Chronic inflammation caused by lymphotoxin is lymphoid neogenesis.

In presenting a unifying concept for chronic inflammation and lymphoid organogenesis, we suggest that lymphotoxin's (LT, LT-alpha, TNF-beta) crucial role in these processes is pivotal and similar. Chronic inflammatory lesions that developed in the kidney and pancreas at the sites of transgene expression in rat insulin promoter-LT (RIP-LT) mice resembled lymph nodes with regard to cellular composition (T cells, B cells, plasma cells, and antigen-presenting cells), delineated T and B cell areas, primary and secondary follicles, characteristic morphologic and antigenic (ICAM-1, VCAM-1, MAdCAM-1, and PNAd) features of high endothelial venules, and ability to respond to antigen and undergo Ig class switching when obtained from mice immunized with SRBC. The vascular changes, with the exception of PNAd, appear to be the direct consequence of transgene derived LT expression, as they were also observed in RIP-LT mice lacking mature T and B cells. These data show that LT-induced chronic inflammation has the characteristics of organized lymphoid tissue.

Protective humoral response against pneumococcal infection in mice elicited by recombinant bacille Calmette-Guérin vaccines expressing pneumococcal surface protein A.

Pneumococcal surface protein A (PspA), a cell-surface protein present on all strains of pneumococci, has been shown to elicit protective antibody responses in mice in the absence of capsular polysaccharide. Whereas PspA is polymorphic, considerable cross-reactivity and cross-protection have been demonstrated among PspA proteins of pneumococci exhibiting different capsular and PspA serotypes. A gene segment encoding the nonrepetitive variable NH2-terminal portion of PspA has been cloned into three distinct recombinant Bacille Calmette-Guérin (rBCG) vectors, allowing for expression of PspA as a cytoplasmic or secreted protein, or a chimeric exported membrane-associated lipoprotein. All rBCG-PspA strains elicited comparable anti-PspA ELISA titers, ranging from 10(4) to 10(5) (reciprocal titers) in both BALB/c and C3H/HeJ mice. However, protective responses were observed only in animals immunized with the rBCG-PspA vaccines expressing PspA as a secreted protein or chimeric exported lipoprotein. In addition, anti-PspA immune sera elicited by the rBCG vaccines passively protected X-linked immunodeficient mice from lethal challenge with the highly virulent, encapsulated WU2 strain of Streptococcus pneumoniae and two additional virulent strains exhibiting heterologous PspA and capsular serotypes. These studies confirm previous PspA immunization studies showing cross-protection against heterologous serotypes of S. pneumoniae and demonstrate a potential for rBCG-based PspA vaccines to elicit protective humoral responses against pneumococcal disease in humans.

Protective immunity elicited by recombinant bacille Calmette-Guerin (BCG) expressing outer surface protein A (OspA) lipoprotein: a candidate Lyme disease vaccine.

The current vaccine against tuberculosis, Mycobacterium bovis strain bacille Calmette-Guerin (BCG), offers potential advantages as a live, innately immunogenic vaccine vehicle for the expression and delivery of protective recombinant antigens (Stover, C.K., V.F. de la Cruz, T.R. Fuerst, J.E. Burlein, L.A. Benson, L.T. Bennett, G.P. Bansal, J.F. Young, M.H. Lee, G.F. Hatfull et al. 1991. Nature [Lond]. 351:456; Jacobs, W.R., Jr., S.B. Snapper, L. Lugosi and B.R. Bloom. 1990. Curr. Top. Microbiol. Immunol. 155:153; Jacobs, W.R., M. Tuckman, and B.R. Bloom. 1987. Nature [Lond.]. 327:532); but as an attenuated intracellular bacterium residing in macrophages, BCG would seem to be best suited for eliciting cellular responses and not humoral responses. Since bacterial lipoproteins are often among the most immunogenic of bacterial antigens, we tested whether BCG expression of a target antigen as a membrane-associated lipoprotein could enhance the potential for a recombinant BCG vaccine to elicit high-titered protective antibody responses to target antigens. Immunization of mice with recombinant BCG vaccines expressing the outer surface protein A (OspA) antigen of Borrelia burgdorferi as a membrane-associated lipoprotein resulted in protective antibody responses that were 100-1,000-fold higher than responses elicited by immunization with recombinant BCG expressing OspA cytoplasmically or as a secreted fusion protein. Furthermore, these improved antibody responses were observed in heterogeneous mouse strains that vary in their immune responsiveness to OspA and sensitivity to BCG growth. Thus, expression of protective antigens as chimeric membrane-associated lipoproteins on recombinant BCG may result in the generation of new candidate vaccines against Lyme borreliosis and other human or veterinary diseases where humoral immunity is the protective response.

