Reversible C3 Hypocomplementaemia in Megaloblastic Anaemia due to Vitamin B12 Deficiency

S ummary . Serum C3 and C4 levels have been determined in patients with Addisonian pernicious anaemia (PA) and megaloblastic anaemia due to vitamin B₁₂ deficiency from other causes, before and after treatment, in order to study the interaction between vitamin B₁₂ deficiency and Complement and the role of complement in the pathogenesis of the gastric lesion of PA. C₃ levels are significantly reduced in vitamin B₁₂ deficiency and return to normal on treatment; C₃ levels correlate with the degree of anaemia but not with serum vitamin B₁₂ levels at diagnosis. C₄ levels are normal. These observations suggest that the observed C₃ hypocomplementaenlia is not a consequence of immune mechanisms, but may be due to altered synthesis of C₃ complement component.     

OKT4/OKT8 Ratio in Asymptomatic Heroin Addicts

The OKT₄ (helper) and OKT₈ (suppressor) lymphocytic subpopulations were enumerated in a sample of 60 asymptomatic drug addicts and in 17 controls. No significant differences in the ratio could be found that could not be explained by the action of HIV. It can be concluded that heroin itself was not responsible for any alteration in the T₄/T₈ ratio in the population considered.     

Ethanol Inhibits C6 Cell Growth: Fetal Alcohol Syndrome Model

Maternal consumption of ethanol produces a pattern of malformations, including nervous system abnormalities, in the developing fetus, a state called Fetal Alcohol Syndrome. We report the dose-dependent inhibition by ethanol of the growth of a glioma derived cell line, C6 cells; the effects occur at ethanol concentrations commonly encountered in the blood during human intoxication. The effects occur with different morphological subtypes of the cell line and do not occur when the cells are exposed to iso-osmolar concentrations of other chemicals. The results demonstrate that C6 cells are a model for the study of the effects of ethanol on nervous system cell growth.     

Ethanol Inhibition of Inducible Nitric Oxide Synthase Activity in C6 Glioma Cells

Nitric oxide (NO), a free radical gas, has been implicated in the CNS actions of ethanol. The brain contains several cell types that can produce NO, including neurons and glia. This study examined the effect of acute and chronic ethanol exposure on the activity of the inducible isoform of nitric oxide synthase (iNOS) found in neuroglia. Experiments were performed using intact rat C₆ glioma cells, and NO production was assessed by nitrite accumulation after iNOS induction by coadministration of phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS). Ethanol was inhibitory at high concentrations (IC₅₀= 150 m m ) when acutely present during the 24-hr period subsequent to initiation of enzyme induction. In contrast, cells exposed to ethanol were inhibited chronically at clinically relevant lower concentrations (IC₅₀= 30 m m with 10 days exposure). Chronic inhibition was both time- and concentration-dependent. Inhibition by ethanol seems to be a consequence of interference with LPS signal transduction. Acutely, ethanol did not affect the ability of PMA to synergize with LPS to induce activity, but it attenuated the ability of LPS to synergize with the PMA. Ten days exposure to 50 m m ethanol decreased the LPS potency by 4-fold in the presence of a maximally activating concentration of PMA, although not significantly changing PMA potency. Inhibition by chronic ethanol exposure was long-lasting, being retained over 24 hr in cells returned to control conditions. Thus, chronic ethanol may downregulate key components needed for iNOS expression. These results suggest that acute toxic and chronic ethanol exposure can inhibit NO production in the brain, and considering the ability of NO to protect neurons from free radical injury, this action could contribute to ethanol-in-duced brain damage.     

