AS1411 aptamer/RGD dual functionalized theranostic chitosan-PLGA nanoparticles for brain cancer treatment and imaging.

Conventional chemotherapy and poor targeted delivery in brain cancer resulting to poor treatment and develop resistance to anticancer drugs. Meanwhile, it is quite challenging to diagnose/detection of brain tumor at early stage of cancer which resulting in severity of the disease. Despite extensive research, effective treatment with real-time imaging still remains completely unavailable, yet. In this study, two brain cancer cell specific moieties i.e., AS1411 aptamer and RGD are decorated on the surface of chitosan-PLGA nanoparticles to improve targeted co-delivery of docetaxel (DTX) and upconversion nanoparticles (UCNP) for effective brain tumor therapy and real-time imaging. The nanoparticles were developed by a slightly modified emulsion/solvent evaporation method. This investigation also translates the successful synthesis of TPGS-chitosan, TPGS-RGD and TPGS-AS1411 aptamer conjugates for making PLGA nanoparticle as a potential tool of the targeted co-delivery of DTX and UCNP to the brain cancer cells. The developed nanoparticles have shown an average particle size <200 nm, spherical in shape, high encapsulation of DTX and UCNP in the core of nanoparticles, and sustained release of DTX up to 72 h in phosphate buffer saline (pH 7.4). AS1411 aptamer and RGD functionalized theranostic chitosan-PLGA nanoparticles containing DTX and UCNP (DUCPN-RGD-AS1411) have achieved greater cellular uptake, 89-fold improved cytotoxicity, enhanced cancer cell arrest even at lower drug conc., improved bioavailability with higher mean residence time of DTX in systemic circulation and brain tissues. Moreover, DUCPN-RGD-AS1411 have greatly facilitated cellular internalization and higher accumulation of UCNP in brain tissues. Additionally, DUCPN-RGD-AS1411 demonstrated a significant suppression in tumor growth in brain-tumor bearing xenograft BALB/c nude mice with no impressive sign of toxicities. DUCPN-RGD-AS1411 has great potential to be utilized as an effective and safe theranostic tool for brain cancer and other life-threatening cancer therapies.

Hyaluronic acid-oleylamine and chitosan-oleic acid conjugate-based hybrid nanoparticle delivery via. dissolving microneedles for enhanced treatment efficacy in localized breast cancer.

Microneedle technology offers a minimally invasive treatment strategy to deliver chemotherapeutics to localized tumors. Amalgamating the surface functionalized nanoparticles with microneedle technology can potentially deliver drugs directly to tumors and subsequently target cancer cells via, overexpressed receptors on the cell surface, thereby enhancing the treatment efficacy while reducing side effects. Here, we report cetuximab anchored hyaluronic acid-oleylamine and chitosan-oleic acid-based hybrid nanoparticle (HA-OA/CS-OA NPT)-loaded dissolving microneedles (MN) for targeted delivery of cabazitaxel (CBT) in localized breast cancer tumor. The HA-OA/CS-OA NPT was characterized for their size, surface charge, morphology, physicochemical characteristics, drug release behavior, and in vitro anti-cancer efficacy. The HA-OA/CS-OA NPT were of ~125 nm size, showed enhanced cytotoxicity and cellular uptake, and elicited a superior apoptotic response against MDA-MB-231 cells. Subsequently, the morphology and physicochemical characteristics of HA-OA/CS-OA NPT-loaded MN were also evaluated. The fabricated microneedles were of ~550 µm height and showed loading of nanoparticles equivalent to ~250 µg of CBT. The ex vivo skin permeation study revealed fast dissolution of microneedles upon hydration, while the drug permeation across the skin exhibited ~4-fold improvement in comparison to free drug-loaded MN. In vivo studies performed on DMBA-induced breast cancer in female SD rats showed a marked reduction in tumor volume after administration of drug and nanoparticle-loaded microneedles in comparison to intravenous administration of free drug. However, the HA-OA/CS-OA NPT-MN showed the highest tumor reduction and survival rate, with the lowest body weight reduction in comparison to other treatment groups, indicating its superior efficacy and low systemic toxicity. Overall, the dissolving microneedle-mediated delivery of targeted nanoparticles loaded with chemotherapeutics offers a superior alternative to conventional intravenous chemotherapy.

