Ronald P. Messner

Genome Screening in Human Systemic Lupus Erythematosus: Results from a Second Minnesota Cohort and Combined Analyses of 187 Sib-Pair Families

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a loss of immunologic tolerance to a multitude of self-antigens. Epidemiological data suggest an important role for genes in the etiology of lupus, and previous genetic studies have implicated the HLA locus, complement genes, and low-affinity IgG (Fcgamma) receptors in SLE pathogenesis. In an effort to identify new susceptibility loci for SLE, we recently reported the results of a genomewide microsatellite marker screen in 105 SLE sib-pair families. By using nonparametric methods, evidence for linkage was found in four intervals: 6p11-21 (near the HLA), 16q13, 14q21-23, and 20p12.3 (LOD scores >/=2.0), and weaker evidence in another nine regions. We now report the results of a second complete genome screen in a new cohort of 82 SLE sib-pair families. In the cohort 2 screen, the four best intervals were 7p22 (LOD score 2.87), 7q21 (LOD score 2.40), 10p13 (LOD score 2.24), and 7q36 (LOD score 2.15). Eight additional intervals were identified with LOD scores in the range 1.00-1.67. A combined analysis of MN cohorts 1 and 2 (187 sib-pair families) showed that markers in 6p11-p21 (D6S426, LOD score 4.19) and 16q13 (D16S415, LOD score 3.85) met the criteria for significant linkage. Three intervals (2p15, 7q36, and 1q42) had LOD scores in the range 1.92-2.06, and another 13 intervals had LOD scores in the range of 1.00-1.78 in the combined sample. These data, together with other available gene mapping results in SLE, are beginning to allow a prioritization of genomic intervals for gene discovery efforts in human SLE.

Treatment of early lyme disease

PURPOSEnTo compare the safety and efficacy of azithromycin, amoxicillin/probenecid, and doxycycline for the treatment of early Lyme disease, to identify risk factors for treatment failure, and to describe the serologic response in treated patients.nnnPATIENTS AND METHODSnFifty-five patients with erythema migrans and two patients with flu-like symptoms alone and fourfold changes in antibody titers to Borrelia burgdorferi were randomized to receive (1) oral azithromycin, 500 mg on the first day followed by 250 mg once a day for 4 days; (2) oral amoxicillin 500 mg and probenecid 500 mg, three times a day for each for 10 days; or (3) doxcycline, 100 mg twice a day for 10 days. If symptoms were still present at 10 days, treatment was extended with amoxicillin/probenecid or doxycycline for 10 more days. Evaluations were done at study entry and 10, 30, and 180 days later.nnnRESULTSnThree of the patients who initially had symptoms suggestive of spread of the spirochete to the nervous system, one from each antibiotic treatment group, subsequently developed neurologic abnormalities, but symptoms in the other 54 patients resolved within 3 to 30 days after study entry. Six of the 19 patients (32%) (95% confidence interval, 13% to 57%) given amoxicillin/probenecid developed a drug eruption, whereas none of the patients given azithromycin or doxycycline had this complication. The presence of dysesthesias at study entry was the only risk factor significantly associated with treatment failure (p less than 0.001). By convalescence, 72% of the patients were seropositive, and 56% still had detectable IgM responses to the spirochete 6 months later.nnnCONCLUSIONSnThe three antibiotic regimens tested in this study were generally effective for the treatment of early Lyme disease, but the regimens differ in the frequency of side effects and in ease of administration.

Structural specificity of polyamines in left-handed Z-DNA formation: Immunological and spectroscopic studies

Natural polyamines putrescine, spermidine and spermine are ubiquitous cellular components. Recent studies showed that these compounds are capable of provoking a conformational transition in poly(dG-m5dC).poly(dG-m5dC) from its usual right-handed B-DNA form to a left-handed Z-DNA form at physiologically relevant cationic concentrations. We studied the efficacy of spermidine, six homologs of spermidine (H2N(CH2)nNH(CH2)3NH2, where n = 2 to 8 (n = 4 for spermidine)) and diethylene triamine to provoke the B-DNA to Z-DNA transition of poly(dG-m5dC).poly(dG-m5dC) using a monoclonal anti-Z-DNA antibody and spectroscopic techniques. The concentration of spermidine at the midpoint of B-DNA to Z-DNA transition was 30 +/- 1 microM. Chemical structural effects were significant when the spermidine homologs were used to induce the transition. The midpoint concentration increased as the number of -CH2 groups varied in relation to that of spermidine. We interpret these structural effects on the basis of molecular models of the interaction of polyamines with polynucleotides.

