Site-selective synthesis and pharmacological elucidation of novel semi-synthetic analogues of koenimbine as a potential anti-inflammatory agent.

Koenimbine (1), a carbazole alkaloid isolated from Murraya koenigii, belongs to the Rutaceae family. Various pharmacological effects such as anti-diabetic, melanogenesis inhibition, anti-diarrheal, anti-cancer, and anti-inflammatory properties of koenimbine have already been reported. In the current study, we investigated the anti-inflammatory role of koenimbine (1) and its novel semi-synthetic derivative 8-methoxy-3,3,5-trimethylpyrano[3,2-a] carbazole-11(3H)-yl) (3-(trifluoromethyl) phenyl) methanone (1G) in both in vitro and in vivo biological systems. Our results demonstrated that the anti-inflammatory activity of 1G significantly lowered the production of NO, pro-inflammatory cytokines (IL-6, TNF-α & IL-1ß), LTB4 following LPS stimulation in RAW 264.7 macrophages. Furthermore, 1G significantly attenuated the expression levels of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in a dose dependent manner and also decreased the production of reactive oxygen species (ROS) in LPS-activated RAW 264.7 cells. In addition, the oral administration of 1G reduced the inflammatory response in carrageenan-induced paw edema in BALB/C mice. Moreover, it effectively reduced NO, IL-6, IL-1ß & TNF-α levels, liver markers (AST, ALT), and kidney markers (BUN, CRE, and Urea). Also, 1G reverted the infiltration of inflammatory cells and tissue damage in lungs, liver and kidney enhanced the survival rate in LPS-challenged mice. 1G blocks NF-κB p65 from translocating into the nucleus and activating inflammatory gene transcription. These results illustrated that 1G suppresses the inflammatory effects both in-vitro and in-vivo studies via downregulating the nuclear factor kappa-B (NF-κB) signaling pathway. In conclusion, our results demonstrate that semi-synthetic derivative 1G can effectively attenuate the inflammatory response via NF-κB and MAPK signaling pathways; suggesting 1G is a potential novel anti-inflammatory drug candidate in treating inflammatory disorders.

Anti-inflammatory and anti-arthritic potential of methotrexate in combination with BA-25, an amino analogue of ß-boswellic acid in the treatment of rheumatoid arthritis.

ß- boswellic acid, a pentacyclic triterpene derived from Boswellia serrata is extensively known for its anti-inflammatory potential. BA-25 (3-α-o-acetoxy-4ß-amino-11-oxo-24-norurs-12-ene) is an amino analogue of ß-boswellic acid that has shown anti-inflammatory potential in LPS-induced macrophages and animal models. The present study aims at investigation of the combination of BA-25 with the conventional gold standard DMARD methotrexate (MTX) for its anti-inflammatory and anti-arthritic potential using in vitro and in vivo experimental models. The anti-inflammatory potential of MTX versus the combination (BA-25 + MTX) was investigated for inhibition of NO, ROS and pro-inflammatory cytokines including TNF-α and IL-6 using ELISA in LPS-stimulated RAW-264.7 cells. The results demonstrated significant reduction in NO, ROS, TNF- α and IL-6 production with the combination treatment in comparison to MTX alone. The cytokine inhibition potential of the combination was further validated in-vivo using balb/c wherein the combination restored LPS-induced increase in pro-inflammatory cytokines. The toxicological aspect of the in vivo doses of the combination was also investigated in mice after dosing for 28 days wherein the results suggested no significant change in the hematological parameters and serum biochemical parameters in the combination versus the vehicle group. The effect of BA-25 was also investigated on MTX-induced increase in liver function tests and the expression of Bax and blc2. The results demonstrated decrease in the production of liver enzymes with BA-25 administration along with downregulating the expression of apoptotic protein Bax while increasing the expression of anti-apoptotic protein Bcl2. Furthermore, pharmacokinetic studies of BA-25 were conducted in Balb/c mice wherein the compound showed rapid absorption, high volume of distribution and a t1/2 of 13.08. Finally the anti-arthritic effect of the combination of MTX + BA-25 vs MTX alone was investigated using CIA model in DBA/1 mice wherein the treatment with the combination resulted in significant reduction in paw inflammation, IL-6 and IL-1ß levels. Furthermore, the western blot analysis demonstrated considerable decrease in the expression of p-NF-κB p65 and p-IκB in the ankle-joint tissue of the CIA mice treated with the combination therapy. The results insinuated increased anti-inflammatory and anti-arthritic potential of the combination of MTX with BA-25 as evident from in to vitro and in-vivo studies.

