Fc-Gamma Receptor Polymorphisms Predispose Patients to Infectious Complications After Liver Transplantation.

We investigated the impact of polymorphisms in host innate immunoregulatory genes on the development of infectious complications after liver transplantation (LT). The single-nucleotide polymorphisms (SNPs) of C1QA [276A/G], FCGR2A [131H/R], and FCGR3A [158F/V], genes encoding the Fc gamma receptor (FcγR), were analyzed in 89 living donor LT recipients in relation to the occurrences of postoperative infectious complications within 30 days after LT. Consistent with a lower affinity of the isoform encoded by FCGR3A [158F] to both IgG1 and IgG3, a significantly higher incidence of bloodstream infections (BSI) was observed in the FCGR3A [158F/V or F/F] than in the FCGR3A [158V/V] individuals. The combination of FCGR2A and FCGR3A SNPs further stratified the incidence of BSI, regardless of C1QA SNP. The predominant causative pathogen of BSI in the FCGR3A [158F/F or F/V] patients was gram-positive cocci (73.3%), of which one third was methicillin-resistant Staphylococcus aureus. No differences were observed in the incidence of fungal infections or in cytomegalovirus infections with respect to the three gene polymorphisms. Our findings indicate that FcγR SNPs are predisposing factors for BSI and can predict mortality after LT. This study provides a foundation for further prospective studies on a larger scale.

A simple detection system for adenovirus receptor expression using a telomerase-specific replication-competent adenovirus.

Adenovirus serotype 5 (Ad5) is frequently used as an effective vector for induction of therapeutic transgenes in cancer gene therapy or of tumor cell lysis in oncolytic virotherapy. Ad5 can infect target cells through binding with the coxsackie and adenovirus receptor (CAR). Thus, the infectious ability of Ad5-based vectors depends on the CAR expression level in target cells. There are conventional methods to evaluate the CAR expression level in human target cells, including flow cytometry, western blotting and immunohistochemistry. Here, we show a simple system for detection and assessment of functional CAR expression in human tumor cells, using the green fluorescent protein (GFP)-expressing telomerase-specific replication-competent adenovirus OBP-401. OBP-401 infection induced detectable GFP expression in CAR-expressing tumor cells, but not in CAR-negative tumor cells, nor in CAR-positive normal fibroblasts, 24 h after infection. OBP-401-mediated GFP expression was significantly associated with CAR expression in tumor cells. OBP-401 infection detected tumor cells with low CAR expression more efficiently than conventional methods. OBP-401 also distinguished CAR-positive tumor tissues from CAR-negative tumor and normal tissues in biopsy samples. These results suggest that GFP-expressing telomerase-specific replication-competent adenovirus is a very potent diagnostic tool for assessment of functional CAR expression in tumor cells for Ad5-based antitumor therapy.

Development of endothermic metabolic response in embryos and hatchlings of the emu (Dromaius novaehollandiae).

During hatching, there is a maturation of the mechanisms controlling the respiratory physiology involved in endotherm in precocial avian species. Here we examined the timing of the development of an endothermic response of oxygen uptake (MO2) to an alteration of ambient temperature (T(a)) in a model precocial species, the preterm and hatching emu (Dromaius novaehollandiae). Late stage pre-pipped and pipped embryos and hatchlings were measured for responses of MO2 and shell or skin temperature (T(s)) to altered T(a) (DeltaT(a)). MO2 remained unchanged in pre-pipped and internally pipped (IP) embryos at the end of 1.5h exposure to DeltaT(a) of +/-10 degrees C. Externally pipped (EP) embryos responded to a cooling and a warming exposure with marked increase and decrease in MO2, as hatchlings responded to DeltaT(a) with an endothermic change in MO2. The demonstration of the endothermic inverse metabolic response first appearing in EP embryos suggests that pre-EP embryos may also possess the ability to produce the endothermic inverse metabolic response, but they are restricted by the eggshell gas conductance. Late pre-pipped and IP embryos were measured again for responses of [Formula: see text] to DeltaT(a) in air and then in a 40% O(2) environment. The metabolic response of pre-pipped embryos at 90% of incubation was partially altered by switching from air to hyperoxia. IP embryos responded to DeltaT(a) in 40% O(2) with apparent inverse changes in MO2. The late stage emu embryo possesses the ability to produce an endothermic metabolic response at an earlier stage of development than in chickens, but this response is limited by the eggshell gas conductance.

