the early formation of cell-cell contacts. Whereas the N terminus in ILK mediates localization to cell borders, expression of an ILK deletion mutant incapable of localizing to the cell membrane (ILK 191-452) interferes with translocation of E-cadherin/beta-catenin to cell borders, precluding Ca(2+)-induced AJ formation. Cells expressing ILK 191-452 also fail to form TJ and sealed cell-cell borders and do not form epithelial sheets. Thus, we have uncovered a novel role for ILK in epithelial cell-cell adhesion, independent of its well-established role in integrin-mediated adhesion and migration.

Signalling in the epidermis: the E2F cell cycle regulatory pathway in epidermal morphogenesis, regeneration and transformation.

The epidermis is the outermost layer in the skin, and it is the first line of defence against the environment. The epidermis also provides a barrier against loss of fluids and electrolytes, which is crucial for life. Essential in the maintenance of this tissue is its ability to continually self-renew and regenerate after injury. These two characteristics are critically dependent on the ability of the principal epidermal cell type, the keratinocyte, to proliferate and to respond to differentiation cues. Indeed, the epidermis is a multilayered tissue composed of keratinocyte stem cells and their differentiated progeny. Central for the control of cell proliferation is the E2F transcription factor regulatory network. This signaling network also includes cyclins, cdk, cdk inhibitors and the retinoblastoma (pRb) family of proteins. The biological importance of the E2F/pRb pathway is emphasized by the fact that a majority of human tumours exhibit alterations that disrupt the ability of pRb proteins to inhibit E2F, leading to permanent activation of the latter. Further, E2F is essential for normal epidermal regeneration after injury. Other member of the E2F signaling pathway are also involved in epidermal development and pathophysiology. Thus, whereas the pRb family of proteins is essential for epidermal morphogenesis, abnormal regulation of cyclins and E2F proteins results in tumorgenesis in this tissue. In this review, we discuss the role of each member of this important growth regulatory network in epidermal formation, homeostasis and carcinogenesis.

RGS14 is a microtubule-associated protein.

Heterotrimeric G-proteins and their regulators are emerging as important players in modulating microtubule polymerization dynamics and in spindle force generation during cell division in C. elegans, D. melanogaster and mammals. We recently demonstrated that RGS14 is required for completion of the first mitotic division of the mouse embryo, and that it regulates microtubule organization in vivo. Here, we demonstrate that RGS14 is a microtubule-associated protein and a component of the mitotic spindle that may regulate microtubule polymerization and spindle organization. Taxol-stabilized tubulin, but not depolymerized tubulin coimmunoprecipitates with RGS14 from cell extracts. Furthermore, RGS14 copurifies with tubulin from porcine brain following multiple rounds of microtubule polymerization/depolymerization and binds directly to microtubules formed in vitro from pure tubulin (KD = 1.3 +/- 0.3 microM). Both RGS14 and Galpha(i1) in the presence of exogenous GTP promote tubulin polymerization, which is dependent on additional microtubule-associated proteins. However, preincubation of RGS14 with Galpha(i1)-GDP precludes either from promoting microtubule polymerization, suggesting that a functional GTP/GDP cycle is necessary. Finally, we show that RGS14 is a component of mitotic asters formed in vitro from HeLa cell extracts and that depletion of RGS14 from cell extracts blocks aster formation. Collectively, these results show that RGS14 is a microtubule-associated protein that may modulate microtubule dynamics and spindle formation.

RGS14 is a mitotic spindle protein essential from the first division of the mammalian zygote.

Heterotrimeric G protein alpha subunits, RGS proteins, and GoLoco motif proteins have been recently implicated in the control of mitotic spindle dynamics in C. elegans and D. melanogaster. Here we show that "regulator of G protein signaling-14" (RGS14) is expressed by the mouse embryonic genome immediately prior to the first mitosis, where it colocalizes with the anastral mitotic apparatus of the mouse zygote. Loss of Rgs14 expression in the mouse zygote results in cytofragmentation and failure to progress to the 2-cell stage. RGS14 is found in all tissues and segregates to the nucleus in interphase and to the mitotic spindle and centrioles during mitosis. Alteration of RGS14 levels in exponentially proliferating cells leads to cell growth arrest. Our results indicate that RGS14 is one of the earliest essential product of the mammalian embryonic genome yet described and has a general role in mitosis.