Arteannuin-B and (3-Chlorophenyl)-2-Spiroisoxazoline Derivative Exhibit Anti-Inflammatory Effects in LPS-Activated RAW 264.7 Macrophages and BALB/c Mice-Induced Proinflammatory Responses via Downregulation of NF-κB/P38 MAPK Signaling.

Host inflammatory responses are key to protection against injury; however, persistent inflammation is detrimental and contributes to morbidity and mortality. Herein, we demonstrated the anti-inflammatory role of Arteannuin-B (1) and its new spirocyclic-2-isoxazoline derivative JR-9 and their side effects in acute inflammatory condition in vivo using LPS-induced cytokines assay, carrageenan-induced paw edema, acetic acid-induced writhing and tail immersion. The results show that the spirocyclic-2-isoxazoline derivative is a potent anti-inflammatory agent with minimal cell toxicity as compared to Arteannuin-B. In addition, the efficacies of these compounds were also validated by flow cytometric, computational, and histopathological analysis. Our results show that the anti-inflammatory response of JR-9 significantly reduces the ability of mouse macrophages to produce NO, TNF-α, and IL-6 following LPS stimulation. Therefore, JR-9 is a prospective candidate for the development of anti-inflammatory drugs and its molecular mechanism is likely related to the regulation of NF-κB and MAPK signaling pathway.

Phytochemicals targeting JAK/STAT pathway in the treatment of rheumatoid arthritis: Is there a future?

Rheumatoid arthritis (RA) is a chronic autoimmune disorder and the treatment involves the use of traditional and biological disease modifying anti-rheumatic drugs (DMARDs). Recent studies have shown JAK/STAT signaling pathway as potential target for the treatment of RA. Novel JAK/STAT inhibitors viz tofacitinib and baricitinib have been recently approved by FDA for RA treatment and have attained substantial importance. However, the discernible risks of thromboembolism, gastrointestinal (GIT) perforations, hepatotoxicity and serious infections including tuberculosis, herpes zoster associated with their administration cannot be overlooked. Furthermore, these are highly expensive which limits their application for a broader use. These limitations provide the basis of exploring novel JAK/STAT inhibitors of natural origin with increased tolerability, safety and cost-effectiveness. In this review we confer an account of various natural compounds/phytochemicals that have proved to be beneficial in attenuating inflammation in RA via modulation of JAK/STAT signaling pathway. Some of these natural compounds including resveratrol have clearly indicated biochemical and clinically significant therapeutic effects in ameliorating RA both in vivo and in clinical settings. We further discuss the physicochemical challenges of poor solubility and absorption coupled with the use of natural JAK/STAT inhibitors. We thereafter discuss and summarize various drug delivery systems (DDS) to confront the physicochemical limitations of natural JAK/STAT inhibitors with the aim to enhance the therapeutic efficacy. Overall the review unveils the potential of natural JAK/STAT inhibitors as a cost-effective approach in ameliorating RA without incorporating the risks of adverse repercussions, thus setting the stage for clinical exploration of these compounds that may possibly complement the present RA therapy.

Origanum vulgare L.: In vitro Assessment of Cytotoxicity, Molecular Docking Studies, Antioxidant and Anti-inflammatory Activity in LPS Stimulated RAW 264.7 Cells.

BACKGROUND: Inflammation involves a dynamic network that is highly regulated by signals that initiate the inflammation process as well as signals that downregulate it. However, an imbalance between the two leads to tissue damage. Throughout the world, inflammatory disease becomes common in the aging society. The drugs which are used clinically have serious side effects. Natural products or compounds derived from natural products show diversity in structure and play an important role in drug discovery and development. OBJECTIVE: Oreganum Vulgare is used in traditional medicine for various ailments including respiratory and rheumatic disorders, severe cold, suppression of tumors. The current study aims to evaluate the anti-inflammatory potential by evaluating various in vitro parameters. METHODS: Inflammation-induced in macrophages via LPS is the most accepted model for evaluating the antiinflammatory activity of various plant extracts and lead compounds. RESULTS: The extracts (OVEE, OVEAF) as well as the isolated compound(OVRA)of Oreganum Vulgare inhibit the pro-inflammatory cytokines (IL-6 and TNF-α) and NO without affecting cell viability. CONCLUSION: Our study established that the leaf extracts of Oreganum vulgare L. exhibit anti-inflammatory activity and thus confirm its importance in traditional medicine.

