P. Routray

Targeting nonhealing ulcers of lower extremity in human through autologous bone marrow-derived mesenchymal stem cells.

Bone marrow (BM)-derived mesenchymal stem cells (MSCs) represent a promising population for supporting new concepts in cellular therapy. This study was undertaken to assess the efficacy and feasibility of autologous BM-derived MSCs in the treatment of chronic nonhealing ulcers (diabetic foot ulcers and Buerger disease) of the lower extremities. A total of 24 patients with nonhealing ulcers of the lower limb were enrolled and randomized into implant and control groups. In the implant group, the patients received autologous cultured BM-derived MSCs along with standard wound dressing; the control group received only the standard wound dressing regimen, followed up for at least a 12-week period. Wound size, pain-free walking distance, and biochemical parameters were measured before therapy and at every 2-week interval following intervention. The implant group had significant improvement in pain-free walking distance and reduction in ulcer size as compared to those in the control group. In the implant group for Buerger disease, the ulcer area decreased from 5.04 +/- 0.70 cm(2) to 1.48 +/- 0.56 cm(2) (p < 0.001), whereas the pain-free walking distance increased from 38.33 +/- 17.68 m to 284.44 +/- 212.12 m (p < 0.001). In the diabetic foot ulcer group, the ulcer size decreased from 7.26 +/- 1.41 cm(2) to 2 +/- 0.98 cm(2) (p < 0.001) at 12 weeks. Mononuclear cells were cultured for a minimum of five passages and characterized by cell-surface markers showing CD90+, CD105+, and CD34(-). There was no significant alteration in the biochemical parameters observed during the follow-up period, indicating normal liver and renal function following intervention. Biopsy microsection of implanted tissues showed development of dermal cells (mainly fibroblasts), including mature and immature inflammatory cells. The study indicates that autologous implantation of BM-derived MSCs in nonhealing ulcers accelerates the healing process and improves clinical parameters significantly.

Inductive expression of toll-like receptor 5 (TLR5) and associated downstream signaling molecules following ligand exposure and bacterial infection in the Indian major carp, mrigal (Cirrhinus mrigala)

Toll-like receptors (TLRs) are one of the key components of innate immunity. Among various types of TLRs, TLR5 is involved in recognizing bacterial flagellin and after binding, it triggers myeloid differentiation primary response gene 88 (MyD88)-dependent signaling pathway to induce pro-inflammatory cytokines. In this report, we analyzed the expression profile of TLR5 and its associated downstream signaling molecules like MyD88 and tumor necrosis factor (TNF) receptor-associated factor (TRAF) 6 in the Indian major carp (IMC), mrigal (Cirrhinus mrigala) which is highly commercially important fish species in the Indian subcontinent. Ontogeny analysis of TLR5, MyD88 and TRAF6 revealed constitutive expression of these genes in all embryonic developmental stages, and highlighted the importance of embryonic innate immune defense system in fish. Tissue specific expression analysis of these genes by quantitative real-time PCR (qRT-PCR) revealed their wide distribution in various organs and tissues; highest expression of TLR5 and MyD88 was in liver and TRAF6 was in kidney. Modulation of TLR5, MyD88 and TRAF6 gene expression, and the induction of interleukin (IL)-8 and TNF-α were analyzed in various organs by qRT-PCR following flagellin stimulation, and Aeromonas hydrophila and Edwardsiella tarda infection. In the treated fish, majority of the tested tissues exhibited significant induction of these genes, although with varied intensity among the tissues and with the types of treatments. Among the examined tissues, a significant relationship of TLR5 induction, MyD88 and TRAF6 up-regulation, and enhanced expression of IL-8 and TNF-α gene transcripts was observed in the blood and intestine of both flagellin stimulated and bacteria infected fish. These findings may indicate the involvement of TLR5 in inducing IL-8 and TNF-α, and suggest the important role of TLR5 in augmenting innate immunity in fish in response to pathogenic invasion. This study will enrich the information in understanding the innate immune mechanism in fish and may be helpful in developing preventive measures against infectious diseases in fish.

Parenteral immunization of fish, Labeo rohita with Poly D, L-lactide-co-glycolic acid (PLGA) encapsulated antigen microparticles promotes innate and adaptive immune responses.

Immunogenicity of different antigen preparations of outer membrane proteins (OMP) of Aeromonas hydrophila such as Poly d, l-lactide-co-glycolic acid (PLGA) microparticles, oil emulsion, neat OMP and bacterial whole cells were compared through intra-peritoneal injection in fish, Labeo rohita. Among these preparations, PLGA encapsulated antigen stimulated both innate and adaptive immune parameters and the immunogenicity exhibited by PLGA microparticles was significantly higher (p < 0.05) at both 21 and 42 days post-immunization suggesting that the above delivery system would be a novel antigen carrier for parenteral immunization in fish, Labeo rohita.

