Progesterone and RU486 regulation of uterine complement C3 after prior induction with estradiol. Previous results demonstrated that progesterone (P4) given simultaneously with estradiol (E2) prevented stimulation by E2 of complement C3 expression in the immature rat uterus. Northern blot analysis revealed that simultaneous administration of P4 was able to prevent the E2-stimulated increase in C3 mRNA concentration in the luminal epithelial cells. The purpose of the present investigation was to determine whether progesterone modulates C3 expression after the gene has been induced by prior administration of E2 and also to determine the reversibility of this effect by the concomitant administration of RU38486, 17 beta-hydroxy-11 beta-[4-dimethylaminophenyl]estra-4,9,-dien-3-one (RU486). This regulation was studied by examination of protein synthesis as well as mRNA concentrations. Immature 21-day-old female rats were treated with E2 for 2 days (1 microgram/day), followed 24 h later by P4 (500 micrograms) or vehicle. Uteri were removed 6, 9, and 18 h after progesterone treatment and the radiolabeled secreted proteins were analyzed by SDS-PAGE and immunoprecipitation using a goat anti-rat C3 antibody. In animals treated with vehicle, E2-stimulated C3 synthesis remained elevated at 6 and 9 h and returned to control values by 18 h. In contrast, the administration of P4 resulted in a decrease in C3 synthesis at 6 and 9 h with the greatest decrease observed at 9 h. Similar results were obtained when C3 mRNA concentrations were examined. E2-stimulated C3 mRNA concentrations were decreased in rats treated with progesterone compared to those treated with vehicle alone.2


Maternal transfer of photoperiodic information in Siberian hamsters. V. Effects of melatonin implants are dependent on photoperiod. Photoperiodic information is transferred from female Siberian hamsters to their fetuses during gestation. Although maternal melatonin is known to be essential for the transfer of prenatal photoperiodic information, its specific role is not well defined. The duration of the daily melatonin signal, expressed as an elevation of serum melatonin levels in the maternal circulation, has been hypothesized to convey day length information to the fetus. If this hypothesis is valid, it predicts that identical maternal melatonin signals should affect the fetuses identically, regardless of the prenatal photoperiod. To test this hypothesis, adult females received melatonin in beeswax or beeswax alone. They were paired with males and housed in photoperiods of 12L:12D or 16L:8D. On the day of parturition, mother and young were transferred to constant light (LL). Young males were killed on Day 28 of life, and weights of testes were determined. Prenatal treatment with beeswax alone did not affect the nature of the signal transferred from mother to fetus; young gestated in 12L:12D and reared in LL developed small testes, while those gestated in 16L:8D had large testes. On the other hand, the effect of the prenatal melatonin treatment on postnatal testicular development in LL was inversely dependent on the prenatal photoperiod: testicular growth was stimulated in young gestated in 12L:12D, but inhibited in young gestated in 16L:8D. To verify that the melatonin pellets produced equivalent serum melatonin levels in adult females in 12L:12D and 16L:8D, unmated adult females were killed 6-10 wk after receiving melatonin pellets. Serum levels were elevated in both groups throughout the day and night.(ABSTRACT TRUNCATED AT 250 WORDS)


Hypotaurine requirement for in vitro development of golden hamster one-cell embryos into morulae and blastocysts, and production of term offspring from in vitro-fertilized ova. Almost 30 years after the first successful in vitro fertilization (IVF) in golden hamsters (Mesocricetus auratus), we report that IVF hamster embryos can develop in a chemically defined, protein-free culture medium into morulae and blastocysts, and produce normal offspring after transfer to recipients. When examined 96 h post-insemination, 82% (160/200) of IVF ova had cleaved to at least 2 cells, 55% (97/200) had developed beyond the 4-cell stage, and 22% (38/200) had developed into morulae/blastocysts. In vitro development of IVF embryos to greater than or equal to 8 cells was absolutely dependent on hypotaurine. Twenty living offspring were produced from transfer of IVF embryos to recipients, with an overall success rate of 5% and 17% for oviductal (2-cell) and uterine (8-cell/morulae) transfers, respectively. In vivo-fertilized pronucleate embryos collected 3 h after egg activation were less able to develop in vitro than embryos collected only 6 h later, revealing a critical influence of the oviduct within the first hours of embryo development. Hypotaurine partly compensated for the decreased oviductal exposure of early 1-cell embryos. Establishment of a key role for hypotaurine in hamster embryo development, support of IVF embryos to morula/blastocyst stages in vitro, and production of living offspring after IVF embryo transfer are significant steps towards the goal of obtaining comparative data on preimplantation embryogenesis.


Cloning of the porcine Calbindin-D9k complementary deoxyribonucleic acid by anchored polymerase chain reaction technique. The Calbindin-D9k (CaBP-9k) is a cytosolic calcium binding protein expressed in the mammalian intestine, placenta, and uterus. The protein is probably involved in calcium transport across the intestinal and placental epithelia. In uterus, a function in controlling myometrial activity involving intracellular calcium has been postulated. The amino acid sequence of the porcine CaBP-9k has been determined from intestine. The cDNAs for the bovine, murine, and rat CaBP-9k have been cloned. The objective of this study was the cloning of the porcine cDNA encoding the CaBP-9k. We performed the anchored polymerase chain reaction (PCR) technique using rat and bovine cDNA sequence-derived primers for amplification of intestinal cDNA. Both 5' and 3' amplification products were cloned and sequenced. The sequences revealed the full-length cDNA encoding the porcine CaBP-9k, coding region for 79 amino acids, 57 nucleotides 5' and 149 nucleotides 3' noncoding region. The inferred amino acid sequence is identical to the published amino acid sequence, except for one residue. The porcine CaBP-9k cDNA is 82.8% and 69.1% homologous with the bovine and rat sequences, respectively. Both bovine and porcine cDNAs contain a stretch of approximately 50 nucleotides not found in the rat sequence. Northern analysis showed a 600 nucleotide transcript in intestine, kidney, and uterus.


Flow cytometric analysis of steroidogenic organelles in differentiating granulosa cells. Functional and structural changes accompany the differentiation of granulosa cells during follicular development. We used flow cytometry and fluorescent dyes to characterize two organelles important to the steroidogenic process. Mitochondria, which contain the rate-limiting enzyme responsible for cholesterol conversion to pregnenolone, and lipid droplets, which store cholesterol substrate, were probed in viable hen granulosa cells during differentiation. The fluorescent dye Dio3-C5 (DiO) was used to probe mitochondrial membrane potential, indicative of mitochondrial activity and/or number, during rapid granulosa cell differentiation in a hierarchy of individual developing hen preovulatory follicles (F6, smallest, to F1, largest). Cellular DiO fluorescence, granularity, and cell size were significantly elevated with increasing maturation state. Treatment with LH significantly increased DiO fluorescence in granulosa cells from F1 but not F3. The increased mitochondrial activity/number in granulosa cells that accompanies follicular maturation and is influenced by LH may reflect, at least in part, increased activity or amount of hormone-regulated mitochondrial enzymes controlling steroidogenesis. Flow spectrofluorometry and the metachromatic lipid dye, nile red, were used to probe lipid droplets in differentiating granulosa cells from F6 to F1. There was a dramatic increase in the fluorescence component related to lipid droplets with increasing stages of follicular maturation, suggesting recruitment of lipids into droplets during the differentiation of granulosa cells into hormone-responsive steroidogenic cells. The results demonstrate the dynamic nature of the granulosa cell morphology involved in steroidogenesis during follicular development.(ABSTRACT TRUNCATED AT 250 WORDS)


Synthesis and secretion of retinol-binding protein by cultured rat Sertoli cells. We report here that retinol-binding protein (RBP) is synthesized and secreted by rat Sertoli cells in culture. This was demonstrated in four ways. First, transthyretin (TTR) bound to Sepharose 4B retained a labeled protein from media collected from Sertoli cells provided with 35S-methionine, under the same conditions as authentic RBP is bound. The protein was co-eluted with authentic RBP by pure water. Second, this same radioactive protein co-eluted with pure RBP upon gel filtration. Third, when subjected to SDS-PAGE, the protein again migrated with pure RBP, as shown by radioautography. Finally, Sertoli cells were incubated with 35S-cysteine and the conditioned medium was put over a TTR-Sepharose column to isolate the radioactive protein, as characterized above. Cysteine residues were oxidized to cysteic acid residues, and the protein was submitted for sequencing through the first ten residues. Radioactivity was located only in the fourth residue, where a cysteine residue is found in rat RBP. This indicates that RBP is secreted by the Sertoli cell and may serve as the carrier of retinol to the developing germ cells, which are known to be dependent upon vitamin A.


Prolactin stimulates the expression of luteinizing hormone/chorionic gonadotropin receptor messenger ribonucleic acid in the rat corpus luteum and rescues early pregnancy from bromocriptine-induced abortion. Timed pseudopregnancy (psp) and pregnancy were induced in adult female rats by mating with infertile and fertile males, respectively. Corpora lutea (CL) and the residual parts of the ovaries were isolated and analyzed for luteinizing hormone/chorionic gonadotropin (LH/CG) receptor mRNA by Northern blot and solution hybridization analyses. Several LH/CG receptor mRNA transcripts were detected that could code for an intact functional receptor (6.8, 4.4, and 2.6 kb) as well as several smaller truncated transcripts. LH/CG receptor mRNA abundance in CL varied dramatically during both psp and pregnancy, with peak levels seen during the period of maximal progestational activity (Days 5-10 of psp and Days 7-14 of pregnancy). During the period of functional luteolysis, LH/CG receptor mRNA abundance decreased to low levels. The changes in LH/CG receptor expression could be explained by hormonal regulation. Bromocriptine treatment inhibited pituitary prolactin secretion. This treatment had a potent luteolytic effect by decreasing the levels of LH/CG receptor mRNA and plasma progesterone during early pregnancy, resulting in embryonal resorption in pregnant rats. Exogenous prolactin acted as a anti-luteolysin to reverse these effects by restoring LH/CG receptor mRNA abundance either by increasing gene expression or by stabilizing mRNA transcripts from degradation in young CL.


