Evidence for a switching mechanism in the invasion of erythrocytes by Plasmodium falciparum.

The human malaria parasite Plasmodium falciparum demonstrates variability in its dependence upon erythrocyte sialic acid residues for invasion. Some lines of P. falciparum invade neuraminidase-treated or glycophorin-deficient red blood cells poorly, or not at all, while other lines invade such cells at substantial rates. To explore the molecular basis of non-sialic acid dependent invasion, we selected parasite lines from a clone (Dd2) that initially exhibited low invasion of neuraminidase-treated erythrocytes. After maintaining Dd2 for several cycles in neuraminidase-treated erythrocytes, parasite lines were recovered that invaded both untreated and neuraminidase-treated erythrocytes at equivalently high rates (Dd2/NM). The change in phenotype was maintained after removal of selection pressure. Four subclones of Dd2 were isolated and each readily converted from sialic acid dependence to non-sialic acid dependence during continuous propagation in neuraminidase-treated erythrocytes. The neuraminidase-selected lines and the Dd2 clone demonstrated identical restriction fragment length polymorphism markers indicating that the Dd2 clone was not contaminated during the selection process. Parasite proteins that bound to neuraminidase-treated and untreated erythrocytes were indistinguishable among the parent Dd2 clone and the neuraminidase-selected lines. The ability of the Dd2 parasite to change its invasion requirements for erythrocyte sialic acid suggests a switch mechanism permitting invasion by alternative pathways.

Salmonella vertebral osteomyelitis treated with cefotaxime.

The antimicrobial therapy of systemic salmonella infections is presently limited to ampicillin, chloramphenicol, and sulfamethoxazole and trimethoprim. Infrequently, alternative therapy may be necessary. We report the successful treatment of a case of Salmonella vertebral osteomyelitis with cefotaxime sodium.

The causes of death in patients with human immunodeficiency virus infection: a clinical and pathologic study with emphasis on the role of pulmonary diseases.

The clinical records and autopsy data of 75 patients dying with AIDS were reviewed to determine the frequency of individual diseases diagnosed premortem and postmortem, the significance of pulmonary processes found in the lungs at autopsy, and the clinical and pathologic causes of death. Cytomegalovirus (CMV) infection was identified histologically either premortem or postmortem in 81% of patients. The lungs and adrenal glands were infected most commonly. Only one-half of CMV infections were recognized premortem. Pneumocystis pneumonia and Kaposi sarcoma occurred in 68% and 59% of patients, respectively, but were not unsuspected premortem in any patient. Visceral involvement with Kaposi sarcoma, however, was frequently recognized only at autopsy. While disseminated M. avium-intracellulare infection was common (31% of patients), histologically documented pulmonary disease was uncommon (3% of patients). Cryptococcal infection, diagnosed in 10 patients, was confined to the central nervous system in only 1 patient. Toxoplasma, in contrast, infected the brain of only 6 patients. All 75 patients had one or more disease processes identified in their lungs or pleurae at autopsy. These processes included opportunistic infections in 76% of patients, neoplasms in 37% (Kaposi sarcoma in 36% and lymphoma in 3%), and other processes in 60%. The most prevalent pathogen, CMV was found in pulmonary tissue from 44 patients and caused significant disease in 21 patients. Five patients died due to CMV pneumonia. Pneumocystis carinii was found at autopsy in 24 patients. In spite of treatment, pneumocystis pneumonia was fatal in 11 patients. One patient died with concomitant CMV and pneumocystis pneumonia. Kaposi sarcoma, identified in the lungs of 23 patients, led to death in 5 patients via upper airway obstruction, hemorrhage, or parenchymal destruction. Other fatal pulmonary processes included bacterial pneumonia in 9 patients, idiopathic diffuse alveolar damage in 5, cryptococcosis in 2, and pulmonary hemorrhage in 1. Specific clinical criteria were used to determine the cause of death due to organ system failure. Fifty-one percent of patients died due to respiratory failure; 16% from neurologic disease; 17% from hypotension that was not caused by respiratory, neurologic, or cardiac disease; and 3% from cardiac dysfunction. Thirteen percent of deaths did not meet the clinical criteria defining these 4 categories. This clinical assessment was combined with autopsy data to identify specific diseases as causes of death.(ABSTRACT TRUNCATED AT 400 WORDS)

Restriction polymorphisms and fingerprint patterns from an interspersed repetitive element of Plasmodium falciparum DNA.

