Love-wave bacteria-based sensor for the detection of heavy metal toxicity in liquid medium.

The present work deals with the development of a Love-wave bacteria-based sensor platform for the detection of heavy metals in liquid medium. The acoustic delay-line is inserted in an oscillation loop in order to record the resonance frequency in real-time. A Polydimethylsiloxane (PDMS) chip with a liquid chamber is maintained by pressure above the acoustic wave propagation path. Bacteria (Escherichia coli) were fixed as bioreceptors onto the sensitive surface of the sensor coated with a polyelectrolyte (PE) multilayer using a simple and efficient layer-by-layer (LbL) electrostatic self-assembly procedure. Poly(allylamine hydrochloride) (PAH cation) and poly(styrene sulfonate) (PSS anion) were alternatively deposited so that the strong attraction between oppositely charged polyelectrolytes resulted in the formation of a (PAH-PSS)(n)-PAH molecular multilayer. The real-time characterization of PE multilayer and bacteria deposition is based on the measurement of the resonance frequency perturbation due to mass loading during material deposition. Real-time response to various concentrations of cadmium (Cd(2+)) and mercury (Hg(2+)) has been investigated. A detection limit as low as 10(-12) mol/l has been achieved, above which the frequency increases gradually up to 10(-3) mol/l, after a delay of 60 s subsequent to their introduction onto bacterial cell-based biosensors. Beyond a 10(-3) mol/l a steep drop in frequency was observed. This response has been attributed to changes in viscoelastic properties, related to modifications in bacteria metabolism.

Real time device for biosensing: design of a bacteriophage model using love acoustic waves.

Love wave sensors (ST-cut quartz substrate with interdigital transducers, SiO(2) guiding layer and sensitive coating) have been receiving a great deal of attention for a few years. Indeed, the wave coupled in a guiding layer confers a high gravimetric sensitivity and the shear horizontal (SH) polarization allows to work in liquid media. In this paper, an analytical method is proposed to calculate the Love wave phase velocity and the gravimetric sensitivity for a complete multilayer structure. This allows us to optimize the Love wave devices design in order to improve their gravimetric sensitivity in liquid media. As a model for virus or bacteria detection in liquids (drinking or bathing water, food em leader ) we design a model using M13 bacteriophage. The first step is the anti-M13 (AM13) monoclonal antibody grafting, on the device surface (SiO(2)). The second step is an immunoreaction in between the M13 bacteriophage and the AM13 antibody. The Love wave device allows to detect in real time the graft of the AM13 sensitive coating, as well as the immobilization of the M13 bacteriophages. With a pH change, the M13 bacteriophages can be removed from the sensor surface, in order to be numerated as plaque forming unit (pfu). Results on the sensitivity of Love waves are compared with similar immunological works with bulk acoustic wave devices, and demonstrate the high potentialities of Love waves sensors.

Sequence variations in the amino- and carboxy-terminal parts of the surface envelope glycoprotein of HTLV type 1 induce specific neutralizing antibodies.

The surface envelope glycoprotein gp46 of the human T cell leukemia virus type 1 elicits a strong immune response. Its protective role against HTLV-1 infection in animal models is well established, suggesting that recombinant envelope glycoproteins or synthetic peptides could be used as an effective vaccine. However, reports have indicated that some variations in envelope sequences may induce incomplete cross-neutralization between HTLV-1 strains. To identify amino acid changes that might be involved in induction of specific neutralizing antibodies, we studied sera from three patients (2085, 2555, and 2709) infected by HTLV-1 with surface glycoprotein gp46 harboring variations in amino acid sequence at positions 39, 72, 265, and 290. Inhibition of syncytia induced by parental, chimeric, or point-mutated envelope proteins indicated that sera 2555 and 2709 primarily recognized neutralizable epitopes located in N- and C-terminal parts of the gp46 glycoprotein. Amino acids changes at positions 39, 265, and 290 greatly impaired recognition of neutralizing epitopes recognized by these two sera. These results demonstrate that amino acid changes in envelope glycoprotein gp46 can induce strain-specific neutralizing antibodies in some patients. On the other hand, the neutralizing activity of serum 2085 was not affected by amino acid changes at positions 39, 265, and 290, suggesting that the neutralizing antibodies present in this serum were directed against epitopes located in other parts of the molecule, possibly those located in the central domain of the molecule, which has the same amino acid sequence in the three viruses.

