RANKL inhibition is an effective adjuvant for docetaxel in a prostate cancer bone metastases model

BACKGROUND: Docetaxel induces an anti-tumor response in men with advanced prostate cancer (PCa); however, the side effects associated with docetaxel treatment can be severe, resulting in discontinuation of therapy. Thus, identification of an effective adjuvant therapy to allow lower doses of docetaxel is needed. Advanced PCa is typically accompanied by skeletal metastasis. Receptor activator of NFkB ligand (RANKL) is a key pro-osteoclastic factor. Targeting RANKL decreases establishment and progression of PCa growth in bone in murine models. METHODS: The efficacy of inhibiting RANKL, using a recombinant soluble RANK extracellular domain fused with the immunoglobulin Fc domain (RANK-Fc), was tested as an adjuvant therapy with docetaxel for PCa bone metastasis in a murine intra-tibial model. RESULT: The combination of RANK-Fc and docetaxel reduced tumor burden in bone greater than either treatment alone. CONCLUSION: The combination of docetaxel with a RANKL-inhibiting agent merits further investigation for treatment of advance PCa.     

A decreased subchondral trabecular bone tissue elastic modulus is associated with pre-arthritic cartilage damage.

In osteoarthritis, one postulate is that changes in the mechanical properties of the subchondral bone layer result in cartilage damage. The goal of this study was to examine changes in subchondral trabecular bone properties at the calcified tissue level in the early stages of cartilage damage. Finite element models were constructed from microCT scans of trabectilar bone from the proximal tibia of donors with mild cartilage damage and from normal donors. In the donors with cartilage damage, macroscopic damage was present only in the medial compartment. The effective tissue elastic moduli were determined using a combination of finite element models and mechanical testing. The bone tissue modulus was reduced by 60% in the medial condyle of the cases with cartilage damage compared to the control specimens. Neither the presence of cartilage damage nor the anatomic site (medial vs. lateral) affected the elastic modulus at the apparent level. The volume fraction of trabecular bone was higher in the medial compartment compared to the lateral compartment of tibiae with cartilage damage (but not the controls), suggesting that mechanical properties were preserved in part at the apparent level by an increase in the bone volume fraction. It seems likely that the normal equilibrium between cartilage properties, bone tissue properties and bone volume fraction is disrupted early in the development of osteoarthritis.     

In vitro elution of tobramycin from bioabsorbable polycaprolactone beads

OBJECTIVES: To compare the in vitro elution characteristics of tobramycin impregnated beads made of polycaprolactone (PCL) and polymethylmethacrylate (PMMA). DESIGN: Six-millimeter PCL and PMMA beads with 6% tobramycin were formed and placed in phosphate-buffered saline or newborn calf serum and incubated at room temperature or 37 degrees C. Aliquots were taken at intervals for eight weeks. Tobramycin levels were determined by fluorescent assay and antibacterial efficacy was assessed by measuring the zones of inhibition against Staphylococcus aureus and Pseudomonas aeruginosa on agar diffusion plates. RESULTS: Tobramycin elution rates at room temperature were similar up to three weeks. At three weeks, elution rates from PCL beads were twice those from PMMA beads, and at eight weeks, elution from PCL was quadruple that from PMMA. At 37 degrees C, tobramycin elution rates from PCL were eight times greater than those from PMMA by eight weeks. Total tobramycin eluted from PCL beads was 38.9% and 20% in PMMA beads. All samples showed bacteriostatic activity against S. aureus and P. aeruginosa at eight weeks. CONCLUSIONS: These in vitro results show that PCL has superior antibiotic elution characteristics compared with PMMA, and this may translate into a more effective antibiotic delivery vehicle. In addition, PCL is a bioabsorbable polymer, which may decrease the need for a second surgical procedure to remove retained beads.     

Growth factor may decrease muscle atrophy secondary to denervation

Despite modern microsurgical techniques, functional outcomes following brachial-plexus reconstruction and peripheral-nerve repair are usually unsatisfactory, because irreversible muscle atrophy develops before reinnervation occurs. Insulin growth factor-1 (IGF-1) has been shown to improve muscle regeneration after injury, and may have a role in muscle preservation following denervation. This study evaluated the histologic, immunohistochemical, and electrophysiologic differences between normal and denervated muscle over an 8-week time period, and also evaluated the effects of injecting IGF-1 into denervated muscle. Denervated mice gastrocnemius muscles demonstrated a decrease in muscle diameter, a decrease in muscle weight, early nuclear proliferation, and a decrease in fast twitch and maximum tetanic strength, compared to normal gastrocnemius muscle up to 8 weeks following denervation. Four weeks after denervated muscle was injected with IGF-1 at time zero, however, relative preservation of muscle diameter and weight, and maintenance of electrophysiologic contractile properties were observed. These preliminary data suggest that IGF-1 may prevent muscle atrophy secondary to denervation.     

