A microwave technique for double indirect immunostaining of human brain tissue cultures with mouse monoclonal antibodies.

The use of 2 monoclonal antibodies during double immunohistochemistry would enable the use of a greater variety of antibody combinations. Here, we demonstrate a simple, cost effective method of double indirect immunostaining of cultured cells using primary antibodies from the same species. This method uses microwaving of cell samples immediately after the application of the first secondary antibody, and significantly reduces the level of nonspecific staining. This technique does not elute the antibodies, nor raise the sample temperature above 37 degrees C.     

Scanning Density Gradient Isoelectric Focusing of Staphylococcus aureus Enterotoxins B and C1

Purified Staphylococcus aureus enterotoxins B and C(1), the causative agents of staphylococcal food poisoning, were resolved into several components by scanning isoelectric focusing in sucrose density gradients in the absence and presence of 6 M urea and 5 mM dithiothreitol. The observed heterogeneity persisted in the denaturing medium, which suggests that the primary structure of protein enterotoxins B and C(1) may be partly responsible for the isoelectric point differences of the various forms. To our knowledge, the scanning isoelectric focusing patterns of staphylococcal enterotoxins B and C(1) are reported for the first time.     

Multidimensional analysis of the symptoms of chronic bronchitis and emphysema

Subjective symptoms and experiences were explored within a group of 146 severe, chronic bronchitis and emphysema patients. Eighty-nine symptoms and experiences, derived from initial interviews with 29 patients, were rated according to the frequency of occurrence during breathing difficulties. Key cluster analyses were used to derive a Bronchitis-Emphysema Symptom Checklist (BESC) measuring 11 symptom categories: Helplessness-Hopelessness, Decathexis, Fatigue, Poor Memory, Peripheral-Sensory Complaints, Dyspnea, Congestion, Sleep Difficulties, Irritability, Anxiety, and Alienation. The BESC symptom categories are highly reliable and the relationships among categories are stable across two subgroups of patients. The BESC provides one way to describe how patients cope with and experience chronic bronchitis and emphysema.     

The effect of stabilization on isokinetic knee extension and flexion torque production

The purpose of this study was to examine the effect of four methods of stabilization on maximal reciprocal isokinetic knee extension and flexion. Left knee extension/flexion was tested at 60°/s in 20 subjects. Warm-up consisted of five submaximal and one maximal effort followed by three maximal efforts in each of four randomized stabilization conditions: 1) Hands and back stabilization; the trunk was strapped to the back rest and the hands grasped the seat. 2) Back stabilization; the trunk was strapped to the back rest and the hands were folded across the chest. 3) Hand stabilization; the hands grasped the seat and the back rest was removed. 4) No stabilization; the hands were folded across the chest and the back rest was removed. One-way repeated measures ANOVA showed a significant effect of stabilization for knee extension (F(3,57)=17.44, p=.0001) and knee flexion (F(3,57)= 5.37, p=.002). Paired, two-tailed student's t-tests with Bonferroni correction showed that, in knee extension, no stabilization was significantly less than all others, p<.001. In addition, back stabilization was less than hands and back stabilization, p<.005. In knee flexion, no stabilization was significantly less than all others, p<.01. In conclusion, the method of trunk stabilization significantly affected maximal reciprocal isokinetic knee extension/flexion strength measurements. Maximal knee extension/flexion torque production was achieved when the trunk was strapped to the back support and when the hands grasped the seat.     

Direct comparison of umbilical cord blood versus bone marrow-derived endothelial precursor cells in mediating neovascularization in response to vascular ischemia.

Endothelial precursor cells (EPCs) cultured from adult bone marrow (BM) have been shown to mediate neovasculogenesis in murine models of vascular injury. We sought to directly compare umbilical cord blood (UCB)- and BM-derived EPC surface phenotypes and in vivo functional capacity. UCB and BM EPCs derived from mononuclear cells (MNC) were phenotyped by surface staining for expression of stromal (Stro-1, CXCR4, CD105, and CD73), endothelial (CD31, CD146, and vascular endothelial [VE]-cadherin), stem cell (CD34 and CD133), and monocyte (CD14) surface markers and analyzed by flow cytometry. The nonobese diabetic/severe combined immunodeficiency murine model of hind-limb ischemia was used to analyze the potential of MNCs and culture-derived EPCs from UCB and BM to mediate neovasculogenesis. Histologic evaluation of the in vivo studies included capillary density as a measure of neovascularization. Surface CXCR4 expression was notably higher on UCB-derived EPCs (64.29%+/-7.41%) compared with BM (19.69%+/-5.49%; P=.021). Although the 2 sources of EPCs were comparable in expression of endothelial and monocyte markers, BM-derived EPCs contained higher proportions of cells expressing stromal cell markers (CD105 and CD73). Injection of UCB- or BM-derived EPCs resulted in significantly improved perfusion as measured by laser Doppler imaging at days 7 and 14 after femoral artery ligation in nonobese diabetic/severe combined immunodeficiency mice compared with controls (P<.05). Injection of uncultured MNCs from BM or UCB showed no significant difference from control mice (P=.119; P=.177). Tissue samples harvested from the lower calf muscle at day 28 demonstrated increased capillary densities in mice receiving BM- or UCB-derived EPCs. In conclusion, we found that UCB and BM-derived EPCs differ in CXCR4 expression and stromal surface markers but mediate equivalent neovasculogenesis in vivo as measured by Doppler flow and histologic analyses.     