Sex ratio manipulation in wild house mice: the effect of fetal resorption in relation to the mode of reproduction. First generation laboratory-born descendants of wild-caught house mice (Mus musculus domesticus Rutty) were bred to produce litters of primipares and of dams that had conceived a second litter either after lactational anestrus or within the postpartum estrus. At the day of birth, pups were sexed and the number of implanted and resorbed embryos was determined to evaluate the influence of mode of reproduction on litter gender composition and its relation to fetal resorption. No significant deviations from an even sex ratio occurred in the sample. The results indicate that primipares produced litters with subnormal dispersion of the gender distribution, but this could not unequivocally be attributed to fetal resorption. No significant bias in the litter gender composition was detectable within litters conceived after lactational anestrus. In contrast, the dispersion of the gender distribution was significantly supernormal in the litters of dams inseminated at postpartum estrus. Within this group, fetal resorption had a significant effect upon the sex ratio, and this relationship was significantly affected by the number of implanted embryos: resorbing dams produced male-biased litters at small and intermediate numbers of implantation sites and female-biased litters when the number of implanted embryos was large. It is argued that this is most likely attributable to sex-selective fetal resorption.


Evidence that most of the radioimmunoassayable inhibin secreted by the corpus luteum of the common marmoset monkey is of a non-dimeric form. Although recent data for several species of primate, including human and marmoset, indicate that the corpus luteum secretes high levels of radioimmunoassayable inhibin, the nature of the immunoreactive (ir) inhibin detected has not been established. In this study, plasma ir-inhibin levels during the ovarian cycle of the marmoset (n = 12 animals) were measured by alpha-subunit-directed inhibin RIA, and values were compared with those estimated by a recently developed two-site immunoradiometric assay (IRMA) specific for inhibin alpha-beta dimer. Consistent with earlier data, plasma levels of ir-inhibin measured by RIA (overall mean value 133 +/- 7 ng/ml; n = 171) reached values 4-fold higher (p less than 0.001) during the luteal phase (222 +/- 20 ng/ml) than during the follicular phase (58 +/- 8 ng/ml), being directly correlated with plasma progesterone levels (r = 0.65; p less than 0.001). In contrast, plasma ir-inhibin levels estimated by IRMA were substantially lower than those measured by RIA (overall mean value 9.62 +/- 1.08 ng/ml; n = 171) and did not vary significantly during the cycle. Administration of a luteolytic dose of cloprostenol during the late luteal phase/early pregnancy led to an abrupt fall in plasma concentrations of progesterone (95%) and alpha-inhibin measured by RIA (82%), whereas dimeric inhibin levels remained unchanged. Analysis of marmoset luteal extracts (n = 5) by RIA, IRMA, and inhibin bioassay yielded inhibin estimates of 102.6 +/- 21.0, 0.632 +/- 0.103, and less than 2.0 ng/mg, respectively, thus confirming that only a very small proportion of the inhibin produced was dimeric (i.e., bioactive) in nature.(ABSTRACT TRUNCATED AT 250 WORDS)


Reversal of reproductive deficiency in the hpg male mouse by neonatal androgenization. Some aspects of reproductive function in the GnRH-deficient hypogonadal (hpg) mutant mouse can be restored by transplanting normal fetal brain tissue containing GnRH cells into the central nervous system of adult hpg mice. However, hpg males showing physiological response to the graft fail to display sexual behavior and are infertile. We hypothesized that the reproductive deficit of these males is due to insufficient perinatal exposure to testicular androgens as a consequence of the GnRH deficiency. To test this hypothesis we androgenized hpg males by giving them neonatal injections of testosterone propionate (TP). Controls consisted of hpg males not androgenized neonatally and of normal males. All three groups received a TP implant in adulthood, and their copulatory behavior and reproductive capability were recorded. In addition, other hpg males, not androgenized neonatally, received fetal brain transplants containing GnRH neurons and were also tested for copulatory behavior and reproductive capability before and after receiving a TP implant. Three of 8 neonatally androgenized hpg males expressed the full repertoire of male sexual behavior, including intromission and ejaculation, and sired several litters. Three of 7 control hpg males that were not androgenized neonatally but received TP implants in adulthood also displayed mounting and intromission, but there was no evidence of ejaculation, and these males failed to impregnate normal females. Of the 8 hpg males that responded to a fetal transplant with testicular growth, only 1 displayed mounting behavior. However, when given a TP implant, 4 of 8 hpg males with grafts displayed mounting and intromissions.(ABSTRACT TRUNCATED AT 250 WORDS)


Testins are localized to the junctional complexes of rat Sertoli and epididymal cells. Testin I and Testin II were originally identified as Sertoli cell products with similar NH2-terminal amino acid sequences. Secretion of testins is stimulated by testosterone in Sertoli cell-enriched cultures. By contrast the secretion of testins from intact seminiferous tubules appears to be inversely related to germ cell number. In the present study testin antiserum that recognized both Testin I and Testin II ("testin") was used to localize these proteins in tissue secretions by immunofluorescence. Testin was localized at the base of the seminiferous epithelium at Sertoli-Sertoli junctions. Fluorescence also appeared to be located at the sites of interaction between spermatoids and Sertoli cells. A punctate pattern of fluorescence was also present in the cytoplasm of Leydig cells; without electron microscopic studies it was not possible to determine which structures the antibodies bound to in these cells. In the epididymis the reaction product was localized at the apices of the epithelial cells adjacent to the lumen at the sites of known junctional complexes. A variety of positive and negative controls indicated that staining was specific for testins. This is the first study to associate testins with junctional complexes. Relative to other junctional proteins, testins are unusual because of their small size and because they are secreted proteins.


Effects of follicle-stimulating hormone on inhibin release by different testicular compartments in the adult ram. The aim of this study was to determine the bidirectional release of immunoreactive inhibin-alpha (irINH-alpha) by different testicular compartments in the adult ram and to assess the effects of FSH on the distribution of inhibin in the testis. Immunoreactive INH-alpha was measured by RIA in fluid samples collected concurrently from the three testicular compartments--the seminiferous tubules, the interstitium, and the vascular system--through catheters inserted surgically into the rete testis, testicular lymphatic duct system, and spermatic veins, respectively. Overall, the concentration of irINH-alpha in rete testis fluid was 25 times the level in testicular lymph and over 500 times the concentration in peripheral blood. The pattern of irINH-alpha concentration in rete testis fluid was inversely related to that in testicular lymph, but i.v. administration of FSH had a decoupling effect on this relationship by depressing inhibin concentration in testicular lymph without affecting inhibin levels in rete testis fluid. Nevertheless, increased flow of testicular lymph more than compensated for the transient fall in irINH-alpha concentration so that, overall, the total output of inhibin via the testicular lymphatic duct system (and the vascular system) increased significantly. No persistent or significant changes were observed in the flow rate of rete testis fluid or concentration of irINH-alpha in the fluid after administration of FSH. The time frame for the response of the testis to FSH is indicative of the involvement of a mediator. Electrophoretic analysis of serially collected testicular lymph samples consistently revealed an FSH-induced release of a series of proteins in the M(r) range of 30,000-32,000.(ABSTRACT TRUNCATED AT 250 WORDS)


Maternal plasma catecholamines in the rhesus macaque during late gestation: effect of photoperiod and timed melatonin infusion. This study tested the hypothesis that changes in photoperiod alter plasma catecholamine concentrations in the rhesus monkey during late gestation. Twelve chronically catheterized pregnant rhesus macaques were acclimated to a 12-h photoperiod (lights-on, 0700-1900 h). Under the control L:D cycle, blood samples were collected at 3-h intervals over 24 h for catecholamine analysis. Plasma concentrations (mean +/- SEM, pg/ml) ranged from 678 +/- 90 to 928 +/- 142 for norepinephrine; 230 +/- 22 to 631 +/- 141 for epinephrine; and 282 +/- 70 to 1090 +/- 362 for dopamine. A diurnal rhythm was observed in epinephrine with peak concentrations during lights-on (0900-1800 h; p less than 0.05, compared to lights-off). After the first sampling protocol, the animals were divided equally between two groups: phase shift, in which lights-on was shifted 11 h (2000-0800 h) and constant light, with lights on continuously. After the phase shift, a parallel shift in the plasma epinephrine rhythm was noted, with peak levels observed between 2200 and 0700 h (p less than 0.05). Constant light abolished the rhythm in epinephrine, with an overall reduction in mean basal levels of all three catecholamines. Daily melatonin infusions (0.2 micrograms/kg/h, 1900-0630 h) under constant light failed to restore the epinephrine rhythm or to return basal catecholamine concentrations to control photoperiod levels. These data suggest that photoperiod entrains the rhythm in epinephrine secretion, but the rhythm is ablated under constant conditions. Further, melatonin does not appear to play a role in the regulation of catecholamine secretion in the pregnant rhesus macaque.


Binding of epididymal proteins to rat spermatozoa in vivo. The secretion of epididymal proteins and their binding to spermatozoa in rats were examined after retrograde perfusion of the superior and inferior epididymal arteries with [35S]methionine. PAGE revealed that the pattern of radioactive proteins in the luminal fluid was markedly different from the well-characterized pattern of secretory proteins obtained by in vitro incubation of epididymal minces with labeled methionine. Of the proteins secreted into the lumen, about 1% were associated with Percoll-purified spermatozoa. More proteins were associated with the spermatozoa in the corpus epididymidis than in the caput. Sequential extraction of spermatozoa with an isotonic buffer, a high-salt buffer, Triton X-100, and SDS revealed that almost half of the radiolabeled proteins could be extracted with the isotonic buffer. The firmly bound radioactive proteins remaining, which were extracted with Triton X-100 or SDS, consisted of one major band of 25 kDa and two minor bands of 30 kDa and 32 kDa. Analysis of the sperm-associated proteins at various times after the isotope was administered indicated that tight binding of proteins to spermatozoa occurs within 3 h after isotope injection.


Pituitary luteinizing hormone and prolactin messenger ribonucleic acid levels are inversely related in laying and incubating turkey hens. The relationships of prolactin (PRL) and LH messenger (m) RNA to serum and pituitary content were determined for turkey hens at different phases of the reproductive cycle. In the nonphotostimulated, reproductively inactive hen, serum and pituitary PRL content and pituitary PRL mRNA levels were low. All three PRL values rose after photostimulation and peaked during the incubation phase. Relative to nonphotostimulated hens, hyperprolactinemic incubating hens showed 220-, 11-, and 57-fold increases in serum PRL, pituitary PRL content, and pituitary PRL mRNA levels, respectively. These peak levels declined 80-, 3-, and 6-fold, respectively, in photorefractory hens. In contrast to PRL levels, serum LH, pituitary LH, and pituitary LH beta-subunit mRNA levels did not change as dramatically. Serum LH showed no significant changes for the different reproductive phases. Pituitary LH peaked after photostimulation and declined to its lowest level in incubating hens. Pituitary LH-beta mRNA abundance was highest in photostimulated and laying hens and lowest in incubating and photorefractory hens. These results demonstrate that the abundance of LH-beta and PRL mRNA shows an inverse relationship in photostimulated/laying and incubating turkey hens.


