Frank F. Richards

Reduced binding and phagocytosis of Pneumocystis carinii by alveolar macrophages from persons infected with HIV-1 correlates with mannose receptor downregulation.

The macrophage mannose receptor, a pattern recognition molecule and component of innate immunity, mediates binding and phagocytosis of Pneumocystis carinii and likely represents an important clearance mechanism in the lungs of immunocompetent hosts. The purpose of this study was to examine the ability of alveolar macrophages from HIV-infected individuals to bind and phagocytose P. carinii, and to investigate the role of the macrophage mannose receptor in mediating this interaction. Compared with healthy individuals, alveolar macrophage phagocytosis of P. carinii from HIV+ persons was reduced up to 74% (P = 0.02), primarily reflecting a reduction in the number of organisms associated with each macrophage (P = 0.019). Furthermore, macrophages from HIV+ individuals demonstrated up to an 80% (P < 0.05) reduction in mannose receptor surface expression and endocytosis. Mannose receptor affinity was unaltered, and mRNA levels were modestly reduced (P < 0.05). Cells from HIV+ individuals with CD4(+) counts < 200 cells/mm3 (representing individuals at high clinical risk for P. carinii pneumonia) demonstrated the lowest levels of P. carinii phagocytosis and mannose receptor endocytosis. In vitro HIV infection of alveolar macrophages from healthy individuals reduced mannose receptor endocytosis to 53.2% (P < 0.05) and P. carinii binding and phagocytosis to 67.4% (P < 0.05) of control. Our studies suggest that HIV infection may alter innate immunity in the lungs, and that impaired alveolar macrophage mannose receptor-mediated binding and phagocytosis of P. carinii may contribute to the susceptibility of HIV-infected individuals to this opportunistic pulmonary pathogen.

Genetic transformation and phylogeny of bacterial symbionts from tsetse.

Two isolates of bacterial endosymbionts, GP01 and GM02, were established in cell free medium from haemolymph of the tsetse, Glossina pallidipes and G. morsitans. These microorganisms appear similar to rickettsia‐like organisms reported previously from various tsetse species. The 16s rRNA sequence analysis, however, placed them within the gamma subdivision of the Proteobacteria, phylogenetically distinct from most members of the Rickettsiaceae which align with the alpha subdivision. Distinct multiple endogenous plasmids are harboured by GP01 and GM02, suggesting that the two isolates are different. Restriction mapping analysis showed that one of the conserved plasmids is present in high copy number and is at least 80 kb in size. A heterologous plasmid pSUP204, which contains the broad host range oriV replication origin, was used to transfect bacterial cultures. The symbiont GM02 was transformed, and it expressed plasmid encoded resistance to the antibiotics ampicillin, tetracycline and chloramphenicol. Transformation of these symbionts may provide a novel means for expressing anti‐parasitic genes within tsetse populations.

The challenge of Pneumocystis carinii culture.

ABSTRACT. Published and unpublished data on the cultivation of P. carinii were reviewed by a panel of investigators convened by the National Institutes of Health. Although several cell culture systems allow propagation of P. carinii for a limited time with modest rates of replication, these have not proved adequate for isolation of P. carinii in sufficient quantity to explore important basic biological investigation. Attempts at cell‐free culture have yielded only transient proliferation. Because much of the unsuccessful work on cultivation of the organism has been unpublished, the panel agreed that these data may be useful to other investigators in designing experimental strategies for cultivation. Therefore, the purpose of this report is to make available this information to researchers, lest others unknowingly repeat unsuccessful methods. It is hoped that by documenting the history and the complexities of Pneumocystis culture, renewed interest and efforts will be directed toward this fundamental scientific challenge.

Speculations how specific are antibodies

Abstract An important assumption underlies some theories of antibody diversity; namely, that a single immunoglobulin is complementary to a single antigen or a group of structurally related antigens. These are thought to bind at a single combining site on the immunoglobulin molecule. This review discusses the evidence for and against this idea. We also consider the biological and genetic implications of the possible existence of immunoglobulins with multiple binding functions.

Quantitative Approach to the Sequential Degradation of Proteins and Peptides

IT has been known for many years that quantitative as well as qualitative information can be derived from the Edman sequential degradation method1,2, but the variable recovery of liberated thiohydantoin derivatives of the constituent amino-acids has made an accurate quantitative analysis of proteins and pep tides difficult. We report here an approach to a quantitative protein degradation method using a volatile Edman reagent (methyl isothiocyanate3), an isotope dilution step for quantitation of the data, and an isotope ratio assay using conventional mass spectrometry4.

