Combination coatings unlock medical device potential.

Employing combination coatings, whether multiple coatings on the same surface or different coatings on different parts of the same device offer medical devices a number of benefits. Examples of what can be achieved are reported here.

Preventing medical device related infections.

Antimicrobial surfaces are a major advance in the ability to design biomaterials that resist colonization and subsequent infection. By reducing device-related infections, infection-resistant biomaterials will greatly improve the effectiveness of the device, which in turn will significantly decrease post-operative health risks and health-care costs. This overview of new surface modification technologies highlights strategies to prevent device-related infections.

Photochemical coatings for the prevention of bacterial colonization.

Biomaterials are being used with increasing frequency for tissue substitution. Implantable, prosthetic devices are instrumental in the saving of patients' lives and enhancing the quality of life for many others. However, the greatest barrier to expanding the use of biomedical devices is the high probability of bacterial adherence and proliferation, causing very difficult and often untreatable medical-device centered infections. The difficulty in treating such infections results in great danger to the patient, and usually retrieval of the device with considerable pain and suffering. Clearly, development of processes that make biomedical devices resistant to bacterial adherence and colonization would have widespread application in the field of biomedical technology. A photochemical surface modification process is being investigated as a generic means of applying antimicrobial coatings to biomedical devices. The photochemical process results in covalent immobilization of coatings to all classes of medical device polymers. A discussion of the photochemical surface modification process and preliminary results demonstrating the success of photochemical coatings in formulating microbial-resistant surfaces are presented in this paper.

Structural relationships among the H-2 D-regions of murine MHC haplotypes.

The number of genes encoding functional Ag-presenting molecules in the D region of the murine MHC differs among haplotypes. For example, the H-2b D region contains a single "D/L" gene, H-2Db, whereas the d-haplotype encodes two, H-2Dd and Ld. Using D/L specific oligonucleotide probes, we have found that, as with H-2d, the q- and v-haplotypes contain two D/L genes, whereas the other haplotype examined have one. Hybridization analysis using cloned probes that map between H-2Dd and Ld revealed similar structures in each of the three haplotypes (d, q, and v) which have "duplicated" D regions. Two approaches were used to examine allelic relationships among the D/L genes. First, the 5' region of the H-2Db gene was sequenced, and found to be more similar to H-2Ld than to H-2Dd. Second, oligonucleotide probes that distinguish H-2Ld from H-2Dd revealed H-2Ld-related genes in several haplotypes, including the duplicated haplotypes H-2q and H-2v. Analogous probes specific for H-2Dd, however, did not detect similar sequences in the other haplotypes. We interpret these results to mean that the three duplicated D regions arose from a common duplication event, and share the five gene structure of the D region cluster defined in H-2d. However, subsequent events have generated sequence divergence at the D-locus.

Tracing the evolution of H-2 D region genes using sequences associated with a repetitive element.

The class I genes in the murine MHC are genetically divided into the K, D, Qa, and T1a region subfamilies. These genes presumably arose by duplication from a common class I ancestor. Oligonucleotide probes specific for sequences associated with a moderately repetitive B2 SINE element, which is inserted into the 3' untranslated region of the H-2D and H-2L genes, were used to examine the evolutionary relationship between these classically defined D region genes (H-2D and H-2L) and the other members of the class I gene family. Hybridization analyses of recombinant cosmid and genomic DNA indicated that the D region genes separated genetically from the other members of the class I gene family 12 to 14 million years ago. The evidence suggests that during this time frame the chromosomal segment harboring the characteristic insertion became fixed in the ancestral population which gave rise to Mus domesticus. Previous studies have shown that the number of genes present in the Qa and T1a regions varies among inbred strains and among laboratory stocks of wild mice derived from more distant species on the genus Mus. No evidence was found in this study to support the hypothesis that variation in class I gene number is the result of recent duplications of the functionally defined class I genes of the D region, H-2D and H-2L.

Genetic analysis of the H-2D region using a new intra-D-region recombinant mouse strain.

A rare D-region recombination event which gave rise to the B10.RQDB major histocompatibility complex haplotype has been examined to ascertain the nature of the crossover and to determine which class I genes are present in the new alignment of D-region genes. Serologic analysis have shown that the B10 . RQDB major histocompatibility complex recombinant mouse inherited the H-2Dd gene from the B10.T(6R) parental line and the H-2Db gene from the B10.A(2R) parental line, representing the first example of an intra-D-region crossover resulting from an intercross. Previous molecular genetic analyses of the d and b haplotypes revealed structural diversity in the organization of their D-region gene clusters. Hence, the D region is comprised of five class I genes in the d haplotype and only one in the b haplotype. Because allelic relationships among the various D-region genes are not defined, either a homologous or nonhomologous alignment of genes has generated the RQDB crossover. Therefore, the possibility that all three D-region antigen-presenting molecules (Dd, Ld, and Db) might be encoded by the RQDB haplotype was examined. Fluorescence-activated cell sorter and cytotoxic T lymphocyte analyses revealed no detectable levels of H-2Ld cell-surface expression, confirming earlier studies with antibody-mediated cytotoxicity and immunoprecipitation. Southern blot analysis localized the recombination point to within a 1-kb region at the centromeric end of the H-2Ld gene on the B10 . T(6R) chromosome in a region of high homology to the H-2Db gene on the B10 . A(2R) chromosome. Together, these studies define the D region of the RQDB haplotype as containing the five class I genes: Dd, D2d, D3d, D4d, and Db. In addition to providing insight into rare recombination events in the D region, the B10.RQDB mouse should be a useful tool for exploring the function of D-region genes.

The diversity of the secondary Salmonella typhimurium-specific B cell repertoire.

