C3H/HeJ Mice Research Articles

Selected contribution: variation in acute hypoxic ventilatory response is linked to mouse chromosome 9.

Genetic determinants confer variation among inbred mouse strains with respect to the magnitude and pattern of breathing during acute hypoxic challenge. Specifically, inheritance patterns derived from C3H/HeJ (C3) and C57BL/6J (B6) parental strains suggest that differences in hypoxic ventilatory response (HVR) are controlled by as few as two genes. The present study demonstrates that at least one genetic determinant is located on mouse chromosome 9. This genotype-phenotype association was established by phenotyping 52 B6C3F2 (F2) offspring for HVR characteristics. A genome-wide screen was performed using microsatellite DNA markers (n = 176) polymorphic between C3 and B6 mice. By computing log-likelihood values (LOD scores), linkage analysis compared marker genotypes with minute ventilation (&Vdot;E), tidal volume (VT), and mean inspiratory flow (VT/TI, where TI is inspiratory time) during acute hypoxic challenge (inspired O2 fraction = 0.10, inspired CO2 fraction = 0.03 in N2). A putative quantitative trait locus (QTL) positioned in the vicinity of D9Mit207 was significantly associated with hypoxic VE (LOD = 4.5), VT (LOD = 4.0), and VT/TI (LOD = 5.1). For each of the three HVR characteristics, the putative QTL explained more than 30% of the phenotypic variation among F(2) offspring. In conclusion, this genetic model of differential HVR characteristics demonstrates that a locus approximately 33 centimorgans from the centromere on mouse chromosome 9 confers a substantial proportion of the variance in VE, VT, and VT/TI during acute hypoxic challenge.

Postnatal and pubertal skeletal changes contribute predominantly to the differences in peak bone density between C3H/HeJ and C57BL/6J mice.

Previous studies have shown that 60-70% of variance in peak bone density is determined genetically. The higher the peak bone density, the less likely an individual is to eventually develop osteoporosis. Therefore, the amount of bone accrued during postnatal and pubertal growth is an important determining factor in the development of osteoporosis. We evaluated the contribution of skeletal changes before, during, and after puberty to the development of peak bone density in C3H/HeJ (C3H) and C57BL/6J (B6) mice. Volumetric bone density and geometric parameters at the middiaphysis of femora were measured by peripheral quantitative computed tomography (pQCT) from days 7 to 56. Additionally, biochemical markers of bone remodeling in serum and bone extracts were quantified. Both B6 and C3H mice showed similar body and femoral weights. B6 mice had greater middiaphyseal total bone area and thinner cortices than did C3H mice. Within strains, males had thicker cortices than did females. C3H mice accumulated more minerals throughout the study, with the most rapid accumulation occurring postnatally (days 7-23) and during pubertal maturation (days 23-31). C3H mice had higher volumetric bone density as early as day 7, compared with B6 mice. Higher serum insulin-like growth factor I (IGF-I) was present in C3H mice postnatally at day 7 and day 14. Until day 31, B6 male and female mice had significantly higher serum osteocalcin than C3H male and female mice, respectively. Alkaline phosphatase (ALP) was found to be significantly higher in the bone extract of C3H mice compared with B6 mice at day 14. These data are consistent with and support the hypothesis that the greater amount of bone accrued during postnatal and pubertal growth in C3H mice compared with B6 mice may be caused by increased cortical thickness, increased endosteal bone formation, and decreased endosteal bone resorption.

Preferential activation of nuclear factor of activated T cells c correlates with mouse strain susceptibility to allergic responses and interleukin-4 gene expression.

