Abby Maizel

Long-term bone marrow culture and its clinical potential in chronic wound healing

Bone marrow‐derived cells have long been regarded to play a crucial role in the homeostasis of skin. We have previously described the clinical benefit of directly applying autologous bone marrow aspirate and cultured bone marrow cells to recalcitrant chronic skin wounds. The initial response to treatment appears to be vascular in nature with the formation of new blood vessels. The difficulty in consistently growing adequate numbers of cells for delivery to patients was, however, a limiting factor. Here, in a subsequent protocol, we describe an improved bone marrow culture system yielding a reliable growth of bone marrow cells and leading to a greater clinical response. Cells expressing markers of endothelial progenitors including CD133, CD146, and particularly CD14 are enhanced in these cultures. CD14‐isolated cells produced colonies in endothelial cell assays and sprouting in matrigel assays. Angiogenic cytokines, including angiogenin, epithelial neutrophil‐activating protein‐78, growth‐regulated oncogene, growth‐regulated oncogene‐α, Interleukin‐8, CXC16, and monocyte chemoattractant protein‐1, were found to be elevated in these cultures. Administration of improved culture cells to patients with chronic wounds present for >1 year lead to an enhanced clinical response.

Characterization of the Interaction between CD45 and CD45-AP

CD45, a leukocyte-specific transmembrane protein tyrosine phosphatase, is required for critical signal transduction pathways in immune responses. To elucidate the molecular interactions of CD45 with other proteins involved in CD45-mediated signal transduction pathways, we have recently cloned a 30-kDa phosphorylated protein, CD45-AP, which specifically associates with CD45. Binding analysis employing several deleted or chimeric forms of CD45-AP and CD45 demonstrated that the potential transmembrane segment of CD45-AP bound to the transmembrane portion of CD45. CD45-AP was found in particulate fractions of lymphocytes along with CD45, indicating that it is likely to be a transmembrane protein. In addition, CD45-AP was resistant to proteolysis by tosylphenylalanyl chloromethyl ketone-treated trypsin applied to intact cells. This is consistent with the most likely membrane orientation of CD45-AP predicted from the amino acid sequence, that is, only a short amino-terminal segment of CD45-AP is extracellular. We propose that CD45-AP interacts with CD45 at the plasma membrane and that the bulk of CD45-AP located in the cytoplasm act as an adapter which directs the interaction between CD45 and other molecules involved in CD45-mediated signal transduction pathways.

Production of human B and T cell growth factors is enhanced by thymic hormones

The thymic preparations thymosin fraction 5 (TF5) and synthetic thymosin alpha 1 (T alpha 1) were examined for their ability to enhance growth factor production by human peripheral blood mononuclear cells (PBMC). The results showed that both TF5 and T alpha 1 were capable of enhancing the production of a B cell growth factor (BCGF-12kD) and T cell growth factor (TCGF; IL-2). Enhancement by T alpha 1 could be obtained at 100-200-fold lower concentrations than that seen with TF5. In contrast, no enhancement of growth factor production was obtained with control preparations of non-thymic tissue extracts at any concentrations used. It was observed that stimulation of BCGF-12kD and IL-2 was most significantly obtained when the PBMC were activated with lectin. Furthermore, no direct effect of thymic hormones on test B and T cells was observed. These observations provide the first direct evidence that production of B cell growth factors can be enhanced by thymic hormones. In addition, these studies suggest that thymic hormones may regulate B cell responses by acting on mature activated T lymphocytes.

Further characterization of a clinically relevant model of melanoma metastasis and an effective vaccine

A major problem in evaluating the effectiveness of tumor cell vaccination and other biological therapies is the variability of experimental models. In this study we have further developed and characterized a model for metastatic melanoma that approximates the major clinical stages of metastatic dissemination: stage I-growth of the primary (local) tumor, stage II-dissemination to regional lymph nodes, and stage III-metastasis to distant organs (lungs). C57BL/6 mice were challenged subcutaneously with B16 F10 murine melanoma cells in the midtail, and within 3 weeks 100% of the mice had local tumors growing in their tails. By 5–7 weeks after challenge, most of the mice had developed metastases to the inguinal lymph nodes and subsequently had metastatic colonies in the lungs and in the bone marrow. Preimmunization of mice with a formalinized extracellular antigen vaccine, derived from B16F10 melanoma cells, provided partial inhibition of the growth of the primary melanoma tumors, as well as reducing the number of metastases to the regional (inguinal) lymph nodes and lungs along with concomitantly increasing survival time. This model for melanoma metastasis provides a reasonable and reproducible test system for the study of anti-melanoma immunity and the different cellular and humoral mechanisms involved.