This report describes the first analysis of the expressed B cell repertoire specific for a bacterium. In this study, responses to an acetone-killed and dried preparation of Salmonella typhimurium strain TML (AKD-TML) are described. The results show that AKD-TML can stimulate splenic B cells from primed CBA/Ca mice over a wide dose range. The average frequency of secondary TML-specific B cells is 16.4 per 10(5) splenic B cells. This frequency is similar to that observed for another complex, natural antigen, the hemagglutinin of influenza virus. The majority of all secondary TML-specific B cells (greater than 70%) secrete immunoglobulin M, but most of these clones also secrete other isotypes of which immunoglobulins G2 and A are the most prevalent. Analysis of the specificity of secondary TML-specific B cells showed that the vast majority of these B cells were specific for the lipopolysaccharide (LPS) molecule. Moreover, fine specificity analysis demonstrated that approximately two-thirds of these anti-LPS-specific B cell clones are directed against the core polysaccharides or lipid A regions of the LPS molecule, while only about one-third are directed toward the O antigen region. Since anti-S. typhimurium serum antibodies are directed primarily against the O antigens, these studies suggest that the serum levels of antibodies to a given epitope on a bacterial antigen may not be a true reflection of the expressed B cell repertoire when analyzed at the single B cell level. These studies also suggest that the role of antibodies to lipid A molecules in the development of protective immunity to S. typhimurium be reevaluated.

Clonal analysis of primary B cells responsive to the pathogenic bacterium Salmonella typhimurium.

In the present study, a modification of the splenic focus system is used to analyze the S. typhimurium strain TML (TML)-specific B cell repertoire. The results show that the frequency of primary TML-specific splenic B cells in CBA/Ca mice is approximately 1 per 10(5) B cells and less than 30% of these B cells are specific for LPS. In contrast, the frequency of memory TML-specific cells is approximately 1 per 5-8 X 10(3) splenic B cells and greater than 95% of these B cells are specific for LPS. These results suggest that the frequency of primary TML-specific B cells is extremely low and that it expands 15-20-fold after antigen exposure. It is interesting that less than 30% of the primary B cells are specific for the LPS molecule since it is considered to be the major antigenic determinant on Salmonella organisms. Furthermore, the majority of the LPS-specific anti-TML antibody-producing clones are directed against the LPS O antigen region. Conversely, more than half to two-thirds of the memory LPS-specific anti-TML B cell clones are directed against the KDO or lipid A region of the LPS molecule. These results indicate that the preferential expansion of LPS-specific B cell clones observed after immunization resides primarily in the B cell subsets responsive to the KDO/lipid A moieties on the LPS molecule. Finally, unlike B cell responses to chemically defined antigens, TML stimulates very little IgG1 antibody. IgG2 and IgA isotypes appear to play a predominant role in anti-TML antibody responses, although all H chain classes are produced to some extent. Collectively, these findings are consistent with the responses reported for two other natural antigens, HA and PC. Hence, the pattern of stimulation by infectious agents, such as S. typhimurium, appears to be distinct from that of synthetic antigens. Thus, the studies presented herein have begun to provide insights into those subsets of B cells responsive to S. typhimurium and other infectious disease organisms.

Antibody-defective, genetically susceptible CBA/N mice have an altered Salmonella typhimurium-specific B cell repertoire.

CBA/N mice, which express the X-linked immunodeficiency gene xid, are susceptible to Salmonella typhimurium. The basis for this susceptibility is currently unknown. However, previous studies (10) from this laboratory have provided evidence that susceptibility may be due to a defective anti-S. typhimurium antibody response. In that report we hypothesized that the defective antibody response may be a reflection of an altered S. typhimurium-specific B cell repertoire. In the studies described here, we have investigated this hypothesis using a modification of the in vitro splenic focus system. The frequency and characteristics of salmonella-specific B cells in normal, innately resistant, CBA/Ca mice have been compared with those of salmonella-susceptible, anti-S. typhimurium antibody-defective CBA/N mice. The results show that CBA/N mice express no primary or secondary S. typhimurium-specific B cell precursors after stimulation with an acetone-killed and dried (AKD) preparation of S. typhimurium strain TML. However, after three immunizations, the CBA/N tertiary frequency of 15.4 per 10(6) splenic B cells was similar to the primary precursor frequency in immunologically normal CBA/Ca mice, but 23-fold lower than the tertiary precursor frequency in CBA/Ca control mice. Moreover, CBA/N mice had an altered isotype distribution pattern after stimulation with AKD-TML. Greater than 70% of the tertiary CBA/N TML-specific B cells secreted IgG2, in contrast to either nonimmune or primed control mice. In addition, 80% of the CBA/N TML-specific B cells secreted only a single isotype, whereas the majority of B cells from primed normal mice secreted multiple isotypes. Fine specificity analysis of the TML-specific B cells indicated that the array of antigenic determinants to which CBA/N B cells could respond was restricted. Although the majority of primed CBA/Ca and primed CBA/N B cells were specific for LPS, the fine specificity pattern exhibited by CBA/N B cells was similar to that observed in unprimed normal mice, i.e., the vast majority were specific for the O antigen region of the LPS molecule. In contrast, a major portion of the LPS-specific B cells in primed CBA/Ca mice were directed against the KDO/lipid A region of the LPS molecule. Therefore, it appears that CBA/N mice lack or are unable to stimulate the B cell subset that predominates in primed, normal mice. Taken together, these studies indicate that the basis for susceptibility of CBA/N mice to S. typhimurium is multifactorial and suggests that the inability of some animals to respond to some infectious agents may be related to holes in their B cell repertoire.