A Novel and Potent Thrombolytic Fusion Protein Consisting of Anti-Insoluble Fibrin Antibody and Mutated Urokinase.

Tissue plasminogen activator (tPA) is used clinically because it has a higher binding specificity for insoluble fibrin (IF) than urokinase (UK), but even pro-tPA has catalytic activity against substrates other than IF. UK has the advantage that it is specifically activated on IF; however, it binds IF weakly. Previously, we established a monoclonal antibody (mAb) that recognizes a pit structure formed only in IF. Here, we developed a new mAb against the pit, 1101, that does not affect coagulation or fibrinolysis, and prepared a fusion protein of UK with humanized 1101 Fab to transport UK selectively to IF. In IF-containing lesions, UK is cleaved by plasmin at two sites, Lys158/Ile159 and Lys135/Lys136. Cleavage of the former leads to activation of UK; however, because activated UK is linked by S-S bonds before and after cleavage, it is not released from the fusion. Cleavage at the latter site causes UK to leave the fusion protein; hence, we mutated Lys135/Lys136 to Gly135/Gly136 to prevent release of UK. This engineered UK-antibody fusion, AMU1114, significantly decreased the reduction of plasma plasminogen levels in vivo relative to UK. In a photochemically induced mouse model of thrombus, the vascular patency rate was 0% (0/10) in the control, 50% (5/10) in the tPA treatment group, and 90% (9/10) in the AMU1114 treatment group. Although no death was observed 1 hour after administration of each thrombolytic agent, some mice died within 24 hours in all treatment groups, including control. These data indicate the need for further basic studies of AMU1114.

The postnatal leptin surge in mice is variable in both time and intensity and reflects nutritional status.

BACKGROUND/OBJECTIVES: The murine postnatal leptin surge occurs within the first 4 weeks of life and is critical for neuronal projection development within hypothalamic feeding circuits. Here we describe the influence of nutritional status on the timing and magnitude of the postnatal leptin surge in mice. METHODS: Plasma leptin concentrations were measured 1-3 times per week for the first 4 weeks of life in C57BL/6J pups reared in litters adjusted to 3 (small), 7-8 (normal), or 11-12 (large) pups per dam fed breeder chow or raised in litters of 7-8 by dams fed high-fat diet (HFD) ad libitum starting either prior to conception or at parturition. RESULTS: Mice raised in small litters become fatter than pups raised in either normal or large litters. The leptin surge in small litter pups starts earlier, lasts longer, and is dramatically larger in magnitude compared to normal litter pups, even when leptin concentrations are normalized to fat mass. In mice reared in large litters, weight gain is diminished and the surge is both significantly delayed and lower in magnitude compared to control pups. Pups reared by HFD-fed dams (starting preconception or at parturition) are fatter and have augmented leptin surge magnitude compared to pups suckled by chow-fed dams. Surge timing varies depending upon nutritional status of the pup; the source of the surge is primarily subcutaneous adipose tissue. At peak leptin surge, within each group, fat mass and plasma leptin are uncorrelated; in comparison with adults, pups overproduce leptin relative to fat mass. Plasma leptin elevation persists longer than previously described; at postnatal day 27 mice continue overproducing leptin relative to fat mass. CONCLUSIONS: In mice, small litter size and maternal HFD feeding during the perinatal period augment the plasma leptin surge whereas large litter size is associated with a delayed surge of reduced magnitude.

Neutrophil extracellular traps (NETs) exacerbate severity of infant sepsis.

