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.     

Synthesis and characterization of CuO@S-doped g-C3N4 based nanocomposites for binder-free sensor applications

This study presents the simultaneous exfoliation and modification of heterostructured copper oxide incorporated sulfur doped graphitic carbon nitride (CuO@S-doped g-C₃N₄) nanocomposites (NCs) synthesized via chemical precipitation and pyrolysis techniques. The results revealed that the approach is feasible and highly efficient in producing 2-dimensional CuO@S-doped g-C₃N₄ NCs. The findings also showed a promising technique for enhancing the optical and electrical properties of bulk g-C₃N₄ by combining CuO nanoparticles (NPs) with S-doped g-C₃N₄. The crystallite and the average size of the NCs were validated using X-ray diffraction (XRD) studies. Incorporation of the cubical structured CuO on flower shaped S-doped-g-C₃N₄ was visualized and characterized through XRD, HR-SEM/EDS/SED, FT-IR, BET, UV-Vis/DRS, PL, XPS and impedance spectroscopy. The agglomerated NCs had various pore sizes, shapes and nanosized crystals, while being photo-active in the UV-vis range. The synergistic effect of CuO and S-doped g-C₃N₄ as co-modifiers greatly facilitates the electron transfer process between the electrolyte and the bare glassy carbon electrode. Specific surface areas of the NCs clearly revealed modification of bulk S-doped g-C₃N₄ when CuO NPs are incorporated with S-doped g-C₃N₄, providing a suitable environment for the binder-free decorated electrode with sensing behavior for hazardous pollutants. This was tested for the preparation of a 4-nitrophenol sensor.

This study presents the simultaneous exfoliation and modification of heterostructured copper oxide incorporated sulfur doped graphitic carbon nitride (CuO@S-doped g-C₃N₄) nanocomposites synthesized via chemical precipitation and pyrolysis techniques.     

Bacteraemia among severely malnourished children in jimma university hospital, ethiopia

Background

Sever acute malnutrition severely suppresses every component of the immune system leading to increased susceptibility and severity to infection. However, symptoms and signs of infections are often unapparent making prompt clinical diagnosis and early treatment very difficult. The aim of the study was to determine the magnitude of bacteraemia and antimicrobial sensitivity among severely malnourished children.

Methods

Severely malnourished children admitted in Jimma University Specialized Hospital were enrolled between October, 2009 to May, 2010. Blood samples were collected, processed and bacterial isolates were identified using standard bacteriological procedures. Then, antibiotic susceptibility pattern of the isolates was determined by using Kirby-Bauer technique.

Results

Bacteraemia was seen in 35 (20.6%) of the 170 study subjects. There were a total of 35 bacterial isolates, Gram positive bacteria constitute 24(68.6%) of the isolates, where Staphylococcus aureus was the leading Gram positive isolate while Klebsiella species were the dominant Gram negative isolates. Twelve (7.1%) children died and 4 (33.3%) of them had bacteraemia. While susceptibility was more than 80% to Gentamicin, Ciprofloxacin and Ceftriaxone, increased level of resistance was documented to commonly used antibiotics, such as Amoxycillin, Co-trimoxazole and Chloramphenicol.

Conclusion

High prevalence of bacteraemia with predominating Gram positive isolates and increased level of resistance to commonly used antibiotics was shown among severely malnourished children in Jimma. Further studies are required to revise the current guideline for antibiotic choice.     

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.     

Clinical efficacy and survival with first-line inhaled iloprost therapy in patients with idiopathic pulmonary arterial hypertension.

AIMS: To describe the long-term clinical efficacy of inhaled iloprost as first-line vasodilator mono-therapy in patients with idiopathic pulmonary arterial hypertension (IPAH). METHODS AND RESULTS: Seventy-six IPAH patients were prospectively identified and treated with inhaled iloprost. Clinical, haemodynamic, and exercise parameters were obtained at baseline, after 3 and 12 months of therapy and yearly thereafter. Four endpoints were prospectively defined as follows: (i) death, (ii) transplantation, (iii) switch to intravenous (i.v.) therapy, or (iv) addition of or switch to other active oral therapy. During follow-up (535+/-61 days), 11 patients died, six were transplanted, 25 were switched to i.v. prostanoids, 16 received additional or other oral therapy, and 12 patients discontinued iloprost inhalation for other reasons. Event-free survival at 3, 12, 24, 36, 48, and 60 months was 81, 53, 29, 20, 17 and 13%, respectively. Among haemodynamic and exercise parameters, mixed venous oxygen saturation (P<0.001), right atrial pressure (P<0.001), and peak oxygen uptake (P=0.002) were associated with event-free survival. CONCLUSION: In this study, only a minority of patients could be stabilized with inhaled iloprost mono-therapy during a follow-up period of up to 5 years. In the presence of multiple treatment options, chronic iloprost inhalation as mono-therapy appears to have a limited role.     