OBP-401-GFP telomerase-dependent adenovirus illuminates and kills high-metastatic more effectively than low-metastatic triple-negative breast cancer in vitro.

We previously described the development of a highly-invasive, triple-negative breast cancer (TNBC) variant using serial orthotopic implantation of MDA-MB-231 human breast cancer in nude mice. The isolated variant is highly invasive in the mammary gland and metastasized to lymph nodes in 10 of 12 mice compared with 2 of 12 of the parental cell line. OBP-401 is a telomerase-dependent cancer-specific, green fluorescent protein (GFP)-expressing adenovirus. OBP-401 was used to infect parental MDA-MB-231P cells and high-metastatic MDA-MB-231H and MDA-MB-231HLN isolated from a lymph node metastasis and MDA-MB-231HLM isolated from a lung metastasis. Time-course imaging showed that OBP-401 labeled MDA-MB-231HP, MDA-MB-231HLN, and MDA-MB-231HLM cells more brightly than MDA-MB-231 parental cells. OBP-401 killed MDA-MB-231H, MDA-MB-231HLN, and MDA-MB-231HLM cells more efficiently than MDA-MB-231P parental cells. These results indicate that OBP-401 could infect, label and then kill high-metastatic MDA-MB-231 more efficiently than low-metastatic MDA-MB-231.

Regulation of glucocorticoid receptor activity by 14--3-3-dependent intracellular relocalization of the corepressor RIP140.

Proteins belonging to the 14--3-3 family interact with various regulatory proteins involved in cellular signaling, cell cycle regulation, or apoptosis. 14--3-3 proteins have been suggested to act by regulating the cytoplasmic/nuclear localization of their target proteins or by acting as molecular scaffolds or chaperones. We have previously shown that overexpression of 14--3-3 enhances the transcriptional activity of the glucocorticoid receptor (GR), which is a member of the nuclear receptor family. In this study, we show that 14--3-3 interacts with the nuclear receptor corepressor RIP140. In transfection assays, RIP140 antagonizes 14--3-3- enhanced GR transactivation. Using colocalization studies we demonstrate that 14--3-3 can export RIP140 out of the nucleus and, interestingly, can also change its intranuclear localization. Moreover, we also observed that 14--3-3 can bind various other nuclear receptors and cofactors. In summary, our findings suggest that 14--3-3-mediated intracellular relocalization of the GR corepressor RIP140 might be a novel mechanism to enhance glucocorticoid responsiveness of target genes. They furthermore indicate a more general role for 14--3-3 protein by influencing the nuclear availability of nuclear receptor-associated cofactors.

Loss of p53 in stromal fibroblasts enhances tumor cell proliferation through nitric-oxide-mediated cyclooxygenase 2 activation.

Overexpression of cyclooxygenase 2 (COX-2) by stromal fibroblasts plays a critical role in the early stage of carcinogenesis. COX-2 expression is thought to be positively or negatively regulated by inflammatory chemical mediators or tumor suppressors. In this study, the contributions of inducible nitric oxide synthase (iNOS) and p53 to COX-2 expression were examined using mouse embryonic fibroblasts (MEFs) from wild-type, p53-deficient, iNOS-deficient, and p53/iNOS-deficient mice. These MEFs were treated with 1 µg/mL of lipopolysaccharide and 100 IU/mL of interferon gamma for up to 72 h. iNOS and COX-2 expression were analyzed by Western blotting. iNOS was induced earlier (16 h) in p53-deficient MEFs than in wild-type MEFs (48 h). Elevated expression of COX-2 was sustained for a longer duration in the p53-deficient MEFs. In contrast, COX-2 expression was reduced earlier in the iNOS-deficient MEFs. Addition of an exogenous NO donor (0.8 mM of S-nitroso-l-glutathione) to the iNOS-deficient MEFs augmented COX-2 expression. Co-culture with stimulated p53-deficient MEFs promoted cell proliferation of mouse rectal polyploid carcinoma CMT93 cells, but treatment with a COX-2-specific inhibitor counteracted this effect. These results suggest that loss of function of the p53 gene in stromal fibroblasts enhances COX-2 expression by enhancing iNOS expression and the resultant production of NO, contributing to the promotion of tumor growth.