Dysregulated expression of the T helper 2 cytokine interleukin (IL)-4 is thought to play a fundamental role in the pathogenesis of allergic asthma. The molecular basis for dysregulated IL-4 production is not well understood. We analyzed in detail the molecular factors involved in regulating IL-4 transcription in a well-characterized mouse model. In this model, A/J mice developed allergen-induced IL-4 cytokine gene expression, airway inflammation, and hyperresponsiveness, whereas C3H/HeJ (C3H) mice did not. Here we report that isolated splenocytes from A/J and C3H mice stimulated ex vivo with concanavalin A reproduced the cytokine phenotype observed in the airway after antigen challenge. We hypothesized that differences in splenocyte IL-4 production involved either polymorphisms in regulatory IL-4 promoter regions, or the expression and activation of transcription factors necessary for promoter transactivation in a strain-dependent manner. To address these questions, we first sequenced ~ 700 base pairs containing well-characterized IL-4 promoter regulatory elements using genomic DNA obtained from C3H and A/J mice. Next, we used electrophoretic mobility shift assays with relevant IL-4 promoter sequences to screen nuclear extracts isolated from A/J and C3H splenocytes for functional transcriptional factor complexes. Here we show that susceptibility to antigen-induced airway hyperresponsiveness is not due to polymorphisms in the IL-4 promoter, but is associated with preferential expression of nuclear factor of activated T cells c in splenocyte nuclear extracts obtained from A/J mice. In conclusion, our data link dysregulated activation of a specific transcription factor with susceptibility to allergic airway inflammation.

Altered behavior and alcohol tolerance in transgenic mice lacking MAO A: a comparison with effects of MAO A inhibitor clorgyline

The influence of deficiency of monoamine oxidase A (MAO A) gene and the lack of enzyme MAO A on the behavior of transgenic mouse strain (Tg8) was studied. It was shown that MAO-A-lacking mice differed from mice of the wild-type strain C3H/HeJ (C3H) by an attenuated acoustic startle response, prepulse inhibition (PPI) was unchanged. In Tg 8 mice, the exploratory nose-poking in the holeboard test as well as exploratory line crossing in the “light–dark” test were decreased. No effect of MAO A deficiency on locomotor activity was found. No alcohol preference or difference between Tg8 and C3H in ethanol consumption in the free-choice test has been found, although an increase in alcohol tolerance has been demonstrated. Ethanol-induced (0.3 g/100 g ip) sleep latency was longer, duration of sleep was shorter and ethanol hypothermia was reduced in MAO-A-lacking mice. Comparison of effects of MAO A knockout with those of irreversible MAO A inhibitor clorgyline (5 and 10 mg/kg ip) on C3H mice showed a similar reducing effect on ethanol-induced sleep, but potentiated ethanol-induced hypothermia. Clorgyline administration provoked a tendency to decrease of exploratory activity in the nose-poking test and decreased the frequency of exploratory rearings in the light–dark test. Clorgyline (5 and 10 mg/kg) did not affect the acoustic startle response, but a dose of 5 mg/kg diminished PPI. Therefore, Tg8 mice exhibited a decreased startle response and exploratory activity and an increased tolerance to ethanol. A similar increase in tolerance to ethanol-induced sleep and a tendency to decrease exploratory behavior were displayed by clorgyline. Other effects on behavior were different, suggesting the influence of long-lasting action of MAO A knockout and the involvement of a compensatory mechanism in Tg8 mice.

Effects of dietary calcium depletion and repletion on dynamic determinants of tibial bone volume in two inbred strains of mice

As an adjunct to our efforts to identify the genes that determine peak bone density, we examined phenotypic differences between two inbred strains of mice, C3H/HeJ (C3H) and C57BL/6J (B6), which are of similar size but differ with respect to peak bone density (e.g., C3H mice have 53% higher femoral bone density than B6 mice). The current studies were intended to compare the skeletal responses of C3H and B6 mice to 2 weeks of dietary calcium (Ca) depletion, followed by 2 weeks of Ca repletion. Initial studies showed that: (a) femur dry weight decreased during Ca depletion in both C3H and B6 mice (by 25% and 19%, respectively, p < 0.001) and most of this loss was recovered during Ca repletion; and (b) serum alkaline phosphatase (ALP) activity increased during Ca depletion, in both strains of mice ( p < 0.001), and returned to normal after Ca repletion. Histological analyses of ground cross sections prepared at the tibiofibular junction showed that Ca-depletion increased medullary area in both C3H and B6 mice (indicating endosteal bone loss, p < 0.01), with reversal during Ca repletion. There were no effects of Ca depletion or repletion on periosteal bone growth. Endosteal bone forming surface and endosteal mineral apposition decreased during Ca depletion and increased during repletion in both C3H and B6 mice ( p < 0.05). Net bone formation decreased during Ca depletion in C3H mice, but not B6 mice ( p < 0.01), and was normal during Ca repletion in both strains. Endosteal bone resorbing surface and net bone resorption increased during Ca depletion and decreased during repletion in both strains ( p < 0.01). A supplemental study (of Ca depletion without repletion) confirmed the effects of Ca depletion on femoral dry weight and serum ALP activity ( p < 0.001 for each). This supplemental study also showed that Ca deficiency increased serum parathyroid hormone (PTH) ( p < 0.05) and decreased (tibial) cortical bone area and cortical mineral content ( p < 0.05 to p < 0.001) in both strains of mice. Together, these data demonstrate that the skeletal responses to Ca depletion and repletion are, qualitatively, similar in C3H and B6 mice.

