CD34 cells in somatic, regenerative and cancer stem cells: Developmental biology, cell therapy, and omics big data perspective.

The transmembrane phosphoglycoprotein protein CD34 has conventionally been regarded as a marker for hematopoietic progenitors. Its expression on these cells has been leveraged for cell therapy applications in various hematological disorders. More recently, the expression of CD34 has also been reported on cells of nonhematopoietic origin. The list includes somatic cells such as endothelial cells, fibrocytes and interstitial cells and regenerative stem cells such as corneal keratocytes, muscle satellite cells, and muscle-derived stem cells. Furthermore, its expression on some cancer stem cells (CSCs) has also been reported. Till date, the functional roles of this molecule have been implicated in a multitude of cellular processes including cell adhesion, signal transduction, and maintenance of progenitor phenotype. However, the complete understanding about this molecule including its developmental origins, its embryonic connection, and associated functions is far from complete. Here, we review our present understanding of the structure and putative functions of the CD34 molecule based upon our literature survey. We also probed various biological databases to retrieve data related to the expression and associated molecular functions of CD34. Such information, upon synthesis, is hence likely to provide the suitability of such cells for cell therapy. Moreover, we have also covered the existing cell therapy and speculated cell therapy applications of CD34+ cells isolated from various lineages. We have also attempted here to speculate the role(s) of CD34 on CSCs. Finally, we discuss number of large-scale proteomics and transcriptomics studies that have been performed using CD34+ cells.

In Vitro Differentiation of Pluripotent Stem Cells into Functional ß Islets Under 2D and 3D Culture Conditions and In Vivo Preclinical Validation of 3D Islets.

Since the advent of pluripotent stem cells, (embryonic and induced pluripotent stem cells), applications of such pluripotent stem cells are of prime importance. Indeed, scientists are involved in studying the basic biology of pluripotent stem cells, but equal impetus is there to direct the pluripotent stem cells into multiple lineages for cell therapy applications. Scientists across the globe have been successful, to a certain extent, in obtaining cells of definitive endoderm and also pancreatic ß islets by differentiating human pluripotent stem cells. Pluripotent stem cell differentiation protocols aim at mimicking in vivo embryonic development. As in vivo embryonic development is a complex process and involves interplay of multiple cytokines, the differentiation protocols also involve a stepwise use of multiple cytokines. Indeed the novel markers for pancreas organogenesis serve as the roadmaps to develop new protocols for pancreatic differentiation from pluripotent stem cells. Earliest developed protocols for pancreas differentiation involved "Nestin selection pathway," a pathway common for both neuronal and pancreatic differentiation lead to the generation of cells that were a combination of cells from neuronal lineage. Eventually with the discovery of hierarchy of ß cell transcription factors like Pdx1, Pax4, and Nkx2.2, forced expression of such transcription factors proved successful in converting a pluripotent stem cell into a ß cell. Protocols developed almost half a decade ago to the recent ones rather involve stepwise differentiations involving various cytokines and could generate as high as 25 % functional insulin-positive cells in vitro. Most advanced protocols for ß islet differentiations from human pluripotent stem cells focused on 3D culture conditions, which reportedly produced 60-65 % functional ß islet cells. Here, we describe the protocol for differentiation of human pluripotent stem cells into functional ß cells under both 2D and 3D culture conditions.

Human embryonic stem cell differentiation into insulin secreting ß-cells for diabetes.