B lymphocytes are essential for the initiation of T cell-mediated autoimmune diabetes: analysis of a new "speed congenic" stock of NOD.Ig mu null mice.

The T lymphocytes mediating autoimmune destruction of pancreatic beta cells in the nonobese diabetic (NOD) mouse model of insulin-dependent diabetes mellitus (IDDM) may be generated due to functional defects in hematopoietically derived antigen-presenting cells (APC). However, it has not been clear which particular subpopulations of APC (B lymphocytes, macrophages, and dendritic cells) contribute to the development and activation of diabetogenic T cells in NOD mice. In the current study we utilized a functionally inactivated immunoglobulin (Ig) mu allele (Ig mu null) to generate a "speed congenic" stock of B lymphocyte-deficient NOD mice that are fixed for linkage markers delineating previously identified diabetes susceptibility (Idd) genes. These B lymphocyte NOD.Ig mu null mice had normal numbers of T cells but were free of overt IDDM and insulitis resistant, while the frequency of disease in the B lymphocyte intact segregants was equivalent to that of standard NOD mice in our colony. Thus, B lymphocytes play a heretofore unrecognized role that is essential for the initial development and/or activation of beta cell autoreactive T cells in NOD mice.

Emv30null NOD-scid mice. An improved host for adoptive transfer of autoimmune diabetes and growth of human lymphohematopoietic cells.

When used as hosts in passive transfer experiments, a stock of NOD/Lt mice congenic for the severe combined immunodeficiency (scid) mutation have provided great insight to the contributions of various T-cell populations in the pathogenesis of autoimmune insulin-dependent diabetes mellitus (IDDM). Moreover, NOD-scid mice support higher levels of human lymphohematopoietic cell growth than the C.B-17-scid strain in which the mutation originated. However, the ability to perform long-term lymphohematopoietic repopulation studies in the NOD-scid stock has been limited by the fact that most of these mice develop lethal thymic lymphomas beginning at 20 weeks of age. These thymic lymphomas are characterized by activation and subsequent genomic reintegrations of Emv30, an endogenous murine ecotropic retrovirus unique to the NOD genome. To test the role of this endogenous retrovirus in thymomagenesis, we produced a stock of Emv30null NOD-scid mice by congenic replacement of the proximal end of chromosome 11 with genetic material derived from the closely related NOR/Lt strain. Thymic lymphomas still initiate in Emv30null NOD-scid females, but their rate of progression is significantly retarded since the frequency of tumors weighing between 170 and 910 mg at 25 weeks of age was reduced to 20.8% vs. 76.2% in Emv30% segregants. The thymic lymphomas that did develop in Emv30null NOD-scid mice were not characterized by a compensatory increase in mink cell focus-forming proviral integrations, which initiate thymomagenesis in other susceptible mouse strains. Significantly, the ability of standard NOD T-cells to transfer IDDM to the Emv30null NOD-scid stock was not impaired.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific detection of Haemophilus influenzae type b lipooligosaccharide by a polymyxin B monoclonal antibody assay.