Genetic regulation of cortical and trabecular bone strength and microstructure in inbred strains of mice.

The inbred strains of mice C57BL/6J (B6) and C3H/HeJ (C3H) have very different femoral peak bone densities and may serve as models for studying the genetic regulation of bone mass. Our objective was to further define the bone biomechanics and microstructure of these two inbred strains. Microarchitecture of the proximal femur, femoral midshaft, and lumbar vertebrae were evaluated in three dimensions using microcomputed tomography (microCT) with an isotropic voxel size of 17 microm. Mineralization of the distal femur was determined using quantitative back-scatter electron (BSE) imaging. MicroCT images suggested that C3H mice had thicker femoral and vertebral cortices compared with B6. The C3H bone tissue also was more highly mineralized. However, C3H mice had few trabeculae in the vertebral bodies, femoral neck, and greater trochanter. The trabecular number (Tb.N) in the C3H vertebral bodies was about half of that in B6 vertebrae (2.8(-1) +/- 0.1 mm(-1) vs. 5.1(-1) +/- 0.2 mm(-1); p < 0.0001). The thick, more highly mineralized femoral cortex of C3H mice resulted in greater bending strength of the femoral diaphysis (62.1 +/- 1.2N vs. 27.4 +/- 0.5N, p < 0.0001). In contrast, strengths of the lumbar vertebra were not significantly different between inbred strains (p = 0.5), presumably because the thicker cortices were combined with inferior trabecular structure in the vertebrae of C3H mice. These results indicate that C3H mice benefit from alleles that enhance femoral strength but paradoxically are deficient in trabecular bone structure in the lumbar vertebrae.

Genetic determinants of acute hypoxic ventilation: patterns of inheritance in mice.

Acutely lowering ambient O(2) tension increases ventilation in many mammalian species, including humans and mice. Inheritance patterns among kinships and between mouse strains suggest that a robust genetic influence determines individual hypoxic ventilatory responses (HVR). Here, we tested specific genetic hypotheses to describe the inheritance patterns of HVR phenotypes among two inbred mouse strains and their segregant and nonsegregant progeny. Using whole body plethysmography, we assessed the magnitude and pattern of ventilation in C3H/HeJ (C3) and C57BL/6J (B6) progenitor strains at baseline and during acute (3-5 min) hypoxic [mild hypercapnic hypoxia, inspired O(2) fraction (FI(O(2))) = 0.10] and normoxic (mild hypercapnic normoxia, FI(O(2)) = 0.21) inspirate challenges in mild hypercapnia (inspired CO(2) fraction = 0.03). First- and second-filial generations and two backcross progeny were also studied to assess response distributions of HVR phenotypes relative to the parental strains. Although the minute ventilation (VE) during hypoxia was comparable between the parental strains, breathing frequency (f) and tidal volume were significantly different; C3 mice demonstrated a slow, deep HVR relative to a rapid, shallow phenotype of B6 mice. The HVR profile in B6C3F(1)/J mice suggested that this offspring class represented a third phenotype, distinguishable from the parental strains. The distribution of HVR among backcross and intercross offspring suggested that the inheritance patterns for f and VE during mild hypercapnic hypoxia are consistent with models that incorporate two genetic determinants. These results further suggest that the quantitative genetic expression of alleles derived from C3 and B6 parental strains interact to significantly attenuate individual HVR in the first- and second-filial generations. In conclusion, the genetic control of HVR in this model was shown to exhibit a relatively simple genetic basis in terms of respiratory timing characteristics.

Determinants of atherosclerosis susceptibility in the C3H and C57BL/6 mouse model: evidence for involvement of endothelial cells but not blood cells or cholesterol metabolism.

