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PubMed2003

Impact of donor infections on outcome of orthotopic liver transplantation.

Infection occurs when microbial agents enter the host, either through airborne transmission or by direct contact of a substance carrying the infectious agent with the host. Human body fluids, solid organs, or other tissues often are ideal vectors to support microbial agents and can transmit infections efficiently from donor to recipient. In the case of blood transfusion and tissue transplantation, the main consequence of such a transmission is infection of the recipient. However, in the case of solid-organ transplantation, and particularly for liver transplantation, donor infections are not only transmitted to the recipient, the donor infection also may affect the donated liver's preservability and subsequent function in the recipient irrespective of the systemic consequences of the infection. In addition, solid organ recipients of infected organs are less able to respond to the infectious agent because of their immunosuppressive treatment. Thus, transmission of infections from organ donor to liver recipient represents serious potential risks that must be weighed against a candidate's mortality risk without the transplant. However, the ever-increasing gap between the number of donors and those waiting for liver grafts makes consideration of every potential donor, regardless of the infection status, essential to minimize waiting list mortality. In this review, we will focus on assessing the risk of transmission of bacterial, fungal, viral, and parasitic infectious agents from cadaveric liver donors to recipients and the effect such a transmission has on liver function, morbidity, and mortality. We will also discuss risk-benefit deliberations for using organs from infected donors for certain types of recipients. These issues are critically important to maximize the use of donated organs but also minimize recipient morbidity and graft dysfunction.

Chagas Disease↗
PubMed2003

The 19-kDa Mycobacterium tuberculosis protein induces macrophage apoptosis through Toll-like receptor-2.

Macrophages infected with Mycobacterium tuberculosis undergo increased rates of apoptosis. Important objectives are to define the microbial factors that cause apoptosis, the mechanisms involved and the impact on infection. The 19-kDa M. tuberculosis glycolipoprotein (p19) is both cell wall-associated and secreted and is a candidate virulence factor. We investigated the potential of recombinant, His-tagged p19 lacking the secretion/acylation signal to induce macrophage apoptosis. The TUNEL assay and annexin V binding to membrane phosphatidylserine were used to measure apoptosis. The results show that p19 does act to induce apoptosis in differentiated THP-1 cells and monocyte-derived macrophages and that this effect is both dose- and time-dependent. Furthermore, this effect of p19 is Toll-like receptor (TLR)-2-mediated because preincubation of either THP-1 cells or TLR-2-expressing CHO cells with anti-TLR-2 mAb inhibited apoptosis induced by p19. Apoptosis of macrophages in response to p19 was found to be caspase-8 dependent and caspase-9 independent consistent with a transmembrane pathway signaling cell death through TLR-2. The viability of M. tuberculosis in cells undergoing apoptosis induced by p19 was significantly reduced suggesting the possibility that this may favor containment of infection. Although native p19 is a mycobacterial glycolipoprotein, based upon the use of recombinant p19 where the acylation signal had been removed, we conclude that it is the polypeptide component of p19 that is responsible for signaling through TLR-2 and that the lipid moiety is not required.

Animals↗
PubMed2005

Inhibition of a vaccine-induced anti-tumor B cell response by soluble protein antigen in the absence of continuing T cell help.

DNA vaccination can elicit the production of anti-tumor antibodies, thus obviating the need to continuously administer passive antibody. This vaccination strategy is particularly important where antibodies have proven to be effective anti-tumor agents. To amplify antibody responses against weak tumor antigens, we previously designed DNA-fusion vaccines incorporating tumor sequences linked to microbial genes. By using a safe idiotypic (Id) antigen from a B cell tumor fused to a fragment C (FrC) sequence from tetanus toxin, we induced both anti-Id and anti-FrC antibodies. It was important to determine whether the antigen itself, either injected or released from residual tumor cells, would boost the antibody response. Id protein not only failed to boost the response, but permanently and rapidly inhibited it by ablating Id-specific memory B cells. In contrast, an Id protein-FrC conjugate boosted both Id-specific and FrC-specific responses. Strikingly, the depletion of CD4+ T cells converted the Id protein-FrC conjugate vaccine into an inhibitor. These findings support the hypothesis that the activation of memory B cells by a DNA vaccine encoding a protein antigen, in the presence of the protein itself, depends completely on T cell help. Furthermore, by using knockout mice, we have shown that inhibition of the Id-specific memory B cells by the Id protein is largely independent of the FcgammaRIIB and, hence, independent of immune complexes. The principles revealed by using a DNA vaccine have implications for all cancer vaccines designed to induce and maintain antibody responses against weak autologous tumor antigens.

