Green tea polyphenols attenuate deterioration of bone microarchitecture in female rats with systemic chronic inflammation.

UNLABELLED: Green tea polyphenols (GTP) are promising agents for preventing bone loss. GTP supplementation sustained microarchitecture and improved bone quality via a decrease in inflammation. Findings suggest a significant role for GTP in skeletal health of patients with chronic inflammation. INTRODUCTION: This study evaluated whether GTP can restore bone microstructure along with a molecular mechanism in rats with chronic inflammation. A 2 [placebo vs. lipopolysaccharide (LPS)]× 2 [no GTP vs. 0.5% GTP (w/v) in drinking water] factorial design was employed. METHODS: Female rats were assigned to four groups: placebo, LPS, placebo + GTP, and LPS + GTP for 12 weeks. Efficacy was evaluated by examining changes in bone microarchitecture using histomorphometric and microcomputed tomographic analyses and by bone strength using the three-point bending test. A possible mechanism was studied by assessing the difference in tumor necrosis factor-α (TNF-α) expression in tibia using immunohistochemistry. RESULTS: LPS lowered trabecular volume fraction, thickness, and bone formation in proximal tibia while increasing osteoclast number and surface perimeter in proximal tibia and eroded surface in endocortical tibial shafts. GTP increased trabecular volume fraction and number in both femur and tibia and periosteal bone formation rate in tibial shafts while decreasing trabecular separation in proximal tibia and eroded surface in endocortical tibial shafts. There was an interaction between LPS and GTP in trabecular number, separation, bone formation, and osteoclast number in proximal tibia, and trabecular thickness and number in femur. GTP improved the strength of femur, while suppressing TNF-α expression in tibia. CONCLUSION: In conclusion, GTP supplementation mitigated deterioration of bone microarchitecture and improved bone integrity in rats with chronic inflammation by suppressing bone erosion and modulating cancellous and endocortical bone compartments, resulting in a larger net bone volume. Such a protective role of GTP may be due to a suppression of TNF-α.

Defining a transcriptional fingerprint of murine splenic B-cell development.

B-cell development occurs in a stepwise fashion that can be followed by the expression of B cell-specific surface markers. In this study, we wished to identify proteins that could contribute to the changes in expression of such markers. By using RNA from freshly isolated B220+ cells, we hoped to reduce the effect of artifacts that occur during the isolation and amplification steps necessary to use flow cytometry analysis-sorted subsets in microarray experiments. Analyses comparing expression patterns from B220+ 2-week bone marrow (pro-B, pre-B, immature B cells), 2-week spleen (predominantly transitional cells) and 8-week spleen (mainly mature B cells) yielded hundreds of genes. We also examined the B cell-activating factor (BAFF)-dependent effects on immature splenic B cells by comparing expression patterns in the spleen between 2-week A/J vs 2-week A/WySnJ mice, which lack functional BAFF receptor signaling. Genes that showed the expression differences between spleen and bone marrow samples were then analyzed through quantitative PCR on B-cell subsets isolated using two different sorting protocols. A comparison of the results from our study with the results from other analyses showed not only some overlap of preferentially expressed genes but also an expansion of other genes potentially involved in B-cell development.

Automated genomic sequence analysis of the three collagen VI genes: applications to Ullrich congenital muscular dystrophy and Bethlem myopathy.

INTRODUCTION: Mutations in the genes encoding collagen VI (COL6A1, COL6A2, and COL6A3) cause Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD). BM is a relatively mild dominantly inherited disorder with proximal weakness and distal joint contractures. UCMD is an autosomal recessive condition causing severe muscle weakness with proximal joint contractures and distal hyperlaxity. METHODS: We developed a method for rapid direct sequence analysis of all 107 coding exons of the COL6 genes using single condition amplification/internal primer (SCAIP) sequencing. We have sequenced all three COL6 genes from genomic DNA in 79 patients with UCMD or BM. RESULTS: We found putative mutations in one of the COL6 genes in 62% of patients. This more than doubles the number of identified COL6 mutations. Most of these changes are consistent with straightforward autosomal dominant or recessive inheritance. However, some patients showed changes in more than one of the COL6 genes, and our results suggest that some UCMD patients may have dominantly acting mutations rather than recessive disease. DISCUSSION: Our findings may explain some or all of the cases of UCMD that are unlinked to the COL6 loci under a recessive model. The large number of single nucleotide polymorphisms which we generated in the course of this work may be of importance in determining the major phenotypic variability seen in this group of disorders.

