Roberta Raeder

Xenogeneic extracellular matrix grafts elicit a TH2-restricted immune response1

Background. Porcine small intestinal submucosa (SIS) is an acellular, naturally derived extracellular matrix (ECM) that has been used for tissue remodeling and repair in numerous xenotransplantations. Although a vigorous immune response to xenogeneic extracellular matrix biomaterials is expected, to date there has been evidence for only normal tissue regeneration without any accompanying rejection. The purpose of this study was to determine the reason for a lack of rejection. Methods. Mice were implanted s.c. with xenogeneic tissue, syngeneic tissue, or SIS, and the graft site analyzed histologically for rejection or acceptance. Additionally, graft site cytokine levels were determined by reverse transcriptase polymerase chain reaction and SIS-specific serum antibody isotype levels were determined by ELISA. Results. Xenogeneically implanted mice showed an acute inflammatory response followed by chronic inflammation and ultimately graft necrosis, consistent with rejection. Syngeneically or SIS implanted mice, however, showed an acute inflammatory response that diminished such that the graft ultimately became indistinguishable from native tissue, observations that are consistent with graft acceptance. Graft site cytokine analysis showed an increase in interleukin-4 and an absence of interferon-&ggr;. In addition, mice implanted with SIS produced a SIS-specific antibody response that was restricted to the IgG1 isotype. Reimplantation of SIS into mice led to a secondary anti-SIS antibody response that was still restricted to IgG1. Similar results were observed with porcine submucosa derived from urinary bladder. To determine if the observed immune responses were T cell dependent, T cell KO mice were implanted with SIS. These mice expressed neither interleukin-4 at the implant site nor anti-SIS-specific serum antibodies but they did accept the SIS graft. Conclusions. Porcine extracellular matrix elicits an immune response that is predominately Th2-like, consistent with a remodeling reaction rather than rejection.

A secreted streptococcal cysteine protease can cleave a surface-expressed M1 protein and alter the immunoglobulin binding properties

Previous studies of recent clinical isolates of serotype M1 group A streptococci indicated that they display two patterns of non-immune human IgG subclass binding reactivity associated with their M1 protein. One group reacted with all four IgG subclasses (type IIo), while the second group expressed an M1 protein reacting preferentially with human IgG3 (type IIb). In this study, we have demonstrated that a cysteine protease, SpeB, present in culture supernatants of M1 serotype group A streptococcal isolates expressing type IIb IgG binding protein, can convert a recombinant Emm1 protein from a type IIo functional profile to a type IIb profile by removal of 24 amino acids from the N-terminus of the mature M1 protein. Furthermore, SpeB can convert bacteria expressing IgG binding proteins of the type IIo phenotype into those expressing type IIb proteins. The role of the cysteine protease as the central bacterial enzyme in this posttranslational modification event was confirmed by generation of an isogenic SpeB-negative mutant.

Absence of SpeB Production in Virulent Large Capsular Forms of Group A Streptococcal Strain 64

ABSTRACT Passage in human blood of group A streptococcal isolate 64p was previously shown to result in the enhanced expression of M and M-related proteins. Similarly, when this isolate was injected into mice via an air sac model for skin infection, organisms recovered from the spleens showed both increased expression of M and M-related proteins and increased skin-invasive potential. We show that these phenotypic changes were not solely the result of increased transcription of the mRNAs encoding the M and M-related gene products. Rather, the altered expression was associated with posttranslational modifications of the M and M-related proteins that occur in this strain, based on the presence or absence of another virulence protein, the streptococcal cysteine protease SpeB. The phenotypic variability also correlates with colony size variation. Large colonies selected by both regimens expressed more hyaluronic acid, which may explain differences in colony morphology. All large-colony variants were SpeB negative and expressed three distinct immunoglobulin G (IgG)-binding proteins in the M and M-related protein family. Small-colony variants were SpeB positive and bound little IgG through their M and M-related proteins because these proteins, although made, were degraded or altered in profile by the SpeB protease. We conclude that passage in either human blood or a mouse selects for a stable, phase-varied strain of group A streptococci which is altered in many virulence properties.

Identification of Key Gene Products Required for Acquisition of Plasmin-like Enzymatic Activity by Group A Streptococci

Group A streptococci incubated in human plasma can acquire a plasmin-like enzymatic activity. This process involves at least two bacterial proteins and two human protein cofactors. In this study, the key bacterial proteins were identified by using a series of isogenic mutants of group A isolate, CS101. These studies confirm a key role for the secreted plasminogen activator, streptokinase, and identify the major surface fibrinogen-binding protein as the product of the mrp gene. The requirement for human fibrinogen and plasminogen as key cofactors was also confirmed.

