Impact of blood transfusion and burn injury on microbial translocation and bacterial survival

The role of the immune system in microbial translocation must be clarified. In these studies, the effect of blood transfusion-related immunosuppression on translocation was investigated in a burn animal model previously known to increase the gut's permeability to 14C-radiolabeled Escherichia coli. In a first experiment, Balb/c mice underwent transfusion (T) with 0.2 mL per mouse of allogeneic C3H/HeJ mouse blood 5 days prior to undergoing 30-percent burn injury (B) and simultaneous gavage (G) with 10(9) E. coli bacteria labeled with 14C. An additional six groups of Balb/c mice underwent different combinations of T, B, and G procedures (TG, BG, TB, T, B, G). Survival rate was recorded for all groups on Day 10. This experiment suggested that B and T, to a lesser extent, were the factors affecting survival, although the combination of T, B, and G clearly showed a synergistic effect on mortality. In a second experiment, 18 Balb/c mice belonging to TBG, BG, TG, and G groups were sacrificed 1, 4, and 24 hours after burn or gavage. The residual radioactivity and the percentage of viable bacteria were computed for mesenteric lymph nodes, spleen, liver, lungs, blood, and peritoneal fluid. Statistical analysis of the radionuclide counts recognized B as the only variable able to enhance the magnitude of 14C E. coli translocation. The percentage of viable bacteria showed that T and, more moderately, B were the factors leading to the failure of bacterial clearance in the tissues.     

Institution-specific versus published in vitro antimicrobial susceptibility data in making formulary decisions

The results of susceptibility testing of 549 isolates of gram-negative organisms to 17 antimicrobial agents were compared with published reports of the sensitivity of those organisms to those agents. All gram-negative bacilli isolated from cultures obtained from hospitalized patients during a three-month period were preserved for antimicrobial sensitivity testing. Standard Kirby-Bauer disk diffusion susceptibility tests were performed using 17 broad-spectrum antimicrobial agents that either were included in the hospital formulary or were being considered for inclusion. Organisms were recorded as being sensitive or resistant to each drug, and the results were compared with the published results of in vitro sensitivity studies. When the results of actual antimicrobial sensitivity testing varied from published results, the discordant results were assigned a ranking of 1 to 4 based on the percentage difference. In 34 of 77 drug-organism pairs tested, the results of susceptibility testing differed by more than 10% cumulative susceptibility from published values; 26 of these represented instances in which the results of actual testing were at least 10% less than published values. For seven of the antimicrobial agents that were being considered for inclusion in the hospital formulary, results indicating unexpectedly suboptimal activity against institutional pathogens were a determinant in eliminating the agents from further consideration. In vitro testing of antimicrobial susceptibility of local pathogens can be a better method of predicting the susceptibility of such pathogens to new antimicrobial agents than relying on published susceptibility data. Pharmacy and therapeutics committees should consider testing prevalent institutional pathogens for susceptibility to all antimicrobial agents that are proposed additions to the formulary.     

Sperm-specific protein kinase A catalytic subunit Calpha2 orchestrates cAMP signaling for male fertility

An unusual cAMP signaling system mediates many of the events that prepare spermatozoa to meet the egg. Its components include the atypical, bicarbonate-stimulated, sperm adenylyl cyclase and a cAMP-dependent protein kinase (PKA) with the unique catalytic subunit termed Calpha(2) or C(s). We generated mice that lack Calpha(2) to determine its importance in the events downstream of cAMP production. Male Calpha(2) null mice produce normal numbers of sperm that swim spontaneously in vitro. Thus, Calpha(2) has no required role in formation of a functional flagellum or the initiation of motility. In contrast, we find that Calpha(2) is required for bicarbonate to speed the flagellar beat and facilitate Ca(2+) entry channels. In addition, Calpha(2) is needed for the protein tyrosine phosphorylation that occurs late in the sequence of sperm maturation and for a negative feedback control of cAMP production, revealed here. Consistent with these specific defects in several important sperm functions, Calpha(2) null males are infertile despite normal mating behavior. These results define several crucial roles of PKA in sperm cell biology, bringing together both known and unique PKA-mediated events that are necessary for male fertility.     