Differential location of growth factors and pancreatic stellate cell activation in chronic pancreatitis.

Pancreatic fibrosis is characterized by the activation of pancreatic stellate cells leading to the expression of smooth muscle actin (α-SMA). Normal pancreatic tissue has predominantly quiescent stellate cells in periductal and perivascular locations, which do not express α-SMA. We aimed at studying the immunohistochemistry (IHC) expression pattern of α-SMA, platelet-derived growth factor (PDGF-BB) and transforming growth factor (TGF-ß) in the resected specimen of chronic pancreatitis. Twenty biopsies from resected specimens of patients with chronic pancreatitis were included. The expression was measured in comparison to positive control biopsies (breast carcinoma for PDGF-BB and TGF-ß and appendicular tissue for α-SMA) and scored based on a semi-quantitative system based on staining intensity. The percentage of positive cells was used for objective scoring, which ranged from 0 to 15. The scoring was done separately for acini, ducts, stroma and islet cell. All patients had undergone surgery for refractory pain and the median duration of symptoms was 48 months. On IHC, α-SMA was not expressed in the acini, ducts or islets, but had high expression in the stromal regions (vs. acini, ducts and islet, p < 0.05), TGF-ß1 was also expressed maximally in islet cells; however, the distribution among all locations was statistically similar. α-SMA expression in the pancreatic stroma is an indicator of the concentration of activated stellate cells in the stroma, a site for genesis of fibrosis under the influence of growth factors in the local milieu.

Acute pancreatitis is characterized by generalized intestinal barrier dysfunction in early stage.

BACKGROUND AND AIMS: The role of intestinal-barrier in acute pancreatitis(AP) is poorly understood. We aimed to assess structural and functional changes in the intestinal-barrier in patients with early AP (time from onset<2 weeks) and the effect of enteral nutrition on them. METHODS: In this prospective observational study, patients with early AP not on enteral nutrition were compared with controls for baseline intestinal-permeability(lactulose: mannitol ratio(L:M)), endotoxinemia(serum IgM/IgG anti-endotoxin antibodies), bacterial-translocation(serum bacterial 16S rRNA) and duodenal epithelial tight-junction structure by immunohistochemistry(IHC) for tight-junction proteins(claudin-2,-3,-4, zonula occludens-1(ZO1), junctional adhesion molecule(JAM) and occludin) and electron microscopy. These parameters were reassessed after 2 weeks enteral feeding in a AP patients subset. RESULTS: 96 patients with AP(age: 38.0 ± 14.5 years; etiology: biliary[46.8%]/alcohol[39.6%]; severe:53.2%, mortality:11.4%) and 40 matched controls were recruited. Patients with AP had higher baseline intestinal permeability(median L:M 0.176(IQR 0.073-0.376) vs 0.049(0.024-0.075) in controls; p < 0.001) and more frequent bacteraemia(positive bacterial 16S rRNA in 24/48 AP vs 0/21 controls; p < 0.001) with trend towards higher serum endotoxinemia(median IgG anti-endotoxin 78(51.2-171.6) GMU/ml vs 51.2(26.16-79.2) in controls; p = 0.061). Claudin-2, claudin-3, ZO1 were downregulated in both duodenal crypts and villi while claudin-4 and JAM were downregulated in duodenal villi and crypts respectively. 22 AP patients reassessed after initiation of enteral nutrition showed trend towards improving intestinal permeability, serum endotoxinemia and bacteraemia, with significant improvement in claudin-2,-3 in duodenal villi. CONCLUSION: Patients with AP have significant disturbances in intestinal barrier structure and function in first 2 weeks from onset that persist despite institution of enteral nutrition.

Demonstration of Gut-Barrier Dysfunction in Early Stages of Non-alcoholic Fatty Liver Disease: A Proof-Of-Concept Study.