A monoclonal antibody (MAb)-based enzyme immunoassay was developed for detection of Haemophilus influenzae type b (Hib) lipooligosaccharides (LOS). The high affinity of polymyxin B for lipid A was used to bind the Hib LOS to microtiter wells. The immobilized LOS was detected with MAbs directed against the oligosaccharide component of Hib endotoxin. Hib LOS concentrations were measured in in vitro samples and in cerebrospinal fluid (CSF) sample obtained from rabbits with experimental Hib meningitis. The sensitivity of the assay was 1 ng LOS/ml sample and the results obtained with this assay correlated significantly with those obtained with the standard Limulus amebocyte lysate assay. This new assay provides a method for specific detection of Hib LOS in CSF samples and in aqueous laboratory fluids. This general methodology should also be useful for experimental research involving specific LPS/LOS molecules.

Progress on development of the live BCG recombinant vaccine vehicle for combined vaccine delivery.

BCG, the current vaccine for tuberculosis, has been administered to approximately three billion people. This live vaccine has a low incidence of serious side effects and can be given at birth. Within the past six years, systems for the manipulation and expression of foreign genes in mycobacteria have been developed, allowing the evaluation of rBCG as a vaccine delivery vehicle for heterologous antigens. Recent studies from our group have shown that rBCG expressing outer surface protein A of Borrelia burgdorferi can completely protect mice from an intradermal challenge with this organism. Immune responses protective against Streptococcus pneumoniae challenge have also been achieved by immunization of mice with rBCG expressing PspA. The simplest means of administering multiple vaccine antigens in a rBCG vehicle would be to coexpress these simultaneously in the same BCG recombinant. Currently two general classes of vectors exist for the expression of foreign proteins in BCG: shuttle plasmid vectors, which replicate extrachromosomally in mycobacteria, and shuttle "phasmid" vectors, which integrate as a single copy into the mycobacterial chromosome by means of vector-encoded integration functions of the lysogenic mycobacteriophage L5. The genetic capacity of the multicopy plasmid vectors may be 20 kb or more, while the potential exists for stable integration of much larger amounts of DNA into the mycobacterial genome (L5 itself is 52 kb). Additionally, these two expression systems can have the compatibility to coexist in a single BCG cell. Otitis media is caused by infections of the middle ear chiefly with either S. pneumoniae or H. influenzae. Thus, an effective vaccine would necessarily include antigens from both these pathogens. Our initial attempt at construction of a BCG multivaccine vehicle was to express proteins from each of these pathogens from the same multicopy plasmid. We have recently succeeded in coexpressing the S. pneumoniae PspA and H. influenzae PAL proteins in BCG. Future work will address how the biochemical characterization of and immune responses to the recombinant antigens of the "bivalent" rBCG:PspA/PAL vaccine compare to those of the respective "monovalent" rBCG vaccines.

Planned Parenthood experience with triphasil.

Triphasil, a low-dose combination oral contraceptive containing levonorgestrel and ethinyl estradiol, was tested in four Planned Parenthood clinics on 317 women between 18 and 34 years of age (mean, 23) for a total of 4,692 cycles, or 361 woman-years of usage. Approximately half these volunteers (165) were nulligravidas, and 309 (97.5%) were white. Despite instructions on proper tablet usage, there were 416 cycles (8.9%) in which one or more tablets were missed. Only one pregnancy occurred, in a cycle in which a total of four tablets was missed, for an uncorrected Pearl index of 0.28 pregnancies per 100 woman-years of usage. No pregnancy resulted from method failure, indicating a 100% efficacy rate for Triphasil when taken properly. The mean length of the menstrual cycle with Triphasil was 27.9 days; the mean length of menses, 4.4 days; and the mean latency period, 2.1 days. Menstrual flow was average in 64.1% of the subjects, light in 34.1%, heavy in 1.3% and variable in 0.5%. Amenorrhea during the tablet-free interval occurred in only 0.6% of the 4,692 cycles in which Triphasil was used. Breakthrough bleeding occurred in 6.9% of first cycles and 3.2% of total cycles; spotting, in 10.7% of first cycles and 4.4% of total cycles. Other symptoms that occurred with an incidence of greater than or equal to 1% were acne (1.0%), appetite increase (1.2%), breast discomfort (2.8%), breast enlargement (1.3%), gastrointestinal symptoms (1.7%), simple headache (1.4%) and nausea (1.1%). A total of 44 women (13.9%) discontinued treatment for medical reasons.(ABSTRACT TRUNCATED AT 250 WORDS)

State also pays for Depo-Provera.