The fungal nature of Pneumocystis

Pneumocystis carinii is an opportunistic protistan pathogen that causes a lethal pneumonia in immunocompromised patients. The organism was described in the early 1900s by Chagas, who suggested that it was a trypanosome [6]. After World War II, P. carinii was recognized as an important cause of morbidity and mortality in malnourished children. With the advent of organ transplantation and the attendant use of immunosuppressive agents, the pathogen again emerged as a significant problem. Drugs were developed to treat P. carinii pneumonia in the 1970s, but these agents have proved to be less effective in managing the disease in patients with AIDS, and most eventually perish from a P.carinii infection [33]. Although P. carinii is now a major medical problem, little is known about the organism and its normal and pathological interactions with mammalian hosts. P. carinii has been refractory to cultivation and this has restricted the application of biochemical and genetic techniques. In the absence of solid biochemical and genetic data, many basic questions remained unanswered. One such question was the delineation of the proper phylogenetic associations of P. carinii. Prior to 1988, classification of P. carinii was based primarily on morphology, ultrastructure, growth requirements and susceptibility or resistance to chemotherapeutic agents. Based on these fairly weak criteria, P. carinii was widely considered to be a protozoan, although some investigators had argued that P. carinii was more similar to the fungi than to the protozoa [31]. The first molecular genetic evidence bearing on the taxonomy of P. carinii was at odds with the prevailing notion. Three groups found that the sequences of P. carinii ribosomal RNAs and ribosomal RNA genes were much more similar to those of fungi than to those of any known protozoan [16, 29, 34]. Over the last 3 years, data have accumulated that support the idea that Pneumocystis is a fungus. However, it is clear that this parasitic fungus is not typical of the fungi most often isolated and studied in either the basic or clinical laboratory.

Spliced leader RNA sequences of Trypanosoma rangeli are organized within the 5S rRNA-encoding genes

The spliced leader RNA(SL RNA)-encoding genes of the salivarian New World trypanosome, Trypanosoma rangeli, are organized within the 5S rRNA tandem repeats. Each repeat contains genes encoding an SL RNA and a 5S rRNA in the same orientation of transcription. This SL-5S organization is also present in the African trypanosome, Trypanosoma vivax. A similar association of SL and 5S genes has been observed in some nematodes, but has not been described previously in trypanosomatids.

Preparation of Anti-GM4 antiserum and its assay by a solid-phase radioimmunoassay

Anti-GM4 antiserum was prepared from rabbits by immunization with pure human brain GM4 ganglioside in complete Freunds adjuvant and methylated bovine serum albumin. None of the immunized animals developed any clinically apparent neurological dysfunction. The antiserum titer and specificity were analyzed by double immunodiffusion and a novel solid-phase radioimmunoassay (RIA). In the latter procedure, microtiter plate wells were coated first with the glycolipid antigen, followed by sequential application of antiserum and [125I]-Staphylococcal Protein A. The absorbed radioactivity in the well was then counted. Employing the RIA procedure, anti-GM4 antibody achieved a titer of 1:1600. The antiserum also exhibited a high degree of specificity to GM4; cross-reactivity with glycolipids of similar structure was negligible. The production of highly specific antiserum to GM4 and the feasibility of detecting antibodies to glycolipid antigens by a convenient solid-phase RIA should be useful to further study the biological and immunological roles of GM4 and other glycolipids in the central nervous system.

Differences in glucose transport between bloodstream and procyclic forms of Trypanosoma brucei rhodesiense

In African trypanosomes the requirements for glucose and its metabolism vary in different stages of the life cycle. Here we present evidence that cultured procyclic trypanosomes of Trypanosoma brucei rhodesiense uptake glucose against a concentration gradient in a time and dose-dependent manner. Moreover, glucose transport is completely inhibited by the sulphydryl inhibitor N-ethylmaleimide, suggesting the presence of a protein moiety as the carrier molecule. Comparison of glucose uptake in bloodstream and procyclic trypanosomes point to the possibility that different transporters may function in the 2 developmental stages. Glucose uptake by bloodstream trypanosomes requires Na+ ions and is inhibited by phlorizin, an inhibitor of Na(+)-dependent glucose transporters in mammalian cells. Conversely, procyclic trypanosomes transport glucose in a Na(+)-dependent manner, and transport is not affected by phlorizin. Finally, the putative procyclic glucose transporter has a higher affinity for glucose (apparent Km 23 microM) than the bloodstream carrier (apparent Km 237 microM).

Trypanosoma congolense: surface glycoproteins of two early bloodstream variants. II. Purification and partial chemical characterization.

Abstract Two sequential variant-specific glycoproteins have been purified from two variants of Trypanosoma congolense expressed during a relapsing infection. Isolation of the two glycoproteins, termed VSG-1 and VSG-2, respectively, employed glycerol lysis followed by purification on concanavalin A, Sephadex G-25, and gradient-eluted DE-52 columns. Partially purified VSG proteins were immunologically cross-reactive, but highly purified VSGs showed no cross-reactivity under the conditions employed. Both VSG-1 and VSG-2 consisted of a triplet of polypeptides. Although each member of a triplet subset could be distinguished by isoelectric focusing, all three gave identical N-terminal amino acid sequences and nearly identical tryptic peptide maps. The members of the VSG-1 polypeptide subset differed from those of the VSG-2 subset both with regard to N-terminal amino acid sequence and in tryptic peptide map patterns. Comparison of N-terminal sequences of VSG-1 and VSG-2 did, however, show that the sequences could be aligned to give a modest degree of amino acid homology (27%). This alignment also produced a minimum in the number of two-base changes, suggesting that the observed homology is not a coincidence and that these two proteins may well have arisen by gene duplication followed by retention of multiple point mutations.