Altered wound healing in mice lacking a functional osteopontin gene (spp1).

Osteopontin (OPN) is an arginine-glycine-aspartate (RGD)- containing glycoprotein encoded by the gene secreted phosphoprotein 1 (spp1). spp1 is expressed during embryogenesis, wound healing, and tumorigenesis; however, its in vivo functions are not well understood. Therefore, OPN null mutant mice were generated by targeted mutagenesis in embryonic stem cells. In OPN mutant mice, embryogenesis occurred normally, and mice were fertile. Since OPN shares receptors with vitronectin (VN), we tested for compensation by creating mice lacking both OPN and VN. The double mutants were also viable, suggesting that other RGD-containing ligands replace the embryonic loss of both proteins. We tested the healing of OPN mutants after skin incisions, where spp1 was upregulated as early as 6 h after wounding. Although the tensile properties of the wounds were unchanged, ultrastructural analysis showed a significantly decreased level of debridement, greater disorganization of matrix, and an alteration of collagen fibrillogenesis leading to small diameter collagen fibrils in the OPN mutant mice. These data indicate a role for OPN in tissue remodeling in vivo, and suggest physiological functions during matrix reorganization after injury.

Mapping loci for pentylenetetrazol-induced seizure susceptibility in mice.

DBA/2J (D2) and C57BL/6J (B6) mice exhibit differential sensitivity to seizures induced by various chemical and physical methods, with D2 mice being relatively sensitive and B6 mice relatively resistant. We conducted studies in mature D2, B6, F1, and F2 intercross mice to investigate behavioral seizure responses to pentylenetetrazol (PTZ) and to map the location of genes that influence this trait. Mice were injected with PTZ and observed for 45 min. Seizure parameters included latencies to focal clonus, generalized clonus, and maximal seizure. Latencies were used to calculate a seizure score that was used for quantitative mapping. F2 mice (n = 511) exhibited a wide range of latencies with two-thirds of the group expressing maximal seizure. Complementary statistical analyses identified loci on proximal (near D1Mit11) and distal chromosome 1 (near D1Mit17) as having the strongest and most significant effects in this model. Another locus of significant effect was detected on chromosome 5 (near D5Mit398). Suggestive evidence for additional PTZ seizure-related loci was detected on chromosomes 3, 4, and 6. Of the seizure-related loci identified in this study, those on chromosomes 1 (distal), 4, and 5 map close to loci previously identified in a similar F2 population tested with kainic acid. Results document that the complex genetic influences controlling seizure response in B6 and D2 mice are partially independent of the nature of the chemoconvulsant stimulus with a locus on distal chromosome 1 being of fundamental importance.

Heterogeneity in the ribosomal RNA genes of the yeast Yarrowia lipolytica; cloning and analysis of two size classes of repeats.

Southern blotting of DNA from the ascomycetous yeast Yarrowia lipolytica revealed two major size classes of DNA units coding for rRNAs, which differ in length by about 1000 bp. We have cloned an rDNA unit of each size class. R-looping experiments revealed that the rRNA genes of both units are uninterrupted; subsequent heteroduplex analysis showed that the size difference both units is located within the nontranscribed spacer. Sequence analysis revealed that a major part of these spacers consists of a complex pattern of repetitions in periodicities of up to about 150 bp and that the difference between both rDNA units are located mainly in this repetitive region. Apart from different lengths of the repetitive regions, both rDNA units also reveal extended microheterogeneity within their homologous parts. Furthermore, no gene for 5S rRNA was observed in the spacer region. Therefore, the organization of the spacer of Yarrowia rDNA is clearly different from that of Saccharomyces cerevisiae.

Genotyping microsatellite polymorphisms by agarose gel electrophoresis with ethidium bromide staining: application to quantitative trait loci analysis of seizure susceptibility in mice.

Agarose gel electrophoresis with ethidium bromide staining (AGE/EBS) is an efficient and reliable method for analyzing microsatellite polymorphisms. We report the use of AGE/EBS for analyzing DNA microsatellite polymorphisms in a preliminary quantitative trait loci (QTL) study of seizure susceptibility in which a candidate gene strategy was used to direct initial mapping efforts. F2 intercross progeny, derived from seizure-sensitive DBA/2J (D2) and seizure-resistant C57BL/6J (B6) inbred strains of mice, were tested for their sensitivity to the seizure-inducing effect of pentylenetetrazol (PTZ), a gamma-aminobutyric acid (GABA) receptor antagonist. A semi-automated method is described, in which DNA microsatellites were amplified by polymerase chain reaction (PCR) to yield products of 100-200 base pair (bp) in length. Alleles were separated on 3-6% MetaPhor agarose gels, stained with ethidium bromide, and visualized by ultraviolet (UV) illumination. Univariate analysis of genotype and phenotype data provides evidence for a seizure-related QTL on chromosome 5, near genes coding for the GABAA receptor subunits alpha 5 and gamma 3. Interestingly, this suggestive QTL derives from the more resistant B6 strain, but it nonetheless provides impetus for the characterization of possible strain differences in these two candidate genes. Overall, these results demonstrate that AGE/EBS can be useful for rapid screening of genomic regions of special interest in QTL mapping studies.

