Molecular Characterization of Mycoplasma pneumoniae Isolates in the United States from 2012 to 2018.

Mycoplasma pneumoniae is a major cause of community-acquired pneumonia. There are limited data in the United States on the molecular epidemiological characteristics of M. pneumoniae We collected 446 M. pneumoniae-positive specimens from 9 states between August 2012 and October 2018. Culture, antimicrobial susceptibility testing, P1 subtyping, and multilocus VNTR (variable-number tandem repeats) analysis (MLVA) were performed to characterize the isolates. Macrolide-resistant M. pneumoniae (MRMp) was detected in 37 (8.3%) specimens. P1 subtype 2 (P1-2) was the predominant P1 subtype (59.8%). P1 subtype distribution did not change significantly chronologically or geographically. The macrolide resistance rate in P1 subtype 1 (P1-1) samples was significantly higher than that in P1-2 (12.9% versus 5.5%). Six P1-2 variants were identified, including two novel types, and variant 2c was predominant (64.6%). P1-2 variants were distributed significantly differently among geographic regions. Classical P1-2 was more frequent in lower respiratory tract specimens and had longer p1 trinucleotide repeats. Classical P1-2 was most common in MRMp (35.7%), while variant 2c was most common in macrolide-susceptible M. pneumoniae (67.5%). Fifteen MLVA types were identified; 3-5-6-2 (41.7%), 4-5-7-2 (35.3%), and 3-6-6-2 (16.6%) were the major types, and four MLVA clusters were delineated. The distribution of MLVA types varied significantly over time and geographic location. The predominant MLVA type switched from 4-5-7-2 to 3-5-6-2 in 2015. MLVA type was associated with P1 subtypes and P1-2 variant types but not with macrolide resistance. To investigate the M. pneumoniae genotype shift and its impact on clinical presentations, additional surveillance programs targeting more diverse populations and prolonged sampling times are required.

Macrolide-Resistant Mycoplasma pneumoniae in the United States as Determined from a National Surveillance Program.

There are sparse data to indicate the extent that macrolide-resistant Mycoplasma pneumoniae (MRMp) occurs in the United States or its clinical significance. Between 2015 and 2018, hospitals in 8 states collected and stored respiratory specimens that tested positive for M. pneumoniae and sent them to the University of Alabama at Birmingham, where real-time PCR was performed for detection of 23S rRNA mutations known to confer macrolide resistance. MRMp was detected in 27 of 360 specimens (7.5%). MRMp prevalence was significantly higher in the South and East (18.3%) than in the West (2.1%). A2063G was the predominant 23S rRNA mutation detected. MICs for macrolide-susceptible M. pneumoniae (MSMp) were ≤0.008 µg/ml, whereas MICs for MRMp were 16 to 32 µg/ml. Patients with MRMp infection were more likely to have a history of immunodeficiency or malignancy. Otherwise, there were no other significant differences in the clinical features between patients infected with MRMp and those infected with MSMp, nor were there any differences in radiographic findings, hospitalization rates, viral coinfections, the mean duration of antimicrobial treatment, or clinical outcomes. There was no significant change in MRMp incidence over time or according to age, sex, race/ethnicity, or status as an inpatient or an outpatient. Patients with MRMp were more likely to have received a macrolide prior to presentation, and their treatment was more likely to have been changed to a fluoroquinolone after presentation. This is the first national surveillance program for M. pneumoniae in the United States. Additional surveillance is needed to assess the clinical significance of MRMp and to monitor changes in MRMp prevalence.

Mycoplasma pneumoniae induces airway epithelial cell expression of MUC5AC in asthma.

As excess mucin expression can contribute to the exacerbation of asthma, the present authors hypothesised that Mycoplasma pneumoniae significantly induces MUC5AC (the major airway mucin) expression in airway epithelial cells isolated directly from asthmatic subjects. A total of 11 subjects with asthma and six normal controls underwent bronchoscopy with airway brushing. Epithelial cells were cultured at an air-liquid interface and incubated with and without M. pneumoniae for 48 h, and in the presence and absence of nuclear factor (NF)-kappaB and a toll-like receptor (TLR)2 inhibitor. Quantitative PCR was performed for MUC5AC and TLR2 mRNA. MUC5AC protein and total protein were determined by ELISA. M. pneumoniae exposure significantly increased MUC5AC mRNA and protein expression after 48 h in epithelial cells isolated from asthmatic, but not from normal control subjects, at all concentrations as compared to unexposed cells. TLR2 mRNA expression was significantly increased in asthmatic epithelial cells at 4 h compared with unexposed cells. NF-kappaB and TLR2 inhibition reduced MUC5AC expression to the level of the unexposed control in both groups. Mycoplasma pneumoniae exposure significantly increased MUC5AC mRNA and protein expression preferentially in airway epithelial cells isolated from asthmatic subjects. The toll-like receptor 2 pathway may be involved in this process.

