Defective pro alpha 2(I) collagen synthesis in a recessive mutation in mice: a model of human osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a heritable disorder of connective tissue associated with fractures, osteopenia, and short stature. OI results from mutations affecting the pro alpha 1 or pro alpha 2 gene of type I collagen. We describe a strain of mice with a nonlethal recessively inherited mutation (oim) that results in phenotypic and biochemical features that simulate moderate to severe human OI. The phenotype of homozygous oim mice includes skeletal fractures, limb deformities, generalized osteopenia, and small body size. Their femurs are smaller and demonstrate marked cortical thinning and fewer medullary trabeculae than those of wild-type mice. Breeding studies show the mutation is inherited in most crosses as a single recessive gene on chromosome 6, near the murine Cola-2 gene. Biochemical analysis of skin and bone, as well as isolated dermal fibroblast cultures, demonstrate that alpha 1(I) homotrimeric collagen accumulates in these tissues and is secreted by fibroblasts. Short labeling studies in fibroblasts demonstrate an absence of pro alpha 2(I) collagen chains. Nucleotide sequencing of the cDNA encoding the COOH-propeptide reveals a G deletion at pro alpha 2(I) nucleotide 3983; this results in an alteration of the sequence of the last 48 amino acids. The oim mouse will facilitate the study of type I collagen-related skeletal disease.     

Ultrastructural studies of bones from patients with osteogenesis imperfecta

Bone samples from patients suffering from osteogenesis imperfecta (OI) types I, II, III and IV, as well as normal controls, were studied by scanning (SEM) and transmission electron microscopy (TEM). SEM views of normal bone at low magnification show coherent structure, with regular striations due to a lamellar plywood-like arrangement of the mineralized collagen fibrils. Compact lamellar bone was also found in various OI specimens, but in limited disconnected regions separated by open spaces. Furthermore, some OI, but not normal, bones have regions of loose unconnected fibers and others of apparently abnormally dense mineral deposition. High resolution TEM studies of OI bone fragments have served to elucidate the structures of these different textures. There appears to be a substantial, though reduced, proportion of normal lamellar bone even in quite severe OI. However, the regions of loose fibers are largely unmineralized and probably contain abnormal collagen. Other regions are overmineralized, with generally small unorganized apatite crystals deposited onto fibril surfaces or in separate clusters. These structural abnormalities, together with the paucity of normal bone, may explain the fragility of OI bones.     

Dietary fat and sex modify heterozygote effects of the rat fatty (fa) allele

Rats carrying one copy of the obesity gene fa may exhibit intermediate phenotypes between lean (+/+) and homozygous mutants (fa/fa). Previous data suggested to us that fa heterozygotes may be more sensitive than wild-type rats to high fat diets. To test this hypothesis, we generated +/+ and fa/+ rats and fed them diets containing 12% or 48% energy as fat for 7 wk. Energy efficiency was significantly greater in males than in females and in high fat-fed vs. low fat-fed rats. Perirenal fat pad weights were significantly greater in males than in females, in high fat-vs. low fat-fed rats and in fa/+ vs. +/+ rats. Adipose and soleus plasma membrane calcium-ATPase concentrations were significantly lower in rats fed the high fat diet. This protein was also lower in soleus of fa/+ rats compared with +/+ rats. There were significant diet x genotype interactions such that the high fat diet had the greatest effect on fat pads and calcium-ATPase in fa/+ rats. The results of the present study show heterozygote effects of the fa allele and suggest that these effects may be modulated by both sex-related factors and dietary manipulation.     