hESC (human embryonic stem cells), when differentiated into pancreatic ß ILC (islet-like clusters), have enormous potential for the cell transplantation therapy for Type 1 diabetes. We have developed a five-step protocol in which the EBs (embryoid bodies) were first differentiated into definitive endoderm and subsequently into pancreatic lineage followed by formation of functional endocrine ß islets, which were finally matured efficiently under 3D conditions. The conventional cytokines activin A and RA (retinoic acid) were used initially to obtain definitive endoderm. In the last step, ILC were further matured under 3D conditions using amino acid rich media (CMRL media) supplemented with anti-hyperglycaemic hormone-Glp1 (glucagon-like peptide 1) analogue Liraglutide with prolonged t(½) and Exendin 4. The differentiated islet-like 3D clusters expressed bonafide mature and functional ß-cell markers-PDX1 (pancreatic and duodenal homoeobox-1), C-peptide, insulin and MafA. Insulin synthesis de novo was confirmed by C-peptide ELISA of culture supernatant in response to varying concentrations of glucose as well as agonist and antagonist of functional 3D ß islet cells in vitro. Our results indicate the presence of almost 65% of insulin producing cells in 3D clusters. The cells were also found to ameliorate hyperglycaemia in STZ (streptozotocin) induced diabetic NOD/SCID (non-obese diabetic/severe combined immunodeficiency) mouse up to 96 days of transplantation. This protocol provides a basis for 3D in vitro generation of long-term in vivo functionally viable islets from hESC.

Revisiting curcumin chemistry part I: a new strategy for the synthesis of curcuminoids.

A new strategy for the synthesis of curcuminoids is described involving the reaction of acetylacetone difluroboronite with an aromatic aldehyde in the presence of n-butylamine as catalyst. The new intermediate products, curcuminoid difluroboronites, of symmetrically substituted curcuminoids like curcumin and bisdemethoxycurcumin are stable, can be isolated and hydrolysed with aq. methanol at pH 5.8 to get the curcuminoids of high purity. The method is applicable for unsymmetrical curcuminoids like demethoxycurcumin also with some modification involving column chromatography. The intermediate curcuminoid difluroboronites, as also the natural ß-diketone pongamol difluroboronite, prepared for the first time were characterized on the basis of physical and chemical properties and spectroscopic data. The advantage of using borontrifluoride to protect the enol group in acetylacetone over the generally used boric oxide is brought out. The importance of conducting biological activity studies using pure curcuminoids is explained.

Cerebral abscess caused by Aggregatibacter aphrophilus.

Aggregatibacter aphrophilus was previously known as Haemophilus aphrophilus and is a rare cause of disease in humans. A recent reclassification of these organisms has placed them in the new genus of Aggregatibacter species. The organism seems to be a normal component of oral flora and has been reported to cause endocarditis, sinusitis, pneumonia, empyema, soft tissue abscess, meningitis, vertebral discitis, and septic arthritis. Brain abscess due to Aggregatibacter is rare. We report a case of cerebral abscess due to Aggregatibacter aphrophilus and discuss the characteristics of this organism.

Investigation of anaphylactic reaction after patent blue V dye injection.

BACKGROUND: Patent blue dye V (PBV) is in widespread use for sentinel node biopsy in breast cancer and melanoma. At present, the best diagnostic approach in investigating possible anaphylaxis due to PBV is not defined. METHOD: We reviewed our experience of patients and the cases reported in the literature that developed an anaphylactic reaction after injection of PBV and suggest a diagnostic protocol. From May 2006 to April 2009 six patients were known to the Cardiff anaesthetics department to have suffered a severe anaphylactic reaction after injection of PBV. We amalgamated the results of the investigations of our patients with those of 42 case reports published in the literature during the last 10 years. RESULTS: Of 40 patients with a documented allergy history 31 patients did not have a past medical history of allergy. The median interval between PBV administration and allergic reaction was 15 min (range 1 min-180 min). Of 20 patients with hypotension 18 received inotropes. 4 patients had a fall in blood pressure as their sole symptom. 23 patients had urticaria or other allergic skin manifestations, 8 had blue wheals. 5 patients had bronchospasm. 2 patients had a cardiac arrest. They were successfully resuscitated. The median dose of PBV was 2 ml (range 0.5 ml-5 ml). Tryptase levels were elevated in 14 of 26 tested patients. Skin prick testing was positive in 24 of 30 tested patients. Intradermal testing was positive in all 13 tested patients. CONCLUSION: Most patients experiencing a severe allergic reaction to PBV have no past medical history of allergy. The value of formal allergy skin testing for PBV-related allergy lies in excluding other agents as the causative factor to avoid their exposure in the future.