Burden of dengue infection in India, 2017: a cross-sectional population based serosurvey.

BACKGROUND: The burden of dengue virus (DENV) infection across geographical regions of India is poorly quantified. We estimated the age-specific seroprevalence, force of infection, and number of infections in India. METHODS: We did a community-based survey in 240 clusters (118 rural, 122 urban), selected from 60 districts of 15 Indian states from five geographical regions. We enumerated each cluster, randomly selected (with an Andriod application developed specifically for the survey) 25 individuals from age groups of 5-8 years, 9-17 years, and 18-45 years, and sampled a minimum of 11 individuals from each age group (all the 25 randomly selected individuals in each age group were visited in their houses and individuals who consented for the survey were included in the study). Age was the only inclusion criterion; for the purpose of enumeration, individuals residing in the household for more than 6 months were included. Sera were tested centrally by a laboratory team of scientific and technical staff for IgG antibodies against the DENV with the use of indirect ELISA. We calculated age group specific seroprevalence and constructed catalytic models to estimate force of infection. FINDINGS: From June 19, 2017, to April 12, 2018, we randomly selected 17 930 individuals from three age groups. Of these, blood samples were collected and tested for 12 300 individuals (5-8 years, n=4059; 9-17 years, n=4265; 18-45 years, n=3976). The overall seroprevalence of DENV infection in India was 48·7% (95% CI 43·5-54·0), increasing from 28·3% (21·5-36·2) among children aged 5-8 years to 41·0% (32·4-50·1) among children aged 9-17 years and 56·2% (49·0-63·1) among individuals aged between 18-45 years. The seroprevalence was high in the southern (76·9% [69·1-83·2]), western (62·3% [55·3-68·8]), and northern (60·3% [49·3-70·5]) regions. The estimated number of primary DENV infections with the constant force of infection model was 12 991 357 (12 825 128-13 130 258) and for the age-dependent force of infection model was 8 655 425 (7 243 630-9 545 052) among individuals aged 5-45 years from 30 Indian states in 2017. INTERPRETATION: The burden of dengue infection in India was heterogeneous, with evidence of high transmission in northern, western, and southern regions. The survey findings will be useful in making informed decisions about introduction of upcoming dengue vaccines in India. FUNDING: Indian Council of Medical Research.

Murrayanine Attenuates Lipopolysaccharide-induced Inflammation and Protects Mice from Sepsis-associated Organ Failure.

Murrayanine (MK) is the main compound isolated from Murraya koenigii, an aromatic plant belonging to the Rutaceae family, also known as curry leaf tree. Murrayanine was reported to possess potential antioxidant, antimycobacterial and antifungal effects. However, its effect in sepsis remains unclear. This study was designed to investigate the anti-inflammatory effect of MK using both in vitro and in vivo assay. Results of this study indicated that MK decreased NO, TNF-α and IL-6 production in both lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and murine peritoneal macrophages. Moreover, iNOS and COX-2 protein expression as well as their downstream product, PGE2, was also decreased effectively in RAW 264.7 cells. Furthermore, MK decreased the phosphorylation of IKB and repressed NF-kB activity in LPS-activated RAW 264.7 cells. Additionally, we evaluated MK efficacy in vivo using LPS-induced sepsis, a systemic inflammation model in mice. Administration of MK inhibits pro-inflammatory cytokines (TNF-α and IL-6) secretion; decreases AST, ALT, BUN and CRE level in mouse sera; mitigates lung, liver and kidney injuries; and also increases LPS-challenged mice survival rate. Collectively, our results suggest that MK exerts potential as a new anti-inflammatory and immunosuppressive drug in sepsis treatment.

