Recombinant human factor VIIa (rFVIIa) in a rabbit stasis model. The thrombogenicity of recombinant human FVIIa (rFVIIa) and FEIBA was studied in a rabbit stasis model. Only minor thrombus formation in isolated vein segments during 10 min. of stasis was seen following the administration of rFVIIa 100-1000 micrograms/kg or FEIBA 50-100 U/kg whereas both compounds caused clear thrombus formation during 30 min. of stasis. RFVIIa caused no change in platelet counts or plasma fibrinogen 3 hours after administration, whereas a small decrease of APTT was seen due to the direct effect of rFVIIa in this assay. In contrast, FEIBA caused a significant and dose-dependent decrease of platelet counts as well as of fibrinogen, and an increase of APTT which demonstrate a general consumption of coagulation factors. In conclusion the study demonstrated a local pharmacological effect of rFVIIa in the absence of a general activation of the coagulation cascade.


Stimulation of PAF-synthesis in pulmonary artery endothelial cells by Staphylococcus aureus alpha-toxin. Staphylococcus aureus alpha-toxin is a pore-forming exotoxin that probably represents a significant virulence factor in staphylococcal infections. Previous studies demonstrated that this agent stimulated arachidonate metabolism with subsequent formation of prostacyclin in endothelial cells and leukotriene B4 in granulocytes. We now examined the effect of alpha-toxin on the synthesis of platelet-activating factor (PAF) in cultured porcine pulmonary artery endothelial cells. PAF was labeled by bioincorporation of tritiated acetate and separated/quantitated using thin-layer chromatography, straight-phase-HPLC, or post-HPLC-bioassay. Alpha-toxin induced synthesis of small amounts of PAF in a time- and dose-dependent manner. The maximal amount of PAF elicited by alpha-toxin was approximately 7% of that observed after application of the ionophore A23187. Staphylococcal alpha-toxin but not the ionophore induced a rapid fall in cellular ATP-content. On kinetic grounds, the decrease in ATP-levels did not explain the differences in stimulated PAF-synthesis. Low amounts of PAF induced by the action of alpha-toxin on endothelial cells may contribute to the development of inflammatory lesions in infectious disease.


Heparin enhances platelet aggregation irrespective of anticoagulation with citrate or with hirudin. The effects of anticoagulation with citrate or hirudin on heparin effects on platelet aggregation was studied with whole blood aggregometry on blood from healthy volunteers. Platelet aggregation was initiated by collagen. The heparin effect was also studied with filtragometry where hirudin was used as the anticoagulant. In citrated blood, a mean collagen dose of 0.42 +/- 0.04 micrograms/ml resulted in an impedance change of 1.1 +/- 0.3 Ohm. Preincubation with heparin doses of 0.5, 2.5 and 5 IU/ml enhanced the impedance induced by the same dose of collagen by 2.9 +/- 1.4, 11.4 +/- 1.6 and 9.9 +/- 2.3 times, respectively (p less than 0.0001, ANOVA). In hirudinized blood a similar degree of change in impedance (1.5 +/- 0.2 Ohm) was achieved at significantly lower concentration of collagen (0.08 +/- 0.006 micrograms/ml, p less than 0.0001). Preincubation with heparin in doses of 0.5, 2.5 and 5 IU/ml increased impedance by 1.5 +/- 0.5, 3.9 +/- 1.6 and 1.3 +/- 0.5 times, respectively (p less than 0.0001, ANOVA). The dose-related increments were smaller in hirudinized blood as compared to citrated blood (p less than 0.04). With filtragometry, heparin dose-dependently shortened the aggregation time (p less than 0.0007). Compared to hirudin alone as anticoagulant, heparin in an equipotent dose in these experiments shortened aggregation time (p less than 0.05). In conclusion, heparin enhanced platelet aggregation both in calcium-chelated blood and in blood anticoagulated with hirudin. Heparin also dose-dependently increased platelet aggregation in filtragometry. Thus the heparin potentiating effect on platelet aggregation seems to be independent of extracellular ionized calcium and be operative at physiological calcium concentrations.


Does fibrinogen contain populations with different degree of sialylation? The reactivity of the galactose specific lectin from Ricinus communis seeds, ricin, towards the fractions of fibrinogen separated by DEAE-cellulose chromatography and their isolated glycopeptides was studied. Ricin is known to differentiate oligosaccharide chains with different degree of sialylation. The results indicated that the fractionation of fibrinogen is not correlated with a different degree of sialylation of its oligosaccharide chains. The higher sialic acid content of the most acidic fraction correlates with a higher neutral sugar content due to the presence of highly glycosylated contaminants. The possibility that the heterogeneity in the degree of sialylation is only of intramolecular nature is discussed.


Coagulation and fibrinolytic system impairment in insulin dependent diabetes mellitus. Selected coagulation and fibrinolytic parameters were assessed in 40 insulin dependent diabetes mellitus patients with varying degrees of metabolic control; 30 healthy subjects matched for age and sex formed the control group. Activated Partial Thromboplastin Time, Prothrombin Time, Fibrinogen, Factor VII, Antithrombin III, Protein C, Plasminogen, alpha 2-Plasmin Inhibitor, Plasminogen Activator Inhibitor-1, tissue-Plasminogen Activator were functionally evaluated. Antigenic levels of tissue-Plasminogen Activator, Thrombin-Antithrombin complexes and fibrinolytic specific product B beta 15-42 were also determined. Compared to the control group diabetic patients displayed significantly higher levels of Fibrinogen (p < 0.01), Factor VII (p < 0.01), Thrombin-Antithrombin complexes (p < 0.01) and Plasminogen Activator Inhibitor-1 activity (p < 0.01). Regardless of the normal level of the tissue-Plasminogen Activator-related antigen, diabetic patients had tissue-Plasminogen Activator activity lower than the control group (p < 0.05). Coagulation Factor VII and Thrombin-Antithrombin complexes were increased only in the patients with poor metabolic control (p < 0.01). Activated Partial Thromboplastin Time, Prothrombin Time, Antithrombin III, Protein C, Plasminogen, alpha 2-Plasmin Inhibitor, B beta 15-42 fibrin peptide were found to be in the normal range. Fibrinogen correlated positively with fasting blood glucose (p < 0.05) and Thrombin-Antithrombin complexes with glycosylated haemoglobin (p < 0.05), whereas Factor VII was positively correlated with glycemia (p < 0.01) and glycosylated haemoglobin (p < 0.05). Higher levels of Fibrinogen were found in patients affected by nephropathy (p < 0.005) or neuropathy (p < 0.05). These results demonstrate an impairment of the haemostatic balance in diabetic patients, that is a possible hypercoagulable state, which represents an important factor in the pathogenesis of atherosclerotic complications.


Reduced functions of intracellular Ca2+ in aggregation, secretion and protein phosphorylation of permeabilized platelets from stroke-prone spontaneously hypertensive rats. Aggregation, secretion and 47kDa protein (P47) phosphorylation by various agonists such as thrombin, ADP and ionophore A23187 were markedly reduced in platelets from stroke-prone spontaneously hypertensive rats (SHRSP) compared with those of age-matched Wistar Kyoto rat (WKY) platelets, suggesting defective functions of intracellular Ca2+ in SHRSP platelets (Tomita et al. Hypertension 1989; 14: 304-315). To clarify the mechanism of the platelet hypofunctions, saponin permeabilized platelets were prepared to compare the responses of platelets from both rats in varying concentrations of extracellular Ca2+. The leakage of lactate dehydrogenase from saponin (15 micrograms/ml)-treated platelets was approx. 5% of total activity; the degree of the leakage in both platelets did not differ. In saponin-treated platelets, extracellular Ca2+ alone did not induce either aggregation or secretion in both strains. However, in the presence of 1-oleoyl-2-acetylglycerol (10 micrograms/ml), Ca2+ dose dependently stimulated both aggregation and secretion. Under this condition, Ca2+ sensitivity of aggregation, secretion and P47 phosphorylation in SHRSP platelets were significantly reduced compared with those in WKY platelets. These results strongly suggest that intracellular Ca2+ functions are impaired in SHRSP platelets.


Reduction of the anticoagulant activity of glycosaminoglycans on the surface of the vascular endothelium by endotoxin and neutrophils: evaluation by an amidolytic assay. The processes that underlie the coagulopathy observed in severe infection are not fully understood, but seem to be due to an imbalance in the antithrombotic, and prothrombotic properties of the vascular endothelium. Sulphated glycosaminoglycans (GAGs) present on the vessel wall represent an important component of the non-thrombogenic nature of the endothelium. We have modified an amidolytic assay to study the functional ability of GAGs on human umbilical vein endothelial cells (HUVECS), and investigate the effect of E. coli endotoxin and neutrophils on HUVEC surface anticoagulant activity (SAA). Neither endotoxin alone, nor separated neutrophils at lower concentrations (less than 10(6) neutrophils per ml), had major effects on endothelial SAA. When activated neutrophils were incubated with HUVECS pre-stimulated with endotoxin, a significant decrease in SAA was seen using either plasma (mean percentage of control 67.8% +/- sem 7.8; p < 0.02) or purified ATIII (mean percentage of control 69% +/- sem 4.6; p < 0.001). We suggest that alterations in endothelial surface GAGs may occur during sepsis and inflammation, and that this may have important consequences for vascular function. This system will allow the further study of the role of GAGs in the intravascular thrombosis of severe sepsis, and other inflammatory diseases.


Purification of rabbit, rat and mouse protein C with the use of monoclonal antibody to human protein C, PC01. Ca(++)-dependent monoclonal antibody specific to gamma-carboxyglutamic acid (Gla) domain of protein C was produced. It did not cross-react to the other vitamin K-dependent plasma proteins but to protein C of the other species. Using this monoclonal antibody, PC01, rabbit (170 micrograms), rat (60 micrograms) and mouse (40 micrograms) protein Cs were isolated from 100 ml of their plasma by affinity chromatography. All of these protein Cs were two chain form linked by disulfide bond as well as human protein C and activated by thrombin-thrombomodulin complex. Rat and mouse protein Cs showed similar characters to human protein C. On the other hand rabbit protein C had different M(r) of heavy and light chains and showed lower anticoagulant activity compared with human protein C.


Evidence for the role of dialysis hypoxemia in the pathogenesis of hemodialysis-induced rise in tissue-type plasminogen activator. It is well known that hemodialysis (HD) causes a rise in plasma tissue-type plasminogen activator (t-PA). Although there have been several suggested mechanisms responsible for this effect of HD, the precise cause has not been well understood yet. Another complication of HD, when performed with acetate-containing dialysate, is hypoxemia, which is commonly observed during the first hour of the session. The purpose of this study was to investigate the relationship between dialysis hypoxemia and HD-induced t-PA changes during the first two hours of HD. HD caused significant increase in plasma t-PA antigen levels. When individual t-PA profiles versus time were examined, two patterns were observed. Whilst ten subjects (%56) experienced minimal or no increase, t-PA antigen level of the remaining eight subjects began to rise at 30 minutes and continued at that level up to 90 minutes, when the last samples were drawn. The courses of pO2 were also different; whilst the former group had "early-onset and short-term" hypoxemia, the latter had "late-onset and prolonged" hypoxemia. The amount of increase in t-PA antigen and the amount of decrease in pO2 were correlated at 60 and 90 minutes of the HD session. Thus, it is concluded that dialysis hypoxemia may contribute to HD-induced rise in plasma t-PA levels. Further studies comparing different dialysates and dialyser membranes are required to confirm this hypothesis.


