Influence of venular prostaglandin release on arteriolar diameter during functional hyperemia.

Indomethacin treatment or removal of the venular endothelium will attenuate functional arteriolar vasodilation in the hamster cremaster muscle. We tested the hypothesis that prostanoid release from venular endothelial cells was responsible for the functional vasodilation of the paired arteriole. The hamster cremaster muscle was prepared for in vivo microscopy and stimulated for 1 minute (10 V, 40 microsec, 1 Hz). Before a second muscle stimulation, the venular endothelium was removed by perfusing the venule with several air bubbles. A third muscle stimulation was performed during prostaglandin inhibition (28 micromol/L indomethacin superfusion). Arterioles (n = 9, 55+/-5 microm) dilated 25+/-4% during the initial muscle stimulation. After removal of the endothelium from the paired venules, there was no effect on resting arteriolar diameters (53+/-4 microm), but the functional arteriolar dilation was attenuated to 15+/-5% (P<.05). The additional indomethacin treatment had a significant effect on resting diameter (50+/-4 microm) but did not alter the magnitude of the functional vasodilation (11+/-4%, P>.05). In a second set of experiments, the order of the experimental protocol was reversed. Muscle stimulation resulted in a 23+/-2% increase in diameter (47+/-2 to 57+/-2 microm). Indomethacin treatment significantly attenuated the functional dilation to 8+/-3% (45+/-2 to 48+/-2 microm). Arteriolar diameter was significantly smaller after disruption of the venular endothelium with air bubbles (40+/-2 microm), but there was no effect on the functional vasodilation, 8+/-3% increase in diameter (to 43+/-2 microm). These results suggest that the arteriolar dilatory response to muscle stimulation is mediated, in part, by prostanoid release from the venular endothelium.

Intestinal cryptosporidiosis: pathophysiologic alterations and specific cellular and humoral immune responses in rnu/+ and rnu/rnu (athymic) rats.

In order to develop an experimental model of symptomatic cryptosporidiosis in an immunosuppressed mammal, we investigated the pathophysiology of infection with Cryptosporidium and the humoral and cellular host responses in rnu/rnu (athymic) rats and their heterozygous (rnu/+) littermates by challenging suckling rats with greater than or equal to 2.5 x 10(6) Cryptosporidium oocytes oro-gastrically. Normal and immunodeficient animals were followed for onset and duration of infection (fecal oocysts), physiologic consequences (diarrhea, impaired weight gain, brush-border enzyme activities), and immunologic response (both B- and T-lymphocyte-mediated). Homozygosity for the rnu gene was associated with protracted cryptosporidial infections; shedding for up to 52 days occurred, and delay in weight gain was noted in rnu/rnu-infected compared with rnu/rnu-uninfected rats (p less than 0.05). In contrast, cryptosporidial challenge of rnu/+ rats resulted in self-resolving infections, occasionally with transient diarrhea lasting four days or less occurring 10-15 days after oro-gastric challenge. The latter animals mounted a cell-mediated immune response to Cryptosporidium: three months after challenge, five of five rnu/+ rats demonstrated positive skin test responses to a subcutaneous 3.5 micrograms dose of cryptosporidial antigen. Further, sera from 6 rnu/+ rats taken two to three months after oro-gastric oocyst challenge exhibited specific anticryptosporidial immunoglobulin binding (A405 = 0.96), compared to that of seven uninfected rnu/+ controls (A405 = 0.09, P less than 0.02). Macromolecules of 150, 105, and 88 kD in the Cryptosporidium antigen preparation were bound by serum immunoglobulin from previously infected, recovered rnu/+ rats. Two brush-border enzymes (lactase and alkaline phosphatase) were markedly reduced in the ileum 8-10 days after oro-gastric challenge in rats with diarrhea and oocyst shedding. We find the rnu/rnu (athymic, nude) rat provides a useful model for study of prolonged cryptosporidial infection with impaired weight loss, brush-border enzyme alteration and intermittent diarrhea. These studies further suggest that a T-lymphocyte population is involved in recovery from Cryptosporidium infection and that this recovery is associated with both cellular and humoral immune responses to specific cryptosporidial antigenic macromolecules. This model should open further avenues for the study of the pathogenesis and protective immunity in cryptosporidial infection.