BACKGROUND: Neutrophil extracellular traps (NETs) are innate defense mechanisms that are also implicated in the pathogenesis of organ dysfunction. However, the role of NETs in pediatric sepsis is unknown. METHODS: Infant (2 weeks old) and adult (6 weeks old) mice were submitted to sepsis by intraperitoneal (i.p.) injection of bacteria suspension or lipopolysaccharide (LPS). Neutrophil infiltration, bacteremia, organ injury, and concentrations of cytokine, NETs, and DNase in the plasma were measured. Production of reactive oxygen and nitrogen species and release of NETs by neutrophils were also evaluated. To investigate the functional role of NETs, mice undergoing sepsis were treated with antibiotic plus rhDNase and the survival, organ injury, and levels of inflammatory markers and NETs were determined. Blood samples from pediatric and adult sepsis patients were collected and the concentrations of NETs measured. RESULTS: Infant C57BL/6 mice subjected to sepsis or LPS-induced endotoxemia produced significantly higher levels of NETs than the adult mice. Moreover, compared to that of the adult mice, this outcome was accompanied by increased organ injury and production of inflammatory cytokines. The increased NETs were associated with elevated expression of Padi4 and histone H3 citrullination in the neutrophils. Furthermore, treatment of infant septic mice with rhDNase or a PAD-4 inhibitor markedly attenuated sepsis. Importantly, pediatric septic patients had high levels of NETs, and the severity of pediatric sepsis was positively correlated with the level of NETs. CONCLUSION: This study reveals a hitherto unrecognized mechanism of pediatric sepsis susceptibility and suggests that NETs represents a potential target to improve clinical outcomes of sepsis.

Hematological and biochemical profile of BALB/c nude and C57BL/6 SCID female mice after ovarian xenograft.

Hematological and biochemical profile studies help to evaluate functional changes of animals used in experiments. The aim of this study was to determine the hematological and biochemical profile of immunosuppressed BALB/c nude and C57BL/6 SCID mice after bovine ovarian xenotransplantation. Therefore, a total of 74 female mice were divided into four groups: non-xenotransplanted animals, xenotransplanted animals, xenotransplanted animals treated with eCG and xenotransplanted animals treated with FSH + LH. After anesthesia, blood samples were collected and hematologic and biochemical values were evaluated. The results showed no significant differences (p ≤ 0.05) for hematological parameters between the control group and the treatment groups of both strains. However, considering the biochemical profile, it was observed an increase of AST concentrations (p ≤ 0.05) in both strains and a decrease of ALT concentrations (p ≤ 0.05) only in C57BL/6 SCID strain of the groups subjected to hormonal treatment compared with those non subjected. Additionally, the values of the renal enzymes, urea and creatinine, did not differ (p ≤ 0.05) between the groups. Our findings suggest that the xenotransplantation procedure as well as the hormonal dosages had no significant effect on the well-being of the animals considering the evaluated hematological and biochemical profile.

Postoperative cellular stress in the kidney is associated with an early systemic γδ T-cell immune cell response.

BACKGROUND: Basic science data suggest that acute kidney injury (AKI) induced by ischemia-reperfusion injury (IRI) is an inflammatory process involving the adaptive immune response. Little is known about the T-cell contribution in the very early phase, so we investigated if tubular cellular stress expressed by elevated cell cycle biomarkers is associated with early changes in circulating T-cell subsets, applying a bedside-to-bench approach. METHODS: Our observational pilot study included 20 consecutive patients undergoing endovascular aortic repair for aortic aneurysms affecting the renal arteries, thereby requiring brief kidney hypoperfusion and reperfusion. Clinical-grade flow cytometry-based immune monitoring of peripheral immune cell populations was conducted perioperatively and linked to tubular cell stress biomarkers ([TIMP-2]•[IGFBP7]) immediately after surgery. To confirm clinical results and prove T-cell infiltration in the kidney, we simulated tubular cellular injury in an established mouse model of mild renal IRI. RESULTS: A significant correlation between tubular cell injury and a peripheral decline of γδ T cells, but no other T-cell subpopulation, was discovered within the first 24 hours (r = 0.53; p = 0.022). Turning to a mouse model of kidney warm IRI, a similar decrease in circulating γδ T cells was found and concomitantly was associated with a 6.65-fold increase in γδ T cells (p = 0.002) in the kidney tissue without alterations in other T-cell subsets, consistent with our human data. In search of a mechanistic driver of IRI, we found that the damage-associated molecule high-mobility group box 1 protein HMGB1 was significantly elevated in the peripheral blood of clinical study subjects after tubular cell injury (p = 0.019). Correspondingly, HMGB1 RNA content was significantly elevated in the murine kidney. CONCLUSIONS: Our investigation supports a hypothesis that γδ T cells are important in the very early phase of human AKI and should be considered when designing clinical trials aimed at preventing kidney damage. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01915446 . Registered on 5 Aug 2013.

