Hibernation in reptiles—II. Changes in blood cell glucose,haemoglobin, red blood cell count,protein and non-protein nitrogen

• 1.1. The level of blood glucose, red blood cell count, haemoglobin, serum protein and non-protein nitrogen were studied during summer and winter periods.
• 2.2. During hibernation, the first four blood constituents were respectively decreased by 62·35 per cent, 34·18 per cent, 35·33 per cent and 7·19 per cent as there is no food supply during the inactive hibernation season.
• 3.3. Non-protein nitrogen was increased by 115·27 per cent during hibernation. This is due to the accumulation of nitrogenous excretory end metabolites as excretion of the animal is impaired during this period.

     

Genetic studies of water buffalo blood markers. I. Red cell acid phosphatase,albumin, catalase,red cell α-esterase-3, group-specific component,and protease inhibitor

We have developed the methodologies for typing and family studies to establish the modes of inheritance of water buffalo red cell acid phosphatase (Acp), protease inhibitor (Pi), and group-specific component (Gc) on isoelectric focusing and albumin (Alb), red cell -esterase-3 (Est-3), and catalase (Cat) on polyacrylamide gel electrophoresis. Family studies showed that Pi, Gc, Alb, and Cat are coded by autosomal genes with two codominant alleles, while Est-3 is autosomal with two codominant alleles and a recessive null allele and Acp exhibits three codominant alleles.This project was funded by the Australian Centre for International Agricultural Research through Grant PN 8364 and the Malaysian programme for Intensification of Research in Priority Areas through Grant IRPA 1-07-05-057.     

Identification of potential biomarkers for post-traumatic complications released after trauma–hemorrhage from murine Kupffer cells and its investigation in lung and liver

Context: Early diagnosis of complications after severe trauma by specific biomarkers remains difficult.

Objective: Identify potential new biomarkers for early diagnosis of post-traumatic complications.

Material and methods: Mice underwent pressure-controlled hemorrhage or sham procedure. Four hours later, genome-wide expression of isolated Kupffer cells was compared with controls using Affymetrix-Genechip-Expression-Analysis and real-time-PCR.

Results: Expression analysis and real-time-PCR revealed a significant increase of gene expression of Cxcl10, Il4ra, Csf2rb2, Lcn2, and Gbp5.

Conclusion: Cxcl10, Il4ra, Csf2rb2, Lcn2, and Gbp5 might represent new biomarkers for early diagnosis of post-traumatic complications, if they are linked to the development of post-traumatic complications.     

A risk-based approach for cell line development,manufacturing and characterization of genetically engineered,induced pluripotent stem cell–derived allogeneic cell therapies

Advances in cellular reprogramming and gene-editing approaches have opened up the potential for a new class of ex vivo cell therapies based on genetically engineered, induced pluripotent stem cell (iPSC)-derived allogeneic cells. While these new therapies share some similarities with their primary cell-derived autologous and allogeneic cell therapy predecessors, key differences exist in the processes used for generating genetically engineered, iPSC-derived allogeneic therapies. Specifically, in iPSC-derived allogeneic therapies, donor selection and gene-editing are performed once over the lifetime of the product as opposed to as part of the manufacturing of each product batch. The introduction of a well-characterized, fully modified, clonally derived master cell bank reduces risks that have been inherent to primary-cell derived autologous and allogeneic therapies. Current regulatory guidance, which was largely developed based on the learnings gained from earlier generation therapies, leaves open questions around considerations for donor eligibility, starting materials and critical components, cell banking and genetic stability. Here, a risk-based approach is proposed to address these considerations, while regulatory guidance continues to evolve.     

Dynamics of blood flow: modeling of Fåhraeus and Fåhraeus–Lindqvist effects using a shear-induced red blood cell migration model

Blood flow in micro capillaries of diameter approximately 15–500 μm is accompanied with a lower tube hematocrit level and lower apparent viscosity as the diameter decreases. These effects are termed the Fåhraeus and Fåhraeus–Lindqvist effects, respectively. Both effects are linked to axial accumulation of red blood cells. In the present investigation, we extend previous works using a shear-induced model for the migration of red blood cells and adopt a model for blood viscosity that accounts for the suspending medium viscosity and local hematocrit level. For fully developed hematocrit profiles (i.e., independent of axial location), the diffusion fluxes due to particle collision frequency and viscosity gradients are of equal magnitude and opposite directions. The ratio of the diffusion coefficients for the two fluxes affects both the Fåhraeus and Fåhraeus–Lindqvist effects and is found related to the capillary diameter and discharge hematocrit using a well-known data-fit correlation for apparent blood viscosity. The velocity and hematocrit profiles were determined numerically as functions of radial coordinate, tube diameter, and discharge hematocrit. The velocity profile determined numerically is consistent with the derived analytical expression and the results are in good agreement with published numerical results and experimental data for hematocrit ratio and hematocrit and velocity profiles.     