Animals↗
PubMed1999

A chemically synthesized version of the insect antibacterial glycopeptide, diptericin, disrupts bacterial membrane integrity.

Insects protect themselves against bacterial infection by secreting a battery of antimicrobial peptides into the hemolymph. Despite recent progress, important mechanistic questions, such as the precise bacterial targets, the nature of any cooperation that occurs between peptides, and the purpose of multiple peptide isoforms, remain largely unanswered. We report herein the chemical synthesis and preliminary mechanistic investigation of diptericin, an 82 residue glycopeptide that contains regions similar to two different types of antibacterial peptides. A revised, highly practical synthesis of the precursor N(alpha)-Fmoc-Thr(Ac(3)-alpha-D-GalNAc) allowed us to produce sufficient quantities of the glycopeptide for mechanistic assays. The synthetic, full-length polypeptide proved to be active in growth inhibition assays with an IC(50) of approximately 250 nM, a concentration similar to that found in the insect hemolymph. Biological analysis of diptericin fragments indicated that the main determinant of antibacterial activity lay in the C-terminal region that is similar to the attacin peptides, although the N-terminal segment, related to the proline-rich family of antibacterial peptides, augmented that activity by 100-fold. In all assays, activity appeared glycosylation independent. Circular dichroism of unglycosylated diptericin indicated that the peptide lacked structure both in plain buffer and in the presence of liposomes. Diptericin increased the permeability of the outer and inner membranes of Escherichia coli D22 cells, suggesting possible mechanisms of action. The ability to access glycopeptides of this type through chemical synthesis will facilitate further mechanistic studies.

Amino Acid Sequence↗
PubMed1964

DRUG RESISTANCE OF ENTERIC BACTERIA. 3. ACQUISITION OF TRANSFERABILITY OF NONTRANSMISSIBLE R(TC) FACTOR IN COOPERATION WITH F FACTOR AND FORMATION OF FR(TC).

Harada, Kenji (Gunma University, Maebashi, Japan), Mitsuo Kameda, Mitsue Suzuki, and Susumu Mitsuhashi. Drug resistance of enteric bacteria. III. Acquisition of transferability of nontransmissible R(TC) factor in cooperation with F factor and formation of FR(TC). J. Bacteriol. 88:1257-1265. 1964.-Transmissible drug-resistance factor R, which confers resistance to tetracycline, chloramphenicol, streptomycin, and sulfonamide, was previously found to be transduced in the system of the Salmonella E group with phage epsilon. The R factor of R(+) transductants was nontransmissible by cell-to-cell contact, and it was not eliminated by treatment with acridine dye. When R(+) transductants were infected with F factor, the nontransmissible R factor acquired transferability by conjugation. The R(+) conjugants, to which only the R factor was separately transmitted by conjugation from the (F(+)R(+)) donor, were still unable to transfer their R factor by conjugation. However, the (FR)(+) conjugants, to which both F and R factor were transmitted simultaneously by conjugation, were also capable of transferring their F and R factors by conjugation. From the present study, it was concluded that the recombinant (FR) factor was formed as a result of an interaction between F and R factors present in a host bacterium, and that one of the mechanisms of acquisition of transferability is accounted for by the formation of recombinant (FR) factor. The recombinant (FR) factor was transferable by conjugation, and it conferred both the drug-resistance and F(+) characters to the recipient cells. The (FR) factor was eliminated by treatment with acridine dye and also transduced as one unit into Escherichia coli K-12 by P1kc phage.

Acridines↗
PubMed2004

The potential impact of early exposures to geohelminth infections on the development of atopy.