Association between Helicobacter pylori infection and platelet count in mice.

Strong evidence for an association between idiopathic thrombocytopenic purpura (ITP) and Helicobacter pylori (HP) infection has been reported in humans. Chronic ITP is known to be improved by the eradication of HP. The purpose of this study was to reproduce these events by the experimental infection of several strains of mice with HP. BALB/c, C57BL/6, and DBA/2 mice were untreated or orally inoculated with HP. Two months later, platelet counts were compared in samples from HP-infected and noninfected mice. Platelet counts (mean ± SD, × 104 cells/µl) in blood samples from HP-infected BALB/c, C57BL/6, and DBA/2 mice were 102.28 ± 14.71, 99.65 ± 17.00, and 111.57 ± 16.20, respectively; the respective counts from noninfected mice were 121.80 ± 13.30, 104.35 ± 18.20, and 107.84 ± 14.33. A significant difference in platelet counts between HP-infected and noninfected mice was observed in BALB/c mice (P≤0.01) but was not observed in DBA/2 mice, even though the histocompatibility (H)-2 type of the DBA/2 was the same as that of BALB/c mice. According to ELISA results, the optical density value for the anti-HP antibody in HP-infected BALB/c mice was not correlated with the number of platelets (P>0.50). These results suggest that the decrease in platelet count caused by HP infection is not related to antibody titer and histocompatibility-2 type. Experimental infection of BALB/c mice with HP can reproduce the relationship between HP and ITP and serves as a good model to investigate the mechanistic basis for the effectiveness of HP eradication therapy for ITP treatment.

Impact of bedding volume on physiological and behavioural parameters in laboratory mice.

The standard housing temperature in animal facilities is substantially below the lower critical temperature of mice. This does not only endanger animal welfare, it can also jeopardize scientific research as cold stress has a major impact on mouse physiology. There is some evidence that deep bedding, comparable to nesting material, can help mice to reduce heat loss. Whenever changes are applied to the cage environment, the potential impact on experimental results, including variation, needs to be assessed. An increased variation can result in a conflict between reduction and refinement, when more animals are needed for significance due to the housing design. The aim of this study was to assess the impact of different bedding volumes (0.5 L, 1.5 L and 6 L per type III cage) on mean values and coefficient of variation (CV) of physiological (pentobarbital sleeping time, blood and anatomical parameters) and behavioural parameters (open-field and novel object recognition tests) of group-housed female and male BALB/c and C57BL/6 mice. A larger bedding volume did not interfere with the CVs, but influenced mean values of organ weights and tail lengths. Mice housed on deeper bedding showed a significant reduction in adrenal, liver, kidney and heart weights as well as an increase in tail lengths; these anatomical changes are akin to warm adaptation, and were previously observed for mice housed under warmer environments. A larger bedding volume appears to be a sensible way to reduce cold stress for laboratory mice without increasing variation in experimental results.

Evaluation of mouse red blood cell and platelet counting with an automated hematology analyzer.

An evaluation of mouse red blood cell (RBC) and platelet (PLT) counting with an automated hematology analyzer was performed with three strains of mice, C57BL/6 (B6), BALB/c (BALB) and DBA/2 (D2). There were no significant differences in RBC and PLT counts between manual and automated optical methods in any of the samples, except for D2 mice. For D2, RBC counts obtained using the manual method were significantly lower than those obtained using the automated optical method (P<0.05), and PLT counts obtained using the manual method were higher than those obtained using the automated optical method (P<0.05). An automated hematology analyzer can be used for RBC and PLT counting; however, an appropriate method should be selected when D2 mice samples are used.

Assessment of endogenous 25-hydroxyvitamin D serum concentrations by liquid chromatography-tandem mass spectrometry in various animal species.