Thrombospondin-1–CD47 blockade and exogenous nitrite enhance ischemic tissue survival,blood flow and angiogenesis via coupled NO–cGMP pathway activation

Tissue ischemia and ischemia–reperfusion (I/R) remain sources of cell and tissue death. Inability to restore blood flow and limit reperfusion injury represents a challenge in surgical tissue repair and transplantation. Nitric oxide (NO) is a central regulator of blood flow, reperfusion signaling and angiogenesis. De novo NO synthesis requires oxygen and is limited in ischemic vascular territories. Nitrite (NO_2?) has been discovered to convert to NO via heme-based reduction during hypoxia, providing a NO synthase independent and oxygen-independent NO source. Furthermore, blockade of the matrix protein thrombospondin-1 (TSP1) or its receptor CD47 has been shown to promote downstream NO signaling via soluble guanylate cyclase (sGC) and cGMP-dependant kinase. We hypothesized that nitrite would provide an ischemic NO source that could be potentiated by TSP1–CD47 blockade enhancing ischemic tissue survival, blood flow and angiogenesis. Both low dose nitrite and direct blockade of TSP1–CD47 interaction using antibodies or gene silencing increased acute blood flow and late tissue survival in ischemic full thickness flaps. Nitrite and TSP1 blockade both enhanced in vitro and in vivo angiogenic responses. The nitrite effect could be abolished by inhibition of sGC and cGMP signaling. Potential therapeutic synergy was tested in a more severe ischemic flap model. We found that combined therapy with nitrite and TSP1–CD47 blockade enhanced flap perfusion, survival and angiogenesis to a greater extent than either agent alone, providing approximately 100% flap survival. These data provide a new therapeutic paradigm for hypoxic NO signaling through enhanced cGMP mediated by TSP1–CD47 blockade and nitrite delivery.     

Stable-isotope dilution GC–MS approach for nitrite quantification in human whole blood,erythrocytes, and plasma using pentafluorobenzyl bromide derivatization: Nitrite distribution in human blood

Previously, we reported on the usefulness of pentafluorobenzyl bromide (PFB-Br) for the simultaneous derivatization and quantitative determination of nitrite and nitrate in various biological fluids by GC–MS using their ¹⁵N-labelled analogues as internal standards. As nitrite may be distributed unevenly in plasma and blood cells, its quantification in whole blood rather than in plasma or serum may be the most appropriate approach to determine nitrite concentration in the circulation. So far, GC–MS methods based on PFB-Br derivatization failed to measure nitrite in whole blood and erythrocytes because of rapid nitrite loss by oxidation and other unknown reactions during derivatization. The present article reports optimized and validated procedures for sample preparation and nitrite derivatization which allow for reliable quantification of nitrite in human whole blood and erythrocytes. Essential measures for stabilizing nitrite in these samples include sample cooling (0–4 °C), hemoglobin (Hb) removal by precipitation with acetone and short derivatization of the Hb-free supernatant (5 min, 50 °C). Potassium ferricyanide (K₃Fe(CN)₆) is useful in preventing Hb-caused nitrite loss, however, this chemical is not absolutely required in the present method. Our results show that accurate GC–MS quantification of nitrite as PFB derivative is feasible virtually in every biological matrix with similar accuracy and precision. In EDTA-anticoagulated venous blood of 10 healthy young volunteers, endogenous nitrite concentration was measured to be 486 ± 280 nM in whole blood, 672 ± 496 nM in plasma (C_P), and 620 ± 350 nM in erythrocytes (C_E). The C_E-to-C_P ratio was 0.993 ± 0.188 indicating almost even distribution of endogenous nitrite between plasma and erythrocytes. By contrast, the major fraction of nitrite added to whole blood remained in plasma. The present GC–MS method is useful to investigate distribution and metabolism of endogenous and exogenous nitrite in blood compartments under basal conditions and during hyperemia.     

