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.     

The GAP-related domain of tuberin, the product of the TSC2 gene, is a target for missense mutations in tuberous sclerosis

Tuberous sclerosis is an autosomal dominant trait in which the dysregulation of cellular proliferation and differentiation results in the development of hamartomatous growths in many organs. The TSC2 gene is one of two genes determining tuberous sclerosis. Inactivating germline mutations of TSC2 in patients with tuberous sclerosis and somatic loss of heterozygosity at the TSC2 locus in the associated hamartomas indicate that TSC2 functions as a tumour suppressor gene and that loss of function is critical to expression of the tuberous sclerosis phenotype. The TSC2 product, tuberin, has a region of homology with the GTPase activating protein rap1GAP and stimulates the GTPase activity of rap1a and rab5a in vitro. Here we show that the region of homology between tuberin and human rap1GAP and the murine GAP mSpa1 is more extensive than previously reported and spans approximately 160 amino acid residues encoded within exons 34-38 of the TSC2 gene. Single strand conformation polymorphism analysis of these exons in 173 unrelated patients with tuberous sclerosis and direct sequencing of variant conformers together with study of additional family members enabled characterisation of disease associated mutations in 14 cases. Missense mutations, which occurred in exons 36, 37 and 38 were identified in eight cases, four of whom shared the same recurrent change P1675L. Each of the five different missense mutations identified was shown to occur de novo in at least one sporadic case of tuberous sclerosis. The high proportion of missense mutations detected in the region of the TSC2 gene encoding the GAP-related domain supports its key role in the regulation of cellular growth.      

Assisted reproductive technology and complex chromosomal rearrangements: the limits of ICSI

Complex chromosomal rearrangements are very rare events in the human population. According to our knowledge on the consequences of simple reciprocal translocations for male fertility, translocations involving three or more chromosomes are thought to lead to severe reproductive impairments in terms of meiotic disturbance or chromosomal imbalance of gametes. We report the case of a 48 year old man whose sperm count revealed either oligozoospermia (<10(3) spermatozoa/ml) or azoospermia. He was referred to the laboratory for in-vitro fertilization after intracytoplasmic sperm injection. Cytogenetic investigations showed a complex chromosomal rearrangement involving firstly a translocation between the short arm of chromosome 7 and the long arm of chromosome 13 and secondly a translocation between the short arm of the same chromosome 13 and the short arm of chromosome 9. Diagnosis was ascertained by fluorescence in-situ hybridization and staining of the nucleolar organizer regions. Theoretical study of the translocated chromosomes predicted a 'chain' configuration of the hexavalent at the pachytene stage of meiosis. In all, 32 modes of segregation were considered and only one resulted either in a normal or a balanced gamete karyotype. Genetic counselling and choice of appropriate artificial reproduction technique are discussed.      

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.     

The morphological differentiation of the internal sphincter muscle of the anus in the human embryo and fetus (26.01.1990)

Conclusion The opening of the anal canal appears to be the factor which initiated the differentiation of the sphincter apparatus.The internal sphincter m. of the anus is entirely composed of smooth muscle as distinct from the striated fibers of the m. puborectalis, and the external sphincter which is a mixture of smooth and striated fibers (of skeletal type). It develops in the terminal part of the internal circular layer of the rectal m., outside which are longitudinal fibers which descend early to form the external sphincter (beginning around the third month).This study shows that the internal sphincter is scarcely evident before 12 SA. Thus continence between 10 and 12 SA (after the closure of the anal membrane) is closely related to the other components of the sphincter apparatus. On the other hand, the internal sphincter has become well formed after 28 to 30 SA and then plays a direct role in maintaining continence.     

Cbl-b differentially regulates activation-induced apoptosis in T helper 1 and T helper 2 cells

We previously reported that ligation of CD3 induces antiapoptotic signals in T helper 2 (Th2) cells, and in contrast causes Th1 cells to undergo apoptosis. Here we show that Cbl-b is accountable for the unequal response, revealing a previously unknown cell-specific regulatory function for the molecule. Absence of Cbl-b resulted in resistance to activation-induced apoptosis in murine Th1 cells following CD3 ligation, akin to what is observed in Th2 cells containing Cbl-b. Concurrent with the apoptosis profile, CD3 ligation in the absence of Cbl-b induced raft mobilization and cytoskeletal rearrangement in Th1 cells. Despite their ability to signal from CD3, Th2 cells did not aggregate their rafts, providing an explanation for cell-specific activity of Cbl-b.     

Use of a selective enrichment broth to recover Clostridium difficile from stool swabs stored under different conditions

The recovery of Clostridium difficile from the stools of patients with C. difficile-associated diarrhea was evaluated by use of an enrichment broth (cycloserine-cefoxitin fructose broth supplemented with 0.1% sodium taurocholate [TCCFB]) and was compared to that from selective agar (cycloserine-cefoxitin fructose agar [CCFA]) and alcohol shock followed by inoculation onto blood agar (AS-BA). TCCFB was superior to CCFA and AS-BA, and neither the storage time nor the storage temperature affected the recovery rate.     

Bladder acellular matrix as a substrate for studying in vitro bladder smooth muscle-urothelial cell interactions

The objective of this study was to evaluate the ability of bladder acellular matrix (BAM) to support the individual and combined growth of primary porcine bladder smooth muscle (SMC) and urothelial (UEC) cells. An in vitro co-culture system was devised to evaluate the effect of UEC on (i) SMC-mediated contraction of BAM discs, and (ii) SMC invasiveness into BAM. Cells were seeded onto BAM discs under 4 different culture conditions. Constructs were incubated for 1, 7, 14 and 28 days. Samples were then harvested for evaluation of matrix contraction. Immunohistochemistry (IHC) was utilized to examine cellular organization within the samples and conditioned media supernatants analyzed for net gelatinase activity. BAM contraction was significantly increased with co-culture. The same side co-culture configuration lead to a greater reduction in surface area than opposite side co-culture. IHC revealed enhanced SMC infiltration into BAM when co-culture was utilized. A significant increase in net gelatinase activity was also observed with the co-culture configuration. Enhanced infiltration and contractile ability of bladder SMCs with UEC co-culture may, in part, be due to an increase in gelatinase activity. The influence of bladder UECs on SMC behaviour in vitro indicates that BAM may contain some key inductive factors that serve to promote important bladder cell-cell and cell-matrix interactions.     

The Personality Assessment Inventory in individuals with traumatic brain injury.

This study examined the Personality Assessment Inventory (PAI) in 95 individuals who had suffered a traumatic brain injury (TBI). Participants were recruited from a rehabilitation hospital (n=60) and a military hospital (n=35); despite differences in demographics and injury characteristics groups did not differ on any of the clinical scales and were thus combined. In the combined group, the highest mean clinical scale elevations were on Somatic Complaints, Depression, and Borderline Features and the most common configural profiles, based on cluster analysis, were Cluster 1 (no prominent elevations), Cluster 6 (social isolation and confused thinking), and Cluster 2 (depression and withdrawal). Factor analysis indicated a robust three-factor solution that accounted for 74.86 percent of the variance and was similar to findings from the psychiatric and non-psychiatric populations in the standardization sample. The above findings are compared with the previous literature on psychopathology in TBI, particularly in regards to the Minnesota Multiphasic Personality Inventory-2 (MMPI-2), as well as previous psychometric research on the PAI.