Analysis of the variable antigen composition of Trypanosoma brucei brucei metacyclic trypanosomes using monoclonal antibodies

The metacyclic trypanosomes of a Trypanosoma brucei brucei clone (ILTat 2.1) were analysed with regard to their variable antigen (VAT) composition using monoclonal antibodies. The metacyclic population was antigenically heterogeneous. Despite the heterogeneity, however, the overall VAT composition of the metacyclic population appeared to be limited in number. A similar pattern of reactivity was observed when the monoclonal antibodies were tested on metacyclics of another clone (IL Tat 2.2) derived from a rabbit 30 days after infection with IL Tat 2.1 as well as those of the parent stock (STIB 247). The VAT characteristics of the metacyclics of this serodeme were consistent regardless of whether they were transmitted by Glossina morsitans morsitans or G.m. centralis. The monoclonal antibodies also reacted with some of the bloodstream VATs isolated within 72 h from cyclically infected mice. None of the monoclonal antibodies, however, reacted with metacyclics of a different stock (LUMP 227).     

Filaments of Trypanosoma brucei: some notes on differences in origin and structure in two strains of Trypanosoma (Trypanozoon) brucei rhodesiense.

Filaments attached to trypanosomes of two strains of T. (T.) brucei were studied by electron microscopy and two distinct types identified: short-thick and long-thin. The former are associated with stumpy trypanosomes and are secretions, via the flagellar pocket, which originate in the area of the Golgi complex, during the infection of the host. They are referred to as 'secretory filaments'. Their diameter is 0.09 to 0.14 mum. The long-thin filaments are associated with slender forms of trypanosome in various artificial situations; those shown by negative staining are believed to be cytoplasmic extrusions from the anatomically weak extremities of the parasite and are referred to as 'plasmanemes'. Their diameter is 0.06 mum. Both types appear to maintain their structure without the aid of the normal type of unit membrane as myelin formations.     

Susceptibility of severe combined immuno-deficient (SCID) mice to Trypanosoma brucei gambiense and T. b. rhodesiense

Susceptibility of severe combined immunodeficient (SCID) mice to 7 isolates of Trypanosoma brucei gambiense and 2 isolates of T. b. rhodesiense was examined in terms of their infectivity, course of parasitaemia, packed cell volume (PCV) and survival period in comparison with that of normal immunocompetent (BALB/c) mice. All isolates of T. b. gambiense and T. b. rhodesiense caused high (> 1 × 10⁸ parasites/ml) parasitaemia in the SCID mice, the survival periods ranged from 5 to 47 days. On the other hand, 5 of 7 isolates of T. b. gambiense developed chronic infection in the BALB/c mice with sporadic but persistent parasitaemia with less than 5 × 10⁶ parasites/ml. All the mice tested in this group survived more than 60 days after infection. In contrast, the 2 remaining isolates of T. b. gambiense and both isolates of T. b. rhodesiense showed high virulence in the BALB/c mice and killed all of them within 30 days after infection. The results demonstrate that the SCID mice, in which functional B- and T-cell-mediated immunities are congenitally lacking, are highly susceptible for 'low-virulence' T. b. gambiense . This makes SCID mice useful tools for the isolation of parasites from T. b. gambiense sleeping sickness patients and the propagation of large amounts of such parasites.     

Detection of Trypanosoma brucei rhodesiense in animals from sleeping sickness foci in East Africa using the serum resistance associated (SRA) gene

The human serum resistance associated (SRA) gene has been found exclusively in Trypanosoma brucei rhodesiense, allowing the unequivocal detection of this pathogen in reservoir hosts and the tsetse vector without recourse to laborious strain characterisation procedures. We investigated the presence of the SRA gene in 264 T. brucei ssp. isolates from humans, domestic animals and Glossina pallidipes from foci of human trypanosomiasis in Kenya and Uganda. The SRA gene was present in all isolates that were resistant to human serum, and absent from all serum sensitive isolates tested. Further, the gene was present in all isolates that had previously been shown to be identical to human infective trypanosomes by isoenzyme characterisation. The SRA gene was detected in isolates from cattle, sheep, pigs, dog, reedbuck, hyena and G. pallidipes from sleeping sickness foci, but was not found in Trypanosoma evansi or in Trypanosoma brucei gambiense isolates. The present study indicates that the SRA gene may be invaluable in detecting and differentiating T. brucei rhodesiense from other T. brucei ssp. in reservoir hosts and tsetse.     

