In vitro wound healing activity of 1-hydroxy-5,7-dimethoxy-2-naphthalene-carboxaldehyde (HDNC) and other isolates of Aegle marmelos L.: Enhances keratinocytes motility via Wnt/β-catenin and RAS-ERK pathways

Wound healing is a complex process in which injured skin and tissues repaired by interaction of a complex cascade of cellular events that generates resurfacing, reconstitution and restoration of the tensile strength of injured skin. It follows β-catenin, extracellular signal regulated kinase (ERK) and Akt signaling pathways. Aegle marmelos L., generally known as bael is found to act as anti-inflammatory, antioxidant and anti-ulcer agent. Furthermore, studies have demonstrated that this Indian traditional medicinal plant, A. marmelos flower extract (AMF) was used for wound injury. Henceforth, the current study was investigated to ascertain the effect of its active constituents in vitro wound healing with mechanism involve in migration of cells and activation of β-catenin in keratinocytes, inhibition of PGE₂ in macrophages and production of collagen in fibroblasts. We have taken full thickness wound of rats and applied AMF for 2 weeks. Cutaneous wound healing activity was performed using HaCaT keratinocytes, Hs68 dermal fibroblasts and RAW264.7 macrophages to determine cell viability, nitric oxide production, collagen expression, cell migration and β-catenin activation. Results shows that AMF treated rats demonstrated reduced wound size and epithelisation was improved, involved in keratinocytes migration by regulation of Akt signaling, beta-catenin and extracellular signal-regulated kinase (ERK) pathways. AMF and its active constituent’s increased mRNA expression, inhibited nitric oxide, PGE₂ release, mRNA expression of mediators in RAW 264.7 macrophages and enhances the motility of HaCaT keratinocytes in vitro wound healing of rats.     

Effect of hydroxylated solvents on the active constituents of Salvadora persica root “Siwak”

Previously, the antimicrobial activity of Salvadora persica was traced to benzyl isothiocyanate. In the present study known inactive compounds were isolated from extracts obtained by different solvents including β-amyrin, β-sitosterol, stigmasterol glucoside, benzyl cyanide and sulphur. However, some inactive compounds were present only in the ethanol and methanol extracts. This observation indicated that these compounds most likely are artifacts resulted from interaction with the solvents used in extraction. Pure benzyl isothiocyanate was kept with different solvents for 72?h and after TLC study they were heated under reflux for 8?h to explore the possibility of interactions. Only solvents with OH groups reacted with benzyl isothiocyanate and gave products similar to those isolated from the alcohol extracts. In conclusion extraction of S. persica with hydroxylated solvents will alter the structure of the active compound benzyl isothiocyanate and leads to loss of antimicrobial activity.     

Low-dose recombinant properdin provides substantial protection against Streptococcus pneumoniae and Neisseria meningitidis infection