An educational intervention to reduce ventilator-associated pneumonia in an integrated health system: a comparison of effects

STUDY OBJECTIVES: To determine whether an educational initiative could decrease rates of ventilator-associated pneumonia in a regional health-care system. SETTING: Two teaching hospitals (one adult, one pediatric) and two community hospitals in an integrated health system. DESIGN: Preintervention and postintervention observational study. PATIENTS: Patients admitted to the four participating hospitals between January 1, 1999, and June 30, 2002, who acquired ventilator-associated pneumonia. INTERVENTION: An educational program for respiratory care practitioners and ICU nurses emphasizing correct practices for the prevention of ventilator-associated pneumonia. The program included a self-study module on risk factors for, and strategies to prevent, ventilator-associated pneumonia and education-based in-services. Fact sheets and posters reinforcing the information were posted throughout the ICU and respiratory care departments. MEASUREMENTS AND RESULTS: Completion rates for the module were calculated by job title at each hospital. Rates of ventilator-associated pneumonia per 1,000 ventilator days were calculated for all hospitals combined and for each hospital separately. Overall 635 of 792 ICU nurses (80.1%) and 215 of 239 respiratory therapists (89.9%) completed the study module. There were 874 episodes of ventilator-associated pneumonia at the four hospitals during the 3.5-year study period out of 129,527 ventilator days. Ventilator-associated pneumonia rates for all four hospitals combined dropped by 46%, from 8.75/1,000 ventilator days in the year prior to the intervention to 4.74/1,000 ventilator days in the 18 months following the intervention (p < 0.001). Statistically significant decreased rates were observed at the pediatric hospital and at two of the three adult hospitals. No change in rates was seen at the community hospital with the lowest rate of study module completion among respiratory therapists (56%). CONCLUSIONS: Educational interventions can be associated with decreased rates of ventilator-associated pneumonia in the ICU setting. The involvement of respiratory therapy staff in addition to ICU nurses is important for the success of educational programs aimed at the prevention of ventilator-associated pneumonia.     

Biallelic expression of the H19 gene during spontaneous hepatocarcinogenesis in the albumin SV40 T antigen transgenic rat

Previous studies in this laboratory have demonstrated that the earliest cytogenetic alteration in the development of hepatic neoplasms in a transgenic strain of rats bearing the albumin Simian virus 40 T antigen (Alb SV40 T Ag) construct was a duplication of the chromosome 1q4.1-1q4.2 band. In this region, in the rat genome a cluster of linked imprinted genes occurs. One of these imprinted genes, H19, which is expressed in fetal liver but not in adult liver, was found to be expressed in virtually all neoplasms investigated. A single-nucleotide polymorphic marker in the H19 coding sequence was identified in two rat strains and utilized for the investigation of H19 imprinting. Our results reveal monoallelic expression of the maternal gene in fetal liver, but biallelic expression of the H19 gene in liver neoplasms, thus demonstrating the basis for the deregulation of the imprinted gene expression during hepatocarcinogenesis. These results suggest that the loss of genomic imprinting of the H19 gene found in the liver neoplasms of the Alb SV40 T Ag rat may result not from allelic loss, but from adverse changes in the epigenetic imprints present in the 5'-upstream region of the H19 promoter of the parental alleles.     