HLA-G polymorphism patterns show lack of detectable association with recurrent spontaneous abortion.

Human leukocyte antigen-G (HLA-G) is a class I non-classical molecule that is predominantly expressed on the extravillous cytotrophoblasts at foetal-maternal interface during pregnancy. We recruited 143 recurrent spontaneous abortion (RSA) and 150 control couples for the study. DNA-based typing of the HLA-G was carried out to explore if we can validate the patterns of association reported elsewhere or find association of novel HLA-G alleles with RSA in the Indian population. We also evaluated the role of allele sharing in couples with RSA. We did not find association of any of the HLA-G alleles with RSA in our study. There is a general trend of increase in sharing among the RSA couples, but the increase is not significant. The results suggest that the HLA-G alleles or the allele sharing by couples may not play a significant role in the manifestation of RSA in the Indian context albeit more studies are required before making any definitive statement.

Fulminant varicella zoster infection with multiorgan involvement: a case report.

Varicella zoster infections are considered to be mild and ubiquitous infections predominantly affecting the paediatric population. However, in adults and in specific groups of patients, such as those who are immunosuppressed, varicella infections can be fulminant and life threatening. We here present a case report of a young female patient with a normal immune system who had a fulminant varicella infection with multiorgan involvement.

Comparison of the analgesic efficacy and respiratory effects of morphine, tramadol and codeine after craniotomy.

Pain after craniotomy remains a significant problem. The effect of morphine and tramadol patient-controlled analgesia (PCA) on arterial carbon dioxide tension is unknown in patients having such surgery. Sixty craniotomy patients were randomly allocated to receive morphine PCA, tramadol PCA or codeine phosphate 60 mg intramuscularly. Baseline values of pain score (0-10), sedation and arterial carbon dioxide tension were recorded at the time of first analgesic administration and at 30 min, 1, 4, 8, 12, 18 and 24 h. Patient satisfaction was assessed at 24 h. There were no differences in arterial carbon dioxide tension or sedation between groups at any time, but in all three groups some patients had rises greater than 1 kPa. Morphine produced significantly better analgesia than tramadol at all time points (p < 0.005) and better analgesia than codeine at 4, 12 and 18 h. Patients were more satisfied with morphine than with codeine or tramadol (p < 0.001). Vomiting and retching occurred in 50% of patients with tramadol, compared with 20% with morphine and 29% with codeine.

Predictive value of IL-18 and SC5b-9 for neurocognitive dysfunction after cardiopulmonary bypass.

BACKGROUND: Neurological injury after cardiopulmonary bypass (CPB) continues to be a major problem after cardiac surgery. The aim of this study was to investigate the predictive value of Interleukin-18 (IL-18) and SC5b-9 as biochemical markers of neurocognitive dysfunction after cardiac surgery. METHODS: A total of 30 patients undergoing elective cardiac surgery using CPB were recruited. Blood samples were obtained for IL-18 and SC5b-9 concentrations before induction, 24, 48, 72, 96 and 120 h post-CPB and 6 weeks after operation. In addition, patients underwent a standard battery of neuropsychometric tests before operation and at day 5 and 6 weeks after operation. RESULTS: Serum concentration of IL-18, but not SC5b-9, was significantly different between patients with and without neurocognitive dysfunction; serum IL-18 concentration significantly increased in patients with neurocognitive dysfunction (P = 0.018). Neurological outcome was significantly dependent on peak difference in IL-18 concentration at day 5 (P = 0.033), but not on peak difference in SC5b-9 concentration (P = 0.16). Eight patients had neurocognitive dysfunction at day 5 and three had neurocognitive dysfunction at 6 weeks. In a very small number of patients, no significant association was demonstrated between IL-18 or SC5b-9 concentrations and neurocognitive dysfunction at 6 weeks. CONCLUSIONS: IL-18 has the potential as a useful marker of neurological dysfunction, requiring further investigation.