Modern medicine has established three central antimicrobial therapeutic concepts: vaccination, antibiotics, and, recently, the use of active immunotherapy to enhance the immune response toward specific pathogens. The efficacy of vaccination and antibiotics is limited by the emergence of new pathogen strains and the increased incidence of antibiotic resistance. To date, immunotherapy development has focused mainly on cytokines. Here we report the successful therapeutic application of a complement component, a recombinant form of properdin (P_n), with significantly higher activity than native properdin, which promotes complement activation via the alternative pathway, affording protection against N. menigitidis and S. pneumoniae. In a mouse model of infection, we challenged C57BL/6 WT mice with N. menigitidis B-MC58 6 h after i.p. administration of P_n (100 µg/mouse) or buffer alone. Twelve hours later, all control mice showed clear symptoms of infectious disease while the P_n treated group looked healthy. After 16 hours, all control mice developed sepsis and had to be culled, while only 10% of P_n treated mice presented with sepsis and recoverable levels of live Meningococci. In a parallel experiment, mice were challenged intranasally with a lethal dose of S. pneumoniae D39. Mice that received a single i.p. dose of P_n at the time of infection showed no signs of bacteremia at 12 h postinfection and had prolonged survival times compared with the saline-treated control group (P < 0.0001). Our findings show a significant therapeutic benefit of P_n administration and suggest that its antimicrobial activity could open new avenues for fighting infections caused by multidrug-resistant neisserial or streptococcal strains.Pneumococcal and meningococcal infectious diseases remain a serious threat to public health. Streptococcus pneumoniae is the leading cause of community-acquired pneumonia and a major cause of otitis media, septicemia, and meningitis (1, 2). S. pneumoniae is responsible for ∼1.2 million deaths per year worldwide, with young children and immunocompromised patients at particular risk (3). Neisseria meningitidis causes epidemic bacterial meningitis and septicemia, with high mortality in children and young adults (4). The impact of meningococcal disease on human health is defined by both the risk and the severity of invasive meningococcal infections, with unacceptably high mortality rates, ranging from 10% in patients under optimal clinical therapy with the latest generation of antibiotics to up to 40% in patients with untreated septicemia. Almost one-third of those who survive invasive infections are left with long-term disabilities and long-term morbidity. Globally, the World Health Organization estimates that ∼1.2 million cases of invasive meningococcal infections occur annually, leading to more than 135,000 fatalities (5).Vaccination programs have reduced the rates of infection in developed countries, but neonates and elderly adults remain especially vulnerable (6, 7). The efficacy of vaccination is further limited by the emergence of new strains of S. pneumoniae and N. meningitidis.The complement system plays a major role in the host resistance to both pathogens (8–13). Complement is activated via three routes: the classical pathway, the lectin pathway, and the alternative pathway. Activation of the classical and lectin pathways is mediated by specific recognition molecules. Binding of C1q to the bacterial surface or the Fc region of antibody initiates the classical pathway. The lectin pathway is initiated by carbohydrate recognition molecules, including mannan-binding lectin, ficolins, and collectin 11, which bind directly to bacterial polysaccharides. Activation of the classical or lectin pathway leads to the formation of a C3 convertase (C4b2a), which splits C3 into the biologically active fragments, C3b and C3a. C3b can bind covalently to an activating surface, and hundreds of molecules of C3b can be deposited in close proximity to the C3 convertase complex. Accumulation of C3b close to C4b2a forms the classical pathway C5 convertase C4b2a(3b)_n, in which C4b and C3b form a binding site for C5, orienting it for cleavage by C2a (14, 15).The mechanisms initiating the alternative pathway are less well understood. It is widely accepted that the alternative pathway maintains a continuous state of low-rate activation, which is held in check by potent negative regulators of activation on nonactivating surfaces, such as the surface of host cells. Turnover of the alternative pathway is initiated either by the provision of C3b via the classical pathway, the lectin pathway, or complement-independent proteolysis of C3 or by the spontaneous hydrolysis of C3 to form C3(H₂O). C3b or C3(H₂O) bind factor B to form either the C3bB or C3(H₂O)B zymogen complex. In this complex, factor B is cleaved by factor D, releasing a Ba fragment. The activated C3bBb or C3(H₂O)Bb fragments are themselves C3 convertases, which in turn cleave more C3 into C3a and C3b. Unchecked, the accumulation of C3b rapidly leads to the formation of more alternative pathway convertase complexes, resulting in a physiologically critical positive feedback mechanism—the amplification loop of complement activation (16). The alternative pathway thus amplifies complement activation initiated by any of the three pathways, making it an attractive target for therapeutic intervention designed to modulate complement-mediated immunity and/or inflammatory processes (17).Deposition of C3b and iC3b on the bacterial surface is a key step in the immune response against S. pneumoniae, because complement-mediated opsonisation is essential for clearance of S. pneumoniae through phagocytosis (8). Lysis of bacteria, owing to formation of the membrane attack complex complex, is the critically important biological activity of complement in the defense against N. meningitidis (10). Inherited or acquired deficiencies of the alternative pathway are associated with a high risk of recurrent bacterial infection. Factor B deficiencies significantly increase the risk of S. pneumoniae and Pseudomonas aeruginosa infection (9, 18). In a mouse model of properdin deficiency, the severity of polymicrobial peritonitis was significantly greater in deficient mice compared with their WT littermates (19). Properdin deficiency in humans has been associated with a high risk of meningococcal infections, especially with unusual infective serotypes, such as W-135 and Y (10, 20, 21). In addition, opsonophagocytosis of S. pneumoniae was found to be severely compromised in properdin-deficient sera, and reconstitution of properdin-deficient sera with purified properdin restored the opsonic activity and killing of S. pneumoniae by polymorphonuclear leukocytes (22, 23).Properdin is the only known positive physiological regulator of complement activation. It stabilizes and extends the half-life of the surface-bound C3 convertase C3bBb, and inhibits its degradation by factor I (24–26). In their pioneering 1954 work, Pillemer et al. (26) first described properdin as a serum protein that mediates complement activation and antimicrobial activity in absence of antibodies.Properdin is present in serum at a concentration of ∼5–15 μg/mL (27). Unlike most other complement components, properdin is not synthesized in the liver but rather is expressed by other cells, including monocytes, T cells, mast cells, and granulocytes (19, 28–30). Properdin monomers can assemble into dimers (P₂), trimers (P₃), and tetramers (P₄), formed by head-to-tail association of monomers (each ∼53 kDa) (31, 32). Properdin aggregates, so-called “activated” properdin (P_n), are considered artificial higher-order oligomers formed during the purification of properdin from plasma or during subsequent freeze–thaw cycles (33). The functional activity of properdin increases with the size of the polymers formed (34). By increasing the half-life of the alternative pathway C3 convertase, properdin antagonizes the functional activity of complement factor H, an abundantly expressed plasma component, which promotes inactivation of the alternative pathway C3 convertase and of all C5 convertases of complement by accelerating the decay of these enzyme complexes through binding to complex-bound C3b and by serving as a cofactor in the factor I-mediated conversion of C3b to its inactive form, termed iC3b (35). Interestingly, the two pathogens used in this study were previously shown to express distinct microbial surface components that sequester factor H from host plasma, leading to resistance to the complement-mediated immune clearance of these pathogens (36, 37).In the present study, we addressed the role of the alternative pathway and the effect of administration of recombinant properdin as a tool for boosting alternative pathway activity to augment the immune response against S. pneumoniae or N. meningitidis.     

