Soluble antigen therapy induces apoptosis of autoreactive T cells preferentially in the target organ rather than in the peripheral lymphoid organs

The administration of soluble myelin proteins is an effective way of down-regulating the inflammation in the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. To shed more light on the mechanism of this antigen-specific therapy, we determined the effect of the intraperitoneal (i.p.) injection of soluble myelin basic protein (MBP) on Tcell apoptosis in the CNS and peripheral lymphoid organs of Lewis rats with EAE induced by inoculation with MBP and complete Freund's adjuvant. In particular we assessed the level of apoptosis of Vbeta8.2+ Tcells, which constitute the predominant encephalitogenic MBP-reactive T cell population in the Lewis rat. The daily i.p. injection of MBP for 3 days from the onset of neurological signs inhibited the further development of neurological signs of EAE. Using two-color flow cytometry we found that a single i.p. injection of MBP increased the level of apoptosis of the Vbeta8.2+ T cell population in the CNS to 26.2% compared to 7.4% in saline-treated rats and 7.6% in ovalbumin-treated rats. In contrast, treatment with MBP did not increase the level of apoptosis of the Vbeta8.2+ population in the popliteal lymph node draining the inoculation site (1.4%) or in the spleen (1.6%) above that occurring in saline-treated rats (1.6% and 1.1%, respectively). Limiting dilution analysis revealed that the frequency of T cells reactive to the major encephalitogenic epitope, MBP72-89, was decreased in the CNS but not in the popliteal lymph node by this treatment. Three-color flow cytometry in MBP-treated rats demonstrated that CNS Vbeta8.2+ T cells expressing Fas (CD95) and Fas ligand were highly vulnerable to apoptosis compared to Vbeta8.2+ Tcells not expressing these proteins. We conclude that the i.p. injection of MBP increases the spontaneously occurring Fas-mediated activation-induced apoptosis of autoreactive T cells in the CNS in EAE and that this contributes to the therapeutic effect of the injection.     

Frequency of cervical intraepithelial neoplasia following large loop excision of the transformation zone

Acute ophthalmoparesis (AO) is a monophasic disease characterized by acute onset of paresis of the extraocular muscles without ataxia or areflexia. Here we report a case of AO with gadolinium enhancement in the cisternal portion of the abducens nerves using contrast-enhanced three-dimensional magnetic resonance imaging.     

DNA minor groove recognition by a tetrahydropyrimidinium analogue of hoechst 33258: NMR and molecular dynamics studies of the complex with d(GGTAATTACC)2

Hoechst 43254 (H43254), a 2,3,4,5-tetrahydropyrimidin-1-ium analogue of the bis-benzimidazole minor groove binding agent Hoechst 33258 (H33258), has been studied by NMR and restrained molecular dynamics in its complex with d(GGTAATTACC)2. We investigate the origin of the enhanced complex stability afforded by the replacement of the N-methylpiperazine ring of H33258 with the tetrahydropyrimidinium ring of H43254, the latter presenting the opportunity for specific minor groove-directed recognition through a pyrimidinium NH. A set of 25 drug-DNA NOEs define the binding site with some precision and are used as part of the structural analysis using restrained molecular dynamics simulations considering explicit solvation and the treatment of electrostatic interactions using the particle mesh Ewald method within AMBER 4.1. Starting with three different initial structures with the drug located at different sites in the groove (pairwise RMSD 4.3-12.6 A) we arrive at three very similar structures (pairwise RMSD 0.80-1.34 A) representing one converged binding site at the centre of the AATT tract. Two of the three structures show the tetrahydropyrimidinium ring to be suitably positioned for an -NH to adenine N3 hydrogen bond suggesting that electrostatic interactions may play an important role in the enhanced affinity as well as imparting additional A-T specificity. The NMR data show that the pyrimidinium NH interaction is dynamic since signal averaging from the two sides of the ring indicate rapid rotations in the bound form.     