Inhibitory effect of activated protein C on platelet aggregation induced by the prothrombin-converting reaction. The present study was undertaken to elucidate the effect on platelet aggregation of the prothrombin-converting reaction on platelets with or without activated protein C (APC). A reaction mixture of washed platelets from human individuals, Factor Xa and prothrombin markedly induced platelet aggregation; maximum aggregation rates, 31.3-92.5%, and times to reach to maximum aggregation, 11.6 to 20.1 min. This aggregation was inhibited by the addition of APC with 50% inhibition concentration (IC50) value of 14.4 U/ml. APC also inhibited thrombin generation in the reaction mixture in a dose-dependent manner with IC50 value of 0.96 U/ml. However, APC did not inhibit the thrombin (0.1 CU/ml)-induced platelet aggregation at concentrations of up to 30 U/ml. These findings suggest that APC has no direct inhibitory effect on platelet aggregation and that APC inhibits platelet aggregation through inhibition of thrombin generation.


Interactions between thrombolytic agents and platelets: effects of plasmin on platelet glycoproteins Ib and IIb/IIIa. The mechanisms by which thrombolytic agents affect platelet function are not yet elucidated. The aim of the present study was to investigate the effects of plasmin, generated by thrombolytic agents in plasma, on platelet glycoproteins (GP) Ib and IIb/IIIa. Platelet-rich plasma was incubated with pharmacological amounts of streptokinase, anistreplase and tissue-type plasminogen activator and the platelet surface GP's were investigated with a panel of monoclonal antibodies using flow cytometry. As assessed from the mean fluorescence intensity of incubated and control platelets, no significant changes in the binding of antibodies to GP Ib and GP IIb/IIIa were found. The functional integrity of these glycoproteins was severely impaired by treatment with the thrombolytic agents, as shown by significant inhibition of ADP- and ristocetin-induced platelet aggregation. Experiments with purified plasmin and washed platelets indicated significant degradation of GP IIb/IIIa and upregulation of GP Ib, which is in agreement with previous findings. In addition, platelet activation by plasmin was shown using two monoclonal antibodies to activation-specific antigens. We conclude that degradation of platelet GP's by plasmin offers no likely explanation for the defect in platelet function, which is induced by thrombolytic agents in platelet-rich plasma.


Inhibition of plasma kallikrein. Kininase and kinin-like activities of preparations from the medicinal leeches. The medicinal leech salivary gland secretion deprived of hirudin antithrombin activity inhibits amidolytic (substrate S-2302) and kininogenase (substrate kininogen) activities of plasma kallikrein, the main component of the intrinsic mechanism of blood coagulation. It therefore possesses high anticoagulant properties. Kininase (substrate bradykinin) activity of leech saliva and extracts from the medicinal leeches, as well as kinin-like effects of extracts heated at 100 degrees C have been detected. The last one is correlated with the hyperalgetic property of the heated extract. An analgetic effect was observed with the unheated extract but not with leech saliva after intranasal administration to rats.


[Neospora abortion in cattle in The Netherlands]. Three cases of bovine protozoal abortion at three different dairy farms are reported. Multiple abortions had occurred, on two farms, and a sporadic abortion on the third farm. Histopathological examination of foetal tissues revealed a distinctive multifocal necrotising encephalitis as well as focal inflammatory lesions in the myocardium, liver, kidney and lung. In one foetus protozoal tissue cysts were found in the brain and individual and clustered tachyzoites were seen in brain, and lung. In another foetus a cluster of tachyzoites was seen in a section of a placentoma. Immunohistochemically, tissue cysts as well as tachyzoites reacted with Neospora caninum antiserum. Tachyzoites were also detected in the brain lesions of the other two foetuses. The preliminary results of a histopathological survey of aborted bovine foetuses indicate that this protozoal infection may be an important cause of bovine abortion in The Netherlands.


[Vesicular swine disease in The Netherlands]. The clinical signs, diagnosis and epizootiology of swine vesicular disease (SVD) are described. The clinical appearance is illustrated by photographs of experimentally and naturally infected pigs. Special attention is paid to differences between SVD and foot-and-mouth disease (FMD) and to the choice of disinfectants.


[The effect of feeding mangel-wurzels on blood beta-hydroxybutyrate level of milk-producing cows]. The effect of feeding dairy cattle mangel-wurzels on the serum increased concentration of beta-hydroxybutyrate was investigated in serum is normally indicative of an increased catabolism of fats in the liver as a result of an energy shortage. Feeding cattle large amounts of mangel-wurzels can cause the serum beta-hydroxybutyrate concentration to increase above normal reference values, thus seemingly indicating that there is a (sub)clinical acetonaemia. In fact the increased concentration of beta-hydroxybutyrate is probably the result of an increased production of butyrate in the rumen.


[Rickets-like bone changes in goat kids fed with artificial milk intended for calves]. In a large commercial goat farm rickets-like symptoms were diagnosed in goat kids. The disease was probably caused by the synthetic milk which was freely available to the kids. The milk feed was for calves and had a calcium-phosphorus ratio of 0.83:1.


[Carpal ligament lesions: a underrated problem in dogs]. Traumatic hyperextension of the carpus in the dog may result in severe lesions of the palmar ligaments, which cannot be treated by coaptation splinting alone. Treatment in three dogs by partial arthrodesis using plate osteosynthesis is discussed.


[Premature infants and hepatitis B vaccination. Study Group Prevention Neonatal Hepatitis B]. Since 1986 health authorities in the Netherlands advise to vaccinate preterm infants at similar age as term infants, without correction for their shortened gestational age. This advice was based on a study, which showed comparable immune responses after DTP vaccination in preterm and term infants. To assess the immunogenicity of hepatitis B vaccination not corrected for gestational age in preterm infants, we compared the antiHBs titer after hepatitis B vaccination in 44 preterm infants with the antiHBs titer in 829 term infants. More than 95% of the preterm infants developed an adequate immune response (> 10 IU/l antiHBs), irrespective the vaccination scheme which varied in vaccine dose, the number of vaccinations and the onset of the first vaccination. The percentage preterm infants with an antiHBs titer > 10 IU/l (98%) was not different of the corresponding percentage of term infants (98%). Similarly, no difference between preterm and term infants was observed when an antiHBs value of 100 IU/l was considered a positive response, neither when active immunisation started at month 0 or month 3. The geometric mean titre at 12 months of age was considered.


[Intracranial germinoma in children].

[Intracranial germinoma in children]. Two cases of intracranial germinoma with different clinical expression, are described. The clinical symptomatology, the diagnosis and the treatment of this tumor are discussed. The symptoms depend on the localization of the tumor: in the suprasellar germinoma endocrinologic manifestations prevail while the symptoms in germinomas which are located in the pineal region, are mainly due to increased intracranial pressure. The diagnosis is suggested by the findings on CT-scan and MRI of the brain, but for the definitive diagnosis, pathologic examination of the tumor remains necessary. Blood HCG and alpha-fetoprotein are useful markers for follow-up; the value of angiotensin converting enzyme (ACE) as a marker, is still unclear. The ideal treatment of germinoma consists of surgical removal, postoperative chemotherapy and, afterwards, local radiotherapy. On the whole, the prognosis of this tumor is good.


[Independence within reach. Ergotherapy treatment of children with a congenital reduction defect]. The occupational therapy department of the University Hospital of Leiden treats a relatively large number of children, born with a dysmelia of the forearm. This article has been written to give information about the possibilities to supply these children with a prosthesis, and how this effects their level of independence. A short description of occupational therapy and the working of a myoelectric prosthesis will be followed by a case-study of a boy with plural congenital reduction defects. He needs several prosthesis and aids to become (mainly) independent of the help from others. It will be described how aids are made and tested, and how the prosthesis is used. The case-study illustrates that the different demands and desires which are demanded from prosthesis and aids, are related to the age and developmental level of the child.


[Tropical malaria and sickle-cell anemia: a tropical surprise]. A case of a very sick 2.5-year-old, Ghanese boy with fever, who fell in coma in the emergency room, is described. He was diagnosed as having blackwater fever (BWF) on clinical grounds. He also had a sickle cell anaemia. A short review of BWF is given and we discuss possible causes of the anaemia in this case. The anaemia in sickle cell anaemia is described and it appears that malaria tropica can lead to a severe hemolytic sickle cell crises. We discuss the combination of sickle cell anaemia and malaria tropica.


[Intraosseous infusions in children].

[Intraosseous infusions in children]. Tibial intraosseous infusions can be invaluable in the management of the pediatric patient who is in cardiac arrest or severe shock when vascular access by the intravenous route is unattainable. Recently there has been renewed interest in this procedure. There is a small risk of complications.


[Quantitative and qualitative stock-taking of scientific research in pediatrics in The Netherlands (1986-1990)]. All Dutch academic and non-academic pediatric training centres were requested to give a survey of all scientific publications produced from 1986 to 1990. The initiative came from the board of the Dutch Pediatric Association. During the observation period (1986-1990) the number of international publications with an external reference system was twice when compared with 1981-1985. The quality of the publications was similar. In at least four of the participating academic pediatric hospitals most publications came from the subspecialty area of metabolic diseases, oncology/hematology, immunology/infectious diseases, and cardiology, both during the first and the second observation period. From 1986 to 1990 the total number of theses in which a pediatrician was the main promoter or promoter in charge came to 84.


Linkage disequilibrium between DPA1 and DPB1 alleles among Norwegian Caucasoids and Japanese. Associations between alleles at the DPA1 and DPB1 loci were analyzed in 181 Norwegian caucasoids. Associations were observed between the DPA1*0201 and the DPB1*0101, *0901, *1001 and *1101 alleles. An association between DPA1*0201 and the DPB1*0901 allele was also observed among 23 healthy Japanese and 24 Japanese MS patients. In addition, it appeared that among Japanese the DPA1*0201 allele may be associated to the DPB1*0501. In conclusion, alleles at the DPA1 and DPB1 loci display different associations among Norwegian caucasoids and Japanese.


