Superantigen responses and co-stimulation: CD28 and CTLA-4 have opposing effects on T cell expansion in vitro and in vivo

Co-stimulation via the CD28/CTLA-4 system appears critical forT cell proliferation to peptide antigens presented in associationwith MHC. In this study, we examine the roles of CD28 and CTLA-4in the response of murine T cells to the superantigen staphylococcalenterotoxin B (SEB). In vitro, antibodies against B7-1/B7-2or Fab fragments of anti-CD28 antibodies significantly inhibitthe response of splenocytes to SEB. Conversely, Fab fragmentsof anti-CTLA-4 antibodies augment the proliferative response.Further, addition of blocking antibodies directed against B7-1/B7-2augment proliferation co-stimulated by intact anti-CD28 antibodies.These data support the hypothesis that CD28 and CTLA-4 exertopposing effects upon early T cell activation. In vivo, Intactanti-CD28 antibodies and non-stimulatory Fab fragments of anti-CD28appear to have similar inhibitory effects upon the expansionof V_ß8⁺ T cells. In contrast, both intact and Fabfragments of anti-CTLA-4 appear to amplify this expansion. Weconclude that the SEB response is significantly augmented byCD28-derived signaling and this in turn may be attenuated bysignals through CTLA-4.     

Residual cholesterol synthesis and simvastatin induction of cholesterol synthesis in Smith-Lemli-Opitz syndrome fibroblasts

Smith-Lemli-Opitz syndrome (RSH/SLOS) is an autosomal recessive, malformation syndrome caused by mutations in the 3beta-hydroxysterol delta7-reductase gene (DHCR7). DHCR7 catalyzes the reduction of 7-dehydrocholesterol (7DHC) to cholesterol. We report the mutation analysis and determination of residual cholesterol synthesis in 47 SLOS patients, and the effects of treatment of SLOS skin fibroblasts with simvastatin. Using deuterium labeling we have quantified the amount of synthesized cholesterol and 7DHC in homozygote, heterozygote, and control fibroblast cell lines. In SLOS fibroblasts, the fraction of synthesized cholesterol to total sterol synthesis ranged from undetectable to over 50%. This establishes that different mutant alleles encode enzymes with varying degrees of residual activity. There was a correlation between increased phenotypic severity and decreased residual cholesterol synthesis (r(2)=0.45, p<0.0001). Simvastatin treatment of SLOS fibroblasts with residual DHCR7 enzymatic activity decreased 7DHC levels and increased cholesterol synthesis. This increase in cholesterol synthesis is due to increased expression of a mutant allele with residual function. Determination of residual enzymatic activity for specific DHCR7 mutant alleles will help in understanding the processes underlying the broad phenotypic spectrum found in this disorder and will be useful in identifying patients who may benefit from simvastatin therapy.     

Kinetics of estimated human muscle capillary blood flow during recovery from exercise

The kinetic characteristics of muscle capillary blood flow (Qcap) during recovery from exercise are controversial (e.g. one versus two phases). Furthermore, it is not clear how the overall Qcap kinetics are temporally associated with muscle oxygen uptake (VO2m) kinetics. To address these issues, we examined the kinetics of Qcap estimated from the rearrangement of the Fick equation (Qcap=VO2m/C(a-v)O2) using the kinetics of pulmonary VO2 (VO2p, primary component) and deoxy-haemoglobin concentration ([HHb]) as indices of VO2m and C(a - v)O2 (arterio-venous oxygen difference) kinetics, respectively. VO2p (l min-1) was measured breath by breath and [HHb] (microm) was measured by near infrared spectroscopy during moderate (M; below lactate threshold, LT) and heavy exercise (H, above LT) in nine subjects. The kinetics of Qcap were biphasic, with an initial fast phase (tauI; M=9.3+/-4.9 s and H=6.0+/-3.8 s) followed by a slower phase 2 (tauP; M=29.9+/-8.6 s and H=47.7+/-26.0 s). For moderate exercise, the overall kinetics of Qcap (mean response time [MRT], 36.1+/-8.6 s) were significantly slower than the kinetics of VO2p (tauP; 27.8+/-5.3 s) and [HHb] (MRT for [HHb]; 16.2+/-6.3 s). However, for heavy exercise, there was no significant difference between MRT-[HHb] (34.7+/-10.4 s) and tauP for VO2p (32.3+/-6.7 s), while MRT for Qcap (48.7+/-21.8 s) was significantly slower than MRT for [HHb] and tauP for VO2p. In conclusion, during recovery from exercise the estimated Qcap kinetics were biphasic, showing an early rapid decrease in blood flow. In addition, the overall kinetics of Qcap were slower than the estimated VO2m kinetics.     

