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HPTLC (High Performance Thin Layer Chromatography) is a powerful analytical technique in the process of drug discovery and evaluation. Due to its dependability and simplicity, it is used as a tool for the identification, authentication, and quality control of herbal drugs. Mandukaparni- Centella asiatica (Linn.) Urban. is a significant and well-known drug expound discussed in the Ayurvedic classics. It is a prostrate, stoloniferous herb with enriched therapeutic potential. The herb is rich in terpinoids, flavonoids, iso-flavonoids, alkanes tannins, saponins, aminoacids and phenylpropanoids, which accounts for its multiple beneficial action in the therapeutics. It has strong anti-inflammatory and antioxidant properties. The Abhavitha choorna (dried powder) and Bhavitha choorna (powder soaked in Mandukaparni swarasa) of dried whole plant of Mandukaparni- Centella asiatica (Linn.) Urban. was subjected to High performance thin layer chromatography and comparative analysis of different peaks in both the Choornas were obtained. The results suggest that change in concentration of phytochemical constituents occur on Abhavitha choorna (powder) to Bhavitha (soaked in Mandukaparni swarasa).

The drug Mandukaparni (Centella asiatica Linn. Urban) has been extensively used as medicine and also used for in homely preparations. In Ayurveda, Bhavana is a principal samskara (pharmaceutical procedure) being used to potentiate the drug and induce additional therapeutic properties in it. This process was carried out in the powdered whole plant (abhavitha choorna) of Mandukaparni (Centella asiatica Linn.Urban), by soaking it overnight into its own swarasa (fresh juice). The preliminary phytochemical evaluation of abhavitha choorna and bhavitha choorna (processed powder) of Mandukaparni (Centella asiatica Linn.Urban) was done and the water insoluble ash, tannin, extractive values and successive solvent extractive values were found more in bhavitha choorna when compared to abhavitha choorna of the drug, which revealed that the bhavana process can increase the potency and efficacy of the drug. The aim of present study is to compare the findings of phytochemical analysis of the both abhavitha and bhavitha choorna of the drug.

Traditional medicine involves the use of different bioactive constituents present in medicinal plants. The present study was carried out on preliminary phytochemicals, metal binding capacity and on floral extract of Solanum nigrum plant. The alcoholic extract was tested for the presence of alkaloids, flavanoids, tannins, phenol, saponins, steroids, glycosides, quinones and coumarins. The UV–VIS spectrum analysis was done to study metal binding capacity of floral extract of Solanum nigrum with different metal chlorides. For this comparative study is done by measuring absorption intensities of the extract, metal chlorides and mixture of extract – metal chloride at wavelength ranging from 200 to 800 nm. Phytochemical screening highlighted the presence of flavanoids, tannins, phenol, steroids, glycosides, quinones and coumarins bioactive constituents in Solanum nigrum which can be used for further investigation of bioactive compounds. UV absorption studies showed the binding potential of floral extract of Solanum nigrum with various metal chlorides.

Eukaryotic cells utilize multiple endocytic pathways for specific uptake of ligands or molecules, and these pathways are commonly hijacked by pathogens to enable host cell invasion. Escherichia coli K1, a pathogenic bacterium that causes neonatal meningitis, invades the endothelium of the blood-brain barrier, but the entry route remains unclear. Here, we demonstrate that the bacteria trigger an actin-mediated uptake route, stimulating fluid phase uptake, membrane ruffling and macropinocytosis. The route of uptake requires intact lipid rafts as shown by cholesterol depletion. Using a variety of perturbants we demonstrate that small Rho GTPases and their downstream effectors have a significant effect on bacterial invasion. Furthermore, clathrin-mediated endocytosis appears to play an indirect role in E. coli K1 uptake. The data suggest that the bacteria effect a complex interplay between the Rho GTPases to increase their chances of uptake by macropinocytosis into human brain microvascular endothelial cells.