A recombinant DNA clone, pC4.H32, identifies distinguishable restriction fragment patterns from different Plasmodium falciparum clones. Analysis of these DNA fingerprint patterns from parasites cultivated over several years and from progeny of a P. falciparum cross showed the fingerprints to be mitotically and meiotically stable. Restriction fragments from the parents of the cross possessed sufficient polymorphism and number to generate 14 unique fingerprint patterns in 16 independent recombinant progeny. The pC4.H32 insert contains a 0.5-kb imperfectly repeated sequence found in subtelomeric regions of multiple chromosomes. Restriction site variations both within and outside of the 0.5-kb repeat contribute to the fingerprint polymorphisms. Fingerprint analysis can serve to type P. falciparum clones and can detect mislabeling and cross-contamination of parasite stocks.

An RFLP map of the Plasmodium falciparum genome, recombination rates and favored linkage groups in a genetic cross.

We report a genetic linkage map of the Plasmodium falciparum genome, using the inheritance patterns of nearly 90 RFLP markers in a genetic cross. Markers were assigned to polymorphic loci on all 14 nuclear chromosomes. Genetic recombination between parental markers was detected in each of the progeny, indicating that progeny from cross-fertilization events were favored over progeny from self-fertilization of either parent alone. Inheritance patterns among the markers suggested that certain parental linkage groups on chromosomes 2, 3, 12 and 13 were favored in the cross. Recombination frequencies on five chromosomes indicated an approximate map unit size of 15-30 kb per centiMorgan for P. falciparum.

Glycophorin B as an EBA-175 independent Plasmodium falciparum receptor of human erythrocytes.

Invasion of erythrocytes by malaria parasites involves multiple receptor-ligand interactions. To elucidate these pathways, we made use of four parasite clones with differing specificities for invasion, erythrocytes that are mutant for either glycophorin A or B, and enzyme modification of the erythrocyte surface with neuraminidase and trypsin. Neuraminidase alone abolishes invasion of two parasite clones (Dd2, FCR3/A2); these invade after trypsin treatment alone. A third clone (7G8) is unable to invade trypsin-treated erythrocytes. The fourth clone (HB3) can invade after either neuraminidase or trypsin treatment. The receptor for invasion of trypsin-treated erythrocytes was explored in two ways: treatment of trypsin-treated normal cells with neuraminidase, and trypsin treatment of glycophorin B-deficient cells. Both treatments eliminated invasion by all clones, indicating that the trypsin-independent pathway uses sialic acid and glycophorin B. To identify parasite proteins involved in the different pathways, erythrocyte binding assays were performed with soluble parasite proteins from each clone. Based on binding assays using erythrocytes that lack glycophorin A, the parasite protein known as EBA-175 appears to bind predominantly to glycophorin A. In contrast, the glycophorin B pathway does not appear to involve EBA-175, as binding of EBA-175 was similarly reduced to trypsin-treated normal and trypsin-treated glycophorin B-deficient erythrocytes. Thus, the glycophorin B-dependent, sialic acid-dependent invasion of trypsin-treated normal erythrocytes uses a different parasite ligand, indicating two or more sialic-dependent pathways for invasion. Clone 7G8, which cannot invade trypsin-treated erythrocytes, may be missing the ligand for invasion via glycophorin B.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of enteropathogenic bacteria by human milk whey in vitro.

Diarrhea caused by enteropathogenic bacteria is a leading cause of childhood mortality world-wide, particularly in less developed regions. Breast-feeding has been advocated to protect infants and children from infectious illnesses. We examined the antibacterial activity of human whey in vitro against multiple strains of the following species of enteropathogenic bacteria, Campylobacter jejuni, Escherichia coli, Salmonella typhimurium, Shigella flexneri, Shigella sonnei and Vibrio cholerae, all isolated from humans with diarrheal illness. In vitro human whey inhibited the growth of most but not all of the enteropathogenic strains tested. The test strains multiplied in both bacterial growth medium and commercial infant-feeding formula controls, with the exception of the Campylobacter strains, which were markedly inhibited in formula. These results confirm and extend previous observations that human whey contains components capable of inhibiting enteropathogenic bacteria and which may be associated with the protective effects of breast feeding. The precise mechanisms of these inhibitory effects merit further study.

Measuring disadvantage: changes in the underprivileged area, Townsend, and Carstairs scores 1981-91.

OBJECTIVE: To compare the intercensal change for each of the underprivileged area (UPA), Townsend, and Carstairs scores calculated from 1981 and 1991 census data. SETTING: England and Wales. METHODS: The method described enables comparison of change in composite scores such as the UPA, Townsend, and Carstairs scores which are derived from normalised variables. The national values of equivalent variables derived from the censuses are calculated and normalised on the same baseline of the 1981 electoral ward mean and SD values. The resultant change in composite scores for different censuses can then be compared directly. MAIN OUTCOME MEASURE: Change in the composite score values for the 1991 census when compared with the 1981 census. RESULTS: For England and Wales, the UPA score increased by 5.62 units (0.35 of the SD) but the Townsend and Carstairs scores fell by 2.39 and 1.13 units respectively (0.71 and 0.33 of the SDs). CONCLUSION: The Townsend and Carstairs scores are good measures of material deprivation and show a general improvement as such between 1981 and 1991. The UPA score, however, includes additional factors relating to family structure, social deprivation, and health need and shows a decline in the overall situation.