In situ localization and characterization of bone marrow-derived cells in the decidua of normal murine pregnancy. The study reported here was designed to examine the in situ distribution and characteristics of hemopoietically derived decidual cells during normal pregnancy in mice prenatally reconstituted with bone marrow cells carrying a transgenic marker. Bone marrow cells from a transgenic CD-1 strain (CD-1 beta; carrying 1000 copies of beta-globin genes in tandem) were injected into the yolk sac of Day 17 conventional CD-1 embryos. The pregnant females were allowed to deliver normally, and the female offspring raised to puberty were mated with CD-1 males and then killed on Day 12 of gestation. The extent of chimerism in sections of their spleens, uteri, and other organs was evaluated by in situ hybridization of the sections with a biotinylated cDNA probe specific for the beta-globin genes followed by avidin-biotin-peroxidase staining. Tissue controls were provided by CD-1 beta and CD-1 mice, respectively. Tissues were also processed without the application of the probe or with the application of biotinylated lambda DNA as specificity controls. Reconstituted mice exhibited variable degrees of hemopoietic chimerism as indicated by labeling of their splenic lymphocytes (18-54%; mean 42%) as well as hemopoietic cells in other organs. Variable cellular labeling was also noted in their decidua basalis and metrial glands. Labeled cells in these tissues were identified as typical decidual cells, macrophages, and granulated metrial gland (GMG) cells. Labeling of typical decidual cells varied extensively among implantation sites in the same chimera, the average labeling ranging from 17% to 33% (mean 24%) in various chimeras. Labeling was also noted in GMG cells, lymphocytes, and some decidual cells migrating out of metrial gland explants after 24-h culture. The non-pregnant uterus of a chimeric mouse revealed significant labeling of endometrial stromal cells indicative of their hemopoietic origin. These results revealed a hemopoietic origin of certain typical decidual cells and GMG cells identified in situ during normal murine pregnancy and a hemopoietic origin of certain endometrial stromal cells that may represent precursors of decidual cells. The precise timing of the predecidual stem cell migration from the bone marrow to the uterus remains to be defined.


Impaired transport and fertilization in vivo of calcium-treated spermatozoa from +/+ or congenic tw32/+ mice. To determine whether calcium alters processes important for fertilization in vivo, mouse (+/+) spermatozoa were incubated in medium with 1.0-1.7 mM calcium prior to artificial insemination (AI) into the cervix of hormonally primed females. Spermatozoa from congenic tw32/+ mice were also tested because their flagella are hypersensitive to calcium. As a control, spermatozoa were incubated in calcium-deficient medium prior to AI. Spermatozoa from mice of both genotypes incubated in calcium-containing medium fertilized significantly fewer eggs after AI than did spermatozoa incubated in calcium-deficient medium. In addition, calcium-treated spermatozoa from tw32/+ mice fertilized significantly fewer eggs than calcium-treated +/+ spermatozoa. Pretreatment with calcium also reduced the number of spermatozoa in the oviducts 0.5-4.5 h after AI, and the oviducts of females inseminated with calcium-treated spermatozoa from tw32/+ mice contained significantly fewer spermatozoa than those of females inseminated with calcium-treated +/+ spermatozoa. These results suggest that preincubation in millimolar levels of calcium changes the physiology of epididymal spermatozoa in such a way as to impair sperm transport to the oviduct and fertilization in vivo.


Calcium alters capacitation and progressive motility of uterine spermatozoa from +/+ and congenic tw32/+ mice. The importance of calcium-dependent sperm processes for fertilization in vitro is well known, but their interaction with sperm transport in vivo is not yet clear. To determine whether exposure to calcium alters sperm physiology after incubation in the uterus, spermatozoa from +/+ mice were incubated in medium with 1.7 mM calcium prior to artificial insemination (AI). Spermatozoa from congenic tw32/+ mice were also tested because their flagella are hypersensitive to calcium. As a control, spermatozoa were incubated in calcium-deficient medium before AI. When recovered from the uterus 60 min post-AI, neither prior exposure to calcium nor genotype affected numbers of spermatozoa, or percentage of motile or acrosome-reacted spermatozoa. However, significantly more calcium-treated spermatozoa were capacitated and significantly fewer were progressively motile than spermatozoa preincubated without calcium. In addition, significantly fewer spermatozoa from tw32/+ mice than from +/+ mice were progressively motile. These results suggest that uterine sperm physiology is changed by prior exposure of sperm to calcium. Since the level of progressive motility of spermatozoa recovered from the uterus was correlated with their ability to reach the oviduct (as determined in a previous study), these data support the hypothesis that progressive motility of uterine spermatozoa is important for passage to the oviduct and fertility.


Nuclear totipotency of cultured rabbit morulae to support full-term development following nuclear transfer. The rabbit was used as a model for nuclear transfer. A critical step in nuclear transfer is oocyte activation, which was evaluated in this research. Optimal field strength of an electric stimulus for activation was examined. A significantly higher activation rate in all criteria tested was achieved when oocytes were activated electrically with a field strength of 2.4 kV/cm versus 1.2 or 1.8 kV/cm. Also, electrical stimulation with combined alternating current (AC) and direct current (DC) was superior to DC stimulation alone for activation. In another study involving 586 oocytes, exposure of oocytes to cytochalasin B for 1 h followed by activation with electrical stimulation significantly improved development of the oocytes to blastocyst stage compared to oocytes without cytochalasin B pre-exposure (38% vs 26%, p less than 0.05). Cytochalasin B exposure alone (control), however, had no effect on activation. Exposing oocytes to activation medium without electrical stimulation also activated some oocytes. In the nuclear transfer experiment, blastomeres from 8-cell embryos cultured for 20-24 h to the 32-64-cell stage were used as nuclear donor cells. Of 491 oocytes used, 459 (93%) survived the enucleation and fusion procedure, 370 (81%) fused, and 284 (77%) developed into 2-4-cell embryos. A total of 243 of these 2-4-cell embryos were transferred to 15 pseudopregnant recipients and produced 8 young (3%). Although the efficiency is low, this study demonstrated that rabbit morulae cultured for 20-24 h to the 32-64-cell stage as nuclear donors for transfer remain totipotent.


Steroid hormone content of the gonads of the tammar wallaby during sexual differentiation. The gonads of the tammar wallaby, Macropus eugenii, are sexually indifferent at birth (Day 0) despite the fact that phenotypic sexual differentiation has already commenced as evidenced by the presence of a scrotum in males and mammary anlagen in females. The seminiferous cords of the testis first become clearly recognizable on Day 2 of pouch life, and ovarian differentiation is recognizable by Day 10. To monitor the endocrine development of the gonads during sexual differentiation of the urogenital tract, we measured the steroid hormone content in 92 pools of gonads from male and female tammar pouch young from the day of birth to 206 days of pouch life. Progesterone, estradiol, and dihydrotestosterone concentrations were low (less than 0.05 ng/mg protein) in both ovaries and testes at all stages examined, and testosterone concentrations were uniformly low in ovaries. Testosterone concentrations in testes were low on Days 0-4, averaging about 0.2 ng/mg protein; they rose by Days 5-10 to an average of 0.9 ng/mg protein, remained elevated until about Day 40, and thereafter fell to values similar to those in the ovaries. The phallus and urogenital sinus were able to convert testosterone to dihydrotestosterone from the earliest stages examined (Days 10 and 11). Thus in the tammar wallaby, as in eutherian mammals, testosterone is the androgen secreted by the developing testis, and dihydrotestosterone is formed in certain androgen target tissues.(ABSTRACT TRUNCATED AT 250 WORDS)


Follicular heterogeneity and oocyte maturation in vitro in pigs. Experiments were conducted to confirm that co-culture of follicular shells and immature pig oocytes improved the rate of male pronuclear (MPN) formation and to test the hypothesis that the maturational state of the follicle used in co-culture would significantly affect the quality of oocytes matured in vitro. In a preliminary experiment, co-culture of oocyte complexes with follicular shells did not affect nuclear maturation, slightly inhibited penetrability (p = 0.04) but greatly enhanced (p = 0.0004) MPN rate. In the main experiment, oocyte complexes (10-15 per dish), obtained from follicles 36 h after eCG injection of prepubertal gilts, were co-cultured with single 36-h small (3.5-5.0 mm), 36-h large (6-9 mm), 72-h small (4-7 mm), or 72-h large (7.5-11.0 mm) follicular shells prior to insemination. MPN formation in penetrated oocytes was significantly affected by follicular size (small: 60.14% vs. large: 72.08%, p = 0.014) but not by time of recovery (36 h: 72.32% vs. 72 h: 59.90%, p = 0.39). Overall, MPN formation rate was significantly correlated with follicular diameter (r = 0.45, p = 0.005), follicular fluid progesterone (r = 0.37, p = 0.02), and estradiol (r = 0.40, p = 0.01), and to the ratios of follicular fluid progesterone:testosterone (r = 0.39, p = 0.018) and follicular fluid estradiol:testosterone (r = 0.43, p = 0.007). There were no simple correlations between rate of MPN formation and steroid concentrations and their ratios in culture media collected at the completion of culture.(ABSTRACT TRUNCATED AT 250 WORDS)


Leydig cells express neural cell adhesion molecules in vivo and in vitro. The neural cell adhesion molecule (NCAM) polypeptides are expressed by numerous tissues during embryonic development, where they are involved in cell-cell interactions. In the adult, NCAM expression is confined to a few cell types, including neurons and peptide-hormone-producing cells. Here we demonstrate that the Leydig cells of the adult rat, mouse, and hamster testes express NCAM as well. Western blotting showed that an NCAM of approximately 120 kDa was present in the adult testes of all three species investigated. This form was also found in freshly isolated mouse Leydig cells and in Leydig cells after 2 days in culture. After 4 days in culture, mouse Leydig cells expressed additional NCAM isoforms of approximately 140 and 180 kDa, indicating changes in alternative splicing of NCAM primary transcripts. Also, NCAM mRNA of all isoforms, as detected by S1-nuclease protection assays, increased with time in culture. The expression of the cell adhesion molecule NCAM by adult Leydig cells may explain the aggregation of Leydig cells in clusters in rodent testes, which could be a prerequisite for functional coordination of groups of Leydig cells. Furthermore, the presence of this neural and endocrine marker may indicate a closer relationship between Leydig cells and neural and peptide-hormone-producing cells than is considered to exist at the present time.


