Veno-right ventricular cannulation reduces recirculation in extracorporeal membrane oxygenation.

BACKGROUND: Veno-venous extracorporeal membrane oxygenation has several advantages over veno-arterial support for patients with severe reversible respiratory failure. However, recirculation can limit oxygen delivery as pump flow increases. This could be ameliorated by placing the return catheter in the right ventricle instead of the central veins. We compared recirculation in veno-right ventricular support with that in conventional veno-venous support and its relationship with pump flow. METHODS: Five greyhound dogs were sequentially cannulated percutaneously for both veno-venous and veno-right ventricular support. Recirculation was measured by comparing oxygen levels in the circuit drainage and return lines before and immediately after a sudden increase in circuit oxygenation at pump flows between 0.5 L/min and 4 L/min for both modalities. RESULTS: Recirculation was reduced in veno-right ventricular support compared with conventional veno-venous support at 4 L/min pump flow (8.4% versus 37.9%, p=0.0076) and increased less with increases in pump flow (2.9% per 1 L/min vs. 11.1% per 1 L/min, p<0.0001). CONCLUSIONS: Recirculation can be dramatically reduced by returning blood into the right ventricle, which improves oxygen delivery to the lungs and the systemic circulation. The design of specialized catheters may facilitate percutaneous ventricular cannulation, improve safety and further reduce recirculation.

Antitumor effects of ajulemic acid (CT3), a synthetic non-psychoactive cannabinoid.

One of the endogenous transformation products of tetrahydrocannabinol (THC) is THC-11-oic acid, and ajulemic acid (AJA; dimethylheptyl-THC-11-oic acid) is a side-chain synthetic analog of THC-11-oic acid. In preclinical studies, AJA has been found to be a potent anti-inflammatory agent without psychoactive properties. Based on recent reports suggesting antitumor effects of cannabinoids (CBs), we assessed the potential of AJA as an antitumor agent. AJA proved to be approximately one-half as potent as THC in inhibiting tumor growth in vitro against a variety of neoplastic cell lines. However, its in vitro effects lasted longer. The antitumor effect was stereospecific, suggesting receptor mediation. Unlike THC, however, whose effect was blocked by both CB(1) and CB(2) receptor antagonists, the effect of AJA was inhibited by only the CB(2) antagonist. Additionally, incubation of C6 glioma cells with AJA resulted in the formation of lipid droplets, the number of which increased over time; this effect was noted to a much greater extent after AJA than after THC and was not seen in WI-38 cells, a human normal fibroblast cell line. Analysis of incorporation of radiolabeled fatty acids revealed a marked accumulation of triglycerides in AJA-treated cells at concentrations that produced tumor growth inhibition. Finally, AJA, administered p.o. to nude mice at a dosage several orders of magnitude below that which produces toxicity, inhibited the growth of subcutaneously implanted U87 human glioma cells modestly but significantly. We conclude that AJA acts to produce significant antitumor activity and effects its actions primarily via CB(2) receptors. Its very favorable toxicity profile, including lack of psychoactivity, makes it suitable for chronic usage. Further studies are warranted to determine its optimal role as an antitumor agent.

Association of increased phosphatidylinositol 3-kinase signaling with increased invasiveness and gelatinase activity in malignant gliomas.

