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Results for “Monitoring, Immunologic”

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PubMed2003

Immune monitoring in whole blood using real-time PCR.

There is a need for simple and sensitive assays to assess innate and adaptive immune responses to microbial agents and vaccines. Herein, we describe a whole blood method allowing to measure the induction of cytokine synthesis at the mRNA level. The originality of this method consists in the combination of PAXgene tubes containing an mRNA stabilizer for blood collection, the MagNA Pure instrument as an automated system for mRNA extraction and RT-PCR reagent mix preparation, and the real-time PCR methodology on the Lightcycler for accurate and reproducible quantification of transcript levels. We first demonstrated that this method is adequate to measure the induction of interleukin (IL)-1beta and IL-1 receptor antagonist (IL-1 RA) mRNA upon the addition of bacterial lipopolysaccharide (LPS) to whole blood. We then showed that this approach is also suitable to detect the production of mRNA encoding T cell-derived cytokines in whole blood incubated with tetanus toxoid as a model of in vitro immune response to a recall antigen. Finally, we demonstrated that this methodology can be used successfully to assess inflammatory as well as T cell responses in vivo, as it allowed to detect the induction of IL-1beta and IL-1 RA after injection of LPS in healthy volunteers, and also the induction of IL-2 upon recall immunisation with tetanus vaccine.

Adult↗

PubMed1999

CD4+ T cell enumeration in HIV infection with limited resources.

The incidence of human immunodeficiency virus (HIV) infection continues to increase in South Africa. Limited resources are available for diagnosis and management of the disease and the development of affordable strategies is required. Absolute CD4 counts are used locally predominantly to monitor disease progression and institute prophylaxis against opportunistic infections. A dramatic increase in demand for CD4 counts prompted an investigation for a more cost-effective flow cytometry method than those currently recommended by the Centers for Disease Control (CDC). CD4 counts generated by two different single tube methods using CD3/CD4/CD8 [1(3)] and CD4 [1(1)] antibodies, respectively, were compared to the CDC recommended 6 tube 2 colour panel [6(2)]. Whole blood analysis using the Coulter Multi-Q-Prep system and an Epics XL Flow Cytometer (Coulter, Hialeah, FL) was performed for each of the three methods. Random samples from HIV positive adult patients were compared. A mean difference in the absolute CD4 counts of less than 10x10(6)/l was generated by both of the alternative panels when compared with the 6(2) panel. The precision of the three methods is comparable. In reagents alone, the 1(3) and 1(1) methods represent a cost saving of 76% and 93%, respectively, over the 6(2) method. The 1(3) and 1(1) panels would permit more affordable CD4 counts to be determined by the gold standard methodology of flow cytometry with no clinically significant sacrifices in accuracy or precision.

Adult↗

PubMed1989

Two-color immunofluorescence flow cytometric analysis of lymphocytes in long-term renal allograft recipients.

We used 2-color immunofluorescence flow cytometry (FACS 440) and monoclonal antibodies (Becton-Dickinson) to study peripheral blood lymphocytes subsets from 26 patients with well functioning renal allografts for longer than 5 years. Comparisons were made to a group of 25 healthy volunteers and 25 dialysis patients awaiting renal transplantation. As anticipated, there was no significant difference with respect to the absolute number or percentage of activated (T11+ and HLA-DR+) and cytotoxic (Leu-2+ and Leu-15-)T lymphocytes among the 3 groups. However, there was a significantly decreased absolute number (p equals 0.0001) and percentage (p equals 0.0001) of suppressor cells (Leu-2+ and Leu-15+) in the transplant patients compared to the healthy control group. No significant differences existed between the transplant and dialysis groups. There also was an increase in the percentage (p equals 0.000) but not in the absolute number of T helper lymphocytes (Leu-3+ and Leu-8-) in the transplant population compared to healthy controls. No significant difference existed between the transplant and dialysis groups. These findings suggest that a normal value of cytotoxic (Leu-2+ and Leu-15-) and activated (T11+ and HLA-DR+) T lymphocytes may be a good prognostic indicator of long-term survival of renal allografts. Also, the highly significant decrease in the number of Leu-2+ and Leu-15+ cells in long-term, well functioning allografts indicates that additional functional characterization of this subset may be necessary.

Adult↗

PubMed1985

Improved allograft survival in nonidentical living related donor transplants using donor-specific blood transfusions.

A total of 78 consecutive HLA nonidentical living related donor transplant recipients with moderate to high stimulating mixed lymphocyte culture indexes underwent a deliberate donor-specific blood transfusion protocol. Of the patients 67 were first and 11 were second allograft recipients. Patients were monitored for immunological responses by cytotoxic B-cold, B-warm and T-warm antibody responses to a random panel of 30 donors, and by serial crossmatches to their donor subset B and T lymphocytes at 5C and 37C before beginning the protocol and after each donor-specific blood transfusion. Patients were followed from 3 months to 2 1/2 years, with allograft survival rates reported by the actuarial method. Survival rates of first allograft recipients were 96.0 plus or minus 2.77, 93.97 plus or minus 3.37, 93.97 plus or minus 3.37 and 90.68 plus or minus 4.50 per cent at 3 and 6 months, and 1 and 2 years, respectively. Of the patients entering the protocol for a primary transplant 20.18 per cent had persistently positive crossmatches. With increasing numbers of previous random blood transfusions a statistically significant sensitization rate was noted. Patient sensitization showed a general pattern of initial development of B-warm lymphocytotoxins resulting in positive B-warm crossmatches, which progressed to T-warm lymphocytotoxins and positive T-warm crossmatches if donor-specific blood transfusions continued. However, on development of B-warm positive crossmatches reversion to a negative crossmatch with successful transplantation was possible upon cessation of transfusions. No patient in the study was rendered nontransplantable due to donor-specific blood transfusions. All 5 patients who were completely disparate suffered amnestic type rejection episodes but following control of the rejection the course mimicked that of mixed lymphocyte culture identical living related donor transplants. Donor-specific blood transfusion is highly successful among first allograft recipients and success in extending the procedure to more disparate relatives is noted.

Blood Transfusion↗

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