Microbial exposures in early life may provide important signals for immune maturation and the development of an antiinflammatory network thereby preventing the development of dysregulated immune responses such as that associated with allergic disease. The human immune system has evolved in the presence of intense helminth infections and has developed regulatory mechanisms to limit the harmful inflammation that can be caused by the potent allergens secreted by these chronic pathogens. Geohelminth infections are highly prevalent childhood infections, and there is strong evidence that chronic geohelminth infections provide protection against atopy in the rural tropics. Because the early environmental exposures that may lead to the development of atopy are likely to occur in the first few years of life, geohelminth infections may exert their protective effects at this time. Early exposures to geohelminth antigens could occur transplacentally, through breast milk, or through early infant exposures, and could induce tolerance to parasite antigens resulting in suppressed allergic responses to the parasite. Tolerization to parasite antigens could suppress allergic responses to inhalant allergens through bystander effects or through tolerization of crossreactive epitopes that are shared between geohelminth parasites and inhalant allergens. Tolerization to crossreactive allergens could occur by thymic deletion or through peripheral mechanisms such as regulatory T cells. Immunologic studies of the mechanisms by which early exposures to geohelminth infections affect immune polarization to inhalant allergens are likely to provide important insights into the early regulation of the immune response and may lead to the design of novel interventions for the prevention of allergic disease.

Environmental Exposure↗
PubMed1999

Mycobacterium wolinskyi sp. nov. and Mycobacterium goodii sp. nov., two new rapidly growing species related to Mycobacterium smegmatis and associated with human wound infections: a cooperative study from the International Working Group on Mycobacterial Taxonomy.

Previous investigations demonstrated three taxonomic groups among 22 clinical isolates of Mycobacterium smegmatis. These studies were expanded to 71 clinical isolates, of which 35 (49%) (group 1) were identical to five ATCC reference strains including the type strain ATCC 19420T. Twenty-eight isolates (39%) were group 2, and eight isolates (11%) were group 3. Isolates of groups 2 and 3 were most often associated with post-traumatic or post-surgical wound infections including osteomyelitis, were susceptible to sulfamethoxazole, amikacin, imipenem and the tetracyclines, variably resistant to clarithromycin, and susceptible (group 1), intermediately resistant (group 2) or resistant (group 3) to tobramycin. The three groups were similar by routine biochemical and growth characteristics, but had different mycolic acid dimethoxy-4-coumarinylmethyl ester elution patterns by HPLC and different PCR-restriction enzyme patterns of a 439 bp fragment of the hsp-65 gene. Group 3 isolates differed from group 1 by 18 bp by 16S rRNA sequencing and exhibited < 25% homology by DNA-DNA hybridization, being most closely related to Mycobacterium mageritense. The 16S rRNA of group 1 and group 2 isolates differed by only 3 bp, but by DNA-DNA hybridization they exhibited only 40% homology. The following names are proposed: Mycobacterium goodii sp. nov. for group 2 isolates (type strain ATCC 700504T = MO69T), Mycobacterium wolinskyi sp. nov. for group 3 isolates (type strain ATCC 700010T = MO739T) and Mycobacterium smegmatis sensu stricto for group 1 isolates.

Adolescent↗
PubMed2004

Phenylpropanoids, phenylalanine ammonia lyase and peroxidases in elicitor-challenged cassava (Manihot esculenta) suspension cells and leaves.