A twin-family study of self-report symptoms of panic-phobia and somatization

Self-report symptoms of anxiety are widely used in mental health and social science research as an index of current psychiatric state. Previous twin studies have suggested that genetic factors account for a significant proportion of the variance in these symptoms. To replicate and extend these findings, we examined self-report symtoms of panic-phobia and somatization in the “Virginia 30,000” twin-family sample. Model fitting applied to 80 unique relationships in the twin-family pedigree produced the following major results: (i) genetic effects were significant for both symptom factors, accounting for between 25 and 49% of the total variance, with the exception of symptoms of panic-phobia in females, where they accounted for 15–16% of the variance; (ii) familial environmental effects were absent for symptoms of somatization, while for symptoms of panic-phobia they accounted for a very small proportion of variance in males (≤1.2%) and a modest proportion in females (6–17%) (iii) spousal correlations were present for both factors, ranging from +0.05 to +0.20; (iv) genetic factors which influenced symptoms were generally the same in males and females, although their effect was greater in males; (v) heritability estimates were lower in the population-based than in the volunteer sample; and (vi) when test-retest reliability was included in the model, results suggest that genetic factors account for at least half of the stable variance for all symptom factors, except panic-phobia in females. Our results support the validity of previous twin studies of self-report symptoms of anxiety and suggest that genetic factors significantly influence these symptoms but familial-environmental factors play little or no etiologic role.     

Evidence for projections from the rostral medullary raphe onto medullary catecholamine neurons in the rat

Following the iontophoretic deposition of Phaseolus vulgaris leucoagglutinin (PHA-L) into the rostral medullary raphe, which included portions of the caudal nucleus raphe magnus, rostral nucleus raphe pallidus, rostral nucleus raphe obscurus and rostral nucleus reticularis paragigantocellularis, two-color immunoperoxidase staining was employed to demonstrate contiguity between PHA-L-immunoreactive (PHA-LI) varicose fibers and boutons and medullary catecholamine (CA) cells. Raphe projections were contiguous with phenylethanolamine N-methyltransferase-immunoreactive (PNMTI) neurons in the C1, C2 and C3 cell groups and with tyrosine hydroxylase-immunoreactive (THI) neurons in the A1 and A2 cell groups. Contiguity between PHA-LI processes and medullary CA cells was observed most frequently in the C1 cell group. Preliminary findings of this study have been presented previously.     

Nonstimulatory peptides contribute to antigen-induced CD8-T cell receptor interaction at the immunological synapse

It is unclear if the interaction between CD8 and the T cell receptor (TCR)-CD3 complex is constitutive or antigen induced. Here, fluorescence resonance energy transfer microscopy between fluorescent chimeras of CD3zeta and CD8beta showed that this interaction was induced by antigen recognition in the immunological synapse. Nonstimulatory endogenous or exogenous peptides presented simultaneously with antigenic peptides increased the CD8-TCR interaction. This finding indicates that the interaction between the intracellular regions of a TCR-CD3 complex recognizing its cognate peptide-major histocompatibility complex (MHC) antigen, and CD8 (plus the kinase Lck), is enhanced by a noncognate CD8-MHC interaction. Thus, the interaction of CD8 with a nonstimulatory peptide-MHC complex helps mediate T cell recognition of antigen, improving the coreceptor function of CD8.     

Ultrastructural Pathology of Murine Cytomegalovirus Infection in Cultured Mouse Nervous System Tissue

Mouse spinal cord-ganglia cultures were innoculated with murine cytomegalo-virus 14 days after explantation. Intranuclear virus was first observed 4 days after infection. The viruses, which occurred in four forms, were observed in increasing numbers during the ensuing 4 days. Differences were noted in the relative prevalence of certain of these forms in older as compared to younger cultures. This suggests that variations in virus form are related to virus maturation. Cytoplasmic viruses were occasionally observed, but their site of origin is not certain. A variety of cytoplasmic inclusions were seen, particularly in the older cultures. It seems likely that they represent specific cell responses to the presence of the virus. They were not observed in the control cultures, even though some of the latter did show severe degenerative changes.     

Poly(ethylene glycol)-containing Hydrogels for Oral Protein Delivery Applications

Novel pH-sensitive hydrogels were developed as suitable candidates for carriers in bioMEMS devices as well as for oral delivery of therapeutic peptides and proteins due to their ability to respond to environmental pH change. Macromonomers containing various PEG molecular weights were synthesized and used to prepare P(MAA-g-EG) hydrogels were by photopolymerization. P(MAA-g-EG) hydrogels showed a drastic change of the equilibrium swelling ratio between pH 2.2 and 7.0. At pH 7.0, hydrogels with PEGMA2000 exhibited higher swelling ratio than hydrogels with PEGMA1000. For both hydrogels with PEGMA1000 and PEGMA2000, the swelling mechanism became more relaxation-controled as the environmental pH changed from 2.2 to 7.0 due to the ionization of the functional groups in polymer networks at high pH. In vitro release studies of insulin were conducted. P(MAA-g-EG) hydrogels exhibited drastic increase of insulin release as the pH of the medium was changed from acidic to basic. Insulin release from P(MAA-g-EG) hydrogels with PEGMA2000 was slower than from hydrogels with PEGMA1000 at both low and high pH. These results were used to design and improve protein release behavior from these carriers.