HLA-DR,DQ nucleotide sequence polymorphisms in five Melanesian populations. HLA-DRB1 nucleotide sequence polymorphisms have been examined in 304 Melanesians from the Papua New Guinean coast (Madang), islands (Rabaul) and highlands (Goroka), and from New Caledonia and Fiji. A total of 20 HLA-DRB1 alleles were detected by oligonucleotide hybridizations of exon 2 HLA-DRB1 polymerase chain reaction products, in a typing protocol designed to detect all 42 officially-designated HLA-DRB1 alleles. DRB1*1502 and 1101 alleles were the most common alleles in coastal and island Melanesians, while DRB1*1501, 1502 and 1408 predominated in Papua New Guinean highlanders. Undefined mixed lymphocyte reaction determinants in earlier studies of Melanesians could be accounted for in the present study as DRB1*0410, 1407 and 1408 in Papua New Guinean highlanders and as DRB1*1104 and 1602 in coastal people. Nucleotide sequence polymorphisms at HLA-DQA1, -DQB1, -DRB3 and -DRB5 were also determined for estimating HLA-DR,DQ allelic disequilibrium relationships; unusual haplotypes in Melanesians included DBR1*1502, DRB5*0101 and DRB1*0410, DQB1*0402. Previous claims of limited heterogeneity in the HLA-DR allele repertoire in Melanesians are now seen to reflect limitations of early typing reagents rather than any dramatic restriction in HLA-DR allelic diversity.


DNA typing of HLA class II genes in B-lymphoblastoid cell lines homozygous for HLA. The HLA class II genotypes were determined in the B-lymphoblastoid cell lines selected for the Tenth International Histocompatibility Workshop. The HLA class II genes were determined by the PCR-SSOP method using the reagents provided by the Eleventh Histocompatibility Workshop. Additional studies have been performed for further characterization of HLA class II polymorphism on these cell lines. It is observed that several cell lines have HLA class II haplotypes with the same DRB1, DQA1 and DQB1 alleles on both haplotypes but different alleles at the other class II loci, confirming that these cell lines are not truly HLA class II-homozygous. Other cell lines carried HLA class II haplotypes which were only different at the DRB1 gene. These results suggest double recombination events or gene conversion-like events in generation of HLA DR, DQ haplotypes. These cell lines provide an important tool as references for HLA DNA typing.


Adult onset spinocerebellar ataxia linked to HLA in a South African kindred of mixed ancestry. Hereditary spinocerebellar ataxia (SCA) is a relatively common disorder in the Western Cape region of South Africa. At present there are no genetic markers available for prenatal or presymptomatic diagnosis. A large kindred of mixed ancestry with late onset SCA was studied in which the disorder segregated in an autosomal dominant fashion. HLA typing was undertaken on 44 family members, and the HLA haplotypes were assigned on the basis of segregation. The LIPED computer program, with a correction factor allowing for the age of onset, was used to analyze the pedigree for linkage to HLA. Of 22 individuals in whom disease status could be definitely assessed, only one recombinant between HLA and the SCA locus occurred. The lod score reached a maximum of 4.13 at a recombination fraction of 0.05, indicating the odds to be approximately 13,500 to 1 in favor of linkage between HLA and the putative disease allele for SCA. A possible recombination within the HLA region suggested that the disease allele lies telomeric of the HLA region. In view of the recent demonstration of tight linkage between SCA1 and D6S89, however, HLA should not be used for presymptomatic diagnosis or genetic counselling.


Do human leukocyte antigens have a role to play in differential manifestation of multibacillary leprosy: a study on multibacillary leprosy patients from north India. 118 multibacillary leprosy patients with differential manifestations were studied for the antigens they expressed at MHC loci to investigate the role of human leukocyte antigens in the differential response to the same causative agent. While the lepromatous leprosy (LL) patients showed a significant increase of Bw60, DR2, DRw8 and DQw1, borderline lepromatous (BL) patients had Bw52, DR9 and DQw7 significantly more often as compared to the normal controls. A comparison of LL, BL and mid-borderline (BB) patients showed a significantly higher frequency of Bw60 in LL patients as compared to the BL. However, Bw52, Bw53, DR9 and DQw7 were found significantly more often in the BL patients as compared to the LL patients but the difference failed to reach significance after pc. A comparison of HLA antigens in BB patients with those of either the LL or BL patients did not show any significant differences.


Distribution of HLA class II alleles among Scandinavian patients with systemic lupus erythematosus (SLE): an increased risk of SLE among non[DRB1*03,DQA1*0501,DQB1*0201] class II homozygotes? HLA-DRB1, -DRB3, -DQA1 and -DQB1 alleles were determined by DNA typing in 51 Scandinavian patients with systemic lupus erythematosus (SLE) and 129 controls. DRB1*03,DRB3*0101,DQA1*0501,DQB1*0201 were significantly increased in the patient group, with relative risks (RR) of 2.80, 3.07, 3.55 and 2.12, respectively. These alleles are in strong linkage disequilibrium, and their possible relative contributions in predisposition to SLE are difficult to distinguish. The strongest association was found for DQA1*0501, which is in linkage disequilibrium with DRB1*03 as well as DRB1*11,12 (DR5). An increased frequency of DRB1*11,12 was observed (RR = 1.89, ns). No association with DRB1*15,16 (DR2) was found. The patients had a higher frequency of HLA class II homozygosity than the controls (RR = 5.05, p = 0.0005). When compared to the low-risk group (nonDRB1*03 class II heterozygotes), the cases homozygous for DRB1*03,DQA1*0501,DQB1*0201, known to be in linkage disequilibrium with the complement allele C4A*Q0, had the highest relative risk of developing SLE (RR = 16.39, p = 0.0002). However non[DRB1*03,DQA1*0501,DQB1*0201] class II homozygotes had a higher relative risk (RR = 4.68, p = 0.0147) than DRB1*03,DQA1*0501,DQB1*0201 heterozygotes, known to carry the C4A*Q0 allele (RR = 2.72, p = 0.0088). This may suggest that HLA class II molecules are directly involved in susceptibility to SLE.


Distribution of HLA-DQA1, -DQB1 and DRB1 alleles in black IDDM patients and controls from Zimbabwe. We have used the XI Histocompatibility Workshop sequence-specific oligonucleotide probes to determine the DRB1, DQA1 and DQB1 genotypes by dot-blot hybridization of polymerase chain reaction (pcr)-amplified material from a homogenous black population in Zimbabwe. The DR4 subtype DRB1*0405, the DR3 subtype DRB1*0301, DQB1*0201 and DQB1*0302 and DQA1*0301 and DQA1*0501 were significantly increased in the IDDM group compared to the controls, whereas DRB1*11, DQB1*0602 and DQA1*0102 were significantly decreased. Taken together, the data show that susceptibility and resistance to IDDM are associated both with particular haplotypes and DQA1-DQB1 heterodimers without one or other being overriding.


Cell cultures from cryopreserved human lung tissue. To assess gene induction in primary human fibroblasts, we have developed a method for cryopreservation of lung biopsies in liquid nitrogen. Fresh biopsies (n = 10) were chopped into 5 x 5 mm pieces and transferred into an ice-cold freezing medium. Biopsies were kept on ice for 15 min, followed by further cooling of the tissue to -70 degrees C. With this method, lung biopsies were preserved for more than 1 year before they were used for generating cell cultures. There was no significant difference in the biological responsiveness of fibroblasts generated from immediately cultured lung biopsies compared with those from cryopreserved tissue. The doubling rate of fibroblasts from fresh tissue was 23.6 +/- 1.1 hr; compared to 23.5 +/- 1.5 hr for fibroblasts generated from cryopreserved tissue. PDGF-BB enhanced de novo synthesis of DNA 100 times, in both the immediately cultured fibroblasts and those generated from cryopreserved biopsies. Macrophages, dendritic cells and endothelial cells could also be recovered from cryopreserved lung tissue. This method permits long-term storage of lung tissue and the possibility of establishing primary cell lines from the same tissue at later times without appreciable changes in their cellular biological characteristics.


The regulated expression of mRNA for Clara cell protein in the developing airways of the rat, as revealed by tissue in situ hybridization. Tissue in situ hybridization has been used on sections of developing rat lung to follow the cellular sites of mRNA expression for a protein identified only in bronchiolar Clara cells. The mRNA for this Clara cell protein (CCP) was first detected on gestational day 16 in only one of the two types of tubules existing in the lung at this developmental stage. During the next 2 days CCP mRNA expression increased uniformly only in the epithelium lining the respiratory tubules. By gestational day 19, CCP mRNA expression became limited to secretory epithelial cells lining the bronchi, and terminal bronchioles. By neonatal day 1, an intense hybridization signal was observed along all of the conducting airways, but it was irregular due to the fact that expression of the CCP gene was limited to the secretory epithelial cells. In adult rats, CCP mRNA was expressed not only in secretory cells of the intrapulmonary airways at all anatomical levels, but also in secretory epithelial cells lining the trachea and its glands, as well as in specific alveolar cells thought to be type II pneumocytes. These findings demonstrate that the regulation of the CCP gene during lung development is a complicated process and that the expression of CCP mRNA does not parallel exactly the sequential development of the airways.


Absence of sex differences in size of the genital ducts of the rat prior to embryonic day 15.5-16.0. Sexual dimorphisms of the rat brain are generally believed to be brought about by the presence of testosterone during a critical period starting at embryonic day (ED) 17/18. In contrast, sex differences of diencephalic and mesencephalic dopaminergic neurons were observed to develop in cell cultures raised from ED 14 rat brains. This was interpreted as evidence indicating that sexual differentiation of certain neural systems may occur independently of gonadal hormones. To substantiate this claim, it was felt necessary to examine the rat embryo for clues to a possible existence of sex differences in hormonal environment prior to ED 17. Morphometry was applied to compare the development of male and female Wolffian and Müllerian ducts, both primary targets of hormones secreted from the male gonad. Diameters of serially cross-sectioned Wolffian and Müllerian ducts were measured in rats of ED 15.0 to ED 16.5. Females had thicker Müllerian ducts from ED 15.5 on. The first step of differentiation in males was the widening of the lumen and a slight increase of the outer diameter of the Wolffian duct at ED 16.0. The size differences of both ducts were most obvious in the vicinity of the lower half of the gonad. Except in Wolffian ducts of ED 16.5, sex differences were absent in the caudal parts of the ducts. It appears that gonadal androgen and Müllerian inhibiting substance do not affect the development of their classical target organs prior to ED 16.0 and ED 15.5, respectively. Furthermore, the first effects are paracrine in nature. There is no evidence for sex differences in systemic androgen environment until ED 16.5.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of exercise and food restriction on rat skeletal muscles. Studies were undertaken to compare the effects of exercise and food restriction on body weight (BW), muscle weight (MW), muscle fiber size, and proportion of muscle fiber types. 20 male Fischer 344 rats were randomly assigned to four equal groups: ad libitum-fed control (AC), ad libitum-fed exercise (AE), food restricted control (RC) and food restricted exercise (RE). From 6 weeks of age, RC and RE rats received 60% of the daily food intake of AC and AE rats, respectively. At 7 months of age, AE and RE rats began 40-50 min of daily treadmill exercise. Running speed increased from 1.2 to 1.6 miles/hour and the grade increased to 15% during the first 2 weeks of training. After 10 weeks of training, rats were weighed, sacrificed, and the soleus (SOL), plantaris (PLN) and extensor digitorum longus (EDL) muscles were removed at in situ rest length, weighed, and quick-frozen. Standard histochemical assays were performed, and muscle fiber cross-sectional area was determined planimetrically. Training had little effect on MW or BW, but food restriction greatly reduced BW. This resulted in greater MW/BW ratio in RC and RE than AC and AE rats, respectively. Exercise also increased SOL muscle fiber area in ad libitum-fed but not food restricted rats resulting in smaller fibers in SOL of RE than AE. No changes in percentage of SOL fiber types occurred with food restriction or exercise. In PLN, the percentage of fast-twitch oxidative fibers of AE and RE was greater than in AC and RC, but there was no effect of food restriction or exercise on fiber area.(ABSTRACT TRUNCATED AT 250 WORDS)


