Flow Cytometry Analysis Of Lymphocytes Research Articles

HIV associated cell death: Peptide-induced apoptosis restricts viral transmission.

The human immunodeficiency virus (HIV) is still a global pandemic and despite the successful use of anti-retroviral therapy, a well-established cure remains to be identified. Viral modulation of cell death has a significant role in HIV pathogenesis. Here we sought to understand the major mechanisms of HIV-induced death of lymphocytes and the effects on viral transmission. Flow cytometry analysis of lymphocytes from five latent HIV-infected patients, and HIV IIIB-infected MT2 cells demonstrated both necrosis and apoptosis to be the major mechanisms of cell death in CD4+ and CD4-/CD8- lymphocytes. Significantly, pro-apoptotic tumor necrosis factor (TNF) peptide (P13) was found to inhibit HIV-related cell death and reduced viral transmission. Whereas pro-necrotic TNF peptide (P16) had little effect on HIV-related cell death and viral transmission. Understanding mechanisms by which cell death can be manipulated may provide additional drug targets to reduce the loss of CD4+ cells and the formation of a viral reservoir in HIV infection.

A Multiple Antigen Peptide Vaccine Containing CD4+ T Cell Epitopes Enhances Humoral Immunity against Trichinella spiralis Infection in Mice.

Multiepitope peptide vaccine has some advantages over traditional recombinant protein vaccine due to its easy and fast production and possible inclusion of multiple protective epitopes of pathogens. However, it is usually poorly immunogenic and needs to conjugate to a large carrier protein. Peptides conjugated to a central lysine core to form multiple antigen peptides (MAPs) will increase the immunogenicity of peptide vaccine. In this study, we constructed a MAP consisting of CD4+ T cell and B cell epitopes of paramyosin (Pmy) of Trichinella spiralis (Ts-Pmy), which has been proved to be a good vaccine candidate in our previous work. The immunogenicity and induced protective immunity of MAP against Trichinella spiralis (T. spiralis) infection were evaluated in mice. We demonstrated that mice immunized with MAP containing CD4+ T cell and B cell epitopes (MAP-TB) induced significantly higher protection against the challenge of T. spiralis larvae (35.5% muscle larva reduction) compared to the MAP containing B cell epitope alone (MAP-B) with a 12.4% muscle larva reduction. The better protection induced by immunization of MAP-TB was correlated with boosted antibody titers (both IgG1 and IgG2a) and mixed Th1/Th2 cytokine production secreted by the splenocytes of immunized mice. Further flow cytometry analysis of lymphocytes in spleens and draining lymph nodes demonstrated that mice immunized with MAP-TB specifically enhanced the generation of T follicular helper (Tfh) cells and germinal center (GC) B cells, while inhibiting follicular regulatory CD4+ T (Tfr) cells and regulatory T (Treg) cells. Immunofluorescence staining of spleen sections also confirmed that MAP-TB vaccination enhanced the formation of GCs. Our results suggest that CD4+ T cell epitope of Ts-Pmy is crucial in vaccine component for inducing better protection against T. spiralis infection.

Natural killer (NK) cells are associated with reduced relapse without increased graft-versus-host disease (GVHD) after allogeneic hematopoetic stem-cell transplantation (HSCT)

6542 Background: Natural killer cells have potential for anti-leukemia activity after haplo-identical HSCT. However, the role of reconstituted NK cells in relapse and survival after matched HLA-identical HSCT is unknown. We investigated the correlation between NK reconstitution and death, relapse, and GVHD after HSCT. Methods: We prospectively studied NK reconstitution in 123 patients with predominantly high-risk hematologic malignancies. Peripheral blood absolute NK cell count (ANK) was determined by flow cytometry analysis of lymphocytes co-expressing bright staining CD56+ and CD16+. Cluster analysis distinguished patients at engraftment with high ANK (>12.2/mm3) (n=80) and low ANK (n=43), and at 60 days with high ANK (>20/mm3) (n=64) and low ANK (n=26). Primary study endpoints were death, relapse and acute grade II-IV GVHD with median follow up of 360 days (28–1967). A cox proportional hazard model was adjusted for important covariates, including age, transplant conditioning (myeloablative vs. non-myeloablative), graft source (peripheral blood vs. bone marrow), and donor-type (HLA-matched related vs. unrelated), dendritic cell level and absolute neutrophil count. Results: A low ANK at engraftment was significantly associated with death (p=0.03) after non-myeloablative HSCT. Using a selection model, only NK remained in the model, and a low ANK was associated with a 2.5 hazard ratio for subsequent death (p=0.04). Additionally, for the overall cohort, ANK at 60 days was strongly associated with time to death (92% in high vs 65% in low group; LR p=0.0007), at 310 days after HSCT. ANK level at 60 days was strongly associated with time to relapse (n=82, (LR p=0.0001). In a multivariate model, low ANK at 60 days was associated with a 4.2 fold relative risk (RR) of death (1.9, 9.1) and 8.6 RR for time to relapse (3.1, 24.3). Conclusions: High NK cell reconstitution is associated with reduced relapse and death without increased incidence of GVHD after HLA-identical HSCT. Our study suggests that NK may have both a potential anti-tumor effect, as well as be tolerogenic. Measuring NK after HSCT may have novel prognostic and therapeutic implications. No significant financial relationships to disclose.