Adaptive immunity critically depends on the functional compartmentalization of secondary lymphoid organs. Mesenchymal stromal cells create and maintain specialized niches that support survival, activation, and expansion of T and B cells, and integrated analysis of lymphocytes and their niche has been instrumental in understanding adaptive immunity. Lymphoid organs are also home to type 3 innate lymphoid cells (ILC3), innate effector cells essential for barrier immunity. However, a specialized stromal niche for ILC3 has not been identified. A novel lineage-tracing approach now identifies a subset of murine fetal lymphoid tissue organizer cells that gives rise exclusively to adult marginal reticular cells. Moreover, both cell types are conserved from mice to humans and colocalize with ILC3 in secondary lymphoid tissues throughout life. In sum, we provide evidence that fetal stromal organizers give rise to adult marginal reticular cells and form a dedicated stromal niche for innate ILC3 in adaptive lymphoid organs.

Rituximab is an anti-CD20 mAb used in the treatment of B cell malignancies. Loss of surface CD20 Ag from the surface of target cells is thought to be one mechanism governing resistance to rituximab, but how this occurs is not completely understood. Two explanations for this have been proposed: antigenic modulation whereby mAb:CD20 complexes are internalized in a B cell intrinsic process and shaving, in which mAb:CD20 complexes undergo trogocytic removal by effector cells, such as macrophages. However, there is conflicting evidence as to which predominates in clinical scenarios and hence the best strategies to overcome resistance. In this study, we investigated the relative importance of modulation and shaving in the downregulation of surface mAb:CD20. We used both murine and human systems and treated ex vivo macrophages with varying concentrations of non–FcγR-interacting beads to achieve differential macrophage saturation states, hence controllably suppressing further phagocytosis of target cells. We then monitored the level and localization of mAb:CD20 using a quenching assay. Suppression of phagocytosis with bead treatment decreased shaving and increased modulation, suggesting that the two compete for surface rituximab:CD20. Under all conditions tested, modulation predominated in rituximab loss, whereas shaving represented an epiphenomenon to phagocytosis. We also demonstrate that the nonmodulating, glycoengineered, type II mAb obinutuzumab caused a modest but significant increase in shaving compared with type II BHH2 human IgG1 wild-type mAb. Therefore, shaving may represent an important mechanism of resistance when modulation is curtailed, and glycoengineering mAb to increase affinity for FcγR may enhance resistance because of shaving.

Background: To compare the efficacy, safety and user satisfaction of levonorgestrel intrauterine system (LNG-IUS) with oral progestogen in medical management of heavy menstrual bleeding (HMB).Methods: This prospective study was conducted on 80 women with HMB in age group of 35-55 years. Patients were divided into two groups of 40 each and followed for six months. In Group I, LNG-IUS was inserted in post menstrual period and in Group II, patients received oral norethisterone 5 mg twice a day during 5th-25th day of cycle for 6 months. Patients were evaluated about amount and duration of blood loss by pictorial blood assessment chart (PBAC) along with haemoglobin estimation on each follow up visit. MMAS (Menorrhagia Multi-Attribute Scale) score comparison between two groups was done to measure the improvement in quality of life. The results were analysed by using Chi-square test and Student t-test.Results: Percentage reduction in PBAC score after six cycles of treatment was 89.3% in LNG IUS group as compared to 68% in norethisterone group. LNG-IUS was found to be more efficient in correcting anemia, lowering duration of bleeding and improving MMAS score as compared to norethisterone.Conclusions: Present study showed that LNG IUS was superior to oral progestogen in decreasing quantity and duration of bleeding and improving overall quality of life over time.

Nonhematopoietic stromal cells of secondary lymphoid organs form important scaffold and fluid transport structures, such as lymph node (LN) trabeculae, lymph vessels, and conduits. Furthermore, through the production of chemokines and cytokines, these cells generate a particular microenvironment that determines lymphocyte positioning and supports lymphocyte homeostasis. IL-7 is an important stromal cell-derived cytokine that has been considered to be derived mainly from T-cell zone fibroblastic reticular cells. We show here that lymphatic endothelial cells (LECs) are a prominent source of IL-7 both in human and murine LNs. Using bacterial artificial chromosome transgenic IL-7–Cre mice, we found that fibroblastic reticular cells and LECs strongly up-regulated IL-7 expression during LN remodeling after viral infection and LN reconstruction after avascular transplantation. Furthermore, IL-7–producing stromal cells contributed to de novo formation of LyveI-positive lymphatic structures connecting reconstructed LNs with the surrounding tissue. Importantly, diphtheria toxin–mediated depletion of IL-7–producing stromal cells completely abolished LN reconstruction. Taken together, this study identifies LN LECs as a major source of IL-7 and shows that IL-7–producing stromal cells are critical for reconstruction and remodeling of the distinct LN microenvironment.