Divergence of the hyperthermophilic archaea Pyrococcus furiosus and P. horikoshii inferred from complete genomic sequences.

Divergence of the hyperthermophilic Archaea, Pyrococcus furiosus and Pyrococcus horikoshii, was assessed by analysis of complete genomic sequences of both species. The average nucleotide identity between the genomic sequences is 70-75% within ORFs. The P. furiosus genome (1.908 mbp) is 170 kbp larger than the P. horikoshii genome (1.738 mbp) and the latter displays significant deletions in coding regions, including the trp, his, aro, leu-ile-val, arg, pro, cys, thr, and mal operons. P. horikoshii is auxotrophic for tryptophan and histidine and is unable to utilize maltose, unlike P. furiosus. In addition, the genomes differ considerably in gene order, displaying displacements and inversions. Six allelic intein sites are common to both Pyrococcus genomes, and two intein insertions occur in each species and not the other. The bacteria-like methylated chemotaxis proteins form a functional group in P. horikoshii, but are absent in P. furiosus. Two paralogous families of ferredoxin oxidoreductases provide evidence of gene duplication preceding the divergence of the Pyrococcus species.

Effects of dietary selenium on UVB-induced skin carcinogenesis and epidermal antioxidant status.

Low plasma selenium levels have been linked to increased risk of non-melanoma skin cancer in humans. The present study examined the relationship between selenium level in the diet and development of skin tumors induced by ultraviolet radiation in female Skh:HR-1 hairless mice. Animals were maintained on a torula yeast-based diet containing either 0, 0.1, or 0.5 mg/kg selenium as Na2SeO3. Ultraviolet light at a dose of 90 mJ/cm2, three times weekly for 20 weeks, resulted in skin tumors in all groups. Following cessation of ultraviolet light exposure, tumors continued to increase in selenium-deficient mice and those fed only 0.1 mg/kg, but leveled off for those on 0.5 mg/kg. During the carcinogenesis process, epidermal antioxidant enzymes catalase, superoxide dismutase, and glutathione peroxidase were monitored. Selenium deficiency decreased glutathione peroxidase and resulted in an early increase in superoxide dismutase and catalase in response to ultraviolet light treatment. These results indicate that dietary Se may be an important chemopreventive agent for skin cancer.

Non-promoting effects of iron from beef in the rat colon carcinogenesis model.

Significant alarm has existed among the general public in the past few years that eating red meat may cause human colon cancer. Iron in beef has been hypothesized as one of the factors in the etiology of this cancer. The present study was designed to test the hypothesis that dietary iron solely from beef would enhance colon tumorigenesis induced in rats. Tumors were induced in Sprague-Dawley rats with 1,2-dimethylhydrazine (20 mg/kg body weight for 10 weeks). Seventy male weanling rats were randomized to two dietary treatment groups with two iron sources (very lean beef vs. iron citrate) as the factor. The rats were allowed free access to the respective diet and deionized water for 27 weeks. At termination of the study, the rats were examined for location, size and type of colon or extracolonic lesions. No significant differences were found in total incidence and number of colon tumors between the beef (51.7%, 0.8 tumors/rat) and casein (62.1%, 0.9 tumors/rat) diets, although the serum iron levels of rats fed the beef diet were higher than for those fed the casein diet. The results demonstrate that, when lean beef is used as an iron source, the risk for colon carcinogenesis is not increased.

Translocation t(15;17) in acute myelogenous leukemia with atypical megakaryoblastic features: diagnostic, clinical, and therapeutic implications.

We describe the first reported case of acute myelogenous leukemia with characteristics of megakaryoblastic differentiation and the t(15;17) chromosomal translocation, which has been associated with promyelocytic leukemia. The diagnostic, clinical, and therapeutic implications are discussed.

Born unto trouble.

We describe unexpected adverse psychological and social results in three adult men with severe Little's disease who were made more mobile by successful surgery.

Replacement of the femoral head by open operation in severe adolescent slipping of the upper femoral epiphysis.