Cervical lymph node infections with non‐tuberculous mycobacteria in preschool children: interferon gamma deficiency as a possible cause of clinical infection

Epidemiological studies have demonstrated a prevalence of positive PPD reactions to non‐tuberculous mycobacteria (NTM) in 9% of 4–5‐y‐old children in the Göteborg area. Very few children of this age develop suppurative infections in lymph nodes that require surgical procedures. The hypothesis was that these children might have T cell deficiencies with abnormalities of macrophage functions, particularly with type 1 cytokines. Twenty‐four children who needed operations were investigated immunologically and compared to 10 children of the same age operated on for non‐infectious reasons. The methods used were flow cytometry analysis of lymphocytes in blood, blood lymphocyte stimulation assays and interferon gamma analyses. The patients had significantly lower levels of interferon gamma than the controls after stimulation with Candida antigens or Con A. The numbers of T and B lymphocytes were higher in patients than in controls. Conclusion: Children with necrotic lymph node infections in the cervical region due to NTM had lower interferon gamma production after stimulation than healthy age‐matched controls.

Regulation of B cell responses to the variant surface glycoprotein molecule in trypanosomiasis. II. Down-regulation of idiotype expression is associated with the appearance of lymphocytes expressing antiidiotypic receptors.

The current study examines the idiotypic expression and regulation of variant surface glycoprotein (VSG)-specific B cell responses during African trypanosomiasis. Utilizing competitive inhibition RIA analysis, we detected antibodies in the serum of BALB/cByJ mice infected with Trypanosoma brucei rhodesiense clone LouTat 1.5 that recognized the same VSG epitopes as three VSG 1.5-specific mAb. These epitope-specific antibody responses were detectable by day 5 of infection, peaked by day 10, and then declined slowly through day 15 of infection. VSG-specific antibodies detectable in the serum of infected BALB/cByJ mice included those that were idiotypically cross-reactive with the VSG 1.5-specific mAb. These idiotypically defined, VSG-specific antibody responses appeared to peak around day 7 of infection, but then declined to near preimmune levels by day 15 of infection, demonstrating that the aggregate epitope-specific response was composed only in part by the idiotypically cross-reactive responses. Although corresponding antiidiotypic antibodies could not be detected in infected sera during periods of up- or down-regulation of idiotypically defined antibodies, flow cytometry analysis of lymphocytes isolated from the spleens of LouTat 1.5-infected BALB/cByJ mice revealed the presence of antiidiotypic receptor-bearing cells. These cells were detectable primarily during days 10 to 12 of infection and subsequently down-regulated their receptors, or declined in numbers, to near preimmune levels by day 15 of infection. The appearance of these antiidiotypic receptor-bearing cells coincides with the decline of idiotypic antibody present in the serum of the LouTat 1.5-infected mice and may represent nascent evidence for idiotypic regulation of trypanosome-specific immune responses in infected animals.

Two-color immunofluorescence and flow cytometry analysis of lymphocytes in minimal change nephrotic syndrome

Two-color immunofluorescence and flow cytometry analysis of lymphocytes in minimal change nephrotic syndrome

Two-color immunofluorescence and flow cytometry analysis of lymphocytes in long-term renal allotransplant recipients: identification of a major Leu-7+/Leu-3+ subpopulation.

Using two-color immunofluorescence with fluorescein isothiocyanate (FITC)- and phycoerythrin (PE)-labeled monoclonal antibodies to human lymphocyte antigens and flow cytometry, we studied lymphocyte subsets in 16 long-term renal allotransplant recipients at risk for a mean of 78 +/- 15 mo. The absolute number of Leu-1+, Leu-2a+, and Leu-3a+ lymphocytes is significantly decreased compared with a control population, whereas Leu-7+ and Leu-15+ subsets remain unchanged despite standard chronic immunosuppression (azathioprine and prednisone). Within the Leu-7+ subset, we found various phenotypes. Doubly fluorescent lymphocytes Leu-7+/Leu-1+ and Leu-7+/Leu-2a+ are not significantly different in the transplant population compared with a normal control population. The Leu-7+/Leu-3a+ subpopulation is seen to be significantly elevated, and the Leu-7+/Leu-15+ subpopulation decreases significantly. The relationship between the modification of these two phenotypes within the Leu-7 subset may be an important correlate of decreased NK cell activity in long-term renal allotransplant recipients. These Leu-7+/Leu-3a+ cells, normally less than 1% of peripheral blood lymphocytes, have no known functional activity.