Background Inflammation and oxidative stress play critical roles in patients with chronic obstructive pulmonary disease (COPD). Mitochondrial oxidative stress might be involved in driving the oxidative stress–induced pathology. Objective We sought to determine the effects of oxidative stress on mitochondrial function in the pathophysiology of airway inflammation in ozone-exposed mice and human airway smooth muscle (ASM) cells. Methods Mice were exposed to ozone, and lung inflammation, airway hyperresponsiveness (AHR), and mitochondrial function were determined. Human ASM cells were isolated from bronchial biopsy specimens from healthy subjects, smokers, and patients with COPD. Inflammation and mitochondrial function in mice and human ASM cells were measured with and without the presence of the mitochondria-targeted antioxidant MitoQ. Results Mice exposed to ozone, a source of oxidative stress, had lung inflammation and AHR associated with mitochondrial dysfunction and reflected by decreased mitochondrial membrane potential (ΔΨm), increased mitochondrial oxidative stress, and reduced mitochondrial complex I, III, and V expression. Reversal of mitochondrial dysfunction by the mitochondria-targeted antioxidant MitoQ reduced inflammation and AHR. ASM cells from patients with COPD have reduced ΔΨm, adenosine triphosphate content, complex expression, basal and maximum respiration levels, and respiratory reserve capacity compared with those from healthy control subjects, whereas mitochondrial reactive oxygen species (ROS) levels were increased. Healthy smokers were intermediate between healthy nonsmokers and patients with COPD. Hydrogen peroxide induced mitochondrial dysfunction in ASM cells from healthy subjects. MitoQ and Tiron inhibited TGF-β–induced ASM cell proliferation and CXCL8 release. Conclusions Mitochondrial dysfunction in patients with COPD is associated with excessive mitochondrial ROS levels, which contribute to enhanced inflammation and cell hyperproliferation. Targeting mitochondrial ROS represents a promising therapeutic approach in patients with COPD.

The lymph node follicular dendritic cell (FDC) network is derived from the expansion and differentiation of marginal reticular cells, as are the new FDCs generated during an immune response., Follicular dendritic cells (FDCs) regulate B cell function and development of high affinity antibody responses but little is known about their biology. FDCs associate in intricate cellular networks within secondary lymphoid organs. In vitro and ex vivo methods, therefore, allow only limited understanding of the genuine immunobiology of FDCs in their native habitat. Herein, we used various multicolor fate mapping systems to investigate the ontogeny and dynamics of lymph node (LN) FDCs in situ. We show that LN FDC networks arise from the clonal expansion and differentiation of marginal reticular cells (MRCs), a population of lymphoid stromal cells lining the LN subcapsular sinus. We further demonstrate that during an immune response, FDCs accumulate in germinal centers and that neither the recruitment of circulating progenitors nor the division of local mature FDCs significantly contributes to this accumulation. Rather, we provide evidence that newly generated FDCs also arise from the proliferation and differentiation of MRCs, thus unraveling a critical function of this poorly defined stromal cell population.

Schistosomes are blood-dwelling parasitic helminths which produce eggs in order to facilitate transmission. Intestinal schistosomes lay eggs in the mesenteries, however, it is unclear how their eggs escape the vasculature to exit the host. Using a murine model of infection, we reveal that Schistosoma mansoni exploits Peyer's Patches (PP) gut lymphoid tissue as a preferential route of egress for their eggs. Egg deposition is favoured within PP as a result of their more abundant vasculature. Moreover, the presence of eggs causes significant vascular remodeling leading to an expanded venule network. Egg deposition results in a decrease in stromal integrity and lymphoid cellularity, including secretory IgA producing lymphocytes, and the focal recruitment of macrophages. In mice lacking PP, egg excretion is significantly impaired, leading to greater numbers of ova being entrapped in tissues and consequently, exacerbated morbidity. Thus, we demonstrate how schistosomes directly facilitate transmission from the host by targeting lymphoid tissue. For the host, PP-dependency of egg egress represents a trade-off, as limiting potentially life-threatening morbidity is balanced by loss of PP structure and perturbed PP IgA production., Author Summary Schistosomes are parasitic helminths that parasitise >200 million people worldwide. Adult worm pairs of intestinal schistosomes lay their eggs in the mesenteric veins from which the eggs need to pass into the lumen prior to excretion and completion of their life cycle. However, it is not known how eggs transfer from the intestinal vasculature to reach the gut lumen. Here, we reveal using a mouse model of infection, that Schistosoma mansoni exploits Peyer's Patches (PP) lymphoid tissues in the wall of the small intestine as a preferential route of egg egress. The eggs cause vascular remodelling in the PP leading to an expanded venule network, reduced stromal integrity, and decreased lymphoid cellularity. Most significantly, in mice rendered deficient in PP, egg excretion is impaired (despite intact immune responses), leading to greater numbers of eggs entrapped in tissues, and consequently exacerbated host morbidity. Thus, we demonstrate how schistosomes directly facilitate transmission from the host by targeting lymphoid tissue. For the host, this represents a trade-off as limiting life-threatening morbidity is balanced by loss of PP structure and function. The requirement of PP for efficient schistosome egress may be a significant risk factor of developing severe disease within heavily infected human populations.