OBJECT: Glioblastoma multiforme is the most malignant of the primary brain tumors and aggressively infiltrates surrounding brain tissue, resulting in distant foci within the central nervous system, thereby rendering this tumor surgically incurable. The recent findings that both phosphatidylinositol 3-kinase (PI 3-K) and the phosphatase and tensin homolog (PTEN) regulate tumor cell invasiveness have led the authors to surmise that these lipid signaling molecules might play a role in regulating matrix metalloproteinases (MMPs), which are essential for tumor cell invasion. METHODS: Using the C6 glioma cell line, which does not express measurable amounts of PTEN protein and in which in vitro invasiveness is MMP dependent, the authors determined that in vitro glioma cell invasiveness was significantly reduced when cells were preincubated overnight with LY294002 or wortmannin, two specific inhibitors of PI 3-K signaling. Next, using gelatin zymography, it was noted that these compounds significantly inhibited MMP-2 and MMP-9 activities. Moreover, the decrease in MMP activity correlated with the decrease in PI 3-K activity, as assessed by Akt phosphorylation. Finally, using semiquantitative reverse transcriptase-polymerase chain reaction, the authors demonstrated that LY294002 decreased messenger (m)RNA levels for both MMPs. Thus, these in vitro data indicate that PI 3-K signaling modulates gelatinase activity at the level of mRNA. Using immunostaining of phosphorylated Akt (p-Akt) as a measure of PI 3-K activity, the authors next assessed rat brains implanted with C6 cells. Compared with surrounding brain, there was marked p-Akt staining in C6 glioma cells and in neurons immediately adjacent to the tumor, but not in normal brain. The p-Akt staining in tumors was especially intense in perivascular areas. Using double-labeling techniques, colocalization of p-Akt with MMP-2 and MMP-9 was also noted in perivascular tumor areas. CONCLUSIONS: The increase in p-Akt staining within these PTEN-deficient gliomas is consistent with what would be predicted from unchecked PI 3-K signaling. Furthermore, the immunohistochemically detected colocalization of p-Akt and MMP-2 and MMP-9 supports the authors' in vitro studies and the proposed linkage between PI 3-K signaling and MMP activity in gliomas.

Immunotoxin sensitivity of Chinese hamster ovary cells expressing human transferrin receptors with differing internalization rates.

Previous studies have shown that immunotoxin action is dependent upon selective binding to the target cell, internalization and then passage into the cytosol. It is important to define precisely how these critical steps are controlled so that the underlying relationship of each to high cytotoxic effectiveness is understood. In order to evaluate the contribution of internalization rate and receptor number on immunotoxin potency, the effects of an anti-(transferrin receptor, TfR)/ricin A chain immunotoxin, 7D3-A, were assessed on a parent Chinese hamster ovary cell line developed in our laboratory with no TfR (TfRneg) and two lines transfected with either wild-type TfR (Tfrwt) or an internalization-deficient (TfR(delta 7-58del)) mutated human TfR. Potent, receptor-mediated cytotoxicity resulted from the action of 7D3-A on TfRwt cells (ID50 < 1 nM) while both TfRneg cells and TfR(delta 7-58del) were only minimally affected (ID50 > 100 nM). Butyrate up-regulation substantially increased receptor expression on the TfRwt and TfR(delta 7-58del) cells, but no corresponding rise in sensitivity to 7D3-A was observed. In contrast, immunotoxin potency was increased by co-treatment of TfRwt cells with the carboxylic ionophore monensin and the effect was even more pronounced for TfR(delta 7-58del) cells. We conclude that internalization rate or intracellular destination is a much more important determinant of immunotoxin efficacy than receptor number.

Isolation and quantitation of several new peptides from the canine neurotensin/neuromedin N precursor.

Using antisera towards the bioactive peptides, neurotensin and neuromedin N, as well as towards the N-terminal and C-terminal regions of their shared 170-residue precursor, peptides representing various portions of the precursor were isolated from extracts of canine ileum. In total, seven peptides were isolated, two of which had not been previously identified. One was the C-terminal tail of the precursor (Gly-Ser-Tyr-Tyr-Tyr) and the other was the tail peptide extended N-terminally to include neurotensin (Glp-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-Lys-Arg-Gly-Ser-Tyr -Tyr- Tyr). By comparing the measured concentrations for each of the identified peptides, it was established that processing at the three Lys-Arg cleavage sites within the precursor did not occur to the same extent. Cleavage at the N-terminus of neuromedin N was approximately 10% complete, that between neurotensin and the tail was approximately 90% complete, and that between neuromedin N and neurotensin was approximately 95% complete. Three immunoreactive proteins were also identified by immunochemical and chromatographic analyses: N-terminally extended neuromedin N (125 residues), N-terminally extended neurotensin (140 residues), and the entire 147-residue precursor. It was concluded that neurotensin, tail and large molecular neuromedin N were the primary products of processing for this precursor in canine ileum, while minor products included neuromedin N, neurotensin tail, and large molecular neurotensin.