Elevated white blood cell synthesis of leukotriene C4 in chronic myelogenous leukaemia but not in polycythaemia vera

Leukotriene (LT) formation was studied in ionophore A23187-stimulated white blood cell (WBC) preparations from patients with chronic myelogenous leukaemia (CML; n = 14), polycythaemia vera (PV; n = 10) and two control groups consisting of patients with non-malignant inflammatory disease (n = 4) and normal healthy donors (n = 25). The synthesized products were identified and quantitated using high-performance liquid chromatography combined with computerized UV-spectroscopy. White blood cell preparations from the CML patients produced more LTC4 (40.2 +/- 7.9 pmol/10(6) WBC, mean +/- SEM) than WBC from the healthy donors (9.0 +/- 1.8), P less than 0.0005. In contrast, the formation of LTB4 was normal and there was no increase in the total leukotriene synthesis (the sum of LTC4, LTB4, 20-OH-LTB4 and the delta 6-trans-isomers of LTB4). The ratio between leukotrienes C4 and B4 was strongly elevated in the CML group; 1.67 +/- 0.25 v. 0.37 +/- 0.07 in the controls, P less than 0.0005. No significant correlation was observed between the levels of LTC4 and the number of known LTC4 producing cells (such as monocytes, eosinophils and basophils) in the CML WBC preparations. In contrast, a correlation was found between the sum of neutrophilic granulocytes and metamyelocytes in these suspensions and the amount of LTB4 formed; r = 0.600, P less than 0.05. A number of other laboratory or clinical variables of the CML patients (including total white blood cell and platelet counts, differential counts, previous cytotoxic treatment, time from diagnosis, time from last treatment, post study survival and age) did not significantly correlate with the formation of leukotrienes. No abnormality in the production of LTB4 or LTC4 was observed in granulocyte and WBC preparations from the patients with polycythaemia vera and non-malignant inflammatory disease, respectively. The results indicate a selectively increased LTC4 producing capacity in CML.     

T4 Thyrotoxicosis with Normal or Low Serum T3 Concentration

Summary: : T₄ thyrotoxicosis with normal or low serum T₃ concentration. A. Joasoo, Aust. N.Z. J. Med , 1975, 5, pp. 432–434.     

Iron Absorption, Iron Loss and Iron Retention in Man: Studies after Oral Administration of a Tracer Dose of 59FeSO4 and 131BaSO4

The absorption of iron administered as ferrosulphate was studied in normal subjects, in patients with iron deficiency and in patients with idiopathic and secondary haemosiderosis. By using a test dose combining radioactive iron (⁵⁹Fe) with radioactive barium sulphate (¹³¹Ba) as inert indicator, it was possible to determine how much iron was initially absorbed and whether a proportion of this was later excreted in the faeces.
The (initial) iron absorption, the iron loss as a percentage of the iron absorbed, and the (ultimate) iron retention 2 weeks after administration of the test dose were calculated.
In normal subjects and patients with secondary haemosiderosis, a large proportion of the initially absorbed iron later returned to the intestinal lumen. In patients with iron deficiency and those with idiopathic haemosiderosis, however, nearly all the initially absorbed iron was retained in the organism. In differential diagnosis between idiopathic and secondary haemosiderosis, therefore, determination of the iron absorption and iron retention percentages from a test dose may be important.
In patients with secondary haemosiderosis there seemed to be a negative correlation between the degree of haemosiderosis and the amount of iron from the test dose ultimately retained in the organism.     

Incorporation of 32PO4 into Phospholipids of Blood Platelets

Human or rabbit platelets in an artificial medium without phosphate were incubated with carrier-free [³²P]orthophosphate for 1 h, washed and re-suspended in Tyrode-albumin solution containing unlabelled phosphate. Aliquots of platelet suspension were subjected to lipid extraction at various times and the extracted phospholipids were separated by thin layer chromatography. The specific radioactivities of triphosphoinositide (TPI) and diphosphoinositide (DPI) were highest during the labelling period and then declined rapidly after the platelets were resuspended in the medium containing unlabelled phosphate. The percentage of total phospholipid ³²P in TPI and DPI decreased from over 95% at 1 h to less than 50% at 12 h with either rabbit or human platelets. The specific radioactivity of monophosphoinositide (MPI) reached its greatest value 6–8 h after labelling. In the in vitro incubation studies, phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE) were negligibly labelled in the initial hours but at 12 h (rabbit or human platelets) PC contained more than 30% of the radioactivity. PE and PS contained less than 10% of the total phospholipid-bound radioactivity at the end of the incubation period. The pattern of incorporation of [³²P]orthophosphate into the inositol phospholipids, PC, PS and PE that had been found in the in vitro studies was confirmed by an in vivo study in which rabbit platelets labelled with ³²P in vitro were infused into rabbits and harvested after 35 h. These findings indicate that the phosphates of all the phospholipids studied in these experiments turn over in vitro and in vivo but the phosphates of the inositol phospholipids turn over most rapidly.     

