Human monoclonal antibodies against Plasmodium falciparum: production, stabilization and characterization

Nine human monoclonal antibodies (MoAbs) recognizing 7 different antigenic structures of blood-stages of the human malarial parasite P. falciparum (Pf) were produced by Epstein-Barr virus transformed B-cell lines (EBV-TCL) with or without fusion to the lymphoblastoid cell line KR4. The peripheral blood B-lymphocytes were obtained from 8 Gambian donors immune to Pf malaria. Two of the EBV-TCL could be expanded and maintained for more than 6 months but neither one could be cloned. Six additional EBV-TCL were stabilized after fusion with the KR4 lymphoblastoid cell line. All resulting hybridomas permitted easy cloning. Some of the MoAbs produced distinct fluorescent staining patterns of asexual Pf blood-stage parasites when using high-resolution digitized video-intensified fluorescence microscopy. Antigens on 195 kD and 155 kD proteins were recognized by 3 and 1 MoAb, respectively, using Western blotting and immunoprecipitation techniques.     

Influence of Different Conditioning Methods on Force and Fatigue Resistance in Chronically Stimulated Skeletal Muscles

A study was undertaken to compare different conditioning methods for the transformation of latissimus dorsi muscle into a fatigue resistant one for application in circulatory assist. In ten sheep four electrodes were sutured to the epineurium of the left thoracodorsal nerve for indirect electrical stimulation of the latissimus dorsi muscle. In six sheep a "carousel stimulation, " a special multichannel stimulation, in combination with a recently developed conditioning protocol was used for muscle conditioning ( multichannel method ). In four sheep, a conventional stimulation protocol using single channel stimulation was applied for transformation of the muscle (single channel method). The final experiments were carried out when fatigue resistance was obtained. The maximum tetanic forces at different preloads were determined and fatigue resistance was tested during 20 minutes of continuous stimulation. Both conditioning patterns led to fatigue-free chronic stimulation. Muscles conditioned by multichannel stimulation exhibited between 20% and 33% less force than the contralateral unconditioned muscles, whereas in the single channel group this loss was between 32% and 43%. Thus, the multichannel method revealed relatively superior in preserving muscle force for chronic stimulation.     

Immunofluorescent localization of type IV collagen and laminin in human skin and its application in junctional zone pathology

Purified antibodies against type IV collagen and laminin were used to localize basement membranes by indirect immunofluorescence in various anatomical regions of normal and diseased human skin. The two proteins showed extensive codistribution. A continuous linear staining was found along the epidermal-dermal junction and around hair follicles, sebaceous gland acini and small capillaries. The same proteins also surrounded individual cells such as those found in vessels, hair erector muscles and subcutaneous tissue. Blister formation in bullous pemphigoid left type IV collagen and laminin on the floor of the blister, while the bullous pemphigoid antigen as detected by human autoantibodies was found on both sides of the blister. In solid basal cell carcinoma a strong staining was found around all tumour islands as well as focally within the cell clusters. This suggests that the tumour cells produce these basement membrane proteins but have lost, at least in part, control of polar deposition.     

Beta2-adrenergic receptor allele frequencies in the Quechua, a high altitude native population

The beta₂‐adrenergic receptor is involved in the control of numerous physiological processes and, as the primary catecholamine receptor in the lungs, is of particular importance in the regulation of pulmonary function. There are several polymorphic loci in the beta₂‐adrenergic receptor gene that have alleles that alter receptor function, including two (A/G⁴⁶, G/C⁷⁹) that increase agonist sensitivity. As such a phenotype may increase vaso and bronchial dilation, thereby facilitating air and blood flow through the lungs, we hypothesized that selection may have favoured these alleles in high altitude populations as part of an adaptive strategy to deal with the hypoxic conditions characteristic of such environments. We tested this hypothesis by determining the allele frequencies for these two polymorphisms, as well one additional missense mutation (C/T⁴⁹¹) and two silent mutations (G/A²⁵² and C/A⁵²³) in 63 Quechua speaking natives from communities located between 3200 and 4200 m on the Peruvian altiplano. These frequencies were compared with those of two lowland populations, one native American (Na‐Dene from the west coast of Canada) and one Caucasian of Western European descent. The Quechua manifest many of the pulmonary characteristics of high altitude populations and differences in allele frequencies between the Quechua and lowlanders could be indicative of a selective advantage conferred by certain genotypes in high altitude environments. Allele frequencies varied between populations at some loci and patterns of linkage disequilibrium differed between the old‐world and new‐world samples; however, as these populations are not closely related, significant variation would be expected due to stochastic effects alone. Neither of the alleles associated with increased receptor sensitivity (A⁴⁶, G⁷⁹) was significantly over‐represented in the Quechua compared with either lowland group. The Quechua were monomorphic for the C allele at base 79. This variant has been associated with body mass index; however no clearly defined metabolic phenotype has been established. In addition, we sequenced the coding region of the gene in three unrelated Quechua to determine if there were any other polymorphisms common in this population. None were detected.     

