Carina Lundqvist

Cytokine profiles for human Vγ9+ T cells stimulated by Plasmodium falciparum

Vγ9+ T cells from malaria non‐exposed donors make proliferative responses to Plasmodium falciparum on in vitro stimulation. Vγ9+ cells are strongly activated by components of the schizont stage of the parasite and by antigens released into the culture upon schizogony, while CD4+ Vγ9‐ cells are stimulated by the earlier stages of the parasite. Using reverse transcriptase‐polymerase chain reaction (RT‐PCR) we determined mRNA expression for 14 cytokines in highly purified Vγ9+ cells enriched by positive selection after in vitro stimulation with P. falciparum schizont antigens. Interferon‐γ (IFN‐γ) and Tumor Necrosis Factor‐α (TNF‐α) were detected in all samples tested. The majority of samples also expressed TNF‐β, transforming growth factor‐β (TGF‐β) and Interleukin‐8 (IL‐8). Only occasional samples expressed IL‐2, IL‐5 and IL‐10. Using the ELISPOT assay we found that a large fraction of the reactive Vγ9+ cells produced IFN‐γ and that γδ T cells are the major producers of IFN‐γ in cultures stimulated with schizont antigens. The majority of Vγ9+ cells in these cultures also express the membrane‐bound form of TNF‐α. Expression of these cytokines speaks for a cytolytic and/or inflammatory role of γδ cells in the response to malaria in non‐exposed individuals.

Isolation of functionally active intraepithelial lymphocytes and enterocytes from human small and large intestine.

A mild purification method has been developed for the isolation of human intraepithelial lymphocytes (IEL) and enterocytes from the same individual. The isolation procedure includes mechanical disruption of the mucosal layer, treatment with reducing agent and sedimentation followed by Percoll gradient centrifugation. Finally, epithelial cells are removed from the IEL fraction using magnetic beads coated with the anti-epithelial antigen monoclonal antibody (mAb) BerEP4. Leucocytes are removed from the enterocyte fraction using magnetic beads coated with mAbs directed against common leucocyte antigen (CD45). Using this procedure IEL and enterocytes have been isolated from apparently normal jejunal, ileal and colonic tissue specimens. Recoveries of IEL were 7 x 10(5), 4 x 10(5) and 1 x 10(5)/cm2 mucosa from jejunum, ileum and colon respectively. 1-2 x 10(6) enterocytes/cm2 mucosa were recovered from small intestine while the corresponding value for colonic biopsies was approximately 2 x 10(5) enterocytes/cm2. The IEL fraction was pure as judged by the low percentages of B cells, macrophages and BerEP4 positive cells (less than 4%) present in the purified fraction. The enterocyte fraction contained less than 2% CD45+ cells. The two cell fractions were viable and expanded in vitro. Enterocytes expanded spontaneously while IEL required initial stimulation with mitogens. The isolation procedure described here will make it possible to study the function of human IEL, interactions between IEL and enterocytes and the role of both cell types in local immunity.

On the Early Mechanisms of Bone Formation after Maxillary Sinus Membrane Elevation: An Experimental Histological and Immunohistochemical Study

BACKGROUND Previous studies have shown predictable bone formation in the maxillary sinus after membrane elevation. However, how and where the bone is formed is not well understood. PURPOSE The aim of the study was to histologically and immunohistochemically study the early bone formation events in primates after membrane elevation in the maxillary sinus. MATERIALS AND METHODS Nine adult male tufted capuchin primates (Cebus apella) were included in the study. Eight animals were subjected to bilateral maxillary sinus membrane elevation using a lateral replaceable bone window technique. One oxidized dental implant was placed into the maxillary sinus cavity on both sides. In four animals, one sinus was left without any additional treatment, whereas the contralateral sinus was filled with autologous bone grafts from the tibia. In two animals, the implants were inserted under the elevated sinus membrane on both sides. In two animals, the sinus membrane was totally removed. The animals were euthanized after 10 or 45 days. One nonoperated animal representing pristine tissue conditions served as control. The maxillary sinuses with implants were retrieved and further processed for light microscopic ground sections or decalcified sections for immune-histochemical analyses. RESULTS Bone formation started from the bottom of the sinus floor, sprouting into the granulation tissue along the implant surface under the elevated membrane irrespective of time and surgical technique. Bone formation was not seen in direct conjunction with the sinus membrane. A distinct expression of osteopontin was observed in the serous glands of the lamina propria close to the implant within all groups. CONCLUSION Bone formation after sinus membrane elevation with or without additional bone grafts starts at the sinus floor and sprouts into the elevated space along the implant surface. The sinus membrane does not seem to present osteoinductive potential in sinus membrane elevation procedures in this study.