In the bone marrow (BM), stromal cells constitute a supportive tissue indispensable for the generation of pro-B/pre-BI, pre-BII, and immature B lymphocytes. IL-7–producing stromal cells constitute a cellular niche for pro-B/pre-BI cells, but no specific stromal cell microenvironment was identified for pre-BII cells expressing a functional pre-B cell receptor (pre-BCR). However expression of the pre-BCR represents a crucial checkpoint during B-cell development. We recently demonstrated that the stromal cell derived-galectin1 (GAL1) is a ligand for the pre-BCR, involved in the proliferation and differentiation of normal mouse pre-BII cells. Here we show that nonhematopoietic osteoblasts and reticular cells in the BM express GAL1. We observed that pre-BII cells, unlike the other B-cell subsets, were specifically localized in close contact with GAL1+ reticular cells. We also determined that IL-7+ and GAL1+ cells represent 2 distinct mesenchymal populations with different BM localization. These results demonstrate the existence of a pre-BII specific stromal cell niche and indicate that early B cells move from IL-7+ to GAL1+ supportive BM niches during their development.

Receptor tyrosine kinases are involved in multiple cellular processes, and drugs that inhibit their action are used in the clinic to treat several types of cancer. However, the value of receptor tyrosine kinase inhibitors (RTKIs) for treating infectious disease has yet to be explored. Here, we have shown in mice that administration of the broad-spectrum RTKI sunitinib maleate (Sm) blocked the vascular remodeling and progressive splenomegaly associated with experimental visceral leishmaniasis. Furthermore, Sm treatment restored the integrity of the splenic microarchitecture. Although restoration of splenic architecture was accompanied by an increase in the frequency of IFN-gamma+CD4+ T cells, Sm treatment alone was insufficient to cause a reduction in tissue parasite burden. However, preconditioning by short-term Sm treatment proved to be successful as an adjunct therapy, increasing the frequency of IFN-gamma+ and IFN-gamma+TNF+CD4+ T cells, enhancing NO production by splenic macrophages, and providing dose-sparing effects when combined with a first-line immune-dependent anti-leishmanial drug. We propose, therefore, that RTKIs may prove clinically useful as agents to restore immune competence before the administration of chemo- or immunotherapeutic drugs in the treatment of visceral leishmaniasis or other diseases involving lymphoid tissue remodeling, including cancer.

NADPH oxidase (NOX2) is a multisubunit membrane-bound enzyme complex that, upon assembly in activated cells, catalyses the reduction of free oxygen to its superoxide anion, which further leads to reactive oxygen species (ROS) that are toxic to invading pathogens, for example, the fungusAspergillus fumigatus. Polymorphonuclear cells (PMNs) employ both nonoxidative and oxidative mechanisms to clear this fungus from the lung. The oxidative mechanisms mainly depend on the proper assembly and function of NOX2. We identified for the first time the NAD(P)H-dependent enzymes involved in such oxidative mechanisms by means of biexponential NAD(P)H-fluorescence lifetime imaging (FLIM). A specific fluorescence lifetime of3670±140picoseconds as compared to 1870 picoseconds for NAD(P)H bound to mitochondrial enzymes could be associated with NADPH bound to oxidative enzymes in activated PMNs. Due to its predominance in PMNs and due to the use of selective activators and inhibitors, we strongly believe that this specific lifetime mainly originates from NOX2. Our experiments also revealed the high site specificity of the NOX2 assembly and, thus, of the ROS production as well as the dynamic nature of these phenomena. On the example of NADPH oxidase, we demonstrate the potential of NAD(P)H-based FLIM in selectively investigating enzymes during their cellular function.