Color-coding cancer and stromal cells with genetic reporters in a patient-derived orthotopic xenograft (PDOX) model of pancreatic cancer enhances fluorescence-guided surgery.

Precise fluorescence-guided surgery (FGS) for pancreatic cancer has the potential to greatly improve the outcome in this recalcitrant disease. To achieve this goal, we have used genetic reporters to color code cancer and stroma cells in a patient-derived orthotopic xenograft (PDOX) model. The telomerase-dependent green fluorescent protein (GFP)-containing adenovirus OBP-401 was used to label the cancer cells of a pancreatic cancer PDOX. The PDOX was previously grown in a red fluorescent protein (RFP) transgenic mouse that stably labeled the PDOX stroma cells bright red. The color-coded PDOX model enabled FGS to completely resect the pancreatic tumors including stroma. Dual-colored FGS significantly prevented local recurrence, which bright-light surgery or single-color FGS could not. FGS, with color-coded cancer and stroma cells has important potential for improving the outcome of recalcitrant-cancer surgery.

A case of recurrent malignant pheochromocytoma complicated by watery diarrhea, hypokalemia, achlorhydria syndrome.

A patient is reported who had undergone right adrenalectomy for pheochromocytoma and 15 yr later developed a recurrence in the same site complicated by the watery diarrhea, hypokalemia, achlorhydria syndrome. This tumor was histologically defined as a composite malignant pheochromocytoma-ganglioneuroblastoma (well differentiated type). Vasoactive intestinal polypeptide and catecholamine concentrations were elevated in both plasma and the tumor. The tumor somatostatin content also was high. The tumor was immunohistochemically determined to contain both vasoactive intestinal polypeptide and somatostatin.

Vitamin D receptor gene polymorphisms affect secondary hyperparathyroidism in hemodialyzed patients.

Adynamic bone disease unrelated to aluminum deposition, with low parathyroid hormone (PTH) levels, has increased in patients with end-stage renal failure. Some patients present with severe secondary hyperparathyroidism despite calcitriol administration and phosphate restriction. Because therapeutic and environmental factors are now similar among hemodialyzed patients, the variable incidence of secondary hyperparathyroidism may be caused by genetic heterogeneity. To examine this possibility, we analyzed restriction fragment length polymorphisms of the vitamin D receptor (VDR) gene in 877 Japanese hemodialysis patients. VDR allelic polymorphism was determined by the method of Morrison et al. Polymerase chain reaction (PCR) amplification and a BsmI endonuclease restriction site at the 5' end of the VDR gene defined BB (absence of restriction site on both alleles), Bb (heterozygous), or bb (restriction site on both alleles). The mean serum PTH level was lower in BB patients (86 +/- 102 pg/mL) than in bb patients (148 +/- 217 pg/mL; P < 0.05). The serum osteocalcin level was also lower in BB than in bb patients (P < 0.05). If results were re-analyzed excluding patients with a history of dialysis exceeding 10 years or those with non-insulin-dependent diabetes mellitus (NIDDM) or who had undergone parathyroidectomy, the differences in serum PTH levels were greater. However, there was no significant difference in serum PTH levels among the VDR genotypes, only for patients with NIDDM. The present study shows that patients with the b allele for the VDR gene have more severe secondary hyperparathyroidism than patients without the b allele. However, NIDDM or a long history of hemodialysis has a stronger power to influence PTH secretion.

pI(Cln) and cytosolic F-actin constitute a heteromeric complex with a constant molecular mass in rat skeletal muscles.

To elucidate the function of pI(Cln), its localization in subcellular organellae was investigated. A specific polyclonal anti-pI(Cln) antibody detected the soluble 38-kDa pI(Cln) exclusively in the cytosols of rat heart, lung, liver, spleen, skeletal muscle, testis, and brain, but not rat kidney. pI(Cln)-associated proteins in skeletal muscle were also analyzed. Native-gradient PAGE showed a single 340-kDa protein band reactive to anti-pI(Cln) antibody. This band also stained with anti-actin antibody. Two-dimensional PAGE and immunoprecipitation analysis indicated that all of the pI(Cln) was present in association with actin of a constant length: the molecular ratio of pI(Cln) to actin was roughly 1:7. In addition, all actin in the cytosol fractions was found in association with pI(Cln). These results suggest the possibility that skeletal muscle pI(Cln) controls the length of cytosolic F-actin.