Lipids, monocytes, and arterial wall cells are primary components involved in atherogenesis. Using the inbred mouse strains C57BL/6J (B6) and C3H/HeJ (C3H), which have been extensively studied as models of the genetic control of diet-induced atherosclerosis, we examined which of these components determine genetic susceptibility. To test whether dietary responsiveness is involved, a congenic strain of C3H carrying an apoE-null allele (apoE(-/-)) was constructed. Although C3H.apoE(-/-) mice had higher plasma cholesterol levels, they developed much smaller lesions than their B6.apoE(-/-) counterpart on either chow or Western diets. Reciprocal bone marrow transplantation between the strains, with congenics carrying the same H-2 haplotype, was performed to examine the role of monocytes. The atherosclerosis susceptibility was not altered in the recipient mice, indicating that variations in monocyte function were not involved. Endothelial cells isolated from the aorta of B6 mice exhibited a dramatic induction of monocyte chemotactic protein-1, macrophage colony-stimulating factor, vascular cell adhesion molecule-1, and heme oxygenase-1 in response to minimally modified LDL, whereas endothelial cells from C3H mice showed little or no induction. In a set of recombinant inbred strains derived from the B6 and C3H parental strains, endothelial responses to minimally modified LDL cosegregated with aortic lesion size. These data provide strong evidence that endothelial cells, but not monocytes or plasma lipid levels, account for the difference in susceptibility to atherosclerosis between the 2 mouse strains.

Protection elicited by native outer membrane protein Oms66 (p66) against host-adapted Borrelia burgdorferi: conformational nature of bactericidal epitopes.

Oms66 is a Borrelia burgdorferi outer membrane porin protein whose role in Lyme disease pathogenesis and immunity has not been well established. Oms66 was solubilized from whole-cell lysates of strain B313 (which is derived from B31 but lacks OspA, -B, -C, and -D) and purified to homogeneity by fast-protein liquid chromatography. Purified native Oms66 (nOms66), which retained the ability to form large channels in a planar lipid bilayer model membrane system, and denatured Oms66 (hOms66) were used to immunize New Zealand White rabbits. The resulting Oms66 antisera were tested in a complement-dependent borreliacidal assay in parallel with basal serum and with serum from rabbits immune to reinfection with B. burgdorferi (IRS). IRS showed high-titer complement-dependent killing of both strains B31 and B313. Sera from animals immunized with nOms66 showed high-titer complement-dependent killing activity against strain B313 but exhibited no killing of B31. By comparison, serum generated from immunizations with hOms66 showed no killing activity against either strain. Following adsorption of antiserum to nOms66 with recombinant Oms66 (rOms66), the serum antibodies no longer bound to rOms66 or to nOms66 that had been denatured with 8 M urea. However, the antibodies still bound to nOms66 and killing activity against B313 was retained, thus suggesting that native, conformational epitopes are targets of this bactericidal activity. Six C3H HeJ mice were immunized with nOms66 and were challenged using "host-adapted" B. burgdorferi B31 by skin implantation of infected mouse ear tissue. Four of the six mice were protected against both localized and disseminated infection. These findings indicate that native Oms66 can elicit potent bactericidal activity and significant protective immunity against host-adapted organisms.

Gender- and androgen-related influence on the expression of proto-oncogene and apoptotic factor mRNAS in lacrimal glands of autoimmune and non-autoimmune mice

Our previous studies have shown that the mRNA levels of c-myb, c-myc, bcl-2 and p53 are higher, and partial Fas antigen (i.e. exons 1–2) lower, in lacrimal tissues of female, as compared to male, MRL/lpr mice, which are a model of Sjögren's syndrome. We have also found that this gender-related difference in bcl-2 and c-myb expression appears to be due to the influence of androgens. To extend these findings, we sought to determine: first, whether these gender- and/or hormone-associated variations in mRNA content are unique to MRL/lpr mice, or are also present in lacrimal glands of other murine strains, including autoimmune NZB/NZW F1 (F1) and non-obese diabetic (NOD), as well as non-autoimmune C3H/HeJ (C3H) and BALB/c, mice; and second, whether the levels of these apoptotic factor mRNAs are altered in lacrimal tissues of mice (i.e. testicular feminized (Tfm) with dysfunctional androgen receptors, as compared to glandular amounts in their ‘normal’ controls (i.e. Tabby). Lacrimal tissues were obtained from adult mice, which were either untreated or treated with placebo or testosterone for 21 days. Glands were processed for the analysis of proto-oncogene mRNAs by RT-PCR (at exponential phase of amplification) and data were standardized to the corresponding levels of β-actin mRNA. Our results demonstrate that Fas antigen, Fas ligand, c-myb, c-myc, bcl-2, Bax and p53 mRNAs are present in lacrimal tissues of F1, NOD, C3H, BALB/c, Tabby and Tfm mice. The relative levels of Fas antigen mRNA are consistently higher in glands of males, whereas amounts of bcl-2 mRNA are greater in tissues of F1, C3H and BALB/c females. Testosterone administration induced a significant increase in the lacrimal gland content of Bax mRNA, but a striking decrease in the lacrimal tissue level of bcl-2 mRNA in F1 and C3H mice. Lacrimal glands of Tfm mice contained elevated amounts of bcl-2 mRNA, as compared to values in tissues of their Tabby controls. In summary, our findings show that fundamental gender-related differences exist in the expression of genes associated with programmed cell death in lacrimal glands of autoimmune and normal mice. In addition, some of these differences may be due, at least in part, to the effect of androgens.