Nicotinic acetylcholine receptors on basophils and mast cells.

Anaphylaxis in response to drugs administered during anaesthesia is a rare but potentially catastrophic event. The anaesthetic drugs most commonly associated with anaphylaxis are neuromuscular blocking agents. As these drugs act on the nicotinic acetylcholine receptor of the neuromuscular junction, potentiation of anaphylaxis by a nicotinic receptor on basophils and mast cells is plausible. The aim of this study was to investigate whether nicotinic acetylcholine receptors are present on a human basophil and mast cell lines as their presence may suggest a mechanism of associated anaphylaxis. Nicotinic receptors were demonstrated on a basophil and a mast cell line using an alpha-bungarotoxin-fluorescein conjugate by flow cytometry and by both conventional and confocal microscopic techniques. The identity of this receptor was confirmed by reverse transcriptase PCR and quantitative PCR.

Haemodynamic effects of the prone position: a comparison of propofol total intravenous and inhalation anaesthesia.

The haemodynamic changes of the prone position were investigated in 40 ASA I-II patients undergoing lumbar spine surgery. Patients were randomly assigned, following propofol intravenous induction, to receive maintenance of anaesthesia using either isoflurane 1-1.2% in air or target controlled propofol 3 microg.ml(-1) infusion. Measurements of non-invasive blood pressure, heart rate and cardiac output were made in the supine position. The patient was then turned prone onto a Montreal pattern mattress and measurements repeated. Cardiac output measurements were made using a non-invasive cardiac output monitor. We found a significant reduction in cardiac index in both groups and a significantly greater change with propofol compared to isoflurane on turning supine to prone (CI change 0.4 vs 0.7 l.min(-1).m(-2) p = 0.001 and SVRI change 89 vs 177 dyne.s(-1).cm(-5), p = 0.041). We conclude that turning healthy patients prone produces a clinically significant reduction in cardiac output, the change being greater during maintenance of anaesthesia using propofol compared to isoflurane.

Histamine release during adult cardiopulmonary bypass.

Histamine, an inflammatory mediator in its own right, may also be a marker for a more widespread systemic inflammatory process. In this study we have examined variations in plasma histamine concentrations produced during the course of cardiac surgery involving cardiopulmonary bypass, the relationship between these variations and intra-operative events. By assays of serum tryptase and CD-63 expression we have also attempted to identify the source of histamine. Histamine concentrations that were significantly raised from baseline level were demonstrated. These were elevated from the time of aortic cross-clamping and continued to be raised for 24 h postoperatively (p < 0.00625). This was associated with an increase in CD-63 expression (p < 0.025) (but not an increase in tryptase concentration) following aortic cross-clamping and protamine administration, suggesting that basophils are the source of histamine. 41% of patients had arrhythmias in the post bypass period. The rise in histamine levels was not related to the incidence of cardiac arrhythmias.

Flow cytometric investigation of peri-anaesthetic anaphylaxis using CD63 and CD203c.

The investigation of anaphylactic reactions in the peri-operative period is difficult. Elevation of serum tryptase levels is a good indicator of an anaphylactic event but the ability of subsequent investigations to identify the drug(s) responsible for the reaction is still potentially unreliable. The aim of this study was to examine basophil activation as an investigative tool. We performed flow cytometric analysis of the expression on the cell surface of the basophil activation markers CD63 and CD203c and measured histamine release in 21 patients who were referred with possible peri-operative anaphylaxis. The sensitivity of CD63, CD203c, basophil histamine release and skin prick for the muscle relaxants was found to be 79%, 36%, 36% and 64%, respectively; the specificity was found to be 100%. These results demonstrate the difficulty in investigating the cause of an unexpected clinical event following drug administration, but the higher sensitivity of neo-expression on the cell surface of CD63 suggests that flow cytometric analysis of its neo-expression on basophils in vitro may be a diagnostic aid.