应用单克隆抗体(McAb)技术分析鼻疽菌与类鼻疽菌表面抗原的初步研究

用7个抗鼻疽菌的单克隆抗体初步分析研究了17株鼻疽菌与29株类鼻疽菌的表面抗原。发现(1)鼻疽菌表面抗原的表达比较稳定,大多数菌株趋于一种类型;(2)类鼻疽菌的表达变异性较大,用单克隆抗体2A5和1A9可将其分成三个群,用单克隆抗体1A10和4D4又能将其中的一群和二群再各分成二个亚群;1a、1b和2a、2b;(3)只有1a亚群与鼻疽菌表面抗原的表达类型交叉,类鼻疽菌与鼻疽菌的鉴别率达到86.15％。     

Production of human monoclonal IgG antibodies against Rhesus (D) antigen.

An Epstein-Barr virus (EBV)-transformed human B-cell line ( LB4r ) producing anti-Rhesus [Rho(D) antigen] antibody was fused with a non-immunoglobulin-producing mouse-human heteromyeloma ( SHM - D33 ) and selected in hypoxanthine/aminopterin/thymidine medium containing 0.5 microM ouabain. Surviving hybrids found to secrete specific anti-Rho(D) antibody were cloned by limiting dilution. Two clones (D4-B2 and E10-C1) producing high levels (12 and 20 micrograms/ml per 10(6) cells per 24 hr, respectively) of monospecific antibody (IgG3, lambda chain) were selected for expansion and further characterization. Compared to the parental cell line ( LB4r ), these hybridoma cell lines presented several advantages: antibody production was increased 10-fold, cloning efficiency was improved, and the EBV genome was not retained. Antibody production has been stable for greater than 8 months. These human monoclonal anti-Rho(D) antibodies have demonstrated utility in routine blood-group typing. They may also prove useful in the biochemical and genetic characterization of the Rh antigen system. Most important, they offer a source of Rh-immune globulin for the prevention of Rh immunization and alloimmune hemolytic disease of the newborn.     

Absence of the LiTat 1.3 (CATT antigen) gene in Trypanosoma brucei gambiense stocks from Cameroon.

Antibodies to the variable antigen type (VAT) designated LiTat 1.3 are common in sera from parasitologically confirmed patients with gambian sleeping sickness. For this reason, LiTat 1.3 has been considered a suitable antigen for detecting Trypanosoma brucei gambiense in the Card Agglutination Test for Trypanosomiasis (CATT; Testryp-CATT, Smith Kline-RIT). However, surveys in the T.b. gambiense endemic focus of Fontem in Cameroon have suggested that expression of LiTat 1.3 might be rare or absent. We show here that the gene for LiTat 1.3 was indeed absent from some T.b. gambiense stocks isolated from this focus, and a LiTat 1.3-like gene was present in others. The divergent gene differed from the cloned version of LiTat 1.3. In addition, antibodies to LiTat 1.3 could not be detected in rabbits infected with either of the two kinds of T.b. gambiense from the Fontem area. We suggest that the absence of LiTat 1.3 expression in this focus may have important implications for the epidemiology and control of sleeping sickness, especially if heavy reliance is placed on the CATT.     

Characterization of murine monoclonal antibodies against the common acute leukemia antigen (CALLA)

This study presents two murine monoclonal antibodies which react with the Common Acute Lymphoblastic Leukemia Antigen (CALLA). Both antibodies can be used for the diagnosis of common ALL (cALL). Indirect immunofluorescence studies (FACS-analysis) showed that the antibodies react with granulocytes and different human cell lines (Nalm-6, Reh, Raji, CCRF-CEM). The monoclonal antibodies BL-CALLA/1 and BL-CALLA/2 identify a single polypeptide chain of 95 kD. Both antibodies recognize the same or closely related epitope of the CALLA-molecule and are able to modulate in vitro the antigen on the CALLA-positive cell line Reh.     

