A defined medium for a fastidious Spiroplasma.

A defined medium (H-1) was developed for cultivation of the suckling mouse cataract agent, Spiroplasma mirum, a fastidious member of the class Mollicutes that causes cataracts and chronic brain infection in inoculated neonate mice. The H-1 medium was used to show the importance of sphingomyelin as a growth factor for the culture of the spiroplasma in vitro. The growth of Spiroplasma mirum and the pathology it induces in sphingomyelin-rich tissues in vivo may be related to this dependency.

Disruption of the human SCL locus by "illegitimate" V-(D)-J recombinase activity.

A fusion complementary DNA in the T cell line HSB-2 elucidates a provocative mechanism for the disruption of the putative hematopoietic transcription factor SCL. The fusion cDNA results from an interstitial deletion between a previously unknown locus, SIL (SCL interrupting locus), and the 5' untranslated region of SCL. Similar to 1;14 translocations, this deletion disrupts the SCL 5' regulatory region. This event is probably mediated by V-(D)-J recombinase activity, although neither locus is an immunoglobulin or a T cell receptor. Two other T cell lines, CEM and RPMI 8402, have essentially identical deletions. Thus, in lymphocytes, growth-affecting genes other than immune receptors risk rearrangements.

Cultivation of the Drosophila sex-ratio spiroplasma.

Uncultivable for more than 25 years, the sex-ratio spiroplasma of Drosophila willistoni grew in a tissue culture medium (H-2) containing an embryo-derived lepidopteran cell line (IPLB-TN-R(2)). After adaptation, it grew in a cell-free H-2 medium. This success demonstrates the usefulness of cell culture systems for cultivation of fastidious microorganisms and facilitates study of the sex-ratio trait in Drosophila.

Lesions of the medial prefrontal cortex enhance the early phase of psychogenic fever to unexpected sucrose concentration reductions, promote recovery from negative contrast and enhance spontaneous recovery of sucrose-entrained anticipatory activity.

Two groups of rats, one bearing bilateral excitotoxic lesions of the medial prefrontal cortex (mPFC) and one sham-lesioned group, were run in a successive negative contrast paradigm. Both groups had telemeters implanted to monitor core temperature and activity. After ad libitum baseline and food restriction to 85% body weights, rats received a sucrose solution once daily for 5 min and 30 s at 10:30 h. They received their preshift 32% sucrose solution for 14 days followed by a sucrose concentration reduction (downshift) to 4% sucrose for 12 days. Rats were then upshifted to 32% for six additional days before being downshifted to 4% for the next 6 days. There were no differences in intake of the 32% sucrose during the preshift. All rats showed profound suppression of intake upon the shift to 4% sucrose. On the first day of the unexpected 4% sucrose, lesioned rats showed an enhanced psychogenic fever compared with Shams, whereas on the second day of 4% sucrose they showed an impaired ability to blunt that fever compared with Shams. In addition, lesioned rats showed greater rates of recovery and asymptotic drinking of the subsequent 4% sucrose solution than Shams, indicating impairments in the encoding or retrieval of the shift. In addition, lesioned rats showed enhanced entrainment to the 32% sucrose meals, normal damping of anticipation, and enhanced spontaneous recovery of anticipatory thermal responses to the calorically impoverished 4% solutions. These failures to inhibit responding point to a failure in interference learning in rats bearing lesions of the mPFC.

Use of allopurinol with low-dose 6-mercaptopurine in inflammatory bowel disease to achieve optimal active metabolite levels: a review of four cases and the literature.

BACKGROUND: At least one-third of patients with inflammatory bowel disease do not respond or are intolerant to therapy with 6-mercaptopurine (6-MP). A subgroup fails to attain optimal levels of 6-thioguanine nucleotide (6-TGN) and instead shunts to 6-methylmercaptopurine nucleotide (6-MMPN). PATIENTS AND METHODS: A retrospective chart review was conducted, and four patients are described who had been previously unable to achieve optimal 6-TGN metabolite levels until allopurinol was added to their treatment. RESULTS: All four patients achieved optimal 6-TGN levels and undetectable 6-MMPN with a mean 6-MP dose of 0.49 mg/kg. Three achieved steroid-free clinical remission. Two of those three patients had normalization of liver enzymes; one patient had baseline normal liver enzymes despite an initial 6-MMPN level of 27,369 pmol/8 x 10(8) red blood cells. Two patients experienced reversible leukopenia. CONCLUSIONS: Combination allopurinol and low-dose 6-MP is an effective means to achieve optimal metabolite levels and steroid-free clinical remission in previously refractory patients. Caution is advised.

