Preterm delivery in mice with renal abscess.

OBJECTIVE: Our purpose was to develop a mouse model of renal abscess to study the effect of extrauterine infection on preterm delivery. METHODS: Escherichia coli or sterile medium was injected into the left kidney of 70 pregnant mice that had completed approximately 75% of gestation. Preterm delivery rates were recorded for various inocula. Kidney specimens were obtained and examined grossly and histologically for abscess formation. RESULTS: Thirty-one of 51 animals (60.8%) infected with 1 x 10(5)-9 x 10(6) bacteria and none of 19 uninfected animals delivered prematurely (P < .001). Renal abscess was induced in 100% of mice receiving bacterial inoculation but in none receiving sterile medium. CONCLUSION: Kidney injection provides a reliable method for inducing renal abscess in pregnant mice. Renal abscess induces preterm delivery at a stable rate across a wide range of bacterial inocula. This model of extrauterine infection may be particularly useful in investigations of infection-induced preterm delivery.

Development of an infection-resistant LVAD driveline: a novel approach to the prevention of device-related infections.

BACKGROUND: Infection remains the single most important challenge to extended left ventricular assist device (LVAD) use and often arises from the percutaneous driveline exit site. We evaluated the ability of an LVAD driveline prototype impregnated with chlorhexidine, triclosan, and silver sulfadiazine to resist bacterial and fungal colonization. METHODS: The spectrum and duration of antimicrobial activity were evaluated in vitro by daily transfer of driveline segments embedded on agar plates inoculated with 10(8) colony-forming units (CFU) of Staphylococcus aureus (S. aureus), Staphlococcus epidermidis, Enterobacter aerogenes, Psuedomonas aeruginosa, and Candida albicans, and then measuring zones of inhibition around the sample subsequent to 24 hours of incubation at 37 degrees C. Antimicrobial activity was demonstrated against all organisms for greater than 14 days, and for over 21 days for gram-positive bacteria. To demonstrate in vivo efficacy of the treated driveline, 3-cm segments of driveline were implanted in the dorsal and ventral surface of rats. The exit site was inoculated with 10(6) CFU of S. aureus. After 7 days, driveline segments were aseptically explanted and assayed for bacterial colonization and retention of antimicrobial activity. One hundred percent of control segments were colonized (10(5) CFU S. aureus/cm) as against 13% of the test explants (< or = 330 CFU/cm; p < 0.0001). RESULTS: Subcultures of the insertion site and driveline pocket tissue resulted in 10(3) to 10(5) CFU per swab culture for control rats and 0 to 10(2) CFU/swab for test animals. Test drivelines retained 80% of anti-S. aureus activity. Gross and histological examination of the driveline and surrounding pocket revealed minimal tissue reactivity with positive signs of tissue ingrowth. CONCLUSION: An antimicrobial driveline may prevent early infections and facilitate ingrowth of tissue to provide long-term stability and protection against late infection.

Inflammatory cytokines in a murine model of infection-induced preterm labor: cause or effect?