BACKGROUND AND AIMS: Control of diseases in the key tropical staple, cassava, is dependent on resistant genotypes, but the innate mechanisms are unknown. The aim was to study phenylpropanoids and associated enzymes as possible defence components. METHODS: Phenylalanine ammonia-lyase (PAL), phenylpropanoids and peroxidases (POD) were investigated in elicited cassava suspension cells and leaves. Yeast elicitor was the most effective of several microbial and endogenous elicitors. Fungitoxicity was determined against the cassava pathogens Fusarium solani, F. oxysporum and the saprotroph Trichoderma harzianum. KEY RESULTS: A single and rapid (> or =2-3 min) oxidative burst, measured as hydrogen peroxide, occurred in elicited cells. PAL activity was induced maximally at 15 h and was preceded by PAL mRNA accumulation, which peaked at 9 h. Symplasmic POD activity increased four-fold in cells, 48 h post-elicitation. POD isoforms (2-7 isoforms, pI 3.1-8.8) were detected in elicited and unelicited cells, extracellular medium and leaves but two extracellular isoforms were enhanced post-elicitation. Also expression of a cassava peroxidase gene MecPOD1 increased in elicited cells. Only anionic forms oxidized scopoletin, with highest activity by isoform pI 3.6, present in all samples. Unidentified phenolics and possibly scopolin increased post-elicitation, but there was no enhancement of scopoletin, rutin or kaempferol-3-O-rutinoside concentration. Fungal germ tube elongation was inhibited more than germination by esculetin, ferulic acid, quercetin and scopoletin. T. harzianum was generally more sensitive than the pathogens and was inhibited by > or =50 microg mL(-1) of ferulic acid and quercetin and > or =10 microg mL(-1) of scopoletin. CONCLUSIONS: Phenolic levels in cells were not enhanced and were, theoretically, too low to be inhibitory. However, in combination and when oxidized they may contribute to defence, because oxidation of esculetin and scopoletin by peroxidase and of esculetin by tyrosinase enhanced their fungitoxicity up to 20-fold.

Cells, Cultured↗
PubMed2004

Metallo-beta-lactamase-producing gram-negative bacilli: laboratory-based surveillance in cooperation with 13 clinical laboratories in the Kinki region of Japan.

A total of 19,753 strains of gram-negative rods collected during two 6-month periods (October 2000 to March 2001 and November 2001 to April 2002) from 13 clinical laboratories in the Kinki region of Japan were investigated for the production of metallo-beta-lactamases (MBLs). MBLs were detected in 96 (0.5%) of the 19,753 isolates by the broth microdilution method, the 2-mercaptopropionic acid inhibition test, and PCR and DNA sequencing analyses. MBL-positive isolates were detected in 9 of 13 laboratories, with the rate of detection ranging between 0 and 2.6% for each laboratory. Forty-four of 1,429 (3.1%) Serratia marcescens, 22 of 6,198 (0.4%) Pseudomonas aeruginosa, 21 of 1,108 (1.9%) Acinetobacter spp., 4 of 544 (0.7%) Citrobacter freundii, 3 of 127 (2.4%) Providencia rettgeri, 1 of 434 (0.2%) Morganella morganii, and 1 of 1,483 (0.1%) Enterobacter cloacae isolates were positive for MBLs. Of these 96 MBL-positive strains, 87 (90.6%), 7 (7.3%), and 2 (2.1%) isolates carried the genes for IMP-1-group MBLs, IMP-2-group MBLs, and VIM-2-group MBLs, respectively. The class 1 integrase gene, intI1, was detected in all MBL-positive strains, and the aac (6')-Ib gene was detected in 37 (38.5%) isolates. Strains with identical PCR fingerprint profiles in a random amplified polymorphic DNA pattern analysis were isolated successively from five separate hospitals, suggesting the nosocomial spread of the organism in each hospital. In conclusion, many species of MBL-positive gram-negative rods are distributed widely in different hospitals in the Kinki region of Japan. The present findings should be considered during the development of policies and strategies to prevent the emergence and further spread of MBL-producing bacteria.

Anti-Bacterial Agents↗
PubMed2003

[Pseudomonas aeruginosa: a multicenter study in 136 hospitals in Spain].