Suppression of human lymphocyte mitogenesis mediated by phagocyte-released reactive oxygen species: Comparative activities in normals and in chronic granulomatous disease☆

Human blood monocytes and neutrophils stimulated in vitro with phorbol myristate acetate, N-formyl-L-methionyl-L-leucyl-L-phenylalanine, activated zymosan, or heat aggregated gamma-globulin were found to suppress lymphocyte mitogenic responses. In activated phagocyte-lymphocyte cocultures, both blast transformation and [3H]-thymidine incorporation were reduced while numbers of dead cells were increased, thus suggesting a cytolethal suppressive mechanism. Suppression was prevented by catalase but not by other oxygen radical scavengers nor by cyclooxygenase inhibitors, thus implicating H2O2 as the suppressive mediator. Activated monocytes and neutrophils but not lymphocytes released measurable quantities of H2O2 into cell supernatants. However, transfer of an inhibitory effect with these supernatants was not routinely achieved. Finally, as opposed to normals, lymphocyte blastogenesis in chronic granulomatous disease patients was not inhibited by their activated phagocytes. However, catalase -reversible suppression could be restored in cocultures of normal phagocytes and patient lymphocytes. In conclusion, these studies demonstrate a potentially important mechanism whereby activated phagocytes may alter lymphocyte reactivity.

Development and evaluation of an integrated musculoskeletal disease course for medical students

BACKGROUNDnA recent survey of medical and surgical residents in the United States suggested that our current training of physicians may be inadequate to meet the increasing demand for diagnosis and treatment of musculoskeletal disorders. In response, we developed an integrated, multidisciplinary course to teach knowledge and skills related to musculoskeletal disease to second-year medical students. A three-year prospective outcomes study was conducted to evaluate the new course.nnnMETHODSnThe primary outcomes that were studied during the first year of the new course were the gains in knowledge, changes in levels of confidence, and long-term retention of skills. Secondary outcomes consisted of student and faculty satisfaction. Ten-item pre-tests and post-tests covering core course concepts were administered to students. A matched-pairs t test was used to evaluate the difference between pre-test and post-test scores. Students were also asked to rate, on a 10-point scale, how much confidence they had in their ability to perform the musculoskeletal physical examination before and after the institution of the new curriculum. A general linear model analysis with post hoc pairwise comparisons (F test) was used to evaluate the changes in the confidence levels of the students. Also, a knee examination station was organized to compare students scores before and after revision of the course. At the conclusion of the course, students rated each aspect of it on a scale of 1 to 5. Instructors were asked to rate the effectiveness of all elements of the course on the same scale.nnnRESULTSnOn the basis of student satisfaction and confidence and faculty satisfaction, the most effective changes in the curriculum were the introduction of a physical examination workshop and simulated clinical situations. Students knowledge increased significantly (p < 0.001), and their level of confidence increased significantly in thirteen specifically targeted areas (p < 0.0001). On the end-of-the-year examination assessing retention of physical examination skills, the scores for the skills emphasized in the revised course increased significantly whereas the scores for a skill not emphasized in the course remained the same. Revisions made in the second and third years after implementation of the course expanded the more successful elements and further improved student ratings.nnnCONCLUSIONSnIntegration of the three clinical disciplines related to musculoskeletal disease--orthopaedics, rheumatology, and physical medicine and rehabilitation--resulted in a highly effective introductory course for second-year medical students. The heuristic strategy of introducing core content through lectures and workshops followed by small-group teaching sessions for practice with the new knowledge effectively increased students knowledge, confidence, and satisfaction.

A transient defect in leukocytic bactericidal capacity

Abstract Leukocytes from three previously healthy patients with overwhelming infection were found to be defective in their ability to kill Staphylococcus aureus . Leukocytes from two patients with infections due to Staphylococcus aureus were unable to kill either the infecting organism or a laboratory strain of S. aureus . One of these patients retained the ability to kill Escherichia coli . The third patient had severe E. coli and Bacteroides infection. His leukocytes could kill the infecting E. coli normally, but failed to kill the laboratory strain of S. aureus . After recovery leukocytes from all three patients killed all bacteria normally. The defective leukocytes had normal surface receptors for IgG and complement, stained normally for myeloperoxidase, and reduced nitro blue tetrazoleum dye normally. In one patient the defect was found to be in phagocytosis rather than intracellular killing of the organism.