The stages in adolescent slipping of the upper femoral epiphysis are classified in relation to treatment. The operation of open replacement of the displaced femoral head is described, and the results of a personal series of seventy-three such operations are presented. Open replacement is excellent treatment for severe chronic slipping so long as the growth plate is still open. The greater incidence of avascular necrosis in acute-on-chronic cases is probably due to damage to the blood supply of the head at the time of the acute slip or kinking of the vessels before replacement. Prolonged traction before operation may increase the risk of chondrolysis. Late onset of osteoarthritis when neither avascular necrosis nor chondrolysis has occurred may be due to misfitting of the articular cartilage because of inaccurate reduction.

Prophylactic pinning in slipped upper femoral epiphysis. Prevention of complications.

We report the complications of prophylactic pinning of slipped upper femoral epiphysis with Crawford Adams pins in 95 cases. Complications of pin placement were seen in 13.7%. Although seven hips had penetration of the joint, there were no cases of chondrolysis or avascular necrosis. Excavation of the lateral femoral cortex was required at pin removal in 12.5% of cases. Analysis of the growth around pins allowed recommendations to be made regarding pin protrusion. The use of improved fixation devices may reduce the need for multiple pins.

Open reduction of the severely slipped upper femoral epiphysis.

The results of open reduction of the severely slipped upper femoral epiphysis are reported for 115 hips with an average follow-up of 12 years 11 months (range 2 to 33 years). In 70 hips with a chronic slip and an open growth plate the incidence of complications was low: two developed avascular necrosis, five chondrolysis, and one had both. There were more complications in the 38 hips with an acute-on-chronic slip: six developed avascular necrosis, one chondrolysis, and three had both. Of the seven hips operated upon with a partially fused plate, only one did well. All these complications were obvious within the first year but there were also three hips in the series in which osteoarthritis developed between 10 and 20 years after operation.

Notoedric mange in an ocelot (Felis pardalis) from southern Texas.

Notoedric mange was diagnosed in a free-ranging adult male ocelot (Felis pardalis) found dead in April 1994 in southern Texas (USA). The emaciated carcass had no body fat. The heart was enlarged and flaccid. There was a nonpurulent serosanguineous pericardial and peritoneal exudate. Severe encrusted skin lesions and alopecia extended from the head posteriorly to the shoulders. The forelegs and feet were less severely affected. We observed a massive infection of Notoedres cati in skin scrapings. Epidermal lesions included hyperkeratosis and parakeratosis with necrotic debris and foci of acute inflammatory cells surrounding mites and their eggs in the stratum corneum. There was mild acanthosis and spongiosis of the stratum germativum, but no chronic inflammation in the dermis. This is the first confirmed case in the ocelot, but notoedric manage has been reported from the bobcat (Felis rufus) in southern Texas. Thus, notoedric mange could pose an additional threat to the already endangered remnant population of the ocelot.

Developing the clinical skills of nurses working in the field of AIDS palliative care.

The study reported here investigated the value of the Self Assessment, Identification of Need and Teaching (SAINT) system in assessing and teaching qualified nurses at work in the field of AIDS palliative care. Primary and community nurses were asked to use the SAINT system to make an assessment of their own nursing skills, identify learning needs and the teaching which they felt was required. Each wrote down their own findings and presented them individually to the researcher; one at a time and, within a group. Under both conditions these nurses were able to assess their skills, identify their needs and suggest teaching. Identification of needs and teaching were found to be more readily available when findings were presented to a group.

Pneumococcal Induced T-activation with Resultant Thrombotic Microangiopathy.

Thrombotic microangiopathies are disorders resulting from platelet thromboses forming in the microvasculature with resultant schistocyte forms. Hemolytic uremic syndrome (HUS) is a microangiopathic hemolytic anemia often complicated by acute renal failure in children. HUS is typically caused by bacterial infection, most commonly enterohemorrhagic Escherichia coli. Neuraminidase-producing organisms, such as Streptococcus pneumoniae have also been reported as potential etiologies. The pathogenesis in these cases involves cleavage of sialic acid residues from the surfaces of erythrocytes, platelets, and glomerular capillary endothelial cells, exposing the Thomsen-Friedenreich antigen, a process known as T-activation. We describe a 2-year-old girl who presented with pneumococcal pneumonia and sepsis ultimately resulting in a thrombotic microangiopathy with acute renal failure, most consistent with HUS. The patient's direct antiglobulin test was positive. Polyagglutination was observed with human adult serum, but not with umbilical cord serum. Her red blood cells (RBCs) were reactive against peanut and soybean lectins, but not Salvia sclarea or Salvia horminum lectins. These findings are consistent with T-activation. Clinicians should be cognizant of the possibility of T-activation with resultant HUS in patients infected with neuraminidase-producing bacteria. Such patients may be difficult to identify using monoclonal typing antisera, as these typically do not have anti-T antibodies. Whether such patients are at risk for transfusion-associated hemolysis is debatable.

