Recombinant PhpA protein, a unique histidine motif-containing protein from Streptococcus pneumoniae, protects mice against intranasal pneumococcal challenge.

The multivalent pneumococcal conjugate vaccine is effective against both systemic disease and otitis media caused by serotypes contained in the vaccine. However, serotypes not covered by the current conjugate vaccine may still cause pneumococcal disease. To address these serotypes and the remaining otitis media due to Streptococcus pneumoniae, we have been evaluating antigenically conserved proteins from S. pneumoniae as vaccine candidates. A previous report identified a 20-kDa protein with putative human complement C3-proteolytic activity. By utilizing the publicly released pneumococcal genomic sequences, we found the gene encoding the 20-kDa protein to be part of a putative open reading frame of approximately 2,400 bp. We recombinantly expressed a 79-kDa fragment (rPhpA-79) that contains a repeated HxxHxH motif and evaluated it for vaccine potential. The antibodies elicited by the purified rPhpA-79 protein were cross-reactive to proteins from multiple strains of S. pneumoniae and were against surface-exposed epitopes. Immunization with rPhpA-79 protein adjuvanted with monophosphoryl lipid A (for subcutaneous immunization) or a mutant cholera toxin, CT-E29H (for intranasal immunization), protected CBA/N mice against death and bacteremia, as well as reduced nasopharyngeal colonization, following intranasal challenge with a heterologous pneumococcal strain. In contrast, immunization with the 20-kDa portion of the PhpA protein did not protect mice. These results suggest that rPhpA-79 is a potential candidate for use as a vaccine against pneumococcal systemic disease and otitis media.

RGD-mediated adhesion in fungal pathogens of humans, plants and insects.

Progress in the understanding of fungal adhesion has led to the identification of novel proteins recognizing the RGD tripeptide in matrix proteins and to the characterization of what appears to be an emerging subset of fungal adhesins that themselves contain an RGD sequence.

C3 as substrate for adhesion of Streptococcus pneumoniae.

The ability of choline-binding protein A (CbpA) of Streptococcus pneumoniae to bind the third component of complement (C3) suggests possible interactions with opsonic C3 in the bloodstream or with C3 secreted by epithelial cells. The latter possibility was investigated by measuring C3 in supernatants of resting and cytokine-activated monolayers of type II pulmonary epithelial cells (A549 cells). Expression of C3 on the epithelial cell surface was confirmed by immunofluorescence. Epithelially produced C3 bound to CbpA, as determined by Western blot test. cbpa(-) mutants and lysates therefrom failed to bind C3, were completely deficient in adhesion to a matrix in which C3 was the sole substrate, and demonstrated a moderate yet significant decrease in adhesion to type II pulmonary epithelial cells. These results confirm the interaction of the pneumococcal protein CbpA and its substrate, C3, in 2 in vitro models of adhesion.

Using ROC curves to test models of recognition memory: the relationship between presentation duration and slope.

Many theories of memory predict that the slope of the z-transformed receiver-operating characteristic (ROC) curve should decrease with increases in the memory strength of old items. While several prior studies have failed to demonstrate this effect, the results of two experiments demonstrate that increasing presentation duration can reduce the slope of the z-transformed ROC curve. These results raise questions about the generality of prior results and of dual-process theories designed to accommodate those results. We close by emphasizing that determining the experimental circumstances that affect whether or not the slope of the z-transformed ROC curve decreases will provide important constraints on theories of recognition memory.

Novel purification scheme and functions for a C3-binding protein from Streptococcus pneumoniae.

To isolate microbial proteins capable of binding the third component of complement (C3), we coupled the free sulfhydryl group of methylamine-inactivated C3 to a thiolSepharose matrix. This simple technique facilitated the purification of the first C3-binding protein isolated from a bacterium (Streptococcus pneumoniae). Both metastable (native) and thioester-disrupted C3 were recognized by this protein; binding of C3 was noncovalent, independent of thioester conformation, and preferential for the C3 alpha-chain. Sequencing of amino-terminal and internal peptides from the C3-binding protein disclosed a proline-rich region spanning approximately 20 amino acids and a signal peptide that had not been previously reported. The gene was isolated from a library of genomic DNA from laboratory strain CP1200 by screening with a 1200 bp PCR product amplified from degenerate oligonucleotides encoding the amino terminal sequence and the internal proline-rich sequence. The open reading frame spanned 1692 bp; all peptide sequences were identified in the translated gene product, which also contained at least three choline-binding repeats at the carboxy-terminus. The gene was conserved, and the translated protein was functionally active in pneumococcal clinical isolates of serotypes 1, 3, 4, 14, and 19F. Serum from a patient recovering from acute pneumococcal infection contained IgG antibodies specific for this protein by immunoblot. Wide conservation among clinical isolates, saturable binding of C3, and the ability to stimulate the human immune response have not previously been reported for this choline-binding protein. A similar biochemical approach should enable the identification of other C3-binding proteins in microorganisms able to elude complement-mediated host defense.