Human macrophage tumor necrosis factor (TNF)-alpha production induced by Trypanosoma brucei gambiense and the role of TNF-alpha in parasite control.

Trypanosoma brucei gambiense, a causative agent of sleeping sickness, induced a dose-dependent production of tumor necrosis factor (TNF)-alpha by human macrophages in vitro. TNF-alpha was also induced in the Mono Mac 6 cell line, which indicates a direct effect of parasite components on macrophages. Parasite-soluble factors were also potent inducers of TNF-alpha. The addition of anti-TNF-alpha to cocultures of macrophages and parasites increased the number of trypanosomes and their life span, whereas irrelevant antibodies had no effect. TNF-alpha may have a direct role (i.e., direct trypanolytic activity) and/or an indirect one, such as TNF-alpha-mediated induction of cytotoxic molecules. A direct dose-dependent lytic effect of TNF-alpha on purified parasites was observed. This lytic effect was inhibited by anti-TNF-alpha. These data suggest that, as in experimental trypanosomiasis, TNF-alpha is involved in parasite growth control in human African trypanosomiasis.

High variability of HTLV-I in a remote population of Gabon as compared to that of a similar population of French Guiana.

An anomalous high frequency of ATL was observed in a remote 'noir maroons' village of French Guiana. Since it is not clear if HTLV-I is responsible for different frequencies of disease in different geographical areas, we undertook a comparison of the population with a similar one located in Gabon. We found a much higher degree of gp46 surface envelope glycoprotein sequence conservation in the Guianese village than in the Gabonese one.

Amino acid changes at positions 173 and 187 in the human T-cell leukemia virus type 1 surface glycoprotein induce specific neutralizing antibodies.

The nucleotide sequence of human T-cell leukemia virus type 1 (HTLV-1) is highly conserved, most strains sharing at least 95% sequence identity. This sequence conservation is also found in the viral env gene, which codes for the two envelope glycoproteins that play a major role in the induction of a protective immune response against the virus. However, recent reports have indicated that some variations in env sequences may induce incomplete cross-reactivity between HTLV-1 strains. To identify the amino acid changes that might be involved in the antigenicity of neutralizable epitopes, we constructed expression vectors coding for the envelope glycoproteins of two HTLV-1 isolates (2060 and 2072) which induced human antibodies with different neutralization patterns. The amino acid sequences of the envelope glycoproteins differed at four positions. Vectors coding for chimeric or point-mutated envelope proteins were derived from 2060 and 2072 HTLV-1 env genes. Syncytium formation induced by the wild-type or mutated envelope proteins was inhibited by human sera with different neutralizing specificities. We thus identified two amino acid changes, I173-->V and A187-->T, that play an important role in the antigenicity of neutralizable epitopes located in this region of the surface envelope glycoprotein.

Nucleotide sequence analysis of human T-cell lymphotropic virus type I pX and LTR regions from patients with sicca syndrome

Human T-cell lymphotropic virus type I (HTLV-I) is associated with adult T-cell leukemia (ATL) and tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM). Other inflammatory disorders may occur in HTLV-I-infected patients, such as sicca syndrome resembling Sjogren's syndrome. The sicca syndrome may be the unique clinical manifestation of HTLV-I infection, but is associated frequently with TSP/HAM, which could suggest that sicca syndrome might be an early event in disease progression to TSP/HAM in some cases. We investigated whether peculiar pX and LTR mutations could be related to sicca syndrome, or might argue the existence of clinical progression to TSP/HAM. pX, especially pX(I), pX(II), and pX(IV) ORFs corresponding to Tax cytotoxic T-lymphocyte epitopes, and LTR regions from Caribbean patients who have sicca sydrome with or without TSP/HAM, ATL patients, and healthy carriers were sequenced. The sequences were aligned and compared with ATK-1 prototype and published sequences. LTR sequences exhibited 1.5-2.4 percent of divergence with ATK-1. pX-sequenced regions showed a lower homology within p12(I) encoding sequences. Only few mutations were found within functionally important regions, but were not associated specifically with the clinical status. Finally, no existence of clinical progression to TSP/HAM were found. It would be of interest to study the clinical evolution of HTLV-I-sicca syndrome in patients and to determine HTLV-I sequences from peripheral blood and salivary glands at different stages. Copyright 1999 Wiley-Liss, Inc.(Au)

