Transplantation of a New Biological Product in Rare Diseases, Such as Epidermolysis Bullosa: Response and Clinical Outcome.

BACKGROUND: Epidermolysis bullosa (EB) is a phenotypically diverse group of hereditary blistering disorders involving mutations in 20 different genes. Those debilitating disorders are currently incurable; however, there are a number of promising preclinical trials, where some treatments already approach the stage of early clinical trial. In this paper we introduce a novel surgical approach to the treatment of EB-induced ulcerations. The purpose of our study was to evaluate the safety and efficacy of a new biological dressing in the form of an allogenic human skin equivalent graft before using multipotent stem cells, classified as an advanced therapy medicinal product. METHODS: Implanted human acellular dermal matrices were prepared from the superficial layers of donated human skin. Scaffold sterilization was conducted via irradiation with the use of a linear electron accelerator. Following water-knife debridement, wounds were surgically covered with accordingly prepared grafts and dressed in burn-injury fashion. Subsequently, the wounds were monitored for infection and viability. RESULTS: Our data indicate that grafting as a potential new medicinal product was safe and effective in patients with rare diseases, such as EB, and may be used for stem cells to create new Advanced Therapy Medicinal Products. During a 200-day follow-up, we proved the safety of using human scaffolds (allogeneic graft) by observing no apparent infection or necrosis. Instead, we noted fewer required dressing changes, promoted wound healing, pain reduction, and an overall improvement in the quality of life in patients with EB. CONCLUSION: The protocol for grafting allogenic acellular epidermal sheets is the most promising treatment for severely affected skin areas in EB patients to date.

New Treatment of Wound Healing With Allogenic Acellular Human Skin Graft: Preclinical Assessment and In Vitro Study.

BACKGROUND: Nonhealing wounds can be a major clinical problem. Impaired wound healing is often related to massive tissue injury, concomitant wound healing deficiencies (chronic wounds), burn injury, or congenital conditions. We propose a novel biological dressing as an alternative surgical approach. The dressing is a form of an allogenic human skin graft equivalent with further use of allogeneic stem cells classified as an advanced therapy medicinal product. This new allogenic acellular human skin graft has been specifically developed to address the clinical indications for dressing wound lesions and promoting tissue repair in specific rare genetic diseases. METHODS: This case report illustrates the use of an acellular human skin allograft seeded with multipotent stem cells in the treatment of tissue injuries (burns), congenital conditions, and chronic wounds. Donor-tissue processing yields an acellular dermal matrix with integral collagen bundling and organization, as well as an intact basement membrane complex. RESULTS: Preclinical observations show prolonged viability of acellular human skin grafts with multipotent stem cells. This was confirmed with histological and electron-microscopic evaluation of biopsies, which demonstrated host-cell infiltration and neovascularization of the biological dressing. Moreover, the dressings were characterized by low immunogenicity, as confirmed by histology exam and T-cell proliferation assays in vitro. CONCLUSION: Our data confirmed the safety and efficacy of the evaluated acellular human skin grafts, which may be used in patients with rare diseases, such as epidermolysis bullosa, burn injuries, and chronic wounds.

Short-term in vitro effects of bisphenol A activity on phenotype and function of peripheral blood immune system cells.

Bisphenol A (BPA) is a phenolic compound being constituent of numerous everyday-used products. Additionally, good absorption through skin and gastrointestinal tract is an unquestionable evidence for high risk of BPA exposure and its possible influence on health. Effects of BPA action on cells and tissues are associated with its structural and functional similarities to steroid hormones. Here we investigated whether BPA could possibly influence immune cells through steroid receptors present on majority of these cells. In in vitro experiments with 200 nM and 1000 nM BPA concentrations we found that high levels affect activation of lymphocytes through increased CD25 expression, with no changes in functional response based on IFN-gamma production. We demonstrated that BPA influence not only phenotype of monocytes with increased frequency of CD14++CD16- subtypes, but also activation inhibition with decline of HLADR expression within monocytes. Similarly to lymphocytes, no changes were observed in context of monocytes function in BPA-exposed cells. Our study revealed that BPA actions are also associated with its direct role in modulation of immune system cells. We presume that further experiments would allow establishing connection between presented data and increased risk of cancer and metabolic diseases in subject exposed to bisphenol A.

