Prophylactic indomethacin infusion increases fractional cerebral oxygen extraction in ELBW neonates.

OBJECTIVE: Intraventricular hemorrhage (IVH) occurs in up to 25% of very low birth weight (VLBW) preterm neonates. Previous studies found that indomethacin administered in the first 6 h of life reduces the incidence of severe IVH in VLBW neonates and decreases cerebral blood flow, suggesting a decrease in cerebral oxygen delivery. Using near-infrared spectroscopy (NIRS), we monitored cerebral oxygenation before, during and after slow indomethacin infusion in extremely low birth weight (ELBW) neonates to determine whether indomethacin decreases cerebral oxygen saturation and increases cerebral oxygen extraction. STUDY DESIGN: Twenty-seven ELBW neonates less than 30 weeks gestational age treated with indomethacin for IVH prophylaxis were monitored for arterial oxygen saturation (SaO(2)) and NIRS-determined regional cerebral oxygen saturation (rSO(2)). At 30 to 60 s intervals, SaO(2), rSO(2) and mean arterial pressure (MAP) were recorded using a VitalSync. Average fractional cerebral oxygen extraction was calculated for the hour before indomethacin infusion, during the infusion and 2 h after infusion. RESULT: Fractional cerebral oxygen extraction increased from baseline after indomethacin administration from 0.23±0.11 to 0.25±0.10 (P=0.034). CONCLUSION: Fractional cerebral oxygen extraction increased 9% with indomethacin 0.1 mg kg(-1) given over 1 to 2 h. However, the clinical implications of this small increase in extraction, likely representative of decreased cerebral perfusion, are unknown but may be harmful to the developing brain.

Differential inhibition of the Fas- and granule-mediated cytolysis pathways by the orthopoxvirus cytokine response modifier A/SPI-2 and SPI-1 protein.

Cytotoxic T lymphocytes are important effectors of antiviral immunity, and they induce target cell death either by secretion of cytoplasmic granules containing perforin and granzymes or by signaling through the Fas cell surface antigen. Although it is not known whether the granule-mediated and Fas-mediated cytolytic mechanisms share common components, proteinase activity has been implicated as an important feature of both pathways. The orthopoxviruses cowpox virus and rabbitpox virus each encode three members of the serpin family of proteinase inhibitors, designated SPI-1, SPI-2, and SPI-3. Of these, SPI-2 (also referred to as cytokine response modifier A in cowpox virus) has been shown to inhibit the proteolytic activity of both members of the interleukin 1 beta converting enzyme family and granzyme B. We report here that cells infected with cowpox or rabbitpox viruses exhibit resistance to cytolysis by either cytolytic mechanism. Whereas mutation of the cytokine response modifier A/SPI-2 gene was necessary to relieve inhibition of Fasmediated cytolysis, in some cell types mutation of SPI-1, in addition to cytokine response modifier A/SPI-2, was necessary to completely abrogate inhibition. In contrast, viral inhibition of granule-mediated killing was unaffected by mutation of cytokine response modifier A/SPI-2 alone, and it was relieved only when both the cytokine response modifier A/SPI-2 and SPI-1 genes were inactivated. These results suggest that an interleukin 1 beta converting enzyme-like enzymatic activity is involved in both killing mechanisms and indicate that two viral proteins, SPI-1 and cytokine response modifier A/SPI-2, are necessary to inhibit both cytolysis pathways.

Activation of recombinant murine cytotoxic cell proteinase-1 requires deletion of an amino-terminal dipeptide.

Murine cytotoxic cell proteinase-1 (granzyme B) is a member of a family of novel serine proteinases that have been implicated to participate in destruction of target cells by cytotoxic T lymphocytes. Comparison of the sequence of the cDNA with the sequence of the protein isolated from cytoplasmic granules of cytotoxic T lymphocytes suggested that this protein may be synthesized as a preproenzyme containing an amino-terminal activation dipeptide. Here we show that this activation dipeptide regulates the activity of the enzyme in hydrolysis of its preferred substrate tert-butyloxycarbonyl-Ala-Ala-Asp-thiobenzyl ester. Lysates of COS cells transfected with a vector expressing the unmodified cytotoxic cell proteinase-1 cDNA were unable to hydrolyze this substrate, whereas lysates of cells transfected with a construct in which the activation dipeptide codons has been deleted were able to hydrolyze the substrate. In each case Western blotting of the lysates revealed a form of the proteinase with an apparent molecular weight of 27,000. We conclude that the activation dipeptide regulates activity of the enzyme. This is the first report of production of an enzymatically active recombinant cytotoxic T cell serine proteinase. The strategy for successful expression of an activated form of cytotoxic cell proteinase-1 may be applicable to other members of this proteinase family.

Incidence of antibiotic resistance and characterization of plasmids in Campylobacter jejuni strains isolated from clinical sources in Alberta, Canada.

Antibiotic susceptibilities of 382 strains of Campylobacter jejuni isolated in Alberta, Canada, in 1980 and 1981 were determined. Although none were resistant to erythromycin or gentamicin, 5.4 and 22% of strains were resistant to ampicillin in 1980 and 1981, respectively. Tetracycline resistance was noted in 6.8% of strains in 1980 and in 8.6% in 1981. Moreover, resistance to high-level tetracycline (32-128 micrograms/mL) was always mediated by a plasmid of 45-50 kilobases. Three transmissible plasmids from the Alberta strains were compared with the prototype plasmid pMAK175 by restriction enzyme analysis and some minor differences in restriction sites were noted between pMAK175 and pUA183. The two other plasmids pUA142 and pUA143 were 4 kilobases larger than pMAK175 and contained additional restriction sites. However, in all plasmids examined, the HincII and AccI fragments where the tetracycline-resistance determinant was located were shown to be conserved.

Characterization of tetracycline resistance plasmids from Campylobacter jejuni and Campylobacter coli.

Tetracycline resistance in strains of Campylobacter jejuni and Campylobacter coli was mediated by plasmids. Intra- and interspecies transfer was demonstrated within the genus Campylobacter. Buoyant densities of plasmid DNAs ranged from 1.691 to 1.694 g/cm3 (31 to 33% guanine plus cytosine). Restriction enzymes AccI, BclI, BglII, and PstI were found to be most useful for comparing the plasmids. The molecular weight of C. jejuni plasmid pMAK175 was 44.7 kilobases (29 X 10(6), and the other plasmids had similar sizes. Two plasmids from Belgian isolates of C. coli of human origin had very similar restriction enzyme profiles and are probably identical. Plasmids from human isolates of C. jejuni originating in Canada and the animal isolate of C. coli showed greater diversity. DNA homology among the campylobacter plasmids was assessed by probing the digests with a nick-translated campylobacter plasmid, pMAK175. All restriction fragments showed significant homology with pMAK175 probe DNA. No homology was noted between campylobacter plasmid DNA and plasmids specifying the four classes of tetracycline resistance determinants found in Enterobacteriaceae.