[The molecular action profile of intravenous anesthetics]. A close look at the molecular interactions of intravenous anaesthetics reveals a complex and non-uniform picture. A multitude of membrane structures are affected by the individual anaesthetic compounds, although with specific differential profiles of molecular action for the different groups of anaesthetic substances. This is consistent with the hypothesis of a multi-mechanistic mode of action, where anaesthesia results from the superposition and integration of several anaesthetic actions on the molecular level. This overall concept is illustrated by using the well characterised voltage-activated human brain sodium channel protein and its interactions with various intravenous anaesthetics as an example for other membrane proteins. Using the novel lipid bilayer "voltage-clamp" technique, single sodium channels were incorporated into artificial lipid bilayers and their single channel properties studied. Compared to controls, all studied anaesthetics (propofol, pentobarbital, ketamine, midazolam) interacted in a dose-dependent manner with two key sodium channel functions: 1. reduction of the voltage-independent fractional channel open-time and 2. interaction with the voltage-dependent steady-state activation process. Only propofol and pentobarbital demonstrated these effects at clinically relevant concentrations; hence, the sodium channel might serve as a molecular site of action for these substances only. Differences between clinical anaesthetics and their clinical actions may thus correlate with differential molecular sites and modes of action.


[Early detection of traumatic aortic aneurysms using transesophageal Doppler echocardiography]. The early diagnosis of traumatic aortic dissections and aneurysms involving the thoracic aorta is of crucial importance for the outcome of patients suffering chest injuries. This subject is presented using two case reports; one illustrates the problems resulting from an overlooked traumatic aneurysm of the thoracic aorta and the second shows the possibilities of detecting such potentially lethal injuries early in patients with thoracic or multiple injuries, using the transesophageal Doppler echocardiography (TDE). The advantages and disadvantages of using TDE as compared to the more conventional methods such as the precordial Doppler echocardiography, computed tomography and angiography, are discussed. The particular advantage of this hardly invasive or burdening method is its immediate availability in the emergency room to assess patients with evident thorax trauma. In the intensive care unit TDE may be of value in screening polytraumatised patients who offered no primary evidence for chest injury upon arrival.


[Extreme duration of action of diazepam as a cause of respiratory insufficiency in a female geriatric patient]. A 77-year-old woman who had undergone surgery of a non-malignant extracerebral retro-orbital tumor suffered postoperatively from respiratory failure due to impaired respiratory drive and unconsciousness of unknown origin and required artificial ventilation for 14 days. After cerebral and endocrinological causes had been excluded, the residual effects of diazepam were taken into consideration, since the patient had received this substance for premedication and during 3 days postoperatively (total 165 mg). After the application of flumazenil (Anexate), a specific benzodiazepine receptor antagonist, she awoke immediately and was extubated with sufficient spontaneous breathing. The hypothesis that diazepam was the causative agent for the respiratory failure and impaired consciousness was supported by the detection of high serum concentrations of diazepam and its active metabolite desmethyldiazepam. During the following 3 days repetitive injections of flumazenil were necessary to counteract recurring depression of respiration and vigilance. Thereafter further application of flumazenil was not necessary.


[The anesthetic site of action: from the intact brain to the isolated protein. An overview]. To the present day there is no consensus of opinion as to which molecular events lead to the complex clinical state generally referred to as anaesthesia. The multitude of simultaneous physiological changes are also responsible for this lack of consensus. During anaesthesia, for example, consciousness, perception, memory, pain, and muscle relaxation are altered. The actions of anaesthetics correlate very well with their partitioning into lipophilic phases. Lipophilic interactions are non-specific. Therefore, it is likely that anaesthetics act at many different sites. Cell membranes are not the only possible sites but also membrane proteins and the various hydrophobic domains they contain. The multitude of clinical anaesthetic effects is paralleled by the complexity of molecular interactions. A better understanding of anaesthesia requires an integrated view of anaesthetic effects at the various systemic levels, beginning with the molecular domain and ending with the intact central nervous system. Anaesthesia research could be directed more towards man because it has now become possible to conduct molecular in-vitro studies with human cells as well as non-invasive studies of the intact human central nervous system.


[The spontaneous and evoked EEG in anesthesia]. Clear differentiation between nociception and pain, or, better, between anti-nociception and pain relief, is essential for understanding the analgesic mechanisms in anaesthesia. Nociception is a neuronal activity in the pain-mediating and pain-processing nervous system, i.e. in the peripheral axons, in the spinal short cord and in certain brain structures. The nociceptive system has been well documented by experiments, especially in animals. If this system is specifically blocked, there will be no transmission and hence no sensation of pain if we leave aside the rare and complicated instances of "psychogenic pain". Nociceptive activity is blocked or at least attenuated by anti-nociceptive drugs, such as surface anaesthetics acting in the periphery by blocking the sodium channels in the conductive nerve membrane. Opiates are another example of drugs which develop an effect on specific nociceptive neurons in the spinal cord and in the brain, thus suppressing pain transmission. An ideally effective anaesthetic should act similarly, that is by specifically and selectively suppressing nociceptive activity induced by surgery. However, general anaesthesia is based on entirely different mechanisms. It lowers the arousal level, or the vigilance of the patient. Since pain is the conscious processing of nociceptive information, attenuating the vigilance also alleviates pain; there is no pain without consciousness. Most of the centrally acting analgesics will also reduce vigilance; e.g. driving is not permitted under analgesia with opiates. Alcohol is another example, although it minimally affects the nociceptive system, it substantially lowers the vigilance, thereby alleviating pain.(ABSTRACT TRUNCATED AT 250 WORDS)


[Acute respiratory failure--support of gas exchange using extracorporeal or implanted oxygenators--present status and future development]. In acute respiratory failure gas exchange can be supported or even maintained in an "alternative" way to mechanical ventilation using extracorporeal techniques (extracorporeal membrane oxygenation ECMO, extracorporeal CO2-removal ECCO2R), or intravenacaval oxygenators (IVOX). These techniques, which are currently in use in neonatology, pediatrics, and adult intensive care medicine, or techniques at present in clinical evaluation (IVOX), are reviewed with their indications, contraindications, differences, problems, worldwide results, and possible future applications.


[Potential risks of high-dose adrenaline for resuscitation following short-term heart arrest in animal experiments]. We compared the haemodynamic effects of epinephrine 10 micrograms/kg iv (group A, n = 8) and 50 micrograms/kg iv (group B, n = 8) in a porcine CPR-model after 3 min of circulatory arrest induced by ventricular fibrillation. All animals of group A were successfully resuscitated after 4.9 +/- 2.8 min and 2.8 +/- 1.6 defibrillations, in group B only 6 of 8 animals were successfully resuscitated after 6.3 +/- 1.1 min and 4.0 +/- 2.7 defibrillations (Mean +/- SD). Cardiac output, left ventricular systolic pressure and mean arterial pressure during CPR were nearly identical in both groups. The first hour of restored spontaneous circulation in group B was characterised by a significantly increased heart rate combined with significantly lower values for cardiac inotropy, cardiac output, left ventricular systolic pressure and mean arterial pressure. It is concluded that in acute or short-term cardiac arrest the currently recommended epinephrine dosages are sufficient. Higher doses of epinephrine for CPR seem to be recommendable only after prolonged cardiac arrest and/or during prolonged resuscitation.


[Intubation aid by a rigid endoscope].

[Intubation aid by a rigid endoscope]. A method has been developed making it possible to look around intubation obstacles with the aid of rigid angular-lens systems. A glottis that is not or is only partly visible in direct laryngoscopy can be visualized endoscopically. The normal intubation technique is retained. A rigid endoscope is additionally introduced into the oropharynx, the glottis located endoscopically, and the endoscope secured to the laryngoscope in the position. The free hand can then be used to advance the tube through the glottis into the trachea under endoscopic control. This endoscopic method is intended to augment the physician's instrumental repertoire.


[Initial experiences with rigid angled optical systems as intubation aids in difficult intubation]. Referring to a classification by Cormack, difficult laryngoscopy of Grade 3 (only the epiglottis or a part of it can be seen) was simulated in 16 patients by lowering the blade of the laryngoscope, so that the epiglottis was pushed down and thus covered up the vocal cords. The object of the study was to test whether a newly developed rigid endoscope is a useful tool during intubation in cases of laryngoscopical view Grade 3. After simulation of Grade 3 as mentioned above, using a clip, an angle optic was fixed to the vertical part of the blade, so that the movement of the optic in the sagittal level was still possible. If an improvement of the laryngoscopical view was possible, the tracheal tube was inserted via the nasal route until the top of the tube could be seen in the oropharynx. The tracheal tube was inserted into the trachea, under endoscopic control. With this new method, naso-tracheal intubation under endoscopic control in all 16 patients was successful, without affecting the pharynx and the vocal cords.


[Curare and its successors. A 50-year's evolution]. The introduction of curare into clinical anaesthesia by Griffith and Johnson in 1942 contributed to the termination of the era where anaesthesia was a reversible intoxication rather than the result of controlled drug action. Curare allowed general anaesthesia to be reduced to a lighter level, thereby conferring a significant safety factor to the patient. Both the shortage in supply of crude curare and its variable composition led the search for synthetic curare analogues conferring well defined pharmacodynamic and pharmacokinetic properties. Based on the chemical structure of tubocurarine which has been known since 1935 the efforts concentrated on bisquaternary ammonium compounds. Gallamine was the only synthetic curare analogue to contain three quaternary ammonium groups. This drug had significant undesired vagolytic effects. In 1951 succinylbischoline appeared to be the ideal muscle relaxant, particularly with respect to its fast onset and short duration of action. The disadvantages of its depolarising mechanism of action which were appreciated during the years to follow prevented the concept of depolarising neuromuscular blockade to be pursued further. With other muscle relaxants, including curare itself, histamine release, vagal blockade and ganglionic blockade were undesired effects to be eliminated in future compounds. Improved understanding of structure-activity relationships turned out to be an indispensable tool for future research. This in turn required more elaborate methods in chemical analysis, in electrophysiology of the motor endplate, and in ultrastructural research. As a result, alcuronium and pancuronium became available in the late sixties and early seventies. Both muscle relaxants had a non-depolarising mechanism of action with reduced side effects relative to curare. From now on better techniques for pharmacodynamic and pharmacokinetic research became available resulting in research activity with particular emphasis in this field. Researchers became aware that new muscle relaxants should be designed for larger volumes of distribution and more rapid biodegradation than those currently available. Concurrently, anaesthesia techniques had changed in a way to use intubation and mechanical ventilation as a routine procedure. The risk of intraoperative hypoventilation and hypoxemia was eliminated, yet, due to the lack of adequate monitoring techniques the slow recovery from curare, alcuronium or pancuronium neuromuscular blockade was hardly appreciated.(ABSTRACT TRUNCATED AT 400 WORDS)


[Spontaneous bacterial peritonitis without ascites]. A 34-year-old woman with acute pain in the lower abdomen and a history of non-A-non-B-hepatitis underwent laparotomy. A diffuse light redness of the small bowel without ascites was the only abnormal finding. An appendectomy was performed. The patient deteriorated into a sepsis during the next 60 hours. Relaparotomy established acute diffuse peritonitis with ascites and without any apparent intra-abdominal source of infection. Tracheal, blood, and intraperitoneal cultures of both procedures grew group A streptococci and proved a haematogenous spread of the infection. The sepsis was successfully treated with antibiotics and peritoneal lavage. The course of the infection and the findings are discussed and the case is interpreted as a spontaneous bacterial peritonitis without ascites.