Cytokine Network & NETs.

The process of forming and releasing neutrophil extracellular traps (NETs) can be regulated by exogenous and endogenous factors, including cytokines. The study aimed to assess the impact of proinflammatory cytokines, IL-15, IL-17, IL-18, and anti-inflammatory IL-10 on the formation of NETs, all in comparison to IL-8 and pathogenic factors: LPS, fMLP. Also, the expression of myeloperoxidase (MPO), one of the main elements of neutrophil traps, was evaluated. After isolating the neutrophils with Polymorphprep™, the cells were sorted using CD16 MACS® microbeads and incubated with selected factors. The formation of NETs was registered using a BD Pathway 855 microscope system and the expression of MPO was evaluated using flow cytometry. The amounts of circulating DNA in cell supernatants was fluorescently quantified. Microscopic photographs indicated that rhIL-15, rhIL-17, rhIL-18 and fMLP induce formation and release of NETs at a similar timespan, while in the presence of rhIL-10, the formation of the traps was delayed. The presence of the studied cytokines indicated two populations of neutrophils displaying differing MPO expression (MPOlow and MPOhigh). Moreover, stimulation of neutrophils with LPS and fMLP revealed two populations of these cells that differed not only in the expression of MPO, but also in size.

High-dose bee venom exposure induces similar tolerogenic B-cell responses in allergic patients and healthy beekeepers.

BACKGROUND: The involvement of B cells in allergen tolerance induction remains largely unexplored. This study investigates the role of B cells in this process, by comparing B-cell responses in allergic patients before and during allergen immunotherapy (AIT) and naturally exposed healthy beekeepers before and during the beekeeping season. METHODS: Circulating B cells were characterized by flow cytometry. Phospholipase A2 (PLA)-specific B cells were identified using dual-color staining with fluorescently labeled PLA. Expression of regulatory B-cell-associated surface markers, interleukin-10, chemokine receptors, and immunoglobulin heavy-chain isotypes, was measured. Specific and total IgG1, IgG4, IgA, and IgE from plasma as well as culture supernatants of PLA-specific cells were measured by ELISA. RESULTS: Strikingly, similar responses were observed in allergic patients and beekeepers after venom exposure. Both groups showed increased frequencies of plasmablasts, PLA-specific memory B cells, and IL-10-secreting CD73- CD25+ CD71+ BR 1 cells. Phospholipase A2-specific IgG4-switched memory B cells expanded after bee venom exposure. Interestingly, PLA-specific B cells showed increased CCR5 expression after high-dose allergen exposure while CXCR4, CXCR5, CCR6, and CCR7 expression remained unaffected. CONCLUSIONS: This study provides the first detailed characterization of allergen-specific B cells before and after bee venom tolerance induction. The observed B-cell responses in both venom immunotherapy-treated patients and naturally exposed beekeepers suggest a similar functional immunoregulatory role for B cells in allergen tolerance in both groups. These findings can be investigated in other AIT models to determine their potential as biomarkers of early and successful AIT responses.

Activation of the complement cascade enhances motility of leukemic cells by downregulating expression of HO-1.