Investigation of the urge-to-cough sensation in healthy volunteers

Background and objective: Recently, there has been interest in the sensation of irritation that precedes the motor act of coughing, which has been termed the urge‐to‐cough (UTC). The aim of this study was to perform the largest evaluation to date of the UTC threshold (C_u) in a healthy population. The specific aims were to investigate the relationship between C_u and cough reflex sensitivity, to evaluate gender differences in the UTC and to assess the reproducibility of measurements of C_u. Methods: Standard capsaicin cough challenge methodology was employed to measure cough reflex sensitivity in 100 healthy adult non‐smokers (50 females) with the additional measurement of C_u. A subgroup of 40 subjects (20 males) underwent repeat cough challenges after 1 week to examine the reproducibility of the measurements. Results: All 100 subjects demonstrated motor cough in response to capsaicin. Twenty‐one subjects (10 females) did not show a discernible C_u, as the motor cough event preceded a UTC sensation unaccompanied by cough. Although cough reflex sensitivity, as measured by the concentration of capsaicin inducing five or more coughs (C₅), was enhanced in women, there was no gender difference in C_u. Similar to standard cough reflex sensitivity measurements, the measurement of C_u was highly reproducible. Conclusions: These results demonstrate that the UTC threshold can be effectively and reproducibly measured using a modification of standard cough challenge methodology. Given its clinical significance as a prevalent symptom, UTC, as measured by C_u, represents an additional relevant end point for studies investigating the effects of pharmacological and other interventions in cough and cough reflex sensitivity.     

Phase II study of weekly docetaxel alone or in combination with trastuzumab in patients with metastatic breast cancer

This study was designed to determine the efficacy and toxicity of weekly docetaxel in metastatic breast cancer when given alone (for HER2/neu negative disease) or with trastuzumab (for HER2/neu overexpressing disease). Patients with metastatic breast carcinoma received docetaxel given on 2 different schedules (group 1A, 33 mg/m2 weekly [n = 21]; group 1B, 40 mg/m2 weekly for 3 weeks with 1 week off [n = 14]). Patients with HER2/neu overexpressing disease also received trastuzumab 4 mg/kg on day 1, then 2 mg/kg on days 8 and 15 of each 28-day cycle (group 2). Fifty-two patients were treated with docetaxel alone (group 1A/B, n = 35) or in combination with trastuzumab (group 2, n = 17). Prior taxane therapy given every 3 weeks had been used for metastatic disease in 19 of 35 patients (54%) in group 1A/B and in 2 of 17 patients (12%) in group 2. The mean delivered dose intensity of docetaxel was 29 mg/m2 per week. Partial response occurred in 7 of 35 patients (21%; 95% exact binomial confidence interval [CI], 9%-38%) treated with docetaxel alone, including 3 of 19 taxane-pretreated patients (16%) and 4 of 16 taxane-naive patients (25%). Partial response occurred in 10 of 17 patients (59%; 95% CI, 34%-82%) treated with docetaxel/trastuzumab. The most common grade 3/4 toxicities, occurring in more than or equal to 10% of patients, included neutropenia (21%), pulmonary toxicity (12%), and hyperglycemia (10%). The median times to disease progression were 4.5 months (95% CI, 2.5-6.5 months) in the docetaxel group and 8.5 months (95% CI, 4.5-12.5 months) in the docetaxel/trastuzumab group. Weekly docetaxel/trastuzumab is an effective regimen for patients with HER2/neu overexpressing metastatic breast cancer. Weekly docetaxel may be effective in as many as 20% of patients who had progressive disease after treatment with taxanes given every 3 weeks.