Pepsin-mediated processing of synthetic precursor-like sequence yields neurotensin-like peptide.

A 15 amino acid synthetic peptide, which spanned the dibasic cleavage site C-terminal to neurotensin (NT), in its 170-residue canine precursor, was synthesized by solid-phase methods. Using this substrate in combination with a radioimmunoassay specific for the C-terminal region of NT, a simple assay was developed to monitor protease-mediated cleavage of the Leu8-Lys9 bond in the substrate. Hog pepsin and the related enzymes, rhizopus pepsin, bovine cathepsin D, and mouse renin, were found to be effective in this assay, pepsin cleaving only this bond to liberate the NT-like sequence. The pH dependence of the reaction indicated that pepsin, cathepsin D, and renin exhibited significant activity at pH's characteristic for secretory vesicles (pH 5.5-6.5). In addition, pepsin and cathepsin D were shown to process the native precursor at pH's as high as 5.5. These results, although not proof, are consistent with the idea that endoproteases with pepsin-like specificity may be involved in the processing of the NT precursor in neural/endocrine cells.

Neurotensin elevates hematocrit and plasma levels of the leukotrienes, LTB4, LTC4, LTD4 and LTE4, in anesthetized rats.

The IV injection of neurotensin (NT) into anesthetized rats produced a marked increase in hematocrit, labored breathing and peripheral blood stasis with cyanosis. This effect could also be produced by the NT-related peptides, neuromedin-N and xenopsin; however, it was not observed when nine other biologically active peptides, including bradykinin and substance P, were tested. Associated with these responses were increases in the plasma levels of histamine (measured radioenzymatically) and the leukotrienes, LTB4, LTC4, LTD4, and LTE4 (measured by RIA and HPLC). The increment in hematocrit after varying doses of NT correlated to the increase in plasma levels of LTC4. Histamine and LTC4 were both capable of elevating hematocrit when given IV; however, LTC4 was approximately 1000 times more potent than histamine and active doses of histamine elevated LTC4 levels. Furthermore, the effects of NT on plasma LTC4 and hematocrit were reduced by pretreating animals with antagonists to histamine and serotonin. Pretreatment with the specific mast cell degranulating agent, compound 48/80, also blocked NT's ability to elevate plasma levels of histamine, LTB4 and LTC4 and prevented the increased hematocrit and cyanosis. These results indicate that NT-related peptides are very potent and specific stimulators of leukotriene release and that this action is mediated by mast cells and associated with loss of plasma volume and blood stasis. A working hypothesis is that histamine, released from mast cells in response to NT, stimulates LTC4 production by other cells.

Effects of cadmium chloride on ovulation and on induction of sterility in the female golden hamster.

The effects of single subcutaneous injections of cadmium chloride (CdCl2) on ovulation, egg transport and early pregnancy in the golden hamster were studied. While a single dose of 1.25 or 2.5 mg/kg of CdCl2 imposed none to marginal effects, hamsters treated with 5 or 10 mg/kg CdCl2 experienced a period of sterility ranging from 11-69 (5 mg/kg) or 46-71 (10 mg/kg) days, followed by a normal pregnancy. Administration of CdCl2 also induced ovulation inhibition which was dose-and time-dependent. A minimum dose of 5 mg/kg CdCl2 was needed to inhibit ovulation. When CdCl2 was given closer to the time of the luteinizing hormone (LH) surge on the day of proestrus, a more pronounced effect on ovulation was recorded. The incidence of failure of ovulation was associated with decreased progesterone levels in serum and inflammation, hemorrhages and necrosis in the ovary. However, the ovarian lesions lasted less than 4 days. The results indicate that CdCl2 inhibits ovulation when administered close to the time of ovulation, whereas its influence on pregnancy is pronounced but temporary.

Antifertility effects of gossypol in male hamsters.