Molecular characterization of nucleotide binding and oligomerization domain (NOD)-2, analysis of its inductive expression and down-stream signaling following ligands exposure and bacterial infection in rohu (Labeo rohita)

Nucleotide-binding and oligomerization domain (NOD)-2 is a cytoplasmic pattern recognition receptor (PRR) and is a member of NOD like receptor (NLR) family. It senses a wide range of bacteria and viruses or their products and is involved in innate immune responses. In this report, NOD-2 gene was cloned and characterized from rohu (Labeo rohita) which is highly commercially important fish species in the Indian subcontinent. The full length rohu NOD-2 (rNOD-2) cDNA comprised of 3176 bp with a single open reading frame (ORF) of 2949 bp encoding a polypeptide of 982 amino acids (aa) with an estimated molecular mass of 109.65 kDa. The rNOD-2 comprised two N-terminal CARD domains (at 4-91 aa and 111-200 aa), one NACHT domain (at 271-441 aa) and seven C-terminal leucine rich repeat (LRR) regions. Phylogenetically, rNOD-2 was closely related to grass carp NOD-2 (gcNOD2) and exhibited significant similarity (94.2%) and identity (88.6%) in their amino acids. Ontogeny analysis of rNOD-2 showed its constitutive expression across the developmental stages, and highlighted the embryonic innate defense system in fish. Tissue specific analysis of rNOD-2 by quantitative real-time PCR (qRT-PCR) revealed its wide distribution; highest expression was in liver followed by blood. In response to PGN and LTA stimulation, Aeromonas hydrophila and Edwardsiella tarda infection, and poly I:C treatment, expression of rNOD-2 and its associated downstream molecules RICK and IFN-γ were significantly enhanced in the treated fish compared to control. These findings suggested the key role of NOD-2 in augmenting innate immunity in fish in response to bacterial and viral infection. This study may be helpful for the development of preventive measures against infectious diseases in fish.

Recent advances in carp seed production and milt cryopreservation

The fish-seed production industry in India has recorded remarkable growth over the last three decades. The hypophysation technique was successfully introduced into India in 1957 and steady progress towards the refinement of the technique has been registered, which has revolutionized carp seed production in the Indian subcontinent. Advancement of carp maturity through brood stock management and multiple breeding has enabled spawn production well ahead of the monsoon and even beyond, ensuring seed availability throughout the year. The quality of seed is an important consideration for commercial aquaculturists. So, partial stock replenishment in carp hatcheries is practised to overcome the problem of inbreeding, which otherwise leads to poor growth of carps. Similarly, gametes of improved stock are cryopreserved and utilized for quality seed production as well as upgrading the brood stock of carps. The gamete cryopreservation protocol for carps is the focus of this review. An attempt has also been made to incorporate information on carp brood stock management, inducing agents, and seed production, including hatchery management.

Use of the Non-Toxic Cryoprotectant Trehalose Enhances Recovery and Function of Fish Embryonic Stem Cells Following Cryogenic Storage

Fish embryonic stem (ES) cells derived from of blastulae (64 cell stage embryo) of Labeo rohita were propagated in culture and retained their ES cell-like properties after cryogenic storage (-196 degrees C, i.e., liquid nitrogen). Toxic effect of DMSO (dimethyl sulphoxide) on stem cells during preservation process has been reported to restrict therapeutic applications. In this study we reduced the concentration of DMSO and added the non-toxic cryoprotective agent (CPA) trehalose. Cryopreservation of ES cell colonies was done at 5, 25 and 52 passages with 0.2 M trehalose and 0.8 M (DMSO). A combination of both the cryoprotective agents (non-toxic and toxic) demonstrated better survival and recovery of ES cells than the DMSO used alone. Use of this CPA combination in the freezing media gave an optimum viability of more than 83 % in a slow freezing protocol. Trehalose showed a definite advantage over DMSO in terms of viability and intactness of ES cell colonies with evenly distributed morphology. There was no significant difference observed in the expression levels of cell surface markers like stage specific embryonic antigen-1 (SSEA-1) and alkaline phosphatase (ALP) between early and late passages after 60 days of post-thawing. More than 90 % of the ES cell colonies showed extensive expression of ALP and positive expression of SSEA-1 from an early stage of ES cells culture up to passage 52 (in our study) in the presence of leukemia inhibitory factor (LIF) and without feeder cells. Further, thawed ES cells showed a normal karyotype and maintained an undifferentiated state through out the study. This study on ES cell cryopreservation and subsequent retention of stem cell properties without feeder cells using a non-toxic cryoprotectant trehalose would be highly useful for future in vitro differentiation, manipulation of fish ES cells and as a model for mammalian ES cell culture.