BACKGROUND: Mass spectrometry (MS) has become the preferential method for the analysis of vitamin D in the clinic, yet no single platform is utilized for preclinical species in drug development studies. For vitamin D, the MS platform can provide certain benefits such as applicability of a single assay for multiple species, low cost, and high specificity. OBJECTIVES: A quantitative liquid chromatography-tandem MS (LC-MS/MS) assay for 25-hydroxyvitamin D3 (25OHD3 ) and D2 (25OHD2 ) was validated for rat, dog, mouse, and monkey, and suitability for drug development studies was assessed. METHODS: Standards were used to determine intra- and inter-assay accuracy and precision for LC-MS/MS. Extraction recovery and carryover due to instrumentation were determined. Repeat analyses of pooled serum samples from rat, dog, mouse, and monkey were assessed for precision, and other serum samples were used to determine the normal range in each species and detect biologically relevant changes. RESULTS: For both 25OHD3 and 25OHD2 , inaccuracy was ≤ 6%, and imprecision was ≤ 13%. Extraction recovery was 75% for 25OHD3 and 72% for 25OHD2 , and carryover was ≤ 0.1%. Measurable concentrations of 25OHD3 were recorded in serum samples from all species tested, but no 25OHD2 as diets were only fortified with 25OHD3 . This dataset provides preliminary information for the determination of RIs for 25OHD3 in rat, dog, mouse, and monkey with the LC-MS/MS platform. CONCLUSIONS: The LC-MS/MS assay was accurate and precise for determination of endogenous concentrations of 25OHD3 in serum samples from drug development studies in rat, dog, mouse, and monkey.

Evaluation of hematologic variables in newborn C57/BL6 mice up to day 35.

BACKGROUND: Hematologic variables are often analyzed in animal analogs during the investigation of complex disease etiologies such as necrotizing enterocolitis. However, reference intervals (RI) can vary depending on animal strain, age, and sampling site. Reference intervals have been published for adult C57BL/6J mice, but not newborn C57BL/6J mice. OBJECTIVES: The purpose of the present study was to determine hematologic RI in newborn C57BL/6J mice up to day 35. METHODS: C57BL/6J mice founders from The Jackson Laboratory were bred at the University of Iowa. Blood samples were obtained via facial vein sampling at postnatal days 0 (p0), p7, p14, p21, p28, or young adulthood (p35). CBCs were determined with the Sysmex XT-2000iV analyzer within 30 minutes of blood collection at a 1:10 dilution. Statistics were determined using nonparametric methods following ASVCP guidelines. RESULTS: Hematologic RI were determined for each of the 6 groups (n = 247, n ≥ 39 per group). Significantly higher values for HGB, RBC, and PLT counts were observed with advancing developmental age. Total WBC counts remained relatively stable during the first 35 days of life. However, WBC differential counts were dominated by neutrophils and lymphocytes in the younger mice, with a trend toward a lymphocytic leukogram on day 35. CONCLUSIONS: These results illustrate the dynamic changes in hematologic variables during murine development after birth. Utilization of age-specific RI is advised when evaluating data derived from experimental perinatal mouse models.

Reduced sulfate plasma concentrations in the BTBR T+tf/J mouse model of autism.

Clinical studies have shown that children diagnosed with autism show abnormal sulfate chemistry, which is critical for cellular and metabolic processes. To determine if the inbred BTBR T+tf/J mouse shows autism-relevant aberrations in sulfate chemistry, the present study examined plasma sulfate concentrations in BTBR T+tf/J, inbred C57BL/6J, and outbred CD-1 mice. Results showed that the BTBR T+tf/J mouse exhibits significantly lower plasma sulfate concentrations in comparison to both C57BL/6J and CD-1 mice. These results suggest that the BTBR mouse shows autism-relevant abnormalities in sulfate chemistry and may serve additional utility in examining the role of sulfate and sulfate-dependent systems in relation to autism-relevant behavioral aberrations.

Methods for long-term 17ß-estradiol administration to mice.