Thymus transplantation, a critical factor for correction of autoimmune disease in aging MRL/+mice

MRL/MP-+/+ (MRL/+) mice develop pancreatitis and sialoadenitis after they reach 7 months of age. Conventional bone marrow transplantation has been found to be ineffective in the treatment of these forms of apparent autoimmune disease. Old MRL/+ mice show a dramatic thymic involution with age. Hematolymphoid reconstitution is incomplete when fetal liver cells (as a source of hemopoietic stem cells) plus fetal bone (FB; which is used to recruit stromal cells) are transplanted from immunologically normal C57BL/6 donor mice to MRL/+ female recipients. Embryonic thymus from allogeneic C57BL/6 donors was therefore engrafted along with either bone marrow or fetal hematopoietic cells (FHCs) plus fragments of adult or fetal bone. More than seventy percent of old MRL/+ mice (> 7 months) that had been given a fetal thymus (FT) transplant plus either bone marrow or FHCs and also bone fragments survived more than 100 days after treatment. The mice that received FHCs, FB, plus FT from allogeneic donors developed normal T cell and B cell functions. Serum amylase levels decreased in these mice whereas they increased in the mice that received FHCs and FB but not FT. The pancreatitis and sialoadenitis already present at the time of transplantations were fully corrected according to histological analysis by transplants of allogeneic FHCs, FB and FT in the MRL/+ mice. These findings are taken as an experimental indication that perhaps stem cell transplants along with FT grafts might represent a useful strategy for treatment of autoimmune diseases in aged humans.     

Changes of bone-perfusion in osteosynthesis of the femur with a marrow-nail (author's transl)

The perfusion of the bone in the hind leg after osteosynthesis (nailing of the bone-marrow) was studied. In 11 shepherd dogs (bastards) an osteotomy of the femur was done; it was treated with a marrow-nail without boring the marrow-cavity. With the "tracer-microsphere"-method the perfusion of femur, tibia and talus of both hind legs was measured. Measurements were performed before and after surgery, in 10 dogs 2 weeks and in 8 dogs 6 weeks after surgery. Immediately after the operation the perfusion was reduced considerably in all the examined bones of the operated leg. Two weeks later the perfusion was increased in all bones of both hind limbs. In the cancellous bone of the femur the perfusion reached the original preoperative values after 6 weeks; in cortical bone a further increase of the perfusion was noted. This increase was most marked in the cortical bone of the operated femur; it was less in the cortical bone of the other bones.     

Heterozygous osteopetrotic (op) mutation reduces atherosclerosis in LDL receptor- deficient mice

Previous studies of osteopetrotic (op) mice lacking macrophage colony-stimulating factor (M-CSF) have revealed an inhibition of atherosclerosis development in the apolipoprotein E (apo E)-deficient model and in a diet-induced model. Using LDL receptor-deficient mice, we now show that atheroma development depends on M-CSF concentration, as not only did homozygous osteopetrotic (op/op) mice have dramatically reduced lesions (approximately 0.3% of control lesion size) but heterozygous (op/+) mice had lesions < 1% of controls. Mice heterozygous for the op mutation (op/+) had plasma levels of M-CSF about half those in controls (+/+). The finding that an approximately 2-fold reduction in M-CSF expression reduced lesion size approximately 100-fold suggests the requirement for a threshold level of M-CSF. The effect of M-CSF on atherosclerosis did not appear to be mediated either by changes in plasma lipoprotein levels or alterations in the number of circulating monocytes, since both op/op and op/+ mice exhibited higher levels of atherogenic lipoprotein particles and (op/+) mice showed a near normal number of circulating monocytes. LDL receptor-null littermates of genotypes from op/op, op/+, to +/+ showed monocyte differentials of approximately 4.5, 8, and 10%, respectively. Taken together, these results suggest that the effects of M-CSF on atherogenesis may not be mediated by expression of M-CSF systemically or by modulation of the number of circulating monocytes. These studies support the conclusion that M-CSF participates critically in fatty streak formation and progression to a complex fibrous lesion.     

The lymphopenia (lyp) gene controls the intrathymic cytokine ratio in congenic BioBreeding rats