Long-term regulation of renal urea transporter protein expression in rat

To test the hypothesis that the abundance of the apical urea transporter of the inner medullary collecting duct (IMCD) is regulated in vivo by factors associated with altered water balance, immunoblots of rat inner medullary membrane fractions were probed with rabbit polyclonal antibodies against the renal urea transporter (RUT) gene product. In inner medullas of Brattleboro rats, which manifest severe chronic water diuresis, a 117-kD band was seen, in addition to the previously described 97-kD band. These two bands were detectable by antibodies directed against two different regions of the RUT sequence. When Brattleboro rats were treated with a 5-d infusion of arginine vasopressin (AVP) by osmotic minipump, the 117-kD band was markedly diminished, whereas the 97-kD band was unchanged. Simultaneous infusion of the diuretic agent furosemide prevented the AVP-induced decrease in the 117-kD band. In AVP-infused Sprague Dawley rats, the 117-kD band was barely perceptible. However, when AVP-treated rats were infused with furosemide for 5 d, the 117-kD band was markedly accentuated, whereas the 97-kD band was unchanged. The abundance of the 117-kD RUT protein in the renal papilla was inversely correlated with dietary protein intake. Further immunoblotting studies revealed that the 117-kD protein is heavily expressed in IMCD cells and not in non-collecting duct components of the inner medulla, and is present in low-density microsome fractions from inner medulla. From this study, the following conclusions can be made: (1) The collecting duct urea transporter is present in at least two forms (97 and 117 kD) in the IMCD. (2) The expression level of the 117-kD urea transporter protein is regulated and is inversely correlated with medullary osmolality and urea concentration, but does not correlate with circulating AVP level. (3) Although AVP regulates RUT function on a short-term basis, long-term changes in AVP levels do not increase RUT abundance.     

Double pessary use in grade 4 uterine and vaginal prolapse

BACKGROUND: Certain situations dictate conservative management of grade 4 uterine or vaginal vault prolapse in women. The reduction of a prolapse may not be possible if the vagina cannot retain a single pessary. We have used double pessaries in the management of this condition. TECHNIQUE: With the patient in the dorsal lithotomy position, either a Donut or Inflatoball (Milex Products Inc., Chicago, IL) pessary is inserted and pushed into the vagina as far as is comfortable. A second pessary, either a flexible Gellhorn or Shaatz (Milex Products Inc.), then is placed caudad to the first. EXPERIENCE: We have used this approach in five women who have been followed for 7-15 months. There have been no erosions or vaginitis and all the women experienced symptomatic relief. CONCLUSION: In women who want or require conservative management of grade 4 prolapse and are unable to retain a single pessary, the placement of two pessaries often will be successful.     

Diagnosis of Trichomonas vaginalis infection by PCR using vaginal swab samples

Trichomonas vaginalis infection is the most prevalent nonviral sexually transmitted disease (STD) in the world. A PCR test using vaginal swab samples for the detection of T. vaginalis was developed to add T. vaginalis infection to the growing list of STDs that can be detected by DNA amplification techniques. A primer set, BTUB 9/2, was designed to target a well-conserved region in the beta-tubulin genes of T. vaginalis. All strains (15 of 15) of T. vaginalis tested were successfully detected by PCR giving a single predicted product of 112 bp in gel electrophoresis. No such targeted product was amplified with DNA from Trichomonas tenax, Trichomonas gallinae, Chlamydia trachomatis, Neisseria gonorrhoeae, Giardia lamblia, Chilomastix sulcatus, Dientamoeba fragilis, and Entamoeba histolytica. An optimal analytical sensitivity of one T. vaginalis organism per PCR was achieved. Culture, performed with the Inpouch TV culture system, was examined daily with a light microscope to identify T. vaginalis. Twenty-three of 350 (6.6%) vaginal swab samples from women attending an army medical clinic were culture positive for T. vaginalis. Of these culture positive specimens, PCR detected 22 of 23 (96%) with primer set BTUB 9/2, and wet preparation detected only 12 of 23 (52%). Seventeen specimens were BTUB 9/2-PCR positive and culture negative. Ten of these discordant specimens were determined to be as true positive by PCR using primer sets TVA 5-1/6 and/or AP65 A/B, which target different regions in the T. vaginalis genome, and seven were determined to be false positive. The sensitivity of BTUB 9/2-PCR was 97% and the specificity was 98%. The sensitivities of culture and wet preparation were 70 and 36%, respectively. The diagnosis of T. vaginalis infection by PCR is a sensitive and specific method that could be incorporated into a joint strategy for the screening of multiple STDs by using molecular amplification methods.     

Obese gene expression in porcine adipose tissue is reduced by food deprivation but not by maintenance or submaintenance intake

The wide distribution of corticotropin-releasing factor (CRF) and substance P (SP)-immunoreactive cell bodies, nerve terminals and corresponding receptors in pressor nuclei controlling emotion and stress implies that CRF and SP may play important roles in pressor responses of these nuclei; hence CRF or SP was microinjected into these nuclei respectively in Wistar male rats anesthetized with urethane to test this possibility. Microinjection of CRF into nucleus amygdaloideus centralis, nucleus paraventricularis, nucleus ventromedialis, lateral hypothalamus-perifornical region, periaqueductal gray matter, nucleus parabrachialis, locus coeruleus or rostral ventrolateral medulla respectively could evoke pressor responses (but CRF injection into nucleus dorsomedialis could not elicit significant pressor responses). Injection of substance P into all the above nuclei could also elicit hypertensive responses of different magnitudes, whereas normal saline injection into these nuclei had no effect. These results indicate that both CRF and SP in the above mentioned nuclei may play important roles in hypertension induced by prolonged emotional stress.     