Triiodothyronine-induced thyrotoxicosis increases mononuclear leukocyte beta-adrenergic receptor density in man.

beta-Adrenergic receptors are increased in some tissues of experimentally thyrotoxic animals but are reported to be unchanged in mononuclear leukocytes of spontaneously thyrotoxic humans. We examined the effects of triiodothyronine (100 mug/d for 7 d) and placebo on high-affinity mononuclear leukocyte beta-adrenergic receptors in 24 normal human subjects, using a double-blind design. beta-Adrenergic receptors were assessed by specific binding of the antagonist (-)[(3)H]dihydroalprenolol. Triiodothyronine administration resulted in objective evidence of moderate thyrotoxicosis and an increase in mean (-)[(3)H]dihydroalprenolol binding from 25+/-3 to 57+/-9 fmol/mg protein (P < 0.001). The latter was attributable, by Scatchard analysis, to an increase in beta-adrenergic receptor density (967 +/- 134 to 2250 +/- 387 sites per cell, P < 0.01); apparent dissociation constants did not change. Placebo administration had no effects. Marked inter- and intraindividual variation in mononuclear leukocyte beta-adrenergic receptor density was also noted. Because this was approximately threefold greater than analytical variation, it is largely attributable to biologic variation. Thus, we conclude: (a) The finding of a triiodothyronine-induced increase in mononuclear leukocyte beta-adrenergic receptor density in human mononuclear leukocytes, coupled with similar findings in tissues of experimentally thyrotoxic animals, provides support for the use of mononuclear leukocytes to assess receptor status in man. (b) There is considerable biologic variation in beta-adrenergic receptor density in man. (c) The findings of thyroid hormone-induced increments in beta-adrenergic receptor density provide a plausible mechanism for the putative enhanced responsiveness to endogenous catecholamines of patients with thyrotoxicosis.

Mapping brain c-Fos immunoreactivity after insulin-induced voluntary lard intake: insulin- and lard-associated patterns.

In addition to the inhibitory role of central insulin on food intake, insulin also acts to promote lard intake. We investigated the neural pathways involved in this facet of insulin action. Insulin or saline was infused into either the superior mesenteric or right external jugular veins of streptozotocin-diabetic rodents with elevated steady-state circulating corticosterone concentrations. After postsurgical recovery, rats were offered the choice of chow or lard to eat. Irrespective of the site of venous infusion, insulin increased lard and decreased chow intake. After 4 days, lard was removed for 8 h. On return for 1 h, only insulin infused into the superior mesenteric vein resulted in lard intake. This facilitated distinction between the effects of circulating insulin concentrations (similar in the two insulin-infused groups) and lard ingestion on the patterns of c-Fos(+) cells in the brain, termed insulin- and lard-associated patterns, respectively. Insulin-associated changes in c-Fos(+) cell numbers were evident in the arcuate nucleus, bed nucleus of the stria terminalis and substantia nigra pars compacta, concomitant with elevated leptin levels and reduced chow intake. Lard-associated changes in c-Fos(+) cell numbers were observed in the nucleus of the tractus solitarius, lateral parabrachial nucleus, central nucleus of the amygdala, ventral tegmental area, nucleus accumbens shell and the prefrontal cortex, and were associated with lower levels of triglycerides and free fatty acids. The anterior paraventricular thalamic nucleus exhibited both patterns. These data collectively fit into a framework for food intake and reward and provide targets for pharmacological manipulation to influence the choice of food intake.

Limited brain diffusion of the glucocorticoid receptor agonist RU28362 following i.c.v. administration: implications for i.c.v. drug delivery and glucocorticoid negative feedback in the hypothalamic-pituitary-adrenal axis.