Motility and fertility of cryopreserved semen in Persian sturgeon,Acipenser persicus,stored for 30–60 min after thawing

We investigated the effect of storage times of frozen–thawed Persian sturgeon (Acipenser persicus) semen on the duration of sperm motility, percentage of motile sperm, and fertilization and hatching rates of fresh sperm and sperm stored for 0, 30, and 60 min at 4 °C post-thawing. Frozen thawed semen analyzed immediately after thawing had similar quality characteristics as fresh semen. For cryopreserved semen stored for 30 min after thawing the characteristics did not differ to fresh semen and cryopreserved semen. For cryopreserved semen stored for 60 min a significant decline in the parameters was observed. Fertilization and hatching rates were not affected by storage times of maximally 30 min of storage.     

Elastic behavior of a red blood cell with the membrane’s nonuniform natural state: equilibrium shape,motion transition under shear flow,and elongation during tank-treading motion

Direct numerical simulations of the mechanics of a single red blood cell (RBC) were performed by considering the nonuniform natural state of the elastic membrane. A RBC was modeled as an incompressible viscous fluid encapsulated by an elastic membrane. The in-plane shear and area dilatation deformations of the membrane were modeled by Skalak constitutive equation, while out-of-plane bending deformation was formulated by the spring model. The natural state of the membrane with respect to in-plane shear deformation was modeled as a sphere ( \(\alpha =0\) ), biconcave disk shape ( \(\alpha =1\) ) and their intermediate shapes ( \(0<\alpha <1\) ) with the nonuniformity parameter \(\alpha \) , while the natural state with respect to out-of-plane bending deformation was modeled as a flat plane. According to the numerical simulations, at an experimentally measured in-plane shear modulus of \(2.5\times 10^{-6}\,\hbox {N}/\hbox {m}\) and an out-of-plane bending rigidity of \(2.0\times 10^{-19}\,\hbox {N}\cdot \hbox {m}\) of the cell membrane, the following results were obtained. (i) The RBC shape at equilibrium was biconcave discoid for \(\alpha >0.22\) and cupped otherwise; (ii) the experimentally measured fluid shear stress at the transition between tumbling and tank-treading motions under shear flow was reproduced for \(0.05<\alpha <0.34\) ; (iii) the elongation deformation of the RBC during tank-treading motion from the simulation was consistent with that from in vitro experiments, irrespective of the \(\alpha \) value. Based on our RBC modeling, the three phenomena (i), (ii), and (iii) were mechanically consistent for \(0.22<\alpha <0.34\) . The condition \(0.05<\alpha <0.22\) precludes a biconcave discoid shape at equilibrium (i); however, it gives appropriate fluid shear stress at the motion transition under shear flow (ii), suggesting that a combined effect of \(\alpha \) and the natural state with respect to out-of-plane bending deformation is necessary for understanding details of the RBC mechanics at equilibrium. Our numerical results demonstrate that moderate nonuniformity in a membrane’s natural state with respect to in-plane shear deformation plays a key role in RBC mechanics.     

Dipeptidyl peptidase-IV inhibition prevents blood–retinal barrier breakdown,inflammation and neuronal cell death in the retina of type 1 diabetic rats

Diabetic retinopathy, a leading cause of vision loss in working-age population, is often associated with inflammation and apoptosis. We have previously reported that sitagliptin, a DPP-IV inhibitor, exerts beneficial effects in the retina of type 2 diabetic animals. The present study aimed to evaluate whether sitagliptin can exert protective effects in the retina of type 1 diabetic animals by a mechanism independent of insulin secretion and glycemia normalization.Streptozotocin-induced diabetic rats were treated orally with sitagliptin (5 mg/kg/day) for the last two weeks of 4 weeks of diabetes. Sitagliptin treatment did not change the weight and glucose, HbA_1c or insulin levels. However, it prevented the diabetes-induced increase in DPP-IV/CD26 activity and levels in serum and retina. Sitagliptin also prevented the increase in blood–retinal barrier (BRB) permeability and inhibited the changes in immunoreactivity and endothelial subcellular distribution of occludin, claudin-5 and ZO-1 proteins induced by diabetes. Furthermore, sitagliptin decreased the retinal inflammatory state and neuronal apoptosis.Sitagliptin inhibited the BRB breakdown in a type 1 diabetic animal model, by a mechanism independent of normalization of glycemia, by preventing changes in tight junctions (TJs) organization. Sitagliptin also exerted protective effects against inflammation and pro-apoptotic state in the retina of diabetic rats. Altogether, these results suggest that sitagliptin might be envisaged to be used to prevent or delay some of the alterations associated with the development of diabetic retinopathy.     