Defects of T-cell effector function and post-thymic maturation in X-linked hyper-IgM syndrome.

X-linked hyper-IgM syndrome (XHIM) results from mutations in the gene encoding for CD40 ligand (CD154). Patients with the syndrome suffer from infections with opportunistic pathogens such as Cryptosporidium and Pneumocystis carinii. In this study, we demonstrate that activated T cells from patients with XHIM produce markedly reduced levels of IFN-gamma, fail to induce antigen-presenting cells to synthesize IL-12, and induce greatly reduced levels of TNF-alpha. In addition, we show that the patients' circulating T lymphocytes of both the CD4(+) and CD8(+) subsets contain a markedly reduced antigen-primed population, as determined by CD45RO expression. Finally, we demonstrate that the defects in antigen priming are likely due to the lack of CD154 expression and insufficient costimulation of T cells by CD80/CD86 interactions. Taken together, this study offers a basis for the increased susceptibility of patients with XHIM to certain opportunistic infections.

Translocation of phospholipase C-gamma 2 induced by in vitro activation of protein tyrosine kinase activity in mast cell lysates.

Aggregation of the high-affinity receptor for IgE (Fc eta RI) on the surface of intact or permeabilized rodent mast cells results in tyrosine phosphorylation of phospholipase C-gamma 1 (PLC gamma 1) and PLC gamma 2, and translocation of both isozymes to the particulate fraction. We report here that activation of resident tyrosine kinases by the addition of adenosine triphosphate (ATP), orthovanadate, and Mg2+ to rat basophilic leukemia cell (RBL) lysates induces an association of PLC gamma 2 with the Triton-insoluble particulate fraction, with a parallel increase in tyrosine phosphorylation of cellular proteins. Both PLC gamma 2 translocation and tyrosine phosphorylation are supported by millimolar Mg2+ or Mn2+ but not by Ca2+. Both tyrosine phosphorylation and PLC gamma 2 translocation are inhibited by genistein. These data suggest that in vitro activation of tyrosine kinase activity in broken cell preparations induces the formation of association between PLC gamma 2 and ligands with the Triton-insoluble fraction.

ICAM-3 (CD50) cross-linking augments signaling in CD3-activated peripheral human T lymphocytes.

ICAM-3 is a pan-hematopoietic, constitutive adhesion molecule. ICAM-3 binds to LFA-1 on antigen-presenting cells (APC) stabilizing the T cell-APC interaction, facilitating signaling through the CD3/TCR complex. However, recent evidence using cultured and transformed T cells suggests ICAM-3 may also function in signaling. Because ICAM-3 is constitutively expressed on resting T cells, we postulated that signaling through ICAM-3 in resting T cells represents an important costimulatory mechanism in these cells. In purified resting human T cells, cross-linking both ICAM-3 and CD3 with plate-bound antibodies resulted in a marked increase in cell size (consistent with blastogenesis), synergistically increased surface expression of CD25 and CD69, and increased T cell metabolism. Similarly, concomitant ICAM-3 and CD3 stimulation significantly (P < 0.001) increased resting human T cell phosphatidylinositol hydrolysis and phospholipase C-gamma1 phosphorylation. These results indicate that ICAM-3 augments signaling through CD3, functioning as a costimulatory molecule for resting T cells in the initial activation step.

Phosphatidylinositol hydrolysis in freshly isolated human T lymphocytes.