Immunological detection of B-DNA to Z-DNA transition of polynucleotides by immobilization of the DNA conformation on a solid support☆

We studied the B-DNA to Z-DNA transition of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) in the presence of NaCl using an enzyme immunoassay. The polynucleotides were coated on microtiter plates at varying concentrations of NaCl and treated with a monoclonal anti-Z-DNA antibody, Z22. The plates were subsequently treated with alkaline phosphatase conjugated polyvalent mouse immunoglobulins and the enzyme substrate, p-nitrophenyl phosphate. The color development due to the enzyme-substrate reaction was quantitated using a microplate autoreader. Our results show that the antibody does not recognize the polynucleotides in the B-DNA conformation and binds strongly to the Z-DNA conformation. A smooth transition curve is obtained at intermediate concentrations of the counterions. From the transition curves, we determined the concentration of the counterions at the midpoint of B-DNA to Z-DNA transition. The midpoint concentrations for poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) are 2.3 and 0.74 M NaCl, respectively. Using the immunological method, we also examined the B-DNA to Z-DNA transition of poly(dG-m5dC).poly(dG-m5dC) in the presence of naturally occurring polyamines. The midpoint concentrations of the polyamines are as follows: putrescine, 2.5 mM; spermidine, 34 microM; spermine, 1.8 microM. The midpoint values determined by the enzyme immunoassay are in good agreement with those determined by circular dichroism and ultraviolet absorption spectroscopic measurements. These results demonstrate that immobilization of a preexisting conformation or a mixture of conformations of DNA on a solid support followed by a titration of the DNA conformations using a monoclonal anti-DNA antibody is an excellent method to study the conformational dynamics of DNA.

Hexammineruthenium (III) chloride: a highly efficient promoter of the B-DNA to Z-DNA transition of poly-(dG-m5dC)·poly(dG-m5dC) and poly(dG-dC)·poly(dG-dC)

The effects of Ru(NH3)(3+)6 on the conformation of poly(dG-m5dC).poly(dG-m5dC) and poly(dG-dC).poly(dG-dC) were studied by circular dichroism (CD) spectroscopy. Ru(NH3)(3+)6 at very low concentrations provokes the Z-DNA conformation in both polynucleotides. In the presence of 50 mM NaCl, the concentration of Ru(NH3)(3+)6 at the midpoint of B to Z transition of poly(dG-m5dC).poly(dG-m5dC) is 4 microM compared to 5 microM for Co(NH3)(3+)6. The half-lives of B to Z transition of poly(dG-m5dC).poly(dG-m5dC) in the presence of 10 microM Ru(NH3)(3+)6 and Co(NHG3)(3+)6 are at 23 and 30 min, respectively. The concentration of Ru(NH3)(3+)6 at the midpoint of B to Z transition of poly(dG-dC).poly(dG-dC) is 50 microM. These results demonstrate that Ru(NH3)(3+)6 is a highly efficient trivalent cation for the induction of B to Z transition in poly(dG-m5dC).poly(dG-m5dC) and poly(dG-dC).poly(dG-dC). In contrast, Ru(NH3)(3+)6 has no significant effect on the conformation of calf thymus DNA, poly(dA-dT).poly(dA-dT) and poly(dA-dC).poly(dG-dT).

Multiple Viral Determinants Mediate Myopathogenicity in Coxsackievirus B1-Induced Chronic Inflammatory Myopathy

ABSTRACT Mice infected with myopathic coxsackievirus B1 Tucson (CVB1T) develop chronic inflammatory myopathy (CIM) consisting of hind limb weakness and inflammation. Amyopathic virus variants are infectious but attenuated for CIM. In this report, viral clones, chimeras, and sequencing were used to identify viral determinants of CIM. Chimeras identified several regions involved in CIM and localized a weakness determinant to nucleotides 2493 to 3200 of VP1. Sequencing of multiple clones and viruses identified five candidate determinants that were strictly conserved in myopathic viruses with one located in the 5′ untranslated region (UTR), three in the VP1 capsid, and one in the 3C protease. Taken together, these studies implicate Tyr-87 and/or Val-136 as candidate determinants of weakness. They also indicate that there are at least two determinants of inflammation and one additional determinant of weakness encoded by myopathic CVB1T.

INTERACTIONS AMONG RHEUMATOID FACTORS, γG ANTIBODIES, LYMPHOCYTES, AND PHAGOCYTES

I t has now been clearly established that anti-7-globulins are indeed antibodies with specificity for autologous 7-globulin. Besides the originally described 19s human rheumatoid factors,; over the past few years various types of anti-7globulins of different immunoglobulin classes,z5 showing an interesting heterogeneity of specificites,Gg have been reported. The real task now at hand seems to he definition of the physiological role of human anti-7-globulins. In approaching this problem, we have turned to studies on the interaction of isolated human or rabbit anti-7-globulins with two basic cellular phenomena, phagocytosis and lymphocyte transformation. It was hoped that these studies would elucidate mechanisms that might be modified, or in some way directed, through interactions between circulating or tissue-bound anti-7-globulins and cells which participate directly in tissue lesions of disease.