Antibody-bearing liposomes as multicolor immunofluorescence markers for flow cytometry and imaging

Loposomes covalently coupled to monoclonal antibodies retain the specificity of the antibody and bind only to cells bearing the appropriate determinant. As opposed to directly labeled antibodies which generally have fluorochrome to protein ratio of between 2–5, the entrapped space inside liposome can contain several hundred to several thousand molecules of fluorochromes in a space chemically isolated from the outside environment, thus providing the potential for an amplified fluorescence signal. We have prepared small unilamellar liposomes containing the soluble fluorochromes carboxyfluorescein (CF), which fluoresces in the green and sulforhodamine (SR), which fluoresces in the red, and covalently coupled a series of monoclonal antibodies using a heterobifunctional reagent. We were able to detect, on an Epics 753 flow cytometer equipped with an argon ion and a dye laser and by fluorescence microscopy, both single and double labeled mouse spleen lymphocyte subsets, fibroblast L cells and Raji cells. Complete color separation was obtained with CF-labeled cells being detected only by the green photomultiplier and SR-labeled cells by the red photomultiplier. Cells labeled with both were detected by both photomultipliers. Liposomes bearing anti-Ia antibodies bound only to B lymphocytes whereas those with anti-H-2K antibody bound both to T and B lymphocytes. In another system, single and dual color immunofluorescence made possible the simultaneous detection of HLA and H-2K molecules on transfected murine fibroblast L cells. The signal-to-noise ratio was more favorable for the liposome-labeled reagents than reagents labeled with fluorescein isothiocyanate. Cells labeled with antibody-bearing liposomes could be fixed with paraformaldehyde or glutaraldehyde without adversely affecting the original staining patterns. Apart from the two fluorochromes described above, other markers of choice could be encapsulated without any adverse effect on the antibody-liposome coupling procedure or on the specificity of the conjugated antibody. Since the fluorochrome is not directly coupled to the protein, there is no requirement for protein conjugation sites in order for it be usefully encapsulated inside liposomes. Therefore, this system provides new opportunities to exploit different, as yet untapped fluorochromes for use in flow cytometry and imaging.     

CD4 coating, but not CD4 depletion, is a predictor of efficacy with primatized monoclonal anti-CD4 treatment of active rheumatoid arthritis

OBJECTIVE: Double blind studies were conducted with the anti-CD4 monoclonal antibody (Mab) keliximab in patients with active, stable rheumatoid arthritis (RA), to confirm preliminary evidence of efficacy and safety from open. uncontrolled studies. METHODS: We enrolled 136 and 186 patients into 2 consecutive, randomized, double blind trials, with similar populations [apart from inclusion of disease modifying antirheumatic drug (DMARD)-na?ve patients in Study 2]. Patients received 4 weeks intravenous placebo or keliximab [40, 80, 120, or 140 mg twice weekly (bw), or 240 mg once weekly (ow)].The primary endpoint was the American College of Rheumatology (ACR) 20 response criteria, one week after the end of treatment. RESULTS: ACR 20 response rates in Study I were 19%, 42%, 51%*, and 69%* (*p < 0.05 compared to placebo), with placebo, 40, 80, or 140 mg keliximab bw, respectively. The response rates in Study 2 were 30%, 39%, 46% and 47% with placebo, 80 or 120 mg bw, or 240 mg keliximab ow, respectively. In the 2 studies, there was a dose dependent increase in peripheral blood CD4+ T cell coating with keliximab, but a different pattern of CD4 depletion was seen. While only 12% of keliximab treated patients in Study I had CD4 counts below 250 cells/mm3 at the end of the treatment period, 47% fell below this level in Study 2. Clinical response was not correlated with CD4 depletion, but was correlated with CD4+ T cell coating with keliximab. CONCLUSION: Coating of peripheral blood CD4+ T cells with keliximab, but not CD4 depletion, is a determinant of clinical response.     

Predictors of adherence to antiretroviral therapy among people living with HIV/AIDS in resource-limited setting of southwest ethiopia

Background  

Good adherence to antiretroviral therapy is necessary to achieve the best virological response, lower the risk that drug resistance will develop, and reduce morbidity and mortality. Little is known about the rate and predictors of adherence in Ethiopia. Therefore this study determines the magnitude and predictors of adherence to antiretroviral therapy among people living with HIV/AIDS in Southwest Ethiopia.     

The surgical treatment of the diastasis recti abdominis: an original technique of prosthesis repair of the abdominal wall

The Authors talk about on the surgical correction of the diastasis recti abdominis and underline its indications and aims. Firstly, they specify the possibilities and define the limits of the traditional surgical method. Secondly, they illustrate the rational of an innovating and original technique of prosthesis repair of the abdominal anterior wall setted up to treat the important diastasis recti abdominis. Particularly, this technique is the result of a kind of eclecticism and integration of some phases of the Quénu's self-plastic surgery and of the Welti-Eudel and Chevrel's technique. Thirdly, the authors describe the sequence of the times of the new technique and present the preliminary clinical experience carried out with it. Therefore, they determine gratifying and encouraging the findings of this method as regards the immediate and enduring curative efficacy (cosmetic and functional), the security and the compliance of the patient. Finally, in accordance with the outcomes, the authors decide to defend the undoubted reliability of the prosthesis repair of the abdominal wall to treat the big diastasis recti abdominis. Moreover, they intend to pass definitive judgement on the method after further clinical experiences on larger series of cases.     

Candidatus Rickettsia hoogstraalii in Ethiopian Argas persicus ticks

Ethiopian soft ticks Argas persicus, hard ticks including both Amblyomma variegatum and Rhipicephalus (Boophilus) spp., and fleas were collected from livestock, traditional human dwellings, and cracks and crevices of trees. They were assessed in pools for the presence of Rickettsia using PCR-based methods. The extracted tick DNA was subjected to molecular screening for Rickettsia, which revealed 50.5% of the pooled samples to be positive for Rickettsia spp. These were then subjected to multi-gene analysis using both outer surface proteins and housekeeping genes with proven discriminatory potential. Sequencing of the citrate synthase and outer membrane genes clearly led to the identification of three distinct rickettsial species, Candidatus Rickettsia hoogstraalii in Argas persicus ticks; R. africae in hard tick pools, and R. felis in fleas. Furthermore, we demonstrated the presence of the plasmid-borne small heat-shock protein gene hsp2 in DNA from A. persicus ticks suggesting that Candidatus R. hoogstraalii carried by these ticks possess a plasmid. Unlike chromosomal gene sequences, the hsp2 gene failed to cluster with Candidatus R. hoogstraalii, instead falling into an isolated separate clade, suggesting a different origin for the plasmid.     

Altitude-dependent Bartonella quintana genotype C in head lice, Ethiopia

To determine the presence of Bartonella quintana in head and body lice from persons in different locations in Ethiopia, we used molecular methods. B. quintana was found in 19 (7%) genotype C head lice and in 76 (18%) genotype A body lice. B. quintana in head lice was positively linked to altitude (p = 0.014).