A cooperative group of 136 Spanish hospitals identified 1014 isolates of P. aeruginosa in one week. It was estimated that Spanish microbiology laboratories identified 168 P. aeruginosa isolates per 100,000 inhabitants population and year (25 isolates for every 1,000 hospital admissions/year). P. aeruginosa was recovered in 5.3% of all the samples with bacterial isolates. Seventy-five percent of samples containing P. aeruginosa came from the lower respiratory tract, wound exudates, abscesses and urine. The three most common serotypes present in Spain were found to be 0:1, 0:4 and 0:11 and constituted more than 50% of all isolates. The antimicrobials active against more than 85% of all the isolates included: ceftazidime (85.2%), piperacillin-tazobactam (92.8%), imipenem (86.2%), meropenem (92.2%) amikacin (91.4%) and tobramycin (91.2%). The study showed a high rate of resistance to ciprofloxacin (22.7%) and gentamicin (31.1%). Of the 529 patients who underwent clinical follow-up, 25.5% showed P. aeruginosa colonization and the remaining 74.5% had clinical infections. We estimated an incidence rate of 88.4 patients infected with P. aeruginosa per 100,000 inhabitants and year (13.8 cases per 1000 hospital admissions and year). Overall, 42% were community acquired. The overall mortality in this study was 15%, and mortality attributable to P. aeruginosa infections was 5%. After logistical regression analysis, the two independent predictors of mortality were the presence of a rapidly fatal underlying condition and the presence of bacteremia. In Spain, P. aeruginosa is much more than a cause of severe nosocomial infections in immunocompromised patients.

Anti-Bacterial Agents↗
PubMed1992

Bundling of microtubules by synapsin 1. Characterization of bundling and interaction of distinct sites in synapsin 1 head and tail domains with different sites in tubulin.

Synapsin 1 is a nerve terminal phosphoprotein whose role seems to encompass the linking of small synaptic vesicles to the cytoskeleton. Synapsin 1 can join small synaptic vesicles to neuronal spectrin, microfilaments and microtubules; it can also bundle microtubules and microfilaments. In this paper, the mode of interaction between synapsin 1 and microtubules has been investigated. Bundling is shown to be highly cooperative: the apparent Hill coefficient is 3.06 +/- 0.3, and bundling is half-maximal at 0.63 +/- 0.02 microM. Bundling occurs either when whole synapsin 1 preparations (containing monomers and oligomers) or when monomeric synapsin 1 is added to microtubules. However, it is not clear that synapsin 1 remains monomeric in the presence of microtubules. Synapsin 1-microtubule mixtures contain two types of filament. One type is characterised by microtubules often with synapsin 1 bound to their surface. The other type is composed of filaments of diameter 15 +/- 5 nm. This filament type is granular and made up in part of 14-nm-diameter particles. These dimensions are consistent with their being made up of polymerised synapsin 1. It is possible that microtubules induce the polymerisation of synapsin 1. Synapsin 1 had independent tubulin binding sites in the N-terminal head domain and in the C-terminal tail domain. Whole synapsin 1 can interact with tubulin after it has been digested to remove the tubulin C terminus (des-C-terminal tubulin). The interaction of des-C-terminal tubulin with synapsin 1 appears to be via the head domain, since 125I-des-C-terminal tubulin only shows specific binding to the head domain on gel blots. By contrast intact tubulin binds to both head and tail domains. Binding to the tail domain can be inhibited by a synthetic peptide representing the microtubule-associated protein 2 (MAP2) binding site of class II beta tubulin. These results suggest a model for microtubule bundling by synapsin 1 in which independent sites in the head and tail domains of synapsin 1 cross-link microtubules by interactions with two distinct sites in tubulin.

Animals↗
PubMed1996

Biodiversity prospecting and benefit-sharing: perspectives from the field.

Searching for new biologically active compounds from natural sources starts, obviously, in the field. Plant, microbial or animal materials to be sought and investigated may be selected through a number of approaches. No matter what selection criterion(a) is (are) used, the first step in obtaining the organism concerned is to undertake field collecting work to search for and to collect the organism. Good knowledge on the ecogeographic distribution and precision in the taxonomic identification of the organism(s) sought are crucial if the field work involves the search for a pre-determined organism or set of organisms. Such knowledge and precision during field work are of secondary importance, however, if the search and collection are based on biodiversity or ethnomedical uses, since accurate taxonomic identification may be made at a later date, in a Museum or Herbarium environment. When an individual or institution from a biotechnologically developed country wishes to obtain indigenous raw biological material from a biotechnologically less developed country, an agreement for the procurement of such raw material may be negotiated. Since the effort to search and develop a biologically active compound(s) from natural sources is a long-term process that involves teamwork between field and laboratory scientists, the success of the endeavor will depend in large part on the continued flow of raw material from the field. Goodwill to maintain such a flow may be achieved through appropriate scientific and monetary compensations, both in real-time and in long-term sharing of the benefits of discovery. Only with the prospect of financial return to the supplying country will there be an incentive for the protection of the natural resources towards sustainable use and development, as well as to allow time for continuing explorations and discoveries.