Gαs proteins activate p72Syk and p60-c-Src tyrosine kinases to mediate sickle red blood cell adhesion to endothelium via LW-αvβ3 and CD44–CD44 interactions

G protein-coupled receptors (GPCRs) have been suggested as new drug targets to treat a variety of diseases. In sickle cell disease (SCD), the LW erythrocyte adhesion receptor can be activated by stimulation of β2 adrenergic receptors (β2ARs), to mediate sickle erythrocyte (SSRBC) adhesion to endothelium. However, the involvement of tyrosine protein kinases in β2AR signaling to activate SSRBC adhesion to endothelium has not been thoroughly elucidated. Either direct activation with Cholera toxin of Gαs protein, which acts downstream of β2ARs, or inhibition with Pertussis toxin of Gαi, mediating suppression of adenylyl cyclase, increased SSRBC adhesion to endothelium over baseline adhesion. This effect involved the non-receptor tyrosine kinases, p72^Syk and p60-c-Src, which were more abundant in SSRBCs than in normal erythrocytes. In contrast, Pertussis toxin and Cholera toxin failed to increase adhesion of normal erythrocytes. SSRBC Gαi inhibition also increased phosphorylation of p72^Syk and p60-c-Src. Further, we investigated the relevance of activation of p72^Syk and p60-c-Src, and identified LW (ICAM-4, CD242) and CD44 as the erythroid adhesion molecules both physically interacting with activated p60-c-Src. As a result, SSRBC LW underwent increased tyrosine phosphorylation, leading to SSRBC LW and CD44 binding to endothelial αvβ3 integrin and CD44, respectively. These data provide in vitro mechanistic evidence that p60-c-Src, which could act downstream of Gαs/p72^Syk, associates with LW and CD44 on SSRBCs leading to their interactions with endothelial αvβ3 and CD44, respectively. Thus, increased activation of these signaling mechanisms in SSRBCs could initiate or exacerbate vascular occlusion, the hallmark of SCD.     

The novel EpCAM-targeting monoclonal antibody 3–17I linked to saporin is highly cytotoxic after photochemical internalization in breast,pancreas and colon cancer cell lines

The epithelial cell adhesion molecule (EpCAM) is expressed by a wide range of human carcinomas, making it an attractive diagnostic and therapeutic target in oncology. Its recent identification on cancer stem cells has raised further interest in its use for tumor targeting and therapy. Here, we present the characterization and therapeutic potential of 3–17I, a novel human EpCAM-targeting monoclonal antibody. Strong reaction of 3–17I was observed in all lung, colon, and breast human tumor biopsies evaluated. By flow cytometry and confocal fluorescence microscopy, we demonstrate that 3–17I specifically targets EpCAM-positive cell lines. We also show evidence for mAb-sequestration in endo-/lysosomes, suggesting internalization of 3–17I by receptor-mediated endocytosis. The ribosomal-inactivating toxin saporin was linked to 3–17I, creating the per se non-toxic immunotoxin 3–17I-saporin, a promising candidate for the drug delivery technology photochemical internalization (PCI). PCI is based on a light-controlled destruction of endolysosomal membranes and subsequent cytosolic release of the sequestered payload upon light exposure. EpCAM-positive human cancer cell lines MCF7 (breast), BxPC-3 (pancreas), WiDr (colon), and the EpCAM-negative COLO320DM (colon), were treated with 3–17I-saporin in combination with the clinically relevant photosensitizer TPCS2a (Amphinex), followed by exposure to light. No cytotoxicity was observed after treatment with 3–17I-saporin without light exposure. However, cell viability, proliferation and colony-forming capacity was strongly reduced in a light-dependent manner after PCI of 3–17I. Our results show that 3–17I is an excellent candidate for diagnosis of EpCAM-positive tumors and for development of clinically relevant antibody-drug conjugates, using PCI for the treatment of localized tumors.