Nucleotide sequence analysis of human T-cell lymphotropic virus type I pX and LTR regions from patients with sicca syndrome.

Human T-cell lymphotropic virus type I (HTLV-I) is associated with adult T-cell leukemia (ATL) and tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM). Other inflammatory disorders may occur in HTLV-I-infected patients, such as sicca syndrome resembling Sjögren's syndrome. The sicca syndrome may be the unique clinical manifestation of HTLV-I infection, but is associated frequently with TSP/HAM, which could suggest that sicca syndrome might be an early event in disease progression to TSP/HAM in some cases. We investigated whether peculiar pX and LTR mutations could be related to sicca syndrome, or might argue the existence of clinical progression to TSP/HAM. pX, especially pX(I), pX(II), and pX(IV) ORFs corresponding to Tax cytotoxic T-lymphocyte epitopes, and LTR regions from Caribbean patients who have sicca syndrome with or without TSP/HAM, ATL patients, and healthy carriers were sequenced. The sequences were aligned and compared with ATK-1 prototype and published sequences. LTR sequences exhibited 1.5-2.4% of divergence with ATK-1. pX-sequenced regions showed a lower homology within p12(I) encoding sequences. Only few mutations were found within functionally important regions, but were not associated specifically with the clinical status. Finally, no mutations that could be related to sicca syndrome or argue the existence of clinical progression to TSP/HAM were found. It would be of interest to study the clinical evolution of HTLV-I-sicca syndrome in patients and to determine HTLV-I sequences from peripheral blood and salivary glands at different stages.

Different HTLV-I neutralization patterns among sera of patients infected with cosmopolitan HTLV-I.

To determine if sequence variations observed in cosmopolitan HTLV-I interfered with viral recognition by neutralizing antibodies, we evaluated the neutralization potential of sera from persons infected by HTLV-I of this clade selected for amino acid changes in their eny glycoproteins. Each serum was used to neutralize three previously described HTLV-I isolates, 2060, 2072, and 1010, that possess amino acid env sequences differing at several positions, one of them being located in the immunodominant and neutralizable domain (aa 187-199). The results obtained in syncytia and/or reporter gene inhibition assays showed that the neutralization pattern of the sera clearly differed and could be classified in three categories. Five sera completely neutralized the three viruses with an equivalent titer, two sera gave a maximum inhibition, with higher ID50 on the 2072 virus than on the 2060 or 1010 viruses, and three sera had a stronger neutralization potential toward the 1010 virus than toward the 2060 virus. One of these sera partially neutralized the virus produced by 2072 cells, whereas neutralizing antibodies in the other two recognized the neutralizable epitopes on the 1010 or 2072 viruses equally well. Identification of amino acid sequences involved in induction of neutralizing antibodies with different recognition capacities could help identify new neutralizable epitopes of HTLV-I envelope glycoproteins and to better define the component(s) of an effective vaccine.

HTLV-I associated sicca syndrome in Guadeloupe: lack of relation with a peculiar encoding sequence of surface envelope glycoprotein.

We previously reported a strikingly high prevalence of ocular diseases in HTLV-I infected patients in Guadeloupe (Caribbean basin). We sequenced the surface envelope encoding region of 7 HTLV-I proviruses from guadeloupean patients (5 with sicca syndrome, 2 with TSP/HAM). No relation between sequence and disease was observed. These 7 sequences are the first described from Guadeloupe.

