Growth of Porphyromonas gingivalis, Treponema denticola, T. pectinovorum, T. socranskii, and T. vincentii in a chemically defined medium.

A chemically defined medium, OMIZ (Oral Microbiology and Immunology, Zürich)-W1 was developed. Medium OMIZ-W1 supports the long-term proliferation of a wide range of oral anaerobes, including representative strains of four Treponema species and Porphyromonas gingivalis. High concentrations of ascorbic acid and ammonium ions proved to be important for the growth of these organisms. T. denticola CD-1 grew in the absence of polyamines and long-chain fatty acids, T. pectinovorum and T. socranskii required polyamines, whereas T. vincentii depended on both polyamines and lecithin for growth. Specific requirements for purines and/or pyrimidines were detected, and these requirements could be used to distinguish Haemophilus-Actinobacillus group organisms. Some strains of P. gingivalis grew without vitamin K, while others were not satisfied by menadione but required its precursor 1,4-dihydroxy-2-naphthoic acid. Protoporphyrin IX or hemin equally satisfied the porphyrin requirements of P. gingivalis and Bacteroides forsythus, whereas ferrous sulfate was more efficiently used as a source of iron than was hemin. The cellular cohesiveness of P. gingivalis increased with high concentrations of hemin in the growth medium. Prevotella intermedia, B. forsythus, and several strains of P. gingivalis were more fastidious and required a protein or serum supplement to grow in medium OMIZ-W1.     

Immuno-proteomic analysis of Trichinella spiralis,T. pseudospiralis,and T. papuae extracts recognized by human T. spiralis-infected sera

Parasitology Research - The present study explored potentially immunogenic proteins of the encapsulated (Trichinella spiralis) and non-encapsulated (T. pseudospiralis, T. papuae) species within the...     

A comparative study of the proteolytic enzymes of Trypanosoma brucei, T. equiperdum, T. evansi, T. vivax, Leishmania tarentolae and Crithidia fasciculata

Four types of proteolytic activity were detected in the bloodstream form of each of the four Trypanosoma species: (i) HPAase, active on hide powder azure and detected on polyacrylamide gels containing denatured haemoglobin; (ii) AZCase, active on azocasein; (iii) type 1, active on the chromogenic peptide N-benzoyl-L-prolyl-L-phenylalanyl-L-arginine p-nitroanilide in the presence of dithiothreitol, and (iv) type 2, active against several nitroanilide derivatives in the absence of dithiothreitol. Studies of the pH optimum, dithiothreitol requirement and inhibitor sensitivities of the proteolytic activities suggested that: (a) HPAase and type 1 activities could be due to the same enzymes, probably a family of cysteine proteinases; (b) AZCase had some characteristics of a cysteine proteinase, but was not identical to HPAase, and (c) type 2 activity could be due to a serine proteinase. Procyclic T. brucei contained relatively low cysteine proteinase activities (HPAase, AZCase and type 1) but high type 2 activity. Their proteolytic enzymes thus were apparently more similar to those in Crithidia fasciculata and Leishmania tarentolae promastigotes than those in T. brucei bloodstream forms.     

T2 lipopolysaccharide antigen of Salmonella: genetic determination of T2 and properties of the T2, T2,S, and T2,SR Forms.

The T2 antigenic form of Salmonella bareilly was examined. The absence of O specificity in this strain was shown to be due to its nonfunctional rfb genes; when the rfb gene cluster was replaced by the rfb cluster derived from smooth donor strains, T2,S and T2,SR recombinants were produced that expressed both T2 and either 0-6,7 or 0-4,12 specificity, depending on O antigen of the donor strain. The T2, T2,S, and T2,SR forms were all unstable on culture and segregated T2-negative forms (R, S, and SR, respectively) at a high rate. In all these respects the T2 antigen closely resembled the other T-form antigen, T1. The genes responsible for the T2 antigen, rfu, were not close to rfb, but their precise location and relation to rft (which determines T1 antigen) could not be discovered because of the instability to the T2 form and low recombination frequency in the necessary interspecies crosses.     

PCR identification of dermatophyte fungi Trichophyton rubrum, T. soudanense and T. gourvilii

Diagnosis of dermatophytosis employing conventional laboratory procedures has been complicated by the slow growth and varied morphological features shown by dermatophytes. After analysis of the nucleotide base sequences of a 1.2-kb fragment amplified from a dermatophyte fungus Trichophyton rubrum by arbitrarily primed PCR with random primer OPD18, a pair of primers (TRIF and TR1R) was designed and evaluated for specific identification of T. rubrum. The sensitivity of the primers TR1F and TR1R was high, as a specific PCR band of c. 600 bp was detected from as little as 7 pg of T. rubrum DNA. By examining 92 dermatophyte strains and clinical isolates, it was found that this pair of primers reacted in PCR with T. rubrum, T. soudanense and T. gourvilii through formation of the specific fragment of 600 bp, but not with any other of the dermatophyte species or varieties, fungi, yeasts or bacteria tested. As T rubrum is one of the most frequently isolated dermatophyte fungi, and T. soudanense and T. gourvilii are relatively uncommon in many parts of the world, these primers can be used for rapid, sensitive and specific identification and differentiation of T. rubrum from other fungi and micro-organisms.     