The fungal pathogens Aspergillus fumigatus and Candida albicans are major health threats for immune-compromised patients. Normally, macrophages and neutrophil granulocytes phagocytose inhaled Aspergillus conidia in the two-dimensional (2-D) environment of the alveolar lumen or Candida growing in tissue microabscesses, which are composed of a three-dimensional (3-D) extracellular matrix. However, neither the cellular dynamics, the per-cell efficiency, the outcome of this interaction, nor the environmental impact on this process are known. Live imaging shows that the interaction of phagocytes with Aspergillus or Candida in 2-D liquid cultures or 3-D collagen environments is a dynamic process that includes phagocytosis, dragging, or the mere touching of fungal elements. Neutrophils and alveolar macrophages efficiently phagocytosed or dragged Aspergillus conidia in 2-D, while in 3-D their function was severely impaired. The reverse was found for phagocytosis of Candida. The phagocytosis rate was very low in 2-D, while in 3-D most neutrophils internalized multiple yeasts. In competitive assays, neutrophils primarily incorporated Aspergillus conidia in 2-D and Candida yeasts in 3-D despite frequent touching of the other pathogen. Thus, phagocytes show activity best in the environment where a pathogen is naturally encountered. This could explain why “delocalized” Aspergillus infections such as hematogeneous spread are almost uncontrollable diseases, even in immunocompetent individuals., Author Summary Aspergillus fumigatus and Candida albicans are the most common of all human pathogenic fungal germs. Normally, inhaled Aspergillus spores are destroyed by alveolar macrophages and polymorphonuclear neutrophils (PMNs), both of which are lung phagocytes, i.e., cells that kill inhaled microbes by ingestion. In contrast, C. albicans is a normal constituent of the human gut flora that is controlled by tissue-resident PMNs. If immune control is lost, both fungi grow into the surrounding tissue and cause life-threatening infections. To investigate how phagocytes function in the disparate environments of lung air sacs (lacking a definite matrix-composition [two-dimensional (2-D)]) or mucosal tissues (providing a three-dimensional [3-D] space), the authors mimicked 2-D and 3-D environments and analyzed the process of ingestion, called phagocytosis, by PMNs and other phagocytes. Phagocytosis was a dynamic cellular process where distinct cells showed vastly different behavior. The environmental setup (2-D versus 3-D) had a profound impact on the cell's ability to phagocytose. Aspergillus conidia were much better ingested in 2-D systems, while Candida yeasts were only ingested in 3-D systems, even if the other pathogen was present. This was true for different 2-D and 3-D systems and for both cells of mice and humans. Besides providing a comprehensive analysis of the cellular movements underlying phagocytosis, the results also suggest an evolution of phagocytes to optimally recognize fungal pathogens in the environment of natural infection.

Dengue, an important human arboviral infection, is endemic in many parts of India. Outbreaks are now reported quite frequently from different parts of the country. We report here findings of serological investigation of serum samples received during a recent outbreak of dengue infection in Delhi from September to November 2003. Acute phase serum samples from suspected cases of dengue infection (including dengue fever, dengue haemorrhagic fever and dengue shock syndrome) of duration 5 days, were tested for dengue specific IgM antibodies by enzyme linked immuno sorbant assay (ELISA). Of the 874 serum samples tested, 456 (52.3% ) were positive for dengue specific IgM antibodies. The maximum number of positive cases was reported in October, peaking in second and third week. Patients in the age group of 21-30 yr accounted for 34.2 per cent of positive cases. This outbreak was less severe as compared to the previous 1996 outbreak and showed a shift towards an older age group.