Screening of phage-displayed antibody libraries.

The problem of amplifying a specific antibody in a population of millions of other antibodies has been solved by the immune system using the process of clonal selection Binding of an antigen to an IgM receptor on the surface of B-lymphocytes stimulates the proliferation and differentiation of the lymphocyte until it matures to an IgG-producing plasma cell. To mimic the first step of this process in bacteria, vectors have been constructed for the expression of antibodies on the surface of bacteria and phages (for review see Chapter 32 ).

Method for the purification of tissue DNA suitable for PCR after fixation with Bouin's fluid. Uses and limitations in microsatellite typing.

Paraffin-embedded tissues are often the only available material to perform polymerase chain reaction (PCR)-based analysis in various medical purposes. Unfortunately, the use in many countries of acid fixatives such as Bouin's fluid limits the use of such a material for molecular analysis. This article reports the methodological details of a DNA purification technique from Bouin-fixed and paraffin-embedded samples based on a double washing, in an alcohol then in an aqueous medium, of the DNA, which enables PCR reactions from this material. Comparison of the results with those obtained by organic solvent purification of DNA from frozen tissue fragments showed excellent reproducibility in terms of detection of an amplification product on agarose gel. However, differences between the methods were quite frequently seen in the allelic typing profile of microsatellite sequences (CA repeats), either as neo-alleles or by the loss of normal alleles in the fixed materials that constitute a limitation in using DNA from Bouin-fixed tissue as a substrate for fine allelotyping.

Neutralizing activity and antibody reactivity toward immunogenic regions of the human T cell leukemia virus type I surface glycoprotein in sera of infected patients with different clinical states.

The induction of specific neutralizing antibodies is an important part of vaccine strategy against human T cell leukemia virus type I (HTLV-I). A recently developed reporter gene induction assay was used to detect and quantify neutralizing antibodies in sera of HTLV-I-infected patients with different clinical states: Most sera (73/89) displayed an inhibitory activity. Neutralizing antibodies were more frequently detected in sera of patients with tropical spastic paraparesis/HTLV-associated myelopathy (TSP/HAM) or sicca syndrome (SS) (100%) than in sera of patients with adult T cell leukemia (ATL; 50%) or of asymptomatic carriers (AS; 83%). The mean titers in the different groups were significantly different (ATL < AS < TSP/HAM and SS). The antibody reactivity detected by the reporter gene inhibition assay was significantly related to the recognition of the neutralizable immunodominant domain (aa 175-199) of the surface envelope glycoprotein, indicating the importance of this region for potential vaccines.

Establishment of HTLV-I-infected cell lines from French, Guianese and West Indian patients and isolation of a proviral clone producing viral particles.

Human T-cell leukemia virus (HTLV-I) induces adult T cell leukemia/lymphoma (ATL) and a chronic neurological disease named either tropical spastic paraparesis (TSP) or HTLV-I associated myelopathy (HAM). We report here the establishment and characterization of eight HTLV-I-infected lymphoid cell lines derived either from patients with TSP (5) or from asymptomatic carriers (1). Southern blot analysis of T cell beta chain gene rearrangements indicates that all cell lines are composed of clonal populations. The same type of analysis performed with HTLV-I-specific probes showed that they harbor 1 to 5 copies of full length proviruses often associated with deleted proviruses with a restriction map for BamHI, HindIII, PstI and SacI restriction enzymes resembling those of HTLV-I previously isolated from Japan and Caribbean area. One of the cell lines, 2060, derived from a TSP patient was shown to express a relative large amount of virus easily transmissible to fresh peripheral and cord blood lymphocytes. The full length proviral genome contained in this cell line was cloned and used in transient expression experiments. We showed that the cloned provirus was able to direct the synthesis of the major structural viral proteins, the protease and the tax and rex regulatory proteins. The structural viral proteins could be assembled into free particles detected in the culture medium of transfected cells. Although the infectivity of these viral particles remains to be determined, this new clone can be employed to examine the cell types in which this TSP-derived provirus directs viral protein synthesis and eventually replicates. It should also prove of value in studies on the early cellular events induced by viral products.