Ultrastructural morphometry of matrical changes induced by exercise and food restriction in the rat calcaneal tendon. The ultrastructural morphometry of collagen fibril populations in 24 calcaneal tendons obtained from 12 Fischer 344 rats were studied to elucidate matrical changes induced by food restriction and/or endurance exercise. Rats were randomly assigned to four equal groups: ad libitum control (AC), ad libitum exercise (AE), restricted diet control (RC) and restricted diet exercise (RE) groups. Beginning from 6 weeks of age, animals in the two food restriction groups were fed 60% of the mean food consumption of ad libitum fed rats. Then, starting from 6-7 months of age, the rats in the two exercise groups performed 40-50 min of treadmill running at 1.2-1.6 miles h-1 every day for a total of 10 weeks. Endurance training did not significantly alter body weight, but food restriction with or without exercise resulted in a significant loss of body weight. In ad libitum fed controls, food restriction alone did not significantly alter the mean collagen fibril CSA, but predisposed a preponderance of small-sized collagen fibrils. Endurance training per se induced a significant (32%) increase in mean fibril CSA (P less than 0.05), but this adaptive response to exercise was prevented by food restriction, as indicated by a 33% decline in fibril CSA (P less than 0.05). These findings demonstrate that dietary restriction modifies the adaptation of tendon collagen morphometry in response to endurance training, and that weight loss is better achieved with food restriction than endurance exercise.


Ultrastructural morphometry of membrane-bound intracytoplasmic collagen fibrils in tendon fibroblasts exposed to He:Ne laser beam. Collagen fibrils are not found in fibroblast cytoplasm except in certain pathological conditions or in the presence of drugs and other agents that accelerate collagen turnover. Because low energy laser photostimulation is both a non-pathogenic and non-chemical accelerator of collagen synthesis, its effects were studied on four groups of calcaneal tendons from 18 rabbits (1) to test the hypothesis that vacuolar fibrils are not produced exclusively by diseases and chemical agents, and (2) to compare the morphometry of matrical and vacuolar fibrils. The right calcaneal tendons of nine rabbits were surgically tenotomized and repaired; six of these were transcutaneously irradiated with He:Ne laser everyday. The right calcaneal tendon of six of the remaining nine rabbits were similarly irradiated with laser, but without prior tenotomy and repair. 21 days later, all tendons were fixed in situ and processed for electron microscopy. Fibril-bearing vacuoles were found only in fibroblasts of tenotomized laser-irradiated tendons. Similar vacuoles were not seen in non-tenotomized laser-irradiated tendons nor in non-irradiated tendons whether tenotomized or not. Mann-Whitney U tests revealed no statistically significant differences in the cross-sectional areas or diameters of matrical and vacuolar fibrils. These findings suggest (a) that matrical and vacuolar fibrils have a common origin, and (b) that vacuolar fibrils can be induced by a non-pathologic, non-chemical accelerator of collagen synthesis.


Ultrastructural and mechanical properties of electrically inexcitable skeletal muscle fibers of the crustacean Atya lanipes. Examination of the ultrastructure and mechanical activation of the ventro-abdominal flexor muscle of the freshwater crustacean Atya lanipes shows that the fibers are of the long sarcomere, tonic type. The fibers possess an ample and well-organized internal membrane system, with extensive regions of T/SR dyad contacts near the ends of the A bands. An orbit of 10-12 thin filaments surrounds each thick filament. The lanthanum tracer method reveals a highly regular organization of the T-system, Z-tubules, and multiple internal clefts. Tension generation responds to extracellular potassium in a concentration dependent manner and is very slow. Mechanical activation is strictly dependent on extracellular Ca2+, even though these muscle fibers do not generate Ca2+ currents when depolarized. Tension development responds to caffeine and is also dependent on extracellular Na+, suggesting that Ca2+ release from the SR and Ca2+ influx via the Na/Ca exchanger intervene in mechanical activation.


The innervation of the closer muscle of the mesothoracic spiracle of the locust. The closer muscle of the mesothoracic spiracle of the locust, Schistocerca gregaria is innervated by two excitatory motoneurones and also by processes of a peripherally located neurosecretory cell. Within the muscle, ultrastructural studies show the presence of two types of excitatory nerve terminal which differ in the content of dense cored vesicles and in their distribution. The ventral segment of the muscle is innervated predominantly by terminals with small clear vesicles and only an occasional dense-cored vesicle. The central part of the muscle is innervated predominantly by terminals with small clear vesicles and larger numbers of dense-cored vesicles. The dorsal segment of the muscle is innervated exclusively by a neurosecretory type innervation. The small neurohaemal organ of the median nerve close to the spiracle muscle is immunoreactive to an antibody raised against bovine pancreatic polypeptide but no immunoreactive processes enter the muscle itself. The muscle possesses specific octopaminergic receptors that increase cyclic AMP levels and the possibility that the neurosecretory input to the muscle is provided by either a central or peripheral octopamine containing neurone is discussed.


Quantitation of Sertoli cell-germ cell desmosome gap junctions in relation to meiotic divisions in the male rat. Desmosome-gap (D-G) junctions were quantified in relation to germ cell meiosis in the male, specifically to test the hypothesis that the loss of these junctions is related to successful passage of cells through diplotene phase of Meiosis I and the two cytokineses that follow. Such a hypothesis has been proposed as the cause for the resumption of meiosis that occurs prior to ovulation in the female. D-G junctions were quantified in pachytene spermatocytes (stage XII), diplotene spermatocytes (stage XII), secondary spermatocytes (stage XIV) and step 1 spermatids (stage I). These were referred to as the cells of interest as compared with spermatocytes (zygotene spermatocytes, zygotene spermatocytes, pachytene spermatocytes, pachytene spermatocytes) in the same stages, respectively, that served as controls termed control cells. Since gap junctions are not easily recognized in the average sectioned profile of a desmosome-gap junction, only the desmosomal component was quantified. The data were expressed as both numbers and length of junctions per tubule, per cell profile and per unit lineal membrane length to overcome errors inherent in the methodologies utilized. There was no indication that numbers of junctions changed specifically in the cells of interest after passage through diplotene suggesting that these junctions do not have a comparable role in meiotic continuance in the male as proposed for the female. Interestingly, the control cells always showed greater numbers and length of junctions than the cells of interest suggesting that junction may relate more to the period of initiation of meiosis than to its continuance.


Morphology of the liver of the brook lamprey, Lampetra lamottenii before and during infection with the nematode, Truttaedacnitis stelmioides, hepatocytes, sinusoids, and perisinusoidal cells. Routine light microscopy and transmission and scanning electron microscopy were used to describe and compare the livers of larval lampreys, Lampetra lamottenii before and during infection of the bile ducts by the nematode, Truttaedacnitis stelmioides. The hepatocytes of uninfected animals differ from other lamprey species in that they contain abundant glycogen, smooth endoplasmic reticulum (SER), and lipoprotein indicating that the liver may be involved in glucose metabolism. Infestation of the biliary tree by T. stelmioides coincides with alterations to the hepatocytes. These changes include dilation of the bile canaliculi, smooth endoplasmic reticulum (SER), rough endoplasmic reticulum (RER), and Golgi apparatus, swollen mitochondria in cells showing a high degree of hypertrophy, and an abundance of dense bodies. Following infection, the sinusoidal lumina became dilated and contain a moderate electron-dense precipitate, an abundance of melanomacrophages, lipocytes, and mononuclear cells. There is also a widening of the fenestrae of the sinusoidal endothelium following infection. Many of the changes in hepatocytes and sinusoids following parasite infections closely resemble those observed in hepatocytes in various pathologies and following experimental bile duct ligation and, therefore, are likely a consequence of increased biliary pressure due to bile duct obstruction.


Role of the dermal tracts in the pigment pattern of the frog. The pigment pattern of the ventral skin of the frog Rana esculenta is compared in skin fragments grown for 24 hr with or without antiserum directed to fibronectin (anti-FN). Melanocyte-stimulating hormone (MSH) was added to the medium during the last hour in culture in order to enhance visibility of melanophores in the ventral region of the frog skin. Comparison of these two treatments provides information regarding the precise localization of melanophores in the dermal tracts and their involvement in the pigment pattern of the ventral frog skin. In this regard, the whitish pigment pattern of skin fragments is compared to the tiny black spots found on anti-FN treated skin fragments and the abundant blotchy spots found on skin cultured alone. The distribution of melanophores in the dermal tracts observed in vertical semithin sections is found to be related to the three different levels of the dermal tracts. This report demonstrates the importance of fibronectin as a substrate for the melanophore migration, the importance of the tract level for the melanophore localization both involved in the pigment pattern of the ventral skin.


Receptor-mediated endocytosis of insulin by cultured endothelial cells. Label-fracture immunochemistry and pre-embedding indirect immunocytochemistry were applied to investigate insulin uptake by endothelial cells. Freeze fracture replicas showed that a small percentage of native insulin receptors are associated with non-coated pits (4%) and coated pits (2%). After warming, receptor bound insulin became increasingly associated with such endocytotic vesicles. After 2 min the percentage of detectable insulin associated with non-coated and coated pits increased to 16% and 8%, respectively. Pre-embedding immunocytochemical localization of insulin gave results consistent with those obtained from the label-fracture studies. Both non-coated and coated vesicles appeared labelled after 5 min of warming. Non-coated vesicles contained 25% of the cell associated insulin while 9% was associated with coated pits and vesicles. After 10 min of warming, 9% of label was located in non-coated vesicles and 7% in coated vesicles. A large proportion (29%) of the label was found in tubular-vesicular endosomes at this time. After 15 min of warming, 30% of the remaining cell-associated gold label was found in multivesicular bodies. These experiments demonstrate that insulin uptake by endothelium is mediated by both coated and non-coated vesicles and that, once internalized, insulin is routed through endosomal pathways that primarily result in transcytosis.


A simple in vitro method for raising monoclonal antibodies to cochlear proteins. Monoclonal antibodies have been produced to mammalian hair cell antigens using a simple in vitro kit. Antigen was crudely prepared from dissected cochlear tissue by detergent extraction. There was no need to purify hair cells. Hybridoma supernatents were screened most efficiently on dissociated cells fixed with acetone. The immunisation method is sensitive to nanograms of antigen and can generate responses to conserved or weak antigens. The kit requires very little previous experience with cell culture and generates monoclonal antibodies within 3-4 weeks. It has overcome a number of problems with production of antibodies to hair cells and it should prove to be a very valuable tool in many laboratories.


