Transplantation of mesenchymal stem cells genetically engineered to overexpress interleukin-10 promotes alternative inflammatory response in rat model of traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) is a major cause for long-term disability, yet the treatments available that improve outcomes after TBI limited. Neuroinflammatory responses are key contributors to determining patient outcomes after TBI. Transplantation of mesenchymal stem cells (MSCs), which release trophic and pro-repair cytokines, represents an effective strategy to reduce inflammation after TBI. One such pro-repair cytokine is interleukin-10 (IL-10), which reduces pro-inflammatory markers and trigger alternative inflammatory markers, such as CD163. In this study, we tested the therapeutic effects of MSCs that were engineered to overexpress IL-10 when transplanted into rats following TBI in the medial frontal cortex. METHODS: Thirty-six hours following TBI, rats were transplanted with MSCs and then assessed for 3 weeks on a battery of behavioral tests that measured motor and cognitive abilities. Histological evaluation was then done to measure the activation of the inflammatory response. Additionally, immunomodulatory effects were evaluated by immunohistochemistry and Western blot analyses. RESULTS: A significant improvement in fine motor function was observed in rats that received transplants of MSCs engineered to overexpress IL-10 (MSCs + IL-10) or MSCs alone compared to TBI + vehicle-treated rats. Although tissue spared was unchanged, anti-inflammatory effects were revealed by a reduction in the number of glial fibrillary acidic protein cells and CD86 cells in both TBI + MSCs + IL-10 and TBI + MSC groups compared to TBI + vehicle rats. Microglial activation was significantly increased in the TBI + MSC group when compared to the sham + vehicle group. Western blot data suggested a reduction in tumor necrosis factor-alpha in the TBI + MSCs + IL-10 group compared to TBI + MSC group. Immunomodulatory effects were demonstrated by a shift from classical inflammation expression (CD86) to an alternative inflammation state (CD163) in both treatments with MSCs and MSCs + IL-10. Furthermore, co-labeling of both CD86 and CD163 was detected in the same cells, suggesting a temporal change in macrophage expression. CONCLUSIONS: Overall, our findings suggest that transplantation of MSCs that were engineered to overexpress IL-10 can improve functional outcomes by providing a beneficial perilesion environment. This improvement may be explained by the shifting of macrophage expression to a more pro-repair state, thereby providing a possible new therapy for treating TBI.

Prompt diagnosis of Scedosporium apiospermum soft tissue infection: Life-saving in a renal transplant recipient.

Scedosporium apiospermum, an ubiquitous filamentous fungus, a known cause of mycetoma, is emerging as an opportunistic pathogen in immunocompromised individuals. We report a case of painful foot abscess in a renal allograft recipient on immunosuppressive therapy, which was clinically diagnosed as a suppurative bacterial abscess. Pus was aspirated, which showed septate, branching hyphal elements and culture on Sabouraud's dextrose agar yielded S. apiospermum, which was identified based on its macroscopic and microscopic features. There are very few reports of scedosporiasis from India. High index of suspicion for unusual fungal infection helps in prompt etiological diagnosis in a transplant recipient and rapid management prevents further dissemination.

Targeted deletion of GD3 synthase protects against MPTP-induced neurodegeneration.

Parkinson's disease is a debilitating neurodegenerative condition for which there is no cure. Converging evidence implicates gangliosides in the pathogenesis of several neurodegenerative diseases, suggesting a potential new class of therapeutic targets. We have shown that interventions that simultaneously increase the neuroprotective GM1 ganglioside and decrease the pro-apoptotic GD3 ganglioside - such as inhibition of GD3 synthase (GD3S) or administration of sialidase - are neuroprotective in vitro and in a number of preclinical models. In this study, we investigated the effects of GD3S deletion on parkinsonism induced by 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP was administered to GD3S-/- mice or controls using a subchronic regimen consisting of three series of low-dose injections (11 mg/kg/day × 5 days each, 3 weeks apart), and motor function was assessed after each. The typical battery of tests used to assess parkinsonism failed to detect deficits in MPTP-treated mice. More sensitive measures - such as the force-plate actimeter and treadmill gait parameters - detected subtle effects of MPTP, some of which were absent in mice lacking GD3S. In wild-type mice, MPTP destroyed 53% of the tyrosine-hydroxylase (TH)-positive neurons in the substantia nigra pars compacta (SNc) and reduced striatal dopamine 60.7%. In contrast, lesion size was only 22.5% in GD3S-/- mice and striatal dopamine was reduced by 37.2%. Stereological counts of Nissl-positive SNc neurons that did not express TH suggest that neuroprotection was complete but TH expression was suppressed in some cells. These results show that inhibition of GD3S has neuroprotective properties in the MPTP model and may warrant further investigation as a therapeutic target.

