Identifying amino acid residues that influence plasma clearance of murine IgG1 fragments by site-directed mutagenesis

Site-directed mutagenesis has been used to change amino acid residues of a recombinant Fc-hinge fragment derived from the murine immunoglobulin (Ig)G1 molecule, and the effects of these mutations on the pharmacokinetics of the Fc-hinge fragment have been determined. Specifically, Ile-253, His-310 and Gln-311 of the CH2 domain and His-433 and Asn-434 of the CH3 domain have been changed. In the three dimensional structure of an antibody, these amino acids are in close proximity to each other at the CH2-CH3 domain interface. The mutated Fc-hinge fragments have been purified from recombinant Escherichia coli cells and their pharmacokinetic parameters determined in mice and compared with those of the wild-type Fc-hinge fragment. The results show that the site of the IgG1 molecule that controls the catabolic rate (the ‘catabolic site’) is located at the CH2-CH3 domain interface and overlaps with the Staphylococcal protein A binding site.     

Detection of human perforin by ELISpot and ELISA: ex vivo identification of virus-specific cells

The perforin (PFN) protein is essential for the elimination of target cells by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. The study of cells releasing PFN has been hampered by a lack of sensitive methods. We therefore produced PFN-reactive monoclonal antibodies (mAb) and developed capture enzyme-linked immunosorbent (ELISA) and enzyme-linked immunospot (ELISpot) assays. Three mAbs were generated and shown to react with unique determinants of PFN. All mAbs recognized intracellular PFN in human peripheral blood mononuclear cell (PBMC) as assessed by flow cytometry and immunohistochemistry. Functional PFN capture ELISA and ELISpot assays were developed utilizing two of the mAbs for capture and the third mAb for detection. When examining PFN release by the YT lymphoma cell line, the ELISpot displayed a greater detection sensitivity than the ELISA. Assessment of PFN release by a CTL clone using ELISpot gave results consistent with a parallel (51)Cr-release cytotoxicity assay. Moreover, PFN release by PBMC could be quantified by ELISpot and ELISA after ex vivo stimulation with defined CTL epitopes from common viruses. These novel immunoassays will be valuable for further investigations of the mechanisms underlying granule-mediated apoptosis. In addition, the capture immunoassays could provide tools for studying CTL responses in infectious and tumor diseases as well as for vaccine development.     

Integration of the biopsychosocial model: perspectives of medical students and residents.

PURPOSE: To examine residents' and medical students' attitudes toward the incorporation of psychosocial factors in diagnosis and treatment and to identify barriers to the integration of evidence-based, mind-body methods. METHOD: A random sample of third- and fourth-year medical students and residents was drawn from the Masterfiles of the American Medical Association. A total of 661 medical students and 550 residents completed a survey, assessing attitudes toward the role of psychosocial factors and the clinical application of behavioral/mind-body methods. RESULTS: The response rate was 40%. Whereas a majority of students and residents seem to recognize the need to address psychosocial factors, 30%-40% believe that addressing such factors leads to minimal or no improvements in outcomes. The majority of students and residents reports that their training in these areas was ineffective, yet relatively few indicate interest in receiving further training.Females are more likely to believe in the need to address psychosocial factors. Additional factors associated with greater openness to addressing psychosocial factors include (1) the perception that training in these areas was helpful, and (2) personal use of behavioral/mind-body methods to care for one's own health. CONCLUSIONS: There is a need for more comprehensive training during medical school and residency regarding both the role of psychosocial factors in health and the application of evidence-based, behavioral/mind-body methods. The current health care structure-particularly insufficient time and inadequate reimbursement for addressing psychosocial factors-may be undermining efforts to improve patient care through inconsistent or nonexistent application of the biopsychosocial model.     

Heritability and clinical features of multigenerational families with obsessive-compulsive disorder and hoarding.

To date, only one complete genome screen for obsessive-compulsive disorder (OCD) has been published. That study identified a region of suggestive linkage (maximum lod score of 2.25) with a relatively small sample size (N = 56; 27 with OCD). Additional complete genome screens are needed to confirm this finding and identify other regions of linkage. We present the clinical characteristics and power to detect linkage of 11 multigenerational families with OCD and hoarding (N = 92; 44 with OCD), as well as heritability estimates for several quantitative traits. Families with at least two individuals with OCD were identified through probands with childhood-onset OCD. Expected lod scores were calculated for simulated genetic marker data under an additive and two dominant models assuming a dense SNP marker map. All affected individuals had an early age of onset (18 or younger). Hoarding was present in 46% of subjects. Obsessive-compulsive symptoms and hoarding were highly heritable. The maximum mean expected lod score was 3.31 for OCD and 1.39 for hoarding. We found reasonable power to detect regions of interest (lod = 2) for OCD in these families, but will need to expand our family collection to have adequate power to detect regions of interest for hoarding.     