OBJECTIVE: To characterize the expression of inflammatory cytokines in a murine model of preterm delivery induced by heat-killed bacteria. METHODS: The right uterine horns of female CD-1 mice on day 14.5 of 19-20 days of gestation were inoculated with either sterile media or killed Escherichia coli bacteria (10(5)-10(10) organisms per mouse). The incidence of preterm delivery was recorded. The concentrations of cytokines (interleukin [IL-] 1 alpha, IL-1 beta, IL-1 receptor antagonist [IL-1ra], IL-6, and tumor necrosis factor alpha [TNF alpha]) within maternal and fetal tissue homogenates were determined by enzyme-linked immunosorbent assay at various times after inoculation. RESULTS: Killed E. coli induced preterm delivery in a dose-dependent fashion. Inoculation with 10(10) bacteria (sufficient to cause delivery in all mice) produced increases in IL-1 alpha, IL-1 beta, IL-6, and TNF alpha within uteri and fetal membranes, but not within placentas, fetal bodies, and maternal serum. Maximum mean uterine levels of IL-1 and IL-6 exceeded those of fetal tissues (membranes, placentas, and fetal bodies) by greater than 15-fold. Maximal uterine IL-1 and TNF alpha levels following inoculation with 10(10) bacteria exceeded those that followed inoculation with 10(7) bacteria (below the threshold for delivery) by 2.5- to 5-fold. The anti-inflammatory cytokine IL-1ra was expressed in higher concentrations in fetal than in maternal tissues and was unaltered by the bacterial inoculum. CONCLUSIONS: E. coli induce labor in mice even in the absence of bacterial viability. Although IL-1 and TNF alpha were upregulated by bacterial inocula causing delivery, peak levels were only 2.5- to 5-fold higher than those that occurred with inocula below the threshold for delivery (1000-fold fewer bacteria). Whether IL-1 and TNF alpha mediate labor during in vivo infection, or whether the upregulation of these cytokines merely represents an epiphenomenon accompanying infection, remains unknown.

Chromosomal localization of the loci responsible for dystrophic cardiac calcinosis in DBA/2 mice.

Dystrophic cardiac calcinosis (DCC) occurs in certain inbred strains of mice, including DBA/2 and C3H/He, and is generally found as an incidental lesion in adult animals at necropsy. Preliminary genetic studies into the cause of DCC have been performed in DBA/2 mice and suggest that DCC is inherited as an autosomal recessive trait involving three or four unlinked genes. To investigate the genetics of DCC further, we produced myocardial cell death by freeze-thaw injury to induce DCC. Experiments were conducted with three F1 hybrids made using three inbred strains of mice (DBA/2J and C3H/HeJ, DCC-susceptible strains; C57BL/6J, DCC-resistant strain) to compare the genetic factors in the development of DCC. We found that DBA/2 and C3H/He mice share the same gene pattern(s) that is responsible for DCC. We determined by backcross linkage analysis in DBA/2 and C57BL/6 mice that at least one recessive locus is responsible for DCC. A haplotype analysis of the backcross data demonstrated that the recessive locus, designated dyscalc1, is located on Chromosome 7, 20.5 cM distal to the centromere. The likely candidate genes for dyscalc1 are discussed. Further understanding of the structure and function of these mutant genes will be beneficial in explaining the molecular pathogenesis of DCC.

A murine model of renal abscess formation.

We developed a murine model of kidney abscess by direct renal injection of either Escherichia coli (1 x 10(6) to 7 x 10(6) organisms) or sterile medium. Bacterial infection produced renal abscesses, bacteremia, and late-onset leukocytosis in all animals. Controls were unaffected. This model may be useful for the study of various sequelae of kidney infection.

Identification and cloning of a truncated isoform of the cardiac sodium-calcium exchanger in the BALB/c mouse heart.

A truncated isoform of the cardiac sodium-calcium exchanger (NCX1) was identified and cloned from BALB/c mouse heart. This cDNA clone has an AATAAA polyadenylation signal in the 3' untranslated region that caused the 3344-bp clone to stop at a premature termination site and encode for a 940-amino acid (AA) protein, in contrast to the wild-type C57BL/6 mouse, which has a 970-AA protein. Comparing the predicted AA sequence of NCX1 between the two mouse strains by hydropathic plot, we found that the BALB/c NCX1 protein has only 11 extracellular domains, missing one domain at the COOH terminal, while C57BL/6 mice have 12 extracellular domains, similar to other species. Using the mouse mapping gene panel BCB, the NCX1 gene was mapped to the distal end of mouse chromosome 17 (51.3 cM), confirming the data published from the rat probe.

Experimental autoimmune encephalomyelitis is exacerbated in mice lacking the NOS2 gene.