Changes of the Golgi apparatus and lysosomes during decidualization in mice. The Golgi apparatus of the endometrial stromal cells of pregnant mice increases in size simultaneously with the differentiation of stromal cells into decidual cells. The activity of acid phosphatase in this organelle increases during this stage. On the other hand, the involuting decidual cells show morphological and cytochemical signs of Golgi regression (dilated cisternae, lack of enzymatic activity) together with the finding of numerous, pleomorphic lysosomes that have intense cytochemical label. These results confirm morphological data suggesting that decidual cell death occurs by autophagic degeneration.


Perinuclear cytoskeleton of acrosome-less spermatids in the blind sterile mutant mouse. The perinuclear cytoskeleton of mammalian spermatids is thought to play a major role in nucleus-acrosome association and in shape changes of the head during spermiogenesis. To test these hypotheses acrosome-less spermatids in blind-sterile mutant mice were investigated for the development of the subacrosomal layer. Immunogold procedures were used for the detection of actin and calmodulin. In addition to various other abnormalities many acrosome-less round and elongating spermatids developed a subacrosomal layer with an actin and calmodulin distribution similar to that observed in normal spermatids. However, in mutant elongating spermatids the apical part of the nucleus was truncated and/or folded. The expected elongation and shaping of the nucleus only occurred in its caudal part associated with an hypertrophied and somewhat ectopic manchette. These abnormalities and those previously observed in mutant and experimental models indicated that the subacrosomal layer may form independently of the acrosome. It is suggested that the subacrosomal filamentous actin is a transitory scaffolding which might be involved in the assemblage of other proteins of the perinuclear cytoskeleton. However, by itself, this layer is not sufficient to ensure a normal shaping of the nucleus. Acrosome-nucleus interactions mediated by the subacrosomal layer seem necessary to shape the cranial spermatid head. The manchette appears to be involved only in the caudal nuclear shaping.


Demonstration of the capacity of nacre to induce bone formation by human osteoblasts maintained in vitro. Nacre implanted in vivo in bone is osteogenic suggesting that it may possess factor(s) which stimulate bone formation. The present study was undertaken to test the hypothesis that nacre can induce mineralization by human osteoblasts in vitro. Nacre chips were placed on a layer of first passage human osteoblasts. None of the chemical inducers generally required to obtain bone formation in vitro was added to the cultures. Osteoblasts proliferated and were clearly attracted by nacre chips to which they attached. Induction of mineralization appeared preferentially in bundles of osteoblasts surrounding the nacre chips. Three-dimensional nodules were formed by a dense osteoid matrix with cuboidal osteoblasts at the periphery and osteocytic-like cells in the center. These nodules contained foci with features of mineralized structures and bone-like structures, both radiodense to X-ray. Active osteoblasts (e.m.) with abundant rough endoplasmic reticulum, extrusion of collagen fibrils and budding of vesicles were observed. Matrix vesicles induced mineral deposition. Extracellular collagen fibrils appeared cross-banded and electrodense indicating mineralization. These results demonstrate that a complete sequence of bone formation is reproduced when human osteoblasts are cultured in the presence of nacre. This model provides a new approach to study the steps of osteoblastic differentiation and the mechanisms of induction of mineralization.


Ultrastructure of carotid baroreceptors in the goat. The initial segment of the occipital artery of the goat appears modified under the electron microscope; endothelial cells are low cuboidal, the tunica media contains many elastic lamellae, and there are few smooth muscle cells. Free afferent nerve endings are seen in this modified arterial wall. They closely resemble the presumptive baroreceptor endings reported in other mammals in their mitochondrial content and abnormal organelles and are interpreted as solely adapting baroreceptors. Special topographical relations between the endings and elastic or collagen fibres do not occur, but microfilaments are present in the vicinity of these endings. Presumptive adrenergic efferent endings are not found in the region of the baroreceptors. We suggest that this zone of the occipited artery is homologous to the carotid sinus.


Stellate cells of aortic intima: I. Human and rabbit. Stellate cells hitherto accounted exclusively in the innermost elastic-hyperplastic layer were already reported to inhabit human aortic intima. The present paper shows that most of these cells are situated just beneath the endothelium. Stellate cells also appear in the deendothelialization-induced myointimal thickening of rabbit aorta. In the myointimal thickening these cells were revealed in the direct proximity to the endothelium. A conclusion is available that the previously demonstrated polymorphism of human aortic intimal cells may be reproduced in a simple experimental model, which gives new possibilities for the study of the cellular polymorphism in the vessel wall.


Calcium-binding proteins in chemoreceptors of Xenopus laevis. Calcium-binding proteins were investigated immunohistochemically in chemo-receptors of the olfactory epithelium and taste buds of the clawed frog, Xenopus laevis. Calmodulin-, S-100- and calbindin-immunoreactive material were found in sensory cells of the olfactory epithelium; however, parvalbumin-like material was absent in these cells. Taste buds of the palate showed calmodulin-, S-100- and parvalbumin-immunoreactive material in sensory cells, while calbindin-immunoreactive material in supporting cells. Merkel cells, surrounding the base of the taste buds in a ring-like manner, exhibited calmodulin- and S-100-immunoreactive material.


The role of the rat submandibular gland in the excretion of bis (tributyltin) oxide: electron microscopy, X-ray microanalysis and atomic absorption analysis. The role of the submandibular glands in the excretion of parenterally administered bis (tributyltin) oxide (TBTO) was studied. Fine structural alterations of the submandibular glands were observed with an electron microscope. Accumulation sites of TBTO were determined with an X-ray microanalyzer and tin concentrations in saliva and blood were measured by atomic absorption spectrophotometry.


[Birth injuries in calves and perinatal calf losses in a large dairy herd]. Post-mortem examinations were carried out on 65 calves which were stillborn or died within 24 hours of birth. All calves came from a large dairy unit with about one thousand cattle of the Schwarzbuntes Milchrind breed. To evaluate the perinatal losses, the farm records of parturitions from 1985-1990 were used. The calves had gross damage of the CNS, the musculature, subcutaneous tissue and internal organs such as lung and intestines. It may be assumed that a lesser degree of similar damage would also be present in the surviving calves. The level of perinatal losses is determined particularly by deaths following prolonged parturition due to foetal oversize in heifers and young cows, but also by the birth of immature calves of low weight after pregnancy of normal or decreased duration and by twin pregnancies with high total calf mass and relative immaturity of the calves.


[Concentration of Fe, Fe-binding capacity, Cu and Zn in the plasma of piglets before and after oral administration of iron sulfate solution]. In piglets, the influence of the ingestion of Fe-enriched faeces, of the oral application of Fe-dextran (containing 150, 200 or 250 mg Fe) up to the 8th hour as well as the intramuscular injection of Fe-dextran (150 mg Fe) on the 2nd and 3rd day after birth on Fe-resorption was analysed. On the 5th to the 7th and on the 12th to the 14th day after birth the highest increase in the Fe-concentration and the highest values of the Fe-binding capacity of the plasma after the oral application of a solution of Fe-sulphate (containing 20 mg Fe/kg body weight) were confirmed in the piglets with the ingestion of Fe-enriched faeces and in the control piglets without Fe-supplementation. The increase of the iron concentration in the plasma induced a decrease in the concentration of Cu in some cases and of Zn in the plasma in most cases. The biggest increase in body weight was established in the piglets that ingested Fe-enriched faeces.


[Ocular manifestations of systemic diseases in birds. Part 2]. Among the representatives of the class aves, ocular lesions may be a particularly strong indicator of systemic disorders. A causative diagnosis of ocular lesions may be vital for the avian patient, not only as a basis for effective therapy of the primary disease, but also for saving the vision, as birds orientate themselves primarily by vision. The following paper presents a synopsis of important ocular disorders in avian patients that are either pathognomonic for certain disorders or, when considered in conjunction with other organic diseases, enable a specific diagnosis. Bacterial and parasitic infections were already described in part 1 of this paper. Part 2 gives outlines of possible viral etiologies and noninfectious diseases.


[Respiratory support techniques in foals in a newborn intensive care unit for large animals]. Various techniques for support and control of respiration in neonatal foals are described. It is crucial to evaluate respiration through frequent arterial blood gas analysis. Details for blood sampling from the metatarsal arteries and interpretation of results are provided. Typical diseases in newborn foals, which cause hypoxemia and/or hypercapnia and can be indications for respiratory support are apnea, hypopnea, pulmonary atelectasis, surfactant deficiency, meconium-, fetal fluid- and milk aspiration, maladjustment syndrome, cardiovascular abnormalities, anemia, airway obstruction, compromised lung expansion, increased abdominal pressure and pneumonias. Oxygen insufflation can be delivered through an intranasal tube. Positive pressure ventilation is best accomplished via an endotracheal tube. A nasogastric tube is inserted for enteral nutrition, application of drugs and checking for gastric reflux. Details for insertion of endotracheal and nasogastric tubes are given. Positive pressure ventilation can be achieved by manual ventilation with a rebreathing resp. resuscitator bag and mechanical ventilation with a respirator. Management and control of mechanical ventilation as well as intensive care and monitoring of foals are described.


[Riding arenas and different riding track surfaces in relation to the airway contamination in horses. Laboratory studies on riding surfaces]. 29 samples of commonly used surfaces were tested for their water characteristics (litre weight, water capacity, water binding, water evaporation) and their contribution to airborne fungal spores (dust formation, dust setting). The results are discussed in comparison to the literature with regard to the environment. The results are: 1. Any surface--no matter of what material--eventually causes air pollution with fungal spores and dust. 2. Correct watering prevents air pollution by any surface. 3. Artificial products have no advantage over natural materials in the parameters tested. 4. The question of proper disposal of old surface material has to be clarified before purchase. The results show that a mixture of sand and wood shavings should be recommended as a surface for indoor arenas, especially in regard to environmental protection and proper disposal.


[Pathology of the retina].

[Pathology of the retina]. Degenerations (atrophies) of the retina are divided into primary (hereditary) and secondary forms including glaucomatous retinopathy of retinal atrophy. The pathogenesis of inflammation of the retina is considered. This condition generally appears in association with inflammation of the choroid (chorioretinitis), but can also occur as an isolated inflammatory condition during infectious disease (distemper). The functional conditions for retinal detachment, its rhegmatogenous and nonrhegmatogenous types, as well as the consequences of retinal detachment are described. Comparison of the pathology of retinal tumors shows that retinoblastoma is not known in animals. However, neuroepithelial tumors like the so-called acquired adenoma and adenocarcinoma of the mature ciliary epithelium as well as the rare congenital tumors of the embryonic neuroepithelium i.e. the medulloepithelioma and the ganglioneuroma are seen in animals. Finally, the rare but not unusual parasitic retinopathies are mentioned.