Analysis of interactions between regulator of G-protein signaling-14 and microtubules.

Microtubules are dynamic polymers essential for mitosis and cell division, intracellular transport, and maintaining cell organization and structure. Microtubule dynamics are tightly controlled in a context-specific manner by a myriad of microtubule-associated proteins. We have identified regulator of G-protein signaling-14 (RGS14) as a microtubule-associated protein. RGS14 is a component of the mitotic apparatus that binds directly to and stabilizes microtubules in vitro and is essential for the first cell division in the mouse embryo. This article describes methods used for examining the impact of the RGS14/microtubule interaction in vivo and in vitro.

The DP-1 transcription factor is required for keratinocyte growth and epidermal stratification.

The epidermis is a stratified epithelium constantly replenished through the ability of keratinocytes in its basal layer to proliferate and self-renew. The epidermis arises from a single-cell layer ectoderm during embryogenesis. Large proliferative capacity is central to ectodermal cell and basal keratinocyte function. DP-1, a heterodimeric partner of E2F transcription factors, is highly expressed in the ectoderm and all epidermal layers during embryogenesis. To investigate the role of DP-1 in epidermal morphogenesis, we inhibited DP-1 activity through exogenous expression of a dominant-negative mutant (dnDP-1). Expression of the dnDP-1 mutant interferes with binding of E2F/DP-1 heterodimers to DNA and inhibits DNA replication, as well as cyclin A mRNA and protein expression. Chromatin immunoprecipitation analysis demonstrated that the cyclin A promoter is predominantly bound in proliferating keratinocytes by complexes containing E2F-3 and E2F-4. Thus, the mechanisms of decreased expression of cyclin A in the presence of dnDP-1 seem to involve inactivation of DP-1 complexes containing E2F-3 and E2F-4. To assess the consequences on epidermal morphogenesis of inhibiting DP-1 activity, we expressed dnDP-1 in rat epithelial keratinocytes in organotypic culture and observed that DP-1 inhibition negatively affected stratification of these cells. Likewise, expression of dnDP-1 in embryonic ectoderm explants produced extensive disorganization of subsequently formed epidermal basal and suprabasal layers, interfering with normal epidermal formation. We conclude that DP-1 activity is required for normal epidermal morphogenesis and ectoderm-to-epidermis transition.

Vascular smooth muscle cells orchestrate the assembly of type I collagen via alpha2beta1 integrin, RhoA, and fibronectin polymerization.

Assembly of collagen into fibrils is widely studied as a spontaneous and entropy-driven process. To determine whether vascular smooth muscle cells (SMCs) impact the formation of collagen fibrils, we microscopically tracked the conversion of soluble to insoluble collagen in human SMC cultures, using fluorescent type I collagen at concentrations less than that which supported self-assembly. Collagen microaggregates were found to form on the cell surface, initially as punctate collections and then as an increasingly intricate network of fibrils. These fibrils displayed 67-nm periodicity and were found in membrane-delimited cellular invaginations. Fibril assembly was inhibited by an anti-alpha2beta1 integrin antibody and accelerated by an alpha2beta1 integrin antibody that stimulates a high-affinity binding state. Newly assembled collagen fibrils were also found to co-localize with newly assembled fibronectin fibrils. Moreover, inhibition of fibronectin assembly with an anti-alpha5beta1 integrin antibody completely inhibited collagen assembly. Collagen fibril formation was also linked to the cytoskeleton. Fibrils formed on the stretched tails of SMCs, ran parallel to actin microfilament bundles, and formed poorly on SMCs transduced with retrovirus containing cDNA for dominant-negative RhoA and robustly on SMCs expressing constitutively active RhoA. Lysophosphatidic acid, which activates RhoA and stimulates fibronectin assembly, stimulated collagen fibril formation, establishing for the first time that collagen polymerization can be regulated by soluble agonists of cell function. Thus, collagen fibril formation is under close cellular control and is dynamically integrated with fibronectin assembly, opening new possibilities for modifying collagen deposition.