Retroviral gene transduction of adult peripheral blood or marrow-derived CD34+ cells for six hours without growth factors or on autologous stroma does not improve marking efficiency assessed in vivo.

Our previous work in patients undergoing autologous transplant for multiple myeloma (MM) or breast cancer (BC) has shown that retroviral transduction of adult CD34+ cells for 72 hours in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor (SCF) resulted in .01% to 1% long-term marking of peripheral blood and marrow cells (Blood 85:3948, 1995). In this study we compare these previous studies to transduction with no added growth factors, previously shown to result in higher levels of marking in children (Lancet 342:1134, 1993) or transduction in the presence of an autologous stromal layer. Peripheral blood (PB) mononuclear cells were collected via apheresis after high-dose cyclophosphamide and granulocyte colony-stimulating factor. Bone marrow (BM) was also harvested in all patients. One third of both BM and PB collections were enriched for CD34+ cells and transduced with one of two marking vectors containing the neomycin-resistance gene to distinguish cells originating from BM and PB posttransplantation. Cells from 3 MM and 2 BC patients were transduced without growth factors for 6 hours and cells from 2 MM and 2 BC patients were transduced in the presence of autologous marrow stroma. Immediately posttransduction, the percentage of Neo-resistant PB and BM progenitors (colony-forming units) were: 0% to 19% in the 6-hour no growth factor group and 0% to 36% in the autologous stroma group. After conditioning therapy, both transduced and untransduced PB and BM fractions were infused into the patients. Semi-quantitative nested DNA polymerase chain reaction was performed on total, mononuclear, and granulocyte fractions of PB and BM at 1, 3, 6, 9, 12, and 18 months. Poor marking has been observed in both groups, with no consistently positive patients. These results compare unfavorably with our prior experience using growth factors during transduction. Further optimization of transduction conditions and vectors needs to be developed to improve transduction efficiency of adult human repopulating hematopoietic cells.

T lymphocyte-directed gene therapy for ADA- SCID: initial trial results after 4 years.

In 1990, a clinical trial was started using retroviral-mediated transfer of the adenosine deaminase (ADA) gene into the T cells of two children with severe combined immunodeficiency (ADA- SCID). The number of blood T cells normalized as did many cellular and humoral immune responses. Gene treatment ended after 2 years, but integrated vector and ADA gene expression in T cells persisted. Although many components remain to be perfected, it is concluded here that gene therapy can be a safe and effective addition to treatment for some patients with this severe immunodeficiency disease.

Morphological differences in Neisseria meningitidis pili.

Disease and carrier isolates of Neisseria meningitidis were examined for their ability to adhere to human buccal epithelial cells and human cell lines and to hemagglutinate human erythrocytes, properties thought to be associated with the presence of pili. Seventy percent (7 of 10) of carrier isolates were found to be highly adherent to human buccal epithelial cells and to agglutinate human A, B, O, Rh-, and Rh+ erythrocytes. In contrast, 60% of the disease isolates adhered poorly to human buccal epithelial cells and 80% failed to agglutinate human erythrocytes. No adherence of either disease or carrier isolates was observed when several human cell lines were tested. When the meningococcal strains were examined by electron microscopy, 7 of 10 disease isolates were found to possess large bundles of aggregated pili (alpha-type pili), while 7 of 10 carrier isolates were found to have numerous unaggregated pili (beta-type pili). A monoclonal antibody against meningococcal pili and one against gonococcal pili reacted with 6 of 10 piliated carrier isolates and 4 of 10 piliated disease isolates. These results suggest that meningococci, like gonococci, possess different types of pili which differ in morphological, antigenic, and binding properties. In addition, antigenic and morphological differences between pili from carrier and disease isolates were observed as well as differences in adherence and hemagglutinating properties.

Measurement of bacterial cell wall in tissues by solid-phase radioimmunoassay: correlation of distribution and persistence with experimental arthritis in rats.

We have developed sensitive and specific solid-phase radioimmunoassays to quantitate the distribution and persistence of bacterial antigen in rats developing arthritis in response to a single injection of streptococcal cell wall material. Three separate assays were specific for either the A polysaccharide (N-acetyl-D-glucosamine), A-variant polysaccharide (polyrhamnose), or peptidoglycan (D-ala-D-ala) moieties of the streptococcal cell wall. Antigen was detected in all tissues surveyed, although the greatest amount was in the liver and spleen. By using three fractions of cell wall separated by size, we have shown that the development of arthritis correlates with the degree of cell wall deposited and persisting in the joints. Further statistical analyses suggested differences in metabolism by different tissues and differential metabolism of different antigenic epitopes in some cases.

Antibody response to streptococcal cell wall antigens associated with experimental arthritis in rats.

The antibody response to group A streptococcal cell wall components was measured in rats during the development of chronic, remittent experimental arthritis. The arthritis was induced by a single intraperitoneal injection of an aqueous suspension of group A streptococcal cell wall fragments and antibodies were measured by a radioactive antigen-binding assay. Antibodies in serum against both peptidoglycan and A polysaccharide reached maximum levels at 1 or 2 weeks and declined to preimmunization levels by day 63. The kinetics and magnitude of the antibody responses were similar in neonatally thymectomized and non-thymectomized rats. A relationship between chronic joint lesions and anti-peptidoglycan concentration in serum was indicated, since all rats which produced high levels of antibody developed severe chronic arthritis. However, 46% of the rats which produced very low levels of antibody also developed moderate to severe arthritis. There was no correlation between anti-A polysaccharide antibodies and joint disease, although the concentration of this antibody was 10- to 100-fold greater than the anti-peptidoglycan. We conclude that antibody can be a component in the pathogenesis of this experimental model of arthritis, but its role requires further elucidation.

Modulation of the susceptibility of inbred and outbred rats to arthritis induced by cell walls of group A streptococci.

Inbred Buffalo rats were resistant to the induction of experimental arthritis induced by systemic injection of cell wall fragments in a crude whole-cell sonic extract of group A streptococci. This was in contrast to the susceptibility of outbred Sprague-Dawley and certain other inbred strains. Preliminary breeding studies indicated that genetic control of resistance of susceptibility is multigenic. When Buffalo rats were infected with a saline suspension of isolated cell wall fragments, chronic remittent arthritis developed. Suspension of isolated cell wall fragments, chronic remittent arthritis developed. Suspension of the isolated cell walls in the supernatant fraction of group A streptococci solubilized by sonication eliminated the arthropathogenicity in Buffalo rats. Thus, a component separable from the cell wall fraction can modulate the arthropathogenicity of cell walls in rats, but the effect depends upon the genetic background of the rat. The antibody response of Buffalo rats to the polysaccharide antigen of cell walls was also affected by the supernatant fraction of sonicated group A streptococci.