Hematopoietic stem cell transplantation for infantile osteopetrosis

Infantile osteopetrosis is a lethal disorder resulting from a severe defect in the ability of osteoclasts to resorb bone. The only therapy shown to be capable of providing lasting benefit is allogeneic hematopoietic stem cell transplantation (HCT). We report the outcome of 10 patients with infantile malignant osteopetrosis treated with HCT from an HLA A, B, DRB1 matched (n=6) or A or B locus mismatched (n=4) family member or unrelated donor at the University of Minnesota between 1978 and 1997. Eight of 10 patients achieved primary engraftment; secondary graft failure was seen in two patients. Five of 10 patients survive; three with full or partial donor chimerism and two with autologous hematological recovery. Transient or partial donor chimerism can be sufficient to correct the hematological manifestations of osteopetrosis. We recommend early referral for consideration of HCT with a related or unrelated donor as neurosensory manifestations of osteopetrosis are generally not reversible. Donor engraftment may be easier to achieve early in the course of the disease.     

HER-2/c-erbB2 is phosphorylated by calmodulin-dependent protein kinase II on a single site in the cytoplasmic tail at threonine-1172

Calmodulin-dependent protein kinase II (Cam kinase II) is known to desensitise epidermal growth factor receptor (HER-1) tyrosine kinase activity by a process involving phosphorylation at serines 1046/47 in the cytoplasmic tail. We have developed an experimental system to investigate phosphorylation of the related HER-2/c-erbB2 proto-oncogene utilising purified Cam kinase II and recombinant glutathione-S-transferase (GST) fusion proteins. The cDNA for rat Cam kinase II-alpha was transfected into human embryonic kidney (HEK) 293 fibroblasts and the expressed protein purified to homogeneity by calmodulin-agarose affinity chromatography. A GST fusion protein comprising residues 1126-1255 of HER-2 was phosphorylated by purified Cam kinase II, in contrast to a GST protein comprising residues 1005-1125. Phosphoamino-acid analysis and site-directed mutagenesis indicated that HER-2 was phosphorylated on a single site at threonine-1172 which resides within a consensus Cam kinase II phosphorylation site (RAKT). HER-2 (threonine-1172-alanine), in the form of a ligand-inducible chimaera HER-1/2, was co-transfected into HEK-293 fibroblasts with a constitutively active form of Cam kinase II, followed by in vivo labelling of these cells with 32 P-orthophosphate. Immunoprecipitation of ligand-activated receptors followed by two-dimensional phosphopeptide mapping indicated that threonine-1172 in HER-2 is a newly identified in vivo site which can be hyper-phosphorylated by constitutively active Cam kinase II. In addition, when over-expressed in HEK-293 fibroblasts, HER-1/2 (threonine-1172-alanine) showed a defect in desensitisation and underwent a more sustained EGF-induced receptor autophosphorylation compared to wild-type HER-1/2.     

Molecular recognition with C-clamp porphyrins: synthesis, structural, and complexation studies

The cholesterolaemic effect of 2 hypercholesterolaemic diets was tested in 12 rat inbred strains. Diet I is a commercial diet supplemented with 2.0% (w/w) cholesterol and 5.0% (w/w) olive oil; diet II is identical to diet I with addition of 0.5% (w/w) sodium cholate. Strains with the highest plasma cholesterol response after diet I (BN and LEW) also had the highest cholesterol response after diet II (hyperresponders, mean response > 3.5 mmol/l). In the strains DA, SHR, BC, WAG, LOU, PVG and BUF the strain mean cholesterol response remained below 1.3 mmol/l after both diets (hyporesponders). Strains F344 and OM had an intermediate cholesterol response after both diets (normoresponders, mean response between 1.3 and 3.5 mmol/l). Only in the strains LOU, PVG and SHR there appeared to be a significant higher cholesterol response after diet II when compared with the cholesterol response after diet I. In the strain WKY this difference was of a borderline significance (P = 0.052) and this strain turned from a normoresponder after diet I into a hyperresponder after diet II. Liver cholesterol levels as measured after feeding diet II for two weeks also appeared to be strain-specific. No correlation was found between the plasma cholesterol response after diet II and the liver cholesterol levels. Changes in plasma phospholipid and triglyceride levels have been measured for both diet I and diet II. For group means a correlation between the cholesterol response and the change in phospholipid levels was found (r = 0.86 for diet I, P < 0.001 and r = 0.76 for diet II, P < 0.01). No such correlation was found for triglyceride levels.     

A microbubble-powered bioparticle actuator

We present the results of a device that uses controllable microbubble actuation to manipulate bioparticles. In order to create a useful device for controlling the position of bioparticles, predictable microfluidic actuation is crucial. The goal of this project was to develop fundamental technology that can be used to manipulate single bioparticles (e.g., cells). We use a thermal bubble actuation method to accomplish this goal. Microbubbles have the advantages of relatively simple electronics and fabrication but can be difficult to control. In this paper, we describe two specific accomplishments: the use of micromachined nucleation cavities to precisely localize thermal bubbles and to achieve controllable bubble formation temperatures and bubble dissipation and the demonstration of controllable microbubbles in a new device for particle sorting.     

Protective effects of green tea extracts (polyphenon E and EGCG) on human cervical lesions

We investigated clinical efficacy of green tea extracts (polyphenon E; poly E and (-)-epigallocatechin-3-gallate [EGCG]) delivered in a form of ointment or capsule in patients with human papilloma virus (HPV) infected cervical lesions. Fifty-one patients with cervical lesions (chronic cervicitis, mild dysplasia, moderate dysplasia and severe dysplasia) were divided into four groups, as compared with 39 untreated patients as a control. Poly E ointment was applied locally to 27 patients twice a week. For oral delivery, a 200 mg of poly E or EGCG capsule was taken orally every day for eight to 12 weeks. In the study, 20 out of 27 patients (74%) under poly E ointment therapy showed a response. Six out of eight patients under poly E ointment plus poly E capsule therapy (75%) showed a response, and three out of six patients (50%) under poly E capsule therapy showed a response. Six out of 10 patients (60%) under EGCG capsule therapy showed a response. Overall, a 69% response rate (35/51) was noted for treatment with green tea extracts, as compared with a 10% response rate (4/39) in untreated controls (P<0.05). Thus, the data collected here demonstrated that green tea extracts in a form of ointment and capsule are effective for treating cervical lesions, suggesting that green tea extracts can be a potential therapy regimen for patients with HPV infected cervical lesions.     

Anti-inflammatory action of dapsone: inhibition of neutrophil adherence is associated with inhibition of chemoattractant-induced signal transduction

Dapsone has clinical utility as an anti-inflammatory agent but the mechanism of this action remains unknown. We have previously reported that dapsone inhibits beta2 integrin (CD11b/CD18)-mediated adherence of human neutrophils in vitro and now describe studies designed to discover how dapsone-mediated inhibition of this neutrophil function occurs. Results indicate that dapsone interferes with the activation or function of the G-protein (Gi type) that initiates the signal transduction cascade common to chemotactic stimuli. They also show that dapsone-mediated suppression of this pathway inhibits the generation of second messengers essential to the activation of beta2 integrin molecules, as well as respiratory and secretory functions of neutrophils exposed to chemoattractants. We propose that the inhibition of chemoattractant-induced signal transduction by dapsone suppresses neutrophil recruitment and local production of toxic respiratory and secretory products in the affected skin of dermatitis herpetiformis and other neutrophilic dermatoses.