Evaluation of the Cytotoxic, Anti-Inflammatory, and Immunomodulatory Effects of Withaferin A (WA) against Lipopolysaccharide (LPS)-Induced Inflammation in Immune Cells Derived from BALB/c Mice.

(1) Background: Inflammation is one of the primary responses of the immune system and plays a key role in the pathophysiology of various diseases. Recent reports suggest that various phytochemicals exhibit promising anti-inflammatory and immunomodulation activities with relatively few undesirable effects, thus offering a viable option to deal with inflammation and associated diseases. The current study evaluates the anti-inflammatory and immunomodulatory effects of withaferin A (WA) in immune cells extracted from BALB/c mice. (2) Methods: MTT assays were performed to assess the cell viability of splenocytes and anti-inflammatory doses of WA. Under aseptic conditions, the isolation of macrophages and splenocytes from BALB/c mice was performed to investigate the anti-inflammatory effects of WA. Analysis of the expression of proinflammatory cytokines and associated signaling mediators was performed using proinflammatory assay kits, real-time polymerase chain reaction (RT-PCR), and immunoblotting, while the quantification of B and T cells was performed by flow cytometry. (3) Results: Our results demonstrated that WA exhibits anti-inflammatory and immunomodulatory effects in LPS-stimulated macrophages and splenocytes derived from BALB/c mice, respectively. Mechanistically, we found that WA promotes an anti-inflammatory effect on LPS-stimulated macrophages by attenuating the secretion and expression of proinflammatory cytokines TNF-α, IL-1ß, IL-6, and the inflammation modulator NO, both at the transcriptional and translational level, respectively. Further, WA inhibits LPS-stimulated inflammatory signaling by dephosphorylation of p-Akt-Ser473 and p-ERK1/2. This dephosphorylation does not allow IĸB-kinase activation to disrupt IĸB-NF-ĸB interaction. The consistent interaction of IĸB with NF-ĸB in WA-treated cells attenuates the activation of downstream inflammatory signaling mediators Cox-2 and iNOS expression, which play crucial roles in inflammatory signaling. Additionally, we observed significant immunomodulation of LPS-stimulated spleen-derived lymphocytes by suppression of B (CD19) and T (CD4+/CD8+) cell populations after treatment with WA. (4) Conclusion: WA exhibits anti-inflammatory and immunomodulatory activity by modulating Akt/ERK/NF-kB-mediated inflammatory signaling in macrophages and immunosuppression of B (CD19) and T cell (CD4+/CD8+) populations in splenocytes after LPS stimulation. These results suggest that WA could act as a potential anti-inflammatory/immunomodulatory molecule and support its use in the field of immunopharmacology to modulate immune system cells.

Use of Saliva as an Alternative Matrix to Serum/Plasma for Therapeutic Drug Monitoring Using Reverse-Phase HPLC.

PURPOSE: This study was conducted to examine and verify the use of saliva as an alternative matrix for monitoring phenytoin drug levels in patients with epilepsy. Drug concentrations are measured to evaluate whether a suitable drug level has been achieved to minimize the risk for toxicity, inadequate efficacy, or therapy resistance and compliance issues. METHODS: Quantitative analysis was performed by using reverse-phase HPLC after sample pretreatment with acetonitrile. Seventy-eight patients who met the inclusion/exclusion criteria were examined in this study. Trough concentrations of both saliva and serum were taken at steady state. FINDINGS: Of the 78 patients enrolled, only 11 (14.1%) had normal levels. Twenty-eight patients (35.9%) had subtherapeutic levels, and 39 (50%) had toxic levels. Simultaneously, salivary phenytoin levels were analyzed; only 13 patients (17.3%) had therapeutic levels, 25 patients (33.3%) had subtherapeutic levels, and 37 (49.3%) had toxic levels. Among the study population, most of the patients were aged 31 to 40 years (25.6%) followed by the age group 21 to 30 years (19.2%). The lowest percentage of patients were in the age groups 71 to 80 years and >80 years (1.3%) each. This study found a statistically significant relationship between free serum and salivary phenytoin levels (P < 0.001). A very weak and insignificant correlation was observed between serum/salivary phenytoin levels and sex/age of the study population. The results of the present study support the use of saliva as an alternative to serum/plasma for monitoring phenytoin therapy. IMPLICATIONS: The free concentration of a drug represents the freely diffusible drug fraction, which is the therapeutically active form. Accordingly, the free drug concentration correlates to clinical efficacy and drug toxicity better than total concentration.

Metastatic Breast Cancer, Organotropism and Therapeutics: A Review.

The final stage of breast cancer involves spreading breast cancer cells to the vital organs like the brain, liver lungs and bones in the process called metastasis. Once the target organ is overtaken by the metastatic breast cancer cells, its usual function is compromised causing organ dysfunction and death. Despite the significant research on breast cancer metastasis, it's still the main culprit of breast cancer-related deaths. Exploring the complex molecular pathways associated with the initiation and progression of breast cancer metastasis could lead to the discovery of more effective ways of treating the devastating phenomenon. The present review article highlights the recent advances to understand the complexity associated with breast cancer metastases, organotropism and therapeutic advances.