Kaempferol-3-o-ß-d-glucuronate exhibit potential anti-inflammatory effect in LPS stimulated RAW 264.7 cells and mice model.

Kaempferol-3-O-ß-d-glucuronide (K3G) having various pharmacological effects was explored for its anti-inflammatory effect in LPS induced RAW 264.7 cells and mice model. K3G significantly inhibited various pro-inflammatory mediators like IL-1ß, NO, PGE2, and LTB4. It upregulated the secretion of anti-inflammatory cytokine IL-10. K3G is found to reduce inflammation when studied for parameters like phagocytic index, carrageenan induced paw edema in mice and organ weight. It reduced inflammation in a dose dependent manner both in-vitro and in-vivo. Further molecular insights into the study reveal that K3G blocks the phosphorylation of NF-kB which is key regulator of inflammation, thereby exhibiting anti-inflammatory potential. Hence, this study permits further investigation to develop K3G as anti-inflammatory drug.

The amino analogue of ß-boswellic acid efficiently attenuates the release of pro-inflammatory mediators than its parent compound through the suppression of NF-κB/IκBα signalling axis.

Natural product derivatives have proven to be cutting edge window for drug discovery and development. BA-25 (3-α-o-acetoxy-4ß-amino-11-oxo-24-norurs-12-ene) an amino analogue of ß-boswellic acid exhibited inhibition of TNF-α and IL-6 in THP-1 cells as demonstrated previously, however, the effect on principal inflammatory mediators such as cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and the pathways that mediate this function remains unknown. This study was designed to examine the comparative anti-inflammatory activity of BA-25 with its parent compound, ß boswellic acid both in vitro and in vivo. The effect of BA and BA-25 on suppression of NO, PGE2, LTB4, COX-2 in LPS-stimulated RAW 264.7 cells was determined by ELISA, RT-PCR and ROS by flow cytometry. Phosphorylation of NF-kBp65, IKB degradation was determined by western blotting and also the nuclear localization of NF-kBp65 was assessed by immunofluorescence. Furthermore, this study was extended on Carrageenan induced paw oedema modelled BALB/c mice. A novel derivative BA-25, reported first time notably decreased the LPS (1 µg/mL) induced upregulation in the transcription of TNF-α, IL-6, iNOS and COX-2. Also the protein expression of iNOS and COX-2 as well as their downstream products NO and PGE2 respectively, were also decreased efficiently at a concentration of 10 µM than BA. Moreover, LPS upregulated NF-kB p65 expression and IκB degradation was significantly decreased after BA-25 treatment. In addition, the treatment of BA-25 also restored the paw oedema and decreased the magnitude of histopathological alterations. Our data together suggested that BA-25 might be regarded as prospective therapeutic anti-inflammatory alternative and demands further investigation in pharmacological studies.

Anti-inflammatory potential of hentriacontane in LPS stimulated RAW 264.7 cells and mice model.

Hentriacontane, has various pharmacological effects including anti-inflammatory, antitumor and antimicrobial activities. Its anti-inflammatory potential has been demonstrated in peritoneal macrophages. However detailed studies on other models elucidating the mechanistic description of the mode of action has not been done. Hence, the aim of the present study is to evaluate the anti-inflammatory potential of hentriacontane both in-vivo (Balb/c mice) and in-vitro (RAW 264.7 cells). Cytokine inhibition of both pro-inflammatory (TNF-α, IL-6, MCP-1 and IL-1ß) and anti-inflammatory (IL-10) cytokines was studied in RAW 264.7 cells and Balb/c mice. Suppressive potential of hentriacontane on NO, PGE2, LTB4 and on LPS induced translocation of NF-κB in RAW 264.7 cells was studied. Further investigations on the effect of hentriacontane on phagocytic index, carrageenan induced paw oedema in mice and on organ weight were done. It was found that hentriacontane significantly reduced all the parameters of inflammation in the experiments under study at all the concentrations, 10µM, 5µM and 1µM (in-vitro) and 5mg/kg, 2mg/kg and 1mg/kg (in-vivo). The highest concentration used in the two models presented the most significant results. The results indicate that hentriacontane is a potent suppressor of inflammatory cytokines and other mediators. Moreover it also has regulatory effect on NF-κB. Hence, hentriacontane is a potential candidate for investigations to develop anti-inflammatory drug.

