Enhanced production of functional extracellular single chain variable fragment against HIV-1 matrix protein from Escherichia coli by sequential simplex optimization.

The optimal culture condition for extracellular recombinant single chain variable fragment anti HIV-1 p17 protein (scFv anti-p17) production in Escherichia coli HB2151 was investigated by the sequential simplex optimization (SS) method. Five variable parameters were submitted in the fermentation process. The most favorable condition obtained from 19 independent experiments was as followed: 58 µM of IPTG induction to 1.7 OD600 nm at 25.5°C for 16 h with 202 rpm agitation rate. The amount of secreted scFv anti-p17 at the optimal condition was 38% higher than under the control condition. The binding activity of soluble extracellular scFv anti-p17 protein increased 95.5% and 73.2% in comparison with the control condition and non-optimized condition respectively. The soluble scFv anti-p17 from crude HB2151 lysated was subsequently purified by immobilized metal ion affinity chromatography (IMAC) with His-tag. The purified scFv anti-p17 was intact and retained its antigen-binding affinity against HIV-1 p17. We demonstrated that the sequential simplex optimization method was a key for exertion of high yield with fewer experimental requirements for acquiring of large scale secretory protein production.

Rapid identification of heterozygous and homozygous hemoglobin constant spring by SYTO9 with a high resolution melting analysis.

BACKGROUND: Gel-electrophoresis and ethidium bromide are not ideally suited to large scale analysis in clinical laboratories. METHODS: Amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) specific for Hb CS was performed in 10 blood samples from normal individuals and 61 samples containing a peak of Hb CS when analyzed by capillary electrophoresis. Heterozygosity of Hb CS was identified using SYTO9 and high resolution melting (HRM) analysis method. RESULTS: Specific peak heights of amplified fragments of wild type and Hb CS alleles were observed in the heterozygote. Only one peak height of amplified fragments of the wild type allele was observed in the normal individual while only one peak height of amplified fragments of Hb CS allele was observed in the homozygote. HRM analysis interpretation results were completely consistent with the interpretation results from gel-electrophoresis. CONCLUSIONS: SYTO9 HRM analysis may be used as an alternative for rapid diagnosis of heterozygosity of Hb CS.

Comparison between capillary electrophoresis and high performance liquid chromatography for detection and quantification of Hb constant spring [Hb CS; α142, Term→Gln (TAA>CAA IN α2)].

Hb Constant Spring [Hb CS; α142, Term→Gln (TAA>CAA in α2)] is often missed by routine laboratory testing since its mRNA as well as gene product are unstable and presented at a low level in peripheral blood. This study aimed to analyze the efficacy of capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) for detecting and quantifying Hb CS in 19 heterozygotes and 14 homozygotes with Hb CS as well as 10 Hb H-CS disease subjects who were detected by molecular analysis. In the CE electrophoregram, Hb CS was seen at zone 2 and was observed in all samples, while the chromatogram of Hb CS peaks was found in 26.32% heterozygotes, 42.86% homozygotes and 90% Hb H-CS disease subjects, respectively. In addition, the Hb CS levels in each group of subjects quantified by CE were significantly higher than those quantified by HPLC. Based on the CE method, the lowest Hb CS level was found in the heterozygotes, whereas the highest level was found in the Hb H-CS disease patients. Therefore, the CE method was superior to the HPLC method for detecting Hb CS. Furthermore, the level of Hb CS quantified by CE proved useful in screening heterozygotes and homozygotes with Hb CS as well as Hb H-CS disease.

Method for analysis of surface molecule alteration upon phagocytosis by flow cytometry.

In this study, we introduce an application of flow cytometry for the concurrent detection of phagocytotic cells and surface molecules involved in the phagocytic process. E. coli expressing green fluorescent protein (GFP) were applied as the phagocytosable particles. Blood samples were incubated with E. coli expressing GFP, followed by indirect immunofluorescence using four candidate monoclonal antibodies (mAbs). Granulocytes that had phagocytosed E. coli exhibited high levels of GFP intensity, in contrast to the nonphagocytosed cells. By comparing the level of expression of molecules expressed on phagocytosed granulocytes with that of nonphagocytosed cells by flow cytometry, it enabled the determination of the expression and alteration of the cell surface molecules upon phogocytosis. Of the four mAbs used in this study, upon phagocytosis, molecules recognized by mAbs WK13, COSA5A and COSA33NL were up-regulated. However, CD15 recognized by mAb VIMD5 was downregulated. The proposed method will benefit the study of phagocytic mechanisms in the future.