As a crucial arm of innate immunity, the complement cascade (ComC) is involved both in mobilization of normal hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood and in their homing to BM. Despite the fact that ComC cleavage fragments alone do not chemoattract normal HSPCs, we found that leukemia cell lines as well as clonogenic blasts from chronic myeloid leukemia and acute myeloid leukemia patients respond robustly to C3 and C5 cleavage fragments by chemotaxis and increased adhesion. This finding was supported by the detection of C3a and C5a receptors in cells from human malignant hematopoietic cell lines and patient blasts at the mRNA (reverse transcriptase-polymerase chain reaction) and protein level (fluorescence-activated cell sorting), and by the demonstration that these receptors respond to stimulation by C3a and C5a by phosphorylation of p42/44 and p38 mitogen-activated protein kinases (MAPK), and protein kinase B (PKB/AKT). We also found that inducible heme oxygenase 1 (HO-1) is a negative regulator of ComC-mediated trafficking of leukemic cells, and that stimulation of leukemic cells by C3 or C5 cleavage fragments activates p38 MAPK, which downregulates HO-1 expression, rendering cells more mobile. We conclude that activation of the ComC in leukemia/lymphoma patients (for example, as a result of accompanying infections) enhances the motility of malignant cells and contributes to their spread in a p38 MAPK-HO-1-dependent manner. Therefore, inhibition of p38 MAPK or upregulation of HO-1 by small-molecule modulators would have a beneficial effect on ameliorating cell migration-mediated expansion of leukemia/lymphoma cells when the ComC becomes activated.

The Role of Different Monocyte Subsets in the Pathogenesis of Atherosclerosis and Acute Coronary Syndromes.

The inflammation underlying both atherosclerosis and acute coronary syndromes is strongly related to monocyte-related actions. However, different monocyte subsets can play differential roles in the formation and destabilization of atherosclerotic plaque as well as healing of damaged myocardial tissue. Monocytes are currently being divided into three functionally distinct subsets with different levels of CD14 (cluster of differentiation 14) and CD16 expression. Thus, there are classical CD14++CD16-, intermediate CD14++CD16+ and non-classical CD14+CD16++ monocytes. Here, we summarize the current knowledge on complex activities of different monocyte subsets in atherosclerosis and acute coronary syndromes. Moreover, we discuss which monocyte subsets can serve either as predictive biomarkers of cardiovascular risk or as potential targets used in atherosclerosis and its complications.

Prognostic significance of ligands belonging to tumour necrosis factor superfamily in acute lymphoblastic leukaemia.

Altered activities of ligands belonging to tumour necrosis factor (TNF) superfamily, namely B-cell activating factor (BAFF), a proliferation-inducing ligand (APRIL) and apoptosis inducing ligand (TRAIL) were demonstrated in several haematological diseases including acute lymphoblastic leukaemia (ALL). BAFF, APRIL and TRAIL provide crucial survival signals to immature, naive and activated B cells. These ligands are capable of activating a broad spectrum of intracellular signalling cascades that can either induce apoptosis or protect from programmed cell death. BAFF and APRIL, which can directly activate the NF-κB pathway, have been identified as crucial survival factors for ALL cells. Here, we have analyzed serum BAFF, APRIL and TRAIL concentrations in 48 patients with newly diagnosed ALL and 44 healthy volunteers. The levels of APRIL and BAFF were significantly higher in ALL patients as compared to healthy volunteers. In contrast, concentrations of TRAIL were significantly lower in ALL patients. Moreover, following induction, the levels of APRIL, but not BAFF or TRAIL, were significantly lower in a group of patients with complete remission (CR) as compared to non-respondent (NR) ALL patients. Furthermore, we demonstrated statistically significant differences in concentrations of APRIL between CR MRD-negative and CR, MRD-positive ALL patients. Notably detection of higher concentrations of APRIL was associated with shorter leukaemia-free survival and overall survival. Altogether, our data indicate that APRIL can play an important role in the pathogenesis of ALL and the measurement of APRIL levels can improve prognostication in ALL patients.

The effect of inhaled corticosteroids on the concentration of soluble CD163 in induced sputum of allergic asthma patients.