Background/Aims: Gut-barrier dysfunction is well recognized in pathogenesis of both non-alcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD). However, comparison of components of this dysfunction between the two etiologies remains unexplored especially in early stages of NAFLD. Methods: Components of gut-barrier dysfunction like alterations in intestinal permeability (IP) by lactulose mannitol ratio (LMR) in urine, systemic endotoxemia (IgG and IgM anti-endotoxin antibodies), systemic inflammation (serum tumor necrosis factor alpha [TNF-α] and interleukin-1 [IL-1] levels), tight junction (TJ) proteins expression in duodenal biopsy and stool microbiota composition using Oxford Nanopore MinION device were prospectively evaluated in patients with NAFLD (n = 34) with no cirrhosis, ALD (n = 28) and were compared with disease free controls (n = 20). Results: Patients with ALD had more advanced disease than those with NAFLD (median liver stiffness -NAFLD:7.1 kPa [5.9-8.9] vs. ALD:14.3 kPa [9.6-24], P < 0.001]. Median LMR was significantly higher in NAFLD and ALD group when compared to controls (NAFLD 0.054 [0.037-0.17] vs. controls 0.027 [0.021-0.045] (P = 0.001)) and ALD 0.043 [0.03-0.068] vs. controls 0.027 [0.021-0.045] (P = 0.019)]. Anti-endotoxin antibody titer (IgM) (MMU/mL) was lowest in NAFLD 72.9 [3.2-1089.5] compared to ALD 120.6 [20.1-728]) (P = 0.042) and controls 155.3 [23.8-442.9]) (P = 0.021). Median TNF-α (pg/mL) levels were elevated in patients with NAFLD (53.3 [24.5-115]) compared to controls (16.1 [10.8-33.3]) (P < 0.001) and ALD (12.3 [10.1-42.7]) (P < 0.001). Expression of zonulin-1 and claudin-3 in duodenal mucosa was lowest in NAFLD. On principal co-ordinate analysis (PCoA), the global bacterial composition was significantly different across the three groups (PERMANOVA test, P < 0.001). Conclusion: While remaining activated in both etiologies, gut-barrier dysfunction abnormalities were more pronounced in NAFLD at early stages compared to ALD despite more advanced disease in the latter.

Silica nanoparticles as novel sustainable approach for plant growth and crop protection.

Agriculture crops encounter several biotic and abiotic stresses, including pests, diseases, nutritional deficits, and climate change, which necessitate the development of new agricultural technologies. By developing nano-based fertilizers, insecticides and herbicides, and early disease diagnostics, nanotechnology may help to increase agricultural crop quality and production. The application of silica nanoparticles (SiNPs) may be the solution for increasing the yield to combat the agriculture crisis in the near future. SiNPs have unique physiological properties, such as large surface area, aggregation, reactivity, penetrating ability, size, and structure, which enable them to penetrate plants and regulate their metabolic processes. Pesticide delivery, enhanced nutrition supply, disease management, and higher photosynthetic efficiency and germination rate are all attributed to SiNPs deposition on plant tissue surfaces. SiNPs have been demonstrated to be non-toxic in nature, making them suitable for usage in agriculture. In this regard, the current work provides the most important and contemporary applications of SiNPs in agriculture as well as biogenic and non-biogenic synthetic techniques. As a result, this review summarizes the literature on SiNPs and explores the use of SiNPs in a variety of agricultural disciplines.

Acute Variceal Bleed in Cirrhosis is Associated With Reversible Changes in Tight Junction Protein Expression in the Intestine: A Proof-of-Concept Study.