Biopharmaceutic parameters, pharmacokinetics, transport and CYP-mediated drug interactions of IIIM-017: A novel nitroimidazooxazole analogue with anti-tuberculosis activity.

Nitroimidazoles are emerging as a new class of therapeutic agents with potent anti-tubercular activity. CSIR-IIIM has synthesized a novel nitrohydroimidazooxazole (NHIO) analogue, IIIM-017 with a MIC of 0.37µg/ml (against H37Rv). Here, we aim at further exploration of physicochemical properties and preclinical absorption, metabolism, disposition and pharmacokinetics of IIIM-017. In this study, in silico physicochemical parameters, lipophilicity, permeability, transport, hepatotoxicity, CYP mediated drug interactions and pharmacokinetics of IIIM-017 were investigated. The results demonstrated that IIIM-017 exhibited good physicochemical properties, comparable to PA-824 and OPC-67683. Caco-2 transport studies revealed that the compound was highly permeable with Papp of 8.85×10-6 (A-B) and 27.69×10-6 (B-A) cm/s. Caco-2 cells were also used to study P-gp mediated transport and inhibition. IIM-017 exhibited very low intrinsic clearance and no substantial hepatotoxicity in vitro. The compound did not have any inhibitory effect on human CYPs 1A2, 2C9, 2D6, 3A4 and 2C19 up to concentration of 30µM. In vivo pharmacokinetics was performed on balb/c mice at 5mg/kg (p.o) and 2.5mg/kg (i.v.) and plasma drug concentrations were determined by LC-MS/MS. The compound showed satisfactory PK parameters in mice. The results insinuate that IIIM-017 should undergo further development as a potential treatment for tuberculosis.

Preclinical comprehensive physicochemical and pharmacokinetic profiling of novel nitroimidazole derivative IIIM-019 - A potential oral treatment for tuberculosis.

New compounds against tuberculosis are urgently needed to combat the crisis of drug resistance in tuberculosis (TB). We have identified a nitrodihydroimidazooxazole analog, IIIM-019 as a new anti-tubercular agent with a MIC of 0.23 µM against H37Rv. Physicochemical properties, in-vitro pharmacokinetics and in-vivo multiple-doses pharmacokinetics were studied for the compound. In silico physicochemical parameters and Lipinski's violations were determined for drug like properties. Lipophilicity was determined experimentally as Octanol-PBS partition coefficient (log P). Passive and active permeability of the compound was determined by PAMPA and Caco-2 cell permeability analysis, respectively. Plasma protein binding was determined by Rapid equilibrium dialysis. Metabolism by liver microsomes revealed the t1/2 and intrinsic clearance of the compound. Hepatotoxicity of IIIM-019 was determined alone and in combination to first line anti-tubercular drugs. The compound was also estimated for nuclear DNA damage. Single doses of IIIM-019 (2.5, 10, 25 and 100 mg/kg) were administered orally to Balb/c mice and the blood samples were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). IIIM-019 exhibited very good lipophilicity (log P) of 2.47 which makes it optimal for oral administration. The compound showed low solubility and permeability and high plasma protein binding. However, it was highly stable in rat liver microsomes with t1/2 > 2 h and very low intrinsic clearance. It was found to be non-hepatotoxic and did not induce any significant DNA damage at high concentrations even up to 100 µM. IIIM-019 showed satisfactory in-vivo pharmacokinetic properties. By increasing the dose from 2.5 mg/kg to 10 mg/kg, AUC0-t increased from 14935 ng h/ml to 81,478 ng h/ml. However the exposure of IIIM-019 in plasma suggested that the levels reached saturation at higher concentrations. The compound showed a good oral bioavailability of 58.7%. The results insinuate that IIIM-019 should undergo further development as a potential treatment for tuberculosis.

Development and validation of a highly sensitive LC-MS/MS-ESI method for quantification of IIIM-019-A novel nitroimidazole derivative with promising action against Tuberculosis: Application to drug development.