Dose-Dependent Destruction of A1 Cells by Anti-A1

Abstract. In a patient of subgroup A₂ the serum contained an unusually potent anti-A₁, giving the following reactions with A₁ red cells in vitro: agglutination of saline-suspended cells up to a temperature of 32°C; a positive indirect antiglobulin test (complement only) at 37°C and lysis of enzyme-treated cells at 37°C. A series of tests was carried out to estimate the ability of the antibody to destroy varying amounts of A₁ red cells in vivo . When about 0.55 ml of red cells was injected, about 65% of the cells were destroyed within 30 min; 2 days later when 18.9 ml of cells were injected, only about 45% were destroyed within 30 min; 5 days after this when a whole unit of A₁ red cells was transfused, survival at 24 h was about 90%. This last figure may indicate that destruction of red cells by anti-A₁ was negligible since at the time of the transfusion of the whole unit the patient was bleeding into her gastrointestinal tract. On the other hand, the titre of anti-A₁ appeared to be declining spontaneously during the period in which tests were carried out so that, if the whole unit of A₁ blood had been transfused at the beginning of this period, survival might have been less good. Nevertheless, from the observed difference in survival between the 0.55 ml and 18.9 ml doses it seems safe to conclude that, even if the unit had been transfused at the time when the antibody concentration was maximal, the percentage of cells destroyed would have been small.     

Hepatoprotective action of prostaglandin A2 analogs under CCl4-induced liver injury in vitro

Aim: The cytoprotective effects of six novel synthetic prostaglandin A(2) analogs against carbon tetrachloride (CCl(4)) as a toxic agent were studied with isolated rat liver hepatocytes in vitro. Results: It was found that hepatocytes treatment with CCl(4) induced: (i) a significant increase of lactic dehydrogenase (LDH) release from cytoplasm; (ii) leakage of glutamate dehydrogenase (GDH) and acid phosphatase from mitochondria and lysosomes, respectively; (iii) 10-fold increase of trien conjugates formation; and (iv) a reduction of free SH-groups by 50%. Prostanoids U-26, U-9 and U-34 decreased cytotoxic index of CCl(4) on average by 1.5-2.0 times and were more effective than PGI(2), the well-known hepatoprotector of prostanoids type. The protective action of the prostanoids was not a cAMP- or Ca(2+)-dependent process. However, prostanoids U-26, U-9 and U-34 normalized intracellular content of SH-groups, reduced trien conjugates formation by 60-80% and strongly prevented enzyme leakage through cellular membranes. They were also able to inhibit CCl(4) effects via decreasing cytochrome P(450)2E1 activity. Conclusion: The results obtained demonstrate that prostanoids provide cytoprotective effects on liver hepatocytes through the prevention of lipid peroxidation of the plasma and the cellular membranes and maintenance of their barrier function.     