Genetic Heterogeneity of Escherichia coli O157:H7 in Scotland and its Utility in Strain Subtyping

 From April 1994 to March 1995, seven outbreaks of Escherichia coli O157 : H7 infection occurred throughout Scotland, including the largest milk-borne outbreak to date worldwide. Various vehicles of infection were identified, and there were 144 confirmed cases in total. All isolates associated with the outbreaks were subjected to detailed subtyping: phage typing, testing for carriage of verotoxin genes (VT), and pulsed-field gel electrophoresis. The outbreak strains were of three different phage types (2, 4, and 28). Those of phage type 2 and 28 were VT1–/VT2+, those of phage type 4 were VT1+/VT2+. To discriminate outbreak-associated isolates from the high sporadic background, real-time pulsed-field gel electrophoresis analyses were performed. The results demonstrated that, within each of the seven outbreak groups, the macrorestriction profiles observed were indistinguishable, whereas profiles for sporadic isolates were not. The consistent genetic heterogeneity observed within the Scottish Escherichia coli O157 population can be exploited in epidemiological investigations.     

Neurobehavioral Symptoms and Family Functioning in Traumatically Brain-Injured Adults

Traumatic brain injury (TBI) often results in a myriad of symptoms across physical, cognitive, and neurobehavioral domains. Despite inherent limitations associated with physical or cognitive impairments, the extant literature suggests that neurobehavioral symptoms tend to be the most distressing symptoms for the family and are more strongly related to poor outcome for the patient. The Neuropsychology Behavior and Affect Profile (NBAP) along with the General Functioning subscale of the Family Assessment Device (FAD-GF) and the Perceived Stress Scale were administered to 153 family members of persons who had sustained a TBI. The results provide new normative data and statistical support for the NBAP as a promising measure of neurobehavioral symptomatology following TBI. The correlation of.54 (p <.01) between FAD-GF and Full Scale NBAP scores provides powerful support for the hypothesis that family dysfunction is related to the presence of neurobehavioral symptoms in the patient. NBAP domains of Depression, Inappropriateness, Pragnosia, and Indifference appear most strongly related to family functioning and also bear a significant relationship to caregiver stress level and patient unemployment, whereas injury severity had little impact on either family functioning or neurobehavioral symptoms. The findings reinforce the significance of neurobehavioral symptoms and fortify their proposed link to family dysfunction post-TBI.     

Subpopulations of motor and sensory neurons respond differently to brain-derived neurotrophic factor depending on the presence of the skeletal muscle.

The aim of our study was to assess the ability of brain-derived neurotrophic factor (BDNF) to rescue motor and sensory neurons from programmed cell death. It is clearly demonstrated that the administration of a single injection of a putative neurotrophic factor to mouse embryos in utero on embryonic day (E) 14.5 is sufficient to significantly reduce the death of motor neurons when assessed on E18.5. However, the trophic requirements of somatic neurons have not been unequivocally determined in a mammalian species in vivo. Indeed, the unexpectedly high numbers of surviving neurons observed in neurotrophin and tyrosine kinase receptor knockout mice are probably the consequence of functional redundancy between the neurotrophins and their receptors. We studied spinal cord and facial motor nucleus neurons and proprioceptive neurons in the dorsal root ganglion and mesencephalic nucleus. The action of BDNF was assessed in wild-type fetuses to gain insight into its ability to rescue neurons from naturally occurring programmed cell death. In addition, we used Myf5(-/-):MyoD(-/-) embryos, which completely lack skeletal musculature, to assess the ability of BDNF to rescue neurons from excessively occurring programmed cell death. We found that BDNF differentially rescued neurons from naturally vs. excessively occurring cell death and that its ability to do so varied among neuronal subpopulations.     