BACKGROUND AND OBJECTIVE: CD163 is a monocyte/macrophage-specific molecule whose expression is induced by corticosteroids and IL-10. The aim of this study was to evaluate the concentration of soluble CD163 (sCD163) in the induced sputum of asthmatic patients before and after therapy with inhaled corticosteroids (ICSs). PATIENTS AND METHODS: The study was performed in 24 patients with mild allergic asthma (AAs) and 10 healthy controls (HCs). In 18 AAs, induced sputum and serum samples were obtained before ICS therapy (T0) and 7 days later (T7). In the 6 AAs not treated with ICSs the procedures were performed at To and T7. The concentration of sCD163 in sputum and serum samples was evaluated using ELISA. RESULTS: There was no significant difference in mean (SD) baseline serum sCD163 concentration between AAs (1030 [449] ng/mL) and HCs (930 [334.5] ng/mL, P = .530). However, at To the mean sputum sCD163 concentration was significantly greater in AAs (4.78 [3.34] ng/mL) than in HCs (1.8 [0.41] ng/mL, P =.009). Treatment with ICSs resulted in a significant increase in sCD163 concentration in sputum (P < .0001) but not in serum (P =.679). No change in sputum or serum sCD163 concentration was detected in AAs who were not treated with ICSs. The change in sputum sCD163 concentration inversely correlated with changes in sputum eosinophilia or exhaled nitric oxide concentration. CONCLUSIONS: ICS therapy leads to local upregulation of sCD163 expression, which in turn may participate in the anti-inflammatory effects of ICS therapy.

The effect of inhaled corticosteroids on the concentration of soluble CD163 in induced sputum of allergic asthma patients

Background and Objective: CD163 is a monocyte/macrophage-specific molecule whose expression is induced by corticosteroids and IL-10. The aim of this study was to evaluate the concentration of soluble CD163 (sCD163) in the induced sputum of asthmatic patients before and after therapy with inhaled corticosteroids (ICSs). Patients and Methods: The study was performed in 24 patients with mild allergic asthma (AAs) and 10 healthy controls (HCs). In 18 AAs, induced sputum and serum samples were obtained before ICS therapy (T0) and 7 days later (T7). In the 6 AAs not treated with ICSs the procedures were performed at T0 and T7. The concentration of sCD163 in sputum and serum samples was evaluated using ELISA. Results: There was no significant difference in mean (SD) baseline serum sCD163 concentration between AAs (1030 [449] ng/mL) and HCs (930 [334.5] ng/mL, P=.530). However, at T0 the mean sputum sCD163 concentration was significantly greater in AAs (4.78 [3.34] ng/mL) than in HCs (1.8 [0.41] ng/mL, P=.009). Treatment with ICSs resulted in a significant increase in sCD163 concentration in sputum (P<.0001) but not in serum (P=.679). No change in sputum or serum sCD163 concentration was detected in AAs who were not treated with ICSs. The change in sputum sCD163 concentration inversely correlated with changes in sputum eosinophilia or exhaled nitric oxide concentration. Conclusions: ICS therapy leads to local upregulation of sCD163 expression, which in turn may participate in the anti-inflammatory effects of ICS therapy (AU)

Antecedentes y Objetivo: El CD163 es una molécula específica de monocitos/macrófagos cuya expresión es inducida por los corticosteroides y la interleucina 10 (IL-10). El objetivo de este estudio fue evaluar la concentración de sCD163 en el esputo inducido de pacientes asmáticos, antes y después del tratamiento con corticosteroides inhalados (ICS). Pacientes y Métodos: El estudio se realizó en 24 pacientes con asma alérgica leve (AA) y 10 controles sanos (HC). En 18 AA, se obtuvieron muestras de esputo y suero antes (T0) y a los 7 días (T7) tras la introducción del tratamiento con ICS. Asimismo, en 6 AA no tratados con ICS se llevaron a cabo los mismos procedimientos en T0 y T7. Se evaluó la concentración de sCD163 en esputo y suero de las muestras mediante ELISA. Resultados: No hubo diferencia en la concentración sérica basal media de sCD163 entre AA (1.030± 449 ng/ml) y los HC (930 ± 334,5 ng/ml, p = 0,530). Sin embargo, en T0 la concentración media de sCD163 esputo fue significativamente mayor en los AA (4,78 ± 3,34 ng/ml) que en HC (1,8 ± 0,41 ng/ml, p = 0,009). El tratamiento con ICS dio lugar a un aumento significativo de la concentración en el esputo de sCD163 (p < 0,0001), pero no en el suero (p = 0,679). No se detectaron diferencias en las concentraciones de sCD163 ni en el suero ni en el esputo del grupo de AA que no fueron tratados con ICS. La concentración sCD163 en esputo se correlacionó inversamente con la eosinofilia en esputo y la concentración NO exhalado. Conclusiones: El tratamiento con ICS induce la expresión local de sCD163, que a su vez puede mediar en el mecanismo anti-inflamatorio de este tratamiento (AU)