Animals↗
PubMed1993

Experimental endocarditis: a review of its relevance to human endocarditis.

Bacterial endocarditis is a difficult-to-cure infection, owing to (i) poor penetration of antibiotics into infected vegetations; (ii) altered metabolic state of bacteria within the lesion; (iii) absence of adequate host-defence cellular response which could cooperate with antibiotic action. The contribution of infection models to definition and improvement of therapeutic regimens of endocarditis in man remains of great importance because of the difficulties encountered in clinical trials. The advantage of the experimental model is that besides the fact that it closely simulates the characteristics of the infection in humans, it provides clear endpoints which allow statistical comparisons among different therapeutic regimens: number of bacteria/g of tissue, frequency of emergence of resistance, positivity of blood cultures, death vs survival rates, and percentage of relapses after treatment has been stopped. All these parameters are more sensitive and more easy to use than in man. The infection model has definitively established that bactericidal therapy is warranted and that in-vitro susceptibility tests, especially those evaluating the killing rate, have a good predictive value for the therapeutic outcome. Three main aspects are discussed for their influence on human therapy (i) the kinetics of antibiotic diffusion into vegetations, with a special reference to the data obtained by autoradiography, (ii) the specificity of some pharmacodynamic aspects of antibiotics in endocarditis, including the clinical consequences of these two parameters on antibiotic dosing regimens and length of therapy, and (iii) in-vivo synergy. This phenomenon involves a variety of mechanisms which are difficult or even impossible to analyse on the sole basis of in-vitro data: enhanced bactericidal activity (beta-lactam-aminoglycoside), prevention of emergence of resistance (as demonstrated for rifampin, quinolones or fosfomycin) and, as shown with rifampin or quinolones, 'pharmacokinetic synergy'. Animal models have helped to define the importance of antibiotic dosing strategies to achieve in-vivo synergy which appears as essential to increase the rate of both bacteriological and clinical cure rate.

Animals↗
PubMed1979

[Quantitative evaluation methods of the chemoantibiotic associations (author's transl)].

In the introductory part a new classification of joint drug actions is submitted, according which three fundamental types are distincted, named respectively interference, cooperation and true interaction. In its turn, interaction is subdivided in three classes (uni-effectual, bis-ineffectual, bis-effectual) in the last of which is placed the most relevant of the interactions, that is synergism, subclassified, at its turn, as additive, super and infra-additive. The second part is devoted to the classification of the bacteriological techniques hitherto proposed in order to evaluate in vitro and in vivo the antibacterial interaction of chemoantibiotics. The third part is devoted to the classification and critical analysis of biometrical techniques hitherto applied to above quoted bacteriological techniques in order to obtain a quantitative evaluation of interaction. Criticism versus isobolic model is pointed out. In the final part a new procedure, named isoeffectual, is proposed. According to such a procedure a close grid of single and joint concentrations of a couple of chemoantibiotics, broad enough in order to cover the whole of the effects to be explored, is tested in vitro adopting the one-center agar diffusion test or a liquid medium. The experimental data so obtained are related in a planar diagram to the log of the sum obtained by adding to the concentrations of the first the concentrations of the second agent, converted into equi-effectual concentrations of the first. By this way a series of curvilinear regressions is obtained, which may be all explained by a mathematical formula according which the data may be submitted to statistical analysis and elaborated in order to draw the parameters able to define quantitatively the interaction. The model so applied is discussed as a general model for joint drug action.

Anti-Bacterial Agents↗
PubMed1998

CD95 (Fas)-based, superantigen-dependent, CD4+ T cell-mediated down-regulation of human in vitro immunoglobulin responses.