Histomorphometric studies show that bone formation and bone mineral apposition rates are greater in C3H/HeJ (high-density) than C57BL/6J (low-density) mice during growth

High-density C3H/HeJ (C3H) and low-density C57BL/6J (B6) mice, with femoral bone density differing by 50%, were chosen as a model to investigate the mechanisms controlling peak bone density and to map peak bone density genes. The present longitudinal study was undertaken to further establish the bone biologic phenotypes of these two inbred strains of mice. To evaluate phenotypic differences in bone formation parameters in C3H and B6 mice between the ages of 6 and 26 weeks, undecalcified ground sections from the diaphyses of the tibia and femur were prepared from mice receiving two injections of tetracycline. Histomorphometric analyses revealed that the cortical bone area was significantly greater (16%–56%, p < 0.001) in both the femur and tibia of the C3H mice than in the B6 mice at all timepoints. This difference in cortical bone area was due to significantly smaller medullary areas in the C3H mice than in the B6 mice. The bone formation rates (BFR) at the endosteum in both the femur and tibia were significantly greater (28%–117%, p < 0.001) in the young C3H mice (6–12 weeks old) than in B6 mice. The higher bone formation in C3H mice was associated with higher values of the bone mineral apposition rate (25%–94%, p < 0.001), and was not associated with higher values of the forming surface length as measured by tetracycline label length. Similar interstrain differences in mineral apposition and bone formation rates were observed in the periosteum of the femur and tibia. In conclusion, the greater bone area in the high-density C3H mice vs. the low-density B6 mice was, in part, due to the greater periosteal and endosteal bone formation rates during growth in the C3H mice. Because the C3H and B6 mice were maintained under identical environmental conditions (diet, lighting, etc.), the observed interstrain differences in bone parameters were the result of the action of genetic factors. Consequently, these two inbred strains of mice are suitable as a model to identify genetic factors responsible for high bone formation rates.

Strain differences in bone density and calcium metabolism between c3h/hej and c57bl/6j mice

Previous reports indicate that peak bone density is significantly higher in C3H/HeJ (C3H) than in C57BL/6J (C57BL) mice, making these two inbred strains useful models for studying the genetic basis for peak bone density. The following study was undertaken to examine whether strain differences in the bone density of C3H and C57BL mice are associated with differences in intestinal calcium (Ca) absorption. Calcium absorption was measured by the balance technique and animals received two injections of fluorochromes 5 days apart before killing. Subsequently, the femurs were removed and, following measurement of volumetric density, the left femur was divided into three equal parts and the middle third served as the femoral cortical diaphysis. Femur diaphyseal volumetric bone density, ash, and Ca content were 10%, 29%, and 29% higher in C3H than in C57BL mice ( p < 0.001), respectively. Bone length, periosteal mineral apposition rate, and periosteal bone formation rate of femoral diaphyseal cortical bone were not significantly different between the two strains of mice, but the marrow area of C57BL mice was almost twofold that of C3H mice ( p < 0.0001). Intestinal Ca absorption and 1,25-dihydroxyvitamin D [1,25(OH) 2D]-stimulated Ca 2+ uptake by intestinal mucosal cells were 38% and 51% higher in C3H than in C57BL mice p < 0.001), respectively. Serum Ca and 1,25(OH) 2D levels were 6% and 32% higher in C3H than in C57BL mice ( p < 0.001), respectively, and the number of intestinal-occupied vitamin D receptors was 51% higher in C3H than in C57BL mice ( p < 0.01). In a second experiment, three groups of C3H mice and three groups of C57BL mice were fed diets that contained 0.4%, 0.1%, or 0.02% Ca, and serum Ca, 1,25(OH) 2D, parathyroid hormone (PTH), and intestinal Ca absorption measured. At all dietary Ca levels, C3H mice maintained positive Ca absorption and absorbed significantly more Ca than C57BL mice. In contrast, at low dietary Ca levels (0.1% and 0.02% Ca), C57BL mice maintained negative Ca absorption. Low dietary Ca increased serum PTH significantly in C57BL but not in C3H mice, and decreased serum 1,25(OH) 2D and Ca levels in both strains of mice. Our findings indicate that the C57BL mice relied more on the mobilization of Ca from bone to maintain extracellular Ca homeostasis than the C3H mice. We conclude that strain differences in bone mass and density between C3H and C57BL mice is expressed, in part, through the vitamin D and PTH endocrine systems and their effects on the maintenance of extracellular Ca homeostasis.