BACKGROUND: Tight junction proteins (TJPs) play an important role in gut-barrier dysfunction in cirrhosis and its complications such as acute variceal bleed (AVB). However, the dynamics of TJPs expression after AVB, its relation to bacterial translocation, and impact on clinical outcome is largely unknown. AIMS: The aim of this study was to study the expression of TJPs in cirrhosis and assess its dynamic changes in AVB. In addition, the relation of TJP expression to endotoxemia and clinical outcomes was assessed. METHODS: In this prospective pilot study, 17 patients of cirrhosis with AVB, 59 patients of cirrhosis without AVB (non-AVB cirrhosis), and 20 controls were assessed for claudin-2 and claudin-4 expression in the duodenal biopsy. In the AVB-cirrhosis group, additional biopsies were obtained after 3 weeks. Endotoxemia was assessed by measuring IgG anti-endotoxin antibody levels. Claudin expression was correlated with a 6-month survival. RESULTS: Claudin-2 expression was downregulated in patients with AVB and non-AVB cirrhosis in villi (P < 0.001 and 0.013) and crypts (P < 0.001 and 0.012), respectively, compared with the controls. Claudin-4 expression was similar in villi (P = 0.079), but lower in crypts (P = 0.007) in patients with cirrhosis. Claudin-2 expression was upregulated on serial biopsies in both villi and crypts (P = 0.003 and 0.001, respectively) in AVB-cirrhosis with postbleed expression comparable with those with non-AVB cirrhosis. IgG anti-endotoxin antibody levels were elevated in cirrhosis with no correlation with claudin-2/4 expression. Claudin-2 expression independently predicted survival at 6 months. CONCLUSION: Both claudin-2 and claudin-4 expression are downregulated in cirrhosis. AVB is associated with dynamic changes in TJPs expression. Gut-barrier dysfunction might predict outcomes independent of bacterial endotoxemia in cirrhosis.

Application of agro-waste-mediated silica nanoparticles to sustainable agriculture.

Use of green agronomic techniques for plant development and crop protection is essential for environmental sustainability. The current research investigates a more efficient and long-term technique of manufacturing silica nanoparticles (SiO2 NPs) from agricultural waste (sugarcane bagasse and corn cob). SiO2 NPs were synthesized by calcinations of waste residues in muffle furnace with varying temperatures (400-1000 °C)/2 h in the present of static air. Field emission scanning electron microscopy (FESEM), Fourier transmission infrared spectroscopy (FTIR), X-ray diffraction (XRD), and energy dispersive X-ray spectroscopy (EDX) were used to characterize SiO2 NPs and assessed for their antifungal activity simultaneously investigated the effects of various concentrations of produced SiO2 NPs on Eruca sativa (E. sativa) physiological and biochemical. With SiO2 NPs treatment at 1000 µg L-1 concentration, the seed germination rate was found to be up to 95.5%, and growth characteristics were enhanced compared to control. Accordingly, the ones treated with SiO2 NPs grew better than the control ones. The treatment of plant with SiO2 NPs (500 µg L-1) increased the protein content by 14.8 mg g-1, and chlorophyll level was also increased by 4.08 mg g-1 in leaves compared to untreated plant. Disc diffusion experiment was conducted to test the efficiency of SiO2 NPs against Fusarium oxysporum and Aspergillus niger for antifungal activities. Highest mycelia growth inhibition was obtained with 73.42% and 58.92% for F. oxysporum and A. niger, respectively. The result shows that the SiO2 NPs have a favorable effect on E. sativa growth and germination, enhancing plant production which helps to improve the sustainable agriculture farming and acting as a possible antifungal agent against plant pathogenic fungi.

Comparison of Efficacy of Two Different Concentrations of Intralesional Amphotericin B in the Treatment of Cutaneous Leishmaniasis; A Randomized Controlled Trial.

INTRODUCTION: Cutaneous leishmaniasis is a vector borne disease caused by Leishmania major and Leishmania tropica. Bikaner is an endemic pocket for cutaneous leishmaniasis caused by Leishmania tropica. MATERIALS AND METHODS: A prospective study was done to evaluate the efficacy of different concentrations of intralesional amphotericin B as a treatment modality for cutaneous leishmaniasis in Bikaner, Rajasthan, India from January 2016 to June 2017. Fifty patients were randomized into two groups, A and B. Twenty-five patients from group A, received intralesionl amphotericin B (2.5 mg/ml) 0.5 ml/cm2, weekly for 8 weeks. Another group of 25 patients were treated by intralesional amphotericin B (5.0 mg/ml) weekly for same period. The cases were followed-up for response, side effects, and recurrence of disease. RESULTS: The results at the end of 8 weeks, showed complete response in 18 (72%) patients, partial response in 5 (20%) and 2 (8%) patients were non responders in group A. In group B, complete response was observed in 14 (56%), partial response in 7 (28%) patients and 4 (16%) patients did not show response. The difference was statistically insignificant (P > 0.05). No side effects were observed in both groups. CONCLUSION: The difference between the efficacy of 5 mg/ml and 2.5 mg/ml concentrations of Amphotericin B injections was found to be statistically insignificant. So, weekly injections of amphotericin B looks promising, however, larger sample size is required to assess the efficacy of both concentrations in the treatment of cutaneous leishmaniasis.