PIP: A mail survey of 50 U.S. states revealed that of 32 who responded, 15 will pay for Depo-Provera under the Medicaid program, for contraception. Another 2 pay for contraceptive visits, which may include any type of contraceptive. As of May 1991, no state now requires prior approval in writing to use Depo-Provera as a contraceptive. Contraception is an unlabeled indication for Depo-Provera, but it is legally acceptable to use the drug for an unlabeled indication. PHP and Blue Shield have reimbursed users for Depo-Provera prescriptions in Minnesota. Minnesota's Medicaid (Medical Assistance) has paid for Depo-Provera since July 1990. The author added that she has never seen a failure of this method in 30 years of use when given correctly, that is 150 mg every 90 days.^ieng

Quantification of basal and stimulated ROS levels as predictors of islet potency and function.

We have developed a luminol-based assay using intact islets, which allows for quantification of reactive oxygen species (ROS). In addition, an index capable of characterizing metabolic and mitochondrial integrity prior to transplantation was created based on the capacity of islets to respond to high glucose and rotenone (mitochondrial respiratory chain complex I inhibitor) by production of ROS. To validate this assay, lipid peroxidation and antioxidative defense capacity were evaluated by detection of malondialdehyde (MDA) levels and glutathione peroxidase activity (GPx), respectively. Also, flow cytometric analyses of ROS (dihydroethidine), apoptosis (Annexin V, active caspases), necrosis (Topro3), and mitochondrial membrane potential (JC-1) were done in parallel to correlate with changes in luminol-measured ROS. ATP/ADP ratios were quantified by HPLC and the predictive value of ROS measurement on islet functional potency was correlated with capacity to reverse diabetes in a streptozotocin-induced diabetic NOD.scid mouse model as well as in human transplant recipients. Our data demonstrate that levels of ROS in islets correlate with the percentage of apoptotic cells and their functional potency in vivo. The ROS indices following glucose and rotenone exposure are indicative of metabolic potency and mitochondrial integrity and can be used as surrogate markers to evaluate the quality of islets prior to transplantation.

Molecular cloning, partial purification, and characterization of a haemin-binding lipoprotein from Haemophilus influenzae type b.

A library of genomic DNA fragments from Haemophilus influenzae type b (Hib) DL42 was constructed in plasmid pBR322, transformed into Escherichia coli strain RR1, and screened for recombinant clones with haemin-binding activity by plating onto haemin-containing agar. Expression of haemin-binding activity by clones correlated with the expression of a protein with an apparent molecular weight of 51,000 (51K) that was also recognized by anti-Hib strain DL42 serum in immunoblots. One recombinant clone, designated pHM2, with the smallest DNA insert (3.62 kb) was characterized further. Ethanol inhibition of expression of pHM2 in minicells revealed that the 51K protein was the result of a processing event involving a larger precursor. E. coli RR1(pHM2) adsorbed haemin in liquid suspensions as well as from solid media. Subcloning of a 2.6 kb fragment of pHM2 into a shuttle vector permitted the construction of a recombinant Hib clone, DL42(pHM1002), which overexpressed the 51K haemin-binding protein. This 51K protein appears to be peripherally associated with the inner, and possibly outer, membranes of Hib. Affinity chromatography on haemin-agarose was utilized to purify the haemin-binding protein from both E. coli RR1(pHM2) and Hib DL42(pHM1002) to near homogeneity. The use of the antibiotic globomycin in a minicell expression system and radioimmunoprecipitation analysis of Hib proteins intrinsically radiolabelled with [3H]-palmitate indicated that the 51K haemin-binding protein is a lipoprotein.