The experiments described herein present a method for tracking diffusion of the glucocorticoid receptor agonist RU28362 in brain following i.c.v. drug administration. A useful property of glucocorticoid receptor is that it is primarily cytoplasmic when unbound and rapidly translocates to the nucleus when bound by ligand. Thus, removal of endogenous glucocorticoids by adrenalectomy allows us to identify brain regions with activated glucocorticoid receptor after i.c.v. glucocorticoid receptor agonist treatment by examining the presence or absence of nuclear glucocorticoid receptor immunostaining. We have previously demonstrated that an i.p. injection of 150 microg/kg RU28362 1 h prior to restraint stress is sufficient to suppress stress-induced hypothalamic-pituitary-adrenal axis hormone secretion [Ginsberg AB, Campeau S, Day HE, Spencer RL (2003) Acute glucocorticoid pretreatment suppresses stress-induced hypothalamic-pituitary-adrenal axis hormone secretion and expression of corticotropin-releasing hormone hnRNA but does not affect c-fos mRNA or fos protein expression in the paraventricular nucleus of the hypothalamus. J Neuroendocrinol 15:1075-1083]. We report here, however, that in rats i.c.v. treatment with a high-dose of RU28362 (1 microg) 1 h prior to stressor onset does not suppress stress-induced hypothalamic-pituitary-adrenal axis activity. We then performed a series of experiments to examine the possible differences in glucocorticoid receptor activation patterns in brain and pituitary after i.c.v. or i.p. treatment with RU28362. In a dose-response study we found that 1 h after i.c.v. injection of RU28362 (0.001, 0.1 and 1.0 microg) glucocorticoid receptor nuclear immunoreactivity was only evident in brain tissue immediately adjacent to the lateral or third ventricle, including the medial but not more lateral portion of the medial parvocellular paraventricular nucleus of the hypothalamus. In contrast, i.p. injection of RU28362 produced a uniform predominantly nuclear glucocorticoid receptor immunostaining pattern throughout all brain tissue. I.c.v. injection of the endogenous glucocorticoid receptor agonist, corticosterone (1 microg) also had limited diffusion into brain tissue. Time-course studies indicated that there was not a greater extent of nuclear glucocorticoid receptor immunostaining present in brain after shorter (10 or 30 min) or longer (2 or 3 h) intervals of time after i.c.v. RU28362 injection. Importantly, time-course studies found that i.c.v. RU28362 produced significant increases in nuclear glucocorticoid receptor immunostaining in the anterior pituitary that were evident within 10 min after injection and maximal after 1 h. These studies support an extensive literature indicating that drugs have very limited ability to diffuse out of the ventricles into brain tissue after i.c.v. injection, while at the same time reaching peripheral tissue sites. In addition, these studies indicate that significant occupancy of some glucocorticoid receptor within the paraventricular nucleus of the hypothalamus and pituitary is not necessarily sufficient to suppress stress-induced hypothalamic-pituitary-adrenal axis activity.

Specific and time-dependent effects of glucocorticoid receptor agonist RU28362 on stress-induced pro-opiomelanocortin hnRNA, c-fos mRNA and zif268 mRNA in the pituitary.

This study examined the effects of the glucocorticoid receptor (GR) agonist RU28362 on stress-induced gene expression in the pituitary of rats to investigate mechanisms of glucocorticoid negative feedback in vivo. In an initial experiment, acute restraint stress produced rapid (within 15 min) induction of c-fos mRNA, zif268 mRNA and pro-opiomelanocortin (POMC) hnRNA within the anterior and intermediate/posterior pituitary as determined by quantitative real-time polymerase chain reaction. Treatment with RU28362 (150 microg/kg, i.p.) 60 min before restraint inhibited adrenocorticotrophic hormone (ACTH) and corticosterone secretion and selectively suppressed the stress-induced increase in POMC hnRNA in the anterior pituitary gland. The failure of RU28362 to surpress the stress-induced rise in c-fos and expression of zif268 mRNA suggests that the central release of ACTH secretagogues was not affected at this time point by treatment with the GR agonist. Rather, the inhibition of ACTH release appeared to be due to a direct effect of RU28362 within the pituitary. A follow-up time-course study varied the interval (10, 60 or 180 min) between RU28362 pretreatment and the onset of restraint. The stress-induced increase in POMC hnRNA was completely blunted by RU28362 treatment within 10 min of treatment, although the stress induced hormone secretion, c-fos mRNA and zif268 mRNA were unaffected. The rapid inhibition of the stress-induced rise in POMC hnRNA in the anterior pituitary appears to reflect direct, GR-mediated suppression of POMC gene expression. RU28362 pretreatment 180 min before restraint onset was sufficient to suppress the stress-induced expression in the anterior pituitary gland of all three genes examined. Thus, the delayed negative feedback effects on hypothalamic-pituitary-adrenal axis activity that emerged after 180 min after glucocorticoid treatment were not evident at 60 min. Taken together, the data suggest that the inhibition of the stress-induced release of ACTH apparent within the first hour of glucocorticoid exposure is effected at the level of the pituitary gland. The delayed glucocorticoid effects evident 180 min after RU28362 treatment may include glucocorticoid actions in the brain and additional actions within the pituitary.