Evaluation of the role of substrate and albumin on Pseudomonas aeruginosa biofilm morphology through FESEM and FTIR studies on polymeric biomaterials.

Bacterial biofilms pose the greatest challenge to implant surgeries leading to device-related infections and implant failure. Our present study aims at monitoring the variation in the biofilm architecture of a clinically isolated strain and ATCC 27853 strain of Pseudomonas aeruginosa on two polymeric biomaterials, used in implants. The perspective of our study is to recognize the potential of these two biomaterials to create biofilm infections and develop the understanding regarding their limitations of use and handle patients with this deeper insight. The final goal, however, is an accurate interpretation of substrate-microbe interactions in the two biomaterials, which will provide us the knowledge of possible surface modifications to develop of an efficacious anti-biofilm therapy for deterring implant infections. The reference strain ATCC 27853 and a clinical isolate of P. aeruginosa collected from urinary catheters of patients suffering from urinary tract infections, have been used as microbes while clinical grades of polypropylene and high density polyethylene, have been used as 'substrates' for biofilm growth. The variation in the nature of the 'substrate' and 'conditioning layer' of BSA have been found to affect the biofilm architecture as well as the physiology of the biofilm-forming bacteria, accompanied by an alteration in the nature and volume of EPS (extracellular polysaccharide) matrices.

Use of an Innovative Simple Method for Anaerobiosis in the Diagnosis and Management of Infections in Two Unusual Cases.

Technical limitations restrict routine anaerobe isolation from clinical materials in resource-limited laboratories. An innovative two steps combustion candle jar technique may be suitable for such setup. This system was tried with one case of chronic osteomyelitis developed on supracondyler compound fracture. Porphyromonas spp. was isolated and identified. Vancomycin was recommended based on in vitro sensitivity test, but the leg was amputed after receiving a resistant drug gentamycin. While in another child with hydrocephalous, V-P shunt associated infection by Peptostreptococcus anaerobius was successfully controlled by sensitive drug vancomycin. These two eye-opener cases insisted us for large scale application of the technique.

Anaerobic culture on growth efficient bi-layered culture plate in a modified candle jar using a rapid and slow combustion system.

Success for maximum isolation of anaerobes depends on maintaining critically low oxygen levels throughout and growth in a reduced medium with exclusion of inhibitory substances. Hence a dual system was used equipped with candle combustion for instant exhaustion of major part of oxygen from a sealed jar, along with acidified steel wool for residual oxygen purging. For inhibitory substances removal, test anaerobes were grown on anaerobic medium layered on buffer charcoal agar bed. After 48 hours incubation average colony sizes were compared with that of growths in conventional Gas-Pak system. Better growths were noted in the innovative system.

Standardized ileal amino acid digestibility in egg from hyperimmunized hens fed to weaned pigs.