Impact of Antibiotic-Resistant Bacterial and Fungal Infections in Outcome of Acute Pancreatitis.

OBJECTIVES: The knowledge about pathogens and their antibiotic susceptibility patterns is essential to select an appropriate antibiotic. METHODS: We investigated the microbiological profile in pancreatic and extrapancreatic infections, and antibiotic sensitivity pattern in patients with acute pancreatitis. RESULTS: Of 556 patients with acute pancreatitis, only 189 developed bacterial infection; however, bacteremia was present in 42 patients (7.6%). Culture-proven infected pancreatic necrotic collection was present in 161 patients (29%). Escherichia coli and Klebsiella pneumoniae were the most common organisms. Among the bacterial infection cohort, 164 patients developed multidrug-resistant bacterial infection. Infection with multidrug-resistant bacteria, especially at multiple sites, increased mortality. Nearly 50% of patients (n = 94) acquired extremely drug-resistant bacterial infection at some time and emerged as key reason for prolonged hospital and intensive care unit stay. Colistin resistance and tigecycline resistance were documented in 2.1% and 17.2% of the specimens at admission and in 4.6% and 21% of specimens during the hospital stay. Of 556 patients, 102 patients developed fungal infection and 28 patients had only fungal infection without bacterial infection. CONCLUSIONS: Colistin and tigecycline are best reserved as last-resort antibiotics. Fungal infection was found to be associated with increased mortality, median hospital stay, and intensive care unit stay.

Comparison of Various Scoring Systems and Biochemical Markers in Predicting the Outcome in Acute Pancreatitis.

OBJECTIVE: Early risk assessment is important in acute pancreatitis (AP). The primary objective of this study was to compare various scores and biochemical markers done on the day of admission in predicting the outcome. METHODS: Demographic, clinical, and laboratory data of patients presenting within 2 weeks of onset were collected. Various scores were calculated and biochemical markers were measured on the day of admission. Optimum cutoffs were identified through receiver operating curve analysis. Multivariate analysis was used to identify predictors of outcome. RESULTS: Of 343 patients included, 202 (59%) were male; mean (SD) age was 38.7 (15.5) years. Acute pancreatitis was severe in 170 (49.6%) patients. Twenty-eight percent of the patients developed infected pancreatic necrosis and 18% died. An Acute Physiology and Chronic Health Evaluation (APACHE II) score of at least 7, bedside index for severity of AP (BISAP) of at least 2, systemic inflammatory response syndrome score of at least 3, and C-reactive protein of at least 82 ng/mL predicted severity. Predictors of infected pancreatic necrosis were as follows: PANC 3 score of at least 1, BISAP score of at least 2, and Marshall score of at least 2, whereas C-reactive protein of greater than 98, BISAP score of at least 2, APACHE score of at least 10, and a blood urea nitrogen of at least 17 predicted mortality. CONCLUSIONS: Both BISAP and APACHE II are comparable in predicting outcome, but BISAP predicted all 3 outcomes with the same cutoff and hence is a robust scoring system.

NFIA and GATA3 are crucial regulators of embryonic articular cartilage differentiation.