Chemokine-adjuvanted electroporated DNA vaccine induces substantial protection from simian immunodeficiency virus vaginal challenge.

There have been encouraging results for the development of an effective HIV vaccine. However, many questions remain regarding the quality of immune responses and the role of mucosal antibodies. We addressed some of these issues by using a simian immunodeficiency virus (SIV) DNA vaccine adjuvanted with plasmid-expressed mucosal chemokines combined with an intravaginal SIV challenge in rhesus macaque (RhM) model. We previously reported on the ability of CCR9 and CCR10 ligand (L) adjuvants to enhance mucosal and systemic IgA and IgG responses in small animals. In this study, RhMs were intramuscularly immunized five times with either DNA or DNA plus chemokine adjuvant delivered by electroporation followed by challenge with SIVsmE660. Sixty-eight percent of all vaccinated animals (P<0.01) remained either uninfected or had aborted infection compared with only 14% in the vaccine naïve group. The highest protection was observed in the CCR10L chemokines group, where six of nine animals had aborted infection and two remained uninfected, leading to 89% protection (P<0.001). The induction of mucosal SIV-specific antibodies and neutralization titers correlated with trends in protection. These results indicate the need to further investigate the contribution of chemokine adjuvants to modulate immune responses and the role of mucosal antibodies in SIV/HIV protection.

Innovative clinical trial designs to rationalize TB vaccine development.

A recent trial of a leading tuberculosis (TB) vaccine candidate in 3000 South African infants failed to show protection over that from BCG alone, and highlights the difficulties in clinical development of TB vaccines. Progression of vaccine candidates to efficacy trials against TB disease rests on demonstration of safety and immunogenicity in target populations and protection against challenge in preclinical models, but immunologic correlates of protection are unknown, and animal models may not be predictive of results in humans. Even in populations most heavily affected by TB the sample sizes required for Phase 2b efficacy trials using TB disease as an endpoint are in the thousands. Novel clinical trial models have been developed to evaluate candidate TB vaccines in selected populations using biologically relevant outcomes and innovative statistical approaches. Such proof of concept studies can be used to more rationally select vaccine candidates for advancement to large scale trials against TB disease.

beta 2- and alpha 2-adrenergic receptors and receptor coupling to adenylate cyclase in human mononuclear leukocytes and platelets in relation to physiological variations of sex steroids.

In view of evidence, largely in animals, indicating effects of sex steroids on adrenergic receptors, we measured mononuclear leukocyte (MNL) beta 2-adrenergic receptors and adenylate cyclase sensitivity to stimulation by isoproterenol as well as platelet alpha 2-adrenergic receptors and sensitivity of sodium fluoride-stimulated adenylate cyclase to inhibition by epinephrine in 3 groups of normal humans with physiologically disparate levels of testosterone, estradiol, and progesterone (10 normal men and 10 normal women, the latter sampled in both the follicular and luteal phases of their menstrual cycles). Differences in testosterone, estradiol, and progesterone were as expected; testosterone levels were 10-fold higher in men, and progesterone levels were 20-fold higher in luteal phase women. T4, cortisol , and norepinephrine levels did not differ. Basal plasma epinephrine concentrations were slightly but significantly higher in luteal phase women [34 +/- 5 (+/-SE) pg/ml] than in follicular phase women (16 +/- 3 pg/ml; P less than 0.01) or men (20 +/- 3 pg/ml; P less than 0.05). There were no significant differences among these 3 groups in the densities or affinities of MNL beta 2-adrenergic or platelet alpha 2-adrenergic receptors or in the corresponding MNL and platelet adenylate cyclase sensitivities. Thus, there is not a generalized effect of physiological variations of testosterone, estradiol, and progesterone on adrenergic receptors or adenylate cyclase. To the extent that the adrenergic receptors and adenylate cyclase activities of circulating cells reflect those of extravascular catecholamine target cells, these data provide no support for a role of physiological variations of testosterone, estradiol, or progesterone in the regulation of catecholamine action in humans.