The lymphopenia gene (lyp) on rat chromosome 4 is closely linked to autoimmune diabetes in the BioBreeding (BB) rat. Lyp controls the number of peripheral lymphocytes by reducing T cells of the RT6+ phenotype by almost 90%. Following nine cycle of marker-assisted cross-intercross breeding we have developed congenic lyp/lyp, lyp/+ and +/+ (wildtype) rats on the background of DR rats. Prediabetic and insulitis free lyp/lyp, lyp/+ and +/+ rats were used to determine the effect of lyp on cytokine expression in the thymus. In situ hybridization of thymus cryosections showed that the interferon gamma (IFN gamma) mRNA expression was highest in lyp/lyp rats and the hybridization signal was restricted to the medullary compartment. The frequency of IFN gamma and interleukin (IL)-10 mRNA expressing cells in isolated thymocytes determined by quantitative image analysis, demonstrated an increased IFN gamma: IL-10 ratio in thymocytes from lyp/lyp homozygotes compared to lyp/+ and +/+ rats. This confirmed a lyp gene dose-dependent segregation of the IFN gamma high phenotype. Recombinant human glutamic acid decarboxylase (GAD65) increased the number of IFN gamma and IL-10 mRNA expressing thymocytes after in vitro culture. We conclude that the quantitative ratio of cytokine producing thymocytes is associated with the lyp genotype. These potentially autoreactive thymocytes may explain the establishment of beta-cell directed autoimmunity in the BB rat despite peripheral lymphopenia.     

Effect of 5-hydroxytryptamine on bioelectric properties of airway epithelial cells in culture

Waardenburg syndrome type 1 is caused by mutations in PAX3. Over 50 human PAX3 mutations that lead to hearing, craniofacial, limb, and pigmentation anomalies have been identified. A PAX3 mutant allele, segregating in a family, can show reduced penetrance and variable expressivity that cannot be explained by the nature of the mutation alone. The Mus musculus Pax3 mutation Spd (Splotch-delayed, Pax3Spd), coisogenic on the C57BL/6J (B6) genetic background, produces in heterozygotes a white belly spot with 100% penetrance and very few other anomalies. By contrast, many Spd/+ BC1 progeny [F1 female Spd/+ (female Spd/+ B6 x male +/+ Mus spretus) x male +/+ B6] exhibit highly variable craniofacial and pigmentary anomalies. Of the BC1 Spd/+ progeny, 23.9% are estimated to be nonviable, and 32.1% are nonpenetrant for the white belly spot. The penetrance and expressivity of the Spd/+ genotype are controlled in part by the genetic background and the sex of the individual. A minimum of two genes interact with Spd to influence the craniofacial features of these mice. One of these genes may be either X-linked or sex-influenced, while the other is autosomal. The A-locus (Agouti) or a gene closely linked to A also plays a role in determining craniofacial features. At least one additional gene, possibly the A-locus or a gene linked to A, interacts with Spd and determines the presence and size of the white belly spot. The viability of BC1 mice is influenced by at least three factors: Spd, A-locus alleles or a gene closely linked to the A-locus, and the sex of the mouse. These BC1 mice provide an opportunity to identify genes that interact with and modify the expression of Pax3 and serve as a model to identify the genes that modify the expression of human PAX3 mutations.     

The impact of muscular dystrophy on limb bone growth and scaling in mice

Muscular loading affects bone growth and the factors determining size and shape. However, it is not known what epigenetic impact muscular dystrophy (dystrophia muscularis) has on limb bone growth or ontogenetic scaling. To assess the effects of two types of muscular dystrophy (genotypes dy/dy and dy2J/dy2J) on limb bone growth, we measured lengths and widths of the right humerus, femur and tibia, and lengths of the ulna and radius from dorsal/ventral radiographs of mice taken over a period of 270 days. Radiographs were taken approximately 3 times a week, and the sampling frequency was gradually reduced to once a month. We plotted measurements from each individual against time and fit a Gompertz equation to the growth of each bone. Parameters of the equation were compared using ANOVA across genotypes and between sexes. Slopes of length versus width were calculated for the limb bones of each individual using linear regression. Slope differences among genotypes and between sexes were tested using ANOVA. Control and dy2J values were significantly longer than those of dy mice in all bones, but there was considerable variation across genotypes for the various width measurements. Sexual dimorphism was found in several measurements, where males were always larger than females. There were few significant differences in limb scaling (lengths vs. widths) among genotypes and almost no scaling differences between sexes despite the size differences. Differences among widths suggest that muscular dystrophy affects different parts of limb bones in different ways. This may be the effect of the type and number of muscular attachments, as well as the usage of the limb. The sexually dimorphic measurements suggest that there are size differences in the skeleton between sexes, regardless of the genotype. Our ontogenetic allometry results indicate that size is affected by the muscular dystrophic condition and by sexual dimorphism, while shape remains largely unchanged.     