A pneumococcal protein that elicits interleukin-8 from pulmonary epithelial cells.

To understand how neutrophils are recruited to the lung in pneumococcal pneumonia, the ability of pneumococcal components to elicit the chemokine interleukin (IL)-8 from monolayers of cultured human type II cells was assessed. Heat-killed clinical and laboratory strains of Streptococcus pneumoniae and secreted proteins from exponentially growing pneumococci elicited significant quantities of IL-8 from A549 cells. All strains that elicited IL-8 production secreted a protein ( approximately 90 kDa) that comigrated on SDS-PAGE with a C3-binding protein previously identified in S. pneumoniae. As little as 7 pmol of the purified 90-kDa protein readily elicited levels of IL-8 production equivalent to those obtained with 1 U of IL-1alpha. Supernatant proteins and heat-killed cells of an isogenic mutant that failed to produce the C3-binding protein elicited significantly less IL-8 than did supernatant proteins or heat-killed cells of the parent strain. These results implicate the C3-binding protein of S. pneumoniae in a novel pathway of pulmonary inflammation.

Fungal morphogenesis and virulence.

Phenotypic variability in pathogenic fungi has long been correlated with virulence, but specific genetic and molecular mechanisms are only recently being unraveled. Fungal morphogenesis, reflecting the expression of several regulated genes, and the capacity of the rising forms or phases to cause disease has been focused on at the XIVth Congress of the International Society for Human and Animal Mycology. Three experimental models of pathogenic fungi have been discussed. In Cryptococcus neoformans, phenotypic variability or switching represents controlled and programmed changes rather than random mutations. Evaluated phenotypic traits were the capsular polysaccharide, cell and colony morphology and virulence. In the dimorphic Paracoccidioides brasiliensis, the serine-thiol proteinase from the yeast phase cleaves the main components of the basal membrane, thus being potentially relevant in fungal dissemination. In Candida albicans, relationships between adhesion proteins and those of lymphocytes and neutrophils are related to fungal pathogenicity. Regulation of the directional growth of hyphae and its tropic responses are correlated with the invasive potential of C. albicans.

Effect of INT1 gene on Candida albicans murine intestinal colonization.

BACKGROUND: Increased intestinal colonization with Candida albicans is believed to be a major factor predisposing immunocompromised and postsurgical patients to systemic candidiasis, although the mechanisms facilitating C. albicans colonization remain unclear. Because previous studies have linked the C. albicans INT1 gene to filament formation, epithelial adherence, and mouse virulence, experiments were designed to evaluate the effect of INT1 on intestinal colonization. MATERIALS AND METHODS: Mice were orally inoculated with either the parent strain (CAF2, INT/INT1), an int1 heterozygote (CAG1, INT1/int1), an int1 homozygote (CAG3, int1/int1), or a reintegrant (CAG5, int1/int1 + INT1), and sacrificed 3 and 7 days later for quantitative analysis of cecal C. albicans. RESULTS: Following oral inoculation with 10(3) C. albicans, only small numbers of each strain were recovered from the cecal flora of normal mice. However, in mice pretreated with oral antibiotics, cecal colonization of each strain was increased (P < 0.01). In addition, cecal colonization was reduced for all int1 mutant strains compared with the parent strain (P < 0.05). By light microscopy, all four C. albicans strains were easily observed in the ileal lumen as both budding yeast and filamentous forms, although only occasional yeast forms appeared adherent to the intestinal epithelium. CONCLUSIONS: C. albicans readily colonized and replicated in the ceca of antibiotic-treated mice. The presence of two functional copies of INT1 appeared to facilitate C. albicans cecal colonization, suggesting that intestinal colonization may be another virulence factor associated with INT1 and that the gene product may be an attractive target to control C. albicans intestinal colonization.