Naturally occurring microbial superantigens (SAg) have been implicated in several human idiopathic disorders, and a compelling argument for the role of SAg in autoantibody-associated disorders, such as systemic lupus erythematosus, has been proposed. To test the effects of SAg on human in vitro Ig responses, CD4+ T cell + B cell cultures were stimulated with graded doses of staphylococcal enterotoxin B (SEB). Ig-secreting cell (IgSC) responses were very weak in CD4+ T cell + B cell cultures stimulated with SEB at the optimal mitogenic concentration (high dose SEB; 100 ng/ml) but were strong in parallel cultures stimulated with low dose SEB (0.01 ng/ml). High dose SEB actually enhanced B cell differentiation in the presence of CD4+ T cell soluble helper factors as long as the B cells were prevented from physically contacting the CD4+ T cells. However, when cell-cell contact between CD4+ T cells and B cells was permitted, high dose, but not low dose, SEB promoted increased CD4+ T cell-mediated B cell apoptosis with resulting decreases in viable CD20+ B cells and IgSC. High dose, but not low dose, SEB triggered increased levels of soluble CD95 ligand, and down-regulation of IgSC responses and incremental apoptosis of activated B cells were prevented by antagonist anti-CD95 mAb. This strongly suggests that CD4+ T cell-mediated CD95-based killing of activated B cells plays a major role in controlling SEB-driven IgSC responses. Defects in SAg-based down-regulation may contribute to autoimmune disorders such as SLE.

Antibodies, Monoclonal↗
PubMed1996

In vitro inhibition by intravenous immunoglobulin of human T cell-dependent B cell differentiation induced by staphylococcal superantigens.

Treatment with intravenous Ig (IVIG) is efficacious not only in humoral immunodeficiency diseases but in several nonimmunodeficiency disorders as well. Since microbial superantigens (SAg) have been postulated to play a role in promoting in vivo pathogenic autoantibody production and since IVIG preparations are rich in anti-SAg antibodies, we tested whether IVIG could inhibit in vitro SAg-driven human T cell-dependent B cell differentiation. We demonstrate that IVIG inhibits such B cell differentiation by at least three different mechanisms. Early addition of IVIG inhibits B cell differentiation not only in SAg-stimulated PBMC cultures but in anti-CD3- and pokeweed mitogen (PWM)-stimulated cultures as well, pointing to a SAg-nonspecific inhibitory effect. However, anti-SAg antibodies contained in IVIG can also effect SAg-specific inhibition, since polyclonal rabbit anti-SAg antisera added early to peripheral blood mononuclear cell (PBMC) cultures inhibit neither anti-CD3- nor PWM-driven B cell differentiation and inhibit B cell differentiation triggered only by the specific SAg against which the individual antiserum was raised. Finally, late addition of IVIG at a time at which B cells have already committed to terminal differentiation inhibits SAg-driven, but not anti-CD3- or PWM-driven, generation of Ig-secreting cells (IgSC). This late inhibition is associated with enhanced SAg-dependent cytolytic activity against Raji cell targets which is dramatic in PBMC cultures but is often not detectable in T + B cell cultures. Reconstitution of T + B cell cultures with natural killer cells restores the enhancing capacity of IVIG on SAg-dependent cytolytic activity as well as the late inhibitory effects of IVIG on IgSC generation. Understanding the multiple mechanisms through which IVIG can inhibit SAg-driven B cell differentiation may offer a rational basis for determining which patients are likely to favorably respond to IVIG administration.

Antibodies, Monoclonal↗
PubMed1975

A transforming marker that produces merodiploids with high efficiency and stable transformants with low efficiency in Streptococcus.