Effect of hydrocortisone on total body calcium in rats

Administration of 5 mg. of hydrocortisone acetate to rats every other day for 2 weeks resulted in growth retardation and weight loss as indicated by body weights of experimental animals, which averaged 33 per cent lower than those of the controls, and a significant decrease in the length of the tibiae and femurs (p less than 0.01 for treated vs controls). However, despite the smaller size of the treated animals, the values for total body calcium (TBCa) and the calcium in the tibia and femur did not differ significantly from control values. Thus, there was more calcium per unit length of bone, resulting in an increase in the skeletal density of treated rats. This finding was confirmed by x-ray examination of these bones. The net intestinal absorption of calcium (rate of initial entry) calculated from plasma levels following an oral and intravenous dose of 47Ca and 85Sr, respectively, was not significantly different in hydrocortisone-treated rats compared to controls. This would indicate that the rate of intestinal absorption of calcium is unimpaired despite the administration of massive doses and corticosteroids. When the animals were placed on a calcium-deficient diet, both TBCa and tibia and femur calcium levels were decreased. Subsequent administration and hydrocortisone did not alter the calcium values. The results of this study are compatible with the hypothesis that hydrocortisone promotes weight loss, retards growth, but inhibits the rate of bone resorption.     

Physical activity and bone mass: exercises in futility?

Growing bone responds to low or moderate exercise through significant additions of new bone in both cortical and trabecular moieties and results in adaptation through periosteal expansion and endocortical contraction. Intracortical activation frequency declines in growing bone in response to exercise, reducing porosity and the remodelling space. These adaptations can be maintained into and throughout adulthood. Young bones have a greater potential for periosteal expansion than aging bone, allowing them to adapt more rapidly and efficiently to an acute need for increased strength, but a threshold level of activity exists above which some bones respond negatively by suppressing normal growth and modelling activity, reducing geometric, mechanical and material properties in cortical and trabecular bone. From cross-sectional studies, differences in bone mass between exercising and non-exercising adults are generally less than 10%, but do not account for exercise history which may be very important, and often fail to consider important confounding variables. There is sufficient longitudinal data to demonstrate that moderate to intensive training can bring about modest increases of about 1-3% in bone mineral content (BMC) of men and premenopausal women. In young adults very strenuous training may increase BMC of the tibia up to 11% and its bone density (BD) by 7%, but may represent periosteal woven bone formation in response to excessive strain. Some evidence shows that exercise can also add bone mass to the post-menopausal skeleton, although the amounts are site-specific and relatively modest. Increases as high as 5-8% can be found after 1-2 years of intensive exercise, but additions of bone to the femur and radius are generally less than 2%, well within the range of the remodelling space and measurement precision. Although increases in bone mass of the post-menopausal skeleton may be extremely modest, physical activity is important to preserve bone mass and muscle function. Detraining reduces any bone mass increase to pre-existing values so that long-term benefits are only retained with continuing exercise. Most importantly, the amount of bone gain that can be achieved appears dependent primarily on the initial bone mass suggesting that individuals with extremely low initial bone mass may have more to gain from exercise than those with moderately reduced bone mass.     

Poststorage diastolic abnormalities of heart transplants: is vascular dysfunction or myocardial contracture the culprit?