Antigen receptor-mediated activation of T and B lymphocytes results in activation of phospholipase C-gamma isozymes with subsequent hydrolysis of membrane inositol phospholipids. As a method of screening autoimmune or immunodeficient patients for early receptor signaling defects, we have developed a rapid technique for studying phosphatidylinositol (PI) hydrolysis in cultured cells and fresh clinical specimens resulting from surface receptor crosslinking. Using staphylococcal alpha-toxin, we permeabilized freshly isolated, purified human T lymphocytes to facilitate incorporation of [3H]myoinositol into membrane phospholipids. Aggregation of surface antigen receptors (TCR-CD3 complex and CD28 on T cells) with specific antibodies produced extensive ATP and Mg(2+)-dependent hydrolysis of the membrane inositol phospholipids as measured by release of water soluble inositol phosphates. Anti-human CD3 antibody produced 18.5 +/- 1.6 net % PI hydrolysis and anti-human CD28 antibody produced 4.6 +/- 0.2 net % PI hydrolysis. Simultaneous anti CD3/CD28 crosslinking produced 30.8 +/- 1.2 net % PI hydrolysis, an increase over either stimulus alone (p = 0.0013 two tailed t test). Isotype matched control antibodies produced 11.6 +/- 0.4% PI hydrolysis. The tyrosine phosphatase inhibitor orthovanadate (Na3VO4) was used as a positive control because it induces maximal protein tyrosine kinase-dependent PI hydrolysis in permeabilized cells. Na3VO4 consistently induced hydrolysis of > 50% of the membrane inositol phospholipid pool. These data indicate that costimulation of T cells with antibodies to CD3 and CD28 is synergistic and reinforces the importance of CD28 as an accessory T cell stimulus. This easy technique allows quick evaluation of the integrity of the early signaling cascade in lymphocytes as a screen for autoimmune and immunodeficiency diseases.

Seasonal and altitudinal variation in Pteridium aquilinum (L.) Kuhn: frond and stand types.

The field ecology of Pteridium aquilinum (L.) Kuhn was studied through a growing season in the northern Malvern Hills. At lower altitudes, stands reached a biomass plateau by late July extending through to late September, while at higher altitudes biomass increment was delayed and curtailed by the shorter growing season. Pteridium retained several competitive characteristics even when altitude and exposure restricted its vigour. With increased altitude, biomass allocation favoured the lamina and the underground portion of the stipe; the stipe-with-rachis component was comparatively reduced with a dwarfing of the canopy but a dense packing of pinnae. Adjustment to conditions in the stand, by the emergence and die-back of fronds, caused frond density to change through the growing season and at different altitudes. Two frond and associated stand types at contrasted altitudes were recognized by morphology and biomass.

Anaphylaxis.

The syndrome of anaphylaxis is a life-threatening event in which the potential for patient morbidity and mortality is high. An understanding of the pathophysiology of anaphylaxis, the most serious of the allergic disorders, is paramount for its diagnosis. In addition to these elements, this article discusses newly recognized causes of anaphylaxis and reviews its treatment.

CD2 (OKT11) augments CD3-mediated intracellular signaling events in human T lymphocytes.

CD2 (LFA-2) is expressed on thymocytes, natural killer cells, and virtually all peripheral T cells. CD2 binds to its primary ligand CD58 (LFA-3) on antigen presenting cells (APC) and stabilizes the T cell-APC interaction; this stable interaction then optimizes Ag-specific T-cell activation. We assessed whether CD2-cross-linking by mAb augments the process of T-cell stimulation through the TCR/CD3 complex. Plate-bound anti-CD2 or anti-CD3 mAb alone had no measurable effect on any of the assessed activation parameters of resting T cells. However, concomitant signaling through both CD2 and CD3 by plate-bound antibodies resulted in marked increases in CD69 expression on the T-cell surface and T-cell-cellular metabolism, as assessed by the ability of the cell to reduce 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxylmethoxyphenyl)-2-( 4-sulphophenyl)- 2H-tetrazolium (MTS) to formazen. In addition, simultaneous cross-linking of CD2 and CD3 caused a significant (P < 0.001) increase in phosphatidylinositol hydrolysis in resting T cells compared to stimulation with anti-CD3 mAb alone and anti-CD3 mAb plus anti-CD2 isotype control antibody. These results indicate that CD2 augments signaling through CD3, and consequently functions as a costimulatory molecule for resting T cells in the initial activation step.

CD5 (OKT1) augments CD3-mediated intracellular signaling events in human T lymphocytes.

CD5 is expressed on thymocytes, all mature T cells, and a subset of mature B cells, and probably contributes to T-cell-B-cell adhesion. We assessed whether CD5-crosslinking by mAb augments T-cell stimulation. Plate-bound anti-CD5 or anti-CD3 mAb alone had no effect on any of the assessed activation parameters of resting T cells. However, concomitant signaling through both CD5 and CD3 by plate-bound antibodies resulted in marked increases in T-cell surface CD69 expression and T-cell metabolism, as assessed by the T cell's ability to reduce 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxylmethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) to formazen. In addition, simultaneous cross-linking of CD5 and CD3 caused a significant (p < 0.001) increase in phosphatidylinositol hydrolysis in resting T cells compared to stimulation with anti-CD3 mAb alone or anti-CD3 mAb plus anti-CD5 isotype control antibody. These results indicate that CD5 augments signaling through CD3 and consequently functions as a costimulatory molecule for resting T cells.