Simultaneous acquisition of ECG, BCG, and blood pressure from chick embryos in the egg.

The applicability of the audiocartridge system to simultaneous measurements of the ballistic movements from multiple points of the egg was examined in late chick embryos. Despite direct contact between the cartridges and the egg, minute movements of the egg attributable to cardiac contractions of the embryo (ballistocardiogram, BCG) could be simultaneously detected from at least three points. The results showed that the amplitude of BCG waves was dependent on the site of detection, indicating that the BCG was a directional wave. The cartridge system for BCG measurement was incorporated in the electrocardiography and arterial catheterization to acquire simultaneously electrocardiogram (ECG), BCG, and blood pressure from the embryo confined in the egg. Isochronal comparison of these waves indicated that the ballistic movements of the embryo might be caused not only by the ventricular contraction but also by the atrial excitation, i.e., the BCG comprised waves appearing in the ventricular presystolic, contraction, and ejection periods. For wide changes in the heart rate, the approximate isometric ventricular contraction period remained almost constant.

Is the bone mass of hemodialysis patients genetically determined?

Polymorphism of the vitamin D receptor gene (VDR) has been linked to bone mineral density in twins, postmenopausal osteoporosis, and premenopausal woman. We examined the possibility that the bone mass in hemodialysis (HD) patients might be determined by VDR. The study consisted of 229 HD patients with a mean age of 53.3 years (range 21 to 83), who were dialyzed three times a week for an average of 8.65 (range 0.2 to 24) years. We determined their VDR using DNA of peripheral white blood cells by restriction enzyme BsmI and the polymerase chain reaction-restriction fragment length polymorphism method. Bone mineral content (BMC) was estimated at 1/3 of the radius using dual energy X-ray bone absorptiometry, and expressed in z-scores standardized by gender and age. Distributions of VDR in this hemodialysis population were BB (9.9%), Bb (13.1%), and bb (77.0%), showing no significant different from those in 105 healthy volunteers (BB, 7.6%; Bb, 13.3%; and bb, 79.0%). Multiple regression analysis revealed that gender, age, duration on HD, and serum osteocalcin are major determinants of BMC (r = 0.762, P < 0.001), while VDR and serum parathyroid hormone are not. In a subgroup with younger (< 65 years) patients dialyzed for less than 8.65 years, the z-score of BMC of patients with BB allele was less than those with Bb and bb allele (N = 77, P = 0.020). We conclude that vitamin D receptor polymorphism is not one of the main determinants of BMC of HD patients, though it might partially effect bone mass in a subgroup of younger HD patients with shorter HD histories. Further studies with longitudinal observation will be needed to confirm these possibilities.

Possible prediction of myeloid and lymphoid crises in chronic myelocytic leukemia at onset by determining the methylation status of the major breakpoint cluster region.

To investigate the relationship between the pattern of methylation at the major breakpoint cluster region (M-BCR) and transformation of chronic myelocytic leukemia (CML) from the chronic to the blastic phase, the M-BCR methylation status was examined serially from chronic to blastic phase in 23 CML patients. The DNA of mononuclear cells from bone marrow or peripheral blood was digested with restriction enzymes HpaII and BglII, and hybridized with a 5'M-BCR probe. The methylation status was stable during evolution of CML from chronic to the myeloid blastic phase. Cells in both phases showed consistent methylation patterns consisting of fully methylated rearranged fragments of variable size, 4.8, 3.1/3.0, and 2.7/2.5 kb. Conversely, there was substantial heterogeneity in methylation patterns in patients with lymphoid crisis. All lymphoid-crisis patients studied in blastic phase showed a pattern distinct from that of the chronic phase in the same patient, as well as from the myeloid pattern, suggesting cell lineage-specific M-BCR methylation. Moreover, in four of six patients with lymphoid crisis, the chronic-phase patterns were different from those of cases with myeloid crisis. Ph-positive and -negative acute lymphocytic leukemia (ALL) showed methylation patterns different from those of lymphoid crisis in CML. Although the number of patients with lymphoid crisis studied has been limited, these results suggest that analysis of M-BCR methylation status may be of clinical use in distinguishing lymphoid from myeloid crises and predicting the cell lineage of a crisis when the disease is still in the chronic phase.