Rodent models constitute a cornerstone in the elucidation of the effects and biological mechanisms of 17ß-estradiol. However, a thorough assessment of the methods for long-term administration of 17ß-estradiol to mice is lacking. The fact that 17ß-estradiol has been demonstrated to exert different effects depending on dose emphasizes the need for validated administration regimens. Therefore, 169 female C57BL/6 mice were ovariectomized and administered 17ß-estradiol using one of the two commonly used subcutaneous methods; slow-release pellets (0.18 mg, 60-day release pellets; 0.72 mg, 90-day release pellets) and silastic capsules (with/without convalescence period, silastic laboratory tubing, inner/outer diameter: 1.575/3.175 mm, filled with a 14 mm column of 36 µg 17ß-estradiol/mL sesame oil), or a novel peroral method (56 µg 17ß-estradiol/day/kg body weight in the hazelnut cream Nutella). Forty animals were used as ovariectomized and intact controls. Serum samples were obtained weekly for five weeks and 17ß-estradiol concentrations were measured using radioimmunoassay. The peroral method resulted in steady concentrations within--except on one occasion--the physiological range and the silastic capsules produced predominantly physiological concentrations, although exceeding the range by maximum a factor three during the first three weeks. The 0.18 mg pellet yielded initial concentrations an order of magnitude higher than the physiological range, which then decreased drastically, and the 0.72 mg pellet produced between 18 and 40 times higher concentrations than the physiological range during the entire experiment. The peroral method and silastic capsules described in this article constitute reliable modes of administration of 17ß-estradiol, superior to the widely used commercial pellets.

Clinical biochemistry parameters in C57BL/6J mice after blood collection from the submandibular vein and retroorbital plexus.

Collection of blood from the submandibular vein allows simple and rapid processing of many animals without anesthesia and facilitates rapid recovery with no signs of pain and discomfort in the mice. Here we compared the submandibular vein and retroorbital plexus blood collection methods, to determine the potential effect of the sampling technique on several clinical biochemistry parameters in C57BL/6J mice. We found statistically significant differences for 8 of the 9 biochemical parameters studied between the 2 blood sampling techniques. Compared with samples collected from the retroorbital plexus, blood obtained from the submandibular vein had higher levels of AST, ALT, protein, albumin, triglycerides, total cholesterol, and creatinine. Glucose values of retroorbital blood were higher than those from the submandibular vein. Urea levels were similar for both sampling techniques. Our results demonstrate that the technique used to obtain blood samples affects parameters commonly used to assess animal health. We recommend caution when comparing results of biochemical analysis of blood obtained from the submandibular vein in mice with reference values obtained by other blood sampling techniques.

Comparative hemostatic parameters in BALB/c, C57BL/6 and C3H/He mice.

This study describes micro-methods to determine biological parameters in plasma of three strains of mice. Platelet count was significantly different among strains. C57BL/6 mice showed the highest values (988 x 10(3)/microL) and BALB/c the lowest (782 x 10(3)/microL). Fibrinogen levels were 2.55 (C57BL/6), 2.37 (BALB/c) and 2.28 g/L (C3H/He). Some inter-strain differences were observed in factor XIII (94, 118 and 114%) and plasminogen levels (142, 80 and 135%) in C57BL/6, BALB/c and C3H/He, respectively. Additionally, we observed individual mice factor XIII and plasminogen levels between 80 to 200% and 65 to 180%, respectively, in relation to pooled human plasma; and between 70 to 185% and 70 to 155%, respectively, against pooled mice plasma. To our knowledge, this is first report in the literature in diverse mice strains regarding hemostasis, mainly on factor XIII, plasminogen levels, and a very simple test that allows measurement of endogenous fibrinolytic activity present in the plasma. The different results are discussed in relationship with existing literature regarding if the animals in some studies were maintained under strict pathogen-free conditions, the collection of blood was from the heart or eye and if the analysis method was tested by counting manually or automatically. This work could contribute useful knowledge to the field of investigations regarding hemostatic disorders using mouse models, especially for laboratories that are not well equipped.

Age-Related Reference Intervals of the Main Biochemical and Hematological Parameters in C57BL/6J, 129SV/EV and C3H/HeJ Mouse Strains.