The study was conducted to determine the apparent ileal digestibility (AID) and standardized ileal digestibility (SID) of AA in egg from hens hyperimmunized with Escherichia coli K88 antigens (EGG) fed to weaned pigs. Spray dried porcine plasma (SDPP) was included for comparison. Eight barrows (Yorkshire-Landrace × Duroc; initial BW of 17 ± 1 kg) were fitted with a T-cannula at the distal ileum and fed 2 diets in a completely randomized design to give 4 replicates per diet. The diets were corn (Zea mays) starch based with either EGG or SDPP as the sole source of protein and were formulated to contain 130 g/kg CP. At the end of the study, a 50 g/kg casein diet was fed to all pigs (n = 8) to quantify endogenous N and AA losses to determine SID. Titanium dioxide (3 g/kg) was included in the diets as an indigestible maker. Each period lasted for 7 d. Pigs were adapted to their respective diets for 5 d followed by 12 h of continuous ileal digesta collection on days 6 and 7. Daily feed allowance was set at 4% BW at the beginning of each period and offered in 2 equal portions at 0800 and 1600 h as a dry mash. Pigs had unlimited access to water via low pressure nipple drinkers. The AID (%) of CP and indispensable AA were lower (P < 0.05) in EGG compared with SDPP. The SID (%) of CP (82 vs. 96) and indispensable AA were lower (P < 0.05) in pigs fed EGG compared with SDPP. In conclusion, the average AID and SID of N and indispensable AA in EGG are lower than in SDPP when fed at high levels.

SEMEDS: an important tool for air pollution bio-monitoring.

Plant canopies act as absorbers of air-borne dust particles. Characterization of the dusts present over the leaf surfaces can indicate the nature of contaminant present in the surrounding area and possible sources as well. Dust particulates get adsorbed on both the surfaces of leaves, however more dust particulates get deposited on the upper surface. These dusts contain many inorganic elements, which were analyzed in the present study. For the present investigation, SEMEDS technique used to characterize the dusts adsorbed over the leaf surfaces. Using SEMEDS the samples were analyzed by two types of methods i.e. point analysis and elemental mapping. Both the methods showed the presence of elements like Si, Al, Fe, Mg, and Ca in the dusts adsorbed over the leaf surfaces. Thus, SEMEDS can be used for in situ air pollution monitoring using tree leaves from the area concerned.

Interaction of single-walled carbon nanotubes with poly(propyl ether imine) dendrimers.

We study the complexation of nontoxic, native poly(propyl ether imine) dendrimers with single-walled carbon nanotubes (SWNTs). The interaction was monitored by measuring the quenching of inherent fluorescence of the dendrimer. The dendrimer-nanotube binding also resulted in the increased electrical resistance of the hole doped SWNT, due to charge-transfer interaction between dendrimer and nanotube. This charge-transfer interaction was further corroborated by observing a shift in frequency of the tangential Raman modes of SWNT. We also report the effect of acidic and neutral pH conditions on the binding affinities. Experimental studies were supplemented by all atom molecular dynamics simulations to provide a microscopic picture of the dendrimer-nanotube complex. The complexation was achieved through charge transfer and hydrophobic interactions, aided by multitude of oxygen, nitrogen, and n-propyl moieties of the dendrimer.

Influence of stabilizers on quality of sandesh from buffalo milk.

Buffalo milk standardized to solids-not-fat (SNF) to fat ratio of 1.4 was added separately with 0.1% (w/w) each of carrageenan, sodium alginate and carboxymethyl cellulose and then heated, cooled and coagulated to obtain chhana which was converted into sandesh by adding 1.5% (w/w) wheat flour and 25% (w/w) cane sugar followed by heating (40 min/kg chhana). The treated samples of sandesh were compared with control prepared similarly manner but without stabilizer. Addition of stabilizer decreased hardness, fracturability, adhesiveness, cohesiveness, gumminess and chewiness of sandesh and improved sensory body and texture, colour and appearance as well as overall acceptability of the product when compared with control. Textural and sensory properties of different samples of sandesh indicated that the product made by adding carrageenan proved best. Carrageenan at 0.1% produced better results in terms of textural and sensory profile of sandesh as compared to 0, 0.075 and 0.125% (w/w) of carrageenan.

Chitosan coated PLA nanoparticles for ophthalmic delivery: characterization, in-vitro and in-vivo study in rabbit eye.

