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  The effects of G-6-PDH deficiency on RBC functions during polymicrobial sepsis are not known. Therefore, in the current study, we tested the hypothesis that a moderate degree of G-6-PDH deficiency would increase the magnitude of sepsis-induced RBC ...

        MATERIALS AND METHODS       TOP   ABSTRACT   MATERIALS AND METHODS   RESULTS   DISCUSSION   GRANTS   REFERENCES     Animals.  Breeding pairs of G-6-PDH-mutant mice ( 24, 36, 43 ) were purchased from the Medical Research Council (MRC) of the U...

  Male G-6-PDH-deficient (y/–) and normal (WT, y/+) 10- to 12-wk-old animals were used in the experiments to allow full maturation of all RBCs in these animals (RBC life span is 40 days in mice). Animals used in the experiments were phenotyped by G-6...

   Genotyping.  The mutant G-6-PDH variant was identified as described previously ( 36, 43 ). The particular mutation in the G-6-PDH gene results in the disappearance of a Dde I restriction site in the mutant gene. Dde I results in full digestion of ...

   Cecal ligation and puncture and in vivo treatments.  Polymicrobial sepsis was induced with the cecal ligation and puncture (CLP) model as described previously ( 1, 19 ). Briefly, animals were anesthetized by a subcutaneous injection of Nembutal (5...

   Separation of erythrocyte subpopulations by centrifugal elutriation.  Circulating erythrocytes can be separated into different subpopulations by size, age, and density with counterflow centrifugal elutriation as we described previously with slight...

   Determination of erythrocyte deformability.  RBC deformability in whole blood or isolated RBC subpopulations was analyzed with a laser-assisted ectacytometer (LORCA; RR Mechatronics, Hoorn, The Netherlands; Ref. 14 ). An aliquot containing 30 mill...

   Glutathione determinations.  GSH and GSSG in RBCs and plasma were determined using the protocol of Tietze ( 49 ). In brief, after the in vivo treatments, blood was drawn into heparinized tubes. From the freshly obtained blood, RBCs were sedimented...

  Plasma hemoglobin was determined by the measurement of absorbance at 405-, 410-, 415-, and 430-nm wavelengths. Plasma hemoglobin concentrations were calculated with the molar extinction coefficient of oxyhemoglobin E 415 (1.25 x 10 5 ). Calculation...

   RBC protein distribution.  The distribution of major protein constituents of circulating RBCs was determined from water- and Triton-soluble sample extracts prepared from RBC ghosts as follows. RBC was sedimented from heparinized blood samples (0.5...

   Reagents.  When applicable, cell culture-grade buffers, media, and reagents were used. Hanks' balanced salt solution, without phenol red, and Dulbecco's PBS were purchased from Life Technologies (Grand Island, NY). Buffers were sterile filtered an...

   Statistical analysis.  Statistical calculations were performed with JMP software (SAS Institute, Cary, NC). Results were analyzed by ANOVA followed by t -test for pairwise comparisons or Tukey-Kramer's test for multiple comparisons. Statistically ...

        RESULTS       TOP   ABSTRACT   MATERIALS AND METHODS   RESULTS   DISCUSSION   GRANTS   REFERENCES     Characterization of mouse model.   Figure 1 A shows G-6-PDH activity in whole blood in animals derived from the breeding colony and in two c...

          View larger version (34K):  [in this window]  [in a new window]  Fig. 1. Glucose-6-phosphate (G-6-PDH) activity and genotyping. A : red blood cell (RBC) G-6-PDH activity in littermates of hemizygous (–/y) and wild-type (WT) (+/y) male anima...

        Sepsis-induced changes in hematologic parameters and RBC deformability.  Baseline values of circulating RBC counts and blood hemoglobin content were similar in unchallenged deficient and WT animals. After sepsis (24 h), RBC counts and blood h...

          View larger version (31K):  [in this window]  [in a new window]  Fig. 2. Comparison of hematologic changes in G-6-PDH-deficient (–/y) and WT (+/y) animals after sepsis. Cell counts ( A, D, E, and F ) and hemoglobin ( B and C ) were determin...

        Figure 3 compares the sepsis-induced changes in RBC deformability. Whereas RBC deformability shear stress response curves overlapped in naive unchallenged deficient and WT animals, sepsis resulted in significant decrease in RBC deformability ...

          View larger version (33K):  [in this window]  [in a new window]  Fig. 3. Comparison of RBC deformability between G-6-PDH-deficient and WT animals after sepsis. Erythrocyte deformability in blood from septic or naive G-6-PDH-deficient and WT...

        Alterations in RBC subpopulations and band 3 abundance.  Circulating erythrocytes can be separated into different subpopulations. With centrifugal elutriation, smaller-sized (older) cells elute at lower and larger-sized (younger) cells at hig...