Comparison of the effects of some muscarinic agonists on smooth muscle function and phosphatidylinositol turnover in the guinea-pig taenia caeci.

1. The effects of the muscarinic agonists acetylcholine (ACh), carbachol (CCh), AHR-602, and McN-A-343 on contractility and on inositol phosphate accumulation in the presence of lithium were compared in the taenia of the guinea-pig caecum. 2. Compared to CCh, ACh was a full agonist for contraction but AHR-602 and McN-A-343 were partial agonists producing 80-85% of the maximal response to CCh. Similar to previous findings with CCh, tonic contractions produced by AHR-602 and McN-A-343 were less sensitive to inhibition by nifedipine or verapamil than tonic contractions to ACh. 3. CCh and ACh produced similar increases in inositol phosphate accumulation and the effect of CCh (0.1 mM) was inhibited by atropine (IC50 8.5 nM) and pirenzepine (IC50 450 nM). The accumulation of inositol phosphates in the presence of AHR-602 or McN-A-343 was not significantly different (P greater than 0.05) from basal levels. 4. A concentration of 0.2 mM AHR-602 produced a parallel shift of the concentration-response curve to CCh on inositol phosphate accumulation. The IC50 value for inhibition of CCh (0.1 mM) was greater than 50 fold higher than the EC50 value for contraction produced by the partial agonist. McN-A-343 (20 microM) produced a flattening of the concentration-response curve to CCh for inositol phosphate accumulation. 5. The results suggest that the increase in phosphatidylinositol turnover produced by muscarinic agonists, like the contractile response, involves an M2-muscarinic receptor. AHR-602 and McN-A-343 are partial agonists for the contractile response and while producing no significant increase in phosphatidylinositol turnover inhibit the response to CCh.

Paraoxon-induced desensitization and its reversal by tetrodotoxin.

1. Paraoxon (10 microM for 20 min) induced a desensitization of the taenia caecum of the guinea-pig for contractions produced by a number of cholinomimetics. 2. Tetrodotoxin (0.3 microM) reversed the desensitization suggesting involvement of Na+ channels.

Endothelial seeding of polytetrafluoroethylene popliteal bypasses. A preliminary report.

At St. Vincent Hospital, Indianapolis, 17 patients have undergone femoropopliteal bypass operations with polytetrafluoroethylene (PTFE) grafts that were seeded with enzymatically harvested, autogenous endothelium. Three patients received seeded grafts because satisfactory veins were not available. Twenty-eight patients alternately received seeded or unseeded, externally supported e-PTFE grafts. Graft patency was evaluated by clinical criteria and changes in the Doppler ankle-brachial systolic pressure ratios at 2 and 30 days postoperatively and at 3-month intervals thereafter. Occlusions were defined arteriographically if the clinical situation or the Doppler findings deteriorated. Smoking histories were taken, and carboxyhemoglobin (COHgb) levels were sampled 1 month postoperatively. Cumulative patency after 3 months was 93.3% +/- 6.5% for seeded and 84.0% +/- 10.4% for unseeded grafts. After 1 year it was 81.6% +/- 12.3% for seeded grafts and 30.8% +/- 18.7% for unseeded grafts (p = 0.02). Thus far all but one of the occlusions have occurred in patients with a history of smoking or with a COHgb level greater than 1.5%, whereas all of the seeded grafts in nonsmokers with COHgb levels less than or equal to 1.5% are patent. We conclude that endothelial seeding of PTFE femoropopliteal grafts is feasible. During this preliminary study period, a small number of patients had favorable patency rates with seeded as compared with unseeded grafts, especially among smokers. A multiple-institution study will be needed to establish the role of endothelial cell seeding in the treatment of vascular occlusions of the femoral and popliteal arteries.