Antibodies to vaccinia and measles viruses in multiple sclerosis patients.

A virologic comparison was made of 144 patients with multiple sclerosis, 34 of their healthy siblings, and 40 patients with other neurologic diseases (OND). Antibodies in serum and cerebrospinal fluid (CSF) to vaccinia and measles viruses were measured, and these were correlated in the multiple sclerosis patients with the clinical characteristics of their disease. The CSF antibody to vaccinia virus was more frequent and at a higher titer in multiple sclerosis patients than in either control group. Moreover, a statistically significant increase was found in both frequency and titer of CSF vaccinia antibody in patients with the progressive form of the disease as compared with those classified as relapsing-remitting. Statistically significant differences between multiple sclerosis patients and their siblings were not observed for CSF or serum measles virus antibody, although both groups had significantly higher serum antibody titers than patients with OND.

T-cell alterations in late postpoliomyelitis.

The immunologic status of patients with prior poliomyelitis was studied using two-color flow cytometric analyses. Ten lymphocyte subsets including subsets of CD4+ T cells, CD8+ T cells, B cells, and natural killer cells were examined. Eighteen patients presented with clinical symptoms compatible with the postpolio syndrome. This group was compared with 18 asymptomatic postpolio survivors and 22 age-matched healthy controls. The results demonstrated significant alterations in CD4+ subsets in the postpolio group as a whole when compared with normal controls. These findings reveal definite alterations in the immune status of postpolio survivors and raise the possibility that immunologic factors may contribute to late disease progression.

Agarose electrophoresis of cerebrospinal fluid in multiple sclerosis. A simplified method for demonstrating cerebrospinal fluid oligoclonal immunoglobulin bands.

The gamma globulins in cerebrospinal fluid from almost all patients with multiple sclerosis migrate in agarose electrophoresis as abnormal discrete populations, so-called oligoclonal bands. Such bands have also appeared in cerebrospinal fluid from patients with other types of inflammatory pathology such as neurosyphilis, acute idiopathic polyneuropathy, and subacute sclerosing panencephalitis. The demonstration of cerebrospinal fluid oligoclonal bands may aid in the evaluation of patients with early or atypical multiple sclerosis. This report describes a simple method for demonstrating cerebrospinal fluid oligoclonal bands using readily available commercial reagents and apparatus. Oligoclonal bands were seen in cerebrospinal fluid from all patients with clinically definite multiple sclerosis, even though some had normal cerebrospinal fluid gamma globulin levels, and in most patients with presumptive multiple sclerosis or other inflammatory conditions of the nervous system. They were not seen in cerebrospinal fluid of control patients with a variety of other neurologic diseases.

Life events and the risk of breast cancer: a case-control study.

A case-control study was undertaken to investigate the possibility of an association between stressful life events and the development of breast cancer. Ninety-nine breast cancer cases, and 99 controls matched by age and area of residence, completed a life events inventory to measure life change and distress scores over a 2-year and a 10-year period. Study subjects were also interviewed to establish potential breast cancer risk factors; their height and weight were measured; they completed a food frequency questionnaire and provided a fasting blood sample for hormonal assay. After adjusting for potential confounders, women with a 10-year life change score greater than 210 (i.e. the highest quartile) had 4.67 times the risk of developing breast cancer, compared with those in the lowest quartile (P < 0.05).

Prevalence of cryptosporidiosis in patients undergoing endoscopy: evidence for an asymptomatic carrier state.

PURPOSE: We sought to determine the presence of the parasite cryptosporidium in the duodenal aspirates of patients undergoing routine upper gastrointestinal endoscopy. PATIENTS AND METHODS: The study consisted of 169 patients undergoing upper endoscopy or endoscopic retrograde cholangiopancreatography. Immunocompromised patients were not included in the study population. Samples were aspirated from the second portion of the duodenum. Biopsy specimens were also obtained. Three randomly passed stool samples were obtained from 75% of the patients who were found to have cryptosporidium in the duodenum. RESULTS: Overall, cryptosporidium oocysts were identified in 12.7% of patients. There was no significant difference in the prevalence of the parasite in any subgroup of procedure or symptom complex. Half of those (46.7%) with positive aspirates had demonstrable cryptosporidium in stool samples, although none of the patients had diarrhea. No patient had detectable cryptosporidium in biopsy samples of the duodenum. CONCLUSION: The findings suggest a surprisingly high asymptomatic carrier rate for cryptosporidium.

