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M J Levin

Publications and source records attributed to M J Levin.

At least 37 records · Page 2Linked to original sources
PubMed2001

Frequency of acyclovir-resistant herpes simplex virus in clinical specimens and laboratory isolates.

The proportion of acyclovir (ACV)-resistant herpes simplex virus (HSV) isolates in clinical specimens and laboratory isolates was determined. HSV isolates in clinical specimens and laboratory isolates were cultured in the absence or presence of 5 microg of ACV per ml. The frequency of ACV-resistant HSV was calculated as (average virus titer in the wells with ACV)/(average virus titer in the wells without ACV). The mutation frequency of HSV type 1 isolates in clinical samples (directly from patient lesions) was 7.5 x 10(-4) +/- 2.5 x 10(-4) (mean +/- standard error), and that of HSV type 2 isolates was 15.0 x 10(-4) +/- 4.6 x 10(-4). The mutation frequencies of isolates derived in the laboratory from these clinical samples were very similar. Similarly, the 50% inhibitory concentrations for isolates in clinical samples and laboratory isolates were identical. The frequencies of ACV-resistant HSV types 1 and 2 were in a narrow range of 7.5 x 10(-4) to 15.0 x 10(-4) among isolates in clinical specimens and did not change for laboratory-derived pools of viral isolates.

Acyclovir↗
PubMed2000

The short interspersed repetitive element of Trypanosoma cruzi, SIRE, is part of VIPER, an unusual retroelement related to long terminal repeat retrotransposons.

The short interspersed repetitive element (SIRE) of Trypanosoma cruzi was first detected when comparing the sequences of loci that encode the TcP2beta genes. It is present in about 1,500-3,000 copies per genome, depending on the strain, and it is distributed in all chromosomes. An initial analysis of SIRE sequences from 21 genomic fragments allowed us to derive a consensus nucleotide sequence and structure for the element, consisting of three regions (I, II, and III) each harboring distinctive features. Analysis of 158 transcribed SIREs demonstrates that the consensus is highly conserved. The sequences of 51 cDNAs show that SIRE is included in the 3' end of several mRNAs, always transcribed from the sense strand, contributing the polyadenylation site in 63% of the cases. This study led to the characterization of VIPER (vestigial interposed retroelement), a 2,326-bp-long unusual retroelement. VIPER's 5' end is formed by the first 182 bp of SIRE, whereas its 3' end is formed by the last 220 bp of the element. Both SIRE moieties are connected by a 1,924-bp-long fragment that carries a unique ORF encoding a complete reverse transcriptase-RNase H gene whose 15 C-terminal amino acids derive from codons specified by SIRE's region II. The amino acid sequence of VIPER's reverse transcriptase-RNase H shares significant homology to that of long terminal repeat retrotransposons. The fact that SIRE and VIPER sequences are found only in the T. cruzi genome may be of relevance for studies concerning the evolution and the genome flexibility of this protozoan parasite.

Amino Acid Sequence↗
PubMed2000

Early diagnosis of recurrence of Trypanosoma cruzi infection by polymerase chain reaction after heart transplantation of a chronic Chagas' heart disease patient.

Heart transplantation is contraindicated as an effective treatment for end-stage Chagas' heart disease because of post-operative recurrence of Trypanosoma cruzi infection and reactivation of disease after immunosupression. In a follow-up study of a heart transplanted patient with Chagas' disease, we prospectively evaluated the usefulness of the polymerase chain reaction (PCR) for early diagnosis of reactivation. We monitored post-operative recurrence of Trypanosoma cruzi infection with microscopic observation of the parasite in peripheral blood (Strout's method), endomyocardial biopsies (EMBs), skin lesions, and 2 PCR assays, based on the amplification of specific T cruzi kinetoplastid and nuclear DNA sequences. During follow-up, parasite DNA was amplified in blood samples and EMB sections 41 days before we observed patent parasitemia and cutaneous manifestations of reactivation, proving that PCR is much more sensitive than direct microscopic observation for early diagnosis of disease reactivation in heart-transplanted Chagas' disease patients.

Animals↗
PubMed2000

Modulation of cardiocyte functional activity by antibodies against trypanosoma cruzi ribosomal P2 protein C terminus.