Nitric oxide is believed to be a prominent mediator of inflammation based in part on the correlative expression of the inducible nitric oxide synthase (iNOS) gene in various pathologies. The resulting high output of the highly reactive molecule nitric oxide is then believed to play an important role in the evolving inflammatory response. Studies have shown that iNOS and nitric oxide are present in the tissues of patients with multiple sclerosis (MS). In rodent models of MS, experimental autoimmune encephalomyelitis (EAE), it has been shown that nonspecific NOS inhibitors partially ameliorate the disease. To determine the importance of iNOS in this model of MS, we induced EAE in mice containing a disrupted iNOS (NOS2) gene. Surprisingly, by day 24, the NOS2 knockout mice had a greater incidence of EAE than wild-type control mice (75 vs 12%), and had a higher average severity score (2.42 vs 0.44). These differences appear to result largely from the failure of the disease to remit in NOS2 KO mice. Wild-type mice have a profound ability to reverse EAE (82%) compared with the knockout mice (19%). This result implies that iNOS may in some instances play a protective role in autoimmune-mediated tissue destruction.

Morphologic response of myocardium to freeze-thaw injury in mouse strains with dystrophic cardiac calcification.

To investigate the pathogenesis of dystrophic cardiac calcification in mice, we studied myocardial and skeletal muscle (diaphragm) necrosis induced by freeze-thaw injury through the abdominal portion of the diaphragm in DBA/2, C3H/He, and C57BL/6 (control) mice. Two mice from each mouse strain were euthanized 6, 12, 24, and 36 h after the initial freeze-thaw injury; 6 mice from each strain were euthanized 2, 4, 7, 14, and 28 days after injury. The hearts and diaphragms were studied by light and electron microscopic techniques. Myocardial and diaphragmatic mineralization in response to injury occurred only in DBA/2 and C3H/He mice and was present as early as 2 days after initial myocyte injury. Ultrastructurally the mineralized deposits first accumulated in mitochondria as early as 24 h after injury, with subsequent complete mineralization of the mitochondria and surrounding sarcoplasm by 48 h. These results suggest that the pathogenesis of dystrophic cardiac calcification in DBA/2 and C3H/He mice may be related to disturbed myocyte calcium metabolism, leading to mitochondrial calcium overload and myocardial calcification.

Decreased bacterial adherence and biofilm formation on chlorhexidine and silver sulfadiazine-impregnated central venous catheters implanted in swine.

OBJECTIVE: To determine if antiseptic central venous catheters impregnated with silver sulfadiazine and chlorhexidine (antiseptic) reduce bacterial adherence and biofilm formation without producing local or systemic toxicity. DESIGN: Prospective, randomized, controlled trial. SETTING: Experimental laboratory in a university teaching hospital. SUBJECTS: Ten outbred New Hampshire pigs. INTERVENTIONS: Nonimpregnated (control) and antiseptic-impregnated catheters were inserted intravascularly into swine for 7 days. After explantation, the catheters were assessed for bacterial adherence and biofilm formation, and the surrounding tissue was assessed for signs of toxicity. Before retrieval, systemic concentrations of antimicrobials were determined. MEASUREMENTS AND MAIN RESULTS: Sequential roll plate and centrifuging were used to detect moderately and tightly adherent bacteria on the outer and luminal surfaces of the catheter. The presence of biofilm was detected by scanning electron microscopy. Tissues surrounding the catheters were examined histopathologically; systemic concentrations of chlorhexidine, sulfadiazine, and silver were determined by atomic absorption and high-performance liquid chromatography. As compared with the controls, antiseptic catheters had significantly (p < .01) fewer moderately and tightly adherent bacteria on outer and luminal surfaces, and fewer adherent bacteria when outer surfaces alone were examined (p < .01). Scanning electron microscopy showed bacterial biofilm and adherence on the control catheters but not on the antiseptic catheters. There were no abnormal histopathologic changes associated with the test catheter, and serum concentrations of the antibacterial agents were shown to be within nontoxic ranges. CONCLUSION: The antiseptic-impregnated catheters prevented bacterial adherence and biofilm formation and produced no local or systemic toxicity.