Comparative structural analysis of calmodulins from Trypanosoma brucei, T. congolense, T. vivax, Tetrahymena thermophila and bovine brain

Calmodulin is an intracellular calcium receptor protein utilized extensively by eukaryotic cells to mediate responsiveness to calcium signals. The present study evaluates the effects on protein structure of amino acid substitutions in trypanosome calmodulin. Calmodulin conformation, hydrophobicity and antigenic determinants are compared among Trypanosoma brucei, Trypanosoma congolense, Trypanosoma vivax, Tetrahymena thermophila and bovine brain. Trypanosome calmodulin differs from brain and Tetrahymena calmodulins based upon isoelectric point, retention time on a C-2/C-18 reverse phase column and interaction with polyclonal antibodies against trypanosome calmodulin by radioimmunoassay or Western procedures. These same analyses do not distinguish trypanosome calmodulins from each other. Polyclonal antibodies against Tetrahymena calmodulin are equally specific and do not recognize the trypanosome or brain calmodulins. Calcium-induced exposure of hydrophobic binding sites are quantitated using the fluorescent probe, N-phenyl-1-naphthylamine. All calmodulins, regardless of source, enhance the fluorescence of N-phenyl-1-naphthylamine 3-4 fold in the presence of calcium. These data demonstrate the extent to which functional calmodulins vary in their structures. We conclude that African trypanosomes share a common calmodulin that is structurally distinct from calmodulin of vertebrates or Tetrahymena.     

Immunization with recombinant actin from Trypanosoma evansi induces protective immunity against T. evansi, T. equiperdum and T. b. brucei infection

Actin gene of Trypanosoma evansi (STIB 806) was cloned and expressed in Escherichia coli. The predicted amino acid sequence of T. evansi actin shows 100%, 98.7%, and 93.1%, homology with Trypanosoma equiperdum, Trypanosoma brucei brucei, and Trypanosoma cruzi. Recombinant actin was expressed as inclusion bodies in E. coli. It was purified and renatured for immunological studies. Mice immunized with the renatured recombinant actin were protected from lethal challenge with T. evansi STIB 806, T. equiperdum STIB 818, and T. b. brucei STIB 940, showing 63.3%, 56.7%, and 53.3% protection, respectively. Serum collected from the rabbit immunized with recombinant actin inhibited the growth of T. evansi, T. equiperdum, and T. b. brucei in vitro cultivation. Serum from mice and rabbits immunized with recombinant actin only recognized T. evansi actin but not mouse actin. The results of this study suggest that the recombinant T. evansi actin induces protective immunity against T. evansi, T. equiperdum, and T. b. brucei infection and may be useful in the development of a vaccine with other cytoskeletal proteins to prevent animal trypanosomiasis caused by these three trypanosome species.     

Obituary: PROFESSOR T. R. TREVOR-JONES, OBE

Professor T. R. Trevor-Jones, former Professor and Head of the Department of General Anatomy, Faculty of Dentistry, University of the Witwatersrand, died in Gauteng on 19th September 1996.     

Epidermal T lymphocytes--ontogeny, features and function.

Conclusions The murine epidermis contains a network of Thy-1⁺ dendritic T cells. These T cells arise from early fetal stem cells and differentiate in the fetal or neonatal thymic or epidermal microenvironment. Their lack of expression of CD5, CD4, and CD8 antigens, as well as their virtually exclusive expression of a CD3/TCR V3/V1 complex, distinguishes DETC from the bulk of peripheral T cells.The early appearance of TCR / cells in ontogeny, the lack of expression of CD4 and CD8 antigens, and the relative paucity of and genes compared to and genes, indicates that / T cells provide a phylogenetically primitive, broadly acting, and poorly discriminating immunologic defense system. In this system, recognition of antigen is not restricted by classical MHC class I and class II antigens, but may occur in the context of relatively nonpolymorphic restricting elements, such as Qa [82], Tla [10] or CD1 [62]. This rather primitive immune system provided by DETC may serve to protect the epidermal integrity. Upon recognition of self proteins released following epidermal injury, DETC may become activated and assist in the removal of altered cells. In this limited fashion, the epidermis may be an independently competent immunologic system. However, the fact that the TCR repertoire of DETC does not allow for the recognition of antigenic peptides in conjunction with MHC moieties excludes the possibility that the diverse immune response elicited by topical contact with foreign antigens is mediated by DETC.Whether this statement also applies to the human epidermis cannot be answered at the present time. Let us consider a few plausible concepts concerning derivation and function of human epidermal T cells. First, one could postulate that in early ontogeny, the human epidermis harbors a small, indigenous population of naive T lymphocytes with monomorphic TCR representing an analogue to murine DETC. These cells could function in a manner similar to that proposed for murine DETC. They may even persist into adult life, so far undetected because they would be outnumbered by immigrating polymorphic T cells from peripheral lymphoid organs. Second, it is conceivable that the human epidermis contains an indigenous population of naive T lymphocytes with a polymorphic TCR repertoire representing a phylogenetically advanced analogue to murine DETC. Although equipped with TCR allowing antigen recognition in the context of MHC, their density is probably too low to make them an effective host defense system against the multitude of environmental antigens presented by Langerhans cells. One could rather assume that they proliferate upon recognition of self antigens occurring in a perturbed epidermis. The autoreactivity of these cells may not necessarily be beneficial. Finally, the fact that the entry of circulating HECA-452⁺ memory cells into the skin is dependent upon the injury-induced ELAM-1 expression by endothelial cells of the dermal microvasculature could indicate that all T cells present in adult human epidermis are recruited upon alteration of the skin. Following this reasoning, the human epidermis should not be regarded as a complete, self-sustaining immunologic organ but rather as a homing site for and a target of lymphocytes antigenically sensitized in peripheral lymphoid organs.