BACKGROUND: Although the mouse is the animal model most widely used to study the pathogenesis and treatment of human diseases, reference values for biochemical parameters are scanty or lacking for the most frequently used strains. We therefore evaluated these parameters in the C57BL/6J, 129SV/EV and C3H/HeJ mice. METHODOLOGY/PRINCIPAL FINDINGS: We measured by dry chemistry 26 analytes relative to electrolyte balance, lipoprotein metabolism, and muscle/heart, liver, kidney and pancreas functions, and by automated blood counter 5 hematological parameters in 30 animals (15 male and 15 female) of each mouse strain at three age ranges: 1-2 months, 3-8 months and 9-12 months. Whole blood was collected from the retro-orbital sinus. We used quality control procedures to investigate analytical imprecision and inaccuracy. Reference values were calculated by non parametric methods (median and 2.5(th) and 97.5(th) percentiles). The Mann-Whitney and Kruskal-Wallis tests were used for between-group comparisons. Median levels of GLU, LDH, Chol and BUN were higher, and LPS, AST, ALP and CHE were lower in males than in females (p range: 0.05-0.001). Inter-strain differences were observed for: (1) GLU, t-Bil, K+, Ca++, PO(4)- (p<0.05) and for TAG, Chol, AST, Fe++ (p<0.001) in 4-8 month-old animals; (2) for CK, Crea, Mg++, Na++, K+, Cl- (p<0.05) and BUN (p<0.001) in 2- and in 10-12 month-old mice; and (3) for WBC, RBC, HGB, HCT and PLT (p<0.05) during the 1 year life span. CONCLUSION/SIGNIFICANCE: Our results indicate that metabolic variations in C57BL/6J, 129SV/EV and C3H/HeJ mice after therapeutic intervention should be evaluated against gender- and age-dependent reference intervals.

Genotype-specific environmental impact on the variance of blood values in inbred and F1 hybrid mice.

Mice are important models for biomedical research because of the possibility of standardizing genetic background and environmental conditions, which both affect phenotypic variability. Inbred mouse strains as well as F1 hybrid mice are routinely used as genetically defined animal models; however, only a few studies investigated the variance of phenotypic parameters in inbred versus F1 hybrid mice and the potential interference of the genetic background with different housing conditions. Thus, we analyzed the ranges of clinical chemical and hematologic parameters in C3H and C57BL/6 inbred mice and their reciprocal F1 hybrids (B6C3F1, C3B6F1) in two different mouse facilities. Two thirds of the blood parameters examined in the same strain differed between the facilities for both the inbred strains and the F1 hybrid lines. The relation of the values between inbred and F1 hybrid mice was also affected by the facility. The variance of blood parameters in F1 hybrid mice compared with their parental inbred strains was inconsistent in one facility but generally smaller in the other facility. A subsequent study of F1 hybrid animals derived from the parental strains C3H and BALB/c, which was done in the latter housing unit, detected no general difference in the variance of blood parameters between F1 hybrid and inbred mice. Our study clearly demonstrates the possibility of major interactions between genotype and environment regarding the variance of clinical chemical and hematologic parameters.

Activation of coagulation in C57BL/6 mice given verotoxin 2 (VT2) and the effect of co-administration of LPS with VT2.

To obtain better insight into the pathogenesis of verotoxin-producing Escherichia coli-associated diseases, in this study, we explored the effect of verotoxin 2 (VT2) on coagulation in an animal model. After being given VT2 (50 ng/kg, lethal dose), C57BL/6 mice showed progressively increasing expression of TF mRNA in the kidney and brain and elevated plasma levels of thrombin-antithrombin III complex (TAT), normotest, fibrinogen, and PAI-1 paralleling the disease course over 24 hours; platelet counts were decreased at 48 hours with hemorrhage in the kidney and brain. Co-administration of lipopolysaccharide (LPS, 0.5 mg/kg) with VT2 (50 ng/kg) exhibited more prominant and/or prolonged increase in not only expression of TF and PAI-1 mRNAs in the kidney and brain but also plasma levels of TAT, fibrinogen, and PAI-1 and was associated with more remarkable hemorrhage in the tissues. Although VT2 (5 ng/kg) was not a lethal dose, co-administration of LPS (0.5 mg/kg) with VT2 (5 ng/kg) enhanced the susceptibility to VT2, resulting in more prolonged elevation of TAT levels during the first 24 hours than that in the LPS group and a second elevation at 72 hours, followed by death. Plasma IL-1beta level reached a maximum at 24 hours after VT2 (50 ng/kg) injection prior to the increase in TAT levels, whereas the increase in TNFalpha level immediately after injection was associated with the increase in PAI-1 mRNA. These observations indicate that the activation of coagulation by VT2 may occur through a mechanism different from that used by LPS, since plasma TAT levels rose in the mice immediately after LPS injection and returned to normal over 36 hours.