Chronokinetics of active biliary ampicillin secretion in rats

To estimate the functional maturity of the phagocytic defence in neonatal calves, we analyzed the characteristics of blood phagocytes from calves (n = 10) 1 h post partum (p.p.) and 4 h p.p. At 1 h p.p., all calves were colostrum-deprived, while 5 calves had received colostrum before the 4 h p.p. sampling. The results were compared to those obtained from 3-9-week-old calves (n = 10). Phagocytic and oxidative burst activity of polymorphonuclear leukocytes (PMNL) and monocytes were determined in whole blood and separately analyzed by flow cytometry. In neonates prior to colostrum ingestion (1 h p.p.), phagocytic activity of PMNL against non-preopsonized E. coli was lower when compared to PMNL of 3-9-week-old calves. Opsonization of bacteria with pooled plasma from adult animals only partially restituted this lower PMNL phagocytic activity, indicating that humoral as well as cellular aspects of PMNL phagocytosis are altered in neonatal calves. In contrast to PMNL, monocytes of neonates exhibited an enhanced phagocytic activity. The oxidative burst activity of PMNL, as well as of monocytes was higher in newborn calves. During the first 4 h of life, the activities of blood phagocytes changed. Colostrum ingestion was accompanied by an increase in the percentage of phagocytizing PMNL and monocytes. This increase was absent in colostrum-deprived calves. In contrast, the oxidative burst activity of phagocytes decreased with age. In monocytes, the decrease of oxidative burst activity was only observed in colostrum-fed calves. In conclusion, some blood phagocyte functions in calves were found to be immature at birth, but these functions are presumably compensated by high absolute PMNL numbers and by other the more active mechanisms.     

Work time estimates for ophthalmic diagnoses and procedures. Results from the Eye Care Workforce Study

OBJECTIVE: To compare the clinical presentation, time elapsed to diagnosis, and survival of elderly patients (> or = 65 years) with that in younger patients with malignant primary brain tumors. DESIGN: Retrospective cohort study. SETTING: Four hospitals in Minneapolis, Minn. PATIENTS: Seven hundred fourteen patients diagnosed as having and treated for primary malignant brain tumors between 1980 and 1995; 230 (32%) were 65 years or older. MAIN OUTCOME MEASURES: The type and duration of the chief presenting symptom, the time elapsed to diagnosis, the treatment modalities used, and patient survival were analyzed. RESULTS: Time elapsed from onset of symptom to diagnosis was not longer for elderly patients than younger ones, with the exception of patients aged 18 to 24 years, who had a significantly longer delay in diagnosis (P = .004). Elderly patients were significantly less likely to present with headache or seizure (P<.001), and more likely to present with confusion, aphasia, or memory loss (for each, P<.001). With the single exception of confusion, the duration of all other presenting symptoms was not significantly longer for patients 65 years and older compared with younger patients. Survival is significantly reduced in older patients, and appears to worsen significantly in patients 45 years and older (P<.001). A significantly higher proportion of patients 65 years and older with glioblastoma multiforme received no treatment (P = .004) if diagnosed after 1990. CONCLUSIONS: Elderly patients (> or = 65 years) with malignant brain tumors are diagnosed as promptly as younger patients, although they have a markedly different constellation of symptoms. Since diagnosis of brain tumors continues to improve in the elderly, it may be more difficult to ascribe the steady increase in incidence to artifactual factors.     

Cocaine benzoyl thioester: synthesis, kinetics of base hydrolysis, and application to the assay of cocaine esterases

The synthesis and characterization of diastereomers of cocaine benzoyl thioester is described. Allococaine benzoyl thioester and allopseudococaine benzoyl thioester were synthesized by the conjugate addition of p-methoxytolyl thiol to ecgonine methyl ester followed by debenzylation and benzoylation. The absolute structure of the hydrochloride salt of the major ecgonine p-methoxytolyl sulfide formed was determined by single-crystal diffraction analysis and used to establish the addition geometry. When placed in aqueous solution, the cocaine benzoyl thioester diastereomers hydrolyzed to give thioecgonine methyl ester. The rate of cocaine benzoyl thioester hydrolysis was carefully investigated spectrophotometrically by using the Ellman reagent. At neutral pH, the hydrolysis of the diastereomers was found to proceed at detectable rates. Upon increasing pH, the rate of hydrolysis of cocaine benzoyl thioester diastereomers was increased and the reaction was catalyzed by basic buffer species. In addition to defining the kinetics of hydrolysis in aqueous solution, cocaine benzoyl thioester was utilized as a highly efficient method to monitor the activity of cholinesterases and pig liver esterase. Use of cocaine benzoyl thioester represents a rapid and sensitive way to screen for cocaine esterase activity.