The objective of the present study is to develop a poly (D, L-lactic acid) (PLA) nano-carrier for topical ocular applications. PLA nanoparticles (PLA-NPs) with 5-fluorouracil were prepared using varying concentration and molecular weight of PLA to regulate the particle size. The dimension and shape of nanoparticles were verified by using dynamic light scattering (DLS), atomic force microscope (AFM) and scanning electron microscope (SEM). Ex-vivo permeation study was conducted by goat and rabbit excised cornea. In-vivo experiment was conducted in rabbit eye and 5-FU concentration was measured in aqueous and vitreous humor by HPLC. In-vitro experiments indicated a diffusion controlled release of 5-FU. No significant interaction was observed in between mucin and PLA NPs that measured in terms of viscosity change. Ex-vivo permeation was significantly higher with rabbit cornea as compared to goat cornea. PLA and CH-PLA DNPs showed increased level of 5-FU as comparison to 5-FU solution. In-vivo study showed significantly higher concentration in case of uncoated and CH coated PLA nanoparticles in rabbit eye as compared to free 5-FU solution. PLA nanoparticle was found non-irritant in nature by modified Draize test.

Polymeric nanoparticulate system: a potential approach for ocular drug delivery.

Various efforts in ocular drug delivery have been made to improve the bioavailability and to prolong the residence time of drugs applied topically onto the eye. The potential use of polymeric nanoparticles as drug carriers has led to the development of many different colloidal delivery vehicles. Drug loaded polymeric nanoparticles (DNPs) offer several favorable biological properties, such as biodegradability, nontoxicity, biocompatibility and mucoadhesiveness. These submicron particles are better than conventional ophthalmic dosage forms to enhance bioavailability without blurring the vision. DNPs have been shown to be amenable to targeting of the drug to the site of action, leading to a decrease in the dose required and a decrease in side effects. Additionally, DNPs can be fabricated by simple techniques with better physical stability than liposomes. This unique combination of properties makes DNPs a novel polymeric drug delivery device, which fulfils the requirements for ophthalmic application. This review discusses the polymeric nanoparticles, physiochemical characterization, fabrication techniques, therapeutic significances, patented technology of nanoparticles and future possibility in the field of ocular drug delivery.

Avian- and mammalian-derived antibodies against adherence-associated proteins inhibit host cell colonization by Escherichia coli O157:H7.

AIM: To evaluate the potential for polyclonal antibodies targeting enterohaemorrhagic Escherichia coli (EHEC) virulence determinants to prevent colonization of host cells by E. coli O157:H7. METHODS AND RESULTS: Rats and laying hens were immunized with recombinant proteins from E. coli O157:H7, EspA, C-terminal intimin or EscF. Rat antisera (IgG) or chicken egg powders (IgY) were assessed for their ability to inhibit growth and colonization-associated processes of E. coli O157:H7. Mammalian antisera with antibodies to intimin, EspA or EscF effectively reduced adherence of the pathogen to HeLa cells (P<0.05) and prevented type III secretion of Tir. Similarly, HeLa cells treated with chicken egg powder containing antibodies against intimin or EspA were protected from EHEC adherence (P<0.05). Neither egg nor rat antibody preparations had any antibacterial effect on the growth of EHEC (P>0.05). CONCLUSIONS: Antibody preparations targeting EHEC adherence-associated factors were effective at preventing adhesion and intimate colonization-associated events. SIGNIFICANCE AND IMPACT OF THE STUDY: This work indicates that immunotherapy with anti-adherence antibodies can reduce E. coli O157:H7 colonization of host cells. Passive immunization with specific antibodies may have the potential to reduce E. coli O157:H7 colonization in hosts such as cattle or humans.

Mycetoma caused by a new red grain mycetoma agent in two members of a family.

An 18-year-old woman from rural West Bengal was affected with mycetoma involving her neck, back, and chest. After an interval of eight years, her younger brother developed mycetoma on his left arm. No history of trauma or immune deficiency was present in either case. By microscopic examination of sinus-discharged materials from both the cases, identical rusty red, hard grains were demonstrated. Soluble red pigment-producing colonies grew in Sabouraud dextrose-agar medium. Isolates were positive for casein hydrolysis and negative for hydrolysis test of xanthine, hypoxanthine, tyrosine, and nitrate reduction. Thus it differed from the only known red grain mycetoma agent, Actinomadura pelletieri and was provisionally identified as Actinomadura vinacea. Familial affection in mycetoma, that too caused by a new agent, is reported here for its uniqueness.

Epidemiological aspects of mycetoma from a retrospective study of 264 cases in West Bengal.