Antibodies against the Trypanosoma cruzi ribosomal P2beta protein (TcP2beta) have been associated with the chronic cardiac pathology of Chagas' disease in humans. Using synthetic peptides spanning the entire TcP2beta molecule, we investigated their epitope recognition by antibodies from mice chronically infected with T. cruzi and from mice immunized with two recombinant TcP2betas. We found clear differences in epitope recognition between antibodies from T. cruzi-infected mice and mice immunized with two different recombinant TcP2betas associated with different schedules of immunization. Major epitopes recognized by antibodies from mice immunized with recombinant glutathione S-transferase (GST) or histidine (Hist) fusion TcP2beta (GST-TcP2beta or Hist-TcP2beta) are located in the central and hinge regions of the molecule. Nevertheless, mice immunized with Hist-TcP2beta were also able to elicit antibodies against the TcP2beta C terminus, a region which is highly conserved in both T. cruzi and mammal ribosomal P proteins. Strikingly, antibodies from infected animals recognized only the TcP2beta C terminus. By using these antisera with distinct profiles of epitope recognition, it could be shown that only C terminus-specific antibodies were able to increase the beating frequency of cardiomyocytes from neonatal rats in vitro by selective stimulation of the beta1-adrenergic receptor. Thus, antibodies against the TcP2beta C terminus elicited in the absence of infection are able to modulate a functional activity of host cells through a molecular mimicry mechanism.

Amino Acid Sequence↗
PubMed1999

Functional analysis of the intergenic regions of TcP2beta gene loci allowed the construction of an improved Trypanosoma cruzi expression vector.

TcP2beta ribosomal protein genes in Trypanosoma cruzi are encoded by four different loci, H6.4, H1.8, H1.5 and H1.3. All loci have a similar organization, except for H1.8 that harbors two TcP2beta genes arranged in tandem and separated by a short repetitive sequence, named SIRE (short interspersed repetitive element), which is also found upstream of the first gene of the tandem and downstream of the second. In this locus the trans-splicing signal of TcP2beta is located within the SIRE element, while in the other loci it is positioned within the first 50bases upstream of the AUG with an AG acceptor site at position -12 respective to the initiation codon. Transient transfection experiments were used to evaluate the efficiency of these two different trans-splicing regions to drive CAT activity. The region named HX1 located upstream the TcP2beta H1. 8 gene was clearly more efficient than the SIRE sequence contained in the region named HX2. Therefore, we decided to use the HX1 region to ameliorate the performance of the cryptic trans-splicing signal present in the T. cruzi expression vector pRIBOTEX (Martinez-Calvillo, S., López, I., Hernandez, H., 1997. pRIBOTEX expression vector: a pTEX derivative for a rapid selection of Trypanosoma cruzi transfectants. Gene 199, 71-76). By insertion of the region HX1 downstream of the ribosomal promoter of pRIBOTEX, we constructed pRHX1CAT40 that, in stable transfected cells, was able to drive CAT activity 2760 times more efficiently than the control plasmids. Based on this, a novel plasmid vector was conceived, named pTREX-n, which retains the neo gene of pRIBOTEX as a positive selectable marker and replaces the CAT-SV40 cassette in pRHX1CAT40 by a multiple cloning site.

Animals↗
PubMed1999

Analysis of the distribution of SIRE in the nuclear genome of Trypanosoma cruzi.

The short interspersed repetitive element (SIRE) of the nuclear genome of Trypanosoma cruzi was first detected when comparing the sequences of loci that encode the TcP2beta genes. The present study was designed to assess its distribution and organization in the nuclear genome of the parasite. Southern blots of genomic DNA from different strains demonstrated that each one possesses a defined and characteristic pattern of SIRE distribution. The conservation of the SIRE sequence in T. cruzi strains allowed the development of a rapid inter-SIRE PCR reaction that yields strain-specific amplicon profiles. In the T. cruzi CL Brener clone, we found 1500 copies of the element distributed in all chromosomes. 16 genomic fragments containing SIRE (SZs) were isolated and characterized. In fragments SZ10, SZ12 and SZ31, SIRE was linked to TcRel, a novel repeated sequence that constitutes the 3' end of vp85 genes. SIRE was also linked to an unknown open reading frame in fragments SZ14 and SZ23 which might be related to the subtelomeric regions of T. cruzi chromosomes. Further sequencing of SZ fragments revealed that SIRE was also linked to protein coding genes that have not yet been described in kinetoplastids such as the one coding for PRP22 helicase and a thimet oligopeptidase. To allow the rapid-generation genetic markers associated with SIRE, we developed a SIRE-bubble PCR reaction that provided several such markers for the construction of the physical map of chromosome XVI. The results herein demonstrate that SIRE-associated sites (SAS) may be of great help in physical mapping and interpretation of T. cruzi genomic sequence data.