Glucocorticoids upregulate decreased IL-7 receptor expression in asthmatic patients and simian immunodeficiency virus-infected non-human primates.

Signaling through interleukin-7 receptor (IL-7R) is essential for regulation of T-cell homeostasis and survival. Previously, we and others have found diminished IL-7R levels in simian immunodeficiency virus (SIV) - infected non-human primates and human immunodeficiency virus (HIV) - infected patients. To date, it remains unknown whether changes in IL-7R expression could also be linked to non-infectious inflammatory diseases such as asthma or anti-inflammatory drug use. Here, we investigated through flow cytometry the levels of IL-7R expression on CD4+ and CD4- T-cells in asthmatic patients in relation to disease severity, immune status and glucocorticoid (GC) use. In addition, we sought to evaluate the effects of in vivo and in vitro GC treatment on IL-7R expression in both asthmatic patients and SIV-infected non-human primates. We demonstrated that expression of IL-7R on peripheral blood CD4+ T-cells was significantly decreased in clinically stable GC-naive mild and moderate asthmatic patients. Accordingly, the development of asthmatic reaction following bronchial allergen challenge performed in sensitized subjects was associated with a significant drop in levels of IL-7R on circulating CD4+ T-cells. In contrast, CD4+ T-cells from both, mild and moderate, but not severe asthmatics, treated with inhaled GC displayed levels of IL-7R similar to that seen in healthy controls. We did not find significant differences with serum or sputum interleukin-7 levels among healthy controls and GC-naïve and GC-treated asthmatic patients. Furthermore, both in vitro GC treatment and short-term oral GC administration to asthmatic patients resulted in a significant enhancement of IL-7R. Similarly, we demonstrated that GC-stimulated T-cells from SIV-infected non-human primates up-regulated IL-7R expression. Accordingly, experimental short-term systemic in vivo administration of GC to SIV-infected macaques led to enhancement of IL-7R expression on circulating T-cells. Our data indicate that GC bear potential to recover diminished IL-7R expression, as well in asthma as in lentiviral infection.

Lower proportions of CD4+CD25(high) and CD4+FoxP3, but not CD4+CD25+CD127(low) FoxP3+ T cell levels in children with autoimmune thyroid diseases.

The essence of autoimmune thyroid disease (AITD) is loss of tolerance of own tissues caused by malfunction of T lymphocytes, which affects the production of antibodies reacting with particular cell structures and tissues. Foxp3(+) regulatory T cells (Tregs) take part in the regulation of immune response and play a leading role in developing immune tolerance through active suppression. The aim of the study was to estimate the expression of CD4+CD25(high), CD4+CD25+CD127(low)FoxP3(+) and CD4+ FoxP3 T cells in patients with Graves' disease (GD) (n = 24, median age 15.5 years), in patients with Hashimoto's thyroiditis (HT) (n = 30, median age 15 years) in comparison with sex- and age-matched healthy control subjects (n = 30, median age 15 years). Polychromatic flow cytometry using a FACSCalibur (BD Biosciences) cytometer was applied to delineate T regulatory cell populations. In untreated patients with Graves' disease and HT we observed a significant decrease in CD4+FoxP3 (p < 0.001, p < 0.01) and CD4+CD25(high) (p < 0.016, p < 0.048) T lymphocytes as compared to the healthy control subjects. After 6-12 months of L-thyroxine therapy in HT cases these phenotypes of Tregs were normalized, yet no such changes were observed during GD therapy. The analysis of CD4+CD25+CD127(low)FoxP3+ T cells in the peripheral blood revealed comparable percentages of these cells in patients with thyroid autoimmune diseases to the controls. We conclude that the reduction number of Tregs with CD4+CD25(high) and CD4+FoxP3 phenotype suggests their role in initiation and development of autoimmune process in thyroid disorders.