A mutation (ery-r8) conferring a high level of resistance to erythromycin in the Challis strain of Streptoccus sanguis can be transferred to wild-type erythromycin-sensitive recipients via single molecules of donor DNA. The transformants thus produced are of two types: (1) cells slightly more resistant to erythromycin than wild-type and capable of segregating (at a frequency of 2 X 10(-4)/bacterium/generation) either wild-type or highly-resistant cells like the original donor type; (2) cells phenotypically and genotypically identical to the original donor type. The unstable diploids (ery-r8/+) occur with a frequency equivalent to that obtained with high-efficiency (HE) markers, whereas the stable donor-type (ery-r8) transformants occur with about five hundred times lower frequency. Penetration of the wild-type recipient by more than one molecule of DNA bearing the ery-r8 marker increases by as much as seven times the incidence of stable transformants. UV-irradiation of molecules bearing the ery-r8 marker diminishes their ability to cooperate in producing a stable transformant, although the UV sensitivity of stable transformant production by a single DNA molecule is not different from that of diploid production. Hence, stable transformants do not appear to be produced by a process typical of low efficiency (LE) markers, which are generally highly sensitive to ultraviolet irradiation. Moreover, stable ery-r8 transformants are produced with equally low frequencies in strains of S. pneumoniae that discriminate (hex+) and fail to discriminate (hex--) between HE and LE markers. We postulate that all transformations by the ery-r8 marker result in ery-r8/+ diploids, and that segregation results in the infrequent stable transformants of the original donor type. This hypothesis is supported by the observations that rifampin treatment of ery-r8/+ populations increases the frequency of segregation and similar treatment of wild-type recipients under-going transformation by the ery-r8 marker increases the frequency of stable transformants.--In producing the ery-r8/+ transformant the r8 allele is integrated close to the site of its wild-type homolog, since single molecules of DNA from this transformant can be shown to carry both alleles. Segregation of either the ery-r8 or + allele is not detectably enhanced by acridine orange or thymidine deprivation.--The ery-r8 marker occurs close to a site of mutation (ery-r2) which confers erythromycin resistance upon ribosomes. When the r2 and r8 markers are jointly transferred, ery-r2-r8/+ genomes are produced in which the r2 marker is stably integrated but the r8 marker is unstably adjoined to its wild-type homolog. Thus, the duplicated region can be quite short. When the ery-r8 marker is stably integrated, the region of the marker is refractory to subsequent transformation. Markers with properties like ery-r8 are not particularly rare, being found with a frequency of about 4% among spontaneous mutations to erythromycin resistance.

Diploidy↗
PubMed1996

[A study on prevalence of resistance to antituberculosis drugs in Japan: comparison of results in the local facilities and in the reference laboratory].

During five years since the last survey of drug-resistant tuberculosis in Japan, features of tuberculosis in Japan have been changed. A nationwide survey was conducted by the Tuberculosis Research Committee of Japan. A total of 38 hospitals in various districts of Japan participated in the cooperative study. Each collaborating laboratory sent all mycobacterial cultures isolated during 1 June to 30 November, 1992 to the reference laboratory of the Committee, where species of the isolates were identified and drug susceptibility of Mycobacterium tuberculosis isolates were reexamined. The reference laboratory received a total of 1,236 cultures. Among them, 290 cultures were excluded from further examination by various reasons, such as contamination (52 cultures), non-viability (53), growth of nontuberculous mycobacteria (182) and other reasons (3). Thus, drug susceptibility test results were available for 946 cultures, including 26 cultures from non-Japanese persons. In the local laboratories, two methods, the absolute concentration method using 1% Ogawa egg slant (standard method, 26 hospitals) and its modified method using a microwell plate (microtiter method, 12 hospitals), were used for drug susceptibility testing, and the standard method was used in the reference laboratory. The results in the local laboratories were compared with those in the reference laboratory. The overall coincidence rate between drug susceptibility results reported from the local laboratories and those from the reference laboratory was 92.5%. A high coincidence rate (94.3%) was seen when the standard method was used in both local and reference laboratories. On the other hand, the coincidence rates between the results with the microtiter method in the local laboratory and those with the standard method in the reference laboratory were lower (standard method vs microtiter method; P < 0.01). Out of 19 hospitals, when the isolates were tested by the standard method 17 (89.5%) showed high coincidence rates (over 85%). Three hospitals using the microtiter method showed the coincidence rate over 90%, while other three showed lower rate (less than 80%) with high overestimation rates (over 19%), indicating that there are variations among facilities in performing the microtiter test. A part of the results concerning the resistance patterns to five antituberculosis drugs will be reported elsewhere.

Adolescent↗