Swine pericardium as dermal substrate for human keratinocyte culture.

BACKGROUND: Synthetic skin analogues or living allogeneic or autologous cells are used as dressings for the care of skin wounds, as well as temporary or permanent substitutes for damaged epithelia. OBJECTIVES: To evaluate if keratinocyte growth on a swine pericardium substrate mimics the natural epithelial layers compared with cultures on allogeneic dermis, which is accepted as having appropriate physical and chemical properties for growth and differentiation. METHODS: Keratinocytes were cultured on a swine pericardium substrate and allogeneic dermis, either submerged or at the air-liquid interface. At 7, 14 and 21 days postseeding the cultures were evaluated by light microscopy after both haematoxylin and eosin staining and immunohistochemistry. RESULTS: Cell-substrate interactions led to growth, stratification and differentiation of cells, with the definition of epithelial layers. The submerged system showed a continuous growth rise on both composites, but this was more prominent with the swine pericardium substrate. An increase in the number of layers at the air-liquid interface with the dermis composites, in contrast to the submerged cultures, occurred only from days 7 to 14. The pattern of keratinocyte growth on swine pericardium substrate was much better in the submerged than in the air-liquid interface cultures. CONCLUSIONS: The results indicate that swine pericardium is a better substrate than allogeneic dermis for keratinocyte cultures in submerged but not in air-liquid interface cultures. Swine pericardium as a substrate opens one more possibility for skin restoration after trauma or burns.

CD4 and CD8 distribution profile in individuals infected by Schistosoma mansoni.

RATIONALE: Patients with chronic Schistosoma mansoni infection show lower anti-soluble egg antigen (SEA) proliferation responses and higher responses to soluble worm antigen preparation (SWAP). OBJECTIVE: To compare the activation status and proliferation response of peripheral blood mononuclear cells (PBMC) of infected (XTO) and egg-negative individuals (NI) living in the same endemic area. METHODS: XTO (n = 51) and NI individuals from the same geographical area (n = 37) and healthy blood donors (n = 22) were evaluated before and after stimulation with SEA and SWAP. The expression of activation markers (CD4(+) HLADR(+), CD8(high+)HLA-DR(+) and CD8(+) CD28(+)) and proliferation assay was assessed by flow cytometry. FINDINGS: PBMC from infected patients showed lower frequency of CD4(+) but no change in CD8(+) T cells when compared with the healthy donor group. The ratio CD4(+)/CD8(+) was 1.3, 0.6 and 0.5 in healthy donors, infected and non-infected individuals, respectively. The HLA-DR(+) expression on CD8(+) was higher in PBMC from infected and non-infected individuals than from healthy donors, but similar in both total lymphocytes and CD4(+) populations. No intergroup proliferation response differences were observed in CD4(+) and CD8(+) PBMC unstimulated and stimulated with SEA and SWAP. The SEA but not SWAP-stimulated cells showed a decrease in the expression of phosphorylated extracellular signal-regulated kinase (ERK1/2). CONCLUSIONS: XTO and NI individuals living in the same area presented a smaller per cent of CD4(+) and a higher per cent of CD8(+) cells. The activation by either CD8(high+)HLA-DR(+) or CD8(high+)HLA-DR(+)/CD8(+) was enhanced and decreased in XTO and NI by CD8(+) CD28(+) and CD8(+) CD28(+)/CD8(+) when compared with healthy donor. ERK phosphorylation was attenuated in XTO and NI individuals when stimulated with SEA but not SWAP.

Stage-specific immune responses in human Necator americanus infection.

We describe how hookworms interact with their human hosts by comparing lymphocyte phenotyping, proliferative responses, and cytokine and chemokine secretion patterns in adults who are either mono-infected with Necator americanus or egg-negative controls resident in an area of high transmission in Brazil. Cellular immune responses against crude hookworm antigen extracts from different developmental stages were evaluated simultaneously. Principal component analysis (PCA) was used to reduce the standardized immune responses. Random effects multivariate regression was then used to investigate whether principal components (PC) differ between the two groups once potential confounders and effect modifiers have been accounted for. Although hookworm patients had reduced percentages of T and B cells, they had higher levels of activated CD4(+) T and CD19(+) B cells. This state of 'immune activation' coincided with lower proliferative responses, especially to third-stage larval antigen. Cytokine levels in mono-infected adults were also lower and characterized by a mixed Th1/Th2-type profile. Excretory/secretory antigen from adult worms was a potent modulator of the immune response, resulting in diminished TNF-alpha and IL-10 secretion in peripheral blood mononuclear cells (PBMC) from hookworm infected patients. We propose that the longevity of hookworms in their human hosts results from a stage-specific, down-modulation of the immune response.

Ageing and Toll-like receptor expression by innate immune cells in chronic human schistosomiasis.