[Malignant hyperthermia as a complication of anesthesia in the dog]. A moderate malignant hyperthermia developed in a Labrador Retriever anaesthetized with isoflurane for a femoral shaft fracture repair. Signs of malignant hyperthermia included progressive increases in PETCO2 and rectal temperature up to 39.8 degrees C, tachycardia, cyanosis, and elevated serum levels of potassium, inorganic phosphorus, AST, CK and alkaline phosphatase. Treatment initiated in the early recovery period consisted of hyperventilation with 100% oxygen, stomach lavage with iced water, body surface cooling, and intravenous administration of cold isotonic saline solution. Cooling was continued until the rectal temperature had dropped to 37.3 degrees C. After treatment the dog recovered uneventfully. Clinical signs, pathophysiology, therapy, prevention of malignant hyperthermia and its association with other disorders are discussed.


[The diagnostic value of sonography in the clinical picture of pyometra in the dog]. Experiences of the diagnostic value of ultrasonography of pyometra in the bitch are reported. The different ultrasound scans of the uterus are illustrated. Results are compared with clinical diagnosis and pathophysiological findings after laparotomy and surgical removal of the uteri. Very close correlation (r: 0.985, p < 0.001) was found between the postoperative macroscopic and the ultrasound scan measurements of the uterus. Accuracy of the ultrasound diagnosis and reasons for false and questionable results are discussed. In summary, ultrasonography is an accurate procedure for the qualitative and quantitative examination and diagnosis of canine pyometra.


[The radiologic differential diagnosis in girth increase of the gastric cavity and the body in lizards (Squamata: Sauria)]. The extended girth is one of the few important and obvious symptoms in lizards (Sauria). Radiological examination proved to be the most important method in the clinical diagnostic procedure. Standard and contrast images using oral and cloacal application of barium sulphate or iodophoric contrast media were frequently indicated. Based on 442 X-rays from 162 lizards out of 20 genus the significant radiological, physiological and pathological findings are described. Frequent findings were dystocia in oviparous and viviparous lizards (18.5%), gastritis and enteritis (12.3%), ascites (9.3%), fractures of the spine and osteodystrophia (9.3%), obstipation (6.2%), foreign bodies (4.9%), neoplasia (4.3%) and enteroliths (4.3%). Pneumonia (3.7%), was as frequent as the physiological lung tympany (3.7%). Pregnancy was the most frequent physiological finding (8%). The radiological findings, are described in detail, especially in regard to differentiation between physiological and pathological conditions.


Effects of ethanol on cocaine metabolism: formation of cocaethylene and norcocaethylene. The coabuse of cocaine and ethanol occurs with high frequency and increases the risk of cocaine-related morbidity and mortality. The mechanisms mediating the toxic interactions of cocaine and ethanol are not clearly defined. This study examined the effects of acute ethanol administration on the metabolism of cocaine in the male Wistar rat. Intraperitoneal administration of 2 g/kg ethanol 30 min prior to administration of 25 mg/kg cocaine resulted in the formation of two ethylated derivatives of cocaine, benzoylecgonine ethyl ester (cocaethylene) and benzoylnorecgonine ethyl ester (norcocaethylene) in liver, brain, and serum. Fifteen minutes after cocaine administration, the tissue levels of cocaethylene were 22, 10, and 9% of the cocaine recovered from liver, serum, and brain, respectively. Ethanol pretreatment increased cocaine concentrations in liver and benzoylnorecgonine concentrations in liver and serum. The increased morbidity and hepatotoxicity seen with acute combined administration of cocaine and ethanol may be due to the formation of the toxic ethylated and N-demethylated metabolites of cocaine. Ethanol pretreatment decreased benzoylecgonine concentrations in serum and liver. The most important consequence of ethanol-induced inhibition of the normally rapid hydrolysis of cocaine to benzoylecgonine may be a decrease in benzoylecgonine-mediated vasoconstriction.


Sulfur mustard-induced microvesication in hairless guinea pigs: effect of short-term niacinamide administration. It has been postulated that sulfur mustard (HD) damage may activate poly(ADP-ribose) polymerase (PADPRP), resulting in depletion of cellular NAD+. This biochemical alteration is postulated to result in blister (vesicle) formation. It has been previously demonstrated that niacinamide (NAM), an inhibitor of PADPRP and a precursor for NAD+ synthesis, may be useful as a pretreatment compound to reduce HD-induced microvesication. The present study was undertaken to determine whether niacinamide's protective action could be extended beyond 24 hr and if the degree of microvesication is related to changes in skin NAD+ content. HD exposures were made by vapor cup to hairless guinea pigs. Niacinamide (750 mg/kg, ip) given as a 30-min pretreatment did not reduce the degree of microvesication 72 hr after HD compared to saline controls. However, niacinamide given as a 30-min pretreatment and at 6-, 24-, and 48-hr after HD, exhibited a 28% reduction in microvesication 72 hr after HD. Skin NAD+ content at 72 hr after HD was depleted by approximately 53% in the saline and NAM-treated groups. Skin NAD+ content was depleted despite NAM administration. Niacinamide did not reduce the degree of erythema at 48 or 72 hr. These results suggest that niacinamide's protective effect against HD-induced microvesication may be extended for at least 72 hr, but NAM levels must be sustained during the post-HD period. The link between maintenance of skin NAD+ and reductions in microvesication is still uncertain.


Paraoxon toxicity is not potentiated by prior reduction in blood acetylcholinesterase. The role of blood acetylcholinesterase in moderating the effects of organophosphate challenge in rats was tested. Adult male rats (n = 42) were injected (iv) either with monoclonal antibodies (MAb) to rat acetylcholinesterase (EC 3.1.1.7; AChE) or normal mouse IgG (controls). Two days later, the rats were injected (sc) with either a mild (0.17 mg/kg) or moderate dosage (0.34 mg/kg) of paraoxon or with vehicle. Neurological integrity was assessed by a functional observational battery followed by motor activity, 3 to 4 hr after dosing. Blood, brain, and diaphragm tissues were then collected for determination of AChE activity. MAb treatment reduced whole blood and plasma AChE activity by 32 and 90%, respectively, but did not affect neurobehavioral parameters or the AChE activity of brain or diaphragm. The paraoxon challenge produced dose-related neurobehavioral changes and inhibition of brain and diaphragm AChE activity to the same extent in IgG- and MAb-treated rats. Thus, significant loss in blood AChE alone produced no detectable neurobehavioral deficits and did not alter the subsequent responses to paraoxon challenge.


Hydrolysis of pyrrolizidine alkaloids by guinea pig hepatic carboxylesterases. Two carboxylesterases (GPL1 and GPH1) were isolated from guinea pig hepatic microsomes and assayed for activity using the following pyrrolizidine alkaloids (PAs): seneciphylline (SNP), monocrotaline (MCT), and a mixture of senecionine (SEN) and integerrimine (INT) referred to as SEN-INT. GPH1 was able to effect the hydrolysis of all PAs, however, only minimal activity was seen for SEN-INT. The specific activity of GPL1 for p-nitrophenyl acetate was four times that of GPH1, but the former showed no activity toward PAs. The molecular weights and pIs were determined for both enzymes, and the Michaelis-Menten constants for two PAs, SNP and MCT were obtained using GPH1. The response to inhibitors confirmed GPH1 as a type B serine hydrolase although it was also inhibited by HgCl2. The isolation of a PA active esterase from the guinea pig may help to explain the resistance of this animal to PA intoxication, while enzyme substrate specificity may explain how the guinea pig's susceptibility to PA intoxication can differ toward various PAs.


Modulation of M1 and M2 muscarinic receptor subtypes following repeated organophosphate exposure in rats. Repeated exposure to organophosphates has been shown to cause a decrease in muscarinic cholinergic receptors in the central and peripheral nervous system. The present study measured the modulation of M1 and M2 muscarinic receptor subtypes in rat brain areas during and following a 2-week daily exposure to the organophosphate disulfoton. The radioligands [3H]telenzepine and [3H]AFDX 384 were utilized to label M1 and M2 receptors, respectively. The study found comparable down-regulation in both [3H]telenzepine- and [3H]AFDX 384-labeled receptors in cortex, hippocampus, and striatum during exposure. Recovery of M2 subtype was slower than recovery of M1, especially in the hippocampus. The results suggest that M1 and M2 receptor subtypes may be similarly regulated in response to subchronic exposure to organophosphates, but that recovery of receptor subtypes to control levels may be governed by distinct factors.


Inhibition of acute TCDD toxicity by treatment with anti-tumor necrosis factor antibody or dexamethasone. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) acute toxicity is characterized in part by a wasting syndrome with depletion of adipose tissue. Tumor necrosis factor (TNF) induces a similar response during chronic infection. The similarities of these toxic effects led to a hypothesis that TNF plays a role in TCDD acute toxicity. To test this hypothesis pharmacologic doses of an antibody specific for murine TNF and the potent anti-inflammatory agent Dexamethasone (DEX) were used to inhibit TCDD toxicity in mice. TNF antibody treatment resulted in a 54% reduction in TCDD-mediated mortality while DEX treatment, a glucocorticoid agonist that inhibits transcription of TNF, reduced mortality by 92%. Cyp 1A1 induction, the most commonly measured TCDD-mediated response, was not blocked by DEX, demonstrating separation of this biochemical effect from acute toxic responses to TCDD. These data suggest that TCDD-mediated changes in the TNF pathway may be an important mechanism for acute TCDD toxicity.


Effect of ozone on platelet activating factor metabolism in phorbol-differentiated HL60 cells. The mechanisms of ozone (O3) toxicity in the lung may involve the formation of lipid inflammatory mediators. We have previously demonstrated that exposure to O3 in vitro results in increased accumulation and release of platelet activating factor (PAF) in the macrophage-like cell line HL60 differentiated with phorbol ester (dHL60). In the present study we have examined possible biochemical mechanisms responsible for the O3-induced increase in PAF levels in dHL60 cells. Specifically, we studied the effect of O3 on phospholipase A2 (PLA2), acetyltransferase, acetylhydrolase, and reacylation activities. dHL60 cells were exposed to 1.0 ppm O3 or air alone. O3 exposure was found to significantly decrease dHL60 cell acetylhydrolase activity by 36%. Additional experiments demonstrated that extracellular acetylhydrolase activity, but not intracellular acetylhydrolase activity, was inhibited by O3 exposure of dHL60 cells. O3 exposure resulted in a small (13%) but statistically significant reduction in reacylation activity in dHL60 cells. In addition, a significant (22%) contribution of PLA2 activation to the O3-induced increase in PAF levels was also found. Basal and calcium ionophore-induced acetyltransferase activity was found to be unaffected by exposure of dHL60 cells to O3. These data suggest that in vitro exposure to O3 affects both synthetic and degradative pathways of PAF metabolism in dHL60 cells.