Between 1981 and 2000, 264 cases of mycetoma were diagnosed clinically and microbiologically at Calcutta School of Tropical Medicine. Retrospective analysis of the records revealed that the ratio of actinomycetomas and eumycetomas was 197 : 67; the male to female ratio was 183 : 81. Ninety-four cases occurred in the 1980s and 170 in 1990s, with significantly more infections of Actinomadura spp. (P < 0.01) and fewer with Nocardia caviae (P < 0.01) during the last decade. Pricking was the most common injury associated with eumycetomas (P < 0.01). A total of 196 infections were in exposed body parts and 68 in covered areas. The localization of mycetomas differed significantly (P < 0.01) according to sex, incidence of actinomycetomas or eumycetomas, and obvious history of trauma. Exposed area cases were more common among agricultural workers (P < 0.01), while covered area mycetomas were almost always actinomycetomas with a remarkably lower incidence of N. caviae, A. madurae and Madurella grisea infections. The peak age of onset was between 16 and 25 years. The delay of diagnosis for the 80th percentile of cases was around 6 years for cases caused by N. brasiliensis and Streptomyces spp.; 8 years for N. caviae and N. asteroides; and 10 years for M. grisea and Actinomadura spp. From the history of trauma in 130 patients, the 80th percentile incubation period (IP) was calculated for N. brasiliensis, N. caviae and N. asteroides as 3 years; for Actinomadura spp. 7 years and for M. grisea 9 years. The minimum IP for all organisms was around 3 months.

Suppression of experimental myasthenia gravis, a B cell-mediated autoimmune disease, by blockade of IL-18.

Interleukin-18 (IL-18) is a pleiotropic proinflammatory cytokine that plays an important role in interferon gamma (IFN-gamma) production and IL-12-driven Th1 phenotype polarization. Increased expression of IL-18 has been observed in several autoimmune diseases. In this study we have analyzed the role of IL-18 in an antibody-mediated autoimmune disease and elucidated the mechanisms involved in disease suppression mediated by blockade of IL-18, using experimental autoimmune myasthenia gravis (EAMG) as a model. EAMG is a T cell-regulated, antibody-mediated autoimmune disease in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen. Th1- and Th2-type responses are both implicated in EAMG development. We show that treatment by anti-IL-18 during ongoing EAMG suppresses disease progression. The protective effect can be adoptively transferred to naive recipients and is mediated by increased levels of the immunosuppressive Th3-type cytokine TGF-beta and decreased AChR-specific Th1-type cellular responses. Suppression of EAMG is accompanied by down-regulation of the costimulatory factor CD40L and up-regulation of CTLA-4, a key negative immunomodulator. Our results suggest that IL-18 blockade may potentially be applied for immunointervention in myasthenia gravis.

Rapid imaging of human melanoma xenografts using an scFv fragment of the human monoclonal antibody H11 labelled with 111In.

H11 is a human IgM monoclonal antibody which recognizes a novel tumour-associated antigen expressed on melanoma, glioma, breast cancer, colon cancer, prostate cancer, lung cancer and B-cell lymphoma. In this study, a recombinant single-chain Fv (scFv) fragment of H11 labelled with 111In was investigated for tumour imaging in athymic mice implanted subcutaneously with A-375 human melanoma xenografts. H11 scFv was derivatized with diethylenetriaminepentaacetic acid (DTPA) for labelling with 111In. The immunoreactivity of DTPA-H11 scFv against A-375 cells in vitro ranged from 23% to 36%. 111In-DTPA-H11 scFv was rapidly eliminated from the blood and most normal tissues (except the kidneys) reaching maximum tumour/blood ratios of 12:1 at 48 h post-injection. Tumours were imaged as early as 40 min after injection. The kidneys accumulated the highest concentration of radioactivity (up to 185% injected dose/g). Tumour uptake was 1-3% injected dose/g. The whole-body radiation absorbed dose predicted for administration of 185 MBq of 111In-DTPA-H11 scFv to humans was 37 mSv. The radiation absorbed dose estimates for the kidneys, spleen and intestines were 405 mSv, 698 mSv and 412 mSv, respectively. The results of this preclinical study and a concurrent phase I trial suggest a promising role for H11 scFv for tumour imaging.