Vaccines for hepatitis A and B. The latest recommendations on safe and extended protection.

Hepatitis A vaccines (Havrix and Vaqta), administered in two doses, provide long-term protection. Target groups include international travelers, children in high-risk communities, homosexually active men, injecting drug users, persons who work with nonhuman primates, patients with chronic hepatitis, and recipients of clotting factors. The place of hepatitis A vaccination in the childhood-immunization schedule has not been determined. Postexposure prophylaxis for hepatitis A consists of administration of immune globulin within 2 weeks of exposure. Hepatitis B vaccines (Recombivax HB and Engerix-B), administered in three doses, provide protective antibody levels in more than 95% of recipients. Duration of protection appears to approach 10 years. Booster doses are not currently recommended. Hepatitis B vaccination has been incorporated into the routine childhood-immunization schedule. Additional target groups include medical personnel exposed to blood products, household and sexual contacts of infected persons, injecting drug users, and homosexually active men. Postexposure prophylaxis consists of administration of hepatitis B immune globulin as soon after exposure as possible, along with the initial dose of vaccine if desired.

Clinical evaluation of the plasma chromogenic Limulus assay.

Clinical correlations of the plasma chromogenic Limulus assay were evaluated in 520 septic episodes to assess the diagnostic utility of the assay in a university hospital setting. Otherwise unselected patients undergoing blood culture were studied. An association of plasma Limulus activity with gram negative bacteremia and focal infections was found (p less than .001 and p less than .01, respectively). There was a lesser correlation between a positive assay and the presence of major hepato-intestinal illness (p = .05). The frequency of positive tests was similar for adult and pediatric groups. There were modest correlations of a positive assay with hypotension (p = .02) and thrombocytopenia (p = .04), but only among patients with gram negative infection. Abnormal neutrophil parameters were unassociated with positive assays in any group. The sensitivity and specificity of the test for a condition known to cause endotoxemia--either gram negative infection or major intestinal disease--were low, 21% and 93% respectively. However, the predictive value of a positive test was 79%, indicating utility for the assay.

Activities of five new fluoroquinolones against Pseudomonas pseudomallei.

Thirty-four isolates of Pseudomonas pseudomallei were tested by a broth microdilution technique for susceptibility to amifloxacin, ciprofloxacin, enoxacin, norfloxacin, and ofloxacin. Ciprofloxacin was the most active agent tested, with an MIC for 90% of the strains tested of 8 micrograms/ml. These in vitro results suggest that the fluoroquinolones tested would not be optimal for therapy of melioidosis.

The changing image of Catholic women.

The characteristics of Roman Catholic women in today's society were investigated. Subjects were 154 Catholic women, both religious and lay, who participated on a volunteer basis. The Catholic lay women (111) were divided into two groups: those who attended a Catholic elementary school (63) and those who attended a non-Catholic elementary school (48). Catholic women religious were found to be more dominant and independent minded than in previous research. All of the women studied were found to be more aggressive and more critical of authority than in previous studies. Likewise, Catholic women no longer see themselves in the role of nurturers.

Selection of genetic variants from Plasmodium clones.

Clones of Plasmodium alter their antigenic profile or invasion phenotype when presented with specific challenges. Two examples are reviewed which may represent different genetic mechanisms of adaptation to selection pressures. In one series of experiments, rhesus monkeys were vaccinated with a 143,000/140,000 Mr P. knowlesi merozoite surface protein and then infected with a parasite clone expressing this protein. Primary parasitemia was controlled, but subsequent waves of parasitemia developed from populations of parasites harboring mutations in the 143,000/140,000 Mr gene. Mutations in this gene may be occurring at a continual low rate in the population (as with any normal gene) and particular mutations may have been selected in the vaccinated monkeys. In other experiments, P. falciparum parasite lines were selected from a clone (Dd2) that initially exhibited low rates of invasion into erythrocytes made sialic-acid deficient by neuraminidase treatment. After several growth cycles in neuraminidase-treated erythrocytes, a switch was observed and parasite lines were recovered that invaded neuraminidase-treated and normal erythrocytes at the same rate. The switch mechanism in invasion may represent another aspect of genetic variation, i.e. a programmed response in which certain genes are activated or rearranged. Vaccine trials in the future should include studies on the selection of mutations in the target antigen. Where switching mechanisms exist, knowledge of the genetic mechanisms that produce these adaptive responses will advance analysis of prospective vaccine candidates and contribute to our understanding of parasite biology.