Developmental patterns of heart rate in altricial avian embryos and hatchlings

The aims of this study were to determine the patterns of development of heart rate (fH) in altricial avian embryos and hatchlings, and then to examine how fH is regulated to meet metabolic requirements in altricial embryos. Embryonic mean heart rate (fH-) in 12 altricial species (Passeriformes and Psittaciformes) increased during pre-pipping incubation in all species except the cockatiel (Nymphicus hollandicus), in which fH- tended to decrease prior to pipping. The rate of increase in fH- tripled during the pipping phase in all species, and fH- was significantly higher during the pipping period and in hatchlings than during pre-pipping development. The O2 pulse (O2 consumed per cardiac beat) of altricial embryos increased in direct proportion to embryo mass (loge/loge base), although fH- was often low prior to pipping, implying that stroke volume increases in the second half of incubation. We conclude that fH contributes more than other factors towards supplying the metabolic demands of the embryo during the middle of incubation and the final pipping phase, but less during the intervening period of late incubation.

Oxygen uptake in one-legged and two-legged exercise.

PURPOSE: The purpose of this study was to determine the primary factors causing the differential oxygen uptake (VO2) response at submaximal intensities between one-legged and two-legged exercise, and whether peak oxygen uptake (VO2peak) increases in proportion to the increase in active muscle mass. METHODS: Two different types of exercise were used for this experiment, each requiring a different movement, a different method of stabilizing posture, and, finally, a different limiting VO2peak. In experiment 1, nine male subjects performed one-legged cycling (OLC) and two-legged cycling exercise (TLC) at a pedaling rate of 80 rpm. The exercise intensity was first set at 80 W and was increased by 40 W every 3 min until exhaustion. In experiment 2, six healthy male subjects performed one-legged knee-extension (OKE) and two-legged knee-extension (TKE) exercise at a rate of 50 contractions per minute. The knee-extension exercise was done at constant work rates for a 3-min session in OKE or a 4-min session in TKE. The exercise bouts were performed intermittently at four to seven different submaximal intensities and VO2 was determined at each intensity in all exercises. RESULTS: At submaximal intensities, VO2 in relation to work rate of one-legged exercise was more steep than those of two-legged exercise, and the mean values of VO2 were significantly higher in one-legged exercise than those in two-legged exercise in both knee extension and cycling exercise. Mean values of VO2peak for two-legged exercise were significantly higher than that for one-legged exercise (P < 0.01); however, it was much lower than two times of that for one-legged exercise even in knee extension exercise where the VO2peak would be limited peripherally. CONCLUSION: The findings of this study suggest that the differential VO2 response between one-legged and two-legged exercise would be attributed not only to the difference in force application throughout the exercise movement and to the effect of a postural component but also to the inhibited circulatory response caused by the multiple limb exercise. In addition, it was supposed that VO2peak does not increase in proportion to the exercising muscle mass even during smaller muscle activity where the cardiac pumping capacity has not reached its upper limit.

Microphotometric method for measuring the oxygenation and deoxygenation rate in a single red blood cell.

A new reaction apparatus combining a microscope and a photometric device was developed for kinetic studies of a single red cell. A monolayer of red cells was placed in a closed reaction cuvette set on a microscope stage, a light beam of 5 to 10 mum in diameter was directed into one of the red cells, and the light transmission change in the cell was analyzed. The light beam with a wavelength range shorter than 460 nm was made by placing a narrow iris diaphragm in the light path. The space in the cuvette prevented the red cells from drying thereby providing favorable physiological conditions during measurements. The cuvette was filled with reagent gas mixtures of O2, CO2, and N2 which came in contact with the red cells. Transmission change due to the reaction was detected separately at two wavelengths of 418 and 402 nm by means of two photomultipliers mounted on the microscope. The linearity was tested by comparison between SO2 measured with a Van-Slyke apparatus and the microphotometer. Both SO2 measurements agreed well with each other, but the latter was about 3% greater than the former at around 50% SO2. Using this apparatus the oxygenation and deoxygenation velocities were measured over an entire O2-saturation range. The velocity factors showed good agreement with those obtained by using conventional flow methods.