Hyaluronan and Hyaluronan-Binding Proteins Accumulate in Both Human Type 1 Diabetic Islets and Lymphoid Tissues and Associate With Inflammatory Cells in Insulitis

Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that is present in pancreatic islets, but little is known about its involvement in the development of human type 1 diabetes (T1D). We have evaluated whether pancreatic islets and lymphoid tissues of T1D and nondiabetic organ donors differ in the amount and distribution of HA and HA-binding proteins (hyaladherins), such as inter-α-inhibitor (IαI), versican, and tumor necrosis factor–stimulated gene-6 (TSG-6). HA was dramatically increased both within the islet and outside the islet endocrine cells, juxtaposed to islet microvessels in T1D. In addition, HA was prominent surrounding immune cells in areas of insulitis. IαI and versican were present in HA-rich areas of islets, and both molecules accumulated in diabetic islets and regions exhibiting insulitis. TSG-6 was observed within the islet endocrine cells and in inflammatory infiltrates. These patterns were only observed in tissues from younger donors with disease duration of <10 years. Furthermore, HA and IαI amassed in follicular germinal centers and in T-cell areas in lymph nodes and spleens in T1D patients compared with control subjects. Our observations highlight potential roles for HA and hyaladherins in the pathogenesis of diabetes.     

Human acute leukemia cell line with the t(4;11) chromosomal rearrangement exhibits B lineage and monocytic characteristics

A cell line, designated RS4;11, was established from the bone marrow of a patient in relapse with an acute leukemia that was characterized by the t(4;11) chromosomal abnormality. The cell line and the patient's fresh leukemic cells both had the t(4;11)(q21;q23) and an isochromosome for the long arm of No. 7. Morphologically, all cells were lymphoid in appearance. Ultrastructurally and cytochemically, approximately 30% of the cells possessed myeloid features. The cells were strongly positive for terminal deoxynucleotidyl transferase. They were HLA-DR positive and expressed surface antigens characteristic for B lineage cells, including those detected by anti-B4, BA-1, BA-2, and PI153/3. Immunoglobulin gene analysis revealed rearrangements of the heavy chain and kappa chain genes. The cells lacked the common acute lymphoblastic leukemia antigen and antigenic markers characteristic of T lineage cells. The cells reacted with the myeloid antibody 1G10 but not with other myeloid monoclonal antibodies. Treatment with 12-O-tetradecanoyl- phorbol-13-acetate induced a monocyte-like phenotype demonstrated by cytochemical, functional, immunologic, and electron microscopic studies. The expression of markers of both early lymphoid and early myeloid cells represents an unusual phenotype and suggests that RS4;11 represents a cell with dual lineage capabilities. To our knowledge, RS4;11 is the first cell line established from t(4;11)-associated acute leukemia.     

Use of multiple T cell-directed intact ricin immunotoxins for autologous bone marrow transplantation

The monoclonal antibodies (MoAb) T101, G3.7, 35.1, and TA-1 were conjugated to intact ricin using a thioether linkage. These MoAb detect, respectively, the CD5[gp67], CD7[p41], CD2[p50], and [gp95, 170] determinants that are found in the vast majority of cases of T cell acute lymphocytic leukemia (T-ALL). The resulting immunotoxins (ITs) and an equimolar mixture of these ITs were evaluated as potential purgative reagents for autologous transplantation in T-ALL. Leukemic cell lines were used to compare the kinetics of protein synthesis inactivation mediated by each IT. The cells were treated with IT in the presence of lactose in order to block the native binding of ricin. The observed rates of protein synthesis inactivation correlated with target antigen expression detected by fluorescence-activated cell sorter analysis. Of the four ITs, T101-ricin (T101-R) exhibited the fastest rate of inactivation, followed in order by G3.7-ricin, TA-1-ricin, and 35.1-ricin. At concentrations greater than 300 ng/mL, a cocktail containing an equimolar amount of all four ITs (referred to as the four- IT cocktail) exhibited kinetics that were as fast or faster than those of T101-R. The long-term cytotoxic effects of individual ITs and the four-IT cocktail were evaluated using a sensitive clonogenic assay. Each IT was specifically cytotoxic and inhibited 1 to 4 logs of clonogenic leukemic cells at doses (300 to 600 ng/mL) that can be used clinically. The four-IT cocktail was highly cytotoxic; a concentration of 300 ng/mL inhibited greater than 4 logs of leukemic cells while sparing the majority of committed (CFU-GM, CFU-E) and pluripotent (CFU- GEMM) hematopoietic stem cells. The determination of both short-term kinetics of protein synthesis inactivation and longer-term inhibition of clonogenic growth allowed new insight into cell killing by IT. Our results suggest that ITs continue to act on clonogenic target cells for a period of three to five days. Interestingly, the four-IT cocktail was not as potent against clonogenic leukemic cells as T101-R alone, although it exhibited kinetics of protein synthesis inhibition that were as fast as those of T101-R alone. This finding suggests that internalized ITs may differ in the length of time they remain active within the cell. Our results also demonstrate the importance of using several different assays to evaluate IT reagents.     