Acute glucocorticoid pretreatment suppresses stress-induced hypothalamic-pituitary-adrenal axis hormone secretion and expression of corticotropin-releasing hormone hnRNA but does not affect c-fos mRNA or fos protein expression in the paraventricular nucleus of the hypothalamus.

Corticosterone regulates both basal and stress-induced hypothalamic-pituitary-adrenal (HPA) axis activity in a negative-feedback fashion. However, the cellular and molecular mechanisms of this negative feedback have yet to be explicitly characterized. By comparing stress-induced c-fos and corticotropin-releasing hormone (CRH) expression in the paraventricular nucleus (PVN), we may be able to determine whether acute glucocorticoid treatment affects the net neural excitatory input to the PVN (represented primarily by c-fos mRNA expression) or directly affects the ability of cells in the PVN to respond to that input (represented primarily by CRH hnRNA expression). In the following studies, we observed the effect of acute glucocorticoid (RU28362) treatment on subsequent HPA axis reactivity by measuring stress-induced plasma hormone concentration [corticosterone and adrenocorticotropic hormone (ACTH)] and gene expression (c-fos and CRH) in the PVN. First, we examined the dose-response relationship between systemically administered RU28362 (1-150 microg/kg, i.p) and suppression of the stress-induced corticosterone response. We then confirmed central nervous system access of the maximally suppressive dose of RU28362 (150 microg/kg) by an ex vivo radioligand binding assay. RU28362 selectively occupied the majority of glucocorticoid receptors in the hippocampus and hypothalamus while having no effect on mineralocorticoid receptors. In separate studies, RU28362 (150 microg/kg) and corticosterone (5 mg/kg) were injected i.p. 1 h before restraint stress. Compared to vehicle-treated controls, rats treated with RU28362 and corticosterone had substantially blunted stress-induced corticosterone and ACTH production, respectively. Furthermore, treatment with RU28362 significantly blunted stress-induced CRH hnRNA expression in the PVN. By contrast, neither RU28362 nor corticosterone treatment had an effect on stress-induced neuronal activation as measured by c-fos mRNA and its protein product in the PVN. This dissociation between c-fos and CRH gene expression suggests that glucocorticoid suppression of HPA activity within this time-frame is not a result of decreased excitatory neural input to the PVN, but instead depends on some direct effect of RU28362 on cells intrinsic to the HPA axis.

Long-term effects on feeding and body weight after stimulation of forebrain or hindbrain CRH receptors with urocortin.

Research on the contribution of CRH receptor stimulation to energy homeostasis has focused on forebrain substrates. In this study, we explored the effects of caudal brainstem administration of the CRH receptor agonist, urocortin, on food intake and body weight, and on plasma glucose and corticosterone (CORT) in non-deprived rats. Urocortin (0, 0.3, 1, 3 microg) delivered, respectively, to the fourth and lateral ventricles yielded substantial suppression of food intake measured 2, 4 and 24 h later. A significant but more modest anorexia was observed between 24 and 48 h after injection. Intake responses did not differ between the injection sites, but body weight loss measured 24 h after lateral-i.c.v. injection was substantially greater than that after fourth-i.c.v. injection. Fourth-i.c.v. urocortin administration (3 microg) produced substantial elevations in plasma glucose and CORT that were not distinguishable in magnitude and duration from responses to lateral-i.c.v. delivery. Unilateral microinjection of urocortin into the dorsal vagal complex significantly reduced 24-h food intake at a dose (0.1 microg) that was subthreshold for the response to ventricular administration, suggesting that fourth-i.c.v. effects are mediated in part by stimulation of CRH receptors in this region of the caudal brainstem. The results indicate that similar effects can be obtained from stimulation of anatomically disparate populations of CRH receptors, and that interactions between forebrain and hindbrain structures should be considered in the evaluation of CRH contributions to food intake and body weight control.