Immune response in biocompatibility.

Immune response in biocompatibility. Biocompatibility is concerned with the interactions that occur between biomaterials and host tissues. As foreign objects in that host tissue these materials may initiate several types of response. It has often been postulated that the immune response, by which the host normally defends itself against invasion by foreign organisms, can be involved in the response to biomaterials. This review discusses the mechanisms by which this could occur and the evidence that suggests the immune response is indeed of significance in biocompatibility.


Preparation and characterization of Al2O3 reinforced hydroxyapatite. The microstructure of Al2O3 reinforced hydroxyapatite (HA-Ca5(PO4)3OH) was studied. It was demonstrated that in this composite the Al2O3 particles are uniformly distributed in a matrix of spherical HA agglomerates formed by primary needlelike HA particles present before making green compact. In the HA + 20 wt% Al2O3 system, hydroxyapatite is partially decomposed into alpha-Ca3(PO4)2 and CaO with H2O vapour during sintering. Subsequently, the CaO is combined with Al2O3 to produce calcium aluminates (CaAl2O4 and CaAl4O7).


Heterogeneous synthesis of fluoridated hydroxyapatites. Fluoridated hydroxyapatites were synthesized with two different modes of fluoride supply: by supplying F(-)-free solution initially, followed by a F(-)-containing solution whose fluoride concentration was stoichiometrically equal to that of fluorapatite; and with the order of supply of these solutions reversed. Both of these heterogeneously synthesized fluoridated hydroxyapatites showed typical calcium phosphate X-ray diffraction patterns; and both had similar total fluoride contents (0.94 +/- 0.02 and 0.99 +/- 0.03 mmol/g, respectively), i.e. half of the maximum fluoride content of fluorapatite. However, they differed considerably in their physico-chemical properties. The former had a wider peak breadth of X-ray diffraction and a lower apparent solubility (Ca = 12.2 +/- 0.7 mmol/l) than the latter (Ca = 19.8 +/- 0.4 mmol/l) at 37 degrees C and pH 4.0. Wavelength dispersive spectroscopy attached to scanning electron microscopy gave clearly different spectra. Electron spectroscopy for chemical analysis of apatite pellets made by pressing and heating showed a slightly larger F1s signal at the crystal surface of the former, although the difference was not marked. These results suggest that two different types of heterogeneous fluoridated hydroxyapatites were formed, hydroxyapatite covered with fluorapatite and fluorapatite covered with hydroxyapatite.


Experimental study on osteoconductive properties of a chitosan-bonded hydroxyapatite self-hardening paste. A developmental bone substitute, composed of a chitosan-bonded hydroxyapatite paste and having various possible applications in medical and dental treatments, was evaluated with regard to its osteoconductive properties. Radiographic examination revealed that a bone-like irregular radiopacity appeared in the region of the embedded paste. This was judged histopathologically as the formation of bone tissue with chondral tissue. These data suggest that the paste has osteoconductive properties, and may, therefore, prove clinically useful as a bioactive bone substitute.


Epicardial propranolol administration for ventricular arrhythmias in dogs: matrix formulation and characterization. The effect of propranolol on the prevention of ventricular tachycardia/fibrillation (VT/VF) due to acute coronary ischaemia was studied in dogs. A series of propranolol-polymer controlled release matrices in slab configuration using various polyurethanes and a polyurethane-silicone rubber copolymer were formulated and characterized. In general, drug release in vitro occurred with an initial burst phase followed by an exponentially declining delivery rate; the silicone rubber containing copolymer preparation had more sustained release properties than did pure polyurethane matrices. In the animal studies, dogs underwent 5-hourly 10 min complete occlusions of the left anterior descending coronary artery (LAD), followed by 50 min normal perfusion. During non-drug occlusions VT occurred at a frequency of 1.22 +/- 0.12 episodes/min. A propranolol-polyurethane matrix (30% w/w, 28-42 mg) was placed on the ischaemic zone of the left ventricular epicardium immediately after the fifth occlusion. After an hour of drug delivery a sixth occlusion took place. The number of arrhythmia episodes both before and after drug were quantified and compared. The time to ventricular fibrillation (when present) and the mean blood pressure were also assessed. The drug patch delivered propranolol at a dose of 140 +/- 45 micrograms/kg by the conclusion of the 1 h study period. Therapeutic drug levels were achieved in the peripheral blood samples (8.7-43.7 ng/ml) and were enhanced in coronary venous samples (360.9-556.2 ng/ml). Reduction of blood pressure and proarrhythmic events following epicardial controlled release propranolol administration were noted but were not statistically significant. Arrhythmia episodes before and after propranolol were not found to be significantly different (VT/min 1.02 +/- 0.31 and 1.22 +/- 0.12).(ABSTRACT TRUNCATED AT 250 WORDS)


Stability constants of Ca-alkyl salicylate complexes and their effect on the dissolution of calcium hydroxide dental cements. The proton-ligand stability constants (pKa) and Ca-ligand stability constants (log beta) of the five types of alkyl salicylate were determined and the dissolution of the calcium hydroxide cements prepared from those salicylates was examined in water. The dissolution of the cements containing monoesters decreased with the increase in their pKa and log beta values. The cement containing diester showed lower dissolution than that estimated from those values. This seems to be related to the polymeric structure of the cement matrix consisting of the complex of the diester with calcium.


Analysis of surface cleanliness of three commercial dental implants. Six routinely packaged commercially pure titanium dental implants (three commercial brands) were analysed using secondary ion mass spectrometry to examine the outermost surface layer. The aim of the analysis was to compare the surface purity (99.95%) of the implants with the substrate metal, common to all three brands. The use of a low primary ion current density provided a nominal protection limit of 0.01% or 100 p.p.m. All the implants examined had extremely pure surfaces. However, only one brand of implant had an ultraclean oxide surface in relation to the substrate.


Joint replacement components made of hot-forged and surface-treated Ti-6Al-7Nb alloy. We have developed a titanium-aluminium alloy with the inert alloying element niobium. The optimal composition was found to be Ti-6Al-7Nb (Protasul-100). This custom-made alloy designed for implants shows the same alpha/beta structure as Ti-6Al-4V and exhibits equally good mechanical properties. The corrosion resistance of Ti-6Al-7Nb in sodium chloride solution is equivalent to that of pure titanium and Ti-6Al-4V. This is due to a very dense and stable passive layer. Highly stressed anchorage stems of different hip prosthesis designs have been made from hot-forged Ti-6Al-7Nb. The polished surfaces of hip, knee and wrist joints made of Ti-6Al-7Nb and articulating against polyethylene are surface-treated by means of a very hard and 3-5 microns thick titanium nitride coating (Tribosul-TiN) or by oxygen diffusion hardening (Tribosul-ODH) to a depth of 30 microns.


Studies on immobilization of urease in gelatin by cross-linking. Urease enzyme was immobilized in photographic gelatin by chemical cross-linking using formaldehyde, glutaraldehyde and chromium (III) acetate. The effects of enzyme and cross-linker concentrations, temperature, incubation time and pH on urea hydrolysis were investigated. Effect of reuse on the activity of immobilized enzyme was also studied. Glutaraldehyde (0.004 M) was the most suitable cross-linker; relative activities within 2.5 months after 24 reuses were stable (about 78%).


ESCA investigation of low-temperature ammonia plasma-treated polyethylene substrate for immobilization of protein. A low-temperature radiofrequency plasma excited in anhydrous ammonia was used to modify polyethylene substrate surfaces for covalent immobilization of proteins. Electron spectroscopy for chemical application (ESCA) was used for surface characterization of polyethylene to a depth scale of 7 nm. The data revealed that surface modification is extensive and occurs in seconds at low discharge power. Primary amino functionalities were detected on the polyethylene surface and the level is dependent on plasma parameters. 125I-labelled antibodies covalently attached to amino groups via glutaraldehyde allowed the conditions for optimum level of primary amine to be established. Both ESCA data and protein loadings are in excellent agreement.


Sticking coefficients of adsorbing proteins. The protein sticking coefficient, phi, the fraction of collisions that result in adsorption, is a function of the molecular interactions between the protein and the surface. A random walk and diffusion-to-capture model was used to describe the kinetics of protein adsorption. The assumption of a constant sticking coefficient leads to a first-order model of the kinetics. A solution of the problem of adsorption from a semi-infinite medium with first-order kinetics at the boundary was obtained by numerical simulation on the computer. The results of the computer simulations match the time dependence observed experimentally. A correlation was developed to estimate phi from experimental data. phi has been found to be in the range 10(-5)-10(-8) for several protein adsorption kinetic studies reported in the literature.


In vivo fragmentation of microporous polyurethane- and copolyesterether elastomer-based vascular prostheses. A previous study showed that microporous, compliant and (bio)degradable vascular prostheses prepared from a polyurethane/poly(L-lactic acid) mixture can function as a temporary scaffold for the regeneration of small-calibre arteries. In this study the mechanism of fragmentation of vascular prostheses made of polyurethane, copolyesterether and blends of either polyurethane or copolyesterether with polymers differing in biodegradability, crystallinity and glass transition temperature is investigated. Animal studies revealed that after 6 wk of implantation only the prostheses made of blends containing a second polymer which was non-elastic at 37 degrees C were fragmented extensively, whether the second polymer was (bio)degradable or not. It is concluded that fragmentation of the prostheses is mainly caused by alternating stresses induced by the arterial pulsations and that (bio)degradation plays a minor role.


In vivo degradation of massive poly(alpha-hydroxy acids): validation of in vitro findings. The degradation of various high-molecular-weight aliphatic polyesters derived from glycolic acid and/or lactic acid enantiomers was previously investigated in vitro. It was demonstrated that the bulk degradation mechanism proposed in the literature actually proceeds heterogeneously and proceeds faster in the centre than at the surface of large specimens. In order to compare them, similar compression-moulded specimens were implanted intramuscularly in the backs of rabbits, namely PLA50 (poly(DL-lactic acid)), PLA37.5GA25 (75% DL-lactide and 25% glycolide in the feed) and PLA75GA25 (75% L-lactide and 25% glycolide). These three intrinsically amorphous compounds exhibited faster central degradation. Furthermore, preferential degradation of glycolic acid units and induced crystallization of L-lactic acid enriched fragments were observed in the case of PLA75GA25. These findings are comparable to phenomena observed in vitro and are conclusively supported by the re-examination of some old in vivo results. Accordingly, data reported in this paper validate both the in vitro modelling and new understanding of the degradation of lactic acid/glycolic acid-based aliphatic polyesters reported previously.