Cortical tibial bone volume in two strains of mice: effects of sciatic neurectomy and genetic regulation of bone response to mechanical loading

Although C3H/HeJ (C3H) and C57BL/6J (B6) mice are similar in body size (and adult weight), and have bones of similar external size, C3H mice have higher peak bone densities than B6 mice (e.g., 53% higher peak bone density in the femora). The current studies were intended to assess the role of mechanical loading/unloading as a possible determinant of the bone density difference between these inbred strains of mice and, specifically, to assess the effect of sciatic neurectomy on histomorphometric indices of bone formation and resorption in the tibiae of female C3H and B6 mice. Groups of 10 mice of each strain were subjected to left-side sciatic neurectomy (left hindlimb immobilization) or a sham procedure. The contralateral (right) legs of each mouse were used as controls. Four weeks of immobilization produced no systemic changes in bone formation indices in either strain of mice (i.e., no change in serum alkaline phosphatase or serum osteocalcin). However, histomorphometric assessments at the tibiofibular junction showed that 4 weeks of immobilization caused a time-dependent decrease in the length of the endosteal bone forming perimeter (e.g., 14% of control single-labeled, noneroded surface at 4 weeks, p < 0.005) with a concomitant increase in the length of the endosteal bone resorbing perimeter (i.e., 424% of control eroded surface at 4 weeks, p < 0.005), in the B6 mice. These effects were associated with an increase in medullary area (132% of control, p < 0.05) at this site, in the B6 mice. The pattern of response was different in the tibiae of the C3 mice—a much smaller decrease in bone forming perimeter (88% of control at 4 weeks, p < 0.05), with no associated increase in bone resorbing perimeter, and no change in medullary area. Similar effects were seen at a second cross-sectional sampling site, in the proximal tibia. Together, these findings indicate that B6 mice are more sensitive to endosteal bone loss from hindlimb immobilization than C3H mice.

Activation of natural killer cells in arthritis-susceptible but not arthritis-resistant mouse strains following Borrelia burgdorferi infection.

Infection of susceptible mouse strains with Borrelia burgdorferi, the agent of Lyme disease, results in the development of arthritis. Components of the innate immune system may be important mediators of this pathology. To investigate the potential role of NK cells in development of experimental Lyme arthritis, we examined their activation in vivo in both resistant and susceptible mouse strains. Following inoculation of B. burgdorferi into the footpad, lymph node NK cells from susceptible C3H/HeJ (C3H) mice produced more gamma interferon than NK cells from resistant DBA/2J mice. Lymph node cells from susceptible C3H and AKR mice also had increased ability to lyse YAC-1 target cells 2 days following infection. Antibody depletion of NK cells from susceptible mice, however, did not alter the development of arthritis following B. burgdorferi challenge. In addition, NK cell depletion had little effect on spirochete burden. Thus, there is a marked activation of NK cells in susceptible mouse strains following infection. Although NK cells are not absolutely required for arthritis, events occurring prior to NK cell activation might be important in mediating pathology in experimental Lyme disease.