We tested the hypothesis that mast cells contribute to platelet-activating factor (PAF)-induced airways hyperreactivity and hyperpermeability in mice. Airways reactivity to acetylcholine (ACh) and lung permeability to Evans blue (EB) dye were measured before and after PAF challenge in genetically mast cell-deficient (WBB6F1 W/Wv) and normal congenic (WBB6F1 +/+) mice, as well as mast cell-reconstituted (BMT W/Wv) mice. In addition, prostaglandin D2 (PGD2), a mast cell-specific mediator, was measured in the bronchoalveolar lavage (BAL) from +/+ and W/Wv mice to determine if lung mast cell activation was a consequence of PAF challenge. Genetically PAF-sensitive AKR/J mice were also treated with the mast cell stabilizer nedocromil prior to assessment of PAF effects on ACh reactivity. Intravenous PAF (10 micrograms/kg) induced a significant (P < 0.05) increase in airways reactivity to ACh (25 micrograms/kg) in both +/+ (371 +/- 52%) and W/Wv (122 +/- 24%) mice. There was a significantly greater increase in +/+ compared with W/Wv mice. PAF-induced hyperreactivity to ACh in BMT W/Wv mice (191 +/- 44%) was significantly (P < 0.05) greater than age-matched W/Wv mice (80 +/- 16%), but not significantly different from age-matched +/+ mice (153 +/- 44%). PAF (10 micrograms/kg) also significantly (P < 0.5) increased lung permeability in +/+ and W/Wv mice, but there was no significant difference between groups. BAL PGD2 increased significantly in +/+ mice following PAF challenge (559 +/- 24 ng/ml) compared with vehicle controls (152 +/- 8 pg/ml). There was no significant increase in BAL PGD2 from W/Wv mice. Nedocromil pretreatment significantly (P < 0.05) decreased PAF-induced hyperreactivity in AKR/J mice but not in W/Wv mice (P > 0.05). We conclude that mast cells contribute significantly to PAF-induced hyperreactivity but not hyperpermeability in mice.     

Impaired cutaneous immune responses in Thy-1-deficient mice

Thy-1 is a cell surface glycoprotein expressed mainly on brain and lymphoid tissue. Although the functions of Thy-1 are incompletely understood, evidence exists that Thy-1 participates in T cell activation. To examine the functional role of Thy-1 in cutaneous immune responses in vivo, Thy-1 gene-targeted mice (Thy-1-/-) and wild-type mice (Thy-1+/+) were immunized with the hapten oxazolone. After challenge with oxazolone, contact hypersensitivity responses in Thy-1-/- mice were reduced by 25% compared with Thy-1+/+ mice. Likewise, irritant dermatitis induced by croton oil was also decreased. In addition, Thy-1-/- mice showed a significantly reduced delayed-type hypersensitivity response after injection of allogeneic spleen cells into the hind footpads of allosensitized animals when compared with Thy-1+/+ mice. Moreover, proliferative responses to immobilized anti-CD3 were decreased in peripheral Thy-1-/- lymphocytes; this decrease was associated with a significantly reduced intracellular Ca2+ influx and protein tyrosine phosphorylation, indicating impairment of early lymphocyte activation. In contrast, the T cell proliferation induced by mitogens was normal, suggesting that Thy-1 expression weakly contributes to TCR-mediated T cell activation. Epidermal Langerhans cells and bone marrow-derived dendritic cells from Thy-1-/- mice exhibited a normal expression of costimulatory surface molecules as well as an unaltered ability to stimulate allogeneic T cells. Taken together, these findings demonstrate that a lack of Thy-1 expression does not generally compromise the immune system; however, Thy-1 expression may be involved in the fine-tuning of T cell-mediated immune responses.     

Deficient differentiation of mast cells in the skin of mi/mi mice. Usefulness of in situ hybridization for evaluation of mast cell phenotype

The staining property of skin mast cells changed from Alcian blue+/berberine sulfate- to Alcian blue+/berberine sulfate+ in the skin of normal (+/+) and Wv/Wv mice. In contrast, this change did not occur in the skin of mi/mi mice. Heparin content and histamine content per a mi/mi skin mast cell were estimated to be 34% and 18% those of a +/+ skin mast cell, respectively. The low heparin content of mi/mi skin mast cells seemed to be consistent with the Alcian blue+/berberine sulfate- staining property. Expression of genes encoding mast cell-specific proteolytic enzymes was examined by Northern blotting and in situ hybridization. Messenger RNA of mast cell carboxypeptidase A was expressed most of all by +/+, Wv/Wv, and mi/mi skin mast cells, but mRNA of mouse mast cell protease (MMCP)-6 was expressed by approximately a half of +/+ and Wv/Wv skin mast cells and by only 3% of mi/mi skin mast cells. A significant amount of MMCP-2 mRNA was not expressed in the skin of all +/+, Wv/Wv and mi/mi mice. This shows the presence of at least three phenotypes in skin mast cells of mice: berberine sulfate+/MMCP-6+, berberine sulfate+/MMCP-6-, and berberine sulfate-/MMCP-6-. The in situ hybridization of mRNA of mast cell-specific proteolytic enzymes seemed to be useful to describe abnormalities of mast cell differentiation in the skin of mi/mi mice.     