Derivation and characterization of embryonic stem-like cells of Indian major carp Catla catla

Embryonic stem (ES)-like cells were derived from mid-blastula stage embryos of a freshwater fish, catla Catla catla, under feeder-free condition and designated as CCES cells. The conditioned media was optimized with 10% foetal bovine serum (FBS), fish embryo extract (FEE) having 100 µg ml(-1) protein concentration, 15 ng ml(-1) basic fibroblast growth factor (bFGF) and basic media containing Leibovitz-15, DMEM with 4·5 g l(-1) glucose and Hams F12 (LDF) in 2:1:1 ratio using a primary culture of CCES cells. Cells attached to gelatin-coated plates after 24 h of seeding and ES-like colonies were obtained at day 5 onwards. A stable cell culture was obtained after passage 10 and further maintained up to passage 44. These cells were characterized by their typical morphology, high alkaline phosphatase activity, positive expression of cell-surface antigen SSEA-1, transcription factor Oct4, germ cell marker vasa and consistent karyotype up to extended periods. The undifferentiated state was confirmed by their ability to form embryoid bodies and their differentiation potential.

Effect of age and abstinence on semen quality: A retrospective study in a teaching hospital

Abstract Objective To elucidate the effect of age and sexual abstinence on semen quality (semen volume, total count, progressive motility, vitality and morphology). Methods A total of 730 semen samples were analyzed. Subjects were grouped according to the age (20-29, 30–34, 35–39 and 40–50) and abstinence (2–3, 4–5 and 6–7). Semen parameters were evaluated following WHO standard criteria. Results Analysis of 730 semen samples showed negative correlation of progressive motility ( r =-0.131, P r =-0.173, P r =-0.324, P H = 20.65, P H = 13.53, P H = 15.33, P U test confirmed the changes in semen volume, total count and vitality in paired grouping from 2–7 days ( P P Conclusions In the present study, age negatively affected progressive motility, vitality and morphology of human sperm. Semen samples showed intra varied results within WHO amended abstinence period.

Goat serum as an alternative to establish cell culture from Indian major carp, Cirrhinus mrigala.

Serum from goat, calf, and chicken sources were evaluated in terms of attachment, growth, and proliferation of explants of Indian major carp, Cirrhinus mrigala. The attachment of explants viz. heart, liver, and kidney was directly proportional to the concentration of the serum. Among these sera, the highest percentage of attachment, growth, and proliferation was recorded for 10% goat serum and 15% newborn calf serum without affecting their cell morphology. On contrary to these sera, chicken serum at 15% concentration was found to be mildly toxic for all the explants. The cell count was significantly high for the kidney, liver, and heart at 10% goat serum among all the tested sera as well as concentration. Similarly, the liver, heart, and kidney explants were found to survive up to the tenth, seventh, and ninth passage, respectively. Therefore, the goat serum at 10% concentration can be used as effectively as newborn calf serum for routine culture of fish cells.

Prevalence of abnormal spermatozoa in tobacco chewing sub‑fertile males

AIM: The aim of the following study is to find out the prevalence of abnormal spermatozoa and associated functional parameters in clinical semen samples of sub-fertile males with the tobacco chewing habit. SETTINGS AND DESIGN: Retrospective study was conducted at infertility unit of a tertiary health care center, in a period of 3 years. MATERIALS AND METHOD: Semen of 642 males were analyzed; of them 194 men (30.2%) were tobacco chewers and they were grouped according to their intensity of chewing (<10 and ≥ 10 packets/day). Counts, motility, vitality, and morphology of sperms were analyzed. RESULTS: In tobacco chewers, 66% of subjects were oligozoospermic, 85% asthenozoospermic and 28% teratozoospermic. Sperm counts (odds ratio [OR] =2.2; 95% confidence interval [CI]: 1.5-3.09), motility (OR = 3.2; 95% CI: 2.05-4.9), and normal morphology (OR = 8.4; 95% CI: 4.9-14.6) were significantly affected (P = 0.001) in tobacco chewers than the non-chewing group. Further, in comparison to the intensity of tobacco chewing, patients with the intensive practice of using ≥10 packets/day had a significant effect on sperm morphology (P = 0.003, OR = 2.7; 95% CI = 1.41-5.08) only. Structural defects in head (P = 0.001) and cytoplasmic residues (P = 0.001) were found to be positively correlated with the intensive chewing, but no significant changes were found in anomalies in mid-piece and tail. CONCLUSION: The adverse impact of tobacco chewing on semen parameters was evident even with mild chewers, but with the intensive chewing practice, phenotypes of sperms, mainly defects in the head and cytoplasmic residue were severely affected.