Biodegradable polymers. II. Degradation characteristics of hydrolysis-sensitive poly[(organo)phosphazenes]. Polyphosphazenes with hydrolytic labile substituents have potential as biodegradable materials. By proper choice of the substituents, polymers can be prepared which can degrade to harmless products. The rate of biodegradation and the nature of the degradation products can be widely varied by changing the chemical composition of the polymers. The degradation properties of a series of new polyphosphazene derivatives are discussed. The synthesis of phosphazene polymers with variable amounts of ethyl 2-(O-glycyl)lactate or ethyl 2-(O-alanyl)lactate as cosubstituents was described previously. These polymers were prepared by partial reaction of poly[(dichloro)phosphazene] with the corresponding amine compound. Total halogen replacement was achieved by subsequent introduction of glycine ethyl ester cosubstituents. The degradation characteristics of these polymers in organic solution or in vitro was investigated. It was demonstrated that the introduction of hydrolysis-sensitive side-groups along the polymer chain results in an increased degradability of the poly[(amino acid ester)phosphazenes]. A plausible mechanism for the hydrolysis of these materials is proposed. The main hydrolysis pathway of poly[(amino acid ester)phosphazene] devices in vitro involves release of the amino acid ester side-group followed by hydrolysis of the ester with formation of the amino acid and ethanol. Initial hydrolysis of the ester bond with subsequent release of glycine cannot be excluded but is probably predominant.


Wettability of cross-linked collagenous biomaterials: in vitro study. Collagenous biomaterials can be treated by chemical and physical agents to decrease biodegradation rate. Treatments to collagen may modify surface properties and subsequently cell and platelet behaviour. Collagenous films were either uncross-linked and cross-linked by glutaraldehyde, formaldehyde or cyanamide and/or treated by a severe dehydration. Contact angles, platelet contacting assay and fibroblast morphology were investigated. After severe dehydration, wettability was diminished except for formaldehyde-cross-linked and severely dehydrated films. Glutaraldehyde-cross-linked collagen results in an increase in wettability. Platelets were similarly distributed, except on formaldehyde-cross-linked films that exhibited no platelet aggregation. Fibroblasts were in a spreading phase on most collagenous films. However, cytotoxicity was noticed on some aldehyde-cross-linked films. No direct relationship was found between contact angles and platelet-cell attachment.


Porous polymer implants for repair of full-thickness defects of articular cartilage: an experimental study in rabbit and dog. Full-thickness defects of articular cartilage were repaired by implantation of porous polymer implants in rabbits and dogs. The quality of the repair tissue was determined by collagen typing with antibodies. Implants with varying pore sizes and chemical composition were used. The effect of loading and motion was determined by inserting implants higher than, level with and lower than the surrounding cartilage. It appeared that healing took place by formation of fibrocartilaginous repair tissue containing both type I and type II collagen. Hyaline cartilage was observed in a minority of the rabbits used but not in the dog. Fibrocartilage formation in the dog was simulated by implantation of a porous polymer. Chemical composition of the polymer did not alter the results, neither did loading of the implant. It is concluded that the formation of fibrocartilaginous repair cartilage is stimulated by implantation of a porous polymer. This tissue seemed to function adequately in the dog but did show signs of degeneration in the rabbit.


Chitosan cross-linked with Mo(VI) polyoxyanions: a new gelling system. A procedure for preparing homogeneous chitosan gels by in situ molybdate cross-linking is described. The gels are obtained by dispersing solid MoO3 in a buffered chitosan solution and the polymer is cross-linked by formation of heavily negatively charged molybdate polyoxyanions. The resulting ionic gels are very transparent, thermoirreversible and can be made at low polymer concentrations. Depending on the ionic strength, these gels are able to swell several times their original size in aqueous solutions. Estimates of the degree of cross-linking reveal a very open pore structure which is confirmed by electron micrographs of the gel.


Implantation of p(HEMA)-collagen composite into bone. The replacement of bone defects is very important in clinical practice. This study compares biological properties of poly(2-hydroxyethyl methacrylate)-collagen composite with those exhibited by pure p(HEMA), an insoluble fraction of calf skin collagen (ISC-40) and demineralized bone matrix after implantation into pig or dog femurs. The levels of biodegradation or destruction of implants and healing of bone defects were studied using X-ray photography, histology and enzyme histochemistry. The results indicated a significant effect of collagen on biological destruction of the p(HEMA)-composite implants; even a minute amount of collagen influences this process dramatically. A stimulatory action of collagen on new bone formation may be of importance in bone defect healing.


Refractive index and molar refraction of methacrylate monomers and polymers. The refractive indices of a number of methacrylate monomers have been measured and corresponding molar refraction data calculated. Similar determinations were made on a number of methacrylate polymers. The molar refraction values determined were in excellent agreement with the values calculated from the published molar refraction values of the chemical groups involved. There was little difference between the molar refraction values of monomers and that of the repeat unit in the corresponding polymers, in marked contrast to the molar volumes. The ratio of refractive index to molar volume was reasonably consistent for all methacrylates studied (0.25-0.30 approximately).


Phagocytosis of carbon particles by macrophages in vitro. Particles of known size ranges of carbon fibre-reinforced carbon were presented to in vitro cultures of murine macrophages. Particles of up to 20 microns diameter were phagocytosed. Larger particles were not phagocytosed but became surrounded by aggregations of macrophages, some of which migrated on to the particle surfaces. Mean rates of phagocytosis up to 2.5 particles per hour were observed. Cells presented with a large excess of particles became rounded, detached from the substrate and some underwent lysis. The implications of these findings for the fate of particulates released from implanted medical devices is discussed. It is argued that a mechanism exists where particles in the size range 8-20 microns, released from medical devices, are small enough to be phagocytosed by macrophages and transported to the lymphatics and subsequently to the vascular circulation but large enough to lodge in capillary beds of tissues remote from the implant site.


Cystatins--inhibitors of cysteine proteinases. The cystatin superfamily of proteins, derived from a common ancestor, is comprised of a diverse group of potent cysteine proteinase inhibitors and antibacterial/viral agents grouped into several families. This review concentrates on family 2 cystatins, namely, the human salivary cystatins and cystatin C. Emphasis is given to their physicochemical and functional properties at both the protein and the molecular level. The role of cystatins in disease processes, including those in the oral cavity, is also discussed. Finally, future directions for cystatin research in oral biology are presented.


Autogenous and allogeneic bone grafts in periodontal therapy. This article is limited to a review of bone autografts and allografts, as used in periodontal therapy. The various graft materials are discussed with respect to case reports, controlled clinical trials, and human histology. Other reviewed areas are wound healing with periodontal bone grafts, tissue banking and freeze-dried bone allografts, and the use of bone grafts in guided tissue regeneration.


The cultured diploid fibroblast as a model for the study of cellular aging. The limited proliferative potential of the cultured human diploid fibroblast is now well established. A number of biological correlates suggest that this culture system is a model for the study of aging at the cellular level. The mechanism(s) that causes the loss of proliferative activity is unknown; the results of some recent studies indicate that specific genes may play a pivotal role in cellular aging in vitro. The extent to which changes in proliferative functions are causally related to aging in vivo is currently under investigation.


The sense of taste: neurobiology, aging, and medication effects. The sense of taste is an oral chemical sense in mammals that is involved in the choice of foods. Initial transduction of taste stimuli occurs in taste buds, which are distributed in four discrete fields in the oral cavity. Medications can affect the taste buds and ion channels in taste-bud cell membranes involved in stimulus transduction. The sense of taste gradually declines with aging, with bitter taste most affected. Neural circuits that mediate taste in primates include cranial nerves VII, IX, and X, the solitary nucleus in the brain stem, the ventroposteromedial nucleus of the thalamus, and the insular-opercular cortex. The central taste pathways process taste information about sweet, salty, sour, and bitter stimuli serially and in parallel. Medications associated with "metallic" dysgeusia and taste losses affect the taste system via unknown mechanisms.


Some aspects of perfluorochemical emulsion's interaction with blood. FDC/FTPA (7:3) emulsions stabilized by procsanol (Emulsion 1) and by procsanol with yolk phospholipids (Emulsion 2) were incubated with the donor plasma. After the incubation during 6 hours of Emulsions 1 and 2 with plasma the 36% and 50% decrease of the cholesterol content in plasma was found. Analysis of the lipid content of lipoproteins after the Emulsion 2 administration to rats (2.5 ml/100 g of weight) revealed the 50% decrease of the cholesterol amount in the HDL fraction at 3 and 24 hours posttransfusion. The ratio cholesterol/total phospholipids in the erythrocyte membrane diminished up to 50% as well. The equal degree of the cholesterol adsorption by emulsion from plasma, HDL of rats and erythrocyte membrane is an evidence of nonspecific interaction of PFC particles with the blood components containing cholesterol.


Studies on blood substitutes based on hemoglobin and perfluorocarbon. Unmodified, and to a lesser extent, modified stroma-free hemoglobin preparations have been reported to exhibit coronary and renal vasoconstrictor activity in isolated perfused hearts and kidneys. The physiological significance in vivo of such ex vivo demonstrated vasoconstriction has not yet been established. We have conducted a number of in vivo dog experiments designed to elucidate (a) whether free hemoglobin in the plasma phase contributes to diffusive oxygen supply to the tissues and (b) whether excessive vasoconstriction results in functional impairment. Our findings indicate that (a) the infusion of unmodified SFHS does not cause a significant disturbance of central hemodynamics, although it causes an elevation of the arterial blood pressure; the latter is accompanied by vasoconstriction in the skeletal muscle vascular bed and in the renal cortex; (b) there is no significant improvement of diffusive oxygen supply to the tissue at rest; and (c) that glutaraldehyde cross-linked SFHS administered to hypotensive dogs causes a brief further aggravation of hypotension as well as renal vasoconstriction accompanied by renal functional impairment. The findings suggest that coronary autoregulatory mechanisms in vivo can override the vasoconstrictor potency demonstrated in vitro, but the renal effects of SFHS containing unmodified hemoglobin can give rise to significant concern.