In Vivo and In Vitro Photodynamic Studies with Benzochlorin Iminium Salts Delivered by a Lipid Emulsion

Benzochlorin iminium salts (BIs) are hydrophobic photosensitizers based on an octaethylbenzochlorin nucleus that absorb in the near-IR region of the visible spectrum. In these studies the photodynamic activities of the zinc, copper and metal-free BI derivatives were compared in vivo in C3H-HeJ mice bearing a mammary adenocarcinoma tumor line. In vitro studies were also performed with the radiation-induced fibrosarcoma tumor line. An argon-pumped Ti-sapphire laser tuned to deliver light between 710 and 800 nm or an Oriel arc-lamp filtered to deliver broadband light above 590 nm were used as light source. A lipid emulsion was used as the delivery system for sensitizers in all studies. A pronounced solvent dependence was observed for the Q band for each of all iminium salts examined. As an example, the metal-free (BI) derivative had an absorption maximum at 798 nm in dichloromethane and at 727 nm in serum. The action spectra showed a greater PDT response at blue-shifted wavelengths for each of the three iminium salts both in vivo and in vitro. Among the three derivatives, the zinc analog (ZnBI) produced the greatest tumor regression at the low drug/light dose of 0.7 (mumole/kg and 200 J/cm2. These results indicate that iminium salts have characteristics that may make them promising third-generation photosensitizers.

Clearance of Borrelia burgdorferi may not be required for resistance to experimental lyme arthritis.

Infection of inbred mouse strains with Borrelia burgdorferi results in the development of experimental Lyme arthritis. The degree of arthritic pathology has been suggested to correlate with the level of spirochete burden within tissues. To investigate this further, we infected resistant DBA/2 (DBA) and susceptible C3H/HeJ (C3H) mice in the hind footpads and monitored arthritis development for 21 days. To quantitate levels of spirochetes within tissues, we created a competitive PCR molecule containing modified ospA and fla gene segments. C3H mice developed severe arthritis of the tibiotarsal joints, while DBA mice developed only mild inflammation throughout the experimental period. At day 21, when the gross size and histologic composition of ankles revealed significant differences in arthritis between the strains, there was little difference in levels of spirochete DNA as determined by competitive PCR. Cultures of ankle tissue at day 21 were also uniformly positive in both C3H and DBA animals and contained relatively similar levels of spirochetes. These results indicate that the presence of spirochetes in the ankles of experimental animals is not sufficient for arthritis development. Since arthritic and nonarthritic animals can harbor relatively equal spirochete burdens yet retain their distinct phenotypic outcomes, an aberrant or overly exuberant immune response may be an additional requirement for pathology in arthritis-prone mice.

Alkaline phosphatase levels and osteoprogenitor cell numbers suggest bone formation may contribute to peak bone density differences between two inbred strains of mice

Previous studies have shown that C3 H HeJ (C3H) mice have higher peak bone density than C57 BL 6 J (B6) mice, at least in part because of differences in rates of bone resorption. The current studies were intended to examine the alternative, additional hypothesis that the greater bone density in C3H mice might also be a consequence of increased bone formation. To that end, we measured two presumptive, indirect indices of bone formation and osteoblast number in these inbred strains of mice: alkaline phosphatase (ALP) activity in serum, bones, and bone cells; and the number of ALP-positive colony-forming units (CFU) in bone marrow stromal cell cultures. We found that C3H mice had higher serum levels of ALP activity than B6 mice at 6 (118 vs. 100 U/L, p < 0.03) and 32 weeks of age (22.2 vs. 17.2 U/L, p < 0.001). Tibiae from C3H mice also contained higher levels of ALP activity than tibiae from B6 mice at 6 (417 vs. 254 mU/mg protein, p < 0.02) and 14 weeks of age (132 vs. 79 mU/mg protein, p < 0.001), as did monolayer cultures of bone-derived cells from explants of 7.5-week-old C3H calvariae and femora (8.2 times more, p < 0.02, and 4.6 times more, p < 0.001, respectively). Monolayer cell cultures prepared by collagenase digestion of calvariae from newborn and 6-week-old mice also showed similar strain-dependent differences in ALP-specific activity ( p < 0.001 for each). Our studies also showed more ALP-positive CFU in bone marrow stromal cell cultures from 8-week-old C3H mice, compared with B6 mice (72.3 vs. 26.1 ALP-positive CFU/culture dish, p < 0.001). A similar result was seen for ALP-positive CFU production at 6 and 14 weeks of age, and the difference was greatest for the CFU that contained the greatest numbers of ALP-positive cells. Because skeletal ALP activity is a product of osteoblasts and has been shown to correlate with rates of bone formation, and because the number of ALP-positive CFU is believed to reflect the number of osteoprogenitor cells, the current data are consistent with the general hypothesis that bone formation may be greater in C3H than B6 mice because of a difference in osteoblast number. Our data further suggest that peak bone density may be greater in C3H mice than B6 mice due to a combination of decreased bone resorption and increased bone formation.