Allergen challenge differentially affects the number of circulating monocyte subsets.

Peripheral blood monocyte (PBM) subsets play different roles in inflammatory response and tissue remodelling. The aim of this study was to investigate how allergen challenge affects the number of circulating PBMs in Dermatophagoides pteronyssinus (Dp) allergic patients (Dp-APs). Among 34 Dp-APs challenged, in 22 patients significant bronchoconstriction was demonstrated [responders (Rs)], while in 12, only upper respiratory symptoms were seen [non-responders (NRs)]. Twelve healthy, non-atopic subjects were used as controls (HCs). Expression of CD14, CD16 and CCR4 was evaluated by flow cytometry on the whole-blood samples before (T(0) ), 6 h (T(6) ) and 24 h (T(24) ) after the challenge. Plasma concentrations of CCL2, CX3CL1 and CCL17 were evaluated using ELISA. At T(0) , the mean percentage of CD14++ CD16+ PBMs in Rs (35.4%; 95%CI 26.9-43.9%) was significantly greater than in HCs (14.6%; 95%CI 7.3-21.8%; P = 0.006) and in NRs (17.5%; 95%CI 9.6-25.4%; P = 0.001). The baseline number of CD14++ CD16+ PBMs correlated with airway hyper responsiveness (AHR) (r = -0.507; 95%CI -0.834 to -0.432, P < 0.001). At T(24) , the number of CD14++ CD16+ PBMs significantly decreased in Rs but not in NRs and the numbers inversely correlated with plasma CCL17 concentration. Changes in the number of circulating CD14++ CD16+ cells after Dp challenge correlated with AHR (r = 0.706, 95%CI 0.43-0.861; P < 0.001). In all subjects, the greatest expression of CCR4 was found on CD14++ CD16+ PBMs. Expansion of CD14++ CD16+ monocytes in the peripheral blood with subsequent mobilization of those cells after allergen challenge may facilitate the development of AHR in Dp-APs.

Oral glucocorticoid treatment decreases interleukin-10 receptor expression on peripheral blood leucocyte subsets.

Glucocorticoids (GCS) are capable of stimulating the secretion of interleukin (IL)-10 by leucocytes; however, the potential of GCS to modulate leucocyte susceptibility to IL-10-mediated actions has not yet been studied. In the current paper, we performed a detailed cross-sectional analysis of IL-10 receptor (IL-10R) expression by CD4(+) and CD8(+) T cells, natural killer (NK) cells, monocytes and neutrophils. Next, we analysed the effects of short-term oral GCS treatment on surface IL-10R expression by various leucocyte subpopulations in asthmatic patients. All leucocyte subsets studied presented with substantial levels of surface IL-10R. The highest levels of IL-10R were found on monocytes, predominantly with CD14(2+)CD16(+) and CD14(+)CD16(+) phenotypes, and on CD4(+)CD25(high) T cells. In contrast, levels of IL-10R on CD8(+) T cells, NK cells and neutrophils were significantly lower and similar to each other in intensity. GCS treatment resulted in a significant decrease of IL-10R expression on all analysed peripheral blood leucocyte subsets. Our data suggest that down-regulation of IL-10R could counterbalance the otherwise suppressive action of GCS.