Complexation of alkyl glycosides with α-cyclodextrin can have drastically different effects on their conversion by glycoside hydrolases.

Substrates present in aggregated forms, such as micelles, are often poorly converted by enzymes. Alkyl glycosides constitute typical examples and the critical micelle concentration (CMC) decreases with increasing length of the alkyl group. In this study, possibilities to hydrolyse alkyl glycosides by glycoside hydrolases were explored, and α-cyclodextrin was used as an agent to form inclusion complexes with the alkyl glycosides, thereby preventing micelle formation. The cyclodextrin complexes were accepted as substrates by the enzymes to variable extent. The ß-glucosidases originating from Thermotoga neapolitana (Tn Bgl3B) and from almond were not at all able to hydrolyse alkyl ß-glucosides in the presence of 100mM α-cyclodextrin. However, Aspergillus niger amyloglucosidase readily accepted the complexes as substrates. In reactions involving decyl and dodecyl maltosides, the presence of 100mM α-cyclodextrin caused an increase in reaction rate in most cases, especially at high substrate concentrations. Surprisingly, the amyloglucosidase-catalyzed hydrolysis of octyl-ß-maltoside to glucose and ß-octylglucoside was faster in the presence of α-cyclodextrin than without, even at substrate concentrations below CMC. A possible explanation of the observed rate enhancement is that binding sites on the carbohydrate binding domain of amyloglucosidase, known to bind cyclodextrins, help to guide the alkyl glycoside-cyclodextrin complex to the active site, and thereby promote its conversion.

Exploring the synthetic potential of cell bound ß-glycosidase of Pichia etchellsii.

Enzymatic synthesis of oligosaccharides with absolute stereo-selectivity and regio-selectivity provides an economical alternative to classical chemical methods. Here we demonstrate, for the first time, that whole cells of P. etchellsii are highly efficient biocatalysts and can be used for oligosaccharide synthesis using p-nitrophenyl-ß-D-glucopyranoside, o-nitrophenyl-ß-D-glucopyranoside and p-nitrophenyl-ß-D-xylopyranoside as both donors and acceptors. Auto-condensation of p-nitrophenyl-ß-D-glucopyranoside and o-nitrophenyl-ß-D-glucopyranoside resulted in formation of ß-(1→6) linked disaccharide as major products in 4 and 12% yield respectively. By contrast, auto condensation of p-nitrophenyl-ß-D-xylopyranoside exclusively lead to formation of ß-(1→4) linked disaccharide in 24% yield.

Biotransformation of methyl-ß-D-glucopyranoside to higher chain alkyl glucosides by cell bound ß-glucosidase of Pichia etchellsii.

In the present study, we have investigated the use of Pichia etchellsii whole cells for synthesis of long-chain alkyl glucosides. Methyl-ß-d-glucopyranoside (MG) was used in reaction with fatty alcohols, n-hexanol, n-octanol, n-decanol and n-dodecanol to synthesize the respective alkyl glucosides. The initial reaction conditions were first optimized at 2.5 ml scale for synthesis of octyl glucoside (OG) and were 8% water content, 100mM MG and 6h of reaction time and this resulted in ≈ 53% yield. A maximum transglucosylation/hydrolysis ratio of 2.79 was obtained at 100mM MG favoring high product yield. Based on the optimized conditions, a reactor was operated at 50 ml level which resulted in ≈ 60% conversion of MG to OG. A simple high performance liquid chromatography method was developed for quantitation of higher chain glucosides using a refractive index detector. A maximum of 27% and 13% yield was obtained for decyl-, and dodecyl-ß-d-glucopyranoside, respectively.

ß-Glucosidase catalyzed synthesis of octyl-ß-D-glucopyranoside using whole cells of Pichia etchellsii in micro aqueous media.

Octyl-ß-D-glucopyranoside was synthesized by transglucosylation between p-nitrophenyl ß-D-glucopyranoside (pNPG) and octanol as an acceptor using whole cells of thermo tolerant yeast Pichia etchellsii displaying cell wall bound ß-glucosidase. Effect of several parameters such as glucosyl donor concentration, enzyme units and initial water activity was studied to optimize product yield. An initial water activity interval of 0.33-0.64 was favorable and increase in total enzyme units had marginal effect on conversion yield. An empirical model was developed to describe the relationship between various parameters and octyl glucoside yield. These factors were combined in a batch replacement strategy whereby octyl-ß-D-glucopyranoside was synthesized in 4h to a concentration of 30 mM (9.25 mg/ml) with a conversion yield of nearly 70% with pNPG as a glucosyl donor. Quantitative analysis was done by a highly reproducible reverse-phase high-performance liquid chromatography (RP-HPLC) method and detection was achieved using refractive index detector. The structure of the product was confirmed by ¹³C and ¹H NMR spectroscopy. Additional products like octyl diglucoside were also formed, the structure of which was confirmed by mass spectrometry.