Role of emm and mrp Genes in the Virulence of Group A Streptococcal Isolate 64/14 in a Mouse Model of Skin Infection

The virulence of group A streptococcal isolate 64/14 and paired isogenic mutants in which either the emm or mrp gene had been insertionally inactivated was compared in mice. Loss of expression of the emm gene product resulted in a significant loss of virulence when the isolate was injected into the skin but had no significant difference when injected intraperitoneally. By contrast, inactivation of the mrp gene caused the organism to be more virulent in the skin, while having no significant effect intraperitoneally. An isogenic mutant, in which the mga gene was inactivated and neither the emm gene nor the mrp gene was expressed, demonstrated no significant difference in virulence from the wild type organism. Organisms recovered from the spleen of mice lethally infected with the mga mutant expressed all Mga-regulated IgG-binding gene products despite the presence of the spectinomycin-resistance cassette, which was used to inactivate the mga gene, in its original position.

A Pivotal Role for Interferon-γ in Protection against Group A Streptococcal Skin Infection

Administration of exogenous recombinant interleukin-12 (rIL-12) either prophylactically or therapeutically provides significant protection against lethal group A streptococcal skin infection in a mouse model. Treatment of mice with rIL-12 before infection with group A streptococci induced expression of interferon-gamma (IFN-gamma) at the infection site. In vivo neutralization of IFN-gamma increased susceptibility to lethal infection and completely abrogated the protective effects of rIL-12. IFN-gamma knockout mice were also more susceptible to lethal infection. Although IL-12 treatment provided protection, higher doses induced significantly elevated levels of IFN-gamma transcription that were associated with increased susceptibility to lethal infection. These results support the hypothesis that IFN-gamma at the infection site is critical for protection but suggest that increased systemic levels are detrimental to survival after infection with group A streptococci.

Comparative In Vitro Activities of N-Formimidoyl Thienamycin and Moxalactam Against Nonfermentative Aerobic Gram-Negative Rods

N-Formimidoyl thienamycin was the most active drug against strains of Pseudomonas aeruginosa with a 90% minimum inhibitory concentration of 1.25 μg/ml. With the exception of P. maltophilia, thienamycin was as active or more active than moxalactam against other species of pseudomonads and against other genera of nonfermenters.

CHARACTERIZATION OF A GENE CODING FOR A TYPE IIO BACTERIAL IGG-BINDING PROTEIN

Two antigenic classes of non-immune IgG-binding proteins can be expressed by group A streptococci. One antigenic group of proteins is recognized by an antibody prepared against the product of a cloned fcrA gene (anti-FcRA). In this study, the immunogen used to prepare the antibody that defines the second antigenic class was shown to be the product of the emm-like (emmL) gene of M serotype 55 group A isolate, A928. The emmL55 gene expressed in E. coli produced an M(r) approximately 58,000 molecule which bound human IgG1, IgG2, IgG3 and IgG4, as well as horse, rabbit and pig IgG in a non-immune fashion. These properties are characteristic of the previously described type IIo IgG-binding protein isolated from this strain. In addition, the recombinant protein was reactive with human serum albumin and fibrinogen. The emmL 55 gene sequence was analysed and found to have the organization and sequence characteristics of a typical class I emm-like gene.

Treatment of serious infections with moxalactam.

In 93 hospitalized patients, 111 bacterial infections were treated with moxalactam. Eighty-three infections responded well to therapy, nine infections failed to respond to therapy or relapsed, and nine infections showed superinfection with resistant bacteria. The great majority of bacteria isolated had mean inhibitory concentrations below levels readily achieved in plasma, cerebrospinal fluid, bile, abscess fluid, and peritoneal fluid. Among the commonly identified bacteria, only Pseudomonas aeruginosa, enterococci, and Staphylococcus epidermidis had variable sensitivity to moxalactam.

Response of peripheral blood lymphocytes from the (nzb/nzw)f1 mouse to phytohemagglutinin.

Abstract In view of the earlier occurrence and greater prevalence of autoimmune disease in the female New Zealand black/white ( NZB NZW )F 1 hybrid mouse, we have compared the response of peripheral blood lymphocytes (PBL) from male and female F 1 mice to phytohemagglutinin-M (PHA). Between the ages of 6 and 20 weeks, the response of lymphocytes from F 1 hybrid females was significantly greater than that of males. A peak in the response of the female occurred at 12 – 14 weeks of age. The response to PHA in both sexes began decreasing at 22 weeks of age, with the rate of decrease being greater in the female. Additional experiments showed that the plasma from ( NZB NZW )F 1 females enhanced the responsiveness of PBL from ( NZB NZW )F 1 males. In contrast, male plasma suppressed responsiveness of PBL from females. The age at which female mice begin to hyperrespond to PHA correlates with the time at which others have reported loss of suppressor T-cell function in these mice. Our data suggest a difference between the immunological responsiveness of peripheral blood lymphocytes of male and female ( NZB NZW )F 1 mice.