Leptin normalizes the impaired response of proinsulin mRNA to long chain fatty acids in heterozygous Zucker diabetic fatty rats

To determine if underleptinization of islets of Zucker diabetic fatty (ZDF) rats is the proximal cause of their inability to compensate for obesity, we compared the proinsulin/beta-actin mRNA ratio in heterozygous (fa/+) ZDF rats with that of wild-type (+/+) and homozygous (fa/fa) ZDF rats. In +/+ islets cultured with 2 mM free fatty acids (FFA) the proinsulin mRNA ratio rose 2.4-fold at 12 h. In fa/+ islets, the ratio rose only 65% above normal. There was no change in fa/fa islets. The presence of leptin (20 ng/ml) in the culture medium increased the FFA-induced response of proinsulin mRNA of fa/+ islets to that of +/+ islets while reducing FFA incorporation into triglycerides. The leptin-induced improvement in the proinsulin mRNA response was independent of any changes in glucose usage. These findings support a causal relationship between diminished leptin action on islets and the impaired beta-cell response to FFA in ZDF rats.     

Mucociliary clearance in cystic fibrosis knockout mice infected with Pseudomonas aeruginosa

In this study, we examined whether mucociliary clearance differed between cystic fibrosis (CF) knockout mice and wildtype controls. Additionally, we investigated whether infection with Pseudomonas aeruginosa, a common pathogen in the CF lung, affected this important host defence mechanism. Ciliary beat frequency (fcb) and particle transport (PT) were recorded using an in vitro lung explant preparation. Measurements were made from uninfected cystic fibrosis transmembrane conductance regulator (CFTR) knockout (-/-) mice and littermate controls (+/+) and compared to measurements from infected animals. While there were no differences detectable in fcb between CFTR -/- mice and their +/+ controls either in the presence or absence of P. aeruginosa, PT rates were different between these groups; interestingly, PT rates appeared dependent on both CFTR and infection status, with uninfected CFTR +/+ animals demonstrating higher rates of PT than their -/- littermates, while CFTR +/+ P. aeruginosa-infected mice demonstrated lower PT than knockout mice. These data demonstrate differences in mucociliary clearance between cystic fibrosis transmembrane conductance regulator knockout mice and controls, and further that Pseudomonas aeruginosa infection affects mucociliary clearance in the peripheral airways of mice. Additionally, the observed differences in particle transport suggest that cystic fibrosis transmembrane conductance regulator knockout mice demonstrate different mucociliary responses to infection.     

Superoxide-mediated cytotoxicity in superoxide dismutase-deficient fetal fibroblasts

To investigate the roles of CuZn superoxide dismutase (CuZnSOD) and Mn superoxide dismutase (MnSOD) in oxygen radical-mediated cytotoxicity and to distinguish the actions of these two enzymes, fetal fibroblasts were derived from mouse fetuses that are either deficient in CuZnSOD (Sod1-/+ and -/-) or MnSOD (Sod2-1+ and -/-) for in vitro studies. Whereas the phenotype of the Sod1 mutant animals did not differ from that of their normal littermates, the growth of Sod1-/- fetal fibroblasts was only 25% of that of the -/+ and +/+ cells. On the other hand, although almost all homozygous Sod2 mutant animals (-/-) died within 10 days after birth, cultivation of Sod2-/- fetal fibroblasts was possible and their growth was about 60% that of -/+ and +/+ cells. When cultured cells were subjected to treatment with paraquat to assess their ability to grow in the presence of high levels of superoxide radicals, Sod1-/- cells were 80 times more sensitive and Sod2-/- cells were 12 times more sensitive to paraquat than wild-type cells. In addition, whereas the loss of 50% CuZnSOD rendered Sod1-/+ cells almost twice more sensitive to paraquat than +/+ cells, loss of 50% MnSOD had no effect on paraquat sensitivity. Our results suggest that CuZnSOD-deficient cells are more sensitive to oxygen toxicity than are MnSOD-deficient cells, that paraquat causes free radical-induced damage in both the mitochondria and cytoplasm, and that SOD compartmentalized in the cytosol cannot compensate for the loss of SOD in the mitochondria and vice versa.     