The study aims to illustrate an analytical validation of a rapid and sensitive liquid chromatography (LC) coupled to tandem mass spectrometry (MS-MS) and electrospray ionization (ESI) method for quantification of IIIM-019 (a novel nitroimidazole derivative with potential activity against Tuberculosis) in mice plasma. The extraction of the analyte and the internal standard (Tolbutamide) from the plasma samples involves protein precipitation using acetonitrile. The chromatographic separation was accomplished using a gradient mode and the mobile phase comprised of acetonitrile and 0.1% formic acid in water. The flow rate used was 0.7 ml/min on a C18e high performance Chromolith column. IIIM-019 and Tolbutamide (IS) were analyzed by combined reversed-phase LC/MS-MS with positive ion electrospray ionization. The MS-MS ion transitions used were 533>170.1, 533>198 for IIIM-019 and 271>74, 271>155 for internal standard (IS) respectively. The method was linear over a concentration range of 0.5-1000 ng/ml and the lower limit of quantification was 0.50 ng/ml. The entire study was validated for accuracy, precision, linearity, range, selectivity, lower limit of quantification (LLOQ), recovery, and matrix effect in accordance with the FDA guidelines of method validation. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The intra and inter-day precisions were in the range of 0.51-11.18% and 0.51-7.55%. The pharmacokinetics was performed on male Balb/c mice by oral (2.5mg/kg), intraperitoneal (2.5mg/kg) and intravenous (1mg/kg) routes. The oral bioavailability of IIIM-019 was 51.6%. The method was also applied successfully in determining microsomal stability wherein the compound was found to be very slightly metabolized by rat liver microsomes.

Analogues of boswellic acids as inhibitors of pro-inflammatory cytokines TNF-α and IL-6.

A library of boswellic acid analogues were synthesized and tested for their anti-inflammatory potential on key inflammatory mediators, TNF-α and IL-6. The study led to the identification of lead compounds showing significant inhibition of the cytokines, TNF-α and IL-6 both in vitro and in vivo.

Elevated levels of vitamin D and deficiency of mannose binding lectin in dengue hemorrhagic fever.

BACKGROUND: Altered plasma concentrations of vitamin D and mannose binding lectin (MBL), components of innate immunity, have been shown to be associated with the pathogenesis of viral infections. The objective of the present study was to find out whether plasma concentrations of MBL and vitamin D are different in patients with dengue fever (DF) and dengue hemorrhagic fever (DHF). THE RESULTS: The plasma concentrations of vitamin D and MBL were assessed in 48 DF cases, 45 DHF cases and 20 apparently healthy controls using ELISA based methods. Vitamin D concentrations were found to be higher among both DF and DHF cases as compared to healthy controls (P < 0.005 and P < 0.001). Vitamin D concentrations were not different between DF and DHF cases. When the dengue cases were classified into primary and secondary infections, secondary DHF cases had significantly higher concentrations of vitamin D as compared to secondary DF cases (P < 0.050). MBL concentrations were not significantly different between healthy controls and dengue cases. MBL concentrations were observed to be lower in DHF cases as compared to DF cases (P < 0.050). Although MBL levels were not different DF and DHF cases based on immune status, the percentage of primary DHF cases (50%) having MBL levels lower than 500 ng/ml were less compared to primary DF cases (P = 0.038). CONCLUSIONS: The present study suggests that higher concentrations of vitamin D might be associated with secondary DHF while deficiency of MBL may be associated with primary DHF.

In silico identification of viper phospholipaseA2 inhibitors: validation by in vitro, in vivo studies.

Snake venom, particularly of vipers from the Indian subcontinent, contains Phospholipase A2 (PLA2) as one its constituents which is widely implicated in hemorrhagic, cardiac arrest and death. Development of inhibitors of the protein can facilitate the weakening or annihilation of the venom toxicity and save many human lives. In the present communication, our studies relate to the design and development of structure-based ligands as inhibitors of PLA2 of Viper venom. The study involves the computational approach towards evaluating a library of molecules comprising of natural products, and synthetic molecules through docking studies on the venom protein PDB ID: 1OXL (a dimer, available in the literature). In silico experiments have resulted in the identification of several of them as PLA2 inhibitors. The inhibitory effect of PLA2 by these compounds is attributed to a great extent to their interaction with the residues Phe 46 and Val47 of chain B of the target protein and hence these two residues are identified as the key contributor for the said activity. In order to validate the in silico findings, a selected panel of compounds have been tested by in vitro and in vivo experiments against the venom, which has led to the observance of significant corroboration between the wet lab and in silico findings, validating thereby the in silico approach used in the present study.