Alpha 1-antitrypsin deficiency in a child with X-linked lymphoproliferative disease.

An 18-month-old white male infant with X-linked lymphoproliferative disease was evaluated for persistent hepatic dysfunction following primary Epstein-Barr virus infection. A liver biopsy revealed cirrhosis with a dense mononuclear cell infiltrate. These findings were confounding because cirrhosis is not a typical finding in either normal or immunodeficient individuals following infection with Epstein-Barr virus. An alpha 1-antitrypsin level obtained shortly after biopsy was spuriously within the lower limits of the physiologic range. Further investigation demonstrated a homozygous Z phenotype, the classic protease inhibitor variant described in alpha 1-antitrypsin deficiency. A repeat liver biopsy confirmed the presence of a second hereditary disease. This is a unique concurrence of two uncommon genetic disorders.

Chronic granulomatous disease in two children with recurrent infections: family studies using dihydrorhodamine-based flow cytometry.

We employed a recently published technique, flow cytometry using the cell permeant dye dihydrorhodamine, to analyze families of two patients with X-linked chronic granulomatous disease. The results illustrate the utility of this method in the diagnosis of this serious immunodeficiency disease and also in the identification of carriers.

In vitro release of histamine from murine mast cells by block co-polymers composed of polyoxyethylene and polyoxypropylene.

A series of block co-polymers composed of polyoxyethylene and polyoxypropylene were investigated for their ability to induce in vitro activation of mouse mast cells. We found that six of these co-polymers could cause histamine release from mouse mast cells in vitro. At low concentrations, the most efficacious co-polymer, T130R2, caused rapid and extensive concentration-dependent release of histamine from mouse mast cells. The release process was not cytotoxic; it required metabolic energy and was not accompanied by release of lactate dehydrogenase. Optimal release of histamine was dependent on both calcium and sodium ions in the extracellular medium. The degree of in vitro histamine release correlated with in vivo inflammation and in vitro ionophore activity. We believe that this represents the first report of the activation of mediator-containing cells by an ionophore selective for monovalent cations. These copolymers may therefore represent new reagents for investigations of cellular excitation.

Histamine release from human basophils by synthetic block co-polymers composed of polyoxyethylene and polyoxypropylene and synergy with immunologic and non-immunologic stimuli.

Co-polymers composed of polyoxyethylene and polyoxypropylene have been shown previously to trigger histamine release from mouse peritoneal mast cells; this property quantitatively is directly related to the ionophorous ability of these compounds to cause a functional exchange of intracellular K+ for extracellular Na+ across the cell membrane. We investigated the effect of an inflammatory copolymer, T130R2, on human basophils. The data demonstrate that T130R2 can cause calcium-dependent histamine release from human basophils in vitro. Further, at concentrations that do not cause histamine release, this co-polymer markedly augments release by suboptimal concentrations of the lectin Con A or anti-IgE antibody and the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate but not the calcium ionophore A23187. Thus, these co-polymers induce mediator release from cells of both rodents and humans. In both instances it is likely that calcium-dependent cell triggering is the result of an influx of sodium ions with concomitant depolarization of the transmembrane potential. In common with the calcium ionophore A23187, the co-polymer T130R2 has the ability to synergize with stimuli which trigger the IgE receptor as well as those which directly activate the cellular calcium- and phospholipid-dependent protein kinase.

A novel type II complement C2 deficiency allele in an African-American family.

A 9-yr-old African-American male presenting with severe recurrent pyogenic infections was found to have C2 deficiency (C2D). Analysis of his genomic DNA demonstrated that he carried one type I C2D allele associated with the HLA-A25, B18, DR15 haplotype. Screening all 18 exons of the C2 gene by exon-specific PCR/single-strand conformation polymorphism indicated abnormal bands in exons 3, 7, and 6, the latter apparently caused by the 28-bp deletion of the typical type I C2D allele. Nucleotide (nt) sequencing of the PCR-amplified exons 3 and 7 revealed a heterozygous G to A transition at nt 392, causing a C111Y mutation, and a heterozygous G to C transversion at nt 954, causing a E298D mutation and a polymorphic MaeII site. Cys111 is the invariable third half-cystine of the second complement control protein module of C2. Pulse-chase biosynthetic labeling experiments indicated that the C111Y mutant C2 was retained by transfected COS cells and secreted only in minimal amounts. Therefore, this mutation causes a type II C2D. In contrast, the E298D mutation affected neither the secretion of C2 from transfected cells nor its specific hemolytic activity. Analysis of genomic DNA from members of the patient's family indicated that 1) the proband as well as one of his sisters inherited the type I C2D allele from their father and the novel type II C2D allele from their mother; 2) the polymorphic MaeII site caused by the G954C transversion is associated with the type I C2D allele; and 3) the novel C111Y mutation is associated in this family with the haplotype HLA-A28, B58, DR12.