There has been no systematic study of the immune response of individuals aged over 60 years living in Schistosomiasis mansoni-endemic areas, although senescence is reportedly associated with susceptibility to infection and progressive decline in immune function. We have shown previously, in two endemic areas in Minas Gerais, Brazil, that the frequency of individuals aged over 60 years with chronic schistosomiasis is no longer negligible. Moreover, several elderly individuals who have always lived in these endemic areas stay protected from infection. An important question for studies of ageing and disease control in developing countries is which differences in the immunological profile of these negatively tested (non-infected) individuals can account for their resistance to either infection or reinfection. We show, in the present study, that non-infected (negative) elderly individuals develop innate immune mechanisms of protection that replace the age-associated decline in T cell function. Non-infected elderly individuals from endemic areas of schistosome infection present an increase in the frequency of the natural killer (NK) CD56(low) subset of NK cells expressing Toll-like receptors (TLR)-1, -2, -3 and -4 as determined by flow cytometry analysis. In addition, the proportion of dendritic cells expressing TLR-1 is elevated as well as the frequency of monocytes expressing TLR-1 and -4. These results suggest that TLR expression by cells of the innate immune system may be related to the negative status of infection in some elderly individuals who are constantly exposed to S. mansoni. Developing mechanisms of protection from infection may represent a biomarker for healthy ageing in this population.

Natural versus drug-induced resistance in Schistosoma mansoni infection.

Rodrigo Corrêa-Oliveira, Iramaya Rodrigues Caldas and Giovanni Gazzinelli here focus on the immune response of individuals with natural resistance to schistosomiasis, which differs significantly from that of post-treatment resistant and infected individuals. They suggest that the activation of T helper type 1 (Th1) and Th2 cells is needed for the induction of natural resistance against Schistosoma mansoni infection.

CD4+ T cells of schistosomiasis naturally resistant individuals living in an endemic area produce interferon-gamma and tumour necrosis factor-alpha in response to the recombinant 14KDA Schistosoma mansoni fatty acid-binding protein.

Cellular immune responses to recombinant (r) Sm14 were examined in chronic, treated patients and uninfected individuals living in an endemic area for schistosomiasis. The lymphocyte proliferative responses and cytokine profile to this antigen were evaluated. Peripheral blood mononuclear cells (PBMC) of all groups studied proliferated to rSm14. However, the highest proliferation index to rSm14 was detected in uninfected endemic normal (EN) individuals who are naturally resistant to schistosomiasis. Regarding the cytokines produced, the levels of interleukin (IL)-5 and IL-10, known as Th2 cytokines, were not statistically different among all groups studied. In contrast, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha were produced in significantly higher amounts by PBMC of EN individuals following rSm14 stimulation. Additionally, we have determined by flow cytometry that CD4+ T cells from these individuals are the main lymphocyte subpopulation producing IFN-gamma and TNF-alpha. Moreover, we have used rIL-10 or rIFN-gamma, or monoclonal antibodies (MoAb) against these two cytokines to determine their role on cellular reactivity to rSm14. Exogenous IL-10 suppressed T-cell proliferation and neutralization of endogenous IL-10 restored lymphocyte activation and enhanced IFN-gamma and TNF-alpha production in chronically infected patients. In contrast, the addition of anti-IFN-gamma totally abrogated the PBMC proliferation within the EN group. This study demonstrated that IL-10 is an important cytokine down-regulating T-cell responses in chronic schistosomiasis, whereas lymphocyte proliferation in the uninfected resistant group is dependent on IFN-gamma. Taken together these results suggest that Th1 type of immune response induced in EN individuals to a specific schistosome antigen might be associated with resistance to infection and also highlighted the importance of Sm14 as a potential vaccine candidate.

Susceptibility and resistance to Schistosoma mansoni reinfection: parallel cellular and isotypic immunologic assessment.

Cellular and humoral immune responses to Schistosoma mansoni antigen preparations were evaluated in individuals presumed to be susceptible or resistant to reinfection after chemotherapeutic cure. A consistent proliferative increase in the response to soluble egg antigen (SEA) was observed post-treatment in both the susceptible and resistant groups. However, this change was not related to resistance. Isotype studies showed that IgM antibody levels to soluble worm antigen preparation (SWAP) and cercariae antigens were significantly higher in the resistant group than in the susceptible group. Post-treatment, an increase in IgE anti-SWAP and anti-schistosomular tegument (STEG) responses and a decrease in IgG4 anti-SEA and anti-STEG responses were observed in the resistant group. These finding are similar to those we have reported previously for a putative resistant group termed endemic normals, and are compatible with immunologic studies in different endemic areas. Together, these findings indicate that even on the population level, high IgE specificities coupled with low IgG4 specificities correlate well with documented resistance to reinfection.