Influx of [3H,14C]cholesterol-labelled lipoprotein into re-endothelialized and de-endothelialized areas of ballooned aortas in normal-fed and cholesterol-fed rabbits

The entry of [3H]- and [3H,14C]cholesterol-labelled lipoprotein into de-endothelialized and re-endothelialized areas of balloon-injured rabbit aortas was studied in normal-fed and cholesterol-fed rabbits. Studies were carried out 11-15 weeks after the initial injury when endothelial regeneration involved approximately half of the aortic area. The entry into the aorta of 3H-labelled free and ester cholesterol in lipoprotein over a 72-h period was studied following the ingestion of a single dose of 3H-labelled cholesterol. The entry of double labelled [3H,14C]cholesterol-labelled lipoprotein was also studied over a 6-h period following the injection of plasma from donor rabbits. The accumulation of cholesterol and cholesterol ester in the aorta in both the normal- and cholesterol-fed rabbits was significantly greater for the re-endothelialized (white) areas than for the de-endothelialized (blue) areas or the sham-operated aortas. Where the rabbits were cholesterol-fed 4-10 times the amount of cholesterol accumulated in re-endothelialized intima compared to normal intima. Both entry (micrograms/day/100 mg wet weight aortic intima) and clearance (mu 1 plasma/day/cm2) of free and ester cholesterol were increased in the neointima compared with the normal intima for both normal-fed and cholesterol-fed rabbits. Hydrolysis of cholesterol ester occurred in the neointima and was greater than in the corresponding de-endothelialized area but less than for the sham-operated intima. Synthesis of cholesterol ester was minimal in all areas. Removal of labelled cholesterol and cholesterol ester from the intima during a 20-h efflux period following the initial 72-h loading period indicated that for aortas of both normal-fed and cholesterol-fed rabbits, there was greater removal for normal intima than for either re-endothelialized or de-endothelialized intima. However, no clear difference between the blue and white areas was observed. It is concluded that the accumulation of cholesterol in neointima after balloon injury is associated with a marked increase in permeability to lipoprotein of the neointima as well as to possible binding of lipoprotein to glycosaminoglycan in the artery.     

Enzyme-linked immunosorbent assays for the detection of adhesion factor antigens of enterotoxigenic Escherichia coli

Two hundred forty-four specimens of Escherichia coli isolated in Bangladesh and Thailand and identified as enterotoxin producers were tested for the presence of adhesion antigens by mannose-resistant hemagglutination, immunodiffusion, and enzyme-linked immunosorbent assays (ELISAs). Specific antisera to the antigens colonization factor antigen (CFA)/I, CFA/II (consisting of coli surface antigens [CS] 1, 2, and 3), and putative colonization factor antigen (PCF) 8775 (consisting of CS4, 5, and 6) were used in immunodiffusion tests and ELISAs. The results showed that the antigens could be detected in more strains by ELISA than by immunodiffusion. Twenty-nine percent of specimens of E. coli from Thailand and 47% from Bangladesh carried an adhesion antigen. Many of the strains had lost the ability to produce enterotoxins. Forty percent of strains from Thailand and 64% from Bangladesh that were still enterotoxigenic carried adhesion factors. These antigens were found on strains with heat-stable or heat-stable and heat-labile enterotoxin but not on strains producing only heat-labile enterotoxin. PCF8775 antigens were associated mainly with strains from Bangladesh, where 10 strains that produced only CS6 were detected.     

Quantitative assessment of the role of O6-methylguanine in the initiation of carcinogenesis by methylating agents.

Induction of transformation, cell lethality, and DNA lesions were quantitatively compared in Syrian hamster embryo cells (HEC) treated with three different methylating agents: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N-nitrosourea (MNU), or methyl methanesulfonate (MMS). Each induced transformation in a dose-dependent manner. On a molar basis, MNNG was approximately equal to 100- and 500-fold more effective than MNU and MMS, respectively. For each carcinogen the induction and repair of O6- and N7-methylguanine (O6- and N7-MeGua) relative to total guanine content was compared. At concentrations that induced equivalent transformation frequencies, the induction of O6-MeGua was the same for all three carcinogens, but N7-MeGua induction was 30-fold higher with MMS than with MNNG or MNU. The capacity to repair methylation lesions in HEC is limited because only between 50% and 70% of both O6- and N7-MeGua lesions were removed from the DNA within 24 hr after treatment, independent of methylating carcinogen. No consistent effect on either the rate of DNA replication or the size distribution of nascent strands correlated with O6-MeGua induction. These data support the hypothesis that O6-MeGua is the critical lesion for initiation of carcinogenesis by methylating agents. The frequency of transformation relative to O6-MeGua induction is 40- to 750-fold more than that of mutation. Based on the quantitative data for induction of O6-MeGua and transformation, the target size for initiation of carcinogenesis was calculated as a minimum of 10(4) nucleotides. This suggests that one of many genes can initiate carcinogenesis or that initiation is not the result of a single base mutation.