Blood substitutes made on the basis of perfluorocarbons inhibit intracellular energy generation. Although perfluorocarbons (PFC) are chemically inert, toxic reactions are observed on using them as fluorocarbon emulsions in blood substitutes. Six to twelve hours after exchanging about half of the circulating blood of conscious rats pathobiochemical reactions occur despite a high interarterial oxygen pressure. They indicate the disturbance of intracellular energy generation, which is characterized by a decrease in ATP, increase in ADP, inorganic phosphate and potassium, increase in NADH and lactate. Impurities of perfluorocarbons and effects of the surfactant were excluded to be causes of this disturbance. The following hypotheses were proposed: Storing of perfluorocarbons in the mitochondrial membrane decreases the ATP-forming proton gradient on the membrane and the electron transport in cytochromes is disturbed, respectively.


Perfluoroalkylated surfactants: relationships between structure and acute toxicity in mice. New single chain neutral, and single and double chain zwitterionic perfluoroalkylated surfactants or co-surfactants have been evaluated for in vivo applications. A study of the relationship between structure and acute toxicity in mice is presented. Acute toxicity evaluations i.v. in mice indicate the following trends (increasing tolerance): zwitterionic single-chain less than neutral single-chain less than zwitterionic double-chain and, where the polar head is concerned, single-chain compounds: phosphocholine less than phosphoramidate less than trehalose approximately sucrose less than maltose less than xylitol.


Retention of perfluorochemicals in rat liver and spleen. The uptake and retention of perfluorochemicals (PFCs) into rat liver and spleen have been measured for up to 28 d following injection of either the commercial PFC emulsion, Fluosol, or a novel perfluorodecalin (FDC)-based emulsion. Both quantitative and qualitative differences in the retention of individual PFCs were observed, depending on composition of emulsion administered. Nevertheless, uptake of PFCs into the spleen was consistently greater than into the liver, irrespective of formulation injected.


Lymphoid tissue responses to concentrated perfluorochemical emulsions in rats. The effects of injecting perfluorochemical (PFC) emulsions of varying concentrations on lymphoid tissues have been studied in rats. Tissue weights were increased in proportion with quantity of PFC injected, with spleen responses consistently greater than those of the liver. PFC droplets recovered from tissues had mean diameters in the 1-10 microns range, with those from the spleen being larger than from the liver. Recovered droplet diameters were considerably greater than freshly-prepared emulsion mean particle sizes (0.20-0.25 microns). This suggests that coalescence of emulsion droplets following accumulation in tissues is a pre-requisite to the eventual excretion of PFC vapour through the lungs.


Acute effects of moderate Fluosol-DA hemodilution on hepatic microsomal and nonmicrosomal metabolism in rats. Fluosol has been shown to alter the disposition of several drugs immediately after its administration. Investigations in this laboratory established that the disposition of several drug markers requiring the hepatic microsomal cytochrome P-450 isoenzymes was time dependent for 72 hours. It was an additional purpose of the research to determine if the nonmicrosomal sulfation and acetylation pathways were also influenced by Fluosol hemodilution in a time dependent manner. Rats were moderately hemodiluted with Fluosol and received an intravenous dose of a drug marker 24, 48, or 72 hours after hemodilution. The formation clearance (ClF) of specific metabolites was used as the pharmacokinetic measure of a specific enzymatic activity. 3-Hydroxymethyl antipyrine ClF (phenobarbital inducible microsomal cytochrome P-450 isoenzymes) increased 300% only at 48 hours. Acetylsulfamethazine ClF (nonmicrosomal acetylation) increased 287% and 162% at 24 and 48 hours, respectively. Acetaminophen sulfate ClF (nonmicrosomal sulfation) decreased 30% only at 48 hours. Substantial evidence shows that cytochrome P-450 content is induced at 72 hours and remains induced for an unprecedented length of time by the PFCs in Fluosol. Therefore, it was unexpected that 3-hydroxymethyl antipyrine ClF was not increased at 72 hours. Several possible explanations are discussed for the unexpected findings.


Effect of a high concentration perfluorocarbon emulsion on platelet function. Perfluorocarbon (PFC) and lipid emulsions (eg. Fluosol, Intralipid,) containing phospholipid have been reported to modify platelet function after intravenous infusion. Platelet activation might be responsible for the generation of mediators responsible for PFC-induced side effects. In view of this, we investigated the effect of a highly concentrated perfluorocarbon emulsion containing 90% (w/v) perfluorooctylbromide (perflubron; PFOB), on porcine and human platelet activation and function. We measured both: a) stimulated ex-vivo porcine platelet aggregation pre and post infusion of either perflubron or control (vehicle only) emulsions, and b) stimulated in vitro human platelet calcium flux in the presence of perflubron emulsion or control emulsions. Platelet aggregation stimulated by collagen, ADP or arachidonic acid (AA) was inhibited in ex-vivo porcine platelets following infusion of perflubron emulsion at a dose of 3 ml/kg (0.2 ml/kg/min). Inhibition was dose dependent and was decreased when the dose of perflubron emulsion was reduced to 1.0 or 0.3 ml/kg. Infusion of saline or "vehicle" emulsions had little or no effect on stimulated ex-vivo pig platelet aggregation. Fluosol, infusion was associated with inconsistent inhibition of platelet aggregation. A23187- or AA-stimulated calcium flux of human platelets in vitro was inhibited in the presence of 1% (v/v) perflubron emulsion. Similar effects were seen with Fluosol, or Intralipid. This inhibition was agonist dose-dependent.(ABSTRACT TRUNCATED AT 250 WORDS)


Prolonged shelf stability and biocompatibility of a concentrated injectable fluorocarbon emulsion. A four-year-old 100% w/v concentrated emulsion of perfluorooctylbromide (PFOB, perflubron) stored at 5 degrees C, when submitted to the close-to-total exchange-perfusion test in conscious rats (Hct 3-5%) still resulted in 75% survival. Particle size and size distribution, viscosity, pH were still in the acceptable range.


Complete blood substitution during profound hypothermic cardiac arrest in dogs. This study compared the effects of 3 hours of cardiac arrest performed at 2 different levels of profound hypothermia in totally exsanguinated, blood substituted dogs. Dogs (N = 10) were anesthetized and esophageal temperature was lowered to 24 degrees at which time exsanguination began. Once exsanguination was complete and the heart had arrested, continuous whole body perfusion of an oxygenated blood substitute solution was performed for 3 hours. Core temperature during this period remained below 10 degrees C and reached nadirs of 1.3 degrees C in Group 1 (N = 5) versus 7.3 degrees C in Group II (N = 5). Once the dog was allowed to rewarm to 10 degrees C replacement of the perfusate with blood was begun. All dogs survived the procedure but 2 dogs from each group died by 4 days following the experiment. The remaining 6 dogs were observed for between 10 and 85 days. The group perfused with blood substitute at the warmer nadir had a faster recovery of motor behavior and showed smaller changes from normal in several biochemical and hematological parameters. These findings indicate that profound hypothermia and total blood substitution can be successfully achieved at either temperature nadir, but the warmer level appears to be associated with a faster recovery.


Arterial blood gases and brain oxygen availability following infusion of intratracheal fluorocarbon neat liquids. Eight adult New Zealand Swiss rabbits (3-5 kg) having previously implanted chronic bilateral platinum electrodes in the visual cerebral cortex and subcutaneous silver reference electrodes were tranquilized and monitored in multiple 2-3 hour sessions using voltammetric techniques. Six of these were given intratracheal neat liquid fluorocarbons ranging in boiling point from 132 degrees C to 215 degrees C at doses of 2 or 4cc/kg. Each animal received only one fluorocarbon liquid. Two additional rabbits were match-studied as controls. Half of the rabbits have survived more than five months. Both controls and two experimental rabbits were sacrificed after more than seven months due to gastric hairballs. The period of daytime monitoring sessions, when cathodic brain oxygen currents (aO2), arterial blood gases and pH were obtained, was between 34 and 263 days. In some animals the arterial pCO2 was increased during the first week but the pO2 and pH remained nearly normal in all eight animals throughout. The two best fluorocarbons for liquid breathing on the basis of this limited but intensive work are F-methyldecalin and F-5,6H-dec-5-ene (F44E).


Response of the rabbit lung as a criterion of safety for fluorocarbon breathing and blood substitutes. From the first liquid breathing experiments until now, the lung, not surprisingly, has played a central role in the evolution of fluorocarbon blood substitutes. The first breathable fluorocarbon, a mixture of F-alkylfurans(FC75), bp 102 degrees C, while a poor solvent for the lung's lining and a good solvent for oxygen and carbon dioxide, proved to cause a characteristic gas/vapor microbubble embolism following intravenous administration as an emulsion. Higher boiling fluorocarbons, e.g. F-tributylamine (FC47), bp 174 degrees C, do not produce such gas-vapor emboli. However, intermediate boiling compounds such as F-decalin (PP5), bp 141 degrees C, produce lungs which, although they certainly appear not to contain microbubble emboli, do not collapse when the thorax is opened. Such hyperinflated non-collapsible lungs (HNCL) occur in the rabbit after the intravenous infusion of F-decalin emulsions as well as after the intratracheal infusion of F-decalin neat liquid. F-decalin induced HNCL retain their appearance and low specific gravity for many weeks, gradually returning toward normal after many months. F-methyl decalin, bp 165 degrees C, does not cause HNCL after intravascular or intratracheal administration. Fluorocarbons having boiling points between 140 degrees C and 165 degrees C are being tested in order to find a perfluorinate with the highest transpiration rate, and hence vapor pressure, compatible with an acceptable body dwell time. We have given fluorocarbons intratracheally to 75, intravenously to 221 and both intratracheally and intravenously to 8 rabbits. Free radical trapping agents, antineutrophil, antiinflammatory and other drugs have been administered without appreciable decrease of HNCL. Fluorocarbon critical solution temperature, lipid solubility, emulsifiability, and other physicochemical properties may mediate the pulmonary effect. One method of preventing and treating low dose F-decalin-induced HNCL in rabbits is described.