Contractile depression and expression of proinflammatory cytokines and iNOS in viral myocarditis.

We assessed two strains of mice [CD-1 and C3H.HeJ (C3H)] with different responses to coxsackievirus B3 (CVB3) infection at 7, 14, and 21 days after inoculation with 10(5) pfu of CVB3. CD-1 mice developed inflammatory lesions at 7 days that nearly recovered by 21 days; C3H mice demonstrated persistence of infiltrates. Although there were differences in the baseline fractional shortening, it was further reduced at 7 and 14 days in both strains. It recovered in CD-1 mice but remained depressed at 21 days in C3H mice. Interleukin-6 and tumor necrosis factor-alpha transcripts were increased in both strains at 7 days. Levels dropped to near control in CD-1 mice at 21 days but remained elevated in C3H mice. Interleukin-1 beta was minimally elevated in CD-1 mice but increased progressively in C3H mice. mRNA for the inducible form of NO synthase (iNOS) was increased at 7 days in the CD-1 mice, returning to baseline by 14 days; it rose progressively in C3H mice, with a fivefold increase at 21 days. We conclude that mice infected with CVB3 show increased expression of proinflammatory cytokines as well as iNOS associated with reduced contractile performance. In more susceptible mice, contractile depression and cytokine and iNOS expression are more pronounced.

Antitumor activity of (10 E, 12 Z)-9-hydroxy-10,12-octadecadienoic acid from rice bran

Two types of hydroxy acids, (10 E, 12 Z)-9-hydroxy-10,12-octadecadienoic acid [1] and (9 Z, 11 E)-13-hydroxy-9,11-octadecadienoic acid [2], were obtained as cytotoxic compounds from a water extract of rice bran. The cytotoxicity of acid 1 was stronger than that of acid 2 in vitro, against P388 mouse leukemia cells. Acid 1 slightly inhibited the growth of a Balb c mouse 3T3 fibroblast cells ( BALB c 3T3), but strongly inhibited that of simian virus 40-transformed 3T3 cells (SV-T2); 1 was therefore, found to have cytotoxicity against cancer cells. On the other hand, 2 inhibited neither normals cells nor transformed cells. Intraperitoneal injection of acid 1 effectively prolonged the survival time of C3H HeJ mice after intraperitoneal transplantation of MM46 mammary tumor cells. The difference in antitumor activity between 1 and 2 was also observed in vivo.

In Vivo and In Vitro Photodynamic Studies with Benzochlorin Iminium Salts Delivered by a Lipid Emulsion

Benzochlorin iminium salts (Bis) are hydrophobic photosensitizers based on an octaethylbenzochlorin nucleus that absorb in the near-IR region of the visible spectrum. In these studies the photodynamic activities of the zinc, copper and metal-free BI derivatives were compared in vivo in C3H-HeJ mice bearing a mammary adenocarcinoma tumor line. In vitro studies were also performed with the radiation-induced fibrosarcoma tumor line. An argon-pumped Ti-sapphire laser tuned to deliver light between 710 and 800 nm or an Oriel arc-lamp filtered to deliver broadband light above 590 nm were used as light source. A lipid emulsion was used as the delivery system for sensitizers in all studies. A pronounced solvent dependence was observed for the Q band for each of all iminium salts examined. As an example, the metal-free (BI) derivative had an absorption maximum at 798 nm in dichloromethane and at 727 nm in serum. The action spectra showed a greater PDT response at blue-shifted wavelengths for each of the three iminium salts both in vivo and in vitro. Among the three derivatives, the zinc analog (ZnBI) produced the greatest tumor regression at the low drug/light dose of 0.7 (μ mole/kg and 200 J/cm2. These results indicate that iminium salts have characteristics that may make them promising third-generation photosensitizers.