To the Editor: Delhi, in the northern part of India, has had outbreaks of dengue caused by various dengue virus types in 1967, 1970, 1982, 1988, and 1996 (1–5). In 1988, for the first time, a few cases of dengue hemorrhagic fever (DHF) were seen (4). Subsequently, we reported the largest outbreak of DHF/dengue shock syndrome (DSS) in Delhi in 1996 and confirmed dengue virus type 2 as the etiologic agent (5). We report the results of virologic testing of samples received at the All India Institute of Medical Sciences from patients with suspected dengue fever or denguelike illness from Delhi and its adjoining areas during a 2003 outbreak of dengue. According to the World Health Organization (6), 2,185 laboratory-confirmed cases were reported during this outbreak. Of the blood samples received by the virology laboratory, 42 were received on ice from patients with acute denguelike illness. Serum was separated aseptically and stored at –70°C. The standard method of virus cultivation, which used the C6/36 clone of the Aedes albopictus cell line, was followed with some modifications (7). On days 5 and 10, harvested cells were tested by an indirect immunofluorescence assay (IFA) using monoclonal antibodies to dengue virus types 1–4 (provided by the Centers for Disease Control and Prevention, Atlanta, Georgia, USA, during the 1996 outbreak). If IFA results were negative for dengue viruses on first passage, a second passage was made, and cells were again harvested on days 5 and 10 for IFA. The 4 dengue virus types (obtained from the National Institute of Virology, Pune, India) were included as positive controls, and uninfected C6/36 cells were kept as negative controls. Dengue virus could be isolated in C6/36 cells from 8 (19%) of 42 samples processed for virus isolation (Table). Of the 8 isolates, two each were identified as dengue virus types 1 and 2, three as type 3, and one as type 4. All but one isolate were from patients with uncomplicated dengue fever. One dengue type 2 isolate was obtained from a 7-year-old boy with secondary dengue infection and DHF/DSS. The ages of culture-positive patients ranged from 5 to 62 years, with a median of 22 years. These patients were equally distributed between children (

Oral lichen planus (OLP) is a chronic inflammatory mucocutaneous disease that frequently involves the oral mucosa. The authors report a long-term follow-up of a case which presented clinically as lichen planus with apparently benign features but histologically exhibited features of early invasive squamous cell carcinoma. Although a common lesion, the matter concerning malignant potential of OLP and its differentiation from lichenoid reaction still remains unresolved. There is no universally accepted, documented, and approved modality of treatment to prevent the occurrence of cancer development and also literature search reveals no standard methods which can predict cancer development of potentially malignant disorders. Hence every reported case should be well documented. This case report emphasizes the need for histological confirmation, with continuous monitoring 3-6 times annually of clinical lesions that have lichenoid features.

The investigation of physiological phenomena at molecular level within vital cells, for instance metabolic processes, represents a major challenge in life sciences. In this context, measuring methods should be non-invasive for the system under study and should provide high sensitivity and specificity. We demonstrate inhere the power of marker-free, spatially and timely resolved intracellular detection of NAD(P)H-dependent enzymes in the study of phenomena of particular biomedical relevance like phagocytosis or autoimmune reactions. The technique is based on biexponential two-photon laser scanning fluorescence lifetime microscopy (TPLSM FLIM) of the physiologic fluorophore NAD(P)H, which exhibits a fluorescence lifetime τ that is strongly dependent on its chemical environment, i.e. on the enzyme to which NAD(P)H is bound to (free NAD(P)H: τ ∼ 400 ps, enzyme-bound NAD(P)H: τ ∼ 2 ns). In order to verify the feasibility of the method, an in-vitro study with different enzymes in solution was performed. The measured fluorescence lifetimes of enzyme-bound NAD(P)H strongly varied from τ = 960±27 ps for malic dehydrogenase to τ = 3640±101 ps for 3α hydroxysteroid dehydrogenase, which made us confident, that an intracellular selective enzyme detection is possible by means of NAD(P)H-FLIM. We indeed succeeded for the first time to specifically and intracellularly detect the NADPH oxidase, a multi-subunit membrane-bound enzyme complex that catalyzes the reduction of free oxygen to its superoxide anion. Thereby, we selectively monitored the NADPH oxidase during its function within differently activated murine polymorphonuclear leucocytes (PMNs). The experiments revealed a specific fluorescence lifetime of 3670±140 ps for NADPH bound to this enzyme both in humorally activated PMNs, i.e. activation with PMA, and in PMNs phagocytosing the fungus Aspergillus fumigatus, confirming the assembly of NADPH oxidase as highly site-specific.