Reversed diurnal variation in depression: associations with a differential antidepressant response, tryptophan: large neutral amino acid ratio and serotonin transporter polymorphisms

BACKGROUND: Although diurnal variation of mood is a widely recognized symptom of depression, the clinical, neurobiological and psychopharmacological significance of this symptom has not previously been reported. METHOD: A total of 195 depressed out-patients underwent a detailed clinical and neurobiological assessment, and were then randomized to treatment with either fluoxetine or nortriptyline. RESULTS: Of the 195 depressed patients, 62 had a pattern of reversed diurnal variation (i.e. worse in the evening). Those with reversed diurnal variation had a poorer response to a serotonergic anti-depressant, were less likely to have bipolar II disorder, had a higher tryptophan: large neutral amino acid ratio and had different allele frequencies of the polymorphisms in the promoter region of the serotonin transporter. CONCLUSIONS: These findings raise the possibility of serotonergic influence on diurnal variation, and that the symptom of reversed diurnal variation is of relevance to antidepressant prescribing.     

Influence of adjuvants on the quantity, affinity, isotype and epitope specificity of murine antibodies

Five adjuvants were compared to Freund's adjuvant for production of mouse polyclonal antibodies and monoclonal antibodies (McAbs) to human serum albumin (HSA) and interleukin-1 alpha (IL-1 alpha). Parameters examined were titer, affinity, concentration, isotype, epitope specificity and neutralizing activity of sera and hybridoma supernatants. Freund's adjuvant, while producing high titers and concentrations of antibodies in sera, was inferior to other adjuvants for eliciting antibodies with particular qualities. The adjuvants Quil A and A1(OH)3/[Thr1]muramyldipeptide elicited the highest affinity antibodies to HSA. Syntex adjuvant formulation-1 (SAF-1) elicited the highest percentage of 'protective' IgG2a antibodies to HSA. All adjuvants, particularly Quil A and Ribi adjuvant system, where superior to Freund's adjuvant in eliciting antibodies which bound native versus denatured HSA. In a comparison of SAF-1 and Freund's adjuvant, SAF-1 was superior to Freund's adjuvant in eliciting polyclonal and hybridoma antibodies which neutralized the biological activity of IL-1 alpha. These results show that adjuvants selectively and independently enhance different qualities of the antibody response. Furthermore, immunization with the appropriate adjuvant can optimize production of McAbs with desired qualities.     

Medical students' attitudes in a PBL curriculum: trust, altruism, and cynicism.

PURPOSE: Studies have shown that medical students become more cynical and less altruistic as they advance in training. However, these studies were conducted in traditional medical schools, and many used unvalidated tools. This study examined students' attitudes in a problem-based learning (PBL) curriculum using reliable and valid measures. METHOD: Medical students and PGY-1 residents at Mercer University School of Medicine in Macon, Georgia, completed Wrightsman's Philosophies of Human Natures Scale (PHNS) in 1999 and 2000. Chronbach's alpha assessed internal reliability among subscales, and test-retest reliability coefficients confirmed acceptable reliability. For 114 students who completed both surveys, changes in PHNS scores were analyzed, with particular attention to the subscales of trustworthiness, altruism, and cynicism. RESULTS: Students assessed at the beginning of their second year increased the extent to which they believed people are trustworthy and increased their beliefs in how altruistic people are. They also showed a significant decrease in cynicism. There was not a significant change in trustworthiness, altruism, or cynicism among the participants beyond first year. In general, female students held less cynical views about others and believed people to be more trustworthy. CONCLUSIONS: Contrary to prior reports, this study found that more advanced trainees were not more cynical or less altruistic than their more junior counterparts. Indeed, a significant and positive change of attitudes among the participants during their first year of medical school refuted earlier reports. Thus, results of earlier studies and the effect of a PBL curriculum on attitudes of medical students need to be re-examined.     

Cotinine levels in follicular fluid and serum of IVF patients: effect on granulosa-luteal cell function in vitro

Cigarette smoking is accepted as a risk factor for pregnancybut its effect on fertility is uncertain. In this study we determinedthe concentration of cotinine, a nicotine metabolite, in follicularfluid and serum from women participating in an In-vitro fertilizationand embryo transfer (IVF-ET) programme. Cotinine was undetectableIn serum and follicular fluid of non-smokers but ranged from<5 to 371 ng/ml in follicular fluid and from 24 to 245 ng/mlin serum of smokers. Granulosa-luteal cells, obtained from IVFpatients and cultured for 4 days, secreted progesterone and,when an aromatizable androgen was added, oestradiol-17. Theaddition of cotinine or nicotine did not alter progesteroneor oestradlol-17 secretion. However, the presence of cotininein follicular fluid of women smokers provides evidence for accessof at least one component of cigarette smoke to the developinggamete and the cells of the follicle. Further work is requiredto determine whether fertllity is compromised by the presence,In follicular fluid, of contaminants derived from cigarettesmoke.