Animals↗
PubMed1999

Clinical practice guidelines for the management of chronic sinusitis in children.

Chronic sinusitis in children is a common and vexing disease for clinicians and the public. There are insufficient data in the literature to develop an evidence-based clinical guideline. Experience in managing pediatric chronic sinusitis has been gained through a multidisciplinary clinic at our institution during the past 3 years. A panel of experts was formed, and with the guidance of a guideline methodologist, the development of a rigorous outcome-based guideline was undertaken. Symptom-improvement and recurrence estimates for a variety of medical and surgical treatments were assessed. Wide probability estimates were made by the panelists in most cases. Although we refrained from making specific recommendations, we developed a ranked series of practical treatment options taking into account side effects and costs.

Anti-Bacterial Agents↗
PubMed1999

Physical mapping of a 670-kb region of chromosomes XVI and XVII from the human protozoan parasite Trypanosoma cruzi encompassing the genes for two immunodominant antigens.

As part of the Trypanosoma cruzi Genome Initiative, we have mapped a large portion of the chromosomal bands XVI (2.3 Mb) and XVII (2.6 Mb) containing the highly repetitive and immunodominant antigenic gene families h49 and jl8. Restriction mapping of the isolated chromosomal bands and hybridization with chromosome specific gene probes showed that genes h49 and jl8 are located in a pair of size-polymorphic homologous chromosomes. To construct the integrated map of the chromosomes harboring the h49 and jl8 loci, we used YAC, cosmid, and lambda phage overlapping clones, and long range restriction analysis using a variety of probes (i.e., known gene sequences, ESTs, polymorphic repetitive sequences, anonymous sequences, STSs generated from the YAC ends). The total length covered by the YAC contig was approximately 670 kb, and its map agreed and was complementary to the one obtained by long-range restriction fragment analysis. Average genetic marker spacing in a 105 kb region around h49 and jl8 genes was estimated to be 6.2 kb/marker. We have detected some polymorphism in the H49/JL8 antigens-encoding chromosomes, affecting also the coding regions. The physical map of this region, together with the isolation of specific chromosome markers, will contribute in the global effort to sequence the nuclear genome of this parasite.

Animals↗
PubMed1999

Evaluation of recombinant antigens for serodiagnosis of Chagas' disease in South and Central America.

The commercially available diagnostic tests for Chagas' disease employ whole extracts or semipurified fractions of Trypanosoma cruzi epimastigotes. Considerable variation in the reproducibility and reliability of these tests has been reported by different research laboratories, mainly due to cross-reactivity with other pathogens and standardization of the reagents. The use of recombinant antigens for the serodiagnosis of Chagas' disease is recommended to increase the sensitivity and specificity of serological tests. Expressed in Escherichia coli, as fusion products with glutathione S-transferase, six T. cruzi recombinant antigens (H49, JL7, A13, B13, JL8, and 1F8) were evaluated in an enzyme-linked immunosorbent assay for Chagas' disease. The study was carried out with a panel of 541 serum samples of chagasic and nonchagasic patients from nine countries of Latin America (Argentina, Bolivia, Brazil, Chile, Colombia, El Salvador, Guatemala, Honduras, and Venezuela). The optimal concentration of each recombinant antigen for coating of plates was determined with the help of 125I-labelled recombinant proteins. While the specificity of the epimastigote antigen was 84% because of false positives from leishmaniasis cases, for the recombinant antigens it varied from 96.2 to 99.6%. Recombinant antigens reacted with 79 to 100% of serum samples from chronic chagasic patients. In this way, it is proposed that a mixture of a few T. cruzi recombinant antigens should be employed in a diagnostic kit to minimize individual variation and promote high sensitivity in the diagnosis of Chagas' disease.

Animals↗
PubMed1999

Photoreceptor ultrastructure of the amphipod, Cyamus ceti (Linné, 1758), an ectoparasite of bowhead, right and gray whales.

