Defective assembly of membrane proteins in erythroid precursors of beta- thalassemic mice

beta-Thalassemic mice provide a useful model for studying the pathophysiology of human beta-thalassemia in that one can perform experiments that are difficult to perform in humans. The ease of access to beta-thalassemic mouse marrow provided the opportunity to explore the cause of the ineffective erythropoiesis that characterizes severe beta-thalassemia in mouse and man. We hypothesized that the accumulation of excess alpha-globin might interfere with the normal assembly of red blood cell (RBC) membrane proteins, thus contributing to the severe intramedullary lysis. Femoral marrow was obtained from normal and beta-thalassemic mice, and RBC precursors were purified (> 90%) by panning and harvesting CD45- cells. The assembly of RBC membrane proteins was assessed by observing immunofluorescence patterns obtained on fixed permeabilized precursors using rabbit polyclonal antibodies directed against human spectrin, and band 4.1, and murine band 3. The distribution of the proteins was shown with a fluorescein- tagged goat antirabbit antibody. In contrast to normal mice, about 30% of intermediate and late stage erythroblasts in beta-thalassemic mice appear abnormal. Neither spectrin nor band 4.1 formed crisp rim fluorescence in these erythroid precursors of thalassemic mice, whereas assembly of band 3 appeared normal. Therefore, the assembly of membrane skeletal proteins is abnormal in murine beta-thalassemic erythroid precursors perhaps because of the deposition of unmatched alpha-globin chains.     

Erythrocyte membrane proteins reactive with IgG (warm-reacting) anti- red blood cell autoantibodies: II. Antibodies coprecipitating band 3 and glycophorin A

In our initial immunochemical study of the red blood cell (RBC) membrane proteins targeted in 20 cases of warm-antibody autoimmune hemolytic anemia (AHA), RBC eluates of 6 patients mediated immunoprecipitation (IP) of both band 3 and glycophorin A (GPA). This dual IP pattern had previously been observed with murine monoclonal antibodies (MoAbs) against the high frequency blood group antigen, Wrb (Wright), suggesting that the Wrb epitope may depend on a band 3-GPA interaction. Earlier, anti-Wrb had been identified serologically as a prominent non-Rh specificity of AHA autoantibodies. In the present study, 6 autoantibody eluates immunoprecipitating band 3 and GPA from common Wr(b+) RBCs were retested, in parallel with murine anti-Wrb MoAbs, against very rare Wr(a+b-)En(a+)RBCs. One patient's autoantibodies were unreactive with the Wr(b-) RBCs by either IP or indirect antiglobulin test (IAT) and were judged to have "pure" anti- Wrb specificity. Two other patients' autoantibodies displayed both IP and serologic evidence for anti-Wrb as a major component in combination with one or more additional specificities. However, among 3 other patients whose autoantibodies coprecipitated band 3 and GPA, there was no reduction in IP or IAT reactivity with Wr(b-) RBCs in 2 and only slight reduction in the third. We conclude (1) that human anti-Wrb autoantibodies, like their murine monoclonal counterparts, coprecipitate band 3 and GPA from human RBCs; but (2) that not all antibodies with this IP behavior have anti-Wrb serologic specificity, as defined by this donor's Wr(b-) RBCs. The possibility of an additional (non-Wrb) RBC epitope dependent on a band 3-GPA interaction is raised.     

Defective platelet-fibrinogen interaction in hereditary canine thrombopathia

A unique, intrinsic, hereditary canine platelet disorder attributable to abnormal fibrinogen receptor availability is described. Thrombopathic platelets from 13 severely affected basset hounds failed to aggregate in response to all agonists tested except thrombin. Normal platelet interaction with the various stimuli was inferred on the basis of their ability to elicit unimpaired shape change in thrombopathic platelets. No quantitative differences in major platelet membrane glycoproteins, intraplatelet fibrinogen, adenine nucleotides, or serotonin uptake were detected. Dense granule secretion was impaired. The ultrastructural appearance of thrombopathic platelets was normal. Fibrinogen-platelet interaction was evaluated by reacting platelet-rich plasma (PRP) with fibrinogen coupled to polymeric acrylonitrile beads and scoring the extent of stimulus-induced agglutination. The aggregatory responses of normal and thrombopathic platelets were closely correlated with fibrinogen receptor availability. In contrast to human platelets, epinephrine-stimulated canine platelets did not interact with immobilized fibrinogen, and arachidonate generally induced only weak agglutination. Thrombopathic platelets agglutinated fibrinogen beads at reduced rates when stimulated with physiologic doses of thrombin and high-dose calcium ionophore, A23187. Our data suggest that thrombin-mediated induction of canine platelet fibrinogen receptors may proceed by pathway(s) alternate to those shared by other platelet agonists, and/or that secreted granule constituents may act synergistically with thrombin to overcome inhibition of signal-response- coupled reactions mediating the interaction of fibrinogen with its receptor. This congenital platelet defect provides further evidence, in a species other than human, for the pivotal role of fibrinogen receptor induction in platelet aggregation.     