Relapse of major depression after complete and partial remission during a 2-year follow-up

BACKGROUND: Rates of remission and relapse were studied over more than 2 years in a sample of Spanish outpatients with DSM-III-R criteria of unipolar major depressive episodes. METHODS: Patients were treated following standardised pharmacological protocols at our centre. In the first visit, the structured clinical interview for DSM-III-R (SCID) was used. The following visits were held monthly. Phases of evolution were recorded using the Hamilton Depression Rating Scale (HDRS), applying the Frank criteria. RESULTS: A significantly greater proportion of relapse was observed in the partial remission group compared to the complete remission one. The rate of relapses for patients in complete remission was 15.18%, while for patients in partial remission was 67.61%. Partial remission was significantly associated with relapses. LIMITATIONS: The short duration of the study and the decreasing sample size during the follow-up. CONCLUSIONS: Partial remission after a depressive episode seems to be strongly associated with relapses. Moreover, this clinical factor could by itself fully predict short-term relapses. CLINICAL RELEVANCE: The study shows the importance of reaching complete remission to decrease the rate of short-term relapses.     

SK&F 95654-induced acute cardiovascular toxicity in Sprague-Dawley rats--histopathologic, electron microscopic, and immunohistochemical studies

The characteristics and pathogenesis of the cardiovascular toxicity induced by the type III selective phosphodiesterase inhibitor SK&F 95654 were examined in 2 studies. Sprague-Dawley rats received either a single sc injection of 50, 100, or 200 mg/kg SK&F 95654 and were euthanized at 24 hours after administration of the drug (Study 1), or were given a single subcutaneous (sc) injection of 100 mg/kg SK&F 95654 and euthanized at 1, 2, 4, 6, 8,12, 24 hours, or 2 weeks after treatment (Study 2). Control rats received either DMSO or saline. Myocardial lesions and vascular lesions of the mesentery, spleen, and pancreas were seen 24 hours after dosing with either 50,100, or 200 mg/kg SK&F 95654. The frequency and severity of these lesions (evaluated after the 100 mg/kg dose) increased with time over a period of 1 to 24 hours. By 2 weeks, the lesions subsided. Cardiac lesions consisted of myocyte necrosis with hypercontraction bands, inflammatory cell infiltration, interstitial hemorrhage, and interstitial edema. Vascular lesions of the mesentery were most prominent and consisted of vasodilatation and inflammation in the small-sized vessels, arterial medial necrosis and hemorrhage, and venous thrombosis. The vascular lesions included: leukocyte adhesion to endothelial cells, transendothelial migration of leukocytes, and inflammatory cell infiltration into vessel walls. Affected vessels included arteries, terminal arterioles, capillaries, postcapillary venules, and veins. Apoptosis of endothelial and smooth muscle cells was detected in the mesenteric vasculature by both TUNEL assay and electron microscopy. Evidence of endothelial cell activation in the mesenteric arteries and veins was also observed by electron microscopy. Immunohistochemical staining detected enhanced endothelial cell expression of intercellular adhesion molecule- 1 (ICAM- 1) and von Willebrand factor (vWF) in the mesenteric arteries and veins. Mast cells were noted to be more prevalent in affected mesenteric tissue from drug-treated animals. The present findings suggest that apoptosis of endothelial and smooth muscle cells, activation of endothelial cells, recruitment of mast cells, and increased expression of adhesion molecules are important factors to the overall pathogenesis of SK&F 95654-induced vasculitis.     

Differential scanning calorimetric study of the complexes of modified myosin subfragment 1 with ADP and vanadate or beryllium fluoride

Summary The effects of various modifications of rabbit skeletal myosin subfragment 1 on thermal denaturation of subfragment 1 in ternary complexes with Mg-ADP and orthovanadate (V_i) or beryllium fluoride (BeF_x) have been studied by differential scanning calorimetry. It has been shown that specific modifications of SH₁ group of Cys-707 by different sulfhydryl reagents, trinitrophenylation of Lys-83, and reductive methylation of lysine residues promote the decomposition of the S1·ADP·V_i complex and change the character of structural transitions of the subfragment 1 molecule induced by the formation of this complex, but they have much less or no influence on subfragment 1 thermal stability in the S1·ADP·BeF_x complex. Thus, the differential scanning calorimetric studies on modified subfragment 1 preparations reveal a significant difference between S1·ADP·V_i and S1·ADP·BeF_x complexes. It is suggested that S1·ADP·V_i and S1·ADP·BeF_x complexes represent structural analogues of different transition states of the ATPase cycle, namely the intermediate states S1^**·ADP·P_i and S1^*·ATP, respectively. It is also proposed that during formation of the S1·ADP·V_i complex the region containing both Cys-707 and Lys-83 plays an important role in the spread of conformational changes from the active site of subfragment 1 ATPase throughout the structure of the entire subfragment 1 molecule. In such a case, the effects of reductive methylation of lysine residues on the subfragment 1 structure in the S1·ADP·V_i complex are related to the modification of Lys-83.     

Stability of Th1 and Th2 populations

Using an in vitro model for the development of IFN-y-producIng(Th1) and IL-4-produclng (Th1) cells from CD4 T lymphocytesexpressing a transgenlc TCR, we show that IL-12 and IL-4 arethe most potent stimuli for the differentiation of naive T cellsto effector populations. When combinations of cytokines arepresent during T cell priming, the effect of IL-4 Is dominant.Furthermore, differentiated Th1 cells can be converted intoIL-4 producers by exposure to IL-4, but the Th2 phenotype Isnot reversible. The stability of Th2 populations may limit theability to regulate Th2-domlnant responses In pathologic situations.