Blood group A1 and A2 revisited: an immunochemical analysis

Background and Objective  The basis of blood group A₁ and A₂ phenotypes has been debated for many decades, and still the chemical basis is unresolved. The literature generally identifies the glycolipid chemical differences between blood group A₁ and A₂ phenotypes as being poor or no expression of A type 3 and A type 4 structures on A₂ red cells, although this assertion is not unanimous.
Materials and Methods  Using purified glycolipids and specific monoclonal antibodies, we revisited the glycolipid basis of the A₁ and A₂ phenotypes. Purified glycolipids were extracted from four individual A₁ and four individual A₂ blood units. One blood unit from an A weak subgroup was also included. Monoclonal anti-A reagents including those originally used to define the basis of A₁ and A₂ phenotypes were used in a thin layer chromatography – enzyme immunoassay to identify the presence of specific glycolipids.
Results  A type 3 glycolipid structures were found to be present in large amounts in all phenotypes. In contrast, the A type 4 glycolipid structure was virtually undetectable in the A₂ phenotype, but was present in the A₁ and A subgroup samples.
Conclusion  The major glycolipid difference between the A₁ and A₂ phenotypes is the dominance of A type 4 glycolipids in the A₁ phenotype.     

Novel enzymatic abnormalities in AML and CML in blast crisis: elevated leucocyte leukotriene C4 synthase activity paralleled by deficient leukotriene biosynthesis from endogenous substrate

Leukotrienes (LT) are inflammatory mediators which can also exert regulatory effects on human myelopoiesis. We have studied the LT-producing capacity of freshly isolated leucocyte suspensions (containing blast cells in variable proportions) from 41 patients with acute myeloid leukaemia (AML) or chronic myeloid leukaemia (CML) in blast crisis (CMLbc) at diagnosis or relapse/resistant disease. Leucocyte suspensions from 19/29 AML patients (66%), and 2/12 CMLbc patients (17%; P  = 0.012) demonstrated deficient capacity to synthesize LT from endogenous substrate after ionophore A23187 stimulation. Thus, these cells produced < 8 pmol LTB₄ + LTC₄/10⁶ cells (< 20% of mean LT formation in leucocyte suspensions from 18 healthy subjects). Addition of exogenous arachidonic acid did not normalize the LT synthesis in poor-producing cell suspensions. Purified, morphologically mature granulocytes from two AML patients also failed to produce normal amounts of LT. In leucocyte suspensions from the remaining 20 AML/CMLbc patients A23187 provoked LT biosynthesis, with markedly increased production of LTC₄, but decreased LTB₄ formation. Furthermore, elevated conversion of exogenous LTA₄ to LTC₄ was noted in the patient samples, independent of their capacity to produce LT after A23187 stimulation. The percentage of blast cells in patient white blood cell differential counts correlated inversely with ionophore-induced LT synthesis, but positively with the conversion of exogenous LTA₄ to LTC₄. The results suggest elevated LTC₄ synthase activity and suppressed 5-lipoxygenase activity as novel enzymatic features of myeloid leukaemia patients with immature phenotype.     

Prenatal Diagnosis of β Thalassaemia by Fetal Red Cell Enrichment with NH4Cl-NH4HCO3 Differential Lysis of Maternal Cells

S ummary . Prenatal diagnosis with globin chain synthesis analysis on fetal red blood cells concentrated by NH₄Cl-NH₂HCO₃ differential lysis of maternal cells (Ørskov lysis) was carried out in 27 pregnancies at risk for β thalassaemia and one at risk for sickle cell β⁰ thalassaemia. The β/γ globin chain synthesis ratio was also determined after anti-i differential agglutination (12 cases), in almost pure fetal samples (six cases) and by extrapolation (one case). Differential lysis permitted the study of samples drawn by placental aspiration containing as little as 3.2% fetal red blood cells. There was no consistent difference between the β/γ ratios observed after differential lysis and those determined after the use of the other approaches. A presumptive diagnosis of homozygous β thalassaemia was made in nine cases. All but one of these pregnancies was terminated. The absence of β chain synthesis was confirmed by the study of fetal blood after abortion in four cases with suitable samples. Of the remaining pregnancies, six proceeded to term and non-homozygous infants were delivered. The others are still in progress. No fetal loss occurred. ørskov lysis seems to be a very reliable method for prenatal diagnosis of β chain abnormalities. Moreover it can minimize the number and duration of placental aspirations required and thus the risk to the fetus.