During appendicular skeletal development, the bi-potential cartilage anlagen gives rise to transient cartilage, which is eventually replaced by bone, and to articular cartilage that caps the ends of individual skeletal elements. While the molecular mechanism that regulates transient cartilage differentiation is relatively well understood, the mechanism of articular cartilage differentiation has only begun to be unraveled. Furthermore, the molecules that coordinate the articular and transient cartilage differentiation processes are poorly understood. Here, we have characterized in chick the regulatory roles of two transcription factors, NFIA and GATA3, in articular cartilage differentiation, maintenance and the coordinated differentiation of articular and transient cartilage. Both NFIA and GATA3 block hypertrophic differentiation. Our results suggest that NFIA is not sufficient but necessary for articular cartilage differentiation. Ectopic activation of GATA3 promotes articular cartilage differentiation, whereas inhibition of GATA3 activity promotes transient cartilage differentiation at the expense of articular cartilage. We propose a novel transcriptional circuitry involved in embryonic articular cartilage differentiation, maintenance and its crosstalk with the transient cartilage differentiation program.

Intestinal Fatty Acid Binding Protein and Citrulline as Markers of Gut Injury and Prognosis in Patients With Acute Pancreatitis.

OBJECTIVES: Severe acute pancreatitis (AP) is associated with high mortality due to systemic inflammatory response syndrome in the early phase and secondary infection in the later phase. Concomitant intestinal ischemia often results in gut injury. We studied intestinal fatty acid binding protein (IFABP) and citrulline levels as markers of gut injury to predict prognosis in AP. METHODS: Acute pancreatitis patients at admission and controls were studied. Serum IFABP was measured by enzyme-linked immunosorbent assay and plasma citrulline by high-performance liquid chromatography technique. Ultrastructural changes in duodenal biopsy were also compared between the 2 groups. RESULTS: The IFABP concentration was significantly higher in AP cases (n = 94) compared with controls (n = 100) (mean [standard deviation], 592.5 [753.6] vs 87.8 [67.6] pg/mL; P < 0.001) and in patients with severe AP versus mild AP (738.3 [955.3] vs 404.0 [263.3] pg/ mL, P = 0.03). Citrulline concentration was lower in AP versus controls (29.9 [33.8] vs 83.9 [60.1] µg/L, P < 0.001). We propose a model by which these biomarkers (IFABP >350 pg/mL and citrulline <18 µg/L) are able to predict poor prognosis in 33.9% of patients with AP. The gut injury was also validated via ultrastructural changes. CONCLUSIONS: Intestinal fatty acid binding protein is a promising prognostic marker in acute pancreatitis.

Mechanism of Increased Intestinal Permeability in Acute Pancreatitis: Alteration in Tight Junction Proteins.

BACKGROUND AND AIMS: Intestinal permeability (IP) has been shown to be increased in acute pancreatitis (AP) and is considered to be responsible for development of septic complications. However, the mechanism of increase in IP is not well studied. We studied whether alteration in tight junction proteins (TJP) has any role in altered IP in patients with AP. MATERIALS AND METHODS: This is a prospective study conducted at a tertiary care referral center. Twenty consecutive moderate and severe AP patients fulfilling the study criteria were included along with 20 controls that underwent gastroduodenoscopy for dyspepsia. IP was measured with lactulose mannitol (LM) ratio and TJP were studied by measuring expression of claudin-2 and claudin-4 in duodenal biopsy samples. Statistical analysis was done with STATA 13.0. RESULTS: IP as depicted by LM ratio was significantly higher in AP patients as compared with controls (4.659±10.4 vs. 0.101±0.297; P<0.001). Claudin-4 expression was reduced in duodenal biopsies in AP patients (P<0.001 for crypt intercellular junction and P=0.007 for crypt cytoplasm). However, LM ratio was not associated with either mortality (P=0.12) or development of infected pancreatic necrosis (P=0.3). CONCLUSIONS: IP is increased in AP. Alteration in TJP in the form of reduced claudin-4 expressions could be the possible mechanism for increased IP.

Are alterations of tight junctions at molecular and ultrastructural level different in duodenal biopsies of patients with celiac disease and Crohn's disease?

