The instability of the membrane skeleton in thalassemic red blood cells

The thalassemias are a heterogeneous group of disorders characterized by accumulation either of unmatched alpha or beta globin chains. These in turn cause the intramedullary and peripheral hemolysis that leads to varying anemia. A partial explanation for the hemolysis came our of our studies on material properties that showed that beta-thalassemia (beta- thal) intermedia ghosts were very rigid but unstable. A clue to this instability came from the observation that the spectrin/band 3 ratio was low in red blood cells (RBCs) of splenectomized beta-thal intermedia patients. The possible explanations for the apparent decrease in spectrin content included deficient or defective spectrin synthesis in thalassemic erythroid precursors or globin chain-induced membrane changes that lead to spectrin dissociation from the membrane during ghost preparation. To explore the latter alternative, samples from different thalassemic variants were obtained, ie, beta-thal intermedia, HbE/beta-thal, HbH (alpha-thal-1/alpha-thal-2), HbH/Constant Spring (CS), and homozygous HbCS/CS. We searched for the presence of spectrin in the first lysate of the standard ghost preparation. Normal individuals and patients with autoimmune hemolytic anemia, sickle cell anemia, and anemia due to chemotherapy served as controls. Using gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, no spectrin was detected in identical aliquots of the supernatants of normals and these control samples. Varying amounts of spectrin were detected in the first lysate supernatants of almost all thalassemic patients. The identification of spectrin was confirmed by Western blotting using an affinity-purified, monospecific, rabbit polyclonal antispectrin antibody. Relative amounts of spectrin detected were as follows in decreasing order: splenectomized beta-thal intermedia including HbE/beta-thal; HbCS/CS; nonsplenectomized beta-thal intermedia, HbH/CS; and, lastly, HbH. These findings were generally confirmed when we used an enzyme-linked immunosorbent assay technique to measure spectrin in the first lysate. Subsequent analyses showed that small amounts of actin and band 4.1 also appeared in lysates of thalassemic RBCs. Therefore, the three major membrane skeletal proteins are, to a varying degree, unstably attached in severe thalassemia. From these studies we could postulate that membrane association of abnormal or partially oxidized alpha- globin chains has a more deleterious effect on the membrane skeleton than do beta-globin chains.     

On the interaction of rabbit antithrombin III with the luminal surface of the normal and deendothelialized rabbit thoracic aorta in vitro

Pure rabbit antithrombin III was isotope labeled (with 125I or 3H) by two different methods; neither procedure caused a loss of antithrombin activity although both methods affected the affinity of the protein for Sepharose-heparin. From segments from freshly excised rabbit aorta, the uptake of isotope-labeled antithrombin III by the endothelium was rapid and saturable, although relatively small compared to the uptake of thrombin; binding of 3H-antithrombin III to the endothelium resembled that of 125I-antithrombin III. Transendothelial passage of antithrombin III into the subendothelial layers (intima-media) was slow and progressive. Endothelium binding was not affected by pretreating the vessel with either heparin, thrombin, or glycosaminoglycan-specific enzymes. Endothelium-bound antithrombin III was not selectively displaced by either heparin or thrombin. In contrast, endothelium-bound thrombin was rapidly dislodged by antithrombin III as a thrombin- antithrombin III complex. The surface of the deendothelialized aorta (ie, subjected to a balloon catheter) bound antithrombin III avidly. Pretreatment of the deendothelialized vessel with glycosaminoglycan- specific enzymes, particularly heparitinase, decreased intima-media binding by up to 80%. 125I-antithrombin III, when bound to the deendothelialized vessel surface, was actively displaced by either heparin, thrombin, or by unlabeled antithrombin III. The relatively poor binding of antithrombin III compared with that of thrombin by the endothelium in vitro supports an earlier proposal (Lollar P, Owen WG: J Clin Invest 66:1222-1230, 1980) that thrombin bound to high-affinity sites, possibly pericellular proteoglycan, of the endothelium is inactivated by plasma antithrombin III in vivo. Such a situation probably holds for large arteries at least.     

Establishing a children's orthopaedic hospital for Malawi: A review after 10 years

Background

BEIT CURE International Hospital (BCIH) opened in 2002 providing orthopaedic surgical services to children in Malawi. This study reviews the hospital''s progress 10 years after establishment of operational services. In addition we assess the impact of the hospital''s Malawi national clubfoot programme (MNCP) and influence on orthopaedic training.

Methods

All operative paediatric procedures performed by BCIH services in the 10th operative year were included. Data on clubfoot clinic locations and number of patients treated were obtained from the MNCP. BCIH records were reviewed to identify the number of healthcare professionals who have received training at the BCIH.