Biological effects of photoactivated-HPD and cholesteryl hemisuccinate on erythroid differentiation. In order to establish the ability of porphyrins to distinguish between differentiated and undifferentiated cells of the erythropoietic pathway, the cytotoxic effects of hematoporphyrin derivative (HPD) on Friend erythroleukemic cells (FLC) were derivative (HPD) on Friend erythroleukemic cells (FLC) were studied. Since cholesterol affects the fluidity of cell membranes, the inhibitory effect of cholesteryl hemisuccinate (CHS) on HPD was similarly tested. FLC were induced with either dimethyl sulfoxide (DMSO), hemin, or both. The cells responded to DMSO-treatment by the synthesis of a large amount of hemoglobin and a decrease in both the cell volume and rate of cell-growth. Hemin, on the other hand, did not drive FLC to synthesize hemoglobin and reduced only moderately the growth rate compared to untreated cultures. The combined effect of DMSO and hemin led to a profound inhibition of the growth rate, but did not increase the synthesis of hemoglobin compared to the level observed in cells treated with DMSO only. The binding property of HPD to FLC was also determined. DMSO-treatment significantly reduced the amount of HPD-binding, and combined treatment with DMSO and hemin resulted in even a lower level of HPD-binding. On the other hand, induced as well as non-induced cells showed the same sensitivity to photoactivated HPD when both DNA and protein synthesis were examined. Pretreatment of both differentiated and undifferentiated cells with CHS reduced the cytotoxicity of photoactivated HPD to the same level. We conclude that the decreased binding of HPD to differentiated cells is a result of a reduction in their cell volume. Thus, the differentiation process per se does not protect cells against the photodynamic effect of HPD. Moreover, CHS may act as a scavenger or first acceptor of singlet oxygen blocking the photodynamic effect.


Blood substitutes based on modified hemoglobin prepared by encapsulation or crosslinking: an overview. Modified hemoglobin consists of (1) encapsulated hemoglobin and (2) crosslinked hemoglobin (polyhemoglobin, intramolecularly cross-linked hemoglobin and conjugated hemoglobin). There have been new advances in all types of modified hemoglobins. Modified hemoglobins are effective in hemorrhagic shock. However, it is important to define hemorrhagic shock models and experimental designs. Important progress has been made in research on vasoactivities, organ perfusion, organ preservation, biodistribution, hematology, complement activation immunology and other areas. A preclinical screening test may bridge the gap between animal safety studies and injection into human. Potential new sources of hemoglobin included bovine hemoglobin, recombinant human hemoglobin and synthetic heme.


Overview of progress in the fluorocarbon approach to in vivo oxygen delivery. The development of fluorocarbon-based oxygen carriers has experienced rapid progress over the past few years. Fluosol has been approved for use during percutaneous transluminal coronary angioplasty (PTCA) for high-risk patients. Its clinical evaluation is being pursued as an adjunct to cancer therapy and for treatment of myocardial infarction in conjunction with thrombolytic therapy. O2-delivery efficacy has been achieved with the development of the new highly concentrated (4 to 5 times more concentrated than Fluosol), fluid, emulsions of perfluorooctyl bromide (perflubron), trade-named Oxygen. The stability of fluorocarbon emulsions has also improved considerably and the new emulsions can be stored unfrozen and are ready for use. The side-effect profile of these emulsions has been characterized as being the normal response of the body's phagocytes to the injection of particles, a response that is considered physiological rather than pathological in nature; it involves some products of arachidonic acid metabolism and can be controlled pharmacologically. Means of further stabilizing fluorocarbon emulsions, involving molecular-diffusion-controlling additives or fluorinated surfactants, including mixed fluorocarbon-hydrocarbon compounds, have been devised. Increased control over in vivo particle recognition, intravascular persistence and side effects, and at adapting emulsion characteristics to specific applications, is being investigated. The range of therapeutic applications is expanding. The concentrated emulsions will be able to serve as a temporary red blood cell substitute in many situations. Acute normovolemic hemodilution with fluorocarbon emulsions, used in conjunction with homologous predonation and other blood-sparing techniques, should afford greater flexibility, increase the margin of safety, and reduce or alleviate the need for autologous blood transfusion during surgical procedures. Fluorocarbon applications in the cardiovascular field include use during PTCA, for cardioplegia and reperfusion, and the treatment of myocardial infarction. Significant tumor growth delay has been achieved when concentrated emulsions are used in conjunction with cancer radio- or chemotherapy. Liquid ventilation has potential as a unique treatment for the adult and infant respiratory distress syndromes and for drug delivery. The radiopaque and versatile perflubron can also be used in contrast agents for diagnosis with computed X-ray tomography, magnetic resonance imaging and ultrasound, allowing the early detection and staging of cancer. Other potential applications investigated include the treatment of cerebral ischemia, organ and limb preservation, use as a tamponade during retinal repair, etc.


Potential clinical applications for blood substitutes. In the coming decade, it is likely that oxygen-carrying alternatives to red blood cells will become available for clinical use. The driving force behind their development is the risk of transfusion of homologous blood, which includes transmission of viral disease (HIV and hepatitis) and transfusion reactions as well as the expense of collecting and storing human blood. A number of clinical applications for these products can be anticipated now, but when available, it is likely that the list will grow. How widely these products will be used depends on their safety. In addition to these clinical applications, blood substitutes will be useful in furthering our understanding of basic oxygen transport physiology.


Potential clinical applications of the oxygen carrying solutions. Two major areas of research of the oxygen carrying solutions are 1) utilization of hemoglobin and 2) perfluorochemicals. Even though they are not perfect red blood cell substitutes, the "oxygen carrying solutions" have many potential clinical applications because they will reach tissues more easily than normal human red cells and can deliver oxygen directly to tissues. In this paper, important examples of such usages are suggested. Additional clinical and non-clinical applications of the oxygen carrying solutions may be added in the near future.


Preparation and characterization of crosslinked and polymerized hemoglobin solutions. In 1982 we synthesized 2-Nor-2-formylpyridoxal 5'-phosphate (NFPLP) and subsequently showed that coupling of the beta chains of hemoglobin (Hb) by this organic phosphate compound according to Benesch et al. (1) lowers the oxygen affinity and prolongs the retention time in the circulation of rats and rabbits with a factor 3 by prevention of excretion via the kidneys. Optimal conditions for the purification of HbNFPLP either by ion-exchange chromatography or by heat treatment were established with recoveries of 70% and 85%, respectively. By extrapolation from the data in rats and rabbits a half life of about 8 hours can be expected in the circulation of humans. However, under some conditions a further prolongation is required. The aim of further modification of HbNFPLP was to achieve a retention time of about 24 hours. Polymerization with glutaraldehyde to polyHbNFPLP resulted in a mixture of polymers of different size. We determined the optimal degree of polymerization with respect to the effects on vascular retention time, oncotic activity, viscosity and oxygen affinity. Depending on the degree of polymerization we found in rats a 5- to 7- fold increase in vascular half-life compared to native Hb. The change in oxygen affinity was found to be independent of the polymer size (P50 = 18-22 mmHg). A limiting factor for polymerization is the increase in viscosity, which was dramatic when large polymers (greater than 300 kD) were present in the preparation.(ABSTRACT TRUNCATED AT 250 WORDS)


Hemoglobin tetramers stabilized with polyaspirins. Organic acids activated by esterification with 3,5-dibromosalicylate react preferentially either with the beta 82 lysines or the alpha 99 lysines of hemoglobin. The versatility and site specificity of these polysapirins and the usage of both human and bovine hemoglobins allowed the construction of a family of oxygen carriers with various P50 ranging from 10 to 50 mmHg. These derivatives are obtained in pure homogeneous form by column chromatography. They are stabilized tetramers where the dissociation into dimers is inhibited. The latest addition is Tri-(3,5,dibromosalicyl)-benzenetricarboxylate, which crosslinks both human and bovine hemoglobin across the beta subunits, decreasing the oxygen affinity of both proteins. The crosslinked hemoglobins have a normal Bohr effect, more expanded in the alkaline region, and are sensitive to chlorides but not to polyphosphates. Solutions of stabilized tetramers, infused into rats or cats up to 25-50% blood replacement, do not produce altered renal and cardiac function. In the cat isovolemic hemodilution increases cerebral flow in controls treated with albumin solutions, when an oxygen carrier is used the cerebral flow remains normal.


Synthesis and properties of polymerized, diaspirin cross-linked hemoglobins. During the course of our studies it became clear that there were therapeutic applications for which a polymeric hemoglobin having an extended half-life in circulation would be appropriate. Therefore, a process for the glutaraldehyde-polymerization of diaspirin cross-linked hemoglobin (DCLHb) was developed and used to prepare glutaraldehyde-polymerized DCLHb (GP-DCLHb) in lactated Ringer's solution in sufficient quantities for biological testing. Both isovolemic exchange-transfusion and "top-load" studies (rats; primates and swine, respectively) were completed in which a broad spectrum of physiologic, histopathologic and analytical parameters were monitored and assessed. In general, GP-DCLHb in lactated Ringer's solution was well-tolerated physiologically. When compared to DCLHb, GP-DCLHb offers the advantages of reduced renal clearance of hemoglobin and an extended half-life in circulation. GP-DCLHb has the disadvantages that (1) glutaraldehyde is an ineffective virucidal agent under the conditions of the polymerization reaction and a separate virus inactivation step is required; (2) low-endotoxin (LAL-negative) GP-DCLHb solutions are pyrogenic (rabbits); and (3) unusual deposition of hemoglobin-containing material in the small arterioles of the liver and kidney (rats) was sometimes seen even after a period of time (2 weeks) during which treatment-related organ pathologies are usually resolved, a finding peculiar to GP-DCLHb among the various hemoglobin derivatives we have tested.


Effects of hypothermic conditions on the oxygen carrying capacity of crosslinked hemoglobins. In view of the potential application for hemoglobin-based oxygen carriers (HBOCs) in organ perfusion under hypothermic conditions, we examined the temperature dependence of oxygen equilibrium curves (OECs) at 15-37 degrees C of three HBOCs: HbA-FMDA and HbBv-FMDA, produced by the reaction of human or bovine oxyHb with fumaryl mono-dibromoaspirin, and HbA-DBBF, produced by the reaction of human deoxyHb with bis(3,5-dibromosalicyl) fumarate. OECs for HbA-DBBF, HbA-FMDA and HbBv-FMDA at 37 degrees C were right shifted (P50 = 24.5, 17 and 35 torr, respectively). van't Hoff's rule gave HbA-DBBF (-12.2 +/- 2.8), HbA-FMDA (-12.0 +/- 2.0), HbBv-FMDA (-10.5 +/- 1.8); these values do not significantly differ from that for native HbAo (-11.5 +/- 2.4). Among the hemoglobins included in this study, HbBv-FMDA had the most favorable oxygenation characteristics at low temperatures (a P50 of 6.0 torr at 15 degrees C as compared to only 2-3 torr for the other hemoglobins in the study). Recently, however, a human hemoglobin crosslinked with bis-pyridoxyl tetraphosphate was reported to have a P50 of 15 torr at 16 degrees C (Keipert et al, Transfusion 1989; 29: 768-773). Therefore, precise knowledge of the oxygen delivering capacity of any potential HBOC should be explored under hypothermic conditions as performance under these conditions may determine its usefulness as an organ perfusate.