Multiparametric MRI with diffusion-weighted imaging in predicting response to chemotherapy in cases of osteosarcoma and Ewing’s sarcoma

Objective:To evaluate the multiparametric MRI in predicting chemotherapy response in pathologically proven cases of osteosarcoma and Ewing’s sarcoma. Correlation between the tumor size changes and internal breakdown using RECIST 1.1, modified RECIST, quantitative apparent diffusion coefficient (ADC) and tumor volume as well as dynamic contrast-enhanced MRI (DCE-MRI).Methods:The study included 104 patients pathologically proved osteosarcoma (53) and Ewing`s sarcoma (51) underwent MRI examinations; before and after chemotherapy. All patients were assessed using the RECIST 1.1 criteria, m-RECIST, quantitative ADC, and tumor volume evaluation. 21 patients underwent DCE-MRI curve type with quantitative parameters. Correlation between the different evaluations was carried out. Results were correlated with the post-operative pathology in 42 patients who underwent surgery and for statistical evaluation, Those patients were classified into responders (≥90% necrosis) and non-responders (<90% necrosis).Results:The initial mean ADC of 104 patients of osteosarcoma and Ewing’s sarcoma (0.90 ± 0.29) and (0.71 ± 0.16) respectively, differed significantly from that after treatment (1.62 ± 0.46) and (1.6 ± 0.39) respectively with (p<0.001).ADC variations (ADC%) in the non-progressive group were higher than those of the progressive group (128.3 ± 63.49 vs 36.34 ± 78.7) % with (p<0.001).ADC values and ADC variations were inversely correlated with morphologic changes, regardless of the effectiveness of chemotherapy expressed as changes in tumor size based on (RECIST 1.1, RECIST, and 3D volume). Linear regression analysis revealed a Pearson correlation coefficient of r=-0.427, -0.498 and -0.408, respectively with (p<0.001).An increase in the ADC value was not always associated with a reduction in tumor volume. The disease control rate (defined as the percentage of CR+PR+SD patients) was 89.4% and 93.9% according to RECIST 1.1 and m-RECIST respectively.42 out of the 104 patients had postsurgical histological evaluation as regards the chemotherapeutic response divided into two groups. ADC values showed a statistically significant difference between Group A and Group B being more evident with minimum ADC% (p<0.001).Conclusion:Quantitative diffusion-weighted imaging with ADC mapping and ADC % after chemotherapy allows a detailed analysis of the treatment response in osteosarcoma and Ewing’s sarcoma. The therapeutic response can be underestimated using RECIST 1.1, so the modified RECIST should be also considered.Advances in knowledge:Quantitative ADC especially ADC% provided an accurate non-invasive tool in the assessment of post-therapeutic cases of osteosarcoma and Ewing''s sarcoma     

Performance of risk assessment instruments for predicting osteoporotic fracture risk: a systematic review

Summary

We systematically reviewed the literature on the performance of osteoporosis absolute fracture risk assessment instruments. Relatively few studies have evaluated the calibration of instruments in populations separate from their development cohorts, and findings are mixed. Many studies had methodological limitations making susceptibility to bias a concern.

Introduction

The aim of this study was to systematically review the literature on the performance of osteoporosis clinical fracture risk assessment instruments for predicting absolute fracture risk, or calibration, in populations other than their derivation cohorts.