Light and electron microscopic findings are presented for the first time on a species of amphipod whale louse, Cyamus ceti, with reference to the paired photoreceptor structures of the head. Whale lice are crustacean ectoparasites of large, slow moving whales. Twenty-two known species parasitize mainly baleens but also toothed whales. Samples were collected from Alaskan bowhead whales during an Eskimo hunt. The photoreceptors, or compound eyes, were sessile and located on the dorsal cephalon. Each photoreceptor contained approximately 50 visual ommatidial units. The general organization was similar to other amphipod crustacean compound eyes. The single ommatidial unit consisted of 1) an overlying cuticle, 2) crystalline cone secreted by cone cells, 3) a fusiform layered rhabdom, 4) surrounding pigmented retinular cells, and 5) basal lamina and axon. The cuticle over each eye was translucent, convex, and thinner than the rest of the cranial cuticle. The photosensitive rhabdom contained a central core of alternating microvillous projections, and the rhabdomere unit was formed by five or six retinular cells. There were numerous electron-dense and electron-lucent granules within the cytoplasm of the retinular cells, corresponding to visual pigment or reflecting granules. The eyes most likely play a role in detecting light direction, duration and/or intensity, which may direct molting, reproduction, or other functions of this cetacean ectoparasite.

Animals↗
PubMed1999

[Contribution of the Trypanosoma cruzi Genome Project to the understanding of the pathogenesis of Chagas disease].

The Trypanosoma cruzi Genome Project (TcGP) is developed by a consortium of laboratories brought together by two international agencies, WHO/TDR and CYTED. The TcGP is an important tool for the study of Chagas disease. It provides researchers and clinicians with information about expressed parasite genes (see TcGP database, http:@gene.dbbm.fiocruz.br) and with an important number of genomic libraries and probes (ESTs). We show with four examples how the TcGP contributes to the understanding of the pathogenesis of this infection. 1) We demonstrate how to derive antigens from ESTs, an important feature regarding the generation of anti-cardiac receptor antibodies in chagasic patients. 2) We introduce concepts such as proteome vaccinome and pathonome that include all techniques needed to analyze quickly the growing amount of genomic information. 3) We show that the genomic regions that have been already sequenced give clues as to the plastic nature of the parasite nuclear genome. Finally, 4) we show how the genome sequences can be used to study the parasite DNA that is associated to the cardiac lesions.

Animals↗
PubMed1998

Expression of a recombinant Fab antibody fragment against cruzipain, the major cysteine proteinase of Trypanosoma cruzi.

Cruzipain, the major proteinase of Trypanosoma cruzi, plays an important role in the biology of this parasite. This study reports the development of a recombinant Fab antibody, using RNA isolated from the anti-Ag163B6 hybridoma against cruzipain. This procedure involves the use of cDNAs obtained with the aid of a specific set of primers complementary to the complete light kappa chain (L kappa) and the first two domains of the IgG1 heavy chain (VH/CH1). These products were subsequently cloned in the pComb3 system, from which the gIII gene had been removed, and expressed in Escherichia coli cells. The recombinant Fab molecule recognized cruzipain by ELISA, in a fashion similar to the original mAb anti-Ag163B6. Nucleotide sequence analysis of the recombinant molecule, together with its immunological recognition by specific anti-mouse IgG (Fab)2, indicated the immunoglobulin nature of the recombinant product. Moreover, both the mAb anti-Ag163B6 and the soluble Fab fragment described here react similarly with the intact parasite surface, as observed in an indirect immunofluorescence assay. In conclusion, our recombinant Fab anti-Ag 163B6 allows the possible use of this molecule for diagnosis, antigen purification, and eventually treatment of Chagas-afflicted individuals.

Animals↗
PubMed1998

Mapping of B cell epitopes in an immunodominant antigen of Trypanosoma cruzi using fusions to the Escherichia coli LamB protein.

The JL8 protein antigen from Trypanosoma cruzi, a dominant immunogen in man, has been characterized as containing tandem amino acid repeats. Here, we describe the use of the LamB protein of Escherichia coli as a carrier of JL8 derived sequences in order to map the immunodominant B cell epitopes in this antigen. Five different sequences of JL8 were inserted in the LamB protein and the JL8-LamB fusion proteins were tested by ELISA with human chronic chagasic sera. The fusion carrying the sequence AEKQKAAEATKVAE was recognized by most sera. This protein was also capable of inhibiting the binding of human chagasic antibodies to GST-JL8 in competitive ELISA suggesting that it contains an immunodominant B cell epitope of JL8.

Animals↗
PubMed1998

Use of a live attenuated varicella vaccine to boost varicella-specific immune responses in seropositive people 55 years of age and older: duration of booster effect.