Drug injecting in patients in New Zealand Methadone Maintenance Treatment programs: An anonymous survey

Introduction and Aims. To investigate the prevalence and nature of injecting behaviour among patients on Methadone Maintenance Treatment (MMT) programs. Design and Methods. A self‐reported questionnaire was handed to 423 patients enrolled in MMT across six clinics in the lower North Island of New Zealand. Results. A total of 151 patients responded, giving a 35.6% response rate. One hundred and twenty (79.5%) respondents reported they had injected methadone while enrolled in MMT, 84 (55.6%) had injected methadone in the last year and of those 43 (35.8%) had injected methadone in the last week. Reasons given for injecting of methadone included: rapid onset of effect, needle fixation and euphoria. Time on the methadone programme was negatively associated with ever injecting methadone [odds ratio (95% CI) 0.92 (0.85–0.99), P = 0.029] and injecting other substances [odds ratio (95% CI) 0.93(0.87–1.0), P = 0.046]. More frequent pharmacy‐observed consumption was associated with increased injecting of other substances [odds ratio (95% CI) 1.32 (1.09–1.59), P = 0.005] but not methadone. The time a person had been enrolled on the methadone programme was associated with decreased use of other substances [odds ratio (95% CI) 0.93 (0.87–1.0), P = 0.046]. Discussion and Conclusions. Many individuals on MMT continue to inject their methadone. In this sample, the frequency of injection of methadone did not correlate with prescribed dose or takeaway arrangements. The beneficial impact of time on the programme emphasises the importance of retention in treatment. It is suggested that these results also indicate a need for routine education concerning safe injecting.[Judson G, Bird R, O'Connor P, Bevin T, Loan R, Schroder M, McGrath R, Weatherall M, Moriarty H, Robinson G. Drug injecting in patients in New Zealand Methadone Maintenance Treatment programs: An anonymous survey. Drug Alcohol Rev 2009]     

Long-Term Results of Nervous Tissue Alterations Caused by Epineurial Electrode Application: An Experimental Study in Rat Sciatic Nerve

In order to evaluate the long-term effects of epineurial electrode application for functional electrical stimulation (FES) the left sciatic nerve of seven rats was exposed. Four ring-shaped stainless steel wire electrodes were sutured to the epineurium of each nerve in the same manner as performed clinically for carrousel stimulation in man. The nerves were reexposed 1 year after implantation and the stimulation threshold to obtain a tetanic contraction in the lower limb was determined for each electrode. Afterwards the animals were sacrificed. The electrodes were excised and cross sections of the sciatic nerve directly at site of the electrodes, 2-mm proximal and 2-mm distal to them were harvested for histologic and planimetric assessment of nerve lesions. The area of damaged neural tissue was expressed as a percentage of the total cross-sectional area within the perineural sheath. The sciatic nerves of the right side served as controls. The values for the stimulation thresholds ranged between 0.1 and 1.0 mA (mean 0.43 mA). By morphometric examination five of seven nerves were seen altered, the altered areas captured between 1% and 4.8% of the total cross-sectional area of the nerves within the perineural sheath. Besides two specimens, all altered nerve segments exhibited distinct signs of nerve fiber regeneration. The clinical implications of the results for long-term electrical stimulation, such as phrenic pacing, are discussed.     

Radioimmunoassay for type III procollagen peptide and its application to human liver disease

A sensitive and specific radioimmunoassay was developed for the precursor-specific peptide segment located at the amino end of bovine type III procollagen. Human material showed high cross-reactivity in this assay. Two forms of human procollagen peptides were detected in body fluids. The larger peptide (45K) was found in serum and ascites, and resembled the whole precursor-specific segment which is presumably released from human type III procollagen by a single enzymatic cleavage. The smaller peptide (10K) was found mainly in urine indicating that further degradation of circulating procollagen peptides is required prior to their passage through the kidney. Compared to peptide concentrations in normal human serum two to twenty-fold increases were observed in all patients with alcoholic liver disease, in fifteen of seventeen patients with acute hepatitis, and in ten of fourteen patients with chronic active hepatitis. Much higher levels were detected in ascites fluid. Patients with rheumatoid arthritis and other diseases showed far smaller elevations of the serum peptide. In alcoholic liver disease peptide levels correlated well with inflammation and necrosis observed in liver biopsies, but not with other laboratory parameters.     

Comparative Analysis of the Immunophenotypes of Decidual and Peripheral Blood Large Granular Lymphocytes and T Cells During Early Human Pregnancy

PROBLEM : The functional role of the leukocytes in the decidua is not clear. They may regulate the maternal immune response to the fetal allograft. However, the factors controlling maternal and fetal communication have not yet been identified. METHOD : A comparative analysis of the phenotypes of decidual and peripheral blood large granular lymphocytes (LGLs) and T lymphocytes in early human pregnancy was performed on decidual tissue and blood samples obtained from ten patients at therapeutic abortion. RESULTS : Whereas most of the decidual LGLs were found to have a CD56^bright++ phenotype, most of the peripheral blood NK cells (90%) showed the classical CD56^dim+ phenotype, and only a small proportion were CD56^bright+ cells. Another striking difference was found in the expression of very late antigen 1 (VLA-1, CD49a): Almost all the decidual CD56^bright++ LGLs, but virtually none of the peripheral blood CD56⁺ NK cells expressed this antigen. Further differences were found in the expression of CD16, CD44, CD45RA, CD54, and CD57. There were also differences in phenotype between T cells derived from decidual tissue and those derived from peripheral blood. Approximately 31% of the CD3⁺ decidual T cells expressed VLA-1, but this antigen was virtually absent on peripheral blood T cells. A further difference was seen in the expression of HLA-DR. This activation antigen was found on 32 ± 13% of the decidual T cells but only 8 ± 5% of the peripheral blood T cells. Additionally, the proportion of cells expressing CD38 was higher among decidual than peripheral blood T cells. CONCLUSION : The findings suggest that both decidual LGLs and a subset of decidual T cells are activated and possibly play a role in the control of trophoblast growth and placental development.