Physiologically based pharmacokinetic model for methanol in rats, monkeys, and humans. The pharmacokinetics of methanol and formate were characterized in male Fischer-344 rats and rhesus monkeys exposed to methanol vapor concentrations between 50 and 2000 ppm for 6 hr. End-of-exposure blood methanol concentrations were not directly proportional to the atmospheric concentration. The methanol exposures did not cause an elevation in blood formate concentrations. After an intravenous dose of [14C]methanol in rats, metabolism, exhalation, and renal excretion contributed 96.6, 2.6, and 0.8%, respectively, to the elimination of blood methanol concentrations. These values and the calculated renal methanol extraction efficiency (0.007) are nearly identical to those for humans after low doses of methanol. A physiologically based pharmacokinetic model was developed to simulate the in vivo data. In order to simulate the observed blood methanol concentrations in the inhalation studies in rats, a double pathway for methanol metabolism to formaldehyde was used. One path used rodent catalase Km and Vmax values and the other used a smaller Km and Vmax to simulate an enzyme with a higher affinity and lower capacity. The lack of proportionality observed in end-of-exposure blood methanol concentrations may be due to saturation of an enzyme with higher affinity and lower capacity than catalase. The physiologically based pharmacokinetic model was modified to simulate the monkey data and was scaled-up for humans. In order to simulate the monkey blood methanol concentrations, the use of rodent catalase parameters for methanol metabolism was required. This finding suggests that primates and rodents may be similar in the initial step of methanol metabolism after low methanol doses. Previously published human urinary methanol excretion data was successfully simulated by the model. The models were used to predict the atmospheric methanol concentration range over which the laboratory species exhibit quantitative similarities with humans. Below 1200 ppm, all three species exhibit similar end-of-exposure blood methanol concentrations and a linear relationship between atmospheric and blood methanol concentrations. At higher atmospheric concentrations, external and internal methanol concentrations increase desparately, suggesting that delivered dose rather than exposure concentration should be used in interpreting data from high-dose studies.


Acute ozone-induced lung injury in rats: structural-functional relationships of developing alveolar edema. As part of a study on the effects of acute ozone stress on the lung surfactant system, we correlated morphometric, biochemical, and functional indices of lung injury using male rats exposed to 3 ppm ozone for 1, 2, 4, and 8 hr. Evaluation of lung mechanics, using the Pulmonary Evaluation and Diagnostic Laboratory System, revealed a significant decrease in dynamic lung compliance (ml/cmH2O/kg) from a control value of 0.84 +/- 0.02 (SEM) to 0.72 +/- 0.04 and 0.57 +/- 0.06 at 4 and 8 hr, respectively. At 2 hr there was a transient increase in PaO2 to 116 torr (control = 92 torr) followed by a decrease at 4 hr (65 torr) and 8 hr (55 torr). Morphometry of lung tissue, fixed by perfusion of fixative via the pulmonary artery at 12 cm H2O airway distending pressure, demonstrated an increase in the area of the intravascular compartment at 8 hr, in association with a 65 and 39% replacement of the alveolar area by fluid in ventral and dorsal lung regions, respectively. There was a positive correlation (r = 0.966) between alveolar edema and transudated proteins in lavage fluid. A stepwise multiple regression model, with edema as the dependent variable, suggested that pulmonary vasodilatation, hypoxemia, and depletion of surfactant tubular myelin in lavage fluid were indices for predicting alveolar edema. In a second model, with lavage protein concentration as the dependent variable, decreasing dynamic compliance and hypoxemia were predictors of progressive, intraalveolar transudation of plasma proteins. The above structural-functional relationships support the concept that ozone-induced high-protein alveolar edema is pathogenetically linked to pulmonary hyperemia, deficiency of surfactant tubular myelin, and associated lung dysfunctions.


Mercury distribution in the rat brain after mercury vapor exposure. Brown Norwegian rats were exposed to mercury vapor at a concentration of approximately 1 mg/m3 for 5 weeks 24 hr/day 7 days a week and 6 hr/day 3 days a week, respectively. The total mercury absorption was calculated to 264 and 35 micrograms per week and 100 g body weight. The mean blood mercury concentration was 0.25 +/- 0.03 and 0.09 +/- 0.01 microgram/g, and the total concentration in the brain was 5.03 +/- 0.73 and 0.71 +/- 0.10 microgram/g tissue, respectively. The mercury distribution in the brains was examined using a method based on chemographic principles. Mercury was found primarily in the neocortex, in the basal nuclei, and in the cerebellar Purkinje cells. This distribution pattern corresponded to the pattern of inorganic mercury described after exposure to methyl mercury. Distribution of mercury after administration of different mercury compounds is discussed.


Comparison of cell-permeable calpain inhibitors and E64 in reduction of cataract in cultured rat lenses. E64, an inhibitor of calpain (EC 3.4.22.17) and other cysteine proteases, slows the rate of formation of cataract in cultured rat lenses. The purpose of this study was to determine (1) why E64, a charged compound with little cell permeability, was effective in reducing cataract in cultured lens and (2) whether uncharged more permeable protease inhibitors are more effective than E64 in preventing cataract. Results showed that E64 entered the lens, but only after the lens was treated with the calcium ionophore, A23187, or sodium selenite, both of which cause cataracts. Therefore, the uptake and subsequent effectiveness of E64 may be related to a generalized increase in membrane permeability during induction of cataract in culture. Three protease inhibitors, reported to have improved cell permeability, were compared with E64 for their ability to prevent cataracts in cultured lenses. cBz-ValPheH, calpain inhibitors I and II, are uncharged-aldehyde inhibitors of calpain. Calpain inhibitors I and II even at high concentrations were not effective at reducing lens opacity caused by calcium ionophore and were toxic to the lens. cBz-ValPheH, which is slightly toxic to the lens, was able to significantly reduce lens opacity induced by calcium ionophore. The presented data suggest that while E64 decreases cataract formation in cultured lens, the more cell permeable inhibitor, cBz-ValPheH, may have greater efficacy as an anticataract drug in vivo.


Determination of mutagenicity in tissues of transgenic mice following exposure to 1,3-butadiene and N-ethyl-N-nitrosourea. 1,3-Butadiene (BD) is carcinogenic in the B6C3F1 mouse in multiple organs, including lung and liver. We conducted a study to measure the frequency of BD mutations in mouse tissues using a transgenic mouse (Muta mouse; MM). MM is a BALB/c x DBA/2 (CD2F1) mouse that has a bacteriophage lambda shuttle vector with the target gene lacZ integrated into the mouse genome. Mice were exposed by inhalation to 625 ppm BD (6 hr/day) for 5 days and the lacZ- mutant frequency (mf) was determined in lung, bone marrow, and liver. The lacZ- mf in lung increased twofold above air-exposed control animals, but the bone marrow and liver samples did not exhibit an increase above background. N-ethyl-N-nitrosourea (250 mg/kg ip) was mutagenic in all three tissues examined. Studies on the biotransformation of BD using MM liver microsomes showed that the ratio between the rates of BD bioactivation to BD monoepoxide (BMO) and hydrolysis of BMO by epoxide hydrolases was approximately 40% less than this ratio using B6C3F1 mouse liver microsomes. Quantitation of adducts of BMO to N-terminal valine in hemoglobin (Hb) in the MM revealed an adduct level of 3.7 pmol/mg globin. Using this value, the predicted Hb adduct level in MM would be approximately one-half of that measured in the B6C3F1 mouse following similar exposures. These results indicate that BD induces mutations in vivo in a known murine target tissue, but strain differences in the biotransformation of BD should be considered in comparing the susceptibility of transgenic mouse strains to mutation.


Dietary 2,3,7,8-tetrachlorodibenzofuran in rainbow trout: accumulation, disposition, and hepatic mixed-function oxidase enzyme induction. Juvenile rainbow trout (Oncorhynchus mykiss) were exposed to dietary 2,3,7,8-[3H]tetrachlorodibenzofuran (TCDF) (0.36 to 42.8 ng g-1) and accumulation, tissue distribution, biotransformation, and hepatic monooxygenase enzyme (MO) induction were studied. The assimilation efficiency of TCDF ranged from 49 to 62% in 30-day exposures and was independent of the TCDF level in the diet. Depuration half-lives (whole body) of TCDF following 30-day exposure ranged from 40 to 77 days and were significantly more rapid in fish exposed to 42.8 ng g-1. Liver somatic index (LSI) and rate of increase in liver weight were elevated in fish exposed to 42.8 ng g-1 TCDF compared to controls. Exposure to 9.2 ng g-1 TCDF in the diet for 140 days also resulted in higher LSI values, as well as increased mortality (16%), but had no significant effects on growth. [3H]TCDF was found mainly in the carcass (63-74%) and GI tract (18-31%), with lesser amounts in liver (0.6-2.3%) during the 140-day exposure, primarily (> 98%) in the form of the parent compound. Radioactivity in bile was found mainly as a single polar transformation product by reverse-phase HPLC. Glucuronidase hydrolysis yielded a product with the retention time expected of hydroxylated TCDF, suggesting the presence of a glucuronide conjugate. MO enzyme induction measured by ethoxyresorufin-O-deethylase (EROD) activity in liver (postmitochondrial supernatant) was 137.5 and 15 times higher than that in control fish after 30 days dietary exposure to 42.8 and 9.2 ng g-1, respectively. EROD activities were correlated with TCDF concentrations in liver (R2 = 0.59, N = 45).


Rat lung metallothionein and heme oxygenase gene expression following ozone and zinc oxide exposure. We have conducted exposures in rats to determine pulmonary responses following inhalation of two common components of welding fumes, zinc oxide and ozone. To examine their effects on target-inducible gene expression, we measured mRNA levels of two metal-responsive genes, metallothionein (MT) and heme oxygenase (HO), in lung tissue by RNA slot-blot analysis. A 3-hr exposure to ZnO fume via a combustion furnace caused a substantial elevation in lung MT mRNA at all concentrations tested. Exposures to 5 and 2.5 mg/m3 ZnO resulted in peak 8-fold increases in MT mRNA levels (compared to air-exposed control animal values) immediately after exposure, while 1 mg/m3 ZnO exposure caused a 3.5-fold elevation in MT mRNA. These levels returned to approximate control gene expression values 24 hr after exposure. In addition, ZnO exposure caused an immediate elevation in lung HO gene expression levels, with 8-, 11-, and 5-fold increases observed after the same ZnO exposure levels (p < 0.05). Like MT gene induction, HO mRNA values returned to approximate control levels 24 hr after exposure. In striking contrast to the induction of MT and HO gene expression after ZnO exposures, there was no elevation in gene expression following a 6-hr exposure to 0.5 and 1 ppm ozone, even when lungs were examined as late as 72 hr after exposure. Our results demonstrate the induction of target gene expression following the inhalation of ZnO at concentrations equal to, and below, the current recommended threshold limit value of 5 mg/m3 ZnO. Furthermore, the lack of effect of ozone exposure on MT and HO gene expression suggests no involvement of these genes in the acute respiratory response to this oxidant compound.