Systemic infection following intravenous inoculation of mice with Candida albicans int1 mutant strains.

The Candida albicans gene INT1 is associated with epithelial adhesion, hyphal formation, and virulence. C. albicans strains carrying two, one, or no functional INT1 alleles were used to assess the association between mortality and C. albicans persistence in the liver and kidney of intravenously inoculated mice. Mice were injected with 10(5) C. albicans CAF2 (parent strain, INT1/INT1), C. albicans CAG3 (homozygous disruptant, Int1/int1), or C. albicans CAG5 (heterozygous reintegrant, int1/int1 + INT1). Mortality was monitored and mice were sacrificed on Days 1, 7, 14, and 21 for quantitative analysis of kidney and liver microbes, with histologic analysis of these tissues as well. Mortality was highest for mice injected with the wild-type strain CAF2 (INT1/INT1) and lowest for mice injected with the homozygous disruptant CAG3 (int/int1). Yeast were readily cleared from the liver of all mice injected with any of the three C. albicans strains. Although the mutant strains CAG3 and CAG5 are defective for hyphal formation in vitro, there was histological evidence of abundant hyphal formation in the renal pelvis of mice injected with these strains. Compared to the wild-type strain, mutant strains were associated with reduced mortality but increased C. albicans persistence in the kidney. Thus, the absolute ability to form hyphae in the kidney did not appear to modulate either C. albicans-induced mortality or the course of progressive infection in the kidney. In addition, reduced virulence was paradoxically associated with increased, not decreased, persistence of C. albicans in the kidney.

Infectious diseases in internationally adopted children: findings in children from China, Russia, and eastern Europe.

Conditions in many orphanages provide a fertile environment for infectious diseases uncommonly encountered in industrialized nations. Moreover, health care providers may be unfamiliar with the need to test for these conditions in internationally adopted children. Pediatric infectious disease specialists provide much-needed expertise for parents and providers alike.

Massive splenomegaly and Epstein-Barr virus-associated infectious mononucleosis in a patient with Gaucher disease.

PURPOSE: Gaucher disease should be considered in the differential diagnosis of a patient with Epstein-Barr virus (EBV) infection who has unexplained or disproportionate splenomegaly. PATIENTS AND METHODS: A previously asymptomatic adolescent with EBV-associated infectious mononucleosis and massive splenomegaly is described. He was found to have Gaucher disease on bone marrow biopsy, which was performed to exclude a hematologic malignancy. The diagnosis was confirmed by assay of beta-glucosidase enzyme activity. RESULTS: Regression of splenomegaly and improving hematologic indices. CONCLUSION: Patients with infectious mononucleosis and disproportionate organomegaly should be investigated to exclude a hematologic malignancy or an underlying storage disorder such as Gaucher disease.

Integrin-like proteins in Candida spp. and other microorganisms.

The vertebrate integrins provide a paradigm for cell surface proteins involved in adhesion and morphogenesis. However, homologs of integrins have been found in more primitive organisms. This review will discuss the evidence for surface proteins in Candida albicans and Candida tropicalis that contain motifs reminiscent of integrins and will analyze the contributions of one of these proteins, Int1p, to adhesion, morphogenesis, and virulence. Other microorganisms thought to express integrin-like proteins will also be addressed.

AMPA/kainate antagonist LY293558 reduces capsaicin-evoked hyperalgesia but not pain in normal skin in humans.