A comparison of Doppler flow measurements, ultrasonic and arteriographic imaging in the evaluation of stenoses of the carotid artery.

To determine the value of Doppler periorbital flow measurements (DOP) and real-time ultrasonic in the analysis of carotid artery occlusive disease, 1,203 carotid arteries were evaluated in 669 consecutive patients from 1 June 1980 to 1 July 1981. The results of DOP and US were compared with biplane carotid arteriograms using the Seldinger technique. The stenoses were graded: Grade I (zero to 25 per cent), Grade II (25 to 50 per cent), Grade III (50 to 75 per cent) and Grade IV (75 to 100 per cent). Both US and CA agreed in 71.3 per cent of 209 arteries. US and DOP were performed together in 1,203 arteries and agreed in 83.2 per cent. The flow surfaces of an additional 29 arteries were visually inspected at operation to determine the capacity of US to predict ulceration. All of the arteries demonstrated visible ulceration, but only six (20.7 per cent) were predicted by US. The deepest ulcerations that were not detected by US were 2 millimeters in depth. US images were unsatisfactory in 44 arteries (3.7 per cent) because of excessive sound reflection and in 60 (5.0 per cent) because of a "high bifurcation." We conclude that Doppler periorbital flow studies are not as accurate as carotid sonography nor do they add to the accuracy of sonography when the two examinations are combined. Sonography identified significant stenoses but failed to grade the stenoses well and was unreliable in detecting shallow to moderately deep ulcerations.

Expression of the Bacillus subtilis glutamine synthetase gene in Escherichia coli.

The structural gene for glutamine synthetase (glnA) in Bacillus subtilis ( glnAB ) cloned in the lambda vector phage Charon 4A was used to transduce a lysogenic glutamine auxotrophic Escherichia coli strain to prototrophy. The defective E. coli gene ( glnAE ) was still present in the transductant since it could be transduced. In addition, curing of the prototroph resulted in the restoration of glutamine auxotrophy. Proteins in crude extracts of the transductant were examined by a "Western blotting" procedure for the presence of B. subtilis or E. coli glutamine synthetase antigen; only the former was detected. Growth of the strain in media without glutamine was not curtailed even when the bacteriophage lambda pL and pRM promoters were hyperrepressed . The specific activities and patterns of derepression of glutamine synthetase in the transductant were similar to those of B. subtilis, with no evidence for adenylylation. The information necessary for regulation of glnAB must be closely linked to the gene and appears to function in E. coli.

Timing of stages and changes in numbers of multiply-infected erythrocytes during the asexual cycle of Plasmodium cynomolgi bastianellii.

Previous evaluations of the timing of developing stages in the erythrocytic cycle of the simian malaria parasite, Plasmodium cynomolgi, a frequently used model for relapsing malaria in man, have depended on procurement of blood samples at 4- or 6-hourly intervals. This brief report describes the blood cycle as determined from hourly bleedings, thus providing a more discriminating sequence usable for immunochemical or biochemical study of specific stages. The approximate timing of development for five defined stages in the asexual cycle was determined to be 12-14 hours for young trophozoites, 22-24 hours for developing trophozoites, 5-7 hours for mature trophozoites, 5-6 hours for early schizonts, and 3-5 hours for late schizonts. It was also demonstrated that the rate of erythrocytic multiple infection declined in proportion to the maturity of the parasite. Young and developing trophozoites presented the most numerous cases of multiple infection whereas very few schizonts were seen multiply infecting a host cell.

Cultivation in vitro of the vivax-type malaria parasite Plasmodium cynomolgi.

The vivax-type simian malaria parasite Plasmodium cynomologi was cultured in vitro by both the candle jar method and the continuous flow technique, with rhesus monkey erythrocytes and RPMI 1640 medium supplemented with Hepes buffer and human serum. After 6 weeks in culture, the growth of the parasite had permitted a 5 X 10(6) cumulative dilution of the original inoculum. Cultured parasites remained infective to rhesus monkeys and exhibited a reversible decrease in the ameboid behavior of their trophozoites.