Varicella-zoster virus (VZV)-specific T cell immunity was measured in 130 persons > or = 55 years of age 6 years after they received a live attenuated VZV vaccine. Circulating T cells, which proliferated in vitro in response to VZV antigen, were enumerated (VZV responder cell frequency assay). Six years after the booster vaccination, the VZV-responding cell frequency (1/61,000 circulating cells) was still significantly (P < .05) improved over the baseline measurements (1/70,000) and appears to have diminished the expected decline in frequency as these vaccinees aged (to 1/86,000). Ten herpes-zoster--like clinical events were recorded. Although the frequency of these events, approximately 1/100 patient-years, is within the expected range of such events for this age cohort, the number of lesions was small, there was very little pain, and there was no postherpetic neuralgia. These results support the development of a vaccine to prevent or attenuate herpes zoster.

Aged↗
PubMed1998

Cytokine production in varicella-zoster virus-stimulated cultures of human blood lymphocytes.

Estimates of responder cell frequency (RCF) based on limiting dilution analyses are laborious, and alternative means to quantitate cell-mediated immunity to immunogens are desirable. It was shown that levels of interleukin (IL)-2 in the supernatant of varicella-zoster virus-stimulated blood lymphocytes from immune adults peaked at 48 h of culture and correlated partially with estimates of RCF (r = .74, P = .003). Levels of gamma-interferon, IL-4, and IL-10 increased through the first 4 days of culture, and gamma-interferon levels showed some correlation with peak IL-2 levels (r = .48, P = .03). Nevertheless, correlations between levels of these cytokines and RCF did not reach statistically significant levels.

Adult↗
PubMed1998

Cellular immunity to varicella-zoster virus in patients with major depression.

The incidence of herpes zoster increases markedly with advancing age, and this appears to be causally related to an age-dependent decline in varicella-zoster virus (VZV)-specific cellular immunity. Psychologic stress has also been linked to the occurrence of herpes zoster, but the mechanism involved has not been investigated. This study examined the relationship between major depression and VZV-specific cellular immunity by comparing VZV-specific responder cell frequency (RCF) in adults with major depression (n = 11) to that in age- and sex-matched nondepressed controls (n = 11) and in a larger group of nondepressed adults who were > or = 60 years old. VZV-specific RCF in depressed patients was markedly reduced compared with the RCF in matched controls (t = 2.7, P < .02). In fact, the levels of VZV-specific RCF in the depressed patients were comparable in magnitude to the low levels found in adults > or = 60 years of age. These data indicate that major depression is associated with a marked decline in VZV-specific cellular immunity.

Adult↗
PubMed1998

Cell-to-cell spread of poliovirus in the spinal cord of bonnet monkeys (Macaca radiata).

In order to study the spread of poliovirus in the spinal cord of bonnet monkeys, 10(8) TCID50 Mahoney strain of poliovirus was inoculated into the ulnar nerves of monkeys that were subsequently autopsied on days 1, 2, 3, 6, 9, 12, 14, 15 and 16 postinoculation (p.i.). Virus spread in the spinal cord, the accompanying histopathological changes and paralysis occurred in a cervico-thoraco-lumbar direction. Virus reached the cervical region of the spinal cord within the first 3 days and subsequently spread to all segments of the spinal cord. In situ hybridization demonstrated viral RNA initially in the cervical neurons on day 3 p.i. and in the anterior horn neurons of lumbar segments of the spinal cord by day 6 p.i. Loss of Nissl substance in some of the anterior horn neurons was apparent on day 3 p.i. in the cervical and thoracic regions and by day 6 p.i. in the lumbar region. In the lumbar region, neuronophagia was a consistent feature which was observed on days 6-9 p.i., followed by neuronal dropouts on day 12 p.i. and thereafter. In the cervical and thoracic region, reappearance of Nissl substance was apparent from day 12 p.i. Upper limb paralysis preceded lower limb paralysis (5.5+/-1.73 vs 8.18+/-2.18, P = 0.046), further suggesting that virus spread within the spinal cord was via an intraneural route despite persistent viraemia detectable from day 2 p.i. onwards. The temporal distribution of the virus spread, distribution of viral RNA, histopathological and clinical changes indicate a cell-to-cell spread of poliovirus in the CNS, having gained access to the CNS from the peripheral nerve.

Animals↗