BACKGROUND: Animal studies suggest that alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-kainate (AMPA-KA) receptors are involved in pain processing. The effects of the competitive AMPA-KA antagonist LY293558 in two types of experimental pain in human volunteers, brief pain sensations in normal skin, and mechanical allodynia-pinprick hyperalgesia were studied after the injection of intradermal capsaicin. METHODS: Brief intravenous infusions of the competitive AMPA-KA antagonist LY293558 were given to 25 healthy volunteers to examine acute toxicity and analgesic effects. Fifteen volunteers then entered a double-blinded, three-period crossover study. In a Phase II study, LY293558 infusions (100% maximally tolerated dose vs. 33% maximally tolerated dose vs. placebo) began 10 min after intradermal injection of 250 microg capsaicin in volar forearm. Spontaneous pain, areas of mechanical allodynia and pinprick hyperalgesia, and side effects were determined every 5 min for 60 min. RESULTS: The median maximally tolerated dose was 1.3 +/- 0.4 (range, 0.9-2.0) mg/kg. Tests of cognitive and neurological function were unchanged. Dose-limiting side effects were hazy vision in 95% of volunteers and sedation in 40%. There were no significant changes in electrical or warm-cool detection and pain thresholds or heat pain thresholds. LY293558 had little effect on brief pain sensations in normal skin. Both high and low doses of LY293558 significantly reduced pain intensity, pain unpleasantness, and the area in which light brush evoked pain after intradermal capsaicin. There was a trend toward a dose-response effect of LY293558 on the area in which pinprick evoked pain after intradermal capsaicin, which did not reach statistical significance. CONCLUSIONS: The authors infer that AMPA-KA receptor blockade reduces the spinal neuron sensitization that mediates capsaicin-evoked pain and allodynia. The low incidence of side effects at effective doses of LY293558 suggests that this class of drugs may prove to be useful in clinical pain states.

Linkage of adhesion, filamentous growth, and virulence in Candida albicans to a single gene, INT1.

Adhesion and the ability to form filaments are thought to contribute to the pathogenicity of Candida albicans, the leading cause of fungal disease in immunocompromised patients. Int1p is a C. albicans surface protein with limited similarity to vertebrate integrins. INT1 expression in Saccharomyces cerevisiae was sufficient to direct the adhesion of this normally nonadherent yeast to human epithelial cells. Furthermore, disruption of INT1 in C. albicans suppressed hyphal growth, adhesion to epithelial cells, and virulence in mice. Thus, INT1 links adhesion, filamentous growth, and pathogenicity in C. albicans and Int1p may be an attractive target for the development of antifungal therapies.

Health of children adopted from the former Soviet Union and Eastern Europe. Comparison with preadoptive medical records.

CONTEXT: Children born in the countries of the former Soviet Union and Eastern Europe are now a main source of international adoptions in the United States, but often little information is available about these children prior to adoption. OBJECTIVE: To analyze the preadoptive medical reports of children adopted from Eastern Europe and the former Soviet Union and to compare these reports with their evaluations after arrival in the United States. DESIGN: Case series. SUBJECTS AND SETTING: A total of 56 children adopted from Eastern Europe and the former Soviet Union were evaluated in 2 international adoption clinics. Preadoptive medical records were available for 47 of these children. RESULTS: There were 43 (91%) of 47 medical reports available from the children's birth countries that included multiple unfamiliar neurologic diagnoses. Evaluations in the International Adoption Clinics frequently revealed growth delays (zscore < or = -1 for weight in 44% of children, height in 68%, and head circumference in 43%). Children had 1 month of linear growth lag for every 5 months in an orphanage (r=-0.48, P<.001). Developmental delays were also common (gross motor delays in 70% of children, fine motor in 82%, language in 59%, and social-emotional in 53%). While serious medical problems were found or corroborated in 11 (20%) of the 56 children evaluated in our clinics, neurologic diagnoses cited in preadoptive medical reports were not confirmed. CONCLUSIONS: Preadoptive medical records from these international adoptees included multiple diagnoses suggesting severe neurologic impairment. Although these diagnoses were not confirmed when the children were evaluated in the United States, substantial growth and developmental delays were identified.

Congenital toxoplasmosis transmitted from an immunologically competent mother infected before conception.

Congenital transmission of Toxoplasma gondii from a mother who was apparently immunologically competent and who had toxoplasmic lymphadenitis 2 months before conception is described. Since no T. gondii-specific serological data were available for this mother from the time her lymph node biopsy specimen was obtained, the specimen was studied by polymerase chain reaction (PCR) to determine whether the T. gondii B1 gene was present. The predictive diagnostic value of histologic findings previously considered to be classic signs of T. gondii lymphadenitis also was studied. This was done by correlation of serological tests diagnostic of acute acquired T. gondii infection and presence of characteristic findings in biopsy specimens from persons without known immunocompromise. Both PCR and review of the characteristic features of her lymph node biopsy specimen confirmed the diagnosis of preconceptual infection in the mother. We also discuss two other cases in which apparently immunologically competent mothers with preconceptually acquired infection transmitted this parasite to their fetuses.