Hematological alterations and thymic function in newborns of HIV-infected mothers receiving antiretroviral drugs.

OBJECTIVE: To investigate the effects of antiretroviral (ARV) drugs on hematological parameters and thymic function in HIV-uninfected newborns of HIV-infected mothers. STUDY DESIGN: Cross sectional study. SETTINGS: Chiang-Mai University Hospital, Chiang-Mai, Thailand. PARTICIPANTS /PATIENTS: 49 HIV-uninfected and 26 HIV-infected pregnancies. METHODS: Cord blood samples of newborns from HIV-uninfected and HIV-infected mothers were collected. Hematological parameters were measured using automatic blood cell count. T-cell receptor excision circles (TRECs) levels in cord blood mononuclear cells (CBMCs), CD4+ and CD8+ T-cells were quantified using real-time PCR.. MAIN OUTCOME MEASURES: Hemotological parameters and thymic function. RESULTS: Newborn of HIV-infected mother tended to have lower mean levels of hemoglobin than those of HIV-uninfected mother (137 ±22 vs 146 ±17 g/L, P = 0.05). Furthermore, mean of red blood cell (RBC) counts and hematocrit and median of TRECs in CD4+ T-cells in the newborns of the former were significantly lower than those of the latter [3.6 ±0.7 vs 4.8 ±0.6 x 1012 cells/L, P <0.001; 0.40 ±0.07 vs 0.46 ±0.05 L/L, P < 0.001 and 0.53 (IQR: 0.03-5.76) vs 13.20 (IQR: 2.77-27.51) x 10-3 pg/uL, P = 0.02, respectively]. CONCLUSION: ARV drugs altered hematological parameters and thymic function (TRECs CD4+ T-cells) in HIV-uninfected newborns of HIV-infected mothers.

Treatment of opportunistic infections prior to HAART initiation does not affect immune reconstitution in HIV-infected patients.

In patients receiving highly active antiretroviral therapy (HAART), increase of naive T-cell production, as measured by T-cell receptor rearrangement excision circles (TRECs), is an indicator of immune reconstitution. Our objective was to assess whether treating opportunistic infections (OIs) prior to HAART initiation affects CD4 T-cells recovery and TRECs in patients on HAART. HIV-infected patients presenting no OIs or treated OIs were prospectively enrolled prior to HAART initiation and followed-up over 12 months of HAART. CD4 T-cells and TRECs were measured at baseline, 6 and 12 months HAART and compared between patients presenting no OIs and those with treated OIs. Univariate and multivariate logistic regression models were used to identify potential factors associated with low TREC increase after 12 months HAART. Forty-four HIV-infected patients, 31 presenting no OIs and 13 with treated OIs at HAART initiation were enrolled. Patients presenting no OIs tended to have higher CD4 T-cell gain than those with treated OIs (151 vs 89 cells/µL; p = 0.05) after 6 months HAART but not after 12 months HAART (120 vs 149 cells/µL; p = 0.84). Among patients presenting no OIs, TREC levels significantly increased from baseline through 12 months HAART while among those with treated OIs, there was a trend for increase only after 12 months. Our study indicates that treatment of OIs prior to HAART does not lead to impaired CD4 T-cells recovery and thymic outputs.

Detection of α-thalassemia-1 Southeast Asian and Thai type deletions and ß-thalassemia 3.5-kb deletion by single-tube multiplex real-time PCR with SYBR Green1 and high-resolution melting analysis.

BACKGROUND: Prevention and control of thalassemia requires simple, rapid, and accurate screening tests for carrier couples who are at risk of conceiving fetuses with severe thalassemia. METHODS: Single-tube multiplex real-time PCR with SYBR Green1 and high-resolution melting (HRM) analysis were used for the identification of α-thalassemia-1 Southeast Asian (SEA) and Thai type deletions and ß-thalassemia 3.5-kb gene deletion. The results were compared with those obtained using conventional gap-PCR. DNA samples were derived from 28 normal individuals, 11 individuals with α-thalassemia-1 SEA type deletion, 2 with α-thalassemia-1 Thai type deletion, and 2 with heterozygous ß-thalassemia 3.5-kb gene deletion. RESULTS: HRM analysis indicated that the amplified fragments from α-thalassemia-1 SEA type deletion, α-thalassemia-1 Thai type deletion, ß-thalassemia 3.5-kb gene deletion, and the wild-type ß-globin gene had specific peak heights at mean melting temperature (T(m)) values of 86.89℃, 85.66℃, 77.24℃, and 74.92℃, respectively. The results obtained using single-tube multiplex real-time PCR with SYBR Green1 and HRM analysis showed 100% consistency with those obtained using conventional gap-PCR. CONCLUSIONS: Single-tube multiplex real-time PCR with SYBR Green1 and HRM analysis is a potential alternative for routine clinical screening of the common types of α- and ß-thalassemia large gene deletions, since it is simple, cost-effective, and highly accurate.