Retroviral vector production in the National Gene Vector Laboratory at Indiana University.

The National Gene Vector Laboratory (NGVL) is a US National Institutes of Health initiative charged with providing clinical grade vectors for gene therapy trials. The program was started in 1995 and Indiana University has served as the production site for retroviral vectors and is also accepting applications for production of lentiviral vectors. The facility is designed to produce vectors for Phase I and Phase II clinical trials with the specific mandate to facilitate investigator-initiated research for academic institutions. To date, the facility has generated over 30 Master Cell Banks for gene therapy investigators throughout the United States. This required the facility to develop a system that can adapt to the varied needs of investigators, most of whom request different vector backbones, packaging cell lines, final product volumes, and media. In this review, we will illustrate some of the experiences of the Indiana University NGVL during the generation of retroviral vectors using murine-based packaging cell lines.

Autonomic cardiovascular neuropathy in primary Sjögren's syndrome.

The case of a 65-year-old female with primary Sjögren's syndrome and severe autonomic cardiovascular neuropathy, manifested by incapacitating postural hypotension, is presented. It is postulated that the patient's Sjögren's syndrome was responsible for the autonomic neuropathy, probably via a vasculitic mechanism. Treatment with hydrocortisone resulted in improvement in the patient's symptoms but it did not significantly alter the objective findings of autonomic dysfunction. We suggest that it is probably worth studying Sjögren's patients for evidence of autonomic neuropathy.

Delayed wound healing in aged rats is associated with increased collagen gel remodeling and contraction by skin fibroblasts, not with differences in apoptotic or myofibroblast cell populations.

Aging has been anecdotally reported to result in prolonged wound healing. Measurement of punch biopsy wound closure in young (4 month old) and old (36 month old) rats indicated there was a significant delay in wound closure by old rats during the early phase of repair, after which closure rates were equivalent. The delay in granulation tissue accumulation in older animals could involve premature programmed cell death (apoptosis); however, apoptotic fibroblasts in sponge granulation tissue and tissue culture were less abundant in samples from old rats relative to young rats. Myofibroblasts express alpha-smooth muscle actin, and they are believed to be important in wound contraction. There were no significant differences in overall abundance or distribution of alpha-smooth muscle actin containing myofibroblasts in granulation tissue and in cultured granulation tissue fibroblasts regardless of the age of the donor rat. The spatial distribution of myofibroblasts and apoptotic cells was distinct. Fibroblasts from granulation tissue and skin explants were placed in a collagen gel contraction assay prior to the 5th passage to determine their in vitro contractility. While granulation tissue fibroblasts from young and old rats showed similar collagen gel contractility, skin fibroblasts from old rats displayed greater collagen gel contractile behavior than young skin fibroblasts. Greater gel contractility of fibroblasts from old rats appeared to result, in large part, from the ability of those cells to cause generalized gel degradation. Gelatin zymography indicated a greater abundance of matrix metalloproteinase-2 in supernatants from gels containing skin fibroblasts from old rats. Taken together, these results suggest that the age-associated healing delay in the rat may not be related to the appearance or abundance of distinct myofibroblast or apoptotic cell populations. Proteolysis may have a significant role in delayed wound healing in aged animals.

Bronchial asthma in the medical literature of Greek antiquity.

The actual term asthma is a Greek word that is derived from the verb aazein, meaning to exhale with open mouth, to pant. The expression asthma appeared for the first time in the Iliad, with the meaning of a short-drawn breath, but the earliest text where the word is found as a medical term is the Corpus Hippocraticum. However it is difficult to determine whether in referring to "asthma," Hippocrates and his school (460-360 B.C.) meant an autonomous clinical entity or simply a symptom. The best clinical description of asthma in later antiquity is offered by the master clinician, Aretaeus of Cappadocia (1st century A.D.). The numerous mentions of "asthma" in the extensive writings of Galen (130-200 A.D.) appear to be in general agreement with the Hippocratic texts and to some extent with the statements of Aretaeus.

Neurosyphilis presenting as schizophrenialike psychosis.