Studies on the effect of vanadate on endocytosis and shape changes in human red blood cells and ghosts

When amphipathic cationic drugs are added to intact human RBCs, the RBCs first undergo a stomatocytic shape change and then, if relatively large amounts of drug are added and if the metabolic state of the RBC is appropriate, endocytic vacuoles form. Vanadate has a structural similarity to the transition state of phosphate, which presumably accounts for its ability to inhibit phosphohydrolases, although other actions of vanadate have been described. Vanadate inhibited three forms of drug-induced endocytosis in intact RBCs despite the fact that the three drugs chosen (primaquine, chlorpromazine, and vinblastine) are known to have differing requirements for RBC ATP. Vanadate also inhibited the stomatocytic shape change produced by primaquine, chlorpromazine, and vinblastine, but not the stomatocytosis produced by low pH. Vanadate had no effect on RBC echinocytosis produced by lysophosphatidylcholine. In studying endocytosis in hypotonic, leaky, "white" ghosts, we discovered that vanadate inhibited only the endocytosis produced by Mg-ATP and not the endocytosis produced by manipulations that directly attack the cytoskeletal proteins. These findings suggest that ATP hydrolysis has a role in some forms of amphipathic cation-induced stomatocytosis and endocytosis in intact RBCs. In addition, studies in ghosts support the idea that Mg-ATP does indeed produce "energized" endocytosis dependent on utilization or hydrolysis of ATP.     

Primary tuberculous nasal polypi—A case report

Primary tuberculous pathology in nasolpolypi is a rare condition. A case of bilateral ethmoidal polypi with tubercular lesion diagnosed on histopathologlcal examination is being reported and the available relevant literature has been reviewed.     

Risk factors associated with having psoriatic arthritis in patients with cutaneous psoriasis

The aim of this study was to determine if the following characteristics were associated with the presence of psoriatic arthritis in a sample of psoriasis patients: race, family history of psoriasis and psoriatic arthritis, age of onset of psoriasis, smoking, alcohol consumption and the maximum body surface area (BSA) affected by psoriasis. This was a case–control study involving 400 psoriasis patients who attended the Psoriasis and Photo‐medicine clinic in the National Skin Center of Singapore over a 1‐year period. Cases were psoriasis patients with psoriatic arthritis while controls were psoriasis patients without psoriatic arthritis. The diagnosis of psoriatic arthritis was made by rheumatologists and participants completed a self‐administered standardized questionnaire. The maximum BSA involved was determined from the case notes. Psoriatic arthritis was not significantly associated with sex, race, age of onset of psoriasis, a family history of psoriasis, smoking and alcohol consumption but was significantly associated with a family history of psoriatic arthritis (P < 0.001) and the maximum body surface involved (P = 0.05). Using multivariate analysis to control for variables, the presence of psoriatic arthritis was significantly associated with a family history of psoriatic arthritis (odds ratio [OR] = 20.5; 95% confidence interval [CI] = 2.49–169.10) and the maximum BSA involved (OR = 2.52; 95% CI = 1.33–4.75). Indian psoriatic patients were more likely to have psoriatic arthritis compared to the other races. A family history of psoriatic arthritis and a greater maximum body surface involved may be associated with having psoriatic arthritis in this study population of psoriasis patients.     

KTP laser assisted excision of glomus tympanicum

A 39-year-old female with a two-year history of mild hearing loss and discomfort on air flight descent was found to have a pulsatile mass behind an intact tympanic membrane. A suspected diagnosis of glomus tympanicum was confirmed by computed tomography (CT) scan imaging. The lesion filled the mesotympanum and hypotympanum but the jugular bony plate was intact, confirming the tympanic site of the lesion. This very vascular tumour was exposed by a tympanomeatal flap and the KTP laser used to shrink and coagulate the tumour progressively with minimal haemorrhage and blood loss. Complete excision of the lesion was achieved without the need for bony removal, and with minimal blood loss. The use of the KTP laser to coagulate this vascular lesion allowed safe removal of the tumour and avoided the need for extended facial recess or hypotympanotomy surgery.     

Nociceptive neurones in the superficial dorsal horn of cat lumbar spinal cord and their primary afferent inputs

Summary The morphology, background activity and responses to stimulation of primary afferent inputs of small neurones in the superficial dorsal horn which could only be excited from the skin by noxious stimulation were investigated by intracellular recording and ionophoresis of HRP. Neurones which gave similar responses to afferent stimulation were morphologically heterogeneous with respect to dendritic tree geometry and axonal projection, but were located around the lamina I/II border. Cutaneous excitatory receptive fields responding to noxious stimulation were generally small; most neurones had more extensive inhibitory fields responding to innocuous mechanical stimulation, in many cases overlapping the excitatory fields. Generally, stimulation of the excitatory field resulted in depolarization of the neurone and increased action potential firing, and stimulation of the inhibitory field resulted in hyperpolarization. Electrical stimulation of peripheral nerves revealed the existence of converging excitatory inputs carried by different fibre groups, and all neurones received an inhibitory input activated at low threshold. Excitatory responses were short-lived and occurred consistently in response to repeated stimulation. Central delay measurements gave evidence of a number of A monosynaptic inputs but only one A monosynaptic input; inhibitory inputs along A fibres were polysynaptic. The constant latency and regularity of the C response suggested monosynaptic connections. Low intensity stimulation of inhibitory inputs elicited a short-lived i.p.s.p. which increased in amplitude with increasing stimulus strength until it disappeared into a more prolonged hyperpolarization. This was associated with inhibition of background action potentials, and increased in duration with increasing stimulus strength up to C levels, indicating an A and C component. It is suggested that the level of excitability of these neurones depends on the relative amounts of concurrent noxious and innocuous stimulation, and that the resultant output, which is conveyed mainly to other neurones within the spinal cord, could modulate reflex action at the spinal level as well as affecting components of ascending sensory pathways.Supported by grant no. 11853/1.5 from the Wellcome Trust