Improvement of bone quality in gonad-intact middle-aged male rats by long-chain n-3 polyunsaturated fatty acid.

The effect of long-chain n-3 polyunsaturated fatty acid (PUFA) on bone measurements was evaluated in gonad-intact middle-aged male rats. Seven rats were killed on day 0 of dietary intervention to determine bone parameters at baseline. Experimental rats (7/group) were fed one of the following lipid treatments (g/kg diet): 167 g safflower oil + 33 g menhaden oil (N6+N3 diet, control), 200 g safflower oil (N6 diet), or 190 menhaden oil + 10 g corn oil (N3 diet). After 20 weeks of dietary treatment, all groups had lower values for peak load and ultimate stiffness in femurs compared to baseline values. Rats fed the N3 diet had the highest values for peak load, ultimate stiffness, and Young's modulus compared with those fed the N6 and control diets. Compared to baseline, all dietary treatment groups had significantly lower values for trabecular thickness and number in proximal tibia but higher values for trabecular separation and formation rate in proximal tibia and endocortical bone formation rate in tibial shaft. Compared with the control group, rats fed the N3 diet had lower values for formation rate, osteoclast number, and eroded surface in proximal tibia but higher values for periosteal mineral apposition and formation rates in tibia shaft. These findings indicate that a diet rich in long-chain n-3 PUFA mitigate aging-induced loss of bone integrity in intact middle-aged male rats through reducing bone turnover rate by suppressing both bone formation and resorption as a result of a larger net bone volume and modulating endocortical and cancellous bone compartments.

Improved molecular diagnosis of dystrophinopathies in an unselected clinical cohort.

Mutations in the DMD gene result in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). Readily available clinical tests detect only deletions of one exon or greater, which are found in approximately 60% of cases. Mutational analysis of other types of DMD mutations, such as premature stop codons and small frameshifting insertions or deletions, has historically been hampered by the large size of the gene. We have recently reported a method that allows the rapid and economical sequencing of the entire coding region of the DMD gene, and that is more sensitive than methods based on single-strand conformational polymorphism (SSCP) screening or other preliminary screening steps. Here we use single condition amplification/internal primer (SCAIP) sequencing analysis, in combination with multiplex amplifiable probe hybridization (MAPH) analysis of duplications, to report the frequency of mutations in a large cohort of unselected dystrophinopathy patients from a single clinic. Our results indicate that 7% of dystrophinopathy patients do not have coding region mutations, suggesting that intronic mutations are not uncommon. The availability of rapid and thorough mutation analysis from peripheral blood samples, along with an improved estimate of the percentage of non-coding region mutations, will be of benefit for improved genetic counseling and in identification of cohorts for clinical trials.

Effects of ascorbate on insoluble elastin accumulation and cross-link formation in rabbit pulmonary artery smooth muscle cultures.

Cultured pulmonary artery smooth muscle cells derived from the medial vessel layer of weanling rabbits were grown in the presence or absence of sodium ascorbate. The connective tissue elements insoluble elastin and collagen were identified and quantified. Formation and accumulation of alpha-aminoadipic acid gamma-semialdehyde (allysine) and the intermolecular cross-links desmosine (Des), isodesmosine (Ides), and aldol condensation product (Aldol) were evaluated from [14C]lysine pulse-chase experiments. [14C]Des, [14C]Ides, peptide-bound [14C]lysine, [14C]allysine, and [14C]Aldol were determined from amino acid analysis. The latter two components were determined after reduction with NaBH4. [14C]Proline conversion to hydroxy[14C]proline and collagenase susceptibility were used to identify and quantify collagen synthesis. Ascorbate dramatically affects insoluble elastin synthesis, accumulation, and cross-link formation. Cells grown in the presence of ascorbate synthesize and accumulate significantly less insoluble elastin than non-ascorbate cultures. Those elastin molecules which do become incorporated into the extracellular matrix in the presence of ascorbate contain a slightly elevated content of hydroxyproline and lysine and, most importantly, are turned over more rapidly.