Merocyanine 540-sensitized photoinactivation of human erythrocytes parasitized by Plasmodium falciparum.

The purpose of this study was to evaluate the photosensitizing dye merocyanine 540 (MC540) as a means for extracorporeal purging of Plasmodium falciparum-infected erythrocytes from human blood. Parasitized red blood cells bound more dye than nonparasitized cells, and exposure to MC540 and light under conditions that are relatively well tolerated by normal erythrocytes and normal pluripotent hematopoietic stem cells reduced the concentration of parasitized cells by as much as 1,000-fold. Cells parasitized by the chloroquine-sensitive HB3 clone and the chloroquine-resistant Dd2 clone of P falciparum were equally susceptible to MC540-sensitized photolysis. These data suggest the potential usefulness of MC540 in the purging of P falciparum-infected blood.

Molecular cloning and characterization of a cDNA for the highly conserved HMG-like protein (Pf16) gene of Plasmodium falciparum.

A cDNA clone (PfHB3-2-4) of 1538 bp corresponding to the highly conserved HMG-like protein (Pf16) was isolated. However, northern analysis suggests that the mRNA is about 2.2 to 2.3 kb. Analysis by RT-PCR indicated that the 0.6 to 0.7 kb sequence missing in the cDNA maps to the 3' end, suggesting that the cDNA is terminated within the 26 adenosine residues that are in the middle of the Pf16 sequence. The most unique feature about this cDNA is the presence of two open reading frames (ORF), one from nucleotides 91 to 927 and the other starting from 1421. The second ORF corresponds to Pf16. Expression of the cDNA clones in Escherichia coli and translation in rabbit reticulocytes of RNA transcribed from the T7 promoter of the cDNA clones revealed that only the 3' end Pf16 is translated from this mRNA. Further experiments with antisense oligonucleotides specific for Pf16 indicated that the Pf16 protein serves an important function in the life cycle of the parasite.

A family of erythrocyte binding proteins of malaria parasites.

Malaria erythrocyte binding proteins use the Duffy blood group antigen (Plasmodium vivax and Plasmodium knowlesi) and sialic acid (Plasmodium falciparum) on the erythrocyte surface as receptors. We had previously cloned the one P. vivax gene, the one P. falciparum gene, and part of one of the three P. knowlesi genes encoding these erythrocyte binding proteins and described the homology between the P. knowlesi and P. vivax genes. We have completed the cloning and sequencing of the three P. knowlesi genes and identified introns in the P. vivax and P. falciparum genes that correct the previously published deduced amino acid sequences. All have similar structures, with one or two exons encoding the signal sequence and the erythrocyte binding domain, an exon encoding the transmembrane domain, and two exons encoding the cytoplasmic domain with the exception of the P. knowlesi beta gene. The regions of amino acid sequence homology among all the genes are the 5' and 3' cysteine-rich regions of the erythrocyte binding domain. On the basis of gene structure and amino acid homology, we propose that the Duffy binding proteins and the sialic acid binding protein are members of a gene family. The level of conservation (approximately 70%) of the deduced amino acid sequences in the 5' cysteine-rich region between the P. vivax protein and the three P. knowlesi proteins is as great as between the three P. knowlesi proteins themselves; the P. knowlesi beta protein just 3' to this cysteine-rich region is homologous to the P. vivax protein but not to the other P. knowlesi proteins. Conservation of amino acid sequences among these organisms, separated in evolution, may indicate the regions where the adhesin function resides.

Plasmodium falciparum protein associated with the invasion junction contains a conserved oxidoreductase domain.

The merozoite cap protein-1 (MCP-1) of Plasmodium falciparum follows the distribution of the moving junction during invasion of erythrocytes. We have cloned the gene encoding this protein from a cDNA library using a monoclonal antibody. The protein lacks a signal sequence and has no predicted transmembrane domains; none of the antisera reacts with the surfaces of intact merozoites, indicating that the cap distribution is submembranous. MCP-1 is divided into three domains. The N-terminal domain includes a 52-amino-acid region that is highly conserved in a large family of bacterial and eukaryotic proteins. Based on the known functions of two proteins of this family and the pattern of amino acid conservation, it is predicted that this domain may possess oxido-reductase activity, since the active cysteine residue of this domain is invariant in all proteins of the family. The other two domains of MCP-1 are not found in any other members of this protein family and may reflect the specific function of MCP-1 in invasion. The middle domain is negatively charged and enriched in glutamate; the C-terminal domain is positively charged and enriched in lysine. By virtue of its positive charge, the C-terminal domain resembles domains in some cytoskeleton-associated proteins and may mediate the interaction of MCP-1 with cytoskeleton in Plasmodium.