Comparison of polymerase chain reaction and immunohistochemistry for the detection of Mycoplasma pulmonis in paraffin-embedded tissue.

The polymerase chain reaction (PCR) is a reliable method of detecting specific DNA sequences. The purpose of this study was to develop a PCR method of detecting Mycoplasma pulmonis in paraffin-embedded tissue sections and to compare the sensitivity of this method with that of the avidin-biotin complex (ABC) immunohistochemistry technique. We infected mice intranasally with 10(7) M. pulmonis and sacrificed them 28 days later. Using a published oligonucleotide primer sequence for M. pulmonis, we examined 8-microns paraffin sections by PCR for a 710 base-pair segment of the genome. A sequential 8-microns paraffin section was stained for M. pulmonis, using standard ABC immunohistochemistry methods. By PCR and dot blot hybridization, 60 of 62 paraffin-embedded lungs were positive for M. pulmonis, whereas only 17 of 62 lungs were positive, using the ABC method. In this study, we demonstrated that PCR of paraffin-embedded tissues followed by dot blot hybridization of the PCR products was much more sensitive than the ABC method in identifying mouse lungs infected with M. pulmonis.

Gastric extramedullary plasmacytoma in a dog.

A 10-year-old mixed-breed dog was examined because of a 6-week history of daily vomiting and sporadic diarrhea. On gastroscopy, a crateriform mass was observed on the greater curvature of the stomach. Partial gastrectomy and lymphadenectomy of a large mesenteric lymph node was performed. Gastric plasmacytoma with lymph node metastasis was diagnosed by histologic and immunoperoxidase methods, and chemotherapy was initiated with doxorubicin hydrochloride and diphenhydramine hydrochloride. The dog remains clinically normal 30 months after initial diagnosis. Although gastric plasmacytomas are rare in dogs, long-term survival appears to be better with this disease than with other types of gastric neoplasia.

Laboratory assessment of chronic hepatitis in Syrian hamsters.

Clinical chemistry studies in the diagnosis of hamster diseases have received little attention. Although normal values exist for serum constituents, the effects of disease on these values are not well documented. Chronic hepatitis is endemic in several Syrian hamster (Mesocricetus auratus) colonies and is reported mainly through routine histologic examination. We investigated whether any differences in serum clinical chemistries were present in animals with hepatobiliary disease versus unaffected hamsters. Only serum alanine aminotransferase (ALT) and bile acids were significantly elevated in hamsters with chronic hepatitis only. In hamsters that had both chronic hepatitis and biliary disease, the serum ALT, alkaline phosphatase, cholesterol, and bile acids were significantly elevated. The results of this study indicated that serum clinical chemistries may be a useful antemortem diagnostic test for chronic hepatobiliary disease in hamsters.

Identification of immunoglobulin light chains in canine extramedullary plasmacytomas by thioflavine T and immunohistochemistry.

Extramedullary plasmacytomas were studied in 29 dogs. The site at which tumors occurred and the age and sex of the dogs were similar to those in previous reports. The skin of the digits, chin, ear, and lip represented the most common (17/29) tumor sites. Males and females were equally represented, and tumors occurred in middle-aged to old dogs (mean age, 9.0 years). A breed predilection was seen in the Cocker Spaniel (n = 7; 24%); Cocker Spaniels represented only 4% (210/4,725) of the submissions during the same period. Tumors were stained with immunohistochemical markers (lambda light chain, K light chain) and thioflavine T. Immunoreactivity was limited to either lambda or K light chains, consistent with a monoclonal plasma cell population. The majority of tumors expressed lambda light chains, consistent with previously reported canine plasma cell dyscrasias. Thioflavine T cytoplasmic fluorescence was seen in the majority (18/29) of plasmacytomas and with inflammatory plasma cells present in control specimens. Other round cell neoplasms (lymphosarcoma, histiocytoma, and mastocytoma) were negative with thioflavine T, indicating that positive staining with thioflavine T was specific for plasma cells (neoplastic and inflammatory). This study confirms by immunohistochemistry that canine extramedullary plasmacytomas disproportionately express lambda light chains and establishes thioflavine T staining as a rapid histochemical method for diagnosis of these tumors.