Frequencies of circulating CD4+CD25+CD127low cells in atopics are altered by bronchial allergen challenge.

BACKGROUND: Recent studies in rodents revealed that regulatory T cells (T reg cells) with CD4+CD25+ phenotype can exert suppressive effects on experimentally-induced allergic airway inflammation and airway hyper-reactivity. It is unclear however, whether modulations of bronchoconstriction responses in human subjects might be related to T reg cells. We report here for the first time the changes in frequency of circulating lymphocytes with putative T reg cell phenotype (CD4+CD25+CD127low) in relation to bronchoconstriction phenotype following an intrabronchial allergen challenge. MATERIAL AND METHODS: Thirty-one house dust mite sensitive patients were challenged with Dermatophagoides pteronyssinus extract (Dp). Eleven isolated early responders (IER) were compared with nine dual (early and late) responders (DR) and to 11 non-responders (NR). Frequencies of peripheral blood CD4+CD25+CD127low lymphocytes were assessed in all groups of patients by using three-parameter flow cytometry before, and six and 24 h, after allergen inhalation. RESULTS: At baseline, frequencies of CD4+CD25+CD127low lymphocytes were not statistically different among NR, IER and DR. When all individuals were analyzed together, a statistically significant decrease in frequency of CD4+CD25+CD127low lymphocytes was observed 6 h after the bronchial challenge. Interestingly, such a pattern was found consistently only in NR, while IER and DR displayed varying responses resulting in a trend similar to that of NR. Twenty-four hours after the bronchial challenge, frequencies of CD4+CD25+CD127low lymphocytes in all groups tended to return to baseline values. CONCLUSIONS: Our data indicate that bronchial allergen inhalation in sensitive patients (predominantly in those who did not develop significant bronchoconstriction) is associated with a decrease of proportion of peripheral lymphocytes with regulatory T cell phenotype.

Impairment of Gag-specific CD8(+) T-cell function in mucosal and systemic compartments of simian immunodeficiency virus mac251- and simian-human immunodeficiency virus KU2-infected macaques.

The identification of several simian immunodeficiency virus mac251 (SIV(mac251)) cytotoxic T-lymphocyte epitopes recognized by CD8(+) T cells of infected rhesus macaques carrying the Mamu-A*01 molecule and the use of peptide-major histocompatibility complex tetrameric complexes enable the study of the frequency, breadth, functionality, and distribution of virus-specific CD8(+) T cells in the body. To begin to address these issues, we have performed a pilot study to measure the virus-specific CD8(+) and CD4(+) T-cell response in the blood, lymph nodes, spleen, and gastrointestinal lymphoid tissues of eight Mamu-A*01-positive macaques, six of those infected with SIV(mac251) and two infected with the pathogenic simian-human immunodeficiency virus KU2. We focused on the analysis of the response to peptide p11C, C-M (Gag 181), since it was predominant in most tissues of all macaques. Five macaques restricted viral replication effectively, whereas the remaining three failed to control viremia and experienced a progressive loss of CD4(+) T cells. The frequency of the Gag 181 (p11C, C-->M) immunodominant response varied among different tissues of the same animal and in the same tissues from different animals. We found that the functionality of this virus-specific CD8(+) T-cell population could not be assumed based on the ability to specifically bind to the Gag 181 tetramer, particularly in the mucosal tissues of some of the macaques infected by SIV(mac251) that were progressing to disease. Overall, the functionality of CD8(+) tetramer-binding T cells in tissues assessed by either measurement of cytolytic activity or the ability of these cells to produce gamma interferon or tumor necrosis factor alpha was low and was even lower in the mucosal tissue than in blood or spleen of some SIV(mac251)-infected animals that failed to control viremia. The data obtained in this pilot study lead to the hypothesis that disease progression may be associated with loss of virus-specific CD8(+) T-cell function.