Interference of hemoglobin Hope on beta-thalassemia diagnosis by the capillary electrophoresis Method.

The ß-chain hemoglobin (Hb) variants interfere with the diagnosis of ß-thalassemia trait using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). We analyzed the effect of Hb Hope, a ß-chain Hb variant frequently found in the Thai population, on ß-thalassemia trait diagnosis. HPLC and CE were used to quantify the level of HbA(2) in 11 whole blood samples containing Hb Hope. The levels of Hb Hope detected by both methods were similar. An elevated HbA(2) level was found in all samples analyzed by the CE method, while 1 was increased when analyzed by HPLC, which was a compound heterozygous of Hb Hope and α-thalassemia-1 SEA-type deletion. Of 11 samples, 6 had mean corpuscular volumes within the reference range. All samples showed negative results for molecular analysis of ß(0)-thalassemia codon 17, 41/42, and 71/72 mutations and ß-thalassemia 3.5-kb deletion. Therefore, Hb Hope interfered with the diagnosis of ß-thalassemia trait analyzed by CE but not by HPLC.

Hematological Alterations and Thymic Function in Newborns of HIV-Infected Mothers Receiving Antiretroviral Drugs.

Objectives: To investigate the effects of antiretroviral (ARV) drugs on hematological parameters and thymic function in HIVuninfected newborns of HIV-infected mothers. Study design: Cross sectional study. Setting: Chiang-Mai University Hospital, Chiang-Mai, Thailand. Participants/Patients: 49 HIV-uninfected and 26 HIV-infected pregnancies. Methods: Cord blood samples of newborns from HIVuninfected and HIV-infected mothers were collected. Hematological parameters were measured using automatic blood cell count. T-cell receptor excision circles (TRECs) levels in cord blood mononuclear cells (CBMCs), CD4+ and CD8+ T-cells were quantified using real-time PCR. Main Outcome Measures: Hemotological parameters and thymic function. Results: Newborn of HIV-infected mother tended to have lower mean levels of hemoglobin than those of HIV-uninfected mother (137 ± 22 vs 146 ± 17 g/L, P = 0.05). Furthermore, mean of red blood cell (RBC) counts and hematocrit and median of TRECs in CD4+ T-cells in the newborns of the former were significantly lower than those of the latter [3.6 ± 0.7 vs 4.8 ± 0.6 x 1012 cells/L, P <0.001; 0.40 ± 0.07 vs 0.46 ± 0.05 L/L, P <0.001 and 0.53 (IQR: 0.03-5.76) vs 13.20 (IQR: 2.77-27.51) x 10-3 pg/μL, P = 0.02, respectively]. Conclusion: ARV drugs altered hematological parameters and thymic function (TRECs CD4+ T-cells) in HIV-uninfected newborns of HIV-infected mothers.

Method for analysis of surface molecule alteration upon phagocytosis by flow cytometry.

In this study, we introduce an application of flow cytometry for the concurrent detection of phagocytotic cells and surface molecules involved in the phagocytic process. E. coli expressing green fluorescent protein (GFP) were applied as the phagocytosable particles. Blood samples were incubated with E. coli expressing GFP, followed by indirect immunofluorescence using four candidate monoclonal antibodies (mAbs). Granulocytes that had phagocytosed E. coli exhibited high levels of GFP intensity, in contrast to the non-phagocytosed cells. By comparing the level of expression of molecules expressed on phagocytosed granulocytes with that of non-phagocytosed cells by flow cytometry, it enabled the determination of the expression and alteration of the cell surface molecules upon phogocytosis. Of the four mAbs used in this study, upon phagocytosis, molecules recognized by mAbs WK13, COSA5A and COSA33NL were up-regulated. However, CD15 recognized by mAb VIMD5 was down-regulated. The proposed method will benefit the study of phagocytic mechanisms in the future.