Results

609 new patients were operated on in the 10th year of hospital service. Patients were treated from all regions; however 60% came from Southern regions compared with the 48% in the 5th year. Clubfoot, burn contracture and angular lower limb deformities were the three most common pathologies treated surgically. In total BCIH managed 9,842 patients surgically over a 10-year period. BCIH helped to establish and co-ordinate the MNCP since 2007. At present the program has a total of 29 clinics, which have treated 5748 patients. Furthermore, BCIH has overseen the full or partial training of 5 orthopaedic surgeons and 82 orthopaedic clinical officers in Malawi.

Conclusion

The BCIH has improved the care of paediatric patients in a country that prior to its establishment had no dedicated paediatric orthopaedic service, treating almost 10,000 patients surgically and 6,000 patients in the MNCP. This service has remained consistent over a 10-year period despite times of global austerity. Whilst the type of training placement offered at BCIH has changed in the last 10 years, the priority placed on training has remained paramount. The strategic impact of long-term training commitments are now being realised, in particular by the addition of Orthopaedic surgeons serving the nation.     

Radial keratotomy. 1. The wound healing process and measurement of incisional gape in two animal models using in vivo confocal microscopy.

Using in vivo confocal microscopy, corneal wound healing was evaluated in both rabbit and cat eyes after radial keratotomy. A total of six rabbit and six cat eyes were evaluated sequentially over time for 1 mo after surgery by in vivo confocal microscopy, and quantitative measurements of changes in incisional wound gape were determined. In vivo histopathologic changes were correlated with conventional histopathologic findings in 18 rabbit and 4 cat eyes; the animals were killed at various intervals from 0-30 days after surgery. In the rabbit, in vivo corneal wound healing was characterized by the initial ingrowth of corneal epithelium followed by persistence within the wound without a marked fibrotic response. Measurement of incisional wound gape showed increasing gape from 144 +/- 32 microns on day 0 to 976 +/- 155 microns on day 26 at a distance of 2.4 mm from the optical zone. These in vivo measurements were not significantly different (P = 0.996) from those obtained using conventional histopathologic techniques which showed an incisional wound gape of 252 +/- 112 microns on day 0 and 917 +/- 216 microns on day 26 at 2.5 mm from the optical zone. In the cat eyes, healing of radial keratotomy wounds showed an initial increase in incisional wound gape from 135 +/- 56 microns on day 0 to 245 +/- 88 microns on day 7 at a distance of 2.4 mm from the optical zone. Starting at day 14 and continuing to day 30, there was a progressive decrease in incisional wound gape from 198 +/- 41 microns to 92 +/- 35 microns. Sequential, in vivo histopathologic analyses indicated that increasing incisional wound gape correlated with the retention of corneal epithelium in the wound. Initiation of decreasing incisional wound gape was associated with replacement of the incisional epithelial plug with fibroblastic tissue. These changes in the incisional wound gape observed in the cat suggest that healing of radial keratotomy wounds involves contraction of the wound in response to the ingrowth of fibroblastic cells. Furthermore, the contractile response appears to be biphasic involving a precontractile and contractile phase. Overall these data indicated that in vivo confocal microscopy provides quantitative histopathologic data on living tissue comparable with that obtained with conventional techniques on dead, fixed, and sectioned tissue. Additionally, the absence of wound fibrosis in the rabbit radial keratotomy model raises important questions as to the appropriateness of this experimental model for human radial keratotomy.     

In vivo confocal microscopy in clinical dental research: An initial appraisal

Until recently, the in vivo microscopic investigation of intraoral tissues at high resolution has been virtually impossible. Confocal microscopy enables high-resolution imaging to be achieved below semitransparent surfaces in intact living specimens, but this may still be impractical for intraoral applications because of the need to stabilize the sample. The development of a steadying objective (x 240 overall mag.) which is held against the sample surface and is focused by moving internal elements, avoids the need for fine adjustment of the living sample under the microscope to achieve a change of focus. It is therefore more comfortable and also reduces the problems of movement due to the pulse. The objective was used with a tandem scanning microscope, with images recorded via a SIT video camera. Using this system internal tooth structure (e.g. enamel prisms/adhesive restoration interfaces) and the lining cells of the gingival crevice through to the junctional epithelium may be examined. It is also possible to image the oral mucous membrane, focusing to the capillary loops in the basal layers, where streaming red blood cells can be seen. Access is limited to the anterior regions as far back as the premolar teeth. Applications could include caries research, soft and hard tissue responses to biomaterials (e.g. implants), wound healing and monitoring the effect of periodontal treatment regimens. This new technique offers numerous exciting opportunities for the microscopic investigation of many clinical operative procedures in vivo, allowing the response of the tissues to be non-destructively monitored, over time, at high resolution.