Liver-specific pRB loss results in ectopic cell cycle entry and aberrant ploidy

The liver exhibits an exquisitely controlled cell cycle, wherein hepatocytes are maintained in quiescence until stimulated to proliferate. The retinoblastoma tumor suppressor, pRB, plays a central role in proliferative control by inhibiting inappropriate cell cycle entry. In many cases, liver cancer arises due to aberrant cycles of proliferation, and correspondingly, pRB is functionally inactivated in the majority of hepatocellular carcinomas. Therefore, to determine how pRB loss may provide conditions permissive for deregulated hepatocyte proliferation, we investigated the consequence of somatic pRB inactivation in murine liver. We show that liver-specific pRB loss results in E2F target gene deregulation and elevated cell cycle progression during post-natal growth. However, in adult livers, E2F targets are repressed and hepatocytes become quiescent independent of pRB, suggesting that other factors may compensate for pRB loss. Therefore, to probe the consequences of acute pRB inactivation in livers of adult mice, we gave adenoviral-Cre by i.v. injection. We show that acute pRB loss is sufficient to elicit E2F target gene expression and cell cycle entry in adult liver, demonstrating a critical role for pRB in maintaining hepatocyte quiescence. Finally, we show that liver-specific pRB loss results in the development of nuclear pleomorphism associated with elevated ploidy that is evident in adult mice harboring both acute and chronic pRB loss. Together, these results show the crucial role played by pRB in maintaining hepatocyte quiescence and ploidy in adult liver in vivo and underscore the critical importance of delineating the consequences of acute pRB loss in adult animals.     

In vivo induction of type 1 and 2 immune responses against protein antigens

Polarization of the immune response towards Th1 or Th2 profiles is under the control of several, not yet well known, mechanisms. The present study was undertaken to investigate whether immune responses generated against major protein antigens, of parasitic (Schistosoma mansoni) and bacterial (Clostridium tetani) origin, present characteristic Th profiles. Mice were immunized with a single dose of S. mansoni 28 kDa glutathione-S-transferase (Sm28-GST) or tetanus toxin fragment c (TTc) in combination with different adjuvants, or Salmonelia typhimurium expressing these antigens as a fusion protein. Antigen- specific IgG isotypes and cytokine mRNA expression in vivo, as well as cytokine secretion after in vitro antigen stimulation were studied. Immunizations with either protein in aluminum hydroxide induced a strong Th2-associated antibody (IgG1) and cytokine (IL-4) response. In contrast, the recombinant S. typhimurium, expressing the TTc/Sm28-GST fusion protein, induced a Th1-like response, associated with the production of IFN-gamma and IgG2a antibodies against both antigens. When complete Freund's adjuvant was used, a non-polarized profile was observed, characterized by expression of both IL-4 and IFN-gamma, as well as strong specific IgG1 and IgG2a antibody responses. These results indicated that some protein antigens play a weak role in polarizing the immune response and that contrasting cytokine profiles could be induced against the same antigen, depending on the adjuvant employed.      

Distribution and survival of Escherichia coli translocating from the intestine after thermal injury.

The present investigation was performed to study the kinetics of tissue distribution and deposition of Escherichia coli and endotoxin translocating from the intestine after thermal injury. Escherichia coli was grown in the presence of 14C glucose and both labeled bacteria and endotoxin prepared from the labeled bacteria were used as translocation probes. Escherichia coli (10(8) to 10(10) bacteria) and E. coli endotoxin (100 micrograms per animal) were gavaged into the stomach immediately before a 30% burn injury was inflicted in mice. Animals were killed 1, 4 and 24 hours after burn injury. Translocation occurred extensively within 1 hour after burn injury. Expressed as amount of radioactivity per gram of tissue, translocation was greatest in the mesenteric lymph node (MLN) followed by spleen, lung, and liver. Translocation of endotoxin was similar to translocation of intact bacteria, with the exception that less radioactivity could be found in the peritoneal cavity and more in the liver. Both intact E. coli and endotoxin translocated directly through the intact bowel wall. Killing of bacteria was greatest in the MLN and spleen, approximating 95% to more than 99% of translocating bacteria. Killing efficiency was lowest in the lungs. It is concluded that estimation of translocation by viable bacterial counts in tissues grossly underestimates the extent of translocation of bacteria and ignores the extent of translocation of endotoxin. Translocation of endotoxin may have biologic significance that is independent of and in addition to translocation of intact bacteria.