The story of CGP 40 215: studies on its efficacy and pharmacokinetics in African green monkey infected with Trypanosoma brucei rhodesiense

CGP 40 215 is an inhibitor of S-adenosylmethionine decarboxylase, a key enzyme in trypanosomal polyamine biosynthesis. It is highly active against Trypanosoma brucei rhodesiense and T. b. gambiense in vitro and in the corresponding rodent models, and therefore was a promising candidate for further development as a new drug against human African trypanosomiasis. We conducted initial pharmacokinetic and efficacy studies in African green monkeys: based on two dose-finding studies, an infection-treatment and a pharmacokinetic study in eight monkeys infected with T. b. rhodesiense in the 1st stage of infection. PK analysis revealed curative drug levels in the serum but complete absence of the drug in the cerebrospinal fluid. No adverse effects of the drug were observed, although in rats CGP 40 215 had caused hypotension. The following PK parameters were calculated using a two-compartment model: t1/2=1.8 h, VSS/f=0.4 l/kg, CL/f=3.0 ml/min x kg and AUC=21 900 ng x h/ml. Six of the eight monkeys were cured, one animal relapsed on day 222 and one animal died of unknown reasons, but was aparasitaemic. The study confirmed the curative potential of CGP 40 215 for 1st stage T. b. rhodesiense infection. Unfortunately, it was also found that the compound did not pass the blood-brain barrier, a pre-requisite for cure of 2nd stage (CNS) infection. As the majority of sleeping sickness patients seeking treatment are in the 2nd stage of the disease, further development of the compound was stopped.     

Characterization of a membrane antigen from rabbit testis and sperm isolated by using monoclonal antibodies and effect of its antiserum on fertility.

An antigen was isolated from deoxycholate-solubilized rabbit testis and sperm by using an immunosorbent column containing IgG from a monoclonal antibody (8C10.5) that inhibits fertility. Elution was by stepwise increases in pH (8.0, 10.0, and 11.4), with the pH 11.4 fraction after recycling through the column showing a single band at 63 kilodaltons in slab NaDodSO4/PAGE with a silver stain. The antigen molecule was composed of two subunits, which on two-dimensional PAGE showed many spots within the same molecular size range (50-70 kilodaltons) but differing in charge. The antigen isolated either from testis or sperm showed mainly the same spots. The antigen is periodic acid/Schiff positive and contained 21% carbohydrate. An asialo-derivative of the antigen did not change its characteristics on NaDodSO4/PAGE. This glycoprotein resolved into two types of polypeptides, those binding and those not binding to a lens culinaris lectin column; some of the polypeptides appeared common to both fractions. Murine antiserum against the antigen neither agglutinated nor immobilized rabbit sperm but in immunofluorescence reacted with the plasma membrane of viable rabbit sperm as well as with murine and human sperm. Fertilization of female rabbits inseminated with treated sperm was not affected, but, by 9 days, fertility was significantly reduced (21% of controls). The postfertilization antifertility effect was not due to parthenogenic activation or to polyspermy. The antiserum reacted with one specific band in the one- and two-dimensional gel electrophoretic transfer blot procedure and was unaffected by absorption with different somatic tissues.     

Assay of factor VIII antigen (VIII:CAg) in 294 Haemophilia A patients by a new commercial ELISA using monoclonal antibodies