Salivary gland oncocytes in African hedgehogs (Atelerix albiventris) mimicking cytomegalic inclusion disease.

The salivary glands from three African hedgehogs contained multiple foci of cytomegalic cells, which occasionally had a mild to moderate infiltrate of lymphocytes at the periphery. The cytomegalic cells were 35 to 40 microns in diameter with abundant acidophilic granular to hyalin cytoplasm. The nuclei were enlarged with clumped marginalized chromatin and a large, (6 to 8 microns in diameter) central, brightly eosinophilic nucleolus that had the appearance of an inclusion body by light microscopy. Histochemically most of the cytomegalic cells contained cytoplasmic metachromatic granules with Feyrter's thionine inclusion stain. Scattered cells at the periphery of the cytomegalic foci contained periodic acid-Schiff-positive cytoplasmic granules. Ultrastructurally the cytomegalic cells contained numerous tightly-packed, often bizarre, enlarged mitochondria that completely filled the cytoplasm. The nucleus consisted of a dense central core of chromatin associated with the nucleolus and the remaining chromatin was clumped and marginalized. Nuclear and cytoplasmic virions consistent with cytomegalovirus were not present. Histochemical stains of the nucleus for heavy metals were negative. The ultrastructural and histochemical findings of the cytomegalic cells were consistent with oncocytes. Previous reports in the literature of similar cells in the salivary glands of insectivores appear to have been erroneously described as cytomegalovirus infections.

Dystrophic cardiac calcinosis in mice: abnormal myocardial response to freeze-thaw injury.

Dystrophic cardiac calcinosis (DCC) is a frequent finding in DBA/2, C3H and BALB/c mice and its etiology is not known. Previous studies have speculated that myocardial necrosis is involved in the pathogenesis of DCC. In this study, cardiac necrosis was induced in DBA/2, C3H and C57BL/6 mice by freeze-thaw injury through the abdominal diaphragm. Four weeks after freeze-thawing, the mice were sacrificed and the hearts and diaphragms were examined. In response to injury, cardiac mineralization was present only in DBA/2 and C3H mice. The myocardium of C57BL/6 mice (control strain) healed by fibrosis without mineralization, the normal response of the myocardium to injury. Calcified diaphragms also were present at the site of freeze-thaw injury in DBA/2 and C3H mice, which is supportive evidence that a systemic abnormality is involved in the pathogenesis of DCC. The conclusion from this study is that the pathogenesis of DCC in DBA/2 and C3H mice is multifactorial and involves both myocardial necrosis and an abnormal response to injury.

Differential expression of glutathione S-transferase, glutathione peroxidase and glutathione reductase in normal and malignant human breast tissues.

In the present study we have compared the levels of glutathione (GSH) S-transferase, GSH peroxidase and GSH reductase in human breast tumors and adjacent normal tissues obtained from the same individuals. We have also quantitated GST pi type antigen in these samples by western blotting. GST pi activity towards 1-chloro-2,4-dinitrobenzene was found to be elevated in tumors from three out of six patients (patient nos. 2, 4 and 5), whereas this activity was suppressed in tumor from patient no. 1. Results of Western blotting using antibodies raised against GST pi of human placenta were in agreement with the GST activity data. GSH peroxidase activity with cumene hydroperoxide as substrate was found to be elevated in four tumor samples (patient nos. 2, 4, 5, and 6) but suppressed in tumor from patient no. 1. On the other hand, GSH reductase activity was elevated in three samples (patients nos. 2, 4 and 5) and downregulated in the remaining three samples (patients nos. 1, 3 and 6). These results indicate that GSH-related enzymes are differentially altered in human breast tumors and GST pi type isoenzyme(s), unlike certain other human carcinomas such as colonic, are not uniformly elevated in human breast tumors.