Methods

We performed a systematic review, and MEDLINE, Embase, Cochrane Library, and multiple other literature sources were searched. Inclusion and exclusion criteria were applied and data extracted, including information about study participants, study design, potential sources of bias, and predicted and observed fracture probabilities.

Results

A total of 19,949 unique records were identified for review. Fourteen studies met inclusion criteria. There was substantial heterogeneity among included studies. Six studies assessed the WHO’s Fracture Risk Assessment (FRAX) instrument in five separate cohorts, and a variety of risk assessment instruments were evaluated in the remainder of the studies. Approximately half found good instrument calibration, with observed fracture probabilities being close to predicted probabilities for different risk categories. Studies that assessed the calibration of FRAX found mixed performance in different populations. A similar proportion of studies that evaluated simple risk assessment instruments (≤5 variables) found good calibration when compared with studies that assessed complex instruments (>5 variables). Many studies had methodological features making them susceptible to bias.

Conclusions

Few studies have evaluated the performance or calibration of osteoporosis fracture risk assessment instruments in populations separate from their development cohorts. Findings are mixed, and many studies had methodological limitations making susceptibility to bias a possibility, raising concerns about use of these tools outside of the original derivation cohorts. Further studies are needed to assess the calibration of instruments in different populations prior to widespread use.     

Hepatoprotective Effect of Cymbopogon Citratus Aqueous Extract Against Hydrogen Peroxide-Induced Liver Injury in Male Rats

Background

Cymbopogon citratus (Poaceae) a tropical perennial herb plant that is widely cultivated to be eaten either fresh with food or dried in tea or soft drink has been reported to possess a number of medicinal and aromatic properties. This study aimed at evaluating the protective effects of C. citratus aqueous extract against liver injury induced by hydrogen peroxide (H₂O₂), in male rats.

Materials and Methods

Twenty-five rats were randomly divided into five different groups of five animals in each group; (1) Control. (2) Received H₂O₂ (0.5%) with drinking water. (3), and (4) received H₂O₂ and C. citratus (100 mg·kg⁻¹ b wt), vitamin C (250 mg·kg⁻¹ b wt) respectively. (5), was given C. citratus alone. The treatments were administered for 30 days. Blood samples were collected and serum was used for biochemical assay including liver enzymes activities, total protein, total bilirubin and malonaldehyde, glutathione in serum and liver homogenates. Liver was excised and routinely processed for histological examinations.

Results

C. citratus attenuated liver damage due to H₂O₂ administration as indicated by the significant reduction (p<0.05), in the elevated levels of ALT, AST, ALP, LDH, TB, and MDA in serum and liver homogenates; increase in TP and GSH levels in serum and liver homogenates; and improvement of liver histo-pathological changes. These effects of the extract were similar to that of vitamin C which used as antioxidant reference.

Conclusion

C. citratus could effectively ameliorate H₂O₂-induced oxidative stress and prevent liver injury in male rats.     

The preventive effects of natural adjuvants,G2 and G2F on tracheal responsiveness and serum IL-4 and IFN-γ (th1/th2 balance) in sensitized guinea pigs

OBJECTIVE:

The effects of natural adjuvants on lung inflammation and tracheal responsiveness were examined in sensitized guinea pigs.

METHODS:

The responses of guinea pig tracheal chains and the serum levels of interleukin-4 and interferon-gamma were examined in control pigs and three other groups of guinea pigs: the sensitized group and two other sensitized groups treated with either adjuvant G2 or adjuvant G2F (n = 7 for each group). Sensitization of the animals was achieved by injection and inhalation of ovalbumin.

RESULTS:

The results showed that sensitized animals had increased tracheal responsiveness and increased serum levels of interleukin-4 and interferon-gamma compared to controls (p<0.05 to p<0.001). Treatments with either G2 or G2F prevented the increase in tracheal responsiveness and serum interleukin-4 (p<0.01 to p<0.001). However, the serum levels of interferon-gamma and the interleukin-4-to-interferon-gamma ratio was increased in the treated groups (p<0.001 for all cases).

CONCLUSIONS:

These results indicate important preventive effects of two natural adjuvants, particularly G2, on the changes in tracheal responsiveness, serum cytokines and the interleukin-4-to-interferon-gamma ratio (T helper 1/T helper 2 balance) in sensitized guinea pigs.     

Optimization of Hormonal Combinations for In Vitro Regeneration of Lesser Periwinkle (Vinca minor L.) and Assessment of Genetic Homogeneity

Proceedings of the National Academy of Sciences, India Section B: Biological Sciences - Lesser Periwinkle (Vinca minor L.) is an important source of valuable secondary metabolites. To survey the...