In the male hamster, administration of 10 or 15 mg/kg/day of gossypol for 5 weeks did not alter the serum concentration of testosterone (T), prolactin (PRL), and luteinizing hormone (LH) or fertility. However, the total sperm population was significantly reduced in the males treated with 15 mg/kg/day of gossypol, and total sterility occurred within 8 weeks. On the other hand, males treated with 10 mg/kg/day of gossypol were sterile after 10 weeks of treatment. The T and total sperm population along the reproductive tract in these infertile males was significantly reduced. During the treatment period no change in body weight or weight of testes or seminal vesicles was observed. A full regaining of fertility, serum T level, and sperm population to the normal range were recorded within 8 nd 14 weeks (15 and 10 mg/kg/day, respectively) after the cessation of treatment.

The concentration of progesterone, 20 alpha-dihydroprogesterone, testosterone, oestrone and oestradiol-17 beta in serum, amniotic fluid and placental tissue of pregnant rabbits.

The concentrations of progesterone (delta 4P), 20 alpha-dihydroprogesterone (20 alpha-DHP), testosterone (T), oestrone (E1) and oestradiol-17 beta (E2 beta) in peripheral blood serum (PBS), amniotic fluid (AF) and placental tissue of rabbits during gestation were determined by radioimmunoassay. The placenta of the 10-day pregnant rabbit was fragile and composed mainly of maternal tissue. By the 12th day of pregnancy it was separable into maternal and foetal placentae. The mean concentration of delta 4P in PBS rose from 200 pg/ml (day 1 pregnancy) to 17--21 ng/ml (days 10--15) and decreased gradually to 1 ng/ml a few hours before parturition. The 20 alpha-DHP in PBS also showed an increase from 1.5 ng/mg (day 1) to 12 ng/ml (day 6) but fluctuated thereafter. The concentration of 20 alpha-DHP in the PBS tended to be lower than that of delta 4 P during pregnancy until the regression of the corpus luteum. An interesting observation was an increase of T on days 6--8 of pregnancy, the time when implantation occurs. The concentrations of E1 and E2 beta in PBS remained very low throughout pregnancy. delta 4P and 20 alpha-DHP in AF ranged between 25 pg to 1 ng/ml and in no case during the course of pregnancy were the levels of T, E1 and E2 beta in AF higher than in PBS. Where the maternal placental delta 4P content remained between 1--2 ng/placenta, the foetal placenta delta 4P rose to a level of 15 ng/placenta by day 31 of pregnancy. A similar trend was recorded for 20 alpha-DHP content. It is concluded that although a parallelism between PBS and myometrial steroid concentration was observed, no relationship could be drawn between the concentrations of steroid in PBS and those of the placental tissue and AF.

Effects of 17 beta-hydroxy-7 alpha-methylandrost-5-en-3-one on early pregnancy in the rat.

A single sc injection of 1 mg 17-beta-hydroxy-7 alpha-methylandrost-5-en-3-one (RMI-12,936) given a few hours after mating interrupted pregnancy in all the treated rats. Circulating progesterone (delta 4P) levels were higher in RMI-12,936 treated females than in controls on the corresponding days during the course of termination of pregnancy. Higher levels of delta 4P were recorded on day-4 (P less than 0.01) and day-6 (P less than 0.05) of pregnancy. In addition to the changes in serum delta 4P, an acceleration of egg transport was encountered. The eggs were prematurely expelled from the uterus within 48 h of the treatment. Although the oestrous cycle of the RMI-12,935 treated females was disturbed, they were found sexually receptive. Successful matings resulting in normal gestation and morphologically normal foetuses were recorded 20-26 days after RMI-12,936 induced pregnancy termination. These results suggest that in addition to its mid-pregnancy terminating effect, RMI-12,936 is capable of interrupting early pregnancy when given soon after mating in the rat. The safety and efficacy of this compound as a post-coital contraceptive deserves further investigation.

Gossypol: its toxicological and endocrinological effects in male rabbits.