Identification and characterization of E. coli type-1 pilus tip adhesion protein.

The type-1 pilus of Escherichia coli is the prototype of this class of hair-like, multimeric adhesive organelles. This pilus mediates adherence to mannose-containing receptors on mucosal epithelia and other cells. The type-1 pilus, in one of several serological variants, is expressed by nearly all E. coli strains, and its promotion of colonization by pathogenic bacteria and the protective effects of purified pilus vaccines suggest that it is important as a bacterial virulence factor. Both the adhesive function and the serological variation of the type-1 pilus have been attributed to the thousand or so pilin protein monomers making up the pilus rods. This idea has been contradicted by our earlier observations on an E. coli strain expressing adhesion-defective pili. More recent genetic evidence also indicates that auxiliary pilus proteins are required for adhesive function. We report here the identification of three previously undetected integral minor proteins on the type-1 pilus, and show that one of them is the receptor-binding adhesin. This protein is antigenically conserved among strains with different pilin serotypes and is located at the pilus tip.

The hbpA gene of Haemophilus influenzae type b encodes a heme-binding lipoprotein conserved among heme-dependent Haemophilus species.

A membrane-associated lipoprotein of Haemophilus influenzae type b has previously been shown to bind heme in vitro and to promote binding of this compound by Escherichia coli recombinants expressing this protein. The H. influenzae type b heme-binding protein A (HbpA) was found to be highly conserved with respect to both antigenicity and apparent molecular weight among heme-requiring Haemophilus species pathogenic for humans. To further the characterization of the structure and function of HbpA, the complete nucleotide sequence of its gene, hbpA, was determined. Analysis of the nucleotide sequence revealed a single large open reading frame of 1,638 bp encoding a protein of 546 amino acid residues, with a molecular weight of 60,695. The sequence of the amino-terminal end of this protein contained a potential site for lipid acylation and for cleavage by signal peptidase II, consistent with earlier biochemical evidence which indicated that HbpA is a lipoprotein. A search of GenBank for proteins with amino acid sequence similarity to HbpA revealed that the periplasmic dipeptide transport protein of E. coli, DppA, has 53% sequence identity to HbpA.

Purification of the Escherichia coli type 1 pilin and minor pilus proteins and partial characterization of the adhesin protein.

Type 1 pili of Escherichia coli contain three integral minor proteins with apparent molecular weights (Mr) of 28,000 (28K protein), 16,500, and 14,500 attached to rods composed of Mr-17,000 pilin subunits (Hanson and Brinton, Nature [London] 322:265-268). We describe here an improvement on our earlier method of pilus purification, which gives higher yields and higher purity. Also reported are methods allowing fractionation of intact type 1 pili into rods of pure pilin and free minor proteins, as well as fractionation of the 28K tip adhesion protein from the 16.5K and 14.5K proteins. We have determined the amino acid composition and amino-terminal sequence of the adhesion protein. This sequence shows limited homology with the amino-terminal sequences of several E. coli pilins, including type 1.

Expression of the heat-modifiable major outer membrane protein of Haemophilus influenzae type b is unrelated to virulence.