Abnormalities of transmembrane and cytoplasmic proteins of tight junctions (TJ) have been implicated in pathogenesis of both celiac (CeD) and Crohn's diseases (CD). Since disease pathogenesis in CeD and CD are different, we planned to study if there is any differential expression pattern of TJ marker proteins and ultrastructural changes, respectively, in duodenal villi vs crypts. Endoscopic duodenal biopsies from treatment naïve patients with CeD (n = 24), active CD (n = 28), and functional dyspepsia (as controls, n = 15), both at baseline and 6 months after treatment, were subjected to light microscopic analysis (modified Marsh grading); immune-histochemical staining and Western blot analysis to see the expression of key TJ proteins [trans-membrane proteins (claudin-2, claudin-3, claudin-4, occludin, and JAM) and cytoplasmic protein (ZO-1)]. Transmission electron microscopy and image analysis of the TJs were also performed. There was significant overexpression of claudin-2 (pore-forming) and occludin (protein maintaining cell polarity) with under-expression of claudin-3 and claudin-4 (pore-sealing proteins) in treatment naïve CeD and active CD with simultaneous alteration in ultrastructure of TJs such as loss of penta-laminar structure and TJ dilatation. Normalization of some of these TJ proteins was noted 6 months after treatment. These changes were not disease specific and were not different in duodenal villi and crypts. Overexpression of pore-forming and under-expression of pore-sealing TJ proteins lead to dilatation of TJ. These changes are neither disease specific nor site specific and the end result of mucosal inflammation.

Comparative tight junction protein expressions in colonic Crohn's disease, ulcerative colitis, and tuberculosis: a new perspective.

We intended to see the pattern of TJ protein expression along with ultrastructural changes in colonic biopsies from patients with Crohn's disease (CD), ulcerative colitis (UC), and tuberculosis (cTB). Colonic biopsies from 11 patients with active CD and ten patients each with active UC and untreated cTB were taken along with biopsies from six patients with irritable bowel syndrome as controls. These were evaluated for expression pattern of key TJ proteins which included claudin-2 as TJ pore-forming protein, claudin-4 as pore-sealing protein, ZO-1 as scaffold protein, and occludin as TJ protein related to cell migration and polarity. Claudin-2 expression was upregulated along the whole length of intercellular junction (ICJ) in biopsies from patients with active CD and UC in comparison to the biopsies from cTB patients and controls, where its expression was limited to the uppermost part of ICJ. There was reduced expression of ZO-1 in UC, CD, and cTB. On transmission electron microscopic examination, the pentalaminar structure of TJs was destroyed in patients with CD and UC but no significant change was seen in those with cTB and in controls. The expression of claudin-2 was distinctly different in active CD and UC in comparison to its expression pattern in patients with cTB and in controls. The redistribution of claudin-2 expression was in accordance with the TJ ultrastructural changes in patients with UC, CD, and cTB. Altered claudin-2 expression, along with destroyed TJs, may result in loss of selective permeability in patients with UC and CD.

Clinical, endoscopic, and histological differentiations between Crohn's disease and intestinal tuberculosis.