Phospholipase C-gamma 1 is translocated to the membrane of rat basophilic leukemia cells in response to aggregation of IgE receptors.

Aggregation of the high affinity receptor for IgE (Fc epsilon RI) on the surface of mast cells results in the rapid hydrolysis of membrane inositol phospholipids by phospholipase C (PLC). Although at least seven isoenzymes of PLC have been characterized in different mammalian cells, the isoenzyme involved in Fc epsilon RI-mediated signal transduction and the mechanism of its activation have not been demonstrated. We now report that PLC-gamma 1 is translocated to the membrane of mast cells after aggregation of Fc epsilon RI. Activation of rat basophilic leukemia cells, a rat mast cell line, with oligomeric IgE resulted in an increase in PLC activity in washed membrane preparations in a cell free assay containing exogenous [3H]phosphatidylinositol (PI). The increase in PLC activity has the same dose-response to oligomeric IgE as receptor mediated hydrolysis of inositol lipids (PI hydrolysis) in intact cells. Analysis by Western blot probed with anti-PLC-gamma 1 antibody revealed that there is a three- to fourfold increase in PLC-gamma 1 in membranes from activated cells. The increase in PLC activity is augmented a further 20% by the addition of orthovanadate to the incubation medium suggesting that a tyrosine phosphatase is involved in the down-regulation of this phenomenon. These findings demonstrate translocation of PLC-gamma 1 to the membrane following activation of a receptor which does not contain intrinsic tyrosine kinase activity. Activation of PLC-gamma 1 by this pathway may account for Fc epsilon RI-mediated PI hydrolysis.

Orthovanadate induces translocation of phospholipase C-gamma 1 and -gamma 2 in permeabilized mast cells.

Rapid activation of phospholipase C (PLC) with a resultant increase in phosphatidylinositol hydrolysis occurs after aggregation of the high affinity receptor for IgE (Fc epsilon RI) on the surface of mast cells. We previously described an increase in PLC activity associated with the particulate fraction of rat basophilic leukemia (RBL) cells after Fc epsilon RI aggregation, and this redistribution of enzyme activity correlated with an increase in immunoreactivity of the gamma 1 isozyme of PLC in the particulate fraction by Western blot analysis (J. Immunol. 148:2194-2200, 1992). We now report that the tyrosine phosphatase inhibitor orthovanadate mimics Fc epsilon RI-mediated activation of PLC-gamma 1 in RBL cells after permeabilization with Staphylococcus aureus alpha-toxin. Orthovanadate treatment of permeabilized cells induced: 1) a large increase in phosphoinositide hydrolysis in endogenously labeled cells; 2) an increase in PLC activity associated with the particulate fraction; and 3) an increase in immunoreactivity of PLC-gamma 1 in Western blots of the particulate fraction. In addition, incubation of RBL cells with either oligomeric IgE or orthovanadate results in the translocation of PLC-gamma 2 from the cytosol to the particulate fraction. All of the above effects were qualitatively similar to those seen after Fc epsilon RI aggregation. These data suggest that translocation and activation of PLC in mast cells are controlled by tyrosine phosphorylation of either the enzyme itself or some regulatory component. The equilibrium can be shifted to the phosphorylated state during either receptor-mediated activation of a tyrosine kinase or by blockade of dephosphorylation.

Evidence for GTP-binding protein involvement in the tyrosine phosphorylation of the T cell receptor zeta chain.

The zeta subunit of the T cell receptor (TCR) is a prominent substrate for a TCR-activated tyrosine kinase. Tyrosine phosphorylation of the zeta subunit in response to antibody-mediated receptor cross-linking was synergized in permeabilized T cells by either of two non-hydrolyzable GTP analogues, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) or guanosine 5'-[beta, gamma-imido]triphosphate Gpp(NH)p. ATP analogues did not significantly affect antibody-induced tyrosine phosphorylation. Unlike the GTP analogues, the GDP analogue guanosine 5'-[beta-thio]diphosphate (GDP beta S) did not enhance phosphorylation of zeta. The effect induced by the GTP analogues required TCR occupancy and was independent of protein kinase C. Taken together these observations implicate a GTP-binding protein in the modulation of TCR-induced tyrosine phosphorylation.