The heat-modifiable major outer membrane protein (P1) of Haemophilus influenzae type b (Hib) has been shown to be both exposed on the cell surface and capable of inducing the synthesis of antibodies protective against experimental Hib disease. Chemical mutagenesis of a recombinant plasmid containing the Hib gene encoding P1 resulted in inactivation of P1 expression by this plasmid. The mutated P1 gene was transformed into Hib to obtain an isogenic mutant lacking only the ability to synthesize this surface protein. In addition, the P1 gene was inserted into a plasmid shuttle vector and used to construct a recombinant Hib strain that overexpressed the P1 protein. Lack of P1 expression did not affect the ability of Hib to grow in vitro. Neither the absence nor the overproduction of P1 affected expression of capsular polysaccharide and lipooligosaccharide by Hib. The P1-negative mutant and the P1-overexpressing strain were both as susceptible to the bactericidal activity of pooled normal human serum as was the wild-type parent strain, while the P1-negative mutant was as resistant to the bactericidal activity of normal infant rat serum as was the wild-type parent strain. The P1-negative mutant was no less virulent than was the wild-type parent strain in an animal model system, such that both the numbers of animals infected by this mutant and the mean magnitudes of the resultant bacteremias were essentially identical to those obtained with challenge by the wild-type parent strain. Similarly, overexpression of P1 did not detectably affect the virulence of Hib. These data indicate that this protective protein antigen plays no detectable role in the expression of virulence by Hib, as assessed in an animal model system.

Efficacy and safety of live recombinant BCG vaccines.

BCG has a long history of safe use in humans and is one of the best adjuvants known. The use of newer production methods may further reduce the risk of adverse side-effects. Early results with experimental animals have shown BCG to be an effective live recombinant delivery vehicle for several foreign vaccine antigens. Additional refinements to the safety and efficacy of the recombinant BCG vaccine vehicle are under development.

Protective immunity elicited by rBCG vaccines.

The live attenuated Mycobacterium bovis strain Bacille Calmette-Guerin (BCG) is still widely used as a vaccine against tuberculosis and therefore offers potential advantages as a safe, live vaccine vehicle for the expression and delivery of protective recombinant antigens. As an attenuated intracellular bacterium residing in macrophages, BCG would seem to be particularly suited for eliciting cellular responses and not humoral responses. Efforts to improve the potential for recombinant BCG (rBCG) vaccines to elicit protective humoral responses were undertaken by developing vectors systems which export or secrete foreign antigens. Expression of the OspA antigen of Borrelia burgdorferi, as a chimaeric membrane-associated lipoprotein, resulted in high-titred protective humoral responses when compared to cytoplasmic or secreted expression of OspA. Expression of the PspA antigen of Streptococcus pneumoniae as a secreted or membrane-associated lipoprotein by rBCG did not result in higher-titred humoral responses in comparison to PspA expressed cytoplasmically but apparently improved the quality of protective PspA specific antibodies. Based on these pre-clinical studies, rBCG vaccines for Lyme and pneumococcal diseases are being developed for clinical trials in humans. The potential for eliciting mucosal responses to antigens delivered by rBCG was also investigated. Nasal immunization was superior at eliciting substantial lasting mucosal responses at multiple mucosal sites and also resulted in systemic responses comparable to those obtained with parenteral immunization.

Identification and characterization of SirA, an iron-regulated protein from Staphylococcus aureus.

The acquisition of iron by pathogenic bacteria is often a crucial step in establishing infection. To accomplish this, many bacteria, including Staphylococcus aureus, produce low-molecular-weight iron-chelating siderophores. However, the secretion and transport of these molecules in gram-positive organisms are poorly understood. The sequence, organization, and regulation of genes involved in siderophore transport are conserved among gram-negative bacteria. We used this information to identify a putative siderophore transport locus from an S. aureus genomic sequence database. This locus contains three predicted open reading frames with a high degree of homology to genes involved in siderophore uptake in several bacterial species, in particular the cbr locus of the plant pathogen Erwinia chrysanthemi. The first gene in the locus, which we have designated sir for staphylococcal iron regulated, encodes a putative lipoprotein with a molecular mass of 37 kDa. The open reading frame is preceded by a 19-bp region of dyad symmetry with homology for operator sequences controlling iron-regulated expression of genes in other bacteria. Fur titration experiments indicate that this region of dyad symmetry is sufficient for Fur-dependent regulation in Escherichia coli. The expression of this gene was repressed, in a dose-dependent manner, by the addition of iron to the S. aureus culture medium. sir-encoded proteins may be involved in iron acquisition in vivo and therefore may be targets for antimicrobial agents.