          View larger version (27K):  [in this window]  [in a new window]  Fig. 4. Comparison of erythrocyte subpopulations between G-6-PDH-deficient and WT animals after sepsis. Whole blood samples from naive or septic (24 h after CLP) animals were ...

       Because alterations in band 3 protein organization have been shown to be important in oxidative RBC damage, aging, and clearance, we tested the effects of G-6-PDH deficiency and sepsis on the association between band 3 and the cytoskeleton. Fi...

          View larger version (58K):  [in this window]  [in a new window]  Fig. 5. Elevated association between band 3 and spectrin in G-6-PDH-deficient RBCs. Triton-soluble protein distribution was determined in RBC ghosts as described in MATERIALS ...

        Role of oxidative stress.  In a separate set of experiments, we compared sepsis-induced alterations in blood glutathione metabolism between deficient and WT animals ( Fig. 6 ). Sepsis resulted in an increase in plasma total glutathione (predo...

          View larger version (40K):  [in this window]  [in a new window]  Fig. 6. Sepsis-induced alterations in RBC glutathione content in G-6-PDH-deficient and WT animals. GSH and GSSG were determined in RBC as described in MATERIALS AND METHODS. A...

       The elevated plasma glutathione levels indicated that sepsis-induced oxidative stress was greater in G-6-PDH-deficient than WT animals. To further elucidate the potential role of oxidative stress in the observed alterations in RBC deformabilit...

          View larger version (20K):  [in this window]  [in a new window]  Fig. 7. N -acetyl-l-cysteine (NAC) in vivo alleviates RBC deformability decrease in septic G-6-PDH-deficient animals. Sham-operated or septic animals were treated by subcutane...

             DISCUSSION       TOP   ABSTRACT   MATERIALS AND METHODS   RESULTS   DISCUSSION   GRANTS   REFERENCES    This study demonstrates for the first time that G-6-PDH deficiency exacerbates the magnitude of decreased RBC deformability after pol...

  It is well known that the life span of circulating erythrocytes is 60 days (half of normal) in the class III human deficiencies, even in otherwise healthy individuals ( 34 ). In mice, the life span of circulating erythrocytes is 40 days, and it is ...

  The more pronounced decrease in erythrocyte deformability in septic deficient vs. WT animals was readily detectible in whole blood ( Fig. 3 ) as well as after comparing erythrocyte subpopulations ( Fig. 4 H ). However, the differences in decreased ...

  Elevated membrane abundance of band 3 tetramers is accompanied by increased associations between band 3 and the cytoskeleton. Elevated band 3 association with the cytoskeleton has been shown to parallel the decrease in RBC deformability ( 21, 39 ) ...

  The role of oxidative stress in causing decreased RBC deformability during the physiological aging process of erythrocytes is well documented ( 5, 13 ). In fact, during the normal process of oxygen exchange, erythrocytes are exposed to oxidative st...

  The notion that oxidative stress is greater in G-6-PDH-deficient animals is further supported by our findings that NAC alleviated the decrease in erythrocyte deformability in septic deficient animals. However, this observation must be interpreted w...

  It is evident from our findings that polymicrobial sepsis resulted in a similar decrease in the number of circulating neutrophils, lymphocytes, and platelets in deficient and WT animals at 24 h after CLP, although the G-6-PDH-deficient animals deve...

  Infection-induced anemia in critically ill patients is a common occurrence with important clinical implications ( 37, 45, 48, 50 ). In previous studies using the CLP septic model, anemia was observed in BALB/c mice ( 20 ) and young (4–5 wk old) C57...

  Previous investigations on severely injured G-6-PDH-deficient trauma patients indicated that these patients had an increased incidence of sepsis, a longer duration of the systemic inflammatory response syndrome, augmented monocyte activation, blunt...

  The role of G-6-PDH deficiency in the protection against malaria is well accepted; however, the mechanism of protection remains only partially elucidated ( 10, 12, 42 ). Our current observations may have potential implications in this context as we...

  Together, our observations indicating a pronounced decrease in RBC deformability in septic G-6-PDH-deficient animals suggest a role of RBC dysfunction in the immunomodulatory effects of G-6-PDH deficiency. The observed RBC dysfunction may aggravate...

        GRANTS       TOP   ABSTRACT   MATERIALS AND METHODS   RESULTS   DISCUSSION   GRANTS   REFERENCES    This study was supported by National Institute of General Medical Sciences Grant GM-55005. 

   

         ACKNOWLEDGMENTS    We thank Pietro Antoneli for technical assistance in the studies. 

         FOOTNOTES     var u = "spolaric", d = "umdnj.edu"; document.getElementById("em0").innerHTML = ' ' + u + '@' + d + '<\/a>'//--> ).

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           REFERENCES       TOP   ABSTRACT   MATERIALS AND METHODS   RESULTS   DISCUSSION   GRANTS   REFERENCES       Baker CC, Chaudry IH, Gaines HO, and Baue AE. Evaluation of factors affecting mortality rate after sepsis in a murine cecal ligation...

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