OBJECTIVE: The authors present a 30-year-old pregnant and previously healthy woman with sudden onset of agitation, delusions, and frontal lobe dementia. Serum and cerebrospinal fluid findings revealed the presence of untreated syphilis. BACKGROUND: Neurosyphilis can present a variety of behavioral symptoms, including mania, depression, and psychosis. METHOD: A neuropsychiatric and neurologic workup was performed before the start of antibiotic treatment. The status of the patient was observed throughout the course of her hospital stay for 7 months. Additionally, a neuropsychological evaluation was administered before treatment, and 2 weeks and 4 months after treatment. RESULTS: After treatment of neurosyphilis, steady improvements were noted in psychotic and cognitive symptoms. By the end of 7 months, and after discontinuation of antipsychotic medication, no psychiatric symptoms were evident. CONCLUSIONS: This case emphasizes the importance of considering neurosyphilis in the differential diagnosis of acute psychosis. Furthermore, this case shows the dramatic improvement in psychiatric symptoms and cognitive dysfunction in response to the treatment of neurosyphilis.

Autonomic cardiovascular neuropathy in Sjögren's syndrome. A controlled study.

OBJECTIVE: To test patients with primary Sjögren's syndrome (SS) for evidence of autonomic neuropathy. METHODS: Thirty-two patients with primary SS and 22 age and sex matched healthy individuals were asked specific questions about symptoms suggestive of autonomic neuropathy, and were subjected to a battery of 5 cardiovascular tests: response of blood pressure to sustained hand grip, Valsalva maneuver, heart rate response to deep breathing, and heart rate and blood pressure response to standing up. The chi-squared test with Yates' correction and 95% confidence intervals were used for statistical analysis of the results. RESULTS: Sixteen patients (50%) had symptoms of autonomic neuropathy when specifically asked versus none of the controls (p < 0.0005). The frequency of abnormal responses to the tests was 68.8% in patients and 12.7% in controls (p < 0.0001). Severe autonomic cardiovascular neuropathy was found in 87.5% of the patients but in none of the healthy individuals (p < 0.0001). CONCLUSION: Our results suggest that autonomic neuropathy is a feature of a significant portion of the SS population, and such patients should have appropriate evaluation. Similarly, patients with unexplained autonomic neuropathy should be investigated for evidence of SS.

MGMT expression in murine bone marrow is a major determinant of animal survival after alkylating agent exposure.

Myelosuppression is commonly observed after alkylating agent chemotherapy due to low levels of O(6)-alkylguanine DNA alkyltransferase protein (AGT) in hematopoietic progenitors. Mice that lack AGT in all organs, O(6)-methylguanine-DNA methyltransferase gene knockout (MGMT(-/-)) mice are extremely hypersensitive to the methylating agent N-methyl-N-nitrosourea (MNU) and exhibit a 10-fold reduction in the LD(90). To determine whether bone marrow damage was the cause of the increased lethality, we transplanted 1 x 10(6) wild-type marrow into MGMT(-/-) mice and MGMT(-/-) marrow into wild-type mice and observed survival after MNU. Lethally irradiated MGMT(-/-) mice given > or = 25 mg/kg MNU 3 weeks after transplant of wild-type cells survived > 30 days (n = 11), whereas this dose was lethal to control MGMT(-/-) mice 9-12 days post treatment (n = 5). Conversely, lethally irradiated wild-type mice transplanted with MGMT(-/-) cells died after only 20-60 mg/kg MNU within 8-12 days (n = 6). No significant toxicities were found in other organs. Additionally, in an in vivo post transplant competition model, wild-type long-term repopulating cells had a > 200-fold competitive survival advantage over MGMT(-/-) cells, and after MNU treatment completely repopulated the mouse when transplanted at only one-tenth the cell number. We also observed a strong selection for transplanted marrow-derived wild-type stromal elements in the MGMT(-/-) background after drug treatment. These data indicate that alkylating agent hypersensitivity of MGMT(-/-) mice results from hematopoietic damage at the stem level. Thus, DNA repair involving AGT in hematopoietic cells is required for normal host survival following exposure to methylating and chloroethylating agents.

Relationship between cytokine-dependent cell cycle progression and MHC class II antigen expression by human CD34+ HLA-DR- bone marrow cells.