Biotransformation and toxicity of acetaminophen in congenic RHA rats with or without a hereditary deficiency in bilirubin UDP-glucuronosyltransferase. Acetaminophen is eliminated primarily by glucuronidation, thereby avoiding cytochrome P450-catalyzed bioactivation to a toxic reactive intermediate. Previous studies have shown that UDP-glucuronosyltransferase-deficient Gunn rats are more susceptible to acetaminophen toxicity than normal Wistar controls, from which the Gunn strain was derived. However, the Gunn and Wistar strains are not congenic, and differences in toxicologic susceptibility could be due in part to genetic differences other than UDP-glucuronosyltransferase activity. Accordingly, acetaminophen (750 mg/kg, ip) was administered to congenic RHA rats with normal (homozygous, RHA/++), moderately deficient (heterozygous, RHA/j+), and severely deficient (homozygous jaundiced, RHA/jj) activities of bilirubin UDP-glucuronosyltransferase. Acetaminophen metabolites were measured by high-performance liquid chromatography and production of the acetaminophen glucuronide conjugate was quantified by the area under plasma concentration-time curve (AUC) from 0 to 2 hr, standardized by the AUC value for acetaminophen in the same animal (glucuronidation ratio = AUC acetaminophen glucuronide/AUC acetaminophen). The 0- to 2-hr time period for AUC calculations was necessitated by the accumulation at later time points of glucuronide and sulfate conjugates in the plasma of animals experiencing severe nephrotoxicity. Acetaminophen bioactivation was quantified by the 24-hr urinary recovery of glutathione-derived conjugates. Hepatotoxicity and nephrotoxicity were assessed respectively by the peak concentrations of plasma alanine aminotransferase (ALT) and blood urea nitrogen (BUN). Glucuronidation of acetaminophen in RHA/jj rats (0.065 +/- 0.005) (mean +/- SE) was reduced 63% compared to the RHA/++ controls (0.17 +/- 0.01) (p < 0.05). RHA/jj rats demonstrated respective 230- and 7-fold increases in the peak plasma concentrations of ALT (17144 +/- 1014 vs 75 +/- 10) and BUN (128 +/- 23 vs 18.4 +/- 0.2) compared to congenic normal controls (RHA/++) (p < 0.05). Heterozygous animals (RHA/j+) demonstrated intermediary toxicity for both parameters (ALT = 2029 +/- 1581, BUN = 41 +/- 16, p < 0.05). Decreased glucuronide production correlated with elevations in ALT (r = -0.86, p < 0.001), while increased acetaminophen bioactivation correlated directly with both elevated ALT (r = 0.93, p < 0.001) and BUN (r = 0.83, p = 0.001). These results using congenic controls demonstrate that the enhanced susceptibility of UDP-glucuronosyltransferase-deficient rats to acetaminophen toxicity is due to decreased glucuronidation resulting in enhanced bioactivation, rather than to other unappreciated genetic differences.


Methylene dianiline: acute toxicity and effects on biliary function. 4,4'-Methylene dianiline (4,4'-diaminodiphenylmethane, DAPM), which is used in the polymer industry, causes hepatobiliary damage in exposed humans. Our objectives were to characterize the acute toxicity of DAPM in liver, particularly on secretion of biliary constituents and on biliary epithelial cell gamma-glutamyl transpeptidase (GGT) activity. Biliary cannulas were positioned in Sprague-Dawley male rats under pentobarbital anesthesia. After 1 hr of control bile collection, each rat was given 250 mg DAPM/kg (50 mg/ml) po in 35% ethanol or 35% ethanol only; bile was collected for a further 4 hr. Groups of rats were also examined for liver injury and biliary function at 8 and 24 hr after DAPM. Four hours after DAPM administration, main bile duct cells were severely damaged with minimal damage to peripheral bile ductule cells. Focal periportal hepatocellular necrosis and extensive cytolysis of cortical thymocytes occurred by 24 hr. Serum indicators of liver injury were elevated by 4 hr and continued to rise through 24 hr. By 4 hr, biliary protein concentration was increased 4-fold while concentrations of biliary bile salt, bilirubin, and glutathione were decreased by approximately 80, 50, and 200%, respectively. DAPM also induced a striking effect on biliary glucose with an approximately 20-fold increase. Histochemical staining of main bile duct GGT was absent by 8 hr after DAPM. Bile flow was diminished by 40% at 4 hr; three of five rats had no bile flow by 8 hr and none had any bile flow by 24 hr. These results indicate that DAPM rapidly diminishes bile flow and alters the secretion of biliary constituents and is highly injurious to biliary epithelial cells.


The relationship between nickel chloride-induced peroxidation and DNA strand breakage in rat liver. Inorganic nickel chloride induces hepatic DNA strand breaks, chromosome aberrations, and lipid peroxidation under in vitro and in vivo conditions. The objective of this research was to determine if a relationship exists between NiCl2 genotoxicity and lipid peroxidation in vivo. Male Sprague-Dawley rats (210-250 g) were dosed with 0.56 or 0.75 mmol/kg NiCl2 subcutaneously and euthanized after specific time periods, ranging from 30 min to 24 hr. Livers were perfused and excised for the measurement of nickel content using atomic absorption spectrometry, lipid peroxidation using a thiobarbituric acid assay, and DNA strand breakage using single-stranded DNA extraction and the diaminobenzoic acid assay. The lower dose (0.56 mmol/kg) did not induce lipid peroxidation or strand breakage. The higher dose (0.75 mmol/kg) induced DNA strand breakage at 4 hr and lipid peroxidation at 12 hr in rat liver. Nickel was seen to accumulate in liver nuclei of rats receiving 0.75 mmol/kg. Deferoxamine (1 g/kg, ip, 15 min before the NiCl2 injection) completely inhibited DNA strand breakage at 4 hr but had no effect on lipid peroxidation. This suggests that lipid peroxidation is not causally related to genetic damage. NiCl2-induced DNA strand breakage may be caused by the induction of the Fenton reaction, generating hydroxyl radicals.


Plants with a reputation against snakebite. Many plants are recommended in traditional medicine as active against various effects of snakebite. Few attempts have been made to investigate the veracity of these assertions in controlled experiments. Several workers, mainly Oriental, have investigated the reputation of such plants by performing in vitro and in vivo experiments in order to demonstrate whether there was any protective effect, using drugs or mixtures of drugs prepared using traditional formulae. In some studies, these extracts were administered to mice before or after treatment with different elapid or crotalid venoms. Other papers deal with selected compounds isolated from Schumanniophyton magnificum, Eclipta prostrata or Aristolochia shimadai, and their capacity to inhibit phospholipase A2 or other enzymes (e.g. ATPase) or for physiological and biochemical properties (such as effects on uterine tone or the protection of mitochondrial membranes). Japanese workers have described the antihaemorrhagic effect of persimmon tannin from Diospyros kaki. Atropine has been attributed a life-prolonging effect after black mamba venom treatment. Prolonged survival was also observed after pretreatment with extracts of Diodia scandens and Andrographis paniculata. Some authors have found little or no beneficial effects. The papers collected so far show that there are no systematic investigations in this field.


Paralytic shellfish poison (saxitoxin family) bioassays: automated endpoint determination and standardization of the in vitro tissue culture bioassay, and comparison with the standard mouse bioassay. Mouse neuroblastoma cells swell and eventually lyse upon exposure to veratridine, which, when added together with ouabain, enhances sodium ion influx. In the presence of saxitoxin (STX), which blocks sodium channels, the action of the other two compounds is inhibited and the cells remain morphologically normal. A tissue culture bioassay using mouse neuroblastoma cells, developed by Kogure and colleagues, takes advantage of these principles; in this bioassay, the fraction of the cells protected from the actions of ouabain and veratridine is in direct proportion to the concentration of STX and its analogues. We have modified this bioassay, improving its convenience and speed by eliminating the need to count individual cells to determine the saxitoxin equivalents, and instead have employed a microplate reader for automated determinations of absorbances of crystal violet from stained neuroblastoma cells. When these changes and other minor technical modifications were tested in the tissue culture bioassay systematically, we found the lower detection limit to be around 10 ng STX equivalents (eq) per ml of extract ( = 2.0 micrograms STX eq/100 g shellfish tissue). Our version of the tissue culture bioassay was compared with the standard mouse bioassay using 10 acid extracts of dinoflagellates (Alexandrium excavata and A. fundyense) and 47 AOAC extracts of shellfish tissues. The tissue culture bioassay provided results virtually identical to those obtained with the mouse bioassay (r > 0.96), and moreover, was considerably more sensitive. The results gained from high performance liquid chromatographic (HPLC) analysis of 12 of the same extracts were less consistent when compared with the results from both bioassay methods. The automated tissue culture (neuroblastoma cell) bioassay may be a valid alternative to live animal testing for paralytic shellfish poisoning.


Identification of anatoxin-A in benthic cyanobacteria (blue-green algae) and in associated dog poisonings at Loch Insh, Scotland. Dog deaths occurred in 1990 and 1991 after the animals drank water containing blooms of benthic cyanobacteria along the shoreline of Loch Insh, Scotland. Signs of poisoning in the affected animals and the high neurotoxicity of bloom extracts in laboratory bioassays indicated acute poisoning due to cyanobacterial neurotoxin(s). The neurotoxic blooms consisted largely of benthic Oscillatoria species which were also observed in the stomach contents of the poisoned dogs. Stomach contents were also neurotoxic in bioassays with the same signs of poisoning as the Oscillatoria blooms. The cyanobacterial alkaloid neurotoxin anatoxin-a was identified in bloom extracts and poisoned dog stomach contents by high-performance liquid chromatography and gas chromatography-mass spectrometry. A species of benthic Oscillatoria has been isolated from the neurotoxic bloom material and shown to produce anatoxin-a in laboratory culture. These findings are the first to associate anatoxin-a toxicoses with benthic, rather than planktonic, cyanobacteria. Procedures for anatoxin-a extraction and identification from the blooms and animal material are also detailed.


Production of a monoclonal antibody against the thrombin-like enzyme, habutobin, from Trimeresurus flavoviridis venom. We succeeded in producing a monoclonal antibody to the thrombin-like enzyme, habutobin, which was purified from crude venom of the snake Trimeresurus flavoviridis. The monoclonal antibody obtained belonged to IgG1, and its light chain consisted of a kappa-chain. The monoclonal antibody reacted specifically with habutobin and crude venom from T. flavoviridis but did not react with human thrombin or bovine thrombin on Western blotting. The concentration of habutobin and crude venom of T. flavoviridis, in vitro, could be measured by means of ELISA using the monoclonal antibody. Furthermore, the ELISA-double sandwich method employing this monoclonal antibody may represent a reliable method for determining the habutobin levels in the circulating blood.


Variation and secretion of toxins in gastropod mollusc Niotha clathrata. Nearly 300 specimens of the gastropod mollusc Niotha clathrata were collected in South Taiwan. All specimens were assayed for toxicity by the official method for tetrodotoxin (TTX). The frequency value of toxic specimens in N. clathrata was 30.0%, 68.0% and 80.4% at Anping, Chiating and Tungkang, respectively. The highest lethal potency of a gastropod specimen was 1900 mouse units (MU). The specimens collected from Tungkang showed the highest frequency of toxic specimens and toxicity, followed by those from Chiating and Anping. The specimens collected in autumn and spring showed higher toxicity than those collected in other seasons. Moreover, another 17 specimens of N. clathrata were collected for testing the toxin secretion by electric shock treatment. It is found that the gastropod did not secrete any additional toxin when electric shock treatment was performed twice at approximately 1-hr intervals. The toxicity of secreted toxin was 206 MU.