Mathematical formulation of CO2 dissociation curve and buffer line of human blood at rest.

The CO2 content and pH of tonometered blood were measured in nine healthy subjects. The CO2 content in the whole blood (Cb) was found to be expressed by an exponential function of PCO2 including only one parameter (B) as follows: Cb = 1.15 . B-2.548 . PBCO2. The B value was specific to the sampled blood and ranged from 0.4 to 0.45 in the deoxygenated and from 0.45 to 0.52 in the oxygenated blood. The relationship between pH and log PCO2 was also expressed by using one characteristic parameter (D) as follows: for the deoxygenated blood, log PCO2 = 2.144-D . (pH-7.045), and for the oxygenated blood, log PCO2 = 2.037-D . (pH-7.085). The D values were in a range of 1.38 to 1.58. The linear relation between log [HCO3-] and pH was also expressed by using only one parameter. Next, between Cb and [HCO3-] obtained at the same PCO2 of 40 mmHg, a high correlation was observed: the regression line was given, independently of O2 saturation, by Cb40 = 1.942 . [HCO3-]40-3.193, where [HCO3-] was expressed in mM. Using the above equations, it was possible to evaluate the approximate B and D values from a pair of pH and PCO2 measurements and subsequently to depict the CO2 dissociation curve as well as the buffer line in the true plasma.

Relationship between venoarterial CO2 content difference and venoalveolar PCO2 difference in acute hypercapnia in dogs.

When mixed venous blood is oxygenated in alveolar air with higher PCO2, the PCO2 within the red cell is though to exceed the alveolar PCO2 due to the Haldane effect and to block the inward CO2 diffusion. If the direction of the CO2 diffusion is not reversed during the contact time, the HCO2-gain in the plasma will not exceed the amount estimated from venoalveolar PCO2 difference by using a CO2 dissociation curve of separated plasma. In order to clarify the validity of the above thought, the venoarterial CO2 content difference was measured by using a van Slyke apparatus and a PCO2 electrode at various alveolar PCO2 levels in rebreathing dogs. The HCO3-rise in the whole blood was obviously reduced when acute hypercapnia was administered in both normoxia and hyperoxia. Quantitatively, the decrease of CO2 content under hypercapnia corresponded to the difference in CO2 content between the true and separated plasma. The reduction, however, was slightly stronger in normoxia than in hyperoxia with alveolar PO2 of 300 to 420 mmHg. These data seem to support the following explanation: When venous blood was oxygenated in normoxic air with PCO2 higher than true venous, the inward CO2 diffusion was inhibited by the Haldane effect and the reversed diffusion after the oxygenation could also be disregarded during the contact time. Because the oxygenation was accelerated in hyperoxia and the direction of the CO2 diffusion was reversed earlier than in normoxia, the plasma CO2 content became higher in hyperoxia than in normoxia.

Derivation of theoretical equations of the CO2 dissociation curve and the carbamate fraction in the Haldane effect.

The simultaneous Henderson-Hasselbalch equations in plasma and red cell were solved in order to obtain the CO2 dissociation curve of oxygenated blood. In order to solve the above two equations the following equation was added, in which the relationship between the intracellular (delta pHC) and the extracellular pH change (delta pHP) was defined as follows: pHC = (1 + sigma) delta pHP, where 1 + sigma is a factor to be determined from experimental data on Donnan's ratio for H+. From the solution, the ratio of bicarbonate shift to the CO2 quantity released out of or combined with hemoglobin was calculated. The solution was validated by comparing the above ratio between the theoretical and experimental data. The CO2 contents calculated at 12 Torr in whole blood, red cell, and plasma compartments show good agreement with the respective analyzed values. When the buffer values of hemoglobin and plasma buffer protein were 70.0 and 7.5 mmol/(liter plasma X pH), respectively, sigma = -0.21 + 0.05 X delta pHP, and the Donnan's ratio for HCO3- was assumed to be 0.7 at pH = 7.33, the theoretical CO2 dissociation curve fitted well with the experimental curve. The CO2 dissociation curve of deoxygenated blood was expressed by adding the measured Haldane effect to the CO2 content of oxygenated blood. This additive characteristic in turn made it possible to estimate carbamate contribution in the Haldane effect.