Human CD34+ HLA-DR- bone marrow cells constitute a phenotypically homogeneous population of quiescent cells. More than 97% of CD34+ HLA-DR- cells reside in the G0/G1 phase of the cell cycle. The in vitro effects of two cytokines, IL-1 alpha and IL-3, alone or in combination, on the viability, cell cycle status and acquisition of HLA-DR by this cell population were examined. Cell viability was preserved in cultures receiving cytokines, but declined steadily in cultures deprived of exogenous IL. Over a period of 4 days, IL-3 progressively induced the expression of HLA-DR although driving corresponding numbers of cells into S and G2 + M. Although IL-1 alpha induced the expression of HLA-DR, it was not as effective as IL-3 in promoting the exit of these cells from G0/G1. Combinations of IL-1 alpha and IL-3, however, exerted an even greater effect on promoting both HLA-DR expression and entry of cells into active phases of the cell cycle. Simultaneous measurement of HLA-DR expression and cell cycle status in response to IL-1 alpha and IL-3 indicated that the majority of de novo expression of HLA-DR occurred in cells that remained in G0/G1. CD34+ HLA-DR- cells cultured with IL-1 alpha and IL-3 but arrested in G0/G1 by hydroxyurea were still capable of expressing HLA-DR, demonstrating that the acquisition of HLA-DR was independent of the entry of these cells into active phases of the cell cycle. These data indicate that the survival, HLA-DR expression, and cell cycle status of human CD34+ HLA-DR- bone marrow cells are governed by regulatory cytokines such as IL-1 alpha and IL-3. In addition, the entry of these cells into active phases of the cell cycle does not seem to be a prerequisite for the expression of HLA-DR, nor does it seem that the acquisition of HLA-DR by hematopoietic progenitor cells is a marker of cells entering the S phase of the cell cycle.

Ziprasidone-associated mania: a case series and review of the mechanism.

Atypical antipsychotics are now commonly used in the treatment of bipolar disorder, as they have been shown to have effects on mania as well as psychosis. Shortly after the introduction of atypical antipsychotics, several cases of associated hypomania and mania were reported. Ziprasidone is an atypical antipsychotic recently approved by the Food and Drug Administration for the treatment of psychosis. Although ziprasidone has also been shown to be effective in treating mania, it may be associated with the induction of mania or hypomania. We report four cases of mania associated with initiation of ziprasidone, which, to our knowledge, are the first reported for this drug in bipolar patients. As ziprasidone has substantial serotonergic and noradrenergic action, we hypothesize, it may more likely induce mania than other atypical antipsychotics. We advocate future studies to evaluate ziprasidone's efficacy in treating bipolar disorder and caution clinicians that induction of mania or hypomania may be possible with this agent.

Relationship between psychiatric disease and neuropsychological impairment in HIV seropositive individuals.

Neuropsychological impairment and DSM-III-R Axis I psychiatric diagnoses were evaluated in a heterogenous group of HIV seropositive individuals and seronegative individuals with similar risk factors for HIV infection. Neuropsychological and psychiatric disorders were common in the HIV seropositive group, but there were no relationships between these two aspects of neuropsychiatric dysfunction in seropositive patients. Results indicate that psychiatric disorders in HIV seropositive individuals tend to predate infection and decrease over time following knowledge of seroconversion, suggesting that they are primarily a function of psychosocial factors. Neuropsychological disorders are specific to HIV seropositive patients and tend to increase over time following seroconversion, suggesting that they are due to neurological effects of HIV-infection.

Mapping murine loci for seizure response to kainic acid.

Mature DBA/2J (D2) mice are very sensitive to seizures induced by various chemical and physical stimuli, whereas C57BL/6J (B6) mice are relatively seizure resistant. We have conducted a genome-wide search for quantitative trait loci (QTLs) influencing the differential sensitivity of these strains to kainic acid (KA)-induced seizures by studying an F2 intercross population. Parental, F1, and F2 mice (8-10 weeks of age) were injected subcutaneously with 25 mg/kg of KA and observed for 3 h. Latencies to focal and generalized seizures and status epilepticus were recorded and used to calculate an overall seizure score. Results of seizure testing indicated that the difference in susceptibility to KA-induced seizures between D2 and B6 mice is a polygenic phenomenon with at least 65% of the variance due to genetic factors. First-pass genome screening (10-cM marker intervals) in F2 progeny (n = 257) documented a QTL of moderate effect on Chromosome (Chr) 1 with a peak LOD score of 5.5 (17% of genetic variance explained) localized between D1Mit30 and D1Mit16. Provisional QTLs of small effect were detected on Chr 11 (D11Mit224-D11Mit14), 15 (D15Mit6-D15Mit46) and 18 (D18Mit9-D18Mit144). Multiple locus models generally confirmed the Mapmaker/QTL results and also provided evidence for another QTL on Chr 4 (D4Mit9). Multilocus analysis of seizure severity suggested that additional loci on Chrs 5 (D5Mit11), 7 (D7Mit66), and 15 (D15Nds2) might also contribute to KA-induced seizure response. Overall, our results document a complex genetic determinism for KA-induced seizures in these mouse strains with contributions from as many as eight QTLs.