OBJECTIVES: The clinical, endoscopic, and histological features of Crohn's disease (CD) and intestinal tuberculosis mimic each other so much that it becomes difficult to differentiate between them. The aim was to find out clinical, endoscopic, and histological predictor features for differentiation between CD and intestinal tuberculosis. METHODS: We recruited 106 patients, 53 each with CD and intestinal tuberculosis, in this study. The clinical, histological, and endoscopic features were subjected to univariate, bivariate, and multivariate analyses. On the basis of regression coefficients of the final multivariate logistic model, a score to discriminate between CD and intestinal tuberculosis was devised. For the validation of the score, the same model was tested on 20 new patients, each with CD and intestinal tuberculosis. RESULTS: On univariate analysis, although longer duration of disease, chronic diarrhea, blood in stool, perianal disease, extra-intestinal manifestations, involvement of left colon, skip lesions, aphthous ulcers, cobblestoning, longitudinal ulcers, focally enhanced colitis, and microgranulomas were significantly more common in CD, partial intestinal obstruction, constipation, presence of nodular lesions, higher number, and larger granulomas were significantly more common in intestinal tuberculosis. On multivariate analysis, blood in stool (odds ratio (OR) 0.1 (confidence interval (CI) 0.04-0.5)), weight loss (OR 9.8 (CI 2.2-43.9)), histologically focally enhanced colitis (OR 0.1 (CI 0.03-0.5)), and involvement of sigmoid colon (OR 0.07(0.01-0.3)) were independent predictors of intestinal tuberculosis. On the basis of regression coefficients of the final multivariate logistic model, a score that varied from 0.3 to 9.3 was devised. Higher score predicted more likelihood of intestinal tuberculosis. Once the cutoff was set at 5.1, then the sensitivity, specificity, and ability to correctly classify the two diseases were 83.0, 79.2, and 81.1%, respectively. Area under the curve for receiver-operating characteristic (ROC) to assess the ability of these features to discriminate between CD and intestinal tuberculosis was 0.9089. The area under ROC in the validation data set was 89.2% (95% CI 0.79-0.99). With a similar cutoff score of 5.1, sensitivity and specificity in the validation model were 90% (95% CI 66.9-98.2) and 60% (95% CI 36.4-80.0), respectively. CONCLUSIONS: Blood in stool, weight loss, focally enhanced colitis, and involvement of the sigmoid colon were the most important features in differentiating CD from intestinal tuberculosis.

Phage displayed scFv: pIII scaffold may fine tune binding specificity.

The fine specificity of antibodies is important for their discriminating powers during diagnostics and in vivo therapy. We have attempted to isolate human scFv antibodies to the oncofetal antigen, the placental isozyme of alkaline phosphatase (PLAP) in which it is important to distinguish between the closely related intestinal alkaline phosphatase (IAP) and bone alkaline phosphatase (BAP) isozymes. As the antibodies are selected in the phage displayed form and might be finally used as different entities, including the soluble scFv form, it may be important to look at the influence of scaffolds in determining specificity. There have been earlier reports of the role of the constant region and other scaffolding proteins in determining specificity. In this paper, we report isolation of one such clone, E6, which showed specificity to PLAP in phage antibody form but lost the specificity when soluble scFv was tested for same, and showed partial cross reactivity to BAP. We suggest that the altered specificity of scFv might be the result of loss of phage pIII scaffold, which is present in phage-displayed antibody and may help the displayed antibody to assume specific conformational structure, which may govern binding characteristics of the same.

The probiotic preparation, VSL#3 induces remission in patients with mild-to-moderately active ulcerative colitis.

BACKGROUND & AIMS: Probiotics can maintain ulcerative colitis (UC) in remission effectively, but little is known of their ability to induce remission. We conducted a multicenter, randomized, double-blind, placebo-controlled trial of a high-potency probiotic, VSL#3, for the treatment of mild-to-moderately active UC. METHODS: Adult patients with mild-to-moderate UC were assigned randomly to groups that were given 3.6 x 10(12) CFU VSL#3 (n = 77) or placebo (n = 70), twice daily for 12 weeks. The primary end point was a 50% decrease in the Ulcerative Colitis Disease Activity Index (UCDAI) at 6 weeks. The secondary end points included remission by 12 weeks and reduction in total individual UCDAI parameters from baseline at 12 weeks. Intention-to-treat analysis was performed. RESULTS: At week 6, the percentage of patients with an improvement in UCDAI score that was greater than 50% was significantly higher in the group given VSL#3 (25; 32.5%) than the group given placebo (7; 10%) (P = .001). At week 12, there were 33 patients given VSL#3 (42.9%) who achieved remission, compared with 11 patients given placebo (15.7%) (P < .001). Furthermore, significantly more patients given VSL#3 (40; 51.9%) achieved a decrease in their UCDAI that was greater than 3 points, compared with those given placebo (13; 18.6%) (P < .001). The VSL#3 group had significantly greater decreases in UCDAI scores and individual symptoms at weeks 6 and 12, compared with the placebo group. CONCLUSIONS: VSL#3 is safe and effective in achieving clinical responses and remissions in patients with mild-to-moderately active UC.