Detection of dengue-4 virus in pune, western india after an absence of 30 years--its association with two severe cases.

BACKGROUND: Difference in severity of dengue outbreaks has been related to virus serotype, genotype and clades within genotypes. Till the 1980 s, India and Sri Lanka reported low number of dengue hemorrhagic fever (DHF) cases despite circulation of all four serotypes of dengue virus (DENV). Since the 1990 s the occurrence of DHF has increased. The increase has been attributed to changes in virus lineage especially with regard to DENV-2 and DENV-3. DENV-1 has been associated with dengue fever (DF) outbreaks and DENV-4 reports have been rare. The emergence of DENV-4 was reported recently in 2003 in Delhi and in 2007 in Hyderabad. The last report of DENV-4 from Maharashtra was in 1975 from Amalner. RESULTS: We report on the detection of DENV-4 in Pune, Maharashtra after an absence of almost 30 years. Two cases were detected in 2009-10, serotyped by multiplex reverse transcriptase polymerase chain reaction (RT-PCR). Both the cases were recorded as severe dengue (Category 3) requiring intensive care unit (ICU) level of treatment. Depending on the hemagglutination inhibiting (HI) antibody titres the 2009 case was characterized as a primary infection and the 2010 case as a secondary infection. Both the cases presented plasma leakage and neither showed any kind of haemorrhage. The 2009 case survived while the 2010 case was fatal. An isolate was obtained from the 2009 case. Based on envelope (E) gene sequence analysis, the virus belonged to genotype I of DENV-4, and clustered with isolates from India and Sri Lanka and was distant from the isolates from Thailand. The nucleotide and amino acid diversity of the E gene of the Indian isolates increased from 1996 to 2007 to 2009 in context of the E gene sequences of other isolates belonging to genotype I. CONCLUSION: The increasing diversity in the circulating DENV-4 calls for close monitoring of the DENV-4 serotype.

Clinical findings and pro-inflammatory cytokines in dengue patients in Western India: a facility-based study.

BACKGROUND: Descriptions of dengue immunopathogenesis have largely relied on data from South-east Asia and America, while India is poorly represented. This study characterizes dengue cases from Pune, Western India, with respect to clinical profile and pro-inflammatory cytokines. METHODOLOGY/PRINCIPAL FINDINGS: In 2005, 372 clinically suspected dengue cases were tested by MAC-ELISA and RT-PCR for dengue virus (DENV) aetiology. The clinical profile was recorded at the hospital. Circulating levels of IFN-gamma, TNF-alpha, IL-6, and IL-8 were assessed by ELISA and secondary infections were defined by IgM to IgG ratio. Statistical analysis was carried out using the SPSS 11.0 version. Of the 372 individuals, 221 were confirmed to be dengue cases. Three serotypes, DENV-1, 2 and 3 were co-circulating and one case of dual infection was identified. Of 221 cases, 159 presented with Dengue fever (DF) and 62 with Dengue hemorrhagic fever (DHF) of which six had severe DHF and one died of shock. There was a strong association of rash, abdominal pain and conjunctival congestion with DHF. Levels of IFN-gamma were higher in DF whereas IL-6 and IL-8 were higher in DHF cases (p<0.05). The mean levels of the three cytokines were higher in secondary compared to primary infections. Levels of IFN-gamma and IL-8 were higher in early samples collected 2-5 days after onset than late samples collected 6-15 days after onset. IFN-gamma showed significant decreasing time trend (p = 0.005) and IL-8 levels showed increasing trend towards significance in DHF cases (interaction p = 0.059). There was a significant association of IL-8 levels with thrombocytopenia and both IFN-gamma and IL-8 were positively associated with alanine transaminase levels. CONCLUSIONS/SIGNIFICANCE: Rash, abdominal pain and conjunctival congestion could be prognostic symptoms for DHF. High levels of IL-6 and IL-8 were shown to associate with DHF. The time trend of IFN-gamma and IL-8 levels had greater significance than absolute values in DHF pathogenesis.