Monoclonal antibodies (MoAbs 833 and D4H1) directed against human factor VIII (FVIII) have been produced on a large scale to measure VIII:CAg by two-site ELISA (Asserachrom VIII:CAg, Diagnostica Stago). F(ab’)₂ from MoAb 833 were used for coating and bound VIII:CAg was revealed with MoAb D4H1 coupled to peroxidase. Control plasma (100 VIII:CAg U dL^?1 by comparing with the International Standard) was used as reference. The assay sensitivity was 0.1 U dL^?1 VIII:CAg. No apparent effect of the plasma proteins was observed provided plasma dilution was 5. Thus this ELISA allowed us to estimate VIII:CAg levels of 0.5 U dL^?1 in plasma. Levels of VIII:CAg were similar to those of VIII:C (correlation coefficient r= 0.87) in plasma from normal individuals (32 cases) and in patients with von Willebrand disease of various types (30 cases). Among 294 patients with Haemophilia A (HA), 161 had severe HA (VIII:C < 1 U dL^?1). Among those patients, 124 were cross-reacting material (CRM) negative with undetectable VIII:CAg and 37 were CRM+ (VIII:CAg 1– 31 U dL^?1). In 42 patients with moderate HA (VIII:C 1– 5 U dL^?1), 33 were CRM reduced (VIII:CAg 0.5–8 U dL^?1) and nine were CRM+ with a VIII:CAg/VIII:C ratio of 6–91 (mean 34.3). In mild HA (91 cases with VIII:C 6 U dL^?1), 29 patients were classified as CRM+ (VIII:C 6–57 U dL^?1, VIII:CAg 17–130 U dL^?1 and VIII:CAg/VIII:C ratio 1.8–13.7 (mean 4.51)). In 62 CRM reduced patients there was a linear correlation between VIII:C (6–39 U dL^?1) and VIII:CAg (2–36 U dL^?1) levels (r= 0.88). In conclusion, this sensitive assay allows us to distinguish the quantitative CRM reduced and negative from the qualitative (CRM+) abnormalities in Haemophilia A.     

Localization of factor VIII-procoagulant antigen: an immunohistological survey of the human body using monoclonal antibodies

Various organs, including liver, spleen, heart, lung, kidney, intestines, lymph nodes, pancreas, bone marrow, and thymus, were investigated for the presence of factor VIII-procoagulant antigen (VIIICAg) and factor VIII-related antigen (VIIIRAg), using a panel of monoclonal antibodies directed to factor VIII-von Willebrand factor in combination with a sensitive immunoperoxidase staining technique. In addition to hepatic sinusoidal endothelial cells, the presence of VIIICAg was demonstrated in mononuclear cells sporadically present in lymph nodes, in the alveolar septa of lung, and in the red pulp of spleen. The identity of these mononuclear cells could not be unequivocally determined. Based on morphological criteria, however, it is tentatively concluded that these cells are nonlymphoid and belong to the mononuclear phagocyte system. The presence of VIII-RAg was confined to vascular endothelial cells, hepatic sinusoidal endothelial cells, cells lining the venous sinuses of the red pulp of the spleen, cells lining renal glomeruli and lung capillaries, platelets, and megakaryocytes.     

Diagnosis of varicella-zoster virus (VZV) infection by using FITC-labeled monoclonal antibodies

Anti-varicella zoster virus (VZV) mouse monoclonal antibodies conjugated with fluorescein isothiocyanate were evaluated for their usefulness as a practical diagnostic tool in the clinical field by examining cells infected with isolated herpes viruses and 431 clinical samples. The kit stained clearly the cells infected with 14 isolated VZV strains without cross reaction to 15 isolated herpes simplex virus type-1 strains (HSV-1) and 14 type-2 (HSV-2) strains. In clinical specimens, viral antigens of VZV were detected in 92/105 (87.6%) cases of varicella and in 176/190 (92.6%) cases of herpes zoster. Specific fluorescence of VZV was also observed in 5 out of 96 cases diagnosed as HSV infections, although these samples had no specific reaction to HSV when tested by the commercially available diagnostic kit. In 24 cases which could not be clinically diagnosed as herpes zoster or herpes simplex, the VZV antigen was demonstrated in 9 cases. All 109 VZV-positive cases in virus isolation by culture were also judged VZV-antigen positive by the kit, while all 69 HSV-positive cases in virus isolation were VZV-antigen negative. Furthermore, the VZV antigen was detected by the kit in 53/60 clinical diagnoses of varicella or herpes zoster without successful virus isolation. These results clearly indicate the usefulness of the kit as a practical VZV diagnostic reagent, especially in terms of specific sensitivity and easy technical manipulation.