The effects of different doses of orally administered polyphenolic compound 'Gossypol' on semen quality, circulating testosterone (T) and fertility of Dutch-belted male rabbits were studied. Bucks fed daily with 80, 40, 20 mg/kg day gossypol died within 8-17, 23-35 or 35-84 days, respectively, after initiation of treatment. Following gossypol treatment at 80, 40 or 20 mg/kg/day, animals lost appetite and body weight, developed hind limb paralysis, breathing difficulties and collapsed while sitting in their cages. At autopsy, the liver and lungs were found congested while the stomach and intestines contained gases. On the other hand, rabbits fed daily with 10 mg/kg/day gossypol exhibited a survival time ranging from 77 to 250 days. Despite the severe side effects resulting in eventual deaths, weekly semen samples from all treated animals did not show any apparent change in sperm motility, morphology or population. Likewise, gossypol-treated males mated to estrous does exhibited a fertility comparable to vehicle-treated controls. Gossypol fed at a dose of 10 mg/kg/day for up to 35 weeks failed to induce sterility. Male rabbits, fed with either 20 or 10 mg/kg/day gossypol, that survived for longer periods of time had substantially reduced T levels by 12-20 weeks depending upon the dose but were fertile at all times. When the in vitro release of T from the rat testes mince in the presence of hCG and gossypol was evaluated, an inhibiton of T release was recorded. It is concluded that although gossypol has been shown to be an effective antifertility agent in several mammalian species, it failed to exhibit such an effect in Dutch-belted rabbits, although serum T levels were reduced. In addition, gossypol exhibited a wide spectrum of toxicity. The in vitro study demonstrated that high concentration of gossypol can directly inhibit the T synthesis in the testis.

Termination of pregnancy in the rat by RMI 12,936.

Administration of a steroid RMI 12,936 (2 MG) ON DAY 8 OF PREGNANCY RESULted in an interruption of gestation within 5 days. No significant changes in progesterone (delta 4P), 20 alpha-dihydroprogesterone (20 alpha-DHP) and pregnenolone (delta 5P) were recorded during the course of termination of pregnancy by RMI 12,936. However, a significant rise in serum delta 4P and hypertropic corpora lutea were evident on day 13 of pregnancy when all treated females showed dead conceptus. The placental tissue was still present at the time of pregnancy termination. On the other hand, serum LH levels was significantly suppressed 24 hr after the treatment and maintained at a very low level. The reduction of LH might be due to a lowered hypophyseal sensitivity towards LH-RH. The occurrence of in utero dead fetus without interference of delta 4P production suggests that in the rat RMI 12,936-induced termination of pregnancy is due to its embryotoxic rather than luteolytic effect. Although the treated females did not return to normal estrous cycles for a period of 2 weeks after pregnancy termination, they were sexually receptive. Successful mating resulted in normal pregnancy which occurred 30-35 days after RMI 12,936 treatment.

RMI 12,936 and pseudopregnancy in rats.

RMI 12,936, when injected subcutaneously at a dose of 2 mg, either on Day 6 of psuedopregnancy (PSP-6) or on PSP-6,7 and 8, shortened the duration of PSP from 12.3 +/- 0.3 (control) to 8.3 +/- 0.1 or 8.7 +/- 0.2 days, respectively. During PSP, plasma progesterone (delta 4P) levels in the peripheral plasma showed a trend of decrease by PSP-9 (52.0 +/- 4.6 on PSP-8 to 42.3 +/- 3.1 ng/ml on PSP-9). Administration of RMI 12,936 on PSP-6 resulted in a significant decrease in delta 4P and pregnenolone (delta 5P) within 24 hr after treatment but caused no apparent changes in 20 alpha-dihydroprogesterone (20 alpha-DHP) levels. However, a significant decrease of delta 4P/20 alpha DHP ratio was encountered 24 hr after RMI 12,936 treatment. The persistent occurrence of proestrous smear after PSP termination, but absence of estrous vaginal cytology, might be attributed to the slight estrogenicity of RMI 12,936. After the premature termination of pseudopregnancy induced by RMI 12,936, the female rats were sexually receptive for at least 3 weeks but failed to conceive, suggesting that this compound has a prolonged contraceptive effect.