The presence of particles resembling human T-cell leukemia virus type I at ultrastructural examination of lymphomatous cells in a case of T-cell leukemia/lymphoma.

BACKGROUND: Cases of adult T-cell leukemia/lymphoma (ATLL) resulting from human T-cell leukemia virus type I (HTLV-I) have been observed mainly in the southern part of Japan. Recently, the authors performed a second examination of cutaneous, muscle, and nerve biopsy specimens from a French white woman who died of ATLL in 1979. METHODS: A 67-year-old white woman had a lymphoma diagnosed on a lymph node biopsy. She then had acute pains and a thickened skin on both legs. Blood examination showed a leukocyte count of 16,000/ml with 75% leukemia T-cells. Biopsies were performed on the antero-external surface of the right leg. She died after 2 years of illness. RESULTS: Lymphomatous infiltrates of T-cell origin were seen in the dermis, between muscle fibers, and in a peripheral nerve. The recent ultrastructural examination of a few vacuoles located in the cytoplasm of certain lymphomatous cells showed rounded structures mixed with larger virus-like formations having a central nucleoid and spike material around the envelope. Polymerase chain reaction experiments performed on deparaffinized sections demonstrated the presence of a tax sequence homologous to that of HTLV-I. Other structural genes were not detected. CONCLUSIONS: These results contrast with other ultrastructural studies in which HTLV-I was detected only after cultivation of leukemia cells from patients with ATLL. This case probably resulted from an HTLV-I variant.

A new leukemogenic retrovirus isolated from tumor cells derived from a radio-induced lymphoma of C57BL/6 mice: analysis of the env and LTR sequences.

We report the cloning and characterization of a new ecotropic provirus encountered in a radio-induced thymic lymphoma of the C57BL/6 mouse. The provirus with an abnormally long LTR was inserted in the chromosomal DNA within the Pvt-1/MLVi-1/Mis-1 region which is a common integration site for MCF virus in mice and for Mo-MuLV in rats. This new ecotropic provirus was molecularly cloned and found to be infectious and competent for replication after transfection of murine cells. The recovered virus termed T3651/B was B-ecotropic, T-lymphotropic (in vivo) and highly leukemogenic for newborn C57BL/6 mice and for adult mice provided they were submitted to a subleukemogenic dose of irradiation. As compared to the AKV prototype N-ecotropic endogenous retrovirus, the T3651/B env proteins are only affected by few scattered point mutations. In contrast, the LTR has five repeats of enhancer sequences containing consensus motifs specific of the nuclear factors NF1-like, LVa, LVb and SEF1. Since a virus with such properties was encountered only once in 31 radio-induced tumors and isolated at a fourth tumor passage, a direct role of T3651/B virus in tumor genesis after irradiation is uncertain. Nevertheless, it is clear that T3651/B virus is a new leukemogenic retrovirus with a particular LTR structure which fits well with the model proposed by Rassart et al. (J. Virol. 58, 96-106, 1986) for the emergence of a thymotropic highly leukemogenic RadLV.

Murine thymic lymphomas after infection with a B-ecotropic murine leukemia virus and/or X-irradiation: proviral organization and RNA expression.

The role of retroviruses in murine radioleukemogenesis was reinvestigated using a protocol associating the injection of a non-pathogenic retrovirus (T1223/B virus) and a subleukemogenic dose of X-radiation (2 X 1.75 Gy). Using the Southern blotting technique we studied MuLV proviral organization and RNA expression in thymic lymphomas induced by the combined effect of virus and irradiation or irradiation alone. A recombinant provirus was detected in the chromosomal DNA of every tumor induced by associating virus and radiation whereas it was unconstantly found in radio-induced tumors. In every instance, the provirus was not integrated at a common site. No relationship was observed between viral RNA expression and tumor induction. Trisomy 15 was observed in all metaphases irrespective of the protocol of tumor induction. The G-banding technique revealed an extra-band in several thymic lymphomas induced by irradiation and T1223/B virus injection.