Malignant anorectal melanoma

Bone marrow cell responses to injections of nitrogen mustard, oncovin, procarbazine, hydrocortisone, and a regimen of all four drugs (MOPH) were evaluated in CRF1 and C57B1/6 mice by determining bone marrow cellularity and content of transplantable colony forming units (CFU) after treatment. The study was done to determine whether the combined regimen, which is widely used clinically in treatment of disseminated Hodgkin's disease, is more or less detrimental to the hemopoietic system than the same drugs used as single agents. Nitrogen mustard and procarbazine used clinically as single drugs are given in three and two times, respectively, greater doses than in the combined regimen. Hydrocortisone, given singly, was least toxic of the drugs, reducing the CFU/femur to 63% and 71% of control values. MOPH appeared slightly more toxic than hydrocortisone, resulting in 41% and 52% of the CFU/femur surviving, and was about equally as toxic as oncovin alone. Nitrogen mustard and procarbazine, administered as single drugs in high doses, were highly suppressive, resulting in only 10-19% survival of CFU/femur, whereas, reduced doses of the two drugs as used in the MOPH regimen spared 30-45% of the CFU/femur. Survival of CFU after MOPH treatment was three to four times greater than after high doses of nitrogen mustard or procarbazine alone. The component drugs of the combined regimen did not act on separate populations of stem cells to produce an additive effect but appeared to inactivate the same population of cells.     

Is incarceration during pregnancy associated with infant birthweight?

To address the hypothesis that tumor necrosis factor (TNF)-alpha has a role in obesity-associated insulin resistance or the regulation of in vivo lipid metabolism, mice with targeted disruption of the TNF-alpha gene were generated and studied. The absence of TNF-alpha protein in TNF-null (-/-) mice was confirmed. Lean or obese (gold-thioglucose [GTG]-injected) homozygous (-/-) mice were compared with lean or obese age- and sex-matched wild-type (+/+) mice derived from the same line at 13, 19, and 28 weeks of age. The following parameters were significantly affected in lean -/- versus +/+ mice: Body weight was not affected until week 28 (decreased by 14%); epididymal fat pad weight also decreased (25%) at this time, as did percentage body fat (16%), while percentage body protein was increased 13%. Fed plasma insulin levels decreased 47% (28 weeks), triglyceride levels decreased (all three ages; maximum 35% at 19 weeks), and fed plasma leptin decreased 33% (28 weeks). Fasting glucose was slightly (10%) reduced, but the glucose response to an oral glucose tolerance test (OGTT) was not affected. There was a trend (NS) toward increased total adipose tissue lipoprotein lipase in -/- versus +/+ mice. GTG-treatment resulted in obese -/- and +/+ mice with equal mean body weights (42 and 58% increased weight versus lean mice). The following parameters were significantly different in obese -/- mice: fasting plasma glucose decreased 13% (28 weeks), fed plasma insulin decreased 67% (28 weeks), and insulin response to OGTT was decreased by 50%. For both groups of obese mice, glucose levels during the OGTT were substantially increased compared with those in lean mice; however, mean stimulated glucose levels were 20% lower in obese -/- versus +/+ mice. We conclude 1) that TNF-alpha functions to regulate plasma triglycerides and body adiposity and 2) that although TNF-alpha contributes to reduced insulin sensitivity in older or obese mice, the absence of TNF-alpha is not sufficient to substantially protect against insulin resistance in the GTG hyperphagic model of rodent obesity.